#include "pysam.h" #include #include #include #include #include #include #include "sam.h" #include "faidx.h" #include "kstring.h" static inline int printw(int c, FILE *fp) { char buf[16]; int l, x; if (c == 0) return fputc('0', fp); for (l = 0, x = c < 0? -c : c; x > 0; x /= 10) buf[l++] = x%10 + '0'; if (c < 0) buf[l++] = '-'; buf[l] = 0; for (x = 0; x < l/2; ++x) { int y = buf[x]; buf[x] = buf[l-1-x]; buf[l-1-x] = y; } fputs(buf, fp); 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_head) { putchar('^'); putchar(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 (ref) { int rb = pos < ref_len? ref[pos] : 'N'; if (c == '=' || bam_nt16_table[c] == bam_nt16_table[rb]) c = bam1_strand(p->b)? ',' : '.'; else c = bam1_strand(p->b)? tolower(c) : toupper(c); } else { if (c == '=') c = bam1_strand(p->b)? ',' : '.'; else c = bam1_strand(p->b)? tolower(c) : toupper(c); } putchar(c); } else putchar(p->is_refskip? (bam1_strand(p->b)? '<' : '>') : '*'); if (p->indel > 0) { putchar('+'); printw(p->indel, stdout); for (j = 1; j <= p->indel; ++j) { int 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) { printw(p->indel, stdout); for (j = 1; j <= -p->indel; ++j) { int c = (ref && (int)pos+j < ref_len)? ref[pos+j] : 'N'; putchar(bam1_strand(p->b)? tolower(c) : toupper(c)); } } if (p->is_tail) putchar('$'); } #include #include "bam2bcf.h" #include "sample.h" #define MPLP_GLF 0x10 #define MPLP_NO_COMP 0x20 #define MPLP_NO_ORPHAN 0x40 #define MPLP_REALN 0x80 #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, max_indel_depth; int openQ, extQ, tandemQ, min_support; // for indels double min_frac; // for indels char *reg, *pl_list; faidx_t *fai; void *bed, *rghash; } mplp_conf_t; typedef struct { bamFile fp; bam_iter_t iter; bam_header_t *h; int ref_id; char *ref; const mplp_conf_t *conf; } mplp_aux_t; typedef struct { int n; int *n_plp, *m_plp; bam_pileup1_t **plp; } mplp_pileup_t; static int mplp_func(void *data, bam1_t *b) { extern int bam_realn(bam1_t *b, const char *ref); extern int bam_prob_realn_core(bam1_t *b, const char *ref, int); extern int bam_cap_mapQ(bam1_t *b, char *ref, int thres); mplp_aux_t *ma = (mplp_aux_t*)data; int ret, skip = 0; do { 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->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.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 ignore_rg) { int i, j; memset(m->n_plp, 0, m->n * sizeof(int)); for (i = 0; i < n; ++i) { for (j = 0; j < n_plp[i]; ++j) { const bam_pileup1_t *p = plp[i] + j; uint8_t *q; int id = -1; 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); if (id < 0 || id >= m->n) { assert(q); // otherwise a bug fprintf(pysamerr, "[%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]); } m->plp[id][m->n_plp[id]++] = *p; } } } static int mpileup(mplp_conf_t *conf, int n, char **fn) { 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, 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; void *rghash = 0; bcf_callaux_t *bca = 0; bcf_callret1_t *bcr = 0; bcf_call_t bc; bcf_t *bp = 0; bcf_hdr_t *bh = 0; bam_sample_t *sm = 0; kstring_t buf; mplp_pileup_t gplp; memset(&gplp, 0, sizeof(mplp_pileup_t)); memset(&buf, 0, sizeof(kstring_t)); memset(&bc, 0, sizeof(bcf_call_t)); data = calloc(n, sizeof(void*)); plp = calloc(n, sizeof(void*)); n_plp = calloc(n, sizeof(int*)); sm = bam_smpl_init(); // 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 = 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); 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; bam_index_t *idx; idx = bam_index_load(fn[i]); if (idx == 0) { fprintf(pysamerr, "[%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(pysamerr, "[%s] malformatted region or wrong seqname for %d-th input.\n", __func__, i+1); exit(1); } if (i == 0) tid0 = tid, 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); } } gplp.n = sm->n; gplp.n_plp = calloc(sm->n, sizeof(int)); gplp.m_plp = calloc(sm->n, sizeof(int)); gplp.plp = calloc(sm->n, sizeof(void*)); fprintf(pysamerr, "[%s] %d samples in %d input files\n", __func__, sm->n, n); // write the VCF header if (conf->flag & MPLP_GLF) { kstring_t s; bh = calloc(1, sizeof(bcf_hdr_t)); s.l = s.m = 0; s.s = 0; bp = bcf_open("-", (conf->flag&MPLP_NO_COMP)? "wu" : "w"); for (i = 0; i < h->n_targets; ++i) { kputs(h->target_name[i], &s); kputc('\0', &s); } bh->l_nm = s.l; bh->name = malloc(s.l); memcpy(bh->name, s.s, s.l); s.l = 0; for (i = 0; i < sm->n; ++i) { kputs(sm->smpl[i], &s); kputc('\0', &s); } bh->l_smpl = s.l; bh->sname = malloc(s.l); memcpy(bh->sname, s.s, s.l); 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); bca = bcf_call_init(-1., conf->min_baseQ); bcr = calloc(sm->n, sizeof(bcf_callret1_t)); bca->rghash = rghash; bca->openQ = conf->openQ, bca->extQ = conf->extQ, bca->tandemQ = conf->tandemQ; 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(pysamerr, "(%s) Max depth is above 1M. Potential memory hog!\n", __func__); if (max_depth * sm->n < 8000) { max_depth = 8000 / sm->n; fprintf(pysamerr, "<%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 (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 = 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 total_depth, _ref0, ref16; bcf1_t *b = calloc(1, sizeof(bcf1_t)); 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_call_glfgen(gplp.n_plp[i], gplp.plp[i], ref16, bca, bcr + i); bcf_call_combine(gplp.n, bcr, ref16, &bc); bcf_call2bcf(tid, pos, &bc, b, (conf->flag&(MPLP_FMT_DP|MPLP_FMT_SP))? bcr : 0, (conf->flag&MPLP_FMT_SP), 0, 0); bcf_write(bp, bh, b); bcf_destroy(b); // call indels 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) { b = calloc(1, sizeof(bcf1_t)); bcf_call2bcf(tid, pos, &bc, b, (conf->flag&(MPLP_FMT_DP|MPLP_FMT_SP))? bcr : 0, (conf->flag&MPLP_FMT_SP), bca, ref); bcf_write(bp, bh, b); bcf_destroy(b); } } } else { 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*"); // 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'); 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); } 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'); } } bcf_close(bp); bam_smpl_destroy(sm); free(buf.s); 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); bcf_hdr_destroy(bh); bcf_call_destroy(bca); free(bc.PL); free(bcr); 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; } #define MAX_PATH_LEN 1024 static int read_file_list(const char *file_list,int *n,char **argv[]) { char buf[MAX_PATH_LEN]; int len, nfiles; char **files; FILE *fh = fopen(file_list,"r"); if ( !fh ) { fprintf(pysamerr,"%s: %s\n", file_list,strerror(errno)); return 1; } // Speed is not an issue here, determine the number of files by reading the file twice nfiles = 0; while ( fgets(buf,MAX_PATH_LEN,fh) ) nfiles++; if ( fseek(fh, 0L, SEEK_SET) ) { fprintf(pysamerr,"%s: %s\n", file_list,strerror(errno)); return 1; } files = calloc(nfiles,sizeof(char*)); nfiles = 0; while ( fgets(buf,MAX_PATH_LEN,fh) ) { len = strlen(buf); while ( len>0 && isspace(buf[len-1]) ) len--; if ( !len ) continue; files[nfiles] = malloc(sizeof(char)*(len+1)); strncpy(files[nfiles],buf,len); files[nfiles][len] = 0; nfiles++; } fclose(fh); if ( !nfiles ) { fprintf(pysamerr,"No files read from %s\n", file_list); return 1; } *argv = files; *n = nfiles; return 0; } #undef MAX_PATH_LEN int bam_mpileup(int argc, char *argv[]) { int c; const char *file_list = NULL; char **fn = NULL; 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_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, "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); if (mplp.fai == 0) return 1; break; case 'd': mplp.max_depth = atoi(optarg); break; case 'r': mplp.reg = 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; 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 'Q': mplp.min_baseQ = atoi(optarg); break; case 'b': file_list = 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(pysamerr, "(%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(pysamerr, "\n"); fprintf(pysamerr, "Usage: samtools mpileup [options] in1.bam [in2.bam [...]]\n\n"); fprintf(pysamerr, "Input options:\n\n"); fprintf(pysamerr, " -6 assume the quality is in the Illumina-1.3+ encoding\n"); fprintf(pysamerr, " -A count anomalous read pairs\n"); fprintf(pysamerr, " -B disable BAQ computation\n"); fprintf(pysamerr, " -b FILE list of input BAM files [null]\n"); fprintf(pysamerr, " -C INT parameter for adjusting mapQ; 0 to disable [0]\n"); fprintf(pysamerr, " -d INT max per-BAM depth to avoid excessive memory usage [%d]\n", mplp.max_depth); fprintf(pysamerr, " -E extended BAQ for higher sensitivity but lower specificity\n"); fprintf(pysamerr, " -f FILE faidx indexed reference sequence file [null]\n"); fprintf(pysamerr, " -G FILE exclude read groups listed in FILE [null]\n"); fprintf(pysamerr, " -l FILE list of positions (chr pos) or regions (BED) [null]\n"); fprintf(pysamerr, " -M INT cap mapping quality at INT [%d]\n", mplp.max_mq); fprintf(pysamerr, " -r STR region in which pileup is generated [null]\n"); fprintf(pysamerr, " -R ignore RG tags\n"); fprintf(pysamerr, " -q INT skip alignments with mapQ smaller than INT [%d]\n", mplp.min_mq); fprintf(pysamerr, " -Q INT skip bases with baseQ/BAQ smaller than INT [%d]\n", mplp.min_baseQ); fprintf(pysamerr, "\nOutput options:\n\n"); fprintf(pysamerr, " -D output per-sample DP in BCF (require -g/-u)\n"); fprintf(pysamerr, " -g generate BCF output (genotype likelihoods)\n"); fprintf(pysamerr, " -O output base positions on reads (disabled by -g/-u)\n"); fprintf(pysamerr, " -s output mapping quality (disabled by -g/-u)\n"); fprintf(pysamerr, " -S output per-sample strand bias P-value in BCF (require -g/-u)\n"); fprintf(pysamerr, " -u generate uncompress BCF output\n"); fprintf(pysamerr, "\nSNP/INDEL genotype likelihoods options (effective with `-g' or `-u'):\n\n"); fprintf(pysamerr, " -e INT Phred-scaled gap extension seq error probability [%d]\n", mplp.extQ); fprintf(pysamerr, " -F FLOAT minimum fraction of gapped reads for candidates [%g]\n", mplp.min_frac); fprintf(pysamerr, " -h INT coefficient for homopolymer errors [%d]\n", mplp.tandemQ); fprintf(pysamerr, " -I do not perform indel calling\n"); fprintf(pysamerr, " -L INT max per-sample depth for INDEL calling [%d]\n", mplp.max_indel_depth); fprintf(pysamerr, " -m INT minimum gapped reads for indel candidates [%d]\n", mplp.min_support); fprintf(pysamerr, " -o INT Phred-scaled gap open sequencing error probability [%d]\n", mplp.openQ); fprintf(pysamerr, " -P STR comma separated list of platforms for indels [all]\n"); fprintf(pysamerr, "\n"); fprintf(pysamerr, "Notes: Assuming diploid individuals.\n\n"); return 1; } if (file_list) { if ( read_file_list(file_list,&nfiles,&fn) ) return 1; mpileup(&mplp,nfiles,fn); for (c=0; c