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#include "alnout.h"
//-------------------------------------------------------------------//
//------ Streamed SAM generation -----------------------------------//
//-------------------------------------------------------------------//
AlnOut::AlnOut(std::vector<CountType> lns, std::vector<CountType> tls, CountType cycl) : cycle(cycl) {
// Fill list of specified barcodes
std::vector<std::string> barcodes;
for ( unsigned i = 0; i < globalAlignmentSettings.get_barcode_vector().size(); i++ ) {
barcodes.push_back(globalAlignmentSettings.get_barcode_string(i));
}
// Get the finished cycles for each mate
for ( CountType mate = 1; mate <= globalAlignmentSettings.get_mates(); mate++ ) {
mateCycles.push_back( getMateCycle( mate, cycle ) );
}
// Add a waiting task for all lanes and tiles.
for ( auto ln : lns ) {
for ( auto tl : tls ) {
add_task(Task(ln,tl,cycle), WAITING);
}
}
}
AlnOut::~AlnOut() {
if ( !is_finalized() && !finalize() ) {
std::cerr << "Could not finish output for cycle " << cycle << "." << std::endl;
}
}
void AlnOut::init() {
std::lock_guard<std::mutex> lock(if_lock);
if ( initialized )
return;
barcodes = globalAlignmentSettings.get_barcode_string_vector();
// Init the bamIOContext (the same object can be used for all output streams)
bfos.set_context(idx->getSeqNames(), idx->getSeqLengths());
// Init the header (the same object can be used for all output streams)
seqan2::BamHeader header = getBamHeader();
// Init output stream for each barcode (plus undetermined if keep_all_barcodes is set)
for ( unsigned barcode=0; barcode < (barcodes.size() + 1); barcode ++) {
if ( barcode < barcodes.size() || globalAlignmentSettings.get_keep_all_barcodes() ) {
std::string barcode_string = ( barcode == barcodes.size() ) ? "undetermined" : barcodes[barcode];
// Open file in Bam output stream and write the header
bfos.emplace_back( getBamTempFileName(barcode_string, cycle).c_str() );
bfos[barcode].writeHeader(header);
}
}
initialized = true;
}
bool AlnOut::set_task_status( Task t, ItemStatus status ) {
std::lock_guard<std::mutex> lock(tasks_lock);
if ( tasks.find(t) == tasks.end() )
return false;
tasks[t] = status;
return true;
}
bool AlnOut::set_task_status_from_to( Task t, ItemStatus oldStatus, ItemStatus newStatus ) {
std::lock_guard<std::mutex> lock(tasks_lock);
if ( tasks.find(t) == tasks.end() )
return false;
if ( tasks[t] == oldStatus ) {
tasks[t] = newStatus;
return true;
}
return false;
}
Task AlnOut::get_next( ItemStatus getStatus, ItemStatus setToStatus ) {
std::lock_guard<std::mutex> lock(tasks_lock);
for ( auto it = tasks.begin(); it != tasks.end(); ++it ) {
if ( it->second == getStatus ) {
tasks[it->first] = setToStatus;
return it->first;
}
}
return NO_TASK;
}
bool AlnOut::add_task( Task t, ItemStatus status ) {
std::lock_guard<std::mutex> lock(tasks_lock);
if ( tasks.find(t) != tasks.end() )
return false;
tasks[t] = status;
return true;
}
bool AlnOut::sort_tile( CountType ln, CountType tl, CountType mate, CountType cycle, bool overwrite ) {
std::string in_fname = get_align_fname(ln, tl, cycle, mate);
std::string out_fname = get_align_fname(ln, tl, cycle, mate) + ".sorted";
// Stop if sorted file already exist
if ( file_exists( out_fname ) && !overwrite )
return true;
iAlnStream input ( globalAlignmentSettings.get_block_size(), globalAlignmentSettings.get_compression_format() );
input.open(in_fname);
// Check for expected input file content
if ( input.get_cycle() != cycle ) {
throw std::runtime_error("Unexpected cycle number in input file when sorting alignments.");
}
if ( input.get_lane() != ln ) {
throw std::runtime_error("Unexpected lane number in input file when sorting alignments.");
}
if ( input.get_tile() != tl ) {
throw std::runtime_error("Unexpected tile number in input file when sorting alignments.");
}
uint32_t num_reads = input.get_num_reads();
oAlnStream output(ln, tl, cycle, input.get_rlen(), num_reads, globalAlignmentSettings.get_block_size(), globalAlignmentSettings.get_compression_format());
output.open(out_fname);
for ( uint32_t i = 0; i < num_reads; i++ ) {
try {
ReadAlignment * ra = input.get_alignment();
ra->sort_seeds_by_as();
output.write_alignment(ra);
delete ra;
} catch (const std::exception & ex) {
return false;
}
}
if (!(input.close() && output.close()))
return false;
return true;
}
CountType AlnOut::openiAlnStream( CountType lane, CountType tile, CountType mateCycle, CountType mate, iAlnStream* istream) {
// Check if mate is valid.
if ( globalAlignmentSettings.get_seq_by_mate(mate) == NULLSEQ )
return 1;
// Check if mate cycle is >0
if ( mateCycle == 0 )
return 2;
// Sort file if necessary
if ( ! sort_tile( lane, tile, mate, mateCycle, globalAlignmentSettings.get_force_resort() ) )
return 3;
// Open sorted alignment file
std::string alignment_fname = get_align_fname(lane, tile, mateCycle, mate) + ".sorted";
// Check if sorted file exist
if ( !file_exists(alignment_fname) ) {
return 3;
}
// Open the stream
istream->open(alignment_fname);
return 0;
}
std::vector<iAlnStream*> AlnOut::openiAlnStreams( CountType lane, CountType tile, bool filter_exist, unsigned filter_size) {
std::vector<iAlnStream*> alignmentFiles;
unsigned numberOfAlignments = 0;
for (unsigned mateIndex = 1; mateIndex <= mateCycles.size(); mateIndex++) {
// Init the stream object
iAlnStream* input = new iAlnStream( globalAlignmentSettings.get_block_size(), globalAlignmentSettings.get_compression_format());
// Try to open the stream
CountType ret = openiAlnStream(lane, tile, mateCycles[mateIndex-1], mateIndex, input);
// On success
if ( ret == 0 ) {
// compare number of reads in alignment file with number of reads in filter file, if filter file exists
if ( filter_exist && input->get_num_reads() != filter_size ) {
std::stringstream msg;
msg << "Unequal number of reads in filter file (" << filter_size << ") and alignment file (" << input->get_num_reads() << ")";
throw std::length_error(msg.str());
}
// compare number of reads in alignment file with number of reads in previous alignment file
if (mateIndex != 1 && input->get_num_reads() != numberOfAlignments) {
throw std::length_error("Unequal number of reads (between mates)");
}
alignmentFiles.push_back(input);
numberOfAlignments = input->get_num_reads();
continue;
}
// Throw an exception if a mate is not valid (should not happen)
else if ( ret == 1 ) {
throw std::runtime_error("Mate number is not valid: " + mateIndex);
}
// Mate not sequenced yet (mateCycle==0)
else if ( ret == 2 ) {
continue;
}
// Sorted file is not available
else if ( ret == 3 ) {
throw std::runtime_error("Sorted align file not found.");
}
// Undefined return value
else {
throw std::logic_error("Undefined return value of function AlnOut::openiAlnStream: " + ret);
}
}
return alignmentFiles;
}
void AlnOut::write_tile_to_bam ( Task t ) {
if ( !is_initialized() )
init();
try {
__write_tile_to_bam__ (t);
set_task_status( t, FINISHED );
} catch ( const std::exception& e) {
set_task_status( t, FAILED );
std::cerr << "Writing of task " << t << " failed: " << e.what() << std::endl;
}
}
void AlnOut::__write_tile_to_bam__ ( Task t ) {
//TODO: Write real paired-end output (?)
CountType lane = t.lane;
CountType tile = t.tile;
////////////////////////////////////////////////////
// Main loop //////////////////////////////////////
////////////////////////////////////////////////////
// set the filter file
std::string filter_fname = get_filter_fname(lane, tile);
FilterParser filters;
if (file_exists(filter_fname)) {
filters.open(filter_fname);
}
// Open the input streams for the sorted alignment files.
std::vector<iAlnStream*> alignmentFiles = openiAlnStreams( lane, tile, file_exists(filter_fname), filters.size());
unsigned numberOfAlignments = alignmentFiles[0]->get_num_reads();
// for all reads in a tile
/////////////////////////////////////////////////////////////////////////////
for (uint64_t i = 0; i < numberOfAlignments; i++) {
std::vector<ReadAlignment*> mateAlignments;
for (auto e:alignmentFiles) {
mateAlignments.push_back(e->get_alignment());
}
std::vector<std::vector<seqan2::BamAlignmentRecord>> mateRecords(mateAlignments.size(), std::vector<seqan2::BamAlignmentRecord>(0));
// if the filter file is available and the filter flag is 0 then skip
if (filters.size() != 0 && filters.next() == false)
continue;
// compute barcode sequence as it should be written to BC tag
std::string barcode = globalAlignmentSettings.format_barcode(mateAlignments[mateAlignments.size()-1]->getBarcodeString());
// Barcode index for the read
CountType barcodeIndex = mateAlignments[mateAlignments.size()-1]->getBarcodeIndex();
// If read has undetermined barcode and keep_all_barcodes is not set, skip this read
if ( barcodeIndex == UNDETERMINED && !globalAlignmentSettings.get_keep_all_barcodes() )
continue;
else if ( barcodeIndex == UNDETERMINED )
barcodeIndex = barcodes.size(); // this is the index for the "undetermined" output stream
// setup QNAME
// Read name format <instrument‐name>:<run ID>:<flowcell ID>:<lane‐number>:<tile‐number>:<x‐pos>:<y‐pos>
// readname << "<instrument>:<run-ID>:<flowcell-ID>:" << ln << ":" << tl << ":<xpos>:<ypos>:" << i;
//TODO: Get coordinates from clocs file, run-ID from runInfo.xml, flowcell ID from runInfo.xml, instrument from runInfo.xml
std::stringstream readname;
readname << "lane." << lane << "|tile." << tile << "|read." << i;
// Track equivalent alignments for the same mate (similar positions from different seeds -> only keep the best one)
// TODO: implement equivalent alignment window as user parameter
PositionType equivalentAlignmentWindow = 10;
// TODO: Improve the prevention of reporting similar alignments.
// std::set<PositionType> alignmentPositions;
std::set<GenomePosType> alignmentPositions;
// for all mates
/////////////////////////////////////////////////////////////////////////////
for (unsigned mateAlignmentIndex=0; mateAlignmentIndex < mateAlignments.size(); ++mateAlignmentIndex) {
// Decrease the ReadAlignment cycle since it is automatically increased when loading the file TODO: this should be changed.
mateAlignments[mateAlignmentIndex]->cycle -= 1;
// Init record with information about the read
seqan2::BamAlignmentRecord mate_record;
mate_record.qName = readname.str();
mate_record.flag = 0;
// Set correct segment (paired) flags for the record
if ( mateAlignments.size() > 1) { // if there are at least two mates already sequenced
mate_record.flag = addSAMFlag(mate_record.flag, SAMFlag::MULT_SEG);
if (mateAlignmentIndex == 0) {
mate_record.flag |= addSAMFlag(mate_record.flag, SAMFlag::FIRST_SEG);
} else if (mateAlignmentIndex == mateAlignments.size()-1) {
mate_record.flag = addSAMFlag(mate_record.flag, SAMFlag::LAST_SEG);
} else {
mate_record.flag = addSAMFlag(mate_record.flag, SAMFlag::FIRST_SEG);
mate_record.flag = addSAMFlag(mate_record.flag, SAMFlag::LAST_SEG);
}
}
// Add read specific information to the BAM record
mateAlignments[mateAlignmentIndex]->addReadInfoToRecord(mate_record);
// Alignment disabled or no seeds
if ( mateAlignments[mateAlignmentIndex]->is_disabled() || mateAlignments[mateAlignmentIndex]->seeds.size() == 0 ) {
// Don't report disabled reads if their sequences are not kept.
if ( mateAlignments[mateAlignmentIndex]->is_disabled() && !globalAlignmentSettings.get_keep_all_sequences() )
continue;
// Report unmapped reads if activated
if ( globalAlignmentSettings.get_report_unmapped() ) {
mate_record.flag = addSAMFlag(mate_record.flag, SAMFlag::SEG_UNMAPPED);
mateRecords[mateAlignmentIndex].push_back(mate_record);
}
continue;
}
// Variables for output modes
ScoreType first_seed_score = 0;
ScoreType last_seed_score = 0;
CountType num_diff_scores = 0;
// Number of printed alignments for the current mate.
unsigned printedMateAlignments = 0;
// Unique mode interruption
// TODO: Think about reporting of unmapped and non-unique reads if report-unmapped is activated
// TODO: this filtering approach is problematic if similar seeds exist ...
if ( globalAlignmentSettings.is_mode(UNIQUE) && (
mateAlignments[mateAlignmentIndex]->seeds.size() > 1 ||
(mateAlignments[mateAlignmentIndex]->seeds.size() == 1 && mateAlignments[mateAlignmentIndex]->seeds.front()->getNumPositions() > 1))) {
continue;
}
std::vector<uint8_t> mapqs = mateAlignments[mateAlignmentIndex]->getMAPQs();
auto mapqs_it = mapqs.begin();
// for all seeds
/////////////////////////////////////////////////////////////////////////////
for (SeedVecIt it = mateAlignments[mateAlignmentIndex]->seeds.begin(); it != mateAlignments[mateAlignmentIndex]->seeds.end(); ++it, ++mapqs_it) {
ScoreType curr_seed_score = (*it)->get_as();
// If no alignment was printed before, the current one has the best "score"
if ( printedMateAlignments == 0 )
first_seed_score = curr_seed_score;
// Stop in all best mode when AS:i score is lower than the first
if( globalAlignmentSettings.is_mode(ALLBEST) && first_seed_score > curr_seed_score ) {
goto nextmate;
}
// Don't write this seed if the user-specified score or softclip ratio is not fulfilled
CountType softclip_length = (*it)->get_softclip_length();
if ( curr_seed_score < globalAlignmentSettings.get_min_as() || softclip_length > globalAlignmentSettings.get_max_softclip_ratio()*mateCycles[mateAlignmentIndex]) {
continue;
}
// get CIGAR-String
seqan2::String<seqan2::CigarElement<> > cigar = (*it)->returnSeqanCigarString();
// Get NM:i value
unsigned nm = (*it)->get_nm();
// check if cigar string sums up to read length
// TODO Potentially conflicts with the 'eachMateAligned' flag if done here.
unsigned cigarElemSum = 0;
unsigned deletionSum = 0;
unsigned supposed_cigar_length = mateCycles[mateAlignmentIndex];
for (seqan2::Iterator<seqan2::String<seqan2::CigarElement<> > >::Type elem = seqan2::begin(cigar); elem != end(cigar); ++elem) {
if ((elem->operation == 'M') || (elem->operation == 'I') || (elem->operation == 'S') || (elem->operation == '=') || (elem->operation == 'X'))
cigarElemSum += elem->count;
if (elem->operation == 'D') {
deletionSum += elem->count;
}
}
if (cigarElemSum != supposed_cigar_length) {
it = mateAlignments[mateAlignmentIndex]->seeds.erase(it);
continue;
}
if (deletionSum >= supposed_cigar_length) {
it = mateAlignments[mateAlignmentIndex]->seeds.erase(it);
continue;
}
// Get positions for the current seed
std::vector<GenomePosType> pos_list;
if ( globalAlignmentSettings.is_mode(ANYBEST) ) {
pos_list = (*it)->getPositions(0, 1); // retrieve only one position from the index
} else
pos_list = (*it)->getPositions(); // retrieve all positions from the index
// handle all positions
for ( auto p = pos_list.begin(); p != pos_list.end(); ++p ) {
// Stop in any best mode when first alignment was already written
if( globalAlignmentSettings.is_mode(ANYBEST) && printedMateAlignments >= 1 ) {
goto nextmate;
}
// Stop in bestn mode if first n alignments were already written
if ( globalAlignmentSettings.is_mode(BESTN) && printedMateAlignments >= globalAlignmentSettings.get_best_n() ) {
goto nextmate;
}
seqan2::BamAlignmentRecord record = mate_record;
record.rID = CountType(p->gid / 2);
record.beginPos = mateAlignments[mateAlignmentIndex]->get_SAM_start_pos(*p, *it);
record.mapQ = *mapqs_it;
// skip invalid positions
if (record.beginPos < 0 || PositionType(record.beginPos) == std::numeric_limits<PositionType>::max()) {
continue;
}
// skip positions that were already written (equivalent alignments). This can be done because the best alignment for this position is written first.
if ( alignmentPositions.find(GenomePosType(p->gid, record.beginPos - ( record.beginPos % equivalentAlignmentWindow ) )) != alignmentPositions.end() ||
alignmentPositions.find(GenomePosType(p->gid, record.beginPos + (equivalentAlignmentWindow - ( record.beginPos % equivalentAlignmentWindow ) ) ) ) != alignmentPositions.end()) {
continue;
}
record.cigar = cigar;
if ( idx->isReverse(p->gid) )
seqan2::reverse(record.cigar);
if ( printedMateAlignments > 0 ) { // if current seed is secondary alignment
record.flag = addSAMFlag(record.flag, SAMFlag::SEC_ALIGNMENT);
seqan2::clear(record.seq);
seqan2::clear(record.qual);
}
if ( idx->isReverse(p->gid) ) { // if read matched reverse complementary
seqan2::reverseComplement(record.seq);
seqan2::reverse(record.qual);
record.flag = addSAMFlag(record.flag, SAMFlag::SEQ_RC);
}
// Dictionary for additional SAM tags
seqan2::BamTagsDict dict;
// Alignment Score
seqan2::appendTagValue(dict, "AS", curr_seed_score);
// Barcode sequence
if (barcode!="")
seqan2::appendTagValue(dict, "BC", barcode);
// Number of mismatches
seqan2::appendTagValue(dict, "NM", nm);
// MD:Z string
std::string mdz = (*it)->getMDZString();
if ( idx->isReverse(p->gid))
mdz = reverse_mdz(mdz);
seqan2::appendTagValue(dict, "MD", mdz);
record.tags = seqan2::host(dict);
// fill records list
mateRecords[mateAlignmentIndex].push_back(record);
// set variables for mode selection
if ( last_seed_score != curr_seed_score || num_diff_scores == 0 )
++num_diff_scores;
last_seed_score = curr_seed_score;
++printedMateAlignments;
alignmentPositions.insert(GenomePosType(p->gid, record.beginPos - ( record.beginPos % equivalentAlignmentWindow )));
}
}
nextmate: {
// Report unmapped reads if activated
if ( mateRecords[mateAlignmentIndex].size()==0 && globalAlignmentSettings.get_report_unmapped() ) {
mate_record.flag = addSAMFlag(mate_record.flag, SAMFlag::SEG_UNMAPPED);
mateRecords[mateAlignmentIndex].push_back(mate_record);
}
};
}
// Set flags related to the next mate.
setMateSAMFlags(mateRecords);
// Write all records as a group to keep suboptimal alignments and paired reads together.
bfos[barcodeIndex].writeRecords(mateRecords);
for (auto e:mateAlignments)
delete e;
}
for (auto e:alignmentFiles)
{
e->close();
delete e;
}
return;
}
void AlnOut::setMateSAMFlags( std::vector<std::vector<seqan2::BamAlignmentRecord>> & mateRecords ) {
for ( CountType i=0; i<mateRecords.size(); i++ ) {
// Stop if not paired
if ( mateRecords.size() <= 1 )
break;
// Set related mate index
CountType next = i==mateRecords.size()-1 ? 0 : i+1;
for ( auto & record_pointer : mateRecords[i] ) {
if ( mateRecords[next].size() > 0 ) {
CountType mateFlag = mateRecords[next][0].flag;
// set next mate rID
record_pointer.rNextId = mateRecords[next][0].rID;
// set next mate pos
record_pointer.pNext = mateRecords[next][0].beginPos;
// other mate entry is reverse complemented
if ( hasSAMFlag( mateFlag, SAMFlag::SEQ_RC ) ) {
record_pointer.flag = addSAMFlag(record_pointer.flag, SAMFlag::NEXT_SEQ_RC);
}
// other mate entry is flagged as unmapped
if ( hasSAMFlag( mateFlag, SAMFlag::SEG_UNMAPPED ) ) {
record_pointer.flag = addSAMFlag(record_pointer.flag, SAMFlag::NEXT_SEG_UNMAPPED);
record_pointer.pNext = record_pointer.beginPos;
record_pointer.rNextId = record_pointer.rID;
// the current read is flagged as unmapped but the mate is not
} else if ( hasSAMFlag(record_pointer.flag, SAMFlag::SEG_UNMAPPED) ) {
record_pointer.beginPos = mateRecords[next][0].beginPos;
record_pointer.rID = mateRecords[next][0].rID;
}
// No record for the mate available (this implies, that it is unmapped)
} else {
record_pointer.flag = addSAMFlag(record_pointer.flag, SAMFlag::NEXT_SEG_UNMAPPED);
}
}
}
}
Task AlnOut::write_next ( ) {
Task t = get_next ( BCL_AVAILABLE, RUNNING );
if ( t != NO_TASK ) {
write_tile_to_bam ( t );
return t;
} else {
return NO_TASK;
}
}
CountType AlnOut::get_task_status_num ( ItemStatus getStatus ) {
CountType num = 0;
std::lock_guard<std::mutex> lock(tasks_lock);
for ( auto it = tasks.begin(); it != tasks.end(); ++it ) {
if ( it->second == getStatus ) {
num += 1;
}
}
return num;
}
bool AlnOut::finalize() {
std::lock_guard<std::mutex> lock(if_lock);
if ( finalized )
return true;
// Don't finish if there are unfinished tasks.
if ( !is_finished() )
return false;
bool success = true;
bfos.clear();
// Move all output files to their final location.
for ( unsigned barcode=0; barcode < barcodes.size() + 1; barcode ++) {
if ( barcode < barcodes.size() || globalAlignmentSettings.get_keep_all_barcodes() ) {
std::string barcode_string = ( barcode == barcodes.size() ) ? "undetermined" : barcodes[barcode];
int rename = atomic_rename(getBamTempFileName(barcode_string, cycle).c_str(), getBamFileName(barcode_string, cycle).c_str());
if ( rename == -1 ) {
std::cerr << "Renaming temporary output file " << getBamTempFileName(barcode_string, cycle).c_str() << " to " << getBamFileName(barcode_string, cycle).c_str() << " failed." << std::endl;
success = false;
}
}
}
// If it comes here, it counts as finalized independently from the success state (otherwise, contradictory error messages may occur).
finalized = true;
return success;
}
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