Repository: walaj/svaba Branch: master Commit: 645fab67c906 Files: 399 Total size: 131.2 MB Directory structure: gitextract_ugvk6jc2/ ├── CLAUDE.md ├── CMakeLists.txt ├── Dockerfile ├── LICENSE ├── R/ │ └── archive_non_functional/ │ ├── create-databases.R │ ├── gen_quals.R │ ├── svaba-annotate.R │ ├── svaba-asqg2pdf.R │ ├── svaba-bam-qcplot.R │ ├── svaba-benchmark.R │ ├── svaba-bps-to-maflite.R │ ├── svaba-circos.R │ ├── svaba-create-pon.R │ ├── svaba-event-plot.R │ ├── svaba-histogram.R │ ├── svaba-nozzle.R │ ├── svaba-sig.R │ └── svaba-vcf-to-maflite.R ├── README.md ├── docs/ │ ├── README.md │ ├── alignments_viewer.html │ ├── app.js │ ├── bps_explorer.html │ ├── bps_viewer.html │ ├── comparison.html │ ├── index.html │ ├── learn_explorer.html │ ├── r2c_explorer.html │ ├── runtime_explorer.html │ └── styles.css ├── notes ├── opt/ │ ├── jemalloc_test.sh │ ├── memprof.sh │ ├── memprof_osx.sh │ ├── memusg.sh │ ├── profiler.sh │ └── runtime.R ├── scripts/ │ ├── combine_blacklists.sh │ ├── extract_by_qname.sh │ ├── extract_discordants.sh │ ├── extract_pairs_by_seq.sh │ ├── filter_contig_supporting_reads.sh │ ├── gcloud_teardown.sh │ ├── mosdepth_lowmapq_blacklist.sh │ ├── plot_learn.sh │ ├── r2c_for_contig.sh │ ├── search_sequence.sh │ ├── sort_and_dedupe_bps_old.sh │ ├── sort_bps.sh │ ├── svaba_cloud.sh │ ├── svaba_local_function.sh │ ├── svaba_postprocess.sh │ └── update_svaba_image.sh ├── src/ │ ├── SGA/ │ │ ├── Algorithm/ │ │ │ ├── ClusterProcess.cpp │ │ │ ├── ClusterProcess.h │ │ │ ├── ConnectProcess.cpp │ │ │ ├── ConnectProcess.h │ │ │ ├── DPAlignment.cpp │ │ │ ├── DPAlignment.h │ │ │ ├── ErrorCorrectProcess.cpp │ │ │ ├── ErrorCorrectProcess.h │ │ │ ├── ExtensionDP.cpp │ │ │ ├── ExtensionDP.h │ │ │ ├── FMMergeProcess.cpp │ │ │ ├── FMMergeProcess.h │ │ │ ├── GapFillProcess.cpp │ │ │ ├── GapFillProcess.h │ │ │ ├── HaplotypeBuilder.cpp │ │ │ ├── HaplotypeBuilder.h │ │ │ ├── KmerOverlaps.cpp │ │ │ ├── KmerOverlaps.h │ │ │ ├── LRAlignment.cpp │ │ │ ├── LRAlignment.h │ │ │ ├── Makefile.am │ │ │ ├── Makefile.in │ │ │ ├── OverlapAlgorithm.cpp │ │ │ ├── OverlapAlgorithm.h │ │ │ ├── OverlapBlock.cpp │ │ │ ├── OverlapBlock.h │ │ │ ├── OverlapTools.cpp │ │ │ ├── OverlapTools.h │ │ │ ├── QCProcess.cpp │ │ │ ├── QCProcess.h │ │ │ ├── ReadCluster.cpp │ │ │ ├── ReadCluster.h │ │ │ ├── SearchHistory.cpp │ │ │ ├── SearchHistory.h │ │ │ ├── SearchSeed.cpp │ │ │ ├── SearchSeed.h │ │ │ ├── StatsProcess.cpp │ │ │ ├── StatsProcess.h │ │ │ ├── StringGraphGenerator.cpp │ │ │ ├── StringGraphGenerator.h │ │ │ ├── StringThreader.cpp │ │ │ ├── StringThreader.h │ │ │ ├── VariationBuilderCommon.cpp │ │ │ └── VariationBuilderCommon.h │ │ ├── Bigraph/ │ │ │ ├── Bigraph.cpp │ │ │ ├── Bigraph.h │ │ │ ├── Edge.cpp │ │ │ ├── Edge.h │ │ │ ├── EdgeDesc.cpp │ │ │ ├── EdgeDesc.h │ │ │ ├── GraphCommon.h │ │ │ ├── Makefile.am │ │ │ ├── Makefile.in │ │ │ ├── Vertex.cpp │ │ │ └── Vertex.h │ │ ├── SGA/ │ │ │ ├── Makefile.am │ │ │ ├── Makefile.in │ │ │ ├── OverlapCommon.cpp │ │ │ ├── OverlapCommon.h │ │ │ ├── SGACommon.h │ │ │ ├── index.cpp │ │ │ ├── index.h │ │ │ ├── overlap.cpp │ │ │ └── overlap.h │ │ ├── SQG/ │ │ │ ├── ASQG.cpp │ │ │ ├── ASQG.h │ │ │ ├── Makefile.am │ │ │ ├── Makefile.in │ │ │ ├── SQG.cpp │ │ │ └── SQG.h │ │ ├── StringGraph/ │ │ │ ├── CompleteOverlapSet.cpp │ │ │ ├── CompleteOverlapSet.h │ │ │ ├── GraphSearchTree.h │ │ │ ├── Makefile.am │ │ │ ├── Makefile.in │ │ │ ├── RemovalAlgorithm.cpp │ │ │ ├── RemovalAlgorithm.h │ │ │ ├── SGAlgorithms.cpp │ │ │ ├── SGAlgorithms.h │ │ │ ├── SGSearch.cpp │ │ │ ├── SGSearch.h │ │ │ ├── SGUtil.cpp │ │ │ ├── SGUtil.h │ │ │ ├── SGVisitors.cpp │ │ │ ├── SGVisitors.h │ │ │ ├── SGWalk.cpp │ │ │ └── SGWalk.h │ │ ├── SuffixTools/ │ │ │ ├── BWT.h │ │ │ ├── BWTAlgorithms.cpp │ │ │ ├── BWTAlgorithms.h │ │ │ ├── BWTCABauerCoxRosone.cpp │ │ │ ├── BWTCABauerCoxRosone.h │ │ │ ├── BWTCARopebwt.cpp │ │ │ ├── BWTCARopebwt.h │ │ │ ├── BWTDiskConstruction.cpp │ │ │ ├── BWTDiskConstruction.h │ │ │ ├── BWTIndexSet.h │ │ │ ├── BWTInterval.h │ │ │ ├── BWTIntervalCache.cpp │ │ │ ├── BWTIntervalCache.h │ │ │ ├── BWTReader.cpp │ │ │ ├── BWTReader.h │ │ │ ├── BWTReaderAscii.cpp │ │ │ ├── BWTReaderAscii.h │ │ │ ├── BWTReaderBinary.cpp │ │ │ ├── BWTReaderBinary.h │ │ │ ├── BWTTraverse.cpp │ │ │ ├── BWTTraverse.h │ │ │ ├── BWTWriter.cpp │ │ │ ├── BWTWriter.h │ │ │ ├── BWTWriterAscii.cpp │ │ │ ├── BWTWriterAscii.h │ │ │ ├── BWTWriterBinary.cpp │ │ │ ├── BWTWriterBinary.h │ │ │ ├── FMMarkers.h │ │ │ ├── GapArray.cpp │ │ │ ├── GapArray.h │ │ │ ├── HitData.h │ │ │ ├── InverseSuffixArray.cpp │ │ │ ├── InverseSuffixArray.h │ │ │ ├── Makefile.am │ │ │ ├── Makefile.in │ │ │ ├── Occurrence.cpp │ │ │ ├── Occurrence.h │ │ │ ├── PopulationIndex.cpp │ │ │ ├── PopulationIndex.h │ │ │ ├── QuickBWT.cpp │ │ │ ├── QuickBWT.h │ │ │ ├── RLBWT.cpp │ │ │ ├── RLBWT.h │ │ │ ├── RLUnit.h │ │ │ ├── RankProcess.cpp │ │ │ ├── RankProcess.h │ │ │ ├── SACAInducedCopying.cpp │ │ │ ├── SACAInducedCopying.h │ │ │ ├── SAReader.cpp │ │ │ ├── SAReader.h │ │ │ ├── SAWriter.cpp │ │ │ ├── SAWriter.h │ │ │ ├── SBWT.cpp │ │ │ ├── SBWT.h │ │ │ ├── STCommon.cpp │ │ │ ├── STCommon.h │ │ │ ├── STGlobals.h │ │ │ ├── SampledSuffixArray.cpp │ │ │ ├── SampledSuffixArray.h │ │ │ ├── SparseGapArray.h │ │ │ ├── SuffixArray.cpp │ │ │ ├── SuffixArray.h │ │ │ ├── SuffixCompare.cpp │ │ │ └── SuffixCompare.h │ │ └── Util/ │ │ ├── Alphabet.cpp │ │ ├── Alphabet.h │ │ ├── BWT4Codec.h │ │ ├── BWTCodec.h │ │ ├── Bamreader.cpp │ │ ├── BitChar.cpp │ │ ├── BitChar.h │ │ ├── BitVector.cpp │ │ ├── BitVector.h │ │ ├── BloomFilter.cpp │ │ ├── BloomFilter.h │ │ ├── ClusterReader.cpp │ │ ├── ClusterReader.h │ │ ├── Contig.cpp │ │ ├── Contig.h │ │ ├── CorrectionThresholds.cpp │ │ ├── CorrectionThresholds.h │ │ ├── DNACodec.h │ │ ├── DNADouble.h │ │ ├── DNAString.cpp │ │ ├── DNAString.h │ │ ├── EncodedString.h │ │ ├── HashMap.h │ │ ├── Interval.cpp │ │ ├── Interval.h │ │ ├── IntervalTree.h │ │ ├── KmerDistribution.cpp │ │ ├── KmerDistribution.h │ │ ├── Makefile.am │ │ ├── Makefile.in │ │ ├── Match.cpp │ │ ├── Match.h │ │ ├── Metrics.h │ │ ├── MultiAlignment.cpp │ │ ├── MultiAlignment.h │ │ ├── MultiOverlap.cpp │ │ ├── MultiOverlap.h │ │ ├── NoCodec.h │ │ ├── Pileup.cpp │ │ ├── Pileup.h │ │ ├── PrimerScreen.cpp │ │ ├── PrimerScreen.h │ │ ├── Profiler.h │ │ ├── Quality.cpp │ │ ├── Quality.h │ │ ├── QualityCodec.h │ │ ├── QualityTable.cpp │ │ ├── QualityTable.h │ │ ├── QualityVector.cpp │ │ ├── QualityVector.h │ │ ├── Read2Contig.h │ │ ├── ReadInfoTable.cpp │ │ ├── ReadInfoTable.h │ │ ├── ReadTable.cpp │ │ ├── ReadTable.h │ │ ├── ReadTableNew.cpp │ │ ├── ReadTableNew.h │ │ ├── ReadTableS.h │ │ ├── SGAStats.cpp │ │ ├── SGAStats.h │ │ ├── SeqCoord.cpp │ │ ├── SeqCoord.h │ │ ├── SeqReader.cpp │ │ ├── SeqReader.h │ │ ├── SimpleAllocator.h │ │ ├── SimplePool.h │ │ ├── StdAlnTools.cpp │ │ ├── StdAlnTools.h │ │ ├── Timer.h │ │ ├── Util.cpp │ │ ├── Util.h │ │ ├── VCFUtil.cpp │ │ ├── VCFUtil.h │ │ ├── VariantIndex.cpp │ │ ├── VariantIndex.h │ │ ├── Verbosity.h │ │ ├── bamreader.h │ │ ├── bucketSort.cpp │ │ ├── bucketSort.h │ │ ├── gzstream.C │ │ ├── gzstream.h │ │ ├── mkqs.h │ │ ├── old.AlignedContig.h │ │ ├── stdaln.c │ │ └── stdaln.h │ ├── svaba/ │ │ ├── AlignedContig.cpp │ │ ├── AlignedContig.h │ │ ├── AlignmentFragment.cpp │ │ ├── AlignmentFragment.h │ │ ├── BamStats.cpp │ │ ├── BamStats.h │ │ ├── BreakPoint.cpp │ │ ├── BreakPoint.h │ │ ├── ContigAlignmentScore.cpp │ │ ├── ContigAlignmentScore.h │ │ ├── DBSnpFilter.cpp │ │ ├── DBSnpFilter.h │ │ ├── DiscordantCluster.cpp │ │ ├── DiscordantCluster.h │ │ ├── DiscordantRealigner.cpp │ │ ├── DiscordantRealigner.h │ │ ├── Histogram.cpp │ │ ├── Histogram.h │ │ ├── KmerFilter.cpp │ │ ├── KmerFilter.h │ │ ├── LearnBamParams.cpp │ │ ├── LearnBamParams.h │ │ ├── ReadToContigAligner.h │ │ ├── STCoverage.cpp │ │ ├── STCoverage.h │ │ ├── SvabaASQG.cpp │ │ ├── SvabaASQG.h │ │ ├── SvabaAssemble.cpp │ │ ├── SvabaAssemble.h │ │ ├── SvabaAssemblerConfig.h │ │ ├── SvabaAssemblerEngine.cpp │ │ ├── SvabaAssemblerEngine.h │ │ ├── SvabaBamWalker.cpp │ │ ├── SvabaBamWalker.h │ │ ├── SvabaDebug.h │ │ ├── SvabaFileLoader.cpp │ │ ├── SvabaFileLoader.h │ │ ├── SvabaLogger.cpp │ │ ├── SvabaLogger.h │ │ ├── SvabaModels.cpp │ │ ├── SvabaModels.h │ │ ├── SvabaOptions.cpp │ │ ├── SvabaOptions.h │ │ ├── SvabaOutputWriter.cpp │ │ ├── SvabaOutputWriter.h │ │ ├── SvabaOverlapAlgorithm.cpp │ │ ├── SvabaOverlapAlgorithm.h │ │ ├── SvabaPostprocess.cpp │ │ ├── SvabaPostprocess.h │ │ ├── SvabaRead.cpp │ │ ├── SvabaRead.h │ │ ├── SvabaRegionProcessor.cpp │ │ ├── SvabaRegionProcessor.h │ │ ├── SvabaSharedConfig.h │ │ ├── SvabaThreadUnit.cpp │ │ ├── SvabaThreadUnit.h │ │ ├── SvabaUtils.cpp │ │ ├── SvabaUtils.h │ │ ├── refilter.cpp │ │ ├── refilter.h │ │ ├── run_svaba.cpp │ │ ├── svaba.cpp │ │ ├── test_svaba.cpp │ │ ├── threadpool.h │ │ ├── tovcf.cpp │ │ ├── vcf.cpp │ │ └── vcf.h │ └── svabautils/ │ ├── AssemblyBamWalker.cpp │ ├── AssemblyBamWalker.h │ ├── BamSplitter.cpp │ ├── BamSplitter.h │ ├── Fractions.cpp │ ├── Fractions.h │ ├── Makefile.am │ ├── Makefile.in │ ├── PowerLawSim.cpp │ ├── PowerLawSim.h │ ├── ReadSim.cpp │ ├── ReadSim.h │ ├── SeqFrag.cpp │ ├── SeqFrag.h │ ├── SimGenome.cpp │ ├── SimGenome.h │ ├── SimTrainerWalker.cpp │ ├── SimTrainerWalker.h │ ├── assembly2vcf.cpp │ ├── assembly2vcf.h │ ├── benchmark.cpp │ ├── benchmark.h │ ├── configure │ ├── configure.ac │ ├── snowmanutils.cpp │ ├── snowtools.cpp │ ├── splitcounter.cpp │ └── splitcounter.h ├── svaba_jemalloc └── tracks/ ├── README.md ├── genome.hg38.sorted.bed ├── hg38.bed ├── hg38.blacklist.sorted.bed ├── hg38.combined_blacklist.bed ├── hg38.high_runtime.bed ├── hg38.manual.blacklist.bed ├── hg38.nonstd_chr.blacklist.bed ├── hg38.rmsk.simple_repeat.bed ├── hg38_arms_excl_centromeres.bed ├── lowmap30perc.bed ├── lowmap50perc.bed ├── lowmap70perc.bed └── region_generator.R ================================================ FILE CONTENTS ================================================ ================================================ FILE: CLAUDE.md ================================================ # CLAUDE.md — svaba working notes This file captures conventions, file landmarks, and open investigations for the svaba SV/indel caller project so future sessions can pick up quickly. Update it as understanding changes — it's the crash-safety net, not the README. ## Project at a glance svaba is a structural variant (SV) and indel caller that uses local assembly + read realignment to call variants from short-read BAMs. The canonical use case is tumor/normal somatic calling, but it also supports germline and multi-sample modes. Top-level layout: - `src/svaba/` — main C++ sources. Entry point is `run_svaba.cpp`; assembly, realignment, breakpoint scoring, VCF output, and postprocess all live here. File-naming convention is **PascalCase** — e.g. `SvabaOptions.cpp`, `SvabaOutputWriter.h`. A few intentional exceptions: `refilter.cpp/h`, `run_svaba.cpp`, `svaba.cpp`, `threadpool.h`, `tovcf.cpp`, `vcf.cpp/h`. - `src/SGA/` — String Graph Assembler sources (vendored). - `SeqLib/` — vendored htslib/bwa/fermi-lite wrapper used for BAM I/O, alignment, and assembly primitives. See "Build system" for how its flags get set. - `bin/`, `build/` — build artifacts; don't edit by hand. - `R/`, `viewer/`, `tracks/` — downstream analysis/visualization helpers. - `tests/`, `example_data/` — test fixtures. - `scripts/` — post-processing and utility shell helpers, all kept here (not at the repo root): `svaba_postprocess.sh`, `combine_blacklists.sh`, `extract_discordants.sh`, `filter_contig_supporting_reads.sh`, `r2c_for_contig.sh`, `sort_bps.sh`, `svaba_cloud.sh`. Profiling helpers (`memprof*.sh`) live under `opt/` (the user's ad-hoc tooling dir). - `somlod_maxlod_analysis.html` — deep-dive writeup of the somatic log-odds scoring model. See "Statistical model" below. Scripts that used to live here and are gone, in case you're looking for them: `sort_output.sh` and `sort_and_deduplicate_bps.sh` were subsumed into the unified `svaba_postprocess.sh`. ## Build system `cmake -B build && cmake --build build` defaults to `CMAKE_BUILD_TYPE = RelWithDebInfo`, which gives you **`-O2 -g -DNDEBUG -fno-omit-frame-pointer`** for the svaba/SeqLib/SGA C++ code. Not O0, not O3. Critical gotcha: **the `-O2` is hardcoded across all of the vendored submodules**, and CMake's build-type doesn't reach them: - `SeqLib/bwa/Makefile`: `CFLAGS = -g -Wall -Wno-unused-function -O2` (hardcoded; doesn't read the parent CMake). Makes `libbwa.a`, owner of ~38% of wall-time in real runs. - `SeqLib/fermi-lite/Makefile`: `CFLAGS = -g -Wall -O2 -Wno-unused-function` (same story). Makes `libfml.a`, ~27% of wall-time. - `SeqLib/CMakeLists.txt:10` does `set(CMAKE_CXX_FLAGS "${CMAKE_CXX_FLAGS} -O2")` — appends `-O2` to whatever you passed, and because later `-O` wins on gcc/clang, this silently defeats any top-level attempt to set `-O3` via `CMAKE_CXX_FLAGS` unless you pass it via `CMAKE_CXX_FLAGS_` (which is appended after, and therefore wins). - htslib is external; whoever built yours picked its flags. To push the submodules to `-O3 -mcpu=native` (the single biggest free-perf knob on Apple Silicon — 65% of compute is at -O2 generic right now): ```bash make -C SeqLib/bwa clean make -C SeqLib/fermi-lite clean make -C SeqLib/bwa -j CFLAGS="-g -O3 -mcpu=native -fno-omit-frame-pointer -Wall -Wno-unused-function" make -C SeqLib/fermi-lite -j CFLAGS="-g -O3 -mcpu=native -fno-omit-frame-pointer -Wall -Wno-unused-function" cd build && make -j ``` Sanity-check: `make VERBOSE=1 2>&1 | grep -oE -- "-O[0-9sg]" | sort | uniq -c`. ## Statistical model — the files that matter For anything related to how variants are scored (LOD, somatic vs germline, error model), the two files to read first are: - `src/svaba/SvabaModels.cpp` / `.h` — self-contained statistical primitives. - `LogLikelihood(d, a, f, e_fwd, e_rev)` (~lines 11-63) is the per-sample two-state error model: `p_ref = (1-f)(1-e_fwd) + f*e_rev` and `p_alt = f(1-e_rev) + (1-f)*e_fwd`. Returns log10 likelihood of observing `a` alt reads out of `d`. This is the primitive every higher-level score is built from. - `SomaticLOD(...)` (~lines 70-83) — public wrapper; forwards to the split-error implementation. - `SomaticLOD_withSplitErrors(...)` (~lines 86-189) — the active somatic model. Enumerates sub-hypotheses: `SOM_true`, `SOM_art`, `GERM_het`, `GERM_hom`, `GERM_art`, `GERM_shared`. Returns `log10( P(somatic) / P(any non-somatic) )`. - Line ~79: `const double eN_fwd = std::min(e_art_fwd, 0.005);` — hard cap on the normal-sample forward error rate. This cap is important: it means even in regions where the artifact model infers a high error rate, the normal sample is assumed to be clean. That's the knob you'd relax if you want somlod to be "artifact-aware" on the normal side. - Lines ~138-147: `GERM_shared` free-MLE branch. This is the sub-hypothesis that fits a single pooled allele fraction across tumor+normal. It exists to catch LOH (loss of heterozygosity) germline events where tumor VAF can be much higher than 0.5 while normal is still ~0.5. It is also the main reason `somlod` asymptotes slowly as tumor alt-support grows (see below). - `src/svaba/BreakPoint.cpp` / `.h` — per-breakpoint scoring glue. Points of interest: - `BreakPoint::score_somatic()` at ~line 975 is the entry point that sets `LO_s` (the somatic LOD). Calls `SvabaModels::SomaticLOD(scaled_alt_n, a_cov_n, scaled_alt_t, a_cov_t, error_fwd, error_rev)` around lines 1028-1031. - `SampleInfo::modelSelection()` (~lines 1562-1674) computes the per-sample `LO = ll_alt - ll_err` at line ~1610. These are unnormalized log10 likelihoods — absolute value is not meaningful, the difference is. - `max_lod` is computed at lines ~198-200 and ~1196-1198 as the max of `al.LO` across samples. This is the "is this artifact or not" score; it does grow with additional supporting reads because it compares a variant hypothesis to a pure-error hypothesis with no germline branch. - Lines ~1072-1073: the current INDEL somatic gate only tests `somlod` — it does not use `maxlod` as a co-gate. This is one of the levers in the proposed fixes. ## The somlod / maxlod investigation (still open) **Problem statement.** Users observe that `maxlod` grows with tumor alt support, but `somlod` barely moves once tumor alt-support gets high. For a clean normal (`aN=0` or `aN=1`) you'd naively expect `somlod` to also keep climbing as tumor evidence accumulates, but it asymptotes around ~9 for `dN ≈ 30`. **Diagnosis.** - There is a real statistical ceiling on somlod roughly equal to `dN · log10(1/(1-fT_hat))`. With 30 normal reads you can only ever rule out a shared germline hypothesis so hard — it's a finite amount of evidence. - The `GERM_shared` free-MLE branch makes `somlod` *approach* that ceiling slowly at sub-clonal tumor VAFs: when the MLE of a pooled AF lands in a germline-plausible band, it provides a strong non-somatic explanation that the somatic hypothesis has to beat. - You cannot simply delete `GERM_shared` because doing so makes LOH germline cases (e.g. `aN=15/dN=30, aT=285/dT=300`) return `somlod ≈ +36`, a false positive. Verified numerically while writing the analysis. - The `eN_fwd ≤ 0.005` cap means that in truly high-artifact regions, the model refuses to "forgive" 2-3 alt reads in the normal as artifacts, which is both good (prevents somatic false positives where normal is contaminated by real signal) and limiting (prevents somatic calls where the artifact model really does explain the normal reads). **Proposed fixes (see `somlod_maxlod_analysis.html` for the full writeup).** - Fix 1 — disjunction gate on `GERM_shared`: only let the free-MLE pooled branch influence somlod when `shared_is_germline_plausible || normal_evidence > 1.0`, where `normal_evidence = LL_N(dN, aN, f_n_mle) - LL_N(dN, aN, 0)`. This is error-rate aware and is the fix I'd land first. - Fix 2 — loosen `eN_fwd` cap in known high-artifact regions. - Fix 3 — BIC penalty on the free-MLE branch (1 free parameter costs ~`0.5 log10(dN+dT)` nats ≈ bits of evidence). - Fix 4 — joint `maxlod` + `somlod` gate for INDELs (require both above threshold), since `maxlod` moves freely with tumor depth. - Fix 5 — debug dump of sub-hypothesis LLs when `somlod` is within some epsilon of the gate, to make future failures diagnosable. **Related fix landed already (SvABA2.0 v3 split-coverage gate):** the old `both_split && homlen > 0` / `one_split && homlen == 0` branching in `BreakPoint::splitCoverage` was removed. A read is now credited as a split-supporter iff (a) its r2c alignment scores strictly higher than its native alignment (`r2c_score > native_score`, no percentage margin — see `src/svaba/SvabaOptions.h`), and (b) it spans at least one breakend on the contig. Long junction homology → r2c and native tie → read doesn't credit either sample, which is the correct conservative behavior (rather than the old "homology=0 one_split is fine, homology>0 you need both_split" which nuked normal support specifically when homology was long, biasing toward somatic calls). The repeat_seq-length padding on the buffers is also gone — same rationale, subsumed by the comparative score gate. See the user- facing bp-id (v3 schema) work for how to trace a specific read's current support attribution end-to-end. **v3.1 fix — remove T_R2C_MIN_MARGIN (set to 0):** the 10% `T_R2C_MIN_MARGIN` was killing all tumor alt-supporting reads for small indels. A 1bp deletion on a 150bp read gives r2c=150 vs native=143, a 4.9% improvement — mathematically impossible to clear the 10% threshold. Traced via the `SVABA_TRACE_CONTIG` system on contig `c_fermi_chr2_215869501_215894501_13C` (CIGAR `392M1D530M`): every tumor read hit TP8 with r2c=150 vs threshold=157.3 → SKIP → 0 split support → LOWLOD → hasMinimal fail → variant dropped. Fix: set `T_R2C_MIN_MARGIN` to 0.0 in `SvabaOptions.h` — same as normal, strict greater-than only. Any percentage margin is inherently read-length-dependent: a 1bp del gives 4.9% improvement on 150bp reads but only 2.9% on 250bp reads, so any fixed percentage either blocks long reads or is too loose for short reads. There's no percentage that works across all read lengths. The margin was belt-and-suspenders on top of the LOD model. In the junction-homology case it was designed for: if both tumor and normal credit borderline reads equally (r2c barely > native by 1-2 points), the downstream LOD model sees similar split support in both samples → low somlod → correctly not called somatic. Normal already used margin=0, so the asymmetry was the only thing preventing normal from crediting those reads — and it wasn't, because N_R2C_MIN_MARGIN was always 0. The somatic/germline distinction is the LOD model's job. **Important correctness notes (earned the hard way):** - Don't propose `aN >= 2` style hard count gates without an error-rate adjustment. In a high-artifact region, 2-3 normal alt reads can be genuine artifacts, and gating on raw counts overcalls the case away. Always reason about `normal_evidence` (LL delta against `f=0`) instead of raw `aN`. - A LL ratio reported by `LogLikelihood` is log10. Multiplying by ~3.32 gives bits. Per-read surprise is `(LL_alt - LL_ref) / d`. Absolute LL values have no meaning — always compare two hypotheses at the same data. - The ~9 ceiling at `dN=30` is a real statistical bound; no reformulation of the somatic test can push above it. `GERM_shared` changes the *slope* of approach, not the asymptote. ## Postprocess pipeline Everything post-`svaba run` is orchestrated by `scripts/svaba_postprocess.sh`. It's the unified replacement for the old `sort_output.sh` + `sort_and_deduplicate_bps.sh` pair, which no longer exist. Five steps per invocation, all idempotent (missing inputs log a one-liner and continue): 1. **Merge per-thread BAMs** — `${ID}.thread*.${suffix}.bam` → `${ID}.${suffix}.bam` for `discordant` / `weird` / `corrected`. Single-file inputs are moved; no-file inputs silently skipped. 2. **`svaba postprocess`** — the C++ subcommand in `src/svaba/SvabaPostprocess.cpp`. For each suffix (`weird`, `corrected`, `discordant`, `contigs`): - `samtools sort -@ per_job_threads -m MEM` (shell out — htslib doesn't expose its sort as a library call). **Auto-skipped** when the BAM already declares `@HD SO:coordinate` — `isCoordinateSorted()` inspects the header via `readHeaderOnly()` and logs "already coordinate-sorted; skipping sort" so reruns are a no-op. - Native streaming dedup (only for `weird`/`corrected`/`discordant`): reads BAM via SeqLib::BamReader, collapses exact (qname, flag) duplicates at each locus, and **unions their `bi:Z` / `bz:Z` comma-token lists** so alt-supporting-contig evidence isn't lost when the same read got emitted by two overlapping assembly windows. Key function: `mergeCommaTokens` — boundary-aware union, mirrors `SvabaOutputWriter::stamp_tag`. Both the reader and writer have the htslib BGZF thread pool enabled via a new `SeqLib::BamReader::SetThreads(int)` / `SeqLib::BamWriter::SetThreads(int)` API that calls `hts_set_threads(fp, n)`. `streamDedup` accepts a `threads` parameter (wired from the full postprocess budget, not the per-suffix slice — see two-phase note below) and applies it on both sides. Without this pool, BGZF decompress + compress is single-threaded and dominates wall time (40 GB BAM → ~2 hours). With `-t 4..8` this typically drops to 25–40 min, a 3–5× speedup. **Buckets-clear gotcha** (landed alongside the thread-pool fix): the per-locus `idx_by_key` is a `std::unordered_map`. `unordered_map::clear()` is **O(bucket_count)** — it memsets the entire bucket array even when size is 0, and `bucket_count` only grows, never shrinks. One pileup locus (centromere / simple repeat / HLA) inflates buckets to 100k+ for the rest of the BAM, and every subsequent locus transition (hundreds of millions of them) pays that memset. Pre-fix perf showed 95% of main-thread CPU going to `__memset_evex_unaligned_erms` called from `unordered_map::clear`. `flushLocus` now swaps with a fresh small map when `bucket_count() > 256`, paying the inflated-map destructor cost ONCE per pileup exit rather than once per locus. Lesson: never trust `unordered_map::clear()` in a transient reuse pattern where the map briefly inflates. **Two-phase driver.** `svaba postprocess` runs in two phases so the thread budget lands where it actually helps: Phase 1 (PARALLEL): samtools sort across all active suffixes concurrently, each worker with `o.threads / n_active` threads. Sort is disk+CPU bound and scales linearly across files. Phase 2 (SERIAL): dedup + reheader + index, one BAM at a time, each with the full `o.threads` as its BGZF pool. Serial here is deliberate — running dedup in parallel across suffixes would oversubscribe (each BAM needs its own read+write BGZF pool), and BGZF parallelism has diminishing returns so `4 workers × 2 threads` is worse wall-clock than `1 worker × 8 threads` iterated four times. **Idempotency.** Every phase has its own auto-skip so rerunning the pipeline on an already-finished BAM is essentially instant: - Phase 1 sort: `isCoordinateSorted()` inspects `@HD SO:coordinate` and bypasses the sort when already done. - Phase 2 dedup: `hasSvabaPostprocessPg()` scans the @PG chain for any `svaba_postprocess` (or uniquified `.1`, `.2` variants); if present, dedup AND the subsequent reheader step are both skipped (only the `.bai` is rebuilt, which is cheap and covers the missing-index case). The first successful `streamDedup` stamps the PG line, so a second `svaba_postprocess.sh` run on the same outputs no-ops almost entirely. - The shell-layer merge step (`scripts/svaba_postprocess.sh` step 1) is already a no-op when per-thread `.thread*.bam` files aren't present (nothing to merge), so all three steps compose naturally. - **@PG stamp**: writes an `@PG ID:svaba_postprocess PN:svaba VN: CL: PP:` line into the output header. For dedup-eligible suffixes this is free (done in the writer during dedup); for `contigs` (no dedup) it's done via `samtools reheader`. ID is auto-uniquified if `svaba postprocess` has been run before on the same BAM. - **BAI index** via `sam_index_build(fn, 0)` directly from htslib — one less subprocess. - Intermediate filenames use a `.postprocess.*.tmp.bam` suffix and are renamed over on success / unlinked on failure. End state per suffix is exactly `${ID}.${suffix}.bam(.bai)`; no `.sorted` / `.deduped` flotsam. 3. **Sort + dedup + PASS-filter `bps.txt.gz`** — external-memory sort via GNU sort (`gsort` on macOS from homebrew coreutils). Sort keys: `chr1(V), pos1(n), strand1, chr2(V), pos2(n), strand2, maxlod(gr)` — maxlod descending so the "best SV per junction" survives the dedup. Produces three files: `.bps.sorted.txt.gz`, `.bps.sorted.dedup.txt.gz` (one row per unique breakpoint pair), `.bps.sorted.dedup.pass.txt.gz` (col 32 == "PASS" only). - Column positions hard-coded: `col 30 = cname (contig_and_region)`, `col 32 = confidence`, `col 38 = maxlod`. These come from `BreakPoint::toFileString` — change there and the script breaks. 4. **Filter `r2c.txt.gz` to PASS contigs (+ PASS-somatic subset)** — resolves PASS cnames from `bps.txt.gz` (col 32 == "PASS") and the PASS-somatic subset (also col 37, `somlod`, >= 1) in one pass. Then one pass over `r2c.txt.gz` writes two outputs via awk pipe-to-command (gzip compressors run in parallel as child processes): - `${ID}.r2c.pass.txt.gz` (all PASS) - `${ID}.r2c.pass.somatic.txt.gz` (PASS ∩ somlod >= 1) Both are cname-keyed; contig and read rows survive together since they share col 2. Either is suitable for `bps_explorer.html`'s alignments sub-panel; the somatic subset is the lighter load when you only care about the tumor-specific calls. 5. **Optional split-by-source** — `--split-by-source` (or env `SPLIT_BY_SOURCE=1`) demuxes the deduped BAMs by the first 4 chars of each QNAME into `${ID}.${suffix}.${prefix}.bam`. CLI: `scripts/svaba_postprocess.sh -t THREADS -m MEM [other flags] `. Flags: `-t/--threads`, `-m/--mem`, `--sort-buffer`, `--split-by-source`, `--input-dir`, `--output-dir`, `--svaba`, `--keep-tmp`, `--skip-bam`, `--skip-dedup`, `--skip-bps`, `--skip-r2c`, `--skip-split`, `-h/--help`. `--skip-dedup` maps to `--sort-only` on the C++ CLI: keeps sort + @PG + index but skips the dedup pass. Combined with the C++'s auto-skip of sort when the BAM header already declares `@HD SO:coordinate` (`isCoordinateSorted()` in `SvabaPostprocess.cpp`), a rerun on already-postprocessed files is effectively instant — useful for refreshing just the index or PG stamp. Env fallbacks for backward compat: `THREADS`, `MEM`, `BUFFER_SIZE`, `SVABA`, `SAM`, `INPUT_DIR`, `OUTPUT_DIR`, `SPLIT_BY_SOURCE`, `KEEP_TMP`. Gotcha: `zcat` on macOS is BSD zcat (looks for `.Z`), not GNU. This script uses `gzip -dc` everywhere for portability — if you add a new decompression step, do the same or it'll fail on Mac. ## `svaba tovcf` subcommand Standalone converter: pre-deduplicated `bps.txt.gz` → two VCFv4.5 files (`${id}.sv.vcf.gz` + `${id}.indel.vcf.gz`). Lives in `src/svaba/tovcf.cpp`; dispatch wired into `src/svaba/svaba.cpp`. Runs the VCFFile parser with `skip_dedup=true` because the postprocess pipeline has already sorted and deduplicated the bps.txt.gz upstream — the internal interval-tree dedup would just be wasted work and can spuriously dup things via its SV-pileup blacklist. Design calls made (see `docs/vcf-design-decisions.md` if created; for now this is the reference): - **VCF spec:** declares `##fileformat=VCFv4.5` (latest formal spec, Oct 2024). Backwards-compatible at the record level with anything that accepts 4.2+. - **File structure:** one SV VCF + one indel VCF per sample-set. Every record (somatic + germline) goes into both files; somatic rows carry the `SOMATIC` flag INFO so `bcftools view -f .,PASS -i 'INFO/SOMATIC'` peels them off cleanly. Replaces the older 4-file split (sv.somatic / sv.germline / indel.somatic / indel.germline). - **SV representation:** intrachrom events with unambiguous orientation become single-record symbolic alleles — `+/+` or `-/-` → ``, `-/+` → ``, `+/-` → ``. Inter-chrom and anything else falls through to paired BND records with mate-bracket notation. Override with `--always-bnd` to force BND for every SV (useful if downstream tooling gets confused by symbolic alleles). Classification lives in `classify_symbolic_kind()` in `vcf.cpp`. - **EVENT grouping:** both BND records of a pair get `EVENT=` (taken from col 52 of bps.txt.gz, the v3 per-BP identifier). This uses the same namespace as `r2c.txt.gz`'s `split_bps`/`disc_bps` and the BAM `bi:Z` tag, so a user can follow a single variant across all svaba outputs with one key. - **QUAL column:** defaults to `.` (missing). QUAL was historically the Phred of `Σ per-sample LO`, which is strongly correlated with `INFO/MAXLOD` but can mislead users into filtering on QUAL when they should be filtering on SOMLOD / MAXLOD / FILTER. `.` is VCF-spec-valid missing and makes downstream tools fall through to the INFO fields where the canonical scores live. Override with `--qual maxlod` (writes `round(10 * maxlod)` capped at 99) or `--qual sum` (legacy). - **SVCLAIM:** per VCF 4.5. `J` for pure assembly-only SVs, `DJ` for ASDIS (both assembly + discordant evidence), `D` for discordant-only. svaba is junction-native so most calls end up `J` or `DJ`. Flag surface: ``` svaba tovcf -i BPS.txt.gz -b BAM -a ID [options] --sv-out FILE override SV path (default ${ID}.sv.vcf.gz) --indel-out FILE override indel path (default ${ID}.indel.vcf.gz) --plain write plain .vcf (no bgzip) --always-bnd force BND for every SV --qual MODE missing (default) | maxlod | sum --include-nonpass include FILTER != PASS records --dedup re-run legacy interval-tree dedup -v / --verbose -h / --help ``` Gotcha: the BAM is required only for the chromosome name/length table (used by contig `##contig` lines in the VCF header and by the BreakPoint parser to turn chrom-name strings into chr IDs). No reads are actually read from it — any BAM that shares the reference is fine. Not-yet-done on this subcommand: bgzip-proper (current `--plain=false` output is plain gzip, which bcftools accepts but tabix doesn't index correctly). For now, pipe through `bcftools sort -Oz` + `tabix -p vcf` after. If this becomes painful, revisit with htslib's `hts_open` + `vcf_write` path instead of ogzstream. ## BreakPoint IDs (v3 schema) Every BP gets a unique stable identifier of the form `bpTTTNNNNNNNN` where `TTT` is the 3-digit zero-padded worker thread ID and `NNNNNNNN` is an 8-digit zero-padded per-thread running counter. Generation is lock-free: the counter lives on `svabaThreadUnit` (see `next_bp_id()` in `src/svaba/SvabaThreadUnit.h`). Assignment happens exactly once per BP in `SvabaRegionProcessor::process()` right before the pointer is pushed into `unit.m_bps`. Because `AlignedContig` holds BPs via `shared_ptr`, the id mutation is immediately visible through every reference (global, multi-map, indel breaks). The id lands as the 52nd core column of `bps.txt.gz` (right after `flipped`, before per-sample blocks — this is the v3 schema; v2 had 51 cols, LEGACY had 41). It's also carried into `r2c.txt.gz` (see next section) so a read's support attribution is unambiguously linked to the exact BP row in bps.txt, eliminating the old "which BP on this contig did this read actually support?" puzzle. `svaba_bps_cols` (from `scripts/svaba_local_function.sh`) documents the full layout; column 52 is the bp_id field. **BAM aux tags `bi:Z` / `bz:Z` (v3).** The two aux tags svaba stamps on weird/discordant/corrected BAM outputs now live in *different* identifier namespaces — choose the right one for the join you want: - `bi:Z` — comma-joined list of **bp_ids** this read supports as ALT. Matches the per-BP resolution of `r2c.txt.gz`'s `split_bps` / `disc_bps` columns and `bps.txt.gz`'s col 52. Pre-v3 this carried cnames (contig-level), which couldn't disambiguate a contig that hosted multiple BPs (global + multi + indel). To pull every ALT-supporting read for a specific variant row: `samtools view corrected.bam | grep bi:Z:bp00100000042`. The tag is populated in `SvabaRegionProcessor::process` at the BP finalization loop (`tag_with_bp_id` lambda), mirrored into `svabaThreadUnit::alt_bp_ids_by_name` for the corrected-BAM restamp path in `SvabaOutputWriter::writeUnit`. - `bz:Z` — comma-joined list of **cnames** this read r2c-aligned to. Stays cname-keyed because "which contigs did this read align to" is a contig-level concept. Populated inside the r2c loop in `SvabaRegionProcessor` alongside `svabaRead::AddR2C(...)`, mirrored into `svabaThreadUnit::all_cnames_by_name`. Superset of `bi:Z` in the sense that every ALT-supporter r2c-aligned to its contig, but uses a different key namespace, so join back to bps.txt by col 30 (cname) here vs col 52 (bp_id) for `bi:Z`. `scripts/r2c_for_contig.sh` defaults to `bz:Z` to pull all r2c'd reads for a contig; set `TAG=bi` and pass a bp_id instead of a cname if you want the ALT-supporter subset for a specific variant. ## r2c TSV format (re-plot-able alignments) `${ID}.r2c.txt.gz` is the structured, re-plot-able alignment dump. It replaces the old pre-rendered `${ID}.alignments.txt.gz` ASCII output, which has been removed entirely — anything that lived implicitly in the ASCII art (fragment orientation, leading/trailing soft-clip bases, etc.) is now available as an explicit field in the TSV, and `viewer/r2c_explorer.html` re-plots it in-browser on demand. **Per-thread emission + postprocess merge.** Each svaba worker writes its own `${ID}.thread${N}.r2c.txt.gz` during the run (stream lives in `svabaThreadUnit::r2c_out_`; opened in the ctor, closed in the dtor, gated on `opts.dump_alignments`). The write happens in `SvabaOutputWriter::writeUnit` **before** `writeMutex_` is acquired — each thread's gzip stream is independent, so deflate runs in parallel across all workers. The first worker (threadId == 1; the worker pool in `threadpool.h` numbers threads 1..N, so there is no thread 0) writes the column-header line on open; other threads start with data. `scripts/svaba_postprocess.sh` step 1 merges the per-thread files via `cat`: gzip is concatenation-safe per RFC 1952, and the postprocess step numerically sorts `.threadN.r2c.txt.gz` so thread 1 is first in the cat, which means the merged file has exactly one header at the top. This is the same architectural pattern as the per-thread BAM writers; for the rationale, see "Perf notes" — in short, a mutex-shared gzip stream serializes `deflate()` across all threads, losing ~15/16 of compression parallelism at `-p 16`. Gotcha: the header-writing branch is keyed to `threadId == 1` in `SvabaThreadUnit::svabaThreadUnit`. Older revisions checked `threadId == 0`; that never fired because the pool hands out 1..N, which silently produced headerless `r2c.txt.gz` files. If you ever change the worker-numbering base, that line must change with it. Schema (documented by `AlignedContig::r2cTsvHeader()` in `src/svaba/AlignedContig.cpp`) — 21 tab-separated columns, record-type discriminated: ``` record_type contig_name contig_len contig_seq frags bps n_reads # contig-only fields above — NA on read rows read_id read_chrom read_pos read_flag r2c_cigar r2c_start r2c_rc r2c_nm support split_bps disc_bps r2c_score native_score read_seq # read-only fields — NA on the contig row ``` `split_bps` / `disc_bps` are comma-joined `bp_id` lists — the unambiguous per-BP attribution of each read. The categorical `support` field (`split` / `disc` / `both` / `none`) is derived from these (whichever is non-NA) and kept for grep-friendliness. The `bps` field on the contig row also carries each BP's id as the 2nd subfield so viewers can join back without a second file. On older r2c files without these columns (pre-v3 emitter) the viewer falls back to the categorical support only. One `contig` row per variant-bearing contig, followed by one `read` row per r2c-aligned read. `contig_name` is the shared join key (same value as bps.txt's col-30 `cname`), so grouping reads back to their contig is O(n). Format details: - `frags`: `|`-separated per fragment; within a frag, `:`-separated `chr:pos:strand:cigar:mapq:cpos_break1:cpos_break2:gpos_break1:gpos_break2:flipped`. - `bps`: `|`-separated; within a bp, `:`-separated `kind:chr1:pos1:strand1:chr2:pos2:strand2:span:insertion`. `kind ∈ {global, multi, indel}`. Insertion is `.` when absent so the field count stays fixed at 9. - All cell values are TSV-escaped (tab/CR/LF → space) via `r2cEscape`. - Per-sample support classification (`split`/`disc`/`both`/`none`) and the scores are computed **identically** to the ASCII emitter — same `r2c_score > native_score` gate, same `corrected_native_cig` precedence, same `split_supporters`/`disc_supporters` sets. The two emitters share enough code to prevent drift. ## Blacklist-aware region pruning `run_svaba.cpp` drops any queued region that is 100% covered by the blacklist BEFORE the region is sent to a worker thread. Lives right after `loader.countJobs(regionsToRun)`, before `sc.total_regions_to_process` is set. Rule: for each `r` in `regionsToRun`, if `sc.blacklist.FindOverlapWidth(r, true) >= r.Width()`, drop it. This was a measured win. Previously each fully-blacklisted chunk (e.g. a 25 kb window on a decoy/alt/random contig) paid the full pipeline cost: `QueryRegion` on the ref, `walker->SetRegion` + `readBam` (which decompresses every BGZF block overlapping the region, parses each bam1_t, allocates an `svabaRead`) — only to have `sc.blacklist.CountOverlaps` drop every read at `SvabaBamWalker.cpp:181-182`. Now those regions never hit a thread. Safe because `sc.blacklist` has had `MergeOverlappingIntervals()` + `CreateTreeMap()` called, so `FindOverlapWidth` can't double-count and wrongly drop a partially-callable region. The per-read and per-BP blacklist checks at `SvabaRegionProcessor.cpp:74, 818` still run for regions that **partially** overlap — this prune only short-circuits the 100%-covered case. Pruned regions don't get a `runtime.txt` row. That's intentional and actually makes the runtime file cleaner (only regions that did work). ## Options surface `src/svaba/SvabaOptions.h`: - `dump_weird_reads` is **compile-time-only** (`static constexpr bool`). Flip the literal in the header and rebuild to enable; there is deliberately no CLI path. Because it's `static constexpr false` by default, the compiler dead-code-eliminates every `if (sc.opts.dump_weird_reads) { ... }` branch at -O2+. - `dump_discordant_reads`, `dump_corrected_reads`, and `dump_alignments` default **false** and are all enabled together by the single `--dump-reads` runtime flag (switch-case 1800 in `SvabaOptions.cpp`). They control, respectively: - `${ID}.discordant.bam` - `${ID}.corrected.bam` - `${ID}.r2c.txt.gz` (per-thread; merged by the postprocess step) The fields are kept separate so individual callsites can key off their own concern (e.g. `svabaThreadUnit` gates `r2c_out_` opening on `dump_alignments` only), but there's no runtime path to toggle them individually — all three flip as a unit under `--dump-reads`. - Without `--dump-reads`, svaba produces the lean output set only: `bps.txt.gz`, VCFs, `contigs.bam`, `runtime.txt`, `discordant.txt.gz` (cluster-level, tiny). No per-thread `r2c.txt.gz`, no `corrected.bam`, no `discordant.bam`. This is the production default because the gated outputs can run to tens of gigabytes on deep samples. - **`alignments.txt.gz` is gone.** The pre-rendered ASCII viewer output was replaced in full by `r2c.txt.gz` (same information, not pre-formatted). `AlignedContig::printToAlignmentsFile` and `BreakPoint::printDeletionMarksForAlignmentsFile` were removed; the surviving `AlignmentFragment::printToAlignmentsFile` is kept only because one `std::cerr` debug print in `BreakPoint.cpp` still calls it. `viewer/alignments_viewer.html` still exists and still works on old `.alignments.txt.gz` files from previous runs, but new runs don't produce that file — use the r2c sub-panel in `viewer/bps_explorer.html` instead. ## Viewer suite (`viewer/`) Entry point is `viewer/index.html`, a card grid pointing at the sub-viewers. All client-side, no server required. - **`bps_explorer.html`** — primary viewer. Sortable table of bps rows, numeric filters (somlod/maxlod/qual/span/etc.), chip filters (counts are live — they reflect the current filter, not the full dataset), per-sample detail panel, small histograms for somlod/maxlod/span, IGV-navigation links in the IGV1/IGV2 columns (fires `fetch('http://localhost:60151/goto?locus=…', {mode:'no-cors'})`; requires IGV running with port 60151 enabled). r2c re-plot sub-panel was removed — that capability now lives in the standalone `r2c_explorer.html` below. - **`r2c_explorer.html`** — standalone re-plotter for the structured r2c TSV (emitted by `svaba run --dump-reads`, or filtered to PASS / PASS-somatic by `scripts/svaba_postprocess.sh`). Upload an `.r2c.txt.gz` / `.r2c.pass.txt.gz` / `.r2c.pass.somatic.txt.gz`, type or pick a contig name in the search box (browser `` autocomplete, capped at 5000 entries), and get the alignment plot rendered in-browser: 10bp ruler, contig sequence, per-BWA-fragment summaries with `>/<` orientation rendering, per-breakpoint summary rows, and per-read gap-expanded CIGAR rendering with lowercase leading/trailing soft-clip bases placed at `start - lead_clip_len` (exact mirror of the old `AlignedContig::printToAlignmentsFile`). Prev/Next/Random navigation, imperfect-only and per-support-kind toggles, click-to-hide source-prefix legend. Load-once, explore many contigs — no need to load a bps.txt.gz first. - **`alignments_viewer.html`** — legacy ASCII-plot viewer for `alignments.txt.gz` outputs produced by svaba runs *before* the r2c migration. New runs no longer produce `alignments.txt.gz`; `r2c_explorer.html` is the replacement. Kept in the tree so historical outputs still render. - **`runtime_explorer.html`** — explorer for `runtime.txt` (the per-region timing TSV produced by `svabaTimer::logRuntime`, schema in `SvabaUtils.cpp`). Sortable table, region-range filter, runtime/contigs/discordant filters, IGV-click on the region column, prominent runtime histogram (defaults to log10 because runtime distributions are always long-tailed — see "Perf notes"). 17-column schema hardcoded from `SvabaUtils.cpp::svabaTimer::header`. - **`comparison.html`** — side-by-side of two bps runs. - **`bps_viewer.html`** — legacy light-theme viewer, uses external `app.js` + `styles.css`. ## tracks/ and blacklists `tracks/hg38.combined_blacklist.bed` is the one to feed `svaba run --blacklist`. It's a **regeneratable artifact** — produced by `scripts/combine_blacklists.sh` from the component files in the same dir. Don't hand-edit it; edit a component file and re-run the script. Components (as of last pass): - `hg38.blacklist.sorted.bed` — ENCODE-style high-signal regions. - `hg38.high_runtime.bed` — regions empirically slow to assemble. - `hg38.manual.blacklist.bed` — ad-hoc bad-list, curated. - `hg38.nonstd_chr.blacklist.bed` — full-contig entries for every chrUn/*_decoy/*_alt/*_random/chrEBV/HLA-* contig in the reference, generated from `tracks/chr` (a GRCh38 fasta-header dump). - `hg38.rmsk.simple_repeat.bed` — UCSC RepeatMasker simple-repeat regions. `scripts/combine_blacklists.sh` has three modes: plain concat (default), `--merge` (sort + `bedtools merge` with distinct-label aggregation), and `--clip GENOME` (clip interval ends to real contig length, because some input BEDs use oversize-end sentinels like `end=250000000` that would otherwise inflate covered-bp totals into the trillions — ask me how I know). Preferred invocation for refreshing the combined blacklist: ```bash ./scripts/combine_blacklists.sh --merge --clip tracks/chr \ -o tracks/hg38.combined_blacklist.bed \ tracks/hg38.blacklist.sorted.bed \ tracks/hg38.high_runtime.bed \ tracks/hg38.manual.blacklist.bed \ tracks/hg38.nonstd_chr.blacklist.bed \ tracks/hg38.rmsk.simple_repeat.bed ``` ## Perf notes (empirical, from profiling the HLA region on M3 Ultra) From a `sample`-based CPU profile with `-p 16`: - **96% of worker time is inside `SvabaRegionProcessor::process`**. Four buckets soak it up: - BWA alignment: **~38%** (`mem_align1_core`, `mem_chain`, seed lookups `bwt_sa`/`bwt_occ*`, `ksw_extend2`) - Fermi-lite assembly: **~27%** (`fml_assemble`, `fml_fmi2mag*`, `rld_rank2a`, `unitig_unidir`) - BFC error correction: **~17%** (`SeqLib::BFC::ErrorCorrect/Train`, `kmer_correct`, `bfc_*`). User-controllable via the ec flags. - BAM walking: **~9%** (`svabaBamWalker::readBam`) - **IPC ≈ 3.0** on Apple Silicon P-cores (theoretical max ~8, typical real workloads 1.2–2.5). The code is already running near the silicon's sustained envelope; algorithmic rewrites have diminishing returns. - **Voluntary context switches ≈ 42 across a 56s run** — i.e. essentially zero. Which means **there is no allocator contention, no mutex contention, no I/O blocking** to fix. jemalloc/mimalloc on macOS **lost to libmalloc by 5-10×** in an A/B test (DYLD interposition overhead + Apple's nanomalloc fast path for small allocs). Save the allocator swap for the Linux production box — on Linux it's often a 10–20% win; on macOS don't bother. - **BWA and fermi-lite default to `n_threads = 1` internally** (`mem_opt_init` at `bwamem.c:100`, `fml_opt_init` at `bfc.c:21`) and svaba never mutates them. `kt_for` samples in the profile are the same worker thread running inline, not spawned threads. So svaba's `-p N` is 1:1 with worker threads; there's no multiplicative thread fan-out. Thread-count guidance on this 20 P-core + 8 E-core box: - `-p 20` is the default sweet spot. One worker per P-core; E-cores absorb macOS background work, jemalloc bg thread, etc. - `-p 26` or `-p 28` is possible but the extra threads land on E-cores and become tail latency (E-core is ~50% the speed of a P-core). Usually a wash or slight loss vs `-p 20`. - Load imbalance is the main remaining utilization gap. On a 3 Mb test region, utilization was ~63%; on a 10 Mb region it climbed to ~78%; on full chromosome / WGS expect 85–90%. Tail regions (HLA, centromere, high-coverage weird spots) dominate wall clock. Build flag recommendation for production runs (see "Build system"): `-O3 -mcpu=native` on bwa + fermi-lite + (via `CMAKE_CXX_FLAGS_RELWITHDEBINFO`) the svaba C++. Measured 5–15% wall-time gain expected on top of the default RelWithDebInfo build. ## Cloud scatter-gather (`scripts/svaba_cloud.sh`) `svaba_cloud.sh` parallelizes a WGS run across multiple GCP VMs — one per chromosome partition — sharing a single read-only persistent disk. Each VM runs `svaba run -k ` independently; outputs go to a GCS bucket; an optional `--merge` step concatenates the per-partition `bps.txt.gz` files and runs `svaba_postprocess.sh`. Architecture rationale: svaba's bottleneck is BWA FM-index random lookups, which are latency-bound and NUMA-hostile. Multi-socket servers waste half their threads on cross-socket access. Small single-socket VMs (e.g. `c3d-highcpu-30`, AMD Genoa, ~128 MB L3, no NUMA) give each worker full-speed local memory. Horizontal scaling across VMs beats vertical scaling to more threads on a big box. On dual-socket Xeon servers (measured on 2×20-core Xeon @ 2.8 GHz), jemalloc is the single biggest optimization — 37% wall-time reduction at 38 threads by eliminating glibc arena lock contention. NUMA pinning (`numactl --cpunodebind --membind`) is second-order at ≤40 threads on a 2-socket box but matters more on 4-socket or at higher thread counts. Disk I/O is ~9% of wall time (sequential BAM streaming). NVMe is unnecessary; standard persistent disk or even gcsfuse over a GCS bucket is fine. Interchromosomal SVs: both breakends get assembled independently by whichever partition contains the discordant read pileup. The merge + dedup step in `svaba_postprocess.sh` pairs them. No calls are lost. ## Conventions - **File naming**: `src/svaba/Svaba*.{cpp,h}` for the svaba-specific files (PascalCase, first letter capitalized). Intentional lowercase / snake_case exceptions live at the top of `src/svaba/` (`refilter`, `run_svaba`, `svaba`, `threadpool`, `tovcf`, `vcf`). Don't introduce new lowercase-first names; if you rename something on a case-insensitive filesystem (macOS), do it via `git rm --cached && git add ` or git's `core.ignorecase` will hide the rename from the index. - **C++ style**: snake_case methods inside ClassName, 2-space indent, header/impl split. Don't introduce new formatting unless asked. - **Statistical code lives in `SvabaModels.*`**; breakpoint glue lives in `BreakPoint.*`. Keep statistical primitives in the models file, not inlined into BreakPoint. - **LL/LOD values in this codebase are always log10**, not natural log. - **`aN, dN, aT, dT`** = alt count / depth in normal and tumor; **`f`** = allele fraction; **`e_fwd`/`e_rev`** = forward/reverse error rates from the artifact model. These names are used consistently in the analysis HTML too. - **Option codes** in `SvabaOptions.cpp::longOpts`: 1001-1099 = mode, 1100s = assembly, 1200s = EC, 1300s = discordant, 1400s = filter, 1500 = chunking, 1600s = bwa-mem tuning, 1700s = output/DBs, 1800 = dump-reads. Keep the ranges coherent when adding new options. ## Mate-region lookup pipeline When svaba encounters discordant reads (insert size too large or wrong orientation), it collects their mate loci and considers doing a secondary "mate-region" assembly to catch the other breakend of an SV. **Six gates** a mate candidate must pass (in order): 1. **Primary MAPQ** (`minMateMAPQ`, default -1 = no gate): the discordant read itself must have MAPQ ≥ this. Set to e.g. 10 to skip multi-mapped primaries. 2. **Chromosome ID** (`maxMateChrID`, default 23): mate must land on chr ≤ this ID (0-indexed: 0=chr1 .. 22=chrX, 23=chrY). Skips chrM/alt/decoy in human. Set to -1 (via `--non-human`) to disable entirely for non-human genomes. 3. **Blacklist**: mate locus checked against `sc.blacklist`. 4. **Min count** (`mateRegionMinCount`, default 2): merged region must have ≥ N supporting reads to survive the BamWalker filter. 5. **Somatic mateLookupMin** (default 3, `MATE_LOOKUP_MIN`): in `SvabaRegionProcessor`, only look up regions with ≥ this many somatic-only reads. 6. **Max regions** (6): cap at 6 mate regions per assembly window. All constants live in `SvabaOptions.h` as `inline constexpr` with runtime overrides in the `SvabaOptions` class: ``` --min-mate-mapq N (default -1, no gate) --max-mate-chr N (default 23, through chrY; set -1 for no limit) --mate-min-count N (default 2) --non-human (sets maxMateChrID = -1, removes human assumptions) ``` Code: `SvabaBamWalker.cpp::calculateMateRegions()`. ## Compile-time read & contig tracing Two zero-cost compile-time trace systems for debugging why a specific read was or wasn't credited / a contig was or wasn't called: **`SVABA_TRACE_READ`** — traces a single read (by QNAME) through the entire pipeline: BamWalker intake → BFC correction → r2c alignment → native realignment → splitCoverage scoring → output tagging. ```bash cmake .. -DCMAKE_CXX_FLAGS='-DSVABA_TRACE_READ="\"LH00306:129:227V5CLT4:6:1204:38807:7191\""' ``` Trace points (19 total across 3 files): - `SvabaBamWalker.cpp`: initial read filter decisions (existing) - `SvabaRegionProcessor.cpp`: BFC correction result, r2c alignment per-contig, native realignment reuse/done/miss, bi:Z tagging - `BreakPoint.cpp`: splitCoverage entry, TP8 r2c-vs-native comparison, TP9 del/ins near break, TP10 span check, TP11 del covers, CREDITED/NOT CREDITED final decision **`SVABA_TRACE_CONTIG`** — traces a single contig through assembly, alignment, and scoring: ```bash cmake .. -DCMAKE_CXX_FLAGS='-DSVABA_TRACE_CONTIG="\"c_fermi_chr2_215869501_215894501_13C\""' ``` Both can be combined. Both are `#ifdef`-guarded so they compile to nothing when not defined. See `src/svaba/SvabaDebug.h` for the macro definitions and `README.md` for full recipes. ## Useful jump points - Somatic LOD calc: `src/svaba/SvabaModels.cpp:86` - Per-sample LO: `src/svaba/BreakPoint.cpp:1610` - Somatic LOD entry: `src/svaba/BreakPoint.cpp:975` - INDEL somatic gate: `src/svaba/BreakPoint.cpp:1072` - Region-queue blacklist prune: `src/svaba/run_svaba.cpp` (right after `loader.countJobs(regionsToRun)`) - Per-read blacklist filter: `src/svaba/SvabaBamWalker.cpp:181-182` - r2c TSV emitter: `src/svaba/AlignedContig.cpp::printToR2CTsv` + `::r2cTsvHeader` - Postprocess (C++): `src/svaba/SvabaPostprocess.cpp` - Postprocess (shell orchestration): `scripts/svaba_postprocess.sh` - `svaba tovcf` driver: `src/svaba/tovcf.cpp::runToVCF` - VCF engine (parse + dedup + emit): `src/svaba/vcf.cpp` + `vcf.h` - Symbolic SV classifier: `vcf.cpp::classify_symbolic_kind` - Single-file VCF writers: `vcf.cpp::writeSvsSingleFile` + `writeIndelsSingleFile` - Blacklist combiner: `scripts/combine_blacklists.sh` - Runtime-file schema: `src/svaba/SvabaUtils.cpp::svabaTimer::header` - Options parsing: `src/svaba/SvabaOptions.cpp::SvabaOptions::parse` - Analysis writeup (somlod/maxlod): `somlod_maxlod_analysis.html` - Mate-region lookup: `src/svaba/SvabaBamWalker.cpp::calculateMateRegions` - Mate-region constants: `src/svaba/SvabaOptions.h` (lines 126-141) - Read trace macro: `src/svaba/SvabaDebug.h` - Read trace (BFC/r2c/native): `src/svaba/SvabaRegionProcessor.cpp` - Read trace (splitCoverage): `src/svaba/BreakPoint.cpp` - Debugging recipes: `README.md` (in svaba_opt root) ================================================ FILE: CMakeLists.txt ================================================ cmake_minimum_required(VERSION 3.10) # Set the C++ standard required for the project project(svaba) # LANGUAGES CXX) set(CMAKE_CXX_STANDARD 17) set(CMAKE_CXX_STANDARD_REQUIRED ON) set(CMAKE_CXX_EXTENSIONS OFF) if(NOT CMAKE_BUILD_TYPE AND NOT CMAKE_CONFIGURATION_TYPES) set(CMAKE_BUILD_TYPE RelWithDebInfo CACHE STRING "Default build type" FORCE) endif() add_compile_options(-fno-omit-frame-pointer) add_subdirectory(${CMAKE_CURRENT_SOURCE_DIR}/SeqLib) # Include directories for headers include_directories( ${CMAKE_CURRENT_SOURCE_DIR} ${CMAKE_CURRENT_SOURCE_DIR}/SeqLib ${CMAKE_CURRENT_SOURCE_DIR}/SeqLib/bwa ${CMAKE_CURRENT_SOURCE_DIR}/SeqLib/fermi-lite ) # Look for htslib on the system find_package(htslib QUIET) # Find required system level type libraries find_package(Threads REQUIRED) find_package(ZLIB REQUIRED) ## LZMA find_path(LZMA_INCLUDE_DIR NAMES lzma.h) find_library(LZMA_LIBRARY NAMES lzma) if(NOT LZMA_INCLUDE_DIR OR NOT LZMA_LIBRARY) message(FATAL_ERROR "LZMA library or headers not found!") endif() ## BZip2 find_package(BZip2 REQUIRED) ## HTSLIB # # Detection order: # 1. find_package(htslib) — works only if htslib ships a # htslibConfig.cmake (rare outside conda/vcpkg installs). # 2. pkg-config — system-installed htslib typically provides a # htslib.pc, so `pkg-config --libs htslib` works. # 3. find_path/find_library for htslib/hts.h and libhts on the # default system paths (/usr/include, /usr/local/include, # /opt/homebrew/include, etc.). # 4. Explicit -DHTSLIB_DIR=/path/to/htslib for non-standard installs. # # The first three paths auto-detect a system install, so a vanilla # `cmake .. && make` works when htslib is installed system-wide. Only # fall back to HTSLIB_DIR when all auto-detections fail, matching the # pattern used here for ZLIB / BZip2 (also auto-detected). set(SVABA_HTSLIB_FOUND FALSE) set(SVABA_HTSLIB_LIBRARIES "") if (htslib_FOUND) message(STATUS "htslib: found via find_package()") set(SVABA_HTSLIB_FOUND TRUE) set(SVABA_HTSLIB_LIBRARIES hts) endif() if (NOT SVABA_HTSLIB_FOUND) find_package(PkgConfig QUIET) if (PkgConfig_FOUND) pkg_check_modules(HTSLIB_PC QUIET htslib) if (HTSLIB_PC_FOUND) message(STATUS "htslib: found via pkg-config (version ${HTSLIB_PC_VERSION})") include_directories(${HTSLIB_PC_INCLUDE_DIRS}) link_directories(${HTSLIB_PC_LIBRARY_DIRS}) set(SVABA_HTSLIB_FOUND TRUE) set(SVABA_HTSLIB_LIBRARIES ${HTSLIB_PC_LIBRARIES}) endif() endif() endif() if (NOT SVABA_HTSLIB_FOUND) find_path(HTSLIB_INCLUDE_DIR NAMES htslib/hts.h DOC "Directory containing htslib/hts.h") find_library(HTSLIB_LIBRARY NAMES hts DOC "Path to the hts library") if (HTSLIB_INCLUDE_DIR AND HTSLIB_LIBRARY) message(STATUS "htslib: found headers at ${HTSLIB_INCLUDE_DIR}, lib at ${HTSLIB_LIBRARY}") include_directories(${HTSLIB_INCLUDE_DIR}) set(SVABA_HTSLIB_FOUND TRUE) set(SVABA_HTSLIB_LIBRARIES ${HTSLIB_LIBRARY}) endif() endif() if (NOT SVABA_HTSLIB_FOUND) set(HTSLIB_DIR "" CACHE PATH "Path to HTSLib root directory") if (HTSLIB_DIR) message(STATUS "htslib: using explicit HTSLIB_DIR=${HTSLIB_DIR}") include_directories(${HTSLIB_DIR}/include) link_directories(${HTSLIB_DIR}/lib) set(SVABA_HTSLIB_FOUND TRUE) set(SVABA_HTSLIB_LIBRARIES hts) else() message(FATAL_ERROR "htslib not found. Install htslib system-wide (e.g. " "`brew install htslib` or `apt install libhts-dev`) so " "pkg-config / find_library locate it automatically, " "or pass -DHTSLIB_DIR=/path/to/htslib to point at a " "manual build.") endif() endif() # Find all source files set(SOURCES src/svaba/run_svaba.cpp src/svaba/SvabaOutputWriter.cpp src/svaba/BreakPoint.cpp src/svaba/ContigAlignmentScore.cpp src/svaba/AlignedContig.cpp src/svaba/AlignmentFragment.cpp src/svaba/DiscordantCluster.cpp src/svaba/DBSnpFilter.cpp src/svaba/SvabaUtils.cpp src/svaba/svaba.cpp src/svaba/SvabaAssemblerEngine.cpp src/svaba/tovcf.cpp src/svaba/test_svaba.cpp src/svaba/vcf.cpp src/svaba/DiscordantRealigner.cpp src/svaba/SvabaOverlapAlgorithm.cpp src/svaba/SvabaASQG.cpp src/svaba/SvabaAssemble.cpp src/svaba/KmerFilter.cpp src/svaba/SvabaThreadUnit.cpp src/svaba/SvabaRegionProcessor.cpp src/svaba/SvabaBamWalker.cpp src/svaba/refilter.cpp src/svaba/SvabaPostprocess.cpp src/svaba/LearnBamParams.cpp src/svaba/STCoverage.cpp src/svaba/SvabaModels.cpp ##src/svaba/Histogram.cpp ##src/svaba/BamStats.cpp src/svaba/SvabaRead.cpp src/svaba/SvabaOptions.cpp src/svaba/SvabaLogger.cpp src/svaba/SvabaFileLoader.cpp src/SGA/SuffixTools/STCommon.cpp src/SGA/SuffixTools/Occurrence.cpp src/SGA/SuffixTools/SuffixArray.cpp src/SGA/SuffixTools/SuffixCompare.cpp src/SGA/SuffixTools/InverseSuffixArray.cpp src/SGA/SuffixTools/SACAInducedCopying.cpp src/SGA/SuffixTools/BWTAlgorithms.cpp src/SGA/SuffixTools/BWTReader.cpp src/SGA/SuffixTools/BWTWriter.cpp src/SGA/SuffixTools/SAReader.cpp src/SGA/SuffixTools/SAWriter.cpp src/SGA/SuffixTools/SBWT.cpp src/SGA/SuffixTools/RLBWT.cpp src/SGA/SuffixTools/BWTWriterBinary.cpp src/SGA/SuffixTools/BWTReaderBinary.cpp src/SGA/SuffixTools/BWTWriterAscii.cpp src/SGA/SuffixTools/BWTReaderAscii.cpp src/SGA/SuffixTools/BWTIntervalCache.cpp src/SGA/SuffixTools/SampledSuffixArray.cpp src/SGA/Algorithm/OverlapAlgorithm.cpp src/SGA/Algorithm/DPAlignment.cpp src/SGA/Algorithm/SearchSeed.cpp src/SGA/Algorithm/OverlapBlock.cpp src/SGA/Algorithm/SearchHistory.cpp src/SGA/Algorithm/OverlapTools.cpp src/SGA/Bigraph/Bigraph.cpp src/SGA/Bigraph/Vertex.cpp src/SGA/Bigraph/Edge.cpp src/SGA/Bigraph/EdgeDesc.cpp src/SGA/SGA/OverlapCommon.cpp src/SGA/SQG/SQG.cpp src/SGA/SQG/ASQG.cpp src/SGA/StringGraph/SGUtil.cpp src/SGA/StringGraph/SGAlgorithms.cpp src/SGA/StringGraph/SGVisitors.cpp src/SGA/StringGraph/CompleteOverlapSet.cpp src/SGA/StringGraph/RemovalAlgorithm.cpp src/SGA/StringGraph/SGSearch.cpp src/SGA/StringGraph/SGWalk.cpp src/SGA/Util/Util.cpp src/SGA/Util/stdaln.c src/SGA/Util/Alphabet.cpp src/SGA/Util/Contig.cpp src/SGA/Util/ReadTable.cpp src/SGA/Util/ReadInfoTable.cpp src/SGA/Util/SeqReader.cpp src/SGA/Util/DNAString.cpp src/SGA/Util/Match.cpp src/SGA/Util/Pileup.cpp src/SGA/Util/Interval.cpp src/SGA/Util/SeqCoord.cpp src/SGA/Util/QualityVector.cpp src/SGA/Util/Quality.cpp src/SGA/Util/PrimerScreen.cpp src/SGA/Util/CorrectionThresholds.cpp src/SGA/Util/ClusterReader.cpp src/SGA/Util/QualityTable.cpp src/SGA/Util/gzstream.C src/SGA/Util/BitChar.cpp src/SGA/Util/MultiOverlap.cpp ) include_directories( ${CMAKE_CURRENT_BINARY_DIR} src/SGA/Algorithm src/SGA/StringGraph src/SGA/SGA src/SGA/SuffixTools src/SGA/Util src/SGA/SQG src/SGA/Bigraph src/svaba src/SGA ) # --------------------------------------------------------------------------- # Optional compile-time contig tracing (SvabaDebug.h). # # cmake .. -DSVABA_TRACE_CONTIG="c_fermi_chr2_215869501_215894501_11C" # # Prints detailed stderr trace for every decision point that contig # touches. Pass -DSVABA_TRACE_ALL=ON to trace ALL contigs (very noisy). # --------------------------------------------------------------------------- set(SVABA_TRACE_CONTIG "" CACHE STRING "Contig name to trace (empty=off)") option(SVABA_TRACE_ALL "Trace ALL contigs (very noisy)" OFF) set(SVABA_TRACE_READ "" CACHE STRING "Read QNAME to trace (empty=off)") option(SVABA_TRACE_ALL_READS "Trace ALL reads (extremely noisy)" OFF) # Generate the executable add_executable(svaba ${SOURCES}) if (NOT "${SVABA_TRACE_CONTIG}" STREQUAL "") message(STATUS "svaba: tracing contig '${SVABA_TRACE_CONTIG}'") target_compile_definitions(svaba PRIVATE SVABA_TRACE_CONTIG="${SVABA_TRACE_CONTIG}") endif() if (SVABA_TRACE_ALL) message(STATUS "svaba: tracing ALL contigs (noisy!)") target_compile_definitions(svaba PRIVATE SVABA_TRACE_ALL=1) endif() if (NOT "${SVABA_TRACE_READ}" STREQUAL "") message(STATUS "svaba: tracing read '${SVABA_TRACE_READ}'") target_compile_definitions(svaba PRIVATE SVABA_TRACE_READ="${SVABA_TRACE_READ}") endif() if (SVABA_TRACE_ALL_READS) message(STATUS "svaba: tracing ALL reads (extremely noisy!)") target_compile_definitions(svaba PRIVATE SVABA_TRACE_ALL_READS=1) endif() # If the submodules already have Makefiles, you can use custom commands to invoke make # in those directories. add_custom_target( COMMAND make WORKING_DIRECTORY ${CMAKE_CURRENT_SOURCE_DIR} ) # --------------------------------------------------------------------------- # Optional jemalloc support. # # On Linux with high thread counts (-p 16+), jemalloc eliminates glibc # arena-lock contention that can cost 30-40% wall time. Link it at # compile time so no LD_PRELOAD is needed at runtime. # # cmake .. -DUSE_JEMALLOC=ON # link jemalloc (recommended on Linux) # cmake .. -DUSE_JEMALLOC=OFF # use system malloc (default) # # macOS users should NOT enable this — Apple's libmalloc with its # nanomalloc fast path outperforms jemalloc on this workload. # --------------------------------------------------------------------------- option(USE_JEMALLOC "Link against jemalloc for reduced allocator contention" OFF) set(JEMALLOC_LIBRARIES "") if (USE_JEMALLOC) find_library(JEMALLOC_LIB NAMES jemalloc PATHS /usr/lib/x86_64-linux-gnu /usr/lib /usr/local/lib DOC "Path to libjemalloc") if (JEMALLOC_LIB) message(STATUS "jemalloc: found at ${JEMALLOC_LIB}") set(JEMALLOC_LIBRARIES ${JEMALLOC_LIB}) else() message(FATAL_ERROR "USE_JEMALLOC=ON but libjemalloc not found. " "Install it (apt install libjemalloc-dev) or set " "-DJEMALLOC_LIB=/path/to/libjemalloc.so") endif() endif() # Linking target_link_libraries(svaba seqlib ${CMAKE_CURRENT_SOURCE_DIR}/SeqLib/bwa/libbwa.a ${CMAKE_CURRENT_SOURCE_DIR}/SeqLib/fermi-lite/libfml.a Threads::Threads ${SVABA_HTSLIB_LIBRARIES} ZLIB::ZLIB ${CURL_LIBRARY} ${LZMA_LIBRARY} BZip2::BZip2 ${JEMALLOC_LIBRARIES} ) # --------------------------------------------------------------------------- # Install support. # # `make install` (equivalent: `cmake --install build`) copies the svaba # binary to ${CMAKE_INSTALL_PREFIX}/bin. GNUInstallDirs sets the usual # distro-friendly defaults for BINDIR / LIBDIR / etc., so this follows # standard Unix conventions. # # Default CMAKE_INSTALL_PREFIX is /usr/local (may need sudo). To stage # into a non-system location, pass -DCMAKE_INSTALL_PREFIX=/some/path at # cmake time, or use `cmake --install build --prefix /some/path` at # install time. To drop the binary into this repo's `bin/` directory: # cmake --install build --prefix ${CMAKE_SOURCE_DIR} # --------------------------------------------------------------------------- include(GNUInstallDirs) install(TARGETS svaba RUNTIME DESTINATION ${CMAKE_INSTALL_BINDIR}) install(PROGRAMS ${CMAKE_SOURCE_DIR}/scripts/svaba_postprocess.sh DESTINATION ${CMAKE_INSTALL_BINDIR}) ================================================ FILE: Dockerfile ================================================ # Start with an Ubuntu image FROM ubuntu:20.04 # Avoid prompts with tzdata (timezones) ENV DEBIAN_FRONTEND=noninteractive # Install dependencies for htslib and svaba RUN apt update && apt install -y \ autoconf \ automake \ make \ gcc \ g++ \ git \ perl \ zlib1g-dev \ libbz2-dev \ liblzma-dev \ libcurl4-gnutls-dev \ libssl-dev \ cmake \ && rm -rf /var/lib/apt/lists/* # Clone and install htslib WORKDIR /opt RUN git clone --recursive https://github.com/samtools/htslib.git && \ cd htslib && \ autoheader && \ autoconf && \ ./configure && \ make && \ make install # Ensure shared libraries are noticed RUN ldconfig # Clone svaba WORKDIR /opt RUN git clone --recursive https://github.com/walaj/svaba.git && cd svaba && mkdir build # Compile svaba with htslib WORKDIR /opt/svaba/build RUN cmake .. \ -DHTSLIB_DIR=/usr/local && \ make # Add svaba to system PATH ENV PATH="/opt/svaba/build:${PATH}" ================================================ FILE: LICENSE ================================================ GNU GENERAL PUBLIC LICENSE Version 3, 29 June 2007 Copyright (C) 2007 Free Software Foundation, Inc. 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But first, please read . ================================================ FILE: R/archive_non_functional/create-databases.R ================================================ #################### ## load CCDS and name table ccds <- fread("/xchip/gistic/Jeremiah/tracks/ccdsGene.hg19.txt") ## load and format name table (CCDS to gene name conversion) nm <- fread("/xchip/gistic/Jeremiah/tracks/name_conversion.tsv") setnames(nm, colnames(nm), gsub(" ","_",colnames(nm))) app_sym <- rep(nm$Approved_Symbol, times=nm[, length(strsplit(CCDS_IDs, ",")[[1]]), by=HGNC_ID]$V1) nmm <- data.table(ccds=gsub(" ", "", unlist(strsplit(nm$CCDS_IDs,","))), gene=app_sym) setkey(nmm, ccds) ## merge names with CCDS ccds[, ccds := gsub("(.*?)\\..*", "\\1", name)] setkey(ccds, ccds) ccds <- nmm[ccds] ## switch frames 1 and 2, so that 2 refers to 2/3 of codon, 1 to 1/3 ## eg. ++***+++***+++ starts with 2/3 of codon in its sense dir, so frame is 2 ccds$exonFrames <- gsub("1","A", ccds$exonFrames) ccds$exonFrames <- gsub("2","1", ccds$exonFrames) ccds$exonFrames <- gsub("A","2", ccds$exonFrames) ## dedupe on gene, taking the largest one ccds[, negc := -exonCount] setkey(ccds, gene, negc) ccds <- ccds[!duplicated(gene)] ccds[, negc := NULL] ## create exon intron track ccds[, exon_start := min(as.numeric(strsplit(exonStarts, ",")[[1]])), by=c("name", "chrom", "txStart")] ccds[, exon_end := max(as.numeric(strsplit(exonEnds, ",")[[1]])), by=c("name", "chrom", "txStart")] ccds[, intron_ends := { ll <- as.numeric(strsplit(exonStarts, ",")[[1]]); paste(ll[seq(2, length(ll), length.out=ifelse(length(ll) > 1, length(ll)-1, 0))], collapse=",")} , by=c("name", "chrom", "txStart")] ccds[, intron_starts := { ll <- as.numeric(strsplit(exonEnds, ",")[[1]]); paste(ll[seq(1, length(ll)-1, length.out=ifelse(length(ll) > 1, length(ll)-1, 0))], collapse=",")} , by=c("name", "chrom", "txStart")] ccds[, intron_frame := { ll <- as.numeric(strsplit(exonFrames, ",")[[1]]) if (length(ll) == 1) ## no exons "" else if (strand == '+') ## in sense dir, give frame to exon after paste(ll[seq(2, length(ll))], collapse=",") else ## exons ordered in + direction, so sense exon after intron is actually the one BEFORE intron for this ordering paste(ll[seq(1, length(ll)-1)], collapse=",") } , by=c("name", "chrom", "txStart")] intrEnds <- as.numeric(unlist(strsplit(ccds$intron_ends, ","))) intrStarts <- as.numeric(unlist(strsplit(ccds$intron_starts, ","))) intrFrame <- as.numeric(unlist(strsplit(ccds$intron_frame, ","))) intrName <- rep(ccds$name, times=as.numeric(ccds$exonCount)- 1) intrChr <- gsub("chr", "", rep(ccds$chrom, times=as.numeric(ccds$exonCount)-1)) intrNums <- as.numeric(ccds[, { s <- seq(length.out=ifelse(as.numeric(exonCount)==1, 0, as.numeric(exonCount)-1)); if (strand == '+') { s } else { rev(s) } }, by=c("name","chrom","txStart")]$V1) exonNums <- as.numeric(ccds[, { s <- seq(as.numeric(exonCount)); if (strand=='+') { s } else { rev(s) }}, by=c("name","chrom","txStart")]$V1) intrGene <- rep(ccds$gene, times=as.numeric(ccds$exonCount)-1) exonGene <- rep(ccds$gene, times=as.numeric(ccds$exonCount)) ccdsGene <- as.numeric(ccds[, seq(as.numeric(exonCount)), by=c("name","chrom","txStart")]$V1) ccds[, exon_end := max(as.numeric(strsplit(exonEnds, ",")[[1]])), by=c("name", "chrom", "txStart")] exonFrames <- as.numeric(unlist(strsplit(ccds$exonFrames, ","))) exonStarts <- as.numeric(unlist(strsplit(ccds$exonStarts, ","))) exonEnds <- as.numeric(unlist(strsplit(ccds$exonEnds, ","))) ccdsName <- rep(ccds$name, times=as.numeric(ccds$exonCount)) exonStrand <- rep(ccds$strand, times=as.numeric(ccds$exonCount)) #exonStarts[exonStrand == '+'] <- exonStarts[exonStrand == '+'] + 1 ## off by one? exonStarts <- exonStarts + 1 ## off by one? #exonEnds[exonStrand == '-'] <- exonEnds[exonStrand == '-'] + 1 ## off by one? intrStrand <- rep(ccds$strand, times=as.numeric(ccds$exonCount)-1) ccdschrom <- gsub("chr","",rep(ccds$chrom, times=as.numeric(ccds$exonCount))) gr.exons <- sort(gr.fix(GRanges(ccdschrom, IRanges(exonStarts, exonEnds), strand=exonStrand, name=ccdsName, num=exonNums, frame=exonFrames, gene=exonGene), si)) gr.introns <- sort(gr.fix(GRanges(intrChr, IRanges(intrStarts, intrEnds), strand=intrStrand, name=intrName, num=intrNums, frame=intrFrame, gene=intrGene), si)) #### genes gr.genes = sort(gr.fix(gr.nochr(with(fread("/xchip/gistic/Jeremiah/tracks/genes.hg19.ucsc.txt", sep="\t"), GRanges(chr, IRanges(beg, end), gene=symbol))), si)) gr.genes <- gr.genes[!grepl("^ULK|^NBPF|^MIR|^LOC|^OR|^SNO|^FAM|^ZNF|^SMN|^NF1P2|^POTEB|^RNF|^RGPD5|^RGPD2|^SNAR|^NBPF", gr.genes$gene) & width(gr.genes) < 3e6] gr.genes$gene <- as.character(gr.genes$gene) gg <- grbind(gr.exons, gr.introns) #gg <- gr.val(gg, gr.genes, 'gene', sep="_") #gg$gene[nchar(gg$gene) > 50] <- "MANYGENES" ## get complement gg <- gr.stripstrand(gr.fix(gg, si)) gg$gene <- as.character(gg$gene) gene.comp <- setdiff(gr.stripstrand(si2gr(si)), gg) + 1 fo <- gr2dt(gr.findoverlaps(gene.comp, gg)) fo <- fo[query.id %in% as.numeric(names(table(fo$query.id)[table(fo$query.id) == 2]))] ## now each intergenic region has front/back overlap fo[, right := gg$gene[subject.id[2]], by=query.id] fo[, left := gg$gene[subject.id[1]], by=query.id] gene.comp$left <- gene.comp$right <- "" gene.comp$left[fo$query.id] <- fo$left gene.comp$right[fo$query.id] <- fo$right gene.comp <- gene.comp - 1 ## tubio ff <- fread("/xchip/gistic/Jeremiah/tracks/master_db_29062016_ranges.txt") setnames(ff, c("V1","V2","V3","V4","V5","V6"), c("seqnames","start","end","dir","type","sample")) ff$strand <- ifelse(ff$dir == "plus","+","-") gr.tub <- dt2gr(ff) ================================================ FILE: R/archive_non_functional/gen_quals.R ================================================ require(data.table) f <- fread("grep -v ^# /broad/broadsv/NA12878/GCAT/gcat_illumina_150x/snowman/v115/v115.snowman.indel.vcf", sep='\t') hdr <- fread("grep ^# /broad/broadsv/NA12878/GCAT/gcat_illumina_150x/snowman/v115/v115.snowman.indel.vcf", sep="\t", header=FALSE) ## rescale the quality score f[, SL := gsub(".*?:.*?:.*?:.*?:.*?:.*?:.*?:.*?:.*?:(.*?)","\\1", V10)] f[, V6 := SL] p <- c(hdr$V1, f[, paste(V1,V2,V3,V4,V5,V6,V7,V8,V9,V10,sep="\t")]) writeLines(p, "/broad/hptmp/jwala/test.vcf") ================================================ FILE: R/archive_non_functional/svaba-annotate.R ================================================ #!/usr/bin/env Rscript library(optparse) option_list = list( make_option(c("-i", "--input"), type = "character", default = NULL, help = "Input VCF file"), make_option(c("-s", "--style"), type = "character", default = "ncbi", help = "[nbci] for 1,2,...X, or [ucsc] for chr1, chr2, ...chrX"), make_option(c("-g", "--genome"), type = "character", default = NULL, help = "genome build (hg19 or hg38)"), make_option(c("-b", "--db"), type = "character", default = "refseq", help = "[refseq] gene database to use (must be refseq or gencode)"), make_option(c("-o", "--output"), type = "character", default = "no_id", help = "Output annotation name") ) parseobj = OptionParser(option_list=option_list) opt = parse_args(parseobj) if (is.null(opt$input)) stop(print_help(parseobj)) if (!tolower(opt$style) %in% c("ncbi","ucsc")) stop("Must specify style as ncbi or ucsc") if (!tolower(opt$genome) %in% c("hg38","hg19")) stop("Must specify --genome as hg19 or hg38") write("...loading required packages\n", stderr()) library(roverlaps) ## can find at https://github.com/walaj/roverlaps ##library(plyr) library(RMySQL) library(data.table) ## setup the connect to UCSC write("...downloading annotation tracks from UCSC\n", stderr()) assembly <- opt$genome mychannel <- dbConnect(MySQL(), user="genome", host="genome-mysql.cse.ucsc.edu") query <- function(...) dbGetQuery(mychannel, ...) if (tolower(opt$db) == tolower("refseq")) { ## download the exons and their gene names from RefSeq (UCSC track) ## - reminder that refseq ids can be interpreted: ## https://www.ncbi.nlm.nih.gov/books/NBK50679/#RefSeqFAQ.what_is_the_difference_between ## - (from above): Accession numbers that begin with the prefix XM_ (mRNA), XR_ (non-coding RNA), and XP_ (protein) ## are model RefSeqs produced either by NCBIs genome annotation pipeline or copied from ## computationally annotated submissions to the INSDC.NCBI ## ...subsequently curated RefSeq records (with NM_, NR_, or NP_ accession prefixes) genes <- suppressWarnings(data.table::as.data.table(query(paste0("SELECT name, name2, chrom, txStart, txEnd, strand, exonStarts, exonEnds, exonCount, exonFrames FROM ", assembly, ".refGene")))) genes[, prefix := gsub("([A-Z]+)_.*","\\1", name)] genes[, longest := { a=rep(FALSE, .N); a[which.max(txEnd - txStart)] <- TRUE; a }, by=name2] genes[, numTranscriptAnnotations := .N, by=name2] genes <- genes[txEnd - txStart >= 100] ## only genes longer than 100 bp genes <- genes[genes$longest] ## just take the longest transcriopt write('...defaulting to annotating only the longest transcript') genes[, longest := NULL] setnames(genes, c("name2","name"), c("geneSymbol","id")) } else if (tolower(opt$db) == "gencode") { ## if want to use GENCODE instead, need to get table to convert GENCODE ids to gene names genes <- suppressWarnings(data.table::as.data.table(query(paste0("SELECT name, chrom, txStart, txEnd, strand, exonStarts, exonEnds, exonCount FROM ", assembly, ".knownGene")))) codes <- suppressWarnings(data.table::as.data.table(query(paste0("SELECT kgID, mRNA, geneSymbol, spID, refSeq FROM ", assembly, ".kgXref")))) ## merge the gene ids to gene names setnames(genes, c("name"), c("kgID")) setkey(genes, kgID) setkey(codes, kgID) genes <- codes[genes] stopifnot(!any(duplicated(genes$kgID))) genes[, geneSymbol := toupper(geneSymbol)] setnames(genes, "kgID", "id") } else { stop("db option must be either GENCODE or RefSeq") } write("...parsing downloaded annotation data\n", stderr()) ## convert to key'ed format setnames(genes, c("chrom", "txStart","txEnd"), c("seqnames","start","end")) if (tolower(opt$style) == "ncbi") genes[, seqnames := gsub("^chr", "", seqnames)] ## make the exons structure starts <- strsplit(genes[, exonStarts], ",") ends <- strsplit(genes[, exonEnds], ",") frames <- strsplit(genes[, exonFrames], ",") stopifnot(sum(sapply(starts, length)) == sum(sapply(ends, length))) stopifnot(sum(sapply(starts, length)) == sum(sapply(frames, length))) stopifnot(sum(sapply(starts, length)) == sum(genes$exonCount)) exons <- data.table(id = genes[, rep(id, exonCount)], geneSymbol = genes[, rep(geneSymbol, exonCount)], exonCount = genes[, rep(exonCount, exonCount)], seqnames = genes[, rep(seqnames, exonCount)], frames = as.numeric(unlist(frames)), start = as.numeric(unlist(starts)), end = as.numeric(unlist(ends)), strand = genes[, rep(strand, exonCount)]) exons[, exonNum := ifelse(strand=="+", seq(.N), rev(seq(.N))), by=id] ## make an introns track ## for the frames, define the intron from as the one of the previous exon, respecting strand ## EEEEiiiiiiiEEEEiiiiiiiiEEEEEiiiiiEEE <<< intron frame is frame of RIGHT exon (iiiiEEEE) ## EEEEiiiiiiiEEEEiiiiiiiiEEEEEiiiiiEEE >>> intron from is frame of LEFT exon (EEEEiii) ends <- exons[exonCount > 1, start[seq(2, .N)], by=id] starts <- exons[exonCount > 1, end[seq(1, .N-1)], by=id] if ("exonFrames" %in% colnames(genes)) { frames <- exons[exonCount > 1, { if (strand[1]=="+") frames[seq(1,.N-1)] else frames[seq(2,.N)] }, by=id] stopifnot(nrow(ends) == nrow(frames)) } stopifnot(nrow(ends) == nrow(starts)) stopifnot(all(ends$id == starts$id)) stopifnot(min(as.numeric(names(table(exons[exonCount>1, exonCount])))) > 1) if ("exonFrames" %in% colnames(genes)) { introns <- data.table(id=ends$id, start=starts$V1, end=ends$V1, frame=frames$V1) } else { introns <- data.table(id=ends$id, start=starts$V1, end=ends$V1) } short <- exons[!duplicated(id), .(id, geneSymbol, seqnames, strand, exonCount)] setkey(introns, id) setkey(short, id) introns <- short[introns] introns[, intronCount := exonCount - 1] introns[, exonCount := NULL] introns[, intronNum := ifelse(strand == "+", seq(.N), rev(seq(.N))), by=id] ## make the inter-genic trackk #ro <- roverlaps(genes, genes) #ro[,max(end), by=query.id]$V1 #setkey(genes, seqnames, start) #starts <- genes[,end[seq(2,.N)], by=seqnames]$V1 #ends <- genes[,start[seq(1,.N-1)], by=seqnames]$V1 ## read in file write("...reading VCF file\n", stderr()) if (grepl("gz$", opt$input)) { print(paste("gunzip -c", opt$input, "| grep -v ^#")) vcf <- data.table::fread(cmd=paste("gunzip -c", opt$input, "| grep -v ^#"), sep="\t") } else { vcf <- data.table::fread(cmd=paste("grep -v ^#", opt$input), sep="\t") } setnames(vcf, paste0("V", seq(9)), c("seqnames","start","id","ref","alt","qual","filter","info","geno")) ## bail early if nothing found if (nrow(vcf) == 0) { write("No rearrangements found\n", stderr()) quit(status=0) } else { write(paste("Found", nrow(vcf), "rearrangements\n"), stderr()) } ## grl.ix is unique ID for each rearrangement ## grl.iix is either 1 or 2 (for each side of the rearrangement) vcf[, grl.ix := gsub("([0-9]+):[0-9]","\\1",id)] vcf[, grl.iix := as.numeric(gsub("[0-9]+:([0-9])","\\1",id))] ## set the strand info for BND format vcf[grepl("SVTYPE=BND", info), strand := ifelse(grepl("^\\[", alt) | grepl("^\\]", alt), '-', '+')] vcf[, inv := strand[1] == strand[2], by=grl.ix] vcf[, altstrand := rev(strand), by=grl.ix] vcf[, altpos := as.integer(gsub(".*?:([0-9]+).*", "\\1", alt))] vcf[, altchr := gsub(".*?(\\[|\\])(.*?):([0-9]+).*", "\\2", alt)] vcf[, span := ifelse(seqnames==altchr, abs(start - altpos), -1)] ## annotate with gene overlaps ro <- roverlaps::roverlaps(vcf, genes) ro[, geneSymbol := genes$geneSymbol[subject.id]] ro[, geneSymbol := paste(unique(geneSymbol), collapse="_"), by=query.id] ro[, geneTranscriptCount := genes$numTranscriptAnnotations[subject.id]] ro[, geneStrand := genes$strand[subject.id]] vcf[ro$query.id, geneSymbol := ro$geneSymbol] vcf[ro$query.id, geneStrand := ro$geneStrand] vcf[, geneSymbolAlt := geneSymbol[rev(grl.iix)], by=grl.ix] vcf[, geneStrandAlt := geneStrand[rev(grl.iix)], by=grl.ix] vcf[ro$query.id, geneTranscriptCount := ro$geneTranscriptCount] ## annotate with genes within 20kb PAD <- 20e3 genes20 <- data.table::copy(genes) genes20[, start := start - PAD/2] genes20[, end := end + PAD/2] ro <- roverlaps::roverlaps(vcf, genes20) ro[, geneSymbol20kb := genes$geneSymbol[subject.id]] ro[, geneSymbol20kb := paste(unique(geneSymbol20kb), collapse="_"), by=query.id] vcf[ro$query.id, geneSymbol20kb := ro$geneSymbol20kb] vcf[, geneSymbol20kbAlt := geneSymbol20kb[rev(grl.iix)], by=grl.ix] ## overlap introns ro <- roverlaps::roverlaps(vcf, introns) ro[, intronNum := introns$intronNum[subject.id]] ro[, intronGene := introns$geneSymbol[subject.id]] ro[, intronStart := ifelse(introns$strand[subject.id] == "+", introns$start[subject.id], introns$end[subject.id])] ro[, intronGene := paste(unique(intronGene), collapse="_"), by=query.id] vcf[ro$query.id, intronGene := ro$intronGene] vcf[ro$query.id, intronNum := ro$intronNum] vcf[ro$query.id, intronStart := ro$intronStart] vcf[, intronGeneAlt := intronGene[rev(grl.iix)], by=grl.ix] vcf[, intronNumAlt := intronNum[rev(grl.iix)], by=grl.ix] vcf[, intronBpsIn := abs(intronStart - start)] if ("frame" %in% colnames(introns)) { ro[, cdsFrame := introns$frame[subject.id]] vcf[ro$query.id, cdsFrame := ro$cdsFrame] } vcf[!is.na(intronNum), bk_msg := paste(intronBpsIn, "bp_into_intron", intronNum, "of", intronGene, sep="_")] ## overlap exons ro <- roverlaps(vcf, exons) ro[, exonNum := exons$exonNum[subject.id]] ro[, exonGene := exons$geneSymbol[subject.id]] ro[, exonStart := ifelse(exons$strand[subject.id] == "+", exons$start[subject.id], exons$end[subject.id])] ro[, exonGene := paste(unique(exonGene), collapse="_"), by=query.id] vcf[ro$query.id, exonGene := ro$exonGene] vcf[ro$query.id, exonNum := ro$exonNum] vcf[ro$query.id, exonStart := ro$exonStart] vcf[, exonGeneAlt := exonGene[rev(grl.iix)], by=grl.ix] vcf[, exonNumAlt := exonNum[rev(grl.iix)], by=grl.ix] vcf[, exonBpsIn := abs(exonStart - start)] vcf[!is.na(exonNum), bk_msg := paste(exonBpsIn, "bp_into_exon", exonNum, "of", exonGene, sep="_")] ## annotate fusions vcf[, fusion := { f <- sum(grepl("intron|exon", bk_msg)) == 2 && (geneSymbol[1] != geneSymbol[2]); if (is.na(f)) { FALSE } else { f } } , by = grl.ix] vcf[, sense := all(geneStrand != "") && ((geneStrand[1] == geneStrand[2] && strand[1] != strand[2]) || (strand[1] != strand[2] && geneStrand[1] == geneStrand[2])), by = grl.ix] ## DECIDE WHICH PIECE IS "first" in the fusion gene (where Tx starts) ## RAR_STRAND GENE_STRAND ## ++ or -- must be +- or -+. Starts on + ## +- or -- msut be -- or ++. Starts on side where gstrand == rarstrand if (any(vcf$fusion)) { vcf[vcf$fusion, gorder := { mk <- c(NA,NA) ## inversion if (strand[1] == strand[2]) { if (geneStrand[1] == '+') mk <- c(2,1) else mk <- c(1,2) } ## non type if (geneStrand[1] == geneStrand[1]) mk <- c(1,2) else mk <- c(2,1) mk }, by=grl.ix] } else { vcf[, gorder := 1] ##dummy } ## decide if in frame if ("cdsFrame" %in% colnames(vcf)) { vcf[, in_frame := { f = (cdsFrame[gorder[2]] - cdsFrame[gorder[1]]); xbases <- 0; ##xbases <- nchar(INSERTION[1]) - nchar(HOMSEQ[1]); if (is.na(f[1])) FALSE else if (all(grepl('intron', bk_msg)) && sense[1]) ## intron to intron f == 0 else f + xbases %% 3 == 0 }, by=grl.ix] ## make the fusion messages vcf[, in_frame_fusion := in_frame && fusion && sense, by=grl.ix] vcf[vcf$in_frame_fusion, msg := paste("In-frame sense fusion from:", bk_msg[gorder[1]], "to", bk_msg[gorder[2]]), by=grl.ix] vcf[!vcf$in_frame_fusion & vcf$fusion & vcf$sense, msg := paste("Out-of-frame sense fusion between:", bk_msg[1], "and", bk_msg[2]), by=grl.ix] vcf[!vcf$sense, msg := paste("Anti-sense fusion between:", bk_msg[1], "and", bk_msg[2]), by=grl.ix] } vcf[, c("fusion","gorder") := NULL] ## writing to stdout write.table(vcf[,.(seqnames, start, start, altchr, altpos, altpos, strand, altstrand, id, ref, alt, qual, filter, info, grl.ix, grl.iix, span, geneSymbol, geneStrand, geneSymbol20kb, intronGene, intronNum, bk_msg, exonGene, exonNum, in_frame, in_frame_fusion, msg)], row.names=FALSE, col.names=TRUE, quote=FALSE, sep="\t", file=stdout()) ## ##llr$bk_msg[!grepl("intron|exon", llr$bk_msg)] <- "" ## write("...annotating intergenic") ## fo <- gr.findoverlaps(llr, gene.comp) ## if (nrow(fo)) { ## fo[, left := gene.comp$left[subject.id], by=query.id] ## fo[, right := gene.comp$right[subject.id], by=query.id] ## fo[, right_start := end(gene.comp)[subject.id], by=subject.id] ## fo[, left_end := start(gene.comp)[subject.id], by=subject.id] ## fo[, left_dist := start - left_end, by=query.id] ## fo[, right_dist := right_start - end, by=query.id] ## fo[, mmm := paste(left_dist, "bp to right of", left, "and", right_dist, "bp to left of", right)] ## llr$msg[!nchar(llr$msg)] <- "" ## llr$bk_msg[fo$query.id] <- ifelse(!nchar(llr$bk_msg[fo$query.id]), fo$mmm, llr$bk_msg[fo$query.id]) ## } ## ############## make the circos plot ## write("...making Circos plot") ## library(RCircos) ## data(UCSC.HG19.Human.CytoBandIdeogram); ## chr.exclude <- NULL; ## cyto.info <- UCSC.HG19.Human.CytoBandIdeogram; ## tracks.inside <- 10; ## tracks.outside <- 0; ## RCircos.Set.Core.Components(cyto.info, chr.exclude, tracks.inside, tracks.outside); ## ## get the gene label dat ## #genes <- genes[width(genes) < 2e6] ## #fo1 <- gr.findoverlaps(gr1+10e3, genes) ## #fo2 <- gr.findoverlaps(gr2+10e3, genes) ## ## annoying bug with seqinfo clash on 'c' ## #if (length(fo1) && length(fo2)) { ## # fo <- c(fo1,fo2) ## #} else if (length(fo1)) { ## # fo <- fo1 ## #} else { ## # fo <- fo2 ## #} ## write("...constructing Circos plot") ## ## set the gene labels ## gene.dat <- data.frame(Chromsome=c(dt.bks$chr1, dt.bks$chr2), chromStart=c(dt.bks$pos1, dt.bks$pos2), ## chromEnd=c(dt.bks$pos1,dt.bks$pos2), Gene=as.character(c(dt.bks$gene1, dt.bks$gene2)), ## stringsAsFactors=FALSE) ## if (nrow(gene.dat)) ## gene.dat <- gene.dat[nchar(gene.dat$Gene) > 0,] ## if (nrow(gene.dat)) ## gene.dat <- gene.dat[!duplicated(gene.dat$Gene),] ## links = data.frame() ## if (length(bks)) ## links = with(dt.bks, data.frame(Chromosome=chr1, chromStart=pos1, chromEnd=pos1, Chromsome.1=chr2, chromStart.1=pos2, chromEnd.1=pos2)) ## ## plot the PDF ## pdf(file=paste0(opt$output,".pdf"), height=opt$height, width=opt$width, compress=TRUE); ## RCircos.Set.Plot.Area(); ## RCircos.Chromosome.Ideogram.Plot(); ## if (opt$genes != 0 && nrow(gene.dat) > 0) { ## track.num <- 1 ## RCircos.Gene.Connector.Plot(gene.dat, track.num, "in"); ## track.num <- 2; ## name.col <- 4; ## RCircos.Gene.Name.Plot(gene.dat, name.col, track.num, "in"); ## } ## if (nrow(links) > 0) { ## track.num = 1 ## RCircos.Link.Plot(links, track.num, by.chromosome=TRUE) ## by.chromosome is for color ## } ## dev.off() ================================================ FILE: R/archive_non_functional/svaba-asqg2pdf.R ================================================ #!/usr/bin/env Rscript library(optparse) option_list = list( make_option(c("-i", "--input"), type = "character", default = NULL, help = "Input asqg file from snowman run --write-asqg or sga"), make_option(c("-c", "--inputcc"), type = "character", default = NULL, help = "Input cc file from snowman run --write-asqg or sga"), make_option(c("-o", "--output"), type = "character", default = "graph.pdf", help = "Output pdf to write the graph"), make_option(c("-d", "--height"), type = "numeric", default = 20, help = "Height"), make_option(c("-w", "--width"), type = "numeric", default = 20, help = "Width"), make_option(c("-m", "--mincount"), type = "numeric", default = 1, help = "Remove nodes with fewer than m components") ) parseobj = OptionParser(option_list=option_list) opt = parse_args(parseobj) if (is.null(opt$input)) stop(print_help(parseobj)) require(igraph) require(data.table) #opt$input ="/xchip/gistic/Jeremiah/Projects/SnowmanPaper/Benchmark/150830/tmp.graph.after.asqg" vert.file = paste(opt$input, "vert", sep=".") ##reads.file = file.path(dir, 'plots', 'readsForR_som.txt') ##reads <- import.snowman.reads(reads.file) #cont = 'c_8:43487254-43492254_11' ##cont = 'c_8:43487254-43492254_0' ##this_reads <- reads$rname[as.character(seqnames(reads)) %in% cont] ## read the edges ln= readLines(opt$input, n = 1000) nskip = sum(grepl('^VT|^HT', ln)) tab.e <- read.delim(opt$input, skip=nskip, strings=FALSE, header=FALSE, sep=" ") tab.e$V1 <- substring(tab.e$V1, 4) colnames(tab.e) <- c('seq1', 'seq2', 'overlap_start1', 'overlap_end1', 'len1', 'overlap_start2', 'overlap_end2', 'len2', 'orientation', 'numdiff') edges <- data.frame(from=tab.e$seq1, to=tab.e$seq2) ## read the verts cmd = paste("grep", '"VT"', opt$input, ">", vert.file) system(cmd) tab <- read.delim(vert.file, skip=0, strings=FALSE, header=FALSE, sep="\t") if (ncol(tab) == 4) { colnames(tab) <- c("V", "rname", 'seq', 'ss') } else if (ncol(tab) == 3) { colnames(tab) <- c("V", "rname", 'seq') } verts <- data.frame(verts=unique(c(as.character(tab$rname), as.character(edges$to), as.character(edges$from)))) g <- graph.data.frame(edges, directed=FALSE, vertices=verts) ## vert lengths vert.lens <- structure(nchar(tab$seq), names=tab$rname) ## open the cc file if (file.info(opt$inputcc)$size > 0) { cc <- fread(opt$inputcc) cc$col <- sample(colors(), length(unique(cc$V2)))[match(cc$V2, unique(cc$V2))] setkey(cc, col) V(g)$color = "black" for (x in cc$col) { V(g)$color[verts$verts %in% cc[x]$V1] <- x } } ## format the verts V(g)$names = paste(as.character(verts$verts), "len:", vert.lens[as.character(verts$verts)]) ##V(g)$color[V(g)$names %in% this_reads] <- 'red' ## format the edges E(g)$lab <- tab.e$overlap_end2 - tab.e$overlap_start2 ## cluster V(g)$community <- membership(cl<-clusters(g)) good_communities <- which(cl$csize >= opt$mincount) g <- delete.vertices(g, V(g)[!community %in% good_communities]) pdf(opt$output, height=opt$height, width=opt$width) plot(g, vertex.color=V(g)$color, vertex.label=V(g)$names, edge.label=E(g)$lab, vertex.size=2) dev.off() ================================================ FILE: R/archive_non_functional/svaba-bam-qcplot.R ================================================ #!/usr/bin/env Rscript library(optparse) option_list = list( make_option(c("-i", "--input"), type = "character", default = "qcreport.txt", help = "Input txt file from a snowman preprocess qcreport.txt"), make_option(c("-o", "--output"), type = "character", default = "qcreport.pdf", help = "Output pdf to generate") ) parseobj = OptionParser(option_list=option_list) opt = parse_args(parseobj) if (is.null(opt$input)) stop(print_help(parseobj)) if (!file.exists(opt$input)) { print(print_help(parseobj)) stop(paste("Input file does not exist", opt$input, ". Must supply path to valid qcreport.txt file (generated from snowman preprocess or snowman run")) } print(opt) require(ggplot2) require(reshape2) require(gridExtra) ## read the table con <- file(opt$input, open = "r") df.mapq <- df.nm <- df.isize <- df.as <- df.xp <- df.len <- df.phred <- df.clip <- data.frame() rg <- list() while (length(line <- readLines(con, n = 1, warn = FALSE)) > 0) { ## found a new one if (grepl("READGROUP", line)) { thisrg = gsub("READGROUP:BI:(.*)", "\\1", line) rg[[thisrg]] <- data.frame(readgroup = thisrg) } else if (grepl("total", line)) { rg[[thisrg]]$total = as.numeric(gsub("total,([0-9]+)", "\\1", line)) } else if (grepl("unmap", line)) { rg[[thisrg]]$unmap = as.numeric(gsub("unmap,([0-9]+)", "\\1", line)) } else if (grepl("qcfail", line)) { rg[[thisrg]]$qcfail = as.numeric(gsub("qcfail,([0-9]+)", "\\1", line)) } else if (grepl("duplicate", line)) { rg[[thisrg]]$duplicate = as.numeric(gsub("duplicate,([0-9]+)", "\\1", line)) } else if (grepl("supplementary", line)) { rg[[thisrg]]$supp = as.numeric(gsub("supplementary,([0-9]+)", "\\1", line)) } else if (grepl("mapq", line)) { df.mapq <- rbind(df.mapq, as.numeric(strsplit(gsub("mapq,([0-9]+)", "\\1", line), ",")[[1]])) } else if (grepl("nm", line)) { df.nm <- rbind(df.nm, as.numeric(strsplit(gsub("nm,([0-9]+)", "\\1", line), ",")[[1]])) } else if (grepl("isize", line)) { df.isize <- rbind(df.isize, as.numeric(strsplit(gsub("isize,([0-9]+)", "\\1", line), ",")[[1]])) } else if (grepl("as", line)) { df.as <- rbind(df.as, as.numeric(strsplit(gsub("as,([0-9]+)", "\\1", line), ",")[[1]])) } else if (grepl("xp", line)) { df.xp <- rbind(df.xp, as.numeric(strsplit(gsub("xp,([0-9]+)", "\\1", line), ",")[[1]])) } else if (grepl("clip", line)) { df.clip <- rbind(df.clip, as.numeric(strsplit(gsub("clip,([0-9]+)", "\\1", line), ",")[[1]])) } else if (grepl("len", line)) { df.len <- rbind(df.len, as.numeric(strsplit(gsub("len,([0-9]+)", "\\1", line), ",")[[1]])) } else if (grepl("phred", line)) { df.phred <- rbind(df.phred, as.numeric(strsplit(gsub("phred,([0-9]+)", "\\1", line), ",")[[1]])) } else { stop(paste("Failed to read file at line:", line)) } } close(con) colnames(df.mapq) <- seq(from=0,to=60) colnames(df.nm) <- seq(from=0,to=ncol(df.nm)-1) colnames(df.isize) <- seq(from=0,to=ncol(df.isize)-1) colnames(df.xp) <- seq(from=0, to=ncol(df.xp)-1) colnames(df.as) <- seq(from=0, to=ncol(df.as)-1) colnames(df.len) <- seq(from=0, to=ncol(df.len)-1) colnames(df.phred) <- seq(from=0, to=ncol(df.phred)-1) colnames(df.clip) <- seq(from=0, to=ncol(df.clip)-1) readg <- sapply(rg, function(x) x$readgroup) df.mapq$readgroup <- readg df.nm$readgroup <- readg df.isize$readgroup <- readg df.xp$readgroup <- readg df.as$readgroup <- readg df.phred$readgroup <- readg df.len$readgroup <- readg df.clip$readgroup <- readg g.mapq <- ggplot(df.mapq.m <- melt(df.mapq, id='readgroup'), aes(x=as.numeric(variable), y=pmax(log(value,10),0), group=readgroup, color=readgroup)) + geom_line() + theme_bw() + scale_x_continuous(limits=c(30,61)) + ylab('Reads') + xlab('Mapping Quality') + scale_y_continuous(breaks=seq(0,8), labels=parse(text=paste('10', seq(0,8), sep='^'))) + theme(legend.text = element_text(size=6), legend.title = element_text(size=6), text = element_text(size=9)) g.nm <- ggplot(df.nm.m <- melt(df.nm, id='readgroup'), aes(x=as.numeric(variable), y=pmax(log(value,10),0), group=readgroup, color=readgroup)) + geom_line() + theme_bw() + scale_x_continuous(limits=c(0,50)) + ylab('Reads') + xlab('NM Tag') + scale_y_continuous(breaks=seq(0,8), labels=parse(text=paste('10', seq(0,8), sep='^'))) + theme(legend.text = element_text(size=6), legend.title = element_text(size=6), text = element_text(size=9)) g.isize <- ggplot(df.isize.m <- melt(df.isize, id='readgroup'), aes(x=as.numeric(variable), y=pmax(log(value,10),0), group=readgroup, color=readgroup)) + geom_line() + theme_bw() + scale_x_continuous(limits=c(10,2001)) + ylab('Reads') + xlab('InsertSize') + scale_y_continuous(breaks=seq(0,8), labels=parse(text=paste('10', seq(0,8), sep='^'))) + theme(legend.text = element_text(size=6), legend.title = element_text(size=6), text = element_text(size=9)) g.xp <- ggplot(df.xp.m <- melt(df.xp, id='readgroup'), aes(x=as.numeric(variable), y=pmax(log(value,10),0), group=readgroup, color=readgroup)) + geom_line() + theme_bw() + scale_x_continuous(limits=c(0,100)) + ylab('Reads') + xlab('XP Tag') + scale_y_continuous(breaks=seq(0,8), labels=parse(text=paste('10', seq(0,8), sep='^'))) + theme(legend.text = element_text(size=6), legend.title = element_text(size=6), text = element_text(size=9)) g.as <- ggplot(df.as.m <- melt(df.as, id='readgroup'), aes(x=as.numeric(variable), y=pmax(log(value,10),0), group=readgroup, color=readgroup)) + geom_line() + theme_bw() + scale_x_continuous(limits=c(0,100)) + ylab('Reads') + xlab('AS Tag') + scale_y_continuous(breaks=seq(0,8), labels=parse(text=paste('10', seq(0,8), sep='^'))) + theme(legend.text = element_text(size=6), legend.title = element_text(size=6), text = element_text(size=9)) g.len <- ggplot(df.len.m <- melt(df.len, id='readgroup'), aes(x=as.numeric(variable), y=pmax(log(value,10),0), group=readgroup, color=readgroup)) + geom_line() + theme_bw() + scale_x_continuous(limits=c(20,102)) + ylab('Reads') + xlab('Read Length') + scale_y_continuous(breaks=seq(0,8), labels=parse(text=paste('10', seq(0,8), sep='^'))) + theme(legend.text = element_text(size=6), legend.title = element_text(size=6), text = element_text(size=9)) g.clip <- ggplot(df.clip.m <- melt(df.clip, id='readgroup'), aes(x=as.numeric(variable), y=pmax(log(value,10),0), group=readgroup, color=readgroup)) + geom_line() + theme_bw() + scale_x_continuous(limits=c(0,85)) + ylab('Reads') + xlab('Clipped bases') + scale_y_continuous(breaks=seq(0,8), labels=parse(text=paste('10', seq(0,8), sep='^'))) + theme(legend.text = element_text(size=6), legend.title = element_text(size=6), text = element_text(size=9)) g.phred <- ggplot(df.phred.m <- melt(df.phred, id='readgroup'), aes(x=as.numeric(variable), y=pmax(log(value,10),0), group=readgroup, color=readgroup)) + geom_line() + theme_bw() + scale_x_continuous(limits=c(0,43)) + ylab('Reads') + xlab('Mean read Phred quality') + scale_y_continuous(breaks=seq(0,8), labels=parse(text=paste('10', seq(0,8), sep='^'))) + theme(legend.text = element_text(size=6), legend.title = element_text(size=6), text = element_text(size=9)) df.isize.m$variable <- as.numeric(as.character(df.isize.m$variable)) df.disco <- df.isize.m[df.isize.m$variable > 0 & df.isize.m$variable < 800, ] g.isize <- ggplot(df.disco, aes(x=variable, y=value, color=readgroup)) + geom_line() + theme_bw() + ylab('Reads') + xlab('Insert Size') + theme(legend.text = element_text(size=6), legend.title = element_text(size=6), text = element_text(size=9)) ## get percentages # disc, proper df.disc.pie <- data.frame(rbind(rowSums(df.isize[, (800+1):(2000+1)]), rowSums(df.isize[, 1:800])), class=c("\"Discordant\" (I > 800)", "\"Proper\"(I < 800)")) colnames(df.disc.pie) <- c(as.character(levels(readg)), "Class") df.disc.pie.m <- melt(df.disc.pie, id='Class') g.disc.pie <- ggplot(df.disc.pie.m, aes(x=factor(1), y=value, fill=Class)) + geom_bar(stat='identity', position="fill") + facet_wrap( ~ variable) + coord_polar(theta="y") + xlab("") + ylab("") + theme(legend.text = element_text(size=6), legend.title = element_text(size=6), text = element_text(size=9), legend.position="bottom") df.clip.pie <- data.frame(rbind(rowSums(df.clip[, 6:102]), rowSums(df.clip[, 1:5])), class=c("\"Clipped\" (clip >= 5)", "\"Matched\"(clip < 5)")) colnames(df.clip.pie) <- c(as.character(levels(readg)), "Class") df.clip.pie.m <- melt(df.clip.pie, id='Class') g.clip.pie <- ggplot(df.clip.pie.m, aes(x=factor(1), y=value, fill=Class)) + geom_bar(stat='identity', position="fill") + facet_wrap( ~ variable) + coord_polar(theta="y") + xlab("") + ylab("") + theme(legend.text = element_text(size=6), legend.title = element_text(size=6), text = element_text(size=9), legend.position="bottom") pdf(opt$output, width=22, height=12) print(grid.arrange(g.mapq, g.nm, g.isize, g.len, g.clip, g.phred, g.disc.pie, g.clip.pie, ncol=3)) dev.off() ================================================ FILE: R/archive_non_functional/svaba-benchmark.R ================================================ #!/use/bin/env/ Rscript require(ggplot2) require(data.table) .libPaths = c("/xchip/gistic/Jeremiah/R", "/broad/software/free/Linux/redhat_6_x86_64/pkgs/r_3.1.1-bioconductor-3.0/lib64/R/library") library(optparse) AVAIL_MODES <- c("realign-test") option_list = list( make_option(c("-i", "--input"), type = "character", default = NULL, help = "Input SV VCF file"), make_option(c("-o", "--output"), type = "character", default = "no_id", help = "Output annotation name"), make_option(c("-m", "--mode"), type = "character", default = "realign-test", help = "Benchmarking mode to analyze") ) parseobj = OptionParser(option_list=option_list) opt = parse_args(parseobj) if (is.null(opt$input)) stop(print_help(parseobj)) if (!opt$mode %in% AVAIL_MODES) stop(paste("Mode", opt$mode, "must be one of", paste(AVAIL_MODES, collapse=", "))) .format_flag_df <- function(dfh2) { dfh2 <- data.table(dfh[dfh$del_size == "0" & dfh$snv_rate == "0" & dfh$ins_size != "0", ]) dfh2$index = seq(nrow(dfh2)) dfh2[, total := sum(c(wrong_align, too_align, no_align, correct)), by=index] dfh2 = data.frame(dfh2) dfh2[, c("no_align", "wrong_align", "too_align","correct")] = sweep(dfh2[, c('no_align', 'wrong_align', 'too_align', 'correct')], 1, dfh2$total, "/") dfh2 <- melt(dfh2, id.vars=c("width","ins_size", "del_size", "snv_rate", "total", "index")) return (dfh2) } if (opt$mode == "realign-test") { ff <- fread(opt$input, header=TRUE) df <- data.frame(); dfh <- data.frame(); for (j in unique(ff$width)) { for (k in unique(ff$snv_rate)) { for (d in unique(ff$del_size)) { for (ii in unique(ff$ins_size)) { #ii = ifelse(d == 0, 0, ii) ## if del is zero, then make ins zero too, since just want to evaluate zeros #d = ifelse(ii == 0, 0, d) ## if ins is zero, then make del zero too, since we just want to evaulate zeros if (ii == 0 || d == 0) { ix <- ff$width == j & ff$snv_rate == k & ff$ins_size == d & ff$del_size == ii if (sum(ix)) { cf <- ecdf(ff$num_align[ix]) s <- seq(0,max(ff$num_align)) df <- rbind(df, data.frame(num_align=s, cdf=cf(s), width=as.character(j), snv_rate=as.character(k), del_size = as.character(d), ins_size=as.character(ii), one.x = 0, one.y = cf(1))) dfh <- rbind(dfh, data.frame(no_align=sum(ix & ff$num_align == 0), wrong_align=sum(ix & ff$correct_hit_num == -1 & ff$num_align == 1), too_align=sum(ix & ff$num_align > 1), correct=sum(ix & ff$correct_hit_num == 0), width=as.character(j), del_size = as.character(d), ins_size = as.character(ii), snv_rate = as.character(k))) } } } } } } ## SNV ONLY df2 <- df[df$ins_size == "0" & df$del_size == "0", ] g <- ggplot(data=df2) + geom_line(aes(x=num_align, y=cdf, color=width)) + geom_point(aes(x=one.x, y=one.y, color=width)) + theme_bw() + xlab("Number of alignments") + ylab("CDF") + facet_wrap(~ snv_rate, nrow=1) + scale_y_continuous(limits=c(min(df2$one.y)-0.1,1), breaks=seq(0,1,by=0.05)) + labs(color="Sequence Length") pdf("~/public_html/realign_test_snv_cigcheck.pdf", width=7, height=2) print(g) dev.off() ## DEL ONLY df2 <- df[df$ins_size == "0" & df$snv_rate == "0", ] g <- ggplot(data=df2) + geom_line(aes(x=num_align, y=cdf, color=width)) + geom_point(aes(x=one.x, y=one.y, color=width)) + theme_bw() + xlab("Number of alignments") + ylab("CDF") + facet_wrap(~ del_size, nrow=1) + scale_y_continuous(limits=c(min(df2$one.y)-0.1,1), breaks=seq(0,1,by=0.2)) + labs(color="Sequence Length") pdf("~/public_html/realign_test_del_cigcheck.pdf", width=7, height=2) print(g) dev.off() ## INS ONLY df2 <- df[df$del_size == "0" & df$snv_rate == "0" & df$ins_size != "0", ] g <- ggplot(data=df2) + geom_line(aes(x=num_align, y=cdf, color=width)) + geom_point(aes(x=one.x, y=one.y, color=width)) + theme_bw() + xlab("Number of alignments") + ylab("CDF") + facet_wrap(~ ins_size, nrow=1) + scale_y_continuous(limits=c(min(df2$one.y)-0.1,1), breaks=seq(0,1,by=0.2)) + labs(color="Sequence Length") pdf("~/public_html/realign_test_ins_cigcheck.pdf", width=7, height=2); print(g); dev.off() dfh2 <- .format_flag_df(df2) g2 <- ggplot(data=dfh2,aes(x=factor(width),y=value,fill=factor(variable))) + geom_bar(position="stack", stat='identity') + facet_wrap(~ ins_size, nrow=1) + scale_fill_manual(values=c("no_align"="black", "wrong_align"="red", "too_align"="purple","correct"="dark green"), labels=c("Unmapped", "Incorrect alignment", "> 1 alignment", "Accurate"), name="Alignment") + scale_y_continuous(breaks=seq(0,1,by=0.25), labels=c("0", "25", "50", "75", "100"), name="Percentage") + xlab("Sequence Length") pdf("~/public_html/realign_ins_flag.pdf", width=7, height=2); print(g2); dev.off() #g <- ggplot(data=ff) + geom_histogram(aes(x=num_aligns)) + scale_y_log10(limits=c(1, 10)) + theme_bw() pdf("~/public_html/realign_test.pdf", width=7, height=7) print(g) dev.off() } if (FALSE) { snow <- ra_breaks("/xchip/gistic/Jeremiah/Projects/SnowmanPaper/Benchmark/snow2/chr1.broad-snowman.DATECODE.somatic.sv.vcf") simd <- fread("/xchip/gistic/Jeremiah/Projects/SnowmanPaper/Benchmark/connections.tsv") gr.sim <- with(simd, GRanges(c(V1,V1)+1, IRanges(c(V2,V4), width=1), strand=ifelse(c(V3, V5)=='+', '+', '-'), id=rep(seq(nrow(simd)),each=2))) grl.sim <- split(gr.sim, gr.sim$id) ra.overlaps(snow, grl.sim, pad=10, ignore.strand=TRUE) ## read it dt <- fread("/xchip/gistic/Jeremiah/Projects/SnowmanPaper/150805benchmark.csv") dt [, mean_cc := mean(contig_coverage), by=c('kmer_corr', 'coverage', 'error_rate')] dt [, se_cc := sd(contig_coverage), by=c('kmer_corr', 'coverage', 'error_rate')] setkey(dt, kmer_corr, coverage, error_rate) dt <- unique(dt) dt <- readRDS("/xchip/gistic/Jeremiah/tracks/100map.dt.rds") ## relabel names en <- c("0"="Error Rate: 0", "0.001"="Error Rate: 1e-3", "0.005"="Error Rate 5e-3", "0.01"="Error Rate: 0.01", "0.03"="Error Rate: 0.03", "0.05"="Error Rate: 0.05", "0.1"="Error Rate: 0.1") .labeller <- function(variable,value){ return(en[value]) } ## plot it df = data.frame(dt) df$error_rate = as.character(df$error_rate) df$kmer_corr = factor(df$kmer_corr, levels=c(0,1)) g <- ggplot(df) + geom_line(aes(x=coverage, y=mean_cc, color=kmer_corr), size=1) + geom_errorbar(aes(x=coverage,ymin=mean_cc-se_cc, ymax=mean_cc+se_cc, color=kmer_corr), width=1) + theme_bw() + xlab("Coverage") + ylab("Percent Re-assembled") + scale_x_continuous(breaks=seq(0,40,by=5)) + facet_grid(error_rate ~ ., labeller=.labeller) + coord_cartesian(ylim=c(0,1.3)) + scale_y_continuous(breaks=seq(0,1,by=0.25)) pdf("~/public_html/plot.pdf", width=6, height=12); print(g); dev.off() } ================================================ FILE: R/archive_non_functional/svaba-bps-to-maflite.R ================================================ #!/usr/bin/env Rscript library(optparse) suppressMessages(suppressWarnings(require(VariantAnnotation, quietly=TRUE))) option_list = list( make_option(c("-i", "--input"), type = "character", default = NULL, help = "Input bps.txt.gz file"), make_option(c("-o", "--output"), type = "character", default = "graph.pdf", help = "Output MAFLITE") ) parseobj = OptionParser(option_list=option_list) opt = parse_args(parseobj) if (is.null(opt$input)) stop(print_help(parseobj)) v <- readVcf(opt$input, "hg19") inf <- info(v) gr <- rowData(v) ## remove non indel lines gr <- gr[inf$EVDNC=="INDEL",] inf <- inf[inf$EVDNC=="INDEL",] ## remove unnecesry columns #tab <- data.frame(chr=as.character(seqnames(gr)), startr=start(gr), endr=end(gr), ref=gr$REF, alt=gr$ALT) tab <- data.frame(chr=as.character(seqnames(gr)), startr=start(gr), end=end(gr), ref=as.character(gr$REF), alt=as.character(gr$ALT), stringsAsFactors=FALSE) del = sapply(tab$ref, nchar) > sapply(tab$alt, nchar) ins = sapply(tab$ref, nchar) < sapply(tab$alt, nchar) tab$alt[ins] <- inf$INSERTION[ins] tab$ref[del] <- sapply(tab$ref[del], function(x) substr(x, 2, nchar(x))) tab$alt[del] = "-" tab$ref[ins] = "-" tab <- cbind(tab, inf[, c("SCTG", "SPAN", "MAPQ", "TSPLIT", "NSPLIT")]) writeLines(tab, opt$output) ================================================ FILE: R/archive_non_functional/svaba-circos.R ================================================ #!/usr/bin/env Rscript ## set the right library paths .libPaths = c("/xchip/gistic/Jeremiah/R", "/broad/software/free/Linux/redhat_6_x86_64/pkgs/r_3.1.1-bioconductor-3.0/lib64/R/library") require(data.table) ra_breaks <- function(rafile, keep.features = T, seqlengths = hg_seqlengths(), chr.convert = T, snowman = FALSE, breakpointer = FALSE, seqlevels = NULL, get.loose = FALSE ## if TRUE will return a list with fields $junctions and $loose.ends ) { if (is.character(rafile)) { if (grepl('(vcf$)|(vcf.gz$)', rafile)) { library(VariantAnnotation) vcf = readVcf(rafile, Seqinfo(seqnames = names(seqlengths), seqlengths = seqlengths)) if (!('SVTYPE' %in% names(info(vcf)))) { warning('Vcf not in proper format. Is this a rearrangement vcf?') return(GRangesList()); } vgr = rowData(vcf) ## parse BND format ## no events if (length(vgr) == 0) return (GRangesList()) ## fix mateids if not included if (!"MATEID"%in%colnames(mcols(vgr))) { nm <- vgr$MATEID <- names(vgr) ix <- grepl("1$",nm) vgr$MATEID[ix] = gsub("(.*?)(1)$", "\\12", nm[ix]) vgr$MATEID[!ix] = gsub("(.*?)(2)$", "\\11", nm[!ix]) vgr$SVTYPE="BND" } if (!any(c("MATEID", "SVTYPE") %in% colnames(mcols(vgr)))) stop("MATEID or SVTYPE not included. Required") vgr$mateid = info(vcf)$MATEID vgr$svtype = info(vcf)$SVTYPE if (!is.null(info(vcf)$SCTG)) vgr$SCTG = info(vcf)$SCTG if (sum(vgr$svtype == 'BND')==0) stop('Vcf not in proper format. Will only process rearrangements in BND format') if (!all(vgr$svtype == 'BND')) warning(sprintf('%s rows of vcf do not have svtype BND, ignoring these'), sum(vgr$svtype != 'BND')) bix = which(vgr$svtype == "BND") vgr = vgr[bix] vgr$first = !grepl('^(\\]|\\[)', vgr$ALT) ## ? is this row the "first breakend" in the ALT string (i.e. does the ALT string not begin with a bracket) vgr$right = grepl('\\[', vgr$ALT) ## ? are the (sharp ends) of the brackets facing right or left vgr$coord = as.character(paste(seqnames(vgr), ':', start(vgr), sep = '')) vgr$mcoord = as.character(gsub('.*(\\[|\\])(.*\\:.*)(\\[|\\]).*', '\\2', vgr$ALT)) vgr$mcoord = gsub('chr', '', vgr$mcoord) if (all(is.na(vgr$mateid))) if (!is.null(names(vgr)) & !any(duplicated(names(vgr)))) { warning('MATEID tag missing, guessing BND partner by parsing names of vgr') vgr$mateid = paste(gsub('::\\d$', '', names(vgr)), (sapply(strsplit(names(vgr), '\\:\\:'), function(x) as.numeric(x[length(x)])))%%2 + 1, sep = '::') } else if (!is.null(vgr$SCTG)) { warning('MATEID tag missing, guessing BND partner from coordinates and SCTG') require(igraph) ucoord = unique(c(vgr$coord, vgr$mcoord)) vgr$mateid = paste(vgr$SCTG, vgr$mcoord, sep = '_') if (any(duplicated(vgr$mateid))) { warning('DOUBLE WARNING! inferred mateids not unique, check VCF') bix = bix[!duplicated(vgr$mateid)] vgr = vgr[!duplicated(vgr$mateid)] } } else stop('MATEID tag missing') vgr$mix = as.numeric(match(vgr$mateid, names(vgr))) pix = which(!is.na(vgr$mix)) vgr.pair = vgr[pix] if (length(vgr.pair)==0) stop('No mates found despite nonzero number of BND rows in VCF') vgr.pair$mix = match(vgr.pair$mix, pix) vix = which(1:length(vgr.pair)0, '+', '-') if (is.numeric(rafile$str2)) rafile$str2 = ifelse(rafile$str2>0, '+', '-') rafile$rowid = 1:nrow(rafile) bad.ix = is.na(rafile$chr1) | is.na(rafile$chr2) | is.na(rafile$pos1) | is.na(rafile$pos2) | is.na(rafile$str1) | is.na(rafile$str2) | rafile$str1 == '*'| rafile$str2 == '*' | rafile$pos1<0 | rafile$pos2<0 rafile = rafile[which(!bad.ix), ] if (nrow(rafile)==0) return(GRanges()) seg = rbind(data.frame(chr = rafile$chr1, pos1 = rafile$pos1, pos2 = rafile$pos1, strand = rafile$str1, ra.index = rafile$rowid, ra.which = 1, stringsAsFactors = F), data.frame(chr = rafile$chr2, pos1 = rafile$pos2, pos2 = rafile$pos2, strand = rafile$str2, ra.index = rafile$rowid, ra.which = 2, stringsAsFactors = F)) if (chr.convert) seg$chr = gsub('25', 'M', gsub('24', 'Y', gsub('23', 'X', seg$chr))) out = seg2gr(seg, seqlengths = seqlengths)[, c('ra.index', 'ra.which')]; out = split(out, out$ra.index) } else if (!is.null(rafile$start1) & !is.null(rafile$start2) & !is.null(rafile$end1) & !is.null(rafile$end2)) { ra1 = gr.flip(GRanges(rafile$chr1, IRanges(rafile$start1, rafile$end1), strand = rafile$str1)) ra2 = gr.flip(GRanges(rafile$chr2, IRanges(rafile$start2, rafile$end2), strand = rafile$str2)) out = grl.pivot(GRangesList(ra1, ra2)) } if (keep.features) values(out) = rafile[, ] return(out) } grl.pivot <- function(x) { if (length(x) == 0) return(GRangesList(GRanges(seqlengths = seqlengths(x)), GRanges(seqlengths = seqlengths(x)))) return(split(unlist(x), rep(1:length(x[[1]]), length(x)))) } read_hg <- function(hg19 = T, fft = F) { if (fft) return(readRDS(REFGENE.FILE.HG19.FFT)) else { require(BSgenome) if (hg19) library(BSgenome.Hsapiens.UCSC.hg19) else library(BSgenome.Hsapiens.UCSC.hg18) } return(Hsapiens) } hg_seqlengths <- function(hg19 = T, chr = F, include.junk = F) { require(BSgenome) hg = read_hg(hg19) sl = seqlengths(hg) if (!include.junk) sl = sl[c(paste('chr', 1:22, sep = ''), 'chrX', 'chrY', 'chrM')] if (!chr) names(sl) = gsub('chr', '', names(sl)) return(sl) } grl.unlist <- function(grl) { if (length(grl) == 0) ## JEREMIAH return(GRanges()) # return(grl) names(grl) = NULL as.df = as.data.frame(grl) el = as.df$element if (is.null(el)) el = as.df$group out = unlist(grl) out$grl.ix = el tmp = rle(el) out$grl.iix = unlist(sapply(tmp$lengths, function(x) 1:x)) values(out) = cbind(values(grl)[out$grl.ix, , drop = FALSE], values(out)) return(out) } grdt <- function(x) { require(data.table) ## new approach just directly instantiating data table cmd = 'data.table('; if (is(x, 'GRanges')) { was.gr = TRUE f = c('seqnames', 'start', 'end', 'strand', 'width') f2 = c('as.character(seqnames', 'c(start', 'c(end', 'as.character(strand', 'as.numeric(width') cmd = paste(cmd, paste(f, '=', f2, '(x))', sep = '', collapse = ','), sep = '') value.f = names(values(x)) } else { was.gr = FALSE value.f = names(x) } if (length(value.f)>0) { if (was.gr) cmd = paste(cmd, ',', sep = '') class.f = sapply(value.f, function(f) eval(parse(text=sprintf("class(x$'%s')", f)))) .StringSetListAsList = function(x) ### why do I need to do this, bioconductor peeps?? { tmp1 = as.character(unlist(x)) tmp2 = rep(1:length(x), elementLengths(x)) return(split(tmp1, tmp2)) } ## take care of annoying S4 / DataFrame / data.frame (wish-they-were-non-)issues as.statement = ifelse(grepl('Integer', class.f), 'as.integer', ifelse(grepl('Character', class.f), 'as.character', ifelse(grepl('StringSetList', class.f), '.StringSetListAsList', ifelse(grepl('StringSet$', class.f), 'as.character', ifelse(grepl('List', class.f), 'as.list', ifelse(grepl('List', class.f), 'as.list', 'c')))))) cmd = paste(cmd, paste(value.f, '=', as.statement, "(x$'", value.f, "')", sep = '', collapse = ','), sep = '') } cmd = paste(cmd, ')', sep = '') return(eval(parse(text =cmd))) } gr.findoverlaps <- function(query, subject, ignore.strand = T, first = F, qcol = NULL, ## any query meta data columns to add to result scol = NULL, ## any subject meta data columns to add to resultx max.chunk = 1e13, foverlaps = ifelse(is.na(as.logical(Sys.getenv('GRFO_FOVERLAPS'))), TRUE, as.logical(Sys.getenv('GRFO_FOVERLAPS'))) & exists('foverlaps'), pintersect = NA, verbose = F, type = 'any', by = NULL, mc.cores = 1, return.type = 'same', ...) { if (type != 'any') { foverlaps = FALSE pintersect = FALSE } if (nchar(foverlaps)==0) foverlaps = TRUE if (is.na(foverlaps)) foverlaps = TRUE isdt <- any(class(query) == 'data.table' ) if (return.type == 'same') return.type <- ifelse(isdt, 'data.table', 'GRanges') if (!((inherits(subject, 'GRanges') | inherits(subject, 'data.table')) & (inherits(query, 'GRanges') | inherits(query, 'data.table')))) stop('both subject and query have to be GRanges or data.table') if (is.na(pintersect)) if (isdt) pintersect <- length(unique(query$seqnames)) > 50 & length(unique(subject$seqnames)) > 50 else pintersect <- seqlevels(query) > 50 && seqlevels(subject) > 50 if (is.na(pintersect)) pintersect <- FALSE if (!is.null(qcol)) if (!all(qcol %in% names(values(query)))) stop('Some qcol are not present in meta data of query') if (!is.null(scol)) if (!all(scol %in% names(values(subject)))) stop('Some scol are not present in meta data of subject') if (!is.null(by)) if (!(by %in% names(values(query)) & by %in% names(values(subject)))) stop('"by" field must be meta data column of both query and subject') if ((as.numeric(length(query)) * as.numeric(length(subject))) > max.chunk) { if (verbose) cat('Overflow .. computing overlaps in chunks. Adjust max.chunk parameter to gr.findoverlaps to avoid chunked computation\n') chunk.size = floor(sqrt(max.chunk)); ix1 = c(seq(1, length(query), chunk.size), length(query)+1) ix2 = c(seq(1, length(subject), chunk.size), length(subject)+1) ij = cbind(rep(1:(length(ix1)-1), length(ix2)-1), rep(1:(length(ix2)-1), each = length(ix1)-1)) if (verbose) print(paste('Number of chunks:', nrow(ij))) out = do.call('c', mclapply(1:nrow(ij), function(x) { if (verbose) cat(sprintf('chunk i = %s-%s (%s), j = %s-%s (%s)\n', ix1[ij[x,1]], ix1[ij[x,1]+1]-1, length(query), ix2[ij[x,2]], (ix2[ij[x,2]+1]-1), length(subject))) i.chunk = ix1[ij[x,1]]:(ix1[ij[x,1]+1]-1) j.chunk = ix2[ij[x,2]]:(ix2[ij[x,2]+1]-1) out = gr.findoverlaps(query[i.chunk], subject[j.chunk], ignore.strand = ignore.strand, first = first, pintersect=pintersect, by = by, qcol = qcol, verbose = verbose, foverlaps = foverlaps, scol = scol, type = type, ...) out$query.id = i.chunk[out$query.id] out$subject.id = j.chunk[out$subject.id] return(out) }, mc.cores=mc.cores)) convert = FALSE if ((return.type == 'same' & is(query, 'data.table')) | return.type == 'data.table') out = grdt(out) return(out) } if (foverlaps) { if (verbose) print('overlaps by data.table::foverlaps') if (ignore.strand) by = c(by, 'seqnames', 'start', 'end') else by = c(by, 'seqnames', 'strand', 'start', 'end') if (!is.data.table(query)) { names(query) = NULL querydt = grdt(query[, setdiff(by, c('seqnames', 'start', 'end', 'strand'))]) } else { if (!all(by %in% names(query))) stop(paste('the following columns are missing from query:', paste(by, collapse = ','))) querydt = query[, by, with = FALSE] } if (!is.data.table(subject)) { names(subject) = NULL subjectdt = grdt(subject[, setdiff(by, c('seqnames', 'start', 'end', 'strand'))]) } else { if (!all(by %in% names(subject))) stop(paste('the following columns are missing from subejct:', paste(by, collapse = ','))) subjectdt = subject[, by, with = FALSE] } ix1 = querydt$query.id = 1:nrow(querydt) ix2 = subjectdt$subject.id = 1:nrow(subjectdt) querydt = querydt[start<=end, ] subjectdt = subjectdt[start<=end, ] querydt = querydt[, c('query.id', by), with = F] subjectdt = subjectdt[, c('subject.id', by), with = F] setkeyv(querydt, by) setkeyv(subjectdt, by) h.df = foverlaps(querydt, subjectdt, by.x = by, by.y = by, mult = 'all', type = 'any', verbose = verbose) h.df = h.df[!is.na(subject.id) & !is.na(query.id), ] h.df[, start := pmax(start, i.start)] h.df[, end := pmin(end, i.end)] if (verbose) cat(sprintf('Generated %s overlaps\n', nrow(h.df))) } else { if (isdt) { sn1 <- query$seqnames sn2 <- subject$seqnames } else { sn1 = as.character(seqnames(query)) sn2 = as.character(seqnames(subject)) } if (is.null(by)) { ix1 = which(sn1 %in% sn2) ix2 = which(sn2 %in% sn1) } else { by1 = values(query)[, by] by2 = values(subject)[, by] ix1 = which(sn1 %in% sn2 & by1 %in% by2) ix2 = which(sn2 %in% sn1 & by2 %in% by1) by1 = by1[ix1] by2 = by2[ix2] } query.ix = query[ix1] subject.ix = subject[ix2] sn1 = sn1[ix1] sn2 = sn2[ix2] if (pintersect) { if (verbose) print('overlaps by pintersect') require(data.table) if (length(sn1)>0 & length(sn2)>0) { if (is.null(by)) { dt1 <- data.table(i=seq_along(sn1), sn=sn1, key="sn") dt2 <- data.table(j=seq_along(sn2), sn=sn2, key="sn") ij <- merge(dt1, dt2, by = 'sn', allow.cartesian=TRUE) } else { dt1 <- data.table(i=seq_along(sn1), sn=sn1, by = by1, key=c("sn", "by")) dt2 <- data.table(j=seq_along(sn2), sn=sn2, by = by2, key=c("sn", "by")) ij <- merge(dt1, dt2, by = c('sn', 'by'), allow.cartesian=TRUE) } if (ignore.strand && isdt) subject$strand <- '*' else if (ignore.strand) strand(subject) = '*' qr <- query.ix[ij$i] sb <- subject.ix[ij$j] if (!isdt) { seqlengths(qr) <- rep(NA, length(seqlengths(qr))) seqlengths(sb) <- rep(NA, length(seqlengths(sb))) } if (!isdt && any(as.character(seqnames(qr)) != as.character(seqnames(sb)))) warning('gr.findoverlaps: violated pintersect assumption') ## changed to ranges(qr) etc rather than just GRanges call. Major problem if too many seqlevels if (isdt) { rqr <- IRanges(start=qr$start, end=qr$end) rsb <- IRanges(start=sb$start, end=sb$end) } else { rqr <- ranges(qr) rsb <- ranges(sb) } tmp <- pintersect(rqr, rsb, resolve.empty = 'start.x', ...) names(tmp) = NULL non.empty = which(width(tmp)!=0) h.df = as.data.frame(tmp[non.empty]) if (isdt) h.df$seqnames <- qr$seqnames[non.empty] else h.df$seqnames <- as.character(seqnames(qr))[non.empty] h.df$query.id = ij$i[non.empty] h.df$subject.id = ij$j[non.empty] } else h.df = data.frame() } else { if (verbose) print('overlaps by findOverlaps') if (isdt) { rqr <- IRanges(start=query.ix$start, end=query.ix$end) rsb <- IRanges(start=subject.ix$start, end=subject.ix$end) } else { rqr <- ranges(query.ix) rsb <- ranges(subject.ix) } h <- findOverlaps(rqr, rsb, type = type) r <- ranges(h, rqr, rsb) h.df <- data.frame(start = start(r), end = end(r), query.id = queryHits(h), subject.id = subjectHits(h), stringsAsFactors = F); # sn.query = as.character(seqnames(query))[h.df$query.id] # sn.subject = as.character(seqnames(subject))[h.df$subject.id] sn.query <- sn1[h.df$query.id] sn.subject <- sn2[h.df$subject.id] if (is.null(by)) keep.ix <- sn.query == sn.subject else { by.query <- by1[h.df$query.id] by.subject <- by2[h.df$subject.id] keep.ix <- sn.query == sn.subject & by.query == by.subject } h.df <- h.df[keep.ix, ] h.df$seqnames <- sn.query[keep.ix]; } if (!ignore.strand) { h.df$strand <- str.query <- as.character(strand(query)[ix1[h.df$query.id]]) str.subject <- as.character(strand(subject)[ix2[h.df$subject.id]]) h.df <- h.df[which(str.query == str.subject | str.query == '*' | str.subject == '*'),] } else if (nrow(h.df)>0) h.df$strand = '*' } if (first) h.df = h.df[!duplicated(h.df$query.id), ] if (return.type=='GRanges') if (nrow(h.df)>0) { if (('strand' %in% names(h.df))) out.gr = GRanges(h.df$seqnames, IRanges(h.df$start, h.df$end), query.id = ix1[h.df$query.id], subject.id = ix2[h.df$subject.id], strand = h.df$strand, seqlengths = seqlengths(query)) else out.gr = GRanges(h.df$seqnames, IRanges(h.df$start, h.df$end), query.id = ix1[h.df$query.id], subject.id = ix2[h.df$subject.id], seqlengths = seqlengths(query)) if (!is.null(qcol)) values(out.gr) = cbind(values(out.gr), values(query)[out.gr$query.id, qcol, drop = FALSE]) if (!is.null(scol)) values(out.gr) = cbind(values(out.gr), values(subject)[out.gr$subject.id, scol, drop = FALSE]) return(out.gr) } else return(GRanges(seqlengths = seqlengths(query))) else if (nrow(h.df)>0) { if (!is.data.table(h.df)) h.df = as.data.table(h.df) h.df$query.id <- ix1[h.df$query.id] h.df$subject.id <- ix2[h.df$subject.id] if (!is.null(qcol)) h.df = cbind(h.df, as.data.table(as.data.frame(values(query))[h.df$query.id, qcol, drop = FALSE])) if (!is.null(scol)) h.df = cbind(h.df, as.data.table(as.data.frame(values(subject))[h.df$subject.id, scol, drop = FALSE])) if ('i.start' %in% colnames(h.df)) h.df[, i.start := NULL] if ('i.end' %in% colnames(h.df)) h.df[, i.end := NULL] return(h.df) } else { return(data.table()) } } library(optparse) option_list = list( make_option(c("-i", "--input"), type = "character", default = NULL, help = "Input SV VCF file"), make_option(c("-o", "--output"), type = "character", default = "circos", help = "Output basename of pdf to write the graph"), make_option(c("-g", "--genes"), type = "logical", default = TRUE, help = "Add genes to the plot?"), make_option(c("-H", "--height"), type = "numeric", default = 10, help = "Height"), make_option(c("-W", "--width"), type = "numeric", default = 10, help = "Width") ) parseobj = OptionParser(option_list=option_list) opt = parse_args(parseobj) if (is.null(opt$input)) stop(print_help(parseobj)) bks <- ra_breaks(opt$input) ## filter out discorant only bks <- bks[-which(mcols(bks)$EVDNC == "DSCRD")] require(RCircos) data(UCSC.HG19.Human.CytoBandIdeogram); chr.exclude <- NULL; cyto.info <- UCSC.HG19.Human.CytoBandIdeogram; tracks.inside <- 10; tracks.outside <- 0; RCircos.Set.Core.Components(cyto.info, chr.exclude, tracks.inside, tracks.outside); b <- grl.unlist(bks) ## get the gene label dat basedir <- '/xchip/gistic/Jeremiah/tracks' genes <- readRDS(file.path(basedir, 'gr.allgenes.rds')) genes <- genes[width(genes) < 2e6] fo <- gr.findoverlaps(b+10e3, genes) fo <- fo[!duplicated(fo$subject.id)] gene.dat = data.frame(Chromosome = seqnames(genes[fo$subject.id]), chromStart=start(genes[fo$subject.id]), chromEnd=end(genes[fo$subject.id]), Gene=genes$gene[fo$subject.id]) print(gene.dat) gename.col <- 4; side <- "in"; track.num <- 1; x1 = b$grl.iix==1 x2 = b$grl.iix==2 links = data.frame(Chromosome=seqnames(b[x1]), chromStart=start(b[x1]), chromEnd=end(b[x1]), Chromosome.1=seqnames(b[x2]), chromStart.1=start(b[x2]), chromeEnd.1=end(b[x2])) ## plot the PDF pdf(file=paste0(opt$output,".pdf"), height=opt$height, width=opt$width, compress=TRUE); RCircos.Set.Plot.Area(); RCircos.Chromosome.Ideogram.Plot(); if (opt$genes && nrow(gene.dat) > 0) { RCircos.Gene.Connector.Plot(gene.dat, track.num, side); track.num <- 2; name.col <- 4; RCircos.Gene.Name.Plot(gene.dat, name.col,track.num, side); } if (nrow(links) > 0) RCircos.Link.Plot(links, track.num, by.chromosome=TRUE) ## by.chromosome is for color dev.off() ================================================ FILE: R/archive_non_functional/svaba-create-pon.R ================================================ #!/usr/bin/env Rscript library(optparse) option_list = list( make_option(c("-i", "--input"), type = "character", default = "qcreport.txt", help = "Input file containing paths to germline "), make_option(c("-o", "--output"), type = "character", default = "qcreport.pdf", help = "Output panel of normals file") ) parseobj = OptionParser(option_list=option_list) opt = parse_args(parseobj) if (is.null(opt$input)) stop(print_help(parseobj)) if (!file.exists(opt$input)) { print(print_help(parseobj)) stop(paste("Input file does not exist", opt$input, ". Must supply path to valid qcreport.txt file (generated from snowman preprocess or snowman run")) } print(opt) ## default opt$input = '/home/unix/jwala/lung_wgs_pon_list.txt' opt$output = "/home/unix/jwala/lung_wgs_pon.txt" ## opt$vcf_files = read.delim(opt$input, stringsAsFactors=FALSE)[,1] dt.all <- mclapply(opt$vcf_files, function(x) { print(x) if (!file.exists(x)) { warning("File does not exist") return (-1) } ab <- read_vcf(x) ab$file = x return(gr2dt(ab)) }, mc.cores=15) dt <- rbindlist(dt.all) ## need to add indel tye, also svss ================================================ FILE: R/archive_non_functional/svaba-event-plot.R ================================================ #!/usr/bin/env Rscript ## source all the required packages source.all <- function() { githome <- Sys.getenv('GIT_HOME') if (!nchar(githome)) githome <- '/xchip/gistic/Jeremiah/GIT' suppressMessages(suppressWarnings(require(ff, quietly=TRUE))) suppressMessages(suppressWarnings(require(VariantAnnotation, quietly=TRUE))) suppressMessages(suppressWarnings(require(rtracklayer, quietly=TRUE))) suppressMessages(suppressWarnings(require(data.table, quietly=TRUE))) suppressMessages(suppressWarnings(require(plyr, quietly=TRUE))) print('...sourced 5 packages') suppressMessages(suppressWarnings(require(ggplot2, quietly=TRUE))) suppressMessages(suppressWarnings(require(reshape2, quietly=TRUE))) suppressMessages(suppressWarnings(require(GenomicRanges, quietly=TRUE))) suppressMessages(suppressWarnings(require(popbio, quietly=TRUE))) suppressMessages(suppressWarnings(require(BSgenome.Hsapiens.UCSC.hg19, quietly=TRUE))) print('...sourced 10 packages') suppressMessages(suppressWarnings(require(bitops, quietly=TRUE))) suppressMessages(suppressWarnings(require(seqinr, quietly=TRUE))) suppressMessages(suppressWarnings(require(Rsamtools, quietly=TRUE))) suppressMessages(suppressWarnings(require(ff, quietly=TRUE))) suppressMessages(suppressWarnings(require(multicore, quietly=TRUE))) print('...sourced 15 packages') suppressMessages(suppressWarnings(require(Biostrings, quietly=TRUE))) suppressMessages(suppressWarnings(require(rtracklayer, quietly=TRUE))) suppressMessages(suppressWarnings(require(lattice, quietly=TRUE))) suppressMessages(suppressWarnings(require(RColorBrewer, quietly=TRUE))) suppressMessages(suppressWarnings(require(Matrix, quietly=TRUE))) print('...sourced 20 packages') source(file.path(githome, "grUtils", "grUtils.R")) source(file.path(githome, "trackData", "trackData.R")) source(file.path(githome, "marcin", "R", "functions.R")) source(file.path(githome, "marcin", "R", "db.R")) source(file.path(githome, "isva", "TaigaSig", "sigUtils.R")) print('...sourced grUtils.R, trackData.R, marcin/functions.R, marcin/db.R, sigUtils.R') } source.all() ################## ## PARSE OPTIONS ################## library(optparse) option_list = list( make_option(c("-f", "--FHworkspace"), type = "character", default = NULL, help = "Firehose workspace to retrieve data from"), make_option(c("-o", "--outdir"), type = "character", default = getwd(), help = "Firehose workspace to retrieve data from"), make_option(c("-p", "--FHpairset"), type = "character", default = NULL, help = "Firehose pairset to retreive data from"), make_option(c("-a", "--FHannotation"), type = "character", default = NULL, help = "Firehose annoation to retreive data from"), make_option(c("-c", "--cores"), type = "numeric", default = 1, help = "Number of cores to use"), make_option(c("-v", "--VCFlist"), type = "character", default = "/home/unix/jwala/test.vcflist.txt", help = "File containing a list of VCFs")) parseobj = OptionParser(option_list=option_list) opt = parse_args(parseobj) ### test data opt$outdir = '/xchip/gistic/Jeremiah/tmp_sig' dir.create(opt$outdir, showWarnings=FALSE) opt$VCFlist = '/xchip/gistic/Jeremiah/Projects/Significance/Sanger578/list.txt' setwd(opt$outdir) ################### ################### ================================================ FILE: R/archive_non_functional/svaba-histogram.R ================================================ #!/usr/bin/env Rscript ## set the right library paths .libPaths = c("/xchip/gistic/Jeremiah/R", "/broad/software/free/Linux/redhat_6_x86_64/pkgs/r_3.1.1-bioconductor-3.0/lib64/R/library") file.dir <- function(paths) { return(gsub('(^|(.*\\/))?([^\\/]*)$', '\\2', paths)) } require(data.table) suppressMessages(suppressWarnings(require(GenomicRanges, quietly = TRUE))) suppressMessages(suppressWarnings(require(gplots, quietly = TRUE))) ra_breaks <- function(rafile, keep.features = T, seqlengths = hg_seqlengths(), chr.convert = T, snowman = FALSE, breakpointer = FALSE, seqlevels = NULL, get.loose = FALSE ## if TRUE will return a list with fields $junctions and $loose.ends ) { if (is.character(rafile)) { if (grepl('(vcf$)|(vcf.gz$)', rafile)) { library(VariantAnnotation) vcf = readVcf(rafile, Seqinfo(seqnames = names(seqlengths), seqlengths = seqlengths)) if (!('SVTYPE' %in% names(info(vcf)))) { warning('Vcf not in proper format. Is this a rearrangement vcf?') return(GRangesList()); } vgr = rowData(vcf) ## parse BND format ## no events if (length(vgr) == 0) return (GRangesList()) ## fix mateids if not included if (!"MATEID"%in%colnames(mcols(vgr))) { nm <- vgr$MATEID <- names(vgr) ix <- grepl("1$",nm) vgr$MATEID[ix] = gsub("(.*?)(1)$", "\\12", nm[ix]) vgr$MATEID[!ix] = gsub("(.*?)(2)$", "\\11", nm[!ix]) vgr$SVTYPE="BND" } if (!any(c("MATEID", "SVTYPE") %in% colnames(mcols(vgr)))) stop("MATEID or SVTYPE not included. Required") vgr$mateid = info(vcf)$MATEID vgr$svtype = info(vcf)$SVTYPE if (!is.null(info(vcf)$SCTG)) vgr$SCTG = info(vcf)$SCTG if (sum(vgr$svtype == 'BND')==0) stop('Vcf not in proper format. Will only process rearrangements in BND format') if (!all(vgr$svtype == 'BND')) warning(sprintf('%s rows of vcf do not have svtype BND, ignoring these'), sum(vgr$svtype != 'BND')) bix = which(vgr$svtype == "BND") vgr = vgr[bix] vgr$first = !grepl('^(\\]|\\[)', vgr$ALT) ## ? is this row the "first breakend" in the ALT string (i.e. does the ALT string not begin with a bracket) vgr$right = grepl('\\[', vgr$ALT) ## ? are the (sharp ends) of the brackets facing right or left vgr$coord = as.character(paste(seqnames(vgr), ':', start(vgr), sep = '')) vgr$mcoord = as.character(gsub('.*(\\[|\\])(.*\\:.*)(\\[|\\]).*', '\\2', vgr$ALT)) vgr$mcoord = gsub('chr', '', vgr$mcoord) if (all(is.na(vgr$mateid))) if (!is.null(names(vgr)) & !any(duplicated(names(vgr)))) { warning('MATEID tag missing, guessing BND partner by parsing names of vgr') vgr$mateid = paste(gsub('::\\d$', '', names(vgr)), (sapply(strsplit(names(vgr), '\\:\\:'), function(x) as.numeric(x[length(x)])))%%2 + 1, sep = '::') } else if (!is.null(vgr$SCTG)) { warning('MATEID tag missing, guessing BND partner from coordinates and SCTG') require(igraph) ucoord = unique(c(vgr$coord, vgr$mcoord)) vgr$mateid = paste(vgr$SCTG, vgr$mcoord, sep = '_') if (any(duplicated(vgr$mateid))) { warning('DOUBLE WARNING! inferred mateids not unique, check VCF') bix = bix[!duplicated(vgr$mateid)] vgr = vgr[!duplicated(vgr$mateid)] } } else stop('MATEID tag missing') vgr$mix = as.numeric(match(vgr$mateid, names(vgr))) pix = which(!is.na(vgr$mix)) vgr.pair = vgr[pix] if (length(vgr.pair)==0) stop('No mates found despite nonzero number of BND rows in VCF') vgr.pair$mix = match(vgr.pair$mix, pix) vix = which(1:length(vgr.pair)0, '+', '-') if (is.numeric(rafile$str2)) rafile$str2 = ifelse(rafile$str2>0, '+', '-') rafile$rowid = 1:nrow(rafile) bad.ix = is.na(rafile$chr1) | is.na(rafile$chr2) | is.na(rafile$pos1) | is.na(rafile$pos2) | is.na(rafile$str1) | is.na(rafile$str2) | rafile$str1 == '*'| rafile$str2 == '*' | rafile$pos1<0 | rafile$pos2<0 rafile = rafile[which(!bad.ix), ] if (nrow(rafile)==0) return(GRanges()) seg = rbind(data.frame(chr = rafile$chr1, pos1 = rafile$pos1, pos2 = rafile$pos1, strand = rafile$str1, ra.index = rafile$rowid, ra.which = 1, stringsAsFactors = F), data.frame(chr = rafile$chr2, pos1 = rafile$pos2, pos2 = rafile$pos2, strand = rafile$str2, ra.index = rafile$rowid, ra.which = 2, stringsAsFactors = F)) if (chr.convert) seg$chr = gsub('25', 'M', gsub('24', 'Y', gsub('23', 'X', seg$chr))) out = seg2gr(seg, seqlengths = seqlengths)[, c('ra.index', 'ra.which')]; out = split(out, out$ra.index) } else if (!is.null(rafile$start1) & !is.null(rafile$start2) & !is.null(rafile$end1) & !is.null(rafile$end2)) { ra1 = gr.flip(GRanges(rafile$chr1, IRanges(rafile$start1, rafile$end1), strand = rafile$str1)) ra2 = gr.flip(GRanges(rafile$chr2, IRanges(rafile$start2, rafile$end2), strand = rafile$str2)) out = grl.pivot(GRangesList(ra1, ra2)) } if (keep.features) values(out) = rafile[, ] return(out) } grl.pivot <- function(x) { if (length(x) == 0) return(GRangesList(GRanges(seqlengths = seqlengths(x)), GRanges(seqlengths = seqlengths(x)))) return(split(unlist(x), rep(1:length(x[[1]]), length(x)))) } read_hg <- function(hg19 = T, fft = F) { if (fft) return(readRDS(REFGENE.FILE.HG19.FFT)) else { require(BSgenome) if (hg19) library(BSgenome.Hsapiens.UCSC.hg19) else library(BSgenome.Hsapiens.UCSC.hg18) } return(Hsapiens) } hg_seqlengths <- function(hg19 = T, chr = F, include.junk = F) { require(BSgenome) hg = read_hg(hg19) sl = seqlengths(hg) if (!include.junk) sl = sl[c(paste('chr', 1:22, sep = ''), 'chrX', 'chrY', 'chrM')] if (!chr) names(sl) = gsub('chr', '', names(sl)) return(sl) } grl.unlist <- function(grl) { if (length(grl) == 0) ## JEREMIAH return(GRanges()) # return(grl) names(grl) = NULL as.df = as.data.frame(grl) el = as.df$element if (is.null(el)) el = as.df$group out = unlist(grl) out$grl.ix = el tmp = rle(el) out$grl.iix = unlist(sapply(tmp$lengths, function(x) 1:x)) values(out) = cbind(values(grl)[out$grl.ix, , drop = FALSE], values(out)) return(out) } grdt <- function(x) { require(data.table) ## new approach just directly instantiating data table cmd = 'data.table('; if (is(x, 'GRanges')) { was.gr = TRUE f = c('seqnames', 'start', 'end', 'strand', 'width') f2 = c('as.character(seqnames', 'c(start', 'c(end', 'as.character(strand', 'as.numeric(width') cmd = paste(cmd, paste(f, '=', f2, '(x))', sep = '', collapse = ','), sep = '') value.f = names(values(x)) } else { was.gr = FALSE value.f = names(x) } if (length(value.f)>0) { if (was.gr) cmd = paste(cmd, ',', sep = '') class.f = sapply(value.f, function(f) eval(parse(text=sprintf("class(x$'%s')", f)))) .StringSetListAsList = function(x) ### why do I need to do this, bioconductor peeps?? { tmp1 = as.character(unlist(x)) tmp2 = rep(1:length(x), elementLengths(x)) return(split(tmp1, tmp2)) } ## take care of annoying S4 / DataFrame / data.frame (wish-they-were-non-)issues as.statement = ifelse(grepl('Integer', class.f), 'as.integer', ifelse(grepl('Character', class.f), 'as.character', ifelse(grepl('StringSetList', class.f), '.StringSetListAsList', ifelse(grepl('StringSet$', class.f), 'as.character', ifelse(grepl('List', class.f), 'as.list', ifelse(grepl('List', class.f), 'as.list', 'c')))))) cmd = paste(cmd, paste(value.f, '=', as.statement, "(x$'", value.f, "')", sep = '', collapse = ','), sep = '') } cmd = paste(cmd, ')', sep = '') return(eval(parse(text =cmd))) } gr.findoverlaps <- function(query, subject, ignore.strand = T, first = F, qcol = NULL, ## any query meta data columns to add to result scol = NULL, ## any subject meta data columns to add to resultx max.chunk = 1e13, foverlaps = ifelse(is.na(as.logical(Sys.getenv('GRFO_FOVERLAPS'))), TRUE, as.logical(Sys.getenv('GRFO_FOVERLAPS'))) & exists('foverlaps'), pintersect = NA, verbose = F, type = 'any', by = NULL, mc.cores = 1, return.type = 'same', ...) { if (type != 'any') { foverlaps = FALSE pintersect = FALSE } if (nchar(foverlaps)==0) foverlaps = TRUE if (is.na(foverlaps)) foverlaps = TRUE isdt <- any(class(query) == 'data.table' ) if (return.type == 'same') return.type <- ifelse(isdt, 'data.table', 'GRanges') if (!((inherits(subject, 'GRanges') | inherits(subject, 'data.table')) & (inherits(query, 'GRanges') | inherits(query, 'data.table')))) stop('both subject and query have to be GRanges or data.table') if (is.na(pintersect)) if (isdt) pintersect <- length(unique(query$seqnames)) > 50 & length(unique(subject$seqnames)) > 50 else pintersect <- seqlevels(query) > 50 && seqlevels(subject) > 50 if (is.na(pintersect)) pintersect <- FALSE if (!is.null(qcol)) if (!all(qcol %in% names(values(query)))) stop('Some qcol are not present in meta data of query') if (!is.null(scol)) if (!all(scol %in% names(values(subject)))) stop('Some scol are not present in meta data of subject') if (!is.null(by)) if (!(by %in% names(values(query)) & by %in% names(values(subject)))) stop('"by" field must be meta data column of both query and subject') if ((as.numeric(length(query)) * as.numeric(length(subject))) > max.chunk) { if (verbose) cat('Overflow .. computing overlaps in chunks. Adjust max.chunk parameter to gr.findoverlaps to avoid chunked computation\n') chunk.size = floor(sqrt(max.chunk)); ix1 = c(seq(1, length(query), chunk.size), length(query)+1) ix2 = c(seq(1, length(subject), chunk.size), length(subject)+1) ij = cbind(rep(1:(length(ix1)-1), length(ix2)-1), rep(1:(length(ix2)-1), each = length(ix1)-1)) if (verbose) print(paste('Number of chunks:', nrow(ij))) out = do.call('c', mclapply(1:nrow(ij), function(x) { if (verbose) cat(sprintf('chunk i = %s-%s (%s), j = %s-%s (%s)\n', ix1[ij[x,1]], ix1[ij[x,1]+1]-1, length(query), ix2[ij[x,2]], (ix2[ij[x,2]+1]-1), length(subject))) i.chunk = ix1[ij[x,1]]:(ix1[ij[x,1]+1]-1) j.chunk = ix2[ij[x,2]]:(ix2[ij[x,2]+1]-1) out = gr.findoverlaps(query[i.chunk], subject[j.chunk], ignore.strand = ignore.strand, first = first, pintersect=pintersect, by = by, qcol = qcol, verbose = verbose, foverlaps = foverlaps, scol = scol, type = type, ...) out$query.id = i.chunk[out$query.id] out$subject.id = j.chunk[out$subject.id] return(out) }, mc.cores=mc.cores)) convert = FALSE if ((return.type == 'same' & is(query, 'data.table')) | return.type == 'data.table') out = grdt(out) return(out) } if (foverlaps) { if (verbose) print('overlaps by data.table::foverlaps') if (ignore.strand) by = c(by, 'seqnames', 'start', 'end') else by = c(by, 'seqnames', 'strand', 'start', 'end') if (!is.data.table(query)) { names(query) = NULL querydt = grdt(query[, setdiff(by, c('seqnames', 'start', 'end', 'strand'))]) } else { if (!all(by %in% names(query))) stop(paste('the following columns are missing from query:', paste(by, collapse = ','))) querydt = query[, by, with = FALSE] } if (!is.data.table(subject)) { names(subject) = NULL subjectdt = grdt(subject[, setdiff(by, c('seqnames', 'start', 'end', 'strand'))]) } else { if (!all(by %in% names(subject))) stop(paste('the following columns are missing from subejct:', paste(by, collapse = ','))) subjectdt = subject[, by, with = FALSE] } ix1 = querydt$query.id = 1:nrow(querydt) ix2 = subjectdt$subject.id = 1:nrow(subjectdt) querydt = querydt[start<=end, ] subjectdt = subjectdt[start<=end, ] querydt = querydt[, c('query.id', by), with = F] subjectdt = subjectdt[, c('subject.id', by), with = F] setkeyv(querydt, by) setkeyv(subjectdt, by) h.df = foverlaps(querydt, subjectdt, by.x = by, by.y = by, mult = 'all', type = 'any', verbose = verbose) h.df = h.df[!is.na(subject.id) & !is.na(query.id), ] h.df[, start := pmax(start, i.start)] h.df[, end := pmin(end, i.end)] if (verbose) cat(sprintf('Generated %s overlaps\n', nrow(h.df))) } else { if (isdt) { sn1 <- query$seqnames sn2 <- subject$seqnames } else { sn1 = as.character(seqnames(query)) sn2 = as.character(seqnames(subject)) } if (is.null(by)) { ix1 = which(sn1 %in% sn2) ix2 = which(sn2 %in% sn1) } else { by1 = values(query)[, by] by2 = values(subject)[, by] ix1 = which(sn1 %in% sn2 & by1 %in% by2) ix2 = which(sn2 %in% sn1 & by2 %in% by1) by1 = by1[ix1] by2 = by2[ix2] } query.ix = query[ix1] subject.ix = subject[ix2] sn1 = sn1[ix1] sn2 = sn2[ix2] if (pintersect) { if (verbose) print('overlaps by pintersect') require(data.table) if (length(sn1)>0 & length(sn2)>0) { if (is.null(by)) { dt1 <- data.table(i=seq_along(sn1), sn=sn1, key="sn") dt2 <- data.table(j=seq_along(sn2), sn=sn2, key="sn") ij <- merge(dt1, dt2, by = 'sn', allow.cartesian=TRUE) } else { dt1 <- data.table(i=seq_along(sn1), sn=sn1, by = by1, key=c("sn", "by")) dt2 <- data.table(j=seq_along(sn2), sn=sn2, by = by2, key=c("sn", "by")) ij <- merge(dt1, dt2, by = c('sn', 'by'), allow.cartesian=TRUE) } if (ignore.strand && isdt) subject$strand <- '*' else if (ignore.strand) strand(subject) = '*' qr <- query.ix[ij$i] sb <- subject.ix[ij$j] if (!isdt) { seqlengths(qr) <- rep(NA, length(seqlengths(qr))) seqlengths(sb) <- rep(NA, length(seqlengths(sb))) } if (!isdt && any(as.character(seqnames(qr)) != as.character(seqnames(sb)))) warning('gr.findoverlaps: violated pintersect assumption') ## changed to ranges(qr) etc rather than just GRanges call. Major problem if too many seqlevels if (isdt) { rqr <- IRanges(start=qr$start, end=qr$end) rsb <- IRanges(start=sb$start, end=sb$end) } else { rqr <- ranges(qr) rsb <- ranges(sb) } tmp <- pintersect(rqr, rsb, resolve.empty = 'start.x', ...) names(tmp) = NULL non.empty = which(width(tmp)!=0) h.df = as.data.frame(tmp[non.empty]) if (isdt) h.df$seqnames <- qr$seqnames[non.empty] else h.df$seqnames <- as.character(seqnames(qr))[non.empty] h.df$query.id = ij$i[non.empty] h.df$subject.id = ij$j[non.empty] } else h.df = data.frame() } else { if (verbose) print('overlaps by findOverlaps') if (isdt) { rqr <- IRanges(start=query.ix$start, end=query.ix$end) rsb <- IRanges(start=subject.ix$start, end=subject.ix$end) } else { rqr <- ranges(query.ix) rsb <- ranges(subject.ix) } h <- findOverlaps(rqr, rsb, type = type) r <- ranges(h, rqr, rsb) h.df <- data.frame(start = start(r), end = end(r), query.id = queryHits(h), subject.id = subjectHits(h), stringsAsFactors = F); # sn.query = as.character(seqnames(query))[h.df$query.id] # sn.subject = as.character(seqnames(subject))[h.df$subject.id] sn.query <- sn1[h.df$query.id] sn.subject <- sn2[h.df$subject.id] if (is.null(by)) keep.ix <- sn.query == sn.subject else { by.query <- by1[h.df$query.id] by.subject <- by2[h.df$subject.id] keep.ix <- sn.query == sn.subject & by.query == by.subject } h.df <- h.df[keep.ix, ] h.df$seqnames <- sn.query[keep.ix]; } if (!ignore.strand) { h.df$strand <- str.query <- as.character(strand(query)[ix1[h.df$query.id]]) str.subject <- as.character(strand(subject)[ix2[h.df$subject.id]]) h.df <- h.df[which(str.query == str.subject | str.query == '*' | str.subject == '*'),] } else if (nrow(h.df)>0) h.df$strand = '*' } if (first) h.df = h.df[!duplicated(h.df$query.id), ] if (return.type=='GRanges') if (nrow(h.df)>0) { if (('strand' %in% names(h.df))) out.gr = GRanges(h.df$seqnames, IRanges(h.df$start, h.df$end), query.id = ix1[h.df$query.id], subject.id = ix2[h.df$subject.id], strand = h.df$strand, seqlengths = seqlengths(query)) else out.gr = GRanges(h.df$seqnames, IRanges(h.df$start, h.df$end), query.id = ix1[h.df$query.id], subject.id = ix2[h.df$subject.id], seqlengths = seqlengths(query)) if (!is.null(qcol)) values(out.gr) = cbind(values(out.gr), values(query)[out.gr$query.id, qcol, drop = FALSE]) if (!is.null(scol)) values(out.gr) = cbind(values(out.gr), values(subject)[out.gr$subject.id, scol, drop = FALSE]) return(out.gr) } else return(GRanges(seqlengths = seqlengths(query))) else if (nrow(h.df)>0) { if (!is.data.table(h.df)) h.df = as.data.table(h.df) h.df$query.id <- ix1[h.df$query.id] h.df$subject.id <- ix2[h.df$subject.id] if (!is.null(qcol)) h.df = cbind(h.df, as.data.table(as.data.frame(values(query))[h.df$query.id, qcol, drop = FALSE])) if (!is.null(scol)) h.df = cbind(h.df, as.data.table(as.data.frame(values(subject))[h.df$subject.id, scol, drop = FALSE])) if ('i.start' %in% colnames(h.df)) h.df[, i.start := NULL] if ('i.end' %in% colnames(h.df)) h.df[, i.end := NULL] return(h.df) } else { return(data.table()) } } write.htab <- function(tab, file = NULL, title = NULL, # text to be written in bold above the table footer = NULL, # text to be writen in bold below the table highlight = NULL, #vector of row indices of the table to highlight row.names = TRUE, # includes row labels col.names = TRUE, # includes col labels high.color = 'yellow', # highlight color to use row.colors = c('lightgray', 'white'), # alternating colors to shade data rows header.colors = c('#4A4A4A', 'white'), # two element vector specifying background and text colors for header row, respectively, data.size = 15, # font size in px for data, title, and footer title.size = 15, footer.size = 20, header.size = round(1.1*data.size)) { require(hwriter) require(gplots) if (!is.data.frame(tab)) tab = as.data.frame(tab) if (is.null(rownames(tab))) row.names = F; if (!is.null(file)) { #if (!grepl('(^~)|(^\\/)', file)) # file = paste('~/public_html/', file, sep = '') } else file = DEFAULT.HTAB.FILE for (nm in names(tab)) tab[[nm]] = as.character(tab[[nm]]) tab[is.na(tab)] = ''; tab = tab[1:nrow(tab), , drop = FALSE]; #not sure why this is necessary, but deflects occasional weird R bug if (any(lix <<- sapply(names(tab), function(x) is.list(tab[, x])))) for (i in which(lix)) tab[, i] = sapply(tab[, i], function(x) paste(x, collapse = ',')) dir.create(dirname(normalizePath(file.dir(file))), recursive=T, showWarnings = F) p = openPage(file, link.css = 'hwriter.css') if (!is.null(title)) hwrite(title, p, style = sprintf('font-weight:bold; font-size:%spx; margin-top;50px', title.size), center = TRUE, div = TRUE, br = TRUE); row.bgcolor = as.list(as.character(col2hex(row.colors)[(1:nrow(tab))%%length(row.colors)+1])); names(row.bgcolor) = rownames(tab) if (!is.null(highlight)) row.bgcolor[rownames(tab[highlight,, drop = FALSE])] = list(col2hex(high.color)); row.bgcolor = c(col2hex(header.colors[1]), row.bgcolor) # if (row.names) col.bgcolor = col2hex(header.colors[1]) col.style = sprintf('font-weight:bold; font-size:%spx; color:%s; text-align:center', header.size, col2hex(header.colors[2])); row.style = rep(sprintf('font-size:%spx; text-align:center', data.size), nrow(tab)) names(row.style) = rownames(tab) row.style = c(list(sprintf('font-weight:bold; font-size:%spx; color:%s; text-align:center', header.size, col2hex(header.colors[2]))), row.style) hwrite(tab, p, row.style = row.style, col.style = col.style, col.bgcolor = col.bgcolor, row.names = row.names, col.names = col.names, row.bgcolor = row.bgcolor, table.frame = 'void', table.style = 'margin-left: 30px; margin-top: 30px', br = TRUE) if (!is.null(footer)) hwrite(footer, p, style = sprintf('font-weight:bold; text-align:center; font-size:%spx; margin-top;50px', footer.size), center = TRUE, div = TRUE); closePage(p) } gr2dt <- function(gr, basic=FALSE) { if (any(class(gr)=='data.table')) return(gr) out <- with(gr, data.table(seqnames=as.character(seqnames(gr)), start=start(gr), end=end(gr), strand=as.character(strand(gr)))) if (!basic && ncol(mcols(gr))) out <- cbind(out, as.data.frame(mcols(gr))) return(out) } library(optparse) option_list = list( make_option(c("-i", "--input"), type = "character", default = NULL, help = "Input SV VCF file"), make_option(c("-o", "--output"), type = "character", default = "no_id", help = "Output annotation name"), make_option(c("-t", "--splitsupport"), type = "numeric", default = 4, help = "Minimum number of tumor supporting reads for ASSMB"), make_option(c("-d", "--discsupport"), type = "numeric", default = 10, help = "Minimum number of tumor support discordant for DSCRD"), make_option(c("-g", "--genes"), type = "logical", default = TRUE, help = "Add genes to the plot?"), make_option(c("-H", "--height"), type = "numeric", default = 10, help = "Height"), make_option(c("-W", "--width"), type = "numeric", default = 10, help = "Width") ) parseobj = OptionParser(option_list=option_list) opt = parse_args(parseobj) if (is.null(opt$input)) stop(print_help(parseobj)) require(GenomicRanges) args <- commandArgs(trailingOnly = TRUE) files <- args #files <- c(ind$snowman_somatic_vcf,ind$snowman_somatic_indel_vcf)[!grepl("DATECODE",c(ind$snowman_somatic_vcf,ind$snowman_somatic_indel_vcf))] files <- c(ind$snowman_somatic_vcf,ind$snowman_somatic_indel_vcf) ## load the SV files snow.sv <- lapply(files, function(x) { if (grepl("sv.vcf$",x)) { print(paste(basename(x), ":", match(x, files),"of",length(files))) if (file.exists(x)) { y=ra_breaks(x) mcols(y)$file = basename(x) return(y) } else { return (GRangesList()) } } return(GRangesList()) }) snow.sv <- do.call('c', snow.sv) mcols(snow.sv)$analysis_id = gsub("(.*?).snowman.somatic.sv.vcf",'\\1',mcols(snow.sv)$file) ## load the indel files snow.indel <- lapply(files, function(x) { if (grepl("indel.vcf$",x)) { print(paste(basename(x), ":", match(x, files),"of",length(files))) if (file.exists(x)) { browser() y=rowData(readVcf(x, "hg19")) mcols(y)$file = basename(x) return(y) } else { print(paste("File does not exist:",x)) return (GRanges()) } } return (GRanges()) }) events <- do.call('grbind',c(lapply(snow.sv, function(x) {y=unlist(x); y$span = mcols(x)$SPAN; return (y) }), snow.indel[sapply(snow.indel, length) > 0])) events$span[is.na(events$span)] = 0 events$span <- pmax(width(events), events$span) spans <- c(unlist(lapply(snow.sv, function(x) mcols(x)$SPAN)), unlist(lapply(snow.indel, function(x) if (length(x))width(x)))) spans <- events$span df <- data.frame(x=log10(spans[spans > 5])) ppdf(print(ggplot() + geom_histogram(data=df, aes(x=x), binwidth=0.1) + theme_bw() + xlab("Distance (bp)") + ylab("Num Events") + scale_x_continuous(breaks=1:8, label=parse(text=paste("10", 1:8,sep="^")))), width=10, height=6) ================================================ FILE: R/archive_non_functional/svaba-nozzle.R ================================================ #!/usr/bin/env Rscript #### # load the libraries ### .nozzleLibraries <- function() { print('...loading libraries') RLIBDIR = '/xchip/gistic/Jeremiah/R/x86_64-unknown-linux-gnu-library/3.1/' GIT.HOME = '/xchip/gistic/Jeremiah/GIT/' ISVA.HOME = paste(Sys.getenv('GIT_HOME'), '/isva/', sep = ""); .libPaths(c(.libPaths(), RLIBDIR)) suppressMessages(suppressWarnings(require(methods, quietly=TRUE))) suppressMessages(suppressWarnings(require(optparse, quietly=TRUE))) suppressMessages(suppressWarnings(require(data.table, quietly=TRUE))) suppressMessages(suppressWarnings(require(Rsamtools, quietly=TRUE))) suppressMessages(suppressWarnings(require(Matrix, quietly=TRUE))) suppressMessages(suppressWarnings(require(bitops, quietly=TRUE))) suppressMessages(suppressWarnings(require(GenomicAlignments, quietly=TRUE))) #source(file.path(GIT.HOME,"isva", "sigUtils.R")) #source(file.path(GIT.HOME,"isva", "sigUtils.R")) source(file.path(GIT.HOME,"dev/gChain/", "gChain.R")) ##source(file.path(GIT.HOME,"grUtilts", "grUtils.R")) source(file.path(GIT.HOME,"dev/gTrack/gTrack/R/", "gTrack.R")) suppressMessages(suppressWarnings(require(Rsamtools, quietly=TRUE))) suppressMessages(suppressWarnings(require(RColorBrewer, quietly=TRUE))) suppressMessages(suppressWarnings(require(Nozzle.R1, quietly=TRUE))) suppressMessages(suppressWarnings(require(BSgenome.Hsapiens.UCSC.hg19, quietly=TRUE))) #source('/home/unix/jwala/GIT/isva/grUtils.R') #source('/home/unix/jwala/GIT/isva/trackData.R') } ### # get the base filename ### file.name = function(paths) return(gsub('(^|(.*\\/))?([^\\/]*)', '\\3', paths)) ### # load the breakpoint files ### .loadBreakpoints <- function(opt) { ## test #fp <- file.path("/xchip/gistic/Jeremiah/Projects/SnowmanWithIndel_140218","bps.txt.gz") #tab = read.table(gzfile(fp), header=T, sep='\t', stringsAsFactors=FALSE) #tab$reads = "" gr <- GRanges(c(tab$chr1, tab$chr2), IRanges(c(tab$pos1,tab$pos2), width=1)) #, strand=ifelse(c(tab$strand1, tab$strand2)=='+', 1,-1)) strand(gr) = c(tab$strand1, tab$strand2) grl <- split(gr, tab$contig) ## debug tabr = table(tab$contig) grl = grl[names(grl) %in% names(tabr[tabr==1])] mcols(grl) = tab[tab$contig %in% names(tabr[tabr==1]), ] return(list(som=grl, ger=GRangesList())) ## load the breakpoints #breakpoints.somatic.file = file.path(opt$indir, 'breakpoints', 'breakpoints.somatic.txt') #print(paste('Loading the breakpoints:', breakpoints.somatic.file)) #grl.sno <- sig.load.snow(breakpoints.somatic.file)[[1]] #breakpoints.germline.file = file.path(opt$indir, 'breakpoints', 'breakpoints.germline.txt') #print(paste('Loading the breakpoints:', breakpoints.germline.file)) #grl.sno.g <- sig.load.snow(breakpoints.germline.file)[[1]] #return(list(som=grl.sno, ger=grl.sno.g)) } ### # ### .initializeReport <- function(r) { intro.str = "Somatic Analysis -- SnowmanSV (Somatic/germline structural variation detction by string graph assembly, Wala et al in prep) is a tool for detecting germline and somatic structural variations (SVs) using whole genome sequencing (WGS) data. The engine of Snowman is a modified version of String Graph Assembler (SGA) [Simpson & Durbin, Genome Research 2012]. Snowman peforms a rolling tiled assembly across the genome in slightly overlapping 5kb windows. For efficiency, only reads which are clipped, unmapped or discordant are used in the assembly. These assembled contigs are then aligned to the genome with BWA-MEM [Heng Li, 2013], and are scored based on their alignment properties and the number of supporting sequencing reads. The final result is a list of junctions and their read support." if (opt$germline) intro.str = "Germline Analysis -- SnowmanSV (Somatic/germline structural variation detction by string graph assembly, Wala et al in prep) is a tool for detecting germline and somatic structural variations (SVs) using whole genome sequencing (WGS) data. The engine of Snowman is a modified version of String Graph Assembler (SGA) [Simpson & Durbin, Genome Research 2012]. Snowman peforms a rolling tiled assembly across the genome in slightly overlapping 5kb windows. For efficiency, only reads which are clipped, unmapped or discordant are used in the assembly. These assembled contigs are then aligned to the genome with BWA-MEM [Heng Li, 2013], and are scored based on their alignment properties and the number of supporting sequencing reads. The final result is a list of junctions and their read support." files.str = "Snowman produces a series of output files, which are useful for visualizing and understanding the junctions. Two core bam files are produced at the end of 'snowman run' -- r2c_clean.bam and all_bwa.bam. r2c_clean.bam is a BAM containing only reads with mappings to assembled contigs. Reads which do not assemble into contigs are discarded. Each read is in its originally aligned position, and is given a CN tag (contig) and AL tag (alignment-on-contig). all_bwa.bam is an alignment of all of the contigs to the human reference genome. r2c_final.bam and contigs_final.bam are trimmed versions of these which contain only high-confidence read-to-contig mappings, and only contigs which support either a germline or somatic event. In the 'breakpoints' folder, there are VCFs and txt files providing the breakpoints, both somatic and germline. The 'alignments' folder contains ASCII plots of the contigs and their read support. The 'plots' folder is for internal use (required to make nozzle output)." r <- setReportSubTitle(r, "Jeremiah Wala") r <- addToIntroduction(r, newParagraph(intro.str)) r <- addToIntroduction(r, newParagraph(files.str)) #r <- addToSummary(r, newParagraph(intro.str)) return (r) } ### # ### .overviewFigure <- function(grl.sno, opt, r) { print('...making the overview trackData figure') grsno = grl.sno$som suffix = 'somatic' if (opt$germline) { grsno = grl.sno$ger suffix = 'germline' } td <- trackData() td$xaxis.newline=FALSE td$xaxis.chronly=TRUE td$xaxis.nticks = 0 overview.fig = file.path(opt$outdir, paste('breakpoint_overview_', suffix, '.png', sep='')); png(overview.fig, width=6000, height=800) display(td, links=grsno, windows=gr.all()) suppressMessages(dev.off()) fig = newFigure(file.name(overview.fig), fileHighRes = file.name(overview.fig), exportId = "FIGURE_1", "Track view of entire JaBbA graph, with unincorporated rearrangements removed. Segments are assigned a copy number and plotted on the y axis. Reference edges are shown in very light gray. Red edges represent aberrant junctions and their weight corresponds to their inferred copy number. Loose ends are shown as dangling dotted blue edges. The bottom track shows genome-wide normalized coverage") r = addToResults(r, fig) return (r) } ### # ### .makeCircos <- function(grl.sno, opt, r) { if (!opt$germline) { ## add the somatic circos figure circos.fig <- file.path(opt$outdir, 'circos_somatic.png') circos.conf <- file.path(opt$outdir, 'circos_somatic.conf') circos.svg <- file.path(opt$outdir, 'circos_somatic.svg') gr2circos(opt$outdir, ra=grl.sno$som) system(paste('cd', opt$outdir, '; /home/unix/marcin/Software/circos/circos-0.62-1/bin/circos')) system(paste('mv', file.path(opt$outdir, 'circos.png'), circos.fig)) system(paste('mv', file.path(opt$outdir, 'circos.svg'), circos.svg)) system(paste('mv', file.path(opt$outdir, 'circos.conf'), circos.conf)) fig = newFigure(file.name(circos.fig), fileHigRes=file.name(circos.fig), exportId = "FIGURE_2", "Somatic events") r = addToResults(r, fig) } else { ## add the germline circos figure circos.conf <- file.path(opt$outdir, 'circos_germline.conf') circos.svg <- file.path(opt$outdir, 'circos_germline.svg') circos.fig <- file.path(opt$outdir, 'circos_germline.png') gr2circos(opt$outdir, ra=grl.sno$ger) system(paste('cd', opt$outdir, '; /home/unix/marcin/Software/circos/circos-0.62-1/bin/circos')) system(paste('mv', file.path(opt$outdir, 'circos.png'), circos.fig)) system(paste('mv', file.path(opt$outdir, 'circos.svg'), circos.svg)) system(paste('mv', file.path(opt$outdir, 'circos.conf'), circos.conf)) fig = newFigure(file.name(circos.fig), fileHigRes=file.name(circos.fig), exportId = "FIGURE_2", "Germline events") r = addToResults(r, fig) } return(r) } ### # ### .getContigs <- function(opt) { ## read in the contigs snow.contigs <- file.path(opt$indir, 'contigs.bam') if (!file.exists(snow.contigs)) snow.contigs <- file.path(opt$indir, 'all_bwa.bam') if (!file.exists(snow.contigs)) stop('Cannot find contigs bam file') print(paste('...importing contigs from:', snow.contigs)) gr.contigs <- import.snowman.contigs(snow.contigs, paste(snow.contigs, "bai", sep='.')) uniq = unique(mcols(grl.sno$som)$contig) if (opt$germline) uniq = unique(mcols(grl.sno$ger)$cname) gr.contigs <- gr.contigs[gr.contigs$qname %in% uniq] ## keep only contigs in somatic breaks suppressWarnings(gr.contigs <- gr.contigs[order(nchar(gr.contigs$seq), decreasing=TRUE)]) ## put longest seqs in front, to deal with potential hard clipping ix <- !is.na(gr.contigs$cigar) gr.contigs <- gr.contigs[ix] return(gr.contigs) } ### # ### .getReads <- function(opt) { ## read in the reads #snow.reads <- file.path(opt$indir, 'plots/readsForR_som.txt') #if (opt$germline) # snow.reads <- file.path(opt$indir, 'plots/readsForR_ger.txt') #if (!file.exists(snow.reads)) # stop(paste('Cannot find', snow.reads)) #print(paste('`...importing reads from:', snow.reads)) #gr.reads <- import.snowman.reads(snow.reads) bam = file.path(opt$indir, "r2c_clean.bam") bami = file.path(opt$indir, "r2c_clean.bam.bai") gr.reads = read.bam(bam, bami=bami, tag=c("AL", "SW", "CN", "SR", "TS"), pairs.grl=FALSE) #gr.reads$tn = substring(gr.reads$rname, 1,1) #gr.reads$rqname = gsub("[a-z]+[0-9]+_?(.*)", "\\1", gr.reads$rname) #gr.reads$rheader = gsub("[^_]+($)", "\\1", gr.reads$rname) #gr.reads$rqname = substring(gr.reads$rname, nchar(gr.reads$rheader)+1, nchar(gr.reads$rname)) #gr.reads$flag = as.numeric(gsub("[a-z]+([0-9]+)_?(.*)", "\\1", gr.reads$rheader)) gr.reads <- gr.reads[!is.na(gr.reads$AL) & !is.na(gr.reads$CN) & !is.na(gr.reads$SR)] al <- unlist(strsplit(gr.reads$AL, "x")) cn <- unlist(strsplit(gr.reads$CN, "x")) len <- sapply(strsplit(gr.reads$AL, "x"), length) seq <- rep(gr.reads$TS, len) seq[is.na(seq)] = gr.reads$seq[is.na(seq)] #ix = !is.na(as.integer(al)) ##debug grr <- GRanges(seqnames=cn, IRanges(as.integer(al), width=nchar(seq)), seq=seq) grr$cigar = paste(nchar(seq), "M", sep="") grr$rname = rep(gr.reads$qname, len) return(grr) } ### # ### .getTrackData <- function(cgc, r2g, gr.contigs.this, gr.reads.this) { ## subet them cgc_t <- gSubset(cgc, xnames=unique(gr.contigs.this$qname)) r2g_t <- gSubset(r2g, xnames=gr.reads.this$rname) if (length(links(r2g_t)$x) == 0 || length(links(cgc_t)$x) == 0) return (trackData()) umap <- gr.reads.this$rname[bitAnd(gr.reads.this$flag, 4) != 0] ix = !duplicated(gr.contigs.this$qname) cseq.set <- DNAStringSet(gr.contigs.this$seq[ix]) names(cseq.set) <- gr.contigs.this$qname[ix] ix <- !duplicated(gr.reads.this$rname) rseq.set <- DNAStringSet(gr.reads.this$seq[ix]) names(rseq.set) <- gr.reads.this$rname[ix] grl.contig.seq <- seq2grl(cseq.set) ## max because of hard clipping issue grs.contig <- lift(cgc_t, grl.contig.seq) td.c2g <- do.call('trackData', c(list(data=grs.contig, track.name='Contig', labels.suppress=TRUE))) grs.reads <- seq2grl(rseq.set, sn=names(rseq.set)) reads2genome <- lift(r2g_t, grs.reads, pintersect=TRUE) r2g.umap <- reads2genome[ names(reads2genome) %in% umap] r2g.map <- reads2genome[!names(reads2genome) %in% umap] td.r2g.u <- do.call('trackData', c(list(data=r2g.umap, track.name='UMap', labels.suppress=TRUE))) td.r2g.m <- do.call('trackData', c(list(data=r2g.map, track.name='Map', labels.suppress=TRUE))) td.r2g <- c(td.r2g.u, td.r2g.m) ## lift the contig onto the genome gr.cdum <- GRanges(names(cseq.set),IRanges(1,width(cseq.set)),strand='+') suppressWarnings(rr <- lift(cgc_t, gr.cdum)) rr$mapq <- values(cgc)$mapq[rr$link.id] rr$col[!is.na(rr$mapq)] <- alpha('darkred', rr$mapq[!is.na(rr$mapq)]/60) rr$col[ is.na(rr$mapq)] <- 'yellow' rr$border <- 'black' rr.split <- split(rr, rr$query.id) names(rr.split) = names(cseq.set) ## make contig mapq track data td.c <- do.call('trackData', c(list(data=rr.split, labels.suppress=TRUE, draw.paths=TRUE))) td.r2g$height = 10 td.c2g$height = 1 td.c$height = 1 tds <- c(td.r2g, td.c2g, td.c) tds$xaxis.nticks=2 tds$xaxis.newline=FALSE tds$xaxis.cex = 2.0 tds$xaxis.unit = 1; tds$xaxis.suffix = "" return(tds) } .getN50 <- function(opt, r) { ## load the contigs print('...reading contig fasta') contigs.file = file.path(opt$indir, 'all_contigs_bootstrap.fa') if (!file.exists(contigs.file)) { print(paste("Contigs file for estimating N50 does not exist:", contigs.file)) return (r) } awk.cmd = paste("awk 'NR % 2 == 0 {print length}'", contigs.file) ab <- as.numeric(read.table(pipe(awk.cmd),header=FALSE)$V1) n50 <- Biostrings::N50(ab) ymax = max(table(cut(log(ab,10),breaks=seq(0,4,by=0.01)))) lab = seq(from=2, to=3.5, by=0.1) p <- ggplot() + geom_histogram(data=data.frame(len=ab), aes(x=log(len,10)), binwidth=0.01, color='black', fill=NA) + geom_line(data=data.frame(x=rep(log(n50,10),2), y=c(0, ymax)), aes(x=x, y=y), color='red') + geom_text(data=data.frame(x=0.1 + log(n50,10), y=ymax), aes(x=x, y=y, label=paste("N50: ", n50, 'bp', sep='')), color='red') + theme_bw() + xlab('Contig length') + ylab('Count') + scale_x_continuous(breaks=lab, labels=parse(text=paste('10', lab, sep='^'))) png(file.path(opt$outdir, 'N50hist.png'), width=800, height=400) print(p) dev.off() fig = newFigure('N50hist.png', fileHigRes='N50hist.png', exporId="FIGURE_1000", paste("Distribution of lengths of assembled contigs. The N50 is", n50)) r = addToResults(r, fig) return (r) } .getTrackDataAlign <- function(cgc, r2g, gr.contigs.this, gr.reads.this) { cgc_t <- gSubset(cgc, xnames=unique(gr.contigs.this$qname)) r2g_t <- gSubset(r2g, xnames=gr.reads.this$rname) if (length(links(r2g_t)$x) == 0 || length(links(cgc_t)$x) == 0) return (trackData()) gr.reads.this$col <- 'gray' gr.reads.this$border <- 'black' ## isolate discordant read pairs tab <- table(gr.reads.this$rname) gr.reads.this$col[gr.reads.this$rname %in% names(tab[tab==2])] <- 'blue' ## isolate the unmapped reads gr.reads.this$border[bitAnd(gr.reads.this$flag, 4) != 0] <- 'red' ## make the new reads struct grr <- GRanges(gr.reads.this$rname, IRanges(1, nchar(gr.reads.this$seq))) grr$col <- gr.reads.this$col grr$border <- gr.reads.this$border grr$rname <- gr.reads.this$rname grl.grr <- split(grr, grr$rname) reads2genome <- lift(r2g_t, grl.grr) tdo <- trackData(reads2genome, labels.suppress=TRUE) return(tdo) } ##################################### ########### RUN NOZZLE ############## ##################################### ## load the libraries .nozzleLibraries() option_list = list( make_option(c("-i", "--indir"), type = "character", default = './', help = "Path to breakpoints file breakpoints.somatic.text in Snowman output directory, or root Snowman output directory"), make_option(c("-g", "--germline"), type = "character", default = 'false', help = "Set this flag to true to output germline snowman results instead of somatic. Default: false"), make_option(c("-m", "--maxpngs"), type = "integer", default = 1e8, help = "Maximum PNGS to produce per report subheading"), make_option(c("-c", "--cores"), type = "integer", default = 1, help = "Number of cores to use"), make_option(c("-o", "--outdir"), type = "character", default = './', help = "Directory to dump output into") ) parseobj = OptionParser(option_list=option_list) opt = parse_args(parseobj) ## make the output directory if (!grepl("nozzle", opt$outdir)) opt$outdir = file.path(opt$outdir, 'nozzle') dir.create(opt$outdir, showWarnings=FALSE) opt$germline = grepl("t|T", opt$germline) ## load the breakpoints grl.sno = .loadBreakpoints(opt) ##debug grl.sno$ger = grl.sno$som # Phase 1: create report elements r <- newReport( "SnowmanSV -- Structural variation detection by genome-wide local assembly" ) r <- .initializeReport(r) ## add the overview figure r <- .overviewFigure(grl.sno, opt, r) ## add the contig N50 figure #r <- .getN50(opt, r) ## make the circos figures r <- .makeCircos(grl.sno, opt, r) ## grab the contigs and reads gr.contigs <- .getContigs(opt) gr.reads <- .getReads(opt) gr.reads <- gr.reads[as.logical(seqnames(gr.reads) %in% gr.contigs$qname)] gr.contigs <- gr.contigs[gr.contigs$qname %in% unique(as.character(seqnames(gr.reads)))] ## make the trackData suppressWarnings(cgc <- cgChain(gr.contigs, sn=gr.contigs$qname)) suppressWarnings(pac <- cgChain(gr.reads, sn=gr.reads$rname)) suppressWarnings(r2g <- gMultiply(cgc, pac, pintersect=TRUE)) suppressWarnings(tdr <- track.refgene()) ## loop through and run nams <- unique(gr.contigs$qname) suffix = 'somatic' sn <- as.character(seqnames(gr.reads)) if (opt$germline) suffix = 'germline' grl_this = grl.sno$som if (opt$germline) grl_this = grl.sno$ger out <- mclapply(seq_along(nams), function(x) { print(paste("Working on contig: ", x, "of", length(nams))) outfile.png = file.path(opt$outdir, paste("contig_", x, '_', suffix, '.png', sep='')) outfile.pdf = file.path(opt$outdir, paste("contig_", x, '_', suffix, '.pdf', sep='')) gr.contigs.this <- gr.contigs[gr.contigs$qname == nams[x]] gr.reads.this <- gr.reads[sn == nams[x]] ## get the sequences track data tds <- suppressWarnings(.getTrackData(cgc, r2g, gr.contigs.this, gr.reads.this)) tds <- c(tds, tdr) ## get the alignment track data tda <- suppressWarnings(.getTrackDataAlign(cgc, r2g, gr.contigs.this, gr.reads.this)) ## set the window win <- streduce(gr.contigs.this) ## make the sequences plot png(outfile.png, width=1500, height=600) display(c(tda, tds), windows=win, links=grl_this, cex.ylabel=1) dev.off() pdf(outfile.pdf, width=15, height=10) display(c(tda, tds), windows=win, links=grl_this, cex.ylabel=1) dev.off() ## make the alignment plot mc <- mcols(grl_this) mc <- mc[mc$cname == nams[x],] ## add the figure to the plot expid = sprintf("BREAKPOINT_FIGURE_%s", x) break1 = paste(mc$chr1, ":", mc$pos1, "(", mc$strand1, "){MAPQ: ", mc$mapq1, "}", sep="") break2 = paste(mc$chr2, ":", mc$pos2, "(", mc$strand2, "){MAPQ: ", mc$mapq2, "}", sep="") lead = sprintf("Breakpoint plot of junction %s. From the top down: The red arrows at the top give the orientation of the junction, with the arrowhead pointing towards the joined DNA (away from the junction). The contig is then shown below, colored with respect to MAPQ, with white = MAPQ 0 and dark red = MAPQ 60. The contig sequence is also shown. The individual reads supporting this contig are displayed as mapped to the genome *through* their mapping to the contig. The reads were identified as potentially supporting this contig within 'snowman run', and were more thouroughly Smith-Waterman realigned in 'snowman gather'. Reads with > 6 bases on both sides of the junction are 'split' reads.", x) caption = paste(lead, "Breakpoint plot of junction", x, "[ Name:", mc$cname, "] -- [ Somatic breakpoint junction:", x, "] -- [ Tumor Split Reads:", mc$tsplit, "] -- [ Normal Split Reads:", mc$nsplit, "] -- [ Break1:", break1, "] -- [ Break2:", break2, "] -- [ Span:", mc$span, "] -- [ Homology:", mc$homology, "] -- [ Insertion:", mc$insertion, "] -- [ Num Times Break Found:", mc$num.dups+1, "] -- [ Number of Alignments:", mc$num.parts, "]") fig = newFigure(file.name(outfile.png), fileHigRes=file.name(outfile.png), exportId = expid, caption) return(fig); }, mc.cores=opt$cores) for (i in seq_along(out)) r = addToResults(r, out[[i]]) # Phase 3: render report to file print('...done with report sending to file') outpath <- file.path(opt$outdir, paste("snowman_report", suffix, sep='_')) writeReport(r, filename=outpath); # w/o extension ## test if (FALSE) { gr.reads$tn = substring(gr.reads$rname, 1,1) gr.reads$rqname = gsub("[a-z]+[0-9]+_?(.*)", "\\1", gr.reads$rname) gr.reads$rheader = gsub("[^_]+($)", "\\1", gr.reads$rname) gr.reads$rqname = substring(gr.reads$rname, nchar(gr.reads$rheader)+1, nchar(gr.reads$rname)) gr.reads$flag = as.numeric(gsub("[a-z]+([0-9]+)_?(.*)", "\\1", gr.reads$rheader)) gr.reads.per <- split(gr.reads, seqnames(gr.reads)) ## grab the discordant reads gr.test = gr.reads.per[[1]] tab = table(gr.test$rqname) gr.test.disc = gr.test[gr.test$rqname %in% names(tab[tab==2])] grl.test.disc = GRangesList() if (length(gr.test.disc)) grl.test.disc = split(gr.test.disc, gr.test.disc$rqname) } ================================================ FILE: R/archive_non_functional/svaba-sig.R ================================================ #!/usr/bin/env Rscript library(optparse) require(VariantAnnotation) option_list = list( make_option(c("-i", "--input"), type = "character", default = "qcreport.txt", help = "Input txt file from a snowman preprocess qcreport.txt"), make_option(c("-o", "--output"), type = "character", default = "qcreport.pdf", help = "Output pdf to generate"), make_option(c("-f", "--FHworkspace"), type = "character", default = NULL, help = "Firehose workspace to retrieve data from"), make_option(c("-p", "--FHpairset"), type = "character", default = NULL, help = "Firehose pairset to retreive data from"), make_option(c("-a", "--FHannotation"), type = "character", default = NULL, help = "Firehose annoation to retreive data from") ) parseobj = OptionParser(option_list=option_list) opt = parse_args(parseobj) fhsum = sum(c(!is.null(opt$FHworkspace), !is.null(opt$FHpairset), !is.null(opt$FHannotation))) GETFH = fhsum == 3 if (fhsum %in% c(1,2)) print(print_help(parseobj)) stop("If importing from FH, must specify fully the FHworkspace, FHpairset and FHannotation") } if (is.null(opt$input)) stop(print_help(parseobj)) if (!file.exists(opt$input)) { print(print_help(parseobj)) stop(paste("Input file does not exist", opt$input, ". Must supply path to valid qcreport.txt file (generated from snowman preprocess or snowman run")) } source('/home/unix/jwala/GIT/isva/Taiga/R/sourceall.R') ######################## ## some functions ######################## source.all <- function() { suppressMessages(suppressWarnings(require(ff, quietly=TRUE))) suppressMessages(suppressWarnings(require(VariantAnnotation, quietly=TRUE))) suppressMessages(suppressWarnings(require(rtracklayer, quietly=TRUE))) suppressMessages(suppressWarnings(require(data.table, quietly=TRUE))) suppressMessages(suppressWarnings(require(plyr, quietly=TRUE))) suppressMessages(suppressWarnings(require(ggplot2, quietly=TRUE))) suppressMessages(suppressWarnings(require(reshape2, quietly=TRUE))) suppressMessages(suppressWarnings(require(GenomicRanges, quietly=TRUE))) suppressMessages(suppressWarnings(require(popbio, quietly=TRUE))) suppressMessages(suppressWarnings(require(BSgenome.Hsapiens.UCSC.hg19, quietly=TRUE))) suppressMessages(suppressWarnings(require(bitops, quietly=TRUE))) suppressMessages(suppressWarnings(require(seqinr, quietly=TRUE))) suppressMessages(suppressWarnings(require(Rsamtools, quietly=TRUE))) suppressMessages(suppressWarnings(require(ff, quietly=TRUE))) suppressMessages(suppressWarnings(require(multicore, quietly=TRUE))) suppressMessages(suppressWarnings(require(Biostrings, quietly=TRUE))) suppressMessages(suppressWarnings(require(rtracklayer, quietly=TRUE))) suppressMessages(suppressWarnings(require(lattice, quietly=TRUE))) suppressMessages(suppressWarnings(require(RColorBrewer, quietly=TRUE))) suppressMessages(suppressWarnings(require(Matrix, quietly=TRUE))) } round.n <- function(x, n) n * round(x / n) lm_eqn = function(m){ eq <- substitute(italic(y) == a + b %.% italic(x)*","~~italic(r)^2~"="~r2, list(a = format(coef(m)[1], digits = 2), b = format(coef(m)[2], digits = 2), r2 = format(summary(m)$r.squared, digits = 3))) as.character(as.expression(eq)); } ## power.law.plot power.law.plot <- function(pspan, plotname) { print(paste('...making power-law plot for', plotname)) ex <- ecdf(pspan[pspan > 0]) #grid <- c(seq(0,10000,5), seq(10000,10^7,100)) df = data.frame(pspan = pspan, cdf = ex(pspan), pspan.log = log(pspan,10), cdf.log = log(ex(pspan), 10)) df <- df[df$cdf.log > -1000,] ## get rid of -Inf lm.s <- lm(df$cdf.log ~ df$pspan.log) lm.eq = lm_eqn(lm.s) yb = c(0.01,0.02,0.03,0.04,0.05,0.10,0.2, 0.3, 0.4,0.5,0.75,1) pdf(file.path(opt$outdir, plotname), width=5,height=5) print(g <- ggplot() + geom_point(data=df, aes(x=pspan.log, y=cdf.log), size=1, color="blue") + geom_smooth(data=df, aes(x=pspan.log, y=cdf.log), method="lm",se=F) + geom_text(aes(x=4.5,y=log(0.01,10), label=lm.eq), label=lm.eq, parse=T) + theme_bw() + ylab("CDF") + xlab("Span (bp)") + scale_y_continuous(breaks=log(yb,10), labels=format(yb,digits=2), limits=c(log(yb[1],10), 0)) + scale_x_continuous(breaks=seq(0,7), labels=parse(text=paste('10', seq(0,7), sep='^')))) dev.off() } ################################## ################ load from data ## ################################## if (GETFH) { ind <- fiss_get(opt$FHpairset, wkspace=opt$FHworkspace, type='pair') vcf <- ind[, opt$FHannotation] } else { } print('...loading VCF files') toload = file.exists(vcf) cols = sample(colors(), sum(toload)) ra <- lapply(vcf[toload], function(x) { ab = ra_breaks(x) mcols(ab)$individual = basename(x) mcols(ab)$border = mcols(ab)$col = cols[match(x, vcf)] return (ab) }) ## place in data table form ra.bound = do.call('grlbind', ra) ra.ul <- grl.unlist(ra.bound) ra.dt <- gr2dt(ra.ul) ## get the spans span = ra.dt[, abs(end[1]-end[2]) * (seqnames[1] == seqnames[2]), by=grl.ix]$V1 mcols(ra.bound)$span = span mcols(ra.bound)$span[mcols(ra.bound)$span == 0] = NA ## set the logical for methods s = mcols(ra.bound)$CALLER == "S" d = mcols(ra.bound)$CALLER == "D" ds = mcols(ra.bound)$CALLER == "DS" ############################ ## make the overlap pie chart ############################ cols = c(ovlp.hue, dran.hue, snow.hue) levs = c("Both", "dRanger", "SnowmanSV") df <- data.frame(Caller=c("SnowmanSV", "dRanger", "Both"), counts=c(sum(s, na.rm=T), sum(d, na.rm=T), sum(ds, na.rm=T)), stringsAsFactors=FALSE) g <- ggplot(df, aes(x=factor(1), y=counts, fill=Caller)) + geom_bar(stat='identity', position="fill") + coord_polar(theta="y") + xlab("") + ylab("") + scale_fill_manual(values=cols, breaks=levs) pdf(file.path(opt$outdir, "caller_overlap_pie.pdf")); print(g); dev.off() ############################ ## make the 1/L distribution ############################ power.law.plot(span, "power_law_DS.pdf") power.law.plot(span[which(s)], "power_law_S.pdf") power.law.plot(span[which(d)], "power_law_D.pdf") ############################################### ## check if anything overlaps with cancer genes ############################################### cgc.genes <- track.load('cgc') fo <- gr.findoverlaps(ra.ul, gr.pad(cgc.genes, 5e4)) tab <- table(fo$subject.id) gene.nums = as.numeric(names(tab)) names(tab) <- cgc.genes$gene[gene.nums] tab.lenscale = tab / width(cgc.genes[gene.nums]) ## generate PDFs for all of these td.rg.cgc = track.refgene(genes = cgc.genes$gene, height=3) ord = order(tab.lenscale, decreasing=T) dir.create(file.path(opt$outdir, "CGCgenes"), showWarnings=FALSE) dum <- sapply(seq_along(ord), function(x) { gene = names(tab.lenscale[ord[x]]) ## find the partners gene.window = gr.pad(cgc.genes[cgc.genes$gene == gene], 5e4) rar.hits = ra.bound[ceiling(gr.findoverlaps(ra.ul, gene.window)$query.id/2)] windows <- streduce(grbind(gr.pad(streduce(rar.hits), 5e4), gene.window)) nindiv = length(unique(mcols(rar.hits)$individual)) print(paste("plotting CGC gene", gene)) pdf(file.path(opt$outdir, "CGCgenes", paste("rank_",sprintf("%04d",x),"_",names(tab.lenscale[ord[x]]), "_NIndiv_", nindiv, ".pdf", sep=""))) #pdf(file.path(opt$outdir, "ROS1_special.pdf")) td.rg.cgc$xaxis.newline = T td.rg.cgc$xaxis.cex = 0.5 td.rg.cgc$xaxis.cex.label = 0.5 td.rg.cgc$xaxis.nticks = 2 display(td.rg.cgc, links=rar.hits, window=windows) dev.off() }) ############################## ## matrix plot ############################## grt = gr.tile(gr.all(), w=10e6) mat <- sig.tri.matrix(ra.bound, grt, log=TRUE) td.mat <- do.call('trackData', c(allopts, list(grt, mdata=mat, triangle=TRUE, cmap.min=min(mat), cmap.max=max(mat)+0.1, track.name='Triangle', height=25, sep.lwd=0.5, m.bg.col='white', track.name='Breakpoint-pair Heatmap', islog=TRUE, xaxis.nticks=0, xaxis.prefix="", xaxis.chronly=TRUE))) pdf(file.path(opt$outdir, "overview_matrix.pdf"), width=12, height=12) display(td.mat, windows=streduce(gr.all())) dev.off() ## per chrom grt = gr.tile(gr.all(), w=10e6) mat <- sig.tri.matrix(ra.bound, grt, log=TRUE) td.mat <- do.call('trackData', c(allopts, list(grt, mdata=mat, triangle=TRUE, cmap.min=min(mat)+0.1, cmap.max=max(mat)+0.1, track.name='Triangle', height=25, sep.lwd=0.5, m.bg.col='white', track.name='Breakpoint-pair Heatmap', islog=TRUE, xaxis.nticks=0, xaxis.prefix="", xaxis.chronly=TRUE))) dum <- lapply(seq(23), function(x) { print(paste("plotting for chr", x)) pdf(file.path(opt$outdir, paste("chr",x,"_matrix.pdf",sep="")), width=12, height=12) display(td.mat, windows=streduce(gr.all()[x])) dev.off() }) ################################################ ## Start the significance testing ## ################################################ ## load data for 1e6 binned mapping dload <- sig.load.bins('1e6') grt <- dload$grt mat.background = sig.2d.map.background(dt2gr(grt), nomap=FALSE) fo <- sig.bin.counts(ra.bound, dt2gr(grt)) ## place events into bins fo[, sample.counts := nrow(.SD), by='sample'] ## get total number of binned-events per sample setkey(fo, sample) ## sort by sample count M <- structure(as.numeric(fo$sample.counts[!duplicated(fo)]), names=fo$sample[!duplicated(fo)]) ## M is number mutations per sample M <- M[order(names(M))] exclude <- names(M[M < 1]) ## drop really weak samples M <- M[!names(M) %in% exclude] ra.bound <- ra.bound[!mcols(ra.bound)$individual %in% exclude] ## normalize background probablity matrix by total intra/inter chrom rate empirical.background.prob <- sig.generate.normalized.matrices(ra.bound, dload$mat, M, single.mat=TRUE) ## get the total vector of background F-scores system.time(f.back <- sig.2d.permute(empirical.background.prob, M, num.permute = 1000, method='1d')) f.back2 <- f.back[f.back > 0] ## plot the distribution of f scores f.back2 <- f.back[f.back > 0] pdf(file.path(opt$outdir, "fscore_distribution.pdf"), height=3.5, width=10) hist(f.back2, col='black', breaks=300, xlab='F-score', ylab='Count', main='F-score distribution under NULL') dev.off() ## get a trackData for the bin probabilities #all.bin.probs <- colSums(do.call('rbind', lapply(all.mats, colSums))) / length(all.mats) all.bin.probs <- colSums(empirical.background.prob) grt$binprobs <- all.bin.probs grt$col <- grt$border <- chr_colors[as.character(seqnames(dt2gr(grt)))] grt$binprobs <- -log(grt$binprobs, 10) td.binprob <- trackData(dt2gr(grt), y.field='binprobs', xaxis.newline=FALSE, xaxis.chronly=TRUE, track.name='-Log(Bin Background Probability)', y0=3.3, y1=3.9, circles=TRUE, lwd.border=0.5, xaxis.nticks = 0, xaxis.prefix="") pdf(file.path(opt$outdir, "td_bin_background_probabiltiies.pdf"), width=10) display(td.binprob, windows=gr.all()) dev.off() ## get the actual f-score f.real <- sig.fscore(grl, dt2gr(grt), empirical.background.prob) ## get the p and q values pq = sig.fscore2pval(f.back, f.real) grt$pval <- pq$pval grt$qval <- pq$qval ## make the qq-plot pdf(file.path(opt$outdir, "qqplot.pdf"), width=5, height=5) qq_pval(grt$pval) dev.off() grt$n.log10.q <- -log(grt$qval, 10) grt$col <- grt$border <- chr_colors[as.character(seqnames(dt2gr(grt)))] td.sig <- trackData(dt2gr(grt), y.field='n.log10.q', circles=TRUE, track.name='-log10(Q-value)', lwd.border=0.5, xaxis.chronly=T, y0=0, xaxis.prefix="", xaxis.nticks=0) ppdf(display(td.sig, windows=gr.all())) td.refgenes <- track.refgene() dir.create(file.path(opt$outdir, "topsighits"), showWarnings=FALSE) ### plot all the hits above -log10(q) = 1 which.hits <- which(grt$n.log10.q) > 1 dum <- lapply(seq_along(which.hits), function(x) { y = which.hits[x] window = dt2gr(grt)[y] genes <- unique(gr.genes$gene[gr.findoverlaps(gr.genes, window)$query.id]) genes.string <- paste(genes, collapse = " ") print(paste("Plotting top significance bin", x, "of", sum(grt$n.log10.q > 1), "which is region", grt$seqnames[y],":", grt$start[y], "-", grt$end[y], "which contains genes", genes.string)) rar.hits <- ra.bound[ceiling(gr.findoverlaps(ra.ul, window)$query.id/2)] disp.window <- streduce(grbind(gr.pad(streduce(rar.hits),5e4),window)) pdf(file.path(opt$outdir, "topsighits", paste("hit_", x, "q_",sprintf("%04f",grt$n.log10.q[y]),".pdf", sep=""))) td.rg.cgc$xaxis.newline = T td.rg.cgc$xaxis.cex = 0.5 td.rg.cgc$xaxis.cex.label = 0.5 td.rg.cgc$xaxis.nticks = 2 td.rg.cgc$cex.tick = 0.5 td.rg.cgc$track.name = "Cancer Genes" td.refgenes$track.name = "All Genes" display(c(td.rg.cgc, td.refgenes), links=rar.hits, window=disp.window) dev.off() hitnum = hitnum + 1 }) ================================================ FILE: R/archive_non_functional/svaba-vcf-to-maflite.R ================================================ #!/usr/bin/env Rscript suppressMessages(suppressWarnings(require(optparse, quietly=TRUE))) option_list = list( make_option(c("-i", "--input"), type = "character", default = NULL, help = "Input bps.txt.gz file"), make_option(c("-o", "--output"), type = "character", default = "vars.lite.maf", help = "Output MAFLITE") ) parseobj = OptionParser(option_list=option_list) opt = parse_args(parseobj) if (is.null(opt$input)) stop(print_help(parseobj)) print('...loading VariantAnnotation package') suppressMessages(suppressWarnings(require(VariantAnnotation, quietly=TRUE))) print('...reading VCF') v <- readVcf(opt$input, "hg19") inf <- info(v) gr <- rowData(v) ## remove non indel lines gr <- gr[inf$EVDNC=="INDEL",] inf <- inf[inf$EVDNC=="INDEL",] ## remove unnecesry columns #tab <- data.frame(chr=as.character(seqnames(gr)), startr=start(gr), endr=end(gr), ref=gr$REF, alt=gr$ALT) tab <- data.frame(chr=as.character(seqnames(gr)), startr=start(gr), end=end(gr), ref=as.character(gr$REF), alt=as.character(gr$ALT), stringsAsFactors=FALSE) del = sapply(tab$ref, nchar) > sapply(tab$alt, nchar) ins = sapply(tab$ref, nchar) < sapply(tab$alt, nchar) tab$alt[ins] <- inf$INSERTION[ins] tab$ref[del] <- sapply(tab$ref[del], function(x) substr(x, 2, nchar(x))) tab$alt[del] = "-" tab$ref[ins] = "-" tab <- cbind(tab, inf[, c("SCTG", "SPAN", "MAPQ", "TSPLIT", "NSPLIT")]) write.table(tab, file=opt$output, quote=FALSE, sep="\t", row.names=FALSE, col.names = FALSE) ## oncotate it if (grepl("oncotator", Sys.getenv("PATH"))) { cmd = paste("oncotator", opt$output, "vars.maf", "hg19") print("Oncotate this MAF lite with the following command:") print(cmd) } ================================================ FILE: README.md ================================================ ## *SvABA* — Structural variation and indel analysis by assembly SvABA (formerly *Snowman*) is an SV and indel caller for short-read BAMs. It performs genome-wide local assembly with a vendored SGA, realigns contigs with BWA-MEM, and scores variants by reassembled read support. Tumor/normal, trios, and single-sample modes are supported; variants are emitted as VCF plus a verbose tab-delimited companion (`bps.txt.gz`) that carries the full per-sample evidence. **License:** [GNU GPLv3](./LICENSE). Uses the [SeqLib][seqlib] API for BAM I/O, BWA-MEM alignment, interval trees, and the assembly front-end. ## Install CMake is required; htslib is an external dependency (no bundled copy). If htslib is installed system-wide, svaba auto-detects it via pkg-config or the default include/lib search paths: ```bash git clone --recursive https://github.com/walaj/svaba cd svaba && mkdir build && cd build cmake .. && make -j ``` For a non-standard htslib install location, point at it explicitly: ```bash cmake .. -DHTSLIB_DIR=/path/to/htslib-1.xx make -j ``` To link jemalloc at compile time (recommended on Linux for `-p 16+` runs — eliminates allocator contention, no `LD_PRELOAD` needed): ```bash cmake .. -DUSE_JEMALLOC=ON make -j ``` The binary lands at `build/svaba`. To install it system-wide (or into this repo's `bin/`), use CMake's install target — the default prefix is `/usr/local` and needs sudo, or override it with `-DCMAKE_INSTALL_PREFIX`: ```bash make install # /usr/local/bin/svaba cmake --install build --prefix $(pwd)/.. # repo-local bin/svaba ``` Build type defaults to `RelWithDebInfo` (`-O2 -g -DNDEBUG`). The vendored `SeqLib/bwa` and `SeqLib/fermi-lite` hardcode `-O2` in their Makefiles; see `CLAUDE.md` for how to push them to `-O3 -mcpu=native` (typically a 5–15% wall-time win). ### Assembler selection By default svaba assembles contigs with **fermi-lite** (ropebwt2-based, the faster path). The vendored SGA (String Graph Assembler) is still fully supported but off by default — switch it on at compile time by passing `-DSVABA_ASSEMBLER_FERMI=0` to CMake: ```bash cmake .. -DSVABA_ASSEMBLER_FERMI=0 make -j ``` The choice is a single compile-time symbol in `src/svaba/SvabaAssemblerConfig.h`; you can also flip it by editing that file directly and rebuilding. Runtime logs report the active assembler under `svaba::kAssemblerName`, so you can always confirm which engine a build was compiled with. ### jemalloc (Linux, high thread count) On Linux, running svaba with **jemalloc** as the allocator typically wins 10–20 % wall-time at `-p 16+` (large WGS, tumor/normal). svaba's per-thread assembly loop generates heavy malloc/free traffic and glibc's default allocator serializes on its arena locks under that contention pattern; jemalloc's thread-local arenas sidestep the problem. The repo ships a drop-in wrapper at `./svaba_jemalloc` that `LD_PRELOAD`s the system `libjemalloc.so.2` and then exec's svaba: ```bash # one-liner replacement for `svaba run ...`: ./svaba_jemalloc run -t tumor.bam -n normal.bam -G ref.fa -a my_run -p 16 \ --blacklist tracks/hg38.combined_blacklist.bed ``` Install jemalloc first (`apt install libjemalloc2`, `yum install jemalloc`, `dnf install jemalloc`). The wrapper auto-detects the library under the standard distro paths; if yours is elsewhere, set `JEMALLOC_LIB=/path/to/libjemalloc.so.2`. If the svaba binary isn't on your `$PATH`, set `SVABA=/path/to/svaba`. For very high concurrency (`-p 24+`), also pass jemalloc's own tuning knobs via `MALLOC_CONF`: ```bash MALLOC_CONF=background_thread:true,narenas:24,dirty_decay_ms:10000 \ ./svaba_jemalloc run -p 24 ... ``` `narenas` should match or modestly exceed the thread count; `background_thread:true` asks jemalloc to reclaim dirty pages on a background thread instead of in the hot path. **macOS users should not use jemalloc.** Apple's native libmalloc (with its nanomalloc fast path for small allocations) and the DYLD_INSERT_LIBRARIES mechanism combine to run 5–10× slower than system malloc on this exact workload in our A/B tests. The wrapper refuses to run on Darwin for that reason. Stick with the system allocator on macOS. ## Quick start Three steps. Run the caller, merge + dedup + index the per-thread outputs, then convert the deduped `bps.txt.gz` to VCF. The bundled combined blacklist is strongly recommended — it keeps svaba out of well-known pileup / high-complexity regions that would otherwise dominate wall-clock time with no real calls to show for it. See `tracks/README.md` for customizing or extending the blacklist. ```bash # 1. Call: tumor/normal on chr22 with 4 threads, bundled blacklist svaba run -t tumor.bam -n normal.bam -G ref.fa -a my_run -p 4 \ -k chr22 \ --blacklist tracks/hg38.combined_blacklist.bed # 2. Post-process: merge per-thread BAMs, sort+dedup+index, # sort+dedup bps.txt.gz, filter r2c to PASS. scripts/svaba_postprocess.sh -t 8 -m 4G my_run # 3. Convert the deduped bps.txt.gz to VCFv4.5 (SV + indel) svaba tovcf -i my_run.bps.sorted.dedup.txt.gz -b tumor.bam -a my_run ``` A single-sample call drops `-n`. Any number of cases/controls can be jointly assembled; prefix on the sample ID drives case/control routing (`t*` = case, `n*` = control). ## Subcommands SvABA is a multi-tool binary. `svaba help` lists everything. The main ones: `svaba run` performs the whole assembly + variant-calling pipeline. Takes a BAM (or many) plus a reference, a blacklist, and an output ID. Emits `bps.txt.gz`, per-sample VCFs, `contigs.bam`, `runtime.txt`, and (with `--dump-reads`) per-thread `*.discordant.bam`, `*.corrected.bam`, `*.weird.bam`, and `*.r2c.txt.gz`. `svaba postprocess` sorts, deduplicates, @PG-stamps, and indexes the per-thread BAMs and the `bps.txt.gz` emitted by `svaba run`. Typically invoked via `scripts/svaba_postprocess.sh` which also merges per-thread files and builds the PASS / PASS-somatic r2c subsets. `svaba tovcf` converts a deduplicated `bps.txt.gz` into VCFv4.5 output (one SV VCF, one indel VCF; somatic distinguished by the `SOMATIC` INFO flag). Clean intrachrom events emit as symbolic ``/``/ ``; everything else stays paired BND. Input is assumed already sorted/deduped by `svaba_postprocess.sh` — use `--dedup` to opt back into the legacy internal dedup. The `SOMATIC` flag is stamped when a record's somatic LOD (`INFO/SOMLOD`) meets or exceeds a configurable cutoff; the default is **1.0**, which is a reasonable "confident somatic" gate and keeps marginal calls out of the somatic set. Tune it with `--somlod`: ```bash svaba tovcf -i deduped.bps.txt.gz -b tumor.bam -a my_run # default somlod >= 1 svaba tovcf ... --somlod 2.0 # stricter: only strong somatic calls svaba tovcf ... --somlod 0.0 # permissive: flag anything with LO_s >= 0 ``` The raw score is always written to `INFO/SOMLOD` regardless of the cutoff, so downstream `bcftools view -i 'INFO/SOMLOD >= 3'` still works if you want to re-threshold after the fact without regenerating the VCF. `svaba refilter` re-runs the LOD cutoffs / PASS logic on an existing `bps.txt.gz` with new thresholds, regenerating VCFs without rerunning assembly. Use it when you want to tune sensitivity/specificity after the fact. ## Output files `${ID}.bps.txt.gz` is the primary output — one row per breakpoint, with a v3 schema of 52 core columns + per-sample blocks (see `BreakPoint::header` for column names). The 52nd column is a unique `bp_id` of the form `bpTTTNNNNNNNN` that joins back to the BAM aux tags and the VCF `EVENT=` field. `${ID}.contigs.bam` holds every assembled contig, `${ID}.runtime.txt` holds per-region timing, and `${ID}.log` carries the run log. The VCF files (`${ID}.sv.vcf.gz`, `${ID}.indel.vcf.gz`) declare `VCFv4.5`, use symbolic alleles where unambiguous, and carry the canonical scoring in INFO: `MAXLOD` (variant-vs-error, per-sample max), `SOMLOD` (somatic LLR), `SOMATIC` (flag), and `SVCLAIM` (evidence class). VCF `QUAL` defaults to `.` — filter on `FILTER=PASS` or the two LOD fields, not QUAL. See `CLAUDE.md` for the full scoring model. Opt-in outputs (gated behind `--dump-reads`): `${ID}.r2c.txt.gz` is a structured, re-plottable dump of every contig + its r2c-aligned reads; `${ID}.corrected.bam` / `${ID}.weird.bam` / `${ID}.discordant.bam` carry per-read evidence streams. These can run to tens of GB on deep samples, so they're off by default. ## Post-processing pipeline `svaba run` emits per-thread, unsorted BAMs and a raw per-thread `r2c.txt.gz`. Merge + sort + dedup + filter them with one command: ```bash scripts/svaba_postprocess.sh -t 8 -m 4G my_run ``` Five idempotent steps: merge per-thread BAMs and r2c files, run `svaba postprocess` (sort + stream-dedup + @PG-stamp + index), sort and dedup `bps.txt.gz` with PASS filter, emit PASS / PASS-somatic subsets of `r2c.txt.gz`, and (optional) demux the BAMs by source prefix. See `CLAUDE.md` for the full flag surface. ## Viewers All-HTML, no server, drop files in from `file://`. Entry point: `viewer/index.html`. The primary viewer is `bps_explorer.html` — sortable `bps.txt.gz` table with chip filters, per-sample detail panel, log10 histograms for somlod/maxlod/span, and click-to-IGV navigation. `r2c_explorer.html` re-plots the structured r2c TSV in browser (contig ruler, fragment rows, indel `||` marker rows with labels, per-read gap-expanded CIGAR, bp_id filter dropdown). `runtime_explorer.html` visualizes `runtime.txt`; `comparison.html` does side-by-side diffs of two runs. `viewer/alignments_viewer.html` still renders the legacy `alignments.txt.gz` ASCII output, but new runs don't produce that file — `r2c_explorer.html` is the replacement. ## Blacklists `tracks/hg38.combined_blacklist.bed` is the ready-made blacklist; feed it to `svaba run --blacklist`. It is a regeneratable union of component BED files in `tracks/` (ENCODE, high-runtime regions, manual curations, simple repeats, non-standard contigs, and a low-mappability blacklist derived from paired mosdepth runs). See `tracks/README.md` for the recipe. ## Debugging recipes ### Trace a specific read through the entire pipeline Compile with `-DSVABA_TRACE_READ` to get per-decision-point stderr output for a single read (by QNAME) from BAM ingestion through to output tagging. Zero runtime cost when not compiled in (all trace macros compile to no-ops). ```bash cmake -B build -DCMAKE_BUILD_TYPE=RelWithDebInfo \ -DCMAKE_CXX_FLAGS='-DSVABA_TRACE_READ="\"LH00306:129:227V5CLT4:6:1204:38807:7191\""' cmake --build build -j$(nproc) # Run on just the region containing the read (faster iteration) svaba run -t tumor.bam -n normal.bam -G ref.fa -p 4 \ -k chr2:215000000-216000000 --dump-reads -a trace_run 2> read_trace.log grep READ_TRACE read_trace.log ``` The trace covers every gate the read hits: | Stage | Log prefix | What it tells you | |-------|-----------|-------------------| | BAM read & filter | (various, from SvabaBamWalker) | Dup/QC/blacklist gates, rule_pass, NM salvage, adapter filter, quality trim, buffer admission | | BFC error correction | `BFC corrected:` | Whether BFC changed the sequence and by how much | | R2C alignment | `R2C SKIP:` / `R2C HIT:` | Perfect-ref-match skip, or which contigs the read aligned to (AS score, CIGAR) | | Native realignment | `NATIVE_REALIGN:` | Whether the corrected seq was re-aligned to ref or reused the BAM CIGAR | | Split coverage scoring | `SPLIT_COV enter` | Entry into per-BP scoring with r2c coords and breakpoint positions | | R2C vs native gate | `SPLIT_COV TP8` | The critical score comparison: r2c_score, native_score, both CIGARs, NM values | | Indel-at-break check | `SPLIT_COV TP9` | Whether an r2c CIGAR indel lands at the breakpoint | | Span check | `SPLIT_COV TP10` | Whether the read spans the breakpoint(s), with exact coord bounds | | DEL-covers-break | `SPLIT_COV TP11` | Whether an r2c deletion masks the breakpoint | | Final verdict | `SPLIT_COV CREDITED` / `NOT CREDITED` / `SKIPPED` | Did this read count as a variant supporter? | | Output tagging | `OUTPUT TAG bi:Z` | BP id stamped on the read, confidence, somatic status | Example trace (abridged) for a read supporting a somatic 1bp deletion: ``` [READ_TRACE:SvabaBamWalker.cpp:203] read=LH00306:129:... flag=163 mapq=60 pos=chr2:215869800 ... [READ_TRACE:SvabaBamWalker.cpp:236] PASS mr.isValid rule_pass=1 [READ_TRACE:SvabaBamWalker.cpp:340] ADDED to read buffer (n=847) [READ_TRACE:SvabaRegionProcessor.cpp:377] BFC corrected: changed=NO ... [READ_TRACE:SvabaRegionProcessor.cpp:808] R2C HIT: contig=c_fermi_chr2_... AS=150 rc=0 CIGAR=75M1D74M [READ_TRACE:SvabaRegionProcessor.cpp:978] NATIVE_REALIGN: reusing BAM CIGAR (seq unchanged by BFC) [READ_TRACE:BreakPoint.cpp:379] SPLIT_COV enter contig=c_fermi_chr2_... r2c_start=340 r2c_end=489 ... [READ_TRACE:BreakPoint.cpp:476] SPLIT_COV TP8 PASS r2c>native r2c_score=150 native_score=143 ... [READ_TRACE:BreakPoint.cpp:601] SPLIT_COV TP10 span check issplit1=1 issplit2=1 one_split=1 [READ_TRACE:BreakPoint.cpp:697] SPLIT_COV CREDITED as variant supporter sample=t000 tumor=1 [READ_TRACE:SvabaRegionProcessor.cpp:1346] OUTPUT TAG bi:Z bp_id=bp00100000042 confidence=PASS somatic=1 ``` ### Trace a specific contig ```bash cmake -B build -DCMAKE_BUILD_TYPE=RelWithDebInfo \ -DCMAKE_CXX_FLAGS='-DSVABA_TRACE_CONTIG="\"c_fermi_chr2_215869501_215894501_13C\""' cmake --build build -j$(nproc) ``` Shows assembly filtering (TP1–TP5), variant identification (TP6), per-read split-coverage scoring (TP8–TP11), breakpoint confidence (TP13–TP16, TP23). ### Trace both a read AND a contig simultaneously ```bash cmake -B build \ -DCMAKE_CXX_FLAGS='-DSVABA_TRACE_READ="\"LH00306:129:227V5CLT4:6:1204:38807:7191\"" \ -DSVABA_TRACE_CONTIG="\"c_fermi_chr2_215869501_215894501_13C\""' cmake --build build -j$(nproc) ``` Independent prefixes (`[READ_TRACE:...]` vs `[TRACE:...]`) — grep for either. ### Trace ALL reads or ALL contigs (very noisy) ```bash cmake ... -DCMAKE_CXX_FLAGS='-DSVABA_TRACE_ALL_READS=1' # every read cmake ... -DCMAKE_CXX_FLAGS='-DSVABA_TRACE_ALL=1' # every contig ``` Best combined with a small `-k` region. ### Finding a read's QNAME to trace ```bash # From bps.txt.gz — get contig name (col 30) and bp_id (col 52) zcat results.bps.txt.gz | awk -F'\t' '$1=="chr2" && $2 > 215869000 && $2 < 215870000' # From the corrected BAM — reads tagged with a specific bp_id samtools view results.corrected.bam chr2:215869000-215870000 | grep "bi:Z:.*bp00100000042" # From the r2c TSV — all reads for a contig zcat results.r2c.txt.gz | awk -F'\t' '$2 == "c_fermi_chr2_215869501_215894501_13C" && $1 == "read"' | cut -f8 ``` ### Restrict assembly to reads containing a specific kmer ```bash cmake -B build -DCMAKE_BUILD_TYPE=RelWithDebInfo \ -DCMAKE_CXX_FLAGS='-DSVABA_KMER_RESTRICT="\"CCATGCAGAGTGTTGAAGAAAAGGC\""' cmake --build build -j$(nproc) ``` After BFC error correction, only reads whose corrected sequence contains the specified kmer (or its reverse complement) survive. All other reads get `to_assemble = false` — they won't enter fermi assembly, r2c alignment, corrected.bam, or scoring. The log prints kept/dropped counts per region. Use case: you see a suspicious contig and want to know whether assembly still produces it when restricted to reads from a particular locus. Pick a 25-mer unique to that locus, compile with `SVABA_KMER_RESTRICT`, and run on the same region: ```bash svaba run -t tumor.bam -n normal.bam -G ref.fa -k chr11:5000000-5100000 \ -a kmer_test --dump-reads -p 1 ``` If the chimeric contig still assembles, the kmer-carrying reads are sufficient to produce it. If it disappears, reads from elsewhere were required. Can be combined with read/contig tracing: ```bash cmake -B build \ -DCMAKE_CXX_FLAGS='-DSVABA_KMER_RESTRICT="\"CCATGCAGAGTGTTGAAGAAAAGGC\"" \ -DSVABA_TRACE_CONTIG="\"c_fermi_chr11_5000001_5100001_3C\""' ``` ### Disable the r2c-vs-native gate (for debugging only) ```bash cmake ... -DCMAKE_CXX_FLAGS='-DSVABA_R2C_NATIVE_GATE=0' ``` Restores old behavior where any r2c-spanning read credits as variant supporter. Will reintroduce false-positive somatic calls from homology-trap reads. --- ## Alt-contig demotion BWA-MEM sometimes places a contig fragment on an alt contig (e.g. `chr11_JH159136v1_alt`) when chr11 proper has an equally good alignment. If the alt later gets blacklisted, the real breakpoint is lost. svaba now requests secondary alignments for contigs and runs a post-alignment step (`preferStandardChromosomes`): for each primary/supplementary fragment on a non-standard chromosome (ChrID > `maxMateChrID`, default 23), it looks for a secondary alignment on a standard chromosome that: - Covers ≥ 80% of the same query range (reciprocal overlap) - Has AS ≥ 95% of the non-standard alignment's AS If found, the standard-chr alignment is promoted (gets the primary/supplementary flag) and the non-standard one is demoted to secondary. The contig trace (`SVABA_TRACE_CONTIG`) logs each swap with both chromosome IDs and alignment scores. This is controlled by `--max-mate-chr` (same constant used for mate-region lookup). `--non-human` sets it to -1, which disables alt-demotion entirely (no chromosome is "non-standard"). --- ## Recipes Germline-only. Raise the mate-lookup threshold so only larger discordant clusters trigger a cross-region lookup — more conservative and usually appropriate for a single-sample germline run where we don't have a built-in control to guard against mapping artifacts (a tumor would typically see smaller, subclonal clusters we want to chase down). Add `--single-end` to disable mate lookups entirely if you want to be even more conservative: ```bash svaba run -t germline.bam -p 8 --mate-min 6 -a germline_run \ -G ref.fa \ --blacklist tracks/hg38.combined_blacklist.bed ``` Targeted assembly over a list of regions (BED, chr, or IGV-style string): ```bash svaba run -t sample.bam -k targets.bed -a exome_cap -G ref.fa svaba run -t sample.bam -k chr17:7,541,145-7,621,399 -a TP53 -G ref.fa ``` Dump all per-read evidence (large output, only for debugging a specific call): ```bash svaba run -t sample.bam -G ref.fa -a debug_run --dump-reads scripts/svaba_postprocess.sh -t 8 -m 4G debug_run ``` Non-human genome (e.g. mouse, zebrafish, _C. elegans_). By default svaba assumes a human reference: mate-region lookups skip chromosomes past chrY (ChrID > 23), and insert-size learning samples only the primary assembly (chr1–chrY). `--non-human` removes these gates so every contig in the reference is treated equally: ```bash svaba run -t mouse.bam -G mm39.fa -a mouse_run --non-human -p 16 ``` The flag sets `maxMateChrID = -1` internally. You can also fine-tune individually: `--max-mate-chr 19` (mouse has 19 autosomes + X + Y = ChrIDs 0–20 in a typical reference), `--min-mate-mapq 10` (require MAPQ ≥ 10 on the primary read before chasing its mate). Snapshot where a long-running job currently is: ```bash tail my_run.log ``` ## Further reading `CLAUDE.md` at the repo root is the crash-safety-net doc — conventions, file landmarks, build-system quirks (the `-O2` hardcoding in submodules), the somatic LOD model, the postprocess pipeline details, performance notes, and open investigations. Always update `CLAUDE.md` when you change something non-obvious. `scripts/svaba_local_function.sh` is a sourceable bash helper library with `svaba_*` utilities for grepping contigs, following a bp_id through the output set, opening locations in IGV, etc. Source it from your shell rc file to use. ## Issues and contact Please file bug reports, feature requests, and questions on the GitHub issues tracker: https://github.com/walaj/svaba/issues. ## Attributions SvABA is developed by Jeremiah Wala in the Rameen Berkoukhim lab at Dana-Farber Cancer Institute, in collaboration with the Cancer Genome Analysis team at the Broad Institute. Particular thanks to Cheng-Zhong Zhang (HMS DBMI) and Marcin Imielinski (Weill Cornell / NYGC). Additional thanks to Jared Simpson for SGA, Heng Li for htslib and BWA, and the SeqLib contributors. The SvABA 2.0 release and its documentation were built with the assistance of OpenAI Codex and Anthropic Claude, with extensive human-in-the-loop review, testing, and decision-making throughout. [seqlib]: https://github.com/walaj/SeqLib ================================================ FILE: docs/README.md ================================================ # SvABA BPS Viewer This folder contains a standalone static viewer for SvABA `bps.txt` and `bps.txt.gz` files. ## Files - `index.html`: app shell - `styles.css`: layout and visual styling - `app.js`: parser, filters, table, and detail pane - `svaba/am.bps.txt.gz`: bundled example file already present in this repo ## Usage You can open `index.html` directly and use the file picker for local files. If you want the bundled example button to work, serve this folder over HTTP: ```bash cd /Users/jeremiahwala/git/svaba/viewer python3 -m http.server 8000 ``` Then open [http://localhost:8000](http://localhost:8000). ## Current filter set The UI supports: - free-text search across locus, contig, alleles, confidence, DBSNP, and sample fields - numeric filtering for `somlod`, `maxlod`, `qual`, split support, ALT support, and discordant support - button filters for derived event type, raw evidence (`type`), confidence (`conf`), and somatic state - export of the currently filtered rows as TSV ## Notes - The raw header contains duplicate `alt` names. In the viewer these are treated as `alt_allele` and `alt_count`. - The raw `type` column is evidence (`INDEL`, `ASDIS`, `DSCRD`, `ASSMB`). The viewer also derives a separate event class to make filtering more usable. ================================================ FILE: docs/alignments_viewer.html ================================================ svaba alignments viewer

svaba alignments viewer

bps (not set) aln (not set)
bps matches click a row to load its contig
No bps results yet. Set the bps file and enter a breakpoint (chr:pos or chr:pos1-pos2) or a contig name.
alignments support:
No alignments loaded. Pick a contig above or drop/paste a block.
================================================ FILE: docs/app.js ================================================ (function () { "use strict"; const BASE_COLUMNS = [ "chr1", "pos1", "strand1", "chr2", "pos2", "strand2", "ref", "alt_allele", "span", "split", "alt_count", "cov", "cigar", "cigar_near", "dmq1", "dmq2", "dcn", "dct", "mapq1", "mapq2", "nm1", "nm2", "as1", "as2", "sub1", "sub2", "homol", "insert", "repeat", "contig_and_region", "naln", "conf", "evidence", "qual", "secondary", "somatic", "somlod", "maxlod", "dbsnp" ]; const NUMERIC_FIELDS = new Set([ "pos1", "pos2", "span", "split", "alt_count", "cov", "cigar", "cigar_near", "dmq1", "dmq2", "dcn", "dct", "mapq1", "mapq2", "nm1", "nm2", "as1", "as2", "sub1", "sub2", "naln", "qual", "secondary", "somlod", "maxlod" ]); const SORT_DEFAULTS = { key: "maxlod", direction: "desc" }; const state = { fileName: "", originalHeader: [], sampleHeaders: [], rows: [], filteredRows: [], selectedId: null, page: 1, pageSize: 100, sort: { ...SORT_DEFAULTS }, filters: { search: "", somlodMin: null, somlodMax: null, maxlodMin: null, maxlodMax: null, qualMin: null, splitMin: null, altMin: null, discordantMin: null, dbsnp: "all", eventTypes: new Set(), evidence: new Set(), confidence: new Set(), somatic: new Set() } }; const els = { fileInput: document.getElementById("file-input"), loadExample: document.getElementById("load-example"), exportButton: document.getElementById("export-button"), resetFilters: document.getElementById("reset-filters"), searchInput: document.getElementById("search-input"), somlodMin: document.getElementById("somlod-min"), somlodMax: document.getElementById("somlod-max"), maxlodMin: document.getElementById("maxlod-min"), maxlodMax: document.getElementById("maxlod-max"), qualMin: document.getElementById("qual-min"), splitMin: document.getElementById("split-min"), altMin: document.getElementById("alt-min"), discordantMin: document.getElementById("discordant-min"), dbsnpFilter: document.getElementById("dbsnp-filter"), pageSize: document.getElementById("page-size"), tableBody: document.getElementById("table-body"), detailContent: document.getElementById("detail-content"), statusText: document.getElementById("status-text"), resultsTitle: document.getElementById("results-title"), resultsSummary: document.getElementById("results-summary"), prevPage: document.getElementById("prev-page"), nextPage: document.getElementById("next-page"), pageSummary: document.getElementById("page-summary"), statTotal: document.getElementById("stat-total"), statShown: document.getElementById("stat-shown"), statPass: document.getElementById("stat-pass"), statTopEvidence: document.getElementById("stat-top-evidence"), eventTypeChips: document.getElementById("event-type-chips"), evidenceChips: document.getElementById("evidence-chips"), confidenceChips: document.getElementById("confidence-chips"), somaticChips: document.getElementById("somatic-chips") }; document.querySelectorAll(".sort-button").forEach((button) => { button.addEventListener("click", () => { const key = button.dataset.sort; if (state.sort.key === key) { state.sort.direction = state.sort.direction === "asc" ? "desc" : "asc"; } else { state.sort.key = key; state.sort.direction = key === "locus" || key === "eventType" || key === "conf" || key === "evidence" || key === "contig_and_region" ? "asc" : "desc"; } render(); }); }); document.querySelectorAll(".preset-button").forEach((button) => { button.addEventListener("click", () => { const target = document.getElementById(button.dataset.target); target.value = button.dataset.value; syncFilterState(); render(); }); }); els.fileInput.addEventListener("change", async (event) => { const file = event.target.files && event.target.files[0]; if (!file) { return; } await loadFromBlob(file, file.name); }); els.loadExample.addEventListener("click", async () => { await loadBundledExample(); }); els.exportButton.addEventListener("click", () => { exportFilteredRows(); }); els.resetFilters.addEventListener("click", () => { resetFilters(); render(); }); els.prevPage.addEventListener("click", () => { if (state.page > 1) { state.page -= 1; renderTable(); } }); els.nextPage.addEventListener("click", () => { const totalPages = Math.max(1, Math.ceil(state.filteredRows.length / state.pageSize)); if (state.page < totalPages) { state.page += 1; renderTable(); } }); [ "searchInput", "somlodMin", "somlodMax", "maxlodMin", "maxlodMax", "qualMin", "splitMin", "altMin", "discordantMin", "dbsnpFilter", "pageSize" ].forEach((key) => { els[key].addEventListener("input", () => { syncFilterState(); render(); }); els[key].addEventListener("change", () => { syncFilterState(); render(); }); }); resetFilters(); render(); function resetFilters() { state.pageSize = 100; state.filters.search = ""; state.filters.somlodMin = null; state.filters.somlodMax = null; state.filters.maxlodMin = null; state.filters.maxlodMax = null; state.filters.qualMin = null; state.filters.splitMin = null; state.filters.altMin = null; state.filters.discordantMin = null; state.filters.dbsnp = "all"; state.filters.eventTypes = new Set(); state.filters.evidence = new Set(); state.filters.confidence = new Set(); state.filters.somatic = new Set(); state.page = 1; els.searchInput.value = ""; els.somlodMin.value = ""; els.somlodMax.value = ""; els.maxlodMin.value = ""; els.maxlodMax.value = ""; els.qualMin.value = ""; els.splitMin.value = ""; els.altMin.value = ""; els.discordantMin.value = ""; els.dbsnpFilter.value = "all"; els.pageSize.value = String(state.pageSize); } function syncFilterState() { state.filters.search = els.searchInput.value.trim().toLowerCase(); state.filters.somlodMin = parseNullableNumber(els.somlodMin.value); state.filters.somlodMax = parseNullableNumber(els.somlodMax.value); state.filters.maxlodMin = parseNullableNumber(els.maxlodMin.value); state.filters.maxlodMax = parseNullableNumber(els.maxlodMax.value); state.filters.qualMin = parseNullableNumber(els.qualMin.value); state.filters.splitMin = parseNullableNumber(els.splitMin.value); state.filters.altMin = parseNullableNumber(els.altMin.value); state.filters.discordantMin = parseNullableNumber(els.discordantMin.value); state.filters.dbsnp = els.dbsnpFilter.value; state.pageSize = Number(els.pageSize.value) || 100; state.page = 1; } async function loadBundledExample() { setStatus("Loading bundled example from ./svaba/am.bps.txt.gz ..."); try { const response = await fetch("./svaba/am.bps.txt.gz"); if (!response.ok) { throw new Error("Could not fetch ./svaba/am.bps.txt.gz"); } const text = await readResponseText(response, "am.bps.txt.gz"); ingestText(text, "svaba/am.bps.txt.gz"); } catch (error) { setStatus( "Bundled example load failed. If you opened index.html directly as a file, use the file picker or serve viewer/ over HTTP." ); console.error(error); } } async function loadFromBlob(blob, fileName) { setStatus("Reading " + fileName + " ..."); try { const text = await readBlobText(blob, fileName); ingestText(text, fileName); } catch (error) { setStatus("Could not read " + fileName + ". " + error.message); console.error(error); } finally { els.fileInput.value = ""; } } async function readBlobText(blob, fileName) { if (fileName.endsWith(".gz")) { if (typeof DecompressionStream === "undefined") { throw new Error("This browser does not support gzip decompression. Use a plain .txt file here."); } const stream = blob.stream().pipeThrough(new DecompressionStream("gzip")); return await new Response(stream).text(); } return await blob.text(); } async function readResponseText(response, fileName) { if (fileName.endsWith(".gz")) { if (typeof DecompressionStream === "undefined") { throw new Error("This browser does not support gzip decompression."); } const stream = response.body.pipeThrough(new DecompressionStream("gzip")); return await new Response(stream).text(); } return await response.text(); } function ingestText(text, fileName) { const lines = text.replace(/\r\n?/g, "\n").split("\n").filter((line) => line.length > 0); if (lines.length < 2) { throw new Error("Expected a header and at least one row."); } const originalHeader = lines[0].split("\t"); if (originalHeader.length < BASE_COLUMNS.length) { throw new Error("Unexpected header width: " + originalHeader.length); } const sampleHeaders = originalHeader.slice(BASE_COLUMNS.length); const rows = []; for (let index = 1; index < lines.length; index += 1) { const line = lines[index]; const cells = line.split("\t"); if (cells.length !== originalHeader.length) { continue; } rows.push(makeRow(index, cells, sampleHeaders)); } state.fileName = fileName; state.originalHeader = originalHeader; state.sampleHeaders = sampleHeaders; state.rows = rows; resetFilters(); state.selectedId = rows.length ? rows[0].id : null; state.page = 1; state.sort = { ...SORT_DEFAULTS }; render(); setStatus("Loaded " + rows.length.toLocaleString() + " calls from " + fileName + "."); } function makeRow(index, cells, sampleHeaders) { const row = { id: index, originalCells: cells }; BASE_COLUMNS.forEach((field, fieldIndex) => { const value = cells[fieldIndex]; row[field] = NUMERIC_FIELDS.has(field) ? parseMaybeNumber(value) : value; }); row.chr1 = stripLeadingHash(row.chr1); row.locus = row.chr1 + ":" + row.pos1 + " -> " + row.chr2 + ":" + row.pos2; row.sampleInfo = sampleHeaders.map((header, offset) => parseSampleField(header, cells[BASE_COLUMNS.length + offset], row.evidence)); row.eventType = deriveEventType(row); row.spanAbs = row.span === null || row.span === -1 ? Infinity : Math.abs(row.span); row.supportPeak = Math.max( zeroIfNull(row.split), zeroIfNull(row.alt_count), zeroIfNull(row.cigar), zeroIfNull(row.dcn), zeroIfNull(row.dct) ); row.hasDbsnp = Boolean(row.dbsnp && row.dbsnp !== "x"); row.searchText = [ row.locus, row.eventType, row.evidence, row.conf, row.ref, row.alt_allele, row.contig_and_region, row.insert, row.repeat, row.homol, row.dbsnp, row.somatic ] .concat(row.sampleInfo.map((sample) => sample.label + " " + sample.raw)) .join(" ") .toLowerCase(); return row; } function deriveEventType(row) { if (row.evidence === "INDEL") { const refLength = (row.ref || "").length; const altLength = (row.alt_allele || "").length; if (altLength > refLength) { return "Insertion"; } if (altLength < refLength) { return "Deletion"; } return "Substitution"; } if (row.chr1 !== row.chr2) { return "Translocation"; } if (row.strand1 === row.strand2) { return "Inversion"; } if (row.strand1 === "-" && row.strand2 === "+") { return "Duplication-like"; } if (row.strand1 === "+" && row.strand2 === "-") { return "Deletion-like"; } return "Rearrangement"; } function parseSampleField(header, raw, evidence) { const label = header.split("_")[0]; const parts = raw.split(":"); const isIndel = evidence === "INDEL"; return { label, header, raw, genotype: parts[0] || "", altSupport: parseMaybeNumber(parts[1]), depth: parseMaybeNumber(parts[2]), splitSupport: parseMaybeNumber(parts[3]), pairedSupport: parseMaybeNumber(parts[4]), supportLabel: isIndel ? "cigar" : "discordant", gq: parseMaybeNumber(parts[5]), pl: parts[6] || "", lod: parseMaybeNumber(parts[7]), lodNormal: parseMaybeNumber(parts[8]) }; } function rebuildChipGroups() { renderChipList(els.eventTypeChips, countBy(state.rows, "eventType"), "eventTypes"); renderChipList(els.evidenceChips, countBy(state.rows, "evidence"), "evidence"); renderChipList(els.confidenceChips, countBy(state.rows, "conf"), "confidence"); renderChipList(els.somaticChips, countBy(state.rows, "somatic"), "somatic"); } function renderChipList(container, counts, filterKey) { const entries = Array.from(counts.entries()).sort((left, right) => { if (right[1] !== left[1]) { return right[1] - left[1]; } return left[0].localeCompare(right[0]); }); if (!entries.length) { container.innerHTML = 'Load a file to populate this filter.'; return; } container.innerHTML = ""; entries.forEach(([value, count]) => { const button = document.createElement("button"); button.type = "button"; button.className = "chip"; button.textContent = value + " (" + count.toLocaleString() + ")"; if (state.filters[filterKey].has(value)) { button.classList.add("active"); } button.addEventListener("click", () => { toggleSetValue(state.filters[filterKey], value); state.page = 1; render(); }); container.appendChild(button); }); } function render() { state.filteredRows = applyFilters(state.rows); sortRows(state.filteredRows); reconcileSelection(); updateSummary(); renderTable(); renderDetail(); rebuildChipGroups(); } function applyFilters(rows) { return rows.filter((row) => { if (state.filters.search && !row.searchText.includes(state.filters.search)) { return false; } if (!passesNumericRange(row.somlod, state.filters.somlodMin, state.filters.somlodMax)) { return false; } if (!passesNumericRange(row.maxlod, state.filters.maxlodMin, state.filters.maxlodMax)) { return false; } if (!passesNumericMinimum(row.qual, state.filters.qualMin)) { return false; } if (!passesNumericMinimum(row.split, state.filters.splitMin)) { return false; } if (!passesNumericMinimum(row.alt_count, state.filters.altMin)) { return false; } if (!passesNumericMinimum(Math.max(zeroIfNull(row.dcn), zeroIfNull(row.dct)), state.filters.discordantMin)) { return false; } if (state.filters.dbsnp === "present" && !row.hasDbsnp) { return false; } if (state.filters.dbsnp === "absent" && row.hasDbsnp) { return false; } if (state.filters.eventTypes.size && !state.filters.eventTypes.has(row.eventType)) { return false; } if (state.filters.evidence.size && !state.filters.evidence.has(row.evidence)) { return false; } if (state.filters.confidence.size && !state.filters.confidence.has(row.conf)) { return false; } if (state.filters.somatic.size && !state.filters.somatic.has(row.somatic)) { return false; } return true; }); } function sortRows(rows) { const direction = state.sort.direction === "asc" ? 1 : -1; const key = state.sort.key; rows.sort((left, right) => { const leftValue = sortableValue(left, key); const rightValue = sortableValue(right, key); if (typeof leftValue === "number" || typeof rightValue === "number") { const leftNumber = normalizeSortNumber(leftValue); const rightNumber = normalizeSortNumber(rightValue); if (leftNumber !== rightNumber) { return (leftNumber - rightNumber) * direction; } } else { const compare = String(leftValue).localeCompare(String(rightValue)); if (compare !== 0) { return compare * direction; } } return left.id - right.id; }); } function sortableValue(row, key) { if (key === "locus") { return row.locus; } if (key === "spanAbs") { return row.spanAbs; } return row[key]; } function updateSummary() { const totalRows = state.rows.length; const shownRows = state.filteredRows.length; const passCount = state.filteredRows.filter((row) => row.conf === "PASS").length; const topEvidence = topCountValue(state.filteredRows, "evidence"); const selected = findSelectedRow(); els.statTotal.textContent = totalRows.toLocaleString(); els.statShown.textContent = shownRows.toLocaleString(); els.statPass.textContent = passCount.toLocaleString(); els.statTopEvidence.textContent = topEvidence || "-"; els.resultsTitle.textContent = totalRows ? state.fileName + " (" + totalRows.toLocaleString() + " calls)" : "No variants loaded"; els.resultsSummary.textContent = totalRows ? shownRows.toLocaleString() + " rows after filters. Sorted by " + state.sort.key + " (" + state.sort.direction + ")." : "Load a file to start browsing calls."; els.exportButton.disabled = shownRows === 0; if (selected && !state.filteredRows.length) { state.selectedId = null; } } function renderTable() { const totalPages = Math.max(1, Math.ceil(state.filteredRows.length / state.pageSize)); if (state.page > totalPages) { state.page = totalPages; } const start = (state.page - 1) * state.pageSize; const end = start + state.pageSize; const rows = state.filteredRows.slice(start, end); if (!rows.length) { els.tableBody.innerHTML = 'No rows match the current filter set.'; } else { const html = rows .map((row) => { const isSelected = row.id === state.selectedId ? " selected" : ""; const spanLabel = row.span === -1 ? "interchrom" : formatNumber(row.span); const support = [ "split " + formatNumber(row.split), "alt " + formatNumber(row.alt_count), "disc " + formatNumber(Math.max(zeroIfNull(row.dcn), zeroIfNull(row.dct))) ].join(" / "); return ( '' + '
' + escapeHtml(row.locus) + '' + escapeHtml(row.ref + " -> " + row.alt_allele) + "
" + "" + escapeHtml(row.eventType) + "" + "" + escapeHtml(row.evidence) + "" + "" + escapeHtml(row.conf) + "" + "" + formatNumber(row.qual) + "" + "" + formatNumber(row.somlod) + "" + "" + formatNumber(row.maxlod) + "" + "" + escapeHtml(support) + "" + "" + escapeHtml(spanLabel) + "" + "" + escapeHtml(row.contig_and_region) + "" + "" ); }) .join(""); els.tableBody.innerHTML = html; els.tableBody.querySelectorAll("tr[data-row-id]").forEach((rowElement) => { rowElement.addEventListener("click", () => { state.selectedId = Number(rowElement.dataset.rowId); renderTable(); renderDetail(); }); }); } els.prevPage.disabled = state.page <= 1 || !state.filteredRows.length; els.nextPage.disabled = state.page >= totalPages || !state.filteredRows.length; els.pageSummary.textContent = "Page " + state.page + " of " + totalPages; } function renderDetail() { const row = findSelectedRow(); if (!row) { els.detailContent.className = "detail-content detail-empty"; els.detailContent.textContent = "Select a row to inspect the raw breakpoint call, support metrics, and sample-level values."; return; } const confidenceClass = row.conf === "PASS" ? "pass" : row.conf === "LOWLOD" || row.conf === "LOWMAPQ" ? "warn" : "low"; const sampleCards = row.sampleInfo .map((sample) => { return ( '
' + "" + escapeHtml(sample.label) + "" + '
' + escapeHtml(sample.header) + "
" + "
" + detailPair("GT", sample.genotype) + detailPair("AD", formatNumber(sample.altSupport)) + detailPair("DP", formatNumber(sample.depth)) + detailPair("SR", formatNumber(sample.splitSupport)) + detailPair(sample.supportLabel, formatNumber(sample.pairedSupport)) + detailPair("GQ", formatNumber(sample.gq)) + detailPair("LO", formatNumber(sample.lod)) + detailPair("LO_n", formatNumber(sample.lodNormal)) + "
" + "
" ); }) .join(""); els.detailContent.className = "detail-content"; els.detailContent.innerHTML = '
' + "

" + escapeHtml(row.locus) + "

" + '

' + escapeHtml(row.eventType + " / " + row.evidence + " / " + row.conf) + "

" + '
' + '' + escapeHtml("conf " + row.conf) + "" + '' + escapeHtml("somatic " + row.somatic) + "" + '' + escapeHtml("somlod " + formatNumber(row.somlod)) + "" + '' + escapeHtml("maxlod " + formatNumber(row.maxlod)) + "" + "
" + "
" + '
' + detailCard("Reference", row.ref) + detailCard("Alternate", row.alt_allele) + detailCard("Breakends", row.chr1 + ":" + row.pos1 + " " + row.strand1 + " -> " + row.chr2 + ":" + row.pos2 + " " + row.strand2) + detailCard("Support", "split " + formatNumber(row.split) + ", alt " + formatNumber(row.alt_count) + ", cigar " + formatNumber(row.cigar) + ", dcn " + formatNumber(row.dcn) + ", dct " + formatNumber(row.dct)) + detailCard("Mapping and alignment", "mapq " + formatNumber(row.mapq1) + "/" + formatNumber(row.mapq2) + ", AS " + formatNumber(row.as1) + "/" + formatNumber(row.as2) + ", NM " + formatNumber(row.nm1) + "/" + formatNumber(row.nm2)) + detailCard("Context", "homol " + presentOrX(row.homol) + ", insert " + presentOrX(row.insert) + ", repeat " + presentOrX(row.repeat)) + detailCard("Contig", row.contig_and_region) + detailCard("Flags", "qual " + formatNumber(row.qual) + ", secondary " + formatNumber(row.secondary) + ", span " + (row.span === -1 ? "interchrom" : formatNumber(row.span)) + ", dbsnp " + presentOrX(row.dbsnp)) + "
" + '

Per-sample fields

' + '
' + sampleCards + "
" + '

Raw row

' + '
' +
      escapeHtml(state.originalHeader.join("\t")) +
      "\n" +
      escapeHtml(row.originalCells.join("\t")) +
      "
"; } function detailCard(label, value) { return "
" + escapeHtml(label) + "
" + escapeHtml(value) + "
"; } function detailPair(label, value) { return "
" + escapeHtml(label) + "
" + escapeHtml(value) + "
"; } function reconcileSelection() { const selectedStillVisible = state.filteredRows.some((row) => row.id === state.selectedId); if (!selectedStillVisible) { state.selectedId = state.filteredRows.length ? state.filteredRows[0].id : null; } } function findSelectedRow() { return state.filteredRows.find((row) => row.id === state.selectedId) || state.rows.find((row) => row.id === state.selectedId) || null; } function exportFilteredRows() { if (!state.filteredRows.length) { return; } const lines = [state.originalHeader.join("\t")].concat(state.filteredRows.map((row) => row.originalCells.join("\t"))); const blob = new Blob([lines.join("\n")], { type: "text/tab-separated-values;charset=utf-8" }); const url = URL.createObjectURL(blob); const anchor = document.createElement("a"); const baseName = state.fileName.replace(/\.gz$/i, "").replace(/\.txt$/i, ""); anchor.href = url; anchor.download = baseName + ".filtered.tsv"; anchor.click(); URL.revokeObjectURL(url); } function countBy(rows, key) { const map = new Map(); rows.forEach((row) => { const value = row[key]; map.set(value, (map.get(value) || 0) + 1); }); return map; } function topCountValue(rows, key) { const counts = countBy(rows, key); let bestValue = ""; let bestCount = -1; counts.forEach((count, value) => { if (count > bestCount) { bestCount = count; bestValue = value; } }); return bestValue; } function toggleSetValue(set, value) { if (set.has(value)) { set.delete(value); } else { set.add(value); } } function setStatus(message) { els.statusText.textContent = message; } function passesNumericRange(value, min, max) { if (min === null && max === null) { return true; } if (value === null || !Number.isFinite(value)) { return false; } if (min !== null && value < min) { return false; } if (max !== null && value > max) { return false; } return true; } function passesNumericMinimum(value, min) { if (min === null) { return true; } if (value === null || !Number.isFinite(value)) { return false; } return value >= min; } function parseNullableNumber(raw) { if (raw === "" || raw === null || raw === undefined) { return null; } const value = Number(raw); return Number.isFinite(value) ? value : null; } function parseMaybeNumber(raw) { if (raw === "" || raw === "NA" || raw === "x" || raw === "NOTSET") { return null; } const value = Number(raw); return Number.isFinite(value) ? value : null; } function formatNumber(value) { if (value === null || value === undefined || value === Infinity) { return "-"; } if (!Number.isFinite(value)) { return String(value); } return Math.abs(value) >= 100 || Number.isInteger(value) ? value.toLocaleString() : value.toFixed(3).replace(/\.?0+$/, ""); } function presentOrX(value) { return value && value !== "x" ? value : "x"; } function normalizeSortNumber(value) { if (value === Infinity) { return Number.MAX_SAFE_INTEGER; } if (value === null || value === undefined || Number.isNaN(value)) { return -Number.MAX_SAFE_INTEGER; } return Number(value); } function zeroIfNull(value) { return value === null || value === undefined ? 0 : value; } function stripLeadingHash(value) { return typeof value === "string" ? value.replace(/^#/, "") : value; } function escapeHtml(value) { return String(value) .replace(/&/g, "&") .replace(//g, ">") .replace(/"/g, """) .replace(/'/g, "'"); } })(); ================================================ FILE: docs/bps_explorer.html ================================================ SvABA BPS Explorer

SvABA BPS Explorer

No file loaded
Drop or click — .bps.txt / .bps.txt.gz
Drop or click — .bed / .bed.gz / .gtf / .gtf.gz (optional, gene annotation)
================================================ FILE: docs/bps_viewer.html ================================================ SvABA BPS Viewer

SvABA raw call explorer

Inspect bps.txt and bps.txt.gz calls in the browser.

Load a breakpoint table, filter it by LOD, confidence, event type, and evidence, then drill into per-sample support without leaving the file.

No file loaded yet. Use the file picker or the bundled example in viewer/svaba/am.bps.txt.gz.

Rows 0
Shown 0
PASS 0
Evidence -

Filters

Slice by score, event class, and support

somlod presets
maxlod presets
Variant type Derived event class
Evidence Raw type field
Confidence Raw conf field
Somatic state Raw somatic field

Calls

No variants loaded

Load a file to start browsing calls.
No data yet. Load a bps.txt or bps.txt.gz file.
================================================ FILE: docs/comparison.html ================================================ SvABA BPS Comparison

SvABA BPS Comparison

📄
Drop or click to load
📄
Drop or click to load
0
-50
0
-50
10
↑
Load two bps files (any format), then click Compare
================================================ FILE: docs/index.html ================================================ SvABA Viewers

SvABA Viewers

Browser-based tools for exploring svaba output. All run client-side — no server required.
BPS Explorer bps.txt.gz
Dark, compact table-first viewer for bps.txt.gz files. Sortable columns, numeric filters (somlod, maxlod, qual, span range), genomic region search, chip filters for type/evidence/conf/somatic, summary histograms, per-sample detail panel. Handles both legacy 41-col and SvABA2.0 51-col formats. Drag-and-drop or file picker.
R2C Explorer r2c.txt.gz
Browser-side re-plotter for the structured r2c TSV emitted by svaba run --dump-reads (or the PASS / PASS-somatic subsets from svaba_postprocess.sh). Load the file, type/search for a contig name (autocomplete from the file's own list of cnames), and get the alignments-style plot generated fresh from the TSV: ruler, contig sequence, per-fragment summaries, breakpoint rows, and per-read gap-expanded CIGAR renderings with support-kind color-coding. Prev/Next/Random navigation plus imperfect-only and support-kind filters. Successor to the old alignments.txt.gz ASCII dump.
Alignments Viewer (legacy) alignments.txt.gz
Original ASCII-plot viewer for *.alignments.txt.gz from svaba runs before the r2c migration. New runs no longer produce alignments.txt.gz — prefer the R2C Explorer above. Kept for rendering historical outputs.
Learn Explorer learn.tsv.gz
Interactive viewer for BAM learning data — per-read-group insert-size distributions emitted by svaba run as ${ID}.*.learn.tsv.gz. Overlaid + faceted histograms, sortable summary table (n, mean, median, SD, percentiles), RG chip filters, click-to-solo. Drag-and-drop the gzipped TSV to explore.
Runtime Explorer runtime.txt
Explore per-region timing from ${ID}.runtime.txt. Sort regions by runtime, contigs, or breakpoint count; filter by chromosome, region, runtime threshold, or contig / discordant-read minimums. Prominent runtime-distribution histogram plus auxiliary contig / discordant histograms with independent log10 toggles. Click a region's locus to jump IGV to it (requires IGV on port 60151). Standard-chr filter drops decoy / alt / random / HLA contigs.
Run Comparison 2× bps.txt.gz
Side-by-side comparison of two svaba runs (e.g. before/after a parameter change, or tumor vs matched). Load two bps.txt.gz files, match calls by locus, and see which are shared vs unique to each run. Charts for agreement rates and score distributions.
BPS Viewer (original) legacy
The original light-theme BPS viewer. Filter by LOD, confidence, event type, and evidence. Per-sample support drill-down. Requires app.js and styles.css alongside it.
Files: bps_viewer.html + app.js + styles.css
SvABA — SV and indel detection by assembly — github.com/walaj/svaba
================================================ FILE: docs/learn_explorer.html ================================================ SvABA Learn Explorer

SvABA Learn Explorer

No file loaded
Drop or click — *.learn.tsv.gz (columns: bam, rg, isize)
Insert size distribution (all selected RGs overlaid)
SvABA — Learn Explorer — ← all viewers — github.com/walaj/svaba
================================================ FILE: docs/r2c_explorer.html ================================================ SvABA R2C Explorer

SvABA R2C Explorer

No file loaded
Drop or click — .r2c.txt.gz / .r2c.pass.txt.gz / .r2c.pass.somatic.txt.gz
================================================ FILE: docs/runtime_explorer.html ================================================ SvABA Runtime Explorer

SvABA Runtime Explorer

No file loaded — drop a runtime log to begin
Drop or click — SvABA runtime log (TSV, 17 cols: chromosome start end … runtime_seconds …)
================================================ FILE: docs/styles.css ================================================ :root { --bg: #f3ece1; --bg-deep: #d7e2df; --panel: rgba(255, 251, 246, 0.85); --panel-strong: rgba(255, 253, 250, 0.94); --text: #162430; --muted: #5d6a72; --line: rgba(22, 36, 48, 0.12); --shadow: 0 24px 60px rgba(41, 54, 61, 0.14); --accent: #c65a31; --accent-ink: #7c2d12; --accent-soft: rgba(198, 90, 49, 0.12); --teal: #235b60; --teal-soft: rgba(35, 91, 96, 0.12); --green-soft: rgba(74, 122, 76, 0.14); --danger-soft: rgba(164, 65, 65, 0.12); --mono: "SFMono-Regular", "Menlo", "Monaco", "Liberation Mono", monospace; --sans: "Avenir Next", "Segoe UI", "Helvetica Neue", sans-serif; --serif: "Iowan Old Style", "Palatino Linotype", "Book Antiqua", Georgia, serif; } * { box-sizing: border-box; } html { font-size: 16px; } body { margin: 0; min-height: 100vh; color: var(--text); font-family: var(--sans); background: radial-gradient(circle at top left, rgba(217, 153, 96, 0.25), transparent 32%), linear-gradient(160deg, var(--bg) 0%, #f8f4ee 45%, var(--bg-deep) 100%); } code, pre, th, td, .stat-value, .chip, .status, .field input, .field select, .detail-grid dd { font-family: var(--mono); } .background-glow { position: fixed; inset: auto; width: 32rem; height: 32rem; border-radius: 50%; filter: blur(48px); opacity: 0.45; pointer-events: none; z-index: 0; } .background-glow-a { top: -10rem; right: -8rem; background: rgba(198, 90, 49, 0.2); } .background-glow-b { bottom: -12rem; left: -10rem; background: rgba(35, 91, 96, 0.18); } .page { position: relative; z-index: 1; width: min(1500px, calc(100vw - 2rem)); margin: 0 auto; padding: 1rem 0 2rem; } .panel { background: var(--panel); border: 1px solid rgba(255, 255, 255, 0.45); box-shadow: var(--shadow); border-radius: 1.5rem; backdrop-filter: blur(14px); } .hero, .filters, .table-panel, .detail-panel { padding: 1.4rem; } .hero { display: grid; gap: 1.2rem; grid-template-columns: minmax(0, 1.5fr) minmax(280px, 0.8fr); align-items: start; } .hero-copy h1, .panel-heading h2 { margin: 0; font-family: var(--serif); font-weight: 700; line-height: 1.04; } .hero-copy h1 { font-size: clamp(2rem, 4vw, 3.6rem); max-width: 12ch; } .hero-text { margin: 1rem 0 0; max-width: 62ch; color: var(--muted); font-size: 1.02rem; line-height: 1.6; } .eyebrow { margin: 0 0 0.45rem; text-transform: uppercase; letter-spacing: 0.18em; font-size: 0.72rem; font-weight: 700; color: var(--teal); } .hero-actions, .pagination, .panel-heading, .chip-heading, .preset-group, .table-meta { display: flex; align-items: center; gap: 0.75rem; } .hero-actions { margin-top: 1.2rem; flex-wrap: wrap; } .button { appearance: none; border: 0; border-radius: 999px; padding: 0.82rem 1.1rem; font: inherit; cursor: pointer; transition: transform 140ms ease, box-shadow 140ms ease, background 140ms ease; } .button:hover:not(:disabled), .button:focus-visible:not(:disabled) { transform: translateY(-1px); } .button:disabled { cursor: not-allowed; opacity: 0.45; } .button-primary { background: linear-gradient(135deg, #12222f, #264053); color: #fdf7f1; box-shadow: 0 16px 32px rgba(18, 34, 47, 0.22); } .button-secondary { background: rgba(255, 255, 255, 0.8); color: var(--text); border: 1px solid var(--line); } .button-ghost { background: transparent; color: var(--text); border: 1px solid var(--line); } .button-small { padding: 0.55rem 0.85rem; } .file-picker input { display: none; } .status { margin: 1rem 0 0; color: var(--muted); line-height: 1.5; } .hero-stats { display: grid; gap: 0.75rem; } .stat-card { padding: 1rem 1.1rem; border-radius: 1.15rem; background: linear-gradient(160deg, var(--panel-strong), rgba(255, 248, 240, 0.8)); border: 1px solid rgba(255, 255, 255, 0.55); } .stat-label { display: block; font-size: 0.8rem; color: var(--muted); } .stat-value { display: block; margin-top: 0.45rem; font-size: clamp(1.4rem, 3vw, 2.2rem); } .filters { margin-top: 1rem; } .filter-grid { display: grid; grid-template-columns: repeat(11, minmax(0, 1fr)); gap: 0.9rem; margin-top: 1rem; } .field { display: flex; flex-direction: column; gap: 0.45rem; min-width: 0; } .field:first-child { grid-column: span 3; } .field-label { font-size: 0.78rem; font-weight: 700; color: var(--muted); } .field input, .field select { width: 100%; border: 1px solid rgba(22, 36, 48, 0.14); background: rgba(255, 255, 255, 0.8); border-radius: 0.95rem; padding: 0.82rem 0.92rem; color: var(--text); } .field input:focus-visible, .field select:focus-visible { outline: 2px solid rgba(198, 90, 49, 0.24); outline-offset: 1px; } .preset-row { display: flex; flex-wrap: wrap; gap: 1rem; margin-top: 1rem; } .preset-group { flex-wrap: wrap; } .preset-label { font-size: 0.78rem; color: var(--muted); font-weight: 700; } .chip-section { display: grid; grid-template-columns: repeat(2, minmax(0, 1fr)); gap: 1rem; margin-top: 1.2rem; } .chip-block { padding: 1rem; border-radius: 1.2rem; background: rgba(255, 255, 255, 0.58); border: 1px solid rgba(255, 255, 255, 0.45); } .chip-heading { justify-content: space-between; margin-bottom: 0.8rem; } .chip-heading .eyebrow { margin: 0; } .chip-list { display: flex; flex-wrap: wrap; gap: 0.55rem; } .chip { padding: 0.55rem 0.75rem; border-radius: 999px; border: 1px solid var(--line); background: rgba(255, 255, 255, 0.78); color: var(--text); font-size: 0.82rem; cursor: pointer; transition: background 120ms ease, border-color 120ms ease, transform 120ms ease; } .chip:hover, .chip:focus-visible { transform: translateY(-1px); } .chip.active { background: linear-gradient(135deg, rgba(198, 90, 49, 0.18), rgba(35, 91, 96, 0.12)); border-color: rgba(198, 90, 49, 0.4); color: var(--accent-ink); } .workspace { display: grid; grid-template-columns: minmax(0, 1.4fr) minmax(320px, 0.8fr); gap: 1rem; margin-top: 1rem; } .panel-heading { justify-content: space-between; align-items: end; } .panel-heading h2 { font-size: 1.55rem; } .table-meta, .detail-content, .detail-grid dd, .detail-grid dt, .table-shell td, .table-shell th { font-size: 0.9rem; } .table-shell { margin-top: 1rem; overflow: auto; max-height: 72vh; border-radius: 1rem; border: 1px solid var(--line); background: rgba(255, 255, 255, 0.55); } table { width: 100%; border-collapse: collapse; } thead th { position: sticky; top: 0; background: rgba(246, 241, 234, 0.94); z-index: 1; } th, td { padding: 0.82rem 0.88rem; border-bottom: 1px solid var(--line); text-align: left; vertical-align: top; } tbody tr { cursor: pointer; transition: background 120ms ease; } tbody tr:hover { background: rgba(198, 90, 49, 0.06); } tbody tr.selected { background: linear-gradient(90deg, rgba(198, 90, 49, 0.12), rgba(35, 91, 96, 0.08)); } .sort-button { border: 0; background: transparent; padding: 0; font: inherit; font-weight: 700; cursor: pointer; color: inherit; } .empty-table, .detail-empty { padding: 1.4rem; color: var(--muted); } .pagination { justify-content: flex-end; margin-top: 1rem; } .detail-content { margin-top: 1rem; } .detail-header h3 { margin: 0; font-family: var(--serif); font-size: 1.5rem; } .detail-subtitle { margin: 0.5rem 0 0; color: var(--muted); } .detail-tags { display: flex; flex-wrap: wrap; gap: 0.45rem; margin-top: 0.9rem; } .tag { padding: 0.38rem 0.62rem; border-radius: 999px; background: rgba(255, 255, 255, 0.78); border: 1px solid var(--line); font-size: 0.78rem; } .tag.pass { background: var(--green-soft); } .tag.warn { background: var(--accent-soft); } .tag.low { background: var(--danger-soft); } .detail-grid { display: grid; grid-template-columns: repeat(2, minmax(0, 1fr)); gap: 0.85rem 1rem; margin-top: 1.15rem; } .detail-grid div, .sample-card { padding: 0.9rem; border-radius: 1rem; background: rgba(255, 255, 255, 0.62); border: 1px solid var(--line); } .detail-grid dt, .sample-card dt { color: var(--muted); margin-bottom: 0.3rem; } .detail-grid dd, .sample-card dd { margin: 0; word-break: break-word; } .section-title { margin: 1.2rem 0 0.7rem; font-size: 0.82rem; text-transform: uppercase; letter-spacing: 0.14em; color: var(--teal); } .sample-grid { display: grid; grid-template-columns: repeat(auto-fit, minmax(220px, 1fr)); gap: 0.8rem; } .sample-card dl { display: grid; grid-template-columns: repeat(2, minmax(0, 1fr)); gap: 0.45rem 0.75rem; margin: 0.6rem 0 0; } .raw-block { margin-top: 1rem; padding: 0.95rem; border-radius: 1rem; background: #162430; color: #eff4f3; overflow: auto; line-height: 1.55; font-size: 0.82rem; } .locus-cell strong { display: block; } .locus-cell span, .mini-note { color: var(--muted); } .badge { display: inline-flex; align-items: center; padding: 0.2rem 0.45rem; border-radius: 999px; font-size: 0.75rem; background: rgba(35, 91, 96, 0.1); color: var(--teal); } .reveal { animation: rise-in 360ms ease both; } @keyframes rise-in { from { opacity: 0; transform: translateY(10px); } to { opacity: 1; transform: translateY(0); } } @media (max-width: 1100px) { .hero, .workspace, .chip-section { grid-template-columns: 1fr; } .filter-grid { grid-template-columns: repeat(2, minmax(0, 1fr)); } .field:first-child { grid-column: span 2; } } @media (max-width: 700px) { .page { width: min(100vw - 1rem, 1500px); padding-top: 0.5rem; } .hero, .filters, .table-panel, .detail-panel { padding: 1rem; } .filter-grid, .detail-grid { grid-template-columns: 1fr; } .field:first-child { grid-column: span 1; } .panel-heading, .pagination { align-items: flex-start; flex-direction: column; } } ================================================ FILE: notes ================================================ AlignedContig constructor from BamRecordPtrVector --- Makes the alignment fragments ------ Makes the indel bps from each AlignmentFragment --- Make the SV BreakPoints ------- Makes each BreakEnd -- this will take BamRecordPtr data from AlignmentFragment into the BreakEnd (e.g. nm) ------- Makes the "local" breakpoints and "secondaries" breakpoints ------- Sets the "global" breakpoint ----------- Calls BreakPoint "set_homologies_insertions" for all global and local bps ----------- Calls BreakPoint "order" for all ----------- Sets the BreakPoint SVType ----------- Instantiates a new Prefix : SampleInfo map Directly from BreakPoints --- scoreBreakpoints ------- "set_evidence" that sets DSCRD, INDEL, ASDIS, etc ------- calls SampleInfo -> modelSelection --------------- sets the alt cov and total cov --------------- calls the __log_likelihood for alt or err --------------- calls the __log_likelihood for ref or alt --------------- calls the __genotype_likelihoods for 0/0, 0/1 or 1/1 --------------- calls "score_somatic" that sets somatic_lod ================================================ FILE: opt/jemalloc_test.sh ================================================ REGION=chr12:10,000,000-12,000,000 T=/Users/jeremiahwala/Desktop/svaba_compare/SG.wgs.UCLA.2025.01.tumor_cleaned.recal.bam N=/Users/jeremiahwala/Desktop/svaba_compare/blood.recal.bam # prime the page cache so cold-I/O doesn't dominate REF=${HOME}/ref_genome/Homo_sapiens_assembly38.fasta for f in $REF ${REF}.bwt ${REF}.sa ${REF}.pac do cat "$f" > /dev/null; done # baseline (system malloc) 3 trials for t in 1 2 3; do /usr/bin/time -l ~/git/svaba/build/svaba run \ -t $T -n $N -G $REF \ -k $REGION -p 16 -a base_$t 2>&1 \ | tail -15 | grep -E "real|user|sys|maximum resident" done # jemalloc 3 trials for t in 1 2 3; do /usr/bin/time -l env \ DYLD_INSERT_LIBRARIES=$(brew --prefix jemalloc)/lib/libjemalloc.dylib \ DYLD_FORCE_FLAT_NAMESPACE=1 \ MALLOC_CONF=background_thread:true,narenas:24,dirty_decay_ms:10000 \ ~/git/svaba/build/svaba run \ -t $T -n $N -G $REF \ -k $REGION -p 16 -a jem_$t 2>&1 \ | tail -15 | grep -E "real|user|sys|maximum resident" done ================================================ FILE: opt/memprof.sh ================================================ #!/usr/bin/env bash set -euo pipefail if (( $# < 1 )); then echo "Usage: $0 [args]" exit 1 fi OUT_LOG="memlog.txt" PLOT="memory.png" # Clean slate : > "$OUT_LOG" rm -f "$PLOT" # Start the users command "$@" & PID=$! echo "[$(date)] Started PID $PID: $*" >&2 START=$(date +%s) LAST_PLOT=$START # Monitor until the process exits while kill -0 "$PID" 2>/dev/null; do NOW=$(date +%s) ELAPSED=$(( NOW - START )) # Get PSS in KB PSS=$(awk '/^Pss:/ { print $2 }' /proc/"$PID"/smaps_rollup 2>/dev/null || echo 0) echo "$ELAPSED $PSS" >> "$OUT_LOG" # RSS=$(ps -o rss= -p "$PID" | awk '{print $1}') #echo "$ELAPSED $RSS" >> "$OUT_LOG" # every 30s replot if (( ELAPSED - (LAST_PLOT - START) >= 30 )); then LAST_PLOT=$NOW Rscript --vanilla - <&2 fi sleep 1 done # One final log entry (in case it just exited) NOW=$(date +%s) ELAPSED=$(( NOW - START )) PSS=$(awk '/^Pss:/ { print $2 }' /proc/"$PID"/smaps_rollup 2>/dev/null || echo 0) echo "$ELAPSED $PSS" >> "$OUT_LOG" # Final plot Rscript --vanilla - <&2 wait "$PID" exit $? ================================================ FILE: opt/memprof_osx.sh ================================================ #!/usr/bin/env bash set -euo pipefail if (( $# < 1 )); then echo "Usage: $0 [args]" exit 1 fi OUT_LOG="memlog.txt" PLOT="memory.png" : > "$OUT_LOG" rm -f "$PLOT" "$@" & PID=$! echo "[$(date)] Started PID $PID: $*" >&2 START=$(date +%s) LAST_PLOT=$START while kill -0 "$PID" 2>/dev/null; do NOW=$(date +%s) ELAPSED=$(( NOW - START )) # macOS: footprint reports physical memory in bytes (matches Activity Monitor) MEM_MB=$(footprint -p "$PID" 2>/dev/null \ | awk '/^[[:space:]]*phys_footprint:/ { val=$2; unit=$3; if (unit == "B") val=val/1024/1024; else if (unit == "KB") val=val/1024; else if (unit == "GB") val=val*1024; # MB stays as-is printf "%d\n", val; exit }') MEM_MB=${MEM_MB:-0} # Convert to KB to match the original log format (R script divides by 1024/1024 for GB) MEM=$(( MEM_MB * 1024 )) echo "$ELAPSED $MEM" >> "$OUT_LOG" if (( NOW - LAST_PLOT >= 30 )); then LAST_PLOT=$NOW if (( $(wc -l < "$OUT_LOG") >= 2 )); then Rscript --vanilla - <&2 fi fi sleep 1 done wait "$PID" || true ================================================ FILE: opt/memusg.sh ================================================ #!/usr/bin/env bash # memusg -- Measure memory usage of processes # Usage: memusg COMMAND [ARGS]... # # Author: Jaeho Shin # Created: 2010-08-16 set -um # check input [ $# -gt 0 ] || { sed -n '2,/^#$/ s/^# //p' <"$0"; exit 1; } # TODO support more options: peak, footprint, sampling rate, etc. pgid=`ps -o pgid= $$` # make sure we're in a separate process group if [ $pgid = $(ps -o pgid= $(ps -o ppid= $$)) ]; then cmd= set -- "$0" "$@" for a; do cmd+="'${a//"'"/"'\\''"}' "; done exec bash -i -c "$cmd" fi # detect operating system and prepare measurement case `uname` in Darwin|*BSD) sizes() { /bin/ps -o rss= -g $1; } ;; Linux) sizes() { /bin/ps -o rss= -$1; } ;; *) echo "`uname`: unsupported operating system" >&2; exit 2 ;; esac # monitor the memory usage in the background. ( peak=0 while sizes=`sizes $pgid` do set -- $sizes sample=$((${@/#/+})) let peak="sample > peak ? sample : peak" sleep 0.1 done echo "memusg: peak=$peak" >&2 ) & monpid=$! # run the given command exec "$@" ================================================ FILE: opt/profiler.sh ================================================ #!/usr/bin/env bash "$@" & # Run the given command line in the background. pid=$! peak=0 rm mem.log ## make the Rscript rm -f memplot.R echo "require(ggplot2); df = read.table('mem.log', header=FALSE); df\$time = seq(from=10,to=10*nrow(df), by=10);" >> memplot.R; echo "pdf('memgraph.pdf'); g <- ggplot(df, aes(x=time, y=V1/1024)) + geom_line() + xlab('Time (s)') + ylab('Memory (Mb)') + theme_bw(); print(g); dev.off();" >> memplot.R; chmod +x memplot.R; while true; do sleep 0.1 sample="$(ps -o rss= $pid 2> /dev/null)" || break let peak='sample > peak ? sample : peak' echo "$sample" >> mem.log done echo "Peak: $peak" 1>&2 Rscript memplot.R ================================================ FILE: opt/runtime.R ================================================ #!/usr/bin/env Rscript suppressPackageStartupMessages({ library(data.table) library(ggplot2) library(ggrepel) }) # Parse arguments args <- commandArgs(trailingOnly = TRUE) if (length(args) < 2) { stop("Usage: ./runtime_plot.R input.tsv output.pdf") } input_file <- args[1] output_file <- args[2] # Read data dt <- fread(input_file) # Only keep chromosomes 1-22 and X allowed_chr <- paste0("chr", c(1:22, "X")) dt <- dt[chromosome %in% allowed_chr] # Convert start to Mb dt[, start_mb := start / 1e6] # Region label for top runtime labels dt[, region_label := paste0(chromosome, ":", start, "-", end)] # For each chromosome, find top 10 regions by runtime top10 <- dt[, .SD[order(-runtime_seconds)][1:10], by = chromosome] # Plot with facet by chromosome p <- ggplot(dt, aes(x = start_mb, y = runtime_seconds)) + geom_point(size = 0.7) + geom_line(aes(group = 1), alpha = 0.7) + # explicitly group the line geom_text_repel( data = top10, aes(label = region_label), size = 2, max.overlaps = Inf, box.padding = 0.5 ) + facet_wrap(~chromosome, scales = "free_x", ncol = 5) + labs( title = "Runtime per Genomic Region by Chromosome", x = "Genomic Start Position (Mb)", y = "Wall Time (seconds)" ) + scale_y_continuous(limits=c(0,200)) + theme_minimal(base_size = 9) + theme(strip.text = element_text(face = "bold")) + scale_x_continuous(expand = expansion(add = c(0.01, 0.01))) # avoid zero-width panels # Save a large PDF ggsave(output_file, plot = p, width = 20, height = 16, dpi = 300) ================================================ FILE: scripts/combine_blacklists.sh ================================================ #!/usr/bin/env bash # # combine_blacklists.sh — combine multiple BED blacklists into one file. # # "Combining" blacklists means a UNION over intervals: a region ends up in the # output if ANY of the input files covered it. This is what you almost always # want when you're assembling a single --blacklist argument for svaba run out # of several sources (high-signal regions, low-mappability, simple repeats, # non-standard contigs, high-runtime regions, ad-hoc bad list, etc.). # # (If you actually need the set intersection — regions blacklisted by ALL # sources simultaneously — this is not that script. For that, use # `bedtools multiinter -i FILES | awk '$4==NFILES'` or iterated # `bedtools intersect`. Intersect-of-blacklists is almost never the right # semantics; you'd end up with a tiny output.) # # What this script does: # 1. cat every input BED to stdout with a 5th-column source tag = basename. # Any existing 4th-column label is preserved. # 2. Filter out obvious non-BED noise: empty lines, `#` comments, # `track`/`browser` UCSC header lines. # 3. By default: sort by (chrom, start) and emit as a 5-column BED. # 4. With --merge: run `bedtools merge` to collapse overlapping/adjacent # intervals into one, and collapse the set of contributing source tags # into a comma-separated list in column 4. # # Usage: # ./combine_blacklists.sh -o OUT.bed [--merge [--slop N]] FILE1.bed FILE2.bed ... # # Flags: # -o, --output OUT output path (required) # -m, --merge sort + bedtools merge the concatenation (union of # overlapping intervals, with label-distinct column). # Requires `bedtools` on PATH. # -d, --merge-dist N pass to bedtools merge -d (intervals within N bp # are merged). Default 0 (touching/overlapping only). # --slop N grow each interval by N bp on both sides before # merging (expands the blacklist). Default 0. # Requires `bedtools` and --merge. # -c, --clip GENOME clip every interval's end to the real contig length # in GENOME, and drop intervals whose start is past the # contig. GENOME may be a .fai, a two-column tsv # (chromlength), or svaba's tracks/chr fasta- # header dump (>chrN AC:... LN:... ...). Unknown # contigs are passed through unchanged (with a # warning). This is what saves you from a 250,000,000- # end sentinel in one of the input BEDs blowing the # reported covered-bp up to absurd levels. # --no-label drop the label column(s); emit 3-col BED only # -q, --quiet suppress progress to stderr # -h, --help this message # # Typical invocation (regenerate the project's combined blacklist): # # ./combine_blacklists.sh --merge \ # -o tracks/hg38.combined_blacklist.bed \ # tracks/hg38.blacklist.bed \ # tracks/hg38.high_runtime.bed \ # tracks/hg38.extra_badlist.bed \ # tracks/hg38.nonstd_chr.blacklist.bed \ # tracks/hg38.rmsk.simple_repeat.bed # # The script is idempotent — re-running it always rebuilds OUT from current # inputs. Change `high_runtime.bed`, rerun, and the combined file updates. set -euo pipefail OUT="" MERGE=0 MERGE_DIST=0 SLOP=0 CLIP="" NO_LABEL=0 QUIET=0 INPUTS=() log() { [[ $QUIET -eq 1 ]] || echo "$@" >&2; } usage() { sed -n '2,60p' "$0" | sed 's/^# \{0,1\}//'; exit "${1:-0}"; } # ---- arg parsing ---- while (( $# )); do case "$1" in -o|--output) OUT="$2"; shift 2 ;; -m|--merge) MERGE=1; shift ;; -d|--merge-dist) MERGE_DIST="$2"; shift 2 ;; --slop) SLOP="$2"; shift 2 ;; -c|--clip) CLIP="$2"; shift 2 ;; --no-label) NO_LABEL=1; shift ;; -q|--quiet) QUIET=1; shift ;; -h|--help) usage 0 ;; --) shift; while (( $# )); do INPUTS+=("$1"); shift; done ;; -*) echo "Unknown flag: $1" >&2; usage 2 ;; *) INPUTS+=("$1"); shift ;; esac done [[ -z "$OUT" ]] && { echo "ERROR: -o OUTPUT is required" >&2; usage 2; } [[ ${#INPUTS[@]} -lt 1 ]] && { echo "ERROR: no input BED files given" >&2; usage 2; } # ---- sanity on inputs ---- for f in "${INPUTS[@]}"; do [[ -f "$f" ]] || { echo "ERROR: input not found: $f" >&2; exit 1; } done if [[ $MERGE -eq 1 || $SLOP -gt 0 ]]; then command -v bedtools >/dev/null 2>&1 \ || { echo "ERROR: --merge/--slop require 'bedtools' on PATH" >&2; exit 1; } fi if [[ $SLOP -gt 0 && $MERGE -ne 1 ]]; then echo "ERROR: --slop requires --merge (expanded intervals need to be merged to stay collapsed)" >&2 exit 2 fi # ---- work in a tempdir ---- TMPDIR_LOCAL="$(mktemp -d)" trap 'rm -rf "$TMPDIR_LOCAL"' EXIT CAT="$TMPDIR_LOCAL/cat.bed" SORTED="$TMPDIR_LOCAL/sorted.bed" GENOME_TSV="$TMPDIR_LOCAL/genome.tsv" # ---- optional: parse clip-genome file into a flat chromlength TSV ---- # # Accepts three input shapes; we auto-detect: # .fai : namelenoffsetlinebaseslinewidth # genome tsv : namelen (optional extra cols ignored) # tracks/chr : >chrN AC:... LN:NNNN ... (fasta-header dump) if [[ -n "$CLIP" ]]; then [[ -f "$CLIP" ]] || { echo "ERROR: --clip file not found: $CLIP" >&2; exit 1; } awk -v OFS='\t' ' /^>/ { name=$1; sub(/^>/, "", name) ln="" for (i=2;i<=NF;i++) if ($i ~ /^LN:/) { ln=substr($i,4); break } if (ln!="" && ln+0>0) print name, ln next } /^[[:space:]]*$/ {next} /^[[:space:]]*#/ {next} { # .fai or 2-col genome. col1=name, col2=length. if ($2 ~ /^[0-9]+$/ && $2+0 > 0) print $1, $2 } ' "$CLIP" > "$GENOME_TSV" CLIP_N="$(wc -l < "$GENOME_TSV" | awk '{print $1}')" [[ "$CLIP_N" -gt 0 ]] || { echo "ERROR: --clip file $CLIP has no usable (name, length) entries" >&2; exit 1; } log "combine_blacklists.sh: clip genome loaded from $CLIP ($CLIP_N contigs)" fi log "combine_blacklists.sh: building $OUT from ${#INPUTS[@]} source(s)" # ---- step 1: concatenate with a 5th-column source tag ---- : > "$CAT" for f in "${INPUTS[@]}"; do src="$(basename "$f")" # Drop blank / comment / UCSC-header lines. For each record, collapse leading # whitespace and ensure we end up tab-separated. Keep cols 1-3, promote any # existing col 4 to the label, and append src as col 5. awk -v src="$src" ' BEGIN{OFS="\t"} /^[[:space:]]*$/ {next} /^[[:space:]]*#/ {next} /^[[:space:]]*(track|browser)[[:space:]]/ {next} { # Normalize: the input may already be tab-separated (BED spec) or # whitespace-separated. Use awk default FS (runs of whitespace) but # write tabs on output. chr=$1; start=$2; end=$3 if (start !~ /^[0-9]+$/ || end !~ /^[0-9]+$/) next label="" if (NF >= 4) { label=$4 for (i=5;i<=NF;i++) label=label" "$i } if (label=="") print chr, start, end, "-", src else print chr, start, end, label, src } ' "$f" >> "$CAT" log " + $(printf "%-45s" "$src") $(wc -l < "$f" | awk '{print $1}') lines" done RAW_LINES="$(wc -l < "$CAT" | awk '{print $1}')" log " = $RAW_LINES raw intervals before sort/merge" # ---- step 2: sort ---- # LC_ALL=C to keep sort output stable across locales and compatible with # bedtools' lexicographic chrom ordering. LC_ALL=C sort -k1,1 -k2,2n "$CAT" > "$SORTED" # ---- step 2b: optional clip-to-genome ---- # # Applied BEFORE merge and slop so downstream steps see real coordinates. # Intervals on unknown contigs are passed through unchanged (with a single # warning). Intervals whose start is beyond the contig length are dropped; # intervals whose end overshoots are clipped to the contig length. if [[ -n "$CLIP" ]]; then CLIPPED="$TMPDIR_LOCAL/clipped.bed" awk -v OFS='\t' -v genomefile="$GENOME_TSV" ' BEGIN { while ((getline < genomefile) > 0) { len[$1] = $2 } close(genomefile) dropped=0; clipped=0; passthrough=0; unknown_n=0 } { chr=$1; s=$2; e=$3 if (!(chr in len)) { if (!(chr in unknown_seen)) { unknown_seen[chr]=1; unknown_n++ } passthrough++ print next } L = len[chr] if (s+0 >= L) { dropped++; next } if (e+0 > L) { e = L; clipped++ } $2=s; $3=e print } END { fmt=" · clip: %d clipped to contig length, %d dropped past contig end, %d intervals on %d unknown contig(s) passed through\n" printf fmt, clipped, dropped, passthrough, unknown_n > "/dev/stderr" if (unknown_n > 0) { n=0 for (c in unknown_seen) { if (n < 5) printf " · unknown contig example: %s\n", c > "/dev/stderr" n++ } if (unknown_n > 5) printf " · ... (%d unknown contigs total)\n", unknown_n > "/dev/stderr" } } ' "$SORTED" > "$CLIPPED" SORTED="$CLIPPED" fi # ---- step 3: emit ---- if [[ $MERGE -eq 1 ]]; then # bedtools merge collapses overlapping / touching intervals. We carry the # source tag (col 5) into the collapsed output as a comma-separated set # of contributing sources via -c 5 -o distinct; and also collapse the # original labels via col 4 so downstream tools still see a human-readable # label column. # # Layout of the intermediate we feed to bedtools: # chr start end label src # After merge with -c 4,5 -o distinct,distinct we get: # chr start end label_set src_set if [[ $SLOP -gt 0 ]]; then GEN="$TMPDIR_LOCAL/genome.tsv" # Try to derive a genome file from the first input's chroms as max(end). # This is a permissive fallback; if you have a real .fai / .genome file # available, you can swap this with `bedtools slop -g your.genome`. awk 'BEGIN{OFS="\t"} {if($3>m[$1]) m[$1]=$3} END{for (k in m) print k, m[k]}' \ "$SORTED" > "$GEN" SLOPPED="$TMPDIR_LOCAL/slopped.bed" bedtools slop -i "$SORTED" -g "$GEN" -b "$SLOP" > "$SLOPPED" LC_ALL=C sort -k1,1 -k2,2n "$SLOPPED" > "$SORTED" log " · slopped +/-${SLOP}bp (genome inferred from max end per chrom)" fi MERGED="$TMPDIR_LOCAL/merged.bed" bedtools merge -i "$SORTED" -d "$MERGE_DIST" -c 4,5 -o distinct,distinct \ > "$MERGED" MERGED_LINES="$(wc -l < "$MERGED" | awk '{print $1}')" log " · bedtools merge (d=$MERGE_DIST) : $RAW_LINES -> $MERGED_LINES intervals" if [[ $NO_LABEL -eq 1 ]]; then cut -f1-3 "$MERGED" > "$OUT" else # 5 cols: chr start end label-set source-set cp "$MERGED" "$OUT" fi else if [[ $NO_LABEL -eq 1 ]]; then cut -f1-3 "$SORTED" > "$OUT" else # 5 cols: chr start end label src (pre-merge, duplicates preserved) cp "$SORTED" "$OUT" fi fi FINAL_LINES="$(wc -l < "$OUT" | awk '{print $1}')" FINAL_BP="$(awk 'BEGIN{s=0} {s+=$3-$2} END{print s+0}' "$OUT")" log "combine_blacklists.sh: wrote $OUT ($FINAL_LINES intervals, $(printf "%'d" "$FINAL_BP") bp covered)" # Sanity check: the human genome is ~3.1 Gbp (~3.2 Gbp incl. alts). If the # reported coverage is larger than a loose 10 Gbp threshold, the input almost # certainly contains synthetic "end-of-contig" sentinels (e.g. end=250000000 # on short alt contigs) and the user should rerun with --clip GENOME to get # a meaningful number. if [[ $FINAL_BP -gt 10000000000 ]]; then log "" log "WARNING: $(printf "%'d" $FINAL_BP) bp is larger than any reasonable reference." if [[ -z "$CLIP" ]]; then log " Likely cause: an input BED uses oversized end coordinates as a 'whole" log " contig' shorthand (e.g. end=250000000 on short alt contigs). Rerun" log " with --clip tracks/chr (or a .fai / genome tsv) to clip each" log " interval to its real contig length before the bp tally." else log " --clip was used; the oversize is probably legitimate (very large" log " genome) or caused by intervals on contigs missing from your clip" log " genome file — check the 'unknown contig' lines above." fi fi ================================================ FILE: scripts/extract_by_qname.sh ================================================ #!/bin/bash # ============================================================ # Extract all reads from a target BAM whose QNAMEs appear in # a query BAM. # # Both inputs can be coordinate-sorted. We extract QNAMEs from # the query BAM into a hash set, then stream the target BAM and # keep any record whose QNAME is in the set. No requirement that # the BAMs are sorted the same way, or at all. # # Usage: # ./extract_by_qname.sh # # Example: # ./extract_by_qname.sh matches.bam /mnt/ssd/tumor.recal.bam out.bam # ============================================================ set -euo pipefail if [ $# -ne 3 ]; then echo "Usage: $0 " >&2 echo " Extracts from target.bam all reads whose QNAME appears" >&2 echo " in query.bam. Writes sorted, indexed BAM to output.bam." >&2 exit 1 fi QUERY_BAM="$1" TARGET_BAM="$2" OUT_BAM="$3" THREADS=${THREADS:-8} # ---- Sanity checks ---- for f in "$QUERY_BAM" "$TARGET_BAM"; do if [ ! -f "$f" ]; then echo "ERROR: BAM not found: $f" >&2 exit 1 fi done if ! command -v samtools >/dev/null 2>&1; then echo "ERROR: samtools not found in PATH" >&2 exit 1 fi # ---- Use a scratch dir for intermediate files ---- TMPDIR=$(mktemp -d -t extract_qname.XXXXXX) trap "rm -rf $TMPDIR" EXIT QNAME_LIST="$TMPDIR/qnames.txt" echo "Temp dir: $TMPDIR" >&2 # ---- Step 1: extract unique QNAMEs from query BAM ---- echo "[1/3] Extracting QNAMEs from $QUERY_BAM..." >&2 samtools view -@ "$THREADS" "$QUERY_BAM" \ | awk '{print $1}' \ | sort -u \ > "$QNAME_LIST" N_QNAMES=$(wc -l < "$QNAME_LIST") echo " Found $N_QNAMES unique QNAMEs" >&2 if [ "$N_QNAMES" -eq 0 ]; then echo "ERROR: No QNAMEs found in query BAM" >&2 exit 1 fi # ---- Step 2: stream target BAM, filter by QNAME ---- echo "[2/3] Filtering $TARGET_BAM..." >&2 # Use awk with a hash lookup - O(1) per read, linear in target BAM size samtools view -@ "$THREADS" -h "$TARGET_BAM" \ | awk -v list="$QNAME_LIST" ' BEGIN { while ((getline line < list) > 0) { qnames[line] = 1 } close(list) } /^@/ { print; next } ($1 in qnames) { print } ' \ | samtools view -@ "$THREADS" -b -o "$TMPDIR/unsorted.bam" - # ---- Step 3: sort and index ---- echo "[3/3] Sorting and indexing output..." >&2 samtools sort -@ "$THREADS" -o "$OUT_BAM" "$TMPDIR/unsorted.bam" samtools index -@ "$THREADS" "$OUT_BAM" # ---- Report ---- N_OUT=$(samtools view -c "$OUT_BAM") echo "" >&2 echo "Done." >&2 echo " Query QNAMEs: $N_QNAMES" >&2 echo " Output reads: $N_OUT" >&2 echo " (Output includes all alignments per QNAME - primary, secondary," >&2 echo " supplementary, and both mates of pairs.)" >&2 echo " Output: $OUT_BAM + ${OUT_BAM%.bam}.bam.bai" >&2 ================================================ FILE: scripts/extract_discordants.sh ================================================ #!/bin/bash prefix=$1 ##prefix="${1%%.*}" /usr/bin/samtools view -h ${prefix}.weird.bam | grep -E '^@|DC:Z' | /usr/bin/samtools view -b -o tmp.filtered.bam /usr/bin/samtools sort -o ${prefix}.dc.bam tmp.filtered.bam /usr/bin/samtools index ${prefix}.dc.bam rm tmp.filtered.bam TARGET=$2 if [[ -n "$TARGET" ]]; then /usr/bin/samtools view -h ${prefix}.dc.bam | \ awk -v pat="${TARGET}" 'BEGIN{OFS="\t"} /^@/ {print; next} $0 ~ pat {print}' | \ /usr/bin/samtools view -b -o dc.${TARGET}.bam /usr/bin/samtools index dc.${TARGET}.bam fi ================================================ FILE: scripts/extract_pairs_by_seq.sh ================================================ #!/bin/bash # ============================================================ # Extract read pairs from a BAM where either mate contains any # of the given sequences (or their reverse complements). # # Two-pass approach on the target BAM: # Pass 1: stream through BAM, collect QNAMEs of reads whose # SEQ matches any query sequence or its reverse complement # Pass 2: stream through BAM again, extract ALL reads (both mates, # plus supplementary/secondary) whose QNAME was collected # # Usage: # ./extract_pairs_by_seq.sh [seq2 ...] # # Example: # ./extract_pairs_by_seq.sh tumor.bam fusion_reads.bam \ # ACGTACGTACGT TTACGATCGATC # ============================================================ set -euo pipefail # ---- Args ---- if [ $# -lt 3 ]; then echo "Usage: $0 [seq2 ...]" >&2 echo " Extracts read pairs where either mate matches any sequence" >&2 echo " (or its reverse complement)." >&2 echo " Output: sorted, indexed BAM + BAI." >&2 exit 1 fi TARGET_BAM="$1" OUT_BAM="$2" shift 2 SEQS=("$@") THREADS=${THREADS:-8} AWK_BIN=$(command -v mawk || command -v awk) # ---- Sanity checks ---- if [ ! -f "$TARGET_BAM" ]; then echo "ERROR: Target BAM not found: $TARGET_BAM" >&2 exit 1 fi if ! command -v samtools >/dev/null 2>&1; then echo "ERROR: samtools not found in PATH" >&2 exit 1 fi # ---- Scratch dir ---- TMPDIR=$(mktemp -d -t extract_pairs.XXXXXX) trap "rm -rf $TMPDIR" EXIT QNAME_LIST="$TMPDIR/qnames.txt" echo "Target: $TARGET_BAM" >&2 echo "Output: $OUT_BAM" >&2 echo "Temp: $TMPDIR" >&2 echo "Awk: $AWK_BIN" >&2 echo "Threads: $THREADS" >&2 echo "" >&2 # ---- Build combined regex: seq1|revcomp1|seq2|revcomp2|... ---- revcomp() { echo "$1" | tr 'acgtnACGTN' 'tgcanTGCAN' | rev } PATTERNS=() echo "Query sequences (with reverse complements):" >&2 for seq in "${SEQS[@]}"; do seq_up=$(echo "$seq" | tr 'a-z' 'A-Z' | tr -d '[:space:]') rc=$(revcomp "$seq_up") PATTERNS+=("$seq_up" "$rc") echo " $seq_up (rc: $rc)" >&2 done PATTERN=$(IFS='|'; echo "${PATTERNS[*]}") echo "" >&2 # ---- PASS 1: find QNAMEs whose SEQ matches ---- echo "[Pass 1/2] Scanning $TARGET_BAM for matching sequences..." >&2 samtools view -@ "$THREADS" "$TARGET_BAM" \ | "$AWK_BIN" -v pat="$PATTERN" '$10 ~ pat {print $1}' \ | sort -u \ > "$QNAME_LIST" N_QNAMES=$(wc -l < "$QNAME_LIST") echo " Matching QNAMEs: $N_QNAMES" >&2 if [ "$N_QNAMES" -eq 0 ]; then echo "No matches found. Exiting without creating output." >&2 exit 0 fi # ---- PASS 2: extract all alignments for those QNAMEs (both mates) ---- echo "[Pass 2/2] Extracting all alignments for matching QNAMEs..." >&2 samtools view -@ "$THREADS" -h "$TARGET_BAM" \ | "$AWK_BIN" -v list="$QNAME_LIST" ' BEGIN { while ((getline line < list) > 0) { qnames[line] = 1 } close(list) } /^@/ { print; next } ($1 in qnames) { print } ' \ | samtools view -@ "$THREADS" -b -o "$TMPDIR/unsorted.bam" - # ---- Sort and index ---- echo "Sorting and indexing..." >&2 samtools sort -@ "$THREADS" -o "$OUT_BAM" "$TMPDIR/unsorted.bam" samtools index -@ "$THREADS" "$OUT_BAM" # ---- Report ---- N_OUT=$(samtools view -c "$OUT_BAM") N_PRIMARY=$(samtools view -c -F 2304 "$OUT_BAM") # exclude secondary + supplementary echo "" >&2 echo "Done." >&2 echo " Matching QNAMEs (read pairs): $N_QNAMES" >&2 echo " Total alignments in output: $N_OUT" >&2 echo " Primary alignments (both mates): $N_PRIMARY" >&2 echo " Output: $OUT_BAM + ${OUT_BAM%.bam}.bam.bai" >&2 ================================================ FILE: scripts/filter_contig_supporting_reads.sh ================================================ #!/usr/bin/env bash # # filter_contig_supporting_reads.sh # # Filter a svaba *.corrected.bam down to just the reads whose r2c (read-to- # contig) BWA alignment landed on a single named contig. The contig name is # matched against the `bi` aux tag that svaba writes onto each corrected # read (one tag value per r2c hit, comma-separated). Output is a coordinate- # sorted+indexed BAM for easy IGV inspection. set -euo pipefail PROG="$(basename "$0")" usage() { cat <&2; usage >&2; exit 2 ;; *) POS+=("$1"); shift ;; esac done # Positional fallback: ID CONTIG if [[ -z "$ID" && ${#POS[@]} -ge 1 ]]; then ID="${POS[0]}"; fi if [[ -z "$CONTIG" && ${#POS[@]} -ge 2 ]]; then CONTIG="${POS[1]}"; fi if [[ -z "$ID" || -z "$CONTIG" ]]; then echo "${PROG}: missing required argument(s)" >&2 usage >&2 exit 2 fi # --- resolve input/output paths -------------------------------------------- if [[ "$ID" == *.bam ]]; then IN_BAM="$ID" ID_BASE="${ID%.corrected.bam}" ID_BASE="${ID_BASE%.bam}" else IN_BAM="${ID}.corrected.bam" ID_BASE="$ID" fi if [[ ! -f "$IN_BAM" ]]; then echo "${PROG}: input BAM not found: ${IN_BAM}" >&2 exit 1 fi if [[ -z "$OUT" ]]; then OUT="${ID_BASE}.${CONTIG}.bam" fi # --- check tools ------------------------------------------------------------ command -v samtools >/dev/null 2>&1 || { echo "${PROG}: samtools not found on PATH" >&2; exit 127; } # --- filter ----------------------------------------------------------------- echo "${PROG}: ID = ${ID_BASE}" >&2 echo "${PROG}: input = ${IN_BAM}" >&2 echo "${PROG}: contig = ${CONTIG}" >&2 echo "${PROG}: output = ${OUT}" >&2 echo "${PROG}: threads = ${THREADS}" >&2 samtools view -h -e "[bi]=~\"${CONTIG}\"" "${IN_BAM}" \ | samtools sort -@"${THREADS}" -o "${OUT}" - samtools index "${OUT}" echo "${PROG}: wrote ${OUT} (+ ${OUT}.bai)" >&2 ================================================ FILE: scripts/gcloud_teardown.sh ================================================ #!/usr/bin/env bash # # gcloud_teardown.sh — kill svaba cloud workers and clean up the bucket. # # Usage: # gcloud_teardown.sh [options] # # --id STR analysis ID (default: my_run) # --partitions N number of workers (default: 5) # --zone ZONE GCP zone (default: us-central1-a) # --bucket gs://... GCS bucket to clean (default: gs://osteosarc-private/svaba-run) # --keep-bucket don't delete bucket contents # -h, --help this message # set -euo pipefail ID="my_run" PARTITIONS=5 ZONE="us-central1-a" BUCKET="gs://osteosarc-private/svaba-run" CLEAN_BUCKET=1 while [[ $# -gt 0 ]]; do case "$1" in --id) ID="$2"; shift 2 ;; --partitions) PARTITIONS="$2"; shift 2 ;; --zone) ZONE="$2"; shift 2 ;; --bucket) BUCKET="$2"; shift 2 ;; --keep-bucket) CLEAN_BUCKET=0; shift ;; -h|--help) sed -n '2,14p' "$0" | sed 's/^# \{0,1\}//'; exit 0 ;; *) echo "unknown option: $1" >&2; exit 2 ;; esac done GCP_ID=$(echo "$ID" | tr '[:upper:]' '[:lower:]' | tr '_' '-' | sed 's/[^a-z0-9-]//g') # 1. Delete worker VMs echo "deleting $PARTITIONS workers (${GCP_ID})..." for i in $(seq 1 "$PARTITIONS"); do vm="svaba-${GCP_ID}-worker-${i}" if gcloud compute instances describe "$vm" --zone="$ZONE" &>/dev/null; then echo " deleting $vm" gcloud compute instances delete "$vm" --zone="$ZONE" --quiet & else echo " $vm not found, skipping" fi done wait # 2. Clean up the bucket if [[ $CLEAN_BUCKET -eq 1 ]]; then echo "cleaning bucket ${BUCKET}/..." gsutil -m rm "${BUCKET}/**" 2>/dev/null || true echo "bucket cleaned" else echo "keeping bucket contents (--keep-bucket)" fi echo "teardown complete" ================================================ FILE: scripts/mosdepth_lowmapq_blacklist.sh ================================================ #!/usr/bin/env bash # # mosdepth_lowmapq_blacklist.sh — flag regions dominated by multi-mappers. # # Given paired mosdepth regions.bed.gz outputs, one unfiltered by MAPQ and # one MAPQ-filtered, emit a BED of genomic windows where the fraction of # reads passing the MAPQ filter is low enough that the locus is effectively # "unmapped" for any caller that only trusts unique placements. These are # prime candidate blacklist intervals for svaba. # # The input pair must come from the SAME BAM with the SAME --by, differing # only in --mapq. Typical generation: # # mosdepth --by 1000 --fast-mode --mapq 0 --threads 4 unf SAMPLE.bam # mosdepth --by 1000 --fast-mode --mapq 30 --threads 4 filt SAMPLE.bam # # mosdepth writes half-open 0-based BED with 4 columns: # chrom start end mean_depth # # This script joins the two files row-by-row (they are bin-aligned), computes # ratio = filt_mean_depth / unf_mean_depth # and flags windows that pass BOTH of: # unf_mean_depth >= --min-cov (skip near-empty bins where 0/0 dominates) # ratio < --max-ratio (most reads are multi-mappers) # # Flagged per-bin rows are then bedtools-merged with --merge-gap into # contiguous intervals, carrying the worst ratio and peak coverage forward # so the merged output has useful label columns. # # The merged BED plugs straight into `combine_blacklists.sh` alongside your # other component BEDs (tracks/hg38.blacklist.sorted.bed, high_runtime.bed, # rmsk.simple_repeat.bed, etc.). # # Usage: # mosdepth_lowmapq_blacklist.sh [OPTIONS] UNF.regions.bed.gz FILT.regions.bed.gz # # Options: # --min-cov N minimum unfiltered mean depth for a bin to be # considered (default 10). Bins below this get skipped # entirely — you do not want a 0/1 accidental flag on # an empty region. # --max-ratio R flag if filt/unf < R (default 0.5). Lower = stricter. # Typical blacklist tuning range is 0.3 – 0.7. # --merge-gap N bedtools merge -d value (default 1000). Flagged bins # within N bp of each other get coalesced into one # interval. # --out PREFIX output prefix (default ./lowmapq). # --dump-per-region also emit ${PREFIX}.per_region.tsv.gz with every # considered bin (chr start end unf filt ratio). Off # by default — on a WGS BAM this file is ~coverage- # file-sized. # -h | --help show this help # # Outputs (always): # ${PREFIX}.flagged.bed one row per flagged bin, cols: # chr start end ratio unf_cov_rounded # ${PREFIX}.flagged.merged.bed bedtools-merged intervals, cols: # chr start end min_ratio max_cov # (min_ratio = worst ratio inside the merged # block; max_cov = peak unfiltered coverage) # ${PREFIX}.summary.txt coverage + flag tallies # # Outputs (optional): # ${PREFIX}.per_region.tsv.gz with --dump-per-region # # Requires: gzip, awk, sort, bedtools on PATH. set -euo pipefail MIN_COV=10 MAX_RATIO=0.5 MERGE_GAP=1000 OUT_PREFIX=lowmapq DUMP_PER=0 usage() { cat <<'EOF' mosdepth_lowmapq_blacklist.sh — flag regions dominated by multi-mappers. Usage: mosdepth_lowmapq_blacklist.sh [OPTIONS] UNF.regions.bed.gz FILT.regions.bed.gz The two inputs must come from the SAME BAM with the SAME --by on mosdepth, differing only in --mapq. Example generation: mosdepth --by 1000 --fast-mode --mapq 0 --threads 4 unf sample.bam mosdepth --by 1000 --fast-mode --mapq 30 --threads 4 filt sample.bam Options: --min-cov N minimum unf mean-depth for a bin to be considered (default 10) --max-ratio R flag if filt/unf < R (default 0.5); 0.3 stricter, 0.7 looser --merge-gap N bedtools merge -d value (default 1000 bp) --out PREFIX output prefix (default lowmapq) --dump-per-region also write ${PREFIX}.per_region.tsv.gz -h, --help show this message Outputs: ${PREFIX}.flagged.bed flagged bins: chr start end ratio unf_cov ${PREFIX}.flagged.merged.bed merged: chr start end min_ratio max_cov ${PREFIX}.summary.txt coverage + flag tallies See the header comment in this script for the full rationale / column docs. EOF exit 0 } while [[ $# -gt 0 ]]; do case "$1" in --min-cov) MIN_COV=$2; shift 2 ;; --max-ratio) MAX_RATIO=$2; shift 2 ;; --merge-gap) MERGE_GAP=$2; shift 2 ;; --out) OUT_PREFIX=$2; shift 2 ;; --dump-per-region) DUMP_PER=1; shift ;; -h|--help) usage ;; --) shift; break ;; -*) echo "unknown option: $1" >&2; exit 2 ;; *) break ;; esac done if [[ $# -ne 2 ]]; then echo "usage: $0 [opts] UNF.regions.bed.gz FILT.regions.bed.gz" >&2 echo " (try -h for full docs)" >&2 exit 2 fi UNF=$1 FILT=$2 for t in gzip awk sort bedtools; do command -v "$t" >/dev/null || { echo "ERROR: $t not on PATH" >&2; exit 1; } done for f in "$UNF" "$FILT"; do [[ -f $f ]] || { echo "ERROR: file not found: $f" >&2; exit 1; } done # Make sure the output directory exists if the user passed a pathy prefix. OUT_DIR=$(dirname -- "$OUT_PREFIX") [[ -d "$OUT_DIR" ]] || mkdir -p "$OUT_DIR" echo "mosdepth_lowmapq_blacklist:" echo " unf=$UNF" echo " filt=$FILT" echo " min_cov=$MIN_COV max_ratio=$MAX_RATIO merge_gap=$MERGE_GAP" echo " out_prefix=$OUT_PREFIX dump_per_region=$DUMP_PER" # ---- 1. Join and filter ----------------------------------------------------- # Both mosdepth files were emitted from the same --by on the same reference, # so their row-1..row-N bins are identical. We paste them and sanity-check # the coords on every row — a mismatch usually means the user accidentally # paired files from different BAMs or different --by values, which makes the # ratio meaningless. flagged="${OUT_PREFIX}.flagged.bed" tmp_flag=$(mktemp) tmp_per=$(mktemp) trap 'rm -f "$tmp_flag" "$tmp_per" "${tmp_per}.gz"' EXIT paste <(gzip -dc -- "$UNF") <(gzip -dc -- "$FILT") \ | awk -F'\t' -v OFS='\t' \ -v mc="$MIN_COV" -v mr="$MAX_RATIO" \ -v flag="$tmp_flag" -v per="$tmp_per" -v dump="$DUMP_PER" ' BEGIN { total = 0 considered = 0 flagged_cnt = 0 } { # cols 1-4: unf (chrom start end meanDepth) # cols 5-8: filt (chrom start end meanDepth) if ($1 != $5 || $2 != $6 || $3 != $7) { printf "ERROR: row %d coordinate mismatch: %s:%s-%s vs %s:%s-%s\n" \ " likely --by mismatch or different BAMs.\n", NR, $1, $2, $3, $5, $6, $7 > "/dev/stderr" exit 1 } total++ u = $4 + 0 f = $8 + 0 if (u < mc) next considered++ r = (u > 0 ? f / u : 0) if (dump == 1) { printf "%s\t%s\t%s\t%.3f\t%.3f\t%.4f\n", $1, $2, $3, u, f, r > per } if (r < mr) { # Col 4 = ratio (merge-friendly numeric), col 5 = rounded unf cov # so a `bedtools merge -c 4,5 -o min,max` gives "worst ratio in # block / peak coverage in block" — both useful for downstream # inspection / QC. printf "%s\t%s\t%s\t%.4f\t%d\n", $1, $2, $3, r, int(u + 0.5) > flag flagged_cnt++ } } END { printf "scan: rows_total=%d rows_considered=%d rows_flagged=%d\n", total, considered, flagged_cnt > "/dev/stderr" } ' # ---- 2. Sort + write per-bin flagged BED ------------------------------------ # bedtools merge requires a sorted BED. mosdepth output is usually already in # BAM-SQ order (which is often `chr1,chr2,...,chrX,chrY,chrM`), but we don't # rely on that — re-sort with LC_ALL=C for deterministic output. LC_ALL=C sort -k1,1 -k2,2n "$tmp_flag" > "$flagged" n_flag=$(wc -l < "$flagged" | tr -d ' ') echo "[1/3] wrote $flagged ($n_flag flagged bins)" # ---- 3. Merge --------------------------------------------------------------- merged="${OUT_PREFIX}.flagged.merged.bed" if [[ $n_flag -gt 0 ]]; then bedtools merge -i "$flagged" -d "$MERGE_GAP" \ -c 4,5 -o min,max > "$merged" else : > "$merged" fi n_merged=$(wc -l < "$merged" | tr -d ' ') echo "[2/3] wrote $merged ($n_merged merged intervals)" # ---- 4. Summary ------------------------------------------------------------- summary="${OUT_PREFIX}.summary.txt" flag_bp=$(awk '{s+=$3-$2} END {print s+0}' "$flagged") merged_bp=$(awk '{s+=$3-$2} END {print s+0}' "$merged") { echo "mosdepth_lowmapq_blacklist ($(date -u +%Y-%m-%dT%H:%M:%SZ))" echo "inputs:" echo " unf: $UNF" echo " filt: $FILT" echo "parameters:" echo " min_cov: $MIN_COV" echo " max_ratio: $MAX_RATIO" echo " merge_gap: $MERGE_GAP" echo "" printf "flagged bins: %8d (%12d bp, %7.1f Mb)\n" \ "$n_flag" "$flag_bp" "$(awk -v x="$flag_bp" 'BEGIN{printf "%.1f", x/1e6}')" printf "merged intervals: %8d (%12d bp, %7.1f Mb)\n" \ "$n_merged" "$merged_bp" "$(awk -v x="$merged_bp" 'BEGIN{printf "%.1f", x/1e6}')" echo "" echo "Top 20 widest merged intervals:" sort -k1,1 -k2,2n "$merged" \ | awk 'BEGIN{OFS="\t"} {print $1, $2, $3, $3-$2, $4, $5}' \ | sort -k4,4nr \ | head -20 \ | awk 'BEGIN{OFS="\t"; print "#chrom","start","end","width","min_ratio","max_cov"} {print}' } > "$summary" echo "[3/3] wrote $summary" # ---- 5. Optional per-region dump ------------------------------------------- if [[ $DUMP_PER -eq 1 ]]; then out="${OUT_PREFIX}.per_region.tsv.gz" # Prepend header, then gzip. Keep it streaming so we don't materialize the # full file uncompressed. { printf "#chrom\tstart\tend\tunf_cov\tfilt_cov\tratio\n"; cat "$tmp_per"; } \ | gzip -c > "$out" echo " wrote $out" fi echo cat "$summary" ================================================ FILE: scripts/plot_learn.sh ================================================ #!/usr/bin/env bash # plot_learn.sh — plot per-RG insert-size distributions from svaba learn data # # Usage: # plot_learn.sh [output.pdf] # # If the argument ends in .tsv or .tsv.gz, treat it as the learn file directly. # Otherwise treat it as a run ID and glob for ${ID}.*.learn.tsv.gz in the # current directory. # # Output PDF defaults to ${ID}.learn.pdf or ${stem}.learn.pdf. # # Requires: Rscript, R packages ggplot2 + data.table (both on CRAN). set -euo pipefail usage() { cat < [output.pdf] ID_or_file Either a svaba run ID (will find *.learn.tsv.gz files) or a path to a .tsv / .tsv.gz learn file directly. output.pdf Optional output path. Defaults to .learn.pdf Examples: $(basename "$0") my_run $(basename "$0") my_run.tumor.learn.tsv.gz $(basename "$0") my_run custom_output.pdf EOF exit 1 } [[ $# -lt 1 ]] && usage INPUT="$1" OUTPDF="${2:-}" # Determine the list of learn files FILES=() ID="" if [[ "$INPUT" == *.tsv.gz || "$INPUT" == *.tsv ]]; then # Direct file path [[ -f "$INPUT" ]] || { echo "ERROR: file not found: $INPUT" >&2; exit 1; } FILES=("$INPUT") # derive stem for default output name stem=$(basename "$INPUT") stem="${stem%.gz}" stem="${stem%.tsv}" stem="${stem%.learn}" ID="$stem" else # Treat as run ID — glob for learn files ID="$INPUT" shopt -s nullglob FILES=( ${ID}.*.learn.tsv.gz ${ID}.learn.tsv.gz ) shopt -u nullglob if [[ ${#FILES[@]} -eq 0 ]]; then echo "ERROR: no learn files found for ID '$ID'" >&2 echo " looked for: ${ID}.*.learn.tsv.gz, ${ID}.learn.tsv.gz" >&2 exit 1 fi fi [[ -z "$OUTPDF" ]] && OUTPDF="${ID}.learn.pdf" echo "Input files: ${FILES[*]}" echo "Output PDF: $OUTPDF" # Build a comma-separated list of files for R (no quoting — paths with # commas would break, but that's exotic enough to not worry about) FILE_LIST=$(IFS=,; echo "${FILES[*]}") LEARN_FILES="$FILE_LIST" Rscript --vanilla - "$OUTPDF" <<'REOF' suppressPackageStartupMessages({ library(data.table) library(ggplot2) }) args <- commandArgs(trailingOnly = TRUE) outpdf <- args[1] # The file list is passed via environment variable file_list <- strsplit(Sys.getenv("LEARN_FILES"), ",")[[1]] # Read and combine all files dt <- rbindlist(lapply(file_list, function(f) { d <- fread(f, sep = "\t") # add a source column from the filename stem <- basename(f) stem <- sub("\\.learn\\.tsv\\.gz$", "", stem) stem <- sub("\\.learn\\.tsv$", "", stem) d[, source := stem] d })) cat(sprintf("Loaded %s isize observations across %d RGs from %d files\n", format(nrow(dt), big.mark = ","), uniqueN(dt$rg), length(file_list))) # Summary stats per RG per source stats <- dt[, .( n = .N, mean = mean(isize), median = as.double(median(isize)), sd = sd(isize), p01 = quantile(isize, 0.01), p99 = quantile(isize, 0.99) ), by = .(source, rg)] cat("\nPer-RG summary:\n") print(stats[order(source, rg)]) # Trim extreme tails for plotting (1st-99th percentile per RG) dt_trim <- dt[, .SD[isize >= quantile(isize, 0.01) & isize <= quantile(isize, 0.99)], by = .(source, rg)] # Create label with n for facets dt_trim[, facet_label := paste0(rg, " (n=", format(.N, big.mark = ","), ")"), by = .(source, rg)] # Determine a reasonable number of columns for facet_wrap n_facets <- uniqueN(dt_trim[, paste(source, rg)]) ncol_facet <- min(4, ceiling(sqrt(n_facets))) # Plot p <- ggplot(dt_trim, aes(x = isize, fill = source)) + geom_histogram(bins = 80, alpha = 0.7, color = "grey30", linewidth = 0.2) + facet_wrap(~ facet_label, scales = "free_y", ncol = ncol_facet) + labs( title = "Insert size distribution per read group", subtitle = paste("Sources:", paste(unique(dt_trim$source), collapse = ", ")), x = "Insert size (bp)", y = "Count", fill = "BAM" ) + theme_minimal(base_size = 10) + theme( strip.text = element_text(size = 7), legend.position = if (uniqueN(dt_trim$source) > 1) "bottom" else "none" ) # Size the PDF to fit the facets n_rows <- ceiling(n_facets / ncol_facet) pdf_h <- max(4, 1.5 + n_rows * 2.5) pdf_w <- max(6, ncol_facet * 3) ggsave(outpdf, p, width = pdf_w, height = pdf_h, limitsize = FALSE) cat(sprintf("\nWrote %s (%d x %d inches)\n", outpdf, as.integer(pdf_w), as.integer(pdf_h))) REOF ================================================ FILE: scripts/r2c_for_contig.sh ================================================ #!/usr/bin/env bash # r2c_for_contig.sh # # Given a svaba contig name, reconstruct the per-contig read-to-contig # (r2c) alignment BAM for IGV. Uses the `bz:Z` aux tag that svaba writes # on every r2c-aligned read (cname-keyed) to pick the right reads out # of corrected.bam. # # Usage: # r2c_for_contig.sh CNAME CONTIGS_BAM CORRECTED_BAM [OUT_DIR] # # Example: # r2c_for_contig.sh c_chr12_10180001_10181001_0C \ # T2_fermi.contigs.bam T2_fermi.corrected.bam \ # r2c_debug/ # # Output (in OUT_DIR): # contig.fa single-contig reference FASTA (+ BWA & faidx indices) # reads.fq corrected reads tagged bz:Z: # r2c.bam(.bai) sorted+indexed read-to-contig BAM — load this in IGV # alongside contig.fa # # Tag semantics (v3): # bz:Z = cname list — every contig this read r2c-aligned to. This is # the right key for a per-contig read pull. # bi:Z = bp_id list — the specific variant rows this read supports # as ALT (matches r2c.txt.gz split_bps/disc_bps and bps.txt.gz # col 52). Pre-v3 this carried cnames; it no longer does. # If you want the ALT-supporter subset, set TAG=bi and pass # a bp_id as the first argument instead of a cname. set -euo pipefail if [[ $# -lt 3 ]]; then echo "usage: $0 CNAME CONTIGS_BAM CORRECTED_BAM [OUT_DIR]" >&2 exit 2 fi CNAME=$1 CONTIGS_BAM=$2 CORRECTED_BAM=$3 OUT_DIR=${4:-r2c_${CNAME}} for t in samtools bwa awk; do command -v "$t" >/dev/null || { echo "ERROR: $t not on PATH" >&2; exit 1; } done mkdir -p "$OUT_DIR" cd "$OUT_DIR" # ---- 1. Pull the contig sequence out of contigs.bam ----------------------- # svaba writes each contig as a single BAM record whose QNAME == cname. Take # SEQ as the reference. If the contig is reverse-complemented in the BAM # (FLAG & 16), revcomp it back so the coordinate system matches cname. # NB: use arithmetic bit extraction (int(flag/16)%2) instead of gawk's # `and(flag,16)` so this works with BSD awk on macOS as well as gawk. samtools view "../$CONTIGS_BAM" 2>/dev/null \ | awk -v c="$CNAME" ' BEGIN { found=0 } $1==c && !found { flag=$2+0; seq=$10 if (int(flag/16) % 2 == 1) { # revcomp n=length(seq); out="" for (i=n;i>=1;i--) { b=substr(seq,i,1) if (b=="A") out=out"T" else if (b=="T") out=out"A" else if (b=="C") out=out"G" else if (b=="G") out=out"C" else if (b=="a") out=out"t" else if (b=="t") out=out"a" else if (b=="c") out=out"g" else if (b=="g") out=out"c" else out=out"N" } seq=out } print ">"c; print seq found=1 } END { if (!found) exit 1 } ' > contig.fa || { echo "ERROR: contig $CNAME not found in $CONTIGS_BAM" >&2 exit 1 } echo "contig length: $(awk 'NR==2{print length($0)}' contig.fa) bp" # ---- 2. Build mini BWA index + faidx -------------------------------------- bwa index contig.fa 2> bwa_index.log samtools faidx contig.fa # ---- 3. Pull corrected reads tagged for this contig (boundary-aware) ------ # Default TAG=bz selects by cname (all r2c'd reads). TAG=bi selects by bp_id # (ALT-supporter subset only) — in that mode, the CNAME positional arg is # treated as a bp_id instead. See the v3 tag-semantics note in the header. TAG=${TAG:-bz} samtools view -h "../$CORRECTED_BAM" \ | awk -v c="$CNAME" -v tag="$TAG" 'BEGIN{OFS="\t"; pfx="^"tag":Z:"} /^@/ { print; next } { for (i=12;i<=NF;i++) if ($i ~ pfx) { v=substr($i,6); n=split(v,a,",") for (j=1;j<=n;j++) if (a[j]==c) { print; next } } }' \ | samtools fastq -@2 -n - > reads.fq 2> /dev/null n_reads=$(( $(wc -l < reads.fq) / 4 )) echo "reads tagged ${TAG}:Z:${CNAME}: $n_reads" if [[ "$n_reads" -eq 0 ]]; then echo "ERROR: no reads tagged for $CNAME in $CORRECTED_BAM" >&2 exit 1 fi # ---- 4. Re-align reads to the single-contig reference --------------------- # -k 15 lowers the minimum seed to pick up more partial matches on a small # reference; -a outputs secondary hits so you can spot reads that map to # multiple places on the contig (e.g. tandem repeats). Drop both if you # want svaba-equivalent alignments. bwa mem -t 4 -k 15 -a contig.fa reads.fq 2> bwa_mem.log \ | samtools sort -@4 -o r2c.bam - samtools index r2c.bam echo echo "Done. IGV: load contig.fa as Genome, then r2c.bam as track." echo "Files in $PWD:" ls -la r2c.bam r2c.bam.bai contig.fa contig.fa.fai reads.fq ================================================ FILE: scripts/search_sequence.sh ================================================ #!/bin/bash # ============================================================ # Search a BAM for reads matching any of the given sequences # (or their reverse complements) and write matches to a new BAM. # # Usage: # ./bam_grep.sh [seq2 ...] # # Example: # ./bam_grep.sh tumor.bam egfr_vIII ACTGACTGACTG TGCATGCATGCA # -> produces egfr_vIII.bam + egfr_vIII.bam.bai # ============================================================ set -euo pipefail # ---- Args ---- if [ $# -lt 3 ]; then echo "Usage: $0 [seq2 ...]" >&2 echo " Searches for each sequence and its reverse complement." >&2 echo " Output: .bam + .bam.bai" >&2 exit 1 fi INPUT_BAM="$1" OUT_PREFIX="$2" shift 2 SEQS=("$@") OUT_BAM="${OUT_PREFIX}.bam" THREADS=${THREADS:-4} # ---- Sanity checks ---- if [ ! -f "$INPUT_BAM" ]; then echo "ERROR: Input BAM not found: $INPUT_BAM" >&2 exit 1 fi if ! command -v samtools >/dev/null 2>&1; then echo "ERROR: samtools not found in PATH" >&2 exit 1 fi # ---- Build the combined regex: seq1|revcomp1|seq2|revcomp2|... ---- revcomp() { # Uppercase, complement, reverse echo "$1" | tr 'acgtnACGTN' 'tgcanTGCAN' | rev } PATTERNS=() echo "Searching for sequences (and reverse complements):" >&2 for seq in "${SEQS[@]}"; do # Uppercase and strip whitespace seq_up=$(echo "$seq" | tr 'a-z' 'A-Z' | tr -d '[:space:]') rc=$(revcomp "$seq_up") PATTERNS+=("$seq_up" "$rc") echo " $seq_up (rc: $rc)" >&2 done # Join patterns with | for egrep PATTERN=$(IFS='|'; echo "${PATTERNS[*]}") echo "" >&2 echo "Input: $INPUT_BAM" >&2 echo "Output: $OUT_BAM" >&2 echo "Threads: $THREADS" >&2 echo "" >&2 # ---- Filter ---- # samtools view streams SAM (header + reads), awk keeps headers and any read # whose sequence column (field 10) matches the pattern, samtools view -b # re-encodes BAM. samtools view -@ "$THREADS" -h "$INPUT_BAM" \ | awk -v pat="$PATTERN" ' /^@/ { print; next } $10 ~ pat { print } ' \ | samtools view -@ "$THREADS" -b -o "$OUT_BAM" - # ---- Sort (safer - preserves coordinate order for indexing) ---- # If input was already sorted, output is too (we only filter), but re-sort # defensively in case the original had unsorted regions. samtools sort -@ "$THREADS" -o "${OUT_BAM}.sorted" "$OUT_BAM" mv "${OUT_BAM}.sorted" "$OUT_BAM" # ---- Index ---- samtools index -@ "$THREADS" "$OUT_BAM" # ---- Report ---- N_READS=$(samtools view -c "$OUT_BAM") echo "Done. Matched reads: $N_READS" >&2 echo "Output: $OUT_BAM + ${OUT_BAM}.bai" >&2 ================================================ FILE: scripts/sort_and_dedupe_bps_old.sh ================================================ #!/usr/bin/env bash # # sort_and_dedupe_bps_old.sh — sort + dedup + PASS-filter for legacy # (pre-2.0) svaba bps.txt.gz files. # # Same logic as the v3 postprocess step but with auto-detected column # positions from the header row. Works on any bps.txt.gz schema as # long as the header has #chr1, confidence, and either maxlod or tlod. # # Usage: # sort_and_dedupe_bps_old.sh [options] # # -o DIR output directory (default: same as input) # -t THREADS parallel sort threads (default: 4) # -S SIZE sort buffer size (default: 4G) # -h, --help this message # # Outputs: # .sorted.txt.gz # .sorted.dedup.txt.gz # .sorted.dedup.pass.txt.gz # set -exo pipefail THREADS=4 SORT_BUFFER="4G" OUT_DIR="" print_usage() { sed -n '2,20p' "$0" | sed 's/^# \{0,1\}//'; } while [[ $# -gt 0 ]]; do case "$1" in -o) OUT_DIR="$2"; shift 2 ;; -t) THREADS="$2"; shift 2 ;; -S) SORT_BUFFER="$2"; shift 2 ;; -h|--help) print_usage; exit 0 ;; -*) echo "unknown option: $1" >&2; exit 2 ;; *) break ;; esac done if [[ $# -lt 1 ]]; then echo "usage: sort_and_dedupe_bps_old.sh [options] " >&2 exit 2 fi INPUT="$1" if [[ ! -r "$INPUT" ]]; then echo "cannot read: $INPUT" >&2 exit 1 fi # Determine reader case "$INPUT" in *.gz) READER="gzip -dc" ;; *) READER="cat" ;; esac # Derive output names BASENAME=$(basename "$INPUT" .txt.gz) BASENAME=$(basename "$BASENAME" .txt) [[ -z "$OUT_DIR" ]] && OUT_DIR=$(dirname "$INPUT") OUT_SORTED="${OUT_DIR}/${BASENAME}.sorted.txt.gz" OUT_DEDUP="${OUT_DIR}/${BASENAME}.sorted.dedup.txt.gz" OUT_PASS="${OUT_DIR}/${BASENAME}.sorted.dedup.pass.txt.gz" # Pick GNU sort if command -v gsort >/dev/null 2>&1; then SORT_BIN=gsort elif sort --version 2>&1 | grep -qi 'gnu' 2>/dev/null; then SORT_BIN=sort else echo "GNU sort not found (macOS: 'brew install coreutils' for gsort)" >&2 exit 1 fi # --- Auto-detect column positions from header --- HEADER=$($READER "$INPUT" | head -n 1 || true) if [[ "${HEADER:0:1}" != "#" ]]; then echo "ERROR: first line doesn't start with '#' — not a bps.txt.gz header?" >&2 echo " got: ${HEADER:0:80}..." >&2 exit 1 fi # Split header into array, find columns by name find_col() { local name="$1" local col col=$(echo "$HEADER" | awk -F'\t' -v name="$name" '{ for (i=1; i<=NF; i++) if ($i == name) { print i; exit } }') if [[ -z "$col" ]]; then return 1 fi echo "$col" } CONF_COL=$(find_col "confidence") || { echo "ERROR: 'confidence' column not found in header" >&2; exit 1; } # Try maxlod first (v3), then tlod (legacy) if LOD_COL=$(find_col "maxlod" 2>/dev/null); then LOD_NAME="maxlod" elif LOD_COL=$(find_col "tlod" 2>/dev/null); then LOD_NAME="tlod" else echo "ERROR: neither 'maxlod' nor 'tlod' column found in header" >&2 exit 1 fi echo "=== sort_and_dedupe_bps_old.sh ===" echo " input: $INPUT" echo " confidence: col $CONF_COL" echo " ${LOD_NAME}: col $LOD_COL" echo " sort: $SORT_BIN buffer=$SORT_BUFFER parallel=$THREADS" echo "" TMP_DIR=$(mktemp -d "${TMPDIR:-/tmp}/svaba_sort_bps.XXXXXXXX") trap 'rm -rf "$TMP_DIR"' EXIT INT TERM # --- 1) sort --- echo "[1/3] sorting..." { # Emit header first, then sort the data rows printf '%s\n' "$HEADER" $READER "$INPUT" | tail -n +2 | \ LC_ALL=C "$SORT_BIN" \ -t $'\t' \ -k1,1V \ -k2,2n \ -k3,3 \ -k4,4V \ -k5,5n \ -k6,6 \ -k${LOD_COL},${LOD_COL}gr \ -S "$SORT_BUFFER" \ --parallel="$THREADS" \ -T "$TMP_DIR" } | gzip -c > "$OUT_SORTED" echo " wrote $OUT_SORTED" # --- 2) dedup by breakpoint pair (first row per key wins = highest lod) --- echo "[2/3] deduplicating..." gzip -dc "$OUT_SORTED" | awk -F'\t' -v OFS='\t' ' NR == 1 && $0 ~ /^#/ { print; next } { key = $1 FS $2 FS $3 FS $4 FS $5 FS $6 if (!(key in seen)) { seen[key] = 1; print } } ' | gzip -c > "$OUT_DEDUP" echo " wrote $OUT_DEDUP" # --- 3) PASS-filter --- echo "[3/3] PASS-filtering..." gzip -dc "$OUT_DEDUP" | awk -F'\t' -v OFS='\t' -v CCOL="$CONF_COL" ' NR == 1 && $0 ~ /^#/ { print; next } $CCOL == "PASS" { print } ' | gzip -c > "$OUT_PASS" echo " wrote $OUT_PASS" # Summary N_SORTED=$(gzip -dc "$OUT_SORTED" | tail -n +2 | wc -l) N_DEDUP=$(gzip -dc "$OUT_DEDUP" | tail -n +2 | wc -l) N_PASS=$(gzip -dc "$OUT_PASS" | tail -n +2 | wc -l) echo "" echo " sorted: $N_SORTED dedup: $N_DEDUP PASS: $N_PASS" echo "=== done ===" ================================================ FILE: scripts/sort_bps.sh ================================================ #!/usr/bin/env bash # # sort_bps.sh — sort a svaba bps.txt.gz file by (chr1, pos1, chr2, pos2, -somlod) # # Reads: ${ID}.bps.txt.gz # Writes: ${ID}.bps.sorted.txt.gz # # Preserves the leading "#chr1 ..." header row, then sorts the body by: # 1) chr1 (natural/version order) ascending # 2) pos1 (numeric) ascending # 3) chr2 (natural/version order) ascending # 4) pos2 (numeric) ascending # 5) somlod (general numeric) DESCENDING (higher first) # # The sort is external — GNU sort spills sorted chunks to a scratch directory # and merges them, so memory stays bounded even on 10+ GB inputs. The scratch # directory is auto-created with mktemp and cleaned up on exit. Works on both # Linux and macOS; on macOS you'll want GNU coreutils installed (`brew install # coreutils`) so `gsort` is available — BSD sort does not support -V / --parallel. # # Usage: # ./sort_bps.sh # ./sort_bps.sh -h | --help # # Options via environment variables (all optional): # BUFFER_SIZE per-chunk memory for sort (default: 2G) # PARALLEL threads used by the sort phase (default: 4) # INPUT_DIR where to look for the input (default: .) # OUTPUT_DIR where to write the output (default: $INPUT_DIR) # KEEP_TMP 1 = don't delete the scratch dir on exit (for debugging) # # Examples: # ./sort_bps.sh tumor_vs_normal # BUFFER_SIZE=8G PARALLEL=8 ./sort_bps.sh tumor_vs_normal # INPUT_DIR=/data/run42 OUTPUT_DIR=/tmp ./sort_bps.sh run42 # set -euo pipefail # ------------------------------------------------------------------ usage --- print_usage() { cat <<'EOF' sort_bps.sh — sort a svaba bps.txt.gz file Usage: sort_bps.sh sort_bps.sh -h | --help Reads /.bps.txt.gz Writes /.bps.sorted.txt.gz Sort keys (in order): 1. chr1 ascending (natural / version order — chr1, chr2, ..., chrX) 2. pos1 ascending 3. chr2 ascending 4. pos2 ascending 5. somlod DESCENDING (higher somatic LOD first; "NA" sorts last) Options (environment variables): BUFFER_SIZE per-chunk memory for sort (default: 2G) PARALLEL threads used by the sort phase (default: 4) INPUT_DIR where to look for the input (default: .) OUTPUT_DIR where to write the output (default: $INPUT_DIR) KEEP_TMP 1 = keep the scratch dir for debugging (default: unset) Examples: sort_bps.sh tumor_vs_normal BUFFER_SIZE=8G PARALLEL=8 sort_bps.sh tumor_vs_normal INPUT_DIR=/data/run42 OUTPUT_DIR=/tmp sort_bps.sh run42 Notes: * Uses GNU sort. On macOS install it via `brew install coreutils` — the script will pick up `gsort` automatically. BSD sort does not support -V or --parallel and will not work. * Memory is bounded by BUFFER_SIZE; scratch disk usage is roughly the uncompressed size of the input (often 6-10x the .gz size). EOF } # ---------------------------------------------------------------- args ----- if [[ $# -eq 0 ]]; then print_usage exit 1 fi case "${1:-}" in -h|--help) print_usage exit 0 ;; -*) echo "sort_bps.sh: unknown option '$1'" >&2 echo "Run 'sort_bps.sh --help' for usage." >&2 exit 2 ;; esac ID=$1 if [[ -z "$ID" ]]; then echo "sort_bps.sh: is required" >&2 print_usage exit 2 fi INPUT_DIR=${INPUT_DIR:-.} OUTPUT_DIR=${OUTPUT_DIR:-$INPUT_DIR} BUFFER_SIZE=${BUFFER_SIZE:-2G} PARALLEL=${PARALLEL:-4} IN="${INPUT_DIR%/}/${ID}.bps.txt.gz" OUT="${OUTPUT_DIR%/}/${ID}.bps.sorted.txt.gz" if [[ ! -r "$IN" ]]; then echo "sort_bps.sh: cannot read input '$IN'" >&2 exit 3 fi mkdir -p "$(dirname "$OUT")" # ----------------------------------------------------------- tool lookup --- # Prefer `gsort` (GNU coreutils on macOS via homebrew); fall back to `sort` # if it reports itself as GNU. Bail out if neither works. pick_sort() { if command -v gsort >/dev/null 2>&1; then echo gsort return 0 fi if sort --version 2>/dev/null | head -n 1 | grep -qi 'gnu'; then echo sort return 0 fi return 1 } if ! SORT_BIN=$(pick_sort); then echo "sort_bps.sh: GNU sort not found." >&2 echo " On macOS: brew install coreutils (provides 'gsort')" >&2 echo " On Linux: install the 'coreutils' package" >&2 exit 4 fi # zcat on macOS appends .Z instead of reading .gz — use `gzip -dc` to stay # portable. ZCAT="gzip -dc" # ---------------------------------------------------------- scratch dir --- # mktemp -d is portable across Linux and macOS. The template form with a # trailing XXXXXX works on both. TMP=$(mktemp -d "${TMPDIR:-/tmp}/svaba_bps_sort.XXXXXXXX") cleanup() { if [[ "${KEEP_TMP:-0}" == "1" ]]; then echo "sort_bps.sh: leaving scratch dir at $TMP" >&2 else rm -rf "$TMP" fi } trap cleanup EXIT INT TERM # ------------------------------------------------------------- the sort --- echo "sort_bps.sh: sorting $IN -> $OUT" >&2 echo "sort_bps.sh: sort=$SORT_BIN buffer=$BUFFER_SIZE parallel=$PARALLEL tmp=$TMP" >&2 # Extract the header once (if present) so we don't have to zcat twice. HEADER_FILE="$TMP/header.txt" BODY_PIPE=0 # Peek at the first line. If it starts with '#', save it; otherwise there is # no header and we just sort the whole file. FIRST_LINE=$($ZCAT "$IN" | head -n 1 || true) if [[ "${FIRST_LINE:0:1}" == "#" ]]; then printf '%s\n' "$FIRST_LINE" > "$HEADER_FILE" BODY_PIPE=1 fi { if [[ $BODY_PIPE -eq 1 ]]; then cat "$HEADER_FILE" $ZCAT "$IN" | tail -n +2 else $ZCAT "$IN" fi | { # The header (if any) is the first line; pass it through untouched, # then pipe the rest into sort. Using `read` here keeps everything # streaming — no intermediate uncompressed file on disk. if [[ $BODY_PIPE -eq 1 ]]; then IFS= read -r hdr printf '%s\n' "$hdr" fi LC_ALL=C "$SORT_BIN" \ -t $'\t' \ -k1,1V -k2,2n -k4,4V -k5,5n -k37,37gr \ -S "$BUFFER_SIZE" \ --parallel="$PARALLEL" \ -T "$TMP" } } | gzip -c > "$OUT" echo "sort_bps.sh: done -> $OUT" >&2 ================================================ FILE: scripts/svaba_cloud.sh ================================================ #!/usr/bin/env bash # # svaba_cloud.sh — scatter svaba across GCP VMs, one partition per VM. # # Takes the svaba run command you'd normally run locally and distributes # it across N worker VMs, each handling a chromosome partition. The # script manages -k (regions) and -a (analysis ID) per worker — you # provide everything else exactly as you would on the command line. # # Workers boot from a pre-built image (--image) that has svaba, htslib, # jemalloc, and the reference genome baked in. A shared read-only data # disk provides the BAM files. # # Usage: # svaba_cloud.sh [cloud options] -- # # Cloud options (before the --): # --id STR analysis ID prefix (required) # --data-disk NAME GCP persistent disk name for BAMs (required) # --bucket gs://... GCS bucket for outputs (required) # --partitions N number of VMs / partitions (default: 6) # --machine TYPE GCP machine type (default: n2-highcpu-16) # --zone ZONE GCP zone (default: us-central1-a) # --image NAME GCP image for workers (default: svaba-worker-image) # --mount-point DIR where data disk mounts (default: /mnt/data) # --boot-disk-size STR boot disk size (default: 50GB) # --regions-file FILE custom partition file (one comma-sep line per partition) # --merge merge + postprocess after all workers finish # --keep-vms don't delete worker VMs on completion # --dry-run print commands without executing # -h, --help this message # # Everything after -- is passed verbatim to `svaba run` on each worker, # with -k and -a injected by the script. Do NOT include -k or -a in # your svaba args. # # Examples: # # 5-way scatter, n2-highcpu-16, merge at the end # svaba_cloud.sh \ # --id my_run --data-disk svaba-data \ # --bucket gs://my-bucket/svaba-run \ # --partitions 5 --machine n2-highcpu-16 --merge \ # -- \ # svaba run \ # -t /mnt/data/tumor.bam -n /mnt/data/normal.bam \ # -G /home/user/ref/hg38.fa -p 14 \ # --blacklist /home/user/tracks/blacklist.bed # # # Dry run # svaba_cloud.sh --id test --data-disk d1 \ # --bucket gs://b/r --dry-run \ # -- svaba run -t /mnt/data/t.bam -G /ref/hg38.fa -p 8 # set -euo pipefail # ----------------------------------------------------------- defaults --- MACHINE="n2-highcpu-16" ZONE="us-central1-a" PARTITIONS=6 MOUNT="/mnt/data" BOOT_DISK_SIZE="50GB" IMAGE="svaba-worker-image" DO_MERGE=0 KEEP_VMS=0 DRY_RUN=0 REGIONS_FILE="" # Required DATA_DISK="" BUCKET="" ID="" # ---------------------------------------------------------------- help --- print_usage() { sed -n '2,50p' "$0" | sed 's/^# \{0,1\}//' } # -------------------------------------------------------------- parse --- # Everything before -- is cloud options; everything after is svaba args. SVABA_ARGS=() while [[ $# -gt 0 ]]; do case "$1" in --id) ID="$2"; shift 2 ;; --data-disk) DATA_DISK="$2"; shift 2 ;; --bucket) BUCKET="$2"; shift 2 ;; --partitions) PARTITIONS="$2"; shift 2 ;; --machine) MACHINE="$2"; shift 2 ;; --zone) ZONE="$2"; shift 2 ;; --image) IMAGE="$2"; shift 2 ;; --mount-point) MOUNT="$2"; shift 2 ;; --boot-disk-size) BOOT_DISK_SIZE="$2"; shift 2 ;; --regions-file) REGIONS_FILE="$2"; shift 2 ;; --merge) DO_MERGE=1; shift ;; --keep-vms) KEEP_VMS=1; shift ;; --dry-run) DRY_RUN=1; shift ;; -h|--help) print_usage; exit 0 ;; --) shift; SVABA_ARGS=("$@"); break ;; -*) echo "svaba_cloud.sh: unknown option '$1'" >&2 echo "run 'svaba_cloud.sh --help' for usage" >&2 exit 2 ;; *) # No -- separator; assume everything from here on is svaba args SVABA_ARGS=("$@"); break ;; esac done # ------------------------------------------------------- validate args --- missing=() [[ -z "$ID" ]] && missing+=("--id") [[ -z "$DATA_DISK" ]] && missing+=("--data-disk") [[ -z "$BUCKET" ]] && missing+=("--bucket") [[ ${#SVABA_ARGS[@]} -eq 0 ]] && missing+=("svaba run args (after --)") if [[ ${#missing[@]} -gt 0 ]]; then echo "svaba_cloud.sh: missing required: ${missing[*]}" >&2 exit 2 fi BUCKET=${BUCKET%/} # Sanitize ID for GCP resource names GCP_ID=$(echo "$ID" | tr '[:upper:]' '[:lower:]' | tr '_' '-' | sed 's/[^a-z0-9-]//g') if [[ "$GCP_ID" != "$ID" ]]; then echo "note: sanitized ID for GCP names: '$ID' -> '$GCP_ID'" fi # Strip "svaba run" prefix if present (we add it back ourselves) if [[ "${SVABA_ARGS[0]:-}" == "svaba" ]]; then SVABA_ARGS=("${SVABA_ARGS[@]:1}") fi if [[ "${SVABA_ARGS[0]:-}" == "run" ]]; then SVABA_ARGS=("${SVABA_ARGS[@]:1}") fi # Build the svaba args string for embedding in the startup script SVABA_ARGS_STR="" for arg in "${SVABA_ARGS[@]}"; do # Quote args that contain spaces if [[ "$arg" == *" "* ]]; then SVABA_ARGS_STR+="\"${arg}\" " else SVABA_ARGS_STR+="${arg} " fi done # Extract -p value from svaba args for the merge step (default 8) THREADS=8 for i in "${!SVABA_ARGS[@]}"; do if [[ "${SVABA_ARGS[$i]}" == "-p" ]]; then THREADS="${SVABA_ARGS[$((i+1))]}" break fi done # ---------------------------------------- default hg38 6-way partition --- default_regions() { local n=$1 case $n in 1) echo "chr1,chr2,chr3,chr4,chr5,chr6,chr7,chr8,chr9,chr10,chr11,chr12,chr13,chr14,chr15,chr16,chr17,chr18,chr19,chr20,chr21,chr22,chrX" ;; 2) echo "chr1,chr2,chr3,chr4,chr5,chr6,chr7" echo "chr8,chr9,chr10,chr11,chr12,chr13,chr14,chr15,chr16,chr17,chr18,chr19,chr20,chr21,chr22,chrX" ;; 3) echo "chr1,chr2,chr3" echo "chr4,chr5,chr6,chr7,chr8" echo "chr9,chr10,chr11,chr12,chr13,chr14,chr15,chr16,chr17,chr18,chr19,chr20,chr21,chr22,chrX" ;; 4) echo "chr1,chr2" echo "chr3,chr4,chr5" echo "chr6,chr7,chr8,chr9,chr10" echo "chr11,chr12,chr13,chr14,chr15,chr16,chr17,chr18,chr19,chr20,chr21,chr22,chrX" ;; 5) echo "chr1,chr2" echo "chr3,chr4,chr5" echo "chr6,chr7,chr8,chr9" echo "chr10,chr11,chr12,chr13,chr14" echo "chr15,chr16,chr17,chr18,chr19,chr20,chr21,chr22,chrX" ;; 6) echo "chr1,chr2" echo "chr3,chr4,chr5" echo "chr6,chr7,chr8,chr9" echo "chr10,chr11,chr12,chr13" echo "chr14,chr15,chr16,chr17,chr18" echo "chr19,chr20,chr21,chr22,chrX" ;; *) for c in chr{1..22} chrX; do echo "$c"; done | head -n "$n" ;; esac } declare -a REGIONS if [[ -n "$REGIONS_FILE" ]]; then while IFS= read -r line; do REGIONS+=("$line"); done < "$REGIONS_FILE" PARTITIONS=${#REGIONS[@]} else while IFS= read -r line; do REGIONS+=("$line"); done < <(default_regions "$PARTITIONS") fi if [[ ${#REGIONS[@]} -ne $PARTITIONS ]]; then echo "svaba_cloud.sh: got ${#REGIONS[@]} region lines but expected $PARTITIONS" >&2 exit 2 fi # ----------------------------------------------------------- run/exec --- run_cmd() { if [[ $DRY_RUN -eq 1 ]]; then echo "[dry-run] $*" else "$@" fi } # ================================================================ # STEP 1: Create worker VMs # ================================================================ echo "============================================================" echo "svaba_cloud.sh: launching $PARTITIONS workers" echo " machine=$MACHINE zone=$ZONE image=$IMAGE" echo " data_disk=$DATA_DISK bucket=$BUCKET id=$ID" echo " svaba args: ${SVABA_ARGS_STR}" echo "============================================================" SCRIPT_TMPDIR=$(mktemp -d) trap 'rm -rf "$SCRIPT_TMPDIR"' EXIT VM_NAMES=() for i in $(seq 1 "$PARTITIONS"); do vm="svaba-${GCP_ID}-worker-${i}" VM_NAMES+=("$vm") SCRIPT_FILE="${SCRIPT_TMPDIR}/startup_${i}.sh" cat > "$SCRIPT_FILE" < /var/log/svaba_startup.log 2>&1 set -euxo pipefail echo "=== svaba worker ${i} starting at \$(date) ===" # --- mount data disk --- mkdir -p ${MOUNT} /mnt/output echo "waiting for data disk..." for attempt in \$(seq 1 30); do [[ -b /dev/sdb ]] && break echo " attempt \${attempt}: /dev/sdb not yet available" sleep 2 done if [[ ! -b /dev/sdb ]]; then echo "FATAL: /dev/sdb not found after 60s" echo "available block devices:" lsblk exit 1 fi MOUNT_DEV=/dev/sdb [[ -b /dev/sdb1 ]] && MOUNT_DEV=/dev/sdb1 echo "mounting \${MOUNT_DEV} -> ${MOUNT}" mount -o ro,noload \${MOUNT_DEV} ${MOUNT} || mount -o ro \${MOUNT_DEV} ${MOUNT} echo "data disk mounted, contents:" ls ${MOUNT}/ # --- run svaba --- PART_ID="${ID}_part${i}" cd /mnt/output echo "=== starting svaba at \$(date) ===" svaba run \\ ${SVABA_ARGS_STR} \\ -k ${REGIONS[$((i-1))]} \\ -a \${PART_ID} \\ 2>&1 | tee \${PART_ID}.startup.log echo "=== svaba finished at \$(date) ===" echo "output files:" ls -lh /mnt/output/ # --- upload results --- echo "uploading to ${BUCKET}/" gsutil -m cp \\ \${PART_ID}.bps.txt.gz \\ \${PART_ID}.log \\ \${PART_ID}.startup.log \\ \${PART_ID}.contigs.bam \\ \${PART_ID}.runtime.txt \\ ${BUCKET}/ # Upload optional outputs if they exist (--dump-reads) for f in \${PART_ID}.discordant.bam \${PART_ID}.corrected.bam \${PART_ID}.r2c.txt.gz; do [[ -f "\$f" ]] && gsutil cp "\$f" ${BUCKET}/ || true done # Signal completion echo "=== uploading done marker at \$(date) ===" echo "DONE" | gsutil cp - ${BUCKET}/.done_part${i} echo "=== worker ${i} complete ===" STARTUP_EOF echo "[${i}/${PARTITIONS}] creating $vm regions=${REGIONS[$((i-1))]}" run_cmd gcloud compute instances create "$vm" \ --zone="$ZONE" \ --machine-type="$MACHINE" \ --image="$IMAGE" \ --disk="name=${DATA_DISK},mode=ro,device-name=svaba-data" \ --boot-disk-size="$BOOT_DISK_SIZE" \ --scopes=storage-rw \ --metadata-from-file=startup-script="$SCRIPT_FILE" # Stagger VM creation to avoid disk-attach races [[ $i -lt $PARTITIONS ]] && sleep 5 done echo "all $PARTITIONS VMs created" # ================================================================ # STEP 2: Wait for all workers to finish # ================================================================ echo "waiting for workers to complete..." echo "(checking ${BUCKET}/.done_part* every 60s)" poll_done() { local expected=$1 while true; do local count count=$(gsutil ls "${BUCKET}/.done_part*" 2>/dev/null | grep -c '\.done_part' || true) if [[ $count -ge $expected ]]; then echo "all $expected partitions complete" return 0 fi echo " $(date +%H:%M:%S) $count / $expected done" sleep 60 done } if [[ $DRY_RUN -eq 0 ]]; then poll_done "$PARTITIONS" fi # ================================================================ # STEP 3: Tear down workers (unless --keep-vms) # ================================================================ if [[ $KEEP_VMS -eq 0 ]]; then echo "deleting worker VMs..." for vm in "${VM_NAMES[@]}"; do run_cmd gcloud compute instances delete "$vm" \ --zone="$ZONE" --quiet & done wait echo "all workers deleted" else echo "keeping VMs (--keep-vms): ${VM_NAMES[*]}" fi # Clean up done markers if [[ $DRY_RUN -eq 0 ]]; then gsutil -m rm "${BUCKET}/.done_part*" 2>/dev/null || true fi # ================================================================ # STEP 4: Merge + postprocess (optional, --merge) # ================================================================ if [[ $DO_MERGE -eq 1 ]]; then if [[ $DRY_RUN -eq 1 ]]; then echo "[dry-run] would merge ${PARTITIONS} partitions from ${BUCKET}/" echo "svaba_cloud.sh: finished" exit 0 fi echo "============================================================" echo "MERGE: downloading and postprocessing" echo "============================================================" MERGE_DIR="${ID}_cloud_merge" mkdir -p "$MERGE_DIR" cd "$MERGE_DIR" # Download all bps.txt.gz files for i in $(seq 1 "$PARTITIONS"); do gsutil cp "${BUCKET}/${ID}_part${i}.bps.txt.gz" . done # Concatenate (gzip is concat-safe per RFC 1952) echo "concatenating ${PARTITIONS} bps.txt.gz files..." cat ${ID}_part*.bps.txt.gz > "${ID}.bps.txt.gz" # Merge contigs BAMs echo "downloading and merging contigs BAMs..." CONTIG_BAMS=() for i in $(seq 1 "$PARTITIONS"); do gsutil cp "${BUCKET}/${ID}_part${i}.contigs.bam" . CONTIG_BAMS+=("${ID}_part${i}.contigs.bam") done samtools merge -f "${ID}.contigs.bam" "${CONTIG_BAMS[@]}" rm -f "${CONTIG_BAMS[@]}" # Merge runtime.txt (just cat, first file has the header) echo "merging runtime.txt..." gsutil cp "${BUCKET}/${ID}_part1.runtime.txt" "${ID}.runtime.txt" for i in $(seq 2 "$PARTITIONS"); do gsutil cp "${BUCKET}/${ID}_part${i}.runtime.txt" tmp_rt.txt tail -n +2 tmp_rt.txt >> "${ID}.runtime.txt" rm -f tmp_rt.txt done # Merge optional r2c.txt.gz if present R2C_FILES=() for i in $(seq 1 "$PARTITIONS"); do if gsutil -q stat "${BUCKET}/${ID}_part${i}.r2c.txt.gz" 2>/dev/null; then gsutil cp "${BUCKET}/${ID}_part${i}.r2c.txt.gz" . R2C_FILES+=("${ID}_part${i}.r2c.txt.gz") fi done if [[ ${#R2C_FILES[@]} -gt 0 ]]; then echo "merging ${#R2C_FILES[@]} r2c.txt.gz files..." cat "${R2C_FILES[@]}" > "${ID}.r2c.txt.gz" rm -f "${R2C_FILES[@]}" fi # Postprocess SCRIPT_DIR="$(cd "$(dirname "$0")" && pwd)" if [[ -x "${SCRIPT_DIR}/svaba_postprocess.sh" ]]; then echo "running svaba_postprocess.sh..." "${SCRIPT_DIR}/svaba_postprocess.sh" -t "$THREADS" -m 4G "$ID" else echo "svaba_postprocess.sh not found at ${SCRIPT_DIR}; skipping" echo "run manually: scripts/svaba_postprocess.sh -t 8 -m 4G ${ID}" fi echo "============================================================" echo "DONE. Merged outputs in: $(pwd)" echo " ${ID}.bps.sorted.dedup.txt.gz" echo " ${ID}.contigs.bam" echo " ${ID}.runtime.txt" echo "============================================================" fi echo "svaba_cloud.sh: finished" ================================================ FILE: scripts/svaba_local_function.sh ================================================ # svaba_local_function.sh — sourceable bash helpers for common svaba tasks. # # Designed to be dot-sourced into an interactive shell (bash/zsh), NOT # executed as a script. Usage: # # # in your ~/.bashrc or ~/.zshrc (or run once per shell session): # source ~/git/svaba/scripts/svaba_local_function.sh # # # then use any of the svaba_* helpers: # svaba_help # svaba_grep_contig fermi4.r2c.pass.txt.gz c_fermi_chr19_48755001_48780001_13C # # Every function is prefixed `svaba_` to stay out of the way of anything # else on your PATH. Every function responds to `-h` / `--help` with its # own usage block, so you don't need to remember signatures — just type # the name and `-h`. # # Set `SVABA_FUNCTIONS_QUIET=1` before sourcing to suppress the banner. # --------------------------------------------------------------------- # svaba_help — list all svaba_* functions defined here with one-liners. # --------------------------------------------------------------------- svaba_help() { cat <<'EOF' svaba_* helper functions (from scripts/svaba_local_function.sh). Call any function with -h / --help for detailed usage. svaba_help this message svaba_grep_contig FILE CNAME [OUTFILE] pull one contig's rows out of an r2c TSV, preserving the header so the result loads in viewer/r2c_explorer.html svaba_pass_cnames FILE print cnames of all PASS variants in a bps.txt[.gz] svaba_pass_somatic_cnames FILE same, restricted to PASS with somlod >= 1 svaba_bps_cols print the bps.txt.gz column-index reference svaba_igv LOCUS [PORT] tell a running IGV (default port 60151) to jump to LOCUS svaba_swap_bps IN.bps.txt.gz OUT.bps.txt.gz sanity-check helper: randomly (50%/row) swap bp1<->bp2 fields across all paired columns, so downstream compare code can be tested for orientation-invariance svaba_bam_grep IN.bam SEQUENCE OUT.bam [REGION] pull reads whose SEQ contains SEQUENCE (or its reverse complement) into a sub-BAM. Optional REGION restricts the scan to a samtools-style locus first (requires indexed IN.bam) EOF } # --------------------------------------------------------------------- # svaba_grep_contig — the most frequent ask: pull one contig's rows out # of an r2c TSV, preserving the header row so the sliced file loads in # viewer/r2c_explorer.html without falling back to the canonical schema. # --------------------------------------------------------------------- svaba_grep_contig() { if [[ "${1:-}" == "-h" || "${1:-}" == "--help" || $# -lt 2 ]]; then cat <<'EOF' svaba_grep_contig FILE CNAME [OUTFILE] Extract the "contig" row plus all "read" rows for a given cname from an r2c TSV produced by svaba run --dump-reads (or the PASS / PASS- somatic subsets from svaba_postprocess.sh). The header line is always preserved so the output loads directly in viewer/r2c_explorer.html. FILE input r2c TSV — .txt.gz (auto-decompressed) or plain .txt CNAME contig name (awk regex; a prefix or unique substring works) OUTFILE output path (default: CNAME.txt — note: plain text, not .gz) Examples: svaba_grep_contig fermi4_full.r2c.pass.txt.gz c_fermi_chr19_48755001_48780001_13C svaba_grep_contig fermi4.r2c.pass.txt.gz c_fermi_chr7 /tmp/chr7_contigs.txt EOF return 1 fi local file="$1" cname="$2" out="${3:-${2}.txt}" if [[ ! -r "$file" ]]; then echo "svaba_grep_contig: cannot read $file" >&2 return 2 fi # Decompress if .gz, cat otherwise. `gzip -dc` works on macOS where # plain `zcat` expects a .Z suffix rather than .gz. local reader case "$file" in *.gz) reader="gzip -dc" ;; *) reader="cat" ;; esac $reader "$file" | awk -v cname="$cname" 'NR==1 || $0 ~ cname' > "$out" local n n=$(wc -l < "$out" | awk '{print $1}') echo "svaba_grep_contig: wrote $out ($n lines incl. header)" >&2 } # --------------------------------------------------------------------- # svaba_pass_cnames — one-liner for pulling the cnames of PASS variants # out of bps.txt[.gz]. Useful for feeding into svaba_grep_contig in a # loop, or for sanity-checking counts. # --------------------------------------------------------------------- svaba_pass_cnames() { if [[ "${1:-}" == "-h" || "${1:-}" == "--help" || $# -lt 1 ]]; then cat <<'EOF' svaba_pass_cnames FILE Print cnames (bps.txt col 30 = contig_and_region) for every PASS row (col 32 == "PASS") in FILE. Output is sorted + unique. FILE ${ID}.bps.txt.gz (or plain .txt) Example: svaba_pass_cnames tumor_normal.bps.txt.gz | wc -l EOF return 1 fi local file="$1" reader case "$file" in *.gz) reader="gzip -dc" ;; *) reader="cat" ;; esac $reader "$file" | awk -F'\t' 'NR>1 && $32=="PASS" {print $30}' | LC_ALL=C sort -u } # --------------------------------------------------------------------- # svaba_pass_somatic_cnames — same as above but restricted to the # PASS + somlod >= 1 subset (i.e. what ends up in r2c.pass.somatic). # --------------------------------------------------------------------- svaba_pass_somatic_cnames() { if [[ "${1:-}" == "-h" || "${1:-}" == "--help" || $# -lt 1 ]]; then cat <<'EOF' svaba_pass_somatic_cnames FILE Print cnames for every PASS + somatic variant (col 32 == "PASS" AND col 37 somlod >= 1) in FILE. Non-numeric somlod values ("NA", missing) coerce to 0 and are excluded. Output is sorted + unique. FILE ${ID}.bps.txt.gz (or plain .txt) EOF return 1 fi local file="$1" reader case "$file" in *.gz) reader="gzip -dc" ;; *) reader="cat" ;; esac $reader "$file" | awk -F'\t' 'NR>1 && $32=="PASS" && $37+0 >= 1 {print $30}' | LC_ALL=C sort -u } # --------------------------------------------------------------------- # svaba_bps_cols — quick printable reference for the bps.txt column # layout. Useful when you're writing one-off awks and need to remember # "which column is somlod again?" # --------------------------------------------------------------------- svaba_bps_cols() { cat <<'EOF' svaba bps.txt column layout (from BreakPoint::toFileString). Column numbers are 1-indexed (awk $1 == chr1). col name notes --- --------------------- ----------------------------------------- 1 chr1 2 pos1 3 strand1 4 chr2 5 pos2 6 strand2 7 ref 8 alt 9 span 10 split 11 alt_count 12 cov 13 cigar 14 cigar_near 15 dmq1 discordant-MAPQ, side 1 16 dmq2 discordant-MAPQ, side 2 17 dcn discordant count, normal 18 dct discordant count, tumor 19 mapq1 contig-alignment MAPQ, side 1 20 mapq2 21 nm1 22 nm2 23 as1 24 as2 25 sub1 26 sub2 27 homol 28 insert 29 repeat 30 contig_and_region <- cname, used as r2c join key 31 naln 32 conf <- "PASS" / "LOWLOD" / etc. 33 type (older files: named "evidence") 34 qual 35 2ndary (older files: named "secondary") 36 somatic 37 somlod <- "somatic LOD" 38 maxlod <- max over per-sample LO 39 dbsnp 40 contig_conf1 41 contig_conf2 42 cpos1 v2 only (post-SvABA2.0 refilter round-trip) 43 cpos2 v2 only 44 lmatch v2 only 45 rmatch v2 only 46 scov1 v2 only 47 scov2 v2 only 48 local1 v2 only 49 local2 v2 only 50 ctglen v2 only 51 flipped v2 only 52 bp_id v3 only — unique per-BP identifier, format "bpTTTNNNNNNNN" (thread TTT, counter NNNNNNNN). Cross-references to r2c.txt.gz's split_bps / disc_bps columns so you can tell which BP on a contig a given read actually supports. ("." on very old v2 files rewritten through a v3 emitter that left id unset.) 53+ per-sample blocks FORMAT: GT:AD:DP:SR:DR:GQ:PL:LO:LO_n (one block per BAM, order from the header row) EOF } # --------------------------------------------------------------------- # svaba_igv — nudge a running IGV to a locus via its HTTP command port. # Requires IGV's "Enable port" preference to be on (default port 60151). # Useful in loops while eyeballing variants. # --------------------------------------------------------------------- svaba_igv() { if [[ "${1:-}" == "-h" || "${1:-}" == "--help" || $# -lt 1 ]]; then cat <<'EOF' svaba_igv LOCUS [PORT] POST a goto request to a running IGV instance so it navigates to LOCUS. IGV must have the "Enable port" preference turned on. LOCUS anything IGV understands: chr:pos, chr:start-end, gene name, ... PORT IGV command port (default 60151) Examples: svaba_igv chr7:55249071 svaba_igv chr17:7675000-7687000 EOF return 1 fi local locus="$1" port="${2:-60151}" # Use curl (widely available on macOS + linux). encodeURIComponent-style # minimal escaping: swap spaces for %20 so gene/locus queries work. local enc enc=$(printf '%s' "$locus" | sed 's/ /%20/g') if ! curl -sS --max-time 3 "http://localhost:${port}/goto?locus=${enc}" >/dev/null; then echo "svaba_igv: IGV not reachable at localhost:${port} (is IGV running with 'Enable port' on?)" >&2 return 3 fi echo "svaba_igv: IGV -> $locus" } # --------------------------------------------------------------------- # svaba_swap_bps — sanity-check helper. Randomly (50% per row) swap all # bp1-side <-> bp2-side paired columns in a bps.txt[.gz], producing an # orientation-randomized file. Downstream compare code that's supposed # to be orientation-invariant should treat in.bps.txt.gz and its # swapped version as equivalent. # --------------------------------------------------------------------- svaba_swap_bps() { if [[ "${1:-}" == "-h" || "${1:-}" == "--help" || $# -lt 2 ]]; then cat <<'EOF' svaba_swap_bps IN.bps.txt[.gz] OUT.bps.txt.gz Read IN, preserve the header, then for each data row independently flip a fair coin: with prob 1/2, swap every bp1-side column with its bp2-side partner. Output is gzip-compressed regardless of the input form. Paired columns swapped together (so rows stay internally consistent): 1/4 chr1 <-> chr2 2/5 pos1 <-> pos2 3/6 strand1 <-> strand2 15/16 dmq1 <-> dmq2 19/20 mapq1 <-> mapq2 21/22 nm1 <-> nm2 23/24 as1 <-> as2 25/26 sub1 <-> sub2 40/41 contig_conf1 <-> contig_conf2 42/43 cpos1 <-> cpos2 (v2 only — auto-skipped on v1 files) 44/45 lmatch <-> rmatch (v2 only) 46/47 scov1 <-> scov2 (v2 only) 48/49 local1 <-> local2 (v2 only) Non-paired side-agnostic columns (ref, alt, span, somlod, ...) are left alone. Example: svaba_swap_bps fermi4_full.bps.txt.gz fermi4_full.bp_swapped.bps.txt.gz EOF return 1 fi local in="$1" out="$2" reader case "$in" in *.gz) reader="gzip -dc" ;; *) reader="cat" ;; esac $reader "$in" | awk 'BEGIN{ FS=OFS="\t"; srand() split("1,4 2,5 3,6 15,16 19,20 21,22 23,24 25,26 40,41 42,43 44,45 46,47 48,49", P, " ") } /^#/ { print; next } { if (rand() < 0.5) { for (k in P) { split(P[k], q, ",") a = q[1] + 0; b = q[2] + 0 if (a <= NF && b <= NF) { t = $a; $a = $b; $b = t } } } print }' | gzip -c > "$out" echo "svaba_swap_bps: wrote $out" >&2 } # --------------------------------------------------------------------- # svaba_bam_grep — pull reads whose SEQ contains a target sequence (or # its reverse complement) into a sub-BAM. Useful for fishing for reads # carrying a specific motif, breakpoint junction, inserted sequence, # etc. Decodes BAM -> SAM, filters col 10 (SEQ) via awk's index(), then # re-encodes to BAM. # --------------------------------------------------------------------- svaba_bam_grep() { if [[ "${1:-}" == "-h" || "${1:-}" == "--help" || $# -lt 3 ]]; then cat <<'EOF' svaba_bam_grep IN.bam SEQUENCE OUT.bam [REGION] Filter IN.bam to reads whose SEQ field contains SEQUENCE or its reverse complement. Writes OUT.bam (with the original header). Match is case-insensitive (SEQUENCE is uppercased, reverse-complemented using the standard A<->T / C<->G / N<->N mapping); both strands are checked in one pass so you don't need to submit both orientations. IN.bam input BAM (reads will be decompressed + re-encoded; coordinate-sorted input yields coordinate-sorted output) SEQUENCE DNA motif to grep for. ACGTN only. OUT.bam output path. REGION optional samtools-style region (e.g. "chr7:55000000-56000000") to restrict the scan. Requires IN.bam to be indexed (.bai). Depends on `samtools` being on PATH. Scanning is linear in the number of reads in the region (full-BAM scan of a 30x WGS takes ~15-30 min on a single core; use REGION to localize when you can). Short sequences (< ~8 bp) will match nearly every read — a warning is printed if you pass one. Examples: # Find reads carrying a specific junction/insert sequence anywhere svaba_bam_grep tumor.bam TTAGGGTTAGGGTTAGGG telomere_reads.bam # Same, but only scan chr17 to speed things up svaba_bam_grep tumor.bam CATCGATCGATC chr17_hits.bam chr17 # Short sequence — will match lots; use a longer motif if you can svaba_bam_grep tumor.bam ACGT lots.bam EOF return 1 fi local in="$1" seq="$2" out="$3" region="${4:-}" if [[ ! -r "$in" ]]; then echo "svaba_bam_grep: cannot read $in" >&2 return 2 fi if ! command -v samtools >/dev/null 2>&1; then echo "svaba_bam_grep: samtools not on PATH" >&2 return 3 fi if [[ -z "$seq" ]]; then echo "svaba_bam_grep: empty SEQUENCE" >&2 return 4 fi # Validate: ACGTN only, any case. if ! printf '%s' "$seq" | grep -qE '^[ACGTNacgtn]+$'; then echo "svaba_bam_grep: SEQUENCE must contain only ACGTN (got: $seq)" >&2 return 5 fi # Uppercase via tr (works on bash 3.2, unlike ${seq^^}). local upper_seq rc_seq upper_seq=$(printf '%s' "$seq" | tr '[:lower:]' '[:upper:]') # Reverse-complement: complement via tr, then reverse. rc_seq=$(printf '%s' "$upper_seq" | tr 'ACGTN' 'TGCAN' | rev) if [[ ${#upper_seq} -lt 8 ]]; then echo "svaba_bam_grep: warning — sequence length ${#upper_seq} is short; expect many incidental matches" >&2 fi echo "svaba_bam_grep: matching '$upper_seq' (or rc '$rc_seq') in $in${region:+ region=$region}" >&2 # samtools view -h emits header (@) + records; awk keeps header as-is # and data rows only if SEQ (col 10) contains either orientation. # index() is a substring search — fast, no regex compile per row. # Re-encode via `samtools view -b -o OUT -` so output is a valid BAM. # If the user passed a REGION, include it after the input path; that's # samtools' native locus-restriction syntax and it only works if the # input has a .bai (or .csi) alongside. if [[ -n "$region" ]]; then samtools view -h "$in" "$region" else samtools view -h "$in" fi | awk -v fwd="$upper_seq" -v rc="$rc_seq" ' /^@/ { print; next } index($10, fwd) || index($10, rc) { print } ' | samtools view -b -o "$out" - # Report count. Try to build a .bai if the output ended up coord-sorted; # silently skip if it didn't (e.g. name-sorted input BAM). local n n=$(samtools view -c "$out" 2>/dev/null) if samtools index "$out" 2>/dev/null; then echo "svaba_bam_grep: wrote $out ($n matching reads, indexed)" >&2 else echo "svaba_bam_grep: wrote $out ($n matching reads; not coord-sorted, no .bai built)" >&2 fi } # --------------------------------------------------------------------- # Banner — suppressible with SVABA_FUNCTIONS_QUIET=1. Sent to stderr so # a non-interactive shell piping output isn't polluted. # --------------------------------------------------------------------- if [[ "${SVABA_FUNCTIONS_QUIET:-0}" != "1" ]]; then echo "svaba_local_function.sh sourced. Try 'svaba_help' for the list." >&2 fi ================================================ FILE: scripts/svaba_postprocess.sh ================================================ #!/usr/bin/env bash # # svaba_postprocess.sh — one-stop post-processing of a svaba run. # # Replaces (and subsumes) the older sort_output.sh and # sort_and_deduplicate_bps.sh. Given a svaba analysis ID, this script: # # 1. Merges per-thread output BAMs into one BAM per suffix. # ${ID}.thread*.${suffix}.bam -> ${ID}.${suffix}.bam # # 2. Runs `svaba postprocess` (the C++ subcommand) to coord-sort, stream- # dedup on exact (qname,flag) duplicates, stamp a @PG "svaba_postprocess" # line onto every final BAM header, and build a .bai index. Suffixes: # weird, corrected, discordant, contigs. # Final state per suffix is ${ID}.${suffix}.bam(.bai); no # .sorted / .deduped intermediate files remain on the user path. # # 3. Sorts, deduplicates, and PASS-filters ${ID}.bps.txt.gz. Emits: # ${ID}.bps.sorted.txt.gz — full sorted list # ${ID}.bps.sorted.dedup.txt.gz — best SV (max maxlod) per # (chr1,pos1,strand1,chr2,pos2,strand2) # ${ID}.bps.sorted.dedup.pass.txt.gz — dedup restricted to PASS rows # ${ID}.bps.sorted.dedup.pass.somatic.txt.gz — PASS rows that are also # somatic (somlod >= 0 for v3 # format, somatic_state == 1 for # legacy format) # # 4. Filters ${ID}.r2c.txt.gz down to PASS breakpoints — writes both # ${ID}.r2c.pass.txt.gz (all PASS calls) and # ${ID}.r2c.pass.somatic.txt.gz (PASS calls with somlod >= 1, # i.e. the somatic subset) for interactive use in bps_explorer.html # (the full r2c.txt.gz is typically too heavy to load). # # 5. Optionally (--split-by-source) demultiplexes the deduped BAMs by the # first 4 chars of each read's QNAME into per-source BAMs. # # Every step is idempotent — missing inputs cause that step to log a short # note and continue, so rerunning this after regenerating one of the inputs # Just Works. The --skip-* flags let you skip a stage explicitly when you # only want to refresh one phase. # # Usage: # svaba_postprocess.sh [options] # # Options (flags take precedence over equivalent env vars): # -t, --threads N total threads budget (default: 4) # -m, --mem STR per-samtools-sort-thread memory (default: 2G) # --sort-buffer STR per-chunk memory for the bps sorter (default: 2G) # --split-by-source also split BAMs by QNAME source prefix # --input-dir DIR look for inputs in DIR (default: .) # --output-dir DIR write outputs to DIR (default: INPUT_DIR) # --svaba PATH path to svaba binary (default: ~/git/svaba/build/svaba) # --keep-tmp keep scratch dirs for debugging # --skip-bam skip BAM merge/sort/dedup/index (steps 1 & 2) # --skip-dedup keep step 2 but tell svaba postprocess to skip the # dedup substep (passes --sort-only through). Useful # for quick reruns of sort + @PG stamp + index # without redoing the expensive dedup pass. Sort is # also auto-skipped when the BAM's header already # declares @HD SO:coordinate, so a repeat rerun is # effectively a no-op except for index rebuild. # --skip-bps skip bps sort/dedup/PASS (step 3) # --skip-r2c skip r2c PASS filter (step 4) # --skip-split force-disable split-by-source # -h, --help this message # # Environment variable fallbacks (used only if the matching flag is absent): # THREADS, MEM, BUFFER_SIZE (→ --sort-buffer), SVABA, SAM, INPUT_DIR, # OUTPUT_DIR, SPLIT_BY_SOURCE, KEEP_TMP # # Examples: # svaba_postprocess.sh tumor_vs_normal # svaba_postprocess.sh -t 16 -m 4G tumor_vs_normal # svaba_postprocess.sh --split-by-source --output-dir /tmp/run42 run42 # svaba_postprocess.sh --skip-bam --skip-bps tumor_vs_normal # only r2c refresh # # Notes: # * Step 3 needs GNU sort (or `gsort` on macOS via `brew install coreutils`). # * Steps 1+2 need samtools on PATH; step 2 needs a working svaba binary. set -euo pipefail # ----------------------------------------------------------- defaults --- THREADS=${THREADS:-4} MEM=${MEM:-2G} SORT_BUFFER=${BUFFER_SIZE:-${SORT_BUFFER:-2G}} SVABA=${SVABA:-~/git/svaba/build/svaba} SAM=${SAM:-samtools} SPLIT_BY_SOURCE=${SPLIT_BY_SOURCE:-0} INPUT_DIR=${INPUT_DIR:-.} OUTPUT_DIR=${OUTPUT_DIR:-} KEEP_TMP=${KEEP_TMP:-0} SKIP_BAM=0 SKIP_DEDUP=0 SKIP_BPS=0 SKIP_R2C=0 print_usage() { sed -n '2,65p' "$0" | sed 's/^# \{0,1\}//' } # ---------------------------------------------------------- arg parse --- while [[ $# -gt 0 ]]; do case "$1" in -t|--threads) THREADS="$2"; shift 2 ;; -m|--mem) MEM="$2"; shift 2 ;; --sort-buffer) SORT_BUFFER="$2"; shift 2 ;; --split-by-source) SPLIT_BY_SOURCE=1; shift ;; --skip-split) SPLIT_BY_SOURCE=0; shift ;; --input-dir) INPUT_DIR="$2"; shift 2 ;; --output-dir) OUTPUT_DIR="$2"; shift 2 ;; --svaba) SVABA="$2"; shift 2 ;; --keep-tmp) KEEP_TMP=1; shift ;; --skip-bam) SKIP_BAM=1; shift ;; --skip-dedup) SKIP_DEDUP=1; shift ;; --skip-bps) SKIP_BPS=1; shift ;; --skip-r2c) SKIP_R2C=1; shift ;; -h|--help) print_usage; exit 0 ;; --) shift; break ;; -*) echo "svaba_postprocess.sh: unknown option '$1'" >&2 echo "run 'svaba_postprocess.sh --help' for usage" >&2 exit 2 ;; *) break ;; esac done if [[ $# -lt 1 ]]; then print_usage exit 2 fi ID=$1 shift OUTPUT_DIR=${OUTPUT_DIR:-$INPUT_DIR} if [[ ! -d "$INPUT_DIR" ]]; then echo "svaba_postprocess.sh: INPUT_DIR does not exist: $INPUT_DIR" >&2 exit 3 fi mkdir -p "$OUTPUT_DIR" # Human-readable echo of the plan so logs tell you what was about to run. echo "svaba_postprocess.sh: id=$ID threads=$THREADS mem=$MEM sort_buffer=$SORT_BUFFER" echo " : input_dir=$INPUT_DIR output_dir=$OUTPUT_DIR" echo " : split_by_source=$SPLIT_BY_SOURCE skip_bam=$SKIP_BAM skip_dedup=$SKIP_DEDUP skip_bps=$SKIP_BPS skip_r2c=$SKIP_R2C" # Useful for absolute-path filenames in the steps below. Use trailing-slash- # free form so we always concatenate `${DIR}/${name}`. INPUT_DIR=${INPUT_DIR%/} OUTPUT_DIR=${OUTPUT_DIR%/} # ======================================================================== # STEP 1: Merge per-thread BAMs and per-thread r2c.txt.gz. # # svaba run with multiple threads produces ${ID}.thread*.${suffix}.bam for # suffix in {discordant,weird,corrected}, and (with --dump-reads) # ${ID}.thread*.r2c.txt.gz. This step coalesces each family into a single # ${ID}.${suffix}.bam / ${ID}.r2c.txt.gz. # # Per-thread BAMs are merged with `samtools merge` — standard BAM merge # with header reconciliation. # # Per-thread r2c.txt.gz files are merged with plain `cat`: gzip is a # concatenation-safe format (RFC 1952), so concatenating multiple .gz # streams produces a valid .gz whose decompressed output is the # concatenation of each member's decompressed output. svaba guarantees # only the first worker (threadId == 1; the worker pool numbers # threads 1..N) emits the TSV column-header line, and the awk|sort # below places that file first in the merge, so the merged file has # exactly one header at the top regardless of thread count. # # Single-file inputs (only one thread) are renamed. No-file inputs are # silently skipped — this happens when the svaba run was single-threaded, # or when --dump-reads wasn't set (no r2c files to merge). # ======================================================================== if [[ $SKIP_BAM -eq 0 ]]; then echo "[1/5] merging per-thread BAMs" for suffix in discordant weird corrected; do target="${INPUT_DIR}/${ID}.${suffix}.bam" shopt -s nullglob bam_files=("${INPUT_DIR}/${ID}".thread*."${suffix}".bam) shopt -u nullglob if [[ ${#bam_files[@]} -gt 1 ]]; then echo " merge ${#bam_files[@]} thread BAMs for '$suffix' -> $target" "$SAM" merge -f -@ "$THREADS" "$target" "${bam_files[@]}" rm -f "${bam_files[@]}" elif [[ ${#bam_files[@]} -eq 1 ]]; then echo " mv ${bam_files[0]} -> $target" mv "${bam_files[0]}" "$target" fi done # Merge per-thread r2c.txt.gz → ${ID}.r2c.txt.gz. gzip is concat-safe, # so `cat a.gz b.gz > c.gz` is valid. Thread 1's file is first in the # numeric sort order (worker pool numbers threads 1..N; see # threadpool.h), and thread 1 is the one that emits the TSV header, # so its header line ends up at the top of the merged file. target="${INPUT_DIR}/${ID}.r2c.txt.gz" shopt -s nullglob r2c_files=("${INPUT_DIR}/${ID}".thread*.r2c.txt.gz) shopt -u nullglob if [[ ${#r2c_files[@]} -gt 1 ]]; then echo " merge ${#r2c_files[@]} thread r2c files -> $target" # Sort by thread index so thread 1 (with the header) is first. # Filenames are ${ID}.threadN.r2c.txt.gz — awk pulls the numeric # thread index out, sort -k1,1n orders it numerically, cut drops # the index so we get just the filenames back in sorted order. # # NB: avoid `mapfile` / `readarray` — they're bash 4+, and macOS # ships bash 3.2 as /bin/bash, which `#!/usr/bin/env bash` often # resolves to. The `while read` loop below is fully portable. r2c_sorted=() while IFS= read -r f; do r2c_sorted+=("$f") done < <( printf '%s\n' "${r2c_files[@]}" \ | awk -F'.thread|.r2c.txt.gz' '{print $(NF-1)"\t"$0}' \ | sort -k1,1n \ | cut -f2- ) cat "${r2c_sorted[@]}" > "$target" rm -f "${r2c_sorted[@]}" elif [[ ${#r2c_files[@]} -eq 1 ]]; then echo " mv ${r2c_files[0]} -> $target" mv "${r2c_files[0]}" "$target" fi else echo "[1/5] skipping BAM + r2c merge (--skip-bam)" fi # ======================================================================== # STEP 2: svaba postprocess — sort, stream-dedup, stamp @PG, build .bai. # # Heavy lifting lives in the C++ subcommand. We just pass it the thread / # memory budget; svaba postprocess splits THREADS across suffixes and passes # -m to samtools sort per-thread. # # --skip-dedup at this layer maps to `--sort-only` on the C++ CLI, which # still runs the sort + @PG-stamp + index phases but skips the # stream-dedup pass. Combined with the C++'s auto-skip-sort-when-already- # sorted check (via @HD SO:coordinate), a rerun with --skip-dedup on # already-postprocessed files is effectively a no-op other than # re-stamping PG + rebuilding the .bai. # ======================================================================== if [[ $SKIP_BAM -eq 0 ]]; then pp_args=(-i "$ID" -t "$THREADS" -m "$MEM") if [[ $SKIP_DEDUP -eq 1 ]]; then pp_args+=(--sort-only) dedup_banner="(dedup skipped via --skip-dedup)" else dedup_banner="" fi echo "[2/5] svaba postprocess: sort + dedup + @PG + BAI index (threads=$THREADS, mem=$MEM) $dedup_banner" # Indent all sub-output and suppress the harmless htslib "idx_find_and_load" # warnings (index files don't exist yet when the reader opens the BAM — # they're built as the final step of each suffix's pipeline). Also strip # the NOTE line about those warnings since we're filtering them here. # Use awk instead of grep -v so a zero-match case doesn't trigger pipefail. (cd "$INPUT_DIR" && "$SVABA" postprocess "${pp_args[@]}" 2>&1) \ | awk '!/idx_find_and_load/ && !/Could not retrieve index file.*expected and can be ignored/' \ | sed 's/^/ /' else echo "[2/5] skipping svaba postprocess (--skip-bam)" fi # ======================================================================== # STEP 3: Sort, dedup, and PASS-filter the bps.txt. # # Three outputs: sorted, sorted+dedup (one row per unique breakpoint pair, # keeping the row with highest maxlod), sorted+dedup+PASS. Preserves the # leading "#chr1..." header row in every output. Uses GNU sort (or gsort # on macOS) for external-memory sort; --parallel=THREADS, -S SORT_BUFFER. # # Column positions in bps.txt (hard-coded from BreakPoint::toFileString): # col 32 = confidence (PASS / LOWMAPQ / BLACKLIST / ...) # col 38 = maxlod (numeric; "NA" sorts last with -g) # # Sort keys in order: chr1(V), pos1(n), strand1, chr2(V), pos2(n), strand2, # maxlod(gr, descending). # ======================================================================== BPS_GZ="${INPUT_DIR}/${ID}.bps.txt.gz" BPS_TXT="${INPUT_DIR}/${ID}.bps.txt" OUT_SORTED="${OUTPUT_DIR}/${ID}.bps.sorted.txt.gz" OUT_DEDUP="${OUTPUT_DIR}/${ID}.bps.sorted.dedup.txt.gz" OUT_PASS="${OUTPUT_DIR}/${ID}.bps.sorted.dedup.pass.txt.gz" OUT_PASS_SOM="${OUTPUT_DIR}/${ID}.bps.sorted.dedup.pass.somatic.txt.gz" # Column positions — auto-detected from the header when available (see # detect_columns below). These are the v3 defaults; the old format has # different positions (e.g. confidence at col 26 instead of 32). CONF_COL=32 SOMATIC_COL=36 SOMLOD_COL=37 MAXLOD_COL=38 CNAME_COL=30 pick_sort() { if command -v gsort >/dev/null 2>&1; then echo gsort; return 0 fi if sort --version 2>/dev/null | head -n 1 | grep -qi 'gnu'; then echo sort; return 0 fi return 1 } # Auto-detect column positions from the header line. Looks for known # column names and sets CONF_COL, SOMATIC_COL, SOMLOD_COL, MAXLOD_COL. # Falls back to v3 defaults if the header is missing or unrecognised. detect_columns() { local header_line="$1" # strip leading # if present local clean="${header_line#\#}" # Use awk to find column indices by name. Tries both old and new # column names (e.g. "somatic_score" vs the unnamed v3 col, # "contig" vs "cname"). eval "$(echo "$clean" | awk -F'\t' '{ for (i = 1; i <= NF; i++) { col = $i # strip whitespace gsub(/^[ \t]+|[ \t]+$/, "", col) # normalise to lowercase for matching lcol = tolower(col) if (lcol == "confidence") printf "CONF_COL=%d\n", i if (lcol == "somatic_score") printf "SOMATIC_COL=%d\n", i if (lcol == "somatic_lod") printf "SOMLOD_COL=%d\n", i if (lcol == "tlod" || lcol == "maxlod" || lcol == "max_lod") printf "MAXLOD_COL=%d\n", i if (lcol == "contig" || lcol == "cname") printf "CNAME_COL=%d\n", i } }')" } if [[ $SKIP_BPS -eq 0 ]]; then if [[ -r "$BPS_GZ" ]]; then IN_BPS="$BPS_GZ"; READER="gzip -dc" elif [[ -r "$BPS_TXT" ]]; then IN_BPS="$BPS_TXT"; READER="cat" else IN_BPS=""; fi if [[ -z "$IN_BPS" ]]; then echo "[3/5] skipping bps sort/dedup/PASS: $BPS_GZ (or .bps.txt) not found" elif ! SORT_BIN=$(pick_sort); then echo "[3/5] skipping bps sort/dedup/PASS: GNU sort not on PATH" >&2 echo " (macOS: 'brew install coreutils' for gsort)" >&2 else echo "[3/5] sort + dedup + PASS-filter $IN_BPS" echo " sort=$SORT_BIN buffer=$SORT_BUFFER parallel=$THREADS" TMP_BPS=$(mktemp -d "${TMPDIR:-/tmp}/svaba_pp_bps.XXXXXXXX") # Single cleanup trap for the whole script; registered the first time # we need a tempdir. Registered here because step 3 creates the first # tempdir used. trap 'if [[ "${KEEP_TMP:-0}" == "1" ]]; then echo "svaba_postprocess.sh: leaving scratch at: $TMP_BPS${TMP_SPLIT:+ $TMP_SPLIT}" >&2 else rm -rf "$TMP_BPS" "${TMP_SPLIT:-}" fi' EXIT INT TERM # Peek at the first line to decide whether to split off a header. FIRST_LINE=$($READER "$IN_BPS" | head -n 1 || true) HAS_HEADER=0 [[ "${FIRST_LINE:0:1}" == "#" ]] && HAS_HEADER=1 # Auto-detect column positions from the header. if [[ $HAS_HEADER -eq 1 ]]; then detect_columns "$FIRST_LINE" fi echo " columns: confidence=$CONF_COL cname=$CNAME_COL somatic=$SOMATIC_COL somlod=$SOMLOD_COL maxlod=$MAXLOD_COL" # --- 3a) sort --- { if [[ $HAS_HEADER -eq 1 ]]; then printf '%s\n' "$FIRST_LINE" $READER "$IN_BPS" | tail -n +2 else $READER "$IN_BPS" fi | { if [[ $HAS_HEADER -eq 1 ]]; then IFS= read -r hdr printf '%s\n' "$hdr" fi LC_ALL=C "$SORT_BIN" \ -t $'\t' \ -k1,1V \ -k2,2n \ -k3,3 \ -k4,4V \ -k5,5n \ -k6,6 \ -k${MAXLOD_COL},${MAXLOD_COL}gr \ -S "$SORT_BUFFER" \ --parallel="$THREADS" \ -T "$TMP_BPS" } } | gzip -c > "$OUT_SORTED" echo " wrote $OUT_SORTED" # --- 3b) dedup by breakpoint pair (first row per key wins = highest maxlod) --- gzip -dc "$OUT_SORTED" | awk -F'\t' -v OFS='\t' ' NR == 1 && $0 ~ /^#/ { print; next } { key = $1 FS $2 FS $3 FS $4 FS $5 FS $6 if (!(key in seen)) { seen[key] = 1; print } } ' | gzip -c > "$OUT_DEDUP" echo " wrote $OUT_DEDUP" # --- 3c) PASS-filter the dedup --- gzip -dc "$OUT_DEDUP" | awk -F'\t' -v OFS='\t' -v CCOL="$CONF_COL" ' NR == 1 && $0 ~ /^#/ { print; next } $CCOL == "PASS" { print } ' | gzip -c > "$OUT_PASS" echo " wrote $OUT_PASS" # --- 3d) PASS + somatic filter --- # Use somatic_score == "1" as the sole somatic gate. The old format's # "somatic_lod" column is actually n.LO_n (normal variant LO) — high # values mean strong normal evidence, which argues AGAINST somatic. # The v3 "somlod" is a proper somatic log-odds, but somatic_score is # set correctly in both formats, so we use it uniformly. gzip -dc "$OUT_PASS" | awk -F'\t' -v OFS='\t' \ -v SCOL="$SOMATIC_COL" ' NR == 1 && $0 ~ /^#/ { print; next } $SCOL == "1" { print } ' | gzip -c > "$OUT_PASS_SOM" echo " wrote $OUT_PASS_SOM" fi else echo "[3/5] skipping bps sort/dedup/PASS (--skip-bps)" fi # ======================================================================== # STEP 4: Filter r2c.txt.gz to PASS contigs (+ PASS-somatic subset). # # The structured r2c TSV emitted by `svaba run` contains one row per # assembled contig + one row per r2c-aligned read, for every variant- # bearing contig. At full genome scale this can be tens of millions of # rows — too heavy for the interactive viewer. Here we produce TWO # filtered outputs from a single pass over r2c.txt.gz: # # ${ID}.r2c.pass.txt.gz - rows for all PASS variants # ${ID}.r2c.pass.somatic.txt.gz - rows for PASS variants that are also # somatic (somlod >= 1 for v3 format, # somatic_score == 1 for legacy) # # Both are keyed on cname (col 30 in bps.txt.gz, col 2 in r2c.txt.gz); # contig and read rows survive together since they share col 2. # # The somatic subset is a strict subset of the pass set, and both are # written by the same awk invocation over r2c.txt.gz via awk's pipe-to- # command output — one decompress+iterate pass, two gzip compressors # running in parallel as child processes. Avoids reading the multi-GB # r2c file twice. # ======================================================================== R2C_GZ="${INPUT_DIR}/${ID}.r2c.txt.gz" R2C_OUT="${OUTPUT_DIR}/${ID}.r2c.pass.txt.gz" R2C_OUT_SOM="${OUTPUT_DIR}/${ID}.r2c.pass.somatic.txt.gz" if [[ $SKIP_R2C -eq 0 ]]; then if [[ ! -f "$BPS_GZ" ]]; then echo "[4/5] skipping r2c PASS filter: $BPS_GZ not found" elif [[ ! -f "$R2C_GZ" ]]; then echo "[4/5] skipping r2c PASS filter: $R2C_GZ not found" echo " (svaba run must be invoked with --dump-reads to produce it;" echo " or this is an older svaba run from before r2c.txt.gz existed.)" else echo "[4/5] filter r2c to PASS contigs (+ PASS-somatic subset)" pass_list="$(mktemp -t "svaba.${ID}.pass_cnames.XXXXXX")" som_list="$(mktemp -t "svaba.${ID}.som_cnames.XXXXXX")" # Column positions are auto-detected from the header in step 3 above # (CONF_COL, CNAME_COL, SOMLOD_COL, SOMATIC_COL). If step 3 was # skipped, re-detect from bps.txt.gz's own header here. if [[ $SKIP_BPS -eq 1 ]]; then BPS_HEADER=$(gzip -dc "$BPS_GZ" | head -n 1 || true) [[ "${BPS_HEADER:0:1}" == "#" ]] && detect_columns "$BPS_HEADER" echo " columns: confidence=$CONF_COL cname=$CNAME_COL somatic=$SOMATIC_COL somlod=$SOMLOD_COL" fi # One awk pass builds both cname sets so we only decompress bps.txt.gz # once. NB: use `gzip -dc` (not `zcat`) so this works on macOS — BSD # zcat hunts for a .Z suffix rather than .gz. # Somatic criterion: somatic_score == "1" (reliable in both old and v3 formats). gzip -dc "$BPS_GZ" \ | awk -F'\t' -v pass_f="$pass_list" -v som_f="$som_list" \ -v CCOL="$CONF_COL" -v NCOL="$CNAME_COL" \ -v SCOL="$SOMATIC_COL" ' NR>1 && $CCOL=="PASS" { print $NCOL >> pass_f if ($SCOL == "1") { print $NCOL >> som_f } } ' LC_ALL=C sort -u "$pass_list" -o "$pass_list" LC_ALL=C sort -u "$som_list" -o "$som_list" n_pass=$(wc -l < "$pass_list" | awk '{print $1}') n_som=$(wc -l < "$som_list" | awk '{print $1}') if [[ "$n_pass" -eq 0 ]]; then echo " no PASS breakpoints — skipping both r2c.pass and r2c.pass.somatic." rm -f "$pass_list" "$som_list" else # Single-pass r2c filter writing both outputs via awk's pipe-to- # command feature. The two `gzip -c > file` child processes run in # parallel, so compression of the two outputs happens concurrently # on separate cores. Each output gets the same header row and then # whichever filtered rows match its cname set. gzip -dc "$R2C_GZ" \ | awk -F'\t' -v pass_f="$pass_list" -v som_f="$som_list" \ -v pass_out="$R2C_OUT" -v som_out="$R2C_OUT_SOM" ' BEGIN { while ((getline line < pass_f) > 0) keep_pass[line] = 1 close(pass_f) while ((getline line < som_f) > 0) keep_som[line] = 1 close(som_f) pass_cmd = "gzip -c > " pass_out som_cmd = "gzip -c > " som_out } NR == 1 { print | pass_cmd print | som_cmd next } ($1 == "contig" || $1 == "read") { if ($2 in keep_pass) print | pass_cmd if ($2 in keep_som) print | som_cmd } END { close(pass_cmd) close(som_cmd) } ' n_pass_rows=$(gzip -dc "$R2C_OUT" | wc -l | awk '{print $1}') n_som_rows=$(gzip -dc "$R2C_OUT_SOM" | wc -l | awk '{print $1}') echo " wrote $R2C_OUT ($n_pass PASS contigs, $n_pass_rows rows incl. header)" echo " wrote $R2C_OUT_SOM ($n_som PASS+somatic [somlod>=1] contigs, $n_som_rows rows incl. header)" rm -f "$pass_list" "$som_list" fi fi else echo "[4/5] skipping r2c PASS filter (--skip-r2c)" fi # ======================================================================== # STEP 5 (optional): Split deduped BAMs by QNAME source prefix. # # Enabled by --split-by-source (or SPLIT_BY_SOURCE=1). Routes each record # into ${ID}.${suffix}.${prefix}.bam based on the first 4 chars of QNAME # (the "source tag" like t001/n001), then sorts+indexes each output. # Runs for discordant/weird/corrected only (not contigs). # ======================================================================== if [[ "$SPLIT_BY_SOURCE" == "1" ]]; then echo "[5/5] splitting BAMs by QNAME source prefix" for suffix in discordant weird corrected; do bam="${INPUT_DIR}/${ID}.${suffix}.bam" if [[ ! -f "$bam" ]]; then echo " skip: $bam not found" continue fi echo " splitting $bam" TMP_SPLIT=$(mktemp -d "${TMPDIR:-/tmp}/svaba_pp_split.XXXXXXXX") header="${TMP_SPLIT}/header.sam" "$SAM" view -H "$bam" > "$header" # Single-pass route by first 4 chars of QNAME into per-prefix SAM files. "$SAM" view "$bam" | awk -v tmpdir="$TMP_SPLIT" '{ prefix = substr($1, 1, 4) out = tmpdir "/body." prefix ".sam" print >> out }' shopt -s nullglob bodies=("${TMP_SPLIT}"/body.*.sam) shopt -u nullglob n_bodies=${#bodies[@]} if [[ $n_bodies -gt 0 ]]; then per_job_threads=$(( THREADS / n_bodies )) [[ $per_job_threads -lt 1 ]] && per_job_threads=1 max_parallel=$(( THREADS < n_bodies ? THREADS : n_bodies )) echo " reassembling $n_bodies per-prefix BAMs (max_parallel=$max_parallel, threads/job=$per_job_threads)" pids=() for body in "${bodies[@]}"; do while [[ ${#pids[@]} -ge $max_parallel ]]; do new_pids=() for pid in "${pids[@]}"; do kill -0 "$pid" 2>/dev/null && new_pids+=("$pid") done pids=("${new_pids[@]}") [[ ${#pids[@]} -ge $max_parallel ]] && sleep 0.1 done base=$(basename "$body" .sam) prefix="${base#body.}" out="${OUTPUT_DIR}/${ID}.${suffix}.${prefix}.bam" echo " writing $out" ( cat "$header" "$body" \ | "$SAM" view -@ "$per_job_threads" -u -bS - \ | "$SAM" sort -@ "$per_job_threads" -m "$MEM" -o "$out" - \ && "$SAM" index -@ "$per_job_threads" "$out" ) & pids+=("$!") done wait fi if [[ "${KEEP_TMP:-0}" != "1" ]]; then rm -rf "$TMP_SPLIT" TMP_SPLIT="" fi done else echo "[5/5] skipping split-by-source (pass --split-by-source to enable)" fi echo "svaba_postprocess.sh: done." ================================================ FILE: scripts/update_svaba_image.sh ================================================ #!/usr/bin/env bash # # update_svaba_image.sh — rebuild the svaba worker image from the builder VM. # # Stops the builder, deletes the old image, creates a new one, restarts # the builder. Takes ~2-3 minutes. # # Usage: # update_svaba_image.sh [options] # # --builder NAME builder VM name (default: svaba-image-builder) # --image NAME image name (default: svaba-worker-image) # --zone ZONE GCP zone (default: us-central1-a) # -h, --help this message # set -euo pipefail BUILDER="svaba-image-builder" IMAGE="svaba-worker-image" ZONE="us-central1-a" while [[ $# -gt 0 ]]; do case "$1" in --builder) BUILDER="$2"; shift 2 ;; --image) IMAGE="$2"; shift 2 ;; --zone) ZONE="$2"; shift 2 ;; -h|--help) sed -n '2,14p' "$0" | sed 's/^# \{0,1\}//'; exit 0 ;; *) echo "unknown option: $1" >&2; exit 2 ;; esac done echo "=== updating image '${IMAGE}' from '${BUILDER}' ===" echo "1. stopping ${BUILDER}..." gcloud compute instances stop "$BUILDER" --zone="$ZONE" echo "2. deleting old image '${IMAGE}'..." gcloud compute images delete "$IMAGE" --quiet 2>/dev/null || echo " (no existing image to delete)" echo "3. creating new image..." gcloud compute images create "$IMAGE" \ --source-disk="$BUILDER" \ --source-disk-zone="$ZONE" echo "4. restarting ${BUILDER}..." gcloud compute instances start "$BUILDER" --zone="$ZONE" echo "=== done. image '${IMAGE}' is ready ===" ================================================ FILE: src/SGA/Algorithm/ClusterProcess.cpp ================================================ ///----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // ClusterProcess - Merge unambiguously overlapping sequences // #include "ClusterProcess.h" #include "SGAlgorithms.h" #include "SGVisitors.h" // ClusterProcess::ClusterProcess(ClusterParameters params) : m_parameters(params) { // Check parameters are set assert(m_parameters.pOverlapper != NULL); assert(m_parameters.pMarkedReads != NULL); assert(m_parameters.minOverlap >= 12); } // ClusterProcess::~ClusterProcess() { } // ClusterResult ClusterProcess::process(const SequenceWorkItem& item) { ReadCluster cluster(m_parameters.pOverlapper, m_parameters.minOverlap); ClusterNode node = cluster.addSeed(item.read.seq.toString(), true); assert(node.interval.isValid()); // Check if this read is already part of a cluster. If so, return an empty result for(int64_t i = node.interval.lower; i <= node.interval.upper; ++i) { if(m_parameters.pMarkedReads->test(i)) { ClusterResult result; return result; // already part of a cluster, return nothing } } // Add sequences to be used to stop extension, if requested if(m_parameters.pLimitKmers != NULL) cluster.setLimitKmers(m_parameters.pLimitKmers, m_parameters.limitK); // Run the clustering process cluster.run(m_parameters.maxClusterSize, 0); // If some work was performed, update the bitvector so other threads do not try to merge the same set of reads. // As a given set of reads should all be merged together, we only need to make sure we atomically update // the bit for the read with the lowest index in the set. ClusterResult result; result.clusterNodes = cluster.getOutput(); // Check if the bit in the vector has already been set for the lowest read index // If it has some other thread has already output this set so we do nothing int64_t lowestIndex = result.clusterNodes.front().interval.lower; bool currentValue = m_parameters.pMarkedReads->test(lowestIndex); bool updateSuccess = false; if(currentValue == false) { // Attempt to update the bit vector with an atomic CAS. If this returns false // the bit was set by some other thread updateSuccess = m_parameters.pMarkedReads->updateCAS(lowestIndex, currentValue, true); } if(updateSuccess) { // We successfully atomically set the bit for the first read in this set // to true. We can safely update the rest of the bits and keep the merged sequences // for output. std::vector::const_iterator iter = result.clusterNodes.begin(); for(; iter != result.clusterNodes.end(); ++iter) { for(int64_t i = iter->interval.lower; i <= iter->interval.upper; ++i) { if(i == lowestIndex) //already set continue; currentValue = m_parameters.pMarkedReads->test(i); if(currentValue) { // This value should not be true, emit a warning std::cout << "Warning: Bit " << i << " was unexpectedly set by a different thread\n"; } else { m_parameters.pMarkedReads->updateCAS(i, currentValue, true); } } } } else { // Some other thread merged these reads already, discard the intermediate // data and set the result to false result.clusterNodes.clear(); } return result; } // Generate a new cluster from a previously build cluster ClusterResult ClusterProcess::process(const ClusterVector& inSequences) { ReadCluster cluster(m_parameters.pOverlapper, m_parameters.minOverlap); // Add seeds to the cluster generator for(size_t i = 0; i < inSequences.size(); ++i) cluster.addSeed(inSequences[i].sequence, false); // Add sequences to be used to stop extension, if requested if(m_parameters.pLimitKmers != NULL) cluster.setLimitKmers(m_parameters.pLimitKmers, m_parameters.limitK); cluster.run(m_parameters.maxClusterSize, m_parameters.maxIterations); ClusterResult result; result.clusterNodes = cluster.getOutput(); return result; } // ClusterPostProcess::ClusterPostProcess(std::ostream* pWriter, size_t minClusterSize, BitVector* pMarkedReads) : m_minClusterSize(minClusterSize), m_numClusters(0), m_numTotalReads(0), m_numTotalReadsClustered(0), m_pWriter(pWriter), m_pMarkedReads(pMarkedReads) { } // ClusterPostProcess::~ClusterPostProcess() { printf("[sga cluster] Clustered %zu reads into %zu clusters (%zu total reads input)\n", m_numTotalReadsClustered, m_numClusters, m_numTotalReads); } // This just dispatches to main post-process function void ClusterPostProcess::process(const SequenceWorkItem& /*item*/, const ClusterResult& result) { process(result); } // void ClusterPostProcess::process(const ClusterVector& /*in*/, const ClusterResult& result) { process(result); } void ClusterPostProcess::process(const ClusterResult& result) { m_numTotalReads += 1; if(result.clusterNodes.size() > 0) { // Compute the cluster size size_t clusterSize = 0; for(size_t i = 0; i < result.clusterNodes.size(); ++i) { if(result.clusterNodes[i].interval.isValid()) clusterSize += result.clusterNodes[i].interval.size(); else clusterSize += 1; } if(clusterSize >= m_minClusterSize) { for(size_t i = 0; i < result.clusterNodes.size(); ++i) *m_pWriter << "cluster-" << m_numClusters << "\t" << clusterSize << "\t" << result.clusterNodes[i].sequence << "\t" << result.clusterNodes[i].interval << "\n"; m_numClusters += 1; m_numTotalReadsClustered += clusterSize; } } } ================================================ FILE: src/SGA/Algorithm/ClusterProcess.h ================================================ ///----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // ClusterProcess - Compute clusters of overlapping reads // #ifndef CLUSTERPROCESS_H #define CLUSTERPROCESS_H #include "Util.h" #include "OverlapAlgorithm.h" #include "SequenceProcessFramework.h" #include "BitVector.h" #include "Bigraph.h" #include "SGUtil.h" #include "ReadCluster.h" #include "ClusterReader.h" struct ClusterParameters { const OverlapAlgorithm* pOverlapper; int minOverlap; size_t maxClusterSize; int maxIterations; BitVector* pMarkedReads; // shared between threads const std::set* pLimitKmers; size_t limitK; }; struct ClusterResult { std::vector clusterNodes; }; // Compute the overlap blocks for reads class ClusterProcess { public: ClusterProcess(ClusterParameters params); ~ClusterProcess(); // Generate a cluster from a single sequence ClusterResult process(const SequenceWorkItem& item); // Generate a new cluster from a previously build cluster ClusterResult process(const ClusterVector& inSequences); private: ClusterParameters m_parameters; }; // Write the cluster results to a temporary output file class ClusterPostProcess { public: ClusterPostProcess(std::ostream* pWriter, size_t minClusterSize, BitVector* pMarkedReads); ~ClusterPostProcess(); void process(const SequenceWorkItem& item, const ClusterResult& result); void process(const ClusterVector& inSequences /*in*/, const ClusterResult& result); void process(const ClusterResult& result); private: size_t m_minClusterSize; size_t m_numClusters; size_t m_numTotalReads; size_t m_numTotalReadsClustered; std::ostream* m_pWriter; BitVector* m_pMarkedReads; }; #endif ================================================ FILE: src/SGA/Algorithm/ConnectProcess.cpp ================================================ ///----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // ConnectProcess - Algorithm to resolve the sequence // connecting two ends of a paired end read // #include "ConnectProcess.h" #include "StringGraphGenerator.h" // // // ConnectProcess::ConnectProcess(const OverlapAlgorithm* pOverlapper, int minOverlap, int maxDistance) : m_pOverlapper(pOverlapper), m_minOverlap(minOverlap), m_maxDistance(maxDistance) { } // ConnectProcess::~ConnectProcess() { } // ConnectResult ConnectProcess::process(const SequenceWorkItemPair& workItemPair) { assert(getPairID(workItemPair.first.read.id) == workItemPair.second.read.id); ConnectResult result; StringGraphGenerator localGraph(m_pOverlapper, workItemPair.first.read, workItemPair.second.read, m_minOverlap, ED_SENSE, m_maxDistance); SGWalkVector walks = localGraph.searchWalks(); //std::cout << "Found " << walks.size() << " walk between " << workItemPair.first.read.id << " and " << workItemPair.second.read.id << "\n"; if(walks.size() == 1) { SGWalk& solution = walks.front(); result.resolvedSequence = solution.getString(SGWT_START_TO_END); //std::cout << "Solution has length " << solution.getStartToEndDistance() << "\n"; } return result; } // // // ConnectPostProcess::ConnectPostProcess(std::ostream* pWriter, std::ostream* pUnconnectedWriter) : m_pWriter(pWriter), m_pUnconnectedWriter(pUnconnectedWriter), m_numPairsTotal(0), m_numPairsResolved(0) { } // ConnectPostProcess::~ConnectPostProcess() { printf("connect: Resolved %d out of %d pairs (%lf)\n", m_numPairsResolved, m_numPairsTotal, (double)m_numPairsResolved / m_numPairsTotal); } // void ConnectPostProcess::process(const SequenceWorkItemPair& workItemPair, const ConnectResult& result) { ++m_numPairsTotal; if(result.resolvedSequence.length() > 0) { ++m_numPairsResolved; SeqRecord record; record.id = getPairBasename(workItemPair.first.read.id); record.seq = result.resolvedSequence; record.write(*m_pWriter); } else { workItemPair.first.read.write(*m_pUnconnectedWriter); workItemPair.second.read.write(*m_pUnconnectedWriter); } } ================================================ FILE: src/SGA/Algorithm/ConnectProcess.h ================================================ ///----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // ConnectProcess - Wrapper to resolve the sequence // connecting two ends of a paired end read // #ifndef CONNECTROCESS_H #define CONNECTPROCESS_H #include "Util.h" #include "OverlapAlgorithm.h" #include "SequenceProcessFramework.h" #include "SequenceWorkItem.h" #include "MultiOverlap.h" #include "Metrics.h" class ConnectResult { public: DNAString resolvedSequence; }; // class ConnectProcess { public: ConnectProcess(const OverlapAlgorithm* pOverlapper, int minOverlap, int maxDistance); ~ConnectProcess(); ConnectResult process(const SequenceWorkItemPair& workPair); private: const OverlapAlgorithm* m_pOverlapper; const int m_minOverlap; const int m_maxDistance; }; // Write the results from the overlap step to an ASQG file class ConnectPostProcess { public: ConnectPostProcess(std::ostream* pWriter, std::ostream* pUnconnectedWriter); ~ConnectPostProcess(); void process(const SequenceWorkItemPair& workPair, const ConnectResult& result); private: std::ostream* m_pWriter; std::ostream* m_pUnconnectedWriter; int m_numPairsTotal; int m_numPairsResolved; }; #endif ================================================ FILE: src/SGA/Algorithm/DPAlignment.cpp ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // DPAlignment - Generic class for dynamic programming alignments // #include "DPAlignment.h" DPAlignment::DPAlignment(const std::string& s1, const std::string& s2, DPAlignMode mode, DPScoringScheme scoring) : m_mode(mode), m_scoring(scoring) { if(s1.empty() || s2.empty()) return; m_dimM = s1.size() + 1; // = num rows m_dimN = s2.size() + 1; // = num columns initializeDPMatrix(); fillMatrix(s1, s2); //printMatrix(s1, s2); } // int DPAlignment::getNumRows() const { return m_dimM; } // int DPAlignment::getNumColumns() const { return m_dimN; } // int DPAlignment::getScore(int i, int j) const { assert(i < m_dimM && j < m_dimN); return m_matrix[i][j]; } // DPScoringScheme DPAlignment::getScoringScheme() const { return m_scoring; } // Setup the dynamic programming matrix for overlap computation void DPAlignment::initializeDPMatrix() { m_matrix.resize(m_dimM); // rows for(int i = 0; i < m_dimM; ++i) m_matrix[i].resize(m_dimN); // columns // Set score[i][0] (first column) to be zero for all i if in overlap mode. // This is so we don't penalize the portion of S1 that // S2 does not overlap for(int i = 0; i < m_dimM; ++i) m_matrix[i][0] = (m_mode == DPM_OVERLAP) ? 0 : i * gap_penalty(); // Set score [0][j] (first row) to be j for all j // This represents not matching the first j bases of S1 for(int j = 0; j < m_dimN; ++j) m_matrix[0][j] = gap_penalty() * j; } // Fill in the DP matrix void DPAlignment::fillMatrix(const std::string& s1, const std::string& s2) { // Update the value of each cell for all i and j greater than zero for(int i = 1; i < m_dimM; ++i) { for(int j = 1; j < m_dimN; ++j) { int up = m_matrix[i - 1][j] + gap_penalty(); // skip a base of S1 int left = m_matrix[i][j - 1] + gap_penalty(); // skip a base of S2 int idx1 = i - 1; int idx2 = j - 1; int diag = m_matrix[i - 1][j - 1] + match_score(s1[idx1], s2[idx2]); m_matrix[i][j] = compare3(up, left, diag); } } } // Print the best alignment starting from the given cell void DPAlignment::printAlignment(const std::string& s1, const std::string& s2, int startI, int startJ) const { if(m_matrix.empty()) return; if(startI == -1 || startJ == -1) { startI = m_dimM - 1; startJ = m_dimN - 1; } int score = m_matrix[startI][startJ]; DPPath backtrack = calculatePath(s1, s2, startI, startJ); // Print the match strings std::string m1; std::string m2; std::string os; for(DPPath::iterator iter = backtrack.begin(); iter != backtrack.end(); ++iter) { int i = iter->i; int j = iter->j; //printf("(%d %d)\n", i, j); switch(iter->op) { case DPO_MATCH: case DPO_SUB: assert(i >= 0 && i < (int)s1.size() && j >= 0 && j < (int)s2.size()); m1.push_back(s1[i]); m2.push_back(s2[j]); os.push_back(iter->op == DPO_MATCH ? '.' : 'S'); break; case DPO_INSERT: assert(j >= 0 && j < (int)s2.size()); m1.push_back('-'); m2.push_back(s2[j]); os.push_back('I'); break; case DPO_DELETE: assert(i >= 0 && i < (int)s1.size()); m1.push_back(s1[i]); m2.push_back('-'); os.push_back('D'); break; case DPO_NOOP: assert(false); break; } } std::cout << "M1: " << m1 << "\n"; std::cout << "M2: " << m2 << "\n"; std::cout << "OS: " << os << "\n"; std::cout << "Score: " << score << "\n"; } // Get the operation that was performed to arrive at cell i,j // If there are multiple possible paths, the // precedence is diagonal, above, left DPOperation DPAlignment::getPathOperationToCell(const std::string& s1, const std::string& s2, int i, int j) const { assert((i > 0 || j > 0) && i < m_dimM && j < m_dimN); // Get the score of cell i,j int currScore = getScore(i,j); // Check diagonal first int a = i - 1; int b = j - 1; if(a >= 0 && b >= 0) { if(currScore == m_matrix[a][b] + match_score(s1[a],s2[b])) return (s1[a] == s2[b]) ? DPO_MATCH : DPO_SUB; } // Check above element a = i - 1; b = j; if(a >= 0 && b >= 0) { // If this is overlap mode, there is no deletion // penalty in the first column int above_score = (m_mode == DPM_OVERLAP && b == 0) ? 0 : gap_penalty(); if(currScore == m_matrix[a][b] + above_score) return DPO_DELETE; } // Check left element a = i; b = j - 1; if(a >= 0 && b >= 0) { if(currScore == m_matrix[a][b] + gap_penalty()) return DPO_INSERT; } assert(false); return DPO_NOOP; } // Calculate an optimal path through the alignment starting from the given cell DPPath DPAlignment::calculatePath(const std::string& s1, const std::string& s2, int startI, int startJ) const { assert((int)s1.size() == m_dimM - 1 && (int)s2.size() == m_dimN - 1); DPPath out; DPPathNode currNode; currNode.i = startI; currNode.j = startJ; currNode.op = DPO_NOOP; while(currNode.i != 0 || currNode.j != 0) { // Set the node to be the cell that preceded the current one DPPathNode nextNode; // The path node op is the operation leading away from the cell nextNode.op = getPathOperationToCell(s1, s2, currNode.i, currNode.j); // Set the cell coordinates based on the op switch(nextNode.op) { case DPO_MATCH: case DPO_SUB: nextNode.i = currNode.i - 1; nextNode.j = currNode.j - 1; break; case DPO_DELETE: nextNode.i = currNode.i - 1; nextNode.j = currNode.j; break; case DPO_INSERT: nextNode.i = currNode.i; nextNode.j = currNode.j - 1; break; case DPO_NOOP: assert(false); } out.push_back(nextNode); currNode = nextNode; } out.reverse(); return out; } // void DPAlignment::printMatrix(const std::string& s1, const std::string& s2) const { // Print the DP matrix std::cout << " \t-\t"; for(int j = 1; j < m_dimN; ++j) { std::cout << s2[j - 1] << "\t"; } std::cout << "\n"; for(int i = 0; i < m_dimM; ++i) { if(i == 0) std::cout << "-\t"; else std::cout << s1[i - 1] << "\t"; for(int j = 0; j < m_dimN; ++j) { std::cout << m_matrix[i][j] << "\t"; } std::cout << "\n"; } } ================================================ FILE: src/SGA/Algorithm/DPAlignment.h ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // DPAlignment - Generic class for dynamic programming alignments // #ifndef DPALIGNMENT_H #define DPALIGNMENT_H #include "Match.h" #include inline int min3(int a, int b, int c) { return std::min(std::min(a,b), c); } inline int max3(int a, int b, int c) { return std::max(std::max(a,b), c); } // Scoring scheme used to calculate the minimum edit distance // between two strings class EditDistanceScoring { public: static int match_score(char a, char b) { return (a == b) ? 0 : 1; }; static int compare3(int a, int b, int c) { return min3(a,b,c); } static int gap_penalty() { return 1; } }; // Scoring scheme used to calculate the similarity of two strings class SimilarityScoring { public: static int match_score(char a, char b) { return (a == b) ? 2 : -1; }; static int compare3(int a, int b, int c) { return max3(a,b,c); } static int gap_penalty() { return -1; } }; // Path data structures enum DPOperation { DPO_MATCH, DPO_SUB, DPO_INSERT, DPO_DELETE, DPO_NOOP }; // struct DPPathNode { DPPathNode() : i(0), j(0), op(DPO_MATCH) {} int i; int j; // The operation leading to the next cell, not to this one DPOperation op; }; typedef std::list DPPath; // enum DPAlignMode { DPM_OVERLAP, DPM_ALIGNMENT }; // enum DPScoringScheme { DPSS_EDIT, DPSS_SIMILARITY }; class DPAlignment { public: // DPAlignment(const std::string& s1, const std::string& s2, DPAlignMode mode, DPScoringScheme scoring); // void printMatrix(const std::string& s1, const std::string& s2) const; void printAlignment(const std::string& s1, const std::string& s2, int startI = -1, int startJ = -1) const; DPPath calculatePath(const std::string& s1, const std::string& s2, int startI, int startJ) const; // int getNumRows() const; int getNumColumns() const; int getScore(int i, int j) const; DPOperation getPathOperationToCell(const std::string& s1, const std::string& s2, int i, int j) const; // DPScoringScheme getScoringScheme() const; private: // inline int match_score(char a, char b) const { switch(m_scoring) { case DPSS_SIMILARITY: return (a == b) ? 7 : -3; case DPSS_EDIT: return (a == b) ? 0 : 1; default: assert(false); } return 0; } // inline int gap_penalty() const { switch(m_scoring) { case DPSS_SIMILARITY: return -10; case DPSS_EDIT: return 1; default: assert(false); } return 0; } // GCC 4.7 warns on this #pragma GCC diagnostic ignored "-Wuninitialized" inline int compare3(int a, int b, int c) const { switch(m_scoring) { case DPSS_SIMILARITY: return max3(a,b,c); case DPSS_EDIT: return min3(a,b,c); default: assert(false); return a; } return a; } void initializeDPMatrix(); void fillMatrix(const std::string& s1, const std::string& s2); typedef std::vector IntVector; typedef std::vector IntMatrix; IntMatrix m_matrix; int m_dimM; int m_dimN; DPAlignMode m_mode; DPScoringScheme m_scoring; }; #endif ================================================ FILE: src/SGA/Algorithm/ErrorCorrectProcess.cpp ================================================ ///----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // ErrorCorrectProcess - Wrapper to perform error correction // for a sequence work item // #include "ErrorCorrectProcess.h" #include "CorrectionThresholds.h" #include "HashMap.h" #include "multiple_alignment.h" #include "KmerOverlaps.h" #include "StringThreader.h" //#define KMER_TESTING 1 // // // ErrorCorrectProcess::ErrorCorrectProcess(const ErrorCorrectParameters params) : m_params(params) { m_params.depthFilter = 10000; } // ErrorCorrectProcess::~ErrorCorrectProcess() { } // ErrorCorrectResult ErrorCorrectProcess::process(const SequenceWorkItem& workItem) { ErrorCorrectResult result = correct(workItem); if(!result.kmerQC && !result.overlapQC && m_params.printOverlaps) std::cout << workItem.read.id << " failed error correction QC\n"; return result; } ErrorCorrectResult ErrorCorrectProcess::correct(const SequenceWorkItem& workItem) { switch(m_params.algorithm) { case ECA_HYBRID: { ErrorCorrectResult result = kmerCorrection(workItem); if(!result.kmerQC) return overlapCorrectionNew(workItem); else return result; break; } case ECA_KMER: { return kmerCorrection(workItem); break; } case ECA_OVERLAP: { return overlapCorrectionNew(workItem); break; } case ECA_THREAD: { return threadingCorrection(workItem); break; } default: { assert(false); } } ErrorCorrectResult result; return result; } ErrorCorrectResult ErrorCorrectProcess::overlapCorrection(const SequenceWorkItem& workItem) { // Overlap based correction static const double p_error = 0.01f; bool done = false; int rounds = 0; ErrorCorrectResult result; SeqRecord currRead = workItem.read; std::string originalRead = workItem.read.seq.toString(); bool bQCPass = false; while(!done) { // Compute the set of overlap blocks for the read m_blockList.clear(); m_params.pOverlapper->overlapRead(currRead, m_params.minOverlap, &m_blockList); int sumOverlaps = 0; // Sum the spans of the overlap blocks to calculate the total number of overlaps this read has for(OverlapBlockList::iterator iter = m_blockList.begin(); iter != m_blockList.end(); ++iter) { assert(iter->ranges.interval[0].size() == iter->ranges.interval[1].size()); sumOverlaps += iter->ranges.interval[0].size(); } if(m_params.depthFilter > 0 && sumOverlaps > m_params.depthFilter) { result.num_prefix_overlaps = sumOverlaps; result.num_suffix_overlaps = sumOverlaps; result.correctSequence = currRead.seq; result.overlapQC = true; break; } // Convert the overlap block list into a multi-overlap MultiOverlap mo = blockListToMultiOverlap(currRead, m_blockList); if(m_params.printOverlaps) mo.printMasked(); result.num_prefix_overlaps = 0; result.num_suffix_overlaps = 0; mo.countOverlaps(result.num_prefix_overlaps, result.num_suffix_overlaps); // Perform conflict-aware consensus correction on the read result.correctSequence = mo.consensusConflict(p_error, m_params.conflictCutoff); ++rounds; if(rounds == m_params.numOverlapRounds || result.correctSequence == currRead.seq) { // Correction has converged or the number of rounds was exceeded. // Check if the sequence of the read passes QC in the multioverlap mo.updateRootSeq(result.correctSequence.toString()); bQCPass = mo.qcCheck(); done = true; } else { currRead.seq = result.correctSequence; } } result.overlapQC = bQCPass; if(m_params.printOverlaps) { std::string corrected_seq = result.correctSequence.toString(); std::cout << "OS: " << originalRead << "\n"; std::cout << "CS: " << corrected_seq << "\n"; std::cout << "DS: " << getDiffString(originalRead, corrected_seq) << "\n"; std::cout << "QS: " << currRead.qual << "\n"; std::cout << "QC: " << (result.overlapQC ? "pass" : "fail") << "\n"; std::cout << "\n"; } return result; } // ErrorCorrectResult ErrorCorrectProcess::overlapCorrectionNew(const SequenceWorkItem& workItem) { assert(m_params.indices.pBWT != NULL); assert(m_params.indices.pSSA != NULL); ErrorCorrectResult result; SeqRecord currRead = workItem.read; std::string current_sequence = workItem.read.seq.toString(); std::string consensus; int num_rounds = m_params.numOverlapRounds; for(int round = 0; round < num_rounds; ++round) { // Construct the multiple alignment MultipleAlignment multiple_alignment = KmerOverlaps::buildMultipleAlignment(current_sequence, m_params.kmerLength, m_params.minOverlap, m_params.minIdentity, 20, m_params.indices); multiple_alignment.filterByCount(m_params.conflictCutoff); bool last_round = (round == num_rounds - 1); if(last_round) consensus = multiple_alignment.calculateBaseConsensus(10000, 0); else current_sequence = multiple_alignment.calculateBaseConsensus(10000, 0); if(m_params.printOverlaps) multiple_alignment.print(200); } if(!consensus.empty()) { result.correctSequence = consensus; result.overlapQC = true; } else { // Return the unmodified query sequence result.correctSequence = workItem.read.seq.toString(); result.overlapQC = false; } return result; } // Correct a read with a k-mer based corrector ErrorCorrectResult ErrorCorrectProcess::kmerCorrection(const SequenceWorkItem& workItem) { assert(m_params.indices.pBWT != NULL); assert(m_params.indices.pCache != NULL); ErrorCorrectResult result; typedef std::map KmerCountMap; KmerCountMap kmerCache; SeqRecord currRead = workItem.read; std::string readSequence = workItem.read.seq.toString(); #ifdef KMER_TESTING std::cout << "Kmer correcting read " << workItem.read.id << "\n"; #endif if((int)readSequence.size() < m_params.kmerLength) { // The read is shorter than the kmer length, nothing can be done result.correctSequence = readSequence; result.kmerQC = false; return result; } int n = readSequence.size(); int nk = n - m_params.kmerLength + 1; // Are all kmers in the read well-represented? bool allSolid = false; bool done = false; int rounds = 0; int maxAttempts = m_params.numKmerRounds; // For each kmer, calculate the minimum phred score seen in the bases // of the kmer std::vector minPhredVector(nk, 0); for(int i = 0; i < nk; ++i) { int end = i + m_params.kmerLength - 1; int minPhred = std::numeric_limits::max(); for(int j = i; j <= end; ++j) { int ps = workItem.read.getPhredScore(j); if(ps < minPhred) minPhred = ps; } minPhredVector[i] = minPhred; } while(!done && nk > 0) { // Compute the kmer counts across the read // and determine the positions in the read that are not covered by any solid kmers // These are the candidate incorrect bases std::vector countVector(nk, 0); std::vector solidVector(n, 0); for(int i = 0; i < nk; ++i) { std::string kmer = readSequence.substr(i, m_params.kmerLength); // First check if this kmer is in the cache // If its not, find its count from the fm-index and cache it int count = 0; KmerCountMap::iterator iter = kmerCache.find(kmer); if(iter != kmerCache.end()) { count = iter->second; } else { count = BWTAlgorithms::countSequenceOccurrences(kmer, m_params.indices); kmerCache.insert(std::make_pair(kmer, count)); } // Get the phred score for the last base of the kmer int phred = minPhredVector[i]; countVector[i] = count; // std::cout << i << "\t" << phred << "\t" << count << "\n"; // Determine whether the base is solid or not based on phred scores int threshold = CorrectionThresholds::Instance().getRequiredSupport(phred); if(count >= threshold) { for(int j = i; j < i + m_params.kmerLength; ++j) solidVector[j] = 1; } } allSolid = true; for(int i = 0; i < n; ++i) { #ifdef KMER_TESTING std::cout << "Position[" << i << "] = " << solidVector[i] << "\n"; #endif if(solidVector[i] != 1) allSolid = false; } #ifdef KMER_TESTING std::cout << "Read " << workItem.read.id << (allSolid ? " is solid\n" : " has potential errors\n"); #endif // Stop if all kmers are well represented or we have exceeded the number of correction rounds if(allSolid || rounds++ > maxAttempts) break; // Attempt to correct the leftmost potentially incorrect base bool corrected = false; for(int i = 0; i < n; ++i) { if(solidVector[i] != 1) { // Attempt to correct the base using the leftmost covering kmer int phred = workItem.read.getPhredScore(i); int threshold = CorrectionThresholds::Instance().getRequiredSupport(phred); int left_k_idx = (i + 1 >= m_params.kmerLength ? i + 1 - m_params.kmerLength : 0); corrected = attemptKmerCorrection(i, left_k_idx, std::max(countVector[left_k_idx], threshold), readSequence); if(corrected) break; // base was not corrected, try using the rightmost covering kmer size_t right_k_idx = std::min(i, n - m_params.kmerLength); corrected = attemptKmerCorrection(i, right_k_idx, std::max(countVector[right_k_idx], threshold), readSequence); if(corrected) break; } } // If no base in the read was corrected, stop the correction process if(!corrected) { assert(!allSolid); done = true; } } if(allSolid) { result.correctSequence = readSequence; result.kmerQC = true; } else { result.correctSequence = workItem.read.seq.toString(); result.kmerQC = false; } return result; } // Attempt to correct the base at position idx in readSequence. Returns true if a correction was made // The correction is made only if the count of the corrected kmer is at least minCount bool ErrorCorrectProcess::attemptKmerCorrection(size_t i, size_t k_idx, size_t minCount, std::string& readSequence) { assert(i >= k_idx && i < k_idx + m_params.kmerLength); size_t base_idx = i - k_idx; char originalBase = readSequence[i]; std::string kmer = readSequence.substr(k_idx, m_params.kmerLength); size_t bestCount = 0; char bestBase = '$'; #if KMER_TESTING std::cout << "i: " << i << " k-idx: " << k_idx << " " << kmer << " " << reverseComplement(kmer) << "\n"; #endif for(int j = 0; j < DNA_ALPHABET::size; ++j) { char currBase = ALPHABET[j]; if(currBase == originalBase) continue; kmer[base_idx] = currBase; size_t count = BWTAlgorithms::countSequenceOccurrences(kmer, m_params.indices); #if KMER_TESTING printf("%c %zu\n", currBase, count); #endif if(count > bestCount && count >= minCount) { // Multiple corrections exist, do not correct if(bestBase != '$') return false; bestCount = count; bestBase = currBase; } } if(bestCount >= minCount) { assert(bestBase != '$'); readSequence[i] = bestBase; return true; } return false; } // // // ErrorCorrectResult ErrorCorrectProcess::threadingCorrection(const SequenceWorkItem& workItem) { ErrorCorrectResult result; // Find the initial seed to start the threading from // This is the prefix of the read containing right-most kmer that is present at least x times SeqRecord currRead = workItem.read; std::string query = workItem.read.seq.toString(); int k = m_params.kmerLength; int nk = query.size() - m_params.kmerLength + 1; int right_index = -1; size_t x = 3; for(int i = 0; i < nk; ++i) { std::string kmer = query.substr(i, k); size_t count = BWTAlgorithms::countSequenceOccurrences(kmer, m_params.indices); if(count >= x) right_index = i; else break; } if(right_index == -1) { // No way to correct this read result.correctSequence = ""; result.kmerQC = false; return result; } if(right_index == nk - 1) { printf("Read does not need correction\n"); // This read does not need correction result.correctSequence = query; result.kmerQC = true; return result; } // Correct the read with the threader int seed_endpoint = k + right_index; printf("Correcting %s [%d %d]\n", query.c_str(), right_index, seed_endpoint); StringThreaderResultVector thread_results; StringThreader threader(query.substr(0, seed_endpoint), &query, seed_endpoint, k, m_params.indices.pBWT); threader.run(thread_results); printf("Found %zu correction threads\n", thread_results.size()); int best_score = 0; int best_index = -1; for(size_t i = 0; i < thread_results.size(); ++i) { int score = StdAlnTools::globalAlignment(query, thread_results[i].thread, false); if(score > best_score) { best_score = score; best_index = i; } } if(best_score > 0) { result.correctSequence = thread_results[best_index].thread; result.kmerQC = true; } return result; } // // // ErrorCorrectPostProcess::ErrorCorrectPostProcess(std::ostream* pCorrectedWriter, std::ostream* pDiscardWriter, bool bCollectMetrics) : m_pCorrectedWriter(pCorrectedWriter), m_pDiscardWriter(pDiscardWriter), m_bCollectMetrics(bCollectMetrics), m_totalBases(0), m_totalErrors(0), m_readsKept(0), m_readsDiscarded(0), m_kmerQCPassed(0), m_overlapQCPassed(0), m_qcFail(0) { } // ErrorCorrectPostProcess::~ErrorCorrectPostProcess() { std::cout << "Reads passed kmer QC check: " << m_kmerQCPassed << "\n"; std::cout << "Reads passed overlap QC check: " << m_overlapQCPassed << "\n"; std::cout << "Reads failed QC: " << m_qcFail << "\n"; } // void ErrorCorrectPostProcess::writeMetrics(std::ostream* pWriter) { m_positionMetrics.write(pWriter, "Bases corrected by position\n", "pos"); m_originalBaseMetrics.write(pWriter, "\nOriginal base that was corrected\n", "base"); m_precedingSeqMetrics.write(pWriter, "\nkmer preceding the corrected base\n", "kmer"); m_qualityMetrics.write(pWriter, "\nBases corrected by quality value\n\n", "quality"); std::cout << "ErrorCorrect -- Corrected " << m_totalErrors << " out of " << m_totalBases << " bases (" << (double)m_totalErrors / m_totalBases << ")\n"; std::cout << "Kept " << m_readsKept << " reads. Discarded " << m_readsDiscarded << " reads (" << (double)m_readsDiscarded / (m_readsKept + m_readsDiscarded)<< ")\n"; } // void ErrorCorrectPostProcess::process(const SequenceWorkItem& item, const ErrorCorrectResult& result) { // Determine if the read should be discarded bool readQCPass = true; if(result.kmerQC) { m_kmerQCPassed += 1; } else if(result.overlapQC) { m_overlapQCPassed += 1; } else { readQCPass = false; m_qcFail += 1; } // Collect metrics for the reads that were actually corrected if(m_bCollectMetrics && readQCPass) { collectMetrics(item.read.seq.toString(), result.correctSequence.toString(), item.read.qual); } SeqRecord record = item.read; record.seq = result.correctSequence; if(readQCPass || m_pDiscardWriter == NULL) { record.write(*m_pCorrectedWriter); ++m_readsKept; } else { record.write(*m_pDiscardWriter); ++m_readsDiscarded; } } void ErrorCorrectPostProcess::collectMetrics(const std::string& originalSeq, const std::string& correctedSeq, const std::string& qualityStr) { size_t precedingLen = 2; for(size_t i = 0; i < originalSeq.length(); ++i) { char qc = !qualityStr.empty() ? qualityStr[i] : '\0'; char ob = originalSeq[i]; ++m_totalBases; m_positionMetrics.incrementSample(i); if(!qualityStr.empty()) m_qualityMetrics.incrementSample(qc); m_originalBaseMetrics.incrementSample(ob); std::string precedingMer; if(i > precedingLen) { precedingMer = originalSeq.substr(i - precedingLen, precedingLen); m_precedingSeqMetrics.incrementSample(precedingMer); } if(originalSeq[i] != correctedSeq[i]) { m_positionMetrics.incrementError(i); if(!qualityStr.empty()) m_qualityMetrics.incrementError(qc); m_originalBaseMetrics.incrementError(ob); if(!precedingMer.empty()) { m_precedingSeqMetrics.incrementError(precedingMer); } ++m_totalErrors; } } } ================================================ FILE: src/SGA/Algorithm/ErrorCorrectProcess.h ================================================ ///----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // ErrorCorrectProcess - Wrapper to perform error correction // for a sequence work item // #ifndef CORRECTPROCESS_H #define CORRECTPROCESS_H #include "Util.h" #include "OverlapAlgorithm.h" #include "SequenceProcessFramework.h" #include "SequenceWorkItem.h" #include "MultiOverlap.h" #include "Metrics.h" #include "BWTIndexSet.h" #include "SampledSuffixArray.h" #include "multiple_alignment.h" enum ErrorCorrectAlgorithm { ECA_HYBRID, // hybrid kmer/overlap correction ECA_KMER, // kmer correction ECA_OVERLAP, // overlap correction ECA_THREAD // thread the read through a de Bruijn graph }; enum ECFlag { ECF_NOTCORRECTED, ECF_CORRECTED, ECF_AMBIGIOUS, ECF_DUPLICATE }; // Parameter object for the error corrector struct ErrorCorrectParameters { ErrorCorrectAlgorithm algorithm; // const OverlapAlgorithm* pOverlapper; BWTIndexSet indices; // Overlap-based corrector params int minOverlap; int numOverlapRounds; double minIdentity; int conflictCutoff; int depthFilter; // k-mer based corrector params int numKmerRounds; int kmerLength; // output options bool printOverlaps; }; class ErrorCorrectResult { public: ErrorCorrectResult() : num_prefix_overlaps(0), num_suffix_overlaps(0), kmerQC(false), overlapQC(false) {} DNAString correctSequence; ECFlag flag; // Metrics size_t num_prefix_overlaps; size_t num_suffix_overlaps; bool kmerQC; bool overlapQC; }; // class ErrorCorrectProcess { public: ErrorCorrectProcess(const ErrorCorrectParameters params); ~ErrorCorrectProcess(); ErrorCorrectResult process(const SequenceWorkItem& item); ErrorCorrectResult correct(const SequenceWorkItem& item); ErrorCorrectResult kmerCorrection(const SequenceWorkItem& item); ErrorCorrectResult overlapCorrection(const SequenceWorkItem& workItem); ErrorCorrectResult overlapCorrectionNew(const SequenceWorkItem& workItem); ErrorCorrectResult threadingCorrection(const SequenceWorkItem& workItem); private: bool attemptKmerCorrection(size_t i, size_t k_idx, size_t minCount, std::string& readSequence); OverlapBlockList m_blockList; ErrorCorrectParameters m_params; }; // Write the results from the overlap step to an ASQG file class ErrorCorrectPostProcess { public: ErrorCorrectPostProcess(std::ostream* pCorrectedWriter, std::ostream* pDiscardWriter, bool bCollectMetrics); ~ErrorCorrectPostProcess(); void process(const SequenceWorkItem& item, const ErrorCorrectResult& result); void writeMetrics(std::ostream* pWriter); private: void collectMetrics(const std::string& originalSeq, const std::string& correctedSeq, const std::string& qualityStr); std::ostream* m_pCorrectedWriter; std::ostream* m_pDiscardWriter; bool m_bCollectMetrics; ErrorCountMap m_qualityMetrics; ErrorCountMap m_positionMetrics; ErrorCountMap m_originalBaseMetrics; ErrorCountMap m_precedingSeqMetrics; size_t m_totalBases; size_t m_totalErrors; size_t m_readsKept; size_t m_readsDiscarded; size_t m_kmerQCPassed; size_t m_overlapQCPassed; size_t m_qcFail; }; #endif ================================================ FILE: src/SGA/Algorithm/ExtensionDP.cpp ================================================ ///---------------------------------------------- // Copyright 2011 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // // ExtensionDP - Class implementing an iterative // dynamic programming alignment. The alignment // starts by globally constructing an alignment // between two strings in a seed region. This initial // alignment can then be extended row-by-row as one of the // strings is extended. This is the core data structure for the // StringThreader class // #include "ExtensionDP.h" #include #include #include #include static const int MINUS_INF = -1073741823; static const int POSITIVE_INF = 1073741823; // Return the largest of the 3 values inline int max3(int a, int b, int c) { return std::max(std::max(a,b), c); } // Return the smalleset of the 3 values inline int min3(int a, int b, int c) { return std::min(std::min(a,b), c); } // Constructor BandedDPColumn::BandedDPColumn(int ci, int maxRows, int bandwidth, const BandedDPColumn* prevColumn) { // Calculate the lower and upper band indices m_bandwidth = bandwidth; // the requested bandwidth, may be greater than actual m_colIdx = ci; m_rowStartIdx = std::max(0, m_colIdx - (bandwidth / 2) - 1); m_rowEndIdx = std::min(maxRows - 1, m_colIdx + (bandwidth / 2)); m_maxRows = maxRows; assert(m_rowStartIdx <= m_rowEndIdx); int numRows = m_rowEndIdx - m_rowStartIdx + 1; m_cells.resize(numRows); m_pPrevColumn = prevColumn; } // Get the score for row int BandedDPColumn::getRowScore(int row) const { int vec_idx = getVectorIndex(row); if(vec_idx == -1) return POSITIVE_INF; else return m_cells[vec_idx].score; } // Get the type of the row char BandedDPColumn::getRowType(int row) const { int vec_idx = getVectorIndex(row); if(vec_idx == -1) return FROM_M; else return m_cells[vec_idx].ctype; } // Return the index of the best-scoring row int BandedDPColumn::getBestRowIndex() const { int bestScore = POSITIVE_INF; int bestIdx = -1; for(int i = m_rowStartIdx; i <= m_rowEndIdx; ++i) { int s = getRowScore(i); if(s < bestScore) { bestScore = s; bestIdx = i; } } return bestIdx; } // Return the pointer to the previous column const BandedDPColumn* BandedDPColumn::getPreviousColumn() const { return m_pPrevColumn; } // Set the score and type for row void BandedDPColumn::setRowScore(int row, int score, char ctype) { int vec_idx = getVectorIndex(row); if(vec_idx != -1) { m_cells[vec_idx].score = score; m_cells[vec_idx].ctype = ctype; } } // Return the score for the requested cell int BandedDPColumn::getCellScore(int colIdx, int rowIdx) const { // If accessing the col/row with negative index, return // the initial gap penalties. For now, we allow free gaps // for the first row if(colIdx == -1 || rowIdx == -1) return 0; const BandedDPColumn* pColumn; if(colIdx < m_colIdx) pColumn = m_pPrevColumn; else pColumn = this; assert(pColumn != NULL); return pColumn->getRowScore(rowIdx); } // Calculate the score for the given row in the column using an edit distance scheme void BandedDPColumn::fillRowEditDistance(int rowIdx, int matchScore) { int vec_idx = getVectorIndex(rowIdx); if(vec_idx == -1) return; // out of band, ignore // Get the scores for the cell above, to the left and diagonal int above = getCellScore(m_colIdx, rowIdx - 1) + 1; int diag = getCellScore(m_colIdx - 1, rowIdx - 1) + matchScore; // Give free insertions if the last row // JS Apr/12 do not give free insertions in the last row //int insert_score = rowIdx < (m_maxRows - 1) ? 1 : 0; int insert_score = 1; int left = getCellScore(m_colIdx - 1, rowIdx) + insert_score; int score = min3(above, left, diag); char ctype; if(score == diag) ctype = FROM_M; else if(score == above) ctype = FROM_D; else ctype = FROM_I; // printf("Cell(%d, %d) a: %d d: %d l: %d s: %d t: %d\n", m_colIdx, rowIdx, above, diag, left, score, ctype); setRowScore(rowIdx, score, ctype); } // Transform a row index into an index in the scores vector int BandedDPColumn::getVectorIndex(int rowIdx) const { if(rowIdx < m_rowStartIdx || rowIdx > m_rowEndIdx) return -1; else return rowIdx - m_rowStartIdx; } // Initialize the extension DP by computing a global alignment between extendable and fixed // This function allocates memory and stores the created pointers in outPtrVec, which // should initially be empty void ExtensionDP::createInitialAlignment(const std::string& extendable, const std::string& fixed, int bandwidth, BandedDPColumnPtrVector& outPtrVec) { assert(outPtrVec.empty()); // Initialize the zero-th column size_t numRows = fixed.size() + 1; size_t numCols = extendable.size() + 1; // Set the score of column zero BandedDPColumn* pZeroCol = new BandedDPColumn(0, numRows, bandwidth, NULL); pZeroCol->setRowScore(0, 0, FROM_M); for(size_t i = 1; i < numRows; ++i) pZeroCol->setRowScore(i, i, FROM_I); outPtrVec.push_back(pZeroCol); BandedDPColumn* pPrevCol = outPtrVec.back(); for(size_t colIdx = 1; colIdx < numCols; ++colIdx) { BandedDPColumn* pCurrCol = createNewColumn(extendable[colIdx - 1], fixed, pPrevCol); outPtrVec.push_back(pCurrCol); pPrevCol = pCurrCol; } } // Create a new column, representing the extension of the alignment to char b BandedDPColumn* ExtensionDP::createNewColumn(char b, const std::string& fixed, const BandedDPColumn* pPrevColumn) { int numRows = fixed.size() + 1; int colIdx = pPrevColumn->getColIdx() + 1; BandedDPColumn* pCurrCol = new BandedDPColumn(colIdx, numRows, pPrevColumn->getBandwidth(), pPrevColumn); // Set the first row score pCurrCol->setRowScore(0, colIdx, FROM_D); // Fill in the rest of the rows int startRow = std::max(1, pCurrCol->getMinRow()); for(int rowIdx = startRow; rowIdx <= pCurrCol->getMaxRow(); ++rowIdx) { int match_score = b == fixed[rowIdx - 1] ? 0 : 1; pCurrCol->fillRowEditDistance(rowIdx, match_score); } return pCurrCol; } // Calculate the edit percentage over the last numBases bases double ExtensionDP::calculateLocalEditPercentage(const BandedDPColumn* pColumn, int numBases) { // Get the row with in the start column with the best score int rowIdx = pColumn->getBestRowIndex(); assert(rowIdx != -1); int colIdx = pColumn->getColIdx(); int startScore = pColumn->getRowScore(rowIdx); int alignLength = 1; while(alignLength < numBases) { char ctype = pColumn->getRowType(rowIdx); switch(ctype) { case FROM_M: pColumn = pColumn->getPreviousColumn(); --colIdx; --rowIdx; break; case FROM_D: --rowIdx; break; case FROM_I: pColumn = pColumn->getPreviousColumn(); --colIdx; break; } alignLength += 1; } int endScore = pColumn->getRowScore(rowIdx); assert(startScore >= endScore); int numDiffs = startScore - endScore; return static_cast(numDiffs) / alignLength; } // Returns the error percentage over the entire alignment double ExtensionDP::calculateGlobalEditPercentage(const BandedDPColumn* pStartColumn) { int edits, alignLength; countEditsAndAlignLength(pStartColumn, edits, alignLength); return (double)edits / alignLength; } // Returns the number of edits and the total alignment length for the submatrix starting at pColumn void ExtensionDP::countEditsAndAlignLength(const BandedDPColumn* pColumn, int& edits, int& alignLength) { // Get the row with in the start column with the best score int rowIdx = pColumn->getBestRowIndex(); assert(rowIdx != -1); int colIdx = pColumn->getColIdx(); edits = pColumn->getRowScore(rowIdx); alignLength = 1; while(rowIdx != 0 && colIdx != 0) { char ctype = pColumn->getRowType(rowIdx); switch(ctype) { case FROM_M: pColumn = pColumn->getPreviousColumn(); --colIdx; --rowIdx; break; case FROM_D: --rowIdx; break; case FROM_I: pColumn = pColumn->getPreviousColumn(); --colIdx; break; } alignLength += 1; } } // Calculate the best alignment through the matrix. Assumes that // path_t* is pre-allocated with max_path entries. void ExtensionDP::backtrack(const BandedDPColumn* pLastColumn, path_t* path, int* path_len, const int maxPathLength) { const BandedDPColumn* pCurrColumn = pLastColumn; // Calculate the starting row and column int colIdx = pLastColumn->getColIdx(); int rowIdx = pLastColumn->getBestRowIndex(); int pathLength = 0; path_t* p = &path[pathLength]; char ctype = pLastColumn->getRowType(rowIdx); p->ctype = ctype; p->i = rowIdx; p->j = colIdx; pathLength += 1; do { assert(pathLength < maxPathLength); assert(pCurrColumn != NULL); p = &path[pathLength]; // Update indices switch (ctype) { case FROM_M: pCurrColumn = pCurrColumn->getPreviousColumn(); --colIdx; --rowIdx; break; case FROM_D: --rowIdx; break; case FROM_I: pCurrColumn = pCurrColumn->getPreviousColumn(); --colIdx; break; } ctype = pCurrColumn->getRowType(rowIdx); p->ctype = ctype; p->i = rowIdx; p->j = colIdx; pathLength += 1; } while (rowIdx || colIdx); // We ignore the path character from the first column/row, like stdaln *path_len = pathLength - 1; } // Return true if the extension can proceed no further. // This is based on two conditions: // 1) There are n insertions at the end of the sequence // 2) The bandwidth in this column is one bool ExtensionDP::isExtensionTerminated(const BandedDPColumn* pLastColumn, int insertionThreshold) { if(pLastColumn->getMinRow() == pLastColumn->getMaxRow()) return true; // Backtrack up to n steps checking for insertions const BandedDPColumn* pCurrColumn = pLastColumn; int rowIdx = pLastColumn->getBestRowIndex(); int colIdx = pLastColumn->getColIdx(); char ctype = pCurrColumn->getRowType(rowIdx); do { assert(pCurrColumn != NULL); switch (ctype) { case FROM_M: case FROM_D: return false; case FROM_I: pCurrColumn = pCurrColumn->getPreviousColumn(); --colIdx; insertionThreshold -= 1; if(insertionThreshold == 0) return true; } ctype = pCurrColumn->getRowType(rowIdx); } while (rowIdx || colIdx); return false; } // Parse the score matrix and return a trimmed alignment between the query and target strings. // The trimming condition requires the last minMatches bases of the two sequences to be perfectly aligned ExtensionDPAlignment ExtensionDP::findTrimmedAlignment(const BandedDPColumn* pLastColumn, int minMatches) { const BandedDPColumn* pBestColumn = pLastColumn; bool columnFound = false; // Iterate backwards over the columns searching for one that // has minMatches matches to the query at the end while(!columnFound) { // Check if the best alignment starting from this column // ends with n matches. int matches = 0; int mismatches = 0; const BandedDPColumn* pCurrColumn = pBestColumn; int rowIdx = pCurrColumn->getBestRowIndex(); int colIdx = pCurrColumn->getColIdx(); int startScore = pCurrColumn->getRowScore(rowIdx); char ctype = pCurrColumn->getRowType(rowIdx); do { if(matches == minMatches) { // Ensure the edit distance is the same columnFound = true; break; } switch (ctype) { case FROM_M: pCurrColumn = pCurrColumn->getPreviousColumn(); --colIdx; --rowIdx; matches += 1; break; case FROM_D: case FROM_I: mismatches += 1; columnFound = false; break; } ctype = pCurrColumn->getRowType(rowIdx); // Update mismatches if(pCurrColumn->getRowScore(rowIdx) != startScore) ++mismatches; } while ((rowIdx || colIdx) && pCurrColumn != NULL && mismatches == 0); if(!columnFound) pBestColumn = pBestColumn->getPreviousColumn(); } // set the result output ExtensionDPAlignment result; if(pBestColumn != NULL) { result.target_align_length = pBestColumn->getColIdx(); result.query_align_length = pBestColumn->getBestRowIndex(); } else { result.target_align_length = 0; result.query_align_length = 0; } return result; } // Parse the score matrix and return a the best alignment that includes the entire query string. ExtensionDPAlignment ExtensionDP::findGlocalAlignment(const BandedDPColumn* pLastColumn) { const BandedDPColumn* pCurrColumn = pLastColumn; int lastRowIdx = pLastColumn->getQueryRows() - 1; const BandedDPColumn* pBestColumn = NULL; int best_score = std::numeric_limits::max(); // Iterate backwards over the columns searching for one that // has minMatches matches to the query at the end while(pCurrColumn != NULL && pCurrColumn->getMaxRow() >= lastRowIdx) { // Get the score for the last row of the matrix int score = pCurrColumn->getRowScore(lastRowIdx); if(score < best_score) { best_score = score; pBestColumn = pCurrColumn; } pCurrColumn = pCurrColumn->getPreviousColumn(); } // set the result output ExtensionDPAlignment result; if(pBestColumn != NULL) { result.target_align_length = pBestColumn->getColIdx(); result.query_align_length = lastRowIdx; } else { result.target_align_length = 0; result.query_align_length = 0; } return result; } // Print the alignment starting from the given column void ExtensionDP::printAlignment(const std::string& extendable, const std::string& fixed, const BandedDPColumn* pLastColumn) { // allocate space for the path int maxPathLength = extendable.size() + fixed.size(); path_t* path = new path_t[maxPathLength]; int path_len; // Perform the backtrack backtrack(pLastColumn, path, &path_len, maxPathLength); // Print the strings std::string paddedF, paddedE, paddedMatch; StdAlnTools::makePaddedStringsFromPath(fixed, extendable, path, path_len, paddedF, paddedE, paddedMatch); StdAlnTools::printPaddedStrings(paddedF, paddedE, paddedMatch); std::cout << "CIGAR: " << StdAlnTools::makeCigar(path, path_len) << "\n"; delete [] path; } // Print the full scoring matrix void ExtensionDP::printMatrix(const BandedDPColumnPtrVector& columnPtrVec) { int numCols = columnPtrVec.size(); int numRows = columnPtrVec[numCols - 1]->getMaxRow() + 1; printf("c:\t"); for(int j = 0; j < numCols; ++j) { printf("%d\t", j); } printf("\n"); for(int i = 0; i < numRows; ++i) { printf("%d:\t", i); for(int j = 0; j < numCols; ++j) { int score = columnPtrVec[j]->getRowScore(i); printf("%d\t", score); } printf("\n"); } } ================================================ FILE: src/SGA/Algorithm/ExtensionDP.h ================================================ ///---------------------------------------------- // Copyright 2011 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // // ExtensionDP - Class implementing an iterative // dynamic programming alignment. The alignment // starts by globally constructing an alignment // between two strings in a seed region. This initial // alignment can then be extended row-by-row as one of the // strings is extended. This is the core data structure for the // StringThreader class // #ifndef EXTENSION_DP_H #define EXTENSION_DP_H #include #include "StdAlnTools.h" // Structure holding the score for a single cell // of the DP matrix struct DPCell { DPCell() : score(0), ctype('\0') {} int score; char ctype; }; typedef std::vector CellVector; // Result object providing the coordinates // of the endpoints of the alignment struct ExtensionDPAlignment { int query_align_length; int target_align_length; }; // Core class providing a single column of the dynamic // programming matrix class BandedDPColumn { public: // // Functions // BandedDPColumn(int ci, int maxRows, int bandwidth, const BandedDPColumn* prevColumn); // Simple getters int getColIdx() const { return m_colIdx; } int getBandwidth() const { return m_bandwidth; } int getRowScore(int row) const; char getRowType(int row) const; int getCellScore(int colIdx, int rowIdx) const; int getMinRow() const { return m_rowStartIdx; } int getMaxRow() const { return m_rowEndIdx; } int getQueryRows() const { return m_maxRows; } const BandedDPColumn* getPreviousColumn() const; // Set the cell information for a given row in the column void setRowScore(int row, int score, char ctype); // Returns the row index of the best scoring cell int getBestRowIndex() const; // Calculate and set the score for the row void fillRowEditDistance(int rowIdx, int matchScore); private: // // Functions // int getVectorIndex(int rowIdx) const; // // Data // int m_colIdx; int m_maxRows; int m_rowStartIdx; int m_rowEndIdx; int m_bandwidth; CellVector m_cells; const BandedDPColumn* m_pPrevColumn; }; typedef std::vector BandedDPColumnPtrVector; // Algorithms to use/extend the extensionDP functionality namespace ExtensionDP { // Initialize the extension DP by computing a global alignment between extendable and fixed // This function allocates memory and stores the created pointers in outPtrVec void createInitialAlignment(const std::string& extendable, const std::string& fixed, int bandwidth, BandedDPColumnPtrVector& outPtrVec); // Create a new column, representing the extended alignment from pPrevColumn to char b BandedDPColumn* createNewColumn(char b, const std::string& fixed, const BandedDPColumn* pPrevColumn); // Calculate the best alignment through the matrix. Assumes that // path_t* is pre-allocated with maxPathLength entries. void backtrack(const BandedDPColumn* pLastColumn, path_t* path, int* pPathLen, const int maxPathLength); // Check if the extension has terminated. This is true if there are more // than insertionThreshold insertions at the end of the alignment. bool isExtensionTerminated(const BandedDPColumn* pLastColumn, int insertionThreshold); // Return the best trimmed alignment of the extensionDP object requiring minMatches matches // at the end of the alignment ExtensionDPAlignment findTrimmedAlignment(const BandedDPColumn* pLastColumn, int minMatches); // Return the best alignment that contains the entire fixed string ExtensionDPAlignment findGlocalAlignment(const BandedDPColumn* pLastColumn); // Calculate mismatch/error rates between the query and the string up to pStartColumn double calculateLocalEditPercentage(const BandedDPColumn* pStartColumn, int numBases); double calculateGlobalEditPercentage(const BandedDPColumn* pStartColumn); void countEditsAndAlignLength(const BandedDPColumn* pStartColumn, int& edits, int& alignLength); // Print the alignment starting from the given column void printAlignment(const std::string& s1, const std::string& s2, const BandedDPColumn* pLastColumn); // Print the full scoring matrix void printMatrix(const BandedDPColumnPtrVector& columnPtrVec); }; #endif ================================================ FILE: src/SGA/Algorithm/FMMergeProcess.cpp ================================================ ///----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // FMMergeProcess - Merge unambiguously overlapping sequences // #include "FMMergeProcess.h" #include "SGAlgorithms.h" #include "SGVisitors.h" // FMMergeProcess::FMMergeProcess(const OverlapAlgorithm* pOverlapper, int minOverlap, BitVector* pMarkedReads) : m_pOverlapper(pOverlapper), m_minOverlap(minOverlap), m_pMarkedReads(pMarkedReads) { } // FMMergeProcess::~FMMergeProcess() { } // FMMergeResult FMMergeProcess::process(const SequenceWorkItem& item) { // Calculate the intervals in the forward FM-index for this read const BWT* pBWT = m_pOverlapper->getBWT(); // Find the interval in the fm-index containing the read std::string readString = item.read.seq.toString(); BWTInterval readInterval = BWTAlgorithms::findInterval(pBWT, readString); // Update the interval by looking for the $ to map the interval into indices in the bit array BWTAlgorithms::updateInterval(readInterval, '$', pBWT); // The read must be present in the index assert(readInterval.isValid()); // Check if this read has been used yet bool used = false; for(int64_t i = readInterval.lower; i <= readInterval.upper; ++i) { if(m_pMarkedReads->test(i)) { used = true; break; } } FMMergeResult result; if(!used) { // Construct a new local graph around this read StringGraph* pGraph = new StringGraph; std::stringstream ssID; ssID << "IDX-" << readInterval.lower; std::string rootID = ssID.str(); Vertex* pVertex = new(pGraph->getVertexAllocator()) Vertex(rootID, readString); pGraph->addVertex(pVertex); // Add the root vertex to the result structure result.usedIntervals.push_back(readInterval); // Enqueue the read for overlap detection in both directions FMMergeQueue queue; // Construct the overlap block list for this node SeqRecord record; record.id = pVertex->getID(); record.seq = pVertex->getSeq().toString(); OverlapBlockList blockList; m_pOverlapper->overlapRead(record, m_minOverlap, &blockList); removeContainmentBlocks(pVertex->getSeqLen(), &blockList); // Construct the initial list of candidates from the block list addCandidates(pGraph, pVertex, NULL, &blockList, queue); while(!queue.empty()) { FMMergeCandidate currCandidate = queue.front(); queue.pop(); // Determine whether this is a valid vertex to merge or not. // It is valid if it has a single edge in the direction of the vertex // that added it to the candidate list SeqRecord record; record.id = currCandidate.pVertex->getID(); record.seq = currCandidate.pVertex->getSeq().toString(); OverlapBlockList candidateBlockList; m_pOverlapper->overlapRead(record, m_minOverlap, &candidateBlockList); removeContainmentBlocks(currCandidate.pVertex->getSeqLen(), &candidateBlockList); bool validMergeNode = checkCandidate(currCandidate, &candidateBlockList); if(validMergeNode) { addCandidates(pGraph, currCandidate.pVertex, currCandidate.pEdge, &candidateBlockList, queue); result.usedIntervals.push_back(currCandidate.interval); } else { // Mark this vertex for later removal currCandidate.pVertex->setColor(GC_RED); } } // The graph has now been constructed. Remove all the nodes that are marked invalid for merging pGraph->sweepVertices(GC_RED); SGDuplicateVisitor dupVisit(true); pGraph->visit(dupVisit); // Merge nodes pGraph->simplify(); // If there was a cycle in the graph, it is possible that more than 1 vertex // remains in the graph. Copy the vertex sequences into the result object. pGraph->getVertexSequences(result.mergedSequences); result.isMerged = true; delete pGraph; } else { result.isMerged = false; } if(result.isMerged) { // If some work was performed, update the bitvector so other threads do not try to merge the same set of reads. // This uses compare-and-swap instructions to ensure the uppdate is atomic. // If some other thread has merged this set (and updated // the bitvector), we discard all the merged data. // As a given set of reads should all be merged together, we only need to make sure we atomically update // the bit for the read with the lowest index in the set. // Sort the intervals into ascending order and remove any duplicate intervals (which can occur // if the subgraph has a simple cycle) std::sort(result.usedIntervals.begin(), result.usedIntervals.end(), BWTInterval::compare); std::vector::iterator newEnd = std::unique(result.usedIntervals.begin(), result.usedIntervals.end(), BWTInterval::equal); result.usedIntervals.erase(newEnd, result.usedIntervals.end()); // Check if the bit in the vector has already been set for the lowest read index // If it has some other thread has already output this set so we do nothing int64_t lowestIndex = result.usedIntervals.front().lower; bool currentValue = m_pMarkedReads->test(lowestIndex); bool updateSuccess = false; if(currentValue == false) { // Attempt to update the bit vector with an atomic CAS. If this returns false // the bit was set by some other thread updateSuccess = m_pMarkedReads->updateCAS(lowestIndex, currentValue, true); } if(updateSuccess) { // We successfully atomically set the bit for the first read in this set // to true. We can safely update the rest of the bits and keep the merged sequences // for output. std::vector::const_iterator iter = result.usedIntervals.begin(); for(; iter != result.usedIntervals.end(); ++iter) { for(int64_t i = iter->lower; i <= iter->upper; ++i) { if(i == lowestIndex) //already set continue; currentValue = m_pMarkedReads->test(i); if(currentValue) { // This value should not be true, emit a warning std::cout << "Warning: Bit " << i << " was set outside of critical section\n"; } else { m_pMarkedReads->updateCAS(i, currentValue, true); } } } } else { // Some other thread merged these reads already, discard the intermediate // data and set the result to false result.mergedSequences.clear(); result.usedIntervals.clear(); result.isMerged = false; } } return result; } // Check if the candidate node can be merged with the node it is linked to. Returns true if so bool FMMergeProcess::checkCandidate(const FMMergeCandidate& candidate, const OverlapBlockList* pBlockList) const { // Get the direction of the edge back to the node that generated this candidate // pEdge is the edge TO the candidate vertex so the twin direction is the direction away // from the candidate vertex EdgeDir mergeDir = candidate.pEdge->getTwinDir(); size_t mergeDirCount = 0; for(OverlapBlockList::const_iterator iter = pBlockList->begin(); iter != pBlockList->end(); ++iter) { if(iter->getEdgeDir() == mergeDir) mergeDirCount += 1; } assert(mergeDirCount > 0); return mergeDirCount == 1; } // Add the edges starting from pX as candidate vertices // using the blockList. // Precondition: pX is a valid vertex in the merge graph. In other words, there is a // unique assembly that includes pX and the root vertex. void FMMergeProcess::addCandidates(StringGraph* pGraph, const Vertex* pX, const Edge* pEdgeToX, const OverlapBlockList* pBlockList, FMMergeQueue& candidateQueue) { // Count the number of edges in each direction. size_t numAnti = 0; size_t numSense = 0; for(OverlapBlockList::const_iterator iter = pBlockList->begin(); iter != pBlockList->end(); ++iter) { if(iter->getEdgeDir() == ED_SENSE) ++numSense; if(iter->getEdgeDir() == ED_ANTISENSE) ++numAnti; } // For each edge block, if it is unique for the direction add the vertex it describes as a candidate for(OverlapBlockList::const_iterator iter = pBlockList->begin(); iter != pBlockList->end(); ++iter) { EdgeDir currDir = iter->getEdgeDir(); if((currDir == ED_SENSE && numSense == 1) || (currDir == ED_ANTISENSE && numAnti == 1)) { // Skip edges in the direction to X if(pEdgeToX != NULL && pEdgeToX->getTwinDir() == currDir) continue; // Construct new candidate vertices and add them to the graph std::string vertexID = iter->toCanonicalID(); assert(vertexID != pX->getID()); std::string vertexSeq = iter->getFullString(pX->getSeq().toString()); Overlap ovrXY = iter->toOverlap(pX->getID(), vertexID, pX->getSeqLen(), vertexSeq.length()); // The vertex may already exist in the graph if the graph contains a loop Vertex* pY = pGraph->getVertex(vertexID); // Generate the new vertex if(pY == NULL) { pY = new(pGraph->getVertexAllocator()) Vertex(vertexID, vertexSeq); pGraph->addVertex(pY); } // Construct a description of the edge based on the overlap EdgeDesc ed = SGAlgorithms::overlapToEdgeDesc(pY, ovrXY); // If an edge with the same description as XY exists for X do not add a new edge or candidate if(!pX->hasEdge(ed)) { // Construct the found edge and add it to the graph Edge* pXY = SGAlgorithms::createEdgesFromOverlap(pGraph, ovrXY, false); // Add the new vertex as a candidate FMMergeCandidate candidate; candidate.pVertex = pY; candidate.pEdge = pXY; candidate.interval = iter->getCanonicalInterval(); candidateQueue.push(candidate); } } } } // FMMergePostProcess::FMMergePostProcess(std::ostream* pWriter, BitVector* pMarkedReads) : m_numMerged(0), m_numTotal(0), m_totalLength(0), m_pWriter(pWriter), m_pMarkedReads(pMarkedReads) { } // FMMergePostProcess::~FMMergePostProcess() { printf("[sga fm-merge] Merged %zu reads into %zu sequences\n", m_numTotal, m_numMerged); printf("[sga fm-merge] Reduction factor: %lf\n", (double)m_numTotal / m_numMerged); printf("[sga fm-merge] Mean merged size: %lf\n", (double)m_totalLength / m_numMerged); } // void FMMergePostProcess::process(const SequenceWorkItem& item, const FMMergeResult& result) { (void)item; m_numTotal += 1; if(result.isMerged) { // Write out the merged sequences for(std::vector::const_iterator iter = result.mergedSequences.begin(); iter != result.mergedSequences.end(); ++iter) { std::stringstream nameSS; nameSS << "merged-" << m_numMerged++; SeqRecord record; record.id = nameSS.str(); record.seq = *iter; record.write(*m_pWriter); m_totalLength += iter->size(); } } } ================================================ FILE: src/SGA/Algorithm/FMMergeProcess.h ================================================ ///----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // FMMergeProcess - Merge unambiguously overlapping sequences // #ifndef FMMERGEPROCESS_H #define FMMERGEPROCESS_H #include "Util.h" #include "OverlapAlgorithm.h" #include "SequenceProcessFramework.h" #include "BitVector.h" #include "Bigraph.h" #include "SGUtil.h" struct FMMergeResult { std::vector mergedSequences; std::vector usedIntervals; bool isMerged; }; // A merge candidate is a read that is a unique extension // from an existing read. If it has a single edge // in the direction pointing back to the existing read // it can be merged with that read. if it has multiple // edges back to the read it cannot and will be marked as // invalid. // struct FMMergeCandidate { Vertex* pVertex; Edge* pEdge; // Edge from the existing vertex to pVertex BWTInterval interval; // interval containing this candidate }; typedef std::queue FMMergeQueue; // Compute the overlap blocks for reads class FMMergeProcess { public: FMMergeProcess(const OverlapAlgorithm* pOverlapper, int minOverlap, BitVector* pMarkedReads); ~FMMergeProcess(); FMMergeResult process(const SequenceWorkItem& item); private: // Add the edges of X as candidates to the graph void addCandidates(StringGraph* pGraph, const Vertex* pX, const Edge* pEdgeToX, const OverlapBlockList* pBlockList, FMMergeQueue& candidateQueue); // Check whether the candidate can be merged into the current graph bool checkCandidate(const FMMergeCandidate& candidate, const OverlapBlockList* pBlockList) const; // std::string makeVertexID(BWTInterval interval); const OverlapAlgorithm* m_pOverlapper; const int m_minOverlap; BitVector* m_pMarkedReads; }; // Write the results from the overlap step to an ASQG file class FMMergePostProcess { public: FMMergePostProcess(std::ostream* pWriter, BitVector* pMarkedReads); ~FMMergePostProcess(); void process(const SequenceWorkItem& item, const FMMergeResult& result); private: size_t m_numMerged; size_t m_numTotal; size_t m_totalLength; std::ostream* m_pWriter; BitVector* m_pMarkedReads; }; #endif ================================================ FILE: src/SGA/Algorithm/GapFillProcess.cpp ================================================ ///---------------------------------------------- // Copyright 2011 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // GapFillProcess - Fill in gaps in a scaffold // #include #include "GapFillProcess.h" #include "BWTAlgorithms.h" #include "SGAlgorithms.h" #include "SGSearch.h" #include "StdAlnTools.h" #include "HaplotypeBuilder.h" #include "MultiAlignment.h" // GapFillStats::GapFillStats() { numGapsAttempted = numGapsFilled = 0; for(size_t i = 0; i < GFRC_NUM_CODES; ++i) numFails[i] = 0; } // void GapFillStats::print() const { printf("Num gaps attempted: %zu\n", numGapsAttempted); printf("Num gaps filled: %zu\n", numGapsFilled); printf(" Failed -- no anchor: %zu\n", numFails[GFRC_NO_ANCHOR]); printf(" Failed -- no haplotype path: %zu\n", numFails[GFRC_NO_HAPLOTYPE_PATH]); printf(" Failed -- no haplotype, search aborted: %zu\n", numFails[GFRC_NO_HAPLOTYPE_ABORTED]); printf(" Failed -- no haplotype, walk failed: %zu\n", numFails[GFRC_NO_HAPLOTYPE_WALK_FAIL]); printf(" Failed -- ambiguous solution: %zu\n", numFails[GFRC_AMBIGUOUS]); printf(" Failed -- assembled size differs from estimate: %zu\n", numFails[GFRC_BAD_SIZE]); printf(" Failed -- anchor sequence mismatch: %zu\n", numFails[GFRC_BAD_TRIM]); } // // // GapFillProcess::GapFillProcess(const GapFillParameters& params) : m_parameters(params) { } // GapFillProcess::~GapFillProcess() { m_stats.print(); } // Process the given scaffold, filling in any gaps found GapFillResult GapFillProcess::processScaffold(const std::string& scaffold) const { if(m_parameters.verbose > 0) std::cout << "Processing scaffold of length " << scaffold.length() << "\n"; GapFillResult result; size_t len = scaffold.length(); size_t currIdx = 0; while(currIdx < len) { char b = scaffold[currIdx]; if(b != 'N') { // Append this non-gap character into the output scaffold result.scaffold.append(1,b); currIdx += 1; } else { // Found the start of a gap size_t gapLength = 0; while(scaffold[currIdx + gapLength] == 'N') gapLength += 1; if(m_parameters.verbose >= 1) printf("Constructing gap at position %zu GapLength: %zu\n", currIdx, gapLength); GapFillReturnCode code = GFRC_UNKNOWN; // Attempt to fill this gap starting with a long kmer, then relaxing the process for(size_t k = m_parameters.startKmer; k >= m_parameters.endKmer; k -= m_parameters.stride) { // Calculate the left-anchor using the sequence of the scaffold already appended AnchorSequence leftAnchor = findAnchor(k, result.scaffold, result.scaffold.length() - k, true); // Calculate the right anchor using the sequence of the input scaffold AnchorSequence rightAnchor = findAnchor(k, scaffold, currIdx + gapLength, false); // Estimate the size of the assembled sequence, including the flanking anchors int leftFlanking = result.scaffold.length() - leftAnchor.position; int rightFlankingPlusGap = rightAnchor.position + k - currIdx; int estimatedSize = leftFlanking + rightFlankingPlusGap; // Attempt to build the gap sequence std::string gapSequence; code = processGap(k, estimatedSize, leftAnchor, rightAnchor, gapSequence); if(code == GFRC_OK) { // Successfully resolved the gap. Calculate the amount of the anchor sequence // that is already present in the scaffold and must be removed. We remove this amount // of sequence from the current scaffold, not the gapSequence, to account // for the case that the gap sequence is significantly different than the sequence // after the left anchor. size_t trimLength = result.scaffold.length() - leftAnchor.position; result.scaffold.replace(leftAnchor.position, trimLength, gapSequence); // We need to update currIdx to point to the next base in the // input scaffold that is not already assembled. This is given // by the position of the rightAnchor, plus a kmer currIdx = rightAnchor.position + k; m_stats.numGapsFilled += 1; break; } } if(code != GFRC_OK) { // Failed to resolve the gap. Append the gap into the growing scaffold m_stats.numFails[code] += 1; while(scaffold[currIdx] == 'N') { result.scaffold.append(1, 'N'); currIdx += 1; } } m_stats.numGapsAttempted += 1; } } if(m_parameters.verbose >= 2) StdAlnTools::globalAlignment(scaffold, result.scaffold, true); return result; } // Fill in the specified gap GapFillReturnCode GapFillProcess::processGap(size_t k, int estimatedSize, const AnchorSequence& startAnchor, const AnchorSequence& endAnchor, std::string& outSequence) const { if(m_parameters.verbose > 0) { std::cout << "\tSTART: " << startAnchor << "\n"; std::cout << "\tEND: " << endAnchor << "\n"; } if(startAnchor.sequence.empty() || endAnchor.sequence.empty() || startAnchor.sequence == endAnchor.sequence) return GFRC_NO_ANCHOR; HaplotypeBuilder builder; builder.setTerminals(startAnchor, endAnchor); builder.setIndex(m_parameters.pBWT, m_parameters.pRevBWT); builder.setKmerParameters(k, m_parameters.kmerThreshold); HaplotypeBuilderReturnCode code = builder.run(); HaplotypeBuilderResult result; // The search was successfull, build strings from the walks if(code == HBRC_OK) code = builder.parseWalks(result); if(code == HBRC_OK) { assert(!result.haplotypes.empty()); // Calculate the estimated size of the sequence, including the anchors return selectGapSequence(estimatedSize, result.haplotypes, outSequence); } else { // Map the haplotype builder code to a gap fill code assert(code != HBRC_OK); if(code == HBRC_NO_PATH) return GFRC_NO_HAPLOTYPE_PATH; else if(code == HBRC_TOO_MANY_VERTICES) return GFRC_NO_HAPLOTYPE_ABORTED; else if(code == HBRC_WALK_FAILED) return GFRC_NO_HAPLOTYPE_WALK_FAIL; } // should not reach here return GFRC_UNKNOWN; } // Find an anchor sequence to start the process of building the gap sequence AnchorSequence GapFillProcess::findAnchor(size_t k, const std::string& scaffold, int64_t position, bool upstream) const { AnchorSequence anchor; int64_t stride = upstream ? -1 : 1; int MAX_DISTANCE = 50; int64_t stop = upstream ? position - MAX_DISTANCE : position + MAX_DISTANCE; // Cap the travel distance to avoid out of bounds if(stop < 0) stop = 0; if(stop > (int64_t)(scaffold.length() - k)) stop = scaffold.length() - k; for(; position != stop; position += stride) { assert(position >= 0); std::string testSeq = scaffold.substr(position, k); std::transform(testSeq.begin(), testSeq.end(), testSeq.begin(), ::toupper); if(testSeq.find_first_not_of("ACGT") != std::string::npos) continue; size_t count = BWTAlgorithms::countSequenceOccurrencesWithCache(testSeq, m_parameters.pBWT, m_parameters.pBWTCache); if(count > m_parameters.kmerThreshold) { anchor.sequence = testSeq; anchor.count = count; anchor.position = position; return anchor; } } anchor.sequence = ""; anchor.count = -1; return anchor; } // Attempt to select one of the passed in strings as the gap sequence. If none fit the constraints, // this sets gapSequence to the empty string and returns an error code GapFillReturnCode GapFillProcess::selectGapSequence(int estimatedSize, const StringVector& sequences, std::string& gapSequence) const { assert(!sequences.empty()); int selectedIdx = -1; int selectedSizeDiff = std::numeric_limits::max(); for(size_t i = 0; i < sequences.size(); ++i) { int diff = abs(sequences[i].size() - estimatedSize); //printf("ES: %d S: %zu D: %d\n", estimatedSize, sequences[i].size(), diff); if(diff < selectedSizeDiff) { selectedSizeDiff = diff; selectedIdx = i; } } // Perform checks on the quality of the gap sequences int MAX_SIZE_DIFF = 100; if(selectedSizeDiff > MAX_SIZE_DIFF) { gapSequence = ""; return GFRC_BAD_SIZE; } gapSequence = sequences[selectedIdx]; return GFRC_OK; } ================================================ FILE: src/SGA/Algorithm/GapFillProcess.h ================================================ ///---------------------------------------------- // Copyright 2011 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // GapFillProcess - Fill in gaps in a scaffold // #ifndef GAPFILL_PROCESS_H #define GAPFILL_PROCESS_H #include #include #include #include "BWT.h" #include "BWTInterval.h" #include "SGUtil.h" #include "SGWalk.h" #include "BitVector.h" #include "HaplotypeBuilder.h" #include "SequenceProcessFramework.h" #include "BWTIntervalCache.h" #include "SampledSuffixArray.h" // Structures and typedefs // Parameters structure struct GapFillParameters { // BWTS const BWT* pBWT; const BWT* pRevBWT; const BWTIntervalCache* pBWTCache; const BWTIntervalCache* pRevBWTCache; size_t startKmer; size_t endKmer; size_t stride; size_t kmerThreshold; int verbose; }; // struct GapFillResult { std::string scaffold; }; enum GapFillReturnCode { GFRC_UNKNOWN, GFRC_OK, GFRC_NO_HAPLOTYPE_PATH, GFRC_NO_HAPLOTYPE_ABORTED, GFRC_NO_HAPLOTYPE_WALK_FAIL, GFRC_NO_ANCHOR, GFRC_AMBIGUOUS, GFRC_BAD_SIZE, GFRC_BAD_TRIM, GFRC_NUM_CODES }; // Statistics tracking object struct GapFillStats { GapFillStats(); size_t numGapsAttempted; size_t numGapsFilled; // Failure stats size_t numFails[GFRC_NUM_CODES]; void print() const; }; // // // class GapFillProcess { public: // // Functions // GapFillProcess(const GapFillParameters& params); ~GapFillProcess(); // Generate haplotypes from chromosome refName, position [start, end] GapFillResult processScaffold(const std::string& scaffold) const; private: // // Functions // // Attempt to fill in the sequence between the two anchors GapFillReturnCode processGap(size_t k, int estimatedSize, const AnchorSequence& leftAnchor, const AnchorSequence& rightAnchor, std::string& outSequence) const; // Find an anchor sequence to start the process of building the gap sequence AnchorSequence findAnchor(size_t k, const std::string& scaffold, int64_t position, bool upstream) const; // Attempt to select one of the passed in strings as the gap sequence. If none fit the constraints, // this sets gapSequence to the empty string and returns an error code GapFillReturnCode selectGapSequence(int estiamtedSize, const StringVector& sequences, std::string& gapSequence) const; // // Data // GapFillParameters m_parameters; mutable GapFillStats m_stats; }; #if 0 // class GapFillPostProcess { public: GapFillPostProcess(const std::string& filename); ~GapFillPostProcess(); //void process(const SequenceWorkItem& item, const GraphCompareResult& result); private: std::ostream* m_pWriter; }; #endif #endif ================================================ FILE: src/SGA/Algorithm/HaplotypeBuilder.cpp ================================================ ///---------------------------------------------- // Copyright 2011 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // HaplotypeBuilder - Construct candidate // haplotypes from a pair of k-mer seeds. // #include "HaplotypeBuilder.h" #include "BWTAlgorithms.h" #include "SGSearch.h" #include "SGAlgorithms.h" #include "Profiler.h" // // // HaplotypeBuilder::HaplotypeBuilder() : m_pStartVertex(NULL), m_pJoinVertex(NULL), m_kmerThreshold(1), m_kmerSize(51) { m_pGraph = new StringGraph; } // HaplotypeBuilder::~HaplotypeBuilder() { delete m_pGraph; } // void HaplotypeBuilder::setKmerParameters(size_t k, size_t t) { m_kmerSize = k; m_kmerThreshold = t; } // The source string is the string the bubble starts from void HaplotypeBuilder::setTerminals(const AnchorSequence& leftAnchor, const AnchorSequence& rightAnchor) { // Create a new vertex for the source sequence // As we are creating a de Bruijn graph, we use the sequence // of the vertex as its ID assert(leftAnchor.sequence != rightAnchor.sequence); Vertex* pLeftVertex = new(m_pGraph->getVertexAllocator()) Vertex(leftAnchor.sequence, leftAnchor.sequence); addVertex(pLeftVertex, leftAnchor.count); Vertex* pRightVertex = new(m_pGraph->getVertexAllocator()) Vertex(rightAnchor.sequence, rightAnchor.sequence); addVertex(pRightVertex, rightAnchor.count); // Add the vertex to the extension queue m_queue.push(BuilderExtensionNode(pLeftVertex, ED_SENSE)); m_pStartVertex = pLeftVertex; m_pJoinVertex = pRightVertex; } // The source index is the index that the contains the source string void HaplotypeBuilder::setIndex(const BWT* pBWT, const BWT* pRBWT) { m_pBWT = pBWT; m_pRevBWT = pRBWT; } // Run the bubble construction process HaplotypeBuilderReturnCode HaplotypeBuilder::run() { PROFILE_FUNC("HaplotypeBuilder::run") assert(m_queue.size() == 1); assert(m_pJoinVertex != NULL); assert(m_pBWT != NULL); size_t MAX_ITERATIONS = 2000; size_t MAX_SIMULTANEOUS_BRANCHES = 20; size_t MAX_TOTAL_BRANCHES = 50; // Tracking stats size_t total_branches = 0; size_t iterations = 0; while(!m_queue.empty()) { if(iterations > MAX_ITERATIONS || m_queue.size() > MAX_SIMULTANEOUS_BRANCHES || total_branches > MAX_TOTAL_BRANCHES) return HBRC_TOO_MANY_VERTICES; iterations += 1; BuilderExtensionNode curr = m_queue.front(); m_queue.pop(); // Calculate de Bruijn extensions for this node std::string vertStr = curr.pVertex->getSeq().toString(); AlphaCount64 extensionCounts; if(m_pRevBWT != NULL) extensionCounts = BWTAlgorithms::calculateDeBruijnExtensions(vertStr, m_pBWT, m_pRevBWT, curr.direction); else extensionCounts = BWTAlgorithms::calculateDeBruijnExtensionsSingleIndex(vertStr, m_pBWT, curr.direction); size_t num_added = 0; for(size_t i = 0; i < DNA_ALPHABET::size; ++i) { char b = DNA_ALPHABET::getBase(i); size_t count = extensionCounts.get(b); if(count < m_kmerThreshold) continue; std::string newStr = makeDeBruijnVertex(vertStr, b, curr.direction); Vertex* pVertex = m_pGraph->getVertex(newStr); // Check if we have found the vertex we are assembling to bool joinFound = pVertex != NULL && pVertex == m_pJoinVertex; if(!joinFound && pVertex == NULL) { pVertex = new(m_pGraph->getVertexAllocator()) Vertex(newStr, newStr); addVertex(pVertex, count); m_queue.push(BuilderExtensionNode(pVertex, curr.direction)); num_added += 1; } // Create the new edge in the graph addDeBruijnEdges(curr.pVertex, pVertex, curr.direction); // If we've found the join vertex, we have completed the target half of the bubble if(joinFound) return HBRC_OK; } if(num_added > 0) total_branches += (num_added - 1); } // no path between the nodes found return HBRC_NO_PATH; } // After the bubble has been built into the graph, this function // finds and compares the two sequences HaplotypeBuilderReturnCode HaplotypeBuilder::parseWalks(HaplotypeBuilderResult& results) const { // Parse walks from the graph that go through the bubbles SGWalkVector outWalks; bool success = SGSearch::findWalks(m_pStartVertex, m_pJoinVertex, ED_SENSE, 10000, // max distance to search 10000, // max nodes to search true, // exhaustive search outWalks); if(!success) return HBRC_WALK_FAILED; // Convert the walks into strings for(size_t i = 0; i < outWalks.size(); ++i) { std::string walkStr = outWalks[i].getString(SGWT_START_TO_END); results.haplotypes.push_back(walkStr); } return HBRC_OK; } // Add a vertex to the graph void HaplotypeBuilder::addVertex(Vertex* pVertex, int coverage) { m_pGraph->addVertex(pVertex); m_vertexCoverageMap[pVertex->getSeq().toString()] = coverage; } // Add a new edge to the graph denoting the relationship between pX and pY. // Assumes pX and pY are already present in the m_pGraph void HaplotypeBuilder::addDeBruijnEdges(const Vertex* pX, const Vertex* pY, EdgeDir direction) { assert(pX->getSeq().length() == pY->getSeq().length()); // overlap length for a de bruijn edge size_t p = pX->getSeq().length() - 1; // Construct an overlap object for this relationship Overlap o; o.id[0] = pX->getID(); o.id[1] = pY->getID(); o.match.isReverse = false; o.match.numDiff = 0; if(direction == ED_SENSE) { // pX -> pY o.match.coord[0].interval.start = 1; o.match.coord[1].interval.start = 0; } else { // pY -> pX o.match.coord[0].interval.start = 0; o.match.coord[1].interval.start = 1; } o.match.coord[0].interval.end = o.match.coord[0].interval.start + p - 1; // inclusive coordinate o.match.coord[1].interval.end = o.match.coord[1].interval.start + p - 1; o.match.coord[0].seqlen = p + 1; o.match.coord[1].seqlen = p + 1; Edge* e = SGAlgorithms::createEdgesFromOverlap(m_pGraph, o, false); assert(e != NULL); } // Make the sequence of a new deBruijn vertex using the edge details std::string HaplotypeBuilder::makeDeBruijnVertex(const std::string& v, char edgeBase, EdgeDir direction) { std::string w; size_t p = v.size() - 1; if(direction == ED_SENSE) { w = v.substr(1, p); w.append(1, edgeBase); } else { w.append(1, edgeBase); w.append(v.substr(0, p)); } return w; } // Count the number of extensions of a de Bruijn node that are above // the required k-mer coverage size_t HaplotypeBuilder::countValidExtensions(const AlphaCount64& ac) const { size_t n = 0; for(size_t i = 0; i < DNA_ALPHABET::size; ++i) { char b = DNA_ALPHABET::getBase(i); size_t count = ac.get(b); if(count >= m_kmerThreshold) n += 1; } return n; } ================================================ FILE: src/SGA/Algorithm/HaplotypeBuilder.h ================================================ ///---------------------------------------------- // Copyright 2011 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // HaplotypeBuilder - Construct candidate // haplotypes from a pair of k-mer seeds. // #ifndef HAPLOTYPE_BUILDER_H #define HAPLOTYPE_BUILDER_H #include "BWT.h" #include "BWTInterval.h" #include "SGUtil.h" #include "SGWalk.h" #include "VariationBuilderCommon.h" #include // Structs struct AnchorSequence { std::string sequence; int position; int count; friend std::ostream& operator<<(std::ostream& out, const AnchorSequence& a) { out << a.position << " " << a.sequence << " " << a.count; return out; } }; typedef std::vector AnchorVector; // The result, a set of haplotypes struct HaplotypeBuilderResult { StringVector haplotypes; }; // Return codes to indicate why the process may have failed enum HaplotypeBuilderReturnCode { HBRC_OK, HBRC_TOO_MANY_VERTICES, HBRC_NO_PATH, HBRC_WALK_FAILED, }; // // Class to build a variant bubble starting at a particular sequence // class HaplotypeBuilder { public: HaplotypeBuilder(); ~HaplotypeBuilder(); void setTerminals(const AnchorSequence& leftAnchor, const AnchorSequence& rightAnchor); void setIndex(const BWT* pBWT, const BWT* pRBWT); // Set the threshold of kmer occurrences to use it as an edge void setKmerParameters(size_t k, size_t t); // Run the bubble construction process // Returns true if the graph was successfully built between the two sequences HaplotypeBuilderReturnCode run(); // Parse walks from the constructed graph HaplotypeBuilderReturnCode parseWalks(HaplotypeBuilderResult& results) const; private: // Add a vertex to the graph and record the sequence coverage value void addVertex(Vertex* pVertex, int coverage); // Make the sequence of a new deBruijn vertex using the edge details std::string makeDeBruijnVertex(const std::string& v, char edgeBase, EdgeDir direction); void addDeBruijnEdges(const Vertex* pX, const Vertex* pY, EdgeDir direction); // Count the number of extensions of a de Bruijn node that are above // the required k-mer coverage size_t countValidExtensions(const AlphaCount64& ac) const; // // Data // const BWT* m_pBWT; const BWT* m_pRevBWT; StringGraph* m_pGraph; StrIntMap m_vertexCoverageMap; BuilderExtensionQueue m_queue; Vertex* m_pStartVertex; Vertex* m_pJoinVertex; // size_t m_kmerThreshold; size_t m_kmerSize; }; #endif ================================================ FILE: src/SGA/Algorithm/KmerOverlaps.cpp ================================================ ///----------------------------------------------- // Copyright 2012 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // KmerOverlaps - Overlap computation functions // seeded by exact kmer matches // #include "KmerOverlaps.h" #include "HashMap.h" #include "BWTAlgorithms.h" #include "Profiler.h" #include "Timer.h" #include "Verbosity.h" // MultipleAlignment KmerOverlaps::buildMultipleAlignment(const std::string& query, size_t k, int min_overlap, double min_identity, int bandwidth, const BWTIndexSet& indices) { SequenceOverlapPairVector overlap_vector = retrieveMatches(query, k, min_overlap, min_identity, bandwidth, indices); MultipleAlignment multiple_alignment; multiple_alignment.addBaseSequence("query", query, ""); for(size_t i = 0; i < overlap_vector.size(); ++i) multiple_alignment.addOverlap("null", overlap_vector[i].sequence[1], "", overlap_vector[i].overlap); return multiple_alignment; } // Struct to hold a partial match in the FM-index // The position field is the location in the query sequence of this kmer. // The index field is an index into the BWT. // The is_reverse flag indicates the strand of the partial match struct KmerMatch { size_t position:16; size_t index:47; size_t is_reverse:1; friend bool operator<(const KmerMatch& a, const KmerMatch& b) { if(a.index == b.index) return a.is_reverse < b.is_reverse; else return a.index < b.index; } friend bool operator==(const KmerMatch& a, const KmerMatch& b) { return a.index == b.index && a.is_reverse == b.is_reverse; } }; // Return a hash key for a KmerMatch struct KmerMatchKey { size_t operator()(const KmerMatch& a) const { return a.index; } }; typedef std::set KmerMatchSet; typedef HashMap KmerMatchMap; // SequenceOverlapPairVector KmerOverlaps::retrieveMatches(const std::string& query, size_t k, int min_overlap, double min_identity, int bandwidth, const BWTIndexSet& indices) { PROFILE_FUNC("OverlapHaplotypeBuilder::retrieveMatches") assert(indices.pBWT != NULL); assert(indices.pSSA != NULL); static size_t n_calls = 0; static size_t n_candidates = 0; static size_t n_output = 0; static double t_time = 0; Timer timer("test", true); n_calls++; int64_t max_interval_size = 200; SequenceOverlapPairVector overlap_vector; // Use the FM-index to look up intervals for each kmer of the read. Each index // in the interval is stored individually in the KmerMatchMap. We then // backtrack to map these kmer indices to read IDs. As reads can share // multiple kmers, we use the map to avoid redundant lookups. // There is likely a faster algorithm which performs direct decompression // of the read sequences without having to expand the intervals to individual // indices. The current algorithm suffices for now. KmerMatchMap prematchMap; size_t num_kmers = query.size() - k + 1; for(size_t i = 0; i < num_kmers; ++i) { std::string kmer = query.substr(i, k); BWTInterval interval = BWTAlgorithms::findInterval(indices, kmer); if(interval.isValid() && interval.size() < max_interval_size) { for(int64_t j = interval.lower; j <= interval.upper; ++j) { KmerMatch match = { i, static_cast(j), false }; prematchMap.insert(std::make_pair(match, false)); } } kmer = reverseComplement(kmer); interval = BWTAlgorithms::findInterval(indices, kmer); if(interval.isValid() && interval.size() < max_interval_size) { for(int64_t j = interval.lower; j <= interval.upper; ++j) { KmerMatch match = { i, static_cast(j), true }; prematchMap.insert(std::make_pair(match, false)); } } } // Backtrack through the kmer indices to turn them into read indices. // This mirrors the calcSA function in SampledSuffixArray except we mark each entry // as visited once it is processed. KmerMatchSet matches; for(KmerMatchMap::iterator iter = prematchMap.begin(); iter != prematchMap.end(); ++iter) { // This index has been visited if(iter->second) continue; // Mark this as visited iter->second = true; // Backtrack the index until we hit the starting symbol KmerMatch out_match = iter->first; while(1) { char b = indices.pBWT->getChar(out_match.index); out_match.index = indices.pBWT->getPC(b) + indices.pBWT->getOcc(b, out_match.index - 1); // Check if the hash indicates we have visited this index. If so, stop the backtrack KmerMatchMap::iterator find_iter = prematchMap.find(out_match); if(find_iter != prematchMap.end()) { // We have processed this index already if(find_iter->second) break; else find_iter->second = true; } if(b == '$') { // We've found the lexicographic index for this read. Turn it into a proper ID out_match.index = indices.pSSA->lookupLexoRank(out_match.index); matches.insert(out_match); break; } } } // Refine the matches by computing proper overlaps between the sequences // Use the overlaps that meet the thresholds to build a multiple alignment for(KmerMatchSet::iterator iter = matches.begin(); iter != matches.end(); ++iter) { std::string match_sequence = BWTAlgorithms::extractString(indices.pBWT, iter->index); if(iter->is_reverse) match_sequence = reverseComplement(match_sequence); // Ignore identical matches if(match_sequence == query) continue; // Compute the overlap. If the kmer match occurs a single time in each sequence we use // the banded extension overlap strategy. Otherwise we use the slow O(M*N) overlapper. SequenceOverlap overlap; std::string match_kmer = query.substr(iter->position, k); size_t pos_0 = query.find(match_kmer); size_t pos_1 = match_sequence.find(match_kmer); assert(pos_0 != std::string::npos && pos_1 != std::string::npos); // Check for secondary occurrences if(query.find(match_kmer, pos_0 + 1) != std::string::npos || match_sequence.find(match_kmer, pos_1 + 1) != std::string::npos) { // One of the reads has a second occurrence of the kmer. Use // the slow overlapper. overlap = Overlapper::computeOverlap(query, match_sequence); } else { overlap = Overlapper::extendMatch(query, match_sequence, pos_0, pos_1, bandwidth); } n_candidates += 1; bool bPassedOverlap = overlap.getOverlapLength() >= min_overlap; bool bPassedIdentity = overlap.getPercentIdentity() / 100 >= min_identity; if(bPassedOverlap && bPassedIdentity) { SequenceOverlapPair op; op.sequence[0] = query; op.sequence[1] = match_sequence; op.overlap = overlap; op.is_reversed = iter->is_reverse; overlap_vector.push_back(op); n_output += 1; } } t_time += timer.getElapsedCPUTime(); if(Verbosity::Instance().getPrintLevel() > 6 && n_calls % 100 == 0) printf("[kmer overlaps] n: %zu candidates: %zu valid: %zu (%.2lf) time: %.2lfs\n", n_calls, n_candidates, n_output, (double)n_output / n_candidates, t_time); return overlap_vector; } struct SeedEdit { SeedEdit(int i, char b) : index(i), base(b) {} int index; char base; }; typedef std::vector SeedEditVector; struct ApproxSeed { int query_index; // the index of the last query base included in the interval BWTInterval interval; int length; SeedEditVector edits; friend std::ostream& operator<<(std::ostream& o, ApproxSeed& a) { o << "QI: " << a.query_index << " IV: " << a.interval; o << " Edits: "; for(size_t i = 0; i < a.edits.size(); i++) o << a.edits[i].index << ":" << a.edits[i].base << ","; return o; } }; void _approximateSeededMatch(const std::string& in_query, int min_overlap, double min_identity, int bandwidth, int max_interval, bool do_reverse, const BWTIndexSet& indices, SequenceOverlapPairVector& out_vector) { Timer timer("test", true); assert(indices.pBWT != NULL); assert(indices.pSSA != NULL); assert(indices.pCache != NULL); static size_t n_calls = 0; static size_t n_candidates = 0; static size_t n_output = 0; static double t_time = 0; n_calls++; int target_seed_length = 41; int seed_stride = target_seed_length / 2; size_t d = 1; SequenceOverlapPairVector overlap_vector; std::string strand_query = do_reverse ? reverseComplement(in_query) : in_query; // Initialize seeds int seed_end = strand_query.size(); int q = indices.pCache->getCachedLength(); std::queue seeds; while(seed_end > target_seed_length) { // For the last q bases of the seed, create all strings within edit distance d std::string qmer = strand_query.substr(seed_end - q, q); assert((int)qmer.size() == q); // 0-distance seed ApproxSeed seed; seed.query_index = seed_end - q; seed.interval = indices.pCache->lookup(qmer.c_str()); seed.length = q; seeds.push(seed); for(int i = 0; i < q; ++i) { // Switch base to other 3 symbols char o = qmer[i]; for(size_t j = 0; j < 4; ++j) { char b = "ACGT"[j]; if(b != o) { qmer[i] = b; ApproxSeed seed; seed.query_index = seed_end - q; seed.interval = indices.pCache->lookup(qmer.c_str()); seed.length = q; seed.edits.push_back(SeedEdit(i + seed.query_index, b)); seeds.push(seed); } } qmer[i] = o; } seed_end -= seed_stride; } // Extend seeds std::vector finished_seeds; while(!seeds.empty()) { ApproxSeed& seed = seeds.front(); // query_index is the index of the last base // in the seed. get the next base char qb = strand_query[seed.query_index - 1]; // Branch to inexact match if(seed.edits.size() < d) { for(size_t j = 0; j < 4; ++j) { char b = "ACGT"[j]; if(b != qb) { ApproxSeed new_seed; new_seed.query_index = seed.query_index - 1; new_seed.interval = seed.interval; BWTAlgorithms::updateInterval(new_seed.interval, b, indices.pBWT); if(new_seed.interval.isValid()) { new_seed.length = seed.length + 1; new_seed.edits = seed.edits; new_seed.edits.push_back(SeedEdit(new_seed.query_index, b)); if(new_seed.length < target_seed_length && new_seed.query_index > 0) seeds.push(new_seed); else finished_seeds.push_back(new_seed); } } } } // Extend with the actual query base without branching seed.query_index = seed.query_index - 1; seed.length += 1; BWTAlgorithms::updateInterval(seed.interval, qb, indices.pBWT); if(!seed.interval.isValid() || seed.length >= target_seed_length || seed.query_index == 0) { if(seed.interval.isValid()) finished_seeds.push_back(seed); seeds.pop(); } } std::set rank_set; for(size_t i = 0; i < finished_seeds.size(); ++i) { if(finished_seeds[i].interval.size() > max_interval) continue; //std::cout << finished_seeds[i] << "\n"; std::string query_seed = strand_query.substr(finished_seeds[i].query_index, target_seed_length); std::string match_seed = query_seed; // Apply edits to the new sequence for(size_t j = 0; j < finished_seeds[i].edits.size(); ++j) match_seed[finished_seeds[i].edits[j].index - finished_seeds[i].query_index] = finished_seeds[i].edits[j].base; // Flip the seeds to match the strand of the query if(do_reverse) { query_seed = reverseComplement(query_seed); match_seed = reverseComplement(match_seed); } // Extract the prefix of every occurrence of this seed RankedPrefixVector extensions = BWTAlgorithms::extractRankedPrefixes(indices.pBWT, finished_seeds[i].interval); // Extend the seeds to the full-length string for(size_t j = 0; j < extensions.size(); ++j) { size_t rank = extensions[j].rank; // The second element of the returned pair is // false if the set already contains this rank if(!rank_set.insert(rank).second) continue; // Extract the reminder of the read std::string& prefix = extensions[j].prefix; int64_t start_index_of_read = indices.pSSA->lookupLexoRank(rank); std::string suffix = BWTAlgorithms::extractUntilInterval(indices.pBWT, start_index_of_read, finished_seeds[i].interval); std::string match_sequence = prefix + suffix; // Ignore identical matches if(match_sequence == strand_query) continue; // Change strands if(do_reverse) match_sequence = reverseComplement(match_sequence); // Compute the overlap SequenceOverlap overlap; size_t pos_0 = in_query.find(query_seed); size_t pos_1 = match_sequence.find(match_seed); assert(pos_0 != std::string::npos); assert(pos_1 != std::string::npos); if(in_query.find(query_seed, pos_0 + 1) != std::string::npos || match_sequence.find(match_seed, pos_1 + 1) != std::string::npos) { // One of the reads has a second occurrence of the kmer. Use // the slow overlapper. overlap = Overlapper::computeOverlap(in_query, match_sequence); } else { overlap = Overlapper::extendMatch(in_query, match_sequence, pos_0, pos_1, bandwidth); } n_candidates += 1; bool bPassedOverlap = overlap.getOverlapLength() >= min_overlap; bool bPassedIdentity = overlap.getPercentIdentity() / 100 >= min_identity; if(bPassedOverlap && bPassedIdentity) { //printf("Rank\t%zu\t%zu\t%s\t%.2lf\t%d\n", n_calls, // rank, match_sequence.c_str(), overlap.getPercentIdentity(), overlap.getOverlapLength()); SequenceOverlapPair op; op.sequence[0] = in_query; op.sequence[1] = match_sequence; op.overlap = overlap; op.is_reversed = do_reverse; out_vector.push_back(op); n_output += 1; } } } t_time += timer.getElapsedCPUTime(); if(Verbosity::Instance().getPrintLevel() > 6 && n_calls % 100 == 0) printf("[approx seeds] n: %zu candidates: %zu valid: %zu (%.2lf) time: %.2lfs\n", n_calls, n_candidates, n_output, (double)n_output / n_candidates, t_time); } // SequenceOverlapPairVector KmerOverlaps::approximateMatch(const std::string& query, int min_overlap, double min_identity, int bandwidth, int max_interval, const BWTIndexSet& indices) { SequenceOverlapPairVector opv; _approximateSeededMatch(query, min_overlap, min_identity, bandwidth, max_interval, false, indices, opv); _approximateSeededMatch(query, min_overlap, min_identity, bandwidth, max_interval, true, indices, opv); return opv; } ================================================ FILE: src/SGA/Algorithm/KmerOverlaps.h ================================================ ///----------------------------------------------- // Copyright 2012 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // KmerOverlaps - Overlap computation functions // seeded by exact kmer matches // #ifndef KMER_OVERLAPS_H #define KMER_OVERLAPS_H #include "multiple_alignment.h" #include "BWTIndexSet.h" #include "SampledSuffixArray.h" // A pair of sequences and an overlap matching them struct SequenceOverlapPair { std::string sequence[2]; bool is_reversed; SequenceOverlap overlap; static bool sortByOverlapLengthDesc(const SequenceOverlapPair& a, const SequenceOverlapPair& b) { return a.overlap.getOverlapLength() > b.overlap.getOverlapLength(); } }; typedef std::vector SequenceOverlapPairVector; namespace KmerOverlaps { // Build a multiple alignment for the query, based on initial exact k-matches MultipleAlignment buildMultipleAlignment(const std::string& query, size_t k, int min_overlap, double min_identity, int bandwidth, const BWTIndexSet& indices); // Retrieve matches to the query sequence SequenceOverlapPairVector retrieveMatches(const std::string& query, size_t k, int min_overlap, double min_identity, int bandwidth, const BWTIndexSet& indices); SequenceOverlapPairVector approximateMatch(const std::string& query, int min_overlap, double min_identity, int bandwidth, int max_interval, const BWTIndexSet& indices); }; #endif ================================================ FILE: src/SGA/Algorithm/LRAlignment.cpp ================================================ //----------------------------------------------- // Copyright 2011 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // LRAlignment - Collection of algorithms for performing // long read matches against an FM-index // #include "LRAlignment.h" #include "QuickBWT.h" #include "MultiAlignment.h" #include "StdAlnTools.h" //#define BWA_COMPAT_DEBUG 1 //#define BWA_COMPAT_DEBUG_RESOLVE 1 namespace LRAlignment { static const int MINUS_INF = -0x3fffffff; #define MASK_LEVEL 0.90f // Initialize the LRCell with default values void LRCell::initializeDefault() { interval.lower = 0; interval.upper = 0; I = MINUS_INF; D = MINUS_INF; G = MINUS_INF; parent_cidx = 0; q_len = 0; t_len = 0; parent_idx = -1; u_idx = -1; clearChildren(); } // Reset the child indices for the cell void LRCell::clearChildren() { children_idx[0] = -1; children_idx[1] = -1; children_idx[2] = -1; children_idx[3] = -1; } // Returns true if any child of the cell is uninitialized bool LRCell::hasUninitializedChild() const { return children_idx[0] == -1 || children_idx[1] == -1 || children_idx[2] == -1 || children_idx[3] == -1; } // Implementation of bwa-sw algorithm. // Roughly follows Heng Li's implementation void bwaswAlignment(const std::string& query, const BWT* pTargetBWT, const SampledSuffixArray* pTargetSSA, const LRParams& params, LRHitVector& outHits) { // Construct an FM-index of the query sequence BWT* pQueryBWT = NULL; SuffixArray* pQuerySA = NULL; createQuickBWT(query, pQueryBWT, pQuerySA); // Interval hash table used to mark cells as being duplicate LRHash dupHash; // Initialize the hash table of DAWG nodes LRHash dawgHash; initializeDAWGHash(pQueryBWT, dawgHash); // Initialize a stack of elements with a single entry for the root node // of the query DAWG LRStack stack; // High scoring alignments are stored as LRHits in these vectors // positionHitsVector stores up to 2 hits starting at every base of the query sequence // terminalHitsVector stores hits to sequence prefixes LRHitVector positionHitsVector(2*query.size()); LRHitVector terminalHitsVector; // Each dawg node is added to the pendingVector initially // Once all predecessors of the node have been visited, // the node is moved to the stack LRPendingVector pendingVector; size_t num_pending = 0; // Initialize a stack entry for the root node with an empty scoring cell LRStackEntry* pInitial = new LRStackEntry; pInitial->interval.lower = 0; pInitial->interval.upper = pQueryBWT->getBWLen() - 1; // LRCell x; x.initializeDefault(); x.G = 0; x.interval.lower = 0; x.interval.upper = pTargetBWT->getBWLen() - 1; pInitial->cells.push_back(x); stack.push(pInitial); // The main loop of the algorithm - traverse the DAWG while(!stack.empty()) { LRStackEntry* v = stack.top(); stack.pop(); size_t old_n = v->cells.size(); // TODO: bandwidth test and max depth ? // Descend into the children of the current dawg node // If the interval update succeeds, calculate scores between // the child node and all the LRCells of the current node for(int qci = 0; qci < DNA_ALPHABET::size; ++qci) { char query_child_base = DNA_ALPHABET::getBase(qci); BWTInterval child_interval = v->interval; BWTAlgorithms::updateInterval(child_interval, query_child_base, pQueryBWT); if(!child_interval.isValid()) continue; // Create an new array of cells for the scores between the child node // and all the nodes in the prefix tree LRStackEntry* u = new LRStackEntry; u->interval = child_interval; // Loop over the nodes in v for(size_t i = 0; i < v->cells.size(); ++i) { LRCell* p = &v->cells[i]; if(p->interval.upper == -1) continue; // duplicate that has been deleted LRCell x; // the cell being calculated LRCell* c[4]; // pointers to cells required to calculate x c[0] = &x; x.G = MINUS_INF; x.u_idx = p->u_idx = -1; bool add_x_to_u = false; if(p->parent_idx >= 0) { int upos = v->cells[p->parent_idx].u_idx; // Set pointers to the cell c[1] = upos >= 0 ? &u->cells[upos] : NULL; c[2] = p; c[3] = &v->cells[p->parent_idx]; int match_score = qci == p->parent_cidx ? params.alnParams.match : -params.alnParams.mismatch; int score = fillCells(params, match_score, c); if(score > 0) { // this cell has a positive score // set the x's parent position in u and set the // parent's child position to x x.parent_idx = upos; p->u_idx = u->cells.size(); // x will be added to u in this position if(x.parent_idx >= 0) u->cells[upos].children_idx[p->parent_cidx] = p->u_idx; add_x_to_u = true; } } else { if(p->D > p->G - params.alnParams.gap_open) x.D = p->D - params.alnParams.gap_ext; // extend gap else x.D = p->G - params.alnParams.gap_open_extend; // open a new gap if(x.D > 0) { x.G = x.D; x.I = MINUS_INF; x.parent_idx = -1; p->u_idx = u->cells.size(); add_x_to_u = true; } } if(add_x_to_u) { // set the remaining fields in the current cell x.clearChildren(); x.parent_cidx = p->parent_cidx; x.interval = p->interval; x.q_len = p->q_len + 1; x.t_len = p->t_len; u->cells.push_back(x); // TODO: heap heuristics from bwa-sw? } // Check if we should descend into another node of the prefix trie of the target if( (x.G > params.alnParams.gap_open_extend /* && heap test */) || i < old_n) { if(p->hasUninitializedChild()) { for(int tci = 0; tci < DNA_ALPHABET::size; ++tci) { if(p->children_idx[tci] != -1) continue; // already added char target_child_base = DNA_ALPHABET::getBase(tci); BWTInterval target_child_interval = p->interval; BWTAlgorithms::updateInterval(target_child_interval, target_child_base, pTargetBWT); if(!target_child_interval.isValid()) // child with this extension base does not exist { p->children_idx[tci] = -2; continue; } // Create new entry on array v LRCell y; y.G = y.I = y.D = MINUS_INF; y.interval = target_child_interval; y.parent_cidx = tci; y.q_len = p->q_len; y.t_len = p->t_len + 1; y.parent_idx = i; y.clearChildren(); p->children_idx[tci] = v->cells.size(); v->cells.push_back(y); p = &v->cells[i]; // p may have been invalidated in the push, update } // for tcl } // if has uninitialized } // if X.h } // for all v // Save high-scoring cells as hits if(!u->cells.empty()) { saveBestPositionHits(pQuerySA, u, params.alnParams.threshold, positionHitsVector); //saveAllHits(pQuerySA, pTargetSSA, pTargetBWT, u, params.threshold, terminalHitsVector); //saveTerminalHits(pQuerySA, pTargetSSA, pTargetBWT, u, params.threshold, terminalHitsVector); } // Update the stack by adding u or pushing it to the pending vector num_pending += updateStack(&stack, u, &pendingVector, &dawgHash, params); } // for qci // done with v delete v; } // for all stack assert(num_pending == 0); // Append the two hits vectors together positionHitsVector.insert(positionHitsVector.end(), terminalHitsVector.begin(), terminalHitsVector.end()); terminalHitsVector.clear(); resolveDuplicateHits(pTargetBWT, pTargetSSA, positionHitsVector, 2); // set the output hits outHits.insert(outHits.end(), positionHitsVector.begin(), positionHitsVector.end()); delete pQueryBWT; delete pQuerySA; } // Update the stack to contain entry u, after considering any possible merges // with StackEntries from the pending vector int updateStack(LRStack* pStack, LRStackEntry* u, LRPendingVector* pPendingVector, LRHash* pDawgHash, const LRParams& params) { // Find the iterator for u in the dawgHash LRHash dupHash; uint64_t key = u->interval.lower << 32 | u->interval.upper; LRHash::iterator hashIter = pDawgHash->find(key); assert(hashIter != pDawgHash->end() && (uint32_t)hashIter->second > 0); --hashIter->second; uint32_t count = (uint32_t)hashIter->second; uint32_t position = hashIter->second >> 32; // Check if an entry in the pending array exists for this DAWG node int change = 0; if(position > 0) { LRStackEntry* w = (*pPendingVector)[position - 1]; // An entry in the pending array has been created for this query substing // Merge u into the interval if(!u->cells.empty()) { // Swap so w is the StackEntry wth more cells if(w->cells.size() < u->cells.size()) { w = u; u = (*pPendingVector)[position - 1]; (*pPendingVector)[position - 1] = w; } mergeStackEntries(w, u); } if(count == 0) { // this node in the dawg will not be visited again // move the stack entry from the pending list to the stack removeDuplicateCells(w, dupHash); cutTail(w, params); pStack->push(w); (*pPendingVector)[position - 1] = 0; change -= 1; } // u is empty or merged, it is no longer needed delete u; } else if(count > 0) { // Create an entry in the pending queue for the current node of the DAWG if(!u->cells.empty()) { pPendingVector->push_back(u); change += 1; // Save the position of u in the pending vector into the hash // all subsequent traversals that visit this node of the dawg // will get merged into this position. index + 1 is stored // so that position == 0 indicates the empty case hashIter->second = (uint64_t)pPendingVector->size() << 32 | count; } else { // u has no cells to calculate, discard it delete u; } } else // count == 0, pos == 0 { // This substring is unique, push u straight onto the stack cutTail(u, params); pStack->push(u); } return change; } // Process a list of cells and save a best hit for each position void saveBestPositionHits(const SuffixArray* pQuerySA, LRStackEntry* u, int threshold, LRHitVector& hits) { for(size_t i = 0; i < u->cells.size(); ++i) { LRCell* p = &u->cells[i]; if(p->G < threshold) continue; for(int64_t k = u->interval.lower; k <= u->interval.upper; ++k) { // Calculate the beginning of the alignment using the suffix array // of the query SAElem e = pQuerySA->get(k); int beg = e.getPos(); int end = beg + p->q_len; // Save the best hit for alignments starting at beg in positions hits[2*beg] // and the second best hit in hits[2*beg+1] LRHit* q = NULL; if(p->G > hits[beg*2].G) { // move the previous best to the second best slot hits[beg*2+1] = hits[beg*2]; q = &hits[2*beg]; } else if(p->G > hits[beg*2+1].G) { // the current hit is the second best at this position q = &hits[2*beg+1]; } if(q) { q->interval = p->interval; q->length = p->t_len; q->G = p->G; q->q_start = beg; q->q_end = end; q->flag = 0; q->num_seeds = 0; } } } } // Process a list of cells and save alignment hits meeting the threshold void saveAllHits(const SuffixArray* pQuerySA, const SampledSuffixArray* /*pTargetSSA*/, const BWT* /*pTargetBWT*/, LRStackEntry* u, int threshold, LRHitVector& hits) { for(size_t i = 0; i < u->cells.size(); ++i) { LRCell* p = &u->cells[i]; if(p->G < threshold) continue; for(int64_t k = u->interval.lower; k <= u->interval.upper; ++k) { // Calculate the beginning of the alignment using the suffix array // of the query SAElem e = pQuerySA->get(k); int beg = e.getPos(); int end = beg + p->q_len; // Save the best hit for alignments starting at beg in positions hits[2*beg] // and the second best hit in hits[2*beg+1] LRHit q; q.interval = p->interval; q.length = p->t_len; q.G = p->G; q.q_start = beg; q.q_end = end; q.flag = 0; q.num_seeds = 0; hits.push_back(q); } } } // Process all the cells in the stack entry and save hits to cells that represent // terminal intervals (those intervals containing the start of some reads) void saveTerminalHits(const SuffixArray* pQuerySA, const SampledSuffixArray* /*pTargetSSA*/, const BWT* pTargetBWT, LRStackEntry* u, int threshold, LRHitVector& hits) { for(size_t i = 0; i < u->cells.size(); ++i) { LRCell* p = &u->cells[i]; if(p->G < threshold) continue; // If the target bwt interval contains a '$' symbol, push the hit AlphaCount64 extCount = BWTAlgorithms::getExtCount(p->interval, pTargetBWT); if(extCount.get('$') > 0) { // Create a hit for every beginning position in the query for(int64_t k = u->interval.lower; k <= u->interval.upper; ++k) { // Calculate the beginning of the alignment using the suffix array // of the query SAElem e = pQuerySA->get(k); int beg = e.getPos(); int end = beg + p->q_len; LRHit q; q.interval = p->interval; q.length = p->t_len; q.G = p->G; q.q_start = beg; q.q_end = end; q.flag = 0; q.num_seeds = 0; hits.push_back(q); } } } } // Initialize the hash table of DAWG nodes by inserting an element // into the hash table for each distinct substring of the query void initializeDAWGHash(BWT* pQueryBWT, LRHash& hashTable) { std::stack stack; stack.push(pQueryBWT->getBWLen() - 1); while(!stack.empty()) { uint64_t x = stack.top(); stack.pop(); BWTInterval interval(x >> 32, (uint32_t)x); AlphaCount64 lower = pQueryBWT->getFullOcc(interval.lower - 1); AlphaCount64 upper = pQueryBWT->getFullOcc(interval.upper); for(int ci = 0; ci < DNA_ALPHABET::size; ++ci) { char b = DNA_ALPHABET::getBase(ci); size_t pb = pQueryBWT->getPC(b); interval.lower = pb + lower.get(b); interval.upper = pb + upper.get(b) - 1; if(!interval.isValid()) continue; x = (uint64_t)interval.lower << 32 | interval.upper; // insert (or update) this key in the hash LRHash::iterator iter = hashTable.find(x); if(iter == hashTable.end()) { hashTable.insert(std::make_pair(x,1)); stack.push(x); } else { iter->second++; } } } } // Merge two stack entries which represent the same node in the DAWG void mergeStackEntries(LRStackEntry* u, LRStackEntry* v) { // Update parent index and child entries for v cell's to reflect // their new positions at the end of array of u size_t num_u = u->cells.size(); for (size_t i = 0; i != v->cells.size(); ++i) { LRCell* p = &v->cells[i]; if(p->parent_idx >= 0) p->parent_idx += num_u; for(size_t j = 0; j < 4; ++j) if (p->children_idx[j] >= 0) p->children_idx[j] += num_u; } u->cells.insert(u->cells.end(), v->cells.begin(), v->cells.end()); } // Remove duplicated cells from the stack entry void removeDuplicateCells(LRStackEntry* u, LRHash& hash) { hash.clear(); int n = 0; for(size_t i = 0; i < u->cells.size(); ++i) { int j = -1; // element to be deleted LRCell* p = &u->cells[i]; if(p->interval.upper == -1) continue; // already deleted uint64_t key = p->interval.lower << 32 | p->interval.upper; LRHash::iterator iter = hash.find(key); if(iter != hash.end()) { // Check if the score of p is greater than the stored cell // with the same key. Set j to be the index of u with the lower // score. uint32_t currScore = iter->second; if((int)currScore >= p->G) { j = i; } else { j = iter->second >> 32; iter->second = i << 32 | p->G; } } else { // initialize hash for cell i hash[key] = i << 32 | p->G; } if(j >= 0) { #ifdef BWA_COMPAT_DEBUG printf("marking p [%zu %zu] as deleted cidx: %d [DUP]\n", p->interval.lower, p->interval.upper, p->parent_cidx); #endif p = &u->cells[j]; p->interval.lower = -1; p->interval.upper = -1; p->G = 0; if(p->parent_idx >= 0) u->cells[p->parent_idx].children_idx[p->parent_cidx] = -3; n += 1; } } #ifdef BWA_COMPAT_DEBUG printf("removed %d duplicate entries\n", n); #endif } // Fill the values of C[0] depending on the values in the other 3 cells int fillCells(const LRParams& params, int match_score, LRCell* c[4]) { int G = c[3] ? c[3]->G + match_score : MINUS_INF; if(c[1]) { if(c[1]->I > c[1]->G - params.alnParams.gap_open) c[0]->I = c[1]->I - params.alnParams.gap_ext; // extend gap else c[0]->I = c[1]->G - params.alnParams.gap_open_extend; // open new gap if (c[0]->I > G) G = c[0]->I; // new best score } else { c[0]->I = MINUS_INF; } if(c[2]) { if(c[2]->D > c[2]->G - params.alnParams.gap_open) c[0]->D = c[2]->D - params.alnParams.gap_ext; // extend gap else c[0]->D = c[2]->G - params.alnParams.gap_open_extend; // open new gap if (c[0]->D > G) G = c[0]->D; // new best score } else { c[0]->D = MINUS_INF; } return(c[0]->G = G); } // Remove duplicate hits to the same target sequence from the hits vector int resolveDuplicateHitsByID(const BWT* pTargetBWT, const SampledSuffixArray* pTargetSSA, LRHitVector& hits, int /*IS*/) { if(hits.empty()) return 0; assert(pTargetBWT != NULL); assert(pTargetSSA != NULL); // Convert each hit interval to target coordinates LRHitVector newHits; for(size_t i = 0; i < hits.size(); ++i) { LRHit* p = &hits[i]; #ifdef BWA_COMPAT_DEBUG_RESOLVE printf("Total hits: %zu\n", hits.size()); printf("Reallocating hits array to size: %d\n", n); #endif if(!p->interval.isValid() || p->G <= 0) continue; for(int64_t k = p->interval.lower; k <= p->interval.upper; ++k) { LRHit tmp = *p; SAElem elem = pTargetSSA->calcSA(k, pTargetBWT); tmp.targetID = elem.getID(); tmp.t_start = elem.getPos(); tmp.interval.lower = 0; tmp.interval.upper = -1; #ifdef BWA_COMPAT_DEBUG_RESOLVE printf("Created new hit at position (%d, %d)\n", tmp.targetID, tmp.position); #endif newHits.push_back(tmp); } } // Swap the new hits structure with the old hits hits.swap(newHits); // sort hits by targetID, then score std::sort(hits.begin(), hits.end(), LRHit::compareIDandG); // Loop over the hits, zeroing all but the highest scoring hit for each target uint64_t prevID = std::numeric_limits::max(); size_t new_n = 0; for(size_t i = 0; i < hits.size(); ++i) { // std::cout << "ID: " << hits[i].targetID << " score: " << hits[i].G << "\n"; if(prevID == hits[i].targetID) hits[i].G = 0; else new_n += 1; prevID = hits[i].targetID; } std::sort(hits.begin(), hits.end(), LRHit::compareG); hits.resize(new_n); std::cout << "FINAL HITS: " << new_n << "\n"; #ifdef BWA_COMPAT_DEBUG_RESOLVE printf("Final number of hits after duplicate removal: %zu\n", hits.size()); #endif return hits.size(); } // Remove duplicated hits from the hits vector int resolveDuplicateHits(const BWT* pTargetBWT, const SampledSuffixArray* pTargetSSA, LRHitVector& hits, int IS) { if(hits.empty()) return 0; assert(pTargetBWT != NULL); if(pTargetBWT != NULL && pTargetSSA != NULL) { // Convert each hit to target coordinates int old_n = hits.size(); int n = 0; // Count the number of valid hits LRHitVector newHits; for(size_t i = 0; i < hits.size(); ++i) { LRHit* p = &hits[i]; if(p->interval.isValid() && p->interval.size() <= IS) n += p->interval.size(); else if(!p->interval.isValid()) // bwa compatibility hack n += 1; else if(p->G > 0) n += 1; } #ifdef BWA_COMPAT_DEBUG_RESOLVE printf("Total hits: %zu\n", hits.size()); printf("Reallocating hits array to size: %d\n", n); #endif newHits.resize(n); // Populate the newHits vector with the hits to keep int j = 0; for(int i = 0; i < old_n; ++i) { LRHit* p = &hits[i]; if(p->interval.isValid() && p->interval.size() <= IS) { for(int64_t k = p->interval.lower; k <= p->interval.upper; ++k) { newHits[j] = *p; SAElem elem = pTargetSSA->calcSA(k, pTargetBWT); newHits[j].targetID = elem.getID(); newHits[j].t_start = elem.getPos(); newHits[j].interval.lower = 0; newHits[j].interval.upper = -1; #ifdef BWA_COMPAT_DEBUG_RESOLVE printf("Created new hit at position (%d, %d)\n", newHits[j].targetID, newHits[j].position); #endif ++j; } } else if(p->G > 0) { newHits[j] = *p; SAElem elem = pTargetSSA->calcSA(p->interval.lower, pTargetBWT); newHits[j].targetID = elem.getID(); newHits[j].t_start = elem.getPos(); newHits[j].interval.lower = 0; newHits[j].interval.upper = -1; newHits[j].flag |= 1; #ifdef BWA_COMPAT_DEBUG_RESOLVE printf("Created new hit at position (%d, %d)\n", newHits[j].targetID, newHits[j].position); #endif ++j; } } // Swap the new hits structure with the old hits hits.swap(newHits); } // sort hits by score std::sort(hits.begin(), hits.end(), LRHit::compareG); int i,j; // Remove hits with significant overlaps on the query or target sequences for(i = 1; i < (int)hits.size(); ++i) { LRHit* p = &hits[i]; if(p->G == 0) break; for(j = 0; j < i; ++j) { LRHit* q = &hits[j]; bool compatible = true; // hit q has already been removed if(q->G == 0) continue; // if the hits are to different targets they can't overlap if(p->targetID != q->targetID) continue; if(p->interval.upper == -1 && q->interval.upper == -1) { // Calculate the overlap length on the query int qol = (p->q_end < q->q_end? p->q_end : q->q_end) - (p->q_start > q->q_start? p->q_start : q->q_start); if(qol < 0) qol = 0; if((float)qol / (p->q_end - p->q_start) > MASK_LEVEL || (float)qol / (q->q_end - q->q_start) > MASK_LEVEL) { int64_t tol = 0; if(p->t_start + p->length < q->t_start + q->length) tol = p->t_start + p->length; else tol = q->t_start + q->length; if(p->t_start > q->t_start) tol -= p->t_start; else tol -= q->t_start; if((double)tol / p->length > MASK_LEVEL || (double)tol / q->length > MASK_LEVEL) compatible = false; #ifdef BWA_COMPAT_DEBUG_RESOLVE printf(" idx = (%d,%d) id=(%d,%d) G=(%d,%d) qol: %d tol: %zu compat: %d\n", i,j, (int)p->targetID, (int)q->targetID, p->G, q->G, qol, tol, compatible); #endif if(!compatible) { p->G = 0; break; } } } } } int new_n = i; for(i = j = 0; i < new_n; ++i) { if(hits[i].G == 0) continue; if(i != j) hits[j++] = hits[i]; else ++j; } hits.resize(j); #ifdef BWA_COMPAT_DEBUG_RESOLVE printf("Final number of hits after duplicate removal: %zu\n", hits.size()); #endif return hits.size(); } // Remove cells from a stack entry based on some criteria void cutTail(LRStackEntry* u, const LRParams& params) { switch(params.cutTailAlgorithm) { case LRCA_Z_BEST: cutTailByZBest(u, params); break; case LRCA_Z_BEST_STRATA: cutTailByStratifiedZBest(u, params); break; case LRCA_SCORE_FRAC: cutTailByScorePercent(u, params); break; case LRCA_NONE: return; default: assert(false); } } // Remove cells from a stack entry (query dawg node) by their score // as expressed as a fraction of the maximum possible score for the // query void cutTailByScorePercent(LRStackEntry* u, const LRParams& params) { //printf("cutTail starting split score: %2.lf\n", split); for(size_t i = 0; i < u->cells.size(); ++i) { LRCell* p = &u->cells[i]; double cellScore = (double)p->G * 100.0f / (p->q_len * 1); if(cellScore < params.percentCutoff) { #ifdef BWA_COMPAT_DEBUG if(p->interval.upper > -1) printf("marking p [%d %d] as deleted [CT]\n", (int)p->interval.lower, (int)p->interval.upper); #endif //printf("cutTail cutting cell with qlen: %d %d %2.1lf\n", p->q_len, p->G, cellScore); p->interval.lower = 0; p->interval.upper = -1; p->G = 0; if(p->parent_idx >= 0) u->cells[p->parent_idx].children_idx[p->parent_cidx] = -1; } // else // printf("cutTail keeping cell with qlen: %d %d %2.1lf\n", p->q_len, p->G, cellScore); } } // Remove cells from a stack entry, only keeping the top zBest scoring nodes void cutTailByZBest(LRStackEntry* u, const LRParams& params) { assert(params.zBest > 0); // Save an int vector of scores IntVector scores; for(size_t i = 0; i < u->cells.size(); ++i) { LRCell* p = &u->cells[i]; if(p->interval.upper != -1 && p->G > 0) scores.push_back(-p->G); } if((int)scores.size() <= params.zBest) return; // Partially sort the scores to select the T-th best score std::nth_element(scores.begin(), scores.begin() + params.zBest, scores.end()); double split = -scores[params.zBest]; #ifdef BWA_COMPAT_DEBUG printf("[CT] split score: %2.lf\n", split); #endif int n = 0; //printf("cutTail starting split score: %2.lf\n", split); for(size_t i = 0; i < u->cells.size(); ++i) { LRCell* p = &u->cells[i]; if(p->G == split) ++n; if(p->G < split || (p->G == split && n >= params.zBest)) { #ifdef BWA_COMPAT_DEBUG if(p->interval.upper > -1) printf("marking p [%d %d] as deleted [CT]\n", (int)p->interval.lower, (int)p->interval.upper); #endif //printf("cutTail cutting cell with qlen: %d %d %2.1lf\n", p->q_len, p->G, cellScore); p->interval.lower = 0; p->interval.upper = -1; p->G = 0; if(p->parent_idx >= 0) u->cells[p->parent_idx].children_idx[p->parent_cidx] = -1; } /*else printf("cutTail keeping cell with qlen: %d %d %2.1lf\n", p->q_len, p->G, cellScore); */ } } // Remove cells from a stack entry, only keeping the top zBest scoring nodes for each query length void cutTailByStratifiedZBest(LRStackEntry* u, const LRParams& params) { assert(params.zBest > 0); if(u->cells.empty()) return; // Calculate the range of query lengths int minQ = std::numeric_limits::max(); int maxQ = 0; for(size_t i = 0; i < u->cells.size(); ++i) { LRCell* p = &u->cells[i]; if(p->q_len < minQ) minQ = p->q_len; if(p->q_len > maxQ) maxQ = p->q_len; } // Calculate cut points for each strata int span = maxQ - minQ + 1; assert(span > 0); // std::vector scores(span); IntVector cutoffs(span); IntVector counts(span); for(size_t i = 0; i < u->cells.size(); ++i) { LRCell* p = &u->cells[i]; int strata = p->q_len - minQ; assert(strata >= 0 && strata < (int)scores.size()); if(p->interval.upper != -1 && p->G > 0) scores[strata].push_back(-p->G); } // Perform a partial sort of each strata and determine cutoff points for(int i = 0; i < span; ++i) { if((int)scores[i].size() <= params.zBest) { cutoffs[i] = 0; // keep all positively-scored nodes continue; } // Partially sort the scores to select the T-th best score std::sort(scores[i].begin(), scores[i].end()); cutoffs[i] = -scores[i][params.zBest]; } #ifdef BWA_COMPAT_DEBUG printf("[CT] split score: %2.lf\n", split); #endif //printf("cutTail starting split score: %2.lf\n", split); for(size_t i = 0; i < u->cells.size(); ++i) { LRCell* p = &u->cells[i]; int strata = p->q_len - minQ; int c = cutoffs[strata]; if(p->G == c) ++counts[strata]; if(p->G < c || (p->G == c && counts[strata] >= params.zBest)) { #ifdef BWA_COMPAT_DEBUG if(p->interval.upper > -1) printf("marking p [%d %d] as deleted [CT]\n", (int)p->interval.lower, (int)p->interval.upper); #endif //printf("cutTail cutting cell with qlen: %d %d %2.1lf\n", p->q_len, p->G, cellScore); p->interval.lower = 0; p->interval.upper = -1; p->G = 0; if(p->parent_idx >= 0) u->cells[p->parent_idx].children_idx[p->parent_cidx] = -1; } /*else printf("cutTail keeping cell with qlen: %d %d %2.1lf\n", p->q_len, p->G, cellScore); */ } } // Construct a multiple alignment from a vector of hits MultiAlignment convertHitsToMultiAlignment(const std::string& query, const BWT* pTargetBWT, const SampledSuffixArray* /*pTargetSSA*/, const LRParams& params, const LRHitVector& hits) { // Set up stdaln data structures size_t max_target = StdAlnTools::calculateMaxTargetLength(query.size(), params.alnParams); path_t* path = (path_t*)calloc(max_target + query.size(), sizeof(path_t)); AlnParam par; int matrix[25]; StdAlnTools::setAlnParam(par, matrix, params.alnParams); MAlignDataVector mAlignVec; uint8_t* pQueryT = StdAlnTools::createPacked(query); for(size_t i = 0; i < hits.size(); ++i) { LRHit tempHit = hits[i]; assert(tempHit.targetID != (uint64_t)-1); std::string target = BWTAlgorithms::extractString(pTargetBWT, tempHit.targetID); uint8_t* pTargetT = StdAlnTools::createPacked(target); //extendHitFullLength(tempHit, pQueryT, pTargetT, query.size(), target.size(), &par); int q_start_pos, q_end_pos, t_start_pos, t_end_pos; q_start_pos = tempHit.q_start; q_end_pos = tempHit.q_end; // exclusive t_start_pos = tempHit.t_start; t_end_pos = t_start_pos + tempHit.length; // exclusive // Get pointers to the substrings of interest int ql = q_end_pos - q_start_pos; uint8_t* pQuerySub = pQueryT + q_start_pos; int tl = t_end_pos - t_start_pos; uint8_t* pTargetSub = pTargetT + t_start_pos; int path_len = 0; /*int score =*/ aln_global_core(pTargetSub, tl, pQuerySub, ql, &par, path, &path_len); int cigarLen = 0; uint32_t* pCigar = aln_path2cigar32(path, path_len, &cigarLen); // MAlignData maData; maData.str = target.substr(t_start_pos, t_end_pos - t_start_pos); maData.position = q_start_pos; // Add initial padding to cigar maData.expandedCigar.append(maData.position, 'S'); // Add alignment symbols for (int j = 0; j != cigarLen; ++j) maData.expandedCigar.append(pCigar[j]>>4, "MID"[pCigar[j]&0xf]); /* printf("AlignmentDP (%zu)\n", hits[i].targetID); printf("Coords: q[%d, %d] t[%d, %d]\n", q_start_pos, q_end_pos, t_start_pos, t_end_pos); printf("C: %s\n", maData.expandedCigar.c_str()); */ mAlignVec.push_back(maData); // Cleanup delete [] pTargetT; free(pCigar); } // Cleanup free(path); delete [] pQueryT; return MultiAlignment(query, mAlignVec); } // Attempt to extend a hit to the left and right using aln_extend_core // from stdaln void extendHitFullLength(LRHit& hit, uint8_t* pQueryPacked, uint8_t* pTargetPacked, int q_length, int t_length, AlnParam* pStdAlnPar) { int q_start = hit.q_start; int q_end = hit.q_end; // exclusive int t_start = hit.t_start; int t_end = t_start + hit.length; // exclusive // Attempt left-extension if(t_start != 0 && q_start != 0) { int q_diff = q_start; int t_diff = t_start; int d = std::min(q_diff, t_diff); q_start -= d; t_start -= d; } // Attempt right-extension if(q_end < q_length && t_end < t_length) { // extension match lengths int tel = t_length - t_start; int qel = q_length - q_start; uint8_t* pTargetPackedSub = pTargetPacked + t_start; uint8_t* pQueryPackedSub = pQueryPacked + q_start; path_t path; int score = aln_extend_core(pTargetPackedSub, tel, pQueryPackedSub, qel, pStdAlnPar, &path, 0, hit.G, NULL); if(score > hit.G) { hit.G = score; hit.length = path.i; hit.q_end = path.j + hit.q_start; } } } }; ================================================ FILE: src/SGA/Algorithm/LRAlignment.h ================================================ //----------------------------------------------- // Copyright 2011 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // LRAlignment - Collection of algorithms for performing // long read matches against an FM-index // #ifndef LRALIGNMENT_H #define LRALIGNMENT_H #include "BWT.h" #include "SampledSuffixArray.h" #include "BWTAlgorithms.h" #include "HashMap.h" #include "MultiAlignment.h" #include "StdAlnTools.h" #include "stdaln.h" #include namespace LRAlignment { // Enum of identifiers for cell-filtering heuristics enum CutAlgorithm { LRCA_DEFAULT, LRCA_Z_BEST, LRCA_Z_BEST_STRATA, LRCA_SCORE_FRAC, LRCA_NONE }; // Parameters object struct LRParams { LRParams() { setDefaults(); } void setDefaults() { alnParams.setDefaults(); zBest = 20; percentCutoff = 0.90f; cutTailAlgorithm = LRCA_Z_BEST; } // void setPacBio() { setDefaults(); alnParams.setPacBio(); } GlobalAlnParams alnParams; // Cell filtering heuristics int zBest; double percentCutoff; CutAlgorithm cutTailAlgorithm; }; // Structure holding an alignment between a query sequence and a BWT of a collection of sequences struct LRHit { LRHit() : interval(0,-1), flag(0), num_seeds(0), targetID(-1), t_start(-1), length(0), G(0), q_start(0), q_end(0) {} BWTInterval interval; uint32_t flag; uint32_t num_seeds; uint64_t targetID; // ID of target sequence int t_start; // alignment start position on target int length; // length of the target alignment int G; int q_start; // alignment start on query int q_end; // alignment end (exclusive) on query static bool compareG(const LRHit& a, const LRHit& b) { return a.G > b.G; } static bool compareIDandG(const LRHit& a, const LRHit& b) { if(a.targetID == b.targetID) return a.G > b.G; else return a.targetID < b.targetID; } }; typedef std::vector LRHitVector; // Structure holding the score between a node of the prefix DAWG of the query // and the prefix trie of the target struct LRCell { // Functions void initializeDefault(); void clearChildren(); bool hasUninitializedChild() const; // Data Members // interval on the target bwt BWTInterval interval; // scores int I; int D; int G; // Character code from parent uint8_t parent_cidx; // Query and target lengths int q_len; int t_len; // Index of the parent in cell array of the stack entry int parent_idx; // Index of this cell in the cell array int u_idx; // Indices of the children in the cell array int children_idx[4]; }; typedef std::vector LRCellVector; // typedef HashMap LRHash; // A stack entry holds the an interval into the query BWT // and an array of cells with the scores for that interval struct LRStackEntry { BWTInterval interval; LRCellVector cells; }; // typedef std::stack LRStack; typedef std::vector LRPendingVector; // Core alignment function - align the sequence query // against all sequences in pTargetBWT void bwaswAlignment(const std::string& query, const BWT* pTargetBWT, const SampledSuffixArray* pTargetSSA, const LRParams& params, LRHitVector& outHits); // MultiAlignment convertHitsToMultiAlignment(const std::string& query, const BWT* pTargetBWT, const SampledSuffixArray* pTargetSSA, const LRParams& params, const LRHitVector& hits); // // Helper functions // // Initialize a hash table of BWT intervals representing // the nodes in a DAWG void initializeDAWGHash(BWT* pQueryBWT, LRHash& dawgHash); // Merge the cells of the two stack entries void mergeStackEntries(LRStackEntry* u, LRStackEntry* v); // Update the given LRStack to contain the new StackEntry after // performing any necessary merges with pending Stacks int updateStack(LRStack* pStack, LRStackEntry* u, LRPendingVector* pPendingVector, LRHash* pDawgHash, const LRParams& params); // Cull duplicated cells in the given stack entry void removeDuplicateCells(LRStackEntry* u, LRHash& hash); // Filter duplicated hits from hitsVector using their position on the query sequence int resolveDuplicateHits(const BWT* pTargetBWT, const SampledSuffixArray* pTargetSSA, LRHitVector& hits, int IS); // Filter hits out of the hits vector using their ID // At most 1 hit per target sequence is kept int resolveDuplicateHitsByID(const BWT* pTargetBWT, const SampledSuffixArray* pTargetSSA, LRHitVector& hits, int IS); // Save the best hits at each position void saveBestPositionHits(const SuffixArray* pQuerySA, LRStackEntry* u, int threshold, LRHitVector& hits); // add hits to the vector for cells that score above threshold void saveAllHits(const SuffixArray* pQuerySA, const SampledSuffixArray* pTargetSSA, const BWT* pTargetBWT, LRStackEntry* u, int threshold, LRHitVector& hits); // save hits to cells that contain intervals that represent prefixes of reads void saveTerminalHits(const SuffixArray* pQuerySA, const SampledSuffixArray* pTargetSSA, const BWT* pTargetBWT, LRStackEntry* u, int threshold, LRHitVector& hits); 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static const AlignFlags prePreAF(false, true, true); static const AlignFlags sufSufAF(true, false, true); static const AlignFlags preSufAF(true, true, false); //#define DEBUGOVERLAP 2 // Perform the overlap OverlapResult OverlapAlgorithm::overlapRead(const SeqRecord& read, int minOverlap, OverlapBlockList* pOutList) const { OverlapResult r; if(static_cast(read.seq.length()) < minOverlap) return r; if(!m_exactModeOverlap) r = overlapReadInexact(read, minOverlap, pOutList); else r = overlapReadExact(read, minOverlap, pOutList); return r; } // OverlapResult OverlapAlgorithm::overlapReadInexact(const SeqRecord& read, int minOverlap, OverlapBlockList* pOBOut) const { OverlapResult result; OverlapBlockList obWorkingList; std::string seq = read.seq.toString(); #ifdef DEBUGOVERLAP std::cout << "\n\n***Overlapping read " << read.id << " suffix\n"; std::cout << "***Overlapping seq " << read.seq.toString() << " suffix\n"; #endif // Match the suffix of seq to prefixes // findInexact returns false is the maximum search time was exceeded. In this // case we dont run any of the subsequent commands and return no overlaps. bool valid = true; valid = findOverlapBlocksInexact(seq, m_pBWT, m_pRevBWT, sufPreAF, minOverlap, &obWorkingList, pOBOut, result); if(valid) valid = findOverlapBlocksInexact(complement(seq), m_pRevBWT, m_pBWT, prePreAF, minOverlap, &obWorkingList, pOBOut, result); #ifdef DEBUGOVERLAP std::cout << "WORKING OverlapBlockList Length SUFFIX: " << obWorkingList.size() << std::endl; #endif if(valid) { if(m_bIrreducible) { computeIrreducibleBlocks(m_pBWT, m_pRevBWT, &obWorkingList, pOBOut); obWorkingList.clear(); } else { pOBOut->splice(pOBOut->end(), obWorkingList); assert(obWorkingList.empty()); } } #ifdef DEBUGOVERLAP std::cout << "\n\n***Overlapping read " << read.id << " prefix\n"; #endif // Match the prefix of seq to suffixes if(valid) valid = findOverlapBlocksInexact(reverseComplement(seq), m_pBWT, m_pRevBWT, sufSufAF, minOverlap, &obWorkingList, pOBOut, result); if(valid) valid = findOverlapBlocksInexact(reverse(seq), m_pRevBWT, m_pBWT, preSufAF, minOverlap, &obWorkingList, pOBOut, result); #ifdef DEBUGOVERLAP std::cout << "WORKING OverlapBlockList Length PREFIX: " << obWorkingList.size() << std::endl; #endif if(valid) { if(m_bIrreducible) { computeIrreducibleBlocks(m_pBWT, m_pRevBWT, &obWorkingList, pOBOut); obWorkingList.clear(); } else { pOBOut->splice(pOBOut->end(), obWorkingList); assert(obWorkingList.empty()); } } if(!valid) { pOBOut->clear(); result.isSubstring = false; result.searchAborted = true; return result; } #ifdef DEBUGOVERLAP unsigned N = 1; if (pOBOut->size() > N) { std::cout << read.id << " OverlapBlockList Length: " << pOBOut->size() << std::endl; std::list::iterator it = pOBOut->begin(); std::advance(it, N); //std::cout << "Olen: " << it->overlapLen << " numDiff: " << it->numDiff << std::endl; std::cout << " BWT1L: " << it->ranges.interval[0].lower; std::cout << " BWT1U: " << it->ranges.interval[0].upper; std::cout << " BWT2L: " << it->ranges.interval[1].lower; std::cout << " BWT2U: " << it->ranges.interval[1].upper; std::cout << " RBWT1L: " << it->rawRanges.interval[0].lower; std::cout << " RBWT1U: " << it->rawRanges.interval[0].upper; std::cout << " RBWT2L: " << it->rawRanges.interval[1].lower; std::cout << " RBWT2U: " << it->rawRanges.interval[1].upper << std::endl; } #endif return result; } // OverlapResult OverlapAlgorithm::alignReadDuplicate(const SeqRecord& read, OverlapBlockList* pOBOut) const { OverlapResult result; OverlapBlockList obWorkingList; std::string seq = read.seq.toString(); int readLength = seq.length(); findOverlapBlocksInexact(seq, m_pBWT, m_pRevBWT, sufPreAF, readLength, &obWorkingList, pOBOut, result); findOverlapBlocksInexact(complement(seq), m_pRevBWT, m_pBWT, prePreAF, readLength, &obWorkingList, pOBOut, result); return result; } // Construct the set of blocks describing irreducible overlaps with READ // and write the blocks to pOBOut OverlapResult OverlapAlgorithm::overlapReadExact(const SeqRecord& read, int minOverlap, OverlapBlockList* pOBOut) const { OverlapResult result; // The complete set of overlap blocks are collected in obWorkingList // The filtered set (containing only irreducible overlaps) are placed into pOBOut // by calculateIrreducibleHits OverlapBlockList obWorkingList; std::string seq = read.seq.toString(); // We store the various overlap blocks using a number of lists, one for the containments // in the forward and reverse index and one for each set of overlap blocks OverlapBlockList oblFwdContain; OverlapBlockList oblRevContain; OverlapBlockList oblSuffixFwd; OverlapBlockList oblSuffixRev; OverlapBlockList oblPrefixFwd; OverlapBlockList oblPrefixRev; // Match the suffix of seq to prefixes findOverlapBlocksExact(seq, m_pBWT, m_pRevBWT, sufPreAF, minOverlap, &oblSuffixFwd, &oblFwdContain, result); findOverlapBlocksExact(complement(seq), m_pRevBWT, m_pBWT, prePreAF, minOverlap, &oblSuffixRev, &oblRevContain, result); // Match the prefix of seq to suffixes findOverlapBlocksExact(reverseComplement(seq), m_pBWT, m_pRevBWT, sufSufAF, minOverlap, &oblPrefixFwd, &oblFwdContain, result); findOverlapBlocksExact(reverse(seq), m_pRevBWT, m_pBWT, preSufAF, minOverlap, &oblPrefixRev, &oblRevContain, result); // Remove submaximal blocks for each block list including fully contained blocks // Copy the containment blocks into the prefix/suffix lists oblSuffixFwd.insert(oblSuffixFwd.end(), oblFwdContain.begin(), oblFwdContain.end()); oblPrefixFwd.insert(oblPrefixFwd.end(), oblFwdContain.begin(), oblFwdContain.end()); oblSuffixRev.insert(oblSuffixRev.end(), oblRevContain.begin(), oblRevContain.end()); oblPrefixRev.insert(oblPrefixRev.end(), oblRevContain.begin(), oblRevContain.end()); // Perform the submaximal filter removeSubMaximalBlocks(&oblSuffixFwd, m_pBWT, m_pRevBWT); removeSubMaximalBlocks(&oblPrefixFwd, m_pBWT, m_pRevBWT); removeSubMaximalBlocks(&oblSuffixRev, m_pRevBWT, m_pBWT); removeSubMaximalBlocks(&oblPrefixRev, m_pRevBWT, m_pBWT); // Remove the contain blocks from the suffix/prefix lists removeContainmentBlocks(seq.length(), &oblSuffixFwd); removeContainmentBlocks(seq.length(), &oblPrefixFwd); removeContainmentBlocks(seq.length(), &oblSuffixRev); removeContainmentBlocks(seq.length(), &oblPrefixRev); // Join the suffix and prefix lists oblSuffixFwd.splice(oblSuffixFwd.end(), oblSuffixRev); oblPrefixFwd.splice(oblPrefixFwd.end(), oblPrefixRev); // Move the containments to the output list pOBOut->splice(pOBOut->end(), oblFwdContain); pOBOut->splice(pOBOut->end(), oblRevContain); // Filter out transitive overlap blocks if requested if(m_bIrreducible) { computeIrreducibleBlocks(m_pBWT, m_pRevBWT, &oblSuffixFwd, pOBOut); computeIrreducibleBlocks(m_pBWT, m_pRevBWT, &oblPrefixFwd, pOBOut); } else { pOBOut->splice(pOBOut->end(), oblSuffixFwd); pOBOut->splice(pOBOut->end(), oblPrefixFwd); } return result; } // Write overlap results to an ASQG file void OverlapAlgorithm::writeResultASQG(std::ostream& writer, const SeqRecord& read, const OverlapResult& result) const { ASQG::VertexRecord record(read.id, read.seq.toString()); record.setSubstringTag(result.isSubstring); record.write(writer); } // Write overlap blocks out to a file void OverlapAlgorithm::writeOverlapBlocks(std::ostream& writer, size_t readIdx, bool isSubstring, const OverlapBlockList* pList) const { // Write the header info size_t numBlocks = pList->size(); writer << readIdx << " " << isSubstring << " " << numBlocks << " "; //std::cout << readIdx << " " << isSubstring << " " << numBlocks << " "; //std::cout << " idx: " << count << " count: " << numBlocks << "\n"; for(OverlapBlockList::const_iterator iter = pList->begin(); iter != pList->end(); ++iter) { writer << *iter << " "; //std::cout << *iter << " "; } writer << "\n"; //std::cout << "\n"; } //JEREMIAH void OverlapAlgorithm::writeOverlapBlocks(std::stringstream& writer, size_t readIdx, bool isSubstring, const OverlapBlockList* pList) const { // Write the header info size_t numBlocks = pList->size(); writer << readIdx << " " << isSubstring << " " << numBlocks << " "; //std::cout << readIdx << " " << isSubstring << " " << numBlocks << " "; for(OverlapBlockList::const_iterator iter = pList->begin(); iter != pList->end(); ++iter) { writer << *iter << " "; //std::cout << *iter << " "; } writer << "\n"; //std::cout << "\n"; } // Calculate the ranges in pBWT that contain a prefix of at least minOverlap basepairs that // overlaps with a suffix of w. The ranges are added to the pOBList void OverlapAlgorithm::findOverlapBlocksExact(const std::string& w, const BWT* pBWT, const BWT* pRevBWT, const AlignFlags& af, int minOverlap, OverlapBlockList* pOverlapList, OverlapBlockList* pContainList, OverlapResult& result) const { // The algorithm is as follows: // We perform a backwards search using the FM-index for the string w. // As we perform the search we collect the intervals // of the significant prefixes (len >= minOverlap) that overlap w. BWTIntervalPair ranges; size_t l = w.length(); int start = l - 1; BWTAlgorithms::initIntervalPair(ranges, w[start], pBWT, pRevBWT); // Collect the OverlapBlocks for(size_t i = start - 1; i >= 1; --i) { // Compute the range of the suffix w[i, l] BWTAlgorithms::updateBothL(ranges, w[i], pBWT); int overlapLen = l - i; if(overlapLen >= minOverlap) { // Calculate which of the prefixes that match w[i, l] are terminal // These are the proper prefixes (they are the start of a read) BWTIntervalPair probe = ranges; BWTAlgorithms::updateBothL(probe, '$', pBWT); // The probe interval contains the range of proper prefixes if(probe.interval[1].isValid()) { assert(probe.interval[1].lower > 0); pOverlapList->push_back(OverlapBlock(probe, ranges, overlapLen, 0, af)); } } } // Determine if this sequence is contained and should not be processed further BWTAlgorithms::updateBothL(ranges, w[0], pBWT); // Ranges now holds the interval for the full-length read // To handle containments, we output the overlapBlock to the final overlap block list // and it will be processed later // Two possible containment cases: // 1) This read is a substring of some other read // 2) This read is identical to some other read // Case 1 is indicated by the existance of a non-$ left or right hand extension // In this case we return no alignments for the string AlphaCount64 left_ext = BWTAlgorithms::getExtCount(ranges.interval[0], pBWT); AlphaCount64 right_ext = BWTAlgorithms::getExtCount(ranges.interval[1], pRevBWT); if(left_ext.hasDNAChar() || right_ext.hasDNAChar()) { result.isSubstring = true; } else { BWTIntervalPair probe = ranges; BWTAlgorithms::updateBothL(probe, '$', pBWT); if(probe.isValid()) { // terminate the contained block and add it to the contained list BWTAlgorithms::updateBothR(probe, '$', pRevBWT); assert(probe.isValid()); pContainList->push_back(OverlapBlock(probe, ranges, w.length(), 0, af)); } } //OverlapBlockList containedWorkingList; //partitionBlockList(w.length(), &workingList, pOverlapList, &containedWorkingList); // Terminate the contained blocks //terminateContainedBlocks(containedWorkingList); // Move the contained blocks to the final contained list //pContainList->splice(pContainList->end(), containedWorkingList); return; } // Seeded blockwise BWT alignment of prefix-suffix for reads // Each alignment is given a seed region and a block region // The seed region is the terminal portion of w where maxDiff + 1 seeds are created // at least 1 of these seeds must align exactly for there to be an alignment with // at most maxDiff differences between the prefix/suffix. Only alignments within the // range [block_start, block_end] are output. The block_end coordinate is inclusive. bool OverlapAlgorithm::findOverlapBlocksInexact(const std::string& w, const BWT* pBWT, const BWT* pRevBWT, const AlignFlags& af, int minOverlap, OverlapBlockList* pOverlapList, OverlapBlockList* pContainList, OverlapResult& result) const { int len = w.length(); int overlap_region_left = len - minOverlap; SearchSeedVector* pCurrVector = new SearchSeedVector; SearchSeedVector* pNextVector = new SearchSeedVector; OverlapBlockList workingList; SearchSeedVector::iterator iter; // Create and extend the initial seeds int actual_seed_length = m_seedLength; int actual_seed_stride = m_seedStride; #ifdef DEBUGOVERLAP std::cout << "SeedLen: " << m_seedLength << " SeedStride: " << m_seedStride << std::endl; #endif if(actual_seed_length == 0) { // Calculate a seed length and stride that will guarantee all overlaps // with error rate m_errorRate will be found calculateSeedParameters(w, minOverlap, actual_seed_length, actual_seed_stride); } assert(actual_seed_stride != 0); createSearchSeeds(w, pBWT, pRevBWT, actual_seed_length, actual_seed_stride, pCurrVector); extendSeedsExactRight(w, pBWT, pRevBWT, ED_RIGHT, pCurrVector, pNextVector); pCurrVector->clear(); pCurrVector->swap(*pNextVector); assert(pNextVector->empty()); int num_steps = 0; // Perform the inexact extensions bool fail = false; while(!pCurrVector->empty()) { if(m_maxSeeds != -1 && (int)pCurrVector->size() > m_maxSeeds) { fail = true; break; } iter = pCurrVector->begin(); while(iter != pCurrVector->end()) { SearchSeed& align = *iter; // If the current aligned region is right-terminal // and the overlap is greater than minOverlap, try to find overlaps // or containments if(align.right_index == len - 1) { double align_error = align.calcErrorRate(); // Check for overlaps if(align.left_index <= overlap_region_left && isErrorRateAcceptable(align_error, m_errorRate)) { int overlapLen = len - align.left_index; BWTIntervalPair probe = align.ranges; BWTAlgorithms::updateBothL(probe, '$', pBWT); // The probe interval contains the range of proper prefixes if(probe.interval[1].isValid()) { assert(probe.interval[1].lower > 0); OverlapBlock nBlock(probe, align.ranges, overlapLen, align.z, af, align.historyLink->getHistoryVector()); workingList.push_back(nBlock); } } // Check for containments // If the seed is left-terminal and there are [ACGT] left/right extensions of the sequence // this read must be a substring of another read if(align.left_index == 0) { AlphaCount64 left_ext = BWTAlgorithms::getExtCount(align.ranges.interval[0], pBWT); AlphaCount64 right_ext = BWTAlgorithms::getExtCount(align.ranges.interval[1], pRevBWT); if(left_ext.hasDNAChar() || right_ext.hasDNAChar()) result.isSubstring = true; } } // Extend the seed to the right/left if(align.dir == ED_RIGHT) extendSeedInexactRight(align, w, pBWT, pRevBWT, pNextVector); else extendSeedInexactLeft(align, w, pBWT, pRevBWT, pNextVector); ++iter; //pCurrVector->erase(iter++); } pCurrVector->clear(); assert(pCurrVector->empty()); pCurrVector->swap(*pNextVector); // Remove identical seeds after we have performed seed_len steps // as there now might be redundant seeds if(num_steps % actual_seed_stride == 0) { std::sort(pCurrVector->begin(), pCurrVector->end(), SearchSeed::compareLeftRange); SearchSeedVector::iterator end_iter = std::unique(pCurrVector->begin(), pCurrVector->end(), SearchSeed::equalLeftRange); pCurrVector->resize(end_iter - pCurrVector->begin()); } ++num_steps; } if(!fail) { // parse the working list to remove any submaximal overlap blocks // these blocks correspond to reads that have multiple valid overlaps. // we only keep the longest removeSubMaximalBlocks(&workingList, pBWT, pRevBWT); OverlapBlockList containedWorkingList; partitionBlockList(len, &workingList, pOverlapList, &containedWorkingList); // Terminate the contained blocks terminateContainedBlocks(containedWorkingList); // Move the contained blocks to the final contained list pContainList->splice(pContainList->end(), containedWorkingList); } delete pCurrVector; delete pNextVector; return !fail; } // Build forward history for the blocks void OverlapAlgorithm::buildForwardHistory(OverlapBlockList* pList) const { OverlapBlockList terminalList; OverlapBlockList potentialContainedList; while(!pList->empty()) { // The terminalBlock list contains all the blocks that became right-terminal // in the current extension round. // Perform a single round of extension, any terminal blocks // are moved to the terminated list extendActiveBlocksRight(m_pBWT, m_pRevBWT, *pList, terminalList, potentialContainedList); } *pList = terminalList; pList->splice(pList->end(), potentialContainedList); } // Calculate the single right extension to the '$' for each the contained blocks // so that the interval ranges are consistent void OverlapAlgorithm::terminateContainedBlocks(OverlapBlockList& containedBlocks) const { for(OverlapBlockList::iterator iter = containedBlocks.begin(); iter != containedBlocks.end(); ++iter) BWTAlgorithms::updateBothR(iter->ranges, '$', iter->getExtensionBWT(m_pBWT, m_pRevBWT)); } // Calculate the seed length and stride to ensure that we will find all // all overlaps with error rate at most m_errorRate. // To overlap two reads allowing for d errors, we create d+1 seeds so that at least one seed will match // exactly. d is a function of the overlap length so we define the seed length using the minimum overlap // parameter. We then tile seeds across the read starting from the end such that for every overlap length // x, there are at least floor(x * error_rate) + 1 seeds. void OverlapAlgorithm::calculateSeedParameters(const std::string& w, const int minOverlap, int& seed_length, int& seed_stride) const { int read_len = w.length(); seed_length = 0; // The maximum possible number of differences occurs for a fully-aligned read int max_diff_high = static_cast(m_errorRate * read_len); // Calculate the seed length to use // If the error rate is so low that no differences are possible just seed // over the entire minOverlap region if(max_diff_high > 0) { // Calculate the maximum number of differences between two sequences that overlap // by minOverlap int max_diff_low = static_cast(m_errorRate * minOverlap); if(max_diff_low == 0) max_diff_low = 1; int seed_region_length = static_cast(ceil(max_diff_low / m_errorRate)); int num_seeds_low = max_diff_low + 1; seed_length = static_cast(seed_region_length / num_seeds_low); if(seed_length > static_cast(minOverlap)) seed_length = minOverlap; } else { seed_length = minOverlap; } seed_stride = seed_length; } // Create and intialize the search seeds int OverlapAlgorithm::createSearchSeeds(const std::string& w, const BWT* pBWT, const BWT* pRevBWT, int seed_length, int seed_stride, SearchSeedVector* pOutVector) const { // Start a new chain of history links SearchHistoryLink rootLink = SearchHistoryNode::createRoot(); // The maximum possible number of differences occurs for a fully-aligned read int read_len = w.length(); int max_diff_high = static_cast(m_errorRate * read_len); // Start the seeds at the end of the read int seed_start = read_len - seed_length; while(seed_start >= 0) { SearchSeed seed; seed.left_index = seed_start; seed.right_index = seed_start; seed.dir = ED_RIGHT; seed.seed_len = seed_length; seed.z = 0; seed.maxDiff = max_diff_high; seed.historyLink = rootLink; // Initialize the left and right suffix array intervals char b = w[seed.left_index]; BWTAlgorithms::initIntervalPair(seed.ranges, b, pBWT, pRevBWT); pOutVector->push_back(seed); seed_start -= seed_stride; // Only create one seed in the exact case if(max_diff_high == 0) break; } return seed_length; } // Extend all the seeds in pInVector to the right over the entire seed range void OverlapAlgorithm::extendSeedsExactRightQueue(const std::string& w, const BWT* /*pBWT*/, const BWT* pRevBWT, ExtendDirection /*dir*/, const SearchSeedVector* pInVector, SearchSeedQueue* pOutQueue) const { for(SearchSeedVector::const_iterator iter = pInVector->begin(); iter != pInVector->end(); ++iter) { SearchSeed align = *iter; bool valid = true; while(align.isSeed()) { ++align.right_index; char b = w[align.right_index]; BWTAlgorithms::updateBothR(align.ranges, b, pRevBWT); if(!align.isIntervalValid(RIGHT_INT_IDX)) { valid = false; break; } } if(valid) pOutQueue->push(align); } } // Extend all the seeds in pInVector to the right over the entire seed range void OverlapAlgorithm::extendSeedsExactRight(const std::string& w, const BWT* /*pBWT*/, const BWT* pRevBWT, ExtendDirection /*dir*/, const SearchSeedVector* pInVector, SearchSeedVector* pOutVector) const { for(SearchSeedVector::const_iterator iter = pInVector->begin(); iter != pInVector->end(); ++iter) { SearchSeed align = *iter; bool valid = true; while(align.isSeed()) { ++align.right_index; char b = w[align.right_index]; BWTAlgorithms::updateBothR(align.ranges, b, pRevBWT); if(!align.isIntervalValid(RIGHT_INT_IDX)) { valid = false; break; } } //std::cout << "Initial seed: "; //align.print(w); if(valid) pOutVector->push_back(align); } } // void OverlapAlgorithm::extendSeedInexactRight(SearchSeed& seed, const std::string& w, const BWT* /*pBWT*/, const BWT* pRevBWT, SearchSeedVector* pOutVector) const { // If this alignment has run all the way to the end of the sequence // switch it to be a left extension sequence if(seed.right_index == int(w.length() - 1)) { seed.dir = ED_LEFT; pOutVector->push_back(seed); return; } ++seed.right_index; if(!seed.allowMismatches()) { char b = w[seed.right_index]; BWTAlgorithms::updateBothR(seed.ranges, b, pRevBWT); if(seed.isIntervalValid(RIGHT_INT_IDX)) pOutVector->push_back(seed); } else { // Calculating the AlphaCounts is the heavy part of the computation so we cache // the value outside the loop, it is the same for all bases AlphaCount64 lower = pRevBWT->getFullOcc(seed.ranges.interval[1].lower - 1); AlphaCount64 upper = pRevBWT->getFullOcc(seed.ranges.interval[1].upper); for(int i = 0; i < 4; ++i) { char b = ALPHABET[i]; BWTIntervalPair probe = seed.ranges; BWTAlgorithms::updateBothR(probe, b, pRevBWT, lower, upper); if(probe.interval[RIGHT_INT_IDX].isValid()) { SearchSeed branched = seed; branched.ranges = probe; if(b != w[seed.right_index]) { ++branched.z; branched.historyLink = seed.historyLink->createChild(w.length() - seed.right_index, b); } pOutVector->push_back(branched); } } } } // void OverlapAlgorithm::extendSeedInexactLeft(SearchSeed& seed, const std::string& w, const BWT* pBWT, const BWT* /*pRevBWT*/, SearchSeedVector* pOutVector) const { //printf("ProcessingLEFT: "); align.print(w); --seed.left_index; if(seed.left_index >= 0) { if(!seed.allowMismatches()) { // Extend exact char b = w[seed.left_index]; BWTAlgorithms::updateBothL(seed.ranges, b, pBWT); if(seed.isIntervalValid(LEFT_INT_IDX)) pOutVector->push_back(seed); } else { // Calculating the AlphaCounts is the heavy part of the computation so we cache // the value outside the loop, it is the same for all bases AlphaCount64 lower = pBWT->getFullOcc(seed.ranges.interval[0].lower - 1); AlphaCount64 upper = pBWT->getFullOcc(seed.ranges.interval[0].upper); for(int i = 0; i < 4; ++i) { char b = ALPHABET[i]; BWTIntervalPair probe = seed.ranges; BWTAlgorithms::updateBothL(probe, b, pBWT, lower, upper); if(probe.interval[LEFT_INT_IDX].isValid()) { SearchSeed branched = seed; branched.ranges = probe; if(b != w[seed.left_index]) { ++branched.z; // The history coordinates are wrt the right end of the read // so that each position corresponds to the length of the overlap // including that position branched.historyLink = seed.historyLink->createChild(w.length() - seed.left_index, b); //branched.history.add(w.length() - seed.left_index, b); } pOutVector->push_back(branched); } } } } } // Extend the seed to the right, return true if the seed is still a valid range bool OverlapAlgorithm::extendSeedExactRight(SearchSeed& seed, const std::string& w, const BWT* /*pBWT*/, const BWT* pRevBWT) const { // If this alignment has run all the way to the end of the sequence // switch it to be a left extension sequence if(seed.right_index == int(w.length() - 1)) { seed.dir = ED_LEFT; return true; } ++seed.right_index; char b = w[seed.right_index]; BWTAlgorithms::updateBothR(seed.ranges, b, pRevBWT); if(seed.isIntervalValid(RIGHT_INT_IDX)) return true; else return false; } // bool OverlapAlgorithm::extendSeedExactLeft(SearchSeed& seed, const std::string& w, const BWT* pBWT, const BWT* /*pRevBWT*/) const { --seed.left_index; if(seed.left_index >= 0) { char b = w[seed.left_index]; BWTAlgorithms::updateBothL(seed.ranges, b, pBWT); if(seed.isIntervalValid(LEFT_INT_IDX)) return true; else return false; } else { return false; } } // Calculate the irreducible blocks from the vector of OverlapBlocks void OverlapAlgorithm::computeIrreducibleBlocks(const BWT* pBWT, const BWT* pRevBWT, OverlapBlockList* pOBList, OverlapBlockList* pOBFinal) const { // processIrreducibleBlocks requires the pOBList to be sorted in descending order pOBList->sort(OverlapBlock::sortSizeDescending); #ifdef DEBUGOVERLAP std::cout << " m_exactModeIrreducible " << m_exactModeIrreducible << "\n"; #endif if(m_exactModeIrreducible) _processIrreducibleBlocksExactIterative(pBWT, pRevBWT, *pOBList, pOBFinal); else _processIrreducibleBlocksInexact(pBWT, pRevBWT, *pOBList, pOBFinal); pOBList->clear(); } // iterate through obList and determine the overlaps that are irreducible. This function is recursive. // The final overlap blocks corresponding to irreducible overlaps are written to pOBFinal. // Invariant: the blocks are ordered in descending order of the overlap size so that the longest overlap is first. // Invariant: each block corresponds to the same extension of the root sequence w. void OverlapAlgorithm::_processIrreducibleBlocksExactIterative(const BWT* pBWT, const BWT* pRevBWT, OverlapBlockList& inList, OverlapBlockList* pOBFinal) const { if(inList.empty()) return; // We store the overlap blocks in groups of blocks that have the same right-extension. // When a branch is found, the groups are split based on the extension typedef std::list BlockGroups; BlockGroups blockGroups; blockGroups.push_back(inList); int numExtensions = 0; int numBranches = 0; while(!blockGroups.empty()) { // Perform one extenion round for each group. // If the top-level block has ended, push the result // to the final list and remove the group from processing BlockGroups::iterator groupIter = blockGroups.begin(); BlockGroups incomingGroups; // Branched blocks are placed here while(groupIter != blockGroups.end()) { OverlapBlockList& currList = *groupIter; bool bEraseGroup = false; // Count the extensions in the top level (longest) blocks first int topLen = currList.front().overlapLen; AlphaCount64 ext_count; OBLIter blockIter = currList.begin(); while(blockIter != currList.end() && blockIter->overlapLen == topLen) { ext_count += blockIter->getCanonicalExtCount(pBWT, pRevBWT); ++blockIter; } // Three cases: // 1) The top level block has ended as it contains the extension $. Output TLB and end. // 2) There is a singular unique extension base for all the blocks. Update the blocks and continue. // 3) There are multiple extension bases, split the block group and continue. // If some block other than the TLB ended, it must be contained within the TLB and it is not output // or considered further. // Likewise if multiple distinct strings in the TLB ended, we only output the top one. The rest // must have the same sequence as the top one and are hence considered to be contained with the top element. #ifdef DEBUGOVERLAP std::cout << "ext_gcount.get($) " << ext_count.get('$') << "\n"; #endif if(ext_count.get('$') > 0) { // An irreducible overlap has been found. It is possible that there are two top level blocks // (one in the forward and reverse direction). Since we can't decide which one // contains the other at this point, we output hits to both. Under a fixed // length string assumption one will be contained within the other and removed later. OBLIter tlbIter = currList.begin(); while(tlbIter != currList.end() && tlbIter->overlapLen == topLen) { // Ensure the tlb is actually terminal and not a substring block AlphaCount64 test_count = tlbIter->getCanonicalExtCount(pBWT, pRevBWT); if(test_count.get('$') == 0) { std::cerr << "Error: substring read found during overlap computation.\n"; std::cerr << "Please run sga rmdup before sga overlap\n"; exit(EXIT_FAILURE); } // Perform the final right-update to make the block terminal OverlapBlock branched = *tlbIter; BWTAlgorithms::updateBothR(branched.ranges, '$', branched.getExtensionBWT(pBWT, pRevBWT)); pOBFinal->push_back(branched); #ifdef DEBUGOVERLAP std::cout << "[IE] TLB of length " << branched.overlapLen << " has ended\n"; std::cout << "[IE]\tBlock data: " << branched << "\n"; #endif ++tlbIter; } // Set the flag to erase this group, it is finished bEraseGroup = true; } else { // Count the extension for the rest of the blocks while(blockIter != currList.end()) { ext_count += blockIter->getCanonicalExtCount(pBWT, pRevBWT); ++blockIter; } if(ext_count.hasUniqueDNAChar()) { // Update all the blocks using the unique extension character // This character is in the canonical representation wrt to the query char b = ext_count.getUniqueDNAChar(); updateOverlapBlockRangesRight(pBWT, pRevBWT, currList, b); numExtensions++; bEraseGroup = false; } else { for(size_t idx = 0; idx < DNA_ALPHABET_SIZE; ++idx) { char b = ALPHABET[idx]; if(ext_count.get(b) > 0) { numBranches++; OverlapBlockList branched = currList; updateOverlapBlockRangesRight(pBWT, pRevBWT, branched, b); incomingGroups.push_back(branched); bEraseGroup = true; } } } } if(bEraseGroup) groupIter = blockGroups.erase(groupIter); else ++groupIter; } // Splice in the newly branched blocks, if any blockGroups.splice(blockGroups.end(), incomingGroups); } } // Classify the blocks in obList as irreducible, transitive or substrings. The irreducible blocks are // put into pOBFinal. The remaining are discarded. // Invariant: the blocks are ordered in descending order of the overlap size so that the longest overlap is first. void OverlapAlgorithm::_processIrreducibleBlocksInexact(const BWT* pBWT, const BWT* pRevBWT, OverlapBlockList& activeList, OverlapBlockList* pOBFinal) const { if(activeList.empty()) return; #ifdef DEBUGOVERLAP std::cout << " activeList.size() " << activeList.size() << "\n"; #endif // The activeList contains all the blocks that are not yet right terminal // Count the extensions in the top level (longest) blocks first bool all_eliminated = false; while(!activeList.empty() && !all_eliminated) { // The terminalBlock list contains all the blocks that became right-terminal // in the current extension round. OverlapBlockList terminalList; OverlapBlockList potentialContainedList; // Perform a single round of extension, any terminal blocks // are moved to the terminated list extendActiveBlocksRight(pBWT, pRevBWT, activeList, terminalList, potentialContainedList); // Compare the blocks in the contained list against the other terminal and active blocks // If they are a substring match to any of these, discard them OverlapBlockList::iterator containedIter = potentialContainedList.begin(); for(; containedIter != potentialContainedList.end(); ++containedIter) { if(!isBlockSubstring(*containedIter, terminalList, m_errorRate) && !isBlockSubstring(*containedIter, activeList, m_errorRate)) { // Not a substring, move to terminal list terminalList.push_back(*containedIter); //std::cout << "Contained block kept: " << containedIter->overlapLen << "\n"; } else { //std::cout << "Contained block found and removed: " << containedIter->overlapLen << "\n"; } } // Using the terminated blocks, mark as eliminated any active blocks // that form a valid overlap to the terminal block. These are transitive edges // We do not compare two terminal blocks, we don't consider these overlaps to be // transitive #ifdef DEBUGOVERLAP std::cout << " terminalList.sizee() " << terminalList.size() << "\n"; #endif OverlapBlockList::iterator terminalIter = terminalList.begin(); for(; terminalIter != terminalList.end(); ++terminalIter) { #ifdef DEBUGOVERLAP std::cout << "[II] ***TLB of length " << terminalIter->overlapLen << " has ended\n"; #endif all_eliminated = true; OverlapBlockList::iterator activeIter = activeList.begin(); for(; activeIter != activeList.end(); ++activeIter) { if(activeIter->isEliminated) continue; // skip previously marked blocks // Two conditions must be met for a block to be transitive wrt terminal: // 1) It must have a strictly shorter overlap than the terminal block // 2) The error rate between the block and terminal must be less than the threshold double inferredErrorRate = calculateBlockErrorRate(*terminalIter, *activeIter); if(activeIter->overlapLen < terminalIter->overlapLen && isErrorRateAcceptable(inferredErrorRate, m_errorRate)) { #ifdef DEBUGOVERLAP_2 std::cout << "Marking block of length " << activeIter->overlapLen << " as eliminated\n"; #endif activeIter->isEliminated = true; } else { all_eliminated = false; } } // Move this block to the final list if it has not been previously marked eliminated if(!terminalIter->isEliminated) { #ifdef DEBUGOVERLAP std::cout << "[II] Adding block " << *terminalIter << " to final list\n"; //std::cout << " extension: " << terminalIter->forwardHistory << "\n"; #endif pOBFinal->push_back(*terminalIter); } } } activeList.clear(); } // Extend all the blocks in activeList by one base to the right // Move all right-terminal blocks to the termainl list. If a block // is terminal and potentially contained by another block, add it to // containedList void OverlapAlgorithm::extendActiveBlocksRight(const BWT* pBWT, const BWT* pRevBWT, OverlapBlockList& activeList, OverlapBlockList& terminalList, OverlapBlockList& /*containedList*/) const { OverlapBlockList::iterator iter = activeList.begin(); OverlapBlockList::iterator next; while(iter != activeList.end()) { next = iter; ++next; // Check if block is terminal AlphaCount64 ext_count = iter->getCanonicalExtCount(pBWT, pRevBWT); if(ext_count.get('$') > 0) { // Only consider this block to be terminal irreducible if it has at least one extension // or else it is a substring block if(iter->forwardHistory.size() > 0) { OverlapBlock branched = *iter; BWTAlgorithms::updateBothR(branched.ranges, '$', branched.getExtensionBWT(pBWT, pRevBWT)); terminalList.push_back(branched); #ifdef DEBUGOVERLAP_2 std::cout << "Block of length " << iter->overlapLen << " moved to terminal\n"; #endif } } int curr_extension = iter->forwardHistory.size(); // Perform the right extensions // Best case, there is only a single extension character // Handle this case specially so we don't need to copy the potentially // large OverlapBlock structure and its full history if(ext_count.hasUniqueDNAChar()) { // Get the extension character with respect to the queried sequence char canonical_base = ext_count.getUniqueDNAChar(); // Flip the base into the frame of reference for the block char block_base = iter->flags.isQueryComp() ? complement(canonical_base) : canonical_base; // Update the block using the base in its frame of reference BWTAlgorithms::updateBothR(iter->ranges, block_base, iter->getExtensionBWT(pBWT, pRevBWT)); // Add the base to the history in the frame of reference of the query read // This is so the history is consistent when comparing between blocks from different strands iter->forwardHistory.add(curr_extension, canonical_base); } else { for(size_t idx = 0; idx < DNA_ALPHABET_SIZE; ++idx) { char canonical_base = ALPHABET[idx]; char block_base = iter->flags.isQueryComp() ? complement(canonical_base) : canonical_base; if(ext_count.get(canonical_base) == 0) continue; // Branch the sequence. This involves copying the entire history which can be large // if the input sequences are very long. This could be avoided by using the SearchHistoyNode/Link // structure but branches are infrequent enough to not have a large impact OverlapBlock branched = *iter; BWTAlgorithms::updateBothR(branched.ranges, block_base, branched.getExtensionBWT(pBWT, pRevBWT)); assert(branched.ranges.isValid()); // Add the base in the canonical frame branched.forwardHistory.add(curr_extension, canonical_base); // Insert the new block after the iterator activeList.insert(iter, branched); } // Remove the original block, which has been superceded by the branches activeList.erase(iter); } iter = next; // this skips the newly-inserted blocks } } // Return true if the terminalBlock is a substring of any member of blockList bool OverlapAlgorithm::isBlockSubstring(OverlapBlock& terminalBlock, const OverlapBlockList& blockList, double maxER) const { OverlapBlockList::const_iterator iter = blockList.begin(); size_t right_extension_length = terminalBlock.forwardHistory.size(); for(; iter != blockList.end(); ++iter) { if(terminalBlock.overlapLen == iter->overlapLen && right_extension_length == iter->forwardHistory.size()) { continue; // same length, cannot be a substring } // Calculate error rate between blocks double er = calculateBlockErrorRate(terminalBlock, *iter); if(isErrorRateAcceptable(er, maxER)) return true; } return false; } // Calculate the error rate between two overlap blocks using their history double OverlapAlgorithm::calculateBlockErrorRate(const OverlapBlock& terminalBlock, const OverlapBlock& otherBlock) const { int back_max = std::min(terminalBlock.overlapLen, otherBlock.overlapLen); int backwards_diff = SearchHistoryVector::countDifferences(terminalBlock.backHistory, otherBlock.backHistory, back_max); // We compare the forward (right) extension only up to the last position of the terminated block's extension int forward_len = terminalBlock.forwardHistory.size(); int forward_max = forward_len - 1; int forward_diff = SearchHistoryVector::countDifferences(terminalBlock.forwardHistory, otherBlock.forwardHistory, forward_max); // Calculate the length of the inferred overlap int trans_overlap_length = back_max + forward_len; double er = static_cast(backwards_diff + forward_diff) / trans_overlap_length; #ifdef DEBUGOVERLAP_2 std::cout << "OL: " << terminalBlock.overlapLen << "\n"; std::cout << "TLB BH: " << terminalBlock.backHistory << "\n"; std::cout << "TB BH: " << otherBlock.backHistory << "\n"; std::cout << "TLB FH: " << terminalBlock.forwardHistory << "\n"; std::cout << "TB FH: " << otherBlock.forwardHistory << "\n"; std::cout << "BM: " << back_max << " FM: " << forward_max << "\n"; std::cout << "IOL: " << trans_overlap_length << " TD: " << (backwards_diff + forward_diff) << "\n"; std::cout << "Block of length " << otherBlock.overlapLen << " has ier: " << er << "\n"; #endif return er; } // Update the overlap block list with a righthand extension to b, removing ranges that become invalid void OverlapAlgorithm::updateOverlapBlockRangesRight(const BWT* pBWT, const BWT* pRevBWT, OverlapBlockList& obList, char canonical_base) const { OverlapBlockList::iterator iter = obList.begin(); while(iter != obList.end()) { char relative_base = iter->flags.isQueryComp() ? complement(canonical_base) : canonical_base; BWTAlgorithms::updateBothR(iter->ranges, relative_base, iter->getExtensionBWT(pBWT, pRevBWT)); // remove the block from the list if its no longer valid if(!iter->ranges.isValid()) { iter = obList.erase(iter); } else { // Add the base to the extension history int currExtension = iter->forwardHistory.size(); iter->forwardHistory.add(currExtension, canonical_base); ++iter; } } } ================================================ FILE: src/SGA/Algorithm/OverlapAlgorithm.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // OverlapAlgorithm - This class implements all the logic // for finding and outputting overlaps for sequence reads // #ifndef OVERLAPALGORITHM_H #define OVERLAPALGORITHM_H // be explicit because there are two bwt.h (in bwa) and BWT.h (in SGA) #include "SuffixTools/BWT.h" #include "OverlapBlock.h" #include "SearchSeed.h" #include "BWTAlgorithms.h" #include "Util.h" enum OverlapMode { OM_OVERLAP, OM_FULLREAD }; struct OverlapResult { OverlapResult() : isSubstring(false), searchAborted(false) {} bool isSubstring; bool searchAborted; }; class OverlapAlgorithm { public: OverlapAlgorithm(const BWT* pBWT, const BWT* pRevBWT, double er, int seedLen, int seedStride, bool irrOnly, int maxSeeds = -1) : m_pBWT(pBWT), m_pRevBWT(pRevBWT), m_errorRate(er), m_seedLength(seedLen), m_seedStride(seedStride), m_bIrreducible(irrOnly), m_exactModeOverlap(false), m_exactModeIrreducible(false), m_maxSeeds(maxSeeds) {} // Perform the overlap // This function is threaded so everything must be const OverlapResult overlapRead(const SeqRecord& read, int minOverlap, OverlapBlockList* pOutList) const; // Perform an irreducible overlap OverlapResult overlapReadExact(const SeqRecord& read, int minOverlap, OverlapBlockList* pOBOut) const; // Find duplicate blocks for this read OverlapResult alignReadDuplicate(const SeqRecord& read, OverlapBlockList* pOBOut) const; // Perform an inexact overlap OverlapResult overlapReadInexact(const SeqRecord& read, int minOverlap, OverlapBlockList* pOBOut) const; // Write the result of an overlap to an ASQG file void writeResultASQG(std::ostream& writer, const SeqRecord& read, const OverlapResult& result) const; // Write all the overlap blocks pList to the filehandle void writeOverlapBlocks(std::ostream& writer, size_t readIdx, bool isSubstring, const OverlapBlockList* pList) const; void writeOverlapBlocks(std::stringstream& writer, size_t readIdx, bool isSubstring, const OverlapBlockList* pList) const; //JEREMIAH // Build the forward history structures for the blocks void buildForwardHistory(OverlapBlockList* pList) const; // Set flag to use exact-match algorithms only void setExactModeOverlap(bool b) { m_exactModeOverlap = b; } void setExactModeIrreducible(bool b) { m_exactModeIrreducible = b; } // const BWT* getBWT() const { return m_pBWT; } const BWT* getRBWT() const { return m_pRevBWT; } private: // Calculate the ranges in pBWT that contain a prefix of at least minOverlap basepairs that // overlaps with a suffix of w. void findOverlapBlocksExact(const std::string& w, const BWT* pBWT, const BWT* pRevBWT, const AlignFlags& af, const int minOverlap, OverlapBlockList* pOBTemp, OverlapBlockList* pOBFinal, OverlapResult& result) const; // Same as above while allowing mismatches bool findOverlapBlocksInexact(const std::string& w, const BWT* pBWT, const BWT* pRevBWT, const AlignFlags& af, const int minOverlap, OverlapBlockList* pOBList, OverlapBlockList* pOBFinal, OverlapResult& result) const; // inline bool extendSeedExactRight(SearchSeed& seed, const std::string& w, const BWT* pBWT, const BWT* pRevBWT) const; inline bool extendSeedExactLeft(SearchSeed& seed, const std::string& w, const BWT* pBWT, const BWT* pRevBWT) const; inline void branchSeedRight(const SearchSeed& seed, const std::string& w, const BWT* pBWT, const BWT* pRevBWT, SearchSeedQueue* pQueue) const; inline void branchSeedLeft(const SearchSeed& seed, const std::string& w, const BWT* pBWT, const BWT* pRevBWT, SearchSeedQueue* pQueue) const; // inline void extendSeedInexactRight(SearchSeed& seed, const std::string& w, const BWT* pBWT, const BWT* pRevBWT, SearchSeedVector* pOutVector) const; // inline void extendSeedInexactLeft(SearchSeed& seed, const std::string& w, const BWT* pBWT, const BWT* pRevBWT, SearchSeedVector* pOutVector) const; // inline void calculateSeedParameters(const std::string& w, const int minOverlap, int& seed_length, int& seed_stride) const; // inline int createSearchSeeds(const std::string& w, const BWT* pBWT, const BWT* pRevBWT, int seed_length, int seed_stride, SearchSeedVector* pOutVector) const; // inline void extendSeedsExactRightQueue(const std::string& w, const BWT* pBWT, const BWT* pRevBWT, ExtendDirection dir, const SearchSeedVector* pInVector, SearchSeedQueue* pOutQueue) const; // inline void extendSeedsExactRight(const std::string& w, const BWT* pBWT, const BWT* pRevBWT, ExtendDirection dir, const SearchSeedVector* pInVector, SearchSeedVector* pOutVector) const; // Calculate the terminal extension for the contained blocks to make the intervals consistent void terminateContainedBlocks(OverlapBlockList& containedBlocks) const; // // Irreducible-only processing algorithms // // Reduce the block list pOBList by removing blocks that correspond to transitive edges void computeIrreducibleBlocks(const BWT* pBWT, const BWT* pRevBWT, OverlapBlockList* pOBList, OverlapBlockList* pOBFinal) const; // these recursive functions do the actual work of computing the irreducible blocks void _processIrreducibleBlocksExact(const BWT* pBWT, const BWT* pRevBWT, OverlapBlockList& obList, OverlapBlockList* pOBFinal) const; void _processIrreducibleBlocksExactIterative(const BWT* pBWT, const BWT* pRevBWT, OverlapBlockList& inList, OverlapBlockList* pOBFinal) const; // void _processIrreducibleBlocksInexact(const BWT* pBWT, const BWT* pRevBWT, OverlapBlockList& obList, OverlapBlockList* pOBFinal) const; // Update the overlap block list with a righthand extension to b, removing ranges that become invalid void updateOverlapBlockRangesRight(const BWT* pBWT, const BWT* pRevBWT, OverlapBlockList& obList, char b) const; // void extendActiveBlocksRight(const BWT* pBWT, const BWT* pRevBWT, OverlapBlockList& activeList, OverlapBlockList& terminalList, OverlapBlockList& containedList) const; double calculateBlockErrorRate(const OverlapBlock& terminalBlock, const OverlapBlock& otherBlock) const; bool isBlockSubstring(OverlapBlock& terminalBlock, const OverlapBlockList& blockList, double maxER) const; // Data const BWT* m_pBWT; const BWT* m_pRevBWT; double m_errorRate; int m_seedLength; int m_seedStride; bool m_bIrreducible; bool m_exactModeOverlap; bool m_exactModeIrreducible; // Optional parameter to limit the amount of branching that is performed int m_maxSeeds; }; #endif ================================================ FILE: src/SGA/Algorithm/OverlapBlock.cpp ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // OverlapBlock - Data structures holding // the result of the alignment of a sequence read // to a BWT // #include "OverlapBlock.h" #include "BWTAlgorithms.h" //#define DEBUG_RESOLVE 1 // OverlapBlock::OverlapBlock(BWTIntervalPair r, BWTIntervalPair rawI, int ol, int nd, const AlignFlags& af, const SearchHistoryVector& backHist) : ranges(r), rawRanges(rawI), overlapLen(ol), numDiff(nd), flags(af), isEliminated(false), backHistory(backHist) { backHistory.normalize(af.isQueryComp()); } // Return a pointer to the BWT that should be used to extend the block // this is the opposite BWT that was used in the backwards search const BWT* OverlapBlock::getExtensionBWT(const BWT* pBWT, const BWT* pRevBWT) const { if(!flags.isTargetRev()) return pRevBWT; else return pBWT; } // AlphaCount64 OverlapBlock::getCanonicalExtCount(const BWT* pBWT, const BWT* pRevBWT) const { AlphaCount64 out = BWTAlgorithms::getExtCount(ranges.interval[1], getExtensionBWT(pBWT, pRevBWT)); if(flags.isQueryComp()) out.complement(); return out; } // Returns 0 if the BWT used for the overlap step was the forward BWT int OverlapBlock::getCanonicalIntervalIndex() const { if(!flags.isTargetRev()) return 0; else return 1; } // BWTInterval OverlapBlock::getCanonicalInterval() const { return ranges.interval[getCanonicalIntervalIndex()]; } // Get the string corresponding to the overlap block. This is the string found // during the backwards search std::string OverlapBlock::getOverlapString(const std::string& original) const { std::string transformed = backHistory.transform(original, flags.isQueryRev()); // If the query was reversed, we take the first overlapLen (the search // was from the front of the sequence) otherwise we take the last overlapLen if(flags.isQueryRev()) return transformed.substr(0, overlapLen); else return transformed.substr(transformed.length() - overlapLen); } // Get the full string corresponding to this overlapblock using the forward history std::string OverlapBlock::getFullString(const std::string& original) const { std::string str = getOverlapString(original); std::string history = forwardHistory.getBaseString(); if(history.empty() && overlapLen != (int)original.size()) { WARN_ONCE("getFullString() called on block with no history") } /* std::cout << "OVERLAP: " << str << "\n"; std::cout << "HIST: " << history << "\n"; std::cout << "QREV: " << flags.isQueryRev() << "\n"; std::cout << "RC: " << flags.isReverseComplement() << "\n"; std::cout << "QC: " << flags.isQueryComp() << "\n"; */ if(!flags.isQueryRev()) { str.append(history); } else { history = reverse(history); history.append(str); str.swap(history); } if(flags.isReverseComplement()) str = reverseComplement(str); return str; } EdgeDir OverlapBlock::getEdgeDir() const { if(flags.isQueryRev()) return ED_ANTISENSE; else return ED_SENSE; } // Overlap OverlapBlock::toOverlap(const std::string queryID, const std::string targetID, int queryLen, int targetLen) const { // Compute the sequence coordinates int s1 = queryLen - overlapLen; int e1 = s1 + overlapLen - 1; SeqCoord sc1(s1, e1, queryLen); int s2 = 0; // The start of the second hit must be zero by definition of a prefix/suffix match int e2 = s2 + overlapLen - 1; SeqCoord sc2(s2, e2, targetLen); // The coordinates are always with respect to the read, so flip them if // we aligned to/from the reverse of the read if(flags.isQueryRev()) { sc1.flip(); } if(flags.isTargetRev()) { sc2.flip(); } bool isRC = flags.isReverseComplement(); Overlap o(queryID, sc1, targetID, sc2, isRC, numDiff); return o; } // std::string OverlapBlock::toCanonicalID() const { std::stringstream ss; int ci = getCanonicalIntervalIndex(); ss << "IDX-" << ranges.interval[ci].lower; return ss.str(); } // void printBlockList(const OverlapBlockList* pList) { for(OverlapBlockList::const_iterator i = pList->begin(); i != pList->end(); ++i) { std::cout << "Block: " << *i << "\n"; } } // void removeSubMaximalBlocks(OverlapBlockList* pList, const BWT* pBWT, const BWT* pRevBWT) { // This algorithm removes any sub-maximal OverlapBlocks from pList // The list is sorted by the left coordinate and iterated through // if two adjacent blocks overlap they are split into maximal contiguous regions // with resolveOverlap. The resulting list is merged back into pList. This process // is repeated until each block in pList is a unique range // The bookkeeping in the intersecting case could be more efficient // but the vast vast majority of the cases will not have overlapping // blocks. pList->sort(OverlapBlock::sortIntervalLeft); OverlapBlockList::iterator iter = pList->begin(); OverlapBlockList::iterator last = pList->end(); --last; while(iter != pList->end()) { OverlapBlockList::iterator next = iter; ++next; if(next == pList->end()) break; // Check if iter and next overlap if(Interval::isIntersecting(iter->ranges.interval[0].lower, iter->ranges.interval[0].upper, next->ranges.interval[0].lower, next->ranges.interval[0].upper)) { OverlapBlockList resolvedList = resolveOverlap(*iter, *next, pBWT, pRevBWT); // Merge the new elements in and start back from the beginning of the list pList->erase(iter); pList->erase(next); pList->merge(resolvedList, OverlapBlock::sortIntervalLeft); iter = pList->begin(); //std::cout << "After splice: \n"; //printList(pList); } else { ++iter; } } } // A tracing interval maps an interval of an overlap block representing // a smaller overlap to an overlap block with a larger overlap. This is // used to determine which intervals are redundant and can be removed. struct TracingInterval { int64_t foundPosForward; int64_t sourcePosReverse; BWTInterval tracing; BWTIntervalPair updateRanges; }; typedef std::list TracingIntervalList; // In rare cases, the overlap blocks may represent sub-maximal overlaps between reads // we need to distinguish these cases and remove the sub-optimal hits. This // function splits two overlapping OverlapBlocks into up to three distinct // blocks, keeping the maximal (longest) overlap at each stage. OverlapBlockList resolveOverlap(const OverlapBlock& A, const OverlapBlock& B, const BWT* pBWT, const BWT* pRevBWT) { OverlapBlockList outList; // Check if A and B have the same overlap length, if so they must be // identical blocks (resulting from different seeds) and we can remove one if(A.overlapLen == B.overlapLen) { if(A.ranges.interval[0].lower == B.ranges.interval[0].lower && A.ranges.interval[0].upper == B.ranges.interval[0].upper) { outList.push_back(A); return outList; } else { std::cerr << "Error in resolveOverlap: Overlap blocks with same length do not " "the have same coordinates\n"; assert(false); } } // A and B must have different overlap lengths assert(A.overlapLen != B.overlapLen); // Determine which of A and B have a higher overlap const OverlapBlock* pHigher; const OverlapBlock* pLower; if(A.overlapLen > B.overlapLen) { pHigher = &A; pLower = &B; } else { pHigher = &B; pLower = &A; } // Complicated logic follows // We always want the entirity of the block with the longer // overlap so it is added to outList unmodified outList.push_back(*pHigher); // The lower block can be split into up to two pieces: // Case 1: // Lower ------ // Higher ------ // Result --- // // Case 2: // Lower ----------- // Higher ------ // Result --- -- // // Case 3: // Lower ------ // Higher ------ // Result (empty set) // It is unclear whether case 2 can happen in reality but we handle it // here anyway. Further complicating matters is that the BWTIntervalPair // keeps track of both the BWT coordinates for the backwards search // and forward search and we must take care to ensure that both intervals // are updated and the mapping between them is correct. We do this // by calculating the new forward interval using interval intersections // and directly recalculating the coordinate of the reverse interval // OverlapBlock split = *pLower; // Left-hand split if( (pLower->ranges.interval[0].lower < pHigher->ranges.interval[0].lower) || (pLower->ranges.interval[0].upper > pHigher->ranges.interval[0].upper) ) { // The intervals do not perfectly overlap and must be recalculated. // We start from the raw intervals in the lower block (the intervals representing // overlaps that are not capped by '$' symbols) and search backwards through the // bwt until the start of the sequence has been found. This maps the source reverse // index position to the forward index position. We can then decide which intervals // are redundant and can be removed. // // If the index has duplicates, it is possible that a given source reverse position // will map to multiple forward positions. To handle this case, we record the used // forward positions in a std::map so we can lookup the next lowest index that is available. // // A better algorithm (that doesn't required so many interval calculations) probably exists // but this case is very rare so simplicity wins here. // #ifdef DEBUG_RESOLVE std::cout << "LOWER -- capped: " << pLower->ranges << "\n"; std::cout << "LOWER -- raw: " << pLower->rawRanges << "\n"; std::cout << "HIGHER -- capped: " << pHigher->ranges << "\n"; std::cout << "HIGHER -- raw: " << pHigher->rawRanges << "\n"; #endif std::map usedMap; // Remap every reverse position to a forward position TracingIntervalList tracingList; int64_t j = pLower->ranges.interval[1].lower; while(j <= pLower->ranges.interval[1].upper) { TracingInterval ti; ti.sourcePosReverse = j; ti.tracing.lower = j; ti.tracing.upper = j; ti.updateRanges = pLower->rawRanges; bool done = false; while(!done) { char trace_base = pRevBWT->getChar(ti.tracing.lower); if(trace_base == '$') { BWTAlgorithms::updateBothL(ti.updateRanges, '$', pBWT); done = true; } BWTAlgorithms::updateInterval(ti.tracing, trace_base, pRevBWT); BWTAlgorithms::updateBothR(ti.updateRanges, trace_base, pRevBWT); } if(ti.updateRanges.interval[0].lower == ti.updateRanges.interval[0].upper) { // This read is not duplicated ti.foundPosForward = ti.updateRanges.interval[0].lower; } else { // This read is duplicated, look up its value in the map int64_t basePos = ti.updateRanges.interval[0].lower; if(usedMap.find(basePos) != usedMap.end()) { // Use the value in the map and update it ti.foundPosForward = usedMap[basePos]; assert(ti.foundPosForward > ti.updateRanges.interval[0].lower && ti.foundPosForward <= ti.updateRanges.interval[0].upper); usedMap[basePos]++; } else { // Use the base value and initialize the map ti.foundPosForward = basePos; usedMap[basePos] = basePos + 1; } } ++j; tracingList.push_back(ti); } // Reset the mapping between blocks std::list retainedIntervals; TracingIntervalList::iterator tracingIter = tracingList.begin(); while(tracingIter != tracingList.end()) { // Check if the forward position intersects the higher block, if so this block // is redundant and can be removed. if(!Interval::isIntersecting(tracingIter->foundPosForward, tracingIter->foundPosForward, pHigher->ranges.interval[0].lower, pHigher->ranges.interval[0].upper)) { BWTIntervalPair retained; retained.interval[0].lower = tracingIter->foundPosForward; retained.interval[0].upper = tracingIter->foundPosForward; retained.interval[1].lower = tracingIter->sourcePosReverse; retained.interval[1].upper = tracingIter->sourcePosReverse; #ifdef DEBUG_RESOLVE std::cout << "Retained coords: " << retained << "\n"; #endif retainedIntervals.push_back(retained); } ++tracingIter; } // Write out the final blocks std::list::iterator iter = retainedIntervals.begin(); while(iter != retainedIntervals.end()) { #ifdef DEBUG_RESOLVE std::cout << "OUTPUT: " << *iter << "\n"; #endif split.ranges = *iter; // Sanity check assert(split.ranges.interval[0].size() == split.ranges.interval[1].size()); assert(split.ranges.interval[0].isValid()); assert(split.ranges.interval[1].isValid()); outList.push_back(split); ++iter; } } // Sort the outlist by left coordinate outList.sort(OverlapBlock::sortIntervalLeft); return outList; } // Partition the overlap block list into two lists, // one for the containment overlaps and one for the proper overlaps void partitionBlockList(int readLen, OverlapBlockList* pCompleteList, OverlapBlockList* pOverlapList, OverlapBlockList* pContainList) { OverlapBlockList::iterator iter = pCompleteList->begin(); while(iter != pCompleteList->end()) { if(iter->overlapLen == readLen) pContainList->splice(pContainList->end(), *pCompleteList, iter++); else pOverlapList->splice(pOverlapList->end(), *pCompleteList, iter++); } } // Filter out full-length (containment) overlaps from the block list void removeContainmentBlocks(int readLen, OverlapBlockList* pList) { OverlapBlockList::iterator iter = pList->begin(); while(iter != pList->end()) { if(iter->overlapLen == readLen) iter = pList->erase(iter); else ++iter; } } // MultiOverlap blockListToMultiOverlap(const SeqRecord& record, OverlapBlockList& blockList) { std::string read_idx = record.id; std::string read_seq = record.seq.toString(); std::string qual = record.qual; MultiOverlap out(read_idx, read_seq, qual); for(OverlapBlockList::iterator iter = blockList.begin(); iter != blockList.end(); ++iter) { std::string overlap_string = iter->getOverlapString(read_seq); // Compute the endpoints of the overlap int s1 = read_seq.length() - iter->overlapLen; int e1 = s1 + iter->overlapLen - 1; SeqCoord sc1(s1, e1, read_seq.length()); int s2 = 0; // The start of the second hit must be zero by definition of a prefix/suffix match int e2 = s2 + iter->overlapLen - 1; SeqCoord sc2(s2, e2, overlap_string.length()); // The coordinates are always with respect to the read, so flip them if // we aligned to/from the reverse of the read if(iter->flags.isQueryRev()) sc1.flip(); if(iter->flags.isTargetRev()) sc2.flip(); bool isRC = false; // since we transformed the original sequence, they are never RC if(sc1.isContained()) continue; // skip containments // Add an overlap for each member of the block for(int64_t i = iter->ranges.interval[0].lower; i <= iter->ranges.interval[0].upper; ++i) { Overlap o(read_idx, sc1, makeIdxString(i), sc2, isRC, -1); out.add(overlap_string, o); } } return out; } // make an id string from a read index std::string makeIdxString(int64_t idx) { std::stringstream ss; ss << idx; return ss.str(); } ================================================ FILE: src/SGA/Algorithm/OverlapBlock.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL license //----------------------------------------------- // // OverlapBlock - Data structures holding // the result of the alignment of a sequence read // to a BWT // #ifndef OVERLAPBLOCK_H #define OVERLAPBLOCK_H #include "BWTInterval.h" #include "SearchHistory.h" #include "BitChar.h" #include "SuffixTools/BWT.h" #include "SearchHistory.h" #include "GraphCommon.h" #include "MultiOverlap.h" // Flags indicating how a given read was aligned to the FM-index // Used for internal bookkeeping struct AlignFlags { public: AlignFlags() {} AlignFlags(bool qr, bool tr, bool qc) { setQueryRev(qr); setTargetRev(tr); setQueryComp(qc); } bool isQueryRev() const { return data.test(QUERYREV_BIT); } bool isTargetRev() const { return data.test(TARGETREV_BIT); } bool isQueryComp() const { return data.test(QUERYCOMP_BIT); } // Returns true if the relationship between the query and target is reverse complement bool isReverseComplement() const { return isTargetRev() != isQueryRev(); } friend std::ostream& operator<<(std::ostream& out, const AlignFlags& af) { out << af.data; return out; } friend std::istream& operator>>(std::istream& in, AlignFlags& af) { in >> af.data; return in; } void write(std::ostream& out) { data.write(out); } void read(std::istream& in) { data.read(in); } private: void setQueryRev(bool b) { data.set(QUERYREV_BIT, b); } void setTargetRev(bool b) { data.set(TARGETREV_BIT, b); } void setQueryComp(bool b) { data.set(QUERYCOMP_BIT, b); } static const uint8_t QUERYREV_BIT = 0; static const uint8_t TARGETREV_BIT = 1; static const uint8_t QUERYCOMP_BIT = 2; BitChar data; }; // A BWTInterval pair and associated alignment data struct OverlapBlock { OverlapBlock() {} OverlapBlock(BWTIntervalPair r, BWTIntervalPair rawI, int ol, int nd, const AlignFlags& af) : ranges(r), rawRanges(rawI), overlapLen(ol), numDiff(nd), flags(af), isEliminated(false) {} OverlapBlock(BWTIntervalPair r, BWTIntervalPair rawI, int ol, int nd, const AlignFlags& af, const SearchHistoryVector& backHist); // Returns the string that corresponds to this overlap block. // This is constructed by transforming the original string using the back // history while correcting for the reverse-complement searches std::string getOverlapString(const std::string& original) const; // Get the full string that is indicated by this overlap // The overlap cannot be a containment and it must have a // forward extension history. The returned string is the string // of the actual read that forms the overlap with original. In other words // it might not be the same strand as the original read. std::string getFullString(const std::string& original) const; // Return a pointer to the BWT that should be used to extend the block // this is the opposite BWT that was used in the backwards search const BWT* getExtensionBWT(const BWT* pBWT, const BWT* pRevBWT) const; // Return the spectrum of extensions given by the interval in ranges // The counts are given in the canonical frame, which means that // if the query string was reversed, we flip the counts AlphaCount64 getCanonicalExtCount(const BWT* pBWT, const BWT* pRevBWT) const; // Return the index of the interval corresponding to the frame of // reference for the original read int getCanonicalIntervalIndex() const; // Return the canonical interval. BWTInterval getCanonicalInterval() const; // Return the direction of the edge that this overlap block describes EdgeDir getEdgeDir() const; // Construct an overlap record from this overlap block Overlap toOverlap(const std::string queryID, const std::string targetID, int queryLen, int targetLen) const; // Construct an ID string describing this overlap block std::string toCanonicalID() const; // Comparison operator, compare by lower coordinate of 0 friend bool operator<(const OverlapBlock& a, const OverlapBlock& b) { return a.ranges.interval[0].lower < b.ranges.interval[0].lower; } static bool sortSizeDescending(const OverlapBlock& ob1, const OverlapBlock& ob2) { return ob1.overlapLen > ob2.overlapLen; } static bool sortIntervalLeft(const OverlapBlock& ob1, const OverlapBlock& ob2) { return ob1.ranges.interval[0].lower < ob2.ranges.interval[0].lower; } // I/O friend std::ostream& operator<<(std::ostream& out, const OverlapBlock& obl) { out << obl.ranges << " " << obl.rawRanges << " " << obl.overlapLen << " " << obl.numDiff << " " << obl.flags; return out; } friend std::istream& operator>>(std::istream& in, OverlapBlock& obl) { in >> obl.ranges >> obl.rawRanges >> obl.overlapLen >> obl.numDiff >> obl.flags; return in; } // Data // The ranges member holds the suffix array interval of the overlapping // string + the terminating $ symbol. This allows us to either find the extension // of the overlap (using the reverse interval) or lookup the index of the read. BWTIntervalPair ranges; // The raw interval of the overlap, not capped with '$' symbols is required // to recompute the forward/reverse intervals if two overlap blocks intersect. // This can happen when two reads/sequences have multiple possible overlaps BWTIntervalPair rawRanges; int overlapLen; int numDiff; AlignFlags flags; bool isEliminated; // The sequence of divergent bases during the backward and forward search steps SearchHistoryVector backHistory; SearchHistoryVector forwardHistory; }; // Collections typedef std::list OverlapBlockList; typedef OverlapBlockList::iterator OBLIter; // Global Functions void printBlockList(const OverlapBlockList* pList); // Ensure all the overlap blocks in the list are distinct void removeSubMaximalBlocks(OverlapBlockList* pList, const BWT* pBWT, const BWT* pRevBWT); // Given the overlapping blocks A and B, construct a list of blocks where the index ranges do not intersect OverlapBlockList resolveOverlap(const OverlapBlock& A, const OverlapBlock& B, const BWT* pBWT, const BWT* pRevBWT); // Partition the overlap block list into two lists, // one for the containment overlaps and one for the proper overlaps void partitionBlockList(int readLen, OverlapBlockList* pCompleteList, OverlapBlockList* pOverlapList, OverlapBlockList* pContainList); // Remove containment blocks from the list void removeContainmentBlocks(int readLen, OverlapBlockList* pList); // Convert an overlap block list into a multiple overlap MultiOverlap blockListToMultiOverlap(const SeqRecord& record, OverlapBlockList& blockList); // std::string makeIdxString(int64_t idx); #endif ================================================ FILE: src/SGA/Algorithm/OverlapTools.cpp ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // OverlapTools - Wrapper for the overlap machinery // to perform an overlap computation for two strings // #include "OverlapTools.h" #include "SuffixArray.h" //#define DPOVERLAPPRINT 1 // Find a suffix of s1 that matches a prefix of s2 with minimal edit distance bool OverlapTools::boundedOverlapDP(const std::string& s1, const std::string& s2, int minOverlap, int maxOverlap, double maxErrorRate, Match& outMatch) { const size_t MAX_REGION_LENGTH = 500; // do not try to align strings that are longer than this number if(s1.empty() || s2.empty()) return false; if(maxOverlap > (int)MAX_REGION_LENGTH) { std::cerr << "Maximum requested overlap " << maxOverlap << " is larger than search limit (" << MAX_REGION_LENGTH << "), not aligning\n"; return false; } // The dynamic programming is too heavy to compute matches for very large strings // so we only try to align the ends of s1 and s2 up to MAX_REGION_LENGTH bases. size_t sublen = min3(maxOverlap, s1.size(), s2.size()); if(sublen > MAX_REGION_LENGTH) return false; std::string sub1 = s1.substr(s1.size() - sublen); std::string sub2 = s2.substr(0, sublen); DPAlignment dpAlign(sub1, sub2, DPM_OVERLAP, DPSS_SIMILARITY); Match match; bool validMatch = findBestOverlapByScore(sub1, sub2, minOverlap, maxErrorRate, dpAlign, match); if(validMatch) { // If we are matching a substring of both s1 and s2, and the match // is a containment (one string was completely matched) we call the // match invalid. if(match.isContainment() && sublen < s1.size() && sublen < s2.size()) { return false; } else { // A valid match has been found which is maximal // Expand the matches to the coordinates of s1 and s2 and return int s1_add = s1.size() - sublen; match.coord[0].interval.start += s1_add; match.coord[0].interval.end += s1_add; match.coord[0].seqlen = s1.size(); match.coord[1].seqlen = s2.size(); #ifdef DPOVERLAPPRINT match.printMatch(s1, s2); #endif outMatch = match; return true; } } else { return false; } } // bool OverlapTools::findBestOverlapByScore(const std::string& s1, const std::string& s2, int minOverlap, double maxErrorRate, const DPAlignment& dpAlign, Match& outMatch) { assert(dpAlign.getScoringScheme() == DPSS_SIMILARITY); // The last row of the table holds the edit distance for the prefix-suffix overlaps of S1 // Iterate over this row and calculate the length and edit % of each overlap int overlapRowIdx = dpAlign.getNumRows() - 1; int numCols = dpAlign.getNumColumns(); int bestOverlapLen = -1; int bestScore = 0; for(int j = minOverlap; j < numCols; ++j) { int overlapLen = j; // Do not allow overlaps to end with insertions // For example, this case is a valid alignment but would be disallowed: // S1 AGTACATTTACA-- // S2 ACATTTACATA // This is an overlap of length 11 but the "true" overlap is only 9 bases // Under a reasonable scoring system this should not happen DPOperation lastOp = dpAlign.getPathOperationToCell(s1, s2, overlapRowIdx, j); if(lastOp != DPO_INSERT) { int score = dpAlign.getScore(overlapRowIdx,j); if(score > bestScore) { // A higher scoring overlap has been found. Make sure that it is valid // by checking that the error rate and overlap length are within the expected // counts and that the coordinates are extreme w.r.t both sequences int i_match = 0; int num_edits = 0; DPPath overlapPath = dpAlign.calculatePath(s1, s2, overlapRowIdx, overlapLen); for(DPPath::iterator iter = overlapPath.begin(); iter != overlapPath.end(); ++iter) { if(iter->j == 0 && iter->i > i_match) i_match = iter->i; // Count the mismatches but don't count deletions in the first column which are "free" if(iter->op != DPO_MATCH && !(iter->j == 0 && iter->op == DPO_DELETE)) ++num_edits; } Match currMatch; currMatch.isReverse = false; currMatch.coord[0].interval.start = i_match; currMatch.coord[0].interval.end = overlapRowIdx - 1; // match coords are inclusive currMatch.coord[0].seqlen = s1.size(); currMatch.coord[1].interval.start = 0; currMatch.coord[1].interval.end = overlapLen - 1; currMatch.coord[1].seqlen = s2.size(); currMatch.setNumDiffs(num_edits); double errorRate = (double)num_edits / currMatch.getMinOverlapLength(); bool isValid = errorRate < maxErrorRate && currMatch.getMinOverlapLength() >= minOverlap && currMatch.coord[0].isExtreme() && currMatch.coord[1].isExtreme(); if(isValid) { bestOverlapLen = overlapLen; bestScore = score; outMatch = currMatch; } } /* std::cout << "\n"; dpAlign.printAlignment(s1, s2, overlapRowIdx, j); std::cout << "OL: " << overlapLen << "\n"; std::cout << "LO: " << (int)lastOp << " ED: " << score << "\n"; */ } } if(bestOverlapLen > 0) { #ifdef DPOVERLAPPRINT std::cout << "\nBest overlap: " << bestOverlapLen << "\n"; dpAlign.printAlignment(s1, s2, overlapRowIdx, bestOverlapLen); outMatch.printMatch(s1, s2); #endif return true; } else { return false; } } ================================================ FILE: src/SGA/Algorithm/OverlapTools.h ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // OverlapTools - Wrapper for the overlap machinery // to perform an overlap computation for two strings // #ifndef OVERLAPTOOLS_H #define OVERLAPTOOLS_H #include "Match.h" #include "DPAlignment.h" #include namespace OverlapTools { // Datatypes typedef std::vector IntVector; typedef std::vector DPMatrix; //typedef EditDistanceScoring DPScoring; typedef SimilarityScoring DPScoring; // Find a match between s1 and s2 using a dynamic programming alignment // This is something of a heavy function and the longest possible match must be provided // If there is a match between minOverlap and maxOverlap with edit distance less than maxErrorRate, it will be returned. // If there are multiple valid matches like this, the highest scoring match will be returned under the DPSS_SIMILARITY // scoring scheme. If true is returned, a valid match has been found and outMatch is filled in appropriately. // This function should not be used to find overlaps greater than 500bp bool boundedOverlapDP(const std::string& s1, const std::string& s2, int minOverlap, int maxOverlap, double maxErrorRate, Match& outMatch); // Find the best overlap using the dpAlign structure. Returns true // if a valid overlap has been found and fills in the match structure bool findBestOverlapByScore(const std::string& s1, const std::string& s2, int minOverlap, double maxErrorRate, const DPAlignment& dpAlign, Match& outMatch); }; #endif ================================================ FILE: src/SGA/Algorithm/QCProcess.cpp ================================================ ///----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // QCProcess - Process to perform quality checks // for a sequence work item // #include "QCProcess.h" #include "BWTAlgorithms.h" // struct KmerWindow { int64_t getCount(bool bothStrand) const { int64_t fwdCount = (fwdIntervals.interval[0].isValid()) ? fwdIntervals.interval[0].size() : 0; int64_t rcCount = (rcIntervals.interval[0].isValid()) ? rcIntervals.interval[0].size() : 0; if (bothStrand) return std::min(fwdCount,rcCount); else return fwdCount + rcCount; } int start; int end; BWTIntervalPair fwdIntervals; BWTIntervalPair rcIntervals; bool isInitialized; }; // // // QCProcess::QCProcess(QCParameters params) : m_params(params) { } // QCProcess::~QCProcess() { } // QCResult QCProcess::process(const SequenceWorkItem& workItem) { QCResult result; if(m_params.checkDuplicates) { DuplicateCheckResult dupCheckResult = performDuplicateCheck(workItem); if(!m_params.substringOnly) result.dupPassed = dupCheckResult == DCR_UNIQUE; else result.dupPassed = dupCheckResult != DCR_SUBSTRING; } else { result.dupPassed = true; } // Only perform the more expensive k-mer test if the dup check succeeded if(m_params.checkKmer && result.dupPassed) result.kmerPassed = performKmerCheck(workItem); else result.kmerPassed = true; if(result.kmerPassed && result.dupPassed && m_params.checkHPRuns) result.hpPassed = performHomopolymerCheck(workItem); else result.hpPassed = true; if(m_params.checkDegenerate && result.dupPassed && result.kmerPassed && result.hpPassed) result.degenPassed = performDegenerateCheck(workItem); else result.degenPassed = true; return result; } bool QCProcess::performKmerCheck(const SequenceWorkItem& workItem) { // Perform a k-mer filter on the read // Each k-mer must be seen at least m times for the read to be kept // Naively, this would required k*(l - k + 1) calls to the BWT // if each k-mer was computed independently. We improve this by performing // a two-stage test. If the kmer k_i is seen at least m times, we extend // it by one base. If this k+1-mer is also seen at least m times, it implies // that the kmer k_(i+1) is also seen at least m times so it does not need // to be recomputed. If the (k+1)-mer is seen less than m times, we recompute // kmer k_(i+1) as a final check. Using this optimistic algorithm (checking // longer kmers than is required) is significantly faster. QCResult result; std::string w = workItem.read.seq.toString(); // Ensure the read is longer than the k-mer length if((int)w.size() < m_params.kmerLength) return false; int k = m_params.kmerLength; int n = w.size(); int nk = n - k + 1; int threshold = m_params.kmerThreshold; // Are all kmers in the read well-represented? bool allSolid = true; int i = 0; KmerWindow window; window.start = 0; window.end = 0; window.isInitialized = false; while(i < nk) { // Determine whether the kmer at position i // has been seen more than m times. if(window.isInitialized) { // Extend the current window by 1 base // to see if the spanning larger // kmer is seen more than m times. // If so, we don't need to check this kmer // individually int next = window.end + k; assert(next < (int)w.size()); char b = w[next]; char cb = complement(b); if(window.fwdIntervals.interval[0].isValid()) BWTAlgorithms::updateBothR(window.fwdIntervals, b, m_params.pRevBWT); if(window.rcIntervals.interval[1].isValid()) BWTAlgorithms::updateBothR(window.rcIntervals, cb, m_params.pBWT); int64_t count = window.getCount(m_params.kmerBothStrand); if(count <= threshold) { // The extended kmer didn't meet the threshold // recompute the interval for this kmer window.isInitialized = false; } else { window.end += 1; } } if(!window.isInitialized) { // initialize the window by computing the // BWTIntervals for the kmer starting at // i and its reverse complement char b = w[i]; char cb = complement(b); BWTAlgorithms::initIntervalPair(window.fwdIntervals, b, m_params.pBWT, m_params.pRevBWT); BWTAlgorithms::initIntervalPair(window.rcIntervals, cb, m_params.pRevBWT, m_params.pBWT); for(int j = i + 1; j < i + k; ++j) { // Update intervals rightwards b = w[j]; cb = complement(b); if(window.fwdIntervals.interval[0].isValid()) BWTAlgorithms::updateBothR(window.fwdIntervals, b, m_params.pRevBWT); if(window.rcIntervals.interval[1].isValid()) BWTAlgorithms::updateBothR(window.rcIntervals, cb, m_params.pBWT); } // record the start/end indices of the kmers spanned by this position window.start = i; window.end = i; window.isInitialized = true; } // Check the count of this interval int64_t count = window.getCount(m_params.kmerBothStrand); if(count <= threshold) { allSolid = false; break; } else { i += 1; } } return allSolid; } // Perform duplicate check // Look up the interval of the read in the BWT. If the index of the read DuplicateCheckResult QCProcess::performDuplicateCheck(const SequenceWorkItem& workItem) { assert(m_params.pSharedBV != NULL); std::string w = workItem.read.seq.toString(); std::string rc_w = reverseComplement(w); // Look up the interval of the sequence and its reverse complement BWTIntervalPair fwdIntervals = BWTAlgorithms::findIntervalPair(m_params.pBWT, m_params.pRevBWT, w); BWTIntervalPair rcIntervals = BWTAlgorithms::findIntervalPair(m_params.pBWT, m_params.pRevBWT, rc_w); // Check if this read is a substring of any other // This is indicated by the presence of a non-$ extension in the left or right direction AlphaCount64 fwdECL = BWTAlgorithms::getExtCount(fwdIntervals.interval[0], m_params.pBWT); AlphaCount64 fwdECR = BWTAlgorithms::getExtCount(fwdIntervals.interval[1], m_params.pRevBWT); AlphaCount64 rcECL = BWTAlgorithms::getExtCount(rcIntervals.interval[0], m_params.pBWT); AlphaCount64 rcECR = BWTAlgorithms::getExtCount(rcIntervals.interval[1], m_params.pRevBWT); if(fwdECL.hasDNAChar() || fwdECR.hasDNAChar() || rcECL.hasDNAChar() || rcECR.hasDNAChar()) { // Substring reads are always removed so no need to update the bit vector return DCR_SUBSTRING; } // Calculate the lexicographic intervals for the fwd and reverse intervals BWTAlgorithms::updateBothL(fwdIntervals, '$', m_params.pBWT); BWTAlgorithms::updateBothL(rcIntervals, '$', m_params.pBWT); // Calculate the canonical index for this string - the lowest // value in the two lexicographic index assert(fwdIntervals.interval[0].isValid() || rcIntervals.interval[0].isValid()); int64_t fi = fwdIntervals.interval[0].isValid() ? fwdIntervals.interval[0].lower : std::numeric_limits::max(); int64_t ri = rcIntervals.interval[0].isValid() ? rcIntervals.interval[0].lower : std::numeric_limits::max(); int64_t canonicalIdx = std::min(fi, ri); // Check if the bit reprsenting the canonical index is set in the shared bit vector if(!m_params.pSharedBV->test(canonicalIdx)) { // This read is not a duplicate // Attempt to atomically set the bit from false to true if(m_params.pSharedBV->updateCAS(canonicalIdx, false, true)) { // Call succeed, return that this read is not a duplicate return DCR_UNIQUE; } else { // Call failed, some other thread set the bit before // this thread. Return that the reead is a duplicate return DCR_FULL_LENGTH_DUPLICATE; } } else { // this read is duplicate return DCR_FULL_LENGTH_DUPLICATE; } } // Perform homopolymer filter bool QCProcess::performHomopolymerCheck(const SequenceWorkItem& item) { std::string w = item.read.seq.toString(); size_t k = m_params.hpKmerLength; // Skip if the read length is less than the kmer size // used for this test if(w.size() < k) return true; size_t maxRunLength = 0; size_t maxRunStart = 0; // Count the longest homopolymer run in the read size_t currRunStart = 0; size_t currRunLength = 1; char prev = w[0]; char runChar = prev; for(size_t i = 1; i < w.size(); ++i) { if(w[i] == prev) currRunLength += 1; if(w[i] != prev || i == w.size() - 1) { if(currRunLength > maxRunLength) { maxRunLength = currRunLength; maxRunStart = currRunStart; runChar = prev; } currRunLength = 1; prev = w[i]; currRunStart = i; } } // If the run length is above the threshold, find // the frequency of the covering k-mer and k-mers with // different lengths of the homopolymer if(maxRunLength >= m_params.hpMinLength && maxRunLength < k / 2) { // Extract the kmer covering this run int hprMiddle = maxRunStart + (maxRunLength / 2); int estimatedKmerStart = hprMiddle - (k / 2); int trueKmerStart = estimatedKmerStart; // If the calculated start position of the covering k-mer //is before the start of the read, or past the end, shift it if(estimatedKmerStart < 0) trueKmerStart = 0; if(estimatedKmerStart + k > w.size()) trueKmerStart = w.size() - k; std::string prefix = w.substr(trueKmerStart, maxRunStart - trueKmerStart); std::string suffix = w.substr(maxRunStart + maxRunLength, trueKmerStart + k - (maxRunStart + maxRunLength)); // Do not attempt this check if we do not have enough context preceding or following the HPR if(prefix.size() < m_params.hpMinContext || suffix.size() < m_params.hpMinContext) return true; size_t highestCountLength = 0; size_t highestCount = 0; size_t actualCount = 0; for(size_t l = maxRunLength - 2; l <= maxRunLength + 2; ++l) { std::string composite = prefix + std::string(l, runChar) + suffix; size_t count = BWTAlgorithms::countSequenceOccurrences(composite, m_params.pBWT); if(l == maxRunLength) actualCount = count; if(count > highestCount) { highestCount = count; highestCountLength = l; } } double proportion = (double)actualCount / (double)highestCount; if(highestCountLength == maxRunLength || actualCount >= m_params.hpHardAcceptCount || proportion >= m_params.hpMinProportion) { return true; } else { if(m_params.verbose > 0) { printf("Read failed homopolymer filter %s\n", w.c_str()); printf("Filtered read with poly-%c run. DL: %zu DC: %zu. AL: %zu AC: %zu P: %lf\n", runChar, highestCountLength, highestCount, maxRunLength, actualCount, proportion); } return false; } } return true; } // Check if sequence is composed of predominantely a single base // Returns true if the sequence is not degenrate bool QCProcess::performDegenerateCheck(const SequenceWorkItem& item) { std::string w = item.read.seq.toString(); AlphaCount64 bc; for(size_t i = 0; i < w.size(); ++i) { bc.increment(w[i]); } size_t maxCount = bc.getMaxCount(); double prop = (double)maxCount / w.size(); if(prop > m_params.degenProportion) { if(m_params.verbose > 0) std::cout << "Read " << w << " failed degenerate filter\n"; return false; } return true; } // // // QCPostProcess::QCPostProcess(std::ostream* pCorrectedWriter, std::ostream* pDiscardWriter) : m_pCorrectedWriter(pCorrectedWriter), m_pDiscardWriter(pDiscardWriter), m_readsKept(0), m_readsDiscarded(0), m_readsFailedKmer(0), m_readsFailedDup(0), m_readsFailedHP(0), m_readsFailedDegen(0) { } // QCPostProcess::~QCPostProcess() { std::cout << "Reads kept: " << m_readsKept << "\n"; std::cout << "Reads discarded: " << m_readsDiscarded << "\n"; std::cout << "Reads failed kmer check: " << m_readsFailedKmer << "\n"; std::cout << "Reads failed duplicate check: " << m_readsFailedDup << "\n"; std::cout << "Reads failed homopolymer check: " << m_readsFailedHP << "\n"; std::cout << "Reads failed degenerate check: " << m_readsFailedDegen << "\n"; } // void QCPostProcess::process(const SequenceWorkItem& item, const QCResult& result) { SeqRecord record = item.read; if(result.kmerPassed && result.dupPassed && result.hpPassed && result.degenPassed) { record.write(*m_pCorrectedWriter); ++m_readsKept; } else { // To be able to rebuild the index after discarding the read, we need to write // the rank of the string (its position in the original read file into the read name) std::stringstream newID; newID << item.read.id << ",seqrank=" << item.idx; record.id = newID.str(); record.write(*m_pDiscardWriter); ++m_readsDiscarded; if(!result.kmerPassed) m_readsFailedKmer += 1; else if(!result.dupPassed) m_readsFailedDup += 1; else if(!result.hpPassed) m_readsFailedHP += 1; else if(!result.degenPassed) m_readsFailedDegen += 1; } } ================================================ FILE: src/SGA/Algorithm/QCProcess.h ================================================ ///----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // QCProcess - Process to perform quality checks // for a sequence work item // #ifndef QCPROCESS_H #define QCPROCESS_H #include "Util.h" #include "BWT.h" #include "SequenceProcessFramework.h" #include "SequenceWorkItem.h" #include "BitVector.h" // Parameters struct QCParameters { QCParameters() { setDefaults(); } // Set reasonable default values for the qc filters void setDefaults() { checkDuplicates = true; checkKmer = true; kmerBothStrand = false; checkHPRuns = true; checkDegenerate = true; verbose = 0; pBWT = NULL; pRevBWT = NULL; pSharedBV = NULL; kmerLength = 27; kmerThreshold = 2; hpHardAcceptCount = 10; hpMinProportion = 0.1f; hpKmerLength = 51; hpMinLength = 6; hpMinContext = 5; degenProportion = 0.90; } const BWT* pBWT; const BWT* pRevBWT; BitVector* pSharedBV; // Control parameters bool checkDuplicates; bool checkKmer; bool kmerBothStrand; bool checkHPRuns; bool checkDegenerate; bool substringOnly; int verbose; // // Kmer frequency filtering parameters // int kmerLength; int kmerThreshold; // // Homopolymer filter parameters // // The length a run must be to trigger filtering size_t hpMinLength; // If the k-mer covering the HP run is seen // at least this many times, it is accepted size_t hpHardAcceptCount; // The hp run length in the read must be // at least this fraction of the dominant // run length to be kept double hpMinProportion; // The context k-mer length for the homopolymer // filter. size_t hpKmerLength; // The minimum amount of sequence around the homopolymer // to trigger filtering size_t hpMinContext; // // Degenerate filter parameters // // If the most frequent base in the read // makes up more than this proportion, the read // is discarded double degenProportion; }; // Results object class QCResult { public: QCResult() : kmerPassed(false), dupPassed(false), hpPassed(false), degenPassed(false) {} bool kmerPassed; bool dupPassed; bool hpPassed; bool degenPassed; }; enum DuplicateCheckResult { DCR_UNIQUE, DCR_SUBSTRING, DCR_FULL_LENGTH_DUPLICATE }; // Perform quality checks on the input stream of reads class QCProcess { public: QCProcess(QCParameters params); ~QCProcess(); QCResult process(const SequenceWorkItem& item); // Discard reads with low-frequency kmers bool performKmerCheck(const SequenceWorkItem& item); // Discard reads that are identical to, or a substring of, some other read DuplicateCheckResult performDuplicateCheck(const SequenceWorkItem& item); // Check whether the sequence has a homopolymer sequencing error. This // check finds a kmer covering a homopolymer run then modifies it to check // for a shorter/longer run of higher frequency bool performHomopolymerCheck(const SequenceWorkItem& item); // Check if the sequence read is degenerate // A degenerate read is define to be reads consisting almost // entirely of a single nucleotide bool performDegenerateCheck(const SequenceWorkItem& item); private: const QCParameters m_params; }; // Write the results from the overlap step to an ASQG file class QCPostProcess { public: QCPostProcess(std::ostream* pCorrectedWriter, std::ostream* pDiscardWriter); ~QCPostProcess(); void process(const SequenceWorkItem& item, const QCResult& result); private: std::ostream* m_pCorrectedWriter; std::ostream* m_pDiscardWriter; size_t m_readsKept; size_t m_readsDiscarded; size_t m_readsFailedKmer; size_t m_readsFailedDup; size_t m_readsFailedHP; size_t m_readsFailedDegen; }; #endif ================================================ FILE: src/SGA/Algorithm/ReadCluster.cpp ================================================ ///----------------------------------------------- // Copyright 2011 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // ReadCluster - Generate a cluster of overlapping // reads using the FM-index // #include "ReadCluster.h" // ReadCluster::ReadCluster(const OverlapAlgorithm* pOverlapper, int minOverlap) : m_pOverlapper(pOverlapper), m_minOverlap(minOverlap), m_pLimitKmers(NULL), m_limitK(0) { } // void ReadCluster::setLimitKmers(const std::set* pLimitKmers, size_t limitK) { m_pLimitKmers = pLimitKmers; m_limitK = limitK; } // Add a seed read to the cluster. Overlaps will be found for // each seed read to grow the cluster ClusterNode ReadCluster::addSeed(const std::string& sequence, bool bCheckInIndex) { // Check if this read is a substring SeqRecord tempRecord; tempRecord.id = "cluster-seed"; tempRecord.seq = sequence; OverlapBlockList tempBlockList; OverlapResult overlapResult = m_pOverlapper->alignReadDuplicate(tempRecord, &tempBlockList); if(overlapResult.isSubstring) { // If bCheckInIndex is true, then we are extending clusters from reads that are in the FM-index // In this case, seeding a substring read is an error and we abort. If the seed is NOT in the index // we just emit a warning and ignore the seed if(bCheckInIndex) { std::cerr << "Error: The seed used for sga-cluster-extend is a substring of some read.\n"; std::cerr << "Please run sga rmdup before cluster\n"; std::cerr << "Sequence: " << sequence << "\n"; exit(1); } else { std::cerr << "Warning: The cluster sequence to extend is a substring of some read. This seed is being skipped\n"; std::cerr << "Sequence: " << sequence << "\n"; ClusterNode emptyNode; // Set an invalid interval emptyNode.interval.lower = 0; emptyNode.interval.upper = -1; return emptyNode; } } // Find the interval in the fm-index containing the read const BWT* pBWT = m_pOverlapper->getBWT(); BWTInterval readInterval = BWTAlgorithms::findInterval(pBWT, sequence); BWTAlgorithms::updateInterval(readInterval, '$', pBWT); // When building primary clusters, we require each read to be in the index. if(bCheckInIndex) { if(!readInterval.isValid()) { std::cerr << "sga cluster error: The seed read is not part of the FM-index.\n"; exit(EXIT_FAILURE); } } ClusterNode node; node.sequence = sequence; node.interval = readInterval; node.isReverseInterval = false; m_usedIndex.insert(readInterval.lower); m_queue.push(node); return node; } // Run the cluster process. If the number of total nodes // exceeds max, abort the search. void ReadCluster::run(size_t max_size, int max_iterations) { int iteration = 0; do { // Perform one iteration of extending each sequence in the queue ClusterNodeQueue next_queue; bool aborted = false; // Have we performed enough iterations? if(max_iterations > 0 && iteration++ > max_iterations) aborted = true; while(!aborted && !m_queue.empty()) { if(m_queue.size() + m_outCluster.size() > max_size) { // Abort the search m_outCluster.clear(); aborted = true; break; } ClusterNode node = m_queue.front(); m_queue.pop(); // Add this node to the output m_outCluster.push_back(node); // If we are using limit kmers, only try to extend this sequence if it does not contain a limit kmer bool extend_read = canExtendRead(node); if(!extend_read) continue; // Find overlaps for the current node SeqRecord tempRecord; tempRecord.id = "cluster"; tempRecord.seq = node.sequence; OverlapBlockList blockList; m_pOverlapper->overlapRead(tempRecord, m_minOverlap, &blockList); // Parse each member of the block list and potentially expand the cluster for(OverlapBlockList::const_iterator iter = blockList.begin(); iter != blockList.end(); ++iter) { // Check if the reads in this block are part of the cluster already BWTInterval canonicalInterval = iter->getCanonicalInterval(); int64_t canonicalIndex = canonicalInterval.lower; if(m_usedIndex.count(canonicalIndex) == 0) { // This is a new node that isn't in the cluster. Add it. m_usedIndex.insert(canonicalIndex); ClusterNode newNode; newNode.sequence = iter->getFullString(node.sequence); newNode.interval = canonicalInterval; newNode.isReverseInterval = iter->flags.isTargetRev(); next_queue.push(newNode); } } } // Clear the queue if(aborted) { while(!m_queue.empty()) m_queue.pop(); } else { // Swap the reads to extend in the next iteration m_queue = next_queue; } } while(!m_queue.empty()); } // Check if the given node contains a kmer that we stop extension at. // Returns true if we can extend the cluster using the given node bool ReadCluster::canExtendRead(const ClusterNode& node) const { // No limiting kmers if(m_pLimitKmers == NULL) return true; // Node too short if(node.sequence.size() < m_limitK) return false; size_t nk = node.sequence.size() - m_limitK + 1; for(size_t i = 0; i < nk; ++i) { std::string kmer = node.sequence.substr(i, m_limitK); if(m_pLimitKmers->find(kmer) != m_pLimitKmers->end()) return false; // this read contains a limiting kmer // check reverse complement too kmer = reverseComplement(kmer); if(m_pLimitKmers->find(kmer) != m_pLimitKmers->end()) return false; // this read contains a limiting kmer } return true; } // ClusterNodeVector ReadCluster::getOutput() const { // Sort the intervals into ascending order and remove any duplicate intervals (which can occur // if the subgraph has a simple cycle) ClusterNodeVector retVector = m_outCluster; std::sort(retVector.begin(), retVector.end(), ClusterNode::compare); std::vector::iterator newEnd = std::unique(retVector.begin(), retVector.end(), ClusterNode::equal); retVector.erase(newEnd, retVector.end()); return retVector; } ================================================ FILE: src/SGA/Algorithm/ReadCluster.h ================================================ ///----------------------------------------------- // Copyright 2011 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // ReadCluster - Generate a cluster of overlapping // reads using the FM-index // #ifndef READCLUSTER_H #define READCLUSTER_H #include "Util.h" #include "OverlapAlgorithm.h" // A node in the queue of reads to use to expand the cluster struct ClusterNode { std::string sequence; int64_t fwdCanonicalID; BWTInterval interval; bool isReverseInterval; static inline bool compare(const ClusterNode& a, const ClusterNode& b) { return BWTInterval::compare(a.interval, b.interval); } static inline bool equal(const ClusterNode& a, const ClusterNode& b) { return BWTInterval::equal(a.interval, b.interval); } }; typedef std::queue ClusterNodeQueue; typedef std::vector ClusterNodeVector; typedef std::set ClusterIntervalSet; // class ReadCluster { public: // ReadCluster(const OverlapAlgorithm* pOverlapper, int minOverlap); // To avoid extending the cluster through repetitive sequence, the extension // process can optionally take in kmers that we use to block extension. If a // read contains a kmer in this set, we do not extend the cluster using the read. void setLimitKmers(const std::set* pLimitKmers, size_t limitK); // Add a new sequence to extend the cluster from ClusterNode addSeed(const std::string& sequence, bool bCheckInIndex); // Run the cluster process. If the number of total nodes // exceeds max, abort the search. void run(size_t max_size, int max_iterations); // ClusterNodeVector getOutput() const; private: // Check if the sequence of the given node contains a limit kmer, which blocks extension bool canExtendRead(const ClusterNode& node) const; ClusterNodeQueue m_queue; const OverlapAlgorithm* m_pOverlapper; int m_minOverlap; const std::set* m_pLimitKmers; size_t m_limitK; ClusterNodeVector m_outCluster; ClusterIntervalSet m_usedIndex; }; #endif ================================================ FILE: src/SGA/Algorithm/SearchHistory.cpp ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL // // SearchHistory - data structures to track // the history of a FM-index search // //----------------------------------------------- #include "SearchHistory.h" #include #include // // Link // SearchHistoryLink::SearchHistoryLink() : pNode(NULL) { } // SearchHistoryLink::SearchHistoryLink(SearchHistoryNode* ptr) : pNode(ptr) { if(pNode != NULL) pNode->increment(); } // We need to handle the copy constructor and the assignment operator // for the reference counting to be correct SearchHistoryLink::SearchHistoryLink(const SearchHistoryLink& link) : pNode(link.pNode) { if(pNode != NULL) pNode->increment(); } // SearchHistoryLink& SearchHistoryLink::operator=(SearchHistoryLink const& link) { SearchHistoryNode* pOld = pNode; pNode = link.pNode; if(pNode != NULL) pNode->increment(); if(pOld != NULL) { pOld->decrement(); if(pOld->getCount() == 0) delete pOld; } return *this; } // SearchHistoryLink::~SearchHistoryLink() { if(pNode != NULL) { pNode->decrement(); if(pNode->getCount() == 0) delete pNode; } } // // Node // // adding a child of the node automatically increments the refCount of this node // through the child's Link to this node. Once all the children of this node // have been removed it will automatically be deleted SearchHistoryLink SearchHistoryNode::createChild(int var_pos, char var_base) { return SearchHistoryLink(new SearchHistoryNode(this, var_pos, var_base)); } // The root has NULL as a parent SearchHistoryLink SearchHistoryNode::createRoot() { return SearchHistoryLink(new SearchHistoryNode(NULL, -1, ROOT_CHAR)); } // Return the search history up to the root node SearchHistoryVector SearchHistoryNode::getHistoryVector() { SearchHistoryVector out; SearchHistoryLink pCurr(this); bool done = false; while(!done) { if(pCurr->m_variant.base == ROOT_CHAR) { // root found, terminate the search done = true; } else { out.add(pCurr->m_variant); pCurr = pCurr->m_parentLink; } } return out; } // // SearchHistoryVector // // Normalize the search history so that the positions are in ascending (left to right) // order and the bases are wrt. the original strand of the forward sequence // This is required to compare two SearchHistories that represent alignments to different // strands void SearchHistoryVector::normalize(bool doComplement) { std::sort(m_history.begin(), m_history.end(), SearchHistoryItem::sortPos); if(doComplement) { for(size_t i = 0; i < m_history.size(); ++i) { m_history[i].base = complement(m_history[i].base); } } } // void SearchHistoryVector::add(int pos, char base) { SearchHistoryItem item(pos, base); m_history.push_back(item); } // void SearchHistoryVector::add(SearchHistoryItem& item) { m_history.push_back(item); } // int SearchHistoryVector::countDifferences(const SearchHistoryVector& a, const SearchHistoryVector& b, int maxPos) { size_t na = a.m_history.size(); size_t nb = b.m_history.size(); size_t i = 0; size_t j = 0; int count = 0; while(i < na && j < nb) { const SearchHistoryItem& itemA = a.m_history[i]; const SearchHistoryItem& itemB = b.m_history[j]; if(itemA.pos > maxPos || itemB.pos > maxPos) break; if(itemA.pos == itemB.pos) { if(itemA.base != itemB.base) ++count; ++i; ++j; } else if(itemA.pos < itemB.pos) { ++count; ++i; } else if(itemB.pos < itemA.pos) { ++count; ++j; } else { assert(false); } } // Count the remaining elements in A and B that are less than maxpos while(i < na && a.m_history[i].pos <= maxPos) { ++count; ++i; } // Count the remaining elements in A and B that are less than maxpos while(j < nb && b.m_history[j].pos <= maxPos) { ++count; ++j; } return count; } // Transform the original string using the history std::string SearchHistoryVector::transform(const std::string& original, bool queryReversed) const { std::string transformed = original; int l = transformed.size(); for(size_t i = 0; i < m_history.size(); ++i) { const SearchHistoryItem& item = m_history[i]; // Transform the position onto the read - if the queryReversed flag // is set, the numbering starts from the left at one int t_pos = (queryReversed) ? item.pos - 1 : l - item.pos; assert(t_pos >= 0 && t_pos < l); assert(transformed[t_pos] != item.base); // pos is 1-based starting from the right side of original transformed[t_pos] = item.base; } return transformed; } // Return the string of bases that make up the history std::string SearchHistoryVector::getBaseString() const { std::string out; out.reserve(m_history.size()); for(size_t i = 0; i < m_history.size(); ++i) out.push_back(m_history[i].base); return out; } // std::ostream& operator<<(std::ostream& out, const SearchHistoryVector& hist) { std::copy(hist.m_history.begin(), hist.m_history.end(), std::ostream_iterator(out, "\t")); return out; } ================================================ FILE: src/SGA/Algorithm/SearchHistory.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL // // SearchHistory - data structures to track // the history of a FM-index search //----------------------------------------------- #ifndef SEARCHHISTORY_H #define SEARCHHISTORY_H #include "Util.h" // Base, Position pair indicating a divergence during the search struct SearchHistoryItem { SearchHistoryItem(int p, char b) : pos(p), base(b) {} int pos; char base; static bool sortPos(const SearchHistoryItem& a, const SearchHistoryItem& b) { return a.pos < b.pos; } friend std::ostream& operator<<(std::ostream& out, const SearchHistoryItem& hi) { out << hi.pos << "," << hi.base; return out; } }; typedef std::vector HistoryItemVector; // A vector of history items that can be compared with other histories class SearchHistoryVector { public: // SearchHistoryVector() {} // void add(int pos, char base); void add(SearchHistoryItem& item); void normalize(bool doComplement); size_t size() const { return m_history.size(); } std::string getBaseString() const; // Transform the original string using the history into a string representing // the sequence of bases searched to create the history std::string transform(const std::string& original, bool queryReversed) const; // static int countDifferences(const SearchHistoryVector& a, const SearchHistoryVector& b, int maxPos); // friend std::ostream& operator<<(std::ostream& out, const SearchHistoryVector& hist); private: HistoryItemVector m_history; }; class SearchHistoryNode; // A SearchHistoryLink is a reference-counted wrapper of a // search node. This is the external interface to the SearchHistoryNodes // This allows the SearchHistoryNodes to be automatically cleaned up when // they are no longer referred to class SearchHistoryLink { public: // SearchHistoryLink(); SearchHistoryLink(SearchHistoryNode* ptr); // We need to handle the copy constructor and the assignment operator // for the reference counting to be correct SearchHistoryLink(const SearchHistoryLink& link); SearchHistoryLink& operator=(SearchHistoryLink const& link); ~SearchHistoryLink(); SearchHistoryNode* operator->() const { assert(pNode != NULL); return pNode; } SearchHistoryNode& operator*() const { assert(pNode != NULL); return *pNode; } private: SearchHistoryNode* pNode; }; // A search history node is one link in a chain of history items. createChild makes a // new element in the chain, indicating a divergence from the parent history class SearchHistoryNode { public: SearchHistoryLink createChild(int var_pos, char var_base); static SearchHistoryLink createRoot(); // Create the root node of the history tree SearchHistoryVector getHistoryVector(); private: friend class SearchHistoryLink; friend class SearchTree; // The nodes should only be constructed/destructed through the links SearchHistoryNode(SearchHistoryNode* pParent, int var_pos, char var_base) : m_parentLink(pParent), m_variant(var_pos, var_base), m_refCount(0) {} ~SearchHistoryNode() { assert(m_refCount == 0); } inline void increment() { ++m_refCount; } inline void decrement() { --m_refCount; } inline int getCount() const { return m_refCount; } SearchHistoryLink m_parentLink; SearchHistoryItem m_variant; int m_refCount; static const char ROOT_CHAR = '0'; }; #endif ================================================ FILE: src/SGA/Algorithm/SearchSeed.cpp ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // SearchSeed.h - Data structure holding a partial // alignment to a FM-index. // #include #include "SearchSeed.h" // void SearchSeed::print() const { printf("li: %d ri: %d sl: %d dir: %d z: %d lrl: %d lru: %d rlr: %d rlu: %d\n", left_index, right_index, seed_len, dir, z, (int)ranges.interval[0].lower, (int)ranges.interval[0].upper, (int)ranges.interval[1].lower, (int)ranges.interval[1].upper); } // void SearchSeed::print(const std::string& w) const { int range = (int)ranges.interval[0].upper - (int)ranges.interval[0].lower; printf("sub: %s li: %d ri: %d sl: %d dir: %d z: %d lrl: %d lru: %d range: %d rlr: %d rlu: %d\n", w.substr(left_index, length()).c_str(), left_index, right_index, seed_len, dir, z, (int)ranges.interval[0].lower, (int)ranges.interval[0].upper, range, (int)ranges.interval[1].lower, (int)ranges.interval[1].upper); } ================================================ FILE: src/SGA/Algorithm/SearchSeed.h ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // SearchSeed.h - Data structure holding a partial // alignment to a FM-index. // #ifndef SEARCHSEED_H #define SEARCHSEED_H #include #include #include "BWTInterval.h" #include "SearchHistory.h" // types enum ExtendDirection { ED_LEFT, ED_RIGHT }; // A search seed describes a partial alignment to a BWT struct SearchSeed { // // Functions // inline int length() const { return right_index - left_index + 1; } inline bool isSeed() const { return length() < seed_len; } inline bool isIntervalValid(int idx) { return ranges.interval[idx].isValid(); } inline bool allowMismatches() const { return z < maxDiff; } inline double calcErrorRate() const { return static_cast(z) / static_cast(length()); } // Sort the alignments based on their r_lower/r_upper static inline bool compareLeftRange(const SearchSeed& a, const SearchSeed& b) { return BWTInterval::compare(a.ranges.interval[0], b.ranges.interval[0]); } // Compare for equality based on the left range // If the length of the alignment is equal, then if the left ranges // are a match, the two alignment objects are redundant and one can be removed static inline bool equalLeftRange(const SearchSeed& a, const SearchSeed& b) { #ifdef VALIDATE if(BWTInterval::equal(a.ranges.inteval[0], b.ranges.interval[0])) assert(a.length() == b.length() && a.z == b.z); #endif return BWTInterval::equal(a.ranges.interval[0], b.ranges.interval[0]); } friend bool operator==(const SearchSeed& a, const SearchSeed& b) { return a.ranges == b.ranges && a.left_index == b.left_index && a.right_index == b.right_index && a.z == b.z && a.dir == b.dir; } // void print() const; void print(const std::string& w) const; // // Data // // BWT interval coordinates, the first element of the pair is the left range BWTIntervalPair ranges; SearchHistoryLink historyLink; // Index range is inclusive on both ends int left_index; int right_index; int seed_len; // The direction the alignment is currently being extended ExtendDirection dir; // The current number of mismatches in the block int z; // The total number of allowed mismatches int maxDiff; }; // Collections typedef std::vector SearchSeedVector; typedef std::queue SearchSeedQueue; #endif ================================================ FILE: src/SGA/Algorithm/StatsProcess.cpp ================================================ ///----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // StatsProcess - Compute statistics about the reads // #include "StatsProcess.h" #include "BWTAlgorithms.h" #include "MultiOverlap.h" // // // StatsProcess::StatsProcess(const BWT* pBWT, const BWT* pRBWT, int kmerLength, int minOverlap, int branchCutoff, bool bNoOverlap) : m_pBWT(pBWT), m_pRBWT(pRBWT), m_kmerLength(kmerLength), m_minOverlap(minOverlap), m_branchCutoff(branchCutoff), m_bNoOverlap(bNoOverlap), m_pAllOverlapper(NULL) { if (!m_bNoOverlap) { assert(m_pRBWT); m_pAllOverlapper = new OverlapAlgorithm(m_pBWT, m_pRBWT, 0.05, 16, 16, false, m_branchCutoff); } } // StatsProcess::~StatsProcess() { if(m_pAllOverlapper) { delete m_pAllOverlapper; m_pAllOverlapper = NULL; } } // StatsResult StatsProcess::process(const SequenceWorkItem& workItem) { StatsResult result; std::string readSequence = workItem.read.seq.toString(); // // Compute the kmer distribution // int k = m_kmerLength; int n = readSequence.size(); int nk = n - m_kmerLength + 1; for(int i = 0; i < nk; ++i) { std::string kmer = readSequence.substr(i, k); int count = BWTAlgorithms::countSequenceOccurrences(kmer, m_pBWT); result.kmerCoverage.push_back(count); } // // Compute the number of implied errors in the read // if(!m_bNoOverlap) { SeqRecord currRead = workItem.read; OverlapBlockList blockList; OverlapResult overlap_result = m_pAllOverlapper->overlapRead(currRead, m_minOverlap, &blockList); // Convert the overlap block list into a multi-overlap if(!overlap_result.searchAborted) { MultiOverlap mo = blockListToMultiOverlap(currRead, blockList); int covered = mo.countBasesCovered(); int wrong = mo.countPotentialIncorrect(m_errorThreshold); result.mean_depth = mo.getMeanDepth(); result.bases_counted += covered; result.bases_wrong += wrong; } } return result; } // // // StatsPostProcess::StatsPostProcess(bool bPrintKmer) : m_bPrintKmer(bPrintKmer), m_basesCounted(0), m_basesWrong(0), m_depthSum(0.0f), m_numReads(0), m_numPerfect(0) { } // StatsPostProcess::~StatsPostProcess() { std::cout << "\n*** Stats: \n"; if(m_bPrintKmer) { int max = 100; m_kmerDist.print(max); } printf("%d out of %d bases are potentially incorrect (%lf)\n", m_basesWrong, m_basesCounted, (double)m_basesWrong/m_basesCounted); printf("%d reads out of %d are perfect (%lf)\n", m_numPerfect, m_numReads, (double)m_numPerfect/m_numReads); printf("Mean overlap depth: %.2lf\n", m_depthSum / m_numReads); } // void StatsPostProcess::process(const SequenceWorkItem& /*item*/, const StatsResult& result) { for(size_t i = 0; i < result.kmerCoverage.size(); ++i) m_kmerDist.add(result.kmerCoverage[i]); m_basesCounted += result.bases_counted; m_basesWrong += result.bases_wrong; m_depthSum += result.mean_depth; m_numReads += 1; m_numPerfect += (result.bases_wrong == 0 ? 1 : 0); } ================================================ FILE: src/SGA/Algorithm/StatsProcess.h ================================================ ///----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // StatsProcess - Compute statistics about the reads // #ifndef STATSPROCESS_H #define STATSPROCESS_H #include "Util.h" #include "BWT.h" #include "SequenceProcessFramework.h" #include "SequenceWorkItem.h" #include "OverlapAlgorithm.h" #include "KmerDistribution.h" class StatsResult { public: StatsResult() : bases_counted(0), bases_wrong(0), mean_depth(0.0f) {} std::vector kmerCoverage; int bases_counted; int bases_wrong; double mean_depth; }; // class StatsProcess { public: StatsProcess(const BWT* pBWT, const BWT* pRBWT, int kmerLength, int minOverlap, int branchCutoff, bool bNoOverlap); ~StatsProcess(); StatsResult process(const SequenceWorkItem& item); private: const BWT* m_pBWT; const BWT* m_pRBWT; const int m_kmerLength; const int m_minOverlap; const int m_branchCutoff; const bool m_bNoOverlap; static const int m_errorThreshold = 3; const OverlapAlgorithm* m_pAllOverlapper; }; // Write the results from the overlap step to an ASQG file class StatsPostProcess { public: StatsPostProcess(bool bPrintKmer); ~StatsPostProcess(); void process(const SequenceWorkItem& item, const StatsResult& result); private: KmerDistribution m_kmerDist; bool m_bPrintKmer; int m_basesCounted; int m_basesWrong; double m_depthSum; int m_numReads; int m_numPerfect; }; #endif ================================================ FILE: src/SGA/Algorithm/StringGraphGenerator.cpp ================================================ ///----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // StringGraphGenerator - Iteratively construct // a local String Graph using the FM-index // #include "StringGraphGenerator.h" #include "SGAlgorithms.h" #include "SGVisitors.h" //#define DEBUGGENERATE 1 StringGraphGenerator::StringGraphGenerator(const OverlapAlgorithm* pOverlapper, const SeqRecord& startRead, const SeqRecord& endRead, int minOverlap, EdgeDir startDir, int maxDistance) : m_pOverlapper(pOverlapper), m_minOverlap(minOverlap), m_pGraph(NULL), m_startDir(startDir), m_maxDistance(maxDistance) { m_pGraph = new StringGraph; // Add the start and end vertices to the graph m_pStartVertex = addTerminalVertex(startRead); m_pEndVertex = addTerminalVertex(endRead); // Set the color of the starting node to be UNEXPLORED // and the color of the end node to be EXPLORED. // This indicates to the subsequent search which vertices // should be expanded m_pStartVertex->setColor(UNEXPLORED_COLOR); m_pEndVertex->setColor(EXPLORED_COLOR); // Set up the expansion frontier FrontierQueue queue; GraphFrontier node; node.pVertex = m_pStartVertex; node.dir = m_startDir; node.distance = m_pStartVertex->getSeqLen(); queue.push(node); buildGraph(queue); //m_pGraph->writeDot("local.dot"); SGDuplicateVisitor dupVisit(true); m_pGraph->visit(dupVisit); // If the terminal vertices are marked as contained, reset the containment flags so they will not be removed resetContainmentFlags(m_pStartVertex); resetContainmentFlags(m_pEndVertex); SGContainRemoveVisitor containVisit; m_pGraph->visit(containVisit); //m_pGraph->writeDot("local-final.dot"); } // StringGraphGenerator::~StringGraphGenerator() { delete m_pGraph; m_pGraph = NULL; // m_pStartVertex and m_pEndVertex are deleted by the graph destructor // so they do not need to be explicitly freed here. } // Build the graph by expanding nodes on the frontier void StringGraphGenerator::buildGraph(FrontierQueue& queue) { while(!queue.empty()) { if(queue.size() > 200) break; GraphFrontier node = queue.front(); queue.pop(); if(node.pVertex->getColor() == EXPLORED_COLOR) continue; // node has been visited already // Search the FM-index for the current vertex SeqRecord record; record.id = node.pVertex->getID(); record.seq = node.pVertex->getSeq().toString(); OverlapBlockList blockList; assert(blockList.empty()); m_pOverlapper->overlapRead(record, m_minOverlap, &blockList); // Update the graph and the frontier queue with newly found vertices updateGraphAndQueue(node, queue, blockList); node.pVertex->setColor(EXPLORED_COLOR); } m_pGraph->setColors(GC_WHITE); } // Search for walks between the start and end vertex SGWalkVector StringGraphGenerator::searchWalks() { SGWalkVector walks; SGSearch::findWalks(m_pStartVertex, m_pEndVertex, m_startDir, m_maxDistance, true, 10000, walks); return walks; } // void StringGraphGenerator::updateGraphAndQueue(GraphFrontier& currNode, FrontierQueue& queue, OverlapBlockList& blockList) { // Partition the block list into containment blocks and extension (valid) blocks // We do not add containment edges to the graph so the containments are discarded OverlapBlockList containList; OverlapBlockList overlapList; Vertex* pX = currNode.pVertex; //partitionBlockList(pX->getSeqLen(), &blockList, &overlapList, &containList); // Process the overlap blocks, adding new vertices and edges where necessary for(OverlapBlockList::iterator iter = blockList.begin(); iter != blockList.end(); ++iter) { if(iter->getEdgeDir() != currNode.dir) continue; std::string vertexID = iter->toCanonicalID(); if(vertexID == pX->getID()) continue; // skip self-edges std::string vertexSeq = iter->getFullString(pX->getSeq().toString()); Overlap o = iter->toOverlap(pX->getID(), vertexID, pX->getSeqLen(), vertexSeq.length()); /* #if DEBUGGENERATE std::cout << "has overlap to: " << vertexID << " len: " << iter->overlapLen << " flags: " << iter->flags << "\n"; std::cout << "Overlap string: " << iter->getOverlapString(pX->getSeq().toString()) << "\n"; #endif */ // Check if a vertex with endVertexID exists in the graph Vertex* pVertex = m_pGraph->getVertex(vertexID); if(pVertex == NULL) { #if DEBUGGENERATE std::cout << "Vertex with ID: " << vertexID << " does not exist, creating\n"; std::cout << "Vertex sequence: " << vertexSeq << "\n"; #endif // Generate the new vertex vertexSeq = iter->getFullString(pX->getSeq().toString()); //pVertex = new(m_pGraph->getVertexAllocator()) Vertex(vertexID, vertexSeq); pVertex = new Vertex(vertexID, vertexSeq); pVertex->setColor(UNEXPLORED_COLOR); m_pGraph->addVertex(pVertex); } // Construct the found edge Edge* pXY = SGAlgorithms::createEdgesFromOverlap(m_pGraph, o, true); // If the endpoint vertex is unexplored, queue it if(pVertex->getColor() == UNEXPLORED_COLOR) { GraphFrontier node; node.pVertex = pVertex; node.dir = !pXY->getTwin()->getDir(); // continuation direction node.distance = currNode.distance + pXY->getSeqLen(); if(node.distance <= m_maxDistance) queue.push(node); } } } // Vertex* StringGraphGenerator::addTerminalVertex(const SeqRecord& record) { assert(m_pGraph != NULL); // Build the vertex by performing a full-length search for the // sequence in the FM-index. We set the ID of the vertex to be the // lowest index in the returned block list. OverlapBlockList endBlockList; m_pOverlapper->alignReadDuplicate(record, &endBlockList); // Search the block list for the exact match to the end read. This must exist OverlapBlockList::iterator matchIter = endBlockList.begin(); while(matchIter != endBlockList.end()) { if(matchIter->numDiff == 0 && !matchIter->flags.isQueryRev()) break; // this block corresponds to the actual sequence of endRead } assert(matchIter != endBlockList.end()); // Construct the canonical ID from the matching interval std::string endID = matchIter->toCanonicalID(); Vertex* pVertex = m_pGraph->getVertex(endID); if(pVertex == NULL) { //pVertex = new(m_pGraph->getVertexAllocator()) Vertex(endID, record.seq.toString()); pVertex = new Vertex(endID, record.seq.toString()); m_pGraph->addVertex(pVertex); } return pVertex; } // void StringGraphGenerator::resetContainmentFlags(Vertex* pVertex) { if(!pVertex->isContained()) return; pVertex->setContained(false); // Set the containment flag for all the vertices that have containment edges with this vertex EdgePtrVec edges = pVertex->getEdges(); for(size_t i = 0; i < edges.size(); ++i) { Edge* pEdge = edges[i]; if(pEdge->getOverlap().isContainment()) pEdge->getEnd()->setContained(true); } } ================================================ FILE: src/SGA/Algorithm/StringGraphGenerator.h ================================================ ///----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // StringGraphGenerator - Iteratively construct // a local String Graph using the FM-index // #ifndef STRINGGRAPHGENERATOR_H #define STRINGGRAPHGENERATOR_H #include #include "OverlapAlgorithm.h" #include "SGUtil.h" #include "GraphCommon.h" #include "SGSearch.h" // The GraphFrontier is a node that is on the edge // of the graph - it can be used to search the FM-index // to expand the graph outwards. struct GraphFrontier { Vertex* pVertex; EdgeDir dir; int distance; // The distance from the starting node }; typedef std::queue FrontierQueue; class StringGraphGenerator { public: StringGraphGenerator(const OverlapAlgorithm* pOverlapper, const SeqRecord& startRead, const SeqRecord& endRead, int minOverlap, EdgeDir startDir, int maxDistance); ~StringGraphGenerator(); // Find walks between the start vertex and the end vertex SGWalkVector searchWalks(); private: // void buildGraph(FrontierQueue& queue); void updateGraphAndQueue(GraphFrontier& currNode, FrontierQueue& queue, OverlapBlockList& blockList); Vertex* addTerminalVertex(const SeqRecord& record); void resetContainmentFlags(Vertex* pVertex); // Data const OverlapAlgorithm* m_pOverlapper; int m_minOverlap; StringGraph* m_pGraph; Vertex* m_pStartVertex; Vertex* m_pEndVertex; EdgeDir m_startDir; int m_maxDistance; static const GraphColor UNEXPLORED_COLOR = GC_WHITE; static const GraphColor EXPLORED_COLOR = GC_BLACK; }; #endif ================================================ FILE: src/SGA/Algorithm/StringThreader.cpp ================================================ ///---------------------------------------------- // Copyright 2011 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // StringThreader - Iteratively construct a // string representing a walk through an assembly graph // matching a query sequence. // // The assembly graph is abstractly represented as // an FM-index. // #include "StringThreader.h" #include "BWTAlgorithms.h" #include "LRAlignment.h" #include "StdAlnTools.h" // // StringThreaderNode // StringThreaderNode::StringThreaderNode(const std::string* pQuery, StringThreaderNode* parent) : m_pQuery(pQuery), m_pParent(parent) { } // Destructor, recurisvely delete the children of the node StringThreaderNode::~StringThreaderNode() { // Delete children for(STNodePtrList::iterator iter = m_children.begin(); iter != m_children.end(); ++iter) delete *iter; // Delete alignment columns for(size_t i = 0; i < m_alignmentColumns.size(); ++i) delete m_alignmentColumns[i]; } // Return a suffix of length l of the path from the root to this node std::string StringThreaderNode::getSuffix(size_t l) const { size_t n = m_label.size(); if(l <= n) { return m_label.substr(n - l, l); } else { assert(m_pParent != NULL); return m_pParent->getSuffix(l - n) + m_label; } } // Return the full string of the path from the root to this node std::string StringThreaderNode::getFullString() const { if(m_pParent == NULL) return m_label; else return m_pParent->getFullString() + m_label; } // Create a new child node with the given label. Returns a pointer to the new node. StringThreaderNode* StringThreaderNode::createChild(const std::string& label) { StringThreaderNode* pAdded = new StringThreaderNode(m_pQuery, this); m_children.push_back(pAdded); assert(!m_alignmentColumns.empty()); pAdded->computeExtendedAlignment(label, m_alignmentColumns.back()); return pAdded; } // Extend the label of this node void StringThreaderNode::extend(const std::string& ext) { assert(!ext.empty()); assert(!m_alignmentColumns.empty()); computeExtendedAlignment(ext, m_alignmentColumns.back()); } // Update the alignment columns for this node. void StringThreaderNode::computeExtendedAlignment(const std::string& ext, const BandedDPColumn* pPrevColumn) { // Calculate a new alignment column for each base of the label for(size_t i = 0; i < ext.size(); ++i) { BandedDPColumn* pNewColumn = ExtensionDP::createNewColumn(ext[i], *m_pQuery, pPrevColumn); m_alignmentColumns.push_back(pNewColumn); pPrevColumn = pNewColumn; } m_label.append(ext); } // Initialize the alignment columns. void StringThreaderNode::computeInitialAlignment(const std::string& initialLabel, int queryAlignmentEnd, int bandwidth) { // Create the initial alignment columnds between label and query m_label = initialLabel; assert(!m_label.empty()); ExtensionDP::createInitialAlignment(m_label, m_pQuery->substr(0, queryAlignmentEnd), bandwidth, m_alignmentColumns); } // Calculate error rate over last context bases of the alignment double StringThreaderNode::getLocalErrorRate(int context) const { return ExtensionDP::calculateLocalEditPercentage(m_alignmentColumns.back(), context); } // Calculate error rate over the entire alignment double StringThreaderNode::getGlobalErrorRate() const { return ExtensionDP::calculateGlobalEditPercentage(m_alignmentColumns.back()); } // Calculate the edit distance between the thread and query int StringThreaderNode::getEditDistance() const { int edits, alignLength; ExtensionDP::countEditsAndAlignLength(m_alignmentColumns.back(), edits, alignLength); return edits; } // Returns true if the extension has terminated bool StringThreaderNode::hasExtensionTerminated() const { return ExtensionDP::isExtensionTerminated(m_alignmentColumns.back(), 2); } // Return the best alignment between the string represented by this node and the query StringThreaderResult StringThreaderNode::getAlignment() const { ExtensionDPAlignment alignment = ExtensionDP::findGlocalAlignment(m_alignmentColumns.back()); StringThreaderResult result; result.query_align_length = alignment.query_align_length; result.thread = getFullString().substr(0, alignment.target_align_length); return result; } // Print the alignment void StringThreaderNode::printFullAlignment() const { std::string fullString = getFullString(); ExtensionDP::printAlignment(fullString, *m_pQuery, m_alignmentColumns.back()); } // Print the string(s) represented by this node and its children void StringThreaderNode::printAllStrings(const std::string& parent) const { if(m_children.empty()) { std::cout << "S: " << parent + m_label << "\n"; } else { for(STNodePtrList::const_iterator iter = m_children.begin(); iter != m_children.end(); ++iter) (*iter)->printAllStrings(parent + m_label); } } // // StringTheader // StringThreader::StringThreader(const std::string& seed, const std::string* pQuery, int queryAlignmentEnd, int kmer, const BWT* pBWT) : m_pBWT(pBWT), m_kmer(kmer), m_pQuery(pQuery) { // Create the root node containing the seed string m_pRootNode = new StringThreaderNode(pQuery, NULL); m_pRootNode->computeInitialAlignment(seed, queryAlignmentEnd, 50); m_leaves.push_back(m_pRootNode); } // StringThreader::~StringThreader() { // Recursively destroy the tree delete m_pRootNode; } // Run the threading algorithm void StringThreader::run(StringThreaderResultVector& results) { // Extend the leaf nodes while(!m_leaves.empty()) { extendLeaves(); cullLeavesByEdits(); checkTerminated(results); } } // Print the string represented by every node void StringThreader::printAll() { std::cout << "Print all: \n"; m_pRootNode->printAllStrings(""); } // Extend each leaf node void StringThreader::extendLeaves() { STNodePtrList newLeaves; for(STNodePtrList::iterator iter = m_leaves.begin(); iter != m_leaves.end(); ++iter) { StringVector extensions = getDeBruijnExtensions(*iter); // Either extend the current node or branch it // If no extension, do nothing and this node // is no longer considered a leaf if(extensions.size() == 1) { // Single extension, do not branch (*iter)->extend(extensions.front()); newLeaves.push_back(*iter); } else if(extensions.size() > 1) { // Branch for(size_t i = 0; i < extensions.size(); ++i) { StringThreaderNode* pAdded = (*iter)->createChild(extensions[i]); newLeaves.push_back(pAdded); } } } m_leaves.clear(); m_leaves = newLeaves; } // Remove leaves that are have a high local error rate // These leaves probably represent branches that are not // the query sequence. void StringThreader::cullLeavesByLocalError() { STNodePtrList newLeaves; int context = 20; double threshold = 0.3f; // Calculate the local error rate of the alignments to each new leaf // If it is less than threshold, add the leaf to the node for(STNodePtrList::iterator iter = m_leaves.begin(); iter != m_leaves.end(); ++iter) { double ler = (*iter)->getLocalErrorRate(context); if(ler < threshold) newLeaves.push_back(*iter); } m_leaves = newLeaves; } // Remove leaves that have an edit distance much higher than // the best leaf void StringThreader::cullLeavesByEdits() { STNodePtrList newLeaves; // Calculate the local error rate of the alignments to each new leaf // If it is less than threshold, add the leaf to the node int bestEdits = std::numeric_limits::max(); IntVector editsVector; for(STNodePtrList::iterator iter = m_leaves.begin(); iter != m_leaves.end(); ++iter) { int edits = (*iter)->getEditDistance(); if(edits < bestEdits) bestEdits = edits; editsVector.push_back(edits); } int leafID = 0; int threshold = 1; for(STNodePtrList::iterator iter = m_leaves.begin(); iter != m_leaves.end(); ++iter) { if(editsVector[leafID] <= bestEdits + threshold) newLeaves.push_back(*iter); leafID += 1; } m_leaves = newLeaves; } // Check for leaves whose extension has terminated. If the leaf has // terminated, its alignment result is pushed to the result vector void StringThreader::checkTerminated(StringThreaderResultVector& results) { STNodePtrList newLeaves; for(STNodePtrList::iterator iter = m_leaves.begin(); iter != m_leaves.end(); ++iter) { if((*iter)->hasExtensionTerminated()) results.push_back((*iter)->getAlignment()); else newLeaves.push_back(*iter); } m_leaves = newLeaves; } // Calculate the successors of this node in the implicit deBruijn graph StringVector StringThreader::getDeBruijnExtensions(StringThreaderNode* pNode) { // Get the last k-1 bases of the node std::string pmer = pNode->getSuffix(m_kmer - 1); AlphaCount64 extensions = BWTAlgorithms::calculateDeBruijnExtensionsSingleIndex(pmer, m_pBWT, ED_SENSE, NULL); // Loop over the DNA symbols, if there is are more than two characters create a branch // otherwise just perform an extension. bool hasExtension = extensions.hasDNAChar(); StringVector out; if(hasExtension) { for(int i = 0; i < DNA_ALPHABET::size; ++i) { char b = DNA_ALPHABET::getBase(i); if(extensions.get(b) >= 3) { // extend to b std::string tmp; tmp.append(1,b); out.push_back(tmp); } } } return out; } ================================================ FILE: src/SGA/Algorithm/StringThreader.h ================================================ ///---------------------------------------------- // Copyright 2011 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // StringThreader - Iteratively construct a // string representing a walk through an assembly graph // matching a query sequence. // // The assembly graph is abstractly represented as // an FM-index. // #ifndef STRING_THREADER_H #define STRING_THREADER_H #include #include "BWT.h" #include "ExtensionDP.h" // Typedefs class StringThreaderNode; typedef std::list STNodePtrList; // Object to hold the result of the threading process struct StringThreaderResult { std::string thread; int query_align_length; }; typedef std::vector StringThreaderResultVector; // A node in the threading tree class StringThreaderNode { public: // // Functions // StringThreaderNode(const std::string* pQuery, StringThreaderNode* parent); ~StringThreaderNode(); // Add a child node to this node with the given label // Returns a pointer to the created node StringThreaderNode* createChild(const std::string& label); // Extend the label of this node by l void extend(const std::string& ext); // Return a suffix of length l of the string represented by this node std::string getSuffix(size_t l) const; // Return the complete sequence of the string represented by the branch std::string getFullString() const; // Get alignment mismatch/error rate metrics double getLocalErrorRate(int context) const; double getGlobalErrorRate() const; int getEditDistance() const; // Initialize or update the alignment data void computeInitialAlignment(const std::string& initialLabel, int queryAlignmentEnd, int bandwidth); void computeExtendedAlignment(const std::string& ext, const BandedDPColumn* pPrevColumn); // Return the best alignment for the string this node represents StringThreaderResult getAlignment() const; // Check if this node can be extended any further bool hasExtensionTerminated() const; // Print the alignment between the string represented by the // path from the root of the tree to this node void printFullAlignment() const; // Recursive function to print all the strings represented // by this node and all its children. void printAllStrings(const std::string& parent) const; private: // // Data // // The extension string from the parent std::string m_label; // The query string being threaded through the graph const std::string* m_pQuery; // The parent node, can be NULL StringThreaderNode* m_pParent; STNodePtrList m_children; // Alignment information between the label of this node and the query sequence // One column per label base BandedDPColumnPtrVector m_alignmentColumns; }; class StringThreader { public: // // Functions // StringThreader(const std::string& seed, const std::string* pQuery, int queryAlignmentEnd, int kmer, const BWT* pBWT); ~StringThreader(); // Run the threading process. Valid alignments are pushed to the results // vector void run(StringThreaderResultVector& results); // Print all the strings represented by the tree void printAll(); private: // // Functions // void extendLeaves(); // Leaf removal heuristics void cullLeavesByLocalError(); void cullLeavesByEdits(); // Check if the leaves can be extended no further // If so, the best alignment is pushed to results void checkTerminated(StringThreaderResultVector& results); // Calculate the successors of this node in the implicit deBruijn graph StringVector getDeBruijnExtensions(StringThreaderNode* pNode); // // Data // const BWT* m_pBWT; int m_kmer; const std::string* m_pQuery; StringThreaderNode* m_pRootNode; STNodePtrList m_leaves; }; #endif ================================================ FILE: src/SGA/Algorithm/VariationBuilderCommon.cpp ================================================ ///---------------------------------------------- // Copyright 2011 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // VariantionVariationBuilderCommon -- Common functions and data // structures for the abstract graph builders // #include "VariationBuilderCommon.h" #include "SGAlgorithms.h" // Count the number of extensions above the given threshold size_t VariationBuilderCommon::countValidExtensions(const AlphaCount64& ac, size_t threshold) { size_t n = 0; for(size_t i = 0; i < DNA_ALPHABET::size; ++i) { char b = DNA_ALPHABET::getBase(i); size_t count = ac.get(b); if(count >= threshold) n += 1; } return n; } // Filter out low counts in AlphaCount using a coverage threshold // relative to the most frequent count. Returns the number of // surviving counts size_t VariationBuilderCommon::filterLowFrequency(AlphaCount64& ac, double alpha) { size_t n = 0; size_t max = ac.getMaxCount(); if(max == 0) return 0; for(size_t i = 0; i < DNA_ALPHABET::size; ++i) { char b = DNA_ALPHABET::getBase(i); size_t count = ac.get(b); double ratio = (double)count / max; if(ratio >= alpha) n += 1; else ac.set(b,0); } return n; } // Make a de Bruijn graph string std::string VariationBuilderCommon::makeDeBruijnVertex(const std::string& v, char edgeBase, EdgeDir direction) { std::string w; size_t p = v.size() - 1; if(direction == ED_SENSE) { w = v.substr(1, p); w.append(1, edgeBase); } else { w.append(1, edgeBase); w.append(v.substr(0, p)); } return w; } // Add a de Bruijn graph edge to the given graph betwee pX and pY. // Assumes the sequences are from the same strand (not reverse complements) void VariationBuilderCommon::addSameStrandDeBruijnEdges(StringGraph* pGraph, const Vertex* pX, const Vertex* pY, EdgeDir direction) { assert(pX->getSeq().length() == pY->getSeq().length()); // overlap length for a de bruijn edge size_t p = pX->getSeq().length() - 1; // Construct an overlap object for this relationship Overlap o; o.id[0] = pX->getID(); o.id[1] = pY->getID(); o.match.isReverse = false; o.match.numDiff = 0; if(direction == ED_SENSE) { // pX -> pY o.match.coord[0].interval.start = 1; o.match.coord[1].interval.start = 0; } else { // pY -> pX o.match.coord[0].interval.start = 0; o.match.coord[1].interval.start = 1; } o.match.coord[0].interval.end = o.match.coord[0].interval.start + p - 1; // inclusive coordinate o.match.coord[1].interval.end = o.match.coord[1].interval.start + p - 1; o.match.coord[0].seqlen = p + 1; o.match.coord[1].seqlen = p + 1; Edge* e = SGAlgorithms::createEdgesFromOverlap(pGraph, o, false); assert(e != NULL); } ================================================ FILE: src/SGA/Algorithm/VariationBuilderCommon.h ================================================ ///---------------------------------------------- // Copyright 2011 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // VariationBuilderCommon - Data structures // commons to various algorithms for assembling // variation haplotypes // #ifndef VARIATION_BUILDER_COMMON_H #define VARIATION_BUILDER_COMMON_H #include "BWT.h" #include "BWTInterval.h" #include "SGUtil.h" #include "SGWalk.h" #include // Result of the bubble construction // Used to track statistics enum BubbleResultCode { BRC_UNKNOWN, BRC_OK, BRC_SOURCE_BROKEN, BRC_SOURCE_BRANCH, BRC_TARGET_BROKEN, BRC_TARGET_BRANCH, BRC_WALK_FAILED, BRC_HB_FAILED, BRC_NO_SOLUTION }; // The actual result structure struct BubbleResult { std::string targetString; std::string sourceString; double targetCoverage; double sourceCoverage; BubbleResultCode returnCode; }; // A directed node in a graph that // has not had it's neighbors visited struct BuilderExtensionNode { BuilderExtensionNode(Vertex* pX, EdgeDir d) : pVertex(pX), direction(d), distance(0) {} BuilderExtensionNode(Vertex* pX, EdgeDir dir, int dist) : pVertex(pX), direction(dir), distance(dist) {} Vertex* pVertex; // the vertex to extend EdgeDir direction; // the direction to extend to int distance; // the total number of nodes from the start to this node. }; typedef std::queue BuilderExtensionQueue; typedef std::map StrIntMap; // Common functions namespace VariationBuilderCommon { // Count the number of extensions above the given threshold size_t countValidExtensions(const AlphaCount64& ac, size_t threshold); // Returns true if there are multiple characters above the threshold bool hasMultipleBranch(const AlphaCount64& ac, size_t threshold); // Filter out low counts in AlphaCount using a coverage threshold // relative to the most frequent count. Returns the number of // surviving counts size_t filterLowFrequency(AlphaCount64& ac, double alpha); // Make a de Bruijn graph string std::string makeDeBruijnVertex(const std::string& v, char edgeBase, EdgeDir direction); // Add a de Bruijn graph edge to the given graph betwee pX and pY. void addSameStrandDeBruijnEdges(StringGraph* pGraph, const Vertex* pX, const Vertex* pY, EdgeDir direction); }; #endif ================================================ FILE: src/SGA/Bigraph/Bigraph.cpp ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // Bidirectional graph // #include #include #include #include #include "Bigraph.h" #include "Timer.h" #include "ASQG.h" // // // Bigraph::Bigraph() : m_hasContainment(false), m_hasTransitive(false), m_isExactMode(false), m_minOverlap(0), m_errorRate(0.0f) { //JEREMIAH // Set up the memory pools for the graph //m_pEdgeAllocator = new SimpleAllocator(); //m_pVertexAllocator = new SimpleAllocator(); //m_vertices.set_deleted_key(""); // JEREMIAH //WARN_ONCE("HARDCODED HASH TABLE MAX SIZE"); //m_vertices.resize(600000000); } // // // Bigraph::~Bigraph() { VertexPtrMap::iterator iter = m_vertices.begin(); for(; iter != m_vertices.end(); ++iter) { delete iter->second; iter->second = NULL; } //JEREMIAH // Clean up the memory pools //delete m_pEdgeAllocator; //delete m_pVertexAllocator; } // // Add a vertex // void Bigraph::addVertex(Vertex* pVert) { // std::pair result = // m_vertices_tokeep.insert(std::make_pair(pVert->getID(), pVert));//JEREMIAH std::pair result = m_vertices.insert(std::make_pair(pVert->getID(), pVert)); if(!result.second) { std::cerr << "Error: Attempted to insert vertex into graph with a duplicate id: " << pVert->getID() << "\n"; std::cerr << "All reads must have a unique identifier\n"; exit(1); } } // // Remove a vertex that is not connected to any other // void Bigraph::removeIslandVertex(Vertex* pVertex) { assert(pVertex->countEdges() == 0); // Remove the vertex from the collection VertexID id = pVertex->getID(); delete pVertex; m_vertices.erase(id); //m_vertices_tokeep.erase(id); //JEREMIAH } // // Remove a vertex that is not connected to any other // void Bigraph::removeIslandVertexNoErase(Vertex* pVertex) { assert(pVertex->countEdges() == 0); // Remove the vertex from the collection VertexID id = pVertex->getID(); delete pVertex; m_vertices.erase(id); } // // Remove a vertex // void Bigraph::removeConnectedVertex(Vertex* pVertex) { // Remove the edges pointing to this Vertex pVertex->deleteEdges(); // Remove the vertex from the collection VertexID id = pVertex->getID(); delete pVertex; m_vertices.erase(id); } // // Check for the existance of a vertex // bool Bigraph::hasVertex(VertexID id) { VertexPtrMapIter iter = m_vertices.find(id); return iter != m_vertices.end(); } // // Get a vertex // Vertex* Bigraph::getVertex(VertexID id) const { VertexPtrMapConstIter iter = m_vertices.find(id); if(iter == m_vertices.end()) return NULL; return iter->second; } // // Add an edge // void Bigraph::addEdge(Vertex* pVertex, Edge* pEdge) { assert(pEdge->getStart() == pVertex); pVertex->addEdge(pEdge); } // // Remove an edge // void Bigraph::removeEdge(const EdgeDesc& ed) { ed.pVertex->removeEdge(ed); } // // High level merge function that does not specify an edge // void Bigraph::mergeVertices(VertexID id1, VertexID id2) { Vertex* pVert1 = getVertex(id1); // Get the edges from vertex1 to vertex2 EdgePtrVec edgesTo = pVert1->findEdgesTo(id2); if(edgesTo.empty()) { std::cerr << "mergeVertices: vertices are not connected\n"; return; } if(edgesTo.size() > 1) { std::cerr << "mergeVertces: cannot merge because of ambigious edges\n"; return; } // There is a single unique edge between the vertices Edge* mergeEdge = *edgesTo.begin(); // Call the real merging function merge(pVert1, mergeEdge); } // // Merge two vertices along the specified edge // void Bigraph::merge(Vertex* pV1, Edge* pEdge) { Vertex* pV2 = pEdge->getEnd(); //std::cout << "Merging " << pV1->getID() << " with " << pV2->getID() << "\n"; // jeremiah connected component tracking if (m_get_components) { int lab1 = m_connected_components[pV1->getID()]; int lab2 = m_connected_components[pV2->getID()]; ++m_label; m_connected_components[pV1->getID()] = m_label; m_connected_components[pV2->getID()] = m_label; for (auto& i : m_connected_components) if (i.second == lab1 || i.second == lab2) i.second = m_label; } //JEREMIAH //pV1->addParent(pV2->getID()); //std::cerr << "LEN: " << pV1->getParentID().size() << std::endl; // Merge the data pV1->merge(pEdge); // Get the twin edge (the edge in v2 that points to v1) Edge* pTwin = pEdge->getTwin(); // Ensure v2 has the twin edge assert(pV2->hasEdge(pTwin)); // Get the edge set opposite of the twin edge (which will be the new edges in this direction for V1) EdgePtrVec transEdges = pV2->getEdges(!pTwin->getDir()); // Move the edges from pV2 to pV1 for(EdgePtrVecIter iter = transEdges.begin(); iter != transEdges.end(); ++iter) { Edge* pTransEdge = *iter; // Remove the edge from V2, this does not destroy the edge pV2->removeEdge(pTransEdge); // Join pEdge to the start of transEdge // This updates the starting point of pTransEdge to be V1 // This calls Edge::extend on the twin edge pTransEdge->join(pEdge); assert(pTransEdge->getDir() == pEdge->getDir()); pV1->addEdge(pTransEdge); // add to V1 // Notify the edges they have been updated pTransEdge->update(); pTransEdge->getTwin()->update(); } // Remove the edge from pV1 to pV2 pV1->removeEdge(pEdge); delete pEdge; pEdge = 0; // Remove the edge from pV2 to pV1 pV2->removeEdge(pTwin); delete pTwin; pEdge = 0; // Remove V2 // It is guarenteed to not be connected removeIslandVertexNoErase(pV2); //validate(); } // int Bigraph::sweepVertices(GraphColor c) { int numRemoved = 0; VertexPtrMapIter iter = m_vertices.begin(); while(iter != m_vertices.end()) { VertexPtrMapIter next = iter; ++next; if(iter->second->getColor() == c) { removeConnectedVertex(iter->second); ++numRemoved; } iter = next; } return numRemoved; } // int Bigraph::sweepEdges(GraphColor c) { int numRemoved = 0; for(VertexPtrMapIter iter = m_vertices.begin(); iter != m_vertices.end(); ++iter) numRemoved += iter->second->sweepEdges(c); return numRemoved; } // Simplify the graph by compacting singular edges void Bigraph::simplify() { assert(!hasContainment()); simplify(ED_SENSE); simplify(ED_ANTISENSE); } // Simplify the graph by compacting edges in the given direction void Bigraph::simplify(EdgeDir dir) { bool graph_changed = true; while(graph_changed) { graph_changed = false; VertexPtrMapIter iter = m_vertices.begin(); while(iter != m_vertices.end()) { // Get the edges for this direction EdgePtrVec edges = iter->second->getEdges(dir); //std::cout << "--VERTEX: dir " << dir << " " << iter->first << " seq len " << iter->second->getSeqLen() << " eges size " << edges.size() << std::endl; // for (auto& i : edges) // std::cerr << " EDGE: " << i->getStartID() << " " << i->getEndID()<< std::endl; // If there is a single edge in this direction, merge the vertices // Don't merge singular self edges though if(edges.size() == 1 && !edges.front()->isSelf()) { // Check that the edge back is singular as well Edge* pSingle = edges.front(); Edge* pTwin = pSingle->getTwin(); Vertex* pV2 = pSingle->getEnd(); //std::cerr << "...edge back edge counts " << pV2->countEdges(pTwin->getDir()) << std::endl; if(pV2->countEdges(pTwin->getDir()) == 1) { //std::cerr << "merging " << std::endl; merge(iter->second, pSingle); graph_changed = true; } } ++iter; } } } // // Rename the vertices to have a sequential idx // starting with prefix. This requires extra memory // as we need to keep a vector of vertex pointers to reconstruct // the graph after the vertices are renamed. This should // only be done after the string graph has been simplified or // else it could require a lot of memory. // void Bigraph::renameVertices(const std::string& prefix) { size_t currIdx = 0; size_t numVertices = m_vertices.size(); std::vector vertexPtrVec(numVertices, 0); VertexPtrMapIter iter = m_vertices.begin(); while(iter != m_vertices.end()) { std::stringstream ss; ss << prefix << currIdx; std::string newlab = ss.str(); // jeremiah if (m_get_components) { int label = m_connected_components[iter->second->getID()]; if (label) { // is a connected component for (auto& i : m_connected_components) { if (i.second == label) m_reads_on_contigs[newlab].push_back(i.first); // i.first is read name of merged read } } } iter->second->setID(ss.str()); vertexPtrVec[currIdx] = iter->second; ++iter; ++currIdx; } // Clear the old graph m_vertices.clear(); // Re-add the vertices for(size_t i = 0; i < numVertices; ++i) addVertex(vertexPtrVec[i]); } // // Sort the adjacency list for each vertex by length // void Bigraph::sortVertexAdjListsByLen() { VertexPtrMapIter iter = m_vertices.begin(); for(; iter != m_vertices.end(); ++iter) iter->second->sortAdjListByLen(); } // // Sort the adjacency list for each vertex by ID // void Bigraph::sortVertexAdjListsByID() { VertexPtrMapIter iter = m_vertices.begin(); for(; iter != m_vertices.end(); ++iter) iter->second->sortAdjListByID(); } // // Validate that the graph is sane // void Bigraph::validate() { VertexPtrMapIter iter = m_vertices.begin(); for(; iter != m_vertices.end(); ++iter) { iter->second->validate(); } } // // Flip a vertex // void Bigraph::flip(VertexID /*id*/) { assert(false); #if 0 // TODO: update this code Vertex* pVertex = getVertex(id); EdgePtrVec edges = pVertex->getEdges(); for(EdgePtrVecIter iter = edges.begin(); iter != edges.end(); ++iter) { // Get the old twin GraphEdgeType twin = iter->getTwin(); GraphEdgeType flipped = *iter; flipped.flip(); // Remove the edge from the source ver pVertex->removeEdge(*iter); pVertex->addEdge(flipped); // Update the partner by deleting the old twin and Vertex* pV2 = getVertex(twin.getStart()); pV2->removeEdge(twin); pV2->addEdge(flipped.getTwin()); } #endif } // // Get the IDs of the vertices that do not branch (both sense/antisense degree <= 1) // VertexIDVec Bigraph::getNonBranchingVertices() const { VertexIDVec out; VertexPtrMapConstIter iter = m_vertices.begin(); for(; iter != m_vertices.end(); ++iter) { int senseEdges = iter->second->countEdges(ED_SENSE); int antisenseEdges = iter->second->countEdges(ED_ANTISENSE); if(antisenseEdges <= 1 && senseEdges <= 1) { out.push_back(iter->second->getID()); } } return out; } // // Get all the paths corresponding to the linear components of the graph // Precondition: all vertices have in/out degree at most 1 (no branching) // PathVector Bigraph::getLinearComponents() { PathVector outPaths; setColors(GC_WHITE); VertexPtrMapIter iter = m_vertices.begin(); for(; iter != m_vertices.end(); ++iter) { // Output the linear path containing this vertex if it hasnt been visited already if(iter->second->getColor() != GC_BLACK) { outPaths.push_back(constructLinearPath(iter->second->getID())); } } assert(checkColors(GC_BLACK)); return outPaths; } // // Return all the path of nodes that can be linearally reached from this node // The path expands in both directions so the first node in the path is not necessarily the source // Path Bigraph::constructLinearPath(VertexID id) { Path sensePath; Path antisensePath; followLinear(id, ED_SENSE, sensePath); followLinear(id, ED_ANTISENSE, antisensePath); // Construct the final path Path final = reversePath(antisensePath); final.insert(final.end(), sensePath.begin(), sensePath.end()); return final; } // // Recursively follow the graph in the specified direction without branching // outPath is an out-parameter of the edges that were followed // void Bigraph::followLinear(VertexID id, EdgeDir dir, Path& outPath) { Vertex* pVertex = getVertex(id); EdgePtrVec edges = pVertex->getEdges(dir); // Color the vertex pVertex->setColor(GC_BLACK); if(edges.size() == 1) { Edge* pSingle = edges.front(); outPath.push_back(pSingle); // Correct the direction for the comp assert(pSingle->getDir() == dir); EdgeDir corrected_dir = correctDir(pSingle->getDir(), pSingle->getComp()); // Recurse followLinear(pSingle->getEndID(), corrected_dir, outPath); } } // Path Bigraph::reversePath(const Path& path) { Path out; for(Path::const_reverse_iterator iter = path.rbegin(); iter != path.rend(); ++iter) out.push_back((*iter)->getTwin()); return out; } // Vertex* Bigraph::getFirstVertex() const { if(m_vertices.empty()) return NULL; else return m_vertices.begin()->second; } // Returns a vector of pointers to the vertices VertexPtrVec Bigraph::getAllVertices() const { VertexPtrVec out; VertexPtrMapConstIter iter = m_vertices.begin(); for(; iter != m_vertices.end(); ++iter) out.push_back(iter->second); return out; } // Returns a vector of pointers to the vertices /*VertexPtrVec Bigraph::getAllKeepVertices() const { VertexPtrVec out; VertexPtrMapConstIter iter = m_vertices_tokeep.begin(); for(; iter != m_vertices_tokeep.end(); ++iter) out.push_back(iter->second); return out; }*/ // // Append vertex sequences to the vector void Bigraph::getVertexSequences(std::vector& outSequences) const { VertexPtrMapConstIter iter = m_vertices.begin(); for(; iter != m_vertices.end(); ++iter) outSequences.push_back(iter->second->getSeq().toString()); } // // Visit each vertex and call the function pointer // The function returns TRUE if it modifies the graph // bool Bigraph::visit(VertexVisitFunction f) { bool modified = false; VertexPtrMapConstIter iter = m_vertices.begin(); for(; iter != m_vertices.end(); ++iter) { modified = f(this, iter->second) || modified; } return modified; } // // Set all the vertices and edges in the graph to the given color // void Bigraph::setColors(GraphColor c) { VertexPtrMapIter iter = m_vertices.begin(); for(; iter != m_vertices.end(); ++iter) { iter->second->setColor(c); iter->second->setEdgeColors(c); } } // // Get/Set the containment flag // void Bigraph::setContainmentFlag(bool b) { m_hasContainment = b; } // bool Bigraph::hasContainment() const { return m_hasContainment; } // // Get/Set the exact mode flag // void Bigraph::setExactMode(bool b) { m_isExactMode = b; } // bool Bigraph::isExactMode() const { return m_isExactMode; } // // Get/Set the transitive flag // void Bigraph::setTransitiveFlag(bool b) { m_hasTransitive = b; } // bool Bigraph::hasTransitive() const { return m_hasTransitive; } // // // void Bigraph::setMinOverlap(int mo) { m_minOverlap = mo; } // int Bigraph::getMinOverlap() const { return m_minOverlap; } // // // void Bigraph::setErrorRate(double er) { m_errorRate = er; } // double Bigraph::getErrorRate() const { return m_errorRate; } // // Check if all the vertices in the graph are the given color // bool Bigraph::checkColors(GraphColor c) { VertexPtrMapIter iter = m_vertices.begin(); for(; iter != m_vertices.end(); ++iter) { if(iter->second->getColor() != c) { std::cerr << "Warning vertex " << iter->second->getID() << " is color " << iter->second->getColor() << " expected " << c << "\n"; return false; } } return true; } // // Output simple stats // void Bigraph::stats() const { int numVerts = 0; int numEdges = 0; VertexPtrMapConstIter iter = m_vertices.begin(); for(; iter != m_vertices.end(); ++iter) { numEdges += iter->second->countEdges(); ++numVerts; } std::cout << "Graph has " << numVerts << " vertices and " << numEdges << " edges\n"; } // // // size_t Bigraph::getNumVertices() const { return m_vertices.size(); } // // Output mem stats // void Bigraph::printMemSize() const { size_t numVerts = 0; size_t vertMem = 0; size_t numEdges = 0; size_t edgeMem = 0; VertexPtrMapConstIter iter = m_vertices.begin(); for(; iter != m_vertices.end(); ++iter) { ++numVerts; vertMem += iter->second->getMemSize(); EdgePtrVec edges = iter->second->getEdges(); for(EdgePtrVecIter edgeIter = edges.begin(); edgeIter != edges.end(); ++edgeIter) { ++numEdges; edgeMem += (*edgeIter)->getMemSize(); } } //printf("num verts: %zu using %zu bytes (%.2lf per vert)\n", numVerts, vertMem, double(vertMem) / numVerts); //printf("num edges: %zu using %zu bytes (%.2lf per edge)\n", numEdges, edgeMem, double(edgeMem) / numEdges); //printf("total: %zu\n", edgeMem + vertMem); } // // Write the graph to a dot file // void Bigraph::writeDot(const std::string& filename, int dotFlags) const { std::ofstream out(filename.c_str()); std::string graphType = (dotFlags & DF_UNDIRECTED) ? "graph" : "digraph"; out << graphType << " G\n{\n"; VertexPtrMapConstIter iter = m_vertices.begin(); for(; iter != m_vertices.end(); ++iter) { VertexID id = iter->second->getID(); std::string label = (dotFlags & DF_NOID) ? "" : id; out << "\"" << id << "\" [ label=\"" << label << "\" "; if(dotFlags & DF_COLORED) out << " style=\"filled\" fillcolor=\"" << getColorString(iter->second->getColor()) << "\" "; out << "];\n"; iter->second->writeEdges(out, dotFlags); } out << "}\n"; out.close(); } // // Write the graph to an ASQG file // void Bigraph::writeASQG(const std::string& filename) const { std::ostream* pWriter = createWriter(filename); // Header ASQG::HeaderRecord headerRecord; headerRecord.setOverlapTag(m_minOverlap); headerRecord.setErrorRateTag(m_errorRate); headerRecord.setTransitiveTag(m_hasTransitive); headerRecord.setContainmentTag(m_hasContainment); headerRecord.write(*pWriter); VertexPtrMapConstIter iter; // Vertices for(iter = m_vertices.begin(); iter != m_vertices.end(); ++iter) { ASQG::VertexRecord vertexRecord(iter->second->getID(), iter->second->getSeq().toString()); vertexRecord.write(*pWriter); } // Edges for(iter = m_vertices.begin(); iter != m_vertices.end(); ++iter) { EdgePtrVec edges = iter->second->getEdges(); for(EdgePtrVecIter edgeIter = edges.begin(); edgeIter != edges.end(); ++edgeIter) { // We write one record for every bidirectional edge so only write edges // that are in canonical form (where id1 < id2) Overlap ovr = (*edgeIter)->getOverlap(); if(ovr.id[0] <= ovr.id[1]) { // Containment edges are in both directions so only output one // record if it is a containment if(!ovr.isContainment() || ((*edgeIter)->getDir() == ED_SENSE)) { ASQG::EdgeRecord edgeRecord(ovr); edgeRecord.write(*pWriter); } } } } delete pWriter; } // std::string Bigraph::getColorString(GraphColor c) { switch(c) { case GC_WHITE: return "white"; case GC_GRAY: return "gray"; case GC_BLACK: return "black"; case GC_BLUE: return "blue"; case GC_RED: return "red"; default: return "black"; } } ================================================ FILE: src/SGA/Bigraph/Bigraph.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // Bidirectional graph // #ifndef BIGRAPH_H #define BIGRAPH_H #include #include #include #include #include "GraphCommon.h" #include "Vertex.h" #include "Edge.h" #include "HashMap.h" #include // // Typedefs // //typedef std::map VertexPtrMap; //typedef __gnu_cxx::hash_map VertexPtrMap; //typedef std::tr1::unordered_map VertexPtrMap; // JEREMIAH commented to switch to normal unordered typedef std::unordered_map VertexPtrMap; //typedef SparseHashMap VertexPtrMap;// JEREMIAH typedef VertexPtrMap::iterator VertexPtrMapIter; typedef VertexPtrMap::const_iterator VertexPtrMapConstIter; class Bigraph; typedef bool(*VertexVisitFunction)(Bigraph*, Vertex*); typedef EdgePtrVec Path; // alias typedef std::vector PathVector; typedef std::vector VertexIDVec; typedef std::vector VertexPtrVec; class Bigraph { public: Bigraph(); ~Bigraph(); // Add a vertex void addVertex(Vertex* pVert); // Remove a vertex // removeIslandVertex removes a vertex that is guarenteed to not have edges // removeConnectedVertex removes a (possibly) connected vertex and all the edges to/from it void removeIslandVertex(Vertex* pVertex); void removeIslandVertexNoErase(Vertex* pVertex); void removeConnectedVertex(Vertex* pVertex); // Check if a vertex exists bool hasVertex(VertexID id); // Get a vertex Vertex* getVertex(VertexID id) const; // Add an edge void addEdge(Vertex* pVertex, Edge* pEdge); // Remove an edge void removeEdge(const EdgeDesc& ed); // Remove all edges marked by color c int sweepVertices(GraphColor c); int sweepEdges(GraphColor c); // Merge vertices void mergeVertices(VertexID id1, VertexID id2); // Merge vertices that are joined by the specified edge void merge(Vertex* pV1, Edge* pEdge); // Rename all the vertices in the graph void renameVertices(const std::string& prefix = ""); // Simplify the graph by removing transitive edges void simplify(); // Validate that the graph is sane void validate(); // Flip a given vertex void flip(VertexID id); // Sort all the vertex adjacency lists void sortVertexAdjListsByLen(); void sortVertexAdjListsByID(); // Get the IDs of the vertices that do not branch (both sense/antisense degree <= 1) VertexIDVec getNonBranchingVertices() const; // Get the linear components of a non-branching graph PathVector getLinearComponents(); // Return all the path of nodes that can be linearally reached from this node // The path expands in both directions so the first node in the path is not necessarily the source Path constructLinearPath(VertexID id); // Reverse a path static Path reversePath(const Path& path); // Returns an iterator to the first vertex in the graph. // If the graph is empty, a null pointer is returned Vertex* getFirstVertex() const; // Print simple summary statistics to stdout void stats() const; void printMemSize() const; size_t getNumVertices() const; // Visit each vertex in the graph and perform the visit function bool visit(VertexVisitFunction f); bool visit(VertexVisitFunction f, std::stringstream &stream); // VertexPtrVec getAllVertices() const; VertexPtrVec getAllKeepVertices() const; // Append each vertex sequence to the vector of strings void getVertexSequences(std::vector& outSequences) const; // Visit each vertex in the graph and call the visit functor object template bool visit(VF& vf) { bool modified = false; vf.previsit(this); VertexPtrMapConstIter iter = m_vertices.begin(); for(; iter != m_vertices.end(); ++iter) { modified = vf.visit(this, iter->second) || modified; } vf.postvisit(this); return modified; } // Set the colors for the entire graph void setColors(GraphColor c); // Check the colors for the entire graph bool checkColors(GraphColor c); // Get/Set the containment flag void setContainmentFlag(bool b); bool hasContainment() const; // void setTransitiveFlag(bool b); bool hasTransitive() const; // void setMinOverlap(int mo); int getMinOverlap() const; // void setErrorRate(double er); double getErrorRate() const; void setExactMode(bool b); bool isExactMode() const; // Write the graph to a file void writeDot(const std::string& filename, int dotFlags = 0) const; void writeASQG(const std::string& filename) const; // Returns an allocator for the edges of the graph SimpleAllocator* getEdgeAllocator() { return m_pEdgeAllocator; } // Returns an allocator for the vertices of the graph SimpleAllocator* getVertexAllocator() { return m_pVertexAllocator; } // Return a string for a color code static std::string getColorString(GraphColor c); // return the vertex map JEREMIAH //VertexPtrMap getVertexMap() { return m_vertices; } //JEREMIAH bool m_get_components = false; std::unordered_map m_connected_components; int m_label = 0; std::unordered_map> m_reads_on_contigs; private: // Simplify the graph by compacting edges in the given direction void simplify(EdgeDir dir); void followLinear(VertexID id, EdgeDir dir, Path& outPath); // // data // VertexPtrMap m_vertices; //VertexPtrMap m_vertices_tokeep; //JEREMIAH // Graph parameters bool m_hasContainment; bool m_hasTransitive; bool m_isExactMode; int m_minOverlap; double m_errorRate; // Memory management SimpleAllocator* m_pVertexAllocator; SimpleAllocator* m_pEdgeAllocator; }; #endif ================================================ FILE: src/SGA/Bigraph/Edge.cpp ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // Base bidirectional edge class // #include "Edge.h" #include "Vertex.h" // EdgeDesc Edge::getTwinDesc() const { return EdgeDesc(getStart(), getTwinDir(), getComp()); } // Get the edge's label std::string Edge::getLabel() const { const Edge* pTwin = getTwin(); const Vertex* pEndpoint = m_pEnd; // get the unmatched coordinates in V2 SeqCoord unmatched = pTwin->getMatchCoord().complement(); std::string seq = unmatched.getSubstring(pEndpoint->getStr()); if(getComp() == EC_REVERSE) seq = reverseComplement(seq); return seq; } // Join the edge pEdge into this edge, adding to the start void Edge::join(const Edge* pEdge) { // Update the match coordinate Match m12 = pEdge->getMatch(); Match m23 = getMatch(); m_matchCoord = m12.inverseTranslate(m23.coord[0]); if(pEdge->getComp() == EC_REVERSE) flip(); // Now, update the twin of this edge to extend to the twin of pEdge m_pTwin->extend(pEdge->getTwin()); } // Extend this edge by adding pEdge to the end void Edge::extend(const Edge* pEdge) { if(pEdge->getComp() == EC_REVERSE) flipComp(); m_pEnd = pEdge->getEnd(); } // return the mapping from V1 to V2 via this edge // If necessary, flip the SC of V2 so that it is // in the same coordinate system as V1 Match Edge::getMatch() const { const SeqCoord& sc = getMatchCoord(); const SeqCoord& tsc = m_pTwin->getMatchCoord(); return Match(sc, tsc, getComp() == EC_REVERSE, -1); } // The overlap structure implied by this edge Overlap Edge::getOverlap() const { return Overlap(getStartID(), getEndID(), getMatch()); } // Get the matching portion of V1 described by this edge std::string Edge::getMatchStr() const { return m_matchCoord.getSubstring(getStart()->getStr()); } // Return the length of the sequence size_t Edge::getSeqLen() const { SeqCoord unmatched = m_pTwin->getMatchCoord().complement(); return unmatched.length(); } void Edge::offsetMatch(int offset) { m_matchCoord.interval.start += offset; m_matchCoord.interval.end += offset; } void Edge::extendMatch(int ext_len) { m_matchCoord.interval.end += ext_len; } // Bump the edges of the match outwards so it covers the entire // sequence void Edge::extendMatchFullLength() { if(m_matchCoord.isLeftExtreme()) m_matchCoord.interval.end = m_matchCoord.seqlen - 1; else m_matchCoord.interval.start = 0; } void Edge::updateSeqLen(int newLen) { m_matchCoord.seqlen = newLen; } // Validate that the edge members are sane void Edge::validate() const { const Edge* pTwin = getTwin(); std::string m_v1 = getMatchStr(); std::string m_v2 = pTwin->getMatchStr(); if(getComp() == EC_REVERSE) m_v2 = reverseComplement(m_v2); bool error = false; if(m_v1.length() != m_v2.length()) { std::cerr << "Error, matching strings are not the same length\n"; error = true; } if(error) { std::cerr << "V1M: " << m_v1 << "\n"; std::cerr << "V2M: " << m_v2 << "\n"; std::cerr << "V1MC: " << getMatchCoord() << "\n"; std::cerr << "V2MC: " << pTwin->getMatchCoord() << "\n"; std::cerr << "V1: " << getStart()->getSeq() << "\n"; std::cerr << "Validation failed for edge " << *this << "\n"; assert(false); } } // Output std::ostream& operator<<(std::ostream& out, const Edge& obj) { out << obj.m_pEnd->getID() << "," << obj.getDir() << "," << obj.getComp(); return out; } ================================================ FILE: src/SGA/Bigraph/Edge.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL license //----------------------------------------------- // // Base bidirectional edge class // #ifndef EDGE_H #define EDGE_H #include #include "Match.h" #include "Util.h" #include "GraphCommon.h" #include "EdgeDesc.h" #include "Vertex.h" #include "BitChar.h" #include "SimpleAllocator.h" // Packed structure holding the direction and comp of an edge // The EdgeDir/EdgeComp enums (which only have values 0/1) are used // as the interface for this class so the set flags are cast to/from // these types. struct EdgeData { public: EdgeData() {} // Setters void setDir(EdgeDir dir) { if(dir == ED_ANTISENSE) m_data.set(DIR_BIT, true); else m_data.set(DIR_BIT, false); } void setComp(EdgeComp comp) { if(comp == EC_REVERSE) m_data.set(COMP_BIT, true); else m_data.set(COMP_BIT, false); } void flipDir() { m_data.flip(DIR_BIT); } void flipComp() { m_data.flip(COMP_BIT); } // Getters inline EdgeDir getDir() const { return m_data.test(DIR_BIT) ? ED_ANTISENSE : ED_SENSE; } inline EdgeComp getComp() const { return m_data.test(COMP_BIT) ? EC_REVERSE : EC_SAME; } private: static const size_t DIR_BIT = 0; static const size_t COMP_BIT = 1; BitChar m_data; }; class Edge { public: Edge(Vertex* end, EdgeDir dir, EdgeComp comp, SeqCoord m) : m_pEnd(end), m_pTwin(NULL), m_matchCoord(m), m_color(GC_WHITE), isTrusted(false) { m_edgeData.setDir(dir); m_edgeData.setComp(comp); } ~Edge() { } // High level modification functions // Join merges pEdge into this edge, with pEdge describing the starting point void join(const Edge* pEdge); // Extend merged pEdge into this edge, with pEdge describing the endpoint void extend(const Edge* pEdge); // Post merge update function void update() {} // Sequence Coordinate functions // update functions void extendMatchFullLength(); void extendMatch(int ext_len); void offsetMatch(int offset); void updateSeqLen(int newLen); // access functions size_t getSeqLen() const; size_t getMatchLength() const { return m_matchCoord.length(); } const SeqCoord& getMatchCoord() const { return m_matchCoord; } std::string getMatchStr() const; Match getMatch() const; Overlap getOverlap() const; // setters void setTwin(Edge* pEdge) { m_pTwin = pEdge; } void setColor(GraphColor c) { m_color = c; } // getters VertexID getStartID() const { return getStart()->getID(); } VertexID getEndID() const { return m_pEnd->getID(); } inline Vertex* getStart() const { assert(m_pTwin != NULL); return m_pTwin->getEnd(); } inline Vertex* getEnd() const { return m_pEnd; } inline EdgeDir getDir() const { return m_edgeData.getDir(); } inline EdgeComp getComp() const { return m_edgeData.getComp(); } inline Edge* getTwin() const { assert(m_pTwin != NULL); return m_pTwin; } EdgeDesc getTwinDesc() const; std::string getLabel() const; bool isSelf() const { return getStart() == getEnd(); } inline GraphColor getColor() const { return m_color; } size_t getMemSize() const { return sizeof(*this); } // Returns the direction of an edge that continues in the same direction // as this edge, corrected for complementary inline EdgeDir getTransitiveDir() const { return (getComp() == EC_SAME) ? getDir() : !getDir(); } // Make the direction of the edge that its twin should point along inline EdgeDir getTwinDir() const { return (getComp() == EC_SAME) ? !getDir() : getDir(); } inline EdgeDesc getDesc() const { return EdgeDesc(getEnd(), getDir(), getComp()); } // Flip the edge inline void flipComp() { m_edgeData.flipComp(); } inline void flipDir() { m_edgeData.flipDir(); } void flip() { flipComp(); flipDir(); } // Memory management //void* operator new(size_t /*size*/, SimpleAllocator* pAllocator) //{ // return pAllocator->alloc(); //} //void operator delete(void* /*target*/, size_t /*size*/) //{ // Deletions are handled at the graph/pool level. The lifetime of an edge // is as long as the graph it belongs to, even if it is deleted before // the graph. //} // Validate that the edge is sane void validate() const; // Output friend std::ostream& operator<<(std::ostream& out, const Edge& obj); protected: // Global new is not allowed, allocation must go through the memory pool // belonging to the graph. //void* operator new(size_t size) { return malloc(size); } Edge() {}; // Default constructor is not allowed Vertex* m_pEnd; Edge* m_pTwin; SeqCoord m_matchCoord; GraphColor m_color; EdgeData m_edgeData; // dir/comp member public: bool isTrusted; }; #endif ================================================ FILE: src/SGA/Bigraph/EdgeDesc.cpp ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // EdgeDesc - A unique description of an edge // #include "EdgeDesc.h" #include "Vertex.h" // Operators bool EdgeDesc::operator<(const EdgeDesc& obj) const { assert(pVertex != NULL && obj.pVertex != NULL); if(pVertex->getID() < obj.pVertex->getID()) return true; else if(pVertex->getID() > obj.pVertex->getID()) return false; else if(dir < obj.dir) return true; else if(dir > obj.dir) return false; else if(comp < obj.comp) return true; else if(comp > obj.comp) return false; return false; } bool EdgeDesc::operator==(const EdgeDesc& obj) const { assert(pVertex != NULL && obj.pVertex != NULL); return pVertex->getID() == obj.pVertex->getID() && dir == obj.dir && comp == obj.comp; } std::ostream& operator<<(std::ostream& out, const EdgeDesc& ed) { assert(ed.pVertex != NULL); out << ed.pVertex->getID() << "," << ed.dir << "," << ed.comp; return out; } ================================================ FILE: src/SGA/Bigraph/EdgeDesc.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // EdgeDesc - A unique description of an edge // #ifndef EDGEDESC_H #define EDGEDESC_H #include "GraphCommon.h" class Vertex; struct EdgeDesc { EdgeDesc() : pVertex(NULL) {} EdgeDesc(Vertex* pV, EdgeDir d, EdgeComp c) : pVertex(pV), dir(d), comp(c) {} Vertex* pVertex; EdgeDir dir; EdgeComp comp; // inline EdgeDir getTransitiveDir() const { return (comp == EC_SAME) ? dir : !dir; } inline EdgeDir getTwinDir() const { return (comp == EC_SAME) ? !dir : dir; } // Operators bool operator<(const EdgeDesc& obj) const; bool operator==(const EdgeDesc& obj) const; friend std::ostream& operator<<(std::ostream& out, const EdgeDesc& ed); }; #endif ================================================ FILE: src/SGA/Bigraph/GraphCommon.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // GraphCommon - Definitions and functions common // to the grpah implementation // #ifndef GRAPHCOMMON_H #define GRAPHCOMMON_H #include #include "Util.h" // The directions an edge can take. // Do not change the values enum EdgeDir { ED_SENSE = 0, ED_ANTISENSE = 1, ED_COUNT = 2 }; // Flag indicating whether the sequences linked by an edge // are from the same strand or not. // Do not change the values enum EdgeComp { EC_SAME = 0, EC_REVERSE = 1 }; // Array holding the directions, for iteration const EdgeDir EDGE_DIRECTIONS[ED_COUNT] = { ED_SENSE, ED_ANTISENSE }; // Flags specifying how the dot file should be drawn enum DotFlags { DF_UNDIRECTED = 0x01, DF_ANNOTATIONS = 0x02, DF_COLORED = 0x04, DF_NOID = 0x08 }; // GraphColors are generic flags that can be used to indicate state typedef uint8_t GraphColor; const GraphColor GC_WHITE = 0; const GraphColor GC_GRAY = 1; const GraphColor GC_BLACK = 2; const GraphColor GC_BLUE = 3; const GraphColor GC_RED = 4; // Typedefs typedef std::string VertexID; typedef std::vector VertexIDVec; // // Edge Operations // inline EdgeDir operator!(const EdgeDir& dir) { return (dir == ED_SENSE) ? ED_ANTISENSE : ED_SENSE; } inline EdgeComp operator!(const EdgeComp& comp) { return (comp == EC_SAME) ? 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for(; iter != m_edges.end(); ++iter) { delete *iter; *iter = NULL; } } // Merging two string vertices has two parts // First, the sequence of the vertex is extended // by the the content of the edge label // Then, all the edges that are pointing to this node // must be updated to contain the extension of the vertex void Vertex::merge(Edge* pEdge) { Edge* pTwin = pEdge->getTwin(); //std::cout << "Adding label to " << getID() << " str: " << pSE->getLabel() << "\n"; // Merge the sequence DNAEncodedString label = pEdge->getLabel(); size_t label_len = label.length(); pEdge->updateSeqLen(m_seq.length() + label_len); bool prepend = false; if(pEdge->getDir() == ED_SENSE) { m_seq.append(label); } else { label.append(m_seq); std::swap(m_seq, label); prepend = true; } // Update the coverage value of the vertex m_coverage += pEdge->getEnd()->getCoverage(); pEdge->extendMatch(label_len); pTwin->extendMatchFullLength(); // All the SeqCoords for the edges must have their seqlen field updated // Also, if we prepended sequence to this edge, all the matches in the // SENSE direction must have their coordinates offset size_t newLen = m_seq.length(); for(EdgePtrVecIter iter = m_edges.begin(); iter != m_edges.end(); ++iter) { Edge* pUpdateEdge = *iter; pUpdateEdge->updateSeqLen(newLen); if(prepend && pUpdateEdge->getDir() == ED_SENSE && pEdge != pUpdateEdge) pUpdateEdge->offsetMatch(label_len); } #ifdef VALIDATE VALIDATION_WARNING("Vertex::merge") validate(); #endif } void Vertex::validate() const { for(EdgePtrVecConstIter iter = m_edges.begin(); iter != m_edges.end(); ++iter) { (*iter)->validate(); /* std::string label = pSE->getLabel(); StringVertex* pEnd = SV_CAST(pSE->getEnd()); std::string vertSeq = pEnd->getSeq(); EdgeDir suffixDir = (pSE->getComp() == EC_SAME) ? pSE->getDir() : !pSE->getDir(); std::string vertSuffix = (suffixDir == ED_SENSE) ? vertSeq.substr(vertSeq.length() - label.length()) : vertSeq.substr(0, label.length()); if(pSE->getComp() == EC_REVERSE) vertSuffix = reverseComplement(vertSuffix); if(vertSuffix != label) { std::cerr << "Warning edge label " << label << " does not match vertex suffix " << vertSuffix << "\n"; } */ } } // void Vertex::sortAdjListByID() { EdgeIDComp comp; std::sort(m_edges.begin(), m_edges.end(), comp); } void Vertex::sortAdjListByLen() { EdgeLenComp comp; std::sort(m_edges.begin(), m_edges.end(), comp); } // Mark duplicate edges with dupColor // Returns true if a duplicate has been marked bool Vertex::markDuplicateEdges(GraphColor dupColor) { // Sort the edge lists by length sortAdjListByLen(); bool hasDup = false; hasDup = markDuplicateEdges(ED_SENSE, dupColor) || hasDup; hasDup = markDuplicateEdges(ED_ANTISENSE, dupColor) || hasDup; return hasDup; } // Mark duplicate edges in the specified direction bool Vertex::markDuplicateEdges(EdgeDir dir, GraphColor dupColor) { for(EdgePtrVecIter iter = m_edges.begin(); iter != m_edges.end(); ++iter) { Edge* pEdge = *iter; if(pEdge->getDir() == dir) { Vertex* pY = pEdge->getEnd(); if(pY->getColor() == GC_BLACK) { //std::cerr << getID() << " has a duplicate edge to " << pEdge->getEndID() << " in direction " << dir << "\n"; // This vertex is the endpoint of some other (potentially longer) edge // Delete the edge Edge* pTwin = pEdge->getTwin(); pTwin->setColor(dupColor); pEdge->setColor(dupColor); } else { assert(pY->getColor() == GC_WHITE); pY->setColor(GC_BLACK); } } } // Reset vertex colors for(EdgePtrVecIter iter = m_edges.begin(); iter != m_edges.end(); ++iter) (*iter)->getEnd()->setColor(GC_WHITE); return true; } // Get a multioverlap object representing the overlaps for this vertex MultiOverlap Vertex::getMultiOverlap() const { MultiOverlap mo(getID(), getSeq().toString()); for(size_t i = 0; i < m_edges.size(); ++i) { Edge* pEdge = m_edges[i]; mo.add(pEdge->getEnd()->getSeq().toString(), pEdge->getOverlap()); } return mo; } // Add an edge void Vertex::addEdge(Edge* ep) { assert(ep->getStart() == this); #ifdef VALIDATE for(EdgePtrVecConstIter iter = m_edges.begin(); iter != m_edges.end(); ++iter) { if((*iter)->getEndID() == ep->getEndID()) { std::cout << "Attempted to add duplicate edge with ID: " << ep->getEndID() << " to vertex: " << ep->getStartID() << "\n"; std::cout << "Added in desc: " << ep->getDesc() << " curr desc: " << (*iter)->getDesc() << "\n"; //assert(false); } } #endif m_edges.push_back(ep); } // Remove an edge from the edge list of the vertex // and free the storage. This does not remove the twin void Vertex::deleteEdge(Edge* pEdge) { removeEdge(pEdge); delete pEdge; pEdge = NULL; } // Remove an edge but do not destroy it void Vertex::removeEdge(Edge* pEdge) { EdgePtrVecIter iter = m_edges.begin(); while(iter != m_edges.end()) { if(*iter == pEdge) break; ++iter; } assert(iter != m_edges.end()); m_edges.erase(iter); } // void Vertex::removeEdge(const EdgeDesc& ed) { EdgePtrVecIter iter = findEdge(ed); if(iter == m_edges.end()) { std::cout << "EDGE NOT FOUND: " << ed << "\n"; } assert(iter != m_edges.end()); m_edges.erase(iter); } // Delete all the edges, and their twins, from this vertex void Vertex::deleteEdges() { EdgePtrVecIter iter = m_edges.begin(); for(; iter != m_edges.end(); ++iter) { Edge* pEdge = *iter; Edge* pTwin = pEdge->getTwin(); Vertex* pPartner = pEdge->getEnd(); pPartner->removeEdge(pTwin); delete pEdge; pEdge = NULL; delete pTwin; pTwin = NULL; *iter = NULL; } m_edges.clear(); } // Delete edges that are marked // This only deletes the edge and not its twin int Vertex::sweepEdges(GraphColor c) { int numRemoved = 0; EdgePtrVecIter iter = m_edges.begin(); while(iter != m_edges.end()) { Edge* pEdge = *iter; if(pEdge->getColor() == c) { delete pEdge; pEdge = NULL; iter = m_edges.erase(iter); ++numRemoved; } else ++iter; } return numRemoved; } // Return the iterator to the edge matching edgedesc EdgePtrVecIter Vertex::findEdge(const EdgeDesc& ed) { for(EdgePtrVecIter iter = m_edges.begin(); iter != m_edges.end(); ++iter) { if((*iter)->getDesc() == ed) return iter; } return m_edges.end(); } // EdgePtrVecConstIter Vertex::findEdge(const EdgeDesc& ed) const { for(EdgePtrVecConstIter iter = m_edges.begin(); iter != m_edges.end(); ++iter) { if((*iter)->getDesc() == ed) return iter; } return m_edges.end(); } // Check for the presence of an edge bool Vertex::hasEdge(Edge* pEdge) const { return hasEdge(pEdge->getDesc()); } // bool Vertex::hasEdge(const EdgeDesc& ed) const { return findEdge(ed) != m_edges.end(); } // bool Vertex::hasEdgeTo(const Vertex* pY) const { assert(pY != NULL); EdgePtrVecConstIter iter = m_edges.begin(); for(; iter != m_edges.end(); ++iter) if((*iter)->getEnd() == pY) return true; return false; } // Return the edge matching the descriptions Edge* Vertex::getEdge(const EdgeDesc& ed) { EdgePtrVecIter i = findEdge(ed); assert(i != m_edges.end()); return *i; } // Find edges to the specified vertex EdgePtrVec Vertex::findEdgesTo(VertexID id) { EdgePtrVecConstIter iter = m_edges.begin(); EdgePtrVec outEdges; for(; iter != m_edges.end(); ++iter) { if((*iter)->getEndID() == id) outEdges.push_back(*iter); } return outEdges; } // Returns the edge with the longest overlap length // in direction dir // Returns NULL if the vertex has no edges Edge* Vertex::getLongestOverlapEdge(EdgeDir dir) const { Edge* pOut = NULL; int maxOL = 0; EdgePtrVecConstIter iter = m_edges.begin(); for(; iter != m_edges.end(); ++iter) { if((*iter)->getDir() != dir) continue; int currOL = (*iter)->getMatchLength(); if(currOL > maxOL) { pOut = *iter; maxOL = currOL; } } return pOut; } // // Get the edges in a particular direction // This preserves the ordering of the edges // EdgePtrVec Vertex::getEdges(EdgeDir dir) const { EdgePtrVecConstIter iter = m_edges.begin(); EdgePtrVec outEdges; for(; iter != m_edges.end(); ++iter) { if((*iter)->getDir() == dir) outEdges.push_back(*iter); } return outEdges; } // Get the edges EdgePtrVec Vertex::getEdges() const { EdgePtrVec outEdges(m_edges.begin(), m_edges.end()); return outEdges; } void Vertex::setEdgeColors(GraphColor c) { for(EdgePtrVecIter iter = m_edges.begin(); iter != m_edges.end(); ++iter) (*iter)->setColor(c); } // Count the edges // This function is not necessarily constant time size_t Vertex::countEdges() const { return m_edges.size(); } // size_t Vertex::countEdges(EdgeDir dir) { EdgePtrVec ev = getEdges(dir); return ev.size(); } // Calculate the difference in overlap lengths between // the longest and second longest edge int Vertex::getOverlapLengthDiff(EdgeDir dir) const { int longest_len = 0; int second_longest_len = 0; EdgePtrVecConstIter iter = m_edges.begin(); for(; iter != m_edges.end(); ++iter) { if((*iter)->getDir() != dir) continue; int currOL = (*iter)->getMatchLength(); if(currOL > longest_len) { second_longest_len = longest_len; longest_len = currOL; } else if(currOL > second_longest_len) { second_longest_len = currOL; } } return longest_len - second_longest_len; } // Return the amount of memory this vertex is using, in bytes size_t Vertex::getMemSize() const { return sizeof(*this) + (m_edges.size() * sizeof(Edge*)) + m_seq.getMemSize(); } // Output edges in graphviz format void Vertex::writeEdges(std::ostream& out, int dotFlags) const { EdgePtrVecConstIter iter = m_edges.begin(); for(; iter != m_edges.end(); ++iter) { if(dotFlags & DF_UNDIRECTED) { if((*iter)->getStartID() < (*iter)->getEndID()) { out << "\"" << (*iter)->getStartID() << "\" -- \"" << (*iter)->getEndID() << "\""; } } else { out << "\"" << (*iter)->getStartID() << "\" -> \"" << (*iter)->getEndID(); std::string color = ((*iter)->getDir() == ED_SENSE) ? "black" : "red"; std::string label = ((*iter)->getComp() == EC_SAME) ? "S" : "F"; out << "\" [color=\"" << color << "\" "; out << "label=\"" << (*iter)->getMatchLength() << "\""; out << "];"; } out << "\n"; } } bool EdgeIDComp::operator()(const Edge* pA, const Edge* pB) { return pA->getEndID() < pB->getEndID(); } // Compare string edge points by length bool EdgeLenComp::operator()(const Edge* pA, const Edge* pB) { return pA->getSeqLen() < pB->getSeqLen(); } ================================================ FILE: src/SGA/Bigraph/Vertex.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL license //----------------------------------------------- // // Vertex - Generic vertex class for bigraph // #ifndef VERTEX_H #define VERTEX_H // Includes #include #include #include #include #include #include #include #include #include "GraphCommon.h" #include "QualityVector.h" #include "EncodedString.h" #include "SimpleAllocator.h" #include "EdgeDesc.h" #include "MultiOverlap.h" // Forward declare class Edge; // Default edge sorting function, by ID struct EdgeIDComp { bool operator()(const Edge* pA, const Edge* pB); }; // Edge sorting function, by length struct EdgeLenComp { bool operator()(const Edge* pA, const Edge* pB); }; // Typedefs typedef std::map EdgePtrMap; typedef std::vector EdgePtrVec; typedef std::set EdgeDescSet; typedef std::list EdgePtrList; typedef EdgePtrMap::iterator EdgePtrMapIter; typedef EdgePtrMap::const_iterator EdgePtrMapConstIter; typedef EdgePtrVec::iterator EdgePtrVecIter; typedef EdgePtrVec::const_iterator EdgePtrVecConstIter; typedef EdgePtrList::iterator EdgePtrListIter; typedef EdgePtrList::const_iterator EdgePtrListConstIter; class Vertex { public: Vertex(VertexID id, const std::string& s) : m_id(id), m_seq(s), m_color(GC_WHITE), m_coverage(1), m_isContained(false), m_isSuperRepeat(false) { //m_parent_id.push_back(id); //JEREMIAH /*std::cerr << "ID: " << id << "Seq: " << s << std::endl;*/ } ~Vertex(); // High-level modification functions // Merge another vertex into this vertex, as specified by pEdge void merge(Edge* pEdge); // sort the edges by the ID of the vertex they point to void sortAdjListByID(); // sort the edges by the length of the label of the edge void sortAdjListByLen(); // Ensure that all the edges are unique bool markDuplicateEdges(GraphColor dupColor); //JEREMIAH //void addParent(VertexID mid) { m_parent_id.push_back(mid);} ; //std::vector getParentID() { return m_parent_id; } ; // Get a multioverlap object representing the overlaps for this vertex MultiOverlap getMultiOverlap() const; // Edge list operations void addEdge(Edge* ep); void removeEdge(Edge* pEdge); void removeEdge(const EdgeDesc& ed); void deleteEdge(Edge* pEdge); void deleteEdges(); int sweepEdges(GraphColor c); bool hasEdge(Edge* pEdge) const; bool hasEdge(const EdgeDesc& ed) const; bool hasEdgeTo(const Vertex* pY) const; Edge* getEdge(const EdgeDesc& ed); EdgePtrVec findEdgesTo(VertexID id); EdgePtrVec getEdges(EdgeDir dir) const; EdgePtrVec getEdges() const; EdgePtrVecIter findEdge(const EdgeDesc& ed); EdgePtrVecConstIter findEdge(const EdgeDesc& ed) const; Edge* getLongestOverlapEdge(EdgeDir dir) const; size_t countEdges() const; size_t countEdges(EdgeDir dir); // Calculate the difference in overlap lengths between // the longest and second longest edge int getOverlapLengthDiff(EdgeDir dir) const; // Ensure the vertex data is sane void validate() const; // setters void setID(VertexID id) { m_id = id; } void setEdgeColors(GraphColor c); void setSeq(const std::string& s) { m_seq = s; } void setColor(GraphColor c) { m_color = c; } void setContained(bool c) { m_isContained = c; } void setSuperRepeat(bool b) { m_isSuperRepeat = b; } // getters VertexID getID() const { return m_id; } GraphColor getColor() const { return m_color; } const DNAEncodedString& getSeq() const { return m_seq; } std::string getStr() const { return m_seq.toString(); } size_t getSeqLen() const { return m_seq.length(); } size_t getMemSize() const; bool isContained() const { return m_isContained; } bool isSuperRepeat() const { return m_isSuperRepeat; } uint16_t getCoverage() const { return m_coverage; } // Memory management //void* operator new(size_t /*size*/, SimpleAllocator* pAllocator) //{ // return pAllocator->alloc(); //} //void operator delete(void* /*target*/, size_t /*size*/) //{ // delete does nothing since all allocations go through the memory pool // belonging to the graph. 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std::istringstream convertor(hitString); sumBlockSize = 0; // Read the overlap blocks for a read size_t numBlocks; convertor >> readIdx >> isSubstring >> numBlocks; for(size_t i = 0; i < numBlocks; ++i) { // Read the block OverlapBlock record; convertor >> record; //std::cout << "\t" << record << "\n"; // Iterate through the range and write the overlaps for(int64_t j = record.ranges.interval[0].lower; j <= record.ranges.interval[0].upper; ++j) { sumBlockSize += 1; const SuffixArray* pCurrSAI = (record.flags.isTargetRev()) ? pRevSAI : pFwdSAI; const ReadInfo& queryInfo = pQueryRIT->getReadInfo(readIdx); int64_t saIdx = j; // The index of the second read is given as the position in the SuffixArray index const ReadInfo& targetInfo = pTargetRIT->getReadInfo(pCurrSAI->get(saIdx).getID()); //std::cout << " HERE RER " << queryInfo.id << " " << targetInfo.id << " " << queryInfo.length << " " << targetInfo.length << "\n"; // Skip self alignments and non-canonical (where the query read has a lexo. higher name) if(queryInfo.id != targetInfo.id) { Overlap o = record.toOverlap(queryInfo.id, targetInfo.id, queryInfo.length, targetInfo.length); //std::cout << queryInfo.id << " " << targetInfo.id << " " << queryInfo.length << " " << targetInfo.length << "\n"; // The alignment logic above has the potential to produce duplicate alignments // To avoid this, we skip overlaps where the id of the first coord is lexo. lower than // the second or the match is a containment and the query is reversed (containments can be // output up to 4 times total). if(bCheckIDs && (o.id[0] < o.id[1] || (o.match.isContainment() && record.flags.isQueryRev()))) continue; outVector.push_back(o); } } } } ================================================ FILE: src/SGA/SGA/OverlapCommon.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // OverlapCommon - Common functions used in overlap methods // #ifndef OVERLAPCOMMON_H #define OVERLAPCOMMON_H #include "Util.h" #include "overlap.h" #include "SuffixArray.h" #include "SGACommon.h" #include "Timer.h" #include "ReadInfoTable.h" namespace OverlapCommon { // Parse a line from a .hits file into a variety of overlap-related data void parseHitsString(const std::string& hitString, const ReadInfoTable* pQueryRIT, const ReadInfoTable* pTargetRIT, const SuffixArray* pFwdSAI, const SuffixArray* pRevSAI, bool bCheckIDs, size_t& readIdx, size_t& sumBlockSize, OverlapVector& outVector, bool& isSubstring); }; #endif ================================================ FILE: src/SGA/SGA/SGACommon.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL license //----------------------------------------------- // // SGACommon.h - Common definitions used in the // SGA programs // #ifndef SGACOMMON_H #define SGACOMMON_H // File extensions #define OVR_EXT ".ovr" #define HITS_EXT ".hits" #define RMDUPHITS_EXT ".rmhits" #define GMAPHITS_EXT ".gmhits" #define CTN_EXT ".ctn" #define ASQG_EXT ".asqg" #define SA_EXT ".sa" #define RSA_EXT ".rsa" #define BWT_EXT ".bwt" #define RBWT_EXT ".rbwt" #define SAI_EXT ".sai" #define RSAI_EXT ".rsai" #define SSA_EXT ".ssa" #define POPIDX_EXT ".popidx" // Default values #define DEFAULT_MIN_OVERLAP 45 #define DEFAULT_EXTRACT_LEN 100 #endif ================================================ FILE: src/SGA/SGA/index.cpp ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // index - Build a BWT/FM-index for a set of reads // #include #include #include #include "SGACommon.h" #include "Util.h" #include "index.h" #include "SuffixArray.h" #include "SeqReader.h" #include "SACAInducedCopying.h" #include "BWTDiskConstruction.h" #include "BWT.h" #include "Timer.h" #include "BWTCABauerCoxRosone.h" #include "BWTCARopebwt.h" #include "SampledSuffixArray.h" // // Getopt // #define SUBPROGRAM "index" static const char *INDEX_VERSION_MESSAGE = SUBPROGRAM " Version " PACKAGE_VERSION "\n" "Written by Jared Simpson.\n" "\n" "Copyright 2009 Wellcome Trust Sanger Institute\n"; static const char *INDEX_USAGE_MESSAGE = "Usage: " PACKAGE_NAME " " SUBPROGRAM " [OPTION] ... READSFILE\n" "Index the reads in READSFILE using a suffixarray/bwt\n" "\n" " -v, --verbose display verbose output\n" " --help display this help and exit\n" " -a, --algorithm=STR BWT construction algorithm. STR can be:\n" " sais - induced sort algorithm, slower but works for very long sequences (default)\n" " ropebwt - very fast and memory efficient. use this for short (<200bp) reads\n" " -d, --disk=NUM use disk-based BWT construction algorithm. The suffix array/BWT will be constructed\n" " for batchs of NUM reads at a time. To construct the suffix array of 200 megabases of sequence\n" " requires ~2GB of memory, set this parameter accordingly.\n" " -t, --threads=NUM use NUM threads to construct the index (default: 1)\n" " -c, --check validate that the suffix array/bwt is correct\n" " -p, --prefix=PREFIX write index to file using PREFIX instead of prefix of READSFILE\n" " --no-reverse suppress construction of the reverse BWT. Use this option when building the index\n" " for reads that will be error corrected using the k-mer corrector, which only needs the forward index\n" " --no-forward suppress construction of the forward BWT. Use this option when building the forward and reverse index separately\n" " --no-sai suppress construction of the SAI file. This option only applies to -a ropebwt\n" " -g, --gap-array=N use N bits of storage for each element of the gap array. Acceptable values are 4,8,16 or 32. Lower\n" " values can substantially reduce the amount of memory required at the cost of less predictable memory usage.\n" " When this value is set to 32, the memory requirement is essentially deterministic and requires ~5N bytes where\n" " N is the size of the FM-index of READS2.\n" " The default value is 8.\n" "\nReport bugs to " PACKAGE_BUGREPORT "\n\n"; namespace opt { static unsigned int verbose; static std::string readsFile; static std::string prefix; static std::string algorithm = "sais"; static int numReadsPerBatch = 2000000; static int numThreads = 1; static bool bDiskAlgo = false; static bool bBuildReverse = true; static bool bBuildForward = true; static bool bBuildSAI = true; static bool validate; static int gapArrayStorage = 4; } static const char* shortopts = "p:a:m:t:d:g:cv"; enum { OPT_HELP = 1, OPT_VERSION, OPT_NO_REVERSE, OPT_NO_FWD, OPT_NO_SAI }; static const struct option longopts[] = { { "verbose", no_argument, NULL, 'v' }, { "check", no_argument, NULL, 'c' }, { "prefix", required_argument, NULL, 'p' }, { "threads", required_argument, NULL, 't' }, { "disk", required_argument, NULL, 'd' }, { "gap-array", required_argument, NULL, 'g' }, { "algorithm", required_argument, NULL, 'a' }, { "no-reverse", no_argument, NULL, OPT_NO_REVERSE }, { "no-forward", no_argument, NULL, OPT_NO_FWD }, { "no-sai", no_argument, NULL, OPT_NO_SAI }, { "help", no_argument, NULL, OPT_HELP }, { "version", no_argument, NULL, OPT_VERSION }, { NULL, 0, NULL, 0 } }; int indexMain(int argc, char** argv) { Timer t("sga index"); parseIndexOptions(argc, argv); if(!opt::bDiskAlgo) { if(opt::algorithm == "sais") indexInMemorySAIS(); else if(opt::algorithm == "bcr") indexInMemoryBCR(); else if(opt::algorithm == "ropebwt") indexInMemoryRopebwt(); } else { indexOnDisk(); } return 0; } // void indexInMemoryBCR() { std::cout << "Building index for " << opt::readsFile << " in memory using BCR\n"; if(opt::bBuildForward || opt::bBuildReverse) { // Parse the initial read table std::vector readSequences; SeqReader reader(opt::readsFile); SeqRecord sr; while(reader.get(sr)) readSequences.push_back(sr.seq.toString()); if(opt::bBuildForward) { BWTCA::runBauerCoxRosone(&readSequences, opt::prefix + BWT_EXT, opt::prefix + SAI_EXT); } if(opt::bBuildReverse) { // Reverse all the reads for(size_t i = 0; i < readSequences.size(); ++i) readSequences[i] = reverse(readSequences[i].toString()); BWTCA::runBauerCoxRosone(&readSequences, opt::prefix + RBWT_EXT, opt::prefix + RSAI_EXT); } } } // void indexInMemoryRopebwt() { std::cout << "Building index for " << opt::readsFile << " in memory using ropebwt\n"; bool use_threads = opt::numThreads >= 4; if(opt::bBuildForward) { std::string bwt_filename = opt::prefix + BWT_EXT; std::string sai_filename = opt::prefix + SAI_EXT; BWTCA::runRopebwt(opt::readsFile, bwt_filename, use_threads, false); if(opt::bBuildSAI) { std::cout << "\t done bwt construction, generating .sai file\n"; BWT* pBWT = new BWT(bwt_filename); SampledSuffixArray ssa; ssa.buildLexicoIndex(pBWT, opt::numThreads); ssa.writeLexicoIndex(sai_filename); delete pBWT; } } if(opt::bBuildReverse) { std::string rbwt_filename = opt::prefix + RBWT_EXT; std::string rsai_filename = opt::prefix + RSAI_EXT; BWTCA::runRopebwt(opt::readsFile, rbwt_filename, use_threads, true); if(opt::bBuildSAI) { std::cout << "\t done rbwt construction, generating .rsai file\n"; BWT* pRBWT = new BWT(rbwt_filename); SampledSuffixArray ssa; ssa.buildLexicoIndex(pRBWT, opt::numThreads); ssa.writeLexicoIndex(rsai_filename); delete pRBWT; } } } // void indexInMemorySAIS() { std::cout << "Building index for " << opt::readsFile << " in memory using SAIS\n"; if(opt::bBuildForward || opt::bBuildReverse) { // Parse the initial read table ReadTable* pRT = new ReadTable(opt::readsFile); // Create and write the suffix array for the forward reads if(opt::bBuildForward) { buildIndexForTable(opt::prefix, pRT, false); } if(opt::bBuildReverse) { // Reverse all the reads pRT->reverseAll(); // Build the reverse suffix array buildIndexForTable(opt::prefix, pRT, true); } delete pRT; } } // void indexOnDisk() { std::cout << "Building index for " << opt::readsFile << " on disk\n"; BWTDiskParameters parameters; parameters.inFile = opt::readsFile; parameters.outPrefix = opt::prefix; parameters.bwtExtension = BWT_EXT; parameters.saiExtension = SAI_EXT; parameters.numReadsPerBatch = opt::numReadsPerBatch; parameters.numThreads = opt::numThreads; parameters.storageLevel = opt::gapArrayStorage; parameters.bBuildReverse = false; parameters.bUseBCR = (opt::algorithm == "bcr"); if(opt::bBuildForward) { buildBWTDisk(parameters); } if(opt::bBuildReverse) { parameters.bwtExtension = RBWT_EXT; parameters.saiExtension = RSAI_EXT; parameters.bBuildReverse = true; buildBWTDisk(parameters); } } // void buildIndexForTable(std::string prefix, const ReadTable* pRT, bool isReverse) { // Create suffix array from read table SuffixArray* pSA = new SuffixArray(pRT, opt::numThreads); if(opt::validate) { std::cout << "Validating suffix array\n"; pSA->validate(pRT); } std::string bwt_filename = prefix + (!isReverse ? BWT_EXT : RBWT_EXT); pSA->writeBWT(bwt_filename, pRT); std::string sufidx_filename = prefix + (!isReverse ? SAI_EXT : RSAI_EXT); pSA->writeIndex(sufidx_filename); delete pSA; pSA = NULL; } // // Handle command line arguments // void parseIndexOptions(int argc, char** argv) { bool die = false; for (char c; (c = getopt_long(argc, argv, shortopts, longopts, NULL)) != -1;) { std::istringstream arg(optarg != NULL ? optarg : ""); switch (c) { case 'p': arg >> opt::prefix; break; case '?': die = true; break; case 'c': opt::validate = true; break; case 'd': opt::bDiskAlgo = true; arg >> opt::numReadsPerBatch; break; case 't': arg >> opt::numThreads; break; case 'g': arg >> opt::gapArrayStorage; break; case 'a': arg >> opt::algorithm; break; case 'v': opt::verbose++; break; case OPT_NO_REVERSE: opt::bBuildReverse = false; break; case OPT_NO_FWD: opt::bBuildForward = false; break; case OPT_NO_SAI: opt::bBuildSAI = false; break; case OPT_HELP: std::cout << INDEX_USAGE_MESSAGE; exit(EXIT_SUCCESS); case OPT_VERSION: std::cout << INDEX_VERSION_MESSAGE; exit(EXIT_SUCCESS); } } // Transform algorithm parameter to lower case std::transform(opt::algorithm.begin(), opt::algorithm.end(), opt::algorithm.begin(), ::tolower); if (argc - optind < 1) { std::cerr << SUBPROGRAM ": missing arguments\n"; die = true; } else if (argc - optind > 1) { std::cerr << SUBPROGRAM ": too many arguments\n"; die = true; } if(opt::gapArrayStorage != 4 && opt::gapArrayStorage != 8 && opt::gapArrayStorage != 16 && opt::gapArrayStorage != 32) { std::cerr << SUBPROGRAM ": invalid argument, --gap-array,-g must be one of 4,8,16,32 (found: " << opt::gapArrayStorage << ")\n"; die = true; } if(opt::numThreads <= 0) { std::cerr << SUBPROGRAM ": invalid number of threads: " << opt::numThreads << "\n"; die = true; } if(opt::algorithm != "sais" && opt::algorithm != "bcr" && opt::algorithm != "ropebwt") { std::cerr << SUBPROGRAM ": unrecognized algorithm string " << opt::algorithm << ". --algorithm must be sais, bcr or ropebwt\n"; die = true; } if(opt::algorithm == "ropebwt" && opt::bDiskAlgo) { std::cerr << SUBPROGRAM ": the options -a ropebwt and -d are not compatible, please only use one.\n"; die = true; } if (die) { std::cout << "\n" << INDEX_USAGE_MESSAGE; exit(EXIT_FAILURE); } // Parse the input filenames opt::readsFile = argv[optind++]; if(opt::prefix.empty()) opt::prefix = stripFilename(opt::readsFile); // Check if input file is empty size_t filesize = getFilesize(opt::readsFile); if(filesize == 0) { std::cerr << SUBPROGRAM ": input file is empty\n"; exit(EXIT_FAILURE); } } ================================================ FILE: src/SGA/SGA/index.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL license //----------------------------------------------- // // index - Build a BWT/FM-index for a set of reads // #ifndef INDEX_H #define INDEX_H #include #include "config.h" #include "SuffixArray.h" int indexMain(int argc, char** argv); void indexInMemorySAIS(); void indexInMemoryBCR(); void indexInMemoryRopebwt(); void indexOnDisk(); void buildIndexForTable(std::string outfile, const ReadTable* pRT, bool isReverse); void parseIndexOptions(int argc, char** argv); #endif ================================================ FILE: src/SGA/SGA/overlap.cpp ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // overlap - compute pairwise overlaps between reads // #include #include #include #include #include "Util.h" #include "overlap.h" #include "SuffixArray.h" #include "BWT.h" #include "SGACommon.h" #include "OverlapCommon.h" #include "Timer.h" #include "BWTAlgorithms.h" #include "ASQG.h" #include "gzstream.h" #include "SequenceProcessFramework.h" #include "OverlapProcess.h" #include "ReadInfoTable.h" // enum OutputType { OT_ASQG, OT_RAW }; // Functions size_t computeHitsSerial(const std::string& prefix, const std::string& readsFile, const OverlapAlgorithm* pOverlapper, int minOverlap, StringVector& filenameVec, std::ostream* pASQGWriter); size_t computeHitsParallel(int numThreads, const std::string& prefix, const std::string& readsFile, const OverlapAlgorithm* pOverlapper, int minOverlap, StringVector& filenameVec, std::ostream* pASQGWriter); // void convertHitsToASQG(const std::string& indexPrefix, const StringVector& hitsFilenames, std::ostream* pASQGWriter); // // Getopt // #define SUBPROGRAM "overlap" static const char *OVERLAP_VERSION_MESSAGE = SUBPROGRAM " Version " PACKAGE_VERSION "\n" "Written by Jared Simpson.\n" "\n" "Copyright 2009 Wellcome Trust Sanger Institute\n"; static const char *OVERLAP_USAGE_MESSAGE = "Usage: " PACKAGE_NAME " " SUBPROGRAM " [OPTION] ... READSFILE\n" "Compute pairwise overlap between all the sequences in READS\n" "\n" " --help display this help and exit\n" " -v, --verbose display verbose output\n" " -t, --threads=NUM use NUM worker threads to compute the overlaps (default: no threading)\n" " -e, --error-rate the maximum error rate allowed to consider two sequences aligned (default: exact matches only)\n" " -m, --min-overlap=LEN minimum overlap required between two reads (default: 45)\n" " -p, --prefix=PREFIX use PREFIX for the names of the index files (default: prefix of the input file)\n" " -f, --target-file=FILE perform the overlap queries against the reads in FILE\n" " -x, --exhaustive output all overlaps, including transitive edges\n" " --exact force the use of the exact-mode irreducible block algorithm. This is faster\n" " but requires that no substrings are present in the input set.\n" " -l, --seed-length=LEN force the seed length to be LEN. By default, the seed length in the overlap step\n" " is calculated to guarantee all overlaps with --error-rate differences are found.\n" " This option removes the guarantee but will be (much) faster. As SGA can tolerate some\n" " missing edges, this option may be preferable for some data sets.\n" " -s, --seed-stride=LEN force the seed stride to be LEN. This parameter will be ignored unless --seed-length\n" " is specified (see above). This parameter defaults to the same value as --seed-length\n" " -d, --sample-rate=N sample the symbol counts every N symbols in the FM-index. Higher values use significantly\n" " less memory at the cost of higher runtime. This value must be a power of 2 (default: 128)\n" "\nReport bugs to " PACKAGE_BUGREPORT "\n\n"; static const char* PROGRAM_IDENT = PACKAGE_NAME "::" SUBPROGRAM; namespace opt { static unsigned int verbose; static int numThreads = 1; static OutputType outputType = OT_ASQG; static std::string readsFile; static std::string targetFile; static std::string outFile; static std::string prefix; static double errorRate = 0.0f; static unsigned int minOverlap = DEFAULT_MIN_OVERLAP; static int seedLength = 0; static int seedStride = 0; static int sampleRate = BWT::DEFAULT_SAMPLE_RATE_SMALL; static bool bIrreducibleOnly = true; static bool bExactIrreducible = false; } static const char* shortopts = "m:d:e:t:l:s:o:f:p:vix"; enum { OPT_HELP = 1, OPT_VERSION, OPT_EXACT }; static const struct option longopts[] = { { "verbose", no_argument, NULL, 'v' }, { "threads", required_argument, NULL, 't' }, { "min-overlap", required_argument, NULL, 'm' }, { "sample-rate", required_argument, NULL, 'd' }, { "outfile", required_argument, NULL, 'o' }, { "target-file", required_argument, NULL, 'f' }, { "prefix", required_argument, NULL, 'p' }, { "error-rate", required_argument, NULL, 'e' }, { "seed-length", required_argument, NULL, 'l' }, { "seed-stride", required_argument, NULL, 's' }, { "exhaustive", no_argument, NULL, 'x' }, { "exact", no_argument, NULL, OPT_EXACT }, { "help", no_argument, NULL, OPT_HELP }, { "version", no_argument, NULL, OPT_VERSION }, { NULL, 0, NULL, 0 } }; // // Main // int overlapMain(int argc, char** argv) { parseOverlapOptions(argc, argv); // Prepare the output ASQG file assert(opt::outputType == OT_ASQG); // Open output file std::ostream* pASQGWriter = createWriter(opt::outFile); // Build and write the ASQG header ASQG::HeaderRecord headerRecord; headerRecord.setOverlapTag(opt::minOverlap); headerRecord.setErrorRateTag(opt::errorRate); headerRecord.setInputFileTag(opt::readsFile); headerRecord.setContainmentTag(true); // containments are always present headerRecord.setTransitiveTag(!opt::bIrreducibleOnly); headerRecord.write(*pASQGWriter); // Compute the overlap hits StringVector hitsFilenames; // Determine which index files to use. If a target file was provided, // use the index of the target reads std::string indexPrefix; if(!opt::prefix.empty()) indexPrefix = opt::prefix; else { if(!opt::targetFile.empty()) indexPrefix = stripFilename(opt::targetFile); else indexPrefix = stripFilename(opt::readsFile); } BWT* pBWT = new BWT(indexPrefix + BWT_EXT, opt::sampleRate); BWT* pRBWT = new BWT(indexPrefix + RBWT_EXT, opt::sampleRate); OverlapAlgorithm* pOverlapper = new OverlapAlgorithm(pBWT, pRBWT, opt::errorRate, opt::seedLength, opt::seedStride, opt::bIrreducibleOnly); pOverlapper->setExactModeOverlap(opt::errorRate <= 0.0001); pOverlapper->setExactModeIrreducible(opt::errorRate <= 0.0001); Timer* pTimer = new Timer(PROGRAM_IDENT); pBWT->printInfo(); // Make a prefix for the temporary hits files std::string outPrefix; outPrefix = stripFilename(opt::readsFile); if(!opt::targetFile.empty()) { outPrefix.append(1, '.'); outPrefix.append(stripFilename(opt::targetFile)); } if(opt::numThreads <= 1) { printf("[%s] starting serial-mode overlap computation\n", PROGRAM_IDENT); computeHitsSerial(outPrefix, opt::readsFile, pOverlapper, opt::minOverlap, hitsFilenames, pASQGWriter); } else { printf("[%s] starting parallel-mode overlap computation with %d threads\n", PROGRAM_IDENT, opt::numThreads); computeHitsParallel(opt::numThreads, outPrefix, opt::readsFile, pOverlapper, opt::minOverlap, hitsFilenames, pASQGWriter); } // Get the number of strings in the BWT, this is used to pre-allocated the read table delete pOverlapper; delete pBWT; delete pRBWT; // Parse the hits files and write the overlaps to the ASQG file convertHitsToASQG(indexPrefix, hitsFilenames, pASQGWriter); // Cleanup delete pASQGWriter; delete pTimer; if(opt::numThreads > 1) pthread_exit(NULL); return 0; } // Compute the hits for each read in the input file without threading // Return the number of reads processed size_t computeHitsSerial(const std::string& prefix, const std::string& readsFile, const OverlapAlgorithm* pOverlapper, int minOverlap, StringVector& filenameVec, std::ostream* pASQGWriter) { std::string filename = prefix + HITS_EXT + GZIP_EXT; filenameVec.push_back(filename); OverlapProcess processor(filename, pOverlapper, minOverlap); OverlapPostProcess postProcessor(pASQGWriter, pOverlapper); size_t numProcessed = SequenceProcessFramework::processSequencesSerial(readsFile, &processor, &postProcessor); return numProcessed; } // Compute the hits for each read in the SeqReader file with threading // The way this works is we create a vector of numThreads OverlapProcess pointers and // pass this to the SequenceProcessFramework which wraps the processes // in threads and distributes the reads to each thread. // The number of reads processsed is returned size_t computeHitsParallel(int numThreads, const std::string& prefix, const std::string& readsFile, const OverlapAlgorithm* pOverlapper, int minOverlap, StringVector& filenameVec, std::ostream* pASQGWriter) { std::string filename = prefix + HITS_EXT + GZIP_EXT; std::vector processorVector; for(int i = 0; i < numThreads; ++i) { std::stringstream ss; ss << prefix << "-thread" << i << HITS_EXT << GZIP_EXT; std::string outfile = ss.str(); filenameVec.push_back(outfile); OverlapProcess* pProcessor = new OverlapProcess(outfile, pOverlapper, minOverlap); processorVector.push_back(pProcessor); } // The post processing is performed serially so only one post processor is created OverlapPostProcess postProcessor(pASQGWriter, pOverlapper); size_t numProcessed = SequenceProcessFramework::processSequencesParallel(readsFile, processorVector, &postProcessor); for(int i = 0; i < numThreads; ++i) delete processorVector[i]; return numProcessed; } // void convertHitsToASQG(const std::string& indexPrefix, const StringVector& hitsFilenames, std::ostream* pASQGWriter) { // Load the suffix array index and the reverse suffix array index // Note these are not the full suffix arrays SuffixArray* pFwdSAI = new SuffixArray(indexPrefix + SAI_EXT); SuffixArray* pRevSAI = new SuffixArray(indexPrefix + RSAI_EXT); // Load the ReadInfoTable for the queries to look up the ID and lengths of the hits ReadInfoTable* pQueryRIT = new ReadInfoTable(opt::readsFile); // If the target file is not the query file, load its ReadInfoTable ReadInfoTable* pTargetRIT; if(!opt::targetFile.empty() && opt::targetFile != opt::readsFile) pTargetRIT = new ReadInfoTable(opt::targetFile); else pTargetRIT = pQueryRIT; bool bIsSelfCompare = pTargetRIT == pQueryRIT; // Convert the hits to overlaps and write them to the asqg file as initial edges for(StringVector::const_iterator iter = hitsFilenames.begin(); iter != hitsFilenames.end(); ++iter) { printf("[%s] parsing file %s\n", PROGRAM_IDENT, iter->c_str()); std::istream* pReader = createReader(*iter); // Read each hit sequentially, converting it to an overlap std::string line; while(getline(*pReader, line)) { size_t readIdx; size_t totalEntries; bool isSubstring; OverlapVector ov; OverlapCommon::parseHitsString(line, pQueryRIT, pTargetRIT, pFwdSAI, pRevSAI, bIsSelfCompare, readIdx, totalEntries, ov, isSubstring); std::cout << line << " " << bIsSelfCompare << " " << readIdx << " " << totalEntries << " " << isSubstring << "\n"; for(OverlapVector::iterator iter = ov.begin(); iter != ov.end(); ++iter) { ASQG::EdgeRecord edgeRecord(*iter); edgeRecord.write(*pASQGWriter); } } delete pReader; // delete the hits file //unlink(iter->c_str()); } // Deallocate data if(pTargetRIT != pQueryRIT) delete pTargetRIT; delete pFwdSAI; delete pRevSAI; delete pQueryRIT; } // // Handle command line arguments // void parseOverlapOptions(int argc, char** argv) { bool die = false; for (char c; (c = getopt_long(argc, argv, shortopts, longopts, NULL)) != -1;) { std::istringstream arg(optarg != NULL ? optarg : ""); switch (c) { case 'm': arg >> opt::minOverlap; break; case 'o': arg >> opt::outFile; break; case 'p': arg >> opt::prefix; break; case 'e': arg >> opt::errorRate; break; case 't': arg >> opt::numThreads; break; case 'l': arg >> opt::seedLength; break; case 's': arg >> opt::seedStride; break; case 'd': arg >> opt::sampleRate; break; case 'f': arg >> opt::targetFile; break; case OPT_EXACT: opt::bExactIrreducible = true; break; case 'x': opt::bIrreducibleOnly = false; break; case '?': die = true; break; case 'v': opt::verbose++; break; case OPT_HELP: std::cout << OVERLAP_USAGE_MESSAGE; exit(EXIT_SUCCESS); case OPT_VERSION: std::cout << OVERLAP_VERSION_MESSAGE; exit(EXIT_SUCCESS); } } if (argc - optind < 1) { std::cerr << SUBPROGRAM ": missing arguments\n"; die = true; } else if (argc - optind > 1) { std::cerr << SUBPROGRAM ": too many arguments\n"; die = true; } if(opt::numThreads <= 0) { std::cerr << SUBPROGRAM ": invalid number of threads: " << opt::numThreads << "\n"; die = true; } if(!IS_POWER_OF_2(opt::sampleRate)) { std::cerr << SUBPROGRAM ": invalid parameter to -d/--sample-rate, must be power of 2. got: " << opt::sampleRate << "\n"; die = true; } if (die) { std::cout << "\n" << OVERLAP_USAGE_MESSAGE; exit(EXIT_FAILURE); } // Validate parameters if(opt::errorRate <= 0) opt::errorRate = 0.0f; if(opt::seedLength < 0) opt::seedLength = 0; if(opt::seedLength > 0 && opt::seedStride <= 0) opt::seedStride = opt::seedLength; // Parse the input filenames opt::readsFile = argv[optind++]; if(opt::outFile.empty()) { std::string prefix = stripFilename(opt::readsFile); if(!opt::targetFile.empty()) { prefix.append(1,'.'); prefix.append(stripFilename(opt::targetFile)); } opt::outFile = prefix + ASQG_EXT + GZIP_EXT; } } ================================================ FILE: src/SGA/SGA/overlap.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // overlap - Overlap reads using a bwt // #ifndef OVERLAP_H #define OVERLAP_H #include //#include "config.h" #include "SuffixTools/BWT.h" #include "Match.h" #include "BWTAlgorithms.h" #include "OverlapAlgorithm.h" #include // functions // int overlapMain(int argc, char** argv); // options void parseOverlapOptions(int argc, char** argv); #endif ================================================ FILE: src/SGA/SQG/ASQG.cpp ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // ASQG - Definitions and functions // for handling ASGQ files // #include "ASQG.h" #include namespace ASQG { const int HEADER_VERSION = 1; // Record ID tags static constexpr int RECORD_TAG_SIZE = 2; // do not include null terminator static char HEADER_TAG[] = "HT"; static char VERTEX_TAG[] = "VT"; static char EDGE_TAG[] = "ED"; // Header tags const int FIELD_TAG_SIZE = 2; static char VERSION_TAG[] = "VN"; static char OVERLAP_TAG[] = "OL"; static char INPUT_FILE_TAG[] = "IN"; static char ERROR_RATE_TAG[] = "ER"; static char CONTAINMENT_TAG[] = "CN"; // 1 if the graph has containment edges/vertices static char TRANSITIVE_TAG[] = "TE"; // 1 if the graph has transitive edges // Vertex tags static char SUBSTRING_TAG[] = "SS"; // // Header Record // HeaderRecord::HeaderRecord() { setVersionTag(HEADER_VERSION); } // HeaderRecord::HeaderRecord(const std::string& recordLine) { parse(recordLine); } // void HeaderRecord::setVersionTag(int version) { m_versionTag.set(version); } // void HeaderRecord::setOverlapTag(int overlapLen) { m_overlapTag.set(overlapLen); } // void HeaderRecord::setInputFileTag(const std::string& name) { m_infileTag.set(name); } // void HeaderRecord::setErrorRateTag(float errorRate) { m_errorRateTag.set(errorRate); } // void HeaderRecord::setContainmentTag(int v) { m_containmentTag.set(v); } // void HeaderRecord::setTransitiveTag(int v) { m_transitiveTag.set(v); } // void HeaderRecord::write(std::ostream& out) { StringVector fields; fields.push_back(HEADER_TAG); // Check for mandatory tags if(!m_versionTag.isInitialized()) { std::cerr << "Error: Header version tag not set, aborting." << std::endl; exit(EXIT_FAILURE); } else { fields.push_back(m_versionTag.toTagString(VERSION_TAG)); } if(m_errorRateTag.isInitialized()) fields.push_back(m_errorRateTag.toTagString(ERROR_RATE_TAG)); if(m_overlapTag.isInitialized()) fields.push_back(m_overlapTag.toTagString(OVERLAP_TAG)); if(m_infileTag.isInitialized()) fields.push_back(m_infileTag.toTagString(INPUT_FILE_TAG)); if(m_containmentTag.isInitialized()) fields.push_back(m_containmentTag.toTagString(CONTAINMENT_TAG)); if(m_transitiveTag.isInitialized()) fields.push_back(m_transitiveTag.toTagString(TRANSITIVE_TAG)); writeFields(out, fields); } //JEREMIAH void HeaderRecord::write(std::stringstream& out) { StringVector fields; fields.push_back(HEADER_TAG); // Check for mandatory tags if(!m_versionTag.isInitialized()) { std::cerr << "Error: Header version tag not set, aborting." << std::endl; exit(EXIT_FAILURE); } else { fields.push_back(m_versionTag.toTagString(VERSION_TAG)); } if(m_errorRateTag.isInitialized()) fields.push_back(m_errorRateTag.toTagString(ERROR_RATE_TAG)); if(m_overlapTag.isInitialized()) fields.push_back(m_overlapTag.toTagString(OVERLAP_TAG)); if(m_infileTag.isInitialized()) fields.push_back(m_infileTag.toTagString(INPUT_FILE_TAG)); if(m_containmentTag.isInitialized()) fields.push_back(m_containmentTag.toTagString(CONTAINMENT_TAG)); if(m_transitiveTag.isInitialized()) fields.push_back(m_transitiveTag.toTagString(TRANSITIVE_TAG)); writeFields(out, fields); } // void HeaderRecord::parse(const std::string& record) { if(record.size() < 2) { std::cerr << "Error: Record is not valid\n"; exit(EXIT_FAILURE); } // Tokenize record StringVector tokens = SQG::tokenizeRecord(record); // Ensure the first token indicates this is a valid header record if(tokens[0].compare(0, RECORD_TAG_SIZE, HEADER_TAG) != 0) { std::cerr << "Error: Record does not have a header tag" << std::endl; std::cerr << "Record: " << record << std::endl; exit(EXIT_FAILURE); } for(size_t i = 1; i < tokens.size(); ++i) { static char VERSION_TAG[] = "VN"; static char OVERLAP_TAG[] = "OL"; static char INPUT_FILE_TAG[] = "IN"; static char ERROR_RATE_TAG[] = "ER"; if(tokens[i].compare(0, FIELD_TAG_SIZE, VERSION_TAG) == 0) m_versionTag.fromString(tokens[i]); if(tokens[i].compare(0, FIELD_TAG_SIZE, OVERLAP_TAG) == 0) m_overlapTag.fromString(tokens[i]); if(tokens[i].compare(0, FIELD_TAG_SIZE, INPUT_FILE_TAG) == 0) m_infileTag.fromString(tokens[i]); if(tokens[i].compare(0, FIELD_TAG_SIZE, ERROR_RATE_TAG) == 0) m_errorRateTag.fromString(tokens[i]); if(tokens[i].compare(0, FIELD_TAG_SIZE, CONTAINMENT_TAG) == 0) m_containmentTag.fromString(tokens[i]); if(tokens[i].compare(0, FIELD_TAG_SIZE, TRANSITIVE_TAG) == 0) m_transitiveTag.fromString(tokens[i]); } } // // Vertex Record // // VertexRecord::VertexRecord(const std::string& recordLine) { parse(recordLine); } // void VertexRecord::setSubstringTag(bool b) { m_substringTag.set(b); } // void VertexRecord::write(std::ostream& out) { StringVector fields; fields.push_back(VERTEX_TAG); fields.push_back(m_id); fields.push_back(m_seq); if(m_substringTag.isInitialized()) fields.push_back(m_substringTag.toTagString(SUBSTRING_TAG)); writeFields(out, fields); } //JEREMIAH void VertexRecord::write(std::stringstream& out) { StringVector fields; fields.push_back(VERTEX_TAG); fields.push_back(m_id); fields.push_back(m_seq); if(m_substringTag.isInitialized()) fields.push_back(m_substringTag.toTagString(SUBSTRING_TAG)); writeFields(out, fields); } // void VertexRecord::parse(const std::string& record) { if(record.size() < 2) { std::cerr << "Error: Record is not valid\n"; exit(EXIT_FAILURE); } // Tokenize record StringVector tokens = SQG::tokenizeRecord(record); if(tokens.size() < 3) { std::cerr << "Error: Vertex record is incomplete.\n"; std::cerr << "Record: " << record << std::endl; exit(EXIT_FAILURE); } // Ensure the first token indicates this is a valid vertex record if(tokens[0].compare(0, RECORD_TAG_SIZE, VERTEX_TAG) != 0) { std::cerr << "Error: Record does not have a vertex tag" << std::endl; std::cerr << "Record: " << record << std::endl; exit(EXIT_FAILURE); } m_id = tokens[1]; m_seq = tokens[2]; for(size_t i = 3; i < tokens.size(); ++i) { if(tokens[i].compare(0, FIELD_TAG_SIZE, SUBSTRING_TAG) == 0) m_substringTag.fromString(tokens[i]); } } // // EdgeRecord // EdgeRecord::EdgeRecord(const std::string& recordLine) { parse(recordLine); } // void EdgeRecord::write(std::ostream& out) { StringVector fields; fields.push_back(EDGE_TAG); std::stringstream ss; ss << m_overlap; fields.push_back(ss.str()); writeFields(out, fields); } // void EdgeRecord::parse(const std::string& record) { if(record.size() < 2) { std::cerr << "Error: Record is not valid\n"; exit(EXIT_FAILURE); } // Tokenize record StringVector tokens = SQG::tokenizeRecord(record); if(tokens.size() < 2) { std::cerr << "Error: Edge record is incomplete.\n"; std::cerr << "Record: " << record << std::endl; exit(EXIT_FAILURE); } // Ensure the first token indicates this is a valid edge record if(tokens[0].compare(EDGE_TAG) != 0) { std::cerr << "Error: Record does not have an edge tag" << std::endl; std::cerr << "Record: " << record << std::endl; exit(EXIT_FAILURE); } // Parse the overlap std::stringstream ssparser(tokens[1]); ssparser >> m_overlap; } // // // RecordType getRecordType(const std::string& record) { // the record type is the first two characters of the record if(record.size() < 2) { std::cerr << "Error: record does not have a valid record-type tag" << std::endl; std::cerr << "Record: " << record << std::endl; exit(EXIT_FAILURE); } char recordTag[RECORD_TAG_SIZE]; record.copy(recordTag, RECORD_TAG_SIZE); if(strncmp(recordTag, HEADER_TAG, RECORD_TAG_SIZE) == 0) { return RT_HEADER; } if(strncmp(recordTag, VERTEX_TAG, RECORD_TAG_SIZE) == 0) { return RT_VERTEX; } if(strncmp(recordTag, EDGE_TAG, RECORD_TAG_SIZE) == 0) { return RT_EDGE; } // Unknown tag std::cerr << "Error: Unknown ASQG file record tag: " << recordTag << std::endl; exit(EXIT_FAILURE); } void writeFields(std::ostream& out, const StringVector& fields) { for(size_t i = 0; i < fields.size(); ++i) { out << fields[i]; if(i != fields.size() - 1) out << SQG::FIELD_SEP; } out << "\n"; } } ================================================ FILE: src/SGA/SQG/ASQG.h ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // ASQG - Definitions and functions // for handling ASGQ files // #ifndef ASQG_H #define ASQG_H #include "SQG.h" #include "Match.h" namespace ASQG { enum RecordType { RT_HEADER = 0, RT_VERTEX, RT_EDGE }; // A header record is just a tag:value pairs struct HeaderRecord { public: HeaderRecord(); HeaderRecord(const std::string& recordLine); void setOverlapTag(int overlapLen); void setInputFileTag(const std::string& name); void setErrorRateTag(float errorRate); void setContainmentTag(int v); void setTransitiveTag(int v); const SQG::IntTag& getVersionTag() const { return m_versionTag; } const SQG::FloatTag& getErrorRateTag() const { return m_errorRateTag; } const SQG::StringTag& getInfileTag() const { return m_infileTag; } const SQG::IntTag& getOverlapTag() const { return m_overlapTag; } const SQG::IntTag& getContainmentTag() const { return m_containmentTag; }; const SQG::IntTag& getTransitiveTag() const { return m_transitiveTag; }; void write(std::ostream& out); void write(std::stringstream& out); // JEREMIAH void parse(const std::string& record); private: void setVersionTag(int version); SQG::IntTag m_versionTag; SQG::FloatTag m_errorRateTag; SQG::StringTag m_infileTag; SQG::IntTag m_overlapTag; SQG::IntTag m_containmentTag; SQG::IntTag m_transitiveTag; }; // A vertex record is an id, sequence and an array of // tag:value struct VertexRecord { public: VertexRecord() {} VertexRecord(const std::string& recordLine); VertexRecord(const std::string& i, const std::string& s) : m_id(i), m_seq(s) {} void setSubstringTag(bool b); const std::string& getID() const { return m_id; } const std::string& getSeq() const { return m_seq; } const SQG::IntTag& getSubstringTag() const { return m_substringTag; } void write(std::ostream& out); void write(std::stringstream& out); void parse(const std::string& record); private: std::string m_id; std::string m_seq; SQG::IntTag m_substringTag; }; // An edge record is just an overlap object and tag:values struct EdgeRecord { public: EdgeRecord() {} EdgeRecord(const std::string& recordLine); EdgeRecord(const Overlap& o) : m_overlap(o) {} const Overlap& getOverlap() const { return m_overlap; } void write(std::ostream& out); void parse(const std::string& record); 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} // StringVector tokenizeTagValue(const std::string& tagValue) { return split(tagValue, TAG_SEP); } } ================================================ FILE: src/SGA/SQG/SQG.h ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // SQG - Common definitions and functions for parsing // assembly and sequence graph files // #ifndef SQG_H #define SQG_H #include #include #include "Util.h" namespace SQG { // const char FIELD_SEP = '\t'; const char TAG_SEP = ':'; // getTypeCode must be defined for every type that TagValue can take static inline char getTypeCode(int) { return 'i'; } static inline char getTypeCode(char) { return 'A'; } static inline char getTypeCode(const std::string&) { return 'Z'; } static inline char getTypeCode(float) { return 'f'; } // Tokenize functions StringVector tokenizeRecord(const std::string& record); StringVector tokenizeTagValue(const std::string& tagValue); // Two-state TagValue that allows the data value to be not set template class TagValue { public: TagValue() : m_isInitialized(false) {} TagValue(const T& v) : m_isInitialized(true), m_value(v) {} T get() const { assert(m_isInitialized); return m_value; } void set(T v) { m_isInitialized = true; m_value = v; } bool isInitialized() const { return m_isInitialized; } std::string toTagString(const char* tag) { std::stringstream ss; char type_code = getTypeCode(m_value); ss << tag << TAG_SEP << type_code << TAG_SEP << m_value; return ss.str(); } void fromString(const std::string& str) { StringVector tokens = tokenizeTagValue(str); if(tokens.size() != 3) { std::cerr << "Incorrect format for tagged value\n"; std::cerr << "Field: " << str << "\n"; exit(EXIT_FAILURE); } if(tokens[1].size() != 1 && tokens[1][0] != getTypeCode(m_value)) { std::cerr << "Unexpected type in tagged value. Expected: " << getTypeCode(m_value); std::cerr << " Found: " << tokens[1] << "\n"; exit(EXIT_FAILURE); } std::stringstream ss(tokens[2]); ss >> m_value; m_isInitialized = true; } private: T m_value; bool m_isInitialized; }; // These are the valid tags that can be used typedef TagValue CharTag; typedef TagValue IntTag; typedef TagValue FloatTag; typedef TagValue StringTag; }; #endif ================================================ FILE: src/SGA/StringGraph/CompleteOverlapSet.cpp ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // CompleteOverlapSet - A collection of all // valid overlaps for a given vertex. This is // inferred from the structure of a transitive reduced // graph // #include "CompleteOverlapSet.h" // CompleteOverlapSet::CompleteOverlapSet(const Vertex* pVertex, double maxER, int minLength) : m_pX(pVertex), m_maxER(maxER), m_minLength(minLength) { m_cost = 0; //iterativeConstruct(); constructBFS(); //constructMap(); } // Perform a breadth-first search of the graph, accumulating all the valid // overlaps of reads to m_pX. // Precondition: All vertices in the graph are colored GC_WHTE void CompleteOverlapSet::constructBFS() { EdgeDescList markedList; ExploreQueue queue; EdgePtrVec edges = m_pX->getEdges(); for(size_t i = 0; i < edges.size(); ++i) { Edge* pEdge = edges[i]; EdgeDesc ed = pEdge->getDesc(); Overlap ovr = pEdge->getOverlap(); ed.pVertex->setColor(GC_GRAY); queue.push(ExploreElement(ed, ovr)); } while(!queue.empty()) { ExploreElement ee = queue.front(); queue.pop(); // EdgeDesc& edXY = ee.ed; Vertex* pY = edXY.pVertex; Overlap& ovrXY = ee.ovr; int overlapLen = ovrXY.getOverlapLength(0); markedList.push_back(edXY); // Check if the overlap between this node and m_pX is valid if(overlapLen >= m_minLength) { double error_rate = SGAlgorithms::calcErrorRate(m_pX, pY, ovrXY); if(isErrorRateAcceptable(error_rate, m_maxER)) { // Mark the vertex as valid pY->setColor(GC_BLACK); m_overlapMap.insert(std::make_pair(edXY, ovrXY)); } else { pY->setColor(GC_RED); } } else { pY->setColor(GC_RED); } // Enqueue neighbors EdgePtrVec neighborEdges = pY->getEdges(); for(size_t i = 0; i < neighborEdges.size(); ++i) { Edge* pEdgeYZ = neighborEdges[i]; Vertex* pZ = pEdgeYZ->getEnd(); if(pZ == m_pX || pZ->getColor() != GC_WHITE) continue; Overlap ovrYZ = pEdgeYZ->getOverlap(); // Check that this vertex actually overlaps pX if(SGAlgorithms::hasTransitiveOverlap(ovrXY, ovrYZ)) { Overlap ovrXZ = SGAlgorithms::inferTransitiveOverlap(ovrXY, ovrYZ); EdgeDesc edXZ = SGAlgorithms::overlapToEdgeDesc(pZ, ovrXZ); if(ovrXZ.getOverlapLength(0) >= m_minLength) { pZ->setColor(GC_GRAY); queue.push(ExploreElement(edXZ, ovrXZ)); } } } } // reset colors for(EdgeDescList::iterator iter = markedList.begin(); iter != markedList.end(); ++iter) iter->pVertex->setColor(GC_WHITE); } // Perform a breadth-first search of the graph, accumulating all the valid // overlaps of reads to m_pX void CompleteOverlapSet::iterativeConstruct() { m_cost = 0; ExplorePriorityQueue queue; // We store the overlaps that do not meet the overlap parameters // in this map. The neighbors of these vertices might have valid // overlaps to m_pX so they must still be explored. SGAlgorithms::EdgeDescOverlapMap exclusionMap; // Add first-order overlaps EdgePtrVec edges = m_pX->getEdges(); for(size_t i = 0; i < edges.size(); ++i) { Edge* pEdge = edges[i]; EdgeDesc ed = pEdge->getDesc(); Overlap ovr = pEdge->getOverlap(); double error_rate = SGAlgorithms::calcErrorRate(m_pX, pEdge->getEnd(), ovr); int overlapLen = ovr.getOverlapLength(0); if(isErrorRateAcceptable(error_rate, m_maxER) && overlapLen >= m_minLength) { m_overlapMap.insert(std::make_pair(ed, ovr)); queue.push(ExploreElement(ed, ovr)); } else { exclusionMap.insert(std::make_pair(ed, ovr)); queue.push(ExploreElement(ed, ovr)); } } while(!queue.empty()) { ExploreElement ee = queue.top(); queue.pop(); EdgeDesc& edXY = ee.ed; Overlap& ovrXY = ee.ovr; // Add the overlaps of Y Vertex* pY = edXY.pVertex; EdgePtrVec neighborEdges = pY->getEdges(); for(size_t i = 0; i < neighborEdges.size(); ++i) { Edge* pEdgeYZ = neighborEdges[i]; Vertex* pZ = pEdgeYZ->getEnd(); if(pZ == m_pX) continue; Overlap ovrYZ = pEdgeYZ->getOverlap(); // Check that this vertex actually overlaps pX if(SGAlgorithms::hasTransitiveOverlap(ovrXY, ovrYZ)) { ++m_cost; Overlap ovrXZ = SGAlgorithms::inferTransitiveOverlap(ovrXY, ovrYZ); EdgeDesc edXZ = SGAlgorithms::overlapToEdgeDesc(pZ, ovrXZ); double error_rate = SGAlgorithms::calcErrorRate(m_pX, pZ, ovrXZ); int overlapLen = ovrXZ.getOverlapLength(0); if(isErrorRateAcceptable(error_rate, m_maxER) && overlapLen >= m_minLength) { // Overlap is valid SGAlgorithms::EdgeDescOverlapMap::iterator findIter = m_overlapMap.find(edXZ); if(findIter == m_overlapMap.end()) { m_overlapMap.insert(std::make_pair(edXZ, ovrXZ)); queue.push(ExploreElement(edXZ, ovrXZ)); } else if(ovrXZ.getOverlapLength(0) > findIter->second.getOverlapLength(0)) { findIter->second = ovrXZ; queue.push(ExploreElement(edXZ, ovrXZ)); } } else { // Overlap is invalid SGAlgorithms::EdgeDescOverlapMap::iterator findIter = exclusionMap.find(edXZ); if(findIter == exclusionMap.end()) { exclusionMap.insert(std::make_pair(edXZ, ovrXZ)); queue.push(ExploreElement(edXZ, ovrXZ)); } else if(overlapLen > findIter->second.getOverlapLength(0)) { findIter->second = ovrXZ; queue.push(ExploreElement(edXZ, ovrXZ)); } } } } } } // void CompleteOverlapSet::constructMap() { EdgePtrVec edges = m_pX->getEdges(); // Add the primary overlaps to the map, and all the nodes reachable from the primaries for(size_t i = 0; i < edges.size(); ++i) { Edge* pEdge = edges[i]; EdgeDesc ed = pEdge->getDesc(); Overlap ovr = pEdge->getOverlap(); if(ovr.isContainment()) continue; m_overlapMap.insert(std::make_pair(ed, ovr)); // Recursively add neighbors recursiveConstruct(ed, ovr, 1, ovr.getOverlapLength(0)); } } // Recursively add overlaps to pX inferred from the edges of pY to outMap void CompleteOverlapSet::recursiveConstruct(const EdgeDesc& edXY, const Overlap& ovrXY, int depth, int) { //std::cout << "depth: " << depth << " " << distance << " " << ovrXY << "\n"; Vertex* pY = edXY.pVertex; EdgePtrVec neighborEdges = pY->getEdges(); for(size_t i = 0; i < neighborEdges.size(); ++i) { Edge* pEdgeYZ = neighborEdges[i]; Vertex* pZ = pEdgeYZ->getEnd(); if(pZ != m_pX) { Overlap ovrYZ = pEdgeYZ->getOverlap(); if(ovrYZ.isContainment()) continue; // Check that this vertex actually overlaps pX if(SGAlgorithms::hasTransitiveOverlap(ovrXY, ovrYZ)) { Overlap ovrXZ = SGAlgorithms::inferTransitiveOverlap(ovrXY, ovrYZ); EdgeDesc edXZ = SGAlgorithms::overlapToEdgeDesc(pZ, ovrXZ); if(edXZ.dir != edXY.dir || ovrXZ.isContainment()) continue; double error_rate = SGAlgorithms::calcErrorRate(m_pX, pZ, ovrXZ); int overlapLen = ovrXZ.getOverlapLength(0); //std::cout << "ovrXY " << ovrXY << "\n"; //std::cout << "ovrYZ " << ovrYZ << "\n"; //std::cout << "ovrXZ " << ovrXZ << "\n"; if(isErrorRateAcceptable(error_rate, m_maxER) && overlapLen >= m_minLength) { SGAlgorithms::EdgeDescOverlapMap::iterator findIter = m_overlapMap.find(edXZ); if(findIter == m_overlapMap.end()) { m_overlapMap.insert(std::make_pair(edXZ, ovrXZ)); recursiveConstruct(edXZ, ovrXZ, depth + 1, ovrXZ.getOverlapLength(0)); } else if(ovrXZ.getOverlapLength(0) > findIter->second.getOverlapLength(0)) { findIter->second = ovrXZ; recursiveConstruct(edXZ, ovrXZ, depth + 1, ovrXZ.getOverlapLength(0)); } } } } } } // Compare the actual edges of m_pX with the edges in the overlap map void CompleteOverlapSet::getDiffMap(SGAlgorithms::EdgeDescOverlapMap& missingMap, SGAlgorithms::EdgeDescOverlapMap& extraMap) { missingMap = m_overlapMap; EdgePtrVec edges = m_pX->getEdges(); for(size_t i = 0; i < edges.size(); ++i) { EdgeDesc ed = edges[i]->getDesc(); SGAlgorithms::EdgeDescOverlapMap::iterator iter = missingMap.find(edges[i]->getDesc()); if(iter != missingMap.end()) { missingMap.erase(iter); } else { Overlap ovr = edges[i]->getOverlap(); extraMap.insert(std::make_pair(ed, ovr)); } } } // Reset the overlap parameters and filter edges void CompleteOverlapSet::resetParameters(double maxER, int minLength) { m_maxER = maxER; m_minLength = minLength; // Filter out overlaps no longer meeting the criteria SGAlgorithms::EdgeDescOverlapMap::iterator iter = m_overlapMap.begin(); while(iter != m_overlapMap.end()) { double er = SGAlgorithms::calcErrorRate(m_pX, iter->first.pVertex, iter->second); bool acceptER = isErrorRateAcceptable(er, m_maxER); bool acceptLen = iter->second.getOverlapLength(0) >= m_minLength; if(!acceptER || !acceptLen) { //std::cout << "Filtering " << iter->second << " flags: " << acceptER << acceptLen << "\n"; //std::cout << "er: " << er << " len: " << iter->second.getOverlapLength(0) << "\n"; m_overlapMap.erase(iter++); } else ++iter; } } // Remove all overlaps to a particular vertex void CompleteOverlapSet::removeOverlapsTo(Vertex* pRemove) { EdgeDesc ed; ed.pVertex = pRemove; for(int i = 0; i != ED_COUNT; ++i) { ed.dir = (EdgeDir)i; ed.comp = EC_SAME; m_overlapMap.erase(ed); ed.comp = EC_REVERSE; m_overlapMap.erase(ed); } } // Remove all the transitive and containment relationships, leaving only the irreducible void CompleteOverlapSet::computeIrreducible(SGAlgorithms::EdgeDescOverlapMap* pTransitive, SGAlgorithms::EdgeDescOverlapMap* pContainments) { SGAlgorithms::EdgeDescOverlapMap temp; partitionOverlaps(&temp, pTransitive, pContainments); m_overlapMap = temp; } // Partition the OverlapMap into edges that are containments, irreducible and transitive // If the pointer for an output map is NULL, simply discard the edges void CompleteOverlapSet::partitionOverlaps(SGAlgorithms::EdgeDescOverlapMap* pIrreducible, SGAlgorithms::EdgeDescOverlapMap* pTransitive, SGAlgorithms::EdgeDescOverlapMap* pContainment) const { if(pIrreducible == NULL && pTransitive == NULL && pContainment == NULL) return; // Nothing to do m_cost = 0; SGAlgorithms::EdgeDescOverlapMap workingMap; // Stage 1, remove containments for(SGAlgorithms::EdgeDescOverlapMap::const_iterator iter = m_overlapMap.begin(); iter != m_overlapMap.end(); ++iter) { if(iter->second.isContainment()) { if(pContainment != NULL) pContainment->insert(*iter); } else { workingMap.insert(*iter); } } // Stage 2, remove transitive edges SGAlgorithms::partitionTransitiveOverlaps(&workingMap, pTransitive, m_maxER, m_minLength); *pIrreducible = workingMap; } ================================================ FILE: src/SGA/StringGraph/CompleteOverlapSet.h ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // CompleteOverlapSet - A collection of all // valid overlaps for a given vertex. This is // inferred from the structure of a transitive reduced // graph // #ifndef COMPLETEOVERLAPSET_H #define COMPLETEOVERLAPSET_H #include "Bigraph.h" #include "SGAlgorithms.h" // Structure used for iterative exploration of the graph // The exploration starts at some vertex X, each element // holds a possible overlap between X and another vertex Y struct ExploreElement { ExploreElement(const EdgeDesc& e, const Overlap& o) : ed(e), ovr(o) {} EdgeDesc ed; Overlap ovr; }; // Comparison operator used to compare ExploreElements // by the length of the overlap on vertex X struct CompareExploreElemOverlapLength { bool operator()(const ExploreElement& elemXY, const ExploreElement& elemXZ) { return elemXY.ovr.match.coord[0].length() < elemXZ.ovr.match.coord[0].length(); } }; // typedef std::priority_queue, CompareExploreElemOverlapLength> ExplorePriorityQueue; typedef std::list EdgeDescList; typedef std::queue ExploreQueue; class CompleteOverlapSet { public: CompleteOverlapSet(const Vertex* pVertex, double maxER, int minLength); void getDiffMap(SGAlgorithms::EdgeDescOverlapMap& missingMap, SGAlgorithms::EdgeDescOverlapMap& extraMap); void removeOverlapsTo(Vertex* pRemove); // Remove all the transitive and containment relationships, leaving only the irreducible // If the input pointers are not NULL, the transitive and containment overlaps will be placed // in the appropriate map void computeIrreducible(SGAlgorithms::EdgeDescOverlapMap* pTransitive, SGAlgorithms::EdgeDescOverlapMap* pContainments); void constructMap(); void recursiveConstruct(const EdgeDesc& edXY, const Overlap& ovrXY, int depth, int distance); // Partition the OverlapMap into edges that are containments, irreducible and transitive // If the pointer for an output map is NULL, simply discard the edges void partitionOverlaps(SGAlgorithms::EdgeDescOverlapMap* pIrreducible, SGAlgorithms::EdgeDescOverlapMap* pTransitive, SGAlgorithms::EdgeDescOverlapMap* pContainment) const; void resetParameters(double maxER, int minLength); SGAlgorithms::EdgeDescOverlapMap getOverlapMap() const { return m_overlapMap; } size_t size() const { return m_overlapMap.size(); } size_t getCost() const { return m_cost; } private: // functions void iterativeConstruct(); void constructBFS(); // data SGAlgorithms::EdgeDescOverlapMap m_overlapMap; // the vertex this set is centered on const Vertex* m_pX; double m_maxER; int m_minLength; mutable size_t m_cost; }; #endif ================================================ FILE: src/SGA/StringGraph/GraphSearchTree.h ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // GraphSearchTree - Generic data structure used for implementing // a breadth-first search of a bidirectional graph. It is designed // to return all possible walks between the given start // and end vertices, up to a given distance. Used to search a // string graph or scaffold graph. // #ifndef GRAPHSEARCHTREE_H #define GRAPHSEARCHTREE_H #include "Bigraph.h" #include "SGWalk.h" #include #include template class GraphSearchNode { // Typedefs public: typedef std::deque* > GraphSearchNodePtrDeque; typedef std::vector _EDGEPtrVector; public: GraphSearchNode(VERTEX* pVertex, EdgeDir expandDir, GraphSearchNode* pParent, EDGE* pEdgeFromParent, int distance); ~GraphSearchNode(); // Reduce the child count by 1 void decrementChildren(); // Create the children of this node and place pointers to their nodes // on the queue. Returns the number of children created; int createChildren(GraphSearchNodePtrDeque& outQueue, const DISTANCE& distanceFunc); GraphSearchNode* getParent() const { return m_pParent; } VERTEX* getVertex() const { return m_pVertex; } int64_t getDistance() const { return m_distance; } EDGE* getEdgeFromParent() const { return m_pEdgeFromParent; } int getNumChildren() const { return m_numChildren; } private: // data VERTEX* m_pVertex; EdgeDir m_expandDir; GraphSearchNode* m_pParent; EDGE* m_pEdgeFromParent; int m_numChildren; int64_t m_distance; }; template class GraphSearchTree { // typedefs typedef GraphSearchNode _SearchNode; typedef typename _SearchNode::GraphSearchNodePtrDeque _SearchNodePtrDeque; typedef typename std::set<_SearchNode*> _SearchNodePtrSet; typedef std::vector WALK; // list of edges defines a walk through the graph typedef std::vector WALKVector; // vector of walks typedef std::vector VertexPtrVector; typedef std::vector VertexPtrVectorVector; public: GraphSearchTree(VERTEX* pStartVertex, VERTEX* pEndVertex, EdgeDir searchDir, int64_t distanceLimit, size_t nodeLimit); ~GraphSearchTree(); // Find connected components in the graph // Takes in a vector of all the vertices in the graph static void connectedComponents(VertexPtrVector allVertices, VertexPtrVectorVector& connectedComponents); // Returns true if the search has converged on a single vertex. In // other words, all walks from the start node share a common vertex, // which is represented by one of the nodes waiting expansion. // This function is the key to finding collapsed walks that represent // complex variation bubbles. It is a heavy operation however, as the // entire tree is search for each node in the expand queue. It should // only be used on small trees. // Returns true if the search converged and the pointer to the vertex // is return in pConvergedVertex. bool hasSearchConverged(VERTEX*& pConvergedVertex); // build walks to a given vertex template void buildWalksContainingVertex(VERTEX* pTarget, BUILDER& walkBuilder); // Construct walks representing every path from the start node template void buildWalksToAllLeaves(BUILDER& walkBuilder); // Construct walks representing every path from the start vertex to the goal vertex template void buildWalksToGoal(BUILDER& walkBuilder); // Expand all nodes in the queue a single time // Returns false if the search has stopped bool stepOnce(); // Check whether the search was aborted or not bool wasSearchAborted() const { return m_searchAborted; } private: // Search the branch from pNode to the root for pX. bool searchBranchForVertex(_SearchNode* pNode, VERTEX* pX, _SearchNode*& pFoundNode) const; // Build the walks from the root to the leaves in the queue template void _buildWalksToLeaves(const _SearchNodePtrDeque& queue, BUILDER& walkBuilder); // void addEdgesFromBranch(_SearchNode* pNode, WALK& outEdges); // Build a queue with all the leaves in it void _makeFullLeafQueue(_SearchNodePtrDeque& completeQueue) const; // print the branch sequence void printBranch(_SearchNode* pNode) const; // We keep the pointers to the search nodes // in one of three queues. // The goal queue contains the nodes representing the vertex we are searching for. // The expand queue contains nodes that have not yet been explored. // The done queue contains non-goal nodes that will not be expanded further. // Together, they represent all leaves of the tree _SearchNodePtrDeque m_goalQueue; _SearchNodePtrDeque m_expandQueue; _SearchNodePtrDeque m_doneQueue; size_t m_totalNodes; // The total number of nodes in the search tree _SearchNode* m_pRootNode; VERTEX* m_pGoalVertex; EdgeDir m_initialDir; int64_t m_distanceLimit; size_t m_nodeLimit; // Flag indicating the search was aborted bool m_searchAborted; // Distance functor DISTANCE m_distanceFunc; }; // template GraphSearchNode::GraphSearchNode(VERTEX* pVertex, EdgeDir expandDir, GraphSearchNode* pParent, EDGE* pEdgeFromParent, int distance) : m_pVertex(pVertex), m_expandDir(expandDir), m_pParent(pParent), m_pEdgeFromParent(pEdgeFromParent), m_numChildren(0) { // Set the extension distance if(m_pParent == NULL) { // this is the root node with distance 0 m_distance = 0; } else { assert(m_pEdgeFromParent != NULL); m_distance = m_pParent->m_distance + distance; } } // Delete this node and decrement the number of children // in the parent node. All children of a node must // be deleted before the parent template GraphSearchNode::~GraphSearchNode() { assert(m_numChildren == 0); if(m_pParent != NULL) m_pParent->decrementChildren(); } // template void GraphSearchNode::decrementChildren() { assert(m_numChildren != 0); m_numChildren -= 1; } // creates nodes for the children of this node // and place pointers to them in the queue. // Returns the number of nodes created template int GraphSearchNode::createChildren(GraphSearchNodePtrDeque& outDeque, const DISTANCE& distanceFunc) { assert(m_numChildren == 0); _EDGEPtrVector edges = m_pVertex->getEdges(m_expandDir); for(size_t i = 0; i < edges.size(); ++i) { EdgeDir childExpandDir = !edges[i]->getTwin()->getDir(); GraphSearchNode* pNode = new GraphSearchNode(edges[i]->getEnd(), childExpandDir, this, edges[i], distanceFunc(edges[i])); outDeque.push_back(pNode); m_numChildren += 1; } return edges.size(); } // // GraphSearchTree // template GraphSearchTree::GraphSearchTree(VERTEX* pStartVertex, VERTEX* pEndVertex, EdgeDir searchDir, int64_t distanceLimit, size_t nodeLimit) : m_pGoalVertex(pEndVertex), m_distanceLimit(distanceLimit), m_nodeLimit(nodeLimit), m_searchAborted(false) { // Create the root node of the search tree m_pRootNode = new GraphSearchNode(pStartVertex, searchDir, NULL, NULL, 0); // add the root to the expand queue m_expandQueue.push_back(m_pRootNode); m_totalNodes = 1; } template GraphSearchTree::~GraphSearchTree() { // Delete the tree // We delete each leaf and recurse up the tree iteratively deleting // parents with a single child node. This ensure that each parent is // deleted after all its children _SearchNodePtrDeque completeLeafNodes; _makeFullLeafQueue(completeLeafNodes); size_t totalDeleted = 0; for(typename _SearchNodePtrDeque::iterator iter = completeLeafNodes.begin(); iter != completeLeafNodes.end(); ++iter) { _SearchNode* pCurr = *iter; // loop invariant: pCurr is a deletable node // the loop stops when the parent is NULL or has // a child other than pCurr do { assert(pCurr->getNumChildren() == 0); _SearchNode* pNext = pCurr->getParent(); delete pCurr; // decrements pNext's child count totalDeleted += 1; pCurr = pNext; } while(pCurr && pCurr->getNumChildren() == 0); } assert(totalDeleted == m_totalNodes); } // Perform one step of the BFS template bool GraphSearchTree::stepOnce() { if(m_expandQueue.empty()) return false; if(m_totalNodes > m_nodeLimit) { // Move all nodes in the expand queue to the done queue m_doneQueue.insert(m_doneQueue.end(), m_expandQueue.begin(), m_expandQueue.end()); m_expandQueue.clear(); // Set a flag indicating the search was aborted m_searchAborted = true; return false; } // Iterate over the expand queue. If the path to the node // is outside the depth limit, move that node to the done queue. It cannot // yield a valid path to the goal. Otherwise, add the children of the node // to the incoming queue _SearchNodePtrDeque incomingQueue; while(!m_expandQueue.empty()) { _SearchNode* pNode = m_expandQueue.front(); m_expandQueue.pop_front(); if(pNode->getVertex() == m_pGoalVertex) { // This node represents the goal, add it to the goal queue m_goalQueue.push_back(pNode); continue; } if(pNode->getDistance() > m_distanceLimit) { // Path to this node is too long, expand it no further m_doneQueue.push_back(pNode); } else { // Add the children of this node to the queue int numCreated = pNode->createChildren(incomingQueue, m_distanceFunc); m_totalNodes += numCreated; if(numCreated == 0) { // No children created, add this node to the done queue m_doneQueue.push_back(pNode); } } } m_expandQueue = incomingQueue; return true; } // Return true if all the walks from the root converge // to one vertex (ie if the search from pX converged // to pY, then ALL paths from pX must go through pY). template bool GraphSearchTree::hasSearchConverged(VERTEX*& pConvergedVertex) { // Construct a set of all the leaf nodes _SearchNodePtrDeque completeLeafNodes; _makeFullLeafQueue(completeLeafNodes); // Search all the tree for all the nodes in the expand queue for(typename _SearchNodePtrDeque::iterator iter = m_expandQueue.begin(); iter != m_expandQueue.end(); ++iter) { _SearchNode* pNode = *iter; // If this node has the same vertex as the root skip it // We do not want to collapse at the root if(pNode->getVertex() == m_pRootNode->getVertex()) continue; bool isInAllBranches = true; for(typename _SearchNodePtrDeque::iterator leafIter = completeLeafNodes.begin(); leafIter != completeLeafNodes.end(); ++leafIter) { // Search the current branch from this leaf node to the root _SearchNode* pFoundNode = NULL; bool isInBranch = searchBranchForVertex(*leafIter, pNode->getVertex(), pFoundNode); if(!isInBranch) { isInAllBranches = false; break; } } // search has converted if(isInAllBranches) { pConvergedVertex = pNode->getVertex(); return true; } } // search has not converted pConvergedVertex = NULL; return false; } // Construct walks representing every path from the start node template template void GraphSearchTree::buildWalksToAllLeaves(BUILDER& walkBuilder) { // Construct a queue with all leaf nodes in it _SearchNodePtrDeque completeLeafNodes; _makeFullLeafQueue(completeLeafNodes); _buildWalksToLeaves(completeLeafNodes, walkBuilder); } // Construct walks representing every path from the start vertex to the goal vertex template template void GraphSearchTree::buildWalksToGoal(BUILDER& walkBuilder) { _buildWalksToLeaves(m_goalQueue, walkBuilder); } // Build all the walks that contain pTarget. template template void GraphSearchTree::buildWalksContainingVertex(VERTEX* pTarget, BUILDER& walkBuilder) { _SearchNodePtrDeque completeLeafNodes; _makeFullLeafQueue(completeLeafNodes); // Search upwards from each leaf until pTarget is found. // When it is found, insert the pointer to the search node // in the set _SearchNodePtrSet leafSet; // Find pTarget in each branch of the graph for(typename _SearchNodePtrDeque::const_iterator iter = completeLeafNodes.begin(); iter != completeLeafNodes.end(); ++iter) { _SearchNode* pFoundNode = NULL; searchBranchForVertex(*iter, pTarget, pFoundNode); assert(pFoundNode != NULL); leafSet.insert(pFoundNode); } // Construct all the walks to the found leaves completeLeafNodes.clear(); completeLeafNodes.insert(completeLeafNodes.end(), leafSet.begin(), leafSet.end()); _buildWalksToLeaves(completeLeafNodes, walkBuilder); } // Main function for constructing a vector of walks from a set of leaves template template void GraphSearchTree::_buildWalksToLeaves(const _SearchNodePtrDeque& queue, BUILDER& walkBuilder) { for(typename _SearchNodePtrDeque::const_iterator iter = queue.begin(); iter != queue.end(); ++iter) { // Recursively travel the tree from the leaf to the root collecting the edges in the vector WALK currWalk; addEdgesFromBranch(*iter, currWalk); // Reverse the walk and write it to the output structure walkBuilder.startNewWalk(m_pRootNode->getVertex()); for(typename WALK::reverse_iterator iter = currWalk.rbegin(); iter != currWalk.rend(); ++iter) walkBuilder.addEdge(*iter); walkBuilder.finishCurrentWalk(); } } // Return true if the vertex pX is found somewhere in the branch // from pNode to the root. If it is found, pFoundNode is set // to the furtherest instance of pX from the root. template bool GraphSearchTree::searchBranchForVertex(_SearchNode* pNode, VERTEX* pX, _SearchNode*& pFoundNode) const { if(pNode == NULL) { pFoundNode = NULL; return false; } if(pNode->getVertex() == pX && pNode != m_pRootNode) { pFoundNode = pNode; return true; } return searchBranchForVertex(pNode->getParent(), pX, pFoundNode); } // template void GraphSearchTree::addEdgesFromBranch(_SearchNode* pNode, WALK& outEdges) { // Terminate the recursion at the root node and dont add an edge if(pNode->getParent() != NULL) { outEdges.push_back(pNode->getEdgeFromParent()); return addEdgesFromBranch(pNode->getParent(), outEdges); } } // template void GraphSearchTree::_makeFullLeafQueue(_SearchNodePtrDeque& completeQueue) const { completeQueue.insert(completeQueue.end(), m_expandQueue.begin(), m_expandQueue.end()); completeQueue.insert(completeQueue.end(), m_goalQueue.begin(), m_goalQueue.end()); completeQueue.insert(completeQueue.end(), m_doneQueue.begin(), m_doneQueue.end()); } // template void GraphSearchTree::printBranch(_SearchNode* pNode) const { if(pNode != NULL) { std::cout << pNode->getVertex()->getID() << ","; printBranch(pNode->getParent()); } } template void GraphSearchTree::connectedComponents(VertexPtrVector allVertices, VertexPtrVectorVector& connectedComponents) { // Set the color of each vertex to be white signalling its not visited typename VertexPtrVector::iterator iter = allVertices.begin(); for(; iter != allVertices.end(); ++iter) { assert((*iter)->getColor() == GC_WHITE); (*iter)->setColor(GC_WHITE); } // iter = allVertices.begin(); for(; iter != allVertices.end(); ++iter) { // Do nothing if this vertex is already part of a CC if((*iter)->getColor() == GC_BLACK) continue; // Start a new CC VertexPtrVector currComponent; std::queue exploreQueue; (*iter)->setColor(GC_GRAY); // queued color exploreQueue.push(*iter); while(!exploreQueue.empty()) { VERTEX* pCurr = exploreQueue.front(); exploreQueue.pop(); assert(pCurr->getColor() != GC_BLACK); currComponent.push_back(pCurr); pCurr->setColor(GC_BLACK); //done with this vertex // Enqueue edges if they havent been visited already std::vector edges = pCurr->getEdges(); for(size_t i = 0; i < edges.size(); ++i) { EDGE* pEdge = edges[i]; VERTEX* pNext = pEdge->getEnd(); if(pNext->getColor() == GC_WHITE) { pNext->setColor(GC_GRAY); // queued exploreQueue.push(pNext); } } } connectedComponents.push_back(currComponent); } iter = allVertices.begin(); for(; iter != allVertices.end(); ++iter) { (*iter)->setColor(GC_WHITE); } // Sanity check size_t totalVertices = allVertices.size(); size_t totalInComponents = 0; for(size_t i = 0; i < connectedComponents.size(); ++i) { totalInComponents += connectedComponents[i].size(); } assert(totalVertices == totalInComponents); //std::cout << "[CC] total: " << totalVertices << " num components: " << connectedComponents.size() << "\n"; //std::cout << "[CC] total vertices in components: " << totalInComponents << "\n"; } #endif ================================================ FILE: src/SGA/StringGraph/Makefile.am ================================================ noinst_LIBRARIES = libstringgraph.a libstringgraph_a_CPPFLAGS = \ -I$(top_srcdir)/src/SGA/Bigraph \ -I$(top_srcdir)/src/SGA/Util \ -I$(top_srcdir)/src/SGA/Thirdparty \ -I$(top_srcdir)/src/SGA/Algorithm \ -I$(top_srcdir)/src/SGA/SQG \ -I$(top_srcdir)/SeqLib libstringgraph_a_SOURCES = \ SGUtil.cpp SGUtil.h \ SGAlgorithms.cpp SGAlgorithms.h \ SGVisitors.h SGVisitors.cpp \ CompleteOverlapSet.h CompleteOverlapSet.cpp \ RemovalAlgorithm.h RemovalAlgorithm.cpp \ SGSearch.h SGSearch.cpp \ GraphSearchTree.h \ SGWalk.h SGWalk.cpp ================================================ FILE: src/SGA/StringGraph/Makefile.in ================================================ # Makefile.in generated by automake 1.16.1 from Makefile.am. # @configure_input@ # Copyright (C) 1994-2018 Free Software Foundation, Inc. # This Makefile.in is free software; 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SGAlgorithms::EdgeDescOverlapMap outMap; findPotentialOverlaps(pVertex, pRemovalEdge, maxER, minLength, outMap); eliminateReachableEdges(pVertex, pRemovalEdge, maxER, minLength, outMap); return outMap; } // Find edges of pRemovalEdge->getEnd() that are potentially irreducible edges of pX once pRemovalEdge // has been eliminated from the graph void RemovalAlgorithm::findPotentialOverlaps(const Vertex* pX, const Edge* pRemovalEdge, double maxER, int minLength, SGAlgorithms::EdgeDescOverlapMap& outMap) { ExploreQueue queue; Vertex* pY = pRemovalEdge->getEnd(); Overlap ovrXY = pRemovalEdge->getOverlap(); EdgeDesc edXY = pRemovalEdge->getDesc(); // Get the edges of pY that face in the opposite direction of edge X <-- Y // New edges of pX must be in this direction. Edges in the direction of X // must have a longer overlap with X than Y does and therefore cannot be transitive // wrt to Y. EdgeDescSet visitedSet; EdgeDir dirY = !pRemovalEdge->getTwin()->getDir(); enqueueEdges(pY, dirY, ovrXY, edXY, minLength, queue, &visitedSet); int extra_extension = 0; while(!queue.empty()) { ExploreElement ee = queue.front(); queue.pop(); // If the overlap between this element and X is valid, add it to the output map // and proceed no further. If the overlap is above minLength but the error rate // with X is higher than the threshold, do not add it to the map but add its neighbors // as they may be valid overlaps to X EdgeDesc& edXZ = ee.ed; Vertex* pZ = edXZ.pVertex; Overlap& ovrXZ = ee.ovr; int overlapLen = ovrXZ.getOverlapLength(0); if(pZ == pX) continue; if(overlapLen >= minLength) { double error_rate = SGAlgorithms::calcErrorRate(pX, pZ, ovrXZ); if(isErrorRateAcceptable(error_rate, maxER)) { outMap.insert(std::make_pair(edXZ, ovrXZ)); } else { // Enqueue neighbors of pZ EdgeDir dirZ = edXZ.getTransitiveDir(); enqueueEdges(pZ, dirZ, ovrXZ, edXZ, minLength, queue, &visitedSet); ++extra_extension; } } } // Remove non-maximal overlaps from the collection SGAlgorithms::removeSubmaximalOverlaps(&outMap); } // Using the edges of pVertex, eliminate edges from outMap if they are transitive void RemovalAlgorithm::eliminateReachableEdges(const Vertex* pVertex, const Edge* pRemovalEdge, double maxER, int minLength, SGAlgorithms::EdgeDescOverlapMap& outMap) { // During the enqueue process, edges may have been added to outMap that are transitive to another // edge in outMap. We get rid of these first. SGAlgorithms::partitionTransitiveOverlaps(&outMap, NULL, maxER, minLength); // Now, eliminate any edges in outMap that are transitive wrt some edge reachable from pVertex // We control the depth of search using the shortest overlap in outMap // Get the length of the shortest overlap on pX in outMap int shortestOverlap = -1; for(SGAlgorithms::EdgeDescOverlapMap::iterator iter = outMap.begin(); iter != outMap.end(); ++iter) { int currOverlap = iter->second.getOverlapLength(0); if(shortestOverlap == -1 || currOverlap < shortestOverlap) { shortestOverlap = currOverlap; // std::cout << "SHORT: " << iter->second << "\n"; } } // Avoid loops by only enqueuing vertices that have not been visited before EdgeDescSet visitedSet; ExploreQueue queue; // Enqueue the initial overlaps of pX to the queue if they are longer than the shortest overlap EdgeDir dirX = pRemovalEdge->getDir(); EdgePtrVec edges = pVertex->getEdges(dirX); for(size_t i = 0; i < edges.size(); ++i) { Edge* pEdge = edges[i]; // Skip the edge that is being removed if(pEdge == pRemovalEdge) continue; EdgeDesc ed = pEdge->getDesc(); Overlap ovr = pEdge->getOverlap(); if(ovr.getOverlapLength(0) >= shortestOverlap && !ovr.isContainment()) { queue.push(ExploreElement(ed, ovr)); visitedSet.insert(ed); } } // The elements in the queue represent the irreducible overlaps of pX and any overlaps // that are transitive wrt the irreducible edges. If the edges in outMap are transitive // wrt to this edge set, remove them from the map. Any remaining edges are new irreducible edges. int num_ext = 0; while(!queue.empty()) { if(outMap.empty()) return; ExploreElement ee = queue.front(); queue.pop(); // If the overlap between this element and X is valid, add it to the output map // and proceed no further. If the overlap is above minLength but the error rate // with X is higher than the threshold, do not add it to the map but add its neighbors // as they may be valid overlaps to X EdgeDesc& edXY = ee.ed; Vertex* pY = edXY.pVertex; Overlap& ovrXY = ee.ovr; //std::cout << "OVRXY: " << ovrXY << "\n"; int overlapLen = ovrXY.getOverlapLength(0); assert(overlapLen >= shortestOverlap); (void)overlapLen; // Check all the overlaps in the map to see if they are transitive wrt pY SGAlgorithms::EdgeDescOverlapMap::iterator iter = outMap.begin(); while(iter != outMap.end()) { bool eliminate = false; const EdgeDesc& edXZ = iter->first; const Overlap& ovrXZ = iter->second; if(ovrXY.getOverlapLength(0) >= ovrXZ.getOverlapLength(0)) { if(edXY.pVertex == edXZ.pVertex) eliminate = true; else eliminate = SGAlgorithms::isOverlapTransitive(edXY.pVertex, edXZ.pVertex, ovrXY, ovrXZ, maxER, minLength); } if(eliminate) { outMap.erase(iter++); } else ++iter; } // If any overlaps remain in the map, enqueue the neighbors of this vertex // that have an overlap with X that is at least as long as shortest overlap if(!outMap.empty()) { num_ext++; enqueueEdges(pY, edXY.getTransitiveDir(), ovrXY, edXY, shortestOverlap, queue, &visitedSet); } } } // Add the edges of pY in direction dirY to the explore queue if they have a valid overlap with X void RemovalAlgorithm::enqueueEdges(const Vertex* pY, EdgeDir dirY, const Overlap& ovrXY, const EdgeDesc& /*edXY*/, int minOverlap, ExploreQueue& outQueue, EdgeDescSet* pSeenSet) { EdgePtrVec edges = pY->getEdges(dirY); for(size_t i = 0; i < edges.size(); ++i) { Edge* pEdge = edges[i]; Vertex* pZ = pEdge->getEnd(); // Compute the edgeDesc and overlap on pX for this edge Overlap ovrYZ = pEdge->getOverlap(); if(!ovrYZ.isContainment() && SGAlgorithms::hasTransitiveOverlap(ovrXY, ovrYZ)) { Overlap ovrXZ = SGAlgorithms::inferTransitiveOverlap(ovrXY, ovrYZ); EdgeDesc edXZ = SGAlgorithms::overlapToEdgeDesc(pZ, ovrXZ); if((pSeenSet == NULL || pSeenSet->count(edXZ) == 0) && ovrXZ.getOverlapLength(0) >= minOverlap) { outQueue.push(ExploreElement(edXZ, ovrXZ)); if(pSeenSet != NULL) pSeenSet->insert(edXZ); } } } } ================================================ FILE: src/SGA/StringGraph/RemovalAlgorithm.h ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // RemovalAlgorithm - Determine the set of edges that // must be added to a given vertex to keep the structure of the // graph intact when a vertex that it shares an edge with // is going to be removed from the graph. // This is similar to the CompleteOverlapSet but does // not perform an exhaustive exploration of the edges // #ifndef REMOVALALGORITHM_H #define REMOVALALGORITHM_H #include "Bigraph.h" #include "SGAlgorithms.h" #include "CompleteOverlapSet.h" namespace RemovalAlgorithm { // Returns the set of overlaps that must be added to the graph // if the vertex at the end of pRemovalEdge is going to be deleted SGAlgorithms::EdgeDescOverlapMap computeRequiredOverlaps(const Vertex* pVertex, const Edge* pRemovalEdge, double maxER, int minLength); void findPotentialOverlaps(const Vertex* pX, const Edge* pRemovalEdge, double maxER, int minLength, SGAlgorithms::EdgeDescOverlapMap& outMap); void eliminateReachableEdges(const Vertex* pVertex, const Edge* pRemovalEdge, double maxER, int minLength, SGAlgorithms::EdgeDescOverlapMap& outMap); void enqueueEdges(const Vertex* pY, EdgeDir dirY, const Overlap& ovrXY, const EdgeDesc& edXY, int minOverlap, ExploreQueue& outQueue, EdgeDescSet* pSeenSet); }; #endif ================================================ FILE: src/SGA/StringGraph/SGAlgorithms.cpp ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // SGAlgorithms - Collection of algorithms for operating on string graphs // #include "SGAlgorithms.h" #include "SGUtil.h" #include "CompleteOverlapSet.h" #include "RemovalAlgorithm.h" #include // add edges to the graph for the given overlap Edge* SGAlgorithms::createEdgesFromOverlap(StringGraph* pGraph, const Overlap& o, bool allowContained, size_t maxEdges) { // Initialize data and perform checks Vertex* pVerts[2]; //Vertex* pVerts = new Vertex[2]; //JEREMIAH PUT ON HEAP EdgeComp comp = (o.match.isRC()) ? EC_REVERSE : EC_SAME; bool isContainment = o.match.isContainment(); assert(allowContained || !isContainment); (void)allowContained; for(size_t idx = 0; idx < 2; ++idx) { pVerts[idx] = pGraph->getVertex(o.id[idx]); // If one of the vertices is not in the graph, skip this edge // This can occur if one of the verts is a strict substring of some other vertex so it will // never be added to the graph if(pVerts[idx] == NULL) return NULL; } // Check if this is a substring containment, if so mark the contained read // but do not create edges for(size_t idx = 0; idx < 2; ++idx) { if(!o.match.coord[idx].isExtreme()) { std::cerr << "contained "; //JEREMIAH size_t containedIdx = 1 - idx; assert(o.match.coord[containedIdx].isExtreme()); pVerts[containedIdx]->setColor(GC_RED); pGraph->setContainmentFlag(true); return NULL; } } // If either vertex has the maximum number of edges, // do not add any more. This is to protect against ultra-dense // regions of the graph inflating memory usage. The nodes that reach // this limit, and nodes connected to them are marked as super repeats. // After loading the graph, all edges to super repeats are cut to prevent // misassembly. size_t num_edges_0 = pVerts[0]->countEdges(); size_t num_edges_1 = pVerts[1]->countEdges(); if(num_edges_0 > maxEdges || num_edges_1 > maxEdges) { //WARN_ONCE("Edge limit reached for vertex when loading graph"); pVerts[0]->setSuperRepeat(true); pVerts[1]->setSuperRepeat(true); return NULL; } if(!isContainment) { Edge* pEdges[2]; for(size_t idx = 0; idx < 2; ++idx) { EdgeDir dir = o.match.coord[idx].isLeftExtreme() ? ED_ANTISENSE : ED_SENSE; const SeqCoord& coord = o.match.coord[idx]; //pEdges[idx] = new(pGraph->getEdgeAllocator()) Edge(pVerts[1 - idx], dir, comp, coord); // JEREMIAH pEdges[idx] = new Edge(pVerts[1 - idx], dir, comp, coord); // JEREMIAH } pEdges[0]->setTwin(pEdges[1]); pEdges[1]->setTwin(pEdges[0]); pGraph->addEdge(pVerts[0], pEdges[0]); pGraph->addEdge(pVerts[1], pEdges[1]); return pEdges[0]; } else { // Contained edges don't have a direction, they can be travelled from // one vertex to the other in either direction. Hence, we // add two edges per vertex. Later during the contain removal // algorithm this is important to determine transitivity Edge* pEdges[4]; for(size_t idx = 0; idx < 2; ++idx) { const SeqCoord& coord = o.match.coord[idx]; // pEdges[idx] = new(pGraph->getEdgeAllocator()) Edge(pVerts[1 - idx], ED_SENSE, comp, coord); // pEdges[idx + 2] = new(pGraph->getEdgeAllocator()) Edge(pVerts[1 - idx], ED_ANTISENSE, comp, coord); pEdges[idx] = new Edge(pVerts[1 - idx], ED_SENSE, comp, coord); pEdges[idx + 2] = new Edge(pVerts[1 - idx], ED_ANTISENSE, comp, coord); } // Twin the edges and add them to the graph pEdges[0]->setTwin(pEdges[1]); pEdges[1]->setTwin(pEdges[0]); pEdges[2]->setTwin(pEdges[3]); pEdges[3]->setTwin(pEdges[2]); pGraph->addEdge(pVerts[0], pEdges[0]); pGraph->addEdge(pVerts[0], pEdges[2]); pGraph->addEdge(pVerts[1], pEdges[1]); pGraph->addEdge(pVerts[1], pEdges[3]); // Set containment flags updateContainFlags(pGraph, pVerts[0], pEdges[0]->getDesc(), o); return pEdges[0]; } } // Find new edges for pVertex that are required if pDeleteEdge is removed from the graph void SGAlgorithms::remodelVertexForExcision(StringGraph* pGraph, Vertex* pVertex, Edge* pDeleteEdge) { assert(pVertex == pDeleteEdge->getStart()); // If the edge is a containment edge, nothing needs to be done. No edges can be transitive // through containments if(pDeleteEdge->getOverlap().isContainment()) return; double maxER = pGraph->getErrorRate(); int minLength = pGraph->getMinOverlap(); EdgeDescOverlapMap addMap = RemovalAlgorithm::computeRequiredOverlaps(pVertex, pDeleteEdge, maxER, minLength); for(EdgeDescOverlapMap::iterator iter = addMap.begin(); iter != addMap.end(); ++iter) { //std::cout << "Adding edge " << iter->second << " during removal of " << pDeleteEdge->getEndID() << "\n"; createEdgesFromOverlap(pGraph, iter->second, false); } /* // Set the contain flags based on newly discovered edges updateContainFlags(pGraph, pVertex, containMap); */ } // Set containment flags in the graph based on the overlap map void SGAlgorithms::updateContainFlags(StringGraph* pGraph, Vertex* pVertex, EdgeDescOverlapMap& containMap) { for(EdgeDescOverlapMap::iterator iter = containMap.begin(); iter != containMap.end(); ++iter) { updateContainFlags(pGraph, pVertex, iter->first, iter->second); } } // void SGAlgorithms::updateContainFlags(StringGraph* pGraph, Vertex* pVertex, const EdgeDesc& ed, const Overlap& ovr) { assert(ovr.isContainment()); // Determine which of the two vertices is contained Vertex* pOther = ed.pVertex; if(ovr.getContainedIdx() == 0) pVertex->setContained(true); else pOther->setContained(true); pGraph->setContainmentFlag(true); } // Calculate the error rate between the two vertices double SGAlgorithms::calcErrorRate(const Vertex* pX, const Vertex* pY, const Overlap& ovrXY) { int num_diffs = ovrXY.match.countDifferences(pX->getSeq().toString(), pY->getSeq().toString()); return static_cast(num_diffs) / static_cast(ovrXY.match.getMinOverlapLength()); } // Infer an overlap from two edges // The input edges are between X->Y Y->Z // and the returned overlap is X->Z Overlap SGAlgorithms::inferTransitiveOverlap(const Overlap& ovrXY, const Overlap& ovrYZ) { // Construct the match Match match_yx = ovrXY.match; match_yx.swap(); Match match_yz = ovrYZ.match; // Infer the match_ij based match_i and match_j Match match_xz = Match::infer(match_yx, match_yz); match_xz.expand(); // Convert the match to an overlap Overlap ovr(ovrXY.id[0], ovrYZ.id[1], match_xz); return ovr; } // Returns true if, given overlaps X->Y, X->Z, the overlap X->Z is transitive bool SGAlgorithms::isOverlapTransitive(const Vertex* pY, const Vertex* pZ, const Overlap& ovrXY, const Overlap& ovrXZ, const double maxER, const int minOverlap) { // Ensure the overlaps are in the correct order, the overlap with Y should be at least // as large as the overlap with Z assert(ovrXY.getOverlapLength(0) >= ovrXZ.getOverlapLength(0)); assert(pY != pZ); // Compute the overlap YZ Overlap ovrYX = ovrXY; ovrYX.swap(); Overlap ovrYZ = SGAlgorithms::inferTransitiveOverlap(ovrYX, ovrXZ); // If ovrYZ is a containment, then Z is not transitive wrt Y if(ovrYZ.match.isContainment()) return false; // If the overlap between Y and Z is not long enough then Z is not transitive if(ovrYZ.getOverlapLength(0) < minOverlap) return false; // Finally, check that the error rate is below the threshold double error_rate = SGAlgorithms::calcErrorRate(pY, pZ, ovrYZ); if(isErrorRateAcceptable(error_rate, maxER)) return true; else return false; } // The following algorithms use these local types // defining an edge/overlap pair. typedef std::pair EdgeDescOverlapPair; // Compare two edges by their overlap length struct EDOPairCompare { bool operator()(const EdgeDescOverlapPair& edpXY, const EdgeDescOverlapPair& edpXZ) { return edpXY.second.match.coord[0].length() < edpXZ.second.match.coord[0].length(); } }; // typedefs typedef std::priority_queue, EDOPairCompare> EDOPairQueue; // Move the transitive edges from pOverlapMap to pTransitive void SGAlgorithms::partitionTransitiveOverlaps(EdgeDescOverlapMap* pOverlapMap, EdgeDescOverlapMap* pTransitive, double maxER, int minLength) { EDOPairQueue overlapQueue; for(SGAlgorithms::EdgeDescOverlapMap::iterator iter = pOverlapMap->begin(); iter != pOverlapMap->end(); ++iter) { overlapQueue.push(*iter); } // Traverse the list of overlaps in order of length and move elements from // the irreducible map to the transitive map while(!overlapQueue.empty()) { EdgeDescOverlapPair edoPair = overlapQueue.top(); overlapQueue.pop(); EdgeDesc& edXY = edoPair.first; Overlap& ovrXY = edoPair.second; assert(!ovrXY.match.isContainment()); SGAlgorithms::EdgeDescOverlapMap::iterator iter = pOverlapMap->begin(); while(iter != pOverlapMap->end()) { bool move = false; const EdgeDesc& edXZ = iter->first; const Overlap& ovrXZ = iter->second; // Four conditions must be met to mark an edge X->Z transitive through X->Y // 1) The overlaps must be in the same direction // 2) The overlap X->Y must be strictly longer than X->Z // 3) The overlap between Y->Z must not be a containment // 4) The overlap between Y->Z must be within the error and length thresholds if(!(edXZ == edXY) && edXY.dir == edXZ.dir && ovrXY.getOverlapLength(0) > ovrXZ.getOverlapLength(0)) { move = SGAlgorithms::isOverlapTransitive(edXY.pVertex, edXZ.pVertex, ovrXY, ovrXZ, maxER, minLength); } if(move) { //std::cout << "Marking overlap: " << iter->second << " as trans via " << ovrXY << "\n"; if(pTransitive != NULL) pTransitive->insert(*iter); pOverlapMap->erase(iter++); } else { ++iter; } } } } void SGAlgorithms::removeSubmaximalOverlaps(EdgeDescOverlapMap* pOverlapMap) { EDOPairQueue overlapQueue; for(SGAlgorithms::EdgeDescOverlapMap::iterator iter = pOverlapMap->begin(); iter != pOverlapMap->end(); ++iter) { overlapQueue.push(*iter); } // Traverse the list of overlaps in order of length // Only add the first seen overlap for each vertex pOverlapMap->clear(); VertexIDSet seenVerts; // the irreducible map to the transitive map while(!overlapQueue.empty()) { EdgeDescOverlapPair edoPair = overlapQueue.top(); overlapQueue.pop(); EdgeDesc& edXY = edoPair.first; if(seenVerts.count(edXY.pVertex->getID()) == 0) { seenVerts.insert(edXY.pVertex->getID()); pOverlapMap->insert(edoPair); } } } // Return a descriptor of the edge describing ovrXY EdgeDesc SGAlgorithms::overlapToEdgeDesc(Vertex* pY, const Overlap& ovrXY) { EdgeDesc edXY; edXY.pVertex = pY; edXY.comp = (ovrXY.match.isRC()) ? EC_REVERSE : EC_SAME; edXY.dir = ovrXY.match.coord[0].isLeftExtreme() ? ED_ANTISENSE : ED_SENSE; // X -> Y return edXY; } // Return true if XZ has an overlap bool SGAlgorithms::hasTransitiveOverlap(const Overlap& ovrXY, const Overlap& ovrYZ) { Match match_yx = ovrXY.match; match_yx.swap(); Match match_yz = ovrYZ.match; return Match::doMatchesIntersect(match_yx, match_yz); } // EdgeDesc SGAlgorithms::getEdgeDescFromEdge(Edge* pEdge) { return pEdge->getDesc(); } void SGAlgorithms::printOverlapMap(const EdgeDescOverlapMap& overlapMap) { for(EdgeDescOverlapMap::const_iterator iter = overlapMap.begin(); iter != overlapMap.end(); ++iter) { std::cout << " Overlap:" << iter->second << "\n"; } } ================================================ FILE: src/SGA/StringGraph/SGAlgorithms.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // SGAlgorithms - Algorithms for manipulating // the string graph // #ifndef SGALGORITHMS_H #define SGALGORITHMS_H #include "Bigraph.h" #include "SGUtil.h" #include namespace SGAlgorithms { // // Helper data structures // typedef std::set VertexIDSet; typedef std::set EdgeDescSet; typedef std::map EdgeDescOverlapMap; // Remodel the graph by finding new edges for the given vertex to avoid // causing a disconnection when removing pDeleteEdge void remodelVertexForExcision(StringGraph* pGraph, Vertex* pVertex, Edge* pDeleteEdge); // Create the edges in pGraph described by the overlap // A pointer to the first edge of the edge/edge twin is returned or NULL // if the edges cannot be added Edge* createEdgesFromOverlap(StringGraph* pGraph, const Overlap& o, bool allowContained, size_t maxEdges = -1); // Calculate the error rate between the two vertex sequences double calcErrorRate(const Vertex* pX, const Vertex* pY, const Overlap& ovrXY); // Update the containment flags in the graph using the described overlaps void updateContainFlags(StringGraph* pGraph, Vertex* pVertex, EdgeDescOverlapMap& containMap); void updateContainFlags(StringGraph* pGraph, Vertex* pVertex, const EdgeDesc& ed, const Overlap& ovr); // // Overlap inference algorithms // // Infer an overlap from two edges // The input overlaps are between X->Y Y->Z // and the returned overlap is X->Z Overlap inferTransitiveOverlap(const Overlap& ovrXY, const Overlap& ovrYZ); EdgeDesc overlapToEdgeDesc(Vertex* pY, const Overlap& ovrXY); // Returns true if given overlaps X->Y, X->Z, the overlap X->Z is transitive bool isOverlapTransitive(const Vertex* pY, const Vertex* pZ, const Overlap& ovrXY, const Overlap& ovrXZ, const double maxER, const int minOverlap); // Returns true if XZ has a non-zero length overlap bool hasTransitiveOverlap(const Overlap& ovrXY, const Overlap& ovrYZ); // Partition a set of overlaps into transitive and irreducible edges void partitionTransitiveOverlaps(EdgeDescOverlapMap* pOverlapMap, EdgeDescOverlapMap* pTransitive, double maxER, int minLength); // Each read can have at most one edge to any other read in a given direction // This function removes any duplicates void removeSubmaximalOverlaps(EdgeDescOverlapMap* pOverlapMap); // Simple getters for std::transform EdgeDesc getEdgeDescFromEdge(Edge* pEdge); void printOverlapMap(const EdgeDescOverlapMap& overlapMap); }; #endif ================================================ FILE: src/SGA/StringGraph/SGSearch.cpp ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // SGSearch - Algorithms and data structures // for searching a string graph // #include "SGSearch.h" #include // SGWalkBuilder::SGWalkBuilder(SGWalkVector& outWalks, bool bIndexWalk) : m_outWalks(outWalks), m_pCurrWalk(NULL), m_bIndexWalk(bIndexWalk) { } // SGWalkBuilder::~SGWalkBuilder() { // The pointer to the current walk should be NULL // or else finishCurrentWalk() was not called for the last // walk and the graph search algorithm has a bug assert(m_pCurrWalk == NULL); } // void SGWalkBuilder::startNewWalk(Vertex* pStartVertex) { m_pCurrWalk = new SGWalk(pStartVertex, m_bIndexWalk); } // void SGWalkBuilder::addEdge(Edge* pEdge) { m_pCurrWalk->addEdge(pEdge); } // void SGWalkBuilder::finishCurrentWalk() { m_outWalks.push_back(*m_pCurrWalk); delete m_pCurrWalk; m_pCurrWalk = NULL; } // Find all the walks between pX and pY that are within maxDistance // If the exhaustive flag is set, only return walks if all the possible // solutions have been found. If exhaustive is false, any walks found will be // returned in outWalks even if the search is aborted. // Returns true if all the possible walks were found. bool SGSearch::findWalks(Vertex* pX, Vertex* pY, EdgeDir initialDir, int maxDistance, size_t maxNodes, bool exhaustive, SGWalkVector& outWalks) { SGSearchTree searchTree(pX, pY, initialDir, maxDistance, maxNodes); // Iteravively perform the BFS using the search tree. while(searchTree.stepOnce()) { } // If the search was aborted, do not return any walks // because we do not know if there are more valid paths from pX // to pY that we could not find because the search space was too large if(!searchTree.wasSearchAborted() || !exhaustive) { // Extract the walks from the graph as a vector of edges SGWalkBuilder builder(outWalks, false); searchTree.buildWalksToGoal(builder); } return !searchTree.wasSearchAborted(); } // Search the graph for a set of walks that represent alternate // versions of the same sequence. These walks are found by searching // the graph for a set of walks that start/end at a common vertex and cover // every vertex inbetween the start/end points. Additionally, the internal // vertices cannot have an edge to any vertex that is not in the set of vertices // indicated by the walks. Finally, all walks must have the same orientation for the // end vertex. // If these conditions are met, we can retain one of the walks and cleanly remove // the others. void SGSearch::findVariantWalks(Vertex* pX, EdgeDir initialDir, int maxDistance, size_t maxWalks, SGWalkVector& outWalks) { findCollapsedWalks(pX, initialDir, maxDistance, 500, outWalks); if(outWalks.size() <= 1 || outWalks.size() > maxWalks) { outWalks.clear(); return; } Edge* pLastEdge = outWalks.front().getLastEdge(); Vertex* pLastVertex = pLastEdge->getEnd(); EdgeDir lastDir = pLastEdge->getTwinDir(); // Validate that any of the returned walks can be removed cleanly. // This means that for all the internal vertices on each walk (between // the common endpoints) the only links are to other vertices in the set, // and that the last vertex has the same orientation for all walks. // Construct the set of vertex IDs std::set completeVertexSet; bool sameOrientation = true; for(size_t i = 0; i < outWalks.size(); ++i) { if (outWalks[i].getLastEdge()->getTwinDir() != lastDir) { sameOrientation = false; break; } VertexPtrVec verts = outWalks[i].getVertices(); for(size_t j = 0; j < verts.size(); ++j) { completeVertexSet.insert(verts[j]); } } // Return if the walks do not have the same orientation for the last vertex if(!sameOrientation) { outWalks.clear(); return; } // Check that each vertex in the internal nodes only has // edges to the vertices in the set bool cleanlyRemovable = true; // Ensure that all the vertices linked to the start vertex // in the specified dir are present in the set. EdgePtrVec epv = pX->getEdges(initialDir); cleanlyRemovable = checkEndpointsInSet(epv, completeVertexSet); // Ensure that all the vertex linked to the last vertex // in the incoming direction are preset epv = pLastVertex->getEdges(lastDir); cleanlyRemovable = cleanlyRemovable && checkEndpointsInSet(epv, completeVertexSet); // Check that each vertex connected to an interval vertex is also present for(std::set::iterator iter = completeVertexSet.begin(); iter != completeVertexSet.end(); ++iter) { Vertex* pY = *iter; if(pY == pX || pY == pLastVertex) continue; epv = pY->getEdges(); cleanlyRemovable = cleanlyRemovable && checkEndpointsInSet(epv, completeVertexSet); } if(!cleanlyRemovable) { outWalks.clear(); } return; } // Check that all the endpoints of the edges in the edge pointer vector // are members of the set bool SGSearch::checkEndpointsInSet(EdgePtrVec& epv, std::set& vertexSet) { for(size_t i = 0; i < epv.size(); ++i) { if(vertexSet.find(epv[i]->getEnd()) == vertexSet.end()) return false; } return true; } // Return a set of walks that all start from pX and join together at some later vertex // If no such walk exists, an empty set is returned void SGSearch::findCollapsedWalks(Vertex* pX, EdgeDir initialDir, int maxDistance, size_t maxNodes, SGWalkVector& outWalks) { SGSearchTree searchTree(pX, NULL, initialDir, maxDistance, maxNodes); // Iteravively perform the BFS using the search tree. After each step // we check if the search has collapsed to a single vertex. bool done = false; while(!done) { done = !searchTree.stepOnce(); if(searchTree.wasSearchAborted()) break; Vertex* pCollapsedVertex; bool isCollapsed = searchTree.hasSearchConverged(pCollapsedVertex); if(isCollapsed) { assert(pCollapsedVertex != NULL); // pCollapsedVertex is common between all walks. // Check that the extension distance for any walk is no longer than maxDistance. // If this is the case, we truncate all walks so they end at pCollapsedVertex and return // all the non-redundant walks in outWalks SGWalkBuilder builder(outWalks, true); searchTree.buildWalksContainingVertex(pCollapsedVertex, builder); return; } } // no collapsed walk found, return empty set outWalks.clear(); } // Count the number of reads that span the junction described by edge XY // X -------------- // Y ------------ // Z ----------- // W ---------- // In this case Z spans the junction but W does not. int SGSearch::countSpanningCoverage(Edge* pXY, size_t maxQueue) { (void)pXY; (void)maxQueue; assert(false && "deprecated"); return 0; } ================================================ FILE: src/SGA/StringGraph/SGSearch.h ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // SGSearch - Algorithms and data structures // for searching a string graph // #ifndef SGSEARCH_H #define SGSEARCH_H #include "Bigraph.h" #include "SGWalk.h" #include "GraphSearchTree.h" #include // Returns the extension distance indicated // by the given edge struct SGDistanceFunction { int operator()(const Edge* pEdge) const { return pEdge->getSeqLen(); } }; // typedef GraphSearchTree SGSearchTree; // struct SGWalkBuilder { public: SGWalkBuilder(SGWalkVector& outWalks, bool bIndexWalk); ~SGWalkBuilder(); // These three functions must be provided by the builder object // the generic graph code calls these to describe the walks through // the graph void startNewWalk(Vertex* pStartVertex); void addEdge(Edge* pEdge); void finishCurrentWalk(); private: SGWalkVector& m_outWalks; SGWalk* m_pCurrWalk; bool m_bIndexWalk; }; // String Graph searching algorithms namespace SGSearch { // bool findWalks(Vertex* pX, Vertex* pY, EdgeDir initialDir, int maxDistance, size_t maxNodes, bool exhaustive, SGWalkVector& outWalks); void findVariantWalks(Vertex* pX, EdgeDir initialDir, int maxDistance, size_t maxWalks, SGWalkVector& outWalks); void findCollapsedWalks(Vertex* pX, EdgeDir initialDir, int maxDistance, size_t maxNodes, SGWalkVector& outWalks); // Count the number of vertices that span the sequence junction // described by edge XY. Returns -1 if the search was not completed int countSpanningCoverage(Edge* pXY, size_t maxQueue); // Returns true if all the endpoints of the edges in epv are in vertexSet bool checkEndpointsInSet(EdgePtrVec& epv, std::set& vertexSet); }; #endif ================================================ FILE: src/SGA/StringGraph/SGUtil.cpp ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // SGUtils - Data structures/Functions related // to building and manipulating string graphs // #include "SGUtil.h" #include "SeqReader.h" #include "SGAlgorithms.h" #include "SGVisitors.h" StringGraph* SGUtil::loadASQG(const std::string& filename, const unsigned int minOverlap, bool allowContainments, size_t maxEdges) { // Initialize graph StringGraph* pGraph = new StringGraph; std::istream* pReader = createReader(filename); int stage = 0; int line = 0; std::string recordLine; while(getline(*pReader, recordLine)) { ASQG::RecordType rt = ASQG::getRecordType(recordLine); switch(rt) { case ASQG::RT_HEADER: { if(stage != 0) { std::cerr << "Error: Unexpected header record found at line " << line << "\n"; exit(EXIT_FAILURE); } ASQG::HeaderRecord headerRecord(recordLine); const SQG::IntTag& overlapTag = headerRecord.getOverlapTag(); if(overlapTag.isInitialized()) pGraph->setMinOverlap(overlapTag.get()); else pGraph->setMinOverlap(0); const SQG::FloatTag& errorRateTag = headerRecord.getErrorRateTag(); if(errorRateTag.isInitialized()) pGraph->setErrorRate(errorRateTag.get()); const SQG::IntTag& containmentTag = headerRecord.getContainmentTag(); if(containmentTag.isInitialized()) pGraph->setContainmentFlag(containmentTag.get()); else pGraph->setContainmentFlag(true); // conservatively assume containments are present const SQG::IntTag& transitiveTag = headerRecord.getTransitiveTag(); if(!transitiveTag.isInitialized()) { std::cerr << "Warning: ASQG does not have transitive tag\n"; pGraph->setTransitiveFlag(true); } else { pGraph->setTransitiveFlag(transitiveTag.get()); } break; } case ASQG::RT_VERTEX: { // progress the stage if we are done the header if(stage == 0) stage = 1; if(stage != 1) { std::cerr << "Error: Unexpected vertex record found at line " << line << "\n"; exit(EXIT_FAILURE); } ASQG::VertexRecord vertexRecord(recordLine); const SQG::IntTag& ssTag = vertexRecord.getSubstringTag(); // Vertex* pVertex = new(pGraph->getVertexAllocator()) Vertex(vertexRecord.getID(), vertexRecord.getSeq()); Vertex* pVertex = new Vertex(vertexRecord.getID(), vertexRecord.getSeq()); if(ssTag.isInitialized() && ssTag.get() == 1) { // Vertex is a substring of some other vertex, mark it as contained pVertex->setContained(true); pGraph->setContainmentFlag(true); } pGraph->addVertex(pVertex); break; } case ASQG::RT_EDGE: { if(stage == 1) stage = 2; if(stage != 2) { std::cerr << "Error: Unexpected edge record found at line " << line << "\n"; exit(EXIT_FAILURE); } ASQG::EdgeRecord edgeRecord(recordLine); const Overlap& ovr = edgeRecord.getOverlap(); // Add the edge to the graph if(ovr.match.getMinOverlapLength() >= (int)minOverlap) SGAlgorithms::createEdgesFromOverlap(pGraph, ovr, allowContainments, maxEdges); break; } } ++line; } // Completely delete the edges for all nodes that were marked as super-repetitive in the graph SGSuperRepeatVisitor superRepeatVisitor; pGraph->visit(superRepeatVisitor); // Remove any duplicate edges SGDuplicateVisitor dupVisit; pGraph->visit(dupVisit); SGGraphStatsVisitor statsVisit; pGraph->visit(statsVisit); // Remove identical vertices // This is much cheaper to do than remove via // SGContainRemove as no remodelling needs to occur /* SGIdenticalRemoveVisitor irv; pGraph->visit(irv); // Remove substring vertices while(pGraph->hasContainment()) { SGContainRemoveVisitor crv; pGraph->visit(crv); } */ delete pReader; return pGraph; } //JEREMIAH StringGraph* SGUtil::loadASQG(std::stringstream& pReader, const unsigned int minOverlap, bool allowContainments, size_t maxEdges) { // Initialize graph StringGraph* pGraph = new StringGraph; //std::istream* pReader = createReader(filename); int stage = 0; int line = 0; std::string recordLine; while(getline(pReader, recordLine)) { ASQG::RecordType rt = ASQG::getRecordType(recordLine); switch(rt) { case ASQG::RT_HEADER: { if(stage != 0) { std::cerr << "Error: Unexpected header record found at line " << line << "\n"; exit(EXIT_FAILURE); } ASQG::HeaderRecord headerRecord(recordLine); const SQG::IntTag& overlapTag = headerRecord.getOverlapTag(); if(overlapTag.isInitialized()) pGraph->setMinOverlap(overlapTag.get()); else pGraph->setMinOverlap(0); const SQG::FloatTag& errorRateTag = headerRecord.getErrorRateTag(); if(errorRateTag.isInitialized()) pGraph->setErrorRate(errorRateTag.get()); const SQG::IntTag& containmentTag = headerRecord.getContainmentTag(); if(containmentTag.isInitialized()) pGraph->setContainmentFlag(containmentTag.get()); else pGraph->setContainmentFlag(true); // conservatively assume containments are present const SQG::IntTag& transitiveTag = headerRecord.getTransitiveTag(); if(!transitiveTag.isInitialized()) { std::cerr << "Warning: ASQG does not have transitive tag\n"; pGraph->setTransitiveFlag(true); } else { pGraph->setTransitiveFlag(transitiveTag.get()); } break; } case ASQG::RT_VERTEX: { // progress the stage if we are done the header if(stage == 0) stage = 1; if(stage != 1) { std::cerr << "Error: Unexpected vertex record found at line " << line << "\n"; exit(EXIT_FAILURE); } ASQG::VertexRecord vertexRecord(recordLine); const SQG::IntTag& ssTag = vertexRecord.getSubstringTag(); // Vertex* pVertex = new(pGraph->getVertexAllocator()) Vertex(vertexRecord.getID(), vertexRecord.getSeq()); Vertex* pVertex = new Vertex(vertexRecord.getID(), vertexRecord.getSeq()); if(ssTag.isInitialized() && ssTag.get() == 1) { // Vertex is a substring of some other vertex, mark it as contained pVertex->setContained(true); pGraph->setContainmentFlag(true); } pGraph->addVertex(pVertex); break; } case ASQG::RT_EDGE: { if(stage == 1) stage = 2; if(stage != 2) { std::cerr << "Error: Unexpected edge record found at line " << line << "\n"; exit(EXIT_FAILURE); } ASQG::EdgeRecord edgeRecord(recordLine); const Overlap& ovr = edgeRecord.getOverlap(); // Add the edge to the graph if(ovr.match.getMinOverlapLength() >= (int)minOverlap) SGAlgorithms::createEdgesFromOverlap(pGraph, ovr, allowContainments, maxEdges); break; } } ++line; } // Completely delete the edges for all nodes that were marked as super-repetitive in the graph SGSuperRepeatVisitor superRepeatVisitor; pGraph->visit(superRepeatVisitor); // Remove any duplicate edges SGDuplicateVisitor dupVisit; pGraph->visit(dupVisit); //SGGraphStatsVisitor statsVisit; //pGraph->visit(statsVisit); // Remove identical vertices // This is much cheaper to do than remove via // SGContainRemove as no remodelling needs to occur /* SGIdenticalRemoveVisitor irv; pGraph->visit(irv); // Remove substring vertices while(pGraph->hasContainment()) { SGContainRemoveVisitor crv; pGraph->visit(crv); } */ //delete pReader; return pGraph; } // Load a graph (with no edges) from a fasta file StringGraph* SGUtil::loadFASTA(const std::string& filename) { StringGraph* pGraph = new StringGraph; SeqReader reader(filename); SeqRecord record; while(reader.get(record)) { //Vertex* pVertex = new(pGraph->getVertexAllocator()) Vertex(record.id, record.seq.toString()); Vertex* pVertex = new Vertex(record.id, record.seq.toString()); pGraph->addVertex(pVertex); } return pGraph; } ================================================ FILE: src/SGA/StringGraph/SGUtil.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL license //----------------------------------------------- // // SGUtils - Data structures/Functions related // to building and manipulating string graphs // #ifndef SGUTIL_H #define SGUTIL_H #include "Bigraph.h" #include "ASQG.h" // typedefs typedef Bigraph StringGraph; namespace SGUtil { // Main string graph loading function // The allowContainments flag forces the string graph to retain identical vertices // Vertices that are substrings of other vertices (SS flag = 1) are never kept StringGraph* loadASQG(const std::string& filename, const unsigned int minOverlap, bool allowContainments = false, size_t maxEdges = -1); StringGraph* loadASQG(std::stringstream& pReader, const unsigned int minOverlap, bool allowContainments = false, size_t maxEdges = -1); // Load a string graph from a fasta file. // Returns a graph where each sequence in the fasta is a vertex but there are no edges in the graph. StringGraph* loadFASTA(const std::string& filename); }; #endif ================================================ FILE: src/SGA/StringGraph/SGVisitors.cpp ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // SGVisitors - Algorithms that visit // each vertex in the graph and perform some // operation // #include "SGVisitors.h" #include "CompleteOverlapSet.h" #include "SGSearch.h" #include "stdaln.h" // // SGFastaVisitor - output the vertices in the graph in // fasta format // bool SGFastaVisitor::visit(StringGraph* /*pGraph*/, Vertex* pVertex) { m_fileHandle << ">" << pVertex->getID() << " " << pVertex->getSeq().length() << " " << 0 << "\n"; m_fileHandle << pVertex->getSeq() << "\n"; return false; } // jwala added bool SGVisitorContig::visit(StringGraph* /*pGraph*/, Vertex* pVertex) { m_ct.push_back(SeqLib::UnalignedSequence(pVertex->getID(), pVertex->getSeq().toString(),std::string())); return false; } // // SGTransRedVisitor - Perform a transitive reduction about this vertex // This uses Myers' algorithm (2005, The fragment assembly string graph) // Precondition: the edge list is sorted by length (ascending) void SGTransitiveReductionVisitor::previsit(StringGraph* pGraph) { // The graph must not have containments assert(!pGraph->hasContainment()); // Set all the vertices in the graph to "vacant" pGraph->setColors(GC_WHITE); pGraph->sortVertexAdjListsByLen(); marked_verts = 0; marked_edges = 0; } // bool SGTransitiveReductionVisitor::visit(StringGraph* /*pGraph*/, Vertex* pVertex) { size_t trans_count = 0; static const size_t FUZZ = 10; // see myers for(size_t idx = 0; idx < ED_COUNT; idx++) { EdgeDir dir = EDGE_DIRECTIONS[idx]; EdgePtrVec edges = pVertex->getEdges(dir); // These edges are already sorted if(edges.size() == 0) continue; for(size_t i = 0; i < edges.size(); ++i) (edges[i])->getEnd()->setColor(GC_GRAY); Edge* pLongestEdge = edges.back(); size_t longestLen = pLongestEdge->getSeqLen() + FUZZ; // Stage 1 for(size_t i = 0; i < edges.size(); ++i) { Edge* pVWEdge = edges[i]; Vertex* pWVert = pVWEdge->getEnd(); EdgeDir transDir = !pVWEdge->getTwinDir(); if(pWVert->getColor() == GC_GRAY) { EdgePtrVec w_edges = pWVert->getEdges(transDir); for(size_t j = 0; j < w_edges.size(); ++j) { Edge* pWXEdge = w_edges[j]; size_t trans_len = pVWEdge->getSeqLen() + pWXEdge->getSeqLen(); if(trans_len <= longestLen) { if(pWXEdge->getEnd()->getColor() == GC_GRAY) { // X is the endpoint of an edge of V, therefore it is transitive pWXEdge->getEnd()->setColor(GC_BLACK); } } else break; } } } // Stage 2 for(size_t i = 0; i < edges.size(); ++i) { Edge* pVWEdge = edges[i]; Vertex* pWVert = pVWEdge->getEnd(); EdgeDir transDir = !pVWEdge->getTwinDir(); EdgePtrVec w_edges = pWVert->getEdges(transDir); for(size_t j = 0; j < w_edges.size(); ++j) { Edge* pWXEdge = w_edges[j]; size_t len = pWXEdge->getSeqLen(); if(len < FUZZ || j == 0) { if(pWXEdge->getEnd()->getColor() == GC_GRAY) { // X is the endpoint of an edge of V, therefore it is transitive pWXEdge->getEnd()->setColor(GC_BLACK); } } else { break; } } } for(size_t i = 0; i < edges.size(); ++i) { if(edges[i]->getEnd()->getColor() == GC_BLACK) { // Mark the edge and its twin for removal if(edges[i]->getColor() != GC_BLACK || edges[i]->getTwin()->getColor() != GC_BLACK) { edges[i]->setColor(GC_BLACK); edges[i]->getTwin()->setColor(GC_BLACK); marked_edges += 2; trans_count++; } } edges[i]->getEnd()->setColor(GC_WHITE); } } if(trans_count > 0) ++marked_verts; return false; } // Remove all the marked edges void SGTransitiveReductionVisitor::postvisit(StringGraph* pGraph) { //printf("TR marked %d verts and %d edges\n", marked_verts, marked_edges); pGraph->sweepEdges(GC_BLACK); pGraph->setTransitiveFlag(false); assert(pGraph->checkColors(GC_WHITE)); } // // SGIdenticalRemoveVisitor - Removes identical vertices // from the graph. This algorithm is less complex // than SGContainRemoveVisitor because we do not have to // remodel the graph in this case because no irreducible // edges need to be moved. // void SGIdenticalRemoveVisitor::previsit(StringGraph* pGraph) { pGraph->setColors(GC_WHITE); count = 0; } // bool SGIdenticalRemoveVisitor::visit(StringGraph* /*pGraph*/, Vertex* pVertex) { if(!pVertex->isContained()) return false; // Check if this vertex is identical to any other vertex EdgePtrVec neighborEdges = pVertex->getEdges(); for(size_t i = 0; i < neighborEdges.size(); ++i) { Edge* pEdge = neighborEdges[i]; Vertex* pOther = pEdge->getEnd(); if(pVertex->getSeqLen() != pOther->getSeqLen()) continue; Overlap ovr = pEdge->getOverlap(); if(!ovr.isContainment() || ovr.getContainedIdx() != 0) continue; if(pVertex->getSeq() == pOther->getSeq()) { pVertex->setColor(GC_BLACK); ++count; break; } } return false; } void SGIdenticalRemoveVisitor::postvisit(StringGraph* pGraph) { pGraph->sweepVertices(GC_BLACK); } // // SGContainRemoveVisitor - Removes contained // vertices from the graph // void SGContainRemoveVisitor::previsit(StringGraph* pGraph) { pGraph->setColors(GC_WHITE); // Clear the containment flag, if any containments are added // during this algorithm the flag will be reset and another // round must be re-run pGraph->setContainmentFlag(false); } // bool SGContainRemoveVisitor::visit(StringGraph* pGraph, Vertex* pVertex) { if(!pVertex->isContained()) return false; //cout << pVertex->getID() << endl; // debug // Add any new irreducible edges that exist when pToRemove is deleted // from the graph EdgePtrVec neighborEdges = pVertex->getEdges(); // If the graph has been transitively reduced, we have to check all // the neighbors to see if any new edges need to be added. If the graph is a // complete overlap graph we can just remove the edges to the deletion vertex if(!pGraph->hasTransitive() && !pGraph->isExactMode()) { // This must be done in order of edge length or some transitive edges // may be created EdgeLenComp comp; std::sort(neighborEdges.begin(), neighborEdges.end(), comp); for(size_t j = 0; j < neighborEdges.size(); ++j) { Vertex* pRemodelVert = neighborEdges[j]->getEnd(); Edge* pRemodelEdge = neighborEdges[j]->getTwin(); SGAlgorithms::remodelVertexForExcision(pGraph, pRemodelVert, pRemodelEdge); } } // Delete the edges from the graph for(size_t j = 0; j < neighborEdges.size(); ++j) { Vertex* pRemodelVert = neighborEdges[j]->getEnd(); Edge* pRemodelEdge = neighborEdges[j]->getTwin(); pRemodelVert->deleteEdge(pRemodelEdge); pVertex->deleteEdge(neighborEdges[j]); } pVertex->setColor(GC_BLACK); return false; } void SGContainRemoveVisitor::postvisit(StringGraph* pGraph) { pGraph->sweepVertices(GC_BLACK); } // // Validate the structure of the graph by detecting missing // or erroneous edges // // bool SGValidateStructureVisitor::visit(StringGraph* pGraph, Vertex* pVertex) { SGAlgorithms::EdgeDescOverlapMap irreducibleMap; SGAlgorithms::EdgeDescOverlapMap transitiveMap; // Construct the set of overlaps reachable within the current parameters CompleteOverlapSet vertexOverlapSet(pVertex, pGraph->getErrorRate(), pGraph->getMinOverlap()); vertexOverlapSet.computeIrreducible(NULL, NULL); SGAlgorithms::EdgeDescOverlapMap missingMap; SGAlgorithms::EdgeDescOverlapMap extraMap; vertexOverlapSet.getDiffMap(missingMap, extraMap); if(!missingMap.empty()) { std::cout << "Missing irreducible for " << pVertex->getID() << ":\n"; SGAlgorithms::printOverlapMap(missingMap); } if(!extraMap.empty()) { std::cout << "Extra irreducible for " << pVertex->getID() << ":\n"; SGAlgorithms::printOverlapMap(extraMap); } return false; } // // SGTrimVisitor - Remove "dead-end" vertices from the graph // void SGTrimVisitor::previsit(StringGraph* pGraph) { num_island = 0; num_terminal = 0; pGraph->setColors(GC_WHITE); } // Mark any nodes that either dont have edges or edges in only one direction for removal bool SGTrimVisitor::visit(StringGraph* /*pGraph*/, Vertex* pVertex) { if(pVertex->countEdges() == 0) { // Is an island, remove if the sequence length is less than the threshold if(pVertex->getSeqLen() < m_minLength) { pVertex->setColor(GC_BLACK); ++num_island; } } else { // Check if this node is a dead-end for(size_t idx = 0; idx < ED_COUNT; idx++) { EdgeDir dir = EDGE_DIRECTIONS[idx]; if(pVertex->countEdges(dir) == 0 && pVertex->getSeqLen() < m_minLength) { pVertex->setColor(GC_BLACK); ++num_terminal; } } } return false; } // Remove all the marked edges void SGTrimVisitor::postvisit(StringGraph* pGraph) { pGraph->sweepVertices(GC_BLACK); //printf("StringGraphTrim: Removed %d island and %d dead-end short vertices\n", num_island, num_terminal); } // // SGDuplicateVisitor - Detect and remove duplicate edges // void SGDuplicateVisitor::previsit(StringGraph* pGraph) { assert(pGraph->checkColors(GC_WHITE)); (void)pGraph; m_hasDuplicate = false; } bool SGDuplicateVisitor::visit(StringGraph* /*pGraph*/, Vertex* pVertex) { m_hasDuplicate = pVertex->markDuplicateEdges(GC_RED) || m_hasDuplicate; return false; } void SGDuplicateVisitor::postvisit(StringGraph* pGraph) { assert(pGraph->checkColors(GC_WHITE)); //if(m_hasDuplicate) // { //int numRemoved = pGraph->sweepEdges(GC_RED); //if(!m_bSilent) // std::cerr << "Warning: removed " << numRemoved << " duplicate edges\n"; //} } // // Small repeat resolver - Remove edges induced from small (sub-read length) // repeats // void SGSmallRepeatResolveVisitor::previsit(StringGraph*) { } // bool SGSmallRepeatResolveVisitor::visit(StringGraph* /*pGraph*/, Vertex* pX) { bool changed = false; // If the vertex has more than MAX_EDGES, do not // attempt to resolve size_t MAX_EDGES = 10; for(size_t idx = 0; idx < ED_COUNT; idx++) { EdgeDir dir = EDGE_DIRECTIONS[idx]; EdgePtrVec x_edges = pX->getEdges(dir); // These edges are already sorted if(x_edges.size() < 2 || x_edges.size() > MAX_EDGES) continue; // Try to eliminate the shortest edge from this vertex (let this be X->Y) // If Y has a longer edge than Y->X in the same direction, we remove X->Y // Edges are sorted by length so the last edge is the shortest Edge* pXY = x_edges.back(); size_t xy_len = pXY->getOverlap().getOverlapLength(0); size_t x_longest_len = x_edges.front()->getOverlap().getOverlapLength(0); if(xy_len == x_longest_len) continue; Edge* pYX = pXY->getTwin(); Vertex* pY = pXY->getEnd(); EdgePtrVec y_edges = pY->getEdges(pYX->getDir()); if(y_edges.size() > MAX_EDGES) continue; size_t yx_len = pYX->getOverlap().getOverlapLength(0); size_t y_longest_len = 0; for(size_t i = 0; i < y_edges.size(); ++i) { Edge* pYZ = y_edges[i]; if(pYZ == pYX) continue; // skip Y->X size_t yz_len = pYZ->getOverlap().getOverlapLength(0); if(yz_len > y_longest_len) y_longest_len = yz_len; } if(y_longest_len > yx_len) { // Delete the edge if the difference between the shortest and longest is greater than minDiff int x_diff = x_longest_len - xy_len; int y_diff = y_longest_len - yx_len; if(x_diff > m_minDiff && y_diff > m_minDiff) { pX->deleteEdge(pXY); pY->deleteEdge(pYX); changed = true; } } } return changed; } // void SGSmallRepeatResolveVisitor::postvisit(StringGraph* pGraph) { pGraph->sweepEdges(GC_RED); } // // OverlapRatio Visitor - Only keep edges in the graph when the ratio // between their length and the length of the longest overlap meets a minimum cutoff // void SGOverlapRatioVisitor::previsit(StringGraph*) { } // bool SGOverlapRatioVisitor::visit(StringGraph* /*pGraph*/, Vertex* pX) { bool changed = false; for(size_t idx = 0; idx < ED_COUNT; idx++) { EdgeDir dir = EDGE_DIRECTIONS[idx]; EdgePtrVec x_edges = pX->getEdges(dir); // These edges are already sorted if(x_edges.size() < 2) continue; size_t x_longest_len = x_edges.front()->getOverlap().getOverlapLength(0); for(size_t i = 1; i < x_edges.size(); ++i) { size_t curr_len = x_edges[i]->getOverlap().getOverlapLength(0); double ratio = (double)curr_len / x_longest_len; if(ratio < m_minRatio) { x_edges[i]->setColor(GC_RED); x_edges[i]->getTwin()->setColor(GC_RED); changed = true; } } } return changed; } // void SGOverlapRatioVisitor::postvisit(StringGraph* pGraph) { pGraph->sweepEdges(GC_RED); } // // SGSuperRepeatVisitor // // Remove all edges of nodes that have been marked as super repeats void SGSuperRepeatVisitor::previsit(StringGraph*) { m_num_superrepeats = 0; } // bool SGSuperRepeatVisitor::visit(StringGraph*, Vertex* pVertex) { if(pVertex->isSuperRepeat()) { pVertex->deleteEdges(); m_num_superrepeats += 1; return true; } return false; } // void SGSuperRepeatVisitor::postvisit(StringGraph*) { //printf("Deleted edges for %zu super repetitive vertices\n", m_num_superrepeats); } // // SGSmoothingVisitor - Find branches in the graph // which arise from variation and remove them // void SGSmoothingVisitor::previsit(StringGraph* pGraph) { pGraph->setColors(GC_WHITE); m_simpleBubblesRemoved = 0; m_complexBubblesRemoved = 0; } // bool SGSmoothingVisitor::visit(StringGraph* pGraph, Vertex* pVertex) { (void)pGraph; if(pVertex->getColor() == GC_RED) return false; bool found = false; for(size_t idx = 0; idx < ED_COUNT; idx++) { EdgeDir dir = EDGE_DIRECTIONS[idx]; EdgePtrVec edges = pVertex->getEdges(dir); if(edges.size() <= 1) continue; for(size_t i = 0; i < edges.size(); ++i) { if(edges[i]->getEnd()->getColor() == GC_RED) return false; } //std::cout << "Smoothing " << pVertex->getID() << "\n"; const int MAX_WALKS = 10; const int MAX_DISTANCE = 5000; bool bIsDegenerate = false; bool bFailGapCheck = false; bool bFailDivergenceCheck = false; bool bFailIndelSizeCheck = false; SGWalkVector variantWalks; SGSearch::findVariantWalks(pVertex, dir, MAX_DISTANCE, MAX_WALKS, variantWalks); if(variantWalks.size() > 0) { found = true; size_t selectedIdx = -1; size_t selectedCoverage = 0; // Calculate the minimum amount overlapped on the start/end vertex. // This is used to properly extract the sequences from walks that represent the variation. int minOverlapX = std::numeric_limits::max(); int minOverlapY = std::numeric_limits::max(); for(size_t i = 0; i < variantWalks.size(); ++i) { if(variantWalks[i].getNumEdges() <= 1) bIsDegenerate = true; // Calculate the walk coverage using the internal vertices of the walk. // The walk with the highest coverage will be retained size_t walkCoverage = 0; for(size_t j = 1; j < variantWalks[i].getNumVertices() - 1; ++j) walkCoverage += variantWalks[i].getVertex(j)->getCoverage(); if(walkCoverage > selectedCoverage || selectedCoverage == 0) { selectedIdx = i; selectedCoverage = walkCoverage; } Edge* pFirstEdge = variantWalks[i].getFirstEdge(); Edge* pLastEdge = variantWalks[i].getLastEdge(); if((int)pFirstEdge->getMatchLength() < minOverlapX) minOverlapX = pFirstEdge->getMatchLength(); if((int)pLastEdge->getTwin()->getMatchLength() < minOverlapY) minOverlapY = pLastEdge->getTwin()->getMatchLength(); } // Calculate the strings for each walk that represent the region of variation StringVector walkStrings; for(size_t i = 0; i < variantWalks.size(); ++i) { Vertex* pStartVertex = variantWalks[i].getStartVertex(); Vertex* pLastVertex = variantWalks[i].getLastVertex(); assert(pStartVertex != NULL && pLastVertex != NULL); std::string full = variantWalks[i].getString(SGWT_START_TO_END); int posStart = 0; int posEnd = 0; if(dir == ED_ANTISENSE) { // pLast ----------- // pStart ------------ // full -------------------- // out ---- posStart = pLastVertex->getSeqLen() - minOverlapY; posEnd = full.size() - (pStartVertex->getSeqLen() - minOverlapX); } else { // pStart -------------- // pLast ----------- // full --------------------- // out ---- posStart = pStartVertex->getSeqLen() - minOverlapX; // match start position posEnd = full.size() - (pLastVertex->getSeqLen() - minOverlapY); // match end position } std::string out; if(posEnd > posStart) out = full.substr(posStart, posEnd - posStart); walkStrings.push_back(out); } assert(selectedIdx != (size_t)-1); SGWalk& selectedWalk = variantWalks[selectedIdx]; assert(selectedWalk.isIndexed()); // Check the divergence of the other walks to this walk StringVector cigarStrings; std::vector maxIndel; std::vector gapPercent; // percentage of matching that is gaps std::vector totalPercent; // percent of total alignment that is mismatch or gap cigarStrings.resize(variantWalks.size()); gapPercent.resize(variantWalks.size()); totalPercent.resize(variantWalks.size()); maxIndel.resize(variantWalks.size()); for(size_t i = 0; i < variantWalks.size(); ++i) { if(i == selectedIdx) continue; // We want to compute the total gap length, total mismatches and percent // divergence between the two paths. int matchLen = 0; int totalDiff = 0; int gapLength = 0; int maxGapLength = 0; // We have to handle the degenerate case where one internal string has zero length // this can happen when there is an isolated insertion/deletion and the walks are like: // x -> y -> z // x -> z if(walkStrings[selectedIdx].empty() || walkStrings[i].empty()) { matchLen = std::max(walkStrings[selectedIdx].size(), walkStrings[i].size()); totalDiff = matchLen; gapLength = matchLen; } else { AlnAln *aln_global; aln_global = aln_stdaln(walkStrings[selectedIdx].c_str(), walkStrings[i].c_str(), &aln_param_blast, 1, 1); // Calculate the alignment parameters while(aln_global->outm[matchLen] != '\0') { if(aln_global->outm[matchLen] == ' ') totalDiff += 1; matchLen += 1; } std::stringstream cigarSS; for (int j = 0; j != aln_global->n_cigar; ++j) { char cigarOp = "MID"[aln_global->cigar32[j]&0xf]; int cigarLen = aln_global->cigar32[j]>>4; if(cigarOp == 'I' || cigarOp == 'D') { gapLength += cigarLen; if(gapLength > maxGapLength) maxGapLength = gapLength; } cigarSS << cigarLen; cigarSS << cigarOp; } cigarStrings[i] = cigarSS.str(); aln_free_AlnAln(aln_global); } double percentDiff = (double)totalDiff / matchLen; double percentGap = (double)gapLength / matchLen; if(percentDiff > m_maxTotalDivergence) bFailDivergenceCheck = true; if(percentGap > m_maxGapDivergence) bFailGapCheck = true; if(maxGapLength > m_maxIndelLength) bFailIndelSizeCheck = true; gapPercent[i] = percentGap; totalPercent[i] = percentDiff; maxIndel[i] = maxGapLength; } if(bIsDegenerate || bFailGapCheck || bFailDivergenceCheck || bFailIndelSizeCheck) continue; // Write the selected path to the variants file as variant 0 int variantIdx = 0; std::string selectedSequence = selectedWalk.getString(SGWT_START_TO_END); std::stringstream ss; ss << "variant-" << m_numRemovedTotal << "/" << variantIdx++; writeFastaRecord(&m_outFile, ss.str(), selectedSequence); // The vertex set for each walk is not necessarily disjoint, // the selected walk may contain vertices that are part // of other paths. We handle this be initially marking all // vertices of the for(size_t i = 0; i < variantWalks.size(); ++i) { if(i == selectedIdx) continue; SGWalk& currWalk = variantWalks[i]; for(size_t j = 0; j < currWalk.getNumEdges() - 1; ++j) { Edge* currEdge = currWalk.getEdge(j); // If the vertex is also on the selected path, do not mark it Vertex* currVertex = currEdge->getEnd(); if(!selectedWalk.containsVertex(currVertex->getID())) { currEdge->getEnd()->setColor(GC_RED); } } // Write the variant to a file std::string variantSequence = currWalk.getString(SGWT_START_TO_END); std::stringstream variantID; std::stringstream ss; ss << "variant-" << m_numRemovedTotal << "/" << variantIdx++; ss << " IGD:" << (double)gapPercent[i] << " ITD:" << totalPercent[i] << " MID: " << maxIndel[i] << " InternalCigar:" << cigarStrings[i]; writeFastaRecord(&m_outFile, ss.str(), variantSequence); } if(variantWalks.size() == 2) m_simpleBubblesRemoved += 1; else m_complexBubblesRemoved += 1; ++m_numRemovedTotal; } } return found; } // Remove all the marked edges void SGSmoothingVisitor::postvisit(StringGraph* pGraph) { pGraph->sweepVertices(GC_RED); assert(pGraph->checkColors(GC_WHITE)); //printf("VariationSmoother: Removed %d simple and %d complex bubbles\n", m_simpleBubblesRemoved, m_complexBubblesRemoved); } // // SGGraphStatsVisitor - Collect summary stasitics // about the graph // void SGGraphStatsVisitor::previsit(StringGraph* /*pGraph*/) { num_terminal = 0; num_island = 0; num_monobranch = 0; num_dibranch = 0; num_simple = 0; num_edges = 0; num_vertex = 0; sum_edgeLen = 0; } // Find bubbles (nodes where there is a split and then immediate rejoin) and mark them for removal bool SGGraphStatsVisitor::visit(StringGraph* /*pGraph*/, Vertex* pVertex) { int s_count = pVertex->countEdges(ED_SENSE); int as_count = pVertex->countEdges(ED_ANTISENSE); if(s_count == 0 && as_count == 0) { ++num_island; } else if(s_count == 0 || as_count == 0) { ++num_terminal; } if(s_count > 1 && as_count > 1) ++num_dibranch; else if(s_count > 1 || as_count > 1) ++num_monobranch; if(s_count == 1 || as_count == 1) ++num_simple; num_edges += (s_count + as_count); ++num_vertex; EdgePtrVec edges = pVertex->getEdges(); for(size_t i = 0; i < edges.size(); ++i) sum_edgeLen += edges[i]->getSeqLen(); return false; } // void SGGraphStatsVisitor::postvisit(StringGraph* /*pGraph*/) { printf("Vertices: %d Edges: %d Islands: %d Tips: %d Monobranch: %d Dibranch: %d Simple: %d\n", num_vertex, num_edges, num_island, num_terminal, num_monobranch, num_dibranch, num_simple); } ================================================ FILE: src/SGA/StringGraph/SGVisitors.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // SGVisitors - Algorithms that visit // each vertex in the graph and perform some // operation // #include "SGAlgorithms.h" #include "SGUtil.h" #include "Util.h" //#include "contigs.h" #include "SeqLib/UnalignedSequence.h" #ifndef SGVISITORS_H #define SGVISITORS_H // Visit each node, writing it to a file as a fasta record struct SGFastaVisitor { // constructor SGFastaVisitor(std::string filename) : m_fileHandle(filename.c_str()) {} ~SGFastaVisitor() { m_fileHandle.close(); } // functions void previsit(StringGraph* /*pGraph*/) {} bool visit(StringGraph* pGraph, Vertex* pVertex); void postvisit(StringGraph* /*pGraph*/) {} // data std::ofstream m_fileHandle; }; // Visit each node, writing it to a file as a fasta record // jwala modified struct SGVisitorContig { // constructor SGVisitorContig() {} ~SGVisitorContig() {} // functions void previsit(StringGraph* /*pGraph*/) {} bool visit(StringGraph* pGraph, Vertex* pVertex); void postvisit(StringGraph* /*pGraph*/) {} // data SeqLib::UnalignedSequenceVector m_ct; }; // Visit each node, writing it to a file as a fasta record // Run the Myers transitive reduction algorithm on each node struct SGTransitiveReductionVisitor { SGTransitiveReductionVisitor() {} void previsit(StringGraph* pGraph); bool visit(StringGraph* pGraph, Vertex* pVertex); void postvisit(StringGraph*); int marked_verts; int marked_edges; }; // Remove identical vertices from the graph struct SGIdenticalRemoveVisitor { SGIdenticalRemoveVisitor() {} void previsit(StringGraph* pGraph); bool visit(StringGraph* pGraph, Vertex* pVertex); void postvisit(StringGraph* pGraph); int count; }; // Remove contained vertices from the graph struct SGContainRemoveVisitor { SGContainRemoveVisitor() {} void previsit(StringGraph* pGraph); bool visit(StringGraph* pGraph, Vertex* pVertex); void postvisit(StringGraph* pGraph); }; // Validate that the graph does not contain // any extra edges or missing irreducible edges struct SGValidateStructureVisitor { SGValidateStructureVisitor() {} void previsit(StringGraph*) {} bool visit(StringGraph* pGraph, Vertex* pVertex); void postvisit(StringGraph*) {} }; // Remodel the graph to infer missing edges or remove erroneous edges struct SGSmallRepeatResolveVisitor { SGSmallRepeatResolveVisitor(int minDiff) : m_minDiff(minDiff) {} void previsit(StringGraph* pGraph); bool visit(StringGraph* pGraph, Vertex* pVertex); void postvisit(StringGraph*); int m_minDiff; }; // Remove edges from the graph when the ratio between an edge's overlap length // and the longest overlap for the vertex is less than the given parameter struct SGOverlapRatioVisitor { SGOverlapRatioVisitor(double minRatio) : m_minRatio(minRatio) {} void previsit(StringGraph* pGraph); bool visit(StringGraph* pGraph, Vertex* pVertex); void postvisit(StringGraph*); double m_minRatio; }; // Detects and removes small "tip" vertices from the graph // when they are less than minLength in size struct SGTrimVisitor { SGTrimVisitor(size_t minLength) : m_minLength(minLength) {} void previsit(StringGraph* pGraph); bool visit(StringGraph* pGraph, Vertex* pVertex); void postvisit(StringGraph*); size_t m_minLength; int num_island; int num_terminal; }; // Detect and remove duplicate edges struct SGDuplicateVisitor { SGDuplicateVisitor(bool silent = false) : m_bSilent(silent) {} void previsit(StringGraph*); bool visit(StringGraph* pGraph, Vertex* pVertex); void postvisit(StringGraph*); bool m_hasDuplicate; bool m_bSilent; }; // Remove the edges of super-repetitive vertices in the graph struct SGSuperRepeatVisitor { SGSuperRepeatVisitor() {} void previsit(StringGraph* pGraph); bool visit(StringGraph* pGraph, Vertex* pVertex); void postvisit(StringGraph*); size_t m_num_superrepeats; }; // Smooth out variation in the graph struct SGSmoothingVisitor { SGSmoothingVisitor(std::string filename, double maxGapDiv, double maxTotalDiv, int maxIndelLength) : m_numRemovedTotal(0), m_maxGapDivergence(maxGapDiv), m_maxTotalDivergence(maxTotalDiv), m_maxIndelLength(maxIndelLength), m_outFile(filename.c_str()) {} void previsit(StringGraph* pGraph); bool visit(StringGraph* pGraph, Vertex* pVertex); void postvisit(StringGraph*); int m_simpleBubblesRemoved; int m_complexBubblesRemoved; int m_numRemovedTotal; double m_maxGapDivergence; double m_maxTotalDivergence; int m_maxIndelLength; std::ofstream m_outFile; }; // Compile summary statistics for the graph struct SGGraphStatsVisitor { SGGraphStatsVisitor() {} void previsit(StringGraph* pGraph); bool visit(StringGraph* pGraph, Vertex* pVertex); void postvisit(StringGraph*); int num_terminal; int num_island; int num_monobranch; int num_dibranch; int num_simple; int num_edges; int num_vertex; size_t sum_edgeLen; }; #endif ================================================ FILE: src/SGA/StringGraph/SGWalk.cpp ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // SGWalk - Data structure holding a walk through // the graph // #include "SGWalk.h" // SGWalk::SGWalk(Vertex* pStartVertex, bool bIndexWalk) : m_pStartVertex(pStartVertex), m_extensionDistance(0), m_extensionFinished(false) { if(bIndexWalk) m_pWalkIndex = new WalkIndex; else m_pWalkIndex = NULL; } // SGWalk::~SGWalk() { if(m_pWalkIndex != NULL) { delete m_pWalkIndex; m_pWalkIndex = NULL; } } // SGWalk::SGWalk(const SGWalk& other) { m_pStartVertex = other.m_pStartVertex; m_edges = other.m_edges; m_extensionDistance = other.m_extensionDistance; m_extensionFinished = other.m_extensionFinished; m_pWalkIndex = NULL; if(other.m_pWalkIndex != NULL) m_pWalkIndex = new WalkIndex(*other.m_pWalkIndex); } // Construct the walk structure from a vector of edges SGWalk::SGWalk(const EdgePtrVec& edgeVec, bool bIndexWalk) : m_extensionDistance(0), m_extensionFinished(false) { assert(!edgeVec.empty()); if(bIndexWalk) m_pWalkIndex = new WalkIndex; else m_pWalkIndex = NULL; // The start vector is the start vertex of the first edge Edge* first = edgeVec.front(); m_pStartVertex = first->getStart(); for(EdgePtrVec::const_iterator iter = edgeVec.begin(); iter != edgeVec.end(); ++iter) { addEdge(*iter); } } // SGWalk& SGWalk::operator=(const SGWalk& other) { if(&other == this) return *this; // self-assign m_pStartVertex = other.m_pStartVertex; m_edges = other.m_edges; m_extensionDistance = other.m_extensionDistance; if(m_pWalkIndex != NULL) delete m_pWalkIndex; if(other.m_pWalkIndex != NULL) m_pWalkIndex = new WalkIndex(*other.m_pWalkIndex); return *this; } // void SGWalk::addEdge(Edge* pEdge) { m_edges.push_back(pEdge); m_extensionDistance += pEdge->getSeqLen(); // Add the vertex ID to the index if necessary if(m_pWalkIndex != NULL) m_pWalkIndex->insert(m_edges.back()->getEndID()); } // void SGWalk::setFinished(bool b) { m_extensionFinished = b; } bool SGWalk::isFinished() const { return m_extensionFinished; } // void SGWalk::popLast() { m_edges.pop_back(); } // Vertex* SGWalk::getStartVertex() const { return m_pStartVertex; } // size_t SGWalk::getNumVertices() const { return getNumEdges() + 1; } // size_t SGWalk::getNumEdges() const { return m_edges.size(); } bool SGWalk::isIndexed() const { return m_pWalkIndex != NULL; } // bool SGWalk::containsVertex(const VertexID& id) const { if(m_pWalkIndex == NULL) assert(false); return m_pWalkIndex->count(id) > 0; } // void SGWalk::truncate(const VertexID& id) { EdgePtrVec::iterator iter = m_edges.begin(); while(iter != m_edges.end()) { if((*iter)->getEndID() == id) break; ++iter; } assert(iter != m_edges.end()); m_edges.erase(iter + 1, m_edges.end()); } // Construct the extension string corresponding to the path std::string SGWalk::getString(SGWalkType type, SGWalkVertexPlacementVector* pPlacementVector) const { std::string out; // Append the full length of the starting vertex to the walk if(type == SGWT_START_TO_END || type == SGWT_INTERNAL) { out.append(m_pStartVertex->getSeq().toString()); // Add the first record to the placement vector if required if(pPlacementVector != NULL) { SGWalkVertexPlacement firstPlace; firstPlace.pVertex = m_pStartVertex; firstPlace.position = 0; // 0-based coordinates firstPlace.isRC = false; pPlacementVector->push_back(firstPlace); } } // Determine if the string should go to the end of the last vertex // in the path size_t stop = m_edges.size(); // The first edge is always in correct frame of reference // so the comp is EC_SAME. This variable tracks where the // string that is being added is different from the starting sequence // and needs to be flipped EdgeComp currComp = EC_SAME; // If the walk direction is antisense, we reverse every component and then // reverse the entire string to generate the final string bool reverseAll = !m_edges.empty() && m_edges[0]->getDir() == ED_ANTISENSE; if(reverseAll) out = reverse(out); for(size_t i = 0; i < stop; ++i) { Edge* pYZ = m_edges[i]; // Append in the extension string std::string edge_str = pYZ->getLabel(); assert(edge_str.size() != 0); // Determine whether this node is reverse complement wrt the string // we are building if(currComp == EC_REVERSE) edge_str = reverseComplement(edge_str); if(reverseAll) edge_str = reverse(edge_str); // Calculate the next comp, between X and Z EdgeComp ecYZ = pYZ->getComp(); EdgeComp ecXZ; if(ecYZ == EC_SAME) ecXZ = currComp; else ecXZ = !currComp; out.append(edge_str); // Add this record into the placement vector if(pPlacementVector != NULL) { SGWalkVertexPlacement placement; placement.pVertex = pYZ->getEnd(); placement.isRC = ecXZ == EC_REVERSE; placement.position = out.size() - pYZ->getEnd()->getSeqLen(); pPlacementVector->push_back(placement); } currComp = ecXZ; } // If we want the internal portion of the string (which does not contain the endpoints // perform the truncation now. This needs to be done before the reversal. if(type == SGWT_INTERNAL) { if(pPlacementVector != NULL) { std::cerr << "Error: Vertex placement not supported for SGWT_INTERNAL walk types\n"; exit(EXIT_FAILURE); } Edge* pFirstEdge = getFirstEdge(); Edge* pLastEdge = getLastEdge(); if(pFirstEdge == NULL || pLastEdge == NULL) { out.clear(); } else { Vertex* pStart = m_pStartVertex; Vertex* pLast = getLastVertex(); int start = pStart->getSeqLen() - pFirstEdge->getMatchLength(); int end = out.size() - (pLast->getSeqLen() - pLastEdge->getMatchLength()); if(end <= start) out.clear(); else { std::string ss = out.substr(start, end - start); out = ss; } } } if(out.empty()) std::cout << "No output for walk: " << pathSignature() << "\n"; if(reverseAll) { out = reverse(out); // Reverse the placement vector too, including flipping the alignment coordinates if(pPlacementVector != NULL) { std::reverse(pPlacementVector->begin(), pPlacementVector->end()); for(size_t i = 0; i < pPlacementVector->size(); ++i) { SGWalkVertexPlacement& item = pPlacementVector->at(i); item.position = out.size() - item.position - item.pVertex->getSeqLen(); } } } return out; } // Returns a vector of EdgeComps of the orientation of each // vertex in the path with respect to the start of the walk std::vector SGWalk::getOrientationsToStart() const { std::vector out; if(m_edges.empty()) return out; // Tracking variable of the orientation between X (the start) // and the next vertex in the walk EdgeComp compXY = m_edges[0]->getComp(); out.push_back(compXY); for(size_t i = 1; i < m_edges.size(); ++i) { // Calculate the orientation of XZ using YZ EdgeComp compYZ = m_edges[i]->getComp(); // The direction flips if the XY/YZ are different EdgeComp compXZ = (compXY == compYZ) ? EC_SAME : EC_REVERSE; out.push_back(compXZ); compXY = compXZ; } return out; } // Get the substring of the full path string starting from position fromX // to position toY on the first and last vertices, respectively. // dirX is the direction along contig X towards vertex Y, vis-versa for dirY std::string SGWalk::getFragmentString(const Vertex* pX, const Vertex* pY, int fromX, int toY, EdgeDir dirX, EdgeDir dirY) const { std::string out; // Calculate the portion of X that we should include in the string // If dirX is SENSE, we take the everything after position fromX // otherwise we take everything up to and including fromX SeqCoord xCoord(0,0,pX->getSeqLen()); if(dirX == ED_SENSE) { xCoord.interval.start = fromX; xCoord.interval.end = pX->getSeqLen() - 1; } else { xCoord.interval.start = 0; xCoord.interval.end = fromX; } // Handle the trivial case where pX == pY and the walk is found immediately if(m_edges.empty() && pX == pY) { if(dirY == ED_SENSE) { xCoord.interval.start = toY; } else { xCoord.interval.end = toY; } } if(!xCoord.isValid()) return ""; // out.append(m_pStartVertex->getSeq().substr(xCoord.interval.start, xCoord.length())); // Determine if the string should go to the end of the last vertex // in the path size_t stop = m_edges.size(); // The first edge is always in correct frame of reference // so the comp is EC_SAME. This variable tracks where the // string that is being added is different from the starting sequence // and needs to be flipped EdgeComp currComp = EC_SAME; // If the walk direction is antisense, we reverse every component and then // reverse the entire string to generate the final string bool reverseAll = !m_edges.empty() && m_edges[0]->getDir() == ED_ANTISENSE; if(reverseAll) out = reverse(out); for(size_t i = 0; i < stop; ++i) { Edge* pYZ = m_edges[i]; bool isLast = i == (stop - 1); if(!isLast) { // Append the extension string without modification std::string edge_str = pYZ->getLabel(); assert(edge_str.size() != 0); if(currComp == EC_REVERSE) edge_str = reverseComplement(edge_str); if(reverseAll) edge_str = reverse(edge_str); out.append(edge_str); } else { // const Edge* pZY = pYZ->getTwin(); // get the unmatched coordinates on pY SeqCoord unmatched = pZY->getMatchCoord().complement(); // Now, we have to shrink the unmatched interval on Y to // only incude up to toY if(dirY == ED_SENSE) unmatched.interval.start = toY; else unmatched.interval.end = toY; if(!unmatched.isValid()) return ""; std::string seq = unmatched.getSubstring(pY->getStr()); if(pYZ->getComp() != currComp) seq = reverseComplement(seq); if(reverseAll) seq = reverse(seq); out.append(seq); } // Calculate the next comp, between X and Z EdgeComp ecYZ = pYZ->getComp(); EdgeComp ecXZ; if(ecYZ == EC_SAME) ecXZ = currComp; else ecXZ = !currComp; currComp = ecXZ; } if(reverseAll) out = reverse(out); return out; } // int SGWalk::getExtensionDistance() const { return m_extensionDistance; } // This is equivalent to the extension distance int SGWalk::getEndToEndDistance() const { return m_extensionDistance; } // int SGWalk::getStartToEndDistance() const { return m_pStartVertex->getSeqLen() + getEndToEndDistance(); } // Returns the distance from the end of pStart to the beginning of the last vertex in the path // This can be negative if they overlap int SGWalk::getEndToStartDistance() const { if(m_edges.empty()) return 0; int len_x = m_pStartVertex->getSeqLen(); int len_y = getLastEdge()->getEnd()->getSeqLen(); return getStartToEndDistance() - (len_x + len_y); } // Edge* SGWalk::getFirstEdge() const { if(m_edges.empty()) return NULL; else return m_edges.front(); } // Edge* SGWalk::getLastEdge() const { if(m_edges.empty()) return NULL; else return m_edges.back(); } // Vertex* SGWalk::getLastVertex() const { if(m_edges.empty()) return NULL; else return getLastEdge()->getEnd(); } // Edge* SGWalk::getEdge(size_t idx) const { return m_edges[idx]; } // Vertex* SGWalk::getVertex(size_t idx) const { assert(idx < getNumVertices()); if(idx == 0) return m_pStartVertex; else return m_edges[idx - 1]->getEnd(); } // VertexPtrVec SGWalk::getVertices() const { VertexPtrVec out; out.push_back(m_pStartVertex); for(EdgePtrVec::const_iterator iter = m_edges.begin(); iter != m_edges.end(); ++iter) out.push_back((*iter)->getEnd()); return out; } // Return a string describing this path std::string SGWalk::pathSignature() const { std::stringstream ss; ss << m_pStartVertex->getID() << ","; for(EdgePtrVec::const_iterator iter = m_edges.begin(); iter != m_edges.end(); ++iter) ss << (*iter)->getEndID() << ", "; return ss.str(); } // void SGWalk::print() const { std::cout << "Walk start: " << m_pStartVertex->getID() << "\nWalk: "; const Vertex* pLast = m_pStartVertex; for(EdgePtrVec::const_iterator iter = m_edges.begin(); iter != m_edges.end(); ++iter) { //std::cout << *(*iter) << " "; std::cout << (*iter)->getStartID() << " -- " << (*iter)->getEndID() << "," << (*iter)->getDir() << "," << (*iter)->getComp() << "\t"; assert((*iter)->getStart() == pLast); pLast = (*iter)->getEnd(); } std::cout << "\n"; } // void SGWalk::printSimple() const { std::cout << pathSignature() << "\n"; } // bool SGWalk::compareByTotalLength(const SGWalk& a, const SGWalk& b) { return a.getStartToEndDistance() > b.getStartToEndDistance(); } ================================================ FILE: src/SGA/StringGraph/SGWalk.h ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // SGWalk - Data structure holding a walk through // the graph // #ifndef SGWALK_H #define SGWALK_H #include "Bigraph.h" #include // A walk on the string graph is given by the starting vertex // then a vector of edges used in the walk enum SGWalkType { SGWT_START_TO_END, // include every vertex of the walk SGWT_EXTENSION, // do not include the start vertex SGWT_INTERNAL // do not include the start and end vertex }; // Placement data for the position of a vertex sequence in a walk struct SGWalkVertexPlacement { Vertex* pVertex; int position; // 0-based bool isRC; }; typedef std::vector SGWalkVertexPlacementVector; class SGWalk { public: SGWalk(Vertex* pStartVertex, bool bIndexWalk = false); SGWalk(const SGWalk& other); SGWalk(const EdgePtrVec& edgeVec, bool bIndexWalk = false); ~SGWalk(); SGWalk& operator=(const SGWalk& other); void addEdge(Edge* pEdge); void popLast(); void setFinished(bool b); bool isFinished() const; VertexPtrVec getVertices() const; Vertex* getStartVertex() const; Vertex* getLastVertex() const; // Returns a vector of EdgeComps of the orientation of each // vertex in the path with respect to the start of the walk std::vector getOrientationsToStart() const; Edge* getFirstEdge() const; Edge* getLastEdge() const; Edge* getEdge(size_t idx) const; Vertex* getVertex(size_t idx) const; size_t getNumVertices() const; size_t getNumEdges() const; // Returns true if the walk contains the specified vertex // If the walk is not indexed, this will assert bool isIndexed() const; bool containsVertex(const VertexID& id) const; // Truncate the walk after the first instance of id void truncate(const VertexID& id); // Get the string this walk represents. This function supports multiple different walk types // (see the enum description). If the pointer to the VertexPlacementVector is not NULL, // the position of the vertices within the walk are written there. std::string getString(SGWalkType type, SGWalkVertexPlacementVector* pPlacementVector = NULL) const; // Get the substring of the full path string starting from position fromX // to position toY on the first and last vertices, respectively std::string getFragmentString(const Vertex* pX, const Vertex* pY, int fromX, int toY, EdgeDir dirX, EdgeDir dirY) const; // Return a string identifying the path through the graph this walk represents std::string pathSignature() const; // distance calculations int getExtensionDistance() const; int getEndToEndDistance() const; int getStartToEndDistance() const; int getEndToStartDistance() const; // Compare a pair of walks by the total string length // Returns true if the walk of a is longer than the walk of b static bool compareByTotalLength(const SGWalk& a, const SGWalk& b); void print() const; void printSimple() const; private: Vertex* m_pStartVertex; EdgePtrVec m_edges; typedef std::set WalkIndex; WalkIndex* m_pWalkIndex; // The distance from the end of pStart to the last vertex in the walk // This equals the length of the extension string // x ----------- // y ------------ // z ---------- // distance ************ int m_extensionDistance; bool m_extensionFinished; }; typedef std::vector SGWalkVector; typedef std::deque WalkQueue; #endif ================================================ FILE: src/SGA/SuffixTools/BWT.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // BWT - All functions that use a BWT include this file // it simple typedefs the BWT name to the implementation // of the BWT that we want, either the uncompressed version // (SBWT) or the run-length encoded version (RLBWT). This could // be done using inheritence but the BWT is so used so much that // overhead of calling virtual functions is unwanted // // #ifndef BWT_H #define BWT_H #include "RLBWT.h" #include "SBWT.h" typedef RLBWT BWT; #endif ================================================ FILE: src/SGA/SuffixTools/BWTAlgorithms.cpp ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // bwt_algorithms.cpp - Algorithms for aligning to a bwt structure // #include "BWTAlgorithms.h" // Find the interval in pBWT corresponding to w // If w does not exist in the BWT, the interval // coordinates [l, u] will be such that l > u BWTInterval BWTAlgorithms::findInterval(const BWT* pBWT, const std::string& w) { int len = w.size(); int j = len - 1; char curr = w[j]; BWTInterval interval; initInterval(interval, curr, pBWT); --j; for(;j >= 0; --j) { curr = w[j]; updateInterval(interval, curr, pBWT); if(!interval.isValid()) return interval; } return interval; } // Find the interval in pBWT corresponding to w // using a cache of short k-mer intervals to avoid // some of the iterations BWTInterval BWTAlgorithms::findIntervalWithCache(const BWT* pBWT, const BWTIntervalCache* pIntervalCache, const std::string& w) { size_t cacheLen = pIntervalCache->getCachedLength(); if(w.size() < cacheLen) return findInterval(pBWT, w); // Compute the interval using the cache for the last k bases int len = w.size(); int j = len - cacheLen; // Check whether the input string has a '$' in it. // We don't cache these strings so if it does // we have to do a direct lookup if(index(w.c_str() + j, '$') != NULL) return findInterval(pBWT, w); BWTInterval interval = pIntervalCache->lookup(w.c_str() + j); j -= 1; for(;j >= 0; --j) { char curr = w[j]; updateInterval(interval, curr, pBWT); if(!interval.isValid()) return interval; } return interval; } // Delegate the findInterval call based on what indices are loaded BWTInterval BWTAlgorithms::findInterval(const BWTIndexSet& indices, const std::string& w) { if(indices.pCache != NULL) return findIntervalWithCache(indices.pBWT, indices.pCache, w); else return findInterval(indices.pBWT, w); } // Find the intervals in pBWT/pRevBWT corresponding to w // If w does not exist in the BWT, the interval // coordinates [l, u] will be such that l > u BWTIntervalPair BWTAlgorithms::findIntervalPair(const BWT* pBWT, const BWT* pRevBWT, const std::string& w) { BWTIntervalPair intervals; int len = w.size(); int j = len - 1; char curr = w[j]; initIntervalPair(intervals, curr, pBWT, pRevBWT); --j; for(;j >= 0; --j) { curr = w[j]; updateBothL(intervals, curr, pBWT); if(!intervals.isValid()) return intervals; } return intervals; } // Find the interval pair corresponding to w using a cached intervals for short substrings BWTIntervalPair BWTAlgorithms::findIntervalPairWithCache(const BWT* pBWT, const BWT* pRevBWT, const BWTIntervalCache* pFwdCache, const BWTIntervalCache* pRevCache, const std::string& w) { size_t cacheLen = pFwdCache->getCachedLength(); if(w.size() < cacheLen) return findIntervalPair(pBWT, pRevBWT, w); // Compute the fwd and reverse interval using the cache for the last k bases BWTIntervalPair ip; int len = w.size(); int j = len - cacheLen; std::string ss = w.substr(j); std::string r_ss = reverse(ss); assert(ss.size() == cacheLen); ip.interval[0] = pFwdCache->lookup(ss.c_str()); ip.interval[1] = pRevCache->lookup(r_ss.c_str()); // Extend the interval to the full length of w as normal j -= 1; for(;j >= 0; --j) { updateBothL(ip, w[j], pBWT); if(!ip.isValid()) return ip; } return ip; } // Count the number of occurrences of string w, including the reverse complement size_t BWTAlgorithms::countSequenceOccurrences(const std::string& w, const BWT* pBWT) { BWTInterval fwd_interval = findInterval(pBWT, w); BWTInterval rc_interval = findInterval(pBWT, reverseComplement(w)); size_t count = 0; if(fwd_interval.isValid()) count += fwd_interval.size(); if(rc_interval.isValid()) count += rc_interval.size(); return count; } // Count the number of occurrences of string w, including the reverse complement using a BWTInterval cache size_t BWTAlgorithms::countSequenceOccurrencesWithCache(const std::string& w, const BWT* pBWT, const BWTIntervalCache* pIntervalCache) { BWTInterval fwd_interval = findIntervalWithCache(pBWT, pIntervalCache, w); BWTInterval rc_interval = findIntervalWithCache(pBWT, pIntervalCache, reverseComplement(w)); size_t count = 0; if(fwd_interval.isValid()) count += fwd_interval.size(); if(rc_interval.isValid()) count += rc_interval.size(); return count; } // size_t BWTAlgorithms::countSequenceOccurrences(const std::string& w, const BWTIndexSet& indices) { assert(indices.pBWT != NULL); if(indices.pCache != NULL) return countSequenceOccurrencesWithCache(w, indices.pBWT, indices.pCache); else return countSequenceOccurrences(w, indices.pBWT); } size_t BWTAlgorithms::countSequenceOccurrencesSingleStrand(const std::string& w, const BWTIndexSet& indices) { assert(indices.pBWT != NULL); assert(indices.pCache != NULL); BWTInterval interval = findIntervalWithCache(indices.pBWT, indices.pCache, w); return interval.isValid() ? interval.size() : 0; } // Return the count of all the possible one base extensions of the string w. // This returns the number of times the suffix w[i, l]A, w[i, l]C, etc // appears in the FM-index for all i s.t. length(w[i, l]) == overlapLen. AlphaCount64 BWTAlgorithms::calculateExactExtensions(const unsigned int overlapLen, const std::string& w, const BWT* pBWT, const BWT* pRevBWT) { // The algorithm is as follows: // We perform a backward search on the FM-index of w. // For each signficant suffix (length w[i,l] >= minOverlap) // we determine the proper prefixes that match w[i,l]. For each proper prefix matching, // we compute the number of extensions of A,C,G,T for those prefix. AlphaCount64 ext_counts; BWTIntervalPair ranges; size_t l = w.length(); int start = l - 1; BWTAlgorithms::initIntervalPair(ranges, w[start], pBWT, pRevBWT); for(int i = start - 1; i >= 0; --i) { // Compute the range of the suffix w[i, l] BWTAlgorithms::updateBothL(ranges, w[i], pBWT); // Break if the suffix is no longer found if(!(ranges.interval[0].isValid() && ranges.interval[1].isValid())) break; if((l - i) == overlapLen) { if(ranges.interval[1].isValid()) { assert(ranges.interval[1].lower > 0); // The count for each extension is the difference between rank(B, upper) and rank(B, lower - 1) AlphaCount64 ac = pRevBWT->getOccDiff(ranges.interval[1].lower - 1, ranges.interval[1].upper); ext_counts += ac; } } } return ext_counts; } // Calculate the 1-base de Bruijn graph extensions of str // The includes the reverse complement AlphaCount64 BWTAlgorithms::calculateDeBruijnExtensions(const std::string str, const BWT* pBWT, const BWT* pRevBWT, EdgeDir direction, const BWTIntervalCache* pFwdCache, const BWTIntervalCache* pRevCache) { size_t k = str.size(); size_t p = k - 1; std::string pmer; // In the sense direction, we extend from the 3' end if(direction == ED_SENSE) pmer = str.substr(1, p); else pmer = str.substr(0, p); assert(pmer.length() == p); std::string rc_pmer = reverseComplement(pmer); // Get the interval for the p-mer and its reverse complement BWTIntervalPair ip; BWTIntervalPair rc_ip; // If pointers to interval caches are available, use them // to speed up the initial calculation if(pFwdCache != NULL && pRevCache != NULL) { ip = BWTAlgorithms::findIntervalPairWithCache(pBWT, pRevBWT, pFwdCache, pRevCache, pmer); rc_ip = BWTAlgorithms::findIntervalPairWithCache(pBWT, pRevBWT, pFwdCache, pRevCache, rc_pmer); } else { ip = BWTAlgorithms::findIntervalPair(pBWT, pRevBWT, pmer); rc_ip = BWTAlgorithms::findIntervalPair(pBWT, pRevBWT, rc_pmer); } assert(ip.isValid() || rc_ip.isValid()); // Get the extension bases AlphaCount64 extensions; AlphaCount64 rc_extensions; // Calculate the interval to use to find the extensions. If extending in the sense // direction this is the reverse interval/reverse bwt for the forward bwt and the forward // interval for the reverse BWT. Vice-versa for anti-sense size_t fwdIdx; if(direction == ED_SENSE) fwdIdx = 1; else fwdIdx = 0; size_t revIdx = 1 - fwdIdx; const BWT* bwts[2]; bwts[0] = pBWT; bwts[1] = pRevBWT; if(ip.interval[fwdIdx].isValid()) extensions += BWTAlgorithms::getExtCount(ip.interval[fwdIdx], bwts[fwdIdx]); if(rc_ip.interval[revIdx].isValid()) rc_extensions = BWTAlgorithms::getExtCount(rc_ip.interval[revIdx], bwts[revIdx]); // Switch the reverse-complement extensions to the same strand as the str rc_extensions.complement(); extensions += rc_extensions; return extensions; } // AlphaCount64 BWTAlgorithms::calculateDeBruijnExtensionsSingleIndex(const std::string str, const BWT* pBWT, EdgeDir direction, const BWTIntervalCache* pFwdCache) { size_t k = str.size(); size_t p = k - 1; std::string pmer; // In the sense direction, we extend from the 3' end if(direction == ED_SENSE) pmer = str.substr(1, p); else pmer = str.substr(0, p); assert(pmer.length() == p); std::string rc_pmer = reverseComplement(pmer); // As we only have a single index, we can only directly look up // the extensions for either the pmer or its reverse complement // In the SENSE extension direction, we directly look up for // the reverse complement. In ANTISENSE we directly look up for // the pmer. // Get the extension bases AlphaCount64 extensions; AlphaCount64 rc_extensions; // Set up pointers to the data to fill in/query // depending on the direction of the extension AlphaCount64* pDirectEC; AlphaCount64* pIndirectEC; std::string* pDirectStr; std::string* pIndirectStr; if(direction == ED_SENSE) { pDirectEC = &rc_extensions; pDirectStr = &rc_pmer; pIndirectEC = &extensions; pIndirectStr = &pmer; } else { pDirectEC = &extensions; pDirectStr = &pmer; pIndirectEC = &rc_extensions; pIndirectStr = &rc_pmer; } // Get the interval for the direct query string BWTInterval interval; // Use interval cache if available if(pFwdCache) interval = BWTAlgorithms::findIntervalWithCache(pBWT, pFwdCache, *pDirectStr); else interval = BWTAlgorithms::findInterval(pBWT, *pDirectStr); // Fill in the direct count if(interval.isValid()) *pDirectEC = BWTAlgorithms::getExtCount(interval, pBWT); // Now, for the non-direct index, query the 4 possible k-mers that are adjacent to the pmer // Setup the query sequence std::string query(k, 'A'); int varIdx = query.size() - 1; query.replace(0, p, *pIndirectStr); for(int i = 0; i < BWT_ALPHABET::size; ++i) { // Transform the query char b = BWT_ALPHABET::getChar(i); query[varIdx] = b; // Perform lookup if(pFwdCache) interval = BWTAlgorithms::findIntervalWithCache(pBWT, pFwdCache, query); else interval = BWTAlgorithms::findInterval(pBWT, query); // Update the extension count if(interval.isValid()) pIndirectEC->add(b, interval.size()); } // Switch the reverse-complement extensions to the same strand as the str rc_extensions.complement(); extensions += rc_extensions; return extensions; } // Return a random string from the BWT std::string BWTAlgorithms::sampleRandomString(const BWT* pBWT) { assert(RAND_MAX > 0x7FFF); size_t n = pBWT->getNumStrings(); size_t idx = rand() % n; return extractString(pBWT, idx); } // Return a random string from the BWT std::string BWTAlgorithms::sampleRandomSubstring(const BWT* pBWT, size_t len) { assert(RAND_MAX > 0x7FFF); size_t tries = 1000; while(1 && tries-- > 0) { size_t n = pBWT->getBWLen(); size_t idx = rand() % n; std::string s = extractString(pBWT, idx, len); if(s.size() == len) return s; } return ""; } // Return the string from the BWT at idx std::string BWTAlgorithms::extractString(const BWT* pBWT, size_t idx) { assert(idx < pBWT->getNumStrings()); // The range [0,n) in the BWT contains all the terminal // symbols for the reads. Search backwards from one of them // until the '$' is found gives a full string. std::string out; BWTInterval interval(idx, idx); while(1) { assert(interval.isValid()); char b = pBWT->getChar(interval.lower); if(b == '$') break; else out.push_back(b); updateInterval(interval, b, pBWT); } return reverse(out); } // Extract the substring from start, start+length of the sequence starting at position idx std::string BWTAlgorithms::extractSubstring(const BWT* pBWT, uint64_t idx, size_t start, size_t length) { std::string s = extractString(pBWT, idx); return s.substr(start, length); } // Return the next len bases of the string starting at index idx of the BWT std::string BWTAlgorithms::extractString(const BWT* pBWT, size_t idx, size_t len) { std::string out; BWTInterval interval(idx, idx); while(out.length() < len) { assert(interval.isValid()); char b = pBWT->getChar(interval.lower); if(b == '$') break; else out.push_back(b); updateInterval(interval, b, pBWT); } return reverse(out); } // Recursive traversal to extract all the strings needed for the above function void _extractRankedPrefixes(const BWT* pBWT, BWTInterval interval, const std::string& curr, RankedPrefixVector* pOutput) { AlphaCount64 extensions = BWTAlgorithms::getExtCount(interval, pBWT); for(size_t i = 0; i < 4; ++i) { char b = "ACGT"[i]; if(extensions.get(b) > 0) { BWTInterval ni = interval; BWTAlgorithms::updateInterval(ni, b, pBWT); _extractRankedPrefixes(pBWT, ni, curr + b, pOutput); } } // If we have extended the prefix as far as possible, stop BWTAlgorithms::updateInterval(interval, '$', pBWT); for(int64_t i = interval.lower; i <= interval.upper; ++i) { // backwards search gives a reversed prefix, fix it RankedPrefix rp = { (size_t)i, reverse(curr) }; pOutput->push_back(rp); } } // Extract all strings found from a backwards search starting at the given interval RankedPrefixVector BWTAlgorithms::extractRankedPrefixes(const BWT* pBWT, BWTInterval interval) { std::string curr; RankedPrefixVector output; output.reserve(interval.size()); _extractRankedPrefixes(pBWT, interval, curr, &output); return output; } std::string BWTAlgorithms::extractUntilInterval(const BWT* pBWT, int64_t start, const BWTInterval& check) { std::string out; BWTInterval interval(start, start); while(interval.lower < check.lower || interval.lower > check.upper) { assert(interval.isValid()); char b = pBWT->getChar(interval.lower); if(b == '$') return ""; else out.push_back(b); updateInterval(interval, b, pBWT); } return reverse(out); } ================================================ FILE: src/SGA/SuffixTools/BWTAlgorithms.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // BWTAlgorithms.h - Algorithms for aligning to a bwt structure // #ifndef BWT_ALGORITHMS_H #define BWT_ALGORITHMS_H #include "STCommon.h" #include "BWTIndexSet.h" #include "BWTInterval.h" #include "GraphCommon.h" #include #include #define LEFT_INT_IDX 0 #define RIGHT_INT_IDX 1 // structures // A (partial) prefix of a string contained in the BWT // and its lexicographic rank struct RankedPrefix { size_t rank; std::string prefix; }; typedef std::vector RankedPrefixVector; // functions namespace BWTAlgorithms { // get the interval(s) in pBWT/pRevBWT that corresponds to the string w using a backward search algorithm BWTInterval findInterval(const BWT* pBWT, const std::string& w); BWTInterval findIntervalWithCache(const BWT* pBWT, const BWTIntervalCache* pIntervalCache, const std::string& w); BWTInterval findInterval(const BWTIndexSet& indices, const std::string& w); BWTIntervalPair findIntervalPair(const BWT* pBWT, const BWT* pRevBWT, const std::string& w); BWTIntervalPair findIntervalPairWithCache(const BWT* pBWT, const BWT* pRevBWT, const BWTIntervalCache* pFwdCache, const BWTIntervalCache* pRevCache, const std::string& w); // Count the number of times the sequence w appears in the collection, including // its reverse complement size_t countSequenceOccurrences(const std::string& w, const BWT* pBWT); size_t countSequenceOccurrencesWithCache(const std::string& w, const BWT* pBWT, const BWTIntervalCache* pIntervalCache); size_t countSequenceOccurrences(const std::string& w, const BWTIndexSet& indices); // Count the occurrences of w, not including the reverse complement size_t countSequenceOccurrencesSingleStrand(const std::string& w, const BWTIndexSet& indices); // Update the given interval using backwards search // If the interval corrsponds to string S, it will be updated // for string bS inline void updateInterval(BWTInterval& interval, char b, const BWT* pBWT) { size_t pb = pBWT->getPC(b); interval.lower = pb + pBWT->getOcc(b, interval.lower - 1); interval.upper = pb + pBWT->getOcc(b, interval.upper) - 1; } // Update the interval pair for the right extension to symbol b. // In this version the AlphaCounts for the upper and lower intervals // have been calculated inline void updateBothR(BWTIntervalPair& pair, char b, const BWT* pRevBWT, AlphaCount64& l, AlphaCount64& u) { AlphaCount64 diff = u - l; pair.interval[0].lower = pair.interval[0].lower + diff.getLessThan(b); pair.interval[0].upper = pair.interval[0].lower + diff.get(b) - 1; // Update the right index directly size_t pb = pRevBWT->getPC(b); pair.interval[1].lower = pb + l.get(b); pair.interval[1].upper = pb + u.get(b) - 1; } // // Update the interval pair for the right extension to symbol b. // inline void updateBothR(BWTIntervalPair& pair, char b, const BWT* pRevBWT) { // Update the left index using the difference between the AlphaCounts in the reverse table AlphaCount64 l = pRevBWT->getFullOcc(pair.interval[1].lower - 1); AlphaCount64 u = pRevBWT->getFullOcc(pair.interval[1].upper); updateBothR(pair, b, pRevBWT, l, u); } // Update the interval pair for the left extension to symbol b. // In this version the AlphaCounts for the upper and lower intervals // have been calculated. inline void updateBothL(BWTIntervalPair& pair, char b, const BWT* pBWT, AlphaCount64& l, AlphaCount64& u) { AlphaCount64 diff = u - l; // Update the left index using the difference between the AlphaCounts in the reverse table pair.interval[1].lower = pair.interval[1].lower + diff.getLessThan(b); pair.interval[1].upper = pair.interval[1].lower + diff.get(b) - 1; // Update the left index directly size_t pb = pBWT->getPC(b); pair.interval[0].lower = pb + l.get(b); pair.interval[0].upper = pb + u.get(b) - 1; } // // Update the interval pair for the left extension to symbol b. // inline void updateBothL(BWTIntervalPair& pair, char b, const BWT* pBWT) { // Update the left index using the difference between the AlphaCounts in the reverse table AlphaCount64 l = pBWT->getFullOcc(pair.interval[0].lower - 1); AlphaCount64 u = pBWT->getFullOcc(pair.interval[0].upper); updateBothL(pair, b, pBWT, l, u); } // Initialize the interval of index idx to be the range containining all the b suffixes inline void initInterval(BWTInterval& interval, char b, const BWT* pB) { interval.lower = pB->getPC(b); interval.upper = interval.lower + pB->getOcc(b, pB->getBWLen() - 1) - 1; } // Initialize the interval of index idx to be the range containining all the b suffixes inline void initIntervalPair(BWTIntervalPair& pair, char b, const BWT* pBWT, const BWT* pRevBWT) { initInterval(pair.interval[LEFT_INT_IDX], b, pBWT); initInterval(pair.interval[RIGHT_INT_IDX], b, pRevBWT); } // Return the counts of the bases between the lower and upper interval in pBWT inline AlphaCount64 getExtCount(const BWTInterval& interval, const BWT* pBWT) { return pBWT->getOccDiff(interval.lower - 1, interval.upper); } // Return the count of all the possible one base extensions of the string w. // This returns the number of times the suffix w[i, l]A, w[i, l]C, etc // appears in the FM-index for all i s.t. length(w[i, l]) >= minOverlap. AlphaCount64 calculateExactExtensions(const unsigned int overlapLen, const std::string& w, const BWT* pBWT, const BWT* pRevBWT); // Calculate de Bruijn graph extensions of the given sequence using an index pair // Returns an AlphaCount64 with the count of each extension base // This function optionally takes in an interval cache to speed up the computation AlphaCount64 calculateDeBruijnExtensions(const std::string str, const BWT* pBWT, const BWT* pRevBWT, EdgeDir direction, const BWTIntervalCache* pFwdCache = NULL, const BWTIntervalCache* pRevCache = NULL); // Calculate de Bruijn graph extensions of the given sequence using a single index // This version is more computationally expensive than above but allows // only one index to be held in memory. // Returns an AlphaCount64 with the count of each extension base // This function optionally takes in an interval cache to speed up the computation AlphaCount64 calculateDeBruijnExtensionsSingleIndex(const std::string str, const BWT* pBWT, EdgeDir direction, const BWTIntervalCache* pFwdCache = NULL); // Extract the complete string starting at idx in the BWT std::string extractString(const BWT* pBWT, size_t idx); // Extract the next len bases of the string starting at idx std::string extractString(const BWT* pBWT, size_t idx, size_t len); // Extract the substring from start, start+length of the sequence starting at position idx std::string extractSubstring(const BWT* pBWT, uint64_t idx, size_t start, size_t length = std::string::npos); // Extract all prefixes of the suffixes for the given interval, along // with their lexicographic rank. RankedPrefixVector extractRankedPrefixes(const BWT* pBWT, BWTInterval interval); // Extract symbols from the starting index of the BWT until the index lies // within the given interval. If the extraction hits the start of a string // without finding a prefix, the empty string is returned. std::string extractUntilInterval(const BWT* pBWT, int64_t start, const BWTInterval& interval); // Returns a randomly chosen string from the BWT std::string sampleRandomString(const BWT* pBWT); // Returns a randomly chosen substring from the BWT std::string sampleRandomSubstring(const BWT* pBWT, size_t len); }; #endif ================================================ FILE: src/SGA/SuffixTools/BWTCABauerCoxRosone.cpp ================================================ //----------------------------------------------- // Copyright 2011 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL license //----------------------------------------------- // // BWTCABauerCoxRosone - In-memory version of Illumina's // BWT construction algorithm #include "BWTCABauerCoxRosone.h" #include "Timer.h" #include "BWTWriterBinary.h" #include #include void BWTCA::runBauerCoxRosone(const DNAEncodedStringVector* pReadSequences, const std::string& bwt_out_name, const std::string& sai_out_name) { size_t num_reads = pReadSequences->size(); size_t num_symbols = 0; for(size_t i = 0; i < num_reads; ++i) num_symbols += pReadSequences->at(i).length(); num_symbols += num_reads; // include 1 sentinal per read printf("Running BCR on %zu symbols, %zu reads\n", num_symbols, num_reads); // Allocate two working BWTs DNAEncodedString read_bwt; DNAEncodedString write_bwt; read_bwt.resize(num_symbols); write_bwt.resize(num_symbols); // Allocate the bcr vector, which tracks the state of the algorithm BCRVector bcrVector(num_reads); // Track the number of suffixes that start with a given symbol AlphaCount64 suffixStartCounts; // Iteration 1: // Output a BWT with the last symbol of every read, in the order they appear in the read table. // This is the ordering of all suffixes that start with the sentinel character outputInitialCycle(pReadSequences, bcrVector, write_bwt, suffixStartCounts); write_bwt.swap(read_bwt); // Iteration 2...n, create new bwts from the bwt of the previous cycle size_t partial_bwt_length = num_reads; Timer timer("cycles", false); size_t maxCycles = pReadSequences->at(0).length(); for(size_t cycle = 2; cycle <= maxCycles; ++cycle) { //std::cout << "Starting cycle " << cycle << "\n"; // Convert relative positions to absolute positions calculateAbsolutePositions(bcrVector, suffixStartCounts); // Sort nvector by the absolute position to insert the symbol std::sort(bcrVector.begin(), bcrVector.end()); //std::cout << " done sorting..." << timer.getElapsedWallTime() << "\n"; // Output the BWT for this cycle and update the vector and suffix start count partial_bwt_length = outputPartialCycle(cycle, pReadSequences, bcrVector, read_bwt, partial_bwt_length, write_bwt, suffixStartCounts); //std::cout << " done writing..." << timer.getElapsedWallTime() << "\n"; // Swap the in/out bwt read_bwt.swap(write_bwt); } // Write the resulting bwt and suffix array index BWTWriterBinary bwtWriter(bwt_out_name); bwtWriter.writeHeader(num_reads, num_symbols, BWF_NOFMI); SAWriter saWriter(sai_out_name); saWriter.writeHeader(num_reads, num_reads); // Calculate the positions of the final insertion symbols and write them directly to the file calculateAbsolutePositions(bcrVector, suffixStartCounts); std::sort(bcrVector.begin(), bcrVector.end()); size_t num_wrote = outputFinalBWT(bcrVector, read_bwt, partial_bwt_length, &bwtWriter, &saWriter); assert(num_wrote == num_symbols); } // Update N and the output BWT for the initial cycle, corresponding to the sentinel suffixes // the symbolCounts vector is updated to hold the number of times each symbol has been inserted // into the bwt void BWTCA::outputInitialCycle(const DNAEncodedStringVector* pReadSequences, BCRVector& bcrVector, DNAEncodedString& bwt, AlphaCount64& suffixSymbolCounts) { AlphaCount64 incomingSymbolCounts; size_t n = pReadSequences->size(); size_t first_read_len = pReadSequences->at(0).length(); for(size_t i = 0; i < n; ++i) { size_t rl = pReadSequences->at(i).length(); // Check that all reads are the same length if(rl != first_read_len) { std::cout << "Error: This implementation of BCR requires all reads to be the same length\n"; exit(EXIT_FAILURE); } char c = pReadSequences->at(i).get(rl - 1); bwt.set(i, c); assert(rl > 1); // Load the elements of the N vector with the next symbol bcrVector[i].sym = c; bcrVector[i].index = i; // Set the relative position of the symbol that is being inserted bcrVector[i].position = incomingSymbolCounts.get(c); // Increment the count of the first base of the suffix of the // incoming strings. This is $ for the initial cycle suffixSymbolCounts.increment('$'); // Update the inserted symbols incomingSymbolCounts.increment(c); } suffixSymbolCounts += incomingSymbolCounts; } // Write out the next BWT for the next cycle. This updates BCRVector // and suffixSymbolCounts. Returns the number of symbols written to writeBWT size_t BWTCA::outputPartialCycle(int cycle, const DNAEncodedStringVector* pReadSequences, BCRVector& bcrVector, const DNAEncodedString& readBWT, size_t total_read_symbols, DNAEncodedString& writeBWT, AlphaCount64& suffixStartCounts) { // We track the rank of each symbol as it is copied/inserted // into the new bwt AlphaCount64 rank; // Counters size_t num_copied = 0; size_t num_inserted = 0; size_t num_wrote = 0; for(size_t i = 0; i < bcrVector.size(); ++i) { BCRElem& ne = bcrVector[i]; // Copy elements from the read bwt until we reach the target position while(num_copied + num_inserted < ne.position) { char c = readBWT.get(num_copied++); writeBWT.set(num_wrote++, c); rank.increment(c); } // Now insert the incoming symbol int rl = pReadSequences->at(ne.index).length(); char c = '$'; // If the cycle number is greater than the read length, we are // on the final iteration and we just add in the '$' characters if(cycle <= rl) c = pReadSequences->at(ne.index).get(rl - cycle); //std::cout << "Inserting " << c << " at position " << num_copied + num_inserted << "\n"; writeBWT.set(num_wrote++, c); num_inserted += 1; // Update the nvector element ne.sym = c; // Record the rank of the inserted symbol ne.position = rank.get(c); // Update the rank and the number of suffixes that start with c rank.increment(c); suffixStartCounts.increment(c); } // Copy any remaining symbols in the bwt while(num_copied < total_read_symbols) writeBWT.set(num_wrote++, readBWT.get(num_copied++)); return num_wrote; } // Write the final BWT to a file. size_t BWTCA::outputFinalBWT(BCRVector& bcrVector, const DNAEncodedString& readBWT, size_t partial_bwt_symbols, BWTWriterBinary* pBWTWriter, SAWriter* pSAWriter) { // Counters size_t num_copied = 0; size_t num_inserted = 0; for(size_t i = 0; i < bcrVector.size(); ++i) { BCRElem& ne = bcrVector[i]; // Copy elements from the read bwt until we reach the target position while(num_copied + num_inserted < ne.position) pBWTWriter->writeBWChar(readBWT.get(num_copied++)); // Write a single $, terminating this string pBWTWriter->writeBWChar('$'); pSAWriter->writeElem(SAElem(ne.index, 0)); num_inserted += 1; } // Copy any remaining symbols in the bwt while(num_copied < partial_bwt_symbols) pBWTWriter->writeBWChar(readBWT.get(num_copied++)); pBWTWriter->finalize(); return num_copied + num_inserted; } void BWTCA::calculateAbsolutePositions(BCRVector& bcrVector, const AlphaCount64& suffixSymbolCounts) { // Calculate a predecessor array from the suffix symbol counts AlphaCount64 predCounts; for(int i = 0; i < BWT_ALPHABET::size; ++i) { char b = RANK_ALPHABET[i]; int64_t pc = suffixSymbolCounts.getLessThan(b); predCounts.set(b, pc); } for(size_t i = 0; i < bcrVector.size(); ++i) bcrVector[i].position += predCounts.get(bcrVector[i].sym); } ================================================ FILE: src/SGA/SuffixTools/BWTCABauerCoxRosone.h ================================================ //----------------------------------------------- // Copyright 2011 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL license //----------------------------------------------- // // BWTCABauerCoxRosone - In-memory version of Illumina's // BWT construction algorithm #ifndef BWTCA_COX_BAUER_ROSONE_H #define BWTCA_COX_BAUER_ROSONE_H #include "SAWriter.h" #include "ReadTable.h" #include "Alphabet.h" #include "EncodedString.h" #include "BWTWriterBinary.h" namespace BWTCA { // This structure tracks the state of each read // It contains a position field, which stores the relative/absolute // position to insert the next symbol of a read into the partial BWT. // It also stores the read index and the symbol to insert struct BCRElem { BCRElem() : index(0), sym('\0') {} // Comparator. Sort first on the symbol, then break ties using the index friend bool operator<(const BCRElem& a, const BCRElem& b) { return a.position < b.position; } friend std::ostream& operator<<(std::ostream& out, const BCRElem& elem) { out << "I: " << elem.index << " S: " << elem.sym << " P: " << elem.position; return out; } // Data uint64_t position; // the relative or absolute position into the next partial bwt to insert a symbol uint32_t index; // the read index this entry represents char sym; // a symbol to be inserted }; typedef std::vector BCRVector; // Construct the burrows-wheeler transform of the set of reads void runBauerCoxRosone(const DNAEncodedStringVector* pReadSequences, const std::string& bwt_out_name, const std::string& sai_out_name); // Run the initial special first iteration of the algorithm void outputInitialCycle(const DNAEncodedStringVector* pReadSequences, BCRVector& bcrVector, DNAEncodedString& bwt, AlphaCount64& suffixSymbolCounts); // Write the final bwt to a file size_t outputFinalBWT(BCRVector& bcrVector, const DNAEncodedString& readBWT, size_t partial_bwt_symbols, BWTWriterBinary* pBWTWriter, SAWriter* pSAWriter); // Output an intermediate bwt size_t outputPartialCycle(int cycle, const DNAEncodedStringVector* pReadSequences, BCRVector& bcrVector, const DNAEncodedString& readBWT, size_t partial_bwt_symbols, DNAEncodedString& writeBWT, AlphaCount64& suffixStartCounts); // Calculate absolute position for each element of the nvector void calculateAbsolutePositions(BCRVector& bcrVector, const AlphaCount64& suffixSymbolCounts); }; #endif ================================================ FILE: src/SGA/SuffixTools/BWTCARopebwt.cpp ================================================ //----------------------------------------------- // Copyright 2012 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // BWTCARopebwt - Construct the BWT for a set of reads // using Heng Li's ropebwt implementation // #include "BWTCARopebwt.h" #include "bcr.h" #include "SeqReader.h" #include "StdAlnTools.h" #include "BWTWriterBinary.h" #include "BWTWriterAscii.h" #include "SAWriter.h" static unsigned char seq_nt6_table[128] = { 0, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 1, 5, 2, 5, 5, 5, 3, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 4, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 1, 5, 2, 5, 5, 5, 3, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 4, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5 }; void BWTCA::runRopebwt(const std::string& input_filename, const std::string& bwt_out_name, bool use_threads, bool do_reverse) { // Initialize ropebwt std::string tmp_name = bwt_out_name + ".tmp"; bcr_t* bcr = bcr_init(use_threads, tmp_name.c_str()); size_t num_sequences = 0; size_t num_bases = 0; SeqReader reader(input_filename); SeqRecord record; while(reader.get(record)) { if(do_reverse) record.seq.reverse(); size_t l = record.seq.length(); // Convert the string into the alphabet encoding expected by ropebwt uint8_t* s = new uint8_t[l]; for(size_t i = 0; i < l; ++i) { char c = record.seq.get(i); s[i] = seq_nt6_table[(int)c]; } // Send the sequence to ropebwt bcr_append(bcr, l, s); num_sequences += 1; num_bases += l; delete [] s; } // Build the BWT bcr_build(bcr); // write the BWT and SAI bcritr_t* itr = bcr_itr_init(bcr); const uint8_t* s; int l; BWTWriterBinary* out_bwt = new BWTWriterBinary(bwt_out_name); size_t num_symbols = num_bases + num_sequences; out_bwt->writeHeader(num_sequences, num_symbols, BWF_NOFMI); // Write each run while( (s = bcr_itr_next(itr, &l)) != 0 ) { for (int i = 0; i < l; ++i) { char c = "$ACGTN"[s[i]&7]; int rl = s[i]>>3; for(int j = 0; j < rl; ++j) out_bwt->writeBWChar(c); } } free(itr); out_bwt->finalize(); delete out_bwt; // Cleanup bcr_destroy(bcr); } ================================================ FILE: src/SGA/SuffixTools/BWTCARopebwt.h ================================================ //----------------------------------------------- // Copyright 2012 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // BWTCARopebwt - Construct the BWT for a set of reads // using Heng Li's ropebwt implementation // #ifndef BWTCA_ROPEBWT_H #define BWTCA_ROPEBWT_H #include namespace BWTCA { void runRopebwt(const std::string& input_filename, const std::string& bwt_out_name, bool use_threads, bool do_reverse); }; #endif ================================================ FILE: src/SGA/SuffixTools/BWTDiskConstruction.cpp ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // BWTDiskConstruction - Implementation of the // BWT disk construction algorithm of Ferragina, // Gagie, Manzini. See Lightweight Data Indexing // and Compression in External Memory // #include "BWTDiskConstruction.h" #include "Util.h" #include "BWTWriter.h" #include "BWTReader.h" #include "SAWriter.h" #include "SAReader.h" #include "GapArray.h" #include "RankProcess.h" #include "SequenceProcessFramework.h" #include "BWTCABauerCoxRosone.h" #include // Definitions and structures static const bool USE_GZ = false; static const int BWT_SAMPLE_RATE = 512; struct MergeItem { int64_t start_index; int64_t end_index; std::string reads_filename; std::string bwt_filename; std::string sai_filename; friend std::ostream& operator<<(std::ostream& out, const MergeItem& item) { out << "[" << item.start_index << "," << item.end_index << "] " << item.bwt_filename; out << " " << item.sai_filename << " " << item.reads_filename; return out; } }; typedef std::vector MergeVector; // Function declarations int64_t merge(SeqReader* pReader, const MergeItem& item1, const MergeItem& item2, const std::string& bwt_outname, const std::string& sai_outname, bool doReverse, int numThreads, int storageLevel); // Initial BWT construction algorithms MergeVector computeInitialSAIS(const BWTDiskParameters& parameters); MergeVector computeInitialBCR(const BWTDiskParameters& parameters); // void writeMergedIndex(const BWT* pBWTInternal, const MergeItem& externalItem, const MergeItem& internalItem, const std::string& bwt_outname, const std::string& sai_outname, const GapArray* pGapArray); void writeRemovalIndex(const BWT* pBWTInternal, const std::string& sai_inname, const std::string& bwt_outname, const std::string& sai_outname, size_t num_strings_remove, size_t num_symbols_remove, const GapArray* pGapArray); void computeGapArray(SeqReader* pReader, size_t n, const BWT* pBWT, bool doReverse, int numThreads, GapArray* pGapArray, bool removeMode, size_t& num_strings_read, size_t& num_symbols_read); // std::string makeTempName(const std::string& prefix, int id, const std::string& extension); std::string makeFilename(const std::string& prefix, const std::string& extension); // The algorithm is as follows. We create M BWTs for subsets of // the input reads. These are created independently and written // to disk. They are then merged either sequentially or pairwise // to create the final BWT void buildBWTDisk(const BWTDiskParameters& parameters) { // Build the initial bwts for subsets of the data MergeVector mergeVector; if(parameters.bUseBCR) mergeVector = computeInitialBCR(parameters); else mergeVector = computeInitialSAIS(parameters); // Phase 2: Pairwise merge the BWTs int groupID = mergeVector.size(); // Initial the name of the next intermediate bwt int round = 1; MergeVector nextMergeRound; while(mergeVector.size() > 1) { std::cout << "Starting round " << round << "\n"; SeqReader* pReader = new SeqReader(parameters.inFile); SeqRecord record; for(size_t i = 0; i < mergeVector.size(); i+=2) { if(i + 1 != mergeVector.size()) { std::string bwt_merged_name = makeTempName(parameters.outPrefix, groupID, parameters.bwtExtension); std::string sai_merged_name = makeTempName(parameters.outPrefix, groupID, parameters.saiExtension); MergeItem item1 = mergeVector[i]; MergeItem item2 = mergeVector[i+1]; // Perform the actual merge int64_t curr_idx = merge(pReader, item1, item2, bwt_merged_name, sai_merged_name, parameters.bBuildReverse, parameters.numThreads, parameters.storageLevel); // pReader now points to the end of item1's block of // reads. Skip item2's reads assert(curr_idx == item2.start_index); while(curr_idx <= item2.end_index) { bool eof = !pReader->get(record); assert(!eof); (void)eof; ++curr_idx; } // Create the merged mergeItem to use in the next round MergeItem merged; merged.start_index = item1.start_index; merged.end_index = item2.end_index; merged.bwt_filename = bwt_merged_name; merged.sai_filename = sai_merged_name; nextMergeRound.push_back(merged); // Done with the temp files, remove them unlink(item1.bwt_filename.c_str()); unlink(item2.bwt_filename.c_str()); unlink(item1.sai_filename.c_str()); unlink(item2.sai_filename.c_str()); ++groupID; } else { // Singleton, pass through to the next round nextMergeRound.push_back(mergeVector[i]); } } delete pReader; mergeVector.clear(); mergeVector.swap(nextMergeRound); ++round; } assert(mergeVector.size() == 1); // Done, rename the files to their final name std::stringstream bwt_ss; bwt_ss << parameters.outPrefix << parameters.bwtExtension << (USE_GZ ? ".gz" : ""); std::string bwt_final_filename = bwt_ss.str(); rename(mergeVector.front().bwt_filename.c_str(), bwt_final_filename.c_str()); std::stringstream sai_ss; sai_ss << parameters.outPrefix << parameters.saiExtension << (USE_GZ ? ".gz" : ""); std::string sai_final_filename = sai_ss.str(); rename(mergeVector.front().sai_filename.c_str(), sai_final_filename.c_str()); } // Compute the initial BWTs for the input file split into blocks of records using the SAIS algorithm MergeVector computeInitialSAIS(const BWTDiskParameters& parameters) { SeqReader* pReader = new SeqReader(parameters.inFile); SeqRecord record; int groupID = 0; size_t numReadTotal = 0; MergeVector mergeVector; MergeItem mergeItem; mergeItem.start_index = 0; // Phase 1: Compute the initial BWTs ReadTable* pCurrRT = new ReadTable; bool done = false; while(!done) { done = !pReader->get(record); if(!done) { // the read is valid SeqItem item = record.toSeqItem(); if(parameters.bBuildReverse) item.seq.reverse(); pCurrRT->addRead(item); ++numReadTotal; } if(pCurrRT->getCount() >= parameters.numReadsPerBatch || (done && pCurrRT->getCount() > 0)) { // Compute the SA and BWT for this group SuffixArray* pSA = new SuffixArray(pCurrRT, 1); // Write the BWT to disk std::string bwt_temp_filename = makeTempName(parameters.outPrefix, groupID, parameters.bwtExtension); pSA->writeBWT(bwt_temp_filename, pCurrRT); std::string sai_temp_filename = makeTempName(parameters.outPrefix, groupID, parameters.saiExtension); pSA->writeIndex(sai_temp_filename); // Push the merge info mergeItem.end_index = numReadTotal - 1; // inclusive mergeItem.reads_filename = parameters.inFile; mergeItem.bwt_filename = bwt_temp_filename; mergeItem.sai_filename = sai_temp_filename; mergeVector.push_back(mergeItem); // Cleanup delete pSA; // Start the new group mergeItem.start_index = numReadTotal; ++groupID; pCurrRT->clear(); } } delete pCurrRT; delete pReader; return mergeVector; } // Compute the initial BWTs for the input file split into blocks of records using the BCR algorithm MergeVector computeInitialBCR(const BWTDiskParameters& parameters) { SeqReader* pReader = new SeqReader(parameters.inFile); SeqRecord record; int groupID = 0; size_t numReadTotal = 0; MergeVector mergeVector; MergeItem mergeItem; mergeItem.start_index = 0; // Phase 1: Compute the initial BWTs DNAEncodedStringVector readSequences; bool done = false; while(!done) { done = !pReader->get(record); if(!done) { // the read is valid SeqItem item = record.toSeqItem(); if(parameters.bBuildReverse) item.seq.reverse(); readSequences.push_back(item.seq.toString()); ++numReadTotal; } if(readSequences.size() >= parameters.numReadsPerBatch || (done && readSequences.size() > 0)) { std::string bwt_temp_filename = makeTempName(parameters.outPrefix, groupID, parameters.bwtExtension); std::string sai_temp_filename = makeTempName(parameters.outPrefix, groupID, parameters.saiExtension); BWTCA::runBauerCoxRosone(&readSequences, bwt_temp_filename, sai_temp_filename); // Push the merge info mergeItem.end_index = numReadTotal - 1; // inclusive mergeItem.reads_filename = parameters.inFile; mergeItem.bwt_filename = bwt_temp_filename; mergeItem.sai_filename = sai_temp_filename; mergeVector.push_back(mergeItem); // Start the new group mergeItem.start_index = numReadTotal; ++groupID; readSequences.clear(); } } delete pReader; return mergeVector; } // Merge the indices for the two independent sets of reads in readsFile1 and readsFile2 void mergeIndependentIndices(const std::string& readsFile1, const std::string& readsFile2, const std::string& outPrefix, const std::string& bwt_extension, const std::string& sai_extension, bool doReverse, int numThreads, int storageLevel) { MergeItem item1; std::string prefix1 = stripGzippedExtension(readsFile1); item1.reads_filename = readsFile1; item1.bwt_filename = makeFilename(prefix1, bwt_extension); item1.sai_filename = makeFilename(prefix1, sai_extension); item1.start_index = 0; item1.end_index = -1; // this tells merge to read the entire file MergeItem item2; std::string prefix2 = stripGzippedExtension(readsFile2); item2.reads_filename = readsFile2; item2.bwt_filename = makeFilename(prefix2, bwt_extension); item2.sai_filename = makeFilename(prefix2, sai_extension); item2.start_index = 0; item2.end_index = -1; // Prepare the input filehandle SeqReader* pReader = new SeqReader(item1.reads_filename); // Build the outnames std::string bwt_merged_name = makeFilename(outPrefix, bwt_extension); std::string sai_merged_name = makeFilename(outPrefix, sai_extension); // Perform the actual merge merge(pReader, item1, item2, bwt_merged_name, sai_merged_name, doReverse, numThreads, storageLevel); delete pReader; } // Construct new indices without the reads in readsToRemove void removeReadsFromIndices(const std::string& allReadsPrefix, const std::string& readsToRemove, const std::string& outPrefix, const std::string& bwt_extension, const std::string& sai_extension, bool doReverse, int numThreads) { std::string bwt_filename = makeFilename(allReadsPrefix, bwt_extension); std::string sai_filename = makeFilename(allReadsPrefix, sai_extension); // Prepare the input filehandle SeqReader* pReader = new SeqReader(readsToRemove); // Build the outnames std::string bwt_out_name = makeFilename(outPrefix, bwt_extension); std::string sai_out_name = makeFilename(outPrefix, sai_extension); // Compute the gap array BWT* pBWT = new BWT(bwt_filename, BWT_SAMPLE_RATE); // Boolean gap array GapArray* pGapArray = createGapArray(1); size_t num_strings_remove; size_t num_symbols_remove; computeGapArray(pReader, (size_t)-1, pBWT, doReverse, numThreads, pGapArray, true, num_strings_remove, num_symbols_remove); //writeRemovalIndex(); writeRemovalIndex(pBWT, sai_filename, bwt_out_name, sai_out_name, num_strings_remove, num_symbols_remove, pGapArray); // Perform the actual merge //merge(pReader, item1, item2, bwt_merged_name, sai_merged_name, doReverse, numThreads); delete pGapArray; delete pReader; delete pBWT; } // Merge two readsFiles together void mergeReadFiles(const std::string& readsFile1, const std::string& readsFile2, const std::string& outPrefix) { // If the outfile is the empty string, append the reads in readsFile2 into readsFile1 // otherwise cat the files together std::ostream* pWriter; if(outPrefix.empty()) { pWriter = createWriter(readsFile1, std::ios_base::out | std::ios_base::app); } else { bool both_fastq = isFastq(readsFile1) && isFastq(readsFile2); bool both_gzip = isGzip(readsFile1) && isGzip(readsFile2); std::string extension = both_fastq ? ".fastq" : ".fa"; if(both_gzip) extension.append(".gz"); std::string out_filename = outPrefix + extension; pWriter = createWriter(out_filename); // Copy reads1 to the outfile SeqReader reader(readsFile1); SeqRecord record; while(reader.get(record)) record.write(*pWriter); } // Copy reads2 to writer SeqReader reader(readsFile2); SeqRecord record; while(reader.get(record)) record.write(*pWriter); delete pWriter; } // Compute the gap array for the first n items in pReader void computeGapArray(SeqReader* pReader, size_t n, const BWT* pBWT, bool doReverse, int numThreads, GapArray* pGapArray, bool removeMode, size_t& num_strings_read, size_t& num_symbols_read) { // Create the gap array size_t gap_array_size = pBWT->getBWLen() + 1; pGapArray->resize(gap_array_size); // The rank processor calculates the rank of every suffix of a given sequence // and returns a vector of ranks. The postprocessor takes in the vector // and updates the gap array RankPostProcess postProcessor(pGapArray); size_t numProcessed = 0; if(numThreads <= 1) { RankProcess processor(pBWT, pGapArray, doReverse, removeMode); numProcessed = SequenceProcessFramework::processSequencesSerial(*pReader, &processor, &postProcessor, n); } else { typedef std::vector RankProcessVector; RankProcessVector rankProcVec; for(int i = 0; i < numThreads; ++i) { RankProcess* pProcess = new RankProcess(pBWT, pGapArray, doReverse, removeMode); rankProcVec.push_back(pProcess); } numProcessed = SequenceProcessFramework::processSequencesParallel(*pReader, rankProcVec, &postProcessor, n); for(int i = 0; i < numThreads; ++i) delete rankProcVec[i]; } num_strings_read = postProcessor.getNumStringsProcessed(); num_symbols_read = postProcessor.getNumSymbolsProcessed(); assert(n == (size_t)-1 || (numProcessed == n)); } // Merge a pair of BWTs using disk storage // Precondition: pReader is positioned at the start of the read block for item1 int64_t merge(SeqReader* pReader, const MergeItem& item1, const MergeItem& item2, const std::string& bwt_outname, const std::string& sai_outname, bool doReverse, int numThreads, int storageLevel) { std::cout << "Merge1: " << item1 << "\n"; std::cout << "Merge2: " << item2 << "\n"; // Load the bwt of item2 into memory as the internal bwt BWT* pBWTInternal = new BWT(item2.bwt_filename, BWT_SAMPLE_RATE); // If end_index is -1, calculate the ranks for every sequence in the file // otherwise only calculate the rank for the next (end_index - start_index + 1) sequences size_t n = (item1.end_index == -1) ? -1 : item1.end_index - item1.start_index + 1; // Calculate the rank of every read from item1.start_index to item1.end_index // and increment the gap counts int64_t curr_idx = item1.start_index; // Compute the gap/rank array GapArray* pGapArray = createGapArray(storageLevel); size_t num_strings_read = 0; size_t num_symbols_read = 0; computeGapArray(pReader, n, pBWTInternal, doReverse, numThreads, pGapArray, false, num_strings_read, num_symbols_read); assert(n == (size_t)-1 || (num_strings_read == n)); // At this point, the gap array has been calculated for all the sequences curr_idx += num_strings_read; assert(item1.end_index == -1 || (curr_idx == item1.end_index + 1 && curr_idx == item2.start_index)); // Write the merged BWT/SAI to disk writeMergedIndex(pBWTInternal, item1, item2, bwt_outname, sai_outname, pGapArray); delete pBWTInternal; delete pGapArray; return curr_idx; } // Merge the internal and external BWTs and the SAIs void writeMergedIndex(const BWT* pBWTInternal, const MergeItem& externalItem, const MergeItem& internalItem, const std::string& bwt_outname, const std::string& sai_outname, const GapArray* pGapArray) { IBWTWriter* pBWTWriter = BWTWriter::createWriter(bwt_outname); IBWTReader* pBWTExtReader = BWTReader::createReader(externalItem.bwt_filename); SAWriter saiWriter(sai_outname); SAReader saiExtReader(externalItem.sai_filename); SAReader saiIntReader(internalItem.sai_filename); // Calculate and write header values size_t disk_strings; size_t disk_symbols; BWFlag flag; pBWTExtReader->readHeader(disk_strings, disk_symbols, flag); size_t total_strings = disk_strings + pBWTInternal->getNumStrings(); size_t total_symbols = disk_symbols + pBWTInternal->getBWLen(); pBWTWriter->writeHeader(total_strings, total_symbols, BWF_NOFMI); // Discard the first header each sai size_t discard1, discard2; saiExtReader.readHeader(discard1, discard2); saiIntReader.readHeader(discard1, discard2); // Write the header of the SAI which is just the number of strings and elements in the SAI saiWriter.writeHeader(total_strings, total_strings); // Calculate and write the actual string // The semantics of the gap array are that we need to write gap_array[i] // symbols to the stream before writing bwtInternal[i] // Each time a '$' symbol is read, it signals the end of some read. We // output one element of the sai from corresponding internal or external // sai file. size_t num_bwt_wrote = 0; size_t num_sai_wrote = 0; for(size_t i = 0; i < pGapArray->size(); ++i) { size_t v = pGapArray->get(i); for(size_t j = 0; j < v; ++j) { char b = pBWTExtReader->readBWChar(); assert(b != '\n'); pBWTWriter->writeBWChar(b); ++num_bwt_wrote; if(b == '$') { // The external indices only need to be copied SAElem e = saiExtReader.readElem(); saiWriter.writeElem(e); ++num_sai_wrote; } } // If this is the last entry in the gap array, do not output a symbol from // the internal BWT if(i != pBWTInternal->getBWLen()) { char b = pBWTInternal->getChar(i); pBWTWriter->writeBWChar(b); ++num_bwt_wrote; if(b == '$') { // The internal indices need to be offset // by the number of strings in the external collection SAElem e = saiIntReader.readElem(); uint64_t id = e.getID(); id += disk_strings; e.setID(id); saiWriter.writeElem(e); ++num_sai_wrote; } } } if(num_bwt_wrote != total_symbols) { printf("Error expected to write %zu symbols, actually wrote %zu\n", total_symbols, num_bwt_wrote); assert(num_bwt_wrote == total_symbols); } assert(num_sai_wrote == total_strings); // Ensure we read the entire bw string from disk char last = pBWTExtReader->readBWChar(); assert(last == '\n'); (void)last; // Finalize the BWT disk file pBWTWriter->finalize(); delete pBWTExtReader; delete pBWTWriter; } // Write a new BWT and SAI that skips the elements marked // by the gap array. This is used to remove entire strings from the // index void writeRemovalIndex(const BWT* pBWTInternal, const std::string& sai_inname, const std::string& bwt_outname, const std::string& sai_outname, size_t num_strings_remove, size_t num_symbols_remove, const GapArray* pGapArray) { IBWTWriter* pBWTWriter = BWTWriter::createWriter(bwt_outname); SAWriter* pSAIWriter = new SAWriter(sai_outname); SAReader* pSAIReader = new SAReader(sai_inname); // Calculate and write header values assert(num_strings_remove <= pBWTInternal->getNumStrings()); assert(num_symbols_remove <= pBWTInternal->getBWLen()); size_t input_strings = pBWTInternal->getNumStrings(); size_t input_symbols = pBWTInternal->getBWLen(); size_t output_strings = input_strings - num_strings_remove; size_t output_symbols = input_symbols - num_symbols_remove; pBWTWriter->writeHeader(output_strings, output_symbols, BWF_NOFMI); // Discard the header of the sai size_t discard1, discard2; pSAIReader->readHeader(discard1, discard2); // Write the header of the SAI which is just the number of strings and elements in the SAI pSAIWriter->writeHeader(output_strings, output_strings); // We need to fix the IDs in the SAI. We do this by tracking // the number of IDs that are removed that are lower than a given // id. std::vector id_vector(pBWTInternal->getNumStrings(), 0); // Calculate and write the actual string // The gap array marks the symbols that should be removed. We // iterate over the gap array, if the value is zero, we output // the BWT symbol. Otherwise we skip the number of elements // indicated by the gap. size_t num_bwt_wrote = 0; size_t num_sai_wrote = 0; size_t i = 0; while(i < input_symbols) { size_t v = pGapArray->get(i); // If v is zero we output a single symbol, otherwise // we skip v elements. size_t num_to_read = (v == 0) ? 1 : v; for(size_t j = 0; j < num_to_read; ++j) { char b = pBWTInternal->getChar(i); if(b == '$') { SAElem e = pSAIReader->readElem(); // This element of the SAI will be removed, // increment the id vector so we can correct // the indices later if(v > 0) { id_vector[e.getID()]++; } } // output if(v == 0) { pBWTWriter->writeBWChar(b); assert(b != '\n'); ++num_bwt_wrote; } } i += num_to_read; } if(num_bwt_wrote != output_symbols) { printf("Error expected to write %zu symbols, actually wrote %zu\n", output_symbols, num_bwt_wrote); assert(num_bwt_wrote == output_symbols); } // Finalize the BWT disk file pBWTWriter->finalize(); // Accumulate the counts for each id int prev = 0; for(size_t i = 0; i < id_vector.size(); ++i) { id_vector[i] += prev; prev = id_vector[i]; } // Read the SAI file again, writing out the // corrected IDs that are not marked for removal delete pSAIReader; pSAIReader = new SAReader(sai_inname); pSAIReader->readHeader(discard1, discard2); for(size_t i = 0; i < input_strings; ++i) { SAElem e = pSAIReader->readElem(); uint64_t id = e.getID(); int prev_count = (id > 0) ? id_vector[id - 1] : 0; int count = id_vector[id]; if(count == prev_count) { // the current element should be output, it wasn't marked for removal id -= count; e.setID(id); pSAIWriter->writeElem(e); ++num_sai_wrote; } } assert(num_sai_wrote == output_strings); delete pSAIReader; delete pSAIWriter; delete pBWTWriter; } // std::string makeTempName(const std::string& prefix, int id, const std::string& extension) { std::stringstream ss; ss << prefix << ".temp-" << id << extension << (USE_GZ ? ".gz" : ""); return ss.str(); } // std::string makeFilename(const std::string& prefix, const std::string& extension) { std::stringstream ss; ss << prefix << extension << (USE_GZ ? ".gz" : ""); return ss.str(); } ================================================ FILE: src/SGA/SuffixTools/BWTDiskConstruction.h ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // BWTDiskConstruction - Implementation of the // BWT disk construction algorithm of Ferragina, // Gagie, Manzini. See Lightweight Data Indexing // and Compression in External Memory // // This implementation does not strictly follow // their method but is modified to handle // large numbers of short strings #ifndef BWTDISKCONSTRUCTION_H #define BWTDISKCONSTRUCTION_H #include "SuffixArray.h" #include "BWT.h" struct BWTDiskParameters { std::string inFile; std::string outPrefix; std::string bwtExtension; std::string saiExtension; size_t numReadsPerBatch; int numThreads; int storageLevel; bool bBuildReverse; bool bUseBCR; }; // Construct the burrows-wheeler transform of reads in in_filename // using the disk storage algorithm void buildBWTDisk(const BWTDiskParameters& parameters); // Merge the indices for the readsFile1 and readsFile2 void mergeIndependentIndices(const std::string& readsFile1, const std::string& readsFile2, const std::string& outPrefix, const std::string& bwt_extension, const std::string& sai_extension, bool doReverse, int numThreads, int storageLevel); // Compute new indices from allReadsFile without the reads in readsToRemove void removeReadsFromIndices(const std::string& allReadsFile, const std::string& readsToRemove, const std::string& outPrefix, const std::string& bwt_extension, const std::string& sai_extension, bool doReverse, int numThreads); // void mergeReadFiles(const std::string& readsFile1, const std::string& readsFile2, const std::string& outPrefix); #endif ================================================ FILE: src/SGA/SuffixTools/BWTIndexSet.h ================================================ //----------------------------------------------- // Copyright 2012 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // BWTIndexSet.h - Struct holding the different // types of index for a set of sequences. Used // to simplify the interface to some functions. // #ifndef BWTINDEXSET_H #define BWTINDEXSET_H #include "BWT.h" #include "BWTIntervalCache.h" #include "SampledSuffixArray.h" #include "PopulationIndex.h" #include "QualityTable.h" // A collection of indices. For some algorithms // all indices are not necessary so some of these // can be NULL. The algorithms will check as preconditions // which indices they need. struct BWTIndexSet { // Constructor BWTIndexSet() : pBWT(NULL), pRBWT(NULL), pCache(NULL), pSSA(NULL), pPopIdx(NULL), pQualityTable(NULL) {} // Data const BWT* pBWT; const BWT* pRBWT; const BWTIntervalCache* pCache; const SampledSuffixArray* pSSA; const PopulationIndex* pPopIdx; const QualityTable* pQualityTable; }; #endif ================================================ FILE: src/SGA/SuffixTools/BWTInterval.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL license //----------------------------------------------- // // BWTInterval - Data structures for holding and manipulating // the coordinates in a BWT/FM-index // #ifndef BWTINTERVAL_H #define BWTINTERVAL_H #include #include #include // A BWTInterval holds a pair of integers which delineate an alignment of some string // to a BWT/Suffix Array struct BWTInterval { // Functions BWTInterval() : lower(0), upper(0) {} BWTInterval(int64_t l, int64_t u) : lower(l), upper(u) {} inline bool isValid() const { return lower <= upper; } inline int64_t size() const { return upper - lower + 1; } static inline bool compare(const BWTInterval& a, const BWTInterval& b) { if(a.lower == b.lower) return a.upper < b.upper; else return a.lower < b.lower; } static inline bool equal(const BWTInterval& a, const BWTInterval& b) { return a.lower == b.lower && a.upper == b.upper; } friend std::ostream& operator<<(std::ostream& out, const BWTInterval& a) { out << a.lower << " " << a.upper; return out; } friend std::istream& operator>>(std::istream& in, BWTInterval& a) { in >> a.lower >> a.upper; return in; } void write(std::ostream& out) { out.write((char*)&lower, sizeof(lower)); out.write((char*)&upper, sizeof(upper)); } void read(std::istream& in) { in.read((char*)&lower, sizeof(lower)); in.read((char*)&upper, sizeof(upper)); } // Data int64_t lower; int64_t upper; }; // A pair of intervals, used for bidirectional searching a bwt/revbwt in lockstep struct BWTIntervalPair { // Functions BWTInterval& get(unsigned int idx) { return interval[idx]; } bool isValid() const { return interval[0].isValid() && interval[1].isValid(); } friend bool operator==(const BWTIntervalPair& a, const BWTIntervalPair& b) { return BWTInterval::equal(a.interval[0], b.interval[0]) && BWTInterval::equal(a.interval[1], b.interval[1]); } // Sort an itnerval pair by interval[0]'s lower coordinate static bool sortFirstLower(const BWTIntervalPair& a, const BWTIntervalPair& b) { return a.interval[0].lower < b.interval[0].lower; } // Sort an itnerval pair by interval[1]'s lower coordinate static bool sortSecondLower(const BWTIntervalPair& a, const BWTIntervalPair& b) { return a.interval[1].lower < b.interval[1].lower; } // I/O friend std::ostream& operator<<(std::ostream& out, const BWTIntervalPair& a) { out << a.interval[0] << " " << a.interval[1]; return out; } friend std::istream& operator>>(std::istream& in, BWTIntervalPair& a) { in >> a.interval[0] >> a.interval[1]; return in; } // Data BWTInterval interval[2]; }; #endif ================================================ FILE: src/SGA/SuffixTools/BWTIntervalCache.cpp ================================================ ///----------------------------------------------- // Copyright 2011 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // BWTIntervalCache - Array of cached bwt intervals for all // substrings of a fixed length // #include "BWTIntervalCache.h" #include "BWTAlgorithms.h" BWTIntervalCache::BWTIntervalCache(size_t k, const BWT* pBWT) : m_kmer(k) { build(pBWT); } // Build the table for the given bwt void BWTIntervalCache::build(const BWT* pBWT) { // Restrict the kmer parameter to something reasonable // so we don't try to allocate an absurdly large array assert(m_kmer <= 12); size_t num_entries = 1 << 2*m_kmer; m_table.resize(num_entries); //debug //std::cerr << "num entries " << num_entries << " m_kmer " << m_kmer << std::endl; // Construct the table for(size_t i = 0; i < num_entries; ++i) { std::string w = int2string(i); //debug //std::cerr << w << std::endl; //printf("i: %zu w: %s o: %zu\n", i, w.c_str(), string2int(w)); BWTInterval interval = BWTAlgorithms::findInterval(pBWT, w); m_table[i] = interval; } //debug //std::cerr << "m_table.size() " << m_table.size() << std::endl; } // Construct the corresponding string for integer i std::string BWTIntervalCache::int2string(size_t i) const { std::string out(m_kmer, 'A'); for(size_t k = 0; k < m_kmer; ++k) { // Get the character as position k (0 = left-most position) size_t code = (i >> 2*(m_kmer - k - 1)) & 3; char b = DNA_ALPHABET::getBase(code); out[k] = b; } return out; } // Return the length of the cached strings size_t BWTIntervalCache::getCachedLength() const { return m_kmer; } ================================================ FILE: src/SGA/SuffixTools/BWTIntervalCache.h ================================================ ///----------------------------------------------- // Copyright 2011 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // BWTIntervalCache - Array of cached bwt intervals for all // substrings of a fixed length // #ifndef BWTINTERVAL_CACHE_H #define BWTINTERVAL_CACHE_H #include "BWT.h" #include "BWTInterval.h" class BWTIntervalCache { public: // BWTIntervalCache(size_t k, const BWT* pBWT); // Look up the bwt interval for the given string inline BWTInterval lookup(const char* w) const { // Convert the string to an integer index in the lookup table size_t idx = str2int(w); return m_table[idx]; } // size_t getCachedLength() const; private: // Build the array for the given BWt void build(const BWT* pBWT); // Map a string to an integer // Precondition: w must be at least m_kmer symbols long inline size_t str2int(const char* w) const { size_t out = 0; for(size_t k = 0; k < m_kmer; ++k) { assert(w[k] != '$'); out |= DNA_ALPHABET::getBaseRank(w[k]) << 2*(m_kmer - k - 1); } return out; } // Map an integer to a string std::string int2string(size_t i) const; size_t m_kmer; std::vector m_table; }; #endif ================================================ FILE: src/SGA/SuffixTools/BWTReader.cpp ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // BWTReader - Abstract class for reading a BWT file // #include "BWTReader.h" #include "BWTReaderBinary.h" #include "BWTReaderAscii.h" // IBWTReader* BWTReader::createReader(const std::string& filename) { return new BWTReaderBinary(filename); } ================================================ FILE: src/SGA/SuffixTools/BWTReader.h ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // BWTReader - Abstract class for reading a BWT file // #ifndef BWTREADER_H #define BWTREADER_H #include "Util.h" #include "STCommon.h" #include "Occurrence.h" #include "EncodedString.h" enum BWIOStage { IOS_NONE, IOS_HEADER, IOS_BWSTR, IOS_PC, IOS_OCC, IOS_DONE }; enum BWFlag { BWF_NOFMI = 0, BWF_HASFMI }; const uint16_t RLBWT_FILE_MAGIC = 0xCACA; const uint16_t BWT_FILE_MAGIC = 0xEFEF; class RLBWT; class IBWTReader { public: IBWTReader() {} IBWTReader(const std::string& /*filename*/) {} virtual ~IBWTReader() {} // virtual void read(RLBWT* pRLBWT) = 0; virtual void readHeader(size_t& num_strings, size_t& num_symbols, BWFlag& flag) = 0; virtual char readBWChar() = 0; }; namespace BWTReader { IBWTReader* createReader(const std::string& filename); }; #endif ================================================ FILE: src/SGA/SuffixTools/BWTReaderAscii.cpp ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // BWTReaderAsciiAscii - Read a BWT file from disk // #include "BWTReaderAscii.h" #include "SBWT.h" #include "RLBWT.h" // BWTReaderAscii::BWTReaderAscii(const std::string& filename) : m_stage(IOS_NONE) { m_pReader = createReader(filename); m_stage = IOS_HEADER; } // BWTReaderAscii::~BWTReaderAscii() { delete m_pReader; } void BWTReaderAscii::read(SBWT* pBWT) { size_t n; BWFlag flag; readHeader(pBWT->m_numStrings, n, flag); pBWT->m_bwStr.resize(n); readBWStr(pBWT->m_bwStr); // Reading the occurrence array from disk is deprecated // we ignore it if it is present } void BWTReaderAscii::read(RLBWT* pRLBWT) { size_t n; BWFlag flag; readHeader(pRLBWT->m_numStrings, n, flag); bool done = false; while(!done) { char b = readBWChar(); if(b != '\n') { pRLBWT->append(b); } else { done = false; break; } } } // void BWTReaderAscii::readHeader(size_t& num_strings, size_t& num_symbols, BWFlag& flag) { assert(m_stage == IOS_HEADER); uint16_t magic_number; // Ensure the file format is sane *m_pReader >> magic_number; if(magic_number != BWT_FILE_MAGIC) { std::cerr << "BWT file is not properly formatted, aborting\n"; exit(EXIT_FAILURE); } *m_pReader >> num_strings; *m_pReader >> num_symbols; int temp; *m_pReader >> temp; flag = static_cast(temp); // We must explicitly set the stream // to the start of the BWStr portion of // the file. The above extraction // does not discard the trailing newline // so we do it here m_pReader->get(); m_stage = IOS_BWSTR; } // void BWTReaderAscii::readBWStr(BWTString& out_str) { assert(m_stage == IOS_BWSTR); char b; size_t idx = 0; while(1) { m_pReader->get(b); if(b != '\n') out_str.set(idx++, b); else break; } assert(idx == out_str.length()); m_stage = IOS_PC; } // Read a single base from the BWStr char BWTReaderAscii::readBWChar() { assert(m_stage == IOS_BWSTR); char b; m_pReader->get(b); if(b == '\n') m_stage = IOS_PC; return b; } // void BWTReaderAscii::readPred(AlphaCount64& out_pc) { assert(m_stage == IOS_PC); *m_pReader >> out_pc; m_stage = IOS_OCC; } // void BWTReaderAscii::readOccurrence(Occurrence& out_occ) { assert(m_stage == IOS_OCC); *m_pReader >> out_occ; m_stage = IOS_DONE; } ================================================ FILE: src/SGA/SuffixTools/BWTReaderAscii.h ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // BWTReaderAsciiAscii.h - Read a plain-text BWT file from disk // #ifndef BWTREADERASCII_H #define BWTREADERASCII_H #include "Util.h" #include "STCommon.h" #include "Occurrence.h" #include "EncodedString.h" #include "BWTReader.h" class SBWT; class RLBWT; class BWTReaderAscii : public IBWTReader { public: BWTReaderAscii(const std::string& filename); ~BWTReaderAscii(); // void read(SBWT* pBWT); void read(RLBWT* pRLBWT); void readHeader(size_t& num_strings, size_t& num_symbols, BWFlag& flag); void readBWStr(BWTString& out_str); char readBWChar(); void readPred(AlphaCount64& out_pc); void readOccurrence(Occurrence& out_icc); private: std::istream* m_pReader; BWIOStage m_stage; }; #endif ================================================ FILE: src/SGA/SuffixTools/BWTReaderBinary.cpp ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // RLBWTReaderBinary - Read a run length encoded BWT file from disk // #include "BWTReaderBinary.h" #include "SBWT.h" #include "RLBWT.h" // BWTReaderBinary::BWTReaderBinary(const std::string& filename) : m_stage(IOS_NONE), m_numRunsOnDisk(0), m_numRunsRead(0) { m_pReader = createReader(filename, std::ios::binary); m_stage = IOS_HEADER; } // BWTReaderBinary::~BWTReaderBinary() { delete m_pReader; } void BWTReaderBinary::read(RLBWT* pRLBWT) { BWFlag flag; readHeader(pRLBWT->m_numStrings, pRLBWT->m_numSymbols, flag); assert(m_numRunsOnDisk > 0); readRuns(pRLBWT->m_rlString, m_numRunsOnDisk); //pRLBWT->printInfo(); //pRLBWT->print(); } void BWTReaderBinary::read(SBWT* pSBWT) { BWFlag flag; size_t numSymbols; readHeader(pSBWT->m_numStrings, numSymbols, flag); size_t numRead = 0; pSBWT->m_bwStr.resize(numSymbols); while(numRead < numSymbols) { char b = readBWChar(); pSBWT->m_bwStr.set(numRead++, b); } assert(m_numRunsOnDisk > 0); } // void BWTReaderBinary::readHeader(size_t& num_strings, size_t& num_symbols, BWFlag& flag) { assert(m_stage == IOS_HEADER); uint16_t magic_number; m_pReader->read(reinterpret_cast(&magic_number), sizeof(magic_number)); if(magic_number != RLBWT_FILE_MAGIC) { std::cerr << "BWT file is not properly formatted, aborting\n"; exit(EXIT_FAILURE); } m_pReader->read(reinterpret_cast(&num_strings), sizeof(num_strings)); m_pReader->read(reinterpret_cast(&num_symbols), sizeof(num_symbols)); m_pReader->read(reinterpret_cast(&m_numRunsOnDisk), sizeof(m_numRunsOnDisk)); m_pReader->read(reinterpret_cast(&flag), sizeof(flag)); //std::cout << "Read magic: " << magic_number << "\n"; //std::cout << "strings:" << num_strings << "\n"; //std::cout << "symbols: " << num_symbols << "\n"; //std::cout << "runs: " << m_numRunsOnDisk << "\n"; m_stage = IOS_BWSTR; } void BWTReaderBinary::readRuns(RLVector& out, size_t numRuns) { out.resize(numRuns); m_pReader->read(reinterpret_cast(&out[0]), numRuns*sizeof(RLUnit)); m_numRunsRead = numRuns; } // Read a single base from the BWStr // The BWT is stored as runs on disk, so this class keeps // an internal buffer of a single run and emits characters from this buffer // and performs reads as necessary. If all the runs have been read, emit // a newline character to signal the end of the BWT char BWTReaderBinary::readBWChar() { assert(m_stage == IOS_BWSTR); if(m_currRun.isEmpty()) { // All runs have been read and emitted, return the end marker if(m_numRunsRead == m_numRunsOnDisk) return '\n'; // Read one run from disk m_pReader->read(reinterpret_cast(&m_currRun), sizeof(RLUnit)); ++m_numRunsRead; } // Decrement the current run and emit its symbol m_currRun.decrementCount(); return m_currRun.getChar(); } ================================================ FILE: src/SGA/SuffixTools/BWTReaderBinary.h ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // BWTWriterBinary - Read a run length encoded binary BWT file from disk // #ifndef BWTREADERBINARY_H #define BWTREADERBINARY_H #include "Util.h" #include "STCommon.h" #include "Occurrence.h" #include "EncodedString.h" #include "BWTReader.h" #include "RLBWT.h" class SBWT; class RLBWT; class BWTReaderBinary : public IBWTReader { public: BWTReaderBinary(const std::string& filename); virtual ~BWTReaderBinary(); // virtual void read(RLBWT* pRLBWT); virtual void read(SBWT* pSBWT); virtual void readHeader(size_t& num_strings, size_t& num_symbols, BWFlag& flag); virtual char readBWChar(); virtual void readRuns(RLVector& out, size_t numRuns); private: std::istream* m_pReader; BWIOStage m_stage; RLUnit m_currRun; size_t m_numRunsOnDisk; size_t m_numRunsRead; }; #endif ================================================ FILE: src/SGA/SuffixTools/BWTTraverse.cpp ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // BWTTraverse - traverse a bwt, outputting all // strings that are length greater than some threshold // #include "BWTTraverse.h" void BWTTraverse::extract(const BWT* pBWT, unsigned int len) { // Keep a stack of the elements to visit and // a string which is the reverse string corresponding // to the current stack elements TraverseStack stack; std::string rev_str; size_t iter_count = 0; size_t output_count = 0; for(size_t i = 0; i < DNA_ALPHABET_SIZE; ++i) { char base_char = ALPHABET[i]; // Initialize the stack and rev_str BWTInterval range; BWTAlgorithms::initInterval(range, base_char, pBWT); AlphaCount ext = BWTAlgorithms::getExtCount(range, pBWT); stack.push(TraverseElem(range, ext)); rev_str.append(1, base_char); while(!stack.empty()) { ++iter_count; TraverseElem& curr = stack.top(); curr.goNext(); bool doPop = !curr.isValid(); // If rev_str is long enough output it and stop the traversal at this depth if(rev_str.length() == len) { if(output_count % 1000000 == 0) std::cerr << "output: " << output_count << "\n"; doPop = true; size_t multiplicity = curr.getRange().size(); printf(">%zu %u %zu\n%s\n", output_count, len, multiplicity, reverse(rev_str).c_str()); ++output_count; } if(doPop) { // Remove one char from the end of the string // and one element from the stack rev_str.erase(rev_str.length() - 1, 1); stack.pop(); } else { // Traverse one level deeper char b = curr.getCurrChar(); BWTInterval subrange = curr.getRange(); BWTAlgorithms::updateInterval(subrange, b, pBWT); AlphaCount subext = BWTAlgorithms::getExtCount(subrange, pBWT); stack.push(TraverseElem(subrange, subext)); rev_str.append(1, b); } } } std::cerr << "Num output: " << output_count << " num iterations: " << iter_count << " count/len: " << (double)iter_count / len << "\n"; } void BWTTraverse::extractSG(const BWT* pBWT, const BWT* pRevBWT, const unsigned int len) { WARN_ONCE("Skipping bwt[0]"); // Keep a bool vector marking which entries in pBWT have been visited size_t n_elems = pBWT->getBWLen(); bool_vec visited(n_elems, false); size_t count = 0; size_t currIdx = 1; while(currIdx < n_elems) { // Invariant: currIdx is the index into pBWT that is the lowest index that has not been visited // Left-extend the entry at currIdx into a string of length len std::string str; str.reserve(len); // Get the first character of the suffix starting at this position char first = pBWT->getF(currIdx); str += first; BWTInterval range(currIdx, currIdx); while(str.length() < len) { char b = pBWT->getChar(range.lower); if(b == '$') break; str += b; BWTAlgorithms::updateInterval(range, b, pBWT); } // The string was built backwards, reverse it str = reverse(str); if(str.length() < len) { visited[currIdx] = true; } else if(!visited[range.lower]) { markVisited(str, visited, pBWT); //std::cout << currIdx << " interval: " << range << " string: " << str << "\n"; extendLeft(len, str, visited, pBWT, pRevBWT); extendRight(len, str, visited, pBWT, pRevBWT, false); printf(">%zu %zu 0\n%s\n", count++, str.length(), str.c_str()); } currIdx++; } } // Extend the sequence by finding a consensus right-ward extension base void BWTTraverse::extendRight(const unsigned int len, std::string& str, bool_vec& visited, const BWT* pBWT, const BWT* pRevBWT, bool isReverse) { // Extract the last len characters unsigned int overlapLen = len - 1; // Initialize the range for str BWTIntervalPair ranges = BWTAlgorithms::findIntervalPair(pBWT, pRevBWT, str); bool done = false; while(!done) { AlphaCount ext_counts = BWTAlgorithms::getExtCount(ranges.interval[1], pRevBWT); if(ext_counts.hasUniqueDNAChar()) { char b = ext_counts.getUniqueDNAChar(); // There is a unique extension from the seed sequence to B // Ensure that the reverse is true and that the sequence we are extending to, extends to this one std::string joined = str + b; std::string back_search = suffix(joined, len); // Get the count of bases back to the ending sequence of seed // We do this in the opposite direction of extension AlphaCount back_count = BWTAlgorithms::calculateExactExtensions(overlapLen, reverse(back_search), pRevBWT, pBWT); if(back_count.hasUniqueDNAChar()) { // Assert back is the character we are expecting assert(back_count.getUniqueDNAChar() == str[str.length() - len]); str = joined; // mark the newly visited l-mers if(!isReverse) { markVisited(back_search, visited, pBWT); } else { markVisited(reverse(back_search), visited, pRevBWT); } BWTAlgorithms::updateBothR(ranges, b, pRevBWT); } else { done = true; } } else { done = true; } } } // Extend the string to the left by reversing it and calling extendRight void BWTTraverse::extendLeft(const unsigned int len, std::string& str, bool_vec& visited, const BWT* pBWT, const BWT* pRevBWT) { str = reverse(str); extendRight(len, str, visited, pRevBWT, pBWT, true); str = reverse(str); } void BWTTraverse::markVisited(const std::string& str, bool_vec& visited, const BWT* pBWT) { BWTInterval range = BWTAlgorithms::findInterval(pBWT, str); for(int64_t i = range.lower; i <= range.upper; ++i) { assert(i >= 0 && i < (int64_t)visited.size()); visited[i] = true; } } ================================================ FILE: src/SGA/SuffixTools/BWTTraverse.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL license //----------------------------------------------- // // BWTTraverse - bwt traversal algorithms // #ifndef BWTTRAVERSE_H #define BWTTRAVERSE_H #include "BWT.h" #include "BWTAlgorithms.h" #include struct TraverseElem { // The current index is set to 0 and we iterate forward until we find the first valid (non-zero) entry // This skips elem 0 which is the $ TraverseElem(const BWTInterval& i, AlphaCount ac) : base_range(i), desc(ac), currIdx(0) { } // Return true if the current position is valid bool isValid() const { return currIdx < ALPHABET_SIZE; } // Return the current char char getCurrChar() const { assert(isValid()); return desc.getBase(currIdx); } // Return the range const BWTInterval& getRange() const { return base_range; } // go to the next character with a non-zero count // this automatically skips index 0, which we want since its the '$' character void goNext() { do { ++currIdx; } while(currIdx < ALPHABET_SIZE && desc.getByIdx(currIdx) == 0); } // The ranges in the BWT that correspond to the string leading up to this element BWTInterval base_range; // The counts of each base {A,C,G,T} that are left-extensions of this range AlphaCount desc; // The index of the current base being processed int currIdx; }; typedef std::stack TraverseStack; typedef std::vector bool_vec; namespace BWTTraverse { // Extract all strings of length len from the BWT void extract(const BWT* pBWT, unsigned int len); // Extract the string graph which minimum component length len from the BWT void extractSG(const BWT* pBWT, const BWT* pRevBWT, const unsigned int len); void extendLeft(const unsigned int len, std::string& str, bool_vec& visited, const BWT* pBWT, const BWT* pRevBWT); void extendRight(const unsigned int len, std::string& str, bool_vec& visited, const BWT* pBWT, const BWT* pRevBWT, bool isReverse); void markVisited(const std::string& str, bool_vec& visited, const BWT* pBWT); }; #endif ================================================ FILE: src/SGA/SuffixTools/BWTWriter.cpp ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // BWTWriter - Abstract class for writing a BWT file to disk // #include "BWTWriter.h" #include "BWTWriterBinary.h" #include "BWTWriterAscii.h" // void IBWTWriter::write(const SuffixArray* pSA, const ReadTable* pRT) { size_t num_symbols = pSA->getSize(); size_t num_strings = pSA->getNumStrings(); writeHeader(num_strings, num_symbols, BWF_NOFMI); for(size_t i = 0; i < num_symbols; ++i) { SAElem saElem = pSA->get(i); const SeqItem& si = pRT->getRead(saElem.getID()); // Get the position of the start of the suffix uint64_t f_pos = saElem.getPos(); uint64_t l_pos = (f_pos == 0) ? si.seq.length() : f_pos - 1; char b = (l_pos == si.seq.length()) ? '$' : si.seq.get(l_pos); writeBWChar(b); } finalize(); } // IBWTWriter* BWTWriter::createWriter(const std::string& filename) { return new BWTWriterBinary(filename); } ================================================ FILE: src/SGA/SuffixTools/BWTWriter.h ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // BWTWriter - Abstract class for writing a BWT file to disk // #ifndef BWTWRITER_H #define BWTWRITER_H #include "Util.h" #include "STCommon.h" #include "Occurrence.h" #include "BWTReader.h" #include "EncodedString.h" #include "SuffixArray.h" class IBWTWriter { public: IBWTWriter() {} IBWTWriter(const std::string& /*filename*/) {} virtual ~IBWTWriter() {} // void write(const SuffixArray* pSA, const ReadTable* pRT); virtual void writeHeader(const size_t& num_strings, const size_t& num_symbols, const BWFlag& flag) = 0; virtual void writeBWChar(char b) = 0; virtual void finalize() = 0; }; namespace BWTWriter { IBWTWriter* createWriter(const std::string& filename); } #endif ================================================ FILE: src/SGA/SuffixTools/BWTWriterAscii.cpp ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // BWTWriterAscii - Write a BWT file to disk // #include "BWTWriterAscii.h" #include "SBWT.h" #include "RLBWT.h" // BWTWriterAscii::BWTWriterAscii(const std::string& filename) : m_stage(IOS_NONE) { m_pWriter = createWriter(filename); m_stage = IOS_HEADER; } // BWTWriterAscii::~BWTWriterAscii() { delete m_pWriter; } void BWTWriterAscii::write(const SBWT* pBWT) { writeHeader(pBWT->m_numStrings, pBWT->m_bwStr.length(), BWF_HASFMI); writeBWStr(pBWT->m_bwStr); writePred(pBWT->m_predCount); writeOccurrence(pBWT->m_occurrence); } void BWTWriterAscii::write(const RLBWT* pRLBWT) { writeHeader(pRLBWT->m_numStrings, pRLBWT->m_numSymbols, BWF_NOFMI); size_t numRuns = pRLBWT->getNumRuns(); for(size_t i = 0; i < numRuns; ++i) { const RLUnit& unit = pRLBWT->m_rlString[i]; char symbol = unit.getChar(); size_t length = unit.getCount(); for(size_t j = 0; j < length; ++j) writeBWChar(symbol); } // Finalize the string writeBWChar('\n'); } // void BWTWriterAscii::writeHeader(const size_t& num_strings, const size_t& num_symbols, const BWFlag& flag) { assert(m_stage == IOS_HEADER); *m_pWriter << BWT_FILE_MAGIC << "\n"; *m_pWriter << num_strings << "\n"; *m_pWriter << num_symbols << "\n"; int temp = flag; *m_pWriter << temp << "\n"; m_stage = IOS_BWSTR; } // void BWTWriterAscii::writeBWStr(const BWTString& str) { assert(m_stage == IOS_BWSTR); size_t n = str.length(); for(size_t i = 0; i < n; ++i) *m_pWriter << str.get(i); finalize(); } // Write a single character of the BWStr // If the char is '\n' we are finished void BWTWriterAscii::writeBWChar(char b) { m_pWriter->put(b); } void BWTWriterAscii::finalize() { m_pWriter->put('\n'); m_stage = IOS_PC; } // void BWTWriterAscii::writePred(const AlphaCount64& pc) { assert(m_stage == IOS_PC); *m_pWriter << pc << "\n"; m_stage = IOS_OCC; } // void BWTWriterAscii::writeOccurrence(const Occurrence& occ) { assert(m_stage == IOS_OCC); *m_pWriter << occ; // delibrately no newline m_stage = IOS_DONE; } ================================================ FILE: src/SGA/SuffixTools/BWTWriterAscii.h ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // BWTWriterAsciiAscii - Write a BWT to a plain text file // #ifndef BWTWRITERASCII_H #define BWTWRITERASCII_H #include "Util.h" #include "STCommon.h" #include "Occurrence.h" #include "BWTWriter.h" #include "EncodedString.h" #include "SuffixArray.h" class SBWT; class RLBWT; class BWTWriterAscii : public IBWTWriter { public: BWTWriterAscii(const std::string& filename); virtual ~BWTWriterAscii(); // virtual void write(const RLBWT* pRLBWT); virtual void writeHeader(const size_t& num_strings, const size_t& num_symbols, const BWFlag& flag); virtual void writeBWChar(char b); virtual void finalize(); void write(const SBWT* pBWT); void writeBWStr(const BWTString& str); void writePred(const AlphaCount64& pc); void writeOccurrence(const Occurrence& icc); private: std::ostream* m_pWriter; BWIOStage m_stage; }; #endif ================================================ FILE: src/SGA/SuffixTools/BWTWriterBinary.cpp ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // BWTWriterBinary - Write a run-length encoded BWT to a binary file // #include "BWTWriterBinary.h" #include "SBWT.h" #include "RLBWT.h" // BWTWriterBinary::BWTWriterBinary(const std::string& filename) : m_numRuns(0), m_runFileOffset(0), m_stage(IOS_NONE) { m_pWriter = createWriter(filename, std::ios::out | std::ios::binary); m_stage = IOS_HEADER; } // BWTWriterBinary::~BWTWriterBinary() { assert(m_stage == IOS_DONE); delete m_pWriter; } // void BWTWriterBinary::writeHeader(const size_t& num_strings, const size_t& num_symbols, const BWFlag& flag) { assert(m_stage == IOS_HEADER); m_pWriter->write(reinterpret_cast(&RLBWT_FILE_MAGIC), sizeof(RLBWT_FILE_MAGIC)); m_pWriter->write(reinterpret_cast(&num_strings), sizeof(num_strings)); m_pWriter->write(reinterpret_cast(&num_symbols), sizeof(num_symbols)); // Here we do not know the number of runs that are going to be written to the file // so we save the offset in the file and write a dummy value. After the bwt string // has been written, we return here and fill in the correct value m_runFileOffset = m_pWriter->tellp(); m_numRuns = 0; m_pWriter->write(reinterpret_cast(&m_numRuns), sizeof(m_numRuns)); assert(flag == BWF_NOFMI); m_pWriter->write(reinterpret_cast(&flag), sizeof(flag)); m_stage = IOS_BWSTR; } // Write a single character of the BWStr // If the char is '\n' we are finished void BWTWriterBinary::writeBWChar(char b) { if(m_currRun.isInitialized()) { if(m_currRun.getChar() == b && !m_currRun.isFull()) { m_currRun.incrementCount(); } else { // Write out the old run and start a new one assert(m_currRun.isInitialized()); writeRun(m_currRun); m_currRun = RLUnit(b); } } else { // Start a new run m_currRun = RLUnit(b); } } // void BWTWriterBinary::writeRun(RLUnit& unit) { //std::cout << "Writing " << unit.getChar() << "," << (int)unit.getCount() << "\n"; m_pWriter->write(reinterpret_cast(&unit.data), sizeof(unit.data)); ++m_numRuns; } // write the final run to the stream and fill in the number of runs void BWTWriterBinary::finalize() { // JS Bugfix 24/05/12 // Allow empty BWTs to be written out if(m_currRun.isInitialized()) writeRun(m_currRun); m_pWriter->seekp(m_runFileOffset); m_pWriter->write(reinterpret_cast(&m_numRuns), sizeof(m_numRuns)); m_pWriter->seekp(std::ios_base::end); m_stage = IOS_DONE; } ================================================ FILE: src/SGA/SuffixTools/BWTWriterBinary.h ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // BWTWriterBinary - Write a run-length encoded BWT to a binary file // #ifndef BWTWRITERBINARY_H #define BWTWRITERBINARY_H #include "Util.h" #include "STCommon.h" #include "Occurrence.h" #include "BWTWriter.h" #include "BWTReaderBinary.h" #include "EncodedString.h" #include "RLBWT.h" class SBWT; class RLBWT; class BWTWriterBinary : public IBWTWriter { public: BWTWriterBinary(const std::string& filename); virtual ~BWTWriterBinary(); // Write an RLBWT file directly from a suffix array and read table virtual void writeHeader(const size_t& num_strings, const size_t& num_symbols, const BWFlag& flag); virtual void writeBWChar(char b); virtual void finalize(); // this method must be called after writing the BW string private: void writeRun(RLUnit& unit); std::ostream* m_pWriter; size_t m_numRuns; std::streampos m_runFileOffset; RLUnit m_currRun; BWIOStage m_stage; }; #endif ================================================ FILE: src/SGA/SuffixTools/FMMarkers.h ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // FMMarkers - Marker classes used in the FM-index // implementation // #ifndef FMMARKERS_H #define FMMARKERS_H // LargeMarker - To allow random access to the // BWT symbols and implement the occurrence array // we keep a vector of symbol counts every D1 symbols. // These counts are the absolute number of times each // symbol has been seen up to that point. // struct LargeMarker { LargeMarker() : unitIndex(0) {} // Calculate the actual position in the uncompressed BWT of this marker // This is the number of symbols preceding this marker inline size_t getActualPosition() const { return counts.getSum(); } void print() const { std::cout << "Large marker actual pos: " << getActualPosition() << "\n"; std::cout << "Marker unit index: " << unitIndex << "\n"; std::cout << "Marker counts: "; for(int i = 0; i < ALPHABET_SIZE; ++i) { std::cout << (int)counts.getByIdx(i) << " "; } std::cout << "\n"; } // Returns true if the data in the markers is identical bool operator==(const LargeMarker& rhs) { for(size_t i = 0; i < ALPHABET_SIZE; ++i) { if(counts.getByIdx(i) != rhs.counts.getByIdx(i)) return false; } return unitIndex == rhs.unitIndex; } // The number of times each symbol has been seen up to this marker AlphaCount64 counts; // The index in the RLVector of the run that starts after // this marker. That is, if C = getActualPosition(), then // the run containing the B[C] is at unitIndex. This is not necessary // a valid index if there is a marker after the last symbol in the BWT size_t unitIndex; }; typedef std::vector LargeMarkerVector; // SmallMarker - Small markers contain the counts // within an individual block of the BWT. In other words // the small marker contains the count for the last D2 symbols // struct SmallMarker { SmallMarker() : unitCount(0) {} // Calculate the actual position in the uncompressed BWT of this marker // This is the number of symbols preceding this marker inline size_t getCountSum() const { return counts.getSum(); } void print() const { for(int i = 0; i < ALPHABET_SIZE; ++i) { std::cout << (int)counts.getByIdx(i) << " "; } std::cout << "\n"; } // The number of times each symbol has been seen up to this marker AlphaCount16 counts; // The number of RL units in this block uint16_t unitCount; }; typedef std::vector SmallMarkerVector; #endif ================================================ FILE: src/SGA/SuffixTools/GapArray.cpp ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // GapArray - Data structure and functions used to count // the number of times a suffix of a given rank occurs in a data set // #include "GapArray.h" #include "SparseGapArray.h" #if 0 // SimpleGapArray SimpleGapArray::SimpleGapArray() { } // void SimpleGapArray::resize(size_t n) { m_data.resize(n); } // void SimpleGapArray::increment(size_t i) { static size_t max_gap_count = std::numeric_limits::max(); assert(i < m_data.size()); assert(m_data[i] < max_gap_count); ++m_data[i]; } // size_t SimpleGapArray::get(size_t i) const { return m_data[i]; } // size_t SimpleGapArray::size() const { return m_data.size(); } #endif // Construct a gap array for the given underlying storage storage GapArray* createGapArray(int storage) { switch(storage) { case 1: return new SparseGapArray1; case 4: return new SparseGapArray4; case 8: return new SparseGapArray8; case 16: return new SparseGapArray16; case 32: return new SparseGapArray32; default: { std::cerr << "Invalid gap array storage parameter: " << storage << "\n"; exit(1); } } } // Increment the gap array for each suffix of seq. Not thread safe. void updateGapArray(const DNAString& w, const BWT* pBWTInternal, GapArray* pGapArray) { (void)w; (void)pBWTInternal; (void)pGapArray; assert(false); #if 0 size_t l = w.length(); int i = l - 1; // Compute the rank of the last character of seq. We consider $ to be implicitly // terminated by a $ character. The first rank calculated is for this and it is given // by the C(a) array in BWTInternal int64_t rank = pBWTInternal->getPC('$'); // always zero pGapArray->incrementSerial(rank); // Compute the starting rank for the last symbol of w char c = w.get(i); rank = pBWTInternal->getPC(c); pGapArray->incrementSerial(rank); --i; // Iteratively compute the remaining ranks while(i >= 0) { char c = w.get(i); rank = pBWTInternal->getPC(c) + pBWTInternal->getOcc(c, rank - 1); pGapArray->incrementSerial(rank); --i; } #endif } // void analyzeGapArray(GapArray* pGapArray) { size_t thresh_8 = 255; size_t thresh_16 = 65535; size_t count_8 = 0; size_t count_16 = 0; size_t count_0 = 0; size_t count_1 = 0; size_t count_2 = 0; size_t count_4 = 0; for(size_t i = 0; i < pGapArray->size(); ++i) { size_t c = pGapArray->get(i); printf("GA\t%zu\t%zu\n", i, c); if(c >= thresh_8) ++count_8; if(c >= thresh_16) ++count_16; if(c == 0) ++count_0; if(c == 1) ++count_1; if(c == 2) ++count_2; if(c >= 16) ++count_4; } printf("Num >= %zu: %zu\n", thresh_8, count_8); printf("Num >= %zu: %zu\n", thresh_16, count_16); printf("Num >= 16: %zu\n", count_4); printf("Num == 0: %zu\n", count_0); printf("Num == 1: %zu\n", count_1); printf("Num == 2: %zu\n", count_2); } ================================================ FILE: src/SGA/SuffixTools/GapArray.h ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // GapArray - Data structure and functions used to count // the number of times a suffix of a given rank occurs in a data set // #ifndef GAPARRAY_H #define GAPARRAY_H #include #include "BWT.h" #include "DNAString.h" // Abstract class for GapArrays class GapArray { public: GapArray() {} virtual ~GapArray() {} virtual void resize(size_t n) = 0; // Attempt to increment the value in the gap array // Call can fail in which case the updates must be serialized // through the call to incrementOverflowSerial virtual bool attemptBaseIncrement(size_t i) = 0; // Update the overflow array of the gap array. This call // is not threadsafe. virtual void incrementOverflowSerial(size_t i) = 0; virtual size_t get(size_t i) const = 0; virtual size_t size() const = 0; }; #if 0 // The simplest representation of a gap array is a vector // of integers. This has a deterministic size // and O(1) increment/get but in most cases will // use (much) more storage than the SparseGapArray since most gap // counts will be very low (<< INT_MAX). See also SparseGapArray.h class SimpleGapArray : public GapArray { public: SimpleGapArray(); void resize(size_t n); void increment(size_t i); size_t get(size_t i) const; size_t size() const; private: typedef uint32_t GAP_TYPE; typedef std::vector GapStorage; GapStorage m_data; }; #endif //typedef uint32_t GAP_TYPE; //typedef std::vector GapArray; GapArray* createGapArray(int storage); void updateGapArray(const DNAString& w, const BWT* pBWTInternal, GapArray* pGapArray); void analyzeGapArray(GapArray* pGapArray); #endif ================================================ FILE: src/SGA/SuffixTools/HitData.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL license //----------------------------------------------- // // HitData - Data structure holding all positions of // alignment hits of a read // #ifndef HITDATA_H #define HITDATA_H #include "STCommon.h" #include "BitChar.h" #include // Deprecated struct Hit { Hit() {} Hit(size_t ri, size_t si, uint32_t qs, uint32_t l, bool tr, bool qr, int nd) : readIdx(ri), saIdx(si), qstart(qs), len(l), targetRev(tr), queryRev(qr), numDiff(nd) {} size_t readIdx; // The index in the read table size_t saIdx; // The index into the suffix array uint32_t qstart; // The query start position uint32_t len; // The overlap length bool targetRev; // Whether the target was reversed bool queryRev; // Whether the query was reversed int numDiff; // the number of differences in the match void setRev(bool tr, bool qr) { targetRev = tr; queryRev = qr; } // Sort hits by saIdx and then by overlap length friend bool operator<(const Hit& h1, const Hit& h2) { if(h1.saIdx != h2.saIdx) return h1.saIdx < h2.saIdx; else return h1.len > h2.len; } friend std::ostream& operator<<(std::ostream& out, const Hit& hit) { out << hit.readIdx << " " << hit.saIdx << " " << hit.qstart << " " << hit.len << " " << hit.targetRev << " " << hit.queryRev << " " << hit.numDiff; return out; } friend std::istream& operator>>(std::istream& in, Hit& hit) { in >> hit.readIdx >> hit.saIdx >> hit.qstart >> hit.len >> hit.targetRev >> hit.queryRev >> hit.numDiff; return in; } }; typedef std::vector HitVector; #endif ================================================ FILE: src/SGA/SuffixTools/InverseSuffixArray.cpp ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // InverseSuffixArray - The inverse of a generalized suffix array // Implemented as a Vector of Vectors // #include "InverseSuffixArray.h" // Constructor which knows the number of strings that will be in the ISA InverseSuffixArray::InverseSuffixArray(const SuffixArray& sa) { // Make a temporary vector to count the number of ranks for each string RankCountMap rcm; // Fill in the max ranks size_t n_sa = sa.getSize(); for(size_t i = 0; i < n_sa; ++i) { size_t id = sa.get(i).getID(); rcm[id]++; } for(RankCountMap::iterator iter = rcm.begin(); iter != rcm.end(); ++iter) { m_data[iter->first].resize(iter->second); } // Fill in the data for(size_t i = 0; i < n_sa; ++i) { size_t id = sa.get(i).getID(); size_t pos = sa.get(i).getPos(); m_data[id][pos] = i; } } // get the rank uint64_t InverseSuffixArray::getRank(size_t id, size_t pos) const { return m_data.find(id)->second[pos]; //return m_data[id][pos]; } // validate void InverseSuffixArray::validate() const { RankVector allRanks; for(RankVectorMap::const_iterator iter = m_data.begin(); iter != m_data.end(); ++iter) { const RankVector& rv = iter->second; for(size_t j = 0; j < rv.size(); ++j) { allRanks.push_back(rv[j]); } } std::sort(allRanks.begin(), allRanks.end()); for(size_t i = 0; i < allRanks.size(); ++i) { assert(i == allRanks[i]); } } // Print the ISA void InverseSuffixArray::print() const { for(RankVectorMap::const_iterator iter = m_data.begin(); iter != m_data.end(); ++iter) { const RankVector& rv = iter->second; for(size_t j = 0; j < rv.size(); ++j) { printf("ISA(%zu, %zu): %zu\n", (size_t)iter->first, j, (size_t)getRank(iter->first, j)); } } } ================================================ FILE: src/SGA/SuffixTools/InverseSuffixArray.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL license //----------------------------------------------- // // InverseSuffixArray - The inverse of a generalized suffix array // Implemented as a Vector of Vectors // #ifndef INVERSESUFFIXARRAY_H #define INVERSESUFFIXARRAY_H #include "STCommon.h" #include "SuffixArray.h" typedef std::vector RankVector; typedef std::map RankCountMap; typedef std::map RankVectorMap; class InverseSuffixArray { public: InverseSuffixArray(const SuffixArray& sa); // uint64_t getRank(size_t id, size_t pos) const; void validate() const; void print() const; private: RankVectorMap m_data; }; #endif ================================================ FILE: src/SGA/SuffixTools/Makefile.am ================================================ noinst_LIBRARIES = libsuffixtools.a libsuffixtools_a_CPPFLAGS = \ -I$(top_srcdir)/src/SGA/Util \ -I$(top_srcdir)/src/SGA/Bigraph \ -I$(top_srcdir)/SeqLib libsuffixtools_a_SOURCES = STCommon.cpp STCommon.h \ STGlobals.h \ Occurrence.cpp Occurrence.h \ SuffixArray.cpp SuffixArray.h \ SuffixCompare.cpp SuffixCompare.h \ InverseSuffixArray.cpp InverseSuffixArray.h \ SACAInducedCopying.h SACAInducedCopying.cpp \ BWTAlgorithms.h BWTAlgorithms.cpp \ ## BWTDiskConstruction.h BWTDiskConstruction.cpp \ BWTReader.h BWTReader.cpp \ BWTWriter.h BWTWriter.cpp \ SAReader.h SAReader.cpp \ SAWriter.h SAWriter.cpp \ ## GapArray.h GapArray.cpp \ ## RankProcess.h RankProcess.cpp \ SBWT.h SBWT.cpp \ RLBWT.h RLBWT.cpp \ BWTReader.h BWTReader.cpp \ BWTWriter.h BWTWriter.cpp \ BWTWriterBinary.h BWTWriterBinary.cpp \ BWTReaderBinary.h BWTReaderBinary.cpp \ BWTWriterAscii.h BWTWriterAscii.cpp \ BWTReaderAscii.h BWTReaderAscii.cpp \ BWTIntervalCache.h BWTIntervalCache.cpp \ ## QuickBWT.h QuickBWT.cpp \ SampledSuffixArray.h SampledSuffixArray.cpp \ ## BWTCABauerCoxRosone.h BWTCABauerCoxRosone.cpp \ ## BWTCARopebwt.h BWTCARopebwt.cpp \ ## PopulationIndex.h PopulationIndex.cpp \ BWT.h \ BWTInterval.h \ BWTIndexSet.h \ HitData.h \ SparseGapArray.h \ FMMarkers.h \ RLUnit.h ================================================ FILE: src/SGA/SuffixTools/Makefile.in ================================================ # Makefile.in generated by automake 1.16.1 from Makefile.am. # @configure_input@ # Copyright (C) 1994-2018 Free Software Foundation, Inc. # This Makefile.in is free software; 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#include "Occurrence.h" #include "SBWT.h" // Initialize the counts from the bwt string b void Occurrence::initialize(const BWTString& bwStr, int sampleRate) { m_sampleRate = sampleRate; m_shift = calculateShiftValue(m_sampleRate); size_t l = bwStr.length(); int num_samples = (l % m_sampleRate == 0) ? (l / m_sampleRate) : (l / m_sampleRate + 1); m_values.resize(num_samples); AlphaCount64 sum; for(size_t i = 0; i < l; ++i) { char currB = bwStr.get(i); sum.increment(currB); if(i % m_sampleRate == 0) m_values[i / m_sampleRate] = sum; } } // int Occurrence::calculateShiftValue(int divisor) { assert(divisor > 0); assert(IS_POWER_OF_2(divisor)); // m_sampleRate is a power of 2, count what bit is set unsigned int v = divisor; unsigned int c = 0; // c accumulates the total bits set in v while(v != 1) { v >>= 1; ++c; } assert(1 << c == divisor); return c; } // void Occurrence::set(char a, size_t i, BaseCount s) { m_values[i].set(a, s); } // size_t Occurrence::getByteSize() const { return m_values.size() * sizeof(AlphaCount64); } // Validate that the sampled occurrence array is correct void Occurrence::validate(const BWTString& bwStr) const { size_t l = bwStr.length(); AlphaCount64 sum; for(size_t i = 0; i < l; ++i) { char currB = bwStr.get(i); sum.increment(currB); AlphaCount64 calculated = get(bwStr, i); for(int i = 0; i < ALPHABET_SIZE; ++i) assert(calculated.get(ALPHABET[i]) == sum.get(ALPHABET[i])); } } std::ostream& operator<<(std::ostream& out, const Occurrence& o) { out << o.m_sampleRate << "\n"; out << o.m_values.size() << "\n"; for(size_t i = 0; i < o.m_values.size(); ++i) out << o.m_values[i] << "\n"; return out; } std::istream& operator>>(std::istream& in, Occurrence& o) { in >> o.m_sampleRate; size_t n; in >> n; o.m_values.resize(n); for(size_t i = 0; i < n; ++i) in >> o.m_values[i]; o.m_shift = Occurrence::calculateShiftValue(o.m_sampleRate); return in; } // void Occurrence::print() const { for(size_t i = 0; i < m_values.size(); i++) { std::cout << m_values[i]; } } ================================================ FILE: src/SGA/SuffixTools/Occurrence.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL license //----------------------------------------------- // // Occurrence.h - Data structure holding the number of times // the letter b appears in the string S before position i // #ifndef OCCURRENCE_H #define OCCURRENCE_H #include "STCommon.h" #include "EncodedString.h" // Power of 2 macros // return true if x is a power of 2 #define IS_POWER_OF_2(x) ((x) & ((x) - 1)) == 0 // return the x % y given that y is a power of 2 #define MOD_POWER_2(x, y) (x) & ((y) - 1) class SBWT; class Occurrence { public: // Constructors Occurrence() : m_sampleRate(1) {} // Initialize the counts from the bwt string b void initialize(const BWTString& bwStr, int sampleRate); // inline const AlphaCount64 get(const BWTString& bwStr, size_t idx) const { // Quick path if((MOD_POWER_2(idx,m_sampleRate)) == 0) return m_values[idx >> m_shift]; // Calculate the nearest sample to this index size_t lower_idx = idx >> m_shift; size_t upper_idx = lower_idx + 1; size_t lower_start = lower_idx << m_shift; size_t upper_start = upper_idx << m_shift; AlphaCount64 sum; // Choose the closest index or force the choice to lower_idx is the upper_idx is invalid if((idx - lower_start < upper_start - idx) || upper_idx == m_values.size()) { for(size_t j = lower_start + 1; j <= idx; ++j) sum.increment(bwStr.get(j)); return m_values[lower_idx] + sum; } else { for(size_t j = idx + 1; j <= upper_start; ++j) sum.increment(bwStr.get(j)); return m_values[upper_idx] - sum; } } // Get the alphacount difference between idx1 and idx0 inline AlphaCount64 getDiff(const BWTString& bwStr, size_t idx0, size_t idx1) const { return get(bwStr, idx1) - get(bwStr, idx0); } inline BaseCount get(const BWTString& bwStr, char a, size_t idx) const { // Quick path if((MOD_POWER_2(idx,m_sampleRate)) == 0) return m_values[idx >> m_shift].get(a); // Calculate the nearest sample to this index size_t lower_idx = idx >> m_shift; size_t upper_idx = lower_idx + 1; size_t lower_start = lower_idx << m_shift; size_t upper_start = upper_idx << m_shift; BaseCount sum = 0; // Choose the closest index or force the choice to lower_idx is the upper_idx is invalid if((idx - lower_start < upper_start - idx) || upper_idx == m_values.size()) { for(size_t j = lower_start + 1; j <= idx; ++j) { if(bwStr.get(j) == a) ++sum; } return m_values[lower_idx].get(a) + sum; } else { for(size_t j = idx + 1; j <= upper_start; ++j) { if(bwStr.get(j) == a) ++sum; } return m_values[upper_idx].get(a) - sum; } //return get(bwStr, idx).get(a); } void set(char a, size_t i, BaseCount s); void print() const; size_t getByteSize() const; size_t size() const { return m_values.size(); } size_t getSampleRate() const { return m_sampleRate; } // Calculate the amount a value should be shifted to perform a division // by divisor. The divisor must be a power of 2 static int calculateShiftValue(int divisor); void validate(const BWTString& bwStr) const; friend std::ostream& operator<<(std::ostream& out, const Occurrence& o); friend std::istream& operator>>(std::istream& in, Occurrence& o); private: int m_shift; int m_sampleRate; std::vector m_values; }; #endif ================================================ FILE: src/SGA/SuffixTools/PopulationIndex.cpp ================================================ //----------------------------------------------- // Copyright 2012 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // PopulationIndex - A structure mapping a read // index to a member of a population // #include "PopulationIndex.h" #include "Util.h" #include PopulationIndex::PopulationIndex(const std::string& filename) { std::istream* reader = createReader(filename); std::string line; while(getline(*reader, line)) { PopulationMember member = str2member(line); m_population.push_back(member); } delete reader; reader = NULL; // Make sure the index is properly formatted assert(m_population.size() != 0); assert(m_population[0].start == 0); for(size_t i = 1; i < m_population.size(); ++i) { assert(m_population[i].start > m_population[i-1].start); assert(m_population[i].start == m_population[i-1].end + 1); } /* Test the index size_t start = 0; size_t end = m_population.back().end + 2; for(size_t i = start; i <= end; ++i) { std::string name = getName(i); printf("%zu = %s\n", i, name.c_str()); } */ } // std::string PopulationIndex::getName(size_t read_index) const { std::vector::const_iterator iter = getIterByReadIndex(read_index); return iter->name; } // size_t PopulationIndex::getSampleIndex(size_t read_index) const { std::vector::const_iterator iter = getIterByReadIndex(read_index); return iter - m_population.begin(); } // std::vector::const_iterator PopulationIndex::getIterByReadIndex(size_t read_index) const { assert(read_index <= m_population.back().end); // Perform a binary search to identify the first element of the population // that is strictly greater than the index. This returns an iterator that is // guarenteed to be one past the individual that index belongs to. PopulationMember search = { read_index, read_index, "" }; std::vector::const_iterator iter = std::upper_bound(m_population.begin(), m_population.end(), search, PopulationMember::compareByStart); assert(iter != m_population.begin()); iter--; assert(read_index >= iter->start && read_index <= iter->end); return iter; } // StringVector PopulationIndex::getSamples() const { StringVector out; for(size_t i = 0; i < m_population.size(); ++i) out.push_back(m_population[i].name); return out; } // void PopulationIndex::mergeIndexFiles(const std::string& file1, const std::string& file2, const std::string& outfile) { std::ostream* writer = createWriter(outfile); // Copy the first index to the output unmodified but track the number of elements read size_t num_file_1 = 0; std::istream* reader = createReader(file1); std::string line; while(getline(*reader, line)) { // Copy *writer << line << "\n"; // Parse PopulationMember member = str2member(line); num_file_1 += (member.end - member.start + 1); } delete reader; // Copy the second index, offsetting by the number of reads in file1 reader = createReader(file2); while(getline(*reader, line)) { PopulationMember member = str2member(line); member.start += num_file_1; member.end += num_file_1; // Copy *writer << member.start << "\t" << member.end << "\t" << member.name << "\n"; } delete reader; delete writer; } // PopulationMember PopulationIndex::str2member(const std::string& line) { std::stringstream parser(line); PopulationMember member; parser >> member.start; parser >> member.end; parser >> member.name; return member; } ================================================ FILE: src/SGA/SuffixTools/PopulationIndex.h ================================================ //----------------------------------------------- // Copyright 2012 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // PopulationIndex - A structure mapping a read // index to a member of a population // #ifndef POPULATION_INDEX_H #define POPULATION_INDEX_H #include #include typedef std::vector StringVector; struct PopulationMember { size_t start; size_t end; std::string name; // static bool compareByStart(const PopulationMember& a, const PopulationMember& b) { return a.start < b.start; } }; class PopulationIndex { public: PopulationIndex(const std::string& filename); // Return the name of the individual for the read at index std::string getName(size_t read_index) const; // Return the index of the sample containing the given read index size_t getSampleIndex(size_t read_index) const; // Get the names of all samples in the collection StringVector getSamples() const; // Returns the number of samples in the population size_t getNumSamples() const { return m_population.size(); } // Merge two population index files into a new one static void mergeIndexFiles(const std::string& file1, const std::string& file2, const std::string& outfile); private: // Returns an iterator pointing to the sample that the contains the given index std::vector::const_iterator getIterByReadIndex(size_t index) const; // Parse a string from a .popidx file into a population member static PopulationMember str2member(const std::string& line); // Data std::vector m_population; }; #endif ================================================ FILE: src/SGA/SuffixTools/QuickBWT.cpp ================================================ //----------------------------------------------- // Copyright 2011 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL license //----------------------------------------------- // // QuickBWT - Construct a simple BWT for a short input string // #include "QuickBWT.h" // Construct a BWT for a small string. // Calling function is responsible for freeing the memory void createQuickBWT(const std::string& str, BWT*& pBWT, SuffixArray*& pSA) { ReadTable rt; SeqItem si = { "a", str }; rt.addRead(si); pSA = new SuffixArray(&rt, 1, true); pBWT = new BWT(pSA, &rt); /* std::cout << "QBWT -- str len: " << str.length() << "\n"; std::cout << "QBWT -- bwt len: " << pBWT->getBWLen() << "\n"; pBWT->printInfo(); */ } ================================================ FILE: src/SGA/SuffixTools/QuickBWT.h ================================================ //----------------------------------------------- // Copyright 2011 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL license //----------------------------------------------- // // QuickBWT - Construct a simple BWT for a short input string // #include "BWT.h" // Construct a BWT/suffix array of the given string // Calling code is responsible for freeing the BWT/SA void createQuickBWT(const std::string& str, BWT*& pBWT, SuffixArray*& pSA); ================================================ FILE: src/SGA/SuffixTools/RLBWT.cpp ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // RLBWT - Run-length encoded Burrows Wheeler transform // #include "RLBWT.h" #include "Timer.h" #include "BWTReader.h" #include "BWTWriter.h" #include "BWTReader.h" #include #include #include // macros #define OCC(c,i) m_occurrence.get(m_bwStr, (c), (i)) #define PRED(c) m_predCount.get((c)) // Parse a BWT from a file RLBWT::RLBWT(const std::string& filename, int sampleRate) : m_numStrings(0), m_numSymbols(0), m_largeSampleRate(DEFAULT_SAMPLE_RATE_LARGE), m_smallSampleRate(sampleRate) { IBWTReader* pReader = BWTReader::createReader(filename); pReader->read(this); initializeFMIndex(); delete pReader; } // Construct the BWT from a suffix array RLBWT::RLBWT(const SuffixArray* pSA, const ReadTable* pRT) { // Set up BWT state size_t n = pSA->getSize(); m_smallSampleRate = DEFAULT_SAMPLE_RATE_SMALL; m_largeSampleRate = DEFAULT_SAMPLE_RATE_LARGE; m_numStrings = pSA->getNumStrings(); m_numSymbols = n; RLUnit currRun; // Set up the bwt string and suffix array from the cycled strings for(size_t i = 0; i < n; ++i) { SAElem saElem = pSA->get(i); const SeqItem& si = pRT->getRead(saElem.getID()); // Get the position of the start of the suffix uint64_t f_pos = saElem.getPos(); uint64_t l_pos = (f_pos == 0) ? si.seq.length() : f_pos - 1; char b = (l_pos == si.seq.length()) ? '$' : si.seq.get(l_pos); // Add to the current run or append in the new char if(currRun.isInitialized()) { if(currRun.getChar() == b && !currRun.isFull()) { currRun.incrementCount(); } else { // Write out the old run and start a new one assert(currRun.isInitialized()); m_rlString.push_back(currRun); currRun = RLUnit(b); } } else { // Start a new run currRun = RLUnit(b); } } assert(currRun.isInitialized()); if(currRun.isInitialized()) m_rlString.push_back(currRun); initializeFMIndex(); } // void RLBWT::append(char b) { bool increment = false; if(!m_rlString.empty()) { RLUnit& lastUnit = m_rlString.back(); if(lastUnit.getChar() == b && !lastUnit.isFull()) { lastUnit.incrementCount(); increment = true; } } if(!increment) { // Must add a new unit to the string RLUnit unit(b); m_rlString.push_back(unit); } ++m_numSymbols; } // Fill in the FM-index data structures void RLBWT::initializeFMIndex() { m_smallShiftValue = Occurrence::calculateShiftValue(m_smallSampleRate); m_largeShiftValue = Occurrence::calculateShiftValue(m_largeSampleRate); // initialize the marker vectors, // LargeMarkers are placed every 2048 bases (by default) containing the absolute count // of symbols seen up to that point. SmallMarkers are placed every 128 bases with the // count over the last 128 symbols. From these relative counts the absolute count // every 128 symbols can be interpolated. size_t num_large_markers = getNumRequiredMarkers(m_numSymbols, m_largeSampleRate); size_t num_small_markers = getNumRequiredMarkers(m_numSymbols, m_smallSampleRate); m_largeMarkers.resize(num_large_markers); m_smallMarkers.resize(num_small_markers); // Fill in the marker values // We wish to place markers every sampleRate symbols however since a run may // not end exactly on sampleRate boundaries, we place the markers AFTER // the run crossing the boundary ends // Place a blank markers at the start of the data m_largeMarkers[0].unitIndex = 0; m_smallMarkers[0].unitCount = 0; // State variables for the number of markers placed, // the next marker to place, etc size_t curr_large_marker_index = 1; size_t curr_small_marker_index = 1; size_t next_small_marker = m_smallSampleRate; size_t next_large_marker = m_largeSampleRate; size_t prev_small_marker_unit_index = 0; size_t running_total = 0; AlphaCount64 running_ac; for(size_t i = 0; i < m_rlString.size(); ++i) { // Update the count and advance the running total RLUnit& unit = m_rlString[i]; char symbol = unit.getChar(); uint8_t run_len = unit.getCount(); running_ac.add(symbol, run_len); running_total += run_len; size_t curr_unit_index = i + 1; bool last_symbol = i == m_rlString.size() - 1; // Check whether to place a new large marker bool place_last_large_marker = last_symbol && curr_large_marker_index < num_large_markers; while(running_total >= next_large_marker || place_last_large_marker) { size_t expected_marker_pos = curr_large_marker_index * m_largeSampleRate; // Sanity checks // The marker position should always be less than the running total unless // the number of symbols is smaller than the sample rate assert(expected_marker_pos <= running_total || place_last_large_marker); assert((running_total - expected_marker_pos) <= RL_FULL_COUNT || place_last_large_marker); assert(curr_large_marker_index < num_large_markers); assert(running_ac.getSum() == running_total); LargeMarker& marker = m_largeMarkers[curr_large_marker_index]; marker.unitIndex = i + 1; marker.counts = running_ac; next_large_marker += m_largeSampleRate; curr_large_marker_index += 1; place_last_large_marker = last_symbol && curr_large_marker_index < num_large_markers; } // Check whether to place a new small marker bool place_last_small_marker = last_symbol && curr_small_marker_index < num_small_markers; while(running_total >= next_small_marker || place_last_small_marker) { // Place markers size_t expected_marker_pos = curr_small_marker_index * m_smallSampleRate; // Sanity checks // The marker position should always be less than the running total unless // the number of symbols is smaller than the sample rate assert(expected_marker_pos <= running_total || place_last_small_marker); assert((running_total - expected_marker_pos) <= RL_FULL_COUNT || place_last_small_marker); assert(curr_small_marker_index < num_small_markers); assert(running_ac.getSum() == running_total); // Calculate the number of rl units that are contained in this block if(curr_unit_index - prev_small_marker_unit_index > std::numeric_limits::max()) { std::cerr << "Error: Number of units in occurrence array block " << curr_small_marker_index << " exceeds the maximum value.\n"; exit(EXIT_FAILURE); } // Calculate the large marker to set the relative count from // This is generally the most previously placed large block except it might // be the second-previous in the case that we placed the last large marker. size_t large_marker_index = expected_marker_pos >> m_largeShiftValue; assert(large_marker_index < curr_large_marker_index); // ensure the last has ben placed LargeMarker& prev_large_marker = m_largeMarkers[large_marker_index]; // Set the 8bit AlphaCounts as the sum since the last large (superblock) marker AlphaCount16 smallAC; for(size_t j = 0; j < ALPHABET_SIZE; ++j) { size_t v = running_ac.getByIdx(j) - prev_large_marker.counts.getByIdx(j); if(v > smallAC.getMaxValue()) { std::cerr << "Error: Number of symbols in occurrence array block " << curr_small_marker_index << " exceeds the maximum value (" << v << " > " << smallAC.getMaxValue() << ")\n"; exit(EXIT_FAILURE); } smallAC.setByIdx(j, v); } // Set the small marker SmallMarker& small_marker = m_smallMarkers[curr_small_marker_index]; small_marker.unitCount = curr_unit_index - prev_large_marker.unitIndex; small_marker.counts = smallAC; // Update state variables next_small_marker += m_smallSampleRate; curr_small_marker_index += 1; prev_small_marker_unit_index = curr_unit_index; place_last_small_marker = last_symbol && curr_small_marker_index < num_small_markers; } } assert(curr_small_marker_index == num_small_markers); assert(curr_large_marker_index == num_large_markers); // Initialize C(a) m_predCount.set('$', 0); m_predCount.set('A', running_ac.get('$')); m_predCount.set('C', m_predCount.get('A') + running_ac.get('A')); m_predCount.set('G', m_predCount.get('C') + running_ac.get('C')); m_predCount.set('T', m_predCount.get('G') + running_ac.get('G')); } // get the number of markers required to cover the n symbols at sample rate of d size_t RLBWT::getNumRequiredMarkers(size_t n, size_t d) const { // we place a marker at the beginning (with no accumulated counts), every m_sampleRate // bases and one at the very end (with the total counts) size_t num_markers = (n % d == 0) ? (n / d) + 1 : (n / d) + 2; return num_markers; } // Print the BWT void RLBWT::print() const { size_t numRuns = getNumRuns(); std::string bwt; for(size_t i = 0; i < numRuns; ++i) { const RLUnit& unit = m_rlString[i]; char symbol = unit.getChar(); size_t length = unit.getCount(); for(size_t j = 0; j < length; ++j) std::cout << symbol; std::cout << " : " << symbol << "," << length << "\n"; bwt.append(length, symbol); } std::cout << "B: " << bwt << "\n"; } // Print information about the BWT void RLBWT::printInfo() const { size_t small_m_size = m_smallMarkers.capacity() * sizeof(SmallMarker); size_t large_m_size = m_largeMarkers.capacity() * sizeof(LargeMarker); size_t total_marker_size = small_m_size + large_m_size; size_t bwStr_size = m_rlString.capacity() * sizeof(RLUnit); size_t other_size = sizeof(*this); size_t total_size = total_marker_size + bwStr_size + other_size; double mb = (double)(1024 * 1024); double total_mb = total_size / mb; printf("\nRLBWT info:\n"); printf("Large Sample rate: %zu\n", m_largeSampleRate); printf("Small Sample rate: %zu\n", m_smallSampleRate); printf("Contains %zu symbols in %zu runs (%1.4lf symbols per run)\n", m_numSymbols, m_rlString.size(), (double)m_numSymbols / m_rlString.size()); printf("Marker Memory -- Small Markers: %zu (%.1lf MB) Large Markers: %zu (%.1lf MB)\n", small_m_size, small_m_size / mb, large_m_size, large_m_size / mb); printf("Total Memory -- Markers: %zu (%.1lf MB) Str: %zu (%.1lf MB) Misc: %zu Total: %zu (%lf MB)\n", total_marker_size, total_marker_size / mb, bwStr_size, bwStr_size / mb, other_size, total_size, total_mb); printf("N: %zu Bytes per symbol: %lf\n\n", m_numSymbols, (double)total_size / m_numSymbols); } // Print the run length distribution of the BWT void RLBWT::printRunLengths() const { typedef std::map DistMap; DistMap rlDist; char prevSym = '\0'; size_t prevRunLen = 0; size_t currLen = 0; size_t adjacentSingletons = 0; size_t numRuns = getNumRuns(); size_t totalRuns = 0; for(size_t i = 0; i < numRuns; ++i) { const RLUnit& unit = m_rlString[i]; size_t length = unit.getCount(); if(unit.getChar() == prevSym) { currLen += unit.getCount(); } else { if(prevSym != '\0') { if(currLen >= 200) rlDist[200]++; else rlDist[currLen]++; totalRuns++; } currLen = length; prevSym = unit.getChar(); } if(length == 1 && prevRunLen == 1) { adjacentSingletons += 1; prevRunLen = 0; } prevRunLen = length; } printf("Run length distrubtion\n"); printf("rl\tcount\tfrac\n"); double cumulative_mb = 0.0f; for(DistMap::iterator iter = rlDist.begin(); iter != rlDist.end(); ++iter) { cumulative_mb += ((double)iter->second / (1024 * 1024)); printf("%zu\t%zu\t%lf\t%lf\n", iter->first, iter->second, double(iter->second) / totalRuns, cumulative_mb); } printf("Total runs: %zu\n", totalRuns); printf("Number of adjacent singleton runs: %zu\n", adjacentSingletons); printf("Minimal runs: %zu\n", totalRuns - adjacentSingletons / 2); } ================================================ FILE: src/SGA/SuffixTools/RLBWT.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // RLBWT - Run-length encoded Burrows Wheeler transform // #ifndef RLBWT_H #define RLBWT_H #include "STCommon.h" #include "Occurrence.h" #include "SuffixArray.h" #include "ReadTable.h" #include "HitData.h" #include "BWTReader.h" #include "EncodedString.h" #include "FMMarkers.h" #include "RLUnit.h" // Defines //#define RLBWT_VALIDATE 1 // // RLBWT // class RLBWT { public: // Constructors RLBWT(const std::string& filename, int sampleRate = DEFAULT_SAMPLE_RATE_SMALL); RLBWT(const SuffixArray* pSA, const ReadTable* pRT); // void initializeFMIndex(); // Append a symbol to the bw string void append(char b); inline char getChar(size_t idx) const { // Calculate the Marker who's position is not less than idx const LargeMarker& upper = getUpperMarker(idx); size_t current_position = upper.getActualPosition(); assert(current_position >= idx); size_t symbol_index = upper.unitIndex; // Search backwards (towards 0) until idx is found while(current_position > idx) { assert(symbol_index != 0); symbol_index -= 1; current_position -= m_rlString[symbol_index].getCount(); } // symbol_index is now the index of the run containing the idx symbol const RLUnit& unit = m_rlString[symbol_index]; assert(current_position <= idx && current_position + unit.getCount() >= idx); return unit.getChar(); } // Get the index of the marker nearest to position in the bwt inline size_t getNearestMarkerIdx(size_t position, size_t sampleRate, size_t shiftValue) const { size_t offset = MOD_POWER_2(position, sampleRate); // equivalent to position % sampleRate size_t baseIdx = position >> shiftValue; if(offset < (sampleRate >> 1)) { return baseIdx; } else { return baseIdx + 1; } } // Get the interpolated marker with position closest to position inline LargeMarker getNearestMarker(size_t position) const { size_t nearest_small_idx = getNearestMarkerIdx(position, m_smallSampleRate, m_smallShiftValue); return getInterpolatedMarker(nearest_small_idx); } // Get the greatest interpolated marker whose position is less than or equal to position inline LargeMarker getLowerMarker(size_t position) const { size_t target_small_idx = position >> m_smallShiftValue; return getInterpolatedMarker(target_small_idx); } // Get the lowest interpolated marker whose position is strictly greater than position inline LargeMarker getUpperMarker(size_t position) const { size_t target_small_idx = (position >> m_smallShiftValue) + 1; return getInterpolatedMarker(target_small_idx); } // Return a LargeMarker with values that are interpolated by adding // the relative count nearest to the requested position to the last // LargeMarker inline LargeMarker getInterpolatedMarker(size_t target_small_idx) const { // Calculate the position of the LargeMarker closest to the target SmallMarker size_t target_position = target_small_idx << m_smallShiftValue; size_t curr_large_idx = target_position >> m_largeShiftValue; LargeMarker absoluteMarker = m_largeMarkers[curr_large_idx]; const SmallMarker& relative = m_smallMarkers[target_small_idx]; alphacount_add16(absoluteMarker.counts, relative.counts); absoluteMarker.unitIndex += relative.unitCount; return absoluteMarker; } inline BaseCount getPC(char b) const { return m_predCount.get(b); } // Return the number of times char b appears in bwt[0, idx] inline BaseCount getOcc(char b, size_t idx) const { // The counts in the marker are not inclusive (unlike the Occurrence class) // so we increment the index by 1. ++idx; const LargeMarker& marker = getNearestMarker(idx); size_t current_position = marker.getActualPosition(); bool forwards = current_position < idx; //printf("cp: %zu idx: %zu f: %d dist: %d\n", current_position, idx, forwards, (int)idx - (int)current_position); size_t running_count = marker.counts.get(b); size_t symbol_index = marker.unitIndex; if(forwards) accumulateForwards(b, running_count, symbol_index, current_position, idx); else accumulateBackwards(b, running_count, symbol_index, current_position, idx); return running_count; } // Return the number of times each symbol in the alphabet appears in bwt[0, idx] inline AlphaCount64 getFullOcc(size_t idx) const { // The counts in the marker are not inclusive (unlike the Occurrence class) // so we increment the index by 1. ++idx; const LargeMarker& marker = getNearestMarker(idx); size_t current_position = marker.getActualPosition(); bool forwards = current_position < idx; AlphaCount64 running_count = marker.counts; size_t symbol_index = marker.unitIndex; if(forwards) accumulateForwards(running_count, symbol_index, current_position, idx); else accumulateBackwards(running_count, symbol_index, current_position, idx); return running_count; } // Adds to the count of symbol b in the range [targetPosition, currentPosition) // Precondition: currentPosition <= targetPosition inline void accumulateBackwards(AlphaCount64& running_count, size_t currentUnitIndex, size_t currentPosition, const size_t targetPosition) const { // Search backwards (towards 0) until idx is found while(currentPosition != targetPosition) { size_t diff = currentPosition - targetPosition; #ifdef RLBWT_VALIDATE assert(currentUnitIndex != 0); #endif --currentUnitIndex; const RLUnit& curr_unit = m_rlString[currentUnitIndex]; currentPosition -= curr_unit.subtractAlphaCount(running_count, diff); } } // Adds to the count of symbol b in the range [currentPosition, targetPosition) // Precondition: currentPosition <= targetPosition inline void accumulateForwards(AlphaCount64& running_count, size_t currentUnitIndex, size_t currentPosition, const size_t targetPosition) const { // Search backwards (towards 0) until idx is found while(currentPosition != targetPosition) { size_t diff = targetPosition - currentPosition; #ifdef RLBWT_VALIDATE assert(currentUnitIndex != m_rlString.size()); #endif const RLUnit& curr_unit = m_rlString[currentUnitIndex]; currentPosition += curr_unit.addAlphaCount(running_count, diff); ++currentUnitIndex; } } // Adds to the count of symbol b in the range [targetPosition, currentPosition) // Precondition: currentPosition <= targetPosition inline void accumulateBackwards(char b, size_t& running_count, size_t currentUnitIndex, size_t currentPosition, const size_t targetPosition) const { // Search backwards (towards 0) until idx is found while(currentPosition != targetPosition) { size_t diff = currentPosition - targetPosition; #ifdef RLBWT_VALIDATE assert(currentUnitIndex != 0); #endif --currentUnitIndex; const RLUnit& curr_unit = m_rlString[currentUnitIndex]; currentPosition -= curr_unit.subtractCount(b, running_count, diff); } } // Adds to the count of symbol b in the range [currentPosition, targetPosition) // Precondition: currentPosition <= targetPosition inline void accumulateForwards(char b, size_t& running_count, size_t currentUnitIndex, size_t currentPosition, const size_t targetPosition) const { // Search backwards (towards 0) until idx is found while(currentPosition != targetPosition) { size_t diff = targetPosition - currentPosition; #ifdef RLBWT_VALIDATE assert(currentUnitIndex != m_rlString.size()); #endif const RLUnit& curr_unit = m_rlString[currentUnitIndex]; currentPosition += curr_unit.addCount(b, running_count, diff); ++currentUnitIndex; } } // Return the number of times each symbol in the alphabet appears ins bwt[idx0, idx1] inline AlphaCount64 getOccDiff(size_t idx0, size_t idx1) const { return getFullOcc(idx1) - getFullOcc(idx0); } inline size_t getNumStrings() const { return m_numStrings; } inline size_t getBWLen() const { return m_numSymbols; } inline size_t getNumRuns() const { return m_rlString.size(); } // Return the first letter of the suffix starting at idx inline char getF(size_t idx) const { size_t ci = 0; while(ci < ALPHABET_SIZE && m_predCount.getByIdx(ci) <= idx) ci++; assert(ci != 0); return RANK_ALPHABET[ci - 1]; } // Print the size of the BWT void printInfo() const; void print() const; void printRunLengths() const; // IO friend class BWTReaderBinary; friend class BWTWriterBinary; friend class BWTReaderAscii; friend class BWTWriterAscii; // Default sample rates for the large (64-bit) and small (8-bit) occurrence markers static const int DEFAULT_SAMPLE_RATE_LARGE = 2; //8192; static const int DEFAULT_SAMPLE_RATE_SMALL = 2; private: // Default constructor is not allowed RLBWT() {} // Calculate the number of markers to place size_t getNumRequiredMarkers(size_t n, size_t d) const; // The C(a) array AlphaCount64 m_predCount; // The run-length encoded string RLVector m_rlString; // The marker vector LargeMarkerVector m_largeMarkers; SmallMarkerVector m_smallMarkers; // The number of strings in the collection size_t m_numStrings; // The total length of the bw string size_t m_numSymbols; // The sample rate used for the markers size_t m_largeSampleRate; size_t m_smallSampleRate; // The amount to shift values by to divide by m_sampleRate int m_smallShiftValue; int m_largeShiftValue; }; #endif ================================================ FILE: src/SGA/SuffixTools/RLUnit.h ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // RLUnit - A run-length encoded unit of the FM-index // #ifndef RLUNIT_H #define RLUNIT_H // #define RL_COUNT_MASK 0x1F //00011111 #define RL_SYMBOL_MASK 0xE0 //11100000 #define RL_FULL_COUNT 31 #define RL_SYMBOL_SHIFT 5 #define RLE_VALIDATE // A unit of the RLBWT is a pair of a symbol and its count // The high 3 bits encodes the symbol to store // The low 5 bits encodes the length of the run struct RLUnit { RLUnit() : data(0) {} RLUnit(char b) : data(1) { setChar(b); } // Returns true if the count cannot be incremented inline bool isFull() const { return (data & RL_COUNT_MASK) == RL_FULL_COUNT; } inline bool isEmpty() const { return (data & RL_COUNT_MASK) == 0; } inline bool isInitialized() const { return data > 0; } // Add this run to the AlphaCount // Only add up to maxCount symbols. Returns the number // of symbols added inline size_t addAlphaCount(AlphaCount64& ac, size_t max) const { size_t count = getCount(); if(count > max) count = max; char symbol = getChar(); ac.add(symbol, count); return count; } // Add this run to the count of base b if it matches // Only add up to maxCount symbols. Returns the number // of symbols in the current run, up to max inline size_t addCount(char b, size_t& base_count, size_t max) const { size_t run_len = getCount(); if(run_len > max) run_len = max; char symbol = getChar(); if(symbol == b) base_count += run_len; return run_len; } // Subtract this run from the AlphaCount // Only subtract up to maxCount symbols. Returns the number // of symbols added inline size_t subtractAlphaCount(AlphaCount64& ac, size_t max) const { size_t count = getCount(); if(count > max) count = max; char symbol = getChar(); ac.subtract(symbol, count); return count; } // Subtract this run from the count of base b if it matches // Only add up to maxCount symbols. Returns the number // of symbols in the current run, up to max inline size_t subtractCount(char b, size_t& base_count, size_t max) const { size_t run_len = getCount(); if(run_len > max) run_len = max; char symbol = getChar(); if(symbol == b) base_count -= run_len; return run_len; } // inline void incrementCount() { #ifdef RLE_VALIDATE assert(!isFull()); #endif ++data; } // inline void decrementCount() { #ifdef RLE_VALIDATE assert(!isEmpty()); #endif --data; } inline uint8_t getCount() const { #ifdef RLE_VALIDATE assert((data & RL_COUNT_MASK) != 0); #endif return data & RL_COUNT_MASK; } // Set the symbol inline void setChar(char symbol) { // Clear the current symbol data &= RL_COUNT_MASK; uint8_t code = BWT_ALPHABET::getRank(symbol); code <<= RL_SYMBOL_SHIFT; data |= code; } // Get the symbol inline char getChar() const { uint8_t code = data & RL_SYMBOL_MASK; code >>= RL_SYMBOL_SHIFT; return BWT_ALPHABET::getChar(code); } // uint8_t data; friend class RLBWTReader; friend class RLBWTWriter; }; typedef std::vector RLVector; #endif ================================================ FILE: src/SGA/SuffixTools/RankProcess.cpp ================================================ ///----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // RankProcess - Compute a vector of BWT ranks for // SequenceWorkItems // #include "RankProcess.h" // // // RankProcess::RankProcess(const BWT* pBWT, GapArray* pSharedGapArray, bool doReverse, bool removeMode) : m_pBWT(pBWT), m_pSharedGapArray(pSharedGapArray), m_doReverse(doReverse), m_removeMode(removeMode) { } // RankProcess::~RankProcess() { } // Calculate the ranks of the given sequence. // We attempt to update the count of the rank in the gap array // using an atomic compare and swap. The update will fail if the // small-storage maximum count is exceeded. In this case // we push the value to the overflow array for a serial // update in the post-processing step. RankResult RankProcess::process(const SequenceWorkItem& workItem) { RankResult out; DNAString w = workItem.read.seq; if(m_doReverse) w.reverse(); size_t l = w.length(); int i = l - 1; // In add mode, the initial rank is zero and we calculate the rank // for the last base of the sequence using just C(a). In remove // mode we use the index of the read (in the original read table) as // the rank so that ranks calculate correspond to the correct // entries in the BWT for the read to remove. int64_t rank = 0; // add mode if(m_removeMode) { // Parse the read index from the read id rank = parseRankFromID(workItem.read.id); } out.numRanksProcessed += 1; if(!m_pSharedGapArray->attemptBaseIncrement(rank)) out.overflowVec.push_back(rank); // Compute the starting rank for the last symbol of w char c = w.get(i); // In the case that the starting rank is zero (default // in add mode, or if we are removing the first read) // there can no occurrence of any characters before this // suffix so we just calculate the rank from C(a) if(rank == 0) rank = m_pBWT->getPC(c); else rank = m_pBWT->getPC(c) + m_pBWT->getOcc(c, rank - 1); out.numRanksProcessed += 1; if(!m_pSharedGapArray->attemptBaseIncrement(rank)) out.overflowVec.push_back(rank); --i; // Iteratively compute the remaining ranks while(i >= 0) { char c = w.get(i); rank = m_pBWT->getPC(c) + m_pBWT->getOcc(c, rank - 1); //std::cout << "c: " << c << " rank: " << rank << "\n"; out.numRanksProcessed += 1; if(!m_pSharedGapArray->attemptBaseIncrement(rank)) out.overflowVec.push_back(rank); --i; } return out; } // Parse the rank of a read from its ID. This must be set by the process // which discards the read. int64_t RankProcess::parseRankFromID(const std::string& id) { static const std::string rank_str = "seqrank="; // Find the position of the rank expression in the string size_t rank_pos = id.rfind(rank_str); if(rank_pos == std::string::npos) { std::cout << "Error: rank token not found in the discarded read with id: " << id << "\n"; exit(EXIT_FAILURE); } assert(rank_pos + rank_str.length() < id.length()); std::stringstream rp(id.substr(rank_pos + rank_str.length())); int64_t rank; rp >> rank; return rank; } // // // RankPostProcess::RankPostProcess(GapArray* pGapArray) : m_pGapArray(pGapArray), num_strings(0), num_symbols(0), num_serial_updates(0) { } RankPostProcess::~RankPostProcess() { //printf("Debug: num serial updates: %zu\n", num_serial_updates); } // void RankPostProcess::process(const SequenceWorkItem& /*item*/, const RankResult& result) { ++num_strings; num_symbols += result.numRanksProcessed; // We update any overflowed ranks here. This call is serial and only updates // the Overflow table in the gap array. for(RankVector::const_iterator iter = result.overflowVec.begin(); iter != result.overflowVec.end(); ++iter) m_pGapArray->incrementOverflowSerial(*iter); num_serial_updates += result.overflowVec.size(); } ================================================ FILE: src/SGA/SuffixTools/RankProcess.h ================================================ ///----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // RankProcess - Compute a vector of BWT ranks for // SequenceWorkItems // #ifndef RANKPROCESS_H #define RANKPROCESS_H #include "Util.h" #include "BWT.h" #include "SequenceWorkItem.h" #include "GapArray.h" typedef std::vector RankVector; struct RankResult { RankResult() : numRanksProcessed(0) {} RankVector overflowVec; size_t numRanksProcessed; }; // Compute the overlap blocks for reads class RankProcess { public: RankProcess(const BWT* pBWT, GapArray* pSharedGapArray, bool doReverse, bool removeMode); ~RankProcess(); RankResult process(const SequenceWorkItem& item); private: int64_t parseRankFromID(const std::string& id); const BWT* m_pBWT; GapArray* m_pSharedGapArray; bool m_doReverse; bool m_removeMode; }; // Update the gap array with class RankPostProcess { public: RankPostProcess(GapArray* pGapArray); ~RankPostProcess(); void process(const SequenceWorkItem& item, const RankResult& result); size_t getNumStringsProcessed() const { return num_strings; } size_t getNumSymbolsProcessed() const { return num_symbols; } private: GapArray* m_pGapArray; size_t num_strings; size_t num_symbols; size_t num_serial_updates; }; #endif ================================================ FILE: src/SGA/SuffixTools/SACAInducedCopying.cpp ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // SACAInducedCopying algorithm // #include "SACAInducedCopying.h" #include "SuffixCompare.h" #include "mkqs.h" #include "bucketSort.h" #include "Util.h" unsigned char mask[]={0x80,0x40,0x20,0x10,0x08,0x04,0x02,0x01}; #define GET_CHAR(i, j) pRT->getChar((i),(j)) #define isLMS(i, j) ((j) > 0 && getBit(type_array, (i), (j)) && !getBit(type_array, (i), (j-1))) #define GET_BKT(c) getBaseRank((c)) // Implementation of induced copying algorithm by // Nong, Zhang, Chan // Follows implementation given as an appendix to their 2008 paper // '\0' is the sentinenl in this algorithm void saca_induced_copying(SuffixArray* pSA, const ReadTable* pRT, int numThreads, bool silent) { // In the multiple strings case, we need a 2D bit array // to hold the L/S types for the suffixes size_t num_strings = pRT->getCount(); char** type_array = new char*[num_strings]; for(size_t i = 0; i < num_strings; ++i) { size_t s_len = pRT->getReadLength(i) + 1; size_t num_bytes = (s_len / 8) + 1; type_array[i] = new char[num_bytes]; assert(type_array[i] != 0); memset(type_array[i], 0, num_bytes); } // Classify each suffix as being L or S type for(size_t i = 0; i < num_strings; ++i) { size_t s_len = pRT->getReadLength(i) + 1; // The empty suffix ($) for each string is defined to be S type // and hence the next suffix must be L type setBit(type_array, i, s_len - 1, 1); setBit(type_array, i, s_len - 2, 0); for(int64_t j = s_len - 3; j >= 0; --j) { char curr_c = GET_CHAR(i, j); char next_c = GET_CHAR(i, j + 1); bool s_type = (curr_c < next_c || (curr_c == next_c && getBit(type_array, i, j + 1) == 1)); setBit(type_array, i, j, s_type); } } // setup buckets const int ALPHABET_SIZE = 5; int64_t bucket_counts[ALPHABET_SIZE]; int64_t buckets[ALPHABET_SIZE]; // find the ends of the buckets countBuckets(pRT, bucket_counts, ALPHABET_SIZE); getBuckets(bucket_counts, buckets, ALPHABET_SIZE, true); // Initialize the suffix array size_t num_suffixes = buckets[ALPHABET_SIZE - 1]; pSA->initialize(num_suffixes, pRT->getCount()); // Copy all the LMS substrings into the first n1 places in the SA size_t n1 = 0; for(size_t i = 0; i < num_strings; ++i) { size_t s_len = pRT->getReadLength(i) + 1; for(size_t j = 0; j < s_len; ++j) { if(isLMS(i,j)) pSA->set(n1++, SAElem(i, j)); } } /* //induceSAl(pRT, pSA, type_array, bucket_counts, buckets, num_suffixes, ALPHABET_SIZE, false); //induceSAs(pRT, pSA, type_array, bucket_counts, buckets, num_suffixes, ALPHABET_SIZE, true); // Compact all the sorted substrings into the first portion of the SA size_t n1 = 0; for(size_t i = 0; i < num_suffixes; ++i) { SAElem elem = pSA->get(i); if(!elem.isEmpty() && isLMS(elem.getID(), elem.getPos())) { pSA->set(n1++, elem); } } */ // Call MKQS, first on the sequence and then on the index in the read table SuffixCompareRadix radix_compare(pRT, 6); SuffixCompareIndex index_compare; //SuffixCompareID id_compare(pRT); if(numThreads <= 1) mkqs2(&pSA->m_data[0], n1, 0, radix_compare, index_compare); //else // parallel_mkqs(&pSA->m_data[0], n1, numThreads, radix_compare, index_compare); //if(!silent) // std::cout << "[saca] mkqs finished\n"; // Induction sort the remaining suffixes for(size_t i = n1; i < num_suffixes; ++i) pSA->set(i, SAElem()); // Find the ends of the buckets getBuckets(bucket_counts, buckets, ALPHABET_SIZE, true); for(int64_t i = n1 - 1; i >= 0; --i) { SAElem elem_i = pSA->get(i); pSA->set(i, SAElem()); // empty char c = GET_CHAR(elem_i.getID(), elem_i.getPos()); pSA->set(--buckets[GET_BKT(c)], elem_i); } induceSAl(pRT, pSA, type_array, bucket_counts, buckets, num_suffixes, ALPHABET_SIZE, false); induceSAs(pRT, pSA, type_array, bucket_counts, buckets, num_suffixes, ALPHABET_SIZE, true); // deallocate t array for(size_t i = 0; i < num_strings; ++i) { delete [] type_array[i]; } delete [] type_array; } void induceSAl(const ReadTable* pRT, SuffixArray* pSA, char** p_array, int64_t* counts, int64_t* buckets, size_t n, int K, bool end) { getBuckets(counts, buckets, K, end); for(size_t i = 0; i < n; ++i) { const SAElem& elem_i = pSA->get(i); if(!elem_i.isEmpty() && elem_i.getPos() > 0) { //std::cout << "Curr: " << elem_i << "\n"; SAElem elem_j(elem_i.getID(), elem_i.getPos() - 1); if(!getBit(p_array, elem_j.getID(), elem_j.getPos())) { char c = GET_CHAR(elem_j.getID(),elem_j.getPos()); //std::cout << "Placing " << elem_j << " at position " << buckets[GET_BKT(c)] << "\n"; pSA->set(buckets[GET_BKT(c)]++, elem_j); } } } } void induceSAs(const ReadTable* pRT, SuffixArray* pSA, char** p_array, int64_t* counts, int64_t* buckets, size_t n, int K, bool end) { getBuckets(counts, buckets, K, end); for(int64_t i = n - 1; i >= 0; --i) { const SAElem& elem_i = pSA->get(i); if(!elem_i.isEmpty() && elem_i.getPos() > 0) { //std::cout << "Curr: " << elem_i << "\n"; SAElem elem_j(elem_i.getID(), elem_i.getPos() - 1); if(getBit(p_array, elem_j.getID(), elem_j.getPos())) { char c = GET_CHAR(elem_j.getID(),elem_j.getPos()); //std::cout << "Placing " << elem_j << " at position " << buckets[GET_BKT(c)] - 1 << "\n"; pSA->set(--buckets[GET_BKT(c)], elem_j); } } } } // Calculate the number of items that should be in each bucket void countBuckets(const ReadTable* pRT, int64_t* counts, int K) { for(int i = 0; i < K; ++i) counts[i] = 0; for(size_t i = 0; i < pRT->getCount(); ++i) { size_t s_len = pRT->getReadLength(i); for(size_t j = 0; j < s_len; ++j) counts[getBaseRank(GET_CHAR(i,j))]++; counts[getBaseRank('\0')]++; } } // If end is true, calculate the end of the buckets, otherwise // calculate the starts void getBuckets(int64_t* counts, int64_t* buckets, int K, bool end) { for(int i = 0; i < K; ++i) buckets[i] = 0; size_t sum = 0; for(int i = 0; i < K; ++i) { sum += counts[i]; buckets[i] = end ? sum : sum - counts[i]; } } // set the element to b void setBit(char** p_array, size_t str_idx, size_t bit_idx, bool b) { char* ba = p_array[str_idx]; ba[bit_idx / 8] = b ? mask[bit_idx % 8] | ba[bit_idx / 8] : ~mask[bit_idx % 8] & ba[bit_idx / 8]; } bool getBit(char** p_array, size_t str_idx, size_t bit_idx) { return p_array[str_idx][bit_idx / 8] & mask[bit_idx % 8] ? 1 : 0; } void printType(const ReadTable* pRT, char** p_array, size_t str_idx) { std::string suf_string = pRT->getRead(str_idx).seq.getSuffixString(0); std::cout << suf_string << "\n"; for(size_t i = 0; i < suf_string.length(); ++i) { std::cout << (getBit(p_array, str_idx, i) == 1 ? "S" : "L"); } std::cout << "\n"; } ================================================ FILE: src/SGA/SuffixTools/SACAInducedCopying.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL license //----------------------------------------------- // // SACAInducedCopying - Implementation of the // induced copying algorithm as described // by Nong, Zhang, Chan (2008) // Modified by JTS to handle multiple strings #ifndef SACA_INDUCED_COPYING_H #include "SuffixArray.h" #include "ReadTable.h" void saca_induced_copying(SuffixArray* pSA, const ReadTable* pRT, int numThreads, bool silent = false); void induceSAl(const ReadTable* pRT, SuffixArray* pSA, char** p_array, int64_t* counts, int64_t* buckets, size_t n, int K, bool end); void induceSAs(const ReadTable* pRT, SuffixArray* pSA, char** p_array, int64_t* counts, int64_t* buckets, size_t n, int K, bool end); void countBuckets(const ReadTable* pRT, int64_t* buckets, int K); void getBuckets(int64_t* counts, int64_t* buckets, int K, bool end); inline void setBit(char** p_array, size_t str_idx, size_t bit_idx, bool b); inline bool getBit(char** p_array, size_t str_idx, size_t bit_idx); void printType(const ReadTable* pRT, char** p_array, size_t str_idx); #endif ================================================ FILE: src/SGA/SuffixTools/SAReader.cpp ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // SAReader.h - Read a suffix array file from disk // #include "SAReader.h" #include "SuffixArray.h" // SAReader::SAReader(const std::string& filename) : m_stage(SAIOS_NONE) { m_pReader = createReader(filename); m_stage = SAIOS_HEADER; } // SAReader::~SAReader() { delete m_pReader; } void SAReader::read(SuffixArray* pSA) { size_t num_elems; readHeader(pSA->m_numStrings, num_elems); pSA->m_data.reserve(num_elems); readElems(pSA->m_data); } // void SAReader::readHeader(size_t& num_strings, size_t& num_elems) { assert(m_stage == SAIOS_HEADER); uint16_t magic_number; // Ensure the file format is sane *m_pReader >> magic_number; if(magic_number != SA_FILE_MAGIC) { std::cerr << "Suffix array file is not properly formatted, aborting\n"; exit(EXIT_FAILURE); } *m_pReader >> num_strings; *m_pReader >> num_elems; // Explicitly set the stream // to the start of the elements m_pReader->get(); m_stage = SAIOS_ELEM; } // void SAReader::readElems(SAElemVector& elemVector) { assert(m_stage == SAIOS_ELEM); size_t cap = elemVector.capacity(); size_t num_read = 0; SAElem e; while(*m_pReader >> e) { elemVector.push_back(e); ++num_read; } assert(cap >= num_read); (void)cap; m_stage = SAIOS_DONE; } // void SAReader::readElems(std::vector& outVector) { assert(m_stage == SAIOS_ELEM); size_t cap = outVector.capacity(); size_t num_read = 0; size_t MAX_ELEMS = std::numeric_limits::max(); SAElem e; while(*m_pReader >> e) { size_t index = e.getID(); size_t pos = e.getPos(); assert(pos == 0); // Ensure this index is representable by the collection if(index > MAX_ELEMS) { std::cerr << "Error: Only " << MAX_ELEMS << " reads are allowed in this program\n"; std::cerr << "Found read with index " << index << "\n"; std::cerr << "Contact sga-users@googlegroups.com for help\n"; exit(EXIT_FAILURE); } outVector.push_back(index); ++num_read; } assert(cap >= num_read); (void)cap; m_stage = SAIOS_DONE; } SAElem SAReader::readElem() { assert(m_stage == SAIOS_ELEM); SAElem e; if(*m_pReader >> e) { return e; } else { std::cerr << "Error: Read past end-of-file in SAReader::readElem()\n"; exit(EXIT_FAILURE); } } ================================================ FILE: src/SGA/SuffixTools/SAReader.h ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // SAReader.h - Read a suffix array file from disk // #ifndef SAREADER_H #define SAREADER_H #include "Util.h" #include "STCommon.h" #include "Occurrence.h" const uint16_t SA_FILE_MAGIC = 0xCACA; enum SAIOStage { SAIOS_NONE, SAIOS_HEADER, SAIOS_ELEM, SAIOS_DONE }; class SuffixArray; class SAReader { public: SAReader(const std::string& filename); ~SAReader(); // void read(SuffixArray* pSA); // Read the header of the file void readHeader(size_t& num_strings, size_t& num_elems); // Read the file into a vector of SAElems void readElems(SAElemVector& elemVector); // Read the file into a vector of unsigned ints storing // the read indices. This is a more compact representation // than storing the full SAElems but only works up to 2**32 values void readElems(std::vector& outVector); // Read a single element SAElem readElem(); private: std::istream* m_pReader; SAIOStage m_stage; }; #endif ================================================ FILE: src/SGA/SuffixTools/SAWriter.cpp ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // SAWriter.h - Read a suffix array file from disk // #include "SAWriter.h" #include "SuffixArray.h" // SAWriter::SAWriter(const std::string& filename) : m_stage(SAIOS_NONE) { m_pWriter = createWriter(filename); m_stage = SAIOS_HEADER; } // SAWriter::~SAWriter() { delete m_pWriter; } void SAWriter::write(const SuffixArray* pSA) { writeHeader(pSA->m_numStrings, pSA->m_data.size()); writeElems(pSA->m_data); } // void SAWriter::writeHeader(const size_t& num_strings, const size_t& num_elems) { assert(m_stage == SAIOS_HEADER); *m_pWriter << SA_FILE_MAGIC << "\n"; *m_pWriter << num_strings << "\n"; *m_pWriter << num_elems << "\n"; m_stage = SAIOS_ELEM; } // void SAWriter::writeElems(const SAElemVector& elemVector) { assert(m_stage == SAIOS_ELEM); for(size_t i = 0; i < elemVector.size(); ++i) writeElem(elemVector[i]); m_stage = SAIOS_DONE; } // void SAWriter::writeElem(const SAElem& elem) { *m_pWriter << elem << "\n"; } ================================================ FILE: src/SGA/SuffixTools/SAWriter.h ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // SAWriter.h - Read a suffix array file from disk // #ifndef SAWRITER_H #define SAWRITER_H #include "Util.h" #include "STCommon.h" #include "Occurrence.h" #include "SAReader.h" class SuffixArray; class SAWriter { public: SAWriter(const std::string& filename); ~SAWriter(); // void write(const SuffixArray* pSA); void writeHeader(const size_t& num_strings, const size_t& num_elems); void writeElems(const SAElemVector& elemVector); void writeElem(const SAElem& elem); private: std::ostream* m_pWriter; SAIOStage m_stage; }; #endif ================================================ FILE: src/SGA/SuffixTools/SBWT.cpp ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // SBWT.cpp - Burrows Wheeler transform of a generalized suffix array // #include "SBWT.h" #include "Timer.h" #include "BWTReaderAscii.h" #include "BWTReaderBinary.h" #include "BWTWriterAscii.h" #include #include #include // macros #define OCC(c,i) m_occurrence.get(m_bwStr, (c), (i)) #define PRED(c) m_predCount.get((c)) // Parse a BWT from a file SBWT::SBWT(const std::string& filename, int sampleRate) { BWTReaderBinary reader(filename); reader.read(this); initializeFMIndex(sampleRate); } // Construct the BWT from a suffix array SBWT::SBWT(const SuffixArray* pSA, const ReadTable* pRT) { size_t n = pSA->getSize(); m_numStrings = pSA->getNumStrings(); m_bwStr.resize(n); // Set up the bwt string and suffix array from the cycled strings for(size_t i = 0; i < n; ++i) { SAElem saElem = pSA->get(i); const SeqItem& si = pRT->getRead(saElem.getID()); // Get the position of the start of the suffix uint64_t f_pos = saElem.getPos(); uint64_t l_pos = (f_pos == 0) ? si.seq.length() : f_pos - 1; char b = (l_pos == si.seq.length()) ? '$' : si.seq.get(l_pos); m_bwStr.set(i, b); } initializeFMIndex(DEFAULT_SAMPLE_RATE); } // Fill in the FM-index data structures void SBWT::initializeFMIndex(int sampleRate) { // initialize the occurance table m_occurrence.initialize(m_bwStr, sampleRate); // Calculate the C(a) array // Calculate the total number of occurances of each character in the BW str AlphaCount64 tmp; for(size_t i = 0; i < m_bwStr.length(); ++i) { tmp.increment(m_bwStr.get(i)); } m_predCount.set('$', 0); m_predCount.set('A', tmp.get('$')); m_predCount.set('C', m_predCount.get('A') + tmp.get('A')); m_predCount.set('G', m_predCount.get('C') + tmp.get('C')); m_predCount.set('T', m_predCount.get('G') + tmp.get('G')); } // Compute the last to first mapping size_t SBWT::LF(size_t idx) const { return m_bwStr.get(idx) != '$' ? PRED(m_bwStr.get(idx)) + OCC(m_bwStr.get(idx), idx) : 0; } // Perform a exact search for the string w using the backwards algorithm void SBWT::backwardSearch(std::string w) const { std::cout << "Searching for " << w << "\n"; int len = w.size(); int j = len - 1; char curr = w[j]; int r_lower = PRED(curr); int r_upper = r_lower + OCC(curr, m_bwStr.length() - 1) - 1; --j; std::cout << "Starting point: " << r_lower << "," << r_upper << "\n"; for(;j >= 0; --j) { curr = w[j]; printf("RL = C(%c) + O(%c,%d) + %zu\n", curr, curr, r_lower - 1, m_numStrings); printf("RU = C(%c) + O(%c,%d)\n", curr, curr, r_upper); printf("RL = %zu + %zu + %zu\n", (size_t)PRED(curr), (size_t)OCC(curr, r_lower - 1), m_numStrings); printf("RU = %zu + %zu\n", (size_t)PRED(curr), (size_t)OCC(curr, r_upper)); r_lower = PRED(curr) + OCC(curr, r_lower - 1); r_upper = PRED(curr) + OCC(curr, r_upper) - 1; printf("Curr: %c, Interval now: %d,%d\n", curr, r_lower, r_upper); } std::cout << "Interval found: " << r_lower << "," << r_upper << "\n"; } // void SBWT::validate() const { std::cerr << "Warning BWT validation is turned on\n"; m_occurrence.validate(m_bwStr); } // Print the BWT void SBWT::print(const ReadTable* pRT, const SuffixArray* pSA) const { std::cout << "i\tL(i)\tF(i)\tO(-,i)\tSUFF\n"; for(size_t i = 0; i < m_bwStr.length(); ++i) { assert(getF(i) == pSA->getSuffix(i, pRT)[0]); std::cout << i << "\t" << m_bwStr.get(i) << "\t" << getF(i) << "\t" << m_occurrence.get(m_bwStr, i) << pSA->getSuffix(i, pRT) << "\n"; } } // Print information about the BWT void SBWT::printInfo() const { size_t o_size = m_occurrence.getByteSize(); size_t p_size = sizeof(m_predCount); size_t bwStr_size = m_bwStr.getMemSize(); size_t offset_size = sizeof(m_numStrings); size_t total_size = o_size + p_size + bwStr_size + offset_size; double total_mb = ((double)total_size / (double)(1024 * 1024)); printf("\nSBWT info\n"); printf("Sample rate: %zu\n", m_occurrence.getSampleRate()); printf("Memory -- OCC: %zu C: %zu Str: %zu Misc: %zu TOTAL: %zu (%lf MB)\n", o_size, p_size, bwStr_size, offset_size, total_size, total_mb); printf("N: %zu Bytes per symbol: %lf\n", m_bwStr.length(), (double)total_size / m_bwStr.length()); } ================================================ FILE: src/SGA/SuffixTools/SBWT.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // SBWT - Uncompressed burrows Wheeler transform // of a set of sequence reads // // #ifndef SBWT_H #define SBWT_H #include "STCommon.h" #include "Occurrence.h" #include "SuffixArray.h" #include "ReadTable.h" #include "HitData.h" #include "BWTReader.h" #include "EncodedString.h" // // BWT // class SBWT { public: // Constructors SBWT(const std::string& filename, int sampleRate = DEFAULT_SAMPLE_RATE); SBWT(const SuffixArray* pSA, const ReadTable* pRT); // void initializeFMIndex(int sampleRate); // Exact match void backwardSearch(std::string w) const; // L[i] -> F mapping size_t LF(size_t idx) const; inline char getChar(size_t idx) const { return m_bwStr.get(idx); } inline BaseCount getPC(char b) const { return m_predCount.get(b); } // Return the number of times char b appears in bwt[0, idx] inline BaseCount getOcc(char b, size_t idx) const { return m_occurrence.get(m_bwStr, b, idx); } // Return the number of times each symbol in the alphabet appears in bwt[0, idx] inline AlphaCount64 getFullOcc(size_t idx) const { return m_occurrence.get(m_bwStr, idx); } // Return the number of times each symbol in the alphabet appears ins bwt[idx0, idx1] inline AlphaCount64 getOccDiff(size_t idx0, size_t idx1) const { return m_occurrence.getDiff(m_bwStr, idx0, idx1); } inline size_t getNumStrings() const { return m_numStrings; } inline size_t getBWLen() const { return m_bwStr.length(); } // Return the first letter of the suffix starting at idx inline char getF(size_t idx) const { size_t ci = 0; while(ci < ALPHABET_SIZE && m_predCount.getByIdx(ci) <= idx) ci++; assert(ci != 0); return RANK_ALPHABET[ci - 1]; } // Print the size of the BWT void printInfo() const; void print(const ReadTable* pRT, const SuffixArray* pSA) const; void printRunLengths() const { std::cout << "Using SimpleBWT - No run lengths\n"; } void validate() const; // IO friend class BWTReaderBinary; friend class BWTWriterBinary; friend class BWTReaderAscii; friend class BWTWriterAscii; static const int DEFAULT_SAMPLE_RATE = 128; static const int DEFAULT_SAMPLE_RATE_SMALL = DEFAULT_SAMPLE_RATE; private: // Default constructor is not allowed SBWT() {} // The O(a,i) array Occurrence m_occurrence; // The C(a) array AlphaCount64 m_predCount; // The bw string BWTString m_bwStr; // The number of strings in the collection size_t m_numStrings; }; #endif ================================================ FILE: src/SGA/SuffixTools/STCommon.cpp ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // STCommon.cpp - Implementation of basic data structures // #include "STCommon.h" #include // // SAId // // Constructors SAElem::SAElem(uint64_t i) { setID(i); } // SAElem::SAElem(uint64_t i, uint64_t p) { setID(i); setPos(p); } // Input std::istream& operator>>(std::istream& in, SAElem& s) { uint64_t i; uint64_t p; in >> i >> p; s.setID(i); s.setPos(p); return in; } // Output std::ostream& operator<<(std::ostream& out, const SAElem& s) { if(!s.isEmpty()) out << s.getID() << " " << s.getPos(); else out << -1; return out; } // // Global functions // // Print a map template void printMap(const std::map& m) { for(typename std::map::const_iterator iter = m.begin(); iter != m.end(); ++iter) { std::cout << iter->first << "\t" << iter->second << "\n"; } } // Print a vector template void printVector(const std::vector& v) { std::copy(v.begin(), v.end(), std::ostream_iterator(std::cout, "\n")); } ================================================ FILE: src/SGA/SuffixTools/STCommon.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // STCommon.h - Base classes and data structures // #ifndef STCOMMON_H #define STCOMMON_H #include "STGlobals.h" #include "Util.h" // // Functions // // Print out a map using cout template void printMap(const std::map& m); // Print a vector template void printVector(const std::vector& v); // // Classes // // // A Generalized SuffixArray ID (SAElem) is a single number where the high n bits represents the // identifier of the string (as the index into a StringDictionary) and the low (64 - n) bits // represents the position in that string // struct SAElem { public: SAElem() : m_val(std::numeric_limits::max()) { } SAElem(uint64_t i); SAElem(uint64_t i, uint64_t p); // inline bool isEmpty() const { return m_val == std::numeric_limits::max(); } // set the id inline void setID(uint64_t i) { // Clear the HIGH bits by ANDing with the low mask m_val &= LOW_MASK; // Shift the new position into place and set the new value i <<= POS_BITS; m_val |= i; } // set the position void setPos(uint64_t i) { // Clear the LOW bits by anding with the high mask m_val &= HIGH_MASK; // Set the new value m_val |= i; } inline uint64_t getID() const { return (m_val & HIGH_MASK) >> POS_BITS; } inline uint64_t getPos() const { return (m_val & LOW_MASK); } // Returns true if the suffix is the full length of the string inline bool isFull() const { return getPos() == 0; } // Input/Output friend std::istream& operator>>(std::istream& in, SAElem& s); friend std::ostream& operator<<(std::ostream& out, const SAElem& s); private: // uint64_t m_val; // Masks static const uint8_t ID_BITS = 36; // Allows up to 68 billion IDs static const uint8_t POS_BITS = 64 - ID_BITS; static const uint64_t HIGH_MASK = (~((uint64_t)(0))) << POS_BITS; static const uint64_t LOW_MASK = ~HIGH_MASK; }; // Typedefs of STL collections of the above classes typedef std::vector SAElemVector; typedef std::pair SAElemPair; typedef std::vector SAElemPairVec; typedef std::set NumericIDSet; #endif ================================================ FILE: src/SGA/SuffixTools/STGlobals.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL license //----------------------------------------------- // // STGlobals.h - Common definitions and declarations for suffix tree, // suffix array and BWT data types // #ifndef STGLOBALS_H #define STGLOBALS_H #include #include #include #include #include #include #include #include "Alphabet.h" // // Basic typedefs // typedef std::string BWStr; typedef uint32_t Label; typedef uint8_t AIdx; typedef std::vector IntVector; typedef std::vector StringVector; #endif ================================================ FILE: src/SGA/SuffixTools/SampledSuffixArray.cpp ================================================ //----------------------------------------------- // Copyright 2011 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // SampledSuffixArray - Data structure holding // a subset of suffix array positions. From the // sampled positions, the other entries of the // suffix array can be calculated. // #include "SampledSuffixArray.h" #include "SAReader.h" #include "SAWriter.h" //#include "config.h" #if HAVE_OPENMP #include #endif static const uint32_t SSA_MAGIC_NUMBER = 12412; #define SSA_READ(x) pReader->read(reinterpret_cast(&(x)), sizeof((x))); #define SSA_READ_N(x,n) pReader->read(reinterpret_cast(&(x)), (n)); #define SSA_WRITE(x) pWriter->write(reinterpret_cast(&(x)), sizeof((x))); #define SSA_WRITE_N(x,n) pWriter->write(reinterpret_cast(&(x)), (n)); // SampledSuffixArray::SampledSuffixArray() { } SampledSuffixArray::SampledSuffixArray(const std::string& filename, SSAFileType filetype) { // Read the sampled suffix array from a file - either from a .ssa or .sai file if(filetype == SSA_FT_SSA) readSSA(filename); else readSAI(filename); } // SAElem SampledSuffixArray::calcSA(int64_t idx, const BWT* pBWT) const { size_t offset = 0; SAElem elem; while(1) { // Check if this position is sampled. If the sample rate is zero we are using the lexo. index only if(m_sampleRate > 0 && idx % m_sampleRate == 0 && !m_saSamples[idx / m_sampleRate].isEmpty()) { // A valid sample is stored for this idx elem = m_saSamples[idx / m_sampleRate]; break; } // A sample does not exist for this position, perform a backtracking step char b = pBWT->getChar(idx); idx = pBWT->getPC(b) + pBWT->getOcc(b, idx - 1); if(b == '$') { // idx (before the update) corresponds to the start of a read. // We can directly look up the saElem for idx from the lexicographic index assert(idx < (int64_t)m_saLexoIndex.size()); elem.setID(m_saLexoIndex[idx]); elem.setPos(0); break; } else { // A backtracking step is performed, increment offset offset += 1; } } elem.setPos(elem.getPos() + offset); return elem; } // Returns the ID of the read with lexicographic rank r size_t SampledSuffixArray::lookupLexoRank(size_t r) const { return m_saLexoIndex[r]; } // void SampledSuffixArray::build(const BWT* pBWT, const ReadInfoTable* pRIT, int sampleRate) { m_sampleRate = sampleRate; size_t numStrings = pRIT->getCount(); m_saLexoIndex.resize(numStrings); size_t MAX_ELEMS = std::numeric_limits::max(); if(numStrings > MAX_ELEMS) { std::cerr << "Error: Only " << MAX_ELEMS << " reads are allowed in the sampled suffix array\n"; std::cerr << "Number of reads in your index: " << numStrings << "\n"; std::cerr << "Contact sga-users@googlegroups.com for help\n"; exit(EXIT_FAILURE); } // Set the size of the sampled vector size_t numElems = (pBWT->getBWLen() / m_sampleRate) + 1; m_saSamples.resize(numElems); // For each read, start from the end of the read and backtrack through the suffix array/BWT. // For every idx that is divisible by the sample rate, store the calculate SAElem for(size_t i = 0; i < numStrings; ++i) { // The suffix array positions for the ends of reads are ordered // by their position in the read information table, therefore // the starting suffix array index is i size_t idx = i; // The ID of the read is i. The position coordinate is inclusive but // since the read information table does not store the '$' symbol // the starting position equals the read length SAElem elem(i, pRIT->getReadLength(i)); while(1) { if(idx % m_sampleRate == 0) { // store this SAElem m_saSamples[idx / m_sampleRate] = elem; } char b = pBWT->getChar(idx); idx = pBWT->getPC(b) + pBWT->getOcc(b, idx - 1); if(b == '$') { // we have hit the beginning of this string // store the SAElem for the beginning of the read // in the lexicographic index if(elem.getPos() != 0) std::cout << "elem: " << elem << " i: " << i << "\n"; assert(elem.getPos() == 0); size_t id = elem.getID(); assert(id < MAX_ELEMS); m_saLexoIndex[idx] = id; break; // done; } else { // Decrease the position of the elem elem.setPos(elem.getPos() - 1); } } } } // A streamlined version of the above function void SampledSuffixArray::buildLexicoIndex(const BWT* pBWT, int num_threads) { int64_t numStrings = pBWT->getNumStrings(); m_saLexoIndex.resize(numStrings); int64_t MAX_ELEMS = std::numeric_limits::max(); assert(numStrings < MAX_ELEMS); (void)num_threads; // Parallelize this computaiton using openmp, if the compiler supports it #if HAVE_OPENMP omp_set_num_threads(num_threads); #pragma omp parallel for #endif for(int64_t read_idx = 0; read_idx < numStrings; ++read_idx) { // For each read, start from the end of the read and backtrack through the suffix array/BWT // to calculate its lexicographic rank in the collection size_t idx = read_idx; while(1) { char b = pBWT->getChar(idx); idx = pBWT->getPC(b) + pBWT->getOcc(b, idx - 1); if(b == '$') { // There is a one-to-one mapping between read_index and the element // of the array that is set - therefore we can perform this operation // without a lock. m_saLexoIndex[idx] = read_idx; break; // done; } } } } // Validate the sampled suffix array values are correct /*void SampledSuffixArray::validate(const std::string filename, const BWT* pBWT) { ReadTable* pRT = new ReadTable(filename); SuffixArray* pSA = new SuffixArray(pRT, 1); std::cout << "Validating sampled suffix array entries\n"; for(size_t i = 0; i < pSA->getSize(); ++i) { SAElem calc = calcSA(i, pBWT); SAElem real = pSA->get(i); if(calc.getID() != real.getID() || calc.getPos() != real.getPos()) { std::cout << "Error SA elements do not match for " << i << "\n"; std::cout << "Calc: " << calc << "\n"; std::cout << "Real: " << real << "\n"; exit(1); } } std::cout << "All calculate SA values are correct\n"; delete pRT; delete pSA; } */ // Save the SA to disc void SampledSuffixArray::writeSSA(std::string filename) { std::ostream* pWriter = createWriter(filename, std::ios::out | std::ios::binary); // Write a magic number SSA_WRITE(SSA_MAGIC_NUMBER) // Write sample rate SSA_WRITE(m_sampleRate) // Write number of lexicographic index entries size_t n = m_saLexoIndex.size(); SSA_WRITE(n) // Write lexo index SSA_WRITE_N(m_saLexoIndex.front(), sizeof(SSA_INT_TYPE) * n) // Write number of samples n = m_saSamples.size(); SSA_WRITE(n) // Write samples SSA_WRITE_N(m_saSamples.front(), sizeof(SAElem) * n) delete pWriter; } // Save just the lexicographic index portion of the SSA to disk as plaintext void SampledSuffixArray::writeLexicoIndex(const std::string& filename) { SAWriter writer(filename); size_t num_strings = m_saLexoIndex.size(); writer.writeHeader(num_strings, num_strings); for(size_t i = 0; i < m_saLexoIndex.size(); ++i) { SAElem elem(m_saLexoIndex[i], 0); writer.writeElem(elem); } } void SampledSuffixArray::readSSA(std::string filename) { std::istream* pReader = createReader(filename, std::ios::binary); // Write a magic number uint32_t magic = 0; SSA_READ(magic) assert(magic == SSA_MAGIC_NUMBER); // Read sample rate SSA_READ(m_sampleRate) // Read number of lexicographic index entries size_t n = 0; SSA_READ(n) m_saLexoIndex.resize(n); // Read lexo index SSA_READ_N(m_saLexoIndex.front(), sizeof(SSA_INT_TYPE) * n) // Read number of samples n = 0; SSA_READ(n) m_saSamples.resize(n); // Read samples SSA_READ_N(m_saSamples.front(), sizeof(SAElem) * n) delete pReader; } void SampledSuffixArray::readSAI(std::string filename) { SAReader reader(filename); size_t num_strings, num_elems; reader.readHeader(num_strings, num_elems); assert(num_strings == num_elems); m_saLexoIndex.reserve(num_strings); reader.readElems(m_saLexoIndex); // Set the sample rate to zero to signify there are no samples m_sampleRate = 0; } // Print memory usage information void SampledSuffixArray::printInfo() const { double mb = (double)(1024*1024); double lexoSize = (double)(sizeof(SSA_INT_TYPE) * m_saLexoIndex.capacity()) / mb; double sampleSize = (double)(sizeof(SAElem) * m_saSamples.capacity()) / mb; printf("SampledSuffixArray info:\n"); printf("Sample rate: %d\n", m_sampleRate); printf("Contains %zu entries in lexicographic array (%.1lf MB)\n", m_saLexoIndex.size(), lexoSize); printf("Contains %zu entries in sample array (%.1lf MB)\n", m_saSamples.size(), sampleSize); printf("Total size: %.1lf\n", lexoSize + sampleSize); } ================================================ FILE: src/SGA/SuffixTools/SampledSuffixArray.h ================================================ //----------------------------------------------- // Copyright 2011 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // SampledSuffixArray - Data structure holding // a subset of suffix array positions. From the // sampled positions, the other entries of the // suffix array can be calculated. // #ifndef SAMPLED_SUFFIX_ARRAY #define SAMPLED_SUFFIX_ARRAY #include "SuffixArray.h" #include "BWT.h" #include "ReadInfoTable.h" typedef uint32_t SSA_INT_TYPE; enum SSAFileType { SSA_FT_SSA, SSA_FT_SAI }; class SampledSuffixArray { public: SampledSuffixArray(); SampledSuffixArray(const std::string& filename, SSAFileType filetype = SSA_FT_SSA); // Calculate the suffix array element for the given index SAElem calcSA(int64_t idx, const BWT* pBWT) const; // Returns the ID of the read with lexicographic rank r size_t lookupLexoRank(size_t r) const; // Construct the sampled SA using the bwt of a set of reads and their lengths void build(const BWT* pBWT, const ReadInfoTable* pRIT, int sampleRate = DEFAULT_SA_SAMPLE_RATE); // Construct the lexicographic index (.sai) from the BWT void buildLexicoIndex(const BWT* pBWT, int num_threads); // Validate using the full suffix array for the given set of reads. Very slow. void validate(std::string readsFile, const BWT* pBWT); void printInfo() const; // I/O void writeLexicoIndex(const std::string& filename); void writeSSA(std::string filename); void readSSA(std::string filename); void readSAI(std::string filename); private: // Unsigned integers indicating the start of every read in the // sequence collection. These elements are in lexicographic order // based on the whole read sequence. Tracing a read backwards through // the suffix array necessarily ends at one of these positions. These // are nominally SAElems representing the full length suffix but // we store them here as unsigned integers to save 4 bytes per entry. // This limits the SampledSuffixArray to represent at most 2**32 strings. // To improve this we could use a polymorphic vector with a runtime-determined // size. std::vector m_saLexoIndex; static const int DEFAULT_SA_SAMPLE_RATE = 64; int m_sampleRate; SAElemVector m_saSamples; }; #endif ================================================ FILE: src/SGA/SuffixTools/SparseGapArray.h ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // SparseGapArray // // The sparse gap array uses a small integer type to // keep the count for each element. A small number // of positions will have a count that is larger // than the maximum value the integer can represent. // These values are automatically overflowed into a // hash map with a larger integer type. In the typical // case this will require far less memory than the // SimpleGapArray. The storage unit is templated // to allow a dynamic tradeoff between the amount // of memory required to store the simple cases // and the overflow rate. // #ifndef SPARSEGAPARRAY_H #define SPARSEGAPARRAY_H #include "HashMap.h" #include "GapArray.h" #include "BitVector.h" // Template base storage for the sparse gap array template class SparseBaseStorage { public: // void resize(size_t n) { m_data.resize(n); } // inline void set(size_t i, IntType c) { m_data[i] = c; } // Attempt to set the value using an atomic update // Returns false if the update fails inline bool setCAS(size_t i, IntType oldV, IntType newV) { assert(oldV != getMax() && newV <= getMax() && oldV != newV); bool success = __sync_bool_compare_and_swap(&m_data[i], oldV, newV); return success; } // inline IntType get(size_t i) const { return m_data[i]; } inline static size_t getMax() { static size_t max = std::numeric_limits::max(); return max; } // size_t size() const { return m_data.size(); } private: // typedef std::vector BaseStorageVector; BaseStorageVector m_data; }; static const uint8_t storage4_offset_mask[]={0xF0,0x0F}; // 11110000, 00001111 static const uint8_t storage4_shift[]={4, 0}; // specialized 4-bit per value base storage for the sparse gap array class SparseBaseStorage4 { public: SparseBaseStorage4() : m_numElems(0) {} // void resize(size_t n) { // With 4 bits per entry, we need n / 2 bytes of storage size_t numBytes = ((n & 1) == 0) ? n / 2 : (n / 2) + 1; m_data.resize(numBytes); m_numElems = n; } // inline void set(size_t i, uint8_t c) { assert(i < m_numElems); assert(c <= getMax()); size_t idx = i >> 1; // equivalent to i / 2 size_t offset = i & 1; // 0 if even, 1 if odd uint8_t mask = storage4_offset_mask[offset]; uint8_t& elem = m_data[idx]; // Clear current value elem &= ~mask; // Set new value elem |= c << storage4_shift[offset]; } // Attempt to set the value using an atomic update // Returns false if the update fails inline bool setCAS(size_t i, uint8_t oldV, uint8_t newV) { assert(i < m_numElems); assert(oldV < getMax() && newV <= getMax()); assert(oldV != newV); size_t idx = i >> 1; // equivalent to i / 2 size_t offset = i & 1; // 0 if even, 1 if odd uint8_t mask = storage4_offset_mask[offset]; uint8_t oldElem = m_data[idx]; // If the count in the stored element has changed since this // function was called, return false uint8_t currV = (oldElem & mask) >> storage4_shift[offset]; if(currV != oldV) return false; // Calculate the new element to swap in uint8_t newElem = oldElem; newElem &= ~mask; newElem |= newV << storage4_shift[offset]; // Perform the update bool success = __sync_bool_compare_and_swap(&m_data[idx], oldElem, newElem); return success; } // inline uint8_t get(size_t i) const { assert(i < m_numElems); size_t idx = i >> 1; // equivalent to i / 2 size_t offset = i & 1; // 0 if even, 1 if odd uint8_t mask = storage4_offset_mask[offset]; uint8_t elem = m_data[idx]; elem &= mask; elem >>= storage4_shift[offset]; assert(elem <= getMax()); return elem; } inline static size_t getMax() { return 15; } // size_t size() const { return m_numElems; } private: // typedef std::vector BaseStorageVector; BaseStorageVector m_data; size_t m_numElems; }; // specialized 1-bit per value base storage for the sparse gap array // used for removing entries from an fm-index when the count // will not exceed 1. class SparseBaseStorage1 { public: SparseBaseStorage1() : m_numElems(0) {} // void resize(size_t n) { m_numElems = n; m_bitVector.resize(n); } // Set bit i from oldV to newV using a compare and swap // Returns false if the update fails inline bool setCAS(size_t i, uint8_t oldV, uint8_t newV) { assert(i < m_numElems); assert(oldV == 0); assert(newV == 1); return m_bitVector.updateCAS(i, oldV, newV); } // inline void set(size_t i, uint8_t c) { assert(i < m_numElems); assert(c <= getMax()); m_bitVector.set(i, true); } // inline uint8_t get(size_t i) const { assert(i < m_numElems); return m_bitVector.test(i) ? 1 : 0; } inline static size_t getMax() { return 1; } // size_t size() const { return m_numElems; } private: // BitVector m_bitVector; size_t m_numElems; }; // The SparseGapArray has two levels of storage. // The first level uses x bits to store counts // up to 2**x for n elements in the array. // If the count for a particular element exceeds 2**x // then an entry in the overflow hash table is created // allowing arbitrary values to be stored. This // class is optimized to allow the base storage to be updated // concurrently with compare and swap operations. Any overflowed // updates must be serialized though incrementOverflowSerial // template class SparseGapArray : public GapArray { public: SparseGapArray() : m_rankZeroCount(0) {} ~SparseGapArray() { //printf("SparseGapArray -- n: %zu overflow: %zu (%lf)\n", m_baseStorage.size(), m_overflow.size(), (double)m_overflow.size() / m_baseStorage.size()); } // void resize(size_t n) { m_baseStorage.resize(n); } // Attempt to increment a value in the GapArray using a compare and swap function // This call can fail to perform the update and return false. In this case // the calling code is responsible for serializing access to this data structure // and calling incrementSerial bool attemptBaseIncrement(size_t i) { assert(i < m_baseStorage.size()); bool success = false; // Rank zero optimization // When merging two indices, all reads // start at rank 0. This would cause many serial // updates to the overflow array so we optimize // for this case by doing a compare and swap update // of a large integer value if the rank is zero. if(i == 0) { do { size_t count = m_rankZeroCount; success = __sync_bool_compare_and_swap(&m_rankZeroCount, count, count+1); } while(!success); return true; } success = false; do { size_t count = m_baseStorage.get(i); if(count == getBaseMax()) return false; success = m_baseStorage.setCAS(i, count, count + 1); } while(!success); return success; } // Increment the value for rank i in the overflow array. This call must be serialized // between any threads. void incrementOverflowSerial(size_t i) { assert(i != 0); assert(i < m_baseStorage.size()); size_t count = m_baseStorage.get(i); assert(count == getBaseMax()); // Check if the overflow map has a value for this index already // if not, enter one if(m_overflow.count(i) == 0) initOverflow(i, count); incrementOverflow(i); } // void initOverflow(size_t i, OverflowStorage c) { m_overflow.insert(std::make_pair(i, c)); } // void incrementOverflow(size_t i) { // precondition: there must be an entry in the hash for this key assert(m_overflow.count(i) != 0); ++m_overflow[i]; } // size_t get(size_t i) const { // rank zero optimization if(i == 0) return m_rankZeroCount; size_t count = m_baseStorage.get(i); if(count == getBaseMax()) { typename OverflowHash::const_iterator iter = m_overflow.find(i); // If there is no entry in the overflow table yet // the count is exactly the maximum value representable // in the base storage if(iter == m_overflow.end()) return count; else return iter->second; } else { return count; } } // size_t getBaseMax() const { return BaseStorage::getMax(); } // size_t size() const { return m_baseStorage.size(); } private: typedef SparseHashMap OverflowHash; OverflowHash m_overflow; BaseStorage m_baseStorage; size_t m_rankZeroCount; }; typedef SparseGapArray SparseGapArray1; typedef SparseGapArray SparseGapArray4; typedef SparseGapArray, size_t> SparseGapArray8; typedef SparseGapArray, size_t> SparseGapArray16; typedef SparseGapArray, size_t> SparseGapArray32; #endif ================================================ FILE: src/SGA/SuffixTools/SuffixArray.cpp ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // SuffixArray - Generalized suffix array // #include "SuffixArray.h" #include "InverseSuffixArray.h" #include "bucketSort.h" #include "SuffixCompare.h" #include "mkqs.h" #include "SACAInducedCopying.h" #include "Timer.h" #include "SAReader.h" #include "SAWriter.h" #include "BWTWriter.h" // Read a suffix array from a file SuffixArray::SuffixArray(const std::string& filename) { SAReader reader(filename); reader.read(this); } // Construct the suffix array for a table of reads SuffixArray::SuffixArray(const ReadTable* pRT, int numThreads, bool silent) { //Timer timer("SuffixArray Construction", silent); saca_induced_copying(this, pRT, numThreads, silent); } // Initialize a suffix array for the strings in RT void SuffixArray::initialize(const ReadTable& rt) { size_t n = rt.countSumLengths() + rt.getCount(); initialize(n, rt.getCount()); // Fill the data table with the linear ordering of the suffixes size_t count = 0; for(size_t i = 0; i < rt.getCount(); ++i) { // + 1 below is for the empty suffix (is it actually needed?) for(size_t j = 0; j < rt.getRead(i).seq.length() + 1; ++j) { m_data[count++] = SAElem(i, j); } } } void SuffixArray::initialize(size_t num_suffixes, size_t num_strings) { m_data.resize(num_suffixes); m_numStrings = num_strings; } // Validate the suffix array using the read table void SuffixArray::validate(const ReadTable* pRT) const { size_t maxIdx = pRT->getCount(); size_t n = m_data.size(); // Exit if there is nothing to do if(n == 0) return; // Compute the ISA InverseSuffixArray isa(*this); // Validate the ISA is a permutation of 1..n, this implies that the id,pos pairs of the SA are valid isa.validate(); size_t empty_count = 0; // Ensure that the suffix at pos i is lexographically lower than the suffix at i + 1 using the full string for(size_t i = 0; i < n - 1; ++i) { SAElem id1 = m_data[i]; SAElem id2 = m_data[i+1]; assert(id1.getID() < maxIdx); std::string suffix1 = pRT->getRead(id1.getID()).seq.getSuffixString(id1.getPos()); std::string suffix2 = pRT->getRead(id2.getID()).seq.getSuffixString(id2.getPos()); if(suffix1.length() == 1) ++empty_count; bool suffixValidated = true; if(suffix1 == suffix2) { suffixValidated = id1.getID() < id2.getID(); } else { suffixValidated = suffix1 < suffix2; } if(!suffixValidated) { std::cerr << "Validation failure: " << suffix1 << " is not less than " << suffix2 << " ids: " << id1.getID() << "," << id2.getID() << "\n"; assert(suffix1 < suffix2); } } assert(m_numStrings == empty_count); } // void SuffixArray::removeReads(const NumericIDSet& idSet) { // Early exit if the idset is empty if(idSet.empty()) return; SAElemVector newData; newData.reserve(m_data.size()); for(size_t idx = 0; idx < m_data.size(); ++idx) { SAElem id = m_data[idx]; if(idSet.find(id.getID()) == idSet.end()) { // not on the delete list newData.push_back(id); } } m_data.swap(newData); m_numStrings -= idSet.size(); } // Get the suffix cooresponding to idx using the read table std::string SuffixArray::getSuffix(size_t idx, const ReadTable* pRT) const { SAElem id = m_data[idx]; return pRT->getRead(id.getID()).seq.getSuffixString(id.getPos()); } // Return the length of the suffix corresponding to elem size_t SuffixArray::getSuffixLength(const ReadTable* pRT, const SAElem elem) const { size_t readLength = pRT->getReadLength(elem.getID()); return readLength - elem.getPos(); } // Print the suffix array void SuffixArray::print(const ReadTable* pRT) const { std::cout << "i\tSA(i)\n"; for(size_t i = 0; i < m_data.size(); ++i) { SAElem id1 = m_data[i]; std::string suffix = !id1.isEmpty() ? getSuffix(i, pRT) : ""; int pos = id1.getPos(); char b; if(pos == 0) b = '$'; else b = pRT->getRead(id1.getID()).seq.get(pos - 1); std::cout << i << "\t" << id1 << "\t" << b << "\t" << suffix << "\n"; } } // Print the suffix array void SuffixArray::print() const { std::cout << "i\tSA(i)\n"; for(size_t i = 0; i < m_data.size(); ++i) { std::cout << i << "\t" << m_data[i] << "\n"; } } // write the suffix array to a file void SuffixArray::write(const std::string& filename) { SAWriter writer(filename); writer.write(this); } // write the suffix array to a file void SuffixArray::writeBWT(const std::string& filename, const ReadTable* pRT) { //RLBWTWriter writer(filename); IBWTWriter* pWriter = BWTWriter::createWriter(filename); pWriter->write(this, pRT); delete pWriter; } // write the index of the suffix array to a file void SuffixArray::writeIndex(std::string& filename) { SAWriter writer(filename); writer.writeHeader(m_numStrings, m_numStrings); for(size_t i = 0; i < m_data.size(); ++i) { if(m_data[i].isFull()) writer.writeElem(m_data[i]); } } /*template inline std::vector erase_indices(const std::vector& data, std::vector& indicesToDelete) { if(indicesToDelete.empty()) return data; std::vector ret; ret.reserve(data.size() - indicesToDelete.size()); std::sort(indicesToDelete.begin(), indicesToDelete.end()); // new we can assume there is at least 1 element to delete. copy blocks at a time. std::vector::const_iterator itBlockBegin = data.begin(); for(std::vector::const_iterator it = indicesToDelete.begin(); it != indicesToDelete.end(); ++ it) { std::vector::const_iterator itBlockEnd = data.begin() + *it; if(itBlockBegin != itBlockEnd) { std::copy(itBlockBegin, itBlockEnd, std::back_inserter(ret)); } itBlockBegin = itBlockEnd + 1; } // copy last block. if(itBlockBegin != data.end()) { std::copy(itBlockBegin, data.end(), std::back_inserter(ret)); } return ret; }*/ //JEREMIAH void SuffixArray::writeIndex() { SAElemVector sae; for(int i = 1; i < m_data.size(); ++i) { if(m_data[i].isFull()) sae.push_back(m_data[i]); } m_data = sae; } // Output operator std::ostream& operator<<(std::ostream& out, const SuffixArray& sa) { // Write the size and number of strings out << sa.m_data.size() << "\n"; out << sa.m_numStrings << "\n"; for(size_t i = 0; i < sa.m_data.size(); ++i) { out << sa.m_data[i] << "\n"; } return out; } // Input operator std::istream& operator>>(std::istream& in, SuffixArray& sa) { // Read the size and number of strings size_t n; in >> n; in >> sa.m_numStrings; sa.m_data.resize(n); size_t i = 0; SAElem id; while(in >> id) sa.m_data[i++] = id; assert(i == n); return in; } ================================================ FILE: src/SGA/SuffixTools/SuffixArray.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL license //----------------------------------------------- // // SuffixArray - Generalized suffix array // #ifndef SUFFIXARRAY_H #define SUFFIXARRAY_H #include "STCommon.h" #include "ReadTable.h" #include "Match.h" class LCPArray; class SuffixArray { public: // SuffixArray() {} SuffixArray(const std::string& filename); SuffixArray(const ReadTable* pRT, int numThreads, bool silent = false); // Construction/Validation functions void initialize(const ReadTable& rt); void initialize(size_t num_suffixes, size_t num_strings); void validate(const ReadTable* pRT) const; void sort(const ReadTable* pRT); // Remove all the suffixes from the SA that have an id in idSet void removeReads(const NumericIDSet& idSet); // Simple accessors inline const SAElem& get(size_t idx) const { assert(idx < m_data.size()); return m_data[idx]; } inline void set(size_t idx, SAElem e) { m_data[idx] = e; } size_t getSize() const { return m_data.size(); } size_t getNumStrings() const { return m_numStrings; } std::string getSuffix(size_t idx, const ReadTable* pRT) const; size_t getSuffixLength(const ReadTable* pRT, const SAElem elem) const; // Operators friend std::ostream& operator<<(std::ostream& out, const SuffixArray& sa); friend std::istream& operator>>(std::istream& in, SuffixArray& sa); // Output the entire suffix array void write(const std::string& filename); // Write the BWT directly to disk void writeBWT(const std::string& filename, const ReadTable* pRT); // Output the suffix array index // The suffix array index are the full-length suffixes (the entire string) // of the suffix array, sorted into lexographical order // The file format is the same as the suffix array // This is used during the BWT indexing and is the only part of the // suffix array that we need void writeIndex(std::string& filename); void writeIndex(); //JEREMIAH // Print funcs void print() const; void print(const ReadTable* pRT) const; // friends friend void saca_induced_copying(SuffixArray* pSA, const ReadTable* pRT, int numThreads, bool silent); friend class SAReader; friend class SAWriter; private: // Data members SAElemVector m_data; size_t m_numStrings; }; #endif ================================================ FILE: src/SGA/SuffixTools/SuffixCompare.cpp ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // SuffixCompareRadix // #include "SuffixCompare.h" // SuffixCompareRadix::SuffixCompareRadix(const ReadTable* pRT) : m_pRT(pRT), m_pNumSuffixLUT(0) { m_bucketOffset = 0; m_bucketLen = 6; initializeNumSuffixLUT(); } // SuffixCompareRadix::SuffixCompareRadix(const ReadTable* pRT, int bucket_len) : m_pRT(pRT), m_bucketOffset(0), m_bucketLen(bucket_len), m_pNumSuffixLUT(0) { if(m_bucketOffset % m_bucketLen != 0) { std::cerr << "Error, bucket offset must be a multiple of bucket length\n"; assert(false); } initializeNumSuffixLUT(); } // SuffixCompareRadix::~SuffixCompareRadix() { delete [] m_pNumSuffixLUT; } void SuffixCompareRadix::initializeNumSuffixLUT() { m_pNumSuffixLUT = new int[m_bucketLen]; for(int i = 0; i < (int)m_bucketLen; ++i) { m_pNumSuffixLUT[i] = calcNumSuffixes(i); } } // Get the bucket for a particular SAElem int SuffixCompareRadix::getBucket(SAElem x) const { //std::cout << "Finding bucket for " << x << "\n"; const DNAString& read = m_pRT->getRead(x.getID()).seq; size_t suffix_start = x.getPos() + m_bucketOffset; const char* suffix = read.getSuffix(suffix_start); size_t suffix_len = read.getSuffixLength(suffix_start); size_t stop = std::min(m_bucketLen, suffix_len); int rank = 0; for(size_t i = 0; i < stop; ++i) { char b = suffix[i]; rank += numPredSuffixes(b, m_bucketLen - i); } return rank; } // void SuffixCompareRadix::printElem(SAElem& x) const { const DNAString& read = m_pRT->getRead(x.getID()).seq; std::cout << x << " = " << read.getSuffixString(x.getPos()) << "\n"; } // void SuffixCompareRadix::setBucketDepth(int depth) { m_bucketOffset = m_bucketLen * depth; } // int SuffixCompareRadix::calcNumSuffixes(int maxLen) const { int r = 0; for(int i = 0; i <= maxLen; ++i) r += (1 << 2*i); return r; } bool SuffixCompareRadix::isBucketDegenerate(int index) const { if(index == 0) return true; else { for(int i = m_bucketLen; i >= 2; --i) { --index; int block_size = calcNumSuffixes(i - 1); if(index % block_size == 0) return true; else { int block = index / block_size; index -= block * block_size; } } } return false; } // int SuffixCompareRadix::getNumBuckets() const { return calcNumSuffixes(m_bucketLen); } // Compare two suffixes by ID // Precondition: the suffixes are sorted by sequence already bool SuffixCompareID::operator()(SAElem x, SAElem y) const { const SeqItem& rx = m_pRT->getRead(x.getID()); const SeqItem& ry = m_pRT->getRead(y.getID()); return rx.id < ry.id; } ================================================ FILE: src/SGA/SuffixTools/SuffixCompare.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL license //----------------------------------------------- // // SuffixCompare - Suffix Comparator classes // Used for histogram/bucket sort, mkqs and std::sort // #ifndef SUFFIXCOMPARE_H #define SUFFIXCOMPARE_H #include "STCommon.h" #include "ReadTable.h" // Suffix comparator object for radix-like sorts (bucket/histogram and MKQS) class SuffixCompareRadix { public: SuffixCompareRadix(const ReadTable* pRT); SuffixCompareRadix(const ReadTable* pRT, int bucket_len); ~SuffixCompareRadix(); // void initializeNumSuffixLUT(); // Bucket function int getBucket(SAElem x) const; // Get the character at position d for the SAElem inline char getChar(SAElem& x, int d) const { return m_pRT->getChar(x.getID(), x.getPos() + d); } // Calculate the number of possible suffixes int calcNumSuffixes(int maxLen) const; // Get the number of possible suffixes of length maxLen using the lookup table inline int getNumSuffixes(int maxLen) const { return m_pNumSuffixLUT[maxLen]; } // Get the suffix character string corresponding to this element inline const char* getChrPtr(SAElem& x) const { return m_pRT->getRead(x.getID()).seq.getSuffix(x.getPos()); } // Calculate the number of suffixes that precede the first instance of b for a // given maximum suffix length inline int numPredSuffixes(char b, int maxLen) const { // base case int rb = getBaseRank(b); if(rb == 0) return 0; int block_size = getNumSuffixes(maxLen - 1); return block_size * (rb - 1) + 1; } // Return the number of buckets needed for bucket sort int getNumBuckets() const; // Return the bucket offset size_t getBucketOffset() const { return m_bucketOffset; } // Get the bucket length size_t getBucketLen() const { return m_bucketLen; } // Set the offset void setBucketDepth(int depth); // Return true if a bucket is degenerate ie it cannot be subdivided any further bool isBucketDegenerate(int index) const; // Print the element void printElem(SAElem& x) const; private: // Disallow default and copy constructor SuffixCompareRadix() {} SuffixCompareRadix(const SuffixCompareRadix& /*other*/) { assert(false); } const ReadTable* m_pRT; size_t m_bucketOffset; size_t m_bucketLen; int* m_pNumSuffixLUT; }; // Compare two suffixes by their ID // This is used for the final pass, after suffixes has been compared by sequence class SuffixCompareID { public: SuffixCompareID(const ReadTable* pRT) : m_pRT(pRT) {} // Comparator function bool operator()(SAElem x, SAElem y) const; private: // default is not accessible SuffixCompareID() : m_pRT(0) {} const ReadTable* m_pRT; }; // Compare two suffixes by their index in the read table // This is used for the final pass, after suffixes has been compared by sequence class SuffixCompareIndex { public: SuffixCompareIndex() {} // Comparator function inline bool operator()(SAElem x, SAElem y) const { return x.getID() < y.getID(); } private: }; #endif ================================================ FILE: src/SGA/Util/Alphabet.cpp ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // Alphabet.h - Abstraction of the alphabet // that is used in the suffix array, fm-index, // etc data structures // #include "Alphabet.h" #include #include // IUPAC alphabet bool IUPAC::isUnambiguous(char c) { switch(c) { case 'A': case 'C': case 'G': case 'T': return true; default: return false; } } // Returns true if c is a valid ambiguity code bool IUPAC::isAmbiguous(char c) { switch(c) { case 'M': case 'R': case 'W': case 'S': case 'Y': case 'K': case 'V': case 'H': case 'D': case 'B': case 'N': return true; default: return false; } } // Returns true if c is a valid symbol in this alphabet bool IUPAC::isValid(char c) { return isUnambiguous(c) || isAmbiguous(c); } // std::string IUPAC::getPossibleSymbols(char c) { switch(c) { case 'A': return "A"; case 'C': return "C"; case 'G': return "G"; case 'T': return "T"; case 'M': return "AC"; case 'R': return "AG"; case 'W': return "AT"; case 'S': return "CG"; case 'Y': return "CT"; case 'K': return "GT"; case 'V': return "ACG"; case 'H': return "ACT"; case 'D': return "AGT"; case 'B': return "CGT"; case 'N': return "ACGT"; default: return ""; } } // AlphaCount template<> const size_t AlphaCount8::maxValue = std::numeric_limits::max(); template<> const size_t AlphaCount16::maxValue = std::numeric_limits::max(); template<> const size_t AlphaCount64::maxValue = std::numeric_limits::max(); ================================================ FILE: src/SGA/Util/Alphabet.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // Alphabet.h - Abstraction of the alphabet // that is used in the suffix array, fm-index, // etc data structures // #ifndef ALPHABET_H #define ALPHABET_H //#define USE_SSE 1 //#define ALPHACOUNT_VALIDATE 1 #include #include #include #include #if USE_SSE #include #include #endif #include #include #include #include "Util.h" // // Constants // // TODO: Refactor these into a namespaced alphabet like DNA_ALPHABET below const uint8_t ALPHABET_SIZE = 5; const char ALPHABET[ALPHABET_SIZE] = {'A', 'C', 'G', 'T', '$'}; const char RANK_ALPHABET[ALPHABET_SIZE] = {'$', 'A', 'C', 'G', 'T'}; const uint8_t DNA_ALPHABET_SIZE = 4; typedef uint64_t BaseCount; namespace DNA_ALPHABET { static const uint8_t s_dnaLexoRankLUT[256] = { 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,1,0,0,0,2,0,0,0,0,0,0,0,0, 0,0,0,0,3,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0 }; inline static uint8_t getBaseRank(char b) { return s_dnaLexoRankLUT[static_cast(b)]; } inline char getBase(size_t idx) { assert(idx < DNA_ALPHABET_SIZE); return ALPHABET[idx]; } static const uint8_t size = 4; }; namespace BWT_ALPHABET { static const uint8_t s_bwtLexoRankLUT[256] = { 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,1,0,2,0,0,0,3,0,0,0,0,0,0,0,0, 0,0,0,0,4,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0 }; static const uint8_t size = 5; inline static uint8_t getRank(char b) { return s_bwtLexoRankLUT[static_cast(b)]; } inline char getChar(size_t idx) { return RANK_ALPHABET[idx]; } }; // IUPAC ambiguity alphabet namespace IUPAC { // Returns true if c is [ACGT] bool isUnambiguous(char c); // Returns true if c is a valid ambiguity code bool isAmbiguous(char c); // Returns true if c is a valid symbol in this alphabet bool isValid(char c); // Returns a string defining the possible unambiguous bases for each symbol // in the alphabet std::string getPossibleSymbols(char c); }; // // A simple class holding the count for each base of a DNA string (plus the terminator) // Note that this uses RANK_ALPHABET template class AlphaCount { public: // inline AlphaCount() { clear(); } inline void clear() { memset(m_counts, 0, ALPHABET_SIZE * sizeof(Storage)); } // inline void set(char b, Storage v) { m_counts[getBaseRank(b)] = v; } // inline void setByIdx(size_t i, Storage v) { m_counts[i] = v; } // inline void increment(char b) { int br = getBaseRank(b); #ifdef ALPHACOUNT_VALIDATE assert(m_counts[br] != maxValue); #endif m_counts[br]++; } // inline void add(char b, Storage v) { int br = getBaseRank(b); #ifdef ALPHACOUNT_VALIDATE assert(m_counts[br] + v <= maxValue); #endif m_counts[br] += v; } // inline void subtract(char b, Storage v) { int br = getBaseRank(b); #ifdef ALPHACOUNT_VALIDATE assert(m_counts[br] > v); #endif m_counts[br] -= v; } // inline Storage get(char b) const { return m_counts[getBaseRank(b)]; } // inline Storage getByIdx(const int i) const { return m_counts[i]; } // Return the base for index i static char getBase(size_t i) { return RANK_ALPHABET[i]; } // Return the maximum possible count for a symbol static size_t getMaxValue() { return maxValue; } // Swap the (A,T) and (C,G) entries, which turns the AlphaCount // into the AlphaCount for the complemented sequence inline void complement() { Storage tmp; // A,T tmp = m_counts[4]; m_counts[4] = m_counts[1]; m_counts[1] = tmp; // C,G tmp = m_counts[3]; m_counts[3] = m_counts[2]; m_counts[2] = tmp; } // Return the sum of the basecounts for characters lexo. lower than b inline size_t getLessThan(char b) const { size_t out = 0; int stop = getBaseRank(b); for(int i = 0; i < stop; ++i) out += m_counts[i]; return out; } // Returns the number of non-zero counts uint8_t getNumNonZero() const { uint8_t count = 0; for(int i = 0; i < ALPHABET_SIZE; ++i) { if(m_counts[i] > 0) count += 1; } return count; } // Returns true if only one of the DNA characters // has a non-zero count inline bool hasUniqueDNAChar() { // index 0 is the '$' character, which we skip bool nonzero = false; for(int i = 1; i < ALPHABET_SIZE; ++i) { if(m_counts[i] > 0) { // if nonzero is set, there is some other nonzero character, return false if(nonzero) return false; else nonzero = true; } } return nonzero; } // Returns true if any DNA char // has a non-zero count inline bool hasDNAChar() { // index 0 is the '$' character, which we skip for(int i = 1; i < ALPHABET_SIZE; ++i) { if(m_counts[i] > 0) return true; } return false; } // inline char getMaxBase() const { char base; Storage val; getMax(base, val); return base; } // inline char getMaxDNABase() const { Storage max = 0; int maxIdx = 0; for(int i = 1; i < ALPHABET_SIZE; ++i) { if(m_counts[i] > max) { max = m_counts[i]; maxIdx = i; } } assert(max > 0); return RANK_ALPHABET[maxIdx]; } // inline Storage getMaxCount() const { char base; Storage val; getMax(base, val); return val; } // inline void getMax(char& base, Storage& val) const { Storage max = 0; int maxIdx = 0; for(int i = 0; i < ALPHABET_SIZE; ++i) { if(m_counts[i] > max) { max = m_counts[i]; maxIdx = i; } } base = RANK_ALPHABET[maxIdx]; val = max; } // inline size_t getSum() const { size_t sum = m_counts[0]; sum += m_counts[1]; sum += m_counts[2]; sum += m_counts[3]; sum += m_counts[4]; return sum; } // Sort the DNA bases into count order and // write them to pOut. This does not include the '$' symbol inline void getSorted(char* pOut, size_t len) const { assert(len >= ALPHABET_SIZE); (void)len; for(size_t i = 0; i < ALPHABET_SIZE; ++i) pOut[i] = RANK_ALPHABET[i]; std::sort(pOut, pOut+ALPHABET_SIZE, AlphaCountCompareDesc(this)); } // Return the unique DNA character described by the alphacount // Returns '$' if no such character exists // Asserts if more than one character is described by the alphacount inline char getUniqueDNAChar() { char r = '$'; for(int i = 1; i < ALPHABET_SIZE; ++i) { if(m_counts[i] > 0) { assert(r == '$'); // lookup the character in the ranked alphabet, since the elements // are stored in lexographic order r = RANK_ALPHABET[i]; } } return r; } // Operators friend std::ostream& operator<<(std::ostream& out, const AlphaCount& ac) { std::copy(ac.m_counts, ac.m_counts+ALPHABET_SIZE, std::ostream_iterator(out, " ")); return out; } friend std::istream& operator>>(std::istream& in, AlphaCount& ac) { for(size_t i = 0; i < ALPHABET_SIZE; ++i) in >> ac.m_counts[i]; return in; } inline friend AlphaCount operator+(const AlphaCount& left, const AlphaCount& right) { AlphaCount out; out.m_counts[0] = left.m_counts[0] + right.m_counts[0]; out.m_counts[1] = left.m_counts[1] + right.m_counts[1]; out.m_counts[2] = left.m_counts[2] + right.m_counts[2]; out.m_counts[3] = left.m_counts[3] + right.m_counts[3]; out.m_counts[4] = left.m_counts[4] + right.m_counts[4]; return out; } inline AlphaCount& operator+=(const AlphaCount& other) { m_counts[0] += other.m_counts[0]; m_counts[1] += other.m_counts[1]; m_counts[2] += other.m_counts[2]; m_counts[3] += other.m_counts[3]; m_counts[4] += other.m_counts[4]; return *this; } // Specialization for add/subtracing a small AlphaCount to/from a large alphacount inline friend void alphacount_add(AlphaCount& lhs, const AlphaCount& rhs); inline friend void alphacount_subtract(AlphaCount& lhs, const AlphaCount& rhs); inline friend void alphacount_add16(AlphaCount& lhs, const AlphaCount& rhs); inline friend void alphacount_subtract16(AlphaCount& lhs, const AlphaCount& rhs); // As the counts are unsigned integers, each value in left // must be larger or equal to value in right. The calling function // must guarentee this. friend AlphaCount operator-(const AlphaCount& left, const AlphaCount& right) { AlphaCount out; out.m_counts[0] = left.m_counts[0] - right.m_counts[0]; out.m_counts[1] = left.m_counts[1] - right.m_counts[1]; out.m_counts[2] = left.m_counts[2] - right.m_counts[2]; out.m_counts[3] = left.m_counts[3] - right.m_counts[3]; out.m_counts[4] = left.m_counts[4] - right.m_counts[4]; return out; } inline friend bool operator==(const AlphaCount& left, const AlphaCount& right) { return left.m_counts[0] == right.m_counts[0] && left.m_counts[1] == right.m_counts[1] && left.m_counts[2] == right.m_counts[2] && left.m_counts[3] == right.m_counts[3] && left.m_counts[4] == right.m_counts[4]; } inline friend bool operator!=(const AlphaCount& left, const AlphaCount& right) { return !(left == right); } private: Storage m_counts[ALPHABET_SIZE]; const static size_t maxValue; struct AlphaCountCompareDesc { AlphaCountCompareDesc(const AlphaCount* p) : pAC(p) {} bool operator()(char a, char b) { return pAC->get(a) > pAC->get(b); } const AlphaCount* pAC; }; }; // Typedef commonly used AlphaCounts typedef AlphaCount AlphaCount64; typedef AlphaCount AlphaCount16; typedef AlphaCount AlphaCount8; // inline void alphacount_add16(AlphaCount64& lhs, const AlphaCount16& rhs) { lhs.m_counts[0] += rhs.m_counts[0]; lhs.m_counts[1] += rhs.m_counts[1]; lhs.m_counts[2] += rhs.m_counts[2]; lhs.m_counts[3] += rhs.m_counts[3]; lhs.m_counts[4] += rhs.m_counts[4]; } inline void alphacount_subtract16(AlphaCount64& lhs, const AlphaCount16& rhs) { // This function should only be used when lhs is larger the rhs // the calling function must guarentee this lhs.m_counts[0] -= rhs.m_counts[0]; lhs.m_counts[1] -= rhs.m_counts[1]; lhs.m_counts[2] -= rhs.m_counts[2]; lhs.m_counts[3] -= rhs.m_counts[3]; lhs.m_counts[4] -= rhs.m_counts[4]; } // inline void alphacount_add(AlphaCount64& lhs, const AlphaCount8& rhs) { lhs.m_counts[0] += rhs.m_counts[0]; lhs.m_counts[1] += rhs.m_counts[1]; lhs.m_counts[2] += rhs.m_counts[2]; lhs.m_counts[3] += rhs.m_counts[3]; lhs.m_counts[4] += rhs.m_counts[4]; } inline void alphacount_subtract(AlphaCount64& lhs, const AlphaCount8& rhs) { // This function should only be used when lhs is larger the rhs // the calling function must guarentee this lhs.m_counts[0] -= rhs.m_counts[0]; lhs.m_counts[1] -= rhs.m_counts[1]; lhs.m_counts[2] -= rhs.m_counts[2]; lhs.m_counts[3] -= rhs.m_counts[3]; lhs.m_counts[4] -= rhs.m_counts[4]; } #endif ================================================ FILE: src/SGA/Util/BWT4Codec.h ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // BWT4Codec - Encoder/decoder for a BWT // string over the alphabet $ACGT using 4 // bits per symbol // #ifndef BWT4CODEC_H #define BWT4CODEC_H #include "Alphabet.h" static const uint8_t bwt4_offset_mask[]={0xF0,0x0F}; static const uint8_t bwt4_offset_shift[]={4, 0}; class BWT4Codec { public: // The data is stored 2 characters per byte // The data pattern is: // 11110000 first symbol // 00001111 second symbol typedef uint8_t UNIT_TYPE; static const int SYMBOLS_PER_UNIT = 2; // Encoded the character b into a value inline uint8_t encode(char b) const { return BWT_ALPHABET::getRank(b); } // Decode the value c into a character inline char decode(uint8_t c) const { return BWT_ALPHABET::getChar(c); } // Returns the number of units required to encode // a string of length n inline size_t getRequiredUnits(size_t n) const { return (n + SYMBOLS_PER_UNIT - 1) / SYMBOLS_PER_UNIT; } // Returns the number of symbols that can be stored // in n units inline size_t getCapacity(size_t n) const { return n * SYMBOLS_PER_UNIT; } // Returns the index of the unit to store the // i-th symbol of the string inline size_t getUnitIndex(const size_t& i) const { return i >> 1; // equivalent to i / 2 } // Returns the position within a unit that the i-th symbol // should be stored in inline size_t getUnitOffset(const size_t& i) const { // this position is the k-th symbol of the unit return i & 1; // equivalent to i % 2 } // Return the amount that a value must be shifted to // store a code at a given offset inline uint8_t getOffsetShift(size_t offset) const { return 4*(1 - offset); } // Store the value v at the i-th encoded position in the data array inline void store(UNIT_TYPE* pData, size_t i, char b) const { UNIT_TYPE& unit = pData[getUnitIndex(i)]; size_t offset = getUnitOffset(i); uint8_t shift = bwt4_offset_shift[offset]; // Clear the currrent position uint16_t mask = bwt4_offset_mask[offset]; unit &= ~mask; // Set position UNIT_TYPE code = encode(b); code <<= shift; unit |= code; } // get the character stored at position i inline char get(const UNIT_TYPE* pData, const size_t& i) const { const UNIT_TYPE& unit = pData[getUnitIndex(i)]; size_t offset = getUnitOffset(i); uint16_t mask = bwt4_offset_mask[offset]; UNIT_TYPE code = unit & mask; uint8_t shift = bwt4_offset_shift[offset]; code >>= shift; return decode(code); } }; #endif ================================================ FILE: src/SGA/Util/BWTCodec.h ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // BWTCodec - Encoder/decoder for a BWT // string over the alphabet $ACGT using // 3 bits per symbol // #ifndef BWTCODEC_H #define BWTCODEC_H #include "Alphabet.h" static const uint16_t bwt_offset_mask[]={0xE000,0x1C00,0x0380,0x0070,0x000E}; static const uint8_t bwt_offset_shift[]={13, 10, 7, 4, 1}; class BWTCodec { public: // The data is stored 5 characters per uint16_t // The data pattern is: // 11100000 00000000 first symbol // 00011100 00000000 second symbol, etc typedef uint16_t UNIT_TYPE; static const int SYMBOLS_PER_UNIT = 5; // Encoded the character b into a value inline uint8_t encode(char b) const { return BWT_ALPHABET::getRank(b); } // Decode the value c into a character inline char decode(uint8_t c) const { return BWT_ALPHABET::getChar(c); } // Returns the number of units required to encode // a string of length n inline size_t getRequiredUnits(size_t n) const { return (n + SYMBOLS_PER_UNIT - 1) / SYMBOLS_PER_UNIT; } // Returns the number of symbols that can be stored // in n units inline size_t getCapacity(size_t n) const { return n * SYMBOLS_PER_UNIT; } // Returns the index of the unit to store the // i-th symbol of the string inline size_t getUnitIndex(size_t i) const { return i / SYMBOLS_PER_UNIT; } // Returns the position within a unit that the i-th symbol // should be stored in inline size_t getUnitOffset(size_t i) const { // this position is the k-th symbol of the unit return i % SYMBOLS_PER_UNIT; } // Return the amount that a value must be shifted to // store a code at a given offset inline uint8_t getOffsetShift(size_t offset) const { return 1 + 3*(4 - offset); } // Store the value v at the i-th encoded position in the data array inline void store(UNIT_TYPE* pData, size_t i, char b) const { UNIT_TYPE& unit = pData[getUnitIndex(i)]; size_t offset = getUnitOffset(i); uint8_t shift = getOffsetShift(offset); // Clear the currrent position uint16_t mask = bwt_offset_mask[offset]; unit &= ~mask; // Set position UNIT_TYPE code = encode(b); code <<= shift; unit |= code; } // get the character stored at position i inline char get(const UNIT_TYPE* pData, size_t i) const { const UNIT_TYPE& unit = pData[getUnitIndex(i)]; size_t offset = getUnitOffset(i); uint16_t mask = bwt_offset_mask[offset]; UNIT_TYPE code = unit & mask; uint8_t shift = bwt_offset_shift[offset]; code >>= shift; return decode(code); } }; #endif ================================================ FILE: src/SGA/Util/Bamreader.cpp ================================================ #include "bamreader.h" bool Bamreader::bamToSeqRecordVector(SeqRecordVector &srv) { if (!m_reader.Open(m_bam) || !reader.SetRegion(m_region)) return false; BamTools::BamAlignment a; while (m_reader.GetNextAlignment()) } bool Bamreader::findBamIndex() { m_bai = m_bam.substr(0, m_bam.size()-3.append("bai")); return m_reader.OpenIndex(m_bai); } bool Bamreader::setBamRegion(int refid1, int refid2, int pos1, int pos2); ================================================ FILE: src/SGA/Util/BitChar.cpp ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // BitChar - Fixed-size bitset of 8 bits // #include #include #include "BitChar.h" static unsigned char bc_mask[]={0x80,0x40,0x20,0x10,0x08,0x04,0x02,0x01}; // void BitChar::set(unsigned char idx, bool v) { if(v) m_data = m_data | bc_mask[idx]; else m_data = m_data & ~bc_mask[idx]; } // bool BitChar::test(unsigned char idx) const { return m_data & bc_mask[idx]; } // void BitChar::flip(unsigned idx) { m_data = m_data ^ bc_mask[idx]; } // void printBinary(std::ostream& out, const BitChar& bc) { for(int i = 7; i >= 0; i--) { if(bc.test(i)) out << "1"; else out << "0"; } } // Update the value of bit idx from oldValue to newValue using an atomic compare and swap operation // Returns true if the update was successfully performed. bool BitChar::updateCAS(unsigned char idx, bool oldValue, bool newValue) { assert(oldValue != newValue); // Iterate attempts of the CAS operation until the desired bit has the correct value. while(1) { // Get the current value of the data. // If any thread updates d after this call the CAS will fail. unsigned char oldData = m_data; // Check if the bit is already set to newValue in our copy of the data. If so, // some other thread must have updated the value already so we return false. bool currValue = (oldData & bc_mask[idx]); if(currValue == newValue) return false; // Calculate the value of the data with the bit set to the correct value unsigned char newData; if(newValue) newData = oldData | bc_mask[idx]; //set the bit else newData = oldData & ~bc_mask[idx]; //clear the bit // perform the atomic CAS. this returns false if the real value of d does not match oldData bool success = __sync_bool_compare_and_swap(&m_data, oldData, newData); // If the update was successfully performed, return true. // Otherwise, some other thread must have updated a bit in this char so we iterate again. if(success) return true; } } // std::ostream& operator<<(std::ostream& out, const BitChar& bc) { int temp = bc.m_data; out << temp; return out; } // std::istream& operator>>(std::istream& in, BitChar& bc) { int temp; in >> temp; bc.m_data = (unsigned char)temp; return in; } // void BitChar::write(std::ostream& out) { out.write((char*)&m_data, sizeof(m_data)); } // void BitChar::read(std::istream& in) { in.read((char*)&m_data, sizeof(m_data)); } ================================================ FILE: src/SGA/Util/BitChar.h ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL license //----------------------------------------------- // // BitChar - Fixed-size bitset of 8 bits // #ifndef BITCHAR_H #define BITCHAR_H #include struct BitChar { public: BitChar() : m_data(0) {} // Update the value of bit idx from oldValue to newValue using an atomic compare and swap operation // Returns true if the update was successfully performed. This is the only thread-safe way to update the BitChar bool updateCAS(unsigned char idx, bool oldValue, bool newValue); // set the bit at idx to the value u void set(unsigned char idx, bool v); // returns true if bit at idx is set bool test(unsigned char idx) const; // flips the bit at idx void flip(unsigned idx); // I/O friend std::ostream& operator<<(std::ostream& out, const BitChar& bc); friend std::istream& operator>>(std::istream& in, BitChar& bc); void write(std::ostream& out); void read(std::istream& in); private: volatile unsigned char m_data; }; #endif ================================================ FILE: src/SGA/Util/BitVector.cpp ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL license //----------------------------------------------- // // BitVector - Vector of bits // #include "BitVector.h" #include #include // BitVector::BitVector() { initializeMutex(); } // BitVector::BitVector(size_t n) { initializeMutex(); resize(n); } void BitVector::initializeMutex() { int ret = pthread_mutex_init(&m_mutex, NULL); if(ret != 0) { std::cerr << "Mutex initialization in BitVector failed with error " << ret << ", aborting" << std::endl; exit(EXIT_FAILURE); } } // BitVector::~BitVector() { int ret = pthread_mutex_destroy(&m_mutex); if(ret != 0) { std::cerr << "Mutex destruction in BitVector failed with error " << ret << ", aborting" << std::endl; exit(EXIT_FAILURE); } } // void BitVector::resize(size_t n) { size_t num_bytes = (n % 8 == 0) ? n / 8 : (n / 8) + 1; m_data.resize(num_bytes); } // void BitVector::lock() { pthread_mutex_lock(&m_mutex); } // void BitVector::unlock() { pthread_mutex_unlock(&m_mutex); } // bool BitVector::updateCAS(size_t i, bool oldValue, bool newValue) { size_t byte = i / 8; assert(byte < m_data.size()); size_t offset = i - byte * 8; return m_data[byte].updateCAS(offset, oldValue, newValue); } // Set bit at position i to value v void BitVector::set(size_t i, bool v) { size_t byte = i / 8; assert(byte < m_data.size()); size_t offset = i - byte * 8; m_data[byte].set(offset, v); } // Test bit i bool BitVector::test(size_t i) const { size_t byte = i / 8; size_t offset = i - byte * 8; return m_data[byte].test(offset); } ================================================ FILE: src/SGA/Util/BitVector.h ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // BitVector - Vector of bits. The structure // can be locked by a mutex to guarentee atomic access. // #ifndef BITVECTOR_H #define BITVECTOR_H #include "BitChar.h" #include class BitVector { public: BitVector(); BitVector(size_t n); ~BitVector(); // Functions to acquire/release the mutex // The client code is responsible for acquiring // the lock before calling set(). Reading a bit with // test() is ok however. void lock(); void unlock(); // Update the bit at position i from oldValue to newValue using a // compare and swap operation. Returns true if the update is successful. bool updateCAS(size_t i, bool oldValue, bool newValue); void resize(size_t n); void set(size_t i, bool v); bool test(size_t i) const; size_t capacity() const { return m_data.size() * 8; } private: void initializeMutex(); std::vector m_data; pthread_mutex_t m_mutex; }; #endif ================================================ FILE: src/SGA/Util/BloomFilter.cpp ================================================ //----------------------------------------------- // Copyright 2012 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- #include "BloomFilter.h" #include #include #include #include "MurmurHash3.h" // BloomFilter::BloomFilter() : m_occupancy(0) { #if TRACK_OCCUPANCY m_test_counter = 0; #endif } // void BloomFilter::initialize(size_t width, size_t num_hashes) { m_width = width; m_bitvector.resize(width); m_occupancy = 0; // Create seeds for murmer hash // TODO: Check how independent this method of selecting // hash function is... m_hashes.resize(num_hashes); for(size_t i = 0; i < m_hashes.size(); ++i) m_hashes[i] = rand(); } // void BloomFilter::add(const void* key, int num_bytes) { // Set h bits for(size_t i = 0; i < m_hashes.size(); ++i) { int64_t h[2]; MurmurHash3_x64_128(key, num_bytes, m_hashes[i], &h); size_t idx = h[0] % m_width; bool was_set = m_bitvector.updateCAS(idx, false, true); #if TRACK_OCCUPANCY // This call uses an atomic update and returns true if the // bit was sucessfully set if(was_set) { // A bit was set, update the occupancy bool set = false; do { size_t old_occ = m_occupancy; size_t new_occ = old_occ + 1; set = __sync_bool_compare_and_swap(&m_occupancy, old_occ, new_occ); } while(!set); } #else (void)was_set; #endif } } // bool BloomFilter::test(const void* key, int num_bytes) const { for(size_t i = 0; i < m_hashes.size(); ++i) { int64_t h[2]; MurmurHash3_x64_128(key, num_bytes, m_hashes[i], &h); size_t idx = h[0] % m_width; if(!m_bitvector.test(idx)) return false; } #if TRACK_OCCUPANCY m_test_counter++; if(m_test_counter % 500000 == 0) { double p = (double)m_occupancy / m_width; printf("WIDTH: %zu OCC: %zu\n", m_width, m_occupancy); printf("Access: %zu Occupancy: %zu P: %1.4lf\n", m_test_counter, m_occupancy, p); } #endif return true; } // void BloomFilter::printOccupancy() const { size_t set_count = 0; for(size_t i = 0; i < m_width; ++i) set_count += m_bitvector.test(i); printf("%zu out of %zu bits are set\n", set_count, m_width); } // void BloomFilter::printMemory() const { size_t bytes = m_bitvector.capacity(); double mb = (double)bytes / (1 << 20); printf("BloomFilter using %.1lf MB\n", mb); } ================================================ FILE: src/SGA/Util/BloomFilter.h ================================================ //----------------------------------------------- // Copyright 2012 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- #ifndef BLOOM_FILTER_H #define BLOOM_FILTER_H #include #include #include #include #include "BitVector.h" //#define TRACK_OCCUPANCY 1 class BloomFilter { public: /** * @brief Default constructor. */ BloomFilter(); BloomFilter(size_t width, size_t num_hashes) { initialize(width, num_hashes); } /** * @brief Initialize the bloom filter. * * @param width The number of bits to use * @param num_hashes The number of hashes to use */ void initialize(size_t width, size_t num_hashes); /** * @brief Add an object to the collection * * @param key A pointer to the key data * @param num_bytes The number of bytes to read from the key */ void add(const void* key, int num_bytes); /** * @brief Test whether an object is in the collection * * @param key A pointer to the key data * @param num_bytes The number of bytes to read from the key * * @return true if the object is in the bloom filter */ bool test(const void* key, int num_bytes) const; /** * @brief Print the amount of memory used to stdout */ void printMemory() const; /** * @brief Count how many bits are set and print to stdout */ void printOccupancy() const; private: BitVector m_bitvector; std::vector m_hashes; size_t m_width; size_t m_occupancy; #if TRACK_OCCUPANCY mutable size_t m_test_counter; #endif }; #endif ================================================ FILE: src/SGA/Util/ClusterReader.cpp ================================================ //----------------------------------------------- // Copyright 2011 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // ClusterReader - Read in a file of read clusters. // This class conforms to the interface needed // for the SequenceProcessFramework concurrency lib // #include "ClusterReader.h" #include "Util.h" // ClusterReader::ClusterReader(const std::string& filename) : m_numConsumedLast(0), m_numConsumedTotal(0) { m_pReader = createReader(filename); } ClusterReader::~ClusterReader() { delete m_pReader; } // Read a cluster from the file and write its members // to out. Returns false if the read failed bool ClusterReader::generate(ClusterVector& out) { out.clear(); // Read a single cluster from the file. ClusterRecord record; bool good = readCluster(record); // Read failed, return false if(!good) return false; out.push_back(record); // Read the remaining records for this cluster int remaining = record.numElements - 1; for(int i = 0; i < remaining; ++i) { bool good = readCluster(record); if(!good) { std::cerr << "Error: expected " << remaining + 1 << " elements in the cluster but only read " << i+1 << "\n"; exit(1); } if(record.clusterID != out.front().clusterID) { std::cerr << "Error: cluster names do not match! " << record.clusterID << " != " << out.front().clusterID << "\n"; exit(1); } out.push_back(record); } m_numConsumedLast = 1; m_numConsumedTotal += 1; return true; } // bool ClusterReader::readCluster(ClusterRecord& record) { std::string line; bool good = getline(*m_pReader, line).good(); if(!good || line.empty()) return false; std::stringstream parser(line); parser >> record.clusterID; parser >> record.numElements; parser >> record.readID; parser >> record.sequence; if(record.clusterID.empty() || record.numElements == 0 || record.readID.empty() || record.sequence.empty()) { std::cerr << "Could not parse cluster record from line: " << line << "\n"; exit(1); } return true; } ================================================ FILE: src/SGA/Util/ClusterReader.h ================================================ //----------------------------------------------- // Copyright 2011 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // ClusterReader - Read in a file of read clusters. // This class conforms to the interface needed // for the SequenceProcessFramework concurrency lib // #ifndef CLUSTER_READER_H #define CLUSTER_READER_H #include #include #include struct ClusterRecord { std::string clusterID; int numElements; std::string readID; std::string sequence; }; typedef std::vector ClusterVector; class ClusterReader { public: ClusterReader(const std::string& filename); ~ClusterReader(); // Read in a cluster from the file and write the records // to out. Returns true if a cluster was successfully read. bool generate(ClusterVector& out); bool readCluster(ClusterRecord& record); // Functions to determine the progress of the read inline size_t getConsumedLast() const { return m_numConsumedLast; } inline size_t getNumConsumed() const { return m_numConsumedTotal; } private: std::istream* m_pReader; size_t m_numConsumedLast; size_t m_numConsumedTotal; }; #endif ================================================ FILE: src/SGA/Util/Contig.cpp ================================================ #include "Contig.h" // // Statics // static const char* IDFIELD = "ID"; static const char* LENFIELD = "LN"; static const char* COVFIELD = "CV"; static const char* SEQFIELD = "SQ"; static const char* UNQFIELD = "UN"; static const char* ANTFIELD = "AN"; Contig::Contig() : m_length(0), m_coverage(0.0f), m_uniqueFlag(UF_UNKNOWN) {} // // // std::istream& readFasta(std::istream& in, Contig& c) { in.ignore(1); // skip ">" in >> c.m_id >> c.m_length >> c.m_coverage; // read header in.ignore(1); // skip newline in >> c.m_seq; // read seq in.ignore(1); // place the ifstream at the next line return in; } std::istream& readCAF(std::istream& in, Contig& c) { std::string line; getline(in, line); std::stringstream tokenizer(line); std::string token; while(tokenizer >> token) { std::string key; std::string value; splitKeyValue(token, key, value); c.setFromKeyValue(key, value); } assert(c.getSequence().size() == c.getLength()); return in; } // // // SequenceVector Contig::getVariants() const { SequenceVector out; out.push_back(getSequence()); out.insert(out.end(), m_variants.begin(), m_variants.end()); return out; } // // // void Contig::addVariants(const SequenceVector& seqs) { m_variants.insert(m_variants.end(), seqs.begin(), seqs.end()); } // // // std::ostream& writeCAF(std::ostream& out, Contig& c) { std::vector fields; // ID fields.push_back(makeKeyValue(IDFIELD, c.m_id)); fields.push_back(makeKeyValue(LENFIELD, c.m_length)); fields.push_back(makeKeyValue(COVFIELD, c.m_coverage)); fields.push_back(makeKeyValue(SEQFIELD, c.m_seq)); fields.push_back(makeKeyValue(UNQFIELD, c.m_uniqueFlag)); std::copy(fields.begin(), fields.end(), std::ostream_iterator(out, "\t")); return out; } // // Set a field based on a key/value pair // void Contig::setFromKeyValue(std::string& key, std::string& value) { std::stringstream parser(value); if(key == IDFIELD) parser >> m_id; else if(key == LENFIELD) parser >> m_length; else if(key == COVFIELD) parser >> m_coverage; else if(key == SEQFIELD) parser >> m_seq; else if(key == ANTFIELD) parser >> m_annotation; else if(key == UNQFIELD) { int v; parser >> v; m_uniqueFlag = (UniqueFlag)v; } else assert(false && "Unknown token found while parsing contig"); } ================================================ FILE: src/SGA/Util/Contig.h ================================================ #ifndef CONTIG_H #define CONTIG_H #include "Util.h" #include #include #include #include // // Enumerations // enum UniqueFlag { UF_UNKNOWN = 0, UF_UNIQUE, UF_REPEAT, UF_NOCALL }; class Contig { public: // // Constructors // Contig(); // // Getters // ContigID getID() const { return m_id; } double getCoverage() const { return m_coverage; } UniqueFlag getUniqueFlag() const { return m_uniqueFlag; } size_t getLength() const { return m_length; } Sequence getSequence() const { return m_seq; } double getNormalizedCoverage() const { return m_coverage / m_length; } bool isUnique() const { return m_uniqueFlag == UF_UNIQUE; } unsigned int getAnnotation() { return m_annotation; } SequenceVector getVariants() const; // // Setters // void setUniqueFlag(UniqueFlag uf) { m_uniqueFlag = uf; } void setFromKeyValue(std::string& key, std::string& value); void addVariants(const SequenceVector& seqs); // // Operators // bool operator()(Contig& c1, Contig& c2) const { return c1.m_seq < c2.m_seq; } // // Readers // friend std::istream& readFasta(std::istream& in, Contig& c); friend std::istream& readCAF(std::istream& in, Contig& c); // // Writers // friend std::ostream& writeCAF(std::ostream& out, Contig& c); private: ContigID m_id; size_t m_length; double m_coverage; UniqueFlag m_uniqueFlag; Sequence m_seq; unsigned int m_annotation; SequenceVector m_variants; }; #endif ================================================ FILE: src/SGA/Util/CorrectionThresholds.cpp ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // CorrectionThresholds - Cutoff values for determining // whether a particular base should be corrected or not // #include "CorrectionThresholds.h" CorrectionThresholds::CorrectionThresholds() { // Set defaults m_minSupportLowQuality = 4; m_minSupportHighQuality = 3; m_highQualityCutoff = 20; } CorrectionThresholds& CorrectionThresholds::Instance() { static CorrectionThresholds instance; return instance; } void CorrectionThresholds::setBaseMinSupport(int ms) { m_minSupportHighQuality = ms; m_minSupportLowQuality = ms + 1; } // int CorrectionThresholds::getRequiredSupport(int phred) { int threshold = m_minSupportLowQuality; if(phred >= m_highQualityCutoff) threshold = m_minSupportHighQuality; return threshold; } ================================================ FILE: src/SGA/Util/CorrectionThresholds.h ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // CorrectionThresholds.h - Singeleton class containing // cutoff values for determining whether a // base should be corrected or not // #ifndef CORRECTION_THRESHOLDS_H #define CORRECTION_THRESHOLDS_H class CorrectionThresholds { public: CorrectionThresholds(); static CorrectionThresholds& Instance(); // Set the base minimum support level (for high-quality reads) void setBaseMinSupport(int ms); int getMinSupportHighQuality() { return m_minSupportHighQuality; } int getMinSupportLowQuality() { return m_minSupportLowQuality; } int getHighQualityCutoff() { return m_highQualityCutoff; } // Returns the support required for a base with phred score phred int getRequiredSupport(int phred); private: int m_highQualityCutoff; int m_minSupportLowQuality; int m_minSupportHighQuality; }; #endif ================================================ FILE: src/SGA/Util/DNACodec.h ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // DNACodec - Encoder/decoder for a DNA alphabet // of A,C,G,T using 2 bits per symbol // #ifndef DNACODEC_H #define DNACODEC_H #include "Alphabet.h" static const unsigned char dna_offset_mask[]={0xC0,0x30,0x0C,0x03}; class DNACodec { public: typedef uint8_t UNIT_TYPE; static const int SYMBOLS_PER_UNIT = 4; // Encoded the character b into a value inline uint8_t encode(char b) { return DNA_ALPHABET::getBaseRank(b); } // Decode the value c into a character inline char decode(uint8_t c) { return DNA_ALPHABET::getBase(c); } // Returns the number of units required to encode // a string of length n inline size_t getRequiredUnits(size_t n) { return (n + SYMBOLS_PER_UNIT - 1) / SYMBOLS_PER_UNIT; } // Returns the number of symbols that can be stored // in n units inline size_t getCapacity(size_t n) { return n * SYMBOLS_PER_UNIT; } // Returns the index of the unit to store the // i-th symbol of the string inline size_t getUnitIndex(size_t i) { return i / SYMBOLS_PER_UNIT; } // Returns the position within a unit that the i-th symbol // should be stored in inline size_t getUnitOffset(size_t i) { // this position is the k-th symbol of the unit return i % SYMBOLS_PER_UNIT; } // Return the amount that a value must be shifted to // store a code at a given offset inline uint8_t getOffsetShift(size_t offset) { return 2*(3 - offset); } // Store the value v at the i-th encoded position in the data array inline void store(UNIT_TYPE* pData, size_t i, char b) { UNIT_TYPE& unit = pData[getUnitIndex(i)]; size_t offset = getUnitOffset(i); uint8_t shift = getOffsetShift(offset); // Clear the unsigned char mask = dna_offset_mask[offset]; // Clear position unit &= ~mask; // Set position uint8_t code = encode(b); code <<= shift; unit |= code; } // get the character stored at position i inline char get(const UNIT_TYPE* pData, size_t i) { const UNIT_TYPE& unit = pData[getUnitIndex(i)]; size_t offset = getUnitOffset(i); unsigned char mask = dna_offset_mask[offset]; UNIT_TYPE code = unit & mask; uint8_t shift = getOffsetShift(offset); code >>= shift; return decode(code); } }; #endif ================================================ FILE: src/SGA/Util/DNADouble.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // DNADouble.h - Simple map from the DNA // alphabet {A,C,G,T} to a double value // #ifndef ALPHADOUBLE_H #define ALPHADOUBLE_H #include "Alphabet.h" class DNADouble { public: // inline DNADouble() { memset(m_vals, 0, DNA_ALPHABET::size * sizeof(double)); } // inline size_t getAlphabetSize() const { return DNA_ALPHABET::size; } // inline void set(char b, double lp) { m_vals[DNA_ALPHABET::getBaseRank(b)] = lp; } // inline double get(char b) const { return m_vals[DNA_ALPHABET::getBaseRank(b)]; } // inline char getMaxBase() const { char base; double val; getMax(base, val); return base; } // inline double getMaxVal() const { char base; double val; getMax(base, val); return val; } // inline void getMax(char& base, double& val) const { double max = -std::numeric_limits::max(); int maxIdx = 0; for(int i = 0; i < DNA_ALPHABET::size; ++i) { if(m_vals[i] > max) { max = m_vals[i]; maxIdx = i; } } base = DNA_ALPHABET::getBase(maxIdx); val = max; } // inline double getByIdx(const int i) const { return m_vals[i]; } // Return the base for index i static char getBase(size_t i) { assert(i < DNA_ALPHABET::size); return DNA_ALPHABET::getBase(i); } // Swap the (A,T) and (C,G) entries inline void complement() { double tmp; // A,T tmp = m_vals[3]; m_vals[3] = m_vals[0]; m_vals[0] = tmp; // C,G tmp = m_vals[2]; m_vals[2] = m_vals[1]; m_vals[1] = tmp; } // Interpret the DNADouble as a log-probabilty vector of some // event occurring and marginalize using a flat prior double marginalize(double prior) const { double direct = 0.0f; for(int i = 0; i < DNA_ALPHABET::size; ++i) { direct += exp(m_vals[i]); } double ld = log(direct) + log(prior); /* double max = -std::numeric_limits::max(); int max_idx = 0; for(int i = 0; i < DNA_ALPHABET::size; ++i) { if(m_vals[i] > max) { max = m_vals[i]; max_idx = i; } } double sum = prior; for(int i = 0; i < DNA_ALPHABET::size; ++i) { if(i != max_idx) sum += exp(m_vals[i] - max) * prior; } double ld = max + log(sum);*/ return ld; } // Add VAL to each element inline void add(double val) { m_vals[0] += val; m_vals[1] += val; m_vals[2] += val; m_vals[3] += val; } // Subtract VAL from each element inline void subtract(double val) { m_vals[0] -= val; m_vals[1] -= val; m_vals[2] -= val; m_vals[3] -= val; } inline DNADouble& operator+=(const DNADouble& other) { assert(DNA_ALPHABET::size == 4); // Manually unrolled m_vals[0] += other.m_vals[0]; m_vals[1] += other.m_vals[1]; m_vals[2] += other.m_vals[2]; m_vals[3] += other.m_vals[3]; return *this; } inline DNADouble& operator-=(const DNADouble& other) { assert(DNA_ALPHABET::size == 4); // Manually unrolled m_vals[0] -= other.m_vals[0]; m_vals[1] -= other.m_vals[1]; m_vals[2] -= other.m_vals[2]; m_vals[3] -= other.m_vals[3]; return *this; } // void printVerbose() const { std::cout << "AP "; for(int i = 0; i < DNA_ALPHABET::size; ++i) { char b = DNA_ALPHABET::getBase(i); std::cout << b << ": " << m_vals[i] << " "; } std::cout << "\n"; } private: double m_vals[DNA_ALPHABET::size]; }; #endif ================================================ FILE: src/SGA/Util/DNAString.cpp ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // DNAString // #include #include "DNAString.h" #include "Util.h" DNAString::DNAString() : m_len(0), m_data(0) {} // DNAString::DNAString(std::string seq) { _alloc(seq.c_str(), seq.length()); } // DNAString::DNAString(const DNAString& other) { // Deep copy _alloc(other.m_data, other.m_len); } // DNAString& DNAString::operator=(const DNAString& dna) { if(&dna == this) return *this; // self-assign _dealloc(); _alloc(dna.m_data, dna.m_len); return *this; } // DNAString& DNAString::operator=(const std::string& str) { _dealloc(); _alloc(str.c_str(), str.length()); return *this; } // bool DNAString::operator==(const DNAString& other) { if(m_len != other.m_len) return false; return strncmp(m_data, other.m_data, m_len) == 0; } // void DNAString::_alloc(const char* pData, size_t l) { m_len = l; size_t alloc_len = m_len + 1; m_data = new char[alloc_len]; strncpy(m_data, pData, m_len); m_data[m_len] = '\0'; } // void DNAString::_dealloc() { if(m_data != NULL) delete [] m_data; m_data = 0; m_len = 0; } // DNAString::~DNAString() { _dealloc(); } // Reverse the sequence void DNAString::reverse() { size_t half = m_len >> 1; for(size_t idx = 0; idx < half; ++idx) { size_t opp = m_len - idx - 1; char tmp = m_data[opp]; m_data[opp] = m_data[idx]; m_data[idx] = tmp; } } void DNAString::reverseComplement() { this->reverse(); for(size_t idx = 0; idx < m_len; ++idx) m_data[idx] = complement(m_data[idx]); } void DNAString::disambiguate() { if(m_len == 0) return; for(size_t idx = 0; idx < m_len; ++idx) { if(m_data[idx] == 'N') { m_data[idx] = randomBase(); } } } // std::string DNAString::getSuffixString(size_t idx) const { std::string str; str.reserve(getSuffixLength(idx) + 1); str += getSuffix(idx); str += "$"; return str; } // std::string DNAString::toString() const { return std::string(m_data, m_len); } ================================================ FILE: src/SGA/Util/DNAString.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL license //----------------------------------------------- // // DNAString - Wrapper for c-string for DNA sequences // It is tailored for fast suffix operations // #ifndef DNASTRING_H #define DNASTRING_H #include #include #include class DNAString { public: // Constructors/Destructors DNAString(); DNAString(const DNAString& other); DNAString(std::string seq); ~DNAString(); // Operators DNAString& operator=(const DNAString& dna); DNAString& operator=(const std::string& str); bool operator==(const DNAString& other); size_t length() const { return m_len; } bool empty() const { return m_len == 0 || m_data[0] == '\0'; } inline const char* getSuffix(size_t idx) const { assert(m_len > 0); // Force the suffix to point to the empty string if out of bounds if(idx <= m_len) return m_data + idx; else return m_data + m_len; } // Return the length of the suffix (not including $) starting at idx size_t getSuffixLength(size_t idx) const { assert(m_len > 0); if(idx <= m_len) return m_len - idx; else return 0; } // Get the character at the given position inline char get(size_t idx) const { assert(idx < m_len + 1); return m_data[idx]; } // Set the character at the given position inline void set(size_t idx, char b) const { assert(idx < m_len + 1); m_data[idx] = b; } // Get the substring of length n starting at position pos std::string substr(size_t pos, size_t n) const { assert(m_data != NULL && pos < m_len && pos + n <= m_len); return std::string(m_data + pos, n); } // randomly change the 'N' bases to one of ACGT so that the string is not ambiguous void disambiguate(); void reverse(); void reverseComplement(); std::string getSuffixString(size_t idx) const; std::string toString() const; private: // functions void _alloc(const char* pData, size_t l); void _dealloc(); // data size_t m_len; char* m_data; }; #endif ================================================ FILE: src/SGA/Util/EncodedString.h ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // EncodedString - Templated class to store // a string from a reduced alphabet. The actual // encoding is done outside this class by the Codec // template parameter. // // Here the storage is in terms of "units" which is // defined by the codec. For a simple 2-bit encoded // DNA string this would be a byte but for more complicated // encodings like the 3-bit BWT this could be a larger // structure like uint16_t // #ifndef ENCODEDSTRING_H #define ENCODEDSTRING_H #include #include #include #include "DNACodec.h" #include "BWTCodec.h" #include "BWT4Codec.h" #include "NoCodec.h" template class EncodedString { typedef typename Codec::UNIT_TYPE StorageUnit; public: // Constructors/Destructors EncodedString() : m_len(0), m_capacity(0), m_data(0) {} // EncodedString(const EncodedString& other) { // deep copy _alloc(other.m_len); _copy(other); } // EncodedString(const std::string& seq) { size_t n = seq.length(); _alloc(n); _copy(seq.c_str(), n); } // ~EncodedString() { _dealloc(); } // Assignment op EncodedString& operator=(const EncodedString& other) { if(&other == this) return *this; // self-assign _dealloc(); _alloc(other.m_len); _copy(other); return *this; } // EncodedString& operator=(const std::string& str) { size_t n = str.length(); _dealloc(); _alloc(n); _copy(str.c_str(), n); return *this; } // Resize this string to n symbols, setting the // new entries to the default value void resize(size_t n) { if(n > m_capacity) _realloc(n); m_len = n; } // Append a std::string void append(const std::string& str) { size_t n = str.length(); size_t num_total = m_len + n; if(num_total > m_capacity) _realloc(num_total); _append(str.c_str(), n); } // Append some other EncodedString void append(const EncodedString& other) { size_t n = other.length(); size_t num_total = m_len + n; if(num_total > m_capacity) _realloc(num_total); _append(other); } // Swap the contents with another encoded string void swap(EncodedString& other) { size_t tmp; tmp = other.m_len; other.m_len = m_len; m_len = tmp; tmp = other.m_capacity; other.m_capacity = m_capacity; m_capacity = tmp; StorageUnit* pTmp = other.m_data; other.m_data = m_data; m_data = pTmp; } // size_t length() const { return m_len; } // size_t capacity() const { return m_capacity; } // bool empty() const { return m_len == 0; } // Get the character at idx inline char get(size_t idx) const { assert(idx < m_len); return s_codec.get(m_data, idx); } // Set the character at idx inline void set(size_t idx, char b) { assert(idx < m_len); s_codec.store(m_data, idx, b); } // std::string toString() const { std::string out(m_len, 'A'); for(size_t i = 0; i < m_len; ++i) out[i] = s_codec.get(m_data, i); return out; } // std::string substr(size_t start) const { assert(start < m_len); std::string out(m_len - start, 'A'); for(size_t i = start; i < m_len; ++i) out[i - start] = s_codec.get(m_data, i); return out; } // std::string substr(size_t start, size_t len) const { assert(start < m_len); assert(start + len <= m_len); std::string out(len, 'C'); for(size_t i = 0; i < len; ++i) out[i] = s_codec.get(m_data, i + start); return out; } // friend bool operator==(const EncodedString& a, const EncodedString& b) { if(a.m_len != b.m_len) return false; // Would be faster to compare entire units at a time size_t n = a.m_len; for(size_t i = 0; i < n; ++i) { if(a.get(i) != b.get(i)) return false; } return true; } // friend std::ostream& operator<<(std::ostream& out, const EncodedString& a) { out << a.toString(); return out; } // Return the amount of space this string is using size_t getMemSize() const { return sizeof(*this) + sizeof(StorageUnit) * s_codec.getRequiredUnits(m_capacity); } private: // functions void _copy(const char* pData, size_t n) { // this assumes that storage for n characters has been // allocated assert(m_capacity >= n); for(size_t i = 0; i < n; ++i) s_codec.store(m_data, i, pData[i]); m_len = n; } // void _copy(const EncodedString& other) { // storage should have been allocated already assert(m_capacity = other.m_capacity); size_t num_units = s_codec.getRequiredUnits(other.m_capacity); _copyUnitData(other.m_data, num_units); m_len = other.m_len; } // Copy num_units from pData into the internal storage void _copyUnitData(StorageUnit* pData, size_t num_units) { assert(s_codec.getRequiredUnits(m_capacity) >= num_units); for(size_t i = 0; i < num_units; ++i) m_data[i] = pData[i]; } // append n symbols from pData into the buffer void _append(const char* pData, size_t n) { assert(m_len + n <= m_capacity); for(size_t i = 0; i < n; ++i) s_codec.store(m_data, m_len + i, pData[i]); m_len += n; } // append void _append(const EncodedString& other) { size_t n = other.m_len; assert(m_len + n <= m_capacity); for(size_t i = 0; i < n; ++i) s_codec.store(m_data, m_len + i, other.get(i)); m_len += n; } // allocate storage for n symbols void _alloc(size_t n) { // Get the number of units that need to be allocated from the codec size_t n_units = s_codec.getRequiredUnits(n); m_data = new StorageUnit[n_units](); // This zeros the memory m_capacity = s_codec.getCapacity(n_units); } // reallocate the storage so that the capacity is at least n symbols // m_len is not changed void _realloc(size_t n) { StorageUnit* oldData = m_data; size_t old_units = s_codec.getRequiredUnits(m_len); _alloc(n); assert(m_capacity >= n); _copyUnitData(oldData, old_units); delete [] oldData; } // deallocate storage void _dealloc() { if(m_data != NULL) delete [] m_data; m_data = 0; m_len = 0; m_capacity = 0; } // data static Codec s_codec; size_t m_len; // the length of the string size_t m_capacity; // the maximum length of the string that can be stored StorageUnit* m_data; }; // Initialize the static member template Codec EncodedString::s_codec; typedef EncodedString DNAEncodedString; typedef EncodedString BWTString; typedef EncodedString NoEncodingString; typedef std::vector DNAEncodedStringVector; #endif ================================================ FILE: src/SGA/Util/HashMap.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // HashMap compability defines. Similar to Shaun Jackman's // compatability code in abyss (see Common/HashMap) // // #ifndef HASHMAP_H #define HASHMAP_H //#include "config.h" #define HAVE_UNORDERED_MAP 1 #if HAVE_UNORDERED_MAP # include # include #define HashMap std::unordered_map #define HashSet std::unordered_set typedef std::hash StringHasher; #elif HAVE_TR1_UNORDERED_MAP #include #include #define HashMap std::tr1::unordered_map #define HashSet std::tr1::unordered_set typedef std::tr1::hash StringHasher; #elif HAVE_EXT_HASH_MAP #define USING_EXT_HASH_MAP 1 # undef __DEPRECATED #include #include #define HashMap __gnu_cxx::hash_map #define HashSet __gnu_cxx::hash_set #else # error No hash map implementation found #endif # if USING_EXT_HASH_MAP # include # include struct StringHasher { size_t operator()( const std::string& x) const { return __gnu_cxx::hash()( x.c_str()); } }; #endif // Ensure the sparse hash is available #if HAVE_GOOGLE_SPARSE_HASH_MAP #include //#define SparseHashMap google::sparse_hash_map // JEREMIAH #else #define SparseHashMap std::tr1::unordered_map //#error The google sparse hash is required #endif #endif ================================================ FILE: src/SGA/Util/Interval.cpp ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // Interval - A pair of integers denoting a range // #include "Interval.h" #if 0 Interval Interval::intersect(const Interval& r1, const Interval& r2) { Interval result; result.start = std::max(r1.start, r2.start); result.end = std::min(r1.end, r2.end); // Check for non-overlap if(result.end <= result.start) { result.start = 0; result.end = 0; } return result; } #endif std::ostream& operator<<(std::ostream& out, const Interval& r) { out << r.start << " " << r.end; return out; } std::istream& operator>>(std::istream& in, Interval& r) { in >> r.start >> r.end; return in; } ================================================ FILE: src/SGA/Util/Interval.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL license //----------------------------------------------- // // Interval - A pair of integers denoting a closed interval // #ifndef INTERVAL_H #define INTERVAL_H #include "Util.h" struct Interval { // constructors Interval() : start(0), end(0) {} Interval(int s, int e) : start(s), end(e) {} // Find the intersection between [s1, e1] [s2, e2] // The intersection is placed in [s3, e3], if the two input // intervals do not intersect then e3 will be strictly less than s3 template static void intersect(const T s1, const T e1, const T s2, const T e2, T& s3, T& e3) { assert(s1 <= e1 && s2 <= e2); s3 = std::max(s1, s2); e3 = std::min(e1, e2); } // Precondition: s1 >= e1 and s2 >= e2 // Return true if the coordinates intersect template static bool isIntersecting(const T s1, const T e1, const T s2, const T e2) { assert(s1 <= e1 && s2 <= e2); if(s2 > e1 || s1 > e2) return false; else return true; } // functions friend std::ostream& operator<<(std::ostream& out, const Interval& i); friend std::istream& operator>>(std::istream& in, Interval& i); // data members int start; int end; }; #endif ================================================ FILE: src/SGA/Util/IntervalTree.h ================================================ #ifndef __INTERVAL_TREE_H #define __INTERVAL_TREE_H #include #include #include #include using namespace std; template class IntervalT { public: K start; K stop; T value; IntervalT() : start(0) , stop(INT_MAX) , value("") { } IntervalT(K s, K e, const T& v) : start(s) , stop(e) , value(v) { } }; template int intervalStart(const IntervalT& i) { return i.start; } template int intervalStop(const IntervalT& i) { return i.stop; } template ostream& operator<<(ostream& out, IntervalT& i) { out << "Interval(" << i.start << ", " << i.stop << "): " << i.value; return out; } template class IntervalStartSorter { public: bool operator() (const IntervalT& a, const IntervalT& b) { return a.start < b.start; } }; template class IntervalTTree { public: typedef IntervalT interval; typedef vector intervalVector; typedef IntervalTTree intervalTree; intervalVector intervals; intervalTree* left; intervalTree* right; int center; IntervalTTree(void) : left(NULL) , right(NULL) , center(0) { } IntervalTTree(const intervalTree& other) { center = other.center; intervals = other.intervals; if (other.left) { left = (intervalTree*) malloc(sizeof(intervalTree)); *left = *other.left; } else { left = NULL; } if (other.right) { right = new intervalTree(); *right = *other.right; } else { right = NULL; } } IntervalTTree& operator=(const intervalTree& other) { center = other.center; intervals = other.intervals; if (other.left) { left = new intervalTree(); *left = *other.left; } else { left = NULL; } if (other.right) { right = new intervalTree(); *right = *other.right; } else { right = NULL; } return *this; } IntervalTTree( intervalVector& ivals, unsigned int depth = 16, unsigned int minbucket = 64, int leftextent = 0, int rightextent = 0, unsigned int maxbucket = 512 ) : left(NULL) , right(NULL) { --depth; if (depth == 0 || (ivals.size() < minbucket && ivals.size() < maxbucket)) { intervals = ivals; } else { if (leftextent == 0 && rightextent == 0) { // sort intervals by start IntervalStartSorter intervalStartSorter; sort(ivals.begin(), ivals.end(), intervalStartSorter); } int leftp = 0; int rightp = 0; int centerp = 0; if (leftextent || rightextent) { leftp = leftextent; rightp = rightextent; } else { leftp = ivals.front().start; vector stops; stops.resize(ivals.size()); transform(ivals.begin(), ivals.end(), stops.begin(), intervalStop); rightp = *max_element(stops.begin(), stops.end()); } //centerp = ( leftp + rightp ) / 2; centerp = ivals.at(ivals.size() / 2).start; center = centerp; intervalVector lefts; intervalVector rights; for (typename intervalVector::iterator i = ivals.begin(); i != ivals.end(); ++i) { interval& interval = *i; if (interval.stop < center) { lefts.push_back(interval); } else if (interval.start > center) { rights.push_back(interval); } else { intervals.push_back(interval); } } if (!lefts.empty()) { left = new intervalTree(lefts, depth, minbucket, leftp, centerp); } if (!rights.empty()) { right = new intervalTree(rights, depth, minbucket, centerp, rightp); } } } void findOverlapping(K start, K stop, intervalVector& overlapping) { if (!intervals.empty() && ! (stop < intervals.front().start)) { for (typename intervalVector::iterator i = intervals.begin(); i != intervals.end(); ++i) { interval& interval = *i; if (interval.stop >= start && interval.start <= stop) { overlapping.push_back(interval); } } } if (left && start <= center) { left->findOverlapping(start, stop, overlapping); } if (right && stop >= center) { right->findOverlapping(start, stop, overlapping); } } void findContained(K start, K stop, intervalVector& contained) { if (!intervals.empty() && ! (stop < intervals.front().start)) { for (typename intervalVector::iterator i = intervals.begin(); i != intervals.end(); ++i) { interval& interval = *i; if (interval.start >= start && interval.stop <= stop) { contained.push_back(interval); } } } if (left && start <= center) { left->findContained(start, stop, contained); } if (right && stop >= center) { right->findContained(start, stop, contained); } } ~IntervalTTree(void) { // traverse the left and right // delete them all the way down if (left) { delete left; } if (right) { delete right; } } }; #endif ================================================ FILE: src/SGA/Util/KmerDistribution.cpp ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL license //----------------------------------------------- // // KmerDistribution - Histogram of kmer frequencies // #include "KmerDistribution.h" #include #include #include #include #include KmerDistribution::KmerDistribution() { } void KmerDistribution::add(int kcount) { m_data[kcount]++; } double KmerDistribution::getCumulativeProportionLEQ(int n) const { std::vector countVector = toCountVector(1000); int64_t sum = 0; for(size_t i = 0; i < countVector.size(); ++i) { sum += countVector[i]; } std::vector cumulativeVector(countVector.size()); int64_t runningSum = 0; for(size_t i = 0; i < cumulativeVector.size(); ++i) { runningSum += countVector[i]; cumulativeVector[i] = (double)runningSum / sum; //std::cout << "CUMUL " << i << " " << cumulativeVector[i] << "\n"; } assert(n < (int)cumulativeVector.size()); return cumulativeVector[n]; } size_t KmerDistribution::getCutoffForProportion(double p) const { int MAX_COUNT = 1000; std::vector countVector = toCountVector(MAX_COUNT); int64_t sum = 0; for(size_t i = 0; i < countVector.size(); ++i) sum += countVector[i]; double c = 0.0f; for(size_t i = 0; i < countVector.size(); ++i) { c += (double)countVector[i] / sum; if(c > p) return i; } return MAX_COUNT; } // int KmerDistribution::findFirstLocalMinimum() const { std::vector countVector = toCountVector(1000); if(countVector.empty()) return -1; std::cout << "CV: " << countVector.size() << "\n"; int prevCount = countVector[1]; double threshold = 0.75; for(size_t i = 2; i < countVector.size(); ++i) { int currCount = countVector[i]; double ratio = (double)currCount / prevCount; std::cout << i << " " << currCount << " " << ratio << "\n"; if(ratio > threshold) return i; prevCount = currCount; } return -1; } int KmerDistribution::getCensoredMode(size_t n) const { std::vector countVector = toCountVector(1000); if(countVector.size() < n) return -1; int modeIdx = -1; int modeCount = -1; for(; n < countVector.size(); ++n) { if(countVector[n] > modeCount) { modeCount = countVector[n]; modeIdx = n; } } return modeIdx; } // Find the boundary of the kmers that are likely erroneous // We do this by finding the value between 1 and the trusted mode // that contributes the fewest int KmerDistribution::findErrorBoundary() const { int mode = getCensoredMode(5); if(mode == -1) return -1; std::cerr << "Trusted kmer mode: " << mode << "\n"; std::vector countVector = toCountVector(1000); if(countVector.empty()) return -1; int runningSum = 0; double minContrib = std::numeric_limits::max(); int idx = -1; for(int i = 1; i < mode; ++i) { runningSum += countVector[i]; double v = (double)countVector[i] / runningSum; if(v < minContrib) { minContrib = v; idx = i; } } return idx; } // Find the boundary of the kmers that are likely erroneous // We do this by finding the value between 1 and the trusted mode // that contributes the fewest int KmerDistribution::findErrorBoundaryByRatio(double ratio) const { int mode = getCensoredMode(5); if(mode == -1) return -1; std::cerr << "Trusted kmer mode: " << mode << "\n"; std::vector countVector = toCountVector(1000); if(countVector.empty()) return -1; for(int i = 1; i < mode - 1; ++i) { int currCount = countVector[i]; int nextCount = countVector[i+1]; double cr = (double)currCount / nextCount; if(cr < ratio) return i; } return -1; } // std::vector KmerDistribution::toCountVector(int max) const { std::vector out; if(m_data.empty()) return out; int min = 0; for(int i = min; i <= max; ++i) { std::map::const_iterator iter = m_data.find(i); int v = (iter != m_data.end()) ? iter->second : 0; out.push_back(v); } return out; } size_t KmerDistribution::getTotalKmers() const { size_t sum = 0; std::map::const_iterator iter = m_data.begin(); for(; iter != m_data.end(); ++iter) sum += iter->second; return sum; } size_t KmerDistribution::getNumberWithCount(size_t c) const { std::map::const_iterator iter = m_data.find(c); if(iter != m_data.end()) return iter->second; else return 0; } // for compatibility with old code void KmerDistribution::print(int max) const { print(stdout, max); } void KmerDistribution::print(FILE* fp, int max) const { fprintf(fp, "Kmer coverage histogram\n"); fprintf(fp, "cov\tcount\n"); int maxCount = 0; std::map::const_iterator iter = m_data.begin(); for(; iter != m_data.end(); ++iter) { if(iter->first <= max) fprintf(fp, "%d\t%d\n", iter->first, iter->second); else maxCount += iter->second; } fprintf(fp, ">%d\t%d\n", max, maxCount); } ================================================ FILE: src/SGA/Util/KmerDistribution.h ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL license //----------------------------------------------- // // KmerDistribution - Histogram of kmer frequencies // #ifndef KMERDISTRIBUTION_H #define KMERDISTRIBUTION_H #include #include #include #include class KmerDistribution { public: KmerDistribution(); ~KmerDistribution() {} // Returns the proportion of the distribution less than or equal to n double getCumulativeProportionLEQ(int n) const; // Returns the smallest value n such that the proportion of the data less // than n is p. 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not export all variables. # Otherwise a system limit (for SysV at least) may be exceeded. .NOEXPORT: ================================================ FILE: src/SGA/Util/Match.cpp ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // Match - A pair of coordinates representing the overlapping // regions of two sequences // #include "Match.h" #include // Constructor Match::Match(const SeqCoord& sc1, const SeqCoord& sc2, bool isRC, int nd) { coord[0] = sc1; coord[1] = sc2; isReverse = isRC; numDiff = nd; } // Constructor Match::Match(int s1, int e1, int l1, int s2, int e2, int l2, bool isRC, int nd) { coord[0] = SeqCoord(s1, e1, l1); coord[1] = SeqCoord(s2, e2, l2); isReverse = isRC; numDiff = nd; } // Swap the order of the elements void Match::swap() { SeqCoord tmp = coord[0]; coord[0] = coord[1]; coord[1] = tmp; } // Flip coord2 so that it is in the same frame as coord1 void Match::canonize() { if(isRC()) coord[1].flip(); isReverse = false; } // Flip coord2 so it is out of frame of coord1 void Match::decanonize() { if(!isRC()) coord[1].flip(); isReverse = true; } // Calculation the translation offset to shift // a coord[0] position to a coord[1]. This must be calculated // using canonical coordinates int Match::calculateTranslation() const { if(!isRC()) return coord[1].interval.start - coord[0].interval.start; else { SeqCoord f = coord[1]; f.flip(); return f.interval.start - coord[0].interval.start; } } // Calculation the translation offset to shift // a coord[1] position to a coord[0]. This must be calculated // using canonical coordinates int Match::calculateInverseTranslation() const { if(!isRC()) return coord[0].interval.start - coord[1].interval.start; else { SeqCoord f = coord[0]; f.flip(); return f.interval.start - coord[1].interval.start; } } // Translate the SeqCoord c from the frame of coord[0] to coord[1] SeqCoord Match::translate(const SeqCoord& c) const { assert(coord[0].length() == coord[1].length()); // ensure translation is valid int t = calculateTranslation(); SeqCoord out; out.seqlen = coord[1].seqlen; out.interval.start = c.interval.start + t; out.interval.end = c.interval.end + t; if(isRC()) out.flip(); return out; } // Translate the SeqCoord c from the frame of coord[1] to coord[0] SeqCoord Match::inverseTranslate(const SeqCoord& c) const { assert(coord[0].length() == coord[1].length()); // ensure translation is valid int t = calculateInverseTranslation(); SeqCoord out; out.seqlen = coord[0].seqlen; out.interval.start = c.interval.start + t; out.interval.end = c.interval.end + t; if(isRC()) out.flip(); return out; } // Translate a single position from c[0] frame to c[1] int Match::translate(int c) const { assert(!isRC()); assert(coord[0].length() == coord[1].length()); // ensure translation is valid int t = calculateTranslation(); return c + t; } // Translate a single position from c[1] frame to c[0] int Match::inverseTranslate(int c) const { assert(!isRC()); assert(coord[0].length() == coord[1].length()); int t = calculateInverseTranslation(); return c + t; } // Given two matches, match_xy and match_xz infer the // match between yz. This requies coord[0] of the input // matches to be referring to the same sequence // This returns the minimal matching region, it could possibly be extended Match Match::infer(const Match& match_xy, const Match& match_xz) { assert(match_xy.coord[0].seqlen == match_xz.coord[0].seqlen); // Calculate the max/min start/end coordinates of coord[0] int s = std::max(match_xy.coord[0].interval.start, match_xz.coord[0].interval.start); int e = std::min(match_xy.coord[0].interval.end, match_xz.coord[0].interval.end); // These are the coordinates in frame X SeqCoord r_y(s, e, match_xy.coord[0].seqlen); SeqCoord r_z(s, e, match_xz.coord[0].seqlen); // Translate into the desired frame SeqCoord t_y = match_xy.translate(r_y); SeqCoord t_z = match_xz.translate(r_z); // Set the new lengths of the seqcoords t_y.seqlen = match_xy.coord[1].seqlen; t_z.seqlen = match_xz.coord[1].seqlen; return Match(t_y, t_z, match_xy.isRC() != match_xz.isRC(), -1); } // Given two matches, match_xy and match_xz, return // true if the match coordinates intersect. Assumes // that the matches have a common basis which is coord[0] bool Match::doMatchesIntersect(const Match& match_xy, const Match& match_xz) { int s = std::max(match_xy.coord[0].interval.start, match_xz.coord[0].interval.start); int e = std::min(match_xy.coord[0].interval.end, match_xz.coord[0].interval.end); if(s > e) return false; else return true; } // Expand the match outwards so one sequence is left terminal and one sequence // is right terminal. This makes it a "proper" overlap // This function assumes the coordinates are valid (see SeqCoord) void Match::expand() { assert(coord[0].isValid() && coord[1].isValid()); // This is simpler if the coordinates are in canonical form, so here were canonize // them and decanonize after bool flipped = false; if(isRC()) { flipped = true; canonize(); } // left expansion if(!coord[0].isLeftExtreme() || !coord[1].isLeftExtreme()) { int dist = std::min(coord[0].getLeftDist(), coord[1].getLeftDist()); coord[0].interval.start -= dist; coord[1].interval.start -= dist; } // right expansion if(!coord[0].isRightExtreme() || !coord[1].isRightExtreme()) { int dist = std::min(coord[0].getRightDist(), coord[1].getRightDist()); coord[0].interval.end += dist; coord[1].interval.end += dist; } if(flipped) decanonize(); assert(coord[0].isValid() && coord[1].isValid()); } // Count the number of differences between the two strings based on the match int Match::countDifferences(const std::string& s1, const std::string& s2) const { std::string matched1 = coord[0].getSubstring(s1); std::string matched2 = coord[1].getSubstring(s2); if(isReverse) matched2 = reverseComplement(matched2); return ::countDifferences(matched1, matched2, matched1.length()); } bool Match::isSubstringContainment() const { return isContainment() && (!coord[0].isContained() || !coord[1].isContained()); } // Print the matched strings void Match::printMatch(const std::string& s1, const std::string& s2) const { std::string matched1 = coord[0].getSubstring(s1); std::string matched2 = coord[1].getSubstring(s2); if(isReverse) matched2 = reverseComplement(matched2); std::cout << "M1: " << matched1 << "\n"; std::cout << "M2: " << matched2 << "\n"; } // Output std::ostream& operator<<(std::ostream& out, const Match& m) { out << m.coord[0] << " " << m.coord[1] << " " << m.isReverse << " " << m.numDiff; return out; } // Input std::istream& operator>>(std::istream& in, Match& m) { in >> m.coord[0] >> m.coord[1] >> m.isReverse >> m.numDiff; return in; } // // Overlap // Overlap::Overlap(const std::string& i1, const std::string& i2, const Match& m) : match(m) { id[0] = i1; id[1] = i2; } Overlap::Overlap(const std::string& i1, const SeqCoord& sc1, const std::string& i2, const SeqCoord& sc2, bool isRC, int nd) { id[0] = i1; id[1] = i2; match = Match(sc1, sc2, isRC, nd); } Overlap::Overlap(const std::string& i1, int s1, int e1, int l1, const std::string& i2, int s2, int e2, int l2, bool isRC, int nd) { id[0] = i1; id[1] = i2; match = Match(s1, e1, l1, s2, e2, l2, isRC, nd); } // Return the index of the contained vertex // Precondition: the overlap is a containment size_t Overlap::getContainedIdx() const { // The verts are mutually contained, return the lexographically lower id if(match.coord[0].isContained() && match.coord[1].isContained()) { if(id[0] < id[1]) return 1; else return 0; } else if(match.coord[0].isContained()) { return 0; } else { assert(match.coord[1].isContained()); return 1; } } // Return the ID of the contained vertex // Precondition: The overlap is a containment std::string Overlap::getContainedID() const { return id[getContainedIdx()]; } // Swap the elements void Overlap::swap() { id[0].swap(id[1]); match.swap(); } // int Overlap::getOverlapLength(size_t idx) const { assert(idx <= 1); return match.coord[idx].length(); } // Output std::ostream& operator<<(std::ostream& out, const Overlap& o) { out << o.id[0] << " " << o.id[1] << " " << o.match; return out; } // Input std::istream& operator>>(std::istream& in, Overlap& o) { in >> o.id[0] >> o.id[1] >> o.match; return in; } ================================================ FILE: src/SGA/Util/Match.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL license //----------------------------------------------- // // Match - A pair of coordinates representing the overlapping // regions of two sequences // #ifndef MATCH_H #define MATCH_H #include "Util.h" #include "SeqCoord.h" struct Match { // functions Match() {} Match(const SeqCoord& sc1, const SeqCoord& sc2, bool isRC, int nd); Match(int s1, int e1, int l1, int s2, int e2, int l2, bool isRC, int nd); // Accessors inline bool isRC() const { return isReverse; } inline int getNumDiffs() const { return numDiff; } inline int getMinOverlapLength() const { return std::min(coord[0].length(), coord[1].length()); } inline int getMaxOverlapLength() const { return std::max(coord[0].length(), coord[1].length()); } void setNumDiffs(int n) { numDiff = n; } // Calculate the translation offset from coord[0] to coord[1] int calculateTranslation() const; int calculateInverseTranslation() const; // Translate the SeqCoord c from the frame of coord[0] to coord[1] SeqCoord translate(const SeqCoord& c) const; // Translate the SeqCoord c from the frame of coord[1] to coord[0] SeqCoord inverseTranslate(const SeqCoord& c) const; // Translate a single position from c[0] frame to c[1] int translate(int c) const; // Translate a single position from c[1] frame to c[0] int inverseTranslate(int c) const; // Swap the coords of this element void swap(); // Infer an overlap between yz given matches xy and xz static Match infer(const Match& match_xy, const Match& match_xz); static bool doMatchesIntersect(const Match& match_xy, const Match& match_xz); // Expand a match outwards so each end is terminal for both coordinates void expand(); // Count the number of differences between the strings assuming no indels int countDifferences(const std::string& s1, const std::string& s2) const; // Print the matched strings void printMatch(const std::string& s1, const std::string& s2) const; // Flip coord[1] if isReverse is true, effectively // bringing the matching strings into the same coordinate system void canonize(); void decanonize(); // Returns true if one overlap is contained within the other, ie they are not equal bool isSubstringContainment() const; bool isContainment() const { return coord[0].isContained() || coord[1].isContained(); } // IO friend std::ostream& operator<<(std::ostream& out, const Match& m); friend std::istream& operator>>(std::istream& in, Match& m); // data SeqCoord coord[2]; bool isReverse; int numDiff; }; // Overlap struct Overlap { // constructors Overlap() {} Overlap(const std::string& i1, const std::string& i2, const Match& m); Overlap(const std::string& i1, const SeqCoord& sc1, const std::string& i2, const SeqCoord& sc2, bool isRC, int nd); Overlap(const std::string& i1, int s1, int e1, int l1, const std::string& i2, int s2, int e2, int l2, bool isRC, int nd); // functions // Swap the order of the elements void swap(); // bool isContainment() const { return match.isContainment(); } bool isSubstringContainment() const { return match.isSubstringContainment(); } // Return the index (0 or 1) of the CONTAINED vertex (the discarded vertex of a containment) size_t getContainedIdx() const; std::string getContainedID() const; // int getOverlapLength(size_t idx) const; friend std::ostream& operator<<(std::ostream& out, const Overlap& o); friend std::istream& operator>>(std::istream& in, Overlap& o); // data std::string id[2]; Match match; }; // Collections typedef std::vector OverlapVector; #endif ================================================ FILE: src/SGA/Util/Metrics.h ================================================ ///----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // Metrics - Data structures to record data metrics // error rates, error distributions, etc // #ifndef METRICS_H #define METRICS_H #include struct ErrorCount { int64_t num_samples; int64_t num_errors; }; template class ErrorCountMap { typedef std::map DataMap; public: ErrorCountMap() {} // void incrementSample(const Key& key) { ++m_data[key].num_samples; } // void incrementError(const Key& key) { ++m_data[key].num_errors; } // void write(std::ostream* pWriter, const std::string& leader, const std::string& header) { *pWriter << leader; *pWriter << header << "\tsamples\terrors\tfraction\n"; for(typename DataMap::iterator iter = m_data.begin(); iter != m_data.end(); ++iter) { *pWriter << iter->first << "\t" << iter->second.num_samples << "\t" << iter->second.num_errors << "\t" << (double)iter->second.num_errors / iter->second.num_samples << "\n"; } } private: DataMap m_data; }; #endif ================================================ FILE: src/SGA/Util/MultiAlignment.cpp ================================================ //----------------------------------------------- // Copyright 2011 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // MultiAlignment - Class constructing a multiple // alignment between a root sequence and a set of sequences // #include #include #include #include #include "MultiAlignment.h" #include "StdAlnTools.h" struct CigarIter { const MAlignData* pData; int symIdx; // the index of the cigar string we are parsing int baseIdx; // the index of the current base char updateAndEmit(char updateMode) { char cigSym = getCigarSymbol(); char outBase; if(updateMode == 'I') { if(cigSym == 'I') { symIdx += 1; outBase = '-'; } else if(cigSym == 'D') { assert(false); } else { symIdx += 1; outBase = getOutputBase(cigSym); if(outBase != '.' && outBase != '-') baseIdx += 1; } } else if(updateMode == 'D') { if(cigSym == 'D') { symIdx += 1; outBase = getOutputBase(cigSym); if(outBase != '.') baseIdx += 1; } else if(cigSym == 'I') { outBase = '-'; } else if(cigSym == 'S') { outBase = '.'; } else { outBase = '-'; //symIdx += 1; } } else { symIdx += 1; outBase = getOutputBase(cigSym); if(outBase != '.') baseIdx += 1; } return outBase; } char getCigarSymbol() { if(symIdx >= (int)pData->expandedCigar.size()) return 'S'; return pData->expandedCigar[symIdx]; } char getOutputBase(char cigSym) { if(cigSym == 'S' || cigSym == 'N') return '.'; else return pData->str[baseIdx]; } static bool sortPosition(const CigarIter& a, const CigarIter& b) { return a.pData->position < b.pData->position; } }; typedef std::vector CigarIterVector; MultiAlignment::MultiAlignment(std::string rootStr, const MAlignDataVector& inData, std::string rootName) { m_verbose = 1; // Build a padded multiple alignment from the pairwise alignments to the root CigarIterVector iterVec; // Create entry for the root MAlignData rootData; rootData.str = rootStr; rootData.expandedCigar = std::string(rootStr.size(), 'M'); rootData.position = 0; rootData.name = rootName; m_alignData.push_back(rootData); m_alignData.insert(m_alignData.end(), inData.begin(), inData.end()); CigarIter tmp = {&rootData, 0, 0}; iterVec.push_back(tmp); if(m_verbose > 1) printf("%d\t%s\n", 0, tmp.pData->expandedCigar.c_str()); for(size_t i = 0; i < inData.size(); ++i) { CigarIter iter = {&inData[i], 0, 0}; iterVec.push_back(iter); if(m_verbose > 1) printf("%zu\t%s\n", i+1, iter.pData->expandedCigar.c_str()); } // Build the padded strings by inserting '-' as necessary bool done = false; while(!done) { // Check if any strings have a deletion or insertion at this base bool hasDel = false; bool hasInsert = false; bool hasMatch = false; for(size_t i = 0; i < iterVec.size(); ++i) { char sym = iterVec[i].getCigarSymbol(); if(sym == 'D') hasDel = true; else if(sym == 'I') hasInsert = true; else if(sym == 'M') hasMatch = true; } done = !hasDel && !hasInsert && !hasMatch; if(done) break; char updateMode = hasDel ? 'D' : (hasInsert ? 'I' : 'M'); for(size_t i = 0; i < iterVec.size(); ++i) { char outSym = iterVec[i].updateAndEmit(updateMode); m_alignData[i].padded.append(1,outSym); } } } // size_t MultiAlignment::getIdxByName(const std::string& name) const { size_t max = std::numeric_limits::max(); size_t idx = max; for(size_t i = 0; i < m_alignData.size(); ++i) { if(m_alignData[i].name == name) { if(idx == max) { idx = i; } else { std::cerr << "Error in MultiAlignment::getIdxByName: duplicate rows found for " << name << "\n"; exit(EXIT_FAILURE); } } } if(idx == max) { std::cerr << "Error in MultiAlignment::getIdxByName: row not found for " << name << "\n"; exit(EXIT_FAILURE); } return idx; } // size_t MultiAlignment::getNumColumns() const { assert(!m_alignData.empty()); return m_alignData.front().padded.size(); } std::string MultiAlignment::getCigar(size_t rowIdx) const { assert(rowIdx < m_alignData.size()); std::cout << "MA has " << m_alignData.size() << " elements\n"; return StdAlnTools::compactCigar(m_alignData[rowIdx].expandedCigar); } // char MultiAlignment::getSymbol(size_t rowIdx, size_t colIdx) const { assert(rowIdx < m_alignData.size()); assert(colIdx < m_alignData[rowIdx].padded.size()); return m_alignData[rowIdx].padded[colIdx]; } size_t MultiAlignment::getBaseIdx(size_t rowIdx, size_t colIdx) const { assert(rowIdx < m_alignData.size()); assert(colIdx < m_alignData[rowIdx].padded.size()); // Convert the column index to the baseIndex in the sequence at // Ensure this is a real base on the target string. assert(getSymbol(rowIdx, colIdx) != '-'); // Substract the number of padding charcters from the column index to get the // base index size_t padSyms = 0; for(size_t i = 0; i < colIdx; ++i) { if(getSymbol(rowIdx, i) == '-') padSyms += 1; } assert(padSyms <= colIdx); return colIdx - padSyms; } // std::string MultiAlignment::getPaddedSubstr(size_t rowIdx, size_t start, size_t length) const { assert(rowIdx < m_alignData.size()); assert(start < m_alignData[rowIdx].padded.size()); assert(start + length <= m_alignData[rowIdx].padded.size()); return m_alignData[rowIdx].padded.substr(start, length); } // Count the length of the homopolymer starting at from size_t MultiAlignment::countHomopolymer(size_t rowIdx, int from, int to) const { assert(rowIdx < m_alignData.size()); assert(from < (int)m_alignData[rowIdx].padded.size()); // if(to == -1) to = m_alignData[rowIdx].padded.size() - 1; // inclusive // Determine iteration direction int step = from <= to ? 1 : -1; // The first or last column of the multiple alignment was requested // This can only be a homopolymer of length 1 if(from == (int)m_alignData[rowIdx].padded.size() - 1 || (from == 0 && step < 0)) return 1; // Get the base of the homopolymer // If it is a padding symbol we use the next non-padded base in the sequence char b = '\0'; int max_position = (int)m_alignData[rowIdx].padded.size() - 1; while(from >= 0 && from <= max_position) { b = getSymbol(rowIdx, from); if(b == '-') from += step; else break; } size_t length = 1; do { from += step; if(from < 0 || from > max_position) return length; char s = getSymbol(rowIdx, from); if(s == '-') continue; if(s == b) length += 1; else break; } while(from != to); return length; } // Generate simple consensus string from the multiple alignment std::string MultiAlignment::generateConsensus() { assert(!m_alignData.empty() && !m_alignData.front().padded.empty()); std::string consensus; std::string paddedConsensus; std::map baseMap; size_t num_rows = m_alignData.size() - 1; // do not include root size_t num_cols = m_alignData.front().padded.size(); for(size_t i = 0; i < num_cols; ++i) { baseMap.clear(); for(size_t j = 1; j < num_rows; ++j) { char b = m_alignData[j].padded[i]; if(b != '.') baseMap[b]++; } int max = 0; char maxBase = '.'; for(std::map::iterator iter = baseMap.begin(); iter != baseMap.end(); ++iter) { if(iter->second > max) { max = iter->second; maxBase = iter->first; } } paddedConsensus.append(1,maxBase); if(maxBase == '.' && consensus.empty()) continue; // skip no-call at beginning else if(maxBase == '.') break; // non-called position in middle of read, stop consensus generation else if(maxBase != '-') // consensus.append(1, maxBase); } if(m_verbose > 0) print(80, &paddedConsensus); return consensus; } // Generate a string representing the columns that differ between the strings std::string MultiAlignment::generateMatchString() const { assert(!m_alignData.empty() && !m_alignData.front().padded.empty()); std::string matchString; size_t num_rows = m_alignData.size(); size_t num_cols = m_alignData.front().padded.size(); for(size_t i = 0; i < num_cols; ++i) { char rootChar = m_alignData[0].padded[i]; bool allMatch = true; for(size_t j = 1; j < num_rows; ++j) { char b = m_alignData[j].padded[i]; if(b != rootChar) allMatch = false; } matchString.append(1, allMatch ? '*' : ' '); } return matchString; } // void MultiAlignment::filterByEditDistance(int max_edit_distance) { size_t num_rows = m_alignData.size(); assert(num_rows > 0); size_t num_cols = m_alignData.front().padded.size(); std::vector keep_vector(num_rows, true); for(size_t i = 1; i < num_rows; ++i) { int current_edit_distance = 0; for(size_t j = 0; j < num_cols; ++j) { char root_symbol = getSymbol(0, j); char seq_symbol = getSymbol(i, j); if(root_symbol != '.' && seq_symbol != '.' && seq_symbol != '-' && root_symbol != seq_symbol) current_edit_distance++; } if(current_edit_distance > max_edit_distance) keep_vector[i] = false; } MAlignDataVector tmp_ma; for(size_t i = 0; i < num_rows; ++i) { if(keep_vector[i]) tmp_ma.push_back(m_alignData[i]); } printf("Removed %zu\n", m_alignData.size() - tmp_ma.size()); m_alignData.swap(tmp_ma); } // void MultiAlignment::print(int col_size, const std::string* pConsensus, bool sorted, bool masked) const { assert(!m_alignData.empty() && !m_alignData.front().padded.empty()); std::string matchString = generateMatchString(); // Create a copy of the m_alignData and sort it by position MAlignDataVector sortedAlignments = m_alignData; if(sorted) std::stable_sort(sortedAlignments.begin(), sortedAlignments.end(), MAlignData::sortPosition); col_size = 100000; size_t len = sortedAlignments[0].padded.size(); for(size_t l = 0; l < len; l += col_size) { // Print the consensus if requested if(pConsensus != NULL) { int diff = pConsensus->size() - l; int stop = diff < col_size ? diff : col_size; if(stop > 0) printf("C\t%s\n", pConsensus->substr(l,stop).c_str()); else printf("C\n"); } // Print each row for(size_t i = 0; i < sortedAlignments.size(); ++i) { const MAlignData& mad = sortedAlignments[i]; int diff = mad.padded.size() - l; int stop = diff < col_size ? diff : col_size; std::string s = mad.padded.substr(l, stop).c_str(); if(masked) { for(size_t j = 0; j < s.size(); ++j) { // get base row symbol char rb = getSymbol(0, l + j); if(s[j] == rb && s[j] != '-') s[j] = '='; } } printf("%zu\t%s\t%s\n", i, s.c_str(), mad.name.c_str()); } // Print the matched columns int diff = matchString.size() - l; int stop = diff < col_size ? diff : col_size; printf("M\t%s\n", matchString.substr(l, stop).c_str()); std::cout << "\n"; } } // Construct a multiple alignment of the input strings from global alignments // to the first element in the vector MultiAlignment MultiAlignmentTools::alignSequencesGlobal(const SeqItemVector& sequences) { assert(!sequences.empty()); const std::string& base = sequences[0].seq.toString(); MAlignDataVector madVector; for(size_t i = 1; i < sequences.size(); ++i) { std::string seq = sequences[i].seq.toString(); std::string cigar = StdAlnTools::globalAlignmentCigar(seq, base); // Initialize the multiple alignment data MAlignData maData; maData.position = 0; maData.str = seq; maData.expandedCigar = StdAlnTools::expandCigar(cigar); maData.name = sequences[i].id; madVector.push_back(maData); } return MultiAlignment(base, madVector, sequences[0].id); } // Construct a multiple alignment of the input strings from local alignments // to the first element of the vector MultiAlignment MultiAlignmentTools::alignSequencesLocal(const SeqItemVector& sequences) { assert(!sequences.empty()); const std::string& base = sequences[0].seq.toString(); MAlignDataVector madVector; for(size_t i = 1; i < sequences.size(); ++i) { // The alignment is (slightly counterintuitively) with respect to the // base string as the query so that all the cigar strings used // are based on edit operations to the base. std::string seq = sequences[i].seq.toString(); LocalAlignmentResult result = StdAlnTools::localAlignment(seq,base); // Initialize the multiple alignment data MAlignData maData; maData.position = result.queryStartIndex; // If the non-base sequence overhangs the base, clip it if(result.targetStartIndex > 0) maData.str = seq.substr(result.targetStartIndex); else maData.str = seq; // Pad out the cigar with reference skip characters maData.expandedCigar = StdAlnTools::expandCigar(result.cigar); maData.name = sequences[i].id; madVector.push_back(maData); } return MultiAlignment(base, madVector, sequences[0].id); } ================================================ FILE: src/SGA/Util/MultiAlignment.h ================================================ //----------------------------------------------- // Copyright 2011 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // MultiAlignment - Class constructing a multiple // alignment between a root sequence and a set of sequences // #ifndef MULTIALIGNMENT_H #define MULTIALIGNMENT_H #include #include #include "Util.h" struct MAlignData { public: std::string str; std::string expandedCigar; // cigar string with symbol counts expanded int position; // start position of the alignment to the root std::string name; // named identifier for this sequence in the MA private: friend class MultiAlignment; std::string padded; // Comparator static bool sortPosition(const MAlignData& a, const MAlignData& b) { return a.position < b.position; } }; typedef std::vector MAlignDataVector; class MultiAlignment { public: MultiAlignment(std::string rootStr, const MAlignDataVector& inData, std::string rootName = "root"); // Experimental function to generate a consensus sequence from the MA std::string generateConsensus(); // Get the index into the m_alignData vector for a named row size_t getIdxByName(const std::string& name) const; size_t getRootIdx() const { return 0; } // Get the symbol at a particular column and row char getSymbol(size_t rowIdx, size_t colIdx) const; // Get the index of the base corresponding to colIdx for sequence at rowIdx size_t getBaseIdx(size_t rowIdx, size_t colIdx) const; // Get a substring of the padded string for the given row std::string getPaddedSubstr(size_t rowIdx, size_t start, size_t length) const; // Count the length of the homopolymer run in the substring [from, to] inclusive // If to is npos, then it is assumed to be until the end of the string // if to < from, then the count proceeds backwards (towards the beginning of the string. size_t countHomopolymer(size_t rowIdx, int from, int to = -1) const; // Return the total number of columns in the MA size_t getNumColumns() const; // Return the cigar string for one of the alignments std::string getCigar(size_t rowIdx) const; // Filter the multiple alignment by removing sequences that are more than max_edit_distance from the root sequence void filterByEditDistance(int max_edit_distance); // Print the multiple alignment, optionally with a consensus sequence void print(int col_size = 80, const std::string* pConsensus = NULL, bool sorted = true, bool masked = false) const; private: // Generate a string representing the columns that differ between the strings std::string generateMatchString() const; // data MAlignDataVector m_alignData; int m_verbose; }; namespace MultiAlignmentTools { // Construct a multiple alignment of the input strings MultiAlignment alignSequencesGlobal(const SeqItemVector& sequences); MultiAlignment alignSequencesLocal(const SeqItemVector& sequences); } #endif ================================================ FILE: src/SGA/Util/MultiOverlap.cpp ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // MultiOverlap.h - Data structure containing a set // of overlaps for a given read // #include #include #include "CorrectionThresholds.h" #include "MultiOverlap.h" #include "Alphabet.h" MultiOverlap::MultiOverlap(const std::string& rootID, const std::string& rootSeq, const std::string rootQual) : m_rootID(rootID), m_rootSeq(rootSeq), m_rootQual(rootQual) { } // void MultiOverlap::add(const std::string& seq, const Overlap& ovr) { MOData mod(seq, ovr); // Swap root read into first position if necessary if(mod.ovr.id[0] != m_rootID) mod.ovr.swap(); assert(mod.ovr.id[0] == m_rootID); // RC the sequence if it is different orientation than the root if(mod.ovr.match.isRC()) { mod.seq = reverseComplement(mod.seq); mod.ovr.match.canonize(); } // Initialize the offset value, the amount that a coordinate // for the non-root sequence must be shifted so that // the sequences are aligned mod.offset = mod.ovr.match.inverseTranslate(0); m_overlaps.push_back(mod); } // void MultiOverlap::add(const MOData& mod) { assert(mod.ovr.id[0] == m_rootID); m_overlaps.push_back(mod); } // void MultiOverlap::updateRootSeq(const std::string& newSeq) { m_rootSeq = newSeq; } // Overlap MultiOverlap::getOverlap(size_t idx) const { assert(idx < m_overlaps.size()); return m_overlaps[idx].ovr; } // std::string MultiOverlap::simpleConsensus() const { std::string out; for(size_t i = 0; i < m_rootSeq.size(); ++i) { Pileup p = getPileup(i); AlphaCount64 ac = p.getAlphaCount(); char maxBase; BaseCount maxCount; ac.getMax(maxBase, maxCount); BaseCount rootCount = ac.get(m_rootSeq[i]); if(rootCount == maxCount) out.push_back(m_rootSeq[i]); else out.push_back(maxBase); } return out; } // int MultiOverlap::countPotentialIncorrect(size_t cutoff) const { int count = 0; for(size_t i = 0; i < m_rootSeq.size(); ++i) { Pileup p = getPileup(i); AlphaCount64 ac = p.getAlphaCount(); char maxBase; BaseCount maxCount; ac.getMax(maxBase, maxCount); BaseCount rootCount = ac.get(m_rootSeq[i]); if(maxBase != m_rootSeq[i] && rootCount < cutoff) ++count; } return count; } // int MultiOverlap::countBasesCovered() const { int count = 0; for(size_t i = 0; i < m_rootSeq.size(); ++i) { Pileup p = getPileup(i); if(p.getDepth() > 1) ++count; } return count; } // Determine if this multi-overlap has a conflicted position, // which is a position in the multioverlap where the second-most // prevalent base has a frequency greater than cutoff bool MultiOverlap::isConflicted(size_t cutoff) const { for(size_t i = 0; i < m_rootSeq.size(); ++i) { Pileup p = getPileup(i); AlphaCount64 ac = p.getAlphaCount(); char order[5]; ac.getSorted(order, 5); // If the second-most prevalent base is greater than the cutoff // we consider the MO to be conflicted if(ac.get(order[1]) > cutoff) return true; } return false; } // Returns true if the overlap at idx has the include flag set int MultiOverlap::getPartition(size_t idx) const { assert(idx < m_overlaps.size()); return m_overlaps[idx].partitionID; } // Returns true if the overlap at idx has the include flag set void MultiOverlap::setPartition(size_t idx, int p) { assert(idx < m_overlaps.size()); m_overlaps[idx].partitionID = p; } // Return the total number of bases in the multioverlap size_t MultiOverlap::getNumBases() const { size_t count = 0; for(size_t i = 0; i < m_rootSeq.size(); ++i) { Pileup p = getPileup(i); count += p.getDepth(); } return count; } // Conflict-aware consensus algorithm // For each position of the read, it calculates whether that position is conflicted (there is // sufficient support for an alternative base at that position). All reads that differ from the // root read at a conflicted position are filtered out. The remaining set of reads match the root read // at all the conflicted positions and the consensus is called from this set. Bases that are not conflicted // are called from the entire set of overlaps. std::string MultiOverlap::consensusConflict(double /*p_error*/, int conflictCutoff) { // Calculate the frequency vector for each base of the read std::vector acVec; for(size_t i = 0; i < m_rootSeq.size(); ++i) { AlphaCount64 ac = getAlphaCount(i); acVec.push_back(ac); } // Sort the alphacounts by frequency StringVector sortedVec; sortedVec.reserve(acVec.size()); for(size_t i = 0; i < acVec.size(); ++i) { char sorted[ALPHABET_SIZE]; acVec[i].getSorted(sorted, ALPHABET_SIZE); sortedVec.push_back(std::string(sorted, ALPHABET_SIZE)); } // Filter out overlaps that do not match the reference // at conflicted positions for(size_t j = 0; j < m_overlaps.size(); ++j) { int numMatch = 0; int numMismatch = 0; int numConflicted = 0; for(size_t i = 0; i < acVec.size(); ++i) { char rootBase = m_rootSeq[i]; std::string& sorted = sortedVec[i]; int second = acVec[i].get(sorted[1]); // If the second-most prevelent base is above the conflict cutoff, // call this position conflicted bool isConflict = second > conflictCutoff; // Get the basecall for read j at position i char b = getMODBase(m_overlaps[j], i); if(isConflict) { // Check if the read matches the root if: // a) the read contains a basecall at this position (it overlaps // the read at this position) // b) the root read base is one of the two most frequent bases // (to filter out sequencing errors at this position in the root). int rootCount = acVec[i].get(rootBase); if(b != '\0' && rootCount > conflictCutoff) { if(b == rootBase) ++numMatch; else ++numMismatch; ++numConflicted; } } } // Set the overlap score to be the fraction of conflict bases that this read // matches the root read at. double frac; if(numConflicted == 0) frac = 1.0f; else frac = (double)numMatch/(double)numConflicted; m_overlaps[j].score = frac; // Filter out the read if there are any conflicted bases // that this read does not match the root read at if(numConflicted > 0 && numMismatch > 0) m_overlaps[j].partitionID = 1; else m_overlaps[j].partitionID = 0; } // Calculate the consensus sequence using all the reads // in partition 1 std::string consensus; std::vector supportVector; for(size_t i = 0; i < m_rootSeq.size(); ++i) { Pileup p0; Pileup p1; getPartitionedPileup(i, p0, p1); AlphaCount64 ac = p0.getAlphaCount(); size_t minSupport = CorrectionThresholds::Instance().getMinSupportLowQuality(); if(!m_rootQual.empty()) { int phredScore = Quality::char2phred(m_rootQual[i]); minSupport = CorrectionThresholds::Instance().getRequiredSupport(phredScore); } size_t callSupport = ac.get(m_rootSeq[i]); if(callSupport >= minSupport) { // This base does not require correction consensus.push_back(m_rootSeq[i]); } else { // Attempt to correct the base with the most frequent base // in the partitioned pileup if it has been seen more often // than the root base char sorted[ALPHABET_SIZE]; ac.getSorted(sorted, ALPHABET_SIZE); size_t bestSupport = ac.get(sorted[0]); bool corrected = false; if(bestSupport > callSupport) { consensus.push_back(sorted[0]); callSupport = bestSupport; corrected = true; } else { // A correction could not be made with the // partitioned pileup, try to use the full pileup // if this base is not conflicted acVec[i].getSorted(sorted, ALPHABET_SIZE); int second = acVec[i].get(sorted[1]); bool isConflict = second > conflictCutoff; if(!isConflict) { bestSupport = acVec[i].get(sorted[0]); if(bestSupport > callSupport) { consensus.push_back(sorted[0]); corrected = true; } } } if(!corrected) { consensus.push_back(m_rootSeq[i]); } } } return consensus; } // bool MultiOverlap::qcCheck() const { for(size_t i = 0; i < m_rootSeq.size(); ++i) { AlphaCount64 ac = getAlphaCount(i); size_t callSupport = ac.get(m_rootSeq[i]); if(callSupport < 2) return false; } return true; } // void MultiOverlap::countOverlaps(size_t& prefix_count, size_t& suffix_count) const { prefix_count = 0; suffix_count = 0; for(size_t i = 0; i < m_overlaps.size(); ++i) { if(!m_overlaps[i].ovr.match.coord[0].isContained()) { if(m_overlaps[i].ovr.match.coord[0].isLeftExtreme()) ++prefix_count; if(m_overlaps[i].ovr.match.coord[0].isRightExtreme()) ++suffix_count; } } } // double MultiOverlap::getMeanDepth() const { double depth = 0.0f; for(size_t i = 0; i < m_rootSeq.size(); ++i) { Pileup p = getPileup(i); depth += p.getDepth(); } return depth / m_rootSeq.size(); } // int MultiOverlap::calculateCoverageOverlap() { int rightmost_prefix = -1; int leftmost_suffix = m_rootSeq.length(); for(size_t i = 0; i < m_overlaps.size(); ++i) { if(!m_overlaps[i].ovr.match.coord[0].isContained()) { if(m_overlaps[i].ovr.match.coord[0].isLeftExtreme()) { // Prefix overlap int end = m_overlaps[i].ovr.match.coord[0].interval.end; if(end > rightmost_prefix) rightmost_prefix = end; } else { // Suffix overlap int start = m_overlaps[i].ovr.match.coord[0].interval.start; if(start < leftmost_suffix) leftmost_suffix = start; } } } if(leftmost_suffix > rightmost_prefix) return 0; else return rightmost_prefix - leftmost_suffix + 1; } std::string MultiOverlap::calculateConsensusFromPartition(double p_error) { std::string out; out.reserve(m_rootSeq.size()); // require the best base call to be above this above to correct it double epsilon = 0.01; for(size_t i = 0; i < m_rootSeq.size(); ++i) { Pileup p0; Pileup p1; getPartitionedPileup(i, p0, p1); DNADouble ap = p0.calculateLikelihoodNoQuality(p_error); char best_c; char curr_c = m_rootSeq[i]; double max; ap.getMax(best_c, max); //printf("%zu best: %c curr: %c max: %lf curr_l: %lf\n", i, best_c, curr_c, max, ap.get(curr_c)); // Require the called value to be substantially better than the // current base if(best_c == curr_c || (best_c != curr_c && max - ap.get(curr_c) < epsilon)) { //std::cout << "using current\n"; out.push_back(curr_c); } else { //std::cout << "corrected\n"; out.push_back(best_c); } } return out; } // Return the base in mod that matches the base at // idx in the root seq. If mod does not overlap // the root at this position, returns '\0' char MultiOverlap::getMODBase(const MOData& mod, int idx) const { int trans_idx = idx - mod.offset; if(trans_idx >= 0 && size_t(trans_idx) < mod.seq.size()) return mod.seq[trans_idx]; else return '\0'; } // Get an AlphaCount64 representing the nucleotides // observed at the given column AlphaCount64 MultiOverlap::getAlphaCount(int idx) const { AlphaCount64 ac; ac.increment(m_rootSeq[idx]); for(size_t i = 0; i < m_overlaps.size(); ++i) { const MOData& curr = m_overlaps[i]; // translate idx into the frame of the current sequence int trans_idx = idx - curr.offset; if(trans_idx >= 0 && size_t(trans_idx) < curr.seq.size()) { ac.increment(curr.seq[trans_idx]); } } return ac; } // Get the "stack" of bases that aligns to // a single position of the root seq, including // the root base Pileup MultiOverlap::getPileup(int idx) const { size_t max_depth = m_overlaps.size() + 1; Pileup p(max_depth); p.add(m_rootSeq[idx]); for(size_t i = 0; i < m_overlaps.size(); ++i) { const MOData& curr = m_overlaps[i]; // translate idx into the frame of the current sequence int trans_idx = idx - curr.offset; if(trans_idx >= 0 && size_t(trans_idx) < curr.seq.size()) { p.add(curr.seq[trans_idx]); } } return p; } // Get the "stack" of bases that aligns to // a single position of the root seq, including // the root base only including the first numElems items Pileup MultiOverlap::getPileup(int idx, int numElems) const { assert((size_t)numElems < m_overlaps.size()); Pileup p; p.add(m_rootSeq[idx]); for(int i = 0; i < numElems; ++i) { const MOData& curr = m_overlaps[i]; // translate idx into the frame of the current sequence int trans_idx = idx - curr.offset; if(trans_idx >= 0 && (size_t)trans_idx < curr.seq.size()) { p.add(curr.seq[trans_idx]); } } return p; } // Get the pileup between the root sequence and one of the sequences in the MO Pileup MultiOverlap::getSingletonPileup(int base_idx, int ovr_idx) const { assert(ovr_idx < (int)m_overlaps.size()); Pileup p; p.add(m_rootSeq[base_idx]); const MOData& curr = m_overlaps[ovr_idx]; // translate idx into the frame of the current sequence int trans_idx = base_idx - curr.offset; if(trans_idx >= 0 && size_t(trans_idx) < curr.seq.size()) { p.add(curr.seq[trans_idx]); } return p; } // Fill in the pileup groups g0 and g1 void MultiOverlap::getPartitionedPileup(int idx, Pileup& g0, Pileup& g1) const { g0.add(m_rootSeq[idx]); for(size_t i = 0; i < m_overlaps.size(); ++i) { const MOData& curr = m_overlaps[i]; // translate idx into the frame of the current sequence int trans_idx = idx - curr.offset; if(trans_idx >= 0 && size_t(trans_idx) < curr.seq.size()) { if(curr.partitionID == 0) g0.add(curr.seq[trans_idx]); else g1.add(curr.seq[trans_idx]); } } } // Fill in the pileup groups g0 and g1 PileupVector MultiOverlap::getPartitionedPileup(int idx, int num_parts) const { assert(false && "untested"); PileupVector pv(num_parts); pv.push_back(Pileup()); // the root always goes in partition 0 pv.back().add(m_rootSeq[idx]); for(size_t i = 0; i < m_overlaps.size(); ++i) { const MOData& curr = m_overlaps[i]; // translate idx into the frame of the current sequence int trans_idx = idx - curr.offset; if(trans_idx >= 0 && size_t(trans_idx) < curr.seq.size()) { assert(curr.partitionID < num_parts); pv[curr.partitionID].add(curr.seq[trans_idx]); } } return pv; } // Print the MultiOverlap groups specified by the IntVec to stdout void MultiOverlap::printGroups() { for(int i = 0; i <= 1; ++i) { MultiOverlap mo_group(m_rootID, m_rootSeq); for(size_t j = 0; j < m_overlaps.size(); ++j) { const MOData& curr = m_overlaps[j]; if(curr.partitionID == i) mo_group.add(curr); } std::cout << "MO GROUP " << i << "\n"; mo_group.print(); } } // Print the MultiOverlap to stdout void MultiOverlap::print(int default_padding, int max_overhang) { std::sort(m_overlaps.begin(), m_overlaps.end(), MOData::sortOffset); std::cout << "\nDrawing overlaps for read " << m_rootID << "\n"; int root_len = int(m_rootSeq.size()); // Print the root row at the bottom printRow(default_padding, max_overhang, root_len, 0, root_len, 0, 0.0f, m_rootSeq, m_rootID); for(size_t i = 0; i < m_overlaps.size(); ++i) { const MOData& curr = m_overlaps[i]; int overlap_len = curr.ovr.match.getMaxOverlapLength(); int nd = curr.ovr.match.countDifferences(m_rootSeq, curr.seq); double score = static_cast(nd) / overlap_len; printRow(default_padding, max_overhang, root_len, curr.offset, overlap_len, nd, score, curr.seq, curr.ovr.id[1].c_str()); } } // Print a single row of a multi-overlap to stdout void MultiOverlap::printRow(int default_padding, int max_overhang, int root_len, int offset, int overlap_len, int pid, double score, const std::string& seq, const std::string& id) { int c_len = seq.length(); // This string runs from c_offset to c_offset + len // Clip the string at -max_overhang to root_len + max_overhang int left_clip = std::max(offset, -max_overhang); int right_clip = std::min(offset + c_len, root_len + max_overhang); // translate the clipping coordinates to the string coords int t_left_clip = left_clip - offset; int t_right_clip = right_clip - offset; // Calculate the length of the left padding int padding = default_padding + left_clip; std::string leader = (t_left_clip > 0) ? "..." : ""; std::string trailer = (t_right_clip < c_len) ? "..." : ""; std::string clipped = seq.substr(t_left_clip, t_right_clip - t_left_clip); padding -= leader.size(); assert(padding >= 0); std::string padding_str(padding, ' '); std::string outstr = padding_str + leader + clipped + trailer; printf("%s\t%d\t%d\t%lf\tID:%s\n", outstr.c_str(), overlap_len, pid, score, id.c_str()); } void MultiOverlap::printMasked() { printf("ROOT\t%s\t%s\n", m_rootSeq.c_str(), m_rootID.c_str()); for(size_t j = 0; j < m_overlaps.size(); ++j) { std::string out; for(size_t i = 0; i < m_rootSeq.length(); ++i) { char b = getMODBase(m_overlaps[j], i); if(b == '\0') out.push_back('.'); else if(b == m_rootSeq[i]) out.push_back('='); else out.push_back(b); } printf("OVRL\t%s\t%s\n", out.c_str(), m_overlaps[j].ovr.id[1].c_str()); } } // Print the MultiOverlap horizontally, in a pileup format void MultiOverlap::printPileup() { std::cout << "\nDrawing overlap pileup for read " << m_rootID << "\n"; for(size_t i = 0; i < m_rootSeq.size(); ++i) { Pileup p = getPileup(i); printf("%zu\t%s\n", i, p.toStr().c_str()); } } // bool MultiOverlap::MOData::sortOffset(const MOData& a, const MOData& b) { return a.offset < b.offset; } // Sort by the ID of the non-root sequence, which is // guarenteed to bethe second id in the overlap structure bool MultiOverlap::MOData::sortID(const MOData& a, const MOData& b) { assert(a.ovr.id[0] == b.ovr.id[0]); return a.ovr.id[1] < b.ovr.id[1]; } ================================================ FILE: src/SGA/Util/MultiOverlap.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // MultiOverlap.h - Data structure containing a set // of overlaps for a given read // #ifndef MULTIOVERLAP_H #define MULTIOVERLAP_H #include "Match.h" #include "Pileup.h" #include "DNADouble.h" class MultiOverlap { public: struct MOData { MOData(const std::string& s, const Overlap& o) : seq(s), ovr(o), offset(0), partitionID(0), score(0.0f) {} static bool sortOffset(const MOData& a, const MOData& b); static bool sortID(const MOData& a, const MOData& b); // data std::string seq; Overlap ovr; int offset; int partitionID; double score; static bool compareScore(const MOData& a, const MOData& b) { return a.score > b.score; } }; typedef std::vector MODVector; public: MultiOverlap(const std::string& rootID, const std::string& rootSeq, const std::string rootQual = ""); /// void add(const std::string& seq, const Overlap& ovr); void add(const MOData& mod); void updateRootSeq(const std::string& newSeq); // Overlap getOverlap(size_t idx) const; size_t getNumBases() const; // bool isConflicted(size_t cutoff) const; std::string simpleConsensus() const; // Count the number of bases that are potentially incorrect // A base is considered to be incorrect if it is not the majority in the column // and it has been seen less than cutoff times int countPotentialIncorrect(size_t cutoff) const; // Count the number of bases in the read that are covered by an overlap int countBasesCovered() const; // Partition the multioverlap into groups int getPartition(size_t idx) const; void setPartition(size_t idx, int p); std::string consensusConflict(double p_error, int conflictCutoff); // Returns true if each base of the root sequence has enough support from // other reads in the multioverlap. The level of support is determined by // the quality score. bool qcCheck() const; // Count the number of prefix and suffix overlaps void countOverlaps(size_t& prefix_count, size_t& suffix_count) const; double getMeanDepth() const; // Calculate the amount of the sequence that is covered // by both prefix and suffix overlaps. For instance: // Read --------------- // OvrP -------------- // OvrX ----------- // xx // In this case the function would return 2. // If there is no overlap, zero is returned // Read --------------- // OvrP --------- // OvrX ------------- int calculateCoverageOverlap(); std::string calculateConsensusFromPartition(double p_error); char getMODBase(const MOData& mod, int idx) const; // IO void print(int default_padding = DEFAULT_PADDING, int max_overhang = DEFAULT_MAX_OVERHANG); void printMasked(); void printPileup(); void printGroups(); private: AlphaCount64 getAlphaCount(int idx) const; Pileup getPileup(int idx) const; Pileup getPileup(int idx, int numElems) const; Pileup getSingletonPileup(int base_idx, int ovr_idx) const; void getPartitionedPileup(int idx, Pileup& g0, Pileup& g1) const; PileupVector getPartitionedPileup(int idx, int num_parts) const; void printRow(int default_padding, int max_overhang, int root_len, int offset, int overlap_len, int pid, double score, const std::string& seq, const std::string& id); // data static const int DEFAULT_PADDING = 20; static const int DEFAULT_MAX_OVERHANG = 3; std::string m_rootID; std::string m_rootSeq; std::string m_rootQual; MODVector m_overlaps; }; #endif ================================================ FILE: src/SGA/Util/NoCodec.h ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // NoCodec - Testing codec for EncodedString // that does not encode anything - it just returns // the numerical value of the char // #ifndef NOCODEC_H #define NOCODEC_H class NoCodec { public: typedef char UNIT_TYPE; static const int SYMBOLS_PER_UNIT = 1; // Encoded the character b into a value inline uint8_t encode(char b) const { return (uint8_t)b; } // Decode the value c into a character inline char decode(uint8_t c) const { return (char)c; } // Returns the number of units required to encode // a string of length n inline size_t getRequiredUnits(size_t n) const { return (n + SYMBOLS_PER_UNIT - 1) / SYMBOLS_PER_UNIT; } // Returns the number of symbols that can be stored // in n units inline size_t getCapacity(size_t n) const { return n * SYMBOLS_PER_UNIT; } // Returns the index of the unit to store the // i-th symbol of the string inline size_t getUnitIndex(size_t i) const { return i / SYMBOLS_PER_UNIT; } // Returns the position within a unit that the i-th symbol // should be stored in inline size_t getUnitOffset(size_t i) const { // this position is the k-th symbol of the unit return i % SYMBOLS_PER_UNIT; } // Return the amount that a value must be shifted to // store a code at a given offset inline uint8_t getOffsetShift(size_t /*offset*/) const { return 0; } // Store the value v at the i-th encoded position in the data array inline void store(UNIT_TYPE* pData, size_t i, char b) const { UNIT_TYPE& unit = pData[getUnitIndex(i)]; unit = encode(b); size_t offset = getUnitOffset(i); uint8_t shift = getOffsetShift(offset); // Clear the currrent position uint8_t mask = 0xFF; unit &= ~mask; // Set position UNIT_TYPE code = encode(b); code <<= shift; unit |= code; } // get the character stored at position i inline char get(const UNIT_TYPE* pData, size_t i) const { const UNIT_TYPE& unit = pData[getUnitIndex(i)]; size_t offset = getUnitOffset(i); (void)offset; uint8_t mask = 0xFF; UNIT_TYPE code = unit & mask; uint8_t shift = 0; code >>= shift; return decode(code); } }; #endif ================================================ FILE: src/SGA/Util/Pileup.cpp ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // Pileup - An array of strings containing all the seen // bases for a given region/read // #include "Pileup.h" #include "Alphabet.h" #include #include // Calculate the consensus base at this position // using a simple model where all bases are treated equally char Pileup::calculateSimpleConsensus() const { assert(!m_data.empty()); AlphaCount64 ac; for(size_t i = 0; i < m_data.size(); ++i) { ac.increment(m_data[i].base); } return ac.getMaxBase(); } // Returns the number of times each base appears in the // pileup string AlphaCount64 Pileup::getAlphaCount() const { AlphaCount64 ac; for(size_t i = 0; i < m_data.size(); ++i) { ac.increment(m_data[i].base); } return ac; } // Calculate the consensus base at this position // using a simple model where all bases are treated equally DNADouble Pileup::calculateSimpleAlphaProb() const { DNADouble ap; assert(!m_data.empty()); WARN_ONCE("Fix Pileup::calculateSimpleDNADouble numerical stability"); assert(false); for(size_t i = 0; i < ap.getAlphabetSize(); ++i) { // Calculate the posterior probability of the data given that // b is the true base char b = ALPHABET[i]; double posterior = 0.0f; for(size_t i = 0; i < m_data.size(); ++i) { if(m_data[i].base == b) posterior += log(1 - exp(m_data[1].lp)); else posterior += m_data[1].lp; } ap.set(b, posterior); } // Calculate the marginal probabilty of the data double marginal = 0.0f; for(size_t i = 0; i < DNA_ALPHABET_SIZE; ++i) { char b = ALPHABET[i]; marginal += exp(ap.get(b)); } // Scale the posterior probabilites by the marginal marginal = log(marginal); for(size_t i = 0; i < ap.getAlphabetSize(); ++i) { char b = DNA_ALPHABET::getBase(i); double lp = ap.get(b); //std::cout << "Marginal: " << marginal << " lp: " << lp << " scaled: " << lp - marginal << "\n"; ap.set(b, lp - marginal); } return ap; } // Calculate the likelihood of the data given the base call is // {A,C,G,T}. DNADouble Pileup::calculateLikelihoodNoQuality(double p_error) const { DNADouble ap; assert(!m_data.empty()); double p_correct = 1.0 - p_error; double log_error = log(p_error); double log_correct = log(p_correct); for(size_t i = 0; i < ap.getAlphabetSize(); ++i) { // Calculate the likelihood of the data given b is the true base char b = ap.getBase(i); double likelihood = 0.0f; for(size_t i = 0; i < m_data.size(); ++i) { if(m_data[i].base == b) likelihood += log_correct; else likelihood += log_error; } ap.set(b, likelihood); } return ap; } // char Pileup::getCount(char base) const { AlphaCount64 ac; for(size_t i = 0; i < m_data.size(); ++i) { ac.increment(m_data[i].base); } return ac.get(base); } // char Pileup::getBase(size_t idx) const { assert(idx < m_data.size()); return m_data[idx].base; } // size_t Pileup::getDepth() const { return m_data.size(); } // std::string Pileup::toStr() const { std::string out; out.reserve(m_data.size()); for(size_t i = 0; i < m_data.size(); ++i) out.append(1, m_data[i].base); return out; } ================================================ FILE: src/SGA/Util/Pileup.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL license //----------------------------------------------- // // Pileup - An array of strings containing all the seen // bases for a given region/read // #ifndef PILEUP_H #define PILEUP_H #include "Util.h" #include "Alphabet.h" #include "DNADouble.h" #define DEFAULT_PROB 0.01 #define DEFAULT_LOG_PROB -4.605170186f struct PUElem { PUElem() : base('A'), lp(0) {} PUElem(char b, double l) : base(b), lp(l) {} char base; // log probability this base is correct double lp; }; typedef std::vector PUElemVector; class Pileup { public: Pileup() {} Pileup(size_t n) { m_data.reserve(n); } char calculateSimpleConsensus() const; AlphaCount64 getAlphaCount() const; DNADouble calculateSimpleAlphaProb() const; DNADouble calculateLikelihoodNoQuality(double p_error) const; inline void add(char b) { m_data.push_back(PUElem(b, DEFAULT_LOG_PROB)); } inline void add(char b, double lp) { m_data.push_back(PUElem(b, lp)); } char getBase(size_t idx) const; char getCount(char base) const; size_t getDepth() const; std::string toStr() const; private: PUElemVector m_data; }; typedef std::vector PileupVector; #endif ================================================ FILE: src/SGA/Util/PrimerScreen.cpp ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // PrimerScreen - Singleton class to filter sequences // that match a database of primer sequences // #include "PrimerScreen.h" // Hardcoded primer sequences that we wish to filter against // Primers used in the Sanger's pcr-free library prep // See Kozarewa et al. (http://www.nature.com/nmeth/journal/v6/n4/full/nmeth.1311.html) // supplemental info. We do not use the whole primer sequences. #define ILLUMINA_SANGER_PCR_FREE_A "AATGATACGGCGACCACCGAGATCTACA" #define ILLUMINA_SANGER_PCR_FREE_B "GATCGGAAGAGCGGTTCAGCAGGAATGC" PrimerScreen::PrimerScreen() { m_db.push_back(ILLUMINA_SANGER_PCR_FREE_A); m_db.push_back(ILLUMINA_SANGER_PCR_FREE_B); } // Check seq against the primer database bool PrimerScreen::containsPrimer(const std::string& seq) { static PrimerScreen screener; // initializes singleton object if necessary // For now we only check if the first 14 // bases of seq is a perfect match to any sequence in the db // This is sufficient to get rid of the vast majority of the primer // contamination const size_t check_size = 14; std::string check = seq.substr(0, check_size); for(size_t i = 0; i < screener.m_db.size(); ++i) { if(screener.m_db[i].find(check) != std::string::npos) return true; } return false; } ================================================ FILE: src/SGA/Util/PrimerScreen.h ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // PrimerScreen - Singleton class to filter sequences // that match a database of primer sequences // #ifndef PRIMERSCREEN_H #define PRIMERSCREEN_H #include "Util.h" class PrimerScreen { public: // Return true if the sequence fails the primer check static bool containsPrimer(const std::string& seq); private: PrimerScreen(); StringVector m_db; }; #endif ================================================ FILE: src/SGA/Util/Profiler.h ================================================ ///---------------------------------------------- // Copyright 2011 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // Profiler.h -- Lightweight macro-based function profiler. // #ifndef PROFILER_H #define PROFILER_H #include #include #include "config.h" //#define USE_PROFILER 1 #if defined(HAVE_CLOCK_GETTIME) && defined(USE_PROFILER) // Change this to determine how often the profile should print #define PROFILE_TICKS_BEFORE_PRINT 1000 // This class writes the lifespan of the object // to the output variable, in nanoseconds class TimeTracker { public: TimeTracker(size_t & output) : m_output(output) { timespec start; clock_gettime(CLOCK_REALTIME, &start); m_start_ns = start.tv_sec * 1000000000 + start.tv_nsec; } ~TimeTracker() { timespec end; clock_gettime(CLOCK_REALTIME, &end); size_t end_ns = end.tv_sec * 1000000000 + end.tv_nsec; // Update the result using an atomic compare and swap size_t diff = end_ns - m_start_ns; while(!__sync_bool_compare_and_swap(&m_output, m_output, m_output + diff)) {} } private: size_t m_start_ns; size_t& m_output; }; // Place this macros at the start of the function you wish the profile // The static variable updates are done via atomic compare and swaps so // the profiling should be threadsafe #define PROFILE_FUNC(x) static std::string __profile_name = x; \ static size_t __profile_iterations = 0; \ static size_t __profile_total_nanoseconds = 0; \ double milli_seconds = (double)__profile_total_nanoseconds / 1000000.0f; \ double avg_per_iteration = milli_seconds / __profile_iterations; \ while(!__sync_bool_compare_and_swap(&__profile_iterations, __profile_iterations, __profile_iterations + 1)) { } \ if(__profile_iterations % PROFILE_TICKS_BEFORE_PRINT == 0) \ printf("[Profile] count: %zu time: %.0lf ms avg: %.0lf ms func: %s\n", __profile_iterations, milli_seconds, avg_per_iteration, __profile_name.c_str()); \ TimeTracker __profile_timer(__profile_total_nanoseconds); #else // Eliminate the macro #define PROFILE_FUNC(x) #endif // #ifdef HAVE_CLOCK_GETTIME #endif // #ifndef PROFILER_H ================================================ FILE: src/SGA/Util/Quality.cpp ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // Quality - functions for manipulating quality values // #include "Quality.h" // Return a uniform log-scaled quality vector of the given size DoubleVector Quality::uniformLogProbVector(double p_error, size_t n) { double lp = log(p_error); DoubleVector dv; dv.reserve(n); for(size_t i = 0; i < n; ++i) dv.push_back(lp); return dv; } // Return a string which encodes the log-scaled double vector as characters std::string Quality::encodeLogProbVector(const DoubleVector& dv) { std::string out; out.reserve(dv.size()); for(size_t i = 0; i < dv.size(); ++i) out.push_back(phred2char(lnprob2phred(dv[i]))); return out; } ================================================ FILE: src/SGA/Util/Quality.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // Quality - functions for manipulating quality values // #ifndef QUALITY_H #define QUALITY_H #include #include #include #include #include #include static const int DEFAULT_QUAL_SCORE = 15; static const int PHRED64_DIFF = 31; typedef std::vector DoubleVector; namespace Quality { // Convert the quality character from phred64 to phred33 encoding inline char phred64toPhred33(char c) { return (int)c - PHRED64_DIFF; } // Returns true if the character c is a valid phred 33 encoding // of a quality value in the range [0, 60] inline bool isValidPhred33(char c) { int p = (int)c - 33; return p >= 0 && p <= 60; } // Phred score transformations inline int char2phred(char b) { uint8_t v = b; assert(v >= 33); return v - 33; } inline char phred2char(int p) { uint8_t v = (p <= 93) ? p : 93; char c = v + 33; return c; } inline int prob2phred(double p) { return static_cast(round(-10.0f * log10(p))); } inline int lnprob2phred(double lp) { // change base static double transform = log(10); lp /= transform; return static_cast(round(-10.0f * lp)); } // Return a uniform log-scaled quality vector of the given size DoubleVector uniformLogProbVector(double p_error, size_t n); // Return a string which encodes the log-scaled double vector as characters std::string encodeLogProbVector(const DoubleVector& dv); }; #endif ================================================ FILE: src/SGA/Util/QualityCodec.h ================================================ //----------------------------------------------- // Copyright 2012 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // QualityCodec - Encoder/decoder for a quality // symbols. Supports downsampling to a small range. // #ifndef QUALITYCODEC_H #define QUALITYCODEC_H #include #include typedef uint8_t QualityStorageUnit; template class QualityCodec { public: typedef QualityStorageUnit UNIT_TYPE; static const int SYMBOLS_PER_UNIT = 8 / BITS; // These values are the lowest and highest quality // values that are stored. The other values are interpolated // between these range static const int PHRED_MIN = 0; static const int PHRED_MAX = 40; static const int QUALITY_RANGE = PHRED_MAX - PHRED_MIN; static const int QUALITY_BINS = 1 << BITS; static const int BIN_SIZE = (QUALITY_RANGE + QUALITY_BINS - 1) / QUALITY_BINS; // ceiling // Encode the character b into a value inline uint8_t encode(char b) const { // Compute the quality bin to place this value in int phred = Quality::char2phred(b); // Clamp values if(phred < 0) phred = 0; UNIT_TYPE bin_idx = (phred - PHRED_MIN) / BIN_SIZE; if(bin_idx >= QUALITY_BINS) bin_idx = QUALITY_BINS - 1; //std::cout << "Q: " << b << " phred: " << phred << " c: " << (int)bin_idx << "\n"; //decode(bin_idx); return bin_idx; } // Decode the value c into a character inline char decode(uint8_t c) const { // Return the midpoint value of the bin int phred = (2 * c + 1) * BIN_SIZE / 2; char b = Quality::phred2char(phred); //std::cout << "c: " << (int)c << " phred: " << phred << " Q: " << b << "\n"; return b; } // Returns the number of units required to encode // a string of length n inline size_t getRequiredUnits(size_t n) const { return (n + SYMBOLS_PER_UNIT - 1) / SYMBOLS_PER_UNIT; } // Returns the number of symbols that can be stored // in n units inline size_t getCapacity(size_t n) const { return n * SYMBOLS_PER_UNIT; } // Returns the index of the unit to store the // i-th symbol of the string inline size_t getUnitIndex(size_t i) const { return i / SYMBOLS_PER_UNIT; } // Returns the position within a unit that the i-th symbol // should be stored in inline size_t getUnitOffset(size_t i) const { // this position is the k-th symbol of the unit return i % SYMBOLS_PER_UNIT; } // Return the amount that a value must be shifted to // store a code at a given offset inline uint8_t getOffsetShift(size_t offset) const { return BITS*(SYMBOLS_PER_UNIT - 1 - offset); } // Return a mask that can be used to select a single value by ANDing a storage unit inline uint8_t getOffsetMask(size_t offset) const { return ((1 << BITS) - 1) << getOffsetShift(offset); } // Store the value v at the i-th encoded position in the data array inline void store(UNIT_TYPE* pData, size_t i, char b) const { UNIT_TYPE& unit = pData[getUnitIndex(i)]; size_t offset = getUnitOffset(i); uint8_t shift = getOffsetShift(offset); // Clear the unsigned char mask = getOffsetMask(offset); // Clear position unit &= ~mask; // Set position uint8_t code = encode(b); code <<= shift; unit |= code; /* std::cout << "E Unit: " << getUnitIndex(i) << "\n"; std::cout << "E Offset: " << offset << "\n"; std::cout << "E Shift: " << (int)shift << "\n"; std::cout << "E Mask: " << (int)mask << "\n"; std::cout << "E UnitV: " << (int)unit << "\n"; */ } // get the character stored at position i inline char get(const UNIT_TYPE* pData, size_t i) const { const UNIT_TYPE& unit = pData[getUnitIndex(i)]; size_t offset = getUnitOffset(i); unsigned char mask = getOffsetMask(offset); UNIT_TYPE code = unit & mask; uint8_t shift = getOffsetShift(offset); code >>= shift; /* std::cout << "D Unit: " << getUnitIndex(i) << "\n"; std::cout << "D Offset: " << offset << "\n"; std::cout << "D Shift: " << (int)shift << "\n"; std::cout << "D Mask: " << (int)mask << "\n"; std::cout << "D UnitV: " << (int)unit << "\n"; */ return decode(code); } }; #endif ================================================ FILE: src/SGA/Util/QualityTable.cpp ================================================ //----------------------------------------------- // Copyright 2012 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // QualityTable - A 0-indexed table of quality strings // #include #include #include "QualityTable.h" #include "QualityCodec.h" #include "SeqReader.h" // Read the sequences from a file QualityTable::QualityTable() : m_bytes_used(0) { int missing_phred = 20; m_missingQualityChar = Quality::phred2char(missing_phred); } QualityTable::~QualityTable() { for(size_t i = 0; i < m_table.size(); ++i) delete m_table[i].encoded_data; } // void QualityTable::loadQualities(const std::string& filename) { SeqReader reader(filename); SeqRecord sr; while(reader.get(sr)) addQualityString(sr.qual); } // void QualityTable::addQualityString(const std::string& qual) { size_t num_bytes_required = m_codec.getRequiredUnits(qual.size()); QualityString incoming; incoming.encoded_data = new QualityStorageUnit[num_bytes_required]; incoming.num_encoded_symbols = qual.size(); for(size_t i = 0; i < qual.size(); ++i) m_codec.store(incoming.encoded_data, i, qual[i]); m_table.push_back(incoming); //std::cout << "Stored: " << qual << "\n"; //std::cout << "Decoded: " << getQualityString(m_table.size() - 1, qual.size()) << "\n"; m_bytes_used += (sizeof(incoming) + num_bytes_required); } // std::string QualityTable::getQualityString(size_t idx, size_t n) const { // If there is no quality string for this index, return default qualities if(idx >= m_table.size()) return std::string(n, m_missingQualityChar); QualityString encoded = m_table[idx]; std::string out; out.reserve(encoded.num_encoded_symbols); for(size_t i = 0; i < encoded.num_encoded_symbols; ++i) out.push_back(m_codec.get(encoded.encoded_data, i)); assert(out.length() == n); return out; } // size_t QualityTable::getCount() const { return m_table.size(); } // void QualityTable::printSize() const { printf("QualityTable: %.2lfGB\n", (double)m_bytes_used / (1000 * 1000 * 1000)); } ================================================ FILE: src/SGA/Util/QualityTable.h ================================================ //----------------------------------------------- // Copyright 2012 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL license //----------------------------------------------- // // QualityTable - A 0-indexed table of quality scores // #ifndef QUALITYTABLE_H #define QUALITYTABLE_H #include "Util.h" #include "SeqReader.h" #include "QualityCodec.h" #include struct QualityString { uint8_t num_encoded_symbols; QualityStorageUnit* encoded_data; }; typedef std::vector QualityStringVector; class QualityTable { public: // QualityTable(); ~QualityTable(); // void loadQualities(const std::string& filename); void addQualityString(const std::string& qual); std::string getQualityString(size_t idx, size_t n) const; size_t getCount() const; void clear(); // void printSize() const; private: QualityCodec<4> m_codec; QualityStringVector m_table; size_t m_bytes_used; char m_missingQualityChar; }; #endif ================================================ FILE: src/SGA/Util/QualityVector.cpp ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // QualityVector - Vector of doubles representing // log-transformed quality values for a sequence // #include #include #include #include "QualityVector.h" // QualityVector::QualityVector() { } // QualityVector::QualityVector(const QualityVector& vec, int start, int size) { assert(!vec.empty()); assert(size >= 0); assert(size + start <= (int)vec.size()); m_data.insert(m_data.end(), vec.m_data.begin() + start, vec.m_data.begin() + start + size); } // void QualityVector::add(DNADouble v) { m_data.push_back(v); } // void QualityVector::set(size_t idx, DNADouble v) { if(m_data.size() < idx) m_data.resize(idx + 1); m_data[idx] = v; } // DNADouble QualityVector::get(size_t idx) const { assert(idx < m_data.size()); return m_data[idx]; } // size_t QualityVector::size() const { return m_data.size(); } // bool QualityVector::empty() const { return m_data.empty(); } // void QualityVector::reverseComplement() { std::reverse(m_data.begin(), m_data.end()); for(size_t i = 0; i < m_data.size(); ++i) m_data[i].complement(); } ================================================ FILE: src/SGA/Util/QualityVector.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // QualityVector - Vector of doubles representing // log-transformed quality values for a sequence // #ifndef QUALITYVECTOR_H #define QUALITYVECTOR_H #include #include "Alphabet.h" #include "DNADouble.h" typedef std::vector APVec; class QualityVector { public: // Constructors QualityVector(); QualityVector(const QualityVector& vec, int start, int size); // void add(DNADouble v); void set(size_t idx, DNADouble v); DNADouble get(size_t idx) const; size_t size() const; bool empty() const; // void reverseComplement(); private: APVec m_data; }; #endif ================================================ FILE: src/SGA/Util/Read2Contig.h ================================================ #ifndef READ2CONTIG_H #define READ2CONTIG_H #include #include #include "api/BamReader.h" #include "api/BamWriter.h" #include struct Read2Contig { // Constructors Read2Contig(std::string tcname, std::string trname, unsigned tpos, std::string tseq) : cname(tcname), rname(trname), pos(tpos), seq(tseq) {} Read2Contig(std::string tcname, std::string trname, unsigned tpos, BamTools::BamAlignment ta, std::string tseq) : cname(tcname), rname(trname), pos(tpos), a(ta), seq(tseq) {} Read2Contig() {} ~Read2Contig() {} //data std::string cname; std::string rname; int pos; BamTools::BamAlignment a; std::string seq; bool valid = true; double sw_score = -1; // set the sort order bool operator < (const Read2Contig& str) const { return (pos < str.pos); } // print it out std::string toString() const { std::stringstream ss; std::string sep = ","; ss << cname << sep << rname << sep << pos << sep << seq << sep << sw_score; return ss.str(); } }; typedef std::vector R2CVec; #endif ================================================ FILE: src/SGA/Util/ReadInfoTable.cpp ================================================ // //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // ReadInfoTable - A 0-indexed table of ID, length pairs // Used to convert suffix array hits to overlaps // #include #include #include "ReadInfoTable.h" #include "SeqReader.h" //JEREMIAH ReadInfoTable::ReadInfoTable(ReadTable *pRT) : m_numericIDs(false) { for (size_t i = 0; i < pRT->getCount(); i++) { m_lengths.push_back(pRT->getReadLength(i)); m_ids.push_back(pRT->getReadID(i)); } } // Read the sequences from a file ReadInfoTable::ReadInfoTable(std::string filename, size_t num_expected, ReadInfoOption option) : m_numericIDs(false) { // Do not store actual ids, use a numeric id equal to the table index if(option == RIO_NUMERICID) m_numericIDs = true; if(num_expected > 0) { m_lengths.reserve(num_expected); if(!m_numericIDs) m_ids.reserve(num_expected); } SeqReader reader(filename); SeqRecord sr; // Load the lengths and ids while(reader.get(sr)) { m_lengths.push_back(sr.seq.length()); if(!m_numericIDs) { m_ids.push_back(sr.id); } } } // ReadInfoTable::~ReadInfoTable() { } // size_t ReadInfoTable::getReadLength(size_t idx) const { assert(idx < m_lengths.size()); return m_lengths[idx]; } // std::string ReadInfoTable::getReadID(size_t idx) const { if(!m_numericIDs) { assert(idx < m_ids.size()); return m_ids[idx]; } else { // Build an identified from the idx std::stringstream idss; idss << idx; return idss.str(); } } // const ReadInfo ReadInfoTable::getReadInfo(size_t idx) const { ReadInfo ri(getReadID(idx), getReadLength(idx)); return ri; } // size_t ReadInfoTable::getCount() const { return m_lengths.size(); } // size_t ReadInfoTable::countSumLengths() const { size_t sum = 0; for(size_t i = 0; i < m_lengths.size(); ++i) sum += m_lengths[i]; return sum; } // void ReadInfoTable::clear() { m_lengths.clear(); m_ids.clear(); } ================================================ FILE: src/SGA/Util/ReadInfoTable.h ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // ReadInfoTable - A 0-indexed table of ID, length pairs // Used to convert suffix array hits to overlaps // #ifndef READINFOTABLE_H #define READINFOTABLE_H #include "Util.h" #include "SeqReader.h" #include "ReadTable.h" #include enum ReadInfoOption { RIO_NONE, RIO_NUMERICID }; struct ReadInfo { ReadInfo() : length(0) {} ReadInfo(const std::string& i, const uint32_t& l) : id(i), length(l) {} std::string id; uint32_t length; }; typedef std::vector InfoVector; class ReadInfoTable { public: // ReadInfoTable() {} ReadInfoTable(ReadTable *pRT); // Load the table using the read in filename // If num_expected > 0, reserve room in the table for num_expected reads ReadInfoTable(std::string filename, size_t num_expected = 0, ReadInfoOption options = RIO_NONE); ~ReadInfoTable(); // const ReadInfo getReadInfo(size_t idx) const; std::string getReadID(size_t idx) const; size_t getReadLength(size_t idx) const; size_t getCount() const; size_t countSumLengths() const; void clear(); private: std::vector m_lengths; std::vector m_ids; bool m_numericIDs; }; #endif ================================================ FILE: src/SGA/Util/ReadTable.cpp ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // ReadTable - A 0-indexed table of reads // #include #include #include "ReadTable.h" //JEREMIAH void ReadTable::setReadSequence(const std::string& seqr) { assert(m_table[idx-1].seq.length() == seqr.length()); m_table[idx-1].seq = seqr; } // JEREMIAH ReadTable::ReadTable(SeqRecordVector &srv) { idx = 0; m_pIndex = NULL; // not built by default for(std::vector::size_type i = 0; i != srv.size(); i++) { m_table.push_back(srv[i].toSeqItem()); } } // JEREMIAH ReadTable::ReadTable(const SeqLib::UnalignedSequenceVector &contigs) { idx = 0; m_pIndex = NULL; // not built by default for (SeqLib::UnalignedSequenceVector::const_iterator it = contigs.begin(); it != contigs.end(); it++) { SeqItem si; si.seq = it->Seq; si.id = it->Name; m_table.push_back(si); } } // std::string ReadTable::getReadID(size_t idx) const { return m_table[idx].id; } //JEREMIAH void ReadTable::setZero() { idx = 0; } // Jeremiah bool ReadTable::getRead(SeqItem &si) { if (idx >= m_table.size()) return false; si = m_table[idx]; idx++; return true; } // ReadTable::~ReadTable() { if(m_pIndex != NULL) delete m_pIndex; } // Populate this read table with the reverse reads from pRT void ReadTable::initializeReverse(const ReadTable* pRT) { size_t numReads = pRT->getCount(); m_table.reserve(numReads); for(size_t i = 0; i < numReads; ++i) { SeqItem read = pRT->getRead(i); read.seq.reverse(); addRead(read); } } // void ReadTable::reverseAll() { for(size_t i = 0; i < getCount(); ++i) m_table[i].seq.reverse(); } // void ReadTable::addRead(const SeqItem& r) { m_table.push_back(r); } // size_t ReadTable::getReadLength(size_t idx) const { return m_table[idx].seq.length(); } // const SeqItem& ReadTable::getRead(size_t idx) const { if (idx >= m_table.size()) { std::cerr << "idx " << idx << " m_table.size() " << m_table.size() << std::endl; assert(idx <= m_table.size()); } assert(idx < m_table.size()); return m_table[idx]; } // indexReadsByID must be called before this function can be used const SeqItem& ReadTable::getRead(const std::string& id) const { assert(m_pIndex != NULL); if(m_pIndex == NULL) { std::cerr << "Error: read table is not indexed (did you forget to call ReadTable::buildIndex?)\n"; assert(false); } ReadIndex::const_iterator i = m_pIndex->find(id); if(i == m_pIndex->end()) { std::cerr << "Read with id " << id << " not found in table\n"; assert(false); } return *i->second; } // build a read id -> *seqitem index void ReadTable::indexReadsByID() { m_pIndex = new ReadIndex; for(size_t i = 0; i < m_table.size(); ++i) { std::pair result = m_pIndex->insert(std::make_pair(m_table[i].id, &m_table[i])); if(!result.second) { std::cerr << "Error: Attempted to insert vertex into table with a duplicate id: " << m_table[i].id << "\n"; std::cerr << "All reads must have a unique identifier\n"; exit(1); } } } // size_t ReadTable::getCount() const { return m_table.size(); } // size_t ReadTable::countSumLengths() const { size_t sum = 0; for(size_t i = 0; i < m_table.size(); ++i) sum += m_table[i].seq.length(); return sum; } // void ReadTable::clear() { m_table.clear(); if(m_pIndex != NULL) { delete m_pIndex; m_pIndex = NULL; } } // std::ostream& operator<<(std::ostream& out, const ReadTable& rt) { size_t numReads = rt.getCount(); for(size_t i = 0; i < numReads; ++i) { const SeqItem& read = rt.getRead(i); out << read.id << "\t" << read.seq.toString() << "\n"; } return out; } ================================================ FILE: src/SGA/Util/ReadTable.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL license //----------------------------------------------- // // ReadTable - A 0-indexed table of reads // #ifndef SNOWS_READTABLE_H #define SNOWS_READTABLE_H #include "Util.h" #include "SeqReader.h" #include "SeqLib/UnalignedSequence.h" #include typedef std::vector ReadVector; typedef std::map ReadIndex; class ReadTable { public: // ReadTable() : idx(0), m_pIndex(NULL) {} //ReadTable(std::string* seq, std::string * id, int length); ReadTable(SeqRecordVector &srv); ReadTable(const SeqLib::UnalignedSequenceVector &contigs); ReadTable(std::string filename, uint32_t reader_flags = 0); ~ReadTable(); // Initialize this read table as the reverse of the passed in read table void initializeReverse(const ReadTable* pRT); // Reverse all the reads in this table void reverseAll(); // Build a readid -> read index void indexReadsByID(); // void addRead(const SeqItem& r); const SeqItem& getRead(size_t idx) const; const SeqItem& getRead(const std::string& id) const; bool getRead(SeqItem &si); // JEREMIAH size_t getReadLength(size_t idx) const; size_t getCount() const; size_t countSumLengths() const; std::string getReadID(size_t idx) const; void setZero(); void clear(); // Get a particular character for a particular read inline char getChar(size_t str_idx, size_t char_idx) const { assert(str_idx < m_table.size()); return m_table[str_idx].seq.get(char_idx); } // I/O friend std::ostream& operator<<(std::ostream& out, const ReadTable& rt); //Jeremiah void setReadSequence(const std::string& seqr); private: ReadVector m_table; unsigned int idx = 0; // Index of readid -> SeqItem // It is not build be default to save memory ReadIndex* m_pIndex; }; #endif ================================================ FILE: src/SGA/Util/ReadTableNew.cpp ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // ReadTable - A 0-indexed table of reads // #include #include #include "ReadTable.h" #include "SeqReader.h" //#include "contigs.h" // Read the sequences from a file ReadTable::ReadTable(std::string filename, uint32_t reader_flags) { idx = 0; m_pIndex = NULL; // not built by default SeqReader reader(filename, reader_flags); SeqRecord sr; while(reader.get(sr)) { addRead(sr.toSeqItem()); } } // ReadTable::~ReadTable() { if(m_pIndex != NULL) delete m_pIndex; } // Populate this read table with the reverse reads from pRT void ReadTable::initializeReverse(const ReadTable* pRT) { size_t numReads = pRT->getCount(); m_table.reserve(numReads); for(size_t i = 0; i < numReads; ++i) { SeqItem read = pRT->getRead(i); read.seq.reverse(); addRead(read); } } // void ReadTable::reverseAll() { for(size_t i = 0; i < getCount(); ++i) m_table[i].seq.reverse(); } // void ReadTable::addRead(const SeqItem& r) { m_table.push_back(r); } // size_t ReadTable::getReadLength(size_t idx) const { return m_table[idx].seq.length(); } // std::string ReadTable::getReadID(size_t idx) const { return m_table[idx].id; } // const SeqItem& ReadTable::getRead(size_t idx) const { assert(idx < m_table.size()); return m_table[idx]; } // JEREMIAH bool ReadTable::getRead(SeqItem &si) { if (idx >= m_table.size()) return false; si = m_table[idx]; idx++; return true; } //JEREMIAH void ReadTable::setZero() { idx = 0; } // indexReadsByID must be called before this function can be used const SeqItem& ReadTable::getRead(const std::string& id) const { assert(m_pIndex != NULL); if(m_pIndex == NULL) { std::cerr << "Error: read table is not indexed (did you forget to call ReadTable::buildIndex?)\n"; assert(false); } ReadIndex::const_iterator i = m_pIndex->find(id); if(i == m_pIndex->end()) { std::cerr << "Read with id " << id << " not found in table\n"; assert(false); } return *i->second; } // build a read id -> *seqitem index void ReadTable::indexReadsByID() { m_pIndex = new ReadIndex; for(size_t i = 0; i < m_table.size(); ++i) { std::pair result = m_pIndex->insert(std::make_pair(m_table[i].id, &m_table[i])); if(!result.second) { std::cerr << "Error: Attempted to insert vertex into table with a duplicate id: " << m_table[i].id << "\n"; std::cerr << "All reads must have a unique identifier\n"; exit(1); } } } // size_t ReadTable::getCount() const { return m_table.size(); } // size_t ReadTable::countSumLengths() const { size_t sum = 0; for(size_t i = 0; i < m_table.size(); ++i) { sum += m_table[i].seq.length(); } return sum; } // void ReadTable::clear() { m_table.clear(); if(m_pIndex != NULL) { delete m_pIndex; m_pIndex = NULL; } } // std::ostream& operator<<(std::ostream& out, const ReadTable& rt) { size_t numReads = rt.getCount(); for(size_t i = 0; i < numReads; ++i) { const SeqItem& read = rt.getRead(i); out << read.id << "\t" << read.seq.toString() << "\n"; } return out; } ================================================ FILE: src/SGA/Util/ReadTableNew.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL license //----------------------------------------------- // // ReadTable - A 0-indexed table of reads // #ifndef READTABLE_H #define READTABLE_H #include "Util.h" #include "SeqReader.h" #include typedef std::vector ReadVector; typedef std::map ReadIndex; class ReadTable { public: //JEREMIAH (added idx(0)) ReadTable() : idx(0), m_pIndex(NULL) {} ReadTable(std::string filename, uint32_t reader_flags = 0); ~ReadTable(); // Initialize this read table as the reverse of the passed in read table void initializeReverse(const ReadTable* pRT); // Reverse all the reads in this table void reverseAll(); // Build a readid -> read index void indexReadsByID(); // void addRead(const SeqItem& r); const SeqItem& getRead(size_t idx) const; const SeqItem& getRead(const std::string& id) const; bool getRead(SeqItem &si); // JEREMIAH size_t getReadLength(size_t idx) const; size_t getCount() const; size_t countSumLengths() const; std::string getReadID(size_t idx) const; void setZero(); void clear(); // Get a particular character for a particular read inline char getChar(size_t str_idx, size_t char_idx) const { assert(str_idx < m_table.size()); return m_table[str_idx].seq.get(char_idx); } // I/O friend std::ostream& operator<<(std::ostream& out, const ReadTable& rt); private: ReadVector m_table; unsigned int idx; // Index of readid -> SeqItem // It is not build be default to save memory ReadIndex* m_pIndex; }; #endif ================================================ FILE: src/SGA/Util/ReadTableS.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL license //----------------------------------------------- // // ReadTable - A 0-indexed table of reads // #ifndef READTABLES_H #define READTABLES_H #include "Util.h" #include "SeqReader.h" #include "contigs.h" #include #include "api/BamReader.h" typedef std::vector ReadVector; typedef std::map ReadIndex; typedef std::vector BamAlignmentVector; class ReadTable { public: // ReadTable() : m_pIndex(NULL) {} //ReadTable(std::string* seq, std::string * id, int length); ReadTable(SeqRecordVector srv); ReadTable(const BamAlignmentVector &bav); ReadTable(const vector &contigs); ReadTable(std::string filename, uint32_t reader_flags = 0); ~ReadTable(); // Initialize this read table as the reverse of the passed in read table void initializeReverse(const ReadTable* pRT); // Reverse all the reads in this table void reverseAll(); // Build a readid -> read index void indexReadsByID(); // void addRead(const SeqItem& r); const SeqItem& getRead(size_t idx) const; const SeqItem& getRead(const std::string& id) const; bool getRead(SeqItem &si); // JEREMIAH size_t getReadLength(size_t idx) const; size_t getCount() const; size_t countSumLengths() const; std::string getReadID(size_t idx) const; void setZero(); void clear(); // Get a particular character for a particular read inline char getChar(size_t str_idx, size_t char_idx) const { assert(str_idx < m_table.size()); return m_table[str_idx].seq.get(char_idx); } // I/O friend std::ostream& operator<<(std::ostream& out, const ReadTable& rt); private: ReadVector m_table; unsigned int idx; // Index of readid -> SeqItem // It is not build be default to save memory ReadIndex* m_pIndex; }; #endif ================================================ FILE: src/SGA/Util/SGAStats.cpp ================================================ //----------------------------------------------- // Copyright 2009-2013 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // SGAStats - Common statistics functions used // throughout the program // #include "SGAStats.h" #include #include #include // // Poisson in log space // double SGAStats::logPoisson(unsigned int k, double m) { double f_k = logFactorial(k); double p = (double)k * log(m) - m - f_k; //std::cout << "k: " << k << " f: " << f_k << " m: " << m << " p: " << p << std::endl; return p; } // // Factorial in log space // double SGAStats::logFactorial(unsigned int k) { double result = 0; while(k > 0) result += log(k--); //slow return result; } // // Log binomial pmf // double SGAStats::logBinomial(unsigned int k, unsigned int n, double p) { assert(k <= n); double b_coeff = logFactorial(n) - logFactorial(k) - logFactorial(n - k); double lp = k * log(p) + (n - k) * log(1 - p); return b_coeff + lp; } // // // double SGAStats::logIntegerBetaDistribution(double x, unsigned int a, unsigned int b) { assert(x >= 0.0f && x <= 1.0f); assert(a > 0 && b > 0); double log_beta_f = logIntegerBetaFunction(a, b); double n = pow(x, a - 1) * pow(1 - x, b - 1); double log_p = log(n) - log_beta_f; return log_p; } // // // double SGAStats::logIntegerBetaFunction(unsigned int a, unsigned int b) { assert(a > 0 && b > 0); return logFactorial(a - 1) + logFactorial(b - 1) - logFactorial(a + b - 1); } // // // double SGAStats::logIntegerBetaBinomialDistribution(unsigned int k, unsigned int n, unsigned int a, unsigned int b) { assert(a > 0 && b > 0 && k <= n); double b_coeff = logFactorial(n) - logFactorial(k) - logFactorial(n - k); return b_coeff + logIntegerBetaFunction(k + a, n - k + b) - logIntegerBetaFunction(a, b); } ================================================ FILE: src/SGA/Util/SGAStats.h ================================================ //----------------------------------------------- // Copyright 2009-2013 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // SGAStats - Common statistics functions used // throughout the program // #ifndef SGASTATS_H #define SGASTATS_H namespace SGAStats { // // Probability // double logPoisson(unsigned int k, double m); double logFactorial(unsigned int k); double logBinomial(unsigned int k, unsigned int n, double p); // The logarithm of the Beta distribution pdf for the special case // that the shape parameters are positive integers double logIntegerBetaDistribution(double x, unsigned int a, unsigned int b); // The logarithm of the integer beta function double logIntegerBetaFunction(unsigned int a, unsigned int b); // The logarithm of the integer beta binomail PMF double logIntegerBetaBinomialDistribution(unsigned int k, unsigned int n, unsigned int a, unsigned int b); } #endif ================================================ FILE: src/SGA/Util/SeqCoord.cpp ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // SeqCoord - A data structure holding the coordinate // of a substring of a sequence which consists of an interval and // the length of the string. Used to build matches and overlaps. // #include "SeqCoord.h" // Return a seqcoord representing the complement of the interval // For example if the seqcoord represents the matched portion of a string, // this returns a seqcoord of the unmatched portion SeqCoord SeqCoord::complement() const { SeqCoord out; out.seqlen = seqlen; if(isFull()) { out.setEmpty(); } else if(isEmpty()) { out.setFull(); } else if(isLeftExtreme()) { out.interval.start = std::max(interval.start, interval.end) + 1; out.interval.end = out.seqlen - 1; } else { assert(isRightExtreme()); out.interval.start = 0; out.interval.end = std::min(interval.start, interval.end) - 1; } assert(out.isValid()); return out; } // Returns the substring of STR described by this seqcoord std::string SeqCoord::getSubstring(const std::string& str) const { assert(isValid()); if(isEmpty()) return std::string(""); else return str.substr(interval.start, length()); } // Returns the subvector described by the seqcoord QualityVector SeqCoord::getSubvector(const QualityVector& vec) const { assert(isValid()); if(isEmpty()) return QualityVector(); else return QualityVector(vec, interval.start, length()); } // std::string SeqCoord::getComplementString(const std::string& str) const { SeqCoord comp = complement(); return comp.getSubstring(str); } // Output std::ostream& operator<<(std::ostream& out, const SeqCoord& sc) { out << sc.interval << " " << sc.seqlen; return out; } // Input std::istream& operator>>(std::istream& in, SeqCoord& sc) { in >> sc.interval >> sc.seqlen; return in; } ================================================ FILE: src/SGA/Util/SeqCoord.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // SeqCoord - A data structure holding the coordinate // of a substring of a sequence which consists of an interval and // the length of the string. Used to build matches and overlaps. // #ifndef SEQCOORD_H #define SEQCOORD_H #include "Util.h" #include "Interval.h" #include "QualityVector.h" struct SeqCoord { // constructor SeqCoord() : seqlen(0) { setEmpty(); } SeqCoord(int s, int e, int l) : interval(s, e), seqlen(l) { assert(isValid()); } // functions inline bool isLeftExtreme() const { return (interval.start == 0); } inline bool isRightExtreme() const { return (interval.end + 1 == seqlen); } inline bool isExtreme() const { return (isLeftExtreme() || isRightExtreme()); } inline bool isContained() const { return (isLeftExtreme() && isRightExtreme()); } inline bool isFull() const { return length() == seqlen; } inline bool isEmpty() const { return interval.start == 0 && interval.end == -1; } inline bool isValid() const { return isEmpty() || (interval.start <= interval.end && interval.start >= 0 && interval.end < seqlen); } // Special coordinate denoting the interval is empty // length() will return 0 inline void setEmpty() { interval.start = 0; interval.end = -1; } // Set the seqcoord to be a full-length match inline void setFull() { interval.start = 0; interval.end = seqlen - 1; } // Return the length of the interval, which is inclusive inline int length() const { return interval.end - interval.start + 1; } // Return the distance from the start to the left end inline int getLeftDist() const { return interval.start; } // Return the distance to the right end inline int getRightDist() const { return seqlen - interval.end - 1; } // Flip mirrors the coordinates so they are on the other strand inline void flip() { int tmp = seqlen - interval.start - 1; interval.start = seqlen - interval.end - 1; interval.end = tmp; assert(interval.start <= interval.end); } // Flip a single position p to the reverse strand for a sequence of length l static inline int flip(int p, int l) { return l - p - 1; } SeqCoord complement() const; // Get the substring described by the interval std::string getSubstring(const std::string& str) const; QualityVector getSubvector(const QualityVector& vec) const; // Get the substring described by the complement of the interval std::string getComplementString(const std::string& str) const; friend std::ostream& operator<<(std::ostream& out, const SeqCoord& sc); friend std::istream& operator>>(std::istream& in, SeqCoord& sc); // data Interval interval; int seqlen; }; #endif ================================================ FILE: src/SGA/Util/SeqReader.cpp ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // SeqReader - Reads fasta or fastq sequence files // #include #include #include "SeqReader.h" #include "Util.h" SeqReader::SeqReader(std::string filename, uint32_t flags) : m_flags(flags) { if(filename == "-") m_pHandle = &std::cin; else m_pHandle = createReader(filename); } SeqReader::~SeqReader() { if(m_pHandle != &std::cin) delete m_pHandle; } // Extract an element from the file // Return true if successful bool SeqReader::get(SeqRecord& sr) { static int warn_count = 0; const int MAX_WARN = 10; RecordType rt = RT_UNKNOWN; std::string header; while(m_pHandle->good()) { getline(*m_pHandle, header); if(header.empty()) continue; if(header[0] == '>') { rt = RT_FASTA; break; } else if(header[0] == '@') { rt = RT_FASTQ; break; } } if(rt == RT_UNKNOWN) { // No valid start found return false; } // Parse the rest of the record bool validRecord = false; std::string seq; std::string qual; if(rt == RT_FASTA) { std::string temp; while(m_pHandle->good() && m_pHandle->peek() != '>' && m_pHandle->peek() != '@') { getline(*m_pHandle, temp); if(m_pHandle->good() && temp.size() > 0) seq.append(temp); } // The record is valid if we extracted at least 1 bp for the sequence // at this point the eof may have been hit but we still want the data validRecord = seq.size() > 0; } else if(rt == RT_FASTQ) { std::string temp; getline(*m_pHandle, seq); getline(*m_pHandle, temp); //discard getline(*m_pHandle, qual); // FASTQ is required to have 4 fields, we must not have hit the EOF by this point if(seq.size() != qual.size() && warn_count++ < MAX_WARN) { std::cerr << "Warning, FASTQ quality string is not the same length as the sequence string for read " << header << "\n"; } // Fix [Issue GH-3]: Handle FASTQ records that have no sequence or quality value. We only // emit a warning here as long as the record is properly formed. if(seq.empty() || qual.empty()) { std::cerr << "Warning, read " << header << " has no sequence or quality values\n"; } validRecord = !m_pHandle->eof(); } if(validRecord) { // Parse the id size_t endPos = std::min(header.find_first_of(' '), header.find_first_of('\t')); if(endPos != std::string::npos) { assert(endPos > 0); sr.id = header.substr(1, endPos - 1); } else { sr.id = header.substr(1); } // Convert the sequence string to upper case if( !(m_flags & SRF_KEEP_CASE) ) std::transform(seq.begin(), seq.end(), seq.begin(), ::toupper); // If the validation flag is set, ensure that there aren't any non-ACGT bases if( !(m_flags & SRF_NO_VALIDATION) ) { if(seq.find_first_not_of("ACGT") != std::string::npos) { std::cerr << "Error: read " << sr.id << " contains non-ACGT characters.\n"; std::cerr << "Please run sga preprocess on the data first.\n"; exit(EXIT_FAILURE); } } sr.seq = seq; sr.qual = qual; } return validRecord; } ================================================ FILE: src/SGA/Util/SeqReader.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // SeqReader - Reads fasta or fastq sequence files // #ifndef SEQREADER_H #define SEQREADER_H #include #include "Util.h" enum RecordType { RT_FASTA, RT_FASTQ, RT_UNKNOWN }; static const uint32_t SRF_NO_VALIDATION = 1; static const uint32_t SRF_KEEP_CASE = 2; // class SeqReader { public: SeqReader(std::string filename, uint32_t flags = 0); ~SeqReader(); bool get(SeqRecord& sr); private: std::istream* m_pHandle; uint32_t m_flags; }; #endif ================================================ FILE: src/SGA/Util/SimpleAllocator.h ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL license //----------------------------------------------- // // SimpleAllocator - High-level manager of SimplePool // memory pools. See SimplePool.h for description of allocation // strategy // #ifndef SIMPLEALLOCATOR_H #define SIMPLEALLOCATOR_H #include #include "SimplePool.h" template class SimpleAllocator { typedef SimplePool StorageType; typedef std::list StorageList; public: SimpleAllocator() {} ~SimpleAllocator() { for(typename StorageList::iterator iter = m_pPoolList.begin(); iter != m_pPoolList.end(); ++iter) { delete *iter; } m_pPoolList.clear(); } void* alloc() { if(m_pPoolList.empty() || m_pPoolList.back()->isFull()) { // new storage must be allocated m_pPoolList.push_back(new StorageType); } // allocate from the last pool return m_pPoolList.back()->alloc(); } void dealloc(void* /*ptr*/) { // deallocation not tracked in this strategy } private: StorageList m_pPoolList; }; #endif ================================================ FILE: src/SGA/Util/SimplePool.h ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL license //----------------------------------------------- // // SimplePool - Zero-overhead templated memory pool // The design is based on the premise that the allocation // of the objects have a lifetime the length of the program's // execution so they are never freed. Should only be used when // the number of objects is bounded so the pool does not constantly grow. // // Not thread-safe. // #ifndef SIMPLEPOOL_H #define SIMPLEPOOL_H template class SimplePool { public: SimplePool() { size_t bytes_per_object = sizeof(T); size_t total_bytes = NUM_OBJECTS * bytes_per_object; m_pPool = malloc(total_bytes); if(m_pPool == NULL) { std::cerr << "SimpleStorage failed to allocate " << total_bytes << " bytes for memory pool, exiting\n"; abort(); } m_capacity = total_bytes; m_used = 0; } ~SimplePool() { free(m_pPool); } // Return a pointer to the next unused block of memory void* alloc() { assert(m_used < m_capacity); void* pNext = (char*)m_pPool + m_used; m_used += sizeof(T); return pNext; } // Do not track deallocations void dealloc(void* /*ptr*/) { // does nothing } bool isFull() { return m_used >= m_capacity; } private: void* m_pPool; size_t m_capacity; size_t m_used; static const size_t NUM_OBJECTS = 50*1024; }; #endif ================================================ FILE: src/SGA/Util/StdAlnTools.cpp ================================================ ///----------------------------------------------- // Copyright 2011 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // StdAlnTools - Collection of wrappers around the // stdaln dynamic programming alignment functions #include #include "StdAlnTools.h" #include "stdaln.h" #include "Alphabet.h" // Perform a global alignment between the given strings int StdAlnTools::globalAlignment(const std::string& target, const std::string& query, bool bPrint) { path_t* path; int path_len = 0; int score = 0; createGlobalAlignmentPath(target, query, &path, &path_len, &score); if(bPrint) { std::string paddedTarget, paddedQuery, paddedMatch; makePaddedStringsFromPath(target, query, path, path_len, paddedTarget, paddedQuery, paddedMatch); printPaddedStrings(paddedTarget, paddedQuery, paddedMatch); std::cout << "CIGAR: " << makeCigar(path, path_len) << "\n"; std::cout << "Global alignment score: " << score << "\n"; } free(path); return score; } // Perform a global alignment between the given strings and return the CIGAR string std::string StdAlnTools::globalAlignmentCigar(const std::string& target, const std::string& query) { std::string cigar; int score = 0; globalAlignment(target, query, cigar, score); return cigar; } // Perform a global alignment between the given strings and return the CIGAR string and score void StdAlnTools::globalAlignment(const std::string& target, const std::string& query, std::string& cigar, int& score) { path_t* path; int path_len = 0; createGlobalAlignmentPath(target, query, &path, &path_len, &score); cigar = makeCigar(path, path_len); free(path); } // Perform a local alignment LocalAlignmentResult StdAlnTools::localAlignment(const std::string& target, const std::string& query) { // Set up global alignment parameters and data structures GlobalAlnParams params; int max_path_length = target.size() + query.size(); path_t *path = (path_t*)calloc(max_path_length, sizeof(path_t)); int path_len; AlnParam par; int matrix[25]; StdAlnTools::setAlnParam(par, matrix, params); // Make a packed version of the query and target uint8_t* pQueryT = createPacked(query); uint8_t* pTargetT = createPacked(target); LocalAlignmentResult result; result.score = aln_local_core(pTargetT, target.size(), pQueryT, query.size(), &par, path, &path_len, 1, 0); assert(path_len <= max_path_length); result.cigar = makeCigar(path, path_len); // Calculate the aligned coordinates // This returns inclusive coordinates of the substrings aligned path_t* p = path + path_len - 1; result.targetStartIndex = (p->i ? p->i : 1) - 1; result.targetEndIndex = path->i - 1; result.queryStartIndex = (p->j ? p->j : 1) - 1; result.queryEndIndex = path->j - 1; // Update cigar if(result.queryStartIndex > 0) { std::stringstream csa; csa << result.queryStartIndex << "S"; result.cigar.insert(0, csa.str()); } int query_left_overhang = query.length() - (result.queryEndIndex + 1); if(query_left_overhang > 0) { std::stringstream csa; csa << query_left_overhang << "S"; result.cigar.append(csa.str()); } // Clean up delete [] pQueryT; delete [] pTargetT; free(path); return result; } // Expand a CIGAR string into a character code for each symbol of the alignment std::string StdAlnTools::expandCigar(const std::string& cigar) { std::string expanded; std::stringstream parser(cigar); int length; char code; while(parser >> length) { bool success = parser >> code; expanded.append(length, code); assert(success); (void)success; } return expanded; } // Compact an expanded CIGAR string into a regular cigar string std::string StdAlnTools::compactCigar(const std::string& expanded_cigar) { if(expanded_cigar.empty()) return ""; std::stringstream parser(expanded_cigar); std::stringstream writer; char prev_code = '\0'; char curr_code; int curr_length = 0; while(parser >> curr_code) { if(curr_code == prev_code) { curr_length += 1; } else { // write the previous cigar character if(prev_code != '\0') writer << curr_length << prev_code; prev_code = curr_code; curr_length = 1; } } // Write the last symbol writer << curr_length << prev_code; return writer.str(); } // Remove padding characters from str std::string StdAlnTools::unpad(const std::string& str) { std::string out; for(size_t i = 0; i < str.size(); ++i) { if(str[i] != '-') out.push_back(str[i]); } return out; } // Create a path array representing the global alignment between target and query // Caller must free the path void StdAlnTools::createGlobalAlignmentPath(const std::string& target, const std::string& query, path_t** path, int* path_len, int* score) { // Set up global alignment parameters and data structures GlobalAlnParams params; int max_path_length = target.size() + query.size(); *path = (path_t*)calloc(max_path_length, sizeof(path_t)); AlnParam par; int matrix[25]; StdAlnTools::setAlnParam(par, matrix, params); // Make a packed version of the query and target uint8_t* pQueryT = createPacked(query); uint8_t* pTargetT = createPacked(target); *score = aln_global_core(pTargetT, target.size(), pQueryT, query.size(), &par, *path, path_len); assert(*path_len <= max_path_length); // Clean up delete [] pQueryT; delete [] pTargetT; } // Convert a std::string into the stdaln required packed format. // This function allocates memory which the caller must free uint8_t* StdAlnTools::createPacked(const std::string& s, size_t start, size_t length) { if(length == std::string::npos) length = s.size(); assert(length <= s.size()); uint8_t* pBuffer = new uint8_t[length]; for(size_t j = 0; j < length; ++j) pBuffer[j] = DNA_ALPHABET::getBaseRank(s[start + j]); return pBuffer; } // Calculate the maximum target length for a query of length ql size_t StdAlnTools::calculateMaxTargetLength(int ql, const GlobalAlnParams& params) { size_t mt = ((ql + 1) / 2 * params.match + params.gap_ext) / params.gap_ext + ql; return mt; } // Fill in the stdaln AlnParam data, using GlobalAlnParams void StdAlnTools::setAlnParam(AlnParam& par, int matrix[25], const GlobalAlnParams& params) { par.matrix = matrix; // Set matrix for (size_t i = 0; i < 25; ++i) par.matrix[i] = -params.mismatch; for (size_t i = 0; i < 4; ++i) par.matrix[i*5+i] = params.match; par.gap_open = params.gap_open; par.gap_ext = params.gap_ext; par.gap_end = params.gap_ext; par.row = 5; par.band_width = params.bandwidth; } // Convert a path to a CIGAR string std::string StdAlnTools::makeCigar(path_t* path, int path_len) { int cigarLen = 0; uint32_t* pCigar = aln_path2cigar32(path, path_len, &cigarLen); std::stringstream cigarSS; for (int j = 0; j != cigarLen; ++j) { cigarSS << (pCigar[j]>>4); cigarSS << "MID"[pCigar[j]&0xf]; } free(pCigar); return cigarSS.str(); } // Make a pair of padded alignment strings from the pair of sequences void StdAlnTools::makePaddedStrings(const std::string& s1, const std::string& s2, std::string& out1, std::string& out2) { path_t* path; int path_len = 0; int score = 0; createGlobalAlignmentPath(s1, s2, &path, &path_len, &score); std::string outm; makePaddedStringsFromPath(s1, s2, path, path_len, out1, out2, outm); free(path); } // Convert a dynamic programming path to a set of padded strings // Algorithm ported from stdaln.c:aln_stdaln_aux void StdAlnTools::makePaddedStringsFromPath(const std::string& s1, const std::string& s2, path_t* path, int path_len, std::string& out1, std::string& out2, std::string& outm) { out1.resize(path_len, 'A'); out2.resize(path_len, 'A'); outm.resize(path_len, 'A'); path_t* p = path + path_len - 1; for (int l = 0; p >= path; --p, ++l) { assert(l < (int)out1.size()); int idx_i = p->i - 1; int idx_j = p->j - 1; switch (p->ctype) { // p->i and p->j are indices into the DP matrix, which is // the string coordinate - 1 case FROM_M: out1[l] = s1[idx_i]; out2[l] = s2[idx_j]; outm[l] = (s1[idx_i] == s2[idx_j] /*&& s1[p->i] != ap->row*/)? '|' : ' '; break; case FROM_I: out1[l] = '-'; out2[l] = s2[idx_j]; outm[l] = ' '; break; case FROM_D: out1[l] = s1[idx_i]; out2[l] = '-'; outm[l] = ' '; break; } } } // void StdAlnTools::printPaddedStrings(const std::string& s1, const std::string& s2, const std::string& m, int colSize) { assert(s1.size() == s2.size() && s1.size() == m.size()); size_t len = s1.size(); for(size_t l = 0; l < len; l += colSize) { int diff = len - l; int stop = diff < colSize ? diff : colSize; printf("1\t%s\n", s1.substr(l, stop).c_str()); printf("2\t%s\n", s2.substr(l, stop).c_str()); printf("m\t%s\n\n", m.substr(l, stop).c_str()); } } ================================================ FILE: src/SGA/Util/StdAlnTools.h ================================================ ///----------------------------------------------- // Copyright 2011 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // StdAlnTools - Collection of wrappers around the // stdaln dynamic programming alignment functions #ifndef STDALNTOOLS_H #define STDALNTOOLS_H #include #include #include #include #include "stdaln.h" // Parameters object struct GlobalAlnParams { GlobalAlnParams() { setDefaults(); } void setDefaults() { gap_open = 5; gap_ext = 2; mismatch = 3; match = 1; gap_open_extend = gap_open + gap_ext; threshold = 30; bandwidth = 50; } // PacBio specific parameters void setPacBio() { setDefaults(); gap_open = 2; gap_ext = 1; mismatch = 5; gap_open_extend = gap_open + gap_ext; } int gap_open; int gap_ext; int mismatch; int match; int gap_open_extend; int threshold; int bandwidth; }; struct LocalAlignmentResult { // Indices of the start/end base in the aligning // substrings. INCLUSIVE coordinates. int64_t targetStartIndex; int64_t targetEndIndex; int64_t queryStartIndex; int64_t queryEndIndex; std::string cigar; int score; friend std::ostream& operator<<(std::ostream& out, const LocalAlignmentResult& a) { out << "S:" << a.score << " [" << a.targetStartIndex << " " << a.targetEndIndex << "] C:" << a.cigar; return out; } }; typedef std::vector LocalAlignmentResultVector; namespace StdAlnTools { // Perform a global alignment between target and query using stdaln // If bPrint is true, the padded alignment is printed // to stdout. // The alignment score is returned. int globalAlignment(const std::string& target, const std::string& query, bool bPrint = false); // Perform a global alignment between the two strings and return a CIGAR string std::string globalAlignmentCigar(const std::string& target, const std::string& query); // Perform global alignment and return the cigar and score as out parameters void globalAlignment(const std::string& target, const std::string& query, std::string& cigar, int& score); // Perform a local alignment LocalAlignmentResult localAlignment(const std::string& target, const std::string& query); // Expand a Cigar string so there is one symbol per code std::string expandCigar(const std::string& cigar); // Compact an expanded cigar string std::string compactCigar(const std::string& cigar); // Convert a std::string into the stdAln required packed format. // This function allocates memory which the caller must free. uint8_t* createPacked(const std::string& s, size_t start = 0, size_t length = std::string::npos); // Create a path array representing the global alignment between target and query // Caller must free the path void createGlobalAlignmentPath(const std::string& target, const std::string& query, path_t** path, int* path_len, int* score); // Calculate the maximum aligned target length possible for a query of length ql // under the scoring scheme given by params size_t calculateMaxTargetLength(int ql, const GlobalAlnParams& params); // Fill in the stdaln AlnParam data, using GlobalAlnParams void setAlnParam(AlnParam& par, int matrix[25], const GlobalAlnParams& params); // Make a padded strings from a global alignment between the pair of sequences void makePaddedStrings(const std::string& s1, const std::string& s2, std::string& out1, std::string& out2); // Convert a stdaln path to a pair of padded strings representing the alignment void makePaddedStringsFromPath(const std::string& s1, const std::string& s2, path_t* path, int path_len, std::string& out1, std::string& out2, std::string& outm); // Returns a new copy of str with padding characters removed std::string unpad(const std::string& str); // Make a cigar string from a path std::string makeCigar(path_t* path, int path_len); // Print the padded aligned strings void printPaddedStrings(const std::string& s1, const std::string& s2, const std::string& m, int colSize = 120); }; #endif ================================================ FILE: src/SGA/Util/Timer.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL license //----------------------------------------------- // // Timer - Simple object to that prints the wallclock // length of its lifetime // #ifndef TIMER_H #define TIMER_H #include #include class Timer { public: Timer(std::string s, bool silent = false) : m_desc(s), m_silent(silent) { reset(); } ~Timer() { if(!m_silent) fprintf(stderr, "[timer - %s] wall clock: %.2lfs CPU: %.2lfs\n", m_desc.c_str(), getElapsedWallTime(), getElapsedCPUTime()); } double getElapsedWallTime() const { timeval now; gettimeofday(&now, NULL); return (now.tv_sec - m_wallStart.tv_sec) + (double(now.tv_usec - m_wallStart.tv_usec) / 1000000); } double getElapsedCPUTime() const { double now = clock(); return (now - m_cpuStart) / CLOCKS_PER_SEC; } void reset() { gettimeofday(&m_wallStart, NULL); m_cpuStart = clock(); } private: std::string m_desc; // Track the wall-clock and CPU times // CPU time includes all threads timeval m_wallStart; double m_cpuStart; bool m_silent; }; #endif ================================================ FILE: src/SGA/Util/Util.cpp ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // Util - Common data structures and functions // #include #include #include #include "Util.h" // // Sequence operations // // Reverse complement a sequence std::string reverseComplement(const std::string& seq) { std::string out(seq.length(), 'A'); size_t last_pos = seq.length() - 1; for(int i = last_pos; i >= 0; --i) { out[last_pos - i] = complement(seq[i]); } return out; } // Reverse complement a sequence using the full iupac alphabet std::string reverseComplementIUPAC(const std::string& seq) { std::string out(seq.length(), 'A'); size_t last_pos = seq.length() - 1; for(int i = last_pos; i >= 0; --i) { out[last_pos - i] = complementIUPAC(seq[i]); } return out; } // Reverse a sequence std::string reverse(const std::string& seq) { return std::string(seq.rbegin(), seq.rend()); } // Complement a sequence std::string complement(const std::string& seq) { std::string out(seq.length(), 'A'); size_t l = seq.length(); for(size_t i = 0; i < l; ++i) out[i] = complement(seq[i]); return out; } // Complement a sequence over the IUPAC alphabet std::string complementIUPAC(const std::string& seq) { std::string out(seq.length(), 'A'); size_t l = seq.length(); for(size_t i = 0; i < l; ++i) out[i] = complementIUPAC(seq[i]); return out; } // std::string prefix(const std::string& seq, const unsigned int len) { assert(seq.length() >= len); return seq.substr(0, len); } // std::string suffix(const std::string& seq, const unsigned int len) { assert(seq.length() >= len); return seq.substr(seq.length() - len); } // // Dust scoring scheme as given by: // Morgulis A. "A fast and symmetric DUST implementation to Mask // Low-Complexity DNA Sequences". J Comp Bio. double calculateDustScore(const std::string& seq) { std::map scoreMap; // Cannot calculate dust scores on very short reads if(seq.size() < 3) return 0.0f; // Slide a 3-mer window over the sequence and insert the sequences into the map for(size_t i = 0; i < seq.size() - 3; ++i) { std::string triMer = seq.substr(i, 3); scoreMap[triMer]++; } // Calculate the score by summing the square of every element in the map double sum = 0; std::map::iterator iter = scoreMap.begin(); for(; iter != scoreMap.end(); ++iter) { int tc = iter->second; double score = (double)(tc * (tc - 1)) / 2.0f; sum += score; } return sum / (seq.size() - 2); } // Returns the window over seq with the highest dust score double maxDustWindow(const std::string& seq, size_t windowSize, size_t minWindow) { double maxScore = 0.0f; for(size_t i = 0; i < seq.size(); i += 1) { size_t r = seq.size() - i; size_t w = r < windowSize ? r : windowSize; if(w >= minWindow) // don't calculate score for small windows { double s = calculateDustScore(seq.substr(i, w)); if(s > maxScore) maxScore = s; } } return maxScore; } // count the differences between s1 and s2 over the first n bases int countDifferences(const std::string& s1, const std::string& s2, size_t n) { int numDiff = 0; for(size_t i = 0; i < n; ++i) { if(s1[i] != s2[i]) numDiff++; } return numDiff; } // count the differences between s1 and s2 over the first n bases std::string getDiffString(const std::string& s1, const std::string& s2) { std::string out; size_t stop = std::min(s1.size(), s2.size()); for(size_t i = 0; i < stop; ++i) out.push_back(s1[i] == s2[i] ? '.' : s1[i]); return out; } // char randomBase() { int i = rand() % 4; switch(i) { case 0: return 'A'; case 1: return 'C'; case 2: return 'G'; case 3: return 'T'; default: assert(false); } return 'A'; } // Strip the leading directories and // the last trailling suffix from a filename std::string stripFilename(const std::string& filename) { std::string out = stripDirectories(filename); // Remove the gzip extension if necessary if(isGzip(out)) out = stripExtension(out); return stripExtension(out); } // Remove a single file extension from the filename std::string stripExtension(const std::string& filename) { size_t suffixPos = filename.find_last_of('.'); if(suffixPos == std::string::npos) return filename; // no suffix else return filename.substr(0, suffixPos); } // Remove an extension from a filename, including the .gz extension // file.fastq will return file // file.fastq.gz will return file std::string stripGzippedExtension(const std::string& filename) { if(isGzip(filename)) return stripExtension(stripExtension(filename)); else return stripExtension(filename); } // Strip the leadering directories from a filename std::string stripDirectories(const std::string& filename) { size_t lastDirPos = filename.find_last_of('/'); if(lastDirPos == std::string::npos) return filename; // no directories else return filename.substr(lastDirPos + 1); } // Return the file extension std::string getFileExtension(const std::string& filename) { size_t suffixPos = filename.find_last_of('.'); if(suffixPos == std::string::npos) return ""; else return filename.substr(suffixPos + 1); } // Write out a fasta record void writeFastaRecord(std::ostream* pWriter, const std::string& id, const std::string& seq, size_t maxLineLength) { *pWriter << ">" << id << " " << seq.length() << "\n"; for(size_t i = 0; i < seq.size(); i += maxLineLength) { size_t span = std::min(seq.size() - i, maxLineLength); *pWriter << seq.substr(i, span) << "\n"; } } // Returns true if the filename has an extension indicating it is compressed bool isGzip(const std::string& filename) { size_t suffix_length = sizeof(GZIP_EXT) - 1; // Assume files without an extension are not compressed if(filename.length() < suffix_length) return false; std::string extension = suffix(filename, suffix_length); return extension == GZIP_EXT; } // Returns true if the filename has an extension indicating it is fastq bool isFastq(const std::string& filename) { return filename.find(".fastq") != std::string::npos || filename.find(".fq") != std::string::npos; } // Returns the size of the file. Code from stackoverflow. std::ifstream::pos_type getFilesize(const std::string& filename) { std::ifstream in(filename.c_str(), std::ifstream::in | std::ifstream::binary); in.seekg(0, std::ifstream::end); return in.tellg(); } // Open a file that may or may not be gzipped for reading // The caller is responsible for freeing the handle std::istream* createReader(const std::string& filename, std::ios_base::openmode mode) { if(isGzip(filename)) { igzstream* pGZ = new igzstream(filename.c_str(), mode); assertGZOpen(*pGZ, filename); return pGZ; } else { std::ifstream* pReader = new std::ifstream(filename.c_str(), mode); assertFileOpen(*pReader, filename); return pReader; } } // Open a file that may or may not be gzipped for writing // The caller is responsible for freeing the handle std::ostream* createWriter(const std::string& filename, std::ios_base::openmode mode) { if(isGzip(filename)) { ogzstream* pGZ = new ogzstream(filename.c_str(), mode); assertGZOpen(*pGZ, filename); return pGZ; } else { std::ofstream* pWriter = new std::ofstream(filename.c_str(), mode); assertFileOpen(*pWriter, filename); return pWriter; } } // Ensure a filehandle is open void assertFileOpen(std::ifstream& fh, const std::string& fn) { if(!fh.is_open()) { std::cerr << "Error: could not open " << fn << " for read\n"; exit(EXIT_FAILURE); } } // Ensure a filehandle is open void assertFileOpen(std::ofstream& fh, const std::string& fn) { if(!fh.is_open()) { std::cerr << "Error: could not open " << fn << " for write\n"; exit(EXIT_FAILURE); } } // void assertGZOpen(gzstreambase& gh, const std::string& fn) { if(!gh.good()) { std::cerr << "Error: could not open " << fn << std::endl; exit(EXIT_FAILURE); } } // Split a string into parts based on the delimiter StringVector split(std::string in, char delimiter) { StringVector out; size_t lastPos = 0; size_t pos = in.find_first_of(delimiter); while(pos != std::string::npos) { out.push_back(in.substr(lastPos, pos - lastPos)); lastPos = pos + 1; pos = in.find_first_of(delimiter, lastPos); } out.push_back(in.substr(lastPos)); return out; } // Split a key-value pair void splitKeyValue(std::string in, std::string& key, std::string& value) { StringVector parts = split(in, CAF_SEP); if(parts.size() != 2) { std::cerr << "Invalid key-value pair " << in << std::endl; assert(false); } key = parts[0]; value = parts[1]; assert(key.size() > 0 && value.size() > 0 && "Invalid key-value pair"); } // Get the shared component of a read pair name // This is the part of the name preceding the "/" std::string getPairBasename(const std::string& id) { assert(!id.empty()); size_t pos = id.find_last_of('/'); if(pos == std::string::npos) return id; else return id.substr(0, pos); } // Get the ID of the pair of a given read std::string getPairID(const std::string& id) { assert(!id.empty()); std::string pid(id); size_t li = id.length() - 1; char last = id[li]; if(last == 'A') pid[li] = 'B'; else if(last == 'B') pid[li] = 'A'; else if(last == '1') pid[li] = '2'; else if(last == '2') pid[li] = '1'; else if(last == 'f') pid[li] = 'r'; else if(last == 'r') pid[li] = 'f'; else pid = ""; return pid; } // Return 0 if the id indicates the first read of a pair, 1 otherwise int getPairIndex(const std::string& id) { size_t li = id.length() - 1; char last = id[li]; if(last == 'A' || last == '1' || last == 'f') return 0; else if(last == 'B' || last == '2' || last == 'r') return 1; else { std::cerr << "Unrecognized pair format: " << id << "\n"; exit(EXIT_FAILURE); } } // Debug function to parse the distance between two reads // based on their names // This assumes is that the read names are just the positions the reads // were sampled from size_t debug_getReadDistFromNames(const std::string& name1, const std::string& name2) { std::string id; std::string pos; StringVector parts = split(name1, ':'); if(parts.size() != 2) return 0; int p1 = atoi(parts[1].c_str()); parts = split(name2, ':'); if(parts.size() != 2) return 0; int p2 = atoi(parts[1].c_str()); int dist = int(abs(p1 - p2)); return dist; } ================================================ FILE: src/SGA/Util/Util.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL license //----------------------------------------------- // // Util - Common data structures and functions // #ifndef UTIL_H #define UTIL_H #include #include #include #include #include #include #include #include #include #include #include #include "DNAString.h" #include "gzstream.h" #include "Quality.h" #define CAF_SEP ':' #define FUNCTION_TIMER Timer functionTimer(__PRETTY_FUNCTION__); #define VALIDATION_WARNING(x) static bool validation_warn = true; if(validation_warn) \ printf("[%s] Warning validation is on\n", (x)); validation_warn = false; #define WARN_ONCE(x) static bool _warn_once = true; if(_warn_once) \ printf("WARNING: [%s]\n", (x)); _warn_once = false; #define GZIP_EXT ".gz" // // Typedef // typedef std::string Sequence; typedef std::string ContigID; typedef std::vector IntVec; typedef std::vector DoubleVec; typedef std::vector StringVector; typedef std::list StringList; typedef std::vector SequenceVector; // SeqItem is just an id, sequence pair struct SeqItem { // data std::string id; DNAString seq; void write(std::ostream& out, const std::string& meta = "") const { out << ">" << id << (meta.empty() ? "" : " ") << meta << "\n"; out << seq.toString() << "\n"; } }; typedef std::vector SeqItemVector; // SeqRecord is a id,sequence pair with an associated quality value struct SeqRecord { SeqItem toSeqItem() const { SeqItem r; r.id = id; r.seq = seq; return r; } // Write the sequence to the file handle. If the void write(std::ostream& out, const std::string& meta = "") const { // If there is a quality string write the record as fastq, otherwise fasta if(!qual.empty()) { out << "@" << id << (meta.empty() ? "" : " ") << meta << "\n"; out << seq.toString() << "\n"; out << "+" << "\n"; out << qual << "\n"; } else { toSeqItem().write(out, meta); } } bool hasQuality() const { return !qual.empty(); } // Get the phred score for base i. If there are no quality string, returns DEFAULT_QUAL_SCORE int getPhredScore(size_t i) const { if(!qual.empty()) { assert(i < qual.size()); return Quality::char2phred(qual[i]); } else { return DEFAULT_QUAL_SCORE; } } // data std::string id; DNAString seq; std::string qual; }; typedef std::vector SeqRecordVector; // // Functions // std::string stripFilename(const std::string& filename); std::string stripExtension(const std::string& filename); std::string stripGzippedExtension(const std::string& filename); std::string stripDirectories(const std::string& filename); std::string getFileExtension(const std::string& filename); bool isGzip(const std::string& filename); bool isFastq(const std::string& filename); std::ifstream::pos_type getFilesize(const std::string& filename); // Write out a fasta record void writeFastaRecord(std::ostream* pWriter, const std::string& id, const std::string& seq, size_t maxLength = 80); // Wrapper function for opening a reader of compressed or uncompressed file std::istream* createReader(const std::string& filename, std::ios_base::openmode mode = std::ios_base::in); std::ostream* createWriter(const std::string& filename, std::ios_base::openmode mode = std::ios_base::out); void assertFileOpen(std::ifstream& fh, const std::string& fn); void assertFileOpen(std::ofstream& fh, const std::string& fn); void assertGZOpen(gzstreambase& gh, const std::string& fn); char randomBase(); // Key-value operations template std::string makeKeyValue(std::string key, C value) { std::stringstream ss; ss << key << CAF_SEP << value; return ss.str(); } StringVector split(std::string in, char delimiter); void splitKeyValue(std::string in, std::string& key, std::string& value); std::string getPairBasename(const std::string& id); std::string getPairID(const std::string& id); // Returns 0 if the id indicates the first read in a pair, 1 otherwise int getPairIndex(const std::string& id); // Debug function to get the distance between two reads based on their names, which // encodes the positions size_t debug_getReadDistFromNames(const std::string& name1, const std::string& name2); // // Return the lexographic value for the given base // static const uint8_t s_lexoRankLUT[256] = { 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,1,0,2,0,0,0,3,0,0,0,0,0,0,0,0, 0,0,0,0,4,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0, 0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0 }; inline static uint8_t getBaseRank(char b) { return s_lexoRankLUT[static_cast(b)]; } // // Sequence operations // std::string reverseComplement(const std::string& seq); std::string complement(const std::string& seq); std::string reverse(const std::string& seq); // Reverse/complement functions, allowing a full IUPAC alphabet std::string reverseComplementIUPAC(const std::string& seq); std::string complementIUPAC(const std::string& seq); // Return the prefix/suffix of seq of length len std::string prefix(const std::string& seq, const unsigned int len); std::string suffix(const std::string& seq, const unsigned int len); // Calculate the dust score for the given sequence double maxDustWindow(const std::string& seq, size_t windowSize = 64, size_t minWindow = 64); double calculateDustScore(const std::string& seq); // Count the number of differences between s1 and s2 over the first n chars int countDifferences(const std::string& s1, const std::string& s2, size_t n); // Construct a string encoding the differences between s1 and s2 std::string getDiffString(const std::string& s1, const std::string& s2); // Complement a base inline char complement(char base) { switch(base) { case 'A': return 'T'; case 'C': return 'G'; case 'G': return 'C'; case 'T': return 'A'; case 'N': return 'N'; default: assert(false && "Unknown base!"); return 'N'; } } // Complement a base using the full IUPAC alphabet // Also allows for lowercase bases inline char complementIUPAC(char c) { char cmp = '\0'; bool is_lc = std::islower(c); switch(std::toupper(c)) { case 'A': cmp = 'T'; break; case 'C': cmp = 'G'; break; case 'G': cmp = 'C'; break; case 'T': cmp = 'A'; break; case 'M': cmp = 'K'; break; case 'R': cmp = 'Y'; break; case 'W': cmp = 'W'; break; case 'S': cmp = 'S'; break; case 'Y': cmp = 'R'; break; case 'K': cmp = 'M'; break; case 'V': cmp = 'B'; break; case 'H': cmp = 'D'; break; case 'D': cmp = 'H'; break; case 'B': cmp = 'V'; break; case 'N': cmp = 'N'; break; default: assert(false); } if(is_lc) cmp = std::tolower(cmp); return cmp; } // Wrapper function to determine whether a calculated error rate is within tolerance of a // threshold. A fixed tolerance is used as this does not need to be super precise, it only // needs to account for small differences in fp calculations inline bool isErrorRateAcceptable(double er, double threshold) { static double tolerance = 0.000001f; if(er - tolerance <= threshold) return true; else return false; } #endif ================================================ FILE: src/SGA/Util/VCFUtil.cpp ================================================ //----------------------------------------------- // Copyright 2011 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // VCFUtil - collection of utility functions // for producing VCF records. // #include #include #include #include #include "VCFUtil.h" #include "StdAlnTools.h" #include "MultiAlignment.h" // // VCFRecord // VCFRecord::VCFRecord() { passStr = "PASS"; quality = 20.0f; } // parse from a line VCFRecord::VCFRecord(const std::string& line) { std::stringstream parser(line); parser >> refName; parser >> refPosition; parser >> id; parser >> refStr; parser >> varStr; // Non-conformant VCF might represent unknown qualities with '.' std::string quality_str; parser >> quality_str; if(quality_str == ".") { quality = 255; } else { std::stringstream qparser(quality_str); qparser >> quality; } parser >> passStr; std::string tmp; parser >> tmp; // Split the comments on the semicolon delimiter comments = split(tmp, ';'); parser >> formatStr; if(!formatStr.empty()) { // parse genotypes while(parser >> tmp) sampleStr.push_back(tmp); } } void VCFRecord::addComment(const std::string& key, const std::string& value) { std::string out = key; out.append(1, '='); out.append(value); comments.push_back(out); } void VCFRecord::addComment(const std::string& key, const int& value) { std::stringstream v_str; v_str << value; addComment(key, v_str.str()); } void VCFRecord::addComment(const std::string& key, const double& value) { std::stringstream v_str; v_str.precision(5); v_str.setf(std::ios::fixed,std::ios::floatfield); v_str << value; addComment(key, v_str.str()); } void VCFRecord::setPassStr(const std::string str) { passStr = str; } void VCFRecord::setQuality(const double q) { quality = q; } void VCFRecord::printVerbose() const { std::cout << "RefName: " << refName << "\n"; std::cout << "Position: " << refPosition << "\n"; std::cout << "RefStr: " << refStr << "\n"; std::cout << "VarStr: " << varStr << "\n"; } // Write the VCF record to the stream std::ostream& operator<<(std::ostream& o, const VCFRecord& record) { o.precision(5); o.setf(std::ios::fixed,std::ios::floatfield); o << record.refName << "\t"; o << record.refPosition << "\t", o << (record.id.empty() ? "." : record.id) << "\t"; o << record.refStr << "\t"; o << record.varStr << "\t"; o << (int)record.quality << "\t"; o << record.passStr << "\t"; for(size_t i = 0; i < record.comments.size(); ++i) { if(i > 0) o << ";"; o << record.comments[i]; } if(record.sampleStr.size()) { o << "\t" << record.formatStr; for(size_t i = 0; i < record.sampleStr.size(); ++i) o << "\t" << record.sampleStr[i]; } return o; } // VCFClassification VCFRecord::classify() const { if(refStr.size() == varStr.size()) return VCF_SUB; if(refStr.size() > varStr.size()) { // If a deletion, varStr should be a prefix of refStr if(refStr.find(varStr) == 0) return VCF_DEL; else return VCF_COMPLEX; } if(refStr.size() < varStr.size()) { // If an insertion, refStr should be a prefix of refStr if(varStr.find(refStr) == 0) return VCF_INS; else return VCF_COMPLEX; } return VCF_COMPLEX; } // // VCFUtil // // See header for description VCFReturnCode VCFUtil::generateVCFFromCancerVariant(const std::string& ref, const std::string& base, const std::string& variant, const std::string& refName, size_t refPosition, const std::string& varName, int minExactMatch, double dustThreshold, int verbose, VCFVector& outRecords) { VCFVector tempRecords; // Construct a multiple alignment from pairwise alignments // of the base and variant strings to the reference // We start by constructing a global alignment between the sequences // and representing the alignment as a Cigar string std::string cigarRefBase = StdAlnTools::globalAlignmentCigar(base, ref); std::string cigarRefVariant = StdAlnTools::globalAlignmentCigar(variant, ref); // Initialize the multiple alignment data MAlignData baseMA; baseMA.position = 0; baseMA.str = base; baseMA.expandedCigar = StdAlnTools::expandCigar(cigarRefBase); baseMA.name = "base"; MAlignData varMA; varMA.position = 0; varMA.str = variant; varMA.expandedCigar = StdAlnTools::expandCigar(cigarRefVariant); varMA.name = "variant"; MAlignDataVector maVector; maVector.push_back(baseMA); maVector.push_back(varMA); MultiAlignment ma(ref, maVector); /* std::cout << "\nMultipleAlignment:\n"; ma.print(); */ // Get the row index of the variant and base strings size_t baseRow = ma.getIdxByName("base"); size_t varRow = ma.getIdxByName("variant"); size_t refRow = ma.getRootIdx(); size_t numCols = ma.getNumColumns(); // Iterate over every column of the multiple alignment and detect variants int leftExactMatch = 0; int rightExactMatch = 0; int eventStart = -1; int eventEnd = -1; bool isIndel = false; bool inLeftExact = true; for(size_t i = 0; i < numCols; ++i) { char baseSymbol = ma.getSymbol(baseRow, i); char varSymbol = ma.getSymbol(varRow, i); bool isVariant = (varSymbol != baseSymbol); // Update the counter of the number of exact matches for the leftmost and rightmost bases if(isVariant) { inLeftExact = false; // stop the count of leftmost exact matches rightExactMatch = 0; // reset the rightmost count } else // this is a good match { rightExactMatch += 1; if(inLeftExact) leftExactMatch += 1; } // Process variants indicated by this portion of the alignment if(isVariant) { if(eventStart == -1) { // Not currently in a variant event, start a new one eventStart = i; eventEnd = i; } else { // Extend the event assert(eventEnd != -1); eventEnd += 1; } isIndel = isIndel || varSymbol == '-' || baseSymbol == '-'; } else { // Check if this is the end of a variant if(eventStart != -1) { if(verbose > 0) { std::cout << "\n\nProcessing variant\n"; ma.print(); } // Extract the substrings of the three sequences describing // this event. // If the event is an indel, we move the start point // of the event backwards until all 3 columns are not padding characters. // This is to avoid the following situation: // AATATTT--CGT ref // AATATTTT-CGT base // AATATTTTTCGT var // or // TTAGGTTTTTTT ref // TTAGG-TTTTTT base // TTAGG--TTTTT var // // In the first case, it is a 1 base insertion wrt base but a 2 base insertion wrt to the ref. // So we need to move the event to the first column with all Ts to properly capture what is happening. // In the second case it is a 1 base deletion wrt base and a 2 base deletion wrt to the ref. // Again, we need to move back to the last full column. if(isIndel) { // It is impossible to extract a reference string if the indel is at the beginning // or end of th multiple alignment so we just fail out here if(eventStart == 0 || eventEnd == (int)numCols - 1) return VCF_INVALID_MULTIALIGNMENT; while(eventStart >= 0) { char rc = ma.getSymbol(refRow, eventStart); char bc = ma.getSymbol(baseRow, eventStart); char vc = ma.getSymbol(varRow, eventStart); if(rc == '-' || bc == '-' || vc == '-') eventStart -= 1; else break; } assert(eventStart >= 0); } size_t eventLength = eventEnd - eventStart + 1; std::string refString = StdAlnTools::unpad(ma.getPaddedSubstr(refRow, eventStart, eventLength)); std::string baseString = StdAlnTools::unpad(ma.getPaddedSubstr(baseRow, eventStart, eventLength)); std::string varString = StdAlnTools::unpad(ma.getPaddedSubstr(varRow, eventStart, eventLength)); if(verbose > 0) { printf("Ref start: %d col: %d eventStart: %d eventEnd: %d\n", (int)refPosition, (int)i, eventStart, eventEnd); std::cout << "RefString " << refString << "\n"; std::cout << "baseString " << baseString << "\n"; std::cout << "varString " << varString << "\n"; } assert(!refString.empty()); //assert(!baseString.empty()); assert(!varString.empty()); if(baseString.empty()) std::cerr << "Warning: Empty base string\n"; // Get the base position in the reference string. This is not necessarily the // same as the eventStart column as the reference may be padded int refBaseOffset = ma.getBaseIdx(refRow, eventStart); // Generate VCF Record VCFRecord record; record.refName = refName; record.refPosition = refPosition + refBaseOffset + 1; // VCF uses 1-based coordinates record.refStr = refString; record.varStr = varString; // Add a comment with the variant name record.addComment("id", varName); // If the base string is not the same as the reference string, not it in the comment // This can happen when a het SNP is mutated or we have some other weird site if(refString != baseString) record.addComment("NS", baseString); // Check if the reference context is a homopolymer int preHP = ma.countHomopolymer(refRow, eventStart, 0); int sufHP = ma.countHomopolymer(refRow, eventEnd); int maxHP = std::max(preHP, sufHP); if(maxHP >= HOMOPOLYMER_ANNOTATE_THRESHOLD) { std::stringstream hps; hps << maxHP; record.addComment("HP", hps.str()); } // Calculate the highest dust score on the reference sequence // using 64-base windows. double globalDustScore = maxDustWindow(ref); if(globalDustScore > dustThreshold) { std::stringstream dts; dts.precision(2); dts << globalDustScore; record.addComment("DT", dts.str()); } if(verbose > 0) std::cout << record << "\n"; tempRecords.push_back(record); // Reset state eventStart = -1; eventEnd = -1; isIndel = false; } } } // Perform the start/end exact match sanity check if(leftExactMatch >= minExactMatch && rightExactMatch >= minExactMatch) { outRecords.insert(outRecords.end(), tempRecords.begin(), tempRecords.end()); return VCF_OK; } else { return VCF_EXACT_MATCH_FAILED; } } // Write a VCF header to the given file handle void VCFUtil::writeHeader(std::ostream* pWriter, const std::string& program, const std::string& inputBAM, const std::string& reference) { // Get and format the current date time_t rawtime; struct tm * timeinfo; static int MAX_DATE_CHARS = 80; char dateBuffer [MAX_DATE_CHARS]; time ( &rawtime ); timeinfo = localtime ( &rawtime ); int copied = strftime(dateBuffer,MAX_DATE_CHARS,"%Y%m%d",timeinfo); assert(copied < MAX_DATE_CHARS); *pWriter << "##fileformat=VCFv4.0\n"; *pWriter << "##fileDate=" << dateBuffer << "\n"; *pWriter << "##command=" << program << "\n"; *pWriter << "##inputBAM=" << inputBAM << "\n"; *pWriter << "##reference=" << reference << "\n"; *pWriter << "#CHROM\tPOS\tID\tREF\tALT\tQUAL\tFILTER\tINFO\n"; } ================================================ FILE: src/SGA/Util/VCFUtil.h ================================================ //----------------------------------------------- // Copyright 2011 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // VCFUtil - collection of utility functions // for producing VCF records. // #ifndef VCFUTIL_H #define VCFUTIL_H #include #include // Enums enum VCFClassification { VCF_SUB, VCF_DEL, VCF_INS, VCF_COMPLEX, VCF_NUM_CLASSIFICATIONS }; // enum VCFReturnCode { VCF_EXACT_MATCH_FAILED, // failed the flanking-base match test VCF_MAP_QUALITY_FAILED, // mapping quality check failed VCF_BASE_MULTIMAP_FAILED, // mapping quality check failed VCF_BASE_PARTIALMAP_FAILED, // the base sequence did not fully align to the reference VCF_INVALID_MULTIALIGNMENT, // failed because the multiple alignment was not valid VCF_OK, // VCF generation succeeded VCF_NUM_RETURN_CODES }; // Structs struct VCFRecord { // functions // initialize an empty record VCFRecord(); // parse a record from a tab-delimited string VCFRecord(const std::string& line); void addComment(const std::string& key, const std::string& value); void addComment(const std::string& key, const int& value); void addComment(const std::string& key, const double& value); void setPassStr(const std::string str); void setQuality(const double q); void printVerbose() const; VCFClassification classify() const; friend std::ostream& operator<<(std::ostream& o, const VCFRecord& record); static bool sort(const VCFRecord& a, const VCFRecord& b) { if(a.refName != b.refName) return a.refName < b.refName; else return a.refPosition < b.refPosition; } bool isMultiAllelic() const { return varStr.find(",") != std::string::npos; } // data std::string refName; size_t refPosition; std::string id; std::string refStr; std::string varStr; double quality; std::string passStr; std::string formatStr; std::vector comments; std::vector sampleStr; }; // Typedefs typedef std::vector VCFVector; typedef std::vector StringVector; // A set of VCF records, with sample names in a predefined order struct VCFCollection { StringVector samples; VCFVector records; }; namespace VCFUtil { // Generate a VCF record from a cancer mutation // Base is the sequence in the individuals normal genome // Variant is the mutated copy of base // Ref is the sequence of the reference that base represents. // Ref will typically be very close to base, but may have some // differences due to SNPs or indels, which we do not want // to call as variants. // The minExactMatch parameter constrains base and variant to have // at least a perfect alignment of minExactMatch at the start // and end of the sequences. This is used as a sanity check for the alignments // If the variants were produced by graph-diff, base and variant // should share exactly k bases at the ends. VCFReturnCode generateVCFFromCancerVariant(const std::string& ref, const std::string& base, const std::string& variant, const std::string& refName, size_t refPosition, const std::string& varName, int minExactMatch, double dustThreshold, int verbose, VCFVector& outRecords); // Write a VCF header to the given file handle void writeHeader(std::ostream* pWriter, const std::string& program, const std::string& bamFile, const std::string& reference); // constants static const int HOMOPOLYMER_ANNOTATE_THRESHOLD = 5; }; #endif ================================================ FILE: src/SGA/Util/VariantIndex.cpp ================================================ //----------------------------------------------------- // Copyright 2014 Ontario Institute for Cancer Research // Written by Jared Simpson (jared.simpson@oicr.on.ca) // Released under the GPL //----------------------------------------------------- // // Data structure for performing proximity queries // against a set of variants // #include #include #include #include "VariantIndex.h" VariantIndex::VariantIndex(const std::string& filename, const ReadTable& refTable) { std::ifstream input(filename.c_str()); std::string line; // Parse the VCF file while(getline(input, line)) { if(line.empty()) continue; if(line[0] == '#') continue; VCFRecord record(line); // Do not allow multi-allelic records if(record.isMultiAllelic()) { std::cerr << "Error: multi-allelic Variant found, please run vcfbreakmulti\n"; exit(EXIT_FAILURE); } // Convert to a minimal representation of the change VariantRecord mvf = { record.refName, record.refStr, record.varStr, record.refPosition }; m_records.push_back(mvf); } buildIndex(refTable); } void VariantIndex::buildIndex(const ReadTable& refTable) { for(size_t i = 0; i < refTable.getCount(); ++i) { const SeqItem& seq_record = refTable.getRead(i); std::string chromosome = seq_record.id; size_t length = seq_record.seq.length(); int buckets = (length / BUCKET_SIZE) + 1; m_map[chromosome].resize(buckets); } for(size_t i = 0; i < m_records.size(); ++i) { VariantIndexMap::iterator iter = m_map.find(m_records[i].reference); assert(iter != m_map.end()); int bucket_id = m_records[i].position / BUCKET_SIZE; assert(bucket_id < (int)iter->second.size()); IntVector& bucket = iter->second[bucket_id]; bucket.push_back(i); } } VariantRecordVector VariantIndex::getNearVariants(const std::string& reference, int position, int distance) const { VariantRecordVector out; VariantIndexMap::const_iterator iter = m_map.find(reference); assert(iter != m_map.end()); const IntVectorVector& chr_buckets = iter->second; int lower_index = std::max(position - distance, 0) / BUCKET_SIZE; int upper_index = std::min((position + distance) / BUCKET_SIZE, (int)chr_buckets.size() - 1); for(int bi = lower_index; bi <= upper_index; ++bi) { const IntVector& bucket = chr_buckets[bi]; for(size_t vi = 0; vi < bucket.size(); ++vi) { const VariantRecord& record = m_records[bucket[vi]]; if(abs(record.position - position) < distance) out.push_back(record); } } return out; } ================================================ FILE: src/SGA/Util/VariantIndex.h ================================================ //----------------------------------------------------- // Copyright 2014 Ontario Institute for Cancer Research // Written by Jared Simpson (jared.simpson@oicr.on.ca) // Released under the GPL //----------------------------------------------------- // // Data structure for performing proximity queries // against a set of variants // #ifndef VARIANTINDEX_H #define VARIANTINDEX_H #include "Util.h" #include "SeqReader.h" #include "VCFUtil.h" #include "ReadTable.h" #include // To save space we store a vcf-like record // with only the required fields struct VariantRecord { std::string reference; std::string ref_sequence; std::string alt_sequence; size_t position; }; typedef std::vector VariantRecordVector; typedef std::vector IntVector; typedef std::vector IntVectorVector; // map from chromosome to a vector of buckets, one every 10kbp typedef std::map VariantIndexMap; class VariantIndex { public: // VariantIndex(const std::string& filename, const ReadTable& refTable); // Return a vector of the variants that are close to the input variant VariantRecordVector getNearVariants(const std::string& reference, int position, int distance = 30) const; private: // void buildIndex(const ReadTable& refTable); // VariantRecordVector m_records; VariantIndexMap m_map; // static const int BUCKET_SIZE = 10000; }; #endif ================================================ FILE: src/SGA/Util/Verbosity.h ================================================ ///---------------------------------------------- // Copyright 2013 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // Verbosity - Singleton object to only // hold the verbosity level for the entire // program // #ifndef VERBOSITY_H #define VERBOSITY_H class Verbosity { public: Verbosity() : m_maxPrintLevel(0) {} // Get singleton object static Verbosity& Instance() { static Verbosity instance; return instance; } // Get/set the print level int getPrintLevel() const { return m_maxPrintLevel; } void setPrintLevel(int l) { m_maxPrintLevel = l; } private: int m_maxPrintLevel; }; #endif ================================================ FILE: src/SGA/Util/bamreader.h ================================================ #ifndef BAMREADER_H #define BAMREADER_h #include "api/BamReader.h" #include "api/BamWriter.h" #include "Util.h" class SVBamReader { public: Bamreader(const std::string filename, const std::string prefix, const int isize = 2000) : m_bam(filename), m_prefix(prefix), m_isize(isize) {} ~Bamreader() { } void bamToSeqRecordVector(SeqRecordVector &srv); bool setBamRegion(int refid1, int refid2, int pos1, int pos2); bool findBamIndex(); private: std::string m_bam; std::string m_prefix; int m_isize; std::string m_index; BamTools::BamReader m_reader; BamTools::BamRegion m_region; } #endif ================================================ FILE: src/SGA/Util/bucketSort.cpp ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // bucketSort - perform a bucket sort over a range of values // ================================================ FILE: src/SGA/Util/bucketSort.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL license //----------------------------------------------- // // bucketSort - perform a bucket sort over a range of values // #ifndef BUCKETSORT_H #define BUCKETSORT_H #include template void bucketSort(IterType start, IterType end, BucketFunctor func) { // Typedef the base value of the iterator typedef typename std::iterator_traits::value_type base_value; typedef std::list base_list; typedef typename base_list::iterator list_iterator; // Get the number of buckets needed int numBuckets = func.getNumBuckets(); // Allocate an array of lists base_list* buckets = new base_list[numBuckets]; // Place the items into buckets for(IterType iter = start; iter != end; ++iter) { int bucket_id = func(*iter); assert(bucket_id < numBuckets); buckets[bucket_id].push_back(*iter); } // Place the elements back into the array IterType curr = start; for(int i = 0; i < numBuckets; ++i) { IterType bucket_start = curr; size_t count = 0; for(list_iterator bucket_iter = buckets[i].begin(); bucket_iter != buckets[i].end(); ++bucket_iter) { ++count; *curr++ = *bucket_iter; } std::sort(bucket_start, curr, func); } assert(curr == end); // delete lists delete [] buckets; } template void histogramSort(T* x, size_t n, int depth, PrimarySorter& primarySorter, const FinalSorter& finalSorter) { // Get the number of buckets needed int numBuckets = primarySorter.getNumBuckets(); // Set the functor's offset primarySorter.setBucketDepth(depth); // Allocate an array for the bucket start points and the bucket end points // TODO: Could remove at least one of these arrays size_t* bucket_starts = new size_t[numBuckets]; size_t* bucket_ends = new size_t[numBuckets]; size_t* bucket_counts = new size_t[numBuckets]; for(int i = 0; i < numBuckets; ++i) { bucket_starts[i] = 0; bucket_ends[i] = 0; bucket_counts[i] = 0; } // Place the items into buckets for(size_t i = 0; i < n; ++i) { int bucket_id = primarySorter.getBucket(x[i]); assert(bucket_id < numBuckets); bucket_counts[bucket_id]++; } // Set up the start/end points size_t sum = 0; for(int i = 0; i < numBuckets; ++i) { size_t val = bucket_counts[i]; bucket_starts[i] = sum; sum += val; bucket_ends[i] = sum; //std::cout << "Bucket count: " << bucket_counts[i] << " start: " << bucket_starts[i] << " end: " << bucket_ends[i] << "\n"; } // Now, consider the bucket_starts positions to be the next (potentially) unsorted position // for each bucket. Iterate through these values cycles the elements into place int curr_bucket = 0; // Start on the first bucket that has element while(bucket_starts[curr_bucket] == bucket_ends[curr_bucket]) ++curr_bucket; while(curr_bucket != numBuckets) { //std::cout << "Curr Bucket: " << curr_bucket << "\n"; //std::cout << "CurrIDX: " << bucket_starts[curr_bucket] << "\n"; int elem_offset = bucket_starts[curr_bucket]; // Get the bucket for this element int bucket_id = primarySorter.getBucket(x[elem_offset]); // if the element is in the correct bucket, just advance the pointer if(bucket_id == curr_bucket) bucket_starts[curr_bucket]++; else { // Cycle the elements until we land back in this bucket, at which point // we have sorted one position further in the current bucket // Hold the info for the element that has been displaced T displaced = x[elem_offset]; bool stop = false; while(!stop) { // Get the offset of the position this element should be in int displaced_bucket = primarySorter.getBucket(displaced); if(displaced_bucket == curr_bucket) { // The displaced element belongs back in the original bucket, place it there x[elem_offset] = displaced; bucket_starts[curr_bucket]++; stop = true; } else { int cycle_offset = bucket_starts[displaced_bucket]; // Make space for the incoming element T swap = x[cycle_offset]; x[cycle_offset] = displaced; displaced = swap; bucket_starts[displaced_bucket]++; } } } // At this point, the bucket_start for current bucket // has moved 1 element // Other buckets may have advanced as well // while(curr_bucket < numBuckets && bucket_starts[curr_bucket] == bucket_ends[curr_bucket]) ++curr_bucket; } // Recompute the start/end points sum = 0; for(int i = 0; i < numBuckets; ++i) { size_t val = bucket_counts[i]; bucket_starts[i] = sum; sum += val; bucket_ends[i] = sum; } // Finally, sort each bucket for(int i = 0; i < numBuckets; ++i) { T* curr_bucket = x + bucket_starts[i]; size_t curr_count = bucket_counts[i]; if(curr_count < 2) continue; assert(curr_count == (bucket_ends[i] - bucket_starts[i])); //std::cout << "Sorting bucket " << i << "\n"; const int max_depth = 100; const size_t min_elements = 1000; // terminate the bucket sort under 3 conditions: // 1) the bucket is degenerate (no more histogram sorting can be done) - this comes from the functor // 2) the number of elements in the bucket is low // 3) the max depth has been hit if(primarySorter.isBucketDegenerate(i)) { std::sort(curr_bucket, curr_bucket + curr_count, finalSorter); } else if(bucket_counts[i] < min_elements || depth >= max_depth) { int next_char = (depth + 1) * primarySorter.getBucketLen(); mkqs2(curr_bucket, curr_count, next_char, primarySorter, finalSorter); //std::sort(curr_bucket, curr_bucket + bucket_counts[i], finalSorter); } else { histogramSort(curr_bucket, curr_count, depth + 1, primarySorter, finalSorter); } } // delete arrays delete [] bucket_counts; delete [] bucket_starts; delete [] bucket_ends; } #endif ================================================ FILE: src/SGA/Util/gzstream.C ================================================ // ============================================================================ // gzstream, C++ iostream classes wrapping the zlib compression library. // Copyright (C) 2001 Deepak Bandyopadhyay, Lutz Kettner // // This library is free software; you can redistribute it and/or // modify it under the terms of the GNU General Public // License as published by the Free Software Foundation; either // version 3 of the License, or (at your option) any later version. // // This library is distributed in the hope that it will be useful, // but WITHOUT ANY WARRANTY; without even the implied warranty of // MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU // General Public License for more details. // // // See src/COPYING for the full terms of the license. // // You should have received a copy of the GNU Lesser General Public // License along with this library; if not, write to the Free Software // Foundation, Inc., 59 Temple Place, Suite 330, Boston, MA 02111-1307 USA // ============================================================================ // // File : gzstream.C // Revision : $Revision: 1.7 $ // Revision_date : $Date: 2003/01/08 14:41:27 $ // Author(s) : Deepak Bandyopadhyay, Lutz Kettner // Modified : Jared Simpson 2010/04/16. Converted license notification to // GPLv3 to be compatible with the rest of the code base as // allowed by LGPLv2.1. No other changes were made. // // Standard streambuf implementation following Nicolai Josuttis, "The // Standard C++ Library". // ============================================================================ #include "gzstream.h" #include #include // for memcpy #ifdef GZSTREAM_NAMESPACE namespace GZSTREAM_NAMESPACE { #endif // ---------------------------------------------------------------------------- // Internal classes to implement gzstream. See header file for user classes. // ---------------------------------------------------------------------------- // -------------------------------------- // class gzstreambuf: // -------------------------------------- gzstreambuf* gzstreambuf::open( const char* name, int open_mode) { if ( is_open()) return (gzstreambuf*)0; mode = open_mode; // no append nor read/write mode if ((mode & std::ios::ate) || (mode & std::ios::app) || ((mode & std::ios::in) && (mode & std::ios::out))) return (gzstreambuf*)0; char fmode[10]; char* fmodeptr = fmode; if ( mode & std::ios::in) *fmodeptr++ = 'r'; else if ( mode & std::ios::out) *fmodeptr++ = 'w'; *fmodeptr++ = 'b'; *fmodeptr = '\0'; file = gzopen( name, fmode); if (file == 0) return (gzstreambuf*)0; opened = 1; return this; } gzstreambuf * gzstreambuf::close() { if ( is_open()) { sync(); opened = 0; if ( gzclose( file) == Z_OK) return this; } return (gzstreambuf*)0; } int gzstreambuf::underflow() { // used for input buffer only if ( gptr() && ( gptr() < egptr())) return * reinterpret_cast( gptr()); if ( ! (mode & std::ios::in) || ! opened) return EOF; // Josuttis' implementation of inbuf int n_putback = gptr() - eback(); if ( n_putback > 4) n_putback = 4; memcpy( buffer + (4 - n_putback), gptr() - n_putback, n_putback); int num = gzread( file, buffer+4, bufferSize-4); if (num <= 0) // ERROR or EOF return EOF; // reset buffer pointers setg( buffer + (4 - n_putback), // beginning of putback area buffer + 4, // read position buffer + 4 + num); // end of buffer // return next character return * reinterpret_cast( gptr()); } int gzstreambuf::flush_buffer() { // Separate the writing of the buffer from overflow() and // sync() operation. int w = pptr() - pbase(); if ( gzwrite( file, pbase(), w) != w) return EOF; pbump( -w); return w; } int gzstreambuf::overflow( int c) { // used for output buffer only if ( ! ( mode & std::ios::out) || ! opened) return EOF; if (c != EOF) { *pptr() = c; pbump(1); } if ( flush_buffer() == EOF) return EOF; return c; } int gzstreambuf::sync() { // Changed to use flush_buffer() instead of overflow( EOF) // which caused improper behavior with std::endl and flush(), // bug reported by Vincent Ricard. if ( pptr() && pptr() > pbase()) { if ( flush_buffer() == EOF) return -1; } return 0; } // -------------------------------------- // class gzstreambase: // -------------------------------------- gzstreambase::gzstreambase( const char* name, int mode) { init( &buf); open( name, mode); } gzstreambase::~gzstreambase() { buf.close(); } void gzstreambase::open( const char* name, int open_mode) { if ( ! buf.open( name, open_mode)) clear( rdstate() | std::ios::badbit); } void gzstreambase::close() { if ( buf.is_open()) if ( ! buf.close()) clear( rdstate() | std::ios::badbit); } #ifdef GZSTREAM_NAMESPACE } // namespace GZSTREAM_NAMESPACE #endif // ============================================================================ // EOF // ================================================ FILE: src/SGA/Util/gzstream.h ================================================ // ============================================================================ // gzstream, C++ iostream classes wrapping the zlib compression library. // Copyright (C) 2001 Deepak Bandyopadhyay, Lutz Kettner // // This library is free software; you can redistribute it and/or // modify it under the terms of the GNU General Public // License as published by the Free Software Foundation; either // version 3 of the License, or (at your option) any later version. // // This library is distributed in the hope that it will be useful, // but WITHOUT ANY WARRANTY; without even the implied warranty of // MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU // General Public License for more details. // // // See src/COPYING for the full terms of the license. // // You should have received a copy of the GNU Lesser General Public // License along with this library; if not, write to the Free Software // Foundation, Inc., 59 Temple Place, Suite 330, Boston, MA 02111-1307 USA // ============================================================================ // // File : gzstream.h // Revision : $Revision: 1.5 $ // Revision_date : $Date: 2002/04/26 23:30:15 $ // Author(s) : Deepak Bandyopadhyay, Lutz Kettner // Modified : Jared Simpson 2010/04/16. Converted license notification to // GPLv3 to be compatible with the rest of the code base as // allowed by LGPLv2.1. No other changes were made. // // // Standard streambuf implementation following Nicolai Josuttis, "The // Standard C++ Library". // ============================================================================ #ifndef GZSTREAM_H #define GZSTREAM_H 1 // standard C++ with new header file names and std:: namespace #include #include #include #ifdef GZSTREAM_NAMESPACE namespace GZSTREAM_NAMESPACE { #endif // ---------------------------------------------------------------------------- // Internal classes to implement gzstream. See below for user classes. // ---------------------------------------------------------------------------- class gzstreambuf : public std::streambuf { private: static const int bufferSize = 47+256; // size of data buff // totals 512 bytes under g++ for igzstream at the end. gzFile file; // file handle for compressed file char buffer[bufferSize]; // data buffer char opened; // open/close state of stream int mode; // I/O mode int flush_buffer(); public: gzstreambuf() : opened(0) { setp( buffer, buffer + (bufferSize-1)); setg( buffer + 4, // beginning of putback area buffer + 4, // read position buffer + 4); // end position // ASSERT: both input & output capabilities will not be used together } int is_open() { return opened; } gzstreambuf* open( const char* name, int open_mode); gzstreambuf* close(); ~gzstreambuf() { close(); } virtual int overflow( int c = EOF); virtual int underflow(); virtual int sync(); }; class gzstreambase : virtual public std::ios { protected: gzstreambuf buf; public: gzstreambase() { init(&buf); } gzstreambase( const char* name, int open_mode); ~gzstreambase(); void open( const char* name, int open_mode); void close(); gzstreambuf* rdbuf() { return &buf; } }; // ---------------------------------------------------------------------------- // User classes. Use igzstream and ogzstream analogously to ifstream and // ofstream respectively. They read and write files based on the gz* // function interface of the zlib. Files are compatible with gzip compression. // ---------------------------------------------------------------------------- class igzstream : public gzstreambase, public std::istream { public: igzstream() : std::istream( &buf) {} igzstream( const char* name, int open_mode = std::ios::in) : gzstreambase( name, open_mode), std::istream( &buf) {} gzstreambuf* rdbuf() { return gzstreambase::rdbuf(); } void open( const char* name, int open_mode = std::ios::in) { gzstreambase::open( name, open_mode); } }; class ogzstream : public gzstreambase, public std::ostream { public: ogzstream() : std::ostream( &buf) {} ogzstream( const char* name, int mode = std::ios::out) : gzstreambase( name, mode), std::ostream( &buf) {} gzstreambuf* rdbuf() { return gzstreambase::rdbuf(); } void open( const char* name, int open_mode = std::ios::out) { gzstreambase::open( name, open_mode); } }; #ifdef GZSTREAM_NAMESPACE } // namespace GZSTREAM_NAMESPACE #endif #endif // GZSTREAM_H // ============================================================================ // EOF // ================================================ FILE: src/SGA/Util/mkqs.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL license //----------------------------------------------- // // mkqs - multikey quicksort // // Perform a ternary quicksort of strings as described in // Bentley and Sedgewick, 1997 // // Example code was downloaded from http://www.cs.princeton.edu/~rs/strings/demo.c // Modified by JTS to take in a comparator and use a generic type // #ifndef MKQS_H #define MKQS_H #include #include #include #include #include //#include "MkqsThread.h" #define mkqs_swap(a, b) { T tmp = x[a]; x[a] = x[b]; x[b] = tmp; } // Swap [i..i+n] and [j..j+n] in x template inline void vecswap(int i, int j, int n, T* x) { while (n-- > 0) { mkqs_swap(i, j); i++; j++; } } #define mkqs_swap2(a, b) { T t = *(a); *(a) = *(b); *(b) = t; } #define ptr2char(p) (primarySorter.getChar(*(p), depth)) #define elem2char(e, d) (primarySorter.getChar((e), (d))) template inline void vecswap2(T* a, T* b, int n) { while (n-- > 0) { T t = *a; *a++ = *b; *b++ = t; } } template T* med3func(T* a, T* b, T* c, int depth, const PrimarySorter& primarySorter) { int va, vb, vc; if ((va=ptr2char(a)) == (vb=ptr2char(b))) return a; if ((vc=ptr2char(c)) == va || vc == vb) return c; return va < vb ? (vb < vc ? b : (va < vc ? c : a ) ) : (vb > vc ? b : (va < vc ? a : c ) ); } #define med3(a, b, c) med3func(a, b, c, depth, primarySorter) template inline void inssort(T* a, int n, int d, const PrimarySorter& primarySorter, const FinalSorter& finalSorter) { T *pi, *pj, s, t; for (pi = a + 1; --n > 0; pi++) { for (pj = pi; pj > a; pj--) { // Inline strcmp: break if *(pj-1) <= *pj T elem_s = *(pj - 1); T elem_t = *pj; const char* s = primarySorter.getChrPtr(elem_s); const char* t = primarySorter.getChrPtr(elem_t); for (s=s+d, t=t+d; *s==*t && *s!=0; s++, t++) ; if (*s < *t || (*s == *t && finalSorter(elem_s, elem_t))) break; mkqs_swap2(pj, pj-1); } } } // template void mkqs2(T* a, int n, int depth, const PrimarySorter& primarySorter, const FinalSorter& finalSorter) { int r, partval; T *pa, *pb, *pc, *pd, *pm, *pn, t; if (n < 10) { inssort(a, n, depth, primarySorter, finalSorter); return; } // chatGPT determinisic method pm = med3(a, a + n/2, a + n - 1); // med3 is already defined above // old rand determined, has hidden mutex lock /* pm = a + (n/2); pn = a + (n-1); int mid_idx = rand() % n; pm = &a[mid_idx];*/ mkqs_swap2(a, pm); partval = ptr2char(a); pa = pb = a + 1; pc = pd = a + n-1; for (;;) { while (pb <= pc && (r = ptr2char(pb)-partval) <= 0) { if (r == 0) { mkqs_swap2(pa, pb); pa++; } pb++; } while (pb <= pc && (r = ptr2char(pc)-partval) >= 0) { if (r == 0) { mkqs_swap2(pc, pd); pd--; } pc--; } if (pb > pc) break; mkqs_swap2(pb, pc); pb++; pc--; } pn = a + n; r = std::min(pa-a, pb-pa); vecswap2(a, pb-r, r); r = std::min(pd-pc, pn-pd-1); vecswap2(pb, pn-r, r); if ((r = pb-pa) > 1) mkqs2(a, r, depth, primarySorter, finalSorter); if (ptr2char(a + r) != 0) mkqs2(a + r, pa-a + pn-pd-1, depth+1, primarySorter, finalSorter); else { int n2 = pa - a + pn - pd - 1; std::sort(a + r, a + r + n2, finalSorter); } if ((r = pd-pc) > 1) mkqs2(a + n-r, r, depth, primarySorter, finalSorter); } // Parallel multikey quicksort. It performs mkqs but will // subdivide the array to sort into sub jobs which can be sorted using threads. /*template void parallel_mkqs(T* pData, int n, int numThreads, const PrimarySorter& primarySorter, const FinalSorter& finalSorter) { typedef MkqsJob Job; typedef std::queue JobQueue; Job initialJob(pData, n, 0); JobQueue queue; queue.push(initialJob); // Create the mutex that guards the queue pthread_mutex_t queue_mutex; int ret = pthread_mutex_init(&queue_mutex, NULL); if(ret != 0) { std::cerr << "Mutex initialization failed with error " << ret << ", aborting" << std::endl; exit(EXIT_FAILURE); } // Calculate the threshold size for performing serial continuation of the sort. Once the chunks // are below this size, it is better to not subdivide the problem into smaller chunks // to avoid the overhead of locking, adding to the queue, etc. int threshold_size = n / numThreads; // Create the semaphore used to signal that data is ready to be processed // Initial value is 1 as there is one item on the queue to start sem_t queue_sem; ret = sem_init( &queue_sem, PTHREAD_PROCESS_PRIVATE, 1 ); if(ret != 0) { std::cerr << "Semaphore initialization failed with error " << ret << "\n"; std::cerr << "You are probably running on OSX which does not provide unnamed semaphores\n"; exit(EXIT_FAILURE); } // Create the semaphore indicating a thread is finished working. // This semaphore is incremented by the threads in their run loop // before waiting for queue items. If the thread takes an item, // this semaphore is decremented. The main thread checks // this semaphore after confirming the queue is empty. If this // semaphore value equals the total number of threads, // no more work can remain the the threads are cleaned up sem_t done_sem; ret = sem_init( &done_sem, PTHREAD_PROCESS_PRIVATE, 0 ); if(ret != 0) { std::cerr << "Semaphore initialization failed with error " << ret << "\n"; std::cerr << "You are probably running on OSX which does not provide unnamed semaphores\n"; exit(EXIT_FAILURE); } // Create and start the threads MkqsThread* threads[numThreads]; for(int i = 0; i < numThreads; ++i) { threads[i] = new MkqsThread(i, &queue, &queue_mutex, &queue_sem, &done_sem, threshold_size, &primarySorter, &finalSorter); threads[i]->start(); } // Check for the end condition bool done = false; while(!done) { // sleep(1); // Check if the queue is empty // If it is and all threads are finished working (all have posted to // the done semaphore), then the threads can be cleaned up. pthread_mutex_lock(&queue_mutex); if(queue.empty()) { int semval; sem_getvalue(&done_sem, &semval); if(semval == numThreads) done = true; } pthread_mutex_unlock(&queue_mutex); } // Signal all the threads to stop, then post to the semaphore they are waiting on // All threads will pick up the stop request after the posts and call pthread exit for(int i = 0; i < numThreads; ++i) { threads[i]->stop(); } for(int i = 0; i < numThreads; ++i) { sem_post(&queue_sem); } // Join and destroy the threads for(int i = 0; i < numThreads; ++i) { threads[i]->join(); delete threads[i]; } // Destroy the semaphore sem_destroy(&queue_sem); sem_destroy(&done_sem); // Destroy the queue mutex ret = pthread_mutex_destroy(&queue_mutex); if(ret != 0) { std::cerr << "Mutex destruction failed with error " << ret << ", aborting" << std::endl; exit(EXIT_FAILURE); } } */ // // Perform a partial sort of the data using the mkqs algorithm // Iterative sort jobs are created and added to pQueue which is // protected by pQueueMutex. After addition, pQueueSem is updated. // /* template void parallel_mkqs_process(MkqsJob& job, std::queue >* pQueue, pthread_mutex_t* pQueueMutex, sem_t* pQueueSem, const PrimarySorter& primarySorter, const FinalSorter& finalSorter) { T* a = job.pData; int n = job.n; int depth = job.depth; int r, partval; T *pa, *pb, *pc, *pd, *pm, *pn, t; if(n < 10) { inssort(a, n, depth, primarySorter, finalSorter); return; } pm = a + (n/2); pn = a + (n-1); int mid_idx = rand() % n; pm = &a[mid_idx]; mkqs_swap2(a, pm); partval = ptr2char(a); pa = pb = a + 1; pc = pd = a + n-1; for (;;) { while (pb <= pc && (r = ptr2char(pb)-partval) <= 0) { if (r == 0) { mkqs_swap2(pa, pb); pa++; } pb++; } while (pb <= pc && (r = ptr2char(pc)-partval) >= 0) { if (r == 0) { mkqs_swap2(pc, pd); pd--; } pc--; } if (pb > pc) break; mkqs_swap2(pb, pc); pb++; pc--; } pn = a + n; r = std::min(pa-a, pb-pa); vecswap2(a, pb-r, r); r = std::min(pd-pc, pn-pd-1); vecswap2(pb, pn-r, r); // Lock the queue and push new items if necessary // If new items are added to the queue, the semaphore is posted to pthread_mutex_lock(pQueueMutex); if ((r = pb-pa) > 1) { MkqsJob job(a, r, depth); pQueue->push(job); sem_post(pQueueSem); } if (ptr2char(a + r) != 0) { MkqsJob job(a + r, pa-a + pn-pd-1, depth + 1); pQueue->push(job); sem_post(pQueueSem); } else { // Finalize the sort int n2 = pa - a + pn - pd - 1; std::sort(a + r, a + r + n2, finalSorter); } if ((r = pd-pc) > 1) { MkqsJob job(a + n-r, r, depth); pQueue->push(job); sem_post(pQueueSem); } // Unlock the mutex pthread_mutex_unlock(pQueueMutex); } */ #endif ================================================ FILE: src/SGA/Util/old.AlignedContig.h ================================================ #include "Util.h" #include "api/BamReader.h" #include "api/BamWriter.h" #include class AlignedContig { public: AlignedContig(const BamTools::BamAlignment align) { m_align.push_back(align); } ~AlignedContig() {} void addAlignement(const BamTools::BamAlignment align) { m_align.push_back(align); m_num++; } void addRead(const SeqRecord sr) { m_sr.push_back(sr); } private: std::vector m_align; std::vector m_sr; unsigned m_num; } ================================================ FILE: src/SGA/Util/stdaln.c ================================================ /* The MIT License Copyright (c) 2003-2006, 2008, 2009, by Heng Li Permission is hereby granted, free of charge, to any person obtaining a copy of this software and associated documentation files (the "Software"), to deal in the Software without restriction, including without limitation the rights to use, copy, modify, merge, publish, distribute, sublicense, and/or sell copies of the Software, and to permit persons to whom the Software is furnished to do so, subject to the following conditions: The above copyright notice and this permission notice shall be included in all copies or substantial portions of the Software. THE SOFTWARE IS PROVIDED "AS IS", WITHOUT WARRANTY OF ANY KIND, EXPRESS OR IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT. IN NO EVENT SHALL THE AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER LIABILITY, WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING FROM, OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS IN THE SOFTWARE. */ #include #include #include #include #include "stdaln.h" /* char -> 17 (=16+1) nucleotides */ unsigned char aln_nt16_table[256] = { 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,16 /*'-'*/,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15, 1,14, 4, 11,15,15, 2, 13,15,15,10, 15, 5,15,15, 15,15, 3, 6, 8,15, 7, 9, 0,12,15,15, 15,15,15,15, 15, 1,14, 4, 11,15,15, 2, 13,15,15,10, 15, 5,15,15, 15,15, 3, 6, 8,15, 7, 9, 0,12,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15, 15,15,15,15 }; char *aln_nt16_rev_table = "XAGRCMSVTWKDYHBN-"; /* char -> 5 (=4+1) nucleotides */ unsigned char aln_nt4_table[256] = { 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 5 /*'-'*/, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 0, 4, 2, 4, 4, 4, 1, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 3, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 0, 4, 2, 4, 4, 4, 1, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 3, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4 }; char *aln_nt4_rev_table = "AGCTN-"; /* char -> 22 (=20+1+1) amino acids */ unsigned char aln_aa_table[256] = { 21,21,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21, 21,21,20,21, 21,22 /*'-'*/,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21, 21, 0,21, 4, 3, 6,13, 7, 8, 9,21,11, 10,12, 2,21, 14, 5, 1,15, 16,21,19,17, 21,18,21,21, 21,21,21,21, 21, 0,21, 4, 3, 6,13, 7, 8, 9,21,11, 10,12, 2,21, 14, 5, 1,15, 16,21,19,17, 21,18,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21, 21,21,21,21 }; char *aln_aa_rev_table = "ARNDCQEGHILKMFPSTWYV*X-"; /* 01234567890123456789012 */ /* translation table. They are useless in stdaln.c, but when you realize you need it, you need not write the table again. */ unsigned char aln_trans_table_eu[66] = { 11,11, 2, 2, 1, 1,15,15, 16,16,16,16, 9,12, 9, 9, 6, 6, 3, 3, 7, 7, 7, 7, 0, 0, 0, 0, 19,19,19,19, 5, 5, 8, 8, 1, 1, 1, 1, 14,14,14,14, 10,10,10,10, 20,20,18,18, 20,17, 4, 4, 15,15,15,15, 10,10,13,13, 21, 22 }; char *aln_trans_table_eu_char = "KKNNRRSSTTTTIMIIEEDDGGGGAAAAVVVVQQHHRRRRPPPPLLLL**YY*WCCSSSSLLFFX"; /* 01234567890123456789012345678901234567890123456789012345678901234 */ int aln_sm_blosum62[] = { /* A R N D C Q E G H I L K M F P S T W Y V * X */ 4,-1,-2,-2, 0,-1,-1, 0,-2,-1,-1,-1,-1,-2,-1, 1, 0,-3,-2, 0,-4, 0, -1, 5, 0,-2,-3, 1, 0,-2, 0,-3,-2, 2,-1,-3,-2,-1,-1,-3,-2,-3,-4,-1, -2, 0, 6, 1,-3, 0, 0, 0, 1,-3,-3, 0,-2,-3,-2, 1, 0,-4,-2,-3,-4,-1, -2,-2, 1, 6,-3, 0, 2,-1,-1,-3,-4,-1,-3,-3,-1, 0,-1,-4,-3,-3,-4,-1, 0,-3,-3,-3, 9,-3,-4,-3,-3,-1,-1,-3,-1,-2,-3,-1,-1,-2,-2,-1,-4,-2, -1, 1, 0, 0,-3, 5, 2,-2, 0,-3,-2, 1, 0,-3,-1, 0,-1,-2,-1,-2,-4,-1, -1, 0, 0, 2,-4, 2, 5,-2, 0,-3,-3, 1,-2,-3,-1, 0,-1,-3,-2,-2,-4,-1, 0,-2, 0,-1,-3,-2,-2, 6,-2,-4,-4,-2,-3,-3,-2, 0,-2,-2,-3,-3,-4,-1, -2, 0, 1,-1,-3, 0, 0,-2, 8,-3,-3,-1,-2,-1,-2,-1,-2,-2, 2,-3,-4,-1, -1,-3,-3,-3,-1,-3,-3,-4,-3, 4, 2,-3, 1, 0,-3,-2,-1,-3,-1, 3,-4,-1, -1,-2,-3,-4,-1,-2,-3,-4,-3, 2, 4,-2, 2, 0,-3,-2,-1,-2,-1, 1,-4,-1, -1, 2, 0,-1,-3, 1, 1,-2,-1,-3,-2, 5,-1,-3,-1, 0,-1,-3,-2,-2,-4,-1, -1,-1,-2,-3,-1, 0,-2,-3,-2, 1, 2,-1, 5, 0,-2,-1,-1,-1,-1, 1,-4,-1, -2,-3,-3,-3,-2,-3,-3,-3,-1, 0, 0,-3, 0, 6,-4,-2,-2, 1, 3,-1,-4,-1, -1,-2,-2,-1,-3,-1,-1,-2,-2,-3,-3,-1,-2,-4, 7,-1,-1,-4,-3,-2,-4,-2, 1,-1, 1, 0,-1, 0, 0, 0,-1,-2,-2, 0,-1,-2,-1, 4, 1,-3,-2,-2,-4, 0, 0,-1, 0,-1,-1,-1,-1,-2,-2,-1,-1,-1,-1,-2,-1, 1, 5,-2,-2, 0,-4, 0, -3,-3,-4,-4,-2,-2,-3,-2,-2,-3,-2,-3,-1, 1,-4,-3,-2,11, 2,-3,-4,-2, -2,-2,-2,-3,-2,-1,-2,-3, 2,-1,-1,-2,-1, 3,-3,-2,-2, 2, 7,-1,-4,-1, 0,-3,-3,-3,-1,-2,-2,-3,-3, 3, 1,-2, 1,-1,-2,-2, 0,-3,-1, 4,-4,-1, -4,-4,-4,-4,-4,-4,-4,-4,-4,-4,-4,-4,-4,-4,-4,-4,-4,-4,-4,-4, 1,-4, 0,-1,-1,-1,-2,-1,-1,-1,-1,-1,-1,-1,-1,-1,-2, 0, 0,-2,-1,-1,-4,-1 }; int aln_sm_blosum45[] = { /* A R N D C Q E G H I L K M F P S T W Y V * X */ 5,-2,-1,-2,-1,-1,-1, 0,-2,-1,-1,-1,-1,-2,-1, 1, 0,-2,-2, 0,-5, 0, -2, 7, 0,-1,-3, 1, 0,-2, 0,-3,-2, 3,-1,-2,-2,-1,-1,-2,-1,-2,-5,-1, -1, 0, 6, 2,-2, 0, 0, 0, 1,-2,-3, 0,-2,-2,-2, 1, 0,-4,-2,-3,-5,-1, -2,-1, 2, 7,-3, 0, 2,-1, 0,-4,-3, 0,-3,-4,-1, 0,-1,-4,-2,-3,-5,-1, -1,-3,-2,-3,12,-3,-3,-3,-3,-3,-2,-3,-2,-2,-4,-1,-1,-5,-3,-1,-5,-2, -1, 1, 0, 0,-3, 6, 2,-2, 1,-2,-2, 1, 0,-4,-1, 0,-1,-2,-1,-3,-5,-1, -1, 0, 0, 2,-3, 2, 6,-2, 0,-3,-2, 1,-2,-3, 0, 0,-1,-3,-2,-3,-5,-1, 0,-2, 0,-1,-3,-2,-2, 7,-2,-4,-3,-2,-2,-3,-2, 0,-2,-2,-3,-3,-5,-1, -2, 0, 1, 0,-3, 1, 0,-2,10,-3,-2,-1, 0,-2,-2,-1,-2,-3, 2,-3,-5,-1, -1,-3,-2,-4,-3,-2,-3,-4,-3, 5, 2,-3, 2, 0,-2,-2,-1,-2, 0, 3,-5,-1, -1,-2,-3,-3,-2,-2,-2,-3,-2, 2, 5,-3, 2, 1,-3,-3,-1,-2, 0, 1,-5,-1, -1, 3, 0, 0,-3, 1, 1,-2,-1,-3,-3, 5,-1,-3,-1,-1,-1,-2,-1,-2,-5,-1, -1,-1,-2,-3,-2, 0,-2,-2, 0, 2, 2,-1, 6, 0,-2,-2,-1,-2, 0, 1,-5,-1, -2,-2,-2,-4,-2,-4,-3,-3,-2, 0, 1,-3, 0, 8,-3,-2,-1, 1, 3, 0,-5,-1, -1,-2,-2,-1,-4,-1, 0,-2,-2,-2,-3,-1,-2,-3, 9,-1,-1,-3,-3,-3,-5,-1, 1,-1, 1, 0,-1, 0, 0, 0,-1,-2,-3,-1,-2,-2,-1, 4, 2,-4,-2,-1,-5, 0, 0,-1, 0,-1,-1,-1,-1,-2,-2,-1,-1,-1,-1,-1,-1, 2, 5,-3,-1, 0,-5, 0, -2,-2,-4,-4,-5,-2,-3,-2,-3,-2,-2,-2,-2, 1,-3,-4,-3,15, 3,-3,-5,-2, -2,-1,-2,-2,-3,-1,-2,-3, 2, 0, 0,-1, 0, 3,-3,-2,-1, 3, 8,-1,-5,-1, 0,-2,-3,-3,-1,-3,-3,-3,-3, 3, 1,-2, 1, 0,-3,-1, 0,-3,-1, 5,-5,-1, -5,-5,-5,-5,-5,-5,-5,-5,-5,-5,-5,-5,-5,-5,-5,-5,-5,-5,-5,-5, 1,-5, 0,-1,-1,-1,-2,-1,-1,-1,-1,-1,-1,-1,-1,-1,-1, 0, 0,-2,-1,-1,-5,-1 }; int aln_sm_nt[] = { /* X A G R C M S V T W K D Y H B N */ -2,-2,-2,-2,-2,-2,-2,-2,-2,-2,-2,-2,-2,-2,-2,-2, -2, 2,-1, 1,-2, 1,-2, 0,-2, 1,-2, 0,-2, 0,-2, 0, -2,-1, 2, 1,-2,-2, 1, 0,-2,-2, 1, 0,-2,-2, 0, 0, -2, 1, 1, 1,-2,-1,-1, 0,-2,-1,-1, 0,-2, 0, 0, 0, -2,-2,-2,-2, 2, 1, 1, 0,-1,-2,-2,-2, 1, 0, 0, 0, -2, 1,-2,-1, 1, 1,-1, 0,-2,-1,-2, 0,-1, 0, 0, 0, -2,-2, 1,-1, 1,-1, 1, 0,-2,-2,-1, 0,-1, 0, 0, 0, -2, 0, 0, 0, 0, 0, 0, 0,-2, 0, 0, 0, 0, 0, 0, 0, -2,-2,-2,-2,-1,-2,-2,-2, 2, 1, 1, 0, 1, 0, 0, 0, -2, 1,-2,-1,-2,-1,-2, 0, 1, 1,-1, 0,-1, 0, 0, 0, -2,-2, 1,-1,-2,-2,-1, 0, 1,-1, 1, 0,-1, 0, 0, 0, -2, 0, 0, 0,-2, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, -2,-2,-2,-2, 1,-1,-1, 0, 1,-1,-1, 0, 1, 0, 0, 0, -2, 0,-2, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, -2,-2, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, -2, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0 }; int aln_sm_read[] = { /* X A G R C M S V T W K D Y H B N */ -17,-17,-17,-17,-17,-17,-17,-17,-17,-17,-17,-17,-17,-17,-17,-17, -17, 2,-17, 1,-17, 1,-17, 0,-17, 1,-17, 0,-17, 0,-17, 0, -17,-17, 2, 1,-17,-17, 1, 0,-17,-17, 1, 0,-17,-17, 0, 0, -17, 1, 1, 1,-17,-17,-17, 0,-17,-17,-17, 0,-17, 0, 0, 0, -17,-17,-17,-17, 2, 1, 1, 0,-17,-17,-17,-17, 1, 0, 0, 0, -17, 1,-17,-17, 1, 1,-17, 0,-17,-17,-17, 0,-17, 0, 0, 0, -17,-17, 1,-17, 1,-17, 1, 0,-17,-17,-17, 0,-17, 0, 0, 0, -17, 0, 0, 0, 0, 0, 0, 0,-17, 0, 0, 0, 0, 0, 0, 0, -17,-17,-17,-17,-17,-17,-17,-17, 2, 1, 1, 0, 1, 0, 0, 0, -17, 1,-17,-17,-17,-17,-17, 0, 1, 1,-17, 0,-17, 0, 0, 0, -17,-17, 1,-17,-17,-17,-17, 0, 1,-17, 1, 0,-17, 0, 0, 0, -17, 0, 0, 0,-17, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, -17,-17,-17,-17, 1,-17,-17, 0, 1,-17,-17, 0, 1, 0, 0, 0, -17, 0,-17, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, -17,-17, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, -17, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0 }; int aln_sm_hs[] = { /* A G C T N */ 91, -31,-114,-123, -44, -31, 100,-125,-114, -42, -123,-125, 100, -31, -42, -114,-114, -31, 91, -42, -44, -42, -42, -42, -43 }; int aln_sm_maq[] = { 11, -19, -19, -19, -13, -19, 11, -19, -19, -13, -19, -19, 11, -19, -13, -19, -19, -19, 11, -13, -13, -13, -13, -13, -13 }; int aln_sm_blast[] = { 1, -3, -3, -3, -2, -3, 1, -3, -3, -2, -3, -3, 1, -3, -2, -3, -3, -3, 1, -2, -2, -2, -2, -2, -2 }; /********************/ /* START OF align.c */ /********************/ AlnParam aln_param_blast = { 5, 2, 2, aln_sm_blast, 5, 50 }; AlnParam aln_param_bwa = { 26, 9, 5, aln_sm_maq, 5, 50 }; AlnParam aln_param_nt2nt = { 8, 2, 2, aln_sm_nt, 16, 75 }; AlnParam aln_param_rd2rd = { 1, 19, 19, aln_sm_read, 16, 75 }; AlnParam aln_param_aa2aa = { 10, 2, 2, aln_sm_blosum62, 22, 50 }; AlnAln *aln_init_AlnAln() { AlnAln *aa; aa = (AlnAln*)malloc(sizeof(AlnAln)); aa->path = 0; aa->out1 = aa->out2 = aa->outm = 0; aa->path_len = 0; return aa; } void aln_free_AlnAln(AlnAln *aa) { free(aa->path); free(aa->cigar32); free(aa->out1); free(aa->out2); free(aa->outm); free(aa); } /***************************/ /* START OF common_align.c */ /***************************/ #define LOCAL_OVERFLOW_THRESHOLD 32000 #define LOCAL_OVERFLOW_REDUCE 16000 #define NT_LOCAL_SCORE int #define NT_LOCAL_SHIFT 16 #define NT_LOCAL_MASK 0xffff #define SET_INF(s) (s).M = (s).I = (s).D = MINOR_INF; #define set_M(MM, cur, p, sc) \ { \ if ((p)->M >= (p)->I) { \ if ((p)->M >= (p)->D) { \ (MM) = (p)->M + (sc); (cur)->Mt = FROM_M; \ } else { \ (MM) = (p)->D + (sc); (cur)->Mt = FROM_D; \ } \ } else { \ if ((p)->I > (p)->D) { \ (MM) = (p)->I + (sc); (cur)->Mt = FROM_I; \ } else { \ (MM) = (p)->D + (sc); (cur)->Mt = FROM_D; \ } \ } \ } #define set_I(II, cur, p) \ { \ if ((p)->M - gap_open > (p)->I) { \ (cur)->It = FROM_M; \ (II) = (p)->M - gap_open - gap_ext; \ } else { \ (cur)->It = FROM_I; \ (II) = (p)->I - gap_ext; \ } \ } #define set_end_I(II, cur, p) \ { \ if (gap_end >= 0) { \ if ((p)->M - gap_open > (p)->I) { \ (cur)->It = FROM_M; \ (II) = (p)->M - gap_open - gap_end; \ } else { \ (cur)->It = FROM_I; \ (II) = (p)->I - gap_end; \ } \ } else set_I(II, cur, p); \ } #define set_D(DD, cur, p) \ { \ if ((p)->M - gap_open > (p)->D) { \ (cur)->Dt = FROM_M; \ (DD) = (p)->M - gap_open - gap_ext; \ } else { \ (cur)->Dt = FROM_D; \ (DD) = (p)->D - gap_ext; \ } \ } #define set_end_D(DD, cur, p) \ { \ if (gap_end >= 0) { \ if ((p)->M - gap_open > (p)->D) { \ (cur)->Dt = FROM_M; \ (DD) = (p)->M - gap_open - gap_end; \ } else { \ (cur)->Dt = FROM_D; \ (DD) = (p)->D - gap_end; \ } \ } else set_D(DD, cur, p); \ } typedef struct { unsigned char Mt:3, It:2, Dt:2; } dpcell_t; typedef struct { int M, I, D; } dpscore_t; /* build score profile for accelerating alignment, in theory */ void aln_init_score_array(unsigned char *seq, int len, int row, int *score_matrix, int **s_array) { int *tmp, *tmp2, i, k; for (i = 0; i != row; ++i) { tmp = score_matrix + i * row; tmp2 = s_array[i]; for (k = 0; k != len; ++k) tmp2[k] = tmp[seq[k]]; } } /*************************** * banded global alignment * ***************************/ int aln_global_core(unsigned char *seq1, int len1, unsigned char *seq2, int len2, const AlnParam *ap, path_t *path, int *path_len) { register int i, j; dpcell_t **dpcell, *q; dpscore_t *curr, *last, *s; path_t *p; int b1, b2, tmp_end; int *mat, end, max; unsigned char type, ctype; int gap_open, gap_ext, gap_end, b; int *score_matrix, N_MATRIX_ROW; /* initialize some align-related parameters. just for compatibility */ gap_open = ap->gap_open; gap_ext = ap->gap_ext; gap_end = ap->gap_end; b = ap->band_width; score_matrix = ap->matrix; N_MATRIX_ROW = ap->row; if (len1 == 0 || len2 == 0) { *path_len = 0; return 0; } /* calculate b1 and b2 */ if (len1 > len2) { b1 = len1 - len2 + b; b2 = b; } else { b1 = b; b2 = len2 - len1 + b; } if (b1 > len1) b1 = len1; if (b2 > len2) b2 = len2; --seq1; --seq2; /* allocate memory */ end = (b1 + b2 <= len1)? (b1 + b2 + 1) : (len1 + 1); dpcell = (dpcell_t**)malloc(sizeof(dpcell_t*) * (len2 + 1)); for (j = 0; j <= len2; ++j) dpcell[j] = (dpcell_t*)malloc(sizeof(dpcell_t) * end); for (j = b2 + 1; j <= len2; ++j) dpcell[j] -= j - b2; curr = (dpscore_t*)malloc(sizeof(dpscore_t) * (len1 + 1)); last = (dpscore_t*)malloc(sizeof(dpscore_t) * (len1 + 1)); /* set first row */ SET_INF(*curr); curr->M = 0; for (i = 1, s = curr + 1; i < b1; ++i, ++s) { SET_INF(*s); set_end_D(s->D, dpcell[0] + i, s - 1); } s = curr; curr = last; last = s; /* core dynamic programming, part 1 */ tmp_end = (b2 < len2)? b2 : len2 - 1; for (j = 1; j <= tmp_end; ++j) { q = dpcell[j]; s = curr; SET_INF(*s); set_end_I(s->I, q, last); end = (j + b1 <= len1 + 1)? (j + b1 - 1) : len1; mat = score_matrix + seq2[j] * N_MATRIX_ROW; ++s; ++q; for (i = 1; i != end; ++i, ++s, ++q) { set_M(s->M, q, last + i - 1, mat[seq1[i]]); /* this will change s->M ! */ set_I(s->I, q, last + i); set_D(s->D, q, s - 1); } set_M(s->M, q, last + i - 1, mat[seq1[i]]); set_D(s->D, q, s - 1); if (j + b1 - 1 > len1) { /* bug fixed, 040227 */ set_end_I(s->I, q, last + i); } else s->I = MINOR_INF; s = curr; curr = last; last = s; } /* last row for part 1, use set_end_D() instead of set_D() */ if (j == len2 && b2 != len2 - 1) { q = dpcell[j]; s = curr; SET_INF(*s); set_end_I(s->I, q, last); end = (j + b1 <= len1 + 1)? (j + b1 - 1) : len1; mat = score_matrix + seq2[j] * N_MATRIX_ROW; ++s; ++q; for (i = 1; i != end; ++i, ++s, ++q) { set_M(s->M, q, last + i - 1, mat[seq1[i]]); /* this will change s->M ! */ set_I(s->I, q, last + i); set_end_D(s->D, q, s - 1); } set_M(s->M, q, last + i - 1, mat[seq1[i]]); set_end_D(s->D, q, s - 1); if (j + b1 - 1 > len1) { /* bug fixed, 040227 */ set_end_I(s->I, q, last + i); } else s->I = MINOR_INF; s = curr; curr = last; last = s; ++j; } /* core dynamic programming, part 2 */ for (; j <= len2 - b2 + 1; ++j) { SET_INF(curr[j - b2]); mat = score_matrix + seq2[j] * N_MATRIX_ROW; end = j + b1 - 1; for (i = j - b2 + 1, q = dpcell[j] + i, s = curr + i; i != end; ++i, ++s, ++q) { set_M(s->M, q, last + i - 1, mat[seq1[i]]); set_I(s->I, q, last + i); set_D(s->D, q, s - 1); } set_M(s->M, q, last + i - 1, mat[seq1[i]]); set_D(s->D, q, s - 1); s->I = MINOR_INF; s = curr; curr = last; last = s; } /* core dynamic programming, part 3 */ for (; j < len2; ++j) { SET_INF(curr[j - b2]); mat = score_matrix + seq2[j] * N_MATRIX_ROW; for (i = j - b2 + 1, q = dpcell[j] + i, s = curr + i; i < len1; ++i, ++s, ++q) { set_M(s->M, q, last + i - 1, mat[seq1[i]]); set_I(s->I, q, last + i); set_D(s->D, q, s - 1); } set_M(s->M, q, last + len1 - 1, mat[seq1[i]]); set_end_I(s->I, q, last + i); set_D(s->D, q, s - 1); s = curr; curr = last; last = s; } /* last row */ if (j == len2) { SET_INF(curr[j - b2]); mat = score_matrix + seq2[j] * N_MATRIX_ROW; for (i = j - b2 + 1, q = dpcell[j] + i, s = curr + i; i < len1; ++i, ++s, ++q) { set_M(s->M, q, last + i - 1, mat[seq1[i]]); set_I(s->I, q, last + i); set_end_D(s->D, q, s - 1); } set_M(s->M, q, last + len1 - 1, mat[seq1[i]]); set_end_I(s->I, q, last + i); set_end_D(s->D, q, s - 1); s = curr; curr = last; last = s; } /* backtrace */ i = len1; j = len2; q = dpcell[j] + i; s = last + len1; max = s->M; type = q->Mt; ctype = FROM_M; if (s->I > max) { max = s->I; type = q->It; ctype = FROM_I; } if (s->D > max) { max = s->D; type = q->Dt; ctype = FROM_D; } p = path; p->ctype = ctype; p->i = i; p->j = j; /* bug fixed 040408 */ ++p; do { switch (ctype) { case FROM_M: --i; --j; break; case FROM_I: --j; break; case FROM_D: --i; break; } q = dpcell[j] + i; ctype = type; switch (type) { case FROM_M: type = q->Mt; break; case FROM_I: type = q->It; break; case FROM_D: type = q->Dt; break; } p->ctype = ctype; p->i = i; p->j = j; ++p; } while (i || j); *path_len = p - path - 1; /* free memory */ for (j = b2 + 1; j <= len2; ++j) dpcell[j] += j - b2; for (j = 0; j <= len2; ++j) free(dpcell[j]); free(dpcell); free(curr); free(last); return max; } /************************************************* * local alignment combined with banded strategy * *************************************************/ int aln_local_core(unsigned char *seq1, int len1, unsigned char *seq2, int len2, const AlnParam *ap, path_t *path, int *path_len, int _thres, int *_subo) { register NT_LOCAL_SCORE *s; register int i; int q, r, qr, tmp_len, qr_shift; int **s_array, *score_array; int e, f; int is_overflow, of_base; NT_LOCAL_SCORE *eh, curr_h, last_h, curr_last_h; int j, start_i, start_j, end_i, end_j; path_t *p; int score_f, score_r, score_g; int start, end, max_score; int thres, *suba, *ss; int gap_open, gap_ext, b; int *score_matrix, N_MATRIX_ROW; /* initialize some align-related parameters. just for compatibility */ gap_open = ap->gap_open; gap_ext = ap->gap_ext; b = ap->band_width; score_matrix = ap->matrix; N_MATRIX_ROW = ap->row; thres = _thres > 0? _thres : -_thres; if (len1 == 0 || len2 == 0) return -1; /* allocate memory */ suba = (int*)malloc(sizeof(int) * (len2 + 1)); eh = (NT_LOCAL_SCORE*)malloc(sizeof(NT_LOCAL_SCORE) * (len1 + 1)); s_array = (int**)malloc(sizeof(int*) * N_MATRIX_ROW); for (i = 0; i != N_MATRIX_ROW; ++i) s_array[i] = (int*)malloc(sizeof(int) * len1); /* initialization */ aln_init_score_array(seq1, len1, N_MATRIX_ROW, score_matrix, s_array); q = gap_open; r = gap_ext; qr = q + r; qr_shift = (qr+1) << NT_LOCAL_SHIFT; tmp_len = len1 + 1; start_i = start_j = end_i = end_j = 0; for (i = 0, max_score = 0; i != N_MATRIX_ROW * N_MATRIX_ROW; ++i) if (max_score < score_matrix[i]) max_score = score_matrix[i]; /* convert the coordinate */ --seq1; --seq2; for (i = 0; i != N_MATRIX_ROW; ++i) --s_array[i]; /* forward dynamic programming */ for (i = 0, s = eh; i != tmp_len; ++i, ++s) *s = 0; score_f = 0; is_overflow = of_base = 0; suba[0] = 0; for (j = 1, ss = suba + 1; j <= len2; ++j, ++ss) { int subo = 0; last_h = f = 0; score_array = s_array[seq2[j]]; if (is_overflow) { /* adjust eh[] array if overflow occurs. */ /* If LOCAL_OVERFLOW_REDUCE is too small, optimal alignment might be missed. * If it is too large, this block will be excuted frequently and therefore * slow down the whole program. * Acually, smaller LOCAL_OVERFLOW_REDUCE might also help to reduce the * number of assignments because it sets some cells to zero when overflow * happens. */ int tmp, tmp2; score_f -= LOCAL_OVERFLOW_REDUCE; of_base += LOCAL_OVERFLOW_REDUCE; is_overflow = 0; for (i = 1, s = eh; i <= tmp_len; ++i, ++s) { tmp = *s >> NT_LOCAL_SHIFT; tmp2 = *s & NT_LOCAL_MASK; if (tmp2 < LOCAL_OVERFLOW_REDUCE) tmp2 = 0; else tmp2 -= LOCAL_OVERFLOW_REDUCE; if (tmp < LOCAL_OVERFLOW_REDUCE) tmp = 0; else tmp -= LOCAL_OVERFLOW_REDUCE; *s = (tmp << NT_LOCAL_SHIFT) | tmp2; } } for (i = 1, s = eh; i != tmp_len; ++i, ++s) { /* prepare for calculate current h */ curr_h = (*s >> NT_LOCAL_SHIFT) + score_array[i]; if (curr_h < 0) curr_h = 0; if (last_h > 0) { /* initialize f */ f = (f > last_h - q)? f - r : last_h - qr; if (curr_h < f) curr_h = f; } if (*(s+1) >= qr_shift) { /* initialize e */ curr_last_h = *(s+1) >> NT_LOCAL_SHIFT; e = ((*s & NT_LOCAL_MASK) > curr_last_h - q)? (*s & NT_LOCAL_MASK) - r : curr_last_h - qr; if (curr_h < e) curr_h = e; *s = (last_h << NT_LOCAL_SHIFT) | e; } else *s = last_h << NT_LOCAL_SHIFT; /* e = 0 */ last_h = curr_h; if (subo < curr_h) subo = curr_h; if (score_f < curr_h) { score_f = curr_h; end_i = i; end_j = j; if (score_f > LOCAL_OVERFLOW_THRESHOLD) is_overflow = 1; } } *s = last_h << NT_LOCAL_SHIFT; *ss = subo + of_base; } score_f += of_base; if (score_f < thres) { /* no matching residue at all, 090218 */ *path_len = 0; goto end_func; } if (path == 0) goto end_func; /* skip path-filling */ /* reverse dynamic programming */ for (i = end_i, s = eh + end_i; i >= 0; --i, --s) *s = 0; if (end_i == 0 || end_j == 0) goto end_func; /* no local match */ score_r = score_matrix[seq1[end_i] * N_MATRIX_ROW + seq2[end_j]]; is_overflow = of_base = 0; start_i = end_i; start_j = end_j; eh[end_i] = ((NT_LOCAL_SCORE)(qr + score_r)) << NT_LOCAL_SHIFT; /* in order to initialize f and e, 040408 */ start = end_i - 1; end = end_i - 3; if (end <= 0) end = 0; /* second pass DP can be done in a band, speed will thus be enhanced */ for (j = end_j - 1; j != 0; --j) { last_h = f = 0; score_array = s_array[seq2[j]]; if (is_overflow) { /* adjust eh[] array if overflow occurs. */ int tmp, tmp2; score_r -= LOCAL_OVERFLOW_REDUCE; of_base += LOCAL_OVERFLOW_REDUCE; is_overflow = 0; for (i = start, s = eh + start + 1; i >= end; --i, --s) { tmp = *s >> NT_LOCAL_SHIFT; tmp2 = *s & NT_LOCAL_MASK; if (tmp2 < LOCAL_OVERFLOW_REDUCE) tmp2 = 0; else tmp2 -= LOCAL_OVERFLOW_REDUCE; if (tmp < LOCAL_OVERFLOW_REDUCE) tmp = 0; else tmp -= LOCAL_OVERFLOW_REDUCE; *s = (tmp << NT_LOCAL_SHIFT) | tmp2; } } for (i = start, s = eh + start + 1; i != end; --i, --s) { /* prepare for calculate current h */ curr_h = (*s >> NT_LOCAL_SHIFT) + score_array[i]; if (curr_h < 0) curr_h = 0; if (last_h > 0) { /* initialize f */ f = (f > last_h - q)? f - r : last_h - qr; if (curr_h < f) curr_h = f; } curr_last_h = *(s-1) >> NT_LOCAL_SHIFT; e = ((*s & NT_LOCAL_MASK) > curr_last_h - q)? (*s & NT_LOCAL_MASK) - r : curr_last_h - qr; if (e < 0) e = 0; if (curr_h < e) curr_h = e; *s = (last_h << NT_LOCAL_SHIFT) | e; last_h = curr_h; if (score_r < curr_h) { score_r = curr_h; start_i = i; start_j = j; if (score_r + of_base - qr == score_f) { j = 1; break; } if (score_r > LOCAL_OVERFLOW_THRESHOLD) is_overflow = 1; } } *s = last_h << NT_LOCAL_SHIFT; /* recalculate start and end, the boundaries of the band */ if ((eh[start] >> NT_LOCAL_SHIFT) <= qr) --start; if (start <= 0) start = 0; end = start_i - (start_j - j) - (score_r + of_base + (start_j - j) * max_score) / r - 1; if (end <= 0) end = 0; } if (_subo) { int tmp2 = 0, tmp = (int)(start_j - .33 * (end_j - start_j) + .499); for (j = 1; j <= tmp; ++j) if (tmp2 < suba[j]) tmp2 = suba[j]; tmp = (int)(end_j + .33 * (end_j - start_j) + .499); for (j = tmp; j <= len2; ++j) if (tmp2 < suba[j]) tmp2 = suba[j]; *_subo = tmp2; } if (path_len == 0) { path[0].i = start_i; path[0].j = start_j; path[1].i = end_i; path[1].j = end_j; goto end_func; } score_r += of_base; score_r -= qr; #ifdef DEBUG /* this seems not a bug */ if (score_f != score_r) fprintf(stderr, "[aln_local_core] unknown flaw occurs: score_f(%d) != score_r(%d)\n", score_f, score_r); #endif if (_thres > 0) { /* call global alignment to fill the path */ score_g = 0; j = (end_i - start_i > end_j - start_j)? end_i - start_i : end_j - start_j; ++j; /* j is the maximum band_width */ for (i = ap->band_width;; i <<= 1) { AlnParam ap_real = *ap; ap_real.gap_end = -1; ap_real.band_width = i; score_g = aln_global_core(seq1 + start_i, end_i - start_i + 1, seq2 + start_j, end_j - start_j + 1, &ap_real, path, path_len); if (score_g == score_r || score_f == score_g) break; if (i > j) break; } if (score_r > score_g && score_f > score_g) { fprintf(stderr, "[aln_local_core] Potential bug: (%d,%d) > %d\n", score_f, score_r, score_g); score_f = score_r = -1; } else score_f = score_g; /* convert coordinate */ for (p = path + *path_len - 1; p >= path; --p) { p->i += start_i - 1; p->j += start_j - 1; } } else { /* just store the start and end */ *path_len = 2; path[1].i = start_i; path[1].j = start_j; path->i = end_i; path->j = end_j; } end_func: /* free */ free(eh); free(suba); /* Suppress -Wfree-nonheap-object locally: the ++s_array[i] below * UNDOES the earlier --s_array[i] at line ~575 (done so the DP * code could use 1-based indexing), so after the increment * s_array[i] points at the original malloc() return. GCC 13+ sees * the explicit pointer arithmetic next to free() and warns even * though the math is a round-trip; the runtime behavior is * correct. Refactoring to avoid the offset trick would touch the * entire aln_local_core DP body, which isn't worth it for a * vendored legacy file. */ #if defined(__GNUC__) # pragma GCC diagnostic push # pragma GCC diagnostic ignored "-Wfree-nonheap-object" #endif for (i = 0; i != N_MATRIX_ROW; ++i) { ++s_array[i]; free(s_array[i]); } #if defined(__GNUC__) # pragma GCC diagnostic pop #endif free(s_array); return score_f; } AlnAln *aln_stdaln_aux(const char *seq1, const char *seq2, const AlnParam *ap, int type, int thres, int len1, int len2) { unsigned char *seq11, *seq22; int score; int i, j, l; path_t *p; char *out1, *out2, *outm; AlnAln *aa; if (len1 < 0) len1 = strlen(seq1); if (len2 < 0) len2 = strlen(seq2); aa = aln_init_AlnAln(); seq11 = (unsigned char*)malloc(sizeof(unsigned char) * len1); seq22 = (unsigned char*)malloc(sizeof(unsigned char) * len2); aa->path = (path_t*)malloc(sizeof(path_t) * (len1 + len2 + 1)); if (ap->row < 10) { /* 4-nucleotide alignment */ for (i = 0; i < len1; ++i) seq11[i] = aln_nt4_table[(int)seq1[i]]; for (j = 0; j < len2; ++j) seq22[j] = aln_nt4_table[(int)seq2[j]]; } else if (ap->row < 20) { /* 16-nucleotide alignment */ for (i = 0; i < len1; ++i) seq11[i] = aln_nt16_table[(int)seq1[i]]; for (j = 0; j < len2; ++j) seq22[j] = aln_nt16_table[(int)seq2[j]]; } else { /* amino acids */ for (i = 0; i < len1; ++i) seq11[i] = aln_aa_table[(int)seq1[i]]; for (j = 0; j < len2; ++j) seq22[j] = aln_aa_table[(int)seq2[j]]; } if (type == ALN_TYPE_GLOBAL) score = aln_global_core(seq11, len1, seq22, len2, ap, aa->path, &aa->path_len); else if (type == ALN_TYPE_LOCAL) score = aln_local_core(seq11, len1, seq22, len2, ap, aa->path, &aa->path_len, thres, &aa->subo); else if (type == ALN_TYPE_EXTEND) score = aln_extend_core(seq11, len1, seq22, len2, ap, aa->path, &aa->path_len, 1, 0); else { free(seq11); free(seq22); free(aa->path); aln_free_AlnAln(aa); return 0; } aa->score = score; if (thres > 0) { out1 = aa->out1 = (char*)malloc(sizeof(char) * (aa->path_len + 1)); out2 = aa->out2 = (char*)malloc(sizeof(char) * (aa->path_len + 1)); outm = aa->outm = (char*)malloc(sizeof(char) * (aa->path_len + 1)); --seq1; --seq2; --seq11; --seq22; p = aa->path + aa->path_len - 1; for (l = 0; p >= aa->path; --p, ++l) { switch (p->ctype) { case FROM_M: out1[l] = seq1[p->i]; out2[l] = seq2[p->j]; outm[l] = (seq11[p->i] == seq22[p->j] && seq11[p->i] != ap->row)? '|' : ' '; break; case FROM_I: out1[l] = '-'; out2[l] = seq2[p->j]; outm[l] = ' '; break; case FROM_D: out1[l] = seq1[p->i]; out2[l] = '-'; outm[l] = ' '; break; } } out1[l] = out2[l] = outm[l] = '\0'; ++seq11; ++seq22; } free(seq11); free(seq22); p = aa->path + aa->path_len - 1; aa->start1 = p->i? p->i : 1; aa->end1 = aa->path->i; aa->start2 = p->j? p->j : 1; aa->end2 = aa->path->j; aa->cigar32 = aln_path2cigar32(aa->path, aa->path_len, &aa->n_cigar); return aa; } AlnAln *aln_stdaln(const char *seq1, const char *seq2, const AlnParam *ap, int type, int thres) { return aln_stdaln_aux(seq1, seq2, ap, type, thres, -1, -1); } /* for backward compatibility */ uint16_t *aln_path2cigar(const path_t *path, int path_len, int *n_cigar) { uint32_t *cigar32; uint16_t *cigar; int i; cigar32 = aln_path2cigar32(path, path_len, n_cigar); cigar = (uint16_t*)cigar32; for (i = 0; i < *n_cigar; ++i) cigar[i] = (cigar32[i]&0xf)<<14 | (cigar32[i]>>4&0x3fff); return cigar; } /* newly added functions (2009-07-21) */ int aln_extend_core(unsigned char *seq1, int len1, unsigned char *seq2, int len2, const AlnParam *ap, path_t *path, int *path_len, int G0, uint8_t *_mem) { int q, r, qr, tmp_len; int32_t **s_array, *score_array; int is_overflow, of_base; uint32_t *eh; int i, j, end_i, end_j; int score, start, end; int *score_matrix, N_MATRIX_ROW; uint8_t *mem, *_p; /* initialize some align-related parameters. just for compatibility */ q = ap->gap_open; r = ap->gap_ext; qr = q + r; score_matrix = ap->matrix; N_MATRIX_ROW = ap->row; if (len1 == 0 || len2 == 0) return -1; /* allocate memory */ mem = _mem? _mem : calloc((len1 + 2) * (N_MATRIX_ROW + 1), 4); _p = mem; eh = (uint32_t*)_p, _p += 4 * (len1 + 2); s_array = calloc(N_MATRIX_ROW, sizeof(void*)); for (i = 0; i != N_MATRIX_ROW; ++i) s_array[i] = (int32_t*)_p, _p += 4 * len1; /* initialization */ aln_init_score_array(seq1, len1, N_MATRIX_ROW, score_matrix, s_array); tmp_len = len1 + 1; start = 1; end = 2; end_i = end_j = 0; score = 0; is_overflow = of_base = 0; /* convert the coordinate */ --seq1; --seq2; for (i = 0; i != N_MATRIX_ROW; ++i) --s_array[i]; /* dynamic programming */ memset(eh, 0, 4 * (len1 + 2)); eh[1] = (uint32_t)G0<<16; for (j = 1; j <= len2; ++j) { int _start, _end; int h1 = 0, f = 0; score_array = s_array[seq2[j]]; /* set start and end */ _start = j - ap->band_width; if (_start < 1) _start = 1; if (_start > start) start = _start; _end = j + ap->band_width; if (_end > len1 + 1) _end = len1 + 1; if (_end < end) end = _end; if (start == end) break; /* adjust eh[] array if overflow occurs. */ if (is_overflow) { int tmp, tmp2; score -= LOCAL_OVERFLOW_REDUCE; of_base += LOCAL_OVERFLOW_REDUCE; is_overflow = 0; for (i = start; i <= end; ++i) { uint32_t *s = &eh[i]; tmp = *s >> 16; tmp2 = *s & 0xffff; if (tmp2 < LOCAL_OVERFLOW_REDUCE) tmp2 = 0; else tmp2 -= LOCAL_OVERFLOW_REDUCE; if (tmp < LOCAL_OVERFLOW_REDUCE) tmp = 0; else tmp -= LOCAL_OVERFLOW_REDUCE; *s = (tmp << 16) | tmp2; } } _start = _end = 0; /* the inner loop */ for (i = start; i < end; ++i) { /* At the beginning of each cycle: eh[i] -> h[j-1,i-1]<<16 | e[j,i] f -> f[j,i] h1 -> h[j,i-1] */ uint32_t *s = &eh[i]; int h = (int)(*s >> 16); int e = *s & 0xffff; /* this is e[j,i] */ *s = (uint32_t)h1 << 16; /* eh[i] now stores h[j,i-1]<<16 */ h += h? score_array[i] : 0; /* this is left_core() specific */ /* calculate h[j,i]; don't need to test 0, as {e,f}>=0 */ h = h > e? h : e; h = h > f? h : f; /* h now is h[j,i] */ h1 = h; if (h > 0) { if (_start == 0) _start = i; _end = i; if (score < h) { score = h; end_i = i; end_j = j; if (score > LOCAL_OVERFLOW_THRESHOLD) is_overflow = 1; } } /* calculate e[j+1,i] and f[j,i+1] */ h -= qr; h = h > 0? h : 0; e -= r; e = e > h? e : h; f -= r; f = f > h? f : h; *s |= e; } eh[end] = h1 << 16; /* recalculate start and end, the boundaries of the band */ if (_end <= 0) break; /* no cell in this row has a positive score */ start = _start; end = _end + 3; } score += of_base - 1; if (score <= 0) { if (path_len) *path_len = 0; goto end_left_func; } if (path == 0) goto end_left_func; if (path_len == 0) { path[0].i = end_i; path[0].j = end_j; goto end_left_func; } { /* call global alignment to fill the path */ int score_g = 0; j = (end_i - 1 > end_j - 1)? end_i - 1 : end_j - 1; ++j; /* j is the maximum band_width */ for (i = ap->band_width;; i <<= 1) { AlnParam ap_real = *ap; ap_real.gap_end = -1; ap_real.band_width = i; score_g = aln_global_core(seq1 + 1, end_i, seq2 + 1, end_j, &ap_real, path, path_len); if (score == score_g) break; if (i > j) break; } if (score > score_g) fprintf(stderr, "[aln_left_core] no suitable bandwidth: %d < %d\n", score_g, score); score = score_g; } end_left_func: /* free */ free(s_array); if (!_mem) free(mem); return score; } uint32_t *aln_path2cigar32(const path_t *path, int path_len, int *n_cigar) { int i, n; uint32_t *cigar; unsigned char last_type; if (path_len == 0 || path == 0) { *n_cigar = 0; return 0; } last_type = path->ctype; for (i = n = 1; i < path_len; ++i) { if (last_type != path[i].ctype) ++n; last_type = path[i].ctype; } *n_cigar = n; cigar = (uint32_t*)malloc(*n_cigar * 4); cigar[0] = 1u << 4 | path[path_len-1].ctype; last_type = path[path_len-1].ctype; for (i = path_len - 2, n = 0; i >= 0; --i) { if (path[i].ctype == last_type) cigar[n] += 1u << 4; else { cigar[++n] = 1u << 4 | path[i].ctype; last_type = path[i].ctype; } } return cigar; } #ifdef STDALN_MAIN int main() { AlnAln *aln_local, *aln_global, *aln_left; int i; aln_local = aln_stdaln("CGTGCGATGCactgCATACGGCTCGCCTAGATCA", "AAGGGATGCTCTGCATCgCTCGGCTAGCTGT", &aln_param_blast, 0, 1); aln_global = aln_stdaln("CGTGCGATGCactgCATACGGCTCGCCTAGATCA", "AAGGGATGCTCTGCATCGgCTCGGCTAGCTGT", &aln_param_blast, 1, 1); // aln_left = aln_stdaln( "GATGCACTGCATACGGCTCGCCTAGATCA", "GATGCTCTGCATCGgCTCGGCTAGCTGT", &aln_param_blast, 2, 1); aln_left = aln_stdaln("CACCTTCGACTCACGTCTCATTCTCGGAGTCGAGTGGACGGTCCCTCATACACGAACAGGTTC", "CACCTTCGACTTTCACCTCTCATTCTCGGACTCGAGTGGACGGTCCCTCATCCAAGAACAGGGTCTGTGAAA", &aln_param_blast, 2, 1); printf(">%d,%d\t%d,%d\n", aln_local->start1, aln_local->end1, aln_local->start2, aln_local->end2); printf("%s\n%s\n%s\n", aln_local->out1, aln_local->outm, aln_local->out2); printf(">%d,%d\t%d,%d\t", aln_global->start1, aln_global->end1, aln_global->start2, aln_global->end2); for (i = 0; i != aln_global->n_cigar; ++i) printf("%d%c", aln_global->cigar32[i]>>4, "MID"[aln_global->cigar32[i]&0xf]); printf("\n%s\n%s\n%s\n", aln_global->out1, aln_global->outm, aln_global->out2); printf(">%d\t%d,%d\t%d,%d\t", aln_left->score, aln_left->start1, aln_left->end1, aln_left->start2, aln_left->end2); for (i = 0; i != aln_left->n_cigar; ++i) printf("%d%c", aln_left->cigar32[i]>>4, "MID"[aln_left->cigar32[i]&0xf]); printf("\n%s\n%s\n%s\n", aln_left->out1, aln_left->outm, aln_left->out2); aln_free_AlnAln(aln_local); aln_free_AlnAln(aln_global); aln_free_AlnAln(aln_left); return 0; } #endif ================================================ FILE: src/SGA/Util/stdaln.h ================================================ /* The MIT License Copyright (c) 2003-2006, 2008, by Heng Li Permission is hereby granted, free of charge, to any person obtaining a copy of this software and associated documentation files (the "Software"), to deal in the Software without restriction, including without limitation the rights to use, copy, modify, merge, publish, distribute, sublicense, and/or sell copies of the Software, and to permit persons to whom the Software is furnished to do so, subject to the following conditions: The above copyright notice and this permission notice shall be included in all copies or substantial portions of the Software. THE SOFTWARE IS PROVIDED "AS IS", WITHOUT WARRANTY OF ANY KIND, EXPRESS OR IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT. IN NO EVENT SHALL THE AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER LIABILITY, WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING FROM, OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS IN THE SOFTWARE. */ /* 2009-07-23, 0.10.0 - Use 32-bit to store CIGAR - Report suboptimal aligments - Implemented half-fixed-half-open DP 2009-04-26, 0.9.10 - Allow to set a threshold for local alignment 2009-02-18, 0.9.9 - Fixed a bug when no residue matches 2008-08-04, 0.9.8 - Fixed the wrong declaration of aln_stdaln_aux() - Avoid 0 coordinate for global alignment 2008-08-01, 0.9.7 - Change gap_end penalty to 5 in aln_param_bwa - Add function to convert path_t to the CIGAR format 2008-08-01, 0.9.6 - The first gap now costs (gap_open+gap_ext), instead of gap_open. Scoring systems are modified accordingly. - Gap end is now correctly handled. Previously it is not correct. - Change license to MIT. */ #ifndef LH3_STDALN_H_ #define LH3_STDALN_H_ #define STDALN_VERSION 0.11.0 #include #define FROM_M 0 #define FROM_I 1 #define FROM_D 2 #define FROM_S 3 #define ALN_TYPE_LOCAL 0 #define ALN_TYPE_GLOBAL 1 #define ALN_TYPE_EXTEND 2 /* This is the smallest integer. It might be CPU-dependent in very RARE cases. */ #define MINOR_INF -1073741823 typedef struct { int gap_open; int gap_ext; int gap_end; int *matrix; int row; int band_width; } AlnParam; typedef struct { int i, j; unsigned char ctype; } path_t; typedef struct { path_t *path; /* for advanced users... :-) */ int path_len; /* for advanced users... :-) */ int start1, end1; /* start and end of the first sequence, coordinations are 1-based */ int start2, end2; /* start and end of the second sequence, coordinations are 1-based */ int score, subo; /* score */ char *out1, *out2; /* print them, and then you will know */ char *outm; int n_cigar; uint32_t *cigar32; } AlnAln; #ifdef __cplusplus extern "C" { #endif AlnAln *aln_stdaln_aux(const char *seq1, const char *seq2, const AlnParam *ap, int type, int do_align, int len1, int len2); AlnAln *aln_stdaln(const char *seq1, const char *seq2, const AlnParam *ap, int type, int do_align); void aln_free_AlnAln(AlnAln *aa); int aln_global_core(unsigned char *seq1, int len1, unsigned char *seq2, int len2, const AlnParam *ap, path_t *path, int *path_len); int aln_local_core(unsigned char *seq1, int len1, unsigned char *seq2, int len2, const AlnParam *ap, path_t *path, int *path_len, int _thres, int *_subo); int aln_extend_core(unsigned char *seq1, int len1, unsigned char *seq2, int len2, const AlnParam *ap, path_t *path, int *path_len, int G0, uint8_t *_mem); uint16_t *aln_path2cigar(const path_t *path, int path_len, int *n_cigar); uint32_t *aln_path2cigar32(const path_t *path, int path_len, int *n_cigar); #ifdef __cplusplus } #endif /******************** * global variables * ********************/ extern AlnParam aln_param_bwa; /* = { 37, 9, 0, aln_sm_maq, 5, 50 }; */ extern AlnParam aln_param_blast; /* = { 5, 2, 2, aln_sm_blast, 5, 50 }; */ extern AlnParam aln_param_nt2nt; /* = { 10, 2, 2, aln_sm_nt, 16, 75 }; */ extern AlnParam aln_param_aa2aa; /* = { 20, 19, 19, aln_sm_read, 16, 75 }; */ extern AlnParam aln_param_rd2rd; /* = { 12, 2, 2, aln_sm_blosum62, 22, 50 }; */ /* common nucleotide score matrix for 16 bases */ extern int aln_sm_nt[], aln_sm_bwa[]; /* BLOSUM62 and BLOSUM45 */ extern int aln_sm_blosum62[], aln_sm_blosum45[]; /* common read for 16 bases. note that read alignment is quite different from common nucleotide alignment */ extern int aln_sm_read[]; /* human-mouse score matrix for 4 bases */ extern int aln_sm_hs[]; #endif ================================================ FILE: src/svaba/AlignedContig.cpp ================================================ #include "AlignedContig.h" #include "SvabaUtils.h" #include "ContigAlignmentScore.h" #include #include #include #include // SvABA2.0: the old ASCII emitter (printToAlignmentsFile → alignments.txt.gz) // and its support header PlottedRead.h have been removed. All alignment-dump // data now goes through the structured r2c TSV (printToR2CTsv / // r2cTsvHeader), which viewers parse and re-plot on demand. // bav is all of the alignments of the contig to the reference AlignedContig::AlignedContig(BamRecordPtrVector& bav, const GenomicRegion& region, const SvabaSharedConfig* sc_) : sc(sc_) { if (!bav.size()) return; // find the longest sequence, taking the first one. // make sure sequence dir is set to same as first alignment for (const auto& i : bav) { if (i->Sequence().length() > m_seq.length()) { if (i->ReverseFlag() == bav.front()->ReverseFlag()) { m_seq = i->Sequence(); } else { m_seq = i->Sequence(); SeqLib::rcomplement(m_seq); } } } // set the sequence. Convention is store as it came off // the assembler for first alignment if (bav.front()->ReverseFlag()) SeqLib::rcomplement(m_seq); // instantiate coverage of reads on the contig aligned_coverage = std::vector(m_seq.length(), 0); // find the number of primary alignments size_t num_align = 0; for (const auto& i : bav) if (!i->SecondaryFlag()) ++num_align; // make the individual alignments and add for (auto& i : bav) { bool flip = (m_seq != i->Sequence()); // if the seq was flipped, need to flip the AlignmentFragment if (!i->SecondaryFlag()) { m_frag_v.emplace_back(AlignmentFragment(i, flip, region, sc)); m_frag_v.back().num_align = num_align; } else if (m_frag_v.size()) { m_frag_v.back().secondaries.emplace_back(AlignmentFragment(i, flip, region, sc)); } else { throw std::runtime_error("Secondary alignment encountered with no primary."); } // set the sequence for the aligned contig in the flipped (or not) orientation //m_frag_v.back().m_seq = m_seq; // set the aligned coverage SeqLib::Cigar cig = i->GetCigar(); size_t p = 0; if (!i->SecondaryFlag()) for (auto& c : cig) { for (size_t j = 0; j < c.Length(); ++j) { if (c.Type() == 'M' || c.Type() == 'I') // consumes contig and not clip ++aligned_coverage[p]; if (c.ConsumesQuery() || c.Type() == 'H') // consumes contig, move iterator ++p; } } } if (!m_frag_v.size()) throw std::runtime_error("AlignedContig - no AlignmentFragments made"); // extract the indels on primary alignments for (auto& f : m_frag_v) f.SetIndels(); // sort fragments by order on fwd-strand contig // underneath this calls AlignmentFragment::operator< // which is defined based on the "flip-convention" // alignment position on the contig, NOT on the genome std::sort(m_frag_v.begin(), m_frag_v.end()); // get rearrangement breaks out of it setMultiMapBreakPairs(); // filter indels that land too close to a multi-map break //filterIndelsAtMultiMapSites(5); } // void AlignedContig::filterIndelsAtMultiMapSites(size_t buff) { // if (m_frag_v.size() < 2) // return; // // make the ranges ON CONIG for the multimaps // SeqLib::GRC grc; // if (m_global_bp->b1.cpos > m_global_bp->b2.cpos) { // grc.add(SeqLib::GenomicRegion(0, m_global_bp->b2.cpos-buff, m_global_bp->b1.cpos+buff)); // homology // } // else { // grc.add(SeqLib::GenomicRegion(0, m_global_bp->b1.cpos-buff, m_global_bp->b2.cpos+buff)); // insertion // } // for (auto& i : m_local_breaks) { // if (i.b1.cpos > i.b2.cpos) { // grc.add(SeqLib::GenomicRegion(0, i.b2.cpos-buff, i.b1.cpos+buff)); // homology // } // else { // grc.add(SeqLib::GenomicRegion(0, i.b1.cpos-buff, i.b2.cpos+buff)); // insertion // } // } // grc.CreateTreeMap(); // // check if // for (auto& i : m_frag_v) { // BPVec new_indel_vec; // for (auto& b : i.m_indel_breaks) { // if (!grc.CountOverlaps(SeqLib::GenomicRegion(0, b.b1.cpos, b.b2.cpos))) // new_indel_vec.push_back(b); // } // i.m_indel_breaks = new_indel_vec; // } // } SeqLib::GenomicRegionVector AlignedContig::getAsGenomicRegionVector() const { SeqLib::GenomicRegionVector g; for (auto& i : m_frag_v) g.push_back(i.m_align->AsGenomicRegion()); return g; } // void AlignedContig::printContigFasta(std::ofstream& os) const { // os << ">" << getContigName() << std::endl; // os << getSequence() << std::endl; // } void AlignedContig::blacklist(SeqLib::GRC &grv) { // loop through the indel breaks and blacklist for (auto& i : m_frag_v) for (auto& j : i.m_indel_breaks) j->checkBlacklist(grv); } void AlignedContig::splitCoverage() { // for (auto& i : m_local_breaks_secondaries) // i.splitCoverage(m_bamreads); // for (auto& i : m_global_bp_secondaries) // i->splitCoverage(m_bamreads); for (auto& i : m_frag_v) for (auto& j : i.m_indel_breaks) j->splitCoverage(m_bamreads); for (auto& i : m_local_breaks) i->splitCoverage(m_bamreads); if (m_global_bp) m_global_bp->splitCoverage(m_bamreads); } // --------------------------------------------------------------------------- // SvABA2.0: structured re-plot format. // // printToR2CTsv emits this contig's info in a tab-separated form that // downstream tools (e.g. viewer/bps_explorer.html) parse and re-plot on // demand. It's the successor to the old printToAlignmentsFile() / // alignments.txt.gz pre-rendered ASCII output, which has been removed. // // The schema is record-type discriminated: one "contig" row per contig, // then one "read" row per r2c-aligned read, all sharing a `contig_name` // key. Contig-only fields (sequence, fragments, BPs, read count) are // populated on the contig row and "NA" on read rows; read-only fields // are populated on read rows and "NA" on the contig row. // // Anything that was encoded only implicitly in the old ASCII art (e.g. // fragment orientation via '>' vs '<', or leading/trailing soft-clip // bases via lowercase letters) is here available as an explicit field // so viewers don't have to reverse-engineer it. // --------------------------------------------------------------------------- namespace { // TSV-escape: replace tab/CR/LF with spaces so a single column value can't // break row parsing. Used everywhere we emit free-form text into a cell. std::string r2cEscape(const std::string& s) { std::string out; out.reserve(s.size()); for (char c : s) { if (c == '\t' || c == '\n' || c == '\r') out.push_back(' '); else out.push_back(c); } return out; } // Serialize a SeqLib::Cigar to the standard string form (e.g. "30M1D15M") // without relying on the overload of operator<< (which is fine but writes // through a stream; we want a plain std::string for cell composition). std::string r2cCigarString(const SeqLib::Cigar& c) { std::ostringstream oss; for (auto it = c.begin(); it != c.end(); ++it) oss << it->Length() << static_cast(it->Type()); return oss.str(); } } // namespace // Header line for the r2c TSV. Callers should write this once before any // rows. Not a row itself — do not prefix with a comment char. std::string AlignedContig::r2cTsvHeader() { static const char* const kCols[] = { "record_type", // "contig" or "read" "contig_name", // shared key: group reads back to their contig "contig_len", // set on both row types for convenience "contig_seq", // assembled sequence [contig only; NA on read] "frags", // BWA hits of contig vs. ref [contig only] "bps", // breakpoints on this contig [contig only] "n_reads", // # read rows that follow this contig [contig only] // read-only: "read_id", // svabaRead UniqueName (sample-prefixed qname) "read_chrom", // chromosome the read originally mapped to (BAM) "read_pos", // BAM alignment position "read_flag", // 0 if r2c forward, 16 if reverse-complemented "r2c_cigar", // read-to-contig CIGAR (e.g. "24M1D15M7I1M7D103M") "r2c_start", // start_on_contig (0-based) "r2c_rc", // 0/1: was r2c alignment reverse-complemented? "r2c_nm", // NM (mismatch count) from r2c alignment "support", // "split" | "disc" | "both" | "none" (categorical) // SvABA2.0: unambiguous per-BP support lists. The categorical // `support` above is the OR over these — use these columns when // you need to know *which* BP(s) a read supports on this contig. // Both are comma-joined lists of bp_id; "NA" if no match. "split_bps", // bp_ids where this read is a split-supporter "disc_bps", // bp_ids where this read (or its mate) is in a // discordant cluster associated with the BP "r2c_score", // alignment score under r2c CIGAR "native_score", // alignment score under BAM CIGAR (or corrected) "read_seq" // raw read sequence (pre-rc, pre-gap-expand) }; std::ostringstream oss; for (size_t i = 0; i < sizeof(kCols) / sizeof(kCols[0]); ++i) { if (i) oss << '\t'; oss << kCols[i]; } return oss.str(); } std::string AlignedContig::printToR2CTsv(const SeqLib::BamHeader& h) const { std::ostringstream out; const std::string cname = this->getContigName(); const std::string NA = "NA"; // --- per-contig fields ------------------------------------------------ // frags: "|"-separated; within a frag, ":"-separated: // chr:pos:strand:cigar:mapq:cpos_break1:cpos_break2:gpos_break1:gpos_break2:flipped std::string frags; for (size_t i = 0; i < m_frag_v.size(); ++i) { const auto& frag = m_frag_v[i]; const auto& a = frag.m_align; if (!a) continue; if (!frags.empty()) frags.push_back('|'); frags += r2cEscape(a->ChrName(sc->header)); frags += ':'; frags += std::to_string(a->Position()); frags += ':'; frags += (a->ReverseFlag() ? '-' : '+'); frags += ':'; frags += r2cEscape(a->CigarString()); frags += ':'; frags += std::to_string(a->MapQuality()); frags += ':'; frags += std::to_string(frag.break1); frags += ':'; frags += std::to_string(frag.break2); frags += ':'; frags += std::to_string(frag.gbreak1); frags += ':'; frags += std::to_string(frag.gbreak2); frags += ':'; frags += (frag.flipped ? '1' : '0'); } if (frags.empty()) frags = NA; // bps: "|"-separated; within a bp, ":"-separated (10 fields): // kind:bp_id:chr1:pos1:strand1:chr2:pos2:strand2:span:insertion // kind ∈ {global, multi, indel}. bp_id is the unique BP identifier // assigned by svabaThreadUnit::next_bp_id() ("." if unset — should // not happen in normal runs). insertion is "." if none. All fields // fixed-position so the field count stays at 10 regardless of row. auto bpCell = [&](const char* kind, const BreakPoint& bp) -> std::string { std::ostringstream os; os << kind << ':' << (bp.id.empty() ? std::string(".") : r2cEscape(bp.id)) << ':' << r2cEscape(bp.b1.gr.ChrName(h)) << ':' << bp.b1.gr.pos1 << ':' << (bp.b1.gr.strand ? bp.b1.gr.strand : '.') << ':' << r2cEscape(bp.b2.gr.ChrName(h)) << ':' << bp.b2.gr.pos1 << ':' << (bp.b2.gr.strand ? bp.b2.gr.strand : '.') << ':' << bp.getSpan() << ':' << (bp.insertion.empty() ? std::string(".") : r2cEscape(bp.insertion)); return os.str(); }; std::string bps; auto appendBp = [&](const std::string& cell) { if (!bps.empty()) bps.push_back('|'); bps += cell; }; if (m_global_bp) appendBp(bpCell("global", *m_global_bp)); for (const auto& b : m_local_breaks) if (b) appendBp(bpCell("multi", *b)); for (const auto& frag : m_frag_v) for (const auto& b : frag.m_indel_breaks) if (b) appendBp(bpCell("indel", *b)); if (bps.empty()) bps = NA; const size_t n_reads = m_bamreads.size(); // --- contig row ------------------------------------------------------- // // Fields in order (21 total, mirroring r2cTsvHeader): // record_type contig_name contig_len contig_seq frags bps n_reads // read_id read_chrom read_pos read_flag r2c_cigar r2c_start r2c_rc // r2c_nm support split_bps disc_bps r2c_score native_score read_seq // On the contig row, all read-specific fields are NA. out << "contig\t" << r2cEscape(cname) << '\t' << m_seq.length() << '\t' << r2cEscape(m_seq) << '\t' << frags << '\t' << bps << '\t' << n_reads << '\t' // read-only columns, 14 of them: << NA << '\t' << NA << '\t' << NA << '\t' << NA << '\t' << NA << '\t' << NA << '\t' << NA << '\t' << NA << '\t' << NA << '\t' << NA << '\t' << NA << '\t' << NA << '\t' << NA << '\t' << NA << '\n'; // --- per-BP support tracking ----------------------------------------- // // Replaces the older flat split_supporters / disc_supporters sets. // For each BP on this contig we collect: // - bp_id: the unique identifier (may be "." if unset — shouldn't // happen in a normal svaba run, but we don't block emission on it) // - split_reads: UniqueNames credited as split-supporters for this // specific BP by BreakPoint::splitCoverage // - disc_reads: UniqueNames (of reads or mates) appearing in this // BP's own DiscordantCluster (populated by // CombineWithDiscordantClusterMap). Note the scope is per-BP, not // per-contig — a contig can have multiple BPs, and a discordant // cluster may be attributed to only some of them. // // Per-read output then walks this vector and concatenates the bp_ids // this read shows up under into `split_bps` / `disc_bps`. A read // supporting two different BPs on the same contig (e.g. a multi-map // contig with a global + a local BP) will show both ids, comma- // joined, instead of the previous unambiguous "split" tag that hid // which BP it actually supported. struct BpSupportEntry { std::string bp_id; std::unordered_set split_reads; std::unordered_set disc_reads; }; std::vector per_bp_support; per_bp_support.reserve(8); for (const auto& bp : getAllBreakPoints()) { if (!bp) continue; BpSupportEntry e; e.bp_id = bp->id.empty() ? std::string(".") : bp->id; for (const auto& un : bp->getAllSupportingReads()) e.split_reads.insert(un); for (const auto& kv : bp->dc.reads) e.disc_reads.insert(kv.first); for (const auto& kv : bp->dc.mates) e.disc_reads.insert(kv.first); per_bp_support.push_back(std::move(e)); } auto fmt_score = [](double s) { std::ostringstream os; os << std::fixed << std::setprecision(1) << s; return os.str(); }; // --- read rows -------------------------------------------------------- for (const auto& i : m_bamreads) { const std::string sr = i->UniqueName(); const r2c this_r2c = i->GetR2C(getContigName()); // r2c CIGAR: empty if no r2c alignment (shouldn't happen for reads // listed here, but guard anyway). std::string r2c_cig_str = r2cCigarString(this_r2c.cig); if (r2c_cig_str.empty()) r2c_cig_str = NA; // Scores: same source-of-truth precedence BreakPoint::splitCoverage // uses for its "r2c better than native" gate — we reproduce it here // so the TSV lets you diagnose split-coverage decisions by field // inspection alone. std::string r2c_score_str = NA; if (this_r2c.cig.size() > 0) { r2c_score_str = fmt_score( svaba::readAlignmentScore(this_r2c.cig, this_r2c.nm)); } std::string native_score_str = NA; { SeqLib::Cigar native_cig; int32_t native_nm = -1; if (i->corrected_native_cig.size() > 0) { native_cig = i->corrected_native_cig; native_nm = i->corrected_native_nm; } else { native_cig = i->GetCigar(); i->GetIntTag("NM", native_nm); } if (native_cig.size() > 0) { native_score_str = fmt_score( svaba::readAlignmentScore(native_cig, native_nm)); } } // Build the per-BP support lists for this specific read. Comma- // joined bp_ids, "NA" if this read doesn't support any BP in that // category on this contig. The categorical `support` is derived // from whether either list is non-empty, so the new columns are // a strict superset of the old info — downstream code keying on // `support == "split"` still works. std::string split_bps_str; std::string disc_bps_str; bool any_split = false, any_disc = false; for (const auto& e : per_bp_support) { if (e.split_reads.count(sr)) { if (!split_bps_str.empty()) split_bps_str.push_back(','); split_bps_str += e.bp_id; any_split = true; } if (e.disc_reads.count(sr)) { if (!disc_bps_str.empty()) disc_bps_str.push_back(','); disc_bps_str += e.bp_id; any_disc = true; } } if (split_bps_str.empty()) split_bps_str = NA; if (disc_bps_str.empty()) disc_bps_str = NA; const char* support_kind = any_split && any_disc ? "both" : any_split ? "split" : any_disc ? "disc" : "none"; // read_chrom: BAM-native chromosome; may be empty for unmapped mates. std::string read_chrom = (i->ChrID() >= 0) ? i->ChrName(h) : std::string("*"); out << "read\t" << r2cEscape(cname) << '\t' << m_seq.length() << '\t' << NA << '\t' // contig_seq << NA << '\t' // frags << NA << '\t' // bps << NA << '\t' // n_reads // read fields: << r2cEscape(sr) << '\t' << r2cEscape(read_chrom) << '\t' << i->Position() << '\t' << (this_r2c.rc ? 16 : 0) << '\t' << r2c_cig_str << '\t' << this_r2c.start_on_contig << '\t' << (this_r2c.rc ? 1 : 0) << '\t' << this_r2c.nm << '\t' << support_kind << '\t' << split_bps_str << '\t' << disc_bps_str << '\t' << r2c_score_str << '\t' << native_score_str << '\t' << r2cEscape(i->Sequence()) << '\n'; } return out.str(); } void AlignedContig::setMultiMapBreakPairs() { // if single mapped contig, nothing to do here if (m_frag_v.size() == 1) return; // walk along the ordered contig list and make the breakpoint pairs // these are the so called "local" breakpoints, meaning they are // AB rearrangemnts without an intervening sequence // so this loop will do AB, BC, CD, etc for (AlignmentFragmentVector::const_iterator it = m_frag_v.begin(); it != m_frag_v.end() - 1; it++) { AlignmentFragmentVector bwa_hits_1, bwa_hits_2; // first add the primary alignments bwa_hits_1.push_back(*it); bwa_hits_2.push_back(*(it+1)); // then the secondaries // bwa_hits_1.insert(bwa_hits_1.end(), // it->secondaries.begin(), // it->secondaries.end()); // bwa_hits_2.insert(bwa_hits_2.end(), // (it+1)->secondaries.begin(), // (it+1)->secondaries.end()); // make all of the local breakpoints // OK so if we turn off secondary alignments, this will // just be one iteration through each loop (A and B) // but that's OK for (auto& a : bwa_hits_1) { for (auto& b : bwa_hits_2) { // initialize the breakpoint BreakPointPtr bp = std::make_shared(sc, &a, &b, this); // add the the vector of breakpoints m_local_breaks.push_back(bp); } } } // end frag iterator loop // if this is a double mapping, we are done if (m_frag_v.size() == 2) { return; } ////////////// // 3+ mappings ////////////// // go through alignments and find start and end that reach mapq // bstart and bend are indices of local alignments // that should contribute to a global // e.g. bstart = 0 = A int bstart = -1; //MAX_CONTIG_SIZE; // dummy value to be overwritten int bend = -1; // loop and find contigs with strong support for (size_t i = 0; i < m_frag_v.size(); i++) { if (m_frag_v[i].m_align->MapQuality() >= 50) { // first good alignment fragment, that's the start if (bstart < 0) bstart = i; bend = i; } } // if nothing good found, then just pick first and last (A-C or whatever) if (bstart == bend || bstart < 0) { bstart = 0; bend = m_frag_v.size() -1; } assert(bend <= m_frag_v.size() && bend > 0); assert(bstart <= m_frag_v.size()); assert(bstart >= 0); // if we had ABC but only AB or BC is good connection // then don't treat as complex if (bend - bstart == 1) { return; } ///// // there are 3+ quality mappings. Set a global break ///// // set some values in the breakpoint for this contig m_global_bp = std::make_shared(sc, &m_frag_v[bstart], &m_frag_v[bend ], this); m_global_bp->svtype = SVType::TSI_GLOBAL; // complex=true; // now, each local breakpoint is a TSI local not a simple rearrangement for (auto& i : m_local_breaks) { i->svtype = SVType::TSI_LOCAL; } } std::string AlignedContig::printDiscordantClusters(const SeqLib::BamHeader& h) const { std::stringstream out; if (m_dc.size() == 0) return "none"; for (const auto& d : m_dc) { out << d.print(h) << " "; } return out.str(); } // bool AlignedContig::checkLocal(const SeqLib::GenomicRegion& window) // { // bool has_loc = false; // for (auto& i : m_frag_v) // if (i.checkLocal(window)) // has_loc = true; // // check for all of the breakpoints of non-indels (already handling indels) // for (auto& i : m_local_breaks) // i.checkLocal(window); // // for (auto& i : m_global_bp_secondaries) // // i.checkLocal(window); // m_global_bp->checkLocal(window); // return has_loc; // } BreakPointPtrVector AlignedContig::getAllBreakPoints() const { BreakPointPtrVector out; // get the indel BreakPoints for (auto& i : m_frag_v) { out.insert(out.end(), i.m_indel_breaks.begin(), i.m_indel_breaks.end()); } // get the global breakpoint if (m_global_bp) //->isEmpty()) out.push_back(m_global_bp); // get the local breakpoints out.insert(out.end(), m_local_breaks.begin(), m_local_breaks.end()); return out; } // std::vector AlignedContig::getAllBreakPointsSecondary() const { // std::vector out; // for (auto& i : m_global_bp_secondaries) // if (!i.isEmpty()) // out.push_back(i); // return out; // } bool AlignedContig::hasVariant() const { if (m_global_bp) //!m_global_bp->isEmpty()) return true; if (m_local_breaks.size()) return true; // if (m_global_bp_secondaries.size()) // return true; // if (m_local_breaks_secondaries.size()) // return true; for (const auto& i : m_frag_v) for (const auto& j : i.m_indel_breaks) if (j->hasMinimal()) return true; return false; } // void AlignedContig::addDiscordantCluster(DiscordantClusterMap& dmap) // { // // loop through the breaks and compare with the map // for (auto& i : m_local_breaks) // i.__combine_with_discordant_cluster(dmap); // if (!m_global_bp->isEmpty()) // m_global_bp->__combine_with_discordant_cluster(dmap); // if (m_global_bp->hasDiscordant()) // m_dc.push_back(m_global_bp->dc); // for (auto& i : m_global_bp_secondaries) // i.__combine_with_discordant_cluster(dmap); // } // void AlignedContig::assessRepeats() { // for (auto& a : m_frag_v) // for (auto& i : a.m_indel_breaks) // i.repeatFilter(); // for (auto& b : m_local_breaks) // b.repeatFilter(); // for (auto& b : m_local_breaks_secondaries) // b.repeatFilter(); // for (auto& b : m_global_bp_secondaries) // b.repeatFilter(); // } // void AlignedContig::refilterComplex() { // if (m_global_bp && m_global_bp->num_align <= 2) // return; // // initialize split counts vec // std::vector scounts(m_local_breaks.size(), 0); // for (size_t i = 0; i < scounts.size() ; ++i) { // for (auto& j : m_local_breaks[i]->allele) // scounts[i] += j.second.split; // } // // every local BP needs to have 4+ split reads // // to be a quality TSI event // // if not, then forget the global / TSI altogether // for (auto& i : scounts) { // if (i < 4) { // m_global_bp = nullptr; // for (auto& i : m_local_breaks) // i->svtype = SVType::ASSMB; // return; // } // } // } std::string AlignedContig::getContigName() const { if (!m_frag_v.size()) return ""; return m_frag_v[0].m_align->Qname(); } int AlignedContig::getMaxMapq() const { int m = -1; for (auto& i : m_frag_v) if (i.m_align->MapQuality() > m) m = i.m_align->MapQuality(); return m; } int AlignedContig::getMinMapq() const { int m = 1000; for (auto& i : m_frag_v) if (i.m_align->MapQuality() < m) m = i.m_align->MapQuality(); return m; } // bool AlignedContig::hasLocal() const { // for (auto& i : m_frag_v) // if (i.local) // return true; // return false; // } // void AlignedContig::writeAlignedReadsToBAM(SeqLib::BamWriter& bw) { // for (const auto& i : m_bamreads) // bw.WriteRecord(*i); // } // void AlignedContig::writeToBAM(SeqLib::BamWriter& bw) const { // for (auto& i : m_frag_v) { // i.writeToBAM(bw); // } // } // std::string AlignedContig::getSequence() const { // assert(m_seq.length()); // return m_seq; // } void AlignedContig::AddAlignedRead(svabaReadPtr& br) { m_bamreads.push_back(br); } ================================================ FILE: src/svaba/AlignedContig.h ================================================ #pragma once #include #include "SeqLib/BWAAligner.h" #include "SeqLib/BamReader.h" #include "SeqLib/BamRecord.h" #include "SeqLib/BamWriter.h" #include "BreakPoint.h" #include "DiscordantCluster.h" #include "AlignmentFragment.h" #include "SvabaRead.h" /*! Contains the mapping of an aligned contig to the reference genome, * along with pointer to all of the reads aligned to this contig, and a * store of all of the breakpoints associated with this contig */ class AlignedContig { friend class AlignmentFragment; friend class BreakPoint; public: AlignedContig() = delete; // prevent default construction // make an AlignedContig from a set of contig alignments AlignedContig(BamRecordPtrVector& bav, const GenomicRegion& region, const SvabaSharedConfig* sc_); // Return as a genomic region vector SeqLib::GenomicRegionVector getAsGenomicRegionVector() const; bool checkLocal(const SeqLib::GenomicRegion& window); // apply repeat filter to each indel break //void assessRepeats(); // Loop through the vector of DiscordantCluster objects // associated with this contig and print std::string printDiscordantClusters(const BamHeader& h) const; // return the name of the contig std::string getContigName() const; // Debug accessors for compile-time trace (SvabaDebug.h) size_t getFragCount() const { return m_frag_v.size(); } bool hasGlobalBP() const { return m_global_bp != nullptr; } size_t getLocalBreakCount() const { return m_local_breaks.size(); } size_t getIndelBreakCount() const { size_t n = 0; for (const auto& f : m_frag_v) n += f.m_indel_breaks.size(); return n; } // Return the max mapping quality from all alignments int getMaxMapq() const; // Return the min mapping quality from all alignments int getMinMapq() const; // Loop through all of the breakpoints and // calculate the split read support for each. Requires // alignedReads to have been run first (will error if not run). void splitCoverage(); // Checks if any of the indel breaks are in a blacklist. If so, mark the // breakpoints of the indels for skipping. That is, hasMinmal() will return false; void blacklist(SeqLib::GRC& grv); // Dump the contigs to a fasta //void printContigFasta(std::ofstream &os) const; // Set the breakpoints on the reference by combining multi-mapped contigs void setMultiMapBreakPairs(); //! return the contig sequence as it came off the assembler //std::string getSequence() const; // SvABA2.0: emit this contig's info as a structured TSV that can be // re-plotted later, rather than pre-rendered into ASCII. One "contig" // row followed by one "read" row per r2c-aligned read. The companion // viewer is bps_explorer.html's r2c re-plot sub-panel. Rows share a // `contig_name` key so reads can be grouped back to their parent contig // without a sorted file. // // Replaced the old printToAlignmentsFile() / alignments.txt.gz output // entirely — same information content, just not pre-formatted. The // header line is emitted once per file by r2cTsvHeader(); for per-thread // streams, only the first worker (threadId == 1; workers are numbered // 1..N by threadpool.h) writes it — see svabaThreadUnit ctor. std::string printToR2CTsv(const SeqLib::BamHeader& h) const; // Column header for the r2c TSV; call once per file, before any rows. static std::string r2cTsvHeader(); // Return if this contig contains a potential variant (indel or multi-map) bool hasVariant() const; // Write all of the contig alignment records to a BAM file //void writeToBAM(SeqLib::BamWriter& bw) const; // Write all of the sequencing reads as aligned to contig to a BAM file //void writeAlignedReadsToBAM(SeqLib::BamWriter& bw); // Remove indels that map extremely close to rearrangement break points //void filterIndelsAtMultiMapSites(size_t buff); // returns whether any of the alignments are local to m_window //bool hasLocal() const; // Retrieves all of the breakpoints by combining indels with global mutli-map break BreakPointPtrVector getAllBreakPoints() const; //std::vector getAllBreakPointsSecondary() const; // void refilterComplex(); std::vector getAllBreakPointPointers() const ; void addDiscordantCluster(DiscordantClusterMap& dmap); std::pair getCoverageAtPosition(int pos) const; // add a new read aligned to this contig // this is a svabaRead (read to genome), but with an r2c in it void AddAlignedRead(svabaReadPtr& br); // return number of bam reads size_t NumBamReads() const { return m_bamreads.size(); } private: // int insertion_against_contig_read_count = 0; // int deletion_against_contig_read_count = 0; // store all of the reads aligned to contig // these are the same alignments as the BAM, but have an r2c as well svabaReadPtrVector m_bamreads; // coverage of each base in contig, whether it has alignment std::vector aligned_coverage; AlignmentFragmentVector m_frag_v; // store all of the individual alignment fragments std::vector m_local_breaks; // store all of the multi-map BreakPoints for this contigs //std::vector m_local_breaks_secondaries; // store all of the multi-map BreakPoints for this contigs BreakPointPtr m_global_bp; // store the single spanning BreakPoing for this contig e //size_t m_index_of_stored_seq = 0; // which alignment did mseq come from? //std::vector m_global_bp_secondaries; // store the single spanning BreakPoing for this contig e std::string m_seq; // sequence of contig as it came off of assembler std::vector m_dc; // collection of all discordant clusters that map to same location as this contig const SvabaSharedConfig* sc; }; typedef std::unordered_map ContigMap; typedef std::vector AlignedContigVec; ================================================ FILE: src/svaba/AlignmentFragment.cpp ================================================ #include "AlignmentFragment.h" #include "AlignedContig.h" #include "SvabaDebug.h" #define MIN_INDEL_MATCH_BRACKET 0 #define MAX_INDELS 10000 #define MAX_INDEL_PER_CONTIG 6 // write the alignment record to a BAM file // void AlignmentFragment::writeToBAM(SeqLib::BamWriter& bw) const { // bw.WriteRecord(*m_align); // } using SeqLib::CigarField; bool AlignmentFragment::operator<(const AlignmentFragment& str) const { return (start < str.start); } // bool AlignmentFragment::checkLocal() const { // assert(region_.pos1 >= 0); // // make a region for this frag // SeqLib::GenomicRegion gfrag(m_align->ChrID(), // m_align->Position(), // m_align->PositionEnd()); // if (region_.GetOverlap(gfrag)) { // return true; // } // return false; // } AlignmentFragment::AlignmentFragment(BamRecordPtr &talign, bool flip, const GenomicRegion& local_region, const SvabaSharedConfig* sc_) : region_(local_region), m_align(talign), sc(sc_) { // orient cigar so it is on the contig orientation. // need to do this to get the right ordering of the contig fragments below // We only flip if we flipped the sequence, and that was determined // by the convention set in AlignedContig::AlignedContig, so we need // to pass that information explicitly // if (flip/*m_align->ReverseFlag()*/) { // m_cigar = m_align->GetReverseCigar(); // } else { // m_cigar = m_align->GetCigar(); // } flipped = flip; // set the left-right breaks unsigned currlen = 0; // NB: CPOS is zero based // NB: for break1 and break2, we use these later in the // for either printing to alignments file, or for // getting the *contig* coordinate breakpoints // So this to account for "flip" convention across // AlignmentFragments const auto& cig = flipped ? m_align->GetReverseCigar() : m_align->GetCigar(); for (auto& i : cig) { // SET THE CONTIG BREAK (treats deletions and leading S differently) // the first M gets the break1, pos on the left if (i.Type() == 'M' && break1 == -1) break1 = currlen; if (i.Type() != 'D') // m_skip deletions but not leading S, but otherwise update currlen += i.Length(); if (i.Type() == 'M') // keeps triggering every M, with pos at the right break2 = currlen; } // assign the genomic coordinates of the break if (m_align->ReverseFlag()) { gbreak2 = m_align->Position() + 1; gbreak1 = m_align->PositionEnd(); } else { gbreak1 = m_align->Position() + 1; gbreak2 = m_align->PositionEnd(); } // shouldn't hit either of the next two conditions // if (break1 >= MAX_CONTIG_SIZE || break2 >= MAX_CONTIG_SIZE || break1 < 0 || break2 < 0) { // throw std::runtime_error( // "Invalid breakpoints: break1 = " + std::to_string(break1) + // ", break2 = " + std::to_string(break2) + // ", context = " + this->print() // ); // } if (break1 < 0 || break2 < 0) { throw std::runtime_error("Negative breakpoint detected in AlignmentFragment "); // + printToAlignmentsFile()); } // find the start position of alignment ON CONTIG start = 0; for (auto& i : cig) { //m_align->GetCigar()) { //m_cigar) { if (i.Type() != 'M') start += i.Length(); else break; } } void AlignmentFragment::SetIndels() { if (m_align->SecondaryFlag()) return; // ignore these const auto& cig = m_align->GetCigar(); const std::string _cname = m_align->Qname(); // Build CIGAR string for trace #ifdef SVABA_TRACING { std::string cig_str; for (const auto& cf : cig) cig_str += std::to_string(cf.Length()) + std::string(1, cf.Type()); SVABA_TRACE(_cname, "TP4 SetIndels: cigar=" << cig_str << " cigar_ops=" << cig.size() << " secondary=" << m_align->SecondaryFlag()); } #endif // loops the alignment fragment to extract indels // start at 1 and end at len-1 because we don't consider indels if they // start the cigar string e.g. 1D5M, not reliable for (size_t i = 1; i < cig.size() - 1; ++i) { const CigarField& c = cig[i]; const CigarField& pre = cig[i-1]; const CigarField& post = cig[i+1]; if ( (c.Type() == 'D' || c.Type() == 'I')) { bool prev_match = pre.Type() == 'M'; bool post_match = post.Type() == 'M'; SVABA_TRACE(_cname, "TP4 indel at cigar[" << i << "]: " << c.Length() << c.Type() << " prev=" << pre.Length() << pre.Type() << " post=" << post.Length() << post.Type() << " prev_match=" << prev_match << " post_match=" << post_match); // only consider indels if if (prev_match && post_match) { // convention is that cpos and gpos for deletions refer to flanking REF sequence. // eg a deletion of 1 bp of base 66 will have gpos1 = 65 and gpos2 = 67 BreakPointPtr bp = std::make_shared(this, i, sc); assert(bp); // add the indel m_indel_breaks.push_back(bp); SVABA_TRACE(_cname, "TP4 -> INDEL BP created"); } } } } std::string AlignmentFragment::printToAlignmentsFile() const { std::stringstream out; // sets the direction to print char jsign = '>'; if (m_align->ReverseFlag()) jsign = '<'; // NB: for printing, this should actually finally // consider if this fragment is "flipped" relative // to the BWA alignment, since we want to sync // orientations across all of the fragments. const auto& cig = flipped ? m_align->GetReverseCigar() : m_align->GetCigar(); // print the cigar value per base for (auto& j : cig) { if (j.Type() == 'M') out << std::string(j.Length(), jsign); else if (j.Type() == 'I') out << std::string(j.Length(), 'I'); else if (j.Type() == 'S' || j.Type() == 'H') out << std::string(j.Length(), '.'); } // print contig and genome breaks out << "\tC[" << break1 << "," << break2 << "] G[" << gbreak1 << "," << gbreak2 << "]"; // print it std::string chr_name = m_align->ChrName(sc->header); assert(chr_name.length()); out << "\tAligned to: " << chr_name << ":" << m_align->Position() << "(" << (m_align->ReverseFlag() ? "-" : "+") << ") CIG: " << m_align->CigarString() << " MAPQ: " << m_align->MapQuality(); // << " SUBN " << sub_n; return out.str(); } // void AlignmentFragment::fillRearrangementBreakEnd(bool left, // BreakEnd& b) { // b.transferContigAlignmentData(m_align); // if (left) { // b.gr = SeqLib::GenomicRegion(m_align->ChrID(), gbreak2, gbreak2); // b.gr.strand = m_align->ReverseFlag() ? '-' : '+'; // b.cpos = break2; // take the right-most breakpoint of the left as the first // } else { // b.gr = SeqLib::GenomicRegion(m_align->ChrID(), gbreak1, gbreak1); // b.gr.strand = m_align->ReverseFlag() ? '+' : '-'; // b.cpos = break1; // take the left-most of the next one // } // assert(b.cpos < MAX_CONTIG_SIZE); // } // assert(i.getSpan() > 0); // // get the hash string in same formate as cigar map (eg. pos_3D) // std::string st = i.getHashString(); // for (auto& c : cmap) { // SeqLib::CigarMap::const_iterator ff = c.second.find(st); // // if it is, add it // if (ff != c.second.end()) { // i.allele[c.first].cigar = ff->second; // } // } // } // } ================================================ FILE: src/svaba/AlignmentFragment.h ================================================ #pragma once #include #include #include #include "SeqLib/BamRecord.h" #include "BreakPoint.h" using SeqLib::BamRecordPtr; using SeqLib::Cigar; using SeqLib::CigarMap; using SeqLib::GenomicRegion; using SeqLib::BamHeader; // forward declare class AlignedContig; /*! This class contains a single alignment fragment from a contig to * the reference. For a multi-part mapping of a contig to the reference, * an object of this class represents just a single fragment from that alignment. */ class AlignmentFragment { friend class BreakPoint; friend class AlignedContig; public: AlignmentFragment() = delete; // prevent default construction /*! Construct an AlignmentFragment from a BWA alignment * @param flip If the contig sequence was flipped (rev of BAM record), need to track this. This flipping occurs in AlignedContig::AlignedContig */ // talign is an alignment of the contig to reference AlignmentFragment(BamRecordPtr &talign, bool flip, const GenomicRegion& local_region, const SvabaSharedConfig* sc_); // sort AlignmentFragment objects by start position bool operator < (const AlignmentFragment& str) const; // print the AlignmentFragment std::string printToAlignmentsFile() const; void fillRearrangementBreakEnd(bool left, BreakEnd& b); // check whether the alignment fragement overlaps with the given windows bool checkLocal() const; //const std::vector& getIndelBreaks() const { return m_indel_breaks; } // write the alignment record to a BAM file //void writeToBAM(SeqLib::BamWriter& bw) const; void SetIndels(); BamRecordPtr m_align; // BWA alignment of contig to reference std::vector secondaries; BreakPointPtrVector m_indel_breaks; // indel variants on this alignment //Cigar m_cigar; //cigar oriented to assembled orientation //std::string m_seq; // seequence, orientated to assembled orientation int break1 = -1; // 0-based breakpoint 1 on contig int break2 = -1; // 0-based breakpoint 2 on contig int gbreak1 = -1; // 0-based breakpoint 1 on reference chr int gbreak2 = -1; // 0-based breakpoint 1 on reference chr const SvabaSharedConfig* sc; //pointer to allow sort later GenomicRegion region_; int start = -1; // where on the contig does this alignment fragment start int num_align = -1; bool flipped = false; }; typedef std::vector AlignmentFragmentVector; ================================================ FILE: src/svaba/BamStats.cpp ================================================ #include "BamStats.h" #include using namespace SeqLib; ================================================ FILE: src/svaba/BamStats.h ================================================ // #pragma once // #include // #include // #include // #include "Histogram.h" // #include "SeqLib/BamRecord.h" // /** Store information pertaining to a given read group * // * // * This class will collect statistics on number of: read, supplementary reads, unmapped reads, qcfail reads, duplicate reads. // * It will also create Histogram objects to store counts of: mapq, nm, isize, clip, mean phred score, length // */ // class BamReadGroup { // friend class BamStats; // public: // /** Construct an empty BamReadGroup */ // BamReadGroup() {} // /** Construct an empty BamReadGroup for the specified read group // * @param name Name of the read group // */ // BamReadGroup(const std::string& name); // /** Display basic information about this read group // */ // friend std::ostream& operator<<(std::ostream& out, const BamReadGroup& rg); // /** Add a BamRecord to this read group */ // void addRead(SeqLib::BamRecord &r); // private: // // count number of reads // size_t reads; // size_t supp; // size_t unmap; // size_t qcfail; // size_t duplicate; // size_t mate_unmap; // int mapq_max = 0; // int realen_max = 0; // std::vector isize_vec; // std::string m_name; // }; // /** Class to store statistics on a BAM file. // * // * BamStats currently stores a map of BamReadGroup objects. Bam statistics // * are collected then on a read-group basis, but can be output in aggregate. See // * BamReadGroup for description of relevant BAM statistics. // */ // class BamStats // { // public: // /** Loop through the BamReadGroup objections and print them */ // friend std::ostream& operator<<(std::ostream& out, const BamStats& qc); // /** Add a read by finding which read group it belongs to and calling the // * addRead function for that BamReadGroup. // */ // void addRead(SeqLib::BamRecord &r); // private: // std::unordered_map m_group_map; // }; ================================================ FILE: src/svaba/BreakPoint.cpp ================================================ #include "BreakPoint.h" #include "SvabaDebug.h" #include #include #include #include #include #include #include #include #include #include #include "gzstream.h" #include "SvabaUtils.h" #include "STCoverage.h" #include "SvabaOptions.h" #include "SeqLib/GenomicRegionCollection.h" #include "SeqLib/GenomicRegion.h" #include "SeqLib/BamHeader.h" #include "AlignmentFragment.h" #include "AlignedContig.h" #include "SvabaModels.h" #include "ContigAlignmentScore.h" // n is the max integer given the int size (e.g. 255). x is string with int #define INTNSTOI(x,n) std::min((int)n, std::stoi(x)); static inline std::string to_string(SVType t) { switch(t) { case SVType::NOTSET: return "NOTSET"; case SVType::TSI_LOCAL: return "TSI_LOCAL"; case SVType::TSI_GLOBAL: return "TSI_GLOBAL"; case SVType::ASSMB: return "ASSMB"; case SVType::ASDIS: return "ASDIS"; case SVType::DSCRD: return "DSCRD"; case SVType::INDEL: return "INDEL"; } return "UNKNOWN_SVTYPE"; } static inline std::string to_string(SomaticState s) { switch(s) { case SomaticState::NOTSET: return "NOTSET"; case SomaticState::NORMAL_LOD: return "0"; case SomaticState::SOMATIC_LOD: return "1"; case SomaticState::FAILED: return "0"; } return "UNKNOWN_SOMATICSTATE"; } std::ostream& operator<<(std::ostream& out, const BreakPoint::SampleInfo& a) { out << " split: " << a.split << " cigar " << a.cigar << " alt " << a.alt << " cov " << a.cov << " disc " << a.disc; return out; } GenomicRegion BreakPoint::BreakEndAsGenomicRegionLeft() const { return b1.gr; } GenomicRegion BreakPoint::BreakEndAsGenomicRegionRight() const { return b2.gr; } bool BreakPoint::isIndel() const { if (svtype == SVType::NOTSET) throw std::runtime_error("BreakPoint without an svtype"); return svtype == SVType::INDEL; } // SvABA2.0: printDeletionMarksForAlignmentsFile() was removed along with // AlignedContig::printToAlignmentsFile / alignments.txt.gz. The deletion- // mark info is available in the r2c TSV via the bps field (per-breakpoint // span + kind), so downstream viewers can reconstruct the marker // positions from structured fields rather than parsing ASCII. HashVector BreakPoint::getBreakEndHashes() { HashVector vec; assert(!confidence.empty()); assert(svtype != SVType::NOTSET); if (svtype != SVType::INDEL) return vec; if (b1.gr.pos1 > 0) { { vec.push_back(b1.hash(0)); vec.push_back(b1.hash(1)); vec.push_back(b1.hash(-1)); } } return vec; } BreakPoint::SampleInfo operator+(const BreakPoint::SampleInfo& a1, const BreakPoint::SampleInfo& a2) { BreakPoint::SampleInfo a; a.disc = a1.disc + a2.disc; a.split = a1.split + a2.split; a.cigar = a1.cigar + a2.cigar; a.cigar_near = a1.cigar_near + a2.cigar_near; a.cov = a1.cov + a2.cov; // add the reads for (auto& i : a1.supporting_reads) a.supporting_reads.insert(i); for (auto& i : a2.supporting_reads) a.supporting_reads.insert(i); //a.alt = std::max((int)a.supporting_reads.size(), a.cigar); if (a.supporting_reads.size()) // we have the read names, so do that (non-refilter run) a.UpdateAltCounts(); else // no read names stored, so just get directly a.alt = a1.alt + a2.alt; return a; } static constexpr double scale_factor = 10.0; static std::unordered_map ERROR_RATES = {{0, scale_factor * 1e-4}, {1, scale_factor * 1e-4}, {2, scale_factor * 1e-4}, {3, scale_factor * 1e-4}, {4, scale_factor * 1e-4}, {5, scale_factor * 2e-4}, {6, scale_factor * 5e-4}, {7, scale_factor * 1e-3}, {8, scale_factor * 2e-3}, {9, scale_factor * 3e-3}, {10, scale_factor * 1e-2}, {11, scale_factor * 2e-2}, {12, scale_factor * 3e-2}}; double __myround(double x) { return std:: floor(x * 10) / 10; } BreakPoint::BreakPoint(const SvabaSharedConfig* _sc, const AlignmentFragment* left, const AlignmentFragment* right, const AlignedContig* alc ) : sc(_sc) { // this is at least of type ASSMB (can upgrade later to ASDIS or TSI) svtype = SVType::ASSMB; // instantiate the SampleInfo objects for each BAM for (auto const& p : sc->prefixes) { // if p isn't already present, constructs SampleInfo(this) in-place allele.try_emplace(p); } assert(alc); // transfer AlignedContig level information seq = alc->m_seq; num_align = alc->m_frag_v.size(); cname = alc->getContigName(); // SvABA2.0: record the contig length so splitCoverage can reject r2c // reads that soft-clip in the middle of the contig (as opposed to at // the contig edge, which is a legitimate geometry). m_seq is already // in assembly-native orientation. We don't set flipped_on_contig here // because SV breakpoints are composed of two AlignmentFragments with // independent flip conventions; splitCoverage's m_seq cpos handling // for SVs uses the per-fragment cpos set by transferContigAlignmentData. contig_len = static_cast(seq.length()); // set the local alignment // int la = 0; // alc->m_frag_v[0].m_align->GetIntTag("LA", la); // if (la > 0) // local = LocalAlignment::NONVAR_LOCAL_REALIGNMENT; // set the break coordinates bool isleft = true; b1.transferContigAlignmentData(left, isleft); b2.transferContigAlignmentData(right, !isleft); // set the insertion / homology set_homologies_insertions(); // set seconary flag secondary = left->m_align->SecondaryFlag() || right->m_align->SecondaryFlag(); } // make the file string std::string BreakPoint::toFileString(const BamHeader& header) const { // make sure we already ran scoring assert(confidence.length()); assert(svtype != SVType::NOTSET); std::string sep = "\t"; std::stringstream ss; double max_lod = 0; for (const auto& [_,al] : allele) max_lod = std::max(max_lod, al.LO); std::string evidence = to_string(svtype); std::string somatic_string = "NA"; std::string somatic_lod_string = "NA"; for (const auto& [pref,_] : allele) { if (pref.at(0) == 'n') { // need to have one normal to call somatic somatic_string = to_string(somatic); somatic_lod_string = std::to_string(std::min((double)99,LO_s)); } } //NB: we are adding +1 to the positions here to be consistent // with SAM/VCF which is 1-indexed, while we are using 0-indexed // positions internally to be consistent with htslib ss << b1.gr.ChrName(header) << sep << (b1.gr.pos1 +1) << sep << b1.gr.strand << sep //1-3 << b2.gr.ChrName(header) << sep << (b2.gr.pos1 +1) << sep << b2.gr.strand << sep //4-6 << ref << sep << alt << sep //7-8 << getSpan() << sep //9 << a.split << sep << a.alt << sep << a.cov << sep << a.cigar << sep << a.cigar_near << sep << dc.mapq1 << sep << dc.mapq2 << sep << dc.ncount << sep << dc.tcount << sep << b1.mapq << sep << b2.mapq << sep << b1.nm << sep << b2.nm << sep << b1.as << sep << b2.as << sep << b1.sub << sep << b2.sub << sep << (homology.length() ? homology : "x") << sep << (insertion.length() ? insertion : "x") << sep << (repeat_seq.length() ? repeat_seq : "x") << sep << cname << sep << num_align << sep << confidence << sep << evidence << sep << quality << sep << secondary << sep << somatic_string << sep << somatic_lod_string << sep << max_lod << sep //<< pon << sep // << (repeat_seq.length() ? repeat_seq : "x") << sep << (rs.length() ? rs : "x") << sep << b1.contig_conf << sep << b2.contig_conf << sep // --- SvABA2.0 additions for refilter round-trip ------------------------ // Contig-relative breakend positions. -1 sentinel means "not set" (e.g. // DSCRD-only breakpoints with no backing contig alignment). << b1.cpos << sep << b2.cpos << sep // Flanking match lengths at each end (drives SHORTALIGNMENT/LOWMATCHLEN). << left_match << sep << right_match << sep // Extent of split-read coverage on the contig (drives DUPREADS/LOWSUPPORT). << split_cov_bounds.first << sep << split_cov_bounds.second << sep // Per-end LocalAlignment enum, serialized as int to keep the parser // identity-simple. See BreakPoint.h for enum values (0..3). << static_cast(b1.local) << sep << static_cast(b2.local) << sep // Contig orientation metadata for cpos_on_m_seq() reconstruction. << contig_len << sep << (flipped_on_contig ? 1 : 0) << sep // SvABA2.0: unique BP identifier. Unset (shouldn't happen if // SvabaRegionProcessor hooked next_bp_id() properly) emits "." // so the column count stays fixed and awk scripts don't skew. << (id.empty() ? "." : id); for (const auto& [_,al] : allele) ss << sep << al.toFileString(svtype); return ss.str(); } // make the file string std::string BreakPoint::printSimple(const SeqLib::BamHeader& h) const { std::stringstream out; // make sure we set everything forthis assert(svtype != SVType::NOTSET); assert(!cname.empty()); assert(b1.gr.chr >= 0); assert(svtype == SVType::DSCRD || b1.nm >= 0); assert(svtype == SVType::DSCRD || b2.nm >= 0); assert(svtype == SVType::DSCRD || b1.as >= 0); assert(svtype == SVType::DSCRD || b2.as >= 0); if (svtype == SVType::INDEL) { out << ">" << (insertion.size() ? "INS: " : "DEL: ") << getSpan() << " " << b1.gr.ToString(h) << " " << cname; for (const auto& [pref, al] : allele) out << " " << pref << ":" << al.split; } else { out << ": " << b1.gr.PointString(h) << " to " << b2.gr.PointString(h) << " SPAN " << getSpan() << " " << cname; for (const auto& [pref, al] : allele) out << " " << pref << ":" << al.split; } return out.str(); } /*std::ostream& operator<<(std::ostream& out, const BreakPoint& b) { if (b.isindel) { out << ">" << (b.insertion.size() ? "INS: " : "DEL: ") << b.getSpan() << " " << b.b1.gr << " " << b.cname << " " << b.evidence; //<< " T/N split: " << b.t.split << "/" << b.n.split << " T/N cigar: " // << b.t.cigar << "/" << b.n.cigar << " T/N Cov " << b.t.cov << "/" << b.n.cov << " DBSNP: " << rs_t; for (auto& i : b.allele) out << " " << i.first << ":" << i.second.split; } else { out << ": " << b.b1.gr.PointString() << " to " << b.b2.gr.PointString() << " SPAN " << b.getSpan() << " " << b.cname << " " << b.evidence; //<< " T/N split: " << b.t.split << "/" << b.n.split << " T/N disc: " // << b.dc.tcount << "/" << b.dc.ncount << " " << b.evidence; for (auto& i : b.allele) out << " " << i.first << ":" << i.second.split; } return out; }*/ void BreakPoint::splitCoverage(svabaReadPtrVector& bav) { // dummy left-most and right-most positions that have an // overlapping read2contig alignment split_cov_bounds = {std::numeric_limits::max(), -1}; // track if first and second mate covers same split. fishy and remove them both std::unordered_map qname_and_num; // keep track of which reads already added std::unordered_set qnames; // keep track of reads to reject std::set reject_qnames; // keep track of which SR tags are valid splits std::unordered_set valid_reads; // SvABA2.0: b1.cpos/b2.cpos are in BAM/genome-forward coordinates, but // the r2c CIGARs and this_r2c.start_on_contig/end_on_contig that we // compare against below are in assembly-native / m_seq coordinates. // For reverse-aligned contigs these are mirror images and the old code // counted reads at the mirror position as "supporting". Use the m_seq // cpos pair everywhere we compare against r2c in this function. const auto [m_b1_cpos, m_b2_cpos] = cpos_on_m_seq(); // SvABA2.0 (v3): homlen is no longer used as a gate input. The old // logic required both_split when homlen > 0 and one_split only when // homlen == 0. That wiped out legitimate spanning reads when the // junction sat inside a long homology region (a common failure mode // for repeat-junction LOH events — tumor looked clean because all // its split reads fell inside the homology and got rejected). // The comparative r2c-vs-native score gate (with per-sample margin) // replaces it: in a long-homology junction, neither alignment wins // decisively, so such reads fail the gate on their own without // special-casing — no "both_split" complication needed. // loop all of the read to contig alignments for this contig for (const auto& j : bav) { r2c this_r2c = j->GetR2C(cname); SVABA_READ_TRACE(j->Qname(), "SPLIT_COV enter contig=" << cname << " r2c_start=" << this_r2c.start_on_contig << " r2c_end=" << this_r2c.end_on_contig << " r2c_rc=" << this_r2c.rc << " r2c_cigar=" << this_r2c.cig << " r2c_nm=" << this_r2c.nm << " b1_cpos=" << m_b1_cpos << " b2_cpos=" << m_b2_cpos << " num_align=" << num_align); bool read_should_be_skipped = false; // SvABA2.0: principled "r2c better than native" gate. // // The fundamental requirement for a read to support a variant is that // the read-to-contig (r2c) alignment must be a *better* explanation // of the read than the read-to-reference alignment that BWA-MEM gave // it in the input BAM. If both alignments are equally clean (the // classic "duplicated reference" trap, where a repeat lets BWA align // the read to a paralogous copy with no clips and no NM, while the // r2c is also clean because the contig was assembled from the same // sequence), then the contig provides no extra explanatory power and // the read should *not* count as a variant supporter. // // svaba::readAlignmentScore() (defined in ContigAlignmentScore.h) is // an SV-focused score that heavily penalizes soft-clips and counts // edit distance, so e.g. // // 80M70S NM=0 -> aligned_bp 80, clip_bp 70 -> score 10 // 150M NM=0 -> aligned_bp 150 -> score 150 // 90M60S NM=0 -> -> score 30 // 75M5I70M NM=5 -> aligned_bp 150, NM 5 -> score 140 // // and the gate is a strict r2c > native comparison. // // This replaces an earlier hard cap on non-edge soft-clip length: // the score-based comparison is more principled (it lets big edge // clips be fine when the native alignment is also bad, and rejects // small interior indels in r2c when the native alignment is actually // clean) and works for both interior and edge clip cases without // needing separate tumor/normal thresholds. // // Guard only on the r2c CIGAR being populated; if it isn't, there's // nothing to score and we let the downstream checks decide. // // Compile-time kill-switch: `SVABA_R2C_NATIVE_GATE` (SvabaOptions.h). // When set to 0, this entire block is elided — reads are no longer // compared r2c-vs-native and the old "any r2c-spanning read credits" // behavior returns. Reintroduces the homology-trap false-positive // somatic bug; flip only to measure the gate's CPU cost. See the // macro's doc comment in SvabaOptions.h. #if SVABA_R2C_NATIVE_GATE if (this_r2c.cig.size() > 0) { // r2c side: use the cached NM on this_r2c (filled in r2c::AddAlignment). // The r2c is computed against the *corrected* read sequence. const double r2c_score = svaba::readAlignmentScore(this_r2c.cig, this_r2c.nm); // Native side: STRONGLY prefer the corrected-read realignment that // SvabaRegionProcessor stashed on the svabaRead. That comparison is // apples-to-apples: same corrected sequence and same svaba-internal // BWA-MEM parameters as the r2c step. Falling back to the original // input-BAM CIGAR/NM is a last resort (e.g. for reads with // to_assemble == false that never went through correction) and is // expected to be biased: pre-correction NM includes sequencing // errors that BFC would have fixed, which inflates native edit // distance and lets r2c look artificially "better". SeqLib::Cigar native_cig; int32_t native_nm = -1; if (j->corrected_native_cig.size() > 0) { native_cig = j->corrected_native_cig; native_nm = j->corrected_native_nm; } else { native_cig = j->GetCigar(); j->GetIntTag("NM", native_nm); } // If neither path produced a usable native CIGAR, fall back to the // read length as a conservative perfect-native upper bound. This // prevents a read with no recoverable native alignment from // auto-passing the gate just because native_score defaulted to 0. double native_score = svaba::readAlignmentScore(native_cig, native_nm); if (native_cig.size() == 0) { native_score = static_cast(j->Length()); } // SvABA2.0 (v3.1): strict greater-than gate, no percentage margin. // r2c must beat native — ties don't credit the read. Both tumor // and normal use margin=0. // // v3 originally had a 10% margin for tumor reads to filter // junction-homology borderline cases, but this was read-length- // dependent and killed real small indels (1bp del on 150bp read = // only 4.9% improvement, impossible to clear 10%; on 250bp read // only 2.9%, can't even clear 3%). Junction-homology cases where // both samples credit borderline reads equally are handled // correctly by the downstream LOD model (similar split support // in both → low somlod → not somatic). const double margin = j->Tumor() ? T_R2C_MIN_MARGIN : N_R2C_MIN_MARGIN; const double threshold = native_score * (1.0 + margin); if (r2c_score <= threshold) { read_should_be_skipped = true; SVABA_TRACE(cname, "TP8 r2c<=native SKIP read=" << j->UniqueName() << " r2c=" << r2c_score << " native=" << native_score << " threshold=" << threshold << " margin=" << margin << " tumor=" << j->Tumor()); SVABA_READ_TRACE(j->Qname(), "SPLIT_COV TP8 SKIP r2c<=native" << " contig=" << cname << " r2c_score=" << r2c_score << " native_score=" << native_score << " threshold=" << threshold << " margin=" << margin << " tumor=" << j->Tumor() << " r2c_cigar=" << this_r2c.cig << " native_cigar=" << native_cig << " r2c_nm=" << this_r2c.nm << " native_nm=" << native_nm); } else { SVABA_TRACE(cname, "TP8 r2c>native PASS read=" << j->UniqueName() << " r2c=" << r2c_score << " native=" << native_score << " threshold=" << threshold); SVABA_READ_TRACE(j->Qname(), "SPLIT_COV TP8 PASS r2c>native" << " contig=" << cname << " r2c_score=" << r2c_score << " native_score=" << native_score << " r2c_cigar=" << this_r2c.cig << " native_cigar=" << native_cig << " r2c_nm=" << this_r2c.nm << " native_nm=" << native_nm); } } #endif // SVABA_R2C_NATIVE_GATE if (num_align == 1) { std::vector del_breaks; std::vector ins_breaks; // SvABA2.0: pos must be in *absolute contig* (m_seq / r2c) coordinates // because we compare it below against m_b1_cpos / m_b2_cpos, which are // also absolute contig coordinates (cpos_on_m_seq() handles the flip). // The previous code initialized pos = 0, which made it relative to the // start of this read's CIGAR, so for any read that didn't start at // contig position 0 the indel-at-variant-location check could never // fire. That allowed the "mirror indel" failure mode through: // a REF-supporting read whose r2c CIGAR has an inserted/deleted base // exactly at the variant locus (because the contig itself carries the // mirror indel) was being credited as variant-supporting. int pos = this_r2c.start_on_contig; // if this is a nasty repeat, don't trust non-perfect alignmentx on r2c alignment // if (checkHomopolymer(j->Sequence())) // read_should_be_skipped = true; // loop through r2c cigar and see positions for(auto& i : this_r2c.cig) { if (i.Type() == 'D') del_breaks.push_back(pos); else if (i.Type() == 'I') ins_breaks.push_back(pos); // update position on contig if (i.ConsumesReference()) pos += i.Length(); // update position on contig } size_t buff = std::max((size_t)3, repeat_seq.length() + 3); // SvABA2.0: the old test was `i > X-buff || i < X+buff`, which is a // tautology (for any i, one side is always true), so it would // reject every read with any D or I in its r2c CIGAR. The intent // is: reject the read if one of its r2c indels lands *at* the // breakpoint position +/- buff (indicating its r2c alignment is // fishy right at the junction we care about). Use AND. We also // check both b1 and b2 so deletions/insertions landing on either // side of the (potentially homologous) junction window are caught. // // NB: copy the structured-binding values (m_b1_cpos, m_b2_cpos) into // plain ints before the lambda. Capturing structured bindings is a // C++20 extension; svaba builds at C++17 so we'd otherwise warn on // -Wc++20-extensions. const int b1c = m_b1_cpos; const int b2c = m_b2_cpos; const int ibuff = static_cast(buff); auto near_break = [b1c, b2c, ibuff](int p) { return (p >= b1c - ibuff && p <= b1c + ibuff) || (p >= b2c - ibuff && p <= b2c + ibuff); }; for (auto& i : del_breaks) if (near_break(i)) { read_should_be_skipped = true; SVABA_TRACE(cname, "TP9 r2c DEL near break SKIP read=" << j->UniqueName() << " del_pos=" << i << " b1c=" << b1c << " b2c=" << b2c << " buff=" << ibuff); SVABA_READ_TRACE(j->Qname(), "SPLIT_COV TP9 SKIP r2c DEL near break" << " contig=" << cname << " del_pos=" << i << " b1_cpos=" << b1c << " b2_cpos=" << b2c << " buff=" << ibuff); } for (auto& i : ins_breaks) if (near_break(i)) { read_should_be_skipped = true; SVABA_TRACE(cname, "TP9 r2c INS near break SKIP read=" << j->UniqueName() << " ins_pos=" << i << " b1c=" << b1c << " b2c=" << b2c << " buff=" << ibuff); SVABA_READ_TRACE(j->Qname(), "SPLIT_COV TP9 SKIP r2c INS near break" << " contig=" << cname << " ins_pos=" << i << " b1_cpos=" << b1c << " b2_cpos=" << b2c << " buff=" << ibuff); } } if (read_should_be_skipped) { // default is r2c does not support var, so don't amend this_r2c SVABA_READ_TRACE(j->Qname(), "SPLIT_COV SKIPPED (gate failed) contig=" << cname); continue; } // get read ID std::string sample_id = j->Prefix(); //substr(0,4); // maybe just make this prefix std::string sr = j->UniqueName(); // SvABA2.0 (v3): small fixed buffer past each breakend on the // contig. The repeat_seq.length() padding on the older code was // part of the homology-era logic and is dropped — the comparative // r2c-vs-native score gate (with per-sample margin) now handles // tandem-repeat ambiguity on its own: when a short indel at the // junction can equally well live anywhere in a tandem repeat, // r2c and native both fit equivalently and neither wins. int this_tbuff = T_SPLIT_BUFF; int this_nbuff = N_SPLIT_BUFF; int rightbreak1 = m_b1_cpos + (j->Tumor() ? this_tbuff : this_nbuff); // read must extend this far right of break1 int leftbreak1 = m_b1_cpos - (j->Tumor() ? this_tbuff : this_nbuff); // read must extend this far left of break1 int rightbreak2 = m_b2_cpos + (j->Tumor() ? this_tbuff : this_nbuff); int leftbreak2 = m_b2_cpos - (j->Tumor() ? this_tbuff : this_nbuff); std::string contig_qname; // for sanity checking // get the alignment position on contig int pos = this_r2c.start_on_contig; int te = this_r2c.end_on_contig; int rightend = te; int leftend = pos; bool issplit1 = (leftend <= leftbreak1) && (rightend >= rightbreak1); bool issplit2 = (leftend <= leftbreak2) && (rightend >= rightbreak2); bool one_split = issplit1 || issplit2; SVABA_TRACE(cname, "TP10 span check read=" << j->UniqueName() << " leftend=" << leftend << " rightend=" << rightend << " b1_cpos=" << m_b1_cpos << " b2_cpos=" << m_b2_cpos << " issplit1=" << issplit1 << " issplit2=" << issplit2 << " one_split=" << one_split); SVABA_READ_TRACE(j->Qname(), "SPLIT_COV TP10 span check" << " contig=" << cname << " leftend=" << leftend << " rightend=" << rightend << " b1_cpos=" << m_b1_cpos << " b2_cpos=" << m_b2_cpos << " lbreak1=" << leftbreak1 << " rbreak1=" << rightbreak1 << " lbreak2=" << leftbreak2 << " rbreak2=" << rightbreak2 << " issplit1=" << issplit1 << " issplit2=" << issplit2 << " one_split=" << one_split << " tumor=" << j->Tumor()); // SvABA2.0 (v3): previously we required both_split when homlen > 0 // (tumor *or* normal) and one_split only when homlen == 0. That // gate threw out legitimate split-supporters inside long junction // homology regions (a common LOH false-somatic pattern). The // principled replacement is the per-sample-prefix r2c > native // score gate (applied above, before this read ever reaches the // valid check). If a read makes it here, its r2c alignment is // strictly better than its native alignment (by the tumor margin // for t*** reads, strictly greater for n*** reads). So we only // need to confirm the read actually spans at least one breakend // on the contig — i.e. it's a "split" in the physical sense of // crossing a junction — and then credit it. // // Note: the giant insertion case is subsumed by this rule too — a // large insertion usually means one_split on at least one side. bool valid = one_split; // check that deletion (in read to contig coords) doesn't cover break point size_t p = pos; // move along on contig, starting at first non-clipped base for (SeqLib::Cigar::const_iterator c = this_r2c.cig.begin(); c != this_r2c.cig.end(); ++c) { if (c->Type() == 'D') { if ( (p >= leftbreak1 && p <= rightbreak1) || (p >= leftbreak2 && p <= rightbreak2) ) { read_should_be_skipped = true; SVABA_TRACE(cname, "TP11 r2c DEL covers break SKIP read=" << j->UniqueName() << " del_pos=" << p << " lb1=" << leftbreak1 << " rb1=" << rightbreak1 << " lb2=" << leftbreak2 << " rb2=" << rightbreak2); SVABA_READ_TRACE(j->Qname(), "SPLIT_COV TP11 SKIP r2c DEL covers break" << " contig=" << cname << " del_pos=" << p); } } if (c->ConsumesReference()) // if it moves it along the contig p += c->Length(); } // add the split reads for each end of the break // a read is split if it is spans both break ends for tumor, one break end for normal (to // be more sensitive to germline) and if it spans both ends for deletion (should be next to // each other), or one end for insertions larger than 10, or this is a complex breakpoint if (!valid) { SVABA_READ_TRACE(j->Qname(), "SPLIT_COV NOT CREDITED (failed span check)" << " contig=" << cname << " one_split=" << one_split << " read_should_be_skipped=" << read_should_be_skipped); } if (valid) { std::string qn = j->Qname(); // if read seen and other read was other mate, then reject if (num_align > 1 && qname_and_num.count(qn) && qname_and_num[qn] != j->FirstFlag()) { // need to reject all reads of this qname // because we saw both first and second in pair hit same split // 2023 - turning this off -- why? Beacuse for very short // insert size distributions, this can still be possible //reject_qnames.insert(qn); } else { // if haven't seen this read, add here. If have, then dont because want to avoid re-setting first/second convention // e.g. if we see a split read with first designation, we want to reject all reads with same qname if at any time // we see one with second mate designation. If we don't have this conditional, we can get fooled if the order is // 1, 2, 1 if (!qname_and_num.count(qn)) qname_and_num[qn] = j->FirstFlag(); // this is a valid read this_r2c.supports_var = true; valid_reads.insert(sr); SVABA_TRACE(cname, "TP10+ CREDITED read=" << sr << " sample=" << sample_id << " tumor=" << j->Tumor()); SVABA_READ_TRACE(j->Qname(), "SPLIT_COV CREDITED as variant supporter" << " contig=" << cname << " sample=" << sample_id << " tumor=" << j->Tumor() << " issplit1=" << issplit1 << " issplit2=" << issplit2); // how much of the contig do these span // for a given read QNAME, get the coverage that split_cov_bounds.first = std::min(split_cov_bounds.first, pos); split_cov_bounds.second = std::max(split_cov_bounds.second, te); } } // update the counters for each break end if (issplit1 && valid) ++b1.split[sample_id]; if (issplit2 && valid) ++b2.split[sample_id]; // add r2c back, but as amended //j.AddR2C(cname, this_r2c); } // end read loop // process valid reads for (auto& i : bav) { r2c this_r2c = i->GetR2C(cname); if (valid_reads.count(i->UniqueName())) { std::string qn = i->Qname(); if (qnames.count(qn)) continue; // don't count support if already added and not a short event // check that it's not a bad 1, 2 split if (reject_qnames.count(qn)) { this_r2c.supports_var = false; i->AddR2C(cname, this_r2c); // update that this actually does not support continue; } reads.push_back(i); // keep track of qnames of split reads qnames.insert(qn); auto prefix = i->Prefix(); if (auto it = allele.find(prefix); it != allele.end()) { auto& sample = it->second; sample.supporting_reads.insert(i->UniqueName()); ++sample.split; } else { throw std::runtime_error("SampleInfo not instantiated with this bam prefix: " + prefix); } } } // adjust the alt count for (auto& [_,al] : allele) { al.UpdateAltCounts(); } #ifdef SVABA_TRACING { int total_split = 0; for (const auto& [pref,al] : allele) total_split += al.split; SVABA_TRACE(cname, "TP_SPLIT_SUMMARY total_split=" << total_split << " reads.size=" << reads.size() << " valid_reads=" << valid_reads.size() << " per_sample:"); for (const auto& [pref,al] : allele) { SVABA_TRACE(cname, " " << pref << ": split=" << al.split << " alt=" << al.alt << " cov=" << al.cov); } } #endif } void BreakPoint::checkBlacklist(GRC &grv) { if (grv.CountOverlaps(b1.gr) || grv.CountOverlaps(b2.gr)) { confidence = "BLACKLIST"; SVABA_TRACE(cname, "TP14 BLACKLIST hit"); } } void BreakPoint::set_homologies_insertions() { try { if (b1.cpos > b2.cpos) homology = seq.substr(b2.cpos, b1.cpos-b2.cpos); else if (b2.cpos > b1.cpos) insertion = seq.substr(b1.cpos, b2.cpos-b1.cpos); //if (insertion.length() == 0) // ;//insertion = "x"; //if (homology.length() == 0) //;//homology = "x"; } catch (...) { std::cerr << "cname: " << cname << " b1.cpos " << b1.cpos << " b2.cpos " << b2.cpos << " seq.length " << seq.length() << std::endl; std::cerr << "Caught error with contig on global-getBreakPairs: " << cname << std::endl; std::cerr << b1.cpos << " " << b2.cpos << " seq.length() " << seq.length() << " num_align " << num_align << std::endl; } } // isleft - this is the left-sided AlignmentFragment, so take the right break // as the breakpoint (and vice versa) void BreakEnd::transferContigAlignmentData(const AlignmentFragment* f, bool isleft) { const BamRecordPtr &r = f->m_align; // get number of suboptimal alignments r->GetIntTag("XS", sub); // sub could be 0 // get number of !SNV! mismatches // this is a svaba internal trick, since we are interested in // indels, so just track number of point mutation differences r->GetIntTag("NM", nm); assert(nm >= 0); nm = std::max(nm - static_cast(r->MaxInsertionBases()) - static_cast(r->MaxDeletionBases()), 0); // get alignment score r->GetIntTag("AS", as); assert(as >= 0); // SvABA2.0: pull the contig-alignment confidence tag. If `zc` wasn't // written recompute // from the record directly so behavior is consistent either way. double cc = svaba::readContigConfTag(*r); if (cc < 0.0) cc = svaba::scoreContigAlignment(*r).confidence; contig_conf = cc; // transfer mapq and match length mapq = r->MapQuality(); matchlen = r->NumMatchBases(); // set the coordinates as right-most (for an isleft=true fragment) // or left-most (for an isleft = false fragment) if (isleft) { gr = GenomicRegion(r->ChrID(), f->gbreak2, f->gbreak2); gr.strand = r->ReverseFlag() ? '-' : '+'; cpos = f->break2; } else { gr = GenomicRegion(r->ChrID(), f->gbreak1, f->gbreak1); gr.strand = r->ReverseFlag() ? '+' : '-'; cpos = f->break1; } } // construct an indel BreakPoint BreakPoint::BreakPoint(const AlignmentFragment* f, const int idx, const SvabaSharedConfig* _sc) : sc(_sc) { svtype = SVType::INDEL; // instantiate the SampleInfo objects for each BAM for (auto const& p : sc->prefixes) { allele.try_emplace(p); } b1.transferContigAlignmentData(f, true); b2.transferContigAlignmentData(f, true); // re-zero the positions, not set yet b1.gr.pos1 = b1.gr.pos2 = -1; b2.gr.pos1 = b2.gr.pos2 = -1; // this alignment fragemnt underlying alignment const BamRecordPtr &r = f->m_align; // SvABA2.0: record the flip-convention of this fragment so that // b1.cpos/b2.cpos (which are computed from the un-flipped BAM CIGAR below, // i.e. in BAM / genome-forward orientation) can be converted to m_seq / // r2c (assembly-native) orientation via BreakPoint::cpos_on_m_seq() at // the sites that need it (split-coverage counting and alignments.txt // rendering). This does NOT change the arithmetic done against // r->Sequence() in this constructor (insertion, homology, repeat_seq), // which legitimately lives in BAM/genome-forward coordinates. flipped_on_contig = f->flipped; contig_len = static_cast(r->Sequence().length()); // assign the contig-wide properties cname = r->Qname(); seq = r->Sequence(); assert(cname.length()); assert(seq.length()); num_align = 1; b1.gr.strand = '+'; // always the case for indels b2.gr.strand = '-'; // set seconary flag secondary = r->SecondaryFlag(); int curr = 0; // current position on the contig (starting at zero) int gcurrlen = 0; // current position on the genome (starting at zero) //debugprint // if (cname == "c_1_49001_74001_14C") // std::cerr << "...constructing... " << idx << // " m_align " << *r << " flipped " << f->flipped << std::endl; //NB: curr is the counter for number of based traversed on the contig // this is actually 1-based in how we use it, since e.g. if we have // a cigar like 1M... then right away curr is 1 and we are looking at the // first based of the contig. A bit confusing since C is 0-based, but // this is effectively how this is used below. 1-based is good since // this is what we want the output to be for VCF / SAM spec // NB: contig position and genome position internally are 0-based // similar to HTSlib size_t count = 0; // count to make sure we are reporting the right indel // NB: don't worry about "flip"" stuff from AlignedContig, // that's only important for comparing across different alignments/ // Since we are parsing indels from a single alignment, which is always // referenced to the forward strand, just go with the raw BamRecord data for (auto& i : f->m_align->GetCigar()) { // update the left match side if (i.Type() == 'M' && count < idx) left_match += i.Length(); // update the right match side if (i.Type() == 'M' && count > idx) right_match += i.Length(); // std::cerr << "LM " << left_match << " RM " << right_match << " count " << // count << " idx " << idx << std::endl; //debug ////// // set the breakpoint positions on the contig ////// // for either insertion or deletion, first base is the one before the // actual variant // NB: recall that curr is the number of bases thus far // that have consumed the contig/query e.g. for 140M5D, curr will be 140 since // the 5D does not consume the query/contig. So in a 0-based system like // we use here internally to be consistent with htslib and C-rules, // we add a -1 so that we count the last base of the contig before the deletion // (e.g. the 140th base, or position 139 in 0-based system) as the first // base on the contig for the deletion (or insertion). if (count == idx && (i.Type() == 'D' || i.Type() == 'I')) { b1.cpos = curr - 1; // 0-based position of first base before the deletion/insertion } // if deletion if (i.Type() == 'D' && count == idx) { b2.cpos = curr; // 0-based position of first base after the deletion } // if insertion if (i.Type() == 'I' && count == idx) { b2.cpos = b1.cpos + i.Length(); // here, we add the +1 to start so that the actual insertion sequence is // at the first base of the insertion. For VCF reporting in the ALT // column, I'll add the leading reference base later // e.g. for a 2 bp insertion, insertion becomes e.g. AG while // in the ALT column of the VCF/bps it is GAG (if G is last reference match) insertion = f->m_align->Sequence().substr(b1.cpos + 1, i.Length()); } // update the contig position traversal -- consumes query // M,I,S if (i.ConsumesQuery()) curr += i.Length(); ////// // set the breakpoint positions on the genome ////// // set the genome breakpoint if (b1.cpos >= 0 && count == idx) { if (i.Type() != 'I' && i.Type() != 'D') throw std::runtime_error("BreakPoint indel parsing - unexpected to get non del/ins CIGAR field"); // here again the -1 is to get htslib position 0-based to SAM format (1-based), since e.g. for // 140M5D, we want the genome position of the start based to be the first // matching base before the deletion, so again have to -1 b1.gr.pos1 = r->Position() + gcurrlen - 1; // for deletions, it's the opposite to contig, so deletion consumes the genome if (i.Type() == 'D') b2.gr.pos1 = b1.gr.pos1 + i.Length(); if (i.Type() == 'I') b2.gr.pos1 = b1.gr.pos1 + 1; } // update the position on the genome // D,M if (i.ConsumesReference()) { gcurrlen += i.Length(); } // if (cname == "c_1_122501_141530_11C") { // std::cerr << " count " << count << " curr " << curr << " gcurr " << gcurrlen << // " C " << i << " insertion " << insertion << " cname " << cname << std::endl; // std::cerr << f->m_seq << std::endl; // std::cerr << "FIPPED " << f->flipped << std::endl; // std::cerr << " b1.gr " << b1.gr << " b2.gr " << b2.gr << std::endl; // } ++count; } // end cigar loop // set the dummy other end b1.gr.pos2 = b1.gr.pos1; b2.gr.pos2 = b2.gr.pos1; // should have been explicitly ordered in the creation above if (!(b1.gr < b2.gr)) { std::cerr << "B1 " << b1.gr << " - " << b2.gr << " " << cname << " b1.cpos " << b1.cpos << " b2.cpos " << b2.cpos << std::endl; std::cerr << f->printToAlignmentsFile() << std::endl; throw std::runtime_error("invalid BreakPoint creation in indels in AlignmentFragment"); } //// add the repeat sequence const std::string& seq = f->m_align->Sequence(); const int REPBUFF = 3; for (const auto& [start, end, rep] : svabaUtils::find_long_homopolymers(seq)) { bool crosses_breakpoint = start < (b1.cpos + REPBUFF) && (b2.cpos - REPBUFF) < end; if (rep.length() > repeat_seq.length() && crosses_breakpoint) repeat_seq = rep; } for (const auto& [start, end, rep] : svabaUtils::find_long_dinuc_repeats(seq)) { bool crosses_breakpoint = start < (b1.cpos + REPBUFF) && (b2.cpos - REPBUFF) < end; if (rep.length() > repeat_seq.length() && crosses_breakpoint) repeat_seq = rep; } /////////// } void BreakPoint::CombineWithDiscordantClusterMap(DiscordantClusterMap& dmap) { // don't operate on indels if (svtype == SVType::INDEL) return; // since discordant clusters are ordered with the // m_reg1 being more left on the genome, we want to // match that convention for the breakpoint. However, // we can't actually change the bp1 and bp2 ordering // since this is set by the alignment orderings on the contig // not on the genome. So can make a dummy swap just for purposes of // finding the matching discorantcluster // on the GENOME const int PAD = 100; GenomicRegion bp1 = (b1.gr < b2.gr) ? b1.gr : b2.gr; GenomicRegion bp2 = (b1.gr < b2.gr) ? b2.gr : b1.gr; bp1.Pad(PAD); bp2.Pad(PAD); assert(! (bp1 > bp2) ); for (auto& [_,d] : dmap) { // checks basic things about discordant cluster if (!d.valid()) continue; assert(d.m_reg1 < d.m_reg2); // do either breakends overlap bool bp1reg1 = bp1.GetOverlap(d.m_reg1) > 0; bool bp2reg2 = bp2.GetOverlap(d.m_reg2) > 0; bool s1 = bp1.strand == d.m_reg1.strand; bool s2 = bp2.strand == d.m_reg2.strand; // get the edge of the cluster // if its a forward alignment discordant read, then get "right" most // if its a reverse alignment discordant read, then get "left" most int pos1 = d.m_reg1.strand == '+' ? d.m_reg1.pos2 : d.m_reg1.pos1; int pos2 = d.m_reg2.strand == '+' ? d.m_reg2.pos2 : d.m_reg2.pos1; // both sides overlap and both orientions agree bool pass = bp1reg1 && bp2reg2 && s1 && s2; // check that the ends are not way off // if (std::abs(pos1 - b1.gr.pos1) > PAD || // std::abs(pos2 - b2.gr.pos1) > PAD) // pass = false; // std::cerr << b1.gr << " - " << b2.gr << std::endl; // std::cerr << " cname " << cname << " pad " << PAD << " pass " << pass << " DC pos1 " << pos1 << " DC pos2 " << pos2 << // " s1 " << s1 << " s2 " << s2 << " bp1reg1 " << bp1reg1 << " bp2reg2 " << bp2reg2 << " diff1 " << std::abs(pos1 - b1.gr.pos1) << // " diff2 " << std::abs(pos2 - b2.gr.pos1) << " bp pos1 " << b1.gr.pos1 << " bp pos2 " << b2.gr.pos1 << " " << // d.toFileString(sc->header, false) << std::endl; if (!pass) continue; // check that we haven't already added a cluster to this breakpoint // if so, chose the one with more normal support first, then more // tumor support second if (dc.isEmpty() || (dc.ncount < d.ncount) || (dc.ncount==d.ncount && dc.tcount < d.tcount)) { dc = d; d.m_contig = cname; // add the read counts from DiscordantCluster to this BreakPoint // the DiscordantCluster "counts" are created at DiscordantCluster constructor assert(!d.counts.empty()); for (auto& c : d.counts) { allele.at(c.first).disc = c.second; } // add the discordant reads names to supporting reads for each sampleinfo // reads for (auto& [_, read] : d.reads) { allele.at(read->Prefix()) .supporting_reads .insert(read->UniqueName()); } // mates (and fix the typo in your assert) for (auto& [_, mate] : d.mates) { allele.at(mate->Prefix()) .supporting_reads .insert(mate->UniqueName()); } // update the variant support read counts per bam // (i.e. per SampleInfo object in our BreakPoint "alleles" structure) for (auto& [_,al] : allele) al.UpdateAltCounts(); } } // make sure not claiming discordant + assembly for small spans if ( (getSpan() < 2 * sc->insertsize && getSpan() >= 0) && b1.gr.strand == '+' && b2.gr.strand == '-') svtype = SVType::ASSMB; else svtype = SVType::ASDIS; } // void BreakPoint::set_evidence() { // // if we are in refilter, then this is already set // if (!evidence.empty()) // return; // bool isdisc = (dc.tcount + dc.ncount) != 0; // if (num_align == 1) // evidence = "INDEL"; // else if ( isdisc && svtype == SVType::REARRANGEMENT) //!complex && num_align > 0) // evidence = "ASDIS"; // else if ( isdisc && num_align < 3) // evidence = "DSCRD"; // else if (svtype == SVType::REARRANGEMENT) // evidence = "ASSMB"; // else if (svtype == SVType::TSI_GLOBAL) ///complex && !complex_local) // is A-C of an ABC // evidence = "TSI_G"; // else if (svtype == SVType::TSI_LOCAL) // //else if (complex && complex_local) // is AB or BC of an ABC // evidence = "TSI_L"; // assert(evidence.length()); // } void BreakPoint::score_assembly_only() { assert(local != LocalAlignment::NOTSET); assert(secondary != -1); int span = getSpan(); int num_split = t.split + n.split; int cov_span = split_cov_bounds.second - split_cov_bounds.first ; // check for high repeats // bool hi_rep = false; // for (auto& rr : hirepr) // if (seq.find(rr) != std::string::npos) // hi_rep = true; float as_frac1 = static_cast(b1.as) / static_cast(b1.matchlen); float sub_frac1 = static_cast(b1.sub) / static_cast(b1.matchlen); float as_frac2 = static_cast(b2.as) / static_cast(b2.matchlen); float sub_frac2 = static_cast(b2.sub) / static_cast(b2.matchlen); const double min_cc = std::min(b1.contig_conf, b2.contig_conf); if (local == LocalAlignment::FROM_DISTANT_REGION && svtype != SVType::TSI_LOCAL) // added this back in v71 // issue is that if a read is secondary aligned, it could be // aligned to way off region. Saw cases where this happend in tumor // and not normal, so false-called germline event as somatic. confidence = "NOLOCAL"; else if (local == LocalAlignment::NONVAR_LOCAL_REALIGNMENT) confidence = "LOCALMATCH"; else if ( num_split > 1 && ( (cov_span <= (sc->readlen + 5 ) && cov_span > 0) || cov_span < 0) ) confidence = "DUPREADS"; // the same sequences keep covering the split else if (homology.length() >= 20 && (span > 1500 || span == -1) && std::max(b1.mapq, b2.mapq) < 60) confidence = "NODISC"; else if ((int)seq.length() < sc->readlen + 30) confidence = "TOOSHORT"; else if (a.split < 7 && (span > 1500 || span == -1)) // large and inter chrom need 7+ confidence = "NODISC"; else if (std::max(b1.mapq, b2.mapq) <= 40 || std::min(b1.mapq, b2.mapq) <= 10) confidence = "LOWMAPQ"; else if ( std::min(b1.mapq, b2.mapq) <= 30 && a.split <= 8 ) confidence = "LOWMAPQ"; else if (std::max(b1.nm, b2.nm) >= 10 || std::min(as_frac1, as_frac2) < 0.8) confidence = "LOWAS"; else if ( (std::max(b1.nm, b2.nm) >= 3 || std::min(as_frac1, as_frac2) < 0.85) && getSpan() < 0 ) confidence = "LOWAS"; //else if ((double)aligned_covered / (double)seq.length() < 0.80) // less than 80% of read is covered by some alignment // confidence = "LOWAS"; else if ( (b1.matchlen < 50 && b1.mapq < 60) || (b2.matchlen < 50 && b2.mapq < 60) ) confidence = "LOWMAPQ"; else if ( std::min(b1.nm, b2.nm) >= 10) confidence = "LOWMAPQ"; else if (a.split <= 3 && span <= 1500 && span != -1) // small with little split confidence = "LOWSPLITSMALL"; else if (b1.gr.chr != b2.gr.chr && std::min(b1.matchlen, b2.matchlen) < 60) // inter-chr, but no disc reads, weird alignment confidence = "LOWICSUPPORT"; else if (b1.gr.chr != b2.gr.chr && std::max(b1.nm, b2.nm) >= 3 && std::min(b1.matchlen, b2.matchlen) < 150) // inter-chr, but no disc reads, and too many nm confidence = "LOWICSUPPORT"; else if (std::min(b1.matchlen, b2.matchlen) < 0.6 * sc->readlen) confidence = "LOWICSUPPORT"; else if (std::min(b1.mapq, b2.mapq) < 50 && b1.gr.chr != b2.gr.chr) // interchr need good mapq for assembly only confidence = "LOWMAPQ"; else if (std::min(b1.matchlen, b2.matchlen) < 40 || (svtype == SVType::TSI_LOCAL && std::min(b1.matchlen, b2.matchlen) < 100)) // not enough evidence confidence = "LOWMATCHLEN"; else if (std::min(b1.matchlen - homology.length(), b2.matchlen - homology.length()) < 40) confidence = "LOWMATCHLEN"; else if ((/*b1.sub_n && */b1.mapq < 30) || (/*b2.sub_n && */b2.mapq < 30)) confidence = "LOWMAPQ"; else if (secondary && std::min(b1.mapq, b2.mapq) < 30) confidence = "SECONDARY"; // else if ((repeat_seq.length() >= 10 && std::max(t.split, n.split) < 7) || hi_rep) // confidence = "WEAKSUPPORTHIREP"; else if (num_split < 6 && getSpan() < 300 && b1.gr.strand==b2.gr.strand) confidence = "LOWQINVERSION"; // else if ( (b1.matchlen - b1.simple < 15 || b2.matchlen - b2.simple < 15) ) // confidence = "SIMPLESEQUENCE"; else if ((int)homology.length() * HOMOLOGY_FACTOR > sc->readlen) // if homology is too high, tough to tell from mis-assemly confidence = "HIGHHOMOLOGY"; else if (min_cc < svaba::kContigConfPassThreshold) confidence = "WEAKCONTIG"; else confidence = "PASS"; assert(confidence.length()); } void BreakPoint::score_somatic(double error_fwd) { // this is LOD of normal being REF vs AF = 0.5+ // We want this to be high for a somatic call // NB: this is almost the flip of LOD alt vs ref. // where high log-odds is more likely there is // a variant. It makes sense, you always think of // log-odds as "higher-is-better", but are asking // two different things: // LO - is this variant versus ref // LO_n - is this ref *in the normal* = somatic // this is a fully Bayesian approach -- if all normals strongly support ref, the sum is huge. // Cons: one bad normal (low or negative LO_n) can be overwhelmed by many good normals. // Commented out since favor conservative approach // double somatic_lod = 0.0; // for (const auto& [pref, al] : allele) { // if (pref[0] == 'n') { // somatic_lod += al.LO_n; // } //} // this is a more conservative approach - if any normal BAM shows evidence for // variant, then get the somatic score from that int thiscov_n = n.cov; if (n.alt >= n.cov) thiscov_n = n.alt; int thiscov_t = t.cov; if (t.alt >= t.cov) thiscov_t = t.alt; // now to be conservative, also count normal near-cigar matches // as alt reads int normal_cigar = n.cigar + n.cigar_near; // adjust the alt count if (n.alt < normal_cigar) n.alt = normal_cigar; if (t.alt < t.split) t.alt = t.split; if (t.alt < t.cigar) t.alt = t.cigar; if (t.alt < t.split) t.alt = t.split; double a_cov_n = (double)thiscov_n * (double)(sc->readlen - 2 * T_SPLIT_BUFF)/sc->readlen; double a_cov_t = (double)thiscov_t * (double)(sc->readlen - 2 * T_SPLIT_BUFF)/sc->readlen; double scaled_alt_n = std::min((double)n.alt, a_cov_n); double scaled_ref_n = a_cov_n- scaled_alt_n; double scaled_alt_t = std::min((double)t.alt, a_cov_t); double scaled_ref_t = a_cov_t- scaled_alt_t; double error_rev = 1e-6; LO_s = SvabaModels::SomaticLOD(scaled_alt_n, a_cov_n, scaled_alt_t, a_cov_t, error_fwd, error_rev); if (false) std::cerr << std::fixed << std::setprecision(6) << "[DEBUG] somatic_lod=" << LO_s << " | n.cov=" << n.cov << " | n.alt=" << n.alt << " | n.cigar=" << n.cigar << " | t.cov=" << t.cov << " | t.alt=" << t.alt << " | t.cigar=" << t.cigar << " | t.split=" << t.split << " | thiscov_n=" << thiscov_n << " | thiscov_t=" << thiscov_t << " | readlen=" << sc->readlen << " | T_SPLIT_BUFF=" << T_SPLIT_BUFF << " | a_cov_n=" << a_cov_n << " | a_cov_t=" << a_cov_t << " | scaled_alt_n=" << scaled_alt_n << " | scaled_ref_n=" << scaled_ref_n << " | scaled_alt_t=" << scaled_alt_t << " | scaled_ref_t=" << scaled_ref_t << " | error_fwd=" << error_fwd << " | error_rev=" << error_rev << " | somatic_LOD=" << LO_s << std::endl; /*for (const auto& [pref, al] : allele) { if (pref[0] == 'n') { LO_s = std::min(somatic_lod, al.LO_n); } }*/ // find the somatic to normal ratio double ratio = n.alt > 0 ? (double)t.alt / (double)n.alt : 100; if (svtype == SVType::INDEL) { // somatic score is just true or false for now // use the specified cutoff for indels, taking into account whether at dbsnp site double cutoff = (rs.empty() || rs=="x") ? sc->opts.lodSomatic : sc->opts.lodSomaticDb; somatic = LO_s > cutoff ? SomaticState::SOMATIC_LOD : SomaticState::NORMAL_LOD; // can't call somatic with 5+ normal reads or <5x more tum than norm ALT //if ((ratio <= 12 && n.cov > 10) || n.alt > 5) //if (n.alt > 5) // for SVs, use LOD and then also a hard cutoff // for gauging somatic vs germline } else { // passes somatic = (LO_s > sc->opts.lodSomatic) ? SomaticState::SOMATIC_LOD : SomaticState::NORMAL_LOD; // require no reads in normal or MAX 1 read and tons of tumor reads if (ratio < MIN_SOMATIC_RATIO || n.split > 2 && dc.ncount > 1) somatic = SomaticState::FAILED; } // set germline if single normal read in discordant clsuter if (svtype == SVType::DSCRD && n.alt > 0 && somatic == SomaticState::SOMATIC_LOD) somatic = SomaticState::FAILED; } void BreakPoint::score_assembly_dscrd() { int this_mapq1 = b1.mapq; int this_mapq2 = b2.mapq; int span = getSpan(); bool germ = dc.ncount > 0 || n.split > 0; int max_a_mapq = std::max(this_mapq1, dc.mapq1); int max_b_mapq = std::max(this_mapq2, dc.mapq2); const double min_cc = std::min(b1.contig_conf, b2.contig_conf); // how much of contig is covered by split reads int cov_span = split_cov_bounds.second - split_cov_bounds.first ; // set the total number of supporting reads for tumor / normal // these alt counts should already be one qname per alt (ie no dupes) int t_reads = 0; int n_reads = 0; for (auto& [pref,al] : allele) { if (pref.at(0) == 't') t_reads += al.alt; else n_reads += al.alt; } int total_count = t_reads + n_reads; //n.split + t.split + dc.ncount + dc.tcount; int disc_count = dc.tcount + dc.ncount; //int hq = dc.tcount_hq + dc.ncount_hq; if ( (max_a_mapq < 30 && b1.local == LocalAlignment::FROM_DISTANT_REGION) || (max_b_mapq < 30 && b2.local == LocalAlignment::FROM_DISTANT_REGION) || (/*b1.sub_n > 7 && */b1.mapq < 10 && b1.local == LocalAlignment::FROM_DISTANT_REGION) || (/*b2.sub_n > 7 && */b2.mapq < 10 && b2.local == LocalAlignment::FROM_DISTANT_REGION) ) confidence = "LOWMAPQ"; else if ( std::min(b1.nm, b2.nm) >= 10) confidence = "LOWMAPQ"; else if ( std::min(b1.mapq, b2.mapq) < 10/* && std::min(dc.mapq1, dc.mapq2) < 10 */) confidence = "LOWMAPQ"; else if ( total_count < 4 || (std::max(t.split, n.split) <= 5 && cov_span < (sc->readlen + 5) && disc_count < 7) ) confidence = "LOWSUPPORT"; else if ( total_count < 15 && germ && span == -1) // be super strict about germline interchrom confidence = "LOWSUPPORT"; else if ( std::min(b1.matchlen, b2.matchlen) < 50 && b1.gr.chr != b2.gr.chr ) confidence = "LOWICSUPPORT"; else if (secondary && getSpan() < 1000) // local alignments are more likely to be false for alignemnts with secondary mappings confidence = "SECONDARY"; /*else if (dc.tcount_hq + dc.ncount_hq < 3) { // multimathces are bad if we don't have good disc support too if ( ((b1.sub_n && dc.mapq1 < 1) || (b2.sub_n && dc.mapq2 < 1)) ) confidence = "MULTIMATCH"; else if ( ( (secondary || b1.sub_n > 1) && !b1.local) && ( std::min(max_a_mapq, max_b_mapq) < 30 || std::max(dc.tcount, dc.ncount) < 10)) confidence = "SECONDARY"; else confidence = "PASS"; }*/ else if (min_cc < svaba::kContigConfPassThreshold) confidence = "WEAKCONTIG"; else confidence = "PASS"; } void BreakPoint::score_dscrd() { t.alt = dc.tcount; n.alt = dc.ncount; int nm_count = std::max(dc.nm1, dc.nm2); int disc_count = dc.ncount + dc.tcount; //int hq_disc_count = dc.ncount_hq + dc.tcount_hq; int disc_cutoff = 8; //int hq_disc_cutoff = disc_count >= 10 ? 3 : 5; // reads with both pair-mates have high MAPQ const int min_dscrd_size = 2000; if (getSpan() > 0 && (getSpan() < min_dscrd_size && b1.gr.strand == '+' && b2.gr.strand == '-')) // restrict span for del (FR) type confidence = "LOWSPANDSCRD"; else if ((disc_count < disc_cutoff || std::min(dc.mapq1, dc.mapq2) < 15)) confidence = "LOWMAPQDISC"; else if (nm_count >= 4) confidence = "HIGHNM"; else if (!dc.m_id_competing.empty()) confidence = "COMPETEDISC"; else if ( disc_count < disc_cutoff) confidence = "WEAKDISC"; else confidence = "PASS"; assert(confidence.length()); } void BreakPoint::score_indel() { assert(b1.mapq == b2.mapq); bool is_refilter = !confidence.empty(); // act differently if this is refilter run // for refilter, only consider ones that were low lod or PASS // ie ones that with a different lod threshold may be changed // if confidence is empty, this is original run so keep going if (confidence != "LOWLOD" && confidence != "PASS" && is_refilter) return; double max_lod = 0; for (const auto& [_,al] : allele) max_lod = std::max(max_lod, al.LO); const double min_cc = std::min(b1.contig_conf, b2.contig_conf); // check if homozygous reference is most likely GT // bool homozygous_ref = true; // for (auto& [_,al] : allele) { // if (al.genotype_likelihoods[0] > al.genotype_likelihoods[1] || // al.genotype_likelihoods[0] > al.genotype_likelihoods[2]) // homozygous_ref = false; // } // get the allelic ractions, just for VLOWAF filter double af_t = t.cov > 0 ? (double)t.alt / (double)t.cov : 0; double af_n = n.cov > 0 ? (double)n.alt / (double)n.cov : 0; double af = std::max(af_t, af_n); assert(b1.local != LocalAlignment::NOTSET); if (b1.local != LocalAlignment::FROM_LOCAL_REGION) confidence="NOLOCAL"; if (b1.mapq < 10) confidence="LOWMAPQ"; //else if (!is_refilter && (double)aligned_covered / (double)seq.length() < 0.80) // less than 80% of read is covered by some alignment // confidence = "LOWAS"; else if ((/*b1.sub_n && */b1.mapq < 30) || (/*b2.sub_n && */b2.mapq < 30)) confidence = "LOWMAPQ"; else if (max_lod < sc->opts.lod && rs.empty()) // non db snp site confidence = "LOWLOD"; else if (max_lod < sc->opts.lodDb && !rs.empty()) // be more permissive for dbsnp site confidence = "LOWLOD"; else if (!is_refilter && std::min(left_match, right_match) < 20) confidence = "SHORTALIGNMENT"; // no conf in indel if match on either side is too small else if (min_cc < svaba::kContigConfPassThreshold) confidence = "WEAKCONTIG"; else confidence="PASS"; SVABA_TRACE(cname, "TP16 score_indel: confidence=" << confidence << " mapq=" << b1.mapq << " max_lod=" << max_lod << " left_match=" << left_match << " right_match=" << right_match << " min_cc=" << min_cc << " af_t=" << af_t << " af_n=" << af_n << " t.split=" << t.split << " n.split=" << n.split << " t.alt=" << t.alt << " t.cov=" << t.cov << " n.alt=" << n.alt << " n.cov=" << n.cov); for (const auto& [pref,al] : allele) { SVABA_TRACE(cname, " " << pref << ": LO=" << al.LO << " split=" << al.split << " alt=" << al.alt << " cov=" << al.cov); } //debugprint // std::cerr <<" MAPQ " << b1.mapq << " b1.sub_n " << b1.sub_n << // " b2.sub_n " << b2.sub_n << " max_lod " << max_lod << " af " << af << " is_refilter " << is_refilter << // std::endl; // for (const auto& [pref,al] : allele) { // std::cerr << " LOD SCORES " << pref << " - " << al.LO << std::endl; // std::cerr << " PL likelihood 0/0 " << al.phred_likelihoods[0] << // " PL likelihood 0/1 " << al.phred_likelihoods[1] << // " PL likelihood 1/1 " << al.phred_likelihoods[2] << std::endl; // } // std::cerr << "AF T " << af_t << " AF N " << af_n << " AF " << af << std::endl; // std::cerr << " confidene " << confidence << std::endl; } // LOD cutoff is just whether this is ref / alt // LOD cutoff dbsnp is same, but at DBSNP site (should be lower threshold since we have prior) // LOD SOM cutoff is cutoff for whether NORMAL BAM(s) are AF = 0 // LOD SOM DBSNP cutoff same as above, but at DBSNP site (should be HIGHER threshold, // since we have prior that it's NOT somatic void BreakPoint::scoreBreakpoint() { // some sanity checking assert(svtype != SVType::NOTSET); if (!confidence.empty() && confidence != "BLACKLIST") throw std::runtime_error("BreakPoint confidence already set (unexpected)"); // set the error rate // depends on teh repeat context double error_rate = (repeat_seq.length() > 10) ? MAX_ERROR : ERROR_RATES[repeat_seq.length()]; // first calculate log-odds and genotype likelihoods for each individual bam std::vector lods; for (auto& [pref,al] : allele) { al.modelSelection(error_rate, sc->readlen); lods.push_back(al.LO); } ///// // Calculate a site-level quality score ///// // Sum the LODs across all your BAMs (assuming independence) to get a combined log-odds double lod_site = std::accumulate(lods.begin(), lods.end(), 0.0); // Convert that back to an error probability double p_err = 1.0 / (1.0 + std::pow(10.0, lod_site)); // Phred-scale (-log10) to get a QUAL score int qual_calc = static_cast(std::lround(-10.0 * std::log10(p_err))); // Cap at 99 quality = std::min({qual_calc, 99}); // combine the all tumor bam support and normal bam support for (auto& [pref, al] : allele) { if (pref.at(0) == 't') { t = t + al; } else { n = n + al; } } // combine all read support regardless of tumor / normal a = t + n; // std::cerr << " T MODEL " << error_rate << std::endl; // t.modelSelection(error_rate, sc->readlen); // std::cerr << " N MODEL " << error_rate << std::endl; // n.modelSelection(error_rate, sc->readlen); // kludge. make sure we have included the DC counts (should have done this arleady...) if (svtype == SVType::DSCRD || svtype == SVType::ASDIS) { t.disc = dc.tcount; n.disc = dc.ncount; } // provide a scaled LOD that accounts for MAPQ. Heuristic, not really used //int mapqr1 = b1.local ? std::max(30, b1.mapq) : b1.mapq; // if local, don't drop below 30 //int mapqr2 = b2.local ? std::max(30, b2.mapq) : b2.mapq; // if local (aligns to within window), don't drop below 30 //double scale = (double)( std::min(mapqr1, mapqr2) - 2 * b1.nm) / (double)60; //for (auto& i : allele) // i.second.SLO = i.second.LO * scale; //t.SLO = t.LO * scale; //n.SLO = n.LO * scale; //a.SLO = a.LO * scale; // // sanity check // int split =0; // for (auto& i : allele) // split += i.second.split; // assert( (split == 0 && t.split == 0 && n.split==0) || // (split > 0 && (t.split + n.split > 0))); SVABA_TRACE(cname, "TP13 scoreBreakpoint: svtype=" << (int)svtype << " confidence=" << confidence << " t.split=" << t.split << " n.split=" << n.split << " dc.t=" << dc.tcount << " dc.n=" << dc.ncount << " quality=" << quality); // if already overlapping blacklist, then we're done if (confidence == "BLACKLIST") return; // do the scoring bool iscomplex = svtype == SVType::TSI_LOCAL || svtype == SVType::TSI_GLOBAL; // score rearrangements of various kinds if (svtype == SVType::ASSMB || (iscomplex && (dc.ncount + dc.tcount)==0)) score_assembly_only(); else if (svtype == SVType::ASDIS || (iscomplex && (dc.ncount + dc.tcount))) score_assembly_dscrd(); else if (svtype == SVType::DSCRD) score_dscrd(); // it failed assembly filters, but might pass discordant filters // if (evidence == "ASDIS" && confidence != "PASS") { // evidence = "DSCRD"; // score_dscrd(min_dscrd_size); // } else if (svtype == SVType::INDEL) score_indel(); else throw std::runtime_error("no scoring method called in BreakPoint::score_breakpoints"); // set the somatic_score field to true or false score_somatic(error_rate); // quality score is odds that read is // non-homozygous reference (max 99) // quality = 0; // for (auto& a : allele) // quality = std::max(a.second.NH_GQ, (double)quality); } void BreakPoint::setRefAlt(const RefGenome* main_rg, const BamHeader& header) { assert(!main_rg->IsEmpty()); assert(ref.empty()); assert(alt.empty()); assert(svtype != SVType::NOTSET); if (svtype != SVType::INDEL) { try { // get the reference for BP1 ref = main_rg->QueryRegion(b1.gr.ChrName(header), b1.gr.pos1, b1.gr.pos1); } catch (const std::invalid_argument& ia) {} try { alt = main_rg->QueryRegion(b2.gr.ChrName(header), b2.gr.pos1, b2.gr.pos1); } catch (const std::invalid_argument& ia) {} } else { // insertion if (!insertion.empty()) { try { // we store b1.gr internally as zero-indexed, // to be consistent with htslib, so since this passed // to htslib query, keep as zero-indexed ref = main_rg->QueryRegion(b1.gr.ChrName(header), b1.gr.pos1, b1.gr.pos1); } catch (const std::invalid_argument& ia) { ref = "N"; std::cerr << "Caught exception in BreakPoint:setRefAlt for indel ref: " << ia.what() << std::endl; } // alt alt = ref + insertion; // deletion } else { // reference assert(b2.gr.pos1 - b1.gr.pos1 - 1 >= 0); try { // again, 0-based, see above comment ref = main_rg->QueryRegion(b1.gr.ChrName(header), b1.gr.pos1, b2.gr.pos2); // if (cname == "c_1_49001_74001_14C") // std::cerr << " REF : " << ref << " b1 " << b1.gr << // " b2 " << b2.gr << " cname " << cname << std::endl; } catch (const std::invalid_argument& ia) { ref = std::string(std::abs(b1.gr.pos1 - b2.gr.pos1), 'N'); std::cerr << "Caught exception in BreakPoint:setRefAlt for indel ref: " << ia.what() << std::endl; } alt = ref.substr(0,1); } } if (ref.empty()) { ref = "N"; } if (alt.empty()) { alt = "N"; } } int BreakPoint::getSpan() const { assert(svtype != SVType::NOTSET); if (svtype == SVType::INDEL && insertion.empty()) return b2.gr.pos1 - b1.gr.pos1; if (svtype == SVType::INDEL && !insertion.empty()) return insertion.length(); if (b1.gr.chr == b2.gr.chr) return abs(b2.gr.pos1-b1.gr.pos1); // interchromosomal else return -1; } // ReducedBreakPoint::ReducedBreakPoint(const std::string &line, const SeqLib::BamHeader& h) { // if (h.isEmpty()) { // std::cerr << "ReducedBreakPoint::ReducedBreakPoint - Must supply non-empty header" << std::endl; // exit(EXIT_FAILURE); // } // std::istringstream iss(line); // std::string val; // size_t count = 0; // ref = nullptr; // alt = nullptr; // cname = nullptr; // evidence = nullptr; // confidence = nullptr; // insertion = nullptr; // homology = nullptr; // //float afn, aft; // std::string ref_s, alt_s, cname_s, insertion_s, homology_s, evidence_s, confidence_s, read_names_s, bxtable_s; // std::string chr1, pos1, chr2, pos2, repeat_s; // char strand1 = '*', strand2 = '*'; // while (std::getline(iss, val, '\t')) { // try{ // switch(++count) { // case 1: chr1 = val; break; // case 2: pos1 = val; break; // case 3: assert(val.length()); strand1 = val.at(0); break; // case 4: chr2 = val; break; // case 5: pos2 = val; break; // case 6: assert(val.length()); strand2 = val.at(0); break; // case 7: // ref_s = val; // break; // case 8: // alt_s = val; // break; // case 9: break; //span = stoi(val); break; // automatically calculated // case 10: //mapq1 // b1 = ReducedBreakEnd(GenomicRegion(chr1, pos1, pos1, h), std::stoi(val)); b1.gr.strand = strand1; break; // case 11: //mapq2 // b2 = ReducedBreakEnd(GenomicRegion(chr2, pos2, pos2, h), std::stoi(val)); b2.gr.strand = strand2; break; // case 12: b1.nm = INTNSTOI(val,255); break; // case 13: b2.nm = INTNSTOI(val,255); break; // case 14: dc.mapq1 = INTNSTOI(val, 255); break; // case 15: dc.mapq2 = INTNSTOI(val, 255); break; // case 16: break; //split (not needed, since in genotype) // case 17: break; //cigar (not needed, since in genotype) // case 18: break; //alt (not needed, since in genotype) // case 19: cov = INTNSTOI(val,65535); break; // case 20: b1.sub_n = INTNSTOI(val,255); break; // case 21: b2.sub_n = INTNSTOI(val,255); break; // case 22: // homology_s = val; // break; // case 23: // insertion_s = val; // break; // case 24: cname_s = val; break; // case 25: num_align = std::min((int)31, std::stoi(val)); break; // case 26: // pass = val == "PASS"; // confidence_s = val; // break; // case 27: // evidence_s = val; // indel = val == "INDEL"; // imprecise = val == "DSCRD"; // break; // case 28: quality = std::stod(val); break; //std::min((int)255,std::stoi(val)); break; // case 29: secondary = val == "1" ? 1 : 0; // case 30: somatic_score = std::stod(val); break; // case 31: LO_s = std::stod(val); break; // case 32: true_lod = std::stod(val); break; // case 33: pon = std::min(255,std::stoi(val)); break; // case 34: repeat_s = val; break; // repeat_seq // case 35: blacklist = (val=="1" ? 1 : 0); break; // case 36: dbsnp = val != "x"; break; // case 37: read_names_s = val; break; //reads // case 38: bxtable_s = val; break; //bx tags // default: // format_s.push_back(val); // } // } catch(...) { // std::cerr << "caught stoi/stod/stof error on: " << val << " for count " << count << std::endl; // std::cerr << line << std::endl; // exit(1); // } // } // confidence = __string_alloc2char(confidence_s, confidence); // evidence = __string_alloc2char(evidence_s, evidence); // insertion = __string_alloc2char(insertion_s, insertion); // homology = __string_alloc2char(homology_s, homology); // cname = __string_alloc2char(cname_s, cname); // ref = __string_alloc2char(ref_s, ref); // alt = __string_alloc2char(alt_s, alt); // repeat = repeat_s.empty() ? nullptr : __string_alloc2char(repeat_s, repeat); // if (somatic_score && confidence_s == "PASS") // read_names = read_names_s; // == "x" ? nullptr : __string_alloc2char(read_names_s, read_names); // bxtable = bxtable_s; // } void BreakPoint::SampleInfo::modelSelection(double er, int readlen) { // can't have more alt reads than total reads // well you can for SVs... // adjust the coverage to be more in line with restrictions on ALT. // namely that ALT reads must overlap the variant site by more than T_SPLIT_BUFF // bases, but the raw cov calc does not take this into account. Therefore, adjust here // if (readlen) { double a_cov = (double)cov * (double)(readlen - 2 * T_SPLIT_BUFF)/readlen; a_cov = a_cov < 0 ? 0 : a_cov; // } else { // a_cov = thiscov; // } af = a_cov > 0 ? (double)alt / (double)a_cov : 1; af = af > 1 ? 1 : af; // mutect log liklihood against error // how likely to see these ALT counts if true AF is af // vs how likely to see these ALT counts if true AF is 0 // and all the ALTs are just errors. // The higher the error rate, the more negative ll_alt will go, // which will drive LO lower and decrease our confidence. // A high er will also drive ll_err up (or less negative), since // it will be more likely to see ALT reads generated by errors // As ALT and COV go higher, we should see LO go higher because // the indiviual calcs will have more confidence. Ultimately, // LO will represent the log likeihood that the variant is AF = af // vs AF = 0 double scaled_alt = std::min((double)alt, a_cov); double scaled_ref = a_cov - scaled_alt; // assume indels don't back-mutate as a sequencing error back to ref // but it's good to have this non-zero, so that with the scaling // tricks above, if we do get a non-zero ref count but var af = 1 // (which technically isn't mathematically allowed), then having some // non-zero chance that the ref is a back-mutate makes the LO not // become uncomputable (which would revert to -1e12 which is not really what we want) double er_back = 0.000001; double ll_alt = SvabaModels::LogLikelihood(scaled_ref, scaled_alt, af, er, er_back); double ll_err = SvabaModels::LogLikelihood(scaled_ref, scaled_alt, 0 , er, er_back); // likelihood that variant is actually true REF //std::cerr << "scaled_ref " << scaled_ref << " scaled_alt " << scaled_alt << " af " << af << " er " << er << " er back " << er_back << " ll_alt " << ll_alt << " ll_err " << ll_err << std::endl; // std::cerr << "SCALD REF " << scaled_ref << " scaled alt " << // scaled_alt << " af " << af << " ll_ALT " << ll_alt << // " ll_err " << ll_err << " scaled af " << scaled_af << std::endl; LO = ll_alt - ll_err; // mutetct log likelihood normal // er = 0.0005; // make this low, so that ALT in REF is rare and NORM in TUM gives low somatic prob // actually, dont' worry about it too much. 3+ alt in ref excludes somatic anyways. // so a high LO_n for the normal means that we are very confident that site is REF only in // normal sample. This is why LO_n from the normal can be used as a discriminant for // germline vs somatic. eg if somatic_lod = normal.LO_n is above a threshold X // or above a larger threshold XX if at DBSNP site, then we accept as somatic // LO_n should not be used for setting the confidence that something is real, just the // confidence that it is somatic // NB: the likelihood that the variant is actually a ref, and any alt reads are explained by errors, is ll_err. // so ll_err = ll_ref_norm // NB: so it's a bit confusing here, but even though we are calculating the LO_n for // any sample (tumor, normal, combined) that is passed, we are only interested in (and will use) // normal.LO_n. This becomes the somatic score, as higher LO_n means it is more likely that the // variant, specifically in the normal, is explained by sequencing errors with a ground-truth of not-present // than by a germline indel. For the tumor, LO_n is not really meaningful, so we look at LO in general for // whether the mutation is around AT ALL. double ll_alt_norm = SvabaModels::LogLikelihood(scaled_ref, scaled_alt, std::max(af, 0.5), er, er_back); // likelihood that variant is at AF >= 0.5 (as expected for germline) LO_n = ll_err - ll_alt_norm; // higher number means more likely to be AF = 0 (ref) than AF = 0.5 (alt). // genotype calculation as provided in // http://bioinformatics.oxfordjournals.org/content/early/2011/09/08/bioinformatics.btr509.full.pdf+html //int scaled_cov = std::floor((double)cov * 0.90); //int this_alt = std::min(alt, scaled_cov); genotype_likelihoods[0] = SvabaModels::GenotypeLikelihoods(2, er, scaled_alt, a_cov); // 0/0 genotype_likelihoods[1] = SvabaModels::GenotypeLikelihoods(1, er, scaled_alt, a_cov); // 0/1 genotype_likelihoods[2] = SvabaModels::GenotypeLikelihoods(0, er, scaled_alt, a_cov); // 1/1 //debugprint //std::cerr << " ALT " << alt << " scaled alt " << scaled_alt << " ER " << er << " A_COV " << a_cov << // " 0/0 " << genotype_likelihoods[0] << " 0/1 " << genotype_likelihoods[1] << // " 1/1 " << genotype_likelihoods[2] << " LOD " << LO << " LO_n " << LO_n << // " af " << af << std::endl; double max_likelihood = *std::max_element(genotype_likelihoods.begin(), genotype_likelihoods.end()); if (max_likelihood == genotype_likelihoods[0]) genotype = "0/0"; else if (max_likelihood == genotype_likelihoods[1]) genotype = "0/1"; else genotype = "1/1"; // compute PLs (phred-scaled likelihoods) for(int i = 0; i < 3; ++i) { // PL = 10 * (GL_i maxGL), rounded to nearest integer phred_likelihoods[i] = int(std::round( -10.0 * (genotype_likelihoods[i] - max_likelihood) )); } //debugprint // std::cerr << " thiscov " << thiscov << " alt " << alt << " cigar " << cigar << " split " << split << // " a_cov " << a_cov << " af " << af << " scaled_alt " << scaled_alt << " ll_alt " << ll_alt << // " ll_err " << ll_err << " LO " << LO << " ll_alt_norm " << // ll_alt_norm << " LO_n " << LO_n << " GT 0/0 " << // genotype_likelihoods[0] << " GT 0/1 " << genotype_likelihoods[1] << // " GT 1/1 " << genotype_likelihoods[2] << std::endl; // get the genotype quality GQ = SvabaModels::GenotypeQuality(phred_likelihoods); // get the genotype quality that it is not hom ref NH_GQ = std::min(phred_likelihoods[0], 99); } void BreakPoint::addCovs(const std::unordered_map& covs) { for (auto& [pref,i] : covs) { int c = 0; for (int j = -COVERAGE_AVG_BUFF; j <= COVERAGE_AVG_BUFF; ++j) { c += i->getCoverageAtPosition(b1.gr.chr, b1.gr.pos1 + j); c += i->getCoverageAtPosition(b2.gr.chr, b2.gr.pos1 + j); } allele.at(pref).cov = c / 2 / (COVERAGE_AVG_BUFF*2 + 1); } } // std::string BreakEnd::print(const SeqLib::BamHeader& h) const { // return gr.ToString(h) + " - " + id + " mapq " + std::to_string(mapq) + " subn " + std::to_string(sub_n); // } /* std::ostream& operator<<(std::ostream& out, const BreakEnd& b) { out << b.gr << " - " << b.id << " mapq " << b.mapq << " subn " << b.sub_n << std::endl; return out; }*/ std::string BreakPoint::SampleInfo::toFileString(SVType svtype) const { std::stringstream pl; pl << phred_likelihoods[0] << ',' << phred_likelihoods[1] << ',' << phred_likelihoods[2]; // std::stringstream gt; // gt << genotype_likelihoods[0] << ',' // << genotype_likelihoods[1] << ',' // << genotype_likelihoods[2]; std::stringstream ss; //GT:AD:DP:SR:DR:GQ:PL:LO:LO_n if (svtype == SVType::INDEL) ss << std::setprecision(4) << genotype << ":" << std::max(alt, cigar) << ":" << cov << ":" << split << ":" << cigar << ":" << GQ << ":" << pl.str() << ":" << LO << ":" << LO_n; else ss << std::setprecision(4) << genotype << ":" << alt << ":" << cov << ":" << split << ":" << disc << ":" << GQ << ":" << pl.str() << ":" << LO << ":" << LO_n; return ss.str(); } void BreakPoint::SampleInfo::FillFromString(const std::string& s, SVType svtype) { std::string val; int count = 0; std::istringstream input(s); while (std::getline(input, val, ':')) { // tmp fix if (val.find("nan") != std::string::npos) val = "0"; switch(++count) { case 1: genotype = val; break; case 2: alt = std::stoi(val); break; case 3: cov = std::stoi(val); break; case 4: split = std::stoi(val); break; case 5: if (svtype == SVType::INDEL) cigar = std::stoi(val); else disc = std::stoi(val); break; case 6: GQ = std::stod(val); break; case 7: phred_likelihoods = svabaUtils::parsePLString(val); break; case 8: LO = std::stod(val); break; case 9: LO_n = std::stod(val); break; } } } // void BreakPoint::__rep(int rep_num, std::string& rseq, bool fwd) { // // move left and right from breakend 1 // //int replen = 0; // //int curr_replen = 1; // rseq = ""; // // return if we are too close to the edge for some reason // if (b1.cpos > (int)seq.length() - 5) // return; // const int REP_BUFF = 50; // std::string sss = seq; // if (!fwd) // std::reverse(sss.begin(), sss.end()); // int cpos = b1.cpos + 1; // if (!fwd) // cpos = seq.length() - b1.cpos; // int i = cpos; //b1.cpos + 1; // int stop = std::min((int)seq.length() - 1, cpos + REP_BUFF); //fwd ? std::min((int)seq.length() - 1, b1.cpos + REP_BUFF) : std::max(0, b1.cpos - REP_BUFF); // int end = -1; // bool no_rep = true; // assert(stop - i < 80); // std::string c = sss.substr(cpos, std::min(rep_num, (int)seq.length() - cpos)); // i += rep_num; //*fr; // for (; i < stop; i+= rep_num) { // if (i >= (int)seq.length() - 1 || i + rep_num > (int)seq.length()) // shouldn't happen, but ensures no substr errors // break; // // terminating // if (c != sss.substr(i,rep_num)) { // || i >= (stop - rep_num)) { // end = i; // break; // } else { // no_rep = false; // //curr_replen += rep_num; // } // } // if (end == -1) // end = stop; // if (!no_rep) // rseq = sss.substr(cpos, std::min(end - cpos, (int)seq.length() - cpos)); // } std::string BreakEnd::hash(int offset) const { return (std::to_string(gr.chr) + ":" + std::to_string(gr.pos1 + offset)); } bool BreakPoint::hasMinimal() const { int count; count = dc.tcount + dc.ncount + t.split + n.split; if (local == LocalAlignment::FROM_DISTANT_REGION) return false; if (count >= 2) return true; return false; } std::string BreakPoint::getHashString() const { if (!isIndel()) return ""; bool isdel = insertion.length() == 0; std::string st = std::to_string(b1.gr.chr) + "_" + std::to_string(b1.gr.pos1) + "_" + std::to_string(this->getSpan()) + (isdel ? "D" : "I"); return st; } void BreakPoint::setLocal(const GenomicRegion& window) { b1.setLocal(window); b2.setLocal(window); assert(b1.local != LocalAlignment::NOTSET); assert(b2.local != LocalAlignment::NOTSET); // if this was already set as a non-variant local re-alignment // during alignedContig construction, then that has precedent if (local == LocalAlignment::NONVAR_LOCAL_REALIGNMENT) return; // set the BreakPoint level local flag if (b1.local == LocalAlignment::FROM_LOCAL_REGION || b2.local == LocalAlignment::FROM_LOCAL_REGION) local = LocalAlignment::FROM_LOCAL_REGION; else local = LocalAlignment::FROM_DISTANT_REGION; } void BreakEnd::setLocal(const GenomicRegion& window) { assert(local == LocalAlignment::NOTSET); if (gr.GetOverlap(window)) local = LocalAlignment::FROM_LOCAL_REGION; else local = LocalAlignment::FROM_DISTANT_REGION; } void BreakPoint::SampleInfo::UpdateAltCounts() { // get unique qnames std::unordered_set qn; for (auto& r : supporting_reads) { size_t posr = r.find("_", 5) + 1; // extract the qname from tXXX_XXX_QNAME qn.insert(r.substr(posr, r.length() - posr)); } // alt count is max of cigar or unique qnames (includes split and discordant) alt = std::max((int)qn.size(), cigar); } // bool ReducedBreakPoint::operator<(const ReducedBreakPoint& bp) const { // //ASDIS > ASSMB > COMP > DSCRD // if (std::strcmp(evidence,bp.evidence) < 0) // < // return true; // else if (std::strcmp(evidence, bp.evidence) > 0) // > // return false; // if (cov > bp.cov) // return true; // else if (cov < bp.cov) // return false; // //if (nsplit > bp.nsplit) // // return true; // //else if (nsplit < bp.nsplit) // // return false; // //if (tsplit > bp.tsplit) // // return true; // //else if (tsplit < bp.tsplit) // // return false; // //if (dc.ncount > bp.dc.ncount) // // return true; // //else if (dc.ncount < bp.dc.ncount) // // return false; // //if (dc.tcount > bp.dc.tcount) // // return true; // //else if (dc.tcount < bp.dc.tcount) // // return false; // // break the tie somehow // if (cname > bp.cname) // return true; // else if (cname < bp.cname) // return false; // return false; // } // std::ostream& operator<<(std::ostream& os, const ReducedBreakEnd& rbe) { // // Example format: [Chr Name: genomic_region (MapQ, SubN, NM)] // // Adjust formatting as needed // os << rbe.gr << " (" // << "MapQ: " << static_cast(rbe.mapq) << ", " // << "SubN: " << static_cast(rbe.sub_n) << ", " // << "NM: " << static_cast(rbe.nm) << ")]"; // return os; // } // make a breakpoint from a discordant cluster BreakPoint::BreakPoint(DiscordantCluster& tdc, DiscordantClusterMap& dmap, const GenomicRegion& region, const SvabaSharedConfig* sc_) : sc(sc_) { svtype = SVType::DSCRD; // instantiate the SampleInfo objects for each BAM for (auto const& p : sc->prefixes) { allele.try_emplace(p); } num_align = 0; dc = tdc; std::string chr_name1, chr_name2; // not based on aligmnent of contig, so set as NA (-1) secondary = -1; try { // if this throw error, it means that there are more chr in // reads than in reference chr_name1 = sc->header.IDtoName(dc.m_reg1.chr); chr_name2 = sc->header.IDtoName(dc.m_reg2.chr); // chr_name1 = bwa->ChrIDToName(dc.m_reg1.chr); //bwa->ChrIDToName(tdc.reads.begin()->second.ChrID()); // chr_name2 = bwa->ChrIDToName(dc.m_reg2.chr); //bwa->ChrIDToName(tdc.reads.begin()->second.ChrID()); } catch (...) { std::cerr << "Warning: Found mismatch between reference genome and BAM genome for discordant cluster " << dc.print(sc->header) << std::endl; chr_name1 = "Unknown"; chr_name2 = "Unknown"; } assert(chr_name1.length()); assert(chr_name2.length()); int pos1 = (dc.m_reg1.strand == '+') ? dc.m_reg1.pos2 : dc.m_reg1.pos1; int pos2 = (dc.m_reg2.strand == '+') ? dc.m_reg2.pos2 : dc.m_reg2.pos1; b1.gr = GenomicRegion(dc.m_reg1.chr, pos1, pos1); b2.gr = GenomicRegion(dc.m_reg2.chr, pos2, pos2); b1.mapq = dc.mapq1; b2.mapq = dc.mapq2; b1.gr.strand = dc.m_reg1.strand; b2.gr.strand = dc.m_reg2.strand; // set the alt counts, counting only unique qnames std::unordered_map> alt_counts; for (auto const& p : sc->prefixes) { alt_counts.try_emplace(p); } // add the supporting read info to alleles auto process = [this, &alt_counts](auto& container){ for (auto& [_, rec] : container) { const auto& prefix = rec->Prefix(); auto ait = allele.find(prefix); ait->second.supporting_reads.insert(rec->UniqueName()); auto act = alt_counts.find(prefix); act->second.insert(rec->Qname()); } }; process(dc.reads); process(dc.mates); // add the alt counts for (auto& [pref,al] : allele) { al.disc = alt_counts[pref].size(); //allele[i.first].supporting_reads.size(); al.UpdateAltCounts(); //i.second.alt = i.second.disc; } // give a unique id (OK to give an empty header here, since used internally) cname = dc.toRegionString(sc->header) + "__" + std::to_string(region.chr+1) + "_" + std::to_string(region.pos1) + "_" + std::to_string(region.pos2) + "D"; // check if another cluster overlaps, but different strands if (getSpan() > 800 || getSpan() == -1) { // only check for large events. for (auto& [_,d] : dmap) { // don't overlap if on different chr, or same event if (dc.m_reg1.chr != d.m_reg1.chr || dc.m_reg2.chr != d.m_reg2.chr || d.ID() == dc.ID()) continue; // isolate and pad GenomicRegion gr1 = d.m_reg1; GenomicRegion gr2 = d.m_reg2; gr1.Pad(100); gr2.Pad(100); if (dc.m_reg1.GetOverlap(gr1) && dc.m_reg2.GetOverlap(gr2)) if (dc.m_reg1.strand != d.m_reg1.strand || dc.m_reg2.strand != d.m_reg2.strand) { dc.m_id_competing = d.ID(); tdc.m_id_competing = d.ID(); d.m_id_competing = dc.ID(); } } } } void BreakPoint::indelCigarCheck(const CigarMapMap& cmap) { if (!isIndel()) return; // get the hash count for this std::string hash = getHashString(); // tally the number of reads that have cigar that support this // NB: the totalling for tumor, normal occur late in scoreBreakpoints for (auto& [sample,si] : allele) { size_t val = cmap.at(sample).count(hash) ? cmap.at(sample).at(hash) : 0; //std::cerr << " OG HASH VAL " << val << " hash " << hash << " sample " << sample << std::endl; si.cigar += val; } int span = getSpan(); // for somatic, be extra careful and score "nearby" cigars // so e.g. if the normal has // this is really weird and inefficient way to do this, but should be so fast it's inconsequential char in_del[2] = {'D', 'I'}; const int CIGAR_NEAR_BUFFER = 4; std::string thischr = std::to_string(b1.gr.chr) + "_"; for (int i = -CIGAR_NEAR_BUFFER; i <= CIGAR_NEAR_BUFFER; i++) { for (int len = 1; len < (span + 5); len++) { std::string lenst = std::to_string(len); for (int ii = 0; ii < 2; ii++) { // loop I or D char c = in_del[ii]; std::string st = thischr + std::to_string(b1.gr.pos1 + i) + "_" + lenst + c; //std::cerr << " cname " << cname << " st " << st << " hash " << hash << " i " << i << " len " << len << " ii " << ii << " span " << span << std::endl; if (hash == st) continue; // don't redo this for (auto& [sample, si] : allele) { size_t val = cmap.at(sample).count(st) ? cmap.at(sample).at(st) : 0; si.cigar_near += val; // std::cerr << " sample " << sample << " cname " << cname << " st " << st << " hash " << hash << " val " << val << std::endl; } } } } } namespace { // split a tab-delimited line (keeps empty fields) inline std::vector split_tabs(const std::string& s) { std::vector out; out.reserve(48); std::string cur; cur.reserve(64); for (char c : s) { if (c == '\t') { out.push_back(cur); cur.clear(); } else if (c != '\r' && c != '\n') { cur.push_back(c); } } out.push_back(cur); return out; } inline long to_long(const std::string& s) { if (s.empty()) return 0; return std::stol(s); } inline int to_int(const std::string& s) { if (s.empty()) return 0; return std::stoi(s); } inline double to_double(const std::string& s) { if (s.empty()) return 0.0; return std::stod(s); } inline char parse_strand(const std::string& s) { return s.empty() ? '.' : s[0]; // expect '+' or '-' } inline std::string upper(std::string v){ std::transform(v.begin(), v.end(), v.begin(), [](unsigned char c){ return std::toupper(c); }); return v; } // Map type string to SVType enum; adjust cases to match your enum names. inline SVType parse_svtype(const std::string& s_in) { const std::string s = upper(s_in); if (s == "NOTSET") return SVType::NOTSET; if (s == "TSI_LOCAL") return SVType::TSI_LOCAL; if (s == "TSI_GLOBAL") return SVType::TSI_GLOBAL; if (s == "ASSMB") return SVType::ASSMB; if (s == "ASDIS") return SVType::ASDIS; if (s == "DSCRD") return SVType::DSCRD; if (s == "INDEL") return SVType::INDEL; return SVType::NOTSET; } // Map somatic field to SomaticState enum; tweak to match your enum. inline SomaticState parse_somatic(const std::string& s_in) { const std::string s = upper(s_in); if (s_in == "1") return SomaticState::SOMATIC_LOD; return SomaticState::NORMAL_LOD; } // stod that tolerates "NA", ".", "". inline double stod_safe(const std::string& s) { if (s.empty()) return 0.0; std::string u; u.reserve(s.size()); for (char c: s) u.push_back(std::toupper(static_cast(c))); if (u == "NA" || u == ".") return 0.0; return std::stod(s); } } // namespace BreakPoint::BreakPoint(const std::string& line, const SvabaSharedConfig* _sc) : sc(_sc) { if (!line.empty() && line[0] == '#') { throw std::invalid_argument("BreakPoint: header/comment line cannot be parsed as data"); } const auto tok = split_tabs(line); // Minimum supported = 41 (legacy pre-SvABA2.0 dumps: core columns through // contig_conf2, no refilter-roundtrip additions). When we see >= 51 we // know the file carries the SvABA2.0 cpos/match/cov/local/flip columns // and we parse them in. This keeps the parser backward-compatible with // bps.txt.gz files produced by older svaba builds. if (tok.size() < 41) { throw std::runtime_error("BreakPoint parse error: expected at least 41 columns, got " + std::to_string(tok.size())); } int i = 0; // Lambda to safely parse an int from a token, tolerating empty / "x" / non- // numeric values that older dumps sometimes emit for unset sentinels. auto to_int_safe = [](const std::string& s, int def) -> int { if (s.empty() || s == "x" || s == "NA" || s == "nan") return def; try { return std::stoi(s); } catch (...) { return def; } }; auto to_double_safe = [](const std::string& s, double def) -> double { if (s.empty() || s == "x" || s == "NA" || s == "nan") return def; try { return std::stod(s); } catch (...) { return def; } }; // ---- fixed columns (1..39) ---- const std::string chr1 = tok[i++]; const long pos1_1idx = to_long(tok[i++]); const char s1 = tok[i++].empty() ? '.' : tok[i-1][0]; const std::string chr2 = tok[i++]; const long pos2_1idx = to_long(tok[i++]); const char s2 = tok[i++].empty() ? '.' : tok[i-1][0]; ref = tok[i++]; // 7 alt = tok[i++]; // 8 /* span */ (void)to_int(tok[i++]); // 9 a.split = to_int(tok[i++]); // 10 a.alt = to_int(tok[i++]); // 11 a.cov = to_int(tok[i++]); // 12 a.cigar = to_int(tok[i++]); // 13 a.cigar_near = to_int(tok[i++]); // 14 dc.mapq1 = to_int(tok[i++]); // 15 dc.mapq2 = to_int(tok[i++]); // 16 dc.ncount = to_int(tok[i++]); // 17 dc.tcount = to_int(tok[i++]); // 18 b1.mapq = to_int(tok[i++]); // 19 b2.mapq = to_int(tok[i++]); // 20 b1.nm = to_int(tok[i++]); // 21 b2.nm = to_int(tok[i++]); // 22 b1.as = to_int(tok[i++]); // 23 b2.as = to_int(tok[i++]); // 24 b1.sub = to_int(tok[i++]); // 25 b2.sub = to_int(tok[i++]); // 26 homology = tok[i++]; if (homology == "x") homology.clear(); // 27 insertion = tok[i++]; if (insertion == "x") insertion.clear(); // 28 repeat_seq = tok[i++]; if (repeat_seq == "x") repeat_seq.clear(); // 29 cname = tok[i++]; // 30 num_align = to_int(tok[i++]); // 31 confidence = tok[i++]; // 32 svtype = parse_svtype(tok[i++]); // 33 quality = to_int(tok[i++]); // 34 secondary = to_int(tok[i++]); // 35 somatic = parse_somatic(tok[i++]); // 36 LO_s = stod_safe(tok[i++]); // 37 max_lod = stod_safe(tok[i++]); // 38 rs = tok[i++]; if (rs == "x") rs.clear(); // 39 // ---- contig-confidence columns (40..41) -- previously skipped by this // ---- parser, which caused the first two trailing tokens to be misread // ---- as sample blocks. Now round-tripped correctly. b1.contig_conf = to_double_safe(tok[i++], 1.0); // 40 b2.contig_conf = to_double_safe(tok[i++], 1.0); // 41 // ---- SvABA2.0 refilter-roundtrip columns (42..51). Optional: only // ---- parse if present, so we remain backward-compatible with older // ---- bps.txt.gz files (which stop at 41 + per-sample blocks). // ---- // ---- Detection heuristic: per-sample blocks are colon-delimited // ---- (GT:AD:DP:SR:...). Core columns 42..51 are plain scalars with // ---- no colons. So we use: "if tok[41] contains no ':' AND there are // ---- at least 10 more tokens, it's the new format". This is robust // ---- against unusual sample counts and doesn't need to know n_bams // ---- at parse time. const bool has_new_core_cols = (tok.size() >= static_cast(i) + 10) && (tok[static_cast(i)].find(':') == std::string::npos); if (has_new_core_cols) { b1.cpos = to_int_safe(tok[i++], -1); // 42 b2.cpos = to_int_safe(tok[i++], -1); // 43 left_match = to_int_safe(tok[i++], -1); // 44 right_match = to_int_safe(tok[i++], -1); // 45 split_cov_bounds.first = to_int_safe(tok[i++], 0); // 46 split_cov_bounds.second = to_int_safe(tok[i++], 0); // 47 const int l1 = to_int_safe(tok[i++], 0); // 48 const int l2 = to_int_safe(tok[i++], 0); // 49 auto to_local = [](int v) -> LocalAlignment { switch (v) { case 1: return LocalAlignment::NONVAR_LOCAL_REALIGNMENT; case 2: return LocalAlignment::FROM_LOCAL_REGION; case 3: return LocalAlignment::FROM_DISTANT_REGION; default: return LocalAlignment::NOTSET; } }; b1.local = to_local(l1); b2.local = to_local(l2); contig_len = to_int_safe(tok[i++], 0); // 50 flipped_on_contig = (to_int_safe(tok[i++], 0) != 0); // 51 // SvABA2.0 v3: optional col 52 = bp_id. Same "no colons" test used // above for cols 42..51: per-sample blocks are colon-delimited // (GT:AD:DP:SR:...), while bp_id is a plain `bpTTTNNNNNNNN` string. // If the next token is non-colon AND there are >= n_samples_expected // tokens left after it, we treat it as the bp_id column. Older dumps // (pre-v3, 51 core cols) skip this and leave bp->id empty; the VCF // emitter falls back to its legacy hash-based id in that case. if (static_cast(i) < tok.size() && tok[static_cast(i)].find(':') == std::string::npos) { const std::string& maybe_bp_id = tok[static_cast(i)]; // bp_id always starts with "bp" or is "." (unset sentinel). Guard // against a malformed input that happens to have a non-colon // token here that isn't actually a bp_id. if (maybe_bp_id == "." || (maybe_bp_id.size() >= 2 && maybe_bp_id[0] == 'b' && maybe_bp_id[1] == 'p')) { id = (maybe_bp_id == "." ? std::string() : maybe_bp_id); // 52 ++i; } } } else { // Legacy dump: leave the new fields at their defaults. The PASS / // assembly-only filters that depend on these will degrade gracefully // (e.g. local=NOTSET, cpos=-1, split_cov_bounds={0,0}), and the // refilter flow should prefer the dumped `confidence` string in that // case rather than recomputing. b1.cpos = -1; b2.cpos = -1; left_match = -1; right_match = -1; split_cov_bounds = {0, 0}; b1.local = LocalAlignment::NOTSET; b2.local = LocalAlignment::NOTSET; contig_len = 0; flipped_on_contig = false; } // Genomic regions (file is 1-based; internal is 0-based) b1.gr.chr = sc->header.Name2ID(chr1); b1.gr.pos1 = static_cast(pos1_1idx - 1); b1.gr.pos2 = b1.gr.pos1; b1.gr.strand = s1; b2.gr.chr = sc->header.Name2ID(chr2); b2.gr.pos1 = static_cast(pos2_1idx - 1); b2.gr.pos2 = b2.gr.pos1; b2.gr.strand = s2; // Top-level `local` is a summary derived from (b1.local, b2.local). The // original scorer sets it as: FROM_LOCAL_REGION if either end is local, // else FROM_DISTANT_REGION (see ~line 2045 in scoreBreakpoint path). // We only set it here if we read a valid per-end local from the dump; // otherwise leave NOTSET so the caller can rescore. if (b1.local != LocalAlignment::NOTSET || b2.local != LocalAlignment::NOTSET) { if (b1.local == LocalAlignment::FROM_LOCAL_REGION || b2.local == LocalAlignment::FROM_LOCAL_REGION) local = LocalAlignment::FROM_LOCAL_REGION; else local = LocalAlignment::FROM_DISTANT_REGION; } else { local = LocalAlignment::NOTSET; } imprecise = 0; pass = 0; // unreliable — downstream code should gate on confidence // ---- trailing per-sample fields (map by order to sc->opts.bams) ---- const size_t rem = tok.size() - static_cast(i); std::vector bam_list; for (const auto& [pref,_] : sc->opts.bams) bam_list.push_back(pref); const size_t nsamples_expected = bam_list.size(); if (rem < nsamples_expected) { std::cerr << "Warning: BreakPoint parse: trailing sample fields fewer than expected (" << rem << " < " << nsamples_expected << ").\n"; } const size_t nsamples = std::min(rem, nsamples_expected); for (size_t j = 0; j < nsamples; ++j) { // sample key = first 4 chars of sc->opts.bams[j] const std::string& bam = bam_list[j]; const size_t key_len = std::min(4, bam.size()); const std::string key = bam.substr(0, key_len); SampleInfo si; si.FillFromString(tok[i + static_cast(j)], svtype); allele[key] = std::move(si); } // Optionally warn if there are extra tokens beyond expected samples if (rem > nsamples_expected) { std::cerr << "Warning: BreakPoint parse: " << (rem - nsamples_expected) << " extra trailing token(s) ignored.\n"; } } ================================================ FILE: src/svaba/BreakPoint.h ================================================ #pragma once #include #include #include #include #include #include #include "DiscordantCluster.h" #include "SeqLib/BamRecord.h" #include "SvabaRead.h" using CigarMapMap = std::unordered_map; enum class LocalAlignment { NOTSET, NONVAR_LOCAL_REALIGNMENT, // contig aligns to local region without variant FROM_LOCAL_REGION, // at least one end of contig bp maps to assembled region, this is good FROM_DISTANT_REGION // neither end maps to local assembly region, this is bad }; enum class SVType { NOTSET, TSI_LOCAL, // TSI_LOCAL TSI_GLOBAL, // TSI_GLOBAL ASSMB, // SIMPLE ASSEMBLY ASDIS, DSCRD, // DISCORDANT ONLY INDEL // INDEL }; enum class SomaticState { NOTSET, SOMATIC_LOD, // somatic by LOD cutoff NORMAL_LOD, // normal by LOD cutoff FAILED, // normal - failed hard filter }; // forward declares class STCoverage; namespace SeqLib { class RefGenome; class BamHeader; } class AlignmentFragment; class AlignedContig; using SeqLib::BamHeader; using SeqLib::BamRecord; using SeqLib::RefGenome; using SeqLib::BamRecordPtr; using SeqLib::GenomicRegion; using SeqLib::GRC; struct BreakPoint; struct BreakEnd; typedef std::vector HashVector; typedef std::vector BPVec; typedef std::shared_ptr BreakPointPtr; typedef std::vector BreakPointPtrVector; typedef std::unordered_set ReadNameSet; struct BreakEnd { BreakEnd() = default; void transferContigAlignmentData(const AlignmentFragment* f, bool isleft); void setLocal(const GenomicRegion& window); std::string printSimple(const BamHeader& h) const; std::string hash(int offset) const; std::string id; GenomicRegion gr; // contig level informaiton set by AlignmentFragment int mapq = -1; ///< mapping quality of alignment int cpos = -1; ///< breakpoint position on the contig (0-based) int nm = -1; ///< number of mismatching bases/indels bases int matchlen = -1; /// number of matching bases in the alignment int as = -1; ///< primary alignment score int sub = -1; ///< best secondary alignment score // Svaba2.0: composite reliability of the contig-alignment fragment backing // this breakend, in [0, 1]. For an SV BreakPoint the two ends can come // from different BWA fragments of the same split-aligned contig, so // each side stores its own score. Default 1.0 so legacy code paths // that never set it don't accidentally demote calls. double contig_conf = 1.0; LocalAlignment local = LocalAlignment::NOTSET; // for high-confidence reads std::unordered_map split; // friend std::ostream& operator<<(std::ostream& out, const BreakEnd& b); }; class BreakPoint { public: // for discordant clusters BreakPoint(DiscordantCluster& tdc, DiscordantClusterMap& dmap, const GenomicRegion& region, const SvabaSharedConfig* _sc); // for rearrangements (including complex) BreakPoint(const SvabaSharedConfig* _sc, const AlignmentFragment* left, const AlignmentFragment* right, const AlignedContig* alc); // for indels BreakPoint(const AlignmentFragment* f, const int idx, // index for which indel on this f alignment we are making const SvabaSharedConfig* _sc); // for readback from file BreakPoint(const std::string &line, const SvabaSharedConfig* _sc); // enforce one of above modalities BreakPoint() = delete; // Disable copy BreakPoint(const BreakPoint&) = delete; BreakPoint& operator=(const BreakPoint&) = delete; // Disable move BreakPoint(BreakPoint&&) = delete; BreakPoint& operator=(BreakPoint&&) = delete; static std::string header() { // Columns 1-41 are historical core fields; 42-51 are SvABA2.0 additions // for refilter round-tripping (contig coords, match lengths, split-cov // bounds, per-end LocalAlignment, contig orientation); 52 is bp_id // (unique thread-stable BreakPoint identifier — see BreakPoint::id). // Per-sample blocks follow. All downstream consumers that index by // NAME (viewer, HTML modules) are header-robust; sort scripts that // hardcode -k37,-k38 still work because somlod/maxlod positions // are unchanged. return std::string( "#chr1\tpos1\tstrand1\tchr2\tpos2\tstrand2\tref\talt\t" "span\tsplit\talt\tcov\tcigar\tcigar_near\t" "dmq1\tdmq2\tdcn\tdct\t" "mapq1\tmapq2\tnm1\tnm2\tas1\tas2\tsub1\tsub2\t" "homol\tinsert\trepeat\t" "contig_and_region\tnaln\tconf\ttype\tqual\t2ndary\t" "somatic\tsomlod\tmaxlod\tdbsnp\tcontig_conf1\tcontig_conf2\t" "cpos1\tcpos2\tlmatch\trmatch\tscov1\tscov2\t" "local1\tlocal2\tctglen\tflipped\t" "bp_id" ); } SomaticState somatic = SomaticState::NOTSET; std::string seq, cname, rs, insertion, homology, repeat_seq, confidence, ref, alt; // SvABA2.0: unique stable identifier for this BreakPoint, assigned // exactly once per BP in SvabaRegionProcessor::process() via // svabaThreadUnit::next_bp_id() right before the pointer is pushed // into unit.m_bps. Format: "bpTTTNNNNNNNN" where TTT is the 3-digit // zero-padded thread ID and NNNNNNNN is an 8-digit zero-padded // per-thread running counter. Because AlignedContig holds its BPs // via shared_ptr, setting id through any reference is visible // everywhere — r2c emission later reads it back through // getAllBreakPoints(). Emitted as the final core column of // bps.txt so downstream tools can cross-reference which BP a read // supports (see r2c's split_bps/disc_bps columns). std::string id; // the evidence per break-end BreakEnd b1, b2; int imprecise = -1; //false; // number of matched bases to left and right // dont want indel matches where there is not confidence alignment // because indel is too close to end int left_match = -1, right_match = -1; // reads spanning this breakpoint (these are read-to-genome, but have an r2c) svabaReadPtrVector reads; // discordant reads supporting this aseembly bp DiscordantCluster dc; int quality = -1; // QUAL score int secondary = -1; // is this a secondary int pass = -1; //false; int num_align = 0; // number of alignments for contigs that generated this // SvABA2.0: for indels only, track whether the underlying AlignmentFragment // had its contig sequence flipped (i.e. the primary contig alignment is on // the reverse strand of the genome). b1.cpos / b2.cpos are computed from // the un-flipped BAM CIGAR (so they are BAM/genome-forward coordinates), // but m_seq in the AlignedContig and the read-to-contig positions from the // r2c BWA index are both in assembly-native orientation. When flipped is // true, these two coordinate systems are mirror images and we must convert // cpos to m_seq coordinates before comparing with r2c positions or // rendering onto m_seq. Use cpos_on_m_seq() below to get the converted // (b1, b2) pair. int contig_len = 0; bool flipped_on_contig = false; // Return (b1.cpos, b2.cpos) converted to m_seq / r2c (assembly-native) // orientation. When flipped_on_contig is false this is the identity. // When flipped, the coordinate system reverses and the "before" / "after" // sense of an indel swaps, so we mirror AND swap. std::pair cpos_on_m_seq() const { if (!flipped_on_contig || contig_len <= 0) return {b1.cpos, b2.cpos}; const int L = contig_len; return {L - 1 - b2.cpos, L - 1 - b1.cpos}; } double LO_s = 0; // log odds of variant being somatic (see svabaModels.cpp - SomaticLOD) double max_lod = 0; // the highest LOD across all samples SVType svtype = SVType::NOTSET; LocalAlignment local = LocalAlignment::NOTSET; const SvabaSharedConfig* sc; // keep track of how much of contig is covered by split // first is left-most position on contig that has a read aligned to it // second is right-most std::pair split_cov_bounds; GenomicRegion BreakEndAsGenomicRegionLeft() const; GenomicRegion BreakEndAsGenomicRegionRight() const; // SvABA2.0: union of UniqueNames of split-supporting reads across all // samples. Used by AlignedContig::printToR2CTsv to tag each read row // with its variant-support kind. SampleInfo is a private nested type, // so we expose this method instead of forcing callers to iterate // `allele` and reach into SampleInfo directly. std::unordered_set getAllSupportingReads() const { std::unordered_set out; for (const auto& kv : allele) for (const auto& un : kv.second.supporting_reads) out.insert(un); return out; } bool isIndel() const; HashVector getBreakEndHashes(); void __combine_alleles(); void __rep(int rep_num, std::string& rseq, bool fwd = true); void setLocal(const GenomicRegion& window); void score_somatic(double error_fwd); void indelCigarCheck(const CigarMapMap& cmap); void addCovs(const std::unordered_map& covs); /** Retrieve the reference sequence at a breakpoint and determine if * it lands on a repeat */ void repeatFilter(); std::string printSimple(const BamHeader& h) const; void CombineWithDiscordantClusterMap(DiscordantClusterMap& dmap); /*! @function determine if the breakpoint has split read support * @param reference to a vector of read smart pointers that have been aligned to a contig * @discussion Note: will cause an error if the AL tag not filled in for the reads. * The AL tag is filled in by AlignedContig::alignReadsToContigs. */ void splitCoverage(svabaReadPtrVector& bav); /*! Determines if the BreakPoint overlays a blacklisted region. If * and overlap is found, sets the blacklist bool to true. * * Note that currently this only is set for the pos1 of indels. * If the BreakPoint object is not an indel, no action is taken. * @param grm An interval tree map created from a BED file containing blacklist regions */ void checkBlacklist(GRC &grv); /*! Score a breakpoint with a QUAL score, and as somatic or germline */ void scoreBreakpoint(); /*! Compute the allelic fraction (tumor and normal) for this BreakPoint. * * The allelic fraction is computed by taking the base-pair level coverage * as the denominator, and the max of number of split reads and number of * cigar supporting reads as the numerator. Note that because, theoretically * but rarely, the number of split reads could be > 0 while the bp-level coverage * at a variant could be exactly zero. This is because unmapped reads could be called split * reads but are not counted in the coverage calculation. In such a case, the allelic fraction is * set to -1. By the same argument, the allelic fraction could rarely be > 1. * @param t_cov Base-pair level Coverage object, with coverage for all reads from Tumor bam(s). * @param n_cov Base-pair level Coverage object, with coverage for all reads from Normal bam(s). */ //void addAllelicFraction(STCoverage * t_cov, STCoverage * n_cov); /*! @function get the span of the breakpoints (in bp). -1 for interchrom * @return int distance between breakpoints */ int getSpan() const; /*! @function get a unique string representation of this breakpoint. * Format for indel is chr_breakpos_type (eg. 0_134134_I) * @return string with breakpoint info */ std::string getHashString() const; bool hasMinimal() const; bool sameBreak(BreakPoint &bp) const; bool isEmpty() const { return (b1.gr.pos1 == 0 && b2.gr.pos1 == 0); } std::string toFileString(const BamHeader& header) const; bool hasDiscordant() const; //bool operator==(const BreakPoint& bp) const; // define how to sort these bool operator<(const BreakPoint& o) const { return std::tie( b1.gr, // first key b2.gr, // then n.split, t.split, dc.ncount, dc.tcount, cname // last key ) < std::tie( o.b1.gr, o.b2.gr, o.n.split, o.t.split, o.dc.ncount, o.dc.tcount, o.cname ); } void score_dscrd(); void score_assembly_only(); void score_assembly_dscrd(); void score_indel(); void set_homologies_insertions(); bool valid() const; void setRefAlt(const RefGenome* main_rg, const BamHeader& header); private: struct SampleInfo { // no default construction: SampleInfo() = default; int split = 0; int cigar = 0; int cigar_near = 0; // cigar matches close (but not same) int alt = 0; int cov = 0; int disc = 0; // genotype info //NB: PL (the Phred-scaled -10*log_10(GL - max(GL)) // rounded to int by convention double GQ = 0; // GQ take, max 99, quality of the genotype tag double NH_GQ = 0; // GQ of 0/0. Higher is more likely to be not hom ref std::string genotype; // GT tag std::vector genotype_likelihoods = {0,0,0}; // GL tag std::vector phred_likelihoods = {0,0,0}; // PL tag double af = 0; // log odds double LO = 0; // log odds of variant vs error double SLO = 0; // MAPQ scaled log odds of variant vs error double LO_n = 0; // log odds of variant at af=0.5 vs ref (af=0) with errors // UniqueName set of supporting reads // not for read tracking but actually needed for breakpoint scoring ReadNameSet supporting_reads; void modelSelection(double err, int readlen); std::string toFileString(SVType svtype) const; void FillFromString(const std::string& s, SVType svtype); void UpdateAltCounts(); }; // Friend declarations so these free functions can see the private nested type friend std::ostream& operator<<( std::ostream& out, const BreakPoint::SampleInfo& a ); friend BreakPoint::SampleInfo operator+( const BreakPoint::SampleInfo& a1, const BreakPoint::SampleInfo& a2 ); public: //tumor allele, normal allele, all allele SampleInfo t, n, a; // ordered to keep in alphabetical order by prefix (e.g. n001) std::map allele; }; // struct ReducedDiscordantCluster { // uint32_t mapq1:8, mapq2:8, tcount:8, ncount:8; // }; // struct ReducedBreakPoint { // // some helper functions // char* __string_alloc2char(const std::string& str, char * p) { // if (!str.empty() && str != "x") { // p = (char*)malloc(str.length() + 1); // strcpy(p, str.c_str()); // return p; // } else { // return nullptr; // } // } // inline void smart_check_free(char * p) { // if (p) // free(p); // } // int getSpan() const { // if (indel && !insertion) // deletion // return (abs((int)b1.gr.pos1 - (int)b2.gr.pos1) - 1); // if (indel) // insertion // return (strlen(insertion)); // insertion // if (b1.gr.chr == b2.gr.chr) // return abs((int)b1.gr.pos1-(int)b2.gr.pos1); // else // return -1; // } // // define how to sort these // bool operator<(const ReducedBreakPoint& bp) const; // // print it with the correct chromsome string // std::string print(const BreakPoint& b, const SeqLib::BamHeader& h) const; // ReducedBreakPoint() {} // ~ReducedBreakPoint() { // smart_check_free(ref); // smart_check_free(alt); // smart_check_free(cname); // smart_check_free(homology); // smart_check_free(insertion); // smart_check_free(evidence); // smart_check_free(confidence); // smart_check_free(repeat); // } // ReducedBreakPoint(const std::string &line, const SeqLib::BamHeader& h); // char * ref; // char * alt; // char * cname; // char * evidence; // char * confidence; // char * insertion; // char * homology; // char * repeat; // std::string read_names, bxtable; // std::vector format_s; // ReducedBreakEnd b1, b2; // double somatic_score = 0; // double somatic_lod = 0; // LogOdds that variant not in normal // double true_lod = 0; // //uint32_t nsplit:8, tsplit:8, // //uint32_t tcov_support:8, ncov_support:8, tcov:8, ncov:8; // uint32_t cov:16, af_n:7, num_align:5, secondary:1, dbsnp:1, pass:1, blacklist:1, indel:1, imprecise:1; // uint32_t tcigar:8, ncigar:8, dummy:8, af_t:8; // float quality; // uint8_t pon; // ReducedDiscordantCluster dc; // }; ================================================ FILE: src/svaba/ContigAlignmentScore.cpp ================================================ // ContigAlignmentScore.cpp // // See header for what this does and where it plugs into svaba. The scoring // rules below map directly to the filter analysis: // // Rule A: n_indel_ops >= 3 is almost certainly mis-alignment in a repeat. // Each additional indel beyond 1 adds a 0.20 penalty. // Rule B: 2+ indels with no single big event (<=20 bp) and noisy flanks // (mm_rate > 0.02) is scattered-small-indel pattern. Flat 0.25 // penalty. A single large indel exempts the contig from this // rule, so real big SVs/indels pass. // Rule C: non-indel mismatch rate. Threshold 1% (well above per-base // sequencing error ~0.1-0.5%), slope 10 so 3% mm = 0.20, // 5% = 0.40, 10% = 0.90. Extra quadratic kicker above 5% so // very noisy alignments crash to zero. // Rule D: as_per_bp < 0.70 means BWA paid a lot of penalties for this // placement. Smooth penalty scaled 2x. // // confidence = clamp(1 - sum(penalties), 0, 1). #include "ContigAlignmentScore.h" #include #include #include #include namespace svaba { namespace { template inline T clip_range(T v, T lo, T hi) { return std::max(lo, std::min(hi, v)); } } // anon ContigAlignScore scoreContigAlignment(const SeqLib::BamRecord& r) { ContigAlignScore s; if (!r.MappedFlag()) return s; // --- parse CIGAR -------------------------------------------------------- const SeqLib::Cigar cig = r.GetCigar(); for (const auto& c : cig) { const int len = static_cast(c.Length()); const char op = c.Type(); switch (op) { case 'M': case '=': case 'X': s.aligned_bp += len; s.ref_span += len; break; case 'I': s.n_indel_ops++; s.total_indel_bp += len; if (len > s.max_indel_bp) s.max_indel_bp = len; s.aligned_bp += len; break; case 'D': s.n_indel_ops++; s.total_indel_bp += len; if (len > s.max_indel_bp) s.max_indel_bp = len; s.ref_span += len; break; case 'N': s.ref_span += len; break; case 'S': s.clip_bp += len; break; // 'H' and 'P' contribute nothing default: break; } } // --- pull NM / AS / XS -------------------------------------------------- int32_t v = 0; if (r.GetIntTag("NM", v)) s.NM = v; if (r.GetIntTag("AS", v)) s.AS = v; if (r.GetIntTag("XS", v)) s.XS = v; if (s.NM >= 0) { // NM in BWA includes mismatches + inserted + deleted bp s.mm = std::max(0, s.NM - s.total_indel_bp); } if (s.aligned_bp > 0 && s.NM >= 0) { s.mm_rate = static_cast(s.mm) / s.aligned_bp; } if (s.AS >= 0) { const int denom = std::max(s.aligned_bp, s.ref_span); if (denom > 0) s.as_per_bp = static_cast(s.AS) / denom; } if (s.AS >= 0 && s.XS >= 0) { s.as_xs_gap = s.AS - s.XS; } // --- composite penalty -------------------------------------------------- double pen = 0.0; // Rule A: multi-indel if (s.n_indel_ops >= 2) { pen += 0.20 * (s.n_indel_ops - 1); if (s.reason.empty()) s.reason = "multi_indel(" + std::to_string(s.n_indel_ops) + ")"; } // Rule B: scattered small indels with noisy flanks. Threshold lowered to // 0.02 to match the new mm-rate sensitivity in Rule C. if (s.n_indel_ops >= 2 && s.max_indel_bp <= 20 && s.mm_rate > 0.02) { pen += 0.25; if (s.reason.empty()) s.reason = "small_indel_cluster_noisy"; } // Rule C: non-indel mismatch rate. A clean sequencing + assembly pipeline // should have ~0.1-0.5% per-base error; 1% is already suspicious, 3% is // almost always mis-assembly or mis-alignment into a repeat/paralog. // // linear slope 10 above 0.01: 1% -> 0, 2% -> 0.10, 3% -> 0.20, // 5% -> 0.40, 10% -> 0.90 // plus quadratic above 0.05: 5% -> +0, 10% -> +0.075, 15% -> +0.30, // 20% -> +0.675 // // Real indels and soft clips are excluded from mm (NM minus indel bp), so // this rule only fires on genuine base mismatches. if (s.mm_rate > 0.01) { pen += 10.0 * (s.mm_rate - 0.01); if (s.mm_rate > 0.05) { pen += 30.0 * (s.mm_rate - 0.05) * (s.mm_rate - 0.05); } if (s.reason.empty() && s.mm_rate > 0.02) s.reason = "high_mm_rate"; } // Rule D: low AS-density (only if AS was actually present) if (s.AS >= 0 && s.as_per_bp < 0.70) { pen += 2.0 * (0.70 - s.as_per_bp); if (s.reason.empty() && s.as_per_bp < 0.60) s.reason = "low_AS_density"; } s.confidence = clip_range(1.0 - pen, 0.0, 1.0); return s; } void tagContigAlignment(SeqLib::BamRecord& r, const ContigAlignScore& s) { // zc:i = round(1000 * confidence), clamped to [0, 1000] // zi:i = n_indel_ops // zm:i = max single-indel length // zr:i = round(1000 * mm_rate), clamped to [0, 1000] if (!r.MappedFlag()) return; // don't touch aux on unmapped/empty records const int32_t zc_val = clip_range(static_cast(std::lround(1000.0 * s.confidence)), int32_t{0}, int32_t{1000}); const int32_t zr_val = clip_range(static_cast(std::lround(1000.0 * s.mm_rate)), int32_t{0}, int32_t{1000}); r.AddIntTag("zc", zc_val); r.AddIntTag("zi", s.n_indel_ops); r.AddIntTag("zm", s.max_indel_bp); r.AddIntTag("zr", zr_val); } double readContigConfTag(const SeqLib::BamRecord& r) { int32_t zc = -1; if (!r.GetIntTag("zc", zc)) return -1.0; // tag truly absent return std::clamp(zc / 1000.0, 0.0, 1.0); } } // namespace svaba ================================================ FILE: src/svaba/ContigAlignmentScore.h ================================================ // ContigAlignmentScore.h // // Composite quantitative reliability score for a single contig-to-genome // alignment. The idea is: once a contig has been aligned back to the // reference (via BWAWrapper in SvabaRegionProcessor), we want a single // float in [0, 1] that summarizes "is this a clean alignment I should // trust" vs "is this a wonky multi-indel pile that BWA forced into a // repeat region". // // The score flows through svaba in three places: // 1. Written to the contig BAM record as tag `zc:i` (milli-confidence, // integer in [0, 1000] so it fits SeqLib's AddIntTag API). // 2. Mirrored onto the BreakEnd struct (field `contig_conf`) inside // BreakEnd::transferContigAlignmentData so every BreakPoint carries // both sides' scores forward. // 3. Emitted as the `contig_conf` column in bps.txt.gz and used as a // co-gate in the PASS/filter logic. #pragma once #include "SeqLib/BamRecord.h" #include namespace svaba { // Raw components + the final composite confidence. struct ContigAlignScore { // CIGAR-derived int n_indel_ops = 0; // count of I + D ops int max_indel_bp = 0; // largest single I or D length int total_indel_bp = 0; // sum of I + D bp int clip_bp = 0; // total soft-clip bp (S ops) int aligned_bp = 0; // query bp in M/I/=/X (excl. clips) int ref_span = 0; // reference bp in M/D/=/X/N // Tag-derived (may be missing; -1 = not present) int NM = -1; int AS = -1; int XS = -1; // Derived metrics int mm = 0; // ~ NM - total_indel_bp (BWA convention) double mm_rate = 0.0; // mm / aligned_bp double as_per_bp = 0.0; // AS / max(aligned_bp, ref_span) int as_xs_gap = 0; // AS - XS (0 if XS absent) // Composite confidence in [0, 1]. 1.0 = pristine single-event alignment. double confidence = 1.0; // First rule that pushed the score down; empty means nothing tripped. // Purely for logging/debug; not used by downstream filters. std::string reason; }; // Pure function: compute the score from a contig alignment record. // Does not mutate the record. ContigAlignScore scoreContigAlignment(const SeqLib::BamRecord& r); // Write the score back onto the record as integer tag `zc` in // milli-confidence units (round(1000 * confidence), clamped to [0, 1000]). // Integer tag keeps us within SeqLib's existing AddIntTag API. // Also writes `zi` (n_indel_ops), `zm` (max_indel_bp), `zr` (mm_rate*1000) // for diagnostic visibility in IGV / samtools view. void tagContigAlignment(SeqLib::BamRecord& r, const ContigAlignScore& s); // Inverse of tagContigAlignment's zc: pull the milli-confidence off a // record and return it as a double in [0, 1]. Returns 1.0 if tag absent // (so legacy records default to "trusted"). double readContigConfTag(const SeqLib::BamRecord& r); // Default PASS threshold. Anything with contig_conf below this is // demoted from PASS. 0.50 is a reasonable default; tune via opts later. inline constexpr double kContigConfPassThreshold = 0.50; // --------------------------------------------------------------------------- // Per-read SV-focused alignment score // --------------------------------------------------------------------------- // // Used by BreakPoint::splitCoverage to decide whether a read's r2c // (read-to-contig) alignment is genuinely "better than" the read's // corrected-native (read-to-reference) alignment. Only when r2c is // strictly better do we credit the read as variant-supporting. Both sides // must use the SAME corrected read sequence and the SAME BWA parameters // (svaba's internal aligner) — see svabaRead::corrected_native_cig. // // Scoring philosophy: an indel-bearing alignment should NOT be allowed to // approach the same score as a clean ungapped one. We therefore use a // BWA-MEM-style gap penalty (gap-open dominant, modest gap-extend) that // is much heavier than the per-bp mismatch cost, so a single indel op // definitively pushes the score below a clean alignment of the same // length. Mismatches are penalized at a smaller per-bp rate. Soft-clips // are penalized 1:1 with matched bp (a clipped base contributes nothing // AND costs as much as it would have earned, mirroring the intuition that // a clip is "wasted" query sequence). // // Penalty schedule (units = "effective matched bp"; loosely modeled on // BWA-MEM defaults match=+1 / mm=-4 / gapO=-6 / gapE=-1): // // score = aligned_bp (M / I / = / X query bp) // - kReadClipPenaltyPerBp * clip_bp (S query bp) // - kReadGapOpenPenalty * indel_ops (count of I + D ops) // - kReadGapExtendPenaltyPerBp * indel_bp (sum of I + D bp) // - kReadMismatchPenaltyPerBp * mismatches (NM - indel_bp; >=0) // // Worked examples (k_clip=1, k_gapO=6, k_gapE=1, k_mm=4): // 150M NM=0 -> 150 clean baseline // 80M70S NM=0 -> 80 - 70 = 10 big clip // 90M60S NM=0 -> 90 - 60 = 30 // 38M3I109M NM=3 -> 150 - 6 - 3 = 141 one 3bp indel, // no mismatches // 75M5I70M NM=5 -> 150 - 6 - 5 = 139 // 75M5D75M NM=5 -> 150 - 6 - 5 = 139 D doesn't consume // query but NM counts // 150M NM=4 -> 150 - 4*4 = 134 4 mismatches, no // indels (still well // above an indel- // bearing alignment // of equal length — // that's the point) // // Decision rule in splitCoverage: accept iff // r2cScore > correctedNativeScore (apples-to-apples comparison). // Strict ">" so ties don't credit the read. // // `nm` may be -1 (tag absent); the mismatch term is dropped in that case // but the CIGAR-derived gap-open / gap-extend / clip terms still apply. inline constexpr double kReadClipPenaltyPerBp = 1.0; inline constexpr double kReadGapOpenPenalty = 6.0; inline constexpr double kReadGapExtendPenaltyPerBp = 1.0; inline constexpr double kReadMismatchPenaltyPerBp = 4.0; inline double readAlignmentScore(const SeqLib::Cigar& cig, int nm) { int aligned_bp = 0; // M / I / = / X (consumes query) int clip_bp = 0; // S int indel_ops = 0; // count of I + D ops int indel_bp = 0; // sum of I + D bp for (const auto& c : cig) { const int len = static_cast(c.Length()); switch (c.Type()) { case 'M': case '=': case 'X': aligned_bp += len; break; case 'I': aligned_bp += len; // I consumes query indel_ops += 1; indel_bp += len; break; case 'D': indel_ops += 1; indel_bp += len; // D doesn't consume query break; case 'S': clip_bp += len; break; // 'N', 'H', 'P' contribute nothing. default: break; } } double score = static_cast(aligned_bp) - kReadClipPenaltyPerBp * clip_bp - kReadGapOpenPenalty * indel_ops - kReadGapExtendPenaltyPerBp * indel_bp; if (nm >= 0) { // BWA convention: NM = mismatches + inserted_bp + deleted_bp. Subtract // out indel_bp to recover the pure mismatch count; clamp at 0 in case // CIGAR and NM disagree (e.g. NM tag absent / stale). const int mm = std::max(0, nm - indel_bp); score -= kReadMismatchPenaltyPerBp * mm; } return score; } } // namespace svaba ================================================ FILE: src/svaba/DBSnpFilter.cpp ================================================ #include "DBSnpFilter.h" #include "gzstream.h" #include "SvabaLogger.h" #include "BreakPoint.h" #include "SeqLib/BamHeader.h" #include "SeqLib/GenomicRegion.h" using SeqLib::BamHeader; using SeqLib::AddCommas; using SeqLib::GenomicRegion; using SeqLib::GRC; DBSnpSite::DBSnpSite(const std::string& tchr, const std::string& pos, const std::string& rs, const std::string& ref, const std::string& alt, const BamHeader& h) { // make the genomic region try { GenomicRegion gr(tchr, pos, pos, h); chr = gr.chr; pos1 = std::stoi(pos); } catch (...) { //std::cerr << "DBSnpSite: Error trying to convert " << tchr << ":" << pos << " to number" << std::endl; } //m_rs = rs; //m_ref = ref; //m_alt = alt; if (ref.length() == 0 || alt.length() == 0) // || m_rs.length() == 0) std::cerr << "DBSnpSite: Is the VCF formated correctly for this entry? Ref " << ref << " ALT " << alt << " rs " << rs << std::endl; // insertion if (ref.length() == 1) pos2 = pos1 + 1; // deletion else pos2 = pos1 + ref.length() + 1; } DBSnpFilter::DBSnpFilter(const std::string& db, const SeqLib::BamHeader& header, SvabaLogger& logger) { logger.log(true, true, "...loading the DBsnp database ", db); // First, try opening the file (gzipped or not) igzstream in(db.c_str()); if (!in) { logger.log(true,true, //toerr, tolog "ERROR: Cannot open DBSNP database file '", db, "' for reading." ); throw std::runtime_error("Cannot open DBSnp database: " + db); } std::string line; std::ostringstream cig; while (std::getline(in, line)) { if (line.empty() || line[0] == '#') continue; std::istringstream iss(line); std::string chr, pos, rs, ref, alt; if (!(iss >> chr >> pos >> rs >> ref >> alt)) { logger.log( true, true, "WARNING: malformed VCF line in ", db, ": '", line, "'. Skipping." ); continue; } // we only care about indels (ref+alt length > 2) if (ref.size() + alt.size() <= 2) continue; DBSnpSite site(chr, pos, rs, ref, alt, header); m_sites.add(site); // build a simple chr_pos hash cig.str(""); cig << chr << "_" << site.pos1; m_int_hash.insert(hasher(cig.str())); } // finalize our index m_sites.CreateTreeMap(); logger.log(true,true, "Loaded ", m_sites.size(), " indel sites from DBSNP file '", db, "'."); } std::ostream& operator<<(std::ostream& out, const DBSnpFilter& d) { out << "DBSnpFilter with a total of " << AddCommas(d.m_sites.size()); return out; } std::ostream& operator<<(std::ostream& out, const DBSnpSite& d) { //out << d.chr << ":" << d.pos1 << "-" << d.pos2 << "\t" << d.m_rs << " REF " << d.m_ref << " ALT " << d.m_alt; out << d.chr << ":" << d.pos1 << "-" << d.pos2;; return out; } bool DBSnpFilter::queryHash(const std::string& h) const { //return m_hash.count(h); return m_int_hash.count(hasher(h)); } bool DBSnpFilter::queryBreakpoint(BreakPoint& bp) { std::vector sub, que; GenomicRegion gr = bp.b1.gr; gr.Pad(2); GRC subject(gr); GRC out = subject.FindOverlaps(m_sites, sub, que, true); // true = ignore_strand if (que.size()) { //bp.rs = m_sites[sub[0]].m_rs; bp.rs = "D"; //for (auto& j : que) { //bp.rs += m_sites[j].m_rs + "_"; //} //bp.rs.pop_back(); // just drop the last comma return true; } return false; } ================================================ FILE: src/svaba/DBSnpFilter.h ================================================ #pragma once #include #include #include #include #include //for std::hash #include "SeqLib/GenomicRegionCollection.h" class BreakPoint; class SvabaLogger; namespace SeqLib { class BamHeader; } class DBSnpSite: public SeqLib::GenomicRegion { public: DBSnpSite(const std::string& tchr, const std::string& pos, const std::string& rs, const std::string& ref, const std::string& alt, const SeqLib::BamHeader& h); //std::string m_rs; //std::string m_ref; //std::string m_alt; friend std::ostream& operator<<(std::ostream& out, const DBSnpSite& d); }; typedef SeqLib::GenomicRegionCollection DBC; class DBSnpFilter { public: DBSnpFilter() {} DBSnpFilter(const std::string& db, const SeqLib::BamHeader& h, SvabaLogger& logger); /** Test whether the variant overlaps a DBSnp site * If it does, fill the BreakPoint rs field */ bool queryBreakpoint(BreakPoint& bp); bool queryRead(const SeqLib::BamRecord& r) const; bool queryHash(const std::string& r) const; friend std::ostream& operator<<(std::ostream& out, const DBSnpFilter& d); private: std::hash hasher; // initialize here once std::stringstream cig; DBC m_sites; std::unordered_set m_hash; std::unordered_set m_int_hash; }; ================================================ FILE: src/svaba/DiscordantCluster.cpp ================================================ #include "DiscordantCluster.h" #include #include #include #include #include "SeqLib/BamWalker.h" #include "SvabaUtils.h" #define DISC_PAD 150 #define MIN_PER_CLUST 2 #define MAX_CLUSTER_WIDTH 3000 #define MAX_REGION_WIDTH 5000 #define DEFAULT_ISIZE_THRESHOLD 2000 // shouldn't be hit if isize was learned //#define DEBUG_CLUSTER 1 namespace SeqLib { class BamHeader; class GenomicRegion; } using SeqLib::GenomicRegion; DiscordantClusterMap DiscordantCluster::clusterReads(svabaReadPtrVector& bav, const SeqLib::GenomicRegion& interval, const SeqLib::BamHeader& header) { // sort by position std::sort(bav.begin(), bav.end(), SeqLib::BamRecordSort::ByReadPositionSharedPtr()); svabaReadClusterVector fwd, rev, fwdfwd, revrev, fwdrev, revfwd; //debug // for (const auto& b : bav) { // if (b->Qname() == "LH00306:129:227V5CLT4:6:2114:6074:25724") // std::cerr << " CLUSTER " << *b << std::endl; // } // make the fwd and reverse READ clusters. dont consider mate yet __cluster_reads(bav, fwd, rev, SeqLib::Orientation::FR); __cluster_reads(bav, fwd, rev, SeqLib::Orientation::FF); __cluster_reads(bav, fwd, rev, SeqLib::Orientation::RF); __cluster_reads(bav, fwd, rev, SeqLib::Orientation::RR); #ifdef DEBUG_CLUSTER for (auto& i : fwd) { std::cerr << "fwd cluster " << std::endl; for (auto& j : i) std::cerr << "fwd " << *j << std::endl; } for (auto& i : rev) { std::cerr << "rev cluster " << std::endl; for (auto& j : i) std::cerr << "rev " << *j << std::endl; } #endif // within the forward read clusters, cluster mates on fwd and rev __cluster_mate_reads(fwd, fwdfwd, fwdrev); // within the reverse read clusters, cluster mates on fwd and rev __cluster_mate_reads(rev, revfwd, revrev); // we have the reads in their clusters. Just convert to discordant reads clusters DiscordantClusterMap dcm; __convertToDiscordantCluster(dcm, fwdfwd, bav, header); __convertToDiscordantCluster(dcm, fwdrev, bav, header); __convertToDiscordantCluster(dcm, revfwd, bav, header); __convertToDiscordantCluster(dcm, revrev, bav, header); #ifdef DEBUG_CLUSTER std::cerr << "----fwd cluster count: " << fwd.size() << std::endl; std::cerr << "----rev cluster count: " << rev.size() << std::endl; std::cerr << "----fwdfwd cluster count: " << fwdfwd.size() << std::endl; std::cerr << "----fwdrev cluster count: " << fwdrev.size() << std::endl; std::cerr << "----revfwd cluster count: " << revfwd.size() << std::endl; std::cerr << "----revrev cluster count: " << revrev.size() << std::endl; for (auto& ii : fwdrev) { std::cerr << " ____________ CLUSTER ______________" << std::endl; for (auto& jj : ii) std::cerr << "FWDREV _____ " << *jj << std::endl; } for (auto& ii : revfwd) { std::cerr << " ____________ CLUSTER ______________" << std::endl; for (auto& jj : ii) std::cerr << "FWDREV _____ " << *jj << std::endl; } #endif // // score by number of maps // for (auto d : dd_clean) { // for (auto& r : d.second.reads) { // double rr = r.second.GetDD(); // d.second.read_score += (rr > 0) ? 1/rr : 1; // } // for (auto& r : d.second.mates) { // double rr = r.second.GetDD(); // d.second.mate_score += (rr > 0) ? 1/rr : 1; // } // } return dcm; } // this reads is reads in the cluster. all_reads is big pile where all the clusters came from DiscordantCluster::DiscordantCluster(const svabaReadPtrVector& this_reads, const svabaReadPtrVector& all_reads, const SeqLib::BamHeader& header) { assert(this_reads.size()); assert(all_reads.size()); // check the orientations, fill the reads auto first_read = this_reads.at(0); auto last_read = this_reads.back(); bool rev = first_read->ReverseFlag(); bool mrev = first_read->MateReverseFlag(); // loop the reads in this cluster for (auto& i : this_reads) { // double check that we did the clustering correctly. All read orientations should be same assert(rev == i->ReverseFlag() && mrev == i->MateReverseFlag()); // add the read to the read map std::string tmp = i->UniqueName(); // this name like t001_165_qname assert(tmp.length()); reads[tmp] = i; // count number of reads per BAM ++counts[i->Prefix()]; if (tmp.at(0) == 't') { ++tcount; } else { ++ncount; } } // loop through the big stack of reads and find the mates addMateReads(all_reads); assert(reads.size()); // set the regions m_reg1 = GenomicRegion(-1, -1, -1); m_reg1.pos1 = INT_MAX; m_reg2 = GenomicRegion(-1, -1, -1); // mate region m_reg2.pos1 = INT_MAX; SeqLib::Orientation por; for (const auto& [_, read] : reads) { por = read->PairOrientation(); m_reg1.strand = read->ReverseFlag() ? '-' : '+'; m_reg2.strand = read->MateReverseFlag() ? '-' : '+'; m_reg1.chr = read->ChrID(); m_reg2.chr = read->MateChrID(); // get left side if (read->Position() < m_reg1.pos1) m_reg1.pos1 = read->Position(); if (read->MatePosition() < m_reg2.pos1) m_reg2.pos1 = read->MatePosition(); // get right side if (read->PositionEnd() > m_reg1.pos2) m_reg1.pos2 = read->PositionEnd(); if (read->MatePosition() > m_reg2.pos2) m_reg2.pos2 = read->MatePosition() + read->Length(); // since don't have mate end // Check region width constraints after building regions if (m_reg1.Width() >= MAX_REGION_WIDTH) { std::cerr << "Warning: Region 1 width (" << m_reg1.Width() << "bp) exceeds " << MAX_REGION_WIDTH << "bp limit. Cluster is too large." << std::endl; } if (m_reg2.Width() >= MAX_REGION_WIDTH) { std::cerr << "Warning: Region 2 width (" << m_reg2.Width() << "bp) exceeds " << MAX_REGION_WIDTH << "bp limit. Cluster is too large." << std::endl; } } mapq1 = __getMeanMapq(reads); mapq2 = __getMeanMapq(mates); nm1 = __getMeanNM(reads); nm2 = __getMeanNM(mates); assert(mapq1 >= 0); assert(mapq2 >= -1); // can have -1 as placeholder if no mate reads (bc didnt do lookup) // orient them correctly so that left end is first if (m_reg2 < m_reg1) { std::swap(m_reg1, m_reg2); std::swap(reads, mates); std::swap(mapq1, mapq2); } // set the ID std::string ortid; switch (por) { case SeqLib::Orientation::FR: ortid = "FR"; break; case SeqLib::Orientation::FF: ortid = "FF"; break; case SeqLib::Orientation::RF: ortid = "RF"; break; case SeqLib::Orientation::RR: ortid = "RR"; break; default: ortid = "UD"; break; // fallback for UD or anything unexpected } int pos1 = m_reg1.strand == '+' ? m_reg1.pos2 : m_reg1.pos1; int pos2 = m_reg2.strand == '+' ? m_reg2.pos2 : m_reg2.pos1; m_id = ortid + "_" + m_reg1.ChrName(header) + "_" + std::to_string(pos1) + "___" + m_reg2.ChrName(header) + "_" + std::to_string(pos2); } void DiscordantCluster::addMateReads(const svabaReadPtrVector& bav) { if (!reads.size()) return; // log the qnames // so qnames is just the same map as reads except: // -- qnames: qname : svabaRead // -- reads: uniquesame : svabaRead // the qnames structure is useful here because when we search the pile // of reads for the mate, the read and mate should have same qname // but then it's good to have the read when we are looking at the mate // to check some things (e.g. read and mate are different members of pair) std::unordered_map qnames; for (auto& [_,read] : reads) qnames.insert({read->Qname(), read}); // OK, so this is necessary because... // we are looping through and trying to fill mate reads by comparing // against qname of reads. BUT this can be problematic if there are secondary // or supplementarty alignments. So use this region to check that mate ALSO agrees // with mate regions auto& first_read = reads.begin()->second; GenomicRegion g(first_read->MateChrID(), first_read->MatePosition(), first_read->MatePosition()); g.Pad(DISC_PAD + 1000); // loop all of the reads and find the mates, // if we have them (don't have to) for (const auto& i : bav) { // first check if this read has same name as one already stored in reads auto it_read = qnames.find(i->Qname()); if (it_read == qnames.end()) // qname not found, so this read can't be a matching mate continue; // now, check if this is the same read as we already now about (in reads) // or if it is not (the its the mate) std::string tmp = i->UniqueName(); auto it = reads.find(tmp); if (it != reads.end()) // shoot, this it just the read as "read", so we're done continue; // now check that the read and this one (i = candidate mate) are opposite in pair if (i->FirstFlag() == it_read->second->FirstFlag()) continue; // check that the orientation if (i->PairOrientation() != it_read->second->PairOrientation()) continue; // don't want to deal with these if (i->SecondaryFlag() || it_read->second->SecondaryFlag()) continue; // agrees with intiial mate orientation and position if (g.GetOverlap(i->AsGenomicRegion()) > 0) mates[tmp] = i; } } double DiscordantCluster::__getMeanMapq(const DiscordantReadMap& m) const { if (m.empty()) return -1.0; // sum them up double total = 0.0; for (auto const& kv : m) { total += kv.second->MapQuality(); } return total / m.size(); } double DiscordantCluster::__getMeanNM(const DiscordantReadMap& m) const { if (m.empty()) return -1.0; // sum them up double total = 0.0; for (auto const& kv : m) { int nm = 0; kv.second->GetIntTag("NM", nm); total += nm; } return total / m.size(); } void DiscordantCluster::labelReads() { for (auto& [_, r] : reads) { std::string dcstring; r->GetZTag("DC", dcstring); if (!dcstring.empty()) dcstring.append("d"); dcstring.append(m_id); r->AddZTag("DC", dcstring); } for (auto& [_, r] : mates) { std::string dcstring; r->GetZTag("DC", dcstring); if (!dcstring.empty()) dcstring.append("d"); dcstring.append(m_id); r->AddZTag("DC", dcstring); } } std::string DiscordantCluster::toRegionString(const SeqLib::BamHeader& h) const { int pos1 = (m_reg1.strand == '+') ? m_reg1.pos2 : m_reg1.pos1; int pos2 = (m_reg2.strand == '+') ? m_reg2.pos2 : m_reg2.pos1; std::stringstream ss; ss << h.IDtoName(m_reg1.chr) << ":" << pos1 << "(" << m_reg1.strand << ")" << "-" << h.IDtoName(m_reg2.chr) << ":" << pos2 << "(" << m_reg2.strand << ")"; return ss.str(); } // define how to print this std::string DiscordantCluster::print(const SeqLib::BamHeader& h) const { std::stringstream ss; ss << toRegionString(h) << " Tcount: " << tcount << " Ncount: " << ncount << " Mean MAPQ: " << mapq1 << " Mean Mate MAPQ: " << mapq2 << " Valid: " << (valid() ? "TRUE" : "FALSE"); return ss.str(); } bool DiscordantCluster::valid() const { // it's OK if the clusters are inter-chromosomal if (m_reg1.chr != m_reg2.chr) return true; // if its RF orientation (<----->) then bc bps are on outside, regions can overlaps if (m_reg1.strand == '-' && m_reg2.strand == '+') return true; // the clusters overlap, doesn't make sense for del and inversion type if (m_reg1.pos2 > m_reg2.pos1) return false; return true; } // define how to print to file std::string DiscordantCluster::toFileString(const SeqLib::BamHeader& h, bool with_read_names) const { std::string sep = "\t"; // get the edge of the cluster int pos1 = m_reg1.strand == '+' ? m_reg1.pos2 : m_reg1.pos1; int pos2 = m_reg2.strand == '+' ? m_reg2.pos2 : m_reg2.pos1; std::string chr1 = isEmpty() ? "NOTSET" : h.IDtoName(m_reg1.chr); std::string chr2 = isEmpty() ? "NOTSET" : h.IDtoName(m_reg2.chr); std::stringstream out; out << chr1 << sep << pos1 << sep << m_reg1.strand << sep << chr2 << sep << pos2 << sep << m_reg2.strand << sep << tcount << sep << ncount << sep << mapq1 << sep << mapq2 << sep << nm1 << sep << nm2 << sep << (m_contig.length() ? m_contig : "x") << sep << m_id; return (out.str()); } // define how to sort theses bool DiscordantCluster::operator<(const DiscordantCluster &b) const { if (m_reg1.chr < b.m_reg1.chr) return true; if (m_reg1.pos1 < b.m_reg1.pos1) return true; return false; } /** * Cluster reads by alignment position * * Checks whether a read belongs to a cluster. If so, adds it. If not, ends * and stores cluster, adds a new one. * * @param cvec Stores the vector of clusters, which grows here * @param clust The current cluster that is being added to * @param a Read to add to cluster * @param mate Flag to specify if we should cluster on mate position instead of read position * @return Description of the return value */ bool DiscordantCluster::__add_read_to_cluster(svabaReadClusterVector& cvec, svabaReadPtrVector& clust, svabaReadPtr& a, bool ismate) { // when ismate is false - just look at this read, ignore the mate // when ismate is true - just consider the mate of this read, ignore this pairmate // last info stores the latest position of the "end" of this cluster std::pair last_info; if (clust.empty()) { last_info = {-1, -1}; } else if (ismate) { last_info = {clust.back()->MateChrID(), clust.back()->MatePosition()}; } else { last_info = {clust.back()->ChrID(), clust.back()->Position()}; } // what is the position of the input read std::pair this_info; if (ismate) this_info = {a->MateChrID(), a->MatePosition()}; else this_info = {a->ChrID(), a->Position()}; // check if the cluster is getting too wide bool too_wide = !__valid_cluster(clust, ismate); // if cluster not too wide, and read is on the same chromosome // and the difference in position is less than the discordant "coupling" // parameter, then add it to this cluster if (!too_wide && this_info.first == last_info.first && (this_info.second - last_info.second) <= DISC_PAD) { clust.push_back(a); return true; // if not, then we're done with the cluster. The key is that the // input reads are position sorted, so that if this didn't make it, then // the rest won't, and we whould start the next cluster with the given read } else { if (clust.size() >= MIN_PER_CLUST) cvec.push_back(clust); clust.clear(); clust.push_back(a); return false; } } void DiscordantCluster::__cluster_mate_reads(svabaReadClusterVector& brcv, svabaReadClusterVector& fwd, svabaReadClusterVector& rev) { // loop the nascent "clustesr" which are just clustered based on // left read, not on the right read yet for (auto& cluster : brcv) { svabaReadPtrVector this_fwd, this_rev; // sort the reads in the cluster std::sort(cluster.begin(), cluster.end(), [](const svabaReadPtr& a, const svabaReadPtr& b) { return (a->MateChrID() < b->MateChrID()) || (a->MateChrID() == b->MateChrID() && a->MatePosition() < b->MatePosition()); }); // loop the reads in the nascent cluster for (svabaReadPtr& r : cluster) { assert(r->dd > 0); // see __cluster_reads for logic. Exactly the same, just now for mates if ( !r->MateReverseFlag()) __add_read_to_cluster(fwd, this_fwd, r, true); else __add_read_to_cluster(rev, this_rev, r, true); } // finish the last clusters if (__valid_cluster(this_fwd, true) && this_fwd.size() >= MIN_PER_CLUST) fwd.push_back(this_fwd); if (__valid_cluster(this_rev, true) && this_rev.size() >= MIN_PER_CLUST) rev.push_back(this_rev); } } void DiscordantCluster::__cluster_reads(svabaReadPtrVector& brv, svabaReadClusterVector& fwd, svabaReadClusterVector& rev, SeqLib::Orientation orientation) { // hold the current cluster svabaReadPtrVector this_fwd, this_rev; std::unordered_set tmp_set; // int pair_orientation = 0; // if (brv.size()) // pair_orientation = brv.front()->PairOrientation(); // cluster in the READ direction, separately for fwd and rev for (svabaReadPtr& i : brv) { // don't want to deal with secondary alignments (multiple mappings, but not split) if (i->SecondaryFlag()) continue; // if (pair_orientation != i->PairOrientation()) { // std::cerr << *i << std::endl; // std::cerr << i->PairOrientation() << std::endl; // std::cerr << pair_orientation << std::endl; // } // not a discordant read, ignore it if (i->dd <= 0) continue; // only cluster FR reads together, RF reads together, FF together and RR together // PairOrientation is invariant to if you are looking at read or mate // - that is, if you are FR from read, then the mate is not read as RF. // - it ensures that the left most read (lowest chr and pos) is the index read if (i->PairOrientation() != orientation) { continue; } // only cluster if not seen before (e.g. left-most is READ, right most is MATE) if (i->PairMappedFlag() && tmp_set.count(i->Qname()) == 0) { //20250911 // right most read acts like the "mate". // the reason to do this is that if I have both the read and it's mate, I dont // want to arbitrarily add one of the two to either fwd or reverse if (!i->IsLeftMostAlignment()) continue; tmp_set.insert(i->Qname()); // forward clustering -- this_fwd is just the most current cluster and // fwd (or rev) is the collection of clusters that grows here // convention here is that the left-most read acts like the "index read" and the if ( !i->ReverseFlag()) __add_read_to_cluster(fwd, this_fwd, i, false); // reverse clustering else __add_read_to_cluster(rev, this_rev, i, false); } } // finish the last clusters if (__valid_cluster(this_fwd, false) && this_fwd.size() >= MIN_PER_CLUST) fwd.push_back(this_fwd); if (__valid_cluster(this_rev, false) && this_rev.size() >= MIN_PER_CLUST) rev.push_back(this_rev); } bool DiscordantCluster::__valid_cluster(svabaReadPtrVector& clust, bool ismate) { if (clust.size() < 2) return true; // check if getting too wide bool too_wide; if (ismate) too_wide = (clust.size() > 1 && (clust.back()->Position() - clust[0]->Position()) > MAX_CLUSTER_WIDTH); else too_wide = (clust.size() > 1 && (clust.back()->Position() - clust[0]->Position()) > MAX_CLUSTER_WIDTH); return !too_wide; } void DiscordantCluster::__convertToDiscordantCluster(DiscordantClusterMap &dd, const svabaReadClusterVector& cvec, const svabaReadPtrVector& bav, const SeqLib::BamHeader& header) { // nothign to do if no clusters if (cvec.size() == 0) return; // always have to be more input reads than clusters assert(bav.size() > cvec.size()); // no reads, nothign to do if (!bav.size()) return; // loop through the clusters for (auto& v : cvec) { DiscordantCluster d(v, bav, header); dd[d.m_id] = d; } } GenomicRegion DiscordantCluster::GetMateRegionOfOverlap(const GenomicRegion& gr) const { if (gr.GetOverlap(m_reg1)) return m_reg2; if (gr.GetOverlap(m_reg2)) return m_reg1; return GenomicRegion(); } bool DiscordantCluster::isEmpty() const { return m_reg1.IsEmpty() || m_reg2.IsEmpty() || m_reg1.chr == -1 || m_reg2.chr == -1; } ================================================ FILE: src/svaba/DiscordantCluster.h ================================================ #pragma once #include #include #include #include #include #include "SvabaRead.h" #include "SvabaSharedConfig.h" class DiscordantCluster; typedef std::unordered_map DiscordantReadMap; typedef std::vector svabaReadClusterVector; typedef std::vector DiscordantClusterVector; typedef std::unordered_map DiscordantClusterMap; /** Class to hold clusters of discordant reads */ class DiscordantCluster { friend struct BreakPoint; public: /** Create an empty cluster */ DiscordantCluster() { m_reg1 = SeqLib::GenomicRegion(); m_reg2 = SeqLib::GenomicRegion(); assert(m_reg1.IsEmpty()); ncount = 0; tcount = 0; mapq1 = -1; mapq2 = -1; } /** Make a cluster from a set of reads (pre-clustered) and look up a larger set to find * their mates * @param this_reads Pre-clustered set of discordant reads (but not their mates) * @param all_reads A pile of reads to search for mates */ DiscordantCluster(const svabaReadPtrVector& this_reads, const svabaReadPtrVector& all_reads, const SeqLib::BamHeader& header); /** Is this discordant cluster empty? */ bool isEmpty() const; /** Return a string representing the output file header */ static std::string header() { return "chr1\tpos1\tstrand1\tchr2\tpos2\tstrand2\ttcount\tncount\t\tmapq1\tmapq2\tnm1\tnm2\tcname\tid"; } bool hasAssociatedAssemblyContig() const { return m_contig.length(); } void addMateReads(const svabaReadPtrVector& bav); /** Return the discordant cluster as a string with just coordinates */ std::string toRegionString(const SeqLib::BamHeader& h) const; /** Return the ID associated with this cluster */ std::string ID() const { return m_id; } /** Print this with region string and read counts and mapq */ //friend std::ostream& operator<<(std::ostream& out, const DiscordantCluster& dc); /** Print this with region string and read counts and mapq */ std::string print(const SeqLib::BamHeader& h) const; /** Return as a string for writing to a file */ std::string toFileString(const SeqLib::BamHeader& h, bool with_read_names) const; /** Sort by coordinate */ bool operator < (const DiscordantCluster& b) const; /** Is this a valid cluster? */ bool valid() const; static std::unordered_map clusterReads(svabaReadPtrVector& bav, const SeqLib::GenomicRegion& interval, const SeqLib::BamHeader& header); void labelReads(); static bool __add_read_to_cluster(svabaReadClusterVector &cvec, svabaReadPtrVector &clust, svabaReadPtr&, bool ismate); static void __cluster_reads(svabaReadPtrVector& brv, svabaReadClusterVector& fwd, svabaReadClusterVector& rev, SeqLib::Orientation orientation); static void __cluster_mate_reads(svabaReadClusterVector& brcv, svabaReadClusterVector& fwd, svabaReadClusterVector& rev); static void __convertToDiscordantCluster(DiscordantClusterMap& dd, const svabaReadClusterVector& cvec, const svabaReadPtrVector& bav, const SeqLib::BamHeader& header); static bool __valid_cluster(svabaReadPtrVector& clust, bool ismate); /** Query an interval against the two regions of the cluster. If the region overlaps * with one region, return the other region. This is useful for finding the partner * region give a query region */ SeqLib::GenomicRegion GetMateRegionOfOverlap(const SeqLib::GenomicRegion& gr) const; int tcount = 0; int ncount = 0; // supporting read counts per sample (e.g. t001 - 4, n001 - 6) std::unordered_map counts; DiscordantReadMap reads, mates; std::string m_contig = ""; double read_score = 0; double mate_score = 0; //int rp_orientation = -1; // FR, FF, RR, RF SeqLib::GenomicRegion m_reg1; SeqLib::GenomicRegion m_reg2; int mapq1; int mapq2; float nm1; float nm2; std::string m_id_competing; // id of discordant cluster with same span, different strands private: std::string m_id; // return the mean mapping quality for this cluster double __getMeanMapq(const DiscordantReadMap& m) const; // return the mean mismatch score for this cluster double __getMeanNM(const DiscordantReadMap& m) const; }; //! vector of AlignmentFragment objects typedef std::vector DiscordantClusterVector; //! Store a set of DiscordantCluster objects, indexed by the "id" field typedef std::unordered_map DiscordantClusterMap; ================================================ FILE: src/svaba/DiscordantRealigner.cpp ================================================ #include "DiscordantRealigner.h" #include "SvabaUtils.h" #include "SeqLib/BWAAligner.h" #ifdef QNAME #define DEBUG(msg, read) \ if (read.Qname() == QNAME && (read.AlignmentFlag() == QFLAG || QFLAG == -1)) { std::cerr << (msg) << " read " << r << std::endl; } #else #define DEBUG(msg, read) #endif //bool DiscordantRealigner::ShouldRealign(const SeqLib::BamRecord& r) const { bool DiscordantRealigner::ShouldRealign(const svabaReadPtr& r) const { //if (r.SR() == "t000_147_H01PEALXX140819:3:1219:25827:26572") // std::cerr << " DD " << r.GetDD() << " fi " << r.FullInsertSize() << " lim " << min_isize_for_discordant_realignment << std::endl; // read was already realigned if (r->GetDD() != 0) return false; // read can't be discordant anyway, since not both mapped if (!r->MappedFlag() || !r->MateMappedFlag()) return false; if (r->Interchromosomal()) return true; // get the insert size int fi = r->FullInsertSize(); // if too small, don't realign if (fi < min_isize_for_discordant_realignment && !r->Interchromosomal()) return false; return true; } //bool DiscordantRealigner::RealignRead(SeqLib::BamRecord& r, const SeqLib::BWAWrapper* bwa) const { bool DiscordantRealigner::RealignRead(svabaReadPtr& r, const std::shared_ptr bwa) const { DEBUG("Realigning original read", r); BamRecordPtrVector als; bwa->alignSequence(r->CorrectedSeq(), r->Qname(), als, false, 0.60, secondary_cap); // no alignments, so label as bad if (!als.size()) { r->SetDD(MAPS_NOWHERE); return false; } // discordant alignments SeqLib::GenomicRegion gr = r->AsGenomicRegion(); SeqLib::GenomicRegion grm = r->AsGenomicRegionMate(); grm.Pad(discordant_realign_mate_pad); bool has_orig = false; bool maps_near_mate = false; for (const auto& i : als) { DEBUG(" Realigned hit (one of) " + std::to_string(als.size()), *i); // if there is another alignment that overlaps with the original // but has a lower MAPQ, take the new mapq if (gr.GetOverlap(i->AsGenomicRegion())) { has_orig = true; r->SetMapQuality(std::min(i->MapQuality(), r->MapQuality())); } DEBUG(" Realigned hit has overlap with mate of N bases: " + std::to_string(r->OverlappingCoverage), i); // if mate is mapped, and read maps to near mate region wo clips, it's not disc if (grm.GetOverlap(i->AsGenomicRegion())) { // if not clipped or overlaps same covered region as original, // then it has a secondary mapping if (r->NumClip() < 20 || r->OverlappingCoverage(*i) >= 20) maps_near_mate = true; DEBUG(" Found realignment hit near mate", i); } } // end als loop // add tags if (!has_orig) r->SetDD(MAPS_NOT_NEAR_ORIG); else if (maps_near_mate) r->SetDD(MAPS_NEAR_MATE); else r->SetDD(als.size()); return r->GetDD() < 0; // was it realigned? } /*void DiscordantRealigner::ReassignRead(svabaRead& r, const svabaRead& s) const { r.Reassign(s); r.SetDD(REASSIGNED_READ); return; }*/ ================================================ FILE: src/svaba/DiscordantRealigner.h ================================================ #pragma once namespace SeqLib { class BWAAligner; } #include "SvabaRead.h" // object that holds functions for re-aligning a discordant read // and checking if it is a true discordant read class DiscordantRealigner { public: DiscordantRealigner() {} ~DiscordantRealigner() {} // check if a read should be realigned //bool ShouldRealign(const SeqLib::BamRecord& r) const; bool ShouldRealign(const svabaReadPtr& r) const; // realign read bool RealignRead(svabaReadPtr& r, const std::shared_ptr bwa) const; // reassign a read if it has a better mapping // r is the read to be reassigned, s source alignment is the new alignment //void ReassignRead(SeqLib::BamRecord& r, const SeqLib::BamRecord& s) const; //void ReassignRead(svabaRead& r, const svabaRead& s) const; static const int MAPS_NOT_NEAR_ORIG = -1; static const int MAPS_NEAR_MATE = -2; static const int MAPS_NOWHERE = -3; static const int MATE_BAD_DISC = -4; static const int REASSIGNED_READ = -5; private: // max number of secondary reads to consider int secondary_cap = 20; // minimum size to check int min_isize_for_discordant_realignment = 1000; // how much to search aroudn mate region for read alignent int discordant_realign_mate_pad = 100; }; ================================================ FILE: src/svaba/Histogram.cpp ================================================ #include "Histogram.h" #include "SeqLib/SeqLibUtils.h" #include #include #include #include #define BINARY_SEARCH 1 #define DEBUG_HISTOGRAM using namespace SeqLib; Histogram::Histogram(const int32_t& start, const int32_t& end, const uint32_t& width) { if (end >= start) throw std::invalid_argument("Histogram end must be > start"); Bin bin; bin.bounds.first = start; int32_t next_end = start + width - 1; // -1 because width=1 is bound.first = bounds.second while (next_end < end) { // finish this bin bin.bounds.second = next_end; m_bins.push_back(bin); m_ind.push_back(bin.bounds.first); // make the index for the lower bound // start a new one bin.bounds.first = next_end+1; next_end += width; } // finish the last bin bin.bounds.second = end; m_bins.push_back(bin); m_ind.push_back(bin.bounds.first); // add a final bin //bin.bounds.first = end+1; //bin.bounds.second = INT_MAX; //m_bins.push_back(bin); //m_ind.push_back(bin.bounds.first); } void Histogram::toCSV(std::ofstream &fs) { for (auto& i : m_bins) fs << i << std::endl; } void Histogram::removeElem(const int32_t& elem) { --m_bins[retrieveBinID(elem)]; } void Histogram::addElem(const int32_t& elem) { ++m_bins[retrieveBinID(elem)]; } std::string Histogram::toFileString() const { std::stringstream ss; for (auto& i : m_bins) if (i.m_count) ss << i.bounds.first << "_" << i.bounds.second << "_" << i.m_count << ","; std::string out = ss.str(); out.pop_back(); // trim off last comma return(out); } size_t Histogram::retrieveBinID(const int32_t& elem) const { if (elem < m_bins[0].bounds.first) { #ifdef DEBUG_HISTOGRAM std::cerr << "removeElem: elem of value " << elem << " is below min bin " << m_bins[0] << std::endl; exit(1); #endif return 0; } if (elem > m_bins.back().bounds.second) { #ifdef DEBUG_HISTOGRAM std::cerr << "removeElem: elem of value " << elem << " is above max bin " << m_bins.back() << std::endl; exit(1); #endif return m_bins.size(); } if (m_bins[0].contains(elem)) return 0; if (m_bins.back().contains(elem)) return m_bins.size(); #ifdef BINARY_SEARCH // binary search std::vector::const_iterator it = std::upper_bound(m_ind.begin(), m_ind.end(), elem); size_t i = it - m_ind.begin()-1; assert(i < m_ind.size()); return i; #else for (size_t i = 0; i < m_bins.size(); i++) { if (m_bins[i].contains(elem)) { return i; } } #endif std::cerr << "bin not found for element " << elem << std::endl; return 0; } /*void Histogram::initialize(size_t num_bins, std::vector* pspanv, size_t min_bin_width) { // ensure that they spans are sorted std::sort(pspanv->begin(), pspanv->end()); // fill the histogram bins with matrix pairs (pre-sorted by distance) Bin bin; // get number of inter-chr events size_t intra = 0; for (auto& i : *pspanv) if (i != INTERCHR) intra++; int bin_cut = 0; try { bin_cut = floor((double)intra / (double)num_bins); if (bin_cut == 0) throw 1; } catch(...) { std::cerr << "Error in determining bin cut. Not enought events or too many bins?" << std::endl; std::cerr << "Events: " << pspanv->size() << " Num Bins " << num_bins << " quantile count (hist height) " << bin_cut << std::endl; } std::cout << "...Events per bin: " << bin_cut << " num bins " << num_bins << std::endl; S last_span = 0; size_t tcount = 0; // count events put into bins // iterate over spans for (auto& span : *pspanv) { if (span != INTERCHR) { ++tcount; // moved into a new bin? (or done?) if (bin.getCount() > bin_cut && span != last_span && (last_span - bin.bounds.first) >= min_bin_width) { // finalize, save old bin bin.bounds.second = last_span; m_bins.push_back(bin); // new bin bin.bounds.first = last_span+1; bin.m_count = 0; } ++bin; if (bin.getCount() >= bin_cut) { last_span = span; } //update the size of current bin bin.bounds.second = span; } } // add the last bin bin.bounds.second = INTERCHR-1; m_bins.push_back(bin); // add a bin for interchr events bin.bounds = {INTERCHR, INTERCHR}; bin.m_count = pspanv->size() - intra; m_bins.push_back(bin); // make the indices of lower bound for (auto& i : m_bins) m_ind.push_back(i.bounds.first); if (m_bins.size() != (num_bins+1)) { //std::cout << " bin cut " << bin_cut << std::endl; //std::cout << " num bins " << num_bins << " bins.size() " << m_bins.size() << std::endl; //assert(bins.size() == (num_bins+1)); } }*/ bool Bin::operator < (const Bin& b) const { return (bounds.first < b.bounds.first || (bounds.first==b.bounds.first && bounds.second < b.bounds.second)); } bool Bin::contains(const int32_t& elem) const { return (elem >= bounds.first && elem <= bounds.second); } Bin& Bin::operator++() { ++m_count; return *this; } Bin& Bin::operator--() { assert(m_count > 0); --m_count; return *this; } ================================================ FILE: src/svaba/Histogram.h ================================================ #ifndef SEQLIB_HISTOGRAM_H__ #define SEQLIB_HISTOGRAM_H__ #include #include #include #include #include #include #include #include "SeqLib/IntervalTree.h" class Bin; typedef SeqLib::TInterval BinInterval; typedef SeqLib::TIntervalTree BinIntervalTree; typedef std::vector BinIntervalVector; #define INTERCHR 250000000 class Histogram; /** Stores one bin in a Histogram */ class Bin { friend class Histogram; public: /** Construct a new object with 0 count and range [0,1] */ Bin() : m_count(0) { bounds = {0,1}; } /** Output the bin in format "start range, end range, count" */ friend std::ostream& operator<<(std::ostream &out, const Bin &b) { out << b.bounds.first << "," << b.bounds.second << "," << b.m_count; return out; } /** Return the number of counts in this histogram bin */ int32_t getCount() const { return m_count; } /** Check if a value fits within the range of this bin * @param dist Distance value to check if its in this range * @return true if the value is within the range */ /** Check if this bin contains a value * @param elem Value to check if its in this range * @return true if the value is within the range */ bool contains(const int32_t& elem) const; /** Define bin comparison operator by location of left bound, then right */ bool operator < (const Bin& b) const; /** Decrement the histogram bin by one. * Note that this is the prefix version only */ Bin& operator--(); /** Increment the histogram bin by one. * Note that this is the prefix version only */ Bin& operator++(); private: int32_t m_count; std::pair bounds; //@! was"bin"; }; /** Class to store histogram of numeric values. * * The bins of the Histogram are not uniformly spaced, and their ranges determined * by partitioning the spans it tablulates into uniform quantiles when initialized * by Histogram::initialSpans(). As elements are added and removed this initial bin * definition remains constant. */ class Histogram { private: std::vector m_ind; public: std::vector m_bins; /** Construct an empty histogram */ Histogram() {} /** Construct a new histogram with bins spaced evenly * @param start Min value covered * @param end Max value covered * @param width Fixed bin width * @exception Throws an invalid_argument if end <= start */ Histogram(const int32_t& start, const int32_t& end, const uint32_t& width); std::string toFileString() const; friend std::ostream& operator<<(std::ostream &out, const Histogram &h) { for (auto& i : h.m_bins) out << i << std::endl; return out; } /** Return iterator to the fist bin */ std::vector::iterator begin() { return m_ind.begin(); } /** Return iterator to the last bin */ std::vector::iterator end() { return m_ind.end(); } /** Initialize histogram from a vector of numeric values */ void Initialize(size_t num_bins, std::vector* pspanv, size_t min_bin_width = 0); /** Add an element to the histogram * @param elem Length of event to add */ void addElem(const int32_t &elem); /** Remove a span from the histogram * @param span Length of event to remove */ void removeElem(const int32_t &elem); /** Output to CSV file like: bin_start,bin_end,count */ void toCSV(std::ofstream &fs); /** Return the total number of elements in the Histogram */ int totalCount() const { int tot = 0; for (auto& i : m_bins) tot += i.getCount(); return tot; } /** Get count for a histogram bin * @param i Bin index * @return number of events in histogram bin */ int32_t binCount(size_t i) { return m_bins[i].getCount(); } /** Get number of bins in histogram * @return Number of bins in histogram */ size_t numBins() { return m_bins.size(); } /** Find bin corresponding to a span * @param elem Event length * @return Bin containing event length */ size_t retrieveBinID(const int32_t& elem) const; }; #endif ================================================ FILE: src/svaba/KmerFilter.cpp ================================================ #include "KmerFilter.h" #include "ReadTable.h" // int KmerFilter::correctReads(svabaReadVector& vec) { // if (!vec.size()) // return 0; // if (!pBWT) // return 0; // cant correct if didnt learn how // int corrected_reads = 0; // //int intervalCacheLength = 10; // SGA defaul // //int intervalCacheLength = 1; //vec[0].Length(); // BWTIntervalCache* pIntervalCache = nullptr; //new BWTIntervalCache(intervalCacheLength, pBWT); // BWTIndexSet indices; //(pBWT, pRBWT, nullptr, pIntervalCache); // indices.pBWT = pBWT; // indices.pCache = pIntervalCache; // KmerCountMap kmerCache; // for (auto& r : vec) { // // non-clipped mapped reads with no mismatches are OK (nothing to correct) // //if (r.GetIntTag("NM") == 0 && r.NumClip() == 0 && r.MappedFlag()) // // continue; // std::string readSequence = r.CorrectedSeq(); //QualityTrimmedSequence(4, dum); // std::string origSequence = readSequence; // int n = readSequence.length(); // if (n < m_kmer_len) // can't correct, too short // continue; // int nk = n - m_kmer_len + 1; // std::vector minPhredVector(nk, 25); // 25 is a dummy value // // Are all kmers in the read well-represented? // bool allSolid = false; // bool done = false; // int rounds = 0; // int maxAttempts = 3; //m_params.numKmerRounds; // while (!done && nk > 0) { // // Compute the kmer counts across the read // // and determine the positions in the read that are not covered by any solid kmers // // These are the candidate incorrect bases // std::vector countVector(nk, 0); // std::vector solidVector(n, 0); // for(int i = 0; i < nk; ++i) // { // std::string kmer; // try { // kmer = readSequence.substr(i, 31); // } catch (...) { // std::cerr << "KmerFilter substr out of bounds. seqlen " << readSequence.length() << // " stat " << i << " length " << 31 << std::endl; // } // int count = 0; // KmerCountMap::iterator iter = kmerCache.find(kmer); // if (iter != kmerCache.end()) { // count = iter->second; // } else { // count = BWTAlgorithms::countSequenceOccurrences(kmer, indices); // kmerCache.insert(std::make_pair(kmer, count)); // } // // Get the phred score for the last base of the kmer // int phred = minPhredVector[i]; // countVector[i] = count; // // Determine whether the base is solid or not based on phred scores // int threshold = CorrectionThresholds::Instance().getRequiredSupport(phred); // if(count >= threshold) // { // for(int j = i; j < i + 31; ++j) // solidVector[j] = 1; // } // } // allSolid = true; // for(int i = 0; i < n; ++i) { // if(solidVector[i] != 1) // allSolid = false; // } // // Stop if all kmers are well represented or we have exceeded the number of correction rounds // if(allSolid || rounds++ > maxAttempts) // break; // // Attempt to correct the leftmost potentially incorrect base // bool corrected = false; // for(int i = 0; i < n; ++i) // { // if(solidVector[i] != 1) // { // // Attempt to correct the base using the leftmost covering kmer // int phred = 25; //workItem.read.getPhredScore(i); // int threshold = CorrectionThresholds::Instance().getRequiredSupport(phred); // int left_k_idx = (i + 1 >= 31 ? i + 1 - 31 : 0); // corrected = attemptKmerCorrection(i, left_k_idx, std::max(countVector[left_k_idx], threshold), readSequence, indices); // if(corrected) // break; // // base was not corrected, try using the rightmost covering kmer // size_t right_k_idx = std::min(i, n - 31); // corrected = attemptKmerCorrection(i, right_k_idx, std::max(countVector[right_k_idx], threshold), readSequence, indices); // if(corrected) // break; // } // } // // If no base in the read was corrected, stop the correction process // if(!corrected) // { // assert(!allSolid); // done = true; // } // } // end while // // if allsolid // if( readSequence != origSequence) // { // ++corrected_reads; // assert(readSequence.length()); // r.AddZTag("KC", readSequence); // //std::cerr << ssi.id << std::endl; // //std::cerr << "**************Read corrected from\to " << std::endl << "\t" << ssi.seq.toString() << std::endl << "\t" << readSequence << std::endl; // //result.correctSequence = readSequence; // //result.kmerQC = true; // } // else // { // //std::cerr << "Read NOT corrected from\to " << std::endl << "\t" << ssi.seq.toString() << std::endl << "\t" << readSequence << std::endl; // //result.correctSequence = workItem.read.seq.toString(); // //result.kmerQC = false; // } // } // delete pIntervalCache; // return corrected_reads; // return 0; // } int KmerFilter::correctReads(svabaReadVector& vec) { if (!vec.size()) return 0; if (!pBWT) return 0; // cant correct if didnt learn how int corrected_reads = 0; //int intervalCacheLength = 10; // SGA defaul //int intervalCacheLength = 1; //vec[0].Length(); BWTIntervalCache* pIntervalCache = nullptr; //new BWTIntervalCache(intervalCacheLength, pBWT); BWTIndexSet indices; //(pBWT, pRBWT, nullptr, pIntervalCache); indices.pBWT = pBWT; indices.pCache = pIntervalCache; KmerCountMap kmerCache; for (auto& r : vec) { // non-clipped mapped reads with no mismatches are OK (nothing to correct) //if (r.GetIntTag("NM") == 0 && r.NumClip() == 0 && r.MappedFlag()) // continue; std::string readSequence = r.CorrectedSeq(); std::string origSequence = readSequence; int n = readSequence.length(); if (n < m_kmer_len) // can't correct, too short continue; int nk = n - m_kmer_len + 1; std::vector minPhredVector(nk, 25); // 25 is a dummy value // Are all kmers in the read well-represented? bool allSolid = false; bool done = false; int rounds = 0; int maxAttempts = 3; //m_params.numKmerRounds; while (!done && nk > 0) { // Compute the kmer counts across the read // and determine the positions in the read that are not covered by any solid kmers // These are the candidate incorrect bases std::vector countVector(nk, 0); std::vector solidVector(n, 0); for(int i = 0; i < nk; ++i) { std::string kmer; try { kmer = readSequence.substr(i, 31); } catch (...) { std::cerr << "KmerFilter substr out of bounds. seqlen " << readSequence.length() << " stat " << i << " length " << 31 << std::endl; } int count = 0; KmerCountMap::iterator iter = kmerCache.find(kmer); if (iter != kmerCache.end()) { count = iter->second; } else { count = BWTAlgorithms::countSequenceOccurrences(kmer, indices); kmerCache.insert(std::make_pair(kmer, count)); } // Get the phred score for the last base of the kmer int phred = minPhredVector[i]; countVector[i] = count; // Determine whether the base is solid or not based on phred scores int threshold = CorrectionThresholds::Instance().getRequiredSupport(phred); if(count >= threshold) { for(int j = i; j < i + 31; ++j) solidVector[j] = 1; } } allSolid = true; for(int i = 0; i < n; ++i) { if(solidVector[i] != 1) allSolid = false; } // Stop if all kmers are well represented or we have exceeded the number of correction rounds if(allSolid || rounds++ > maxAttempts) break; // Attempt to correct the leftmost potentially incorrect base bool corrected = false; for(int i = 0; i < n; ++i) { if(solidVector[i] != 1) { // Attempt to correct the base using the leftmost covering kmer int phred = 25; //workItem.read.getPhredScore(i); int threshold = CorrectionThresholds::Instance().getRequiredSupport(phred); int left_k_idx = (i + 1 >= 31 ? i + 1 - 31 : 0); corrected = attemptKmerCorrection(i, left_k_idx, std::max(countVector[left_k_idx], threshold), readSequence, indices); if(corrected) break; // base was not corrected, try using the rightmost covering kmer size_t right_k_idx = std::min(i, n - 31); corrected = attemptKmerCorrection(i, right_k_idx, std::max(countVector[right_k_idx], threshold), readSequence, indices); if(corrected) break; } } // If no base in the read was corrected, stop the correction process if(!corrected) { assert(!allSolid); done = true; } } // end while // if allsolid if( readSequence != origSequence) { ++corrected_reads; assert(readSequence.length()); //r.AddZTag("KC", readSequence); r.SetCorrectedSeq(readSequence); //std::cerr << ssi.id << std::endl; //std::cerr << "**************Read corrected from\to " << std::endl << "\t" << ssi.seq.toString() << std::endl << "\t" << readSequence << std::endl; //result.correctSequence = readSequence; //result.kmerQC = true; } else { //std::cerr << "Read NOT corrected from\to " << std::endl << "\t" << ssi.seq.toString() << std::endl << "\t" << readSequence << std::endl; //result.correctSequence = workItem.read.seq.toString(); //result.kmerQC = false; } } delete pIntervalCache; return corrected_reads; return 0; } // directly from SGA, Jared Simpson bool KmerFilter::attemptKmerCorrection(size_t i, size_t k_idx, size_t minCount, std::string& readSequence, BWTIndexSet& inds) { assert(i >= k_idx && i < k_idx + m_kmer_len); size_t base_idx = i - k_idx; char originalBase = readSequence[i]; std::string kmer; try { kmer = readSequence.substr(k_idx, m_kmer_len); } catch(...) { std::cerr << "KmerFilter::attemptKmerCorrection substr out of bounds. seqlen " << readSequence.length() << " start " << k_idx << " length " << m_kmer_len << std::endl; } size_t bestCount = 0; char bestBase = '$'; #if KMER_TESTING std::cout << "i: " << i << " k-idx: " << k_idx << " " << kmer << " " << reverseComplement(kmer) << "\n"; #endif for(int j = 0; j < DNA_ALPHABET::size; ++j) { char currBase = ALPHABET[j]; if(currBase == originalBase) continue; kmer[base_idx] = currBase; size_t count = BWTAlgorithms::countSequenceOccurrences(kmer, inds); #if KMER_TESTING printf("%c %zu\n", currBase, count); #endif if(count >= minCount) { // Multiple corrections exist, do not correct if(bestBase != '$') return false; bestCount = count; bestBase = currBase; } } if(bestCount >= minCount) { assert(bestBase != '$'); readSequence[i] = bestBase; return true; } return false; } void KmerFilter::makeIndex(const std::vector& v) { ReadTable pRT; pRT.setZero(); int dd = 0; // make the reads tables for (auto& i : v) { SeqItem si; std::string seq(i); // if the read is good, add it to the table so we can use for kmer index if (seq.length() >= 40 && seq.find("N") == std::string::npos) { si.id = std::to_string(dd); si.seq = seq; pRT.addRead(si); ++dd; } } if (pRT.getCount() == 0) return; // make suffix array pSAf = new SuffixArray(&pRT, 1, false); // make BWT pBWT = new RLBWT(pSAf, &pRT); return; } // /*void KmerFilter::makeIndex(SeqLib::BamRecordVector& vec) { ReadTable pRT; pRT.setZero(); int dd = 0; // make the reads tables for (auto& i : vec) { SeqItem si; std::string sr, seq = ""; seq = i.QualitySequence(); //i.QualityTrimmedSequence(4, dum); // if the read is good, add it to the table so we can use for kmer index if (seq.length() >= 40 && seq.find("N") == std::string::npos) { si.id = std::to_string(dd); si.seq = seq; std::cerr << " seq " << seq << std::endl; pRT.addRead(si); ++dd; } } if (pRT.getCount() == 0) return; // make suffix array pSAf = new SuffixArray(&pRT, 1, false); // make BWT pBWT= new RLBWT(pSAf, &pRT); } // void KmerFilter::makeIndex(svabaReadVector& vec) { ReadTable pRT; pRT.setZero(); int dd = 0; // make the reads tables for (auto& i : vec) { SeqItem si; std::string sr; std::string seq = i.Seq(); // if the read is good, add it to the table so we can use for kmer index if (seq.length() >= 40 && seq.find("N") == std::string::npos) { si.id = std::to_string(dd); si.seq = seq; pRT.addRead(si); ++dd; } } if (pRT.getCount() == 0) return; // make suffix array pSAf = new SuffixArray(&pRT, 1, false); // make BWT pBWT= new RLBWT(pSAf, &pRT); } */ ================================================ FILE: src/svaba/KmerFilter.h ================================================ #pragma once #include #include #include "SeqLib/BamRecord.h" #include "CorrectionThresholds.h" #include "OverlapCommon.h" #include "SvabaRead.h" typedef std::map KmerCountMap; class KmerFilter { public: KmerFilter() : pBWT(nullptr), pSAf(nullptr) {} ~KmerFilter() { delete pBWT; delete pSAf; } int correctReads(SeqLib::BamRecordVector& vec); int correctReads(svabaReadVector& vec); void makeIndex(const std::vector& v); //void makeIndex(SeqLib::BamRecordVector& vec); private: RLBWT* pBWT; SuffixArray* pSAf; int m_kmer_len = 31; bool attemptKmerCorrection(size_t i, size_t k_idx, size_t minCount, std::string& readSequence, BWTIndexSet& inds); }; ================================================ FILE: src/svaba/LearnBamParams.cpp ================================================ // LearnBamParams.cpp #include "LearnBamParams.h" #include "SeqLib/BamRecord.h" #include "SeqLib/BamReader.h" #include "SeqLib/GenomicRegion.h" #include "SvabaSharedConfig.h" #include "SvabaOptions.h" #include "SvabaLogger.h" #include "gzstream.h" #include #include #include #include #include #include #include #include namespace { // helper to extract read group ids from the header std::vector extractReadGroups(const SeqLib::BamHeader& hdr) { std::vector groups; std::istringstream lines(hdr.AsString()); std::string line; while (std::getline(lines, line)) { if (line.rfind("@RG", 0) != 0) continue; std::istringstream fields(line); std::string field; while (std::getline(fields, field, '\t')) { if (field.rfind("ID:", 0) == 0) { groups.push_back(field.substr(3)); break; } } } return groups; } } // anonymous namespace #include std::ostream& operator<<(std::ostream& os, const BamReadGroup& bg) { os << "r=" << bg.reads << " s=" << bg.supp << " u=" << bg.unmap << " qc=" << bg.qcfail << " d=" << bg.duplicate << " mu=" << bg.mate_unmap << " MQ=" << bg.mapq_max << " RL=" << bg.readlen_max << " mIS=" << bg.isize_median << " sdIS=" << bg.sd_isize << " n_isize=" << bg.isize_vec.size(); return os; } LearnBamParams::LearnBamParams(SvabaSharedConfig& sc_, const std::string& bamPath) : sc(sc_), bam_(bamPath) { // make the new BamReader reader_ = std::make_shared(); // open the BAM for learning if (!reader_->Open(bam_)) { throw std::runtime_error("Could not open bam file " + bam_ + " in LearnBamParams"); } } size_t LearnBamParams::consumeReads( size_t max_reads, std::unordered_map& rg_count, std::unordered_set& satisfied_rgs, const std::vector& groups) { size_t consumed = 0; while (auto r = reader_->Next()) { if (consumed >= max_reads) break; // get read group tag std::string rg; if (!r->GetZTag("RG", rg)) rg = "NA"; ++consumed; // already saturated this RG — skip if (satisfied_rgs.count(rg)) continue; // check if this read pushes the RG over the limit size_t& cnt = rg_count[rg]; if (cnt >= static_cast(sc.opts.perRgLearnLimit)) { satisfied_rgs.insert(rg); // check if all header-declared RGs are satisfied if (satisfied_rgs.size() >= groups.size()) break; continue; } // refer to existing BamReadGroup or make new auto& bstats = bam_read_groups[rg]; bstats.addRead(*r); ++cnt; } return consumed; } void LearnBamParams::learnParams() { // gather read group ids from the header auto hdr = reader_->Header(); auto groups = extractReadGroups(hdr); int n_contigs = hdr.NumSequences(); sc.logger.log(true, true, "......header declares ", groups.size(), " read groups across ", n_contigs, " contigs"); // per-RG counts and satisfaction tracking std::unordered_map rg_count; std::unordered_set satisfied_rgs; size_t total_reads = 0; // --- Phase 1: multi-region sampling --- // Build sampling windows at the midpoint of each reference contig. // This ensures we see reads from every part of the genome, catching // read groups that only appear on later chromosomes. // // For standard contigs (chr1-chrY, ~24 contigs), the 1M-read-per-window // budget means we sample ~24M reads total in the worst case. For BAMs // with many small contigs (alt/decoy/random), we skip contigs shorter // than 1 Mb to avoid wasting time on regions with few reads. // // In human mode (default), we also skip non-standard chromosomes // (chrID > MAX_MATE_CHR_ID, i.e. chrM/alt/decoy/random) to focus // sampling on the primary assembly where isize/coverage stats are // representative. --non-human disables this gate. constexpr int32_t MIN_CONTIG_LEN = 1'000'000; // skip tiny contigs constexpr size_t READS_PER_WINDOW = 1'000'000; // reads per sampling window constexpr int32_t WINDOW_HALF = 500'000; // +/- 500kb around midpoint const int max_sample_chr = sc.opts.maxMateChrID; // 23 (through chrY) or -1 if --non-human for (int c = 0; c < n_contigs; ++c) { // early exit if all RGs are satisfied if (satisfied_rgs.size() >= groups.size()) break; // skip non-standard chromosomes in human mode if (max_sample_chr >= 0 && c > max_sample_chr) continue; int32_t clen = hdr.GetSequenceLength(c); if (clen < MIN_CONTIG_LEN) continue; // sample from the midpoint of the contig int32_t mid = clen / 2; int32_t start = std::max(0, mid - WINDOW_HALF); int32_t end = std::min(clen, mid + WINDOW_HALF); SeqLib::GenomicRegion region(c, start, end); if (!reader_->SetRegion(region)) continue; // skip if region query fails (no index?) size_t consumed = consumeReads(READS_PER_WINDOW, rg_count, satisfied_rgs, groups); total_reads += consumed; // log progress every 5 contigs if (c % 5 == 0 || c == n_contigs - 1) { sc.logger.log(sc.opts.verbose > 0, true, "......sampling ", hdr.IDtoName(c), " - ", SeqLib::AddCommas(total_reads), " reads, ", satisfied_rgs.size(), "/", groups.size(), " RGs satisfied"); } } // If some header-declared RGs weren't seen during multi-region // sampling, they're likely absent from the BAM entirely (phantom // header entries from upstream merges, etc.). Don't waste time on // a sequential scan — the multi-region pass already covered every // standard chromosome. if (satisfied_rgs.size() < groups.size()) { sc.logger.log(true, true, "......", satisfied_rgs.size(), "/", groups.size(), " RGs satisfied after multi-region sampling (", SeqLib::AddCommas(total_reads), " reads); ", "skipping sequential fallback — missing RGs likely absent from BAM"); } sc.logger.log(true, true, "......learning complete: ", SeqLib::AddCommas(total_reads), " reads scanned, ", bam_read_groups.size(), " read groups found, ", satisfied_rgs.size(), "/", groups.size(), " header RGs satisfied"); // log any RGs from the header that we never found reads for for (const auto& g : groups) { if (bam_read_groups.find(g) == bam_read_groups.end()) { sc.logger.log(true, true, "......WARNING: read group '", g, "' declared in header but no reads found"); } } // dump raw isize data for R plotting BEFORE computeStats clears the vectors. // Use the BAM filename stem in the output name so multiple BAMs don't clobber. if (!sc.opts.analysisId.empty()) { // extract basename: /path/to/foo.bam -> foo std::string stem = bam_; auto slash = stem.rfind('/'); if (slash != std::string::npos) stem = stem.substr(slash + 1); auto dot = stem.rfind('.'); if (dot != std::string::npos) stem = stem.substr(0, dot); dumpLearnData(sc.opts.analysisId + "." + stem); } // compute isize stats // store the max readlen and mapq for this entire bam across RGs for (auto& br : bam_read_groups) { br.second.computeStats(); readlen_max = std::max(readlen_max, br.second.readlen_max); mapq_max = std::max(mapq_max, br.second.mapq_max); isize_max = std::max(isize_max, br.second.isize_median); } // Log per-RG insert size stats and the derived discordant cutoff. // // Method: // 1. Sample up to perRgLearnLimit (default 1000) FR read pairs per // read group from the midpoint of each standard chromosome. // 2. Discard the top 2% of |isize| values as outliers. // 3. Compute median and population SD on the remaining 98%. // 4. Discordant cutoff = median + SD * sdDiscCutoff // (sdDiscCutoff defaults to 3.92 for tumor, 3.60 for normal). // 5. A read pair with |isize| > cutoff is classified as discordant. // // The cutoff is applied per-RG in svabaBamWalker::getIsizeCutoff(), // used by both readBam (read extraction) and TagDiscordant (read // tagging). Normal uses a lower multiplier (sdDiscCutoffNormal) // so it's more sensitive to marginal discordant reads, preventing // germline events from being mis-called as somatic. sc.logger.log(true, true, "......insert size learning summary (per read group):"); sc.logger.log(true, true, "...... method: median + SD * sdDiscCutoff on bottom 98% of FR pairs"); sc.logger.log(true, true, "...... sdDiscCutoff=", sc.opts.sdDiscCutoff, " (tumor), sdDiscCutoffNormal=", sc.opts.sdDiscCutoffNormal, " (normal); up to ", sc.opts.perRgLearnLimit, " pairs/RG"); for (const auto& [rg, bp] : bam_read_groups) { double cutoff = bp.isize_median + bp.sd_isize * sc.opts.sdDiscCutoff; sc.logger.log(true, true, "...... RG='", rg, "' n_pairs=", bp.n_isize_pairs, " median=", static_cast(bp.isize_median), " sd=", static_cast(bp.sd_isize), " disc_cutoff(tumor)=", static_cast(cutoff), " readlen=", bp.readlen_max); } } void LearnBamParams::dumpLearnData(const std::string& prefix) const { std::string fn = prefix + ".learn.tsv.gz"; ogzstream out(fn.c_str()); if (!out.good()) { sc.logger.log(true, true, "WARNING: could not open ", fn, " for writing learn data"); return; } // header out << "bam\trg\tisize\n"; // one row per isize observation, per RG for (const auto& [rg, brg] : bam_read_groups) { for (uint32_t is : brg.isize_vec) { out << bam_ << '\t' << rg << '\t' << is << '\n'; } } out.close(); sc.logger.log(true, true, "......wrote learning data to ", fn); } void BamReadGroup::addRead(const SeqLib::BamRecord &r) { // track the flags ++reads; if (r.SecondaryFlag()) ++supp; if (r.QCFailFlag()) ++qcfail; if (r.DuplicateFlag()) ++duplicate; if (!r.MappedFlag()) ++unmap; if (!r.MateMappedFlag()) ++mate_unmap; // track the mapq mapq_max = std::max(mapq_max, r.MapQuality()); // track the insert size if (!r.PairMappedFlag() || r.Interchromosomal() || r.PairOrientation() != SeqLib::Orientation::FR) ; else if (!r.Interchromosomal()) isize_vec.push_back(std::abs(r.InsertSize())); // track the read length readlen_max = std::max(readlen_max, r.Length()); } void BamReadGroup::computeStats() { if (isize_vec.empty()) { isize_median = 0.0; sd_isize = 0.0; return; } // Remove the top 2% of values to filter out extreme outliers std::sort(isize_vec.begin(), isize_vec.end()); size_t n = isize_vec.size(); size_t keep = static_cast(std::floor(n * 0.98)); if (keep == 0) { // If 98% rounds down to 0, keep at least one element keep = 1; } isize_vec.resize(keep); n_isize_pairs = keep; // Calculate median (vector is already sorted) if (keep % 2 == 1) { isize_median = isize_vec[keep / 2]; } else { isize_median = (isize_vec[keep / 2 - 1] + isize_vec[keep / 2]) / 2.0; } // Calculate population standard deviation around the median double sq_sum = 0.0; for (uint32_t val : isize_vec) { double diff = val - isize_median; sq_sum += diff * diff; } sd_isize = std::sqrt(sq_sum / keep); // Clean up memory isize_vec.clear(); } ================================================ FILE: src/svaba/LearnBamParams.h ================================================ // LearnBamParams.h #pragma once #include #include #include #include #include #include #include class SvabaSharedConfig; namespace SeqLib { class BamRecord; class BamReader; } /** Store information pertaining to a given read group * * * This class will collect statistics on number of: read, supplementary reads, unmapped reads, qcfail reads, duplicate reads. * It will also create Histogram objects to store counts of: mapq, nm, isize, clip, mean phred score, length */ class BamReadGroup { public: /** Construct an empty BamReadGroup */ BamReadGroup() {} /** Add a BamRecord to this read group */ void addRead(const SeqLib::BamRecord &r); void computeStats(); friend std::ostream& operator<<(std::ostream& os, const BamReadGroup& bg); // count number of reads size_t reads = 0; size_t supp = 0; size_t unmap = 0; size_t qcfail = 0; size_t duplicate = 0; size_t mate_unmap = 0; int mapq_max = 0; int readlen_max = 0; std::vector isize_vec; // statistics (set by computeStats) size_t n_isize_pairs = 0; // number of FR pairs used (after 98% trim) double isize_median = 0; double sd_isize = 0; // SD computed around the median }; /** * LearnBamParams encapsulates logic to learn insert-size and related statistics * from a single BAM file or across multiple BAM files. It: * - Opens and iterates through each read in the BAM (via SeqLib::BamReader) * - Groups reads by read-group and collects metrics (read length, MAPQ, insert-size distribution) * - Computes per-read-group statistics (mean, median, SD of insert size, coverage, clip fraction, etc.) * - Provides a static helper to run this process on a set of BAM files and * produce both per-file (per RG) and global summary statistics (max read length, * max mapQ, and a discordant read size cutoff). * * Sampling strategy (SvABA2.0): coordinate-sorted BAMs cluster read groups * by genomic position, so scanning from the start only sees RGs present in * early chromosomes. To ensure all RGs are represented: * 1. Build sampling windows at the midpoint of each reference contig * 2. For each window, read up to `reads_per_window` reads (100k default) * 3. Track per-RG counts; skip reads from already-saturated RGs * 4. Stop early once all header-declared RGs are satisfied * This replaces the old sequential-from-start scan that missed 20/21 RGs. */ class LearnBamParams { public: /// Learn parameters from a single BAM file LearnBamParams(SvabaSharedConfig& sc_, const std::string& bamPath); // opens the BAM // map of RG : params, for a single bam void learnParams(); // learn the RGs // this is map of RG-name : BamReadGroup std::unordered_map bam_read_groups; // per BAM readlen / mapq max int readlen_max = 0; int mapq_max = 0; double isize_max = 0; /// Write per-RG isize distributions to a TSV for R plotting. /// Called after learnParams() and before computeStats() clears the vectors. /// Output: ${prefix}.learn.tsv.gz with columns: bam, rg, isize void dumpLearnData(const std::string& prefix) const; private: /// Process reads from the current reader position, updating rg_count /// and bam_read_groups. Returns number of reads consumed. size_t consumeReads(size_t max_reads, std::unordered_map& rg_count, std::unordered_set& satisfied_rgs, const std::vector& groups); std::string bam_; // file path of the BAM SvabaSharedConfig& sc; std::shared_ptr reader_; }; ================================================ FILE: src/svaba/ReadToContigAligner.h ================================================ #ifndef SNOWMAN_READ_TO_CONTIG_ALIGNER_H__ #define SNOWMAN_READ_TO_CONTIG_ALIGNER_H__ #include "SnowTools/AlignedContig.h" #include "SnowTools/BamRead.h" #include "SnowTools/BWAWrapper.h" class ReadToContigAligner { public: ReadToContigAligner() {} ReadToContigAligner(const SnowTools::USeqVector& usv, BamReadVector& bav_this, std::vector& this_alc) { private: BamReadVector m_reads; SnowTools::USeqVector m_usv; SnowTools::BWAWrapper& m_bw; std::vector m_alc; }; #endif ================================================ FILE: src/svaba/STCoverage.cpp ================================================ #include "STCoverage.h" #include "SeqLib/SeqLibCommon.h" #include "SeqLib/BamRecord.h" #include #include using SeqLib::BamRecord; using SeqLib::GenomicRegion; using SeqLib::Cigar; void STCoverage::clear() { m_map.clear(); } void STCoverage::addRead(const BamRecord &r, int buff) { //, bool full_length) { int p = -1; int e = -1; // old code to also cover soft clips, not needed /* if (full_length) { Cigar c = r.GetCigar(); // get beginning if (c.size() && c[0].RawType() == BAM_CSOFT_CLIP) p = std::max((int32_t)0, r.Position() - (int32_t)c[0].Length()); // get prefixing S else p = r.Position(); // get end if (c.size() && c.back().RawType() == BAM_CSOFT_CLIP) e = r.PositionEnd() + c.back().Length(); else e = r.PositionEnd(); } else { */ p = r.Position() + buff; e = r.PositionEnd() - buff; // } if (p < 0 || e < 0) return; assert(e - p < 1e6); // limit on read length assert(r.ChrID() >= 0); try { while (p <= e) { ++m_map[r.ChrID()][p]; // add one to this position ++p; } } catch (std::out_of_range &oor) { std::cerr << "Position " << p << " on tid " << r.ChrID() << " is greater than expected max -- skipping" << std::endl; } } uint32_t STCoverage::getCoverageAtPosition(int chr, int pos) const { auto chr_it = m_map.find(chr); if (chr_it == m_map.end()) { return 0; } auto pos_it = chr_it->second.find(pos); if (pos_it == chr_it->second.end()) { return 0; } return pos_it->second; } ================================================ FILE: src/svaba/STCoverage.h ================================================ #pragma once #include #include #include #include #include #include #include #include #include "htslib/hts.h" #include "htslib/sam.h" #include "htslib/bgzf.h" #include "htslib/kstring.h" #include "SeqLib/GenomicRegion.h" #include "SeqLib/GenomicRegionCollection.h" namespace SeqLib { class BamRecord; } typedef std::unordered_map CovMap; /** Hold base-pair or binned coverage across an interval or genome * * Currently stores coverage as an unordered_map. */ class STCoverage { private: std::unordered_map m_map; //[chr [pos,cov] ] public: STCoverage() = default; /** Clear the coverage map */ void clear(); /** Add a read to this coverage track * @param reserve_size Upper bound estimate for size of map. Not a hard * cutoff but improves performance if total number of positions is less than this, * as it will not rehash. */ void addRead(const SeqLib::BamRecord &r, int buff); //, bool full_length); /** Print the entire data */ friend std::ostream& operator<<(std::ostream &out, const STCoverage &c); /** Return the coverage count at a position */ uint32_t getCoverageAtPosition(int chr, int pos) const; }; ================================================ FILE: src/svaba/SvabaASQG.cpp ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // ASQG - Definitions and functions // for handling ASGQ files // #include "SvabaASQG.h" #include namespace svabaASQG { const int HEADER_VERSION = 1; // Record ID tags static constexpr int RECORD_TAG_SIZE = 2; // do not include null terminator static char HEADER_TAG[] = "HT"; static char VERTEX_TAG[] = "VT"; static char EDGE_TAG[] = "ED"; // Header tags const int FIELD_TAG_SIZE = 2; static char VERSION_TAG[] = "VN"; static char OVERLAP_TAG[] = "OL"; static char INPUT_FILE_TAG[] = "IN"; static char ERROR_RATE_TAG[] = "ER"; static char CONTAINMENT_TAG[] = "CN"; // 1 if the graph has containment edges/vertices static char TRANSITIVE_TAG[] = "TE"; // 1 if the graph has transitive edges // Vertex tags static char SUBSTRING_TAG[] = "SS"; // // Header Record // HeaderRecord::HeaderRecord() { setVersionTag(HEADER_VERSION); } // HeaderRecord::HeaderRecord(const std::string& recordLine) { parse(recordLine); } // void HeaderRecord::setVersionTag(int version) { m_versionTag.set(version); } // void HeaderRecord::setOverlapTag(int overlapLen) { m_overlapTag.set(overlapLen); } // void HeaderRecord::setInputFileTag(const std::string& name) { m_infileTag.set(name); } // void HeaderRecord::setErrorRateTag(float errorRate) { m_errorRateTag.set(errorRate); } // void HeaderRecord::setContainmentTag(int v) { m_containmentTag.set(v); } // void HeaderRecord::setTransitiveTag(int v) { m_transitiveTag.set(v); } // void HeaderRecord::write(std::ostream& out) { StringVector fields; fields.push_back(HEADER_TAG); // Check for mandatory tags if(!m_versionTag.isInitialized()) { std::cerr << "Error: Header version tag not set, aborting." << std::endl; exit(EXIT_FAILURE); } else { fields.push_back(m_versionTag.toTagString(VERSION_TAG)); } if(m_errorRateTag.isInitialized()) fields.push_back(m_errorRateTag.toTagString(ERROR_RATE_TAG)); if(m_overlapTag.isInitialized()) fields.push_back(m_overlapTag.toTagString(OVERLAP_TAG)); if(m_infileTag.isInitialized()) fields.push_back(m_infileTag.toTagString(INPUT_FILE_TAG)); if(m_containmentTag.isInitialized()) fields.push_back(m_containmentTag.toTagString(CONTAINMENT_TAG)); if(m_transitiveTag.isInitialized()) fields.push_back(m_transitiveTag.toTagString(TRANSITIVE_TAG)); writeFields(out, fields); } //JEREMIAH void HeaderRecord::write(std::stringstream& out) { StringVector fields; fields.push_back(HEADER_TAG); // Check for mandatory tags if(!m_versionTag.isInitialized()) { std::cerr << "Error: Header version tag not set, aborting." << std::endl; exit(EXIT_FAILURE); } else { fields.push_back(m_versionTag.toTagString(VERSION_TAG)); } if(m_errorRateTag.isInitialized()) fields.push_back(m_errorRateTag.toTagString(ERROR_RATE_TAG)); if(m_overlapTag.isInitialized()) fields.push_back(m_overlapTag.toTagString(OVERLAP_TAG)); if(m_infileTag.isInitialized()) fields.push_back(m_infileTag.toTagString(INPUT_FILE_TAG)); if(m_containmentTag.isInitialized()) fields.push_back(m_containmentTag.toTagString(CONTAINMENT_TAG)); if(m_transitiveTag.isInitialized()) fields.push_back(m_transitiveTag.toTagString(TRANSITIVE_TAG)); writeFields(out, fields); } // void HeaderRecord::parse(const std::string& record) { if(record.size() < 2) { std::cerr << "Error: Record is not valid\n"; exit(EXIT_FAILURE); } // Tokenize record StringVector tokens = SQG::tokenizeRecord(record); // Ensure the first token indicates this is a valid header record if(tokens[0].compare(0, RECORD_TAG_SIZE, HEADER_TAG) != 0) { std::cerr << "Error: Record does not have a header tag" << std::endl; std::cerr << "Record: " << record << std::endl; exit(EXIT_FAILURE); } for(size_t i = 1; i < tokens.size(); ++i) { static char VERSION_TAG[] = "VN"; static char OVERLAP_TAG[] = "OL"; static char INPUT_FILE_TAG[] = "IN"; static char ERROR_RATE_TAG[] = "ER"; if(tokens[i].compare(0, FIELD_TAG_SIZE, VERSION_TAG) == 0) m_versionTag.fromString(tokens[i]); if(tokens[i].compare(0, FIELD_TAG_SIZE, OVERLAP_TAG) == 0) m_overlapTag.fromString(tokens[i]); if(tokens[i].compare(0, FIELD_TAG_SIZE, INPUT_FILE_TAG) == 0) m_infileTag.fromString(tokens[i]); if(tokens[i].compare(0, FIELD_TAG_SIZE, ERROR_RATE_TAG) == 0) m_errorRateTag.fromString(tokens[i]); if(tokens[i].compare(0, FIELD_TAG_SIZE, CONTAINMENT_TAG) == 0) m_containmentTag.fromString(tokens[i]); if(tokens[i].compare(0, FIELD_TAG_SIZE, TRANSITIVE_TAG) == 0) m_transitiveTag.fromString(tokens[i]); } } // // Vertex Record // // VertexRecord::VertexRecord(const std::string& recordLine) { parse(recordLine); } // void VertexRecord::setSubstringTag(bool b) { m_substringTag.set(b); } // void VertexRecord::write(std::ostream& out) { StringVector fields; fields.push_back(VERTEX_TAG); fields.push_back(m_id); fields.push_back(m_seq); if(m_substringTag.isInitialized()) fields.push_back(m_substringTag.toTagString(SUBSTRING_TAG)); writeFields(out, fields); } //JEREMIAH void VertexRecord::write(std::stringstream& out) { StringVector fields; fields.push_back(VERTEX_TAG); fields.push_back(m_id); fields.push_back(m_seq); if(m_substringTag.isInitialized()) fields.push_back(m_substringTag.toTagString(SUBSTRING_TAG)); writeFields(out, fields); } // void VertexRecord::parse(const std::string& record) { if(record.size() < 2) { std::cerr << "Error: Record is not valid\n"; exit(EXIT_FAILURE); } // Tokenize record StringVector tokens = SQG::tokenizeRecord(record); if(tokens.size() < 3) { std::cerr << "Error: Vertex record is incomplete.\n"; std::cerr << "Record: " << record << std::endl; exit(EXIT_FAILURE); } // Ensure the first token indicates this is a valid vertex record if(tokens[0].compare(0, RECORD_TAG_SIZE, VERTEX_TAG) != 0) { std::cerr << "Error: Record does not have a vertex tag" << std::endl; std::cerr << "Record: " << record << std::endl; exit(EXIT_FAILURE); } m_id = tokens[1]; m_seq = tokens[2]; for(size_t i = 3; i < tokens.size(); ++i) { if(tokens[i].compare(0, FIELD_TAG_SIZE, SUBSTRING_TAG) == 0) m_substringTag.fromString(tokens[i]); } } // // EdgeRecord // EdgeRecord::EdgeRecord(const std::string& recordLine) { parse(recordLine); } // void EdgeRecord::write(std::ostream& out) { StringVector fields; fields.push_back(EDGE_TAG); std::stringstream ss; ss << m_overlap; fields.push_back(ss.str()); writeFields(out, fields); } // void EdgeRecord::parse(const std::string& record) { if(record.size() < 2) { std::cerr << "Error: Record is not valid\n"; exit(EXIT_FAILURE); } // Tokenize record StringVector tokens = SQG::tokenizeRecord(record); if(tokens.size() < 2) { std::cerr << "Error: Edge record is incomplete.\n"; std::cerr << "Record: " << record << std::endl; exit(EXIT_FAILURE); } // Ensure the first token indicates this is a valid edge record if(tokens[0].compare(EDGE_TAG) != 0) { std::cerr << "Error: Record does not have an edge tag" << std::endl; std::cerr << "Record: " << record << std::endl; exit(EXIT_FAILURE); } // Parse the overlap std::stringstream ssparser(tokens[1]); ssparser >> m_overlap; } // // // RecordType getRecordType(const std::string& record) { // the record type is the first two characters of the record if(record.size() < 2) { std::cerr << "Error: record does not have a valid record-type tag" << std::endl; std::cerr << "Record: " << record << std::endl; exit(EXIT_FAILURE); } char recordTag[RECORD_TAG_SIZE]; record.copy(recordTag, RECORD_TAG_SIZE); if(strncmp(recordTag, HEADER_TAG, RECORD_TAG_SIZE) == 0) { return RT_HEADER; } if(strncmp(recordTag, VERTEX_TAG, RECORD_TAG_SIZE) == 0) { return RT_VERTEX; } if(strncmp(recordTag, EDGE_TAG, RECORD_TAG_SIZE) == 0) { return RT_EDGE; } // Unknown tag std::cerr << "Error: Unknown ASQG file record tag: " << recordTag << std::endl; exit(EXIT_FAILURE); } void writeFields(std::ostream& out, const StringVector& fields) { for(size_t i = 0; i < fields.size(); ++i) { out << fields[i]; if(i != fields.size() - 1) out << SQG::FIELD_SEP; } out << "\n"; } } ================================================ FILE: src/svaba/SvabaASQG.h ================================================ //----------------------------------------------- // Copyright 2010 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // ASQG - Definitions and functions // for handling ASGQ files // #ifndef SVABA_ASQG_H #define SVABA_ASQG_H #include "SQG.h" #include "Match.h" namespace svabaASQG { enum RecordType { RT_HEADER = 0, RT_VERTEX, RT_EDGE }; // A header record is just a tag:value pairs struct HeaderRecord { public: HeaderRecord(); HeaderRecord(const std::string& recordLine); void setOverlapTag(int overlapLen); void setInputFileTag(const std::string& name); void setErrorRateTag(float errorRate); void setContainmentTag(int v); void setTransitiveTag(int v); const SQG::IntTag& getVersionTag() const { return m_versionTag; } const SQG::FloatTag& getErrorRateTag() const { return m_errorRateTag; } const SQG::StringTag& getInfileTag() const { return m_infileTag; } const SQG::IntTag& getOverlapTag() const { return m_overlapTag; } const SQG::IntTag& getContainmentTag() const { return m_containmentTag; }; const SQG::IntTag& getTransitiveTag() const { return m_transitiveTag; }; void write(std::ostream& out); void write(std::stringstream& out); // JEREMIAH void parse(const std::string& record); private: void setVersionTag(int version); SQG::IntTag m_versionTag; SQG::FloatTag m_errorRateTag; SQG::StringTag m_infileTag; SQG::IntTag m_overlapTag; SQG::IntTag m_containmentTag; SQG::IntTag m_transitiveTag; }; // A vertex record is an id, sequence and an array of // tag:value struct VertexRecord { public: VertexRecord() {} VertexRecord(const std::string& recordLine); VertexRecord(const std::string& i, const std::string& s) : m_id(i), m_seq(s) {} void setSubstringTag(bool b); const std::string& getID() const { return m_id; } const std::string& getSeq() const { return m_seq; } const SQG::IntTag& getSubstringTag() const { return m_substringTag; } void write(std::ostream& out); void write(std::stringstream& out); void parse(const std::string& record); private: std::string m_id; std::string m_seq; SQG::IntTag m_substringTag; }; // An edge record is just an overlap object and tag:values struct EdgeRecord { public: EdgeRecord() {} EdgeRecord(const std::string& recordLine); EdgeRecord(const Overlap& o) : m_overlap(o) {} const Overlap& getOverlap() const { return m_overlap; } void write(std::ostream& out); void parse(const std::string& record); private: Overlap m_overlap; }; // Parsing functions RecordType getRecordType(const std::string& record); HeaderRecord parseHeaderRecord(const std::string& record); VertexRecord parseVertexRecord(const std::string& record); EdgeRecord parseEdgeRecord(const std::string& record); // Writing functions void writeFields(std::ostream& out, const StringVector& fields); }; #endif ================================================ FILE: src/svaba/SvabaAssemble.cpp ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // assemble - convert read overlaps into contigs // #include #include #include "Util.h" #include "SvabaAssemble.h" #include "SGUtil.h" #include "SGAlgorithms.h" #include "SGVisitors.h" #include "EncodedString.h" #include #include #include "SGSearch.h" //#define DEBUG_ASSEMBLY 1 void walkExtra(StringGraph * pGraph, SGWalkVector& outWalks); StringGraph* assemble(std::stringstream& asqg_stream, int minOverlap, int maxEdges, bool bExact, int trimLengthThreshold, bool bPerformTR, bool bValidate, int numTrimRounds, int resolveSmallRepeatLen, int numBubbleRounds, double maxBubbleGapDivergence, double maxBubbleDivergence, int maxIndelLength, int cutoff, std::string prefix, SeqLib::UnalignedSequenceVector &contigs, bool get_components) { AssemblyOptions ao; StringGraph * pGraph = SGUtil::loadASQG(asqg_stream, minOverlap, true, maxEdges); pGraph->m_get_components = get_components; if(bExact) pGraph->setExactMode(true); // Visitor functors SGTransitiveReductionVisitor trVisit; SGGraphStatsVisitor statsVisit; SGTrimVisitor trimVisit(trimLengthThreshold); SGContainRemoveVisitor containVisit; SGValidateStructureVisitor validationVisit; while(pGraph->hasContainment()) pGraph->visit(containVisit); // Remove any extraneous transitive edges that may remain in the graph if(bPerformTR) { std::cout << "Removing transitive edges\n"; pGraph->visit(trVisit); } // Compact together unbranched chains of vertices pGraph->simplify(); if(bValidate) { pGraph->visit(validationVisit); } // Remove dead-end branches from the graph if(numTrimRounds > 0) { int numTrims = numTrimRounds; while(numTrims-- > 0) pGraph->visit(trimVisit); } // Resolve small repeats if(resolveSmallRepeatLen > 0 && false) { SGSmallRepeatResolveVisitor smallRepeatVisit(resolveSmallRepeatLen); } if(numBubbleRounds > 0) { SGSmoothingVisitor smoothingVisit(ao.outVariantsFile, maxBubbleGapDivergence, maxBubbleDivergence, maxIndelLength); int numSmooth = numBubbleRounds; while(numSmooth-- > 0) pGraph->visit(smoothingVisit); pGraph->simplify(); } pGraph->renameVertices(prefix); SGVisitorContig av; pGraph->visit(av); SeqLib::UnalignedSequenceVector tmp = av.m_ct; for (SeqLib::UnalignedSequenceVector::const_iterator it = tmp.begin(); it != tmp.end(); it++) { if ((int)(it->Seq.length()) >= cutoff) { contigs.push_back({it->Name + "C", it->Seq, std::string()}); //postpend with character to distribugish _2 from _22 } } /* if (walk_all) { SGWalkVector outWalks; walkExtra(pGraph, outWalks); for (auto& i : outWalks) { std::string seqr = i.getString(SGWT_START_TO_END); if ((int)seqr.length() >= cutoff) contigs.push_back(Contig(i.pathSignature(), seqr)); } }*/ return pGraph; // delete pGraph; } void walkExtra(StringGraph * pGraph, SGWalkVector& outWalks) { typedef std::vector ComponentVector; VertexPtrVec allVertices = pGraph->getAllVertices(); ComponentVector components; #ifdef DEBUG_ASSEMBLY std::cerr << "selecting connected componsnet" << std::endl; #endif SGSearchTree::connectedComponents(allVertices, components); // Select the largest component int selectedIdx = -1; size_t largestSize = 0; for(size_t i = 0; i < components.size(); ++i) { if(components[i].size() > largestSize) { selectedIdx = i; largestSize = components[i].size(); } } #ifdef DEBUG_ASSEMBLY std::cerr << "looping vertices" << std::endl; #endif assert(selectedIdx != -1); VertexPtrVec selectedComponent = components[selectedIdx]; // Build a vector of the terminal vertices VertexPtrVec terminals; for(size_t i = 0; i < selectedComponent.size(); ++i) { Vertex* pVertex = selectedComponent[i]; size_t asCount = pVertex->getEdges(ED_ANTISENSE).size(); size_t sCount = pVertex->getEdges(ED_SENSE).size(); if(asCount == 0 || sCount == 0) terminals.push_back(pVertex); } #ifdef DEBUG_ASSEMBLY std::cerr << "getting walks on " << terminals.size() << " vertices " << std::endl; #endif // Find walks between all-pairs of terminal vertices SGWalkVector tempWalks; if (terminals.size() > 2 && terminals.size() < 10) { for(size_t i = 0; i < terminals.size(); ++i) { for(size_t j = i + 1; j < terminals.size(); j++) { Vertex* pX = terminals[i]; Vertex* pY = terminals[j]; int maxDistance = 2; SGSearch::findWalks(pX, pY, ED_SENSE, maxDistance, 1000000, false, tempWalks); SGSearch::findWalks(pX, pY, ED_ANTISENSE, maxDistance, 1000000, false, tempWalks); } } } #ifdef DEBUG_ASSEMBLY std::cerr << "deduping walks on " << terminals.size() << " vertices " << std::endl; #endif // Remove duplicate walks std::map walkMap; for(size_t i = 0; i < tempWalks.size(); ++i) { std::string walkString = tempWalks[i].getString(SGWT_START_TO_END); walkMap.insert(std::make_pair(walkString, tempWalks[i])); } // Copy unique walks to the output for(std::map::iterator mapIter = walkMap.begin(); mapIter != walkMap.end(); ++mapIter) outWalks.push_back(mapIter->second); // Sort the walks by string length std::sort(outWalks.begin(), outWalks.end(), SGWalk::compareByTotalLength); int numOutputWalks = 10; if(numOutputWalks > 0 && numOutputWalks < (int)outWalks.size()) { assert(outWalks.begin() + numOutputWalks < outWalks.end()); outWalks.erase(outWalks.begin() + numOutputWalks, outWalks.end()); } } ================================================ FILE: src/svaba/SvabaAssemble.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL license //----------------------------------------------- // // assemble - Assemble reads into contigs // #ifndef SVABA_ASSEMBLE_H #define SVABA_ASSEMBLE_H #include //#include "config.h" #include #include #include #include "Util.h" #include "SGUtil.h" #include "SeqLib/UnalignedSequence.h" struct AssemblyOptions { unsigned int verbose = 1; std::string asqgFile; std::string outContigsFile; std::string outVariantsFile; std::string outGraphFile; unsigned int minOverlap; bool bEdgeStats = false; bool bSmoothGraph = false; int resolveSmallRepeatLen = -1; size_t maxEdges = 128; // Trim parameters int numTrimRounds = 10; size_t trimLengthThreshold = 300; // Bubble parameters int numBubbleRounds = 3; double maxBubbleDivergence = 0.05f; double maxBubbleGapDivergence = 0.01f; int maxIndelLength = 20; // bool bValidate; bool bExact = true; bool bPerformTR = false; }; StringGraph* assemble(std::stringstream& asqg_stream, int minOverlap, int maxEdges, bool bExact, int trimLengthThreshold, bool bPerformTR, bool bValidate, int numTrimRounds, int resolveSmallRepeatLen, int numBubbleRounds, double maxBubbleGapDivergence, double maxBubbleDivergence, int maxIndelLength, int cutoff, std::string prefix, SeqLib::UnalignedSequenceVector &contigs, bool get_components); #endif ================================================ FILE: src/svaba/SvabaAssemblerConfig.h ================================================ #pragma once // --------------------------------------------------------------------------- // svabaAssemblerConfig.h // // Compile-time selection of the local-assembly engine used by svaba. // // SVABA_ASSEMBLER_FERMI (1) -> use fermi2 (ropebwt2-based) assembler // SVABA_ASSEMBLER_FERMI (0) -> use the vendored SGA String Graph Assembler // // This used to live as a bare `#define FERMI 1` at the top of // SvabaRegionProcessor.cpp, which meant nothing else in the build could see // it (so e.g. run_svaba.cpp could not report which assembler was compiled // in). Centralizing it here lets any TU `#include "SvabaAssemblerConfig.h"` // and branch on the same symbol. // // To switch assemblers at build time without editing this header, pass // -DSVABA_ASSEMBLER_FERMI=0 // (or =1) via CMake / CXXFLAGS. // --------------------------------------------------------------------------- #ifndef SVABA_ASSEMBLER_FERMI #define SVABA_ASSEMBLER_FERMI 1 #endif // Back-compat alias: existing code uses `#ifndef FERMI` / `#ifdef FERMI`. #if SVABA_ASSEMBLER_FERMI #ifndef FERMI #define FERMI 1 #endif #else #ifdef FERMI #undef FERMI #endif #endif // Human-readable name of the active assembler, for logging. namespace svaba { #if SVABA_ASSEMBLER_FERMI inline constexpr const char* kAssemblerName = "fermi2"; inline constexpr const char* kAssemblerTag = "fermi"; #else inline constexpr const char* kAssemblerName = "SGA"; inline constexpr const char* kAssemblerTag = "sga"; #endif } ================================================ FILE: src/svaba/SvabaAssemblerEngine.cpp ================================================ #include "SvabaAssemblerEngine.h" #include "SvabaUtils.h" #include #include #include "SGACommon.h" #include "SvabaASQG.h" #include "SvabaAssemble.h" #include "SvabaOverlapAlgorithm.h" #include "SvabaOptions.h" #include "OverlapCommon.h" #include "CorrectionThresholds.h" //#define DEBUG_ENGINE 1 static std::string repeat(char base, int count) { return std::string(count, base); } static const std::string POLYA = repeat('A', 30); static const std::string POLYT = repeat('T', 30); static const std::string POLYC = repeat('C', 30); static const std::string POLYG = repeat('G', 30); static const std::string POLYAT = "ATATATATATATATATATATATATATATATATATATATAT"; static const std::string POLYTC = "TCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTC"; static const std::string POLYAG = "AGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG"; static const std::string POLYCG = "CGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCG"; static const std::string POLYTG = "TGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTG"; static const std::string POLYCA = "CACACACACACACACACACACACACACACACACACACACA"; void svabaAssemblerEngine::fillReadTable(const svabaReadPtrVector& reads) { // make the reads tables for (const auto& i : reads) { // skip if not for assembly if (!i->to_assemble) continue; // get the sequence and unique ID std::string sr = std::to_string(++count); std::string seq = i->CorrectedSeq(); assert(sr.length()); assert(seq.length()); if (hasRepeat(seq) || seq.length() < m_min_overlap) continue; // put onto the foward strand if not if (!i->MappedFlag() && !i->MateReverseFlag()) SeqLib::rcomplement(seq); SeqItem si; si.id = sr; si.seq = seq; m_pRT.addRead(si); } } bool svabaAssemblerEngine::hasRepeat(const std::string& seq) { if (seq.length() < 40) return false; static const std::vector repeatMotifs = { "N", POLYT, POLYA, POLYC, POLYG, POLYCG, POLYAT, POLYTC, POLYAG, POLYCA, POLYTG }; return std::any_of(repeatMotifs.begin(), repeatMotifs.end(), [&seq](const std::string& motif) { return seq.find(motif) != std::string::npos; }); } bool svabaAssemblerEngine::performAssembly(int num_assembly_rounds) { if (m_pRT.getCount() < 2) return false; #ifdef DEBUG_ENGINE std::cout << "Doing assembly on: " << m_id << " with " << m_pRT.getCount() << " reads" << std::endl; #endif SeqLib::UnalignedSequenceVector contigs0; //ContigVector contigs0; #ifdef DEBUG_ENGINE std::cout << "...round 1" << std::endl; #endif // do the first round (on raw reads) doAssembly(&m_pRT, m_contigs, 0); for (int yy = 1; yy != num_assembly_rounds; yy++) { if (m_contigs.size() < 2) continue; // break because too few contigs to assemle #ifdef DEBUG_ENGINE std::cout << "...round " << (yy+1) << std::endl; #endif // do the second round (on assembled contigs) SeqLib::UnalignedSequenceVector tmpc; for (auto& j : m_contigs) if (j.Seq.length() > m_readlen) tmpc.push_back(j); ReadTable pRTc0(tmpc); m_contigs.clear(); doAssembly(&pRTc0, m_contigs, yy); } return true; } // call the assembler void svabaAssemblerEngine::doAssembly(ReadTable *pRT, SeqLib::UnalignedSequenceVector &contigs, int pass) { if (pRT->getCount() == 0) return; // clear the hits stream std::stringstream hits_stream, asqg_stream; double errorRate = m_error_rate; int min_overlap = m_min_overlap; int cutoff = 0; if (pass > 1) { //errorRate = 0.015f; // up to one bp mismatch errorRate = 0; m_error_rate = errorRate; min_overlap = m_min_overlap * 2; } bool exact = errorRate < 0.001f; // remove duplicates if running in exact mode ReadTable * pRT_nd = exact ? removeDuplicates(pRT) : pRT; // forward SuffixArray* pSAf_nd = new SuffixArray(pRT_nd, 1, false); //1 is num threads. false is silent/no RLBWT *pBWT_nd = new RLBWT(pSAf_nd, pRT_nd); // reverse pRT_nd->reverseAll(); SuffixArray * pSAr_nd = new SuffixArray(pRT_nd, 1, false); RLBWT *pRBWT_nd = new RLBWT(pSAr_nd, pRT_nd); pRT_nd->reverseAll(); pSAf_nd->writeIndex(); pSAr_nd->writeIndex(); bool bIrreducibleOnly = true; // default int seedLength = 0; int seedStride = 0; if (!exact) calculateSeedParameters(m_readlen, min_overlap, seedLength, seedStride); svabaOverlapAlgorithm* pOverlapper = new svabaOverlapAlgorithm(pBWT_nd, pRBWT_nd, errorRate, seedLength, seedStride, bIrreducibleOnly); pOverlapper->setExactModeOverlap(exact); pOverlapper->setExactModeIrreducible(exact); svabaASQG::HeaderRecord headerRecord; headerRecord.setOverlapTag(min_overlap); headerRecord.setErrorRateTag(errorRate); headerRecord.setInputFileTag(""); headerRecord.setContainmentTag(true); // containments are always present headerRecord.setTransitiveTag(!bIrreducibleOnly); headerRecord.write(asqg_stream); pRT_nd->setZero(); size_t workid = 0; SeqItem si; size_t ocount = 0; while (pRT_nd->getRead(si) && (++ocount < MAX_OVERLAPS_PER_ASSEMBLY)) { SeqRecord read; read.id = si.id; read.seq = si.seq; OverlapBlockList obl; OverlapResult rr = pOverlapper->overlapRead(read, min_overlap, &obl); pOverlapper->writeOverlapBlocks(hits_stream, workid, rr.isSubstring, &obl); svabaASQG::VertexRecord record(read.id, read.seq.toString()); record.setSubstringTag(rr.isSubstring); record.write(asqg_stream); ++workid; } std::string line; bool bIsSelfCompare = true; ReadInfoTable* pQueryRIT = new ReadInfoTable(pRT_nd); while(std::getline(hits_stream, line)) { size_t readIdx; size_t totalEntries; bool isSubstring; OverlapVector ov; OverlapCommon::parseHitsString(line, pQueryRIT, pQueryRIT, pSAf_nd, pSAr_nd, bIsSelfCompare, readIdx, totalEntries, ov, isSubstring); for(OverlapVector::iterator iter = ov.begin(); iter != ov.end(); ++iter) { svabaASQG::EdgeRecord edgeRecord(*iter); edgeRecord.write(asqg_stream); } } // Get the number of strings in the BWT, this is used to pre-allocated the read table delete pOverlapper; delete pBWT_nd; delete pRBWT_nd; delete pSAf_nd; delete pSAr_nd; if (exact) // only in exact mode did we actually allocate for pRT_nd, otherwise just pRT which we want to keep delete pRT_nd; //#ifdef CLOCK_COUNTER //clock_t ctA_before = clock(); //#endif // PERFORM THE ASSMEBLY trimLengthThreshold = 100; numTrimRounds = 1; StringGraph * oGraph = assemble(asqg_stream, min_overlap, maxEdges, bExact, trimLengthThreshold, bPerformTR, bValidate, numTrimRounds, resolveSmallRepeatLen, numBubbleRounds, gap_divergence, divergence, maxIndelLength, cutoff, m_id + "_", contigs, m_write_asqg); // optionally output the graph structure //if (m_write_asqg) //debug // write_asqg(oGraph, asqg_stream, hits_stream, pass); // this was allocated in assemble delete oGraph; delete pQueryRIT; // remove exact dups remove_exact_dups(contigs); // print out some results #ifdef DEBUG_ENGINE std::cerr << " PASS " << pass << std::endl; print_results(contigs); #endif return; } // not totally sure this works... ReadTable* svabaAssemblerEngine::removeDuplicates(ReadTable* pRT) { // forward SuffixArray* pSAf = new SuffixArray(pRT, 1, false); //1 is num threads. false is silent/no RLBWT *pBWT= new RLBWT(pSAf, pRT); // reverse pRT->reverseAll(); SuffixArray * pSAr = new SuffixArray(pRT, 1, false); RLBWT *pRBWT = new RLBWT(pSAr, pRT); pRT->reverseAll(); svabaOverlapAlgorithm* pRmDupOverlapper = new svabaOverlapAlgorithm(pBWT, pRBWT, 0, 0, 0, false); pRT->setZero(); ReadTable * pRT_nd = new ReadTable(); SeqItem sir; while (pRT->getRead(sir)) { OverlapBlockList OBout; SeqRecord read; read.id = sir.id; read.seq = sir.seq; OverlapBlockList obl; OverlapResult rr = pRmDupOverlapper->alignReadDuplicate(read, &OBout); if (!rr.isSubstring) pRT_nd->addRead(sir); } delete pRmDupOverlapper; delete pBWT; delete pRBWT; delete pSAf; delete pSAr; return pRT_nd; } void svabaAssemblerEngine::write_asqg(const StringGraph* oGraph, std::stringstream& asqg_stream, std::stringstream& hits_stream, int pass) const { // write ASQG to file for visualization std::ofstream ofile(m_id + "pass_" + std::to_string(pass) + ".asqg", std::ios::out); ofile << asqg_stream.str(); ofile.close(); // write the hits stream file std::ofstream ofile_hits(m_id + "pass_" + std::to_string(pass) + ".hits", std::ios::out); ofile_hits << hits_stream.str(); ofile_hits.close(); // write the connected components file std::ofstream ofile_cc(m_id + "pass_" + std::to_string(pass) + ".cc", std::ios::out); for (auto& i : oGraph->m_connected_components) ofile_cc << i.first << "\t" << i.second << std::endl; ofile_cc.close(); // write the labels std::ofstream ofile_lb(m_id + "pass_" + std::to_string(pass) + ".lb", std::ios::out); for (auto& i : oGraph->m_reads_on_contigs) for (auto& r : i.second) ofile_lb << i.first << "\t" << r << std::endl; ofile_lb.close(); } void svabaAssemblerEngine::remove_exact_dups(SeqLib::UnalignedSequenceVector& cc) const { std::set ContigDeDup; SeqLib::UnalignedSequenceVector cvec; for (auto& i : cc) { if (!ContigDeDup.count(i.Seq)) { ContigDeDup.insert(i.Seq); cvec.push_back(i); } else { //std::cerr << "Filtered out a contig for having exact duplicate with another contig" << std::endl; } } cc = cvec; } void svabaAssemblerEngine::print_results(const SeqLib::UnalignedSequenceVector& cc) const { if (cc.size() >= 1) { std::cout << "Contig Count: " << cc.size() << " at " << m_id << std::endl; for (auto& i : cc) std::cout << " " << i.Name << " " << i.Seq.length() << " " << i.Seq << std::endl; } } // lifted from OverlapAlgorithm void svabaAssemblerEngine::calculateSeedParameters(int read_len, int minOverlap, int& seed_length, int& seed_stride) const { seed_length = 0; // The maximum possible number of differences occurs for a fully-aligned read int max_diff_high = static_cast(m_error_rate * read_len); // Calculate the seed length to use // If the error rate is so low that no differences are possible just seed // over the entire minOverlap region if(max_diff_high > 0) { // Calculate the maximum number of differences between two sequences that overlap // by minOverlap int max_diff_low = static_cast(m_error_rate * minOverlap); if(max_diff_low == 0) max_diff_low = 1; int seed_region_length = static_cast(ceil(max_diff_low / m_error_rate)); int num_seeds_low = max_diff_low + 1; seed_length = static_cast(seed_region_length / num_seeds_low); if(seed_length > static_cast(minOverlap)) seed_length = minOverlap; } else { seed_length = minOverlap; } seed_stride = seed_length; } ================================================ FILE: src/svaba/SvabaAssemblerEngine.h ================================================ #ifndef SVABA_ASSEMBLER_ENGINE_H__ #define SVABA_ASSEMBLER_ENGINE_H__ #include "Util.h" //#include "contigs.h" #include "SGUtil.h" #include "ReadTable.h" #include "SeqLib/BamRecord.h" #include "SeqLib/UnalignedSequence.h" #include "SvabaRead.h" class svabaAssemblerEngine { public: svabaAssemblerEngine() {} svabaAssemblerEngine(const std::string& id, double er, size_t mo, size_t rl) : m_id(id), m_error_rate(er), m_min_overlap(mo), m_readlen(rl) {} bool hasRepeat(const std::string& seq); void fillReadTable(const svabaReadPtrVector& reads); //void fillReadTable(const std::vector& r); bool performAssembly(int num_assembly_rounds); //void doAssembly(ReadTable *pRT, ContigVector &contigs, int pass); void doAssembly(ReadTable *pRT, SeqLib::UnalignedSequenceVector &contigs, int pass); void setToWriteASQG() { m_write_asqg = true; } SeqLib::UnalignedSequenceVector getContigs() const { return m_contigs; } //ContigVector getContigs() const { return m_contigs; } void clearContigs() { m_contigs.clear(); } ReadTable* removeDuplicates(ReadTable* pRT); void calculateSeedParameters(int read_len, const int minOverlap, int& seed_length, int& seed_stride) const; private: void print_results(const SeqLib::UnalignedSequenceVector& cc) const; // void remove_exact_dups(ContigVector& cc) const; void remove_exact_dups(SeqLib::UnalignedSequenceVector& cc) const; void write_asqg(const StringGraph * oGraph, std::stringstream& asqg_stream, std::stringstream& hits_stream, int pass) const; std::string m_id; double m_error_rate; size_t m_min_overlap; size_t m_readlen; size_t count = 0; // num sequencecs added size_t numBubbleRounds = 3; float divergence = 0.00; //0.05 float gap_divergence = 0.00; int maxEdges = 128; int numTrimRounds = 0; // int trimLengthThreshold = -1; // doesn't matter bool bPerformTR = false; // transitivie edge reducetion bool bValidate = false; int resolveSmallRepeatLen = -1; int maxIndelLength = 100; bool bExact = true; std::string outVariantsFile = ""; // dummy bool m_write_asqg = false; ReadTable m_pRT; //ContigVector m_contigs; SeqLib::UnalignedSequenceVector m_contigs; }; #endif ================================================ FILE: src/svaba/SvabaBamWalker.cpp ================================================ #include "SvabaBamWalker.h" #include "SvabaRead.h" #include "SvabaSharedConfig.h" #include "SvabaOptions.h" #include "SvabaLogger.h" #include "SvabaThreadUnit.h" #include "SvabaDebug.h" #include #define QNAME "LH00306:129:227V5CLT4:6:1268:41599:27601" #define QFLAG 147 using SeqLib::AddCommas; using SeqLib::UnalignedSequenceVector; using SeqLib::UnalignedSequence; inline void debug_read(std::string_view msg, const svabaReadPtr read, std::string_view qname_filter = {}, int qflag_filter = -1) { if (!read) return; if (!qname_filter.empty() && read->Qname() != qname_filter) return; if (qflag_filter != -1 && read->AlignmentFlag() != qflag_filter) return; std::cerr << "DEBUG: " << msg << " read " << read << '\n'; } static const std::string ILLUMINA_PE_PRIMER_2p0 = "CAAGCAGAAGACGGCAT"; // Detect adapter read-through: the fragment is shorter than the read // length, so the tail of the read sequences into the adapter. Returns // true if the read looks like adapter contamination and should be // skipped. Reads with indels or unmapped mates are exempted — those // might genuinely support an SV even if the geometry looks adapter-ish. static bool hasAdapter(const svabaRead& r) { // keep reads with indels or unmapped mate — could be real SV signal if (r.MaxDeletionBases() || r.MaxInsertionBases() || !r.InsertSize()) return false; // toss if insert size ≈ read length (completely overlapping pair) if (std::abs(r.FullInsertSize() - r.Length()) < 5) return true; // from here, only consider reads that have soft-clips if (!r.NumClip()) return false; // toss if insert size explains the clip length (adapter read-through) int exp_ins_size = r.Length() - r.NumClip(); if (std::abs(exp_ins_size - std::abs(r.InsertSize())) <= 6) return true; // toss if literal Illumina adapter sequence is present if (r.Sequence().find(ILLUMINA_PE_PRIMER_2p0) != std::string::npos) return true; return false; } //static const std::string REV_ADAPTER = "GCTCTTCCGATCT"; svabaBamWalker::svabaBamWalker(SvabaSharedConfig& sc_) : sc(sc_) {} void svabaBamWalker::addCigar(const svabaReadPtr& r) { if (r->CigarSize() == 1) return; // NB: the -1 is NOT a 0- vs 1-based thing. // It's that VCF (and BP) store location of an indel as the base PRIOR to the event. // but accumulating positions here has "pos" starting at the first base of the next // position - which would be the first base of the indel, so we minus 1 to keep // convention c/w BPs file. It's all 0-based as usual int pos = r->Position() - 1; // position ON REFERENCE for (const auto& i : r->GetCigar()) { if (i.Type() == 'D' || i.Type() == 'I') { std::string key = std::to_string(r->ChrID()) + "_" + std::to_string(pos) + "_" + std::to_string(i.Length()) + i.Type(); ++cigmap[key]; // //debug // if (pos > 79347219 & pos < 79347403) // std::cerr << r->Qname() << " pos " << r->Position() << " pos " << pos << // " " << i.Length() << i.Type() << std::endl; } // advance along the reference for alignment types that consume reference if (i.Type() != 'I' && i.Type() != 'S' && i.Type() != 'H') pos += i.Length(); } } /*bool svabaBamWalker::isDuplicate(const SeqLib::BamRecord &r) { // deduplicate by query-bases / position std::string key = r.Sequence() + std::to_string(r.Position()) + "_" + std::to_string(r.MatePosition()); // its not already add, insert if (!seq_set.count(key)) { seq_set.insert(key); return false; } return true; }*/ SeqLib::GRC svabaBamWalker::readBam(svabaThreadUnit& unit) { // keep track of regions where we hit the weird-read limit SeqLib::GRC bad_regions; // buffer to store reads from a region // if we don't hit the limit, then add them to be assembled svabaReadPtrVector read_buffer; UnalignedSequenceVector train_buffer; train_reads.clear(); // member — cleared per readBam call // if we have a LOT of reads (aka whole genome run), keep track for printing size_t countr = 0; size_t seen = 0; // keep track of which region we are in int current_region = this->region_idx_; // should just skip regions that have excessive coverage // cov = readlength + reads / region_width. So 150 * X / 25000 = 10000 to set max coverage to 10000 // so 25'000 * 10'000 / 150 = 1'666'667 for max cov 10'000 const size_t max_coverage_before_ignore_region = 10'000; size_t seen_cutoff = regions_.at(current_region).Width() * max_coverage_before_ignore_region / std::max(1, sc.readlen); sc.logger.log(sc.opts.verbose > 4, false, "...seen cutoff ", SeqLib::AddCommas(seen_cutoff), " region width ", regions_.at(current_region), " read len ", sc.readlen); // loop the reads while ( auto bamrec = this->Next()) { // makes a deep copy. So when bamrec goes out at end of this iteration, s is still safe svabaReadPtr s = std::make_shared(*bamrec, prefix_); ++seen; if (seen % 100'000 == 0) sc.logger.log(sc.opts.verbose > 3, false, "...bamwlker reading read ", s->Brief(), " - ", SeqLib::AddCommas(seen)); // when we move regions, clear the buffer if (this->region_idx_ != current_region) { seen = 0; sc.logger.log(sc.opts.verbose > 3, false, "...svabaBamWalker switched region from ", regions_.at(current_region), " to ", regions_.at(region_idx_)); current_region = region_idx_; // recalculate seen limit for this region if (current_region < regions_.size()) { seen_cutoff = regions_.at(current_region).Width() * max_coverage_before_ignore_region / std::max(1, sc.readlen); sc.logger.log(sc.opts.verbose > 4, false, "...seen cutoff ", SeqLib::AddCommas(seen_cutoff)); } else { // shouldn't really get here seen_cutoff = 0; } // cache the reads from the last region into BamWalker cache reads.insert(reads.end(), read_buffer.begin(), read_buffer.end()); read_buffer.clear(); } // if hit the limit of reads, log it and try next region if ( (read_buffer.size() > m_limit && m_limit > 0) || seen > seen_cutoff) { // don't train on these train_buffer.clear(); // clear the buffer for this region seen = 0; std::string regstr = (regions_.size() ? regions_[region_idx_].ToString(this->Header()) : " whole BAM"); sc.logger.log(sc.opts.verbose > 1, sc.opts.verbose_log, "\tstopping read lookup at ", s->Brief(), " in window ", regstr, "with ", AddCommas(read_buffer.size()), " weird reads. Limit: ", AddCommas(m_limit)); // add this region to the bad regions list if (regions_.size()) bad_regions.add(regions_[region_idx_]); // clear these reads out, not a good region read_buffer.clear(); // force it to try the next region, or return if none left ++region_idx_; // increment to next region if (region_idx_ >= regions_.size()) {/// no more regions left and last one was bad return bad_regions; // totally done } } SVABA_READ_TRACE(s->Qname(), "SEEN flag=" << s->AlignmentFlag() << " mapq=" << s->MapQuality() << " pos=" << s->ChrName(sc.header) << ":" << s->Position() << " isize=" << s->InsertSize() << " cigar=" << s->CigarString() << " len=" << s->Length()); // check if this read passes the rules for potential SV reads if (s->DuplicateFlag() || s->QCFailFlag() || s->NumHardClip() || sc.blacklist.CountOverlaps(s->AsGenomicRegionMate()) || sc.blacklist.CountOverlaps(s->AsGenomicRegion())) { SVABA_READ_TRACE(s->Qname(), "SKIP dup=" << s->DuplicateFlag() << " qcfail=" << s->QCFailFlag() << " hardclip=" << s->NumHardClip() << " blacklist_mate=" << sc.blacklist.CountOverlaps(s->AsGenomicRegionMate()) << " blacklist_self=" << sc.blacklist.CountOverlaps(s->AsGenomicRegion())); continue; } // quality score trim read s->QualityTrimRead(); // copies sequence into svabaRead.seq_corrected SVABA_READ_TRACE(s->Qname(), "AFTER_TRIM corrected_len=" << s->CorrectedSeqLength()); // this is the main rule check (clips, orientation, unmapped mate, etc.) bool rule_pass = sc.mr.isValid(*s); // FR reads with large isize: per-RG discordant cutoff check. // This replaces the old ReadFilterCollection FR isize rule and uses // the same cutoff as TagDiscordant, so the two are always consistent. if (!rule_pass && s->PairMappedFlag() && s->PairOrientation() == SeqLib::Orientation::FR) { std::string rg; if (!s->GetZTag("RG", rg)) rg = "NA"; int cutoff = getIsizeCutoff(rg); if (std::abs(s->FullInsertSize()) >= cutoff) rule_pass = true; } SVABA_READ_TRACE(s->Qname(), "RULE_CHECK rule_pass=" << rule_pass); // special case to also check against high mismatch // --no-nm disables this path entirely for maximum efficiency if (!rule_pass && !sc.opts.noNmSalvage) { int32_t nm_tag = 0; s->GetIntTag("NM", nm_tag); float nm_frac = static_cast(nm_tag) / static_cast(s->Length()); bool is_adapter = hasAdapter(*s); SVABA_READ_TRACE(s->Qname(), "NM_SALVAGE nm=" << nm_tag << " nm_frac=" << nm_frac << " adapter=" << is_adapter << " pass=" << (nm_frac > 0.02 && !is_adapter)); if (nm_frac > 0.02 && !is_adapter) { s->to_assemble = false; read_buffer.push_back(s); SVABA_READ_TRACE(s->Qname(), "ADDED via NM salvage (to_assemble=false)"); } } // if its less than 40, dont even mess with it if (s->CorrectedSeqLength() < 40) { SVABA_READ_TRACE_IF(rule_pass, s->Qname(), "REJECT corrected_len=" << s->CorrectedSeqLength() << " < 40"); rule_pass = false; } // special rule to filter "RF" "discordants" that are just overlaps if (s->PairOrientation() == SeqLib::Orientation::RF && std::abs(s->InsertSize()) < std::max(200, sc.readlen * 2)) { SVABA_READ_TRACE_IF(rule_pass, s->Qname(), "REJECT RF overlap isize=" << s->InsertSize() << " threshold=" << std::max(200, sc.readlen * 2)); rule_pass = false; } // for mate regions, be more strict if (!get_mate_regions && s->MapQuality() == 0) { SVABA_READ_TRACE_IF(rule_pass, s->Qname(), "REJECT mapq=0 in non-mate region"); rule_pass = false; } // add to all reads pile for kmer correction if (get_coverage) { cov.addRead(*s, 0); } // add to weird coverage if (rule_pass && get_coverage) weird_cov.addRead(*s, 0); // add to the cigar map for all non-duplicate reads if (get_mate_regions) // only add cigar for non-mate regions addCigar(s); // add all the reads for kmer correction // TODO this will add reads to training even if this entire buffer is rejected, so need to make a training buffer as well before adding them if (!sc.opts.ecCorrectType.empty() && sc.opts.ecCorrectType != "0") { // if its not a weird read, include for training purposes // but can subsample for speed if (!rule_pass) { uint32_t hashed = __ac_Wang_hash(__ac_X31_hash_string(s->Qname().c_str())); if ((double)(hashed&0xffffff) / 0x1000000 <= kmer_subsample) train_buffer.push_back(UnalignedSequence(s->UniqueName(), s->CorrectedSeq())); } } // if not a weird read, move on if (!rule_pass) { SVABA_READ_TRACE(s->Qname(), "SKIP not weird (rule_pass=false after all gates)"); continue; } // skip adapter read-through (fragment shorter than read length) // unless the read has an indel that could indicate real SV signal if (hasAdapter(*s)) { SVABA_READ_TRACE(s->Qname(), "SKIP adapter isize=" << s->InsertSize() << " len=" << s->Length() << " nclip=" << s->NumClip()); continue; } // label as discordant or not // modifies r in place TagDiscordant(s); // message logging ++countr; if (countr % 25000 == 0) { sc.logger.log(sc.opts.verbose > 1, false, "...read in ", AddCommas(countr), " weird reads for whole genome read-in. At pos ", s->Brief()); } // adding first to a temp buffer, in case hit limit and then // just don't add any reads read_buffer.push_back(s); SVABA_READ_TRACE(s->Qname(), "ADDED as weird read (to_assemble=true) buffer_size=" << read_buffer.size()); } // end the read loop // "reads" is total cache for this walker. // "read_buffer" was temporary cache in readBam for last region /*reads.insert(reads.end(), std::make_move_iterator(read_buffer.begin()), std::make_move_iterator(read_buffer.end())); read_buffer.clear(); */ // shouldn't need to, but avoid "lingering state" reads.insert(reads.end(), read_buffer.begin(), read_buffer.end()); read_buffer.clear(); // stash training reads as member for the pooled BFC in SvabaRegionProcessor train_reads.insert(train_reads.end(), train_buffer.begin(), train_buffer.end()); train_buffer.clear(); // subsamples reads if too high of coverage // if (get_coverage) // subSampleToWeirdCoverage(max_cov); // calculate the mate region if (get_mate_regions && regions_.size() /* don't get mate regions if reading whole bam */) calculateMateRegions(); return bad_regions; } void svabaBamWalker::AddBackReadsToCorrect() { for (const auto& r : reads) { if (!r->to_assemble) continue; // same filter as Train() side bfc.AddSequence(r->CorrectedSeq(), "", r->UniqueName()); } } void svabaBamWalker::ClearTraining() { bfc.ClearReads(); } void svabaBamWalker::Train() { bfc.Train(); } void svabaBamWalker::ErrorCorrect() { bfc.ErrorCorrect(); } void svabaBamWalker::subSampleToWeirdCoverage(double max_coverage) { auto keep = [&](const std::shared_ptr& r) { double cov1 = weird_cov.getCoverageAtPosition(r->ChrID(), r->Position()); double cov2 = weird_cov.getCoverageAtPosition(r->ChrID(), r->PositionEnd()); double this_cov = std::max(cov1, cov2); if (this_cov <= max_coverage) return true; double sample_rate = 1 - (this_cov - max_coverage) / this_cov; uint32_t k = __ac_Wang_hash(__ac_X31_hash_string(r->Qname().c_str()) ^ m_seed); return ((double)(k & 0xffffff) / 0x1000000) <= sample_rate; }; reads.erase(std::remove_if(reads.begin(), reads.end(), [&](const std::shared_ptr& r) { return !keep(r); }), reads.end()); } void svabaBamWalker::calculateMateRegions() { assert(regions_.size()); SeqLib::GenomicRegion main_region = regions_.at(0); // hold candidate regions. Later trim based on count MateRegionVector tmp_mate_regions; // loop the reads and add mate reads to MateRegionVector for (const auto& r : reads) { int dd = r->GetDD(); // throw away reads that have too many different discordant mappings // or are otherwise bad (dd < 0). // // maxMateChrID gate: skip mates on decoy/alt/mito chroms in // human (default 23 = through chrY). Set to -1 via --non-human // or --max-mate-chr to allow all chromosomes. // // minMateMAPQ gate: skip reads below this MAPQ (default -1 = no // gate). Set via --min-mate-mapq to e.g. 1 to exclude MAPQ=0 // multi-mappers from triggering mate lookups. const int maxChr = sc.opts.maxMateChrID; const int minMQ = sc.opts.minMateMAPQ; if (!r->PairedFlag() || (maxChr >= 0 && r->MateChrID() > maxChr) || (minMQ >= 0 && r->MapQuality() < minMQ) || (maxChr >= 0 && r->ChrID() > maxChr) || !r->MappedFlag() || !r->MateMappedFlag() || dd < 0 || dd > MAX_SECONDARY_HIT_DISC) continue; // get the mate region position, will check next if different // from current main region MateRegion mate(r->MateChrID(), r->MatePosition(), r->MatePosition()); mate.Pad(MATE_REGION_PAD); mate.partner = main_region; // if mate not in main interval, add a padded version if (!main_region.GetOverlap(mate) && !local_blacklist.CountOverlaps(mate) && !sc.blacklist.CountOverlaps(mate)) { tmp_mate_regions.add(mate); } } // merge it down to get the mate regions tmp_mate_regions.MergeOverlappingIntervals(); // get the counts by overlapping mate-reads with the newly defined // mate regions. Same MAPQ gate as above so the count is consistent // with the candidate set. const int minMQ_count = sc.opts.minMateMAPQ; for (const auto& r : reads) { SeqLib::GenomicRegion mate(r->MateChrID(), r->MatePosition(), r->MatePosition()); // if mate not in main interval, check which mate regions it's in if (!main_region.GetOverlap(mate) && (minMQ_count < 0 || r->MapQuality() >= minMQ_count)) { for (auto& k : tmp_mate_regions) if (k.GetOverlap(mate)) { ++k.count; continue; } } } #ifdef DEBUG_SVABA_BAMWALKER std::cerr << "SBW: Mate regions are" << std::endl; for (auto& i : tmp_mate_regions) std::cerr << " " << i << " count " << i.count << std::endl; #endif // keep only ones with enough supporting reads const int minCount = sc.opts.mateRegionMinCount; for (auto& i : tmp_mate_regions) { if (i.count >= minCount) mate_regions.add(i); } #ifdef DEBUG_SVABA_BAMWALKER std::cerr << "SBW: Final mate regions are:" << std::endl; for (auto& i : mate_regions) std::cerr << " " << i << " read count " << i.count << std::endl; #endif // hard cutoff on mate regions TODO make more elegant if (mate_regions.size() > 6) { sc.logger.log(sc.opts.verbose > 2, true, "...cutting off mate lookups for region ", regions_.at(0), " with too many mate regions of: ", mate_regions.size()); mate_regions.clear(); } } // bool svabaBamWalker::hasAdapter(const SeqLib::BamRecord& r) const { // // keep it if it has indel or unmapped read // if (r.MaxDeletionBases() || r.MaxInsertionBases() || !r.InsertSize()) // || !r.NumClip()) // return false; // // toss if isize is basically read length (completely overlaping) // if (std::abs(r.FullInsertSize() - r.Length()) < 5) // return true; // // now only consider reads where clip can be explained by isize // if (!r.NumClip()) // return false; // // toss it then if isize explans clip // int exp_ins_size = r.Length() - r.NumClip(); // expected isize if has adapter // if ((exp_ins_size - 6) < std::abs(r.InsertSize()) && (exp_ins_size + 6) > std::abs(r.InsertSize())) // return true; // std::string seq = r.Sequence(); // if (seq.find(ILLUMINA_PE_PRIMER_2p0) != std::string::npos) // return true; // return false; // } void svabaBamWalker::RealignDiscordants(svabaThreadUnit& unit) { size_t realigned_count = 0; size_t realign_attempted = 0; std::unordered_set bad_discordant; for (auto& r : reads) { if (!discordantRealigner.ShouldRealign(r)) continue; ++realign_attempted; if (realign_attempted % 10000 == 0) sc.logger.log(sc.opts.verbose > 3, false, "Realigning read ", r->Brief(), " - ", SeqLib::AddCommas(realign_attempted), " - ", realigned_count); // modifies the read in place if(discordantRealigner.RealignRead(r, unit.bwa_aligner)) { ++realigned_count; bad_discordant.insert(r->Qname()); } } // loop through and set DD < 0 for all read-pairs that have a bad discordant for (auto& r : reads) if (r->GetDD() >= 0 && bad_discordant.count(r->Qname())) r->SetDD(DiscordantRealigner::MATE_BAD_DISC); } int svabaBamWalker::getIsizeCutoff(const std::string& RG) { auto cc = isize_cutoff_per_rg.find(RG); if (cc != isize_cutoff_per_rg.end()) return cc->second; // not cached yet — look up from learned params const auto it = sc.bamStats.find(this->prefix_); if (it == sc.bamStats.end()) { if (!sc.warned.count(this->prefix_)) { sc.logger.log(true, true, "SBW: Can't find LearnBamParams for ", this->prefix_); sc.warned.insert(this->prefix_); } isize_cutoff_per_rg[RG] = DEFAULT_ISIZE_THRESHOLD; return DEFAULT_ISIZE_THRESHOLD; } const auto rgg = it->second.bam_read_groups.find(RG); if (rgg == it->second.bam_read_groups.end()) { if (!sc.warned.count(RG)) sc.warned.insert(RG); isize_cutoff_per_rg[RG] = DEFAULT_ISIZE_THRESHOLD; return DEFAULT_ISIZE_THRESHOLD; } bool is_normal = (!prefix_.empty() && prefix_[0] == 'n'); double sd_mult = is_normal ? sc.opts.sdDiscCutoffNormal : sc.opts.sdDiscCutoff; int cutoff = static_cast( rgg->second.isize_median + rgg->second.sd_isize * sd_mult); isize_cutoff_per_rg[RG] = cutoff; return cutoff; } void svabaBamWalker::TagDiscordant(svabaReadPtr& r) { std::string RG; if (!r->GetZTag("RG", RG)) RG = "NA"; int cutoff = getIsizeCutoff(RG); // accept as discordant if not FR, has large enough isize, or is inter-chromosomal bool weird_orientation = r->PairOrientation() != SeqLib::Orientation::FR; if ( weird_orientation || abs(r->FullInsertSize()) >= cutoff || r->Interchromosomal() ) r->dd = 1; // set as discordant // not discordant if read or mate in blacklist if (sc.blacklist.CountOverlaps(r->AsGenomicRegion()) || sc.blacklist.CountOverlaps(r->AsGenomicRegionMate()) || local_blacklist.CountOverlaps(r->AsGenomicRegion()) || local_blacklist.CountOverlaps(r->AsGenomicRegionMate())) r->dd = 0; //debug // if (r->Qname() == "LH00306:129:227V5CLT4:6:2114:6074:25724") { // std::cerr << " TAG " << r->dd << " -- " << *r << " weird orientation " << // weird_orientation << " cutoff " << cc->second << " INS " << // abs(r->FullInsertSize()) << std::endl; // } return; } ================================================ FILE: src/svaba/SvabaBamWalker.h ================================================ #pragma once #include #include #include #include //#include "malloc.h" //debug #include "SeqLib/BamReader.h" #include "SeqLib/ReadFilter.h" #include "STCoverage.h" #include "SeqLib/BWAAligner.h" #include "DiscordantRealigner.h" #include "SeqLib/BFC.h" class svabaThreadUnit; class SvabaSharedConfig; // storage container for mate lookup-regions class MateRegion: public SeqLib::GenomicRegion { public: MateRegion() {} MateRegion (int32_t c, uint32_t p1, uint32_t p2, char s = '*') : SeqLib::GenomicRegion(c, p1, p2, s) {} size_t count = 0;// read count SeqLib::GenomicRegion partner; }; typedef SeqLib::GenomicRegionCollection MateRegionVector; class svabaBamWalker: public SeqLib::BamReader { public: svabaBamWalker(SvabaSharedConfig& sc_); // read in the reads SeqLib::GRC readBam(svabaThreadUnit& unit); // clear it out void clear() { cov.clear(); weird_cov.clear(); bfc.ClearReads(); cigmap.clear(); mate_regions.clear(); reads.clear(); get_coverage = true; get_mate_regions = true; local_blacklist.clear(); train_reads.clear(); //malloc_trim(0);//debug } void AddBackReadsToCorrect(); // void Train(); void ErrorCorrect(); void ClearTraining(); // set the id for this bam e.g. t001 void SetPrefix(std::string_view pref) { prefix_ = pref; } void RealignDiscordants(svabaThreadUnit& unit); ///bool hasAdapter(const SeqLib::BamRecord& r) const; void addCigar(const svabaReadPtr& r); //bool isDuplicate(const SeqLib::BamRecord &r); void subSampleToWeirdCoverage(double max_coverage); void calculateMateRegions(); void TagDiscordant(svabaReadPtr& r); /// Return the per-RG isize discordant cutoff, lazily cached. int getIsizeCutoff(const std::string& RG); // should we store the mate regions? bool get_mate_regions = true; // place to store reads when we get them svabaReadPtrVector reads; //c // cov is the all-read coverage tracker // weird-cov just tracks coverage of accepted (clip, disc, etc reads) // buffered cov is coverage minus first 8 and last 8 bp. Why? // because when looking for variant-supporting reads, we require // alignment of a read to the variant to overlap it by 8 bp // to reduce false-positive alt reads. So we use the buffered // coverage to compare against this buffered alt cov. STCoverage cov, weird_cov; //c // hash of cigars for indels SeqLib::CigarMap cigmap; //c // mate regions to lookup MateRegionVector mate_regions; //c // object for realigning discordant reads DiscordantRealigner discordantRealigner; //c // maximum coverage of accepted reads, before subsampling size_t max_cov = 100; // should we keep reads for learning correction double kmer_subsample = 0.5; // should we subsample the learning reads? bool do_kmer_filtering = true; // should we get the read coverage bool get_coverage = true; // set a hard limit on how many reads to accept size_t m_limit = 0; // an extra learned region-specific blacklist SeqLib::GRC local_blacklist; SeqLib::BFC bfc; // subsampled non-weird reads kept for BFC training. Populated during // readBam, consumed by the pooled BFC in SvabaRegionProcessor. SeqLib::UnalignedSequenceVector train_reads; private: // for setting the SR tag std::string prefix_; // eg. tumor, normal // might want these in case we are looking for duplicates //std::unordered_set seq_set; //c // seed for the kmer-learning subsampling uint32_t m_seed = 1337; // for logging to console, options etc SvabaSharedConfig& sc; // cache RG cutoffs so don't have to recalculate std::unordered_map isize_cutoff_per_rg; }; ================================================ FILE: src/svaba/SvabaDebug.h ================================================ // SvabaDebug.h — compile-time contig and read tracing. // // Contig tracing: // cmake .. -DSVABA_TRACE_CONTIG='"c_fermi_chr2_215869501_215894501_11C"' // make -j // // This compiles svaba with verbose stderr tracing for every decision // point that contig touches: assembly filtering, BP identification, // r2c scoring, split coverage, confidence assignment, and output gating. // // To disable (default): just don't pass -DSVABA_TRACE_CONTIG. // To trace ALL contigs (very noisy): -DSVABA_TRACE_ALL=1 // // Read tracing: // cmake .. -DSVABA_TRACE_READ='"LH00306:235:22NHGWLT4:6:1329:45742:15246"' // make -j // // Traces a specific read (by QNAME) through the BamWalker read-filter // pipeline: duplicate/QC/blacklist gates, rule_pass, high-NM salvage, // adapter filter, quality trim, and final buffer admission. // // To trace ALL reads (extremely noisy): -DSVABA_TRACE_ALL_READS=1 // #pragma once #include #include // ── Contig tracing ────────────────────────────────────────────────── #ifdef SVABA_TRACE_CONTIG // Match by contig name (cname) inline bool _svaba_trace_match(const std::string& cname) { return cname == SVABA_TRACE_CONTIG; } #define SVABA_TRACE(cname, msg) \ do { if (_svaba_trace_match(cname)) { \ std::cerr << "[TRACE:" << __FILE__ << ":" << __LINE__ << "] " \ << msg << std::endl; \ } } while(0) #define SVABA_TRACE_IF(cond, cname, msg) \ do { if ((cond) && _svaba_trace_match(cname)) { \ std::cerr << "[TRACE:" << __FILE__ << ":" << __LINE__ << "] " \ << msg << std::endl; \ } } while(0) #define SVABA_TRACING 1 #elif defined(SVABA_TRACE_ALL) #define SVABA_TRACE(cname, msg) \ do { std::cerr << "[TRACE:" << __FILE__ << ":" << __LINE__ << "] " \ << "[" << (cname) << "] " << msg << std::endl; \ } while(0) #define SVABA_TRACE_IF(cond, cname, msg) \ do { if (cond) { std::cerr << "[TRACE:" << __FILE__ << ":" << __LINE__ << "] " \ << "[" << (cname) << "] " << msg << std::endl; } \ } while(0) #define SVABA_TRACING 1 #else #define SVABA_TRACE(cname, msg) do {} while(0) #define SVABA_TRACE_IF(cond, cname, msg) do {} while(0) // SVABA_TRACING intentionally not defined #endif // ── Read tracing ──────────────────────────────────────────────────── #ifdef SVABA_TRACE_READ inline bool _svaba_read_trace_match(const std::string& qname) { return qname == SVABA_TRACE_READ; } #define SVABA_READ_TRACE(qname, msg) \ do { if (_svaba_read_trace_match(qname)) { \ std::cerr << "[READ_TRACE:" << __FILE__ << ":" << __LINE__ << "] " \ << msg << std::endl; \ } } while(0) #define SVABA_READ_TRACE_IF(cond, qname, msg) \ do { if ((cond) && _svaba_read_trace_match(qname)) { \ std::cerr << "[READ_TRACE:" << __FILE__ << ":" << __LINE__ << "] " \ << msg << std::endl; \ } } while(0) #define SVABA_READ_TRACING 1 #elif defined(SVABA_TRACE_ALL_READS) #define SVABA_READ_TRACE(qname, msg) \ do { std::cerr << "[READ_TRACE:" << __FILE__ << ":" << __LINE__ << "] " \ << "[" << (qname) << "] " << msg << std::endl; \ } while(0) #define SVABA_READ_TRACE_IF(cond, qname, msg) \ do { if (cond) { std::cerr << "[READ_TRACE:" << __FILE__ << ":" << __LINE__ << "] " \ << "[" << (qname) << "] " << msg << std::endl; } \ } while(0) #define SVABA_READ_TRACING 1 #else #define SVABA_READ_TRACE(qname, msg) do {} while(0) #define SVABA_READ_TRACE_IF(cond, qname, msg) do {} while(0) // SVABA_READ_TRACING intentionally not defined #endif // ── Kmer restriction ──────────────────────────────────────────────── // // Compile-time read filter: after BFC error correction, only reads // whose corrected sequence contains the specified kmer survive into // assembly/r2c/corrected.bam. All others get to_assemble = false. // // Usage: // cmake .. -DCMAKE_CXX_FLAGS='-DSVABA_KMER_RESTRICT="\"CCATGCAGAGTGTTGAAGAAAAGGC\""' // // Also checks the reverse complement so orientation doesn't matter. // Zero cost when not compiled in. #ifdef SVABA_KMER_RESTRICT inline std::string _svaba_revcomp(const std::string& s) { std::string rc(s.size(), 'N'); for (size_t i = 0; i < s.size(); ++i) { switch (s[s.size() - 1 - i]) { case 'A': case 'a': rc[i] = 'T'; break; case 'T': case 't': rc[i] = 'A'; break; case 'C': case 'c': rc[i] = 'G'; break; case 'G': case 'g': rc[i] = 'C'; break; default: rc[i] = 'N'; break; } } return rc; } inline bool _svaba_kmer_match(const std::string& seq) { static const std::string kmer_fwd(SVABA_KMER_RESTRICT); static const std::string kmer_rc = _svaba_revcomp(kmer_fwd); return seq.find(kmer_fwd) != std::string::npos || seq.find(kmer_rc) != std::string::npos; } #define SVABA_KMER_RESTRICTING 1 #endif ================================================ FILE: src/svaba/SvabaFileLoader.cpp ================================================ // SvabaFileLoader.cpp #include #include // for std::unique_ptr #include // for std::istringstream #include // for std::string #include // if you use any std::vector in this file #include "SvabaFileLoader.h" // your own header #include "SvabaSharedConfig.h" #include "SvabaLogger.h" #include "SvabaOptions.h" #include "SeqLib/RefGenome.h" // defines SeqLib::RefGenome #include "SeqLib/GenomicRegionCollection.h" // defines SeqLib::GRC #include "SeqLib/BamHeader.h" // if you use SeqLib::BamHeader anywhere #include "SeqLib/BamReader.h" // if you invoke any BamReader methods namespace fs = std::filesystem; using std::string; SvabaFileLoader::SvabaFileLoader(SvabaSharedConfig& sc_) : sc(sc_) {} std::unique_ptr SvabaFileLoader::loadReference() { auto rg = std::make_unique(); try { rg->LoadIndex(sc.opts.refGenome); } catch (const std::exception& e) { sc.logger.log(true, true, "ERROR: Unable to load reference genome '", sc.opts.refGenome, "': ", e.what()); throw; } sc.logger.log(false, true, "Loaded reference genome: ", sc.opts.refGenome); return rg; } void SvabaFileLoader::loadBedRegions(const std::string& path, SeqLib::GRC& gr) { if (path.empty()) return; try { gr = SeqLib::GRC(path, sc.header); gr.CreateTreeMap(); sc.logger.log(true, true, "...loaded BED file '", path, "' (", gr.size(), " regions)"); } catch (const std::exception& e) { sc.logger.log(true, true, "ERROR: cannot read BED file '", path, "': ", e.what()); throw; } } void SvabaFileLoader::countJobs( SeqLib::GRC& runRegions) { SeqLib::GRC fileRegions; const auto& rf = sc.opts.regionFile; // 1) If its a real BED/region file on disk if (!rf.empty() && fs::exists(rf)) { try { fileRegions = SeqLib::GRC(rf, sc.header); } catch (const std::exception& ex) { sc.logger.log( /*toErr=*/true, /*toLog=*/true, "Could not parse region file '", rf, "': ", ex.what(), ". Skipping region file."); } // 2) If it looks like chr:start-end } else if (rf.find(':') != string::npos && rf.find('-') != string::npos) { fileRegions.add(SeqLib::GenomicRegion(rf, sc.header)); // 3) Bare chromosome name(s), possibly comma-separated (e.g. "chr1,chr2,chr3") } else if (!rf.empty()) { // Split on commas to support -k chr1,chr2,chr3 std::istringstream ss(rf); std::string token; while (std::getline(ss, token, ',')) { if (token.empty()) continue; // Each token could be "chr1" or "chr1:100-200" if (token.find(':') != string::npos && token.find('-') != string::npos) { fileRegions.add(SeqLib::GenomicRegion(token, sc.header)); } else { SeqLib::GenomicRegion gr(token, "1", "1", sc.header); if (gr.chr < 0 || gr.chr >= sc.header.NumSequences()) { throw std::runtime_error("Region '" + token + "' failed to match any chromosome in BAM header"); } gr.pos2 = sc.header.GetSequenceLength(gr.chr); fileRegions.add(gr); } } // 4) else: whole genome } else { for (int i = 0; i < sc.header.NumSequences(); ++i) { fileRegions.add( SeqLib::GenomicRegion(i, 1, sc.header.GetSequenceLength(i)) ); } } if (fileRegions.size() == 0) { throw std::runtime_error("No regions found" + (rf.empty() ? string("") : " in '" + rf + "'")); } // 5) Chunk them up if (sc.opts.chunkSize > 0) { for (auto& r : fileRegions) { // this SeqLib constructor splits a region into windows of size chunkSize with pad SeqLib::GRC pieces(sc.opts.chunkSize, sc.opts.windowpad, r); runRegions.Concat(pieces); } } else { // if chunkSize <= 0, we just do one big pass: runRegions = fileRegions; } // if no explicit regionFile, clear fileRegions to signal whole genome if (rf.empty()) { fileRegions.clear(); } return; } ================================================ FILE: src/svaba/SvabaFileLoader.h ================================================ // SvabaFileLoader.h #pragma once #include #include #include "SeqLib/GenomicRegionCollection.h" class SvabaLogger; class SvabaOptions; class SvabaSharedConfig; namespace SeqLib { class RefGenome; } /// A small helper for all the open this file, bail out if it fails logic. /// Carries around references to your single SvabaLogger and SvabaOptions. class SvabaFileLoader { public: /// You must give it your one-and-only logger and options. SvabaFileLoader(SvabaSharedConfig& sc_); /// Loads a BED style file into a GenomicRegionCollection, or returns empty if no path. void loadBedRegions(const std::string& path, SeqLib::GRC& gr); std::unique_ptr loadReference(); /// load the regions to run file and parese out jobs void countJobs(SeqLib::GRC& runRegions); private: SvabaSharedConfig& sc; }; ================================================ FILE: src/svaba/SvabaLogger.cpp ================================================ #include "SvabaLogger.h" #include "SvabaOptions.h" SvabaLogger::~SvabaLogger() { if (logFile_.is_open()) logFile_.close(); } void SvabaLogger::init(const std::string& filename) { logFile_.open(filename, std::ios::out | std::ios::app); if (!logFile_) { std::cerr << "ERROR: Unable to open log file: " << filename << std::endl; } } void SvabaLogger::welcome(SvabaOptions& opts) { // write to log this->log(true, true, "-----------------------------------------------------------\n", "--- Running svaba SV and indel detection on ", " threads ---", (opts.numThreads >= 10 ? "\n" : "-\n"), "--- Version: ", SVABA_VERSION, " - ", SVABA_DATE, " ---\n", "--- (inspect *.log for real-time progress updates) ---\n", "-----------------------------------------------------------"); // print the options to console if verbose (handled insize SvabaOptions) //opts.printToLogger(*this); } ================================================ FILE: src/svaba/SvabaLogger.h ================================================ #pragma once #include #include #include #include #include class SvabaOptions; /// A simple thread safe logger that writes to a file and (optionally) stderr. class SvabaLogger { public: SvabaLogger() = default; ~SvabaLogger(); /// Must be called once at program startup, before any log() calls. /// Opens (and appends to) the given filename. void init(const std::string& filename); /// Log a message. /// @param toErr if true, also write to std::cerr /// @param toLog if true, write to the log file /// @param args one or more things you can stream into an ostringstream template void log(bool toErr, bool toLog, Args&&... args) { // it no printing at all, just leave if (!toErr && !toLog) return; std::lock_guard lock(mtx_); std::ostringstream oss; // fold expression to stream all args (oss << ... << std::forward(args)); auto msg = oss.str(); if (toLog && logFile_.is_open()) logFile_ << msg << "\n"; if (toErr) std::cerr << msg << "\n"; } void welcome(SvabaOptions& opts); private: std::ofstream logFile_; std::mutex mtx_; }; ================================================ FILE: src/svaba/SvabaModels.cpp ================================================ #include "SvabaModels.h" #include #include #include #include namespace SvabaModels { double LogLikelihood(double ref, double alt, double f, double e_fwd, double e_back) { // less negative log-likelihoods means more likely // eg for low error rate, odds that you see 5 ALT and 5 REF // if you are testing for AF = 0 is going to be very low (eg -40) // To test if something is true, we want to test the log-likelihood that // it's AF is != 0, so we test LL(ref, alt, AF=0, er). If this is // a large negative number, it means that AF = 0 is very unlikely. // If we use a larger error rate, then it is more likely that we will // see ALT reads even if true AF = 0, so as ER goes up, then // LL(ref, alt, AF=0, er) becomes less negative. // Unnormalized log10-likelihood of seeing 'ref' ref-reads and 'alt' alt-reads // under a simple two-state error/mutation model. double ll = 0.0; // Clamp negative counts to zero ref = std::max(0.0, ref); alt = std::max(0.0, alt); // P(read = REF): // (1-f)*(1-e_fwd) true-ref, no forward error // + f*e_back true-alt, mis-read back to ref double p_ref = (1.0 - f) * (1.0 - e_fwd) // prior arg1 + f * e_back; // std::cerr << " pref " << p_ref << " ref " << ref << std::endl; if (p_ref > 0.0) { ll += ref * std::log10(p_ref); } else if (ref > 0) { return -1e12; // zero probability but nonzero observations -> -Inf } // P(read = ALT): // f*(1-e_back) true-alt, no back mutation // + (1-f)*e_fwd true-ref, forward error to alt double p_alt = f * (1.0 - e_back) + (1.0 - f) * e_fwd; // std::cerr << " palt " << p_alt << " alt " << alt << std::endl; if (p_alt > 0.0) { ll += alt * std::log10(p_alt); } else if (alt > 0) { return -1e12; } return ll; } // Forward: keep your public signature e_fwd is the high artifact rate. static inline double SomaticLOD_withSplitErrors(double aN, double dN, double aT, double dT, double e_art_fwd, double e_rev, double eN_fwd, double eT_fwd); double SomaticLOD(double aN, double dN, double aT, double dT, double e_fwd, double e_rev) { // Map old params to split errors: // - e_fwd (caller) is your high artifact rate -> e_art_fwd // - Choose conservative, lower empirical forward error caps for normal/tumor const double e_art_fwd = e_fwd; // You can tune these caps globally if you like const double eN_fwd = std::min(e_art_fwd, 0.005); // 0.5% in NORMAL const double eT_fwd = std::min(std::max(e_art_fwd, 1e-4), 0.02); // 2% in TUMOR return SomaticLOD_withSplitErrors(aN, dN, aT, dT, e_art_fwd, e_rev, eN_fwd, eT_fwd); } // Implementation with split errors static inline double SomaticLOD_withSplitErrors(double aN, double dN, double aT, double dT, double e_art_fwd, double e_rev, double eN_fwd, double eT_fwd) { const double epsilon = 1e-6; const double dN_safe = std::max(1.0, dN); const double dT_safe = std::max(1.0, dT); // MLE tumor AF under H1 double fT_hat_raw = aT / dT_safe; double fT_hat = std::clamp(fT_hat_raw, epsilon, 1.0 - epsilon); // Likelihood helpers (use *different* forward error by sample) auto LL_N = [&](double d, double a, double f) { return LogLikelihood(d - a, a, f, eN_fwd, e_rev); // log10 }; auto LL_T = [&](double d, double a, double f) { return LogLikelihood(d - a, a, f, eT_fwd, e_rev); // log10 }; // Artifact-branch helper: use the generous artifact forward rate auto LL_art = [&](double d, double a, double f) { return LogLikelihood(d - a, a, f, e_art_fwd, e_rev); // log10 }; // ========================= // Artifact allele fraction // ========================= // Keep artifact AF small; rate of errors (e_art_fwd) can be high, but true AF under artifact is low. const double f_art = std::clamp(e_art_fwd, 1e-5, 1e-2); // ========================= // SOMATIC (H1): max of // (A) True somatic: N~0, T~fT_hat // (B) Artifact in both: N~f_art, T~f_art // ========================= double llN_som_true = LL_N(dN_safe, aN, 0.0); double llT_som_true = LL_T(dT_safe, aT, fT_hat); double ll_som_true = llN_som_true + llT_som_true; double llN_som_art = LL_art(dN_safe, aN, f_art); double llT_som_art = LL_art(dT_safe, aT, f_art); double ll_som_art = llN_som_art + llT_som_art; double ll_som = std::max(ll_som_true, ll_som_art); // ========================= // GERMLINE (H0): max of // Het (0.5), Hom-alt (1-eps), Shared MLE f, Artifact (low f) // ========================= const double f_het = std::clamp(0.5, epsilon, 1.0 - epsilon); const double f_hom = std::clamp(1.0, epsilon, 1.0 - epsilon); // Upper edge of "looks like a low-level artifact" VAF band. Anything // above this is either heterozygous (~0.5), homozygous (~1.0), or // an LOH-shifted germline event -- not a low-VAF shared artifact. const double germ_plausible_floor = 0.15; // ========================================================================= // GERM_shared: pooled-MLE allele fraction across N+T. // // Design goals: // - aN = 0 (clean normal) -> GERM_shared must be held back so that a // low-VAF tumor signal can win as somatic. // - aN = 1-2 in a clean normal -> GERM_shared should compete strongly, // because a shared low-VAF artifact is // exactly what that data looks like. // - aN >= ~3 -> GERM_shared dominates normally; shared artifact or // real (possibly LOH) germline event explains the data. // // We use TWO shaping knobs on top of the raw pooled-MLE log-likelihood: // // (1) "Cleanliness" penalty: a decaying function of aN relative to // expected errors in the normal (dN * eN_fwd). Near-maximum penalty // when the normal looks clean (aN at or below the error floor), // drops off steeply as excess normal alt reads accumulate. // // (2) VAF similarity bonus: when the normal does have real alt // evidence, *matching* VAFs between normal and tumor are a strong // "shared" fingerprint. Gated by (1 - cleanliness) so this bonus // only fires once the normal actually has signal -- this prevents // accidentally penalizing low-VAF-tumor somatic calls where the // VAF-ratio would be noisy and meaningless. // // f_shared_hat floats to the true pooled MLE (no 0.15 floor). The old // clamp turned the "free-MLE" branch into a "germline-only-MLE" branch, // which is exactly what we don't want when explaining low-VAF shared // signal. // ========================================================================= // Pooled MLE across both samples, free to land anywhere in (0,1). const double f_shared_hat_raw = (aN + aT) / (dN_safe + dT_safe); const double f_shared_hat = std::clamp(f_shared_hat_raw, epsilon, 1.0 - epsilon); // Start with the raw pooled-MLE log-likelihood -- never -inf, always let // GERM_shared compete; shaping below decides how strongly. double ll_germ_shared = LL_N(dN_safe, aN, f_shared_hat) + LL_T(dT_safe, aT, f_shared_hat); // ----- (1) cleanliness penalty on GERM_shared ----------------------------- // Expected normal alt count under pure forward error at the (capped) // normal error rate. Anything at or below this is indistinguishable from // a clean normal. const double expected_errors_N = dN_safe * eN_fwd; const double excess_alt_N = std::max(0.0, aN - expected_errors_N); // Steep exponential decay: coefficient=3 means a single excess alt read // already drops cleanliness by ~20x. At dN=79, eN_fwd=0.005: // aN=0 -> cleanliness=1.00 (full hold-back, clean somatic wins) // aN=1 -> cleanliness~0.16 (penalty already mostly gone) // aN=2 -> cleanliness~0.008 (essentially no hold-back) // This is the "even 1-2 normal alt reads should steeply consider shared" // behavior from the design discussion. const double kCleanDecay = 3.0; const double cleanliness = std::exp(-kCleanDecay * excess_alt_N); // Max hold-back magnitude at aN=0. const double kCleanPenalty = 2.0; ll_germ_shared -= kCleanPenalty * cleanliness; // ----- (2) VAF similarity bonus -------------------------------------------- // Matching tumor/normal VAFs are the fingerprint of a shared artifact or // a shared (possibly LOH-shifted) germline event. Gated by (1-cleanliness) // so this bonus only really fires once the normal has alt evidence -- // otherwise the ratio is dominated by numerical epsilons. const double f_n_hat = std::clamp(aN / dN_safe, epsilon, 1.0 - epsilon); const double f_t_hat = fT_hat; const double ratio = (std::max(f_n_hat, f_t_hat) > 0.0) ? std::min(f_n_hat, f_t_hat) / std::max(f_n_hat, f_t_hat) : 0.0; const double sim_weight = 1.0 - cleanliness; // ~0 at aN=0, ~1 at aN>=2 const double kSimBonus = 3.0; // log10 ll_germ_shared += sim_weight * ratio * kSimBonus; // ----- (3) "both samples low VAF" shared-artifact bonus -------------------- // If both normal and tumor carry nonzero alt evidence AND both VAFs sit // below the germline-plausible band, that is the canonical signature of a // low-level shared artifact (repeat tract, low-complexity region, etc). // We add a bonus proportional to how similar the two VAFs are. This is // independent of (2) -- (2) rewards *matching*, (3) rewards *both-low*. const double low_vaf_cutoff = germ_plausible_floor; // 0.15 const bool both_nonzero_alt = (aN > 0.0) && (aT > 0.0); const bool both_low_vaf = (f_n_hat < low_vaf_cutoff) && (f_t_hat < low_vaf_cutoff); if (both_nonzero_alt && both_low_vaf) { const double kSharedLowVafBonus = 2.0; // log10 ll_germ_shared += kSharedLowVafBonus * ratio; } // other model options (germline het, germline homozygous, germline artifact) double ll_germ_het = LL_N(dN_safe, aN, f_het) + LL_T(dT_safe, aT, f_het); double ll_germ_hom = LL_N(dN_safe, aN, f_hom) + LL_T(dT_safe, aT, f_hom); double ll_germ_art = LL_art(dN_safe, aN, f_art) + LL_art(dT_safe, aT, f_art); // ========================================================================= // GERM_free: f_N and f_T MLEd independently (NOT tied like GERM_shared). // // Fills the gap where normal has low-but-real variant evidence while the // tumor is near-clonal: LOH, clonal hematopoiesis, tumor-in-normal // contamination, mosaic germline, etc. GERM_shared forces f_N = f_T, so // e.g. (aN=6/dN=33, aT=84/dT=90) -- normal VAF ~0.18, tumor VAF ~0.93 -- // fits NEITHER sample under GERM_shared (pooled f≈0.73 is wrong for // both), GERM_het is also wrong for both, and SOM_true wins despite // clear alt evidence in the normal. // // Parameter count: // SOM_true has 1 free parameter (f_T). // GERM_free has 2 free parameters (f_N, f_T). // Charge a BIC penalty of 0.5·log10(dN+dT) for the 1 net extra parameter. // // Activation gate: aN > expected_errors_N + 1 // Rationale: when aN is at or near the expected number of error-driven // alt reads in the normal (dN·eN_fwd), there is no real variant signal // and the MLE f_N pegs at the error floor. Unconditionally enabling // GERM_free in that regime makes it degenerate to "SOM_true minus BIC" // on every clean-normal event, artificially capping somlod at ~1 bit // regardless of how strong the tumor signal is. The +1 buffer above the // error floor is Poisson-tail slack so a single stray error read in a // clean normal doesn't trip the gate. // ========================================================================= double ll_germ_free = -std::numeric_limits::infinity(); double ll_germ_free_LL_N = 0.0, ll_germ_free_LL_T = 0.0; // for debug dump double kGermFreeBIC = 0.0; double f_n_free_dbg = 0.0, f_t_free_dbg = 0.0; bool germ_free_active = false; if (aN > expected_errors_N + 1.0) { germ_free_active = true; f_n_free_dbg = std::clamp(aN / dN_safe, epsilon, 1.0 - epsilon); f_t_free_dbg = std::clamp(aT / dT_safe, epsilon, 1.0 - epsilon); kGermFreeBIC = 0.5 * std::log10(dN_safe + dT_safe); ll_germ_free_LL_N = LL_N(dN_safe, aN, f_n_free_dbg); ll_germ_free_LL_T = LL_T(dT_safe, aT, f_t_free_dbg); ll_germ_free = ll_germ_free_LL_N + ll_germ_free_LL_T - kGermFreeBIC; } double ll_germ = std::max({ ll_germ_shared, ll_germ_het, ll_germ_hom, ll_germ_art, ll_germ_free }); double lod_somatic_vs_germ = ll_som - ll_germ; // --------- DEBUG DUMP ---------- if (false) { // SOM/H0 winners (for readability) const bool som_true_better = (ll_som_true >= ll_som_art); const char* som_branch = som_true_better ? "SOM:true" : "SOM:art"; double ll_germ_best = ll_germ_het; const char* germ_branch = "GERM:het"; if (ll_germ_hom > ll_germ_best) { ll_germ_best = ll_germ_hom; germ_branch = "GERM:hom"; } if (ll_germ_art > ll_germ_best) { ll_germ_best = ll_germ_art; germ_branch = "GERM:art"; } if (ll_germ_shared > ll_germ_best) { ll_germ_best = ll_germ_shared; germ_branch = "GERM:shared"; } if (ll_germ_free > ll_germ_best) { ll_germ_best = ll_germ_free; germ_branch = "GERM:free"; } std::cerr << "SomaticLOD" << " | aN=" << aN << " dN=" << dN << " aT=" << aT << " dT=" << dT << " e_art_fwd=" << e_art_fwd << " eN_fwd=" << eN_fwd << " eT_fwd=" << eT_fwd << " e_rev=" << e_rev << " eps=" << epsilon << " fT_hat_raw=" << fT_hat_raw << " fT_hat=" << fT_hat << " f_art=" << f_art << " f_shared_raw=" << f_shared_hat_raw << " f_shared=" << f_shared_hat << " expN_err=" << expected_errors_N << " excessN=" << excess_alt_N << " cleanliness=" << cleanliness << " clean_penalty=" << (kCleanPenalty * cleanliness) << " f_n_hat=" << f_n_hat << " f_t_hat=" << f_t_hat << " ratio=" << ratio << " sim_weight=" << sim_weight << " sim_bonus=" << (sim_weight * ratio * kSimBonus) << " both_low=" << (both_nonzero_alt && both_low_vaf ? 1 : 0) << " low_vaf_bonus=" << ((both_nonzero_alt && both_low_vaf) ? (2.0 * ratio) : 0.0) << " || SOM_TRUE(N,T,sum)=(" << llN_som_true << "," << llT_som_true << "," << ll_som_true << ")" << " SOM_ART(N,T,sum)=(" << llN_som_art << "," << llT_som_art << "," << ll_som_art << ")" << " SOM=" << ll_som << " [" << som_branch << "]" << " || GERM_HET(N,T,sum)=(" << (LL_N(dN_safe,aN,f_het)) << "," << (LL_T(dT_safe,aT,f_het)) << "," << ll_germ_het << ")" << " GERM_HOM(N,T,sum)=(" << (LL_N(dN_safe,aN,f_hom)) << "," << (LL_T(dT_safe,aT,f_hom)) << "," << ll_germ_hom << ")" << " GERM_ART(N,T,sum)=(" << (LL_art(dN_safe,aN,f_art)) << "," << (LL_art(dT_safe,aT,f_art)) << "," << ll_germ_art << ")" << " GERM_SHARED(N,T,sum)=(" << (LL_N(dN_safe,aN,f_shared_hat)) << "," << (LL_T(dT_safe,aT,f_shared_hat)) << "," << ll_germ_shared << ")" << " GERM_FREE(active=" << (germ_free_active ? 1 : 0) << ",fN=" << f_n_free_dbg << ",fT=" << f_t_free_dbg << ",N=" << ll_germ_free_LL_N << ",T=" << ll_germ_free_LL_T << ",BIC=" << kGermFreeBIC << ",sum=" << ll_germ_free << ")" << " GERM=" << ll_germ << " [" << germ_branch << "]" << " || LOD10=" << lod_somatic_vs_germ << std::endl; } // ------------------------------- return lod_somatic_vs_germ; // log10 } int GenotypeQuality(const std::vector& PLs) { int best = std::numeric_limits::max(); int second = std::numeric_limits::max(); for (int p : PLs) { if (p < best) { second = best; best = p; } else if (p < second) { second = p; } } return std::min(second, 99); } // g is the number of reference alleles (e.g. g = 2 is homozygous reference) // assumes biallelic model // http://bioinformatics.oxfordjournals.org/content/early/2011/09/08/bioinformatics.btr509.full.pdf+html double GenotypeLikelihoods(int g, double er, int alt, int cov) { // g = number of reference alleles: 2 = hom_ref, 1 = het, 0 = hom_alt // er = per-read error rate (0 <= er <= 1) // alt = count of alt reads // cov = total read depth // Returns log10-likelihood under a biallelic model. // Clamp inputs g = std::min(2, std::max(0, g)); alt = std::max(0, alt); cov = std::max(0, cov); er = std::clamp(er, 0.0, 1.0); if (alt > cov) return -1e12; int ref = cov - alt; double norm = -cov * std::log10(2.0); // accounts for dividing by 2 // Compute numerators (before /2) double num_ref = (2 - g) * er + g * (1.0 - er); double num_alt = (2 - g) * (1.0 - er) + g * er; // True probabilities (divide by 2) double p_ref = num_ref / 2.0; double p_alt = num_alt / 2.0; double ll = norm; // Add ref-read contribution if (p_ref > 0.0) { ll += ref * std::log10(p_ref); } else if (ref > 0) { return -1e12; } // Add alt-read contribution if (p_alt > 0.0) { ll += alt * std::log10(p_alt); } else if (alt > 0) { return -1e12; } return ll; } } ================================================ FILE: src/svaba/SvabaModels.h ================================================ #pragma once #include namespace SvabaModels { double LogLikelihood(double ref, double alt, double f, double e_fwd, double e_back); double SomaticLOD(double aN, double dN, double aT, double dT, double e_fwd, double e_rev); double GenotypeLikelihoods(int g, double er, int alt, int cov); int GenotypeQuality(const std::vector& PLs); } ================================================ FILE: src/svaba/SvabaOptions.cpp ================================================ #include "SvabaOptions.h" #include // for std::ostringstream #include // for std::setw and std::setfill #include #include #include #include "SvabaLogger.h" #include "SvabaAssemblerConfig.h" // SVABA_ASSEMBLER_FERMI for SGA guards void SvabaOptions::printUsage() { std::cout << R"( Usage: svaba run [OPTIONS] General: -h, --help Show this message -v, --verbose Verbosity level (0-4), default 0 -p, --threads Number of threads, default 1 -a, --analysis-id Identifier for this run Input: -t, --case-bam Tumor BAM; may repeat -n, --control-bam Normal BAM; may repeat -G, --reference-genome Indexed reference FASTA -k, --region-file BED or samtools-style regions Mode: --single-end Single-end mode (no mate lookup) --all-contigs Output all assembled contigs --discordant-only Skip assembly, only discordants --override-ref-check Skip BAM vs REF compatibility check )" #if !SVABA_ASSEMBLER_FERMI // SGA-specific knobs. Only surface them to --help when svaba was // compiled with SGA as the active assembler (see // SvabaAssemblerConfig.h). The option codes themselves still parse // under fermi — they just get ignored. << R"( Assembly (SGA): --min-overlap Minimum read overlap for SGA --error-rate SGA fractional error rate --rounds Number of assembly rounds )" #endif << R"( Error-correction: --ec-type s=SGA k-mer; f=Fermi BFC; 0=off --ec-subsample Fraction to sample for EC learning Discordant clustering: --disc-sd SD cutoff, default 3.92 Filtering: --max-cov Max coverage to assemble, default 100 --mate-min Min reads to trigger somatic mate lookup, default 3 --mate-min-count Min reads to form a candidate mate region, default 2 --mate-lim Max reads in mate lookup, default 400 --min-mate-mapq Min MAPQ on primary read for mate-region candidate, default -1 (no gate). Set to e.g. 1 to exclude MAPQ=0 multi-mappers from mate lookup. --max-mate-chr Exclude mate regions on chromosomes with ChrID > N, default 23 (through chrY; skips chrM/alt/decoy). Set to -1 for no limit (see --non-human). --non-human Remove hardcoded human genome assumptions. Currently sets --max-mate-chr -1 (allow all chroms) and samples all contigs during insert-size learning. --no-nm Skip high-NM read salvage (NM/len > 0.02). Faster: fewer reads enter r2c and correction. Trades away rare NM-only SV sensitivity. BWA-MEM tuning: --bw-op Gap-open pen, default 32 --bw-ep Gap-ext pen, default 1 --bw-mm Mismatch pen, default 18 --bw-ms Match score, default 2 --bw-zd Z-drop, default 100 --bw-bw Bandwidth, default 1000 --bw-rt Reseed trigger, default 1.5 --bw-c3 3' clip pen, default 5 --bw-c5 5' clip pen, default 5 Output & DBs: --blacklist BED of blacklisted regions --germline-sv BED of known germline SVs --dbsnp DBSNP VCF of known variants --dump-reads Emit per-read debug/visualization outputs (off by default; large output on deep samples). Four files are produced by this flag together: ${ID}.corrected.bam ${ID}.discordant.bam ${ID}.alignments.txt.gz ${ID}.r2c.txt.gz Without this flag, svaba writes the bps.txt.gz / VCF / contigs.bam / runtime.txt summaries but none of the per-read detail. Weird-reads BAM is compile-time only; see SvabaOptions.h::dump_weird_reads. --always-realign-corrected Force re-alignment of every corrected read to the reference, even if BFC didn't modify it. By default, reads whose sequence is unchanged reuse the input BAM's CIGAR/NM (valid when the BAM was aligned with BWA). Use this flag when the input was aligned with a non-BWA aligner. )" << "\n"; } SvabaOptions SvabaOptions::parse(int argc, char** argv) { static const char* shortOpts = "hv:p:a:t:n:G:k:"; static struct option longOpts[] = { {"help", no_argument, nullptr, 'h'}, {"verbose", required_argument, nullptr, 'v'}, {"threads", required_argument, nullptr, 'p'}, {"analysis-id", required_argument, nullptr, 'a'}, {"case-bam", required_argument, nullptr, 't'}, {"control-bam", required_argument, nullptr, 'n'}, {"reference-genome", required_argument, nullptr, 'G'}, {"region-file", required_argument, nullptr, 'k'}, {"single-end", no_argument, nullptr, 1001}, {"all-contigs", no_argument, nullptr, 1002}, {"discordant-only", no_argument, nullptr, 1003}, {"override-ref-check",no_argument, nullptr, 1004}, {"min-overlap", required_argument, nullptr, 1100}, {"error-rate", required_argument, nullptr, 1101}, {"rounds", required_argument, nullptr, 1102}, {"ec-type", required_argument, nullptr, 1200}, {"ec-subsample",required_argument, nullptr, 1201}, {"disc-sd", required_argument, nullptr, 1300}, {"max-cov", required_argument, nullptr, 1400}, {"mate-min", required_argument, nullptr, 1401}, {"mate-lim", required_argument, nullptr, 1402}, {"no-nm", no_argument, nullptr, 1403}, {"min-mate-mapq", required_argument, nullptr, 1404}, {"max-mate-chr", required_argument, nullptr, 1405}, {"non-human", no_argument, nullptr, 1406}, {"mate-min-count", required_argument, nullptr, 1407}, {"chunk-size", required_argument, nullptr, 1500}, {"bw-op", required_argument, nullptr, 1600}, {"bw-ep", required_argument, nullptr, 1601}, {"bw-mm", required_argument, nullptr, 1602}, {"bw-ms", required_argument, nullptr, 1603}, {"bw-zd", required_argument, nullptr, 1604}, {"bw-bw", required_argument, nullptr, 1605}, {"bw-rt", required_argument, nullptr, 1606}, {"bw-c3", required_argument, nullptr, 1607}, {"bw-c5", required_argument, nullptr, 1608}, {"blacklist", required_argument, nullptr, 1700}, {"germline-sv",required_argument, nullptr, 1701}, {"dbsnp", required_argument, nullptr, 1702}, // --dump-reads: runtime opt-in for corrected + discordant-reads BAMs. // Single flag, both side effects — see comment in SvabaOptions.h. // Weird-reads BAM is deliberately not on this flag (compile-time only). {"dump-reads", no_argument, nullptr, 1800}, {"always-realign-corrected", no_argument, nullptr, 1801}, {nullptr,0,nullptr,0} }; SvabaOptions o; int idx, c; while ((c = getopt_long(argc, argv, shortOpts, longOpts, &idx)) != -1) { switch (c) { case 'h': o.help = true; return o; case 'v': o.verbose = std::stoi(optarg); break; case 'p': o.numThreads = std::stoi(optarg); break; case 'a': o.analysisId = optarg; break; case 't': o.caseBams .push_back(optarg); break; case 'n': o.controlBams.push_back(optarg); break; case 'G': o.refGenome = optarg; break; case 'k': o.regionFile = optarg; break; case 1001: o.singleEnd = true; break; case 1002: o.allContigs = true; break; case 1003: o.discClusterOnly = true; break; case 1004: o.overrideRefCheck = true; break; case 1100: o.sgaMinOverlap = std::stoi(optarg); break; case 1101: o.sgaErrorRate = std::stof(optarg); break; case 1102: o.sgaNumRounds = std::stoi(optarg); break; case 1200: o.ecCorrectType = optarg; break; case 1201: o.ecSubsample = std::stod(optarg); break; case 1300: o.sdDiscCutoff = std::stod(optarg); break; case 1400: o.maxCov = std::stoi(optarg); break; case 1401: o.mateLookupMin = std::stoul(optarg);break; case 1402: o.mateRegionLookupLim = std::stoul(optarg); break; case 1403: o.noNmSalvage = true; break; case 1404: o.minMateMAPQ = std::stoi(optarg); break; case 1405: o.maxMateChrID = std::stoi(optarg); break; case 1406: o.nonHuman = true; break; case 1407: o.mateRegionMinCount = std::stoi(optarg); break; case 1500: o.chunkSize = std::stoi(optarg); break; case 1600: o.bwaGapOpen = std::stoi(optarg); break; case 1601: o.bwaGapExt = std::stoi(optarg); break; case 1602: o.bwaMismatch = std::stoi(optarg); break; case 1603: o.bwaMatchScore = std::stoi(optarg); break; case 1604: o.bwaZdrop = std::stoi(optarg); break; case 1605: o.bwaBandwidth = std::stoi(optarg); break; case 1606: o.bwaReseedTrigger = std::stof(optarg); break; case 1607: o.bwaClip3 = std::stoi(optarg); break; case 1608: o.bwaClip5 = std::stoi(optarg); break; case 1700: o.blacklistFile.push_back(optarg); break; case 1701: o.germlineSvFile = optarg; break; case 1702: o.dbsnpVcf = optarg; break; // --dump-reads is the single runtime knob for all per-read detail // outputs. Flip all three flags together here; any call-site // reading these fields after parse() sees a consistent state. // See SvabaOptions.h for the comment on why these are separate // fields instead of a single bool. case 1800: o.dump_discordant_reads = true; o.dump_corrected_reads = true; o.dump_alignments = true; break; case 1801: o.alwaysRealignCorrected = true; break; case '?': default: throw std::runtime_error("Unknown or malformed option; see --help"); } } // --non-human: remove human-specific assumptions if (o.nonHuman) { o.maxMateChrID = -1; // allow all chromosomes for mate lookup } // post validation if (argc == 1) { o.help = true; } else if (!o.help) { if (o.caseBams.empty()) throw std::runtime_error("Need at least one --case-bam"); if (o.refGenome.empty()) throw std::runtime_error("Must supply --reference-genome"); if (o.analysisId.empty()) o.analysisId = "no_id"; if (o.chunkSize <= 0) o.chunkSize = 25000; } // make the bam map from the file names int tumorIdx = 1; for (const auto& path : o.caseBams) { std::ostringstream ss; ss << 't' << std::setw(3) << std::setfill('0') << tumorIdx++; o.bams[ ss.str() ] = path; } int normalIdx = 1; for (const auto& path : o.controlBams) { std::ostringstream ss; ss << 'n' << std::setw(3) << std::setfill('0') << normalIdx++; o.bams[ ss.str() ] = path; } // set the rules to skip read learning if doing stdin // if (o.bams["t001"] == "-" && !o.rulesJson.empty()) o.rulesJson = R"json( { "global": { "duplicate": false, "qcfail": false }, "": { "rules": [ { "rr": true }, { "ff": true }, { "rf": true }, { "ic": true }, { "clip": 5, "length": 30 }, { "ins": true }, { "del": true }, { "mapped": true, "mate_mapped": false }, { "mate_mapped": true, "mapped": false } ] } } )json"; // if read stream, treat as single-end if (o.bams["t001"] == "-") o.singleEnd = true; // set the "main bam" o.main_bam = o.bams["t001"]; return o; } void SvabaOptions::printLogger(SvabaLogger& logger) const { logger.log(verbose > 1, true, "***************************** PARAMS ****************************"); logger.log(verbose > 1, true, " DBSNP Database file: ", dbsnpVcf); logger.log(verbose > 1, true, " Max cov to assemble: ", maxCov); logger.log(verbose > 1, true, " Error correction mode: ", ecCorrectType); logger.log(verbose > 1, true, " Subsample-rate for correction learning: ", ecSubsample); logger.log(verbose > 1, true, " Assembler: ", svaba::kAssemblerName); #if !SVABA_ASSEMBLER_FERMI // SGA-only parameters; don't confuse fermi-build operators with // values they can't tune without recompiling. logger.log(verbose > 1, true, " ErrorRate (SGA): ", (sgaErrorRate < 0.001f ? std::string("EXACT (0)") : std::to_string(sgaErrorRate))); logger.log(verbose > 1, true, " Num assembly rounds (SGA): ", sgaNumRounds); #endif logger.log(verbose > 1, true, " Discordant SD cutoff (tumor): ", sdDiscCutoff); logger.log(verbose > 1, true, " Discordant SD cutoff (normal): ", sdDiscCutoffNormal); logger.log(verbose > 1, true, " Minimum number of reads for mate lookup: ", mateLookupMin); logger.log(verbose > 1, true, " Min reads to form candidate mate region: ", mateRegionMinCount); logger.log(verbose > 1, true, " Min MAPQ for mate region candidate: ", minMateMAPQ, (minMateMAPQ < 0 ? " (no gate)" : "")); logger.log(verbose > 1, true, " Max ChrID for mate region: ", maxMateChrID, (maxMateChrID < 0 ? " (no limit)" : "")); if (nonHuman) logger.log(true, true, " Non-human genome mode: chromosome gates disabled"); logger.log(verbose > 1, true, " LOD cutoff (non-REF): ", lod); logger.log(verbose > 1, true, " LOD cutoff (non-REF, at DBSNP): ", lodDb); logger.log(verbose > 1, true, " LOD somatic cutoff: ", lodSomatic); logger.log(verbose > 1, true, " LOD somatic cutoff (at DBSNP): ", lodSomaticDb); logger.log(verbose > 1, true, " BWA-MEM params:"); logger.log(verbose > 1, true, " Gap open penalty: ", bwaGapOpen); logger.log(verbose > 1, true, " Gap extension penalty: ", bwaGapExt); logger.log(verbose > 1, true, " Mismatch penalty: ", bwaMismatch); logger.log(verbose > 1, true, " Sequence match score: ", bwaMatchScore); logger.log(verbose > 1, true, " Z-dropoff: ", bwaZdrop); logger.log(verbose > 1, true, " Alignment bandwidth: ", bwaBandwidth); logger.log(verbose > 1, true, " Clip 3 penalty: ", bwaClip3); logger.log(verbose > 1, true, " Clip 5 penalty: ", bwaClip5); logger.log(verbose > 1, true, " Reseed trigger: ", bwaReseedTrigger); if (discClusterOnly) { logger.log(true, true, " ######## ONLY DISCORDANT READ CLUSTERING. NO ASSEMBLY ##############"); } } ================================================ FILE: src/svaba/SvabaOptions.h ================================================ #pragma once #include #include #include #include // version & date // // Bumped to 2.0.0 to mark the SvABA2.0 overhaul: v3 bps.txt schema with // per-BP bp_id (col 52), r2c.txt.gz structured r2c emission replacing the // old alignments.txt.gz, comparative split-coverage gate, standalone // `svaba tovcf` + `svaba postprocess` subcommands, VCFv4.5 output, and // fermi-lite as the default local-assembly engine. See CLAUDE.md for the // full set of changes. inline constexpr char SVABA_VERSION[] = "2.0.0"; inline constexpr char SVABA_DATE[] = "04/2026"; // from AlignmentFragment.h inline constexpr std::size_t MAX_CONTIG_SIZE = 5'000'000; // from run_svaba.cpp inline constexpr int MIN_CLIP_FOR_LOCAL = 40; inline constexpr int MAX_NM_FOR_LOCAL = 10; // from BreakPoint inline constexpr double MAX_ERROR = 0.08; // for very repetitive regions, what is the maximum expected error rate for an indel inline constexpr double MIN_ERROR = 0.0005; inline constexpr int T_SPLIT_BUFF = 5; inline constexpr int N_SPLIT_BUFF = 5; inline constexpr int INSERT_SIZE_TOO_BIG_SPAN_READS = 16; // (Removed in SvABA2.0) R2C_MAX_NONEDGE_SOFTCLIP_{TUMOR,NORMAL} — // replaced by the principled per-read score comparison gate in // BreakPoint::splitCoverage(). See svaba::readAlignmentScore() in // ContigAlignmentScore.h: a read is now credited as a variant supporter // only when its r2c alignment scores strictly higher than its native // read-to-reference alignment, which subsumes both the interior-clip // rejection and the duplicated-reference equally-clean case. // SvABA2.0 (v3): r2c-better-than-native gate. A read is credited as a // split-supporter iff r2c_score > native_score (strict greater-than). // Ties don't credit the read — if the contig provides no better // explanation than the native alignment, the read is not evidence for // the variant. // // v3 originally used a per-sample percentage margin (10% for tumor, // 0% for normal) to filter junction-homology borderline cases on the // tumor side. v3.1 removed the margin entirely: // // - The 10% margin was read-length-dependent and mathematically // impossible to clear for small indels (a 1bp del on a 150bp read // gives only 4.9% improvement; on a 250bp read only 2.9%). // - Junction-homology cases where r2c barely beats native by 1-2 // points: normal already credits these reads (margin was always 0 // for normal). If both samples credit borderline reads equally, // the downstream LOD model sees similar split support in both → // low somlod → correctly not called somatic. The somatic/germline // distinction is the LOD model's job, not the split-coverage gate's. // - The margin was belt-and-suspenders with a worse failure mode // (killing real small indels) than the problem it prevented. // // This replaces the older both_split / one_split / homlen branching // gate — when junction homology is on the order of the read length, // r2c and native tie and such reads fall out naturally via the strict > // comparison, which is the correct conservative behavior. inline constexpr double T_R2C_MIN_MARGIN = 0.0; inline constexpr double N_R2C_MIN_MARGIN = 0.0; // --------------------------------------------------------------------------- // SVABA_R2C_NATIVE_GATE — compile-time kill-switch for the r2c-vs-native // alignment-score split-coverage gate in BreakPoint::splitCoverage. // // 1 (default) — gate enabled. Correct, recommended. Each candidate // split-supporting read is scored against both its r2c // alignment and its native (read→reference) alignment, // and must win by the per-sample-prefix margin above // (T_R2C_MIN_MARGIN / N_R2C_MIN_MARGIN). Fixes the // long-homology false-positive somatic bug. // // 0 (opt-in) — gate disabled. Falls back to counting any r2c-spanning // read as a split supporter regardless of how it // compares to the native alignment. Reintroduces the // homology-trap bug (long junction homology + clean // normal-side reads → spurious somatic calls), so do // NOT use for real calling. Provided solely to isolate // the CPU cost of native_score computation on dense // contigs — flip to 0, rebuild, time a reference // region. Delta vs default-build tells you what the // gate costs. // // Build with: // cmake .. -DCMAKE_CXX_FLAGS='-DSVABA_R2C_NATIVE_GATE=0' // --------------------------------------------------------------------------- #ifndef SVABA_R2C_NATIVE_GATE #define SVABA_R2C_NATIVE_GATE 1 #endif inline constexpr int HOMOLOGY_FACTOR = 4; inline constexpr int MIN_SOMATIC_RATIO = 15; inline constexpr int COVERAGE_AVG_BUFF = 10; // from DiscordantCluster inline constexpr int DISC_PAD = 150; inline constexpr int MIN_PER_CLUST = 3; inline constexpr int DEFAULT_ISIZE_THRESHOLD = 2000; // from run_svaba inline constexpr std::size_t THREAD_READ_LIMIT = 20'000; inline constexpr int THREAD_CONTIG_LIMIT = 5'000; // from svabaAssemblerEngine inline constexpr std::size_t MAX_OVERLAPS_PER_ASSEMBLY = 20'000; inline constexpr int MIN_CONTIG_MATCH = 35; inline constexpr int MATE_LOOKUP_MIN = 3; inline constexpr int SECONDARY_CAP = 10; inline constexpr int MAX_MATE_ROUNDS = 1; inline constexpr std::size_t MAX_NUM_MATE_WINDOWS = 50'000'000; inline constexpr int GERMLINE_CNV_PAD = 10; inline constexpr int GET_MATES = 1; inline constexpr int LARGE_INTRA_LOOKUP_LIMIT = 50'000; inline constexpr double SECONDARY_FRAC = 0.90; // from svabaBamWalker — mate-region lookup // // A discordant read contributes its mate locus as a candidate mate region // iff its own MAPQ >= MIN_MATE_MAPQ AND the mate locus is on a chromosome // with ChrID <= MAX_MATE_CHR_ID (set to -1 to disable the chromosome // gate, e.g. for non-human genomes; default 23 = through chrY). After // candidate regions are merged, // a region must have >= MATE_REGION_MIN_COUNT supporting reads to survive // the initial BamWalker filter, and then >= mateLookupMin (runtime option, // default MATE_LOOKUP_MIN=3) to trigger the actual somatic mate lookup // in SvabaRegionProcessor. // // Defaults: // MIN_MATE_MAPQ = -1 (no MAPQ gate; any mapped read qualifies) // MAX_MATE_CHR_ID = 23 (skip chrM/alt/decoy in human; -1 = no limit) // 0-indexed: 0=chr1 .. 21=chr22, 22=chrX, 23=chrY // MATE_REGION_MIN_COUNT = 2 (at least 2 reads to form a candidate) // MATE_REGION_PAD = 250 (bp padding on each side of mate locus) inline constexpr int MIN_MATE_MAPQ = -1; inline constexpr int MAX_MATE_CHR_ID = 23; inline constexpr int MATE_REGION_MIN_COUNT = 2; inline constexpr int MATE_REGION_PAD = 250; // from svabaBamWalker — other inline constexpr int MIN_DSCRD_READS_DSCRD_ONLY = 6; inline constexpr int MIN_ISIZE_FOR_DISCORDANT_REALIGNMENT = 1'000; inline constexpr int DISC_REALIGN_MATE_PAD = 100; inline constexpr int MAX_SECONDARY_HIT_DISC = 10; // coverage buffer inline constexpr int INFORMATIVE_COVERAGE_BUFFER = 0; // from vcf inline constexpr int VCF_SECONDARY_CAP = 200; inline constexpr int SOMATIC_LOD = 1; inline constexpr int DEDUPEPAD = 200; class SvabaLogger; class SvabaOptions { public: // high-level flags bool help = false; int verbose = 0; int numThreads = 1; std::string analysisId; bool hp = false; int perRgLearnLimit = 1'000; size_t weird_read_limit = 15'000; size_t mate_region_lookup_limit = 5'000; // dumping // // weird reads: compile-time-only toggle. Flip this to `true` here and // rebuild if you really want the weird-reads BAM. Deliberately NOT // exposed on the CLI — it's a large, niche output mostly used for // debugging the read-collection phase, not for routine runs. static constexpr bool dump_weird_reads = false; // All three below are opt-in at runtime via a single --dump-reads flag. // Default off so routine runs don't pay the (substantial) I/O and disk // cost of emitting this per-read detail on deep samples: // // dump_discordant_reads -> ${ID}.discordant.bam // dump_corrected_reads -> ${ID}.corrected.bam // dump_alignments -> ${ID}.alignments.txt.gz // -> ${ID}.r2c.txt.gz // // All three flip together under --dump-reads. The fields are kept // separate so an individual callsite can still key off its own narrow // concern (e.g. SvabaOutputWriter gates the alignments file streams on // dump_alignments only), but there is intentionally no way to toggle // them individually at runtime — that would bloat the CLI surface // without serving a real workflow. bool dump_discordant_reads = false; bool dump_corrected_reads = false; bool dump_alignments = false; // inputs std::vector caseBams; std::vector controlBams; std::string refGenome; std::string regionFile; // make the log verbose, but will invoke a lot of mutex locks bool verbose_log = false; int windowpad = 500; std::string main_bam; // mode flags bool singleEnd = false; bool allContigs = false; bool discClusterOnly = false; bool overrideRefCheck = false; // numeric thresholds double sdDiscCutoff = 3.92; // SD multiplier for tumor discordant cutoff double sdDiscCutoffNormal = 3.60; // SD multiplier for normal (lower = more sensitive) int chunkSize = 25000; int32_t maxReadsPerAssem = -1; // this is site-level Log-odds cutoff for PASS that is variant double lod = 1.0; //8.0; // this is site-level Log-odds cuttof for PASS that is variant, if also has supporting DBSNP site double lodDb = 1.0; //6.0; // this is log-odds that this is REF in the "worst" normal (the one with the most potential alt reads) double lodSomatic = 0.0; //6.0; // same, but be more strict if this somatic variant overlaps a dbsnp site double lodSomaticDb = 2.0; // 10.0; int maxCov = 100; // SGA / assembly int sgaMinOverlap = 0; float sgaErrorRate = 0.0f; int sgaNumRounds = 3; // error correction std::string ecCorrectType = "f"; //s, f, or 0 double ecSubsample = 0.50; // BWA-MEM tuning int bwaGapOpen = 32; int bwaGapExt = 1; int bwaMismatch = 18; int bwaMatchScore = 2; int bwaZdrop = 100; int bwaBandwidth = 1000; float bwaReseedTrigger = 1.5f; int bwaClip3 = 5; int bwaClip5 = 5; // filtering std::string rulesJson; size_t mateLookupMin = MATE_LOOKUP_MIN; size_t mateRegionLookupLim = 400; bool noBadAvoid = true; // Mate-region lookup tuning (runtime overrides for compile-time defaults) int minMateMAPQ = MIN_MATE_MAPQ; // --min-mate-mapq int maxMateChrID = MAX_MATE_CHR_ID; // --max-mate-chr (or -1 via --non-human) int mateRegionMinCount = MATE_REGION_MIN_COUNT; // --mate-min-count // Non-human genome mode: removes all hardcoded human chromosome // assumptions (currently just the mate-lookup ChrID > 23 gate and // the LearnBamParams chr1-chrY sampling preference). // Sets maxMateChrID = -1 (no limit) and logs a banner. bool nonHuman = false; // When true, skip the high-NM salvage path that pulls in reads with // NM/len > 0.02 even when they don't pass the normal weird-read rules. // Those reads are useful for catching NM-only SVs (no clips/discordance) // but substantially increase read count and r2c cost. --no-nm disables // the path entirely for maximum efficiency. bool noNmSalvage = false; // When false (default), reads whose corrected sequence matches the // original BAM sequence reuse the input BAM's CIGAR/NM for the // native-vs-r2c gate in splitCoverage, skipping the expensive // full-reference BWA re-alignment. This is correct when the input // BAM was aligned with BWA-MEM (same scoring model as svaba's // internal aligner). When the input BAM was aligned with a // different aligner (e.g. bowtie2, STAR, minimap2), the native // CIGAR/NM may not be comparable — set this to true to force // re-alignment of every corrected read against the reference. bool alwaysRealignCorrected = false; // external DBs std::vector blacklistFile; std::string germlineSvFile; std::string dbsnpVcf; // BAM map (idpath) // e.g. t001, /path/to/ std::map bams; // ---------------------------------------------------- // Print help/usage static void printUsage(); void printLogger(SvabaLogger& logger) const; // Parse argc/argv into an SvabaOptions; throws on error static SvabaOptions parse(int argc, char** argv); }; ================================================ FILE: src/svaba/SvabaOutputWriter.cpp ================================================ // svabaOutputWriter.cpp #include "SvabaOutputWriter.h" #include "SvabaThreadUnit.h" #include "SvabaUtils.h" #include "BreakPoint.h" #include "DiscordantCluster.h" #include "SvabaSharedConfig.h" #include #include using namespace std; namespace { // one mutex to serialize ALL threads writes static mutex writeMutex_; } // Build a new BamHeader that prepends an @HD line (with the given sort order) // to the @SQ block of `src`. Avoids SeqLib::BamHeader::AsString(), which // blindly dereferences h->text; in modern htslib that field is lazy and is // often NULL until sam_hdr_str() materializes it, causing a segfault. static SeqLib::BamHeader make_header_with_hd(const SeqLib::BamHeader& src, const std::string& so) { std::string txt = "@HD\tVN:1.6\tSO:" + so + "\n"; const bam_hdr_t* h = src.get(); if (h) { // sam_hdr_str forces htslib to regenerate the textual form from its // internal representation and caches it in h->text. const char* cur = sam_hdr_str(const_cast(h)); if (cur) txt += cur; } return SeqLib::BamHeader(txt); } SvabaOutputWriter::SvabaOutputWriter(SvabaLogger& logger_, SvabaOptions& opts_) : logger(logger_), opts(opts_) { } // init(): call once, from run_svaba.cpp immediately after you've parsed // your opts and before you shoot off any threads. void SvabaOutputWriter::init(const string& analysis_id, const SeqLib::BamHeader& b_header) { // keep a local reference to the header bam_header_ = b_header; // open our gzipped text outputs // // Per-thread r2c.txt.gz streams are opened by svabaThreadUnit itself // (see its ctor) — nothing to open here for that output. What's left // on this shared-writer path is only the small coordination outputs // where mutex contention is a non-issue. svabaUtils::fopen(analysis_id + ".bps.txt.gz", os_allbps_); svabaUtils::fopen(analysis_id + ".discordant.txt.gz", os_discordant_); svabaUtils::fopen(analysis_id + ".runtime.txt", os_runtime_); // write the header line for breakpoints file: os_allbps_ << BreakPoint::header(); for (auto &p : opts.bams) os_allbps_ << "\t" << p.first << "_" << p.second; os_allbps_ << "\n"; // write the header line for runtime file os_runtime_ << svabaUtils::svabaTimer::header << "\n"; // write the header line for discordant clusters: os_discordant_ << DiscordantCluster::header() << "\n"; // BAM output for aligned contigs std::string aligned_contigs_bam_path = analysis_id; aligned_contigs_bam_path.append(".contigs.bam"); b_contig_writer_ = SeqLib::BamWriter(SeqLib::BAM); auto hdr_unsorted = make_header_with_hd(b_header, "unsorted"); b_contig_writer_.SetHeader(hdr_unsorted); if (!b_contig_writer_.Open(aligned_contigs_bam_path)) { std::cerr << "ERROR: could not open aligned contig writer " << aligned_contigs_bam_path << std::endl; exit(EXIT_FAILURE); } b_contig_writer_.WriteHeader(); } void SvabaOutputWriter::writeUnit(svabaThreadUnit& unit, SvabaSharedConfig& sc) { // SvABA2.0: stamp a comma-joined aux tag on any record whose // UniqueName (svabaRead) or Qname (bwa-aligned corrected record) // appears in one of the thread-unit maps. The svabaRead pointer // path already tagged weird/discordant records during the r2c and // BP-finalization passes, but the corrected records are newly- // aligned by bwa and need tagging here. We apply uniformly so the // tag is present in every tagged BAM. // // bi:Z — bp_ids of variants this read supports as ALT (subset, // v3 — pre-v3 this was cnames; switched to bp_ids so the // tag matches the per-BP resolution of r2c.txt.gz's // split_bps / disc_bps columns and bps.txt.gz's col 52 // bp_id. `samtools view | grep bi:Z:` pulls the // ALT-supporters for exactly that variant row.) // bz:Z — cnames of contigs this read r2c'd to (superset; the // contig-level grouping key. A read may r2c to a contig // yet not support any of its BPs.) // // A read can appear in both maps at the same time (every // ALT-supporter has an r2c alignment to its contig). The two // tags use different identifier namespaces now: bp_id for bi:Z, // cname for bz:Z. Join back to bps.txt.gz via col 30 (cname) or // col 52 (bp_id) as appropriate. auto stamp_tag = [&](auto& rec, const char* tag, const std::unordered_map& m, const std::string& key) { auto it = m.find(key); if (it == m.end() || it->second.empty()) return; std::string cur; rec.GetZTag(tag, cur); const std::string& to_add = it->second; if (cur.empty()) { rec.AddZTag(tag, to_add); return; } // boundary-aware merge/dedupe std::string merged = cur; size_t s = 0; while (s <= to_add.size()) { size_t e = to_add.find(',', s); if (e == std::string::npos) e = to_add.size(); std::string tok = to_add.substr(s, e - s); if (!tok.empty()) { bool present = false; size_t pos = 0; while ((pos = merged.find(tok, pos)) != std::string::npos) { const bool left_ok = (pos == 0) || merged[pos-1] == ','; const bool right_ok = (pos + tok.size() == merged.size()) || merged[pos + tok.size()] == ','; if (left_ok && right_ok) { present = true; break; } pos += tok.size(); } if (!present) merged += "," + tok; } if (e == to_add.size()) break; s = e + 1; } if (merged != cur) { rec.RemoveTag(tag); rec.AddZTag(tag, merged); } }; auto stamp_bi = [&](auto& rec, const std::string& key) { stamp_tag(rec, "bi", unit.alt_bp_ids_by_name, key); }; auto stamp_bz = [&](auto& rec, const std::string& key) { stamp_tag(rec, "bz", unit.all_cnames_by_name, key); }; // write the weird reads if (sc.opts.dump_weird_reads) { auto it = unit.writers.find("w"); if (it == unit.writers.end()) { std::cerr << " BAM writer for weird reads not found" << std::endl; exit(EXIT_FAILURE); } for (const auto& r : unit.all_weird_reads) { // stamp bi:Z / bz:Z BEFORE SetQname, while UniqueName is still // the map key we indexed under stamp_bi(*r, r->UniqueName()); stamp_bz(*r, r->UniqueName()); // switch out the qname so it indicates also which BAM it was from r->SetQname(r->UniqueName()); r->RemoveTag("RG"); r->RemoveTag("PG"); bool ok = it->second->WriteRecord(*r); if (!ok) std::cerr << "...unable to write weird read record" << std::endl; } } // write the discordant reads if (sc.opts.dump_discordant_reads) { auto it = unit.writers.find("d"); if (it == unit.writers.end()) { std::cerr << " BAM writer for discordant reads not found" << std::endl; } for (const auto& r : unit.all_weird_reads) { if (r->dd > 0) { // stamp bi:Z / bz:Z before qname mangling stamp_bi(*r, r->UniqueName()); stamp_bz(*r, r->UniqueName()); // switch out the qname so it indicates also which BAM it was from r->SetQname(r->UniqueName()); // write it r->RemoveTag("RG"); r->RemoveTag("PG"); bool ok = it->second->WriteRecord(*r); if (!ok) std::cerr << "...unable to write discordant read record" << std::endl; } } } // write the corrected reads if (sc.opts.dump_corrected_reads) { auto it = unit.writers.find("c"); if (it == unit.writers.end()) { std::cerr << " read found with prefix " << " c " << " that is not in the weird read writers\n"; exit(EXIT_FAILURE); } for (const auto& r : unit.all_corrected_reads) { // corrected records are bwa-aligned BamRecords whose Qname was // set to the original svabaRead UniqueName at alignSequence() // time. That's the key both maps are indexed by. stamp_bi(*r, r->Qname()); stamp_bz(*r, r->Qname()); r->RemoveTag("RG"); r->RemoveTag("PG"); bool ok = it->second->WriteRecord(*r); if (!ok) std::cerr << "...unable to write corrected read record" << std::endl; } } // SvABA2.0: per-thread r2c.txt.gz write path. // // Happens BEFORE writeMutex_ is acquired because unit.r2c_out_ is a // thread-local gzip stream (not shared across threads) — no coordination // needed, and keeping the gzip deflate out from under the shared mutex // lets all 16 threads compress in parallel, which is the whole point of // the per-thread split (see CLAUDE.md for the full rationale). Mirrors // the per-thread BAM writes above, which also run outside the lock. // // Gated on opts.dump_alignments (--dump-reads) — without it no stream // was opened and we skip both the serialization and the write. if (opts.dump_alignments) { for (const auto& alc : unit.master_alc) { if (alc.hasVariant()) { // printToR2CTsv emits a contig row followed by one row per // r2c-aligned read (already newline-terminated internally), // so no trailing newline here. // r2c_out_ is unique_ptr, so dereference. It's non-null // here because opts.dump_alignments implies ctor already allocated. *unit.r2c_out_ << alc.printToR2CTsv(bam_header_); } } } lock_guard guard(writeMutex_); // lock the shared writers sc.total_regions_done += unit.processed_since_memory_dump; unit.processed_since_memory_dump = 0; // discordant clusters // hardcoding "false" for readtracking for simplicity for (const auto& kv : unit.m_disc) { const auto& dc = kv.second; if (dc.valid()) os_discordant_ << dc.toFileString(bam_header_, false) << "\n"; } // write contig alignments to BAM for (auto& i : unit.master_contigs) { bool ok = b_contig_writer_.WriteRecord(*i); assert(ok); } // breakpoints for (auto& bp : unit.m_bps) { if ( bp->hasMinimal() ) // && (bp.confidence != "NOLOCAL" || bp.complex_local) ) { os_allbps_ << bp->toFileString(sc.header) << "\n"; } } // discordant reads //std::cerr << " DC " << unit.all_discordant_reads.size() << std::endl; // if (opts.dump_discordant_reads) { // for (const auto& r : unit.all_discordant_reads) { // bool ok = b_discordant_read_writer_.WriteRecord(*r); // assert(ok); // } // } // weird reads // if (opts.dump_weird_reads) { // for (const auto& r : unit.all_weird_reads) { // bool ok = b_weird_read_writer_.WriteRecord(*r); // assert(ok); // } // } // // corrected reads // if (opts.dump_corrected_reads) { // for (const auto& r : unit.all_corrected_reads) { // bool ok = b_corrected_read_writer_.WriteRecord(*r); // assert(ok); // } // } // runtime os_runtime_ << unit.ss.str(); } void SvabaOutputWriter::close() { // SvABA2.0: alignments.txt.gz is gone entirely; r2c.txt.gz is per-thread // (closed in svabaThreadUnit's destructor). Nothing to close here for // those outputs. os_allbps_.close(); os_discordant_.close(); // if (opts.dump_discordant_reads) { // if (!b_discordant_read_writer_.Close()) { // std::cerr << "Unable to close discordant read writer" << std::endl; // } // } // if (opts.dump_weird_reads) { // if (!b_weird_read_writer_.Close()) { // std::cerr << "Unable to close weird read writer" << std::endl; // } // } // if (opts.dump_corrected_reads) { // if (!b_corrected_read_writer_.Close()) { // std::cerr << "Unable to close corrected read writer" << std::endl; // } // } if (!b_contig_writer_.Close()) { std::cerr << "Unable to close contigs bam writer" << std::endl; } } ================================================ FILE: src/svaba/SvabaOutputWriter.h ================================================ // svabaOutputWriter.h #pragma once #include #include #include #include "SvabaThreadUnit.h" #include "SeqLib/BamWriter.h" #include "gzstream.h" #include "SvabaLogger.h" #include "SvabaOptions.h" using std::ofstream; struct SvabaSharedConfig; struct SvabaOutputWriter { public: SvabaOutputWriter(SvabaLogger& logger_, SvabaOptions& opts_); // call this once at startup: void init(const std::string& analysis_id, const SeqLib::BamHeader& b_header); // call this from any thread under a lock: void writeUnit(svabaThreadUnit& unit, SvabaSharedConfig& sc); void close(); private: SvabaLogger& logger; SvabaOptions& opts; // SvABA2.0: alignments.txt.gz (pre-rendered ASCII) and the shared // r2c.txt.gz stream used to live here. Both are gone: // - alignments.txt.gz is removed entirely; its re-plot-able successor // is r2c.txt.gz (AlignedContig::printToR2CTsv + r2cTsvHeader). // - r2c.txt.gz is now emitted per-thread by svabaThreadUnit::r2c_out_ // and merged at postprocess time (gzip is concatenation-safe). // The remaining shared streams below are small enough that mutex // contention isn't measurable (bps + cluster-level discordant + // runtime are KB-MB per run). ogzstream os_allbps_; ogzstream os_discordant_; ofstream os_runtime_; SeqLib::BamWriter b_contig_writer_; SeqLib::BamWriter b_weird_read_writer_; SeqLib::BamWriter b_discordant_read_writer_; SeqLib::BamWriter b_corrected_read_writer_; SeqLib::BamHeader bam_header_; bool disc_cluster_only_ = false; }; ================================================ FILE: src/svaba/SvabaOverlapAlgorithm.cpp ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // This has been modified (slightly) for svaba #include "SvabaOverlapAlgorithm.h" #include "ASQG.h" #include // Collect the complete set of overlaps in pOBOut static const AlignFlags sufPreAF(false, false, false); static const AlignFlags prePreAF(false, true, true); static const AlignFlags sufSufAF(true, false, true); static const AlignFlags preSufAF(true, true, false); //#define DEBUGOVERLAP 2 // Perform the overlap OverlapResult svabaOverlapAlgorithm::overlapRead(const SeqRecord& read, int minOverlap, OverlapBlockList* pOutList) const { OverlapResult r; if(static_cast(read.seq.length()) < minOverlap) return r; if(!m_exactModeOverlap) r = overlapReadInexact(read, minOverlap, pOutList); else r = overlapReadExact(read, minOverlap, pOutList); return r; } // OverlapResult svabaOverlapAlgorithm::overlapReadInexact(const SeqRecord& read, int minOverlap, OverlapBlockList* pOBOut) const { OverlapResult result; OverlapBlockList obWorkingList; std::string seq = read.seq.toString(); #ifdef DEBUGOVERLAP std::cout << "\n\n***Overlapping read " << read.id << " suffix\n"; std::cout << "***Overlapping seq " << read.seq.toString() << " suffix\n"; #endif // Match the suffix of seq to prefixes // findInexact returns false is the maximum search time was exceeded. In this // case we dont run any of the subsequent commands and return no overlaps. bool valid = true; valid = findOverlapBlocksInexact(seq, m_pBWT, m_pRevBWT, sufPreAF, minOverlap, &obWorkingList, pOBOut, result); #ifdef DEBUGOR size_t block_size = pOBOut->size(); if (block_size != pOBOut->size()) { align1 = true; std::cout << "1";//<< std::endl; block_size = pOBOut->size(); } #endif if(valid) valid = findOverlapBlocksInexact(complement(seq), m_pRevBWT, m_pBWT, prePreAF, minOverlap, &obWorkingList, pOBOut, result); #ifdef DEBUGOR if (block_size != pOBOut->size()) { std::cout << "2";// << std::endl; block_size = pOBOut->size(); } #endif #ifdef DEBUGOVERLAP std::cout << "WORKING OverlapBlockList Length SUFFIX: " << obWorkingList.size() << " VALID " << valid << std::endl; #endif if(valid) { if(m_bIrreducible) { computeIrreducibleBlocks(m_pBWT, m_pRevBWT, &obWorkingList, pOBOut); obWorkingList.clear(); } else { pOBOut->splice(pOBOut->end(), obWorkingList); assert(obWorkingList.empty()); } } #ifdef DEBUGOVERLAP std::cout << "\n\n***Overlapping read " << read.id << " prefix\n"; #endif // Match the prefix of seq to suffixes if(valid) valid = findOverlapBlocksInexact(reverseComplement(seq), m_pBWT, m_pRevBWT, sufSufAF, minOverlap, &obWorkingList, pOBOut, result); #ifdef DEBUGOR if (block_size != pOBOut->size()) { std::cout << "3";// << std::endl; block_size = pOBOut->size(); } #endif if(valid) valid = findOverlapBlocksInexact(reverse(seq), m_pRevBWT, m_pBWT, preSufAF, minOverlap, &obWorkingList, pOBOut, result); #ifdef DEBUGOR if (block_size != pOBOut->size()) { std::cout << "4";// << std::endl; block_size = pOBOut->size(); } std::cout<splice(pOBOut->end(), obWorkingList); assert(obWorkingList.empty()); } } if(!valid) { pOBOut->clear(); result.isSubstring = false; result.searchAborted = true; return result; } #ifdef DEBUGOVERLAP unsigned N = 1; if (pOBOut->size() > N) { std::cout << read.id << " OverlapBlockList Length: " << pOBOut->size() << std::endl; std::list::iterator it = pOBOut->begin(); std::advance(it, N); //std::cout << "Olen: " << it->overlapLen << " numDiff: " << it->numDiff << std::endl; std::cout << " BWT1L: " << it->ranges.interval[0].lower; std::cout << " BWT1U: " << it->ranges.interval[0].upper; std::cout << " BWT2L: " << it->ranges.interval[1].lower; std::cout << " BWT2U: " << it->ranges.interval[1].upper; std::cout << " RBWT1L: " << it->rawRanges.interval[0].lower; std::cout << " RBWT1U: " << it->rawRanges.interval[0].upper; std::cout << " RBWT2L: " << it->rawRanges.interval[1].lower; std::cout << " RBWT2U: " << it->rawRanges.interval[1].upper << std::endl; } #endif return result; } // OverlapResult svabaOverlapAlgorithm::alignReadDuplicate(const SeqRecord& read, OverlapBlockList* pOBOut) const { OverlapResult result; OverlapBlockList obWorkingList; std::string seq = read.seq.toString(); int readLength = seq.length(); findOverlapBlocksInexact(seq, m_pBWT, m_pRevBWT, sufPreAF, readLength, &obWorkingList, pOBOut, result); findOverlapBlocksInexact(complement(seq), m_pRevBWT, m_pBWT, prePreAF, readLength, &obWorkingList, pOBOut, result); return result; } // Construct the set of blocks describing irreducible overlaps with READ // and write the blocks to pOBOut OverlapResult svabaOverlapAlgorithm::overlapReadExact(const SeqRecord& read, int minOverlap, OverlapBlockList* pOBOut) const { OverlapResult result; // The complete set of overlap blocks are collected in obWorkingList // The filtered set (containing only irreducible overlaps) are placed into pOBOut // by calculateIrreducibleHits OverlapBlockList obWorkingList; std::string seq = read.seq.toString(); // We store the various overlap blocks using a number of lists, one for the containments // in the forward and reverse index and one for each set of overlap blocks OverlapBlockList oblFwdContain; OverlapBlockList oblRevContain; OverlapBlockList oblSuffixFwd; OverlapBlockList oblSuffixRev; OverlapBlockList oblPrefixFwd; OverlapBlockList oblPrefixRev; // Match the suffix of seq to prefixes findOverlapBlocksExact(seq, m_pBWT, m_pRevBWT, sufPreAF, minOverlap, &oblSuffixFwd, &oblFwdContain, result); findOverlapBlocksExact(complement(seq), m_pRevBWT, m_pBWT, prePreAF, minOverlap, &oblSuffixRev, &oblRevContain, result); // Match the prefix of seq to suffixes findOverlapBlocksExact(reverseComplement(seq), m_pBWT, m_pRevBWT, sufSufAF, minOverlap, &oblPrefixFwd, &oblFwdContain, result); findOverlapBlocksExact(reverse(seq), m_pRevBWT, m_pBWT, preSufAF, minOverlap, &oblPrefixRev, &oblRevContain, result); // Remove submaximal blocks for each block list including fully contained blocks // Copy the containment blocks into the prefix/suffix lists oblSuffixFwd.insert(oblSuffixFwd.end(), oblFwdContain.begin(), oblFwdContain.end()); oblPrefixFwd.insert(oblPrefixFwd.end(), oblFwdContain.begin(), oblFwdContain.end()); oblSuffixRev.insert(oblSuffixRev.end(), oblRevContain.begin(), oblRevContain.end()); oblPrefixRev.insert(oblPrefixRev.end(), oblRevContain.begin(), oblRevContain.end()); // Perform the submaximal filter removeSubMaximalBlocks(&oblSuffixFwd, m_pBWT, m_pRevBWT); removeSubMaximalBlocks(&oblPrefixFwd, m_pBWT, m_pRevBWT); removeSubMaximalBlocks(&oblSuffixRev, m_pRevBWT, m_pBWT); removeSubMaximalBlocks(&oblPrefixRev, m_pRevBWT, m_pBWT); // Remove the contain blocks from the suffix/prefix lists removeContainmentBlocks(seq.length(), &oblSuffixFwd); removeContainmentBlocks(seq.length(), &oblPrefixFwd); removeContainmentBlocks(seq.length(), &oblSuffixRev); removeContainmentBlocks(seq.length(), &oblPrefixRev); // Join the suffix and prefix lists oblSuffixFwd.splice(oblSuffixFwd.end(), oblSuffixRev); oblPrefixFwd.splice(oblPrefixFwd.end(), oblPrefixRev); // Move the containments to the output list pOBOut->splice(pOBOut->end(), oblFwdContain); pOBOut->splice(pOBOut->end(), oblRevContain); // Filter out transitive overlap blocks if requested if(m_bIrreducible) { computeIrreducibleBlocks(m_pBWT, m_pRevBWT, &oblSuffixFwd, pOBOut); computeIrreducibleBlocks(m_pBWT, m_pRevBWT, &oblPrefixFwd, pOBOut); } else { pOBOut->splice(pOBOut->end(), oblSuffixFwd); pOBOut->splice(pOBOut->end(), oblPrefixFwd); } return result; } // Write overlap results to an ASQG file void svabaOverlapAlgorithm::writeResultASQG(std::ostream& writer, const SeqRecord& read, const OverlapResult& result) const { ASQG::VertexRecord record(read.id, read.seq.toString()); record.setSubstringTag(result.isSubstring); record.write(writer); } // Write overlap blocks out to a file void svabaOverlapAlgorithm::writeOverlapBlocks(std::ostream& writer, size_t readIdx, bool isSubstring, const OverlapBlockList* pList) const { // Write the header info size_t numBlocks = pList->size(); writer << readIdx << " " << isSubstring << " " << numBlocks << " "; //std::cout << readIdx << " " << isSubstring << " " << numBlocks << " "; //std::cout << " idx: " << count << " count: " << numBlocks << "\n"; for(OverlapBlockList::const_iterator iter = pList->begin(); iter != pList->end(); ++iter) { writer << *iter << " "; //std::cout << *iter << " "; } writer << "\n"; //std::cout << "\n"; } //JEREMIAH void svabaOverlapAlgorithm::writeOverlapBlocks(std::stringstream& writer, size_t readIdx, bool isSubstring, const OverlapBlockList* pList) const { // Write the header info size_t numBlocks = pList->size(); writer << readIdx << " " << isSubstring << " " << numBlocks << " "; //std::cout << readIdx << " " << isSubstring << " " << numBlocks << " "; for(OverlapBlockList::const_iterator iter = pList->begin(); iter != pList->end(); ++iter) { writer << *iter << " "; //std::cout << *iter << " "; } writer << "\n"; //std::cout << "\n"; } // Calculate the ranges in pBWT that contain a prefix of at least minOverlap basepairs that // overlaps with a suffix of w. The ranges are added to the pOBList void svabaOverlapAlgorithm::findOverlapBlocksExact(const std::string& w, const BWT* pBWT, const BWT* pRevBWT, const AlignFlags& af, int minOverlap, OverlapBlockList* pOverlapList, OverlapBlockList* pContainList, OverlapResult& result) const { // The algorithm is as follows: // We perform a backwards search using the FM-index for the string w. // As we perform the search we collect the intervals // of the significant prefixes (len >= minOverlap) that overlap w. BWTIntervalPair ranges; size_t l = w.length(); int start = l - 1; BWTAlgorithms::initIntervalPair(ranges, w[start], pBWT, pRevBWT); // Collect the OverlapBlocks for(size_t i = start - 1; i >= 1; --i) { // Compute the range of the suffix w[i, l] BWTAlgorithms::updateBothL(ranges, w[i], pBWT); int overlapLen = l - i; if(overlapLen >= minOverlap) { // Calculate which of the prefixes that match w[i, l] are terminal // These are the proper prefixes (they are the start of a read) BWTIntervalPair probe = ranges; BWTAlgorithms::updateBothL(probe, '$', pBWT); // The probe interval contains the range of proper prefixes if(probe.interval[1].isValid()) { assert(probe.interval[1].lower > 0); pOverlapList->push_back(OverlapBlock(probe, ranges, overlapLen, 0, af)); } } } // Determine if this sequence is contained and should not be processed further BWTAlgorithms::updateBothL(ranges, w[0], pBWT); // Ranges now holds the interval for the full-length read // To handle containments, we output the overlapBlock to the final overlap block list // and it will be processed later // Two possible containment cases: // 1) This read is a substring of some other read // 2) This read is identical to some other read // Case 1 is indicated by the existance of a non-$ left or right hand extension // In this case we return no alignments for the string AlphaCount64 left_ext = BWTAlgorithms::getExtCount(ranges.interval[0], pBWT); AlphaCount64 right_ext = BWTAlgorithms::getExtCount(ranges.interval[1], pRevBWT); if(left_ext.hasDNAChar() || right_ext.hasDNAChar()) { result.isSubstring = true; } else { BWTIntervalPair probe = ranges; BWTAlgorithms::updateBothL(probe, '$', pBWT); if(probe.isValid()) { // terminate the contained block and add it to the contained list BWTAlgorithms::updateBothR(probe, '$', pRevBWT); assert(probe.isValid()); pContainList->push_back(OverlapBlock(probe, ranges, w.length(), 0, af)); } } //OverlapBlockList containedWorkingList; //partitionBlockList(w.length(), &workingList, pOverlapList, &containedWorkingList); // Terminate the contained blocks //terminateContainedBlocks(containedWorkingList); // Move the contained blocks to the final contained list //pContainList->splice(pContainList->end(), containedWorkingList); return; } // Seeded blockwise BWT alignment of prefix-suffix for reads // Each alignment is given a seed region and a block region // The seed region is the terminal portion of w where maxDiff + 1 seeds are created // at least 1 of these seeds must align exactly for there to be an alignment with // at most maxDiff differences between the prefix/suffix. Only alignments within the // range [block_start, block_end] are output. The block_end coordinate is inclusive. bool svabaOverlapAlgorithm::findOverlapBlocksInexact(const std::string& w, const BWT* pBWT, const BWT* pRevBWT, const AlignFlags& af, int minOverlap, OverlapBlockList* pOverlapList, OverlapBlockList* pContainList, OverlapResult& result) const { int len = w.length(); int overlap_region_left = len - minOverlap; SearchSeedVector* pCurrVector = new SearchSeedVector; SearchSeedVector* pNextVector = new SearchSeedVector; OverlapBlockList workingList; SearchSeedVector::iterator iter; // Create and extend the initial seeds int actual_seed_length = m_seedLength; int actual_seed_stride = m_seedStride; #ifdef DEBUGOVERLAP std::cout << "SeedLen: " << m_seedLength << " SeedStride: " << m_seedStride << std::endl; #endif if(actual_seed_length == 0) { // Calculate a seed length and stride that will guarantee all overlaps // with error rate m_errorRate will be found calculateSeedParameters(w, minOverlap, actual_seed_length, actual_seed_stride); } assert(actual_seed_stride != 0); createSearchSeeds(w, pBWT, pRevBWT, actual_seed_length, actual_seed_stride, pCurrVector); #ifdef DEBUGOVERLAP std::cout << "SeedVectorLen: " << pCurrVector->size() << std::endl; #endif extendSeedsExactRight(w, pBWT, pRevBWT, ED_RIGHT, pCurrVector, pNextVector); pCurrVector->clear(); pCurrVector->swap(*pNextVector); assert(pNextVector->empty()); int num_steps = 0; // Perform the inexact extensions bool fail = false; while(!pCurrVector->empty()) { if(m_maxSeeds != -1 && (int)pCurrVector->size() > m_maxSeeds) { fail = true; break; } iter = pCurrVector->begin(); while(iter != pCurrVector->end()) { SearchSeed& align = *iter; // If the current aligned region is right-terminal // and the overlap is greater than minOverlap, try to find overlaps // or containments if(align.right_index == len - 1) { double align_error = align.calcErrorRate(); // Check for overlaps if(align.left_index <= overlap_region_left && isErrorRateAcceptable(align_error, m_errorRate)) { int overlapLen = len - align.left_index; BWTIntervalPair probe = align.ranges; BWTAlgorithms::updateBothL(probe, '$', pBWT); // The probe interval contains the range of proper prefixes if(probe.interval[1].isValid()) { assert(probe.interval[1].lower > 0); OverlapBlock nBlock(probe, align.ranges, overlapLen, align.z, af, align.historyLink->getHistoryVector()); workingList.push_back(nBlock); } } // Check for containments // If the seed is left-terminal and there are [ACGT] left/right extensions of the sequence // this read must be a substring of another read if(align.left_index == 0) { AlphaCount64 left_ext = BWTAlgorithms::getExtCount(align.ranges.interval[0], pBWT); AlphaCount64 right_ext = BWTAlgorithms::getExtCount(align.ranges.interval[1], pRevBWT); if(left_ext.hasDNAChar() || right_ext.hasDNAChar()) result.isSubstring = true; } } // Extend the seed to the right/left if(align.dir == ED_RIGHT) extendSeedInexactRight(align, w, pBWT, pRevBWT, pNextVector); else extendSeedInexactLeft(align, w, pBWT, pRevBWT, pNextVector); ++iter; //pCurrVector->erase(iter++); } pCurrVector->clear(); assert(pCurrVector->empty()); pCurrVector->swap(*pNextVector); // Remove identical seeds after we have performed seed_len steps // as there now might be redundant seeds if(num_steps % actual_seed_stride == 0) { std::sort(pCurrVector->begin(), pCurrVector->end(), SearchSeed::compareLeftRange); SearchSeedVector::iterator end_iter = std::unique(pCurrVector->begin(), pCurrVector->end(), SearchSeed::equalLeftRange); pCurrVector->resize(end_iter - pCurrVector->begin()); } ++num_steps; } if(!fail) { // parse the working list to remove any submaximal overlap blocks // these blocks correspond to reads that have multiple valid overlaps. // we only keep the longest removeSubMaximalBlocks(&workingList, pBWT, pRevBWT); OverlapBlockList containedWorkingList; partitionBlockList(len, &workingList, pOverlapList, &containedWorkingList); // Terminate the contained blocks terminateContainedBlocks(containedWorkingList); // Move the contained blocks to the final contained list pContainList->splice(pContainList->end(), containedWorkingList); } delete pCurrVector; delete pNextVector; return !fail; } // Build forward history for the blocks void svabaOverlapAlgorithm::buildForwardHistory(OverlapBlockList* pList) const { OverlapBlockList terminalList; OverlapBlockList potentialContainedList; while(!pList->empty()) { // The terminalBlock list contains all the blocks that became right-terminal // in the current extension round. // Perform a single round of extension, any terminal blocks // are moved to the terminated list extendActiveBlocksRight(m_pBWT, m_pRevBWT, *pList, terminalList, potentialContainedList); } *pList = terminalList; pList->splice(pList->end(), potentialContainedList); } // Calculate the single right extension to the '$' for each the contained blocks // so that the interval ranges are consistent void svabaOverlapAlgorithm::terminateContainedBlocks(OverlapBlockList& containedBlocks) const { for(OverlapBlockList::iterator iter = containedBlocks.begin(); iter != containedBlocks.end(); ++iter) BWTAlgorithms::updateBothR(iter->ranges, '$', iter->getExtensionBWT(m_pBWT, m_pRevBWT)); } // Calculate the seed length and stride to ensure that we will find all // all overlaps with error rate at most m_errorRate. // To overlap two reads allowing for d errors, we create d+1 seeds so that at least one seed will match // exactly. d is a function of the overlap length so we define the seed length using the minimum overlap // parameter. We then tile seeds across the read starting from the end such that for every overlap length // x, there are at least floor(x * error_rate) + 1 seeds. void svabaOverlapAlgorithm::calculateSeedParameters(const std::string& w, const int minOverlap, int& seed_length, int& seed_stride) const { int read_len = w.length(); seed_length = 0; // The maximum possible number of differences occurs for a fully-aligned read int max_diff_high = static_cast(m_errorRate * read_len); // Calculate the seed length to use // If the error rate is so low that no differences are possible just seed // over the entire minOverlap region if(max_diff_high > 0) { // Calculate the maximum number of differences between two sequences that overlap // by minOverlap int max_diff_low = static_cast(m_errorRate * minOverlap); if(max_diff_low == 0) max_diff_low = 1; int seed_region_length = static_cast(ceil(max_diff_low / m_errorRate)); int num_seeds_low = max_diff_low + 1; seed_length = static_cast(seed_region_length / num_seeds_low); if(seed_length > static_cast(minOverlap)) seed_length = minOverlap; } else { seed_length = minOverlap; } seed_stride = seed_length; } // Create and intialize the search seeds int svabaOverlapAlgorithm::createSearchSeeds(const std::string& w, const BWT* pBWT, const BWT* pRevBWT, int seed_length, int seed_stride, SearchSeedVector* pOutVector) const { // Start a new chain of history links SearchHistoryLink rootLink = SearchHistoryNode::createRoot(); // The maximum possible number of differences occurs for a fully-aligned read int read_len = w.length(); int max_diff_high = static_cast(m_errorRate * read_len); // Start the seeds at the end of the read int seed_start = read_len - seed_length; while(seed_start >= 0) { SearchSeed seed; seed.left_index = seed_start; seed.right_index = seed_start; seed.dir = ED_RIGHT; seed.seed_len = seed_length; seed.z = 0; seed.maxDiff = max_diff_high; seed.historyLink = rootLink; // Initialize the left and right suffix array intervals char b = w[seed.left_index]; BWTAlgorithms::initIntervalPair(seed.ranges, b, pBWT, pRevBWT); pOutVector->push_back(seed); seed_start -= seed_stride; // Only create one seed in the exact case if(max_diff_high == 0) break; } return seed_length; } // Extend all the seeds in pInVector to the right over the entire seed range void svabaOverlapAlgorithm::extendSeedsExactRightQueue(const std::string& w, const BWT* /*pBWT*/, const BWT* pRevBWT, ExtendDirection /*dir*/, const SearchSeedVector* pInVector, SearchSeedQueue* pOutQueue) const { for(SearchSeedVector::const_iterator iter = pInVector->begin(); iter != pInVector->end(); ++iter) { SearchSeed align = *iter; bool valid = true; while(align.isSeed()) { ++align.right_index; char b = w[align.right_index]; BWTAlgorithms::updateBothR(align.ranges, b, pRevBWT); if(!align.isIntervalValid(RIGHT_INT_IDX)) { valid = false; break; } } if(valid) pOutQueue->push(align); } } // Extend all the seeds in pInVector to the right over the entire seed range void svabaOverlapAlgorithm::extendSeedsExactRight(const std::string& w, const BWT* /*pBWT*/, const BWT* pRevBWT, ExtendDirection /*dir*/, const SearchSeedVector* pInVector, SearchSeedVector* pOutVector) const { for(SearchSeedVector::const_iterator iter = pInVector->begin(); iter != pInVector->end(); ++iter) { SearchSeed align = *iter; bool valid = true; while(align.isSeed()) { ++align.right_index; char b = w[align.right_index]; BWTAlgorithms::updateBothR(align.ranges, b, pRevBWT); if(!align.isIntervalValid(RIGHT_INT_IDX)) { valid = false; break; } } //std::cout << "Initial seed: "; //align.print(w); if(valid) pOutVector->push_back(align); } } // void svabaOverlapAlgorithm::extendSeedInexactRight(SearchSeed& seed, const std::string& w, const BWT* /*pBWT*/, const BWT* pRevBWT, SearchSeedVector* pOutVector) const { // If this alignment has run all the way to the end of the sequence // switch it to be a left extension sequence if(seed.right_index == int(w.length() - 1)) { seed.dir = ED_LEFT; pOutVector->push_back(seed); return; } ++seed.right_index; if(!seed.allowMismatches()) { char b = w[seed.right_index]; BWTAlgorithms::updateBothR(seed.ranges, b, pRevBWT); if(seed.isIntervalValid(RIGHT_INT_IDX)) pOutVector->push_back(seed); } else { // Calculating the AlphaCounts is the heavy part of the computation so we cache // the value outside the loop, it is the same for all bases AlphaCount64 lower = pRevBWT->getFullOcc(seed.ranges.interval[1].lower - 1); AlphaCount64 upper = pRevBWT->getFullOcc(seed.ranges.interval[1].upper); for(int i = 0; i < 4; ++i) { char b = ALPHABET[i]; BWTIntervalPair probe = seed.ranges; BWTAlgorithms::updateBothR(probe, b, pRevBWT, lower, upper); if(probe.interval[RIGHT_INT_IDX].isValid()) { SearchSeed branched = seed; branched.ranges = probe; if(b != w[seed.right_index]) { ++branched.z; branched.historyLink = seed.historyLink->createChild(w.length() - seed.right_index, b); } pOutVector->push_back(branched); } } } } // void svabaOverlapAlgorithm::extendSeedInexactLeft(SearchSeed& seed, const std::string& w, const BWT* pBWT, const BWT* /*pRevBWT*/, SearchSeedVector* pOutVector) const { //printf("ProcessingLEFT: "); align.print(w); --seed.left_index; if(seed.left_index >= 0) { if(!seed.allowMismatches()) { // Extend exact char b = w[seed.left_index]; BWTAlgorithms::updateBothL(seed.ranges, b, pBWT); if(seed.isIntervalValid(LEFT_INT_IDX)) pOutVector->push_back(seed); } else { // Calculating the AlphaCounts is the heavy part of the computation so we cache // the value outside the loop, it is the same for all bases AlphaCount64 lower = pBWT->getFullOcc(seed.ranges.interval[0].lower - 1); AlphaCount64 upper = pBWT->getFullOcc(seed.ranges.interval[0].upper); for(int i = 0; i < 4; ++i) { char b = ALPHABET[i]; BWTIntervalPair probe = seed.ranges; BWTAlgorithms::updateBothL(probe, b, pBWT, lower, upper); if(probe.interval[LEFT_INT_IDX].isValid()) { SearchSeed branched = seed; branched.ranges = probe; if(b != w[seed.left_index]) { ++branched.z; // The history coordinates are wrt the right end of the read // so that each position corresponds to the length of the overlap // including that position branched.historyLink = seed.historyLink->createChild(w.length() - seed.left_index, b); //branched.history.add(w.length() - seed.left_index, b); } pOutVector->push_back(branched); } } } } } // Extend the seed to the right, return true if the seed is still a valid range bool svabaOverlapAlgorithm::extendSeedExactRight(SearchSeed& seed, const std::string& w, const BWT* /*pBWT*/, const BWT* pRevBWT) const { // If this alignment has run all the way to the end of the sequence // switch it to be a left extension sequence if(seed.right_index == int(w.length() - 1)) { seed.dir = ED_LEFT; return true; } ++seed.right_index; char b = w[seed.right_index]; BWTAlgorithms::updateBothR(seed.ranges, b, pRevBWT); if(seed.isIntervalValid(RIGHT_INT_IDX)) return true; else return false; } // bool svabaOverlapAlgorithm::extendSeedExactLeft(SearchSeed& seed, const std::string& w, const BWT* pBWT, const BWT* /*pRevBWT*/) const { --seed.left_index; if(seed.left_index >= 0) { char b = w[seed.left_index]; BWTAlgorithms::updateBothL(seed.ranges, b, pBWT); if(seed.isIntervalValid(LEFT_INT_IDX)) return true; else return false; } else { return false; } } // Calculate the irreducible blocks from the vector of OverlapBlocks void svabaOverlapAlgorithm::computeIrreducibleBlocks(const BWT* pBWT, const BWT* pRevBWT, OverlapBlockList* pOBList, OverlapBlockList* pOBFinal) const { // processIrreducibleBlocks requires the pOBList to be sorted in descending order pOBList->sort(OverlapBlock::sortSizeDescending); #ifdef DEBUGOVERLAP std::cout << " m_exactModeIrreducible " << m_exactModeIrreducible << "\n"; #endif if(m_exactModeIrreducible) _processIrreducibleBlocksExactIterative(pBWT, pRevBWT, *pOBList, pOBFinal); else _processIrreducibleBlocksInexact(pBWT, pRevBWT, *pOBList, pOBFinal); pOBList->clear(); } // iterate through obList and determine the overlaps that are irreducible. This function is recursive. // The final overlap blocks corresponding to irreducible overlaps are written to pOBFinal. // Invariant: the blocks are ordered in descending order of the overlap size so that the longest overlap is first. // Invariant: each block corresponds to the same extension of the root sequence w. void svabaOverlapAlgorithm::_processIrreducibleBlocksExactIterative(const BWT* pBWT, const BWT* pRevBWT, OverlapBlockList& inList, OverlapBlockList* pOBFinal) const { if(inList.empty()) return; // We store the overlap blocks in groups of blocks that have the same right-extension. // When a branch is found, the groups are split based on the extension typedef std::list BlockGroups; BlockGroups blockGroups; blockGroups.push_back(inList); int numExtensions = 0; int numBranches = 0; while(!blockGroups.empty()) { // Perform one extenion round for each group. // If the top-level block has ended, push the result // to the final list and remove the group from processing BlockGroups::iterator groupIter = blockGroups.begin(); BlockGroups incomingGroups; // Branched blocks are placed here while(groupIter != blockGroups.end()) { OverlapBlockList& currList = *groupIter; bool bEraseGroup = false; // Count the extensions in the top level (longest) blocks first int topLen = currList.front().overlapLen; AlphaCount64 ext_count; OBLIter blockIter = currList.begin(); while(blockIter != currList.end() && blockIter->overlapLen == topLen) { ext_count += blockIter->getCanonicalExtCount(pBWT, pRevBWT); ++blockIter; } // Three cases: // 1) The top level block has ended as it contains the extension $. Output TLB and end. // 2) There is a singular unique extension base for all the blocks. Update the blocks and continue. // 3) There are multiple extension bases, split the block group and continue. // If some block other than the TLB ended, it must be contained within the TLB and it is not output // or considered further. // Likewise if multiple distinct strings in the TLB ended, we only output the top one. The rest // must have the same sequence as the top one and are hence considered to be contained with the top element. #ifdef DEBUGOVERLAP std::cout << "ext_gcount.get($) " << ext_count.get('$') << "\n"; #endif if(ext_count.get('$') > 0) { // An irreducible overlap has been found. It is possible that there are two top level blocks // (one in the forward and reverse direction). Since we can't decide which one // contains the other at this point, we output hits to both. Under a fixed // length string assumption one will be contained within the other and removed later. OBLIter tlbIter = currList.begin(); while(tlbIter != currList.end() && tlbIter->overlapLen == topLen) { // Ensure the tlb is actually terminal and not a substring block AlphaCount64 test_count = tlbIter->getCanonicalExtCount(pBWT, pRevBWT); if(test_count.get('$') == 0) { std::cerr << "Error: substring read found during overlap computation.\n"; std::cerr << "Please run sga rmdup before sga overlap\n"; exit(EXIT_FAILURE); } // Perform the final right-update to make the block terminal OverlapBlock branched = *tlbIter; BWTAlgorithms::updateBothR(branched.ranges, '$', branched.getExtensionBWT(pBWT, pRevBWT)); pOBFinal->push_back(branched); #ifdef DEBUGOVERLAP std::cout << "[IE] TLB of length " << branched.overlapLen << " has ended\n"; std::cout << "[IE]\tBlock data: " << branched << "\n"; #endif ++tlbIter; } // Set the flag to erase this group, it is finished bEraseGroup = true; } else { // Count the extension for the rest of the blocks while(blockIter != currList.end()) { ext_count += blockIter->getCanonicalExtCount(pBWT, pRevBWT); ++blockIter; } if(ext_count.hasUniqueDNAChar()) { // Update all the blocks using the unique extension character // This character is in the canonical representation wrt to the query char b = ext_count.getUniqueDNAChar(); updateOverlapBlockRangesRight(pBWT, pRevBWT, currList, b); numExtensions++; bEraseGroup = false; } else { for(size_t idx = 0; idx < DNA_ALPHABET_SIZE; ++idx) { char b = ALPHABET[idx]; if(ext_count.get(b) > 0) { numBranches++; OverlapBlockList branched = currList; updateOverlapBlockRangesRight(pBWT, pRevBWT, branched, b); incomingGroups.push_back(branched); bEraseGroup = true; } } } } if(bEraseGroup) groupIter = blockGroups.erase(groupIter); else ++groupIter; } // Splice in the newly branched blocks, if any blockGroups.splice(blockGroups.end(), incomingGroups); } } // Classify the blocks in obList as irreducible, transitive or substrings. The irreducible blocks are // put into pOBFinal. The remaining are discarded. // Invariant: the blocks are ordered in descending order of the overlap size so that the longest overlap is first. void svabaOverlapAlgorithm::_processIrreducibleBlocksInexact(const BWT* pBWT, const BWT* pRevBWT, OverlapBlockList& activeList, OverlapBlockList* pOBFinal) const { if(activeList.empty()) return; #ifdef DEBUGOVERLAP std::cout << " activeList.size() " << activeList.size() << "\n"; #endif // The activeList contains all the blocks that are not yet right terminal // Count the extensions in the top level (longest) blocks first bool all_eliminated = false; while(!activeList.empty() && !all_eliminated) { // The terminalBlock list contains all the blocks that became right-terminal // in the current extension round. OverlapBlockList terminalList; OverlapBlockList potentialContainedList; // Perform a single round of extension, any terminal blocks // are moved to the terminated list extendActiveBlocksRight(pBWT, pRevBWT, activeList, terminalList, potentialContainedList); // Compare the blocks in the contained list against the other terminal and active blocks // If they are a substring match to any of these, discard them OverlapBlockList::iterator containedIter = potentialContainedList.begin(); for(; containedIter != potentialContainedList.end(); ++containedIter) { if(!isBlockSubstring(*containedIter, terminalList, m_errorRate) && !isBlockSubstring(*containedIter, activeList, m_errorRate)) { // Not a substring, move to terminal list terminalList.push_back(*containedIter); //std::cout << "Contained block kept: " << containedIter->overlapLen << "\n"; } else { //std::cout << "Contained block found and removed: " << containedIter->overlapLen << "\n"; } } // Using the terminated blocks, mark as eliminated any active blocks // that form a valid overlap to the terminal block. These are transitive edges // We do not compare two terminal blocks, we don't consider these overlaps to be // transitive #ifdef DEBUGOVERLAP std::cout << " terminalList.sizee() " << terminalList.size() << "\n"; #endif OverlapBlockList::iterator terminalIter = terminalList.begin(); for(; terminalIter != terminalList.end(); ++terminalIter) { #ifdef DEBUGOVERLAP std::cout << "[II] ***TLB of length " << terminalIter->overlapLen << " has ended\n"; #endif all_eliminated = true; OverlapBlockList::iterator activeIter = activeList.begin(); for(; activeIter != activeList.end(); ++activeIter) { if(activeIter->isEliminated) continue; // skip previously marked blocks // Two conditions must be met for a block to be transitive wrt terminal: // 1) It must have a strictly shorter overlap than the terminal block // 2) The error rate between the block and terminal must be less than the threshold double inferredErrorRate = calculateBlockErrorRate(*terminalIter, *activeIter); if(activeIter->overlapLen < terminalIter->overlapLen && isErrorRateAcceptable(inferredErrorRate, m_errorRate)) { #ifdef DEBUGOVERLAP_2 std::cout << "Marking block of length " << activeIter->overlapLen << " as eliminated\n"; #endif activeIter->isEliminated = true; } else { all_eliminated = false; } } // Move this block to the final list if it has not been previously marked eliminated if(!terminalIter->isEliminated) { #ifdef DEBUGOVERLAP std::cout << "[II] Adding block " << *terminalIter << " to final list\n"; //std::cout << " extension: " << terminalIter->forwardHistory << "\n"; #endif pOBFinal->push_back(*terminalIter); } } } activeList.clear(); } // Extend all the blocks in activeList by one base to the right // Move all right-terminal blocks to the termainl list. If a block // is terminal and potentially contained by another block, add it to // containedList void svabaOverlapAlgorithm::extendActiveBlocksRight(const BWT* pBWT, const BWT* pRevBWT, OverlapBlockList& activeList, OverlapBlockList& terminalList, OverlapBlockList& /*containedList*/) const { OverlapBlockList::iterator iter = activeList.begin(); OverlapBlockList::iterator next; while(iter != activeList.end()) { next = iter; ++next; // Check if block is terminal AlphaCount64 ext_count = iter->getCanonicalExtCount(pBWT, pRevBWT); if(ext_count.get('$') > 0) { // Only consider this block to be terminal irreducible if it has at least one extension // or else it is a substring block if(iter->forwardHistory.size() > 0) { OverlapBlock branched = *iter; BWTAlgorithms::updateBothR(branched.ranges, '$', branched.getExtensionBWT(pBWT, pRevBWT)); terminalList.push_back(branched); #ifdef DEBUGOVERLAP_2 std::cout << "Block of length " << iter->overlapLen << " moved to terminal\n"; #endif } } int curr_extension = iter->forwardHistory.size(); // Perform the right extensions // Best case, there is only a single extension character // Handle this case specially so we don't need to copy the potentially // large OverlapBlock structure and its full history if(ext_count.hasUniqueDNAChar()) { // Get the extension character with respect to the queried sequence char canonical_base = ext_count.getUniqueDNAChar(); // Flip the base into the frame of reference for the block char block_base = iter->flags.isQueryComp() ? complement(canonical_base) : canonical_base; // Update the block using the base in its frame of reference BWTAlgorithms::updateBothR(iter->ranges, block_base, iter->getExtensionBWT(pBWT, pRevBWT)); // Add the base to the history in the frame of reference of the query read // This is so the history is consistent when comparing between blocks from different strands iter->forwardHistory.add(curr_extension, canonical_base); } else { for(size_t idx = 0; idx < DNA_ALPHABET_SIZE; ++idx) { char canonical_base = ALPHABET[idx]; char block_base = iter->flags.isQueryComp() ? complement(canonical_base) : canonical_base; if(ext_count.get(canonical_base) == 0) continue; // Branch the sequence. This involves copying the entire history which can be large // if the input sequences are very long. This could be avoided by using the SearchHistoyNode/Link // structure but branches are infrequent enough to not have a large impact OverlapBlock branched = *iter; BWTAlgorithms::updateBothR(branched.ranges, block_base, branched.getExtensionBWT(pBWT, pRevBWT)); assert(branched.ranges.isValid()); // Add the base in the canonical frame branched.forwardHistory.add(curr_extension, canonical_base); // Insert the new block after the iterator activeList.insert(iter, branched); } // Remove the original block, which has been superceded by the branches activeList.erase(iter); } iter = next; // this skips the newly-inserted blocks } } // Return true if the terminalBlock is a substring of any member of blockList bool svabaOverlapAlgorithm::isBlockSubstring(OverlapBlock& terminalBlock, const OverlapBlockList& blockList, double maxER) const { OverlapBlockList::const_iterator iter = blockList.begin(); size_t right_extension_length = terminalBlock.forwardHistory.size(); for(; iter != blockList.end(); ++iter) { if(terminalBlock.overlapLen == iter->overlapLen && right_extension_length == iter->forwardHistory.size()) { continue; // same length, cannot be a substring } // Calculate error rate between blocks double er = calculateBlockErrorRate(terminalBlock, *iter); if(isErrorRateAcceptable(er, maxER)) return true; } return false; } // Calculate the error rate between two overlap blocks using their history double svabaOverlapAlgorithm::calculateBlockErrorRate(const OverlapBlock& terminalBlock, const OverlapBlock& otherBlock) const { int back_max = std::min(terminalBlock.overlapLen, otherBlock.overlapLen); int backwards_diff = SearchHistoryVector::countDifferences(terminalBlock.backHistory, otherBlock.backHistory, back_max); // We compare the forward (right) extension only up to the last position of the terminated block's extension int forward_len = terminalBlock.forwardHistory.size(); int forward_max = forward_len - 1; int forward_diff = SearchHistoryVector::countDifferences(terminalBlock.forwardHistory, otherBlock.forwardHistory, forward_max); // Calculate the length of the inferred overlap int trans_overlap_length = back_max + forward_len; double er = static_cast(backwards_diff + forward_diff) / trans_overlap_length; #ifdef DEBUGOVERLAP_2 std::cout << "OL: " << terminalBlock.overlapLen << "\n"; std::cout << "TLB BH: " << terminalBlock.backHistory << "\n"; std::cout << "TB BH: " << otherBlock.backHistory << "\n"; std::cout << "TLB FH: " << terminalBlock.forwardHistory << "\n"; std::cout << "TB FH: " << otherBlock.forwardHistory << "\n"; std::cout << "BM: " << back_max << " FM: " << forward_max << "\n"; std::cout << "IOL: " << trans_overlap_length << " TD: " << (backwards_diff + forward_diff) << "\n"; std::cout << "Block of length " << otherBlock.overlapLen << " has ier: " << er << "\n"; #endif return er; } // Update the overlap block list with a righthand extension to b, removing ranges that become invalid void svabaOverlapAlgorithm::updateOverlapBlockRangesRight(const BWT* pBWT, const BWT* pRevBWT, OverlapBlockList& obList, char canonical_base) const { OverlapBlockList::iterator iter = obList.begin(); while(iter != obList.end()) { char relative_base = iter->flags.isQueryComp() ? complement(canonical_base) : canonical_base; BWTAlgorithms::updateBothR(iter->ranges, relative_base, iter->getExtensionBWT(pBWT, pRevBWT)); // remove the block from the list if its no longer valid if(!iter->ranges.isValid()) { iter = obList.erase(iter); } else { // Add the base to the extension history int currExtension = iter->forwardHistory.size(); iter->forwardHistory.add(currExtension, canonical_base); ++iter; } } } ================================================ FILE: src/svaba/SvabaOverlapAlgorithm.h ================================================ //----------------------------------------------- // Copyright 2009 Wellcome Trust Sanger Institute // Written by Jared Simpson (js18@sanger.ac.uk) // Released under the GPL //----------------------------------------------- // // svabaOverlapAlgorithm - This class implements all the logic // for finding and outputting overlaps for sequence reads // #ifndef SVABA_OVERLAPALGORITHM_H #define SVABA_OVERLAPALGORITHM_H #include "SuffixTools/BWT.h" #include "OverlapBlock.h" #include "OverlapAlgorithm.h" #include "SearchSeed.h" #include "BWTAlgorithms.h" #include "Util.h" /*enum OverlapMode { OM_OVERLAP, OM_FULLREAD }; struct OverlapResult { OverlapResult() : isSubstring(false), searchAborted(false) {} bool isSubstring; bool searchAborted; };*/ class svabaOverlapAlgorithm { public: svabaOverlapAlgorithm(const BWT* pBWT, const BWT* pRevBWT, double er, int seedLen, int seedStride, bool irrOnly, int maxSeeds = -1) : m_pBWT(pBWT), m_pRevBWT(pRevBWT), m_errorRate(er), m_seedLength(seedLen), m_seedStride(seedStride), m_bIrreducible(irrOnly), m_exactModeOverlap(false), m_exactModeIrreducible(false), m_maxSeeds(maxSeeds) {} // Perform the overlap // This function is threaded so everything must be const OverlapResult overlapRead(const SeqRecord& read, int minOverlap, OverlapBlockList* pOutList) const; // Perform an irreducible overlap OverlapResult overlapReadExact(const SeqRecord& read, int minOverlap, OverlapBlockList* pOBOut) const; // Find duplicate blocks for this read OverlapResult alignReadDuplicate(const SeqRecord& read, OverlapBlockList* pOBOut) const; // Perform an inexact overlap OverlapResult overlapReadInexact(const SeqRecord& read, int minOverlap, OverlapBlockList* pOBOut) const; // Write the result of an overlap to an ASQG file void writeResultASQG(std::ostream& writer, const SeqRecord& read, const OverlapResult& result) const; // Write all the overlap blocks pList to the filehandle void writeOverlapBlocks(std::ostream& writer, size_t readIdx, bool isSubstring, const OverlapBlockList* pList) const; void writeOverlapBlocks(std::stringstream& writer, size_t readIdx, bool isSubstring, const OverlapBlockList* pList) const; //JEREMIAH // Build the forward history structures for the blocks void buildForwardHistory(OverlapBlockList* pList) const; // Set flag to use exact-match algorithms only void setExactModeOverlap(bool b) { m_exactModeOverlap = b; } void setExactModeIrreducible(bool b) { m_exactModeIrreducible = b; } // const BWT* getBWT() const { return m_pBWT; } const BWT* getRBWT() const { return m_pRevBWT; } private: // Calculate the ranges in pBWT that contain a prefix of at least minOverlap basepairs that // overlaps with a suffix of w. void findOverlapBlocksExact(const std::string& w, const BWT* pBWT, const BWT* pRevBWT, const AlignFlags& af, const int minOverlap, OverlapBlockList* pOBTemp, OverlapBlockList* pOBFinal, OverlapResult& result) const; // Same as above while allowing mismatches bool findOverlapBlocksInexact(const std::string& w, const BWT* pBWT, const BWT* pRevBWT, const AlignFlags& af, const int minOverlap, OverlapBlockList* pOBList, OverlapBlockList* pOBFinal, OverlapResult& result) const; // inline bool extendSeedExactRight(SearchSeed& seed, const std::string& w, const BWT* pBWT, const BWT* pRevBWT) const; inline bool extendSeedExactLeft(SearchSeed& seed, const std::string& w, const BWT* pBWT, const BWT* pRevBWT) const; inline void branchSeedRight(const SearchSeed& seed, const std::string& w, const BWT* pBWT, const BWT* pRevBWT, SearchSeedQueue* pQueue) const; inline void branchSeedLeft(const SearchSeed& seed, const std::string& w, const BWT* pBWT, const BWT* pRevBWT, SearchSeedQueue* pQueue) const; // inline void extendSeedInexactRight(SearchSeed& seed, const std::string& w, const BWT* pBWT, const BWT* pRevBWT, SearchSeedVector* pOutVector) const; // inline void extendSeedInexactLeft(SearchSeed& seed, const std::string& w, const BWT* pBWT, const BWT* pRevBWT, SearchSeedVector* pOutVector) const; // inline void calculateSeedParameters(const std::string& w, const int minOverlap, int& seed_length, int& seed_stride) const; // inline int createSearchSeeds(const std::string& w, const BWT* pBWT, const BWT* pRevBWT, int seed_length, int seed_stride, SearchSeedVector* pOutVector) const; // inline void extendSeedsExactRightQueue(const std::string& w, const BWT* pBWT, const BWT* pRevBWT, ExtendDirection dir, const SearchSeedVector* pInVector, SearchSeedQueue* pOutQueue) const; // inline void extendSeedsExactRight(const std::string& w, const BWT* pBWT, const BWT* pRevBWT, ExtendDirection dir, const SearchSeedVector* pInVector, SearchSeedVector* pOutVector) const; // Calculate the terminal extension for the contained blocks to make the intervals consistent void terminateContainedBlocks(OverlapBlockList& containedBlocks) const; // // Irreducible-only processing algorithms // // Reduce the block list pOBList by removing blocks that correspond to transitive edges void computeIrreducibleBlocks(const BWT* pBWT, const BWT* pRevBWT, OverlapBlockList* pOBList, OverlapBlockList* pOBFinal) const; // these recursive functions do the actual work of computing the irreducible blocks void _processIrreducibleBlocksExact(const BWT* pBWT, const BWT* pRevBWT, OverlapBlockList& obList, OverlapBlockList* pOBFinal) const; void _processIrreducibleBlocksExactIterative(const BWT* pBWT, const BWT* pRevBWT, OverlapBlockList& inList, OverlapBlockList* pOBFinal) const; // void _processIrreducibleBlocksInexact(const BWT* pBWT, const BWT* pRevBWT, OverlapBlockList& obList, OverlapBlockList* pOBFinal) const; // Update the overlap block list with a righthand extension to b, removing ranges that become invalid void updateOverlapBlockRangesRight(const BWT* pBWT, const BWT* pRevBWT, OverlapBlockList& obList, char b) const; // void extendActiveBlocksRight(const BWT* pBWT, const BWT* pRevBWT, OverlapBlockList& activeList, OverlapBlockList& terminalList, OverlapBlockList& containedList) const; double calculateBlockErrorRate(const OverlapBlock& terminalBlock, const OverlapBlock& otherBlock) const; bool isBlockSubstring(OverlapBlock& terminalBlock, const OverlapBlockList& blockList, double maxER) const; // Data const BWT* m_pBWT; const BWT* m_pRevBWT; double m_errorRate; int m_seedLength; int m_seedStride; bool m_bIrreducible; bool m_exactModeOverlap; bool m_exactModeIrreducible; // Optional parameter to limit the amount of branching that is performed int m_maxSeeds; }; #endif ================================================ FILE: src/svaba/SvabaPostprocess.cpp ================================================ // SvabaPostprocess.cpp — see SvabaPostprocess.h for the design rationale. // // The flow for each suffix runs in its own std::thread: // // [${ID}.${suffix}.bam] // | // | (unless --dedup-only) // v // samtools sort -@ jthreads -m MEM -o *.sort.tmp.bam ${bam} # shell out // rename *.sort.tmp.bam -> ${bam} // | // | (if suffix in DEDUP set, unless --sort-only) // v // native streaming dedup+merge -> *.dedup.tmp.bam # htslib // (writer uses a header with our @PG line appended) // rename *.dedup.tmp.bam -> ${bam} # PG stamped // | // | (only if dedup didn't run for this suffix) // v // samtools reheader ${bam} > *.reheader.tmp.bam # shell out // rename *.reheader.tmp.bam -> ${bam} # PG stamped // | // v // sam_index_build(${bam}, 0) # htslib // → ${bam}.bai // // Every final ${ID}.${suffix}.bam therefore ends up sorted, deduped (where // applicable), carrying an @PG "svaba_postprocess" line chained onto the // existing PG chain, and accompanied by a .bai index. The user never has to // know the intermediate .postprocess.*.tmp.bam files existed. // // The dedup step is NOT a straight drop of duplicate (qname, flag) records // at a locus: overlapping assembly windows can emit the same read twice // with different bi:Z / bz:Z tags naming different supporting contigs. // Dropping one would lose that support evidence. Instead we buffer per // locus and union the comma-token bi/bz lists into the first record, using // the same boundary-aware merge that SvabaOutputWriter.cpp::stamp_tag() // performs during `svaba run`. // // Progress is reported per 1M processed reads, per suffix, serialized through // a shared stderr mutex so lines don't interleave. We avoid any per-record // system call — stat() is only used for file-existence checks and size-based // ETA estimation. #include "SvabaPostprocess.h" #include #include #include #include #include #include #include #include #include #include #include #include #include #include #include #include #include #include #include #include #include "htslib/sam.h" #include "SeqLib/BamHeader.h" #include "SeqLib/BamReader.h" #include "SeqLib/BamRecord.h" #include "SeqLib/BamWriter.h" #include "SvabaOptions.h" // SVABA_VERSION namespace { // Serialize progress/log output across the per-suffix threads. We intentionally // keep the critical section tiny — just the single ostringstream flush — so // workers don't serialize on their own progress prints. std::mutex g_stderr_mu; // Suffixes eligible for dedup. `contigs` is sorted but never deduped because // each contig is emitted exactly once by a single window and there is no // overlap-induced duplication to clean up. const std::vector kDedupSuffixes = { "weird", "corrected", "discordant" }; // Full suffix list to consider, in the same order as sort_output.sh. The // contigs suffix goes last so that any shared stderr verbosity from it doesn't // bury the (more interesting) dedup stats. const std::vector kAllSuffixes = { "weird", "corrected", "discordant", "contigs" }; // ---------- options ---------- struct Opts { std::string id; int threads = 4; std::string mem = "2G"; // per samtools sort thread; matches -m flag int verbose = 1; bool sort_only = false; // skip dedup bool dedup_only = false; // skip sort (assume sorted) }; enum { OPT_SORT_ONLY = 1000, OPT_DEDUP_ONLY, OPT_MEM }; const char* kShortOpts = "hi:t:m:v:"; const struct option kLongOpts[] = { { "help", no_argument, nullptr, 'h' }, { "id", required_argument, nullptr, 'i' }, { "threads", required_argument, nullptr, 't' }, { "mem", required_argument, nullptr, 'm' }, { "verbose", required_argument, nullptr, 'v' }, { "sort-only", no_argument, nullptr, OPT_SORT_ONLY }, { "dedup-only", no_argument, nullptr, OPT_DEDUP_ONLY }, { nullptr, 0, nullptr, 0 } }; void printUsage() { std::cerr << "Usage: svaba postprocess -i [options]\n" "\n" " Coord-sort (via samtools sort) and natively streaming-dedup\n" " svaba's per-suffix output BAMs. Much faster than the legacy\n" " samtools-view | awk | samtools-view pipeline.\n" "\n" " Expects files at ${ID}.${suffix}.bam for suffix in:\n" " weird corrected discordant contigs\n" "\n" " -i, --id Analysis ID (required).\n" " -t, --threads Total threads budget, split across concurrent\n" " per-suffix jobs. [4]\n" " -m, --mem Memory per samtools-sort thread (e.g. 2G). [2G]\n" " --sort-only Only sort; skip dedup.\n" " --dedup-only Only dedup; skip sort. Assumes input is already\n" " coord-sorted; output is undefined otherwise.\n" " -v, --verbose <0-3> Verbosity. [1]\n" " -h, --help This message.\n"; } Opts parseOpts(int argc, char** argv) { Opts o; if (argc <= 2) { printUsage(); std::exit(EXIT_FAILURE); } for (int c; (c = getopt_long(argc, argv, kShortOpts, kLongOpts, nullptr)) != -1;) { std::istringstream arg(optarg ? optarg : ""); switch (c) { case 'h': printUsage(); std::exit(EXIT_SUCCESS); case 'i': arg >> o.id; break; case 't': arg >> o.threads; break; case 'm': arg >> o.mem; break; case 'v': arg >> o.verbose; break; case OPT_SORT_ONLY: o.sort_only = true; break; case OPT_DEDUP_ONLY: o.dedup_only = true; break; default: printUsage(); std::exit(EXIT_FAILURE); } } if (o.id.empty()) { std::cerr << "ERROR: --id is required\n"; printUsage(); std::exit(EXIT_FAILURE); } if (o.sort_only && o.dedup_only) { std::cerr << "ERROR: --sort-only and --dedup-only are mutually exclusive\n"; std::exit(EXIT_FAILURE); } if (o.threads < 1) o.threads = 1; return o; } // ---------- small utilities ---------- bool fileExists(const std::string& p) { struct stat st{}; return ::stat(p.c_str(), &st) == 0 && S_ISREG(st.st_mode); } off_t fileSize(const std::string& p) { struct stat st{}; if (::stat(p.c_str(), &st) != 0) return 0; return st.st_size; } // Thread-safe stderr log. Accepts anything streamable into an ostringstream. template void logLine(Args&&... args) { std::ostringstream oss; (oss << ... << std::forward(args)); std::lock_guard lg(g_stderr_mu); std::cerr << oss.str() << std::endl; } // Quote a path for use inside a /bin/sh command string. Wraps in single // quotes and escapes embedded single quotes as '\''. This is safe against // arbitrary IDs containing spaces or shell metacharacters. std::string shQuote(const std::string& s) { std::string out; out.reserve(s.size() + 2); out.push_back('\''); for (char c : s) { if (c == '\'') out += "'\\''"; else out.push_back(c); } out.push_back('\''); return out; } // Forward decl: defined further down in the "per-suffix pipeline" section. // Declared here so the reheader helper below can use std::rename via the same // diagnostics-rich wrapper the dedup path uses. void renameOrThrow(const std::string& from, const std::string& to); // ---------- @PG stamping ---------- // // Every final BAM produced by `svaba postprocess` gets a new @PG line stamped // onto its header so downstream tools (and humans) can tell at a glance that // postprocess ran and with what arguments. The line chains onto the existing // @PG chain via PP: so samtools' `--pg` resolution keeps working. // // Conventions: // ID: "svaba_postprocess" (or ".1", ".2", ... if already present — IDs must // be unique within a header) // PN: "svaba" // VN: SVABA_VERSION // CL: the original argv reassembled, with "svaba " prepended so the CL // matches what the user actually typed // PP: the tail of the existing PG chain (the ID that is nobody's PP pointer) struct PgChainInfo { std::vector ids; // all @PG IDs, in file order std::string tail; // ID that is nobody else's PP (or "" if none) }; PgChainInfo scanPgChain(const std::string& hdr_text) { PgChainInfo info; std::set is_pp_target; std::istringstream iss(hdr_text); std::string line; while (std::getline(iss, line)) { if (line.rfind("@PG\t", 0) != 0) continue; std::string id, pp; std::istringstream ls(line); std::string field; while (std::getline(ls, field, '\t')) { if (field.rfind("ID:", 0) == 0) id = field.substr(3); else if (field.rfind("PP:", 0) == 0) pp = field.substr(3); } if (!id.empty()) info.ids.push_back(id); if (!pp.empty()) is_pp_target.insert(pp); } // Tail = an ID that no one else's PP points at. If multiple, take the last // one in file order — matches what samtools reheader/markdup do in practice. for (const auto& id : info.ids) { if (is_pp_target.find(id) == is_pp_target.end()) info.tail = id; } return info; } // Idempotency check: has *any* svaba_postprocess @PG line (including // uniquified variants svaba_postprocess.1, .2, ...) already been stamped // onto this header? Used to auto-skip the dedup/reheader phase on // re-runs so `scripts/svaba_postprocess.sh` is safely rerunnable. // // Matches both the bare "svaba_postprocess" ID emitted on the first run // and the "svaba_postprocess." variants uniquifyId() would produce if // the user deliberately runs postprocess twice in a row. Either form // means "dedup+PG-stamp has already happened at least once." bool hasSvabaPostprocessPg(const std::string& hdr_text) { const PgChainInfo info = scanPgChain(hdr_text); for (const auto& id : info.ids) { if (id == "svaba_postprocess") return true; if (id.rfind("svaba_postprocess.", 0) == 0) return true; } return false; } std::string uniquifyId(const std::string& base, const std::vector& existing) { auto has = [&](const std::string& s) { return std::find(existing.begin(), existing.end(), s) != existing.end(); }; if (!has(base)) return base; for (int i = 1; i < 10000; ++i) { std::string cand = base + "." + std::to_string(i); if (!has(cand)) return cand; } return base + ".new"; // extremely unreachable fallback } // Strip tab/newline/CR from a value so it can't break @PG line structure. std::string sanitizeHeaderValue(const std::string& s) { std::string out; out.reserve(s.size()); for (char c : s) { if (c == '\t') out.push_back(' '); else if (c == '\n' || c == '\r') continue; else out.push_back(c); } return out; } // Append a single @PG line to hdr_text. Does not otherwise touch the header. std::string appendSvabaPostprocessPg(const std::string& hdr_text, const std::string& cl) { const PgChainInfo info = scanPgChain(hdr_text); const std::string id = uniquifyId("svaba_postprocess", info.ids); const std::string vn = SVABA_VERSION; const std::string cl_s = sanitizeHeaderValue(cl); std::string line = "@PG\tID:" + id + "\tPN:svaba\tVN:" + vn + "\tCL:" + cl_s; if (!info.tail.empty()) line += "\tPP:" + info.tail; line.push_back('\n'); std::string out = hdr_text; if (!out.empty() && out.back() != '\n') out.push_back('\n'); out += line; return out; } // Build a new SeqLib::BamHeader with our @PG line appended onto `src`. SeqLib::BamHeader stampedHeader(const SeqLib::BamHeader& src, const std::string& cl) { return SeqLib::BamHeader(appendSvabaPostprocessPg(src.AsString(), cl)); } // Read the header of `bam` (without walking records) and return it as a // SeqLib::BamHeader. The BamReader is closed on scope exit. SeqLib::BamHeader readHeaderOnly(const std::string& bam) { SeqLib::BamReader r; if (!r.Open(bam)) throw std::runtime_error("reheader: cannot open " + bam); return r.Header(); } // Return true iff the BAM's header declares it coordinate-sorted via an // @HD line with SO:coordinate. Samtools, SeqLib, and bwa all emit that // form when producing a sorted BAM, so a simple substring check on the // first @HD line is sufficient. // // Used by processSuffix() to short-circuit the sort step when it would be // a no-op — lets the user rerun postprocess on already-sorted inputs // without paying the sort cost again (e.g. after a --skip-dedup rerun). // // Errors (can't open, no @HD at all, @HD without SO:) conservatively // return false so we fall through to running sort, which will produce a // correct result regardless. bool isCoordinateSorted(const std::string& bam) { try { const SeqLib::BamHeader h = readHeaderOnly(bam); const std::string text = h.AsString(); std::istringstream iss(text); std::string line; while (std::getline(iss, line)) { if (line.rfind("@HD\t", 0) == 0) { // @HD line found; check for the SO:coordinate tag. Tags are // tab-separated, any order — a plain substring search on the // line is accurate enough because "SO:coordinate" can't appear // as a substring of any other valid @HD tag value. return line.find("SO:coordinate") != std::string::npos; } // @HD, if present, is required to be the first header line. If // the first non-empty line isn't @HD we can stop looking. if (!line.empty() && line[0] == '@') break; } } catch (...) { // header unreadable — let the sort step run and produce its own // error with better context. } return false; } // Use `samtools reheader` to swap in a new header on an existing BAM. Fast // because samtools reheader streams the BGZF body as opaque blocks. We use // this for the suffixes whose pipeline doesn't already rewrite the file // (i.e. `contigs`, or any suffix when --sort-only is passed). int reheaderBamWithPg(const std::string& bam, const std::string& cl, const std::string& tag, int verbose) { const std::string tmp_hdr = bam + ".postprocess.hdr.tmp.sam"; const std::string tmp_bam = bam + ".postprocess.reheader.tmp.bam"; try { const SeqLib::BamHeader src = readHeaderOnly(bam); const std::string new_text = appendSvabaPostprocessPg(src.AsString(), cl); { std::ofstream ofs(tmp_hdr, std::ios::binary); if (!ofs) throw std::runtime_error("cannot write temp header " + tmp_hdr); ofs << new_text; if (!ofs) throw std::runtime_error("short write on " + tmp_hdr); } const std::string cmd = "samtools reheader " + shQuote(tmp_hdr) + " " + shQuote(bam) + " > " + shQuote(tmp_bam); if (verbose >= 2) logLine("[", tag, "] exec: ", cmd); const auto t0 = std::chrono::steady_clock::now(); const int rc = std::system(cmd.c_str()); const double elapsed = std::chrono::duration(std::chrono::steady_clock::now() - t0).count(); ::unlink(tmp_hdr.c_str()); if (rc != 0) { ::unlink(tmp_bam.c_str()); logLine("[", tag, "] samtools reheader FAILED (exit ", rc, ") after ", std::fixed, std::setprecision(1), elapsed, "s"); return rc; } renameOrThrow(tmp_bam, bam); if (verbose >= 1) logLine("[", tag, "] PG stamped via reheader in ", std::fixed, std::setprecision(1), elapsed, "s"); return 0; } catch (const std::exception& e) { ::unlink(tmp_hdr.c_str()); ::unlink(tmp_bam.c_str()); logLine("[", tag, "] reheader error: ", e.what()); return -1; } } // ---------- BAI indexing ---------- // Build a .bai alongside `bam`. Uses htslib directly rather than shelling out // to `samtools index` — one less fork, and we're already linked against // htslib through SeqLib. `sam_index_build(fn, 0)` emits BAI; min_shift > 0 // would emit CSI (which svaba's consumers don't expect). int indexBam(const std::string& bam, const std::string& tag, int verbose) { const auto t0 = std::chrono::steady_clock::now(); const int rc = sam_index_build(bam.c_str(), 0); const double elapsed = std::chrono::duration(std::chrono::steady_clock::now() - t0).count(); if (rc < 0) { logLine("[", tag, "] indexing FAILED (sam_index_build rc=", rc, ") after ", std::fixed, std::setprecision(2), elapsed, "s"); return rc; } if (verbose >= 1) logLine("[", tag, "] indexed (.bai) in ", std::fixed, std::setprecision(2), elapsed, "s"); return 0; } // ---------- sort step ---------- // Run samtools sort on in_bam producing out_bam. Returns process exit code. // We don't parse samtools' progress output; samtools -v isn't universally // respected and its stderr format changes. Caller emits start/end log lines // with timing. int runSort(const std::string& in_bam, const std::string& out_bam, int sort_threads, const std::string& mem, const std::string& tag, int verbose) { std::string cmd = "samtools sort -@ " + std::to_string(std::max(1, sort_threads)) + " -m " + shQuote(mem) + " -o " + shQuote(out_bam) + " " + shQuote(in_bam); if (verbose >= 2) logLine("[", tag, "] exec: ", cmd); const auto t0 = std::chrono::steady_clock::now(); const int rc = std::system(cmd.c_str()); const double elapsed = std::chrono::duration(std::chrono::steady_clock::now() - t0).count(); if (rc != 0) { logLine("[", tag, "] samtools sort FAILED (exit ", rc, ") after ", std::fixed, std::setprecision(1), elapsed, "s"); return rc; } if (verbose >= 1) logLine("[", tag, "] sort done in ", std::fixed, std::setprecision(1), elapsed, "s"); return 0; } // ---------- dedup step ---------- struct DedupStats { std::size_t in_records = 0; // records read std::size_t kept = 0; // unique (qname, flag) per locus, written out std::size_t merged = 0; // duplicates folded into an earlier record's tags std::size_t tag_edits = 0; // number of bi/bz merges that actually changed tag double seconds = 0; }; // Boundary-aware comma-token union: adds any tokens in `incoming` that aren't // already in `cur` as comma-separated atoms. Mirrors stamp_tag() in // SvabaOutputWriter.cpp so the postprocess dedup produces exactly the same // bi:Z / bz:Z strings svaba run would have written if the two contributing // windows had been merged in-memory. // // Returns true iff cur was modified. bool mergeCommaTokens(std::string& cur, std::string_view incoming) { if (incoming.empty()) return false; if (cur.empty()) { cur.assign(incoming); return true; } bool changed = false; std::size_t s = 0; while (s <= incoming.size()) { std::size_t e = incoming.find(',', s); if (e == std::string_view::npos) e = incoming.size(); std::string_view tok = incoming.substr(s, e - s); if (!tok.empty()) { bool present = false; std::size_t pos = 0; while ((pos = cur.find(tok, pos)) != std::string::npos) { const bool left_ok = (pos == 0) || cur[pos - 1] == ','; const bool right_ok = (pos + tok.size() == cur.size()) || cur[pos + tok.size()] == ','; if (left_ok && right_ok) { present = true; break; } pos += tok.size(); } if (!present) { cur.push_back(','); cur.append(tok); changed = true; } } if (e == incoming.size()) break; s = e + 1; } return changed; } // Pull `tag` off `incoming` and union it into `existing`'s `tag`. Returns // true iff the existing record's tag string actually changed. bool mergeZTagInto(SeqLib::BamRecord& existing, const SeqLib::BamRecord& incoming, const char* tag) { std::string incoming_val; if (!incoming.GetZTag(tag, incoming_val) || incoming_val.empty()) return false; std::string cur; existing.GetZTag(tag, cur); if (!mergeCommaTokens(cur, incoming_val)) return false; existing.RemoveTag(tag); existing.AddZTag(tag, cur); return true; } // Streaming dedup + tag-merge. Reads in_bam, writes out_bam, collapses exact // (qname, flag) duplicates at the same (chr, pos). Duplicates are not simply // discarded: their bi:Z and bz:Z tags (the comma-joined alt-supporting and // all-supporting contig lists) are unioned into the first record's tags // using the same boundary-aware merge that stamp_tag() in // SvabaOutputWriter.cpp uses during `svaba run`. Since a duplicate record // typically comes from an overlapping assembly window, its bi/bz may name // a different contig than the first record — dropping it would lose that // support evidence. Requires input to be coord-sorted. // // Memory: O(reads at a single locus). The per-locus index and record buffer // are cleared whenever (chr, pos) advances, so even pathological pileups // only blow up transiently. DedupStats streamDedup(const std::string& in_bam, const std::string& out_bam, const SeqLib::BamHeader& out_hdr, const std::string& tag, int threads, int verbose) { using clock = std::chrono::steady_clock; DedupStats st; const auto t0 = clock::now(); auto last = t0; const off_t in_size = fileSize(in_bam); SeqLib::BamReader r; if (!r.Open(in_bam)) throw std::runtime_error("dedup: cannot open input " + in_bam); // Enable BGZF decompression thread pool on the reader. Must happen // after Open() (where fp_ is populated) and before the first Next(). // Typical 3–5x end-to-end speedup at threads=4..8 on dense BAMs, // because without it the main thread does every BGZF block inflate // sequentially. const int io_threads = std::max(1, threads); r.SetThreads(io_threads); SeqLib::BamWriter w; if (!w.Open(out_bam)) throw std::runtime_error("dedup: cannot open output " + out_bam); // Matching BGZF compression thread pool on the write side. The // output is the expensive half of this function — every kept record // goes through deflate — so pooling here matters most. w.SetThreads(io_threads); // Use the caller-supplied header (which already carries our @PG stamp) // rather than blindly mirroring the reader's — that's how postprocess // marks its output without a separate reheader pass for dedup suffixes. w.SetHeader(out_hdr); if (!w.WriteHeader()) throw std::runtime_error("dedup: cannot write header " + out_bam); // Per-locus buffer. We must hold records in insertion order so flush // preserves the original coord-sort: same (chr, pos) records keep whatever // secondary ordering samtools sort produced (qname-stable tiebreak). // // keep_buf[i] holds the record we're emitting for the i'th unique key. // idx_by_key[key] is that buffer index, used to fold duplicate tags into // the already-buffered record. std::vector keep_buf; std::unordered_map idx_by_key; keep_buf.reserve(64); idx_by_key.reserve(64); int32_t cur_chr = -2; // -2 is a sentinel that won't match any valid ChrID int32_t cur_pos = -2; // Cached display name for cur_chr. ChrName() hits the header every call, so // we resolve it only on (rare) chromosome change, not per record. std::string cur_chr_name = "*"; // When bucket_count() drifts beyond this threshold (after a pileup locus // inflates the map), flushLocus() swaps in a fresh small map instead of // calling clear(). See comment below for why this matters. constexpr std::size_t kBucketResetThreshold = 256; // Flush buffered records for the current locus in insertion order. // // Perf note: std::unordered_map::clear() is O(bucket_count), NOT O(size) // — it walks the entire bucket array to zero out each slot, even when // the map currently has 0 elements. The bucket array grows with the // max number of entries ever held and NEVER shrinks. So after one // pileup locus (centromere, simple repeat, HLA, etc.) bloats buckets // to ~130k, every subsequent locus transition — hundreds of millions // of them — pays a full memset of that inflated array. A perf profile // showed 95% of main-thread CPU going to this single memset before // this fix landed, explaining why the BGZF thread pools were starving. // // Fix: when the bucket count has grown past kBucketResetThreshold, // swap the inflated map with a fresh small one. The inflated map's // destructor pays the O(bucket_count) cost ONCE (on pileup exit) // instead of once per locus transition. Subsequent small loci see a // small bucket array and their clear() is fast. auto flushLocus = [&]() { for (auto& kept : keep_buf) { if (!w.WriteRecord(kept)) throw std::runtime_error("dedup: WriteRecord failed on " + out_bam); } keep_buf.clear(); if (idx_by_key.bucket_count() > kBucketResetThreshold) { std::unordered_map fresh; fresh.reserve(64); idx_by_key.swap(fresh); // `fresh` (now holding the inflated map) destructs at end of this // lambda — that's where we pay the one-time big memset. } else { idx_by_key.clear(); } }; while (auto opt = r.Next()) { SeqLib::BamRecord& rec = *opt; const int32_t chr = rec.ChrID(); const int32_t pos = rec.Position(); if (chr != cur_chr || pos != cur_pos) { flushLocus(); if (chr != cur_chr) { // Resolve name once per chromosome. Unmapped (chr == -1) stays "*". cur_chr_name = (chr >= 0) ? rec.ChrName(r.Header()) : std::string("*"); } cur_chr = chr; cur_pos = pos; } // Build the dedup key: qname + '\t' + decimal flag. Decimal keeps keys // ASCII / comparable across platforms; '\t' as the separator can never // appear inside a BAM qname. std::string key = rec.Qname(); key.push_back('\t'); { char buf[16]; const int n = std::snprintf(buf, sizeof(buf), "%u", rec.AlignmentFlag()); key.append(buf, n); } auto [it, inserted] = idx_by_key.try_emplace(std::move(key), keep_buf.size()); if (inserted) { // BamRecord is move-only (shared_ptr under the hood); move // it into the buffer so we transfer the htslib pointer without // ref-bumping. keep_buf.emplace_back(std::move(rec)); ++st.kept; } else { // Duplicate at same (chr, pos). Fold its bi/bz tags into the kept copy // so we don't drop alt/contig support from the overlapping window. We // read from rec here (still alive — only moved in the other branch). SeqLib::BamRecord& existing = keep_buf[it->second]; if (mergeZTagInto(existing, rec, "bi")) ++st.tag_edits; if (mergeZTagInto(existing, rec, "bz")) ++st.tag_edits; ++st.merged; } ++st.in_records; // Progress print every 25M reads. Was 1M originally, then 5M; post // BGZF-threading + bucket-clear fix the dedup runs fast enough that // 5M was firing too often on deep BAMs. 25M gives ~a screenful of // lines for a typical 100–500M-read WGS BAM. constexpr std::size_t PROGRESS_EVERY = 25'000'000ULL; if (verbose >= 1 && (st.in_records % PROGRESS_EVERY) == 0) { const auto now = clock::now(); const double elapsed = std::chrono::duration(now - t0).count(); const double dt = std::chrono::duration(now - last).count(); const double inst_rps = dt > 0 ? static_cast(PROGRESS_EVERY) / dt : 0.0; const double avg_rps = elapsed > 0 ? st.in_records / elapsed : 0.0; last = now; // Column widths chosen so the line never shifts as counters grow: // chr: 5 chars right-padded ("chr22" max) // : // pos: left-justified, with thousands separators // (flush against colon; human chr1 max "248,956,422" = 11 chars) // locus total padded to 17 so subsequent columns stay anchored. // counts: 11 chars right-aligned with thousands separators // (fits "999,999,999" = 9 figures + 2 commas) // rps: 6 chars "%6.2f" (up to 999.99 M/s) // elapsed 8 chars "%8.1f" (up to 99999.9 s ≈ 27 hr) // MiB: 7 chars (up to 9,999,999 MiB ≈ 10 TB) auto fmtN = [](std::size_t n) { std::ostringstream oss; oss << std::setw(11) << std::right << SeqLib::AddCommas(n); return oss.str(); }; // Locus = right-padded chr + ':' + left-justified comma'd pos. // Flush to 17 chars total so " | " lands at a fixed column regardless // of chr length or position magnitude. std::ostringstream locus_oss; locus_oss << std::setw(5) << std::right << cur_chr_name << ":" << SeqLib::AddCommas(cur_pos >= 0 ? cur_pos + 1 : 0); std::string locus_str = locus_oss.str(); if (locus_str.size() < 17) locus_str.append(17 - locus_str.size(), ' '); // Input-size hint for a coarse "how far into the job" feel. A true // ETA would need bytes-read from htslib, which SeqLib doesn't expose. std::string size_hint; if (in_size > 0) { std::ostringstream oss; oss << " (input " << std::setw(7) << std::right << (in_size / (1024 * 1024)) << " MiB)"; size_hint = oss.str(); } logLine("[", tag, "] dedup: at ", locus_str, " | ", fmtN(st.in_records), " reads | ", fmtN(st.kept), " kept, ", fmtN(st.merged), " merged (", fmtN(st.tag_edits), " tag-edits) | ", std::fixed, std::setprecision(2), std::setw(6), inst_rps / 1e6, "M/s last-25M, ", std::setw(6), avg_rps / 1e6, "M/s avg, ", std::setprecision(1), std::setw(8), elapsed, "s elapsed", size_hint); } } // Final locus flush. flushLocus(); if (!w.Close()) throw std::runtime_error("dedup: writer Close failed on " + out_bam); st.seconds = std::chrono::duration(clock::now() - t0).count(); return st; } // ---------- per-suffix pipeline ---------- // Move one file to another atomically within the same filesystem, via // std::rename. Throws on failure so the caller can decide what to do. void renameOrThrow(const std::string& from, const std::string& to) { if (std::rename(from.c_str(), to.c_str()) != 0) { throw std::runtime_error("rename " + from + " -> " + to + " failed: " + std::strerror(errno)); } } bool isDedupSuffix(const std::string& s) { for (const auto& d : kDedupSuffixes) if (d == s) return true; return false; } // Run the full sort + dedup + reheader + index pipeline for a single suffix. // Safe to invoke from its own thread. Never throws out — caller joins; any // failure is logged and the pipeline aborts for this suffix only. // // End state on success: the file at ${id}.${suffix}.bam is the final output, // coord-sorted, dedup-collapsed (for dedup-eligible suffixes), @PG-stamped, // and accompanied by a ${id}.${suffix}.bam.bai index. All intermediate // filenames use a .postprocess.*.tmp.bam suffix so anything left behind on // failure is obviously transient and trivially cleanable. // do_finalize controls whether the dedup + reheader + index phase runs. // Phase-splitting the driver lets sort run in parallel across suffixes // (narrow thread budget each) and dedup run serially with the full thread // budget, which is what the user wants since htslib BGZF pools are // per-file — sharing them across concurrent workers oversubscribes. void processSuffix(const std::string& id, const std::string& suffix, int per_job_threads, const std::string& mem, bool do_sort, bool do_dedup, bool do_finalize, const std::string& cl, int verbose) { const std::string bam = id + "." + suffix + ".bam"; // Intermediate temp files. Named so there's no ambiguity about the step // that produced them — anything matching .postprocess.*.tmp.bam is safe // to remove after the run is done. const std::string sort_tmp = id + "." + suffix + ".postprocess.sort.tmp.bam"; const std::string dedup_tmp = id + "." + suffix + ".postprocess.dedup.tmp.bam"; auto cleanup_tmps = [&]() { ::unlink(sort_tmp.c_str()); ::unlink(dedup_tmp.c_str()); }; if (!fileExists(bam)) { logLine("[", suffix, "] skipping: ", bam, " not found"); return; } bool pg_stamped = false; try { // --- Sort ----------------------------------------------------------- // Skip the sort step if the BAM already declares itself coordinate- // sorted. Cheap O(header) check; turns a repeat postprocess run on // an already-postprocessed BAM into an effectively instant no-op // for this suffix. Lets users pass --skip-dedup at the shell layer // (or --sort-only from the C++ CLI) and rerun without paying 30+s // per suffix for a redundant sort. if (do_sort && isCoordinateSorted(bam)) { logLine("[", suffix, "] already coordinate-sorted (per @HD SO:coordinate); " "skipping sort"); } else if (do_sort) { const int rc = runSort(bam, sort_tmp, per_job_threads, mem, suffix, verbose); if (rc != 0) throw std::runtime_error("samtools sort exited " + std::to_string(rc)); renameOrThrow(sort_tmp, bam); } if (!do_finalize) { // Caller wants sort-only (the parallel Phase 1 of the driver); leave // dedup + reheader + index to the caller's Phase 2 invocation. This // split exists so Phase 2 can run serially with the full thread // budget dedicated to BGZF read/write pooling per BAM, instead of // fragmenting threads across concurrent workers. return; } // Read the current header ONCE and reuse it for both the PG-idempotency // check and (if we do run dedup) the stamped output header. Avoids a // second BAM open just to look up PG state. const SeqLib::BamHeader existing_hdr = readHeaderOnly(bam); const std::string existing_txt = existing_hdr.AsString(); const bool already_postprocessed = hasSvabaPostprocessPg(existing_txt); // --- Dedup (also stamps PG on the way) ------------------------------ if (do_dedup && isDedupSuffix(suffix) && !already_postprocessed) { if (verbose >= 1) logLine("[", suffix, "] starting dedup on ", bam); // Append our @PG line to the existing header text, then hand the // stamped header to the writer. This is free — dedup is already // rewriting the file, so we avoid a separate reheader pass. const SeqLib::BamHeader stamped = SeqLib::BamHeader(appendSvabaPostprocessPg(existing_txt, cl)); const DedupStats ds = streamDedup(bam, dedup_tmp, stamped, suffix, per_job_threads, // BGZF read/write pool size verbose); renameOrThrow(dedup_tmp, bam); pg_stamped = true; logLine("[", suffix, "] dedup complete: ", ds.in_records, " in, ", ds.kept, " kept, ", ds.merged, " merged (", (ds.in_records ? 100.0 * ds.merged / static_cast(ds.in_records) : 0.0), "% dup, ", ds.tag_edits, " bi/bz tag-edits) in ", std::fixed, std::setprecision(1), ds.seconds, "s"); } else if (do_dedup && isDedupSuffix(suffix) && already_postprocessed) { // BAM has already been through svaba_postprocess. Skip the expensive // dedup rewrite AND the reheader below — PG is already stamped. logLine("[", suffix, "] already has svaba_postprocess @PG in header; " "skipping dedup (rerun-safe no-op)"); pg_stamped = true; } else if (do_dedup && !isDedupSuffix(suffix)) { if (verbose >= 2) logLine("[", suffix, "] dedup skipped (not a dedup suffix)"); } // --- Reheader to stamp @PG for any suffix that didn't go through ----- // --- dedup (e.g. `contigs`, or anything under --sort-only). Also ----- // --- skipped when `already_postprocessed` set `pg_stamped` above ----- // --- (nothing to add to an already-PG-stamped header). ----- if (!pg_stamped && !already_postprocessed) { const int rc = reheaderBamWithPg(bam, cl, suffix, verbose); if (rc != 0) throw std::runtime_error("reheader failed with rc " + std::to_string(rc)); } // --- Index ---------------------------------------------------------- // Index last so the .bai matches the BGZF offsets of the final file. // Any earlier index would be invalidated by the dedup/reheader rewrite. // Always runs — even on a rerun, the .bai may be missing or stale // (e.g. if the user renamed the BAM after a previous run). Cheap op. (void) indexBam(bam, suffix, verbose); } catch (const std::exception& e) { logLine("[", suffix, "] ERROR: ", e.what()); cleanup_tmps(); } } } // namespace void runPostprocess(int argc, char** argv) { // Reconstruct the invocation string for the @PG CL: tag BEFORE parseOpts // consumes argv via getopt_long. We prepend "svaba" so the CL in the // final BAM headers reads as the user actually typed it (the dispatch in // svaba.cpp strips the "svaba" token before calling us). std::string cl = "svaba"; for (int i = 0; i < argc; ++i) { cl.push_back(' '); cl += argv[i]; } const Opts o = parseOpts(argc, argv); // Pick active suffixes — only the ones whose BAM actually exists. No point // reserving a thread budget slice for a missing file. std::vector active; for (const auto& s : kAllSuffixes) { const std::string bam = o.id + "." + s + ".bam"; if (fileExists(bam)) active.push_back(s); } if (active.empty()) { std::cerr << "No BAMs found for ID '" << o.id << "' (checked suffixes: "; for (size_t i = 0; i < kAllSuffixes.size(); ++i) { if (i) std::cerr << ", "; std::cerr << kAllSuffixes[i]; } std::cerr << ")" << std::endl; return; } // Split the thread budget across concurrent suffix jobs, matching the // shell script's behavior: floor(THREADS / n_active), min 1. If threads < // n_active, cap concurrency to threads so we don't oversubscribe. const int n_active = static_cast(active.size()); const int max_parallel = std::min(o.threads, n_active); const int per_job_sort_t = std::max(1, o.threads / n_active); if (o.verbose >= 1) { std::ostringstream act; for (size_t i = 0; i < active.size(); ++i) { if (i) act << " "; act << active[i]; } std::cerr << "svaba postprocess: id=" << o.id << " threads=" << o.threads << " mem=" << o.mem << " parallel=" << max_parallel << " per-job-sort-threads=" << per_job_sort_t << " sort=" << (o.dedup_only ? "off" : "on") << " dedup=" << (o.sort_only ? "off" : "on") << "\n active: " << act.str() << std::endl; } // One-shot note so users don't chase the htslib "[E::idx_find_and_load]" // lines that will appear below. Those come from SeqLib::BamReader::Open // (called by streamDedup and by readHeaderOnly inside reheaderBamWithPg) // eagerly trying to load a .bai that doesn't exist yet — we sort first, // then rewrite (dedup or reheader), then build the .bai at the very end. // Every read path here is sequential, so the missing index is irrelevant // to correctness. We print this once, before spawning workers, and leave // htslib's own logging alone so genuine warnings (corrupt records, // permission errors, out-of-space, etc.) still surface visibly. std::cerr << "svaba postprocess: NOTE — the following " "\"[E::idx_find_and_load] Could not retrieve index file ...\" " "lines from htslib are expected and can be ignored. " ".bai files are built as the final step of each suffix's " "pipeline, not before the read passes that produce them." << std::endl; // Two-phase execution: // // Phase 1 (PARALLEL): run samtools sort across all active suffixes // concurrently. Sort is disk+CPU bound and perfectly parallelizable // across files, so each worker gets `per_job_sort_t = o.threads / // n_active` threads — total thread usage ≈ o.threads. // // Phase 2 (SERIAL): dedup + reheader + index, one suffix at a time, // each with the FULL `o.threads` budget for its BGZF read/write // pool. Running the dedup phase in parallel would fragment the // thread budget (4 suffixes × 2 threads/pool = 2× oversubscription // once you count read+write pools per suffix), yielding worse wall // time than serial-with-wide-pools because BGZF parallelism has // diminishing returns — 8 threads get ~5x vs single-thread, but 4 // parallel workers with 2 threads each get only 4 × 1.8x / 4 ≈ 1.8x // effective per-BAM. // // Each phase is itself idempotent: Phase 1 skips sort when the BAM is // already @HD SO:coordinate; Phase 2 skips dedup when the header // already carries a `svaba_postprocess` @PG line. Rerunning a // completed postprocess is essentially instant. // --- Phase 1: parallel sort ---------------------------------------- if (!o.dedup_only) { if (o.verbose >= 1) std::cerr << "svaba postprocess: phase 1/2 — parallel sort across " << active.size() << " suffixes (" << per_job_sort_t << " threads each)" << std::endl; std::vector workers; workers.reserve(active.size()); for (const auto& suffix : active) { workers.emplace_back( processSuffix, o.id, suffix, per_job_sort_t, o.mem, /*do_sort=*/ true, /*do_dedup=*/ false, // deferred to phase 2 /*do_finalize=*/false, // phase 1 exits right after sort cl, o.verbose); } for (auto& t : workers) t.join(); } // --- Phase 2: serial dedup + reheader + index ---------------------- // `o.threads` per invocation — one BAM at a time gets the whole budget. if (o.verbose >= 1) std::cerr << "svaba postprocess: phase 2/2 — serial dedup+reheader+index" << " (" << o.threads << " threads per BAM)" << std::endl; for (const auto& suffix : active) { processSuffix( o.id, suffix, o.threads, // full BGZF pool budget o.mem, /*do_sort=*/ false, // already sorted in phase 1 (or skipped by --dedup-only) /*do_dedup=*/ !o.sort_only, /*do_finalize=*/true, cl, o.verbose); } if (o.verbose >= 1) std::cerr << "svaba postprocess: all suffixes done (sorted" << (o.sort_only ? "" : ", deduped where applicable") << ", PG-stamped, indexed)" << std::endl; } ================================================ FILE: src/svaba/SvabaPostprocess.h ================================================ #pragma once // Post-process svaba output BAMs: coordinate-sort via samtools, then run a // native streaming dedup on the suffixes that can accumulate exact duplicate // records from overlapping assembly windows (weird, corrected, discordant). // // Replaces the slow path of sort_output.sh, which went through // `samtools view | awk | samtools view` — that pipeline decompresses every // record to SAM text, maintains a large awk hash, and recompresses on the // way out. This module keeps everything in native htslib / SeqLib records // and resets the dedup set at every locus boundary, so memory is O(reads // per locus) and throughput is close to what the disk+decoder can sustain. // // Scope: // - Sort → shell out to `samtools sort` (already highly tuned; htslib // doesn't expose its sort as a library call). // - Dedup → native streaming, (qname, flag) keyed, per-locus reset. // Stamps an @PG line onto the output header as a free // side-effect of rewriting the file. // - Reheader → for suffixes that don't go through dedup (e.g. `contigs`, // or anything under --sort-only), shell out to // `samtools reheader` to stamp the same @PG line. Streams the // BGZF body as opaque blocks — cheap. // - Index → native `sam_index_build` (htslib) producing a .bai // alongside every final BAM. // - Per-suffix parallelism via std::thread; suffixes run independently. // // End state: ${ID}.${suffix}.bam is the single authoritative output per // suffix — sorted, deduped (where applicable), @PG-stamped, and indexed. // Intermediate files use a .postprocess.*.tmp.bam suffix and are cleaned up // on both success (rename-over) and failure (best-effort unlink). // // Explicitly NOT in scope here (still in sort_output.sh): // - thread-BAM merging (pre-svaba-postprocess step) // - SPLIT_BY_SOURCE qname-prefix demultiplexing // // CLI: svaba postprocess -i [-t THREADS] [-m MEM] [-v V] // [--sort-only | --dedup-only] void runPostprocess(int argc, char** argv); ================================================ FILE: src/svaba/SvabaRead.cpp ================================================ #include "SvabaRead.h" #include // bam_get_seq, bam_seqi svabaRead::svabaRead() = default; void svabaRead::AddR2C(const std::string& contig_name, const r2c& r) { auto it = m_r2c.find(contig_name); if(it != m_r2c.end()) it->second = r; else m_r2c.insert({contig_name, r}); // add as a tag to the read //TODO } r2c svabaRead::GetR2C(const std::string& contig_name) const { //assert(m_r2c); R2CMap::const_iterator ff = m_r2c.find(contig_name); assert(ff != m_r2c.end()); return m_r2c.find(contig_name)->second; } int svabaRead::CorrectedSeqLength() const { if (seq_corrected.length()) return seq_corrected.length(); else return Length(); } std::ostream& operator<<(std::ostream& out, const r2c& a) { out << "[" << a.start_on_contig << "," << a.end_on_contig << "] -- " << "[" << a.start_on_read << "]" << a.cig; return out; } void svabaRead::QualityTrimRead() { int32_t startpoint = 0, endpoint = 0; QualityTrimmedSequence(3, startpoint, endpoint); int32_t new_len = endpoint - startpoint; if (endpoint != -1 && new_len < Length() && new_len > 0 && new_len - startpoint >= 0 && startpoint + new_len <= Length()) { try { SetCorrectedSeq(Sequence().substr(startpoint, new_len)); } catch (...) { std::cerr << "Subsequence failure with sequence of length " << Sequence().length() << " and startpoint " << startpoint << " endpoint " << endpoint << " newlen " << new_len << std::endl; } } else { SetCorrectedSeq(Sequence()); // copies the sequence into private "seq" char } // remove the HTSlib version of qual and sequence // since we store the trimmed sequence in svabaRead //SetSequence(std::string()); } svabaRead::svabaRead(const SeqLib::BamRecord& r, std::string_view prefix) : SeqLib::BamRecord() // base class ctor will init `b` to nullptr { // bam_dup1 will allocate-and-copy a new bam1_t for us: bam1_t* dup = bam_dup1(r.raw()); if (!dup) throw std::runtime_error("svabaRead: failed to duplicate BamRecord"); // wrap it in your shared-ptr with the proper deleter b = SeqPointer(dup, SeqLib::Bam1Deleter()); // now copy over your svabaRead-specific prefix assert(prefix.size() >= 4); p.assign(prefix); } std::string svabaRead::Prefix() const { assert(p.c_str()[0]=='t' || p.c_str()[0] == 'n'); return p; //std::string(p, 4); } std::string svabaRead::CorrectedSeq() const { if (!seq_corrected.length()) return Sequence(); else return seq_corrected; } bool svabaRead::CorrectedSeqChanged() const { // If seq_corrected was never set, it's empty → CorrectedSeq() would // return Sequence(), so by definition nothing changed. if (seq_corrected.empty()) return false; // Fast path: length mismatch means quality trimming shortened the read. const int32_t bam_len = Length(); // b->core.l_qseq if (static_cast(seq_corrected.size()) != bam_len) return true; // Same length: compare seq_corrected against the BAM's 4-bit encoding // character by character, without allocating a Sequence() string. const uint8_t* seq_enc = bam_get_seq(raw()); for (int32_t i = 0; i < bam_len; ++i) { if (seq_corrected[i] != BASES[bam_seqi(seq_enc, i)]) return true; } return false; } void svabaRead::SetCorrectedSeq(std::string_view nseq) { seq_corrected = nseq; } std::string svabaRead::UniqueName() const { return(p + "_" + std::to_string(AlignmentFlag()) + "_" + Qname()); } ================================================ FILE: src/svaba/SvabaRead.h ================================================ #pragma once #include "SeqLib/BamRecord.h" #include #include #include using SeqLib::BamRecordPtr; using SeqLib::BamRecordPtrVector; /** Store information about a read to contig alignment */ struct r2c { int32_t start_on_contig = 0; // start pos on contig (from r.Position()) int32_t end_on_contig = 0; // end pos on contig (from r.PositionEnd()) int32_t start_on_read = 0; // start pos on read (from r.AlignmentPosition()) int32_t end_on_read = 0; // end pos on read (from r.AlignmentPosition()) bool rc = false; // reverse complement wrt contig? SeqLib::Cigar cig; // cigar of read to contig bool supports_var = false; // does this support a variant? bool is_split = false; // is this a split read? int left_or_right = 0; //-1 read aligns on left of contig, 1 on right bool supports_discordant = false; // true if this is part of a discordant pair that supports the break // SvABA2.0: NM tag on the r2c alignment (edit distance to contig). // -1 sentinel means "not set" (legacy paths that filled r2c without // going through AddAlignment). Surfaced in alignments.txt.gz so the // human-readable dump shows per-read edit distance to the contig. int32_t nm = -1; void AddAlignment (const BamRecordPtr& b) { start_on_contig = b->Position(); end_on_contig = b->PositionEnd(); start_on_read = b->AlignmentPosition(); cig = b->GetCigar(); int _nm = 0; if (b->GetIntTag("NM", _nm)) nm = _nm; } friend std::ostream& operator<<(std::ostream& out, const r2c& a); }; typedef std::unordered_map R2CMap; class svabaRead; typedef std::shared_ptr svabaReadPtr; typedef std::vector svabaReadPtrVector; class svabaRead : public SeqLib::BamRecord { public: svabaRead(); svabaRead(const SeqLib::BamRecord& r, std::string_view prefix); // Delete the copy constructor svabaRead(const svabaRead&) = delete; // Optionally also delete copy assignment svabaRead& operator=(const svabaRead&) = delete; // Still allow move operations: svabaRead(svabaRead&&) = default; svabaRead& operator=(svabaRead&&) = default; std::string CorrectedSeq() const; /// True if BFC correction or quality trimming changed the read sequence /// relative to the original BAM record. When false, the original BAM's /// CIGAR/NM is a valid native alignment (assuming the same aligner). /// Compares the stored seq_corrected against the BAM 4-bit encoding /// without constructing a second string for the original. bool CorrectedSeqChanged() const; std::string Prefix() const; void SetPrefix(const std::string_view pref) { p = pref; } void SetCorrectedSeq(const std::string_view nseq); std::string UniqueName() const; int GetDD() const { return dd; } void SetDD(int d) { dd = d; } bool Tumor() const { return p[0] == 't'; } int SeqLength() const; void AddR2C(const std::string& contig_name, const r2c& r); r2c GetR2C(const std::string& contig_name) const; /// Does this read have any r2c alignments? bool HasR2C() const { return !m_r2c.empty(); } /// Trim the read based on quality score and store seq in char void QualityTrimRead(); int CorrectedSeqLength() const; // discordant read status // < 0 is bad discordant read (see DiscordantRealigner.h) // == 0 not discordant // 1 = good int dd = 0; // SvABA2.0: post-BFC re-alignment of the *corrected* read sequence to the // reference, populated by SvabaRegionProcessor before assembly. The point // is to give BreakPoint::splitCoverage's "r2c better than native" gate // an apples-to-apples comparison: both sides use the corrected read // sequence and the same BWA-MEM parameters that svaba uses internally, // rather than mixing svaba-corrected r2c against the input BAM's // pre-correction CIGAR/NM (which was the source of an asymmetric gate // letting reads with mirror r2c indels through as variant supporters). // // Sentinel: corrected_native_nm == -1 means "not populated" (e.g. read // with to_assemble == false, or empty corrected sequence). In that case // splitCoverage falls back to GetCigar()/GetIntTag("NM",...) on the // original BAM record. SeqLib::Cigar corrected_native_cig; int32_t corrected_native_nm = -1; friend class svabaBamWalker; bool to_assemble = true; private: std::string p; // prefix for file ID (e.g. t001) bool train = false; std::string seq_corrected; // quality trimmed and/or error corrected // store the r2c alignment information. key is contig name R2CMap m_r2c; }; typedef std::vector svabaReadVector; ================================================ FILE: src/svaba/SvabaRegionProcessor.cpp ================================================ #include "SvabaRegionProcessor.h" extern "C" { #include "fml.h" } #include // For std::setw and std::right #include // For std::ostringstream #include #include // For memset #include "SvabaDebug.h" #include "SvabaUtils.h" #include "SvabaOutputWriter.h" #include "ContigAlignmentScore.h" #include "SeqLib/BWAAligner.h" #include "SeqLib/GenomicRegion.h" #include "SeqLib/GenomicRegionCollection.h" #include "SeqLib/BFC.h" #include "SeqLib/BamRecord.h" #include "SvabaRead.h" #include "SvabaAssemblerEngine.h" using SeqLib::GenomicRegion; using SeqLib::GRC; using SeqLib::AddCommas; using SeqLib::BFC; using SeqLib::BamRecordVector; using SeqLib::CigarMap; using SeqLib::UnalignedSequenceVector; using SeqLib::BWAIndexPtr; using SeqLib::BWAAligner; using SeqLib::BWAIndex; using SeqLib::BamRecordPtr; using SeqLib::BamRecordPtrVector; using std::vector; using std::unordered_map; using std::unordered_set; using std::string; #include "SvabaAssemblerConfig.h" // --------------------------------------------------------------------------- // Alt-contig demotion: after BWA aligns a contig, prefer standard-chromosome // placements over alt/decoy when the alignment quality is comparable. // // BWA-MEM sometimes places a contig fragment on an alt contig (e.g. // chr11_JH159136v1_alt) when chr11 proper has an equally good alignment. // If the alt later gets blacklisted, the real breakpoint is lost. Fix: for // each primary/supplementary fragment on a non-standard chr, check if a // secondary on a standard chr covers the same query range with comparable // AS. If so, swap their SAM flags so the standard-chr placement becomes // the active fragment and the alt-chr one becomes a secondary. // --------------------------------------------------------------------------- namespace { // Query interval [start, end) consumed by an alignment, in contig // coordinates. Uses the CIGAR to find leading/trailing soft-clip // (hardclip=false so all clips are soft). inline std::pair queryInterval(const SeqLib::BamRecord& r) { const auto cig = r.GetCigar(); int lead_clip = 0, trail_clip = 0, qlen = r.Length(); if (cig.size() > 0) { if (cig.front().Type() == 'S') lead_clip = cig.front().Length(); if (cig.size() > 1 && cig.back().Type() == 'S') trail_clip = cig.back().Length(); } return {lead_clip, qlen - trail_clip}; } // Reciprocal overlap fraction between two intervals. inline double reciprocalOverlap(std::pair a, std::pair b) { int ov = std::max(0, std::min(a.second, b.second) - std::max(a.first, b.first)); int la = a.second - a.first, lb = b.second - b.first; if (la <= 0 || lb <= 0) return 0.0; return std::min(static_cast(ov) / la, static_cast(ov) / lb); } // Number of swaps performed (for logging). size_t preferStandardChromosomes(BamRecordPtrVector& alns, int maxStdChr) { if (maxStdChr < 0) return 0; // --non-human: no preference size_t n_swaps = 0; for (size_t i = 0; i < alns.size(); ++i) { auto& aln = alns[i]; if (!aln || !aln->MappedFlag()) continue; if (aln->SecondaryFlag()) continue; // only consider primary/supp if (aln->ChrID() <= maxStdChr) continue; // already on a standard chr int32_t orig_as = 0; aln->GetIntTag("AS", orig_as); auto orig_qi = queryInterval(*aln); size_t best_j = SIZE_MAX; int32_t best_as = -1; for (size_t j = 0; j < alns.size(); ++j) { auto& sec = alns[j]; if (!sec || !sec->MappedFlag()) continue; if (!sec->SecondaryFlag()) continue; // must be a secondary if (sec->ChrID() > maxStdChr) continue; // also non-standard int32_t sec_as = 0; sec->GetIntTag("AS", sec_as); // Must be within 5% of the non-standard alignment's AS if (orig_as > 0 && sec_as < static_cast(orig_as * 0.95)) continue; // Must cover similar query range (>= 80% reciprocal overlap) if (reciprocalOverlap(orig_qi, queryInterval(*sec)) < 0.80) continue; if (sec_as > best_as) { best_as = sec_as; best_j = j; } } if (best_j != SIZE_MAX) { auto& promoted = alns[best_j]; SVABA_TRACE(aln->Qname(), "ALT_DEMOTION: chr" << aln->ChrID() << " AS=" << orig_as << " -> chr" << promoted->ChrID() << " AS=" << best_as << (aln->SupplementaryFlag() ? " (supp)" : " (pri)")); // Swap SAM flags: demote non-standard to secondary, // promote standard to the role the non-standard held. bool was_supp = aln->SupplementaryFlag(); // Demote: secondary=1, supplementary=0 aln->raw()->core.flag |= BAM_FSECONDARY; aln->raw()->core.flag &= ~BAM_FSUPPLEMENTARY; // Promote: secondary=0, supplementary=was_supp promoted->raw()->core.flag &= ~BAM_FSECONDARY; if (was_supp) promoted->raw()->core.flag |= BAM_FSUPPLEMENTARY; else promoted->raw()->core.flag &= ~BAM_FSUPPLEMENTARY; ++n_swaps; } } return n_swaps; } } // anon namespace SvabaRegionProcessor::SvabaRegionProcessor(SvabaSharedConfig& sh_cf) : sc(sh_cf) { } GRC SvabaRegionProcessor::runMateCollectionLoop(const GenomicRegion& region, svabaThreadUnit& stu) { // store the newly found bad mate regions GRC this_bad_mate_regions; MateRegionVector all_somatic_mate_regions; // add the origional, don't want to double back all_somatic_mate_regions.add(MateRegion(region.chr, region.pos1, region.pos2)); // collect together mate regions from control MateRegionVector normal_mate_regions; for (const auto& [key, walker] : stu.walkers) { if (!key.empty() && key[0] == 'n') { normal_mate_regions.Concat(walker->mate_regions); } } normal_mate_regions.MergeOverlappingIntervals(); normal_mate_regions.CreateTreeMap(); // get the mates from somatic 3+ mate regions // that don't overlap with normal mate region MateRegionVector somatic_mate_regions; for (const auto& [key, walker] : stu.walkers) { if (!key.empty() && key[0] == 't') { for (const auto& i : walker->mate_regions) { if (i.count >= sc.opts.mateLookupMin && !normal_mate_regions.CountOverlaps(i) && //!stu.badd.CountOverlaps(i) && //TODO - don't use this anymore (sc.blacklist.empty() || !sc.blacklist.CountOverlaps(i))) { somatic_mate_regions.add(i); } } } } somatic_mate_regions.MergeOverlappingIntervals(); // if none, then we're done if (somatic_mate_regions.size() == 0) return GRC(); // print out to log for (auto& i : somatic_mate_regions) sc.logger.log(sc.opts.verbose > 1, sc.opts.verbose_log, "......mate region ", i.ToString(sc.header) , " case read count that triggered lookup: ", i.count); // convert MateRegionVector to GRC GRC somatic_mate_region_collection; for (const auto& s : somatic_mate_regions) somatic_mate_region_collection.add( GenomicRegion(s.chr, s.pos1, s.pos2, s.strand)); somatic_mate_region_collection.MergeOverlappingIntervals(); somatic_mate_region_collection.CreateTreeMap(); // convert MateRegionVector to GRC GRC gg; for (auto& s : somatic_mate_regions) gg.add(GenomicRegion(s.chr, s.pos1, s.pos2, s.strand)); // collect the reads for this round for (auto& [key, walker] : stu.walkers) { const int before = walker->reads.size(); walker->m_limit = sc.opts.mate_region_lookup_limit; if (!walker->SetRegions(gg)) continue; walker->get_coverage = false; walker->get_mate_regions = false; walker->mate_regions.clear(); auto prior_end = walker->reads.end(); GRC bad = walker->readBam(stu); // reset walker->m_limit = sc.opts.weird_read_limit; walker->get_coverage = true; walker->get_mate_regions = true; //stu.badd.Concat(bad); //stu.badd.MergeOverlappingIntervals(); //stu.badd.CreateTreeMap(); assert(!key.empty()); auto& count = (key[0] == 't') ? stu.st.mate_read_count.first : stu.st.mate_read_count.second; count += walker->reads.size() - before; } sc.logger.log(sc.opts.verbose > 1, sc.opts.verbose_log, "......Mate region reads: : <", stu.st.mate_read_count.first, ",", stu.st.mate_read_count.second, ">"); return somatic_mate_region_collection; } bool SvabaRegionProcessor::process(const SeqLib::GenomicRegion& region, svabaThreadUnit& unit, size_t threadId) { // SvABA2.0: are any dump-reads BAMs going to be written? Used to skip // bi:Z and bz:Z tag bookkeeping on the read hot path when no dump // output is requested — the tags feed only the weird/discordant/ // corrected BAM writers in SvabaOutputWriter, so under the default // production command line (no --dump-reads) the tag work is pure // waste. Declared at function scope so both the r2c-alignment // (bz:Z) and BP-finalization (bi:Z) blocks see it. const bool need_read_tags = sc.opts.dump_weird_reads || // compile-time constexpr sc.opts.dump_discordant_reads || // both flip together under sc.opts.dump_corrected_reads; // --dump-reads // count for this unit unit.processed_count++; sc.total_regions_done++; if (sc.total_regions_done % 250 == 0) { std::ostringstream msg; msg << "...processing " << std::right << std::setw(5) << SeqLib::AddCommas(sc.total_regions_done) << " of " << std::right << std::setw(6) << SeqLib::AddCommas(sc.total_regions_to_process) << " for thread " << std::right << std::setw(2) << unit.threadId << " for region " << region; sc.logger.log(true, true, msg.str()); } sc.logger.log(sc.opts.verbose > 1, sc.opts.verbose_log, "===Running region ", region.ToString(sc.header), " on thread ", unit.threadId); // start a new timer unit.st = svabaUtils::svabaTimer(); unit.st.gr = region; unit.st.start(); // get the reference sequence of the local region string lregion; try { lregion = unit.ref_genome->QueryRegion(sc.header.IDtoName(region.chr), region.pos1, region.pos2); } catch (...) { std::cerr << " Caught exception for lregion with reg " << region.ToString(sc.header); lregion = ""; } std::vector> repeats_to_avoid = svabaUtils::find_repeats(lregion, 20,10); SeqLib::GRC local_blacklist; for (auto& [key, walker] : unit.walkers) { for (const auto& [st, sp] : repeats_to_avoid) { walker->local_blacklist.add(GenomicRegion(region.chr, region.pos1 + st, region.pos1 + sp)); } walker->local_blacklist.MergeOverlappingIntervals(); walker->local_blacklist.CreateTreeMap(); } if (repeats_to_avoid.size()) sc.logger.log(sc.opts.verbose > 1, sc.opts.verbose_log, " found ref repeats at"); //debug for (const auto& [st,sp] : repeats_to_avoid) { sc.logger.log(sc.opts.verbose > 1, sc.opts.verbose_log, " ",region.ChrName(sc.header), ":",region.pos1 + st, "-",region.pos1+sp); } // holder for Bam: (read : cigar) unordered_map cigmap; // loop all of the BAMs (walkers) CountPair read_counts = {0,0}; //tumor, normal for (auto& [key, walker] : unit.walkers) { // Set regions based on command-line region or file-defined intervals if (!region.IsEmpty()) walker->SetRegion(region); else if (!sc.file_regions.empty()) walker->SetRegions(sc.file_regions); // else: walk entire BAM (default) sc.logger.log(sc.opts.verbose > 1, sc.opts.verbose_log, "---running svabaBamWalker", *walker); // reset the walker params walker->m_limit = sc.opts.weird_read_limit; walker->get_coverage = true; walker->get_mate_regions = true; SeqLib::GRC bad = walker->readBam(unit); //unit.badd.Concat(bad); sc.logger.log(sc.opts.verbose > 3, false, "...finished reading, found ", bad.size(), " bad regions"); // TODO - right now we don't do anything with these bad regions //unit.badd.MergeOverlappingIntervals(); //unit.badd.CreateTreeMap(); // Update read count based on sample type auto& count = (key[0] == 't') ? unit.st.weird_read_count.first : unit.st.weird_read_count.second; count += walker->reads.size(); // Store CIGARs cigmap[key] = walker->cigmap; //debug // for (const auto& [walker_name,cm] : cigmap) { // for (const auto& [key, ccount] : cm) { // std::cerr << "Walker: " << walker_name << " key: " << // key << " count " << ccount << std::endl; // } // } } //end BAM loop sc.logger.log(sc.opts.verbose > 1, sc.opts.verbose_log, "...main region reads ", "<", unit.st.weird_read_count.first, ",", unit.st.weird_read_count.second, ">"); // adjust counts and timer unit.st.stop("r"); // get the mate reads, if this is local assembly and has insert-size distro GRC mate_regions_for_native; if (!region.IsEmpty()) { mate_regions_for_native = runMateCollectionLoop(region, unit); unit.st.stop("m"); } // do the discordant read clustering sc.logger.log(sc.opts.verbose > 1, false, "...discordant read clustering"); // tag the reads by discordant status svabaReadPtrVector all_discordant_reads; for (auto& [_, walker] : unit.walkers) { //walker->RealignDiscordants(unit); for (auto& r : walker->reads) { if (r->dd > 0) all_discordant_reads.push_back(r); } } unit.st.dc_read_count = all_discordant_reads.size(); // do the discordant read clustering across BAMs DiscordantClusterMap dmap = DiscordantCluster::clusterReads(all_discordant_reads, region, sc.header); all_discordant_reads.clear(); unit.st.dc_cluster_count = dmap.size(); // add the discordant clusters to the svabathreadunit for writing later unit.m_disc.insert(dmap.begin(), dmap.end()); // for dumping all reads (weird BAM and/or discordant BAM) if (sc.opts.dump_weird_reads || sc.opts.dump_discordant_reads) { for (auto& [_, dc] : dmap) { dc.labelReads(); // add the discordantcluster label to the read } for (const auto& [_, walker] : unit.walkers) { unit.all_weird_reads.insert(unit.all_weird_reads.end(), walker->reads.begin(), walker->reads.end()); } } // do the discordant read clustering sc.logger.log(sc.opts.verbose > 1, false, "...error correcting"); // do kmer correction — pooled across all walkers (tumor + normal) // so BFC sees the combined k-mer spectrum for better error/signal // discrimination. Matches old svaba behavior. Single Train + single // ErrorCorrect instead of per-walker, halving the BFC cost. // // The BFC object lives on the thread unit (unit.pooled_bfc) so its // k-mer hash table (1M sub-tables at l_pre=20) persists across // regions. On the first Train(), fml_count allocates all 1M tables; // on subsequent calls, fml_count_into clears and reuses them via // kh_clear (memset of flags), eliminating ~2M malloc/free calls // per region — measured at 47% of worker time before this fix. if (sc.opts.ecCorrectType == "f") { // Lazy-init the per-thread BFC on first use if (!unit.pooled_bfc) unit.pooled_bfc = std::make_unique(); BFC& bfc = *unit.pooled_bfc; // Phase 1: pool all training reads + weird reads for (auto& [_, walker] : unit.walkers) { for (const auto& u : walker->train_reads) bfc.AddSequence(u.Seq, "", u.Name); for (const auto& r : walker->reads) { if (!r->to_assemble) continue; bfc.AddSequence(r->CorrectedSeq(), "", r->UniqueName()); } } // Phase 2: train (reuses hash on 2nd+ call), then re-add // only correctable reads and run error correction bfc.Train(); bfc.ClearReads(); for (auto& [_, walker] : unit.walkers) { for (const auto& r : walker->reads) { if (!r->to_assemble) continue; bfc.AddSequence(r->CorrectedSeq(), "", r->UniqueName()); } } bfc.ErrorCorrect(); // Phase 3: drain corrected sequences and distribute back std::unordered_map corrected_by_name; std::string s, nm; while (bfc.GetSequence(s, nm)) corrected_by_name.emplace(std::move(nm), std::move(s)); // Clear reads from the BFC (hash stays alive for next region) bfc.ClearReads(); size_t num_reads_corrected = 0; for (auto& [_, walker] : unit.walkers) { for (auto& r : walker->reads) { if (!r->to_assemble) continue; auto it = corrected_by_name.find(r->UniqueName()); if (it != corrected_by_name.end()) { SVABA_READ_TRACE(r->Qname(), "BFC corrected: changed=" << (r->CorrectedSeq() != it->second ? "YES" : "NO") << " orig_len=" << r->CorrectedSeq().size() << " corr_len=" << it->second.size()); r->SetCorrectedSeq(it->second); ++num_reads_corrected; } else { SVABA_READ_TRACE(r->Qname(), "BFC: not in corrected output (lost in hash?)"); } } } sc.logger.log(sc.opts.verbose > 0, sc.opts.verbose_log, "...BFC corrected ", SeqLib::AddCommas(num_reads_corrected)); #ifdef SVABA_KMER_RESTRICT // Post-correction kmer filter: drop any read whose corrected // sequence doesn't contain the target kmer (or its revcomp). // Dropped reads get to_assemble=false so they won't enter // fermi assembly, r2c alignment, corrected.bam, or scoring. size_t kmer_kept = 0, kmer_dropped = 0; for (auto& [_, walker] : unit.walkers) { for (auto& r : walker->reads) { if (!r->to_assemble) continue; if (_svaba_kmer_match(r->CorrectedSeq())) { ++kmer_kept; } else { r->to_assemble = false; ++kmer_dropped; } } } sc.logger.log(true, true, "...KMER_RESTRICT: kept ", kmer_kept, " dropped ", kmer_dropped, " kmer=", SVABA_KMER_RESTRICT); #endif unit.st.stop("k"); } // SvABA2.0: realign corrected read sequences to the reference so // BreakPoint::splitCoverage's "r2c better than native" gate has an // apples-to-apples comparison: same corrected sequence, same // svaba-internal BWA-MEM parameters, same scoring on both sides. // // Performance optimization: in production mode (no --dump-reads), we // DEFER this to after the r2c alignment loop and only realign reads // that actually hit a contig (HasR2C()). The full-reference BWA call // is the single most expensive operation (~39% of wall time) due to // cache misses on the ~3 GB FM-index, and most reads don't end up // supporting any variant. Reads without r2c hits never enter // splitCoverage, so their native alignment is never consulted. // // In --dump-reads mode we realign ALL reads here (before assembly) // so the corrected.bam output has full reference alignments for IGV. // // Reads that didn't go through correction (to_assemble == false) // keep their default sentinel (corrected_native_nm == -1) and the // gate falls back to the original BAM record for those. if (sc.opts.dump_corrected_reads) { for (const auto& [_, walker] : unit.walkers) { for (const auto& r : walker->reads) { if (!r->to_assemble) continue; BamRecordPtrVector aln; unit.bwa_aligner->alignSequence(r->CorrectedSeq(), r->UniqueName(), aln, false, 0.6, SECONDARY_CAP); // Alt-contig demotion: if BWA placed the primary on a non- // standard chr but a secondary on a standard chr is comparable, // swap them so the corrected.bam shows standard-chr placements. preferStandardChromosomes(aln, sc.opts.maxMateChrID); // Pick the primary (non-supplementary, non-secondary, mapped) // record. If no usable record came back, leave the sentinel. for (const auto& a : aln) { if (!a->MappedFlag()) continue; if ( a->SecondaryFlag()) continue; if ( a->SupplementaryFlag()) continue; int32_t nm = -1; a->GetIntTag("NM", nm); r->corrected_native_cig = a->GetCigar(); r->corrected_native_nm = nm; break; } // Only push non-secondary alignments into the corrected BAM // output — secondaries were requested solely for alt-demotion. for (auto& a : aln) { if (!a->SecondaryFlag()) unit.all_corrected_reads.push_back(a); } } } sc.logger.log(sc.opts.verbose > 0, sc.opts.verbose_log, "...realigned corrected reads to genome (dump mode, all reads)"); } sc.logger.log(sc.opts.verbose > 0, sc.opts.verbose_log, "...running assemblies for region ", region); // set the contig prefix // Include the active assembler tag (fermi / sga) so that contig names // carry a record of which engine produced them, e.g. // c_fermi_chr12_18000000_20000000_0 // c_sga_chr12_18000000_20000000_0 string ctg_prefix = string("c_") + svaba::kAssemblerTag + "_" + region.ChrName(sc.header) + "_" + std::to_string(region.pos1) + "_" + std::to_string(region.pos2); // where to store the AlignedContigs for this region std::unordered_map all_AlignedContigs_this_region; // setup the engine and peform assembly UnalignedSequenceVector all_unaligned_contigs_this_region; #ifndef FERMI { // std::cerr << " SGA" << std::endl; svabaAssemblerEngine engine(ctg_prefix, sc.opts.sgaErrorRate, sc.opts.sgaMinOverlap, sc.readlen); size_t dbg = 0; for (const auto& [_, walker] : unit.walkers) { //debug // if (region.pos1 == 4287501) { // for (const auto& r : walker->reads) // std::cout << " RSEQ " << r->CorrectedSeq() << std::endl; // } engine.fillReadTable(walker->reads); dbg += walker->reads.size(); } sc.logger.log(sc.opts.verbose > 1, false, "...assembling reads with SGA"); // do the actual assembly engine.performAssembly(1/*sc.opts.num_assembly_rounds*/); // retrieve contigs all_unaligned_contigs_this_region = engine.getContigs(); // if (region.pos1 == 4287501) // std::cerr << " contigs " << // all_unaligned_contigs_this_region.size() << std::endl; } #else { // std::cerr << "FERMI" << std::endl; // build the reads structure size_t n_reads = 0; for (const auto& [_, walker] : unit.walkers) { for (const auto& r : walker->reads) n_reads += static_cast(r->to_assemble); } fseq1_t *fseq = (fseq1_t*)calloc(n_reads, sizeof(fseq1_t)); size_t i = 0; for (const auto& [_, walker] : unit.walkers) { for (const auto& r : walker->reads) { // skip reads that we want to track but not assemble if (!r->to_assemble) continue; const std::string& cseq = r->CorrectedSeq(); fseq[i].seq = strdup(cseq.c_str()); fseq[i].l_seq = cseq.length(); // fermi's fml_fltuniq reads qual even when ec_k=-1. // Avoid constructing a std::string just to strdup it — // malloc + memset directly. fseq[i].qual = (char*)malloc(fseq[i].l_seq + 1); memset(fseq[i].qual, 'I', fseq[i].l_seq); fseq[i].qual[fseq[i].l_seq] = '\0'; ++i; } } fml_opt_t opt; fml_opt_init(&opt); // SvABA2.0: loosen fermi-lite defaults for variant-supporting contigs. // Rationale (see fermi-lite/misc.c): // - We already hand fermi CorrectedSeq(); a second EC pass with // min_cnt=4 is prone to "correcting" low-VAF alt k-mers back to // reference. Disable EC entirely (ec_k = -1 skips fml_correct). // - fml_assemble clamps min_ensr/min_insr to [min_cnt, max_cnt]. // Lowering min_cnt lets tips/branches with 2 supporting reads // survive graph cleaning. // - MAG_F_POPOPEN enables aggressive trimming of "open" tips // (dead-end branches). A heterozygous indel whose alt bubble // is shorter than ~2.5x read length reads exactly like an // open tip and gets trimmed. Clear the flag. // - fml_opt_adjust sets min_elen = 2.5 * avg_read_len (~375bp for // 150bp reads). We can't override that through fml_assemble // because it re-runs adjust internally, but disabling POPOPEN // and lowering min_cnt already do most of the work. opt.ec_k = -1; // skip fermi's EC (svaba pre-corrects) opt.min_cnt = 2; // tolerate rare k-mers opt.max_cnt = 8; // keep headroom for high-cov regions opt.mag_opt.flag &= ~MAG_F_POPOPEN; // don't aggressively trim variant tips // optional, very aggressive: opt.mag_opt.flag |= MAG_F_AGGRESSIVE is the // OPPOSITE of what we want (it POPS variant bubbles). Leave it cleared. sc.logger.log(sc.opts.verbose > 1, false, "...assembling reads with Fermi-lite"); // 3) Run the assembler int n_utgs = 0; fml_utg_t *utgs = nullptr; if (n_reads > 0) utgs = fml_assemble(&opt,n_reads,fseq,&n_utgs); // 4) Copy out contig names/sequences into a C++ vector all_unaligned_contigs_this_region.reserve(n_utgs); for (int i = 0; i < n_utgs; i++) { std::string name = ctg_prefix + "_" + std::to_string(i+1) + "C"; std::string seq = utgs[i].seq; SeqLib::UnalignedSequence us(name, seq); all_unaligned_contigs_this_region.push_back(std::move(us)); } // 5) Clean up fml_utg_destroy(n_utgs, utgs); for (int i = 0; i < n_reads; i++) { free(fseq[i].seq); free(fseq[i].qual); } free(fseq); } #endif unit.st.contig_count = all_unaligned_contigs_this_region.size(); sc.logger.log(sc.opts.verbose > 0, sc.opts.verbose_log, "...assembled ", all_unaligned_contigs_this_region.size(), " contigs for ", ctg_prefix); ///////// // SETUP FOR LOCAL ALIGNMENT // get the reference sequence of the local region /* string lregion; try { lregion = unit.ref_genome->QueryRegion(sc.header.IDtoName(region.chr), region.pos1, region.pos2); } catch (...) { std::cerr << " Caught exception for lregion with reg " << region.ToString(sc.header); lregion = ""; } // make a BWA index from the locally retrieved sequence UnalignedSequenceVector local_usv = {{"local", lregion, string()}}; BWAIndexPtr local_bwa_index = std::make_shared(); local_bwa_index->ConstructIndex(local_usv); BWAAligner local_bwa_aligner(local_bwa_index); */ ////////////// // loop and process unaligned contigs //vector alc; // where to put the contigs BamRecordPtrVector all_aligned_contigs_this_region; // contig-to-genome alignments size_t count_contigs_of_size = 0; for (auto& i : all_unaligned_contigs_this_region) { // if too short, skip if ((int)i.Seq.length() < (sc.readlen * 1.2)) { SVABA_TRACE(i.Name, "TP1 SKIP contig too short: len=" << i.Seq.length() << " threshold=" << (int)(sc.readlen * 1.2)); continue; } ++count_contigs_of_size; //// LOCAL REALIGNMENT // align to the local region /* BamRecordVector local_ct_alignments; local_bwa_aligner.alignSequence(i.Seq, i.Name, local_ct_alignments, false, SECONDARY_FRAC, SECONDARY_CAP); // check if it has a non-local alignment bool valid_sv = true; for (auto& aa : local_ct_alignments) { if (aa.NumClip() < MIN_CLIP_FOR_LOCAL) valid_sv = false; // has a non-clipped local alignment. can't be SV. Indel only }*/ // do the main realignment of unaligned contigs to the reference genome BamRecordPtrVector human_alignments; unit.bwa_aligner->alignSequence(i.Seq, i.Name, human_alignments, false, SECONDARY_FRAC, SECONDARY_CAP); SVABA_TRACE(i.Name, "TP2 BWA aligned: " << human_alignments.size() << " alignments, contig_len=" << i.Seq.length()); // Alt-contig demotion: if BWA placed a fragment on a non-standard // chromosome (alt/decoy) but a secondary on a standard chromosome // has a comparable score, swap them so the standard placement wins. // This prevents real breakpoints from being lost when one fragment // of a split-mapped contig lands on e.g. chr11_JH159136v1_alt // instead of chr11 proper. size_t n_demoted = preferStandardChromosomes(human_alignments, sc.opts.maxMateChrID); SVABA_TRACE_IF(n_demoted > 0, i.Name, "TP2b alt-demotion: swapped " << n_demoted << " fragment(s) from non-standard to standard chr"); // Strip secondaries — they were only needed for alt-demotion above. // AlignedContig's constructor assumes non-secondaries come first; // a stray secondary before the first primary/supp causes a throw. human_alignments.erase( std::remove_if(human_alignments.begin(), human_alignments.end(), [](const BamRecordPtr& r) { return r && r->SecondaryFlag(); }), human_alignments.end()); // provide a svaba-specific continuous "alignment score" for (auto& ba : human_alignments) { if (!ba) continue; if (!ba->MappedFlag()) continue; const auto s = svaba::scoreContigAlignment(*ba); svaba::tagContigAlignment(*ba, s); } // sort the alignments by position std::sort(human_alignments.begin(), human_alignments.end(), SeqLib::BamRecordSort::ByReadPositionSharedPtr()); // store all contig alignment all_aligned_contigs_this_region.insert(all_aligned_contigs_this_region.end(), human_alignments.begin(), human_alignments.end()); // add contig alignments to svabaThreadUnit for writing later unit.master_contigs.insert(unit.master_contigs.end(), human_alignments.begin(), human_alignments.end()); // make the AlignedContig object for this contig AlignedContig ac(human_alignments, region, &sc); SVABA_TRACE(ac.getContigName(), "TP5 AlignedContig: frags=" << ac.getFragCount() << " global_bp=" << (ac.hasGlobalBP() ? "YES" : "NO") << " local_breaks=" << ac.getLocalBreakCount() << " hasVariant=" << (ac.hasVariant() ? "YES" : "NO")); // add this all_AlignedContigs_this_region.insert_or_assign(ac.getContigName(), std::move(ac)); } unit.st.aligned_contig_count = all_aligned_contigs_this_region.size(); sc.logger.log(sc.opts.verbose > 0, sc.opts.verbose_log, "...contigs that meet size criteria: ", count_contigs_of_size); // if (region.pos1 == 4287501) { // std::cerr <<" ALIGNED CONTIGS " << // all_aligned_contigs_this_region.size() << std::endl; // for (const auto& r : all_unaligned_contigs_this_region) // std::cout << "PASSED CONTIG " << r.Name << "\t" << // r.Seq << std::endl; // } // didnt get any contigs that made it all the way through if (!all_AlignedContigs_this_region.size()) { unit.flush(); return true; } // Make a BWA mapper of the contigs themselves BWAIndexPtr contig_bwa_index = std::make_shared(); contig_bwa_index->ConstructIndex(all_unaligned_contigs_this_region); BWAAligner contig_bwa_aligner(contig_bwa_index); sc.logger.log(sc.opts.verbose > 0, sc.opts.verbose_log, "...aligning reads to contigs"); // align the reads to the contigs BamRecordPtrVector all_read2contigs; size_t seen = 0; size_t n_ref_match_skipped = 0; for (auto& [_, walker] : unit.walkers) { for (auto& i : walker->reads) { ++seen; if (seen % 1000 == 0) sc.logger.log(sc.opts.verbose > 4, sc.opts.verbose_log, "- r2c ", SeqLib::AddCommas(seen)); // get the sequence const std::string& seq = i->CorrectedSeq(); // Fast pre-filter: if the BFC-corrected sequence is an exact // substring of the local reference, this read is a perfect // reference match and cannot support any variant. Skip the // expensive BWA-MEM r2c alignment entirely. This catches the // common case where high-NM reads (salvaged via the NM/len > // 0.02 path) had their sequencing errors corrected by BFC and // now match the reference perfectly. All reads align to the // forward strand so no RC check is needed. if (!seq.empty() && lregion.find(seq) != std::string::npos) { SVABA_READ_TRACE(i->Qname(), "R2C SKIP: perfect ref match after BFC (seq_len=" << seq.size() << ")"); ++n_ref_match_skipped; continue; } // do the read to contig alignment for a single read BamRecordPtrVector read2contigs; contig_bwa_aligner.alignSequence(seq, i->Qname(), read2contigs, false, 0.60, 1000); // make sure we have only one alignment per contig. // Use a small vector + linear scan instead of unordered_set — // typical read hits 1-3 contigs, so hashing overhead dominates. // Store ChrID (int) instead of contig name strings. std::vector seen_chrids; seen_chrids.reserve(4); // check which ones pass auto keep = [&](const BamRecordPtr& r) { int thisas = 0; r->GetIntTag("AS", thisas); // Inline match-base count from CIGAR to avoid GetCigar() // vector allocation inside NumMatchBases(). uint32_t match_bases = 0; const uint32_t* cig_raw = bam_get_cigar(r->raw()); for (uint32_t ci = 0; ci < r->raw()->core.n_cigar; ++ci) { if (bam_cigar_op(cig_raw[ci]) == BAM_CMATCH) match_bases += bam_cigar_oplen(cig_raw[ci]); } if ((double)match_bases * 0.5 > thisas) return false; int chrid = r->ChrID(); for (int id : seen_chrids) if (id == chrid) return false; seen_chrids.push_back(chrid); return true; }; read2contigs.erase( std::remove_if(read2contigs.begin(), read2contigs.end(), [&](const BamRecordPtr& r) { return !keep(r); }), read2contigs.end()); if (read2contigs.empty()) { SVABA_READ_TRACE(i->Qname(), "R2C: no contig hits (0 alignments passed filters)"); } else { for (const auto& rc : read2contigs) { int as = 0; rc->GetIntTag("AS", as); SVABA_READ_TRACE(i->Qname(), "R2C HIT: contig=" << all_unaligned_contigs_this_region[rc->ChrID()].Name << " AS=" << as << " rc=" << rc->ReverseFlag() << " CIGAR=" << rc->CigarString()); } } // SvABA2.0: boundary-aware comma-append helper (matches the // dedupe semantics used for the bi:Z tag at BP finalization). // `need_read_tags` is declared at function scope above and // gates both this block (bz:Z) and the BP-finalization block // (bi:Z) further down. auto append_unique = [](std::string& cur, const std::string& id) { if (id.empty()) return false; if (cur.empty()) { cur = id; return true; } size_t pos = 0; while ((pos = cur.find(id, pos)) != std::string::npos) { const bool left_ok = (pos == 0) || cur[pos-1] == ','; const bool right_ok = (pos + id.size() == cur.size()) || cur[pos + id.size()] == ','; if (left_ok && right_ok) return false; pos += id.size(); } cur += "," + id; return true; }; // annotate the original read for (auto& r : read2contigs) { all_read2contigs.push_back(r); // add to total r2c this_r2c; // alignment of this read to this contig if (r->ReverseFlag()) this_r2c.rc = true; this_r2c.AddAlignment(r); // doesn't copy any memory const std::string& contig_name = all_unaligned_contigs_this_region[r->ChrID()].Name; i->AddR2C(contig_name, this_r2c); // SvABA2.0: stamp the bz:Z aux tag on the svabaRead pointer // (covers the weird/discordant BAMs, pointer-identity path) and // register for later stamping onto the corrected BAM via the // qname-keyed map. bz:Z is the "aligned to this contig" // superset; bi:Z (set later at BP finalization) is the // "supports a variant on this contig" subset. // // Gated on need_read_tags — the tags and the per-read map are // consumed only by the dump-reads BAM writers. Skipping this // under the default (--dump-reads off) is measurable on // high-coverage contigs; with --dump-reads on, nothing changes. if (need_read_tags) { std::string cur; i->GetZTag("bz", cur); if (append_unique(cur, contig_name)) { i->RemoveTag("bz"); i->AddZTag("bz", cur); } std::string& entry = unit.all_cnames_by_name[i->UniqueName()]; append_unique(entry, contig_name); } // if (region.pos1 == 4287501) { // std::cout << "R2C " << r->Qname() << " - " << // r->Sequence() << " contig " << r->ChrName(sc.header) << // std::endl; // } // add the read to the right contig auto it = all_AlignedContigs_this_region.find(contig_name); if (it != all_AlignedContigs_this_region.end()) { it->second.AddAlignedRead(i); // we are adding the svabaBamRead!! not the cread } } // read2contig alignment loop (per read) } // short read realignment loop (all reads) } // short read realignment loop (all bam walkers) sc.logger.log(sc.opts.verbose > 0, sc.opts.verbose_log, "...done aligning reads to contigs for ", SeqLib::AddCommas(all_read2contigs.size()), " r2c hits from ", SeqLib::AddCommas(seen), " reads (", SeqLib::AddCommas(n_ref_match_skipped), " skipped as perfect ref matches). Processing variants"); // Deferred native realignment (production mode only): now that r2c is // done, realign only the reads that actually hit a contig. This avoids // the expensive full-reference BWA call for reads that will never enter // splitCoverage. In --dump-reads mode this was already done above for // ALL reads (before assembly). // // Additional optimization: when --always-realign-corrected is NOT set // (default), reads whose corrected sequence matches the original BAM // sequence skip BWA entirely — the input BAM's CIGAR/NM is directly // comparable because svaba uses the same BWA-MEM scoring internally. // Only reads that BFC actually modified (or that quality trimming // shortened) need re-alignment. The sentinel corrected_native_nm == -1 // signals splitCoverage to fall back to the original BAM record, which // is exactly what we want for unchanged reads. if (!sc.opts.dump_corrected_reads) { // Build a local-reference BWA index for native realignment of // BFC-modified reads. The full genome FM-index is ~3 GB and every // bwt_sa/bwt_occ call hits main memory (~L3 miss). A local index // covering just this region's reference + all mate regions fits // in L1/L2 cache, making every BWA operation 10-50× cheaper per call. // // Pad each region by readlen on each side so reads near the // boundaries still find their full native alignment. // --always-realign-corrected forces the full genome index for // all reads (for non-BWA-aligned input BAMs). const int pad = std::max(sc.readlen, 200); // Collect reference sequences for the main region + all mate regions. // Each region becomes a separate "chromosome" in the local index so // reads from any source region can map back to their origin. UnalignedSequenceVector local_usv; bool local_build_ok = false; if (!sc.opts.alwaysRealignCorrected) { // Main region { const int32_t local_start = std::max(0, region.pos1 - pad); const int32_t local_end = region.pos2 + pad; try { std::string seq = unit.ref_genome->QueryRegion( sc.header.IDtoName(region.chr), local_start, local_end); if (!seq.empty()) { // Name encodes chr:start-end so alignment coords can be // interpreted if needed, but for NM/CIGAR purposes we // only care about the edit distance, not the absolute // position on the genome. local_usv.push_back({ sc.header.IDtoName(region.chr) + ":" + std::to_string(local_start) + "-" + std::to_string(local_end), std::move(seq), std::string() }); } } catch (...) { /* skip this region */ } } // Mate regions — reads from here are in the walkers too and // need their own reference to align against. for (size_t i = 0; i < mate_regions_for_native.size(); ++i) { const auto& mr = mate_regions_for_native[i]; const int32_t mr_start = std::max(0, mr.pos1 - pad); const int32_t mr_end = mr.pos2 + pad; try { std::string seq = unit.ref_genome->QueryRegion( sc.header.IDtoName(mr.chr), mr_start, mr_end); if (!seq.empty()) { local_usv.push_back({ sc.header.IDtoName(mr.chr) + ":" + std::to_string(mr_start) + "-" + std::to_string(mr_end), std::move(seq), std::string() }); } } catch (...) { /* skip this mate region */ } } } // Build the local index. If it fails (e.g. empty ref), fall back // to the full genome aligner. BWAIndexPtr local_native_idx; std::unique_ptr local_native_aligner; if (!local_usv.empty()) { try { local_native_idx = std::make_shared(); local_native_idx->ConstructIndex(local_usv); local_native_aligner = std::make_unique(local_native_idx); local_build_ok = true; } catch (...) { local_native_aligner.reset(); } } // Choose which aligner to use: local (fast, cache-friendly) or // full genome (--always-realign-corrected, or local index failed). BWAAligner* native_aligner = local_native_aligner ? local_native_aligner.get() : unit.bwa_aligner.get(); size_t n_realigned = 0; size_t n_reused = 0; size_t n_skipped = 0; for (const auto& [_, walker] : unit.walkers) { for (const auto& r : walker->reads) { if (!r->to_assemble) continue; if (!r->HasR2C()) { ++n_skipped; continue; } // If --always-realign-corrected is NOT set and the corrected // sequence is identical to the original BAM sequence, the input // BAM's CIGAR/NM is already a valid native alignment (assuming // BWA was used for the input). Leave the sentinel // (corrected_native_nm == -1) so splitCoverage falls back to // GetCigar()/GetIntTag("NM",...) on the original BAM record. // CorrectedSeqChanged() compares against the BAM's 4-bit // encoding without allocating a Sequence() string. if (!sc.opts.alwaysRealignCorrected && !r->CorrectedSeqChanged()) { SVABA_READ_TRACE(r->Qname(), "NATIVE_REALIGN: reusing BAM CIGAR (seq unchanged by BFC)"); ++n_reused; continue; } BamRecordPtrVector aln; native_aligner->alignSequence(r->CorrectedSeq(), r->UniqueName(), aln, false, 0.6, 0); bool found_native = false; for (const auto& a : aln) { if (!a->MappedFlag()) continue; if ( a->SecondaryFlag()) continue; if ( a->SupplementaryFlag()) continue; int32_t nm = -1; a->GetIntTag("NM", nm); r->corrected_native_cig = a->GetCigar(); r->corrected_native_nm = nm; SVABA_READ_TRACE(r->Qname(), "NATIVE_REALIGN: corrected seq realigned to ref" << " NM=" << nm << " CIGAR=" << a->CigarString()); found_native = true; break; } if (!found_native) { SVABA_READ_TRACE(r->Qname(), "NATIVE_REALIGN: no mapped primary alignment (unmapped after correction?)"); } ++n_realigned; } } sc.logger.log(sc.opts.verbose > 0, sc.opts.verbose_log, "...native realignment: ", SeqLib::AddCommas(n_realigned), " re-aligned", (local_build_ok ? std::string(" (local ref, ") + std::to_string(local_usv.size()) + " seqs)" : std::string(" (full genome)")), ", ", SeqLib::AddCommas(n_reused), " reused BAM, ", SeqLib::AddCommas(n_skipped), " no r2c hit"); } // BPS need a repeat filter // BPS can have addDiscordantFilter(dmap) applied directly // Get contig coverage, discordant matching to contigs, etc for (auto& [_, a] : all_AlignedContigs_this_region) { // right now all break points are stored in an AlignedContig // this routine calls all internally stored BreakPoint and // checks split read coverage, since all read2contig alignments // for this AlignedContig are stored in this AlignedContig a.splitCoverage(); // now that we have all the break support, // check that the complex breaks are OK //a.refilterComplex(); // add to the final structure //alc.push_back(std::move(a)); } // if (region.pos1 == 4287501) { // for (const auto& [_, bbb] : all_AlignedContigs_this_region) { // std::cout << " ALC " << bbb.printToAlignmentsFile(sc.header) << std::endl; // } // } unit.st.stop("as"); // get the breakpoints BreakPointPtrVector bp_glob; for (auto& [_, i] : all_AlignedContigs_this_region) { BreakPointPtrVector tmp_allbreaks = i.getAllBreakPoints(); bp_glob.insert(bp_glob.end(), tmp_allbreaks.begin(), tmp_allbreaks.end()); //debig for (const auto& bp : bp_glob) assert(bp); } // set each interal BreakEnd as to whether it overlaps with the // "local" assembly window for (auto& i : bp_glob) i->setLocal(region); //if (dbsnp_filter && opt::dbsnp.length()) { // WRITELOG("...DBSNP filtering", opt::verbose > 1, false); // for (auto & i : bp_glob) // dbsnp_filter->queryBreakpoint(i); //} // filter against blacklist for (auto& i : bp_glob) { i->checkBlacklist(sc.blacklist); // remove anything in a high-repeat blacklist for (const auto& [_, walker] : unit.walkers) { if (walker->local_blacklist.CountOverlaps(i->b1.gr) || walker->local_blacklist.CountOverlaps(i->b2.gr)) i->confidence = "BLACKLIST"; } } // add discordant reads for (auto& i : bp_glob) { i->CombineWithDiscordantClusterMap(dmap); } // if (region.pos1 == 4287501) { // for (const auto& bbb : bp_glob) { // std::cout << " BPGLOB " << bbb->printSimple(sc.header) << std::endl; // } // } // add in the discordant clusters as breakpoints for (auto& [_, d] : dmap) { // dont send DSCRD if FR and below size /*bool below_size = i.second.m_reg1.strand == '+' && i.second.m_reg2.strand == '-' && (i.second.m_reg2.pos1 - i.second.m_reg1.pos2) < min_dscrd_size_for_variant && i.second.m_reg1.chr == i.second.m_reg2.chr; */ // already associated with an assembly, so don't make a new variant // this information should be added to the DiscordantCluster // during "BreakPoint::CombineWithDiscordantClusterMap()" if (d.hasAssociatedAssemblyContig()) continue; // too little read support, skip if ( (d.tcount + d.ncount) < MIN_DSCRD_READS_DSCRD_ONLY) continue; if (!d.valid()) continue; // make the Discordant cluster BreakPointPtr tmpbp = std::make_shared(d, dmap, region, &sc); assert(tmpbp); bp_glob.push_back(tmpbp); } // de duplicate the breakpoints std::sort(bp_glob.begin(), bp_glob.end(), [](auto const& a, auto const& b){ return *a < *b; }); auto new_end = std::unique( bp_glob.begin(), bp_glob.end(), [](auto const& a, auto const& b){ // return true if *a and *b are "equal" in the sense of your operator< return !(*a < *b) && !(*b < *a); }); bp_glob.erase(new_end, bp_glob.end()); // remove the duplicates after they are sorted to the back // if (region.pos1 == 4287501) { // for (const auto& bbb : bp_glob) { // std::cout << " AFTER BPGLOB " << bbb->printSimple(sc.header) << std::endl; // } // } // add the coverage data to breaks for allelic fraction computation unordered_map covs; for (auto& [key, walker] : unit.walkers) { covs[key] = &walker->cov; } for (auto& i : bp_glob) { assert(i); i->addCovs(covs); } // check the cigar matches for (auto& i : bp_glob) { i->indelCigarCheck(cigmap); } // score the breakpoints for (auto& i : bp_glob) { i->scoreBreakpoint(); } // label somatic breakpoints that intersect directly with normal as NOT somatic unordered_set norm_hash; for (auto& i : bp_glob) { // hash the normals if (i->somatic != SomaticState::SOMATIC_LOD && i->confidence == "PASS") { for (const auto& h : i->getBreakEndHashes()) norm_hash.insert(h); } } // find somatic that intersect with norm. Set somatic = 0; for (auto& i : bp_glob) { // don't need to check normal against normal if (i->somatic != SomaticState::SOMATIC_LOD) continue; // if overlaps then turn off somatic for (const auto& h : i->getBreakEndHashes()) if (norm_hash.count(h)) i->somatic = SomaticState::FAILED; // not somatic } // remove indels at repeats that have multiple variants // unordered_map ccc; // for (auto& i : bp_glob) { // if (i->svtype == SVType::INDEL && i->repeat_seq.length() > 6) { // ++ccc[i->b1.hash()]; // } // } // for (auto& i : bp_glob) { // if (i->svtype == SVType::INDEL && ccc[i->b1.hash()] > 1) // i->confidence = "REPVAR"; // } // remove somatic calls if they have a germline normal SV in // them or indels with // 2+ germline normal in same contig // unordered_set bp_hash; // for (auto& i : bp_glob) { // hash the normals // if (!i->somatic_score && i->svtype != SVType::INDEL && i->confidence == "PASS") { // bp_hash.insert(i->cname); // } // } // for (auto& i : bp_glob) // find somatic that intersect with norm. Set somatic = 0; // if (i->somatic_score && i->num_align > 1 && bp_hash.count(i->cname)) { // i->somatic_score = -2; // } // remove somatic SVs that overlap with germline svs if (sc.germline_svs.size()) { for (auto& i : bp_glob) { // don't filter normals or indels if (i->somatic != SomaticState::SOMATIC_LOD || i->isIndel()) continue; GenomicRegion gr1 = i->BreakEndAsGenomicRegionLeft(); GenomicRegion gr2 = i->BreakEndAsGenomicRegionRight(); gr1.Pad(GERMLINE_CNV_PAD); gr2.Pad(GERMLINE_CNV_PAD); if (sc.germline_svs.OverlapSameInterval(gr1, gr2)) { i->somatic = SomaticState::FAILED; } } } // add the ref and alt tags for (auto& i : bp_glob) i->setRefAlt(unit.ref_genome.get(), sc.header); // transfer local versions to thread store for (const auto& [_, a] : all_AlignedContigs_this_region) { if (a.hasVariant()) { SVABA_TRACE(a.getContigName(), "TP6 hasVariant=YES, pushing to master_alc"); unit.master_alc.push_back(a); //std::move(a)); } else { SVABA_TRACE(a.getContigName(), "TP6 hasVariant=NO — contig dropped from output" << " frags=" << a.getFragCount() << " indel_breaks=" << a.getIndelBreakCount()); } } all_AlignedContigs_this_region.clear(); //for (const auto& d : dmap) // unit.m_disc_reads += d.second.reads.size(); //for (const auto& a : alc) // unit.m_bamreads_count += a.NumBamReads(); for (auto& i : bp_glob) { SVABA_TRACE(i->cname, "TP23 hasMinimal check: dc.t=" << i->dc.tcount << " dc.n=" << i->dc.ncount << " t.split=" << i->t.split << " n.split=" << i->n.split << " local=" << (int)i->local << " confidence=" << i->confidence << " result=" << (i->hasMinimal() ? "PASS" : "FAIL")); if ( i->hasMinimal() ) { // SvABA2.0 (v3): assign this BP a unique, thread-stable ID // exactly once in its lifetime. Must happen BEFORE the bi:Z // stamping below, since the tag payload is now the bp_id (not // the cname). The `id.empty()` guard lets this loop be safely // reentered (e.g. across regions) without re-numbering BPs // that were finalized in a prior region. Because i is a // shared_ptr also referenced by the parent AlignedContig's // m_global_bp / m_local_breaks / fragment indel_breaks, // setting id here is immediately visible to printToR2CTsv // when it walks getAllBreakPoints() during writeUnit. if (i->id.empty()) i->id = unit.next_bp_id(); // SvABA2.0 (v3): tag every alt-supporting read with this // breakpoint's bp_id on the `bi:Z` aux tag. bp_id is a // thread-stable per-BP identifier (see SvabaThreadUnit:: // next_bp_id) and is the same string emitted as col 52 of // bps.txt.gz and in the r2c.txt.gz `split_bps` / `disc_bps` // columns — so `samtools view | grep bi:Z:` returns // exactly the ALT-supporters for that specific variant row. // Pre-v3 the tag carried cnames (contig-level); a single // contig with multiple BPs (global + multi + indel) couldn't // be disambiguated. bz:Z still carries cnames (contig-level // "which contigs did this read r2c to") — see the r2c loop // above where that map is populated. // // Two parallel paths for bi:Z: // (a) stamp the `bi:Z` aux tag on the original svabaRead // shared_ptr. This covers weird/discordant BAM outputs, // which share pointer identity with those records. // (b) record UniqueName -> bp_ids in // `unit.alt_bp_ids_by_name` so `writeUnit` can stamp // the tag on all_corrected_reads (newly-aligned // BamRecords from bwa that don't share pointer // identity). // A read can support multiple BPs (split reads crossing // nearby calls; indel + flanking SV on one contig) so the tag // value is a comma-joined list, dedup'd on exact-match // boundaries. auto append_unique = [](std::string& cur, const std::string& id) { if (id.empty()) return false; if (cur.empty()) { cur = id; return true; } size_t pos = 0; while ((pos = cur.find(id, pos)) != std::string::npos) { const bool left_ok = (pos == 0) || cur[pos-1] == ','; const bool right_ok = (pos + id.size() == cur.size()) || cur[pos + id.size()] == ','; if (left_ok && right_ok) return false; pos += id.size(); } cur += "," + id; return true; }; const std::string& this_bp_id = i->id; auto tag_with_bp_id = [&](auto& r) { if (!r || this_bp_id.empty()) return; SVABA_READ_TRACE(r->Qname(), "OUTPUT TAG bi:Z with bp_id=" << this_bp_id << " contig=" << i->cname << " confidence=" << i->confidence << " somatic=" << (int)i->somatic); // (a) stamp the shared_ptr std::string cur; r->GetZTag("bi", cur); if (append_unique(cur, this_bp_id)) { r->RemoveTag("bi"); r->AddZTag("bi", cur); } // (b) register in name->bp_ids map so the corrected BAM can // be tagged at write time std::string& entry = unit.alt_bp_ids_by_name[r->UniqueName()]; append_unique(entry, this_bp_id); }; // Same need_read_tags gate as the bz:Z stamping in the r2c loop // above — the bi:Z tag is consumed only by the dump-reads BAM // emitters in SvabaOutputWriter, so skipping the per-alt-read // GetZTag/RemoveTag/AddZTag triplets under the default (no // --dump-reads) path is a measurable CPU win on SVs with many // alt supporters. Under --dump-reads nothing changes. if (need_read_tags) { for (auto& r : i->reads) tag_with_bp_id(r); for (auto& [_, r] : i->dc.reads) tag_with_bp_id(r); for (auto& [_, r] : i->dc.mates) tag_with_bp_id(r); } unit.m_bps.push_back(i); } } // end for bp_glob unit.st.bps_count = bp_glob.size(); // if (region.pos1 == 4287501) { // for (const auto& bbb : unit.m_bps/*bp_glob*/) { // std::cout << " FINAL BPGLOB " << bbb->printSimple(sc.header) << std::endl; // } // } // dump if getting to much memory unit.st.stop("pp"); unit.ss << unit.st.logRuntime(sc.header) << "\n"; unit.flush(); // display the run time if (sc.total_regions_done % 1000 == 0) { sc.logger.log(sc.opts.verbose > 0, true, svabaUtils::runTimeString(read_counts.first, read_counts.second, all_AlignedContigs_this_region.size(), region, sc.header, unit.st, sc.start)); } sc.logger.log(sc.opts.verbose > 0, sc.opts.verbose_log, "...done with this region"); return true; } ================================================ FILE: src/svaba/SvabaRegionProcessor.h ================================================ #pragma once #include // for size_t #include "SvabaThreadUnit.h" // for svabaThreadUnit #include "SvabaUtils.h" #include "SeqLib/GenomicRegionCollection.h" // for GRC return type class SvabaLogger; class SvabaOptions; class SvabaOutputWriter; /// Encapsulates everything needed to process one genomic region: /// - shared logger /// - shared run-time options /// - shared output writer /// Its `process()` method is exactly where your old runWorkItem logic goes. namespace SeqLib { class BamHeader; } using svabaUtils::svabaTimer; class SvabaRegionProcessor { public: SvabaRegionProcessor(SvabaSharedConfig& sh_cf); /// Run the SVABA pipeline on `region` using per-thread state in `unit`. /// Returns true on success, false on error. bool process(const SeqLib::GenomicRegion& region, svabaThreadUnit& unit, size_t threadId); /// Run mate-region collection. Returns the merged somatic mate regions /// (as a GRC) so the caller can include their reference in a local BWA /// index for native realignment. SeqLib::GRC runMateCollectionLoop(const SeqLib::GenomicRegion& region, svabaThreadUnit& stu); private: SvabaSharedConfig& sc; }; ================================================ FILE: src/svaba/SvabaSharedConfig.h ================================================ #pragma once #include //#include #include "SeqLib/BWAIndex.h" #include "SeqLib/BamHeader.h" #include "SeqLib/GenomicRegionCollection.h" #include "SeqLib/ReadFilter.h" #include "LearnBamParams.h" #include class SvabaOutputWriter; class SvabaLogger; class SvabaOptions; class SvabaOutputWriter; class DBSnpFilter; class SvabaSharedConfig { public: SvabaSharedConfig(SvabaLogger& _logger, SvabaOptions& _opts, SvabaOutputWriter& _writer) : logger(_logger), opts(_opts), writer(_writer) {} // Delete copy constructor and copy assignment SvabaSharedConfig(const SvabaSharedConfig&) = delete; SvabaSharedConfig& operator=(const SvabaSharedConfig&) = delete; // Delete move constructor and move assignment SvabaSharedConfig(SvabaSharedConfig&&) = delete; SvabaSharedConfig& operator=(SvabaSharedConfig&&) = delete; size_t total_regions_to_process = 0; size_t total_regions_done = 0; struct timespec start; // store which readgroups we already warned about std::unordered_set warned; SvabaLogger& logger; SvabaOptions& opts; SvabaOutputWriter& writer; SeqLib::BWAIndexPtr bwa_idx; SeqLib::BamHeader header; SeqLib::GRC blacklist; SeqLib::GRC file_regions; SeqLib::GRC germline_svs; int readlen = -1; double insertsize = -1; std::shared_ptr dbsnp_filter; SeqLib::Filter::ReadFilterCollection mr; std::unordered_map bamStats; // needed for aligned contig std::set prefixes; std::string args = "svaba"; }; ================================================ FILE: src/svaba/SvabaThreadUnit.cpp ================================================ #include "SvabaThreadUnit.h" #include "SvabaOutputWriter.h" #if defined(__GLIBC__) #include #endif // Build a new BamHeader that prepends an @HD line (with the given sort order) // to the @SQ block of `src`. Avoids SeqLib::BamHeader::AsString(), which // blindly dereferences h->text; in modern htslib that field is lazy and is // often NULL until sam_hdr_str() materializes it, causing a segfault. static SeqLib::BamHeader make_header_with_hd(const SeqLib::BamHeader& src, const std::string& so) { std::string txt = "@HD\tVN:1.6\tSO:" + so + "\n"; const bam_hdr_t* h = src.get(); if (h) { // sam_hdr_str forces htslib to regenerate the textual form from its // internal representation and caches it in h->text. const char* cur = sam_hdr_str(const_cast(h)); if (cur) txt += cur; } return SeqLib::BamHeader(txt); } svabaThreadUnit::svabaThreadUnit(SvabaSharedConfig& sc_, int thread) : sc(sc_), threadId(thread) { // load the samtools faidx ref_genome = std::make_unique(); ref_genome->LoadIndex(sc.opts.refGenome); // set the *shared* memory BWAIndex bwa_aligner = std::make_shared(sc.bwa_idx); //bwa_aligner->allocBuffer(4096); // longest contig // load the .bai files for each bam and set parameters for(const auto& [pref, path] : sc.opts.bams) { // instantiate a new BAM walker auto [it, inserted] = walkers.try_emplace(pref, std::make_shared(sc)); it->second->Open(path); it->second->SetPrefix(pref); } // make the header in case dumping reads for debugging auto hdr_unsorted = make_header_with_hd(sc.header, "unsorted"); // setup the weird read writers if (sc.opts.dump_weird_reads) { // instantiate a new BAM writer std::string bamname =sc.opts.analysisId + ".thread" + std::to_string(threadId) + ".weird.bam"; auto [it, inserted] = writers.try_emplace("w", std::make_shared(SeqLib::BAM)); it->second->SetHeader(hdr_unsorted); if (!it->second->Open(bamname)) { std::cerr << "ERROR: could not open weird read writer for thread " << threadId << " at path " << bamname << "/n"; exit(EXIT_FAILURE); } it->second->WriteHeader(); } // setup the corrected read writers if (sc.opts.dump_corrected_reads) { // instantiate a new BAM writer std::string bamname =sc.opts.analysisId + ".thread" + std::to_string(threadId) + ".corrected.bam"; auto [it, inserted] = writers.try_emplace("c", std::make_shared(SeqLib::BAM)); it->second->SetHeader(hdr_unsorted); if (!it->second->Open(bamname)) { std::cerr << "ERROR: could not open corrected read writer for thread " << threadId << " at path " << bamname << "/n"; exit(EXIT_FAILURE); } it->second->WriteHeader(); } // setup the discordant read writers if (sc.opts.dump_discordant_reads) { // instantiate a new BAM writer std::string bamname =sc.opts.analysisId + ".thread" + std::to_string(threadId) + ".discordant.bam"; auto [it, inserted] = writers.try_emplace("d", std::make_shared(SeqLib::BAM)); it->second->SetHeader(hdr_unsorted); if (!it->second->Open(bamname)) { std::cerr << "ERROR: could not open discordant read writer for thread " << threadId << " at path " << bamname << "/n"; exit(EXIT_FAILURE); } it->second->WriteHeader(); } // SvABA2.0: setup the per-thread r2c.txt.gz stream. Same pattern as the // per-thread BAM writers — each thread writes its own file with no // coordination, postprocess merges them via cat. Exactly one thread // owns the column-header row so the merged file has one header at // the top. r2c_out_ is unique_ptr (see header for why); // heap-allocate only when the flag is on, keep null otherwise. // // We key the header-writing thread to threadId == 1 (NOT 0) because // the worker pool in `threadpool.h` constructs units with `i + 1` // where `i` is the 0-based worker index — so the first worker's // threadId is 1, and there is no thread 0. This also matches the // lexicographic / numeric sort order postprocess step-1 imposes via // `awk | sort -k1,1n`, which places `${ID}.thread1.r2c.txt.gz` // first in the cat merge. If the worker numbering convention ever // changes, this line must change with it (there is no reader unit // test that catches a missing header — empirically, ad-hoc runs do). if (sc.opts.dump_alignments) { std::string r2cname = sc.opts.analysisId + ".thread" + std::to_string(threadId) + ".r2c.txt.gz"; r2c_out_ = std::make_unique(); svabaUtils::fopen(r2cname, *r2c_out_); if (threadId == 1) { *r2c_out_ << AlignedContig::r2cTsvHeader() << "\n"; } } } void svabaThreadUnit::clear() { master_alc.clear(); master_contigs.clear(); m_bps.clear(); m_disc.clear(); //m_bamreads_count = 0; // m_disc_reads = 0; //badd.clear(); all_weird_reads.clear(); all_corrected_reads.clear(); // SvABA2.0: drop the bi:Z / bz:Z lookup tables so the next window // doesn't stamp tags derived from the previous window onto reads. // (bi now stores bp_ids, bz still stores cnames — see // SvabaThreadUnit.h for the rationale.) alt_bp_ids_by_name.clear(); all_cnames_by_name.clear(); ss.str(""); // Clear the string content ss.clear(); #if defined(__GLIBC__) malloc_trim(0); #endif } void svabaThreadUnit::flush() { if (MemoryLimit(THREAD_READ_LIMIT, THREAD_CONTIG_LIMIT)) { sc.writer.writeUnit(*this, sc); // mutex and flush are inside this call clear(); } // clear out the reads and reset the walkers for (auto& [_, walker] : walkers) { walker->clear(); } #if defined(__GLIBC__) malloc_trim(0); #endif } bool svabaThreadUnit::MemoryLimit(size_t readLimit, size_t contLimit) const { size_t stored_reads = all_weird_reads.size() + all_corrected_reads.size(); bool mem_exceeded = stored_reads > readLimit || master_contigs.size() > contLimit; if (mem_exceeded) { sc.logger.log(sc.opts.verbose > 1, false, "...writing files on thread ", threadId, " with limit hit of ", stored_reads, " reads and ", master_contigs.size(), " contigs========================================================="); size_t all_walker_reads = 0; for (const auto& [_, walker] : walkers) { all_walker_reads += walker->reads.size(); } // more memory mapping /* sc.logger.log(true, true, " bad regions: ", badd.size(), " master_alc.size() ", master_alc.size(), " m_disc.size() ", m_disc.size(), " walkers.reads.size() ", all_walker_reads); */ } return mem_exceeded; } svabaThreadUnit::~svabaThreadUnit() { // if (sc.opts.dump_weird_reads) { auto it = writers.find("w"); it->second->Close(); } // if (sc.opts.dump_corrected_reads) { auto it = writers.find("c"); it->second->Close(); } // if (sc.opts.dump_discordant_reads) { auto it = writers.find("d"); it->second->Close(); } // SvABA2.0: close the per-thread r2c stream if it was opened. The // unique_ptr is null when dump_alignments was off, so guard on it // rather than only on the flag (belt-and-braces in case the flag's // value ever gets out of sync with what the ctor actually did). if (r2c_out_) { r2c_out_->close(); } } ================================================ FILE: src/svaba/SvabaThreadUnit.h ================================================ #pragma once #include #include #include #include #include #include #include // std::snprintf in next_bp_id() #include #include "SvabaBamWalker.h" #include "SvabaLogger.h" #include "SvabaOptions.h" #include "AlignedContig.h" #include "DiscordantCluster.h" #include "BreakPoint.h" #include "SvabaUtils.h" #include "gzstream.h" // ogzstream for the per-thread r2c.txt.gz #include "SeqLib/RefGenome.h" #include "SeqLib/BFC.h" #include "SvabaSharedConfig.h" #include "SeqLib/BWAAligner.h" using SeqLib::BamRecordPtrVector; class svabaBamWalker; namespace SeqLib { class BamWriter; } using WalkerMap = std::map>; using WriterMap = std::map>; class svabaThreadUnit { public: //svabaThreadUnit() = default; ~svabaThreadUnit(); svabaThreadUnit(SvabaSharedConfig& sc_, int thread); void flush(); // local version of aligner class, but will hold shared memory index std::shared_ptr bwa_aligner; //(sc.bwa_idx); size_t processed_count = 0; size_t total_count = 0; // total to process size_t processed_since_memory_dump = 0; // results std::vector master_alc; BamRecordPtrVector master_contigs; BreakPointPtrVector m_bps; DiscordantClusterMap m_disc; //size_t m_bamreads_count = 0; //size_t m_disc_reads = 0; //SeqLib::GRC badd; // bad regions int threadId; // SvABA2.0 (v3): map from read UniqueName -> comma-separated list // of bp_ids this read supports as ALT. Populated at BP // finalization so `SvabaOutputWriter::writeUnit` can stamp the // `bi:Z:` BAM aux tag on records in // `all_corrected_reads` (which are *newly aligned* BamRecords // produced by bwa, so they don't share identity with the original // svabaRead pointers and couldn't have been tagged via pointer). // The same map is used to (re)tag weird and discordant records at // write time for consistency. // // Prior to v3 this carried cnames (deterministic contig-window // identifiers). Switched to bp_ids so the tag matches the // granularity of the r2c.txt.gz `split_bps` / `disc_bps` columns: // a single contig can carry several BPs (global + multi + indel), // and a read may support one, some, or all of them. Keying off the // BP, not the contig, removes the "which variant on this contig // does this read support?" ambiguity and makes // `samtools view | grep bi:Z:bp00100000042` return exactly the // ALT-supporting reads for that specific variant row in // bps.txt.gz. cname-level grouping is still available on the // companion `bz:Z` tag (below). std::unordered_map alt_bp_ids_by_name; // SvABA2.0: map from read UniqueName -> comma-separated list of // contig cnames this read aligned to (r2c), regardless of whether // the alignment ended up supporting a variant. Populated inside the // r2c alignment loop in SvabaRegionProcessor right alongside // `svabaRead::AddR2C(...)`, and consumed at write time by // `SvabaOutputWriter::writeUnit` to stamp the `bz:Z:` aux // tag. This stays as cnames because "which contigs did this read // align to" is a contig-level concept — unlike `bi:Z` which is the // per-variant ALT-supporter attribution. The set of reads with a // `bz:Z` tag is a superset of the set with `bi:Z` (every // ALT-supporter aligned to the contig; not every r2c alignment // yields ALT support). std::unordered_map all_cnames_by_name; // very verbose outpout svabaReadPtrVector all_weird_reads; BamRecordPtrVector all_corrected_reads; // time and temp log dump until goes to file svabaUtils::svabaTimer st; std::stringstream ss; // store the BAM .bai indicies for for this thread WalkerMap walkers; WriterMap writers; // SvABA2.0: per-thread counter for generating unique BreakPoint IDs // without cross-thread coordination. Format emitted by next_bp_id() // is "bpTTTNNNNNNNN" (3-digit thread + 8-digit counter). Up to // 1000 threads and ~1e8 BPs per thread, far beyond any realistic // svaba run. If either ever overflows, the resulting ID is still a // unique string — just one digit wider — so downstream tools that // treat it opaquely are unaffected. size_t bp_id_counter = 0; std::string next_bp_id() { char buf[32]; // %03d pads thread ID; %08zu pads the counter. post-increment so // the first BP for thread 1 is bp00100000000 (the worker pool // numbers threads 1..N — see threadpool.h). std::snprintf(buf, sizeof(buf), "bp%03d%08zu", threadId, bp_id_counter++); return std::string(buf); } // SvABA2.0: per-thread r2c TSV stream. Mirrors the per-thread BAM // writers above — when --dump-reads is set (opts.dump_alignments), // each thread writes its variant-bearing contigs + r2c-aligned reads // into ${ID}.thread${N}.r2c.txt.gz directly, no shared handle, no // mutex. Postprocess merges the per-thread files via // cat ${ID}.thread*.r2c.txt.gz > ${ID}.r2c.txt.gz // which produces a valid gzip (gzip is concatenation-safe per // RFC 1952). The first worker (threadId == 1; the pool numbers // workers 1..N) writes the column-header line once at open time // so the merged file has exactly one header at the top. // // Held via unique_ptr because ogzstream inherits from std::ios (which // has deleted copy/move). Embedding it by value would implicitly // delete svabaThreadUnit's move constructor and produce // -Wdefaulted-function-deleted. unique_ptr is trivially movable, so // the `svabaThreadUnit(svabaThreadUnit&&) noexcept = default;` // below stays valid. Same reason the BAM writers live in // shared_ptr inside the `writers` map above. std::unique_ptr r2c_out_; // non-copyable, movable svabaThreadUnit(const svabaThreadUnit&) = delete; svabaThreadUnit& operator=(const svabaThreadUnit&) = delete; svabaThreadUnit(svabaThreadUnit&&) noexcept = default; svabaThreadUnit& operator=(svabaThreadUnit&&) noexcept = delete; void clear(); bool MemoryLimit(size_t readLimit, size_t contLimit) const; // Per-thread BFC object, reused across regions. The k-mer hash // (1M sub-tables at l_pre=20) is allocated once on first Train() // and cleared+reused on subsequent calls via fml_count_into(), // avoiding 2M malloc/free calls per region. unique_ptr because // BFC has raw-pointer members — implicit move would double-free. std::unique_ptr pooled_bfc; // store the faidx index for this thread std::unique_ptr ref_genome; SvabaSharedConfig& sc; private: }; ================================================ FILE: src/svaba/SvabaUtils.cpp ================================================ #include "SvabaUtils.h" #include #include // C++17 namespace fs = std::filesystem; namespace svabaUtils { static std::string POLYA = "AAAAAAAAAAAAAAAAAAA"; static std::string POLYT = "TTTTTTTTTTTTTTTTTTT"; static std::string POLYC = "CCCCCCCCCCCCCCCCCCC"; static std::string POLYG = "GGGGGGGGGGGGGGGGGGG"; static std::string POLYAT = "ATATATATATATATATATATATAT"; static std::string POLYCG = "CGCGCGCGCGCGCGCGCGCGCGCG"; static std::string POLYTG = "TGTGTGTGTGTGTGTGTGTGTGTG"; static std::string POLYCA = "CACACACACACACACACACACACA"; static std::string POLYAG = "AGAGAGAGAGAGAGAGAGAGAGAG"; static std::string POLYTC = "TCTCTCTCTCTCTCTCTCTCTCTC"; const std::string svabaTimer::header = "chromosome\tstart\tend\t" "tumor_weird_reads\tnormal_weird_reads\t" "tumor_mate_reads\tnormal_mate_reads\t" "discordant_reads\tdiscordant_clusters\t" "contigs\tcontigs_pass\tbps\truntime_seconds\t" "pct_r\tpct_m\tpct_k\tpct_as"; std::string svabaTimer::logRuntime(const SeqLib::BamHeader& h) { double total_time = 0; for (const auto& i : times) total_time += i.second; if (total_time == 0) total_time = 1.0; // Avoid division by zero auto get_pct = [&](const std::string& key) -> int { auto it = times.find(key); return (it != times.end()) ? SeqLib::percentCalc(it->second, total_time) : 0; }; // Store percentages pct_r = get_pct("r"); pct_m = get_pct("m"); pct_k = get_pct("k"); pct_as = get_pct("as"); //pct_pp = get_pct("pp"); std::ostringstream oss; oss << gr.ChrName(h) << "\t" << gr.pos1 << "\t" << gr.pos2 << "\t" << weird_read_count.first << "\t" << weird_read_count.second << "\t" << mate_read_count.first << "\t" << mate_read_count.second << "\t" << dc_read_count << "\t" << dc_cluster_count << "\t" << contig_count << "\t" << aligned_contig_count << "\t" << bps_count << "\t" << total_time << "\t" << pct_r << "\t" << pct_m << "\t" << pct_k << "\t" << pct_as; return oss.str(); } std::string fileDateString() { // set the time string time_t t = time(0); // get time now struct tm * now = localtime( & t ); std::stringstream mdate; mdate << (now->tm_year + 1900) << (now->tm_mon + 1 < 10 ? "0": "") << (now->tm_mon + 1) << (now->tm_mday < 10 ? "0" : "") << now->tm_mday; return mdate.str(); } svabaTimer::svabaTimer() { s = {"r", "m", "as", "bw", "pp", "k"}; for (auto& i : s) times[i] = 0; } void svabaTimer::stop(const std::string& part) { auto wall_end = std::chrono::steady_clock::now(); double elapsed = std::chrono::duration(wall_end - wall_start).count(); times[part] += elapsed; wall_elapsed += elapsed; //times[part] += static_cast(clock() - curr_clock) / CLOCKS_PER_SEC; //curr_clock = clock(); } void svabaTimer::start() { wall_start = std::chrono::steady_clock::now(); //curr_clock = clock(); } std::ostream& operator<<(std::ostream &out, const svabaTimer st) { double total_time = 0; for (auto& i : st.times) total_time += i.second; if (total_time == 0) return out; char buffer[140]; auto itr = st.times.find("r"); auto itm = st.times.find("m"); auto ita = st.times.find("as"); auto itk = st.times.find("k"); auto itt = st.times.find("t"); auto itp = st.times.find("pp"); if (total_time) snprintf (buffer, 140, "R: %2d%% M: %2d%% T: %2d%% C: %2d%% A: %2d%% P: %2d%%", SeqLib::percentCalc(itr->second, total_time), SeqLib::percentCalc(itm->second, total_time), SeqLib::percentCalc(itt->second, total_time), SeqLib::percentCalc(itk->second, total_time), SeqLib::percentCalc(ita->second, total_time), SeqLib::percentCalc(itp->second, total_time)); else snprintf (buffer, 140, "NO TIME"); out << std::string(buffer); return out; } // useful replace function std::string myreplace(std::string &s, std::string toReplace, std::string replaceWith) { return(s.replace(s.find(toReplace), toReplace.length(), replaceWith)); } bool hasRepeat(const std::string& seq) { if ((seq.find(POLYT) == std::string::npos) && (seq.find(POLYA) == std::string::npos) && (seq.find(POLYC) == std::string::npos) && (seq.find(POLYG) == std::string::npos) && (seq.find(POLYCG) == std::string::npos) && (seq.find(POLYAT) == std::string::npos) && (seq.find(POLYTC) == std::string::npos) && (seq.find(POLYAG) == std::string::npos) && (seq.find(POLYCA) == std::string::npos) && (seq.find(POLYTG) == std::string::npos)) return false; return true; } int overlapSize(const SeqLib::BamRecord& query, const SeqLib::BamRecordVector& subject) { // get the amount covered by subjet sequences typedef std::pair AP; typedef std::pair AP_wseq; std::vector align_pos; std::string convention_seq = ""; if (subject.size()) { convention_seq = subject[0].Sequence(); // set the orientation convention for(auto& j : subject) { if (j.Sequence() == convention_seq) align_pos.push_back(AP_wseq(AP(j.AlignmentPosition(),j.AlignmentEndPosition()), j.Sequence())); else align_pos.push_back(AP_wseq(AP(j.AlignmentPositionReverse(),j.AlignmentEndPositionReverse()), j.Sequence())); } } bool same_orientation = query.Sequence() == convention_seq; int al1 = same_orientation ? query.AlignmentPosition() : query.AlignmentPositionReverse(); int al2 = same_orientation ? query.AlignmentEndPosition() : query.AlignmentEndPositionReverse(); int max_overlap = 0; for (auto& k : align_pos) { // check each subject alignment to see if it overlaps query AP kk = k.first; max_overlap = std::max(max_overlap, std::max(0, std::min(kk.second, al2) - std::max(kk.first,al1))); // out = true; //if (al1 >= kk.first && al2 <= kk.second) // query_contained = true; } return max_overlap; } void print(std::stringstream& s, std::ofstream& log, bool cerr) { log << s.str(); if (cerr) std::cerr << s.str(); s.str(std::string()); } std::string runTimeString(int num_t_reads, int num_n_reads, int contig_counter, const SeqLib::GenomicRegion& region, const SeqLib::BamHeader& h, const svabaTimer& st, const timespec& start) { std::stringstream ss; if ( (num_t_reads + num_n_reads) > 0 && !region.IsEmpty()) { std::string print1 = SeqLib::AddCommas(region.pos1); std::string print2 = SeqLib::AddCommas(region.pos2); char buffer[180]; snprintf (buffer, 180, "***Ran %2s:%11s-%11s | T: %5d N: %5d C: %5d | ", h.IDtoName(region.chr).c_str(), print1.c_str(),print2.c_str(), (int)num_t_reads, (int)num_n_reads, (int)contig_counter); ss << std::string(buffer) << st << " | "; ss << SeqLib::displayRuntime(start); } else if (num_t_reads + num_n_reads > 0) { char buffer[180]; snprintf (buffer, 180, "Ran Whole Genome | T: %5d N: %5d C: %5d | ", (int)num_t_reads, (int)num_n_reads, (int)contig_counter); ss << std::string(buffer) << st << " | "; ss << SeqLib::displayRuntime(start); } return ss.str(); } // just get a count of how many jobs to run. Useful for limiting threads. Also set the regions int countJobs(const std::string& regionFile, SeqLib::GRC &file_regions, SeqLib::GRC &run_regions, const SeqLib::BamHeader& h, int chunk, int window_pad) { // open the region file if it exists if (!regionFile.empty() && fs::exists(regionFile)) { try { file_regions = SeqLib::GRC(regionFile, h); } catch (const std::exception &exc) { std::cerr << "Found chromosome in region file " << regionFile << " not in reference genome. Skipping.\n" << " Caught error: " << exc.what() << "\n"; } } // parse as a samtools string eg 1:1,000,000-2,000,000 else if (regionFile.find(":") != std::string::npos && regionFile.find("-") != std::string::npos) { file_regions.add(SeqLib::GenomicRegion(regionFile, h)); } // it's a single chromosome else if (!regionFile.empty()) { SeqLib::GenomicRegion gr(regionFile, "1", "1", h); if (gr.chr == -1 || gr.chr >= h.NumSequences()) { std::cerr << "ERROR: Trying to match chromosome " << regionFile << " to one in header, but no match found" << std::endl; exit(EXIT_FAILURE); } else { gr.pos2 = h.GetSequenceLength(gr.chr); //get_()->target_len[gr.chr]; file_regions.add(gr); } } // its empty, so cover whole genome else { // add all chromosomes for (int i = 0; i < h.NumSequences(); i++) { file_regions.add(SeqLib::GenomicRegion(i, 1, h.GetSequenceLength(i))); //h.get()_->target_len[i])); } } // check if the mask was successful if (file_regions.size() == 0) { std::cerr << "ERROR: Cannot read region file: " << regionFile << " or something wrong with bam header ('chr' prefix mismatch?)" << std::endl; exit(EXIT_FAILURE); } // divide it up if (chunk > 0) { // if <= 0, whole genome at once for (auto& r : file_regions) { SeqLib::GRC test(chunk, window_pad, r); run_regions.Concat(test); } } // now clear file regions, to signal that it was empty if (regionFile.empty()) file_regions.clear(); return run_regions.size(); } std::string __bamOptParse(std::map& obam, std::istringstream& arg, int sample_number, const std::string& prefix) { std::stringstream ss; std::string bam; arg >> bam; ss.str(std::string()); ss << prefix << std::setw(3) << std::setfill('0') << sample_number; obam[ss.str()] = bam; return bam; } bool __openWriterBam(const SeqLib::BamHeader& h, const std::string& name, SeqLib::BamWriter& wbam) { if (!wbam.Open(name)) return false; wbam.SetHeader(h);; if (!wbam.WriteHeader()) return false; return true; } /* bool __header_has_chr_prefix(bam_hdr_t * h) { for (int i = 0; i < h.NumSequences()) //->n_targets; ++i) if (h->target_name[i] && std::string(h->target_name[i]).find("chr") != std::string::npos) return true; return false; } */ //http://stackoverflow.com/questions/2114797/compute-median-of-values-stored-in-vector-c double CalcMHWScore(std::vector& scores) { double median; size_t size = scores.size(); std::sort(scores.begin(), scores.end()); if (size % 2 == 0) { median = (scores[size / 2 - 1] + scores[size / 2]) / 2; } else { median = scores[size / 2]; } return median; } int weightedRandom(const std::vector& cs) { // get a weighted random number size_t al = 0; double rand_val = rand() % 1000; while (al < cs.size()) { if (rand_val <= cs[al] * 1000) return al; ++al; } return al; } std::vector tokenize_delimited(const std::string& str, char delim) { std::vector tokens; size_t start = 0; size_t end = str.find(delim); while (end != std::string::npos) { tokens.push_back(str.substr(start, end - start)); start = end + 1; end = str.find(delim, start); } // Add the last token tokens.push_back(str.substr(start)); return tokens; } /// Check that the BAM header and BWA reference header line up. void checkHeaderCompatibility(const SeqLib::BamHeader& bamHeader, const SeqLib::BamHeader& refHeader, SvabaLogger& logger) { bool triggerExplain = false; if (bamHeader.NumSequences() != refHeader.NumSequences()) { triggerExplain = true; logger.log( true, true, //toerr, tolog "!!!!!!!!!!! WARNING !!!!!!!!!!!\n", "!!!!!! Number of sequences in BAM header mismatches reference\n", "!!!!!! BAM: ", bamHeader.NumSequences(), " -- Ref: ", refHeader.NumSequences() ); } if (bamHeader.NumSequences() != refHeader.NumSequences()) { triggerExplain = true; logger.log(true, true, //toerr, tolog "!!!!!!!!!!! WARNING !!!!!!!!!!!\n", "!!!!!! Number of sequences in BAM header mismatches reference\n" "!!!!!! BAM: ", bamHeader.NumSequences(), " -- Ref: ", refHeader.NumSequences()); } const int n = std::min(bamHeader.NumSequences(), refHeader.NumSequences()); for (int i = 0; i < n; ++i) { auto bamName = bamHeader.IDtoName(i); auto refName = refHeader.IDtoName(i); if (bamName != refName) { triggerExplain = true; logger.log(true, true, "!!!!!! BAM sequence id ", i, ": \"", bamName, "\"", " -- Ref sequence id ", i, ": \"", refName, "\""); } } if (triggerExplain) { logger.log(true, true, std::string(100, '!'), "\n", "!!! SvABA is being run with different reference genome than the reads were mapped to.\n", "!!! This can cause a massive failure in variant detection!\n", "!!! If you are *sure* that the two references are functionally equivalent (e.g. chr1 vs 1)\n", "!!! and that the order of the chromosomes is equivalent between the two,\n", "!!! you can override this error with option \"--override-reference-check\"\n", std::string(100, '!')); std::exit(EXIT_FAILURE); } } std::vector> find_repeats(std::string_view seq, size_t single_repeat_count = 0, size_t dinuc_repeat_count = 0) { std::vector> result; size_t n = seq.size(); // Check for single-base repeats (e.g., "AAAA") if (single_repeat_count > 0 && n >= single_repeat_count) { for (size_t i = 0; i <= n - single_repeat_count; ++i) { char base = seq[i]; size_t j = i + 1; while (j < n && seq[j] == base) ++j; size_t len = j - i; if (len >= single_repeat_count) { result.emplace_back(i, j - 1); i = j - 1; // advance to end of this repeat block } } } // Check for dinucleotide repeats (e.g., "AGAGAG") if (dinuc_repeat_count > 0 && n >= 2 * dinuc_repeat_count) { for (size_t i = 0; i <= n - 2 * dinuc_repeat_count; ++i) { std::string_view unit = seq.substr(i, 2); size_t j = i + 2; size_t count = 1; while (j + 1 < n && seq.substr(j, 2) == unit) { ++count; j += 2; } if (count >= dinuc_repeat_count) { result.emplace_back(i, j - 1); i = j - 2; // advance to end of this repeat block } } } return result; } std::vector parsePLString(const std::string& pl_str) { std::vector out; out.reserve(3); std::stringstream ss(pl_str); std::string tok; while (std::getline(ss, tok, ',')) { try { out.push_back(std::stoi(tok)); } catch (const std::exception& e) { throw std::runtime_error("Invalid integer in PL string: " + tok); } } if (out.size() != 3) { throw std::runtime_error("PL string must have exactly 3 comma-separated values"); } return out; } // thresholds constexpr size_t HOMOPOLYMER_MIN = 3; // e.g. 16 the same base constexpr size_t DINUC_REPEAT_MIN = 2; // e.g. 8 a nt motif SubstringList find_long_homopolymers(const std::string& s) { SubstringList results; if (s.size() < HOMOPOLYMER_MIN) return results; size_t run = 1; for (size_t i = 1; i < s.size(); ++i) { if (s[i] == s[i - 1]) { ++run; } else { if (run >= HOMOPOLYMER_MIN) { size_t end = i - 1; size_t start = end - run + 1; results.emplace_back(start, end, std::string(run, s[i - 1])); } run = 1; } } // Catch repeat at end of string if (run >= HOMOPOLYMER_MIN) { size_t end = s.size() - 1; size_t start = end - run + 1; results.emplace_back(start, end, std::string(run, s.back())); } return results; } SubstringList find_long_dinuc_repeats(const std::string& s) { SubstringList results; if (s.size() < 2 * DINUC_REPEAT_MIN) return results; for (size_t i = 0; i + 2 * DINUC_REPEAT_MIN <= s.size(); ++i) { std::string unit = s.substr(i, 2); size_t reps = 1; while (i + 2 * (reps + 1) <= s.size() && s.substr(i + 2 * reps, 2) == unit) { ++reps; } if (reps >= DINUC_REPEAT_MIN) { size_t start = i; size_t end = i + 2 * reps - 1; std::string repeated_seq; for (size_t r = 0; r < reps; ++r) repeated_seq += unit; results.emplace_back(start, end, repeated_seq); i = end - 1; // Skip ahead past this repeat } } return results; } } // end namespace svabaUtils ================================================ FILE: src/svaba/SvabaUtils.h ================================================ #pragma once #include #include #include #include #include #include #include "SvabaLogger.h" #include "SeqLib/BamReader.h" #include "SeqLib/BamWriter.h" #include "SeqLib/RefGenome.h" #include "SeqLib/GenomicRegion.h" typedef std::pair CountPair; typedef std::tuple Substring; typedef std::vector SubstringList; #define SRTAG(r) ((r).GetZTag("SR") + "_" + std::to_string((r).AlignmentFlag()) + "_" + (r).Qname()) namespace svabaUtils { // make a structure to store timing opt struct svabaTimer { static const std::string header; svabaTimer(); std::vector s; //clock_t curr_clock; std::chrono::time_point wall_start; double wall_elapsed = 0; void stop(const std::string& part); void start(); // print it friend std::ostream& operator<<(std::ostream &out, const svabaTimer st); CountPair weird_read_count = {0,0}; // tumor/normal weird reads CountPair mate_read_count = {0,0}; // tumor/normal weird reads size_t dc_read_count = 0; size_t dc_cluster_count = 0; size_t contig_count = 0; size_t aligned_contig_count; size_t bps_count; std::string logRuntime(const SeqLib::BamHeader& h); // run time std::unordered_map times; int pct_r = 0, pct_m = 0, pct_k = 0, pct_as = 0; // pct_pp = 0; SeqLib::GenomicRegion gr; }; std::string myreplace(std::string &s, std::string toReplace, std::string replaceWith); double CalcMHWScore(std::vector& scores); int overlapSize(const SeqLib::BamRecord& query, const SeqLib::BamRecordVector& subject); bool hasRepeat(const std::string& seq); std::string runTimeString(int num_t_reads, int num_n_reads, int contig_counter, const SeqLib::GenomicRegion& region, const SeqLib::BamHeader& h, const svabaTimer& st, const timespec& start); int countJobs(const std::string& regionFile, SeqLib::GRC &file_regions, SeqLib::GRC &run_regions, const SeqLib::BamHeader& h, int chunk, int window_pad); template void fopen(const std::string& s, T& o) { o.open(s.c_str(), std::ios::out); if (!o) { std::cerr << "Failed to open file: " << s << std::endl; exit(EXIT_FAILURE); } } std::string fileDateString(); bool __header_has_chr_prefix(bam_hdr_t * h); /** Generate a weighed random integer * @param cs Weighting for each integer (values must sum to one) * @return Random integer bounded on [0,cs.size()) */ int weightedRandom(const std::vector& cs); std::vector tokenize_delimited(const std::string& str, char delim); void checkHeaderCompatibility(const SeqLib::BamHeader& bamHeader, const SeqLib::BamHeader& refHeader, SvabaLogger& logger); std::vector> find_repeats(std::string_view seq, size_t single_repeat_count, size_t dinuc_repeat_count); std::vector parsePLString(const std::string& pl_str); SubstringList find_long_dinuc_repeats(const std::string& s); SubstringList find_long_homopolymers(const std::string& s); } ================================================ FILE: src/svaba/refilter.cpp ================================================ // refilter.cpp — re-apply LOD / PASS filtering to an existing bps.txt.gz // // The goal here is to avoid re-running assembly when the user only wants to // tweak filtering thresholds. We read bps.txt.gz line-by-line, reconstruct // BreakPoint objects via BreakPoint(line, sc*), rescore, redump, and // regenerate the VCFs. The `dbsnp` column is round-tripped verbatim from // the dump; DBSnp re-querying has been sunset. // // Requirements on the input file: // - Header line present (tab-delimited). Per-sample columns are expected to // begin with 't' (tumor) or 'n' (normal). Core column count is either 41 // (legacy pre-SvABA2.0) or 51 (SvABA2.0 refilter-aware dump). Both are // accepted. // - Column formatting matches BreakPoint::toFileString(). The BreakPoint // parse constructor tolerates sentinel values ("x", "NA", "nan", empty) // for ints/doubles. // // Caveats: // - Without contigs/reads, assembly-derived gates that re-examine sequence // cannot run. We rely on the dumped split/read counts and the scoring // primitives in svabaModels. // - Older dumps may leave per-end LocalAlignment, cpos, match, split_cov // bounds, and contig_len at defaults; those filters will degrade // gracefully rather than erroring out. #include "refilter.h" #include #include #include #include #include "gzstream.h" #include "SeqLib/BamReader.h" #include "vcf.h" #include "BreakPoint.h" #include "SvabaUtils.h" #include "SvabaLogger.h" #include "SvabaOptions.h" #include "SvabaOutputWriter.h" #include "SvabaSharedConfig.h" namespace opt { static std::string input_file; static std::string analysis_id = "refilter"; static std::string bam; // any BAM; used only for the sequence header static int verbose = 1; // Indel probability cutoffs (will be pushed into SvabaOptions before // scoring). Defaults mirror the in-tree SvabaOptions defaults so refilter // is non-destructive when no --lod* flags are passed. static double lod = 1.0; static double lod_db = 1.0; static double lod_somatic = 0.0; static double lod_somatic_db = 2.0; // Assumed mean read length when the dump lacks it. modelSelection uses this // for per-read error scaling; in practice most short-read BAMs are 101-151. static int readlen = 150; } enum { OPT_LOD, OPT_LOD_DB, OPT_LOD_SOMATIC, OPT_LOD_SOMATIC_DB, OPT_READLEN }; static const char* shortopts = "hi:a:v:G:b:"; static const struct option longopts[] = { { "help", no_argument, NULL, 'h' }, { "input-bps", required_argument, NULL, 'i' }, { "bam", required_argument, NULL, 'b' }, { "analysis-id", required_argument, NULL, 'a' }, { "verbose", required_argument, NULL, 'v' }, { "lod", required_argument, NULL, OPT_LOD }, { "lod-dbsnp", required_argument, NULL, OPT_LOD_DB }, { "lod-somatic", required_argument, NULL, OPT_LOD_SOMATIC }, { "lod-somatic-dbsnp", required_argument, NULL, OPT_LOD_SOMATIC_DB }, { "readlen", required_argument, NULL, OPT_READLEN }, { NULL, 0, NULL, 0 } }; static const char *BP_USAGE_MESSAGE = "Usage: svaba refilter [OPTION] -i bps.txt.gz -b \n\n" " Description: re-run LOD/PASS filtering against an existing bps.txt.gz\n" " without re-doing assembly. Samples, coverage, split counts, contig\n" " coordinates, and local-alignment flags are read back from the dump.\n" "\n" " General options\n" " -v, --verbose Verbosity level (0-4). Default: 1\n" " -h, --help Display this help and exit\n" " -a, --analysis-id Analysis ID for output filenames. [refilter]\n" " Required input\n" " -i, --input-bps bps.txt.gz produced by `svaba run`.\n" " -b, --bam Any svaba-input BAM (used for sequence header only).\n" " Optional\n" " --lod LOD for non-REF PASS. [1.0]\n" " --lod-dbsnp LOD for non-REF PASS at DBSnp sites. [1.0]\n" " --lod-somatic LOD for SOMATIC. [0.0]\n" " --lod-somatic-dbsnp LOD for SOMATIC at DBSnp sites. [2.0]\n" " --readlen Assumed read length when dump lacks it. [150]\n" "\n"; // parse the command line options static void parseBreakOptions(int argc, char** argv) { bool die = false; if (argc <= 2) die = true; for (char c; (c = getopt_long(argc, argv, shortopts, longopts, NULL)) != -1;) { std::istringstream arg(optarg != NULL ? optarg : ""); switch (c) { case 'h': die = true; break; case 'i': arg >> opt::input_file; break; case 'v': arg >> opt::verbose; break; case 'a': arg >> opt::analysis_id; break; case 'b': arg >> opt::bam; break; case OPT_LOD: arg >> opt::lod; break; case OPT_LOD_DB: arg >> opt::lod_db; break; case OPT_LOD_SOMATIC: arg >> opt::lod_somatic; break; case OPT_LOD_SOMATIC_DB: arg >> opt::lod_somatic_db; break; case OPT_READLEN: arg >> opt::readlen; break; } } if (opt::input_file.empty()) { std::cerr << "ERROR: --input-bps is required\n"; die = true; } if (opt::bam.empty()) { std::cerr << "ERROR: --bam is required (for the sequence header)\n"; die = true; } if (die) { std::cerr << "\n" << BP_USAGE_MESSAGE; exit(EXIT_FAILURE); } } // Parse a bps.txt sample-column header token like "t001_/path/to.bam" into // (prefix, path). If there is no underscore, the whole token is treated as // the prefix. static std::pair splitSampleHeader(const std::string& tok) { const auto us = tok.find('_'); if (us == std::string::npos) return { tok, "" }; return { tok.substr(0, us), tok.substr(us + 1) }; } void runRefilterBreakpoints(int argc, char** argv) { parseBreakOptions(argc, argv); if (opt::verbose > 0) { std::cerr << "Input bps file: " << opt::input_file << std::endl << "Output analysis id: " << opt::analysis_id << std::endl << " LOD cutoff (non-REF): " << opt::lod << std::endl << " LOD cutoff (non-REF, at DBSNP): " << opt::lod_db << std::endl << " LOD somatic cutoff: " << opt::lod_somatic << std::endl << " LOD somatic cutoff (at DBSNP): " << opt::lod_somatic_db << std::endl; } if (!SeqLib::read_access_test(opt::input_file)) { std::cerr << "ERROR: Cannot read " << opt::input_file << std::endl; exit(EXIT_FAILURE); } // Open the BAM only to grab its header; refilter does not read any reads. SeqLib::BamReader bwalker; if (!bwalker.Open(opt::bam)) { std::cerr << "ERROR: could not open BAM " << opt::bam << std::endl; exit(EXIT_FAILURE); } const SeqLib::BamHeader hdr = bwalker.Header(); // ------------------------------------------------------------------ // Read the bps.txt.gz header and recover sample prefixes. // // Layouts supported: // legacy (41 core cols): ..., contig_conf1, contig_conf2, // svaba2 (51 core cols): ..., contig_conf1, contig_conf2, // cpos1, cpos2, lmatch, rmatch, scov1, scov2, // local1, local2, ctglen, flipped, // // // Sample header tokens are of the form "{prefix}_{bampath}" where prefix // begins with 't' or 'n'. We locate the sample block by scanning for the // first column whose name starts with 't' or 'n' past the fixed-width // prefix (col 34+ is safe: no core column from there starts t/n). // ------------------------------------------------------------------ igzstream infile(opt::input_file.c_str(), std::ios::in); std::string headerLine; if (!std::getline(infile, headerLine)) { std::cerr << "ERROR: bps.txt file is empty or unreadable" << std::endl; exit(EXIT_FAILURE); } const std::vector headerv = svabaUtils::tokenize_delimited(headerLine, '\t'); if (headerv.size() < 41) { std::cerr << "ERROR: bps.txt header has only " << headerv.size() << " columns; expected at least 41 (legacy) or 51 (svaba2)." << " File may be truncated or from an unsupported svaba version." << std::endl; exit(EXIT_FAILURE); } size_t sample_start = 0; for (size_t i = 34; i < headerv.size(); ++i) { if (!headerv[i].empty() && (headerv[i].at(0) == 't' || headerv[i].at(0) == 'n')) { sample_start = i; break; } } if (sample_start == 0) { std::cerr << "ERROR: could not locate sample-name columns in bps.txt header." << " No column starting with 't' or 'n' found after the core block." << std::endl; exit(EXIT_FAILURE); } // Build a minimal SvabaSharedConfig sufficient to drive BreakPoint parsing // and rescoring. SvabaOutputWriter is constructed but not `init`-ed; no // files are opened by it. We open our own bps output stream below. SvabaLogger logger; SvabaOptions opts; SvabaOutputWriter writer(logger, opts); // Push CLI-provided cutoffs into opts; score_indel() reads these. opts.lod = opt::lod; opts.lodDb = opt::lod_db; opts.lodSomatic = opt::lod_somatic; opts.lodSomaticDb = opt::lod_somatic_db; // Populate opts.bams from the header. The BreakPoint parse constructor // iterates sc->opts.bams (a std::map) in sorted key order, so as long as // the dump also iterated opts.bams in sorted order (which it does), the // per-sample token order is round-trip-safe. Key is the sample prefix // (e.g. "t001"); the value (bam path from the header token) is only used // here for the reconstructed header line. std::vector allele_names; // raw header tokens, for info/debug std::vector sample_prefixes; for (size_t i = sample_start; i < headerv.size(); ++i) { allele_names.push_back(headerv[i]); auto [pref, path] = splitSampleHeader(headerv[i]); if (pref.empty() || (pref.at(0) != 't' && pref.at(0) != 'n')) { std::cerr << "ERROR: unexpected sample header token '" << headerv[i] << "' (col " << i << "); " << "expected to start with 't' or 'n'." << std::endl; exit(EXIT_FAILURE); } // Preserve the original bam path from the header (may be empty if the // header token was just a bare prefix). Used only for the re-emitted // header line and (incidentally) as the value in the map iterated by // the BreakPoint parse constructor. opts.bams[pref] = path; sample_prefixes.push_back(pref); } if (opt::verbose > 0) { std::cerr << "...bps.txt has " << headerv.size() << " columns; " << sample_start << " core, " << (headerv.size() - sample_start) << " sample(s): "; for (const auto& p : sample_prefixes) std::cerr << p << " "; std::cerr << std::endl; } // Build the SvabaSharedConfig SvabaSharedConfig sc(logger, opts, writer); sc.header = hdr; sc.readlen = opt::readlen; // (DBSnp filtering has been sunset — refilter no longer supports it. The // `dbsnp` column in bps.txt.gz is round-tripped verbatim from the dump.) // VCF header (minimal) VCFHeader vheader; vheader.filedate = svabaUtils::fileDateString(); vheader.source = "svaba refilter"; vheader.reference = ""; // Open the rewritten bps file and write its header. We re-emit the current // BreakPoint::header() layout so downstream tooling always sees the // newest column set (the reader above was backward-compatible with the // older 41-col dumps, but we prefer to always emit the modern format). const std::string new_bps_file = opt::analysis_id + ".bps.txt.gz"; ogzstream os_bps; svabaUtils::fopen(new_bps_file, os_bps); os_bps << BreakPoint::header(); for (auto& p : opts.bams) os_bps << "\t" << p.first << "_" << p.second; os_bps << "\n"; // --------------------------- main loop --------------------------- size_t line_count = 0; size_t parse_errors = 0; std::string line; while (std::getline(infile, line)) { if (line.empty()) continue; if (line[0] == '#') continue; // defensive if (opt::verbose > 0 && line_count && (line_count % 100000) == 0) std::cerr << "...read " << opt::input_file << " at line " << SeqLib::AddCommas(line_count) << std::endl; std::unique_ptr bp; try { bp.reset(new BreakPoint(line, &sc)); } catch (const std::exception& e) { ++parse_errors; if (parse_errors <= 5) { std::cerr << "WARN: parse error at line " << (line_count + 1) << ": " << e.what() << std::endl; } ++line_count; continue; } // Roll discordant counts from per-sample disc into dc.{tcount,ncount}. // The original scorer sets these during assembly; they may not be // present in the dump's dc fields, so derive from per-sample `disc`. bp->dc.tcount = 0; bp->dc.ncount = 0; for (auto& [pref, si] : bp->allele) { if (!pref.empty() && pref.at(0) == 't') bp->dc.tcount += si.disc; else bp->dc.ncount += si.disc; } // Clear derived fields so scoreBreakpoint() can repopulate from scratch. // The parse sets `confidence` from the dump; scoreBreakpoint() asserts // it's empty (except for BLACKLIST), so blank it here unless already // BLACKLIST (which is a terminal state we want to preserve). if (bp->confidence != "BLACKLIST") bp->confidence.clear(); // Re-score try { bp->scoreBreakpoint(); } catch (const std::exception& e) { std::cerr << "WARN: scoreBreakpoint failed at line " << (line_count + 1) << ": " << e.what() << std::endl; ++line_count; continue; } // toFileString() needs the BAM header to render chromosome names; // read-tracking is no longer a per-call toggle (supporting-read qnames // are stored or stripped elsewhere in the write path). os_bps << bp->toFileString(hdr) << "\n"; ++line_count; } os_bps.close(); if (opt::verbose > 0) { std::cerr << "...refilter read " << line_count << " lines" << " (" << parse_errors << " parse error" << (parse_errors == 1 ? "" : "s") << ")" << std::endl; } // ---- regenerate VCFs from the refiltered bps ---- if (SeqLib::read_access_test(new_bps_file)) { if (opt::verbose > 0) std::cerr << "...making the primary VCFs (unfiltered and filtered) from file " << new_bps_file << std::endl; VCFFile snowvcf(new_bps_file, opt::analysis_id, sc, vheader, true, opt::verbose > 0); const bool onefile = (allele_names.size() == 1); std::string basename = opt::analysis_id + ".svaba.unfiltered."; snowvcf.include_nonpass = true; snowvcf.writeIndels(basename, false, onefile, hdr); snowvcf.writeSVs (basename, false, onefile, hdr); basename = opt::analysis_id + ".svaba."; snowvcf.include_nonpass = false; snowvcf.writeIndels(basename, false, onefile, hdr); snowvcf.writeSVs (basename, false, onefile, hdr); } else { std::cerr << "Failed to make VCF. Could not find rewritten bps file " << new_bps_file << std::endl; } } ================================================ FILE: src/svaba/refilter.h ================================================ #pragma once // Re-run LOD / PASS filtering against an existing bps.txt.gz. See refilter.cpp // for option parsing and the supported input layouts (41-col legacy and // 51-col SvABA2.0). Invoked from svaba.cpp as `svaba refilter ...`. void runRefilterBreakpoints(int argc, char** argv); ================================================ FILE: src/svaba/run_svaba.cpp ================================================ //run_svaba.cpp #include #include #include "threadpool.h" #include #include #include #include #include #include #include #include #include #include "SeqLib/GenomicRegion.h" #include "SeqLib/UnalignedSequence.h" #include "SeqLib/ReadFilter.h" #include "SeqLib/BamHeader.h" #include "SeqLib/BWAAligner.h" #include "SeqLib/ReadFilter.h" #include "SvabaRegionProcessor.h" #include "SvabaLogger.h" #include "KmerFilter.h" #include "vcf.h" #include "DBSnpFilter.h" #include "SvabaUtils.h" #include "SeqLib/BFC.h" #include "SvabaFileLoader.h" #include "SvabaOutputWriter.h" #include "SvabaAssemblerEngine.h" #include "SvabaAssemblerConfig.h" using SeqLib::BWAIndex; using SeqLib::BWAAligner; using SeqLib::BamHeader; using SeqLib::Filter::ReadFilterCollection; using SeqLib::GenomicRegion; using SeqLib::GRC; using SeqLib::BWAIndexPtr; using std::string; using std::cerr; using std::endl; using std::make_shared; // 1 is normal logging // 2 is heavy constexpr inline int log_level = 1; // forward declaration, need non-static and not in // anonymous namespace to keep external linkage void runsvaba (int argc, char** argv); struct SvabaBatchWorkItem { // batch of region + unique region ID pairs std::vector> items; SvabaRegionProcessor& processor; // constructor takes a reference to the shared processor SvabaBatchWorkItem(const std::vector>& items_, SvabaRegionProcessor& proc) : items(items_), processor(proc) {} // call operator matching SvabaWorkItem signature bool operator()(svabaThreadUnit& unit, size_t threadId) const { for (auto& [region, id] : items) { processor.process(region, unit, threadId); } return true; } }; // Modified sendThreads() to submit batches of regions instead of one-by-one jobs void sendThreads(const SeqLib::GRC& regionsToRun, SvabaSharedConfig& sc) { // shared processor instance SvabaRegionProcessor proc(sc); // thread pool now handles SvabaBatchWorkItem jobs ThreadPool pool(sc); size_t batch_size = 1; if (sc.opts.numThreads > 0) { size_t per_thread = sc.total_regions_to_process / sc.opts.numThreads; if (per_thread < batch_size) { batch_size = std::max(1, per_thread); // prevent batch_size = 0 } } std::vector> batch; batch.reserve(batch_size); int count = 0; for (auto& region : regionsToRun) { batch.emplace_back(region, ++count); if (batch.size() == batch_size) { pool.submit(std::make_unique(batch, proc)); batch.clear(); } } // submit any leftover regions as one final batch if (!batch.empty()) { pool.submit(std::make_unique(batch, proc)); } pool.shutdown(); } void makeVCFs(SvabaSharedConfig& sc) { // // make the VCF file string file = sc.opts.analysisId + ".bps.txt.gz"; sc.logger.log(true, true, "...loading the bps file ", file," for conversion to VCF"); // // make the header VCFHeader header; header.filedate = svabaUtils::fileDateString(); header.source = sc.args; header.reference = sc.opts.refGenome; for (int i = 0; i < sc.header.NumSequences(); ++i) header.addContigField(sc.header.IDtoName(i),sc.header.GetSequenceLength(i)); for (auto& b : sc.opts.bams) { string fname = b.second; //bpf.filename(); header.addSampleField(fname); header.colnames += "\t" + fname; } // check if it has a matched control. If so, output "somatic / germline" vcfs bool case_control_run = false; for (auto& b : sc.opts.bams) if (b.first.at(0) == 'n') case_control_run = true; // primary VCFs sc.logger.log(true, true, "...making the primary VCFs (unfiltered and filtered) from file ",file); VCFFile snowvcf(file, sc.opts.analysisId, sc, header, false, sc.opts.verbose > 0); string basename = sc.opts.analysisId + ".svaba.unfiltered."; snowvcf.include_nonpass = true; sc.logger.log(true, true, "...writing unfiltered VCFs"); snowvcf.writeIndels(basename, false, !case_control_run, sc.header); snowvcf.writeSVs(basename, false, !case_control_run, sc.header); sc.logger.log(true, true, "...writing filtered VCFs"); basename = sc.opts.analysisId + ".svaba."; snowvcf.include_nonpass = false; snowvcf.writeIndels(basename, false, !case_control_run, sc.header); snowvcf.writeSVs(basename, false, !case_control_run, sc.header); } void runsvaba(int argc, char** argv) { // parse command line SvabaOptions opts; try { opts = SvabaOptions::parse(argc, argv); if (opts.help) { SvabaOptions::printUsage(); return; } } catch (const std::exception& e) { std::cerr << "ERROR: " << e.what() << "\n"; return; } // instantiate logger, options, writer here. // its just easier then to store refernce in SvabaSharedConfig // to avoid header dependencies SvabaLogger logger; logger.init(opts.analysisId + ".log"); //logger.welcome(opts); // initial message // open the reference genome logger.log(true, true, "...loading the reference sequence for BWA"); BWAIndexPtr bwa_idx = make_shared(); bwa_idx->LoadIndex(opts.refGenome); // get the dictionary from reference BamHeader bwa_header = bwa_idx->HeaderFromIndex(); // open the writer SvabaOutputWriter writer(logger, opts); writer.init(opts.analysisId, bwa_header); // shared information to be passed around thread units SvabaSharedConfig sc(logger, opts, writer); sc.bwa_idx = bwa_idx; sc.header = bwa_header; // start the timer clock_gettime(CLOCK_MONOTONIC, &sc.start); // report which local-assembly engine was compiled in std::cerr << "...local-assembly engine: " << svaba::kAssemblerName #if SVABA_ASSEMBLER_FERMI << " (FERMI assembly enabled)" #else << " (SGA assembly enabled)" #endif << std::endl; // check that the two headers are equivalant // open the main bam to get header info SeqLib::BamReader first_tumor_bam_reader; if (!first_tumor_bam_reader.Open(opts.main_bam)) { cerr << "ERROR: Cannot open main bam file: " << opts.main_bam << endl; exit(EXIT_FAILURE); } SeqLib::BamHeader first_header = first_tumor_bam_reader.Header(); svabaUtils::checkHeaderCompatibility(first_header, bwa_header, logger); // Check input BAM @PG tags for BWA. When --always-realign-corrected is // NOT set, svaba reuses the input BAM's native CIGAR/NM for unchanged // reads in the r2c-vs-native gate. That comparison is only valid if the // input was aligned with BWA (same scoring model as svaba's internal // aligner). If the PG chain doesn't mention "bwa", warn loudly. if (!opts.alwaysRealignCorrected) { std::string hdr_text = first_header.AsString(); // case-insensitive search: look for "bwa" anywhere in @PG lines bool found_bwa = false; std::istringstream hdr_stream(hdr_text); std::string line; while (std::getline(hdr_stream, line)) { if (line.substr(0, 3) != "@PG") continue; // case-insensitive: lowercase the line for matching std::string lower_line = line; std::transform(lower_line.begin(), lower_line.end(), lower_line.begin(), ::tolower); if (lower_line.find("bwa") != std::string::npos) { found_bwa = true; break; } } if (!found_bwa) { logger.log(true, true, ""); logger.log(true, true, "************************************************************"); logger.log(true, true, "* WARNING: No BWA @PG tag found in input BAM header. *"); logger.log(true, true, "* svaba reuses the input BAM's CIGAR/NM for reads that *"); logger.log(true, true, "* BFC did not modify. This is only valid when the input *"); logger.log(true, true, "* was aligned with BWA-MEM (same scoring model as svaba's *"); logger.log(true, true, "* internal aligner). *"); logger.log(true, true, "* *"); logger.log(true, true, "* If this BAM was NOT aligned with BWA, re-run with: *"); logger.log(true, true, "* --always-realign-corrected *"); logger.log(true, true, "* to force re-alignment of every read to the reference. *"); logger.log(true, true, "************************************************************"); logger.log(true, true, ""); } } // open file loader SvabaFileLoader loader(sc); // open the blacklist, load into sc.blacklist for (const auto& path : sc.opts.blacklistFile) { SeqLib::GRC this_blacklist; loader.loadBedRegions(path, this_blacklist); sc.blacklist.Concat(this_blacklist); } sc.blacklist.MergeOverlappingIntervals(); sc.blacklist.CreateTreeMap(); logger.log(true, true, "...loaded ", SeqLib::AddCommas(sc.blacklist.size()), " blacklist regions across ", SeqLib::AddCommas(sc.opts.blacklistFile.size()), " files"); // open the germline sv database loader.loadBedRegions(sc.opts.germlineSvFile, sc.germline_svs); // open the DBSnpFilter if (sc.opts.dbsnpVcf.length()) { sc.dbsnp_filter = std::make_shared(opts.dbsnpVcf, bwa_header, logger); } // needed for aligned contig for (auto& b : opts.bams) sc.prefixes.insert(b.first); // parse the region file, count number of jobs SeqLib::GRC regionsToRun; loader.countJobs(regionsToRun); // SvABA2.0: drop any queued region that is 100% covered by the blacklist. // svaba already filters reads by blacklist *inside* each region (see // SvabaRegionProcessor.cpp:74), so a chunk that sits entirely in a // blacklisted contig (e.g. the chrUn / *_decoy / *_alt contigs in the // nonstd_chr blacklist) would otherwise still get its full region // pipeline run — open the BAM walker, learn params, attempt local // assembly on a stream of reads that all get dropped — just to produce // zero callable bases. Net behavior is unchanged; this is pure // optimization of the queue. // // The blacklist has had MergeOverlappingIntervals() + CreateTreeMap() // called already (a few lines above), so FindOverlapWidth is an O(k log n) // tree walk over the blacklist intervals that touch each region. Runs // once, pre-queue, so it's off the critical path. if (!sc.blacklist.empty() && regionsToRun.size() > 0) { SeqLib::GRC kept; size_t dropped_n = 0; size_t dropped_bp = 0; for (const auto& r : regionsToRun) { const int w = r.Width(); if (w > 0 && static_cast(sc.blacklist.FindOverlapWidth(r, /*ignore_strand=*/true)) >= w) { ++dropped_n; dropped_bp += static_cast(w); continue; } kept.add(r); } if (dropped_n > 0) { logger.log(true, true, "...pruned ", SeqLib::AddCommas(dropped_n), " of ", SeqLib::AddCommas(regionsToRun.size()), " queued regions (", SeqLib::AddCommas(dropped_bp), " bp) that were 100% covered by the blacklist"); regionsToRun = std::move(kept); // No runtime.txt line will be emitted for a pruned region; the // accounting in sc.total_regions_to_process below reflects what // actually gets queued. } } sc.total_regions_to_process = regionsToRun.size(); if (sc.total_regions_to_process < opts.numThreads) { opts.numThreads = sc.total_regions_to_process; } // debug /* regionsToRun.clear(); for (int i = 0; i < 10; i++) regionsToRun.add(GenomicRegion(2,89000000,91500000)); */ // --- learn the insert-sizes --- logger.log(true, true,"...learning insert size distribution across all BAMs; this may take a while"); // learn from the BAM files for (const auto& b : opts.bams) { auto [it, inserted] = sc.bamStats.emplace(b.first, LearnBamParams(sc, b.second)); logger.log(true,true,"......learning BAM: ", b.second); it->second.learnParams(); } // --- report what we learned --- for (const auto& bs : sc.bamStats) { logger.log(opts.verbose > 1, true, "=====", bs.first); for (const auto& brg : bs.second.bam_read_groups) logger.log(opts.verbose > 1, true, brg.first, " - ", brg.second); } int globalReadLen = 0; int globalMaxMapQ = 0; double globalInsertSize = 0; for (const auto& [_, learn_bam_param] : sc.bamStats) { globalReadLen = std::max(globalReadLen, learn_bam_param.readlen_max); globalMaxMapQ = std::max(globalMaxMapQ, learn_bam_param.mapq_max); globalInsertSize = std::max(globalInsertSize, learn_bam_param.isize_max); } sc.readlen = globalReadLen; sc.insertsize = globalInsertSize; // --- set the SGA min overlap if user didn't --- if (opts.sgaMinOverlap == 0) { opts.sgaMinOverlap = std::max(30, int(0.6 * globalReadLen)); } logger.log(true, true, "... found max read length = ", globalReadLen, "; SGA minOverlap = ", opts.sgaMinOverlap, "; max MAPQ = ", globalMaxMapQ ); // --- compute seed parameters --- int seedLength, seedStride; { svabaAssemblerEngine tester( "test", opts.sgaErrorRate, opts.sgaMinOverlap, globalReadLen ); tester.calculateSeedParameters( globalReadLen, opts.sgaMinOverlap, seedLength, seedStride ); } logger.log(opts.verbose > 0, true, "...seedLength = ", seedLength,", readlen=", globalReadLen, ")" ); // FR isize discordant cutoff is now applied per-RG directly in // svabaBamWalker::readBam (via getIsizeCutoff), using the same // formula as TagDiscordant: median + SD * sdDiscCutoff. // No more per-RG FR rules in ReadFilterCollection. // set the ReadFilterCollection to be applied to each region logger.log(opts.verbose > 1, true, opts.rulesJson); sc.mr = ReadFilterCollection(opts.rulesJson, sc.header); logger.log(opts.verbose > 0, true, sc.mr); // put args into string for VCF later sc.args += "(v" + string(SVABA_VERSION) + ") "; for (int i = 0; i < argc; ++i) sc.args += string(argv[i]) + " "; if (regionsToRun.size()) { logger.log(true, true, "...running on ", SeqLib::AddCommas(regionsToRun.size()), " chunks"); } else { logger.log(true, true, "Chunk was <= 0: Reading in whole-genome at once"); } // send the jobs to the queue logger.log(true, true, "Starting detection pipeline"); sendThreads(regionsToRun, sc); // close the writer writer.close(); // make the VCF file //makeVCFs(sc); cerr << SeqLib::displayRuntime(sc.start) << endl; // suggest postprocess command std::string stars(72, '*'); logger.log(true, true, ""); logger.log(true, true, stars); logger.log(true, true, " svaba run complete for: ", opts.analysisId); logger.log(true, true, ""); logger.log(true, true, " Next step — sort, dedup, and filter outputs:"); logger.log(true, true, ""); logger.log(true, true, " svaba_postprocess.sh -t 8 -m 4G ", opts.analysisId); logger.log(true, true, ""); logger.log(true, true, " Then convert to VCF:"); logger.log(true, true, ""); logger.log(true, true, " svaba tovcf -i ", opts.analysisId, ".bps.sorted.dedup.txt.gz -b ", opts.main_bam, " -a ", opts.analysisId); logger.log(true, true, stars); logger.log(true, true, ""); } ================================================ FILE: src/svaba/svaba.cpp ================================================ /* SvABA - Somatic Structural Variation Dectection * Copyright 2014 Broad Institute * Written by Jeremiah Wala (jeremiah.wala@gmail.com) * Released under the included license detailed below. * * SvABA incorportes the core of String Graph Assembler, * -- String Graph Assembler -- Copyright 2009 Wellcome Trust Sanger Institute * -- Written by Jared Simpson * -- Released under the GPL */ // svaba.cpp #include #include #include "SvabaOptions.h" void runToVCF(int argc, char** argv); void runsvaba(int argc, char** argv); void runRefilterBreakpoints(int argc, char** argv); void runPostprocess(int argc, char** argv); // "Secret" benchmark subcommand: deliberately NOT listed in printUsage() // below so it doesn't show up in the user-facing --help. Invoke with // `svaba test -h` to see its own usage. Used to isolate the per-phase // CPU cost of the main hot paths (walk / correct / assemble / align) // independently of the real pipeline's bookkeeping and scoring. void runTest(int argc, char** argv); static void printUsage() { constexpr std::string_view header = "------------------------------------------------------------\n" "-------- SvABA - SV and indel detection by assembly --------\n" "------------------------------------------------------------\n"; std::cout << header << "Program: SvABA\n" << "Version: " << SVABA_VERSION << " - " << SVABA_DATE << "\n\n" << "Usage: svaba [options]\n\n" << "Commands:\n" << " run Run SV and indel detection on BAM(s)\n" << " refilter Re-run LOD/PASS filtering on an existing bps.txt.gz\n" << " postprocess Sort + streaming-dedup per-suffix output BAMs\n" << " tovcf Convert a deduped bps.txt.gz into VCFv4.5 output\n\n" << "Report issues at https://github.com/walaj/svaba/issues\n"; } int main(int argc, char* argv[]) { if (argc < 2) { printUsage(); return EXIT_SUCCESS; } std::string_view cmd = argv[1]; if (cmd == "help" || cmd == "--help") { printUsage(); return EXIT_SUCCESS; } else if (cmd == "run") { // strip off the run before passing to parser return (runsvaba(argc - 1, argv + 1), EXIT_SUCCESS); } else if (cmd == "refilter") { return (runRefilterBreakpoints(argc - 1, argv + 1), EXIT_SUCCESS); } else if (cmd == "postprocess") { return (runPostprocess(argc - 1, argv + 1), EXIT_SUCCESS); } else if (cmd == "tovcf") { return (runToVCF(argc - 1, argv + 1), EXIT_SUCCESS); } // Secret benchmark subcommand — intentionally not in the user-facing // command list. Entry point: `svaba test -h` for its own usage. else if (cmd == "test") { return (runTest(argc - 1, argv + 1), EXIT_SUCCESS); } else { std::cerr << "Unknown command: " << cmd << "\n\n"; printUsage(); return EXIT_FAILURE; } std::cerr << "Done with SvABA" << std::endl; return 0; } ================================================ FILE: src/svaba/test_svaba.cpp ================================================ // test_svaba.cpp // // `svaba test` — a "secret" subcommand (not listed in the top-level help) // that runs a minimal microbenchmark of svaba's five main hot paths: // // 1. walk — read N reads from a BAM // 2. correct — BFC error-correct those reads // 3. realign — BWA-MEM align each *corrected* read back to the reference. // This mirrors the per-read native alignment that // BreakPoint::splitCoverage needs for its r2c-vs-native // comparative gate (SVABA_R2C_NATIVE_GATE). On a real // svaba run this is currently an underrated cost: N // BWA-MEM calls PER WINDOW, where N is a few thousand // on a typical WGS, times ~120k windows for a full run. // 4. assemble — fermi-lite assemble the reads // 5. align — BWA-MEM align the resulting contigs back to a reference // // Each phase is timed independently across a sweep of N values (default // 100, 500, 1 000, 5 000, 10 000, 50 000) so you can eyeball how each // phase scales. Results are not checked — the point is wall-clock per- // phase, not correctness. Repeat each trial -r times; the median is // reported (first-trial cold-cache effects wash out). // // Designed to isolate the bottleneck profile independently of the real // svaba pipeline's bookkeeping, scoring, and BAM writing — so when // someone says "svaba is slow" we can point at a phase. // // Usage: // svaba test -i BAM -G REF [-n N ...] [-k REGION] [-r REPEATS] [--skip-*] // // -i FILE input BAM (any BAM with reads; aligned status is irrelevant) // -G FILE BWA-indexed reference FASTA (needs .bwt / .pac / etc) // -n N reads-per-trial; may repeat. Default sweep is // 100 / 500 / 1000 / 5000 / 10000 / 50000. // -k REGION samtools-style region (chr:start-end) to restrict the BAM // read. Defaults to the beginning of the file. // -r N repetitions per trial (default 3). Reports median. // --skip-walk (benchmark still does the read-gathering; // just doesn't include it in the phase table) // --skip-ec skip BFC error correction // --skip-assemble skip fermi-lite assembly // --skip-align skip BWA-MEM alignment // -v, --verbose // -h, --help #include #include #include #include #include #include #include #include #include #include #include #include "SeqLib/BamReader.h" #include "SeqLib/BamRecord.h" #include "SeqLib/BFC.h" #include "SeqLib/BWAAligner.h" #include "SeqLib/BWAIndex.h" #include "SeqLib/FermiAssembler.h" #include "SeqLib/UnalignedSequence.h" namespace { struct TestOpts { std::string bam; std::string ref; std::string region; std::vector n_sweep; int repeats = 3; bool skip_walk = false; bool skip_ec = false; bool skip_realign = false; bool skip_assemble = false; bool skip_align = false; int verbose = 0; }; constexpr const char* kUsage = "svaba test — microbenchmark the four main svaba hot paths.\n\n" "Usage:\n" " svaba test -i BAM -G REF [-n N ...] [-k REGION] [-r REPEATS] [options]\n\n" " -i, --bam FILE input BAM (reads source)\n" " -G, --reference FILE BWA-indexed reference (for alignment phase)\n" " -n, --num-reads N trial size; may be passed multiple times.\n" " Default sweep: 500, 1000, 2000, 3000, 5000, 10000 —\n" " the 2k-3k range spans typical svaba 25kb-window loads.\n" " -k, --region REGION samtools-style region to restrict BAM reading\n" " -r, --repeats N per-trial repetitions (median reported, default 3)\n" " --skip-walk exclude walk phase from the table (reads are still fetched)\n" " --skip-ec skip BFC error correction\n" " --skip-realign skip BWA-MEM realignment of corrected reads\n" " --skip-assemble skip fermi-lite assembly\n" " --skip-align skip BWA-MEM alignment of the produced contigs\n" " -v, --verbose chatty diagnostics\n" " -h, --help this message\n"; constexpr int OPT_SKIP_WALK = 3001; constexpr int OPT_SKIP_EC = 3002; constexpr int OPT_SKIP_REALIGN = 3003; constexpr int OPT_SKIP_ASSEMBLE = 3004; constexpr int OPT_SKIP_ALIGN = 3005; const struct option LONGOPTS[] = { { "bam", required_argument, nullptr, 'i' }, { "reference", required_argument, nullptr, 'G' }, { "num-reads", required_argument, nullptr, 'n' }, { "region", required_argument, nullptr, 'k' }, { "repeats", required_argument, nullptr, 'r' }, { "skip-walk", no_argument, nullptr, OPT_SKIP_WALK }, { "skip-ec", no_argument, nullptr, OPT_SKIP_EC }, { "skip-realign", no_argument, nullptr, OPT_SKIP_REALIGN }, { "skip-assemble", no_argument, nullptr, OPT_SKIP_ASSEMBLE }, { "skip-align", no_argument, nullptr, OPT_SKIP_ALIGN }, { "verbose", no_argument, nullptr, 'v' }, { "help", no_argument, nullptr, 'h' }, { nullptr, 0, nullptr, 0 } }; TestOpts parseCli(int argc, char** argv) { TestOpts o; bool die = (argc <= 2); optind = 1; for (int c; (c = getopt_long(argc, argv, "hvi:G:n:k:r:", LONGOPTS, nullptr)) != -1;) { switch (c) { case 'h': die = true; break; case 'v': o.verbose = 1; break; case 'i': o.bam = optarg ? optarg : ""; break; case 'G': o.ref = optarg ? optarg : ""; break; case 'k': o.region = optarg ? optarg : ""; break; case 'r': o.repeats = std::max(1, std::atoi(optarg ? optarg : "3")); break; case 'n': { try { o.n_sweep.push_back(std::stoull(optarg ? optarg : "")); } catch (...) { std::cerr << "ERROR: -n expects a positive integer\n"; std::exit(EXIT_FAILURE); } break; } case OPT_SKIP_WALK: o.skip_walk = true; break; case OPT_SKIP_EC: o.skip_ec = true; break; case OPT_SKIP_REALIGN: o.skip_realign = true; break; case OPT_SKIP_ASSEMBLE: o.skip_assemble = true; break; case OPT_SKIP_ALIGN: o.skip_align = true; break; default: die = true; break; } } if (!die) { if (o.bam.empty()) { std::cerr << "ERROR: -i BAM required\n"; die = true; } // Reference is needed for both the contig-align AND the corrected-read // realign phase. Require it unless BOTH are skipped. if ((!o.skip_align || !o.skip_realign) && o.ref.empty()) { std::cerr << "ERROR: -G REF required unless --skip-align AND --skip-realign\n"; die = true; } } if (die) { std::cerr << "\n" << kUsage; std::exit(1); } if (o.n_sweep.empty()) // Default sweep targets the real per-window range (~2k-3k reads/window // on typical WGS) with brackets below and above to show scaling. o.n_sweep = { 500, 1000, 2000, 3000, 5000, 10000 }; return o; } // Read up to N BamRecord from the BAM, optionally restricted to `region`. // Anything shorter than `min_read_len` (e.g. unmapped stubs) is skipped so // the benchmark doesn't get polluted by degenerate records. SeqLib::BamRecordVector readNReads(const std::string& bam, const std::string& region, size_t N, int min_read_len = 30) { SeqLib::BamReader r; if (!r.Open(bam)) { std::cerr << "ERROR: cannot open " << bam << "\n"; std::exit(EXIT_FAILURE); } if (!region.empty()) { SeqLib::GenomicRegion gr(region, r.Header()); if (!r.SetRegion(gr)) { std::cerr << "ERROR: SetRegion failed for '" << region << "'\n"; std::exit(EXIT_FAILURE); } } SeqLib::BamRecordVector out; out.reserve(N); while (auto opt = r.Next()) { SeqLib::BamRecord& rec = *opt; if (rec.Length() < min_read_len) continue; // BamRecord is move-only (holds a shared_ptr); transfer rather // than copy. r.Next() just handed us ownership via the optional, so // we move out of it and drop it before the next loop iteration. out.push_back(std::move(rec)); if (out.size() >= N) break; } return out; } // Per-trial timing result. Fields are wall-clock seconds; 0.0 means the // phase was skipped. struct Phase { double walk = 0.0; double ec = 0.0; double realign = 0.0; // BWA-MEM of corrected reads -> reference double assem = 0.0; // "asm" is GCC-reserved (inline assembly) double align = 0.0; // BWA-MEM of contigs -> reference double total() const { return walk + ec + realign + assem + align; } }; double since(std::chrono::steady_clock::time_point t0) { return std::chrono::duration(std::chrono::steady_clock::now() - t0).count(); } Phase oneTrial(const TestOpts& opts, size_t N, SeqLib::BWAIndexPtr& bwa_idx) { Phase p; using clock = std::chrono::steady_clock; // ---- Phase 1: walk (always runs; --skip-walk just hides it in the table). auto t0 = clock::now(); SeqLib::BamRecordVector reads = readNReads(opts.bam, opts.region, N); p.walk = since(t0); if (reads.empty()) { std::cerr << "WARNING: no reads fetched for N=" << N << "\n"; return p; } // ---- Phase 2: BFC error correction --------------------------------------- // Mirrors what SvabaRegionProcessor does: add sequences, train, correct. // Kept alive across phases so we can drain the corrected sequences for // the realign phase below. SeqLib::BFC bfc; if (!opts.skip_ec) { auto t1 = clock::now(); for (const auto& rec : reads) { bfc.AddSequence(rec.Sequence(), rec.Qualities(0), rec.Qname()); } bfc.Train(); bfc.ErrorCorrect(); p.ec = since(t1); } // ---- Phase 3: realign corrected reads -> reference ----------------------- // Feeds BreakPoint::splitCoverage's r2c-vs-native comparative gate. Real // svaba runs ONE BWA-MEM call per corrected read per window, which on a // typical WGS is a few thousand calls per window × ~120k windows. This // is the phase the user was curious about — the "additional alignment" // that the comparative split-coverage gate introduced. // // We drain the corrected sequences from BFC via GetSequence() rather // than re-reading the original BAM sequence, so timing reflects the // same payload svaba's gate operates on. if (!opts.skip_realign && !opts.skip_ec) { auto t_r = clock::now(); SeqLib::BWAAligner aligner(bwa_idx); aligner.allocBuffer(4096); bfc.ResetGetSequence(); std::string s, name; size_t nr = 0; while (bfc.GetSequence(s, name)) { SeqLib::BamRecordPtrVector hits; // Only need the best hit for a native-score comparison, so keep // maxSecondary low and keepSecFrac at 0. Matches the intent of // the SvabaRegionProcessor corrected_native_cig realign. aligner.alignSequence(s, name.empty() ? std::to_string(nr) : name, hits, /*hardclip=*/false, /*keepSecFrac=*/0.0, /*maxSecondary=*/0); ++nr; } p.realign = since(t_r); if (opts.verbose) { std::cerr << " [N=" << N << "] realigned " << nr << " corrected reads\n"; } } else if (!opts.skip_realign && opts.skip_ec) { // --skip-ec pre-empts realign (no corrected sequences to align). if (opts.verbose) { std::cerr << " [N=" << N << "] realign skipped (requires --skip-ec unset)\n"; } } // ---- Phase 4: fermi-lite assembly ---------------------------------------- // Feed raw BamRecords; the SeqLib wrapper extracts sequences. std::vector contigs; if (!opts.skip_assemble) { auto t2 = clock::now(); SeqLib::FermiAssembler fml; fml.AddReads(reads); fml.PerformAssembly(); contigs = fml.GetContigs(); p.assem = since(t2); if (opts.verbose) { std::cerr << " [N=" << N << "] assembled " << contigs.size() << " contigs\n"; } } // ---- Phase 4: BWA-MEM align the contigs back to the reference ------------ // If no contigs were produced (either --skip-assemble was set or fermi // produced nothing), fall back to aligning the raw read sequences so the // phase still exercises BWA. This is deliberately a coarse measure — // svaba's real align phase has a bunch of contig-sanity logic on top. if (!opts.skip_align) { auto t3 = clock::now(); SeqLib::BWAAligner aligner(bwa_idx); aligner.allocBuffer(4096); std::vector* targets = &contigs; std::vector raw_seqs; if (targets->empty()) { raw_seqs.reserve(reads.size()); for (const auto& rec : reads) raw_seqs.push_back(rec.Sequence()); targets = &raw_seqs; } for (size_t i = 0; i < targets->size(); ++i) { SeqLib::BamRecordPtrVector hits; aligner.alignSequence((*targets)[i], "t" + std::to_string(i), hits, /*hardclip=*/false, /*keepSecFrac=*/0.9, /*maxSecondary=*/10); } p.align = since(t3); } return p; } double median(std::vector v) { if (v.empty()) return 0.0; std::sort(v.begin(), v.end()); const size_t m = v.size() / 2; return (v.size() % 2) ? v[m] : 0.5 * (v[m-1] + v[m]); } // Compact fixed-width table printer. Each row = one (N, phase) cell with // the median timing and a per-read microseconds column so you can eyeball // scaling linearity. Scaling column is the ratio vs the smallest N. void printTable(const TestOpts& opts, const std::vector>>& results) { std::cout << "\n"; std::cout << std::left << std::setw(10) << "phase" << std::right << std::setw(10) << "N" << std::setw(12) << "median(s)" << std::setw(14) << "per-read(us)" << std::setw(10) << "scale" << "\n"; std::cout << std::string(56, '-') << "\n"; struct PhaseInfo { const char* label; double Phase::*field; bool skipped; }; const PhaseInfo phases[] = { { "walk", &Phase::walk, opts.skip_walk }, { "correct", &Phase::ec, opts.skip_ec }, { "realign", &Phase::realign, opts.skip_realign || opts.skip_ec }, { "assemble", &Phase::assem, opts.skip_assemble }, { "align", &Phase::align, opts.skip_align }, }; for (const auto& ph : phases) { if (ph.skipped) continue; double base_per_read = -1.0; // baseline for the scaling column (smallest N) for (const auto& [N, trials] : results) { std::vector v; v.reserve(trials.size()); for (const auto& t : trials) v.push_back(t.*ph.field); const double med = median(v); const double us_per_read = (N > 0) ? 1e6 * med / static_cast(N) : 0.0; if (base_per_read < 0) base_per_read = us_per_read; const double scale = (base_per_read > 0) ? us_per_read / base_per_read : 1.0; std::cout << std::left << std::setw(10) << ph.label << std::right << std::setw(10) << N << std::setw(12) << std::fixed << std::setprecision(4) << med << std::setw(14) << std::fixed << std::setprecision(1) << us_per_read << std::setw(10) << std::fixed << std::setprecision(2) << scale << "\n"; } std::cout << "\n"; } // Totals row: sum of non-skipped phases, median across trials. std::cout << std::left << std::setw(10) << "TOTAL" << std::right << std::setw(10) << " " << std::setw(12) << "median(s)" << std::setw(14) << "per-read(us)" << "\n"; std::cout << std::string(46, '-') << "\n"; for (const auto& [N, trials] : results) { std::vector v; for (const auto& t : trials) v.push_back(t.total()); const double med = median(v); const double us_per_read = (N > 0) ? 1e6 * med / static_cast(N) : 0.0; std::cout << std::left << std::setw(10) << "" << std::right << std::setw(10) << N << std::setw(12) << std::fixed << std::setprecision(4) << med << std::setw(14) << std::fixed << std::setprecision(1) << us_per_read << "\n"; } std::cout << "\nScaling column is (per-read us at this N) / (per-read us at smallest N).\n" << "Near 1.00 = linear scaling. >1 means per-read cost grows with N\n" << "(= superlinear algorithm or cache effects). <1 means setup cost\n" << "amortizes as N grows.\n"; } } // namespace // Forward-declared in svaba.cpp dispatch. void runTest(int argc, char** argv) { const TestOpts opts = parseCli(argc, argv); std::cerr << "svaba test:\n" << " bam: " << opts.bam << "\n" << " ref: " << opts.ref << "\n" << " region: " << (opts.region.empty() ? "(all)" : opts.region) << "\n" << " repeats: " << opts.repeats << "\n" << " N sweep:"; for (auto n : opts.n_sweep) std::cerr << " " << n; std::cerr << "\n"; // Load BWA index once up front; it's large and expensive to load // repeatedly. Reuse across every trial and every N. SeqLib::BWAIndexPtr bwa_idx; if (!opts.skip_align) { std::cerr << "loading BWA index from " << opts.ref << " ...\n"; auto t0 = std::chrono::steady_clock::now(); bwa_idx = std::make_shared(); bwa_idx->LoadIndex(opts.ref); std::cerr << " loaded in " << std::fixed << std::setprecision(1) << since(t0) << "s\n"; } std::vector>> results; results.reserve(opts.n_sweep.size()); for (size_t N : opts.n_sweep) { std::vector trials; trials.reserve(opts.repeats); for (int r = 0; r < opts.repeats; ++r) { if (opts.verbose) std::cerr << " [N=" << N << " trial " << (r+1) << "/" << opts.repeats << "]\n"; trials.push_back(oneTrial(opts, N, bwa_idx)); } results.emplace_back(N, std::move(trials)); } printTable(opts, results); } ================================================ FILE: src/svaba/threadpool.h ================================================ // threadpool.h #pragma once #include #include #include #include #include #include #include #include "SvabaSharedConfig.h" #include "SvabaOutputWriter.h" #include "SvabaOptions.h" #include "SvabaThreadUnit.h" // A simple thread safe queue for unique_ptr jobs template class WorkQueue { public: void push(std::unique_ptr job) { { std::lock_guard lk(mtx_); queue_.push_back(std::move(job)); } cv_.notify_one(); } // pop one job; blocks until one is available or a nullptr sentinel is received std::unique_ptr pop() { std::unique_lock lk(mtx_); cv_.wait(lk, [&]{ return !queue_.empty(); }); auto job = std::move(queue_.front()); queue_.pop_front(); return job; } // send one nullptr for each worker so they can exit void shutdown(size_t numThreads) { for(size_t i=0;i> queue_; }; // A fixed-size thread pool that takes Job functors template class ThreadPool { public: ThreadPool(SvabaSharedConfig& sc) : queue_(), workers_(), flushMutex_(), sc_(sc) { workers_.reserve(sc.opts.numThreads); for(size_t i=0;iflushMutex_, &sc = sc_]() mutable { //[=, &flushMutex = this->flushMutex_, &sc_ref = sc_]() mutable { // per-thread setup - each thread gets its own FASTA read and BAM readers // these should not be shared across threads, even if using const functions only // this also opens the BAMs too in the svabaThreadUnit constructor svabaThreadUnit unit(sc, i + 1); // give it the shared_ptr to the master index unit.total_count = sc.total_regions_to_process; // consume jobs while(auto job = queue_.pop()){ (*job)(unit, std::hash{}(std::this_thread::get_id())); } // final flush std::cerr << "...final flush for thread " << unit.threadId << std::endl; sc_.writer.writeUnit(unit, sc_); // this does the flush and mutex in it unit.clear(); }); } } // submit a new work item void submit(std::unique_ptr job){ queue_.push(std::move(job)); } // tell threads no more work, then join void shutdown(){ queue_.shutdown(workers_.size()); for(auto &t : workers_) t.join(); } private: WorkQueue queue_; std::vector workers_; std::mutex flushMutex_; SvabaSharedConfig& sc_; }; ================================================ FILE: src/svaba/tovcf.cpp ================================================ // tovcf.cpp // // `svaba tovcf` — convert a deduplicated bps.txt.gz into a pair of VCF // files (one for SVs, one for indels). Target spec: VCFv4.5. // // This is the standalone format-conversion entry point. It assumes the // input bps.txt.gz has already been sorted and deduplicated by // scripts/svaba_postprocess.sh — so the VCFFile internal dedup pass is // skipped. All calls (somatic + germline) land in one SV file and one // indel file, with the SOMATIC INFO flag distinguishing somatic rows // for downstream `bcftools filter` / grep workflows. // // Knobs (with defaults chosen for the new pipeline): // --sv-out PATH output SV VCF (default ${id}.sv.vcf.gz) // --indel-out PATH output indel VCF (default ${id}.indel.vcf.gz) // --always-bnd force paired BND records for every SV // (otherwise intrachrom events collapse into // // symbolic records). // --qual MODE missing | maxlod | sum (default: missing). // Controls the VCF QUAL column. "missing" ('.') // pushes users toward INFO/MAXLOD / INFO/SOMLOD // for filtering; "maxlod" puts 10*MAXLOD there; // "sum" is the legacy behavior. // --include-nonpass also emit records with FILTER != PASS. // --dedup opt back in to the legacy interval-tree dedup // (off by default; the postprocess pipeline // already dedupes upstream). // --plain write plain .vcf instead of bgzip'd .vcf.gz. // (Appends .vcf; by default outputs .vcf.gz.) // -v / --verbose chatty diagnostics. // -h / --help show help. // // Required: // -i, --input-bps deduped bps.txt.gz (from svaba_postprocess) // -b, --bam BAM used to recover the chromosome name/length // table; no reads are actually read. // -a, --id-string analysis id for output names. #include #include #include #include #include #include #include "gzstream.h" #include "SeqLib/BamReader.h" #include "BreakPoint.h" #include "SvabaLogger.h" #include "SvabaOptions.h" #include "SvabaOutputWriter.h" #include "SvabaSharedConfig.h" #include "SvabaUtils.h" #include "vcf.h" namespace { struct ToVcfOpts { std::string input_file; std::string bam; std::string analysis_id; std::string sv_out; // explicit path; filled from analysis_id if empty std::string indel_out; bool always_bnd = false; bool include_nonpass = false; bool dedup = false; bool gzip = true; int verbose = 0; QualMode qual_mode = QualMode::MISSING; // Minimum somlod (INFO/SOMLOD) to mark a record with the SOMATIC // INFO flag. 1.0 is the recommended default — strong enough that // `bcftools view -i 'INFO/SOMATIC'` picks out confident somatic // calls without dragging in marginal ones. double somlod_cutoff = 1.0; }; constexpr const char* TOVCF_USAGE = "Usage: svaba tovcf -i BPS.txt.gz -b BAM -a ID [options]\n" "\n" " Convert a deduplicated bps.txt.gz into VCFv4.5 output. Emits a SV\n" " VCF and an indel VCF; all calls (somatic+germline) go into one file\n" " each, with the SOMATIC INFO flag distinguishing somatic rows.\n" "\n" " Required:\n" " -i, --input-bps FILE deduplicated bps.txt.gz\n" " -b, --bam FILE BAM used to source the chrom name/length table\n" " -a, --id-string STR analysis id for default output names\n" "\n" " Output:\n" " --sv-out FILE override SV output path\n" " --indel-out FILE override indel output path\n" " --plain write plain .vcf instead of .vcf.gz\n" "\n" " Format knobs:\n" " --always-bnd force paired BND for every SV\n" " --qual MODE missing | maxlod | sum (default: missing)\n" " --somlod N min INFO/SOMLOD to stamp SOMATIC flag (default 1.0)\n" " --include-nonpass include records with FILTER != PASS\n" " --dedup re-run legacy interval-tree dedup on input\n" "\n" " Misc:\n" " -v, --verbose\n" " -h, --help\n"; constexpr const char* SHORTOPTS = "hi:b:va:"; // Long-only flags are dispatched via the `val` column of longopts; keep // these unique across the option surface. constexpr int OPT_SV_OUT = 2001; constexpr int OPT_INDEL_OUT = 2002; constexpr int OPT_ALWAYS_BND = 2003; constexpr int OPT_QUAL = 2004; constexpr int OPT_INCLUDE_NONPASS = 2005; constexpr int OPT_DEDUP = 2006; constexpr int OPT_PLAIN = 2007; constexpr int OPT_SOMLOD = 2008; const struct option LONGOPTS[] = { { "help", no_argument, nullptr, 'h' }, { "input-bps", required_argument, nullptr, 'i' }, { "bam", required_argument, nullptr, 'b' }, { "id-string", required_argument, nullptr, 'a' }, { "verbose", no_argument, nullptr, 'v' }, { "sv-out", required_argument, nullptr, OPT_SV_OUT }, { "indel-out", required_argument, nullptr, OPT_INDEL_OUT }, { "always-bnd", no_argument, nullptr, OPT_ALWAYS_BND }, { "qual", required_argument, nullptr, OPT_QUAL }, { "include-nonpass", no_argument, nullptr, OPT_INCLUDE_NONPASS }, { "dedup", no_argument, nullptr, OPT_DEDUP }, { "plain", no_argument, nullptr, OPT_PLAIN }, { "somlod", required_argument, nullptr, OPT_SOMLOD }, { nullptr, 0, nullptr, 0 } }; // Parse --qual {missing|maxlod|sum}. Case-insensitive prefixes accepted. QualMode parse_qual_mode(const std::string& s) { if (s == "missing" || s == "." || s == "none" ) return QualMode::MISSING; if (s == "maxlod" || s == "max" || s == "mlod") return QualMode::MAXLOD_PHRED; if (s == "sum" || s == "legacy") return QualMode::SUM_LO_PHRED; std::cerr << "ERROR: --qual must be one of: missing | maxlod | sum (got '" << s << "')\n"; std::exit(EXIT_FAILURE); } ToVcfOpts parse_cli(int argc, char** argv) { ToVcfOpts o; bool die = (argc <= 2); optind = 1; // reset getopt state (svaba.cpp consumes argv[1] before dispatch) for (int c; (c = getopt_long(argc, argv, SHORTOPTS, LONGOPTS, nullptr)) != -1;) { switch (c) { case 'h': die = true; break; case 'i': o.input_file = optarg ? optarg : ""; break; case 'b': o.bam = optarg ? optarg : ""; break; case 'a': o.analysis_id = optarg ? optarg : ""; break; case 'v': o.verbose = 1; break; case OPT_SV_OUT: o.sv_out = optarg ? optarg : ""; break; case OPT_INDEL_OUT: o.indel_out = optarg ? optarg : ""; break; case OPT_ALWAYS_BND: o.always_bnd = true; break; case OPT_QUAL: o.qual_mode = parse_qual_mode(optarg ? optarg : ""); break; case OPT_INCLUDE_NONPASS: o.include_nonpass = true; break; case OPT_DEDUP: o.dedup = true; break; case OPT_PLAIN: o.gzip = false; break; case OPT_SOMLOD: { try { o.somlod_cutoff = std::stod(optarg ? optarg : ""); } catch (const std::exception&) { std::cerr << "ERROR: --somlod expects a floating-point cutoff (got '" << (optarg ? optarg : "") << "')\n"; std::exit(EXIT_FAILURE); } break; } default: die = true; break; } } if (!die) { if (o.input_file.empty()) { std::cerr << "ERROR: -i / --input-bps is required\n"; die = true; } if (o.bam.empty()) { std::cerr << "ERROR: -b / --bam is required (for chrom name/length table)\n"; die = true; } if (o.analysis_id.empty()) { std::cerr << "ERROR: -a / --id-string is required\n"; die = true; } } if (die) { std::cerr << "\n" << TOVCF_USAGE; std::exit(1); } const std::string ext = o.gzip ? ".vcf.gz" : ".vcf"; if (o.sv_out.empty()) o.sv_out = o.analysis_id + ".sv" + ext; if (o.indel_out.empty()) o.indel_out = o.analysis_id + ".indel" + ext; return o; } // Copy of the refilter.cpp helper — splits "prefix_/path/to.bam" header // tokens into (prefix, path). std::pair splitSampleHeader(const std::string& tok) { const auto us = tok.find('_'); if (us == std::string::npos) return { tok, "" }; return { tok.substr(0, us), tok.substr(us + 1) }; } } // namespace // Forward declared in svaba.cpp dispatch. void runToVCF(int argc, char** argv) { const ToVcfOpts o = parse_cli(argc, argv); if (o.verbose > 0) { std::cerr << "svaba tovcf:\n" << " input: " << o.input_file << "\n" << " bam: " << o.bam << "\n" << " sv-out: " << o.sv_out << "\n" << " indel-out: " << o.indel_out << "\n" << " qual: " << (o.qual_mode == QualMode::MISSING ? "missing" : o.qual_mode == QualMode::MAXLOD_PHRED ? "maxlod" : "sum") << "\n" << " sv-format: " << (o.always_bnd ? "BND_ALWAYS" : "SYMBOLIC_WHEN_OBVIOUS") << "\n" << " somlod: " << o.somlod_cutoff << "\n" << " dedup: " << (o.dedup ? "yes" : "no (input assumed pre-deduped)") << "\n" << " nonpass: " << (o.include_nonpass ? "yes" : "no") << "\n"; } if (!SeqLib::read_access_test(o.input_file)) { std::cerr << "ERROR: cannot read " << o.input_file << "\n"; std::exit(EXIT_FAILURE); } // ---- 1. Open BAM only to grab the reference chromosome header ---------- SeqLib::BamReader bwalker; if (!bwalker.Open(o.bam)) { std::cerr << "ERROR: cannot open BAM " << o.bam << "\n"; std::exit(EXIT_FAILURE); } const SeqLib::BamHeader hdr = bwalker.Header(); // ---- 2. Read bps.txt.gz header and discover sample prefixes ---------- // // Matches the logic in refilter.cpp:runRefilterBreakpoints. The sample // block starts at the first header token whose first char is 't' or 'n' // past the fixed-width prefix (scan from col 34 to be safe on all v1-v3 // schemas). Once we know the prefixes, we stuff them into opts.bams so // BreakPoint's line-parser iterates sample tokens in the right order. igzstream infile(o.input_file.c_str(), std::ios::in); std::string headerLine; if (!std::getline(infile, headerLine)) { std::cerr << "ERROR: " << o.input_file << " is empty or unreadable\n"; std::exit(EXIT_FAILURE); } const auto headerv = svabaUtils::tokenize_delimited(headerLine, '\t'); if (headerv.size() < 41) { std::cerr << "ERROR: bps.txt header has " << headerv.size() << " columns; expected >=41 (legacy) or >=52 (v3).\n"; std::exit(EXIT_FAILURE); } size_t sample_start = 0; for (size_t i = 34; i < headerv.size(); ++i) { if (!headerv[i].empty() && (headerv[i].at(0) == 't' || headerv[i].at(0) == 'n')) { sample_start = i; break; } } if (sample_start == 0) { std::cerr << "ERROR: could not locate sample columns in bps.txt header.\n"; std::exit(EXIT_FAILURE); } // ---- 3. Build a minimal SvabaSharedConfig ------------------------------ // // Same pattern refilter.cpp uses: logger/opts/writer on the stack (they // wrap a no-op output writer; we never init it or open any BAMs through // it). sc.header carries the BamHeader so BreakPoint parsing can turn // chrom names into IDs. SvabaLogger logger; SvabaOptions opts; SvabaOutputWriter writer(logger, opts); SvabaSharedConfig sc(logger, opts, writer); sc.header = hdr; // Populate opts.bams (std::map) and the VCFHeader's // sample + colname state in lockstep, so the per-sample VCF column // order matches the sample block order in bps.txt. VCFHeader base_header; base_header.filedate = svabaUtils::fileDateString(); base_header.source = "svaba tovcf " + o.input_file; base_header.reference = ""; // contig lines: chrom + length from the BAM for (int i = 0; i < hdr.NumSequences(); ++i) { base_header.addContigField(hdr.IDtoName(i), hdr.GetSequenceLength(i)); } for (size_t i = sample_start; i < headerv.size(); ++i) { auto [pref, path] = splitSampleHeader(headerv[i]); if (pref.empty() || (pref.at(0) != 't' && pref.at(0) != 'n')) { std::cerr << "ERROR: unexpected sample header token '" << headerv[i] << "' (col " << i << "); expected t***/n***.\n"; std::exit(EXIT_FAILURE); } opts.bams[pref] = path; base_header.addSampleField(pref); base_header.colnames += "\t" + pref; } if (o.verbose > 0) { std::cerr << "...bps.txt has " << headerv.size() << " columns; " << sample_start << " core, " << (headerv.size() - sample_start) << " sample(s)\n"; } // ---- 4. Construct VCFFile --------------------------------------------- // // `nopass=true` in the ctor tells VCFFile to RETAIN non-PASS rows while // parsing; whether they're emitted is a separate decision controlled by // `include_nonpass`. We always ingest everything from disk so the knob // surface below can choose later. // // `skip_dedup_in_ctor` is set from the --dedup flag (default on): the // ctor runs deduplicate() internally, and we need skip_dedup=true to // be in effect BEFORE that call fires. Passing it through the ctor // param gets that ordering right in one place. const bool skip_internal_dedup = !o.dedup; VCFFile vcf(o.input_file, o.analysis_id, sc, base_header, /*nopass=*/true, /*verbose=*/o.verbose > 0, /*skip_dedup_in_ctor=*/skip_internal_dedup); vcf.qual_mode = o.qual_mode; vcf.sv_format = o.always_bnd ? SvFormat::BND_ALWAYS : SvFormat::SYMBOLIC_WHEN_OBVIOUS; vcf.include_nonpass = o.include_nonpass; vcf.somatic_threshold = o.somlod_cutoff; // vcf.skip_dedup is already set from the ctor param above. // ---- 5. Emit -------------------------------------------------------- if (o.verbose > 0) { std::cerr << "...writing SV VCF -> " << o.sv_out << "\n" << "...writing indel VCF -> " << o.indel_out << "\n"; } vcf.writeSvsSingleFile (o.sv_out, o.gzip, hdr); vcf.writeIndelsSingleFile(o.indel_out, o.gzip, hdr); if (o.verbose > 0) { std::cerr << "svaba tovcf: done.\n"; } } ================================================ FILE: src/svaba/vcf.cpp ================================================ // vcf.cpp // // Read svaba breakpoints back from `bps.txt.gz`, deduplicate them, and emit // VCFs. See vcf.h for the external API. Only ~three things change vs the // pre-overhaul vcf.cpp: // // 1. BreakPoint is non-copyable/non-movable, so everything flows through // std::shared_ptr. // 2. BreakPoint::SampleInfo is private; we iterate `bp->allele` via // structured bindings (auto) and only use its public data members. // 3. The `BreakPoint(line, &sc)` ctor now takes a SvabaSharedConfig*, and // the parsed bps row schema (39 fixed cols + per-sample tail) is // described in BreakPoint.cpp at the parse loop. #include "vcf.h" #include #include #include // std::lround (QUAL phred-rescaling) #include #include #include #include #include #include #include #include #include #include #include #include #include "gzstream.h" #include "SeqLib/GenomicRegion.h" #include "SeqLib/GenomicRegionCollection.h" #include "BreakPoint.h" #include "SvabaOptions.h" // --------------------------------------------------------------------------- // File-local constants and helpers. // --------------------------------------------------------------------------- namespace { // Maximum overlaps one breakend can have before we blacklist the whole // region as an SV-pileup and drop it from the output. constexpr int HIGH_OVERLAP_LIMIT = 500; // How many bp of padding to apply when we blacklist a high-overlap region. constexpr int BAD_REGION_PAD = 200; // FORMAT column strings — must stay in sync with BreakPoint::SampleInfo:: // toFileString (see BreakPoint.cpp ~line 1710) and with the ##FORMAT lines // we emit below. const std::string kSvFormat = "GT:AD:DP:SR:DR:GQ:PL:LO:LO_n"; const std::string kIndelFormat = "GT:AD:DP:SR:CR:GQ:PL:LO:LO_n"; // INFO fields that were declared `Type=Flag` in the header — flags are // printed without an `=value` tail. std::unordered_set g_flag_fields; // Track hashed VCFEntryPair ids we've already handed out so we can retry on // collision. File-scope because the constructor consumes it across all pairs. std::unordered_set g_hash_avoid; // Stable 32-bit string hash helper; we avoid pulling in htslib/khash.h just // for this and use std::hash truncated to 32 bits. inline uint32_t hash_string32(const std::string& s) { const size_t h = std::hash{}(s); return static_cast(h ^ (h >> 32)); } // Sort INFO key/value pairs so that READ_ID (if present) always comes last, // and everything else is sorted alphabetically. This matches legacy output. bool compareInfoFields(const std::pair& lhs, const std::pair& rhs) { const bool lhs_rid = (lhs.first == "READ_ID"); const bool rhs_rid = (rhs.first == "READ_ID"); if (rhs_rid && !lhs_rid) return true; if (lhs_rid && !rhs_rid) return false; return lhs.first < rhs.first; } // For the contig-by-length sort in the header emitter. bool pairCompareDesc( const std::pair>& a, const std::pair>& b) { return a.first > b.first; } // SVType → short string used in the EVDNC INFO field. std::string svtype_to_string(SVType t) { switch (t) { case SVType::NOTSET: return "NOTSET"; case SVType::TSI_LOCAL: return "TSI_LOCAL"; case SVType::TSI_GLOBAL: return "TSI_GLOBAL"; case SVType::ASSMB: return "ASSMB"; case SVType::ASDIS: return "ASDIS"; case SVType::DSCRD: return "DSCRD"; case SVType::INDEL: return "INDEL"; } return "UNKNOWN_SVTYPE"; } // Return the maximum per-sample LO across all alleles on a BreakPoint. // Used as the legacy "LOD" INFO field for indels. double max_allele_lo(const BreakPoint& bp) { double m = 0.0; for (const auto& [_, al] : bp.allele) m = std::max(m, al.LO); return m; } } // namespace // =========================================================================== // VCFHeader // =========================================================================== std::ostream& operator<<(std::ostream& out, const VCFHeader& v) { out << "##fileformat=" << v.fileformat << '\n'; out << "##fileDate=" << v.filedate << '\n'; out << "##source=" << v.source << '\n'; out << "##reference=" << v.reference << '\n'; // Emit contigs sorted by length, largest first (matches legacy output). using CC = std::pair; using CI = std::pair; std::vector contig_vec; contig_vec.reserve(v.contigfieldmap.size()); for (const auto& kv : v.contigfieldmap) contig_vec.emplace_back(std::stoi(kv.second), CC(kv.first, kv.second)); std::sort(contig_vec.begin(), contig_vec.end(), pairCompareDesc); for (const auto& c : contig_vec) out << "##contig=\n"; for (const auto& kv : v.infomap) out << "##INFO=\n"; for (const auto& kv : v.filtermap) out << "##FILTER=\n"; for (const auto& kv : v.formatmap) out << "##FORMAT=\n"; for (const auto& kv : v.samplemap) out << "##SAMPLE=\n"; out << v.colnames; return out; } void VCFHeader::addInfoField(std::string field, std::string number, std::string type, std::string description) { if (infomap.find(field) != infomap.end()) { std::cerr << "Warning: Info field already exists: " << field << '\n'; return; } if (type == "Flag") g_flag_fields.insert(field); infomap[field] = "Number=" + number + ",Type=" + type + ",Description=\"" + description + "\""; } void VCFHeader::addFilterField(std::string field, std::string description) { if (filtermap.find(field) != filtermap.end()) { std::cerr << "Warning: Filter field already exists: " << field << '\n'; return; } filtermap[field] = "Description=\"" + description + "\""; } void VCFHeader::addFormatField(std::string field, std::string number, std::string type, std::string description) { if (formatmap.find(field) != formatmap.end()) { std::cerr << "Warning: Format field already exists: " << field << '\n'; return; } formatmap[field] = "Number=" + number + ",Type=" + type + ",Description=\"" + description + "\""; } void VCFHeader::addSampleField(std::string field) { if (samplemap.find(field) != samplemap.end()) { std::cerr << "Warning: Sample field already exists: " << field << '\n'; return; } samplemap[field] = field; } void VCFHeader::addContigField(std::string id, int len) { contigfieldmap[id] = std::to_string(len); } // =========================================================================== // VCFEntry // =========================================================================== std::string VCFEntry::getRefString() const { std::string p = (bp->svtype == SVType::INDEL || id_num == 1) ? bp->ref : bp->alt; if (p.empty()) { std::cerr << "WARNING: Empty ref/alt field for bp\n"; return "N"; } return p; } std::string VCFEntry::getAltString(const SeqLib::BamHeader& header) const { // Indels just use bp->alt as-is (VCF-style insert/delete REF/ALT). if (bp->svtype == SVType::INDEL) { if (bp->alt.empty()) { std::cerr << "WARNING: Empty alt field for indel bp\n"; return "N"; } return bp->alt; } // SvABA2.0: symbolic SV emission for SvFormat::SYMBOLIC_WHEN_OBVIOUS. // The writer classifies each pair before calling; when symbolic_rep // is set we emit `` / `` / `` as the single-record ALT // and rely on END / SVLEN / SVTYPE INFO to describe the event. if (symbolic_rep) return "<" + symbolic_kind + ">"; // SV breakends get the BND-style `N]chr:pos]` mate notation. const std::string ref = getRefString(); std::stringstream ptag; if (id_num == 1) { ptag << bp->b2.gr.ChrName(header) << ':' << bp->b2.gr.pos1; } else { ptag << bp->b1.gr.ChrName(header) << ':' << bp->b1.gr.pos1; } std::stringstream alt; const char s1 = bp->b1.gr.strand; const char s2 = bp->b2.gr.strand; if (s1 == '+' && s2 == '+') { alt << ref << ']' << ptag.str() << ']'; } else if (s1 == '+' && s2 == '-') { if (id_num == 1) alt << ref << '[' << ptag.str() << '['; else alt << ']' << ptag.str() << ']' << ref; } else if (s1 == '-' && s2 == '+') { if (id_num == 1) alt << ']' << ptag.str() << ']' << ref; else alt << ref << '[' << ptag.str() << '['; } else { alt << '[' << ptag.str() << '[' << ref; } return alt.str(); } std::string VCFEntry::getIdString() const { if (bp->svtype != SVType::INDEL) return std::to_string(id) + ':' + std::to_string(id_num); return std::to_string(id); } std::unordered_map VCFEntry::fillInfoFields() const { std::unordered_map info; info["SPAN"] = std::to_string(bp->getSpan()); info["SCTG"] = bp->cname; // SvABA2.0: common per-record metadata, populated for both SVs and // indels so downstream filters don't need to condition on shape. // // SOMATIC flag: gated on the somlod threshold carried per-entry // (`somatic_threshold`, defaulted 1.0, overridable via `svaba tovcf // --somlod N`). The svaba-run-time SomaticState::FAILED state is // honored as a hard veto — something earlier in the pipeline // explicitly said "not somatic", so we don't mark it regardless // of what LO_s looks like. if (bp->LO_s >= somatic_threshold && bp->somatic != SomaticState::FAILED) info["SOMATIC"] = ""; // flag if (!bp->id.empty()) info["EVENT"] = bp->id; // v3 bp_id (col 52 of bps.txt) { std::stringstream ss; ss << std::setprecision(4) << bp->max_lod; info["MAXLOD"] = ss.str(); } { std::stringstream ss; ss << std::setprecision(4) << bp->LO_s; info["SOMLOD"] = ss.str(); } if (bp->svtype != SVType::INDEL) { info["EVDNC"] = svtype_to_string(bp->svtype); // SvABA2.0: SVTYPE is either BND (paired-breakend notation) or a // symbolic type (DEL/DUP/INV) when the writer has opted into // symbolic alleles and this record qualifies. symbolic_rep / // symbolic_kind are set by writeSvsSingleFile before toFileString // runs; when they're unset we fall back to legacy BND. if (symbolic_rep) { info["SVTYPE"] = symbolic_kind; // "DEL" / "DUP" / "INV" // For symbolic alleles we must set END (SV end position) and // SVLEN (positive for INS/DUP, negative for DEL; absolute length // for INV because the inverted segment length is what matters). const int pos1_1idx = bp->b1.gr.pos1 + 1; const int pos2_1idx = bp->b2.gr.pos1 + 1; const int end_pos = std::max(pos1_1idx, pos2_1idx); const int length = std::abs(pos2_1idx - pos1_1idx); info["END"] = std::to_string(end_pos); if (symbolic_kind == "DEL") info["SVLEN"] = std::to_string(-length); else info["SVLEN"] = std::to_string(length); // svaba uses primarily junction evidence (+ optional depth via // discordant pairs). "J" is the honest claim for assembly-only, // "DJ" for ASDIS (assembly+discordant). DSCRD-only is rare for // symbolic-eligible intrachrom events but we'd emit "D" there. if (bp->svtype == SVType::ASSMB) info["SVCLAIM"] = "J"; else if (bp->svtype == SVType::ASDIS) info["SVCLAIM"] = "DJ"; else if (bp->svtype == SVType::DSCRD) info["SVCLAIM"] = "D"; else info["SVCLAIM"] = "J"; } else { info["SVTYPE"] = "BND"; info["SVCLAIM"] = "J"; } } if (!bp->repeat_seq.empty()) info["REPSEQ"] = bp->repeat_seq; // NM / MATENM / MATEID: only for multi-alignment (BND-style) breakpoints. if (bp->num_align != 1) { info["MATEID"] = std::to_string(id) + ':' + std::to_string(id_num == 1 ? 2 : 1); if (id_num == 1) { info["NM"] = std::to_string(bp->b1.nm); info["MATENM"] = std::to_string(bp->b2.nm); } else { info["NM"] = std::to_string(bp->b2.nm); info["MATENM"] = std::to_string(bp->b1.nm); } } else { info["NM"] = std::to_string(bp->b1.nm); } // MAPQ of the breakend this entry represents. info["MAPQ"] = std::to_string(id_num == 1 ? bp->b1.mapq : bp->b2.mapq); if (bp->num_align != 1) { // --- SV-only fields --- if (id_num == 1) { if (bp->b1.sub) info["SUBN"] = std::to_string(bp->b1.sub); else if (bp->b2.sub) info["SUBN"] = std::to_string(bp->b2.sub); } if (!bp->homology.empty()) info["HOMSEQ"] = bp->homology; if (!bp->insertion.empty()) info["INSERTION"] = bp->insertion; info["NUMPARTS"] = std::to_string(bp->num_align); if (bp->imprecise > 0) info["IMPRECISE"] = ""; if (bp->secondary > 0) info["SECONDARY"] = ""; if (info["EVDNC"] != "ASSMB") { info["DISC_MAPQ"] = std::to_string( id_num == 1 ? bp->dc.mapq1 : bp->dc.mapq2); } } else { // --- indel-only fields --- std::stringstream lod_ss; lod_ss << std::setprecision(4) << max_allele_lo(*bp); info["LOD"] = lod_ss.str(); if (!bp->rs.empty()) info["DBSNP"] = bp->rs; } return info; } std::string VCFEntry::toFileString(const SeqLib::BamHeader& header, QualMode qual_mode) const { std::stringstream out; // Sort INFO fields for stable, legacy-compatible output. auto info_map = fillInfoFields(); std::vector> info_vec( info_map.begin(), info_map.end()); std::sort(info_vec.begin(), info_vec.end(), compareInfoFields); std::string info; for (const auto& kv : info_vec) { // Don't emit HOMSEQ/HOMLEN/INSERTION when imprecise (legacy behavior). if (bp->imprecise && (kv.first == "HOMSEQ" || kv.first == "HOMLEN" || kv.first == "INSERTION")) continue; const bool is_flag = (g_flag_fields.count(kv.first) > 0); info += kv.first + (is_flag ? "" : "=") + kv.second + ';'; } if (!info.empty()) info.pop_back(); // trim trailing ';' // SvABA2.0: pick QUAL representation. Legacy (SUM_LO_PHRED) keeps // exactly the pre-2.0 behavior by passing bp->quality through; the // two new modes are for the `svaba tovcf` path, where the canonical // confidence lives in INFO/MAXLOD / INFO/SOMLOD and writing the sum- // of-LOs Phred into QUAL just invites confused downstream filters. std::string qual_str; switch (qual_mode) { case QualMode::MISSING: qual_str = "."; break; case QualMode::MAXLOD_PHRED: { // maxlod is already log10 — scale by 10 for Phred and cap at 99 // to stay inside the conventional VCF QUAL range. const double v = std::max(0.0, bp->max_lod); const long p = std::lround(10.0 * v); qual_str = std::to_string(std::min(p, 99)); break; } case QualMode::SUM_LO_PHRED: default: qual_str = std::to_string(bp->quality); break; } // For symbolic SV records, POS is the lower of the two breakend // positions (the event's 5' anchor); the INFO/END field carries the // other boundary. For BND records, POS is just the breakend this // entry represents (legacy behavior). int pos; std::string chr_name; if (symbolic_rep) { const int p1 = bp->b1.gr.pos1; const int p2 = bp->b2.gr.pos1; pos = std::min(p1, p2); // Both breakends live on the same chrom when symbolic; use b1's. chr_name = bp->b1.gr.ChrName(header); } else { const BreakEnd* be = (id_num == 1) ? &bp->b1 : &bp->b2; pos = be->gr.pos1; chr_name = be->gr.ChrName(header); } const char sep = '\t'; out << chr_name << sep << pos << sep << getIdString() << sep << getRefString() << sep << getAltString(header) << sep << qual_str << sep << bp->confidence << sep << info << sep << (bp->svtype == SVType::INDEL ? kIndelFormat : kSvFormat); // SvABA2.0: per-sample columns (VCF column 10+). Iterating bp->allele // in-order is deterministic because SampleInfo is stored in a // std::map keyed by the 4-char bam prefix (n001, t001, ...). The // header's `colnames` must list samples in the same order for the // VCF to be well-formed — callers that build the header from the // bps.txt.gz row-0 sample list implicitly match this ordering. for (const auto& [_, al] : bp->allele) { out << sep << al.toFileString(bp->svtype); } return out.str(); } bool VCFEntry::operator<(const VCFEntry& v) const { const BreakEnd* a = (id_num == 1) ? &bp->b1 : &bp->b2; const BreakEnd* b = (v.id_num == 1) ? &v.bp->b1 : &v.bp->b2; return a->gr < b->gr; } bool VCFEntry::operator==(const VCFEntry& v) const { const BreakEnd* a = (id_num == 1) ? &bp->b1 : &bp->b2; const BreakEnd* b = (v.id_num == 1) ? &v.bp->b1 : &v.bp->b2; return a->gr == b->gr; } // =========================================================================== // VCFEntryPair // =========================================================================== VCFEntryPair::VCFEntryPair(std::shared_ptr& b) { bp = b; e1.bp = bp; e2.bp = bp; e1.id_num = 1; e2.id_num = 2; // Compose a stable string describing this pair and hash it. Retry on // collision by salting with an increment counter, so ids remain unique // even if two genuinely-distinct pairs happen to collide. uint32_t hashed = 0; int salt = 0; do { const std::string s = std::to_string(bp->b1.gr.pos1) + '-' + std::to_string(bp->b2.gr.pos1) + '-' + std::to_string(bp->b1.gr.chr) + '_' + std::to_string(bp->b2.gr.chr) + bp->cname + std::to_string(salt); hashed = hash_string32(s); ++salt; } while (g_hash_avoid.find(hashed) != g_hash_avoid.end()); g_hash_avoid.insert(hashed); e1.id = hashed; e2.id = hashed; } std::string VCFEntryPair::toFileString(const SeqLib::BamHeader& header, QualMode qual_mode) const { std::stringstream out; // If e1 is marked symbolic_rep, only emit it (single-record SV event). // Otherwise emit both breakends as a paired BND event. out << e1.toFileString(header, qual_mode) << '\n'; if (!e1.symbolic_rep) out << e2.toFileString(header, qual_mode) << '\n'; return out.str(); } // =========================================================================== // VCFFile::VCFFile — read bps.txt.gz, build entries, deduplicate. // =========================================================================== namespace { // All of the boilerplate ##INFO/##FILTER/##FORMAT lines for SV and indel // headers. Kept here rather than inline in the ctor so the ctor stays // readable. void populate_sv_header(VCFHeader& h) { // FILTERs h.addFilterField("NOLOCAL", "Contig realigned to region outside of local assembly region, and no disc support."); h.addFilterField("LOCALMATCH", "Contig realigned to assembly region without clipping"); h.addFilterField("HIGHHOMOLOGY", "Contig realigns with > 25% of readlength of homology. High probaility of assembly/mapping artifact"); h.addFilterField("DUPREADS", "Contig built from what appear to be duplicate reads (split reads all same contig cov))"); h.addFilterField("NODISC", "Rearrangement was not detected independently by assembly"); h.addFilterField("LOWSUPPORT", "Fewer than 2 split reads or < 4 total alt reads for ASDISC"); h.addFilterField("COMPETEDISC", "Discordant cluster found with nearly same breakpoints, but different strands for DSCRD event"); h.addFilterField("LOWSPAN", "Discordant read cluster (no split read support), and less than 10kb span and < 12 reads"); h.addFilterField("LOWMAPQ", "Assembly contig has non 60/60 mapq and no discordant support"); h.addFilterField("LOWMATCHLEN", "Assembly contig has fewer than 40 bases mapping uniquely to a reference locus (<100 if complex mapping or )"); h.addFilterField("SINGLEBX", "Variant is supported by only a single BX tag (if run with 10X Genomics data)"); h.addFilterField("LOWQINVERSION", "Assembly-only inversion of span < 300 and < 6 split reads. Common artifact in Illumina data"); h.addFilterField("LOWMAPQDISC", "Both clusters of reads failed to achieve mean mapq of > 30 for DSCRD"); h.addFilterField("LOWSPLITSMALL", "Fewer than 4 split reads for small events ( < 1500 bp)"); h.addFilterField("LOWICSUPPORT", "Less than 60bp of contig match on one end of an inter-chromosomal break"); h.addFilterField("LOWAS", "Alignment score of one end is less than 80% of contig length, or number of mismatch bases (NM) on one end is >= 10"); h.addFilterField("WEAKSUPPORTHIREP","Fewer then 7 split reads for variant with >= 10 bases of repeat sequence (need to be more strict)"); h.addFilterField("WEAKDISC", "Fewer than 7 supporting discordant reads and no assembly support"); h.addFilterField("TOOSHORT", "Contig alignment for part of this rearrangement has <= 25bp match to reference"); h.addFilterField("PASS", "Strong assembly support, strong discordant support, or combined support. Strong MAPQ"); h.addFilterField("MULTIMATCH", "Low MAPQ and this contig fragment maps well to multiple locations"); h.addFilterField("LOWSPANDSCRD", "Discordant-only cluster is too small given isize distribution to call confidently"); h.addFilterField("SIMPLESEQUENCE", "Major portion of one contig mapping falls in a simple sequence, as given by -R flag. Assembly-only filter"); // INFOs h.addInfoField("REPSEQ", "1", "String", "Repeat sequence near the event"); h.addInfoField("NM", "1", "Integer", "Number of mismatches of this alignment fragment to reference"); h.addInfoField("MATENM", "1", "Integer", "Number of mismatches of partner alignment fragment to reference"); h.addInfoField("SVTYPE", "1", "String", "Type of structural variant"); h.addInfoField("HOMSEQ", "1", "String", "Sequence of base pair identical micro-homology at event breakpoints. Plus strand sequence displayed."); h.addInfoField("IMPRECISE", "0", "Flag", "Imprecise structural variation"); h.addInfoField("SECONDARY", "0", "Flag", "SV calls comes from a secondary alignment"); h.addInfoField("HOMLEN", "1", "Integer", "Length of base pair identical micro-homology at event breakpoints"); h.addInfoField("MAPQ", "1", "Integer", "Mapping quality (BWA-MEM) of this fragement of the contig (-1 if discordant only)"); h.addInfoField("MATEMAPQ", "1", "Integer", "Mapping quality of the partner fragment of the contig"); h.addInfoField("MATEID", "1", "String", "ID of mate breakends"); h.addInfoField("SUBN", "1", "Integer", "Number of secondary alignments associated with this contig fragment"); h.addInfoField("NUMPARTS", "1", "Integer", "If detected with assembly, number of parts the contig maps to. Otherwise 0"); h.addInfoField("EVDNC", "1", "String", "Evidence for variant. ASSMB assembly only, ASDIS assembly+discordant. DSCRD discordant only, TSI_L templated-sequence insertion (local, e.g. AB or BC of an ABC), TSI_G global (e.g. AC of ABC)"); h.addInfoField("SCTG", "1", "String", "Identifier for the contig assembled by svaba to make the SV call"); h.addInfoField("INSERTION", "1", "String", "Sequence insertion at the breakpoint."); h.addInfoField("SPAN", "1", "Integer", "Distance between the breakpoints. -1 for interchromosomal"); h.addInfoField("DISC_MAPQ", "1", "Integer", "Mean mapping quality of discordant reads mapped here"); // SvABA2.0 (v3): VCF 4.5-aligned SV INFO fields. h.addInfoField("SOMATIC", "0", "Flag", "Somatic call per svaba's somatic LOD model (see BreakPoint::score_somatic)"); h.addInfoField("EVENT", "1", "String", "ID of the SV event this breakend belongs to. Paired BND records share the same EVENT; a symbolic SV is its own event. Populated from BreakPoint::id (col 52 of bps.txt) when available."); h.addInfoField("END", "1", "Integer", "End position for symbolic alleles (//). Not set for BND."); h.addInfoField("SVLEN", "1", "Integer", "Length of the SV in bp. Negative for deletions. Only set on symbolic alleles."); h.addInfoField("SVCLAIM", "1", "String", "Evidence type supporting the SV call, per VCFv4.5: J=junction-level (split/assembly), D=depth, DJ=both."); h.addInfoField("MAXLOD", "1", "Float", "Maximum per-sample log-odds (variant vs error) across all samples (see BreakPoint::max_lod)"); h.addInfoField("SOMLOD", "1", "Float", "Somatic log-odds (LLR of somatic vs non-somatic; see SvabaModels::SomaticLOD)"); // FORMATs (SV) h.addFormatField("GT", "1", "String", "Most likely genotype"); h.addFormatField("AD", "1", "Integer", "Allele depth: Number of reads supporting the variant"); h.addFormatField("DP", "1", "Integer", "Depth of coverage: Number of reads covering site."); h.addFormatField("GQ", "1", "String", "Genotype quality (currently not supported. Always 0)"); h.addFormatField("PL", ".", "Float", "Normalized likelihood of the current genotype"); h.addFormatField("SR", "1", "Integer", "Number of spanning reads for this variant"); h.addFormatField("DR", "1", "Integer", "Number of discordant-supported reads for this variant"); h.addFormatField("LO", "1", "Float", "Log-odds that this variant is real vs artifact"); h.addFormatField("LO_n", "1", "Float", "Log-odds that this variant is AF=0 vs AF>=0.5"); } void populate_indel_header(VCFHeader& h) { // FILTERs h.addFilterField("LOWMAPQ", "Assembly contig has less than MAPQ 10"); h.addFilterField("SHORTALIGNMENT", "Matched (M) contig frag to left or right of indel < 20 bp"); h.addFilterField("LOWLOD", "LOD score is less than the cutoff"); h.addFilterField("MULTIMATCH", "Low MAPQ and this contig fragment maps well to multiple locations"); h.addFilterField("LOWAS", "Less than 80% of contig length is covered by a supporting read"); h.addFilterField("NONVAR", "0/0 is the most likely genotype"); h.addFilterField("PASS", "LOD score pass"); h.addFilterField("VLOWAF", "allelic fraction < 0.05"); h.addFilterField("REPVAR", "Multiple conflicting variants at a highly repetitive region"); // INFOs h.addInfoField("SCTG", "1", "String", "Identifier for the contig assembled by svaba to make the indel call"); h.addInfoField("MAPQ", "1", "Integer", "Mapping quality (BWA-MEM) of the assembled contig"); h.addInfoField("SPAN", "1", "Integer", "Size of the indel"); h.addInfoField("REPSEQ", "1", "String", "Repeat sequence near the variant"); h.addInfoField("NM", "1", "Integer", "Number of mismatches of this alignment fragment to reference"); h.addInfoField("READNAMES",".", "String", "IDs of ALT reads"); h.addInfoField("BX", ".", "String", "Table of BX tag counts for supporting reads"); h.addInfoField("DBSNP", "0", "Flag", "Variant found in dbSNP"); h.addInfoField("LOD", "1", "Float", "Log of the odds that variant is real vs artifact"); // SvABA2.0 (v3): VCF 4.5-aligned indel INFO fields. h.addInfoField("SOMATIC", "0", "Flag", "Somatic indel per svaba's somatic LOD model"); h.addInfoField("EVENT", "1", "String", "Stable indel identifier (populated from BreakPoint::id when available)"); h.addInfoField("SVTYPE", "1", "String", "INS / DEL for indels emitted with explicit SVTYPE"); h.addInfoField("SVLEN", "1", "Integer", "Length of the indel (negative for deletions)"); h.addInfoField("MAXLOD", "1", "Float", "Maximum per-sample log-odds (variant vs error) across samples"); h.addInfoField("SOMLOD", "1", "Float", "Somatic log-odds (LLR of somatic vs non-somatic)"); // FORMATs (indel) h.addFormatField("GT", "1", "String", "Most likely genotype"); h.addFormatField("AD", "1", "Integer", "Allele depth: Number of reads supporting the variant"); h.addFormatField("DP", "1", "Integer", "Depth of coverage: Number of reads covering site."); h.addFormatField("GQ", "1", "String", "Genotype quality (currently not supported. Always 0)"); h.addFormatField("PL", ".", "Float", "Normalized likelihood of the current genotype"); h.addFormatField("SR", "1", "Integer", "Number of spanning reads for this variant"); h.addFormatField("CR", "1", "Integer", "Number of cigar-supported reads for this variant"); h.addFormatField("LO", "1", "Float", "Log-odds that this variant is real vs artifact"); h.addFormatField("LO_n", "1", "Float", "Log-odds that this variant is AF=0 vs AF>=0.5"); } } // namespace VCFFile::VCFFile(std::string file, std::string id, const SvabaSharedConfig& sc, const VCFHeader& vheader, bool nopass, bool m_verbose, bool skip_dedup_in_ctor) { filename = file; analysis_id = id; verbose = m_verbose; include_nonpass = nopass; skip_dedup = skip_dedup_in_ctor; // captured before ctor's deduplicate() runs // Seed both headers from the caller-provided base (which carries contig // and sample field lines), then layer on the SV- and indel-specific // INFO/FILTER/FORMAT blocks. sv_header = vheader; indel_header = vheader; populate_sv_header(sv_header); populate_indel_header(indel_header); // Open the gzipped bps file stream. igzstream infile(file.c_str(), std::ios::in); if (!infile) { std::cerr << "Can't read file " << file << " for parsing VCF\n"; std::exit(EXIT_FAILURE); } std::cerr << "...vcf - reading breakpoints: " << file << '\n'; // Per-contig cap: if more than VCF_SECONDARY_CAP pairs share the same // cname, drop additional ones to avoid runaway output at pileups. std::unordered_map cname_count; std::string line; // Skip the first row — it's the column header emitted by BreakPoint::header(). std::getline(infile, line, '\n'); int line_count = 0; while (std::getline(infile, line, '\n')) { if (line.empty() || line[0] == '#') continue; // BreakPoint parser will fail if any chr is unknown. Skip defensively. if (line.find("Unknown") != std::string::npos) continue; // Parse one bps.txt.gz row into a heap BreakPoint. Must be shared_ptr // because BreakPoint is non-copyable/non-movable. std::shared_ptr bp; try { bp = std::make_shared(line, &sc); } catch (const std::exception& e) { std::cerr << "Warning: failed to parse bps line: " << e.what() << '\n'; continue; } // Honor the unfiltered/filtered flag from the caller. Gate on // `confidence` (the FILTER column string) rather than bp->pass — // the parser doesn't repopulate the `pass` bool from the dump, // and the scorer only updates `confidence`, so `confidence` is // the single source of truth for "did this record PASS". if (!include_nonpass && bp->confidence != "PASS") continue; // Per-contig cap: cap the number of entries per contig name. if (++cname_count[bp->cname] >= VCF_SECONDARY_CAP) continue; auto vpair = std::make_shared(bp); if (bp->svtype == SVType::INDEL) indels.emplace(line_count, vpair); else entry_pairs.emplace(line_count, vpair); ++line_count; } std::cerr << "...vcf - read in " << SeqLib::AddCommas(indels.size()) << " indels and " << SeqLib::AddCommas(entry_pairs.size()) << " SVs\n"; std::cerr << "...vcf - SV deduplicating " << SeqLib::AddCommas(entry_pairs.size()) << " events\n"; deduplicate(sc.header); std::cerr << "...vcf - SV deduplicated down to " << SeqLib::AddCommas(entry_pairs.size() - dups.size()) << " break pairs\n"; } // =========================================================================== // Dedupe — SVs via paired-breakend interval-tree matching, indels via hash. // =========================================================================== namespace { // Extension of GenomicRegion carrying the entry_pairs key and a pass flag. class GenomicRegionWithID : public SeqLib::GenomicRegion { public: GenomicRegionWithID(int32_t c, uint32_t p1, uint32_t p2, int i, int p) : SeqLib::GenomicRegion(c, p1, p2), id(i), pass(p) {} uint32_t id : 30; uint32_t pass : 2; }; } // namespace void VCFFile::deduplicate(const SeqLib::BamHeader& header) { // SvABA2.0: `svaba tovcf` receives a bps.txt.gz that has already been // sorted + deduped by scripts/svaba_postprocess.sh (step 3). Rerunning // the internal paired-interval-tree dedup on that input is wasted work // and can introduce its own collisions via the high-overlap blacklist // logic below. Short-circuit when the caller opts in. if (skip_dedup) { if (verbose) std::cerr << "...vcf - skip_dedup set, bypassing internal dedupe\n"; return; } // Build separate interval trees for left (b1) and right (b2) breakends. SeqLib::GenomicRegionCollection grv1; SeqLib::GenomicRegionCollection grv2; for (const auto& kv : entry_pairs) { const auto& bpp = kv.second->bp; // Use the confidence string (not the stale bp->pass bool) to // decide PASS status for the dedup grouping — see BreakPoint.cpp // parser note: pass never gets repopulated from the dumped // confidence column, so it's 0 across all parsed records. const int bp_pass_int = (bpp->confidence == "PASS") ? 1 : 0; grv1.add(GenomicRegionWithID(bpp->b1.gr.chr, bpp->b1.gr.pos1, bpp->b1.gr.pos2, kv.first, bp_pass_int)); grv2.add(GenomicRegionWithID(bpp->b2.gr.chr, bpp->b2.gr.pos1, bpp->b2.gr.pos2, kv.first, bp_pass_int)); } grv1.CreateTreeMap(); grv2.CreateTreeMap(); assert(grv1.size() == grv2.size()); // Regions blacklisted mid-pass because they turned out to be SV pileups. SeqLib::GenomicRegionCollection high_overlap_blacklist; size_t count = 0; for (const auto& kv : entry_pairs) { const int current_key = kv.first; const auto& pair = kv.second; const auto& bpp = pair->bp; // Tighter pad for small intrachrom events, looser for interchrom / large. const int pad = (bpp->b1.gr.chr != bpp->b2.gr.chr || std::abs(bpp->b1.gr.pos1 - bpp->b2.gr.pos1) > DEDUPEPAD * 2) ? DEDUPEPAD : 10; if (verbose && (count % 20000 == 0)) std::cerr << "...deduping at " << SeqLib::AddCommas(count) << '\n'; ++count; // Blacklist check for either breakend. SeqLib::GenomicIntervalVector bad1, bad2; auto fb1 = high_overlap_blacklist.GetTree()->find(bpp->b1.gr.chr); auto fb2 = high_overlap_blacklist.GetTree()->find(bpp->b2.gr.chr); if (fb1 != high_overlap_blacklist.GetTree()->end()) { fb1->second.findOverlapping(bpp->b1.gr.pos1 - pad, bpp->b1.gr.pos1 + pad, bad1); if (!bad1.empty()) { dups.insert(current_key); continue; } } if (fb2 != high_overlap_blacklist.GetTree()->end()) { fb2->second.findOverlapping(bpp->b2.gr.pos1 - pad, bpp->b2.gr.pos1 + pad, bad2); if (!bad2.empty()) { dups.insert(current_key); continue; } } // Find all entries whose b1/b2 fall within pad of this one's b1/b2. SeqLib::GenomicIntervalVector giv1, giv2; auto ff1 = grv1.GetTree()->find(bpp->b1.gr.chr); auto ff2 = grv2.GetTree()->find(bpp->b2.gr.chr); assert(ff1 != grv1.GetTree()->end()); assert(ff2 != grv2.GetTree()->end()); ff1->second.findContained(bpp->b1.gr.pos1 - pad, bpp->b1.gr.pos1 + pad, giv1); ff2->second.findContained(bpp->b2.gr.pos1 - pad, bpp->b2.gr.pos1 + pad, giv2); // If this breakend is in a region hit by more than HIGH_OVERLAP_LIMIT // other breakends, treat it as an SV pileup: blacklist the region and // drop the current entry. if (giv1.size() > HIGH_OVERLAP_LIMIT) { SeqLib::GenomicRegion gr_bad = bpp->b1.gr; std::cerr << "...added " << gr_bad << " with " << giv1.size() << " overlaps to SV-pileup blacklist at dedupe\n" << "NB: Any SV with one breakend occuring more than " << HIGH_OVERLAP_LIMIT << " times will be removed. To change this behavior, " << "adjust HIGH_OVERLAP_LIMIT in vcf.cpp and recompile/run\n"; gr_bad.Pad(BAD_REGION_PAD); high_overlap_blacklist.add(gr_bad); high_overlap_blacklist.CreateTreeMap(); dups.insert(current_key); continue; } if (giv2.size() > HIGH_OVERLAP_LIMIT) { SeqLib::GenomicRegion gr_bad = bpp->b2.gr; std::cerr << "...added " << gr_bad << " with " << giv2.size() << " overlaps to SV-pileup blacklist at dedupe\n"; gr_bad.Pad(BAD_REGION_PAD); high_overlap_blacklist.add(gr_bad); high_overlap_blacklist.CreateTreeMap(); dups.insert(current_key); continue; } assert(giv1.size() <= HIGH_OVERLAP_LIMIT); assert(giv2.size() <= HIGH_OVERLAP_LIMIT); // A hit is a dup if the same entry_pairs key appears on BOTH sides, with // matching strands and matching pass status. Source of truth for // PASS is confidence == "PASS" (see note at top of deduplicate()). const bool is_pass = (pair->e1.bp->confidence == "PASS"); const char s1_here = bpp->b1.gr.strand; const char s2_here = bpp->b2.gr.strand; std::unordered_map> key_count; for (const auto& j : giv1) { const auto& hit = grv1.at(j.value); if (hit.id == current_key) continue; if ((is_pass == static_cast(hit.pass)) && entry_pairs[hit.id]->bp->b1.gr.strand == s1_here) ++key_count[hit.id].first; } for (const auto& j : giv2) { const auto& hit = grv2.at(j.value); if (hit.id == current_key) continue; if ((is_pass == static_cast(hit.pass)) && entry_pairs[hit.id]->bp->b2.gr.strand == s2_here) ++key_count[hit.id].second; } // If this entry has a "twin" with a worse (lower) score than ours, mark // the twin — not ours — as a dup. Ties break by `operator<` on BreakPoint. // Exception: TSI_LOCAL/TSI_GLOBAL annotate the same underlying event in // two ways and should NOT dup each other. for (const auto& kc : key_count) { if (kc.second.first == 0 || kc.second.second == 0) continue; const auto& other = entry_pairs[kc.first]; if (*bpp < *other->bp) { const bool local_global_pair = (bpp->svtype == SVType::TSI_LOCAL && other->bp->svtype == SVType::TSI_GLOBAL) || (bpp->svtype == SVType::TSI_GLOBAL && other->bp->svtype == SVType::TSI_LOCAL); if (local_global_pair) { // don't dup: intentional double-annotation } else { dups.insert(kc.first); } } } } // --- Indel dedupe: cheap hash on (chr, pos, REF, ALT). --- if (verbose) std::cerr << "...vcf - hashing " << SeqLib::AddCommas(indels.size()) << " indels for dedupe\n"; std::unordered_set seen; VCFEntryPairMap deduped_indels; deduped_indels.reserve(indels.size()); for (const auto& kv : indels) { std::string key; try { const auto& b1 = kv.second->e1.bp->b1; key = std::to_string(b1.gr.chr) + ':' + std::to_string(b1.gr.pos1) + '_' + kv.second->e1.getRefString() + '_' + kv.second->e1.getAltString(header); } catch (...) { std::cerr << "indel dedupe: error building hash key for entry " << kv.first << '\n'; continue; } if (seen.insert(key).second) deduped_indels.emplace(kv.first, kv.second); } indels = std::move(deduped_indels); } // =========================================================================== // Writers // =========================================================================== void VCFFile::writeIndels(std::string basename, bool /*zip*/, bool /*onefile*/, const SeqLib::BamHeader& header) const { // The legacy writer emitted a single combined indel VCF regardless of // zip/onefile flags; we preserve that behavior to avoid surprising // downstream consumers. const std::string out_name = basename + "germline.indel.vcf"; std::ofstream out(out_name); if (!out) { std::cerr << "vcf: failed to open " << out_name << " for writing\n"; return; } out << indel_header << '\n'; // Collect e1 of every indel pair into a vector, sort by genomic position. VCFEntryVec rows; rows.reserve(indels.size()); for (const auto& kv : indels) rows.push_back(kv.second->e1); std::sort(rows.begin(), rows.end()); for (const auto& r : rows) { // PASS/FILTER gate: use the confidence string as source of truth // (bp->pass is a stale bool; see parser note in BreakPoint.cpp). if (r.bp->confidence != "PASS" && !include_nonpass) continue; out << r.toFileString(header) << '\n'; } } void VCFFile::writeSVs(std::string basename, bool /*zip*/, bool /*onefile*/, const SeqLib::BamHeader& header) const { const std::string out_name = basename + "germline.sv.vcf"; std::ofstream out(out_name); if (!out) { std::cerr << "vcf: failed to open " << out_name << " for writing\n"; return; } out << sv_header << '\n'; // Pull both breakends of each surviving pair into a flat vector, sort, // then emit. Skip entries marked as dups during dedupe. VCFEntryVec rows; rows.reserve(entry_pairs.size() * 2); for (const auto& kv : entry_pairs) { if (dups.count(kv.first)) continue; rows.push_back(kv.second->e1); rows.push_back(kv.second->e2); } std::sort(rows.begin(), rows.end()); for (const auto& r : rows) { // PASS/FILTER gate: use the confidence string as source of truth // (bp->pass is a stale bool; see parser note in BreakPoint.cpp). if (r.bp->confidence != "PASS" && !include_nonpass) continue; out << r.toFileString(header) << '\n'; } } // =========================================================================== // SvABA2.0: symbolic-allele classifier + single-file writers (tovcf path). // =========================================================================== namespace { // Classify a BreakPoint pair to decide whether it can be emitted as a // single symbolic-allele record (//) instead of two paired // BND records. Conservative: // - both breakends must live on the same chromosome // - INDEL records keep their existing REF/ALT shape (not this path) // - strand pattern determines the symbolic kind: // +/+ or -/- -> // -/+ -> // +/- -> // - anything ambiguous / inter-chrom falls through to BND. // Returns empty string when the pair should stay as paired BND. std::string classify_symbolic_kind(const BreakPoint& bp) { if (bp.svtype == SVType::INDEL) return ""; if (bp.b1.gr.chr != bp.b2.gr.chr) return ""; const char s1 = bp.b1.gr.strand; const char s2 = bp.b2.gr.strand; if (s1 == '+' && s2 == '+') return "DEL"; if (s1 == '-' && s2 == '-') return "DEL"; if (s1 == '-' && s2 == '+') return "DUP"; if (s1 == '+' && s2 == '-') return "INV"; return ""; } // Sort key used by the single-file writers: for a BND entry use its // represented breakend; for a symbolic entry use the lower of the two // endpoints (matching VCF convention for the POS of a symbolic SV). bool entry_lt_for_output(const VCFEntry& a, const VCFEntry& b) { const BreakEnd* ba = a.symbolic_rep ? (a.bp->b1.gr.pos1 < a.bp->b2.gr.pos1 ? &a.bp->b1 : &a.bp->b2) : (a.id_num == 1 ? &a.bp->b1 : &a.bp->b2); const BreakEnd* bb = b.symbolic_rep ? (b.bp->b1.gr.pos1 < b.bp->b2.gr.pos1 ? &b.bp->b1 : &b.bp->b2) : (b.id_num == 1 ? &b.bp->b1 : &b.bp->b2); return ba->gr < bb->gr; } // Open path for writing (gzipped or plain). Returns empty unique_ptr on // failure, with the error already logged to stderr. std::unique_ptr open_vcf_out(const std::string& path, bool gzip) { if (gzip) { auto g = std::make_unique(path.c_str(), std::ios::out); if (!g->good()) { std::cerr << "vcf: failed to open " << path << " for writing\n"; return {}; } return g; } auto f = std::make_unique(path); if (!f->good()) { std::cerr << "vcf: failed to open " << path << " for writing\n"; return {}; } return f; } } // namespace void VCFFile::writeSvsSingleFile(const std::string& path, bool gzip, const SeqLib::BamHeader& header) const { auto out_owner = open_vcf_out(path, gzip); if (!out_owner) return; std::ostream& out = *out_owner; out << sv_header << '\n'; // Build the emit list. For each pair: // - skip dups (if dedup ran) // - classify symbolic eligibility when sv_format == SYMBOLIC_WHEN_OBVIOUS // - emit one e1 (symbolic) or two (BND) entries into rows VCFEntryVec rows; rows.reserve(entry_pairs.size() * 2); for (const auto& kv : entry_pairs) { if (dups.count(kv.first)) continue; VCFEntryPair pair_copy = *kv.second; // shallow copy to stamp symbolic_rep if (sv_format == SvFormat::SYMBOLIC_WHEN_OBVIOUS) { const std::string kind = classify_symbolic_kind(*pair_copy.bp); if (!kind.empty()) { pair_copy.e1.symbolic_rep = true; pair_copy.e1.symbolic_kind = kind; rows.push_back(pair_copy.e1); continue; } } rows.push_back(pair_copy.e1); rows.push_back(pair_copy.e2); } std::sort(rows.begin(), rows.end(), entry_lt_for_output); for (auto& r : rows) { // PASS/FILTER gate: use the confidence string as source of truth // (bp->pass is a stale bool; see parser note in BreakPoint.cpp). if (r.bp->confidence != "PASS" && !include_nonpass) continue; // Stamp the per-entry somlod threshold so fillInfoFields applies // the right cutoff to the SOMATIC flag. r.somatic_threshold = somatic_threshold; out << r.toFileString(header, qual_mode) << '\n'; } } void VCFFile::writeIndelsSingleFile(const std::string& path, bool gzip, const SeqLib::BamHeader& header) const { auto out_owner = open_vcf_out(path, gzip); if (!out_owner) return; std::ostream& out = *out_owner; out << indel_header << '\n'; // Indels are always a single REF/ALT record per event (no BND pair), // so this is simpler than the SV path: collect e1 of every indel, // sort, emit. VCFEntryVec rows; rows.reserve(indels.size()); for (const auto& kv : indels) rows.push_back(kv.second->e1); std::sort(rows.begin(), rows.end()); for (auto& r : rows) { // PASS/FILTER gate: use the confidence string as source of truth // (bp->pass is a stale bool; see parser note in BreakPoint.cpp). if (r.bp->confidence != "PASS" && !include_nonpass) continue; r.somatic_threshold = somatic_threshold; out << r.toFileString(header, qual_mode) << '\n'; } } ================================================ FILE: src/svaba/vcf.h ================================================ #pragma once // vcf.h // // Read a svaba `bps.txt.gz` file back in, dedupe the breakpoint pairs, // and write out somatic/germline SV and indel VCFs. // // Design notes: // * `BreakPoint` is non-copyable and non-movable (see BreakPoint.h), so we // store every breakpoint as a `std::shared_ptr` — both the // `VCFEntry` views and the `VCFEntryPair` aggregate share ownership of // the same underlying object. // * `BreakPoint::SampleInfo` is a private nested type. vcf.cpp therefore // iterates `bp->allele` with `auto` and never names the type directly. // * The bps.txt.gz file is read one line at a time (memory-efficient); only // parsed records that survive the per-contig secondary cap are retained // in memory. // * The external API exposed to `run_svaba.cpp` is preserved verbatim: // `VCFFile(file, id, sc, header, nopass, verbose)` + // `writeIndels / writeSVs(basename, zip, onefile, bam_header)` + // `include_nonpass`. #include #include #include #include #include #include #include #include "SeqLib/BamHeader.h" #include "SeqLib/GenomicRegion.h" #include "BreakPoint.h" #include "SvabaSharedConfig.h" // --------------------------------------------------------------------------- // Header-field dictionaries. // --------------------------------------------------------------------------- using InfoMap = std::unordered_map; using FilterMap = std::unordered_map; using FormatMap = std::unordered_map; using SampleMap = std::unordered_map; using ContigFieldMap = std::unordered_map; // --------------------------------------------------------------------------- // Knobs for the `svaba tovcf` subcommand. // --------------------------------------------------------------------------- // // QualMode — what to put in the VCF QUAL column. // SUM_LO_PHRED (legacy): -10 log10 P(all alt reads are errors), taken from // BreakPoint::quality (col 34 of bps.txt). Keeps // behavior compatible with `svaba run` / refilter. // MAXLOD_PHRED: round(10 * bp->max_lod), cap 99. One-to-one with // INFO/MAXLOD, meaningful as a VCF QUAL field. // MISSING: emit '.' (VCF spec-valid missing value). Forces // users to look at FILTER / INFO for confidence, // which is usually what you want when somlod and // maxlod are the canonical scores. enum class QualMode { SUM_LO_PHRED, MAXLOD_PHRED, MISSING }; // SvFormat — how to serialize an SV call. // BND_ALWAYS (legacy): every SV emits as two paired BND records // with mate-bracket ALT notation. Matches // the current `svaba run` output exactly. // SYMBOLIC_WHEN_OBVIOUS: intrachromosomal events with unambiguous // orientation emit as a single symbolic // record ( / / ). Inter- // chromosomal and complex events stay BND. // Nicer for IGV / samplot / bcftools // pipelines that render symbolic alleles // natively. enum class SvFormat { BND_ALWAYS, SYMBOLIC_WHEN_OBVIOUS }; // --------------------------------------------------------------------------- // VCFHeader — the ##-lines that precede a VCF body. // --------------------------------------------------------------------------- struct VCFHeader { VCFHeader() = default; ~VCFHeader() = default; // VCF spec version this header declares. Defaulted to 4.5 (the most // recent formal spec as of 2024) — backwards-compatible at the record // level with anything accepting 4.2+, and enables the clean symbolic- // SV / EVENT / SVCLAIM idioms the tovcf path uses. std::string fileformat = "VCFv4.5"; std::string filedate; std::string source; std::string reference = "hg19"; std::string colnames = "#CHROM\tPOS\tID\tREF\tALT\tQUAL\tFILTER\tINFO\tFORMAT"; InfoMap infomap; FilterMap filtermap; FormatMap formatmap; SampleMap samplemap; ContigFieldMap contigfieldmap; friend std::ostream& operator<<(std::ostream& out, const VCFHeader& v); void addInfoField (std::string field, std::string number, std::string type, std::string description); void addFilterField(std::string field, std::string description); void addFormatField(std::string field, std::string number, std::string type, std::string description); void addSampleField(std::string field); void addContigField(std::string id, int len); }; // --------------------------------------------------------------------------- // VCFEntry — a single breakend row (one side of a BreakPoint, or an indel). // --------------------------------------------------------------------------- struct VCFEntry { VCFEntry() = default; ~VCFEntry() = default; // Shared ownership with the owning VCFEntryPair. std::shared_ptr bp; // Unique id across all VCFEntryPairs in a VCFFile. `id_num` is 1 for // the left breakend (b1) and 2 for the right (b2); indels always use 1. uint32_t id = 0; uint8_t id_num = 0; // Set by VCFFile::writeSvsSingleFile when the SvFormat:: // SYMBOLIC_WHEN_OBVIOUS mode decides this pair should be emitted as // a single ``/``/`` record instead of two paired BND // records. When true, only the e1 entry of the pair gets serialized // (the caller skips e2 for that pair). Kind is stashed here so // getAltString / fillInfoFields can key off it without reclassifying. bool symbolic_rep = false; std::string symbolic_kind; // "DEL" / "DUP" / "INV" when symbolic_rep is true // Somlod cutoff for stamping the INFO/SOMATIC flag. A record gets the // flag iff bp->LO_s >= somatic_threshold AND bp->somatic != FAILED. // Writers set this per-entry from VCFFile::somatic_threshold before // calling toFileString(); default 1.0 matches the tovcf default. double somatic_threshold = 1.0; std::string getRefString() const; std::string getAltString(const SeqLib::BamHeader& header) const; std::string getIdString () const; // Build the full INFO map (keys → values) for this entry. // `somatic_flag` = true adds `SOMATIC` (a flag INFO field). std::unordered_map fillInfoFields() const; // Serialize the entry as one VCF body line (no trailing newline). // qual_mode — what goes in the QUAL column (see QualMode). // NB: the symbolic/BND choice is read off this->symbolic_rep, // which the writer sets before calling. std::string toFileString(const SeqLib::BamHeader& header, QualMode qual_mode = QualMode::SUM_LO_PHRED) const; // Sort primarily by genomic position of the represented breakend. bool operator< (const VCFEntry& v) const; bool operator==(const VCFEntry& v) const; }; using VCFEntryVec = std::vector; // --------------------------------------------------------------------------- // VCFEntryPair — holds both sides (e1, e2) of a rearrangement, plus the // shared BreakPoint they are built from. Indels still use a pair but only // populate e1 as a real record. // --------------------------------------------------------------------------- struct VCFEntryPair { VCFEntryPair() = default; ~VCFEntryPair() = default; // Build both sides from a shared BreakPoint. Assigns a collision-avoided // hash id to e1.id / e2.id. explicit VCFEntryPair(std::shared_ptr& b); VCFEntry e1, e2; std::shared_ptr bp; std::string toFileString(const SeqLib::BamHeader& header, QualMode qual_mode = QualMode::SUM_LO_PHRED) const; }; using VCFEntryPairMap = std::unordered_map>; // --------------------------------------------------------------------------- // VCFFile — the top-level object, owning all entry pairs and the two headers. // --------------------------------------------------------------------------- struct VCFFile { VCFFile() = default; ~VCFFile() = default; // Read a svaba bps.txt.gz file, parse line-by-line into BreakPoints, // build VCFEntryPairs, and deduplicate. `nopass` controls whether // initially non-PASS records are retained (they can still be filtered // later by writeIndels/writeSVs via `include_nonpass`). // // `skip_dedup_in_ctor` = true has the ctor set `this->skip_dedup` // BEFORE the internal `deduplicate()` call fires, so no dedup runs // on input that was already deduped upstream (the `svaba tovcf` path). // Default false keeps backward-compat with run_svaba and refilter. VCFFile(std::string file, std::string id, const SvabaSharedConfig& sc, const VCFHeader& vheader, bool nopass, bool verbose, bool skip_dedup_in_ctor = false); std::string filename; std::string analysis_id; bool verbose = false; bool include_nonpass = false; // SvABA2.0 tovcf-path knobs. All default to legacy-compatible values so // run_svaba.cpp and refilter.cpp pick up no behavior change. The `svaba // tovcf` entry point flips them to the new defaults (MISSING qual, // SYMBOLIC_WHEN_OBVIOUS sv format, skip_dedup=true). bool skip_dedup = false; // deduplicate() returns immediately when set QualMode qual_mode = QualMode::SUM_LO_PHRED; SvFormat sv_format = SvFormat::BND_ALWAYS; // Minimum somlod (LO_s) to mark a record with the INFO/SOMATIC flag. // Writers stamp this onto each VCFEntry before emission. Default 1.0 // is the recommended somatic-call gate; lower it to be permissive, // raise it to be stricter. double somatic_threshold = 1.0; VCFHeader indel_header; VCFHeader sv_header; VCFEntryPairMap entry_pairs; // SVs VCFEntryPairMap indels; // indels std::unordered_set dups; // keys in entry_pairs marked as duplicates // Dedupe SVs via paired breakend interval-tree matching; dedupe indels // via (chr,pos,ref,alt) hash set. void deduplicate(const SeqLib::BamHeader& header); // Write the body VCFs to disk. `zip` is currently ignored (always writes // plain .vcf); `onefile` merges somatic/germline into one emission. void writeIndels(std::string basename, bool zip, bool onefile, const SeqLib::BamHeader& header) const; void writeSVs(std::string basename, bool zip, bool onefile, const SeqLib::BamHeader& header) const; // SvABA2.0: single-file writers used by `svaba tovcf`. Unlike // writeSVs / writeIndels above, these write EVERY record (somatic and // germline together) to one path, with the SOMATIC INFO flag set on // somatic rows for downstream filtering. Honors sv_format, qual_mode, // and include_nonpass set on the VCFFile. `gzip=true` uses ogzstream; // otherwise a plain ofstream is used. void writeSvsSingleFile (const std::string& path, bool gzip, const SeqLib::BamHeader& header) const; void writeIndelsSingleFile(const std::string& path, bool gzip, const SeqLib::BamHeader& header) const; }; ================================================ FILE: src/svabautils/AssemblyBamWalker.cpp ================================================ #include "AssemblyBamWalker.h" #include "SeqLib/UnalignedSequence.h" #include "SeqLib/ReadFilter.h" #include #include #include "SnowmanUtils.h" #include "run_snowman.h" #include //#define QNAME "5427150" #define MIN_ISIZE_FOR_DISC 700 //static std::vector contig_elems; static int num_to_run; static pthread_mutex_t snow_lock; static ogzstream all_align, os_allbps; //static std::ofstream all_align, os_allbps; static std::string tt, nn; // so hacky static std::shared_ptr ttindex, nnindex; static faidx_t* f; static std::set prefixes; static SeqLib::Filter::ReadFilterCollection * mr; //static ofstream os_allbps; static struct timespec start; static SeqLib::RefGenome * ref_genomeAW, * ref_genome_viral_dummy; bool __good_contig(const SeqLib::BamRecordVector& brv, const SeqLib::GenomicRegionVector& regions, int max_len, int max_mapq) { // NO INDELS /* return (brv.size() && regions.size() && brv.size() < 20 && (brv.size() > 1) && brv[0].Length() < 20000 && brv[0].CigarSize() < 50 && max_len > 250 && max_mapq >= 0); */ // all hard clip? size_t hc = 0; for (auto& i : brv) if (i.NumHardClip()) ++hc; return (brv.size() && hc != brv.size() && regions.size() && brv.size() < 20 && (brv.size() > 1 || brv[0].CigarSize() > 1) && brv[0].Length() < 20000 && brv[0].CigarSize() < 20 && max_len > 101 && max_mapq >= 0); } //bool runAC(SeqLib::BamRecordVector& brv, faidx_t * f, std::shared_ptr pt, std::shared_ptr pn, // const std::string& t, const std::string& n, const SeqLib::GenomicRegionVector& regions) { bool runAC(const ContigElement * c) { SeqLib::GRC trimmed_regions; for (auto& r : c->regions) if (r.chr < 24) trimmed_regions.add(r); if (!trimmed_regions.size()) return true; SnowmanBamWalker twalk, nwalk; if (!tt.empty()) { twalk = SnowmanBamWalker(tt); twalk.prefix = "t000"; twalk.max_cov = 200; twalk.get_mate_regions = false; twalk.SetPreloadedIndex(ttindex); twalk.SetMultipleRegions(trimmed_regions); twalk.readBam(); } if (!nn.empty()) { nwalk = SnowmanBamWalker(nn); nwalk.prefix = "n000"; nwalk.max_cov = 200; nwalk.get_mate_regions = false; nwalk.SetPreloadedIndex(nnindex); nwalk.SetMultipleRegions(trimmed_regions); nwalk.readBam(); } SeqLib::BamRecordVector bav_this; for (auto& q : twalk.reads) bav_this.push_back(q); for (auto& q : nwalk.reads) bav_this.push_back(q); // cluster the reads // set region to empty, just won't double-check that cluster overlaps regino. No big deal DiscordantClusterMap dmap = DiscordantCluster::clusterReads(bav_this, SeqLib::GenomicRegion(), 37, nullptr); std::vector this_alc; // contruct the index SeqLib::UnalignedSequenceVector usv; assert(c->brv[0].Qname().length()); assert(c->brv[0].Sequence().find("N") == std::string::npos); // here we have to flip if it has (-) alignment to reference. // this is because the pipeline assumes it came from a // de novo assembly, which is PRE alignment. Thus, we don't want // to take the i.Sequeunce directly, as this has been reverse-complemented // BY THE ALIGNER. std::string sss = c->brv[0].Sequence(); if (c->brv[0].ReverseFlag()) SeqLib::rcomplement(sss); usv.push_back({c->brv[0].Qname(), sss, std::string()}); SeqLib::BWAWrapper bw; bw.constructIndex(usv); if (!prefixes.size()) { prefixes.insert("t000"); prefixes.insert("n000"); } this_alc.push_back(AlignedContig(c->brv, prefixes)); AlignedContig * ac = &this_alc.back(); for (auto& kk : ac->m_frag_v) kk.m_max_indel = 20; // align the reads alignReadsToContigs(bw, usv, bav_this, this_alc, ref_genomeAW); ac->assignSupportCoverage(); // dummies ac->splitCoverage(); ac->addDiscordantCluster(dmap); // get teh coverages std::unordered_map covs; std::unordered_map clip_covs; covs["t000"] = &twalk.cov; covs["n000"] = &nwalk.cov; clip_covs["t000"] = &twalk.cov; clip_covs["n000"] = &nwalk.cov; std::vector allbreaks = ac->getAllBreakPoints(false); // false says dont worry about "local" for (auto& i : allbreaks) i.repeatFilter(); for (auto& i : allbreaks) i.addCovs(covs, clip_covs); for (auto& i : allbreaks) i.scoreBreakpoint(8, 2.5, 7, 3, 0); for (auto& i : allbreaks) i.setRefAlt(ref_genomeAW, ref_genome_viral_dummy); double region_width = 0; for (auto& i : trimmed_regions) region_width += i.width(); double cov = (double)bav_this.size() / region_width * 250; // print message if (cov > 100 || num_to_run % 1000 == 0) { std::stringstream ss; #ifndef __APPLE__ ss << SeqLib::displayRuntime(start); #endif std::cerr << "..read " << SeqLib::AddCommas(bav_this.size()) << " reads from " << trimmed_regions.size() << " region starting at " << trimmed_regions.at(0) << " Queue-left " << SeqLib::AddCommas(num_to_run) << " " << ss.str() << std::endl; } std::stringstream outr; bool no_reads = true; for (auto& i : allbreaks) outr << i.toFileString(no_reads) << std::endl; // MUTEX LOCKED //////////////////////////////////// pthread_mutex_lock(&snow_lock); --num_to_run; //all_align << (*ac) << std::endl; os_allbps << outr.str(); //////////////////////////////////// // MUTEX UNLOCKED //////////////////////////////////// pthread_mutex_unlock(&snow_lock); return true; } void AssemblyBamWalker::walkDiscovar() { //std::string rules = "global@!hardclip;!duplicate;!qcfail;phred[4,100];length[25,1000]%region@WG%!isize[0,1200];mapq[0,1000]%clip[5,1000]%ins[1,1000];mapq[0,100]%del[1,1000];mapq[1,1000]%mapped;!mate_mapped;mapq[1,1000]%mate_mapped;!mapped"; std::string rules = "{\"global\" : {\"duplicate\" : false, \"qcfail\" : false}, \"\" : { \"rules\" : [{\"isize\" : [MINS,0]},{\"rr\" : true},{\"ff\" : true}, {\"rf\" : true}, {\"ic\" : true}, {\"clip\" : 5, \"phred\" : 4}, {\"ins\" : true}, {\"del\" : true}, {\"mapped\": true , \"mate_mapped\" : false}, {\"mate_mapped\" : true, \"mapped\" : false}]}}"; rules.replace(rules.find("MINS"), 4, std::to_string(MIN_ISIZE_FOR_DISC)); mr = new SeqLib::Filter::ReadFilterCollection(rules, SeqLib::BamHeader()); f = findex; tt = tbam; nn = nbam; nnindex = nindex; ttindex = tindex; SnowmanUtils::fopen(id + ".alignments.txt.gz", all_align); SnowmanUtils::fopen(id + ".bps.txt.gz", os_allbps); os_allbps << BreakPoint::header() << std::endl; SeqLib::BamRecord r; std::cerr << "...starting to walk assembly BAM" << std::endl; std::vector ac_vec; std::unordered_map map; SeqLib::BamRecordVector brv; SeqLib::GenomicRegionVector regions; // start the timer #ifndef __APPLE__ clock_gettime(CLOCK_MONOTONIC, &start); #endif // open the mutex if (pthread_mutex_init(&snow_lock, NULL) != 0) { printf("\n mutex init failed\n"); return; } // Create the queue and consumer (worker) threads wqueue queue; std::vector*> threadqueue; for (int i = 0; i < numThreads; i++) { ConsumerThread* threadr = new ConsumerThread(queue, true); threadr->start(); threadqueue.push_back(threadr); } int count = 0, acc_count = 0; std::string curr_name; std::vector tmp_queue; // open ref genome for extracting sequences ref_genomeAW = new SeqLib::RefGenome(refGenome); int max_mapq = 0, max_len = 0; while(GetNextRecord(r)) { if (++count % 200000 == 0) std::cerr << "...checking contig " << SeqLib::AddCommas(count) << " to see if we should parse" << std::endl; // give each contig a unique prefix //r.SetQname("contig_" + r.Qname()); // first one if (curr_name.empty()) curr_name = r.Qname(); // add the MC tag if (r.ChrID() < br->n_targets && r.ChrID() >= 0) { r.AddZTag("MC", std::string(br->target_name[r.ChrID()])); } else continue; // add to existing if (curr_name == r.Qname()) { SeqLib::GenomicRegion gr = r.asGenomicRegion(); gr.Pad(100); regions.push_back(gr); brv.push_back(r); max_mapq = std::max(r.MapQuality(), max_mapq); max_len = std::max(r.Length(), max_len); } else { #ifdef QNAME if (brv.size()) if (brv[0].Qname() == QNAME || true) { std::cerr << " found it here -- checking " << std::endl; std::cerr << " brv.size() " << brv.size() << " regions.size() " << regions.size() << " brv.CigarSize() " << brv[0].CigarSize() << " brv.Length() " << brv[0].Length() << " max_len " << max_len << " max_mapq " << max_mapq << std::endl; } #endif if (true || __good_contig(brv, regions, max_len, max_mapq)) { ++acc_count; //AssemblyWalkerWorkItem * item = new AssemblyWalkerWorkItem(brv, findex, tindex, nindex, regions, tbam, nbam); AssemblyWalkerWorkItem * item = new AssemblyWalkerWorkItem(new ContigElement(brv, regions)); #ifdef QNAME if (brv[0].Qname() == QNAME) std::cerr << " found it here -- adding " << std::endl; #endif tmp_queue.push_back(item); regions.clear(); } // prepare the next one brv.clear(); brv.push_back(r); curr_name = r.Qname(); regions.clear(); SeqLib::GenomicRegion gr = r.asGenomicRegion(); gr.Pad(1000); regions.push_back(gr); max_mapq = r.MapQuality(); max_len = r.Length(); } } // add the last one if (__good_contig(brv, regions, max_len, max_mapq)) { AssemblyWalkerWorkItem * item = new AssemblyWalkerWorkItem(new ContigElement(brv, regions)); tmp_queue.push_back(item); } std::cerr << "...done adding " << SeqLib::AddCommas(tmp_queue.size()) << " contigs. Firing up " << numThreads << " re-alignment threads" << std::endl; num_to_run = tmp_queue.size(); if (!num_to_run) return; for (auto& i : tmp_queue) queue.add(i); // wait for the threads to finish for (int i = 0; i < numThreads; i++) threadqueue[i]->join(); all_align.close(); os_allbps.close(); } ================================================ FILE: src/svabautils/AssemblyBamWalker.h ================================================ #ifndef SNOWMAN_ASSEMBLY_BAM_WALKER_H__ #define SNOWMAN_ASSEMBLY_BAM_WALKER_H__ #include "SeqLib/BamReader.h" #include "AlignedContig2.h" #include "SnowmanBamWalker.h" /** Walk along a BAM file generated from a de novo assembly, * realigned to the genome (preferably by BWA-MEM). */ class AssemblyBamWalker: public SeqLib::BamReader { public: /** Construct a new read-only walker to move along the assembly bam * @param in File path of the assembly BAM */ AssemblyBamWalker() {} /** Move along a BAM file generated from Discovar and make the AlignedContigs */ void walkDiscovar(); void sendThread(); int numThreads = 1; faidx_t * findex = nullptr; }; struct ContigElement { SeqLib::BamRecordVector brv; SeqLib::GenomicRegionVector regions; ContigElement(const SeqLib::BamRecordVector& b, const SeqLib::GenomicRegionVector& r) : brv(b), regions(r) {} }; //bool runAC(SnowTools::BamReadVector& brv, faidx_t * f, std::shared_ptr pt, std::shared_ptr pn, // const std::string& t, const std::string& n, const SnowTools::GenomicRegionVector& regions); bool runAC(const ContigElement * c); class AssemblyWalkerWorkItem { private: //SnowTools::BamReadVector m_brv; //faidx_t * m_findex; //std::shared_ptr m_tindex, m_nindex; //SnowTools::GenomicRegionVector m_regions; //std::string m_t, m_n; ContigElement * m_contig; public: //AssemblyWalkerWorkItem(SnowTools::BamReadVector& brv, faidx_t *f, std::shared_ptr pt, std::shared_ptr pn, // SnowTools::GenomicRegionVector r, // const std::string& t, const std::string& n) //: m_brv(brv), m_findex(f), m_tindex(pt), m_nindex(pn), m_regions(r), m_t(t), m_n(n) {} AssemblyWalkerWorkItem(ContigElement* c) : m_contig(c) {} //bool run() { return runAC(m_brv, m_findex, m_tindex, m_nindex, m_t, m_n, m_regions); } bool run() { return runAC(m_contig); } ~AssemblyWalkerWorkItem() { delete m_contig; } }; #endif ================================================ FILE: src/svabautils/BamSplitter.cpp ================================================ #include "BamSplitter.h" #include "htslib/khash.h" #define PRINT_MOD 200000 void BamSplitter::splitBam() { assert(m_writers.size() == m_frac.size()); std::vector csum; double cs = 0; for (auto& i : m_frac) { cs += i; csum.push_back(cs); } SeqLib::BamRecord r; std::cerr << "**Starting to split BAM " << __startMessage(); size_t countr = 0; std::vector all_counts(m_writers.size(), 0); while (GetNextRecord(r)) { // print a message ++countr; if (countr % PRINT_MOD == 0) std::cerr << "...splitting BAM at position " << r.Brief() << " with sample rate " << csum[0] << " on " << m_writers.size() << " writers " << std::endl; // put in one BAM or another uint32_t k = __ac_Wang_hash(__ac_X31_hash_string(r.Qname().c_str()) ^ m_seed); double hash_val = (double)(k&0xffffff) / 0x1000000; for (size_t i = 0; i < m_writers.size(); ++i) { // decide whether to keep if (hash_val <= csum[i]/*sample_rate*/) { //r.RemoveAllTags(); m_writers[i].WriteRecord(r); ++all_counts[i]; break; } } } } void BamSplitter::fractionateBam(const std::string& outbam, Fractions& f) { std::cerr << "...setting up output fractionated BAM " << outbam << std::endl; SeqLib::BamWriter w; assert(w.Open(outbam)); w.SetHeader(Header()); w.WriteHeader(); //bam_hdr_t * h = bam_hdr_dup(br.get()); //w.SetWriteHeader(h); //w.OpenWriteBam(outbam); f.m_frc.CreateTreeMap(); SeqLib::BamRecord r; std::cerr << "**Starting to fractionate BAM " << __startMessage(); size_t countr = 0; std::vector all_counts(m_writers.size(), 0); while (GetNextRecord(r)) { // print a message ++countr; if (countr % PRINT_MOD == 0) std::cerr << "...fractionating BAM at position " << r.Brief(); SeqLib::GenomicRegion gr(r.ChrID(), r.Position(), r.Position(), '*'); std::vector qid, sid; SeqLib::GRC grc(gr); grc.FindOverlaps(f.m_frc, sid, qid, true); //f.m_frc.findOverlaps(grc, qid, sid, true); /* std::cerr << gr << " qid.size() " << qid.size() << " sid.size() " << sid.size() << std::endl; for (auto& i : qid) std::cerr << " Q: " << i << std::endl; for (auto& i : sid) std::cerr << " S: " << i << std::endl; */ if (!qid.size()) { if (countr % PRINT_MOD == 0) std::cerr << " -- Not in fraction region. Skipping read " << std::endl; continue; } double sample_rate = f.m_frc.at(qid[0]).frac; if (countr % PRINT_MOD == 0) std::cerr << " -- found frac region " << f.m_frc.at(qid[0]) << " w/rate " << sample_rate << std::endl; // put in one BAM or another uint32_t k = __ac_Wang_hash(__ac_X31_hash_string(r.Qname().c_str()) ^ m_seed); double hash_val = (double)(k&0xffffff) / 0x1000000; // keep sampling the read (if sample_rate < 1, just does this once) size_t num_sampled = 0; // how many times has read been sampled std::string qn; while(sample_rate > 0) { // decide whether to keep if (hash_val <= sample_rate) { //r.RemoveAllTags(); // if super-sampling, give unique qname if (num_sampled) { if (qn.empty()) qn = r.Qname(); r.SetQname("S" + std::to_string(num_sampled) + "_" + qn); } // add tag and remove another, if first time seen read if (!num_sampled) { r.RemoveTag("OQ"); // just for compactedness r.AddZTag("RT", std::to_string(sample_rate)); } w.WriteRecord(r); ++num_sampled; } sample_rate = sample_rate - 1; } } } void BamSplitter::setWriters(const std::vector& writers, const std::vector& fracs) { assert(fracs.size() == writers.size()); for (auto& i : writers) { std::cerr << "...setting up BAM: " << i << std::endl; SeqLib::BamWriter w; assert(w.Open(i)); w.SetHeader(Header()); w.WriteHeader(); // bam_hdr_t * h = bam_hdr_dup(br.get()/*header()*/); //w.SetWriteHeader(h); //w.OpenWriteBam(i); m_writers.push_back(w); } m_frac = fracs; } std::string BamSplitter::__startMessage() const { std::stringstream ss; if (m_region.size() == 1) ss << " on region " << m_region[0] << std::endl; else if (m_region.size() == 0) ss << " on WHOLE GENOME" << std::endl; else ss << " on " << m_region.size() << " regions " << std::endl; return ss.str(); } ================================================ FILE: src/svabautils/BamSplitter.h ================================================ #ifndef SNOWMAN_BAM_SPLITTER_H__ #define SNOWMAN_BAM_SPLITTER_H__ #include "SeqLib/BamReader.h" #include "SeqLib/BamWriter.h" #include "Fractions.h" class BamSplitter: public SeqLib::BamReader { public: BamSplitter() {} BamSplitter(uint32_t seed) : m_seed(seed) {} void setWriters(const std::vector& writers, const std::vector& fracs); void splitBam(); void fractionateBam(const std::string& outbam, Fractions& f); private: std::string __startMessage() const; uint32_t m_seed = 0; std::vector m_frac; std::vector m_writers; }; #endif ================================================ FILE: src/svabautils/Fractions.cpp ================================================ #include "Fractions.h" using namespace SeqLib; FracRegion::FracRegion(const std::string& c, const std::string& p1, const std::string& p2, const SeqLib::BamHeader& h, const std::string& f) : SeqLib::GenomicRegion(c, p1, p2, h) { // convert frac to double try { frac = std::stod(f); } catch (...) { std::cerr << "FracRegion::FracRegion - Error converting fraction " << f << " to double " << std::endl; exit(EXIT_FAILURE); } } size_t Fractions::size() const { return m_frc.size(); } std::ostream& operator<<(std::ostream& out, const FracRegion& f) { out << f.chr << ":" << SeqLib::AddCommas(f.pos1) << "-" << SeqLib::AddCommas(f.pos2) << " Frac: " << f.frac; return out; } void Fractions::readFromBed(const std::string& file, const SeqLib::BamHeader& h) { std::ifstream iss(file.c_str()); if (!iss || file.length() == 0) { std::cerr << "BED file does not exist: " << file << std::endl; exit(EXIT_FAILURE); } std::string line; std::string curr_chr = "-1"; while (std::getline(iss, line, '\n')) { size_t counter = 0; std::string chr, pos1, pos2, f; std::istringstream iss_line(line); std::string val; if (line.find("#") == std::string::npos) { while(std::getline(iss_line, val, '\t')) { switch (counter) { case 0 : chr = val; break; case 1 : pos1 = val; break; case 2 : pos2 = val; break; case 3 : f = val; break; } if (counter >= 3) break; ++counter; if (chr != curr_chr) { //std::cerr << "...reading from BED - chr" << chr << std::endl; curr_chr = chr; } } // construct the GenomicRegion FracRegion ff(chr, pos1, pos2, h, f); //if (ff.valid()) { //gr.pad(pad); //m_grv.push_back(gr); m_frc.add(ff); //} //} } // end "keep" conditional } // end main while } ================================================ FILE: src/svabautils/Fractions.h ================================================ #ifndef SNOWMAN_FRACTIONS_H__ #define SNOWMAN_FRACTIONS_H__ #include "SeqLib/GenomicRegionCollection.h" #include "SeqLib/BamHeader.h" #include /** @brief Extension of GenomicRegion with a fraction of reads to keep on an interval * * Used in conjunction with Fractions and used for selectively sub-sampling BAM files. */ class FracRegion : public SeqLib::GenomicRegion { public: FracRegion() {} FracRegion(const std::string& c, const std::string& p1, const std::string& p2, const SeqLib::BamHeader& h, const std::string& f); friend std::ostream& operator<<(std::ostream& out, const FracRegion& f); double frac; }; /** @brief Genomic intervals and associated sampling fractions */ class Fractions { public: Fractions() {} size_t size() const; void readFromBed(const std::string& file, const SeqLib::BamHeader& h) ; SeqLib::GenomicRegionCollection m_frc; private: }; #endif ================================================ FILE: src/svabautils/Makefile.am ================================================ bin_PROGRAMS = svabautils svabautils_CPPFLAGS = \ -I$(top_srcdir)/src/SGA/Util \ -I$(top_srcdir)/src/SGA/Bigraph \ -I$(top_srcdir)/src/SGA/SuffixTools \ -I$(top_srcdir)/src/SGA/StringGraph \ -I$(top_srcdir)/src/SGA/Algorithm \ -I$(top_srcdir)/src/SGA/SQG \ -I$(top_srcdir)/src/SGA/SGA \ -I$(top_srcdir)/src/Svaba \ -I$(top_srcdir)/SeqLib \ -I$(top_srcdir)/SeqLib/htslib svabautils_LDADD = \ $(top_builddir)/src/SGA/SGA/libsga.a \ $(top_builddir)/src/SGA/StringGraph/libstringgraph.a \ $(top_builddir)/src/SGA/Algorithm/libalgorithm.a \ $(top_builddir)/src/SGA/SuffixTools/libsuffixtools.a \ $(top_builddir)/src/SGA/Bigraph/libbigraph.a \ $(top_builddir)/src/SGA/Util/libutil.a \ $(top_builddir)/src/SGA/SQG/libsqg.a \ $(top_builddir)/SeqLib/src/libseqlib.a \ $(top_builddir)/SeqLib/bwa/libbwa.a \ $(top_builddir)/SeqLib/htslib/libhts.a \ $(top_builddir)/SeqLib/fermi-lite/libfml.a svabautils_LDFLAGS = -pthread -std=c++11 svabautils_SOURCES = snowutils.cpp splitcounter.cpp ../Snowman/BreakPoint2.cpp ../Snowman/AlignedContig2.cpp \ ../Snowman/DiscordantCluster.cpp ../Snowman/DBSnpFilter.cpp ../Snowman/PONFilter.cpp \ ../Snowman/SnowmanUtils.cpp ../Snowman/SnowmanAssemblerEngine.cpp ../Snowman/vcf.cpp ../Snowman/DiscordantRealigner.cpp \ ../Snowman/SnowmanOverlapAlgorithm.cpp ../Snowman/SnowmanASQG.cpp \ ../Snowman/SnowmanAssemble.cpp \ ../Snowman/KmerFilter.cpp ../Snowman/SnowmanBamWalker.cpp \ SeqFrag.cpp SimGenome.cpp BamSplitter.cpp SimTrainerWalker.cpp \ Fractions.cpp ../Snowman/STCoverage.cpp ../Snowman/Histogram.cpp ../Snowman/BamStats.cpp \ PowerLawSim.cpp AssemblyBamWalker.cpp assembly2vcf.cpp ================================================ FILE: src/svabautils/Makefile.in ================================================ # Makefile.in generated by automake 1.11.1 from Makefile.am. # @configure_input@ # Copyright (C) 1994, 1995, 1996, 1997, 1998, 1999, 2000, 2001, 2002, # 2003, 2004, 2005, 2006, 2007, 2008, 2009 Free Software Foundation, # Inc. # This Makefile.in is free software; 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bool verbose = false; // generate the random numbers std::vector rpower = drawFromPower(1, MAX_RAR_SIZE, power_law, num_breaks); // generate again, but for small indels std::vector small_indels2 = drawFromPower(1, 10, -3, num_breaks / 4); // generate inter-chr size_t num_intra = rpower.size() + small_indels2.size(); std::vector inter_chr((double)num_intra / (1-frac_inter) - num_intra, -1); rpower.insert(rpower.end(), small_indels2.begin(), small_indels2.end()); rpower.insert(rpower.end(), inter_chr.begin(), inter_chr.end()); std::random_shuffle(rpower.begin(), rpower.end()); // check for (auto& i : rpower) assert(i <= MAX_RAR_SIZE); const char TCGA[5] = "TCGA"; // get random inserts std::vector inserts; std::vector rsize = drawFromPower(1, 20, -2, num_breaks); for (int i = 0; i < num_breaks; ++i) { if (rand() % 2 == 0) { inserts.push_back(std::string()); continue; } std::string is(rsize[i], 'N'); for (size_t j = 0; j < is.length(); ++j) is[j] = TCGA[rand() % 3]; inserts.push_back(is); } SVEvent e; std::string ename, etype; for (int i = 0; i < num_breaks; ++i) { e.number = i; if (i % 500 == 0) std::cerr << "working on " << i << " of " << num_breaks << std::endl; int rval = rand() % 3; std::string frag, outstring; if (rpower[i] == -1) { etype = "INT"; ename = "tumor-" + std::to_string(i) + "-" + etype; if (verbose) std::cerr << "...generating inter-chr " << std::endl; genRandomSequence(frag, e.reg1, EVENT_BUFFER, findex, grc); std::string frag_ic; do { genRandomSequence(frag_ic, e.reg2, EVENT_BUFFER, findex, grc); } while (e.reg1.chr == e.reg2.chr); outstring = frag + inserts[i] + frag_ic; events << SeqLib::CHR_NAME[e.reg1.chr] << "\t" << e.reg1.pos2 << "\t" << SeqLib::CHR_NAME[e.reg2.chr] << "\t" << e.reg2.pos1 << "\t+\t-\t" << "N\t-1\tINT\t" << (inserts[i].empty() ? "N" : inserts[i]) << "\t" << ename << std::endl; // deletion } else if (rpower[i] <= 50 && rand() % 2) { etype = "del"; ename = "tumor-" + std::to_string(i) + "-" + etype; if (verbose) std::cerr << "...generating deletion of length " << rpower[i] << std::endl; genRandomSequence(frag, e.reg1, EVENT_BUFFER * 2 + rpower[i], findex, grc); try {outstring = frag.substr(0, EVENT_BUFFER) + frag.substr(EVENT_BUFFER + rpower[i], frag.length() - rpower[i] - EVENT_BUFFER);} catch (...) { std::cerr << " len " << (frag.length() - rpower[i] - EVENT_BUFFER) << std::endl; } assert(outstring.length() < EVENT_BUFFER * 10); events << SeqLib::CHR_NAME[e.reg1.chr] << "\t" << (e.reg1.pos1+EVENT_BUFFER) << "\t" << SeqLib::CHR_NAME[e.reg1.chr] << "\t" << (e.reg1.pos1 + EVENT_BUFFER + rpower[i]) << "\t+\t-\tN\t" << rpower[i] << "\tdel\tN\t" << ename << std::endl; // insertion } else if (rpower[i] <= 50) { etype = "ins"; ename = "tumor-" + std::to_string(i) + "-" + etype; if (verbose) std::cerr << "...generating insertion of length " << rpower[i] << std::endl; genRandomSequence(frag, e.reg1, EVENT_BUFFER * 2, findex, grc); std::string ins(rpower[i], 'N'); for (int y = 0; y < rpower[i]; ++y) ins[y] = TCGA[rand() % 3]; outstring = frag.substr(0, EVENT_BUFFER) + ins + frag.substr(EVENT_BUFFER, frag.length() - EVENT_BUFFER); assert(outstring.length() < EVENT_BUFFER * 10); events << SeqLib::CHR_NAME[e.reg1.chr] << "\t" << (e.reg1.pos1+EVENT_BUFFER) << "\t" << SeqLib::CHR_NAME[e.reg1.chr] << "\t" << (e.reg1.pos1 + EVENT_BUFFER + 1) << "\t+\t-\t" << ins << "\t" << rpower[i] << "\tins\tN\t" << ename << std::endl; // TANDEM DUPLICATION } else if (rpower[i] < MAX_DUP_DEL_INV_SIZE && rval == 0) { etype = "DUP"; ename = "tumor-" + std::to_string(i) + "-" + etype; if (verbose) std::cerr << "...generating DUP of length " << rpower[i] << std::endl; genRandomSequence(frag, e.reg1, EVENT_BUFFER * 2 + rpower[i], findex, grc); outstring = frag.substr(0, EVENT_BUFFER + rpower[i]) + inserts[i] + frag.substr(EVENT_BUFFER, frag.length() - EVENT_BUFFER); //rpower[i]) + frag.substr(EVENT_BUFFER, frag.length() - EVENT_BUFFER); assert(outstring.length() < EVENT_BUFFER * 10); events << SeqLib::CHR_NAME[e.reg1.chr] << "\t" << (e.reg1.pos1+EVENT_BUFFER+rpower[i]) << "\t" << SeqLib::CHR_NAME[e.reg1.chr] << "\t" << (e.reg1.pos1 + EVENT_BUFFER) << "\t+\t-\t" << "N" << "\t" << rpower[i] << "\tDUP\t" << (inserts[i].empty() ? "N" : inserts[i]) << "\t" << ename << std::endl; // DELETION } else if (rpower[i] < MAX_DUP_DEL_INV_SIZE && rval == 1) { etype = "DEL"; ename = "tumor-" + std::to_string(i) + "-" + etype; if (verbose) std::cerr << "...generating DEL of length " << rpower[i] << std::endl; genRandomSequence(frag, e.reg1, rpower[i] + EVENT_BUFFER * 2, findex, grc); outstring = frag.substr(0, EVENT_BUFFER) + inserts[i] + frag.substr(EVENT_BUFFER + rpower[i], frag.length() - EVENT_BUFFER - rpower[i]); assert(outstring.length() < EVENT_BUFFER * 10); events << SeqLib::CHR_NAME[e.reg1.chr] << "\t" << (e.reg1.pos1+EVENT_BUFFER) << "\t" << SeqLib::CHR_NAME[e.reg1.chr] << "\t" << (e.reg1.pos1 + EVENT_BUFFER + rpower[i]) << "\t+\t-\tN\t" << rpower[i] << "\tDEL\t" << (inserts[i].empty() ? "N" : inserts[i]) << "\t" << ename << std::endl; // INV } else if (rpower[i] < MAX_DUP_DEL_INV_SIZE && rval == 2 && false) { etype = "INV"; ename = "tumor-" + std::to_string(i) + "-" + etype; if (verbose) std::cerr << "...generating INV of length " << rpower[i] << std::endl; genRandomSequence(frag, e.reg1, rpower[i] + EVENT_BUFFER * 2, findex, grc); std::string inv_frag = frag.substr(EVENT_BUFFER, rpower[i]); std::reverse(inv_frag.begin(), inv_frag.end()); outstring = frag.substr(0, EVENT_BUFFER) + inv_frag + frag.substr(EVENT_BUFFER, frag.length() - EVENT_BUFFER - rpower[i]); assert(outstring.length() < EVENT_BUFFER * 10); events << SeqLib::CHR_NAME[e.reg1.chr] << "\t" << (e.reg1.pos1+EVENT_BUFFER) << "\t" << SeqLib::CHR_NAME[e.reg1.chr] << "\t" << (e.reg1.pos1 + EVENT_BUFFER + rpower[i]) << "\t+\t-\tN\t" << rpower[i] << "\tINV\t" << (inserts[i].empty() ? "N" : inserts[i]) << "\t" << ename << std::endl; } else { etype = "RAR"; ename = "tumor-" + std::to_string(i) + "-" + etype; if (verbose) std::cerr << "...generating RAR of length " << rpower[i] << std::endl; genRandomSequence(frag, e.reg1, EVENT_BUFFER, findex, grc); bool leftfit = e.reg1.pos1 - rpower[i] > 1e6; bool rightfit = e.reg1.pos2 + rpower[i] < SeqLib::CHR_LEN[e.reg1.chr]; int rrr = -1; if (leftfit && rightfit) rrr = rand() % 2; else if (leftfit) rrr = 0; else if (rightfit) rrr = 1; // rearrange to left std::string seq2; if (rrr == 0) { if (verbose) std::cerr << "...generating LEFT RAR of length " << rpower[i] << std::endl; SeqLib::GenomicRegion gr(e.reg1.chr, e.reg1.pos1 - rpower[i] - EVENT_BUFFER, e.reg1.pos1 - rpower[i]); std::string chrstring = SeqLib::CHR_NAME[e.reg1.chr]; int len; char * seq = faidx_fetch_seq(findex, const_cast(chrstring.c_str()), gr.pos1, gr.pos2 - 1, &len); if (!seq) break; else seq2 = std::string(seq); outstring = seq2 + inserts[i] + frag; assert(outstring.length() < EVENT_BUFFER * 10); events << SeqLib::CHR_NAME[gr.chr] << "\t" << gr.pos2 << "\t" << SeqLib::CHR_NAME[e.reg1.chr] << "\t" << e.reg1.pos1 << "\t+\t-\tN\t" << rpower[i] << "\tRAR\t" << (inserts[i].empty() ? "N" : inserts[i]) << "\t" << ename << std::endl; //std::cerr << rpower[i] << " span " << (std::abs(gr.pos2 - e.reg1.pos1) - rpower[i]) << std::endl; assert(rpower[i] == (e.reg1.pos1 - gr.pos2)); // rearrange to right } else if (rrr == 1) { if (verbose) std::cerr << "...generating RIGHT RAR of length " << rpower[i] << std::endl; SeqLib::GenomicRegion gr(e.reg1.chr, e.reg1.pos2 + rpower[i], e.reg1.pos2 + rpower[i] + EVENT_BUFFER); std::string chrstring = SeqLib::CHR_NAME[gr.chr]; int len; char * seq = faidx_fetch_seq(findex, const_cast(chrstring.c_str()), gr.pos1, gr.pos2 - 1, &len); if (!seq) break; else seq2 = std::string(seq); events << SeqLib::CHR_NAME[e.reg1.chr] << "\t" << e.reg1.pos2 << "\t" << SeqLib::CHR_NAME[gr.chr] << "\t" << gr.pos1 << "\t+\t-\tN\t" << rpower[i] << "\tRAR\t" << (inserts[i].empty() ? "N" : inserts[i]) << "\t" << ename << std::endl; outstring = frag + inserts[i] + seq2; assert(outstring.length() < EVENT_BUFFER * 10); //std::cerr << rpower[i] << " span " << (std::abs(gr.pos1 - e.reg1.pos2) - rpower[i]) << std::endl; assert(rpower[i] == gr.pos1 - e.reg1.pos2); // rearrangement doesn't fit } else { if (verbose) std::cerr << "FAILED TO FIT RAR of length " << rpower[i] << " on break " << e.reg1 << std::endl; } } assert(outstring.length() < 50000); if (!outstring.empty()) outfasta << ">" << ename << std::endl << outstring << std::endl; else std::cerr << "...empty string on rar " << i << std::endl; } } std::vector drawFromPower(double x0, double x1, double power, int n_draws) { assert(power != -1); const int PRECISION = 1e6; std::vector rpower(n_draws, 0); for (int i = 0; i < n_draws; ++i) { double r = (double)(rand() % PRECISION)/(double)PRECISION; double t1 = std::pow(x1, power+1); double t2 = std::pow(x0, power+1); double tsum = (t1-t2) * r + t2; rpower[i] = std::floor(std::pow(tsum, 1 / (power + 1))); } return rpower; } void genRandomSequence(std::string& s, SeqLib::GenomicRegion& gr, int width, faidx_t * findex, SeqLib::GRC& grc) { gr.chr = SnowmanUtils::weightedRandom(SeqLib::CHR_CUMSUM_WEIGHT_X); std::string chrstring = SeqLib::CHR_NAME[gr.chr]; char * seq = nullptr; int len; // get the first sequence do { if (seq) free(seq); seq = nullptr; s = std::string(); gr.pos1 = 1e6 + rand() % (int)(SeqLib::CHR_LEN_VEC[gr.chr] - 2e6); gr.pos2 = gr.pos1 + width; seq = faidx_fetch_seq(findex, const_cast(chrstring.c_str()), gr.pos1, gr.pos2 - 1, &len); if (seq) s = std::string(seq); } while (!seq || s.find("N") != std::string::npos || grc.CountOverlaps(gr)); } ================================================ FILE: src/svabautils/PowerLawSim.h ================================================ #ifndef SNOWMAN_POWER_LAW_SIM_HH #define SNOWMAN_POWER_LAW_SIM_HH #include #include "SeqLib/GenomicRegionCollection.h" #include "SeqLib/BWAWrapper.h" void PowerLawSim(faidx_t* findex, int num_breaks, double power_law, SeqLib::GRC& grc, std::ofstream& outfasta, std::ofstream& events); std::vector drawFromPower(double x0, double x1, double power, int n_draws); void genRandomSequence(std::string& s, SeqLib::GenomicRegion& gr, int width, faidx_t * findex, SeqLib::GRC& grc); struct SVEvent { SeqLib::GenomicRegion reg1; SeqLib::GenomicRegion reg2; int break1; int break2; std::string ins; std::string r_ins; std::string etype; int number; }; #endif ================================================ FILE: src/svabautils/ReadSim.cpp ================================================ #include "ReadSim.h" #include #include #include void ReadSim::addAllele(const std::string& s, double af) { if (m_seq.size() && m_seq.back().length() != s.length()) { std::cerr << "ReadSim::addAllele error. Expecting sequences to be same size" << std::endl; exit(EXIT_FAILURE); } if (af <= 0 || af > 1) { std::cerr << "ReadSim::addAllele error. Expecting allelic fraction > 0 and <= 1. value is " << af << std::endl; exit(EXIT_FAILURE); } m_seq.push_back(s); m_frac.push_back(af); } int ReadSim::__random_allele(std::vector& cs) const { // get a random allele size_t al = 0; uint32_t rval = rand() % 100000; double rand_allele = rval % 100000; while (al < cs.size()) { if (rand_allele <= cs[al] * 100000) break; ++al; } return al; } double ReadSim::__get_cumsum(std::vector& cs) const { double csum = 0; for (size_t i = 0; i < m_frac.size(); ++i) { csum += m_frac[i]; cs.push_back(csum); } if (csum < 0.999 || csum > 1.001) { std::cerr << "ReadSim::addSequence: Expecting sum of allelic fractions = 1. Sum is " << csum << std::endl; return csum; } return csum; } void ReadSim::sampleReadsToCoverage(std::vector& reads, int cov, double error_rate, double ins_error_rate, double del_error_rate, int readlen) { // check validity if (m_seq.size() == 0) { std::cerr << "ReadSim::sampleReadsToCoverage: No sequences. Add with ReadSim::addSequence" << std::endl; return; } // how many smaples to get desired coverage? size_t seqlen = m_seq[0].length(); int ns = cov * seqlen / readlen; uint32_t maxstart = seqlen - readlen; // get cumulative sum for sampling std::vector m_frac_cumsum; __get_cumsum(m_frac_cumsum); // loop through num samples and start sampling for (int i = 0; i < ns; ++i) { // random allele int al = __random_allele(m_frac_cumsum); // get a random subsequence //uint32_t rstart; uint32_t rstart = rand() % maxstart; std::string s = m_seq[al].substr(rstart, readlen); // add the errors makeSNVErrors(s, error_rate); // add the insertion errors if (rand() % 100000 > ins_error_rate*100000) makeInsErrors(s); // only make sometimes if (rand() % 100000 > del_error_rate*100000) makeDelErrors(s, rstart, m_seq[al]); reads.push_back(s); } } void ReadSim::samplePairedEndReadsToCoverage(std::vector& reads1, std::vector& reads2, std::vector& qual1, std::vector& qual2, int cov, double error_rate, double ins_error_rate, double del_error_rate, int readlen, double mean_isize, double sd_isize, const std::vector& qual_dist) { // check validity if (m_seq.size() == 0) { std::cerr << "ReadSim::samplePairedEndReadsToCoverage: No sequences. Add with ReadSim::addSequence" << std::endl; return; } // how many samples to get desired coverage? size_t seqlen = m_seq[0].length(); int ns = cov * seqlen / readlen / 2; // get cumulative sum for sampling std::vector m_frac_cumsum; __get_cumsum(m_frac_cumsum); // setup random number generator for isize std::default_random_engine generator(rand()); std::normal_distribution distribution(mean_isize, sd_isize); // loop through num samples and start sampling for (int i = 0; i < ns; ++i) { if (i % 100000 == 0) std::cerr << "...sampling read " << i << " of " << ns << std::endl; // get a random isize int isize = distribution(generator); uint32_t maxstart = seqlen - readlen*2 - isize; // random allele int al = __random_allele(m_frac_cumsum); // get random subsequences uint32_t rstart = rand() % maxstart; std::string s1 = m_seq[al].substr(rstart, readlen); std::string s2 = m_seq[al].substr(rstart + readlen + isize, readlen); std::string q1, q2; size_t rep_spot = std::min(s1.find("AAAAAAAAAA"), s1.find("TTTTTTTTTT")); // homopolymer scrambling if (rep_spot != std::string::npos) { q1 = qual_dist[rand() % qual_dist.size()]; for (size_t i = rep_spot; i < q1.length(); ++i) q1[i] = '#'; } else { q1 = qual_dist[rand() % qual_dist.size()]; } rep_spot = std::min(s2.find("AAAAAAAAAA"), s2.find("TTTTTTTTTT")); // homopolymer scrambling if (rep_spot != std::string::npos) { q2 = qual_dist[rand() % qual_dist.size()]; for (size_t i = rep_spot; i < q2.length(); ++i) q2[i] = '#'; } else { q2 = qual_dist[rand() % qual_dist.size()]; } // dont keep reads with N if (s1.find("N") != std::string::npos || s2.find("N") != std::string::npos) continue; SeqLib::rcomplement(s2); std::reverse(q2.begin(), q2.end()); // add the errors //std::cerr << " " << s1 << " " << rstart << " rlen " << readlen << " ms " << m_seq[al].length() << std::endl; if (error_rate > 0) { makeSNVErrors(s1, error_rate); makeSNVErrors(s2, error_rate); } if (ins_error_rate) { if (rand() % 100000 < ins_error_rate*100000) makeInsErrors(s1); if (rand() % 100000 < ins_error_rate*100000) makeInsErrors(s2); } if (del_error_rate) { if (rand() % 100000 < del_error_rate*100000) makeDelErrors(s1, rstart, m_seq[al]); if (rand() % 100000 < del_error_rate*100000) makeDelErrors(s2, rstart + readlen + isize, m_seq[al]); } reads1.push_back(s1); reads2.push_back(s2); qual1.push_back(q1); qual2.push_back(q2); } } std::ostream& operator<<(std::ostream& out, const Indel& i) { out << i.len << "\t" << i.type << "\t" << i.gr.chr << "\t" << i.gr.pos1 << "\t" << i.gr.pos2 << "\t" << i.frag_id << "\t" << (i.lead_base + i.ref_seq) << "\t" << (i.lead_base + i.alt_seq); return out; } Indel ReadSim::makeDelErrors(std::string& s, int del_size) { std::vector sizer = {del_size}; if (del_size <= 0) sizer = {1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1, 2,2,2,2,2,2,2,2,2,2,3,3,3,3,4,4,4,4,4,5,5,5,6,6, 7,8,9,10,11,12,13,14,15,16,17,18,20,22,24,26}; uint32_t rpos = s.length() + 1; // get a random size //int ds = getRandomIndelSize(); int ds = del_size; size_t failsafe = 0; while (rpos + ds > s.length() && failsafe < 1000) { ++failsafe; // get a random position in read rpos = rand() % (s.length() - 5 - ds); rpos += 5; } // get the replacement sequence //std::cerr << del_size << " rpos " << rpos << " s.length() " << s.length() << std::endl; s = s.substr(0, rpos) + s.substr(rpos + ds, s.length() - rpos - ds); // + refseq.substr(rpos + s.length(), ds); return Indel(); //{ds, 'D', rpos}; } Indel ReadSim::makeDelErrors(std::string& s, int sstart, const std::string& refseq) { const std::vector sizer = {1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1, 2,2,2,2,2,2,2,2,2,2,3,3,3,3,4,4,4,4,4,5,5,5,6,6, 7,8,9,10,11,12,13,14,15,16,17,18,20,22,24,26}; int ds = getRandomIndelSize(); // get a random position in read uint32_t rpos = rand() % (s.length() - 5 - ds); rpos += 5; // make sure we aren't at end if (sstart + s.length() + ds >= refseq.length()) return Indel(); // empty // get the replacement sequence s = s.substr(0, rpos) + s.substr(rpos + ds, s.length() - rpos - ds) + refseq.substr(sstart + s.length(), ds); //return Indel(ds, 'D', ); return Indel(); } int ReadSim::getRandomIndelSize() const { const std::vector s = {1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1, 2,2,2,2,2,2,2,2,2,2,3,3,3,3,4,4,4,4,4,5,5,5,6,6, 2,2,2,2,2,2,2,2,2,2,3,3,3,3,4,4,4,4,4,5,5,5,6,6, 7,7,8,8,9,9,10,10,11,11,12,12,13,13, 14,15,16,17,18,19,20,21,22,23,24,25,26, 27,28,29,30,31,32,33,34,35,36,37,38,39, 40,41,42,43,44,45,46,47,48,49,50, 60,70,80,90,100,110,120,130,140,150,160,170,180,190,200,210,220,230,240,250,260,270,280,290,300,310,320,330,340,350,360,370,380,390,400,410,420,430,440,450,460,470,480,490,500,510,520,530,540,550,560,570,580,590,600,610,620,630,640,650,660,670,680,690,700,710,720,730,740,750,760,770,780,790,800,810,820,830,840,850,860,870,880,890,900,910,920,930,940,950,960,970,980,990,1000, 1010,1020,1030,1040,1050,100,1070,1080,1090,1100,1110,1120,1130,1140,1150,1160,1170,1180,1190,1200,1210,1220,1230,1240,1250,1260,1270,1280,1290,1300,1320,1340,1360,1380,1400,1420,1440,1460,1480,1500,1520,1540,1560,1580,1600,1620,1640,1660,1680,1700,1720,1740,1760,1780,1800,1820,1840,1860,1880,1900,1920,1940,1960,1980,2000}; size_t rr = rand() % s.size(); return s[rr]; } Indel ReadSim::makeInsErrors(std::string& s, bool keep_size, int indel_size) { std::vector sizer = {indel_size}; if (indel_size <= 0) sizer = {1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1, 2,2,2,2,2,2,2,2,3,3,3,3,4,4,4,4,4,5,5,5,6,6, 7,8,9,10,11,12,13,14,15,16,17,18,20,22,24,26}; const char TCGA[5] = "TCGA"; // get a random size //int is = getRandomIndelSize(); int is = indel_size; // generate the random insertion piece std::string ins(is, 'N'); for (int i = 0; i < is; ++i) ins[i] = TCGA[rand() % 3]; // get a random position in read uint32_t rpos = rand() % (s.length() - 10); assert(s.length() - 10 > 0); rpos += 10; if (keep_size) { if (rpos < s.length() / 2) { // trim off back end s = s.substr(0, rpos) + ins + s.substr(rpos, s.length() - rpos - is); } else { // trim off front end s = s.substr(is, rpos - is) + ins + s.substr(rpos, s.length() - rpos + is); } } else { s = s.substr(0, rpos) + ins + s.substr(rpos, s.length() - rpos); } return Indel(); //Indel(is, 'I', rpos); } void ReadSim::makeSNVErrors(std::string& s, double er) { const char T[4] = "ACG"; const char C[4] = "ATG"; const char G[4] = "ACT"; const char A[4] = "TCG"; std::default_random_engine generator(rand()); std::binomial_distribution distribution(s.length(), er); size_t num_errors = distribution(generator); for (size_t i = 0; i < num_errors; ++i) { uint32_t pos = rand() % s.length(); size_t w = rand() % 3; if (s.at(pos) == 'A') s[pos] = A[w]; else if (s.at(pos) == 'T') s[pos] = T[w]; else if (s.at(pos) == 'C') s[pos] = C[w]; else if (s.at(pos) == 'G') s[pos] = G[w]; else if (s.at(pos) == 'N') ; else std::cerr << "ReadSim::makeSNVErrors: Unexpected character in string. Char: " << s.at(pos) << " main seq " << s << std::endl; } } void ReadSim::baseQualityRelevantErrors(std::string& s, const std::string& bq) { char TCGA[5] = "TCGA"; assert(s.length() == bq.length()); for (size_t i = 0; i < s.length(); ++i) { if (bq.at(i) <= 37) { // low quality-ish s[i] = TCGA[rand() % 4]; } } } ================================================ FILE: src/svabautils/ReadSim.h ================================================ #ifndef SNOWMAN_READSIM_H__ #define SNOWMAN_READSIM_H__ #include #include #include #include "SeqLib/GenomicRegion.h" struct Indel { Indel() : len(0), type('N') {} Indel(size_t l, char t, const std::string& rseq, const std::string& aseq, const std::string& lseq) : len(l), type(t) { ref_seq = rseq; alt_seq = aseq; lead_base = lseq; assert(lseq.length() == 1); assert(t == 'D' || alt_seq.length() == l); assert(t == 'I' || ref_seq.length() == l); } size_t len; char type; std::string ref_seq, alt_seq, lead_base; SeqLib::GenomicRegion gr; int frag_id; friend std::ostream& operator<<(std::ostream& out, const Indel& i); }; class ReadSim { public: ReadSim() : m_error(0), m_imean(350), m_isd(50) {} /** Give the sequene string SNP errors at rate of er */ void makeSNVErrors(std::string& s, double er); Indel placeDel(std::string& s, uint32_t rpos, uint32_t ds, const std::string& refseq); void sampleReadsToCoverage(std::vector& reads, int cov, double error_rate, double ins_error_rate, double del_error_rate, int readlen); void samplePairedEndReadsToCoverage(std::vector& reads1, std::vector& reads2, std::vector& qual1, std::vector& qual2, int cov, double error_rate, double ins_error_rate, double del_error_rate, int readlen, double mean_isize, double sd_isize, const std::vector& qual_dist); int getRandomIndelSize() const; void addAllele(const std::string& s, double af); Indel makeInsErrors(std::string& s, bool keep_size = true, int ins_size = -1); Indel makeDelErrors(std::string& s, int sstart, const std::string& refseq); Indel makeDelErrors(std::string& s, int ins_size = -1); void makeClipErrors(std::string& s, double er, int min_clip_len, int max_clip_len); void baseQualityRelevantErrors(std::string& s, const std::string& bq); private: // get the cumulative sum double __get_cumsum(std::vector& cs) const; int __random_allele(std::vector& cs) const; // sequences to simulate from std::vector m_seq; // allelic fractions for each std::vector m_frac; // read SNP error rate double m_error; // insert size distribution double m_imean; double m_isd; }; #endif ================================================ FILE: src/svabautils/SeqFrag.cpp ================================================ #include "SeqFrag.h" #include void SeqFrag::addScrambledEnds(size_t left_len, size_t right_len) { std::vector TCGA = {"T","C","G","A"}; std::string left, right; for (size_t i = 0; i < left_len; ++i) left += TCGA[rand() % 4]; for (size_t i = 0; i < right_len; ++i) right += TCGA[rand() % 4]; left_scramble = left; right_scramble = right; m_seq = left + m_seq + right; } void SeqFrag::spikeMicrobe() { const char* PSUEDO = "CAAAAAGACATTTTTTTTGGGATATTAAGATTTCCTAGGATAGAAATCAAATTATTAGATAATTTTTGTATATATTTTAA" "ATATGTCTTAAAAAAATGTCTTTTTGTCTAAATATATTAAGCTTTTCATTTTTACATCGACAAATTTAGATATTTTTAGA" "CATTTTGGGTATTTTTTTAGACATTTTTAAAATATTTTAGACAGAATTGTATTATGATCCTATTTTTAGACCTAAATATA" "TGCAAAATTATCATATCGTCGTGGAGTGTTACCAAGTAATTCACTATATTGATACAAATCATTACCTCTATAATCTGGTT" "TAAAGGGAAGTTCTTTAATTGGTTCTTTCTTATCCTCTTTTTTATCCTCATCATTACCTTTATGAGCATTGTTTCTACAC" "ATGTATTTGACTAGAAAGAAAAATAATATAAACAATATTATAAGTACTATAAAAGTAAATATACATATTTTGATCATCAT" "TTTGTTACCAATTTCATTACCAACTTCCTTGCCTATAACGCTGAACGCATTATATGCAATGGGATCCATCGCGAAAGGAT" "TTTCCATTACTATCTATTATATCATTACATAATTGTTTATTGTTCTATAAATAATAAACAAATATACACTATTAGATAGT" "TTTTACCATCCAAATTCTTTCATAAACTCATTAATATCTTCTATTGACTTGCATTCTTCTAGTTCAATCCAATATATTGG" "TGTTTCTGGATCAATTTCTTCGTTTTCCTCTTCTTGTTTATCAAGATATTCTATTAGATCATTCTCTGCTCTGTTTCTCA" "ATGATTCAAGTTTTTTCTGTTCTTTTTTCAAGAGTTTTTTACTCACACTAAATTTACCATACATTAATATATAATACTAT" "AAAATACTATATATTAATTATCTTTATATCATTTTATATTAATTTATATTATTTTGATAAATTTTCCACGTATTTATAAT" "AATCATTAAATGTTCCTGATCTTACCGATTGATATTTGTTACGAACAGATTGTTGTTGTTCTTCAGTAGTTTTATCCCAT" "TCATCAACCATAATATTTCTAAAATTGGTTTTTGGTGCGACTATTTCTAATTTTTTTTTGTATGAATTAGCTTCTCTTTT" "GAATTTAGCAATATCACTCAACATAAGATTTTTAAGATCCTTTTCTATGAGTAGTTGTTCTTCCAATTCTGTAACGGTTA" "ATATTTTTTCCCTAGTCGTTATAAAAGCTTTTTCAAGAGCCATCGCTAATTGATCATATGATACAAAATTTTCATCATCT" "ATATTTTCTAATACTTCTTCAGCATTTCTTTTAATTTTTTTAGCTTTACTTGCCATTATATATACTATATTTACATATAA" "TTATTTATCATTATCATTAATAAAAAAATCAACCATTTCTTTTAGATTATTCTTTTTTTTTAGAGTAGATATTAAGTTAT" "AATTTTGTATAATTTAGGTTGTTTATGGGATTTTTTAGGTTTATTTTCTGTAACAGACATGAACATTGATCCACCATGTT" "GATCATTATCATCATCAAGAAGATCATCAACATTATCATTATAATTATCGGCCTCATTTAATTCATTTTCATCAGTATTA" "TAATTTCCAGATGATTGATTATCATCATCACTTTCATTATCACTTTCATCACTGACGTCAAAGATAACTTCTTTTTTTGA" "TTTAACAATCTTTGGTGTTTTTTTAACCATTTTTTTGCCTCTTTTTGACTTAGATTTGACTGGTTCTATCTCAGTTTCTG" "ATTCAACTTCCTCATCGGATTCATTATCAGATTCATTATCAGATTCTTCTGATTCACTTGATTCATCTCTAGATTTTGAT" "ATTTTTTTGGTTGTTCTATTCAATTCCTCTTTCATCATTTTTCTGGTTTTATAAAAAATTGGTTTACTGATTCCAGCTTT" "CTTACAAGCTTGAACCACTGTCAAACCACCTTTCAAATAATATGATTCGGCTTTTTCATATAATATTTTATTTCGTCGGT" "ATCTTAGGTTTTCTAGAACAGACATGTCCTTTATTATACCTAAATAATATTATTTTTATACCAAAACAGATCCTTTATCA" "TATACACATAATTCGGATCCAATCCGATTTTTTACTTTGATAAAAAGGCGAAAAATAATCAAAGTAAAAAGTAAGAAATA" "TTAAATATATGCATAATATAATATTATAATGTCGCATAATTGTCAAAATAGTAATGAACCTGATACAGTGTATTATGATA" "TAGTTATCCCATATAATCCCAATGATTCTGGTTTAAGCCCAGCAACATTCCAAGCACAGTTAACACAACCATTGCTTTAT" "AATCCTGACAAATATTACCTGAGTGTAGTGAGGTTCAGTATTCCGACCCAATATGTACCCTTAATGATACCTGAAATACA" "ACCTTTTCCAAATACTAATGTTAATAATACCATATATTCAGTTACACTAGAGTATAATGGTGTATTTTCATCTCAAACAT" "TTGTACAATATGATGTATCTTTAACTAATCCTAATGATACTCCTCCACCTCCTCCAACAATCAATAATAGGACAGTAGAA" "CCAACAGCCTATTATTATGTTTATAATTTCTCGCCATTTTTACAAATGATCAACAAAGCACTTTCTGATGCTTTTACTGA" "AATTACTATGCCAGTTGGCGCAGTTGCTCCATATTTTGTATACAGTCCGGTCACACAAAGGATTAGTTTAGTAGCACAGA" "GGCAATTTTATGATAGAAATTTAGCGCAACCTATTAGAATATATTGTAATGAGGCATTATTCCCATTTTTAGATGGAATA" "CCATTTGGAGGACTTGATTTTAATAGTGTTGATGGGCGTGATATATTATTCAATGTGGAAAATTTAGGTAATAATTTGGT" "TCAAAATCAGCTAACGGCTCCAGCATATCCTCCTGAATTTATCCAAATGGAACAAGAATATGCTACACTTTCTAACTGGA" "ATGCTATAAAAACTATCCAATTAGTATCCAATCTTTTACCTATTAATAGAGAATTTATCCCATCATTTAGAAATACTAAT" "GTAGGTGTAGTTAATTCACAAGGTATTTTAGCAGACTTTGTACCATTGGTAACACTTGGGCCAGAATCTAGAACCAGTAT" "AGATTATGTTGCAAATGGTCCTTGGAGATTAATAGATATGTTTGGAGGAGTACCTATAACAATGGTTGATTTATCAGTAT" "ATTGGACAGATCAAATAGGAAGACGATTTGTGTTAGATGTACCCAGAGGTAGAATAGCAACCTGTAAATTAATATTCATT" "AAGAAGGATCTGGCAGGTCACACTCTATCTCGGAAATAATAGATAGTTAATGTTTCATCAAAATATAATATAATAATATA" "GTATATTAAGAATATGTCAAATAGTGCTGTACCTTTAAATGTTGTAGCAATTCAAGAACCCCGTCTTGAACTTAATAATG" "AAAGAACTTGGGTAGTGGTTAAAGGTGGTCAACAAGTCACCTATTATCCTTTCCCTTCAACTTCTTTCAGCTCTAATCAA" "TTTAATTTCATTTGTAATCCTCCTTCAGCACAAACTGTATTGGATCGATTAGCATTCATTCAAGTTCCTTATGATATCAC" "TTTCAATGTTAATCCTGCTCATGCTGGTGTAACTGACAATCTTTTACAACCTGGTCGTGATGCTTTCCGTGCTTTCCCTA" "TTAGTTCTATTACTAATACTTTAACTGCAACCATTAATGGTTTCCCTGTGAATATTGAATTAGGACAAATTATTCACGCT" "CTTAGCAGATATCATACTCCTCTTAAACTTAAAAATGGATGGATGTCTATGCAACCTTCTTTTGAAGATAATTATCAATC" "ATATCGTGATGCTGATGCCACTAATAACAATCCTTTAGGTGATTTTACAAGCGCATCTGGTCTTTCTGAGCTTCCAAGAG" "GATCATATTCCATGAATGTTGTGTCAAATACTCCTACTACTGCTAGAATTACTGGTGTACTTTATGAACAAGTATTCCTT" "CCTCCATTTATTTGGGATGAACATCAAGCTGGTGGTCTTGCAAATCTAACCAGTTTAACCTTCAATTGGGTACTAAATAA" "TAATCTTGCTAGAATATGGTCTCATTCAGATATTACTAATGATGTTTCTGGAAACAGTACTATTGGTTCAATGAATGTCA" "GCTTCCAACAACCCTCAATGTATCTTGGATTTGTTACTCCTCGATTAAATATTCCTATTCCTCCCAGAATAACTTATCCT" "TATTTCAAACTTTCAAGATACACTACACAATTCCAAAATACTCTTGCTCCCAATGCAACCAGTACTTACAAATCTAATAT" "TGTTCAACTTGATTCTATTCCAAGGAAACTTTATGTATTCATGAAACAATCTGATTCAGTGATTTATCAAAATCTCAATA" "ATCAAATTACCACTCCTGATGTATTCCTACAAATCAATTCTCTCAATTTAACTTGGAACAATCAACAGGGTGTTCTTTCT" "GGAGCATCTGCACAAAATTTATATGACTTCAGTGTTCAAAATGGTTACAACAAGACTTGGACTGAATTTAATGGTCTTAC" "ACAACAATTATCAGGAGTCTCTGGATCACCTACCAAAGTAATTGGTCTTGAAGGAGGAATAGTTTGTCTCGAATTAGGTA" "AAGATGTAGGATTACGTGACGATGAAGCTGAAGGTGTGCTTGGTAATTTCAATTTACAAGTACAAATGACTTGCACTAAT" "ACTAATCAATATCTCACAATCGTACCTGATATGTATATTATTGCAGTATATGATGGTACTCTTGTTATCTCTAATACTAG" "TGCTATGGCATCAATTGGTGTTGCCTCTAAAGAAGAAGTATTAAATGCTCCCATTAATCATAGCATGTCATATCACGAAT" "TACAAACTGTATATGGAGGAGATTTCTTTAGCACATTTAAGAATTTCCTTGGAAAAGCCGCTAATGTAGCCGGAAAGGTA" "AATAATTTTCTCAAAGATTCAAAAGTAGCTAGTTCTGTGCTAGGAGCTATTCCTCATCCTTATGCTCAAGTTCCTGGACA" "AATTTTAAGGAATATTGGTTATGGTGAAGGTGGAGTATCTGCTGGTGGTGTTGTTGCTGGATCTGGAAGAAGAAAGAAAG" "GTGGAGTATTAGTAGGAGGCAGTGAATGTGAAGGTGGAAGATATATGTCCAAAGCTGAATTAAAGAAAGCTTTACGAATG" "TAAACACTATTAAATATAATATTGGTTAATAATATATTTAATATGAACATCAGAATAAAAGATTATTTCGATCATCCTTT" "TATTAAATTTGATCCAAGGGAAGTTAAACCAATTGAATCATATACTCAAGAACAAATCAGTAATATCAAATTATTATCTG" "TTACTTCTGATCCTACACAAATGGCAAGACCTTTTGGATCTGCTACATATCGTATTCAAAAATATCCTGGAGATTTAGAT" "TTACAAGAGGAATTCATTGATTGTTGTTCCATAAATGAGGTTGTTAAAACATTTGCTAAGAGATTACAAAAAATAGTCAC" "CAATATCAAAAAAAATAAGTTACATTATTTTTCAGAGGTAAAAGCTGGGATTGATATTAGATACGATATTGATATTGGCA" "TAATAACTAGTGGAGTGTATATTCCCAGTTTTAATTTGATCCAAAAAATACAATCAATGTATCGAAAAGGATTATTAGAT" "GATGAAGAATACAATACTTTATCTACAGCATTATCAAAACCTAATTTAGATGGTGATGTATATGACGTCGTTCATCATAT" "TTTGAGAGAACGTAAAATATTGAGATGGTCACAAGAAGAGGTGTTATTGGGTACAAAAAGTCTTCCTTTAGGGAAAAAAA" "TAAAGCTATCTGAAGCATTGAAGGCAAAATCACATGTAAAAATAGACATGATATCACTCATTAATAATCAGTTTGTTGAG" "GTAACTAATTTCTATATTTTGATTTATGTCGATTTGAAGAAAAATACTTTGGAGACTGTTAATTTTAATTTCGATTATCT" "TGATAACAACATACTTACCAAACAATATGATATGCAAATCAAAGAGGAAGTACAAAAATTGTATTACTCTGATATGTATT" "ACAGTGCTTTCAAGATGGTCAAAAGAATGTGGGCATATTCAAGAGCTTTTAGAAATATGGAAGATGTCAATATATTATTA" "CCTATTGTAAGTGGAAATATATCATTATTGTATCAGATTGTGAGTGAATTATCCACTATTGTGAGACTTTATGAAGTCAG" "TAAATCAACTCCCGAAACAACAATAGATAAAAGATTAGATTCATTAGCCTATAAATTAGCTGATGTAACAGAAATAGATA" "AAGAATCATTGATACATATCACAGATATAATTGATAGTTTGAAGAATTATAAAGGAATCAACAAAGCAATCCAAATAAAT" "CAGTTTGTAATTAAACCATTGAAAAGATATATCAATGCTTTAACTATAGATGAATTGGAAGAAATAGGTTTTAATCCTCC" "TCCTCCTAGATTTTTACCATATCCTTTGAAATATGCTCCTATTGTTAGATATCCTTTTGAGGATGTTGAAAACCCTTTGA" "AGCAATACTAGAAGTGTTTATTTGTAAAAAAGCCAAAAAACAAATTAAAACAGATCAAAAGACTATTTGTCTAAAATATT" "TTAAAAATGTCTAAAAAATGTCTAAATTTGTCGATGTAAAAATGAAAAGCTTCATATATTTAGACAAAAAGACATTTTTT" "AGACATTTTTAAGATATATACAAAAATTATCTAATATTTTGATTTCTATCCTGGAAAATCTTAATATCCCAAAAAAAATG" "TCTTTTTGTCTAAATTATTAAGCTTTTCATTTTTTACGCCGATATTTTTGACATTTTAGACAAATAATCATCAAAATAAT" "ATGTGATCAATATGCTAATTTATAATATTTCTTTCTAAGCTTTCTATATTTTGCCTTTAAATGCTCATATTTATTCTCTA" "ATTTATCCATTGCTTCATTGTGTTTCTTTGATTTCTTGTGATTAGAAGAATTACTTTTGGCATAAACTGCTCCACAAATA" "GTACATTTCACTTTTTCCTTGTAATATTCCCTTTGTTTTTCCTTATTTGCTTCATAATATTTTTTATGATACTCATTTTT" "TGATATGTCCATTATATTATAATATATTATTTTATATTAATATAATATTATTTTATATTATTTAGTATTATAGAGTAAAT" "AATATAAAAAATAATTTACTCTATTAAAATGATGAAACAATAATATAGCTAATCTACCTCAATATCTGCTAGTATATCTA" "TTTTTTTAATACCCTTCCAGAACCTAAAAGTGGATCTACCTTCCACTAATTTTAGTTCATCGCGGAGTATATCATTGAAT" "CTGTTTAATGATATATCCTTAAATTCTTTGTGTGAATTTTTAAATTTTTTCATATCAGACACTGGTACTTTATCATTATC" "TTTACCAGTGATTTCATAGTGCTCTTTAATGGTTTCAATAACAATATCATTTTGTTTTGTTTCAGCAGTCCATTTTTCTT" "TGACTTCAGGATCAAATTCAGGCATACCATTTTTCAAATAATCTTGGTATGCATCAAGGAAAAGATGTATAAATCCTTTC" "ATGAAATCTTTGGTCTCAAATTTTGATTCAATATTATTATCTTTGAGTTTGTAATAAGGTTTTTTATCGACATCTTCTTC" "ATCAACAAAAACATAAGGAAATTCATTATATACTAATCTATTCATTACAGCTTGATCAAAAGGTTCAATATTAGGAACAT" "CATTGAACATACAAAAAACAGTAAAATTAGGTTTGAAACTTTCTTCTGATTCTTGATGTTCTCTACCTACAATATTATCT" "CCACCAGATGCAAGTCTTTTTATCATAATACCATCAAGAACAGAACCTTTCATTGGCATTTCATTTGACATTAATATTCT" "AGTATGTGCAAGTTTATATGCCCATCTAAATTTTGCTGCTTCATCTCTTGAGTCATTCTTTTGATATAATAAATTTGCAC" "CATTAAAATCACCAATATAACCTCCAAAACAATATTGTAACATCATAATTAAATATGATTTGCCAGCATTTGATCTCCCA" "GGACAAAAATACATTTTTTTGAGTTTAATATCGCCAGCTAATGCACAAGCTAATGCTGTTATCATAGGTTTTGGATTATC" "AAACAATTTCTTGAATGATAATCGATATGCTTTTTTGACCAAATCTTTGTCATATTTAGGGAATTTGTACGGCACACCAC" "ATTTGAAAACTATTTTTGGATCAAATCCCTCAGTAAATGTTCCAGTCTTGAAATTATATATACCATCTTTAAATAAAAGA" "TATCCTAATGACGATTTTGCAGTTCTTTTTAACCAATTATTATCCTCTGATACGTCTTTAACAAAACCTACAACCTTTTC" "TCTTAATGTATGATCTGTACCATAATTTTTGGTTTTATGAACACCATTTTTATCAGTGGAAATAATAACATTGAGATACA" "TTTGATGTTTTCTCAAATAGCGGAAAAGTATATGATTATTATCATCAAACATACCAGTATTTTCATCAAATATGTATAAC" "TTTTTGTTACAGAATTTGAATTTTTCTGCTCCTTCTAATTCGAACAATTTTTCCTGACAATCACAATCACTGCTAACATA" "AATACTATGATCTGGTACTTCAAAATCCAATTCATAGTTCATTGGTTTTTCTTCTAGATTAATCTTGTATTCTGTCTTAT" "CATAGACATATTTTTCACAACCTTTCAATAAAACCTTAATATCCTCATCAAAGGAGTTAATCAACATACCATCAAAGCAT" "AATGTACTCACTTTAATTTTTTTGCTAGTGAAATAATCATACATTGCCATTAAACACTTATGTTCTAATCGTTGTGCTAA" "TATGGATAATACTACTGCTAATTTATTCTTACAATCATTATTATCCTTGATTTTCTCATAAAGACTTGTTTCTTTTTTAG" "CTACTTTTTCAGCTATGTATTCTGCTTCTTCCTTAAATTTTGTGAGGAATTTTAATTTTTTGGTTGGTGTGTAGCTTTCA" "CCATTATCATTAGTCAATTTATATGAACCAAGATAACATAATTTCAAGATCAGTTCTTTTGCCTTGTCTCTTGATATTTT" "ATGGAAACTACTTATGTCTTCGAGTATCTTTTCTCTTCTATTGACATAATCAACTAAATATGGACACAATATGTCTTTTT" "TTTTACAATATTGTGTAATCAATGTAGGATGTGAGTTTTTCATATCAATATCAGTATTAATTCCATTATTGATAAACCCT" "CTAATACTCTTTTTGAAGGACTGAAGACTTTGTCCCTTCTCAGCATATACCCTACCAGTATCTTCTAATTTATCTGAATA" "GTAATATTTCACCTTGACATATCCTATTTCATCTCCCTCTTCACGTTTTGTCTTGAACACTTTCTTGTAGTATGCACTTA" "ATTGTCTCTGAATGTCTTCATCTAAATCTTCTGCTGATAACAATCTCTTCAAGTTTTTCATACTAATTGTTTCTGTCAGT" "TTAATCACTGGCTTAACCACTTTCTTTTCAATTTTCTTAGTCACTTTCTTCTGTGTCTTACTCATTGTTTACTAGTATAT" "ATGGTACTAGATATATTATTTTTATATCATTTTATATTATTATATAGTGAAAAAATATTCAATATTATTTCTATAGTTTG" "AGCGTAATCTTTCGGCCTTACAATCCAACTTATACCCCCTTTTCTACGCTGTCTCAAAAATTTTGTGATGCGAATATATC" "CGTATCATAAAATTGTCACATGCTCGTTAATGAGCATGTCATGTAAAAATGTCATCAGCTGATTAATAAGCCGCTGAAAA" "ATACCAATATAATGGGAACTGAACAGCCGAAACTTCGGAAATATGACTGATATTTCCGAACCCTCTCCCTGTTCAGTTCC" "TACTACAAATCAACTATATCACGTCCTTATCCATATATCATCATACTCTTTCTTATGACCAAATACCATCATATACACAT" "AATTAAACATACTTATTATATTTGACACTAAATACTCAATCGGATTCATCTTCTTCATCTTCACTAACTAACACTTGATT" "TTTTTTTACAAAGAATGTCGGATATTCATTCTCTAAATATTTGTATTGTTTTTCGGTGATATTACTTATTTTCTTTAATT" "CTGACATTTGGTTTTGTAAATCAAGCATTTGTTTGTACATCTCATTCATATTATGTAATTCATAGATTTTTCTCATATAT" "ATTTGTGTTTGTGATTCATTTTCTTTTTTCAGTTTCTCATTTTCTTTACTGACCTCATTTAATTCCCTTTTCAAATCATT" "ATTTTCTGATTTAATCTCATTCAATTGTCTATGAAACATAATATTATTATCATTCATACTCGTTTGAAGTGATACTCTTA" "GATCATTTTTGTTCTTATTGGTTTGATCAATCTCTTGATACAATGTATTAATACAAGATTGTAATTTTTGTTTACAATCT" "TCGGGTAGCTTTACCAATATTTGTGAGATAATATTATTGATTTCCTCTTTATTTGACATCATATTGATAAGGTTATATTT" "TATATTATTTTTTCAATCAACGGAATATCTCGTAAATGGTACAAACATTCGGCCTTATAAAAATAATATTATGTAATAAT" "ATTACAATGGCTAAATTATATAAACTAGAGCTTGATGATTTGTTAAATGAGGACTTAAATGAGGCTGTCAAAATCAAAAA" "AGGATTTGACAGGAATATTAATTTTATGAAGATTAAGAAAAAATTACTTCGATCATTAAAGGTGAATTGTGCTATTGTCA" "ATGACAAAGAGGAGAGTGATTATAATAAAAGAATATCAACTACTAGAATAATATACATCATCATTGCACTTTTACAACTC" "AGAAATTGTTCCAGAATATCAGAAGCTGTTGCTGCTATCAAGAAATTCTCTGTCAAAAAAAATCTAAATGAAAGGGTGTT" "AGTAAAGATAGCTAAATCTGAAAAAAGAATAGTGGATAGGAAAACAAAGGAAGTATATGAAACTAAGCCAAGATATCGTG" "AAATGATTTTCCCTCTAGCATGGGTTGATAAGAAACTATTCAAAAAAATCATAAAAGATGATCAATGGGCCAAATTCAAT" "AACAATAAAGAACCCAGAAAACGTGTGCTTGACTACCTTTTAAATAATTTTGATTGTAATACTCATTCATTAAGATATGC" "TGGAATTAATTTTATGTTGAATGAAAAAAAGGTTCCTATGAACATTATCGCTAAATTTGTTGGTCATGTTAATACAGAAC" "AACTTGTTACATATACTCAACATCAAGCATTGGATGATGTTTTTGCTATGGATGTGTAAAAAGGCACCTCGATTATCCGG" "GGTATGTAGACATTGTCTTATCTTTTTTTATCTTTTTTTATCTTTGTAATTCTGTTGTACAATGTAGGTGTTTTTGCAAA" "GATAAACATTTTAAATGGTCATCATAGTGATTATCTCGTCTGGCTTGGCTTTATAATATTTACACATCTGATCAATGAAA" "TTTTTGAACTCATATATACTTAAATGCCTCAAAAGTATCCTAATCACACACCATCTACCACACGTTTTAATATCATTTGC" "TCTCTTTTGGAATTTAAATTCGTTGTAATGTAATTCATACGGACACTCTAACAGGAGTAATGATAGATATGGATAATATT" "GGTTTGATATTTTTCTGAAATGTAGAGGAATAAATTTGAGGCTATTATCGGGATATCCTCCATAGCTATTGAAAAATTCT" "ATGGTATTTTCATCTCTCTTGAATAAGCAACACCAGTGACCAAAGTTCTTTTTAGCTTCAAACAATAATATACATGCACC" "ATATGGACCTAATACCTCATCAATAGAATTGTAGTTAATCAAATCTGGATACAAAACTATTTGTGCTTTATTATCTAACA" "TACCTAAAATATCGGTATTTGAAAGGGCTACATTTTCATAATGTTTGATAGTTTTCATTTTATTATAATTATATGATAAT" "AAAATAAAATACTTCATACAGTTAGACCATATATGTTATGCTAAATGGATTGAATCCAGAACCTCCAATACCAGTAGTAA" "ATTGACCAAGATTATTGTCTTTGTAAACAACTATTTGACCATTTGATAATAGTGTTATTAATCCAGGATTAACTGGCCTA" "TTTCCATCATCAATCACAAATATTTCAAAATCTACTTGAATATTGGTGGTTGGTCTTAAATCAGCTGGTAATGCTCCAAT" "AGCTGAATTTATTACAGCTGATGATGTTGCATTTGCTTGGAAAGGAGGAAATGTTAGATTGACAAGATTATTTATTTTAG" "TGTATGCAACATTTTGTACTAATGGTGATGCCCATGGACCTGAAAGAGATAGGGAAAATGATCCAGTTGTCAAAGAAATA" "TTGCTAACTGCTTGAGTAATATCAGAAGCACCATACATTAAATGAGGAGGAGTAAAATTACTATTACTCCAGACTGTATT" "AGTATTTCCTGTTGGATTTGCTGGAACATTTGTCAGAGTATTAGATTTATTATTAATGTTGTACAGATTTGGTTGTAGTA" "ATGATGATATTGACATATATTATTATAATTAGATTCTAAATATATTTAGTGTAAATCCGCTTGCATTGGGTTGTATTTCC" "CCTCCTGAATTTGCAAAAGCGATAAAAATATTGTCTGATGGTGTCATATCAATAATACCATTTAGAGATGCACTCATTTG" "AATTCCAGCTCCTGAATAAATTACTGCCGTATTATAGACAGCTGTTCCACCATTTTTGAATATAGATATGATTAATGATC" "CATCTAATGTACCGATTGAGGTACCAATATATGTTACAGCAACATAAAATGGAGCAGTTTCCGTGGTGGTGAACACACCG" "TCGACATAGGAACAATTACCACCTAATGTTTTTGTGGTAAAAGCAACAGTACCAGTTGATACTTCTGGAATTGATGCTTG" "CGTGCCACCACTTGTTTCATATGTCGGTAAATCTCCAGAAAAATAAGTTCCTGAACCAACTGTTCCACCTGTATTACCTT" "TTGGACCCATTTCACCCTTAATTCCAACCCCAATATCACCTTTATCACCCTTTAACCCGTTAGTTCCGGGATCACCTTTG" "TCACCTTTTGGACCTGATGAACCAGTACCACTATTCAAATCAGCTGAACCAAACATTAAATGAGGCGGATTAAAAGCATT" "ATTACTCCATAAAGTATTGGATGCTGATGTTGGATTGGTGGGTAAATTTGTGAGAGAATGTGATTTAGAATTAATATTAT" "ATGTATTTGGTTGCAATAATATTGATACAGACATAATTATATTATCAAGATGATATAATTATTTCTCATAACTAACCAGG" "AGGAATAAAATATTGTCCTGTTATGTAAATTAAGACTGTTATTGATGTTGATGCTTGTATTTTCATACTTAACACAATCT" "CTGGTGTAGCTGCTGTAGCTGTTACAATACCATATTGTACAACACCTGTAGATGCACCTGTTATTGAAGCTGTTCCAATC" "ACATTATTACCATTAAAATCACCACTAGCAACTGGAATAGAAAAATATATTATTCCTTGTGCCATATTAGCAGGAATTGT" "TGCGTTTGTACAAATACTAAATGTTACAATATTACCTATTCTCGAATAAATAGATGTTGTTGGTGAAACACTTGTAAATA" "TTTGTGTGAATGAAACTGTTGGTGTCCAAATTCCTGATGATAATACTACTGCTGAACCTACATCACCTTTTAATCCCTTA" "TCACCCAAATTACCTTTTAATCCATTTGTACCAGTATCTCCTTTTAAACCTTTATCTCCACTTCCTGTTTCCCCTTTTAA" "ACCATTTATACCTGCATCACCTTTTAATCCTTTATCTCCACTCCCAGGATCTCCCTTTAATCCATTTGATCCAATATCTC" "CTTTGAGACCATCTAAACCTGCATCACCTTTTAATCCTTTATCGCCACTTCCAGGATCTCCTTTTAATCCATTTGATCCA" "ATATCTCCTTTTAATCCATTTGATCCAATATCTCCTTTTAATCCATTTGTTCCAGCTTCTCCTTTATCACCCTTTTCCCC" "TTTGTCTCCACCACCACCACCGCCGTTGGATAAATCTGTGGAACCAAACATTAAATGAGGTGGATTATGAGCATTATTAC" "TCCATATTGTATTACTCAAATTAGTGGGATTTGATGGTAAATTATTAAGAGTATGTGATTTAGAATTAATATTGTATGTA" "TTAGGTGAAAATAATGTGGATAACGACATATATTAATATTATATTATTAAATATGATATTAATTATTCTATTTACTTCTT" "ACTTTTGCATTTTTAATACTTTAAGTATTATTTTTTACCCTTATGATTTTTTCTACTCTGAACAGGTTTTTGCTTAGGTT" "GTTCCTCTTCTTCTTCATCACTTTCTTGTTCAGTCATAATTTTCAATCGGTGTTTTTGGTTCTCTAATTCTTGTCGGTAT" "GCTTTAAATTCTCTATTCATTCTTGATTTAATTTTACTTGTTCGATGTGCTCTGAATTCTCTAAGTTTTTTCTCATCATT" "AATATCTTCAATCAAATCATCTAATTGTTCTGTTGTCATATTGTCAAATAAATTATATAACGCATCCATATCAAATGGTT" "TTTCTACTCGTGGCATACCTCCACTATTGATCACTATCTCATTATTTTCATTATTGAATGGTGTGTAATTCATTATTATA" "TTATATATACATATATTTTTATCCTATTTTGACACATAATAGATACTTAATCTATCCATAAATCATAATATCACGAAGTT" "TTTTATTCATTTTTTGATAGACTTGACATACTTCTGTTTTTCTATGTTTAGGTCTATTGGATCTTGTGTATGTTTTTCCA" "CAAAATTCACAAATCAATACATCATTTGGATGTGCTTTAACTTTTTCTTCACCATATGTTCTCCCATTGTACGAATCTTT" "GTACACATTTTGACCATTTTTTTTCCCAATTGGACCTGGTCTTTGATTGAATCTTAATACACAAGTTGGTTGTCCAGCAT" "CCTCATTACGAATAACTTTATTTTTATATACTGCTTTTGGTACTGGTGTTTGTATATATTTCAATTCTTTTCTATTCATT" "CGAGTTAATTCTTTTTCTTCTGCGAAATCATATGGTATATCTCTTTTTAGCATTTAATATTACCTATAATATTTTTTTAC" "TAGTTACTGACGGAATAGAATTAGTATTAGAATTACTCTCAAATAATAAATATATACATAGGAATTTAATTTATCAACTT" "TTTATATAAAATAAATGTTATCCTACATAAAATATGATAAATTTAGCAAGCCTTTTGCCAAAATCAGTGGAGGGAAATAC" "AAGGGAAAAATGGCATATGTGAATGAAGAAAATGATAAAGGACGTAATGAATTAAAGCTACCCGAGGGTAAATTTGTACC" "ATATCCTAATCCTTTAACTAGAGAAGTAGTTTATGTAGCCGGACAAAGTGGTAGTGGTAAATCTACCTACGCAGCACAAT" "ATATCTACAACTACAAGAAATTATTTCCAGCTAACAAGGTGTTTGTGTTTTCAAGGTTAGAAATGGATCAAATATTAGCA" "TCACTTGGATGTATTCAAATACCTATTGATGAAGAACTATCAGAAATGGACGCAATTAGGGATATAAAGAATGCATTATG" "TTTGTTTGACGATATTGATACAATCAAAGACAAGAATCTAAAGAATTGTGTTTATGATATTCAAAATGATATTCTTGAAA" "CTGGACGTCATAAAAATATATATATTCTTGTCACATCCCATTTGATTAATGGTAATGATAAAAAAAATTCTAGAACTATC" "CTCAATGAAGCCCATAAAGTGACATTCTTTCCAAAGAGTGGTTCGTATGCCATAAATTATTTCCTCAAAAACTACATTGG" "AATACCTAAAAAAGACATAGATGAGATCTTAAAGATAAAATCAAGGTGGATCACTATCAATAAAGGTTATCCTTTGTACA" "TATTTTATGAAACGGGGGCAAAAACTATATAAATTTTATCTTATTATTTACCCCCAACAATCTTGGGGTCGAACAATCTT" "CGGGTATTTAATTGTCTAAAAAAAGGAATTTCGCGCCATTTAACTGATAAACAATCATAATTGATGATATTTTTACTATT" "TACTTTGATGAAATTTCCACTAATAGTAGTAAAAATTTATATTGGACAGAAATCTAGCCAACCCTTTTCTTCTTGGTAGG" "TTTCTTTTTGGATTTCTTCTTGGCCTTTTCTATCTCTTTCTCAGCCTTTTCTGCTTCATTCTCTAGCTTGGTAACAATTT" "TCTTTTGAGCAAGCATTTTCTTAACTAGTTTGTCTTTTTTAACCAATAATGCCTTTAATTTTACCTGTGATGCTTTGATC" "AATGATTTTTTCATAGTATCATCATCGACAATTAATTTAAGGTTTTTATATTGTTTAATCAATATTTTAGCATCATCCTG" "TAATTTCTTATATTTGAGTATTTCTTTGGTCAAATCGAGTTTCTTTTTACCAGCATTCTCCAATAATTTTGGATCAACAC" "TATATCTACCATACATCCTGACTTGATTTGCCTTAAGACACTGCTCTGGAGTACCAGCTTTTTTACCTTTCGGTATCTTT" "CCTATACCACAATACATTTTTAAGATATAATTAGATCATAAAAATGTATAAAGATTACATATTCATTAATTCTTCCAAAA" "TTCTTTGATCATTATTGGACATTTTGGGTGATCGTTTAGTTCTTTTGGATCCTCCTTTCAAAGATTTCTTGACAGGTTTT" "TTCTTAGTAATTTTCTTGGTTTTACTACCAGAACTCTTTTTCTTGGTTGTTTTACCAGATTTCTTTTTCAAAGCTTCATA" "TTGTTTCTTGGCTTTGGGACTTTGCATAGCTTCACCATATTTCATACCATGTTCTTTAGCAAATTTCTGTAAAAATGCTA" "CCCATGGTGATTTCTTAGCACCAGCTTTTGTACCAATACCACCTTTCTTAACCCTACGTTTGACCGTTCTACCACCTGAT" "AATACACCTGCTAAAACACCTCCTACTTCAGTGCCACAATTGGGACAAAAACATCCTCCTCTTGGAGCAGTTCCAGATAA" "TAATACTTTCTTAGACATAATAATATTATATGATATTATTAATAATCACGCATTTACATAATTATGATCATAATTATTGT"; std::string phage(PSUEDO); int start = rand() % (phage.length() - 1000) + 500; int width = 300; std::string to_insert = phage.substr(start, width); // get the center point int center = m_gr.Width() / 2; phage_site = center; phage_string = to_insert; m_seq = m_seq.substr(0, center + left_scramble.length()) + to_insert + m_seq.substr(center + left_scramble.length(), m_seq.length() - center - left_scramble.length()); } void SeqFrag::addIndels(size_t n) { if (n == 0 || m_seq.length() < 5000) return; size_t spacing = m_seq.length() / (n+1); spacing = std::max((size_t)5000, spacing); std::vector breaks; for (size_t i = spacing; i < m_seq.length() - 5000; i += spacing) { breaks.push_back(i); } breaks.push_back(m_seq.length() - 1); if (spacing <= 30) { std::cerr << "Simulated indel spacing < 30. Should be > 30, reduce number of indels" << std::endl; exit(EXIT_FAILURE); } if (breaks.size() < 2) return; if (m_gr.strand == '-') // flip it back to process, then flip at end SeqLib::rcomplement(m_seq); std::vector TCGA = {"T","C","G","A"}; // make a read sim to get random sized del ReadSim rs; // set up for the loop int del_cumsum = 0; int ins_cumsum = 0; std::vector frags; frags.push_back(m_seq.substr(0, spacing)); for (size_t i = 0; i < breaks.size() - 1; ++i) { // random size for events int ds = rs.getRandomIndelSize(); char etype = (rand() % 2 ? 'D' : 'I'); //etype = ds >= 50 ? 'D' ? 'I'; // events above 50 are always dels //char etype = 'D'; // get the bounds int start = etype == 'D' ? breaks[i] + ds : breaks[i]; int end = breaks[i+1]; std::string ins_string = ""; if (etype == 'I') { if (ds < 50) { // random sequences for small insertions for (int i = 0; i < ds; ++i) ins_string += TCGA[rand() % 4]; } else { // tandem duplications for large int len; std::string chrstring = m_gr.ChrName(SeqLib::BamHeader()); char * seq = faidx_fetch_seq(m_index, const_cast(chrstring.c_str()), m_gr.pos1 + breaks[i], m_gr.pos1 + breaks[i] + ds -1, &len); ins_string = std::string(seq); //std::cerr << ins_string.length() << " " << ds << std::endl; } } std::string lead_base = m_seq.substr(breaks[i]-1,1); std::string ref_seq = (etype == 'D' ? m_seq.substr(breaks[i], ds) : ""); std::string alt_seq = (etype == 'D' ? "" : ins_string); // make the indel object Indel ind(ds, etype, ref_seq, alt_seq, lead_base); if (etype == 'D') ind.gr = SeqLib::GenomicRegion(m_gr.chr, m_gr.pos1 + breaks[i]-1, m_gr.pos1 + breaks[i] + ds-1); else ind.gr = SeqLib::GenomicRegion(m_gr.chr, m_gr.pos1 + breaks[i], m_gr.pos1 + breaks[i] + 1); ind.frag_id = frag_id; // check that it's not on a blank region if (breaks[i] < 101 || m_seq.substr(breaks[i] - 100, 200).find("N") != std::string::npos) { start = breaks[i]; // don't put in the del or insert etype = 'D'; } else { // store the indel and update reference counter m_indels.push_back(ind); if (etype == 'D') del_cumsum += ds; else ins_cumsum += ds; } // cut the sequence out if (etype == 'D') { frags.push_back(m_seq.substr(start, end - start)); } else { // make the random insertion frags.push_back(ins_string + m_seq.substr(start, end - start)); } } std::string new_seq; for (auto& i : frags) { new_seq += i; } if (m_gr.strand == '-') // flip back if need be SeqLib::rcomplement(new_seq); m_seq = new_seq; } void SeqFrag::getSeqFromRef(faidx_t * findex) { int len; std::string chrstring = m_gr.ChrName(SeqLib::BamHeader()); char * seq = faidx_fetch_seq(findex, const_cast(chrstring.c_str()), m_gr.pos1-1, m_gr.pos2/*-1*/, &len); if (!seq) { std::cerr << "Failed to get reference sequence at " << m_gr << std::endl; return; } m_seq = std::string(seq); // reverse complement if need if (m_gr.strand == '-') { SeqLib::rcomplement(m_seq); } } int SeqFrag::getLeftSide() const { if (m_gr.strand == '-') return m_gr.pos2; return m_gr.pos1; } int SeqFrag::getRightSide() const { if (m_gr.strand == '-') return m_gr.pos1; return m_gr.pos2; } std::ostream& operator<<(std::ostream& out, const SeqFrag& s) { out << s.m_gr << "\t" << s.m_seq.length() << "\t" << (s.right_scramble.length() ? s.right_scramble : "N"); return out; } ================================================ FILE: src/svabautils/SeqFrag.h ================================================ #ifndef SNOWMAN_SEQFRAG_H__ #define SNOWMAN_SEQFRAG_H__ #include #include "SeqLib/BWAWrapper.h" #include "ReadSim.h" using SeqLib::GenomicRegion; class SeqFrag { public: SeqFrag(const GenomicRegion& gr, faidx_t * findex) : m_gr(gr), m_index(findex) {} void getSeqFromRef(faidx_t * findex); int getLeftSide() const; int getRightSide() const; friend std::ostream& operator<<(std::ostream& out, const SeqFrag& s); std::string m_seq; void addScrambledEnds(size_t left_len, size_t right_len); char getStrand() const { return m_gr.strand; } void addIndels(size_t n); void addIns(); void spikeMicrobe(); std::vector m_indels; int frag_id; std::string left_scramble = ""; std::string right_scramble = ""; GenomicRegion m_gr; int32_t phage_site = -1; std::string phage_string = ""; private: faidx_t * m_index; }; #endif ================================================ FILE: src/svabautils/SimGenome.cpp ================================================ #include "SimGenome.h" #include "SeqLib/GenomicRegionCollection.h" SimGenome::SimGenome(const SeqLib::GenomicRegion& gr, int nbreaks, int ndels, faidx_t * findex, bool scramble, int viral_count) : m_gr(gr) { std::vector index = {0}; size_t ii = 1; double viral_prob = viral_count ? (double)viral_count/((double)(nbreaks+1)) * 2 * 1000: 0; // int size_t avg_width = std::max((size_t)(m_gr.Width() / nbreaks ), (size_t)1000); size_t max_breaks = std::min((size_t)(m_gr.Width() / avg_width), (size_t)nbreaks); std::cerr << "avg width: " << avg_width << " max breaks: " << max_breaks << std::endl; size_t real_vcount = 0; // choose a set of random break points SeqLib::GRC grc; grc.add(SeqLib::GenomicRegion(m_gr.chr, m_gr.pos1, m_gr.pos1, '+')); size_t exp_break = 0; while(grc.size() < (size_t)(nbreaks)) { exp_break += avg_width; uint32_t r = exp_break + (rand() % 400) - 200; //m_gr.Width(); r += m_gr.pos1; char strand = (rand() % 2) ? '+' : '-'; grc.add(SeqLib::GenomicRegion(0, r, r, strand)); index.push_back(ii++); } // setup the scramble probability distribution std::vector scramble_size; for (size_t i = 1; i <= 200; ++i) { if (i <= 100) scramble_size.push_back(0); else scramble_size.push_back(i - 100); } grc.SortAndStretchRight(m_gr.pos2); //grc.sendToBED("segments.bed"); // shuffle the indices if (index.size() > 2) std::random_shuffle(index.begin() + 1, index.end() - 1); // seeded by srand //debug //for (auto& i : index) // std::cerr << i << "\t"; //std::cerr << std::endl; // get the sequence and add insertions / deletions double grcwidth = grc.TotalWidth(); size_t id = 0; for (auto& i : index) { SeqFrag sf(grc.at(i), findex); sf.frag_id = id; ++id; sf.getSeqFromRef(findex); if (rand() % 1000 < viral_prob && grc.at(i).strand == '+') { sf.spikeMicrobe(); ++real_vcount; } else { int nd = ceil((double)sf.m_seq.length() / grcwidth * (double)ndels); sf.addIndels(nd); } // scramble the ends if (scramble) { size_t right = 0; //if (i != 0 && sf.m_gr.strand != '+') // don't scramble left side of + //left = scramble_size[rand() % 200]; //else if (i != index.size() && sf.m_gr.strand != '-') // dont' scramble right side - if (i != index.size()) right = scramble_size[rand() % 200]; sf.addScrambledEnds(0, right); } m_sfv.push_back(sf); m_indels.insert(m_indels.end(), sf.m_indels.begin(), sf.m_indels.end()); } //debug std::ofstream sg; sg.open("segments.tsv", std::ios::out); for (auto& i : m_sfv) { sg << i << std::endl; } sg.close(); //debug size_t mmm = 0; for (auto& i : m_sfv) mmm += i.m_indels.size(); std::cerr << "TOTAL NUMBER OF INDELS IS " << mmm << " AND VIRAL INTEGRATION IS " << real_vcount << std::endl; } std::string SimGenome::printBreaks() const { std::stringstream ss; // MARCIN strand convention here. Left side strand for revComp frag is "-". // right side strand for non-revcomp is "-" for (size_t i = 1; i < m_sfv.size(); ++i) { //ss << m_sfv[i-1].m_gr.chr << "\t" << // m_sfv[i-1].getRightSide() << "\t" << (m_sfv[i-1].getStrand() == '+' ? '-' : '+') << "\t" << m_sfv[i-1].left_scramble << "\t" << //m_sfv[i].getLeftSide() << "\t" << (m_sfv[i].getStrand()) << "\t" << m_sfv[i].right_scramble << std::endl; ss << m_sfv[i-1].m_gr.chr << "\t" << m_sfv[i-1].getRightSide() << "\t" << (m_sfv[i-1].getStrand() == '+' ? '-' : '+') << "\t" << m_sfv[i].getLeftSide() << "\t" << (m_sfv[i].getStrand()) << "\t" << m_sfv[i-1].right_scramble << std::endl; } // loop through the tandem duplications and indels for (auto& i : m_indels) { //if (i.len >= 50 && i.type == 'D') // ss << i.gr.chr << "\t" << i.gr.pos1 << "\t-" << i.gr.pos2 << "\t+" << std::endl; if (i.len >= 50 && i.type == 'I') // tandem dup ss << i.gr.chr << "\t" << i.gr.pos2 << "\t-\t" << i.gr.pos1 << "\t+" << std::endl; } return ss.str(); } std::string SimGenome::printMicrobeSpikes() const { std::stringstream ss; for (auto& i : m_sfv) { if (i.phage_string.length()) { ss << i.m_gr.chr << "\t" << (i.m_gr.pos1 + i.phage_site) << "\t" << i.phage_string << std::endl; } } return ss.str(); } std::string SimGenome::getSequence() const { std::string s; for (auto& i : m_sfv) s += i.m_seq; return s; } std::ostream& operator<<(std::ostream& out, const SimGenome& s) { //for (auto& i : s.m_sfv) { // out << i << std::endl; //} return out; } ================================================ FILE: src/svabautils/SimGenome.h ================================================ #ifndef SNOWMAN_SIMGENOME_H__ #define SNOWMAN_SIMGENOME_H__ #include "SeqFrag.h" #include "ReadSim.h" class SimGenome { public: SimGenome(const SeqLib::GenomicRegion& gr, int nbreaks, int nindels, faidx_t * findex, bool scramble, int viral_count); void addBreak(int b); friend std::ostream& operator<<(std::ostream& out, const SimGenome& s); std::string getSequence() const; std::string printBreaks() const; std::vector m_indels; std::string printMicrobeSpikes() const; private: SeqLib::GenomicRegion m_gr; std::vector m_sfv; }; #endif ================================================ FILE: src/svabautils/SimTrainerWalker.cpp ================================================ #include "SimTrainerWalker.h" void SimTrainerWalker::train() { SeqLib::BamRecord r; size_t count = 0; while (GetNextRecord(r)) { ++count; // check occasionally that we still have read groups if (count % 10000 == 0) assert(r.GetZTag("RG").length()); if (count % 1000000 == 0) std::cerr << "...training on read " << SeqLib::AddCommas(count) << " at read at " << r.Brief() << std::endl; m_bam_stats.addRead(r); } } std::string SimTrainerWalker::printBamStats() const { std::stringstream ss; ss << m_bam_stats; return ss.str(); } ================================================ FILE: src/svabautils/SimTrainerWalker.h ================================================ #ifndef SNOWMAN_SIMTRAINER_WALKER_H__ #define SNOWMAN_SIMTRAINER_WALKER_H__ #include "SeqLib/BamReader.h" #include "BamStats.h" class SimTrainerWalker : public SeqLib::BamReader { public: SimTrainerWalker() {} void train(); std::string printBamStats() const; private: BamStats m_bam_stats; }; #endif ================================================ FILE: src/svabautils/assembly2vcf.cpp ================================================ #include "assembly2vcf.h" #include #include #include #include #include "vcf.h" #include "AssemblyBamWalker.h" #include "SnowmanBamWalker.h" #include "SnowmanUtils.h" #define MIN_ISIZE_FOR_DISC 700 faidx_t * findex; enum { OPT_NO_READS }; struct bidx_delete { void operator()(void* x) { hts_idx_destroy((hts_idx_t*)x); } }; static const char* shortopts = "hi:m:q:p:v:k:z:x:a:r:t:n:G:"; static const struct option longopts[] = { { "help", no_argument, NULL, 'h' }, { "assembly-bam", required_argument, NULL, 'i' }, { "tumor-reads-bam", required_argument, NULL, 't' }, { "normal-reads-bam", required_argument, NULL, 'n' }, { "indel-mask", required_argument, NULL, 'm' }, { "panel-of-normals", required_argument, NULL, 'q' }, { "id-string", required_argument, NULL, 'a' }, { "normal-bam", required_argument, NULL, 'n' }, { "threads", required_argument, NULL, 'p' }, { "region-file", required_argument, NULL, 'k' }, { "rules", required_argument, NULL, 'r' }, { "no-zip", no_argument, NULL, 'z' }, { "no-read-tracking", no_argument, NULL, OPT_NO_READS }, { "no-r2c-bam", no_argument, NULL, 'x' }, { "threads", required_argument, NULL, 'p' }, { "verbose", required_argument, NULL, 'v' }, { "reference-genome", required_argument, NULL, 'G' }, { NULL, 0, NULL, 0 } }; static const char *ASSEMBLY2VCF_USAGE_MESSAGE = "Usage: snowman assembly2vcf -i -r \n\n" " Description: Take a BAM (from de novo assembly) and aligned reads --> Call variants\n" "\n" " General options\n" " -v, --verbose Select verbosity level (0-4). Default: 1 \n" " -h, --help Display this help and exit\n" " -p, --threads Use NUM threads to run snowman. Default: 1\n" " Required input\n" " -i, --assembly-bam BAM from aligned de-novo assembly\n" " -t, --tumor-reads-bam BAM from tumor reads\n" " -n, --normal-reads-bam BAM from normal reads\n" " -G, --reference-genome Path to indexed reference genome to be used by BWA-MEM. Default is Broad hg19 (/seq/reference/...)\n" " Optional input\n" " -a, --id-string String specifying the analysis ID to be used as part of ID common.\n" " -r, --rules VariantBam style rules string to determine which reads to do assembly on. See documentation for default.\n" " -k, --region-file Set a region txt file. Format: one region per line, Ex: 1,10000000,11000000\n" " -q, --panel-of-normals Panel of normals gzipped txt file generated from snowman pon\n" " -m, --indel-mask BED-file with blacklisted regions for indel calling. Default /xchip/gistic/Jeremiah/Projects/HengLiMask/um75-hs37d5.bed.gz\n" " --no-read-tracking Don't track supporting reads. Reduces file size.\n" " --no-zip Don't tabix and gzip the output vcf files.\n" "\n"; namespace opt { static std::string args = "snowman "; static std::string assembly_bam; static std::string tumor_reads_bam; static std::string normal_reads_bam; static std::string analysis_id = "assembly2vcf_noid"; static size_t verbose = 1; static std::string rules = "{\"global\" : {\"duplicate\" : false, \"qcfail\" : false}, \"\" : { \"rules\" : [{\"isize\" : [MINS,0]},{\"rr\" : true},{\"ff\" : true}, {\"rf\" : true}, {\"ic\" : true}, {\"clip\" : 5, \"phred\" : 4}, {\"ins\" : true}, {\"del\" : true}, {\"mapped\": true , \"mate_mapped\" : false}, {\"mate_mapped\" : true, \"mapped\" : false}]}}"; static std::string indel_mask = ""; static std::string pon = ""; static bool no_reads = false; static bool zip = true; static size_t numThreads = 1; static bool no_r2c = false; static std::string regionFile = ""; static std::string refgenome = SnowTools::REFHG19; } void runAssembly2VCF(int argc, char** argv) { // parse the options parseAssembly2VCFOptions(argc, argv); // load the reference findex = fai_load(opt::refgenome.c_str()); // load the reference // set the min isize for FR discordants opt::rules.replace(opt::rules.find("MINS"), 4, std::to_string(MIN_ISIZE_FOR_DISC)); SnowmanBamWalker twalk, nwalk; SnowTools::MiniRulesCollection * mr; if (SnowTools::read_access_test(opt::tumor_reads_bam)) { twalk = SnowmanBamWalker(opt::tumor_reads_bam); twalk.prefix= "t000"; twalk.max_cov = 500; mr = new SnowTools::MiniRulesCollection(opt::rules, twalk.header()); twalk.SetMiniRulesCollection(*mr); } if (SnowTools::read_access_test(opt::normal_reads_bam)) { nwalk = SnowmanBamWalker(opt::normal_reads_bam); nwalk.prefix= "n000"; nwalk.max_cov = 500; mr = new SnowTools::MiniRulesCollection(opt::rules, twalk.header()); nwalk.SetMiniRulesCollection(*mr); } // read in the assembly bam file AssemblyBamWalker awalk(opt::assembly_bam); awalk.twalk = twalk; awalk.nwalk = nwalk; awalk.findex = findex; awalk.id = opt::analysis_id; if (SnowTools::read_access_test(opt::tumor_reads_bam)) awalk.tindex = std::shared_ptr(hts_idx_load(opt::tumor_reads_bam.c_str(), HTS_FMT_BAI), bidx_delete()); if (SnowTools::read_access_test(opt::normal_reads_bam)) awalk.nindex = std::shared_ptr(hts_idx_load(opt::normal_reads_bam.c_str(), HTS_FMT_BAI), bidx_delete()); else opt::normal_reads_bam = std::string(); std::cerr << awalk << std::endl; awalk.tbam = opt::tumor_reads_bam; awalk.nbam = opt::normal_reads_bam; awalk.numThreads = opt::numThreads; awalk.refGenome = opt::refgenome; awalk.walkDiscovar(); // make the VCFs std::cerr << "...loading the bps files for conversion to VCF" << std::endl; std::string file = opt::analysis_id + ".bps.txt.gz"; if (!SnowTools::read_access_test(file)) file = opt::analysis_id + ".bps.txt"; // put args into string for VCF later for (int i = 0; i < argc; ++i) opt::args += std::string(argv[i]) + " "; // make the header VCFHeader header; header.filedate = SnowmanUtils::fileDateString(); header.source = opt::args; header.reference = opt::refgenome; //boost::filesystem::path tfp(opt::tumor_reads_bam); //boost::filesystem::path nfp(opt::normal_reads_bam); // TODO fix this header.addSampleField(opt::tumor_reads_bam); header.colnames += "\t" + opt::tumor_reads_bam; header.addSampleField(opt::normal_reads_bam); header.colnames += "\t" + opt::normal_reads_bam; //header.addSampleField(nfp.filename().string()); //header.colnames += "\t" + nfp.filename().string(); //header.addSampleField(tfp.filename().string()); //header.colnames += "\t" + tfp.filename().string(); bool zip = false; VCFFile snowvcf(file, opt::analysis_id, twalk.header(), header); std::string basename = opt::analysis_id + ".unfiltered."; snowvcf.include_nonpass = true; snowvcf.writeIndels(basename, zip, false); snowvcf.writeSVs(basename, zip, false); basename = opt::analysis_id + ".assembly."; //"assembly."; snowvcf.include_nonpass = false; snowvcf.writeIndels(basename, zip, false); snowvcf.writeSVs(basename, zip, false); } // parse the command line options void parseAssembly2VCFOptions(int argc, char** argv) { bool die = false; if (argc <= 2) die = true; for (char c; (c = getopt_long(argc, argv, shortopts, longopts, NULL)) != -1;) { std::istringstream arg(optarg != NULL ? optarg : ""); switch (c) { case 'p': arg >> opt::numThreads; break; case 'a': arg >> opt::analysis_id; break; case 'm': arg >> opt::indel_mask; break; case 'q': arg >> opt::pon; break; case 'z': opt::zip = false; break; case 'h': die = true; break; case 'x': opt::no_r2c = true; break; case OPT_NO_READS: opt::no_reads = true; break; case 'i': arg >> opt::assembly_bam; break; case 't': arg >> opt::tumor_reads_bam; break; case 'n': arg >> opt::normal_reads_bam; break; case 'k': arg >> opt::regionFile; break; case 'r': arg >> opt::rules; break; case 'v': arg >> opt::verbose; break; case 'G': arg >> opt::refgenome; break; default: die= true; } } if (die) { std::cout << "\n" << ASSEMBLY2VCF_USAGE_MESSAGE; exit(EXIT_FAILURE); } } ================================================ FILE: src/svabautils/assembly2vcf.h ================================================ #ifndef SNOWMAN_ASSEMBLY2VCF_H__ #define SNOWMAN_ASSEMBLY2VCF_H__ void parseAssembly2VCFOptions(int argc, char** argv); void runAssembly2VCF(int argc, char** argv); #endif ================================================ FILE: src/svabautils/benchmark.cpp ================================================ #include "benchmark.h" #include #include #include #include #include #include #include #include "vcf.h" #include "Fractions.h" #include "SeqLib/BWAWrapper.h" #include "SeqLib/SeqLibCommon.h" #include "SeqLib/GenomicRegion.h" #include "ReadSim.h" #include "SeqFrag.h" #include "SimGenome.h" #include "KmerFilter.h" #include "PowerLawSim.h" #include "BamSplitter.h" #include "SnowmanUtils.h" #include "AlignedContig2.h" #include "SimTrainerWalker.h" #include "SnowmanAssemblerEngine.h" static std::vector snv_error_rates; static std::vector del_error_rates; static std::vector ins_error_rates; static std::vector coverages; static std::vector fractions; static SeqLib::GRC regions; static Fractions fractions_bed; static SeqLib::BamReader bwalker; static faidx_t * findex; #define DEFAULT_SNV_RATE 0.01 #define DEFAULT_DEL_RATE 0.05 #define DEFAULT_INS_RATE 0.05 #define DEFAULT_COV 10 namespace opt { static std::string refgenome = "/seq/references/Homo_sapiens_assembly19/v1/Homo_sapiens_assembly19.fasta"; static int mode = -1; static size_t readlen = 101; static int num_runs = 100; static uint32_t seed = 0; static std::string regionFile = ""; static std::string bam = ""; static int isize_mean = 250; static int isize_sd = 50; static int nbreaks = 10; static int nindels = 10; static std::string string_id = "noid"; static std::string frac_bed_file; static bool scramble = false; static int viral_count = 0; static std::string blacklist; static std::set prefixes; } enum { OPT_ASSEMBLY, OPT_SIMBREAKS, OPT_ISIZE_MEAN, OPT_ISIZE_SD, OPT_SPLITBAM, OPT_SCRAMBLE, OPT_POWERSIM, OPT_REALIGN, OPT_BLACKLIST, OPT_REALIGN_SV }; static const char *BENCHMARK_USAGE_MESSAGE = "Usage: snowman benchmark\n\n" " Description: Various benchmarking tests for Snowman\n" "\n" " General options\n" " -v, --verbose Select verbosity level (0-4). Default: 1 \n" " -G, --reference-genome Indexed ref genome for BWA-MEM. Default (Broad): /seq/reference/...)\n" " -s, --seed Seed for the random number generator\n" " -A, --string-id String to name output files with (e.g. _0_01.bam\n" " Choose one of the following:\n" " --test-assembly Generate single-end reads from small contigs to test assembly/remapping\n" " --sim-breaks-power Simulate rearrangements and indels and output paired-end reads\n" " --split-bam Divide up a BAM file into smaller sub-sampled files, with no read overlaps between files. Preserves read-pairs\n" " --realign-test Randomly sample the reference genome and test ability of BWA-MEM to realign to reference for different sizes / error rates\n" " --realign-sv-test Make an SV (rearrangment) and simulate contigs from it. Test size of contigs vs alignment accuracy for SVs\n" " Shared Options for Test and Simulate:\n" " -c, --read-covearge Desired coverage. Input as comma-separated to test multiple (test assembly)\n" " -b, --bam BAM file to train the simulation with\n" " -k, --regions Regions to simulate breaks or test assembly\n" " -E, --snv-error-rate The random SNV error rate per base. Input as comma-separated to test multiple (test assembly)\n" " -I, --ins-error-rate The random insertion error rate per read. Input as comma-separated to test multiple (test assembly)\n" " -D, --del-error-rate The random deletion error rate per read. Input as comma-separated to test multiple (test assembly)\n" " Test Assembly (--test-assembly) Options:\n" " -n, --num-runs Number of random trials to run\n" // " Simulate Breaks (--sim-breaks) Options:\n" //" --isize-mean Desired mean insert size for the simulated reads\n" //" --isize-sd Desired std. dev. forinsert size for the simulated reads\n" //" -M, --viral-integration Number of segments to integrate viruses into. (Reduces amount of space for indels)\n" //" --add-scrambled-inserts Add scrambled inserts at junctions, randomly between 0 and 100 bp with p(0) = 50% and p(1-100) = 50%;\n" " Simulate Breaks with Power Law(--sim-breaks-power) Options:\n" " --blacklist BED file specifying blacklist regions not to put breaks in\n" " -R, --num-rearrangements Number of rearrangements to simulate\n" " -X, --num-indels Number of indels to simulate\n" " Split Bam (--split-bam) Options:\n" " -f, --fractions Fractions to split the bam into\n" "\n"; static const char* shortopts = "haG:c:n:s:k:b:E:I:D:R:X:A:f:M:"; static const struct option longopts[] = { { "help", no_argument, NULL, 'h' }, { "reference-genome", required_argument, NULL, 'G' }, { "string-id", required_argument, NULL, 'A' }, { "seed", required_argument, NULL, 's' }, { "num-runs", required_argument, NULL, 'n' }, { "regions", required_argument, NULL, 'k' }, { "bam", required_argument, NULL, 'b' }, { "read-coverage", required_argument, NULL, 'c' }, { "snv-error-rate", required_argument, NULL, 'E' }, { "del-error-rate", required_argument, NULL, 'D' }, { "ins-error-rate", required_argument, NULL, 'I' }, { "num-rearrangements", required_argument, NULL, 'R' }, { "fractions", required_argument, NULL, 'f' }, { "num-indels", required_argument, NULL, 'X' }, { "viral-integration", required_argument, NULL, 'M' }, { "isize-mean", required_argument, NULL, OPT_ISIZE_MEAN}, { "isize-sd", required_argument, NULL, OPT_ISIZE_SD}, { "test-assembly", no_argument, NULL, OPT_ASSEMBLY}, { "blacklist", required_argument, NULL, OPT_BLACKLIST}, { "sim-breaks-power", no_argument, NULL, OPT_POWERSIM}, { "sim-breaks", no_argument, NULL, OPT_SIMBREAKS}, { "split-bam", no_argument, NULL, OPT_SPLITBAM}, { "realign-sv-test", no_argument, NULL, OPT_REALIGN_SV}, { "add-scrambled-inserts",no_argument, NULL, OPT_SCRAMBLE}, { "realign-test",no_argument, NULL, OPT_REALIGN}, { NULL, 0, NULL, 0 } }; void runBenchmark(int argc, char** argv) { parseBenchmarkOptions(argc, argv); opt::prefixes.insert(opt::bam); std::cerr << "-----------------------------------------" << std::endl << "--- Running Snowman Benchmarking Test ---" << std::endl << "-----------------------------------------" << std::endl; if (opt::mode == OPT_ASSEMBLY) std::cerr << "********* RUNNING ASSEMBLY TEST ***********" << std::endl; else if (opt::mode == OPT_SIMBREAKS) std::cerr << "********* RUNNING SIMULATE BREAKS ***********" << std::endl; else if (opt::mode == OPT_SPLITBAM) std::cerr << "********* RUNNING SPLIT BAM ***********" << std::endl; else if (opt::mode == OPT_REALIGN) std::cerr << "********* RUNNING REALIGN TEST ***********" << std::endl; else if (opt::mode == OPT_REALIGN_SV) std::cerr << "********* RUNNING REALIGN SV TEST ***********" << std::endl; if (opt::mode == OPT_ASSEMBLY || opt::mode == OPT_SIMBREAKS) { std::cerr << " Error rates:" << std::endl; std::cerr << errorRateString(snv_error_rates, "SNV") << std::endl; std::cerr << errorRateString(ins_error_rates, "Del") << std::endl; std::cerr << errorRateString(del_error_rates, "Ins") << std::endl; std::cerr << errorRateString(coverages, "Coverages") << std::endl; std::cerr << " Insert size: " << opt::isize_mean << "(" << opt::isize_sd << ")" << std::endl; } else if (opt::mode == OPT_SPLITBAM) { std::cerr << errorRateString(fractions, "Fractions") << std::endl; } // open the BAM if (opt::bam.length() && SeqLib::read_access_test(opt::bam)) { assert(bwalker.Open(opt::bam)); } else if (opt::mode == OPT_REALIGN || opt::mode == OPT_SPLITBAM || opt::mode == OPT_REALIGN_SV) { std::cerr << "NEED TO INPUT VALID BAM (perhaps just to get header info)" << std::endl; exit(EXIT_FAILURE); } // parse the region file if (opt::regionFile.length()) { if (SeqLib::read_access_test(opt::regionFile)) { regions = SeqLib::GRC(opt::regionFile, bwalker.Header()); regions.MergeOverlappingIntervals(); } // samtools format else if (opt::regionFile.find(":") != std::string::npos && opt::regionFile.find("-") != std::string::npos) { if (bwalker.Header().isEmpty()) { std::cerr << "Error: To parse a samtools style string, need a BAM header. Input bam with -b" << std::endl; exit(EXIT_FAILURE); } regions.add(SeqLib::GenomicRegion(opt::regionFile, bwalker.Header())); } else { std::cerr << "Can't parse the regions. Input as BED file or Samtools style string (requires BAM with -b to for header info)" << std::endl; exit(EXIT_FAILURE); } if (!regions.size()) { std::cerr << "ERROR: Must input a region to run on " << std::endl; exit(EXIT_FAILURE); } } // seed the RNG if (opt::mode != OPT_SPLITBAM) { if (opt::seed == 0) opt::seed = (unsigned)time(NULL); srand(opt::seed); std::cerr << " Seed: " << opt::seed << std::endl; } // read the fractions file if (opt::frac_bed_file.length() && opt::mode == OPT_SPLITBAM) { fractions_bed.readFromBed(opt::frac_bed_file, bwalker.Header()); // fractions_bed.readBEDfile(opt::frac_bed_file.length(), 0, bwalker.header()); } findex = fai_load(opt::refgenome.c_str()); // load the reference SeqLib::GRC blacklist; if (opt::mode == OPT_POWERSIM && !opt::blacklist.empty()) { std::cerr << "...reading blacklist file " << opt::blacklist << std::endl; blacklist = SeqLib::GRC(opt::blacklist, bwalker.Header()); blacklist.CreateTreeMap(); std::cerr << "...read in " << blacklist.size() << " blacklist regions " << std::endl; } // if (opt::mode == OPT_ASSEMBLY) assemblyTest(); else if (opt::mode == OPT_SIMBREAKS) genBreaks(); else if (opt::mode == OPT_SPLITBAM) splitBam(); else if (opt::mode == OPT_REALIGN) realignRandomSegments(); else if (opt::mode == OPT_REALIGN_SV) realignBreaks(); else if (opt::mode == OPT_POWERSIM) { std::cerr << "...opening output" << std::endl; std::ofstream outfasta; SnowmanUtils::fopen("tumor_seq.fa", outfasta); std::ofstream events; SnowmanUtils::fopen("events.txt", events); PowerLawSim(findex, opt::nbreaks, -1.0001, blacklist, outfasta, events); outfasta.close(); events.close(); } else std::cerr << "Mode not recognized. Chose from: --test-assembly, --sim-breaks, --split-bam" << std::endl; } std::string genBreaks() { // train on the input BAM std::vector quality_scores; SeqLib::GenomicRegion v(0, 1000000,2000000); /* std::vector v = { SeqLib::GenomicRegion(0, 1000000, 2000000) SeqLib::GenomicRegion(0, 60000000,70000000), SeqLib::GenomicRegion(1, 1000000, 10000000), SeqLib::GenomicRegion(1, 60000000,70000000), SeqLib::GenomicRegion(2, 1000000, 10000000), SeqLib::GenomicRegion(3, 60000000,70000000), SeqLib::GenomicRegion(16, 1000000,1110000), SeqLib::GenomicRegion(17, 1000000,1110000), SeqLib::GenomicRegion(21, 1000000,1110000) };*/ /* SimTrainerWalker stw(opt::bam); stw.setBamWalkerRegions(v); stw.train(); std::ofstream bamstats("bam_stats.txt"); bamstats << stw.printBamStats() << std::endl; bamstats.close(); */ bwalker.SetRegion(v); SeqLib::BamRecord r; std::cerr << "...sampling reads to learn quality scores" << std::endl; while (bwalker.GetNextRecord(r)) { std::string ss = r.Sequence(); if (ss.find("AAAAAAAA") == std::string::npos && ss.find("TTTTTTTT") == std::string::npos) // already handle homopolymers quality_scores.push_back(r.Qualities()); } std::cerr << "...loading the reference genome" << std::endl; SeqLib::GenomicRegion gg = regions[0]; std::cerr << "--Generating breaks on: " << gg << std::endl; std::cerr << "--Total number of rearrangement breaks: " << opt::nbreaks << std::endl; std::cerr << "--Total (approx) number of indels: " << opt::nindels << std::endl; SimGenome sg(gg, opt::nbreaks, opt::nindels, findex, opt::scramble, opt::viral_count); std::string final_seq = sg.getSequence(); // write the final seq to a file std::ofstream fseq; fseq.open("tumor_seq.fa", std::ios::out); fseq << ">tumor\n" << final_seq << std::endl; fseq.close(); //exit(1); ReadSim rs; std::ofstream ind; ind.open("indels.tsv", std::ios::out); for (auto& i : sg.m_indels) ind << i << std::endl; ind.close(); std::ofstream con; con.open("connections.tsv", std::ios::out); con << sg.printBreaks(); con.close(); std::ofstream mic; mic.open("microbe_spikes.tsv", std::ios::out); mic << sg.printMicrobeSpikes(); mic.close(); exit(0); rs.addAllele(final_seq, 1); // sample paired reads std::vector reads1; std::vector reads2; std::vector qual1; std::vector qual2; std::cerr << "Simulating reads at coverage of " << coverages[0] << " del rate " << del_error_rates[0] << " ins rate " << ins_error_rates[0] << " snv-rate " << snv_error_rates[0] << " isize " << opt::isize_mean << "(" << opt::isize_sd << ")" << std::endl; rs.samplePairedEndReadsToCoverage(reads1, reads2, qual1, qual2, coverages[0], snv_error_rates[0], ins_error_rates[0], del_error_rates[0], opt::readlen, opt::isize_mean, opt::isize_sd, quality_scores); assert(reads1.size() == reads2.size()); // write the paired end fastq. Give random errors std::cerr << "...writing/errorring reads 1" << std::endl; std::ofstream pe1; pe1.open("paired_end1.fastq", std::ios::out); size_t ccc= 0; for (size_t i = 0; i < reads1.size(); ++i) { rs.baseQualityRelevantErrors(reads1[i], qual1[i]); pe1 << "@r" << ccc++ << std::endl << reads1[i] << std::endl << "+\n" << qual1[i] << std::endl; } pe1.close(); std::cerr << "...writing/erroring reads 2" << std::endl; std::ofstream pe2; pe2.open("paired_end2.fastq", std::ios::out); ccc= 0; for (size_t i = 0; i < reads2.size(); ++i) { //std::string qs = quality_scores[rand() % quality_scores.size()]; rs.baseQualityRelevantErrors(reads2[i], qual2[i]); pe2 << "@r" << ccc++ << std::endl << reads2[i] << std::endl << "+\n" << qual2[i] << std::endl; } pe2.close(); std::cerr << "********************************" << std::endl; std::cerr << "Suggest running: " << std::endl; std::cerr << "bwa mem -t 10 $REFHG19 paired_end1.fastq paired_end2.fastq | samtools sort -O bam -T /tmp -l 9 -m 16G > sim.bam && samtools index sim.bam" << std::endl; std::cerr << "snowman benchmark --split-bam -f 0.3,0.6 -b $n11 -k -A norm" << std::endl; std::cerr << "samtools merge sim_wnormal.bam sim.bam norm_0.30_subsampled.bam" << std::endl; std::cerr << "samtools index norm_0.60_subsampled.bam && samtools index sim_wnormal.bam" << std::endl; //std::cerr << "bwa mem $REFHG19 paired_end1.fastq paired_end2.fastq | samtools sort -O bam -T /tmp -l 9 -m 16G > sim.bam && samtools sort sim.bam" << std::endl; //std::cerr << "bwa mem $REFHG19 paired_end1.fastq paired_end2.fastq > sim.sam && samtools view sim.sam -Sb > tmp.bam && " << // "samtools sort -m 4G tmp.bam sim && rm sim.sam tmp.bam && samtools index sim.bam" << std::endl; std::cerr << "********************************" << std::endl; return ""; } void splitBam() { BamSplitter bs(opt::seed); assert(bs.Open(opt::bam)); // set the regions to split on if (regions.size()) bs.SetMultipleRegions(regions); std::cerr << "...set " << regions.size() << " walker regions " << std::endl; if (fractions_bed.size()) { bs.fractionateBam(opt::string_id + ".fractioned.bam", fractions_bed); } else { // set the output bams std::vector fnames; for (auto& i : fractions) { std::stringstream ss; ss.precision(2); ss << opt::string_id << "_" << i << "_subsampled.bam"; fnames.push_back(ss.str()); } bs.setWriters(fnames, fractions); bs.splitBam(); } } void assemblyTest() { SeqLib::GenomicRegion gr(16, 7565720, 7575000); //"chr17:7,569,720-7,592,868"); std::cerr << "...loading the reference genome" << std::endl; findex = fai_load(opt::refgenome.c_str()); // load the reference std::string local_ref = ""; // debug // getRefSequence("16", gr, findex); size_t seqlen = local_ref.length(); if (seqlen * 2 <= opt::readlen) { std::cerr << "**** Read length must be > 2 * sequence length" << std::endl; exit(EXIT_FAILURE); } // make the BWA Wrapper std::cerr << "...constructing local_seq index" << std::endl; SeqLib::BWAWrapper local_bwa; local_bwa.ConstructIndex({{"local_ref", local_ref, std::string()}}); // align local_seq to itself SeqLib::BamRecordVector self_align; local_bwa.AlignSequence(local_ref, "local_ref", self_align, false, false, 0); // write out the index local_bwa.WriteIndex("local_ref.fa"); std::ofstream fa; fa.open("local_ref.fa"); fa << ">local_ref" << std::endl << local_ref << std::endl; fa.close(); std::cout << "coverage\tnumreads\tnumcontigs\tnumfinal\tcontig_coverage\tkmer_corr\terror_rate" << std::endl; for (int rep = 0; rep < opt::num_runs; ++rep) { std::cerr << "...assembly test. Working on iteration " << rep << " of " << opt::num_runs << std::endl; for (int k = 1; k <= 1; ++k) { for (auto& c : coverages) { for (auto& E : snv_error_rates) { for (auto& D : del_error_rates) { for (auto& I : ins_error_rates) { // make the read vector ReadSim rs; rs.addAllele(local_ref, 1); // sample reads randomly //std::cerr << "...making read vector" << std::endl; std::vector reads; rs.sampleReadsToCoverage(reads, c, E, I, D, opt::readlen); // sample paired reads //std::cerr << "...making paired-end read vector" << std::endl; std::vector reads1; std::vector reads2; std::vector qual1; std::vector qual2; std::vector qual = {std::string('I',opt::readlen)}; rs.samplePairedEndReadsToCoverage(qual1, qual2, reads1, reads2, c, E, I, D, opt::readlen, 350, 50, qual); assert(reads1.size() == reads2.size()); // align these reads to the local_seq //std::cerr << "...realigned to local_seq" << std::endl; SeqLib::BamRecordVector reads_to_local; int count = 0; for (auto& i : reads) { if (i.find("N") == std::string::npos) { SeqLib::BamRecordVector read_hits; local_bwa.AlignSequence(i, "read_" + std::to_string(++count), read_hits, false,false, 0); if (read_hits.size()) reads_to_local.push_back(read_hits[0]); } } // kmer filter the reads KmerFilter kmer; if (k == 1) kmer.correctReads(reads_to_local, reads_to_local); //std::cerr << " Attempted align of " << reads.size() << " to local_seq. Got hits on " << reads_to_local.size() << std::endl; // make plot of reads to contig //AlignedContig sa(self_align); //sa.alignReads(reads_to_local); // assemble them //std::cerr << "...assembling" << std::endl; double error_rate = 0; if (k == 0) error_rate = 0.05; int min_overlap = 35; SnowmanAssemblerEngine engine("test", error_rate, min_overlap, opt::readlen); engine.fillReadTable(reads_to_local); engine.performAssembly(2); // align them back SeqLib::BamRecordVector contigs_to_local; for (auto& i : engine.getContigs()) { SeqLib::BamRecordVector ct_alignments; local_bwa.AlignSequence(i.Seq, i.Name, ct_alignments, false, false, 0); AlignedContig ac(ct_alignments, opt::prefixes); //ac.alignReads(reads_to_local); //std::cout << ac; contigs_to_local.insert(contigs_to_local.begin(), ct_alignments.begin(), ct_alignments.end()); } // write the results SeqLib::GRC grc(contigs_to_local); grc.MergeOverlappingIntervals(); double width = 0; for (auto& i : grc) width = std::max(width, (double)i.Width()); width = width / local_ref.length(); std::cout << c << "\t" << reads_to_local.size() << "\t" << engine.getContigs().size() << "\t" << grc.size() << "\t" << width << "\t" << k << "\t" << E << std::endl; if (k == 1 && c == 20 && E == 0.01) { // write out the contig to local ref bam SeqLib::BamWriter bw2; bw2.Open("contigs_to_ref.bam"); bw2.SetHeader(local_bwa.HeaderFromIndex()); bw2.WriteHeader(); for (auto& i : contigs_to_local) bw2.WriteRecord(i); // write the paired end fasta std::ofstream pe1; pe1.open("paired_end1.fa", std::ios::out); size_t ccc= 0; for (auto& i : reads1) pe1 << ">r" << ccc++ << std::endl << i << std::endl; pe1.close(); std::ofstream pe2; pe2.open("paired_end2.fa", std::ios::out); ccc= 0; for (auto& i : reads2) pe2 << ">r" << ccc++ << std::endl << i << std::endl; pe2.close(); // write out the read to local ref aligned bam SeqLib::BamWriter bw; bw.Open("reads_to_ref_" + std::to_string(c) + ".bam"); bw.SetHeader(local_bwa.HeaderFromIndex()); bw.WriteHeader(); for (auto& i : reads_to_local) bw.WriteRecord(i); SeqLib::BamWriter bwk; bwk.Open("k.bam"); bwk.SetHeader(local_bwa.HeaderFromIndex()); bwk.WriteHeader(); for (auto& i : reads_to_local) { std::string kc = i.GetZTag("KC"); if (kc.length()) i.SetSequence(kc); bwk.WriteRecord(i); } } } // end kmer loop } // end error loop } // end coverage } } } } void parseBenchmarkOptions(int argc, char** argv) { bool die = false; if (argc < 2) die = true; std::string del_er; std::string snv_er; std::string ins_er; std::string covs; std::string frac; std::string t; for (char c; (c = getopt_long(argc, argv, shortopts, longopts, NULL)) != -1;) { std::istringstream arg(optarg != NULL ? optarg : ""); switch (c) { case 'h': die = true; break; case OPT_ASSEMBLY: opt::mode = OPT_ASSEMBLY; break; case OPT_REALIGN: opt::mode = OPT_REALIGN; break; case OPT_REALIGN_SV: opt::mode = OPT_REALIGN_SV; break; case OPT_POWERSIM: opt::mode = OPT_POWERSIM; break; case OPT_BLACKLIST: arg >> opt::blacklist; break; case 'G': arg >> opt::refgenome; break; case 'n': arg >> opt::num_runs; break; case 's': arg >> opt::seed; break; case 'c': arg >> covs; break; case 'k': arg >> opt::regionFile; break; case 'b': arg >> opt::bam; break; case 'E': arg >> snv_er; break; case 'R': arg >> opt::nbreaks; break; case 'X': arg >> opt::nindels; break; case 'D': arg >> del_er; break; case 'I': arg >> ins_er; break; case 'A': arg >> opt::string_id; break; case 'f': arg >> frac; break; case 'M': arg >> opt::viral_count; break; case OPT_SIMBREAKS: opt::mode = OPT_SIMBREAKS; break; case OPT_SPLITBAM: opt::mode = OPT_SPLITBAM; break; case OPT_ISIZE_MEAN: arg >> opt::isize_mean; break; case OPT_ISIZE_SD: arg >> opt::isize_sd; break; case OPT_SCRAMBLE: opt::scramble = true; break; default: die= true; } } if (die) { std::cerr << "\n" << BENCHMARK_USAGE_MESSAGE; exit(EXIT_FAILURE); } // parse the error rates snv_error_rates = parseErrorRates(snv_er); del_error_rates = parseErrorRates(del_er); ins_error_rates = parseErrorRates(ins_er); coverages = parseErrorRates(covs); // parse the fractions string or read file if (!SeqLib::read_access_test(frac)) fractions = parseErrorRates(frac); else opt::frac_bed_file = frac; // check that the bam is valid if (opt::mode == OPT_SIMBREAKS && !SeqLib::read_access_test(opt::bam)) { std::cerr << "ERROR: Input BAM required for --sim-breaks" << std::endl; exit(EXIT_FAILURE); } // set the default error rates if (!snv_error_rates.size()) snv_error_rates.push_back(DEFAULT_SNV_RATE); if (!del_error_rates.size()) del_error_rates.push_back(DEFAULT_DEL_RATE); if (!ins_error_rates.size()) ins_error_rates.push_back(DEFAULT_INS_RATE); if (!coverages.size()) coverages.push_back(DEFAULT_COV); if ((!fractions.size() && !opt::frac_bed_file.length()) && opt::mode == OPT_SPLITBAM) { std::cerr << "Error: Must specify fractions to split into with -f (e.g. -f 0.1,0.8), or as BED file" << std::endl; exit(EXIT_FAILURE); } } // parse the error rates string from the options std::vector parseErrorRates(const std::string& s) { std::vector out; std::istringstream is(s); std::string val; while(std::getline(is, val, ',')) { try { out.push_back(std::stod(val)); } catch (...) { std::cerr << "Could not convert " << val << " to number. If you're inputting a file not CSV, then check file exists" << std::endl; exit(EXIT_FAILURE); } } return out; } std::string errorRateString(const std::vector& v, const std::string& name) { std::stringstream ss; ss << " " << name << ":"; for (auto& i : v) ss << " " << i << ","; std::string out = ss.str(); out.pop_back(); return out; } void realignBreaks() { ReadSim rs; std::cerr << "...loading reference genome" << std::endl; SeqLib::BWAWrapper bwa; bwa.LoadIndex(opt::refgenome); std::ofstream results; SnowmanUtils::fopen("realign.sv.results.csv", results); results << "id\tsize\tAlign1\tAlign2\tAlignBoth" << std::endl; const int RUN_NUM = 1000; //size_t count = 0; std::string chrstring1, chrstring2; for (int k = 0; k < RUN_NUM; ++k) { for (int i = 30; i < 500; i += 10) { SeqLib::GenomicRegion gr1, gr2; gr1.Random(); gr2.Random(); gr1.pos2 = gr1.pos1 + i/2; ; //+ k - 1 + (ins == 0 ? iii : 0); // add sequence to deletion ones, because it gets removed later gr2.pos2 = gr2.pos2 + i/2; chrstring1 = bwalker.Header().IDtoName(gr1.chr); //std::to_string(gr.chr+1); chrstring2 = bwalker.Header().IDtoName(gr2.chr); //std::to_string(gr.chr+1); int len; char * seq1 = faidx_fetch_seq(findex, const_cast(chrstring1.c_str()), gr1.pos1, gr1.pos2 - 1, &len); char * seq2 = faidx_fetch_seq(findex, const_cast(chrstring2.c_str()), gr2.pos1, gr2.pos2 - 1, &len); std::string s1, s2; if (seq1) s1 = std::string(seq1); else continue; if (s1.find("N") != std::string::npos) continue; if (seq2) s2 = std::string(seq2); else continue; if (s2.find("N") != std::string::npos) continue; if (rand() % 2) SeqLib::rcomplement(s2); std::string ss = s1 + s2; SeqLib::BamRecordVector aligns; bwa.AlignSequence(ss, std::to_string(i), aligns, false, 0.90, 2); bool a1 = false; bool a2 = false; for (auto& jj : aligns) { if (gr1.GetOverlap(jj.asGenomicRegion())) a1 = true; if (gr2.GetOverlap(jj.asGenomicRegion())) a2 = true; } results << k << "\t" << i << "\t" << a1 << "\t" << a2 << "\t" << (a1 && a2) << std::endl; } } } void realignRandomSegments() { ReadSim rs; std::cerr << "...loading reference genome" << std::endl; SeqLib::BWAWrapper bwa; bwa.LoadIndex(opt::refgenome); std::ofstream results; SnowmanUtils::fopen("realign.results.csv", results); results << "ID" << "\t" << "chr" << "\t" << "pos1" << "\t" << "pos2" << "\t" << "width" << "\t" << "num_aligns" << "\t" << "correct_hit_num" << "\t" << "snv_rate" << "\t" << "del_size" << "\t" << "ins_size" << std::endl; const int RUN_NUM = 1000; size_t tcount = 0; std::string chrstring; std::vector widths = {70, 101, 250}; std::vector snv_rate = {0, 0.01, 0.05}; std::vector indel_size = {0, 1, 5, 20, 50}; for (size_t ins = 0; ins < 2; ++ins) { //insertion or del for (auto& iii : indel_size) { for (auto& snv : snv_rate) { for (auto& k : widths) { ++tcount; for (size_t i = 0; i < RUN_NUM; ++i) { if (i == 0) std::cerr << "...working on width " << k << " and SNV rate " << snv << "\t" << (ins == 1 ? "INS" : "DEL") << "\t" << iii << ". " << tcount << " of " << (2 * widths.size() * snv_rate.size() * indel_size.size()) << std::endl; SeqLib::GenomicRegion gr; gr.Random(); gr.pos2 = gr.pos1 + k - 1 + (ins == 0 ? iii : 0); // add sequence to deletion ones, because it gets removed later chrstring = bwalker.Header().IDtoName(gr.chr); //bwalker.header()->target_name[gr.chr]; //std::to_string(gr.chr+1); int len; char * seq = faidx_fetch_seq(findex, const_cast(chrstring.c_str()), gr.pos1, gr.pos2 - 1, &len); std::string s; if (seq) s = std::string(seq); else continue; if (s.find("N") != std::string::npos) continue; rs.makeSNVErrors(s, snv); if (gr.Width() < iii + 10) continue; if (ins == 1 && iii > 0) rs.makeInsErrors(s, true, iii); else if (iii > 0) rs.makeDelErrors(s, iii); SeqLib::BamRecordVector aligns; bwa.AlignSequence(s, std::to_string(i), aligns, false, 0.90, 50); int align_num = -1; for (size_t j = 0; j < aligns.size(); ++j) { SeqLib::GenomicRegion gr_this = aligns[j].asGenomicRegion(); gr_this.Pad(100); if (gr.GetOverlap(gr_this)) { if (iii == 0) { align_num = j; break; } else { if (ins == 1) { int g = aligns[j].MaxInsertionBases(); if (g && g - 2 < iii && g + 2 > iii) { align_num = j; break; } } else { int d = aligns[j].MaxDeletionBases(); if (d && d - 2 < iii && d + 2 > iii) { align_num = j; break; } } } } } results << i << "\t" << chrstring << "\t" << gr.pos1 << "\t" << gr.pos2 << "\t" << k << "\t" << aligns.size() << "\t" << align_num << "\t" << snv << "\t" << (ins == 0 ? iii : 0) << "\t" << (ins == 1 ? iii : 0) << std::endl; } } } } } results.close(); } ================================================ FILE: src/svabautils/benchmark.h ================================================ #ifndef SNOWMAN_BENCHMARK_H__ #define SNOWMAN_BENCHMARK_H__ #include #include void parseBenchmarkOptions(int argc, char** argv); void runBenchmark(int argc, char** argv); void assemblyTest(); void sampleReads(const std::string& seq, std::vector& reads, int cov, double error_rate); void realignRandomSegments(); void realignBreaks(); std::string genBreaks(); std::vector parseErrorRates(const std::string& s); std::string errorRateString(const std::vector& v, const std::string& name); void splitBam(); #endif ================================================ FILE: src/svabautils/configure ================================================ #! /bin/sh # Guess values for system-dependent variables and create Makefiles. # Generated by GNU Autoconf 2.63 for snowman 1.0. # # Report bugs to . # # Copyright (C) 1992, 1993, 1994, 1995, 1996, 1998, 1999, 2000, 2001, # 2002, 2003, 2004, 2005, 2006, 2007, 2008 Free Software Foundation, Inc. # This configure script is free software; the Free Software Foundation # gives unlimited permission to copy, distribute and modify it. ## --------------------- ## ## M4sh Initialization. ## ## --------------------- ## # Be more Bourne compatible DUALCASE=1; export DUALCASE # for MKS sh if test -n "${ZSH_VERSION+set}" && (emulate sh) >/dev/null 2>&1; then emulate sh NULLCMD=: # Pre-4.2 versions of Zsh do word splitting on ${1+"$@"}, which # is contrary to our usage. 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AC_PREREQ(2.59) ## specificy version of autoconf AC_INIT(snowman, 1.0, jwala@broadinstitute.org) AM_INIT_AUTOMAKE(foreign) AC_CONFIG_SRCDIR([snowmanutils.cpp]) AC_CONFIG_HEADER([config.h]) AM_MAINTAINER_MODE([disable]) # Checks for programs. AC_PROG_CXX ## test for cpp compiler AC_PROG_CC ## test for C compiler AC_PROG_RANLIB ## required if libraries are built in package # Check for headers AC_LANG([C++]) AC_CHECK_HEADER([zlib.h]) # Check for libraries AC_SEARCH_LIBS([gzopen],[z],,[AC_MSG_ERROR([libz not found, please install zlib (http://www.zlib.net/)])]) AC_SEARCH_LIBS([clock_gettime], [rt], [AC_DEFINE([HAVE_CLOCK_GETTIME], [1], [clock_getttime found])], ) AC_ARG_ENABLE(development, AS_HELP_STRING([--enable-development], [Turn on development options, like failing compilation on warnings])) if test "$enable_development"; then fail_on_warning="-Werror" fi # Set compiler flags. AC_SUBST(AM_CXXFLAGS, "-g -Wall -Wextra $fail_on_warning -Wno-unknown-pragmas -std=c++11 -D_GLIBCXX_USE_CXX11_ABI=0") # Check for hash table headers AC_CHECK_HEADERS([ \ google/sparse_hash_set google/sparse_hash_map \ unordered_map tr1/unordered_map ext/hash_map \ unordered_set tr1/unordered_set ext/hash_set \ ]) AC_CONFIG_FILES([Makefile]) AC_OUTPUT ================================================ FILE: src/svabautils/snowmanutils.cpp ================================================ #include "benchmark.h" #include "assembly2vcf.h" #include "splitcounter.h" #define AUTHOR "Jeremiah Wala " static const char *SUTILS_USAGE_MESSAGE = "-----------------------------------------------------------------------\n" "--- Snowman Utils - Utililty methods for analyzing/benchmarking SVs ---\n" "-----------------------------------------------------------------------\n" "Program: SnowmanUtils \n" "Contact: Jeremiah Wala [ jwala@broadinstitute.org ]\n" "Usage: snowmanutils [options]\n\n" "Commands:\n" " benchmark Run benchmarking tests for Snowman\n" " assembly2vcf Run Snowman filtering on BWA-MEM aligned assembly (eg Discovar, SGA, etc) and aligned reads\n" " splitcounter Simple utility to count locations of split-alignment breakpoints (eg for PacBio)\n" " splitfasta Simple utility to split a fasta into smaller subsequences, splitting seq in the middle\n" "\nReport bugs to jwala@broadinstitute.org \n\n"; int main(int argc, char** argv) { if (argc <= 1) { std::cerr << SUTILS_USAGE_MESSAGE; return 0; } else { std::string command(argv[1]); if (command == "help" || command == "--help") { std::cerr << SUTILS_USAGE_MESSAGE; return 0; } else if (command == "benchmark") { runBenchmark(argc-1, argv+1); } else if (command == "splitfasta") { runSplitFasta(argc-1, argv+1); } else if (command == "assembly2vcf") { runAssembly2VCF(argc-1, argv+1); } else if (command == "splitcounter") { runSplitCounter(argc-1, argv+1); } else { std::cerr << SUTILS_USAGE_MESSAGE; return 0; } } std::cerr << "Done with snowman utils" << std::endl; return 0; } ================================================ FILE: src/svabautils/snowtools.cpp ================================================ #include "json/json.h" #include "json/json-forwards.h" #include "SeqLib/MiniRules2.h" #include #include #include #include #include #include using namespace std; const std::vector m_propertyNames = {"reference", "mapQuality", "isReverseStrand"}; bool ParseFilterObject(const string& filterName, const Json::Value& filterObject) { // filter object parsing variables Json::Value null(Json::nullValue); Json::Value propertyValue; // store results map propertyTokens; // iterate over known properties vector::const_iterator propertyNameIter = m_propertyNames.begin(); vector::const_iterator propertyNameEnd = m_propertyNames.end(); for ( ; propertyNameIter != propertyNameEnd; ++propertyNameIter ) { const string& propertyName = (*propertyNameIter); // if property defined in filter, add to token list propertyValue = filterObject.get(propertyName, null); if ( propertyValue != null ) { std::cerr << "[" << propertyName << "] -- " << propertyValue.asString() << std::endl; propertyTokens.insert( make_pair(propertyName, propertyValue.asString()) ); } } // add this filter to engin //m_filterEngine.addFilter(filterName); // add token list to this filter //return AddPropertyTokensToFilter(filterName, propertyTokens); return true; } const string GetScriptContents(string script) { // open script for reading FILE* inFile = fopen(script.c_str(), "rb"); if ( !inFile ) { cerr << "bamtools filter ERROR: could not open script: " << script << " for reading" << endl; return string(); } // read in entire script contents char buffer[1024]; ostringstream docStream(""); while ( true ) { // peek ahead, make sure there is data available char ch = fgetc(inFile); ungetc(ch, inFile); if( feof(inFile) ) break; // read next block of data if ( fgets(buffer, 1024, inFile) == 0 ) { cerr << "bamtools filter ERROR: could not read script contents" << endl; return string(); } docStream << buffer; } // close script file fclose(inFile); // import buffer contents to document, return return docStream.str(); } int main(int argc, char** argv) { std::string script = "/xchip/gistic/Jeremiah/GIT/SeqLib/test.json"; const string document = GetScriptContents(script); // set up JsonCPP reader and attempt to parse script Json::Value root; Json::Reader reader; if ( !reader.parse(document, root) ) { // use built-in error reporting mechanism to alert user what was wrong with the script cerr << "bamtools filter ERROR: failed to parse script - see error message(s) below" << endl; return false; } // see if root object contains multiple filters const Json::Value filters = root["filters"]; cerr << " root size " << root.size() << std::endl; cerr << " filters size " << filters.size() << std::endl; // iterate over any filters found int filterIndex = 0; Json::Value::const_iterator filtersIter = filters.begin(); Json::Value::const_iterator filtersEnd = filters.end(); for ( ; filtersIter != filtersEnd; ++filtersIter, ++filterIndex ) { Json::Value filter = (*filtersIter); // convert filter index to string string filterName; // if id tag supplied const Json::Value id = filter["id"]; if ( !id.isNull() ) filterName = id.asString(); // use array index else { stringstream convert; convert << filterIndex; filterName = convert.str(); } cerr << "filter " << filterName << std::endl; // create & parse filter bool success = true; success &= ParseFilterObject(filterName, filter); } /// make mini rules cerr << " ... maing mini rules " << endl; SeqLib::MiniRulesCollection mrc(script); } ================================================ FILE: src/svabautils/splitcounter.cpp ================================================ #include "splitcounter.h" #include #include #include #include #include "gzstream.h" #include "SnowmanUtils.h" #include "SeqLib/BamReader.h" #include "SeqLib/ReadFilter.h" #define MIN_CLIP_FACTOR 2 #define MIN_MATCH_FACTOR 4 #define MIN_MAPQ 10 #define READ_LEN 151 SeqLib::GRC bks; namespace opt { static std::string bam; static std::string breaks_file; static std::string header; static std::string fasta; static int split_num = 1000; } static const char* shortopts = "hb:B:H:f:n:o:"; static const struct option longopts[] = { { "help", no_argument, NULL, 'h' }, { "bam", required_argument, NULL, 'b' }, { "fasta", required_argument, NULL, 'f' }, { "breaks", required_argument, NULL, 'B' }, { "breaks-header", required_argument, NULL, 'H' }, { "num-bases", required_argument, NULL, 'n' }, { "out-fasta", required_argument, NULL, 'o' }, { NULL, 0, NULL, 0 } }; static const char *RUN_SPLITCOUNTER_MESSAGE = "Usage: snowman splitcounter [OPTION] -b BAM\n\n" " Description: Simple utility to count locations of split-alignment breakpoints (eg for PacBio)\n" "\n" " General options\n" " -h, --help Display this help and exit\n" " Required input\n" " -b, --bam BAM file containing split alignments to count\n" " Optional input\n" " -B, --breaks BED file containing regions to limit analysis to\n" " -H, --breaks-header BAM file to pair with the BED file (to get header from)\n" "\n"; static const char *RUN_FASTASPLIT_MESSAGE = "Usage: snowman fastasplit -f fasta -n num_bases > outfile.fa\n\n" " Description: Divide a fasta into smaller sub-sequences, splitting some seqs in the middle\n" "\n" " General options\n" " -h, --help Display this help and exit\n" " Required input\n" " -f, --fasta BAM file containing split alignments to count\n" " -n, --num-bases Number of bases to split into\n" "\n"; void parseFastaSplitOptions(int argc, char** argv); void runSplitFasta(int argc, char** argv) { parseFastaSplitOptions(argc, argv); std::cerr << " SPLITTING: " << opt::fasta << "\n" << " NUM: " << SeqLib::AddCommas(opt::split_num) << std::endl; // open the fasta igzstream infile(opt::fasta.c_str(), std::ios::in); // confirm that it is open if (!infile) { std::cerr << "Can't read file " << opt::fasta << " for parsing fasta" << std::endl; exit(EXIT_FAILURE); } int line_count = 0; // std::string line; std::string curr_line; size_t this_line_count = 0; // new line count while (std::getline(infile, line, '\n')) { ++this_line_count; if (this_line_count % 20000) std::cerr << "...on fasta file line (new-line separated) " << SeqLib::AddCommas(this_line_count) << std::endl; if (line.empty() || line.find(">") != std::string::npos) continue; // doesn't hit limit if ((int)(line.length() + curr_line.length()) < opt::split_num) curr_line = curr_line + line; // hits limit, but only if adding the new line else if ((int)line.length() <= opt::split_num) { std::cout << ">" << line_count << std::endl; ++line_count; std::cout << curr_line << std::endl; curr_line = line; } // the line itself is too big, split up else if ((int)line.length() > opt::split_num) { int lsize = line.size(); int lstart = 0; while (lstart < (int)line.size()) { std::cout << ">" << line_count << std::endl; ++line_count; std::cout << line.substr(lstart, std::min(opt::split_num, lsize - lstart)) << std::endl; lstart += opt::split_num; } } } } void runSplitCounter(int argc, char** argv) { parseSplitCounterOptions(argc, argv); // open the header BAM std::cerr << "...opening the header BAM " << opt::header << std::endl; SeqLib::BamReader bwh; assert(bwh.Open(opt::header)); // open the breaks std::cerr << "...reading " << opt::breaks_file << std::endl; SnowmanUtils::__open_bed(opt::breaks_file, bks, bwh.Header()); std::cerr << "...read in " << SeqLib::AddCommas(bks.size()) << " break regions " << std::endl; // open the BAM SeqLib::BamReader walk; assert(walk.Open(opt::bam)); SeqLib::BamRecord r; std::string rules_string = "{\"\" : {\"rules\" : [{\"mapq\" : 10}]}}"; std::cerr << "Rules: " << rules_string << std::endl; SeqLib::Filter::ReadFilterCollection mr(rules_string, walk.Header()); // convert qname (big) to in (small) std::unordered_map qname_set; size_t countr = 0; while(walk.GetNextRecord(r)) { if (r.ChrID() > 23) break; if (!mr.isValid(r)) continue; ++countr; SeqLib::Cigar cig = r.GetCigar(); if (cig.size() <= 1) continue; int matchlen = 0; for (auto& c : cig) if (c.Type() == 'M') matchlen += c.Length(); if (matchlen < MIN_MATCH_FACTOR * READ_LEN) continue; if (!qname_set.count(r.Qname())) qname_set[r.Qname()] = qname_set.size(); std::string QN = std::to_string(qname_set[r.Qname()]); if (countr % 100000 == 0 ) std::cerr << "...at split read " << SeqLib::AddCommas(countr) << " at position " << r.Brief() << std::endl; if ( (cig[0].Type() == 'H' || cig[0].Type() == 'S') && cig[0].Length() >= MIN_CLIP_FACTOR * READ_LEN) // split on left std::cout << "L\t" << r.ChrName(walk.Header()) << "\t" << r.Position() << "\t" << (r.ReverseFlag() ? "-" : "+") << "\t" << QN << "\t" << r.MapQuality() << "\t" << (r.SecondaryFlag() ? "SEC" : "PRI") << "\t" << (bks.CountOverlaps(r.asGenomicRegion()) ? "BK" : "NBK") << "\t0" << std::endl; if ( (cig.back().Type() == 'H' || cig.back().Type() == 'S') && cig.back().Length() >= MIN_CLIP_FACTOR * READ_LEN) // split on right std::cout << "R\t" << r.ChrName(walk.Header()) << "\t" << r.PositionEnd() << "\t" << (r.ReverseFlag() ? "-" : "+") << "\t" << QN << "\t" << r.MapQuality() << "\t" << (r.SecondaryFlag() ? "SEC" : "PRI") << "\t" << (bks.CountOverlaps(r.asGenomicRegion()) ? "BK" : "NBK") << "\t0" << std::endl; // look for indels int pos = r.Position(); for (auto& c : cig) { if ((c.Type() == 'D' || c.Type() == 'I') && c.Length() >= 30) std::cout << c.Type() << "\t" << r.ChrName(walk.Header()) << "\t" << pos << "\t+\t" << QN << "\t" << r.MapQuality() << "\t" << (r.SecondaryFlag() ? "SEC" : "PRI") << "\t" << (bks.CountOverlaps(r.asGenomicRegion()) ? "BK" : "NBK") << "\t" << c.Length() << std::endl; if (c.ConsumesReference()) pos += c.Length(); } // try BLAT realignment //SeqLib::BamReadVector brv; //blat.querySequence(r.Qname(), r.Sequence(), brv); //for(auto& a : brv) // w.writeAlignment(a); } std::cerr << "...done" << std::endl; } void parseSplitCounterOptions(int argc, char** argv) { bool die = false; if (argc <= 2) die = true; bool help = false; std::stringstream ss; std::string tmp; for (char c; (c = getopt_long(argc, argv, shortopts, longopts, NULL)) != -1;) { std::istringstream arg(optarg != NULL ? optarg : ""); switch (c) { case 'b': arg >> opt::bam; break; case 'B': arg >> opt::breaks_file; break; case 'H': arg >> opt::header; break; case 'h': help = true; break; } } if (die || help) { std::cerr << "\n" << RUN_SPLITCOUNTER_MESSAGE; die ? exit(EXIT_FAILURE) : exit(EXIT_SUCCESS); } } void parseFastaSplitOptions(int argc, char** argv) { bool die = false; if (argc <= 2) die = true; bool help = false; std::stringstream ss; std::string tmp; for (char c; (c = getopt_long(argc, argv, shortopts, longopts, NULL)) != -1;) { std::istringstream arg(optarg != NULL ? optarg : ""); switch (c) { case 'f': arg >> opt::fasta; break; case 'n': arg >> opt::split_num; break; case 'h': help = true; break; } } if (die || help) { std::cerr << "\n" << RUN_FASTASPLIT_MESSAGE; die ? exit(EXIT_FAILURE) : exit(EXIT_SUCCESS); } } ================================================ FILE: src/svabautils/splitcounter.h ================================================ #ifndef SNOWMAN_SPLITCOUNTER_H__ #define SNOWMAN_SPLITCOUNTER_H__ void runSplitCounter(int argc,char** argv); void parseSplitCounterOptions(int argc, char** argv); void runSplitFasta(int argc, char** argv); #endif ================================================ FILE: svaba_jemalloc ================================================ #!/usr/bin/env bash # # svaba_jemalloc — run svaba with jemalloc forced as the allocator via # LD_PRELOAD. Recommended for Linux workloads at high thread counts # (~12+ threads): jemalloc's thread-local arenas relieve lock # contention the glibc allocator suffers under heavy malloc/free # traffic from the per-thread assembly pipeline. Typical wall-time # win: 10–20 % at -p 16 on a WGS region; less at low -p, sometimes a # wash under -p 4. # # *Do not use this on macOS.* Apple's libmalloc (nanomalloc fast path) # plus DYLD_INSERT_LIBRARIES overhead has empirically run 5–10× SLOWER # than the default allocator on the same BAM. Stick with system malloc # there. # # Usage: # svaba_jemalloc run -t tumor.bam -n normal.bam -G ref.fa ... # # or any other svaba subcommand — args are passed through. # # Optional environment overrides: # JEMALLOC_LIB=/path/to/libjemalloc.so.2 # skip auto-detection # SVABA=/path/to/svaba # skip $PATH lookup # # MALLOC_CONF is passed through if the caller sets it; a reasonable # default for high-concurrency svaba runs is: # MALLOC_CONF=background_thread:true,narenas:24,dirty_decay_ms:10000 # Tune `narenas` to match -p if you go above 24 threads. set -euo pipefail # Refuse to run on macOS — jemalloc loses badly there. case "$(uname -s)" in Darwin) echo "svaba_jemalloc: Linux only. On macOS, Apple's libmalloc is" >&2 echo " faster than jemalloc for this workload." >&2 echo " Just run svaba directly." >&2 exit 1 ;; esac # 1) Resolve libjemalloc path. JEMALLOC_LIB=${JEMALLOC_LIB:-} if [[ -z "$JEMALLOC_LIB" ]]; then for cand in \ /usr/lib/x86_64-linux-gnu/libjemalloc.so.2 \ /usr/lib64/libjemalloc.so.2 \ /usr/lib/libjemalloc.so.2 \ /usr/local/lib/libjemalloc.so.2 \ /opt/homebrew/lib/libjemalloc.so.2 do if [[ -f "$cand" ]]; then JEMALLOC_LIB=$cand; break; fi done fi if [[ -z "$JEMALLOC_LIB" ]]; then # last-ditch: ask ldconfig for a cached match JEMALLOC_LIB=$(ldconfig -p 2>/dev/null | awk '/libjemalloc\.so\.2/ {print $NF; exit}') fi if [[ -z "$JEMALLOC_LIB" || ! -f "$JEMALLOC_LIB" ]]; then echo "svaba_jemalloc: could not find libjemalloc.so.2." >&2 echo " Install it (e.g. 'apt install libjemalloc2' or 'yum install jemalloc')" >&2 echo " or set JEMALLOC_LIB=/path/to/libjemalloc.so.2." >&2 exit 1 fi # 2) Resolve svaba binary. SVABA=${SVABA:-} if [[ -z "$SVABA" ]]; then # Prefer $PATH; fall back to a build/svaba next to this script. if command -v svaba >/dev/null 2>&1; then SVABA=$(command -v svaba) else here=$(cd "$(dirname "${BASH_SOURCE[0]}")" && pwd) for cand in "$here/build/svaba" "$here/bin/svaba"; do if [[ -x "$cand" ]]; then SVABA=$cand; break; fi done fi fi if [[ -z "$SVABA" || ! -x "$SVABA" ]]; then echo "svaba_jemalloc: could not find the svaba binary." >&2 echo " Put svaba on \$PATH, or set SVABA=/path/to/svaba." >&2 exit 1 fi exec env LD_PRELOAD="$JEMALLOC_LIB" "$SVABA" "$@" ================================================ FILE: tracks/README.md ================================================ # svaba tracks/ This directory holds the BED files consumed by `svaba run --blacklist` (and by the scripts that build the combined blacklist from component files). Everything here is a regeneratable artifact — don't hand-edit `hg38.combined_blacklist.bed` or `lowmap*perc.bed`; edit the upstream component or re-run the generator script. ## `hg38.combined_blacklist.bed` — the master blacklist This is the file you point svaba at. It is the union of several component blacklists, produced by `scripts/combine_blacklists.sh` from the components in this same directory. The three primary content sources are the manual blacklist (`hg38.manual.blacklist.bed`), empirically-slow-to-assemble regions (`hg38.high_runtime.bed`), and UCSC RepeatMasker simple repeats (`hg38.rmsk.simple_repeat.bed`). The non-standard-contig file (`hg38.nonstd_chr.blacklist.bed`) and the ENCODE-style high-signal blacklist (`hg38.blacklist.sorted.bed`) are typically folded in as well. To refresh the combined file after editing any component: ```bash ./scripts/combine_blacklists.sh --merge --clip tracks/chr \ -o tracks/hg38.combined_blacklist.bed \ tracks/hg38.manual.blacklist.bed \ tracks/hg38.high_runtime.bed \ tracks/hg38.rmsk.simple_repeat.bed ``` `--merge` runs `bedtools merge` to collapse overlapping intervals, with a comma-separated list of contributing source tags in col 4. `--clip` clips any oversize-end sentinels (e.g. synthetic `end=250000000` rows in some BED files) to real contig lengths. See the header comment in `combine_blacklists.sh` for the full flag surface. ## `lowmap{30,50}perc.bed` — low-mappability blacklists These are blacklists of regions where the vast majority of reads at a locus fail a mapping-quality threshold — i.e. the locus is effectively "unmapped" for any caller that only trusts uniquely-placed reads. They're derived from paired `mosdepth` runs over known-non-tumor BAMs using `scripts/mosdepth_lowmapq_blacklist.sh`, then intersected across two independent non-tumor samples so only regions confirmed as low- mappability in both samples survive. The number in the filename is the ratio cutoff used: `lowmap30perc.bed` keeps bins where fewer than 30% of reads pass MAPQ (stricter), and `lowmap50perc.bed` keeps bins below 50% (the default). The generation recipe has three steps per sample, plus a final intersection. ### Step 1 — mosdepth coverage per sample, unfiltered vs MAPQ-filtered Run mosdepth twice per BAM, differing only in `--mapq`. The unfiltered pass (`--mapq 0`) counts every alignment; the filtered pass (`--mapq 20`) counts only uniquely-placed reads. `--by 300` bins the coverage into 300 bp windows, which is finer than the svaba blacklist's natural resolution and gives us room to merge later. ```bash mosdepth --by 300 --fast-mode --mapq 0 unf_T0 NP_WGS_Normal_DNA.recal.bam mosdepth --by 300 --fast-mode --mapq 20 filt_T0 NP_WGS_Normal_DNA.recal.bam mosdepth --by 300 --fast-mode --mapq 0 unf_T2 .recal.bam mosdepth --by 300 --fast-mode --mapq 20 filt_T2 .recal.bam ``` Each mosdepth invocation produces a `.regions.bed.gz` with `chrom, start, end, mean_depth` columns. ### Step 2 — per-sample ratio + flagging `mosdepth_lowmapq_blacklist.sh` joins the two mosdepth outputs bin by bin, computes `filt/unf` (the fraction of reads that pass MAPQ), and flags bins below a threshold where `unf` is high enough to be statistically meaningful. `--max-ratio` controls strictness: `0.5` means "flag bins where fewer than half of reads pass MAPQ" (default), `0.3` is stricter, `0.7` is looser. ```bash ~/git/svaba/scripts/mosdepth_lowmapq_blacklist.sh --max-ratio 0.5 --out lowmap0.5_T0 unf_T0.regions.bed.gz filt_T0.regions.bed.gz ~/git/svaba/scripts/mosdepth_lowmapq_blacklist.sh --max-ratio 0.5 --out lowmap0.5_T2 unf_T2.regions.bed.gz filt_T2.regions.bed.gz ~/git/svaba/scripts/mosdepth_lowmapq_blacklist.sh --max-ratio 0.7 --out lowmap0.7_T2 unf_T2.regions.bed.gz filt_T2.regions.bed.gz ``` Each run emits `.flagged.bed` (raw per-bin flags), `.flagged.merged.bed` (intervals merged within 1 kb), and a `.summary.txt` with totals. ### Step 3 — cross-sample intersection The per-sample `lowmap0.Xp_T*.flagged.merged.bed` files are noisier than we want as a blacklist input. Taking the `bedtools intersect` of two independent non-tumor samples gives a more conservative result: only regions flagged as low-mappability in BOTH samples survive. ```bash bedtools intersect -a lowmap0.5_T0.flagged.merged.bed -b lowmap0.5_T2.flagged.merged.bed > lowmap50perc.bed bedtools intersect -a lowmap0.3_T0.flagged.merged.bed -b lowmap0.3_T2.flagged.merged.bed > lowmap30perc.bed ``` Both samples must be non-tumor — tumor BAMs have structural variation that can pile up multi-mappers at real break sites, which we don't want mistaken for low-mappability and blacklisted. Intersecting two independent non-tumor samples gets you a much smaller (and more trustworthy) blacklist than either sample's raw output. Per the v3 mosdepth helper's summary: typical single-sample flagged volume on hg38 WGS is 200-400 Mb; after intersection with a second non-tumor sample, the `lowmap50perc.bed` settles in the ~30-50 Mb range, and `lowmap30perc.bed` shrinks further. ## Other component files `hg38.blacklist.sorted.bed` is the ENCODE-style high-signal blacklist. `hg38.high_runtime.bed` is a list of regions empirically slow to assemble; curated from svaba runtime.txt tails. `hg38.manual.blacklist.bed` is the curated ad-hoc bad-list. `hg38.nonstd_chr.blacklist.bed` is full-contig entries for every chrUn/*_decoy/*_alt/*_random/chrEBV/HLA-* contig in the reference (regenerated from `tracks/chr` which is a GRCh38 fasta-header dump). `hg38.rmsk.simple_repeat.bed` is UCSC RepeatMasker simple-repeat regions. `genome.hg38.sorted.bed`, `hg38.bed`, and `hg38_arms_excl_centromeres.bed` are reference-coordinate helpers used by downstream analysis scripts, not by `svaba run` directly. `region_generator.R` is an R script used to regenerate some of the above. ## Adding a new component to the combined blacklist Put the new `.bed` file in this directory, then re-run `combine_blacklists.sh` with it added to the argument list. The script tolerates any well-formed 3-column BED. If the new file has a 4th column with per-interval labels, those get preserved in the merged output's label column. ================================================ FILE: tracks/genome.hg38.sorted.bed ================================================ chr1 0 248956422 chr10 0 133797422 chr11 0 135086622 chr12 0 133275309 chr13 0 114364328 chr14 0 107043718 chr15 0 101991189 chr16 0 90338345 chr17 0 83257441 chr18 0 80373285 chr19 0 58617616 chr2 0 32865900 chr2 32918400 242193529 chr20 0 64444167 chr21 0 46709983 chr22 0 50818468 chr3 0 198295559 chr4 0 190214555 chr5 0 181538259 chr6 0 170805979 chr7 0 159345973 chr8 0 145138636 chr9 0 138394717 chrX 0 156040895 chrY 0 57227415 ================================================ FILE: tracks/hg38.bed ================================================ chr1 0 248956422 chr2 0 32928000 chr2 32953000 242193529 chr3 0 198295559 chr4 0 190214555 chr5 0 181538259 chr6 0 170805979 chr7 0 159345973 chr8 0 145138636 chr9 0 138394717 chr10 0 133797422 chr11 0 135086622 chr12 0 133275309 chr13 0 114364328 chr14 0 107043718 chr15 0 101991189 chr16 0 90338345 chr17 0 83257441 chr18 0 80373285 chr19 0 58617616 chr20 0 64444167 chr21 0 46709983 chr22 0 50818468 chrX 0 156040895 ================================================ FILE: tracks/hg38.blacklist.sorted.bed ================================================ chr1 0 792500 High Signal Region chr1 91386300 91388400 Low Mappability chr1 103594400 103760600 High Signal Region chr1 121605200 124938900 High Signal Region chr1 125067600 125086000 High Signal Region chr1 125130200 143562200 High Signal Region chr1 161423100 161472400 High Signal Region chr1 168348600 168349900 High Signal Region chr1 224010800 224017000 High Signal Region chr1 230872006 230872168 High Signal Region chr1 236713000 236715600 Low Mappability chr1 248932700 248956400 High Signal Region chr10 0 45700 Low Mappability chr10 38481300 38596500 High Signal Region chr10 38782600 38967900 High Signal Region chr10 39901300 41712900 High Signal Region chr10 41838900 42107300 High Signal Region chr10 42279400 42322500 High Signal Region chr10 126946300 126953400 Low Mappability chr10 133625800 133797400 High Signal Region chr11 0 194500 Low Mappability chr11 518900 520700 Low Mappability chr11 584400 586500 High Signal Region chr11 964100 966000 Low Mappability chr11 1015700 1019300 High Signal Region chr11 1091000 1098200 Low Mappability chr11 3652800 3655600 High Signal Region chr11 10506900 10511100 High Signal Region chr11 28206300 28236700 High Signal Region chr11 50813600 54383000 High Signal Region chr11 61084500 61130400 High Signal Region chr11 70370400 70372400 High Signal Region chr11 73509800 73511700 High Signal Region chr11 77885600 77887600 High Signal Region chr11 93417500 93427700 High Signal Region chr11 94232700 94240400 High Signal Region chr11 103408700 103410600 High Signal Region chr11 121175000 121187000 High Signal Region chr11 131679500 131681500 High Signal Region chr11 135075600 135086600 High Signal Region chr12 0 77800 High Signal Region chr12 371800 422400 High Signal Region chr12 2254900 2257000 High Signal Region chr12 2519800 2540500 Low Mappability chr12 5928900 5933000 Low Mappability chr12 20550500 20552400 Low Mappability chr12 20768400 20770300 High Signal Region chr12 29790400 29834600 High Signal Region chr12 34715400 37269100 High Signal Region chr12 41362700 41364600 High Signal Region chr12 61471100 61473000 High Signal Region chr12 66473900 66475800 High Signal Region chr12 101147000 101155000 High Signal Region chr12 113079600 113081500 High Signal Region chr12 124430500 124440300 High Signal Region chr12 124905900 124941800 High Signal Region chr12 130386400 130394100 High Signal Region chr12 131475300 131478600 High Signal Region chr12 131576000 131589700 High Signal Region chr12 132223300 132243400 High Signal Region chr12 132455100 132465200 High Signal Region chr12 133249000 133275300 High Signal Region chr13 16087600 16165300 High Signal Region chr13 16226300 18171400 High Signal Region chr13 18211000 18216100 High Signal Region chr13 57140500 57172500 High Signal Region chr13 109423200 109425200 High Signal Region chr13 114353300 114364300 Low Mappability chr14 0 18670900 High Signal Region chr14 18695400 19724300 High Signal Region chr14 23033300 23098600 High Signal Region chr14 26629300 26634900 High Signal Region chr14 31793800 31798100 High Signal Region chr14 32483400 32486000 High Signal Region chr14 34537100 34562600 High Signal Region chr14 35947200 35950000 High Signal Region chr14 37351000 37356700 High Signal Region chr14 44025100 44027200 High Signal Region chr14 44705100 44709900 High Signal Region chr14 45477100 45482500 High Signal Region chr14 46865300 46866500 High Signal Region chr14 54235600 54240000 High Signal Region chr14 57112100 57118100 High Signal Region chr14 74711700 74729000 High Signal Region chr14 86074000 86076000 High Signal Region chr14 86593300 86595200 High Signal 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65699300 High Signal Region chr3 66273800 66275200 High Signal Region chr3 68076400 68077800 High Signal Region chr3 69047300 69053600 High Signal Region chr3 69475300 69479700 High Signal Region chr3 75630100 75707800 High Signal Region chr3 75736400 75754600 High Signal Region chr3 78948800 78950500 High Signal Region chr3 80876000 80894000 High Signal Region chr3 89345600 89370500 High Signal Region chr3 90156400 90175500 High Signal Region chr3 90455400 91297100 High Signal Region chr3 91516200 93749200 High Signal Region chr3 96616300 96619300 High Signal Region chr3 97905100 97923200 High Signal Region chr3 101674800 101698400 High Signal Region chr3 103224300 103236500 High Signal Region chr3 106665700 106669700 High Signal Region chr3 106975900 106979600 High Signal Region chr3 108751100 108755100 High Signal Region chr3 111019500 111024600 High Signal Region chr3 121933800 121936400 High Signal Region chr3 122414300 122417500 High Signal Region chr3 122735500 122796600 High Signal Region chr3 122837000 122838700 High Signal Region chr3 133177100 133179800 High Signal Region chr3 133551500 133579500 High Signal Region chr3 135437200 135439100 High Signal Region chr3 136954600 136969200 High Signal Region chr3 137168400 137169900 High Signal Region chr3 138575800 138595900 High Signal Region chr3 139190800 139194700 High Signal Region chr3 153236200 153241300 High Signal Region chr3 155544100 155546700 High Signal Region chr3 156279000 156283500 High Signal Region chr3 157080800 157093400 High Signal Region chr3 158511300 158513100 High Signal Region chr3 160941200 160948700 High Signal Region chr3 161001900 161014100 High Signal Region chr3 165573100 165591000 High Signal Region chr3 166228200 166232400 High Signal Region chr3 168012100 168016800 High Signal Region chr3 170567000 170569900 High Signal Region chr3 170864300 170881400 High Signal Region chr3 171626600 171637700 High Signal Region chr3 174829200 174831800 High Signal Region chr3 176828700 176833000 High Signal Region chr3 177660600 177664000 High Signal Region chr3 178926800 178941300 High Signal Region chr3 183016900 183019100 High Signal Region chr3 183955400 183958700 High Signal Region chr3 187893900 187896100 High Signal Region chr3 192739300 192742700 High Signal Region chr3 194323600 194334900 High Signal Region chr3 195477900 195507300 High Signal Region chr3 195616000 195750100 High Signal Region chr3 195775500 195791400 High Signal Region chr3 195914100 196028300 High Signal Region chr3 196249400 196251900 High Signal Region chr3 196897800 196899800 High Signal Region chr3 197030600 197035800 High Signal Region chr3 197383400 197428800 High Signal Region chr3 197454700 197460800 High Signal Region chr3 197598400 197680900 High Signal Region chr3 198099800 198295500 High Signal Region chr4 0 69200 High Signal Region chr4 554100 556500 High Signal Region chr4 1427000 1468900 High Signal Region chr4 6002700 6005700 High Signal Region chr4 7863000 7865000 High Signal Region chr4 9212700 9369600 High Signal Region chr4 40291700 40318200 High Signal Region chr4 49077200 51816100 High Signal Region chr4 55327200 55329200 High Signal Region chr4 77994000 78009600 High Signal Region chr4 119274400 119301700 High Signal Region chr4 146285100 146305300 High Signal Region chr4 162420500 162422400 High Signal Region chr4 166554300 166581300 Low Mappability chr4 181238800 181242300 Low Mappability chr4 189232500 189236300 High Signal Region chr4 189834900 189849700 High Signal Region chr4 189877500 190023700 High Signal Region chr4 190048600 190214500 High Signal Region chr5 0 44100 High Signal Region chr5 548300 564100 High Signal Region chr5 647600 651700 High Signal Region chr5 1326100 1334600 High Signal Region chr5 2144600 2147800 High Signal Region chr5 2489800 2491700 High Signal Region chr5 3322100 3325100 High Signal Region chr5 6967700 6971700 High Signal Region chr5 17516800 17600200 High Signal Region chr5 21477600 21497600 High Signal Region chr5 25381400 25384300 High Signal Region chr5 34177900 34244800 High Signal Region chr5 45522900 45525200 High Signal Region chr5 45743000 45744800 High Signal Region chr5 46433900 46687700 High Signal Region chr5 46708100 50165300 High Signal Region chr5 60759700 60762500 High Signal Region chr5 63320900 63335500 High Signal Region chr5 69540700 71359500 High Signal Region chr5 71850000 71852800 High Signal Region chr5 74685400 74712400 High Signal Region chr5 78452400 78457600 High Signal Region chr5 78848400 78872800 High Signal Region chr5 80649100 80653100 High Signal Region chr5 85641800 85662700 High Signal Region chr5 93947500 93948900 High Signal Region chr5 94567100 94570400 High Signal Region chr5 100045500 100076300 High Signal Region chr5 106425500 106429500 High Signal Region chr5 109259500 109265400 High Signal Region chr5 111302100 111308300 High Signal Region chr5 114156700 114158300 High Signal Region chr5 119904000 119905600 High Signal Region chr5 123760300 123762200 High Signal Region chr5 134922500 134929400 High Signal Region chr5 139005500 139011600 High Signal Region chr5 146610000 146615500 High Signal Region chr5 153071100 153077000 High Signal Region chr5 156658300 156665400 High Signal Region chr5 161606000 161611700 High Signal Region chr5 171083900 171090200 High Signal Region chr5 175904500 176118000 High Signal Region chr5 176590700 176593000 High Signal Region chr5 177636700 177684700 High Signal Region chr5 177960500 177981400 High Signal Region chr5 178584600 178586600 High Signal Region chr5 181172600 181538200 High Signal Region chr6 256500 382800 High Signal Region chr6 861500 864200 High Signal Region chr6 1052800 1054800 High Signal Region chr6 26669200 26832300 High Signal Region chr6 33484600 33486400 High Signal Region chr6 34070600 34074000 High Signal Region chr6 38262000 38301600 High Signal Region chr6 39455800 39460500 High Signal Region chr6 44043600 44080000 High Signal Region chr6 44180600 44182900 High Signal Region chr6 51874900 51901300 High Signal Region chr6 54961900 54967400 High Signal Region chr6 58432200 60242300 High Signal Region chr6 61321800 61493000 High Signal Region chr6 61573200 61575100 Low Mappability chr6 61661900 61673400 High Signal Region chr6 103709000 103715100 High Signal Region chr6 115254900 115256800 High Signal Region chr6 143799900 143801800 High Signal Region chr6 156035300 156040100 High Signal Region chr6 157309500 157324800 High Signal Region chr6 160611700 160647400 High Signal Region chr6 170145300 170147200 High Signal Region chr6 170376900 170401000 High Signal Region chr6 170465400 170468400 High Signal Region chr7 0 49600 High Signal Region chr7 224500 241300 High Signal Region chr7 904700 907100 Low Mappability chr7 1271400 1273500 High Signal Region chr7 45251000 45253000 High Signal Region chr7 56369500 56375600 High Signal Region chr7 57485300 57497800 High Signal Region chr7 57611600 57637700 Low Mappability chr7 58031800 60997400 High Signal Region chr7 61017800 61075200 High Signal Region chr7 61102900 61725200 Low Mappability chr7 62265700 62409500 High Signal Region chr7 62430000 62520600 High Signal Region chr7 65488000 65496500 High Signal Region chr7 100951400 100968300 High Signal Region chr7 100991500 101004600 High Signal Region chr7 102474700 102686400 High Signal Region chr7 142665100 142668500 High Signal Region chr7 144180800 144377300 Low Mappability chr7 145996400 146018600 High Signal Region chr7 152375800 152435100 High Signal Region chr7 158131400 158156200 High Signal Region chr7 158594300 158596200 High Signal Region chr7 158893100 158918100 High Signal Region chr7 159334900 159345900 High Signal Region chr8 7209800 7914700 High Signal Region chr8 7940500 8075700 High Signal Region chr8 8128200 8204600 High Signal Region chr8 12136900 12614300 High Signal Region chr8 43236700 43262600 High Signal Region chr8 43937900 45969600 High Signal Region chr8 46829400 46832000 High Signal Region chr8 57204900 57216100 High Signal Region chr8 59168700 59170400 High Signal Region chr8 67584500 67592700 High Signal Region chr8 69688400 69691100 High Signal Region chr8 71406700 71412400 High Signal Region chr8 75444100 75448200 High Signal Region chr8 81841500 81851900 High Signal Region chr8 85642100 85829300 High Signal Region chr8 88685900 88691700 High Signal Region chr8 96171200 96173100 High Signal Region chr8 99494900 99496800 High Signal Region chr8 105789200 105793800 High Signal Region chr8 141491400 141493500 High Signal Region chr8 141871100 141875200 High Signal Region chr8 143641400 143670500 High Signal Region chr8 144124800 144137600 High Signal Region chr9 319900 322400 High Signal Region chr9 33656600 33660000 High Signal Region chr9 35912600 35915300 High Signal Region chr9 38824200 39089400 High Signal Region chr9 39846200 40771100 High Signal Region chr9 40792500 41323100 High Signal Region chr9 41492300 41635600 High Signal Region chr9 41661300 42119600 Low Mappability chr9 42364000 42410600 High Signal Region chr9 42899400 42901300 High Signal Region chr9 43263100 61518900 High Signal Region chr9 61735300 63548000 High Signal Region chr9 63761400 64027300 High Signal Region chr9 64135000 65390600 High Signal Region chr9 65579400 66874600 High Signal Region chr9 66959000 68398100 High Signal Region chr9 70037200 70039600 High Signal Region chr9 76174300 76176200 High Signal Region chr9 83222900 83226900 High Signal Region chr9 85071600 85075100 High Signal Region chr9 85164800 85166100 High Signal Region chr9 108502000 108506600 High Signal Region chr9 134164500 134185500 High Signal Region chr9 137326800 137330600 High Signal Region chr9 137715200 137722200 Low Mappability chr9 137841200 137846800 Low Mappability chr9 138222000 138394700 High Signal Region chrX 0 329300 High Signal Region chrX 362400 388500 High Signal Region chrX 456500 531800 High Signal Region chrX 723800 739500 High Signal Region chrX 864500 930400 High Signal Region chrX 1049100 1054300 High Signal Region chrX 1085100 1175500 High Signal Region chrX 1200600 1209400 High Signal Region chrX 1249200 1269000 High Signal Region chrX 1289500 1298900 High Signal Region chrX 1365300 1458700 High Signal Region chrX 1480900 1492800 High Signal Region chrX 1816200 1820600 High Signal Region chrX 2223900 2521900 High Signal Region chrX 2580600 2751300 High Signal Region chrX 3966700 3968700 High Signal Region chrX 5481200 5486100 High Signal Region chrX 6933400 6938700 High Signal Region chrX 7587600 7591800 High Signal Region chrX 9403600 9415100 High Signal Region chrX 10785000 10809700 High Signal Region chrX 10966600 10976800 High Signal Region chrX 11218800 11221100 Low Mappability chrX 11840900 11848000 High Signal Region chrX 14085100 14109500 High Signal Region chrX 14286500 14289300 High Signal Region chrX 16361200 16366000 High Signal Region chrX 16498100 16503400 High Signal Region chrX 19940200 19946300 High Signal Region chrX 21340600 21345700 High Signal Region chrX 25773300 25776000 High Signal Region chrX 26176400 26181400 High Signal Region chrX 30767800 30772600 High Signal Region chrX 31077600 31082600 High Signal Region chrX 31511400 31535800 High Signal Region chrX 34416800 34425900 High Signal Region chrX 36465200 36471200 High Signal Region chrX 37628400 37633500 High Signal Region chrX 42872300 42910700 High Signal Region chrX 49317500 49623500 High Signal Region chrX 50019400 50033700 High Signal Region chrX 50056700 50066100 High Signal Region chrX 51202300 51268100 High Signal Region chrX 51427500 51432400 High Signal Region chrX 52175000 52228100 High Signal Region chrX 52442800 52538100 High Signal Region chrX 53761700 53789500 High Signal Region chrX 55180400 55184500 High Signal Region chrX 56754900 56781100 Low Mappability chrX 57712300 57719700 High Signal Region chrX 58467900 62522800 High Signal Region chrX 63129600 63290600 Low Mappability chrX 67311800 67323800 High Signal Region chrX 67626800 67632300 High Signal Region chrX 68217300 68230200 High Signal Region chrX 70600000 70603800 High Signal Region chrX 70640600 70645000 High Signal Region chrX 70963600 70964900 High Signal Region chrX 71978800 71980500 High Signal Region chrX 72489400 72490800 High Signal Region chrX 72743200 73035800 High Signal Region chrX 73381000 73387000 High Signal Region chrX 73887000 73891300 High Signal Region chrX 74660000 74718100 High Signal Region chrX 74789000 74794000 High Signal Region chrX 74952200 74995200 High Signal Region chrX 78802400 78804500 High Signal Region chrX 79765500 79789600 High Signal Region chrX 80534100 80537000 High Signal Region chrX 82849700 82859300 Low Mappability chrX 83752100 83756900 High Signal Region chrX 86046600 86076600 High Signal Region chrX 86395500 86398100 High Signal Region chrX 86970000 86975600 High Signal Region chrX 87220500 87222100 High Signal Region chrX 89060200 89062700 High Signal Region chrX 89202500 89208400 High Signal Region chrX 91332900 91336600 High Signal Region chrX 93618000 93633400 High Signal Region chrX 94863600 94868300 High Signal Region chrX 97509600 97515000 High Signal Region chrX 100135800 100141000 High Signal Region chrX 100257100 100261600 High Signal Region chrX 101471700 101474900 High Signal Region chrX 102188700 102489200 High Signal Region chrX 103851800 103897800 High Signal Region chrX 106755500 106769400 High Signal Region chrX 106813900 106830900 High Signal Region chrX 107515800 107517200 High Signal Region chrX 109034800 109069100 High Signal Region chrX 109114900 109119400 High Signal Region chrX 109520800 109525700 High Signal Region chrX 109985900 109987300 High Signal Region chrX 110816700 110833400 High Signal Region chrX 111416100 111418000 High Signal Region chrX 113141700 113143600 High Signal Region chrX 114701600 114724300 High Signal Region chrX 115725600 115889600 High Signal Region chrX 116557600 116595600 High Signal Region chrX 117874100 117880000 High Signal Region chrX 118009000 118037800 High Signal Region chrX 118070900 118072700 High Signal Region chrX 121263700 121268100 High Signal Region chrX 121299200 121300600 High Signal Region chrX 122528400 122550000 High Signal Region chrX 124584300 124588400 High Signal Region chrX 125927600 125937100 High Signal Region chrX 126463700 126474200 High Signal Region chrX 127116700 127122600 High Signal Region chrX 127362200 127368300 High Signal Region chrX 128785000 128788700 High Signal Region chrX 129337600 129357900 High Signal Region chrX 129388400 129408400 High Signal Region chrX 130567700 130572000 High Signal Region chrX 131152200 131157400 High Signal Region chrX 131378300 131383300 High Signal Region chrX 131664300 131670000 High Signal Region chrX 132284600 132320400 High Signal Region chrX 133108600 133116500 High Signal Region chrX 135718600 135888700 High Signal Region chrX 137074700 137079100 High Signal Region chrX 137436600 137439300 High Signal Region chrX 138300600 138302200 High Signal Region chrX 139437600 139446800 High Signal Region chrX 139621500 139622800 High Signal Region chrX 140722400 140726100 High Signal Region chrX 141000400 141108300 High Signal Region chrX 142478000 142483800 High Signal Region chrX 142892300 142911600 High Signal Region chrX 143352000 143356500 High Signal Region chrX 144404500 144475900 Low Mappability chrX 147281700 147287100 High Signal Region chrX 147653800 147659900 High Signal Region chrX 148123500 148129000 High Signal Region chrX 148347100 148378700 High Signal Region chrX 149437900 149441900 High Signal Region chrX 150024800 150026200 High Signal Region chrX 152173800 152175100 High Signal Region chrX 153251200 153316400 High Signal Region chrX 154870000 154890200 High Signal Region chrX 154938900 154945100 High Signal Region chrX 155299600 155305100 High Signal Region chrX 155454000 155522000 High Signal Region chrX 155700400 155727500 High Signal Region chrX 155983500 156040800 High Signal Region chrY 4343800 4345800 High Signal Region chrY 10246200 11041200 High Signal Region chrY 11072100 11335300 High Signal Region chrY 11486600 11757800 High Signal Region chrY 26637300 57227400 High Signal Region chr1 230872006 230872168 High Signal Region chr1 230872009 230872119 chr2 32928000 32953000 ================================================ FILE: tracks/hg38.nonstd_chr.blacklist.bed ================================================ chr1_KI270706v1_random 0 175055 Unplaced random chr1_KI270707v1_random 0 32032 Unplaced random chr1_KI270708v1_random 0 127682 Unplaced random chr1_KI270709v1_random 0 66860 Unplaced random chr1_KI270710v1_random 0 40176 Unplaced random chr1_KI270711v1_random 0 42210 Unplaced random chr1_KI270712v1_random 0 176043 Unplaced random chr1_KI270713v1_random 0 40745 Unplaced random chr1_KI270714v1_random 0 41717 Unplaced random chr2_KI270715v1_random 0 161471 Unplaced random chr2_KI270716v1_random 0 153799 Unplaced random chr3_GL000221v1_random 0 155397 Unplaced random chr4_GL000008v2_random 0 209709 Unplaced random chr5_GL000208v1_random 0 92689 Unplaced random chr9_KI270717v1_random 0 40062 Unplaced random chr9_KI270718v1_random 0 38054 Unplaced random chr9_KI270719v1_random 0 176845 Unplaced random chr9_KI270720v1_random 0 39050 Unplaced random chr11_KI270721v1_random 0 100316 Unplaced random chr14_GL000009v2_random 0 201709 Unplaced random chr14_GL000225v1_random 0 211173 Unplaced random chr14_KI270722v1_random 0 194050 Unplaced random chr14_GL000194v1_random 0 191469 Unplaced random chr14_KI270723v1_random 0 38115 Unplaced random chr14_KI270724v1_random 0 39555 Unplaced random chr14_KI270725v1_random 0 172810 Unplaced random chr14_KI270726v1_random 0 43739 Unplaced random chr15_KI270727v1_random 0 448248 Unplaced random chr16_KI270728v1_random 0 1872759 Unplaced random chr17_GL000205v2_random 0 185591 Unplaced random chr17_KI270729v1_random 0 280839 Unplaced random chr17_KI270730v1_random 0 112551 Unplaced random chr22_KI270731v1_random 0 150754 Unplaced random chr22_KI270732v1_random 0 41543 Unplaced random chr22_KI270733v1_random 0 179772 Unplaced random chr22_KI270734v1_random 0 165050 Unplaced random chr22_KI270735v1_random 0 42811 Unplaced random chr22_KI270736v1_random 0 181920 Unplaced random chr22_KI270737v1_random 0 103838 Unplaced random chr22_KI270738v1_random 0 99375 Unplaced random chr22_KI270739v1_random 0 73985 Unplaced random chrY_KI270740v1_random 0 37240 Unplaced random chrUn_KI270302v1 0 2274 Unplaced chrUn_KI270304v1 0 2165 Unplaced chrUn_KI270303v1 0 1942 Unplaced chrUn_KI270305v1 0 1472 Unplaced chrUn_KI270322v1 0 21476 Unplaced chrUn_KI270320v1 0 4416 Unplaced chrUn_KI270310v1 0 1201 Unplaced chrUn_KI270316v1 0 1444 Unplaced chrUn_KI270315v1 0 2276 Unplaced chrUn_KI270312v1 0 998 Unplaced chrUn_KI270311v1 0 12399 Unplaced chrUn_KI270317v1 0 37690 Unplaced chrUn_KI270412v1 0 1179 Unplaced chrUn_KI270411v1 0 2646 Unplaced chrUn_KI270414v1 0 2489 Unplaced chrUn_KI270419v1 0 1029 Unplaced chrUn_KI270418v1 0 2145 Unplaced chrUn_KI270420v1 0 2321 Unplaced chrUn_KI270424v1 0 2140 Unplaced chrUn_KI270417v1 0 2043 Unplaced chrUn_KI270422v1 0 1445 Unplaced chrUn_KI270423v1 0 981 Unplaced chrUn_KI270425v1 0 1884 Unplaced chrUn_KI270429v1 0 1361 Unplaced chrUn_KI270442v1 0 392061 Unplaced chrUn_KI270466v1 0 1233 Unplaced chrUn_KI270465v1 0 1774 Unplaced chrUn_KI270467v1 0 3920 Unplaced chrUn_KI270435v1 0 92983 Unplaced chrUn_KI270438v1 0 112505 Unplaced chrUn_KI270468v1 0 4055 Unplaced chrUn_KI270510v1 0 2415 Unplaced chrUn_KI270509v1 0 2318 Unplaced chrUn_KI270518v1 0 2186 Unplaced chrUn_KI270508v1 0 1951 Unplaced chrUn_KI270516v1 0 1300 Unplaced chrUn_KI270512v1 0 22689 Unplaced chrUn_KI270519v1 0 138126 Unplaced chrUn_KI270522v1 0 5674 Unplaced chrUn_KI270511v1 0 8127 Unplaced chrUn_KI270515v1 0 6361 Unplaced chrUn_KI270507v1 0 5353 Unplaced chrUn_KI270517v1 0 3253 Unplaced chrUn_KI270529v1 0 1899 Unplaced chrUn_KI270528v1 0 2983 Unplaced chrUn_KI270530v1 0 2168 Unplaced chrUn_KI270539v1 0 993 Unplaced chrUn_KI270538v1 0 91309 Unplaced chrUn_KI270544v1 0 1202 Unplaced chrUn_KI270548v1 0 1599 Unplaced chrUn_KI270583v1 0 1400 Unplaced chrUn_KI270587v1 0 2969 Unplaced chrUn_KI270580v1 0 1553 Unplaced chrUn_KI270581v1 0 7046 Unplaced chrUn_KI270579v1 0 31033 Unplaced chrUn_KI270589v1 0 44474 Unplaced chrUn_KI270590v1 0 4685 Unplaced chrUn_KI270584v1 0 4513 Unplaced chrUn_KI270582v1 0 6504 Unplaced chrUn_KI270588v1 0 6158 Unplaced chrUn_KI270593v1 0 3041 Unplaced chrUn_KI270591v1 0 5796 Unplaced chrUn_KI270330v1 0 1652 Unplaced chrUn_KI270329v1 0 1040 Unplaced chrUn_KI270334v1 0 1368 Unplaced chrUn_KI270333v1 0 2699 Unplaced chrUn_KI270335v1 0 1048 Unplaced chrUn_KI270338v1 0 1428 Unplaced chrUn_KI270340v1 0 1428 Unplaced chrUn_KI270336v1 0 1026 Unplaced chrUn_KI270337v1 0 1121 Unplaced chrUn_KI270363v1 0 1803 Unplaced chrUn_KI270364v1 0 2855 Unplaced chrUn_KI270362v1 0 3530 Unplaced chrUn_KI270366v1 0 8320 Unplaced chrUn_KI270378v1 0 1048 Unplaced chrUn_KI270379v1 0 1045 Unplaced chrUn_KI270389v1 0 1298 Unplaced chrUn_KI270390v1 0 2387 Unplaced chrUn_KI270387v1 0 1537 Unplaced chrUn_KI270395v1 0 1143 Unplaced chrUn_KI270396v1 0 1880 Unplaced chrUn_KI270388v1 0 1216 Unplaced chrUn_KI270394v1 0 970 Unplaced chrUn_KI270386v1 0 1788 Unplaced chrUn_KI270391v1 0 1484 Unplaced chrUn_KI270383v1 0 1750 Unplaced chrUn_KI270393v1 0 1308 Unplaced chrUn_KI270384v1 0 1658 Unplaced chrUn_KI270392v1 0 971 Unplaced chrUn_KI270381v1 0 1930 Unplaced chrUn_KI270385v1 0 990 Unplaced chrUn_KI270382v1 0 4215 Unplaced chrUn_KI270376v1 0 1136 Unplaced chrUn_KI270374v1 0 2656 Unplaced chrUn_KI270372v1 0 1650 Unplaced chrUn_KI270373v1 0 1451 Unplaced chrUn_KI270375v1 0 2378 Unplaced chrUn_KI270371v1 0 2805 Unplaced chrUn_KI270448v1 0 7992 Unplaced chrUn_KI270521v1 0 7642 Unplaced chrUn_GL000195v1 0 182896 Unplaced chrUn_GL000219v1 0 179198 Unplaced chrUn_GL000220v1 0 161802 Unplaced chrUn_GL000224v1 0 179693 Unplaced chrUn_KI270741v1 0 157432 Unplaced chrUn_GL000226v1 0 15008 Unplaced chrUn_GL000213v1 0 164239 Unplaced chrUn_KI270743v1 0 210658 Unplaced chrUn_KI270744v1 0 168472 Unplaced chrUn_KI270745v1 0 41891 Unplaced chrUn_KI270746v1 0 66486 Unplaced chrUn_KI270747v1 0 198735 Unplaced chrUn_KI270748v1 0 93321 Unplaced chrUn_KI270749v1 0 158759 Unplaced chrUn_KI270750v1 0 148850 Unplaced chrUn_KI270751v1 0 150742 Unplaced chrUn_KI270752v1 0 27745 Unplaced chrUn_KI270753v1 0 62944 Unplaced chrUn_KI270754v1 0 40191 Unplaced chrUn_KI270755v1 0 36723 Unplaced chrUn_KI270756v1 0 79590 Unplaced chrUn_KI270757v1 0 71251 Unplaced chrUn_GL000214v1 0 137718 Unplaced chrUn_KI270742v1 0 186739 Unplaced chrUn_GL000216v2 0 176608 Unplaced chrUn_GL000218v1 0 161147 Unplaced chr1_KI270762v1_alt 0 354444 Alt haplotype chr1_KI270766v1_alt 0 256271 Alt haplotype chr1_KI270760v1_alt 0 109528 Alt haplotype chr1_KI270765v1_alt 0 185285 Alt haplotype chr1_GL383518v1_alt 0 182439 Alt haplotype chr1_GL383519v1_alt 0 110268 Alt haplotype chr1_GL383520v2_alt 0 366580 Alt haplotype chr1_KI270764v1_alt 0 50258 Alt haplotype chr1_KI270763v1_alt 0 911658 Alt haplotype chr1_KI270759v1_alt 0 425601 Alt haplotype chr1_KI270761v1_alt 0 165834 Alt haplotype chr2_KI270770v1_alt 0 136240 Alt haplotype chr2_KI270773v1_alt 0 70887 Alt haplotype chr2_KI270774v1_alt 0 223625 Alt haplotype chr2_KI270769v1_alt 0 120616 Alt haplotype chr2_GL383521v1_alt 0 143390 Alt haplotype chr2_KI270772v1_alt 0 133041 Alt haplotype chr2_KI270775v1_alt 0 138019 Alt haplotype chr2_KI270771v1_alt 0 110395 Alt haplotype chr2_KI270768v1_alt 0 110099 Alt haplotype chr2_GL582966v2_alt 0 96131 Alt haplotype chr2_GL383522v1_alt 0 123821 Alt haplotype chr2_KI270776v1_alt 0 174166 Alt haplotype chr2_KI270767v1_alt 0 161578 Alt haplotype chr3_JH636055v2_alt 0 173151 Alt haplotype chr3_KI270783v1_alt 0 109187 Alt haplotype chr3_KI270780v1_alt 0 224108 Alt haplotype chr3_GL383526v1_alt 0 180671 Alt haplotype chr3_KI270777v1_alt 0 173649 Alt haplotype chr3_KI270778v1_alt 0 248252 Alt haplotype chr3_KI270781v1_alt 0 113034 Alt haplotype chr3_KI270779v1_alt 0 205312 Alt haplotype chr3_KI270782v1_alt 0 162429 Alt haplotype chr3_KI270784v1_alt 0 184404 Alt haplotype chr4_KI270790v1_alt 0 220246 Alt haplotype chr4_GL383528v1_alt 0 376187 Alt haplotype chr4_KI270787v1_alt 0 111943 Alt haplotype chr4_GL000257v2_alt 0 586476 Alt haplotype chr4_KI270788v1_alt 0 158965 Alt haplotype chr4_GL383527v1_alt 0 164536 Alt haplotype chr4_KI270785v1_alt 0 119912 Alt haplotype chr4_KI270789v1_alt 0 205944 Alt haplotype chr4_KI270786v1_alt 0 244096 Alt haplotype chr5_KI270793v1_alt 0 126136 Alt haplotype chr5_KI270792v1_alt 0 179043 Alt haplotype chr5_KI270791v1_alt 0 195710 Alt haplotype chr5_GL383532v1_alt 0 82728 Alt haplotype chr5_GL949742v1_alt 0 226852 Alt haplotype chr5_KI270794v1_alt 0 164558 Alt haplotype chr5_GL339449v2_alt 0 1612928 Alt haplotype chr5_GL383530v1_alt 0 101241 Alt haplotype chr5_KI270796v1_alt 0 172708 Alt haplotype chr5_GL383531v1_alt 0 173459 Alt haplotype chr5_KI270795v1_alt 0 131892 Alt haplotype chr6_GL000250v2_alt 0 4672374 Alt haplotype chr6_KI270800v1_alt 0 175808 Alt haplotype chr6_KI270799v1_alt 0 152148 Alt haplotype chr6_GL383533v1_alt 0 124736 Alt haplotype chr6_KI270801v1_alt 0 870480 Alt haplotype chr6_KI270802v1_alt 0 75005 Alt haplotype chr6_KB021644v2_alt 0 185823 Alt haplotype chr6_KI270797v1_alt 0 197536 Alt haplotype chr6_KI270798v1_alt 0 271782 Alt haplotype chr7_KI270804v1_alt 0 157952 Alt haplotype chr7_KI270809v1_alt 0 209586 Alt haplotype chr7_KI270806v1_alt 0 158166 Alt haplotype chr7_GL383534v2_alt 0 119183 Alt haplotype chr7_KI270803v1_alt 0 1111570 Alt haplotype chr7_KI270808v1_alt 0 271455 Alt haplotype chr7_KI270807v1_alt 0 126434 Alt haplotype chr7_KI270805v1_alt 0 209988 Alt haplotype chr8_KI270818v1_alt 0 145606 Alt haplotype chr8_KI270812v1_alt 0 282736 Alt haplotype chr8_KI270811v1_alt 0 292436 Alt haplotype chr8_KI270821v1_alt 0 985506 Alt haplotype chr8_KI270813v1_alt 0 300230 Alt haplotype chr8_KI270822v1_alt 0 624492 Alt haplotype chr8_KI270814v1_alt 0 141812 Alt haplotype chr8_KI270810v1_alt 0 374415 Alt haplotype chr8_KI270819v1_alt 0 133535 Alt haplotype chr8_KI270820v1_alt 0 36640 Alt haplotype chr8_KI270817v1_alt 0 158983 Alt haplotype chr8_KI270816v1_alt 0 305841 Alt haplotype chr8_KI270815v1_alt 0 132244 Alt haplotype chr9_GL383539v1_alt 0 162988 Alt haplotype chr9_GL383540v1_alt 0 71551 Alt haplotype chr9_GL383541v1_alt 0 171286 Alt haplotype chr9_GL383542v1_alt 0 60032 Alt haplotype chr9_KI270823v1_alt 0 439082 Alt haplotype chr10_GL383545v1_alt 0 179254 Alt haplotype chr10_KI270824v1_alt 0 181496 Alt haplotype chr10_GL383546v1_alt 0 309802 Alt haplotype chr10_KI270825v1_alt 0 188315 Alt haplotype chr11_KI270832v1_alt 0 210133 Alt haplotype chr11_KI270830v1_alt 0 177092 Alt haplotype chr11_KI270831v1_alt 0 296895 Alt haplotype chr11_KI270829v1_alt 0 204059 Alt haplotype chr11_GL383547v1_alt 0 154407 Alt haplotype chr11_JH159136v1_alt 0 200998 Alt haplotype chr11_JH159137v1_alt 0 191409 Alt haplotype chr11_KI270827v1_alt 0 67707 Alt haplotype chr11_KI270826v1_alt 0 186169 Alt haplotype chr12_GL877875v1_alt 0 167313 Alt haplotype chr12_GL877876v1_alt 0 408271 Alt haplotype chr12_KI270837v1_alt 0 40090 Alt haplotype chr12_GL383549v1_alt 0 120804 Alt haplotype chr12_KI270835v1_alt 0 238139 Alt haplotype chr12_GL383550v2_alt 0 169178 Alt haplotype chr12_GL383552v1_alt 0 138655 Alt haplotype chr12_GL383553v2_alt 0 152874 Alt haplotype chr12_KI270834v1_alt 0 119498 Alt haplotype chr12_GL383551v1_alt 0 184319 Alt haplotype chr12_KI270833v1_alt 0 76061 Alt haplotype chr12_KI270836v1_alt 0 56134 Alt haplotype chr13_KI270840v1_alt 0 191684 Alt haplotype chr13_KI270839v1_alt 0 180306 Alt haplotype chr13_KI270843v1_alt 0 103832 Alt haplotype chr13_KI270841v1_alt 0 169134 Alt haplotype chr13_KI270838v1_alt 0 306913 Alt haplotype chr13_KI270842v1_alt 0 37287 Alt haplotype chr14_KI270844v1_alt 0 322166 Alt haplotype chr14_KI270847v1_alt 0 1511111 Alt haplotype chr14_KI270845v1_alt 0 180703 Alt haplotype chr14_KI270846v1_alt 0 1351393 Alt haplotype chr15_KI270852v1_alt 0 478999 Alt haplotype chr15_KI270851v1_alt 0 263054 Alt haplotype chr15_KI270848v1_alt 0 327382 Alt haplotype chr15_GL383554v1_alt 0 296527 Alt haplotype chr15_KI270849v1_alt 0 244917 Alt haplotype chr15_GL383555v2_alt 0 388773 Alt haplotype chr15_KI270850v1_alt 0 430880 Alt haplotype chr16_KI270854v1_alt 0 134193 Alt haplotype chr16_KI270856v1_alt 0 63982 Alt haplotype chr16_KI270855v1_alt 0 232857 Alt haplotype chr16_KI270853v1_alt 0 2659700 Alt haplotype chr16_GL383556v1_alt 0 192462 Alt haplotype chr16_GL383557v1_alt 0 89672 Alt haplotype chr17_GL383563v3_alt 0 375691 Alt haplotype chr17_KI270862v1_alt 0 391357 Alt haplotype chr17_KI270861v1_alt 0 196688 Alt haplotype chr17_KI270857v1_alt 0 2877074 Alt haplotype chr17_JH159146v1_alt 0 278131 Alt haplotype chr17_JH159147v1_alt 0 70345 Alt haplotype chr17_GL383564v2_alt 0 133151 Alt haplotype chr17_GL000258v2_alt 0 1821992 Alt haplotype chr17_GL383565v1_alt 0 223995 Alt haplotype chr17_KI270858v1_alt 0 235827 Alt haplotype chr17_KI270859v1_alt 0 108763 Alt haplotype chr17_GL383566v1_alt 0 90219 Alt haplotype chr17_KI270860v1_alt 0 178921 Alt haplotype chr18_KI270864v1_alt 0 111737 Alt haplotype chr18_GL383567v1_alt 0 289831 Alt haplotype chr18_GL383570v1_alt 0 164789 Alt haplotype chr18_GL383571v1_alt 0 198278 Alt haplotype chr18_GL383568v1_alt 0 104552 Alt haplotype chr18_GL383569v1_alt 0 167950 Alt haplotype chr18_GL383572v1_alt 0 159547 Alt haplotype chr18_KI270863v1_alt 0 167999 Alt haplotype chr19_KI270868v1_alt 0 61734 Alt haplotype chr19_KI270865v1_alt 0 52969 Alt haplotype chr19_GL383573v1_alt 0 385657 Alt haplotype chr19_GL383575v2_alt 0 170222 Alt haplotype chr19_GL383576v1_alt 0 188024 Alt haplotype chr19_GL383574v1_alt 0 155864 Alt haplotype chr19_KI270866v1_alt 0 43156 Alt haplotype chr19_KI270867v1_alt 0 233762 Alt haplotype chr19_GL949746v1_alt 0 987716 Alt haplotype chr20_GL383577v2_alt 0 128386 Alt haplotype chr20_KI270869v1_alt 0 118774 Alt haplotype chr20_KI270871v1_alt 0 58661 Alt haplotype chr20_KI270870v1_alt 0 183433 Alt haplotype chr21_GL383578v2_alt 0 63917 Alt haplotype chr21_KI270874v1_alt 0 166743 Alt haplotype chr21_KI270873v1_alt 0 143900 Alt haplotype chr21_GL383579v2_alt 0 201197 Alt haplotype chr21_GL383580v2_alt 0 74653 Alt haplotype chr21_GL383581v2_alt 0 116689 Alt haplotype chr21_KI270872v1_alt 0 82692 Alt haplotype chr22_KI270875v1_alt 0 259914 Alt haplotype chr22_KI270878v1_alt 0 186262 Alt haplotype chr22_KI270879v1_alt 0 304135 Alt haplotype chr22_KI270876v1_alt 0 263666 Alt haplotype chr22_KI270877v1_alt 0 101331 Alt haplotype chr22_GL383583v2_alt 0 96924 Alt haplotype chr22_GL383582v2_alt 0 162811 Alt haplotype chrX_KI270880v1_alt 0 284869 Alt haplotype chrX_KI270881v1_alt 0 144206 Alt haplotype chr19_KI270882v1_alt 0 248807 Alt haplotype chr19_KI270883v1_alt 0 170399 Alt haplotype chr19_KI270884v1_alt 0 157053 Alt haplotype chr19_KI270885v1_alt 0 171027 Alt haplotype chr19_KI270886v1_alt 0 204239 Alt haplotype chr19_KI270887v1_alt 0 209512 Alt haplotype chr19_KI270888v1_alt 0 155532 Alt haplotype chr19_KI270889v1_alt 0 170698 Alt haplotype chr19_KI270890v1_alt 0 184499 Alt haplotype chr19_KI270891v1_alt 0 170680 Alt haplotype chr1_KI270892v1_alt 0 162212 Alt haplotype chr2_KI270894v1_alt 0 214158 Alt haplotype chr2_KI270893v1_alt 0 161218 Alt haplotype chr3_KI270895v1_alt 0 162896 Alt haplotype chr4_KI270896v1_alt 0 378547 Alt haplotype chr5_KI270897v1_alt 0 1144418 Alt haplotype chr5_KI270898v1_alt 0 130957 Alt haplotype chr6_GL000251v2_alt 0 4795265 Alt haplotype chr7_KI270899v1_alt 0 190869 Alt haplotype chr8_KI270901v1_alt 0 136959 Alt haplotype chr8_KI270900v1_alt 0 318687 Alt haplotype chr11_KI270902v1_alt 0 106711 Alt haplotype chr11_KI270903v1_alt 0 214625 Alt haplotype chr12_KI270904v1_alt 0 572349 Alt haplotype chr15_KI270906v1_alt 0 196384 Alt haplotype chr15_KI270905v1_alt 0 5161414 Alt haplotype chr17_KI270907v1_alt 0 137721 Alt haplotype chr17_KI270910v1_alt 0 157099 Alt haplotype chr17_KI270909v1_alt 0 325800 Alt haplotype chr17_JH159148v1_alt 0 88070 Alt haplotype chr17_KI270908v1_alt 0 1423190 Alt haplotype chr18_KI270912v1_alt 0 174061 Alt haplotype chr18_KI270911v1_alt 0 157710 Alt haplotype chr19_GL949747v2_alt 0 729520 Alt haplotype chr22_KB663609v1_alt 0 74013 Alt haplotype chrX_KI270913v1_alt 0 274009 Alt haplotype chr19_KI270914v1_alt 0 205194 Alt haplotype chr19_KI270915v1_alt 0 170665 Alt haplotype chr19_KI270916v1_alt 0 184516 Alt haplotype chr19_KI270917v1_alt 0 190932 Alt haplotype chr19_KI270918v1_alt 0 123111 Alt haplotype chr19_KI270919v1_alt 0 170701 Alt haplotype chr19_KI270920v1_alt 0 198005 Alt haplotype chr19_KI270921v1_alt 0 282224 Alt haplotype chr19_KI270922v1_alt 0 187935 Alt haplotype chr19_KI270923v1_alt 0 189352 Alt haplotype chr3_KI270924v1_alt 0 166540 Alt haplotype chr4_KI270925v1_alt 0 555799 Alt haplotype chr6_GL000252v2_alt 0 4604811 Alt haplotype chr8_KI270926v1_alt 0 229282 Alt haplotype chr11_KI270927v1_alt 0 218612 Alt haplotype chr19_GL949748v2_alt 0 1064304 Alt haplotype chr22_KI270928v1_alt 0 176103 Alt haplotype chr19_KI270929v1_alt 0 186203 Alt haplotype chr19_KI270930v1_alt 0 200773 Alt haplotype chr19_KI270931v1_alt 0 170148 Alt haplotype chr19_KI270932v1_alt 0 215732 Alt haplotype chr19_KI270933v1_alt 0 170537 Alt haplotype chr19_GL000209v2_alt 0 177381 Alt haplotype chr3_KI270934v1_alt 0 163458 Alt haplotype chr6_GL000253v2_alt 0 4677643 Alt haplotype chr19_GL949749v2_alt 0 1091841 Alt haplotype chr3_KI270935v1_alt 0 197351 Alt haplotype chr6_GL000254v2_alt 0 4827813 Alt haplotype chr19_GL949750v2_alt 0 1066390 Alt haplotype chr3_KI270936v1_alt 0 164170 Alt haplotype chr6_GL000255v2_alt 0 4606388 Alt haplotype chr19_GL949751v2_alt 0 1002683 Alt haplotype chr3_KI270937v1_alt 0 165607 Alt haplotype chr6_GL000256v2_alt 0 4929269 Alt haplotype chr19_GL949752v1_alt 0 987100 Alt haplotype chr6_KI270758v1_alt 0 76752 Alt haplotype chr19_GL949753v2_alt 0 796479 Alt haplotype chr19_KI270938v1_alt 0 1066800 Alt haplotype chrEBV 0 171823 EBV chrUn_KN707606v1_decoy 0 2200 Decoy chrUn_KN707607v1_decoy 0 3033 Decoy chrUn_KN707608v1_decoy 0 3112 Decoy chrUn_KN707609v1_decoy 0 1642 Decoy chrUn_KN707610v1_decoy 0 1393 Decoy chrUn_KN707611v1_decoy 0 1103 Decoy chrUn_KN707612v1_decoy 0 1039 Decoy chrUn_KN707613v1_decoy 0 1619 Decoy chrUn_KN707614v1_decoy 0 3122 Decoy chrUn_KN707615v1_decoy 0 1934 Decoy chrUn_KN707616v1_decoy 0 3111 Decoy chrUn_KN707617v1_decoy 0 2545 Decoy chrUn_KN707618v1_decoy 0 2295 Decoy chrUn_KN707619v1_decoy 0 1551 Decoy chrUn_KN707620v1_decoy 0 2046 Decoy chrUn_KN707621v1_decoy 0 1222 Decoy chrUn_KN707622v1_decoy 0 1535 Decoy chrUn_KN707623v1_decoy 0 3784 Decoy chrUn_KN707624v1_decoy 0 1329 Decoy chrUn_KN707625v1_decoy 0 1238 Decoy chrUn_KN707626v1_decoy 0 5623 Decoy chrUn_KN707627v1_decoy 0 5821 Decoy chrUn_KN707628v1_decoy 0 2960 Decoy chrUn_KN707629v1_decoy 0 1848 Decoy chrUn_KN707630v1_decoy 0 2315 Decoy chrUn_KN707631v1_decoy 0 1945 Decoy chrUn_KN707632v1_decoy 0 1424 Decoy chrUn_KN707633v1_decoy 0 1274 Decoy chrUn_KN707634v1_decoy 0 1007 Decoy chrUn_KN707635v1_decoy 0 1414 Decoy chrUn_KN707636v1_decoy 0 1725 Decoy chrUn_KN707637v1_decoy 0 5354 Decoy chrUn_KN707638v1_decoy 0 2189 Decoy chrUn_KN707639v1_decoy 0 1294 Decoy chrUn_KN707640v1_decoy 0 1831 Decoy chrUn_KN707641v1_decoy 0 1647 Decoy chrUn_KN707642v1_decoy 0 2943 Decoy chrUn_KN707643v1_decoy 0 2857 Decoy chrUn_KN707644v1_decoy 0 1030 Decoy chrUn_KN707645v1_decoy 0 1070 Decoy chrUn_KN707646v1_decoy 0 1735 Decoy chrUn_KN707647v1_decoy 0 1982 Decoy chrUn_KN707648v1_decoy 0 1564 Decoy chrUn_KN707649v1_decoy 0 1775 Decoy chrUn_KN707650v1_decoy 0 1540 Decoy chrUn_KN707651v1_decoy 0 2013 Decoy chrUn_KN707652v1_decoy 0 1176 Decoy chrUn_KN707653v1_decoy 0 1890 Decoy chrUn_KN707654v1_decoy 0 3644 Decoy chrUn_KN707655v1_decoy 0 2785 Decoy chrUn_KN707656v1_decoy 0 1017 Decoy chrUn_KN707657v1_decoy 0 1068 Decoy chrUn_KN707658v1_decoy 0 1007 Decoy chrUn_KN707659v1_decoy 0 2605 Decoy chrUn_KN707660v1_decoy 0 8410 Decoy chrUn_KN707661v1_decoy 0 5534 Decoy chrUn_KN707662v1_decoy 0 2173 Decoy chrUn_KN707663v1_decoy 0 1065 Decoy chrUn_KN707664v1_decoy 0 8683 Decoy chrUn_KN707665v1_decoy 0 2670 Decoy chrUn_KN707666v1_decoy 0 2420 Decoy chrUn_KN707667v1_decoy 0 2189 Decoy chrUn_KN707668v1_decoy 0 2093 Decoy chrUn_KN707669v1_decoy 0 1184 Decoy chrUn_KN707670v1_decoy 0 1205 Decoy chrUn_KN707671v1_decoy 0 2786 Decoy chrUn_KN707672v1_decoy 0 2794 Decoy chrUn_KN707673v1_decoy 0 19544 Decoy chrUn_KN707674v1_decoy 0 2848 Decoy chrUn_KN707675v1_decoy 0 10556 Decoy chrUn_KN707676v1_decoy 0 9066 Decoy chrUn_KN707677v1_decoy 0 7267 Decoy chrUn_KN707678v1_decoy 0 2462 Decoy chrUn_KN707679v1_decoy 0 1774 Decoy chrUn_KN707680v1_decoy 0 1297 Decoy chrUn_KN707681v1_decoy 0 4379 Decoy chrUn_KN707682v1_decoy 0 4208 Decoy chrUn_KN707683v1_decoy 0 4068 Decoy chrUn_KN707684v1_decoy 0 2940 Decoy chrUn_KN707685v1_decoy 0 3938 Decoy chrUn_KN707686v1_decoy 0 2072 Decoy chrUn_KN707687v1_decoy 0 1136 Decoy chrUn_KN707688v1_decoy 0 4248 Decoy chrUn_KN707689v1_decoy 0 5823 Decoy chrUn_KN707690v1_decoy 0 3715 Decoy chrUn_KN707691v1_decoy 0 4885 Decoy chrUn_KN707692v1_decoy 0 4813 Decoy chrUn_KN707693v1_decoy 0 2899 Decoy chrUn_KN707694v1_decoy 0 1228 Decoy chrUn_KN707695v1_decoy 0 3119 Decoy chrUn_KN707696v1_decoy 0 3828 Decoy chrUn_KN707697v1_decoy 0 1186 Decoy chrUn_KN707698v1_decoy 0 1908 Decoy chrUn_KN707699v1_decoy 0 2795 Decoy chrUn_KN707700v1_decoy 0 3703 Decoy chrUn_KN707701v1_decoy 0 6722 Decoy chrUn_KN707702v1_decoy 0 6466 Decoy chrUn_KN707703v1_decoy 0 2235 Decoy chrUn_KN707704v1_decoy 0 2871 Decoy chrUn_KN707705v1_decoy 0 4632 Decoy chrUn_KN707706v1_decoy 0 4225 Decoy chrUn_KN707707v1_decoy 0 4339 Decoy chrUn_KN707708v1_decoy 0 2305 Decoy chrUn_KN707709v1_decoy 0 3273 Decoy chrUn_KN707710v1_decoy 0 5701 Decoy chrUn_KN707711v1_decoy 0 4154 Decoy chrUn_KN707712v1_decoy 0 1243 Decoy chrUn_KN707713v1_decoy 0 1308 Decoy chrUn_KN707714v1_decoy 0 2922 Decoy chrUn_KN707715v1_decoy 0 3044 Decoy chrUn_KN707716v1_decoy 0 2888 Decoy chrUn_KN707717v1_decoy 0 1742 Decoy chrUn_KN707718v1_decoy 0 4969 Decoy chrUn_KN707719v1_decoy 0 3270 Decoy chrUn_KN707720v1_decoy 0 6028 Decoy chrUn_KN707721v1_decoy 0 1105 Decoy chrUn_KN707722v1_decoy 0 2884 Decoy chrUn_KN707723v1_decoy 0 1124 Decoy chrUn_KN707724v1_decoy 0 1454 Decoy chrUn_KN707725v1_decoy 0 2565 Decoy chrUn_KN707726v1_decoy 0 2149 Decoy chrUn_KN707727v1_decoy 0 2630 Decoy chrUn_KN707728v1_decoy 0 14625 Decoy chrUn_KN707729v1_decoy 0 7431 Decoy chrUn_KN707730v1_decoy 0 5776 Decoy chrUn_KN707731v1_decoy 0 4820 Decoy chrUn_KN707732v1_decoy 0 1227 Decoy chrUn_KN707733v1_decoy 0 7503 Decoy chrUn_KN707734v1_decoy 0 9652 Decoy chrUn_KN707735v1_decoy 0 1091 Decoy chrUn_KN707736v1_decoy 0 2467 Decoy chrUn_KN707737v1_decoy 0 1270 Decoy chrUn_KN707738v1_decoy 0 4365 Decoy chrUn_KN707739v1_decoy 0 4284 Decoy chrUn_KN707740v1_decoy 0 10282 Decoy chrUn_KN707741v1_decoy 0 5601 Decoy chrUn_KN707742v1_decoy 0 4758 Decoy chrUn_KN707743v1_decoy 0 1624 Decoy chrUn_KN707744v1_decoy 0 4024 Decoy chrUn_KN707745v1_decoy 0 1276 Decoy chrUn_KN707746v1_decoy 0 5083 Decoy chrUn_KN707747v1_decoy 0 2075 Decoy chrUn_KN707748v1_decoy 0 3553 Decoy chrUn_KN707749v1_decoy 0 7010 Decoy chrUn_KN707750v1_decoy 0 4718 Decoy chrUn_KN707751v1_decoy 0 3546 Decoy chrUn_KN707752v1_decoy 0 2873 Decoy chrUn_KN707753v1_decoy 0 2144 Decoy chrUn_KN707754v1_decoy 0 2243 Decoy chrUn_KN707755v1_decoy 0 5343 Decoy chrUn_KN707756v1_decoy 0 4877 Decoy chrUn_KN707757v1_decoy 0 3034 Decoy chrUn_KN707758v1_decoy 0 2826 Decoy chrUn_KN707759v1_decoy 0 1221 Decoy chrUn_KN707760v1_decoy 0 1169 Decoy chrUn_KN707761v1_decoy 0 2319 Decoy chrUn_KN707762v1_decoy 0 3450 Decoy chrUn_KN707763v1_decoy 0 2674 Decoy chrUn_KN707764v1_decoy 0 3912 Decoy chrUn_KN707765v1_decoy 0 6020 Decoy chrUn_KN707766v1_decoy 0 2303 Decoy chrUn_KN707767v1_decoy 0 2552 Decoy chrUn_KN707768v1_decoy 0 3656 Decoy chrUn_KN707769v1_decoy 0 1591 Decoy chrUn_KN707770v1_decoy 0 1209 Decoy chrUn_KN707771v1_decoy 0 3176 Decoy chrUn_KN707772v1_decoy 0 8915 Decoy chrUn_KN707773v1_decoy 0 4902 Decoy chrUn_KN707774v1_decoy 0 3324 Decoy chrUn_KN707775v1_decoy 0 5997 Decoy chrUn_KN707776v1_decoy 0 2618 Decoy chrUn_KN707777v1_decoy 0 10311 Decoy chrUn_KN707778v1_decoy 0 2440 Decoy chrUn_KN707779v1_decoy 0 12444 Decoy chrUn_KN707780v1_decoy 0 5691 Decoy chrUn_KN707781v1_decoy 0 2717 Decoy chrUn_KN707782v1_decoy 0 5277 Decoy chrUn_KN707783v1_decoy 0 4373 Decoy chrUn_KN707784v1_decoy 0 3224 Decoy chrUn_KN707785v1_decoy 0 2631 Decoy chrUn_KN707786v1_decoy 0 5385 Decoy chrUn_KN707787v1_decoy 0 3678 Decoy chrUn_KN707788v1_decoy 0 1412 Decoy chrUn_KN707789v1_decoy 0 1443 Decoy chrUn_KN707790v1_decoy 0 1098 Decoy chrUn_KN707791v1_decoy 0 3240 Decoy chrUn_KN707792v1_decoy 0 1915 Decoy chrUn_KN707793v1_decoy 0 4667 Decoy chrUn_KN707794v1_decoy 0 7219 Decoy chrUn_KN707795v1_decoy 0 3277 Decoy chrUn_KN707796v1_decoy 0 3473 Decoy chrUn_KN707797v1_decoy 0 4243 Decoy chrUn_KN707798v1_decoy 0 17599 Decoy chrUn_KN707799v1_decoy 0 5095 Decoy chrUn_KN707800v1_decoy 0 2237 Decoy chrUn_KN707801v1_decoy 0 2901 Decoy chrUn_KN707802v1_decoy 0 2666 Decoy chrUn_KN707803v1_decoy 0 5336 Decoy chrUn_KN707804v1_decoy 0 4383 Decoy chrUn_KN707805v1_decoy 0 5446 Decoy chrUn_KN707806v1_decoy 0 6252 Decoy chrUn_KN707807v1_decoy 0 4616 Decoy chrUn_KN707808v1_decoy 0 3021 Decoy chrUn_KN707809v1_decoy 0 3667 Decoy chrUn_KN707810v1_decoy 0 4563 Decoy chrUn_KN707811v1_decoy 0 1120 Decoy chrUn_KN707812v1_decoy 0 3845 Decoy chrUn_KN707813v1_decoy 0 2272 Decoy chrUn_KN707814v1_decoy 0 4764 Decoy chrUn_KN707815v1_decoy 0 5410 Decoy chrUn_KN707816v1_decoy 0 7150 Decoy chrUn_KN707817v1_decoy 0 1762 Decoy chrUn_KN707818v1_decoy 0 1207 Decoy chrUn_KN707819v1_decoy 0 1331 Decoy chrUn_KN707820v1_decoy 0 8307 Decoy chrUn_KN707821v1_decoy 0 2276 Decoy chrUn_KN707822v1_decoy 0 2575 Decoy chrUn_KN707823v1_decoy 0 3970 Decoy chrUn_KN707824v1_decoy 0 1352 Decoy chrUn_KN707825v1_decoy 0 3040 Decoy chrUn_KN707826v1_decoy 0 2070 Decoy chrUn_KN707827v1_decoy 0 2913 Decoy chrUn_KN707828v1_decoy 0 2389 Decoy chrUn_KN707829v1_decoy 0 1835 Decoy chrUn_KN707830v1_decoy 0 4807 Decoy chrUn_KN707831v1_decoy 0 2201 Decoy chrUn_KN707832v1_decoy 0 1265 Decoy chrUn_KN707833v1_decoy 0 1961 Decoy chrUn_KN707834v1_decoy 0 1064 Decoy chrUn_KN707835v1_decoy 0 1932 Decoy chrUn_KN707836v1_decoy 0 3213 Decoy chrUn_KN707837v1_decoy 0 1178 Decoy chrUn_KN707838v1_decoy 0 2926 Decoy chrUn_KN707839v1_decoy 0 1038 Decoy chrUn_KN707840v1_decoy 0 3298 Decoy chrUn_KN707841v1_decoy 0 8992 Decoy chrUn_KN707842v1_decoy 0 6698 Decoy chrUn_KN707843v1_decoy 0 4880 Decoy chrUn_KN707844v1_decoy 0 1766 Decoy chrUn_KN707845v1_decoy 0 3532 Decoy chrUn_KN707846v1_decoy 0 2297 Decoy chrUn_KN707847v1_decoy 0 1234 Decoy chrUn_KN707848v1_decoy 0 1205 Decoy chrUn_KN707849v1_decoy 0 2790 Decoy chrUn_KN707850v1_decoy 0 2006 Decoy chrUn_KN707851v1_decoy 0 4593 Decoy chrUn_KN707852v1_decoy 0 1579 Decoy chrUn_KN707853v1_decoy 0 9597 Decoy chrUn_KN707854v1_decoy 0 10451 Decoy chrUn_KN707855v1_decoy 0 3219 Decoy chrUn_KN707856v1_decoy 0 2300 Decoy chrUn_KN707857v1_decoy 0 5985 Decoy chrUn_KN707858v1_decoy 0 2959 Decoy chrUn_KN707859v1_decoy 0 1340 Decoy chrUn_KN707860v1_decoy 0 3148 Decoy chrUn_KN707861v1_decoy 0 2242 Decoy chrUn_KN707862v1_decoy 0 16513 Decoy chrUn_KN707863v1_decoy 0 7821 Decoy chrUn_KN707864v1_decoy 0 2159 Decoy chrUn_KN707865v1_decoy 0 2114 Decoy chrUn_KN707866v1_decoy 0 4109 Decoy chrUn_KN707867v1_decoy 0 1544 Decoy chrUn_KN707868v1_decoy 0 1005 Decoy chrUn_KN707869v1_decoy 0 8632 Decoy chrUn_KN707870v1_decoy 0 1012 Decoy chrUn_KN707871v1_decoy 0 4728 Decoy chrUn_KN707872v1_decoy 0 2165 Decoy chrUn_KN707873v1_decoy 0 7591 Decoy chrUn_KN707874v1_decoy 0 5202 Decoy chrUn_KN707875v1_decoy 0 4241 Decoy chrUn_KN707876v1_decoy 0 4131 Decoy chrUn_KN707877v1_decoy 0 2272 Decoy chrUn_KN707878v1_decoy 0 2085 Decoy chrUn_KN707879v1_decoy 0 4346 Decoy chrUn_KN707880v1_decoy 0 1208 Decoy chrUn_KN707881v1_decoy 0 4543 Decoy chrUn_KN707882v1_decoy 0 2772 Decoy chrUn_KN707883v1_decoy 0 2490 Decoy chrUn_KN707884v1_decoy 0 4568 Decoy chrUn_KN707885v1_decoy 0 1776 Decoy chrUn_KN707886v1_decoy 0 2699 Decoy chrUn_KN707887v1_decoy 0 3534 Decoy chrUn_KN707888v1_decoy 0 2424 Decoy chrUn_KN707889v1_decoy 0 1747 Decoy chrUn_KN707890v1_decoy 0 1088 Decoy chrUn_KN707891v1_decoy 0 1143 Decoy chrUn_KN707892v1_decoy 0 2530 Decoy chrUn_KN707893v1_decoy 0 8049 Decoy chrUn_KN707894v1_decoy 0 1366 Decoy chrUn_KN707895v1_decoy 0 4284 Decoy chrUn_KN707896v1_decoy 0 33125 Decoy chrUn_KN707897v1_decoy 0 2137 Decoy chrUn_KN707898v1_decoy 0 3840 Decoy chrUn_KN707899v1_decoy 0 3087 Decoy chrUn_KN707900v1_decoy 0 2041 Decoy chrUn_KN707901v1_decoy 0 3344 Decoy chrUn_KN707902v1_decoy 0 2921 Decoy chrUn_KN707903v1_decoy 0 6581 Decoy chrUn_KN707904v1_decoy 0 3968 Decoy chrUn_KN707905v1_decoy 0 2339 Decoy chrUn_KN707906v1_decoy 0 1243 Decoy chrUn_KN707907v1_decoy 0 7776 Decoy chrUn_KN707908v1_decoy 0 19837 Decoy chrUn_KN707909v1_decoy 0 1737 Decoy chrUn_KN707910v1_decoy 0 1098 Decoy chrUn_KN707911v1_decoy 0 1893 Decoy chrUn_KN707912v1_decoy 0 1281 Decoy chrUn_KN707913v1_decoy 0 1527 Decoy chrUn_KN707914v1_decoy 0 2055 Decoy chrUn_KN707915v1_decoy 0 2527 Decoy chrUn_KN707916v1_decoy 0 3275 Decoy chrUn_KN707917v1_decoy 0 1265 Decoy chrUn_KN707918v1_decoy 0 2623 Decoy chrUn_KN707919v1_decoy 0 4850 Decoy chrUn_KN707920v1_decoy 0 3584 Decoy chrUn_KN707921v1_decoy 0 2561 Decoy chrUn_KN707922v1_decoy 0 3041 Decoy chrUn_KN707923v1_decoy 0 1409 Decoy chrUn_KN707924v1_decoy 0 4596 Decoy chrUn_KN707925v1_decoy 0 11555 Decoy chrUn_KN707926v1_decoy 0 1266 Decoy chrUn_KN707927v1_decoy 0 1079 Decoy chrUn_KN707928v1_decoy 0 1087 Decoy chrUn_KN707929v1_decoy 0 1226 Decoy chrUn_KN707930v1_decoy 0 1131 Decoy chrUn_KN707931v1_decoy 0 1199 Decoy chrUn_KN707932v1_decoy 0 1084 Decoy chrUn_KN707933v1_decoy 0 2038 Decoy chrUn_KN707934v1_decoy 0 1070 Decoy chrUn_KN707935v1_decoy 0 1312 Decoy chrUn_KN707936v1_decoy 0 4031 Decoy chrUn_KN707937v1_decoy 0 7445 Decoy chrUn_KN707938v1_decoy 0 1770 Decoy chrUn_KN707939v1_decoy 0 5600 Decoy chrUn_KN707940v1_decoy 0 1882 Decoy chrUn_KN707941v1_decoy 0 1170 Decoy chrUn_KN707942v1_decoy 0 1300 Decoy chrUn_KN707943v1_decoy 0 5325 Decoy chrUn_KN707944v1_decoy 0 2043 Decoy chrUn_KN707945v1_decoy 0 1072 Decoy chrUn_KN707946v1_decoy 0 2463 Decoy chrUn_KN707947v1_decoy 0 1010 Decoy chrUn_KN707948v1_decoy 0 1432 Decoy chrUn_KN707949v1_decoy 0 1162 Decoy chrUn_KN707950v1_decoy 0 1095 Decoy chrUn_KN707951v1_decoy 0 1118 Decoy chrUn_KN707952v1_decoy 0 1383 Decoy chrUn_KN707953v1_decoy 0 2289 Decoy chrUn_KN707954v1_decoy 0 1648 Decoy chrUn_KN707955v1_decoy 0 2203 Decoy chrUn_KN707956v1_decoy 0 3270 Decoy chrUn_KN707957v1_decoy 0 11499 Decoy chrUn_KN707958v1_decoy 0 2474 Decoy chrUn_KN707959v1_decoy 0 2294 Decoy chrUn_KN707960v1_decoy 0 1238 Decoy chrUn_KN707961v1_decoy 0 3410 Decoy chrUn_KN707962v1_decoy 0 1523 Decoy chrUn_KN707963v1_decoy 0 62955 Decoy chrUn_KN707964v1_decoy 0 6282 Decoy chrUn_KN707965v1_decoy 0 3836 Decoy chrUn_KN707966v1_decoy 0 6486 Decoy chrUn_KN707967v1_decoy 0 15368 Decoy chrUn_KN707968v1_decoy 0 9572 Decoy chrUn_KN707969v1_decoy 0 6413 Decoy chrUn_KN707970v1_decoy 0 4104 Decoy chrUn_KN707971v1_decoy 0 12943 Decoy chrUn_KN707972v1_decoy 0 4650 Decoy chrUn_KN707973v1_decoy 0 3080 Decoy chrUn_KN707974v1_decoy 0 3134 Decoy chrUn_KN707975v1_decoy 0 6211 Decoy chrUn_KN707976v1_decoy 0 1126 Decoy chrUn_KN707977v1_decoy 0 1101 Decoy chrUn_KN707978v1_decoy 0 1101 Decoy chrUn_KN707979v1_decoy 0 2648 Decoy chrUn_KN707980v1_decoy 0 2973 Decoy chrUn_KN707981v1_decoy 0 2520 Decoy chrUn_KN707982v1_decoy 0 2318 Decoy chrUn_KN707983v1_decoy 0 2606 Decoy chrUn_KN707984v1_decoy 0 2205 Decoy chrUn_KN707985v1_decoy 0 2929 Decoy chrUn_KN707986v1_decoy 0 3869 Decoy chrUn_KN707987v1_decoy 0 1117 Decoy chrUn_KN707988v1_decoy 0 2960 Decoy chrUn_KN707989v1_decoy 0 1009 Decoy chrUn_KN707990v1_decoy 0 4048 Decoy chrUn_KN707991v1_decoy 0 2193 Decoy chrUn_KN707992v1_decoy 0 1830 Decoy chrUn_JTFH01000001v1_decoy 0 25139 Decoy chrUn_JTFH01000002v1_decoy 0 18532 Decoy chrUn_JTFH01000003v1_decoy 0 15240 Decoy chrUn_JTFH01000004v1_decoy 0 13739 Decoy chrUn_JTFH01000005v1_decoy 0 11297 Decoy chrUn_JTFH01000006v1_decoy 0 10074 Decoy chrUn_JTFH01000007v1_decoy 0 9891 Decoy chrUn_JTFH01000008v1_decoy 0 9774 Decoy chrUn_JTFH01000009v1_decoy 0 9727 Decoy chrUn_JTFH01000010v1_decoy 0 9358 Decoy chrUn_JTFH01000011v1_decoy 0 8920 Decoy chrUn_JTFH01000012v1_decoy 0 8479 Decoy chrUn_JTFH01000013v1_decoy 0 8312 Decoy chrUn_JTFH01000014v1_decoy 0 8261 Decoy chrUn_JTFH01000015v1_decoy 0 8131 Decoy chrUn_JTFH01000016v1_decoy 0 8051 Decoy chrUn_JTFH01000017v1_decoy 0 7832 Decoy chrUn_JTFH01000018v1_decoy 0 7710 Decoy chrUn_JTFH01000019v1_decoy 0 7702 Decoy chrUn_JTFH01000020v1_decoy 0 7479 Decoy chrUn_JTFH01000021v1_decoy 0 7368 Decoy chrUn_JTFH01000022v1_decoy 0 7162 Decoy chrUn_JTFH01000023v1_decoy 0 7065 Decoy chrUn_JTFH01000024v1_decoy 0 7019 Decoy chrUn_JTFH01000025v1_decoy 0 6997 Decoy chrUn_JTFH01000026v1_decoy 0 6994 Decoy chrUn_JTFH01000027v1_decoy 0 6979 Decoy chrUn_JTFH01000028v1_decoy 0 6797 Decoy chrUn_JTFH01000029v1_decoy 0 6525 Decoy chrUn_JTFH01000030v1_decoy 0 6246 Decoy chrUn_JTFH01000031v1_decoy 0 5926 Decoy chrUn_JTFH01000032v1_decoy 0 5914 Decoy chrUn_JTFH01000033v1_decoy 0 5898 Decoy chrUn_JTFH01000034v1_decoy 0 5879 Decoy chrUn_JTFH01000035v1_decoy 0 5834 Decoy chrUn_JTFH01000036v1_decoy 0 5743 Decoy chrUn_JTFH01000037v1_decoy 0 5577 Decoy chrUn_JTFH01000038v1_decoy 0 5413 Decoy chrUn_JTFH01000039v1_decoy 0 5250 Decoy chrUn_JTFH01000040v1_decoy 0 5246 Decoy chrUn_JTFH01000041v1_decoy 0 5118 Decoy chrUn_JTFH01000042v1_decoy 0 5058 Decoy chrUn_JTFH01000043v1_decoy 0 4959 Decoy chrUn_JTFH01000044v1_decoy 0 4853 Decoy chrUn_JTFH01000045v1_decoy 0 4828 Decoy chrUn_JTFH01000046v1_decoy 0 4819 Decoy chrUn_JTFH01000047v1_decoy 0 4809 Decoy chrUn_JTFH01000048v1_decoy 0 4710 Decoy chrUn_JTFH01000049v1_decoy 0 4680 Decoy chrUn_JTFH01000050v1_decoy 0 4645 Decoy chrUn_JTFH01000051v1_decoy 0 4514 Decoy chrUn_JTFH01000052v1_decoy 0 4439 Decoy chrUn_JTFH01000053v1_decoy 0 4416 Decoy chrUn_JTFH01000054v1_decoy 0 4409 Decoy chrUn_JTFH01000055v1_decoy 0 4392 Decoy chrUn_JTFH01000056v1_decoy 0 4359 Decoy chrUn_JTFH01000057v1_decoy 0 4319 Decoy chrUn_JTFH01000058v1_decoy 0 4290 Decoy chrUn_JTFH01000059v1_decoy 0 4242 Decoy chrUn_JTFH01000060v1_decoy 0 4228 Decoy chrUn_JTFH01000061v1_decoy 0 4222 Decoy chrUn_JTFH01000062v1_decoy 0 4216 Decoy chrUn_JTFH01000063v1_decoy 0 4210 Decoy chrUn_JTFH01000064v1_decoy 0 4206 Decoy chrUn_JTFH01000065v1_decoy 0 4102 Decoy chrUn_JTFH01000066v1_decoy 0 4101 Decoy chrUn_JTFH01000067v1_decoy 0 4083 Decoy chrUn_JTFH01000068v1_decoy 0 3967 Decoy chrUn_JTFH01000069v1_decoy 0 3955 Decoy chrUn_JTFH01000070v1_decoy 0 3945 Decoy chrUn_JTFH01000071v1_decoy 0 3930 Decoy chrUn_JTFH01000072v1_decoy 0 3929 Decoy chrUn_JTFH01000073v1_decoy 0 3924 Decoy chrUn_JTFH01000074v1_decoy 0 3919 Decoy chrUn_JTFH01000075v1_decoy 0 3908 Decoy chrUn_JTFH01000076v1_decoy 0 3892 Decoy chrUn_JTFH01000077v1_decoy 0 3890 Decoy chrUn_JTFH01000078v1_decoy 0 3859 Decoy chrUn_JTFH01000079v1_decoy 0 3846 Decoy chrUn_JTFH01000080v1_decoy 0 3835 Decoy chrUn_JTFH01000081v1_decoy 0 3830 Decoy chrUn_JTFH01000082v1_decoy 0 3828 Decoy chrUn_JTFH01000083v1_decoy 0 3825 Decoy chrUn_JTFH01000084v1_decoy 0 3821 Decoy chrUn_JTFH01000085v1_decoy 0 3809 Decoy chrUn_JTFH01000086v1_decoy 0 3801 Decoy chrUn_JTFH01000087v1_decoy 0 3799 Decoy chrUn_JTFH01000088v1_decoy 0 3737 Decoy chrUn_JTFH01000089v1_decoy 0 3701 Decoy chrUn_JTFH01000090v1_decoy 0 3698 Decoy chrUn_JTFH01000091v1_decoy 0 3692 Decoy chrUn_JTFH01000092v1_decoy 0 3686 Decoy chrUn_JTFH01000093v1_decoy 0 3677 Decoy chrUn_JTFH01000094v1_decoy 0 3664 Decoy chrUn_JTFH01000095v1_decoy 0 3613 Decoy chrUn_JTFH01000096v1_decoy 0 3611 Decoy chrUn_JTFH01000097v1_decoy 0 3606 Decoy chrUn_JTFH01000098v1_decoy 0 3584 Decoy chrUn_JTFH01000099v1_decoy 0 3581 Decoy chrUn_JTFH01000100v1_decoy 0 3543 Decoy chrUn_JTFH01000101v1_decoy 0 3528 Decoy chrUn_JTFH01000102v1_decoy 0 3527 Decoy chrUn_JTFH01000103v1_decoy 0 3496 Decoy chrUn_JTFH01000104v1_decoy 0 3493 Decoy chrUn_JTFH01000105v1_decoy 0 3484 Decoy chrUn_JTFH01000106v1_decoy 0 3435 Decoy chrUn_JTFH01000107v1_decoy 0 3391 Decoy chrUn_JTFH01000108v1_decoy 0 3374 Decoy chrUn_JTFH01000109v1_decoy 0 3371 Decoy chrUn_JTFH01000110v1_decoy 0 3361 Decoy chrUn_JTFH01000111v1_decoy 0 3351 Decoy chrUn_JTFH01000112v1_decoy 0 3340 Decoy chrUn_JTFH01000113v1_decoy 0 3320 Decoy chrUn_JTFH01000114v1_decoy 0 3282 Decoy chrUn_JTFH01000115v1_decoy 0 3278 Decoy chrUn_JTFH01000116v1_decoy 0 3260 Decoy chrUn_JTFH01000117v1_decoy 0 3258 Decoy chrUn_JTFH01000118v1_decoy 0 3253 Decoy chrUn_JTFH01000119v1_decoy 0 3247 Decoy chrUn_JTFH01000120v1_decoy 0 3230 Decoy chrUn_JTFH01000121v1_decoy 0 3224 Decoy chrUn_JTFH01000122v1_decoy 0 3216 Decoy chrUn_JTFH01000123v1_decoy 0 3212 Decoy chrUn_JTFH01000124v1_decoy 0 3194 Decoy chrUn_JTFH01000125v1_decoy 0 3189 Decoy chrUn_JTFH01000126v1_decoy 0 3177 Decoy chrUn_JTFH01000127v1_decoy 0 3176 Decoy chrUn_JTFH01000128v1_decoy 0 3173 Decoy chrUn_JTFH01000129v1_decoy 0 3170 Decoy chrUn_JTFH01000130v1_decoy 0 3166 Decoy chrUn_JTFH01000131v1_decoy 0 3163 Decoy chrUn_JTFH01000132v1_decoy 0 3143 Decoy chrUn_JTFH01000133v1_decoy 0 3137 Decoy chrUn_JTFH01000134v1_decoy 0 3116 Decoy chrUn_JTFH01000135v1_decoy 0 3106 Decoy chrUn_JTFH01000136v1_decoy 0 3093 Decoy chrUn_JTFH01000137v1_decoy 0 3079 Decoy chrUn_JTFH01000138v1_decoy 0 3053 Decoy chrUn_JTFH01000139v1_decoy 0 3051 Decoy chrUn_JTFH01000140v1_decoy 0 3015 Decoy chrUn_JTFH01000141v1_decoy 0 3012 Decoy chrUn_JTFH01000142v1_decoy 0 3009 Decoy chrUn_JTFH01000143v1_decoy 0 2997 Decoy chrUn_JTFH01000144v1_decoy 0 2997 Decoy chrUn_JTFH01000145v1_decoy 0 2983 Decoy chrUn_JTFH01000146v1_decoy 0 2979 Decoy chrUn_JTFH01000147v1_decoy 0 2967 Decoy chrUn_JTFH01000148v1_decoy 0 2967 Decoy chrUn_JTFH01000149v1_decoy 0 2966 Decoy chrUn_JTFH01000150v1_decoy 0 2954 Decoy chrUn_JTFH01000151v1_decoy 0 2952 Decoy chrUn_JTFH01000152v1_decoy 0 2934 Decoy chrUn_JTFH01000153v1_decoy 0 2918 Decoy chrUn_JTFH01000154v1_decoy 0 2895 Decoy chrUn_JTFH01000155v1_decoy 0 2887 Decoy chrUn_JTFH01000156v1_decoy 0 2879 Decoy chrUn_JTFH01000157v1_decoy 0 2878 Decoy chrUn_JTFH01000158v1_decoy 0 2872 Decoy chrUn_JTFH01000159v1_decoy 0 2868 Decoy chrUn_JTFH01000160v1_decoy 0 2866 Decoy chrUn_JTFH01000161v1_decoy 0 2865 Decoy chrUn_JTFH01000162v1_decoy 0 2864 Decoy chrUn_JTFH01000163v1_decoy 0 2859 Decoy chrUn_JTFH01000164v1_decoy 0 2854 Decoy chrUn_JTFH01000165v1_decoy 0 2830 Decoy chrUn_JTFH01000166v1_decoy 0 2828 Decoy chrUn_JTFH01000167v1_decoy 0 2824 Decoy chrUn_JTFH01000168v1_decoy 0 2819 Decoy chrUn_JTFH01000169v1_decoy 0 2813 Decoy chrUn_JTFH01000170v1_decoy 0 2809 Decoy chrUn_JTFH01000171v1_decoy 0 2802 Decoy chrUn_JTFH01000172v1_decoy 0 2791 Decoy chrUn_JTFH01000173v1_decoy 0 2783 Decoy chrUn_JTFH01000174v1_decoy 0 2778 Decoy chrUn_JTFH01000175v1_decoy 0 2777 Decoy chrUn_JTFH01000176v1_decoy 0 2770 Decoy chrUn_JTFH01000177v1_decoy 0 2769 Decoy chrUn_JTFH01000178v1_decoy 0 2767 Decoy chrUn_JTFH01000179v1_decoy 0 2763 Decoy chrUn_JTFH01000180v1_decoy 0 2745 Decoy chrUn_JTFH01000181v1_decoy 0 2742 Decoy chrUn_JTFH01000182v1_decoy 0 2736 Decoy chrUn_JTFH01000183v1_decoy 0 2729 Decoy chrUn_JTFH01000184v1_decoy 0 2726 Decoy chrUn_JTFH01000185v1_decoy 0 2719 Decoy chrUn_JTFH01000186v1_decoy 0 2715 Decoy chrUn_JTFH01000187v1_decoy 0 2708 Decoy chrUn_JTFH01000188v1_decoy 0 2704 Decoy chrUn_JTFH01000189v1_decoy 0 2692 Decoy chrUn_JTFH01000190v1_decoy 0 2691 Decoy chrUn_JTFH01000191v1_decoy 0 2690 Decoy chrUn_JTFH01000192v1_decoy 0 2687 Decoy chrUn_JTFH01000193v1_decoy 0 2677 Decoy chrUn_JTFH01000194v1_decoy 0 2668 Decoy chrUn_JTFH01000195v1_decoy 0 2668 Decoy chrUn_JTFH01000196v1_decoy 0 2663 Decoy chrUn_JTFH01000197v1_decoy 0 2655 Decoy chrUn_JTFH01000198v1_decoy 0 2644 Decoy chrUn_JTFH01000199v1_decoy 0 2642 Decoy chrUn_JTFH01000200v1_decoy 0 2632 Decoy chrUn_JTFH01000201v1_decoy 0 2632 Decoy chrUn_JTFH01000202v1_decoy 0 2628 Decoy chrUn_JTFH01000203v1_decoy 0 2623 Decoy chrUn_JTFH01000204v1_decoy 0 2622 Decoy chrUn_JTFH01000205v1_decoy 0 2619 Decoy chrUn_JTFH01000206v1_decoy 0 2605 Decoy chrUn_JTFH01000207v1_decoy 0 2603 Decoy chrUn_JTFH01000208v1_decoy 0 2601 Decoy chrUn_JTFH01000209v1_decoy 0 2598 Decoy chrUn_JTFH01000210v1_decoy 0 2597 Decoy chrUn_JTFH01000211v1_decoy 0 2596 Decoy chrUn_JTFH01000212v1_decoy 0 2594 Decoy chrUn_JTFH01000213v1_decoy 0 2586 Decoy chrUn_JTFH01000214v1_decoy 0 2585 Decoy chrUn_JTFH01000215v1_decoy 0 2583 Decoy chrUn_JTFH01000216v1_decoy 0 2578 Decoy chrUn_JTFH01000217v1_decoy 0 2569 Decoy chrUn_JTFH01000218v1_decoy 0 2569 Decoy chrUn_JTFH01000219v1_decoy 0 2551 Decoy chrUn_JTFH01000220v1_decoy 0 2548 Decoy chrUn_JTFH01000221v1_decoy 0 2548 Decoy chrUn_JTFH01000222v1_decoy 0 2546 Decoy chrUn_JTFH01000223v1_decoy 0 2545 Decoy chrUn_JTFH01000224v1_decoy 0 2534 Decoy chrUn_JTFH01000225v1_decoy 0 2533 Decoy chrUn_JTFH01000226v1_decoy 0 2522 Decoy chrUn_JTFH01000227v1_decoy 0 2522 Decoy chrUn_JTFH01000228v1_decoy 0 2515 Decoy chrUn_JTFH01000229v1_decoy 0 2513 Decoy chrUn_JTFH01000230v1_decoy 0 2507 Decoy chrUn_JTFH01000231v1_decoy 0 2504 Decoy chrUn_JTFH01000232v1_decoy 0 2497 Decoy chrUn_JTFH01000233v1_decoy 0 2471 Decoy chrUn_JTFH01000234v1_decoy 0 2465 Decoy chrUn_JTFH01000235v1_decoy 0 2464 Decoy chrUn_JTFH01000236v1_decoy 0 2459 Decoy chrUn_JTFH01000237v1_decoy 0 2457 Decoy chrUn_JTFH01000238v1_decoy 0 2450 Decoy chrUn_JTFH01000239v1_decoy 0 2435 Decoy chrUn_JTFH01000240v1_decoy 0 2434 Decoy chrUn_JTFH01000241v1_decoy 0 2432 Decoy chrUn_JTFH01000242v1_decoy 0 2427 Decoy chrUn_JTFH01000243v1_decoy 0 2421 Decoy chrUn_JTFH01000244v1_decoy 0 2420 Decoy chrUn_JTFH01000245v1_decoy 0 2414 Decoy chrUn_JTFH01000246v1_decoy 0 2404 Decoy chrUn_JTFH01000247v1_decoy 0 2403 Decoy chrUn_JTFH01000248v1_decoy 0 2402 Decoy chrUn_JTFH01000249v1_decoy 0 2397 Decoy chrUn_JTFH01000250v1_decoy 0 2395 Decoy chrUn_JTFH01000251v1_decoy 0 2394 Decoy chrUn_JTFH01000252v1_decoy 0 2388 Decoy chrUn_JTFH01000253v1_decoy 0 2382 Decoy chrUn_JTFH01000254v1_decoy 0 2381 Decoy chrUn_JTFH01000255v1_decoy 0 2380 Decoy chrUn_JTFH01000256v1_decoy 0 2368 Decoy chrUn_JTFH01000257v1_decoy 0 2364 Decoy chrUn_JTFH01000258v1_decoy 0 2363 Decoy chrUn_JTFH01000259v1_decoy 0 2348 Decoy chrUn_JTFH01000260v1_decoy 0 2339 Decoy chrUn_JTFH01000261v1_decoy 0 2335 Decoy chrUn_JTFH01000262v1_decoy 0 2332 Decoy chrUn_JTFH01000263v1_decoy 0 2331 Decoy chrUn_JTFH01000264v1_decoy 0 2330 Decoy chrUn_JTFH01000265v1_decoy 0 2323 Decoy chrUn_JTFH01000266v1_decoy 0 2319 Decoy chrUn_JTFH01000267v1_decoy 0 2314 Decoy chrUn_JTFH01000268v1_decoy 0 2308 Decoy chrUn_JTFH01000269v1_decoy 0 2306 Decoy chrUn_JTFH01000270v1_decoy 0 2296 Decoy chrUn_JTFH01000271v1_decoy 0 2287 Decoy chrUn_JTFH01000272v1_decoy 0 2279 Decoy chrUn_JTFH01000273v1_decoy 0 2276 Decoy chrUn_JTFH01000274v1_decoy 0 2273 Decoy chrUn_JTFH01000275v1_decoy 0 2262 Decoy chrUn_JTFH01000276v1_decoy 0 2254 Decoy chrUn_JTFH01000277v1_decoy 0 2252 Decoy chrUn_JTFH01000278v1_decoy 0 2245 Decoy chrUn_JTFH01000279v1_decoy 0 2239 Decoy chrUn_JTFH01000280v1_decoy 0 2223 Decoy chrUn_JTFH01000281v1_decoy 0 2220 Decoy chrUn_JTFH01000282v1_decoy 0 2218 Decoy chrUn_JTFH01000283v1_decoy 0 2215 Decoy chrUn_JTFH01000284v1_decoy 0 2213 Decoy chrUn_JTFH01000285v1_decoy 0 2203 Decoy chrUn_JTFH01000286v1_decoy 0 2200 Decoy chrUn_JTFH01000287v1_decoy 0 2197 Decoy chrUn_JTFH01000288v1_decoy 0 2194 Decoy chrUn_JTFH01000289v1_decoy 0 2183 Decoy chrUn_JTFH01000290v1_decoy 0 2179 Decoy chrUn_JTFH01000291v1_decoy 0 2177 Decoy chrUn_JTFH01000292v1_decoy 0 2177 Decoy chrUn_JTFH01000293v1_decoy 0 2177 Decoy chrUn_JTFH01000294v1_decoy 0 2168 Decoy chrUn_JTFH01000295v1_decoy 0 2160 Decoy chrUn_JTFH01000296v1_decoy 0 2155 Decoy chrUn_JTFH01000297v1_decoy 0 2144 Decoy chrUn_JTFH01000298v1_decoy 0 2143 Decoy chrUn_JTFH01000299v1_decoy 0 2136 Decoy chrUn_JTFH01000300v1_decoy 0 2134 Decoy chrUn_JTFH01000301v1_decoy 0 2129 Decoy chrUn_JTFH01000302v1_decoy 0 2128 Decoy chrUn_JTFH01000303v1_decoy 0 2125 Decoy chrUn_JTFH01000304v1_decoy 0 2125 Decoy chrUn_JTFH01000305v1_decoy 0 2122 Decoy chrUn_JTFH01000306v1_decoy 0 2111 Decoy chrUn_JTFH01000307v1_decoy 0 2106 Decoy chrUn_JTFH01000308v1_decoy 0 2094 Decoy chrUn_JTFH01000309v1_decoy 0 2093 Decoy chrUn_JTFH01000310v1_decoy 0 2088 Decoy chrUn_JTFH01000311v1_decoy 0 2086 Decoy chrUn_JTFH01000312v1_decoy 0 2086 Decoy chrUn_JTFH01000313v1_decoy 0 2084 Decoy chrUn_JTFH01000314v1_decoy 0 2080 Decoy chrUn_JTFH01000315v1_decoy 0 2079 Decoy chrUn_JTFH01000316v1_decoy 0 2076 Decoy chrUn_JTFH01000317v1_decoy 0 2071 Decoy chrUn_JTFH01000318v1_decoy 0 2066 Decoy chrUn_JTFH01000319v1_decoy 0 2061 Decoy chrUn_JTFH01000320v1_decoy 0 2055 Decoy chrUn_JTFH01000321v1_decoy 0 2053 Decoy chrUn_JTFH01000322v1_decoy 0 2040 Decoy chrUn_JTFH01000323v1_decoy 0 2036 Decoy chrUn_JTFH01000324v1_decoy 0 2035 Decoy chrUn_JTFH01000325v1_decoy 0 2034 Decoy chrUn_JTFH01000326v1_decoy 0 2032 Decoy chrUn_JTFH01000327v1_decoy 0 2029 Decoy chrUn_JTFH01000328v1_decoy 0 2025 Decoy chrUn_JTFH01000329v1_decoy 0 2021 Decoy chrUn_JTFH01000330v1_decoy 0 2018 Decoy chrUn_JTFH01000331v1_decoy 0 2015 Decoy chrUn_JTFH01000332v1_decoy 0 2009 Decoy chrUn_JTFH01000333v1_decoy 0 2007 Decoy chrUn_JTFH01000334v1_decoy 0 2005 Decoy chrUn_JTFH01000335v1_decoy 0 2003 Decoy chrUn_JTFH01000336v1_decoy 0 2001 Decoy chrUn_JTFH01000337v1_decoy 0 2001 Decoy chrUn_JTFH01000338v1_decoy 0 2000 Decoy chrUn_JTFH01000339v1_decoy 0 1996 Decoy chrUn_JTFH01000340v1_decoy 0 1992 Decoy chrUn_JTFH01000341v1_decoy 0 1985 Decoy chrUn_JTFH01000342v1_decoy 0 1981 Decoy chrUn_JTFH01000343v1_decoy 0 1977 Decoy chrUn_JTFH01000344v1_decoy 0 1971 Decoy chrUn_JTFH01000345v1_decoy 0 1968 Decoy chrUn_JTFH01000346v1_decoy 0 1962 Decoy chrUn_JTFH01000347v1_decoy 0 1961 Decoy chrUn_JTFH01000348v1_decoy 0 1960 Decoy chrUn_JTFH01000349v1_decoy 0 1960 Decoy chrUn_JTFH01000350v1_decoy 0 1954 Decoy chrUn_JTFH01000351v1_decoy 0 1952 Decoy chrUn_JTFH01000352v1_decoy 0 1947 Decoy chrUn_JTFH01000353v1_decoy 0 1944 Decoy chrUn_JTFH01000354v1_decoy 0 1943 Decoy chrUn_JTFH01000355v1_decoy 0 1941 Decoy chrUn_JTFH01000356v1_decoy 0 1937 Decoy chrUn_JTFH01000357v1_decoy 0 1934 Decoy chrUn_JTFH01000358v1_decoy 0 1929 Decoy chrUn_JTFH01000359v1_decoy 0 1924 Decoy chrUn_JTFH01000360v1_decoy 0 1924 Decoy chrUn_JTFH01000361v1_decoy 0 1923 Decoy chrUn_JTFH01000362v1_decoy 0 1921 Decoy chrUn_JTFH01000363v1_decoy 0 1918 Decoy chrUn_JTFH01000364v1_decoy 0 1915 Decoy chrUn_JTFH01000365v1_decoy 0 1915 Decoy chrUn_JTFH01000366v1_decoy 0 1914 Decoy chrUn_JTFH01000367v1_decoy 0 1912 Decoy chrUn_JTFH01000368v1_decoy 0 1910 Decoy chrUn_JTFH01000369v1_decoy 0 1907 Decoy chrUn_JTFH01000370v1_decoy 0 1904 Decoy chrUn_JTFH01000371v1_decoy 0 1897 Decoy chrUn_JTFH01000372v1_decoy 0 1891 Decoy chrUn_JTFH01000373v1_decoy 0 1890 Decoy chrUn_JTFH01000374v1_decoy 0 1888 Decoy chrUn_JTFH01000375v1_decoy 0 1888 Decoy chrUn_JTFH01000376v1_decoy 0 1885 Decoy chrUn_JTFH01000377v1_decoy 0 1881 Decoy chrUn_JTFH01000378v1_decoy 0 1879 Decoy chrUn_JTFH01000379v1_decoy 0 1877 Decoy chrUn_JTFH01000380v1_decoy 0 1876 Decoy chrUn_JTFH01000381v1_decoy 0 1876 Decoy chrUn_JTFH01000382v1_decoy 0 1874 Decoy chrUn_JTFH01000383v1_decoy 0 1872 Decoy chrUn_JTFH01000384v1_decoy 0 1869 Decoy chrUn_JTFH01000385v1_decoy 0 1866 Decoy chrUn_JTFH01000386v1_decoy 0 1865 Decoy chrUn_JTFH01000387v1_decoy 0 1865 Decoy chrUn_JTFH01000388v1_decoy 0 1865 Decoy chrUn_JTFH01000389v1_decoy 0 1862 Decoy chrUn_JTFH01000390v1_decoy 0 1862 Decoy chrUn_JTFH01000391v1_decoy 0 1859 Decoy chrUn_JTFH01000392v1_decoy 0 1856 Decoy chrUn_JTFH01000393v1_decoy 0 1856 Decoy chrUn_JTFH01000394v1_decoy 0 1854 Decoy chrUn_JTFH01000395v1_decoy 0 1850 Decoy chrUn_JTFH01000396v1_decoy 0 1849 Decoy chrUn_JTFH01000397v1_decoy 0 1849 Decoy chrUn_JTFH01000398v1_decoy 0 1847 Decoy chrUn_JTFH01000399v1_decoy 0 1839 Decoy chrUn_JTFH01000400v1_decoy 0 1834 Decoy chrUn_JTFH01000401v1_decoy 0 1821 Decoy chrUn_JTFH01000402v1_decoy 0 1815 Decoy chrUn_JTFH01000403v1_decoy 0 1811 Decoy chrUn_JTFH01000404v1_decoy 0 1808 Decoy chrUn_JTFH01000405v1_decoy 0 1808 Decoy chrUn_JTFH01000406v1_decoy 0 1807 Decoy chrUn_JTFH01000407v1_decoy 0 1807 Decoy chrUn_JTFH01000408v1_decoy 0 1802 Decoy chrUn_JTFH01000409v1_decoy 0 1801 Decoy chrUn_JTFH01000410v1_decoy 0 1800 Decoy chrUn_JTFH01000411v1_decoy 0 1795 Decoy chrUn_JTFH01000412v1_decoy 0 1794 Decoy chrUn_JTFH01000413v1_decoy 0 1792 Decoy chrUn_JTFH01000414v1_decoy 0 1788 Decoy chrUn_JTFH01000415v1_decoy 0 1786 Decoy chrUn_JTFH01000416v1_decoy 0 1782 Decoy chrUn_JTFH01000417v1_decoy 0 1782 Decoy chrUn_JTFH01000418v1_decoy 0 1781 Decoy chrUn_JTFH01000419v1_decoy 0 1781 Decoy chrUn_JTFH01000420v1_decoy 0 1779 Decoy chrUn_JTFH01000421v1_decoy 0 1777 Decoy chrUn_JTFH01000422v1_decoy 0 1764 Decoy chrUn_JTFH01000423v1_decoy 0 1762 Decoy chrUn_JTFH01000424v1_decoy 0 1755 Decoy chrUn_JTFH01000425v1_decoy 0 1749 Decoy chrUn_JTFH01000426v1_decoy 0 1747 Decoy chrUn_JTFH01000427v1_decoy 0 1746 Decoy chrUn_JTFH01000428v1_decoy 0 1745 Decoy chrUn_JTFH01000429v1_decoy 0 1744 Decoy chrUn_JTFH01000430v1_decoy 0 1742 Decoy chrUn_JTFH01000431v1_decoy 0 1740 Decoy chrUn_JTFH01000432v1_decoy 0 1740 Decoy chrUn_JTFH01000433v1_decoy 0 1736 Decoy chrUn_JTFH01000434v1_decoy 0 1735 Decoy chrUn_JTFH01000435v1_decoy 0 1732 Decoy chrUn_JTFH01000436v1_decoy 0 1732 Decoy chrUn_JTFH01000437v1_decoy 0 1730 Decoy chrUn_JTFH01000438v1_decoy 0 1727 Decoy chrUn_JTFH01000439v1_decoy 0 1722 Decoy chrUn_JTFH01000440v1_decoy 0 1718 Decoy chrUn_JTFH01000441v1_decoy 0 1716 Decoy chrUn_JTFH01000442v1_decoy 0 1710 Decoy chrUn_JTFH01000443v1_decoy 0 1708 Decoy chrUn_JTFH01000444v1_decoy 0 1707 Decoy chrUn_JTFH01000445v1_decoy 0 1706 Decoy chrUn_JTFH01000446v1_decoy 0 1705 Decoy chrUn_JTFH01000447v1_decoy 0 1704 Decoy chrUn_JTFH01000448v1_decoy 0 1699 Decoy chrUn_JTFH01000449v1_decoy 0 1698 Decoy chrUn_JTFH01000450v1_decoy 0 1697 Decoy chrUn_JTFH01000451v1_decoy 0 1697 Decoy chrUn_JTFH01000452v1_decoy 0 1695 Decoy chrUn_JTFH01000453v1_decoy 0 1695 Decoy chrUn_JTFH01000454v1_decoy 0 1693 Decoy chrUn_JTFH01000455v1_decoy 0 1687 Decoy chrUn_JTFH01000456v1_decoy 0 1686 Decoy chrUn_JTFH01000457v1_decoy 0 1680 Decoy chrUn_JTFH01000458v1_decoy 0 1679 Decoy chrUn_JTFH01000459v1_decoy 0 1679 Decoy chrUn_JTFH01000460v1_decoy 0 1678 Decoy chrUn_JTFH01000461v1_decoy 0 1674 Decoy chrUn_JTFH01000462v1_decoy 0 1674 Decoy chrUn_JTFH01000463v1_decoy 0 1671 Decoy chrUn_JTFH01000464v1_decoy 0 1669 Decoy chrUn_JTFH01000465v1_decoy 0 1665 Decoy chrUn_JTFH01000466v1_decoy 0 1663 Decoy chrUn_JTFH01000467v1_decoy 0 1657 Decoy chrUn_JTFH01000468v1_decoy 0 1653 Decoy chrUn_JTFH01000469v1_decoy 0 1652 Decoy chrUn_JTFH01000470v1_decoy 0 1650 Decoy chrUn_JTFH01000471v1_decoy 0 1649 Decoy chrUn_JTFH01000472v1_decoy 0 1649 Decoy chrUn_JTFH01000473v1_decoy 0 1640 Decoy chrUn_JTFH01000474v1_decoy 0 1638 Decoy chrUn_JTFH01000475v1_decoy 0 1636 Decoy chrUn_JTFH01000476v1_decoy 0 1632 Decoy chrUn_JTFH01000477v1_decoy 0 1631 Decoy chrUn_JTFH01000478v1_decoy 0 1630 Decoy chrUn_JTFH01000479v1_decoy 0 1627 Decoy chrUn_JTFH01000480v1_decoy 0 1624 Decoy chrUn_JTFH01000481v1_decoy 0 1617 Decoy chrUn_JTFH01000482v1_decoy 0 1616 Decoy chrUn_JTFH01000483v1_decoy 0 1615 Decoy chrUn_JTFH01000484v1_decoy 0 1611 Decoy chrUn_JTFH01000485v1_decoy 0 1611 Decoy chrUn_JTFH01000486v1_decoy 0 1606 Decoy chrUn_JTFH01000487v1_decoy 0 1605 Decoy chrUn_JTFH01000488v1_decoy 0 1605 Decoy chrUn_JTFH01000489v1_decoy 0 1600 Decoy chrUn_JTFH01000490v1_decoy 0 1598 Decoy chrUn_JTFH01000491v1_decoy 0 1598 Decoy chrUn_JTFH01000492v1_decoy 0 1597 Decoy chrUn_JTFH01000493v1_decoy 0 1596 Decoy chrUn_JTFH01000494v1_decoy 0 1595 Decoy chrUn_JTFH01000495v1_decoy 0 1592 Decoy chrUn_JTFH01000496v1_decoy 0 1589 Decoy chrUn_JTFH01000497v1_decoy 0 1585 Decoy chrUn_JTFH01000498v1_decoy 0 1579 Decoy chrUn_JTFH01000499v1_decoy 0 1578 Decoy chrUn_JTFH01000500v1_decoy 0 1577 Decoy chrUn_JTFH01000501v1_decoy 0 1577 Decoy chrUn_JTFH01000502v1_decoy 0 1577 Decoy chrUn_JTFH01000503v1_decoy 0 1576 Decoy chrUn_JTFH01000504v1_decoy 0 1575 Decoy chrUn_JTFH01000505v1_decoy 0 1574 Decoy chrUn_JTFH01000506v1_decoy 0 1572 Decoy chrUn_JTFH01000507v1_decoy 0 1571 Decoy chrUn_JTFH01000508v1_decoy 0 1563 Decoy chrUn_JTFH01000509v1_decoy 0 1561 Decoy chrUn_JTFH01000510v1_decoy 0 1561 Decoy chrUn_JTFH01000511v1_decoy 0 1560 Decoy chrUn_JTFH01000512v1_decoy 0 1560 Decoy chrUn_JTFH01000513v1_decoy 0 1554 Decoy chrUn_JTFH01000514v1_decoy 0 1552 Decoy chrUn_JTFH01000515v1_decoy 0 1548 Decoy chrUn_JTFH01000516v1_decoy 0 1546 Decoy chrUn_JTFH01000517v1_decoy 0 1541 Decoy chrUn_JTFH01000518v1_decoy 0 1536 Decoy chrUn_JTFH01000519v1_decoy 0 1533 Decoy chrUn_JTFH01000520v1_decoy 0 1532 Decoy chrUn_JTFH01000521v1_decoy 0 1532 Decoy chrUn_JTFH01000522v1_decoy 0 1530 Decoy chrUn_JTFH01000523v1_decoy 0 1527 Decoy chrUn_JTFH01000524v1_decoy 0 1526 Decoy chrUn_JTFH01000525v1_decoy 0 1524 Decoy chrUn_JTFH01000526v1_decoy 0 1523 Decoy chrUn_JTFH01000527v1_decoy 0 1523 Decoy chrUn_JTFH01000528v1_decoy 0 1522 Decoy chrUn_JTFH01000529v1_decoy 0 1522 Decoy chrUn_JTFH01000530v1_decoy 0 1519 Decoy chrUn_JTFH01000531v1_decoy 0 1513 Decoy chrUn_JTFH01000532v1_decoy 0 1508 Decoy chrUn_JTFH01000533v1_decoy 0 1508 Decoy chrUn_JTFH01000534v1_decoy 0 1505 Decoy chrUn_JTFH01000535v1_decoy 0 1503 Decoy chrUn_JTFH01000536v1_decoy 0 1496 Decoy chrUn_JTFH01000537v1_decoy 0 1491 Decoy chrUn_JTFH01000538v1_decoy 0 1490 Decoy chrUn_JTFH01000539v1_decoy 0 1490 Decoy chrUn_JTFH01000540v1_decoy 0 1487 Decoy chrUn_JTFH01000541v1_decoy 0 1486 Decoy chrUn_JTFH01000542v1_decoy 0 1485 Decoy chrUn_JTFH01000543v1_decoy 0 1484 Decoy chrUn_JTFH01000544v1_decoy 0 1483 Decoy chrUn_JTFH01000545v1_decoy 0 1479 Decoy chrUn_JTFH01000546v1_decoy 0 1479 Decoy chrUn_JTFH01000547v1_decoy 0 1476 Decoy chrUn_JTFH01000548v1_decoy 0 1475 Decoy chrUn_JTFH01000549v1_decoy 0 1472 Decoy chrUn_JTFH01000550v1_decoy 0 1469 Decoy chrUn_JTFH01000551v1_decoy 0 1468 Decoy chrUn_JTFH01000552v1_decoy 0 1467 Decoy chrUn_JTFH01000553v1_decoy 0 1465 Decoy chrUn_JTFH01000554v1_decoy 0 1464 Decoy chrUn_JTFH01000555v1_decoy 0 1463 Decoy chrUn_JTFH01000556v1_decoy 0 1463 Decoy chrUn_JTFH01000557v1_decoy 0 1459 Decoy chrUn_JTFH01000558v1_decoy 0 1459 Decoy chrUn_JTFH01000559v1_decoy 0 1458 Decoy chrUn_JTFH01000560v1_decoy 0 1458 Decoy chrUn_JTFH01000561v1_decoy 0 1454 Decoy chrUn_JTFH01000562v1_decoy 0 1449 Decoy chrUn_JTFH01000563v1_decoy 0 1449 Decoy chrUn_JTFH01000564v1_decoy 0 1448 Decoy chrUn_JTFH01000565v1_decoy 0 1446 Decoy chrUn_JTFH01000566v1_decoy 0 1442 Decoy chrUn_JTFH01000567v1_decoy 0 1441 Decoy chrUn_JTFH01000568v1_decoy 0 1440 Decoy chrUn_JTFH01000569v1_decoy 0 1439 Decoy chrUn_JTFH01000570v1_decoy 0 1437 Decoy chrUn_JTFH01000571v1_decoy 0 1436 Decoy chrUn_JTFH01000572v1_decoy 0 1429 Decoy chrUn_JTFH01000573v1_decoy 0 1429 Decoy chrUn_JTFH01000574v1_decoy 0 1427 Decoy chrUn_JTFH01000575v1_decoy 0 1426 Decoy chrUn_JTFH01000576v1_decoy 0 1425 Decoy chrUn_JTFH01000577v1_decoy 0 1424 Decoy chrUn_JTFH01000578v1_decoy 0 1424 Decoy chrUn_JTFH01000579v1_decoy 0 1423 Decoy chrUn_JTFH01000580v1_decoy 0 1423 Decoy chrUn_JTFH01000581v1_decoy 0 1423 Decoy chrUn_JTFH01000582v1_decoy 0 1414 Decoy chrUn_JTFH01000583v1_decoy 0 1414 Decoy chrUn_JTFH01000584v1_decoy 0 1413 Decoy chrUn_JTFH01000585v1_decoy 0 1413 Decoy chrUn_JTFH01000586v1_decoy 0 1410 Decoy chrUn_JTFH01000587v1_decoy 0 1409 Decoy chrUn_JTFH01000588v1_decoy 0 1409 Decoy chrUn_JTFH01000589v1_decoy 0 1406 Decoy chrUn_JTFH01000590v1_decoy 0 1405 Decoy chrUn_JTFH01000591v1_decoy 0 1405 Decoy chrUn_JTFH01000592v1_decoy 0 1404 Decoy chrUn_JTFH01000593v1_decoy 0 1404 Decoy chrUn_JTFH01000594v1_decoy 0 1402 Decoy chrUn_JTFH01000595v1_decoy 0 1402 Decoy chrUn_JTFH01000596v1_decoy 0 1402 Decoy chrUn_JTFH01000597v1_decoy 0 1402 Decoy chrUn_JTFH01000598v1_decoy 0 1400 Decoy chrUn_JTFH01000599v1_decoy 0 1398 Decoy chrUn_JTFH01000600v1_decoy 0 1396 Decoy chrUn_JTFH01000601v1_decoy 0 1395 Decoy chrUn_JTFH01000602v1_decoy 0 1394 Decoy chrUn_JTFH01000603v1_decoy 0 1393 Decoy chrUn_JTFH01000604v1_decoy 0 1391 Decoy chrUn_JTFH01000605v1_decoy 0 1389 Decoy chrUn_JTFH01000606v1_decoy 0 1389 Decoy chrUn_JTFH01000607v1_decoy 0 1388 Decoy chrUn_JTFH01000608v1_decoy 0 1387 Decoy chrUn_JTFH01000609v1_decoy 0 1384 Decoy chrUn_JTFH01000610v1_decoy 0 1381 Decoy chrUn_JTFH01000611v1_decoy 0 1381 Decoy chrUn_JTFH01000612v1_decoy 0 1379 Decoy chrUn_JTFH01000613v1_decoy 0 1377 Decoy chrUn_JTFH01000614v1_decoy 0 1376 Decoy chrUn_JTFH01000615v1_decoy 0 1376 Decoy chrUn_JTFH01000616v1_decoy 0 1375 Decoy chrUn_JTFH01000617v1_decoy 0 1374 Decoy chrUn_JTFH01000618v1_decoy 0 1372 Decoy chrUn_JTFH01000619v1_decoy 0 1371 Decoy chrUn_JTFH01000620v1_decoy 0 1370 Decoy chrUn_JTFH01000621v1_decoy 0 1370 Decoy chrUn_JTFH01000622v1_decoy 0 1366 Decoy chrUn_JTFH01000623v1_decoy 0 1363 Decoy chrUn_JTFH01000624v1_decoy 0 1360 Decoy chrUn_JTFH01000625v1_decoy 0 1356 Decoy chrUn_JTFH01000626v1_decoy 0 1355 Decoy chrUn_JTFH01000627v1_decoy 0 1355 Decoy chrUn_JTFH01000628v1_decoy 0 1352 Decoy chrUn_JTFH01000629v1_decoy 0 1345 Decoy chrUn_JTFH01000630v1_decoy 0 1344 Decoy chrUn_JTFH01000631v1_decoy 0 1344 Decoy chrUn_JTFH01000632v1_decoy 0 1342 Decoy chrUn_JTFH01000633v1_decoy 0 1342 Decoy chrUn_JTFH01000634v1_decoy 0 1336 Decoy chrUn_JTFH01000635v1_decoy 0 1334 Decoy chrUn_JTFH01000636v1_decoy 0 1334 Decoy chrUn_JTFH01000637v1_decoy 0 1333 Decoy chrUn_JTFH01000638v1_decoy 0 1332 Decoy chrUn_JTFH01000639v1_decoy 0 1328 Decoy chrUn_JTFH01000640v1_decoy 0 1328 Decoy chrUn_JTFH01000641v1_decoy 0 1328 Decoy chrUn_JTFH01000642v1_decoy 0 1327 Decoy chrUn_JTFH01000643v1_decoy 0 1325 Decoy chrUn_JTFH01000644v1_decoy 0 1322 Decoy chrUn_JTFH01000645v1_decoy 0 1320 Decoy chrUn_JTFH01000646v1_decoy 0 1319 Decoy chrUn_JTFH01000647v1_decoy 0 1318 Decoy chrUn_JTFH01000648v1_decoy 0 1315 Decoy chrUn_JTFH01000649v1_decoy 0 1314 Decoy chrUn_JTFH01000650v1_decoy 0 1313 Decoy chrUn_JTFH01000651v1_decoy 0 1313 Decoy chrUn_JTFH01000652v1_decoy 0 1312 Decoy chrUn_JTFH01000653v1_decoy 0 1310 Decoy chrUn_JTFH01000654v1_decoy 0 1309 Decoy chrUn_JTFH01000655v1_decoy 0 1309 Decoy chrUn_JTFH01000656v1_decoy 0 1307 Decoy chrUn_JTFH01000657v1_decoy 0 1307 Decoy chrUn_JTFH01000658v1_decoy 0 1305 Decoy chrUn_JTFH01000659v1_decoy 0 1304 Decoy chrUn_JTFH01000660v1_decoy 0 1303 Decoy chrUn_JTFH01000661v1_decoy 0 1302 Decoy chrUn_JTFH01000662v1_decoy 0 1302 Decoy chrUn_JTFH01000663v1_decoy 0 1301 Decoy chrUn_JTFH01000664v1_decoy 0 1301 Decoy chrUn_JTFH01000665v1_decoy 0 1300 Decoy chrUn_JTFH01000666v1_decoy 0 1299 Decoy chrUn_JTFH01000667v1_decoy 0 1297 Decoy chrUn_JTFH01000668v1_decoy 0 1295 Decoy chrUn_JTFH01000669v1_decoy 0 1294 Decoy chrUn_JTFH01000670v1_decoy 0 1293 Decoy chrUn_JTFH01000671v1_decoy 0 1291 Decoy chrUn_JTFH01000672v1_decoy 0 1291 Decoy chrUn_JTFH01000673v1_decoy 0 1289 Decoy chrUn_JTFH01000674v1_decoy 0 1288 Decoy chrUn_JTFH01000675v1_decoy 0 1288 Decoy chrUn_JTFH01000676v1_decoy 0 1287 Decoy chrUn_JTFH01000677v1_decoy 0 1287 Decoy chrUn_JTFH01000678v1_decoy 0 1287 Decoy chrUn_JTFH01000679v1_decoy 0 1286 Decoy chrUn_JTFH01000680v1_decoy 0 1283 Decoy chrUn_JTFH01000681v1_decoy 0 1281 Decoy chrUn_JTFH01000682v1_decoy 0 1277 Decoy chrUn_JTFH01000683v1_decoy 0 1274 Decoy chrUn_JTFH01000684v1_decoy 0 1270 Decoy chrUn_JTFH01000685v1_decoy 0 1267 Decoy chrUn_JTFH01000686v1_decoy 0 1266 Decoy chrUn_JTFH01000687v1_decoy 0 1260 Decoy chrUn_JTFH01000688v1_decoy 0 1259 Decoy chrUn_JTFH01000689v1_decoy 0 1258 Decoy chrUn_JTFH01000690v1_decoy 0 1258 Decoy chrUn_JTFH01000691v1_decoy 0 1258 Decoy chrUn_JTFH01000692v1_decoy 0 1256 Decoy chrUn_JTFH01000693v1_decoy 0 1255 Decoy chrUn_JTFH01000694v1_decoy 0 1254 Decoy chrUn_JTFH01000695v1_decoy 0 1254 Decoy chrUn_JTFH01000696v1_decoy 0 1253 Decoy chrUn_JTFH01000697v1_decoy 0 1250 Decoy chrUn_JTFH01000698v1_decoy 0 1249 Decoy chrUn_JTFH01000699v1_decoy 0 1248 Decoy chrUn_JTFH01000700v1_decoy 0 1248 Decoy chrUn_JTFH01000701v1_decoy 0 1247 Decoy chrUn_JTFH01000702v1_decoy 0 1242 Decoy chrUn_JTFH01000703v1_decoy 0 1242 Decoy chrUn_JTFH01000704v1_decoy 0 1241 Decoy chrUn_JTFH01000705v1_decoy 0 1241 Decoy chrUn_JTFH01000706v1_decoy 0 1241 Decoy chrUn_JTFH01000707v1_decoy 0 1239 Decoy chrUn_JTFH01000708v1_decoy 0 1238 Decoy chrUn_JTFH01000709v1_decoy 0 1237 Decoy chrUn_JTFH01000710v1_decoy 0 1236 Decoy chrUn_JTFH01000711v1_decoy 0 1235 Decoy chrUn_JTFH01000712v1_decoy 0 1234 Decoy chrUn_JTFH01000713v1_decoy 0 1234 Decoy chrUn_JTFH01000714v1_decoy 0 1234 Decoy chrUn_JTFH01000715v1_decoy 0 1233 Decoy chrUn_JTFH01000716v1_decoy 0 1232 Decoy chrUn_JTFH01000717v1_decoy 0 1232 Decoy chrUn_JTFH01000718v1_decoy 0 1231 Decoy chrUn_JTFH01000719v1_decoy 0 1230 Decoy chrUn_JTFH01000720v1_decoy 0 1228 Decoy chrUn_JTFH01000721v1_decoy 0 1227 Decoy chrUn_JTFH01000722v1_decoy 0 1227 Decoy chrUn_JTFH01000723v1_decoy 0 1226 Decoy chrUn_JTFH01000724v1_decoy 0 1224 Decoy chrUn_JTFH01000725v1_decoy 0 1224 Decoy chrUn_JTFH01000726v1_decoy 0 1220 Decoy chrUn_JTFH01000727v1_decoy 0 1220 Decoy chrUn_JTFH01000728v1_decoy 0 1219 Decoy chrUn_JTFH01000729v1_decoy 0 1217 Decoy chrUn_JTFH01000730v1_decoy 0 1216 Decoy chrUn_JTFH01000731v1_decoy 0 1215 Decoy chrUn_JTFH01000732v1_decoy 0 1214 Decoy chrUn_JTFH01000733v1_decoy 0 1214 Decoy chrUn_JTFH01000734v1_decoy 0 1214 Decoy chrUn_JTFH01000735v1_decoy 0 1213 Decoy chrUn_JTFH01000736v1_decoy 0 1212 Decoy chrUn_JTFH01000737v1_decoy 0 1209 Decoy chrUn_JTFH01000738v1_decoy 0 1208 Decoy chrUn_JTFH01000739v1_decoy 0 1207 Decoy chrUn_JTFH01000740v1_decoy 0 1207 Decoy chrUn_JTFH01000741v1_decoy 0 1207 Decoy chrUn_JTFH01000742v1_decoy 0 1206 Decoy chrUn_JTFH01000743v1_decoy 0 1206 Decoy chrUn_JTFH01000744v1_decoy 0 1205 Decoy chrUn_JTFH01000745v1_decoy 0 1205 Decoy chrUn_JTFH01000746v1_decoy 0 1204 Decoy chrUn_JTFH01000747v1_decoy 0 1204 Decoy chrUn_JTFH01000748v1_decoy 0 1204 Decoy chrUn_JTFH01000749v1_decoy 0 1203 Decoy chrUn_JTFH01000750v1_decoy 0 1201 Decoy chrUn_JTFH01000751v1_decoy 0 1201 Decoy chrUn_JTFH01000752v1_decoy 0 1200 Decoy chrUn_JTFH01000753v1_decoy 0 1200 Decoy chrUn_JTFH01000754v1_decoy 0 1199 Decoy chrUn_JTFH01000755v1_decoy 0 1198 Decoy chrUn_JTFH01000756v1_decoy 0 1197 Decoy chrUn_JTFH01000757v1_decoy 0 1196 Decoy chrUn_JTFH01000758v1_decoy 0 1195 Decoy chrUn_JTFH01000759v1_decoy 0 1194 Decoy chrUn_JTFH01000760v1_decoy 0 1194 Decoy chrUn_JTFH01000761v1_decoy 0 1191 Decoy chrUn_JTFH01000762v1_decoy 0 1189 Decoy chrUn_JTFH01000763v1_decoy 0 1186 Decoy chrUn_JTFH01000764v1_decoy 0 1186 Decoy chrUn_JTFH01000765v1_decoy 0 1184 Decoy chrUn_JTFH01000766v1_decoy 0 1183 Decoy chrUn_JTFH01000767v1_decoy 0 1183 Decoy chrUn_JTFH01000768v1_decoy 0 1182 Decoy chrUn_JTFH01000769v1_decoy 0 1181 Decoy chrUn_JTFH01000770v1_decoy 0 1181 Decoy chrUn_JTFH01000771v1_decoy 0 1181 Decoy chrUn_JTFH01000772v1_decoy 0 1181 Decoy chrUn_JTFH01000773v1_decoy 0 1179 Decoy chrUn_JTFH01000774v1_decoy 0 1178 Decoy chrUn_JTFH01000775v1_decoy 0 1178 Decoy chrUn_JTFH01000776v1_decoy 0 1177 Decoy chrUn_JTFH01000777v1_decoy 0 1177 Decoy chrUn_JTFH01000778v1_decoy 0 1171 Decoy chrUn_JTFH01000779v1_decoy 0 1171 Decoy chrUn_JTFH01000780v1_decoy 0 1171 Decoy chrUn_JTFH01000781v1_decoy 0 1170 Decoy chrUn_JTFH01000782v1_decoy 0 1170 Decoy chrUn_JTFH01000783v1_decoy 0 1167 Decoy chrUn_JTFH01000784v1_decoy 0 1167 Decoy chrUn_JTFH01000785v1_decoy 0 1167 Decoy chrUn_JTFH01000786v1_decoy 0 1165 Decoy chrUn_JTFH01000787v1_decoy 0 1165 Decoy chrUn_JTFH01000788v1_decoy 0 1162 Decoy chrUn_JTFH01000789v1_decoy 0 1157 Decoy chrUn_JTFH01000790v1_decoy 0 1156 Decoy chrUn_JTFH01000791v1_decoy 0 1156 Decoy chrUn_JTFH01000792v1_decoy 0 1154 Decoy chrUn_JTFH01000793v1_decoy 0 1154 Decoy chrUn_JTFH01000794v1_decoy 0 1151 Decoy chrUn_JTFH01000795v1_decoy 0 1151 Decoy chrUn_JTFH01000796v1_decoy 0 1150 Decoy chrUn_JTFH01000797v1_decoy 0 1150 Decoy chrUn_JTFH01000798v1_decoy 0 1147 Decoy chrUn_JTFH01000799v1_decoy 0 1147 Decoy chrUn_JTFH01000800v1_decoy 0 1146 Decoy chrUn_JTFH01000801v1_decoy 0 1144 Decoy chrUn_JTFH01000802v1_decoy 0 1144 Decoy chrUn_JTFH01000803v1_decoy 0 1143 Decoy chrUn_JTFH01000804v1_decoy 0 1142 Decoy chrUn_JTFH01000805v1_decoy 0 1141 Decoy chrUn_JTFH01000806v1_decoy 0 1141 Decoy chrUn_JTFH01000807v1_decoy 0 1140 Decoy chrUn_JTFH01000808v1_decoy 0 1138 Decoy chrUn_JTFH01000809v1_decoy 0 1134 Decoy chrUn_JTFH01000810v1_decoy 0 1134 Decoy chrUn_JTFH01000811v1_decoy 0 1132 Decoy chrUn_JTFH01000812v1_decoy 0 1131 Decoy chrUn_JTFH01000813v1_decoy 0 1131 Decoy chrUn_JTFH01000814v1_decoy 0 1130 Decoy chrUn_JTFH01000815v1_decoy 0 1127 Decoy chrUn_JTFH01000816v1_decoy 0 1126 Decoy chrUn_JTFH01000817v1_decoy 0 1124 Decoy chrUn_JTFH01000818v1_decoy 0 1122 Decoy chrUn_JTFH01000819v1_decoy 0 1122 Decoy chrUn_JTFH01000820v1_decoy 0 1121 Decoy chrUn_JTFH01000821v1_decoy 0 1119 Decoy chrUn_JTFH01000822v1_decoy 0 1119 Decoy chrUn_JTFH01000823v1_decoy 0 1119 Decoy chrUn_JTFH01000824v1_decoy 0 1119 Decoy chrUn_JTFH01000825v1_decoy 0 1118 Decoy chrUn_JTFH01000826v1_decoy 0 1116 Decoy chrUn_JTFH01000827v1_decoy 0 1116 Decoy chrUn_JTFH01000828v1_decoy 0 1115 Decoy chrUn_JTFH01000829v1_decoy 0 1115 Decoy chrUn_JTFH01000830v1_decoy 0 1115 Decoy chrUn_JTFH01000831v1_decoy 0 1114 Decoy chrUn_JTFH01000832v1_decoy 0 1113 Decoy chrUn_JTFH01000833v1_decoy 0 1113 Decoy chrUn_JTFH01000834v1_decoy 0 1110 Decoy chrUn_JTFH01000835v1_decoy 0 1110 Decoy chrUn_JTFH01000836v1_decoy 0 1109 Decoy chrUn_JTFH01000837v1_decoy 0 1108 Decoy chrUn_JTFH01000838v1_decoy 0 1107 Decoy chrUn_JTFH01000839v1_decoy 0 1107 Decoy chrUn_JTFH01000840v1_decoy 0 1107 Decoy chrUn_JTFH01000841v1_decoy 0 1107 Decoy chrUn_JTFH01000842v1_decoy 0 1106 Decoy chrUn_JTFH01000843v1_decoy 0 1103 Decoy chrUn_JTFH01000844v1_decoy 0 1103 Decoy chrUn_JTFH01000845v1_decoy 0 1103 Decoy chrUn_JTFH01000846v1_decoy 0 1100 Decoy chrUn_JTFH01000847v1_decoy 0 1099 Decoy chrUn_JTFH01000848v1_decoy 0 1098 Decoy chrUn_JTFH01000849v1_decoy 0 1097 Decoy chrUn_JTFH01000850v1_decoy 0 1096 Decoy chrUn_JTFH01000851v1_decoy 0 1096 Decoy chrUn_JTFH01000852v1_decoy 0 1094 Decoy chrUn_JTFH01000853v1_decoy 0 1093 Decoy chrUn_JTFH01000854v1_decoy 0 1090 Decoy chrUn_JTFH01000855v1_decoy 0 1088 Decoy chrUn_JTFH01000856v1_decoy 0 1087 Decoy chrUn_JTFH01000857v1_decoy 0 1086 Decoy chrUn_JTFH01000858v1_decoy 0 1085 Decoy chrUn_JTFH01000859v1_decoy 0 1084 Decoy chrUn_JTFH01000860v1_decoy 0 1084 Decoy chrUn_JTFH01000861v1_decoy 0 1084 Decoy chrUn_JTFH01000862v1_decoy 0 1084 Decoy chrUn_JTFH01000863v1_decoy 0 1083 Decoy chrUn_JTFH01000864v1_decoy 0 1083 Decoy chrUn_JTFH01000865v1_decoy 0 1082 Decoy chrUn_JTFH01000866v1_decoy 0 1082 Decoy chrUn_JTFH01000867v1_decoy 0 1081 Decoy chrUn_JTFH01000868v1_decoy 0 1081 Decoy chrUn_JTFH01000869v1_decoy 0 1079 Decoy chrUn_JTFH01000870v1_decoy 0 1076 Decoy chrUn_JTFH01000871v1_decoy 0 1074 Decoy chrUn_JTFH01000872v1_decoy 0 1073 Decoy chrUn_JTFH01000873v1_decoy 0 1073 Decoy chrUn_JTFH01000874v1_decoy 0 1071 Decoy chrUn_JTFH01000875v1_decoy 0 1069 Decoy chrUn_JTFH01000876v1_decoy 0 1067 Decoy chrUn_JTFH01000877v1_decoy 0 1067 Decoy chrUn_JTFH01000878v1_decoy 0 1067 Decoy chrUn_JTFH01000879v1_decoy 0 1066 Decoy chrUn_JTFH01000880v1_decoy 0 1065 Decoy chrUn_JTFH01000881v1_decoy 0 1065 Decoy chrUn_JTFH01000882v1_decoy 0 1065 Decoy chrUn_JTFH01000883v1_decoy 0 1065 Decoy chrUn_JTFH01000884v1_decoy 0 1065 Decoy chrUn_JTFH01000885v1_decoy 0 1064 Decoy chrUn_JTFH01000886v1_decoy 0 1064 Decoy chrUn_JTFH01000887v1_decoy 0 1064 Decoy chrUn_JTFH01000888v1_decoy 0 1063 Decoy chrUn_JTFH01000889v1_decoy 0 1062 Decoy chrUn_JTFH01000890v1_decoy 0 1062 Decoy chrUn_JTFH01000891v1_decoy 0 1062 Decoy chrUn_JTFH01000892v1_decoy 0 1061 Decoy chrUn_JTFH01000893v1_decoy 0 1060 Decoy chrUn_JTFH01000894v1_decoy 0 1057 Decoy chrUn_JTFH01000895v1_decoy 0 1057 Decoy chrUn_JTFH01000896v1_decoy 0 1056 Decoy chrUn_JTFH01000897v1_decoy 0 1055 Decoy chrUn_JTFH01000898v1_decoy 0 1055 Decoy chrUn_JTFH01000899v1_decoy 0 1055 Decoy chrUn_JTFH01000900v1_decoy 0 1055 Decoy chrUn_JTFH01000901v1_decoy 0 1054 Decoy chrUn_JTFH01000902v1_decoy 0 1051 Decoy chrUn_JTFH01000903v1_decoy 0 1050 Decoy chrUn_JTFH01000904v1_decoy 0 1050 Decoy chrUn_JTFH01000905v1_decoy 0 1049 Decoy chrUn_JTFH01000906v1_decoy 0 1048 Decoy chrUn_JTFH01000907v1_decoy 0 1047 Decoy chrUn_JTFH01000908v1_decoy 0 1046 Decoy chrUn_JTFH01000909v1_decoy 0 1046 Decoy chrUn_JTFH01000910v1_decoy 0 1046 Decoy chrUn_JTFH01000911v1_decoy 0 1045 Decoy chrUn_JTFH01000912v1_decoy 0 1045 Decoy chrUn_JTFH01000913v1_decoy 0 1045 Decoy chrUn_JTFH01000914v1_decoy 0 1044 Decoy chrUn_JTFH01000915v1_decoy 0 1042 Decoy chrUn_JTFH01000916v1_decoy 0 1041 Decoy chrUn_JTFH01000917v1_decoy 0 1039 Decoy chrUn_JTFH01000918v1_decoy 0 1039 Decoy chrUn_JTFH01000919v1_decoy 0 1038 Decoy chrUn_JTFH01000920v1_decoy 0 1036 Decoy chrUn_JTFH01000921v1_decoy 0 1036 Decoy chrUn_JTFH01000922v1_decoy 0 1035 Decoy chrUn_JTFH01000923v1_decoy 0 1035 Decoy chrUn_JTFH01000924v1_decoy 0 1033 Decoy chrUn_JTFH01000925v1_decoy 0 1032 Decoy chrUn_JTFH01000926v1_decoy 0 1031 Decoy chrUn_JTFH01000927v1_decoy 0 1031 Decoy chrUn_JTFH01000928v1_decoy 0 1031 Decoy chrUn_JTFH01000929v1_decoy 0 1027 Decoy chrUn_JTFH01000930v1_decoy 0 1027 Decoy chrUn_JTFH01000931v1_decoy 0 1026 Decoy chrUn_JTFH01000932v1_decoy 0 1026 Decoy chrUn_JTFH01000933v1_decoy 0 1024 Decoy chrUn_JTFH01000934v1_decoy 0 1024 Decoy chrUn_JTFH01000935v1_decoy 0 1022 Decoy chrUn_JTFH01000936v1_decoy 0 1022 Decoy chrUn_JTFH01000937v1_decoy 0 1021 Decoy chrUn_JTFH01000938v1_decoy 0 1020 Decoy chrUn_JTFH01000939v1_decoy 0 1019 Decoy chrUn_JTFH01000940v1_decoy 0 1018 Decoy chrUn_JTFH01000941v1_decoy 0 1018 Decoy chrUn_JTFH01000942v1_decoy 0 1018 Decoy chrUn_JTFH01000943v1_decoy 0 1016 Decoy chrUn_JTFH01000944v1_decoy 0 1010 Decoy chrUn_JTFH01000945v1_decoy 0 1010 Decoy chrUn_JTFH01000946v1_decoy 0 1009 Decoy chrUn_JTFH01000947v1_decoy 0 1008 Decoy chrUn_JTFH01000948v1_decoy 0 1007 Decoy chrUn_JTFH01000949v1_decoy 0 1006 Decoy chrUn_JTFH01000950v1_decoy 0 1005 Decoy chrUn_JTFH01000951v1_decoy 0 1005 Decoy chrUn_JTFH01000952v1_decoy 0 1004 Decoy chrUn_JTFH01000953v1_decoy 0 1004 Decoy chrUn_JTFH01000954v1_decoy 0 1003 Decoy chrUn_JTFH01000955v1_decoy 0 1003 Decoy chrUn_JTFH01000956v1_decoy 0 1003 Decoy chrUn_JTFH01000957v1_decoy 0 1003 Decoy chrUn_JTFH01000958v1_decoy 0 1002 Decoy chrUn_JTFH01000959v1_decoy 0 1002 Decoy chrUn_JTFH01000960v1_decoy 0 1000 Decoy chrUn_JTFH01000961v1_decoy 0 1000 Decoy chrUn_JTFH01000962v1_decoy 0 8358 Decoy chrUn_JTFH01000963v1_decoy 0 7932 Decoy chrUn_JTFH01000964v1_decoy 0 6846 Decoy chrUn_JTFH01000965v1_decoy 0 4591 Decoy chrUn_JTFH01000966v1_decoy 0 4041 Decoy chrUn_JTFH01000967v1_decoy 0 3841 Decoy chrUn_JTFH01000968v1_decoy 0 3754 Decoy chrUn_JTFH01000969v1_decoy 0 3743 Decoy chrUn_JTFH01000970v1_decoy 0 3702 Decoy chrUn_JTFH01000971v1_decoy 0 3625 Decoy chrUn_JTFH01000972v1_decoy 0 3529 Decoy chrUn_JTFH01000973v1_decoy 0 3508 Decoy chrUn_JTFH01000974v1_decoy 0 3359 Decoy chrUn_JTFH01000975v1_decoy 0 3320 Decoy chrUn_JTFH01000976v1_decoy 0 3231 Decoy chrUn_JTFH01000977v1_decoy 0 3220 Decoy chrUn_JTFH01000978v1_decoy 0 3212 Decoy chrUn_JTFH01000979v1_decoy 0 3192 Decoy chrUn_JTFH01000980v1_decoy 0 3092 Decoy chrUn_JTFH01000981v1_decoy 0 3087 Decoy chrUn_JTFH01000982v1_decoy 0 3048 Decoy chrUn_JTFH01000983v1_decoy 0 3005 Decoy chrUn_JTFH01000984v1_decoy 0 3004 Decoy chrUn_JTFH01000985v1_decoy 0 2959 Decoy chrUn_JTFH01000986v1_decoy 0 2934 Decoy chrUn_JTFH01000987v1_decoy 0 2933 Decoy chrUn_JTFH01000988v1_decoy 0 2827 Decoy chrUn_JTFH01000989v1_decoy 0 2794 Decoy chrUn_JTFH01000990v1_decoy 0 2749 Decoy chrUn_JTFH01000991v1_decoy 0 2745 Decoy chrUn_JTFH01000992v1_decoy 0 2733 Decoy chrUn_JTFH01000993v1_decoy 0 2698 Decoy chrUn_JTFH01000994v1_decoy 0 2665 Decoy chrUn_JTFH01000995v1_decoy 0 2634 Decoy chrUn_JTFH01000996v1_decoy 0 2492 Decoy chrUn_JTFH01000997v1_decoy 0 2489 Decoy chrUn_JTFH01000998v1_decoy 0 2468 Decoy chrUn_JTFH01000999v1_decoy 0 2414 Decoy chrUn_JTFH01001000v1_decoy 0 2395 Decoy chrUn_JTFH01001001v1_decoy 0 2356 Decoy chrUn_JTFH01001002v1_decoy 0 2339 Decoy chrUn_JTFH01001003v1_decoy 0 2310 Decoy chrUn_JTFH01001004v1_decoy 0 2288 Decoy chrUn_JTFH01001005v1_decoy 0 2285 Decoy chrUn_JTFH01001006v1_decoy 0 2269 Decoy chrUn_JTFH01001007v1_decoy 0 2253 Decoy chrUn_JTFH01001008v1_decoy 0 2203 Decoy chrUn_JTFH01001009v1_decoy 0 2176 Decoy chrUn_JTFH01001010v1_decoy 0 2159 Decoy chrUn_JTFH01001011v1_decoy 0 2155 Decoy chrUn_JTFH01001012v1_decoy 0 2149 Decoy chrUn_JTFH01001013v1_decoy 0 2129 Decoy chrUn_JTFH01001014v1_decoy 0 2116 Decoy chrUn_JTFH01001015v1_decoy 0 2113 Decoy chrUn_JTFH01001016v1_decoy 0 2098 Decoy chrUn_JTFH01001017v1_decoy 0 2066 Decoy chrUn_JTFH01001018v1_decoy 0 2066 Decoy chrUn_JTFH01001019v1_decoy 0 2059 Decoy chrUn_JTFH01001020v1_decoy 0 2047 Decoy chrUn_JTFH01001021v1_decoy 0 2040 Decoy chrUn_JTFH01001022v1_decoy 0 2030 Decoy chrUn_JTFH01001023v1_decoy 0 2024 Decoy chrUn_JTFH01001024v1_decoy 0 2001 Decoy chrUn_JTFH01001025v1_decoy 0 1992 Decoy chrUn_JTFH01001026v1_decoy 0 1981 Decoy chrUn_JTFH01001027v1_decoy 0 1979 Decoy chrUn_JTFH01001028v1_decoy 0 1957 Decoy chrUn_JTFH01001029v1_decoy 0 1953 Decoy chrUn_JTFH01001030v1_decoy 0 1944 Decoy chrUn_JTFH01001031v1_decoy 0 1936 Decoy chrUn_JTFH01001032v1_decoy 0 1932 Decoy chrUn_JTFH01001033v1_decoy 0 1882 Decoy chrUn_JTFH01001034v1_decoy 0 1878 Decoy chrUn_JTFH01001035v1_decoy 0 1870 Decoy chrUn_JTFH01001036v1_decoy 0 1821 Decoy chrUn_JTFH01001037v1_decoy 0 1813 Decoy chrUn_JTFH01001038v1_decoy 0 1809 Decoy chrUn_JTFH01001039v1_decoy 0 1804 Decoy chrUn_JTFH01001040v1_decoy 0 1797 Decoy chrUn_JTFH01001041v1_decoy 0 1791 Decoy chrUn_JTFH01001042v1_decoy 0 1781 Decoy chrUn_JTFH01001043v1_decoy 0 1766 Decoy chrUn_JTFH01001044v1_decoy 0 1764 Decoy chrUn_JTFH01001045v1_decoy 0 1743 Decoy chrUn_JTFH01001046v1_decoy 0 1741 Decoy chrUn_JTFH01001047v1_decoy 0 1709 Decoy chrUn_JTFH01001048v1_decoy 0 1706 Decoy chrUn_JTFH01001049v1_decoy 0 1701 Decoy chrUn_JTFH01001050v1_decoy 0 1689 Decoy chrUn_JTFH01001051v1_decoy 0 1646 Decoy chrUn_JTFH01001052v1_decoy 0 1641 Decoy chrUn_JTFH01001053v1_decoy 0 1639 Decoy chrUn_JTFH01001054v1_decoy 0 1636 Decoy chrUn_JTFH01001055v1_decoy 0 1632 Decoy chrUn_JTFH01001056v1_decoy 0 1629 Decoy chrUn_JTFH01001057v1_decoy 0 1623 Decoy chrUn_JTFH01001058v1_decoy 0 1622 Decoy chrUn_JTFH01001059v1_decoy 0 1622 Decoy chrUn_JTFH01001060v1_decoy 0 1619 Decoy chrUn_JTFH01001061v1_decoy 0 1606 Decoy chrUn_JTFH01001062v1_decoy 0 1593 Decoy chrUn_JTFH01001063v1_decoy 0 1592 Decoy chrUn_JTFH01001064v1_decoy 0 1558 Decoy chrUn_JTFH01001065v1_decoy 0 1545 Decoy chrUn_JTFH01001066v1_decoy 0 1542 Decoy chrUn_JTFH01001067v1_decoy 0 1540 Decoy chrUn_JTFH01001068v1_decoy 0 1529 Decoy chrUn_JTFH01001069v1_decoy 0 1518 Decoy chrUn_JTFH01001070v1_decoy 0 1515 Decoy chrUn_JTFH01001071v1_decoy 0 1513 Decoy chrUn_JTFH01001072v1_decoy 0 1507 Decoy chrUn_JTFH01001073v1_decoy 0 1504 Decoy chrUn_JTFH01001074v1_decoy 0 1499 Decoy chrUn_JTFH01001075v1_decoy 0 1495 Decoy chrUn_JTFH01001076v1_decoy 0 1495 Decoy chrUn_JTFH01001077v1_decoy 0 1492 Decoy chrUn_JTFH01001078v1_decoy 0 1492 Decoy chrUn_JTFH01001079v1_decoy 0 1489 Decoy chrUn_JTFH01001080v1_decoy 0 1485 Decoy chrUn_JTFH01001081v1_decoy 0 1483 Decoy chrUn_JTFH01001082v1_decoy 0 1473 Decoy chrUn_JTFH01001083v1_decoy 0 1470 Decoy chrUn_JTFH01001084v1_decoy 0 1463 Decoy chrUn_JTFH01001085v1_decoy 0 1460 Decoy chrUn_JTFH01001086v1_decoy 0 1458 Decoy chrUn_JTFH01001087v1_decoy 0 1456 Decoy chrUn_JTFH01001088v1_decoy 0 1453 Decoy chrUn_JTFH01001089v1_decoy 0 1443 Decoy chrUn_JTFH01001090v1_decoy 0 1441 Decoy chrUn_JTFH01001091v1_decoy 0 1426 Decoy chrUn_JTFH01001092v1_decoy 0 1425 Decoy chrUn_JTFH01001093v1_decoy 0 1418 Decoy chrUn_JTFH01001094v1_decoy 0 1413 Decoy chrUn_JTFH01001095v1_decoy 0 1413 Decoy chrUn_JTFH01001096v1_decoy 0 1412 Decoy chrUn_JTFH01001097v1_decoy 0 1407 Decoy chrUn_JTFH01001098v1_decoy 0 1406 Decoy chrUn_JTFH01001099v1_decoy 0 1396 Decoy chrUn_JTFH01001100v1_decoy 0 1390 Decoy chrUn_JTFH01001101v1_decoy 0 1382 Decoy chrUn_JTFH01001102v1_decoy 0 1376 Decoy chrUn_JTFH01001103v1_decoy 0 1375 Decoy chrUn_JTFH01001104v1_decoy 0 1371 Decoy chrUn_JTFH01001105v1_decoy 0 1367 Decoy chrUn_JTFH01001106v1_decoy 0 1364 Decoy chrUn_JTFH01001107v1_decoy 0 1356 Decoy chrUn_JTFH01001108v1_decoy 0 1355 Decoy chrUn_JTFH01001109v1_decoy 0 1352 Decoy chrUn_JTFH01001110v1_decoy 0 1350 Decoy chrUn_JTFH01001111v1_decoy 0 1346 Decoy chrUn_JTFH01001112v1_decoy 0 1345 Decoy chrUn_JTFH01001113v1_decoy 0 1340 Decoy chrUn_JTFH01001114v1_decoy 0 1330 Decoy chrUn_JTFH01001115v1_decoy 0 1329 Decoy chrUn_JTFH01001116v1_decoy 0 1324 Decoy chrUn_JTFH01001117v1_decoy 0 1316 Decoy chrUn_JTFH01001118v1_decoy 0 1307 Decoy chrUn_JTFH01001119v1_decoy 0 1304 Decoy chrUn_JTFH01001120v1_decoy 0 1304 Decoy chrUn_JTFH01001121v1_decoy 0 1303 Decoy chrUn_JTFH01001122v1_decoy 0 1301 Decoy chrUn_JTFH01001123v1_decoy 0 1300 Decoy chrUn_JTFH01001124v1_decoy 0 1297 Decoy chrUn_JTFH01001125v1_decoy 0 1296 Decoy chrUn_JTFH01001126v1_decoy 0 1290 Decoy chrUn_JTFH01001127v1_decoy 0 1284 Decoy chrUn_JTFH01001128v1_decoy 0 1282 Decoy chrUn_JTFH01001129v1_decoy 0 1281 Decoy chrUn_JTFH01001130v1_decoy 0 1280 Decoy chrUn_JTFH01001131v1_decoy 0 1279 Decoy chrUn_JTFH01001132v1_decoy 0 1272 Decoy chrUn_JTFH01001133v1_decoy 0 1267 Decoy chrUn_JTFH01001134v1_decoy 0 1267 Decoy chrUn_JTFH01001135v1_decoy 0 1266 Decoy chrUn_JTFH01001136v1_decoy 0 1264 Decoy chrUn_JTFH01001137v1_decoy 0 1264 Decoy chrUn_JTFH01001138v1_decoy 0 1264 Decoy chrUn_JTFH01001139v1_decoy 0 1263 Decoy chrUn_JTFH01001140v1_decoy 0 1249 Decoy chrUn_JTFH01001141v1_decoy 0 1240 Decoy chrUn_JTFH01001142v1_decoy 0 1239 Decoy chrUn_JTFH01001143v1_decoy 0 1235 Decoy chrUn_JTFH01001144v1_decoy 0 1235 Decoy chrUn_JTFH01001145v1_decoy 0 1233 Decoy chrUn_JTFH01001146v1_decoy 0 1232 Decoy chrUn_JTFH01001147v1_decoy 0 1230 Decoy chrUn_JTFH01001148v1_decoy 0 1226 Decoy chrUn_JTFH01001149v1_decoy 0 1223 Decoy chrUn_JTFH01001150v1_decoy 0 1214 Decoy chrUn_JTFH01001151v1_decoy 0 1213 Decoy chrUn_JTFH01001152v1_decoy 0 1211 Decoy chrUn_JTFH01001153v1_decoy 0 1209 Decoy chrUn_JTFH01001154v1_decoy 0 1202 Decoy chrUn_JTFH01001155v1_decoy 0 1199 Decoy chrUn_JTFH01001156v1_decoy 0 1197 Decoy chrUn_JTFH01001157v1_decoy 0 1193 Decoy chrUn_JTFH01001158v1_decoy 0 1191 Decoy chrUn_JTFH01001159v1_decoy 0 1187 Decoy chrUn_JTFH01001160v1_decoy 0 1186 Decoy chrUn_JTFH01001161v1_decoy 0 1184 Decoy chrUn_JTFH01001162v1_decoy 0 1184 Decoy chrUn_JTFH01001163v1_decoy 0 1182 Decoy chrUn_JTFH01001164v1_decoy 0 1179 Decoy chrUn_JTFH01001165v1_decoy 0 1173 Decoy chrUn_JTFH01001166v1_decoy 0 1169 Decoy chrUn_JTFH01001167v1_decoy 0 1167 Decoy chrUn_JTFH01001168v1_decoy 0 1166 Decoy chrUn_JTFH01001169v1_decoy 0 1165 Decoy chrUn_JTFH01001170v1_decoy 0 1164 Decoy chrUn_JTFH01001171v1_decoy 0 1163 Decoy chrUn_JTFH01001172v1_decoy 0 1158 Decoy chrUn_JTFH01001173v1_decoy 0 1158 Decoy chrUn_JTFH01001174v1_decoy 0 1157 Decoy chrUn_JTFH01001175v1_decoy 0 1157 Decoy chrUn_JTFH01001176v1_decoy 0 1157 Decoy chrUn_JTFH01001177v1_decoy 0 1155 Decoy chrUn_JTFH01001178v1_decoy 0 1154 Decoy chrUn_JTFH01001179v1_decoy 0 1149 Decoy chrUn_JTFH01001180v1_decoy 0 1148 Decoy chrUn_JTFH01001181v1_decoy 0 1148 Decoy chrUn_JTFH01001182v1_decoy 0 1146 Decoy chrUn_JTFH01001183v1_decoy 0 1144 Decoy chrUn_JTFH01001184v1_decoy 0 1140 Decoy chrUn_JTFH01001185v1_decoy 0 1136 Decoy chrUn_JTFH01001186v1_decoy 0 1134 Decoy chrUn_JTFH01001187v1_decoy 0 1133 Decoy chrUn_JTFH01001188v1_decoy 0 1129 Decoy chrUn_JTFH01001189v1_decoy 0 1127 Decoy chrUn_JTFH01001190v1_decoy 0 1127 Decoy chrUn_JTFH01001191v1_decoy 0 1118 Decoy chrUn_JTFH01001192v1_decoy 0 1110 Decoy chrUn_JTFH01001193v1_decoy 0 1104 Decoy chrUn_JTFH01001194v1_decoy 0 1104 Decoy chrUn_JTFH01001195v1_decoy 0 1101 Decoy chrUn_JTFH01001196v1_decoy 0 1098 Decoy chrUn_JTFH01001197v1_decoy 0 1096 Decoy chrUn_JTFH01001198v1_decoy 0 1094 Decoy chrUn_JTFH01001199v1_decoy 0 1091 Decoy chrUn_JTFH01001200v1_decoy 0 1089 Decoy chrUn_JTFH01001201v1_decoy 0 1086 Decoy chrUn_JTFH01001202v1_decoy 0 1085 Decoy chrUn_JTFH01001203v1_decoy 0 1084 Decoy chrUn_JTFH01001204v1_decoy 0 1083 Decoy chrUn_JTFH01001205v1_decoy 0 1083 Decoy chrUn_JTFH01001206v1_decoy 0 1079 Decoy chrUn_JTFH01001207v1_decoy 0 1076 Decoy chrUn_JTFH01001208v1_decoy 0 1069 Decoy chrUn_JTFH01001209v1_decoy 0 1068 Decoy chrUn_JTFH01001210v1_decoy 0 1067 Decoy chrUn_JTFH01001211v1_decoy 0 1067 Decoy chrUn_JTFH01001212v1_decoy 0 1067 Decoy chrUn_JTFH01001213v1_decoy 0 1063 Decoy chrUn_JTFH01001214v1_decoy 0 1062 Decoy chrUn_JTFH01001215v1_decoy 0 1059 Decoy chrUn_JTFH01001216v1_decoy 0 1058 Decoy chrUn_JTFH01001217v1_decoy 0 1058 Decoy chrUn_JTFH01001218v1_decoy 0 1055 Decoy chrUn_JTFH01001219v1_decoy 0 1054 Decoy chrUn_JTFH01001220v1_decoy 0 1054 Decoy chrUn_JTFH01001221v1_decoy 0 1053 Decoy chrUn_JTFH01001222v1_decoy 0 1053 Decoy chrUn_JTFH01001223v1_decoy 0 1052 Decoy chrUn_JTFH01001224v1_decoy 0 1051 Decoy chrUn_JTFH01001225v1_decoy 0 1049 Decoy chrUn_JTFH01001226v1_decoy 0 1047 Decoy chrUn_JTFH01001227v1_decoy 0 1044 Decoy chrUn_JTFH01001228v1_decoy 0 1043 Decoy chrUn_JTFH01001229v1_decoy 0 1043 Decoy chrUn_JTFH01001230v1_decoy 0 1042 Decoy chrUn_JTFH01001231v1_decoy 0 1042 Decoy chrUn_JTFH01001232v1_decoy 0 1041 Decoy chrUn_JTFH01001233v1_decoy 0 1040 Decoy chrUn_JTFH01001234v1_decoy 0 1039 Decoy chrUn_JTFH01001235v1_decoy 0 1038 Decoy chrUn_JTFH01001236v1_decoy 0 1037 Decoy chrUn_JTFH01001237v1_decoy 0 1037 Decoy chrUn_JTFH01001238v1_decoy 0 1035 Decoy chrUn_JTFH01001239v1_decoy 0 1027 Decoy chrUn_JTFH01001240v1_decoy 0 1021 Decoy chrUn_JTFH01001241v1_decoy 0 1021 Decoy chrUn_JTFH01001242v1_decoy 0 1019 Decoy chrUn_JTFH01001243v1_decoy 0 1019 Decoy chrUn_JTFH01001244v1_decoy 0 1016 Decoy chrUn_JTFH01001245v1_decoy 0 1014 Decoy chrUn_JTFH01001246v1_decoy 0 1013 Decoy chrUn_JTFH01001247v1_decoy 0 1009 Decoy chrUn_JTFH01001248v1_decoy 0 1008 Decoy chrUn_JTFH01001249v1_decoy 0 1007 Decoy chrUn_JTFH01001250v1_decoy 0 1004 Decoy chrUn_JTFH01001251v1_decoy 0 1004 Decoy chrUn_JTFH01001252v1_decoy 0 1003 Decoy chrUn_JTFH01001253v1_decoy 0 1001 Decoy chrUn_JTFH01001254v1_decoy 0 1000 Decoy chrUn_JTFH01001255v1_decoy 0 1000 Decoy chrUn_JTFH01001256v1_decoy 0 1000 Decoy chrUn_JTFH01001257v1_decoy 0 17929 Decoy chrUn_JTFH01001258v1_decoy 0 9749 Decoy chrUn_JTFH01001259v1_decoy 0 8053 Decoy chrUn_JTFH01001260v1_decoy 0 7826 Decoy chrUn_JTFH01001261v1_decoy 0 7768 Decoy chrUn_JTFH01001262v1_decoy 0 5691 Decoy chrUn_JTFH01001263v1_decoy 0 5444 Decoy chrUn_JTFH01001264v1_decoy 0 5077 Decoy chrUn_JTFH01001265v1_decoy 0 4990 Decoy chrUn_JTFH01001266v1_decoy 0 4545 Decoy chrUn_JTFH01001267v1_decoy 0 4544 Decoy chrUn_JTFH01001268v1_decoy 0 4202 Decoy chrUn_JTFH01001269v1_decoy 0 4195 Decoy chrUn_JTFH01001270v1_decoy 0 3807 Decoy chrUn_JTFH01001271v1_decoy 0 3741 Decoy chrUn_JTFH01001272v1_decoy 0 3699 Decoy chrUn_JTFH01001273v1_decoy 0 3640 Decoy chrUn_JTFH01001274v1_decoy 0 3531 Decoy chrUn_JTFH01001275v1_decoy 0 3455 Decoy chrUn_JTFH01001276v1_decoy 0 3411 Decoy chrUn_JTFH01001277v1_decoy 0 3387 Decoy chrUn_JTFH01001278v1_decoy 0 3358 Decoy chrUn_JTFH01001279v1_decoy 0 3285 Decoy chrUn_JTFH01001280v1_decoy 0 3273 Decoy chrUn_JTFH01001281v1_decoy 0 3262 Decoy chrUn_JTFH01001282v1_decoy 0 3259 Decoy chrUn_JTFH01001283v1_decoy 0 3222 Decoy chrUn_JTFH01001284v1_decoy 0 3127 Decoy chrUn_JTFH01001285v1_decoy 0 3110 Decoy chrUn_JTFH01001286v1_decoy 0 3104 Decoy chrUn_JTFH01001287v1_decoy 0 3071 Decoy chrUn_JTFH01001288v1_decoy 0 3063 Decoy chrUn_JTFH01001289v1_decoy 0 3059 Decoy chrUn_JTFH01001290v1_decoy 0 2990 Decoy chrUn_JTFH01001291v1_decoy 0 2986 Decoy chrUn_JTFH01001292v1_decoy 0 2928 Decoy chrUn_JTFH01001293v1_decoy 0 2922 Decoy chrUn_JTFH01001294v1_decoy 0 2875 Decoy chrUn_JTFH01001295v1_decoy 0 2859 Decoy chrUn_JTFH01001296v1_decoy 0 2850 Decoy chrUn_JTFH01001297v1_decoy 0 2813 Decoy chrUn_JTFH01001298v1_decoy 0 2785 Decoy chrUn_JTFH01001299v1_decoy 0 2736 Decoy chrUn_JTFH01001300v1_decoy 0 2688 Decoy chrUn_JTFH01001301v1_decoy 0 2658 Decoy chrUn_JTFH01001302v1_decoy 0 2643 Decoy chrUn_JTFH01001303v1_decoy 0 2618 Decoy chrUn_JTFH01001304v1_decoy 0 2605 Decoy chrUn_JTFH01001305v1_decoy 0 2583 Decoy chrUn_JTFH01001306v1_decoy 0 2534 Decoy chrUn_JTFH01001307v1_decoy 0 2512 Decoy chrUn_JTFH01001308v1_decoy 0 2500 Decoy chrUn_JTFH01001309v1_decoy 0 2481 Decoy chrUn_JTFH01001310v1_decoy 0 2478 Decoy chrUn_JTFH01001311v1_decoy 0 2473 Decoy chrUn_JTFH01001312v1_decoy 0 2467 Decoy chrUn_JTFH01001313v1_decoy 0 2442 Decoy chrUn_JTFH01001314v1_decoy 0 2430 Decoy chrUn_JTFH01001315v1_decoy 0 2417 Decoy chrUn_JTFH01001316v1_decoy 0 2408 Decoy chrUn_JTFH01001317v1_decoy 0 2395 Decoy chrUn_JTFH01001318v1_decoy 0 2352 Decoy chrUn_JTFH01001319v1_decoy 0 2337 Decoy chrUn_JTFH01001320v1_decoy 0 2322 Decoy chrUn_JTFH01001321v1_decoy 0 2307 Decoy chrUn_JTFH01001322v1_decoy 0 2306 Decoy chrUn_JTFH01001323v1_decoy 0 2292 Decoy chrUn_JTFH01001324v1_decoy 0 2271 Decoy chrUn_JTFH01001325v1_decoy 0 2265 Decoy chrUn_JTFH01001326v1_decoy 0 2260 Decoy chrUn_JTFH01001327v1_decoy 0 2240 Decoy chrUn_JTFH01001328v1_decoy 0 2238 Decoy chrUn_JTFH01001329v1_decoy 0 2228 Decoy chrUn_JTFH01001330v1_decoy 0 2215 Decoy chrUn_JTFH01001331v1_decoy 0 2205 Decoy chrUn_JTFH01001332v1_decoy 0 2191 Decoy chrUn_JTFH01001333v1_decoy 0 2191 Decoy chrUn_JTFH01001334v1_decoy 0 2190 Decoy chrUn_JTFH01001335v1_decoy 0 2184 Decoy chrUn_JTFH01001336v1_decoy 0 2166 Decoy chrUn_JTFH01001337v1_decoy 0 2165 Decoy chrUn_JTFH01001338v1_decoy 0 2162 Decoy chrUn_JTFH01001339v1_decoy 0 2146 Decoy chrUn_JTFH01001340v1_decoy 0 2116 Decoy chrUn_JTFH01001341v1_decoy 0 2112 Decoy chrUn_JTFH01001342v1_decoy 0 2108 Decoy chrUn_JTFH01001343v1_decoy 0 2106 Decoy chrUn_JTFH01001344v1_decoy 0 2106 Decoy chrUn_JTFH01001345v1_decoy 0 2106 Decoy chrUn_JTFH01001346v1_decoy 0 2097 Decoy chrUn_JTFH01001347v1_decoy 0 2081 Decoy chrUn_JTFH01001348v1_decoy 0 2058 Decoy chrUn_JTFH01001349v1_decoy 0 2055 Decoy chrUn_JTFH01001350v1_decoy 0 2054 Decoy chrUn_JTFH01001351v1_decoy 0 2037 Decoy chrUn_JTFH01001352v1_decoy 0 2032 Decoy chrUn_JTFH01001353v1_decoy 0 2032 Decoy chrUn_JTFH01001354v1_decoy 0 2020 Decoy chrUn_JTFH01001355v1_decoy 0 2018 Decoy chrUn_JTFH01001356v1_decoy 0 2014 Decoy chrUn_JTFH01001357v1_decoy 0 2001 Decoy chrUn_JTFH01001358v1_decoy 0 2001 Decoy chrUn_JTFH01001359v1_decoy 0 1991 Decoy chrUn_JTFH01001360v1_decoy 0 1990 Decoy chrUn_JTFH01001361v1_decoy 0 1983 Decoy chrUn_JTFH01001362v1_decoy 0 1981 Decoy chrUn_JTFH01001363v1_decoy 0 1981 Decoy chrUn_JTFH01001364v1_decoy 0 1979 Decoy chrUn_JTFH01001365v1_decoy 0 1963 Decoy chrUn_JTFH01001366v1_decoy 0 1932 Decoy chrUn_JTFH01001367v1_decoy 0 1929 Decoy chrUn_JTFH01001368v1_decoy 0 1881 Decoy chrUn_JTFH01001369v1_decoy 0 1874 Decoy chrUn_JTFH01001370v1_decoy 0 1849 Decoy chrUn_JTFH01001371v1_decoy 0 1849 Decoy chrUn_JTFH01001372v1_decoy 0 1833 Decoy chrUn_JTFH01001373v1_decoy 0 1832 Decoy chrUn_JTFH01001374v1_decoy 0 1826 Decoy chrUn_JTFH01001375v1_decoy 0 1814 Decoy chrUn_JTFH01001376v1_decoy 0 1814 Decoy chrUn_JTFH01001377v1_decoy 0 1791 Decoy chrUn_JTFH01001378v1_decoy 0 1789 Decoy chrUn_JTFH01001379v1_decoy 0 1786 Decoy chrUn_JTFH01001380v1_decoy 0 1778 Decoy chrUn_JTFH01001381v1_decoy 0 1776 Decoy chrUn_JTFH01001382v1_decoy 0 1762 Decoy chrUn_JTFH01001383v1_decoy 0 1758 Decoy chrUn_JTFH01001384v1_decoy 0 1757 Decoy chrUn_JTFH01001385v1_decoy 0 1754 Decoy chrUn_JTFH01001386v1_decoy 0 1752 Decoy chrUn_JTFH01001387v1_decoy 0 1751 Decoy chrUn_JTFH01001388v1_decoy 0 1749 Decoy chrUn_JTFH01001389v1_decoy 0 1738 Decoy chrUn_JTFH01001390v1_decoy 0 1729 Decoy chrUn_JTFH01001391v1_decoy 0 1726 Decoy chrUn_JTFH01001392v1_decoy 0 1716 Decoy chrUn_JTFH01001393v1_decoy 0 1712 Decoy chrUn_JTFH01001394v1_decoy 0 1711 Decoy chrUn_JTFH01001395v1_decoy 0 1703 Decoy chrUn_JTFH01001396v1_decoy 0 1702 Decoy chrUn_JTFH01001397v1_decoy 0 1699 Decoy chrUn_JTFH01001398v1_decoy 0 1686 Decoy chrUn_JTFH01001399v1_decoy 0 1684 Decoy chrUn_JTFH01001400v1_decoy 0 1680 Decoy chrUn_JTFH01001401v1_decoy 0 1678 Decoy chrUn_JTFH01001402v1_decoy 0 1678 Decoy chrUn_JTFH01001403v1_decoy 0 1677 Decoy chrUn_JTFH01001404v1_decoy 0 1676 Decoy chrUn_JTFH01001405v1_decoy 0 1672 Decoy chrUn_JTFH01001406v1_decoy 0 1669 Decoy chrUn_JTFH01001407v1_decoy 0 1668 Decoy chrUn_JTFH01001408v1_decoy 0 1663 Decoy chrUn_JTFH01001409v1_decoy 0 1660 Decoy chrUn_JTFH01001410v1_decoy 0 1660 Decoy chrUn_JTFH01001411v1_decoy 0 1658 Decoy chrUn_JTFH01001412v1_decoy 0 1656 Decoy chrUn_JTFH01001413v1_decoy 0 1656 Decoy chrUn_JTFH01001414v1_decoy 0 1652 Decoy chrUn_JTFH01001415v1_decoy 0 1647 Decoy chrUn_JTFH01001416v1_decoy 0 1645 Decoy chrUn_JTFH01001417v1_decoy 0 1641 Decoy chrUn_JTFH01001418v1_decoy 0 1638 Decoy chrUn_JTFH01001419v1_decoy 0 1633 Decoy chrUn_JTFH01001420v1_decoy 0 1626 Decoy chrUn_JTFH01001421v1_decoy 0 1614 Decoy chrUn_JTFH01001422v1_decoy 0 1612 Decoy chrUn_JTFH01001423v1_decoy 0 1605 Decoy chrUn_JTFH01001424v1_decoy 0 1603 Decoy chrUn_JTFH01001425v1_decoy 0 1599 Decoy chrUn_JTFH01001426v1_decoy 0 1589 Decoy chrUn_JTFH01001427v1_decoy 0 1588 Decoy chrUn_JTFH01001428v1_decoy 0 1585 Decoy chrUn_JTFH01001429v1_decoy 0 1584 Decoy chrUn_JTFH01001430v1_decoy 0 1584 Decoy chrUn_JTFH01001431v1_decoy 0 1580 Decoy chrUn_JTFH01001432v1_decoy 0 1572 Decoy chrUn_JTFH01001433v1_decoy 0 1570 Decoy chrUn_JTFH01001434v1_decoy 0 1569 Decoy chrUn_JTFH01001435v1_decoy 0 1568 Decoy chrUn_JTFH01001436v1_decoy 0 1567 Decoy chrUn_JTFH01001437v1_decoy 0 1565 Decoy chrUn_JTFH01001438v1_decoy 0 1559 Decoy chrUn_JTFH01001439v1_decoy 0 1559 Decoy chrUn_JTFH01001440v1_decoy 0 1556 Decoy chrUn_JTFH01001441v1_decoy 0 1554 Decoy chrUn_JTFH01001442v1_decoy 0 1549 Decoy chrUn_JTFH01001443v1_decoy 0 1542 Decoy chrUn_JTFH01001444v1_decoy 0 1541 Decoy chrUn_JTFH01001445v1_decoy 0 1538 Decoy chrUn_JTFH01001446v1_decoy 0 1537 Decoy chrUn_JTFH01001447v1_decoy 0 1535 Decoy chrUn_JTFH01001448v1_decoy 0 1530 Decoy chrUn_JTFH01001449v1_decoy 0 1528 Decoy chrUn_JTFH01001450v1_decoy 0 1522 Decoy chrUn_JTFH01001451v1_decoy 0 1514 Decoy chrUn_JTFH01001452v1_decoy 0 1509 Decoy chrUn_JTFH01001453v1_decoy 0 1507 Decoy chrUn_JTFH01001454v1_decoy 0 1500 Decoy chrUn_JTFH01001455v1_decoy 0 1499 Decoy chrUn_JTFH01001456v1_decoy 0 1499 Decoy chrUn_JTFH01001457v1_decoy 0 1497 Decoy chrUn_JTFH01001458v1_decoy 0 1496 Decoy chrUn_JTFH01001459v1_decoy 0 1488 Decoy chrUn_JTFH01001460v1_decoy 0 1486 Decoy chrUn_JTFH01001461v1_decoy 0 1485 Decoy chrUn_JTFH01001462v1_decoy 0 1481 Decoy chrUn_JTFH01001463v1_decoy 0 1479 Decoy chrUn_JTFH01001464v1_decoy 0 1472 Decoy chrUn_JTFH01001465v1_decoy 0 1472 Decoy chrUn_JTFH01001466v1_decoy 0 1470 Decoy chrUn_JTFH01001467v1_decoy 0 1466 Decoy chrUn_JTFH01001468v1_decoy 0 1465 Decoy chrUn_JTFH01001469v1_decoy 0 1461 Decoy chrUn_JTFH01001470v1_decoy 0 1458 Decoy chrUn_JTFH01001471v1_decoy 0 1457 Decoy chrUn_JTFH01001472v1_decoy 0 1448 Decoy chrUn_JTFH01001473v1_decoy 0 1447 Decoy chrUn_JTFH01001474v1_decoy 0 1444 Decoy chrUn_JTFH01001475v1_decoy 0 1443 Decoy chrUn_JTFH01001476v1_decoy 0 1443 Decoy chrUn_JTFH01001477v1_decoy 0 1438 Decoy chrUn_JTFH01001478v1_decoy 0 1432 Decoy chrUn_JTFH01001479v1_decoy 0 1430 Decoy chrUn_JTFH01001480v1_decoy 0 1430 Decoy chrUn_JTFH01001481v1_decoy 0 1429 Decoy chrUn_JTFH01001482v1_decoy 0 1429 Decoy chrUn_JTFH01001483v1_decoy 0 1429 Decoy chrUn_JTFH01001484v1_decoy 0 1426 Decoy chrUn_JTFH01001485v1_decoy 0 1426 Decoy chrUn_JTFH01001486v1_decoy 0 1420 Decoy chrUn_JTFH01001487v1_decoy 0 1416 Decoy chrUn_JTFH01001488v1_decoy 0 1416 Decoy chrUn_JTFH01001489v1_decoy 0 1415 Decoy chrUn_JTFH01001490v1_decoy 0 1415 Decoy chrUn_JTFH01001491v1_decoy 0 1414 Decoy chrUn_JTFH01001492v1_decoy 0 1413 Decoy chrUn_JTFH01001493v1_decoy 0 1410 Decoy chrUn_JTFH01001494v1_decoy 0 1405 Decoy chrUn_JTFH01001495v1_decoy 0 1402 Decoy chrUn_JTFH01001496v1_decoy 0 1398 Decoy chrUn_JTFH01001497v1_decoy 0 1397 Decoy chrUn_JTFH01001498v1_decoy 0 1395 Decoy chrUn_JTFH01001499v1_decoy 0 1392 Decoy chrUn_JTFH01001500v1_decoy 0 1388 Decoy chrUn_JTFH01001501v1_decoy 0 1386 Decoy chrUn_JTFH01001502v1_decoy 0 1382 Decoy chrUn_JTFH01001503v1_decoy 0 1381 Decoy chrUn_JTFH01001504v1_decoy 0 1379 Decoy chrUn_JTFH01001505v1_decoy 0 1376 Decoy chrUn_JTFH01001506v1_decoy 0 1374 Decoy chrUn_JTFH01001507v1_decoy 0 1374 Decoy chrUn_JTFH01001508v1_decoy 0 1373 Decoy chrUn_JTFH01001509v1_decoy 0 1373 Decoy chrUn_JTFH01001510v1_decoy 0 1372 Decoy chrUn_JTFH01001511v1_decoy 0 1370 Decoy chrUn_JTFH01001512v1_decoy 0 1367 Decoy chrUn_JTFH01001513v1_decoy 0 1365 Decoy chrUn_JTFH01001514v1_decoy 0 1364 Decoy chrUn_JTFH01001515v1_decoy 0 1361 Decoy chrUn_JTFH01001516v1_decoy 0 1361 Decoy chrUn_JTFH01001517v1_decoy 0 1355 Decoy chrUn_JTFH01001518v1_decoy 0 1355 Decoy chrUn_JTFH01001519v1_decoy 0 1354 Decoy chrUn_JTFH01001520v1_decoy 0 1353 Decoy chrUn_JTFH01001521v1_decoy 0 1349 Decoy chrUn_JTFH01001522v1_decoy 0 1345 Decoy chrUn_JTFH01001523v1_decoy 0 1344 Decoy chrUn_JTFH01001524v1_decoy 0 1343 Decoy chrUn_JTFH01001525v1_decoy 0 1338 Decoy chrUn_JTFH01001526v1_decoy 0 1338 Decoy chrUn_JTFH01001527v1_decoy 0 1338 Decoy chrUn_JTFH01001528v1_decoy 0 1336 Decoy chrUn_JTFH01001529v1_decoy 0 1333 Decoy chrUn_JTFH01001530v1_decoy 0 1333 Decoy chrUn_JTFH01001531v1_decoy 0 1332 Decoy chrUn_JTFH01001532v1_decoy 0 1324 Decoy chrUn_JTFH01001533v1_decoy 0 1323 Decoy chrUn_JTFH01001534v1_decoy 0 1323 Decoy chrUn_JTFH01001535v1_decoy 0 1320 Decoy chrUn_JTFH01001536v1_decoy 0 1320 Decoy chrUn_JTFH01001537v1_decoy 0 1317 Decoy chrUn_JTFH01001538v1_decoy 0 1316 Decoy chrUn_JTFH01001539v1_decoy 0 1304 Decoy chrUn_JTFH01001540v1_decoy 0 1304 Decoy chrUn_JTFH01001541v1_decoy 0 1303 Decoy chrUn_JTFH01001542v1_decoy 0 1302 Decoy chrUn_JTFH01001543v1_decoy 0 1301 Decoy chrUn_JTFH01001544v1_decoy 0 1300 Decoy chrUn_JTFH01001545v1_decoy 0 1298 Decoy chrUn_JTFH01001546v1_decoy 0 1297 Decoy chrUn_JTFH01001547v1_decoy 0 1295 Decoy chrUn_JTFH01001548v1_decoy 0 1284 Decoy chrUn_JTFH01001549v1_decoy 0 1283 Decoy chrUn_JTFH01001550v1_decoy 0 1283 Decoy chrUn_JTFH01001551v1_decoy 0 1279 Decoy chrUn_JTFH01001552v1_decoy 0 1278 Decoy chrUn_JTFH01001553v1_decoy 0 1271 Decoy chrUn_JTFH01001554v1_decoy 0 1271 Decoy chrUn_JTFH01001555v1_decoy 0 1268 Decoy chrUn_JTFH01001556v1_decoy 0 1264 Decoy chrUn_JTFH01001557v1_decoy 0 1263 Decoy chrUn_JTFH01001558v1_decoy 0 1262 Decoy chrUn_JTFH01001559v1_decoy 0 1261 Decoy chrUn_JTFH01001560v1_decoy 0 1260 Decoy chrUn_JTFH01001561v1_decoy 0 1259 Decoy chrUn_JTFH01001562v1_decoy 0 1259 Decoy chrUn_JTFH01001563v1_decoy 0 1258 Decoy chrUn_JTFH01001564v1_decoy 0 1256 Decoy chrUn_JTFH01001565v1_decoy 0 1253 Decoy chrUn_JTFH01001566v1_decoy 0 1248 Decoy chrUn_JTFH01001567v1_decoy 0 1248 Decoy chrUn_JTFH01001568v1_decoy 0 1246 Decoy chrUn_JTFH01001569v1_decoy 0 1246 Decoy chrUn_JTFH01001570v1_decoy 0 1244 Decoy chrUn_JTFH01001571v1_decoy 0 1238 Decoy chrUn_JTFH01001572v1_decoy 0 1238 Decoy chrUn_JTFH01001573v1_decoy 0 1236 Decoy chrUn_JTFH01001574v1_decoy 0 1234 Decoy chrUn_JTFH01001575v1_decoy 0 1234 Decoy chrUn_JTFH01001576v1_decoy 0 1231 Decoy chrUn_JTFH01001577v1_decoy 0 1231 Decoy chrUn_JTFH01001578v1_decoy 0 1230 Decoy chrUn_JTFH01001579v1_decoy 0 1230 Decoy chrUn_JTFH01001580v1_decoy 0 1228 Decoy chrUn_JTFH01001581v1_decoy 0 1227 Decoy chrUn_JTFH01001582v1_decoy 0 1222 Decoy chrUn_JTFH01001583v1_decoy 0 1222 Decoy chrUn_JTFH01001584v1_decoy 0 1221 Decoy chrUn_JTFH01001585v1_decoy 0 1221 Decoy chrUn_JTFH01001586v1_decoy 0 1220 Decoy chrUn_JTFH01001587v1_decoy 0 1218 Decoy chrUn_JTFH01001588v1_decoy 0 1218 Decoy chrUn_JTFH01001589v1_decoy 0 1216 Decoy chrUn_JTFH01001590v1_decoy 0 1216 Decoy chrUn_JTFH01001591v1_decoy 0 1212 Decoy chrUn_JTFH01001592v1_decoy 0 1210 Decoy chrUn_JTFH01001593v1_decoy 0 1209 Decoy chrUn_JTFH01001594v1_decoy 0 1208 Decoy chrUn_JTFH01001595v1_decoy 0 1208 Decoy chrUn_JTFH01001596v1_decoy 0 1206 Decoy chrUn_JTFH01001597v1_decoy 0 1205 Decoy chrUn_JTFH01001598v1_decoy 0 1205 Decoy chrUn_JTFH01001599v1_decoy 0 1202 Decoy chrUn_JTFH01001600v1_decoy 0 1200 Decoy chrUn_JTFH01001601v1_decoy 0 1199 Decoy chrUn_JTFH01001602v1_decoy 0 1198 Decoy chrUn_JTFH01001603v1_decoy 0 1198 Decoy chrUn_JTFH01001604v1_decoy 0 1198 Decoy chrUn_JTFH01001605v1_decoy 0 1195 Decoy chrUn_JTFH01001606v1_decoy 0 1194 Decoy chrUn_JTFH01001607v1_decoy 0 1191 Decoy chrUn_JTFH01001608v1_decoy 0 1189 Decoy chrUn_JTFH01001609v1_decoy 0 1188 Decoy chrUn_JTFH01001610v1_decoy 0 1180 Decoy chrUn_JTFH01001611v1_decoy 0 1180 Decoy chrUn_JTFH01001612v1_decoy 0 1179 Decoy chrUn_JTFH01001613v1_decoy 0 1172 Decoy chrUn_JTFH01001614v1_decoy 0 1168 Decoy chrUn_JTFH01001615v1_decoy 0 1166 Decoy chrUn_JTFH01001616v1_decoy 0 1157 Decoy chrUn_JTFH01001617v1_decoy 0 1156 Decoy chrUn_JTFH01001618v1_decoy 0 1156 Decoy chrUn_JTFH01001619v1_decoy 0 1155 Decoy chrUn_JTFH01001620v1_decoy 0 1154 Decoy chrUn_JTFH01001621v1_decoy 0 1154 Decoy chrUn_JTFH01001622v1_decoy 0 1149 Decoy chrUn_JTFH01001623v1_decoy 0 1143 Decoy chrUn_JTFH01001624v1_decoy 0 1143 Decoy chrUn_JTFH01001625v1_decoy 0 1140 Decoy chrUn_JTFH01001626v1_decoy 0 1137 Decoy chrUn_JTFH01001627v1_decoy 0 1135 Decoy chrUn_JTFH01001628v1_decoy 0 1135 Decoy chrUn_JTFH01001629v1_decoy 0 1135 Decoy chrUn_JTFH01001630v1_decoy 0 1127 Decoy chrUn_JTFH01001631v1_decoy 0 1127 Decoy chrUn_JTFH01001632v1_decoy 0 1126 Decoy chrUn_JTFH01001633v1_decoy 0 1123 Decoy chrUn_JTFH01001634v1_decoy 0 1123 Decoy chrUn_JTFH01001635v1_decoy 0 1123 Decoy chrUn_JTFH01001636v1_decoy 0 1122 Decoy chrUn_JTFH01001637v1_decoy 0 1122 Decoy chrUn_JTFH01001638v1_decoy 0 1121 Decoy chrUn_JTFH01001639v1_decoy 0 1121 Decoy chrUn_JTFH01001640v1_decoy 0 1119 Decoy chrUn_JTFH01001641v1_decoy 0 1119 Decoy chrUn_JTFH01001642v1_decoy 0 1119 Decoy chrUn_JTFH01001643v1_decoy 0 1118 Decoy chrUn_JTFH01001644v1_decoy 0 1115 Decoy chrUn_JTFH01001645v1_decoy 0 1106 Decoy chrUn_JTFH01001646v1_decoy 0 1106 Decoy chrUn_JTFH01001647v1_decoy 0 1104 Decoy chrUn_JTFH01001648v1_decoy 0 1102 Decoy chrUn_JTFH01001649v1_decoy 0 1101 Decoy chrUn_JTFH01001650v1_decoy 0 1098 Decoy chrUn_JTFH01001651v1_decoy 0 1098 Decoy chrUn_JTFH01001652v1_decoy 0 1096 Decoy chrUn_JTFH01001653v1_decoy 0 1096 Decoy chrUn_JTFH01001654v1_decoy 0 1095 Decoy chrUn_JTFH01001655v1_decoy 0 1093 Decoy chrUn_JTFH01001656v1_decoy 0 1090 Decoy chrUn_JTFH01001657v1_decoy 0 1089 Decoy chrUn_JTFH01001658v1_decoy 0 1087 Decoy chrUn_JTFH01001659v1_decoy 0 1087 Decoy chrUn_JTFH01001660v1_decoy 0 1085 Decoy chrUn_JTFH01001661v1_decoy 0 1085 Decoy chrUn_JTFH01001662v1_decoy 0 1085 Decoy chrUn_JTFH01001663v1_decoy 0 1083 Decoy chrUn_JTFH01001664v1_decoy 0 1080 Decoy chrUn_JTFH01001665v1_decoy 0 1080 Decoy chrUn_JTFH01001666v1_decoy 0 1079 Decoy chrUn_JTFH01001667v1_decoy 0 1079 Decoy chrUn_JTFH01001668v1_decoy 0 1079 Decoy chrUn_JTFH01001669v1_decoy 0 1075 Decoy chrUn_JTFH01001670v1_decoy 0 1074 Decoy chrUn_JTFH01001671v1_decoy 0 1073 Decoy chrUn_JTFH01001672v1_decoy 0 1070 Decoy chrUn_JTFH01001673v1_decoy 0 1068 Decoy chrUn_JTFH01001674v1_decoy 0 1067 Decoy chrUn_JTFH01001675v1_decoy 0 1066 Decoy chrUn_JTFH01001676v1_decoy 0 1066 Decoy chrUn_JTFH01001677v1_decoy 0 1066 Decoy chrUn_JTFH01001678v1_decoy 0 1063 Decoy chrUn_JTFH01001679v1_decoy 0 1063 Decoy chrUn_JTFH01001680v1_decoy 0 1063 Decoy chrUn_JTFH01001681v1_decoy 0 1062 Decoy chrUn_JTFH01001682v1_decoy 0 1058 Decoy chrUn_JTFH01001683v1_decoy 0 1056 Decoy chrUn_JTFH01001684v1_decoy 0 1052 Decoy chrUn_JTFH01001685v1_decoy 0 1051 Decoy chrUn_JTFH01001686v1_decoy 0 1051 Decoy chrUn_JTFH01001687v1_decoy 0 1050 Decoy chrUn_JTFH01001688v1_decoy 0 1048 Decoy chrUn_JTFH01001689v1_decoy 0 1046 Decoy chrUn_JTFH01001690v1_decoy 0 1046 Decoy chrUn_JTFH01001691v1_decoy 0 1045 Decoy chrUn_JTFH01001692v1_decoy 0 1043 Decoy chrUn_JTFH01001693v1_decoy 0 1038 Decoy chrUn_JTFH01001694v1_decoy 0 1036 Decoy chrUn_JTFH01001695v1_decoy 0 1035 Decoy chrUn_JTFH01001696v1_decoy 0 1035 Decoy chrUn_JTFH01001697v1_decoy 0 1035 Decoy chrUn_JTFH01001698v1_decoy 0 1033 Decoy chrUn_JTFH01001699v1_decoy 0 1032 Decoy chrUn_JTFH01001700v1_decoy 0 1031 Decoy chrUn_JTFH01001701v1_decoy 0 1026 Decoy chrUn_JTFH01001702v1_decoy 0 1026 Decoy chrUn_JTFH01001703v1_decoy 0 1026 Decoy chrUn_JTFH01001704v1_decoy 0 1023 Decoy chrUn_JTFH01001705v1_decoy 0 1022 Decoy chrUn_JTFH01001706v1_decoy 0 1020 Decoy chrUn_JTFH01001707v1_decoy 0 1020 Decoy chrUn_JTFH01001708v1_decoy 0 1020 Decoy chrUn_JTFH01001709v1_decoy 0 1019 Decoy chrUn_JTFH01001710v1_decoy 0 1018 Decoy chrUn_JTFH01001711v1_decoy 0 1018 Decoy chrUn_JTFH01001712v1_decoy 0 1017 Decoy chrUn_JTFH01001713v1_decoy 0 1015 Decoy chrUn_JTFH01001714v1_decoy 0 1015 Decoy chrUn_JTFH01001715v1_decoy 0 1015 Decoy chrUn_JTFH01001716v1_decoy 0 1014 Decoy chrUn_JTFH01001717v1_decoy 0 1014 Decoy chrUn_JTFH01001718v1_decoy 0 1013 Decoy chrUn_JTFH01001719v1_decoy 0 1013 Decoy chrUn_JTFH01001720v1_decoy 0 1013 Decoy chrUn_JTFH01001721v1_decoy 0 1012 Decoy chrUn_JTFH01001722v1_decoy 0 1011 Decoy chrUn_JTFH01001723v1_decoy 0 1011 Decoy chrUn_JTFH01001724v1_decoy 0 1009 Decoy chrUn_JTFH01001725v1_decoy 0 1008 Decoy chrUn_JTFH01001726v1_decoy 0 1008 Decoy chrUn_JTFH01001727v1_decoy 0 1007 Decoy chrUn_JTFH01001728v1_decoy 0 1007 Decoy chrUn_JTFH01001729v1_decoy 0 1007 Decoy chrUn_JTFH01001730v1_decoy 0 1006 Decoy chrUn_JTFH01001731v1_decoy 0 1005 Decoy chrUn_JTFH01001732v1_decoy 0 1003 Decoy chrUn_JTFH01001733v1_decoy 0 1001 Decoy chrUn_JTFH01001734v1_decoy 0 1000 Decoy chrUn_JTFH01001735v1_decoy 0 19311 Decoy chrUn_JTFH01001736v1_decoy 0 11713 Decoy chrUn_JTFH01001737v1_decoy 0 11263 Decoy chrUn_JTFH01001738v1_decoy 0 9779 Decoy chrUn_JTFH01001739v1_decoy 0 9568 Decoy chrUn_JTFH01001740v1_decoy 0 9344 Decoy chrUn_JTFH01001741v1_decoy 0 9188 Decoy chrUn_JTFH01001742v1_decoy 0 9100 Decoy chrUn_JTFH01001743v1_decoy 0 8771 Decoy chrUn_JTFH01001744v1_decoy 0 8690 Decoy chrUn_JTFH01001745v1_decoy 0 8566 Decoy chrUn_JTFH01001746v1_decoy 0 8058 Decoy chrUn_JTFH01001747v1_decoy 0 7759 Decoy chrUn_JTFH01001748v1_decoy 0 7585 Decoy chrUn_JTFH01001749v1_decoy 0 7471 Decoy chrUn_JTFH01001750v1_decoy 0 7461 Decoy chrUn_JTFH01001751v1_decoy 0 7342 Decoy chrUn_JTFH01001752v1_decoy 0 7223 Decoy chrUn_JTFH01001753v1_decoy 0 7064 Decoy chrUn_JTFH01001754v1_decoy 0 6916 Decoy chrUn_JTFH01001755v1_decoy 0 6897 Decoy chrUn_JTFH01001756v1_decoy 0 6880 Decoy chrUn_JTFH01001757v1_decoy 0 6857 Decoy chrUn_JTFH01001758v1_decoy 0 6840 Decoy chrUn_JTFH01001759v1_decoy 0 6728 Decoy chrUn_JTFH01001760v1_decoy 0 6688 Decoy chrUn_JTFH01001761v1_decoy 0 6553 Decoy chrUn_JTFH01001762v1_decoy 0 6396 Decoy chrUn_JTFH01001763v1_decoy 0 6345 Decoy chrUn_JTFH01001764v1_decoy 0 6295 Decoy chrUn_JTFH01001765v1_decoy 0 6266 Decoy chrUn_JTFH01001766v1_decoy 0 6173 Decoy chrUn_JTFH01001767v1_decoy 0 6171 Decoy chrUn_JTFH01001768v1_decoy 0 6120 Decoy chrUn_JTFH01001769v1_decoy 0 6105 Decoy chrUn_JTFH01001770v1_decoy 0 6099 Decoy chrUn_JTFH01001771v1_decoy 0 5893 Decoy chrUn_JTFH01001772v1_decoy 0 5829 Decoy chrUn_JTFH01001773v1_decoy 0 5793 Decoy chrUn_JTFH01001774v1_decoy 0 5776 Decoy chrUn_JTFH01001775v1_decoy 0 5759 Decoy chrUn_JTFH01001776v1_decoy 0 5716 Decoy chrUn_JTFH01001777v1_decoy 0 5708 Decoy chrUn_JTFH01001778v1_decoy 0 5590 Decoy chrUn_JTFH01001779v1_decoy 0 5566 Decoy chrUn_JTFH01001780v1_decoy 0 5558 Decoy chrUn_JTFH01001781v1_decoy 0 5418 Decoy chrUn_JTFH01001782v1_decoy 0 5375 Decoy chrUn_JTFH01001783v1_decoy 0 5300 Decoy chrUn_JTFH01001784v1_decoy 0 5255 Decoy chrUn_JTFH01001785v1_decoy 0 5157 Decoy chrUn_JTFH01001786v1_decoy 0 5130 Decoy chrUn_JTFH01001787v1_decoy 0 4978 Decoy chrUn_JTFH01001788v1_decoy 0 4957 Decoy chrUn_JTFH01001789v1_decoy 0 4947 Decoy chrUn_JTFH01001790v1_decoy 0 4897 Decoy chrUn_JTFH01001791v1_decoy 0 4867 Decoy chrUn_JTFH01001792v1_decoy 0 4845 Decoy chrUn_JTFH01001793v1_decoy 0 4678 Decoy chrUn_JTFH01001794v1_decoy 0 4641 Decoy chrUn_JTFH01001795v1_decoy 0 4592 Decoy chrUn_JTFH01001796v1_decoy 0 4543 Decoy chrUn_JTFH01001797v1_decoy 0 4532 Decoy chrUn_JTFH01001798v1_decoy 0 4503 Decoy chrUn_JTFH01001799v1_decoy 0 4495 Decoy chrUn_JTFH01001800v1_decoy 0 4444 Decoy chrUn_JTFH01001801v1_decoy 0 4414 Decoy chrUn_JTFH01001802v1_decoy 0 4409 Decoy chrUn_JTFH01001803v1_decoy 0 4302 Decoy chrUn_JTFH01001804v1_decoy 0 4300 Decoy chrUn_JTFH01001805v1_decoy 0 4277 Decoy chrUn_JTFH01001806v1_decoy 0 4173 Decoy chrUn_JTFH01001807v1_decoy 0 4169 Decoy chrUn_JTFH01001808v1_decoy 0 4136 Decoy chrUn_JTFH01001809v1_decoy 0 4101 Decoy chrUn_JTFH01001810v1_decoy 0 4089 Decoy chrUn_JTFH01001811v1_decoy 0 4015 Decoy chrUn_JTFH01001812v1_decoy 0 4000 Decoy chrUn_JTFH01001813v1_decoy 0 3973 Decoy chrUn_JTFH01001814v1_decoy 0 3732 Decoy chrUn_JTFH01001815v1_decoy 0 3709 Decoy chrUn_JTFH01001816v1_decoy 0 3686 Decoy chrUn_JTFH01001817v1_decoy 0 3676 Decoy chrUn_JTFH01001818v1_decoy 0 3673 Decoy chrUn_JTFH01001819v1_decoy 0 3672 Decoy chrUn_JTFH01001820v1_decoy 0 3633 Decoy chrUn_JTFH01001821v1_decoy 0 3633 Decoy chrUn_JTFH01001822v1_decoy 0 3613 Decoy chrUn_JTFH01001823v1_decoy 0 3605 Decoy chrUn_JTFH01001824v1_decoy 0 3592 Decoy chrUn_JTFH01001825v1_decoy 0 3586 Decoy chrUn_JTFH01001826v1_decoy 0 3584 Decoy chrUn_JTFH01001827v1_decoy 0 3577 Decoy chrUn_JTFH01001828v1_decoy 0 3537 Decoy chrUn_JTFH01001829v1_decoy 0 3510 Decoy chrUn_JTFH01001830v1_decoy 0 3509 Decoy chrUn_JTFH01001831v1_decoy 0 3488 Decoy chrUn_JTFH01001832v1_decoy 0 3473 Decoy chrUn_JTFH01001833v1_decoy 0 3445 Decoy chrUn_JTFH01001834v1_decoy 0 3427 Decoy chrUn_JTFH01001835v1_decoy 0 3395 Decoy chrUn_JTFH01001836v1_decoy 0 3367 Decoy chrUn_JTFH01001837v1_decoy 0 3337 Decoy chrUn_JTFH01001838v1_decoy 0 3324 Decoy chrUn_JTFH01001839v1_decoy 0 3315 Decoy chrUn_JTFH01001840v1_decoy 0 3313 Decoy chrUn_JTFH01001841v1_decoy 0 3283 Decoy chrUn_JTFH01001842v1_decoy 0 3250 Decoy chrUn_JTFH01001843v1_decoy 0 3247 Decoy chrUn_JTFH01001844v1_decoy 0 3237 Decoy chrUn_JTFH01001845v1_decoy 0 3235 Decoy chrUn_JTFH01001846v1_decoy 0 3200 Decoy chrUn_JTFH01001847v1_decoy 0 3195 Decoy chrUn_JTFH01001848v1_decoy 0 3175 Decoy chrUn_JTFH01001849v1_decoy 0 3158 Decoy chrUn_JTFH01001850v1_decoy 0 3143 Decoy chrUn_JTFH01001851v1_decoy 0 3139 Decoy chrUn_JTFH01001852v1_decoy 0 3138 Decoy chrUn_JTFH01001853v1_decoy 0 3136 Decoy chrUn_JTFH01001854v1_decoy 0 3132 Decoy chrUn_JTFH01001855v1_decoy 0 3132 Decoy chrUn_JTFH01001856v1_decoy 0 3095 Decoy chrUn_JTFH01001857v1_decoy 0 3094 Decoy chrUn_JTFH01001858v1_decoy 0 3093 Decoy chrUn_JTFH01001859v1_decoy 0 3059 Decoy chrUn_JTFH01001860v1_decoy 0 2985 Decoy chrUn_JTFH01001861v1_decoy 0 2975 Decoy chrUn_JTFH01001862v1_decoy 0 2967 Decoy chrUn_JTFH01001863v1_decoy 0 2961 Decoy chrUn_JTFH01001864v1_decoy 0 2955 Decoy chrUn_JTFH01001865v1_decoy 0 2935 Decoy chrUn_JTFH01001866v1_decoy 0 2933 Decoy chrUn_JTFH01001867v1_decoy 0 2909 Decoy chrUn_JTFH01001868v1_decoy 0 2904 Decoy chrUn_JTFH01001869v1_decoy 0 2892 Decoy chrUn_JTFH01001870v1_decoy 0 2886 Decoy chrUn_JTFH01001871v1_decoy 0 2885 Decoy chrUn_JTFH01001872v1_decoy 0 2878 Decoy chrUn_JTFH01001873v1_decoy 0 2875 Decoy chrUn_JTFH01001874v1_decoy 0 2861 Decoy chrUn_JTFH01001875v1_decoy 0 2856 Decoy chrUn_JTFH01001876v1_decoy 0 2838 Decoy chrUn_JTFH01001877v1_decoy 0 2801 Decoy chrUn_JTFH01001878v1_decoy 0 2797 Decoy chrUn_JTFH01001879v1_decoy 0 2788 Decoy chrUn_JTFH01001880v1_decoy 0 2773 Decoy chrUn_JTFH01001881v1_decoy 0 2755 Decoy chrUn_JTFH01001882v1_decoy 0 2754 Decoy chrUn_JTFH01001883v1_decoy 0 2743 Decoy chrUn_JTFH01001884v1_decoy 0 2725 Decoy chrUn_JTFH01001885v1_decoy 0 2722 Decoy chrUn_JTFH01001886v1_decoy 0 2682 Decoy chrUn_JTFH01001887v1_decoy 0 2669 Decoy chrUn_JTFH01001888v1_decoy 0 2663 Decoy chrUn_JTFH01001889v1_decoy 0 2652 Decoy chrUn_JTFH01001890v1_decoy 0 2647 Decoy chrUn_JTFH01001891v1_decoy 0 2635 Decoy chrUn_JTFH01001892v1_decoy 0 2633 Decoy chrUn_JTFH01001893v1_decoy 0 2629 Decoy chrUn_JTFH01001894v1_decoy 0 2612 Decoy chrUn_JTFH01001895v1_decoy 0 2599 Decoy chrUn_JTFH01001896v1_decoy 0 2566 Decoy chrUn_JTFH01001897v1_decoy 0 2556 Decoy chrUn_JTFH01001898v1_decoy 0 2551 Decoy chrUn_JTFH01001899v1_decoy 0 2551 Decoy chrUn_JTFH01001900v1_decoy 0 2538 Decoy chrUn_JTFH01001901v1_decoy 0 2538 Decoy chrUn_JTFH01001902v1_decoy 0 2525 Decoy chrUn_JTFH01001903v1_decoy 0 2498 Decoy chrUn_JTFH01001904v1_decoy 0 2496 Decoy chrUn_JTFH01001905v1_decoy 0 2483 Decoy chrUn_JTFH01001906v1_decoy 0 2475 Decoy chrUn_JTFH01001907v1_decoy 0 2469 Decoy chrUn_JTFH01001908v1_decoy 0 2455 Decoy chrUn_JTFH01001909v1_decoy 0 2444 Decoy chrUn_JTFH01001910v1_decoy 0 2437 Decoy chrUn_JTFH01001911v1_decoy 0 2435 Decoy chrUn_JTFH01001912v1_decoy 0 2427 Decoy chrUn_JTFH01001913v1_decoy 0 2419 Decoy chrUn_JTFH01001914v1_decoy 0 2413 Decoy chrUn_JTFH01001915v1_decoy 0 2412 Decoy chrUn_JTFH01001916v1_decoy 0 2400 Decoy chrUn_JTFH01001917v1_decoy 0 2399 Decoy chrUn_JTFH01001918v1_decoy 0 2396 Decoy chrUn_JTFH01001919v1_decoy 0 2393 Decoy chrUn_JTFH01001920v1_decoy 0 2386 Decoy chrUn_JTFH01001921v1_decoy 0 2384 Decoy chrUn_JTFH01001922v1_decoy 0 2382 Decoy chrUn_JTFH01001923v1_decoy 0 2382 Decoy chrUn_JTFH01001924v1_decoy 0 2367 Decoy chrUn_JTFH01001925v1_decoy 0 2366 Decoy chrUn_JTFH01001926v1_decoy 0 2362 Decoy chrUn_JTFH01001927v1_decoy 0 2361 Decoy chrUn_JTFH01001928v1_decoy 0 2353 Decoy chrUn_JTFH01001929v1_decoy 0 2349 Decoy chrUn_JTFH01001930v1_decoy 0 2348 Decoy chrUn_JTFH01001931v1_decoy 0 2340 Decoy chrUn_JTFH01001932v1_decoy 0 2339 Decoy chrUn_JTFH01001933v1_decoy 0 2336 Decoy chrUn_JTFH01001934v1_decoy 0 2333 Decoy chrUn_JTFH01001935v1_decoy 0 2330 Decoy chrUn_JTFH01001936v1_decoy 0 2327 Decoy chrUn_JTFH01001937v1_decoy 0 2318 Decoy chrUn_JTFH01001938v1_decoy 0 2293 Decoy chrUn_JTFH01001939v1_decoy 0 2292 Decoy chrUn_JTFH01001940v1_decoy 0 2287 Decoy chrUn_JTFH01001941v1_decoy 0 2274 Decoy chrUn_JTFH01001942v1_decoy 0 2274 Decoy chrUn_JTFH01001943v1_decoy 0 2267 Decoy chrUn_JTFH01001944v1_decoy 0 2260 Decoy chrUn_JTFH01001945v1_decoy 0 2257 Decoy chrUn_JTFH01001946v1_decoy 0 2240 Decoy chrUn_JTFH01001947v1_decoy 0 2239 Decoy chrUn_JTFH01001948v1_decoy 0 2232 Decoy chrUn_JTFH01001949v1_decoy 0 2230 Decoy chrUn_JTFH01001950v1_decoy 0 2230 Decoy chrUn_JTFH01001951v1_decoy 0 2222 Decoy chrUn_JTFH01001952v1_decoy 0 2216 Decoy chrUn_JTFH01001953v1_decoy 0 2214 Decoy chrUn_JTFH01001954v1_decoy 0 2210 Decoy chrUn_JTFH01001955v1_decoy 0 2203 Decoy chrUn_JTFH01001956v1_decoy 0 2197 Decoy chrUn_JTFH01001957v1_decoy 0 2196 Decoy chrUn_JTFH01001958v1_decoy 0 2196 Decoy chrUn_JTFH01001959v1_decoy 0 2179 Decoy chrUn_JTFH01001960v1_decoy 0 2178 Decoy chrUn_JTFH01001961v1_decoy 0 2178 Decoy chrUn_JTFH01001962v1_decoy 0 2172 Decoy chrUn_JTFH01001963v1_decoy 0 2170 Decoy chrUn_JTFH01001964v1_decoy 0 2167 Decoy chrUn_JTFH01001965v1_decoy 0 2167 Decoy chrUn_JTFH01001966v1_decoy 0 2157 Decoy chrUn_JTFH01001967v1_decoy 0 2153 Decoy chrUn_JTFH01001968v1_decoy 0 2151 Decoy chrUn_JTFH01001969v1_decoy 0 2147 Decoy chrUn_JTFH01001970v1_decoy 0 2145 Decoy chrUn_JTFH01001971v1_decoy 0 2142 Decoy chrUn_JTFH01001972v1_decoy 0 2142 Decoy chrUn_JTFH01001973v1_decoy 0 2136 Decoy chrUn_JTFH01001974v1_decoy 0 2130 Decoy chrUn_JTFH01001975v1_decoy 0 2128 Decoy chrUn_JTFH01001976v1_decoy 0 2126 Decoy chrUn_JTFH01001977v1_decoy 0 2126 Decoy chrUn_JTFH01001978v1_decoy 0 2119 Decoy chrUn_JTFH01001979v1_decoy 0 2107 Decoy chrUn_JTFH01001980v1_decoy 0 2091 Decoy chrUn_JTFH01001981v1_decoy 0 2087 Decoy chrUn_JTFH01001982v1_decoy 0 2086 Decoy chrUn_JTFH01001983v1_decoy 0 2083 Decoy chrUn_JTFH01001984v1_decoy 0 2075 Decoy chrUn_JTFH01001985v1_decoy 0 2075 Decoy chrUn_JTFH01001986v1_decoy 0 2072 Decoy chrUn_JTFH01001987v1_decoy 0 2068 Decoy chrUn_JTFH01001988v1_decoy 0 2067 Decoy chrUn_JTFH01001989v1_decoy 0 2055 Decoy chrUn_JTFH01001990v1_decoy 0 2051 Decoy chrUn_JTFH01001991v1_decoy 0 2050 Decoy chrUn_JTFH01001992v1_decoy 0 2033 Decoy chrUn_JTFH01001993v1_decoy 0 2024 Decoy chrUn_JTFH01001994v1_decoy 0 2016 Decoy chrUn_JTFH01001995v1_decoy 0 2011 Decoy chrUn_JTFH01001996v1_decoy 0 2009 Decoy chrUn_JTFH01001997v1_decoy 0 2003 Decoy chrUn_JTFH01001998v1_decoy 0 2001 Decoy ================================================ FILE: tracks/hg38.rmsk.simple_repeat.bed ================================================ [File too large to display: 53.5 MB] ================================================ FILE: tracks/hg38_arms_excl_centromeres.bed ================================================ chr1 0 121700000 chr1 14300000 12000000 chr1 15400000 58000000 chr1 18700000 24400000 chr1 19000000 17000000 chr1 19500000 16000000 chr1 20500000 35300000 chr1 25800000 15400000 chr1 28000000 25800000 chr1 30000000 10900000 chr1 38200000 22100000 chr1 38600000 16000000 chr1 42900000 51400000 chr1 48100000 47100000 chr1 50700000 46100000 chr1 50700000 58600000 chr1 50700000 39100000 chr1 55800000 34700000 chr1 61900000 248956422 chr1 62700000 43900000 chr1 63400000 58000000 chr1 94000000 87800000 chr1 94000000 48200000 chr1 124600000 91800000 chr2 0 121700000 chr2 14300000 12000000 chr2 15400000 58000000 chr2 18700000 24400000 chr2 19000000 17000000 chr2 19500000 16000000 chr2 20500000 35300000 chr2 25800000 15400000 chr2 28000000 25800000 chr2 30000000 10900000 chr2 38200000 22100000 chr2 38600000 16000000 chr2 42900000 51400000 chr2 48100000 47100000 chr2 50700000 46100000 chr2 50700000 58600000 chr2 50700000 39100000 chr2 55800000 34700000 chr2 61900000 242193529 chr2 62700000 43900000 chr2 63400000 58000000 chr2 94000000 87800000 chr2 94000000 48200000 chr2 124600000 91800000 chr3 0 121700000 chr3 14300000 12000000 chr3 15400000 58000000 chr3 18700000 24400000 chr3 19000000 17000000 chr3 19500000 16000000 chr3 20500000 35300000 chr3 25800000 15400000 chr3 28000000 25800000 chr3 30000000 10900000 chr3 38200000 22100000 chr3 38600000 16000000 chr3 42900000 51400000 chr3 48100000 47100000 chr3 50700000 46100000 chr3 50700000 58600000 chr3 50700000 39100000 chr3 55800000 34700000 chr3 61900000 198295559 chr3 62700000 43900000 chr3 63400000 58000000 chr3 94000000 87800000 chr3 94000000 48200000 chr3 124600000 91800000 chr4 0 121700000 chr4 14300000 12000000 chr4 15400000 58000000 chr4 18700000 24400000 chr4 19000000 17000000 chr4 19500000 16000000 chr4 20500000 35300000 chr4 25800000 15400000 chr4 28000000 25800000 chr4 30000000 10900000 chr4 38200000 22100000 chr4 38600000 16000000 chr4 42900000 51400000 chr4 48100000 47100000 chr4 50700000 46100000 chr4 50700000 58600000 chr4 50700000 39100000 chr4 55800000 34700000 chr4 61900000 190214555 chr4 62700000 43900000 chr4 63400000 58000000 chr4 94000000 87800000 chr4 94000000 48200000 chr4 124600000 91800000 chr5 0 121700000 chr5 14300000 12000000 chr5 15400000 58000000 chr5 18700000 24400000 chr5 19000000 17000000 chr5 19500000 16000000 chr5 20500000 35300000 chr5 25800000 15400000 chr5 28000000 25800000 chr5 30000000 10900000 chr5 38200000 22100000 chr5 38600000 16000000 chr5 42900000 51400000 chr5 48100000 47100000 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0.318 16 chrY 22274700 22275000 0.4872 25 chrY 22288800 22289400 0.1461 52 chrY 22313400 22314000 0.4668 29 chrY 22341900 22342800 0.1696 74 chrY 22344300 22344900 0.1696 74 chrY 22377600 22725900 0 53 chrY 22730700 22734600 0.0225 42 chrY 22735800 22739700 0.0225 42 chrY 22741500 22742400 0.0225 42 chrY 22743600 26311500 0 69 chrY 26345100 26345400 0.5778 31 chrY 26407500 26407800 0.4148 30 chrY 26436900 26437200 0.6871 129 chrY 26440500 26441100 0.3966 170 chrY 26511600 26511900 0.4686 24 chrY 26514600 26514900 0.6505 30 chrY 26589000 26589300 0.444 45 chrY 26637900 26670000 0.06 1063 chrY 26671800 26673300 0.1076 600 chrY 56673000 56771700 0 28529 chrY 56825700 56830500 0.4436 1724 chrY 56832600 56837100 0.4786 3185 chrY 56838900 56839500 0.6016 925 chrY 56842200 56842500 0.6384 926 chrY 56846100 56847300 0.6157 547 chrY 56850300 56851200 0.4243 1367 chrY 56884500 56884800 0.5545 46 ================================================ FILE: tracks/region_generator.R ================================================ library(data.table) options(scipen = 999) # Load hg38 chromosome sizes from UCSC chr_sizes <- fread("https://hgdownload.soe.ucsc.edu/goldenPath/hg38/bigZips/hg38.chrom.sizes", col.names = c("chrom", "size")) chr_sizes <- chr_sizes[chrom %in% paste0("chr", c(1:22, "X"))] # Centromere positions (approximate from UCSC Table Browser / literature) centromeres <- fread(" chrom start end chr1 121700000 124600000 chr2 91800000 94000000 chr3 87800000 94000000 chr4 48200000 50700000 chr5 46100000 50700000 chr6 58600000 63400000 chr7 58000000 62700000 chr8 43900000 48100000 chr9 47100000 50700000 chr10 39100000 42900000 chr11 51400000 55800000 chr12 34700000 38600000 chr13 16000000 19500000 chr14 16000000 19000000 chr15 17000000 20500000 chr16 35300000 38200000 chr17 22100000 25800000 chr18 15400000 18700000 chr19 24400000 28000000 chr20 25800000 30000000 chr21 10900000 14300000 chr22 12000000 15400000 chrX 58000000 61900000 ") # Construct p and q arms bed <- rbindlist(lapply(1:nrow(chr_sizes), function(i) { chrom <- chr_sizes$chrom[i] size <- chr_sizes$size[i] cent <- centromeres[chrom == chrom] if (nrow(cent) == 0) return(NULL) data.table( chrom = c(chrom, chrom), start = c(0, cent$end), end = c(cent$start, size) ) })) # Natural chromosome order: chr1 chr22, chrX bed[, chrom := factor(chrom, levels = paste0("chr", c(1:22, "X")))] # Sort by chromosome and start position setorder(bed, chrom, start) # Write to BED file fwrite(bed, "hg38_arms_excl_centromeres.bed", sep = "\t", col.names = FALSE)