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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--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" ;
char 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:" ;
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},
{ (char *)0, 0, 0, 0}
} ;
struct _sortRead
{
char *id ;
char *read ;
char *qual ;
int minCnt ;
int medianCnt ;
double avgCnt ;
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
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 (barcode != b.barcode)
return barcode < b.barcode ;
else
{
int tmp = strcmp( read, b.read ) ;
if ( tmp != 0 )
return tmp < 0;
else
return strcmp( id, b.id ) < 0 ;
}
}
} ;
struct _quickAnnotateReadsThreadArg
{
int tid ;
int threadCnt ;
SeqSet *refSet ;
std::vector<struct _sortRead> *pSortedReads ;
int readCnt ;
} ;
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( 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 ) ;
}
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 *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 ;
int maxReadLen = -1 ;
int firstReadLen = -1 ;
int trimLevel = 1 ;
bool hasMate = false ;
bool hasBarcode = false ;
bool hasUmi = false ;
int constantGeneEnd = 200 ;
bool keepMissingBarcode = false ;
int threadCnt = 1 ;
bool skipMateExtension = false ;
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 == 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
{
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 ;
}
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 ;
i = 0 ;
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() ;
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 ( 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 ;
int rWeight = 1 ;
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 ( countMyself && i % 100000 == 0 )
PrintLog( "Read in and count kmers for %d reads.", i ) ;
else if ( !countMyself && i % 1000000 == 0 )
PrintLog( "Read in %d reads.", i ) ;
if ( firstReadLen == -1 )
{
firstReadLen = strlen( reads.seq ) ;
}
struct _sortRead mateR ;
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 ( countMyself && i % 100000 == 0 )
PrintLog( "Read in and count kmers for %d reads.", i ) ;
else if ( !countMyself && i % 1000000 == 0 )
PrintLog( "Read in %d reads.", i ) ;
// Check chimeric. After reverse-complement, the mate should be after the current read.
int flen = strlen( mateReads.seq ) ;
int slen = strlen( nr.read ) ;
seqSet.ReverseComplement( mateR.read, mateReads.seq, flen ) ;
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( mateR.read, flen, nr.read, slen, minOverlap, offset, bestMatchCnt, false ) ;
if ( overlapSize >= 0 )
{
// Wrong order happened,
// Only keep the overlapped portion.
nr.read[ overlapSize ] = '\0' ;
if ( nr.qual != NULL )
{
nr.qual[ overlapSize ] = '\0' ;
for ( j = 0 ; j < overlapSize ; ++j )
{
if ( mateR.qual[j + offset] > nr.qual[j] || nr.read[j] == 'N' )
{
nr.read[j] = mateR.read[j + offset] ;
nr.qual[j] = mateR.qual[j + offset] ;
}
}
}
// filter the mate.
free( mateR.read ) ; free( mateR.id ) ; free( mateR.qual ) ;
mateR.read = NULL ;
}
else if ( ( overlapSize =
AlignAlgo::IsMateOverlap( nr.read, slen, mateR.read, flen, minOverlap2, offset, bestMatchCnt ) ) >= 0 )
{
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 ] = mateR.read[j] ;
q[ offset + j ] = mateR.qual[j] ;
}
int len = offset + j ;
for ( j = 0 ; j < slen && j < len ; ++j )
{
if ( j < offset || nr.qual[j] >= q[j] - 14 || r[j] == 'N' )
{
r[j] = nr.read[j] ;
q[j] = nr.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( mateR.read ) ; free( mateR.id ) ; free( mateR.qual ) ;
mateR.read = NULL ;
free( nr.read ) ; free( nr.qual ) ;
nr.read = r ;
nr.qual = q ;
++rWeight ;
}
else
{
// Discard one of the read
bool useFirst = true ;
if ( nr.qual != NULL )
{
double avgQualR = 0, avgQualMate = 0 ;
for ( j = offset ; j < slen ; ++j )
avgQualR += nr.qual[j] - 32 ;
for ( j = flen - 1 ; j >= flen - overlapSize ; --j )
avgQualMate += mateR.qual[j] - 32 ;
avgQualR /= overlapSize ; avgQualMate /= overlapSize ;
if ( avgQualR + 10 < avgQualMate )
useFirst = false ;
}
if ( useFirst )
{
free( mateR.read ) ; free( mateR.id ) ; free( mateR.qual ) ;
mateR.read = NULL ;
}
else
{
free( nr.read ) ; free( nr.qual ) ;
free( mateR.id ) ;
nr.read = mateR.read ;
nr.qual = mateR.qual ;
strcpy( nr.read, mateReads.seq ) ;
mateR.read = NULL ;
}
}
}
else
{
strcpy( mateR.read, mateReads.seq ) ;
}
}
else if ( hasMate )
{
fprintf( stderr, "The two mate-pair read files have different number of reads.\n" ) ;
exit( 1 ) ;
}
if ( !IsLowComplexity( reads.seq ) )
{
sortedReads.push_back( nr ) ;
if ( countMyself )
kmerCount.AddCount( nr.read ) ;
if ( rWeight == 2 )
{
struct _sortRead wr ;
wr = nr ;
wr.read = strdup( nr.read ) ;
if ( nr.qual != NULL )
wr.qual = strdup( nr.qual ) ;
else
wr.qual = NULL ;
int len = strlen( nr.id ) ;
wr.id = ( char * )malloc( sizeof( char ) * ( len + 3 ) ) ;
strcpy( wr.id, nr.id ) ;
wr.id[len] = '.' ;
wr.id[len + 1] = '1' ;
wr.id[len + 2] = '\0' ;
sortedReads.push_back( wr ) ;
if ( countMyself )
kmerCount.AddCount( wr.read ) ;
}
int len = strlen( nr.read ) ;
if ( len > maxReadLen )
maxReadLen = len ;
}
else
{
free( nr.read ) ; free( nr.id ) ; free( nr.qual ) ;
}
if ( mateR.read != NULL )
{
if ( !IsLowComplexity( mateReads.seq ) )
{
sortedReads.push_back( mateR ) ;
if ( countMyself )
kmerCount.AddCount( mateR.read ) ;
int len = strlen( mateR.read ) ;
if ( len > maxReadLen )
maxReadLen = len ;
}
else
{
free( mateR.read ) ; free( mateR.id ) ; free( mateR.qual ) ;
}
}
/*if ( countMyself && i % 100000 == 0 )
PrintLog( "Read in and count kmers for %d reads.", i ) ;
else if ( !countMyself && i % 1000000 == 0 )
PrintLog( "Read in %d reads.", i ) ;*/
}
if ( maxReadLen <= 0 )
{
return 0 ;
}
#ifdef DEBUG
printf( "Finish read in the reads and kmer count.\n") ;
#endif
int readCnt = sortedReads.size() ;
kmerCount.SetBuffer( maxReadLen ) ;
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 )
{
free( sortedReads[i].qual ) ;
sortedReads[i].qual = NULL ;
}
if ( sortedReads[i].read[0] == '\0' )
{
free( sortedReads[i].read ) ;
free( sortedReads[i].id ) ;
sortedReads[i].read = NULL ;
}
}
k = 0 ;
for ( i = 0 ; i < readCnt ; ++i )
{
if ( sortedReads[i].read == NULL )
continue ;
sortedReads[k] = sortedReads[i] ;
sortedReads[k].len = strlen( sortedReads[i].read ) ;
++k ;
}
readCnt = k ;
sortedReads.resize( k ) ;
PrintLog( "Found %i reads.", k ) ;
#ifdef DEBUG
printf( "Finish put in the read kmer count.\n" ) ;
#endif
kmerCount.Release() ;
for ( i = 0 ; i < readCnt ; ++i )
{
sortedReads[i].info = i ;
sortedReads[i].mateIdx = -1 ;
}
for ( i = 0 ; i < readCnt - 1 ; ++i )
{
if ( !strcmp( sortedReads[i].id, sortedReads[i + 1].id ) )
{
sortedReads[i].mateIdx = i + 1 ;
sortedReads[i + 1].mateIdx = i ;
++i ;
}
}
std::sort( sortedReads.begin(), sortedReads.end() ) ;
SimpleVector<int> originToSortedIdx ;
SimpleVector<bool> goodCandidate ; // Use mate pair alignment information to infer
// whether this read could be a good candidate
originToSortedIdx.ExpandTo( readCnt ) ;
goodCandidate.ExpandTo( readCnt ) ;
for ( i = 0 ; i < readCnt ; ++i )
originToSortedIdx[ sortedReads[i].info ] = i ;
for ( i = 0 ; i < readCnt ; ++i )
{
if ( sortedReads[i].mateIdx != -1 )
sortedReads[i].mateIdx = originToSortedIdx[ sortedReads[i].mateIdx ] ;
goodCandidate[i] = false ;
}
PrintLog( "Finish sorting the reads." ) ;
// Quickly annoate the reads.
if ( trimLevel > 1 )
refSet.SetRadius(0) ;
if ( threadCnt <= 1 )
{
struct _overlap geneOverlap[4] ;
for ( i = 0 ; i < readCnt ; ++i )
{
if ( i == 0 || strcmp( sortedReads[i].read, sortedReads[i - 1].read ) )
refSet.AnnotateRead( sortedReads[i].read, 0, geneOverlap, NULL, NULL ) ;
for ( j = 0 ; j < 4 ; ++j )
sortedReads[i].geneOverlap[j] = geneOverlap[j] ;
}
}
else
{
pthread_t *threads = new pthread_t[ threadCnt ] ;
struct _quickAnnotateReadsThreadArg *args = new struct _quickAnnotateReadsThreadArg[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].refSet = &refSet ;
args[i].pSortedReads = &sortedReads ;
args[i].readCnt = readCnt ;
pthread_create( &threads[i], &attr, QuickAnnotateReads_Thread, (void *)( args + i ) ) ;
}
for ( i = 0 ; i < threadCnt ; ++i )
pthread_join( threads[i], NULL ) ;
delete[] threads ;
delete[] args ;
}
PrintLog( "Finish rough annotations." ) ;
if ( trimLevel > 1 && !hasBarcode )
{
// use V gene assignment as barcode to speed up.
for ( i = 0 ; i < readCnt ; ++i )
{
if ( sortedReads[i].geneOverlap[0].seqIdx != -1 && sortedReads[i].geneOverlap[0].similarity > 0.95 )
{
sortedReads[i].barcode = sortedReads[i].geneOverlap[0].seqIdx ;
if ( sortedReads[i].mateIdx != -1 )
sortedReads[ sortedReads[i].mateIdx ].barcode = sortedReads[i].geneOverlap[0].seqIdx ;
}
}
/*for ( i = 0 ; i < readCnt ; ++i )
{
if ( sortedReads[i].barcode == -1 )
{
free( sortedReads[i].id ) ;
free( sortedReads[i].read ) ;
if ( sortedReads[i].qual != NULL )
free( sortedReads[i].qual ) ;
sortedReads[i].read = NULL ;
}
}*/
/*for ( i = 0 ; i < readCnt ; ++i )
{
if ( sortedReads[i].geneOverlap[0].seqIdx == -1 && sortedReads[i].geneOverlap[2].seqIdx == -1 )
{
free( sortedReads[i].id ) ;
free( sortedReads[i].read ) ;
if ( sortedReads[i].qual != NULL )
free( sortedReads[i].qual ) ;
sortedReads[i].read = NULL ;
}
}*/
}
// Remove the redudant sequence before V gene.
for ( i = 0 ; i < readCnt ; ++i )
{
if ( sortedReads[i].read == NULL )
continue ;
struct _overlap *geneOverlap = sortedReads[i].geneOverlap ;
if ( geneOverlap[0].seqIdx == -1 )
continue ;
bool mayTrim = false ;
if ( geneOverlap[0].seqStart < 31 && geneOverlap[0].similarity > 0.9 )
mayTrim = true ;
if ( geneOverlap[0].similarity > 0.95 &&
geneOverlap[0].seqStart <= refSet.GetSeqConsensusLen( geneOverlap[0].seqIdx ) / 3 )
mayTrim = true ;
if ( trimLevel > 1 )
mayTrim = true ;
if ( !mayTrim )
continue ;
int trimBase = geneOverlap[0].readStart ;
if ( trimLevel > 1 && refSet.GetSeqName(geneOverlap[0].seqIdx)[0] == 'T') // Some bad annotation
{
if ( geneOverlap[0].seqEnd + 25 < refSet.GetSeqConsensusLen( geneOverlap[0].seqIdx ) )
trimBase = geneOverlap[0].readEnd + 2 ;
else if ( geneOverlap[0].similarity < 0.97 )
{
trimBase = ( geneOverlap[0].readStart + geneOverlap[0].readEnd ) / 2 ;
}
}
//printf("%s\n", sortedReads[i].read) ;
//printf("0: %lf %d %d %s: %d %d\n", geneOverlap[0].similarity, mayTrim, trimBase,
// refSet.GetSeqName(geneOverlap[0].seqIdx), geneOverlap[0].readStart, geneOverlap[0].readEnd) ;
if ( trimBase <= 0 )
continue ;
if ( geneOverlap[2].seqIdx != -1
&& geneOverlap[2].readStart < trimBase && trimLevel <= 1 )
continue ;
if ( geneOverlap[3].seqIdx != -1
&& geneOverlap[3].readStart < trimBase && trimLevel <= 1 )
continue ;
if ( sortedReads[i].len - trimBase < 31 )
{
free( sortedReads[i].id ) ;
free( sortedReads[i].read ) ;
if ( sortedReads[i].qual != NULL )
free( sortedReads[i].qual ) ;
sortedReads[i].read = NULL ;
continue ;
}
if ( geneOverlap[0].strand >= 0 )
{
// Remove the first bases
int len = sortedReads[i].len ;
for ( j = trimBase ; j <= len ; ++j )
{
sortedReads[i].read[j - trimBase] = sortedReads[i].read[j] ;
if ( sortedReads[i].qual != NULL )
sortedReads[i].qual[j - trimBase] = sortedReads[i].qual[j] ;
}
}
else
{
// Remove the last bases.
int len = sortedReads[i].len ;
sortedReads[i].read[len - trimBase] = '\0' ;
if ( sortedReads[i].qual != NULL )
sortedReads[i].qual[len - trimBase] = '\0' ;
}
for ( j = 0 ; j < 4 ; ++j )
{
if ( geneOverlap[j].seqIdx == -1 )
continue ;
geneOverlap[j].readStart -= trimBase ;
geneOverlap[j].readEnd -= trimBase ;
if ( geneOverlap[j].readStart < 0 )
geneOverlap[j].readStart = 0 ;
if ( geneOverlap[j].readEnd < 0 )
{
geneOverlap[j].readEnd = 0 ;
geneOverlap[j].seqIdx = -1 ;
}
}
sortedReads[i].len -= trimBase ;
}
// Remove redundant sequences after constant gene ; a
// and also constant gene for non IGH genes for long reads.
for ( i = 0 ; i < readCnt ; ++i )
{
int len = sortedReads[i].len ;
int gidx = 3 ;
struct _overlap *geneOverlap = sortedReads[i].geneOverlap ;
if ( sortedReads[i].read == NULL )
continue ;
for ( gidx = 2 ; gidx <= 3 ; ++gidx)
if ( geneOverlap[gidx].seqIdx != -1 )
break ;
if ( gidx > 3 )
continue ;
if ( gidx == 2 && refSet.GetSeqName(geneOverlap[gidx].seqIdx)[2] == 'H' )
{
gidx = 3 ;
if ( geneOverlap[gidx].seqIdx == -1 )
continue ;
}
bool mayTrim = false ;
if ( gidx == 3 && geneOverlap[3].seqStart < indexKmerLength && geneOverlap[3].similarity > 0.95 )
//&& geneOverlap[3].readEnd + indexKmerLength >= sortedReads[i].len
//&& refSet.GetSeqName( geneOverlap[3].seqIdx )[2] != 'H' )
mayTrim = true ;
if ( trimLevel > 1 )
mayTrim = true ;
if ( !mayTrim )
continue ;
int trimBase = len - geneOverlap[gidx].readEnd - 1 ;
if ( trimLevel > 1 && refSet.GetSeqName(geneOverlap[gidx].seqIdx)[0] == 'T') // Some bad annotation
{
if ( geneOverlap[gidx].seqStart >= 25 )
trimBase = len - ( geneOverlap[gidx].readStart - 1 ) ;
else if ( geneOverlap[gidx].similarity < 0.97 )
{
trimBase = len - ( ( geneOverlap[gidx].readStart + geneOverlap[gidx].readEnd ) / 2 ) - 1 ;
}
}
//printf("%s %d %s\n", sortedReads[i].id, gidx, sortedReads[i].read) ;
//printf("%d: %lf %d %d %s: %d %d\n", gidx, geneOverlap[gidx].similarity, mayTrim, trimBase,
// refSet.GetSeqName(geneOverlap[gidx].seqIdx), geneOverlap[gidx].readStart, geneOverlap[gidx].readEnd) ;
/*if ( firstReadLen > 200 && gidx == 3 && trimLevel < 2 && refSet.GetSeqName( geneOverlap[3].seqIdx )[2] != 'H' )
{
trimBase = ( len - geneOverlap[3].readStart ) + geneOverlap[3].seqStart ;
}*/
if ( trimBase <= 0 )
continue ;
if ( gidx == 3 && geneOverlap[2].seqIdx != -1
&& geneOverlap[2].readStart + trimBase >= sortedReads[i].len && trimLevel <= 1 )
continue ;
if ( geneOverlap[0].seqIdx != -1
&& geneOverlap[0].readStart + trimBase >= sortedReads[i].len && trimLevel <= 1 )
continue ;
if ( sortedReads[i].len - trimBase < 31 )
{
free( sortedReads[i].id ) ;
free( sortedReads[i].read ) ;
if ( sortedReads[i].qual != NULL )
free( sortedReads[i].qual ) ;
sortedReads[i].read = NULL ;
continue ;
}
if ( geneOverlap[gidx].strand < 0 )
{
// Remove the first bases
for ( j = trimBase ; j <= len ; ++j )
{
sortedReads[i].read[j - trimBase] = sortedReads[i].read[j] ;
if ( sortedReads[i].qual != NULL )
sortedReads[i].qual[j - trimBase] = sortedReads[i].qual[j] ;
}
geneOverlap[3].seqIdx = -1 ;
}
else
{
// Remove the last bases.
sortedReads[i].read[len - trimBase] = '\0' ;
if ( sortedReads[i].qual != NULL )
sortedReads[i].qual[len - trimBase] = '\0' ;
geneOverlap[3].seqIdx = -1 ;
}
for ( j = 0 ; j < 4 ; ++j )
{
if ( geneOverlap[j].seqIdx == -1 )
continue ;
if ( geneOverlap[j].readStart + trimBase >= len )
{
geneOverlap[j].readStart = len - 1 ;
geneOverlap[j].seqIdx = -1 ;
}
if ( geneOverlap[j].readEnd + trimBase >= len )
geneOverlap[j].readEnd = len - 1 ;
}
//printf("%s\n", sortedReads[i].read);
sortedReads[i].len -= trimBase ;
}
// Remove the reads that are too short if this is a long read data set.
if ( firstReadLen > 200 )
{
for ( i = 0 ; i < readCnt ; ++i )
if ( sortedReads[i].read != NULL && sortedReads[i].len < firstReadLen / 3 )
{
free( sortedReads[i].id ) ;
free( sortedReads[i].read ) ;
if ( sortedReads[i].qual != NULL )
free( sortedReads[i].qual ) ;
sortedReads[i].read = NULL ;
continue ;
}
seqSet.SetIsLongSeqSet( true ) ;
}
// Remove the sequences whose J gene is not associate with C gene.