/* bam_md.c -- calmd subcommand. Copyright (C) 2009-2011, 2014-2015, 2019-2020 Genome Research Ltd. Portions copyright (C) 2009-2011 Broad Institute. 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. */ #include #include #include #include #include #include #include #include "htslib/faidx.h" #include "htslib/sam.h" #include "htslib/kstring.h" #include "htslib/thread_pool.h" #include "sam_opts.h" #include "samtools.h" #define USE_EQUAL 1 #define DROP_TAG 2 #define BIN_QUAL 4 #define UPDATE_NM 8 #define UPDATE_MD 16 #define HASH_QNM 32 int bam_aux_drop_other(bam1_t *b, uint8_t *s); static int bam_fillmd1_core(const char *ref_name, bam1_t *b, char *ref, hts_pos_t ref_len, int flag, int max_nm, int quiet_mode, uint32_t *skipped) { uint8_t *seq = bam_get_seq(b); uint32_t *cigar = bam_get_cigar(b); bam1_core_t *c = &b->core; int i, qpos, matched = 0; hts_pos_t rpos; kstring_t str = KS_INITIALIZE; int32_t old_nm_i = -1, nm = 0; uint32_t err = 0; if (c->l_qseq == 0) { if (!quiet_mode) { if (ref_name) { fprintf(stderr, "[bam_fillmd1] no sequence in alignment " "record for '%s' at %s:%"PRIhts_pos", skipped\n", bam_get_qname(b), ref_name, c->pos + 1); } else { fprintf(stderr, "[bam_fillmd1] no sequence in alignment " "record for '%s', skipped", bam_get_qname(b)); } } if (skipped) (*skipped)++; return 0; } for (i = qpos = 0, rpos = c->pos; i < c->n_cigar; ++i) { int j, oplen = cigar[i]>>4, op = cigar[i]&0xf; if (op == BAM_CMATCH || op == BAM_CEQUAL || op == BAM_CDIFF) { for (j = 0; j < oplen; ++j) { int c1, c2, z = qpos + j; if (rpos+j >= ref_len || z >= c->l_qseq || ref[rpos+j] == '\0') break; // out of bounds c1 = bam_seqi(seq, z); c2 = seq_nt16_table[(uint8_t)ref[rpos+j]]; if ((c1 == c2 && c1 != 15 && c2 != 15) || c1 == 0) { // a match if (flag&USE_EQUAL) seq[z/2] &= (z&1)? 0xf0 : 0x0f; ++matched; } else { err |= kputw(matched, &str) < 0; err |= kputc(toupper(ref[rpos+j]), &str) < 0; matched = 0; ++nm; } } if (j < oplen) break; rpos += oplen; qpos += oplen; } else if (op == BAM_CDEL) { err |= kputw(matched, &str) < 0; err |= kputc('^', &str) < 0; for (j = 0; j < oplen; ++j) { if (rpos+j >= ref_len || ref[rpos+j] == '\0') break; err |= kputc(toupper(ref[rpos+j]), &str) < 0; } matched = 0; rpos += j; nm += j; if (j < oplen) break; } else if (op == BAM_CINS || op == BAM_CSOFT_CLIP) { qpos += oplen; if (op == BAM_CINS) nm += oplen; } else if (op == BAM_CREF_SKIP) { rpos += oplen; } } err |= kputw(matched, &str) < 0; if (err) { print_error_errno("calmd", "Couldn't build new MD string"); goto fail; } // apply max_nm if (max_nm > 0 && nm >= max_nm) { for (i = qpos = 0, rpos = c->pos; i < c->n_cigar; ++i) { int j, oplen = cigar[i]>>4, op = cigar[i]&0xf; if (op == BAM_CMATCH || op == BAM_CEQUAL || op == BAM_CDIFF) { for (j = 0; j < oplen; ++j) { int c1, c2, z = qpos + j; if (rpos+j >= ref_len || z >= c->l_qseq || ref[rpos+j] == '\0') break; // out of bounds c1 = bam_seqi(seq, z); c2 = seq_nt16_table[(uint8_t)ref[rpos+j]]; if ((c1 == c2 && c1 != 15 && c2 != 15) || c1 == 0) { // a match seq[z/2] |= (z&1)? 0x0f : 0xf0; bam_get_qual(b)[z] = 0; } } if (j < oplen) break; rpos += oplen; qpos += oplen; } else if (op == BAM_CDEL || op == BAM_CREF_SKIP) rpos += oplen; else if (op == BAM_CINS || op == BAM_CSOFT_CLIP) qpos += oplen; } } // update NM if ((flag & UPDATE_NM) && !(c->flag & BAM_FUNMAP)) { uint8_t *old_nm = bam_aux_get(b, "NM"); if (old_nm) old_nm_i = bam_aux2i(old_nm); if (!old_nm) { if (bam_aux_append(b, "NM", 'i', 4, (uint8_t*)&nm) < 0) goto aux_fail; } else if (nm != old_nm_i) { if (!quiet_mode) { fprintf(stderr, "[bam_fillmd1] different NM for read '%s': %d -> %d\n", bam_get_qname(b), old_nm_i, nm); } if (bam_aux_del(b, old_nm) < 0) goto aux_fail; if (bam_aux_append(b, "NM", 'i', 4, (uint8_t*)&nm) < 0) goto aux_fail; } } // update MD if ((flag & UPDATE_MD) && !(c->flag & BAM_FUNMAP)) { uint8_t *old_md = bam_aux_get(b, "MD"); if (!old_md) { if (bam_aux_append(b, "MD", 'Z', str.l + 1, (uint8_t*)str.s) < 0) goto aux_fail; } else { int is_diff = 0; if (strlen((char*)old_md+1) == str.l) { for (i = 0; i < str.l; ++i) if (toupper(old_md[i+1]) != toupper(str.s[i])) break; if (i < str.l) is_diff = 1; } else is_diff = 1; if (is_diff) { if (!quiet_mode) { fprintf(stderr, "[bam_fillmd1] different MD for read '%s': '%s' -> '%s'\n", bam_get_qname(b), old_md+1, str.s); } if (bam_aux_del(b, old_md) < 0) goto aux_fail; if (bam_aux_append(b, "MD", 'Z', str.l + 1, (uint8_t*)str.s) < 0) goto aux_fail; } } } // drop all tags but RG if (flag&DROP_TAG) { uint8_t *q = bam_aux_get(b, "RG"); bam_aux_drop_other(b, q); } // reduce the resolution of base quality if (flag&BIN_QUAL) { uint8_t *qual = bam_get_qual(b); for (i = 0; i < b->core.l_qseq; ++i) if (qual[i] >= 3) qual[i] = qual[i]/10*10 + 7; } free(str.s); return 0; aux_fail: if (errno == ENOMEM) { print_error("calmd", "Couldn't add aux tag (too long)"); } else if (errno == EINVAL) { print_error("calmd", "Corrupt aux data"); } else { print_error_errno("calmd", "Couldn't add aux tag"); } fail: free(str.s); return -1; } int bam_fillmd1(bam1_t *b, char *ref, int flag, int quiet_mode) { return bam_fillmd1_core(NULL, b, ref, INT_MAX, flag, 0, quiet_mode, NULL); } int calmd_usage() { fprintf(stderr, "Usage: samtools calmd [-eubrAESQ] \n" "Options:\n" " -e change identical bases to '='\n" " -u uncompressed BAM output (for piping)\n" " -b compressed BAM output\n" " -S ignored (input format is auto-detected)\n" " -A modify the quality string\n" " -Q use quiet mode to output less debug info to stdout\n" " -r compute the BQ tag (without -A) or cap baseQ by BAQ (with -A)\n" " -E extended BAQ for better sensitivity but lower specificity\n" " --no-PG do not add a PG line\n"); sam_global_opt_help(stderr, "-....@-."); return 1; } int bam_fillmd(int argc, char *argv[]) { int c, flt_flag, tid = -2, ret, is_bam_out, is_uncompressed, max_nm, is_realn, capQ, baq_flag, quiet_mode, no_pg = 0; hts_pos_t len; htsThreadPool p = {NULL, 0}; samFile *fp = NULL, *fpout = NULL; sam_hdr_t *header = NULL; faidx_t *fai = NULL; char *ref = NULL, mode_w[8], *ref_file, *arg_list = NULL; const char *ref_name = NULL; bam1_t *b = NULL; sam_global_args ga = SAM_GLOBAL_ARGS_INIT; uint32_t skipped = 0; static const struct option lopts[] = { SAM_OPT_GLOBAL_OPTIONS('-', 0, 0, 0, 0,'@'), {"no-PG", no_argument, NULL, 1}, { NULL, 0, NULL, 0 } }; flt_flag = UPDATE_NM | UPDATE_MD; is_bam_out = is_uncompressed = is_realn = max_nm = capQ = baq_flag = quiet_mode = 0; strcpy(mode_w, "w"); while ((c = getopt_long(argc, argv, "EqQreuNhbSC:n:Ad@:", lopts, NULL)) >= 0) { switch (c) { case 'r': is_realn = 1; break; case 'e': flt_flag |= USE_EQUAL; break; case 'd': flt_flag |= DROP_TAG; break; case 'q': flt_flag |= BIN_QUAL; break; case 'h': flt_flag |= HASH_QNM; break; case 'N': flt_flag &= ~(UPDATE_MD|UPDATE_NM); break; case 'b': is_bam_out = 1; break; case 'u': is_uncompressed = is_bam_out = 1; break; case 'S': break; case 'n': max_nm = atoi(optarg); break; case 'C': capQ = atoi(optarg); break; case 'A': baq_flag |= 1; break; case 'E': baq_flag |= 2; break; case 'Q': quiet_mode = 1; break; case 1: no_pg = 1; break; default: if (parse_sam_global_opt(c, optarg, lopts, &ga) == 0) break; fprintf(stderr, "[bam_fillmd] unrecognized option '-%c'\n\n", c); /* else fall-through */ case '?': return calmd_usage(); } } if (is_bam_out) strcat(mode_w, "b"); else strcat(mode_w, "h"); if (is_uncompressed) strcat(mode_w, "0"); if (optind + (ga.reference == NULL) >= argc) return calmd_usage(); fp = sam_open_format(argv[optind], "r", &ga.in); if (fp == NULL) { print_error_errno("calmd", "Failed to open input file '%s'", argv[optind]); return 1; } if (!no_pg && !(arg_list = stringify_argv(argc+1, argv-1))) { print_error("calmd", "failed to create arg_list"); return 1; } header = sam_hdr_read(fp); if (header == NULL || sam_hdr_nref(header) == 0) { fprintf(stderr, "[bam_fillmd] input SAM does not have header. Abort!\n"); goto fail; } fpout = sam_open_format("-", mode_w, &ga.out); if (fpout == NULL) { print_error_errno("calmd", "Failed to open output"); goto fail; } if (!no_pg && sam_hdr_add_pg(header, "samtools", "VN", samtools_version(), arg_list ? "CL": NULL, arg_list ? arg_list : NULL, NULL)) { print_error("calmd", "failed to add PG line to header"); goto fail; } if (sam_hdr_write(fpout, header) < 0) { print_error_errno("calmd", "Failed to write sam header"); goto fail; } if (ga.nthreads > 0) { if (!(p.pool = hts_tpool_init(ga.nthreads))) { fprintf(stderr, "Error creating thread pool\n"); goto fail; } hts_set_opt(fp, HTS_OPT_THREAD_POOL, &p); hts_set_opt(fpout, HTS_OPT_THREAD_POOL, &p); } ref_file = argc > optind + 1 ? argv[optind+1] : ga.reference; fai = fai_load(ref_file); if (!fai) { print_error_errno("calmd", "Failed to open reference file '%s'", ref_file); goto fail; } b = bam_init1(); if (!b) { fprintf(stderr, "[bam_fillmd] Failed to allocate bam struct\n"); goto fail; } while ((ret = sam_read1(fp, header, b)) >= 0) { if (b->core.tid >= 0) { if (tid != b->core.tid) { free(ref); ref = NULL; len = 0; ref_name = sam_hdr_tid2name(header, b->core.tid); if (ref_name) { ref = fai_fetch64(fai, ref_name, &len); } tid = b->core.tid; if (ref == 0) { // FIXME: Should this always be fatal? fprintf(stderr, "[bam_fillmd] fail to find sequence '%s' in the reference.\n", ref_name ? ref_name : "(unknown)"); if (is_realn || capQ > 10) goto fail; // Would otherwise crash } } if (is_realn) { if (sam_prob_realn(b, ref, len, baq_flag) < -3) { print_error_errno("calmd", "BAQ alignment failed"); goto fail; } } if (capQ > 10) { int q = sam_cap_mapq(b, ref, len, capQ); if (b->core.qual > q) b->core.qual = q; } if (ref) { if (bam_fillmd1_core(ref_name, b, ref, len, flt_flag, max_nm, quiet_mode, &skipped) < 0) goto fail; } } if (sam_write1(fpout, header, b) < 0) { print_error_errno("calmd", "failed to write to output file"); goto fail; } } if (ret < -1) { fprintf(stderr, "[bam_fillmd] Error reading input.\n"); goto fail; } if (skipped) { fprintf(stderr, "[calmd] Warning: %"PRIu32" records skipped due " "to no query sequence\n", skipped); } bam_destroy1(b); sam_hdr_destroy(header); free(arg_list); free(ref); fai_destroy(fai); sam_close(fp); if (sam_close(fpout) < 0) { fprintf(stderr, "[bam_fillmd] error when closing output file\n"); return 1; } if (p.pool) hts_tpool_destroy(p.pool); return 0; fail: free(arg_list); free(ref); if (b) bam_destroy1(b); if (header) sam_hdr_destroy(header); if (fai) fai_destroy(fai); if (fp) sam_close(fp); if (fpout) sam_close(fpout); if (p.pool) hts_tpool_destroy(p.pool); return 1; }