1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503
|
/* bam_import -- Import of FASTQ files.
*
* samtools import -1 a_1.fq -2 a_2.fq --i1 a_i1.fq --i2 a_i2.fq
* samtools import a_1.fq a_2.fq
* samtools import a_interleaved.fq
*
* Copyright (C) 2020 Genome Research Ltd.
*
* Author: James Bonfield <jkb@sanger.ac.uk>
*/
/*
Permission is hereby granted, free of charge, to any person obtaining a copy
of this software and associated documentation files (the "Software"), to deal
in the Software without restriction, including without limitation the rights
to use, copy, modify, merge, publish, distribute, sublicense, and/or sell
copies of the Software, and to permit persons to whom the Software is
furnished to do so, subject to the following conditions:
The above copyright notices and this permission notice shall be included in
all copies or substantial portions of the Software.
THE SOFTWARE IS PROVIDED "AS IS", WITHOUT WARRANTY OF ANY KIND, EXPRESS OR
IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY,
FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT. IN NO EVENT SHALL
THE AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER
LIABILITY, WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING
FROM, OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER
DEALINGS IN THE SOFTWARE.
*/
// TODO: Store other non-aux comments; in new sam tag?
#include <config.h>
#include <ctype.h>
#include "htslib/sam.h"
#include "htslib/thread_pool.h"
#include "samtools.h"
#include "sam_opts.h"
static int usage(FILE *fp, int exit_status) {
fprintf(fp, "Usage: samtools import [options] [file.fastq ...]\n");
fprintf(fp, "\n");
fprintf(fp, "Options:\n");
fprintf(fp, " -s FILE Read paired-ended data from single FILE\n");
fprintf(fp, " -0 FILE Read single-ended data from FILE\n");
fprintf(fp, " -1 FILE Read-1 from FILE\n");
fprintf(fp, " -2 FILE Read-2 from FILE\n");
fprintf(fp, " --i1 FILE Index-1 from FILE\n");
fprintf(fp, " --i2 FILE Index-2 from FILE\n");
fprintf(fp, " -i Parse CASAVA identifier\n");
fprintf(fp, " --barcode-tag TAG\n");
fprintf(fp, " Tag to use with barcode sequences [BC]\n");
fprintf(fp, " --quality-tag TAG\n");
fprintf(fp, " Tag to use with barcode qualities [QT]\n");
fprintf(fp, " -N, --name2 Use 2nd field as read name (SRA format)\n");
fprintf(fp, " -r STRING Build up a complete @RG line\n");
fprintf(fp, " -R STRING Add a simple RG line of \"@RG\\tID:STRING\"\n");
fprintf(fp, " -T TAGLIST Parse tags in SAM format; list of '*' for all\n");
fprintf(fp, " -o FILE Output to FILE instead of stdout\n");
fprintf(fp, " -u Uncompressed output\n");
fprintf(fp, " --order TAG Store Nth record count in TAG\n");
fprintf(fp, "\n");
sam_global_opt_help(fp, "-.O.-@--");
fprintf(fp, "\nA single fastq file will be interpreted as -s, -0 or -1 depending on\n");
fprintf(fp, "file contents, and a pair of fastq files as \"-1 FILE1 -2 FILE2\".\n");
return exit_status;
}
// Order matters here as we want to read index elements before main
// sequences so on reading the seqs we can emit a fully annotated record.
enum fileno {
FQ_I1, FQ_I2, // index seqs for R1 and R2
FQ_R0, // single file and unpaired data (singled-ended tech).
FQ_R1, FQ_R2, // separate read1 and read2 files
FQ_SINGLE, // single file, but with read1 and/or read2 present.
FQ_END
};
typedef struct {
sam_global_args ga;
int no_pg;
char *fn[FQ_END], *fn_out;
int idx_both; // add index to READ2 too, not just READ1
int casava;
char *barcode_seq;
char *barcode_qual;
char *aux;
char *rg;
char *rg_line;
char *order;
int compress_level;
htsThreadPool p;
int name2;
} opts_t;
// Append a sequence and quality string from a BAM record to a BC:Z and
// QT:Z style aux tag string.
static int append_index(kstring_t *s, kstring_t *q, bam1_t *b) {
char *sp, *qp;
if (ks_resize(s, s->l + b->core.l_qseq+1 +1) < 0)
return -1;
if (ks_resize(q, q->l + b->core.l_qseq+1 +1) < 0)
return -1;
sp = s->s + s->l - (s->l > 0);
qp = q->s + q->l - (q->l > 0);
if (s->l)
*sp++ = '-';
if (q->l)
*qp++ = ' ';
int i;
uint8_t *seq = bam_get_seq(b);
uint8_t *qual = bam_get_qual(b);
for (i = 0; i < b->core.l_qseq; i++) {
*sp++ = seq_nt16_str[bam_seqi(seq, i)];
*qp++ = qual[i] + '!';
}
*sp++ = 0;
*qp++ = 0;
s->l = sp - s->s;
q->l = qp - q->s;
return 0;
}
static int import_fastq(int argc, char **argv, opts_t *opts) {
int i, n, ret = 0;
samFile *fp_in[FQ_END] = {NULL};
bam1_t *b = bam_init1();
int ids[FQ_END];
samFile *fp_out = NULL;
sam_hdr_t *hdr_out = NULL;
kstring_t index_str = {0,0};
kstring_t read_str = {0,0};
char *rg = opts->rg;
kstring_t rg_line = {0,0};
uint64_t read_num = 0;
kstring_t idx_seq = {0};
kstring_t idx_qual = {0};
// Any additional arguments are assumed to be r1 r2, as a
// short cut. We support reading index tags out of those too (eg
// Illumina CASAVA format), but if we do that we lack the barcode
// quality string.
//
// We also consider a read name ending in /1 or /2 to be a single
// file containing interleaved fastq records for both ends.
// These will be labeled as fn[FQ_R1] but adjusted during reading.
if (argc == 1)
opts->fn[FQ_SINGLE] = argv[0];
else
for (i = 0; i < 4; i++)
if (argc > i)
opts->fn[FQ_R1+i] = argv[i];
// Open all files
for (i = n = 0; i < FQ_END; i++) {
if (!opts->fn[i])
continue;
fp_in[i] = sam_open_format(opts->fn[i], "r", &opts->ga.in);
if (!fp_in[i]) {
perror(opts->fn[i]);
ret = -1;
goto err;
}
if (opts->p.pool)
hts_set_thread_pool(fp_in[i], &opts->p);
ids[n++] = i;
if (opts->name2)
hts_set_opt(fp_in[i], FASTQ_OPT_NAME2, 1);
if (opts->casava)
hts_set_opt(fp_in[i], FASTQ_OPT_CASAVA, 1);
if (opts->barcode_seq) // for auto-CASAVA parsing
hts_set_opt(fp_in[i], FASTQ_OPT_BARCODE, opts->barcode_seq);
if (opts->aux)
hts_set_opt(fp_in[i], FASTQ_OPT_AUX,
*opts->aux == '*' || *opts->aux == '\0'
? NULL : opts->aux);
switch (i) {
case FQ_I1:
kputs("--i1 I1.fastq ", &read_str);
kputs("i*", &index_str);
break;
case FQ_I2:
kputs("--i2 I2.fastq ", &read_str);
kputs("i*", &index_str);
break;
case FQ_R0:
kputs("-0 unpaired.fastq ", &read_str);
break;
case FQ_R1:
kputs("-1 R1.fastq ", &read_str);
break;
case FQ_R2:
kputs("-2 R2.fastq ", &read_str);
break;
case FQ_SINGLE:
kputs("-N -o paired.fastq ", &read_str);
break;
default:
ks_clear(&read_str); // not reversible
kputs("", &read_str);
}
}
if (n == 0) {
bam_destroy1(b);
return usage(stdout, EXIT_SUCCESS);
}
char out_mode[10] = {'w', 0, 0};
if (opts->compress_level != -1)
out_mode[1] = '0' + opts->compress_level;
sam_open_mode(out_mode+strlen(out_mode), opts->fn_out, NULL);
fp_out = sam_open_format(opts->fn_out, out_mode, &opts->ga.out);
if (!fp_out) {
perror(opts->fn_out);
goto err;
}
autoflush_if_stdout(fp_out, opts->fn_out);
if (opts->p.pool)
hts_set_thread_pool(fp_out, &opts->p);
// Create header
if (ks_len(&read_str)) {
char CO[2100];
if (ks_len(&index_str))
snprintf(CO, sizeof(CO), "@CO\tReverse with: samtools fastq %s "
"--index-format=\"%s\"\n",
ks_str(&read_str), ks_str(&index_str));
else
snprintf(CO, sizeof(CO), "@CO\tReverse with: samtools fastq %s\n",
ks_str(&read_str));
hdr_out = sam_hdr_parse(strlen(CO), CO);
} else {
hdr_out = sam_hdr_init();
}
// Add a version line with the sort order to the output header
if (sam_hdr_add_line(hdr_out, "HD", "VN", SAM_FORMAT_VERSION, "SO", "unsorted", "GO", "query", NULL) < 0) {
fprintf(stderr, "Could not set SO and GO in the header.\n");
goto err;
}
// Read group
if (opts->rg_line) {
if (*opts->rg_line != '@')
ksprintf(&rg_line, "@RG\t%s", opts->rg_line);
else
kputs(opts->rg_line, &rg_line);
} else if (opts->rg) {
ksprintf(&rg_line, "@RG\tID:%s", opts->rg);
}
if (ks_len(&rg_line)) {
if (sam_hdr_add_lines(hdr_out, ks_str(&rg_line), 0) < 0)
goto err;
rg = strstr(ks_str(&rg_line), "\tID:");
if (!rg) {
fprintf(stderr, "\"-r RG-LINE\" option contained no ID field\n");
goto err;
}
rg += 4;
i = 0;
while (rg[i] != '\t' && rg[i] != '\0')
i++;
rg[i] = 0;
}
if ((ret = sam_hdr_write(fp_out, hdr_out)) < 0)
goto err;
// Interleave / combine from n files (ids[0..n-1]).
int res;
int eof = 0;
do {
idx_seq.l = idx_qual.l = 0;
for (i = 0; i < n; i++) {
if ((res = sam_read1(fp_in[ids[i]], NULL, b)) < 0) {
if (res == -1) {
eof++;
continue;
} else
break;
}
// index
if (ids[i] == FQ_I1 || ids[i] == FQ_I2) {
if (append_index(&idx_seq, &idx_qual, b) < 0) {
res = -1;
break;
}
continue;
}
// full read
if (idx_seq.l) {
if (opts->idx_both || ids[i] == FQ_SINGLE ||
ids[i] == FQ_R0 || ids[i] == FQ_R1) {
if (bam_aux_append(b, opts->barcode_seq, 'Z', idx_seq.l,
(uint8_t *)idx_seq.s) ||
bam_aux_append(b, opts->barcode_qual, 'Z', idx_qual.l,
(uint8_t *)idx_qual.s)) {
res = -1;
break;
}
}
}
switch(ids[i]) {
case FQ_R0:
// unpaired; no flags to declare
break;
case FQ_SINGLE:
// paired (but don't know if R1 or R2) or unpaired.
// We rely on the /1 and /2 read suffix parsing in htslib
// to distinguish the two cases, or CASAVA tags if
// explicitly enabled.
break;
case FQ_R1:
if ((b->core.flag & (BAM_FREAD1 | BAM_FREAD2)) == 0)
b->core.flag |= BAM_FREAD1;
b->core.flag |= BAM_FPAIRED;
if (i+1 < n && ids[i+1] == FQ_R2)
b->core.flag |= BAM_FMUNMAP;
break;
case FQ_R2:
b->core.flag |= BAM_FPAIRED | BAM_FREAD2;
if (i > 0 && ids[i-1] == FQ_R1)
b->core.flag |= BAM_FMUNMAP;
break;
}
if (rg) {
if (bam_aux_append(b, "RG", 'Z', strlen(rg)+1,
(uint8_t *)rg) < 0) {
ret = -1;
goto err;
}
}
if (opts->order) {
if (bam_aux_update_int(b, opts->order, read_num++) < 0) {
ret = -1;
goto err;
}
}
res = sam_write1(fp_out, hdr_out, b);
}
} while (res >= 0);
if (res != -1) {
print_error("import", "truncated file. Aborting");
ret = res;
goto err;
}
if (eof != n) {
print_error("import", "input files with differing number of records");
ret = -1;
goto err;
}
// Close and return
ret = 0;
err:
bam_destroy1(b);
sam_hdr_destroy(hdr_out);
ks_free(&rg_line);
ks_free(&index_str);
ks_free(&read_str);
if (fp_out) {
release_autoflush(fp_out);
if (sam_close(fp_out) < 0) {
perror(opts->fn_out);
ret |= -1;
}
}
for (i = 0; i < FQ_END; i++) {
if (fp_in[i] && sam_close(fp_in[i]) < 0) {
perror(opts->fn[i]);
ret |= -1;
}
}
ks_free(&idx_seq);
ks_free(&idx_qual);
return ret;
}
int main_import(int argc, char *argv[]) {
int c;
opts_t opts = {
.no_pg = 0,
.ga = SAM_GLOBAL_ARGS_INIT,
.fn = {NULL},
.fn_out = "-",
.casava = 0,
.barcode_seq = "BC",
.barcode_qual = "QT",
.aux = NULL,
.rg = NULL,
.rg_line = NULL,
.order = NULL,
.compress_level = -1,
.name2 = 0,
};
kstring_t rg = {0};
static const struct option lopts[] = {
SAM_OPT_GLOBAL_OPTIONS('-', 0, 'O', 0, '-', '@'),
{"no-PG", no_argument, NULL, 9},
{"i1", required_argument, NULL, 1},
{"i2", required_argument, NULL, 2},
{"r1", required_argument, NULL, '1'},
{"r2", required_argument, NULL, '2'},
{"rg", required_argument, NULL, 'R'},
{"rg-line", required_argument, NULL, 'r'},
{"order", required_argument, NULL, 3},
{"barcode-tag", required_argument, NULL, 4},
{"quality-tag", required_argument, NULL, 5},
{"name2", no_argument, NULL, 'N'},
{ NULL, 0, NULL, 0 }
};
while ((c = getopt_long(argc, argv, "1:2:s:0:bhiT:r:R:o:O:u@:N", lopts, NULL)) >= 0) {
switch (c) {
case 'b': opts.idx_both = 1; break;
case '0': opts.fn[FQ_R0] = optarg; break;
case '1': opts.fn[FQ_R1] = optarg; break;
case '2': opts.fn[FQ_R2] = optarg; break;
case 1: opts.fn[FQ_I1] = optarg; break;
case 2: opts.fn[FQ_I2] = optarg; break;
case 's': opts.fn[FQ_SINGLE] = optarg; break;
case 'o': opts.fn_out = optarg; break;
case 'i': opts.casava = 1; break;
case 4: opts.barcode_seq = optarg; break;
case 5: opts.barcode_qual = optarg; break;
case 'T': opts.aux = optarg; break;
case 'u': opts.compress_level = 0; break;
case 'R': opts.rg = optarg; break;
case 'r':
if (*optarg != '@' && ks_len(&rg) == 0)
kputs("@RG", &rg);
if (ks_len(&rg))
kputc_('\t', &rg);
kputs(optarg, &rg);
opts.rg_line = rg.s;
break;
case 'N': opts.name2 = 1; break;
case 9: opts.no_pg = 1; break;
case 3: opts.order = optarg; break;
case 'h': return usage(stdout, EXIT_SUCCESS);
case '?': return usage(stderr, EXIT_FAILURE);
default:
if (parse_sam_global_opt(c, optarg, lopts, &opts.ga) != 0)
return usage(stderr, EXIT_FAILURE);
break;
}
}
if (opts.ga.nthreads > 0) {
if (!(opts.p.pool = hts_tpool_init(opts.ga.nthreads))) {
fprintf(stderr, "Failed to create thread pool\n");
if (rg.s)
free(rg.s);
return -1;;
}
}
int ret = import_fastq(argc-optind, argv+optind, &opts) ? 1 : 0;
if (rg.s)
free(rg.s);
if (opts.p.pool)
hts_tpool_destroy(opts.p.pool);
return ret;
}
|