/* bam2depth.c -- depth subcommand. Copyright (C) 2011, 2012 Broad Institute. Copyright (C) 2012-2014 Genome Research Ltd. Author: Heng Li Permission is hereby granted, free of charge, to any person obtaining a copy of this software and associated documentation files (the "Software"), to deal in the Software without restriction, including without limitation the rights to use, copy, modify, merge, publish, distribute, sublicense, and/or sell copies of the Software, and to permit persons to whom the Software is furnished to do so, subject to the following conditions: The above copyright notice and this permission notice shall be included in all copies or substantial portions of the Software. THE SOFTWARE IS PROVIDED "AS IS", WITHOUT WARRANTY OF ANY KIND, EXPRESS OR IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT. IN NO EVENT SHALL THE AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER LIABILITY, WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING FROM, OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS IN THE SOFTWARE. */ /* This program demonstrates how to generate pileup from multiple BAMs * simutaneously, to achieve random access and to use the BED interface. * To compile this program separately, you may: * * gcc -g -O2 -Wall -o bam2depth -D_MAIN_BAM2DEPTH bam2depth.c -lhts -lz */ #include #include #include #include #include "htslib/sam.h" #include "samtools.h" typedef struct { // auxiliary data structure samFile *fp; // the file handle bam_hdr_t *hdr; // the file header hts_itr_t *iter; // NULL if a region not specified int min_mapQ, min_len; // mapQ filter; length filter } aux_t; void *bed_read(const char *fn); // read a BED or position list file void bed_destroy(void *_h); // destroy the BED data structure int bed_overlap(const void *_h, const char *chr, int beg, int end); // test if chr:beg-end overlaps // This function reads a BAM alignment from one BAM file. static int read_bam(void *data, bam1_t *b) // read level filters better go here to avoid pileup { aux_t *aux = (aux_t*)data; // data in fact is a pointer to an auxiliary structure int ret; while (1) { ret = aux->iter? sam_itr_next(aux->fp, aux->iter, b) : sam_read1(aux->fp, aux->hdr, b); if ( ret<0 ) break; if ( b->core.flag & (BAM_FUNMAP | BAM_FSECONDARY | BAM_FQCFAIL | BAM_FDUP) ) continue; if ( (int)b->core.qual < aux->min_mapQ ) continue; if ( aux->min_len && bam_cigar2qlen(b->core.n_cigar, bam_get_cigar(b)) < aux->min_len ) continue; break; } return ret; } int read_file_list(const char *file_list,int *n,char **argv[]); int main_depth(int argc, char *argv[]) { int i, n, tid, beg, end, pos, *n_plp, baseQ = 0, mapQ = 0, min_len = 0, status = EXIT_SUCCESS, nfiles; const bam_pileup1_t **plp; char *reg = 0; // specified region void *bed = 0; // BED data structure char *file_list = NULL, **fn = NULL; bam_hdr_t *h = NULL; // BAM header of the 1st input aux_t **data; bam_mplp_t mplp; // parse the command line while ((n = getopt(argc, argv, "r:b:q:Q:l:f:")) >= 0) { switch (n) { case 'l': min_len = atoi(optarg); break; // minimum query length case 'r': reg = strdup(optarg); break; // parsing a region requires a BAM header case 'b': bed = bed_read(optarg); // BED or position list file can be parsed now if (!bed) { print_error_errno("Could not read file \"%s\"", optarg); return 1; } break; case 'q': baseQ = atoi(optarg); break; // base quality threshold case 'Q': mapQ = atoi(optarg); break; // mapping quality threshold case 'f': file_list = optarg; break; } } if (optind == argc && !file_list) { fprintf(stderr, "\n"); fprintf(stderr, "Usage: samtools depth [options] in1.bam [in2.bam [...]]\n"); fprintf(stderr, "Options:\n"); fprintf(stderr, " -b list of positions or regions\n"); fprintf(stderr, " -f list of input BAM filenames, one per line [null]\n"); fprintf(stderr, " -l read length threshold (ignore reads shorter than )\n"); fprintf(stderr, " -q base quality threshold\n"); fprintf(stderr, " -Q mapping quality threshold\n"); fprintf(stderr, " -r region\n"); fprintf(stderr, "\n"); return 1; } // initialize the auxiliary data structures if (file_list) { if ( read_file_list(file_list,&nfiles,&fn) ) return 1; n = nfiles; argv = fn; optind = 0; } else n = argc - optind; // the number of BAMs on the command line data = calloc(n, sizeof(aux_t*)); // data[i] for the i-th input beg = 0; end = 1<<30; // set the default region for (i = 0; i < n; ++i) { data[i] = calloc(1, sizeof(aux_t)); data[i]->fp = sam_open(argv[optind+i], "r"); // open BAM if (data[i]->fp == NULL) { print_error_errno("Could not open \"%s\"", argv[optind+i]); status = EXIT_FAILURE; goto depth_end; } if (hts_set_opt(data[i]->fp, CRAM_OPT_REQUIRED_FIELDS, SAM_FLAG | SAM_RNAME | SAM_POS | SAM_MAPQ | SAM_CIGAR | SAM_SEQ)) { fprintf(stderr, "Failed to set CRAM_OPT_REQUIRED_FIELDS value\n"); return 1; } if (hts_set_opt(data[i]->fp, CRAM_OPT_DECODE_MD, 0)) { fprintf(stderr, "Failed to set CRAM_OPT_DECODE_MD value\n"); return 1; } data[i]->min_mapQ = mapQ; // set the mapQ filter data[i]->min_len = min_len; // set the qlen filter data[i]->hdr = sam_hdr_read(data[i]->fp); // read the BAM header if (reg) { // if a region is specified hts_idx_t *idx = sam_index_load(data[i]->fp, argv[optind+i]); // load the index if (idx == NULL) { print_error("can't load index for \"%s\"", argv[optind+i]); status = EXIT_FAILURE; goto depth_end; } data[i]->iter = sam_itr_querys(idx, data[i]->hdr, reg); // set the iterator hts_idx_destroy(idx); // the index is not needed any more; free the memory if (data[i]->iter == NULL) { print_error("can't parse region \"%s\"", reg); status = EXIT_FAILURE; goto depth_end; } } } h = data[0]->hdr; // easy access to the header of the 1st BAM if (reg) { beg = data[0]->iter->beg; // and to the parsed region coordinates end = data[0]->iter->end; } // the core multi-pileup loop mplp = bam_mplp_init(n, read_bam, (void**)data); // initialization n_plp = calloc(n, sizeof(int)); // n_plp[i] is the number of covering reads from the i-th BAM plp = calloc(n, sizeof(bam_pileup1_t*)); // plp[i] points to the array of covering reads (internal in mplp) while (bam_mplp_auto(mplp, &tid, &pos, n_plp, plp) > 0) { // come to the next covered position if (pos < beg || pos >= end) continue; // out of range; skip if (bed && bed_overlap(bed, h->target_name[tid], pos, pos + 1) == 0) continue; // not in BED; skip fputs(h->target_name[tid], stdout); printf("\t%d", pos+1); // a customized printf() would be faster for (i = 0; i < n; ++i) { // base level filters have to go here int j, m = 0; for (j = 0; j < n_plp[i]; ++j) { const bam_pileup1_t *p = plp[i] + j; // DON'T modfity plp[][] unless you really know if (p->is_del || p->is_refskip) ++m; // having dels or refskips at tid:pos else if (bam_get_qual(p->b)[p->qpos] < baseQ) ++m; // low base quality } printf("\t%d", n_plp[i] - m); // this the depth to output } putchar('\n'); } free(n_plp); free(plp); bam_mplp_destroy(mplp); depth_end: for (i = 0; i < n && data[i]; ++i) { bam_hdr_destroy(data[i]->hdr); if (data[i]->fp) sam_close(data[i]->fp); hts_itr_destroy(data[i]->iter); free(data[i]); } free(data); free(reg); if (bed) bed_destroy(bed); if ( file_list ) { for (i=0; i