#include #include #include #include #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 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 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; } // 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 void pileup_seq(const bam_pileup1_t *p, int pos, int ref_len, const char *ref) { if (p->is_head) printf("^%c", p->b->core.qual > 93? 126 : p->b->core.qual + 33); if (!p->is_del) { int j, rb, c = bam_nt16_rev_table[bam1_seqi(bam1_seq(p->b), p->qpos)]; rb = (ref && pos < ref_len)? ref[pos] : 'N'; 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) { printf("+%d", p->indel); for (j = 1; j <= p->indel; ++j) { c = bam_nt16_rev_table[bam1_seqi(bam1_seq(p->b), p->qpos + j)]; putchar(bam1_strand(p->b)? tolower(c) : toupper(c)); } } else if (p->indel < 0) { printf("%d", p->indel); for (j = 1; j <= -p->indel; ++j) { c = (ref && (int)pos+j < ref_len)? ref[pos+j] : 'N'; putchar(bam1_strand(p->b)? tolower(c) : toupper(c)); } } } else putchar('*'); 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 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); // print consensus information if required if (d->format & BAM_PLF_CNS) { int ref_q, rb4 = bam_nt16_table[rb]; ref_q = 0; 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; } 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); } // 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; int tmp = p->b->core.qual < d->c->cap_mapQ? p->b->core.qual : d->c->cap_mapQ; rms_aux += tmp * tmp; pileup_seq(p, pos, d->len, d->ref); } // 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) { 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; printf("%d,", x < l/2? x+1 : -((l-1)-x+1)); } } 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 = 0; d->tid = -1; d->mask = BAM_DEF_MASK; d->c = bam_maqcns_init(); d->c->is_soap = 1; // 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:PA")) >= 0) { switch (c) { case 'a': d->c->is_soap = 1; break; case 'A': d->c->is_soap = 0; 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 (fn_list) is_SAM = 1; if (optind == argc) { fprintf(stderr, "\n"); fprintf(stderr, "Usage: samtools pileup [options] |\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 MAQ 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, " -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 SOAPsnp consensus sequence\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); fprintf(stderr, " -G FLOAT prior of an indel between two haplotypes (for -c/-g) [%f]\n", d->ido->r_indel); fprintf(stderr, " -I INT phred prob. of an indel in sequencing/prep. (for -c/-g) [%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); sampileup(fp, d->mask, pileup_func, d); 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 * ***********/ typedef struct { char *reg; faidx_t *fai; } mplp_conf_t; typedef struct { bamFile fp; bam_iter_t iter; } mplp_aux_t; static int mplp_func(void *data, bam1_t *b) { mplp_aux_t *ma = (mplp_aux_t*)data; if (ma->iter) return bam_iter_read(ma->fp, ma->iter, b); return bam_read1(ma->fp, b); } static int mpileup(mplp_conf_t *conf, int n, char **fn) { mplp_aux_t **data; int i, tid, pos, *n_plp, beg0 = 0, end0 = 1u<<29, ref_len, ref_tid; const bam_pileup1_t **plp; bam_mplp_t iter; bam_header_t *h = 0; char *ref; // allocate data = calloc(n, sizeof(void*)); plp = calloc(n, sizeof(void*)); n_plp = calloc(n, sizeof(int*)); // read the header and initialize data for (i = 0; i < n; ++i) { bam_header_t *h_tmp; data[i] = calloc(1, sizeof(mplp_aux_t)); data[i]->fp = bam_open(fn[i], "r"); h_tmp = bam_header_read(data[i]->fp); if (conf->reg) { int beg, end; bam_index_t *idx; idx = bam_index_load(fn[i]); if (idx == 0) { fprintf(stderr, "[%s] fail to load index for %d-th input.\n", __func__, i+1); exit(1); } if (bam_parse_region(h_tmp, conf->reg, &tid, &beg, &end) < 0) { 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; data[i]->iter = bam_iter_query(idx, tid, beg, end); bam_index_destroy(idx); } if (i == 0) h = h_tmp; else { // FIXME: to check consistency bam_header_destroy(h_tmp); } } // mpileup ref_tid = -1; ref = 0; iter = bam_mplp_init(n, mplp_func, (void**)data); 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 (tid != ref_tid) { free(ref); if (conf->fai) ref = fai_fetch(conf->fai, h->target_name[tid], &ref_len); ref_tid = tid; } printf("%s\t%d\t%c", h->target_name[tid], pos + 1, (ref && pos < ref_len)? ref[pos] : 'N'); for (i = 0; i < n; ++i) { int j; printf("\t%d\t", n_plp[i]); if (n_plp[i] == 0) printf("*\t*"); else { for (j = 0; j < n_plp[i]; ++j) pileup_seq(plp[i] + j, pos, ref_len, ref); putchar('\t'); for (j = 0; j < n_plp[i]; ++j) { const bam_pileup1_t *p = plp[i] + j; int c = bam1_qual(p->b)[p->qpos] + 33; if (c > 126) c = 126; putchar(c); } } } putchar('\n'); } bam_mplp_destroy(iter); bam_header_destroy(h); for (i = 0; i < n; ++i) { bam_close(data[i]->fp); if (data[i]->iter) bam_iter_destroy(data[i]->iter); free(data[i]); } free(data); free(plp); free(ref); free(n_plp); return 0; } int bam_mpileup(int argc, char *argv[]) { int c; mplp_conf_t mplp; memset(&mplp, 0, sizeof(mplp_conf_t)); while ((c = getopt(argc, argv, "f:r:")) >= 0) { switch (c) { case 'f': mplp.fai = fai_load(optarg); if (mplp.fai == 0) return 1; break; case 'r': mplp.reg = strdup(optarg); } } if (argc == 1) { fprintf(stderr, "Usage: samtools mpileup [-r reg] [-f in.fa] in1.bam [in2.bam [...]]\n"); return 1; } mpileup(&mplp, argc - optind, argv + optind); free(mplp.reg); if (mplp.fai) fai_destroy(mplp.fai); return 0; }