#include #include #include #include #include "kstring.h" #include "bwamem.h" #include "kvec.h" #include "utils.h" #include "ksw.h" #ifdef USE_MALLOC_WRAPPERS # include "malloc_wrap.h" #endif #define MIN_RATIO 0.8 #define MIN_DIR_CNT 10 #define MIN_DIR_RATIO 0.05 #define OUTLIER_BOUND 2.0 #define MAPPING_BOUND 3.0 #define MAX_STDDEV 4.0 static inline int mem_infer_dir(int64_t l_pac, int64_t b1, int64_t b2, int64_t *dist) { int64_t p2; int r1 = (b1 >= l_pac), r2 = (b2 >= l_pac); p2 = r1 == r2? b2 : (l_pac<<1) - 1 - b2; // p2 is the coordinate of read 2 on the read 1 strand *dist = p2 > b1? p2 - b1 : b1 - p2; return (r1 == r2? 0 : 1) ^ (p2 > b1? 0 : 3); } static int cal_sub(const mem_opt_t *opt, mem_alnreg_v *r) { int j; for (j = 1; j < r->n; ++j) { // choose unique alignment int b_max = r->a[j].qb > r->a[0].qb? r->a[j].qb : r->a[0].qb; int e_min = r->a[j].qe < r->a[0].qe? r->a[j].qe : r->a[0].qe; if (e_min > b_max) { // have overlap int min_l = r->a[j].qe - r->a[j].qb < r->a[0].qe - r->a[0].qb? r->a[j].qe - r->a[j].qb : r->a[0].qe - r->a[0].qb; if (e_min - b_max >= min_l * opt->mask_level) break; // significant overlap } } return j < r->n? r->a[j].score : opt->min_seed_len * opt->a; } void mem_pestat(const mem_opt_t *opt, int64_t l_pac, int n, const mem_alnreg_v *regs, mem_pestat_t pes[4]) { int i, d, max; uint64_v isize[4]; memset(pes, 0, 4 * sizeof(mem_pestat_t)); memset(isize, 0, sizeof(kvec_t(int)) * 4); for (i = 0; i < n>>1; ++i) { int dir; int64_t is; mem_alnreg_v *r[2]; r[0] = (mem_alnreg_v*)®s[i<<1|0]; r[1] = (mem_alnreg_v*)®s[i<<1|1]; if (r[0]->n == 0 || r[1]->n == 0) continue; if (cal_sub(opt, r[0]) > MIN_RATIO * r[0]->a[0].score) continue; if (cal_sub(opt, r[1]) > MIN_RATIO * r[1]->a[0].score) continue; if (r[0]->a[0].rid != r[1]->a[0].rid) continue; // not on the same chr dir = mem_infer_dir(l_pac, r[0]->a[0].rb, r[1]->a[0].rb, &is); if (is && is <= opt->max_ins) kv_push(uint64_t, isize[dir], is); } if (bwa_verbose >= 3) fprintf(stderr, "[M::%s] # candidate unique pairs for (FF, FR, RF, RR): (%ld, %ld, %ld, %ld)\n", __func__, isize[0].n, isize[1].n, isize[2].n, isize[3].n); for (d = 0; d < 4; ++d) { // TODO: this block is nearly identical to the one in bwtsw2_pair.c. It would be better to merge these two. mem_pestat_t *r = &pes[d]; uint64_v *q = &isize[d]; int p25, p50, p75, x; if (q->n < MIN_DIR_CNT) { fprintf(stderr, "[M::%s] skip orientation %c%c as there are not enough pairs\n", __func__, "FR"[d>>1&1], "FR"[d&1]); r->failed = 1; free(q->a); continue; } else fprintf(stderr, "[M::%s] analyzing insert size distribution for orientation %c%c...\n", __func__, "FR"[d>>1&1], "FR"[d&1]); ks_introsort_64(q->n, q->a); p25 = q->a[(int)(.25 * q->n + .499)]; p50 = q->a[(int)(.50 * q->n + .499)]; p75 = q->a[(int)(.75 * q->n + .499)]; r->low = (int)(p25 - OUTLIER_BOUND * (p75 - p25) + .499); if (r->low < 1) r->low = 1; r->high = (int)(p75 + OUTLIER_BOUND * (p75 - p25) + .499); fprintf(stderr, "[M::%s] (25, 50, 75) percentile: (%d, %d, %d)\n", __func__, p25, p50, p75); fprintf(stderr, "[M::%s] low and high boundaries for computing mean and std.dev: (%d, %d)\n", __func__, r->low, r->high); for (i = x = 0, r->avg = 0; i < q->n; ++i) if (q->a[i] >= r->low && q->a[i] <= r->high) r->avg += q->a[i], ++x; r->avg /= x; for (i = 0, r->std = 0; i < q->n; ++i) if (q->a[i] >= r->low && q->a[i] <= r->high) r->std += (q->a[i] - r->avg) * (q->a[i] - r->avg); r->std = sqrt(r->std / x); fprintf(stderr, "[M::%s] mean and std.dev: (%.2f, %.2f)\n", __func__, r->avg, r->std); r->low = (int)(p25 - MAPPING_BOUND * (p75 - p25) + .499); r->high = (int)(p75 + MAPPING_BOUND * (p75 - p25) + .499); if (r->low > r->avg - MAX_STDDEV * r->std) r->low = (int)(r->avg - MAX_STDDEV * r->std + .499); if (r->high < r->avg - MAX_STDDEV * r->std) r->high = (int)(r->avg + MAX_STDDEV * r->std + .499); if (r->low < 1) r->low = 1; fprintf(stderr, "[M::%s] low and high boundaries for proper pairs: (%d, %d)\n", __func__, r->low, r->high); free(q->a); } for (d = 0, max = 0; d < 4; ++d) max = max > isize[d].n? max : isize[d].n; for (d = 0; d < 4; ++d) if (pes[d].failed == 0 && isize[d].n < max * MIN_DIR_RATIO) { pes[d].failed = 1; fprintf(stderr, "[M::%s] skip orientation %c%c\n", __func__, "FR"[d>>1&1], "FR"[d&1]); } } int mem_matesw(const mem_opt_t *opt, const bntseq_t *bns, const uint8_t *pac, const mem_pestat_t pes[4], const mem_alnreg_t *a, int l_ms, const uint8_t *ms, mem_alnreg_v *ma) { extern int mem_sort_dedup_patch(const mem_opt_t *opt, const bntseq_t *bns, const uint8_t *pac, uint8_t *query, int n, mem_alnreg_t *a); int64_t l_pac = bns->l_pac; int i, r, skip[4], n = 0, rid; for (r = 0; r < 4; ++r) skip[r] = pes[r].failed? 1 : 0; for (i = 0; i < ma->n; ++i) { // check which orinentation has been found int64_t dist; r = mem_infer_dir(l_pac, a->rb, ma->a[i].rb, &dist); if (dist >= pes[r].low && dist <= pes[r].high) skip[r] = 1; } if (skip[0] + skip[1] + skip[2] + skip[3] == 4) return 0; // consistent pair exist; no need to perform SW for (r = 0; r < 4; ++r) { int is_rev, is_larger; uint8_t *seq, *rev = 0, *ref = 0; int64_t rb, re; if (skip[r]) continue; is_rev = (r>>1 != (r&1)); // whether to reverse complement the mate is_larger = !(r>>1); // whether the mate has larger coordinate if (is_rev) { rev = malloc(l_ms); // this is the reverse complement of $ms for (i = 0; i < l_ms; ++i) rev[l_ms - 1 - i] = ms[i] < 4? 3 - ms[i] : 4; seq = rev; } else seq = (uint8_t*)ms; if (!is_rev) { rb = is_larger? a->rb + pes[r].low : a->rb - pes[r].high; re = (is_larger? a->rb + pes[r].high: a->rb - pes[r].low) + l_ms; // if on the same strand, end position should be larger to make room for the seq length } else { rb = (is_larger? a->rb + pes[r].low : a->rb - pes[r].high) - l_ms; // similarly on opposite strands re = is_larger? a->rb + pes[r].high: a->rb - pes[r].low; } if (rb < 0) rb = 0; if (re > l_pac<<1) re = l_pac<<1; if (rb < re) ref = bns_fetch_seq(bns, pac, &rb, (rb+re)>>1, &re, &rid); if (a->rid == rid && re - rb >= opt->min_seed_len) { // no funny things happening kswr_t aln; mem_alnreg_t b; int tmp, xtra = KSW_XSUBO | KSW_XSTART | (l_ms * opt->a < 250? KSW_XBYTE : 0) | (opt->min_seed_len * opt->a); aln = ksw_align2(l_ms, seq, re - rb, ref, 5, opt->mat, opt->o_del, opt->e_del, opt->o_ins, opt->e_ins, xtra, 0); memset(&b, 0, sizeof(mem_alnreg_t)); if (aln.score >= opt->min_seed_len && aln.qb >= 0) { // something goes wrong if aln.qb < 0 b.rid = a->rid; b.is_alt = a->is_alt; b.qb = is_rev? l_ms - (aln.qe + 1) : aln.qb; b.qe = is_rev? l_ms - aln.qb : aln.qe + 1; b.rb = is_rev? (l_pac<<1) - (rb + aln.te + 1) : rb + aln.tb; b.re = is_rev? (l_pac<<1) - (rb + aln.tb) : rb + aln.te + 1; b.score = aln.score; b.csub = aln.score2; b.secondary = -1; b.seedcov = (b.re - b.rb < b.qe - b.qb? b.re - b.rb : b.qe - b.qb) >> 1; // printf("*** %d, [%lld,%lld], %d:%d, (%lld,%lld), (%lld,%lld) == (%lld,%lld)\n", aln.score, rb, re, is_rev, is_larger, a->rb, a->re, ma->a[0].rb, ma->a[0].re, b.rb, b.re); kv_push(mem_alnreg_t, *ma, b); // make room for a new element // move b s.t. ma is sorted for (i = 0; i < ma->n - 1; ++i) // find the insertion point if (ma->a[i].score < b.score) break; tmp = i; for (i = ma->n - 1; i > tmp; --i) ma->a[i] = ma->a[i-1]; ma->a[i] = b; } ++n; } if (n) ma->n = mem_sort_dedup_patch(opt, 0, 0, 0, ma->n, ma->a); if (rev) free(rev); free(ref); } return n; } int mem_pair(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], int id, int *sub, int *n_sub, int z[2], int n_pri[2]) { pair64_v v, u; int r, i, k, y[4], ret; // y[] keeps the last hit int64_t l_pac = bns->l_pac; kv_init(v); kv_init(u); for (r = 0; r < 2; ++r) { // loop through read number for (i = 0; i < n_pri[r]; ++i) { pair64_t key; mem_alnreg_t *e = &a[r].a[i]; key.x = e->rb < l_pac? e->rb : (l_pac<<1) - 1 - e->rb; // forward position key.x = (uint64_t)e->rid<<32 | (key.x - bns->anns[e->rid].offset); key.y = (uint64_t)e->score << 32 | i << 2 | (e->rb >= l_pac)<<1 | r; kv_push(pair64_t, v, key); } } ks_introsort_128(v.n, v.a); y[0] = y[1] = y[2] = y[3] = -1; //for (i = 0; i < v.n; ++i) printf("[%d]\t%d\t%c%ld\n", i, (int)(v.a[i].y&1)+1, "+-"[v.a[i].y>>1&1], (long)v.a[i].x); for (i = 0; i < v.n; ++i) { for (r = 0; r < 2; ++r) { // loop through direction int dir = r<<1 | (v.a[i].y>>1&1), which; if (pes[dir].failed) continue; // invalid orientation which = r<<1 | ((v.a[i].y&1)^1); if (y[which] < 0) continue; // no previous hits for (k = y[which]; k >= 0; --k) { // TODO: this is a O(n^2) solution in the worst case; remember to check if this loop takes a lot of time (I doubt) int64_t dist; int q; double ns; pair64_t *p; if ((v.a[k].y&3) != which) continue; dist = (int64_t)v.a[i].x - v.a[k].x; //printf("%d: %lld\n", k, dist); if (dist > pes[dir].high) break; if (dist < pes[dir].low) continue; ns = (dist - pes[dir].avg) / pes[dir].std; q = (int)((v.a[i].y>>32) + (v.a[k].y>>32) + .721 * log(2. * erfc(fabs(ns) * M_SQRT1_2)) * opt->a + .499); // .721 = 1/log(4) if (q < 0) q = 0; p = kv_pushp(pair64_t, u); p->y = (uint64_t)k<<32 | i; p->x = (uint64_t)q<<32 | (hash_64(p->y ^ id<<8) & 0xffffffffU); //printf("[%lld,%lld]\t%d\tdist=%ld\n", v.a[k].x, v.a[i].x, q, (long)dist); } } y[v.a[i].y&3] = i; } if (u.n) { // found at least one proper pair int tmp = opt->a + opt->b; tmp = tmp > opt->o_del + opt->e_del? tmp : opt->o_del + opt->e_del; tmp = tmp > opt->o_ins + opt->e_ins? tmp : opt->o_ins + opt->e_ins; ks_introsort_128(u.n, u.a); i = u.a[u.n-1].y >> 32; k = u.a[u.n-1].y << 32 >> 32; z[v.a[i].y&1] = v.a[i].y<<32>>34; // index of the best pair z[v.a[k].y&1] = v.a[k].y<<32>>34; ret = u.a[u.n-1].x >> 32; *sub = u.n > 1? u.a[u.n-2].x>>32 : 0; for (i = (long)u.n - 2, *n_sub = 0; i >= 0; --i) if (*sub - (int)(u.a[i].x>>32) <= tmp) ++*n_sub; } else ret = 0, *sub = 0, *n_sub = 0; free(u.a); free(v.a); return ret; } void mem_aln2sam(const mem_opt_t *opt, const bntseq_t *bns, kstring_t *str, bseq1_t *s, int n, const mem_aln_t *list, int which, const mem_aln_t *m); #define raw_mapq(diff, a) ((int)(6.02 * (diff) / (a) + .499)) int mem_sam_pe(const mem_opt_t *opt, const bntseq_t *bns, const uint8_t *pac, const mem_pestat_t pes[4], uint64_t id, bseq1_t s[2], mem_alnreg_v a[2]) { extern int mem_mark_primary_se(const mem_opt_t *opt, int n, mem_alnreg_t *a, int64_t id); extern int mem_approx_mapq_se(const mem_opt_t *opt, const mem_alnreg_t *a); extern void mem_reg2sam(const mem_opt_t *opt, const bntseq_t *bns, const uint8_t *pac, bseq1_t *s, mem_alnreg_v *a, int extra_flag, const mem_aln_t *m); extern char **mem_gen_alt(const mem_opt_t *opt, const bntseq_t *bns, const uint8_t *pac, const mem_alnreg_v *a, int l_query, const char *query); int n = 0, i, j, z[2], o, subo, n_sub, extra_flag = 1, n_pri[2], n_aa[2]; kstring_t str; mem_aln_t h[2], g[2], aa[2][2]; str.l = str.m = 0; str.s = 0; memset(h, 0, sizeof(mem_aln_t) * 2); memset(g, 0, sizeof(mem_aln_t) * 2); n_aa[0] = n_aa[1] = 0; if (!(opt->flag & MEM_F_NO_RESCUE)) { // then perform SW for the best alignment mem_alnreg_v b[2]; kv_init(b[0]); kv_init(b[1]); for (i = 0; i < 2; ++i) for (j = 0; j < a[i].n; ++j) if (a[i].a[j].score >= a[i].a[0].score - opt->pen_unpaired) kv_push(mem_alnreg_t, b[i], a[i].a[j]); for (i = 0; i < 2; ++i) for (j = 0; j < b[i].n && j < opt->max_matesw; ++j) n += mem_matesw(opt, bns, pac, pes, &b[i].a[j], s[!i].l_seq, (uint8_t*)s[!i].seq, &a[!i]); free(b[0].a); free(b[1].a); } n_pri[0] = mem_mark_primary_se(opt, a[0].n, a[0].a, id<<1|0); n_pri[1] = mem_mark_primary_se(opt, a[1].n, a[1].a, id<<1|1); if (opt->flag&MEM_F_NOPAIRING) goto no_pairing; // pairing single-end hits if (n_pri[0] && n_pri[1] && (o = mem_pair(opt, bns, pac, pes, s, a, id, &subo, &n_sub, z, n_pri)) > 0) { int is_multi[2], q_pe, score_un, q_se[2]; char **XA[2]; // check if an end has multiple hits even after mate-SW for (i = 0; i < 2; ++i) { for (j = 1; j < n_pri[i]; ++j) if (a[i].a[j].secondary < 0 && a[i].a[j].score >= opt->T) break; is_multi[i] = j < n_pri[i]? 1 : 0; } if (is_multi[0] || is_multi[1]) goto no_pairing; // TODO: in rare cases, the true hit may be long but with low score // compute mapQ for the best SE hit score_un = a[0].a[0].score + a[1].a[0].score - opt->pen_unpaired; //q_pe = o && subo < o? (int)(MEM_MAPQ_COEF * (1. - (double)subo / o) * log(a[0].a[z[0]].seedcov + a[1].a[z[1]].seedcov) + .499) : 0; subo = subo > score_un? subo : score_un; q_pe = raw_mapq(o - subo, opt->a); if (n_sub > 0) q_pe -= (int)(4.343 * log(n_sub+1) + .499); if (q_pe < 0) q_pe = 0; if (q_pe > 60) q_pe = 60; q_pe = (int)(q_pe * (1. - .5 * (a[0].a[0].frac_rep + a[1].a[0].frac_rep)) + .499); // the following assumes no split hits if (o > score_un) { // paired alignment is preferred mem_alnreg_t *c[2]; c[0] = &a[0].a[z[0]]; c[1] = &a[1].a[z[1]]; for (i = 0; i < 2; ++i) { if (c[i]->secondary >= 0) c[i]->sub = a[i].a[c[i]->secondary].score, c[i]->secondary = -2; q_se[i] = mem_approx_mapq_se(opt, c[i]); } q_se[0] = q_se[0] > q_pe? q_se[0] : q_pe < q_se[0] + 40? q_pe : q_se[0] + 40; q_se[1] = q_se[1] > q_pe? q_se[1] : q_pe < q_se[1] + 40? q_pe : q_se[1] + 40; extra_flag |= 2; // cap at the tandem repeat score q_se[0] = q_se[0] < raw_mapq(c[0]->score - c[0]->csub, opt->a)? q_se[0] : raw_mapq(c[0]->score - c[0]->csub, opt->a); q_se[1] = q_se[1] < raw_mapq(c[1]->score - c[1]->csub, opt->a)? q_se[1] : raw_mapq(c[1]->score - c[1]->csub, opt->a); } else { // the unpaired alignment is preferred z[0] = z[1] = 0; q_se[0] = mem_approx_mapq_se(opt, &a[0].a[0]); q_se[1] = mem_approx_mapq_se(opt, &a[1].a[0]); } for (i = 0; i < 2; ++i) { int k = a[i].a[z[i]].secondary_all; if (k >= 0 && k < n_pri[i]) { // switch secondary and primary if both of them are non-ALT assert(a[i].a[k].secondary_all < 0); for (j = 0; j < a[i].n; ++j) if (a[i].a[j].secondary_all == k || j == k) a[i].a[j].secondary_all = z[i]; a[i].a[z[i]].secondary_all = -1; } } if (!(opt->flag & MEM_F_ALL)) { for (i = 0; i < 2; ++i) XA[i] = mem_gen_alt(opt, bns, pac, &a[i], s[i].l_seq, s[i].seq); } else XA[0] = XA[1] = 0; // write SAM for (i = 0; i < 2; ++i) { h[i] = mem_reg2aln(opt, bns, pac, s[i].l_seq, s[i].seq, &a[i].a[z[i]]); h[i].mapq = q_se[i]; h[i].flag |= 0x40<score < opt->T || p->secondary >= 0 || !p->is_alt) continue; g[i] = mem_reg2aln(opt, bns, pac, s[i].l_seq, s[i].seq, p); g[i].flag |= 0x800 | 0x40<= opt->T) which = 0; else if (n_pri[i] < a[i].n && a[i].a[n_pri[i]].score >= opt->T) which = n_pri[i]; } if (which >= 0) h[i] = mem_reg2aln(opt, bns, pac, s[i].l_seq, s[i].seq, &a[i].a[which]); else h[i] = mem_reg2aln(opt, bns, pac, s[i].l_seq, s[i].seq, 0); } if (!(opt->flag & MEM_F_NOPAIRING) && h[0].rid == h[1].rid && h[0].rid >= 0) { // if the top hits from the two ends constitute a proper pair, flag it. int64_t dist; int d; d = mem_infer_dir(bns->l_pac, a[0].a[0].rb, a[1].a[0].rb, &dist); if (!pes[d].failed && dist >= pes[d].low && dist <= pes[d].high) extra_flag |= 2; } mem_reg2sam(opt, bns, pac, &s[0], &a[0], 0x41|extra_flag, &h[1]); mem_reg2sam(opt, bns, pac, &s[1], &a[1], 0x81|extra_flag, &h[0]); if (strcmp(s[0].name, s[1].name) != 0) err_fatal(__func__, "paired reads have different names: \"%s\", \"%s\"\n", s[0].name, s[1].name); free(h[0].cigar); free(h[1].cigar); return n; }