//#define GFF_DEBUG 1 //debugging GFF/GTF loading

#include "rlink.h"

#include "tmerge.h"

#ifndef NOTHREADS

#include "GThreads.h"

#endif

//#define GMEMTRACE 1 //debugging mem allocation

#ifdef GMEMTRACE

#include "proc_mem.h"

#endif

#define VERSION "1.3.5"

//#define DEBUGPRINT 1

#ifdef DEBUGPRINT

#define DBGPRINT(x) GMessage(x)

#define DBGPRINT2(a,b) GMessage(a,b)

#define DBGPRINT3(a,b,c) GMessage(a,b,c)

#define DBGPRINT4(a,b,c,d) GMessage(a,b,c,d)

#define DBGPRINT5(a,b,c,d,e) GMessage(a,b,c,d,e)

#else

#define DBGPRINT(x)

#define DBGPRINT2(a,b)

#define DBGPRINT3(a,b,c)

#define DBGPRINT4(a,b,c,d)

#define DBGPRINT5(a,b,c,d,e)

#endif

#define USAGE "StringTie v" VERSION " usage:\n\

stringtie <input.bam ..> [-G <guide_gff>] [-l <label>] [-o <out_gtf>] [-p <cpus>]\n\

[-v] [-a <min_anchor_len>] [-m <min_tlen>] [-j <min_anchor_cov>] [-f <min_iso>]\n\

[-C <coverage_file_name>] [-c <min_bundle_cov>] [-g <bdist>] [-u]\n\

[-e] [-x <seqid,..>] [-A <gene_abund.out>] [-h] {-B | -b <dir_path>} \n\

Assemble RNA-Seq alignments into potential transcripts.\n\

Options:\n\

--version : print just the version at stdout and exit\n\

-G reference annotation to use for guiding the assembly process (GTF/GFF3)\n\

--rf assume stranded library fr-firststrand\n\

--fr assume stranded library fr-secondstrand\n\

-l name prefix for output transcripts (default: STRG)\n\

-f minimum isoform fraction (default: 0.1)\n\

-m minimum assembled transcript length (default: 200)\n\

-o output path/file name for the assembled transcripts GTF (default: stdout)\n\

-a minimum anchor length for junctions (default: 10)\n\

-j minimum junction coverage (default: 1)\n\

-t disable trimming of predicted transcripts based on coverage\n\

(default: coverage trimming is enabled)\n\

-c minimum reads per bp coverage to consider for transcript assembly\n\

(default: 2.5)\n\

-v verbose (log bundle processing details)\n\

-g gap between read mappings triggering a new bundle (default: 50)\n\

-C output a file with reference transcripts that are covered by reads\n\

-M fraction of bundle allowed to be covered by multi-hit reads (default: 1.0)\n\

-p number of threads (CPUs) to use (default: 1)\n\

-A gene abundance estimation output file\n\

-B enable output of Ballgown table files which will be created in the\n\

same directory as the output GTF (requires -G, -o recommended)\n\

-b enable output of Ballgown table files but these files will be \n\

created under the directory path given as <dir_path>\n\

-e only estimate the abundance of given reference transcripts (requires -G)\n\

-x do not assemble any transcripts on the given reference sequence(s)\n\

-u no multi-mapping correction (default: correction enabled)\n\

-h print this usage message and exit\n\

\n\

Transcript merge usage mode: \n\

stringtie --merge [Options] { gtf_list | strg1.gtf ...}\n\

With this option StringTie will assemble transcripts from multiple\n\

input files generating a unified non-redundant set of isoforms. In this mode\n\

the following options are available:\n\

-G <guide_gff> reference annotation to include in the merging (GTF/GFF3)\n\

-o <out_gtf> output file name for the merged transcripts GTF\n\

(default: stdout)\n\

-m <min_len> minimum input transcript length to include in the merge\n\

(default: 50)\n\

-c <min_cov> minimum input transcript coverage to include in the merge\n\

(default: 0)\n\

-F <min_fpkm> minimum input transcript FPKM to include in the merge\n\

(default: 1.0)\n\

-T <min_tpm> minimum input transcript TPM to include in the merge\n\

(default: 1.0)\n\

-f <min_iso> minimum isoform fraction (default: 0.01)\n\

-g <gap_len> gap between transcripts to merge together (default: 250)\n\

-i keep merged transcripts with retained introns; by default\n\

these are not kept unless there is strong evidence for them\n\

-l <label> name prefix for output transcripts (default: MSTRG)\n\

"

//---- globals ----

FILE* f_out=NULL;

FILE* c_out=NULL;

//#define B_DEBUG 1

#ifdef B_DEBUG

FILE* dbg_out=NULL;

#endif

GStr outfname;

GStr out_dir;

GStr tmp_path;

GStr tmpfname;

GStr genefname;

bool guided=false;

bool trim=true;

bool fast=true;

bool eonly=false; // parameter -e ; for mergeMode includes estimated coverage sum in the merged transcripts

bool nomulti=false;

bool enableNames=false;

bool includecov=false;

bool fr_strand=false;

bool rf_strand=false;

//bool complete=true; // set by parameter -i the reference annotation contains partial transcripts

bool retained_intron=false; // set by parameter -i for merge option

bool geneabundance=false;

//bool partialcov=false;

int num_cpus=1;

int mintranscriptlen=200; // minimum length for a transcript to be printed

//int sensitivitylevel=1;

uint junctionsupport=10; // anchor length for junction to be considered well supported <- consider shorter??

int junctionthr=1; // number of reads needed to support a particular junction

float readthr=2.5; // read coverage per bundle bp to accept it; otherwise considered noise; paper uses 3

uint bundledist=50; // reads at what distance should be considered part of separate bundles

float mcov=1.0; // fraction of bundle allowed to be covered by multi-hit reads; paper used 1

int no_xs=0; // number of records without the xs tag

float fpkm_thr=1;

float tpm_thr=1;

// different options of implementation reflected with the next three options

bool includesource=true;

//bool EM=false;

//bool weight=false;

float isofrac=0.1;

GStr label("STRG");

GStr ballgown_dir;

GStr guidegff;

bool debugMode=false;

bool verbose=false;

bool ballgown=false;

//int maxReadCov=1000000; //max local read coverage (changed with -s option)

//no more reads will be considered for a bundle if the local coverage exceeds this value

//(each exon is checked for this)

bool forceBAM = false; //useful for stdin (piping alignments into StringTie)

bool mergeMode = false; //--merge option

bool keepTempFiles = false; //--keeptmp

int GeneNo=0; //-- global "gene" counter

double Num_Fragments=0; //global fragment counter (aligned pairs)

double Frag_Len=0;

double Cov_Sum=0;

//bool firstPrint=true; //just for writing the GFF header before the first transcript is printed

GffNames* gseqNames=NULL; //used as a dictionary for genomic sequence names and ids

int refseqCount=0;

#ifdef GMEMTRACE

double maxMemRS=0;

double maxMemVM=0;

GStr maxMemBundle;

#endif

#ifndef NOTHREADS

GMutex dataMutex; //manage availability of data records ready to be loaded by main thread

GVec<int> dataClear; //indexes of data bundles cleared for loading by main thread (clear data pool)

GConditionVar haveBundles; //will notify a thread that a bundle was loaded in the ready queue

//(or that no more bundles are coming)

int bundleWork=1; // bit 0 set if bundles are still being prepared (BAM file not exhausted yet)

// bit 1 set if there are Bundles ready in the queue

//GFastMutex waitMutex;

GMutex waitMutex; // controls threadsWaiting (idle threads counter)

int threadsWaiting; // idle worker threads

GConditionVar haveThreads; //will notify the bundle loader when a thread

//is available to process the currently loaded bundle

GConditionVar haveClear; //will notify when bundle buf space available

GMutex queueMutex; //controls bundleQueue and bundles access

GFastMutex printMutex; //for writing the output to file

GFastMutex logMutex; //only when verbose - to avoid mangling the log output

GFastMutex bamReadingMutex;

GFastMutex countMutex;

#endif

GHash<int> excludeGseqs; //hash of chromosomes/contigs to exclude (e.g. chrM)

bool NoMoreBundles=false;

bool moreBundles(); //thread-safe retrieves NoMoreBundles

void noMoreBundles(); //sets NoMoreBundles to true

//--

void processOptions(GArgs& args);

char* sprintTime();

void processBundle(BundleData* bundle);

//void processBundle1stPass(BundleData* bundle); //two-pass testing

void writeUnbundledGuides(GVec<GRefData>& refdata, FILE* fout, FILE* gout=NULL);

#ifndef NOTHREADS

bool noThreadsWaiting();

void workerThread(GThreadData& td); // Thread function

//prepare the next free bundle for loading

int waitForData(BundleData* bundles);

#endif

TInputFiles bamreader;

int main(int argc, char* argv[]) {

// == Process arguments.

GArgs args(argc, argv,

//"debug;help;fast;xhvntj:D:G:C:l:m:o:a:j:c:f:p:g:");

"debug;help;version;keeptmp;bam;fr;rf;merge;exclude=zZSEihvteux:n:j:s:D:G:C:l:m:o:a:j:c:f:p:g:P:M:Bb:A:F:T:");

args.printError(USAGE, true);

processOptions(args);

GVec<GRefData> refguides; // plain vector with transcripts for each chromosome

//table indexes for Ballgown Raw Counts data (-B/-b option)

GPVec<RC_TData> guides_RC_tdata(true); //raw count data or other info for all guide transcripts

GPVec<RC_Feature> guides_RC_exons(true); //raw count data for all guide exons

GPVec<RC_Feature> guides_RC_introns(true);//raw count data for all guide introns

GVec<int> alncounts(30,0); //keep track of the number of read alignments per chromosome [gseq_id]

int bamcount=bamreader.start(); //setup and open input files

#ifndef GFF_DEBUG

if (bamcount<1) {

GError("%sError: no input files provided!\n",USAGE);

}

#endif

#ifdef DEBUGPRINT

verbose=true;

#endif

const char* ERR_BAM_SORT="\nError: the input alignment file is not sorted!\n";

if(guided) { // read guiding transcripts from input gff file

if (verbose) {

printTime(stderr);

GMessage(" Loading reference annotation (guides)..\n");

}

FILE* f=fopen(guidegff.chars(),"r");

if (f==NULL) GError("Error: could not open reference annotation file (%s)!\n",

guidegff.chars());

// transcripts_only sort gffr->gfflst by loc?

GffReader gffr(f, true, true); //loading only recognizable transcript features

gffr.showWarnings(verbose);

// keepAttrs mergeCloseExons noExonAttrs

gffr.readAll(false, true, true);

//the list of GffObj is in gffr.gflst, sorted by chromosome and start-end coordinates

//collect them in other data structures, if it's kept for later call gffobj->isUsed(true)

// (otherwise it'll be deallocated when gffr is destroyed due to going out of scope)

refseqCount=gffr.gseqtable.Count();

if (refseqCount==0 || gffr.gflst.Count()==0) {

GError("Error: could not any valid reference transcripts in %s (invalid GTF/GFF file?)\n",

guidegff.chars());

}

refguides.setCount(refseqCount); //maximum gseqid

uint c_tid=0;

uint c_exon_id=0;

uint c_intron_id=0;

GList<RC_Feature> uexons(true, false, true); //sorted, free items, unique

GList<RC_Feature> uintrons(true, false, true);

//assign unique transcript IDs based on the sorted order

int last_refid=-1;

bool skipGseq=false;

for (int i=0;i<gffr.gflst.Count();i++) {

GffObj* m=gffr.gflst[i];

if (last_refid!=m->gseq_id) {

//chromosome switch

if (ballgown) { //prepare memory storage/tables for all guides on this chromosome

uexons.Clear();

uintrons.Clear();

}

last_refid=m->gseq_id;

skipGseq=excludeGseqs.hasKey(m->getGSeqName());

}

//sanity check: make sure there are no exonless "genes" or other

if (skipGseq) continue;

if (m->exons.Count()==0) {

if (verbose)

GMessage("Warning: exonless GFF %s feature with ID %s found, added implicit exon %d-%d.\n",

m->getFeatureName(), m->getID(), m->start, m->end);

m->addExon(m->start, m->end); //should never happen!

}

//DONE: always keep a RC_TData pointer around, with additional info about guides

RC_TData* tdata=new RC_TData(*m, ++c_tid);

m->uptr=tdata;

guides_RC_tdata.Add(tdata);

if (ballgown) { //already gather exon & intron info for all ref transcripts

tdata->rc_addFeatures(c_exon_id, uexons, guides_RC_exons,

c_intron_id, uintrons, guides_RC_introns);

}

GRefData& grefdata = refguides[m->gseq_id];

grefdata.add(&gffr, m); //transcripts already sorted by location

}

if (verbose) {

printTime(stderr);

GMessage(" %d reference transcripts loaded.\n", gffr.gflst.Count());

}

}

#ifdef GFF_DEBUG

for (int r=0;r<refguides.Count();++r) {

GRefData& grefdata = refguides[r];

for (int k=0;k<grefdata.rnas.Count();++k) {

GMessage("#transcript #%d : %s (%d exons)\n", k, grefdata.rnas[k]->getID(), grefdata.rnas[k]->exons.Count());

grefdata.rnas[k]->printGff(stderr);

}

}

GMessage("GFF Debug mode, exiting...\n");

exit(0);

#endif

// --- here we do the input processing

gseqNames=GffObj::names; //might have been populated already by gff data

gffnames_ref(gseqNames); //initialize the names collection if not guided

GHash<int> hashread; //read_name:pos:hit_index => readlist index

GList<GffObj>* guides=NULL; //list of transcripts on a specific chromosome

int currentstart=0, currentend=0;

int ng_start=0;

int ng_end=-1;

int ng=0;

GStr lastref;

bool no_ref_used=true;

int lastref_id=-1; //last seen gseq_id

// int ncluster=0; used it for debug purposes only

//Ballgown files

FILE* f_tdata=NULL;

FILE* f_edata=NULL;

FILE* f_idata=NULL;

FILE* f_e2t=NULL;

FILE* f_i2t=NULL;

if (ballgown)

Ballgown_setupFiles(f_tdata, f_edata, f_idata, f_e2t, f_i2t);

#ifndef NOTHREADS

//model: one producer, multiple consumers

#define DEF_TSTACK_SIZE 8388608

int tstackSize=GThread::defaultStackSize();

size_t defStackSize=0;

if (tstackSize<DEF_TSTACK_SIZE) defStackSize=DEF_TSTACK_SIZE;

if (verbose) {

if (defStackSize>0){

int ssize=defStackSize;

GMessage("Default stack size for threads: %d (increased to %d)\n", tstackSize, ssize);

}

else GMessage("Default stack size for threads: %d\n", tstackSize);

}

GThread* threads=new GThread[num_cpus]; //bundle processing threads

GPVec<BundleData> bundleQueue(false); //queue of loaded bundles

//the consumers take (pop) bundles out of this queue for processing

//the producer populates this queue with bundles, built from the BAM file

BundleData* bundles=new BundleData[num_cpus+1];

//bundles[0..num_cpus-1] are processed by threads, loading bundles[num_cpus] first

dataClear.setCapacity(num_cpus+1);

for (int b=0;b<num_cpus;b++) {

threads[b].kickStart(workerThread, (void*) &bundleQueue, defStackSize);

bundles[b+1].idx=b+1;

dataClear.Push(b);

}

BundleData* bundle = &(bundles[num_cpus]);

#else

BundleData bundles[1];

BundleData* bundle = &(bundles[0]);

#endif

GBamRecord* brec=NULL;

bool more_alns=true;

TAlnInfo* tinfo=NULL; // for --merge

int prev_pos=0;

bool skipGseq=false;

while (more_alns) {

bool chr_changed=false;

int pos=0;

const char* refseqName=NULL;

char xstrand=0;

int nh=1;

int hi=0;

int gseq_id=lastref_id; //current chr id

bool new_bundle=false;

//delete brec;

if ((brec=bamreader.next())!=NULL) {

if (brec->isUnmapped()) continue;

if (brec->start<1 || brec->mapped_len<10) {

if (verbose) GMessage("Warning: invalid mapping found for read %s (position=%d, mapped length=%d)\n",

brec->name(), brec->start, brec->mapped_len);

continue;

}

refseqName=brec->refName();

xstrand=brec->spliceStrand(); // tagged strand gets priority

if(xstrand=='.' && (fr_strand || rf_strand)) { // set strand if stranded library

if(brec->isPaired()) { // read is paired

if(brec->pairOrder()==1) { // first read in pair

if((rf_strand && brec->revStrand())||(fr_strand && !brec->revStrand())) xstrand='+';

else xstrand='-';

}

else {

if((rf_strand && brec->revStrand())||(fr_strand && !brec->revStrand())) xstrand='-';

else xstrand='+';

}

}

else {

if((rf_strand && brec->revStrand())||(fr_strand && !brec->revStrand())) xstrand='+';

else xstrand='-';

}

}

if (xstrand=='.' && brec->exons.Count()>1) {

no_xs++;

//continue; //skip spliced alignments lacking XS tag (e.g. HISAT alignments) ?

//this is changed in StringTie 2 !

}

if (refseqName==NULL) GError("Error: cannot retrieve target seq name from BAM record!\n");

pos=brec->start; //BAM is 0 based, but GBamRecord makes it 1-based

chr_changed=(lastref.is_empty() || lastref!=refseqName);

if (chr_changed) {

skipGseq=excludeGseqs.hasKey(refseqName);

gseq_id=gseqNames->gseqs.addName(refseqName);

if (guided) {

if (gseq_id>=refseqCount) {

if (verbose)

GMessage("WARNING: no reference transcripts found for genomic sequence \"%s\"! (mismatched reference names?)\n",

refseqName);

}

else no_ref_used=false;

}

if (alncounts.Count()<=gseq_id) {

alncounts.Resize(gseq_id+1, 0);

}

else if (alncounts[gseq_id]>0)

GError(ERR_BAM_SORT);

prev_pos=0;

}

if (pos<prev_pos)

GError(ERR_BAM_SORT);

prev_pos=pos;

if (skipGseq) continue;

alncounts[gseq_id]++;

nh=brec->tag_int("NH");

if (nh==0) nh=1;

hi=brec->tag_int("HI");

if (mergeMode) {

tinfo=new TAlnInfo(brec->name(), brec->tag_int("ZF"));

GStr score(brec->tag_str("ZS"));

if (!score.is_empty()) {

GStr srest=score.split('|');

if (!score.is_empty())

tinfo->cov=score.asDouble();

score=srest.split('|');

if (!srest.is_empty())

tinfo->fpkm=srest.asDouble();

srest=score.split('|');

if (!score.is_empty())

tinfo->tpm=score.asDouble();

}

}

if (!chr_changed && currentend>0 && pos>currentend+(int)bundledist) {

new_bundle=true;

}

}

else { //no more alignments

more_alns=false;

new_bundle=true; //fake a new start (end of last bundle)

}

if (new_bundle || chr_changed) {

//bgeneids.Clear();

hashread.Clear();

if (bundle->readlist.Count()>0) { // process reads in previous bundle

// (readthr, junctionthr, mintranscriptlen are globals)

bundle->getReady(currentstart, currentend);

#ifndef NOTHREADS

//push this in the bundle queue where it'll be picked up by the threads

DBGPRINT2("##> Locking queueMutex to push loaded bundle into the queue (bundle.start=%d)\n", bundle->start);

int qCount=0;

queueMutex.lock(); // FIXME: possible point of contention here between multiple threads

bundleQueue.Push(bundle);

bundleWork |= 0x02; //set bit 1

qCount=bundleQueue.Count();

queueMutex.unlock();

DBGPRINT2("##> bundleQueue.Count()=%d)\n", qCount);

//wait for a thread to pop this bundle from the queue

waitMutex.lock();

DBGPRINT("##> waiting for a thread to become available..\n");

while (threadsWaiting==0) {

haveThreads.wait(waitMutex);

}

waitMutex.unlock();

haveBundles.notify_one();

this_thread::yield();

queueMutex.lock();

while (bundleQueue.Count()==qCount) {

queueMutex.unlock();

haveBundles.notify_one();

this_thread::yield();

queueMutex.lock();

}

queueMutex.unlock();

#else //no threads

//Num_Fragments+=bundle->num_fragments;

//Frag_Len+=bundle->frag_len;

processBundle(bundle);

#endif

// ncluster++; used it for debug purposes only

} //have alignments to process

else { //no read alignments in this bundle?

bundle->Clear();

#ifndef NOTHREADS

dataMutex.lock();

dataClear.Push(bundle->idx);

dataMutex.unlock();

#endif

} //nothing to do with this bundle

if (chr_changed) {

if (guided) {

ng=0;

guides=NULL;

ng_start=0;

ng_end=-1;

if (refguides.Count()>gseq_id && refguides[gseq_id].rnas.Count()>0) {

guides=&(refguides[gseq_id].rnas);

ng=guides->Count();

}

}

lastref=refseqName;

lastref_id=gseq_id;

currentend=0;

}

if (!more_alns) {

if (verbose) {

#ifndef NOTHREADS

GLockGuard<GFastMutex> lock(logMutex);

#endif

if (Num_Fragments) {

printTime(stderr);

GMessage(" %g aligned fragments found.\n", Num_Fragments);

}

//GMessage(" Done reading alignments.\n");

}

noMoreBundles();

break;

}

#ifndef NOTHREADS

int new_bidx=waitForData(bundles);

if (new_bidx<0) {

//should never happen!

GError("Error: waitForData() returned invalid bundle index(%d)!\n",new_bidx);

break;

}

bundle=&(bundles[new_bidx]);

#endif

currentstart=pos;

currentend=brec->end;

if (guides) { //guided and guides!=NULL

ng_start=ng_end+1;

while (ng_start<ng && (int)(*guides)[ng_start]->end < pos) {

// for now, skip guides which have no overlap with current read

ng_start++;

}

int ng_ovl=ng_start;

//add all guides overlapping the current read and other guides that overlap them

while (ng_ovl<ng && (int)(*guides)[ng_ovl]->start<=currentend) { //while guide overlap

if (currentstart>(int)(*guides)[ng_ovl]->start)

currentstart=(*guides)[ng_ovl]->start;

if (currentend<(int)(*guides)[ng_ovl]->end)

currentend=(*guides)[ng_ovl]->end;

if (ng_ovl==ng_start && ng_ovl>0) { //first time only, we have to check back all possible transitive guide overlaps

//char* geneid=(*guides)[ng_ovlstart]->getGeneID();

//if (geneid==NULL) geneid=(*guides)[ng_ovlstart]->getGeneName();

//if (geneid && !bgeneids.hasKey(geneid))

// bgeneids.shkAdd(geneid, &ng); //whatever pointer to int

int g_back=ng_ovl; //start from the overlapping guide, going backwards

int g_ovl_start=ng_ovl;

while (g_back>ng_end+1) {

--g_back;

//if overlap, set g_back_start=g_back and update currentstart

if (currentstart<=(int)(*guides)[g_back]->end) {

g_ovl_start=g_back;

if (currentstart>(int)(*guides)[g_back]->start)

currentstart=(int)(*guides)[g_back]->start;

}

} //while checking previous guides that could be pulled in this bundle

for (int gb=g_ovl_start;gb<=ng_ovl;++gb) {

bundle->keepGuide((*guides)[gb],

&guides_RC_tdata, &guides_RC_exons, &guides_RC_introns);

}

} //needed to check previous guides for overlaps

else

bundle->keepGuide((*guides)[ng_ovl],

&guides_RC_tdata, &guides_RC_exons, &guides_RC_introns);

ng_ovl++;

} //while guide overlap

ng_end=ng_ovl-1; //MUST update ng_end here, even if no overlaps were found

} //guides present on the current chromosome

bundle->refseq=lastref;

bundle->start=currentstart;

bundle->end=currentend;

} //<---- new bundle started

if (currentend<(int)brec->end) {

//current read extends the bundle

//this might not happen if a longer guide had already been added to the bundle

currentend=brec->end;

if (guides) { //add any newly overlapping guides to bundle

bool cend_changed;

do {

cend_changed=false;

while (ng_end+1<ng && (int)(*guides)[ng_end+1]->start<=currentend) {

++ng_end;

//more transcripts overlapping this bundle?

if ((int)(*guides)[ng_end]->end>=currentstart) {

//it should really overlap the bundle

bundle->keepGuide((*guides)[ng_end],

&guides_RC_tdata, &guides_RC_exons, &guides_RC_introns);

if(currentend<(int)(*guides)[ng_end]->end) {

currentend=(*guides)[ng_end]->end;

cend_changed=true;

}

}

}

} while (cend_changed);

}

} //adjusted currentend ; selected overlapping reference transcripts

GReadAlnData alndata(brec, 0, nh, hi, tinfo);

bool ovlpguide=bundle->evalReadAln(alndata, xstrand);

//overlaps with ref transcripts may set xstrand if needed

if (!eonly || ovlpguide) {

// in -e mode: a read alignment is discarded if it doesn't overlap a reference transcript!

if (xstrand=='+') alndata.strand=1;

else if (xstrand=='-') alndata.strand=-1;

//this is changed in StringTie 2 ! (improved transcription strand guessing)

bool XSmissing=(alndata.strand==0 && brec->exons.Count()>1);

if (!XSmissing)

processRead(currentstart, currentend, *bundle, hashread, alndata);

}

} //for each read alignment

//cleaning up

delete brec;

//bamreader.bclose();

bamreader.stop(); //close all BAM files

if (guided && no_ref_used) {

GMessage("WARNING: no reference transcripts were found for the genomic sequences where reads were mapped!\n"

"Please make sure the -G annotation file uses the same naming convention for the genome sequences.\n");

}

#ifndef NOTHREADS

for (int t=0;t<num_cpus;t++)

threads[t].join();

if (verbose) {

printTime(stderr);

GMessage(" All threads finished.\n");

}

delete[] threads;

delete[] bundles;

#else

if (verbose) {

printTime(stderr);

GMessage(" Done.\n");

}

#endif

#ifdef B_DEBUG

fclose(dbg_out);

#endif

if (mergeMode && guided )

writeUnbundledGuides(refguides, f_out);

fclose(f_out);

if (c_out && c_out!=stdout) fclose(c_out);

if(verbose && no_xs>0)

GMessage("Number spliced alignments missing the XS tag (skipped): %d\n",no_xs);

if(!mergeMode) {

if(verbose) {

GMessage("Total count of aligned fragments: %g\n", Num_Fragments);

if (Num_Fragments)

GMessage("Fragment coverage length: %g\n", Frag_Len/Num_Fragments);

}

f_out=stdout;

if(outfname!="stdout") {

f_out=fopen(outfname.chars(), "w");

if (f_out==NULL) GError("Error creating output file %s\n", outfname.chars());

}

fprintf(f_out,"# ");

args.printCmdLine(f_out);

fprintf(f_out,"# StringTie version %s\n",VERSION);

//fprintf(stderr,"cov_sum=%g frag_len=%g num_frag=%g\n",Cov_Sum,Frag_Len,Num_Fragments);

FILE *g_out=NULL;

if(geneabundance) {

g_out=fopen(genefname.chars(),"w");

if (g_out==NULL)

GError("Error creating gene abundance output file %s\n", genefname.chars());

fprintf(g_out,"Gene ID\tGene Name\tReference\tStrand\tStart\tEnd\tCoverage\tFPKM\tTPM\n");

}

FILE* ftmp_in=fopen(tmpfname.chars(),"rt");

if (ftmp_in!=NULL) {

char* linebuf=NULL;

int linebuflen=5000;

GMALLOC(linebuf, linebuflen);

int nl;

int istr;

int tlen;

float tcov;

//float fpkm;

float calc_fpkm;

float calc_tpm;

int t_id;

while(fgetline(linebuf,linebuflen,ftmp_in)) {

//sscanf(linebuf,"%d %d %d %g %g", &nl, &tlen, &t_id, &fpkm, &tcov);

sscanf(linebuf,"%d %d %d %d %g", &istr, &nl, &tlen, &t_id, &tcov);

//for the rare cases tcov < 0, invert it ??

//if (tcov<0) tcov=-tcov; //should not happen

if (tcov<0) tcov=0;

calc_fpkm=tcov*1000000000/Frag_Len;

calc_tpm=tcov*1000000/Cov_Sum;

if(istr) { // this is a transcript

if (ballgown && t_id>0) {

guides_RC_tdata[t_id-1]->fpkm=calc_fpkm;

guides_RC_tdata[t_id-1]->cov=tcov;

}

for(int i=0;i<nl;i++) {

fgetline(linebuf,linebuflen,ftmp_in);

if(!i) {

//linebuf[strlen(line)-1]='\0';

fprintf(f_out,"%s",linebuf);

fprintf(f_out," FPKM \"%.6f\";",calc_fpkm);

fprintf(f_out," TPM \"%.6f\";",calc_tpm);

fprintf(f_out,"\n");

}

else fprintf(f_out,"%s\n",linebuf);

}

}

else { // this is a gene -> different file pointer

fgetline(linebuf, linebuflen, ftmp_in);

fprintf(g_out, "%s\t%.6f\t%.6f\n", linebuf, calc_fpkm, calc_tpm);

}

}

if (guided) {

writeUnbundledGuides(refguides, f_out, g_out);

}

fclose(f_out);

fclose(ftmp_in);

if(geneabundance) fclose(g_out);

GFREE(linebuf);

if (!keepTempFiles) {

remove(tmpfname.chars());

}

}

else {

fclose(f_out);

GError("No temporary file %s present!\n",tmpfname.chars());

}

//lastly, for ballgown, rewrite the tdata file with updated cov and fpkm

if (ballgown) {

rc_writeRC(guides_RC_tdata, guides_RC_exons, guides_RC_introns,

f_tdata, f_edata, f_idata, f_e2t, f_i2t);

}

}

if (!keepTempFiles) {

tmp_path.chomp('/');

remove(tmp_path);

}

gffnames_unref(gseqNames); //deallocate names collection

#ifdef GMEMTRACE

if(verbose) GMessage(" Max bundle memory: %6.1fMB for bundle %s\n", maxMemRS/1024, maxMemBundle.chars());

#endif

} // -- END main

//----------------------------------------

char* sprintTime() {

static char sbuf[32];

time_t ltime; /* calendar time */

ltime=time(NULL);

struct tm *t=localtime(&ltime);

sprintf(sbuf, "%02d_%02d_%02d:%02d:%02d",t->tm_mon+1, t->tm_mday,

t->tm_hour, t->tm_min, t->tm_sec);

return(sbuf);

}

void processOptions(GArgs& args) {

if (args.getOpt('h') || args.getOpt("help")) {

fprintf(stdout,"%s",USAGE);

exit(0);

}

if (args.getOpt("version")) {

fprintf(stdout,"%s\n",VERSION);

exit(0);

}

if (args.getOpt("fr")) {

fr_strand=true;

}

if (args.getOpt("rf")) {

rf_strand=true;

if(fr_strand) GError("Error: --fr and --rf options are incompatible.\n");

}

debugMode=(args.getOpt("debug")!=NULL || args.getOpt('D')!=NULL);

forceBAM=(args.getOpt("bam")!=NULL); //assume the stdin stream is BAM instead of text SAM

mergeMode=(args.getOpt("merge")!=NULL);

keepTempFiles=(args.getOpt("keeptmp")!=NULL);

fast=!(args.getOpt('Z')!=NULL);

verbose=(args.getOpt('v')!=NULL);

if (verbose) {

fprintf(stderr, "Running StringTie " VERSION ". Command line:\n");

args.printCmdLine(stderr);

}

//complete=!(args.getOpt('i')!=NULL);

trim=!(args.getOpt('t')!=NULL);

includesource=!(args.getOpt('z')!=NULL);

//EM=(args.getOpt('y')!=NULL);

//weight=(args.getOpt('w')!=NULL);

GStr s=args.getOpt('m');

if (!s.is_empty()) {

mintranscriptlen=s.asInt();

if (!mergeMode) {

if (mintranscriptlen<30)

GError("Error: invalid -m value, must be >=30)\n");

}

else if (mintranscriptlen<0) GError("Error: invalid -m value, must be >=0)\n");

}

else if(mergeMode) mintranscriptlen=50;

s=args.getOpt('x');

if (!s.is_empty()) {

//split by comma and populate excludeGSeqs

s.startTokenize(" ,\t");

GStr chrname;

while (s.nextToken(chrname)) {

excludeGseqs.Add(chrname.chars(),new int(0));

}

}

/*

s=args.getOpt('n');

if (!s.is_empty()) {

sensitivitylevel=s.asInt();

if(sensitivitylevel<0) {

sensitivitylevel=0;

GMessage("sensitivity level out of range: setting sensitivity level at 0\n");

}

if(sensitivitylevel>3) {

sensitivitylevel=3;

GMessage("sensitivity level out of range: setting sensitivity level at 2\n");

}

}

*/

s=args.getOpt('g');

if (!s.is_empty()) bundledist=s.asInt();

else if(mergeMode) bundledist=250; // should figure out here a reasonable parameter for merge

s=args.getOpt('p');

if (!s.is_empty()) {

num_cpus=s.asInt();

if (num_cpus<=0) num_cpus=1;

}

s=args.getOpt('a');

if (!s.is_empty()) {

junctionsupport=(uint)s.asInt();

}

s=args.getOpt('j');

if (!s.is_empty()) junctionthr=s.asInt();

s=args.getOpt('c');

if (!s.is_empty()) {

readthr=(float)s.asDouble();

if (readthr<0.001 && !mergeMode) {

GError("Error: invalid -c value, must be >=0.001)\n");

}

}

else if(mergeMode) readthr=0;

s=args.getOpt('F');

if (!s.is_empty()) {

fpkm_thr=(float)s.asDouble();

}

//else if(mergeMode) fpkm_thr=0;

s=args.getOpt('T');

if (!s.is_empty()) {

tpm_thr=(float)s.asDouble();

}

//else if(mergeMode) tpm_thr=0;

s=args.getOpt('l');

if (!s.is_empty()) label=s;

else if(mergeMode) label="MSTRG";

s=args.getOpt('f');

if (!s.is_empty()) {

isofrac=(float)s.asDouble();

if(isofrac>=1) GError("Miminum isoform fraction (-f coefficient: %f) needs to be less than 1\n",isofrac);

}

else if(mergeMode) isofrac=0.01;

s=args.getOpt('M');

if (!s.is_empty()) {

mcov=(float)s.asDouble();

}

genefname=args.getOpt('A');

if(!genefname.is_empty()) {

geneabundance=true;

}

tmpfname=args.getOpt('o');

/*

if (args.getOpt('S')) {

// sensitivitylevel=2; no longer supported from version 1.0.3

sensitivitylevel=1;

}

*/

// coverage saturation no longer used after version 1.0.4; left here for compatibility with previous versions

s=args.getOpt('s');

if (!s.is_empty()) {

GMessage("Coverage saturation parameter is deprecated starting at version 1.0.5");

/*

int r=s.asInt();

if (r<2) {

GMessage("Warning: invalid -s value, setting coverage saturation threshold, using default (%d)\n", maxReadCov);

}

else maxReadCov=r;

*/

}

if (args.getOpt('G')) {

guidegff=args.getOpt('G');

if (fileExists(guidegff.chars())>1)

guided=true;

else GError("Error: reference annotation file (%s) not found.\n",

guidegff.chars());

}

enableNames=(args.getOpt('E')!=NULL);

retained_intron=(args.getOpt('i')!=NULL);

nomulti=(args.getOpt('u')!=NULL);

eonly=(args.getOpt('e')!=NULL);

if(eonly && mergeMode) {

eonly=false;