#include #include #include #include #include "bntseq.h" #include "bwa.h" #include "ksw.h" #include "utils.h" int bwa_verbose = 3; char bwa_rg_id[256]; /************************ * Batch FASTA/Q reader * ************************/ #include "kseq.h" KSEQ_DECLARE(gzFile) static inline void trim_readno(kstring_t *s) { if (s->l > 2 && s->s[s->l-2] == '/' && isdigit(s->s[s->l-1])) s->l -= 2, s->s[s->l] = 0; } static inline void kseq2bseq1(const kseq_t *ks, bseq1_t *s) { // TODO: it would be better to allocate one chunk of memory, but probably it does not matter in practice s->name = xstrdup(ks->name.s); s->comment = ks->comment.l? xstrdup(ks->comment.s) : 0; s->seq = xstrdup(ks->seq.s); s->qual = ks->qual.l? xstrdup(ks->qual.s) : 0; s->l_seq = strlen(s->seq); } bseq1_t *bseq_read(int chunk_size, int *n_, void *ks1_, void *ks2_) { kseq_t *ks = (kseq_t*)ks1_, *ks2 = (kseq_t*)ks2_; int size = 0, m, n; bseq1_t *seqs; m = n = 0; seqs = 0; while (kseq_read(ks) >= 0) { if (ks2 && kseq_read(ks2) < 0) { // the 2nd file has fewer reads fprintf(stderr, "[W::%s] the 2nd file has fewer sequences.\n", __func__); break; } if (n >= m) { m = m? m<<1 : 256; seqs = xrealloc(seqs, m * sizeof(bseq1_t)); } trim_readno(&ks->name); kseq2bseq1(ks, &seqs[n]); size += seqs[n++].l_seq; if (ks2) { trim_readno(&ks2->name); kseq2bseq1(ks2, &seqs[n]); size += seqs[n++].l_seq; } if (size >= chunk_size && (n&1) == 0) break; } if (size == 0) { // test if the 2nd file is finished if (ks2 && kseq_read(ks2) >= 0) fprintf(stderr, "[W::%s] the 1st file has fewer sequences.\n", __func__); } *n_ = n; return seqs; } /***************** * CIGAR related * *****************/ void bwa_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++] = -1; // ambiguous base } for (j = 0; j < 5; ++j) mat[k++] = -1; } // Generate CIGAR when the alignment end points are known 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, int *NM) { uint32_t *cigar = 0; uint8_t tmp, *rseq; int i; int64_t rlen; *n_cigar = 0; *NM = -1; 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; } if (l_query == re - rb && w_ == 0) { // no gap; no need to do DP cigar = xmalloc(4); cigar[0] = l_query<<4 | 0; *n_cigar = 1; for (i = 0, *score = 0; i < l_query; ++i) *score += mat[rseq[i]*5 + query[i]]; } else { int w, max_gap, min_w; //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 max_gap = (int)((double)(((l_query+1)>>1) * mat[0] - q) / r + 1.); max_gap = max_gap > 1? max_gap : 1; w = (max_gap + abs(rlen - l_query) + 1) >> 1; w = w < w_? w : w_; min_w = abs(rlen - l_query) + 3; w = w > min_w? w : min_w; // NW alignment *score = ksw_global(l_query, query, rlen, rseq, 5, mat, q, r, w, n_cigar, &cigar); } {// compute NM int k, x, y, n_mm = 0, n_gap = 0; for (k = 0, x = y = 0; k < *n_cigar; ++k) { int op = cigar[k]&0xf; int len = cigar[k]>>4; if (op == 0) { // match for (i = 0; i < len; ++i) if (query[x + i] != rseq[y + i]) ++n_mm; x += len; y += len; } else if (op == 1) x += len, n_gap += len; else if (op == 2) y += len, n_gap += len; } *NM = n_mm + n_gap; } 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; } int bwa_fix_xref(const int8_t mat[25], int q, int r, int w, const bntseq_t *bns, const uint8_t *pac, uint8_t *query, int *qb, int *qe, int64_t *rb, int64_t *re) { int ib, ie, is_rev; int64_t fb, fe, mid = -1; if (*rb < bns->l_pac && *re > bns->l_pac) { // cross the for-rev boundary; actually with BWA-MEM, we should never come to here *qb = *qe = *rb = *re = -1; return -1; // unable to fix } else { fb = bns_depos(bns, *rb < bns->l_pac? *rb : *re - 1, &is_rev); ib = bns_pos2rid(bns, fb); if (fb - bns->anns[ib].offset + (*re - *rb) <= bns->anns[ib].len) return 0; // no need to fix fe = bns_depos(bns, *re - 1 < bns->l_pac? *re - 1 : *rb, &is_rev); ie = bns_pos2rid(bns, fe); if (ie - ib > 1) { // bridge three or more references *qb = *qe = *rb = *re = -1; return -2; // unable to fix } else { int l = bns->anns[ib].offset + bns->anns[ib].len - fb; mid = is_rev? *re - l : *rb + l; } } if (mid >= 0) { int i, score, n_cigar, y, NM; uint32_t *cigar; int64_t x; cigar = bwa_gen_cigar(mat, q, r, w, bns->l_pac, pac, *qe - *qb, query + *qb, *rb, *re, &score, &n_cigar, &NM); for (i = 0, x = *rb, y = *qb; i < n_cigar; ++i) { int op = cigar[i]&0xf, len = cigar[i]>>4; if (op == 0) { if (x <= mid && mid < x + len) { if (mid - *rb > *re - mid) { // the first part is longer if (x == mid) { // need to check the previous operation assert(i); // mid != *rb should always stand if ((cigar[i-1]&0xf) == 1) *qe = y - (cigar[i-1]>>4), *re = x; else if ((cigar[i-1]&0xf) == 2) *qe = y, *re = x - (cigar[i-1]>>4); else abort(); // should not be here } else *qe = y + (mid - x), *re = mid; } else *qb = y + (mid - x), *rb = mid; break; } else x += len, y += len; } else if (op == 1) { // insertion y += len; } else if (op == 2) { // deletion if (x <= mid && mid < x + len) { if (mid - *rb > *re - mid) *qe = y, *re = x; else *qb = y, *rb = x + len; break; } else x += len; } else abort(); // should not be here } free(cigar); } return 1; } /********************* * Full index reader * *********************/ char *bwa_idx_infer_prefix(const char *hint) { char *prefix; int l_hint; FILE *fp; l_hint = strlen(hint); prefix = xmalloc(l_hint + 3 + 4 + 1); strcpy(prefix, hint); strcpy(prefix + l_hint, ".64.bwt"); if ((fp = fopen(prefix, "rb")) != 0) { fclose(fp); prefix[l_hint + 3] = 0; return prefix; } else { strcpy(prefix + l_hint, ".bwt"); if ((fp = fopen(prefix, "rb")) == 0) { free(prefix); return 0; } else { fclose(fp); prefix[l_hint] = 0; return prefix; } } } bwt_t *bwa_idx_load_bwt(const char *hint) { char *tmp, *prefix; bwt_t *bwt; prefix = bwa_idx_infer_prefix(hint); if (prefix == 0) { if (bwa_verbose >= 1) fprintf(stderr, "[E::%s] fail to locate the index files\n", __func__); return 0; } tmp = xcalloc(strlen(prefix) + 5, 1); strcat(strcpy(tmp, prefix), ".bwt"); // FM-index bwt = bwt_restore_bwt(tmp); strcat(strcpy(tmp, prefix), ".sa"); // partial suffix array (SA) bwt_restore_sa(tmp, bwt); free(tmp); free(prefix); return bwt; } bwaidx_t *bwa_idx_load(const char *hint, int which) { bwaidx_t *idx; char *prefix; prefix = bwa_idx_infer_prefix(hint); if (prefix == 0) { if (bwa_verbose >= 1) fprintf(stderr, "[E::%s] fail to locate the index files\n", __func__); return 0; } idx = xcalloc(1, sizeof(bwaidx_t)); if (which & BWA_IDX_BWT) idx->bwt = bwa_idx_load_bwt(hint); if (which & BWA_IDX_BNS) { idx->bns = bns_restore(prefix); if (which & BWA_IDX_PAC) { idx->pac = xcalloc(idx->bns->l_pac/4+1, 1); err_fread_noeof(idx->pac, 1, idx->bns->l_pac/4+1, idx->bns->fp_pac); // concatenated 2-bit encoded sequence err_fclose(idx->bns->fp_pac); idx->bns->fp_pac = 0; } } free(prefix); return idx; } void bwa_idx_destroy(bwaidx_t *idx) { if (idx == 0) return; if (idx->bwt) bwt_destroy(idx->bwt); if (idx->bns) bns_destroy(idx->bns); if (idx->pac) free(idx->pac); free(idx); } /*********************** * SAM header routines * ***********************/ void bwa_print_sam_hdr(const bntseq_t *bns, const char *rg_line) { int i; for (i = 0; i < bns->n_seqs; ++i) err_printf("@SQ\tSN:%s\tLN:%d\n", bns->anns[i].name, bns->anns[i].len); if (rg_line) err_printf("%s\n", rg_line); } static char *bwa_escape(char *s) { char *p, *q; for (p = q = s; *p; ++p) { if (*p == '\\') { ++p; if (*p == 't') *q++ = '\t'; else if (*p == 'n') *q++ = '\n'; else if (*p == 'r') *q++ = '\r'; else if (*p == '\\') *q++ = '\\'; } else *q++ = *p; } *q = '\0'; return s; } char *bwa_set_rg(const char *s) { char *p, *q, *r, *rg_line = 0; memset(bwa_rg_id, 0, 256); if (strstr(s, "@RG") != s) { if (bwa_verbose >= 1) fprintf(stderr, "[E::%s] the read group line is not started with @RG\n", __func__); goto err_set_rg; } rg_line = xstrdup(s); bwa_escape(rg_line); if ((p = strstr(rg_line, "\tID:")) == 0) { if (bwa_verbose >= 1) fprintf(stderr, "[E::%s] no ID at the read group line\n", __func__); goto err_set_rg; } p += 4; for (q = p; *q && *q != '\t' && *q != '\n'; ++q); if (q - p + 1 > 256) { if (bwa_verbose >= 1) fprintf(stderr, "[E::%s] @RG:ID is longer than 255 characters\n", __func__); goto err_set_rg; } for (q = p, r = bwa_rg_id; *q && *q != '\t' && *q != '\n'; ++q) *r++ = *q; return rg_line; err_set_rg: free(rg_line); return 0; }