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package org.broadinstitute.gatk.tools.walkers.annotator;
import htsjdk.variant.variantcontext.Allele;
import htsjdk.variant.variantcontext.VariantContext;
import htsjdk.variant.vcf.VCFFormatHeaderLine;
import htsjdk.variant.vcf.VCFHeaderLine;
import htsjdk.variant.vcf.VCFInfoHeaderLine;
import org.apache.log4j.Logger;
import org.broadinstitute.gatk.engine.GenomeAnalysisEngine;
import org.broadinstitute.gatk.engine.contexts.AlignmentContext;
import org.broadinstitute.gatk.engine.contexts.ReferenceContext;
import org.broadinstitute.gatk.engine.refdata.RefMetaDataTracker;
import org.broadinstitute.gatk.tools.walkers.annotator.interfaces.AnnotatorCompatible;
import org.broadinstitute.gatk.tools.walkers.annotator.interfaces.InfoFieldAnnotation;
import htsjdk.variant.variantcontext.Genotype;
import htsjdk.variant.variantcontext.GenotypesContext;
import org.broadinstitute.gatk.utils.QualityUtils;
import org.broadinstitute.gatk.utils.genotyper.MostLikelyAllele;
import org.broadinstitute.gatk.utils.genotyper.PerReadAlleleLikelihoodMap;
import org.broadinstitute.gatk.utils.pileup.PileupElement;
import org.broadinstitute.gatk.utils.sam.GATKSAMRecord;
import java.util.*;
/**
* Class of tests to detect strand bias.
*/
public abstract class StrandBiasTest extends InfoFieldAnnotation {
private final static Logger logger = Logger.getLogger(StrandBiasTest.class);
@Override
public void initialize(final AnnotatorCompatible walker, final GenomeAnalysisEngine toolkit, final Set<VCFHeaderLine> headerLines) {
boolean hasSBBSannotation = false;
for ( final VCFHeaderLine line : headerLines) {
if ( line instanceof VCFFormatHeaderLine) {
final VCFFormatHeaderLine formatline = (VCFFormatHeaderLine)line;
if ( formatline.getID().equals(StrandBiasBySample.STRAND_BIAS_BY_SAMPLE_KEY_NAME) ) {
hasSBBSannotation = true;
break;
}
}
}
if (hasSBBSannotation) {
logger.info("StrandBiasBySample annotation exists in input VCF header. Attempting to use StrandBiasBySample " +
"values to calculate strand bias annotation values. If no sample has the SB genotype annotation, annotation may still fail.");
return;
}
boolean hasReads = toolkit.getReadsDataSource().getReaderIDs().size() > 0;
if (hasReads) {
logger.info("SAM/BAM data was found. Attempting to use read data to calculate strand bias annotations values.");
return;
}
logger.info(new String("No StrandBiasBySample annotation or read data was found. Strand bias annotations will not be output."));
}
@Override
//template method for calculating strand bias annotations using the three different methods
public Map<String, Object> annotate(final RefMetaDataTracker tracker,
final AnnotatorCompatible walker,
final ReferenceContext ref,
final Map<String,AlignmentContext> stratifiedContexts,
final VariantContext vc,
final Map<String, PerReadAlleleLikelihoodMap> stratifiedPerReadAlleleLikelihoodMap) {
if ( !vc.isVariant() )
return null;
if ( vc.hasGenotypes() ) {
boolean hasSB = false;
for (final Genotype g : vc.getGenotypes()) {
if (g.hasAnyAttribute(StrandBiasBySample.STRAND_BIAS_BY_SAMPLE_KEY_NAME)) {
hasSB = true;
break;
}
}
if (hasSB)
return calculateAnnotationFromGTfield(vc.getGenotypes());
}
//stratifiedContexts can come come from VariantAnnotator, but will be size 0 if no reads were provided
if (vc.isSNP() && stratifiedContexts != null && stratifiedContexts.size() > 0) {
return calculateAnnotationFromStratifiedContexts(stratifiedContexts, vc);
}
//stratifiedPerReadAllelelikelihoodMap can come from HaplotypeCaller call to VariantAnnotatorEngine
else if (stratifiedPerReadAlleleLikelihoodMap != null) {
return calculateAnnotationFromLikelihoodMap(stratifiedPerReadAlleleLikelihoodMap, vc);
}
else
// for non-snp variants, we need per-read likelihoods.
// for snps, we can get same result from simple pileup
return null;
}
protected abstract Map<String, Object> calculateAnnotationFromGTfield(final GenotypesContext genotypes);
protected abstract Map<String, Object> calculateAnnotationFromStratifiedContexts(final Map<String, AlignmentContext> stratifiedContexts,
final VariantContext vc);
protected abstract Map<String, Object> calculateAnnotationFromLikelihoodMap(final Map<String, PerReadAlleleLikelihoodMap> stratifiedPerReadAlleleLikelihoodMap,
final VariantContext vc);
/**
* Create the contingency table by retrieving the per-sample strand bias annotation and adding them together
* @param genotypes the genotypes from which to pull out the per-sample strand bias annotation
* @param minCount minimum threshold for the sample strand bias counts for each ref and alt.
* If both ref and alt counts are above minCount the whole sample strand bias is added to the resulting table
* @return the table used for several strand bias tests, will be null if none of the genotypes contain the per-sample SB annotation
*/
protected int[][] getTableFromSamples( final GenotypesContext genotypes, final int minCount ) {
if( genotypes == null ) { throw new IllegalArgumentException("Genotypes cannot be null."); }
final int[] sbArray = {0,0,0,0}; // reference-forward-reverse -by- alternate-forward-reverse
boolean foundData = false;
for( final Genotype g : genotypes ) {
if( g.isNoCall() || ! g.hasAnyAttribute(StrandBiasBySample.STRAND_BIAS_BY_SAMPLE_KEY_NAME) )
continue;
foundData = true;
final String sbbsString = (String) g.getAnyAttribute(StrandBiasBySample.STRAND_BIAS_BY_SAMPLE_KEY_NAME);
final int[] data = encodeSBBS(sbbsString);
if ( passesMinimumThreshold(data, minCount) ) {
for( int index = 0; index < sbArray.length; index++ ) {
sbArray[index] += data[index];
}
}
}
return ( foundData ? decodeSBBS(sbArray) : null );
}
/**
Allocate and fill a 2x2 strand contingency table. In the end, it'll look something like this:
* fw rc
* allele1 # #
* allele2 # #
* @return a 2x2 contingency table
*/
protected static int[][] getSNPContingencyTable(final Map<String, AlignmentContext> stratifiedContexts,
final Allele ref,
final Allele alt,
final int minQScoreToConsider,
final int minCount ) {
int[][] table = new int[2][2];
for (final Map.Entry<String, AlignmentContext> sample : stratifiedContexts.entrySet() ) {
final int[] myTable = new int[4];
for (final PileupElement p : sample.getValue().getBasePileup()) {
if ( ! isUsableBase(p) ) // ignore deletions and bad MQ
continue;
if ( p.getQual() < minQScoreToConsider || p.getMappingQual() < minQScoreToConsider )
continue;
updateTable(myTable, Allele.create(p.getBase(), false), p.getRead(), ref, alt);
}
if ( passesMinimumThreshold( myTable, minCount ) )
copyToMainTable(myTable, table);
}
return table;
}
/**
Allocate and fill a 2x2 strand contingency table. In the end, it'll look something like this:
* fw rc
* allele1 # #
* allele2 # #
* @return a 2x2 contingency table
*/
public static int[][] getContingencyTable( final Map<String, PerReadAlleleLikelihoodMap> stratifiedPerReadAlleleLikelihoodMap,
final VariantContext vc,
final int minCount) {
if( stratifiedPerReadAlleleLikelihoodMap == null ) { throw new IllegalArgumentException("stratifiedPerReadAlleleLikelihoodMap cannot be null"); }
if( vc == null ) { throw new IllegalArgumentException("input vc cannot be null"); }
final Allele ref = vc.getReference();
final Allele alt = vc.getAltAlleleWithHighestAlleleCount();
final int[][] table = new int[2][2];
for (final PerReadAlleleLikelihoodMap maps : stratifiedPerReadAlleleLikelihoodMap.values() ) {
final int[] myTable = new int[4];
for (final Map.Entry<GATKSAMRecord,Map<Allele,Double>> el : maps.getLikelihoodReadMap().entrySet()) {
final MostLikelyAllele mostLikelyAllele = PerReadAlleleLikelihoodMap.getMostLikelyAllele(el.getValue());
final GATKSAMRecord read = el.getKey();
updateTable(myTable, mostLikelyAllele.getAlleleIfInformative(), read, ref, alt);
}
if ( passesMinimumThreshold(myTable, minCount) )
copyToMainTable(myTable, table);
}
return table;
}
/**
* Helper method to copy the per-sample table to the main table
*
* @param perSampleTable per-sample table (single dimension)
* @param mainTable main table (two dimensions)
*/
private static void copyToMainTable(final int[] perSampleTable, final int[][] mainTable) {
mainTable[0][0] += perSampleTable[0];
mainTable[0][1] += perSampleTable[1];
mainTable[1][0] += perSampleTable[2];
mainTable[1][1] += perSampleTable[3];
}
/**
* Can the base in this pileup element be used in comparative tests?
*
* @param p the pileup element to consider
*
* @return true if this base is part of a meaningful read for comparison, false otherwise
*/
private static boolean isUsableBase(final PileupElement p) {
return !( p.isDeletion() ||
p.getMappingQual() == 0 ||
p.getMappingQual() == QualityUtils.MAPPING_QUALITY_UNAVAILABLE ||
((int) p.getQual()) < QualityUtils.MIN_USABLE_Q_SCORE);
}
private static void updateTable(final int[] table, final Allele allele, final GATKSAMRecord read, final Allele ref, final Allele alt) {
final boolean matchesRef = allele.equals(ref, true);
final boolean matchesAlt = allele.equals(alt, true);
if ( matchesRef || matchesAlt ) {
final int offset = matchesRef ? 0 : 2;
if ( read.isStrandless() ) {
// a strandless read counts as observations on both strand, at 50% weight, with a minimum of 1
// (the 1 is to ensure that a strandless read always counts as an observation on both strands, even
// if the read is only seen once, because it's a merged read or other)
table[offset]++;
table[offset + 1]++;
} else {
// a normal read with an actual strand
final boolean isFW = !read.getReadNegativeStrandFlag();
table[offset + (isFW ? 0 : 1)]++;
}
}
}
/**
* Does this strand data array pass the minimum threshold for inclusion?
*
* @param data the array
* @minCount The minimum threshold of counts in the array
* @return true if it passes the minimum threshold, false otherwise
*/
protected static boolean passesMinimumThreshold(final int[] data, final int minCount) {
// the ref and alt totals must be greater than MIN_COUNT
return data[0] + data[1] + data[2] + data[3] > minCount;
}
/**
* Helper function to parse the genotype annotation into the SB annotation array
* @param string the string that is returned by genotype.getAnnotation("SB")
* @return the array used by the per-sample Strand Bias annotation
*/
private static int[] encodeSBBS( final String string ) {
final int[] array = new int[4];
final StringTokenizer tokenizer = new StringTokenizer(string, ",", false);
for( int index = 0; index < 4; index++ ) {
array[index] = Integer.parseInt(tokenizer.nextToken());
}
return array;
}
/**
* Helper function to turn the SB annotation array into a contingency table
* @param array the array used by the per-sample Strand Bias annotation
* @return the table used by the StrandOddsRatio annotation
*/
private static int[][] decodeSBBS( final int[] array ) {
if(array.length != 4) { throw new IllegalArgumentException("Expecting a length = 4 strand bias array."); }
final int[][] table = new int[2][2];
table[0][0] = array[0];
table[0][1] = array[1];
table[1][0] = array[2];
table[1][1] = array[3];
return table;
}
}