From 44557b96b5c930b9afe281b4f1d626ae4fac14cf Mon Sep 17 00:00:00 2001 From: takutosato Date: Mon, 16 Mar 2020 15:02:32 -0400 Subject: [PATCH] A new tool to randomly sample a fraction of an input bam sorted by UMI. --- .../hellbender/engine/DuplicateSetWalker.java | 153 ++++++++++++++++++ .../consensus/DownsampleByDuplicateSet.java | 103 ++++++++++++ .../tools/walkers/consensus/MoleculeID.java | 67 ++++++++ .../walkers/consensus/ReadsWithSameUMI.java | 106 ++++++++++++ ...wnsampleByDuplicateSetIntegrationTest.java | 100 ++++++++++++ .../walkers/consensus/MoleculeIDUnitTest.java | 33 ++++ .../consensus/ReadsWithSameUMIUnitTest.java | 93 +++++++++++ .../NA12878.grouped.bam | Bin 0 -> 340989 bytes 8 files changed, 655 insertions(+) create mode 100644 src/main/java/org/broadinstitute/hellbender/engine/DuplicateSetWalker.java create mode 100644 src/main/java/org/broadinstitute/hellbender/tools/walkers/consensus/DownsampleByDuplicateSet.java create mode 100644 src/main/java/org/broadinstitute/hellbender/tools/walkers/consensus/MoleculeID.java create mode 100644 src/main/java/org/broadinstitute/hellbender/tools/walkers/consensus/ReadsWithSameUMI.java create mode 100644 src/test/java/org/broadinstitute/hellbender/tools/walkers/consensus/DownsampleByDuplicateSetIntegrationTest.java create mode 100644 src/test/java/org/broadinstitute/hellbender/tools/walkers/consensus/MoleculeIDUnitTest.java create mode 100644 src/test/java/org/broadinstitute/hellbender/tools/walkers/consensus/ReadsWithSameUMIUnitTest.java create mode 100644 src/test/resources/org/broadinstitute/hellbender/tools/downsampleByDuplicateSet/NA12878.grouped.bam diff --git a/src/main/java/org/broadinstitute/hellbender/engine/DuplicateSetWalker.java b/src/main/java/org/broadinstitute/hellbender/engine/DuplicateSetWalker.java new file mode 100644 index 00000000000..152bd687608 --- /dev/null +++ b/src/main/java/org/broadinstitute/hellbender/engine/DuplicateSetWalker.java @@ -0,0 +1,153 @@ +package org.broadinstitute.hellbender.engine; + +import htsjdk.samtools.SAMTag; +import org.apache.commons.lang3.tuple.Pair; +import org.broadinstitute.barclay.argparser.Argument; +import org.broadinstitute.hellbender.engine.filters.ReadFilter; +import org.broadinstitute.hellbender.engine.filters.ReadFilterLibrary; +import org.broadinstitute.hellbender.engine.filters.WellformedReadFilter; +import org.broadinstitute.hellbender.exceptions.UserException; +import org.broadinstitute.hellbender.tools.walkers.consensus.MoleculeID; +import org.broadinstitute.hellbender.tools.walkers.consensus.ReadsWithSameUMI; +import org.broadinstitute.hellbender.utils.read.GATKRead; + +import java.util.ArrayList; +import java.util.List; + +/** + * A walker that processes duplicate reads that share the same Unique molecule Identifier (UMI) as a single unit. + * + * This tool assumes that the input bam has been sorted by UMI (the {@link SAMTag.MI} tag to be specific) with FGBio GroupReadsByUmi: + * http://fulcrumgenomics.github.io/fgbio/tools/latest/GroupReadsByUmi.html + */ +public abstract class DuplicateSetWalker extends ReadWalker { + public static final String MIN_REQUIRED_READS_NAME = "min-reads"; + public static final String MIN_REQUIRED_READS_PER_STRAND_NAME = "min-per-strand-reads"; + + private static final int DEFAULT_MINIMUM_READS_PER_SET = 1; + private static final int DEFAULT_MINIMUM_READS_PER_STRAND = 0; + + @Argument(fullName = MIN_REQUIRED_READS_NAME, doc = "The mininum total number of reads required in the set", optional = true, minValue = 0) + private int minimumRequiredReadsPerUMI = DEFAULT_MINIMUM_READS_PER_SET; + + // The user may choose to only keep read sets containing both strands (i.d. duplex evidence) by setting this argument to a positive number + @Argument(fullName = MIN_REQUIRED_READS_PER_STRAND_NAME, doc = "The mininum total number of reads in each strand", optional = true, minValue = 0) + private int minimumRequiredReadsPerStrand = DEFAULT_MINIMUM_READS_PER_STRAND; + + protected ReadsWithSameUMI currentReadsWithSameUMI = null; + + @Override + public final void traverse(){ + super.traverse(); + processLastReadSet(); + } + + /*** + * FGBio GroupByUMI returns reads sorted by molecule ID: For example, the input bam may look like + * read1: ... MI:Z:0/A ... + * read2: ... MI:Z:0/A ... + * read3: ... MI:Z:0/B ... + * read4: ... MI:Z:0/B ... + * read5: ... MI:Z:1/A ... + * read6: ... MI:Z:1/B ... + * read7: ... MI:Z:1/B ... + * + * Thus it's sufficient to go through the reads in order and collect them in a list until + * we encounter the next molecule ID, at which point we pass the list to the {@code apply} method, + * process the set based on the child class's implementation of the method, and clear the {@code currentDuplicateSet} variable and start collecting reads again. + * + * Notice there are two apply() methods in this class: + * This apply() inherited from ReadWalker is marked final to discourage subclassing. + * A subclass must override the other apply() method that takes in the DuplicateSet. + */ + @Override + public final void apply(GATKRead read, ReferenceContext referenceContext, FeatureContext featureContext) { + if (currentReadsWithSameUMI == null){ // evaluates to true for the very first read + currentReadsWithSameUMI = new ReadsWithSameUMI(read); + return; + } + + final int readMoleculeNumber = MoleculeID.getMoleculeNumberOfRead(read); + final int duplicateSetMoleculeNumber = currentReadsWithSameUMI.getMoleculeNumber(); + + // If the incoming read has the molecule id less than that of the currentDuplicateSet, + // the input bam is not sorted properly by the MI tag + if (duplicateSetMoleculeNumber > readMoleculeNumber){ + throw new UserException(String.format("The input bam must be sorted by the molecule ID (%s) tag.", SAMTag.MI.name())); + } + + if (duplicateSetMoleculeNumber < readMoleculeNumber) { + // The incoming read's molecule ID is greater than that of the current set, meaning we've reached the end of the current set. + if (rejectSet(currentReadsWithSameUMI)){ + currentReadsWithSameUMI = new ReadsWithSameUMI(read); + return; + } + + // Call the apply() method to process the current set and start a new set. + apply(currentReadsWithSameUMI, + new ReferenceContext(reference, currentReadsWithSameUMI.getInterval()), // Will create an empty ReferenceContext if reference or readInterval == null + new FeatureContext(features, currentReadsWithSameUMI.getInterval())); + currentReadsWithSameUMI = new ReadsWithSameUMI(read); + return; + } + + // The incoming read has the same UMI as the current set; simply add to the current set + currentReadsWithSameUMI.addRead(read); + } + + /** + * A subclass must specify how to process the duplicate sets by overriding this method. + * + * @param readsWithSameUMI A set of reads with the matching UMIs with the same fragment start and end + * @param referenceContext A reference context object over the intervals determined by the duplicate set. + * @param featureContext Entries from a secondary feature file (e.g. vcf) if provided + * + */ + public abstract void apply(ReadsWithSameUMI readsWithSameUMI, ReferenceContext referenceContext, FeatureContext featureContext ); + + private void processLastReadSet(){ + if (currentReadsWithSameUMI.getReads().size() > 0){ + apply(currentReadsWithSameUMI, + new ReferenceContext(reference, currentReadsWithSameUMI.getInterval()), + new FeatureContext(features, currentReadsWithSameUMI.getInterval())); + } + } + + /** + * Returns true for duplicate sets that does not meet required criteria for further processing. + * We encourage the user to override this method to meet their needs. + */ + protected boolean rejectSet(final ReadsWithSameUMI readsWithSameUMI){ + // Check that the set contains the minimum required number of reads in each strand + final Pair strandCounts = MoleculeID.countStrands(readsWithSameUMI.getReads()); + if (Math.min(strandCounts.getLeft(), strandCounts.getRight()) < minimumRequiredReadsPerStrand){ + return true; + } + + // Check that the read set is paired (this check may not reject some sets that contain unpaired reads) + if (readsWithSameUMI.getReads().size() % 2 == 1){ + return true; + } + + // Check that the total number of reads (from both strands) exceeds a specified threshold + return readsWithSameUMI.getReads().size() < minimumRequiredReadsPerUMI; + } + + @Override + public List getDefaultReadFilters() { + final List readFilters = new ArrayList<>(6); + readFilters.add(new WellformedReadFilter()); + readFilters.addAll(getDuplicateSetWalkerSpecificReadFilterList()); + return readFilters; + } + + private static List getDuplicateSetWalkerSpecificReadFilterList() { + List filters = new ArrayList<>(5); + filters.add(ReadFilterLibrary.MAPPING_QUALITY_AVAILABLE); + filters.add(ReadFilterLibrary.MAPPED); + filters.add(ReadFilterLibrary.NOT_SECONDARY_ALIGNMENT); + filters.add(ReadFilterLibrary.PASSES_VENDOR_QUALITY_CHECK); + filters.add(ReadFilterLibrary.NON_ZERO_REFERENCE_LENGTH_ALIGNMENT); + return filters; + } +} diff --git a/src/main/java/org/broadinstitute/hellbender/tools/walkers/consensus/DownsampleByDuplicateSet.java b/src/main/java/org/broadinstitute/hellbender/tools/walkers/consensus/DownsampleByDuplicateSet.java new file mode 100644 index 00000000000..ccb5d70edeb --- /dev/null +++ b/src/main/java/org/broadinstitute/hellbender/tools/walkers/consensus/DownsampleByDuplicateSet.java @@ -0,0 +1,103 @@ +package org.broadinstitute.hellbender.tools.walkers.consensus; + +import org.broadinstitute.barclay.argparser.Argument; +import org.broadinstitute.barclay.argparser.BetaFeature; +import org.broadinstitute.barclay.argparser.CommandLineProgramProperties; +import org.broadinstitute.barclay.help.DocumentedFeature; +import org.broadinstitute.hellbender.cmdline.StandardArgumentDefinitions; +import org.broadinstitute.hellbender.engine.DuplicateSetWalker; +import org.broadinstitute.hellbender.engine.FeatureContext; +import org.broadinstitute.hellbender.engine.GATKPathSpecifier; +import org.broadinstitute.hellbender.engine.ReferenceContext; +import org.broadinstitute.hellbender.utils.read.SAMFileGATKReadWriter; +import org.glassfish.jersey.Beta; +import picard.cmdline.programgroups.ReadDataManipulationProgramGroup; + +import java.util.Random; + +@CommandLineProgramProperties( + summary = "Discard a set fraction of duplicate sets from a UMI-grouped bam", + oneLineSummary = "Discard a set fraction of duplicate sets from a UMI-grouped bam", + programGroup = ReadDataManipulationProgramGroup.class +) +/** + * Given a bam grouped by the same unique molecular identifier (UMI), this tool drops a specified fraction of duplicate sets and returns a new bam. + * A duplicate set refers to a group of reads whose fragments start and end at the same genomic coordinate _and_ share the same UMI. + * + * The input bam must first be sorted by UMI using FGBio GroupReadsByUmi (http://fulcrumgenomics.github.io/fgbio/tools/latest/GroupReadsByUmi.html). + * + * Use this tool to create, for instance, an insilico mixture of duplex-sequenced samples to simulate tumor subclones. + * + * Suppose you wish to simulate a tumor sample in which 5% cells share a common set of somatic mutations + * in addition to ones common to the entire cell population. + * + * If you randomly drop 5% of reads in sample A and 95% of reads in sample B and merge the reduced bams, + * the resulting mixture skews the family-size distribution to the left. Here the family size refers to the + * number of sequenced duplicate reads that share the same UMI. + * + * To see this, take a cancer sample, in which 5% of cells share a unique set of somatic mutations, + * that was processed with duplex-UMIs (i.e. UMIs on both adapters) and high rounds of PCR. Suppose we have the sequence-ready + * libraries of this sample attached to and amplified on the flowcell. Now, sort the flowcell lawn such that the reads from the above + * 5% of the cell population moves near the top of the flowcell. This population must have the same family-size distribution as + * the rest of the flowcell, at about 5% of the library complexity compared to the entire flowcell. + * + * Now imagine replacing this population with 5% ramdonly chosen from the *entire* flowcell of another sample that was prepared and sequenced similarly. + * The library complexity of these "graft" reads is higher than that of the original, and, consequently, with other parameters + * such as the number of PCR cycles and sequencing depth fixed, its family distribution would be skewed left---that is, the family size + * would be smaller than it should be. + * + * This tool will help address the above problem by dropping a set fraction of _molecules_, or duplicate sets, rather than reads, at random. + * + * Example Usage: + * + * Keep 95 percent of the molecules. + * + * gatk DownsampleByDuplicateSet \ \ + * -I umiGrouped.bam \ + * --fraction-to-keep 0.95 \ + * -O umiGrouped_0.95.bam + **/ +@BetaFeature +public class DownsampleByDuplicateSet extends DuplicateSetWalker { + @Argument(fullName = StandardArgumentDefinitions.OUTPUT_LONG_NAME, shortName = StandardArgumentDefinitions.OUTPUT_SHORT_NAME, doc = "") + public GATKPathSpecifier outputBam; + + public static final String FRACTION_TO_KEEP_NAME = "fraction-to-keep"; + @Argument(fullName = FRACTION_TO_KEEP_NAME, doc = "This fraction of molecules in the input bam will be retained", minValue = 0.0, maxValue = 1.0) + public double fractionToKeep; + + private static final int RANDOM_SEED = 142; + private final Random rng = new Random(RANDOM_SEED); + private int numDuplicateReadSets; + private int numReads; + private SAMFileGATKReadWriter outputWriter; + + @Override + public void onTraversalStart() { + outputWriter = createSAMWriter(outputBam.toPath(), false); + } + + @Override + public void apply(ReadsWithSameUMI readsWithSameUMI, ReferenceContext referenceContext, FeatureContext featureContext) { + if (rng.nextDouble() < fractionToKeep){ + readsWithSameUMI.getReads().forEach(r -> outputWriter.addRead(r)); + numReads += readsWithSameUMI.getReads().size(); + numDuplicateReadSets += 1; + } + } + + @Override + public Object onTraversalSuccess(){ + outputWriter.close(); + logger.info(String.format("Wrote %d reads", numReads)); + logger.info(String.format("Wrote %d duplicate read sets", numDuplicateReadSets)); + return "SUCCESS"; + } + + @Override + public void closeTool(){ + if (outputWriter != null) { + outputWriter.close(); + } + } +} diff --git a/src/main/java/org/broadinstitute/hellbender/tools/walkers/consensus/MoleculeID.java b/src/main/java/org/broadinstitute/hellbender/tools/walkers/consensus/MoleculeID.java new file mode 100644 index 00000000000..6d2701676b6 --- /dev/null +++ b/src/main/java/org/broadinstitute/hellbender/tools/walkers/consensus/MoleculeID.java @@ -0,0 +1,67 @@ +package org.broadinstitute.hellbender.tools.walkers.consensus; + +import htsjdk.samtools.SAMTag; +import org.apache.commons.lang3.tuple.ImmutablePair; +import org.apache.commons.lang3.tuple.Pair; +import org.broadinstitute.hellbender.utils.read.GATKRead; + +import java.util.List; + +/** + * A container class for the molecule ID, which consists of an integer ID and a binary strand. + * For example, Reads with the tags 12/A and 12/B originated from the same DNA fragment before PCR, + * (i.e. from the same library) but they originated from different strands in that library. + * In other words, one read is F1R2 and the other F2R1. + * + * The word "molecule" here refers to the original DNA fragment with barcode before undergoing + * PCR and sequencing. We amplify this molecule through PCR and end up with many duplicate _fragments_. + */ +public class MoleculeID { + private int moleculeNumber; + private String strand; + + public MoleculeID(final GATKRead read){ + this.moleculeNumber = getMoleculeNumberOfRead(read); + this.strand = getStrandOfRead(read); + } + + public MoleculeID(final int moleculeNumber, final String strand){ + this.moleculeNumber = moleculeNumber; + this.strand = strand; + } + + public int getMoleculeNumber() { + return moleculeNumber; + } + + public String getStrand() { + return strand; + } + + /** Format the molecule ID as stored in the sam/bam/cram file under the {@link SAMTag.MI} tag **/ + public String getSAMField(){ + return moleculeNumber + ReadsWithSameUMI.FGBIO_MI_TAG_DELIMITER + strand; + } + + /** Extracts the molecule number portion of the {@link SAMTag.MI} field of the read **/ + public static int getMoleculeNumberOfRead(final GATKRead read){ + final String MITag = read.getAttributeAsString(SAMTag.MI.name()); + return Integer.parseInt(MITag.split(ReadsWithSameUMI.FGBIO_MI_TAG_DELIMITER)[0]); + } + + /** Extracts the strand portion of the {@link SAMTag.MI} field of the read **/ + public static String getStrandOfRead(final GATKRead read){ + final String MITag = read.getAttributeAsString(SAMTag.MI.name()); + return MITag.split(ReadsWithSameUMI.FGBIO_MI_TAG_DELIMITER)[1]; + } + + /** + * Assumes that the input reads have the same molecule number in the {@link SAMTag.MI} tag + * @returns Counts of reads from each strand, the first element is always larger than the second + **/ + public static Pair countStrands(final List reads){ + final int strandACount = (int) reads.stream().filter(r -> getStrandOfRead(r).equals("A")).count(); + final int strandBCount = (int) reads.stream().filter(r -> getStrandOfRead(r).equals("B")).count(); + return new ImmutablePair<>(strandACount, strandBCount); + } +} diff --git a/src/main/java/org/broadinstitute/hellbender/tools/walkers/consensus/ReadsWithSameUMI.java b/src/main/java/org/broadinstitute/hellbender/tools/walkers/consensus/ReadsWithSameUMI.java new file mode 100644 index 00000000000..0ee31c9472c --- /dev/null +++ b/src/main/java/org/broadinstitute/hellbender/tools/walkers/consensus/ReadsWithSameUMI.java @@ -0,0 +1,106 @@ +package org.broadinstitute.hellbender.tools.walkers.consensus; + +import htsjdk.samtools.SAMTag; +import htsjdk.samtools.util.Locatable; +import org.broadinstitute.hellbender.utils.SimpleInterval; +import org.broadinstitute.hellbender.utils.Utils; +import org.broadinstitute.hellbender.utils.read.GATKRead; + +import java.util.ArrayList; +import java.util.Collections; +import java.util.List; +import java.util.stream.Collectors; + +/** + * A container class for a set of reads that share the same unique molecular identifier (UMI) as judged by + * FGBio GroupReadsByUmi (http://fulcrumgenomics.github.io/fgbio/tools/latest/GroupReadsByUmi.html) + * + * Examples of molecule IDs (MI tag): + * + * "0/A" (The first molecule in the bam, A strand) + * "0/B" (The first molecule in the bam, B strand) + * "99/A" (100th molecule in the bam, A strand) + * + * For a given set of reads with the same molecule number, the strand with a larger number of reads is defined as the A strand. + * i.e. A and B strand doesn't map to top or bottom strand. + * + * I use "top strand" as a synonym for "F1R2". "Bottom strand" is synonymous to "F2R1." + * + * Thus only the integer component is relevant for identifying reads that originated from the same molecule. + * Should the need arise, we could extend this to distinguish between different strands of the same molecule. + * + * All reads in the set must share the same molecular number; this allows for reads that originated from the same fragment before PCR + * but from the different strands to be grouped in the same set. + * For instance, 1/A and 1/B may be in the same set, as they share the same UMI. + * But 2/A and 3/A may not be in the same set. + */ +public class ReadsWithSameUMI implements Locatable { + public static final String FGBIO_MI_TAG_DELIMITER = "/"; + public MoleculeID moleculeID; + + private SimpleInterval interval; + + private List reads; + + public ReadsWithSameUMI(final GATKRead read){ + reads = new ArrayList<>(); + init(read); + } + + private void init(GATKRead read){ + Utils.validate(reads.isEmpty(), String.format("Initializing a non-empty set")); + moleculeID = new MoleculeID(read); + interval = new SimpleInterval(read); + reads.add(read); + } + + public List getReads(){ + return Collections.unmodifiableList(reads); + } + + /** + * Add a read to the set. Throws an error if the molecule ID doens't match. + * **/ + public void addRead(final GATKRead read){ + Utils.validate(reads.isEmpty() || moleculeID.getMoleculeNumber() == MoleculeID.getMoleculeNumberOfRead(read), + String.format("Molecule number of the set and that of the new read don't match: set number = %d, read number = %d", moleculeID.getMoleculeNumber(), MoleculeID.getMoleculeNumberOfRead(read))); + Utils.validate(interval.contigsMatch(read), + String.format("Adding a read from another contig: set contig = %s, read contig = %s", interval.getContig(), read.getContig())); + + if (reads.isEmpty()){ + init(read); + return; + } + + reads.add(read); + interval = interval.spanWith(read); + } + + public boolean isEmpty(){ + return reads.isEmpty(); + } + + public int getMoleculeNumber() { + Utils.nonNull(moleculeID, "Querying a non-initialized set for a molecule number"); + return moleculeID.getMoleculeNumber(); + } + + public SimpleInterval getInterval(){ + return interval; + } + + @Override + public String getContig() { + return interval.getContig(); + } + + @Override + public int getStart() { + return interval.getStart(); + } + + @Override + public int getEnd() { + return interval.getEnd(); + } +} diff --git a/src/test/java/org/broadinstitute/hellbender/tools/walkers/consensus/DownsampleByDuplicateSetIntegrationTest.java b/src/test/java/org/broadinstitute/hellbender/tools/walkers/consensus/DownsampleByDuplicateSetIntegrationTest.java new file mode 100644 index 00000000000..4fa1d3f60f7 --- /dev/null +++ b/src/test/java/org/broadinstitute/hellbender/tools/walkers/consensus/DownsampleByDuplicateSetIntegrationTest.java @@ -0,0 +1,100 @@ +package org.broadinstitute.hellbender.tools.walkers.consensus; + +import htsjdk.samtools.SAMTag; +import org.broadinstitute.hellbender.CommandLineProgramTest; +import org.broadinstitute.hellbender.engine.ReadsDataSource; +import org.broadinstitute.hellbender.exceptions.UserException; +import org.broadinstitute.hellbender.testutils.ArgumentsBuilder; +import org.broadinstitute.hellbender.utils.read.GATKRead; +import org.testng.Assert; +import org.testng.annotations.Test; + +import java.io.File; +import java.nio.file.Paths; +import java.util.*; + +public class DownsampleByDuplicateSetIntegrationTest extends CommandLineProgramTest { + private static final String downsampleByDuplicateSetTestDir = publicTestDir + "org/broadinstitute/hellbender/tools/downsampleByDuplicateSet/"; + private static final String NA12878_GROUPED = downsampleByDuplicateSetTestDir + "NA12878.grouped.bam"; + + @Test + public void testMatesAreTogether() { + final File out = createTempFile("downsampled", "bam"); + final ArgumentsBuilder args = new ArgumentsBuilder() + .add("I", NA12878_GROUPED) + .add("O", out.getAbsolutePath()) + .add(DownsampleByDuplicateSet.FRACTION_TO_KEEP_NAME, "1.0"); + runCommandLine(args, DownsampleByDuplicateSet.class.getSimpleName()); + + try (final ReadsDataSource readsDataSource = new ReadsDataSource(Paths.get(out.getAbsolutePath()))) { + final Iterator iterator = readsDataSource.iterator(); + while (iterator.hasNext()) { + // Make sure that the read and its mate are next to each other in the file + final GATKRead read1 = iterator.next(); + final GATKRead read2 = iterator.next(); + Assert.assertEquals(read1.getName(), read2.getName()); + } + } catch (Exception e) { + throw new UserException("Encountered an IOException writing to " + out, e); + } + } + + /** + * Test that the downsampling rate corresponds to the reduction in the number of duplicates in the output + * file up to sampling noise. + */ + @Test + public void testDownsampleFraction(){ + final File out = createTempFile("downasampled", "bam"); + for (double downsampleRate : Arrays.asList(0.0, 0.1, 0.3, 0.5, 1.0)){ + final ArgumentsBuilder args = new ArgumentsBuilder() + .add("I", NA12878_GROUPED) + .add(DownsampleByDuplicateSet.FRACTION_TO_KEEP_NAME, Double.toString(downsampleRate)) + .add("O", out.getAbsolutePath()); + runCommandLine(args, DownsampleByDuplicateSet.class.getSimpleName()); + + try(final ReadsDataSource originalBam = new ReadsDataSource(Paths.get(NA12878_GROUPED)); + final ReadsDataSource downsampledBam = new ReadsDataSource(Paths.get(out.getAbsolutePath()))){ + + final int originalMoleculeCount = countDuplicateSets(originalBam); + final int downsampledMoleculeCount = countDuplicateSets(downsampledBam); + + if (downsampleRate == 0.0){ // Keep none of the reads + Assert.assertEquals(downsampledMoleculeCount, 0.0); + continue; + } + + if (downsampleRate == 1.0){ // Keep all of the reads + Assert.assertEquals(downsampledMoleculeCount, originalMoleculeCount); + continue; + } + + // In addition to the stochastic noise, there's an additional source of deviation from the mean due to + // the fact that we are downsampling by molecule, whose family size may vary and therefore is not the same thing as + // dropping 5% of the reads, for example. + final double noise = originalMoleculeCount*0.05; // allow for a 5% deviation from the expected + final double deviationFromExpected = Math.abs(downsampleRate * originalMoleculeCount - downsampledMoleculeCount); + Assert.assertTrue(deviationFromExpected < noise); + } catch (Exception e) { + throw new UserException("Encountered an IO error", e); + } + } + } + + private int countDuplicateSets(final ReadsDataSource readsDataSource){ + int count = 0; + String currentMoleculeId = ""; // Note we are duplex aware: 12/A different from 12/B + + final Iterator iterator = readsDataSource.iterator(); + while (iterator.hasNext()){ + final GATKRead read = iterator.next(); + final String moleculeID = read.getAttributeAsString(SAMTag.MI.name()); + if (!moleculeID.equals(currentMoleculeId)){ + count++; + currentMoleculeId = moleculeID; + } + } + + return count; + } +} \ No newline at end of file diff --git a/src/test/java/org/broadinstitute/hellbender/tools/walkers/consensus/MoleculeIDUnitTest.java b/src/test/java/org/broadinstitute/hellbender/tools/walkers/consensus/MoleculeIDUnitTest.java new file mode 100644 index 00000000000..420d22aed0c --- /dev/null +++ b/src/test/java/org/broadinstitute/hellbender/tools/walkers/consensus/MoleculeIDUnitTest.java @@ -0,0 +1,33 @@ +package org.broadinstitute.hellbender.tools.walkers.consensus; + +import htsjdk.samtools.SAMTag; +import org.broadinstitute.hellbender.testutils.BaseTest; +import org.broadinstitute.hellbender.utils.read.ArtificialReadUtils; +import org.broadinstitute.hellbender.utils.read.GATKRead; +import org.testng.Assert; +import org.testng.annotations.Test; + +import java.util.List; + +public class MoleculeIDUnitTest extends BaseTest { + @Test + public void testBasicOperations(){ + final int moleculeNumber = 131; + final String strand = "A"; + final String samField = moleculeNumber + "/" + strand; + final GATKRead read = ArtificialReadUtils.createSamBackedRead( "read", "3", 1000, 1151); + read.setAttribute(SAMTag.MI.name(), samField); + final MoleculeID id = new MoleculeID(read); + Assert.assertEquals(id.getMoleculeNumber(), moleculeNumber); + Assert.assertEquals(id.getStrand(), strand); + Assert.assertEquals(id.getSAMField(), samField); + } + + @Test + public void testCountStrand(){ + final int numReadPairs = 50; + final List reads = ReadsWithSameUMIUnitTest.makeReads(numReadPairs, "3", 10, "read"); + Assert.assertEquals((int) MoleculeID.countStrands(reads).getLeft(), 50); + Assert.assertEquals((int) MoleculeID.countStrands(reads).getRight(), 50); + } +} \ No newline at end of file diff --git a/src/test/java/org/broadinstitute/hellbender/tools/walkers/consensus/ReadsWithSameUMIUnitTest.java b/src/test/java/org/broadinstitute/hellbender/tools/walkers/consensus/ReadsWithSameUMIUnitTest.java new file mode 100644 index 00000000000..4d05620e25e --- /dev/null +++ b/src/test/java/org/broadinstitute/hellbender/tools/walkers/consensus/ReadsWithSameUMIUnitTest.java @@ -0,0 +1,93 @@ +package org.broadinstitute.hellbender.tools.walkers.consensus; + +import htsjdk.samtools.SAMTag; +import org.broadinstitute.hellbender.testutils.BaseTest; +import org.broadinstitute.hellbender.utils.read.ArtificialReadUtils; +import org.broadinstitute.hellbender.utils.read.GATKRead; +import org.testng.Assert; +import org.testng.annotations.Test; + +import java.util.ArrayList; +import java.util.List; + +public class ReadsWithSameUMIUnitTest extends BaseTest { + // Parameters for the default reads + final String contig = "3"; + final int numReadPairs = 30; + final int moleculeNumber = 10; + + @Test + public void testAddRead(){ + final List reads = makeReads(numReadPairs, contig, moleculeNumber, "read"); + final ReadsWithSameUMI set = new ReadsWithSameUMI(reads.get(0)); + reads.stream().skip(1).forEach(r -> set.addRead(r)); + + Assert.assertEquals(set.getReads().size(), reads.size()); + } + + @Test(expectedExceptions = IllegalStateException.class) + public void testAddDifferentMolecule(){ + final List reads = makeReads(1, contig, moleculeNumber, "one_molecule"); + final ReadsWithSameUMI set = new ReadsWithSameUMI(reads.get(0)); + reads.stream().skip(1).forEach(set::addRead); + + // Add reads from a different molecule + final List readsFromAnotherMolecule = makeReads(1, contig, moleculeNumber + 1, "another_molecule"); + readsFromAnotherMolecule.forEach(set::addRead); // Throws an exception + } + + // Adding a read from a different contig (even if the molecule ID matches) must throw an error + @Test(expectedExceptions = IllegalStateException.class) + public void testAddDifferentContig(){ + final List reads = makeReads(1, contig, moleculeNumber, "one_molecule"); + final ReadsWithSameUMI set = new ReadsWithSameUMI(reads.get(0)); + reads.stream().skip(1).forEach(set::addRead); + + // Add reads from a different molecule + final String anotherContig = "4"; + final List readsFromAnotherMolecule = makeReads(1, anotherContig, moleculeNumber + 1, "another_molecule"); + readsFromAnotherMolecule.forEach(set::addRead); // Throws an exception + } + + // Helper method to generate test reads + public static List makeReads(final int numReadPairs, final String contig, final int moleculeNumber, + final String readName){ + final int readLength = 146; + final List reads = new ArrayList<>(numReadPairs); + final List mates = new ArrayList<>(numReadPairs); + + for (int i = 0; i < numReadPairs; i++){ + // Forward read + final int readStart = 3000; + final String readStrand = i % 2 == 0 ? "A" : "B"; + final MoleculeID id = new MoleculeID(moleculeNumber, readStrand); + final GATKRead read = ArtificialReadUtils.createSamBackedRead(readName + i, contig, readStart, readLength); + read.setAttribute(SAMTag.MI.name(), id.getSAMField()); + read.setIsReverseStrand(false); + + // Reverse read + final int insertSize = 1000; + final int mateStart = readStart + insertSize - readLength; + final GATKRead mate = ArtificialReadUtils.createSamBackedRead(readName + "_mate" + i, contig, mateStart, readLength); + final MoleculeID mateId = new MoleculeID(moleculeNumber, readStrand == "A" ? "B" : "A"); + mate.setAttribute(SAMTag.MI.name(), mateId.getSAMField()); + mate.setIsReverseStrand(true); + + if (readStrand.equals("A")){ + read.setIsFirstOfPair(); // F1R2 + mate.setIsSecondOfPair(); + } else { + read.setIsSecondOfPair(); // F2R1 + mate.setIsFirstOfPair(); + } + + reads.add(read); + mates.add(mate); + } + + final List results = new ArrayList<>(numReadPairs*2); + results.addAll(reads); + results.addAll(mates); // Add reads and mates in order so that the resulting list is ordered by coordinate + return results; + } +} \ No newline at end of file diff --git a/src/test/resources/org/broadinstitute/hellbender/tools/downsampleByDuplicateSet/NA12878.grouped.bam b/src/test/resources/org/broadinstitute/hellbender/tools/downsampleByDuplicateSet/NA12878.grouped.bam new file mode 100644 index 0000000000000000000000000000000000000000..ebb92cfff0a49451e0c16dc96d4c3b747edd6cdb GIT binary patch literal 340989 zcmV)WK(48#Ou+@9V}`nGNNFq_rLoNJGF-L}_u&tw8Zii9G82oaG&A_XPOMY-{W;3YyJ@)jb( 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