Examples with NotPrimaryAlignmentFilter htsjdk.samtools.filter.NotPrimaryAlignmentFilter used on opensource projects

Example 1 with NotPrimaryAlignmentFilter

use of htsjdk.samtools.filter.NotPrimaryAlignmentFilter in project gatk by broadinstitute.

the class AllelicCountCollector method collect.

/**
     * Returns an {@link AllelicCountCollection} based on the pileup at sites (specified by an interval list)
     * in a sorted BAM file.  Reads and bases below the specified mapping quality and base quality, respectively,
     * are filtered out of the pileup.  The alt count is defined as the total count minus the ref count, and the
     * alt nucleotide is defined as the non-ref base with the highest count, with ties broken by the order of the
     * bases in {@link AllelicCountCollector#BASES}.
     * @param bamFile           sorted BAM file
     * @param siteIntervals     interval list of sites
     * @param minMappingQuality minimum mapping quality required for reads to be included in pileup
     * @param minBaseQuality    minimum base quality required for bases to be included in pileup
     * @return                  AllelicCountCollection of ref/alt counts at sites in BAM file
     */
public AllelicCountCollection collect(final File bamFile, final IntervalList siteIntervals, final int minMappingQuality, final int minBaseQuality) {
    try (final SamReader reader = readerFactory.open(bamFile)) {
        ParamUtils.isPositiveOrZero(minMappingQuality, "Minimum mapping quality must be nonnegative.");
        ParamUtils.isPositiveOrZero(minBaseQuality, "Minimum base quality must be nonnegative.");
        if (reader.getFileHeader().getSortOrder() != SAMFileHeader.SortOrder.coordinate) {
            throw new UserException.BadInput("BAM file " + bamFile.toString() + " must be coordinate sorted.");
        }
        final int numberOfSites = siteIntervals.size();
        final boolean useIndex = numberOfSites < MAX_INTERVALS_FOR_INDEX;
        final SamLocusIterator locusIterator = new SamLocusIterator(reader, siteIntervals, useIndex);
        //set read and locus filters [note: read counts match IGV, but off by a few from pysam.mpileup]
        final List<SamRecordFilter> samFilters = Arrays.asList(new NotPrimaryAlignmentFilter(), new DuplicateReadFilter());
        locusIterator.setSamFilters(samFilters);
        locusIterator.setEmitUncoveredLoci(true);
        locusIterator.setIncludeNonPfReads(false);
        locusIterator.setMappingQualityScoreCutoff(minMappingQuality);
        locusIterator.setQualityScoreCutoff(minBaseQuality);
        logger.info("Examining " + numberOfSites + " sites in total...");
        int locusCount = 0;
        final AllelicCountCollection counts = new AllelicCountCollection();
        for (final SamLocusIterator.LocusInfo locus : locusIterator) {
            if (locusCount % NUMBER_OF_SITES_PER_LOGGED_STATUS_UPDATE == 0) {
                logger.info("Examined " + locusCount + " sites.");
            }
            locusCount++;
            final Nucleotide refBase = Nucleotide.valueOf(referenceWalker.get(locus.getSequenceIndex()).getBases()[locus.getPosition() - 1]);
            if (!BASES.contains(refBase)) {
                logger.warn(String.format("The reference position at %d has an unknown base call (value: %s). Skipping...", locus.getPosition(), refBase.toString()));
                continue;
            }
            final Nucleotide.Counter baseCounts = getPileupBaseCounts(locus);
            //only include total ACGT counts in binomial test (exclude N, etc.)
            final int totalBaseCount = BASES.stream().mapToInt(b -> (int) baseCounts.get(b)).sum();
            final int refReadCount = (int) baseCounts.get(refBase);
            //we take alt = total - ref instead of the actual alt count
            final int altReadCount = totalBaseCount - refReadCount;
            final Nucleotide altBase = inferAltFromPileupBaseCounts(baseCounts, refBase);
            counts.add(new AllelicCount(new SimpleInterval(locus.getSequenceName(), locus.getPosition(), locus.getPosition()), refReadCount, altReadCount, refBase, altBase));
        }
        logger.info(locusCount + " sites out of " + numberOfSites + " total sites were examined.");
        return counts;
    } catch (final IOException | SAMFormatException e) {
        throw new UserException("Unable to collect allelic counts from " + bamFile);
    }
}

Example 2 with NotPrimaryAlignmentFilter

use of htsjdk.samtools.filter.NotPrimaryAlignmentFilter in project gatk by broadinstitute.

the class CollectOxoGMetrics method doWork.

@Override
protected Object doWork() {
    IOUtil.assertFileIsReadable(INPUT);
    IOUtil.assertFileIsWritable(OUTPUT);
    if (INTERVALS != null)
        IOUtil.assertFileIsReadable(INTERVALS);
    IOUtil.assertFileIsReadable(REFERENCE_SEQUENCE);
    final ReferenceSequenceFileWalker refWalker = new ReferenceSequenceFileWalker(REFERENCE_SEQUENCE);
    final SamReader in = SamReaderFactory.makeDefault().open(INPUT);
    final Set<String> samples = new HashSet<>();
    final Set<String> libraries = new HashSet<>();
    for (final SAMReadGroupRecord rec : in.getFileHeader().getReadGroups()) {
        samples.add(getOrElse(rec.getSample(), UNKNOWN_SAMPLE));
        libraries.add(getOrElse(rec.getLibrary(), UNKNOWN_LIBRARY));
    }
    // Setup the calculators
    final Set<String> contexts = CONTEXTS.isEmpty() ? makeContextStrings(CONTEXT_SIZE) : CONTEXTS;
    final ListMap<String, Calculator> calculators = new ListMap<>();
    for (final String context : contexts) {
        for (final String library : libraries) {
            calculators.add(context, new Calculator(library, context));
        }
    }
    // Load up dbSNP if available
    logger.info("Loading dbSNP File: " + DB_SNP);
    final DbSnpBitSetUtil dbSnp;
    if (DB_SNP != null)
        dbSnp = new DbSnpBitSetUtil(DB_SNP, in.getFileHeader().getSequenceDictionary());
    else
        dbSnp = null;
    // Make an iterator that will filter out funny looking things
    final SamLocusIterator iterator;
    if (INTERVALS != null) {
        final IntervalList intervals = IntervalList.fromFile(INTERVALS);
        iterator = new SamLocusIterator(in, intervals.uniqued(), false);
    } else {
        iterator = new SamLocusIterator(in);
    }
    iterator.setEmitUncoveredLoci(false);
    iterator.setMappingQualityScoreCutoff(MINIMUM_MAPPING_QUALITY);
    final List<SamRecordFilter> filters = new ArrayList<>();
    filters.add(new NotPrimaryAlignmentFilter());
    filters.add(new DuplicateReadFilter());
    if (MINIMUM_INSERT_SIZE > 0 || MAXIMUM_INSERT_SIZE > 0) {
        filters.add(new InsertSizeFilter(MINIMUM_INSERT_SIZE, MAXIMUM_INSERT_SIZE));
    }
    iterator.setSamFilters(filters);
    logger.info("Starting iteration.");
    long nextLogTime = 0;
    int sites = 0;
    for (final SamLocusIterator.LocusInfo info : iterator) {
        // Skip dbSNP sites
        final String chrom = info.getSequenceName();
        final int pos = info.getPosition();
        final int index = pos - 1;
        if (dbSnp != null && dbSnp.isDbSnpSite(chrom, pos))
            continue;
        // Skip sites at the end of chromosomes 
        final byte[] bases = refWalker.get(info.getSequenceIndex()).getBases();
        if (pos < 3 || pos > bases.length - 3)
            continue;
        // Skip non C-G bases
        final byte base = StringUtil.toUpperCase(bases[index]);
        if (base != 'C' && base != 'G')
            continue;
        // Get the context string
        final String context;
        {
            final String tmp = StringUtil.bytesToString(bases, index - CONTEXT_SIZE, 1 + (2 * CONTEXT_SIZE)).toUpperCase();
            if (base == 'C')
                context = tmp;
            else
                /* if G */
                context = SequenceUtil.reverseComplement(tmp);
        }
        final List<Calculator> calculatorsForContext = calculators.get(context);
        // happens if we get ambiguous bases in the reference
        if (calculatorsForContext == null)
            continue;
        for (final Calculator calc : calculatorsForContext) calc.accept(info, base);
        // See if we need to stop
        if (++sites % 100 == 0) {
            final long now = System.currentTimeMillis();
            if (now > nextLogTime) {
                logger.info("Visited " + sites + " sites of interest. Last site: " + chrom + ":" + pos);
                nextLogTime = now + 60000;
            }
        }
        if (sites >= STOP_AFTER)
            break;
    }
    final MetricsFile<CpcgMetrics, Integer> file = getMetricsFile();
    for (final List<Calculator> calcs : calculators.values()) {
        for (final Calculator calc : calcs) {
            final CpcgMetrics m = calc.finish();
            m.SAMPLE_ALIAS = StringUtil.join(",", new ArrayList<>(samples));
            file.addMetric(m);
        }
    }
    file.write(OUTPUT);
    CloserUtil.close(in);
    return null;
}

Example 3 with NotPrimaryAlignmentFilter

use of htsjdk.samtools.filter.NotPrimaryAlignmentFilter in project gatk-protected by broadinstitute.

the class BayesianHetPulldownCalculator method getSamLocusIteratorWithDefaultFilters.

/**
     * Returns a {@link SamLocusIterator} object for a given {@link SamReader} and {@link IntervalList} with filters
     * on minimum base quality and minimum mapping quality
     *
     * @param samReader a SamReader object
     * @return a SamLocusIterator object
     */
private SamLocusIterator getSamLocusIteratorWithDefaultFilters(final SamReader samReader) {
    final SamLocusIterator locusIterator = new SamLocusIterator(samReader, snpIntervals, false);
    /* set read and locus filters */
    final List<SamRecordFilter> samFilters = Arrays.asList(new NotPrimaryAlignmentFilter(), new DuplicateReadFilter());
    locusIterator.setSamFilters(samFilters);
    locusIterator.setEmitUncoveredLoci(false);
    locusIterator.setIncludeNonPfReads(false);
    locusIterator.setMappingQualityScoreCutoff(minMappingQuality);
    locusIterator.setQualityScoreCutoff(minBaseQuality);
    return locusIterator;
}

Example 4 with NotPrimaryAlignmentFilter

use of htsjdk.samtools.filter.NotPrimaryAlignmentFilter in project gatk-protected by broadinstitute.

the class HetPulldownCalculator method getHetPulldown.

/**
     * For a normal or tumor sample, returns a data structure giving (intervals, reference counts, alternate counts),
     * where intervals give positions of likely heterozygous SNP sites.
     *
     * <p>
     *     For a normal sample:
     *     <ul>
     *         The IntervalList snpIntervals gives common SNP sites in 1-based format.
     *     </ul>
     *     <ul>
     *         The p-value threshold must be specified for a two-sided binomial test,
     *         which is used to determine SNP sites from snpIntervals that are
     *         compatible with a heterozygous SNP, given the sample.  Only these sites are output.
     *     </ul>
     * </p>
     * <p>
     *     For a tumor sample:
     *     <ul>
     *         The IntervalList snpIntervals gives heterozygous SNP sites likely to be present in the normal sample.
     *         This should be from {@link HetPulldownCalculator#getNormal} in 1-based format.
     *         Only these sites are output.
     *     </ul>
     * </p>
     * @param bamFile           sorted BAM file for sample
     * @param snpIntervals      IntervalList of SNP sites
     * @param sampleType        flag indicating type of sample (SampleType.NORMAL or SampleType.TUMOR)
     *                          (determines whether to perform binomial test)
     * @param pvalThreshold     p-value threshold for two-sided binomial test, used for normal sample
     * @param minimumRawReads   minimum number of total reads that must be present at a het site
     * @return                  Pulldown of heterozygous SNP sites in 1-based format
     */
private Pulldown getHetPulldown(final File bamFile, final IntervalList snpIntervals, final SampleType sampleType, final double pvalThreshold, final int minimumRawReads) {
    try (final SamReader bamReader = SamReaderFactory.makeDefault().validationStringency(validationStringency).referenceSequence(refFile).open(bamFile);
        final ReferenceSequenceFileWalker refWalker = new ReferenceSequenceFileWalker(refFile)) {
        if (bamReader.getFileHeader().getSortOrder() != SAMFileHeader.SortOrder.coordinate) {
            throw new UserException.BadInput("BAM file " + bamFile.toString() + " must be coordinate sorted.");
        }
        final Pulldown hetPulldown = new Pulldown(bamReader.getFileHeader());
        final int totalNumberOfSNPs = snpIntervals.size();
        final SamLocusIterator locusIterator = new SamLocusIterator(bamReader, snpIntervals, totalNumberOfSNPs < MAX_INTERVALS_FOR_INDEX);
        //set read and locus filters [note: read counts match IGV, but off by a few from pysam.mpileup]
        final List<SamRecordFilter> samFilters = Arrays.asList(new NotPrimaryAlignmentFilter(), new DuplicateReadFilter());
        locusIterator.setSamFilters(samFilters);
        locusIterator.setEmitUncoveredLoci(false);
        locusIterator.setIncludeNonPfReads(false);
        locusIterator.setMappingQualityScoreCutoff(minMappingQuality);
        locusIterator.setQualityScoreCutoff(minBaseQuality);
        logger.info("Examining " + totalNumberOfSNPs + " sites in total...");
        int locusCount = 0;
        for (final SamLocusIterator.LocusInfo locus : locusIterator) {
            if (locusCount % NUMBER_OF_SITES_PER_LOGGED_STATUS_UPDATE == 0) {
                logger.info("Examined " + locusCount + " covered sites.");
            }
            locusCount++;
            //include N, etc. reads here
            final int totalReadCount = locus.getRecordAndOffsets().size();
            if (totalReadCount < minimumRawReads) {
                continue;
            }
            final Nucleotide.Counter baseCounts = getPileupBaseCounts(locus);
            //only include total ACGT counts in binomial test (exclude N, etc.)
            final int totalBaseCount = Arrays.stream(BASES).mapToInt(b -> (int) baseCounts.get(b)).sum();
            if (sampleType == SampleType.NORMAL && !isPileupHetCompatible(baseCounts, totalBaseCount, pvalThreshold)) {
                continue;
            }
            final Nucleotide refBase = Nucleotide.valueOf(refWalker.get(locus.getSequenceIndex()).getBases()[locus.getPosition() - 1]);
            final int refReadCount = (int) baseCounts.get(refBase);
            final int altReadCount = totalBaseCount - refReadCount;
            hetPulldown.add(new AllelicCount(new SimpleInterval(locus.getSequenceName(), locus.getPosition(), locus.getPosition()), refReadCount, altReadCount));
        }
        logger.info(locusCount + " covered sites out of " + totalNumberOfSNPs + " total sites were examined.");
        return hetPulldown;
    } catch (final IOException | SAMFormatException e) {
        throw new UserException(e.getMessage());
    }
}

Example 5 with NotPrimaryAlignmentFilter

use of htsjdk.samtools.filter.NotPrimaryAlignmentFilter in project gatk-protected by broadinstitute.

the class AllelicCountCollector method collect.

/**
     * Returns an {@link AllelicCountCollection} based on the pileup at sites (specified by an interval list)
     * in a sorted BAM file.  Reads and bases below the specified mapping quality and base quality, respectively,
     * are filtered out of the pileup.  The alt count is defined as the total count minus the ref count, and the
     * alt nucleotide is defined as the non-ref base with the highest count, with ties broken by the order of the
     * bases in {@link AllelicCountCollector#BASES}.
     * @param bamFile           sorted BAM file
     * @param siteIntervals     interval list of sites
     * @param minMappingQuality minimum mapping quality required for reads to be included in pileup
     * @param minBaseQuality    minimum base quality required for bases to be included in pileup
     * @return                  AllelicCountCollection of ref/alt counts at sites in BAM file
     */
public AllelicCountCollection collect(final File bamFile, final IntervalList siteIntervals, final int minMappingQuality, final int minBaseQuality) {
    try (final SamReader reader = readerFactory.open(bamFile)) {
        ParamUtils.isPositiveOrZero(minMappingQuality, "Minimum mapping quality must be nonnegative.");
        ParamUtils.isPositiveOrZero(minBaseQuality, "Minimum base quality must be nonnegative.");
        if (reader.getFileHeader().getSortOrder() != SAMFileHeader.SortOrder.coordinate) {
            throw new UserException.BadInput("BAM file " + bamFile.toString() + " must be coordinate sorted.");
        }
        final int numberOfSites = siteIntervals.size();
        final boolean useIndex = numberOfSites < MAX_INTERVALS_FOR_INDEX;
        final SamLocusIterator locusIterator = new SamLocusIterator(reader, siteIntervals, useIndex);
        //set read and locus filters [note: read counts match IGV, but off by a few from pysam.mpileup]
        final List<SamRecordFilter> samFilters = Arrays.asList(new NotPrimaryAlignmentFilter(), new DuplicateReadFilter());
        locusIterator.setSamFilters(samFilters);
        locusIterator.setEmitUncoveredLoci(true);
        locusIterator.setIncludeNonPfReads(false);
        locusIterator.setMappingQualityScoreCutoff(minMappingQuality);
        locusIterator.setQualityScoreCutoff(minBaseQuality);
        logger.info("Examining " + numberOfSites + " sites in total...");
        int locusCount = 0;
        final AllelicCountCollection counts = new AllelicCountCollection();
        for (final SamLocusIterator.LocusInfo locus : locusIterator) {
            if (locusCount % NUMBER_OF_SITES_PER_LOGGED_STATUS_UPDATE == 0) {
                logger.info("Examined " + locusCount + " sites.");
            }
            locusCount++;
            final Nucleotide refBase = Nucleotide.valueOf(referenceWalker.get(locus.getSequenceIndex()).getBases()[locus.getPosition() - 1]);
            if (!BASES.contains(refBase)) {
                logger.warn(String.format("The reference position at %d has an unknown base call (value: %s). Skipping...", locus.getPosition(), refBase.toString()));
                continue;
            }
            final Nucleotide.Counter baseCounts = getPileupBaseCounts(locus);
            //only include total ACGT counts in binomial test (exclude N, etc.)
            final int totalBaseCount = BASES.stream().mapToInt(b -> (int) baseCounts.get(b)).sum();
            final int refReadCount = (int) baseCounts.get(refBase);
            //we take alt = total - ref instead of the actual alt count
            final int altReadCount = totalBaseCount - refReadCount;
            final Nucleotide altBase = inferAltFromPileupBaseCounts(baseCounts, refBase);
            counts.add(new AllelicCount(new SimpleInterval(locus.getSequenceName(), locus.getPosition(), locus.getPosition()), refReadCount, altReadCount, refBase, altBase));
        }
        logger.info(locusCount + " sites out of " + numberOfSites + " total sites were examined.");
        return counts;
    } catch (final IOException | SAMFormatException e) {
        throw new UserException("Unable to collect allelic counts from " + bamFile);
    }
}