broadinstitute · takutosato · Oct 11, 2018 · Oct 4, 2018
diff --git a/src/main/java/org/broadinstitute/hellbender/tools/walkers/annotator/StrandArtifact.java b/src/main/java/org/broadinstitute/hellbender/tools/walkers/annotator/StrandArtifact.java
@@ -9,13 +9,15 @@
 import org.apache.commons.math3.distribution.BetaDistribution;
 import org.broadinstitute.barclay.help.DocumentedFeature;
 import org.broadinstitute.hellbender.engine.ReferenceContext;
+import org.broadinstitute.hellbender.tools.walkers.mutect.SomaticGenotypingEngine;
 import org.broadinstitute.hellbender.utils.*;
 import org.broadinstitute.hellbender.utils.genotyper.ReadLikelihoods;
 import org.broadinstitute.hellbender.utils.help.HelpConstants;
 import org.broadinstitute.hellbender.utils.logging.OneShotLogger;
 import org.broadinstitute.hellbender.utils.variant.GATKVCFConstants;
 
 import java.util.*;
+import java.util.stream.Collectors;
 
 import static org.broadinstitute.hellbender.tools.walkers.annotator.StrandArtifact.ArtifactState.*;
 
@@ -88,13 +90,22 @@ public void annotate(final ReferenceContext ref,
             return;
         }
         final int indexOfMaxTumorLod = MathUtils.maxElementIndex(tumorLods);
-        final Allele altAlelle = vc.getAlternateAllele(indexOfMaxTumorLod);
-
-        final Collection<ReadLikelihoods<Allele>.BestAllele> bestAlleles = likelihoods.bestAllelesBreakingTies(g.getSampleName());
-        final int numFwdAltReads = (int) bestAlleles.stream().filter(ba -> !ba.read.isReverseStrand() && ba.isInformative() && ba.allele.equals(altAlelle)).count();
-        final int numRevAltReads = (int) bestAlleles.stream().filter(ba -> ba.read.isReverseStrand() && ba.isInformative() && ba.allele.equals(altAlelle)).count();
-        final int numFwdReads = (int) bestAlleles.stream().filter(ba -> !ba.read.isReverseStrand() && ba.isInformative()).count();
-        final int numRevReads = (int) bestAlleles.stream().filter(ba -> ba.read.isReverseStrand() && ba.isInformative()).count();
+        final Allele altAllele = vc.getAlternateAllele(indexOfMaxTumorLod);
+
+        final Collection<ReadLikelihoods<Allele>.BestAllele> informativeBestAlleles = likelihoods.bestAllelesBreakingTies(g.getSampleName()).stream().
+                filter(ba -> ba.isInformative()).collect(Collectors.toList());
+        final Map<Strand, List<ReadLikelihoods<Allele>.BestAllele>> altReads = informativeBestAlleles.stream().filter(ba -> ba.allele.equals(altAllele))
+                .collect(Collectors.groupingBy(ba -> ba.read.isReverseStrand() ? Strand.REV : Strand.FWD));
+        final Map<Strand, List<ReadLikelihoods<Allele>.BestAllele>> refReads = informativeBestAlleles.stream().filter(ba -> ba.allele.equals(vc.getReference()))
+                .collect(Collectors.groupingBy(ba -> ba.read.isReverseStrand() ? Strand.REV : Strand.FWD));
+
+        final int numFwdAltReads = countReads(altReads, Strand.FWD);
+        final int numRevAltReads = countReads(altReads, Strand.REV);
+        final int numFwdRefReads = countReads(refReads, Strand.FWD);
+        final int numRevRefReads = countReads(refReads, Strand.REV);
+
+        final int numFwdReads = numFwdRefReads + numFwdAltReads;
+        final int numRevReads = numRevRefReads + numRevAltReads;
         final int numAltReads = numFwdAltReads + numRevAltReads;
         final int numReads = numFwdReads + numRevReads;
 
@@ -162,6 +173,10 @@ public void annotate(final ReferenceContext ref,
         gb.attribute(MAP_ALLELE_FRACTIONS_KEY, estimatedAlleleFractions.values().stream().mapToDouble(Double::doubleValue).toArray());
     }
 
+    private enum Strand {
+        FWD, REV
+    }
+
     @Override
     public List<VCFFormatHeaderLine> getDescriptions() {
         return Arrays.asList(new VCFFormatHeaderLine(POSTERIOR_PROBABILITIES_KEY, 3, VCFHeaderLineType.Float, "posterior probabilities of the presence of strand artifact"),
@@ -177,5 +192,19 @@ private double getIntegrandGivenArtifact(final double f, final double epsilon, f
                 MathUtils.binomialProbability(nNoArtifact, xNoArtifact, f);
     }
 
+    /**
+     *
+     * Count the number of reads in {@param reads}, including the overlapping mates discarded by {@link SomaticGenotypingEngine}.
+     * @param strand is the strand of the reads we're counting. For example, if strand = Strand.FWD, the we look among {@param reads}
+     *               for reverse reads with {@link SomaticGenotypingEngine.DISCARDED_MATE_READ_TAG} set to 1
+     */
+    private int countReads(final Map<Strand, List<ReadLikelihoods<Allele>.BestAllele>> reads, final Strand strand){
+        final Strand strandOfKeptRead = strand == Strand.FWD ? Strand.REV : Strand.FWD;
 
+        // check of the key in case there are no forward or reverse in {@link reads}
+        final int numDiscardedReads = ! reads.containsKey(strandOfKeptRead) ? 0 : (int) reads.get(strandOfKeptRead).stream().filter(ba -> ba.read.hasAttribute(SomaticGenotypingEngine.DISCARDED_MATE_READ_TAG)).count();
+        final int numReads = ! reads.containsKey(strand) ? 0 : reads.get(strand).size();
+        return numReads + numDiscardedReads;
+
+    }
 }
diff --git a/src/main/java/org/broadinstitute/hellbender/tools/walkers/annotator/StrandBiasTest.java b/src/main/java/org/broadinstitute/hellbender/tools/walkers/annotator/StrandBiasTest.java
@@ -6,6 +6,7 @@
 import htsjdk.variant.variantcontext.VariantContext;
 import org.broadinstitute.hellbender.engine.ReferenceContext;
 import org.broadinstitute.hellbender.exceptions.GATKException;
+import org.broadinstitute.hellbender.tools.walkers.mutect.SomaticGenotypingEngine;
 import org.broadinstitute.hellbender.utils.Utils;
 import org.broadinstitute.hellbender.utils.genotyper.ReadLikelihoods;
 import org.broadinstitute.hellbender.utils.pileup.PileupElement;
@@ -170,8 +171,14 @@ private static void updateTable(final int[] table, final Allele allele, final GA
             final int offset = matchesRef ? 0 : ARRAY_DIM;
 
             // a normal read with an actual strand
-            final boolean isFW = !read.isReverseStrand();
-            table[offset + (isFW ? 0 : 1)]++;
+            final boolean isForward = !read.isReverseStrand();
+            table[offset + (isForward ? 0 : 1)]++;
+
+            // This read's mate got discarded by SomaticGenotypingEngine::clipOverlappingReads()
+            if (read.hasAttribute(SomaticGenotypingEngine.DISCARDED_MATE_READ_TAG)){
+                // Note that if this read is forward, then we increment its mate which we assume is reverse
+                table[offset + (isForward ? 1 : 0)]++;
+            }
         }
     }
 

diff --git a/...itute/hellbender/tools/walkers/haplotypecaller/AssemblyBasedCallerArgumentCollection.java b/...itute/hellbender/tools/walkers/haplotypecaller/AssemblyBasedCallerArgumentCollection.java
@@ -24,6 +24,7 @@ public abstract class AssemblyBasedCallerArgumentCollection extends StandardCall
 
     public static final String MIN_BASE_QUALITY_SCORE_LONG_NAME = "min-base-quality-score";
     public static final String SMITH_WATERMAN_LONG_NAME = "smith-waterman";
+    public static final String CORRECT_OVERLAPPING_BASE_QUALITIES_LONG_NAME = "correct-overlapping-quality";
 
     @ArgumentCollection
     public AssemblyRegionTrimmerArgumentCollection assemblyRegionTrimmerArgs = new AssemblyRegionTrimmerArgumentCollection();
@@ -123,4 +124,7 @@ public abstract class AssemblyBasedCallerArgumentCollection extends StandardCall
     @Argument(fullName = SMITH_WATERMAN_LONG_NAME, doc = "Which Smith-Waterman implementation to use, generally FASTEST_AVAILABLE is the right choice", optional = true)
     public SmithWatermanAligner.Implementation smithWatermanImplementation = SmithWatermanAligner.Implementation.JAVA;
 
+    @Argument(fullName = CORRECT_OVERLAPPING_BASE_QUALITIES_LONG_NAME)
+    public boolean doNotCorrectOverlappingBaseQualities = false;
+
 }
diff --git a/...org/broadinstitute/hellbender/tools/walkers/haplotypecaller/AssemblyBasedCallerUtils.java b/...org/broadinstitute/hellbender/tools/walkers/haplotypecaller/AssemblyBasedCallerUtils.java
@@ -64,7 +64,8 @@ public static void finalizeRegion(final AssemblyRegion region,
                                       final boolean dontUseSoftClippedBases,
                                       final byte minTailQuality,
                                       final SAMFileHeader readsHeader,
-                                      final SampleList samplesList) {
+                                      final SampleList samplesList,
+                                      final boolean correctOverlappingBaseQualities) {
         if ( region.isFinalized() ) {
             return;
         }
@@ -98,7 +99,7 @@ public static void finalizeRegion(final AssemblyRegion region,
         Collections.sort(readsToUse, new ReadCoordinateComparator(readsHeader)); // TODO: sort may be unnecessary here
 
         // handle overlapping read pairs from the same fragment
-        cleanOverlappingReadPairs(readsToUse, samplesList, readsHeader);
+        cleanOverlappingReadPairs(readsToUse, samplesList, readsHeader, correctOverlappingBaseQualities);
 
         region.clearReads();
         region.addAll(readsToUse);
@@ -109,12 +110,14 @@ public static void finalizeRegion(final AssemblyRegion region,
      * Clean up reads/bases that overlap within read pairs
      *
      * @param reads the list of reads to consider
+     * @param correctOverlappingBaseQualities
      */
-    private static void cleanOverlappingReadPairs(final List<GATKRead> reads, final SampleList samplesList, final SAMFileHeader readsHeader) {
+    private static void cleanOverlappingReadPairs(final List<GATKRead> reads, final SampleList samplesList, final SAMFileHeader readsHeader,
+                                                  final boolean correctOverlappingBaseQualities) {
         for ( final List<GATKRead> perSampleReadList : splitReadsBySample(samplesList, readsHeader, reads).values() ) {
             final FragmentCollection<GATKRead> fragmentCollection = FragmentCollection.create(perSampleReadList);
             for ( final List<GATKRead> overlappingPair : fragmentCollection.getOverlappingPairs() ) {
-                FragmentUtils.adjustQualsOfOverlappingPairedFragments(overlappingPair);
+                FragmentUtils.adjustQualsOfOverlappingPairedFragments(overlappingPair, correctOverlappingBaseQualities);
             }
         }
     }
@@ -238,7 +241,7 @@ public static AssemblyResultSet assembleReads(final AssemblyRegion region,
                                                   final ReferenceSequenceFile referenceReader,
                                                   final ReadThreadingAssembler assemblyEngine,
                                                   final SmithWatermanAligner aligner){
-        finalizeRegion(region, argumentCollection.errorCorrectReads, argumentCollection.dontUseSoftClippedBases, (byte)(argumentCollection.minBaseQualityScore - 1), header, sampleList);
+        finalizeRegion(region, argumentCollection.errorCorrectReads, argumentCollection.dontUseSoftClippedBases, (byte)(argumentCollection.minBaseQualityScore - 1), header, sampleList, ! argumentCollection.doNotCorrectOverlappingBaseQualities);
         if( argumentCollection.debug) {
             logger.info("Assembling " + region.getSpan() + " with " + region.size() + " reads:    (with overlap region = " + region.getExtendedSpan() + ")");
         }

diff --git a/...va/org/broadinstitute/hellbender/tools/walkers/haplotypecaller/HaplotypeCallerEngine.java b/...va/org/broadinstitute/hellbender/tools/walkers/haplotypecaller/HaplotypeCallerEngine.java
@@ -673,7 +673,7 @@ private List<VariantContext> referenceModelForNoVariation(final AssemblyRegion r
             //TODO - why the activeRegion cannot manage its own one-time finalization and filtering?
             //TODO - perhaps we can remove the last parameter of this method and the three lines bellow?
             if ( needsToBeFinalized ) {
-                finalizeRegion(region);
+                AssemblyBasedCallerUtils.finalizeRegion(region, hcArgs.errorCorrectReads, hcArgs.dontUseSoftClippedBases, minTailQuality, readsHeader, samplesList, ! hcArgs.doNotCorrectOverlappingBaseQualities);
             }
             filterNonPassingReads(region);
 
@@ -723,10 +723,6 @@ public void shutdown() {
 
     }
 
-    private void finalizeRegion(final AssemblyRegion region) {
-        AssemblyBasedCallerUtils.finalizeRegion(region, hcArgs.errorCorrectReads, hcArgs.dontUseSoftClippedBases, minTailQuality, readsHeader, samplesList);
-    }
-
     private Set<GATKRead> filterNonPassingReads( final AssemblyRegion activeRegion ) {
         // TODO: can we unify this additional filtering with makeStandardHCReadFilter()?
 

diff --git a/src/main/java/org/broadinstitute/hellbender/tools/walkers/mutect/M2ArgumentCollection.java b/src/main/java/org/broadinstitute/hellbender/tools/walkers/mutect/M2ArgumentCollection.java
@@ -37,6 +37,7 @@ public class M2ArgumentCollection extends AssemblyBasedCallerArgumentCollection
     public static final String IGNORE_ITR_ARTIFACTS_LONG_NAME = "ignore-itr-artifacts";
     public static final String ARTIFACT_PRIOR_TABLE_NAME = "orientation-bias-artifact-priors";
     public static final String GET_AF_FROM_AD_LONG_NAME = "get-af-from-ad";
+    public static final String ANNOTATE_BASED_ON_READS_LONG_NAME = "annotate-fragments";
 
     public static final double DEFAULT_AF_FOR_TUMOR_ONLY_CALLING = 5e-8;
     public static final double DEFAULT_AF_FOR_TUMOR_NORMAL_CALLING = 1e-6;
@@ -169,4 +170,12 @@ public double getDefaultAlleleFrequency() {
     @Argument(fullName = GET_AF_FROM_AD_LONG_NAME, doc="Use allelic depth to calculate tumor allele fraction; recommended for mitochondrial applications", optional = true)
     public boolean calculateAFfromAD = false;
 
+    /**
+     * If set to true, count an overlapping read pair as two separate reads instead of one for {@link StrandArtifact} and {@link StrandBiasBySample} annotations,
+     * which is the correct behavior for these annotations. Note that doing so would break the independence assumption of reads and over-count the alt depth in these annotations.
+     * On the other hand it could prevent spurious false negatives that could arise if by chance one strand in overlapping pairs is dropped disproportionately
+     */
+    @Argument(fullName = ANNOTATE_BASED_ON_READS_LONG_NAME, doc = "If true, strand-based annotations use the number of reads, not fragments")
+    public boolean annotateBasedOnReads = false;
+
 }
diff --git a/src/main/java/org/broadinstitute/hellbender/tools/walkers/mutect/Mutect2Engine.java b/src/main/java/org/broadinstitute/hellbender/tools/walkers/mutect/Mutect2Engine.java
@@ -190,7 +190,7 @@ public List<VariantContext> callRegion(final AssemblyRegion originalAssemblyRegi
         final AssemblyRegion assemblyActiveRegion = AssemblyBasedCallerUtils.assemblyRegionWithWellMappedReads(originalAssemblyRegion, READ_QUALITY_FILTER_THRESHOLD, header);
         final AssemblyResultSet untrimmedAssemblyResult = AssemblyBasedCallerUtils.assembleReads(assemblyActiveRegion, givenAlleles, MTAC, header, samplesList, logger, referenceReader, assemblyEngine, aligner);
         final SortedSet<VariantContext> allVariationEvents = untrimmedAssemblyResult.getVariationEvents(MTAC.maxMnpDistance);
-        final AssemblyRegionTrimmer.Result trimmingResult = trimmer.trim(originalAssemblyRegion,allVariationEvents);
+        final AssemblyRegionTrimmer.Result trimmingResult = trimmer.trim(originalAssemblyRegion, allVariationEvents);
         if (!trimmingResult.isVariationPresent()) {
             return NO_CALLS;
         }

diff --git a/...main/java/org/broadinstitute/hellbender/tools/walkers/mutect/SomaticGenotypingEngine.java b/...main/java/org/broadinstitute/hellbender/tools/walkers/mutect/SomaticGenotypingEngine.java
@@ -11,6 +11,7 @@
 import org.apache.commons.math3.util.FastMath;
 import org.broadinstitute.hellbender.engine.FeatureContext;
 import org.broadinstitute.hellbender.engine.ReferenceContext;
+import org.broadinstitute.hellbender.tools.walkers.annotator.StrandBiasTest;
 import org.broadinstitute.hellbender.tools.walkers.genotyper.afcalc.AFCalculator;
 import org.broadinstitute.hellbender.tools.walkers.genotyper.afcalc.AFCalculatorProvider;
 import org.broadinstitute.hellbender.tools.walkers.haplotypecaller.AssemblyBasedCallerGenotypingEngine;
@@ -37,6 +38,7 @@ public class SomaticGenotypingEngine extends AssemblyBasedCallerGenotypingEngine
     public final String tumorSample;
     private final String normalSample;
     final boolean hasNormal;
+    public static final String DISCARDED_MATE_READ_TAG = "DM";
 
     // {@link GenotypingEngine} requires a non-null {@link AFCalculatorProvider} but this class doesn't need it.  Thus we make a dummy
     private static final AFCalculatorProvider DUMMY_AF_CALCULATOR_PROVIDER = new AFCalculatorProvider() {
@@ -226,8 +228,8 @@ private PerAlleleCollection<Double> somaticLog10Odds(final LikelihoodMatrix<Alle
     }
 
     private void addGenotypes(final LikelihoodMatrix<Allele> tumorLog10Matrix,
-                                        final Optional<LikelihoodMatrix<Allele>> normalLog10Matrix,
-                                        final VariantContextBuilder callVcb) {
+                              final Optional<LikelihoodMatrix<Allele>> normalLog10Matrix,
+                              final VariantContextBuilder callVcb) {
         final double[] tumorAlleleCounts = getEffectiveCounts(tumorLog10Matrix);
         final int[] adArray = Arrays.stream(tumorAlleleCounts).mapToInt(x -> (int) FastMath.round(x)).toArray();
         final int dp = (int) MathUtils.sum(adArray);
@@ -325,6 +327,13 @@ private void filterOverlappingReads(final ReadLikelihoods<Allele> likelihoods, f
                 if (read.allele.equals(mate.allele)) {
                     // keep the higher-quality read
                     readsToDiscard.add(read.likelihood < mate.likelihood ? read.read : mate.read);
+
+                    // mark the read to indicate that its mate was dropped - so that we can account for it in {@link StrandArtifact}
+                    // and {@link StrandBiasBySample}
+                    if (MTAC.annotateBasedOnReads){
+                        final GATKRead readToKeep = read.likelihood >= mate.likelihood ? read.read : mate.read;
+                        readToKeep.setAttribute(DISCARDED_MATE_READ_TAG, 1);
+                    }
                 } else if (retainMismatches) {
                     // keep the alt read
                     readsToDiscard.add(read.allele.equals(ref) ? read.read : mate.read);

diff --git a/src/main/java/org/broadinstitute/hellbender/utils/fragments/FragmentUtils.java b/src/main/java/org/broadinstitute/hellbender/utils/fragments/FragmentUtils.java
@@ -25,12 +25,13 @@ private FragmentUtils() {}
      * Assumes that firstRead starts before secondRead (according to their soft clipped starts)
      * Sets the qualities of clippedFirstRead and clippedSecondRead to mimic a merged read or
      * nothing if the algorithm cannot create a meaningful one
-     *
-     * @param clippedFirstRead the left most read
+     *  @param clippedFirstRead the left most read
      * @param clippedSecondRead the right most read
+     * @param correctOverlappingBaseQualities
      *
      */
-    public static void adjustQualsOfOverlappingPairedFragments(final GATKRead clippedFirstRead, final GATKRead clippedSecondRead) {
+    public static void adjustQualsOfOverlappingPairedFragments(final GATKRead clippedFirstRead, final GATKRead clippedSecondRead,
+                                                               final boolean correctOverlappingBaseQualities) {
         Utils.nonNull(clippedFirstRead);
         Utils.nonNull(clippedSecondRead);
         Utils.validateArg(clippedFirstRead.getName().equals(clippedSecondRead.getName()), () ->
@@ -51,6 +52,10 @@ public static void adjustQualsOfOverlappingPairedFragments(final GATKRead clippe
         final byte[] secondReadQuals = clippedSecondRead.getBaseQualities();
 
         for ( int i = 0; i < numOverlappingBases; i++ ) {
+            if (! correctOverlappingBaseQualities) {
+                break;
+            }
+
             final int firstReadIndex = firstReadStop + i;
             final byte firstReadBase = firstReadBases[firstReadIndex];
             final byte secondReadBase = secondReadBases[i];
@@ -69,17 +74,17 @@ public static void adjustQualsOfOverlappingPairedFragments(final GATKRead clippe
         clippedSecondRead.setBaseQualities(secondReadQuals);
     }
 
-    public static void adjustQualsOfOverlappingPairedFragments( final List<GATKRead> overlappingPair ) {
+    public static void adjustQualsOfOverlappingPairedFragments(final List<GATKRead> overlappingPair, final boolean correctOverlappingBaseQualities) {
         Utils.validateArg( overlappingPair.size() == 2, () -> "Found overlapping pair with " + overlappingPair.size() + " reads, but expecting exactly 2.");
 
         final GATKRead firstRead = overlappingPair.get(0);
         final GATKRead secondRead = overlappingPair.get(1);
 
         if ( secondRead.getSoftStart() < firstRead.getSoftStart() ) {
-            adjustQualsOfOverlappingPairedFragments(secondRead, firstRead);
+            adjustQualsOfOverlappingPairedFragments(secondRead, firstRead, correctOverlappingBaseQualities);
         }
         else {
-            adjustQualsOfOverlappingPairedFragments(firstRead, secondRead);
+            adjustQualsOfOverlappingPairedFragments(firstRead, secondRead, correctOverlappingBaseQualities);
         }
     }
 }