/* * Copyright (c) 2010 The Broad Institute * * Permission is hereby granted, free of charge, to any person * obtaining a copy of this software and associated documentation * files (the "Software"), to deal in the Software without * restriction, including without limitation the rights to use, * copy, modify, merge, publish, distribute, sublicense, and/or sell * copies of the Software, and to permit persons to whom the * Software is furnished to do so, subject to the following * conditions: * * The above copyright notice and this permission notice shall be * included in all copies or substantial portions of the Software. * * THE SOFTWARE IS PROVIDED "AS IS", WITHOUT WARRANTY OF ANY KIND, * EXPRESS OR IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES * OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE AND * NONINFRINGEMENT. IN NO EVENT SHALL THE AUTHORS OR COPYRIGHT * HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER LIABILITY, * WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING * FROM, OUT OF OR IN CONNECTION WITH THE SOFTWARE OR * THE USE OR OTHER DEALINGS IN THE SOFTWARE. */ package org.broadinstitute.sting.utils.sam; import net.sf.samtools.CigarOperator; import net.sf.samtools.SAMRecord; import net.sf.samtools.Cigar; import net.sf.samtools.CigarElement; import net.sf.samtools.util.StringUtil; import org.broadinstitute.sting.gatk.contexts.ReferenceContext; import org.broadinstitute.sting.utils.pileup.*; import org.broadinstitute.sting.utils.StingException; import org.broadinstitute.sting.utils.BaseUtils; public class AlignmentUtils { private static class MismatchCount { public int numMismatches = 0; public long mismatchQualities = 0; } /** Returns number of mismatches in the alignment r to the reference sequence * refSeq assuming the alignment starts at (ZERO-based) position refIndex on the * specified reference sequence; in other words, refIndex is used in place of alignment's own * getAlignmentStart() coordinate and the latter is never used. However, the structure of the alignment r * (i.e. it's cigar string with all the insertions/deletions it may specify) is fully respected. * * THIS CODE ASSUMES THAT ALL BYTES COME FROM UPPERCASED CHARS. * * @param r alignment * @param refSeq chunk of reference sequence that subsumes the alignment completely (if alignment runs out of * the reference string, IndexOutOfBound exception will be thrown at runtime). * @param refIndex zero-based position, at which the alignment starts on the specified reference string. * @return the number of mismatches */ public static int numMismatches(SAMRecord r, byte[] refSeq, int refIndex) { return getMismatchCount(r, refSeq, refIndex).numMismatches; } public static int numMismatches(SAMRecord r, String refSeq, int refIndex ) { if ( r.getReadUnmappedFlag() ) return 1000000; return numMismatches(r, StringUtil.stringToBytes(refSeq), refIndex); } public static long mismatchingQualities(SAMRecord r, byte[] refSeq, int refIndex) { return getMismatchCount(r, refSeq, refIndex).mismatchQualities; } public static long mismatchingQualities(SAMRecord r, String refSeq, int refIndex ) { if ( r.getReadUnmappedFlag() ) return 1000000; return numMismatches(r, StringUtil.stringToBytes(refSeq), refIndex); } private static MismatchCount getMismatchCount(SAMRecord r, byte[] refSeq, int refIndex) { MismatchCount mc = new MismatchCount(); int readIdx = 0; byte[] readSeq = r.getReadBases(); Cigar c = r.getCigar(); for (int i = 0 ; i < c.numCigarElements() ; i++) { CigarElement ce = c.getCigarElement(i); switch ( ce.getOperator() ) { case M: for (int j = 0 ; j < ce.getLength() ; j++, refIndex++, readIdx++ ) { if ( refIndex >= refSeq.length ) continue; byte refChr = refSeq[refIndex]; byte readChr = readSeq[readIdx]; // Note: we need to count X/N's as mismatches because that's what SAM requires //if ( BaseUtils.simpleBaseToBaseIndex(readChr) == -1 || // BaseUtils.simpleBaseToBaseIndex(refChr) == -1 ) // continue; // do not count Ns/Xs/etc ? if ( readChr != refChr ) { mc.numMismatches++; mc.mismatchQualities += r.getBaseQualities()[readIdx]; } } break; case I: case S: readIdx += ce.getLength(); break; case D: case N: refIndex += ce.getLength(); break; default: throw new StingException("The " + ce.getOperator() + " cigar element is not currently supported"); } } return mc; } /** Returns the number of mismatches in the pileup within the given reference context. * * @param pileup the pileup with reads * @param ref the reference context * @param ignoreTargetSite if true, ignore mismatches at the target locus (i.e. the center of the window) * @return the number of mismatches */ public static int mismatchesInRefWindow(ReadBackedPileup pileup, ReferenceContext ref, boolean ignoreTargetSite) { int mismatches = 0; for ( PileupElement p : pileup ) mismatches += mismatchesInRefWindow(p, ref, ignoreTargetSite); return mismatches; } /** Returns the number of mismatches in the pileup element within the given reference context. * * @param p the pileup element * @param ref the reference context * @param ignoreTargetSite if true, ignore mismatches at the target locus (i.e. the center of the window) * @return the number of mismatches */ public static int mismatchesInRefWindow(PileupElement p, ReferenceContext ref, boolean ignoreTargetSite) { return mismatchesInRefWindow(p, ref, ignoreTargetSite, false); } /** Returns the number of mismatches in the pileup element within the given reference context. * * @param p the pileup element * @param ref the reference context * @param ignoreTargetSite if true, ignore mismatches at the target locus (i.e. the center of the window) * @param qualitySumInsteadOfMismatchCount if true, return the quality score sum of the mismatches rather than the count * @return the number of mismatches */ public static int mismatchesInRefWindow(PileupElement p, ReferenceContext ref, boolean ignoreTargetSite, boolean qualitySumInsteadOfMismatchCount) { int sum = 0; int windowStart = (int)ref.getWindow().getStart(); int windowStop = (int)ref.getWindow().getStop(); char[] refBases = ref.getBases(); byte[] readBases = p.getRead().getReadBases(); byte[] readQualities = p.getRead().getBaseQualities(); Cigar c = p.getRead().getCigar(); int readIndex = 0; int currentPos = p.getRead().getAlignmentStart(); int refIndex = Math.max(0, currentPos - windowStart); for (int i = 0 ; i < c.numCigarElements() ; i++) { CigarElement ce = c.getCigarElement(i); int cigarElementLength = ce.getLength(); switch ( ce.getOperator() ) { case M: for (int j = 0; j < cigarElementLength; j++, readIndex++, currentPos++) { // are we past the ref window? if ( currentPos > windowStop ) break; // are we before the ref window? if ( currentPos < windowStart ) continue; char refChr = refBases[refIndex++]; // do we need to skip the target site? if ( ignoreTargetSite && ref.getLocus().getStart() == currentPos ) continue; char readChr = (char)readBases[readIndex]; if ( Character.toUpperCase(readChr) != Character.toUpperCase(refChr) ) sum += (qualitySumInsteadOfMismatchCount) ? readQualities[readIndex] : 1; } break; case I: case S: readIndex += cigarElementLength; break; case D: case N: currentPos += cigarElementLength; if ( currentPos > windowStart ) refIndex += Math.min(cigarElementLength, currentPos - windowStart); break; default: // fail silently return 0; } } return sum; } /** Returns number of alignment blocks (continuous stretches of aligned bases) in the specified alignment. * This method follows closely the SAMRecord::getAlignmentBlocks() implemented in samtools library, but * it only counts blocks without actually allocating and filling the list of blocks themselves. Hence, this method is * a much more efficient alternative to r.getAlignmentBlocks.size() in the situations when this number is all that is needed. * Formally, this method simply returns the number of M elements in the cigar. * @param r alignment * @return number of continuous alignment blocks (i.e. 'M' elements of the cigar; all indel and clipping elements are ignored). */ public static int getNumAlignmentBlocks(final SAMRecord r) { int n = 0; final Cigar cigar = r.getCigar(); if (cigar == null) return 0; for (final CigarElement e : cigar.getCigarElements()) { if (e.getOperator() == CigarOperator.M ) n++; } return n; } public static String toString(Cigar cig) { StringBuilder b = new StringBuilder(); for ( int i = 0 ; i < cig.numCigarElements() ; i++ ) { char c='?'; switch ( cig.getCigarElement(i).getOperator() ) { case M : c = 'M'; break; case D : c = 'D'; break; case I : c = 'I'; break; } b.append(cig.getCigarElement(i).getLength()); b.append(c); } return b.toString(); } public static String alignmentToString(final Cigar cigar,final String seq, final String ref, final int posOnRef ) { return alignmentToString( cigar, seq, ref, posOnRef, 0 ); } public static String cigarToString(Cigar cig) { if ( cig == null ) return "null"; StringBuilder b = new StringBuilder(); for ( int i = 0 ; i < cig.numCigarElements() ; i++ ) { char c='?'; switch ( cig.getCigarElement(i).getOperator() ) { case M : c = 'M'; break; case D : c = 'D'; break; case I : c = 'I'; break; } b.append(cig.getCigarElement(i).getLength()); b.append(c); } return b.toString(); } public static String alignmentToString(final Cigar cigar,final String seq, final String ref, final int posOnRef, final int posOnRead ) { int readPos = posOnRead; int refPos = posOnRef; StringBuilder refLine = new StringBuilder(); StringBuilder readLine = new StringBuilder(); for ( int i = 0 ; i < posOnRead ; i++ ) { refLine.append( ref.charAt( refPos - readPos + i ) ); readLine.append( seq.charAt(i) ) ; } for ( int i = 0 ; i < cigar.numCigarElements() ; i++ ) { final CigarElement ce = cigar.getCigarElement(i); switch(ce.getOperator()) { case I: for ( int j = 0 ; j < ce.getLength(); j++ ) { refLine.append('+'); readLine.append( seq.charAt( readPos++ ) ); } break; case D: for ( int j = 0 ; j < ce.getLength(); j++ ) { readLine.append('*'); refLine.append( ref.charAt( refPos++ ) ); } break; case M: for ( int j = 0 ; j < ce.getLength(); j++ ) { refLine.append(ref.charAt( refPos++ ) ); readLine.append( seq.charAt( readPos++ ) ); } break; default: throw new StingException("Unsupported cigar operator: "+ce.getOperator() ); } } refLine.append('\n'); refLine.append(readLine); refLine.append('\n'); return refLine.toString(); } public static char[] alignmentToCharArray( final Cigar cigar, final char[] read, final char[] ref ) { final char[] alignment = new char[read.length]; int refPos = 0; int alignPos = 0; for ( int iii = 0 ; iii < cigar.numCigarElements() ; iii++ ) { final CigarElement ce = cigar.getCigarElement(iii); switch( ce.getOperator() ) { case I: case S: for ( int jjj = 0 ; jjj < ce.getLength(); jjj++ ) { alignment[alignPos++] = '+'; } break; case D: case N: refPos++; break; case M: for ( int jjj = 0 ; jjj < ce.getLength(); jjj++ ) { alignment[alignPos] = ref[refPos]; alignPos++; refPos++; } break; default: throw new StingException( "Unsupported cigar operator: " + ce.getOperator() ); } } return alignment; } /** * Due to (unfortunate) multiple ways to indicate that read is unmapped allowed by SAM format * specification, one may need this convenience shortcut. Checks both 'read unmapped' flag and * alignment reference index/start. * @param r record * @return true if read is unmapped */ public static boolean isReadUnmapped(final SAMRecord r) { if ( r.getReadUnmappedFlag() ) return true; // our life would be so much easier if all sam files followed the specs. In reality, // sam files (including those generated by maq or bwa) miss headers alltogether. When // reading such a SAM file, reference name is set, but since there is no sequence dictionary, // null is always returned for referenceIndex. Let's be paranoid here, and make sure that // we do not call the read "unmapped" when it has only reference name set with ref. index missing // or vice versa. if ( ( r.getReferenceIndex() != null && r.getReferenceIndex() != SAMRecord.NO_ALIGNMENT_REFERENCE_INDEX || r.getReferenceName() != null && r.getReferenceName() != SAMRecord.NO_ALIGNMENT_REFERENCE_NAME ) && r.getAlignmentStart() != SAMRecord.NO_ALIGNMENT_START ) return false ; return true; } /** Returns the array of base qualitites in the order the bases were read on the machine (i.e. always starting from * cycle 1). In other words, if the read is unmapped or aligned in the forward direction, the read's own base * qualities are returned as stored in the SAM record; if the read is aligned in the reverse direction, the array * of read's base qualitites is inverted (in this case new array is allocated and returned). * @param read * @return */ public static byte [] getQualsInCycleOrder(SAMRecord read) { if ( isReadUnmapped(read) || ! read.getReadNegativeStrandFlag() ) return read.getBaseQualities(); return BaseUtils.reverse(read.getBaseQualities()); } }