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main.cpp
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main.cpp
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#include <stdio.h>
#include <getopt.h>
#include <string.h>
#include <stdlib.h>
#include <time.h>
#include <stdarg.h>
#include <pthread.h>
#include <vector>
#include "KmerCount.hpp"
#include "SeqSet.hpp"
#include "AlignAlgo.hpp"
char usage[] = "./trust4 [OPTIONS]:\n"
"Required:\n"
"\t-f STRING: fasta file containing the receptor genome sequence\n"
"\t[Read file]\n"
"\t-u STRING: path to single-end read file\n"
"\t\tor\n"
"\t-1 STRING -2 STRING: path to paried-end read files\n"
"\t\tor\n"
"\t-b STRING: path to BAM alignment file\n"
"Optional:\n"
"\t-o STRING: prefix of the output file (default: trust)\n"
"\t-t INT: number of threads (default: 1)\n"
"\t-c STRING: the path to the kmer count file\n"
"\t-k INT: the starting k-mer size for indexing contigs (default: 9)\n"
"\t--minHitLen INT: the minimal hit length for a valid overlap (default: auto)\n"
"\t--skipMateExtension: skip the step of extension assemblies with mate-pair information\n"
///"\t--noV: do not assemble the full length V gene (default: not used)\n"
"\t--trimLevel INT: 0: no trim; 1: trim low quality; 2: trim unmatched (default: 1)\n"
"\t--barcode STRING: the path to the barcode file (default: not used)\n"
"\t--UMI STRING: the path to the UMI file (default: not used)\n"
"\t--keepNoBarcode: assemble the reads with missing barcodes. (default: ignore the reads)\n"
"\t--contigMinCov INT: ignore contigs that have bases covered by fewer than INT reads (default: 0)\n" ;
int nucToNum[26] = { 0, -1, 1, -1, -1, -1, 2,
-1, -1, -1, -1, -1, -1, 0,
-1, -1, -1, -1, -1, 3,
-1, -1, -1, -1, -1, -1 } ;
char numToNuc[26] = {'A', 'C', 'G', 'T'} ;
char buffer[10240] = "" ;
static const char *short_options = "f:u:1:2:b:o:c:t:k:" ;
static struct option long_options[] = {
{ "debug-ns", required_argument, 0, 10000 },
{ "trimLevel", required_argument, 0, 10001 },
{ "barcode", required_argument, 0, 10002 },
{ "keepNoBarcode", no_argument, 0, 10003 },
{ "UMI", required_argument, 0, 10004},
{ "skipMateExtension", no_argument, 0, 10005},
{ "minHitLen", required_argument, 0, 10006},
{ "contigMinCov", required_argument, 0, 10007},
{ (char *)0, 0, 0, 0}
} ;
struct _sortRead
{
char *id ;
char *read ;
char *qual ;
// global k-mer count
int minCnt ;
int medianCnt ;
float avgCnt ;
// barcode-wise k-mer count
int barcodeMinCnt ;
int barcodeMedianCnt ;
float barcodeAvgCnt ;
//double avgQual ;
//int germlineMatchCnt ;
int len ;
int strand ;
int mateIdx ; // the index of its mate pair.
int info ; // some random information, such as it's index in orginal array.
int barcode ; // this is for cell id
int umi ; // this is for mrna id.
struct _overlap geneOverlap[4] ; // Rough annotation of the read
_sortRead()
{
id = read = qual = NULL ;
minCnt = medianCnt = barcodeMinCnt = barcodeMedianCnt = 0 ;
avgCnt = barcodeAvgCnt = 0 ;
len = strand = 0 ;
mateIdx = info = -1 ;
barcode = umi = 1 ;
}
bool operator<( const struct _sortRead &b ) const
{
if ( minCnt != b.minCnt )
return minCnt > b.minCnt ;
else if ( medianCnt != b.medianCnt )
return medianCnt > b.medianCnt ;
else if ( avgCnt != b.avgCnt )
return avgCnt > b.avgCnt ;
else if ( len != b.len )
return len > b.len ;
//else if (avgQual != b.avgQual)
// return avgQual > b.avgQual ;
//else if (germlineMatchCnt != b.germlineMatchCnt)
// return germlineMatchCnt > b.germlineMatchCnt ;
else
{
int tmp = strcmp( read, b.read ) ;
if ( tmp != 0 )
return tmp < 0;
else
return strcmp( id, b.id ) < 0 ;
}
}
} ;
bool CompReadWithBarcode(const struct _sortRead &a, const struct _sortRead &b)
{
if (a.barcode != -1 && a.barcode != b.barcode)
return a.barcode < b.barcode ;
else if ( a.barcode != -1 && b.barcode != -1 && a.barcodeMinCnt != b.barcodeMinCnt )
return a.barcodeMinCnt > b.barcodeMinCnt ;
else
return a < b ;
}
struct _quickAnnotateReadsThreadArg
{
int tid ;
int threadCnt ;
SeqSet *refSet ;
std::vector<struct _sortRead> *pSortedReads ;
int readCnt ;
} ;
// The structure used for threads related to kmer count, including read trimming and barcode-level kmer count
struct _kmerCountThreadArg
{
int tid ;
int threadCnt ;
int countKmer ;
KmerCount *pKmerCount ;
SeqSet *pSeqSet ;
std::vector<struct _sortRead> *pRead1Batch ;
std::vector<struct _sortRead> *pRead2Batch ;
std::vector<struct _sortRead> *pReads ;
int readCnt ;
int maxReadLen ;
int trimLevel ;
} ;
struct _assignReadsThreadArg
{
int tid ;
int threadCnt ;
SeqSet *seqSet ;
std::vector<struct _assignRead> *pAssembledReads ;
int assembledReadCnt ;
} ;
bool CompSortReadById( const struct _Read &a, const struct _Read &b )
{
return strcmp( a.id, b.id ) < 0 ;
}
bool IsLowComplexity( const char *seq )
{
int cnt[5] = {0, 0, 0, 0, 0} ;
int i ;
for ( i = 0 ; seq[i] ; ++i )
{
if ( seq[i] == 'N' )
++cnt[4] ;
else
++cnt[ nucToNum[ seq[i] - 'A' ] ] ;
}
if ( cnt[0] >= i / 2 || cnt[1] >= i / 2 || cnt[2] >= i / 2 || cnt[3] >= i / 2 || cnt[4] >= i / 10 )
return true ;
int lowCnt = 0 ;
for ( i = 0 ; i < 4 ; ++i )
if ( cnt[i] <= 2 )
++lowCnt ;
if ( lowCnt >= 2 )
return true ;
return false ;
}
void PrintLog( const char *fmt, ... )
{
va_list args ;
va_start( args, fmt ) ;
vsprintf( buffer, fmt, args ) ;
time_t mytime = time(NULL) ;
struct tm *localT = localtime( &mytime ) ;
char stime[500] ;
strftime( stime, sizeof( stime ), "%c", localT ) ;
fprintf( stderr, "[%s] %s\n", stime, buffer ) ;
}
// Process one read (pair), and append the result to reads
// This includes readthrough, mate pair merging, and kmer count
// The r1, r2's content can be modified in the process, including release
// origR1, origR2 and the content from the original input
void ProcessRead(struct _sortRead &r1, struct _sortRead &r2, int countKmer, KmerCount &kmerCount, SeqSet &seqSet, std::vector<struct _sortRead> &reads)
{
int j, k ;
int rWeight = 1 ;
// Check chimeric. After reverse-complement, the mate should be after the current read.
if (r2.read != NULL)
{
int flen = strlen(r2.read) ;
int slen = strlen(r1.read) ;
seqSet.ReverseComplementInPlace(r2.read, flen ) ;
if (r2.qual != NULL)
{
// Reverse the mateR's quality score
for (j = 0, k = flen - 1 ; j < k ; ++j, --k)
{
char tmp = r2.qual[j] ;
r2.qual[j] = r2.qual[k] ;
r2.qual[k] = tmp ;
}
}
int minOverlap = ( flen + slen ) / 10 ;
int minOverlap2 = ( flen + slen ) / 20 ;
if ( minOverlap > 31 )
minOverlap = 31 ;
if ( minOverlap2 > 31 )
minOverlap2 = 31 ;
int offset = -1 ;
int bestMatchCnt = -1 ;
/*if ( AlignAlgo::IsMateOverlap( mateR.read, flen, nr.read, slen, minOverlap, offset, bestMatchCnt ) >= 0 )
{
printf( "Outie\n%s\n%s\n", nr.read, mateR.read ) ;
}
else if ( AlignAlgo::IsMateOverlap( nr.read, slen, mateR.read, flen, minOverlap, offset, bestMatchCnt ) >= 0 )
{
printf( "Innie\n%s\n%s\n", nr.read, mateR.read ) ;
}
else
printf( "Uncertain\n" ) ;*/
int overlapSize = AlignAlgo::IsMateOverlap( r2.read, flen, r1.read, slen, minOverlap, offset, bestMatchCnt, false ) ;
if ( overlapSize >= 0 )
{
// Wrong order (read-through) happened,
// Only keep the overlapped portion.
r1.read[ overlapSize ] = '\0' ;
if ( r1.qual != NULL )
{
r1.qual[ overlapSize ] = '\0' ;
for ( j = 0 ; j < overlapSize ; ++j )
{
if ( r2.qual[j + offset] > r1.qual[j] || r1.read[j] == 'N' )
{
r1.read[j] = r2.read[j + offset] ;
r1.qual[j] = r2.qual[j + offset] ;
}
}
}
// Release the mate.
free( r2.read ) ; free( r2.id ) ;
if (r2.qual)
free(r2.qual) ;
r2.read = NULL ;
}
else if ( ( overlapSize =
AlignAlgo::IsMateOverlap( r1.read, slen, r2.read, flen, minOverlap2, offset, bestMatchCnt ) ) >= 0 )
{
// Merge paired-end reads
if ( bestMatchCnt >= 0.95 * overlapSize )
{
char *r = (char *)malloc( sizeof( char ) * ( slen + flen + 1 ) ) ;
char *q = (char *)malloc( sizeof( char ) * ( slen + flen + 1 ) ) ;
for ( j = 0 ; j < flen ; ++j )
{
r[ offset + j ] = r2.read[j] ;
q[ offset + j ] = r2.qual[j] ;
}
int len = offset + j ;
for ( j = 0 ; j < slen && j < len ; ++j )
{
if ( j < offset || r1.qual[j] >= q[j] - 14 || r[j] == 'N' )
{
r[j] = r1.read[j] ;
q[j] = r1.qual[j] ;
}
}
//if ( j > len )
// len = j ;
r[len] = q[len] = '\0' ;
if ( 0 )//len > 4 )
{
for ( j = 2 ; j <= len ; ++j )
{
r[j - 2] = r[j] ;
q[j - 2] = q[j] ;
}
r[len - 4] = '\0' ;
q[len - 4] = '\0' ;
len -= 4 ;
}
free( r2.read ) ; free( r2.id ) ;
if (r2.qual)
free( r2.qual ) ;
r2.read = NULL ;
free( r1.read ) ; free( r1.qual ) ;
r1.read = r ;
r1.qual = q ;
++rWeight ;
}
else
{
// Discard one of the read
bool useFirst = true ;
if ( r1.qual != NULL )
{
double avgQualR = 0, avgQualMate = 0 ;
for ( j = offset ; j < slen ; ++j )
avgQualR += r1.qual[j] - 32 ;
for ( j = flen - 1 ; j >= flen - overlapSize ; --j )
avgQualMate += r2.qual[j] - 32 ;
avgQualR /= overlapSize ; avgQualMate /= overlapSize ;
if ( avgQualR + 10 < avgQualMate )
useFirst = false ;
}
if ( useFirst )
{
free( r2.read ) ; free( r2.id ) ;
if (r2.qual)
free( r2.qual ) ;
r2.read = NULL ;
}
else
{
free( r1.read ) ;
if (r1.qual)
free(r1.qual) ;
free( r2.id ) ;
r1.read = r2.read ;
seqSet.ReverseComplementInPlace(r1.read, flen) ;
r1.qual = r2.qual ;
/* Not sure why, keeping it reversed seems better
if (r1.qual != NULL)
{
for (j = 0, k = flen - 1 ; j < k ; ++j, --k)
{
char tmp = r1.qual[j] ;
r1.qual[j] = r1.qual[k] ;
r1.qual[k] = tmp ;
}
}*/
r2.read = NULL ;
}
}
}
else // Nothing changes
{
seqSet.ReverseComplementInPlace(r2.read, flen) ;
if (r2.qual != NULL)
{
for (j = 0, k = flen - 1 ; j < k ; ++j, --k)
{
char tmp = r2.qual[j] ;
r2.qual[j] = r2.qual[k] ;
r2.qual[k] = tmp ;
}
}
}
} // End of testing read-through or overlapped paired-end.
if ( !IsLowComplexity(r1.read) )
{
reads.push_back( r1 ) ;
if ( countKmer )
kmerCount.AddCount( r1.read ) ;
if ( rWeight == 2 )
{
struct _sortRead wr ;
wr = r1 ;
wr.read = strdup( r1.read ) ;
if ( r1.qual != NULL )
wr.qual = strdup( r1.qual ) ;
else
wr.qual = NULL ;
int len = strlen( r1.id ) ;
wr.id = ( char * )malloc( sizeof( char ) * ( len + 3 ) ) ;
strcpy( wr.id, r1.id ) ;
wr.id[len] = '.' ;
wr.id[len + 1] = '1' ;
wr.id[len + 2] = '\0' ;
reads.push_back( wr ) ;
if ( countKmer )
kmerCount.AddCount( wr.read ) ;
}
}
else
{
free( r1.read ) ; free( r1.id ) ;
if (r1.qual)
free( r1.qual ) ;
}
if (r2.read != NULL )
{
if ( !IsLowComplexity(r2.read) )
{
reads.push_back(r2) ;
if ( countKmer )
kmerCount.AddCount(r2.read ) ;
}
else
{
free( r2.read ) ; free( r2.id ) ;
if (r2.qual)
free( r2.qual ) ;
}
}
}
void *ProcessReads_Thread(void *pArg)
{
struct _kmerCountThreadArg &arg = *((struct _kmerCountThreadArg*)pArg) ;
std::vector< struct _sortRead> &reads = *arg.pReads ;
std::vector<struct _sortRead> &read1Batch = *arg.pRead1Batch ;
std::vector<struct _sortRead> &read2Batch = *arg.pRead2Batch ;
KmerCount &kmerCount = *arg.pKmerCount ;
SeqSet &seqSet = *arg.pSeqSet ;
int readCnt = read1Batch.size() ;
int i, start, end ;
int block = readCnt / arg.threadCnt ;
if (readCnt % arg.threadCnt)
++block ;
start = arg.tid * block ;
end = start + block - 1 ;
if (end >= readCnt)
end = readCnt - 1 ;
for (i = start ; i <= end ; ++i)
ProcessRead(read1Batch[i], read2Batch[i], arg.countKmer, kmerCount, seqSet, reads) ;
pthread_exit(NULL) ;
}
void ProcessReadsWithThreads(std::vector<struct _sortRead> &r1Batch, std::vector<struct _sortRead> &r2Batch, int countKmer, KmerCount &kmerCount, SeqSet &seqSet, std::vector<struct _sortRead> &reads, int threadCnt)
{
int i ;
pthread_t *threads = new pthread_t[ threadCnt ] ;
struct _kmerCountThreadArg *args = new struct _kmerCountThreadArg[threadCnt] ;
pthread_attr_t attr ;
pthread_attr_init( &attr ) ;
pthread_attr_setdetachstate( &attr, PTHREAD_CREATE_JOINABLE ) ;
for ( i = 0 ; i < threadCnt ; ++i )
{
args[i].tid = i ;
args[i].threadCnt = threadCnt ;
args[i].pRead1Batch = &r1Batch ;
args[i].pRead2Batch = &r2Batch ;
args[i].pReads = new std::vector<struct _sortRead> ;
args[i].countKmer = countKmer ;
args[i].pKmerCount = &kmerCount ;
args[i].pSeqSet = &seqSet ;
pthread_create( &threads[i], &attr, ProcessReads_Thread, (void *)( args + i ) ) ;
}
for ( i = 0 ; i < threadCnt ; ++i )
pthread_join( threads[i], NULL ) ;
for (i = 0 ; i < threadCnt ; ++i)
{
reads.insert(reads.end(), args[i].pReads->begin(), args[i].pReads->end()) ;
delete args[i].pReads ;
}
delete[] threads ;
delete[] args ;
}
void *QuickAnnotateReads_Thread( void *pArg )
{
struct _quickAnnotateReadsThreadArg &arg = *( (struct _quickAnnotateReadsThreadArg *)pArg ) ;
int start, end ;
int i, j ;
std::vector< struct _sortRead> &sortedReads = *arg.pSortedReads ;
start = arg.readCnt / arg.threadCnt * arg.tid ;
end = start + arg.readCnt / arg.threadCnt ;
if ( arg.tid == arg.threadCnt - 1 )
end = arg.readCnt ;
struct _overlap geneOverlap[4] ;
for ( i = start ; i < end ; ++i )
{
if ( i == start || strcmp( sortedReads[i].read, sortedReads[i - 1].read ) )
arg.refSet->AnnotateRead( sortedReads[i].read, 0, geneOverlap, NULL, NULL ) ;
for ( j = 0 ; j < 4 ; ++j )
sortedReads[i].geneOverlap[j] = geneOverlap[j] ;
}
pthread_exit( NULL ) ;
}
void *GetReadCountStatsAndTrim_Thread(void *pArg)
{
int i ;
struct _kmerCountThreadArg &arg = *((struct _kmerCountThreadArg*)pArg) ;
std::vector< struct _sortRead> &reads = *arg.pReads ;
int readCnt = arg.readCnt ;
KmerCount &kmerCount = *arg.pKmerCount ;
int *buffer = new int[arg.maxReadLen] ;
for (i = arg.tid ; i < readCnt ; i += arg.threadCnt)
{
if ( arg.trimLevel == 0 )
kmerCount.GetCountStatsAndTrim( reads[i].read, NULL,
reads[i].minCnt, reads[i].medianCnt, reads[i].avgCnt, buffer ) ;
else
kmerCount.GetCountStatsAndTrim( reads[i].read, reads[i].qual,
reads[i].minCnt, reads[i].medianCnt, reads[i].avgCnt, buffer ) ;
if ( reads[i].qual != NULL )
{
free( reads[i].qual ) ;
reads[i].qual = NULL ;
}
if ( reads[i].read[0] == '\0' )
{
free( reads[i].read ) ;
free( reads[i].id ) ;
reads[i].read = NULL ;
}
}
delete[] buffer ;
pthread_exit(NULL) ;
}
void *BarcodeKmerCount_Thread(void *pArg)
{
int i, j, k ;
struct _kmerCountThreadArg &arg = *((struct _kmerCountThreadArg*)pArg) ;
std::vector< struct _sortRead> &reads = *arg.pReads ;
int readCnt = arg.readCnt ;
KmerCount barcodeKmerCount(21, 23) ;
barcodeKmerCount.SetBuffer( arg.maxReadLen ) ;
for (i = 0 ; i < readCnt ; )
{
for (j = i + 1 ; j < readCnt ; ++j)
{
if (reads[i].barcode != reads[j].barcode)
break ;
}
if (reads[i].barcode % arg.threadCnt != arg.tid)
{
i = j ;
continue ;
}
for (k = i ; k < j ; ++k)
barcodeKmerCount.AddCount(reads[k].read) ;
for (k = i ; k < j ; ++k)
barcodeKmerCount.GetCountStatsAndTrim(reads[k].read, NULL,
reads[k].barcodeMinCnt, reads[k].barcodeMedianCnt,
reads[k].barcodeAvgCnt ) ;
barcodeKmerCount.Clear() ;
i = j ;
}
barcodeKmerCount.Release() ;
pthread_exit(NULL) ;
}
void *AssignReads_Thread( void *pArg )
{
struct _assignReadsThreadArg &arg = *( (struct _assignReadsThreadArg *)pArg ) ;
int start, end ;
int i ;
std::vector< struct _assignRead> &assembledReads = *arg.pAssembledReads ;
start = arg.assembledReadCnt / arg.threadCnt * arg.tid ;
end = start + arg.assembledReadCnt / arg.threadCnt ;
if ( arg.tid == arg.threadCnt - 1 )
end = arg.assembledReadCnt ;
struct _overlap assign ;
for ( i = start ; i < end ; ++i )
{
if ( i == start || strcmp( assembledReads[i].read, assembledReads[i - 1].read ) )
arg.seqSet->AssignRead( assembledReads[i].read, assembledReads[i].overlap.strand,
assembledReads[i].barcode, assign ) ;
assembledReads[i].overlap = assign ;
}
pthread_exit( NULL ) ;
}
int main( int argc, char *argv[] )
{
int i, j, k ;
if ( argc <= 1 )
{
fprintf( stderr, "%s", usage ) ;
return 0 ;
}
int c, option_index ;
option_index = 0 ;
int indexKmerLength = 9 ;
int changeKmerLengthThreshold = 4096 ;
SeqSet seqSet( indexKmerLength ) ; // Only hold the novel seq.
SeqSet refSet( 9 ) ;
KmerCount kmerCount( 21 ) ;
char outputPrefix[1024] = "trust" ;
ReadFiles reads ;
ReadFiles mateReads ;
ReadFiles barcodeFile, umiFile ;
bool countMyself = true ; // Count using our procedure, or read from some kmer count tool's output.
int maxReadLen = -1 ;
int firstReadLen = -1 ;
int trimLevel = 1 ;
int minHitLen = -1 ;
bool hasMate = false ;
bool hasBarcode = false ;
bool hasUmi = false ;
int constantGeneEnd = 200 ;
bool keepMissingBarcode = false ;
int threadCnt = 1 ;
bool skipMateExtension = false ;
int contigMinCov = 0 ;
while ( 1 )
{
c = getopt_long( argc, argv, short_options, long_options, &option_index ) ;
if ( c == -1 )
break ;
if ( c == 'f' )
{
//seqSet.InputRefFa( optarg ) ;
refSet.InputRefFa( optarg ) ;
}
else if ( c == 'u' )
{
reads.AddReadFile( optarg, false ) ;
}
else if ( c == '1' )
{
reads.AddReadFile( optarg, true ) ;
hasMate = true ;
}
else if ( c == '2' )
{
mateReads.AddReadFile( optarg, true ) ;
hasMate = true ;
}
else if ( c == 'o' )
{
strcpy( outputPrefix, optarg ) ;
}
else if ( c == 'c' )
{
kmerCount.AddCountFromFile( optarg ) ;
countMyself = false ;
PrintLog( "Read in the kmer count information from %s", optarg ) ;
}
else if ( c == 't' )
{
threadCnt = atoi( optarg ) ;
}
else if ( c == 'k' )
{
indexKmerLength = atoi( optarg ) ;
seqSet.ChangeKmerLength( indexKmerLength ) ;
}
else if ( c == 10000 ) //debug-ns
{
seqSet.InputNovelFa( optarg ) ;
}
else if ( c == 10001) //trimLevel
{
trimLevel = atoi( optarg ) ;
}
else if ( c == 10002 ) // barcode
{
barcodeFile.AddReadFile( optarg, false ) ;
hasBarcode = true ;
}
else if ( c == 10003 ) // keepNoBarcode
{
keepMissingBarcode = true ;
}
else if ( c == 10004 ) // umi
{
umiFile.AddReadFile( optarg, false ) ;
hasUmi = true ;
}
else if ( c == 10005 ) // skipMateExtension
{
skipMateExtension = true ;
}
else if ( c == 10006 ) // minHitLen
{
minHitLen = atoi( optarg ) ;
}
else if ( c == 10007 )
{
contigMinCov = atoi( optarg ) ;
}
else
{
fprintf( stderr, "%s", usage ) ;
return EXIT_FAILURE ;
}
}
/*char align[10000] ;
char t[] = "ACGGGGTTTTTT" ;
char p[] = "ACTTTTTTGGGG" ;
AlignAlgo::GlobalAlignment( t, strlen( t ), p, strlen( p ), align ) ;
AlignAlgo::VisualizeAlignment( t, strlen( t ), p, strlen( p ), align ) ; */
if ( refSet.Size() == 0 )
{
fprintf( stderr, "Need to use -f to specify the receptor genome sequence.\n" ) ;
return EXIT_FAILURE ;
}
PrintLog( "Start to assemble reads." ) ;
if ( trimLevel > 1 )
{
// We need a more careful rough annotation.
refSet.ChangeKmerLength( 7 ) ;
}
refSet.SetHitLenRequired( 17 ) ;
std::vector< struct _sortRead > sortedReads ;
std::map<std::string, int> barcodeStrToInt ;
std::map<std::string, int> umiStrToInt ;
std::vector<std::string> barcodeIntToStr ;
// Relate to barcode release
SimpleVector<int> barcodeTotalReadCount ; // Number of reads for this barcode
SimpleVector<int> barcodeReadCount ;
std::map<int, int> finishedBarcodes ;
std::vector<struct _sortRead> readBuffer, mateReadBuffer ;
i = 0 ;
if (threadCnt > 1)
kmerCount.SetPthreadLocks() ;
while ( reads.Next() )
{
/*struct _overlap geneOverlap[4] ;
refSet.AnnotateRead( reads.seq, 1, geneOverlap, buffer ) ;
if ( geneOverlap[0].seqIdx + geneOverlap[1].seqIdx + geneOverlap[2].seqIdx + geneOverlap[3].seqIdx == -4 )
{
continue ;
}*/
int barcode = -1 ;
int umi = -1 ;
if ( hasBarcode )
{
barcodeFile.Next() ;
if ( !strcmp( barcodeFile.seq, "missing_barcode" ) && !keepMissingBarcode )
{
if ( hasMate )
mateReads.Next() ;
if ( hasUmi )
umiFile.Next() ;
continue ;
}
std::string s( barcodeFile.seq ) ;
if ( barcodeStrToInt.find( s ) != barcodeStrToInt.end() )
barcode = barcodeStrToInt[s] ;
else
{
barcode = barcodeIntToStr.size() ;
barcodeStrToInt[s] = barcode ;
barcodeIntToStr.push_back( s ) ;
}
if (contigMinCov > 0)
{
if (barcode >= barcodeTotalReadCount.Size())
barcodeTotalReadCount.PushBack(1) ;
else
++barcodeTotalReadCount[barcode] ;
}
}
if ( hasUmi )
{
umiFile.Next() ;
std::string s(umiFile.seq) ;
if ( umiStrToInt.find(s) != umiStrToInt.end())
umi = umiStrToInt[s] ;
else
{
umi = umiStrToInt.size() ;
umiStrToInt[s] = umi ;
}
}
struct _sortRead nr, mateR ;
nr.read = strdup( reads.seq ) ;
nr.id = strdup( reads.id ) ;
if ( reads.qual != NULL )
nr.qual = strdup( reads.qual ) ;
else
nr.qual = NULL ;
nr.barcode = barcode ;
nr.umi = umi ;
++i ;
if ( threadCnt == 1 && countMyself && i % 100000 == 0 )
PrintLog( "Read in and count kmers for %d reads.", i ) ;
else if ( threadCnt == 1 && !countMyself && i % 1000000 == 0 )
PrintLog( "Read in %d reads.", i ) ;
if ( firstReadLen == -1 )
{
firstReadLen = strlen( reads.seq ) ;
}
mateR.read = NULL ;
if ( mateReads.Next() )
{
mateR.read = strdup( mateReads.seq ) ;
mateR.id = strdup( mateReads.id ) ;
mateR.barcode = barcode ;
mateR.umi = umi ;
if ( mateReads.qual != NULL )
{
mateR.qual = strdup( mateReads.qual ) ;
}
else
mateR.qual = NULL ;
++i ;
if ( threadCnt == 1 && countMyself && i % 100000 == 0 )
PrintLog( "Read in and count kmers for %d reads.", i ) ;
else if ( threadCnt == 1 && !countMyself && i % 1000000 == 0 )
PrintLog( "Read in %d reads.", i ) ;
}
else if ( hasMate )
{
fprintf( stderr, "The two mate-pair read files have different number of reads.\n" ) ;
exit( 1 ) ;
}
if (threadCnt == 1)
ProcessRead(nr, mateR, countMyself, kmerCount, seqSet, sortedReads) ;
else
{
readBuffer.push_back(nr) ;
mateReadBuffer.push_back(mateR) ;
if ((int)readBuffer.size() >= 100000 * threadCnt)
{
ProcessReadsWithThreads(readBuffer, mateReadBuffer, countMyself, kmerCount, seqSet, sortedReads, threadCnt) ;
readBuffer.clear() ;
mateReadBuffer.clear() ;
if (countMyself)
PrintLog( "Read in and count kmers for %d reads.", i ) ;
else
PrintLog( "Read in %d reads.", i ) ;
}
}
}
if (readBuffer.size() > 0)
{
ProcessReadsWithThreads(readBuffer, mateReadBuffer, countMyself, kmerCount, seqSet, sortedReads, threadCnt) ;
}
int readCnt = sortedReads.size() ;
maxReadLen = 0 ;
for (i = 0 ; i < readCnt ; ++i)
{
sortedReads[i].len = strlen( sortedReads[i].read ) ;
if (sortedReads[i].len > maxReadLen)
maxReadLen = sortedReads[i].len ;
//printf("%s %s %s\n", sortedReads[i].id, sortedReads[i].read, sortedReads[i].qual) ;
}
// empty file
if ( readCnt <= 0 )
{
sprintf( buffer, "%s_raw.out", outputPrefix ) ;
FILE *fp = fopen( buffer, "w" ) ;
fclose(fp) ;
sprintf( buffer, "%s_assembled_reads.fa", outputPrefix ) ;
fp = fopen( buffer, "w" ) ;
fclose(fp) ;
sprintf( buffer, "%s_final.out", outputPrefix ) ;
fp = fopen( buffer, "w" ) ;
fclose(fp) ;
return 0 ;
}
#ifdef DEBUG
printf( "Finish read in the reads and kmer count.\n") ;
#endif
// Remove the reads from the barcode with too few reads
if (contigMinCov > 0)
{
for (i = 0 ; i < readCnt ; ++i)
{
if (sortedReads[i].barcode != -1 && barcodeTotalReadCount[sortedReads[i].barcode] < contigMinCov)
{
free( sortedReads[i].read ) ;
free( sortedReads[i].id ) ;
if (sortedReads[i].qual != NULL)
{
free(sortedReads[i].qual) ;
sortedReads[i].qual = NULL ;
}
sortedReads[i].read = NULL ;
}
}
k = 0 ;
for ( i = 0 ; i < readCnt ; ++i )
{
if ( sortedReads[i].read == NULL )
continue ;
sortedReads[k] = sortedReads[i] ;
++k ;
}
readCnt = k ;
sortedReads.resize( k ) ;
}
kmerCount.SetBuffer( maxReadLen ) ;
if (threadCnt == 1)
{
for ( i = 0 ; i < readCnt ; ++i )
{
if ( trimLevel == 0 )
kmerCount.GetCountStatsAndTrim( sortedReads[i].read, NULL,
sortedReads[i].minCnt, sortedReads[i].medianCnt, sortedReads[i].avgCnt ) ;
else
kmerCount.GetCountStatsAndTrim( sortedReads[i].read, sortedReads[i].qual,
sortedReads[i].minCnt, sortedReads[i].medianCnt, sortedReads[i].avgCnt ) ;
if ( sortedReads[i].qual != NULL )
{
/*char *qual = sortedReads[i].qual ;
double avgQual = 0 ;
for (j = 0 ; qual[j] ; ++j)
avgQual += qual[j] ;
sortedReads[i].avgQual = avgQual / j ;*/
free( sortedReads[i].qual ) ;
sortedReads[i].qual = NULL ;
}
//else
// sortedReads[i].avgQual = 0 ;