#include #include #include #include #include #include #include #include "bam.h" #include "ksort.h" static int g_is_by_qname = 0; static int strnum_cmp(const char *_a, const char *_b) { const unsigned char *a = (const unsigned char*)_a, *b = (const unsigned char*)_b; const unsigned char *pa = a, *pb = b; while (*pa && *pb) { if (isdigit(*pa) && isdigit(*pb)) { while (*pa == '0') ++pa; while (*pb == '0') ++pb; while (isdigit(*pa) && isdigit(*pb) && *pa == *pb) ++pa, ++pb; if (isdigit(*pa) && isdigit(*pb)) { int i = 0; while (isdigit(pa[i]) && isdigit(pb[i])) ++i; return isdigit(pa[i])? 1 : isdigit(pb[i])? -1 : (int)*pa - (int)*pb; } else if (isdigit(*pa)) return 1; else if (isdigit(*pb)) return -1; else if (pa - a != pb - b) return pa - a < pb - b? 1 : -1; } else { if (*pa != *pb) return (int)*pa - (int)*pb; ++pa; ++pb; } } return *pa? 1 : *pb? -1 : 0; } #define HEAP_EMPTY 0xffffffffffffffffull typedef struct { int i; uint64_t pos, idx; bam1_t *b; } heap1_t; #define __pos_cmp(a, b) ((a).pos > (b).pos || ((a).pos == (b).pos && ((a).i > (b).i || ((a).i == (b).i && (a).idx > (b).idx)))) static inline int heap_lt(const heap1_t a, const heap1_t b) { if (g_is_by_qname) { int t; if (a.b == 0 || b.b == 0) return a.b == 0? 1 : 0; t = strnum_cmp(bam1_qname(a.b), bam1_qname(b.b)); return (t > 0 || (t == 0 && (a.b->core.flag&0xc0) > (b.b->core.flag&0xc0))); } else return __pos_cmp(a, b); } KSORT_INIT(heap, heap1_t, heap_lt) static void swap_header_targets(bam_header_t *h1, bam_header_t *h2) { bam_header_t t; t.n_targets = h1->n_targets, h1->n_targets = h2->n_targets, h2->n_targets = t.n_targets; t.target_name = h1->target_name, h1->target_name = h2->target_name, h2->target_name = t.target_name; t.target_len = h1->target_len, h1->target_len = h2->target_len, h2->target_len = t.target_len; } static void swap_header_text(bam_header_t *h1, bam_header_t *h2) { int tempi; char *temps; tempi = h1->l_text, h1->l_text = h2->l_text, h2->l_text = tempi; temps = h1->text, h1->text = h2->text, h2->text = temps; } #define MERGE_RG 1 #define MERGE_UNCOMP 2 #define MERGE_LEVEL1 4 #define MERGE_FORCE 8 /*! @abstract Merge multiple sorted BAM. @param is_by_qname whether to sort by query name @param out output BAM file name @param headers name of SAM file from which to copy '@' header lines, or NULL to copy them from the first file to be merged @param n number of files to be merged @param fn names of files to be merged @discussion Padding information may NOT correctly maintained. This function is NOT thread safe. */ int bam_merge_core2(int by_qname, const char *out, const char *headers, int n, char * const *fn, int flag, const char *reg, int n_threads, int level) { bamFile fpout, *fp; heap1_t *heap; bam_header_t *hout = 0; bam_header_t *hheaders = NULL; int i, j, *RG_len = 0; uint64_t idx = 0; char **RG = 0, mode[8]; bam_iter_t *iter = 0; if (headers) { tamFile fpheaders = sam_open(headers); if (fpheaders == 0) { const char *message = strerror(errno); fprintf(stderr, "[bam_merge_core] cannot open '%s': %s\n", headers, message); return -1; } hheaders = sam_header_read(fpheaders); sam_close(fpheaders); } g_is_by_qname = by_qname; fp = (bamFile*)calloc(n, sizeof(bamFile)); heap = (heap1_t*)calloc(n, sizeof(heap1_t)); iter = (bam_iter_t*)calloc(n, sizeof(bam_iter_t)); // prepare RG tag if (flag & MERGE_RG) { RG = (char**)calloc(n, sizeof(void*)); RG_len = (int*)calloc(n, sizeof(int)); for (i = 0; i != n; ++i) { int l = strlen(fn[i]); const char *s = fn[i]; if (l > 4 && strcmp(s + l - 4, ".bam") == 0) l -= 4; for (j = l - 1; j >= 0; --j) if (s[j] == '/') break; ++j; l -= j; RG[i] = calloc(l + 1, 1); RG_len[i] = l; strncpy(RG[i], s + j, l); } } // read the first for (i = 0; i != n; ++i) { bam_header_t *hin; fp[i] = bam_open(fn[i], "r"); if (fp[i] == 0) { int j; fprintf(stderr, "[bam_merge_core] fail to open file %s\n", fn[i]); for (j = 0; j < i; ++j) bam_close(fp[j]); free(fp); free(heap); // FIXME: possible memory leak return -1; } hin = bam_header_read(fp[i]); if (i == 0) { // the first BAM hout = hin; } else { // validate multiple baf int min_n_targets = hout->n_targets; if (hin->n_targets < min_n_targets) min_n_targets = hin->n_targets; for (j = 0; j < min_n_targets; ++j) if (strcmp(hout->target_name[j], hin->target_name[j]) != 0) { fprintf(stderr, "[bam_merge_core] different target sequence name: '%s' != '%s' in file '%s'\n", hout->target_name[j], hin->target_name[j], fn[i]); return -1; } // If this input file has additional target reference sequences, // add them to the headers to be output if (hin->n_targets > hout->n_targets) { swap_header_targets(hout, hin); // FIXME Possibly we should also create @SQ text headers // for the newly added reference sequences } bam_header_destroy(hin); } } if (hheaders) { // If the text headers to be swapped in include any @SQ headers, // check that they are consistent with the existing binary list // of reference information. if (hheaders->n_targets > 0) { if (hout->n_targets != hheaders->n_targets) { fprintf(stderr, "[bam_merge_core] number of @SQ headers in '%s' differs from number of target sequences\n", headers); if (!reg) return -1; } for (j = 0; j < hout->n_targets; ++j) if (strcmp(hout->target_name[j], hheaders->target_name[j]) != 0) { fprintf(stderr, "[bam_merge_core] @SQ header '%s' in '%s' differs from target sequence\n", hheaders->target_name[j], headers); if (!reg) return -1; } } swap_header_text(hout, hheaders); bam_header_destroy(hheaders); } if (reg) { int tid, beg, end; if (bam_parse_region(hout, reg, &tid, &beg, &end) < 0) { fprintf(stderr, "[%s] Malformated region string or undefined reference name\n", __func__); return -1; } for (i = 0; i < n; ++i) { bam_index_t *idx; idx = bam_index_load(fn[i]); iter[i] = bam_iter_query(idx, tid, beg, end); bam_index_destroy(idx); } } for (i = 0; i < n; ++i) { heap1_t *h = heap + i; h->i = i; h->b = (bam1_t*)calloc(1, sizeof(bam1_t)); if (bam_iter_read(fp[i], iter[i], h->b) >= 0) { h->pos = ((uint64_t)h->b->core.tid<<32) | (uint32_t)((int32_t)h->b->core.pos+1)<<1 | bam1_strand(h->b); h->idx = idx++; } else h->pos = HEAP_EMPTY; } if (flag & MERGE_UNCOMP) level = 0; else if (flag & MERGE_LEVEL1) level = 1; strcpy(mode, "w"); if (level >= 0) sprintf(mode + 1, "%d", level < 9? level : 9); if ((fpout = strcmp(out, "-")? bam_open(out, "w") : bam_dopen(fileno(stdout), "w")) == 0) { fprintf(stderr, "[%s] fail to create the output file.\n", __func__); return -1; } bam_header_write(fpout, hout); bam_header_destroy(hout); if (!(flag & MERGE_UNCOMP)) bgzf_mt(fpout, n_threads, 256); ks_heapmake(heap, n, heap); while (heap->pos != HEAP_EMPTY) { bam1_t *b = heap->b; if (flag & MERGE_RG) { uint8_t *rg = bam_aux_get(b, "RG"); if (rg) bam_aux_del(b, rg); bam_aux_append(b, "RG", 'Z', RG_len[heap->i] + 1, (uint8_t*)RG[heap->i]); } bam_write1_core(fpout, &b->core, b->data_len, b->data); if ((j = bam_iter_read(fp[heap->i], iter[heap->i], b)) >= 0) { heap->pos = ((uint64_t)b->core.tid<<32) | (uint32_t)((int)b->core.pos+1)<<1 | bam1_strand(b); heap->idx = idx++; } else if (j == -1) { heap->pos = HEAP_EMPTY; free(heap->b->data); free(heap->b); heap->b = 0; } else fprintf(stderr, "[bam_merge_core] '%s' is truncated. Continue anyway.\n", fn[heap->i]); ks_heapadjust(heap, 0, n, heap); } if (flag & MERGE_RG) { for (i = 0; i != n; ++i) free(RG[i]); free(RG); free(RG_len); } for (i = 0; i != n; ++i) { bam_iter_destroy(iter[i]); bam_close(fp[i]); } bam_close(fpout); free(fp); free(heap); free(iter); return 0; } int bam_merge_core(int by_qname, const char *out, const char *headers, int n, char * const *fn, int flag, const char *reg) { return bam_merge_core2(by_qname, out, headers, n, fn, flag, reg, 0, -1); } int bam_merge(int argc, char *argv[]) { int c, is_by_qname = 0, flag = 0, ret = 0, n_threads = 0, level = -1; char *fn_headers = NULL, *reg = 0; while ((c = getopt(argc, argv, "h:nru1R:f@:l:")) >= 0) { switch (c) { case 'r': flag |= MERGE_RG; break; case 'f': flag |= MERGE_FORCE; break; case 'h': fn_headers = strdup(optarg); break; case 'n': is_by_qname = 1; break; case '1': flag |= MERGE_LEVEL1; break; case 'u': flag |= MERGE_UNCOMP; break; case 'R': reg = strdup(optarg); break; case 'l': level = atoi(optarg); break; case '@': n_threads = atoi(optarg); break; } } if (optind + 2 >= argc) { fprintf(stderr, "\n"); fprintf(stderr, "Usage: samtools merge [-nr] [-h inh.sam] [...]\n\n"); fprintf(stderr, "Options: -n sort by read names\n"); fprintf(stderr, " -r attach RG tag (inferred from file names)\n"); fprintf(stderr, " -u uncompressed BAM output\n"); fprintf(stderr, " -f overwrite the output BAM if exist\n"); fprintf(stderr, " -1 compress level 1\n"); fprintf(stderr, " -l INT compression level, from 0 to 9 [-1]\n"); fprintf(stderr, " -@ INT number of BAM compression threads [0]\n"); fprintf(stderr, " -R STR merge file in the specified region STR [all]\n"); fprintf(stderr, " -h FILE copy the header in FILE to [in1.bam]\n\n"); fprintf(stderr, "Note: Samtools' merge does not reconstruct the @RG dictionary in the header. Users\n"); fprintf(stderr, " must provide the correct header with -h, or uses Picard which properly maintains\n"); fprintf(stderr, " the header dictionary in merging.\n\n"); return 1; } if (!(flag & MERGE_FORCE) && strcmp(argv[optind], "-")) { FILE *fp = fopen(argv[optind], "rb"); if (fp != NULL) { fclose(fp); fprintf(stderr, "[%s] File '%s' exists. Please apply '-f' to overwrite. Abort.\n", __func__, argv[optind]); return 1; } } if (bam_merge_core2(is_by_qname, argv[optind], fn_headers, argc - optind - 1, argv + optind + 1, flag, reg, n_threads, level) < 0) ret = 1; free(reg); free(fn_headers); return ret; } /*************** * BAM sorting * ***************/ #include typedef bam1_t *bam1_p; static int change_SO(bam_header_t *h, const char *so) { char *p, *q, *beg = 0, *end = 0, *newtext; if (h->l_text > 3) { if (strncmp(h->text, "@HD", 3) == 0) { if ((p = strchr(h->text, '\n')) == 0) return -1; *p = '\0'; if ((q = strstr(h->text, "\tSO:")) != 0) { *p = '\n'; // change back if (strncmp(q + 4, so, p - q - 4) != 0) { beg = q; for (q += 4; *q != '\n' && *q != '\t'; ++q); end = q; } else return 0; // no need to change } else beg = end = p, *p = '\n'; } } if (beg == 0) { // no @HD h->l_text += strlen(so) + 15; newtext = malloc(h->l_text + 1); sprintf(newtext, "@HD\tVN:1.3\tSO:%s\n", so); strcat(newtext, h->text); } else { // has @HD but different or no SO h->l_text = (beg - h->text) + (4 + strlen(so)) + (h->text + h->l_text - end); newtext = malloc(h->l_text + 1); strncpy(newtext, h->text, beg - h->text); sprintf(newtext + (beg - h->text), "\tSO:%s", so); strcat(newtext, end); } free(h->text); h->text = newtext; return 0; } static inline int bam1_lt(const bam1_p a, const bam1_p b) { if (g_is_by_qname) { int t = strnum_cmp(bam1_qname(a), bam1_qname(b)); return (t < 0 || (t == 0 && (a->core.flag&0xc0) < (b->core.flag&0xc0))); } else return (((uint64_t)a->core.tid<<32|(a->core.pos+1)<<1|bam1_strand(a)) < ((uint64_t)b->core.tid<<32|(b->core.pos+1)<<1|bam1_strand(b))); } KSORT_INIT(sort, bam1_p, bam1_lt) typedef struct { size_t buf_len; const char *prefix; bam1_p *buf; const bam_header_t *h; int index; } worker_t; static void write_buffer(const char *fn, const char *mode, size_t l, bam1_p *buf, const bam_header_t *h, int n_threads) { size_t i; bamFile fp; fp = strcmp(fn, "-")? bam_open(fn, mode) : bam_dopen(fileno(stdout), mode); if (fp == 0) return; bam_header_write(fp, h); if (n_threads > 1) bgzf_mt(fp, n_threads, 256); for (i = 0; i < l; ++i) bam_write1_core(fp, &buf[i]->core, buf[i]->data_len, buf[i]->data); bam_close(fp); } static void *worker(void *data) { worker_t *w = (worker_t*)data; char *name; ks_mergesort(sort, w->buf_len, w->buf, 0); name = (char*)calloc(strlen(w->prefix) + 20, 1); sprintf(name, "%s.%.4d.bam", w->prefix, w->index); write_buffer(name, "w1", w->buf_len, w->buf, w->h, 0); free(name); return 0; } static int sort_blocks(int n_files, size_t k, bam1_p *buf, const char *prefix, const bam_header_t *h, int n_threads) { int i; size_t rest; bam1_p *b; pthread_t *tid; pthread_attr_t attr; worker_t *w; if (n_threads < 1) n_threads = 1; if (k < n_threads * 64) n_threads = 1; // use a single thread if we only sort a small batch of records pthread_attr_init(&attr); pthread_attr_setdetachstate(&attr, PTHREAD_CREATE_JOINABLE); w = calloc(n_threads, sizeof(worker_t)); tid = calloc(n_threads, sizeof(pthread_t)); b = buf; rest = k; for (i = 0; i < n_threads; ++i) { w[i].buf_len = rest / (n_threads - i); w[i].buf = b; w[i].prefix = prefix; w[i].h = h; w[i].index = n_files + i; b += w[i].buf_len; rest -= w[i].buf_len; pthread_create(&tid[i], &attr, worker, &w[i]); } for (i = 0; i < n_threads; ++i) pthread_join(tid[i], 0); free(tid); free(w); return n_files + n_threads; } /*! @abstract Sort an unsorted BAM file based on the chromosome order and the leftmost position of an alignment @param is_by_qname whether to sort by query name @param fn name of the file to be sorted @param prefix prefix of the output and the temporary files; upon sucessess, prefix.bam will be written. @param max_mem approxiate maximum memory (very inaccurate) @param full_path the given output path is the full path and not just the prefix @discussion It may create multiple temporary subalignment files and then merge them by calling bam_merge_core(). This function is NOT thread safe. */ void bam_sort_core_ext(int is_by_qname, const char *fn, const char *prefix, size_t _max_mem, int is_stdout, int n_threads, int level, int full_path) { int ret, i, n_files = 0; size_t mem, max_k, k, max_mem; bam_header_t *header; bamFile fp; bam1_t *b, **buf; char *fnout = 0; char const *suffix = ".bam"; if (full_path) suffix += 4; if (n_threads < 2) n_threads = 1; g_is_by_qname = is_by_qname; max_k = k = 0; mem = 0; max_mem = _max_mem * n_threads; buf = 0; fp = strcmp(fn, "-")? bam_open(fn, "r") : bam_dopen(fileno(stdin), "r"); if (fp == 0) { fprintf(stderr, "[bam_sort_core] fail to open file %s\n", fn); return; } header = bam_header_read(fp); if (is_by_qname) change_SO(header, "queryname"); else change_SO(header, "coordinate"); // write sub files for (;;) { if (k == max_k) { size_t old_max = max_k; max_k = max_k? max_k<<1 : 0x10000; buf = realloc(buf, max_k * sizeof(void*)); memset(buf + old_max, 0, sizeof(void*) * (max_k - old_max)); } if (buf[k] == 0) buf[k] = (bam1_t*)calloc(1, sizeof(bam1_t)); b = buf[k]; if ((ret = bam_read1(fp, b)) < 0) break; if (b->data_len < b->m_data>>2) { // shrink b->m_data = b->data_len; kroundup32(b->m_data); b->data = realloc(b->data, b->m_data); } mem += sizeof(bam1_t) + b->m_data + sizeof(void*) + sizeof(void*); // two sizeof(void*) for the data allocated to pointer arrays ++k; if (mem >= max_mem) { n_files = sort_blocks(n_files, k, buf, prefix, header, n_threads); mem = k = 0; } } if (ret != -1) fprintf(stderr, "[bam_sort_core] truncated file. Continue anyway.\n"); // output file name fnout = calloc(strlen(prefix) + 20, 1); if (is_stdout) sprintf(fnout, "-"); else sprintf(fnout, "%s%s", prefix, suffix); // write the final output if (n_files == 0) { // a single block char mode[8]; strcpy(mode, "w"); if (level >= 0) sprintf(mode + 1, "%d", level < 9? level : 9); ks_mergesort(sort, k, buf, 0); write_buffer(fnout, mode, k, buf, header, n_threads); } else { // then merge char **fns; n_files = sort_blocks(n_files, k, buf, prefix, header, n_threads); fprintf(stderr, "[bam_sort_core] merging from %d files...\n", n_files); fns = (char**)calloc(n_files, sizeof(char*)); for (i = 0; i < n_files; ++i) { fns[i] = (char*)calloc(strlen(prefix) + 20, 1); sprintf(fns[i], "%s.%.4d%s", prefix, i, suffix); } bam_merge_core2(is_by_qname, fnout, 0, n_files, fns, 0, 0, n_threads, level); for (i = 0; i < n_files; ++i) { unlink(fns[i]); free(fns[i]); } free(fns); } free(fnout); // free for (k = 0; k < max_k; ++k) { if (!buf[k]) continue; free(buf[k]->data); free(buf[k]); } free(buf); bam_header_destroy(header); bam_close(fp); } void bam_sort_core(int is_by_qname, const char *fn, const char *prefix, size_t max_mem) { bam_sort_core_ext(is_by_qname, fn, prefix, max_mem, 0, 0, -1, 0); } int bam_sort(int argc, char *argv[]) { size_t max_mem = 768<<20; // 512MB int c, is_by_qname = 0, is_stdout = 0, n_threads = 0, level = -1, full_path = 0; while ((c = getopt(argc, argv, "fnom:@:l:")) >= 0) { switch (c) { case 'f': full_path = 1; break; case 'o': is_stdout = 1; break; case 'n': is_by_qname = 1; break; case 'm': { char *q; max_mem = strtol(optarg, &q, 0); if (*q == 'k' || *q == 'K') max_mem <<= 10; else if (*q == 'm' || *q == 'M') max_mem <<= 20; else if (*q == 'g' || *q == 'G') max_mem <<= 30; break; } case '@': n_threads = atoi(optarg); break; case 'l': level = atoi(optarg); break; } } if (optind + 2 > argc) { fprintf(stderr, "\n"); fprintf(stderr, "Usage: samtools sort [options] \n\n"); fprintf(stderr, "Options: -n sort by read name\n"); fprintf(stderr, " -f use as full file name instead of prefix\n"); fprintf(stderr, " -o final output to stdout\n"); fprintf(stderr, " -l INT compression level, from 0 to 9 [-1]\n"); fprintf(stderr, " -@ INT number of sorting and compression threads [1]\n"); fprintf(stderr, " -m INT max memory per thread; suffix K/M/G recognized [768M]\n"); fprintf(stderr, "\n"); return 1; } bam_sort_core_ext(is_by_qname, argv[optind], argv[optind+1], max_mem, is_stdout, n_threads, level, full_path); return 0; }