@Override
protected int doWork() {
IOUtil.assertFileIsReadable(INPUT);
IOUtil.assertFileIsWritable(OUTPUT);
IOUtil.assertFileIsReadable(REFERENCE_SEQUENCE);
// Setup all the inputs
final ProgressLogger progress = new ProgressLogger(log, 10000000, "Processed", "loci");
final ReferenceSequenceFileWalker refWalker =
new ReferenceSequenceFileWalker(REFERENCE_SEQUENCE);
final SamReader in =
SamReaderFactory.makeDefault().referenceSequence(REFERENCE_SEQUENCE).open(INPUT);
final SamLocusIterator iterator = getLocusIterator(in);
final List<SamRecordFilter> filters = new ArrayList<SamRecordFilter>();
final CountingFilter dupeFilter = new CountingDuplicateFilter();
final CountingFilter mapqFilter = new CountingMapQFilter(MINIMUM_MAPPING_QUALITY);
final CountingPairedFilter pairFilter = new CountingPairedFilter();
filters.add(mapqFilter);
filters.add(dupeFilter);
if (!COUNT_UNPAIRED) {
filters.add(pairFilter);
}
filters.add(
new SecondaryAlignmentFilter()); // Not a counting filter because we never want to count
// reads twice
iterator.setSamFilters(filters);
iterator.setEmitUncoveredLoci(true);
iterator.setMappingQualityScoreCutoff(0); // Handled separately because we want to count bases
iterator.setQualityScoreCutoff(0); // Handled separately because we want to count bases
iterator.setIncludeNonPfReads(false);
final int max = COVERAGE_CAP;
final long[] HistogramArray = new long[max + 1];
final long[] baseQHistogramArray = new long[Byte.MAX_VALUE];
final boolean usingStopAfter = STOP_AFTER > 0;
final long stopAfter = STOP_AFTER - 1;
long counter = 0;
long basesExcludedByBaseq = 0;
long basesExcludedByOverlap = 0;
long basesExcludedByCapping = 0;
// Loop through all the loci
while (iterator.hasNext()) {
final SamLocusIterator.LocusInfo info = iterator.next();
// Check that the reference is not N
final ReferenceSequence ref = refWalker.get(info.getSequenceIndex());
final byte base = ref.getBases()[info.getPosition() - 1];
if (base == 'N') continue;
// Figure out the coverage while not counting overlapping reads twice, and excluding various
// things
final HashSet<String> readNames = new HashSet<String>(info.getRecordAndPositions().size());
int pileupSize = 0;
for (final SamLocusIterator.RecordAndOffset recs : info.getRecordAndPositions()) {
if (recs.getBaseQuality() < MINIMUM_BASE_QUALITY) {
++basesExcludedByBaseq;
continue;
}
if (!readNames.add(recs.getRecord().getReadName())) {
++basesExcludedByOverlap;
continue;
}
pileupSize++;
if (pileupSize <= max) {
baseQHistogramArray[recs.getRecord().getBaseQualities()[recs.getOffset()]]++;
}
}
final int depth = Math.min(readNames.size(), max);
if (depth < readNames.size()) basesExcludedByCapping += readNames.size() - max;
HistogramArray[depth]++;
// Record progress and perhaps stop
progress.record(info.getSequenceName(), info.getPosition());
if (usingStopAfter && ++counter > stopAfter) break;
}
// Construct and write the outputs
final Histogram<Integer> histo = new Histogram<Integer>("coverage", "count");
for (int i = 0; i < HistogramArray.length; ++i) {
histo.increment(i, HistogramArray[i]);
}
// Construct and write the outputs
final Histogram<Integer> baseQHisto = new Histogram<Integer>("value", "baseq_count");
for (int i = 0; i < baseQHistogramArray.length; ++i) {
baseQHisto.increment(i, baseQHistogramArray[i]);
}
final WgsMetrics metrics = generateWgsMetrics();
metrics.GENOME_TERRITORY = (long) histo.getSumOfValues();
metrics.MEAN_COVERAGE = histo.getMean();
metrics.SD_COVERAGE = histo.getStandardDeviation();
metrics.MEDIAN_COVERAGE = histo.getMedian();
metrics.MAD_COVERAGE = histo.getMedianAbsoluteDeviation();
final long basesExcludedByDupes = getBasesExcludedBy(dupeFilter);
final long basesExcludedByMapq = getBasesExcludedBy(mapqFilter);
final long basesExcludedByPairing = getBasesExcludedBy(pairFilter);
final double total = histo.getSum();
final double totalWithExcludes =
total
+ basesExcludedByDupes
+ basesExcludedByMapq
+ basesExcludedByPairing
+ basesExcludedByBaseq
+ basesExcludedByOverlap
+ basesExcludedByCapping;
metrics.PCT_EXC_DUPE = basesExcludedByDupes / totalWithExcludes;
metrics.PCT_EXC_MAPQ = basesExcludedByMapq / totalWithExcludes;
metrics.PCT_EXC_UNPAIRED = basesExcludedByPairing / totalWithExcludes;
metrics.PCT_EXC_BASEQ = basesExcludedByBaseq / totalWithExcludes;
metrics.PCT_EXC_OVERLAP = basesExcludedByOverlap / totalWithExcludes;
metrics.PCT_EXC_CAPPED = basesExcludedByCapping / totalWithExcludes;
metrics.PCT_EXC_TOTAL = (totalWithExcludes - total) / totalWithExcludes;
metrics.PCT_1X =
MathUtil.sum(HistogramArray, 1, HistogramArray.length) / (double) metrics.GENOME_TERRITORY;
metrics.PCT_5X =
MathUtil.sum(HistogramArray, 5, HistogramArray.length) / (double) metrics.GENOME_TERRITORY;
metrics.PCT_10X =
MathUtil.sum(HistogramArray, 10, HistogramArray.length) / (double) metrics.GENOME_TERRITORY;
metrics.PCT_15X =
MathUtil.sum(HistogramArray, 15, HistogramArray.length) / (double) metrics.GENOME_TERRITORY;
metrics.PCT_20X =
MathUtil.sum(HistogramArray, 20, HistogramArray.length) / (double) metrics.GENOME_TERRITORY;
metrics.PCT_25X =
MathUtil.sum(HistogramArray, 25, HistogramArray.length) / (double) metrics.GENOME_TERRITORY;
metrics.PCT_30X =
MathUtil.sum(HistogramArray, 30, HistogramArray.length) / (double) metrics.GENOME_TERRITORY;
metrics.PCT_40X =
MathUtil.sum(HistogramArray, 40, HistogramArray.length) / (double) metrics.GENOME_TERRITORY;
metrics.PCT_50X =
MathUtil.sum(HistogramArray, 50, HistogramArray.length) / (double) metrics.GENOME_TERRITORY;
metrics.PCT_60X =
MathUtil.sum(HistogramArray, 60, HistogramArray.length) / (double) metrics.GENOME_TERRITORY;
metrics.PCT_70X =
MathUtil.sum(HistogramArray, 70, HistogramArray.length) / (double) metrics.GENOME_TERRITORY;
metrics.PCT_80X =
MathUtil.sum(HistogramArray, 80, HistogramArray.length) / (double) metrics.GENOME_TERRITORY;
metrics.PCT_90X =
MathUtil.sum(HistogramArray, 90, HistogramArray.length) / (double) metrics.GENOME_TERRITORY;
metrics.PCT_100X =
MathUtil.sum(HistogramArray, 100, HistogramArray.length)
/ (double) metrics.GENOME_TERRITORY;
final MetricsFile<WgsMetrics, Integer> out = getMetricsFile();
out.addMetric(metrics);
out.addHistogram(histo);
if (INCLUDE_BQ_HISTOGRAM) {
out.addHistogram(baseQHisto);
}
out.write(OUTPUT);
return 0;
}
@Override
public void accumulate(final VariantContext ctx) {
logger.record(ctx.getContig(), ctx.getStart());
final String variantChrom = ctx.getContig();
final int variantPos = ctx.getStart();
// Skip anything a little too funky
if (ctx.isFiltered()) return;
if (!ctx.isVariant()) return;
if (SKIP_CHROMS.contains(variantChrom)) return;
for (final MendelianViolationMetrics trio : trios) {
final Genotype momGt = ctx.getGenotype(trio.MOTHER);
final Genotype dadGt = ctx.getGenotype(trio.FATHER);
final Genotype kidGt = ctx.getGenotype(trio.OFFSPRING);
// if any genotype:
// - has a non-snp allele; or
// - lacks a reference allele
//
// then ignore this trio
if (CollectionUtil.makeList(momGt, dadGt, kidGt)
.stream()
.anyMatch(
gt ->
gt.isHetNonRef()
|| Stream.concat(Stream.of(ctx.getReference()), gt.getAlleles().stream())
.anyMatch(a -> a.length() != 1 || a.isSymbolic()))) {
continue;
}
// if between the trio there are more than 2 alleles including the reference, continue
if (Stream.concat(
Collections.singleton(ctx.getReference()).stream(),
CollectionUtil.makeList(momGt, dadGt, kidGt)
.stream()
.flatMap(gt -> gt.getAlleles().stream()))
.collect(Collectors.toSet())
.size()
> 2) continue;
// Test to make sure:
// 1) That the site is in fact variant in the trio
// 2) that the offspring doesn't have a really wacky het allele balance
if (!isVariant(momGt, dadGt, kidGt)) continue;
if (kidGt.isHet()) {
final int[] ad = kidGt.getAD();
if (ad == null) continue;
final List<Integer> adOfAlleles =
kidGt
.getAlleles()
.stream()
.map(a -> ad[ctx.getAlleleIndex(a)])
.collect(Collectors.toList());
final double minAlleleFraction =
Math.min(adOfAlleles.get(0), adOfAlleles.get(1))
/ (double) (adOfAlleles.get(0) + adOfAlleles.get(1));
if (minAlleleFraction < MIN_HET_FRACTION) continue;
}
///////////////////////////////////////////////////////////////
// Determine whether the offspring should be haploid at this
// locus and which is the parental donor of the haploid genotype
///////////////////////////////////////////////////////////////
boolean haploid = false;
Genotype haploidParentalGenotype = null;
if (FEMALE_CHROMS.contains(variantChrom) && trio.OFFSPRING_SEX != Sex.Unknown) {
if (trio.OFFSPRING_SEX == Sex.Female) {
// famale
haploid = false;
} else if (isInPseudoAutosomalRegion(variantChrom, variantPos)) {
// male but in PAR on X, so diploid
haploid = false;
} else {
// male, out of PAR on X, haploid
haploid = true;
haploidParentalGenotype = momGt;
}
}
// the PAR on the male chromosome should be masked so that reads
// align to the female chromosomes instead, so there's no point
// of worrying about that here.
if (MALE_CHROMS.contains(variantChrom)) {
if (trio.OFFSPRING_SEX == Sex.Male) {
haploid = true;
haploidParentalGenotype = dadGt;
} else {
continue;
}
}
// We only want to look at sites where we have high enough confidence that the genotypes we
// are looking at are
// interesting. We want to ensure that parents are always GQ>=MIN_GQ, and that the kid is
// either GQ>=MIN_GQ or in the
// case where kid is het that the phred-scaled-likelihood of being reference is >=MIN_GQ.
if (haploid
&& (haploidParentalGenotype.isNoCall() || haploidParentalGenotype.getGQ() < MIN_GQ))
continue;
if (!haploid
&& (momGt.isNoCall()
|| momGt.getGQ() < MIN_GQ
|| dadGt.isNoCall()
|| dadGt.getGQ() < MIN_GQ)) continue;
if (kidGt.isNoCall()) continue;
if (momGt.isHomRef() && dadGt.isHomRef() && !kidGt.isHomRef()) {
if (kidGt.getPL()[0] < MIN_GQ) continue;
} else if (kidGt.getGQ() < MIN_GQ) continue;
// Also filter on the DP for each of the samples - it's possible to miss hets when DP is too
// low
if (haploid && (kidGt.getDP() < MIN_DP || haploidParentalGenotype.getDP() < MIN_DP)) continue;
if (!haploid && (kidGt.getDP() < MIN_DP || momGt.getDP() < MIN_DP || dadGt.getDP() < MIN_DP))
continue;
trio.NUM_VARIANT_SITES++;
///////////////////////////////////////////////////////////////
// First test for haploid violations
///////////////////////////////////////////////////////////////
MendelianViolation type = null;
if (haploid) {
if (kidGt.isHet()) continue; // Should not see heterozygous calls at haploid regions
if (!haploidParentalGenotype.getAlleles().contains(kidGt.getAllele(0))) {
if (kidGt.isHomRef()) {
type = MendelianViolation.Haploid_Other;
trio.NUM_HAPLOID_OTHER++;
} else {
type = MendelianViolation.Haploid_Denovo;
trio.NUM_HAPLOID_DENOVO++;
}
}
}
///////////////////////////////////////////////////////////////
// Then test for diploid mendelian violations
///////////////////////////////////////////////////////////////
else if (isMendelianViolation(momGt, dadGt, kidGt)) {
if (momGt.isHomRef() && dadGt.isHomRef() && !kidGt.isHomRef()) {
trio.NUM_DIPLOID_DENOVO++;
type = MendelianViolation.Diploid_Denovo;
} else if (momGt.isHomVar() && dadGt.isHomVar() && kidGt.isHet()) {
trio.NUM_HOMVAR_HOMVAR_HET++;
type = MendelianViolation.HomVar_HomVar_Het;
} else if (kidGt.isHom()
&& ((momGt.isHomRef() && dadGt.isHomVar()) || (momGt.isHomVar() && dadGt.isHomRef()))) {
trio.NUM_HOMREF_HOMVAR_HOM++;
type = MendelianViolation.HomRef_HomVar_Hom;
} else if (kidGt.isHom()
&& ((momGt.isHom() && dadGt.isHet()) || (momGt.isHet() && dadGt.isHom()))) {
trio.NUM_HOM_HET_HOM++;
type = MendelianViolation.Hom_Het_Hom;
} else {
trio.NUM_OTHER++;
type = MendelianViolation.Other;
}
}
// Output a record into the family's violation VCF
if (type != null) {
// Create a new Context subsetted to the three samples
final VariantContextBuilder builder = new VariantContextBuilder(ctx);
builder.genotypes(
ctx.getGenotypes()
.subsetToSamples(CollectionUtil.makeSet(trio.MOTHER, trio.FATHER, trio.OFFSPRING)));
builder.attribute(MENDELIAN_VIOLATION_KEY, type.name());
// Copy over some useful attributes from the full context
if (ctx.hasAttribute(VCFConstants.ALLELE_COUNT_KEY))
builder.attribute(ORIGINAL_AC, ctx.getAttribute(VCFConstants.ALLELE_COUNT_KEY));
if (ctx.hasAttribute(VCFConstants.ALLELE_FREQUENCY_KEY))
builder.attribute(ORIGINAL_AF, ctx.getAttribute(VCFConstants.ALLELE_FREQUENCY_KEY));
if (ctx.hasAttribute(VCFConstants.ALLELE_NUMBER_KEY))
builder.attribute(ORIGINAL_AN, ctx.getAttribute(VCFConstants.ALLELE_NUMBER_KEY));
// Write out the variant record
familyToViolations.get(trio.FAMILY_ID).add(builder.make());
}
}
}
/** Combines multiple SAM/BAM files into one. */
@Override
protected int doWork() {
boolean matchedSortOrders = true;
// read interval list if it is defined
final List<Interval> intervalList =
(INTERVALS == null ? null : IntervalList.fromFile(INTERVALS).uniqued().getIntervals());
// map reader->iterator used if INTERVALS is defined
final Map<SamReader, CloseableIterator<SAMRecord>> samReaderToIterator =
new HashMap<SamReader, CloseableIterator<SAMRecord>>(INPUT.size());
// Open the files for reading and writing
final List<SamReader> readers = new ArrayList<SamReader>();
final List<SAMFileHeader> headers = new ArrayList<SAMFileHeader>();
{
SAMSequenceDictionary dict = null; // Used to try and reduce redundant SDs in memory
for (final File inFile : INPUT) {
IOUtil.assertFileIsReadable(inFile);
final SamReader in =
SamReaderFactory.makeDefault().referenceSequence(REFERENCE_SEQUENCE).open(inFile);
if (INTERVALS != null) {
if (!in.hasIndex())
throw new PicardException(
"Merging with interval but Bam file is not indexed " + inFile);
final CloseableIterator<SAMRecord> samIterator =
new SamRecordIntervalIteratorFactory()
.makeSamRecordIntervalIterator(in, intervalList, true);
samReaderToIterator.put(in, samIterator);
}
readers.add(in);
headers.add(in.getFileHeader());
// A slightly hackish attempt to keep memory consumption down when merging multiple files
// with
// large sequence dictionaries (10,000s of sequences). If the dictionaries are identical,
// then
// replace the duplicate copies with a single dictionary to reduce the memory footprint.
if (dict == null) {
dict = in.getFileHeader().getSequenceDictionary();
} else if (dict.equals(in.getFileHeader().getSequenceDictionary())) {
in.getFileHeader().setSequenceDictionary(dict);
}
matchedSortOrders = matchedSortOrders && in.getFileHeader().getSortOrder() == SORT_ORDER;
}
}
// If all the input sort orders match the output sort order then just merge them and
// write on the fly, otherwise setup to merge and sort before writing out the final file
IOUtil.assertFileIsWritable(OUTPUT);
final boolean presorted;
final SAMFileHeader.SortOrder headerMergerSortOrder;
final boolean mergingSamRecordIteratorAssumeSorted;
if (matchedSortOrders
|| SORT_ORDER == SAMFileHeader.SortOrder.unsorted
|| ASSUME_SORTED
|| INTERVALS != null) {
log.info(
"Input files are in same order as output so sorting to temp directory is not needed.");
headerMergerSortOrder = SORT_ORDER;
mergingSamRecordIteratorAssumeSorted = ASSUME_SORTED;
presorted = true;
} else {
log.info("Sorting input files using temp directory " + TMP_DIR);
headerMergerSortOrder = SAMFileHeader.SortOrder.unsorted;
mergingSamRecordIteratorAssumeSorted = false;
presorted = false;
}
final SamFileHeaderMerger headerMerger =
new SamFileHeaderMerger(headerMergerSortOrder, headers, MERGE_SEQUENCE_DICTIONARIES);
final MergingSamRecordIterator iterator;
// no interval defined, get an iterator for the whole bam
if (intervalList == null) {
iterator =
new MergingSamRecordIterator(headerMerger, readers, mergingSamRecordIteratorAssumeSorted);
} else {
// show warning related to https://github.com/broadinstitute/picard/pull/314/files
log.info(
"Warning: merged bams from different interval lists may contain the same read in both files");
iterator = new MergingSamRecordIterator(headerMerger, samReaderToIterator, true);
}
final SAMFileHeader header = headerMerger.getMergedHeader();
for (final String comment : COMMENT) {
header.addComment(comment);
}
header.setSortOrder(SORT_ORDER);
final SAMFileWriterFactory samFileWriterFactory = new SAMFileWriterFactory();
if (USE_THREADING) {
samFileWriterFactory.setUseAsyncIo(true);
}
final SAMFileWriter out = samFileWriterFactory.makeSAMOrBAMWriter(header, presorted, OUTPUT);
// Lastly loop through and write out the records
final ProgressLogger progress = new ProgressLogger(log, PROGRESS_INTERVAL);
while (iterator.hasNext()) {
final SAMRecord record = iterator.next();
out.addAlignment(record);
progress.record(record);
}
log.info("Finished reading inputs.");
for (final CloseableIterator<SAMRecord> iter : samReaderToIterator.values())
CloserUtil.close(iter);
CloserUtil.close(readers);
out.close();
return 0;
}
