#include #include #include #include #include #include "faidx.h" #include "sam.h" #include "kstring.h" #include "kaln.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 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) { uint8_t *old_nm = bam_aux_get(b, "NM"); if (c->flag & BAM_FUNMAP) return; 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) { uint8_t *old_md = bam_aux_get(b, "MD"); if (c->flag & BAM_FUNMAP) return; 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; 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 && 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 fillmd [-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; }