#include #include #include #include #include #ifdef HAVE_PTHREAD #include #endif #include "kstring.h" #include "bwamem.h" #include "bntseq.h" #include "ksw.h" #include "ksort.h" #define MAPQ_COEF 40. int mem_verbose = 3; // 1: error only; 2: error+warning; 3: message+error+warning; >=4: debugging void mem_fill_scmat(int a, int b, int8_t mat[25]) { int i, j, k; for (i = k = 0; i < 4; ++i) { for (j = 0; j < 4; ++j) mat[k++] = i == j? a : -b; mat[k++] = 0; // ambiguous base } for (j = 0; j < 5; ++j) mat[k++] = 0; } mem_opt_t *mem_opt_init() { mem_opt_t *o; o = calloc(1, sizeof(mem_opt_t)); o->a = 1; o->b = 5; o->q = 8; o->r = 1; o->w = 100; o->min_seed_len = 19; o->split_width = 10; o->max_occ = 10000; o->max_chain_gap = 10000; o->max_ins = 10000; o->mask_level = 0.50; o->chain_drop_ratio = 0.50; o->chunk_size = 10000000; o->n_threads = 1; o->pe_dir = 0<<1|1; o->is_pe = 0; mem_fill_scmat(o->a, o->b, o->mat); return o; } /*************************** * SMEM iterator interface * ***************************/ struct __smem_i { const bwt_t *bwt; const uint8_t *query; int start, len; bwtintv_v *matches; // matches; to be returned by smem_next() bwtintv_v *sub; // sub-matches inside the longest match; temporary bwtintv_v *tmpvec[2]; // temporary arrays }; smem_i *smem_itr_init(const bwt_t *bwt) { smem_i *itr; itr = calloc(1, sizeof(smem_i)); itr->bwt = bwt; itr->tmpvec[0] = calloc(1, sizeof(bwtintv_v)); itr->tmpvec[1] = calloc(1, sizeof(bwtintv_v)); itr->matches = calloc(1, sizeof(bwtintv_v)); itr->sub = calloc(1, sizeof(bwtintv_v)); return itr; } void smem_itr_destroy(smem_i *itr) { free(itr->tmpvec[0]->a); free(itr->tmpvec[0]); free(itr->tmpvec[1]->a); free(itr->tmpvec[1]); free(itr->matches->a); free(itr->matches); free(itr->sub->a); free(itr->sub); free(itr); } void smem_set_query(smem_i *itr, int len, const uint8_t *query) { itr->query = query; itr->start = 0; itr->len = len; } const bwtintv_v *smem_next(smem_i *itr, int split_len, int split_width) { int i, max, max_i, ori_start; itr->tmpvec[0]->n = itr->tmpvec[1]->n = itr->matches->n = itr->sub->n = 0; if (itr->start >= itr->len || itr->start < 0) return 0; while (itr->start < itr->len && itr->query[itr->start] > 3) ++itr->start; // skip ambiguous bases if (itr->start == itr->len) return 0; ori_start = itr->start; itr->start = bwt_smem1(itr->bwt, itr->len, itr->query, ori_start, 1, itr->matches, itr->tmpvec); // search for SMEM if (itr->matches->n == 0) return itr->matches; // well, in theory, we should never come here for (i = max = 0, max_i = 0; i < itr->matches->n; ++i) { // look for the longest match bwtintv_t *p = &itr->matches->a[i]; int len = (uint32_t)p->info - (p->info>>32); if (max < len) max = len, max_i = i; } if (split_len > 0 && max >= split_len && itr->matches->a[max_i].x[2] <= split_width) { // if the longest SMEM is unique and long int j; bwtintv_v *a = itr->tmpvec[0]; // reuse tmpvec[0] for merging bwtintv_t *p = &itr->matches->a[max_i]; bwt_smem1(itr->bwt, itr->len, itr->query, ((uint32_t)p->info + (p->info>>32))>>1, itr->matches->a[max_i].x[2]+1, itr->sub, itr->tmpvec); // starting from the middle of the longest MEM i = j = 0; a->n = 0; while (i < itr->matches->n && j < itr->sub->n) { // ordered merge int64_t xi = itr->matches->a[i].info>>32<<32 | (itr->len - (uint32_t)itr->matches->a[i].info); int64_t xj = itr->sub->a[j].info>>32<<32 | (itr->len - (uint32_t)itr->sub->a[j].info); if (xi < xj) { kv_push(bwtintv_t, *a, itr->matches->a[i]); ++i; } else if ((uint32_t)itr->sub->a[j].info - (itr->sub->a[j].info>>32) >= max>>1 && (uint32_t)itr->sub->a[j].info > ori_start) { kv_push(bwtintv_t, *a, itr->sub->a[j]); ++j; } else ++j; } for (; i < itr->matches->n; ++i) kv_push(bwtintv_t, *a, itr->matches->a[i]); for (; j < itr->sub->n; ++j) if ((uint32_t)itr->sub->a[j].info - (itr->sub->a[j].info>>32) >= max>>1 && (uint32_t)itr->sub->a[j].info > ori_start) kv_push(bwtintv_t, *a, itr->sub->a[j]); kv_copy(bwtintv_t, *itr->matches, *a); } return itr->matches; } /******************************** * Chaining while finding SMEMs * ********************************/ #include "kbtree.h" #define chain_cmp(a, b) ((a).pos - (b).pos) KBTREE_INIT(chn, mem_chain_t, chain_cmp) static int test_and_merge(const mem_opt_t *opt, mem_chain_t *c, const mem_seed_t *p) { int64_t qend, rend, x, y; const mem_seed_t *last = &c->seeds[c->n-1]; qend = last->qbeg + last->len; rend = last->rbeg + last->len; if (p->qbeg >= c->seeds[0].qbeg && p->qbeg + p->len <= qend && p->rbeg >= c->seeds[0].rbeg && p->rbeg + p->len <= rend) return 1; // contained seed; do nothing x = p->qbeg - last->qbeg; // always non-negtive y = p->rbeg - last->rbeg; if (y >= 0 && x - y <= opt->w && y - x <= opt->w && x - last->len < opt->max_chain_gap && y - last->len < opt->max_chain_gap) { // grow the chain if (c->n == c->m) { c->m <<= 1; c->seeds = realloc(c->seeds, c->m * sizeof(mem_seed_t)); } c->seeds[c->n++] = *p; return 1; } return 0; // request to add a new chain } static void mem_insert_seed(const mem_opt_t *opt, kbtree_t(chn) *tree, smem_i *itr) { const bwtintv_v *a; while ((a = smem_next(itr, opt->min_seed_len<<1, opt->split_width)) != 0) { // to find all SMEM and some internal MEM int i; for (i = 0; i < a->n; ++i) { // go through each SMEM/MEM up to itr->start bwtintv_t *p = &a->a[i]; int slen = (uint32_t)p->info - (p->info>>32); // seed length int64_t k; if (slen < opt->min_seed_len || p->x[2] > opt->max_occ) continue; // ignore if too short or too repetitive for (k = 0; k < p->x[2]; ++k) { mem_chain_t tmp, *lower, *upper; mem_seed_t s; int to_add = 0; s.rbeg = tmp.pos = bwt_sa(itr->bwt, p->x[0] + k); // this is the base coordinate in the forward-reverse reference s.qbeg = p->info>>32; s.len = slen; if (kb_size(tree)) { kb_intervalp(chn, tree, &tmp, &lower, &upper); // find the closest chain if (!lower || !test_and_merge(opt, lower, &s)) to_add = 1; } else to_add = 1; if (to_add) { // add the seed as a new chain tmp.n = 1; tmp.m = 4; tmp.seeds = calloc(tmp.m, sizeof(mem_seed_t)); tmp.seeds[0] = s; kb_putp(chn, tree, &tmp); } } } } } void mem_print_chain(const bntseq_t *bns, mem_chain_v *chn) { int i, j; for (i = 0; i < chn->n; ++i) { mem_chain_t *p = &chn->a[i]; printf("%d", p->n); for (j = 0; j < p->n; ++j) { bwtint_t pos; int is_rev, ref_id; pos = bns_depos(bns, p->seeds[j].rbeg, &is_rev); if (is_rev) pos -= p->seeds[j].len - 1; bns_cnt_ambi(bns, pos, p->seeds[j].len, &ref_id); printf("\t%d,%d,%ld(%s:%c%ld)", p->seeds[j].len, p->seeds[j].qbeg, (long)p->seeds[j].rbeg, bns->anns[ref_id].name, "+-"[is_rev], (long)(pos - bns->anns[ref_id].offset) + 1); } putchar('\n'); } } mem_chain_v mem_chain(const mem_opt_t *opt, const bwt_t *bwt, int len, const uint8_t *seq) { mem_chain_v chain; smem_i *itr; kbtree_t(chn) *tree; kv_init(chain); if (len < opt->min_seed_len) return chain; // if the query is shorter than the seed length, no match tree = kb_init(chn, KB_DEFAULT_SIZE); itr = smem_itr_init(bwt); smem_set_query(itr, len, seq); mem_insert_seed(opt, tree, itr); kv_resize(mem_chain_t, chain, kb_size(tree)); #define traverse_func(p_) (chain.a[chain.n++] = *(p_)) __kb_traverse(mem_chain_t, tree, traverse_func); #undef traverse_func smem_itr_destroy(itr); kb_destroy(chn, tree); return chain; } /******************** * Filtering chains * ********************/ typedef struct { int beg, end, w; void *p, *p2; } flt_aux_t; #define flt_lt(a, b) ((a).w > (b).w) KSORT_INIT(mem_flt, flt_aux_t, flt_lt) int mem_chain_flt(const mem_opt_t *opt, int n_chn, mem_chain_t *chains) { flt_aux_t *a; int i, j, n; if (n_chn <= 1) return n_chn; // no need to filter a = malloc(sizeof(flt_aux_t) * n_chn); for (i = 0; i < n_chn; ++i) { mem_chain_t *c = &chains[i]; int64_t end; int w = 0, tmp; for (j = 0, end = 0; j < c->n; ++j) { const mem_seed_t *s = &c->seeds[j]; if (s->qbeg >= end) w += s->len; else if (s->qbeg + s->len > end) w += s->qbeg + s->len - end; end = end > s->qbeg + s->len? end : s->qbeg + s->len; } tmp = w; for (j = 0, end = 0; j < c->n; ++j) { const mem_seed_t *s = &c->seeds[j]; if (s->rbeg >= end) w += s->len; else if (s->rbeg + s->len > end) w += s->rbeg + s->len - end; end = end > s->qbeg + s->len? end : s->qbeg + s->len; } w = w < tmp? w : tmp; a[i].beg = c->seeds[0].qbeg; a[i].end = c->seeds[c->n-1].qbeg + c->seeds[c->n-1].len; a[i].w = w; a[i].p = c; a[i].p2 = 0; } ks_introsort(mem_flt, n_chn, a); { // reorder chains such that the best chain appears first mem_chain_t *swap; swap = malloc(sizeof(mem_chain_t) * n_chn); for (i = 0; i < n_chn; ++i) { swap[i] = *((mem_chain_t*)a[i].p); a[i].p = &chains[i]; // as we will memcpy() below, a[i].p is changed } memcpy(chains, swap, sizeof(mem_chain_t) * n_chn); free(swap); } for (i = 1, n = 1; i < n_chn; ++i) { for (j = 0; j < n; ++j) { int b_max = a[j].beg > a[i].beg? a[j].beg : a[i].beg; int e_min = a[j].end < a[i].end? a[j].end : a[i].end; if (e_min > b_max) { // have overlap int min_l = a[i].end - a[i].beg < a[j].end - a[j].beg? a[i].end - a[i].beg : a[j].end - a[j].beg; if (e_min - b_max >= min_l * opt->mask_level) { // significant overlap if (a[j].p2 == 0) a[j].p2 = a[i].p; if (a[i].w < a[j].w * opt->chain_drop_ratio) break; } } } if (j == n) a[n++] = a[i]; // if have no significant overlap with better chains, keep it. } for (i = 0; i < n; ++i) { // mark chains to be kept mem_chain_t *c = (mem_chain_t*)a[i].p; if (c->n > 0) c->n = -c->n; c = (mem_chain_t*)a[i].p2; if (c && c->n > 0) c->n = -c->n; } free(a); for (i = 0; i < n_chn; ++i) { // free discarded chains mem_chain_t *c = &chains[i]; if (c->n >= 0) { free(c->seeds); c->n = c->m = 0; } else c->n = -c->n; } for (i = n = 0; i < n_chn; ++i) { // squeeze out discarded chains if (chains[i].n > 0) { if (n != i) chains[n++] = chains[i]; else ++n; } } return n; } /****************************** * De-overlap single-end hits * ******************************/ #define alnreg_slt(a, b) ((a).score > (b).score || ((a).score == (b).score && ((a).rb < (b).rb || ((a).rb == (b).rb && (a).qb < (b).qb)))) KSORT_INIT(mem_ars, mem_alnreg_t, alnreg_slt) int mem_sort_and_dedup(int n, mem_alnreg_t *a) { int m, i; if (n <= 1) return n; ks_introsort(mem_ars, n, a); for (i = 1; i < n; ++i) { // mark identical hits if (a[i].score == a[i-1].score && a[i].rb == a[i-1].rb && a[i].qb == a[i-1].qb) a[i].qe = a[i].qb; } for (i = 1, m = 1; i < n; ++i) // exclude identical hits if (a[i].qe > a[i].qb) a[m++] = a[i]; return m; } int mem_choose_alnreg_se(const mem_opt_t *opt, int n, mem_alnreg_t *a) // IMPORTANT: must run mem_sort_and_dedup() before calling this function { // similar to the loop in mem_chain_flt() int i, j, m, tmp; if (n <= 1) return n; for (i = 0; i < n; ++i) a[i].sub = 0; tmp = opt->a + opt->b > opt->q + opt->r? opt->a + opt->b : opt->q + opt->r; for (i = 1, m = 1; i < n; ++i) { for (j = 0; j < m; ++j) { int b_max = a[j].qb > a[i].qb? a[j].qb : a[i].qb; int e_min = a[j].qe < a[i].qe? a[j].qe : a[i].qe; if (e_min > b_max) { // have overlap int min_l = a[i].qe - a[i].qb < a[j].qe - a[j].qb? a[i].qe - a[i].qb : a[j].qe - a[j].qb; if (e_min - b_max >= min_l * opt->mask_level) { // significant overlap if (a[j].sub == 0) a[j].sub = a[i].score; if (a[j].score - a[i].score <= tmp) ++a[j].sub_n; break; } } } if (j == m) a[m++] = a[i]; } return m; } /************************ * Pick paired-end hits * ************************/ /**************************************** * Construct the alignment from a chain * ****************************************/ static inline int cal_max_gap(const mem_opt_t *opt, int qlen) { int l = (int)((double)(qlen * opt->a - opt->q) / opt->r + 1.); return l > 1? l : 1; } void mem_chain2aln(const mem_opt_t *opt, int64_t l_pac, const uint8_t *pac, int l_query, const uint8_t *query, const mem_chain_t *c, mem_alnreg_t *a) { // FIXME: in general, we SHOULD check funny seed patterns such as contained seeds. When that happens, we should use a SW or extend more seeds int i, k, csub = 0; int64_t rlen, rmax[2], tmp, max = 0, max_i = 0; const mem_seed_t *s; uint8_t *rseq = 0; mem_alnreg_t best; memset(&best, 0, sizeof(mem_alnreg_t)); // get the max possible span rmax[0] = l_pac<<1; rmax[1] = 0; for (i = 0; i < c->n; ++i) { int64_t b, e; const mem_seed_t *t = &c->seeds[i]; b = t->rbeg - (t->qbeg + cal_max_gap(opt, t->qbeg)); e = t->rbeg + t->len + ((l_query - t->qbeg - t->len) + cal_max_gap(opt, l_query - t->qbeg - t->len)); rmax[0] = rmax[0] < b? rmax[0] : b; rmax[1] = rmax[1] > e? rmax[1] : e; if (t->len > max) max = t->len, max_i = i; } // retrieve the reference sequence rseq = bns_get_seq(l_pac, pac, rmax[0], rmax[1], &rlen); for (k = 0; k < c->n;) { s = &c->seeds[k]; memset(a, 0, sizeof(mem_alnreg_t)); if (s->qbeg) { // left extension uint8_t *rs, *qs; int qle, tle; qs = malloc(s->qbeg); for (i = 0; i < s->qbeg; ++i) qs[i] = query[s->qbeg - 1 - i]; tmp = s->rbeg - rmax[0]; rs = malloc(tmp); for (i = 0; i < tmp; ++i) rs[i] = rseq[tmp - 1 - i]; a->score = ksw_extend(s->qbeg, qs, tmp, rs, 5, opt->mat, opt->q, opt->r, opt->w, s->len * opt->a, &qle, &tle); a->qb = s->qbeg - qle; a->rb = s->rbeg - tle; free(qs); free(rs); } else a->score = s->len * opt->a, a->qb = 0, a->rb = s->rbeg; if (s->qbeg + s->len != l_query) { // right extension of the first seed int qle, tle, qe, re; qe = s->qbeg + s->len; re = s->rbeg + s->len - rmax[0]; a->score = ksw_extend(l_query - qe, query + qe, rmax[1] - rmax[0] - re, rseq + re, 5, opt->mat, opt->q, opt->r, opt->w, a->score, &qle, &tle); a->qe = qe + qle; a->re = rmax[0] + re + tle; } else a->qe = l_query, a->re = s->rbeg + s->len; if (mem_verbose >= 4) printf("[%d] score=%d\t[%d,%d) <=> [%ld,%ld)\n", k, a->score, a->qb, a->qe, (long)a->rb, (long)a->re); // check how many seeds have been covered for (i = k + 1; i < c->n; ++i) { const mem_seed_t *t = &c->seeds[i]; if (t->rbeg + t->len > a->re || t->qbeg + t->len > a->qe) break; } if (a->score >= best.score) csub = best.score, best = *a; if (i >= c->n) break; // all seeds are included; no need to proceed k = i; } if (a->score < best.score) *a = best; a->csub = csub; free(rseq); // compute seedcov if (c->n > 1) { for (i = 0, a->seedcov = 0; i < c->n; ++i) { s = &c->seeds[i]; if (s->qbeg >= a->qb && s->qbeg + s->len <= a->qe && s->rbeg >= a->rb && s->rbeg + s->len <= a->re) // seed fully contained a->seedcov += s->len; // this is not very accurate, but for approx. mapQ, this is good enough } } else a->seedcov = c->seeds[0].len; } /***************************** * Basic hit->SAM conversion * *****************************/ uint32_t *bwa_gen_cigar(const int8_t mat[25], int q, int r, int w_, int64_t l_pac, const uint8_t *pac, int l_query, uint8_t *query, int64_t rb, int64_t re, int *score, int *n_cigar) { uint32_t *cigar = 0; uint8_t tmp, *rseq; int i, w; int64_t rlen; *n_cigar = 0; if (l_query <= 0 || rb >= re || (rb < l_pac && re > l_pac)) return 0; // reject if negative length or bridging the forward and reverse strand rseq = bns_get_seq(l_pac, pac, rb, re, &rlen); if (re - rb != rlen) goto ret_gen_cigar; // possible if out of range if (rb >= l_pac) { // then reverse both query and rseq; this is to ensure indels to be placed at the leftmost position for (i = 0; i < l_query>>1; ++i) tmp = query[i], query[i] = query[l_query - 1 - i], query[l_query - 1 - i] = tmp; for (i = 0; i < rlen>>1; ++i) tmp = rseq[i], rseq[i] = rseq[rlen - 1 - i], rseq[rlen - 1 - i] = tmp; } //printf("[Q] "); for (i = 0; i < l_query; ++i) putchar("ACGTN"[(int)query[i]]); putchar('\n'); //printf("[R] "); for (i = 0; i < re - rb; ++i) putchar("ACGTN"[(int)rseq[i]]); putchar('\n'); // set the band-width w = (int)((double)(l_query * mat[0] - q) / r + 1.); w = w < 1? w : 1; w = w < w_? w : w_; w += abs(rlen - l_query); // NW alignment *score = ksw_global(l_query, query, rlen, rseq, 5, mat, q, r, w, n_cigar, &cigar); if (rb >= l_pac) // reverse back query for (i = 0; i < l_query>>1; ++i) tmp = query[i], query[i] = query[l_query - 1 - i], query[l_query - 1 - i] = tmp; ret_gen_cigar: free(rseq); return cigar; } void bwa_hit2sam(kstring_t *str, const int8_t mat[25], int q, int r, int w, const bntseq_t *bns, const uint8_t *pac, bseq1_t *s, bwahit_t *p, int is_hard) { int score, n_cigar, is_rev = 0, nn, rid, mid, is_unmapped = 0; uint32_t *cigar = 0; int64_t pos; kputs(s->name, str); if (p && p->rb >= 0 && p->re < bns->l_pac<<1) { cigar = bwa_gen_cigar(mat, q, r, w, bns->l_pac, pac, p->qe - p->qb, (uint8_t*)&s->seq[p->qb], p->rb, p->re, &score, &n_cigar); pos = bns_depos(bns, p->rb < bns->l_pac? p->rb : p->re - 1, &is_rev); nn = bns_cnt_ambi(bns, pos, p->re - p->rb, &rid); p->flag |= is_rev? 16 : 0; // reverse p->flag |= p->mb >= 0? 1 : 0; // paired in sequencing p->flag |= n_cigar == 0? 8 : 0; // FIXME: check why this may happen (this has already happened) kputc('\t', str); kputw(p->flag, str); kputc('\t', str); kputs(bns->anns[rid].name, str); kputc('\t', str); kputuw(pos - bns->anns[rid].offset + 1, str); kputc('\t', str); kputw(p->qual, str); kputc('\t', str); if (n_cigar) { int i, clip5, clip3; clip5 = is_rev? s->l_seq - p->qe : p->qb; clip3 = is_rev? p->qb : s->l_seq - p->qe; if (clip5) { kputw(clip5, str); kputc("SH"[(is_hard!=0)], str); } for (i = 0; i < n_cigar; ++i) { kputw(cigar[i]>>4, str); kputc("MIDSH"[cigar[i]&0xf], str); } if (clip3) { kputw(clip3, str); kputc("SH"[(is_hard!=0)], str); } } else kputc('*', str); if (p->mb >= 0 && p->mb < bns->l_pac<<1) { // then print mate pos and isize pos = bns_depos(bns, p->mb < bns->l_pac? p->mb : p->me - 1, &is_rev); nn = bns_cnt_ambi(bns, pos, p->me - p->mb, &mid); kputc('\t', str); if (mid == rid) kputc('=', str); else kputs(bns->anns[mid].name, str); kputc('\t', str); kputuw(pos - bns->anns[mid].offset + 1, str); kputc('\t', str); if (mid != rid) { int64_t p0 = p->rb < bns->l_pac? p->rb : (bns->l_pac<<1) - 1 - p->rb; int64_t p1 = p->mb < bns->l_pac? p->mb : (bns->l_pac<<1) - 1 - p->mb; kputw(abs(p0 - p1), str); } kputc('\t', str); } else kputsn("\t*\t0\t0\t", 7, str); } else { // unaligned is_unmapped = 1; kputw(p? p->flag : 0, str); kputs("\t*\t0\t0\t*\t*\t0\t0\t", str); } if (!is_rev) { // print SEQ and QUAL, the forward strand int i, qb = 0, qe = s->l_seq; if (!is_unmapped && is_hard) qb = p->qb, qe = p->qe; ks_resize(str, str->l + (qe - qb) + 1); for (i = qb; i < qe; ++i) str->s[str->l++] = "ACGTN"[(int)s->seq[i]]; kputc('\t', str); if (s->qual) { // printf qual ks_resize(str, str->l + (qe - qb) + 1); for (i = qb; i < qe; ++i) str->s[str->l++] = s->qual[i]; str->s[str->l] = 0; } else kputc('*', str); } else { // the reverse strand int i, qb = 0, qe = s->l_seq; if (!is_unmapped && is_hard) qb = p->qb, qe = p->qe; ks_resize(str, str->l + (qe - qb) + 1); for (i = qe-1; i >= qb; --i) str->s[str->l++] = "TGCAN"[(int)s->seq[i]]; kputc('\t', str); if (s->qual) { // printf qual ks_resize(str, str->l + (qe - qb) + 1); for (i = qe-1; i >= qb; --i) str->s[str->l++] = s->qual[i]; str->s[str->l] = 0; } else kputc('*', str); } if (!is_unmapped && p->score >= 0) { kputsn("\tAS:i:", 6, str); kputw(p->score, str); } if (!is_unmapped && p->sub >= 0) { kputsn("\tss:i:", 6, str); kputw(p->sub, str); } kputc('\n', str); free(cigar); } /************************ * Integrated interface * ************************/ static inline int approx_mapq_se(const mem_opt_t *opt, const mem_alnreg_t *a) { int mapq, l, sub = a->sub? a->sub : opt->min_seed_len * opt->a; double identity; sub = a->csub > sub? a->csub : sub; l = a->qe - a->qb > a->re - a->rb? a->qe - a->qb : a->re - a->rb; mapq = a->score? (int)(MAPQ_COEF * (1. - (double)sub / a->score) * log(a->seedcov) + .499) : 0; identity = 1. - (double)(l * opt->a - a->score) / (opt->a + opt->b) / l; mapq = identity < 0.95? (int)(mapq * identity * identity + .499) : mapq; if (a->sub_n) mapq -= (int)(4.343 * log(a->sub_n) + .499); if (mapq > 60) mapq = 60; if (mapq < 0) mapq = 0; return mapq; } void mem_sam_se(const mem_opt_t *opt, const bntseq_t *bns, const uint8_t *pac, bseq1_t *s, mem_alnreg_v *a) { int k; kstring_t str; str.l = str.m = 0; str.s = 0; a->n = mem_choose_alnreg_se(opt, a->n, a->a); // NOTE: mem_sort_and_dedup() called in worker1() if (a->n > 0) { for (k = 0; k < a->n; ++k) { bwahit_t h; mem_alnreg_t *p = &a->a[k]; h.rb = p->rb; h.re = p->re; h.qb = p->qb; h.qe = p->qe; h.score = p->score; h.sub = p->sub; h.flag = 0; h.qual = approx_mapq_se(opt, p); h.mb = h.me = -2; bwa_hit2sam(&str, opt->mat, opt->q, opt->r, opt->w, bns, pac, s, &h, opt->is_hard); } } else bwa_hit2sam(&str, opt->mat, opt->q, opt->r, opt->w, bns, pac, s, 0, opt->is_hard); s->sam = str.s; } static mem_alnreg_v find_alnreg(const mem_opt_t *opt, const bwt_t *bwt, const bntseq_t *bns, const uint8_t *pac, bseq1_t *s) { int i; mem_chain_v chn; mem_alnreg_v regs; for (i = 0; i < s->l_seq; ++i) s->seq[i] = nst_nt4_table[(int)s->seq[i]]; chn = mem_chain(opt, bwt, s->l_seq, (uint8_t*)s->seq); chn.n = mem_chain_flt(opt, chn.n, chn.a); if (mem_verbose >= 4) mem_print_chain(bns, &chn); regs.n = regs.m = chn.n; regs.a = malloc(regs.n * sizeof(mem_alnreg_t)); for (i = 0; i < chn.n; ++i) { mem_chain2aln(opt, bns->l_pac, pac, s->l_seq, (uint8_t*)s->seq, &chn.a[i], ®s.a[i]); free(chn.a[i].seeds); } free(chn.a); return regs; } typedef struct { int start, step, n; const mem_opt_t *opt; const bwt_t *bwt; const bntseq_t *bns; const uint8_t *pac; const mem_pestat_t *pes; bseq1_t *seqs; mem_alnreg_v *regs; } worker_t; static void *worker1(void *data) { worker_t *w = (worker_t*)data; int i; for (i = w->start; i < w->n; i += w->step) { w->regs[i] = find_alnreg(w->opt, w->bwt, w->bns, w->pac, &w->seqs[i]); w->regs[i].n = mem_sort_and_dedup(w->regs[i].n, w->regs[i].a); } return 0; } static void *worker2(void *data) { extern void mem_sam_pe(const mem_opt_t *opt, const bntseq_t *bns, const uint8_t *pac, const mem_pestat_t pes[4], bseq1_t s[2], mem_alnreg_v a[2]); worker_t *w = (worker_t*)data; int i; if (!w->opt->is_pe) { for (i = 0; i < w->n; i += w->step) { mem_sam_se(w->opt, w->bns, w->pac, &w->seqs[i], &w->regs[i]); free(w->regs[i].a); } } else { for (i = 0; i < w->n>>1; i += w->step) { // not implemented yet mem_sam_pe(w->opt, w->bns, w->pac, w->pes, &w->seqs[i<<1], &w->regs[i<<1]); free(w->regs[i<<1|0].a); free(w->regs[i<<1|1].a); } } return 0; } int mem_process_seqs(const mem_opt_t *opt, const bwt_t *bwt, const bntseq_t *bns, const uint8_t *pac, int n, bseq1_t *seqs) { int i; worker_t *w; mem_alnreg_v *regs; mem_pestat_t pes[4]; w = calloc(opt->n_threads, sizeof(worker_t)); regs = malloc(n * sizeof(mem_alnreg_v)); for (i = 0; i < opt->n_threads; ++i) { worker_t *p = &w[i]; p->start = i; p->step = opt->n_threads; p->n = n; p->opt = opt; p->bwt = bwt; p->bns = bns; p->pac = pac; p->seqs = seqs; p->regs = regs; p->pes = &pes[0]; } #ifdef HAVE_PTHREAD if (opt->n_threads == 1) { worker1(w); if (opt->is_pe) mem_pestat(opt, bns->l_pac, n, regs, pes); worker2(w); } else { pthread_t *tid; tid = (pthread_t*)calloc(opt->n_threads, sizeof(pthread_t)); for (i = 0; i < opt->n_threads; ++i) pthread_create(&tid[i], 0, worker1, &w[i]); for (i = 0; i < opt->n_threads; ++i) pthread_join(tid[i], 0); if (opt->is_pe) mem_pestat(opt, bns->l_pac, n, regs, pes); for (i = 0; i < opt->n_threads; ++i) pthread_create(&tid[i], 0, worker2, &w[i]); for (i = 0; i < opt->n_threads; ++i) pthread_join(tid[i], 0); free(tid); } #else worker1(w); if (opt->is_pe) mem_pestat(opt, bns->l_pac, n, regs, pes); worker2(w); #endif for (i = 0; i < n; ++i) { fputs(seqs[i].sam, stdout); free(seqs[i].name); free(seqs[i].comment); free(seqs[i].seq); free(seqs[i].qual); free(seqs[i].sam); } free(regs); free(w); return 0; }