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bam_fastq.c
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/* bam_fastq.c -- FASTA and FASTQ file generation
Copyright (C) 2009-2017, 2019-2020, 2023-2024 Genome Research Ltd.
Portions copyright (C) 2009, 2011, 2012 Broad Institute.
Author: Heng Li <[email protected]>
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. */
#include <config.h>
#include <stdlib.h>
#include <string.h>
#include <strings.h>
#include <stdbool.h>
#include <ctype.h>
#include <assert.h>
#include <inttypes.h>
#include <unistd.h>
#include <float.h>
#include "htslib/sam.h"
#include "htslib/klist.h"
#include "htslib/kstring.h"
#include "htslib/bgzf.h"
#include "htslib/thread_pool.h"
#include "htslib/khash.h"
#include "samtools.h"
#include "sam_opts.h"
KHASH_SET_INIT_STR(str)
typedef khash_t(str) strhash_t;
#define DEFAULT_BARCODE_TAG "BC"
#define DEFAULT_QUALITY_TAG "QT"
#define INDEX_SEPARATOR "+"
int8_t seq_comp_table[16] = { 0, 8, 4, 12, 2, 10, 6, 14, 1, 9, 5, 13, 3, 11, 7, 15 };
static void bam2fq_usage(FILE *to, const char *command)
{
int fq = strcasecmp("fastq", command) == 0 || strcasecmp("bam2fq", command) == 0;
fprintf(to,
"Usage: samtools %s [options...] <in.bam>\n", command);
fprintf(to,
"\n"
"Description:\n"
"Converts a SAM, BAM or CRAM to %s format.\n"
"\n"
"Options:\n"
" -0 FILE write reads designated READ_OTHER to FILE\n"
" -1 FILE write reads designated READ1 to FILE\n"
" -2 FILE write reads designated READ2 to FILE\n"
" -o FILE write reads designated READ1 or READ2 to FILE\n"
" note: if a singleton file is specified with -s, only\n"
" paired reads will be written to the -1 and -2 files.\n"
" -d, --tag TAG[:VAL]\n"
" only include reads containing TAG, optionally with value VAL\n"
" -D, --tag-file STR:FILE\n"
" only include reads containing TAG, with a value listed in FILE\n"
" -f, --require-flags INT\n"
" only include reads with all of the FLAGs in INT present [0]\n" // F&x == x
" -F, --excl[ude]-flags INT\n"
" only include reads with none of the FLAGs in INT present [0x900]\n" // F&x == 0
" --rf, --incl[ude]-flags INT\n"
" only include reads with any of the FLAGs in INT present [0]\n" // !(F&x == 0)
" -G INT only EXCLUDE reads with all of the FLAGs in INT present [0]\n" // !(F&x == x)
" -n don't append /1 and /2 to the read name\n"
" -N always append /1 and /2 to the read name\n",
fq ? "FASTQ" : "FASTA");
if (fq) fprintf(to,
" -O output quality in the OQ tag if present\n");
fprintf(to,
" -s FILE write singleton reads designated READ1 or READ2 to FILE\n"
" -t copy RG, BC and QT tags to the %s header line\n",
fq ? "FASTQ" : "FASTA");
fprintf(to,
" -T TAGLIST copy arbitrary tags to the %s header line, '*' for all\n",
fq ? "FASTQ" : "FASTA");
if (fq) fprintf(to,
" -v INT default quality score if not given in file [1]\n"
" -i add Illumina Casava 1.8 format entry to header (eg 1:N:0:ATCACG)\n"
" -c INT compression level [0..9] to use when writing bgzf files [1]\n"
" --i1 FILE write first index reads to FILE\n"
" --i2 FILE write second index reads to FILE\n"
" --barcode-tag TAG\n"
" Barcode tag [" DEFAULT_BARCODE_TAG "]\n"
" --quality-tag TAG\n"
" Quality tag [" DEFAULT_QUALITY_TAG "]\n"
" --index-format STR\n"
" How to parse barcode and quality tags\n\n");
sam_global_opt_help(to, "-.--.@-.");
fprintf(to,
"\n"
"The files will be automatically compressed if the file names have a .gz\n"
"or .bgzf extension. The input to this program must be collated by name.\n"
"Run 'samtools collate' or 'samtools sort -n' to achieve this.\n"
"\n"
"Reads are designated READ1 if FLAG READ1 is set and READ2 is not set.\n"
"Reads are designated READ2 if FLAG READ1 is not set and READ2 is set.\n"
"Otherwise reads are designated READ_OTHER (both flags set or both flags unset).\n"
"Run 'samtools flags' for more information on flag codes and meanings.\n");
fprintf(to,
"\n"
"The index-format string describes how to parse the barcode and quality tags.\n"
"It is made up of 'i' or 'n' followed by a length or '*'. For example:\n"
" i14i8 The first 14 characters are index 1, the next 8 are index 2\n"
" n8i14 Ignore the first 8 characters, and use the next 14 for index 1\n\n"
"If the tag contains a separator, then the numeric part can be replaced with\n"
"'*' to mean 'read until the separator or end of tag', for example:\n"
" i*i* Break the tag at the separator into index 1 and index 2\n"
" n*i* Ignore the left part of the tag until the separator,\n"
" then use the second part of the tag as index 1\n");
fprintf(to,
"\n"
"Examples:\n"
"To get just the paired reads in separate files, use:\n"
" samtools %s -1 pair1.%s -2 pair2.%s -0 /dev/null -s /dev/null -n in.bam\n"
"\nTo get all non-supplementary/secondary reads in a single file, redirect\n"
"the output:\n"
" samtools %s in.bam > all_reads.%s\n",
command, fq ? "fq" : "fa", fq ? "fq" : "fa",
command, fq ? "fq" : "fa");
}
typedef enum { READ_UNKNOWN = 0, READ_1 = 1, READ_2 = 2 } readpart;
typedef enum { FASTA, FASTQ } fastfile;
typedef struct bam2fq_opts {
char *fnse;
char *fnr[3];
char *fn_input; // pointer to input filename in argv do not free
bool has12, has12always, use_oq, copy_tags, illumina_tag;
int flag_on, flag_off, flag_alloff, flag_anyon;
sam_global_args ga;
fastfile filetype;
int def_qual;
char *barcode_tag;
char *quality_tag;
char *index_file[2];
char *index_format;
char *extra_tags;
char compression_level;
const char *filter_tag; // -d opt
strhash_t *filter_tag_vals;
} bam2fq_opts_t;
typedef struct bam2fq_state {
samFile *fp;
samFile *fpse;
samFile *fpr[3];
samFile *fpi[3];
samFile *hstdout;
sam_hdr_t *h;
bool has12, use_oq, copy_tags, illumina_tag;
int flag_on, flag_off, flag_alloff, flag_anyon;
fastfile filetype;
int def_qual;
char *index_sequence;
char compression_level;
htsThreadPool p;
} bam2fq_state_t;
// Adds a single tag value to the filter tag value hash
static int add_tag_value(bam2fq_opts_t *opts, char *val) {
if (!opts->filter_tag_vals) {
if (!(opts->filter_tag_vals = kh_init(str)))
return -1;
}
if (!(val = strdup(val)))
return -1;
int ret = 0;
kh_put(str, opts->filter_tag_vals, val, &ret);
if (ret <= 0)
free(val);
return ret < 0 ? -1 : 0;
}
// Adds multiple values, listed in a file
static int add_tag_file(bam2fq_opts_t *opts, char *fn) {
FILE *fp;
kstring_t ks = {0,0};
if (!(fp = fopen(fn, "r"))) {
print_error_errno("fastq", "failed to open \"%s\" for reading", fn);
return -1;
}
while (ks.l = 0, kgetline(&ks, (kgets_func *)fgets, fp) >= 0) {
if (add_tag_value(opts, ks.s) < 0) {
ks_free(&ks);
return -1;
}
}
ks_free(&ks);
return 0;
}
static readpart which_readpart(const bam1_t *b)
{
if ((b->core.flag & BAM_FREAD1) && !(b->core.flag & BAM_FREAD2)) {
return READ_1;
} else if ((b->core.flag & BAM_FREAD2) && !(b->core.flag & BAM_FREAD1)) {
return READ_2;
} else {
return READ_UNKNOWN;
}
}
static void free_opts(bam2fq_opts_t *opts)
{
if (opts->filter_tag_vals) {
khint_t k;
for (k = 0; k < kh_end(opts->filter_tag_vals); k++)
if (kh_exist(opts->filter_tag_vals, k))
free((char *)kh_key(opts->filter_tag_vals, k));
kh_destroy(str, opts->filter_tag_vals);
}
free(opts);
}
// Make mnemonic distinct values for longoption-only options
#define LONGOPT(c) ((c) + 128)
// return true if valid
static bool parse_opts(int argc, char *argv[], bam2fq_opts_t** opts_out)
{
// Parse args
bam2fq_opts_t* opts = calloc(1, sizeof(bam2fq_opts_t));
opts->has12 = true;
opts->has12always = false;
opts->filetype = FASTQ;
opts->def_qual = 1;
opts->barcode_tag = NULL;
opts->quality_tag = NULL;
opts->index_format = NULL;
opts->index_file[0] = NULL;
opts->index_file[1] = NULL;
opts->extra_tags = NULL;
opts->compression_level = 1;
opts->flag_off = BAM_FSECONDARY|BAM_FSUPPLEMENTARY;
int c;
sam_global_args_init(&opts->ga);
static const struct option lopts[] = {
SAM_OPT_GLOBAL_OPTIONS('-', 0, '-', '-', 0, '@'),
{"require-flags", required_argument, NULL, 'f'},
{"excl-flags", required_argument, NULL, 'F'},
{"exclude-flags", required_argument, NULL, 'F'},
// following the same convention as view: g exists as a longoption_only
// argument, accessible from the command line as --rf/--incl[ude]-flags
{"rf", required_argument, NULL, LONGOPT('g')},
{"incl-flags", required_argument, NULL, LONGOPT('g')},
{"include-flags", required_argument, NULL, LONGOPT('g')},
{"i1", required_argument, NULL, 1},
{"I1", required_argument, NULL, 1},
{"i2", required_argument, NULL, 2},
{"I2", required_argument, NULL, 2},
{"if", required_argument, NULL, 3},
{"IF", required_argument, NULL, 3},
{"index-format", required_argument, NULL, 3},
{"barcode-tag", required_argument, NULL, 'b'},
{"quality-tag", required_argument, NULL, 'q'},
{"tag", required_argument, NULL, 'd'},
{"tag-file", required_argument, NULL, 'D'},
{ NULL, 0, NULL, 0 }
};
while ((c = getopt_long(argc, argv, "0:1:2:o:f:F:G:niNOs:c:tT:v:@:d:D:",
lopts, NULL)) > 0) {
switch (c) {
case 'b': opts->barcode_tag = optarg; break;
case 'q': opts->quality_tag = optarg; break;
case 1 : opts->index_file[0] = optarg; break;
case 2 : opts->index_file[1] = optarg; break;
case 3 : opts->index_format = optarg; break;
case '0': opts->fnr[0] = optarg; break;
case '1': opts->fnr[1] = optarg; break;
case '2': opts->fnr[2] = optarg; break;
case 'o': opts->fnr[1] = optarg; opts->fnr[2] = optarg; break;
case 'f': opts->flag_on |= strtol(optarg, 0, 0); break;
// note that flag_off does not have |= because it has a default
// value of 0x900 which needs to be replaced by the optarg
case 'F': opts->flag_off = strtol(optarg, 0, 0); break;
case 'G': opts->flag_alloff |= strtol(optarg, 0, 0); break;
case LONGOPT('g'): opts->flag_anyon |= strtol(optarg, 0, 0); break;
case 'n': opts->has12 = false; break;
case 'N': opts->has12always = true; break;
case 'O': opts->use_oq = true; break;
case 's': opts->fnse = optarg; break;
case 't': opts->copy_tags = true; break;
case 'i': opts->illumina_tag = true; break;
case 'c':
opts->compression_level = atoi(optarg);
if (opts->compression_level < 0)
opts->compression_level = 0;
if (opts->compression_level > 9)
opts->compression_level = 9;
break;
case 'T': opts->extra_tags = optarg; break;
case 'v': opts->def_qual = atoi(optarg); break;
case 'd':
if (strlen(optarg) < 2 ||
(strlen(optarg) > 2 && optarg[2] != ':')) {
print_error("fastq",
"Invalid \"tag:value\" option: \"%s\"",
optarg);
free_opts(opts);
return false;
}
if (opts->filter_tag && memcmp(opts->filter_tag, optarg, 2)) {
print_error("fastq", "Different tag type specified "
"to before");
free_opts(opts);
return false;
}
if (strlen(optarg) >= 3)
add_tag_value(opts, optarg+3);
opts->filter_tag = optarg;
break;
case 'D':
// Allow ";" as delimiter besides ":" to support MinGW CLI POSIX
// path translation as described at:
// http://www.mingw.org/wiki/Posix_path_conversion
if (strlen(optarg) < 4
|| (optarg[2] != ':' && optarg[2] != ';')) {
print_error("view", "Invalid \"tag:file\" option: \"%s\"",
optarg);
free_opts(opts);
return false;
}
if (opts->filter_tag && memcmp(opts->filter_tag, optarg, 2)) {
print_error("fastq", "Different tag type specified "
"to before");
free_opts(opts);
return false;
}
if (strlen(optarg) >= 3) {
if (add_tag_file(opts, optarg+3) < 0) {
free_opts(opts);
return false;
}
}
opts->filter_tag = optarg;
break;
case '?':
bam2fq_usage(stderr, argv[0]);
free_opts(opts);
return false;
default:
if (parse_sam_global_opt(c, optarg, lopts, &opts->ga) != 0) {
bam2fq_usage(stderr, argv[0]);
free_opts(opts);
return false;
}
break;
}
}
if (opts->fnr[1] || opts->fnr[2]) opts->has12 = false;
if (opts->has12always) opts->has12 = true;
if (!opts->barcode_tag) opts->barcode_tag = DEFAULT_BARCODE_TAG;
if (!opts->quality_tag) opts->quality_tag = DEFAULT_QUALITY_TAG;
int nIndex = 0;
if (opts->index_format) {
char *s;
for (s = opts->index_format; *s; s++) {
if (*s == 'i') nIndex++;
}
}
if (nIndex>2) {
fprintf(stderr,"Invalid index format: more than 2 indexes\n");
bam2fq_usage(stderr, argv[0]);
free_opts(opts);
return false;
}
if (opts->index_file[1] && !opts->index_file[0]) {
fprintf(stderr, "Index one specified, but index two not given\n");
bam2fq_usage(stderr, argv[0]);
free_opts(opts);
return false;
}
if (opts->illumina_tag && !nIndex) {
fprintf(stderr, "You must specify an index format (--index-format) with the Illumina Casava (-i) option\n");
bam2fq_usage(stderr, argv[0]);
free_opts(opts);
return false;
}
if (nIndex==0 && opts->index_file[0]) {
fprintf(stderr, "index_format not specified, but index file given\n");
bam2fq_usage(stderr, argv[0]);
free_opts(opts);
return false;
}
if (opts->def_qual < 0 || 93 < opts->def_qual) {
fprintf(stderr, "Invalid -v default quality %i, allowed range 0 to 93\n", opts->def_qual);
bam2fq_usage(stderr, argv[0]);
free_opts(opts);
return false;
}
const char* type_str = argv[0];
if (strcasecmp("fastq", type_str) == 0 ||
strcasecmp("bam2fq", type_str) == 0) {
opts->filetype = FASTQ;
} else if (strcasecmp("fasta", type_str) == 0) {
opts->filetype = FASTA;
} else {
print_error("bam2fq", "Unrecognised type call \"%s\", this should be impossible... but you managed it!", type_str);
bam2fq_usage(stderr, argv[0]);
free_opts(opts);
return false;
}
if (argc == optind && isatty(STDIN_FILENO)) {
bam2fq_usage(stdout, argv[0]);
free_opts(opts);
return true;
}
if (argc - optind > 1) {
fprintf(stderr, "Too many arguments.\n");
bam2fq_usage(stderr, argv[0]);
free_opts(opts);
return false;
}
opts->fn_input = argc > optind ? argv[optind] : "-";
*opts_out = opts;
return true;
}
void set_sam_opts(samFile *fp, bam2fq_state_t *state,
const bam2fq_opts_t *opts) {
if (state->has12)
hts_set_opt(fp, FASTQ_OPT_RNUM, 1);
if (state->illumina_tag)
hts_set_opt(fp, FASTQ_OPT_CASAVA, 1);
hts_set_opt(fp, FASTQ_OPT_BARCODE, opts->barcode_tag);
if (opts->extra_tags && (*opts->extra_tags == '*' || *opts->extra_tags == '\0'))
hts_set_opt(fp, FASTQ_OPT_AUX, NULL);
else {
kstring_t tag_list = {0,0};
if (state->copy_tags)
kputs("RG,BC,QT", &tag_list);
if (opts->extra_tags) {
if (tag_list.l)
kputc(',', &tag_list);
kputs(opts->extra_tags, &tag_list);
}
if (tag_list.l)
hts_set_opt(fp, FASTQ_OPT_AUX, tag_list.s);
ks_free(&tag_list);
}
}
// Open a file as normal or gzipped based on filename.
// Note we always use bgzf and don't bother to attempt non-blocked
// gzip streams. This is a departure from the old fastq code.
static samFile *sam_open_z(char *fn, char *mode, bam2fq_state_t *state) {
char modez[6];
strcpy(modez, mode);
size_t l = strlen(fn);
if ((l > 3 && strcmp(fn+l-3, ".gz") == 0) ||
(l > 4 && strcmp(fn+l-4, ".bgz") == 0) ||
(l > 5 && strcmp(fn+l-5, ".bgzf") == 0)) {
char m[3] = {'z', state->compression_level+'0', '\0'};
strcat(modez, m);
}
samFile *fp = sam_open(fn, modez);
if (!fp)
return NULL;
if (state->p.pool)
hts_set_thread_pool(fp, &state->p);
return fp;
}
static bool init_state(const bam2fq_opts_t* opts, bam2fq_state_t** state_out)
{
char *mode = opts->filetype == FASTA ? "wF" : "wf";
bam2fq_state_t* state = calloc(1, sizeof(bam2fq_state_t));
if (!state)
return false;
state->flag_on = opts->flag_on;
state->flag_off = opts->flag_off;
state->flag_alloff = opts->flag_alloff;
state->flag_anyon = opts->flag_anyon;
state->has12 = opts->has12;
state->use_oq = opts->use_oq;
state->illumina_tag = opts->illumina_tag;
state->copy_tags = opts->copy_tags;
state->filetype = opts->filetype;
state->def_qual = opts->def_qual;
state->index_sequence = NULL;
state->hstdout = NULL;
state->compression_level = opts->compression_level;
state->fp = sam_open_format(opts->fn_input, "r", &opts->ga.in);
if (state->fp == NULL) {
print_error_errno("bam2fq","Cannot read file \"%s\"", opts->fn_input);
free(state);
return false;
}
state->p.pool = NULL;
if (opts->ga.nthreads > 0) {
if (!(state->p.pool = hts_tpool_init(opts->ga.nthreads))) {
fprintf(stderr, "Failed to create thread pool\n");
free(state);
return false;
}
state->p.qsize = opts->ga.nthreads*2;
hts_set_thread_pool(state->fp, &state->p);
}
uint32_t rf = SAM_QNAME | SAM_FLAG | SAM_SEQ | SAM_QUAL;
if (opts->use_oq || opts->extra_tags || opts->index_file[0])
rf |= SAM_AUX;
if (opts->filter_tag) {
if (memcmp(opts->filter_tag, "NM", 2) == 0 ||
memcmp(opts->filter_tag, "MD", 2) == 0)
rf |= SAM_AUX | SAM_SEQ;
else if (memcmp(opts->filter_tag, "RG", 2) == 0)
rf |= SAM_RGAUX;
else
rf |= SAM_AUX;
}
if (opts->illumina_tag || opts->copy_tags)
rf |= SAM_AUX;
if (hts_set_opt(state->fp, CRAM_OPT_REQUIRED_FIELDS, rf)) {
fprintf(stderr, "Failed to set CRAM_OPT_REQUIRED_FIELDS value\n");
free(state);
return false;
}
if (hts_set_opt(state->fp, CRAM_OPT_DECODE_MD, 0)) {
fprintf(stderr, "Failed to set CRAM_OPT_DECODE_MD value\n");
free(state);
return false;
}
if (opts->fnse) {
if (!(state->fpse = sam_open_z(opts->fnse, mode, state))) {
print_error_errno("bam2fq", "Cannot open singleton file \"%s\"", opts->fnse);
free(state);
return false;
}
set_sam_opts(state->fpse, state, opts);
}
if (opts->ga.reference) {
if (hts_set_fai_filename(state->fp, opts->ga.reference) != 0) {
print_error_errno("bam2fq", "cannot load reference \"%s\"", opts->ga.reference);
free(state);
return false;
}
}
// single, read1, read2
int i, j;
for (i = 0; i < 3; ++i) {
if (opts->fnr[i]) {
for (j = 0; j < i; j++)
if (opts->fnr[j] && strcmp(opts->fnr[j], opts->fnr[i]) == 0)
break;
if (j == i) {
if (!(state->fpr[i] = sam_open_z(opts->fnr[i], mode, state))) {
print_error_errno("bam2fq", "Cannot open r%d file \"%s\"",
i, opts->fnr[i]);
free(state);
return false;
}
set_sam_opts(state->fpr[i], state, opts);
} else {
state->fpr[i] = state->fpr[j];
}
} else {
if (!state->hstdout) {
if (!(state->hstdout = sam_open_z("-", mode, state))) {
print_error_errno("bam2fq", "Cannot open STDOUT");
free(state);
return false;
}
set_sam_opts(state->hstdout, state, opts);
autoflush_if_stdout(state->hstdout, "-");
}
state->fpr[i] = state->hstdout;
}
}
// index 1, index 2
for (i = 0; i < 2; i++) {
state->fpi[i] = NULL;
if (opts->index_file[i]) {
for (j = 0; j < 3; j++)
if (opts->fnr[j] && strcmp(opts->fnr[j], opts->index_file[i]) == 0)
break;
for (j -= 3; j >= 0 && j < i; j++)
if (opts->index_file[j] && strcmp(opts->index_file[j], opts->index_file[i]) == 0)
break;
if (i == j) {
if (!(state->fpi[i] = sam_open_z(opts->index_file[i], mode,
state))) {
print_error_errno("bam2fq", "Cannot open i%d file \"%s\"",
i+1, opts->index_file[i]);
free(state);
return false;
}
set_sam_opts(state->fpi[i], state, opts);
} else if (j < 0) {
state->fpi[i] = state->fpr[j+3];
} else {
state->fpi[i] = state->fpi[j];
}
}
}
state->h = sam_hdr_read(state->fp);
if (state->h == NULL) {
fprintf(stderr, "Failed to read header for \"%s\"\n", opts->fn_input);
free(state);
return false;
}
kstring_t str = KS_INITIALIZE;
if (sam_hdr_find_tag_hd(state->h, "SO", &str) == 0 &&
strcmp(str.s, "coordinate") == 0) {
print_error(opts->filetype == FASTA ? "fasta" : "fastq",
"Coordinate sorted file. "
"Read pairs may be out of order");
}
ks_free(&str);
*state_out = state;
return true;
}
static bool destroy_state(const bam2fq_opts_t *opts, bam2fq_state_t *state, int* status)
{
bool valid = true;
sam_hdr_destroy(state->h);
check_sam_close("bam2fq", state->fp, opts->fn_input, "file", status);
if (state->fpse && sam_close(state->fpse) < 0) {
print_error_errno("bam2fq", "Error closing singleton file \"%s\"", opts->fnse);
valid = false;
}
int i, j;
for (i = 0; i < 3; ++i) {
if (state->fpr[i] != state->hstdout) {
for (j = 0; j < i; j++)
if (state->fpr[i] == state->fpr[j])
break;
if (j == i && sam_close(state->fpr[i])) {
print_error_errno("bam2fq", "Error closing r%d file \"%s\"", i, opts->fnr[i]);
valid = false;
}
}
}
if (state->hstdout) {
release_autoflush(state->hstdout);
if (sam_close(state->hstdout) < 0) {
print_error_errno("bam2fq", "Error closing STDOUT");
valid = false;
}
}
for (i = 0; i < 2; i++) {
for (j = 0; j < 3; j++)
if (state->fpi[i] == state->fpr[j])
break;
for (j -= 3; j >= 0 && j < i; j++)
if (state->fpi[i] == state->fpi[j])
break;
if (j == i && state->fpi[i] && sam_close(state->fpi[i]) < 0) {
print_error_errno("bam2fq", "Error closing i%d file \"%s\"", i+1, opts->index_file[i]);
valid = false;
}
}
free(state->index_sequence);
if (state->p.pool)
hts_tpool_destroy(state->p.pool);
free(state);
return valid;
}
static inline bool filter_it_out(const bam1_t *b, const bam2fq_state_t *state,
bam2fq_opts_t *opts)
{
if (opts->filter_tag) {
uint8_t *s = bam_aux_get(b, opts->filter_tag);
if (!s)
return true;
if (opts->filter_tag_vals) {
char t[32], *val = t;
switch (*s) {
case 'i': case 'I':
case 's': case 'S':
case 'c': case 'C':
if (snprintf(t, 32, "%"PRId64, bam_aux2i(s)) <= 0)
return true;
break;
case 'f':
// Comparing floats is hard.
// Eg (double)0.1 - (double)0.1f is -1.5e-9.
// Given BAM binary encoding is float however, just keep it.
// This means rounding errors will (hopefully) always be the
// same and basic equality still works.
if (snprintf(t, 32, "%f", (float)bam_aux2f(s)) <= 0)
return true;
break;
case 'A':
t[0] = s[1];
t[1] = 0;
break;
case 'Z': case 'H':
val = (char *)s+1;
break;
default:
// Anything unsupported fails the filter match too.
return true;
}
khint_t k = kh_get(str, opts->filter_tag_vals, val);
if (k == kh_end(opts->filter_tag_vals))
return 1; // tag value not found
}
}
return ((b->core.flag&(state->flag_on)) != state->flag_on // or reads indicated by filter flags
|| (b->core.flag&(state->flag_off)) != 0
|| (((b->core.flag&(state->flag_anyon)) == 0) && (state->flag_anyon != 0))
|| (b->core.flag&(state->flag_alloff) && (b->core.flag&(state->flag_alloff)) == state->flag_alloff));
}
int write_index_rec(samFile *fp, bam1_t *b, bam2fq_state_t *state,
bam2fq_opts_t* opts, char *seq, int seq_len,
char *qual, int qual_len) {
if (!fp || !b || !seq_len)
return 0;
int ret = -1;
bam1_t *b2 = bam_init1(); // FIXME: reuse
if (!b2)
return -1;
size_t aux_len = b->data + b->l_data - bam_get_aux(b);
if (bam_set1(b2, b->core.l_qname, bam_get_qname(b),
(b->core.flag | BAM_FUNMAP) & ~BAM_FREVERSE,
-1, -1, 0, // refid, pos, mapq
0, NULL, // cigar
-1, -1, 0, // rnext, pnext, tlen
seq_len, seq, qual,
aux_len) < 0)
goto err;
uint8_t *q = bam_get_qual(b2);
if (qual) {
int i;
for (i = 0; i < seq_len; i++)
q[i] -= '!';
} else {
memset(q, opts->def_qual, seq_len);
}
memcpy(bam_get_aux(b2), bam_get_aux(b), aux_len);
b2->l_data += aux_len;
if (sam_write1(fp, state->h, b2) < 0)
goto err;
ret = 0;
err:
if (b2)
bam_destroy1(b2);
return ret;
}
int output_index(bam1_t *b1, bam1_t *b2, bam2fq_state_t *state,
bam2fq_opts_t* opts) {
bam1_t *b = b1 ? b1 : b2;
char *ifmt = opts->index_format;
if (!ifmt)
ifmt = "i*i*";
// Get seq / qual elements
char *bc = NULL, *qt = NULL;
if (b1)
bc = (char *)bam_aux_get(b1, opts->barcode_tag);
if (b2 && !bc)
bc = (char *)bam_aux_get(b2, opts->barcode_tag);
if (!bc)
return 0;
else
bc++; // skip Z
if (b1)
qt = (char *)bam_aux_get(b1, opts->quality_tag);
if (b2 && !qt)
qt = (char *)bam_aux_get(b2, opts->quality_tag);
if (qt && strlen(bc) != strlen(qt)-1)
qt = NULL;
else if (qt)
qt++;
int inum = 0;
while (inum < 2) {
char fc = *ifmt++;
if (!fc)
break; // ran out of index-format
long len, rem = 0;
if (isdigit(*ifmt)) {
rem = len = strtol(ifmt, &ifmt, 10);
} else {
ifmt++;
len = 0;
}
char *bc_end = bc, *qt_end = qt;
if (qt) {
while (len ? *bc_end && rem-- : isalpha(*bc_end))
bc_end++, qt_end++;
} else {
while (len ? *bc_end && rem-- : isalpha(*bc_end))
bc_end++;
}
switch (fc) {
case 'n':
// skip
bc = bc_end + (len==0);
if (qt)
qt = qt_end + (len==0);
break;
case 'i':
if (write_index_rec(state->fpi[inum], b, state, opts,
bc, bc_end-bc, qt, qt_end-qt) < 0)
return -1;
bc = bc_end + (len==0);
if (qt)
qt = qt_end + (len==0);
inum++;
break;
default:
fprintf(stderr, "Unknown index-format code\n");
return -1;
}
}
return 0;
}
static int flush_rec(bam2fq_state_t *state, bam2fq_opts_t* opts,
bam1_t *b[4], int score[3], int best[3],
int64_t *n_singletons) {
// Paired data, with 1 or 2 ends present.
if (score[1] > 0 && score[2] > 0) {
// If CASAVA tag is required and barcode is only on R1,
// copy it to R2
if (state->illumina_tag) {
char *tag;
if ((tag = (char *)bam_aux_get(b[best[1]],
opts->barcode_tag)))
if (bam_aux_update_str(b[best[2]],
opts->barcode_tag,
strlen(tag), tag+1) < 0)
goto err;
if ((tag = (char *)bam_aux_get(b[best[1]],
opts->quality_tag)))
if (bam_aux_update_str(b[best[2]],
opts->quality_tag,
strlen(tag), tag+1) < 0)
goto err;
}
if (sam_write1(state->fpr[1], state->h, b[best[1]]) < 0)
goto err;
if (sam_write1(state->fpr[2], state->h, b[best[2]]) < 0)
goto err;
if (output_index(b[best[1]], b[best[2]], state, opts) < 0)
goto err;
} else if (score[1] > 0 || score[2] > 0) {
if (state->fpse) {
// print whichever one exists to fpse
if (score[1] > 0) {
if (sam_write1(state->fpse, state->h, b[best[1]]) < 0)
goto err;
} else {
if (sam_write1(state->fpse, state->h, b[best[2]]) < 0)
goto err;
}
++(*n_singletons);
} else {
if (score[1] > 0) {
if (sam_write1(state->fpr[1], state->h, b[best[1]]) < 0)
goto err;
} else {
if (sam_write1(state->fpr[2], state->h, b[best[2]]) < 0)
goto err;
}
}
if (output_index(score[1] > 0 ? b[best[1]] : NULL,
score[2] > 0 ? b[best[2]] : NULL,
state, opts) < 0)
goto err;
}
if (score[0]) { // single ended data (neither READ1 nor READ2)
if (sam_write1(state->fpr[0], state->h, b[best[0]]) < 0)
goto err;
if (output_index(b[best[0]], NULL, state, opts) < 0)
goto err;
}
return 0;
err:
return -1;
}
static bool bam2fq_mainloop(bam2fq_state_t *state, bam2fq_opts_t* opts)
{
int n;
char *current_qname = NULL;
int64_t n_reads = 0, n_singletons = 0; // Statistics
int score[3];
int at_eof;
bool valid = false;
int best[3] = {-1, -1, -1}; // map R0, R1, single to b[] indices;
// indexed by [readpart]
bam1_t *b[4]; // 3 readparts, plus current record
for (n = 0; n < 4; n++) {
if (!(b[n] = bam_init1())) {
perror("[bam2fq_mainloop] Malloc error for bam record buffer.");
return false;
}
}
n = 0;
while (true) {
int res = sam_read1(state->fp, state->h, b[n]);
if (res < -1) {
print_error("bam2fq", "Failed to read bam record");
goto err;
}
at_eof = res < 0;
if (!at_eof && filter_it_out(b[n], state, opts))
continue;
if (!at_eof) {
++n_reads;