+#include <math.h>
#include <stdio.h>
#include <unistd.h>
#include <ctype.h>
-#include "bam.h"
+#include "sam.h"
#include "faidx.h"
#include "bam_maqcns.h"
#include "khash.h"
#define BAM_PLF_CNS 0x02
#define BAM_PLF_INDEL_ONLY 0x04
#define BAM_PLF_GLF 0x08
+#define BAM_PLF_VAR_ONLY 0x10
+#define BAM_PLF_2ND 0x20
+#define BAM_PLF_RANBASE 0x40
+#define BAM_PLF_1STBASE 0x80
+#define BAM_PLF_ALLBASE 0x100
typedef struct {
bam_header_t *h;
uint32_t format;
int tid, len, last_pos;
int mask;
+ int max_depth; // for indel calling, ignore reads with the depth too high. 0 for unlimited
char *ref;
- glfFile fp; // for glf output only
+ glfFile fp_glf; // for glf output only
} pu_data_t;
char **__bam_get_lines(const char *fn, int *_n);
if (d->fai && (int)tid != d->tid) {
if (d->ref) { // then write the end mark
g3->rtype = GLF3_RTYPE_END;
- glf3_write1(d->fp, g3);
+ glf3_write1(d->fp_glf, g3);
}
- glf3_ref_write(d->fp, d->h->target_name[tid], d->h->target_len[tid]); // write reference
+ glf3_ref_write(d->fp_glf, d->h->target_name[tid], d->h->target_len[tid]); // write reference
free(d->ref);
d->ref = fai_fetch(d->fai, d->h->target_name[tid], &d->len);
d->tid = tid;
g3->rtype = GLF3_RTYPE_SUB;
g3->offset = pos - d->last_pos;
d->last_pos = pos;
- glf3_write1(d->fp, g3);
- if (proposed_indels)
- r = bam_maqindel(n, pos, d->ido, pu, d->ref, proposed_indels[0], proposed_indels+1);
- else r = bam_maqindel(n, pos, d->ido, pu, d->ref, 0, 0);
+ glf3_write1(d->fp_glf, g3);
+ if (pos < d->len) {
+ int m = (!d->max_depth || d->max_depth>n) ? n : d->max_depth;
+ if (proposed_indels)
+ r = bam_maqindel(m, pos, d->ido, pu, d->ref, proposed_indels[0], proposed_indels+1);
+ else r = bam_maqindel(m, pos, d->ido, pu, d->ref, 0, 0);
+ }
if (r) { // then write indel line
int het = 3 * n, min;
min = het;
g3->max_len = (abs(r->indel1) > abs(r->indel2)? abs(r->indel1) : abs(r->indel2)) + 1;
g3->indel_seq[0] = strdup(r->s[0]+1);
g3->indel_seq[1] = strdup(r->s[1]+1);
- glf3_write1(d->fp, g3);
+ glf3_write1(d->fp_glf, g3);
bam_maqindel_ret_destroy(r);
}
free(g);
{
pu_data_t *d = (pu_data_t*)data;
bam_maqindel_ret_t *r = 0;
- int i, j, rb, max_mapq = 0, *proposed_indels = 0;
- uint32_t x;
+ int i, j, rb, rms_mapq = -1, *proposed_indels = 0;
+ uint64_t rms_aux;
+ uint32_t cns = 0;
+ // if GLF is required, suppress -c completely
if (d->format & BAM_PLF_GLF) return glt3_func(tid, pos, n, pu, data);
- if (d->hash) { // only output a list of sites
+ // if d->hash is initialized, only output the sites in the hash table
+ if (d->hash) {
khint_t k = kh_get(64, d->hash, (uint64_t)tid<<32|pos);
if (k == kh_end(d->hash)) return 0;
proposed_indels = kh_val(d->hash, k);
}
- if (d->fai && (int)tid != d->tid) { // then update d->ref
+ // update d->ref if necessary
+ if (d->fai && (int)tid != d->tid) {
free(d->ref);
- d->ref = fai_fetch(d->fai, d->h->target_name[tid], &d->len);
+ d->ref = faidx_fetch_seq(d->fai, d->h->target_name[tid], 0, 0x7fffffff, &d->len);
d->tid = tid;
}
rb = (d->ref && (int)pos < d->len)? d->ref[pos] : 'N';
+ // when the indel-only mode is asked for, return if no reads mapped with indels
if (d->format & BAM_PLF_INDEL_ONLY) {
for (i = 0; i < n; ++i)
if (pu[i].indel != 0) break;
if (i == n) return 0;
}
+ // call the consensus and indel
+ if (d->format & BAM_PLF_CNS) { // call consensus
+ if (d->format & (BAM_PLF_RANBASE|BAM_PLF_1STBASE)) { // use a random base or the 1st base as the consensus call
+ const bam_pileup1_t *p = (d->format & BAM_PLF_1STBASE)? pu : pu + (int)(drand48() * n);
+ int q = bam1_qual(p->b)[p->qpos];
+ int mapQ = p->b->core.qual < d->c->cap_mapQ? p->b->core.qual : d->c->cap_mapQ;
+ uint32_t b = bam1_seqi(bam1_seq(p->b), p->qpos);
+ cns = b<<28 | 0xf<<24 | mapQ<<16 | q<<8;
+ } else if (d->format & BAM_PLF_ALLBASE) { // collapse all bases
+ uint64_t rmsQ = 0;
+ uint32_t b = 0;
+ for (i = 0; i < n; ++i) {
+ const bam_pileup1_t *p = pu + i;
+ int q = p->b->core.qual < d->c->cap_mapQ? p->b->core.qual : d->c->cap_mapQ;
+ b |= bam1_seqi(bam1_seq(p->b), p->qpos);
+ rmsQ += q * q;
+ }
+ rmsQ = (uint64_t)(sqrt((double)rmsQ / n) + .499);
+ cns = b<<28 | 0xf<<24 | rmsQ<<16 | 60<<8;
+ } else cns = bam_maqcns_call(n, pu, d->c);
+ }
+ if ((d->format & (BAM_PLF_CNS|BAM_PLF_INDEL_ONLY)) && d->ref && pos < d->len) { // call indels
+ int m = (!d->max_depth || d->max_depth>n) ? n : d->max_depth;
+ if (proposed_indels) // the first element gives the size of the array
+ r = bam_maqindel(m, pos, d->ido, pu, d->ref, proposed_indels[0], proposed_indels+1);
+ else r = bam_maqindel(m, pos, d->ido, pu, d->ref, 0, 0);
+ }
+ // when only variant sites are asked for, test if the site is a variant
+ if ((d->format & BAM_PLF_CNS) && (d->format & BAM_PLF_VAR_ONLY)) {
+ if (!(bam_nt16_table[rb] != 15 && cns>>28 != bam_nt16_table[rb])) { // not a SNP
+ if (!(r && (r->gt == 2 || strcmp(r->s[r->gt], "*")))) { // not an indel
+ if (r) bam_maqindel_ret_destroy(r);
+ return 0;
+ }
+ }
+ }
+ // print the first 3 columns
printf("%s\t%d\t%c\t", d->h->target_name[tid], pos + 1, rb);
- if (d->format & BAM_PLF_CNS) { // consensus
+ // print consensus information if required
+ if (d->format & BAM_PLF_CNS) {
int ref_q, rb4 = bam_nt16_table[rb];
- x = bam_maqcns_call(n, pu, d->c);
ref_q = 0;
- if (rb4 != 15 && x>>28 != 15 && x>>28 != rb4) { // a SNP
- ref_q = ((x>>24&0xf) == rb4)? x>>8&0xff : (x>>8&0xff) + (x&0xff);
+ if (rb4 != 15 && cns>>28 != 15 && cns>>28 != rb4) { // a SNP
+ ref_q = ((cns>>24&0xf) == rb4)? cns>>8&0xff : (cns>>8&0xff) + (cns&0xff);
if (ref_q > 255) ref_q = 255;
}
- printf("%c\t%d\t%d\t%d\t", bam_nt16_rev_table[x>>28], x>>8&0xff, ref_q, x>>16&0xff);
- }
- if ((d->format & (BAM_PLF_CNS|BAM_PLF_INDEL_ONLY)) && d->ref) {
- if (proposed_indels)
- r = bam_maqindel(n, pos, d->ido, pu, d->ref, proposed_indels[0], proposed_indels+1);
- else r = bam_maqindel(n, pos, d->ido, pu, d->ref, 0, 0);
+ rms_mapq = cns>>16&0xff;
+ printf("%c\t%d\t%d\t%d\t", bam_nt16_rev_table[cns>>28], cns>>8&0xff, ref_q, rms_mapq);
}
- // pileup strings
+ // print pileup sequences
printf("%d\t", n);
+ rms_aux = 0; // we need to recalculate rms_mapq when -c is not flagged on the command line
for (i = 0; i < n; ++i) {
const bam_pileup1_t *p = pu + i;
- if (max_mapq < p->b->core.qual) max_mapq = p->b->core.qual;
+ int tmp = p->b->core.qual < d->c->cap_mapQ? p->b->core.qual : d->c->cap_mapQ;
+ rms_aux += tmp * tmp;
if (p->is_head) printf("^%c", p->b->core.qual > 93? 126 : p->b->core.qual + 33);
if (!p->is_del) {
int c = bam_nt16_rev_table[bam1_seqi(bam1_seq(p->b), p->qpos)];
- if (toupper(c) == toupper(rb)) c = bam1_strand(p->b)? ',' : '.';
+ if (c == '=' || toupper(c) == toupper(rb)) c = bam1_strand(p->b)? ',' : '.';
else c = bam1_strand(p->b)? tolower(c) : toupper(c);
putchar(c);
if (p->indel > 0) {
} else putchar('*');
if (p->is_tail) putchar('$');
}
+ // finalize rms_mapq
+ rms_aux = (uint64_t)(sqrt((double)rms_aux / n) + .499);
+ if (rms_mapq < 0) rms_mapq = rms_aux;
putchar('\t');
+ // print quality
for (i = 0; i < n; ++i) {
const bam_pileup1_t *p = pu + i;
int c = bam1_qual(p->b)[p->qpos] + 33;
if (c > 126) c = 126;
putchar(c);
}
+ if (d->format & BAM_PLF_2ND) { // print 2nd calls and qualities
+ const unsigned char *q;
+ putchar('\t');
+ for (i = 0; i < n; ++i) {
+ const bam_pileup1_t *p = pu + i;
+ q = bam_aux_get(p->b, "E2");
+ putchar(q? q[p->qpos + 1] : 'N');
+ }
+ putchar('\t');
+ for (i = 0; i < n; ++i) {
+ const bam_pileup1_t *p = pu + i;
+ q = bam_aux_get(p->b, "U2");
+ putchar(q? q[p->qpos + 1] : '!');
+ }
+ }
+ // print mapping quality if -s is flagged on the command line
if (d->format & BAM_PLF_SIMPLE) {
putchar('\t');
for (i = 0; i < n; ++i) {
}
}
putchar('\n');
- if (r) { // then print indel line
+ // print the indel line if r has been calculated. This only happens if:
+ // a) -c or -i are flagged, AND b) the reference sequence is available
+ if (r) {
printf("%s\t%d\t*\t", d->h->target_name[tid], pos + 1);
if (r->gt < 2) printf("%s/%s\t", r->s[r->gt], r->s[r->gt]);
else printf("%s/%s\t", r->s[0], r->s[1]);
printf("%d\t%d\t", r->q_cns, r->q_ref);
- printf("%d\t%d\t", max_mapq, n);
+ printf("%d\t%d\t", rms_mapq, n);
printf("%s\t%s\t", r->s[0], r->s[1]);
//printf("%d\t%d\t", r->gl[0], r->gl[1]);
- printf("%d\t%d\t%d\t%d\n", r->cnt1, r->cnt2, r->cnt_ambi, r->cnt_anti);
+ printf("%d\t%d\t%d\t", r->cnt1, r->cnt2, r->cnt_anti);
+ printf("%d\t%d\n", r->cnt_ref, r->cnt_ambi);
bam_maqindel_ret_destroy(r);
}
return 0;
int bam_pileup(int argc, char *argv[])
{
- int c;
+ int c, is_SAM = 0;
char *fn_list = 0, *fn_fa = 0, *fn_pos = 0;
pu_data_t *d = (pu_data_t*)calloc(1, sizeof(pu_data_t));
+ d->max_depth = 0;
d->tid = -1; d->mask = BAM_DEF_MASK;
d->c = bam_maqcns_init();
d->ido = bam_maqindel_opt_init();
- while ((c = getopt(argc, argv, "st:f:cT:N:r:l:im:gI:G:")) >= 0) {
+ while ((c = getopt(argc, argv, "st:f:cT:N:r:l:d:im:gI:G:vM:S2aR:")) >= 0) {
switch (c) {
+ case 'a': d->c->is_soap = 1; break;
case 's': d->format |= BAM_PLF_SIMPLE; break;
case 't': fn_list = strdup(optarg); break;
case 'l': fn_pos = strdup(optarg); break;
case 'T': d->c->theta = atof(optarg); break;
case 'N': d->c->n_hap = atoi(optarg); break;
case 'r': d->c->het_rate = atof(optarg); break;
+ case 'M': d->c->cap_mapQ = atoi(optarg); break;
+ case 'd': d->max_depth = atoi(optarg); break;
case 'c': d->format |= BAM_PLF_CNS; break;
case 'i': d->format |= BAM_PLF_INDEL_ONLY; break;
- case 'm': d->mask = atoi(optarg); break;
+ case 'v': d->format |= BAM_PLF_VAR_ONLY; break;
+ case 'm': d->mask = strtol(optarg, 0, 0); break;
case 'g': d->format |= BAM_PLF_GLF; break;
+ case '2': d->format |= BAM_PLF_2ND; break;
case 'I': d->ido->q_indel = atoi(optarg); break;
case 'G': d->ido->r_indel = atof(optarg); break;
+ case 'S': is_SAM = 1; break;
+ case 'R':
+ if (strcmp(optarg, "random") == 0) d->format |= BAM_PLF_RANBASE;
+ else if (strcmp(optarg, "first") == 0) d->format |= BAM_PLF_1STBASE;
+ else if (strcmp(optarg, "all") == 0) d->format |= BAM_PLF_ALLBASE;
+ else fprintf(stderr, "[bam_pileup] unrecognized -R\n");
+ break;
default: fprintf(stderr, "Unrecognizd option '-%c'.\n", c); return 1;
}
}
+ if (fn_list) is_SAM = 1;
if (optind == argc) {
fprintf(stderr, "\n");
fprintf(stderr, "Usage: samtools pileup [options] <in.bam>|<in.sam>\n\n");
fprintf(stderr, "Option: -s simple (yet incomplete) pileup format\n");
+ fprintf(stderr, " -S the input is in SAM\n");
+ fprintf(stderr, " -a use the SOAPsnp model for SNP calling\n");
+ fprintf(stderr, " -2 output the 2nd best call and quality\n");
fprintf(stderr, " -i only show lines/consensus with indels\n");
fprintf(stderr, " -m INT filtering reads with bits in INT [%d]\n", d->mask);
- fprintf(stderr, " -t FILE list of reference sequences (assume the input is in SAM)\n");
+ fprintf(stderr, " -M INT cap mapping quality at INT [%d]\n", d->c->cap_mapQ);
+ fprintf(stderr, " -d INT limit maximum depth for indels [unlimited]\n");
+ fprintf(stderr, " -t FILE list of reference sequences (force -S)\n");
fprintf(stderr, " -l FILE list of sites at which pileup is output\n");
fprintf(stderr, " -f FILE reference sequence in the FASTA format\n\n");
fprintf(stderr, " -c output the maq consensus sequence\n");
- fprintf(stderr, " -g output in the extended GLT format (suppressing -c/-i/-s)\n");
+ fprintf(stderr, " -v print variants only (for -c)\n");
+ fprintf(stderr, " -g output in the GLFv3 format (suppressing -c/-i/-s)\n");
fprintf(stderr, " -T FLOAT theta in maq consensus calling model (for -c/-g) [%f]\n", d->c->theta);
fprintf(stderr, " -N INT number of haplotypes in the sample (for -c/-g) [%d]\n", d->c->n_hap);
fprintf(stderr, " -r FLOAT prior of a difference between two haplotypes (for -c/-g) [%f]\n", d->c->het_rate);
free(fn_list); free(fn_fa); free(d);
return 1;
}
+ if (d->format & (BAM_PLF_RANBASE|BAM_PLF_1STBASE|BAM_PLF_ALLBASE)) d->format |= BAM_PLF_CNS;
if (fn_fa) d->fai = fai_load(fn_fa);
- free(fn_fa);
- bam_maqcns_prepare(d->c);
- if (d->format & BAM_PLF_GLF) {
+ if (d->format & (BAM_PLF_CNS|BAM_PLF_GLF)) bam_maqcns_prepare(d->c); // consensus calling
+ if (d->format & BAM_PLF_GLF) { // for glf output
glf3_header_t *h;
h = glf3_header_init();
- d->fp = bgzf_fdopen(fileno(stdout), "w");
- glf3_header_write(d->fp, h);
+ d->fp_glf = bgzf_fdopen(fileno(stdout), "w");
+ glf3_header_write(d->fp_glf, h);
glf3_header_destroy(h);
}
- if (d->fai == 0)
+ if (d->fai == 0 && (d->format & (BAM_PLF_CNS|BAM_PLF_INDEL_ONLY)))
fprintf(stderr, "[bam_pileup] indels will not be called when -f is absent.\n");
- if (fn_list) { // the input is SAM
- tamFile fp;
- bam1_t *b;
- int ret;
- bam_plbuf_t *buf = bam_plbuf_init(pileup_func, d);
- bam_plbuf_set_mask(buf, d->mask);
- d->h = sam_header_read2(fn_list);
- if (fn_pos) d->hash = load_pos(fn_pos, d->h);
- fp = sam_open(argv[optind]);
- b = (bam1_t*)calloc(1, sizeof(bam1_t));
- while ((ret = sam_read1(fp, d->h, b)) >= 0)
- bam_plbuf_push(b, buf);
- bam_plbuf_push(0, buf);
- bam_plbuf_destroy(buf);
- bam_destroy1(b);
- sam_close(fp);
- } else { // the input is BAM
- bamFile fp;
- fp = (strcmp(argv[optind], "-") == 0)? bam_dopen(fileno(stdin), "r") : bam_open(argv[optind], "r");
- d->h = bam_header_read(fp);
+ if (fn_fa && is_SAM && fn_list == 0) fn_list = samfaipath(fn_fa);
+
+ {
+ samfile_t *fp;
+ fp = is_SAM? samopen(argv[optind], "r", fn_list) : samopen(argv[optind], "rb", 0);
+ if (fp == 0 || fp->header == 0) {
+ fprintf(stderr, "[bam_pileup] fail to read the header: non-exisiting file or wrong format.\n");
+ return 1;
+ }
+ d->h = fp->header;
if (fn_pos) d->hash = load_pos(fn_pos, d->h);
- bam_pileup_file(fp, d->mask, pileup_func, d);
- bam_close(fp);
+ sampileup(fp, d->mask, pileup_func, d);
+ samclose(fp); // d->h will be destroyed here
}
- if (d->format & BAM_PLF_GLF) bgzf_close(d->fp);
+
+ // free
+ if (d->format & BAM_PLF_GLF) bgzf_close(d->fp_glf);
if (fn_pos) { // free the hash table
khint_t k;
for (k = kh_begin(d->hash); k < kh_end(d->hash); ++k)
if (kh_exist(d->hash, k)) free(kh_val(d->hash, k));
kh_destroy(64, d->hash);
}
- free(fn_pos); free(fn_list);
- bam_header_destroy(d->h);
+ free(fn_pos); free(fn_list); free(fn_fa);
if (d->fai) fai_destroy(d->fai);
bam_maqcns_destroy(d->c);
free(d->ido); free(d->ref); free(d);
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