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FastqExtractor.cpp
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FastqExtractor.cpp
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#include <stdio.h>
#include <getopt.h>
#include <string.h>
#include <stdlib.h>
#include <stdarg.h>
#include <pthread.h>
#include "SeqSet.hpp"
#include "ReadFiles.hpp"
#include "BarcodeCorrector.hpp"
char usage[] = "./fastq-extractor [OPTIONS]:\n"
"Required:\n"
"\t-f STRING: fasta file containing the receptor genome sequence\n"
"\t-u STRING: path to single-end read file\n"
"\t\tor\n"
"\t-1 STRING -2 STRING: path to paired-end read files\n"
"Optional:\n"
"\t-o STRING: prefix to the output file (default: toassemble)\n"
"\t-t INT: number of threads (default: 1)\n"
"\t--barcode STRING: path to the raw barcode file (default: not used)\n"
"\t--barcodeStart INT: the start position of barcode in the barcode sequence (default: 0)\n"
"\t--barcodeEnd INT: the end position of barcode in the barcode sequence (default: length-1)\n"
"\t--barcodeRevComp: whether the barcode need to be reverse complemented (default: not used)\n"
"\t--barcodeWhitelist STRING: path to the barcode whitelist (default: not used)\n"
"\t--read1Start INT: the start position of sequence in read 1 (default: 0)\n"
"\t--read1End INT: the end position of sequence in read 1 (default: length-1)\n"
"\t--read2Start INT: the start position of sequence in read 2 (default: 0)\n"
"\t--read2End INT: the end position of sequence in read 2 (default: length-1)\n"
"\t--UMI STRING: path to the raw UMI file (default: not used)\n"
"\t--umiStart INT: the start position of barcode in the barcode sequence (default: 0)\n"
"\t--umiEnd INT: the end position of barcode in the barcode sequence (default: length-1)\n"
"\t--umiRevComp: whether the barcode need to be reverse complemented (default: not used)\n"
;
static const char *short_options = "f:u:1:2:o:t:" ;
static struct option long_options[] = {
{ "barcode", required_argument, 0, 10000},
{ "barcodeStart", required_argument, 0, 10001},
{ "barcodeEnd", required_argument, 0, 10002},
{ "barcodeRevComp", no_argument, 0, 10003},
{ "barcodeWhitelist", required_argument, 0, 10004},
{ "read1Start", required_argument, 0, 10005},
{ "read1End", required_argument, 0, 10006},
{ "read2Start", required_argument, 0, 10007},
{ "read2End", required_argument, 0, 10008},
{ "UMI", required_argument, 0, 10009},
{ "umiStart", required_argument, 0, 10010},
{ "umiEnd", required_argument, 0, 10011},
{ "umiRevComp", no_argument, 0, 10012},
{ (char *)0, 0, 0, 0}
} ;
char buffer[100001] ;
char seqBuffer[100001] ;
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'} ;
struct _threadArg
{
struct _Read *readBatch, *readBatch2, *barcodeBatch ;
int threadCnt ;
int batchSize ;
int batchUsed ;
char *buffer ;
SeqSet *refSet ;
BarcodeCorrector *barcodeCorrector ;
int barcodeStart, barcodeEnd ;
bool barcodeRevComp ;
int umiStart, umiEnd ;
bool umiRevComp ;
int tid ;
} ;
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 ) ;
}
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 ;
}
int IsGoodCandidate( char *read, char *buffer, SeqSet *refSet )
{
if ( !IsLowComplexity( read ) && refSet->HasHitInSet( read ) != 0)
return 1 ;
return 0 ;
}
void OutputSeq( FILE *fp, const char *name, char *seq, char *qual, int start, int end )
{
if ( start == 0 && end == -1 )
{
if ( qual != NULL )
fprintf( fp, "@%s\n%s\n+\n%s\n", name, seq, qual ) ;
else
fprintf( fp, ">%s\n%s\n", name, seq ) ;
}
else
{
int i ;
int s = start ;
int e = ( end == -1 ? strlen( seq ) - 1 : end ) ;
for ( i = s ; i <= e ; ++i )
buffer[i - s] = seq[i] ;
buffer[i - s] = '\0' ;
if (qual == NULL)
{
fprintf( fp, ">%s\n%s\n", name, buffer ) ;
}
else
{
fprintf( fp, "@%s\n%s\n+\n", name, buffer ) ;
for ( i = s ; i <= e ; ++i )
buffer[i - s] = qual[i] ;
buffer[i - s] = '\0' ;
fprintf( fp, "%s\n", buffer ) ;
}
}
}
// Maybe barcode read quality could be useful in future.
void OutputBarcode( FILE *fp, const char *name, char *barcode, char *qual,
int start, int end, bool revcomp, BarcodeCorrector *barcodeCorrector, SeqSet &seqSet )
{
if ( barcode && barcode[0] != '\0')
{
if ( start == 0 && end == -1 && revcomp == false )
{
int result = 0 ;
if ( barcodeCorrector != NULL )
result = barcodeCorrector->Correct( barcode, qual ) ;
if (result >= 0)
fprintf( fp, ">%s\n%s\n", name, barcode ) ;
else
fprintf(fp, ">%s\nmissing_barcode\n", name) ;
}
else
{
int i ;
int s = start ;
int e = ( end == -1 ? strlen( barcode ) - 1 : end ) ;
if ( revcomp == false )
{
for ( i = s ; i <= e ; ++i )
buffer[i - s] = barcode[i] ;
buffer[i - s] = '\0' ;
}
else
seqSet.ReverseComplement( buffer, barcode + s, e - s + 1 ) ;
int result = 0 ;
if ( barcodeCorrector != NULL )
result = barcodeCorrector->Correct( buffer, qual ) ;
if (result >= 0)
fprintf( fp, ">%s\n%s\n", name, buffer ) ;
else
fprintf( fp, ">%s\nmissing_barcode\n", name ) ;
}
}
else
fprintf( fp, ">%s\nmissing_barcode\n", name ) ;
}
void *ProcessReads_Thread( void *pArg )
{
int i, j ;
struct _threadArg &arg = *((struct _threadArg *)pArg ) ;
for ( i = 0 ; i < arg.batchSize ; ++i )
{
if ( i % arg.threadCnt != arg.tid )
continue ;
int goodCandidate = 0 ;
if ( IsGoodCandidate( arg.readBatch[i].seq, arg.buffer, arg.refSet ) )
++goodCandidate ;
if ( !goodCandidate && arg.readBatch2 && IsGoodCandidate( arg.readBatch2[i].seq, arg.buffer, arg.refSet ) )
++goodCandidate ;
if ( !goodCandidate )
arg.readBatch[i].id[0] = '\0' ;
/*else if (arg.barcodeBatch != NULL)
{
// Process the barcode.
char *buffer = arg.buffer ;
char *barcode = arg.barcodeBatch[i].seq ;
//printf("%d: %s %s. %s %s\n", goodCandidate, arg.readBatch[i].id, arg.readBatch[i].seq,
// arg.barcodeBatch[i].id, arg.barcodeBatch[i].seq) ;
if ( arg.barcodeStart == 0 && arg.barcodeEnd == -1 && arg.barcodeRevComp == false )
{
if ( arg.barcodeCorrector != NULL )
strcpy( buffer, barcode ) ;
else
continue ; // no need to process the barcode
}
else
{
int s = arg.barcodeStart ;
int e = ( arg.barcodeEnd == -1 ? strlen( barcode ) - 1 : arg.barcodeEnd ) ;
if ( arg.barcodeRevComp == false )
{
for ( j = s ; j <= e ; ++j )
buffer[j - s] = barcode[j] ;
buffer[j - s] = '\0' ;
}
else
arg.refSet->ReverseComplement( buffer, barcode + s, e - s + 1 ) ;
}
if ( arg.barcodeCorrector != NULL )
{
int result = 0 ;
result = arg.barcodeCorrector->Correct( buffer, arg.barcodeBatch[i].qual ) ;
if (result < 0)
buffer[0] = '\0' ;
}
strcpy( barcode, buffer ) ;
}*/
}
pthread_exit( NULL ) ;
}
int main( int argc, char *argv[] )
{
int i ;
if ( argc <= 1 )
{
fprintf( stderr, "%s", usage ) ;
return 0 ;
}
int c, option_index ;
option_index = 0 ;
FILE *fpRef = NULL ;
char prefix[1024] = "toassemble" ;
int kmerLength = 9 ;
SeqSet refSet( kmerLength ) ;
int threadCnt = 1 ;
ReadFiles reads ;
ReadFiles mateReads ;
ReadFiles barcodeFile ;
ReadFiles umiFile ;
BarcodeCorrector barcodeCorrector ;
bool hasMate = false ;
bool hasBarcode = false ;
bool hasBarcodeWhitelist = false ;
bool hasUmi = false ;
int barcodeStart = 0 ;
int barcodeEnd = -1 ;
int read1Start = 0 ;
int read1End = -1 ;
int read2Start = 0 ;
int read2End = -1 ;
bool barcodeRevComp = false ;
int umiStart = 0 ;
int umiEnd = -1 ;
bool umiRevComp = false ;
while ( 1 )
{
c = getopt_long( argc, argv, short_options, long_options, &option_index ) ;
if ( c == -1 )
break ;
if ( c == 'f' )
{
fpRef = fopen( optarg, "r" ) ;
refSet.InputRefFa( optarg ) ;
}
else if ( c == 'o' )
{
strcpy( prefix, optarg ) ;
}
else if ( c == '1' )
{
reads.AddReadFile( optarg, true ) ;
hasMate = true ;
}
else if ( c == '2' )
{
mateReads.AddReadFile( optarg, true ) ;
hasMate = true ;
}
else if ( c == 'u' )
{
reads.AddReadFile( optarg, false ) ;
}
else if ( c == 't' )
{
threadCnt = atoi( optarg ) ;
}
else if ( c == 10000 ) // barcode
{
hasBarcode = true ;
barcodeFile.AddReadFile( optarg, false ) ;
}
else if ( c == 10001 ) // barcodeStart
{
barcodeStart = atoi( optarg ) ;
}
else if ( c == 10002 ) // barcodeEnd
{
barcodeEnd = atoi( optarg ) ;
}
else if ( c == 10003 ) // barcodeRevComp
{
barcodeRevComp = true ;
}
else if ( c == 10004 ) // barcodeWhitelist
{
hasBarcodeWhitelist = true ;
barcodeCorrector.SetWhitelist( optarg ) ;
}
else if ( c == 10005 ) // read1Start
{
read1Start = atoi( optarg ) ;
}
else if ( c == 10006 ) // read1Start
{
read1End = atoi( optarg ) ;
}
else if ( c == 10007 ) // read1Start
{
read2Start = atoi( optarg ) ;
}
else if ( c == 10008 ) // read1Start
{
read2End = atoi( optarg ) ;
}
else if ( c == 10009 ) // umi
{
hasUmi = true ;
umiFile.AddReadFile( optarg, false ) ;
}
else if ( c == 10010 ) // umiStart
{
umiStart = atoi( optarg ) ;
}
else if ( c == 10011 ) // umiEnd
{
umiEnd = atoi( optarg ) ;
}
else if ( c == 10012 ) // umiRevComp
{
umiRevComp = true ;
}
else
{
fprintf( stderr, "Unknown parameter %s\n", optarg ) ;
return EXIT_FAILURE ;
}
}
if ( fpRef == NULL )
{
fprintf( stderr, "Need to use -f to specify the receptor genome sequence.\n" ) ;
return EXIT_FAILURE ;
}
PrintLog( "Start to extract candidate reads from read files." ) ;
int hitLenRequired = 27 ;
if ( !hasMate )
hitLenRequired = 23 ;
int len = 0 ;
for ( i = 0 ; i < 1000 ; ++i )
{
if ( !reads.Next() )
break ;
len += strlen( reads.seq ) ;
}
if ( i == 0 )
{
fprintf( stderr, "Read file is empty.\n" ) ;
return EXIT_FAILURE ;
}
if ( len / (i * 5) > hitLenRequired )
hitLenRequired = len / (i * 5) ;
refSet.SetHitLenRequired( hitLenRequired ) ;
reads.Rewind() ;
if ( hasBarcode && hasBarcodeWhitelist )
{
barcodeCorrector.CollectBackgroundDistribution(barcodeFile, barcodeStart, barcodeEnd, barcodeRevComp) ;
}
FILE *fp1 = NULL ;
FILE *fp2 = NULL ;
FILE *fpBc = NULL ;
FILE *fpUmi = NULL ;
if ( !hasMate )
{
sprintf( buffer, "%s.fq", prefix ) ;
fp1 = fopen( buffer, "w" ) ;
}
else
{
sprintf( buffer, "%s_1.fq", prefix ) ;
fp1 = fopen( buffer, "w" ) ;
sprintf( buffer, "%s_2.fq", prefix ) ;
fp2 = fopen( buffer, "w" ) ;
}
if ( hasBarcode )
{
sprintf( buffer, "%s_bc.fa", prefix ) ;
fpBc = fopen( buffer, "w" ) ;
}
if ( hasUmi )
{
sprintf( buffer, "%s_umi.fa", prefix ) ;
fpUmi = fopen( buffer, "w" ) ;
}
if ( threadCnt == 1 )
{
while ( reads.Next() )
{
if ( hasMate && !mateReads.Next() )
{
fprintf( stderr, "The two mate-pair read files have different number of reads.\n" ) ;
exit( 1 ) ;
}
if ( hasBarcode && !barcodeFile.Next() )
{
fprintf( stderr, "Read file and barcode file have different number of reads.\n" ) ;
exit( 1 ) ;
}
if ( hasUmi && !umiFile.Next() )
{
fprintf( stderr, "Read file and UMI file have different number of reads.\n" ) ;
exit( 1 ) ;
}
int goodCandidate = 0 ;
if ( IsGoodCandidate( reads.seq, seqBuffer, &refSet ) )
++goodCandidate ;
if ( !goodCandidate && hasMate && IsGoodCandidate( mateReads.seq, seqBuffer, &refSet ) )
++goodCandidate ;
if ( goodCandidate )
{
OutputSeq( fp1, reads.id, reads.seq, reads.qual, read1Start, read1End ) ;
if ( hasMate )
OutputSeq( fp2, reads.id, mateReads.seq, mateReads.qual, read2Start, read2End ) ;
if ( hasBarcode )
OutputBarcode( fpBc, reads.id, barcodeFile.seq, barcodeFile.qual,
barcodeStart, barcodeEnd, barcodeRevComp,
hasBarcodeWhitelist ? &barcodeCorrector : NULL, refSet ) ;
if ( hasUmi )
OutputBarcode( fpUmi, reads.id, umiFile.seq, umiFile.qual,
umiStart, umiEnd, umiRevComp, NULL, refSet ) ;
}
}
}
else
{
int maxBatchSize = 512 * threadCnt ;
int batchSize ;
struct _Read *readBatch = ( struct _Read *)calloc( sizeof( struct _Read ), maxBatchSize ) ;
struct _Read *readBatch2 = NULL ;
struct _Read *barcodeBatch = NULL ;
struct _Read *umiBatch = NULL ;
if ( hasMate )
readBatch2 = ( struct _Read *)calloc( sizeof( struct _Read ), maxBatchSize ) ;
if ( hasBarcode )
barcodeBatch = ( struct _Read *)calloc( sizeof( struct _Read ), maxBatchSize ) ;
if ( hasUmi )
umiBatch = ( struct _Read *)calloc( sizeof( struct _Read ), maxBatchSize ) ;
int fileInd1, fileInd2, fileIndBc, fileIndUmi ;
pthread_t *threads = (pthread_t *)malloc( sizeof( pthread_t ) * threadCnt ) ;
struct _threadArg *args = (struct _threadArg *)malloc( sizeof( struct _threadArg ) * threadCnt ) ;
pthread_attr_t attr ;
pthread_attr_init( &attr ) ;
pthread_attr_setdetachstate( &attr, PTHREAD_CREATE_JOINABLE ) ;
for ( i = 0 ; i < threadCnt ; ++i )
{
args[i].threadCnt = threadCnt ;
args[i].tid = i ;
args[i].readBatch = readBatch ;
args[i].readBatch2 = readBatch2 ;
args[i].barcodeBatch = barcodeBatch ;
args[i].barcodeStart = barcodeStart ;
args[i].barcodeEnd = barcodeEnd ;
args[i].barcodeRevComp = barcodeRevComp ;
if ( hasBarcodeWhitelist )
args[i].barcodeCorrector = &barcodeCorrector ;
else
args[i].barcodeCorrector = NULL ;
args[i].buffer = (char *)malloc( sizeof( char ) * 100001 ) ;
args[i].refSet = &refSet ;
}
while ( 1 )
{
batchSize = reads.GetBatch( readBatch, maxBatchSize, fileInd1, true, true ) ;
if ( hasMate )
{
int tmp = mateReads.GetBatch( readBatch2, maxBatchSize, fileInd2, true, true ) ;
if ( tmp != batchSize )
{
fprintf( stderr, "The two mate-pair read files have different number of reads.\n" ) ;
exit ( 1 ) ;
}
}
if ( hasBarcode )
{
int tmp = barcodeFile.GetBatch( barcodeBatch, maxBatchSize, fileIndBc, true, true ) ;
if ( tmp != batchSize )
{
fprintf( stderr, "Read file and barcode file have different number of reads.\n" ) ;
exit ( 1 ) ;
}
}
if ( hasUmi )
{
int tmp = umiFile.GetBatch( umiBatch, maxBatchSize, fileIndUmi, true, true ) ;
if ( tmp != batchSize )
{
fprintf( stderr, "Read file and umi file have different number of reads.\n" ) ;
exit ( 1 ) ;
}
}
if ( batchSize == 0 )
break ;
//printf( "batchSize=%d\n", batchSize ) ;
for ( i = 0 ; i < threadCnt ; ++i )
{
args[i].batchSize = batchSize ;
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 < batchSize ; ++i )
{
if ( readBatch[i].id[0] == '\0' )
continue ;
OutputSeq( fp1, readBatch[i].id, readBatch[i].seq, readBatch[i].qual, read1Start, read1End ) ;
if ( readBatch2 != NULL )
OutputSeq( fp2, readBatch[i].id, readBatch2[i].seq, readBatch2[i].qual, read2Start, read2End ) ;
if ( hasBarcode )
OutputBarcode( fpBc, readBatch[i].id, barcodeBatch[i].seq, barcodeBatch[i].qual,
barcodeStart, barcodeEnd, barcodeRevComp,
hasBarcodeWhitelist ? &barcodeCorrector : NULL, refSet ) ;
if ( hasUmi )
OutputBarcode( fpUmi, readBatch[i].id, umiBatch[i].seq, umiBatch[i].qual,
umiStart, umiEnd, umiRevComp, NULL, refSet ) ;
}
}
// Release memory.
for ( i = 0 ; i < maxBatchSize ; ++i )
{
int j ;
free( readBatch[i].id ) ;
free( readBatch[i].seq ) ;
if ( readBatch[i].qual )
free( readBatch[i].qual ) ;
if ( readBatch2 != NULL )
{
free( readBatch2[i].id ) ;
free( readBatch2[i].seq ) ;
if ( readBatch2[i].qual )
free( readBatch2[i].qual ) ;
}
if ( barcodeBatch != NULL )
{
free( barcodeBatch[i].id ) ;
free( barcodeBatch[i].seq ) ;
if ( barcodeBatch[i].qual )
free( barcodeBatch[i].qual ) ;
}
if ( umiBatch != NULL )
{
free( umiBatch[i].id ) ;
free( umiBatch[i].seq ) ;
if ( umiBatch[i].qual )
free( umiBatch[i].qual ) ;
}
}
reads.id = NULL ;
reads.seq = NULL ;
reads.qual = NULL ;
if ( hasMate )
{
mateReads.id = NULL ;
mateReads.seq = NULL ;
mateReads.qual = NULL ;
}
if ( hasBarcode )
{
barcodeFile.id = NULL ;
barcodeFile.seq = NULL ;
barcodeFile.qual = NULL ;
}
if ( hasUmi )
{
umiFile.id = NULL ;
umiFile.seq = NULL ;
umiFile.qual = NULL ;
}
free( threads ) ;
for ( i = 0 ; i < threadCnt ; ++i )
free( args[i].buffer ) ;
free( args ) ;
free( readBatch ) ;
if ( hasMate )
free( readBatch2 ) ;
if ( hasBarcode )
free( barcodeBatch ) ;
pthread_attr_destroy( &attr ) ;
}
fclose( fp1 ) ;
if ( hasMate )
fclose( fp2 ) ;
if ( hasBarcode )
fclose( fpBc ) ;
fclose( fpRef ) ;
PrintLog( "Finish extracting reads." ) ;
return 0 ;
}