#include #include #include #include #include #include #include "sam_header.h" #include "sam.h" #include "faidx.h" #include "kstring.h" #include "khash.h" KHASH_SET_INIT_STR(rg) // When counting records instead of printing them, // data passed to the bam_fetch callback is encapsulated in this struct. typedef struct { bam_header_t *header; int64_t *count; // int does overflow for very big BAMs } count_func_data_t; typedef khash_t(rg) *rghash_t; // FIXME: we'd better use no global variables... static rghash_t g_rghash = 0; static int g_min_mapQ = 0, g_flag_on = 0, g_flag_off = 0, g_qual_scale = 0, g_min_qlen = 0; static uint32_t g_subsam_seed = 0; static double g_subsam_frac = -1.; static char *g_library, *g_rg; static void *g_bed; void *bed_read(const char *fn); void bed_destroy(void *_h); int bed_overlap(const void *_h, const char *chr, int beg, int end); static int process_aln(const bam_header_t *h, bam1_t *b) { if (g_qual_scale > 1) { int i; uint8_t *qual = bam1_qual(b); for (i = 0; i < b->core.l_qseq; ++i) { int c = qual[i] * g_qual_scale; qual[i] = c < 93? c : 93; } } if (g_min_qlen > 0) { int k, qlen = 0; uint32_t *cigar = bam1_cigar(b); for (k = 0; k < b->core.n_cigar; ++k) if ((bam_cigar_type(bam_cigar_op(cigar[k]))&1) || bam_cigar_op(cigar[k]) == BAM_CHARD_CLIP) qlen += bam_cigar_oplen(cigar[k]); if (qlen < g_min_qlen) return 1; } if (b->core.qual < g_min_mapQ || ((b->core.flag & g_flag_on) != g_flag_on) || (b->core.flag & g_flag_off)) return 1; if (g_bed && b->core.tid >= 0 && !bed_overlap(g_bed, h->target_name[b->core.tid], b->core.pos, bam_calend(&b->core, bam1_cigar(b)))) return 1; if (g_subsam_frac > 0.) { uint32_t k = __ac_X31_hash_string(bam1_qname(b)) + g_subsam_seed; if ((double)(k&0xffffff) / 0x1000000 >= g_subsam_frac) return 1; } if (g_rg || g_rghash) { uint8_t *s = bam_aux_get(b, "RG"); if (s) { if (g_rg) return (strcmp(g_rg, (char*)(s + 1)) == 0)? 0 : 1; if (g_rghash) { khint_t k = kh_get(rg, g_rghash, (char*)(s + 1)); return (k != kh_end(g_rghash))? 0 : 1; } } } if (g_library) { const char *p = bam_get_library((bam_header_t*)h, b); return (p && strcmp(p, g_library) == 0)? 0 : 1; } return 0; } static char *drop_rg(char *hdtxt, rghash_t h, int *len) { char *p = hdtxt, *q, *r, *s; kstring_t str; memset(&str, 0, sizeof(kstring_t)); while (1) { int toprint = 0; q = strchr(p, '\n'); if (q == 0) q = p + strlen(p); if (q - p < 3) break; // the line is too short; then stop if (strncmp(p, "@RG\t", 4) == 0) { int c; khint_t k; if ((r = strstr(p, "\tID:")) != 0) { r += 4; for (s = r; *s != '\0' && *s != '\n' && *s != '\t'; ++s); c = *s; *s = '\0'; k = kh_get(rg, h, r); *s = c; if (k != kh_end(h)) toprint = 1; } } else toprint = 1; if (toprint) { kputsn(p, q - p, &str); kputc('\n', &str); } p = q + 1; } *len = str.l; return str.s; } // callback function for bam_fetch() that prints nonskipped records static int view_func(const bam1_t *b, void *data) { if (!process_aln(((samfile_t*)data)->header, (bam1_t*)b)) samwrite((samfile_t*)data, b); return 0; } // callback function for bam_fetch() that counts nonskipped records static int count_func(const bam1_t *b, void *data) { if (!process_aln(((count_func_data_t*)data)->header, (bam1_t*)b)) { (*((count_func_data_t*)data)->count)++; } return 0; } static int usage(int is_long_help); int main_samview(int argc, char *argv[]) { int c, is_header = 0, is_header_only = 0, is_bamin = 1, ret = 0, compress_level = -1, is_bamout = 0, is_count = 0; int of_type = BAM_OFDEC, is_long_help = 0, n_threads = 0; int64_t count = 0; samfile_t *in = 0, *out = 0; char in_mode[5], out_mode[5], *fn_out = 0, *fn_list = 0, *fn_ref = 0, *fn_rg = 0, *q; /* parse command-line options */ strcpy(in_mode, "r"); strcpy(out_mode, "w"); while ((c = getopt(argc, argv, "SbBct:h1Ho:q:f:F:ul:r:xX?T:R:L:s:Q:@:m:")) >= 0) { switch (c) { case 's': if ((g_subsam_seed = strtol(optarg, &q, 10)) != 0) { srand(g_subsam_seed); g_subsam_seed = rand(); } g_subsam_frac = strtod(q, &q); break; case 'm': g_min_qlen = atoi(optarg); break; case 'c': is_count = 1; break; case 'S': is_bamin = 0; break; case 'b': is_bamout = 1; break; case 't': fn_list = strdup(optarg); is_bamin = 0; break; case 'h': is_header = 1; break; case 'H': is_header_only = 1; break; case 'o': fn_out = strdup(optarg); break; case 'f': g_flag_on = strtol(optarg, 0, 0); break; case 'F': g_flag_off = strtol(optarg, 0, 0); break; case 'q': g_min_mapQ = atoi(optarg); break; case 'u': compress_level = 0; break; case '1': compress_level = 1; break; case 'l': g_library = strdup(optarg); break; case 'L': g_bed = bed_read(optarg); break; case 'r': g_rg = strdup(optarg); break; case 'R': fn_rg = strdup(optarg); break; case 'x': of_type = BAM_OFHEX; break; case 'X': of_type = BAM_OFSTR; break; case '?': is_long_help = 1; break; case 'T': fn_ref = strdup(optarg); is_bamin = 0; break; case 'B': bam_no_B = 1; break; case 'Q': g_qual_scale = atoi(optarg); break; case '@': n_threads = strtol(optarg, 0, 0); break; default: return usage(is_long_help); } } if (compress_level >= 0) is_bamout = 1; if (is_header_only) is_header = 1; if (is_bamout) strcat(out_mode, "b"); else { if (of_type == BAM_OFHEX) strcat(out_mode, "x"); else if (of_type == BAM_OFSTR) strcat(out_mode, "X"); } if (is_bamin) strcat(in_mode, "b"); if (is_header) strcat(out_mode, "h"); if (compress_level >= 0) { char tmp[2]; tmp[0] = compress_level + '0'; tmp[1] = '\0'; strcat(out_mode, tmp); } if (argc == optind) return usage(is_long_help); // potential memory leak... // read the list of read groups if (fn_rg) { FILE *fp_rg; char buf[1024]; int ret; g_rghash = kh_init(rg); fp_rg = fopen(fn_rg, "r"); while (!feof(fp_rg) && fscanf(fp_rg, "%s", buf) > 0) // this is not a good style, but bear me... kh_put(rg, g_rghash, strdup(buf), &ret); // we'd better check duplicates... fclose(fp_rg); } // generate the fn_list if necessary if (fn_list == 0 && fn_ref) fn_list = samfaipath(fn_ref); // open file handlers if ((in = samopen(argv[optind], in_mode, fn_list)) == 0) { fprintf(stderr, "[main_samview] fail to open \"%s\" for reading.\n", argv[optind]); ret = 1; goto view_end; } if (in->header == 0) { fprintf(stderr, "[main_samview] fail to read the header from \"%s\".\n", argv[optind]); ret = 1; goto view_end; } if (g_rghash) { // FIXME: I do not know what "bam_header_t::n_text" is for... char *tmp; int l; tmp = drop_rg(in->header->text, g_rghash, &l); free(in->header->text); in->header->text = tmp; in->header->l_text = l; } if (!is_count && (out = samopen(fn_out? fn_out : "-", out_mode, in->header)) == 0) { fprintf(stderr, "[main_samview] fail to open \"%s\" for writing.\n", fn_out? fn_out : "standard output"); ret = 1; goto view_end; } if (n_threads > 1) samthreads(out, n_threads, 256); if (is_header_only) goto view_end; // no need to print alignments if (argc == optind + 1) { // convert/print the entire file bam1_t *b = bam_init1(); int r; while ((r = samread(in, b)) >= 0) { // read one alignment from `in' if (!process_aln(in->header, b)) { if (!is_count) samwrite(out, b); // write the alignment to `out' count++; } } if (r < -1) { fprintf(stderr, "[main_samview] truncated file.\n"); ret = 1; } bam_destroy1(b); } else { // retrieve alignments in specified regions int i; bam_index_t *idx = 0; if (is_bamin) idx = bam_index_load(argv[optind]); // load BAM index if (idx == 0) { // index is unavailable fprintf(stderr, "[main_samview] random alignment retrieval only works for indexed BAM files.\n"); ret = 1; goto view_end; } for (i = optind + 1; i < argc; ++i) { int tid, beg, end, result; bam_parse_region(in->header, argv[i], &tid, &beg, &end); // parse a region in the format like `chr2:100-200' if (tid < 0) { // reference name is not found fprintf(stderr, "[main_samview] region \"%s\" specifies an unknown reference name. Continue anyway.\n", argv[i]); continue; } // fetch alignments if (is_count) { count_func_data_t count_data = { in->header, &count }; result = bam_fetch(in->x.bam, idx, tid, beg, end, &count_data, count_func); } else result = bam_fetch(in->x.bam, idx, tid, beg, end, out, view_func); if (result < 0) { fprintf(stderr, "[main_samview] retrieval of region \"%s\" failed due to truncated file or corrupt BAM index file\n", argv[i]); ret = 1; break; } } bam_index_destroy(idx); // destroy the BAM index } view_end: if (is_count && ret == 0) printf("%" PRId64 "\n", count); // close files, free and return free(fn_list); free(fn_ref); free(fn_out); free(g_library); free(g_rg); free(fn_rg); if (g_bed) bed_destroy(g_bed); if (g_rghash) { khint_t k; for (k = 0; k < kh_end(g_rghash); ++k) if (kh_exist(g_rghash, k)) free((char*)kh_key(g_rghash, k)); kh_destroy(rg, g_rghash); } samclose(in); if (!is_count) samclose(out); return ret; } static int usage(int is_long_help) { fprintf(stderr, "\n"); fprintf(stderr, "Usage: samtools view [options] | [region1 [...]]\n\n"); fprintf(stderr, "Options: -b output BAM\n"); fprintf(stderr, " -h print header for the SAM output\n"); fprintf(stderr, " -H print header only (no alignments)\n"); fprintf(stderr, " -S input is SAM\n"); fprintf(stderr, " -u uncompressed BAM output (force -b)\n"); fprintf(stderr, " -1 fast compression (force -b)\n"); fprintf(stderr, " -x output FLAG in HEX (samtools-C specific)\n"); fprintf(stderr, " -X output FLAG in string (samtools-C specific)\n"); fprintf(stderr, " -c print only the count of matching records\n"); fprintf(stderr, " -B collapse the backward CIGAR operation\n"); fprintf(stderr, " -@ INT number of BAM compression threads [0]\n"); fprintf(stderr, " -L FILE output alignments overlapping the input BED FILE [null]\n"); fprintf(stderr, " -t FILE list of reference names and lengths (force -S) [null]\n"); fprintf(stderr, " -T FILE reference sequence file (force -S) [null]\n"); fprintf(stderr, " -o FILE output file name [stdout]\n"); fprintf(stderr, " -R FILE list of read groups to be outputted [null]\n"); fprintf(stderr, " -f INT required flag, 0 for unset [0]\n"); fprintf(stderr, " -F INT filtering flag, 0 for unset [0]\n"); fprintf(stderr, " -q INT minimum mapping quality [0]\n"); fprintf(stderr, " -l STR only output reads in library STR [null]\n"); fprintf(stderr, " -r STR only output reads in read group STR [null]\n"); fprintf(stderr, " -s FLOAT fraction of templates to subsample; integer part as seed [-1]\n"); fprintf(stderr, " -? longer help\n"); fprintf(stderr, "\n"); if (is_long_help) fprintf(stderr, "Notes:\n\ \n\ 1. By default, this command assumes the file on the command line is in\n\ the BAM format and it prints the alignments in SAM. If `-t' is\n\ applied, the input file is assumed to be in the SAM format. The\n\ file supplied with `-t' is SPACE/TAB delimited with the first two\n\ fields of each line consisting of the reference name and the\n\ corresponding sequence length. The `.fai' file generated by `faidx'\n\ can be used here. This file may be empty if reads are unaligned.\n\ \n\ 2. SAM->BAM conversion: `samtools view -bT ref.fa in.sam.gz'.\n\ \n\ 3. BAM->SAM conversion: `samtools view in.bam'.\n\ \n\ 4. A region should be presented in one of the following formats:\n\ `chr1', `chr2:1,000' and `chr3:1000-2,000'. When a region is\n\ specified, the input alignment file must be an indexed BAM file.\n\ \n\ 5. Option `-u' is preferred over `-b' when the output is piped to\n\ another samtools command.\n\ \n\ 6. In a string FLAG, each character represents one bit with\n\ p=0x1 (paired), P=0x2 (properly paired), u=0x4 (unmapped),\n\ U=0x8 (mate unmapped), r=0x10 (reverse), R=0x20 (mate reverse)\n\ 1=0x40 (first), 2=0x80 (second), s=0x100 (not primary), \n\ f=0x200 (failure) and d=0x400 (duplicate). Note that `-x' and\n\ `-X' are samtools-C specific. Picard and older samtools do not\n\ support HEX or string flags.\n\ \n"); return 1; } int main_import(int argc, char *argv[]) { int argc2, ret; char **argv2; if (argc != 4) { fprintf(stderr, "Usage: bamtk import \n"); return 1; } argc2 = 6; argv2 = calloc(6, sizeof(char*)); argv2[0] = "import", argv2[1] = "-o", argv2[2] = argv[3], argv2[3] = "-bt", argv2[4] = argv[1], argv2[5] = argv[2]; ret = main_samview(argc2, argv2); free(argv2); return ret; } int8_t seq_comp_table[16] = { 0, 8, 4, 12, 2, 10, 9, 14, 1, 6, 5, 13, 3, 11, 7, 15 }; int main_bam2fq(int argc, char *argv[]) { bamFile fp; bam_header_t *h; bam1_t *b; int8_t *buf; int max_buf, c, no12 = 0; while ((c = getopt(argc, argv, "n")) > 0) if (c == 'n') no12 = 1; if (argc == 1) { fprintf(stderr, "Usage: samtools bam2fq \n"); return 1; } fp = strcmp(argv[optind], "-")? bam_open(argv[optind], "r") : bam_dopen(fileno(stdin), "r"); if (fp == 0) return 1; h = bam_header_read(fp); b = bam_init1(); buf = 0; max_buf = 0; while (bam_read1(fp, b) >= 0) { int i, qlen = b->core.l_qseq; uint8_t *seq; putchar('@'); fputs(bam1_qname(b), stdout); if (no12) putchar('\n'); else { if ((b->core.flag & 0x40) && !(b->core.flag & 0x80)) puts("/1"); else if ((b->core.flag & 0x80) && !(b->core.flag & 0x40)) puts("/2"); else putchar('\n'); } if (max_buf < qlen + 1) { max_buf = qlen + 1; kroundup32(max_buf); buf = realloc(buf, max_buf); } buf[qlen] = 0; seq = bam1_seq(b); for (i = 0; i < qlen; ++i) buf[i] = bam1_seqi(seq, i); if (b->core.flag & 16) { // reverse complement for (i = 0; i < qlen>>1; ++i) { int8_t t = seq_comp_table[buf[qlen - 1 - i]]; buf[qlen - 1 - i] = seq_comp_table[buf[i]]; buf[i] = t; } if (qlen&1) buf[i] = seq_comp_table[buf[i]]; } for (i = 0; i < qlen; ++i) buf[i] = bam_nt16_rev_table[buf[i]]; puts((char*)buf); puts("+"); seq = bam1_qual(b); for (i = 0; i < qlen; ++i) buf[i] = 33 + seq[i]; if (b->core.flag & 16) { // reverse for (i = 0; i < qlen>>1; ++i) { int8_t t = buf[qlen - 1 - i]; buf[qlen - 1 - i] = buf[i]; buf[i] = t; } } puts((char*)buf); } free(buf); bam_destroy1(b); bam_header_destroy(h); bam_close(fp); return 0; }