X-Git-Url: http://woldlab.caltech.edu/gitweb/?p=samtools.git;a=blobdiff_plain;f=bam_plcmd.c;h=f6381c3d67d9da414bfdd40b4df7a7db9693199a;hp=e3f73aaddd03a14343a82084ee060d9c41ba42d4;hb=9f4bebab2e0917c676ae739b2d05cb22ad6c4ed5;hpb=e3b3a0177339fb8c099346986e965e3bd5b85999 diff --git a/bam_plcmd.c b/bam_plcmd.c index e3f73aa..f6381c3 100644 --- a/bam_plcmd.c +++ b/bam_plcmd.c @@ -437,6 +437,7 @@ int bam_pileup(int argc, char *argv[]) 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"); + fprintf(stderr, "Warning: Please use the `mpileup' command instead `pileup'. `Pileup' is deprecated!\n\n"); free(fn_list); free(fn_fa); free(d); return 1; } @@ -533,20 +534,29 @@ int bam_pileup(int argc, char *argv[]) #define MPLP_FMT_DP 0x100 #define MPLP_FMT_SP 0x200 #define MPLP_NO_INDEL 0x400 +#define MPLP_EXT_BAQ 0x800 +#define MPLP_ILLUMINA13 0x1000 + +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 { @@ -563,18 +573,38 @@ static int mplp_func(void *data, bam1_t *b) mplp_aux_t *ma = (mplp_aux_t*)data; int ret, skip = 0; do { - int has_ref = (ma->ref && ma->ref_id == b->core.tid)? 1 : 0; + 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; } @@ -592,7 +622,11 @@ static void group_smpl(mplp_pileup_t *m, bam_sample_t *sm, kstring_t *buf, q = 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]); @@ -607,12 +641,11 @@ 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, beg0 = 0, end0 = 1u<<29, ref_len, ref_tid, max_depth; + int i, tid, pos, *n_plp, beg0 = 0, end0 = 1u<<29, ref_len, ref_tid, 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; @@ -637,11 +670,10 @@ static int mpileup(mplp_conf_t *conf, int n, char **fn) 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); + data[i]->h = i? h : h_tmp; // for i==0, "h" has not been set yet bam_smpl_add(sm, fn[i], h_tmp->text); rghash = bcf_call_add_rg(rghash, h_tmp->text, conf->pl_list); if (conf->reg) { @@ -672,7 +704,6 @@ static int mpileup(mplp_conf_t *conf, int n, char **fn) 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; @@ -693,7 +724,8 @@ static int mpileup(mplp_conf_t *conf, int n, char **fn) 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); @@ -701,6 +733,8 @@ static int mpileup(mplp_conf_t *conf, int n, char **fn) 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; } ref_tid = -1; ref = 0; iter = bam_mplp_init(n, mplp_func, (void**)data); @@ -709,16 +743,13 @@ static int mpileup(mplp_conf_t *conf, int n, char **fn) 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); @@ -726,8 +757,9 @@ static int mpileup(mplp_conf_t *conf, int n, char **fn) ref_tid = tid; } if (conf->flag & MPLP_GLF) { - int _ref0, ref16; + 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); _ref0 = (ref && pos < ref_len)? ref[pos] : 'N'; ref16 = bam_nt16_table[_ref0]; @@ -739,7 +771,7 @@ static int mpileup(mplp_conf_t *conf, int n, char **fn) 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) { @@ -777,12 +809,6 @@ static int mpileup(mplp_conf_t *conf, int n, char **fn) 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); @@ -796,7 +822,7 @@ static int mpileup(mplp_conf_t *conf, int n, char **fn) } #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; @@ -849,16 +875,17 @@ int bam_mpileup(int argc, char *argv[]) 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)); 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:6")) >= 0) { switch (c) { case 'f': mplp.fai = fai_load(optarg); @@ -866,17 +893,18 @@ int bam_mpileup(int argc, char *argv[]) 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 'B': mplp.flag &= ~MPLP_REALN; break; case 'R': 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 'C': mplp.capQ_thres = atoi(optarg); break; case 'M': mplp.max_mq = atoi(optarg); break; case 'q': mplp.min_mq = atoi(optarg); break; @@ -885,43 +913,68 @@ int bam_mpileup(int argc, char *argv[]) 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, " -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, " -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, " -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