#include #include #include #include #include "bwa.h" #include "bwt.h" #include "bwtgap.h" #include "bntseq.h" #ifndef kroundup32 #define kroundup32(x) (--(x), (x)|=(x)>>1, (x)|=(x)>>2, (x)|=(x)>>4, (x)|=(x)>>8, (x)|=(x)>>16, ++(x)) #endif extern unsigned char nst_nt4_table[256]; extern void seq_reverse(int len, uint8_t *seq, int is_comp); bwa_opt_t bwa_def_opt = { 11, 4, -1, 1, 6, 32, 2, 0.04 }; struct bwa_idx_t { bwt_t *bwt; bntseq_t *bns; uint8_t *pac; }; struct bwa_buf_t { int max_buf; bwa_pestat_t pes; gap_stack_t *stack; gap_opt_t *opt; int *diff_tab; uint8_t *buf; int *logn; }; bwa_idx_t *bwa_idx_load(const char *prefix) { bwa_idx_t *p; int l; char *str; l = strlen(prefix); p = calloc(1, sizeof(bwa_idx_t)); str = malloc(l + 10); strcpy(str, prefix); p->bns = bns_restore(str); strcpy(str + l, ".bwt"); p->bwt = bwt_restore_bwt(str); str[l] = 0; strcpy(str + l, ".sa"); bwt_restore_sa(str, p->bwt); free(str); p->pac = calloc(p->bns->l_pac/4+1, 1); fread(p->pac, 1, p->bns->l_pac/4+1, p->bns->fp_pac); fclose(p->bns->fp_pac); p->bns->fp_pac = 0; return p; } void bwa_idx_destroy(bwa_idx_t *p) { bns_destroy(p->bns); bwt_destroy(p->bwt); free(p->pac); free(p); } bwa_buf_t *bwa_buf_init(const bwa_opt_t *opt, int max_score) { extern gap_opt_t *gap_init_opt(void); extern int bwa_cal_maxdiff(int l, double err, double thres); int i; bwa_buf_t *p; p = malloc(sizeof(bwa_buf_t)); p->stack = gap_init_stack2(max_score); p->opt = gap_init_opt(); p->opt->s_gapo = opt->s_gapo; p->opt->s_gape = opt->s_gape; p->opt->max_diff = opt->max_diff; p->opt->max_gapo = opt->max_gapo; p->opt->max_gape = opt->max_gape; p->opt->seed_len = opt->seed_len; p->opt->max_seed_diff = opt->max_seed_diff; p->opt->fnr = opt->fnr; p->diff_tab = calloc(BWA_MAX_QUERY_LEN, sizeof(int)); for (i = 1; i < BWA_MAX_QUERY_LEN; ++i) p->diff_tab[i] = bwa_cal_maxdiff(i, BWA_AVG_ERR, opt->fnr); p->logn = calloc(256, sizeof(int)); for (i = 1; i != 256; ++i) p->logn[i] = (int)(4.343 * log(i) + 0.499); return p; } void bwa_buf_destroy(bwa_buf_t *p) { gap_destroy_stack(p->stack); free(p->diff_tab); free(p->logn); free(p->opt); free(p); } bwa_sai_t bwa_sai(const bwa_idx_t *idx, bwa_buf_t *buf, const char *seq) { extern int bwt_cal_width(const bwt_t *bwt, int len, const ubyte_t *str, bwt_width_t *width); int i, seq_len, buf_len; bwt_width_t *w, *seed_w; uint8_t *s; gap_opt_t opt2 = *buf->opt; bwa_sai_t sai; seq_len = strlen(seq); // estimate the buffer length buf_len = (buf->opt->seed_len + seq_len + 1) * sizeof(bwt_width_t) + seq_len; if (buf_len > buf->max_buf) { buf->max_buf = buf_len; kroundup32(buf->max_buf); buf->buf = realloc(buf->buf, buf->max_buf); } memset(buf->buf, 0, buf_len); seed_w = (bwt_width_t*)buf->buf; w = seed_w + buf->opt->seed_len; s = (uint8_t*)(w + seq_len + 1); if (opt2.fnr > 0.) opt2.max_diff = buf->diff_tab[seq_len]; // copy the sequence for (i = 0; i < seq_len; ++i) s[i] = nst_nt4_table[(int)seq[i]]; seq_reverse(seq_len, s, 0); // mapping bwt_cal_width(idx->bwt, seq_len, s, w); if (opt2.seed_len >= seq_len) opt2.seed_len = 0x7fffffff; if (seq_len > buf->opt->seed_len) bwt_cal_width(idx->bwt, buf->opt->seed_len, s + (seq_len - buf->opt->seed_len), seed_w); for (i = 0; i < seq_len; ++i) // complement; I forgot why... s[i] = s[i] > 3? 4 : 3 - s[i]; sai.sai = (bwa_sai1_t*)bwt_match_gap(idx->bwt, seq_len, s, w, seq_len <= buf->opt->seed_len? 0 : seed_w, &opt2, &sai.n, buf->stack); return sai; } static void compute_NM(const uint8_t *pac, uint64_t l_pac, uint8_t *seq, int64_t pos, int n_cigar, uint32_t *cigar, int *n_mm, int *n_gaps) { uint64_t x = pos, z; int k, y = 0; *n_mm = *n_gaps = 0; for (k = 0; k < n_cigar; ++k) { int l = cigar[k]>>4; int op = cigar[k]&0xf; if (op == 0) { // match/mismatch for (z = 0; z < l && x + z < l_pac; ++z) { int c = pac[(x+z)>>2] >> ((~(x+z)&3)<<1) & 3; if (c > 3 || seq[y+z] > 3 || c != seq[y+z]) ++(*n_mm); } } if (op == 1 || op == 2) (*n_gaps) += l; if (op == 0 || op == 2) x += l; if (op == 0 || op == 1 || op == 4) y += l; } } void bwa_sa2aln(const bwa_idx_t *idx, bwa_buf_t *buf, const char *seq, uint64_t sa, int n_gaps, bwa_aln_t *aln) { extern bwtint_t bwa_sa2pos(const bntseq_t *bns, const bwt_t *bwt, bwtint_t sapos, int len, int *strand); extern bwa_cigar_t *bwa_refine_gapped_core(bwtint_t l_pac, const ubyte_t *pacseq, int len, const uint8_t *seq, bwtint_t *_pos, int ext, int *n_cigar, int is_end_correct); int strand, seq_len, i, n_gap, n_mm; uint64_t pos3, pac_pos; uint8_t *s[2]; memset(aln, 0, sizeof(bwa_aln_t)); seq_len = strlen(seq); if (seq_len<<1 > buf->max_buf) { buf->max_buf = seq_len<<1; kroundup32(buf->max_buf); buf->buf = realloc(buf->buf, buf->max_buf); } s[0] = buf->buf; s[1] = s[0] + seq_len; for (i = 0; i < seq_len; ++i) s[0][i] = s[1][i] = nst_nt4_table[(int)seq[i]]; seq_reverse(seq_len, s[1], 1); pac_pos = bwa_sa2pos(idx->bns, idx->bwt, sa, seq_len, &strand); if (strand) aln->flag |= 16; if (n_gaps) { // only for gapped alignment int n_cigar; bwa_cigar_t *cigar16; cigar16 = bwa_refine_gapped_core(idx->bns->l_pac, idx->pac, seq_len, s[strand], &pac_pos, strand? n_gaps : -n_gaps, &n_cigar, 1); aln->n_cigar = n_cigar; aln->cigar = malloc(n_cigar * 4); for (i = 0, pos3 = pac_pos; i < n_cigar; ++i) { int op = cigar16[i]>>14; int len = cigar16[i]&0x3fff; if (op == 3) op = 4; // the 16-bit CIGAR is different from the 32-bit CIGAR aln->cigar[i] = len<<4 | op; if (op == 0 || op == 2) pos3 += len; } free(cigar16); } else { // ungapped aln->n_cigar = 1; aln->cigar = malloc(4); aln->cigar[0] = seq_len<<4 | 0; pos3 = pac_pos + seq_len; } aln->n_n = bns_cnt_ambi(idx->bns, pac_pos, pos3 - pac_pos, &aln->ref_id); aln->offset = pac_pos - idx->bns->anns[aln->ref_id].offset; if (pos3 - idx->bns->anns[aln->ref_id].offset > idx->bns->anns[aln->ref_id].len) // read mapped beyond the end of a sequence aln->flag |= 4; // read unmapped compute_NM(idx->pac, idx->bns->l_pac, s[strand], pac_pos, aln->n_cigar, aln->cigar, &n_mm, &n_gap); aln->n_mm = n_mm; aln->n_gap = n_gap; } /************************ * Single-end alignment * ************************/ bwa_one_t *bwa_se(const bwa_idx_t *idx, bwa_buf_t *buf, const char *seq, int gen_cigar) { bwa_one_t *one; int best, cnt, i, seq_len; seq_len = strlen(seq); one = calloc(1, sizeof(bwa_one_t)); one->sai = bwa_sai(idx, buf, seq); if (one->sai.n == 0) return one; // count number of hits; randomly select one alignment best = one->sai.sai[0].score; for (i = cnt = 0; i < one->sai.n; ++i) { bwa_sai1_t *p = &one->sai.sai[i]; if (p->score > best) break; if (drand48() * (p->l - p->k + 1 + cnt) > (double)cnt) { one->which = p; one->sa = p->k + (bwtint_t)((p->l - p->k + 1) * drand48()); } cnt += p->l - p->k + 1; } one->c1 = cnt; for (; i < one->sai.n; ++i) cnt += one->sai.sai[i].l - one->sai.sai[i].k + 1; one->c2 = cnt - one->c1; // estimate single-end mapping quality one->mapQs = -1; if (one->c1 == 0) one->mapQs = 23; // FIXME: is it possible? else if (one->c1 > 1) one->mapQs = 0; else { int diff = one->which->n_mm + one->which->n_gapo + one->which->n_gape; if (diff >= buf->diff_tab[seq_len]) one->mapQs = 25; else if (one->c2 == 0) one->mapQs = 37; } if (one->mapQs < 0) { cnt = (one->c2 >= 255)? 255 : one->c2; one->mapQs = 23 < buf->logn[cnt]? 0 : 23 - buf->logn[cnt]; } one->mapQ = one->mapQs; // compute CIGAR on request one->one.ref_id = -1; if (gen_cigar) bwa_sa2aln(idx, buf, seq, one->sa, one->which->n_gapo + one->which->n_gape, &one->one); return one; } void bwa_one_destroy(bwa_one_t *one) { free(one->sai.sai); free(one->one.cigar); free(one); } /************************ * Paired-end alignment * ************************/ void bwa_pestat(bwa_buf_t *buf, int n, bwa_one_t **o[2]) { } void bwa_pe(const bwa_idx_t *idx, bwa_buf_t *buf, const char *seq[2], bwa_one_t *o[2]) { }