/* realn.c -- BAQ calculation and realignment. Copyright (C) 2009-2011, 2014-2016, 2018, 2021 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. */ #define HTS_BUILDING_LIBRARY // Enables HTSLIB_EXPORT, see htslib/hts_defs.h #include #include #include #include #include #include #include #include #include "htslib/hts.h" #include "htslib/sam.h" int sam_cap_mapq(bam1_t *b, const char *ref, hts_pos_t ref_len, int thres) { uint8_t *seq = bam_get_seq(b), *qual = bam_get_qual(b); uint32_t *cigar = bam_get_cigar(b); bam1_core_t *c = &b->core; int i, y, mm, q, len, clip_l, clip_q; hts_pos_t x; 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 c1, c2, z = y + j; if (x+j >= ref_len || ref[x+j] == '\0') break; // out of bounds c1 = bam_seqi(seq, z), c2 = seq_nt16_table[(unsigned char)ref[x+j]]; 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 (x+j >= ref_len || 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", bam_get_qname(b), t, q); return (int)(t + .499); } static int realn_check_tag(const uint8_t *tg, enum htsLogLevel severity, const char *type, const bam1_t *b) { if (*tg != 'Z') { hts_log(severity, __func__, "Incorrect %s tag type (%c) for read %s", type, *tg, bam_get_qname(b)); return -1; } if (b->core.l_qseq != strlen((const char *) tg + 1)) { hts_log(severity, __func__, "Read %s %s tag is wrong length", bam_get_qname(b), type); return -1; } return 0; } int sam_prob_realn(bam1_t *b, const char *ref, hts_pos_t ref_len, int flag) { int k, bw, y, yb, ye, xb, xe, fix_bq = 0, apply_baq = flag & BAQ_APPLY, extend_baq = flag & BAQ_EXTEND, redo_baq = flag & BAQ_REDO; enum htsRealnFlags system = flag & (0xff << 3); hts_pos_t i, x; uint32_t *cigar = bam_get_cigar(b); bam1_core_t *c = &b->core; // d(I) e(M) band probaln_par_t conf = { 0.001, 0.1, 10 }; // Illumina if (b->core.l_qseq > 1000 || system > BAQ_ILLUMINA) { // Params that work well on PacBio CCS 15k. Unknown if they // help other long-read platforms yet, but likely better than // the short-read tuned ones. // // This function has no access to the SAM header. // Ideally the calling function would check for e.g. // @RG PL = "PACBIO" and DS contains "READTYPE=CCS". // // In the absense of this, we simply auto-detect via a crude // short vs long strategy. conf.d = 1e-7; conf.e = 1e-1; } uint8_t *bq = NULL, *zq = NULL, *qual = bam_get_qual(b); int *state = NULL; if ((c->flag & BAM_FUNMAP) || b->core.l_qseq == 0 || qual[0] == (uint8_t)-1) return -1; // do nothing // test if BQ or ZQ is present, and make sanity checks if ((bq = bam_aux_get(b, "BQ")) != NULL) { if (!redo_baq) { if (realn_check_tag(bq, HTS_LOG_WARNING, "BQ", b) < 0) fix_bq = 1; } ++bq; } if ((zq = bam_aux_get(b, "ZQ")) != NULL) { if (realn_check_tag(zq, HTS_LOG_ERROR, "ZQ", b) < 0) return -4; ++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 (!zq && fix_bq) { // Need to fix invalid BQ tag (by realigning) assert(bq != NULL); bam_aux_del(b, bq-1); bq = 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 } if (xb == -1) // No matches in CIGAR. return -1; // 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 *seq = bam_get_seq(b); uint8_t *tseq; // translated seq A=>0,C=>1,G=>2,T=>3,other=>4 uint8_t *tref; // translated ref uint8_t *q; // Probability of incorrect alignment from probaln_glocal() size_t lref = xe > xb ? xe - xb : 1; size_t align_lqseq; if (extend_baq && lref < c->l_qseq) lref = c->l_qseq; // So we can recycle tseq,tref for left,rght below // Try to make q,tref,tseq reasonably well aligned align_lqseq = ((c->l_qseq + 1) | 0xf) + 1; // Overflow check - 3 for *bq, sizeof(int) for *state if ((SIZE_MAX - lref) / (3 + sizeof(int)) < align_lqseq) { errno = ENOMEM; goto fail; } assert(bq == NULL); // bq was used above, but should now be NULL bq = malloc(align_lqseq * 3 + lref); if (!bq) goto fail; q = bq + align_lqseq; tseq = q + align_lqseq; tref = tseq + align_lqseq; memcpy(bq, qual, c->l_qseq); bq[c->l_qseq] = 0; for (i = 0; i < c->l_qseq; ++i) tseq[i] = seq_nt16_int[bam_seqi(seq, i)]; for (i = xb; i < xe; ++i) { if (i >= ref_len || ref[i] == '\0') { xe = i; break; } tref[i-xb] = seq_nt16_int[seq_nt16_table[(unsigned char)ref[i]]]; } state = malloc(c->l_qseq * sizeof(int)); if (!state) goto fail; if (probaln_glocal(tref, xe-xb, tseq, c->l_qseq, qual, &conf, state, q) == INT_MIN) { goto fail; } 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 (l == 0) continue; if (op == BAM_CMATCH || op == BAM_CEQUAL || op == BAM_CDIFF) { // Sanity check running off the end of the sequence // Can only happen if the alignment is broken if (l > c->l_qseq - y) l = c->l_qseq - y; 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) { // Need sanity check here too. if (l > c->l_qseq - y) l = c->l_qseq - y; 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!) // tseq,tref are no longer needed, so we can steal them to avoid mallocs uint8_t *left = tseq; uint8_t *rght = tref; for (k = 0, x = c->pos, y = 0; k < c->n_cigar; ++k) { int op = cigar[k]&0xf, l = cigar[k]>>4; if (l == 0) continue; if (op == BAM_CMATCH || op == BAM_CEQUAL || op == BAM_CDIFF) { // Sanity check running off the end of the sequence // Can only happen if the alignment is broken if (l > c->l_qseq - y) l = c->l_qseq - y; 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) { // Need sanity check here too. if (l > c->l_qseq - y) l = c->l_qseq - y; 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 } 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(state); } return 0; fail: free(bq); free(state); return -4; }