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/*
* sideRETRO - A pipeline for detecting Somatic Insertion of DE novo RETROcopies
* Copyright (C) 2019-2020 Thiago L. A. Miller <tmiller@mochsl.org.br
*
* This program is free software: you can redistribute it and/or modify
* it under the terms of the GNU General Public License as published by
* the Free Software Foundation, either version 3 of the License, or
* (at your option) any later version.
*
* This program is distributed in the hope that it will be useful,
* but WITHOUT ANY WARRANTY; without even the implied warranty of
* MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the
* GNU General Public License for more details.
*
* You should have received a copy of the GNU General Public License
* along with this program. If not, see <http://www.gnu.org/licenses/>.
*/
#include "config.h"
#include <stdio.h>
#include <time.h>
#include <getopt.h>
#include <assert.h>
#include "wrapper.h"
#include "array.h"
#include "utils.h"
#include "chr.h"
#include "db.h"
#include "log.h"
#include "logger.h"
#include "io.h"
#include "str.h"
#include "thpool.h"
#include "exon.h"
#include "abnormal.h"
#include "dedup.h"
#include "process_sample.h"
#define DEFAULT_MAX_DISTANCE 10000
#define DEFAULT_CACHE_SIZE 200000 /* 200MiB */
#define DEFAULT_THREADS 1
#define DEFAULT_SORTED 0
#define DEFAULT_DEDUPLICATE 0
#define DEFAULT_EXON_FRAC 1e-09
#define DEFAULT_ALIGNMENT_FRAC 1e-09
#define DEFAULT_EITHER 0
#define DEFAULT_RECIPROCAL 0
#define DEFAULT_PREFIX "out"
#define DEFAULT_OUTPUT_DIR "."
#define DEFAULT_LOG_SILENT 0
#define DEFAULT_LOG_LEVEL LOG_INFO
#define DEFAULT_PHRED_QUALITY 8
#define DEFAULT_MAX_BASE_FREQ 0.75
struct _ProcessSample
{
// Mandatory
Array *sam_files;
const char *gff_file;
// I/O
const char *input_file;
const char *output_dir;
const char *prefix;
// Log
Logger *logger;
const char *log_file;
int log_level;
int silent;
// SQLite3
int cache_size;
// Read Quality
float max_base_freq;
int phred_quality;
int deduplicate;
// Processing
int threads;
int sorted;
int max_distance;
float exon_frac;
float alignment_frac;
int alignment_frac_set;
int reciprocal;
int either;
};
typedef struct _ProcessSample ProcessSample;
static void
run (ProcessSample *ps)
{
log_trace ("Inside %s", __func__);
sqlite3 *db = NULL;
sqlite3_stmt *batch_stmt = NULL;
sqlite3_stmt *source_stmt = NULL;
sqlite3_stmt *alignment_stmt = NULL;
sqlite3_stmt *exon_stmt = NULL;
sqlite3_stmt *overlapping_stmt = NULL;
ExonTree *exon_tree = NULL;
ChrStd *cs = NULL;
threadpool thpool = NULL;
const int batch_id = 1;
char timestamp[32] = {};
time_t t = 0;
struct tm *lt = NULL;
const char *sam_file = NULL;
const int num_files = array_len (ps->sam_files);
char *db_file = NULL;
int i = 0;
// Assemble database output filename
xasprintf_concat (&db_file, "%s/%s.db",
ps->output_dir, ps->prefix);
log_info ("Create output dir '%s'", ps->output_dir);
mkdir_p (ps->output_dir);
// Create and connect to database
log_info ("Create and connect to database '%s'", db_file);
db = db_create (db_file);
batch_stmt = db_prepare_batch_stmt (db);
source_stmt = db_prepare_source_stmt (db);
alignment_stmt = db_prepare_alignment_stmt (db);
exon_stmt = db_prepare_exon_stmt (db);
overlapping_stmt = db_prepare_overlapping_stmt (db);
// Increase the cache size
db_cache_size (db, ps->cache_size);
// Begin transaction to speed up
db_begin_transaction (db);
// Get current local datetime as timestamp
// for batch table
t = time (NULL);
lt = localtime (&t);
timestamp[strftime (timestamp, sizeof (timestamp),
"%Y-%m-%d %H:%M:%S", lt)] = '\0';
// Dump batch entry
log_debug ("Dump batch entry %d => %s", batch_id, timestamp);
db_insert_batch (batch_stmt, batch_id, timestamp);
// Get chromosome standardization
cs = chr_std_new ();
// Index protein coding genes into the database
// and its exons into an intervalar tree by
// chromosome
log_info ("Index annotation file '%s'", ps->gff_file);
exon_tree = exon_tree_new (exon_stmt, overlapping_stmt, cs);
exon_tree_index_dump (exon_tree, ps->gff_file);
log_info ("Create thread pool");
thpool = thpool_init (ps->threads);
AbnormalArg *ab_args = xcalloc (num_files, sizeof (AbnormalArg));
for (i = 0; i < num_files; i++)
{
sam_file = array_get (ps->sam_files, i);
// Init abnormal_filter arg
ab_args[i] = (AbnormalArg)
{
.tid = i + 1,
.inc_step = num_files,
.sam_file = sam_file,
.either = ps->either,
.exon_frac = ps->exon_frac,
.alignment_frac = ps->alignment_frac,
.exon_tree = exon_tree,
.cs = cs,
.alignment_stmt = alignment_stmt,
.queryname_sorted = ps->sorted,
.max_distance = ps->max_distance,
.phred_quality = ps->phred_quality,
.max_base_freq = ps->max_base_freq
};
log_debug ("Dump source entry '%s'", sam_file);
db_insert_source (source_stmt, i + 1, batch_id, sam_file);
log_info ("Run abnormal filter for '%s'", sam_file);
thpool_add_work (thpool, (void *) abnormal_filter,
(void *) &ab_args[i]);
}
// Wait all threads to return
thpool_wait (thpool);
// Commit database
db_end_transaction (db);
if (ps->deduplicate)
{
// Begin transaction to speed up
db_begin_transaction (db);
// Run deduplication step
log_info ("Run deduplication step for '%s'", db_file);
dedup (db);
// Commit database
db_end_transaction (db);
}
log_info ("Process Sample at '%s' is finished. "
"Run merge-call command to discover somatic retrocopies",
db_file);
// Time to clean
db_finalize (exon_stmt);
db_finalize (batch_stmt);
db_finalize (source_stmt);
db_finalize (alignment_stmt);
db_finalize (overlapping_stmt);
db_close (db);
xfree (db_file);
xfree (ab_args);
chr_std_free (cs);
exon_tree_free (exon_tree);
thpool_destroy (thpool);
}
static void
print_usage (FILE *fp)
{
int pkg_len = strlen (PACKAGE);
fprintf (fp,
"%s\n"
"\n"
"Usage: %s process-sample [-h] [-q] [-d] [-s] [-l FILE] [-o DIR]\n"
" %*c [-p STR] [-t INT] [-c INT] [-Q INT]\n"
" %*c [-m INT] [-f FLOAT] [-F FLOAT | -r]\n"
" %*c [-D] [-M FLOAT] [-e] [-i FILE]\n"
" %*c -a FILE <FILE> ...\n"
"\n"
"Extract alignments related to event of retrocopy\n"
"\n"
"Examples:\n"
" $ sider ps -l ps.log -a gencode.gff3.gz -o result in.bam\n"
" $ sider ps -t 3 -a gencode.gtf in1.bam in2.sam in3.bam\n"
" $ sider ps -t 5 -m 15000 -Q 20 -F 0.9 -a exon.gtf -i list.txt\n"
"\n"
"Output:\n"
" A SQLite3 database that can be processed at 'merge-call' step\n"
"\n"
"Arguments:\n"
" One or more alignment file in SAM/BAM/CRAM format\n"
"\n"
"Mandatory Options:\n"
" -a, --annotation-file Gene annotation on the reference genome\n"
" in GTF/GFF3 format. %s will look for 'exon'\n"
" with the attribute 'transcript_type=protein_coding'.\n"
" The attributes 'gene_name', 'gene_id' and 'exon_id'\n"
" are also required\n"
" -i, --input-file File containing a newline separated list of\n"
" alignment files in SAM/BAM/CRAM format.\n"
" This option is not mandatory if one or more\n"
" SAM/BAM/CRAM files are passed as argument.\n"
" If 'input-file' and arguments are set\n"
" concomitantly, then the union of all alignment\n"
" files is used\n"
"\n"
"Input/Output Options:\n"
" -h, --help Show help options\n"
" -q, --quiet Decrease verbosity to error messages only\n"
" or suppress terminal outputs at all if\n"
" 'log-file' is passed\n"
" --silent Same as '--quiet'\n"
" -d, --debug Increase verbosity to debug level\n"
" -l, --log-file Print log messages to a file\n"
" -o, --output-dir Output directory. Create the directory if it does\n"
" not exist [default:\"%s\"]\n"
" -p, --prefix Prefix output files [default:\"%s\"]\n"
"\n"
"SQLite3 Options:\n"
" -c, --cache-size Set SQLite3 cache size in KiB [default:\"%d\"]\n"
"\n"
"Read Quality Options:\n"
" -Q, --phred-quality Minimum mapping quality of the reads required\n"
" [default:\"%d\"]\n"
" -M, --max-base-freq Maximum base frequency fraction allowed\n"
" [default:\"%.2f\"]\n"
" -D, --deduplicate Remove duplicated reads. Reads are considered\n"
" duplicates when they share the 5 prime positions\n"
" of both reads and read-pairs\n"
"\n"
"Processing Options:\n"
" -s, --sorted Assume all reads are grouped by queryname, even if\n"
" there is no SAM/BAM/CRAM header tag 'SO:queryname'\n"
" -t, --threads Number of threads [default:\"%d\"]\n"
" -m, --max-distance Maximum distance allowed between paired-end reads\n"
" [default:\"%d\"]\n"
" -f, --exon-frac Minimum overlap required as a fraction of exon\n"
" [default:\"%.0e\"; 1 base]\n"
" -F, --alignment-frac Minimum overlap required as a fraction of\n"
" alignment [default:\"%.0e\"; 1 base]\n"
" -e, --either The minimum fraction must be satisfied for at least\n"
" exon OR alignment. Without '-e', both fractions would\n"
" have to be satisfied\n"
" -r, --reciprocal The fraction overlap must be reciprocal for exon and\n"
" alignment. If '-f' is 0.5, then '-F' will be set to\n"
" 0.5 as well\n"
"\n",
PACKAGE_STRING, PACKAGE, pkg_len, ' ', pkg_len, ' ', pkg_len, ' ', pkg_len, ' ',
PACKAGE, DEFAULT_OUTPUT_DIR, DEFAULT_PREFIX, DEFAULT_CACHE_SIZE, DEFAULT_PHRED_QUALITY,
DEFAULT_MAX_BASE_FREQ, DEFAULT_THREADS, DEFAULT_MAX_DISTANCE,
DEFAULT_EXON_FRAC, DEFAULT_ALIGNMENT_FRAC);
}
static void
print_try_help (FILE *fp)
{
fprintf (fp, "Try '%s process-sample --help' for more information\n",
PACKAGE);
}
static void
process_sample_init (ProcessSample *ps)
{
*ps = (ProcessSample) {
.sam_files = array_new (xfree),
.gff_file = NULL,
.input_file = NULL,
.output_dir = DEFAULT_OUTPUT_DIR,
.prefix = DEFAULT_PREFIX,
.logger = NULL,
.log_file = NULL,
.log_level = DEFAULT_LOG_LEVEL,
.silent = DEFAULT_LOG_SILENT,
.cache_size = DEFAULT_CACHE_SIZE,
.max_base_freq = DEFAULT_MAX_BASE_FREQ,
.phred_quality = DEFAULT_PHRED_QUALITY,
.deduplicate = DEFAULT_DEDUPLICATE,
.threads = DEFAULT_THREADS,
.sorted = DEFAULT_SORTED,
.max_distance = DEFAULT_MAX_DISTANCE,
.exon_frac = DEFAULT_EXON_FRAC,
.alignment_frac = DEFAULT_ALIGNMENT_FRAC,
.alignment_frac_set = 0,
.reciprocal = DEFAULT_RECIPROCAL,
.either = DEFAULT_EITHER
};
}
static void
process_sample_destroy (ProcessSample *ps)
{
if (ps == NULL)
return;
array_free (ps->sam_files, 1);
logger_free (ps->logger);
memset (ps, 0, sizeof (ProcessSample));
}
static int
process_sample_validate (ProcessSample *ps)
{
int rc = EXIT_SUCCESS;
int i = 0;
/*
* Validate arguments and mandatory options
*/
// If no one file was passed, throw an error
if (array_len (ps->sam_files) == 0)
{
fprintf (stderr, "%s: Missing alignment files (SAM/BAM/CRAM)\n", PACKAGE);
print_try_help (stderr);
rc = EXIT_FAILURE; goto Exit;
}
// Test if all alignment files exist
for (i = 0; i < array_len (ps->sam_files); i++)
{
const char *sam_file = array_get (ps->sam_files, i);
if (!exists (sam_file))
{
fprintf (stderr, "%s: alignment file '%s': No such file\n", PACKAGE, sam_file);
rc = EXIT_FAILURE; goto Exit;
}
}
// If no annotation-file was passed, throw an error
if (ps->gff_file == NULL)
{
fprintf (stderr, "%s: Missing annotation file (GTF/GFF3)\n", PACKAGE);
print_try_help (stderr);
rc = EXIT_FAILURE; goto Exit;
}
// Test if annotation file exists
if (!exists (ps->gff_file))
{
fprintf (stderr, "%s: annotation file '%s': No such file\n", PACKAGE, ps->gff_file);
rc = EXIT_FAILURE; goto Exit;
}
/*
* Validate options
*/
// If --reciprocal, set alignment_frac to exon_frac
if (ps->reciprocal)
{
if (ps->alignment_frac_set)
{
fprintf (stderr, "%s: --reciprocal must be set solely with '-f'\n", PACKAGE);
rc = EXIT_FAILURE; goto Exit;
}
ps->alignment_frac = ps->exon_frac;
}
// Validate exon_frac: ]0, 1]
if (ps->exon_frac > 1 || ps->exon_frac <= 0)
{
fprintf (stderr, "%s: --exon-frac must be in the interval of ]0, 1]\n", PACKAGE);
rc = EXIT_FAILURE; goto Exit;
}
// Validate alignment_frac ]0, 1]
if (ps->alignment_frac > 1 || ps->alignment_frac <= 0)
{
fprintf (stderr, "%s: --alignment-frac must be in the interval of ]0, 1]\n", PACKAGE);
rc = EXIT_FAILURE; goto Exit;
}
// Validate threads > 0
if (ps->threads < 1)
{
fprintf (stderr, "%s: --threads must be greater or equal to 1\n", PACKAGE);
rc = EXIT_FAILURE; goto Exit;
}
// Validate cache_size >= DEFAULT_CACHE_SIZE
if (ps->cache_size < DEFAULT_CACHE_SIZE)
{
fprintf (stderr, "%s: --cache-size must be greater or equal to %uKiB\n",
PACKAGE, DEFAULT_CACHE_SIZE);
rc = EXIT_FAILURE; goto Exit;
}
// Validate max_distance >= 0
if (ps->max_distance < 0)
{
fprintf (stderr, "%s: --max-distance must be a positive value\n", PACKAGE);
rc = EXIT_FAILURE; goto Exit;
}
if (ps->max_base_freq <= 0.25 || ps->max_base_freq > 1)
{
fprintf (stderr, "%s: --max-base-frac must be in the range of ]0.25, 1]\n", PACKAGE);
rc = EXIT_FAILURE; goto Exit;
}
// Validate phred_quality >= 0
if (ps->phred_quality < 0)
{
fprintf (stderr, "%s: --phred-quality must be a positive value\n", PACKAGE);
rc = EXIT_FAILURE; goto Exit;
}
/*
* Final settings
*/
// Avoid to include repetitive files
array_uniq (ps->sam_files, cmpstringp);
// If it's silent and no log file
// was passed, then set log_level
// to LOG_ERROR - At least print
// errors
if (ps->silent && ps->log_file == NULL)
{
ps->silent = 0;
ps->log_level = LOG_ERROR;
}
ps->logger = logger_new (ps->log_file, ps->log_level,
ps->silent, 1);
Exit:
return rc;
}
static void
process_sample_print (const ProcessSample *ps)
{
String *msg = string_sized_new (BUFSIZ);
int i = 0;
string_concat_printf (msg, ">> Process Sample step <<\n"
"\n"
"#\n"
"# %s\n"
"#\n"
"\n"
"## Command line parsing with default values\n"
"\n"
"# Input SAM/BAM/CRAM files\n"
"$ cat my-inputfile.txt\n",
PACKAGE_STRING);
for (i = 0; i < array_len (ps->sam_files); i++)
string_concat_printf (msg, "%s\n",
(char *) array_get (ps->sam_files, i));
string_concat_printf (msg,
"\n"
"# Run %s\n"
"$ %s process-sample\n"
" --input-file='my-inputfile.txt' \\\n"
" --annotation-file='%s' \\\n"
" --output-dir='%s' \\\n"
" --prefix='%s' \\\n",
PACKAGE, PACKAGE, ps->gff_file, ps->output_dir, ps->prefix);
if (ps->log_file != NULL)
string_concat_printf (msg, " --log-file='%s' \\\n", ps->log_file);
if (ps->log_level <= LOG_DEBUG)
string_concat_printf (msg, " --debug \\\n");
if (ps->silent)
string_concat_printf (msg, " --silent \\\n");
string_concat_printf (msg,
" --cache-size=%d \\\n"
" --max-base-freq=%.2f \\\n"
" --phred-quality=%d \\\n",
ps->cache_size, ps->max_base_freq, ps->phred_quality);
if (ps->deduplicate)
string_concat_printf (msg, " --deduplicate \\\n");
if (ps->sorted)
string_concat_printf (msg, " --sorted \\\n");
string_concat_printf (msg,
" --threads=%d \\\n"
" --max-distance=%d \\\n"
" --exon-frac=%e \\\n"
" --alignment-frac=%e",
ps->threads, ps->max_distance, ps->exon_frac,
ps->alignment_frac);
if (ps->either)
{
string_concat_printf (msg, " \\\n");
string_concat_printf (msg, " --either");
}
string_concat_printf (msg, "\n");
log_info ("%s", msg->str);
string_free (msg, 1);
}
int
parse_process_sample_command_opt (int argc, char **argv)
{
assert (argc > 1 && argv != NULL && *argv != NULL);
// No options or arguments
// Print usage
if (argc == 2)
{
print_usage (stdout);
return EXIT_SUCCESS;
}
struct option opt[] =
{
{"help", no_argument, 0, 'h'},
{"quiet", no_argument, 0, 'q'},
{"silent", no_argument, 0, 'q'},
{"debug", no_argument, 0, 'd'},
{"log-file", required_argument, 0, 'l'},
{"annotation-file", required_argument, 0, 'a'},
{"output-dir", required_argument, 0, 'o'},
{"prefix", required_argument, 0, 'p'},
{"threads", required_argument, 0, 't'},
{"phred-quality", required_argument, 0, 'Q'},
{"max-distance", required_argument, 0, 'm'},
{"max-base-freq", required_argument, 0, 'M'},
{"cache-size", required_argument, 0, 'c'},
{"sorted", no_argument, 0, 's'},
{"deduplicate", no_argument, 0, 'D'},
{"exon-frac", required_argument, 0, 'f'},
{"alignment-frac", required_argument, 0, 'F'},
{"either", no_argument, 0, 'e'},
{"reciprocal", no_argument, 0, 'r'},
{"input-file", required_argument, 0, 'i'},
{0, 0, 0, 0 }
};
// Init variables to default values
ProcessSample ps = {};
process_sample_init (&ps);
int rc = EXIT_SUCCESS;
int option_index = 0;
int c, i;
while ((c = getopt_long (argc, argv, "hqdsDl:a:o:p:t:m:M:c:Q:f:F:eri:", opt, &option_index)) >= 0)
{
switch (c)
{
case 'h':
{
print_usage (stdout);
goto Exit;
break;
}
case 'q':
{
ps.silent = 1;
break;
}
case 'd':
{
ps.log_level = LOG_DEBUG;
break;
}
case 'l':
{
ps.log_file = optarg;
break;
}
case 'a':
{
ps.gff_file = optarg;
break;
}
case 'o':
{
ps.output_dir = optarg;
break;
}
case 'p':
{
ps.prefix = optarg;
break;
}
case 't':
{
ps.threads = atoi (optarg);
break;
}
case 'c':
{
ps.cache_size = atoi (optarg);
break;
}
case 'Q':
{
ps.phred_quality = atoi (optarg);
break;
}
case 'm':
{
ps.max_distance = atoi (optarg);
break;
}
case 'M':
{
ps.max_base_freq = atof (optarg);
break;
}
case 'D':
{
ps.deduplicate = 1;
break;
}
case 's':
{
ps.sorted = 1;
break;
}
case 'f':
{
ps.exon_frac = atof (optarg);
break;
}
case 'F':
{
ps.alignment_frac = atof (optarg);
ps.alignment_frac_set = 1;
break;
}
case 'e':
{
ps.either = 1;
break;
}
case 'r':
{
ps.reciprocal = 1;
break;
}
case 'i':
{
ps.input_file = optarg;
break;
}
case '?':
case ':':
{
print_try_help (stderr);
rc = EXIT_FAILURE; goto Exit;
break;
}
}
}
// Catch all alignment files passed
// as argument
for (i = optind + 1; i < argc; i++)
array_add (ps.sam_files, xstrdup (argv[i]));
// Catch all alignment files passed into
// --input-file
if (ps.input_file != NULL)
read_file_lines (ps.sam_files, ps.input_file);
// Validate and init logger
rc = process_sample_validate (&ps);
// If no error
if (rc == EXIT_SUCCESS)
{
/*
* RUN FOOLS
*/
process_sample_print (&ps);
run (&ps);
}
Exit:
process_sample_destroy (&ps);
return rc;
}
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