#include <errno.h>
#include "sam.h"
#include "faidx.h"
-#include "bam_maqcns.h"
-#include "khash.h"
-#include "glf.h"
#include "kstring.h"
-typedef int *indel_list_t;
-KHASH_MAP_INIT_INT64(64, indel_list_t)
-
-#define BAM_PLF_SIMPLE 0x01
-#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
-#define BAM_PLF_READPOS 0x200
-#define BAM_PLF_NOBAQ 0x400
-
-typedef struct {
- bam_header_t *h;
- bam_maqcns_t *c;
- bam_maqindel_opt_t *ido;
- faidx_t *fai;
- khash_t(64) *hash;
- uint32_t format;
- int tid, len, last_pos;
- int mask;
- int capQ_thres, min_baseQ;
- int max_depth; // for indel calling, ignore reads with the depth too high. 0 for unlimited
- char *ref;
- glfFile fp_glf; // for glf output only
-} pu_data_t;
-
-char **__bam_get_lines(const char *fn, int *_n);
-void bam_init_header_hash(bam_header_t *header);
-int32_t bam_get_tid(const bam_header_t *header, const char *seq_name);
-
-static khash_t(64) *load_pos(const char *fn, bam_header_t *h)
-{
- char **list;
- int i, j, n, *fields, max_fields;
- khash_t(64) *hash;
- bam_init_header_hash(h);
- list = __bam_get_lines(fn, &n);
- hash = kh_init(64);
- max_fields = 0; fields = 0;
- for (i = 0; i < n; ++i) {
- char *str = list[i];
- int chr, n_fields, ret;
- khint_t k;
- uint64_t x;
- n_fields = ksplit_core(str, 0, &max_fields, &fields);
- if (n_fields < 2) continue;
- chr = bam_get_tid(h, str + fields[0]);
- if (chr < 0) {
- fprintf(stderr, "[load_pos] unknown reference sequence name: %s\n", str + fields[0]);
- continue;
- }
- x = (uint64_t)chr << 32 | (atoi(str + fields[1]) - 1);
- k = kh_put(64, hash, x, &ret);
- if (ret == 0) {
- fprintf(stderr, "[load_pos] position %s:%s has been loaded.\n", str+fields[0], str+fields[1]);
- continue;
- }
- kh_val(hash, k) = 0;
- if (n_fields > 2) {
- // count
- for (j = 2; j < n_fields; ++j) {
- char *s = str + fields[j];
- if ((*s != '+' && *s != '-') || !isdigit(s[1])) break;
- }
- if (j > 2) { // update kh_val()
- int *q, y, z;
- q = kh_val(hash, k) = (int*)calloc(j - 1, sizeof(int));
- q[0] = j - 2; z = j; y = 1;
- for (j = 2; j < z; ++j)
- q[y++] = atoi(str + fields[j]);
- }
- }
- free(str);
- }
- free(list); free(fields);
- return hash;
-}
-
static inline int printw(int c, FILE *fp)
{
char buf[16];
return 0;
}
-// an analogy to pileup_func() below
-static int glt3_func(uint32_t tid, uint32_t pos, int n, const bam_pileup1_t *pu, void *data)
-{
- pu_data_t *d = (pu_data_t*)data;
- bam_maqindel_ret_t *r = 0;
- int rb, *proposed_indels = 0;
- glf1_t *g;
- glf3_t *g3;
-
- if (d->fai == 0) {
- fprintf(stderr, "[glt3_func] reference sequence is required for generating GLT. Abort!\n");
- exit(1);
- }
- if (d->hash) { // only output a list of sites
- 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);
- }
- g3 = glf3_init1();
- if (d->fai && (int)tid != d->tid) {
- if (d->ref) { // then write the end mark
- g3->rtype = GLF3_RTYPE_END;
- glf3_write1(d->fp_glf, g3);
- }
- 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;
- d->last_pos = 0;
- }
- rb = (d->ref && (int)pos < d->len)? d->ref[pos] : 'N';
- g = bam_maqcns_glfgen(n, pu, bam_nt16_table[rb], d->c);
- memcpy(g3, g, sizeof(glf1_t));
- g3->rtype = GLF3_RTYPE_SUB;
- g3->offset = pos - d->last_pos;
- d->last_pos = pos;
- 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;
- if (min > r->gl[0]) min = r->gl[0];
- if (min > r->gl[1]) min = r->gl[1];
- g3->ref_base = 0;
- g3->rtype = GLF3_RTYPE_INDEL;
- memset(g3->lk, 0, 10);
- g3->lk[0] = r->gl[0] - min < 255? r->gl[0] - min : 255;
- g3->lk[1] = r->gl[1] - min < 255? r->gl[1] - min : 255;
- g3->lk[2] = het - min < 255? het - min : 255;
- g3->offset = 0;
- g3->indel_len[0] = r->indel1;
- g3->indel_len[1] = r->indel2;
- g3->min_lk = min < 255? min : 255;
- 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_glf, g3);
- bam_maqindel_ret_destroy(r);
- }
- free(g);
- glf3_destroy1(g3);
- return 0;
-}
-
static inline void pileup_seq(const bam_pileup1_t *p, int pos, int ref_len, const char *ref)
{
int j;
if (p->is_tail) putchar('$');
}
-static int pileup_func(uint32_t tid, uint32_t pos, int n, const bam_pileup1_t *pu, void *data)
-{
- pu_data_t *d = (pu_data_t*)data;
- bam_maqindel_ret_t *r = 0;
- int i, 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 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);
- }
- // update d->ref if necessary
- if (d->fai && (int)tid != d->tid) {
- free(d->ref);
- 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 {
- glf1_t *g = bam_maqcns_glfgen(n, pu, bam_nt16_table[rb], d->c);
- cns = g->depth == 0? (0xfu<<28 | 0xf<<24) : glf2cns(g, (int)(d->c->q_r + .499));
- free(g);
- }
- }
- 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 != 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
- fputs(d->h->target_name[tid], stdout); putchar('\t');
- printw(pos+1, stdout); putchar('\t'); putchar(rb); putchar('\t');
- // print consensus information if required
- if (d->format & BAM_PLF_CNS) {
- putchar(bam_nt16_rev_table[cns>>28]); putchar('\t');
- printw(cns>>8&0xff, stdout); putchar('\t');
- printw(cns&0xff, stdout); putchar('\t');
- printw(cns>>16&0xff, stdout); putchar('\t');
- }
- // print pileup sequences
- printw(n, stdout); putchar('\t');
- for (i = 0; i < n; ++i)
- pileup_seq(pu + i, pos, d->len, d->ref);
- // finalize rms_mapq
- if (d->format & BAM_PLF_CNS) {
- for (i = rms_aux = 0; i < n; ++i) {
- const bam_pileup1_t *p = pu + i;
- int tmp = p->b->core.qual < d->c->cap_mapQ? p->b->core.qual : d->c->cap_mapQ;
- rms_aux += tmp * tmp;
- }
- 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) {
- int c = pu[i].b->core.qual + 33;
- if (c > 126) c = 126;
- putchar(c);
- }
- }
- // print read position
- if (d->format & BAM_PLF_READPOS) {
- putchar('\t');
- for (i = 0; i < n; ++i) {
- int x = pu[i].qpos;
- int l = pu[i].b->core.l_qseq;
- printw(x < l/2? x+1 : -((l-1)-x+1), stdout); putchar(',');
- }
- }
- putchar('\n');
- // 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", 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", 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, 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 = 1024; d->tid = -1; d->mask = BAM_DEF_MASK; d->min_baseQ = 13;
- d->c = bam_maqcns_init();
- d->c->errmod = BAM_ERRMOD_MAQ2; // change the default model
- d->ido = bam_maqindel_opt_init();
- while ((c = getopt(argc, argv, "st:f:cT:N:r:l:d:im:gI:G:vM:S2aR:PAQ:C:B")) >= 0) {
- switch (c) {
- case 'Q': d->c->min_baseQ = atoi(optarg); break;
- case 'C': d->capQ_thres = atoi(optarg); break;
- case 'B': d->format |= BAM_PLF_NOBAQ; break;
- case 'a': d->c->errmod = BAM_ERRMOD_SOAP; break;
- case 'A': d->c->errmod = BAM_ERRMOD_MAQ; break;
- case 's': d->format |= BAM_PLF_SIMPLE; break;
- case 't': fn_list = strdup(optarg); break;
- case 'l': fn_pos = strdup(optarg); break;
- case 'f': fn_fa = 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); d->ido->r_snp = d->c->het_rate; 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 '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 'P': d->format |= BAM_PLF_READPOS; 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 (d->c->errmod != BAM_ERRMOD_MAQ2) d->c->theta += 0.02;
- if (d->c->theta > 1.0) d->c->theta = 1.0;
- 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, " -B disable BAQ computation\n");
- fprintf(stderr, " -A use the original MAQ model for SNP calling (DEPRECATED)\n");
- fprintf(stderr, " -2 output the 2nd best call and quality\n");
- fprintf(stderr, " -i only show lines/consensus with indels\n");
- fprintf(stderr, " -Q INT min base quality (possibly capped by BAQ) [%d]\n", d->c->min_baseQ);
- fprintf(stderr, " -C INT coefficient for adjusting mapQ of poor mappings [%d]\n", d->capQ_thres);
- fprintf(stderr, " -m INT filtering reads with bits in INT [0x%x]\n", d->mask);
- fprintf(stderr, " -M INT cap mapping quality at INT [%d]\n", d->c->cap_mapQ);
- fprintf(stderr, " -d INT limit maximum depth for indels [%d]\n", d->max_depth);
- 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 compute the consensus sequence\n");
- fprintf(stderr, " -v print variants only (for -c)\n");
- fprintf(stderr, " -g output in the GLFv3 format (DEPRECATED)\n");
- fprintf(stderr, " -T FLOAT theta in maq consensus calling model (for -c) [%.4g]\n", d->c->theta);
- fprintf(stderr, " -N INT number of haplotypes in the sample (for -c) [%d]\n", d->c->n_hap);
- fprintf(stderr, " -r FLOAT prior of a difference between two haplotypes (for -c) [%.4g]\n", d->c->het_rate);
- fprintf(stderr, " -G FLOAT prior of an indel between two haplotypes (for -c) [%.4g]\n", d->ido->r_indel);
- fprintf(stderr, " -I INT phred prob. of an indel in sequencing/prep. (for -c) [%d]\n", d->ido->q_indel);
- fprintf(stderr, "\n");
- 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);
- 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_glf = bgzf_fdopen(fileno(stdout), "w");
- glf3_header_write(d->fp_glf, h);
- glf3_header_destroy(h);
- }
- 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_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);
- { // run pileup
- extern int bam_prob_realn(bam1_t *b, const char *ref);
- extern int bam_cap_mapQ(bam1_t *b, char *ref, int thres);
- bam1_t *b;
- int ret, tid, pos, n_plp;
- bam_plp_t iter;
- const bam_pileup1_t *plp;
- b = bam_init1();
- iter = bam_plp_init(0, 0);
- bam_plp_set_mask(iter, d->mask);
- while ((ret = samread(fp, b)) >= 0) {
- int skip = 0;
- if ((int)b->core.tid < 0) break;
- // update d->ref if necessary
- if (d->fai && (int)b->core.tid != d->tid) {
- free(d->ref);
- d->ref = faidx_fetch_seq(d->fai, d->h->target_name[b->core.tid], 0, 0x7fffffff, &d->len);
- d->tid = b->core.tid;
- }
- if (d->ref && (d->format&BAM_PLF_CNS) && !(d->format&BAM_PLF_NOBAQ)) bam_prob_realn(b, d->ref);
- if (d->ref && (d->format&BAM_PLF_CNS) && d->capQ_thres > 10) {
- int q = bam_cap_mapQ(b, d->ref, d->capQ_thres);
- if (q < 0) skip = 1;
- else if (b->core.qual > q) b->core.qual = q;
- } else if (b->core.flag&BAM_FUNMAP) skip = 1;
- else if ((d->format&BAM_PLF_CNS) && (b->core.flag&1) && !(b->core.flag&2)) skip = 1;
- if (skip) continue;
- bam_plp_push(iter, b);
- while ((plp = bam_plp_next(iter, &tid, &pos, &n_plp)) != 0)
- pileup_func(tid, pos, n_plp, plp, d);
- }
- bam_plp_push(iter, 0);
- while ((plp = bam_plp_next(iter, &tid, &pos, &n_plp)) != 0)
- pileup_func(tid, pos, n_plp, plp, d);
- bam_plp_destroy(iter);
- bam_destroy1(b);
- }
- samclose(fp); // d->h will be destroyed here
- }
-
- // 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); free(fn_fa);
- if (d->fai) fai_destroy(d->fai);
- bam_maqcns_destroy(d->c);
- free(d->ido); free(d->ref); free(d);
- return 0;
-}
-
-/***********
- * mpileup *
- ***********/
-
#include <assert.h>
#include "bam2bcf.h"
#include "sample.h"
#define MPLP_FMT_DP 0x100
#define MPLP_FMT_SP 0x200
#define MPLP_NO_INDEL 0x400
+#define MPLP_EXT_BAQ 0x800
+#define MPLP_ILLUMINA13 0x1000
+#define MPLP_IGNORE_RG 0x2000
+#define MPLP_PRINT_POS 0x4000
+#define MPLP_PRINT_MAPQ 0x8000
+
+void *bed_read(const char *fn);
+void bed_destroy(void *_h);
+int bed_overlap(const void *_h, const char *chr, int beg, int end);
typedef struct {
- int max_mq, min_mq, flag, min_baseQ, capQ_thres, max_depth;
- int openQ, extQ, tandemQ;
- char *reg, *fn_pos, *pl_list;
+ int max_mq, min_mq, flag, min_baseQ, capQ_thres, max_depth, max_indel_depth;
+ int openQ, extQ, tandemQ, min_support; // for indels
+ double min_frac; // for indels
+ char *reg, *pl_list;
faidx_t *fai;
- kh_64_t *hash;
+ void *bed, *rghash;
} mplp_conf_t;
typedef struct {
bamFile fp;
bam_iter_t iter;
- int min_mq, flag, ref_id, capQ_thres;
+ bam_header_t *h;
+ int ref_id;
char *ref;
+ const mplp_conf_t *conf;
} mplp_aux_t;
typedef struct {
int has_ref;
ret = ma->iter? bam_iter_read(ma->fp, ma->iter, b) : bam_read1(ma->fp, b);
if (ret < 0) break;
+ if (b->core.tid < 0 || (b->core.flag&BAM_FUNMAP)) { // exclude unmapped reads
+ skip = 1;
+ continue;
+ }
+ if (ma->conf->bed) { // test overlap
+ skip = !bed_overlap(ma->conf->bed, ma->h->target_name[b->core.tid], b->core.pos, bam_calend(&b->core, bam1_cigar(b)));
+ if (skip) continue;
+ }
+ if (ma->conf->rghash) { // exclude read groups
+ uint8_t *rg = bam_aux_get(b, "RG");
+ skip = (rg && bcf_str2id(ma->conf->rghash, (const char*)(rg+1)) >= 0);
+ if (skip) continue;
+ }
+ if (ma->conf->flag & MPLP_ILLUMINA13) {
+ int i;
+ uint8_t *qual = bam1_qual(b);
+ for (i = 0; i < b->core.l_qseq; ++i)
+ qual[i] = qual[i] > 31? qual[i] - 31 : 0;
+ }
has_ref = (ma->ref && ma->ref_id == b->core.tid)? 1 : 0;
skip = 0;
- if (has_ref && (ma->flag&MPLP_REALN)) bam_prob_realn_core(b, ma->ref, 1);
- if (has_ref && ma->capQ_thres > 10) {
- int q = bam_cap_mapQ(b, ma->ref, ma->capQ_thres);
+ if (has_ref && (ma->conf->flag&MPLP_REALN)) bam_prob_realn_core(b, ma->ref, (ma->conf->flag & MPLP_EXT_BAQ)? 3 : 1);
+ if (has_ref && ma->conf->capQ_thres > 10) {
+ int q = bam_cap_mapQ(b, ma->ref, ma->conf->capQ_thres);
if (q < 0) skip = 1;
else if (b->core.qual > q) b->core.qual = q;
- } else if (b->core.flag&BAM_FUNMAP) skip = 1;
- else if (b->core.qual < ma->min_mq) skip = 1;
- else if ((ma->flag&MPLP_NO_ORPHAN) && (b->core.flag&1) && !(b->core.flag&2)) skip = 1;
+ }
+ else if (b->core.qual < ma->conf->min_mq) skip = 1;
+ else if ((ma->conf->flag&MPLP_NO_ORPHAN) && (b->core.flag&1) && !(b->core.flag&2)) skip = 1;
} while (skip);
return ret;
}
static void group_smpl(mplp_pileup_t *m, bam_sample_t *sm, kstring_t *buf,
- int n, char *const*fn, int *n_plp, const bam_pileup1_t **plp)
+ int n, char *const*fn, int *n_plp, const bam_pileup1_t **plp, int ignore_rg)
{
int i, j;
memset(m->n_plp, 0, m->n * sizeof(int));
const bam_pileup1_t *p = plp[i] + j;
uint8_t *q;
int id = -1;
- q = bam_aux_get(p->b, "RG");
+ q = ignore_rg? 0 : bam_aux_get(p->b, "RG");
if (q) id = bam_smpl_rg2smid(sm, fn[i], (char*)q+1, buf);
if (id < 0) id = bam_smpl_rg2smid(sm, fn[i], 0, buf);
- assert(id >= 0 && id < m->n);
+ if (id < 0 || id >= m->n) {
+ assert(q); // otherwise a bug
+ fprintf(stderr, "[%s] Read group %s used in file %s but absent from the header or an alignment missing read group.\n", __func__, (char*)q+1, fn[i]);
+ exit(1);
+ }
if (m->n_plp[id] == m->m_plp[id]) {
m->m_plp[id] = m->m_plp[id]? m->m_plp[id]<<1 : 8;
m->plp[id] = realloc(m->plp[id], sizeof(bam_pileup1_t) * m->m_plp[id]);
extern void *bcf_call_add_rg(void *rghash, const char *hdtext, const char *list);
extern void bcf_call_del_rghash(void *rghash);
mplp_aux_t **data;
- int i, tid, pos, *n_plp, beg0 = 0, end0 = 1u<<29, ref_len, ref_tid, max_depth;
+ int i, tid, pos, *n_plp, tid0 = -1, beg0 = 0, end0 = 1u<<29, ref_len, ref_tid = -1, max_depth, max_indel_depth;
const bam_pileup1_t **plp;
bam_mplp_t iter;
bam_header_t *h = 0;
char *ref;
- khash_t(64) *hash = 0;
void *rghash = 0;
bcf_callaux_t *bca = 0;
for (i = 0; i < n; ++i) {
bam_header_t *h_tmp;
data[i] = calloc(1, sizeof(mplp_aux_t));
- data[i]->min_mq = conf->min_mq;
- data[i]->flag = conf->flag;
- data[i]->capQ_thres = conf->capQ_thres;
data[i]->fp = strcmp(fn[i], "-") == 0? bam_dopen(fileno(stdin), "r") : bam_open(fn[i], "r");
+ data[i]->conf = conf;
h_tmp = bam_header_read(data[i]->fp);
- bam_smpl_add(sm, fn[i], h_tmp->text);
+ data[i]->h = i? h : h_tmp; // for i==0, "h" has not been set yet
+ bam_smpl_add(sm, fn[i], (conf->flag&MPLP_IGNORE_RG)? 0 : h_tmp->text);
rghash = bcf_call_add_rg(rghash, h_tmp->text, conf->pl_list);
if (conf->reg) {
int beg, end;
fprintf(stderr, "[%s] malformatted region or wrong seqname for %d-th input.\n", __func__, i+1);
exit(1);
}
- if (i == 0) beg0 = beg, end0 = end;
+ if (i == 0) tid0 = tid, beg0 = beg, end0 = end;
data[i]->iter = bam_iter_query(idx, tid, beg, end);
bam_index_destroy(idx);
}
gplp.plp = calloc(sm->n, sizeof(void*));
fprintf(stderr, "[%s] %d samples in %d input files\n", __func__, sm->n, n);
- if (conf->fn_pos) hash = load_pos(conf->fn_pos, h);
// write the VCF header
if (conf->flag & MPLP_GLF) {
kstring_t s;
bh->l_smpl = s.l;
bh->sname = malloc(s.l);
memcpy(bh->sname, s.s, s.l);
- bh->l_txt = 0;
+ bh->txt = malloc(strlen(BAM_VERSION) + 64);
+ bh->l_txt = 1 + sprintf(bh->txt, "##samtoolsVersion=%s\n", BAM_VERSION);
free(s.s);
bcf_hdr_sync(bh);
bcf_hdr_write(bp, bh);
bcr = calloc(sm->n, sizeof(bcf_callret1_t));
bca->rghash = rghash;
bca->openQ = conf->openQ, bca->extQ = conf->extQ, bca->tandemQ = conf->tandemQ;
- }
- ref_tid = -1; ref = 0;
+ bca->min_frac = conf->min_frac;
+ bca->min_support = conf->min_support;
+ }
+ if (tid0 >= 0 && conf->fai) { // region is set
+ ref = faidx_fetch_seq(conf->fai, h->target_name[tid0], 0, 0x7fffffff, &ref_len);
+ ref_tid = tid0;
+ for (i = 0; i < n; ++i) data[i]->ref = ref, data[i]->ref_id = tid0;
+ } else ref_tid = -1, ref = 0;
iter = bam_mplp_init(n, mplp_func, (void**)data);
max_depth = conf->max_depth;
if (max_depth * sm->n > 1<<20)
fprintf(stderr, "(%s) Max depth is above 1M. Potential memory hog!\n", __func__);
if (max_depth * sm->n < 8000) {
max_depth = 8000 / sm->n;
- fprintf(stderr, "<%s> Set max per-sample depth to %d\n", __func__, max_depth);
+ fprintf(stderr, "<%s> Set max per-file depth to %d\n", __func__, max_depth);
}
+ max_indel_depth = conf->max_indel_depth * sm->n;
bam_mplp_set_maxcnt(iter, max_depth);
while (bam_mplp_auto(iter, &tid, &pos, n_plp, plp) > 0) {
if (conf->reg && (pos < beg0 || pos >= end0)) continue; // out of the region requested
- if (hash) {
- khint_t k;
- k = kh_get(64, hash, (uint64_t)tid<<32 | pos);
- if (k == kh_end(hash)) continue;
- }
+ if (conf->bed && tid >= 0 && !bed_overlap(conf->bed, h->target_name[tid], pos, pos+1)) continue;
if (tid != ref_tid) {
free(ref); ref = 0;
- if (conf->fai) ref = fai_fetch(conf->fai, h->target_name[tid], &ref_len);
+ if (conf->fai) ref = faidx_fetch_seq(conf->fai, h->target_name[tid], 0, 0x7fffffff, &ref_len);
for (i = 0; i < n; ++i) data[i]->ref = ref, data[i]->ref_id = tid;
ref_tid = tid;
}
if (conf->flag & MPLP_GLF) {
- int _ref0, ref16;
+ int total_depth, _ref0, ref16;
bcf1_t *b = calloc(1, sizeof(bcf1_t));
- group_smpl(&gplp, sm, &buf, n, fn, n_plp, plp);
+ for (i = total_depth = 0; i < n; ++i) total_depth += n_plp[i];
+ group_smpl(&gplp, sm, &buf, n, fn, n_plp, plp, conf->flag & MPLP_IGNORE_RG);
_ref0 = (ref && pos < ref_len)? ref[pos] : 'N';
ref16 = bam_nt16_table[_ref0];
for (i = 0; i < gplp.n; ++i)
bcf_write(bp, bh, b);
bcf_destroy(b);
// call indels
- if (!(conf->flag&MPLP_NO_INDEL) && bcf_call_gap_prep(gplp.n, gplp.n_plp, gplp.plp, pos, bca, ref, rghash) >= 0) {
+ if (!(conf->flag&MPLP_NO_INDEL) && total_depth < max_indel_depth && bcf_call_gap_prep(gplp.n, gplp.n_plp, gplp.plp, pos, bca, ref, rghash) >= 0) {
for (i = 0; i < gplp.n; ++i)
bcf_call_glfgen(gplp.n_plp[i], gplp.plp[i], -1, bca, bcr + i);
if (bcf_call_combine(gplp.n, bcr, -1, &bc) >= 0) {
for (i = 0; i < n; ++i) {
int j;
printf("\t%d\t", n_plp[i]);
- if (n_plp[i] == 0) printf("*\t*");
- else {
+ if (n_plp[i] == 0) {
+ printf("*\t*"); // FIXME: printf() is very slow...
+ if (conf->flag & MPLP_PRINT_POS) printf("\t*");
+ } else {
for (j = 0; j < n_plp[i]; ++j)
pileup_seq(plp[i] + j, pos, ref_len, ref);
putchar('\t');
if (c > 126) c = 126;
putchar(c);
}
+ if (conf->flag & MPLP_PRINT_MAPQ) {
+ putchar('\t');
+ for (j = 0; j < n_plp[i]; ++j) {
+ int c = plp[i][j].b->core.qual + 33;
+ if (c > 126) c = 126;
+ putchar(c);
+ }
+ }
+ if (conf->flag & MPLP_PRINT_POS) {
+ putchar('\t');
+ for (j = 0; j < n_plp[i]; ++j) {
+ if (j > 0) putchar(',');
+ printf("%d", plp[i][j].qpos + 1); // FIXME: printf() is very slow...
+ }
+ }
}
}
putchar('\n');
for (i = 0; i < gplp.n; ++i) free(gplp.plp[i]);
free(gplp.plp); free(gplp.n_plp); free(gplp.m_plp);
bcf_call_del_rghash(rghash);
- if (hash) { // free the hash table
- khint_t k;
- for (k = kh_begin(hash); k < kh_end(hash); ++k)
- if (kh_exist(hash, k)) free(kh_val(hash, k));
- kh_destroy(64, hash);
- }
bcf_hdr_destroy(bh); bcf_call_destroy(bca); free(bc.PL); free(bcr);
bam_mplp_destroy(iter);
bam_header_destroy(h);
}
#define MAX_PATH_LEN 1024
-int read_file_list(const char *file_list,int *n,char **argv[])
+static int read_file_list(const char *file_list,int *n,char **argv[])
{
char buf[MAX_PATH_LEN];
int len, nfiles;
int c;
const char *file_list = NULL;
char **fn = NULL;
- int nfiles = 0;
+ int nfiles = 0, use_orphan = 0;
mplp_conf_t mplp;
memset(&mplp, 0, sizeof(mplp_conf_t));
+ #define MPLP_PRINT_POS 0x4000
mplp.max_mq = 60;
mplp.min_baseQ = 13;
mplp.capQ_thres = 0;
- mplp.max_depth = 250;
+ mplp.max_depth = 250; mplp.max_indel_depth = 250;
mplp.openQ = 40; mplp.extQ = 20; mplp.tandemQ = 100;
+ mplp.min_frac = 0.002; mplp.min_support = 1;
mplp.flag = MPLP_NO_ORPHAN | MPLP_REALN;
- while ((c = getopt(argc, argv, "gf:r:l:M:q:Q:uaORC:BDSd:b:P:o:e:h:I")) >= 0) {
+ while ((c = getopt(argc, argv, "Agf:r:l:M:q:Q:uaRC:BDSd:L:b:P:o:e:h:Im:F:EG:6Os")) >= 0) {
switch (c) {
case 'f':
mplp.fai = fai_load(optarg);
break;
case 'd': mplp.max_depth = atoi(optarg); break;
case 'r': mplp.reg = strdup(optarg); break;
- case 'l': mplp.fn_pos = strdup(optarg); break;
+ case 'l': mplp.bed = bed_read(optarg); break;
case 'P': mplp.pl_list = strdup(optarg); break;
case 'g': mplp.flag |= MPLP_GLF; break;
case 'u': mplp.flag |= MPLP_NO_COMP | MPLP_GLF; break;
case 'a': mplp.flag |= MPLP_NO_ORPHAN | MPLP_REALN; break;
- case 'B': mplp.flag &= ~MPLP_REALN & ~MPLP_NO_ORPHAN; break;
- case 'O': mplp.flag |= MPLP_NO_ORPHAN; break;
- case 'R': mplp.flag |= MPLP_REALN; break;
+ case 'B': mplp.flag &= ~MPLP_REALN; break;
case 'D': mplp.flag |= MPLP_FMT_DP; break;
case 'S': mplp.flag |= MPLP_FMT_SP; break;
case 'I': mplp.flag |= MPLP_NO_INDEL; break;
+ case 'E': mplp.flag |= MPLP_EXT_BAQ; break;
+ case '6': mplp.flag |= MPLP_ILLUMINA13; break;
+ case 'R': mplp.flag |= MPLP_IGNORE_RG; break;
+ case 's': mplp.flag |= MPLP_PRINT_MAPQ; break;
+ case 'O': mplp.flag |= MPLP_PRINT_POS; break;
case 'C': mplp.capQ_thres = atoi(optarg); break;
case 'M': mplp.max_mq = atoi(optarg); break;
case 'q': mplp.min_mq = atoi(optarg); break;
case 'o': mplp.openQ = atoi(optarg); break;
case 'e': mplp.extQ = atoi(optarg); break;
case 'h': mplp.tandemQ = atoi(optarg); break;
+ case 'A': use_orphan = 1; break;
+ case 'F': mplp.min_frac = atof(optarg); break;
+ case 'm': mplp.min_support = atoi(optarg); break;
+ case 'L': mplp.max_indel_depth = atoi(optarg); break;
+ case 'G': {
+ FILE *fp_rg;
+ char buf[1024];
+ mplp.rghash = bcf_str2id_init();
+ if ((fp_rg = fopen(optarg, "r")) == 0)
+ fprintf(stderr, "(%s) Fail to open file %s. Continue anyway.\n", __func__, optarg);
+ while (!feof(fp_rg) && fscanf(fp_rg, "%s", buf) > 0) // this is not a good style, but forgive me...
+ bcf_str2id_add(mplp.rghash, strdup(buf));
+ fclose(fp_rg);
+ }
+ break;
}
}
+ if (use_orphan) mplp.flag &= ~MPLP_NO_ORPHAN;
if (argc == 1) {
fprintf(stderr, "\n");
- fprintf(stderr, "Usage: samtools mpileup [options] in1.bam [in2.bam [...]]\n\n");
- fprintf(stderr, "Options: -f FILE reference sequence file [null]\n");
- fprintf(stderr, " -r STR region in which pileup is generated [null]\n");
- fprintf(stderr, " -l FILE list of positions (format: chr pos) [null]\n");
- fprintf(stderr, " -b FILE list of input BAM files [null]\n");
- fprintf(stderr, " -M INT cap mapping quality at INT [%d]\n", mplp.max_mq);
- fprintf(stderr, " -Q INT min base quality [%d]\n", mplp.min_baseQ);
- fprintf(stderr, " -q INT filter out alignment with MQ smaller than INT [%d]\n", mplp.min_mq);
- fprintf(stderr, " -d INT max per-sample depth [%d]\n", mplp.max_depth);
- fprintf(stderr, " -P STR comma separated list of platforms for indels [all]\n");
- fprintf(stderr, " -o INT Phred-scaled gap open sequencing error probability [%d]\n", mplp.openQ);
- fprintf(stderr, " -e INT Phred-scaled gap extension seq error probability [%d]\n", mplp.extQ);
- fprintf(stderr, " -h INT coefficient for homopolyer errors [%d]\n", mplp.tandemQ);
- fprintf(stderr, " -g generate BCF output\n");
- fprintf(stderr, " -u do not compress BCF output\n");
- fprintf(stderr, " -B disable BAQ computation\n");
- fprintf(stderr, " -D output per-sample DP\n");
- fprintf(stderr, " -S output per-sample SP (strand bias P-value, slow)\n");
- fprintf(stderr, " -I do not perform indel calling\n");
+ fprintf(stderr, "Usage: samtools mpileup [options] in1.bam [in2.bam [...]]\n\n");
+ fprintf(stderr, "Input options:\n\n");
+ fprintf(stderr, " -6 assume the quality is in the Illumina-1.3+ encoding\n");
+ fprintf(stderr, " -A count anomalous read pairs\n");
+ fprintf(stderr, " -B disable BAQ computation\n");
+ fprintf(stderr, " -b FILE list of input BAM files [null]\n");
+ fprintf(stderr, " -C INT parameter for adjusting mapQ; 0 to disable [0]\n");
+ fprintf(stderr, " -d INT max per-BAM depth to avoid excessive memory usage [%d]\n", mplp.max_depth);
+ fprintf(stderr, " -E extended BAQ for higher sensitivity but lower specificity\n");
+ fprintf(stderr, " -f FILE faidx indexed reference sequence file [null]\n");
+ fprintf(stderr, " -G FILE exclude read groups listed in FILE [null]\n");
+ fprintf(stderr, " -l FILE list of positions (chr pos) or regions (BED) [null]\n");
+ fprintf(stderr, " -M INT cap mapping quality at INT [%d]\n", mplp.max_mq);
+ fprintf(stderr, " -r STR region in which pileup is generated [null]\n");
+ fprintf(stderr, " -R ignore RG tags\n");
+ fprintf(stderr, " -q INT skip alignments with mapQ smaller than INT [%d]\n", mplp.min_mq);
+ fprintf(stderr, " -Q INT skip bases with baseQ/BAQ smaller than INT [%d]\n", mplp.min_baseQ);
+ fprintf(stderr, "\nOutput options:\n\n");
+ fprintf(stderr, " -D output per-sample DP in BCF (require -g/-u)\n");
+ fprintf(stderr, " -g generate BCF output (genotype likelihoods)\n");
+ fprintf(stderr, " -O output base positions on reads (disabled by -g/-u)\n");
+ fprintf(stderr, " -s output mapping quality (disabled by -g/-u)\n");
+ fprintf(stderr, " -S output per-sample strand bias P-value in BCF (require -g/-u)\n");
+ fprintf(stderr, " -u generate uncompress BCF output\n");
+ fprintf(stderr, "\nSNP/INDEL genotype likelihoods options (effective with `-g' or `-u'):\n\n");
+ fprintf(stderr, " -e INT Phred-scaled gap extension seq error probability [%d]\n", mplp.extQ);
+ fprintf(stderr, " -F FLOAT minimum fraction of gapped reads for candidates [%g]\n", mplp.min_frac);
+ fprintf(stderr, " -h INT coefficient for homopolymer errors [%d]\n", mplp.tandemQ);
+ fprintf(stderr, " -I do not perform indel calling\n");
+ fprintf(stderr, " -L INT max per-sample depth for INDEL calling [%d]\n", mplp.max_indel_depth);
+ fprintf(stderr, " -m INT minimum gapped reads for indel candidates [%d]\n", mplp.min_support);
+ fprintf(stderr, " -o INT Phred-scaled gap open sequencing error probability [%d]\n", mplp.openQ);
+ fprintf(stderr, " -P STR comma separated list of platforms for indels [all]\n");
fprintf(stderr, "\n");
fprintf(stderr, "Notes: Assuming diploid individuals.\n\n");
return 1;
}
- if ( file_list )
- {
+ if (file_list) {
if ( read_file_list(file_list,&nfiles,&fn) ) return 1;
mpileup(&mplp,nfiles,fn);
for (c=0; c<nfiles; c++) free(fn[c]);
free(fn);
- }
- else
- mpileup(&mplp, argc - optind, argv + optind);
+ } else mpileup(&mplp, argc - optind, argv + optind);
+ if (mplp.rghash) bcf_str2id_thorough_destroy(mplp.rghash);
free(mplp.reg); free(mplp.pl_list);
if (mplp.fai) fai_destroy(mplp.fai);
+ if (mplp.bed) bed_destroy(mplp.bed);
return 0;
}
projects / samtools.git / blobdiff