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fastmin-sg.c
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fastmin-sg.c
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/*
###############################################################################
# Author: Alex Di Genova
# Laboratory: ERABLE/INRIA
# Copyright (c)
# year: 2019
###############################################################################
*/
#include <stdlib.h>
#include <assert.h>
#include <unistd.h>
#include <stdio.h>
#include <zlib.h>
#include <pthread.h>
#include <string.h>
#include <limits.h>
#include <errno.h>
#include "minimap.h"
#include "kseq.h"
#include <time.h>
#include "math.h"
KSEQ_INIT(gzFile, gzread)
//revcomp
//global array for to compute revcomp
char comp_tab[] = {
0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,
64, 'T', 'V', 'G', 'H', 'E', 'F', 'C', 'D', 'I', 'J', 'M', 'L', 'K', 'N', 'O',
'P', 'Q', 'Y', 'S', 'A', 'A', 'B', 'W', 'X', 'R', 'Z', 91, 92, 93, 94, 95,
64, 't', 'v', 'g', 'h', 'e', 'f', 'c', 'd', 'i', 'j', 'm', 'l', 'k', 'n', 'o',
'p', 'q', 'y', 's', 'a', 'a', 'b', 'w', 'x', 'r', 'z', 123, 124, 125, 126, 127
};
//split_str
char** str_split(char* a_str, const char a_delim)
{
char** result = 0;
size_t count = 0;
char* tmp = a_str;
char* last_comma = 0;
char delim[2];
delim[0] = a_delim;
delim[1] = 0;
/* Count how many elements will be extracted. */
while (*tmp)
{
if (a_delim == *tmp)
{
count++;
last_comma = tmp;
}
tmp++;
}
/* Add space for trailing token. */
count += last_comma < (a_str + strlen(a_str) - 1);
/* Add space for terminating null string so caller
knows where the list of returned strings ends. */
count++;
result = malloc(sizeof(char*) * count);
if (result)
{
size_t idx = 0;
char* token = strtok(a_str, delim);
while (token)
{
assert(idx < count);
*(result + idx++) = strdup(token);
token = strtok(0, delim);
}
assert(idx == count - 1);
*(result + idx) = 0;
}
return result;
}
//heng li revcomp this is for printing only
void revcomp(char* kmer, int len) {
int c0, c1;
int klen=len;
for (int i = 0; i < klen>>1; ++i) { // reverse complement sequence
c0 = comp_tab[(int)kmer[i]];
c1 = comp_tab[(int)kmer[klen - 1 - i]];
kmer[i] = c1;
kmer[klen - 1 - i] = c0;
}
if (klen & 1) // complement the remaining base
kmer[klen>>1] = comp_tab[(int)kmer[klen>>1]];
//return kmer;
}
//funtions for rename long reads
static void stk_printstr(const kstring_t *s, unsigned line_len,FILE* outlongr)
{
if (line_len != UINT_MAX && line_len != 0) {
int i, rest = s->l;
for (i = 0; i < s->l; i += line_len, rest -= line_len) {
putc('\n',outlongr);
if (rest > line_len) fwrite(s->s + i, 1, line_len, outlongr);
else fwrite(s->s + i, 1, rest, outlongr);
}
putc('\n',outlongr);
} else {
putc('\n',outlongr);
fputs(s->s,outlongr);
}
}
static inline void stk_printseq_renamed(const kseq_t *s, int line_len, const char *prefix, int64_t n, FILE* outlongr)
{
putc('>',outlongr);
if (n >= 0) {
if (prefix) fputs(prefix, outlongr);
fprintf(outlongr,"%lld", (long long)n);
} else fputs(s->name.s, outlongr);
stk_printstr(&s->seq, line_len,outlongr);
}
#define NELEMS(x) (sizeof(x) / sizeof((x)[0]))
typedef struct{
int std;
int avg;
} pe_stats;
//global variables
int inserts[50];
//total long thread_seqs
int64_t total_long_reads;
//global mutexs
pthread_mutex_t gseq;//for reading sequence
pthread_mutex_t pres;//for printing results
//struct for mapping variables
typedef struct {
int read_len;
int minq;
int kmer_size;
int minimizer_d;
int moving_w;
int qcov;
int numseq2read;//long reads per thread
int min_len_long_read;//minimal long read length
int n_threads;
int pair_rescue;//flag for pair rescue
} aligment_parameters;
//strcuture for threads
typedef struct {
//pointer to read the seq from file
kseq_t* ks;//forward read
kseq_t* kr;//reverse reads
//minimap2 index
mm_idx_t *mi;//pointer to minimap2 database
mm_mapopt_t mopt;//parameters for db construction in minimap
mm_tbuf_t *tbuf; //buffer for minimap2
//output files
int libcount;//number of synthetic libs to create
FILE** outFiles;//synthetic mate pair libraries
FILE* outlong;//renamed long read file
//mapping variables
aligment_parameters *alnp;//pointer to aligment parameters
pe_stats *pes;
/*int read_len,minq,kmer_size,moving_w,qcov;
int numseq2read;//long reads per thread
int min_len_long_read;//minimal long read length*/
} thread_data_struct;
//structure that save temporary a seq in the local thread
typedef struct{
char *name;
char *seq;
int length;
int64_t id;
} thread_seqs;
//we need ks->name.s,d,l,fo,mi->seq[fwdh->rid].name,fwdh->rs,fwdh->mapq,kmer_size,fwdh->mlen,fwdh->blen
//dinamical list that saves the hits founds
typedef struct phit{
//char* lrid;//long read id
int64_t lrid;//long read unique id
char* ctgid;//target name
int32_t plr;
int32_t orientation;//match orientation
int32_t pos;//position in target
int32_t mapq;//mapping quality
int32_t mlen, blen; // seeded exact match length; seeded alignment block length
int32_t qcov;
//kmer seq
char* k_seq;
struct phit *next; //next hit result
} phit_t;
//we create a list to handle the hits
void push_hit(phit_t **head, int64_t lrid, char* ctgid,int32_t plr,int32_t o, int32_t pos, int32_t mapq, int32_t mlen, int32_t blen, char* k_seq, int qcov) {
phit_t * new_node;
new_node = malloc(sizeof(phit_t));
//new_node->val = val;
new_node->plr=plr;
new_node->orientation=o;
new_node->pos=pos;
new_node->mapq=mapq;
new_node->mlen=mlen;
new_node->blen=blen;
new_node->qcov=qcov;
//we allocate the strings and copy them
//new_node->lrid=malloc(sizeof(char) * (strlen(lrid)));
//strcpy(new_node->lrid,lrid);
new_node->lrid=lrid;
new_node->ctgid=malloc(sizeof(char*) * (strlen(ctgid)));
strcpy(new_node->ctgid,ctgid);
new_node->k_seq=malloc(sizeof(char*) * (strlen(k_seq)));
strcpy(new_node->k_seq,k_seq);
//we update the header of the list
new_node->next = *head;
*head = new_node;
}
//thread mapping of long reads
void* maplongreads(void *arg)
{
//we get the given variables
//thread_data_struct *dargs = arg;
thread_data_struct *dargs = (thread_data_struct*)arg;
kseq_t *lks =dargs->ks;
aligment_parameters *alnp = (aligment_parameters*)dargs->alnp;
thread_seqs *localseqs=malloc(sizeof(thread_seqs) * (alnp->numseq2read));
FILE** outFiles = dargs->outFiles;
FILE* outlongr=dargs->outlong;
//we set the mapping variables
int read_len=alnp->read_len;
int minq=alnp->minq;
int kmer_size=alnp->kmer_size;
int moving_w=alnp->moving_w;
int qcov=alnp->qcov;//query coverage
mm_idx_t *mi=dargs->mi;
mm_mapopt_t mopt = dargs->mopt;
mm_tbuf_t *tbuf=dargs->tbuf;
int libcount=dargs->libcount;
//we create a list of hits for each library to store the results
phit_t** headhits = malloc(sizeof(phit_t*) * (libcount) );
for (int i = 0; i < libcount; ++i) headhits[i]=NULL;
//we read the sequence from the reads
int rseq=0;
//we create the fwd rev containers and the kmer_seq
char* fwd=malloc(sizeof(char*) * (read_len));
char* rev=malloc(sizeof(char*) * (read_len));
char* kfwdseq=malloc(sizeof(char*) * (kmer_size));
int fcov=0,rcov=0;
//I set the values to 0 in the memory
memset(fwd,0,read_len);
memset(fwd,0,read_len);
memset(fwd,0,kmer_size);
//pointer to regions
mm_reg1_t *regfwd; //fwd
mm_reg1_t *regrev; //rev
int n_regfwd=0; // number of regions to consider fwd
int n_regrev=0; // number of regions to consider rev
//pointers to hits
mm_reg1_t *fwdh;
mm_reg1_t *revh;
//local threads variables
int64_t total_pairs=0;
int64_t aling_pairs=0;
int64_t number_long_reads=0;
int getjob=1;//flag to control the execution of threads jobs
while(getjob){
pthread_mutex_lock(&gseq);
rseq=0;
//the order is always important
while(rseq < alnp->numseq2read-1 && kseq_read(lks) >=0 ){
//we copy the sequence in
if(lks->seq.l >= alnp->min_len_long_read) {
stk_printseq_renamed(lks, 60, 0, total_long_reads, outlongr);
localseqs[rseq].name = malloc(sizeof(char *) * (lks->name.l));
localseqs[rseq].id = total_long_reads;
memset(localseqs[rseq].name, 0, lks->name.l);
memcpy(localseqs[rseq].name, lks->name.s, lks->name.l);
localseqs[rseq].name[lks->name.l] = '\0';
localseqs[rseq].length = lks->seq.l;
localseqs[rseq].seq = malloc(sizeof(char *) * (lks->seq.l));
memset(localseqs[rseq].seq, 0, lks->seq.l);
memcpy(localseqs[rseq].seq, lks->seq.s, lks->seq.l);
localseqs[rseq].seq[lks->seq.l] = '\0';
//we check that the sequence leenght is equal to the sequence
assert(strlen(localseqs[rseq].seq) == lks->seq.l);
rseq++;
}
total_long_reads++;
}
pthread_mutex_unlock(&gseq);
if(rseq){
//we map the reads;
for(int i=0; i< rseq ; i++){ // each kseq_read() call reads one query sequence
number_long_reads++;
for(int ii=0; ii<libcount; ii++){
int d=inserts[ii];
int max_d = localseqs[i].length - d;
if(max_d < 0){
continue;
}
//create the read of the current library
for(int l=0; l<max_d; l+=moving_w){
//we clean the statics containers
memset(fwd,0,read_len);
memset(rev,0,read_len);
memset(kfwdseq,0,kmer_size);
int pa=l+1;
memcpy(fwd, localseqs[i].seq+pa, read_len);
fwd[read_len]='\0';
int pb=l+d-read_len;
memcpy(rev, localseqs[i].seq+pb, read_len);
rev[read_len]='\0';
revcomp(rev,read_len);
rev[read_len]='\0';
//we check that the length of fwd, rev is equal to the read_len
assert(strlen(rev)==strlen(fwd) && strlen(fwd)==read_len);
n_regfwd=0;
n_regrev=0;
total_pairs++;
//we actually map the reads using minimpa2 API
regfwd = mm_map(mi, read_len, fwd, &n_regfwd, tbuf, &mopt, 0); // get all hits for the query
if(n_regfwd > 0){
regrev = mm_map(mi, read_len, rev, &n_regrev, tbuf, &mopt, 0); // get all hits for the query
if(n_regrev > 0){
if(n_regrev == 1 && n_regfwd == 1){
//we print the aligment results
fwdh=®fwd[0];
revh=®rev[0];
//coverage
fcov=(int)(((float)fwdh->blen/(float)read_len) * 100);
rcov=(int)(((float)revh->blen/(float)read_len) * 100);
//printf("%d %d\n",fcov,rcov );
//we check that the aligment cover the query
if(fwdh->mapq >= minq && revh->mapq >= minq && fcov >=qcov && rcov>=qcov ){
//we add the reverse first to the hits pool
strncpy(kfwdseq, rev, kmer_size);
kfwdseq[kmer_size]='\0';
int fr=0;
if(revh->rev){
fr=16;
}
//printf("%s %s\n",mi->seq[revh->rid].name,mi->seq[fwdh->rid].name);
push_hit(&headhits[ii],localseqs[i].id,mi->seq[revh->rid].name,l,fr,fr > 0 ? revh->re:revh->rs,revh->mapq,revh->mlen,revh->blen,kfwdseq,rcov);
//we add the fwd
int fo=0;
if(fwdh->rev){
fo=16;
}
strncpy(kfwdseq, fwd, kmer_size);
kfwdseq[kmer_size]='\0';
push_hit(&headhits[ii],localseqs[i].id,mi->seq[fwdh->rid].name,l,fo,fo > 0 ? fwdh->re:fwdh->rs,fwdh->mapq,fwdh->mlen,fwdh->blen,kfwdseq,fcov);
aling_pairs++;
}
}
}
}
//we clean the hits
if(n_regfwd > 0)
free(regfwd);
if(n_regrev > 0)
free(regrev);
}
}
}
//we printnt the results
pthread_mutex_lock(&pres);
//we store the hits in the files
phit_t* nh = NULL;//next hit
for (int f = 0; f < libcount; ++f)
{
//this way print the reverse before the foward
//the header is not null
nh=headhits[f];
while(nh != NULL){
//fprintf(outFiles[f],"%lld_FG_%d_%d\t%d\t%s\t%d\t%d\t%dM\t*\t%d\t%d\t%s\t%d\n",
// (long long)nh->lrid,inserts[f],nh->plr,nh->orientation,nh->ctgid,nh->pos,nh->mapq,kmer_size,nh->mlen,nh->blen,nh->k_seq,nh->qcov);
//read_id,orientation,ctg_id,pos,mapq,ksize,qcov
//fprintf(outshort,"%lld\t%d\t%s\t%d\t%d\t%d\t%d\n",
// (long long)nh->lrid,nh->orientation,nh->ctgid,nh->pos,nh->mapq,kmer_size,nh->qcov);
fprintf(outFiles[f],"%lld_FG_%d_%d\t%d\t%s\t%d\t%d\t%d\t%d\n",
(long long)nh->lrid,inserts[f],nh->plr,nh->orientation,nh->ctgid,nh->pos,nh->mapq,kmer_size,nh->qcov);
headhits[f]=headhits[f]->next;
//we free the allocated hits
//free(nh->lrid);
free(nh->ctgid);
free(nh->k_seq);
free(nh);
nh=headhits[f];
}
}
//if(number_long_reads%4995==0)
fprintf(stderr,"Thread_info:Partial LONGREADS=%lld MAPPED_PAIRS=%lld TOTAL_PAIRS=%lld RL=%d MINQ=%d KS=%d MW=%d\n",
(long long)number_long_reads,(long long)aling_pairs,(long long)total_pairs,read_len,minq,kmer_size,moving_w);
//we clean the container of reads before allocate more memory in a single threads fashion
for (size_t i = 0; i < rseq; i++) {
free(localseqs[i].seq);
free(localseqs[i].name);
}
pthread_mutex_unlock(&pres);
}else{
getjob=0;
}
}
//we print some thread information
pthread_mutex_lock(&pres);
fprintf(stderr,"Final_Thread_info:Total LONGREADS=%lld MAPPED_PAIRS=%lld TOTAL_PAIRS=%lld RL=%d MINQ=%d KS=%d MW=%d\n",
(long long)number_long_reads,(long long)aling_pairs,(long long)total_pairs,read_len,minq,kmer_size,moving_w);
pthread_mutex_unlock(&pres);
//all done in this thread
return NULL;
}
int map_long_read_files(char* linserts, char* queryfile, char* prefix, aligment_parameters *alnp, mm_idx_t *mi, mm_mapopt_t mopt){
//we populate the array of libraries
char* token=strtok(linserts,",");
int libcount=0;
while(token != NULL)
{
//printf("IS=%d\n",atoi(token));
inserts[libcount]=atoi(token);
token=strtok(NULL,",");
libcount++;
if(libcount >= 50){
fprintf(stderr,"Max number of synthetic libraries is 50\n");
return 1;
}
}
printf("Query file = %s\n",queryfile);
// open query file for reading; you may use your favorite FASTA/Q parser
gzFile f = gzopen(queryfile, "r");
//printf("%d",f);
if (! f) {
fprintf (stderr, "gzopen of '%s' failed: %s.\n", queryfile,
strerror (errno));
exit (EXIT_FAILURE);
}
//assert(f);
kseq_t *ks = kseq_init(f);
FILE** outFiles = malloc(sizeof(FILE*) * (libcount));
char filename[100];//max length of file name
memset(filename, '\0', sizeof(filename));
sprintf(filename, "longreads.%s.fa",prefix);
FILE* outlong = fopen(filename,"w");
for(int i=0; i<libcount; i++){
//sprintf(filename, "lib.I%d.%s.fastmin-sg.sam", inserts[i],prefix);
sprintf(filename, "%s.I%d.fm.sam", prefix,inserts[i]);
printf("Lib : %s\n",filename);
outFiles[i] = fopen(filename,"w");
}
printf("A total of %d syntethic libraries will be created by FastMin-SG\n",libcount);
//we start the mapping of the reads
//we create the threads
if (pthread_mutex_init(&gseq, NULL) != 0){
printf("\n mutex for file reading failed\n");
return 1;
}
if (pthread_mutex_init(&pres, NULL) != 0){
printf("\n mutex for printing failed\n");
return 1;
}
//we start the mapping
pthread_t *tid=(pthread_t *)malloc(alnp->n_threads * sizeof(pthread_t ));
mm_tbuf_t **tbuf;// = mm_tbuf_init(n_threads); // thread buffer; for multi-threading, allocate one tbuf for each thread
tbuf = (mm_tbuf_t**)calloc(alnp->n_threads, sizeof(mm_tbuf_t*));
for(int i =0; i < alnp->n_threads; ++i) tbuf[i] = mm_tbuf_init();
for(int i=0; i < alnp->n_threads; i++){
thread_data_struct *data = malloc(sizeof *data);
data->ks=ks;
data->outFiles=outFiles;
data->outlong=outlong;
data->mi=mi;
data->libcount=libcount;
data->mopt=mopt;
data->tbuf=tbuf[i];
//mapping variables
data->alnp=alnp;
int err = pthread_create(&tid[i], NULL, &maplongreads, data);
if (err != 0){
printf("\ncan't create thread :[%s]", strerror(err));
}
}
//we wait for the thread to finish
for(int i=0; i< alnp->n_threads; ++i) pthread_join(tid[i], NULL);
//we destroy the thhread buffers
for(int i=0; i<alnp->n_threads; i++) mm_tbuf_destroy(tbuf[i]);
//end of threading code
kseq_destroy(ks); // close the query file
gzclose(f);//flose gzfile
//close sinthetic library files and fasta file of long reads
for(int i=0; i<libcount ; i++){
fclose(outFiles[i]);
}
// todo: be a variable file ?
fclose(outlong);
return 0;
}
int rescue_pair(mm_reg1_t *r1,mm_reg1_t *ra,int n_reg,pe_stats *ilib){
//we check return the pair satisfiying the first distance, otherwise we return a negative number indicating a null result
int d1= r1->rev ? r1->re:r1->rs;
int min=ilib->avg-(int)2.5*ilib->std;
int max=ilib->avg+(int)2.5*ilib->std;
mm_reg1_t *r2;//region that iter for looking for a candidate to rescue
for(int i=0; i<n_reg; i++){
r2=&ra[i];
int d2=r2->rev ? r2->re:r2->rs;
//same contig
if(r1->rid == r2->rid){
int d=abs(d2-d1);
//expected insert size
if(d>=min && d<=max){
//printf("RR:%d %d %d %d %d %d\n",min, d,max,r1->rid,r1->mapq,r2->mapq);
return i;
}
}
}
return -1;
}
//thread mapping of short reads
void* mapshortreads(void *arg) {
//we get the given variables
//thread_data_struct *dargs = arg;
thread_data_struct *dargs = (thread_data_struct*)arg;
kseq_t *lks =dargs->ks;//fwd
kseq_t *lkr =dargs->kr;//rev
aligment_parameters *alnp = (aligment_parameters*)dargs->alnp;
pe_stats *ilib=(pe_stats*)dargs->pes;
//fprintf(stderr,"Average insert size %d std %d [-10 outlayers,]\n",ilib->avg, ilib->std);
//fwd pool
thread_seqs *localseqs=malloc(sizeof(thread_seqs) * (alnp->numseq2read));
//rev pool
thread_seqs *localseqr=malloc(sizeof(thread_seqs) * (alnp->numseq2read));
//FILE** outFiles = dargs->outFiles;
FILE* outshort=dargs->outlong;
//we set the mapping variables
int read_len=0;//todo make it a variable
int minq=alnp->minq;
int kmer_size=alnp->kmer_size;
int moving_w=alnp->moving_w;
int qcov=alnp->qcov;//query coverage
mm_idx_t *mi=dargs->mi;
mm_mapopt_t mopt = dargs->mopt;
mm_tbuf_t *tbuf=dargs->tbuf;
//we have oonly one library with short reads
int libcount=1;
//we create a list of hits for each library to store the results
phit_t** headhits = malloc(sizeof(phit_t*) * (libcount) );
for (int i = 0; i < libcount; ++i) headhits[i]=NULL;
//we read the sequence from the reads
int rseq=0;
//we create the fwd rev containers and the kmer_seq
//char* fwd=malloc(sizeof(char*) * (read_len));
// char* rev=malloc(sizeof(char*) * (read_len));
char* kfwdseq=malloc(sizeof(char*) * (kmer_size));
int fcov=0,rcov=0;
//I set the values to 0 in the memory
//memset(fwd,0,read_len);
//memset(fwd,0,read_len);
memset(kfwdseq,0,kmer_size);
//pointer to regions
mm_reg1_t *regfwd; //fwd
mm_reg1_t *regrev; //rev
int n_regfwd=0; // number of regions to consider fwd
int n_regrev=0; // number of regions to consider rev
int64_t rescue=0;
int64_t prescue=0;
int pair_rescue=alnp->pair_rescue;//flag to set on off pair rescue;
//pointers to hits
mm_reg1_t *fwdh;
mm_reg1_t *revh;
//local threads variables
//int64_t total_pairs=0;
int64_t aling_pairs=0;
int64_t number_read_pairs=0;
//read_len=localseqr[rseq].length;
int getjob=1;//flag to control the execution of threads jobs
while(getjob){
pthread_mutex_lock(&gseq);
rseq=0;
//the order is always important
while(rseq < alnp->numseq2read-1 && kseq_read(lks) >=0 && kseq_read(lkr) >=0 ){
if(read_len == 0){
read_len=lks->seq.l;
}
//we copy the sequence in fwd
localseqs[rseq].name = malloc(sizeof(char *) * (lks->name.l));
localseqs[rseq].id = total_long_reads;
memset(localseqs[rseq].name, 0, lks->name.l);
memcpy(localseqs[rseq].name, lks->name.s, lks->name.l);
localseqs[rseq].name[lks->name.l] = '\0';
localseqs[rseq].length = lks->seq.l;
localseqs[rseq].seq = malloc(sizeof(char *) * (lks->seq.l));
memset(localseqs[rseq].seq, 0, lks->seq.l);
memcpy(localseqs[rseq].seq, lks->seq.s, lks->seq.l);
localseqs[rseq].seq[lks->seq.l] = '\0';
//we check that the sequence leenght is equal to the sequence
assert(strlen(localseqs[rseq].seq) == lks->seq.l);
//we copy the reverse seq
localseqr[rseq].name = malloc(sizeof(char *) * (lkr->name.l));
localseqr[rseq].id = total_long_reads;
memset(localseqr[rseq].name, 0, lkr->name.l);
memcpy(localseqr[rseq].name, lkr->name.s, lkr->name.l);
localseqr[rseq].name[lkr->name.l] = '\0';
localseqr[rseq].length = lkr->seq.l;
localseqr[rseq].seq = malloc(sizeof(char *) * (lkr->seq.l));
memset(localseqr[rseq].seq, 0, lkr->seq.l);
memcpy(localseqr[rseq].seq, lkr->seq.s, lkr->seq.l);
localseqr[rseq].seq[lkr->seq.l] = '\0';
//we check that the sequence leenght is equal to the sequence
assert(strlen(localseqr[rseq].seq) == lkr->seq.l);
//printf("%s %s\n",localseqs[rseq].name,localseqr[rseq].name);
rseq++;
total_long_reads++;
}
pthread_mutex_unlock(&gseq);
if(rseq){
//we map the reads;
for(int i=0; i< rseq ; i++){ // each kseq_read() call reads one query sequence
number_read_pairs++;
//we actually map the reads using minimpa2 API
n_regfwd=0,n_regrev=0;
regfwd = mm_map(mi, localseqs[i].length, localseqs[i].seq, &n_regfwd, tbuf, &mopt, 0); // get all hits for the query
if(n_regfwd > 0){
regrev = mm_map(mi, localseqr[i].length, localseqr[i].seq, &n_regrev, tbuf, &mopt, 0); // get all hits for the query
if(n_regrev > 0){
if(n_regrev == 1 && n_regfwd == 1){
//we print the aligment results
fwdh=®fwd[0];
revh=®rev[0];
//coverage
fcov=(int)(((float)fwdh->blen/(float)localseqs[i].length) * 100);
rcov=(int)(((float)revh->blen/(float)localseqr[i].length) * 100);
//printf("%d %d\n",fcov,rcov );
//we check that the aligment cover the query
if(fwdh->mapq >= minq && revh->mapq >= minq && fcov >=qcov && rcov>=qcov && fcov <=110 && rcov<=110){
//we add the reverse first to the hits pool
strncpy(kfwdseq, localseqr[i].seq, kmer_size);
kfwdseq[kmer_size]='\0';
int fr=0;
if(revh->rev){
fr=16;
}
push_hit(&headhits[0],localseqr[i].id,mi->seq[revh->rid].name,2,fr,fr > 0 ? revh->re:revh->rs,revh->mapq,revh->mlen,revh->blen,kfwdseq,rcov);
//we add the fwd
int fo=0;
if(fwdh->rev){
fo=16;
}
strncpy(kfwdseq, localseqs[i].seq, kmer_size);
kfwdseq[kmer_size]='\0';
push_hit(&headhits[0],localseqs[i].id,mi->seq[fwdh->rid].name,1,fo,fo > 0 ? fwdh->re:fwdh->rs,fwdh->mapq,fwdh->mlen,fwdh->blen,kfwdseq,fcov);
//printf("%lld %s %s %d %d %d %d %d %d %d %d %d %d %d %d\n",localseqs[i].id,mi->seq[fwdh->rid].name,mi->seq[revh->rid].name,
// fwdh->rs,fwdh->re,revh->rs,revh->re,fo,fr,fwdh->qs,fwdh->qe,revh->qs,revh->qe, fo > 0 ? fwdh->re:fwdh->rs, fr > 0 ? revh->re:revh->rs);
aling_pairs++;
}
}else{
//we can try rescue the reads
//printf("%d %d\n",n_regfwd,n_regrev);
if((n_regfwd == 1 || n_regrev == 1) && pair_rescue == 1){
prescue++; //we can rescue 10% of read pairs, focus within a contig rescue only, not between contigs
int p=-1;
//we try to rescue the pair within a contig
//we fix fwd
if(n_regfwd == 1){
p=rescue_pair(®fwd[0],regrev,n_regrev,ilib);
fwdh=®fwd[0];
if(p>=0)
revh=®rev[p];
}else{
//we fix rev
p=rescue_pair(®rev[0],regfwd,n_regfwd,ilib);
revh=®rev[0];
if(p>=0)
fwdh=®fwd[p];
}
//we rescue the pair
if(p>=0){
//printf("Rescue pair %lld %s %s %d %d %d %d %d %d %d %d %d %d %d %d\n",localseqs[i].id,mi->seq[fwdh->rid].name,mi->seq[revh->rid].name,
// fwdh->rs,fwdh->re,revh->rs,revh->re,fwdh->rev,revh->rev,fwdh->qs,fwdh->qe,revh->qs,revh->qe, fwdh->rev > 0 ? fwdh->re:fwdh->rs, revh->rev > 0 ? revh->re:revh->rs);
//coverage
fcov=(int)(((float)fwdh->blen/(float)localseqs[i].length) * 100);
rcov=(int)(((float)revh->blen/(float)localseqr[i].length) * 100);
//printf("%d %d\n",fcov,rcov );
//we check that the aligment cover the query
if(fcov >=qcov && rcov>=qcov && fcov <=110 && rcov<=110 && fwdh->mapq >= minq && revh->mapq >= minq){
//we add the reverse first to the hits pool
strncpy(kfwdseq, localseqr[i].seq, kmer_size);
kfwdseq[kmer_size]='\0';
int fr=0;
if(revh->rev){
fr=16;
}
push_hit(&headhits[0],localseqr[i].id,mi->seq[revh->rid].name,2,fr,fr > 0 ? revh->re:revh->rs,revh->mapq,revh->mlen,revh->blen,kfwdseq,rcov);
//we add the fwd
int fo=0;
if(fwdh->rev){
fo=16;
}
strncpy(kfwdseq, localseqs[i].seq, kmer_size);
kfwdseq[kmer_size]='\0';
push_hit(&headhits[0],localseqs[i].id,mi->seq[fwdh->rid].name,1,fo,fo > 0 ? fwdh->re:fwdh->rs,fwdh->mapq,fwdh->mlen,fwdh->blen,kfwdseq,fcov);
aling_pairs++;
rescue++;
}
}
}
}
}
}
//we clean the hits
if(n_regfwd > 0)
free(regfwd);
if(n_regrev > 0)
free(regrev);
}
//we printnt the results
pthread_mutex_lock(&pres);
//we store the hits in the files
phit_t* nh = NULL;//next hit
for (int f = 0; f < libcount; ++f)
{
//this way print the reverse before the foward
//the header is not null
nh=headhits[f];
while(nh != NULL){
//fprintf(outshort,"%lld/%d\t%d\t%s\t%d\t%d\t%dM\t*\t%d\t%d\t%s\t%d\n",
// nh->lrid,nh->plr,nh->orientation,nh->ctgid,nh->pos,nh->mapq,kmer_size,nh->mlen,nh->blen,nh->k_seq,nh->qcov);
//fprintf(outshort,"%lld\t%d\t%s\t%d\t%d\t%dM\t*\t%d\t%d\t%s\t%d\n",
// (long long)nh->lrid,nh->orientation,nh->ctgid,nh->pos,nh->mapq,kmer_size,nh->mlen,nh->blen,nh->k_seq,nh->qcov);
//read_id,orientation,ctg_id,pos,mapq,ksize,qcov
fprintf(outshort,"%lld\t%d\t%s\t%d\t%d\t%d\t%d\n",
(long long)nh->lrid,nh->orientation,nh->ctgid,nh->pos,nh->mapq,kmer_size,nh->qcov);
headhits[f]=headhits[f]->next;
//we free the allocated hits
//free(nh->lrid);
free(nh->ctgid);
free(nh->k_seq);
free(nh);
nh=headhits[f];
}
}
//if(number_long_reads%4995==0)
if(pair_rescue)
fprintf(stderr,"Thread_info:Partial TOTAL_PAIRS=%lld MAPPED_PAIRS=%lld RL=%d MINQ=%d KS=%d MW=%d PRESCUE=%lld RESCUE=%lld\n",
(long long)number_read_pairs,(long long)aling_pairs,read_len,minq,kmer_size,moving_w,(long long)prescue,(long long)rescue);
else
fprintf(stderr,"Thread_info:Partial TOTAL_PAIRS=%lld MAPPED_PAIRS=%lld RL=%d MINQ=%d KS=%d MW=%d\n",
(long long)number_read_pairs,(long long)aling_pairs,read_len,minq,kmer_size,moving_w);
//we clean the container of reads before allocate more memory in a single threads fashion
for (size_t i = 0; i < rseq; i++) {
free(localseqs[i].seq);
free(localseqs[i].name);
free(localseqr[i].seq);
free(localseqr[i].name);
}
pthread_mutex_unlock(&pres);
}else{
getjob=0;
}
} //endwhile
//we print some thread information
pthread_mutex_lock(&pres);
//print when pair rescue is enabled
if(pair_rescue)
fprintf(stderr,"Final_Thread_info:Total TOTAL_PAIRS=%lld MAPPED_PAIRS=%lld RL=%d MINQ=%d KS=%d MW=%d PRESCUE=%lld RESCUE=%lld\n",
(long long)number_read_pairs,(long long)aling_pairs,read_len,minq,kmer_size,moving_w,(long long)prescue,(long long)rescue);
else
fprintf(stderr,"Final_Thread_info:Total TOTAL_PAIRS=%lld MAPPED_PAIRS=%lld RL=%d MINQ=%d KS=%d MW=%d\n",
(long long)number_read_pairs,(long long)aling_pairs,read_len,minq,kmer_size,moving_w);
pthread_mutex_unlock(&pres);
//all done in this thread
return NULL;
}
int compare (const void * a, const void * b) {
return ( *(int*)a - *(int*)b );
}
pe_stats infer_insert_size(char* fwdfile, char* revfile, aligment_parameters *alnp, mm_idx_t *mi, mm_mapopt_t mopt){
// open query file for reading; you may use your favorite FASTA/Q parser
gzFile fwd = gzopen(fwdfile, "r");
//printf("%d",f);
if (! fwd) {
fprintf (stderr, "gzopen of '%s' failed: %s.\n", fwdfile,
strerror (errno));
exit (EXIT_FAILURE);
}
gzFile rev = gzopen(revfile, "r");
//printf("%d",f);
if (! rev) {
fprintf (stderr, "gzopen of '%s' failed: %s.\n", revfile,
strerror (errno));
exit (EXIT_FAILURE);
}
if (strcmp(fwdfile,revfile) == 0) {
fprintf (stderr, "fwd and rev file are the same fwd=%s rev=%s.\n", fwdfile,
revfile);
exit (EXIT_FAILURE);
}
//assert(f);
kseq_t *ks = kseq_init(fwd);
kseq_t *kr = kseq_init(rev);
//pointer to regions
mm_reg1_t *regfwd; //fwd
mm_reg1_t *regrev; //rev
//pointers to hits
mm_reg1_t *fwdh;
mm_reg1_t *revh;
int n_regfwd=0; // number of regions to consider fwd
int n_regrev=0; // number of regions to consider rev
int rseq=0;
int mpairs=50000;
mm_tbuf_t **tbuf;// = mm_tbuf_init(n_threads); // thread buffer; for multi-threading, allocate one tbuf for each thread
tbuf = (mm_tbuf_t**)calloc(1, sizeof(mm_tbuf_t*));
tbuf[0] = mm_tbuf_init();
int insobs[50000] = {0};
//we read both sequences and know we map them
while(rseq < mpairs && kseq_read(ks) >=0 && kseq_read(kr) >=0 ){
regfwd = mm_map(mi, ks->seq.l, ks->seq.s, &n_regfwd, tbuf[0], &mopt, 0); // get all hits for the query
regrev = mm_map(mi, kr->seq.l, kr->seq.s, &n_regrev, tbuf[0], &mopt, 0); // get all hits for the query
if(n_regfwd == 1 && n_regrev ==1){
fwdh=®fwd[0];
revh=®rev[0];
if(revh->rid == fwdh->rid) {
//coverage
int fcov = (int) (((float) fwdh->blen / (float) ks->seq.l) * 100);
int rcov = (int) (((float) revh->blen / (float) kr->seq.l) * 100);
if (fcov > 65 && rcov > 65 && fwdh->mapq >= 30 && revh->mapq >= 30) {
// us a good pair aligned to the same cluster
int d1= fwdh->rev ? fwdh->re:fwdh->rs;
int d2= revh->rev ? revh->re:revh->rs;
//printf("%d %d %d %d %d\n", fwdh->rid, revh->rid, d1, d2, abs(d1-d2));
if(rseq < mpairs)
insobs[rseq]=abs(d1-d2);
rseq++;// is a good hit
//we save the distance among the pairs
}
}
}
}
//we close the files and destriy the buffer;
//we destroy the thhread buffers
for(int i=0; i<1; i++) mm_tbuf_destroy(tbuf[i]);
//end of threading code
kseq_destroy(ks); // close the fwd file
kseq_destroy(kr); // close the rev file
gzclose(fwd);//flose gzfile
gzclose(rev);//flose gzfile
//we should return the std and avg of the insert size
qsort (insobs, mpairs, sizeof(int), compare);
//we remove 10% outlayers
int beg=(int)(mpairs * 0.1);
int end=mpairs-beg;
//we compute the average
uint64_t avg=0;
for (int n=beg; n<end+1; n++) {
//printf("%d %d\n", n, insobs[n]);
avg+=insobs[n];
}
int average = (int) (avg / (end - beg));
int64_t std=0;
//we compute the std of the sample
for (int n=beg; n<end+1; n++) {
std+=pow(insobs[n]-average,2);
}
int standar_dev=(int)sqrt(std/(end - beg));
fprintf(stderr,"Average insert size %d std %d [-10 outlayers]\n",average, standar_dev);
pe_stats a;
a.avg=average;
a.std=standar_dev;
return a;
}
int map_short_read_files(char* prefix, char* fwdfile, char* revfile, aligment_parameters *alnp, mm_idx_t *mi, mm_mapopt_t mopt){
printf("Forward file = %s\n",fwdfile);
printf("Reverse file = %s\n",revfile);
// open query file for reading; you may use your favorite FASTA/Q parser
gzFile fwd = gzopen(fwdfile, "r");
//printf("%d",f);
if (! fwd) {
fprintf (stderr, "gzopen of '%s' failed: %s.\n", fwdfile,
strerror (errno));
exit (EXIT_FAILURE);
}
gzFile rev = gzopen(revfile, "r");
//printf("%d",f);
if (! rev) {
fprintf (stderr, "gzopen of '%s' failed: %s.\n", revfile,
strerror (errno));
exit (EXIT_FAILURE);
}
if (strcmp(fwdfile,revfile) == 0) {
fprintf (stderr, "fwd and rev file are the same fwd=%s rev=%s.\n", fwdfile,
revfile);
exit (EXIT_FAILURE);
}
//we infer the values of the library from 50000 pairs mapped within contigs
pe_stats ilib=infer_insert_size(fwdfile,revfile,alnp,mi,mopt);
//fprintf(stderr,"Average insert size %d std %d [-10 outlayers,]\n",ilib.avg, ilib.std);
//exit(0);
//assert(f);
kseq_t *ks = kseq_init(fwd);
kseq_t *kr = kseq_init(rev);
char filename[100];//max length of file name
memset(filename, '\0', sizeof(filename));
//sprintf(filename, "libshort.%s.fastmin-sg.sam",prefix);
sprintf(filename, "%s.fm.sam",prefix);
FILE* outlong = fopen(filename,"w");
//we start the mapping of the reads
//we create the threads
if (pthread_mutex_init(&gseq, NULL) != 0){
printf("\n mutex for file reading failed\n");
return 1;
}
if (pthread_mutex_init(&pres, NULL) != 0){
printf("\n mutex for printing failed\n");
return 1;
}
//we start the mapping
pthread_t *tid=(pthread_t *)malloc(alnp->n_threads * sizeof(pthread_t ));
mm_tbuf_t **tbuf;// = mm_tbuf_init(n_threads); // thread buffer; for multi-threading, allocate one tbuf for each thread
tbuf = (mm_tbuf_t**)calloc(alnp->n_threads, sizeof(mm_tbuf_t*));
for(int i =0; i < alnp->n_threads; ++i) tbuf[i] = mm_tbuf_init();
for(int i=0; i < alnp->n_threads; i++){