// returns BAM file pointers to beginning of alignment data
bool BamMultiReaderPrivate::RewindReaders()
{
m_errorString.clear();
bool errorsEncountered = false;
// iterate over readers
std::vector<MergeItem>::iterator readerIter = m_readers.begin();
std::vector<MergeItem>::iterator readerEnd = m_readers.end();
for (; readerIter != readerEnd; ++readerIter) {
MergeItem& item = (*readerIter);
BamReader* reader = item.Reader;
if (reader == 0) continue;
// attempt rewind on BamReader
if (!reader->Rewind()) {
m_errorString.append(1, '\t');
m_errorString.append(reader->GetErrorString());
m_errorString.append(1, '\n');
errorsEncountered = true;
}
}
return !errorsEncountered;
}
// returns BAM file pointers to beginning of alignment data
bool BamMultiReader::Rewind(void) {
bool result = true;
for (vector<pair<BamReader*, BamAlignment*> >::iterator it = readers.begin(); it != readers.end(); ++it) {
BamReader* reader = it->first;
result &= reader->Rewind();
}
return result;
}
void GenericIndividualSnpCall::PyroHMMsnp(Fasta &fastaObj, BamReader &bamObj, int chrID, int leftPosition, int rightPosition, GenericProbabilisticAlignment &probAligner, list<Allele>& allelesInBlock, VariantCallSetting& snpCallSettings, vector<GenericVariant> &variantResults)
{
VariantCallSetting settingForPyroHMMsnp = snpCallSettings;
// allele pool
vector<Allele> allelePool;
for (list<Allele>::iterator allelesInBlockIter=allelesInBlock.begin(); allelesInBlockIter!=allelesInBlock.end(); allelesInBlockIter++)
{
allelePool.push_back(*allelesInBlockIter);
}
// add 10bp flanking segment at each side
int windowLeftPosition = leftPosition - snpCallSettings.m_flankingSize;
int windowRightPosition = rightPosition + snpCallSettings.m_flankingSize;
// genome
string genome;
fastaObj.GetSequence(chrID, windowLeftPosition, windowRightPosition, genome);
int globalDepth;
double globalMapQual;
int globalStrandPos;
int globalStrandNeg;
vector<PyroHMMsnp_Sequence_t> readsInWindow;
// rewind BAM reader
bamObj.Rewind();
// set BAM region
bamObj.SetRegion(chrID, windowLeftPosition, chrID, windowRightPosition);
// read alignment
BamAlignment al;
while (bamObj.GetNextAlignment(al))
{
// skip if it is not a good alignment
if (!GenericBamAlignmentTools::goodAlignment(al))
{
continue;
}
// skip if it is not valid at length
if (!GenericBamAlignmentTools::validReadLength(al, m_minReadLength))
{
continue;
}
// skip if it is not valid at map quality
if (!GenericBamAlignmentTools::validMapQuality(al, m_minMapQuality))
{
continue;
}
// skip if it is not valid at alignment identity
if (!GenericBamAlignmentTools::validReadIdentity(al, m_maxMismatchFrac))
{
continue;
}
// global info
globalDepth += 1;
globalMapQual += al.MapQuality*al.MapQuality;
if (al.IsReverseStrand())
globalStrandNeg += 1;
else
globalStrandPos += 1;
// get local alignment
string t_localRead, t_localGenome;
Cigar t_cigar;
BamMD t_md;
int t_numMismatch, t_numInDel;
GenericBamAlignmentTools::getLocalAlignment(al, windowLeftPosition, windowRightPosition-windowLeftPosition,
t_localRead, t_localGenome, t_cigar, t_md,
t_numMismatch, t_numInDel);
if (t_localRead.empty() || t_localGenome.empty())
continue;
// save into set
PyroHMMsnp_Sequence_t t_seq;
t_seq.t_ID = GenericBamAlignmentTools::getBamAlignmentID(al);
t_seq.t_sequence = t_localRead;
t_seq.t_cigar = t_cigar;
t_seq.t_md = t_md;
t_seq.t_numMismatch = t_numMismatch;
t_seq.t_numInDel = t_numInDel;
t_seq.t_mapQualScore = al.MapQuality;
if (al.Position>windowLeftPosition)
t_seq.t_startPositionShift = al.Position-windowLeftPosition;
else
t_seq.t_startPositionShift = 0;
if (al.GetEndPosition()<windowRightPosition)
t_seq.t_endPositionShift = windowRightPosition-al.GetEndPosition();
else
t_seq.t_endPositionShift = 0;
readsInWindow.push_back(t_seq);
}
int numData = readsInWindow.size();
// construct the consensus sequence graph
GenericDagGraph consensusGraph;
vector<string> consensusGraphReads;
vector<Cigar> consensusGraphReadCigars;
vector<int> consensusGraphReadStarts;
// set of aligned reads to construct the graph
for (int i=0; i<numData; ++i)
{
consensusGraphReads.push_back(readsInWindow[i].t_sequence);
consensusGraphReadCigars.push_back(readsInWindow[i].t_cigar);
consensusGraphReadStarts.push_back(readsInWindow[i].t_startPositionShift);
}
// build up the graph
consensusGraph.buildDagGraph(genome, consensusGraphReads, consensusGraphReadCigars, consensusGraphReadStarts);
consensusGraph.edgePruning(snpCallSettings.m_graphPruneLevel);
// search topK paths, excluding reference
vector<string> topRankConsensusGraphPaths;
vector<list<Vertex>> topRankConsensusGraphPathVertexs;
vector<double> topRankConsensusGraphPathWeights;
consensusGraph.topRankPathsExcludeGenome(30, topRankConsensusGraphPaths, topRankConsensusGraphPathVertexs, topRankConsensusGraphPathWeights);
// change vertex list to vertex set
vector<set<Vertex>> topRankConsensusGraphPathVertexSet;
for (int i=0; i<topRankConsensusGraphPathVertexs.size(); i++)
{
list<Vertex>::iterator vertexIter = topRankConsensusGraphPathVertexs[i].begin();
set<Vertex> vertexSet;
for (; vertexIter!=topRankConsensusGraphPathVertexs[i].end(); vertexIter++)
{
vertexSet.insert(*vertexIter);
}
topRankConsensusGraphPathVertexSet.push_back(vertexSet);
}
// get variant vertices
vector<int> allelePositions;
vector<string> alleleChars;
for (list<Allele>::iterator alleleIter=allelesInBlock.begin(); alleleIter!=allelesInBlock.end(); alleleIter++)
{
Allele allele = *alleleIter;
allelePositions.push_back(allele.m_chrPosition-windowLeftPosition);
alleleChars.push_back(allele.m_allele);
}
// map allele to graph vertex
set<Vertex> variantVertexs;
map<int,Vertex> mapAlleleToVertex;
map<Vertex,int> mapVertexToAllele;
for (int v=0; v<consensusGraph.m_numVertexs; v++)
{
if (consensusGraph.m_skip[v])
continue;
if (!consensusGraph.m_isMismatch[v])
continue;
int gp = consensusGraph.m_genomePosition[v] - 1;
for (int j=0; j<allelePool.size(); j++)
{
int ap = allelePositions[j];
if (ap==gp)
{
if (alleleChars[j]==consensusGraph.m_labels[v])
{
variantVertexs.insert(v);
mapAlleleToVertex[j] = v;
mapVertexToAllele[v] = j;
}
}
}
}
// set up the haplotypes
vector<string> haplotypes;
vector<int> haplotypeToPathIndex;
vector<set<Vertex>> haplotypeVariantVertexs;
haplotypes.push_back(genome);
haplotypeToPathIndex.push_back(-1);
haplotypeVariantVertexs.push_back(set<Vertex>());
int kk = 0;
for (int i=0; i<topRankConsensusGraphPaths.size(); i++)
{
if (kk>=snpCallSettings.m_topK)
continue;
bool hasVariantVertex = false;
int deltaLength = (topRankConsensusGraphPaths[i].length()-genome.length());
deltaLength = abs(deltaLength);
if (deltaLength>5)
continue;
set<Vertex> pathVertexs = topRankConsensusGraphPathVertexSet[i];
set<Vertex> pathVariantVertexs;
for (set<Vertex>::iterator variantIter=variantVertexs.begin(); variantIter!=variantVertexs.end(); variantIter++)
{
if (pathVertexs.find(*variantIter)!=pathVertexs.end())
{
hasVariantVertex = true;
pathVariantVertexs.insert(*variantIter);
}
}
int totalNumberVariantVertexInPath = 0;
for (set<Vertex>::iterator vertexIter=pathVertexs.begin(); vertexIter!=pathVertexs.end(); vertexIter++)
{
int v = *vertexIter;
if (consensusGraph.m_isMismatch[v])
{
totalNumberVariantVertexInPath += 1;
}
}
if (hasVariantVertex && totalNumberVariantVertexInPath<=pathVariantVertexs.size())
{
haplotypes.push_back(topRankConsensusGraphPaths[i]);
haplotypeToPathIndex.push_back(i);
haplotypeVariantVertexs.push_back(pathVariantVertexs);
kk++;
}
}
int numHaplotypes = haplotypes.size();
// skip if there is no variant haplotype
if (numHaplotypes==1)
{
return;
}
// compute haplotype data likelihood
vector<vector<long double>> haplotypeDataLikelihoods(numHaplotypes);
PyroHMMsnpHaplotypeDataLikelihood(probAligner, snpCallSettings.m_band, numHaplotypes, haplotypes, readsInWindow, haplotypeDataLikelihoods);
// genotype
vector<vector<int>> genotypes;
set<set<int>> genotypeDiscovered;
for (int i=0; i<numHaplotypes; i++)
{
vector<int> precedeHaplotypes;
PyroHMMsnpGenotypeSet(snpCallSettings.m_ploidy, i, numHaplotypes, precedeHaplotypes, genotypes, genotypeDiscovered);
}
int numGenotypes = genotypes.size();
// genotype variant vertex
vector<set<Vertex>> genotypeVariantVertexs;
for (int i=0; i<numGenotypes; i++)
{
set<Vertex> variantVertexInGenotype;
for (int j=0; j<settingForPyroHMMsnp.m_ploidy; j++)
{
int haplotype = genotypes[i][j];
set<Vertex> variantVertexInHaplotype = haplotypeVariantVertexs[haplotype];
variantVertexInGenotype.insert(variantVertexInHaplotype.begin(), variantVertexInHaplotype.end());
}
genotypeVariantVertexs.push_back(variantVertexInGenotype);
}
// genotype priors
vector<long double> genotypePriors(numGenotypes);
PyroHMMsnpGenotypePrior(numGenotypes, genotypes, settingForPyroHMMsnp, genotypePriors);
// genotype likelihoods
vector<long double> genotypeLikelihoods(numGenotypes);
PyroHMMsnpGenotypeLikelihood(numGenotypes, genotypes, readsInWindow.size(), haplotypeDataLikelihoods, snpCallSettings, genotypeLikelihoods);
// genotype posteriors
vector<long double> genotypePosteriors(numGenotypes);
PyroHMMsnpGenotypePosterior(numGenotypes, genotypePriors, genotypeLikelihoods, genotypePosteriors);
// search maximal genotype posterior
long double maxGenotypePosterior = 0;
int inferGenotype;
for (int i=1; i<numGenotypes; i++)
{
if (maxGenotypePosterior<genotypePosteriors[i])
{
maxGenotypePosterior = genotypePosteriors[i];
inferGenotype = i;
}
}
// all variant vertexs in the inferred genotype
set<Vertex> inferGenotypeVariantVertexs = genotypeVariantVertexs[inferGenotype];
// count haploid type of variant
map<Vertex,vector<int>> inferGenotypeVariantHaploidType;
set<Vertex>::iterator inferVariantIter = inferGenotypeVariantVertexs.begin();
for (; inferVariantIter!=inferGenotypeVariantVertexs.end(); inferVariantIter++)
{
int v = *inferVariantIter;
vector<int> variantHaploidType;
for (int j=0; j<settingForPyroHMMsnp.m_ploidy; j++)
{
int haplotype = genotypes[inferGenotype][j];
set<Vertex> variantVertexInHaplotype = haplotypeVariantVertexs[haplotype];
if (variantVertexInHaplotype.find(v)==variantVertexInHaplotype.end())
{
variantHaploidType.push_back(0);
}else
{
variantHaploidType.push_back(1);
}
}
inferGenotypeVariantHaploidType[v] = variantHaploidType;
}
// variant score
map<Vertex,long double> inferGenotypeVariantScore;
inferVariantIter = inferGenotypeVariantVertexs.begin();
for (; inferVariantIter!=inferGenotypeVariantVertexs.end(); inferVariantIter++)
{
int v = *inferVariantIter;
long double variantScore = 0;
for (int i=0; i<numGenotypes; i++)
{
set<Vertex> variantVertexInGenotype = genotypeVariantVertexs[i];
if (variantVertexInGenotype.find(v)!=variantVertexInGenotype.end())
variantScore += genotypePosteriors[i];
}
inferGenotypeVariantScore[v] = variantScore;
}
// save variant result
inferVariantIter = inferGenotypeVariantVertexs.begin();
for (; inferVariantIter!=inferGenotypeVariantVertexs.end(); inferVariantIter++)
{
GenericVariant result;
int v = *inferVariantIter;
int a = mapVertexToAllele[v];
int variantChrID;
int variantChrPos;
vector<int> haploidType = inferGenotypeVariantHaploidType[v];
for (int j=0; j<settingForPyroHMMsnp.m_ploidy; j++)
{
if (haploidType[j]==0)
{
int g = consensusGraph.m_genomePosition[v];
Allele allele;
allele.m_allele = consensusGraph.m_labels[g];
result.m_alleles.push_back(allele);
}else
{
Allele allele = allelePool[a];
result.m_alleles.push_back(allele);
variantChrID = allele.m_chrID;
variantChrPos = allele.m_chrPosition;
}
}
result.m_chrID = variantChrID;
result.m_chrPosition = variantChrPos;
result.m_probScoreRef = genotypePosteriors[0];
result.m_probScoreVar = genotypePosteriors[inferGenotype];
result.m_variantType = VARIANT_SNP;
long double variantScore = inferGenotypeVariantScore[v];
if (fabs(1-variantScore)<1e-300)
result.m_quality = 3000;
else if (variantScore<1e-300)
result.m_quality = 0;
else
result.m_quality = -10*log10(1-variantScore);
char refBase;
fastaObj.GetBase(result.m_chrID, result.m_chrPosition, refBase);
result.m_reference = refBase;
for (int i=0; i<result.m_alleles.size(); i++)
{
if (result.m_alleles[i].m_allele==result.m_reference)
result.m_haploidType.push_back(0);
else
result.m_haploidType.push_back(1);
}
// filter
if (result.m_quality>=snpCallSettings.m_variantQualityFilter)
variantResults.push_back(result);
}
}
void GenericIndividualSnpCall::simpleSnpCall(string &fastaObj, BamReader &bamObj, int chrID, int leftPosition, int rightPosition, vector<Allele> &variantCandidates, map<int,list<tuple<char,int,int,double>>> &bamData)
{
set<int> BlockSnpPositions;
vector<Allele> BlockSnpAlleles;
// rewind
bamObj.Rewind();
// set region
bamObj.SetRegion(chrID, leftPosition, chrID, rightPosition);
BamAlignment al;
// search SNP positions in the region
while (bamObj.GetNextAlignment(al))
{
if (!GenericBamAlignmentTools::goodAlignment(al))
continue;
if (!al.HasTag("MD"))
continue;
vector<long> SnpInAlignment;
GenericBamAlignmentTools::getBamAlignmentMismatches(al, SnpInAlignment);
for (int i=0; i<SnpInAlignment.size(); i++)
{
BlockSnpPositions.insert(SnpInAlignment[i]);
}
}
// pileup visitor
SimpleSnpCallPileupVisitor visitor(&fastaObj, chrID, leftPosition, rightPosition, m_downSample, &BlockSnpPositions, &BlockSnpAlleles, &bamData);
PileupEngine SimpleSnpCallPileupEngine;
SimpleSnpCallPileupEngine.AddVisitor(&visitor);
// rewind
bamObj.Rewind();
// set region
bamObj.SetRegion(chrID, leftPosition, chrID, rightPosition);
// load data
while(bamObj.GetNextAlignment(al))
{
if (!GenericBamAlignmentTools::goodAlignment(al))
continue;
if (!GenericBamAlignmentTools::validMapQuality(al, m_minMapQuality))
continue;
if (!GenericBamAlignmentTools::validReadIdentity(al, m_maxMismatchFrac))
continue;
if (!GenericBamAlignmentTools::validReadLength(al, m_minReadLength))
continue;
if (!al.HasTag("MD"))
continue;
SimpleSnpCallPileupEngine.AddAlignment(al);
}
SimpleSnpCallPileupEngine.Flush();
// Filter SNP candidiate
for (int i=0; i<BlockSnpAlleles.size(); i++)
{
Allele allele = BlockSnpAlleles[i];
if (allele.m_alleleDepth < m_minSnpRead)
continue;
if (allele.m_alleleDepth < m_minSnpFrac*allele.m_globalDepth)
continue;
variantCandidates.push_back(allele);
}
}
