#include #include #include #include #include "bntseq.h" #include "bwa.h" #include "ksw.h" #include "utils.h" #include "kstring.h" #ifdef USE_MALLOC_WRAPPERS # include "malloc_wrap.h" #endif 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 = strdup(ks->name.s); s->comment = ks->comment.l? strdup(ks->comment.s) : 0; s->seq = strdup(ks->seq.s); s->qual = ks->qual.l? strdup(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 = realloc(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; kstring_t str; const char *int2base; *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 = malloc(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 and MD int k, x, y, u, n_mm = 0, n_gap = 0; str.l = str.m = *n_cigar * 4; str.s = (char*)cigar; // append MD to CIGAR int2base = rb < l_pac? "ACGTN" : "TGCAN"; for (k = 0, x = y = u = 0; k < *n_cigar; ++k) { int op, len; cigar = (uint32_t*)str.s; op = cigar[k]&0xf, len = cigar[k]>>4; if (op == 0) { // match for (i = 0; i < len; ++i) { if (query[x + i] != rseq[y + i]) { kputw(u, &str); kputc(int2base[rseq[y+i]], &str); ++n_mm; u = 0; } else ++u; } x += len; y += len; } else if (op == 2) { // deletion if (k > 0 && k < *n_cigar - 1) { // don't do the following if D is the first or the last CIGAR kputw(u, &str); kputc('^', &str); for (i = 0; i < len; ++i) kputc(int2base[rseq[y+i]], &str); u = 0; n_gap += len; } y += len; } else if (op == 1) x += len, n_gap += len; // insertion } kputw(u, &str); kputc(0, &str); *NM = n_mm + n_gap; cigar = (uint32_t*)str.s; } 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 is_rev; int64_t cb, ce, fm; bntann1_t *ra; 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 } fm = bns_depos(bns, (*rb + *re) >> 1, &is_rev); // coordinate of the middle point on the forward strand ra = &bns->anns[bns_pos2rid(bns, fm)]; // annotation of chr corresponding to the middle point cb = is_rev? (bns->l_pac<<1) - (ra->offset + ra->len) : ra->offset; // chr start on the mapping strand ce = cb + ra->len; // chr end if (cb > *rb || ce < *re) { // fix is needed int i, score, n_cigar, y, NM; uint32_t *cigar; int64_t x; cb = cb > *rb? cb : *rb; ce = ce < *re? ce : *re; 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 <= cb && cb < x + len) *qb = y + (cb - x), *rb = cb; if (x < ce && ce <= x + len) { *qe = y + (ce - x), *re = ce; break; } else x += len, y += len; } else if (op == 1) { y += len; } else if (op == 2) { if (x <= cb && cb < x + len) *qb = y, *rb = x + len; if (x < ce && ce <= x + len) { *qe = y, *re = x; break; } else x += len; } else abort(); // should not be here } free(cigar); } return (*qb == *qe || *rb == *re)? -2 : 0; } /********************* * Full index reader * *********************/ char *bwa_idx_infer_prefix(const char *hint) { char *prefix; int l_hint; FILE *fp; l_hint = strlen(hint); prefix = malloc(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 = calloc(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 = calloc(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 = calloc(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; extern char *bwa_pg; 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); err_printf("%s\n", bwa_pg); } 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 = strdup(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; }