https://github.com/jstjohn/SeqPrep
Tip revision: 8fed36b33412e26f0ab2322694b7e7a0533619dd authored by John St. John on 29 June 2011, 23:08:15 UTC
Fixed an important bug with forced overlap after adapter alignment. This will result in more reads passing. Also modifying defaults slightly
Fixed an important bug with forced overlap after adapter alignment. This will result in more reads passing. Also modifying defaults slightly
Tip revision: 8fed36b
SeqPrep.c
#include <stdio.h>
#include <stdlib.h>
#include <string.h>
#include <ctype.h>
#include <limits.h>
#include <stdbool.h>
#include <unistd.h>
#include <math.h>
#include <time.h>
#include "utils.h"
#include "stdaln.h"
#define DEF_OL2MERGE_ADAPTER (10)
#define DEF_OL2MERGE_READS (20)
#define DEF_QCUT (10)
#define DEF_MIN_MATCH_ADAPTER (0.6)
#define DEF_MIN_MATCH_READS (0.75)
#define DEF_MIN_READ_LEN (30)
#define DEF_MAX_MISMATCH_ADAPTER (0.2)
#define DEF_MAX_MISMATCH_READS (0.02)
#define DEF_MAX_PRETTY_PRINT (10000)
#define DEF_ADAPTER_SCORE_THRES (28)
#define DEF_READ_SCORE_THRES (-500)
//two revolutions of 4 positions = 5000 reads
#define SPIN_INTERVAL (1250)
//following primer sequences are from:
//http://intron.ccam.uchc.edu/groups/tgcore/wiki/013c0/Solexa_Library_Primer_Sequences.html
//and I validated both with grep, the first gets hits to the forward file only and the second
//gets hits to the reverse file only.
//#define DEF_FORWARD_PRIMER ("AGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCG")
//#define DEF_REVERSE_PRIMER ("AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT")
#define DEF_FORWARD_PRIMER ("AGATCGGAAGAGCGGTTCAG")
#define DEF_REVERSE_PRIMER ("AGATCGGAAGAGCGTCGTGT")
void help ( char *prog_name ) {
fprintf(stderr, "\n\nUsage:\n%s [Required Args] [Options]\n",prog_name );
fprintf(stderr, "NOTE 1: The output is always gziped compressed.\n");
fprintf(stderr, "NOTE 2: If the quality strings in the output contain characters less than ascii 33 on an ascii table (they look like lines from a binary file), try running again with or without the -6 option.\n");
fprintf(stderr, "Required Arguments:\n" );
fprintf(stderr, "\t-f <first read input fastq filename>\n" );
fprintf(stderr, "\t-r <second read input fastq filename>\n" );
fprintf(stderr, "\t-1 <first read output fastq filename>\n" );
fprintf(stderr, "\t-2 <second read output fastq filename>\n" );
fprintf(stderr, "General Arguments (Optional):\n" );
fprintf(stderr, "\t-h Display this help message and exit (also works with no args) \n" );
fprintf(stderr, "\t-6 Input sequence is in phred+64 rather than phred+33 format, the output will still be phred+33 \n" );
fprintf(stderr, "\t-q <Quality score cutoff for mismatches to be counted in overlap; default = %d>\n", DEF_QCUT );
fprintf(stderr, "\t-L <Minimum length of a trimmed or merged read to print it; default = %d>\n", DEF_MIN_READ_LEN );
fprintf(stderr, "Arguments for Adapter/Primer Trimming (Optional):\n" );
fprintf(stderr, "\t-A <forward read primer/adapter sequence to trim as it would appear at the end of a read (recommend about 20bp of this)\n\t\t (should validate by grepping a file); default = %s>\n", DEF_FORWARD_PRIMER );
fprintf(stderr, "\t-B <reverse read primer/adapter sequence to trim as it would appear at the end of a read (recommend about 20bp of this)\n\t\t (should validate by grepping a file); default = %s>\n", DEF_REVERSE_PRIMER );
fprintf(stderr, "\t-O <minimum overall base pair overlap with adapter sequence to trim; default = %d>\n", DEF_OL2MERGE_ADAPTER );
fprintf(stderr, "\t-M <maximum fraction of good quality mismatching bases for primer/adapter overlap; default = %f>\n", DEF_MAX_MISMATCH_ADAPTER );
fprintf(stderr, "\t-N <minimum fraction of matching bases for primer/adapter overlap; default = %f>\n", DEF_MIN_MATCH_ADAPTER );
fprintf(stderr, "\t-b <adapter alignment band-width; default = %d>\n", aln_param_nt2nt.band_width );
fprintf(stderr, "\t-Q <adapter alignment gap-open; default = %d>\n", aln_param_nt2nt.gap_open );
fprintf(stderr, "\t-t <adapter alignment gap-extension; default = %d>\n", aln_param_nt2nt.gap_ext );
fprintf(stderr, "\t-e <adapter alignment gap-end; default = %d>\n", aln_param_nt2nt.gap_end );
fprintf(stderr, "\t-Z <adapter local alignment cutoff score ((2*num_matches) - (gap_open*num_gaps) - (gap_close*num_gaps) - (gap_ext*gap_len)) ; default = %d>\n", DEF_ADAPTER_SCORE_THRES );
fprintf(stderr, "Optional Arguments for Merging:\n" );
fprintf(stderr, "\t-g <print overhang when adapters are present and stripped (use this if reads are different length)>\n");
fprintf(stderr, "\t-s <perform merging and output the merged reads to this file>\n" );
fprintf(stderr, "\t-E <write pretty alignments to this file for visual Examination>\n" );
fprintf(stderr, "\t-x <max number of pretty alignments to write (if -E provided); default = %d>\n", DEF_MAX_PRETTY_PRINT );
fprintf(stderr, "\t-o <minimum overall base pair overlap to merge two reads; default = %d>\n", DEF_OL2MERGE_READS );
fprintf(stderr, "\t-m <maximum fraction of good quality mismatching bases to overlap reads; default = %f>\n", DEF_MAX_MISMATCH_READS );
fprintf(stderr, "\t-n <minimum fraction of matching bases to overlap reads; default = %f>\n", DEF_MIN_MATCH_READS );
fprintf(stderr, "\n");
exit( 1 );
}
static unsigned short spcount = 0;
/**
* Have a nice spinner to give you a false sense of hope
*/
inline void update_spinner(unsigned long long num_reads){
if(num_reads == 0){
fprintf(stderr,"Processing reads... |");
fflush(stderr);
}else if (num_reads % SPIN_INTERVAL == 0){
switch(spcount % 4){
case 0:
fprintf(stderr,"\b/");
fflush(stderr);
break;
case 1:
fprintf(stderr,"\b-");
fflush(stderr);
break;
case 2:
fprintf(stderr,"\b\\");
fflush(stderr);
break;
default:
fprintf(stderr,"\b|");
fflush(stderr);
break;
}
spcount++;
}
}
int main( int argc, char* argv[] ) {
unsigned long long num_pairs;
unsigned long long num_merged;
unsigned long long num_adapter;
unsigned long long num_discarded;
unsigned long long num_too_ambiguous_to_merge;
unsigned long long max_pretty_print = DEF_MAX_PRETTY_PRINT;
unsigned long long num_pretty_print = 0;
int adapter_thresh = DEF_ADAPTER_SCORE_THRES;
int read_thresh = DEF_READ_SCORE_THRES;
clock_t start, end;
//init to 0
num_pairs = num_merged = num_adapter = num_discarded = num_too_ambiguous_to_merge = 0;
extern char* optarg;
bool p64 = false;
char forward_fn[MAX_FN_LEN];
char reverse_fn[MAX_FN_LEN];
char forward_out_fn[MAX_FN_LEN];
char reverse_out_fn[MAX_FN_LEN];
char merged_out_fn[MAX_FN_LEN];
bool do_read_merging = false;
bool print_overhang = false;
char forward_primer[MAX_SEQ_LEN+1];
strcpy(forward_primer, DEF_FORWARD_PRIMER); //set default
char forward_primer_dummy_qual[MAX_SEQ_LEN+1];
char reverse_primer[MAX_SEQ_LEN+1];
strcpy(reverse_primer, DEF_REVERSE_PRIMER); //set default
char reverse_primer_dummy_qual[MAX_SEQ_LEN+1];
int i;
for(i=0;i<MAX_SEQ_LEN+1;i++){
forward_primer_dummy_qual[i] = 'N';//phred score of 45
reverse_primer_dummy_qual[i] = 'N';
}
int ich;
int min_ol_adapter = DEF_OL2MERGE_ADAPTER;
int min_ol_reads = DEF_OL2MERGE_READS;
unsigned short int min_read_len =DEF_MIN_READ_LEN;
float min_match_adapter_frac = DEF_MIN_MATCH_ADAPTER;
float min_match_reads_frac = DEF_MIN_MATCH_READS;
float max_mismatch_adapter_frac = DEF_MAX_MISMATCH_ADAPTER;
float max_mismatch_reads_frac = DEF_MAX_MISMATCH_READS;
unsigned short max_mismatch_adapter[MAX_SEQ_LEN+1];
unsigned short max_mismatch_reads[MAX_SEQ_LEN+1];
unsigned short min_match_adapter[MAX_SEQ_LEN+1];
unsigned short min_match_reads[MAX_SEQ_LEN+1];
char qcut = (char)DEF_QCUT+33;
bool pretty_print = false;
char pretty_print_fn[MAX_FN_LEN+1];
SQP sqp = SQP_init();
/* No args - help! */
if ( argc == 1 ) {
help(argv[0]);
}
int req_args = 0;
while( (ich=getopt( argc, argv, "f:r:1:2:q:A:s:B:O:E:x:M:N:L:o:m:b:Q:t:e:Z:n:6gh" )) != -1 ) {
switch( ich ) {
//REQUIRED ARGUMENTS
case 'f' :
req_args ++;
strcpy( forward_fn, optarg );
break;
case 'r' :
req_args ++;
strcpy( reverse_fn, optarg );
break;
case '1' :
req_args ++;
strcpy(forward_out_fn, optarg);
break;
case '2' :
req_args ++;
strcpy(reverse_out_fn, optarg);
break;
//OPTIONAL GENERAL ARGUMENTS
case 'h' :
help(argv[0]);
break;
case '6' :
p64 = true;
break;
case 'q' :
qcut = atoi(optarg)+33;
break;
case 'L' :
min_read_len = atoi(optarg);
break;
//OPTIONAL ADAPTER/PRIMER TRIMMING ARGUMENTS
case 'A':
strcpy(forward_primer, optarg);
break;
case 'B':
strcpy(reverse_primer, optarg);
break;
case 'O':
min_ol_adapter = atoi(optarg);
break;
case 'M':
max_mismatch_adapter_frac = atof(optarg);
break;
case 'N':
min_match_adapter_frac = atof(optarg);
break;
case 'b':
aln_param_nt2nt.band_width = atoi(optarg);
break;
case 'Q':
aln_param_nt2nt.gap_open = atoi(optarg);
break;
case 't':
aln_param_nt2nt.gap_ext = atoi(optarg);
break;
case 'e':
aln_param_nt2nt.gap_end = atoi(optarg);
break;
case 'Z':
adapter_thresh = atoi(optarg);
break;
//OPTIONAL MERGING ARGUMENTS
case 'g' :
print_overhang = true;
break;
case 's' :
do_read_merging = true;
strcpy( merged_out_fn, optarg );
break;
case 'o':
min_ol_reads = atoi(optarg);
break;
case 'm':
max_mismatch_reads_frac = atof(optarg);
break;
case 'n':
min_match_reads_frac = atof(optarg);
break;
case 'E':
pretty_print = true;
strcpy(pretty_print_fn,optarg);
break;
case 'x':
max_pretty_print = atol(optarg);
break;
default :
help(argv[0]);
}
}
if(req_args < 4){
fprintf(stderr, "Missing a required argument!\n");
help(argv[0]);
}
start = clock();
//allocate alignment memory
// int min_match = 8;
// int ngaps = 1;
// int maxglen = 3;
// AlnParam aln_param_adapter = { 5, 13, 19, aln_sm_read, 16, 75 };
//
//Calculate table matching overlap length to min matches and max mismatches
for(i=0;i<MAX_SEQ_LEN+1;i++){
max_mismatch_reads[i] = floor(((float)i)*max_mismatch_reads_frac);
max_mismatch_adapter[i] = floor(((float)i)*max_mismatch_adapter_frac);
min_match_reads[i] = ceil(((float)i)*min_match_reads_frac);
min_match_adapter[i] = ceil(((float)i)*min_match_adapter_frac);
}
//get length of forward and reverse primers
int forward_primer_len = strlen(forward_primer);
int reverse_primer_len = strlen(reverse_primer);
gzFile ffq = fileOpen(forward_fn, "r");
gzFile ffqw = fileOpen(forward_out_fn,"w");
gzFile rfq = fileOpen(reverse_fn, "r");
gzFile rfqw = fileOpen(reverse_out_fn,"w");
gzFile mfqw = NULL;
gzFile ppaw = NULL;
if(do_read_merging)
mfqw = fileOpen(merged_out_fn,"w");
if(pretty_print)
ppaw = fileOpen(pretty_print_fn,"w");
while(next_fastqs( ffq, rfq, sqp, p64 )){ //returns false when done
update_spinner(num_pairs++);
// //see if we can trim based on read-read overlap alone
// if(adapter_trim(sqp, min_ol_adapter,
// forward_primer, forward_primer_dummy_qual,
// forward_primer_len,
// reverse_primer, reverse_primer_dummy_qual,
// reverse_primer_len,
// min_match_adapter,
// max_mismatch_adapter,
// min_match_reads,
// max_mismatch_reads,
// qcut)){
// //we trimmed the adapter!
// num_adapter++;
// if((sqp->flen < min_read_len) || (sqp->rlen < min_read_len)){
// num_discarded++;
// continue;
// }
// if(!do_read_merging){ //just print
// write_fastq(ffqw, sqp->fid, sqp->fseq, sqp->fqual);
// write_fastq(rfqw, sqp->rid, sqp->rseq, sqp->rqual);
//
// }else{ //force merge
// num_merged++;
// adapter_merge(sqp,false);
// write_fastq(mfqw,sqp->fid,sqp->merged_seq,sqp->merged_qual);
// if(pretty_print && num_pretty_print < max_pretty_print){
// num_pretty_print++;
// pretty_print_alignment(ppaw,sqp,qcut,true); //true b/c merged input sorted
// }
// }
// continue;
// }
AlnAln *faaln, *raaln, *fraln;
faaln = aln_stdaln_aux(sqp->fseq, forward_primer, &aln_param_nt2nt,
ALN_TYPE_LOCAL, adapter_thresh , sqp->flen, forward_primer_len);
raaln = aln_stdaln_aux(sqp->rseq, reverse_primer, &aln_param_nt2nt,
ALN_TYPE_LOCAL, adapter_thresh, sqp->rlen, reverse_primer_len);
//check for direct adapter match.
if(adapter_trim(sqp, min_ol_adapter,
forward_primer, forward_primer_dummy_qual,
forward_primer_len,
reverse_primer, reverse_primer_dummy_qual,
reverse_primer_len,
min_match_adapter,
max_mismatch_adapter,
min_match_reads,
max_mismatch_reads,
qcut) ||
faaln->score >= adapter_thresh ||
raaln->score >= adapter_thresh){
num_adapter++; //adapter present
//print it if user wants
if(pretty_print && num_pretty_print < max_pretty_print){
//void pretty_print_alignment_stdaln(gzFile out, SQP sqp, AlnAln *aln, bool first_adapter, bool second_adapter)
if(faaln->score >= adapter_thresh){
num_pretty_print++;
pretty_print_alignment_stdaln(ppaw,sqp,faaln,true,false,false);
}
if(raaln->score >= adapter_thresh){
num_pretty_print++;
pretty_print_alignment_stdaln(ppaw,sqp,raaln,false,true,false);
}
}
//do stuff to it
//assume full length adapter and squish it down to the read with no gaps
int rpos,fpos;
rpos = fpos = -MAX_SEQ_LEN;
if(faaln->score >= adapter_thresh){
fpos = faaln->start1 - faaln->start2;
}
if(raaln->score >= adapter_thresh){
rpos = raaln->start1 - raaln->start2;
}
if(rpos == -MAX_SEQ_LEN && fpos == -MAX_SEQ_LEN){
rpos = fpos = min(sqp->rlen,sqp->flen);
}else if (rpos == -MAX_SEQ_LEN){
rpos = max(0,min(fpos,sqp->rlen));
}else if (fpos == -MAX_SEQ_LEN){
fpos = max(0,min(rpos,sqp->flen));
}else{
rpos = max(0,min(rpos,min(fpos,sqp->rlen)));
fpos = max(0,min(fpos,min(rpos,sqp->flen)));
}
sqp->rlen = rpos;
sqp->flen = fpos;
if(fpos < min_read_len || rpos < min_read_len){
num_discarded++;
goto CLEAN_ADAPTERS;
}else{ //trim the adapters
sqp->fseq[fpos] = '\0';
sqp->fqual[fpos] = '\0';
sqp->rseq[rpos] = '\0';
sqp->rqual[rpos] = '\0';
sqp->flen = fpos;
sqp->rlen = rpos;
strcpy(sqp->rc_rseq,sqp->rseq); //move RC reads into reg place and reverse them
strcpy(sqp->rc_rqual,sqp->rqual);
rev_qual(sqp->rc_rqual);
revcom_seq(sqp->rc_rseq);
}
//do a nice global alignment between two reads, and print consensus
//from now on we need to clean everythin
fraln = aln_stdaln_aux(sqp->fseq, sqp->rc_rseq, &aln_param_rd2rd,
ALN_TYPE_GLOBAL, 1, sqp->flen, sqp->rlen);
//do we want read merging?
//calculate the minimum score we are willing to accept to merge the reads
read_thresh = sqp->flen + sqp->rlen - ((float)sqp->flen/8.0 * (float)aln_param_rd2rd.gap_ext) - ((float)sqp->rlen/8.0 * (float)aln_param_rd2rd.gap_ext) - aln_param_rd2rd.gap_open*2 - aln_param_rd2rd.gap_end*2;
if(do_read_merging && fraln->score > read_thresh){
//write the merged sequence
fill_merged_sequence(sqp, fraln, true);
if(pretty_print && num_pretty_print < max_pretty_print){
num_pretty_print++;
pretty_print_alignment_stdaln(ppaw,sqp,fraln,false,false,true);
}
if(strlen(sqp->merged_seq) >= min_read_len && strlen(sqp->merged_qual) >= min_read_len)
write_fastq(mfqw,sqp->fid,sqp->merged_seq,sqp->merged_qual);
else{
num_discarded++;
}
}else if(fraln->score > read_thresh){
// we know that the adapters are present, trimmed, and the resulting
// read lengths are both long enough to print.
// We also know that we aren't doing merging.
// Now we just need to print.
if(strlen(sqp->fseq) >= min_read_len &&
strlen(sqp->fqual) >= min_read_len &&
strlen(sqp->rseq) >= min_read_len &&
strlen(sqp->rqual) >= min_read_len){
write_fastq(ffqw, sqp->fid, sqp->fseq, sqp->fqual);
write_fastq(rfqw, sqp->rid, sqp->rseq, sqp->rqual);
}else{
num_discarded++;
}
}else{ //there was a bad looking read-read alignment, so lets not risk it and junk it
num_discarded++;
}
goto CLEAN_ALL;
}else{
//no adapters present
//check for strong read overlap to assist trimming ends of adapters from end of read
if(do_read_merging){
if(read_merge(sqp, min_ol_reads, min_match_reads, max_mismatch_reads, qcut)){
//print merged output
if(strlen(sqp->merged_seq) >= min_read_len &&
strlen(sqp->merged_qual) >= min_read_len){
num_merged++;
write_fastq(mfqw,sqp->fid,sqp->merged_seq,sqp->merged_qual);
if(pretty_print && num_pretty_print < max_pretty_print){
num_pretty_print++;
pretty_print_alignment(ppaw,sqp,qcut,false); //false b/c merged input in fixed order
}
}else{
num_discarded++;
}
}else{
//no significant overlap so just write them
if(strlen(sqp->fseq) >= min_read_len &&
strlen(sqp->fqual) >= min_read_len &&
strlen(sqp->rseq) >= min_read_len &&
strlen(sqp->rqual) >= min_read_len){
write_fastq(ffqw, sqp->fid, sqp->fseq, sqp->fqual);
write_fastq(rfqw, sqp->rid, sqp->rseq, sqp->rqual);
}else{
num_discarded++;
}
}
//done
goto CLEAN_ADAPTERS;
}else{ //just write reads to output fastqs
if(strlen(sqp->fseq) >= min_read_len &&
strlen(sqp->fqual) >= min_read_len &&
strlen(sqp->rseq) >= min_read_len &&
strlen(sqp->rqual) >= min_read_len){
write_fastq(ffqw, sqp->fid, sqp->fseq, sqp->fqual);
write_fastq(rfqw, sqp->rid, sqp->rseq, sqp->rqual);
}else{
num_discarded++;
}
goto CLEAN_ADAPTERS;
}
}
/**
* Section for heirarchial cleanup
*
* In every case we will at least have to free up the alignment between the adapter and two reads.
* however in some cases there will be an additional alignment between the two reads. We can do
* good cleanup in this case with gotos
*/
CLEAN_ALL:
aln_free_AlnAln(fraln);
CLEAN_ADAPTERS:
aln_free_AlnAln(faaln);
aln_free_AlnAln(raaln);
//End the loop over reads
}
end = clock();
double cpu_time_used = ((double) (end - start)) / CLOCKS_PER_SEC;
fprintf(stderr,"\nPairs Processed:\t%lld\n",num_pairs);
fprintf(stderr,"Pairs Merged:\t%lld\n",num_merged);
fprintf(stderr,"Pairs With Adapters:\t%lld\n",num_adapter);
fprintf(stderr,"Pairs Discarded:\t%lld\n",num_discarded);
fprintf(stderr,"CPU Time Used (Minutes):\t%lf\n",cpu_time_used/60.0);
SQP_destroy(sqp);
gzclose(ffq);
gzclose(ffqw);
gzclose(rfq);
gzclose(rfqw);
if(mfqw != NULL)
gzclose(mfqw);
if(ppaw != NULL)
gzclose(ppaw);
return 0;
}