#include #include #include #ifdef HAVE_CONFIG_H #include "config.h" #endif #ifdef HAVE_PTHREAD #include #endif #include "bntseq.h" #include "bwt_lite.h" #include "utils.h" #include "bwtsw2.h" #include "kstring.h" #include "bwa.h" #include "ksw.h" #include "kseq.h" KSEQ_DECLARE(gzFile) #include "ksort.h" #define __left_lt(a, b) ((a).end > (b).end) KSORT_INIT(hit, bsw2hit_t, __left_lt) #ifdef USE_MALLOC_WRAPPERS # include "malloc_wrap.h" #endif extern unsigned char nst_nt4_table[256]; unsigned char nt_comp_table[256] = { 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','T','V','G', 'H','N','N','C', 'D','N','N','M', 'N','K','N','N', 'N','N','Y','S', 'A','N','B','W', 'X','R','N','N', 'N','N','N','N', 'n','t','v','g', 'h','n','n','c', 'd','n','n','m', 'n','k','n','n', 'n','n','y','s', 'a','n','b','w', 'x','r','n','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N' }; extern int bsw2_resolve_duphits(const bntseq_t *bns, const bwt_t *bwt, bwtsw2_t *b, int IS); extern int bsw2_resolve_query_overlaps(bwtsw2_t *b, float mask_level); bsw2opt_t *bsw2_init_opt() { bsw2opt_t *o = (bsw2opt_t*)calloc(1, sizeof(bsw2opt_t)); o->a = 1; o->b = 3; o->q = 5; o->r = 2; o->t = 30; o->bw = 50; o->max_ins = 20000; o->z = 1; o->is = 3; o->t_seeds = 5; o->hard_clip = 0; o->skip_sw = 0; o->mask_level = 0.50f; o->coef = 5.5f; o->qr = o->q + o->r; o->n_threads = 1; o->chunk_size = 10000000; o->max_chain_gap = 10000; o->cpy_cmt = 0; return o; } void bsw2_destroy(bwtsw2_t *b) { int i; if (b == 0) return; if (b->aux) for (i = 0; i < b->n; ++i) free(b->aux[i].cigar); free(b->aux); free(b->hits); free(b); } bwtsw2_t *bsw2_dup_no_cigar(const bwtsw2_t *b) { bwtsw2_t *p; p = calloc(1, sizeof(bwtsw2_t)); p->max = p->n = b->n; if (b->n) { kroundup32(p->max); p->hits = calloc(p->max, sizeof(bsw2hit_t)); memcpy(p->hits, b->hits, p->n * sizeof(bsw2hit_t)); } return p; } #define __gen_ap(par, opt) do { \ int i; \ for (i = 0; i < 25; ++i) (par).matrix[i] = -(opt)->b; \ for (i = 0; i < 4; ++i) (par).matrix[i*5+i] = (opt)->a; \ (par).gap_open = (opt)->q; (par).gap_ext = (opt)->r; \ (par).gap_end = (opt)->r; \ (par).row = 5; (par).band_width = opt->bw; \ } while (0) void bsw2_extend_left(const bsw2opt_t *opt, bwtsw2_t *b, uint8_t *_query, int lq, uint8_t *pac, bwtint_t l_pac, uint8_t *_mem) { int i; bwtint_t k; uint8_t *target = 0, *query; int8_t mat[25]; bwa_fill_scmat(opt->a, opt->b, mat); query = calloc(lq, 1); // sort according to the descending order of query end ks_introsort(hit, b->n, b->hits); target = calloc(((lq + 1) / 2 * opt->a + opt->r) / opt->r + lq, 1); // reverse _query for (i = 0; i < lq; ++i) query[lq - i - 1] = _query[i]; // core loop for (i = 0; i < b->n; ++i) { bsw2hit_t *p = b->hits + i; int lt = ((p->beg + 1) / 2 * opt->a + opt->r) / opt->r + lq; int score, j, qle, tle; p->n_seeds = 1; if (p->l || p->k == 0) continue; for (j = score = 0; j < i; ++j) { bsw2hit_t *q = b->hits + j; if (q->beg <= p->beg && q->k <= p->k && q->k + q->len >= p->k + p->len) { if (q->n_seeds < (1<<13) - 2) ++q->n_seeds; ++score; } } if (score) continue; if (lt > p->k) lt = p->k; for (k = p->k - 1, j = 0; k > 0 && j < lt; --k) // FIXME: k=0 not considered! target[j++] = pac[k>>2] >> (~k&3)*2 & 0x3; lt = j; score = ksw_extend(p->beg, &query[lq - p->beg], lt, target, 5, mat, opt->q, opt->r, opt->bw, 0, -1, p->G, &qle, &tle, 0, 0, 0); if (score > p->G) { // extensible p->G = score; p->k -= tle; p->len += tle; p->beg -= qle; } } free(query); free(target); } void bsw2_extend_rght(const bsw2opt_t *opt, bwtsw2_t *b, uint8_t *query, int lq, uint8_t *pac, bwtint_t l_pac, uint8_t *_mem) { int i; bwtint_t k; uint8_t *target; int8_t mat[25]; bwa_fill_scmat(opt->a, opt->b, mat); target = calloc(((lq + 1) / 2 * opt->a + opt->r) / opt->r + lq, 1); for (i = 0; i < b->n; ++i) { bsw2hit_t *p = b->hits + i; int lt = ((lq - p->beg + 1) / 2 * opt->a + opt->r) / opt->r + lq; int j, score, qle, tle; if (p->l) continue; for (k = p->k, j = 0; k < p->k + lt && k < l_pac; ++k) target[j++] = pac[k>>2] >> (~k&3)*2 & 0x3; lt = j; score = ksw_extend(lq - p->beg, &query[p->beg], lt, target, 5, mat, opt->q, opt->r, opt->bw, 0, -1, 1, &qle, &tle, 0, 0, 0) - 1; // if (score < p->G) fprintf(stderr, "[bsw2_extend_hits] %d < %d\n", score, p->G); if (score >= p->G) { p->G = score; p->len = tle; p->end = p->beg + qle; } } free(target); } /* generate CIGAR array(s) in b->cigar[] */ static void gen_cigar(const bsw2opt_t *opt, int lq, uint8_t *seq[2], int64_t l_pac, const uint8_t *pac, bwtsw2_t *b, const char *name) { int i; int8_t mat[25]; bwa_fill_scmat(opt->a, opt->b, mat); for (i = 0; i < b->n; ++i) { bsw2hit_t *p = b->hits + i; bsw2aux_t *q = b->aux + i; uint8_t *query; int beg, end, score; if (p->l) continue; beg = (p->flag & 0x10)? lq - p->end : p->beg; end = (p->flag & 0x10)? lq - p->beg : p->end; query = seq[(p->flag & 0x10)? 1 : 0] + beg; q->cigar = bwa_gen_cigar(mat, opt->q, opt->r, opt->bw, l_pac, pac, end - beg, query, p->k, p->k + p->len, &score, &q->n_cigar, &q->nm); #if 0 if (name && score != p->G) { // debugging only int j, glen = 0; for (j = 0; j < q->n_cigar; ++j) if ((q->cigar[j]&0xf) == 1 || (q->cigar[j]&0xf) == 2) glen += q->cigar[j]>>4; fprintf(stderr, "[E::%s] %s - unequal score: %d != %d; (qlen, aqlen, arlen, glen, bw) = (%d, %d, %d, %d, %d)\n", __func__, name, score, p->G, lq, end - beg, p->len, glen, opt->bw); } #endif if (q->cigar && (beg != 0 || end < lq)) { // write soft clipping q->cigar = realloc(q->cigar, 4 * (q->n_cigar + 2)); if (beg != 0) { memmove(q->cigar + 1, q->cigar, q->n_cigar * 4); q->cigar[0] = beg<<4 | 4; ++q->n_cigar; } if (end < lq) { q->cigar[q->n_cigar] = (lq - end)<<4 | 4; ++q->n_cigar; } } } } /* this is for the debugging purpose only */ void bsw2_debug_hits(const bwtsw2_t *b) { int i; printf("# raw hits: %d\n", b->n); for (i = 0; i < b->n; ++i) { bsw2hit_t *p = b->hits + i; if (p->G > 0) printf("G=%d, G2=%d, len=%d, [%d,%d), k=%lu, l=%lu, #seeds=%d, is_rev=%d\n", p->G, p->G2, p->len, p->beg, p->end, (long)p->k, (long)p->l, p->n_seeds, p->is_rev); } } static void merge_hits(bwtsw2_t *b[2], int l, int is_reverse) { int i; if (b[0]->n + b[1]->n > b[0]->max) { b[0]->max = b[0]->n + b[1]->n; b[0]->hits = realloc(b[0]->hits, b[0]->max * sizeof(bsw2hit_t)); } for (i = 0; i < b[1]->n; ++i) { bsw2hit_t *p = b[0]->hits + b[0]->n + i; *p = b[1]->hits[i]; if (is_reverse) { int x = p->beg; p->beg = l - p->end; p->end = l - x; p->flag |= 0x10; } } b[0]->n += b[1]->n; bsw2_destroy(b[1]); b[1] = 0; } /* seq[0] is the forward sequence and seq[1] is the reverse complement. */ static bwtsw2_t *bsw2_aln1_core(const bsw2opt_t *opt, const bntseq_t *bns, uint8_t *pac, const bwt_t *target, int l, uint8_t *seq[2], bsw2global_t *pool) { extern void bsw2_chain_filter(const bsw2opt_t *opt, int len, bwtsw2_t *b[2]); bwtsw2_t *b[2], **bb[2], **_b, *p; int k, j; bwtl_t *query; query = bwtl_seq2bwtl(l, seq[0]); _b = bsw2_core(bns, opt, query, target, pool); bwtl_destroy(query); for (k = 0; k < 2; ++k) { bb[k] = calloc(2, sizeof(void*)); bb[k][0] = calloc(1, sizeof(bwtsw2_t)); bb[k][1] = calloc(1, sizeof(bwtsw2_t)); } for (k = 0; k < 2; ++k) { // separate _b into bb[2] based on the strand for (j = 0; j < _b[k]->n; ++j) { bsw2hit_t *q; p = bb[_b[k]->hits[j].is_rev][k]; if (p->n == p->max) { p->max = p->max? p->max<<1 : 8; p->hits = realloc(p->hits, p->max * sizeof(bsw2hit_t)); } q = &p->hits[p->n++]; *q = _b[k]->hits[j]; if (_b[k]->hits[j].is_rev) { int x = q->beg; q->beg = l - q->end; q->end = l - x; } } } b[0] = bb[0][1]; b[1] = bb[1][1]; // bb[*][1] are "narrow SA hits" bsw2_chain_filter(opt, l, b); // NB: only unique seeds are chained for (k = 0; k < 2; ++k) { bsw2_extend_left(opt, bb[k][1], seq[k], l, pac, bns->l_pac, pool->aln_mem); merge_hits(bb[k], l, 0); // bb[k][1] is merged to bb[k][0] here bsw2_resolve_duphits(0, 0, bb[k][0], 0); bsw2_extend_rght(opt, bb[k][0], seq[k], l, pac, bns->l_pac, pool->aln_mem); bsw2_resolve_duphits(0, 0, bb[k][0], 0); b[k] = bb[k][0]; free(bb[k]); } merge_hits(b, l, 1); // again, b[1] is merged to b[0] bsw2_resolve_query_overlaps(b[0], opt->mask_level); bsw2_destroy(_b[0]); bsw2_destroy(_b[1]); free(_b); return b[0]; } /* set ->flag to records the origin of the hit (to forward bwt or reverse bwt) */ static void flag_fr(bwtsw2_t *b[2]) { int i, j; for (i = 0; i < b[0]->n; ++i) { bsw2hit_t *p = b[0]->hits + i; p->flag |= 0x10000; } for (i = 0; i < b[1]->n; ++i) { bsw2hit_t *p = b[1]->hits + i; p->flag |= 0x20000; } for (i = 0; i < b[0]->n; ++i) { bsw2hit_t *p = b[0]->hits + i; for (j = 0; j < b[1]->n; ++j) { bsw2hit_t *q = b[1]->hits + j; if (q->beg == p->beg && q->end == p->end && q->k == p->k && q->len == p->len && q->G == p->G) { q->flag |= 0x30000; p->flag |= 0x30000; break; } } } } typedef struct { int n, max; bsw2seq1_t *seq; } bsw2seq_t; static int fix_cigar(const bntseq_t *bns, bsw2hit_t *p, int n_cigar, uint32_t *cigar) { // FIXME: this routine does not work if the query bridge three reference sequences int32_t coor, refl, lq; int x, y, i, seqid; bns_cnt_ambi(bns, p->k, p->len, &seqid); coor = p->k - bns->anns[seqid].offset; refl = bns->anns[seqid].len; x = coor; y = 0; // test if the alignment goes beyond the boundary for (i = 0; i < n_cigar; ++i) { int op = cigar[i]&0xf, ln = cigar[i]>>4; if (op == 1 || op == 4 || op == 5) y += ln; else if (op == 2) x += ln; else x += ln, y += ln; } lq = y; // length of the query sequence if (x > refl) { // then fix it int j, nc, mq[2], nlen[2]; uint32_t *cn; bwtint_t kk = 0; nc = mq[0] = mq[1] = nlen[0] = nlen[1] = 0; cn = calloc(n_cigar + 3, 4); x = coor; y = 0; for (i = j = 0; i < n_cigar; ++i) { int op = cigar[i]&0xf, ln = cigar[i]>>4; if (op == 4 || op == 5 || op == 1) { // ins or clipping y += ln; cn[j++] = cigar[i]; } else if (op == 2) { // del if (x + ln >= refl && nc == 0) { cn[j++] = (uint32_t)(lq - y)<<4 | 4; nc = j; cn[j++] = (uint32_t)y<<4 | 4; kk = p->k + (x + ln - refl); nlen[0] = x - coor; nlen[1] = p->len - nlen[0] - ln; } else cn[j++] = cigar[i]; x += ln; } else if (op == 0) { // match if (x + ln >= refl && nc == 0) { // FIXME: not consider a special case where a split right between M and I cn[j++] = (uint32_t)(refl - x)<<4 | 0; // write M cn[j++] = (uint32_t)(lq - y - (refl - x))<<4 | 4; // write S nc = j; mq[0] += refl - x; cn[j++] = (uint32_t)(y + (refl - x))<<4 | 4; if (x + ln - refl) cn[j++] = (uint32_t)(x + ln - refl)<<4 | 0; mq[1] += x + ln - refl; kk = bns->anns[seqid].offset + refl; nlen[0] = refl - coor; nlen[1] = p->len - nlen[0]; } else { cn[j++] = cigar[i]; mq[nc?1:0] += ln; } x += ln; y += ln; } } if (mq[0] > mq[1]) { // then take the first alignment n_cigar = nc; memcpy(cigar, cn, 4 * nc); p->len = nlen[0]; } else { p->k = kk; p->len = nlen[1]; n_cigar = j - nc; memcpy(cigar, cn + nc, 4 * (j - nc)); } free(cn); } return n_cigar; } static void write_aux(const bsw2opt_t *opt, const bntseq_t *bns, int qlen, uint8_t *seq[2], const uint8_t *pac, bwtsw2_t *b, const char *name) { int i; // allocate for b->aux if (b->n<<1 < b->max) { b->max = b->n; kroundup32(b->max); b->hits = realloc(b->hits, b->max * sizeof(bsw2hit_t)); } b->aux = calloc(b->n, sizeof(bsw2aux_t)); // generate CIGAR gen_cigar(opt, qlen, seq, bns->l_pac, pac, b, name); // fix CIGAR, generate mapQ, and write chromosomal position for (i = 0; i < b->n; ++i) { bsw2hit_t *p = &b->hits[i]; bsw2aux_t *q = &b->aux[i]; q->flag = p->flag & 0xfe; q->isize = 0; if (p->l == 0) { // unique hit float c = 1.0; int subo; // fix out-of-boundary CIGAR q->n_cigar = fix_cigar(bns, p, q->n_cigar, q->cigar); // compute mapQ subo = p->G2 > opt->t? p->G2 : opt->t; if (p->flag>>16 == 1 || p->flag>>16 == 2) c *= .5; if (p->n_seeds < 2) c *= .2; q->qual = (int)(c * (p->G - subo) * (250.0 / p->G + 0.03 / opt->a) + .499); if (q->qual > 250) q->qual = 250; if (q->qual < 0) q->qual = 0; if (p->flag&1) q->qual = 0; // this is a random hit q->pqual = q->qual; // set the paired qual as qual // get the chromosomal position q->nn = bns_cnt_ambi(bns, p->k, p->len, &q->chr); q->pos = p->k - bns->anns[q->chr].offset; } else q->qual = 0, q->n_cigar = 0, q->chr = q->pos = -1, q->nn = 0; } } static void update_mate_aux(bwtsw2_t *b, const bwtsw2_t *m) { int i; if (m == 0) return; // update flag, mchr and mpos for (i = 0; i < b->n; ++i) { bsw2aux_t *q = &b->aux[i]; q->flag |= 1; // paired if (m->n == 0) q->flag |= 8; // mate unmapped if (m->n == 1) { q->mchr = m->aux[0].chr; q->mpos = m->aux[0].pos; if (m->aux[0].flag&0x10) q->flag |= 0x20; // mate reverse strand if (q->chr == q->mchr) { // set insert size if (q->mpos + m->hits[0].len > q->pos) q->isize = q->mpos + m->hits[0].len - q->pos; else q->isize = q->mpos - q->pos - b->hits[0].len; } else q->isize = 0; } else q->mchr = q->mpos = -1; } // update mapping quality if (b->n == 1 && m->n == 1) { bsw2hit_t *p = &b->hits[0]; if (p->flag & BSW2_FLAG_MATESW) { // this alignment is found by Smith-Waterman if (!(p->flag & BSW2_FLAG_TANDEM) && b->aux[0].pqual < 20) b->aux[0].pqual = 20; if (b->aux[0].pqual >= m->aux[0].qual) b->aux[0].pqual = m->aux[0].qual; } else if ((p->flag & 2) && !(m->hits[0].flag & BSW2_FLAG_MATESW)) { // properly paired if (!(p->flag & BSW2_FLAG_TANDEM)) { // pqual is bounded by [b->aux[0].qual,m->aux[0].qual] b->aux[0].pqual += 20; if (b->aux[0].pqual > m->aux[0].qual) b->aux[0].pqual = m->aux[0].qual; if (b->aux[0].pqual < b->aux[0].qual) b->aux[0].pqual = b->aux[0].qual; } } } } /* generate SAM lines for a sequence in ks with alignment stored in * b. ks->name and ks->seq will be freed and set to NULL in the end. */ static void print_hits(const bntseq_t *bns, const bsw2opt_t *opt, bsw2seq1_t *ks, bwtsw2_t *b, int is_pe, bwtsw2_t *bmate) { int i, k; kstring_t str; memset(&str, 0, sizeof(kstring_t)); if (b == 0 || b->n == 0) { // no hits ksprintf(&str, "%s\t4\t*\t0\t0\t*\t*\t0\t0\t", ks->name); for (i = 0; i < ks->l; ++i) kputc(ks->seq[i], &str); if (ks->qual) { kputc('\t', &str); for (i = 0; i < ks->l; ++i) kputc(ks->qual[i], &str); } else kputs("\t*", &str); kputc('\n', &str); } for (i = 0; b && i < b->n; ++i) { bsw2hit_t *p = b->hits + i; bsw2aux_t *q = b->aux + i; int j, beg, end, type = 0; // print mandatory fields before SEQ if (q->cigar == 0) q->flag |= 0x4; ksprintf(&str, "%s\t%d", ks->name, q->flag | (opt->multi_2nd && i? 0x100 : 0)); ksprintf(&str, "\t%s\t%ld", q->chr>=0? bns->anns[q->chr].name : "*", (long)q->pos + 1); if (p->l == 0 && q->cigar) { // not a repetitive hit ksprintf(&str, "\t%d\t", q->pqual); for (k = 0; k < q->n_cigar; ++k) ksprintf(&str, "%d%c", q->cigar[k]>>4, (opt->hard_clip? "MIDNHHP" : "MIDNSHP")[q->cigar[k]&0xf]); } else ksprintf(&str, "\t0\t*"); if (!is_pe) kputs("\t*\t0\t0\t", &str); else ksprintf(&str, "\t%s\t%d\t%d\t", q->mchr==q->chr? "=" : (q->mchr<0? "*" : bns->anns[q->mchr].name), q->mpos+1, q->isize); // get the sequence begin and end beg = 0; end = ks->l; if (opt->hard_clip && q->cigar) { if ((q->cigar[0]&0xf) == 4) beg += q->cigar[0]>>4; if ((q->cigar[q->n_cigar-1]&0xf) == 4) end -= q->cigar[q->n_cigar-1]>>4; } for (j = beg; j < end; ++j) { if (p->flag&0x10) kputc(nt_comp_table[(int)ks->seq[ks->l - 1 - j]], &str); else kputc(ks->seq[j], &str); } // print base quality if present if (ks->qual) { kputc('\t', &str); for (j = beg; j < end; ++j) { if (p->flag&0x10) kputc(ks->qual[ks->l - 1 - j], &str); else kputc(ks->qual[j], &str); } } else kputs("\t*", &str); // print optional tags ksprintf(&str, "\tAS:i:%d\tXS:i:%d\tXF:i:%d\tXE:i:%d\tNM:i:%d", p->G, p->G2, p->flag>>16, p->n_seeds, q->nm); if (q->nn) ksprintf(&str, "\tXN:i:%d", q->nn); if (p->l) ksprintf(&str, "\tXI:i:%d", p->l - p->k + 1); if (p->flag&BSW2_FLAG_MATESW) type |= 1; if (p->flag&BSW2_FLAG_TANDEM) type |= 2; if (type) ksprintf(&str, "\tXT:i:%d", type); if (opt->cpy_cmt && ks->comment) { int l = strlen(ks->comment); if (l >= 6 && ks->comment[2] == ':' && ks->comment[4] == ':') { kputc('\t', &str); kputs(ks->comment, &str); } } kputc('\n', &str); } ks->sam = str.s; free(ks->seq); ks->seq = 0; free(ks->qual); ks->qual = 0; free(ks->name); ks->name = 0; } static void update_opt(bsw2opt_t *dst, const bsw2opt_t *src, int qlen) { double ll = log(qlen); int i, k; *dst = *src; if (dst->t < ll * dst->coef) dst->t = (int)(ll * dst->coef + .499); // set band width: the query length sets a boundary on the maximum band width k = (qlen * dst->a - 2 * dst->q) / (2 * dst->r + dst->a); i = (qlen * dst->a - dst->a - dst->t) / dst->r; if (k > i) k = i; if (k < 1) k = 1; // I do not know if k==0 causes troubles dst->bw = src->bw < k? src->bw : k; } /* Core routine to align reads in _seq. It is separated from * process_seqs() to realize multi-threading */ static void bsw2_aln_core(bsw2seq_t *_seq, const bsw2opt_t *_opt, const bntseq_t *bns, uint8_t *pac, const bwt_t *target, int is_pe) { int x; bsw2opt_t opt; bsw2global_t *pool = bsw2_global_init(); bwtsw2_t **buf; buf = calloc(_seq->n, sizeof(void*)); for (x = 0; x < _seq->n; ++x) { bsw2seq1_t *p = _seq->seq + x; uint8_t *seq[2], *rseq[2]; int i, l, k; bwtsw2_t *b[2]; l = p->l; update_opt(&opt, _opt, p->l); if (pool->max_l < l) { // then enlarge working space for aln_extend_core() int tmp = ((l + 1) / 2 * opt.a + opt.r) / opt.r + l; pool->max_l = l; pool->aln_mem = realloc(pool->aln_mem, (tmp + 2) * 24); } // set seq[2] and rseq[2] seq[0] = calloc(l * 4, 1); seq[1] = seq[0] + l; rseq[0] = seq[1] + l; rseq[1] = rseq[0] + l; // convert sequences to 2-bit representation for (i = k = 0; i < l; ++i) { int c = nst_nt4_table[(int)p->seq[i]]; if (c >= 4) { c = (int)(drand48() * 4); ++k; } // FIXME: ambiguous bases are not properly handled seq[0][i] = c; seq[1][l-1-i] = 3 - c; rseq[0][l-1-i] = 3 - c; rseq[1][i] = c; } if (l - k < opt.t) { // too few unambiguous bases buf[x] = calloc(1, sizeof(bwtsw2_t)); free(seq[0]); continue; } // alignment b[0] = bsw2_aln1_core(&opt, bns, pac, target, l, seq, pool); for (k = 0; k < b[0]->n; ++k) if (b[0]->hits[k].n_seeds < opt.t_seeds) break; if (k < b[0]->n) { b[1] = bsw2_aln1_core(&opt, bns, pac, target, l, rseq, pool); for (i = 0; i < b[1]->n; ++i) { bsw2hit_t *p = &b[1]->hits[i]; int x = p->beg; p->flag ^= 0x10, p->is_rev ^= 1; // flip the strand p->beg = l - p->end; p->end = l - x; } flag_fr(b); merge_hits(b, l, 0); bsw2_resolve_duphits(0, 0, b[0], 0); bsw2_resolve_query_overlaps(b[0], opt.mask_level); } else b[1] = 0; // generate CIGAR and print SAM buf[x] = bsw2_dup_no_cigar(b[0]); // free free(seq[0]); bsw2_destroy(b[0]); } if (is_pe) bsw2_pair(&opt, bns->l_pac, pac, _seq->n, _seq->seq, buf); for (x = 0; x < _seq->n; ++x) { bsw2seq1_t *p = _seq->seq + x; uint8_t *seq[2]; int i; seq[0] = malloc(p->l * 2); seq[1] = seq[0] + p->l; for (i = 0; i < p->l; ++i) { int c = nst_nt4_table[(int)p->seq[i]]; if (c >= 4) c = (int)(drand48() * 4); seq[0][i] = c; seq[1][p->l-1-i] = 3 - c; } update_opt(&opt, _opt, p->l); write_aux(&opt, bns, p->l, seq, pac, buf[x], _seq->seq[x].name); free(seq[0]); } for (x = 0; x < _seq->n; ++x) { if (is_pe) update_mate_aux(buf[x], buf[x^1]); print_hits(bns, &opt, &_seq->seq[x], buf[x], is_pe, buf[x^1]); } for (x = 0; x < _seq->n; ++x) bsw2_destroy(buf[x]); free(buf); bsw2_global_destroy(pool); } #ifdef HAVE_PTHREAD typedef struct { int tid, is_pe; bsw2seq_t *_seq; const bsw2opt_t *_opt; const bntseq_t *bns; uint8_t *pac; const bwt_t *target; } thread_aux_t; /* another interface to bsw2_aln_core() to facilitate pthread_create() */ static void *worker(void *data) { thread_aux_t *p = (thread_aux_t*)data; bsw2_aln_core(p->_seq, p->_opt, p->bns, p->pac, p->target, p->is_pe); return 0; } #endif /* process sequences stored in _seq, generate SAM lines for these * sequences and reset _seq afterwards. */ static void process_seqs(bsw2seq_t *_seq, const bsw2opt_t *opt, const bntseq_t *bns, uint8_t *pac, const bwt_t *target, int is_pe) { int i; is_pe = is_pe? 1 : 0; #ifdef HAVE_PTHREAD if (opt->n_threads <= 1) { bsw2_aln_core(_seq, opt, bns, pac, target, is_pe); } else { pthread_t *tid; pthread_attr_t attr; thread_aux_t *data; int j; pthread_attr_init(&attr); pthread_attr_setdetachstate(&attr, PTHREAD_CREATE_JOINABLE); data = (thread_aux_t*)calloc(opt->n_threads, sizeof(thread_aux_t)); tid = (pthread_t*)calloc(opt->n_threads, sizeof(pthread_t)); for (j = 0; j < opt->n_threads; ++j) { thread_aux_t *p = data + j; p->tid = j; p->_opt = opt; p->bns = bns; p->is_pe = is_pe; p->pac = pac; p->target = target; p->_seq = calloc(1, sizeof(bsw2seq_t)); p->_seq->max = (_seq->n + opt->n_threads - 1) / opt->n_threads + 1; p->_seq->n = 0; p->_seq->seq = calloc(p->_seq->max, sizeof(bsw2seq1_t)); } for (i = 0; i < _seq->n; ++i) { // assign sequences to each thread bsw2seq_t *p = data[(i>>is_pe)%opt->n_threads]._seq; p->seq[p->n++] = _seq->seq[i]; } for (j = 0; j < opt->n_threads; ++j) pthread_create(&tid[j], &attr, worker, &data[j]); for (j = 0; j < opt->n_threads; ++j) pthread_join(tid[j], 0); for (j = 0; j < opt->n_threads; ++j) data[j]._seq->n = 0; for (i = 0; i < _seq->n; ++i) { // copy the result from each thread back bsw2seq_t *p = data[(i>>is_pe)%opt->n_threads]._seq; _seq->seq[i] = p->seq[p->n++]; } for (j = 0; j < opt->n_threads; ++j) { thread_aux_t *p = data + j; free(p->_seq->seq); free(p->_seq); } free(data); free(tid); } #else bsw2_aln_core(_seq, opt, bns, pac, target, is_pe); #endif // print and reset for (i = 0; i < _seq->n; ++i) { bsw2seq1_t *p = _seq->seq + i; if (p->sam) err_printf("%s", p->sam); free(p->name); free(p->seq); free(p->qual); free(p->sam); p->tid = -1; p->l = 0; p->name = p->seq = p->qual = p->sam = 0; } err_fflush(stdout); _seq->n = 0; } void bsw2_aln(const bsw2opt_t *opt, const bntseq_t *bns, bwt_t * const target, const char *fn, const char *fn2) { gzFile fp, fp2; kseq_t *ks, *ks2; int l, is_pe = 0, i, n; uint8_t *pac; bsw2seq_t *_seq; bseq1_t *bseq; pac = calloc(bns->l_pac/4+1, 1); for (l = 0; l < bns->n_seqs; ++l) err_printf("@SQ\tSN:%s\tLN:%d\n", bns->anns[l].name, bns->anns[l].len); err_fread_noeof(pac, 1, bns->l_pac/4+1, bns->fp_pac); fp = xzopen(fn, "r"); ks = kseq_init(fp); _seq = calloc(1, sizeof(bsw2seq_t)); if (fn2) { fp2 = xzopen(fn2, "r"); ks2 = kseq_init(fp2); is_pe = 1; } else fp2 = 0, ks2 = 0, is_pe = 0; while ((bseq = bseq_read(opt->chunk_size * opt->n_threads, &n, ks, ks2)) != 0) { int size = 0; if (n > _seq->max) { _seq->max = n; kroundup32(_seq->max); _seq->seq = realloc(_seq->seq, _seq->max * sizeof(bsw2seq1_t)); } _seq->n = n; for (i = 0; i < n; ++i) { bseq1_t *b = &bseq[i]; bsw2seq1_t *p = &_seq->seq[i]; p->tid = -1; p->l = b->l_seq; p->name = b->name; p->seq = b->seq; p->qual = b->qual; p->comment = b->comment; p->sam = 0; size += p->l; } fprintf(stderr, "[bsw2_aln] read %d sequences/pairs (%d bp) ...\n", n, size); free(bseq); process_seqs(_seq, opt, bns, pac, target, is_pe); } // free free(pac); free(_seq->seq); free(_seq); kseq_destroy(ks); err_gzclose(fp); if (fn2) { kseq_destroy(ks2); err_gzclose(fp2); } }