#include #include #include #include #include #include #include #ifdef HAVE_CONFIG_H #include "config.h" #endif #include "bwtaln.h" #include "bwtgap.h" #include "utils.h" #include "bwa.h" #ifdef HAVE_PTHREAD #include #endif #ifdef USE_MALLOC_WRAPPERS # include "malloc_wrap.h" #endif gap_opt_t *gap_init_opt() { gap_opt_t *o; o = (gap_opt_t*)calloc(1, sizeof(gap_opt_t)); /* IMPORTANT: s_mm*10 should be about the average base error rate. Voilating this requirement will break pairing! */ o->s_mm = 3; o->s_gapo = 11; o->s_gape = 4; o->max_diff = -1; o->max_gapo = 1; o->max_gape = 6; o->indel_end_skip = 5; o->max_del_occ = 10; o->max_entries = 2000000; o->mode = BWA_MODE_GAPE | BWA_MODE_COMPREAD; o->seed_len = 32; o->max_seed_diff = 2; o->fnr = 0.04; o->n_threads = 1; o->max_top2 = 30; o->trim_qual = 0; return o; } int bwa_cal_maxdiff(int l, double err, double thres) { double elambda = exp(-l * err); double sum, y = 1.0; int k, x = 1; for (k = 1, sum = elambda; k < 1000; ++k) { y *= l * err; x *= k; sum += elambda * y / x; if (1.0 - sum < thres) return k; } return 2; } // width must be filled as zero int bwt_cal_width(const bwt_t *bwt, int len, const ubyte_t *str, bwt_width_t *width) { bwtint_t k, l, ok, ol; int i, bid; bid = 0; k = 0; l = bwt->seq_len; for (i = 0; i < len; ++i) { ubyte_t c = str[i]; if (c < 4) { bwt_2occ(bwt, k - 1, l, c, &ok, &ol); k = bwt->L2[c] + ok + 1; l = bwt->L2[c] + ol; } if (k > l || c > 3) { // then restart k = 0; l = bwt->seq_len; ++bid; } width[i].w = l - k + 1; width[i].bid = bid; } width[len].w = 0; width[len].bid = ++bid; return bid; } void bwa_cal_sa_reg_gap(int tid, bwt_t *const bwt, int n_seqs, bwa_seq_t *seqs, const gap_opt_t *opt) { int i, j, max_l = 0, max_len; gap_stack_t *stack; bwt_width_t *w, *seed_w; gap_opt_t local_opt = *opt; // initiate priority stack for (i = max_len = 0; i != n_seqs; ++i) if (seqs[i].len > max_len) max_len = seqs[i].len; if (opt->fnr > 0.0) local_opt.max_diff = bwa_cal_maxdiff(max_len, BWA_AVG_ERR, opt->fnr); if (local_opt.max_diff < local_opt.max_gapo) local_opt.max_gapo = local_opt.max_diff; stack = gap_init_stack(local_opt.max_diff, local_opt.max_gapo, local_opt.max_gape, &local_opt); seed_w = (bwt_width_t*)calloc(opt->seed_len+1, sizeof(bwt_width_t)); w = 0; for (i = 0; i != n_seqs; ++i) { bwa_seq_t *p = seqs + i; #ifdef HAVE_PTHREAD if (i % opt->n_threads != tid) continue; #endif p->sa = 0; p->type = BWA_TYPE_NO_MATCH; p->c1 = p->c2 = 0; p->n_aln = 0; p->aln = 0; if (max_l < p->len) { max_l = p->len; w = (bwt_width_t*)realloc(w, (max_l + 1) * sizeof(bwt_width_t)); memset(w, 0, (max_l + 1) * sizeof(bwt_width_t)); } bwt_cal_width(bwt, p->len, p->seq, w); if (opt->fnr > 0.0) local_opt.max_diff = bwa_cal_maxdiff(p->len, BWA_AVG_ERR, opt->fnr); local_opt.seed_len = opt->seed_len < p->len? opt->seed_len : 0x7fffffff; if (p->len > opt->seed_len) bwt_cal_width(bwt, opt->seed_len, p->seq + (p->len - opt->seed_len), seed_w); // core function for (j = 0; j < p->len; ++j) // we need to complement p->seq[j] = p->seq[j] > 3? 4 : 3 - p->seq[j]; p->aln = bwt_match_gap(bwt, p->len, p->seq, w, p->len <= opt->seed_len? 0 : seed_w, &local_opt, &p->n_aln, stack); //fprintf(stderr, "mm=%lld,ins=%lld,del=%lld,gapo=%lld\n", p->aln->n_mm, p->aln->n_ins, p->aln->n_del, p->aln->n_gapo); // clean up the unused data in the record free(p->name); free(p->seq); free(p->rseq); free(p->qual); p->name = 0; p->seq = p->rseq = p->qual = 0; } free(seed_w); free(w); gap_destroy_stack(stack); } #ifdef HAVE_PTHREAD typedef struct { int tid; bwt_t *bwt; int n_seqs; bwa_seq_t *seqs; const gap_opt_t *opt; } thread_aux_t; static void *worker(void *data) { thread_aux_t *d = (thread_aux_t*)data; bwa_cal_sa_reg_gap(d->tid, d->bwt, d->n_seqs, d->seqs, d->opt); return 0; } #endif bwa_seqio_t *bwa_open_reads(int mode, const char *fn_fa) { bwa_seqio_t *ks; if (mode & BWA_MODE_BAM) { // open BAM int which = 0; if (mode & BWA_MODE_BAM_SE) which |= 4; if (mode & BWA_MODE_BAM_READ1) which |= 1; if (mode & BWA_MODE_BAM_READ2) which |= 2; if (which == 0) which = 7; // then read all reads ks = bwa_bam_open(fn_fa, which); } else ks = bwa_seq_open(fn_fa); return ks; } void bwa_aln_core(const char *prefix, const char *fn_fa, const gap_opt_t *opt) { int i, n_seqs, tot_seqs = 0; bwa_seq_t *seqs; bwa_seqio_t *ks; clock_t t; bwt_t *bwt; // initialization ks = bwa_open_reads(opt->mode, fn_fa); { // load BWT char *str = (char*)calloc(strlen(prefix) + 10, 1); strcpy(str, prefix); strcat(str, ".bwt"); bwt = bwt_restore_bwt(str); free(str); } // core loop err_fwrite(SAI_MAGIC, 1, 4, stdout); err_fwrite(opt, sizeof(gap_opt_t), 1, stdout); while ((seqs = bwa_read_seq(ks, 0x40000, &n_seqs, opt->mode, opt->trim_qual)) != 0) { tot_seqs += n_seqs; t = clock(); fprintf(stderr, "[bwa_aln_core] calculate SA coordinate... "); #ifdef HAVE_PTHREAD if (opt->n_threads <= 1) { // no multi-threading at all bwa_cal_sa_reg_gap(0, bwt, n_seqs, seqs, opt); } 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) { data[j].tid = j; data[j].bwt = bwt; data[j].n_seqs = n_seqs; data[j].seqs = seqs; data[j].opt = opt; pthread_create(&tid[j], &attr, worker, data + j); } for (j = 0; j < opt->n_threads; ++j) pthread_join(tid[j], 0); free(data); free(tid); } #else bwa_cal_sa_reg_gap(0, bwt, n_seqs, seqs, opt); #endif fprintf(stderr, "%.2f sec\n", (float)(clock() - t) / CLOCKS_PER_SEC); t = clock(); fprintf(stderr, "[bwa_aln_core] write to the disk... "); for (i = 0; i < n_seqs; ++i) { bwa_seq_t *p = seqs + i; err_fwrite(&p->n_aln, 4, 1, stdout); if (p->n_aln) err_fwrite(p->aln, sizeof(bwt_aln1_t), p->n_aln, stdout); } fprintf(stderr, "%.2f sec\n", (float)(clock() - t) / CLOCKS_PER_SEC); bwa_free_read_seq(n_seqs, seqs); fprintf(stderr, "[bwa_aln_core] %d sequences have been processed.\n", tot_seqs); } // destroy bwt_destroy(bwt); bwa_seq_close(ks); } int bwa_aln(int argc, char *argv[]) { int c, opte = -1; gap_opt_t *opt; char *prefix; opt = gap_init_opt(); while ((c = getopt(argc, argv, "n:o:e:i:d:l:k:LR:m:t:NM:O:E:q:f:b012IYB:")) >= 0) { switch (c) { case 'n': if (strstr(optarg, ".")) opt->fnr = atof(optarg), opt->max_diff = -1; else opt->max_diff = atoi(optarg), opt->fnr = -1.0; break; case 'o': opt->max_gapo = atoi(optarg); break; case 'e': opte = atoi(optarg); break; case 'M': opt->s_mm = atoi(optarg); break; case 'O': opt->s_gapo = atoi(optarg); break; case 'E': opt->s_gape = atoi(optarg); break; case 'd': opt->max_del_occ = atoi(optarg); break; case 'i': opt->indel_end_skip = atoi(optarg); break; case 'l': opt->seed_len = atoi(optarg); break; case 'k': opt->max_seed_diff = atoi(optarg); break; case 'm': opt->max_entries = atoi(optarg); break; case 't': opt->n_threads = atoi(optarg); break; case 'L': opt->mode |= BWA_MODE_LOGGAP; break; case 'R': opt->max_top2 = atoi(optarg); break; case 'q': opt->trim_qual = atoi(optarg); break; case 'N': opt->mode |= BWA_MODE_NONSTOP; opt->max_top2 = 0x7fffffff; break; case 'f': xreopen(optarg, "wb", stdout); break; case 'b': opt->mode |= BWA_MODE_BAM; break; case '0': opt->mode |= BWA_MODE_BAM_SE; break; case '1': opt->mode |= BWA_MODE_BAM_READ1; break; case '2': opt->mode |= BWA_MODE_BAM_READ2; break; case 'I': opt->mode |= BWA_MODE_IL13; break; case 'Y': opt->mode |= BWA_MODE_CFY; break; case 'B': opt->mode |= atoi(optarg) << 24; break; default: return 1; } } if (opte > 0) { opt->max_gape = opte; opt->mode &= ~BWA_MODE_GAPE; } if (optind + 2 > argc) { fprintf(stderr, "\n"); fprintf(stderr, "Usage: bwa aln [options] \n\n"); fprintf(stderr, "Options: -n NUM max #diff (int) or missing prob under %.2f err rate (float) [%.2f]\n", BWA_AVG_ERR, opt->fnr); fprintf(stderr, " -o INT maximum number or fraction of gap opens [%d]\n", opt->max_gapo); fprintf(stderr, " -e INT maximum number of gap extensions, -1 for disabling long gaps [-1]\n"); fprintf(stderr, " -i INT do not put an indel within INT bp towards the ends [%d]\n", opt->indel_end_skip); fprintf(stderr, " -d INT maximum occurrences for extending a long deletion [%d]\n", opt->max_del_occ); fprintf(stderr, " -l INT seed length [%d]\n", opt->seed_len); fprintf(stderr, " -k INT maximum differences in the seed [%d]\n", opt->max_seed_diff); fprintf(stderr, " -m INT maximum entries in the queue [%d]\n", opt->max_entries); fprintf(stderr, " -t INT number of threads [%d]\n", opt->n_threads); fprintf(stderr, " -M INT mismatch penalty [%d]\n", opt->s_mm); fprintf(stderr, " -O INT gap open penalty [%d]\n", opt->s_gapo); fprintf(stderr, " -E INT gap extension penalty [%d]\n", opt->s_gape); fprintf(stderr, " -R INT stop searching when there are >INT equally best hits [%d]\n", opt->max_top2); fprintf(stderr, " -q INT quality threshold for read trimming down to %dbp [%d]\n", BWA_MIN_RDLEN, opt->trim_qual); fprintf(stderr, " -f FILE file to write output to instead of stdout\n"); fprintf(stderr, " -B INT length of barcode\n"); fprintf(stderr, " -L log-scaled gap penalty for long deletions\n"); fprintf(stderr, " -N non-iterative mode: search for all n-difference hits (slooow)\n"); fprintf(stderr, " -I the input is in the Illumina 1.3+ FASTQ-like format\n"); fprintf(stderr, " -b the input read file is in the BAM format\n"); fprintf(stderr, " -0 use single-end reads only (effective with -b)\n"); fprintf(stderr, " -1 use the 1st read in a pair (effective with -b)\n"); fprintf(stderr, " -2 use the 2nd read in a pair (effective with -b)\n"); fprintf(stderr, " -Y filter Casava-filtered sequences\n"); fprintf(stderr, "\n"); return 1; } if (opt->fnr > 0.0) { int i, k; for (i = 17, k = 0; i <= 250; ++i) { int l = bwa_cal_maxdiff(i, BWA_AVG_ERR, opt->fnr); if (l != k) fprintf(stderr, "[bwa_aln] %dbp reads: max_diff = %d\n", i, l); k = l; } } if ((prefix = bwa_idx_infer_prefix(argv[optind])) == 0) { fprintf(stderr, "[%s] fail to locate the index\n", __func__); free(opt); return 1; } bwa_aln_core(prefix, argv[optind+1], opt); free(opt); free(prefix); return 0; }