/* bam_md.c -- calmd subcommand. Copyright (C) 2009-2011 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 "htslib/faidx.h" #include "sam.h" #include "htslib/kstring.h" #include "kprobaln.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 const char bam_nt16_nt4_table[] = { 4, 0, 1, 4, 2, 4, 4, 4, 3, 4, 4, 4, 4, 4, 4, 4 }; int bam_aux_drop_other(bam1_t *b, uint8_t *s); void bam_fillmd1_core(bam1_t *b, char *ref, int flag, int max_nm) { uint8_t *seq = bam1_seq(b); uint32_t *cigar = bam1_cigar(b); bam1_core_t *c = &b->core; int i, x, y, u = 0; kstring_t *str; int32_t old_nm_i = -1, nm = 0; str = (kstring_t*)calloc(1, sizeof(kstring_t)); for (i = y = 0, x = c->pos; i < c->n_cigar; ++i) { int j, l = cigar[i]>>4, op = cigar[i]&0xf; if (op == BAM_CMATCH || op == BAM_CEQUAL || op == BAM_CDIFF) { for (j = 0; j < l; ++j) { int z = y + j; int c1 = bam1_seqi(seq, z), c2 = bam_nt16_table[(int)ref[x+j]]; if (ref[x+j] == 0) break; // out of boundary if ((c1 == c2 && c1 != 15 && c2 != 15) || c1 == 0) { // a match if (flag&USE_EQUAL) seq[z/2] &= (z&1)? 0xf0 : 0x0f; ++u; } else { kputw(u, str); kputc(ref[x+j], str); u = 0; ++nm; } } if (j < l) break; x += l; y += l; } else if (op == BAM_CDEL) { kputw(u, str); kputc('^', str); for (j = 0; j < l; ++j) { if (ref[x+j] == 0) break; kputc(ref[x+j], str); } u = 0; if (j < l) break; x += l; nm += l; } else if (op == BAM_CINS || op == BAM_CSOFT_CLIP) { y += l; if (op == BAM_CINS) nm += l; } else if (op == BAM_CREF_SKIP) { x += l; } } kputw(u, str); // apply max_nm if (max_nm > 0 && nm >= max_nm) { for (i = y = 0, x = c->pos; i < c->n_cigar; ++i) { int j, l = cigar[i]>>4, op = cigar[i]&0xf; if (op == BAM_CMATCH || op == BAM_CEQUAL || op == BAM_CDIFF) { for (j = 0; j < l; ++j) { int z = y + j; int c1 = bam1_seqi(seq, z), c2 = bam_nt16_table[(int)ref[x+j]]; if (ref[x+j] == 0) break; // out of boundary if ((c1 == c2 && c1 != 15 && c2 != 15) || c1 == 0) { // a match seq[z/2] |= (z&1)? 0x0f : 0xf0; bam1_qual(b)[z] = 0; } } if (j < l) break; x += l; y += l; } else if (op == BAM_CDEL || op == BAM_CREF_SKIP) x += l; else if (op == BAM_CINS || op == BAM_CSOFT_CLIP) y += l; } } // 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) bam_aux_append(b, "NM", 'i', 4, (uint8_t*)&nm); else if (nm != old_nm_i) { fprintf(stderr, "[bam_fillmd1] different NM for read '%s': %d -> %d\n", bam1_qname(b), old_nm_i, nm); bam_aux_del(b, old_nm); bam_aux_append(b, "NM", 'i', 4, (uint8_t*)&nm); } } // update MD if ((flag & UPDATE_MD) && !(c->flag & BAM_FUNMAP)) { uint8_t *old_md = bam_aux_get(b, "MD"); if (!old_md) bam_aux_append(b, "MD", 'Z', str->l + 1, (uint8_t*)str->s); 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) { fprintf(stderr, "[bam_fillmd1] different MD for read '%s': '%s' -> '%s'\n", bam1_qname(b), old_md+1, str->s); bam_aux_del(b, old_md); bam_aux_append(b, "MD", 'Z', str->l + 1, (uint8_t*)str->s); } } } // 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 = bam1_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); free(str); } void bam_fillmd1(bam1_t *b, char *ref, int flag) { bam_fillmd1_core(b, ref, flag, 0); } int bam_cap_mapQ(bam1_t *b, char *ref, int thres) { uint8_t *seq = bam1_seq(b), *qual = bam1_qual(b); uint32_t *cigar = bam1_cigar(b); bam1_core_t *c = &b->core; int i, x, y, mm, q, len, clip_l, clip_q; double t; if (thres < 0) thres = 40; // set the default mm = q = len = clip_l = clip_q = 0; for (i = y = 0, x = c->pos; i < c->n_cigar; ++i) { int j, l = cigar[i]>>4, op = cigar[i]&0xf; if (op == BAM_CMATCH || op == BAM_CEQUAL || op == BAM_CDIFF) { for (j = 0; j < l; ++j) { int z = y + j; int c1 = bam1_seqi(seq, z), c2 = bam_nt16_table[(int)ref[x+j]]; if (ref[x+j] == 0) break; // out of boundary if (c2 != 15 && c1 != 15 && qual[z] >= 13) { // not ambiguous ++len; if (c1 && c1 != c2 && qual[z] >= 13) { // mismatch ++mm; q += qual[z] > 33? 33 : qual[z]; } } } if (j < l) break; x += l; y += l; len += l; } else if (op == BAM_CDEL) { for (j = 0; j < l; ++j) if (ref[x+j] == 0) break; if (j < l) break; x += l; } else if (op == BAM_CSOFT_CLIP) { for (j = 0; j < l; ++j) clip_q += qual[y+j]; clip_l += l; y += l; } else if (op == BAM_CHARD_CLIP) { clip_q += 13 * l; clip_l += l; } else if (op == BAM_CINS) y += l; else if (op == BAM_CREF_SKIP) x += l; } for (i = 0, t = 1; i < mm; ++i) t *= (double)len / (i+1); t = q - 4.343 * log(t) + clip_q / 5.; if (t > thres) return -1; if (t < 0) t = 0; t = sqrt((thres - t) / thres) * thres; // fprintf(stderr, "%s %lf %d\n", bam1_qname(b), t, q); return (int)(t + .499); } int bam_prob_realn_core(bam1_t *b, const char *ref, int flag) { int k, i, bw, x, y, yb, ye, xb, xe, apply_baq = flag&1, extend_baq = flag>>1&1, redo_baq = flag&4; uint32_t *cigar = bam1_cigar(b); bam1_core_t *c = &b->core; kpa_par_t conf = kpa_par_def; uint8_t *bq = 0, *zq = 0, *qual = bam1_qual(b); if ((c->flag & BAM_FUNMAP) || b->core.l_qseq == 0) return -1; // do nothing // test if BQ or ZQ is present if ((bq = bam_aux_get(b, "BQ")) != 0) ++bq; if ((zq = bam_aux_get(b, "ZQ")) != 0 && *zq == 'Z') ++zq; if (bq && redo_baq) { bam_aux_del(b, bq-1); bq = 0; } if (bq && zq) { // remove the ZQ tag bam_aux_del(b, zq-1); zq = 0; } if (bq || zq) { if ((apply_baq && zq) || (!apply_baq && bq)) return -3; // in both cases, do nothing if (bq && apply_baq) { // then convert BQ to ZQ for (i = 0; i < c->l_qseq; ++i) qual[i] = qual[i] + 64 < bq[i]? 0 : qual[i] - ((int)bq[i] - 64); *(bq - 3) = 'Z'; } else if (zq && !apply_baq) { // then convert ZQ to BQ for (i = 0; i < c->l_qseq; ++i) qual[i] += (int)zq[i] - 64; *(zq - 3) = 'B'; } return 0; } // find the start and end of the alignment x = c->pos, y = 0, yb = ye = xb = xe = -1; for (k = 0; k < c->n_cigar; ++k) { int op, l; op = cigar[k]&0xf; l = cigar[k]>>4; if (op == BAM_CMATCH || op == BAM_CEQUAL || op == BAM_CDIFF) { if (yb < 0) yb = y; if (xb < 0) xb = x; ye = y + l; xe = x + l; x += l; y += l; } else if (op == BAM_CSOFT_CLIP || op == BAM_CINS) y += l; else if (op == BAM_CDEL) x += l; else if (op == BAM_CREF_SKIP) return -1; // do nothing if there is a reference skip } // set bandwidth and the start and the end bw = 7; if (abs((xe - xb) - (ye - yb)) > bw) bw = abs((xe - xb) - (ye - yb)) + 3; conf.bw = bw; xb -= yb + bw/2; if (xb < 0) xb = 0; xe += c->l_qseq - ye + bw/2; if (xe - xb - c->l_qseq > bw) xb += (xe - xb - c->l_qseq - bw) / 2, xe -= (xe - xb - c->l_qseq - bw) / 2; { // glocal uint8_t *s, *r, *q, *seq = bam1_seq(b), *bq; int *state; bq = calloc(c->l_qseq + 1, 1); memcpy(bq, qual, c->l_qseq); s = calloc(c->l_qseq, 1); for (i = 0; i < c->l_qseq; ++i) s[i] = bam_nt16_nt4_table[bam1_seqi(seq, i)]; r = calloc(xe - xb, 1); for (i = xb; i < xe; ++i) { if (ref[i] == 0) { xe = i; break; } r[i-xb] = bam_nt16_nt4_table[bam_nt16_table[(int)ref[i]]]; } state = calloc(c->l_qseq, sizeof(int)); q = calloc(c->l_qseq, 1); kpa_glocal(r, xe-xb, s, c->l_qseq, qual, &conf, state, q); if (!extend_baq) { // in this block, bq[] is capped by base quality qual[] for (k = 0, x = c->pos, y = 0; k < c->n_cigar; ++k) { int op = cigar[k]&0xf, l = cigar[k]>>4; if (op == BAM_CMATCH || op == BAM_CEQUAL || op == BAM_CDIFF) { for (i = y; i < y + l; ++i) { if ((state[i]&3) != 0 || state[i]>>2 != x - xb + (i - y)) bq[i] = 0; else bq[i] = bq[i] < q[i]? bq[i] : q[i]; } x += l; y += l; } else if (op == BAM_CSOFT_CLIP || op == BAM_CINS) y += l; else if (op == BAM_CDEL) x += l; } for (i = 0; i < c->l_qseq; ++i) bq[i] = qual[i] - bq[i] + 64; // finalize BQ } else { // in this block, bq[] is BAQ that can be larger than qual[] (different from the above!) uint8_t *left, *rght; left = calloc(c->l_qseq, 1); rght = calloc(c->l_qseq, 1); for (k = 0, x = c->pos, y = 0; k < c->n_cigar; ++k) { int op = cigar[k]&0xf, l = cigar[k]>>4; if (op == BAM_CMATCH || op == BAM_CEQUAL || op == BAM_CDIFF) { for (i = y; i < y + l; ++i) bq[i] = ((state[i]&3) != 0 || state[i]>>2 != x - xb + (i - y))? 0 : q[i]; for (left[y] = bq[y], i = y + 1; i < y + l; ++i) left[i] = bq[i] > left[i-1]? bq[i] : left[i-1]; for (rght[y+l-1] = bq[y+l-1], i = y + l - 2; i >= y; --i) rght[i] = bq[i] > rght[i+1]? bq[i] : rght[i+1]; for (i = y; i < y + l; ++i) bq[i] = left[i] < rght[i]? left[i] : rght[i]; x += l; y += l; } else if (op == BAM_CSOFT_CLIP || op == BAM_CINS) y += l; else if (op == BAM_CDEL) x += l; } for (i = 0; i < c->l_qseq; ++i) bq[i] = 64 + (qual[i] <= bq[i]? 0 : qual[i] - bq[i]); // finalize BQ free(left); free(rght); } if (apply_baq) { for (i = 0; i < c->l_qseq; ++i) qual[i] -= bq[i] - 64; // modify qual bam_aux_append(b, "ZQ", 'Z', c->l_qseq + 1, bq); } else bam_aux_append(b, "BQ", 'Z', c->l_qseq + 1, bq); free(bq); free(s); free(r); free(q); free(state); } return 0; } int bam_prob_realn(bam1_t *b, const char *ref) { return bam_prob_realn_core(b, ref, 1); } int bam_fillmd(int argc, char *argv[]) { int c, flt_flag, tid = -2, ret, len, is_bam_out, is_sam_in, is_uncompressed, max_nm, is_realn, capQ, baq_flag; samfile_t *fp, *fpout = 0; faidx_t *fai; char *ref = 0, mode_w[8], mode_r[8]; bam1_t *b; flt_flag = UPDATE_NM | UPDATE_MD; is_bam_out = is_sam_in = is_uncompressed = is_realn = max_nm = capQ = baq_flag = 0; mode_w[0] = mode_r[0] = 0; strcpy(mode_r, "r"); strcpy(mode_w, "w"); while ((c = getopt(argc, argv, "EqreuNhbSC:n:Ad")) >= 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': is_sam_in = 1; 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; default: fprintf(stderr, "[bam_fillmd] unrecognized option '-%c'\n", c); return 1; } } if (!is_sam_in) strcat(mode_r, "b"); if (is_bam_out) strcat(mode_w, "b"); else strcat(mode_w, "h"); if (is_uncompressed) strcat(mode_w, "u"); if (optind + 1 >= argc) { fprintf(stderr, "\n"); fprintf(stderr, "Usage: samtools calmd [-eubrS] \n\n"); fprintf(stderr, "Options: -e change identical bases to '='\n"); fprintf(stderr, " -u uncompressed BAM output (for piping)\n"); fprintf(stderr, " -b compressed BAM output\n"); fprintf(stderr, " -S the input is SAM with header\n"); fprintf(stderr, " -A modify the quality string\n"); fprintf(stderr, " -r compute the BQ tag (without -A) or cap baseQ by BAQ (with -A)\n"); fprintf(stderr, " -E extended BAQ for better sensitivity but lower specificity\n\n"); return 1; } fp = samopen(argv[optind], mode_r, 0); if (fp == 0) return 1; if (is_sam_in && (fp->header == 0 || fp->header->n_targets == 0)) { fprintf(stderr, "[bam_fillmd] input SAM does not have header. Abort!\n"); return 1; } fpout = samopen("-", mode_w, fp->header); fai = fai_load(argv[optind+1]); b = bam_init1(); while ((ret = samread(fp, b)) >= 0) { if (b->core.tid >= 0) { if (tid != b->core.tid) { free(ref); ref = fai_fetch(fai, fp->header->target_name[b->core.tid], &len); tid = b->core.tid; if (ref == 0) fprintf(stderr, "[bam_fillmd] fail to find sequence '%s' in the reference.\n", fp->header->target_name[tid]); } if (is_realn) bam_prob_realn_core(b, ref, baq_flag); if (capQ > 10) { int q = bam_cap_mapQ(b, ref, capQ); if (b->core.qual > q) b->core.qual = q; } if (ref) bam_fillmd1_core(b, ref, flt_flag, max_nm); } samwrite(fpout, b); } bam_destroy1(b); free(ref); fai_destroy(fai); samclose(fp); samclose(fpout); return 0; }