bool ReadContainer::ParseRead(const BamTools::BamAlignment& aln,
AlignedRead* aligned_read,
map<pair<string,int>, string>& ref_ext_nucleotides) {
// get read ID
aligned_read->ID = aln.Name;
// get nucleotides
aligned_read->nucleotides = aln.QueryBases;
// get qualities
aligned_read->qualities = aln.Qualities;
// get strand
aligned_read->strand = aln.IsReverseStrand();
// get chrom
aligned_read->chrom = references.at(aln.RefID).RefName;
// get read start
aligned_read->read_start = aln.Position;
// get cigar
aligned_read->cigar_ops = aln.CigarData;
// get if mate pair
if (aln.IsSecondMate()) {
aligned_read->mate = 1;
} else {
aligned_read->mate = 0;
}
// Only process if it is the primary alignment
if (aligned_read->mate) {
return false;
}
// Get all the tag data
// don't process if partially spanning (from old lobSTR)
int partial = 0;
if (GetIntBamTag(aln, "XP", &partial)) {
if (partial == 1) return false;
}
// get read group
if (!GetStringBamTag(aln, "RG", &aligned_read->read_group)) {
stringstream msg;
msg << aln.Name << " Could not get read group.";
PrintMessageDieOnError(msg.str(), ERROR);
}
// get msStart
if (!GetIntBamTag(aln, "XS", &aligned_read->msStart)) {
stringstream msg;
msg << aln.Name << " from group " << aligned_read->read_group << " Could not get STR start coordinate. Did this bam file come from lobSTR?";
PrintMessageDieOnError(msg.str(), ERROR);
}
// get msEnd
if (!GetIntBamTag(aln, "XE", &aligned_read->msEnd)) {
stringstream msg;
msg << aln.Name << " from group " << aligned_read->read_group << " Could not get STR end coordinate. Did this bam file come from lobSTR?";
PrintMessageDieOnError(msg.str(), ERROR);
}
// get mapq. Try unsigned/signed
if (!GetIntBamTag(aln, "XQ", &aligned_read->mapq)) {
stringstream msg;
aligned_read->mapq = 0;
}
// get diff
if (!GetIntBamTag(aln, "XD", &aligned_read->diffFromRef)) {
return false;
}
// get mate dist
if (!GetIntBamTag(aln, "XM", &aligned_read->matedist)) {
aligned_read->matedist = 0;
}
// get STR seq
if (!GetStringBamTag(aln, "XR", &aligned_read->repseq)) {
stringstream msg;
msg << aln.Name << " from group " << aligned_read->read_group << " Could not get repseq.";
PrintMessageDieOnError(msg.str(), ERROR);
}
// get if stitched
if (!GetIntBamTag(aln, "XX", &aligned_read->stitched)) {
aligned_read->stitched = 0;
}
// get ref copy num
if (!GetFloatBamTag(aln, "XC", &aligned_read->refCopyNum)) {
stringstream msg;
msg << aln.Name << " from group " << aligned_read->read_group << " Could not get reference copy number.";
PrintMessageDieOnError(msg.str(), ERROR);
}
// get period
aligned_read->period = aligned_read->repseq.length();
if (include_flank) { // diff is just sum of differences in cigar
CIGAR_LIST cigar_list;
for (vector<BamTools::CigarOp>::const_iterator
it = aligned_read->cigar_ops.begin();
it != aligned_read->cigar_ops.end(); it++) {
CIGAR cig;
cig.num = (*it).Length;
cig.cigar_type = (*it).Type;
cigar_list.cigars.push_back(cig);
}
bool added_s;
bool cigar_had_s;
cigar_list.ResetString();
GenerateCorrectCigar(&cigar_list, aln.QueryBases,
&added_s, &cigar_had_s);
aligned_read->diffFromRef = GetSTRAllele(cigar_list);
}
// apply filters
if (unit) {
if (aligned_read->diffFromRef % aligned_read->period != 0){
filter_counter.increment(FilterCounter::NOT_UNIT);
return false;
}
}
if (abs(aligned_read->diffFromRef) > max_diff_ref) {
filter_counter.increment(FilterCounter::DIFF_FROM_REF);
return false;
}
if (aligned_read->mapq > max_mapq) {
filter_counter.increment(FilterCounter::MAPPING_QUALITY);
return false;
}
if (aligned_read->matedist > max_matedist) {
filter_counter.increment(FilterCounter::MATE_DIST);
return false;
}
// Check if the allele length is valid
if (aligned_read->diffFromRef + (aligned_read->refCopyNum*aligned_read->period) < MIN_ALLELE_SIZE) {
filter_counter.increment(FilterCounter::ALLELE_SIZE);
return false;
}
// check that read sufficiently spans STR
int max_read_start = aligned_read->msStart - min_border;
int min_read_stop = aligned_read->msEnd + min_border;
if (aln.Position > max_read_start || aln.GetEndPosition() < min_read_stop){
filter_counter.increment(FilterCounter::SPANNING_AMOUNT);
return false;
}
// check that both ends of the read contain sufficient perfect matches
if (min_read_end_match > 0){
map<pair<string,int>, string>::iterator loc_iter = ref_ext_nucleotides.find(pair<string,int>(aligned_read->chrom, aligned_read->msStart));
if (loc_iter == ref_ext_nucleotides.end())
PrintMessageDieOnError("No extended reference sequence found for locus", ERROR);
string ref_ext_seq = loc_iter->second;
pair<int,int> num_end_matches = AlignmentFilters::GetNumEndMatches(aligned_read, ref_ext_seq, aligned_read->msStart-extend);
if (num_end_matches.first < min_read_end_match || num_end_matches.second < min_read_end_match){
filter_counter.increment(FilterCounter::NUM_END_MATCHES);
return false;
}
}
// check that the prefix and suffix of the read match maximally compared to proximal reference locations
if (maximal_end_match_window > 0){
map<pair<string,int>, string>::iterator loc_iter = ref_ext_nucleotides.find(pair<string,int>(aligned_read->chrom, aligned_read->msStart));
if (loc_iter == ref_ext_nucleotides.end())
PrintMessageDieOnError("No extended reference sequence found for locus", ERROR);
string ref_ext_seq = loc_iter->second;
bool maximum_end_matches = AlignmentFilters::HasLargestEndMatches(aligned_read, ref_ext_seq, aligned_read->msStart-extend, maximal_end_match_window, maximal_end_match_window);
if (!maximum_end_matches){
filter_counter.increment(FilterCounter::NOT_MAXIMAL_END);
return false;
}
}
// check that both ends of the aligned read have sufficient bases before the first indel
if (min_bp_before_indel > 0){
pair<int, int> num_bps = AlignmentFilters::GetEndDistToIndel(aligned_read);
if (num_bps.first != -1 && num_bps.first < min_bp_before_indel){
filter_counter.increment(FilterCounter::BP_BEFORE_INDEL);
return false;
}
if (num_bps.second != -1 && num_bps.second < min_bp_before_indel){
filter_counter.increment(FilterCounter::BP_BEFORE_INDEL);
return false;
}
}
filter_counter.increment(FilterCounter::UNFILTERED);
return true;
}
void ReadContainer::AddReadsFromFile(const ReferenceSTR& ref_str) {
if (ref_str.chrom != "NA") {
int refid = -1;
if (chrom_to_refid.find(ref_str.chrom) !=
chrom_to_refid.end()) {
refid = chrom_to_refid.at(ref_str.chrom);
}
if (refid == -1) {
PrintMessageDieOnError("Could not locate STR reference chromosome in bam file", ERROR);
}
BamTools::BamRegion bam_region(refid, ref_str.start-extend, refid, ref_str.stop+extend);
if (!reader.SetRegion(bam_region)) {
PrintMessageDieOnError("Could not set bam region", ERROR);
}
}
BamTools::BamAlignment aln;
while (reader.GetNextAlignment(aln)) {
AlignedRead aligned_read;
// get read ID
aligned_read.ID = aln.Name;
// get nucleotides
aligned_read.nucleotides = aln.QueryBases;
// get qualities
aligned_read.qualities = aln.Qualities;
// get strand
aligned_read.strand = aln.IsReverseStrand();
// get chrom
aligned_read.chrom = references.at(aln.RefID).RefName;
// get read start
aligned_read.read_start = aln.Position;
// get cigar
aligned_read.cigar_ops = aln.CigarData;
// get if mate pair
if (aln.IsSecondMate()) {
aligned_read.mate = 1;
} else {
aligned_read.mate = 0;
}
// Only process if it is the primary alignment
if (aligned_read.mate) {
continue;
}
// Get all the tag data
// don't process if partially spanning (from old lobSTR)
int partial = 0;
if (GetIntBamTag(aln, "XP", &partial)) {
if (partial == 1) continue;
}
// get read group
if (!GetStringBamTag(aln, "RG", &aligned_read.read_group)) {
stringstream msg;
msg << aln.Name << " Could not get read group.";
PrintMessageDieOnError(msg.str(), ERROR);
}
// get msStart
if (!GetIntBamTag(aln, "XS", &aligned_read.msStart)) {
stringstream msg;
msg << aln.Name << " from group " << aligned_read.read_group << " Could not get STR start coordinate. Did this bam file come from lobSTR?";
PrintMessageDieOnError(msg.str(), ERROR);
}
// get msEnd
if (!GetIntBamTag(aln, "XE", &aligned_read.msEnd)) {
stringstream msg;
msg << aln.Name << " from group " << aligned_read.read_group << " Could not get STR end coordinate. Did this bam file come from lobSTR?";
PrintMessageDieOnError(msg.str(), ERROR);
}
// get mapq. Try unsigned/signed
if (!GetIntBamTag(aln, "XQ", &aligned_read.mapq)) {
stringstream msg;
aligned_read.mapq = 0;
}
// get diff
if (!GetIntBamTag(aln, "XD", &aligned_read.diffFromRef)) {
if (aligned_read.mate == 0) {
stringstream msg;
msg << aln.Name << " from group " << aligned_read.read_group << " Could not get genotype.";
PrintMessageDieOnError(msg.str(), ERROR);
}
continue;
}
// get mate dist
if (!GetIntBamTag(aln, "XM", &aligned_read.matedist)) {
aligned_read.matedist = 0;
}
// get STR seq
if (!GetStringBamTag(aln, "XR", &aligned_read.repseq)) {
stringstream msg;
msg << aln.Name << " from group " << aligned_read.read_group << " Could not get repseq.";
PrintMessageDieOnError(msg.str(), ERROR);
}
// get if stitched
if (!GetIntBamTag(aln, "XX", &aligned_read.stitched)) {
aligned_read.stitched = 0;
}
// get ref copy num
if (!GetFloatBamTag(aln, "XC", &aligned_read.refCopyNum)) {
stringstream msg;
msg << aln.Name << " from group " << aligned_read.read_group << " Could not get reference copy number.";
PrintMessageDieOnError(msg.str(), ERROR);
}
// get period
aligned_read.period = aligned_read.repseq.length();
if (include_flank) { // diff is just sum of differences in cigar
CIGAR_LIST cigar_list;
for (vector<BamTools::CigarOp>::const_iterator
it = aligned_read.cigar_ops.begin();
it != aligned_read.cigar_ops.end(); it++) {
CIGAR cig;
cig.num = (*it).Length;
cig.cigar_type = (*it).Type;
cigar_list.cigars.push_back(cig);
}
bool added_s;
bool cigar_had_s;
cigar_list.ResetString();
GenerateCorrectCigar(&cigar_list, aln.QueryBases,
&added_s, &cigar_had_s);
aligned_read.diffFromRef = GetSTRAllele(cigar_list);
}
// apply filters
if (unit) {
if (aligned_read.diffFromRef % aligned_read.period != 0) continue;
}
if (abs(aligned_read.diffFromRef) > max_diff_ref) {
continue;
}
if (aligned_read.mapq > max_mapq) {
continue;
}
if (aligned_read.matedist > max_matedist) {
continue;
}
// Add to map
pair<string, int> coord
(aligned_read.chrom, aligned_read.msStart);
if (aligned_str_map_.find(coord) != aligned_str_map_.end()) {
aligned_str_map_.at(coord).push_back(aligned_read);
} else {
list<AlignedRead> aligned_read_list;
aligned_read_list.push_back(aligned_read);
aligned_str_map_.insert(pair< pair<string, int>, list<AlignedRead> >
(coord, aligned_read_list));
}
}
}