void BAMbinSortByCoordinate(uint32 iBin, uint binN, uint binS, uint nThreads, string dirBAMsort, Parameters *P) {
if (binS==0) return; //nothing to do for empty bins
//allocate arrays
char *bamIn=new char[binS];
uint *startPos=new uint[binN*3];
uint bamInBytes=0;
//load all aligns
for (uint it=0; it<nThreads; it++) {
string bamInFile=dirBAMsort+to_string(it)+"/"+to_string((uint) iBin);
ifstream bamInStream (bamInFile.c_str());
bamInStream.read(bamIn+bamInBytes,binS);//read the whole file
bamInBytes += bamInStream.gcount();
bamInStream.close();
remove(bamInFile.c_str());
};
if (bamInBytes!=binS) {
ostringstream errOut;
errOut << "EXITING because of FATAL ERROR: number of bytes expected from the BAM bin does not agree with the actual size on disk: ";
errOut << binS <<" "<< bamInBytes <<" "<< iBin <<"\n";
exitWithError(errOut.str(),std::cerr, P->inOut->logMain, 1, *P);
};
//extract coordinates
for (uint ib=0,ia=0;ia<binN;ia++) {
uint32 *bamIn32=(uint32*) (bamIn+ib);
startPos[ia*3] =( ((uint) bamIn32[1]) << 32) | ( (uint)bamIn32[2] );
startPos[ia*3+2]=ib;
ib+=bamIn32[0]+sizeof(uint32);//note that size of the BAM record does not include the size record itself
startPos[ia*3+1]=*( (uint*) (bamIn+ib) ); //read order
ib+=sizeof(uint);
};
//sort
qsort((void*) startPos, binN, sizeof(uint)*3, funCompareUint2);
BGZF *bgzfBin;
bgzfBin=bgzf_open((dirBAMsort+"/b"+to_string((uint) iBin)).c_str(),("w"+to_string((long long) P->outBAMcompression)).c_str());
outBAMwriteHeader(bgzfBin,P->samHeaderSortedCoord,P->chrName,P->chrLength);
//send ordered aligns to bgzf one-by-one
for (uint ia=0;ia<binN;ia++) {
char* ib=bamIn+startPos[ia*3+2];
bgzf_write(bgzfBin,ib, *((uint32*) ib)+sizeof(uint32) );
};
bgzf_flush(bgzfBin);
bgzf_close(bgzfBin);
//release memory
delete [] bamIn;
delete [] startPos;
};
int main(int argInN, char* argIn[]) {
time(&g_statsAll.timeStart);
Parameters *P = new Parameters; //all parameters
P->inputParameters(argInN, argIn);
*(P->inOut->logStdOut) << timeMonthDayTime(g_statsAll.timeStart) << " ..... Started STAR run\n" <<flush;
//generate genome
if (P->runMode=="genomeGenerate") {
genomeGenerate(P);
(void) sysRemoveDir (P->outFileTmp);
P->inOut->logMain << "DONE: Genome generation, EXITING\n" << flush;
exit(0);
} else if (P->runMode!="alignReads") {
P->inOut->logMain << "EXITING because of INPUT ERROR: unknown value of input parameter runMode=" <<P->runMode<<endl<<flush;
exit(1);
};
Genome mainGenome (P);
mainGenome.genomeLoad();
if (P->genomeLoad=="LoadAndExit" || P->genomeLoad=="Remove")
{
return 0;
};
P->twoPass.pass2=false; //this is the 1st pass
SjdbClass sjdbLoci;
if (P->sjdbInsert.pass1)
{
Parameters *P1=new Parameters;
*P1=*P;
sjdbInsertJunctions(P, P1, mainGenome, sjdbLoci);
};
//calculate genome-related parameters
Transcriptome *mainTranscriptome=NULL;
/////////////////////////////////////////////////////////////////////////////////////////////////START
if (P->runThreadN>1) {
g_threadChunks.threadArray=new pthread_t[P->runThreadN];
pthread_mutex_init(&g_threadChunks.mutexInRead, NULL);
pthread_mutex_init(&g_threadChunks.mutexOutSAM, NULL);
pthread_mutex_init(&g_threadChunks.mutexOutBAM1, NULL);
pthread_mutex_init(&g_threadChunks.mutexOutUnmappedFastx, NULL);
pthread_mutex_init(&g_threadChunks.mutexOutFilterBySJout, NULL);
pthread_mutex_init(&g_threadChunks.mutexStats, NULL);
pthread_mutex_init(&g_threadChunks.mutexBAMsortBins, NULL);
};
g_statsAll.progressReportHeader(P->inOut->logProgress);
if (P->twoPass.yes) {//2-pass
//re-define P for the pass1
Parameters *P1=new Parameters;
*P1=*P;
//turn off unnecessary calculations
P1->outSAMtype[0]="None";
P1->outSAMbool=false;
P1->outBAMunsorted=false;
P1->outBAMcoord=false;
P1->chimSegmentMin=0;
P1->quant.yes=false;
P1->quant.trSAM.yes=false;
P1->quant.geCount.yes=false;
P1->outFilterBySJoutStage=0;
P1->outReadsUnmapped="None";
P1->outFileNamePrefix=P->twoPass.dir;
P1->readMapNumber=P->twoPass.pass1readsN;
// P1->inOut->logMain.open((P1->outFileNamePrefix + "Log.out").c_str());
g_statsAll.resetN();
time(&g_statsAll.timeStartMap);
P->inOut->logProgress << timeMonthDayTime(g_statsAll.timeStartMap) <<"\tStarted 1st pass mapping\n" <<flush;
*P->inOut->logStdOut << timeMonthDayTime(g_statsAll.timeStartMap) << " ..... Started 1st pass mapping\n" <<flush;
//run mapping for Pass1
ReadAlignChunk *RAchunk1[P->runThreadN];
for (int ii=0;ii<P1->runThreadN;ii++) {
RAchunk1[ii]=new ReadAlignChunk(P1, mainGenome, mainTranscriptome, ii);
};
mapThreadsSpawn(P1, RAchunk1);
outputSJ(RAchunk1,P1); //collapse and output junctions
// for (int ii=0;ii<P1->runThreadN;ii++) {
// delete [] RAchunk[ii];
// };
time_t rawtime; time (&rawtime);
P->inOut->logProgress << timeMonthDayTime(rawtime) <<"\tFinished 1st pass mapping\n";
*P->inOut->logStdOut << timeMonthDayTime(rawtime) << " ..... Finished 1st pass mapping\n" <<flush;
ofstream logFinal1 ( (P->twoPass.dir + "/Log.final.out").c_str());
g_statsAll.reportFinal(logFinal1,P1);
P->twoPass.pass2=true;//starting the 2nd pass
P->twoPass.pass1sjFile=P->twoPass.dir+"/SJ.out.tab";
sjdbInsertJunctions(P, P1, mainGenome, sjdbLoci);
//reopen reads files
P->closeReadsFiles();
P->openReadsFiles();
} else {//not 2-pass
//nothing for now
};
if ( P->quant.yes ) {//load transcriptome
mainTranscriptome=new Transcriptome(P);
};
//initialize Stats
g_statsAll.resetN();
time(&g_statsAll.timeStartMap);
*P->inOut->logStdOut << timeMonthDayTime(g_statsAll.timeStartMap) << " ..... Started mapping\n" <<flush;
g_statsAll.timeLastReport=g_statsAll.timeStartMap;
//open SAM/BAM files for output
if (P->outSAMmode != "None") {//open SAM file and write header
ostringstream samHeaderStream;
for (uint ii=0;ii<P->nChrReal;ii++) {
samHeaderStream << "@SQ\tSN:"<< P->chrName.at(ii) <<"\tLN:"<<P->chrLength[ii]<<"\n";
};
if (P->outSAMheaderPG.at(0)!="-") {
samHeaderStream << P->outSAMheaderPG.at(0);
for (uint ii=1;ii<P->outSAMheaderPG.size(); ii++) {
samHeaderStream << "\t" << P->outSAMheaderPG.at(ii);
};
samHeaderStream << "\n";
};
samHeaderStream << "@PG\tID:STAR\tPN:STAR\tVN:" << STAR_VERSION <<"\tCL:" << P->commandLineFull <<"\n";
if (P->outSAMheaderCommentFile!="-") {
ifstream comstream (P->outSAMheaderCommentFile);
while (comstream.good()) {
string line1;
getline(comstream,line1);
if (line1.find_first_not_of(" \t\n\v\f\r")!=std::string::npos) {//skip blank lines
samHeaderStream << line1 <<"\n";
};
};
};
for (uint32 ii=0;ii<P->outSAMattrRGlineSplit.size();ii++) {//@RG lines
samHeaderStream << "@RG\t" << P->outSAMattrRGlineSplit.at(ii) <<"\n";
};
samHeaderStream << "@CO\t" <<"user command line: " << P->commandLine <<"\n";
if (P->outSAMheaderHD.at(0)!="-") {
P->samHeaderHD = P->outSAMheaderHD.at(0);
for (uint ii=1;ii<P->outSAMheaderHD.size(); ii++) {
P->samHeaderHD +="\t" + P->outSAMheaderHD.at(ii);
};
} else {
P->samHeaderHD = "@HD\tVN:1.4";
};
P->samHeader=P->samHeaderHD+"\n"+samHeaderStream.str();
//for the sorted BAM, need to add SO:cooridnate to the header line
P->samHeaderSortedCoord=P->samHeaderHD + (P->outSAMheaderHD.size()==0 ? "" : "\tSO:coordinate") + "\n" + samHeaderStream.str();
if (P->outSAMbool) {//
*P->inOut->outSAM << P->samHeader;
};
if (P->outBAMunsorted){
outBAMwriteHeader(P->inOut->outBAMfileUnsorted,P->samHeader,P->chrName,P->chrLength);
};
// if (P->outBAMcoord){
// outBAMwriteHeader(P->inOut->outBAMfileCoord,P->samHeader,P->chrName,P->chrLength);
// };
if ( P->quant.trSAM.yes ) {
samHeaderStream.str("");
vector <uint> trlength;
for (uint32 ii=0;ii<mainTranscriptome->trID.size();ii++) {
uint32 iex1=mainTranscriptome->trExI[ii]+mainTranscriptome->trExN[ii]-1; //last exon of the transcript
trlength.push_back(mainTranscriptome->exLenCum[iex1]+mainTranscriptome->exSE[2*iex1+1]-mainTranscriptome->exSE[2*iex1]+1);
samHeaderStream << "@SQ\tSN:"<< mainTranscriptome->trID.at(ii) <<"\tLN:"<<trlength.back()<<"\n";
};
for (uint32 ii=0;ii<P->outSAMattrRGlineSplit.size();ii++) {//@RG lines
samHeaderStream << "@RG\t" << P->outSAMattrRGlineSplit.at(ii) <<"\n";
};
outBAMwriteHeader(P->inOut->outQuantBAMfile,samHeaderStream.str(),mainTranscriptome->trID,trlength);
};
};
if (P->chimSegmentMin>0) {
P->inOut->outChimJunction.open((P->outFileNamePrefix + "Chimeric.out.junction").c_str());
P->inOut->outChimSAM.open((P->outFileNamePrefix + "Chimeric.out.sam").c_str());
P->inOut->outChimSAM << P->samHeader;
pthread_mutex_init(&g_threadChunks.mutexOutChimSAM, NULL);
pthread_mutex_init(&g_threadChunks.mutexOutChimJunction, NULL);
};
// P->inOut->logMain << "mlock value="<<mlockall(MCL_CURRENT|MCL_FUTURE) <<"\n"<<flush;
// prepare chunks and spawn mapping threads
ReadAlignChunk *RAchunk[P->runThreadN];
for (int ii=0;ii<P->runThreadN;ii++) {
RAchunk[ii]=new ReadAlignChunk(P, mainGenome, mainTranscriptome, ii);
};
mapThreadsSpawn(P, RAchunk);
if (P->outFilterBySJoutStage==1) {//completed stage 1, go to stage 2
P->inOut->logMain << "Completed stage 1 mapping of outFilterBySJout mapping\n"<<flush;
outputSJ(RAchunk,P);//collapse novel junctions
P->readFilesIndex=-1;
P->outFilterBySJoutStage=2;
if (P->outBAMcoord) {
for (int it=0; it<P->runThreadN; it++) {//prepare the unmapped bin
RAchunk[it]->chunkOutBAMcoord->coordUnmappedPrepareBySJout();
};
};
mapThreadsSpawn(P, RAchunk);
};
//close some BAM files
if (P->inOut->outBAMfileUnsorted!=NULL) {
bgzf_flush(P->inOut->outBAMfileUnsorted);
bgzf_close(P->inOut->outBAMfileUnsorted);
};
if (P->inOut->outQuantBAMfile!=NULL) {
bgzf_flush(P->inOut->outQuantBAMfile);
bgzf_close(P->inOut->outQuantBAMfile);
};
if (P->outBAMcoord && P->limitBAMsortRAM==0) {//make it equal ot the genome size
P->limitBAMsortRAM=P->nGenome+mainGenome.SA.lengthByte+mainGenome.SAi.lengthByte;
};
//no need for genome anymore, free the memory
mainGenome.freeMemory();
if ( P->quant.geCount.yes )
{//output gene quantifications
for (int ichunk=1; ichunk<P->runThreadN; ichunk++)
{//sum counts from all chunks into 0th chunk
RAchunk[0]->chunkTr->quants->addQuants(*(RAchunk[ichunk]->chunkTr->quants));
};
RAchunk[0]->chunkTr->quantsOutput();
};
if (P->runThreadN>1 && P->outSAMorder=="PairedKeepInputOrder") {//concatenate Aligned.* files
RAchunk[0]->chunkFilesCat(P->inOut->outSAM, P->outFileTmp + "/Aligned.out.sam.chunk", g_threadChunks.chunkOutN);
};
if (P->outBAMcoord) {//sort BAM if needed
*P->inOut->logStdOut << timeMonthDayTime() << " ..... Started sorting BAM\n" <<flush;
P->inOut->logMain << timeMonthDayTime() << " ..... Started sorting BAM\n" <<flush;
uint32 nBins=P->outBAMcoordNbins;
//check max size needed for sorting
uint maxMem=0;
for (uint32 ibin=0; ibin<nBins-1; ibin++) {//check akk bins
uint binS=0;
for (int it=0; it<P->runThreadN; it++) {//collect sizes from threads
binS += RAchunk[it]->chunkOutBAMcoord->binTotalBytes[ibin]+24*RAchunk[it]->chunkOutBAMcoord->binTotalN[ibin];
};
if (binS>maxMem) maxMem=binS;
};
P->inOut->logMain << "Max memory needed for sorting = "<<maxMem<<endl;
if (maxMem>P->limitBAMsortRAM) {
ostringstream errOut;
errOut <<"EXITING because of fatal ERROR: not enough memory for BAM sorting: \n";
errOut <<"SOLUTION: re-run STAR with at least --limitBAMsortRAM " <<maxMem+1000000000;
exitWithError(errOut.str(), std::cerr, P->inOut->logMain, EXIT_CODE_PARAMETER, *P);
};
uint totalMem=0;
// P->inOut->logMain << "Started sorting BAM ..." <<endl;
#pragma omp parallel num_threads(P->outBAMsortingThreadNactual)
#pragma omp for schedule (dynamic,1)
for (uint32 ibin1=0; ibin1<nBins; ibin1++) {
uint32 ibin=nBins-1-ibin1;//reverse order to start with the last bin - unmapped reads
uint binN=0, binS=0;
for (int it=0; it<P->runThreadN; it++) {//collect sizes from threads
binN += RAchunk[it]->chunkOutBAMcoord->binTotalN[ibin];
binS += RAchunk[it]->chunkOutBAMcoord->binTotalBytes[ibin];
};
if (binS==0) continue; //empty bin
if (ibin == nBins-1) {//last bin for unmapped reads
BAMbinSortUnmapped(ibin,P->runThreadN,P->outBAMsortTmpDir,P->inOut->outBAMfileCoord, P);
} else {
uint newMem=binS+binN*24;
bool boolWait=true;
while (boolWait) {
#pragma omp critical
if (totalMem+newMem < P->limitBAMsortRAM) {
boolWait=false;
totalMem+=newMem;
};
sleep(0.1);
};
BAMbinSortByCoordinate(ibin,binN,binS,P->runThreadN,P->outBAMsortTmpDir,P->inOut->outBAMfileCoord, P);
#pragma omp critical
totalMem-=newMem;//"release" RAM
};
};
//concatenate all BAM files, using bam_cat
char **bamBinNames = new char* [nBins];
vector <string> bamBinNamesV;
for (uint32 ibin=0; ibin<nBins; ibin++) {
bamBinNamesV.push_back(P->outBAMsortTmpDir+"/b"+to_string((uint) ibin));
struct stat buffer;
if (stat (bamBinNamesV.back().c_str(), &buffer) != 0) {//check if file exists
bamBinNamesV.pop_back();
};
};
for (uint32 ibin=0; ibin<bamBinNamesV.size(); ibin++) {
bamBinNames[ibin] = (char*) bamBinNamesV.at(ibin).c_str();
};
bam_cat(bamBinNamesV.size(), bamBinNames, 0, P->outBAMfileCoordName.c_str());
};
//wiggle output
if (P->outWigFlags.yes) {
*P->inOut->logStdOut << timeMonthDayTime() << " ..... Started wiggle output\n" <<flush;
P->inOut->logMain << timeMonthDayTime() << " ..... Started wiggle output\n" <<flush;
string wigOutFileNamePrefix=P->outFileNamePrefix + "Signal";
signalFromBAM(P->outBAMfileCoordName, wigOutFileNamePrefix, *P);
};
//aggregate output junctions
//collapse splice junctions from different threads/chunks, and output them
outputSJ(RAchunk,P);
g_statsAll.progressReport(P->inOut->logProgress);
P->inOut->logProgress << "ALL DONE!\n"<<flush;
P->inOut->logFinal.open((P->outFileNamePrefix + "Log.final.out").c_str());
g_statsAll.reportFinal(P->inOut->logFinal,P);
*P->inOut->logStdOut << timeMonthDayTime(g_statsAll.timeFinish) << " ..... Finished successfully\n" <<flush;
P->inOut->logMain << "ALL DONE!\n"<<flush;
sysRemoveDir (P->outFileTmp);
P->closeReadsFiles();//this will kill the readFilesCommand processes if necessary
mainGenome.~Genome(); //need explicit call because of the 'delete P->inOut' below, which will destroy P->inOut->logStdOut
delete P->inOut; //to close files
delete P;
return 0;
};
