#include #include #include #include #include "minimap.h" #include "mmpriv.h" #include "ksw2.h" static void ksw_gen_simple_mat(int m, int8_t *mat, int8_t a, int8_t b, int8_t sc_ambi) { int i, j; a = a < 0? -a : a; b = b > 0? -b : b; sc_ambi = sc_ambi > 0? -sc_ambi : sc_ambi; for (i = 0; i < m - 1; ++i) { for (j = 0; j < m - 1; ++j) mat[i * m + j] = i == j? a : b; mat[i * m + m - 1] = sc_ambi; } for (j = 0; j < m; ++j) mat[(m - 1) * m + j] = sc_ambi; } static inline void mm_seq_rev(uint32_t len, uint8_t *seq) { uint32_t i; uint8_t t; for (i = 0; i < len>>1; ++i) t = seq[i], seq[i] = seq[len - 1 - i], seq[len - 1 - i] = t; } static inline void update_max_zdrop(int32_t score, int i, int j, int32_t *max, int *max_i, int *max_j, int e, int *max_zdrop, int pos[2][2]) { if (score < *max) { int li = i - *max_i; int lj = j - *max_j; int diff = li > lj? li - lj : lj - li; int z = *max - score - diff * e; if (z > *max_zdrop) { *max_zdrop = z; pos[0][0] = *max_i, pos[0][1] = i; pos[1][0] = *max_j, pos[1][1] = j; } } else *max = score, *max_i = i, *max_j = j; } static int mm_test_zdrop(void *km, const mm_mapopt_t *opt, const uint8_t *qseq, const uint8_t *tseq, uint32_t n_cigar, uint32_t *cigar, const int8_t *mat) { uint32_t k; int32_t score = 0, max = INT32_MIN, max_i = -1, max_j = -1, i = 0, j = 0, max_zdrop = 0; int pos[2][2] = {{-1, -1}, {-1, -1}}, q_len, t_len; // find the score and the region where score drops most along diagonal for (k = 0, score = 0; k < n_cigar; ++k) { uint32_t l, op = cigar[k]&0xf, len = cigar[k]>>4; if (op == MM_CIGAR_MATCH) { for (l = 0; l < len; ++l) { score += mat[tseq[i + l] * 5 + qseq[j + l]]; update_max_zdrop(score, i+l, j+l, &max, &max_i, &max_j, opt->e, &max_zdrop, pos); } i += len, j += len; } else if (op == MM_CIGAR_INS || op == MM_CIGAR_DEL || op == MM_CIGAR_N_SKIP) { score -= opt->q + opt->e * len; if (op == MM_CIGAR_INS) j += len; else i += len; update_max_zdrop(score, i, j, &max, &max_i, &max_j, opt->e, &max_zdrop, pos); } } // test if there is an inversion in the most dropped region q_len = pos[1][1] - pos[1][0], t_len = pos[0][1] - pos[0][0]; if (!(opt->flag&(MM_F_SPLICE|MM_F_SR|MM_F_FOR_ONLY|MM_F_REV_ONLY)) && max_zdrop > opt->zdrop_inv && q_len < opt->max_gap && t_len < opt->max_gap) { uint8_t *qseq2; void *qp; int q_off, t_off; qseq2 = (uint8_t*)kmalloc(km, q_len); for (i = 0; i < q_len; ++i) { int c = qseq[pos[1][1] - i - 1]; qseq2[i] = c >= 4? 4 : 3 - c; } qp = ksw_ll_qinit(km, 2, q_len, qseq2, 5, mat); score = ksw_ll_i16(qp, t_len, tseq + pos[0][0], opt->q, opt->e, &q_off, &t_off); kfree(km, qseq2); kfree(km, qp); if (score >= opt->min_chain_score * opt->a && score >= opt->min_dp_max) return 2; // there is a potential inversion } return max_zdrop > opt->zdrop? 1 : 0; } static void mm_fix_cigar(mm_reg1_t *r, const uint8_t *qseq, const uint8_t *tseq, int *qshift, int *tshift) { mm_extra_t *p = r->p; int32_t toff = 0, qoff = 0, to_shrink = 0; uint32_t k; *qshift = *tshift = 0; if (p->n_cigar <= 1) return; for (k = 0; k < p->n_cigar; ++k) { // indel left alignment uint32_t op = p->cigar[k]&0xf, len = p->cigar[k]>>4; if (len == 0) to_shrink = 1; if (op == MM_CIGAR_MATCH) { toff += len, qoff += len; } else if (op == MM_CIGAR_INS || op == MM_CIGAR_DEL) { if (k > 0 && k < p->n_cigar - 1 && (p->cigar[k-1]&0xf) == 0 && (p->cigar[k+1]&0xf) == 0) { int l, prev_len = p->cigar[k-1] >> 4; if (op == MM_CIGAR_INS) { for (l = 0; l < prev_len; ++l) if (qseq[qoff - 1 - l] != qseq[qoff + len - 1 - l]) break; } else { for (l = 0; l < prev_len; ++l) if (tseq[toff - 1 - l] != tseq[toff + len - 1 - l]) break; } if (l > 0) p->cigar[k-1] -= l<<4, p->cigar[k+1] += l<<4, qoff -= l, toff -= l; if (l == prev_len) to_shrink = 1; } if (op == MM_CIGAR_INS) qoff += len; else toff += len; } else if (op == MM_CIGAR_N_SKIP) { toff += len; } } assert(qoff == r->qe - r->qs && toff == r->re - r->rs); for (k = 0; k < p->n_cigar - 2; ++k) { // fix CIGAR like 5I6D7I if ((p->cigar[k]&0xf) > 0 && (p->cigar[k]&0xf) + (p->cigar[k+1]&0xf) == 3) { uint32_t l, s[3] = {0,0,0}; for (l = k; l < p->n_cigar; ++l) { // count number of adjacent I and D uint32_t op = p->cigar[l]&0xf; if (op == MM_CIGAR_INS || op == MM_CIGAR_DEL || p->cigar[l]>>4 == 0) s[op] += p->cigar[l] >> 4; else break; } if (s[1] > 0 && s[2] > 0 && l - k > 2) { // turn to a single I and a single D p->cigar[k] = s[1]<<4|MM_CIGAR_INS; p->cigar[k+1] = s[2]<<4|MM_CIGAR_DEL; for (k += 2; k < l; ++k) p->cigar[k] &= 0xf; to_shrink = 1; } k = l; } } if (to_shrink) { // squeeze out zero-length operations int32_t l = 0; for (k = 0; k < p->n_cigar; ++k) // squeeze out zero-length operations if (p->cigar[k]>>4 != 0) p->cigar[l++] = p->cigar[k]; p->n_cigar = l; for (k = l = 0; k < p->n_cigar; ++k) // merge two adjacent operations if they are the same if (k == p->n_cigar - 1 || (p->cigar[k]&0xf) != (p->cigar[k+1]&0xf)) p->cigar[l++] = p->cigar[k]; else p->cigar[k+1] += p->cigar[k]>>4<<4; // add length to the next CIGAR operator p->n_cigar = l; } if ((p->cigar[0]&0xf) == MM_CIGAR_INS || (p->cigar[0]&0xf) == MM_CIGAR_DEL) { // get rid of leading I or D int32_t l = p->cigar[0] >> 4; if ((p->cigar[0]&0xf) == MM_CIGAR_INS) { if (r->rev) r->qe -= l; else r->qs += l; *qshift = l; } else r->rs += l, *tshift = l; --p->n_cigar; memmove(p->cigar, p->cigar + 1, p->n_cigar * 4); } } static void mm_update_cigar_eqx(mm_reg1_t *r, const uint8_t *qseq, const uint8_t *tseq) // written by @armintoepfer { uint32_t n_EQX = 0; uint32_t k, l, m, cap, toff = 0, qoff = 0, n_M = 0; mm_extra_t *p; if (r->p == 0) return; for (k = 0; k < r->p->n_cigar; ++k) { uint32_t op = r->p->cigar[k]&0xf, len = r->p->cigar[k]>>4; if (op == MM_CIGAR_MATCH) { while (len > 0) { for (l = 0; l < len && qseq[qoff + l] == tseq[toff + l]; ++l) {} // run of "="; TODO: N<=>N is converted to "=" if (l > 0) { ++n_EQX; len -= l; toff += l; qoff += l; } for (l = 0; l < len && qseq[qoff + l] != tseq[toff + l]; ++l) {} // run of "X" if (l > 0) { ++n_EQX; len -= l; toff += l; qoff += l; } } ++n_M; } else if (op == MM_CIGAR_INS) { qoff += len; } else if (op == MM_CIGAR_DEL) { toff += len; } else if (op == MM_CIGAR_N_SKIP) { toff += len; } } // update in-place if we can if (n_EQX == n_M) { for (k = 0; k < r->p->n_cigar; ++k) { uint32_t op = r->p->cigar[k]&0xf, len = r->p->cigar[k]>>4; if (op == MM_CIGAR_MATCH) r->p->cigar[k] = len << 4 | MM_CIGAR_EQ_MATCH; } return; } // allocate new storage cap = r->p->n_cigar + (n_EQX - n_M) + sizeof(mm_extra_t); kroundup32(cap); p = (mm_extra_t*)calloc(cap, 4); memcpy(p, r->p, sizeof(mm_extra_t)); p->capacity = cap; // update cigar while copying toff = qoff = m = 0; for (k = 0; k < r->p->n_cigar; ++k) { uint32_t op = r->p->cigar[k]&0xf, len = r->p->cigar[k]>>4; if (op == MM_CIGAR_MATCH) { while (len > 0) { // match for (l = 0; l < len && qseq[qoff + l] == tseq[toff + l]; ++l) {} if (l > 0) p->cigar[m++] = l << 4 | MM_CIGAR_EQ_MATCH; len -= l; toff += l, qoff += l; // mismatch for (l = 0; l < len && qseq[qoff + l] != tseq[toff + l]; ++l) {} if (l > 0) p->cigar[m++] = l << 4 | MM_CIGAR_X_MISMATCH; len -= l; toff += l, qoff += l; } continue; } else if (op == MM_CIGAR_INS) { qoff += len; } else if (op == MM_CIGAR_DEL) { toff += len; } else if (op == MM_CIGAR_N_SKIP) { toff += len; } p->cigar[m++] = r->p->cigar[k]; } p->n_cigar = m; free(r->p); r->p = p; } static void mm_update_extra(mm_reg1_t *r, const uint8_t *qseq, const uint8_t *tseq, const int8_t *mat, int8_t q, int8_t e, int is_eqx, int log_gap) { uint32_t k, l; int32_t qshift, tshift, toff = 0, qoff = 0; double s = 0.0, max = 0.0; mm_extra_t *p = r->p; if (p == 0) return; mm_fix_cigar(r, qseq, tseq, &qshift, &tshift); qseq += qshift, tseq += tshift; // qseq and tseq may be shifted due to the removal of leading I/D r->blen = r->mlen = 0; for (k = 0; k < p->n_cigar; ++k) { uint32_t op = p->cigar[k]&0xf, len = p->cigar[k]>>4; if (op == MM_CIGAR_MATCH) { int n_ambi = 0, n_diff = 0; for (l = 0; l < len; ++l) { int cq = qseq[qoff + l], ct = tseq[toff + l]; if (ct > 3 || cq > 3) ++n_ambi; else if (ct != cq) ++n_diff; s += mat[ct * 5 + cq]; if (s < 0) s = 0; else max = max > s? max : s; } r->blen += len - n_ambi, r->mlen += len - (n_ambi + n_diff), p->n_ambi += n_ambi; toff += len, qoff += len; } else if (op == MM_CIGAR_INS) { int n_ambi = 0; for (l = 0; l < len; ++l) if (qseq[qoff + l] > 3) ++n_ambi; r->blen += len - n_ambi, p->n_ambi += n_ambi; if (log_gap) s -= q + (double)e * mg_log2(1.0 + len); else s -= q + e; if (s < 0) s = 0; qoff += len; } else if (op == MM_CIGAR_DEL) { int n_ambi = 0; for (l = 0; l < len; ++l) if (tseq[toff + l] > 3) ++n_ambi; r->blen += len - n_ambi, p->n_ambi += n_ambi; if (log_gap) s -= q + (double)e * mg_log2(1.0 + len); else s -= q + e; if (s < 0) s = 0; toff += len; } else if (op == MM_CIGAR_N_SKIP) { toff += len; } } p->dp_max = (int32_t)(max + .499); assert(qoff == r->qe - r->qs && toff == r->re - r->rs); if (is_eqx) mm_update_cigar_eqx(r, qseq, tseq); // NB: it has to be called here as changes to qseq and tseq are not returned } static void mm_append_cigar(mm_reg1_t *r, uint32_t n_cigar, uint32_t *cigar) // TODO: this calls the libc realloc() { mm_extra_t *p; if (n_cigar == 0) return; if (r->p == 0) { uint32_t capacity = n_cigar + sizeof(mm_extra_t)/4; kroundup32(capacity); r->p = (mm_extra_t*)calloc(capacity, 4); r->p->capacity = capacity; } else if (r->p->n_cigar + n_cigar + sizeof(mm_extra_t)/4 > r->p->capacity) { r->p->capacity = r->p->n_cigar + n_cigar + sizeof(mm_extra_t)/4; kroundup32(r->p->capacity); r->p = (mm_extra_t*)realloc(r->p, r->p->capacity * 4); } p = r->p; if (p->n_cigar > 0 && (p->cigar[p->n_cigar-1]&0xf) == (cigar[0]&0xf)) { // same CIGAR op at the boundary p->cigar[p->n_cigar-1] += cigar[0]>>4<<4; if (n_cigar > 1) memcpy(p->cigar + p->n_cigar, cigar + 1, (n_cigar - 1) * 4); p->n_cigar += n_cigar - 1; } else { memcpy(p->cigar + p->n_cigar, cigar, n_cigar * 4); p->n_cigar += n_cigar; } } static void mm_align_pair(void *km, const mm_mapopt_t *opt, int qlen, const uint8_t *qseq, int tlen, const uint8_t *tseq, const uint8_t *junc, const int8_t *mat, int w, int end_bonus, int zdrop, int flag, ksw_extz_t *ez) { if (mm_dbg_flag & MM_DBG_PRINT_ALN_SEQ) { int i; fprintf(stderr, "===> q=(%d,%d), e=(%d,%d), bw=%d, flag=%d, zdrop=%d <===\n", opt->q, opt->q2, opt->e, opt->e2, w, flag, opt->zdrop); for (i = 0; i < tlen; ++i) fputc("ACGTN"[tseq[i]], stderr); fputc('\n', stderr); for (i = 0; i < qlen; ++i) fputc("ACGTN"[qseq[i]], stderr); fputc('\n', stderr); } if (opt->max_sw_mat > 0 && (int64_t)tlen * qlen > opt->max_sw_mat) { ksw_reset_extz(ez); ez->zdropped = 1; } else if (opt->flag & MM_F_SPLICE) ksw_exts2_sse(km, qlen, qseq, tlen, tseq, 5, mat, opt->q, opt->e, opt->q2, opt->noncan, zdrop, opt->junc_bonus, flag, junc, ez); else if (opt->q == opt->q2 && opt->e == opt->e2) ksw_extz2_sse(km, qlen, qseq, tlen, tseq, 5, mat, opt->q, opt->e, w, zdrop, end_bonus, flag, ez); else ksw_extd2_sse(km, qlen, qseq, tlen, tseq, 5, mat, opt->q, opt->e, opt->q2, opt->e2, w, zdrop, end_bonus, flag, ez); if (mm_dbg_flag & MM_DBG_PRINT_ALN_SEQ) { int i; fprintf(stderr, "score=%d, cigar=", ez->score); for (i = 0; i < ez->n_cigar; ++i) fprintf(stderr, "%d%c", ez->cigar[i]>>4, MM_CIGAR_STR[ez->cigar[i]&0xf]); fprintf(stderr, "\n"); } } static inline int mm_get_hplen_back(const mm_idx_t *mi, uint32_t rid, uint32_t x) { int64_t i, off0 = mi->seq[rid].offset, off = off0 + x; int c = mm_seq4_get(mi->S, off); for (i = off - 1; i >= off0; --i) if (mm_seq4_get(mi->S, i) != c) break; return (int)(off - i); } static inline void mm_adjust_minier(const mm_idx_t *mi, uint8_t *const qseq0[2], mm128_t *a, int32_t *r, int32_t *q) { if (mi->flag & MM_I_HPC) { const uint8_t *qseq = qseq0[a->x>>63]; int i, c; *q = (int32_t)a->y; for (i = *q - 1, c = qseq[*q]; i > 0; --i) if (qseq[i] != c) break; *q = i + 1; c = mm_get_hplen_back(mi, a->x<<1>>33, (int32_t)a->x); *r = (int32_t)a->x + 1 - c; } else { *r = (int32_t)a->x - (mi->k>>1); *q = (int32_t)a->y - (mi->k>>1); } } static int *collect_long_gaps(void *km, int as1, int cnt1, mm128_t *a, int min_gap, int *n_) { int i, n, *K; *n_ = 0; for (i = 1, n = 0; i < cnt1; ++i) { // count the number of gaps longer than min_gap int gap = ((int32_t)a[as1 + i].y - a[as1 + i - 1].y) - ((int32_t)a[as1 + i].x - a[as1 + i - 1].x); if (gap < -min_gap || gap > min_gap) ++n; } if (n <= 1) return 0; K = (int*)kmalloc(km, n * sizeof(int)); for (i = 1, n = 0; i < cnt1; ++i) { // store the positions of long gaps int gap = ((int32_t)a[as1 + i].y - a[as1 + i - 1].y) - ((int32_t)a[as1 + i].x - a[as1 + i - 1].x); if (gap < -min_gap || gap > min_gap) K[n++] = i; } *n_ = n; return K; } static void mm_filter_bad_seeds(void *km, int as1, int cnt1, mm128_t *a, int min_gap, int diff_thres, int max_ext_len, int max_ext_cnt) { int max_st, max_en, n, i, k, max, *K; K = collect_long_gaps(km, as1, cnt1, a, min_gap, &n); if (K == 0) return; max = 0, max_st = max_en = -1; for (k = 0;; ++k) { // traverse long gaps int gap, l, n_ins = 0, n_del = 0, qs, rs, max_diff = 0, max_diff_l = -1; if (k == n || k >= max_en) { if (max_en > 0) for (i = K[max_st]; i < K[max_en]; ++i) a[as1 + i].y |= MM_SEED_IGNORE; max = 0, max_st = max_en = -1; if (k == n) break; } i = K[k]; gap = ((int32_t)a[as1 + i].y - (int32_t)a[as1 + i - 1].y) - (int32_t)(a[as1 + i].x - a[as1 + i - 1].x); if (gap > 0) n_ins += gap; else n_del += -gap; qs = (int32_t)a[as1 + i - 1].y; rs = (int32_t)a[as1 + i - 1].x; for (l = k + 1; l < n && l <= k + max_ext_cnt; ++l) { int j = K[l], diff; if ((int32_t)a[as1 + j].y - qs > max_ext_len || (int32_t)a[as1 + j].x - rs > max_ext_len) break; gap = ((int32_t)a[as1 + j].y - (int32_t)a[as1 + j - 1].y) - (int32_t)(a[as1 + j].x - a[as1 + j - 1].x); if (gap > 0) n_ins += gap; else n_del += -gap; diff = n_ins + n_del - abs(n_ins - n_del); if (max_diff < diff) max_diff = diff, max_diff_l = l; } if (max_diff > diff_thres && max_diff > max) max = max_diff, max_st = k, max_en = max_diff_l; } kfree(km, K); } static void mm_filter_bad_seeds_alt(void *km, int as1, int cnt1, mm128_t *a, int min_gap, int max_ext) { int n, k, *K; K = collect_long_gaps(km, as1, cnt1, a, min_gap, &n); if (K == 0) return; for (k = 0; k < n;) { int i = K[k], l; int gap1 = ((int32_t)a[as1 + i].y - (int32_t)a[as1 + i - 1].y) - ((int32_t)a[as1 + i].x - (int32_t)a[as1 + i - 1].x); int re1 = (int32_t)a[as1 + i].x; int qe1 = (int32_t)a[as1 + i].y; gap1 = gap1 > 0? gap1 : -gap1; for (l = k + 1; l < n; ++l) { int j = K[l], gap2, q_span_pre, rs2, qs2, m; if ((int32_t)a[as1 + j].y - qe1 > max_ext || (int32_t)a[as1 + j].x - re1 > max_ext) break; gap2 = ((int32_t)a[as1 + j].y - (int32_t)a[as1 + j - 1].y) - (int32_t)(a[as1 + j].x - a[as1 + j - 1].x); q_span_pre = a[as1 + j - 1].y >> 32 & 0xff; rs2 = (int32_t)a[as1 + j - 1].x + q_span_pre; qs2 = (int32_t)a[as1 + j - 1].y + q_span_pre; m = rs2 - re1 < qs2 - qe1? rs2 - re1 : qs2 - qe1; gap2 = gap2 > 0? gap2 : -gap2; if (m > gap1 + gap2) break; re1 = (int32_t)a[as1 + j].x; qe1 = (int32_t)a[as1 + j].y; gap1 = gap2; } if (l > k + 1) { int j, end = K[l - 1]; for (j = K[k]; j < end; ++j) a[as1 + j].y |= MM_SEED_IGNORE; a[as1 + end].y |= MM_SEED_LONG_JOIN; } k = l; } kfree(km, K); } static void mm_fix_bad_ends(const mm_reg1_t *r, const mm128_t *a, int bw, int min_match, int32_t *as, int32_t *cnt) { int32_t i, l, m; *as = r->as, *cnt = r->cnt; if (r->cnt < 3) return; m = l = a[r->as].y >> 32 & 0xff; for (i = r->as + 1; i < r->as + r->cnt - 1; ++i) { int32_t lq, lr, min, max; int32_t q_span = a[i].y >> 32 & 0xff; if (a[i].y & MM_SEED_LONG_JOIN) break; lr = (int32_t)a[i].x - (int32_t)a[i-1].x; lq = (int32_t)a[i].y - (int32_t)a[i-1].y; min = lr < lq? lr : lq; max = lr > lq? lr : lq; if (max - min > l >> 1) *as = i; l += min; m += min < q_span? min : q_span; if (l >= bw << 1 || (m >= min_match && m >= bw) || m >= r->mlen >> 1) break; } *cnt = r->as + r->cnt - *as; m = l = a[r->as + r->cnt - 1].y >> 32 & 0xff; for (i = r->as + r->cnt - 2; i > *as; --i) { int32_t lq, lr, min, max; int32_t q_span = a[i+1].y >> 32 & 0xff; if (a[i+1].y & MM_SEED_LONG_JOIN) break; lr = (int32_t)a[i+1].x - (int32_t)a[i].x; lq = (int32_t)a[i+1].y - (int32_t)a[i].y; min = lr < lq? lr : lq; max = lr > lq? lr : lq; if (max - min > l >> 1) *cnt = i + 1 - *as; l += min; m += min < q_span? min : q_span; if (l >= bw << 1 || (m >= min_match && m >= bw) || m >= r->mlen >> 1) break; } } static void mm_max_stretch(const mm_reg1_t *r, const mm128_t *a, int32_t *as, int32_t *cnt) { int32_t i, score, max_score, len, max_i, max_len; *as = r->as, *cnt = r->cnt; if (r->cnt < 2) return; max_score = -1, max_i = -1, max_len = 0; score = a[r->as].y >> 32 & 0xff, len = 1; for (i = r->as + 1; i < r->as + r->cnt; ++i) { int32_t lq, lr, q_span; q_span = a[i].y >> 32 & 0xff; lr = (int32_t)a[i].x - (int32_t)a[i-1].x; lq = (int32_t)a[i].y - (int32_t)a[i-1].y; if (lq == lr) { score += lq < q_span? lq : q_span; ++len; } else { if (score > max_score) max_score = score, max_len = len, max_i = i - len; score = q_span, len = 1; } } if (score > max_score) max_score = score, max_len = len, max_i = i - len; *as = max_i, *cnt = max_len; } static int mm_seed_ext_score(void *km, const mm_mapopt_t *opt, const mm_idx_t *mi, const int8_t mat[25], int qlen, uint8_t *qseq0[2], const mm128_t *a) { uint8_t *qseq, *tseq; int q_span = a->y>>32&0xff, qs, qe, rs, re, rid, score, q_off, t_off, ext_len = opt->anchor_ext_len; void *qp; rid = a->x<<1>>33; re = (uint32_t)a->x + 1, rs = re - q_span; qe = (uint32_t)a->y + 1, qs = qe - q_span; rs = rs - ext_len > 0? rs - ext_len : 0; qs = qs - ext_len > 0? qs - ext_len : 0; re = re + ext_len < (int32_t)mi->seq[rid].len? re + ext_len : mi->seq[rid].len; qe = qe + ext_len < qlen? qe + ext_len : qlen; tseq = (uint8_t*)kmalloc(km, re - rs); if (opt->flag & MM_F_QSTRAND) { qseq = qseq0[0] + qs; mm_idx_getseq2(mi, a->x>>63, rid, rs, re, tseq); } else { qseq = qseq0[a->x>>63] + qs; mm_idx_getseq(mi, rid, rs, re, tseq); } qp = ksw_ll_qinit(km, 2, qe - qs, qseq, 5, mat); score = ksw_ll_i16(qp, re - rs, tseq, opt->q, opt->e, &q_off, &t_off); kfree(km, tseq); kfree(km, qp); return score; } static void mm_fix_bad_ends_splice(void *km, const mm_mapopt_t *opt, const mm_idx_t *mi, const mm_reg1_t *r, const int8_t mat[25], int qlen, uint8_t *qseq0[2], const mm128_t *a, int *as1, int *cnt1) { // this assumes a very crude k-mer based mode; it is not necessary to use a good model just for filtering bounary exons int score; double log_gap; *as1 = r->as, *cnt1 = r->cnt; if (r->cnt < 3) return; log_gap = log((int32_t)a[r->as + 1].x - (int32_t)a[r->as].x); if ((a[r->as].y>>32&0xff) < log_gap + opt->anchor_ext_shift) { score = mm_seed_ext_score(km, opt, mi, mat, qlen, qseq0, &a[r->as]); if ((double)score / mat[0] < log_gap + opt->anchor_ext_shift) // a more exact format is "score < log_4(gap) + shift" ++(*as1), --(*cnt1); } log_gap = log((int32_t)a[r->as + r->cnt - 1].x - (int32_t)a[r->as + r->cnt - 2].x); if ((a[r->as + r->cnt - 1].y>>32&0xff) < log_gap + opt->anchor_ext_shift) { score = mm_seed_ext_score(km, opt, mi, mat, qlen, qseq0, &a[r->as + r->cnt - 1]); if ((double)score / mat[0] < log_gap + opt->anchor_ext_shift) --(*cnt1); } } static void mm_align1(void *km, const mm_mapopt_t *opt, const mm_idx_t *mi, int qlen, uint8_t *qseq0[2], mm_reg1_t *r, mm_reg1_t *r2, int n_a, mm128_t *a, ksw_extz_t *ez, int splice_flag) { int is_sr = !!(opt->flag & MM_F_SR), is_splice = !!(opt->flag & MM_F_SPLICE); int32_t rid = a[r->as].x<<1>>33, rev = a[r->as].x>>63, as1, cnt1; uint8_t *tseq, *qseq, *junc; int32_t i, l, bw, bw_long, dropped = 0, extra_flag = 0, rs0, re0, qs0, qe0; int32_t rs, re, qs, qe; int32_t rs1, qs1, re1, qe1; int8_t mat[25]; if (is_sr) assert(!(mi->flag & MM_I_HPC)); // HPC won't work with SR because with HPC we can't easily tell if there is a gap r2->cnt = 0; if (r->cnt == 0) return; ksw_gen_simple_mat(5, mat, opt->a, opt->b, opt->sc_ambi); bw = (int)(opt->bw * 1.5 + 1.); bw_long = (int)(opt->bw_long * 1.5 + 1.); if (bw_long < bw) bw_long = bw; if (is_sr && !(mi->flag & MM_I_HPC)) { mm_max_stretch(r, a, &as1, &cnt1); rs = (int32_t)a[as1].x + 1 - (int32_t)(a[as1].y>>32&0xff); qs = (int32_t)a[as1].y + 1 - (int32_t)(a[as1].y>>32&0xff); re = (int32_t)a[as1+cnt1-1].x + 1; qe = (int32_t)a[as1+cnt1-1].y + 1; } else { if (!(opt->flag & MM_F_NO_END_FLT)) { if (is_splice) mm_fix_bad_ends_splice(km, opt, mi, r, mat, qlen, qseq0, a, &as1, &cnt1); else mm_fix_bad_ends(r, a, opt->bw, opt->min_chain_score * 2, &as1, &cnt1); } else as1 = r->as, cnt1 = r->cnt; mm_filter_bad_seeds(km, as1, cnt1, a, 10, 40, opt->max_gap>>1, 10); mm_filter_bad_seeds_alt(km, as1, cnt1, a, 30, opt->max_gap>>1); mm_adjust_minier(mi, qseq0, &a[as1], &rs, &qs); mm_adjust_minier(mi, qseq0, &a[as1 + cnt1 - 1], &re, &qe); } assert(cnt1 > 0); if (is_splice) { if (splice_flag & MM_F_SPLICE_FOR) extra_flag |= rev? KSW_EZ_SPLICE_REV : KSW_EZ_SPLICE_FOR; if (splice_flag & MM_F_SPLICE_REV) extra_flag |= rev? KSW_EZ_SPLICE_FOR : KSW_EZ_SPLICE_REV; if (opt->flag & MM_F_SPLICE_FLANK) extra_flag |= KSW_EZ_SPLICE_FLANK; } /* Look for the start and end of regions to perform DP. This sounds easy * but is in fact tricky. Excessively small regions lead to unnecessary * clippings and lose alignable sequences. Excessively large regions * occasionally lead to large overlaps between two chains and may cause * loss of alignments in corner cases. */ if (is_sr) { qs0 = 0, qe0 = qlen; l = qs; l += l * opt->a + opt->end_bonus > opt->q? (l * opt->a + opt->end_bonus - opt->q) / opt->e : 0; rs0 = rs - l > 0? rs - l : 0; l = qlen - qe; l += l * opt->a + opt->end_bonus > opt->q? (l * opt->a + opt->end_bonus - opt->q) / opt->e : 0; re0 = re + l < (int32_t)mi->seq[rid].len? re + l : mi->seq[rid].len; } else { // compute rs0 and qs0 rs0 = (int32_t)a[r->as].x + 1 - (int32_t)(a[r->as].y>>32&0xff); qs0 = (int32_t)a[r->as].y + 1 - (int32_t)(a[r->as].y>>32&0xff); if (rs0 < 0) rs0 = 0; // this may happen when HPC is in use assert(qs0 >= 0); // this should never happen, or it is logic error rs1 = qs1 = 0; for (i = r->as - 1, l = 0; i >= 0 && a[i].x>>32 == a[r->as].x>>32; --i) { // inspect nearby seeds int32_t x = (int32_t)a[i].x + 1 - (int32_t)(a[i].y>>32&0xff); int32_t y = (int32_t)a[i].y + 1 - (int32_t)(a[i].y>>32&0xff); if (x < rs0 && y < qs0) { if (++l > opt->min_cnt) { l = rs0 - x > qs0 - y? rs0 - x : qs0 - y; rs1 = rs0 - l, qs1 = qs0 - l; if (rs1 < 0) rs1 = 0; // not strictly necessary; better have this guard for explicit break; } } } if (qs > 0 && rs > 0) { l = qs < opt->max_gap? qs : opt->max_gap; qs1 = qs1 > qs - l? qs1 : qs - l; qs0 = qs0 < qs1? qs0 : qs1; // at least include qs0 l += l * opt->a > opt->q? (l * opt->a - opt->q) / opt->e : 0; l = l < opt->max_gap? l : opt->max_gap; l = l < rs? l : rs; rs1 = rs1 > rs - l? rs1 : rs - l; rs0 = rs0 < rs1? rs0 : rs1; rs0 = rs0 < rs? rs0 : rs; } else rs0 = rs, qs0 = qs; // compute re0 and qe0 re0 = (int32_t)a[r->as + r->cnt - 1].x + 1; qe0 = (int32_t)a[r->as + r->cnt - 1].y + 1; re1 = mi->seq[rid].len, qe1 = qlen; for (i = r->as + r->cnt, l = 0; i < n_a && a[i].x>>32 == a[r->as].x>>32; ++i) { // inspect nearby seeds int32_t x = (int32_t)a[i].x + 1; int32_t y = (int32_t)a[i].y + 1; if (x > re0 && y > qe0) { if (++l > opt->min_cnt) { l = x - re0 > y - qe0? x - re0 : y - qe0; re1 = re0 + l, qe1 = qe0 + l; break; } } } if (qe < qlen && re < (int32_t)mi->seq[rid].len) { l = qlen - qe < opt->max_gap? qlen - qe : opt->max_gap; qe1 = qe1 < qe + l? qe1 : qe + l; qe0 = qe0 > qe1? qe0 : qe1; // at least include qe0 l += l * opt->a > opt->q? (l * opt->a - opt->q) / opt->e : 0; l = l < opt->max_gap? l : opt->max_gap; l = l < (int32_t)mi->seq[rid].len - re? l : mi->seq[rid].len - re; re1 = re1 < re + l? re1 : re + l; re0 = re0 > re1? re0 : re1; } else re0 = re, qe0 = qe; } if (a[r->as].y & MM_SEED_SELF) { int max_ext = r->qs > r->rs? r->qs - r->rs : r->rs - r->qs; if (r->rs - rs0 > max_ext) rs0 = r->rs - max_ext; if (r->qs - qs0 > max_ext) qs0 = r->qs - max_ext; max_ext = r->qe > r->re? r->qe - r->re : r->re - r->qe; if (re0 - r->re > max_ext) re0 = r->re + max_ext; if (qe0 - r->qe > max_ext) qe0 = r->qe + max_ext; } assert(re0 > rs0); tseq = (uint8_t*)kmalloc(km, re0 - rs0); junc = (uint8_t*)kmalloc(km, re0 - rs0); if (qs > 0 && rs > 0) { // left extension; probably the condition can be changed to "qs > qs0 && rs > rs0" if (opt->flag & MM_F_QSTRAND) { qseq = &qseq0[0][qs0]; mm_idx_getseq2(mi, rev, rid, rs0, rs, tseq); } else { qseq = &qseq0[rev][qs0]; mm_idx_getseq(mi, rid, rs0, rs, tseq); } mm_idx_bed_junc(mi, rid, rs0, rs, junc); mm_seq_rev(qs - qs0, qseq); mm_seq_rev(rs - rs0, tseq); mm_seq_rev(rs - rs0, junc); mm_align_pair(km, opt, qs - qs0, qseq, rs - rs0, tseq, junc, mat, bw, opt->end_bonus, r->split_inv? opt->zdrop_inv : opt->zdrop, extra_flag|KSW_EZ_EXTZ_ONLY|KSW_EZ_RIGHT|KSW_EZ_REV_CIGAR, ez); if (ez->n_cigar > 0) { mm_append_cigar(r, ez->n_cigar, ez->cigar); r->p->dp_score += ez->max; } rs1 = rs - (ez->reach_end? ez->mqe_t + 1 : ez->max_t + 1); qs1 = qs - (ez->reach_end? qs - qs0 : ez->max_q + 1); mm_seq_rev(qs - qs0, qseq); } else rs1 = rs, qs1 = qs; re1 = rs, qe1 = qs; assert(qs1 >= 0 && rs1 >= 0); for (i = is_sr? cnt1 - 1 : 1; i < cnt1; ++i) { // gap filling if ((a[as1+i].y & (MM_SEED_IGNORE|MM_SEED_TANDEM)) && i != cnt1 - 1) continue; if (is_sr && !(mi->flag & MM_I_HPC)) { re = (int32_t)a[as1 + i].x + 1; qe = (int32_t)a[as1 + i].y + 1; } else mm_adjust_minier(mi, qseq0, &a[as1 + i], &re, &qe); re1 = re, qe1 = qe; if (i == cnt1 - 1 || (a[as1+i].y&MM_SEED_LONG_JOIN) || (qe - qs >= opt->min_ksw_len && re - rs >= opt->min_ksw_len)) { int j, bw1 = bw_long, zdrop_code; if (a[as1+i].y & MM_SEED_LONG_JOIN) bw1 = qe - qs > re - rs? qe - qs : re - rs; // perform alignment if (opt->flag & MM_F_QSTRAND) { qseq = &qseq0[0][qs]; mm_idx_getseq2(mi, rev, rid, rs, re, tseq); } else { qseq = &qseq0[rev][qs]; mm_idx_getseq(mi, rid, rs, re, tseq); } mm_idx_bed_junc(mi, rid, rs, re, junc); if (is_sr) { // perform ungapped alignment assert(qe - qs == re - rs); ksw_reset_extz(ez); for (j = 0, ez->score = 0; j < qe - qs; ++j) { if (qseq[j] >= 4 || tseq[j] >= 4) ez->score += opt->e2; else ez->score += qseq[j] == tseq[j]? opt->a : -opt->b; } ez->cigar = ksw_push_cigar(km, &ez->n_cigar, &ez->m_cigar, ez->cigar, MM_CIGAR_MATCH, qe - qs); } else { // perform normal gapped alignment mm_align_pair(km, opt, qe - qs, qseq, re - rs, tseq, junc, mat, bw1, -1, opt->zdrop, extra_flag|KSW_EZ_APPROX_MAX, ez); // first pass: with approximate Z-drop } // test Z-drop and inversion Z-drop if ((zdrop_code = mm_test_zdrop(km, opt, qseq, tseq, ez->n_cigar, ez->cigar, mat)) != 0) mm_align_pair(km, opt, qe - qs, qseq, re - rs, tseq, junc, mat, bw1, -1, zdrop_code == 2? opt->zdrop_inv : opt->zdrop, extra_flag, ez); // second pass: lift approximate // update CIGAR if (ez->n_cigar > 0) mm_append_cigar(r, ez->n_cigar, ez->cigar); if (ez->zdropped) { // truncated by Z-drop; TODO: sometimes Z-drop kicks in because the next seed placement is wrong. This can be fixed in principle. if (!r->p) { assert(ez->n_cigar == 0); uint32_t capacity = sizeof(mm_extra_t)/4; kroundup32(capacity); r->p = (mm_extra_t*)calloc(capacity, 4); r->p->capacity = capacity; } for (j = i - 1; j >= 0; --j) if ((int32_t)a[as1 + j].x <= rs + ez->max_t) break; dropped = 1; if (j < 0) j = 0; r->p->dp_score += ez->max; re1 = rs + (ez->max_t + 1); qe1 = qs + (ez->max_q + 1); if (cnt1 - (j + 1) >= opt->min_cnt) { mm_split_reg(r, r2, as1 + j + 1 - r->as, qlen, a, !!(opt->flag&MM_F_QSTRAND)); if (zdrop_code == 2) r2->split_inv = 1; } break; } else r->p->dp_score += ez->score; rs = re, qs = qe; } } if (!dropped && qe < qe0 && re < re0) { // right extension if (opt->flag & MM_F_QSTRAND) { qseq = &qseq0[0][qe]; mm_idx_getseq2(mi, rev, rid, re, re0, tseq); } else { qseq = &qseq0[rev][qe]; mm_idx_getseq(mi, rid, re, re0, tseq); } mm_idx_bed_junc(mi, rid, re, re0, junc); mm_align_pair(km, opt, qe0 - qe, qseq, re0 - re, tseq, junc, mat, bw, opt->end_bonus, opt->zdrop, extra_flag|KSW_EZ_EXTZ_ONLY, ez); if (ez->n_cigar > 0) { mm_append_cigar(r, ez->n_cigar, ez->cigar); r->p->dp_score += ez->max; } re1 = re + (ez->reach_end? ez->mqe_t + 1 : ez->max_t + 1); qe1 = qe + (ez->reach_end? qe0 - qe : ez->max_q + 1); } assert(qe1 <= qlen); r->rs = rs1, r->re = re1; if (!rev || (opt->flag & MM_F_QSTRAND)) r->qs = qs1, r->qe = qe1; else r->qs = qlen - qe1, r->qe = qlen - qs1; assert(re1 - rs1 <= re0 - rs0); if (r->p) { if (opt->flag & MM_F_QSTRAND) { mm_idx_getseq2(mi, r->rev, rid, rs1, re1, tseq); qseq = &qseq0[0][qs1]; } else { mm_idx_getseq(mi, rid, rs1, re1, tseq); qseq = &qseq0[r->rev][qs1]; } mm_update_extra(r, qseq, tseq, mat, opt->q, opt->e, opt->flag & MM_F_EQX, !(opt->flag & MM_F_SR)); if (rev && r->p->trans_strand) r->p->trans_strand ^= 3; // flip to the read strand } kfree(km, tseq); kfree(km, junc); } static int mm_align1_inv(void *km, const mm_mapopt_t *opt, const mm_idx_t *mi, int qlen, uint8_t *qseq0[2], const mm_reg1_t *r1, const mm_reg1_t *r2, mm_reg1_t *r_inv, ksw_extz_t *ez) { // NB: this doesn't work with the qstrand mode int tl, ql, score, ret = 0, q_off, t_off; uint8_t *tseq, *qseq; int8_t mat[25]; void *qp; memset(r_inv, 0, sizeof(mm_reg1_t)); if (!(r1->split&1) || !(r2->split&2)) return 0; if (r1->id != r1->parent && r1->parent != MM_PARENT_TMP_PRI) return 0; if (r2->id != r2->parent && r2->parent != MM_PARENT_TMP_PRI) return 0; if (r1->rid != r2->rid || r1->rev != r2->rev) return 0; ql = r1->rev? r1->qs - r2->qe : r2->qs - r1->qe; tl = r2->rs - r1->re; if (ql < opt->min_chain_score || ql > opt->max_gap) return 0; if (tl < opt->min_chain_score || tl > opt->max_gap) return 0; ksw_gen_simple_mat(5, mat, opt->a, opt->b, opt->sc_ambi); tseq = (uint8_t*)kmalloc(km, tl); mm_idx_getseq(mi, r1->rid, r1->re, r2->rs, tseq); qseq = r1->rev? &qseq0[0][r2->qe] : &qseq0[1][qlen - r2->qs]; mm_seq_rev(ql, qseq); mm_seq_rev(tl, tseq); qp = ksw_ll_qinit(km, 2, ql, qseq, 5, mat); score = ksw_ll_i16(qp, tl, tseq, opt->q, opt->e, &q_off, &t_off); kfree(km, qp); mm_seq_rev(ql, qseq); mm_seq_rev(tl, tseq); if (score < opt->min_dp_max) goto end_align1_inv; q_off = ql - (q_off + 1), t_off = tl - (t_off + 1); mm_align_pair(km, opt, ql - q_off, qseq + q_off, tl - t_off, tseq + t_off, 0, mat, (int)(opt->bw * 1.5), -1, opt->zdrop, KSW_EZ_EXTZ_ONLY, ez); if (ez->n_cigar == 0) goto end_align1_inv; // should never be here mm_append_cigar(r_inv, ez->n_cigar, ez->cigar); r_inv->p->dp_score = ez->max; r_inv->id = -1; r_inv->parent = MM_PARENT_UNSET; r_inv->inv = 1; r_inv->rev = !r1->rev; r_inv->rid = r1->rid; r_inv->div = -1.0f; if (r_inv->rev == 0) { r_inv->qs = r2->qe + q_off; r_inv->qe = r_inv->qs + ez->max_q + 1; } else { r_inv->qe = r2->qs - q_off; r_inv->qs = r_inv->qe - (ez->max_q + 1); } r_inv->rs = r1->re + t_off; r_inv->re = r_inv->rs + ez->max_t + 1; mm_update_extra(r_inv, &qseq[q_off], &tseq[t_off], mat, opt->q, opt->e, opt->flag & MM_F_EQX, !(opt->flag & MM_F_SR)); ret = 1; end_align1_inv: kfree(km, tseq); return ret; } static inline mm_reg1_t *mm_insert_reg(const mm_reg1_t *r, int i, int *n_regs, mm_reg1_t *regs) { regs = (mm_reg1_t*)realloc(regs, (*n_regs + 1) * sizeof(mm_reg1_t)); if (i + 1 != *n_regs) memmove(®s[i + 2], ®s[i + 1], sizeof(mm_reg1_t) * (*n_regs - i - 1)); regs[i + 1] = *r; ++*n_regs; return regs; } static inline void mm_count_gaps(const mm_reg1_t *r, int32_t *n_gap_, int32_t *n_gapo_) { uint32_t i; int32_t n_gapo = 0, n_gap = 0; *n_gap_ = *n_gapo_ = -1; if (r->p == 0) return; for (i = 0; i < r->p->n_cigar; ++i) { int32_t op = r->p->cigar[i] & 0xf, len = r->p->cigar[i] >> 4; if (op == MM_CIGAR_INS || op == MM_CIGAR_DEL) ++n_gapo, n_gap += len; } *n_gap_ = n_gap, *n_gapo_ = n_gapo; } double mm_event_identity(const mm_reg1_t *r) { int32_t n_gap, n_gapo; if (r->p == 0) return -1.0f; mm_count_gaps(r, &n_gap, &n_gapo); return (double)r->mlen / (r->blen + r->p->n_ambi - n_gap + n_gapo); } static int32_t mm_recal_max_dp(const mm_reg1_t *r, double b2, int32_t match_sc) { uint32_t i; int32_t n_gap = 0, n_gapo = 0, n_mis; double gap_cost = 0.0; if (r->p == 0) return -1; for (i = 0; i < r->p->n_cigar; ++i) { int32_t op = r->p->cigar[i] & 0xf, len = r->p->cigar[i] >> 4; if (op == MM_CIGAR_INS || op == MM_CIGAR_DEL) { gap_cost += b2 + (double)mg_log2(1.0 + len); ++n_gapo, n_gap += len; } } n_mis = r->blen + r->p->n_ambi - r->mlen - n_gap; return (int32_t)(match_sc * (r->mlen - b2 * n_mis - gap_cost) + .499); } void mm_update_dp_max(int qlen, int n_regs, mm_reg1_t *regs, float frac, int a, int b) { int32_t max = -1, max2 = -1, i, max_i = -1; double div, b2; if (n_regs < 2) return; for (i = 0; i < n_regs; ++i) { mm_reg1_t *r = ®s[i]; if (r->p == 0) continue; if (r->p->dp_max > max) max2 = max, max = r->p->dp_max, max_i = i; else if (r->p->dp_max > max2) max2 = r->p->dp_max; } if (max_i < 0 || max < 0 || max2 < 0) return; if (regs[max_i].qe - regs[max_i].qs < (double)qlen * frac) return; if (max2 < (double)max * frac) return; div = 1. - mm_event_identity(®s[max_i]); if (div < 0.02) div = 0.02; b2 = 0.5 / div; // max value: 25 if (b2 * a < b) b2 = (double)a / b; for (i = 0; i < n_regs; ++i) { mm_reg1_t *r = ®s[i]; if (r->p == 0) continue; r->p->dp_max = mm_recal_max_dp(r, b2, a); if (r->p->dp_max < 0) r->p->dp_max = 0; } } mm_reg1_t *mm_align_skeleton(void *km, const mm_mapopt_t *opt, const mm_idx_t *mi, int qlen, const char *qstr, int *n_regs_, mm_reg1_t *regs, mm128_t *a) { extern unsigned char seq_nt4_table[256]; int32_t i, n_regs = *n_regs_, n_a; uint8_t *qseq0[2]; ksw_extz_t ez; // encode the query sequence qseq0[0] = (uint8_t*)kmalloc(km, qlen * 2); qseq0[1] = qseq0[0] + qlen; for (i = 0; i < qlen; ++i) { qseq0[0][i] = seq_nt4_table[(uint8_t)qstr[i]]; qseq0[1][qlen - 1 - i] = qseq0[0][i] < 4? 3 - qseq0[0][i] : 4; } // align through seed hits n_a = mm_squeeze_a(km, n_regs, regs, a); memset(&ez, 0, sizeof(ksw_extz_t)); for (i = 0; i < n_regs; ++i) { mm_reg1_t r2; if ((opt->flag&MM_F_SPLICE) && (opt->flag&MM_F_SPLICE_FOR) && (opt->flag&MM_F_SPLICE_REV)) { // then do two rounds of alignments for both strands mm_reg1_t s[2], s2[2]; int which, trans_strand; s[0] = s[1] = regs[i]; mm_align1(km, opt, mi, qlen, qseq0, &s[0], &s2[0], n_a, a, &ez, MM_F_SPLICE_FOR); mm_align1(km, opt, mi, qlen, qseq0, &s[1], &s2[1], n_a, a, &ez, MM_F_SPLICE_REV); if (s[0].p->dp_score > s[1].p->dp_score) which = 0, trans_strand = 1; else if (s[0].p->dp_score < s[1].p->dp_score) which = 1, trans_strand = 2; else trans_strand = 3, which = (qlen + s[0].p->dp_score) & 1; // randomly choose a strand, effectively if (which == 0) { regs[i] = s[0], r2 = s2[0]; free(s[1].p); } else { regs[i] = s[1], r2 = s2[1]; free(s[0].p); } regs[i].p->trans_strand = trans_strand; } else { // one round of alignment mm_align1(km, opt, mi, qlen, qseq0, ®s[i], &r2, n_a, a, &ez, opt->flag); if (opt->flag&MM_F_SPLICE) regs[i].p->trans_strand = opt->flag&MM_F_SPLICE_FOR? 1 : 2; } if (r2.cnt > 0) regs = mm_insert_reg(&r2, i, &n_regs, regs); if (i > 0 && regs[i].split_inv && !(opt->flag & MM_F_NO_INV)) { if (mm_align1_inv(km, opt, mi, qlen, qseq0, ®s[i-1], ®s[i], &r2, &ez)) { regs = mm_insert_reg(&r2, i, &n_regs, regs); ++i; // skip the inserted INV alignment } } } *n_regs_ = n_regs; kfree(km, qseq0[0]); kfree(km, ez.cigar); mm_filter_regs(opt, qlen, n_regs_, regs); if (!(opt->flag&MM_F_SR) && !opt->split_prefix && qlen >= opt->rank_min_len) { mm_update_dp_max(qlen, *n_regs_, regs, opt->rank_frac, opt->a, opt->b); mm_filter_regs(opt, qlen, n_regs_, regs); } mm_hit_sort(km, n_regs_, regs, opt->alt_drop); return regs; }