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Blasr.cpp
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Blasr.cpp
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#ifdef __linux__
# include <mcheck.h>
#endif
#include <string>
#include <iostream>
#include <vector>
#include <set>
#include <sstream>
#include <pthread.h>
#include <stdlib.h>
#include <time.h>
#include <signal.h>
#include <execinfo.h>
#include "MappingIPC.h"
#include "MappingSemaphores.h"
#include "RegisterBlasrOptions.h"
#include "CCSSequence.hpp"
#include "SMRTSequence.hpp"
#include "FASTASequence.hpp"
#include "FASTAReader.hpp"
#include "SeqUtils.hpp"
#include "defs.h"
#include "utils.hpp"
#include "tuples/DNATuple.hpp"
#include "tuples/HashedTupleList.hpp"
#include "algorithms/compare/CompareStrings.hpp"
#include "algorithms/alignment/AffineKBandAlign.hpp"
#include "algorithms/alignment/GuidedAlign.hpp"
#include "algorithms/alignment/AffineGuidedAlign.hpp"
#include "algorithms/alignment/FullQVAlign.hpp"
#include "algorithms/alignment/ExtendAlign.hpp"
#include "algorithms/alignment/OneGapAlignment.hpp"
#include "algorithms/alignment/AlignmentUtils.hpp"
#include "algorithms/alignment/QualityValueScoreFunction.hpp"
#include "algorithms/alignment/IDSScoreFunction.hpp"
#include "algorithms/alignment/DistanceMatrixScoreFunction.hpp"
#include "algorithms/alignment/StringToScoreMatrix.hpp"
#include "algorithms/alignment/AlignmentFormats.hpp"
#include "algorithms/anchoring/LISPValue.hpp"
#include "algorithms/anchoring/LISPValueWeightor.hpp"
#include "algorithms/anchoring/LISSizeWeightor.hpp"
#include "algorithms/anchoring/LISQValueWeightor.hpp"
#include "algorithms/anchoring/FindMaxInterval.hpp"
#include "algorithms/anchoring/MapBySuffixArray.hpp"
#include "datastructures/anchoring/ClusterList.hpp"
#include "algorithms/anchoring/ClusterProbability.hpp"
#include "algorithms/anchoring/BWTSearch.hpp"
#include "metagenome/SequenceIndexDatabase.hpp"
#include "metagenome/TitleTable.hpp"
#include "suffixarray/SharedSuffixArray.hpp"
#include "suffixarray/SuffixArrayTypes.hpp"
#include "tuples/TupleCountTable.hpp"
#include "datastructures/anchoring/WeightedInterval.hpp"
#include "datastructures/anchoring/AnchorParameters.hpp"
#include "datastructures/alignment/AlignmentCandidate.hpp"
#include "datastructures/alignment/AlignmentContext.hpp"
#include "MappingMetrics.hpp"
#include "reads/ReadInterval.hpp"
#include "utils/FileOfFileNames.hpp"
#include "utils/RegionUtils.hpp"
#include "utils/TimeUtils.hpp"
#include "qvs/QualityTransform.hpp"
#include "files/ReaderAgglomerate.hpp"
#include "files/CCSIterator.hpp"
#include "files/FragmentCCSIterator.hpp"
#include "HDFRegionTableReader.hpp"
#include "bwt/BWT.hpp"
#include "PackedDNASequence.hpp"
#include "CommandLineParser.hpp"
#include "qvs/QualityValue.hpp"
#include "statistics/VarianceAccumulator.hpp"
#include "statistics/pdfs.hpp"
#include "statistics/cdfs.hpp"
#include "statistics/StatUtils.hpp"
#include "statistics/LookupAnchorDistribution.hpp"
#include "format/StickAlignmentPrinter.hpp"
#include "format/SAMPrinter.hpp"
#include "format/XMLPrinter.hpp"
#include "format/CompareSequencesPrinter.hpp"
#include "format/VulgarPrinter.hpp"
#include "format/IntervalPrinter.hpp"
#include "format/SummaryPrinter.hpp"
#include "format/SAMHeaderPrinter.hpp"
#include "format/BAMPrinter.hpp"
#define MAX_PHRED_SCORE 254
#define MAPQV_END_ALIGN_WIGGLE 5
//#define USE_GOOGLE_PROFILER
#ifdef USE_GOOGLE_PROFILER
#include "gperftools/profiler.h"
#endif
#ifdef USE_PBBAM
#include "pbbam/BamWriter.h"
using namespace PacBio::BAM;
#else
#define BamWriter ostream
#endif
using namespace std;
MappingSemaphores semaphores;
/*
* Declare global structures that are shared between threads.
*/
ostream *outFilePtr = NULL;
BamWriter * bamWriterPtr = NULL;
//ofstream clOut, nsOut, mcOut;
HDFRegionTableReader *regionTableReader = NULL;
typedef SMRTSequence T_Sequence;
typedef FASTASequence T_GenomeSequence;
typedef DNASuffixArray T_SuffixArray;
typedef DNATuple T_Tuple;
typedef LISPValueWeightor<T_GenomeSequence, DNATuple, vector<ChainedMatchPos> > PValueWeightor;
typedef LISSMatchFrequencyPValueWeightor<T_GenomeSequence, DNATuple, vector<ChainedMatchPos> > MultiplicityPValueWeightor;
ReaderAgglomerate *reader = NULL;
typedef MappingData<T_SuffixArray, T_GenomeSequence, T_Tuple> MappingIPC;
class ClusterInformation {
public:
int maxClusterSize;
float meanAnchorBasesPerRead;
float sdAnchorBasesPerRead;
int score;
float pctSimilarity;
int readLength;
float nStdDev ;
int numSignificant;
int numForward, numReverse;
};
class ReadAlignments {
public:
/*
This class stores the alignments from a read. A read may be
aligned in several different modes:
1. Fullread - Treat the read as a unit from start to end
2. Subread - Align each subread independently
3. CCSDeNovo - Only align the CCS sequence from a read
4. CCSAllPass - Align the de novo ccs sequences and then the
subreads to where the denovo ccs aligned.
5. CCSFullPass - Same as allpass, except using only complete
subreads.
6. ZmwSubreads - Align subreads of each zmw to where the longest
subread of the zmw aligned to.
The alignments are a raggad array of n sequences; n is 1 for cases
1 and 3, the number of subreads for cases 2 and 4, and the number
of full length passes for case 5.
A ReadAligments class must only have alignments for a single type
of read in it.
*/
vector<vector<T_AlignmentCandidate*> > subreadAlignments;
vector<SMRTSequence> subreads;
AlignMode alignMode;
SMRTSequence read;
int GetNAlignedSeq() {
return subreadAlignments.size();
}
bool AllSubreadsHaveAlignments() {
int i, nAlignedSeq;
nAlignedSeq = subreadAlignments.size();
for (i = 0; i < nAlignedSeq; i++) {
if (subreadAlignments[i].size() == 0) {
return false;
}
}
return true;
}
void Clear() {
int i;
int nAlignedSeq;
for (i = 0, nAlignedSeq = subreadAlignments.size(); i < nAlignedSeq; i++) {
int nAlignments;
int a;
for (a = 0, nAlignments = subreadAlignments[i].size(); a < nAlignments; a++) {
delete subreadAlignments[i][a];
}
subreadAlignments[i].clear();
}
for (i = 0, nAlignedSeq = subreads.size(); i< nAlignedSeq; i++) {
subreads[i].Free();
}
subreadAlignments.clear();
read.Free();
}
void Resize(int nSeq) {
subreadAlignments.resize(nSeq);
subreads.resize(nSeq);
}
void CheckSeqIndex(int seqIndex) {
if ( seqIndex < 0 or seqIndex >= int(subreads.size()) ) {
cout << "ERROR, adding a sequence to an unallocated position."
<< endl;
assert(0);
}
}
void SetSequence(int seqIndex, SMRTSequence &seq) {
CheckSeqIndex(seqIndex);
subreads[seqIndex] = seq;
}
void AddAlignmentForSeq(int seqIndex, T_AlignmentCandidate *alignmentPtr) {
CheckSeqIndex(seqIndex);
subreadAlignments[seqIndex].push_back(alignmentPtr);
}
void AddAlignmentsForSeq(int seqIndex, vector<T_AlignmentCandidate*> &seqAlignmentPtrs) {
CheckSeqIndex(seqIndex);
subreadAlignments[seqIndex].insert(subreadAlignments[seqIndex].end(), seqAlignmentPtrs.begin(), seqAlignmentPtrs.end());
}
// Copy all T_AlignmentCandidate objects (to which subreadAlignment[seqIndex]
// is pointing) to newly created objects, and then return pointers to the new
// objects.
vector<T_AlignmentCandidate*> CopySubreadAlignments(int seqIndex) {
vector<T_AlignmentCandidate*> ret;
for (int i=0; i<int(subreadAlignments[seqIndex].size()); i++) {
T_AlignmentCandidate * q = new T_AlignmentCandidate();
*q = *(subreadAlignments[seqIndex][i]);
ret.push_back(q);
}
return ret;
}
void Print(ostream &out=cout) {
out << "A ReadAlignments object with "
<< subreadAlignments.size()
<< " groups of subread alignments." << endl;
for (int i = 0; i < int(subreadAlignments.size()); i++) {
out << " subreadAlignment group [" << i << "/"
<< subreadAlignments.size() << "] has "
<< subreadAlignments[i].size() << " alignments." << endl;
for(int j = 0; j < int(subreadAlignments[i].size()); j++) {
out << " [" << i << "][" << j << "/"
<< subreadAlignments[i].size() << "]" << endl;
subreadAlignments[i][j]->Print(out);
}
}
/* subreads may have been freed or not initialized when
* being printed.
for (int i = 0; i < subreads.size(); i++) {
out << " subread [" << i << "/" << subreads.size()
<< "]: ";
subreads[i].Print(out);
} */
out << " read: ";
read.Print(out);
out << endl << endl;
}
~ReadAlignments() {
read.Free();
}
};
string GetMajorVersion() {
return "2.0.0";
}
const string GetVersion(void) {
string perforceVersionString("$Change$");
string version = GetMajorVersion();
if (perforceVersionString.size() > 12) {
version.insert(version.size(), ".");
version.insert(version.size(), perforceVersionString, 9, perforceVersionString.size() - 11);
}
return version;
}
//
// Define a list of buffers that are meant to grow to high-water
// marks, and not shrink down past that. The memory is reused rather
// than having multiple calls to new.
//
class MappingBuffers {
public:
vector<int> hpInsScoreMat, insScoreMat;
vector<int> kbandScoreMat;
vector<Arrow> hpInsPathMat, insPathMat;
vector<Arrow> kbandPathMat;
vector<int> scoreMat;
vector<Arrow> pathMat;
vector<int> affineScoreMat;
vector<Arrow> affinePathMat;
vector<ChainedMatchPos> matchPosList;
vector<ChainedMatchPos> rcMatchPosList;
vector<BasicEndpoint<ChainedMatchPos> > globalChainEndpointBuffer;
vector<Fragment> sdpFragmentSet, sdpPrefixFragmentSet, sdpSuffixFragmentSet;
TupleList<PositionDNATuple> sdpCachedTargetTupleList;
TupleList<PositionDNATuple> sdpCachedTargetPrefixTupleList;
TupleList<PositionDNATuple> sdpCachedTargetSuffixTupleList;
std::vector<int> sdpCachedMaxFragmentChain;
vector<double> probMat;
vector<double> optPathProbMat;
vector<float> lnSubPValueMat;
vector<float> lnInsPValueMat;
vector<float> lnDelPValueMat;
vector<float> lnMatchPValueMat;
vector<int> clusterNumBases;
ClusterList clusterList;
ClusterList revStrandClusterList;
void Reset() {
vector<int>().swap(hpInsScoreMat);
vector<int>().swap(insScoreMat);
vector<int>().swap(kbandScoreMat);
vector<Arrow>().swap(hpInsPathMat);
vector<Arrow>().swap(insPathMat);
vector<Arrow>().swap(kbandPathMat);
vector<int>().swap(scoreMat);
vector<Arrow>().swap(pathMat);
vector<ChainedMatchPos>().swap(matchPosList);
vector<ChainedMatchPos>().swap(rcMatchPosList);
vector<BasicEndpoint<ChainedMatchPos> >().swap(globalChainEndpointBuffer);
vector<Fragment>().swap(sdpFragmentSet);
vector<Fragment>().swap(sdpPrefixFragmentSet);
vector<Fragment>().swap(sdpSuffixFragmentSet);
sdpCachedTargetTupleList.Reset();
sdpCachedTargetPrefixTupleList.Reset();
sdpCachedTargetSuffixTupleList.Reset();
vector<int>().swap(sdpCachedMaxFragmentChain);
vector<double>().swap(probMat);
vector<double>().swap(optPathProbMat);
vector<float>().swap(lnSubPValueMat);
vector<float>().swap(lnInsPValueMat);
vector<float>().swap(lnDelPValueMat);
vector<float>().swap(lnMatchPValueMat);
vector<int>().swap(clusterNumBases);
}
};
int CountZero(unsigned char *ptr, int length) {
int i;
int nZero = 0;
for (i = 0; i < length; i++) {
if (ptr[i] == 0) { ++nZero; }
}
return nZero;
}
bool ReadHasMeaningfulQualityValues(FASTQSequence &sequence) {
if (sequence.qual.Empty() == true) {
return 0;
}
else {
int numZero=0, numNonZero=0;
if (sequence.qual.data == NULL) {
return false;
}
numZero = CountZero(sequence.qual.data, sequence.length);
numNonZero = sequence.length - numZero;
int subNumZero = 0, subNonZero = 0;
if (sequence.substitutionQV.data == NULL) {
return false;
}
subNumZero = CountZero(sequence.substitutionQV.data, sequence.length);
subNonZero = sequence.length - subNumZero;
if (numZero < 0.5*numNonZero and subNumZero < 0.5 * subNonZero) {
return true;
}
else {
return false;
}
}
}
void StoreRankingStats( vector<T_AlignmentCandidate*> &alignments,
VarianceAccumulator<float> &accumPValue,
VarianceAccumulator<float> &accumWeight) {
int i;
for (i = 0; i < int(alignments.size()); i++) {
alignments[i]->pvalVariance = accumPValue.GetVariance();
alignments[i]->pvalNStdDev = accumPValue.GetNStdDev(alignments[i]->clusterScore);
alignments[i]->weightVariance = accumWeight.GetVariance();
alignments[i]->weightNStdDev = accumWeight.GetNStdDev(alignments[i]->clusterWeight);
}
}
template<typename T_TargetSequence, typename T_QuerySequence, typename TDBSequence>
void AlignIntervals(T_TargetSequence &genome, T_QuerySequence &read, T_QuerySequence &rcRead,
WeightedIntervalSet &weightedIntervals,
int mutationCostMatrix[][5],
int ins, int del, int sdpTupleSize,
int useSeqDB, SequenceIndexDatabase<TDBSequence> &seqDB,
vector<T_AlignmentCandidate*> &alignments,
MappingParameters ¶ms,
MappingBuffers &mappingBuffers,
int procId=0) {
vector<T_QuerySequence*> forrev;
forrev.resize(2);
forrev[Forward] = &read;
forrev[Reverse] = &rcRead;
//
// Use an edit distance scoring function instead of IDS. Although
// the IDS should be more accurate, it is more slow, and it is more
// important at this stage to have faster alignments than accurate,
// since all alignments are rerun using GuidedAlignment later on.
//
DistanceMatrixScoreFunction<DNASequence, FASTQSequence> distScoreFn(SMRTDistanceMatrix, params.insertion, params.deletion);
DistanceMatrixScoreFunction<DNASequence, FASTQSequence> distScoreFn2(SMRTDistanceMatrix, ins, ins);
//
// Assume there is at least one interval.
//
if (weightedIntervals.size() == 0)
return;
WeightedIntervalSet::iterator intvIt = weightedIntervals.begin();
int alignmentIndex = 0;
do {
T_AlignmentCandidate *alignment = alignments[alignmentIndex];
alignment->clusterWeight= (*intvIt).size; // totalAnchorSize == size
alignment->clusterScore = (*intvIt).pValue;
//
// Advance references. Intervals are stored in reverse order, so
// go backwards in the list, and alignments are in forward order.
// That should probably be changed.
//
++alignmentIndex;
//
// Try aligning the read to the genome.
//
DNALength matchIntervalStart, matchIntervalEnd;
matchIntervalStart = (*intvIt).start;
matchIntervalEnd = (*intvIt).end;
bool readOverlapsContigStart = false;
bool readOverlapsContigEnd = false;
int startOverlappedContigIndex = 0;
int endOverlappedContigIndex = 0;
if (params.verbosity > 0) {
cout << "aligning interval : " << read.length << " " << (*intvIt).start << " "
<< (*intvIt).end << " " << (*intvIt).qStart << " " << (*intvIt).qEnd
<< " " << matchIntervalStart << " to " << matchIntervalEnd << " "
<< params.approximateMaxInsertionRate << " " << endl;
}
assert(matchIntervalEnd >= matchIntervalStart);
//
// If using a sequence database, check to make sure that the
// boundaries of the sequence windows do not overlap with
// the boundaries of the reads. If the beginning is before
// the boundary, move the beginning up to the start of the read.
// If the end is past the end boundary of the read, similarly move
// the window boundary to the end of the read boundary.
DNALength tAlignedContigStart = 0;
int seqDBIndex = 0;
//
// Stretch the alignment interval so that it is close to where
// the read actually starts.
//
DNALength subreadStart = read.subreadStart;
DNALength subreadEnd = read.subreadEnd;
if ((*intvIt).GetStrandIndex() == Reverse) {
subreadEnd = read.MakeRCCoordinate(read.subreadStart) + 1;
subreadStart = read.MakeRCCoordinate(read.subreadEnd-1);
}
DNALength lengthBeforeFirstMatch = ((*intvIt).qStart - subreadStart) * params.approximateMaxInsertionRate ;
DNALength lengthAfterLastMatch = (subreadEnd - (*intvIt).qEnd) * params.approximateMaxInsertionRate;
if (matchIntervalStart < lengthBeforeFirstMatch or params.doGlobalAlignment) {
matchIntervalStart = 0;
}
else {
matchIntervalStart -= lengthBeforeFirstMatch;
}
if (genome.length < matchIntervalEnd + lengthAfterLastMatch or params.doGlobalAlignment) {
matchIntervalEnd = genome.length;
}
else {
matchIntervalEnd += lengthAfterLastMatch;
}
DNALength intervalContigStartPos, intervalContigEndPos;
if (useSeqDB) {
//
// The sequence db index is the one where the actual match is
// contained. The matchIntervalStart might be before the sequence
// index boundary due to the extrapolation of alignment start by
// insertion rate. If this is the case, bump up the
// matchIntervalStart to be at the beginning of the boundary.
// Modify bounds similarly for the matchIntervalEnd and the end
// of a boundary.
//
seqDBIndex = seqDB.SearchForIndex((*intvIt).start);
intervalContigStartPos = seqDB.seqStartPos[seqDBIndex];
if (intervalContigStartPos > matchIntervalStart) {
matchIntervalStart = intervalContigStartPos;
}
intervalContigEndPos = seqDB.seqStartPos[seqDBIndex+1] - 1;
if (intervalContigEndPos < matchIntervalEnd) {
matchIntervalEnd = intervalContigEndPos;
}
alignment->tName = seqDB.GetSpaceDelimitedName(seqDBIndex);
alignment->tLength = intervalContigEndPos - intervalContigStartPos;
//
// When there are multiple sequences in the database, store the
// index of this sequence. This lets one compare the contigs
// that reads are mapped to, for instance.
//
alignment->tIndex = seqDBIndex;
}
else {
alignment->tLength = genome.length;
alignment->tName = genome.GetName();
intervalContigStartPos = 0;
intervalContigEndPos = genome.length;
//
// When there are multiple sequences in the database, store the
// index of this sequence. This lets one compare the contigs
// that reads are mapped to, for instance.
//
}
alignment->qName = read.title;
//
// Look to see if a read overhangs the beginning of a contig.
//
if (params.verbosity > 2) {
cout << "Check for prefix/suffix overlap on interval: " << (*intvIt).qStart << " ?> " << (*intvIt).start - intervalContigStartPos <<endl;
}
if ( (*intvIt).qStart > (*intvIt).start - intervalContigStartPos) {
readOverlapsContigStart = true;
startOverlappedContigIndex = seqDBIndex;
}
//
// Look to see if the read overhangs the end of a contig.
//
if (params.verbosity > 2) {
cout << "Check for suffix/prefix overlap on interval, read overhang: " << read.length - (*intvIt).qEnd << " ?> " << matchIntervalEnd - (*intvIt).end <<endl;
}
if (read.length - (*intvIt).qEnd > matchIntervalEnd - (*intvIt).end) {
if (params.verbosity > 2) {
cout << "read overlaps genome end." << endl;
}
readOverlapsContigEnd = true;
endOverlappedContigIndex = seqDBIndex;
}
int alignScore;
alignScore = 0;
alignment->tAlignedSeqPos = matchIntervalStart;
alignment->tAlignedSeqLength = matchIntervalEnd - matchIntervalStart;
if ((*intvIt).GetStrandIndex() == Forward) {
alignment->tAlignedSeq.Copy(genome, alignment->tAlignedSeqPos, alignment->tAlignedSeqLength);
alignment->tStrand = Forward;
}
else {
DNALength rcAlignedSeqPos = genome.MakeRCCoordinate(alignment->tAlignedSeqPos + alignment->tAlignedSeqLength - 1);
genome.CopyAsRC(alignment->tAlignedSeq, rcAlignedSeqPos, alignment->tAlignedSeqLength);
// Map forward coordinates into reverse complement.
intervalContigStartPos = genome.MakeRCCoordinate(intervalContigStartPos) + 1;
intervalContigEndPos = genome.MakeRCCoordinate(intervalContigEndPos - 1);
swap(intervalContigStartPos, intervalContigEndPos);
alignment->tAlignedSeqPos = rcAlignedSeqPos;
alignment->tStrand = Reverse;
}
// Configure the part of the query that is aligned. The entire
// query should always be aligned.
alignment->qAlignedSeqPos = 0;
alignment->qAlignedSeq.ReferenceSubstring(read);
alignment->qAlignedSeqLength = alignment->qAlignedSeq.length;
alignment->qLength = read.length;
alignment->qStrand = 0;
if (params.verbosity > 1) {
cout << "aligning read " << endl;
static_cast<DNASequence*>(&(alignment->qAlignedSeq))->PrintSeq(cout);
cout << endl << "aligning reference" << endl;
static_cast<DNASequence*>(&(alignment->tAlignedSeq))->PrintSeq(cout);
cout << endl;
}
//
// The type of alignment that is performed depends on the mode
// blasr is running in. If it is running in normal mode, local
// aligment is performed and guided by SDP alignment. When
// running in overlap mode, the alignments are forced to the ends
// of reads.
//
int intervalSize = 0;
int m;
//
// Check to see if the matches to the genome are sufficiently
// dense to allow them to be used instead of having to redo
// sdp alignment.
//
// First count how much of the read matches the genome exactly.
for (m = 0; m < intvIt->matches.size(); m++) { intervalSize += intvIt->matches[m].l;}
int subreadLength = forrev[(*intvIt).GetStrandIndex()]->subreadEnd - forrev[(*intvIt).GetStrandIndex()]->subreadStart;
if ((1.0*intervalSize) / subreadLength < params.sdpBypassThreshold and !params.emulateNucmer) {
//
// Not enough of the read maps to the genome, need to use
// sdp alignment to define the regions of the read that map.
//
if (params.refineBetweenAnchorsOnly) {
//
// Run SDP alignment only between the genomic anchors,
// including the genomic anchors as part of the alignment.
//
int m;
vector<ChainedMatchPos> *matches;
vector<ChainedMatchPos> rcMatches;
Alignment anchorsOnly;
DNASequence tAlignedSeq;
FASTQSequence qAlignedSeq;
//
// The strand bookkeeping is a bit confusing, so hopefully
// this will set things straight.
//
// If the alignment is forward strand, the coordinates of the
// blocks are relative to the forward read, starting at 0, not
// the subread start.
// If the alignment is reverse strand, the coordinates of the
// blocks are relative to the reverse strand, starting at the
// position of the subread on the reverse strand.
//
// The coordinates of the blocks in the genome are always
// relative to the forward strand on the genome, starting at
// 0.
//
//
// The first step to refining between anchors only is to make
// the anchors relative to the tAlignedSeq.
matches = (vector<ChainedMatchPos>*) &(*intvIt).matches;
tAlignedSeq = alignment->tAlignedSeq;
qAlignedSeq = alignment->qAlignedSeq;
if (alignment->tStrand == 0) {
for (m = 0; m < matches->size(); m++) {
(*matches)[m].t -= alignment->tAlignedSeqPos;
(*matches)[m].q -= alignment->qAlignedSeqPos;
}
}
else {
//
// Flip the entire alignment if it is on the reverse strand.
DNALength rcAlignedSeqPos = genome.MakeRCCoordinate(alignment->tAlignedSeqPos + alignment->tAlignedSeqLength - 1);
for (m = 0; m < matches->size(); m++) {
(*matches)[m].t -= rcAlignedSeqPos;
(*matches)[m].q -= alignment->qAlignedSeqPos;
}
alignment->tAlignedSeq.CopyAsRC(tAlignedSeq);
rcMatches.resize((*intvIt).matches.size());
//
// Make the reverse complement of the match list.
//
// 1. Reverse complement the coordinates.
for (m = 0; m < (*intvIt).matches.size(); m++) {
int revCompIndex = rcMatches.size() - m - 1;
rcMatches[revCompIndex].q = read.MakeRCCoordinate((*intvIt).matches[m].q + (*intvIt).matches[m].l - 1);
rcMatches[revCompIndex].t = tAlignedSeq.MakeRCCoordinate((*intvIt).matches[m].t + (*intvIt).matches[m].l - 1);
rcMatches[revCompIndex].l = (*intvIt).matches[m].l;
}
matches = &rcMatches;
}
/*
Uncomment to get a dot plot
ofstream matchFile;
matchFile.open("matches.txt");
matchFile << "q t l " << endl;
for (m = 0; matches->size() > 0 and m < matches->size() - 1; m++) {
matchFile << (*matches)[m].q << " " << (*matches)[m].t << " " << (*matches)[m].l << endl;
}
*/
DNASequence tSubSeq;
FASTQSequence qSubSeq;
for (m = 0; matches->size() > 0 and m < matches->size() - 1; m++) {
Block block;
block.qPos = (*matches)[m].q;
block.tPos = (*matches)[m].t;
block.length = (*matches)[m].l;
//
// Find the lengths of the gaps between anchors.
//
int tGap, qGap;
tGap = (*matches)[m+1].t - ((*matches)[m].t + (*matches)[m].l);
qGap = (*matches)[m+1].q - ((*matches)[m].q + (*matches)[m].l);
float gapRatio = (1.0*tGap)/qGap;
if (tGap > 0 and qGap > 0) {
DNALength tPos, qPos;
tPos = block.tPos + block.length;
qPos = block.qPos + block.length;
tSubSeq.ReferenceSubstring(tAlignedSeq, tPos, tGap);
qSubSeq.ReferenceSubstring(alignment->qAlignedSeq, qPos, qGap);
Alignment alignmentInGap;
int alignScore;
/*
The following code is experimental code for trying to do
something like affine gap alignment in long gaps. It
would eventually be used in cDNA alignment to align
between exons, but for now is being tested here by using
it to align when there is a big gap between anchors.
*/
if (params.separateGaps == true and
qSubSeq.length > 0 and tSubSeq.length > 0 and
( (1.0*qSubSeq.length)/tSubSeq.length < 0.25 )) {
alignScore = OneGapAlign(qSubSeq, tSubSeq, distScoreFn, mappingBuffers, alignmentInGap);
}
else {
/*
This is the 'normal/default' way to align between
gaps. It is more well tested than OneGapAlign.
*/
alignScore = SDPAlign(qSubSeq, tSubSeq, distScoreFn, params.sdpTupleSize,
params.sdpIns, params.sdpDel, params.indelRate*2,
alignmentInGap, mappingBuffers, Global,
params.detailedSDPAlignment,
params.extendFrontAlignment,
params.recurseOver,
params.fastSDP);
}
//
// Now, splice the fragment alignment into the current
// alignment.
//
if (alignmentInGap.blocks.size() > 0) {
int b;
//
// Configure this block to be relative to the beginning
// of the aligned substring.
//
for (b = 0; b < alignmentInGap.size(); b++) {
alignmentInGap.blocks[b].tPos += tPos + alignmentInGap.tPos;
alignmentInGap.blocks[b].qPos += qPos + alignmentInGap.qPos;
assert(alignmentInGap.blocks[b].tPos < alignment->tAlignedSeq.length);
assert(alignmentInGap.blocks[b].qPos < alignment->qAlignedSeq.length);
}
}
// Add the original block
alignment->blocks.push_back(block);
anchorsOnly.blocks.push_back(block);
// Add the blocks for the refined alignment
alignment->blocks.insert(alignment->blocks.end(),
alignmentInGap.blocks.begin(),
alignmentInGap.blocks.end());
}
}
// Add the last block
m = (*matches).size() - 1;
Block block;
block.qPos = (*matches)[m].q;
block.tPos = (*matches)[m].t;
assert(block.tPos <= alignment->tAlignedSeq.length);
assert(block.qPos <= alignment->qAlignedSeq.length);
block.length = (*matches)[m].l;
alignment->blocks.push_back(block);
anchorsOnly.blocks.push_back(block);
//
// By convention, blocks start at 0, and the
// alignment->tPos,qPos give the start of the alignment.
// Modify the block positions so that they are offset by 0.
alignment->tPos = alignment->blocks[0].tPos;
alignment->qPos = alignment->blocks[0].qPos;
int b;
int blocksSize = alignment->blocks.size();
for (b = 0; b < blocksSize ; b++) {
assert(alignment->tPos <= alignment->blocks[b].tPos);
assert(alignment->qPos <= alignment->blocks[b].qPos);
alignment->blocks[b].tPos -= alignment->tPos;
alignment->blocks[b].qPos -= alignment->qPos;
}
for (b = 0; b < anchorsOnly.blocks.size(); b++) {
anchorsOnly.blocks[b].tPos -= alignment->tPos;
anchorsOnly.blocks[b].qPos -= alignment->qPos;
}
anchorsOnly.tPos = alignment->tPos;
anchorsOnly.qPos = alignment->qPos;
ComputeAlignmentStats(*alignment, alignment->qAlignedSeq.seq, alignment->tAlignedSeq.seq,
distScoreFn);
tAlignedSeq.Free();
qAlignedSeq.Free();
tSubSeq.Free();
qSubSeq.Free();
}
else {
alignScore = SDPAlign(alignment->qAlignedSeq, alignment->tAlignedSeq, distScoreFn,
sdpTupleSize, params.sdpIns, params.sdpDel, params.indelRate*3,
*alignment, mappingBuffers,
Local,
params.detailedSDPAlignment,
params.extendFrontAlignment,
params.recurseOver,
params.fastSDP);
ComputeAlignmentStats(*alignment, alignment->qAlignedSeq.seq, alignment->tAlignedSeq.seq,
distScoreFn);
}
}
else {
//
// The anchors used to anchor the sequence are sufficient to extend the alignment.
//
int m;
for (m = 0; m < (*intvIt).matches.size(); m++ ){
Block block;
block.qPos = (*intvIt).matches[m].q - alignment->qAlignedSeqPos;
block.tPos = (*intvIt).matches[m].t - alignment->tAlignedSeqPos;
block.length = (*intvIt).matches[m].l;
alignment->blocks.push_back(block);
}
}
//
// The anchors/sdp alignments may leave portions of the read
// unaligned at the beginning and end. If the parameters
// specify extending alignments, try and align extra bases at
// the beginning and end of alignments.
if (params.extendAlignments) {
//
// Modify the alignment so that the start and end of the
// alignment strings are at the alignment boundaries.
//
// Since the query sequence is pointing at a subsequence of the
// read (and is always in the forward direction), just reference
// a new portion of the read.
alignment->qAlignedSeqPos = alignment->qAlignedSeqPos + alignment->qPos;
alignment->qAlignedSeqLength = alignment->QEnd();
alignment->qAlignedSeq.ReferenceSubstring(read, alignment->qAlignedSeqPos, alignment->qAlignedSeqLength );
alignment->qPos = 0;
//
// Since the target sequence may be on the forward or reverse
// strand, a copy of the subsequence is made, and the original
// sequence free'd.
//
DNASequence tSubseq;
alignment->tAlignedSeqPos = alignment->tAlignedSeqPos + alignment->tPos;
alignment->tAlignedSeqLength = alignment->TEnd();
tSubseq.Copy(alignment->tAlignedSeq, alignment->tPos, alignment->tAlignedSeqLength);
alignment->tPos = 0;
alignment->tAlignedSeq.Free();
alignment->tAlignedSeq.TakeOwnership(tSubseq);
DNALength maximumExtendLength = 500;
if (alignment->blocks.size() > 0 ) {
int lastAlignedBlock = alignment->blocks.size() - 1;
DNALength lastAlignedQPos = alignment->blocks[lastAlignedBlock].QEnd() + alignment->qPos + alignment->qAlignedSeqPos;
DNALength lastAlignedTPos = alignment->blocks[lastAlignedBlock].TEnd() + alignment->tPos + alignment->tAlignedSeqPos;
T_AlignmentCandidate extendedAlignmentForward, extendedAlignmentReverse;
int forwardScore, reverseScore;
SMRTSequence readSuffix;
DNALength readSuffixLength;
DNASequence genomeSuffix;
DNALength genomeSuffixLength;
SMRTSequence readPrefix;
DNALength readPrefixLength;
DNASequence genomePrefix;
DNALength genomePrefixLength;
//
// Align the entire end of the read if it is short enough.
//
readSuffixLength = min(read.length - lastAlignedQPos, maximumExtendLength);
if (readSuffixLength > 0) {
readSuffix.ReferenceSubstring(read, lastAlignedQPos, readSuffixLength);
}
else {
readSuffix.length = 0;
}
//
// Align The entire end of the genome up to the maximum extend length;
//
genomeSuffixLength = min(intervalContigEndPos - lastAlignedTPos, maximumExtendLength);
if (genomeSuffixLength > 0) {
if (alignment->tStrand == Forward) {
genomeSuffix.Copy(genome, lastAlignedTPos, genomeSuffixLength);
}
else {
static_cast<DNASequence*>(&genome)->CopyAsRC(genomeSuffix, lastAlignedTPos, genomeSuffixLength);
}
}
else {
genomeSuffix.length = 0;
}
forwardScore = 0;
if (readSuffix.length > 0 and genomeSuffix.length > 0) {
forwardScore = ExtendAlignmentForward(readSuffix, 0,
genomeSuffix, 0,
params.extendBandSize,
// Reuse buffers to speed up alignment
mappingBuffers.scoreMat,
mappingBuffers.pathMat,
// Do the alignment in the forward direction.
extendedAlignmentForward,
distScoreFn,
1, // don't bother attempting
// to extend the alignment
// if one of the sequences
// is less than 1 base long
params.maxExtendDropoff);
}
if ( forwardScore < 0 ) {
//
// The extended alignment considers the whole genome, but
// should be modified to be starting at the end of where
// the original alignment left off.
//
if (params.verbosity > 0) {
cout << "forward extended an alignment of score " << alignment->score << " with score " << forwardScore << " by " << extendedAlignmentForward.blocks.size() << " blocks and length " << extendedAlignmentForward.blocks[extendedAlignmentForward.blocks.size()-1].qPos << endl;
}
extendedAlignmentForward.tAlignedSeqPos = lastAlignedTPos;
extendedAlignmentForward.qAlignedSeqPos = lastAlignedQPos;
genomeSuffix.length = extendedAlignmentForward.tPos + extendedAlignmentForward.TEnd();
alignment->tAlignedSeq.Append(genomeSuffix);
alignment->qAlignedSeq.length += extendedAlignmentForward.qPos + extendedAlignmentForward.QEnd();
assert(alignment->qAlignedSeq.length <= read.length);
alignment->AppendAlignment(extendedAlignmentForward);
}
DNALength firstAlignedQPos = alignment->qPos + alignment->qAlignedSeqPos;
DNALength firstAlignedTPos = alignment->tPos + alignment->tAlignedSeqPos;
readPrefixLength = min(firstAlignedQPos, maximumExtendLength);
if (readPrefixLength > 0) {
readPrefix.ReferenceSubstring(read, firstAlignedQPos-readPrefixLength, readPrefixLength);
}
else {
readPrefix.length = 0;
}
genomePrefixLength = min(firstAlignedTPos - intervalContigStartPos, maximumExtendLength);
if (genomePrefixLength > 0) {
if (alignment->tStrand == 0) {
genomePrefix.Copy(genome, firstAlignedTPos - genomePrefixLength, genomePrefixLength);
}
else {
static_cast<DNASequence*>(&genome)->MakeRC(genomePrefix, firstAlignedTPos - genomePrefixLength, genomePrefixLength);
}