示例#1
0
  public Map<String, Object> annotate(
      RefMetaDataTracker tracker,
      AnnotatorCompatible walker,
      ReferenceContext ref,
      Map<String, AlignmentContext> stratifiedContexts,
      VariantContext vc) {
    if (stratifiedContexts.size()
        == 0) // size 0 means that call was made by someone else and we have no data here
    return null;

    if (!vc.isSNP() && !vc.isIndel() && !vc.isMixed()) return null;

    final AlignmentContext context =
        AlignmentContextUtils.joinContexts(stratifiedContexts.values());

    final int contextWingSize = Math.min((ref.getWindow().size() - 1) / 2, MIN_CONTEXT_WING_SIZE);
    final int contextSize = contextWingSize * 2 + 1;

    final int locus =
        ref.getLocus().getStart() + (ref.getLocus().getStop() - ref.getLocus().getStart()) / 2;

    final ReadBackedPileup pileup = context.getBasePileup();

    // Compute all haplotypes consistent with the current read pileup
    final List<Haplotype> haplotypes = computeHaplotypes(pileup, contextSize, locus, vc);

    final MathUtils.RunningAverage scoreRA = new MathUtils.RunningAverage();
    if (haplotypes != null) {
      for (final Genotype genotype : vc.getGenotypes()) {
        final AlignmentContext thisContext = stratifiedContexts.get(genotype.getSampleName());
        if (thisContext != null) {
          final ReadBackedPileup thisPileup = thisContext.getBasePileup();
          if (vc.isSNP())
            scoreRA.add(
                scoreReadsAgainstHaplotypes(
                    haplotypes,
                    thisPileup,
                    contextSize,
                    locus)); // Taking the simple average of all sample's score since the score can
                             // be negative and the RMS doesn't make sense
          else if (vc.isIndel() || vc.isMixed()) {
            Double d = scoreIndelsAgainstHaplotypes(thisPileup);
            if (d == null) return null;
            scoreRA.add(
                d); // Taking the simple average of all sample's score since the score can be
                    // negative and the RMS doesn't make sense
          }
        }
      }
    }

    // annotate the score in the info field
    final Map<String, Object> map = new HashMap<String, Object>();
    map.put(getKeyNames().get(0), String.format("%.4f", scoreRA.mean()));
    return map;
  }
  private Map<String, AlignmentContext> getFilteredAndStratifiedContexts(
      UnifiedArgumentCollection UAC,
      ReferenceContext refContext,
      AlignmentContext rawContext,
      final GenotypeLikelihoodsCalculationModel.Model model) {

    if (!BaseUtils.isRegularBase(refContext.getBase())) return null;

    Map<String, AlignmentContext> stratifiedContexts = null;

    if (model.name().contains("INDEL")) {

      final ReadBackedPileup pileup =
          rawContext.getBasePileup().getMappingFilteredPileup(UAC.MIN_BASE_QUALTY_SCORE);
      // don't call when there is no coverage
      if (pileup.getNumberOfElements() == 0 && UAC.OutputMode != OUTPUT_MODE.EMIT_ALL_SITES)
        return null;

      // stratify the AlignmentContext and cut by sample
      stratifiedContexts = AlignmentContextUtils.splitContextBySampleName(pileup);

    } else if (model.name().contains("SNP")) {

      // stratify the AlignmentContext and cut by sample
      stratifiedContexts =
          AlignmentContextUtils.splitContextBySampleName(rawContext.getBasePileup());

      if (!(UAC.OutputMode == OUTPUT_MODE.EMIT_ALL_SITES
          && UAC.GenotypingMode
              != GenotypeLikelihoodsCalculationModel.GENOTYPING_MODE.GENOTYPE_GIVEN_ALLELES)) {
        int numDeletions = 0;
        for (final PileupElement p : rawContext.getBasePileup()) {
          if (p.isDeletion()) numDeletions++;
        }
        if (((double) numDeletions) / ((double) rawContext.getBasePileup().getNumberOfElements())
            > UAC.MAX_DELETION_FRACTION) {
          return null;
        }
      }
    }

    return stratifiedContexts;
  }
  private VariantCallContext generateEmptyContext(
      RefMetaDataTracker tracker,
      ReferenceContext ref,
      Map<String, AlignmentContext> stratifiedContexts,
      AlignmentContext rawContext) {
    VariantContext vc;
    if (UAC.GenotypingMode
        == GenotypeLikelihoodsCalculationModel.GENOTYPING_MODE.GENOTYPE_GIVEN_ALLELES) {
      VariantContext vcInput =
          UnifiedGenotyperEngine.getVCFromAllelesRod(
              tracker, ref, rawContext.getLocation(), false, logger, UAC.alleles);
      if (vcInput == null) return null;
      vc =
          new VariantContextBuilder(
                  "UG_call",
                  ref.getLocus().getContig(),
                  vcInput.getStart(),
                  vcInput.getEnd(),
                  vcInput.getAlleles())
              .make();
    } else {
      // deal with bad/non-standard reference bases
      if (!Allele.acceptableAlleleBases(new byte[] {ref.getBase()})) return null;

      Set<Allele> alleles = new HashSet<Allele>();
      alleles.add(Allele.create(ref.getBase(), true));
      vc =
          new VariantContextBuilder(
                  "UG_call",
                  ref.getLocus().getContig(),
                  ref.getLocus().getStart(),
                  ref.getLocus().getStart(),
                  alleles)
              .make();
    }

    if (annotationEngine != null) {
      // Note: we want to use the *unfiltered* and *unBAQed* context for the annotations
      final ReadBackedPileup pileup = rawContext.getBasePileup();
      stratifiedContexts = AlignmentContextUtils.splitContextBySampleName(pileup);

      vc = annotationEngine.annotateContext(tracker, ref, stratifiedContexts, vc);
    }

    return new VariantCallContext(vc, false);
  }
  private ArrayList<Allele> computeConsensusAlleles(
      ReferenceContext ref,
      Map<String, AlignmentContext> contexts,
      AlignmentContextUtils.ReadOrientation contextType) {
    Allele refAllele = null, altAllele = null;
    GenomeLoc loc = ref.getLocus();
    ArrayList<Allele> aList = new ArrayList<Allele>();

    HashMap<String, Integer> consensusIndelStrings = new HashMap<String, Integer>();

    int insCount = 0, delCount = 0;
    // quick check of total number of indels in pileup
    for (Map.Entry<String, AlignmentContext> sample : contexts.entrySet()) {
      AlignmentContext context = AlignmentContextUtils.stratify(sample.getValue(), contextType);

      final ReadBackedExtendedEventPileup indelPileup = context.getExtendedEventPileup();
      insCount += indelPileup.getNumberOfInsertions();
      delCount += indelPileup.getNumberOfDeletions();
    }

    if (insCount < minIndelCountForGenotyping && delCount < minIndelCountForGenotyping)
      return aList;

    for (Map.Entry<String, AlignmentContext> sample : contexts.entrySet()) {
      // todo -- warning, can be duplicating expensive partition here
      AlignmentContext context = AlignmentContextUtils.stratify(sample.getValue(), contextType);

      final ReadBackedExtendedEventPileup indelPileup = context.getExtendedEventPileup();

      for (ExtendedEventPileupElement p : indelPileup.toExtendedIterable()) {
        // SAMRecord read = p.getRead();
        GATKSAMRecord read = ReadUtils.hardClipAdaptorSequence(p.getRead());
        if (read == null) continue;
        if (ReadUtils.is454Read(read)) {
          continue;
        }

        /*                if (DEBUG && p.isIndel()) {
                         System.out.format("Read: %s, cigar: %s, aln start: %d, aln end: %d, p.len:%d, Type:%s, EventBases:%s\n",
                                 read.getReadName(),read.getCigar().toString(),read.getAlignmentStart(),read.getAlignmentEnd(),
                                 p.getEventLength(),p.getType().toString(), p.getEventBases());
                     }
        */

        String indelString = p.getEventBases();
        if (p.isInsertion()) {
          boolean foundKey = false;
          if (read.getAlignmentEnd() == loc.getStart()) {
            // first corner condition: a read has an insertion at the end, and we're right at the
            // insertion.
            // In this case, the read could have any of the inserted bases and we need to build a
            // consensus
            for (String s : consensusIndelStrings.keySet()) {
              int cnt = consensusIndelStrings.get(s);
              if (s.startsWith(indelString)) {
                // case 1: current insertion is prefix of indel in hash map
                consensusIndelStrings.put(s, cnt + 1);
                foundKey = true;
                break;
              } else if (indelString.startsWith(s)) {
                // case 2: indel stored in hash table is prefix of current insertion
                // In this case, new bases are new key.
                consensusIndelStrings.remove(s);
                consensusIndelStrings.put(indelString, cnt + 1);
                foundKey = true;
                break;
              }
            }
            if (!foundKey)
              // none of the above: event bases not supported by previous table, so add new key
              consensusIndelStrings.put(indelString, 1);

          } else if (read.getAlignmentStart() == loc.getStart() + 1) {
            // opposite corner condition: read will start at current locus with an insertion
            for (String s : consensusIndelStrings.keySet()) {
              int cnt = consensusIndelStrings.get(s);
              if (s.endsWith(indelString)) {
                // case 1: current insertion is suffix of indel in hash map
                consensusIndelStrings.put(s, cnt + 1);
                foundKey = true;
                break;
              } else if (indelString.endsWith(s)) {
                // case 2: indel stored in hash table is suffix of current insertion
                // In this case, new bases are new key.

                consensusIndelStrings.remove(s);
                consensusIndelStrings.put(indelString, cnt + 1);
                foundKey = true;
                break;
              }
            }
            if (!foundKey)
              // none of the above: event bases not supported by previous table, so add new key
              consensusIndelStrings.put(indelString, 1);

          } else {
            // normal case: insertion somewhere in the middle of a read: add count to hash map
            int cnt =
                consensusIndelStrings.containsKey(indelString)
                    ? consensusIndelStrings.get(indelString)
                    : 0;
            consensusIndelStrings.put(indelString, cnt + 1);
          }

        } else if (p.isDeletion()) {
          indelString = String.format("D%d", p.getEventLength());
          int cnt =
              consensusIndelStrings.containsKey(indelString)
                  ? consensusIndelStrings.get(indelString)
                  : 0;
          consensusIndelStrings.put(indelString, cnt + 1);
        }
      }

      /*            if (DEBUG) {
          int icount = indelPileup.getNumberOfInsertions();
          int dcount = indelPileup.getNumberOfDeletions();
          if (icount + dcount > 0)
          {
              List<Pair<String,Integer>> eventStrings = indelPileup.getEventStringsWithCounts(ref.getBases());
              System.out.format("#ins: %d, #del:%d\n", insCount, delCount);

              for (int i=0 ; i < eventStrings.size() ; i++ ) {
                  System.out.format("%s:%d,",eventStrings.get(i).first,eventStrings.get(i).second);
                  //                int k=0;
              }
              System.out.println();
          }
      }             */
    }

    int maxAlleleCnt = 0;
    String bestAltAllele = "";
    for (String s : consensusIndelStrings.keySet()) {
      int curCnt = consensusIndelStrings.get(s);
      if (curCnt > maxAlleleCnt) {
        maxAlleleCnt = curCnt;
        bestAltAllele = s;
      }
      //            if (DEBUG)
      //                System.out.format("Key:%s, number: %d\n",s,consensusIndelStrings.get(s)  );
    } // gdebug-

    if (maxAlleleCnt < minIndelCountForGenotyping) return aList;

    if (bestAltAllele.startsWith("D")) {
      // get deletion length
      int dLen = Integer.valueOf(bestAltAllele.substring(1));
      // get ref bases of accurate deletion
      int startIdxInReference = (int) (1 + loc.getStart() - ref.getWindow().getStart());

      // System.out.println(new String(ref.getBases()));
      byte[] refBases =
          Arrays.copyOfRange(ref.getBases(), startIdxInReference, startIdxInReference + dLen);

      if (Allele.acceptableAlleleBases(refBases)) {
        refAllele = Allele.create(refBases, true);
        altAllele = Allele.create(Allele.NULL_ALLELE_STRING, false);
      }
    } else {
      // insertion case
      if (Allele.acceptableAlleleBases(bestAltAllele)) {
        refAllele = Allele.create(Allele.NULL_ALLELE_STRING, true);
        altAllele = Allele.create(bestAltAllele, false);
      }
    }
    if (refAllele != null && altAllele != null) {
      aList.add(0, refAllele);
      aList.add(1, altAllele);
    }
    return aList;
  }
  public Allele getLikelihoods(
      RefMetaDataTracker tracker,
      ReferenceContext ref,
      Map<String, AlignmentContext> contexts,
      AlignmentContextUtils.ReadOrientation contextType,
      GenotypePriors priors,
      Map<String, MultiallelicGenotypeLikelihoods> GLs,
      Allele alternateAlleleToUse,
      boolean useBAQedPileup) {

    if (tracker == null) return null;

    GenomeLoc loc = ref.getLocus();
    Allele refAllele, altAllele;
    VariantContext vc = null;

    if (!ref.getLocus().equals(lastSiteVisited)) {
      // starting a new site: clear allele list
      alleleList.clear();
      lastSiteVisited = ref.getLocus();
      indelLikelihoodMap.set(new HashMap<PileupElement, LinkedHashMap<Allele, Double>>());
      haplotypeMap.clear();

      if (getAlleleListFromVCF) {
        for (final VariantContext vc_input : tracker.getValues(UAC.alleles, loc)) {
          if (vc_input != null
              && allowableTypes.contains(vc_input.getType())
              && ref.getLocus().getStart() == vc_input.getStart()) {
            vc = vc_input;
            break;
          }
        }
        // ignore places where we don't have a variant
        if (vc == null) return null;

        alleleList.clear();
        if (ignoreSNPAllelesWhenGenotypingIndels) {
          // if there's an allele that has same length as the reference (i.e. a SNP or MNP), ignore
          // it and don't genotype it
          for (Allele a : vc.getAlleles())
            if (a.isNonReference() && a.getBases().length == vc.getReference().getBases().length)
              continue;
            else alleleList.add(a);

        } else {
          for (Allele a : vc.getAlleles()) alleleList.add(a);
        }

      } else {
        alleleList = computeConsensusAlleles(ref, contexts, contextType);
        if (alleleList.isEmpty()) return null;
      }
    }
    // protect against having an indel too close to the edge of a contig
    if (loc.getStart() <= HAPLOTYPE_SIZE) return null;

    // check if there is enough reference window to create haplotypes (can be an issue at end of
    // contigs)
    if (ref.getWindow().getStop() < loc.getStop() + HAPLOTYPE_SIZE) return null;
    if (!(priors instanceof DiploidIndelGenotypePriors))
      throw new StingException(
          "Only diploid-based Indel priors are supported in the DINDEL GL model");

    if (alleleList.isEmpty()) return null;

    refAllele = alleleList.get(0);
    altAllele = alleleList.get(1);

    // look for alt allele that has biggest length distance to ref allele
    int maxLenDiff = 0;
    for (Allele a : alleleList) {
      if (a.isNonReference()) {
        int lenDiff = Math.abs(a.getBaseString().length() - refAllele.getBaseString().length());
        if (lenDiff > maxLenDiff) {
          maxLenDiff = lenDiff;
          altAllele = a;
        }
      }
    }

    final int eventLength = altAllele.getBaseString().length() - refAllele.getBaseString().length();
    final int hsize = (int) ref.getWindow().size() - Math.abs(eventLength) - 1;
    final int numPrefBases = ref.getLocus().getStart() - ref.getWindow().getStart() + 1;

    haplotypeMap =
        Haplotype.makeHaplotypeListFromAlleles(
            alleleList, loc.getStart(), ref, hsize, numPrefBases);

    // For each sample, get genotype likelihoods based on pileup
    // compute prior likelihoods on haplotypes, and initialize haplotype likelihood matrix with
    // them.
    // initialize the GenotypeLikelihoods
    GLs.clear();

    for (Map.Entry<String, AlignmentContext> sample : contexts.entrySet()) {
      AlignmentContext context = AlignmentContextUtils.stratify(sample.getValue(), contextType);

      ReadBackedPileup pileup = null;
      if (context.hasExtendedEventPileup()) pileup = context.getExtendedEventPileup();
      else if (context.hasBasePileup()) pileup = context.getBasePileup();

      if (pileup != null) {
        final double[] genotypeLikelihoods =
            pairModel.computeReadHaplotypeLikelihoods(
                pileup, haplotypeMap, ref, eventLength, getIndelLikelihoodMap());

        GLs.put(
            sample.getKey(),
            new MultiallelicGenotypeLikelihoods(
                sample.getKey(), alleleList, genotypeLikelihoods, getFilteredDepth(pileup)));

        if (DEBUG) {
          System.out.format("Sample:%s Alleles:%s GL:", sample.getKey(), alleleList.toString());
          for (int k = 0; k < genotypeLikelihoods.length; k++)
            System.out.format("%1.4f ", genotypeLikelihoods[k]);
          System.out.println();
        }
      }
    }

    return refAllele;
  }
  /**
   * Main entry function to calculate genotypes of a given VC with corresponding GL's
   *
   * @param tracker Tracker
   * @param refContext Reference context
   * @param rawContext Raw context
   * @param stratifiedContexts Stratified alignment contexts
   * @param vc Input VC
   * @param model GL calculation model
   * @param inheritAttributesFromInputVC Output VC will contain attributes inherited from input vc
   * @return VC with assigned genotypes
   */
  public VariantCallContext calculateGenotypes(
      final RefMetaDataTracker tracker,
      final ReferenceContext refContext,
      final AlignmentContext rawContext,
      Map<String, AlignmentContext> stratifiedContexts,
      final VariantContext vc,
      final GenotypeLikelihoodsCalculationModel.Model model,
      final boolean inheritAttributesFromInputVC,
      final Map<String, org.broadinstitute.sting.utils.genotyper.PerReadAlleleLikelihoodMap>
          perReadAlleleLikelihoodMap) {

    boolean limitedContext =
        tracker == null || refContext == null || rawContext == null || stratifiedContexts == null;

    // initialize the data for this thread if that hasn't been done yet
    if (afcm.get() == null) {
      afcm.set(AFCalcFactory.createAFCalc(UAC, N, logger));
    }

    // estimate our confidence in a reference call and return
    if (vc.getNSamples() == 0) {
      if (limitedContext) return null;
      return (UAC.OutputMode != OUTPUT_MODE.EMIT_ALL_SITES
          ? estimateReferenceConfidence(vc, stratifiedContexts, getTheta(model), false, 1.0)
          : generateEmptyContext(tracker, refContext, stratifiedContexts, rawContext));
    }

    AFCalcResult AFresult = afcm.get().getLog10PNonRef(vc, getAlleleFrequencyPriors(model));

    // is the most likely frequency conformation AC=0 for all alternate alleles?
    boolean bestGuessIsRef = true;

    // determine which alternate alleles have AF>0
    final List<Allele> myAlleles = new ArrayList<Allele>(vc.getAlleles().size());
    final List<Integer> alleleCountsofMLE = new ArrayList<Integer>(vc.getAlleles().size());
    myAlleles.add(vc.getReference());
    for (int i = 0; i < AFresult.getAllelesUsedInGenotyping().size(); i++) {
      final Allele alternateAllele = AFresult.getAllelesUsedInGenotyping().get(i);
      if (alternateAllele.isReference()) continue;

      // we are non-ref if the probability of being non-ref > the emit confidence.
      // the emit confidence is phred-scaled, say 30 => 10^-3.
      // the posterior AF > 0 is log10: -5 => 10^-5
      // we are non-ref if 10^-5 < 10^-3 => -5 < -3
      final boolean isNonRef =
          AFresult.isPolymorphic(alternateAllele, UAC.STANDARD_CONFIDENCE_FOR_EMITTING / -10.0);

      // if the most likely AC is not 0, then this is a good alternate allele to use
      if (isNonRef) {
        myAlleles.add(alternateAllele);
        alleleCountsofMLE.add(AFresult.getAlleleCountAtMLE(alternateAllele));
        bestGuessIsRef = false;
      }
      // if in GENOTYPE_GIVEN_ALLELES mode, we still want to allow the use of a poor allele
      else if (UAC.GenotypingMode
          == GenotypeLikelihoodsCalculationModel.GENOTYPING_MODE.GENOTYPE_GIVEN_ALLELES) {
        myAlleles.add(alternateAllele);
        alleleCountsofMLE.add(AFresult.getAlleleCountAtMLE(alternateAllele));
      }
    }

    final double PoFGT0 = Math.pow(10, AFresult.getLog10PosteriorOfAFGT0());

    // note the math.abs is necessary because -10 * 0.0 => -0.0 which isn't nice
    final double phredScaledConfidence =
        Math.abs(
            !bestGuessIsRef
                    || UAC.GenotypingMode
                        == GenotypeLikelihoodsCalculationModel.GENOTYPING_MODE
                            .GENOTYPE_GIVEN_ALLELES
                ? -10 * AFresult.getLog10PosteriorOfAFEq0()
                : -10 * AFresult.getLog10PosteriorOfAFGT0());

    // return a null call if we don't pass the confidence cutoff or the most likely allele frequency
    // is zero
    if (UAC.OutputMode != OUTPUT_MODE.EMIT_ALL_SITES
        && !passesEmitThreshold(phredScaledConfidence, bestGuessIsRef)) {
      // technically, at this point our confidence in a reference call isn't accurately estimated
      //  because it didn't take into account samples with no data, so let's get a better estimate
      return limitedContext
          ? null
          : estimateReferenceConfidence(vc, stratifiedContexts, getTheta(model), true, PoFGT0);
    }

    // start constructing the resulting VC
    final GenomeLoc loc = genomeLocParser.createGenomeLoc(vc);
    final VariantContextBuilder builder =
        new VariantContextBuilder(
            "UG_call", loc.getContig(), loc.getStart(), loc.getStop(), myAlleles);
    builder.log10PError(phredScaledConfidence / -10.0);
    if (!passesCallThreshold(phredScaledConfidence)) builder.filters(filter);

    // create the genotypes
    final GenotypesContext genotypes = afcm.get().subsetAlleles(vc, myAlleles, true, ploidy);
    builder.genotypes(genotypes);

    // print out stats if we have a writer
    if (verboseWriter != null && !limitedContext)
      printVerboseData(refContext.getLocus().toString(), vc, PoFGT0, phredScaledConfidence, model);

    // *** note that calculating strand bias involves overwriting data structures, so we do that
    // last
    final HashMap<String, Object> attributes = new HashMap<String, Object>();

    // inherit attributed from input vc if requested
    if (inheritAttributesFromInputVC) attributes.putAll(vc.getAttributes());
    // if the site was downsampled, record that fact
    if (!limitedContext && rawContext.hasPileupBeenDownsampled())
      attributes.put(VCFConstants.DOWNSAMPLED_KEY, true);

    if (UAC.ANNOTATE_NUMBER_OF_ALLELES_DISCOVERED)
      attributes.put(NUMBER_OF_DISCOVERED_ALLELES_KEY, vc.getAlternateAlleles().size());

    // add the MLE AC and AF annotations
    if (alleleCountsofMLE.size() > 0) {
      attributes.put(VCFConstants.MLE_ALLELE_COUNT_KEY, alleleCountsofMLE);
      final int AN = builder.make().getCalledChrCount();
      final ArrayList<Double> MLEfrequencies = new ArrayList<Double>(alleleCountsofMLE.size());
      // the MLEAC is allowed to be larger than the AN (e.g. in the case of all PLs being 0, the GT
      // is ./. but the exact model may arbitrarily choose an AC>1)
      for (int AC : alleleCountsofMLE) MLEfrequencies.add(Math.min(1.0, (double) AC / (double) AN));
      attributes.put(VCFConstants.MLE_ALLELE_FREQUENCY_KEY, MLEfrequencies);
    }

    if (UAC.COMPUTE_SLOD && !limitedContext && !bestGuessIsRef) {
      // final boolean DEBUG_SLOD = false;

      // the overall lod
      // double overallLog10PofNull = AFresult.log10AlleleFrequencyPosteriors[0];
      double overallLog10PofF = AFresult.getLog10LikelihoodOfAFGT0();
      // if ( DEBUG_SLOD ) System.out.println("overallLog10PofF=" + overallLog10PofF);

      List<Allele> allAllelesToUse = builder.make().getAlleles();

      // the forward lod
      VariantContext vcForward =
          calculateLikelihoods(
              tracker,
              refContext,
              stratifiedContexts,
              AlignmentContextUtils.ReadOrientation.FORWARD,
              allAllelesToUse,
              false,
              model,
              perReadAlleleLikelihoodMap);
      AFresult = afcm.get().getLog10PNonRef(vcForward, getAlleleFrequencyPriors(model));
      // double[] normalizedLog10Posteriors =
      // MathUtils.normalizeFromLog10(AFresult.log10AlleleFrequencyPosteriors, true);
      double forwardLog10PofNull = AFresult.getLog10LikelihoodOfAFEq0();
      double forwardLog10PofF = AFresult.getLog10LikelihoodOfAFGT0();
      // if ( DEBUG_SLOD ) System.out.println("forwardLog10PofNull=" + forwardLog10PofNull + ",
      // forwardLog10PofF=" + forwardLog10PofF);

      // the reverse lod
      VariantContext vcReverse =
          calculateLikelihoods(
              tracker,
              refContext,
              stratifiedContexts,
              AlignmentContextUtils.ReadOrientation.REVERSE,
              allAllelesToUse,
              false,
              model,
              perReadAlleleLikelihoodMap);
      AFresult = afcm.get().getLog10PNonRef(vcReverse, getAlleleFrequencyPriors(model));
      // normalizedLog10Posteriors =
      // MathUtils.normalizeFromLog10(AFresult.log10AlleleFrequencyPosteriors, true);
      double reverseLog10PofNull = AFresult.getLog10LikelihoodOfAFEq0();
      double reverseLog10PofF = AFresult.getLog10LikelihoodOfAFGT0();
      // if ( DEBUG_SLOD ) System.out.println("reverseLog10PofNull=" + reverseLog10PofNull + ",
      // reverseLog10PofF=" + reverseLog10PofF);

      double forwardLod = forwardLog10PofF + reverseLog10PofNull - overallLog10PofF;
      double reverseLod = reverseLog10PofF + forwardLog10PofNull - overallLog10PofF;
      // if ( DEBUG_SLOD ) System.out.println("forward lod=" + forwardLod + ", reverse lod=" +
      // reverseLod);

      // strand score is max bias between forward and reverse strands
      double strandScore = Math.max(forwardLod, reverseLod);
      // rescale by a factor of 10
      strandScore *= 10.0;
      // logger.debug(String.format("SLOD=%f", strandScore));

      if (!Double.isNaN(strandScore)) attributes.put("SB", strandScore);
    }

    // finish constructing the resulting VC
    builder.attributes(attributes);
    VariantContext vcCall = builder.make();

    // if we are subsetting alleles (either because there were too many or because some were not
    // polymorphic)
    // then we may need to trim the alleles (because the original VariantContext may have had to pad
    // at the end).
    if (myAlleles.size() != vc.getAlleles().size()
        && !limitedContext) // limitedContext callers need to handle allele trimming on their own to
                            // keep their perReadAlleleLikelihoodMap alleles in sync
    vcCall = VariantContextUtils.reverseTrimAlleles(vcCall);

    if (annotationEngine != null
        && !limitedContext) { // limitedContext callers need to handle annotations on their own by
                              // calling their own annotationEngine
      // Note: we want to use the *unfiltered* and *unBAQed* context for the annotations
      final ReadBackedPileup pileup = rawContext.getBasePileup();
      stratifiedContexts = AlignmentContextUtils.splitContextBySampleName(pileup);

      vcCall =
          annotationEngine.annotateContext(
              tracker, refContext, stratifiedContexts, vcCall, perReadAlleleLikelihoodMap);
    }

    return new VariantCallContext(vcCall, confidentlyCalled(phredScaledConfidence, PoFGT0));
  }