File: denovo_map.pl

package info (click to toggle)
stacks 2.2%2Bdfsg-1
  • links: PTS, VCS
  • area: main
  • in suites: buster
  • size: 3,108 kB
  • sloc: cpp: 36,453; php: 4,059; sh: 2,122; perl: 1,163; python: 497; sql: 389; makefile: 148
file content (636 lines) | stat: -rwxr-xr-x 21,609 bytes parent folder | download
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
504
505
506
507
508
509
510
511
512
513
514
515
516
517
518
519
520
521
522
523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
567
568
569
570
571
572
573
574
575
576
577
578
579
580
581
582
583
584
585
586
587
588
589
590
591
592
593
594
595
596
597
598
599
600
601
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
618
619
620
621
622
623
624
625
626
627
628
629
630
631
632
633
634
635
636
#!/usr/bin/env perl
#
# Copyright 2010-2018, Julian Catchen <jcatchen@illinois.edu>
#
# This file is part of Stacks.
#
# Stacks 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.
#
# Stacks 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 Stacks.  If not, see <http://www.gnu.org/licenses/>.
#

use strict;
use POSIX;
use File::Temp qw/ mktemp /;
use File::Spec;
use constant stacks_version => "_VERSION_";

use constant true  => 1;
use constant false => 0;

my $dry_run      = false;
my $exe_path     = "_BINDIR_";
my $out_path     = "";
my $popmap_path  = "";
my $sample_path  = "";
my $db           = "";
my $min_cov      = 0;
my $min_rcov     = 0;
my $sample_id    = 1;
my $gzip         = false;
my $paired       = false;
my $time         = "";

my @parents;
my @progeny;
my @samples;

my (@_ustacks, @_cstacks, @_sstacks, @_tsv2bam, @_gstacks, @_populations);

my $cmd_str = $0 . " " . join(" ", @ARGV);

parse_command_line();

#
# Check for the existence of the necessary pipeline programs
#
foreach my $prog ("ustacks", "cstacks", "sstacks", "tsv2bam", "gstacks", "populations") {
    die "Unable to find '" . $exe_path . $prog . "'.\n" if (!-e $exe_path . $prog);
}

my ($log, $log_fh, $sample);

my (@sample_list, %pop_ids, %pops, %grp_ids, %grps, %sample_ids);

parse_population_map(\@sample_list, \%pop_ids, \%pops, \%grp_ids, \%grps);

initialize_samples(\@parents, \@progeny, \@samples, \@sample_list, \%pop_ids, \%grp_ids);

#
# Open the log file
#
$log = "$out_path/denovo_map.log";
open($log_fh, ">$log") or die("Unable to open log file '$log'; $!\n");

print $log_fh
        "denovo_map.pl version ", stacks_version, " started at ", strftime("%Y-%m-%d %H:%M:%S", (localtime(time))), "\n",
        $cmd_str, "\n";

execute_stacks($log_fh, $sample_id, \@parents, \@progeny, \@samples, \%sample_ids);

print $log_fh "\ndenovo_map.pl completed at ", strftime("%Y-%m-%d %H:%M:%S", (localtime(time))), "\n";
close($log_fh);

sub check_return_value {
    # $? is a 16 bit int. Exit code is given by `$? & 255` if the process was
    # terminated by a signal, and by `$? >> 8` if it exited normally.
    my ($rv, $log_fh) = @_;
    if ($rv != 0) {
        my $code = ($rv >> 8) & 127;
        if ($rv & 255 || ($rv >> 8) > 127) {
            $code += 128;
        }
        my $msg = "\ndenovo_map.pl: Aborted because the last command failed ($code";
        if ($code == 129 || $code == 130 || $code == 131) {
            $msg .= "/interrupted";
        } elsif ($code == 137 || $code == 143) {
            $msg .= "/killed";
        } elsif ($code == 134) {
            $msg .= "/SIGABRT";
        } elsif ($code == 139) {
            $msg .= "/segmentation fault";
        }
        $msg .= ")";
        print $log_fh ($msg . ".\n");
        print STDERR ($msg . "; see log file.\n");
        exit 1;
    }
}

sub execute_stacks {
    my ($log_fh, $sample_id, $parents, $progeny, $samples, $sample_ids) = @_;

    my (@results, @depths_of_cov);
    my ($pop_cnt, $sample, $num_files, $i, $cmd, $pipe_fh);

    my $minc  = $min_cov  > 0 ? " -m $min_cov"  : "";
    my $minrc = $min_rcov > 0 ? " -m $min_rcov" : $minc;

    $i         = 1;
    $num_files = scalar(@{$parents}) + scalar(@{$progeny}) + scalar(@{$samples});

    #
    # Assemble RAD loci in each individual.
    #
    print STDERR "Indentifying unique stacks...\n";
    print $log_fh "\nustacks\n==========\n";
    foreach $sample (@parents, @progeny, @samples) {

        print $log_fh "\nSample $i of $num_files '$sample->{'file'}'\n----------\n";

        if (scalar(keys %{$sample_ids}) > 0) {
            $sample_id = $sample_ids->{$sample->{'file'}};
        }

        $cmd = $exe_path . "ustacks -t $sample->{'fmt'} -f $sample->{'path'} -o $out_path -i $sample_id";
        if ($sample->{'type'} eq "sample") {
            $cmd .= $minc;
        } elsif ($sample->{'type'} eq "parent") {
            $cmd .= $minc;
        } elsif ($sample->{'type'} eq "progeny") {
            $cmd = $minrc;
        }
        if ($sample->{'path'} !~ /$sample->{'file'}.$sample->{'suffix'}$/) {
            # Guessing the sample name from the input path won't work.
            $cmd .= " --name " . $sample->{'file'};
        }
        foreach (@_ustacks) {
            $cmd .= " " . $_;
        }
        print STDERR  "  $cmd\n";
        print $log_fh "$cmd\n";

        if ($dry_run == false) {
            open($pipe_fh, "$time $cmd 2>&1 |");
            @results = ();
            while (<$pipe_fh>) {
                print $log_fh $_;
                push(@results, $_);
            }
            close($pipe_fh);
            check_return_value($?, $log_fh);

            #
            # Pull the depth of coverage from ustacks.
            #
            my $depthline = (grep(/^Final coverage/, @results))[0];
            my ($depth) = ($depthline =~ /mean=([^;]+)/);
            push(@depths_of_cov, [$sample->{'file'}, $depth]);
        }

        $i++;
        $sample_id++;
    }

    write_depths_of_cov(\@depths_of_cov, $log_fh);
    print STDERR "\n";

    #
    # Generate catalog of RAD loci.
    #
    print STDERR "Generating catalog...\n";
    print $log_fh "\ncstacks\n==========\n";

    $cmd = $exe_path . "cstacks -P $out_path " . join(" ", @_cstacks);
    print STDERR  "  $cmd\n\n";
    print $log_fh "$cmd\n\n";

    if ($dry_run == false) {
        open($pipe_fh, "$time $cmd 2>&1 |");
        while (<$pipe_fh>) {
            print $log_fh $_;
            if ($_ =~ /failed/i) { print STDERR "Catalog construction failed.\n"; exit(1); }
        }
        close($pipe_fh);
        check_return_value($?, $log_fh);
    }

    #
    # Match parents, progeny, or samples to the catalog.
    #
    print STDERR "Matching samples to the catalog...\n";
    print $log_fh "\nsstacks\n==========\n";

    $cmd      = $exe_path . "sstacks -P $out_path " . join(" ", @_sstacks);
    print STDERR  "  $cmd\n\n";
    print $log_fh "$cmd\n\n";

    if ($dry_run == false) {
        open($pipe_fh, "$time $cmd 2>&1 |");
        while (<$pipe_fh>) {
            print $log_fh $_;
        }
        close($pipe_fh);
        check_return_value($?, $log_fh);
    }

    #
    # Sort the reads according by catalog locus / run tsv2bam.
    #
    print STDERR "Sorting reads by RAD locus...\n";
    print $log_fh "\ntsv2bam\n==========\n";

    my $file_paths = "";
    if (length($popmap_path) == 0) {
        foreach $sample (@parents, @progeny, @samples) {
            $file_paths .= " -s $sample->{'file'}";
        }
    }

    $cmd = $exe_path . "tsv2bam -P $out_path $file_paths " . join(" ", @_tsv2bam);
    print STDERR  "  $cmd\n\n";
    print $log_fh "$cmd\n\n";

    if (!$dry_run) {
        open($pipe_fh, "$time $cmd 2>&1 |");
        while (<$pipe_fh>) {
            print $log_fh $_;
        }
        close($pipe_fh);
        check_return_value($?, $log_fh);
    }

    #
    # Call genotypes / run gstacks.
    #
    print STDERR "Calling variants, genotypes and haplotypes...\n";
    print $log_fh "\ngstacks\n==========\n";

    $cmd = $exe_path . "gstacks -P $out_path " . join(" ", @_gstacks);
    print STDERR  "  $cmd\n\n";
    print $log_fh "$cmd\n\n";

    if (!$dry_run) {
        open($pipe_fh, "$time $cmd 2>&1 |");
        while (<$pipe_fh>) {
            print $log_fh $_;
        }
        close($pipe_fh);
        check_return_value($?, $log_fh);
    }

    printf(STDERR "Calculating population-level summary statistics\n");
    print $log_fh "\npopulations\n==========\n";

    $cmd = $exe_path . "populations" . " -P $out_path " . join(" ", @_populations);
    print STDERR  "  $cmd\n\n";
    print $log_fh "$cmd\n\n";

    if ($dry_run == 0) {
        open($pipe_fh, "$time $cmd 2>&1 |");
        while (<$pipe_fh>) {
            print $log_fh $_;
        }
        close($pipe_fh);
        check_return_value($?, $log_fh);
    }

    print STDERR  "denovo_map.pl is done.\n";
    print $log_fh "denovo_map.pl is done.\n";
}

sub parse_population_map {
    my ($sample_list, $pop_ids, $pops, $grp_ids, $grps) = @_;

    my ($fh, @parts, $line, $sample);

    return if (length($popmap_path) == 0);

    open($fh, "<$popmap_path") or die("Unable to open population map, '$popmap_path', $!\n");

    while ($line = <$fh>) {
        chomp $line;
        next if ($line =~ /^\s*#/);

        @parts = split(/\t/, $line);
        if (scalar(@parts) != 2 and scalar(@parts) != 3) {
            die("Unable to parse population map, '$popmap_path' (expected 2 or 3 columns, found " . scalar(@parts) . "); at line:\n$line\n");
        }

        foreach my $part (@parts) {
            $part =~ s/^\s*|\s*$//g;
        }

        push(@{$sample_list}, $parts[0]);
        $pop_ids->{$parts[0]} = $parts[1];
        $pops->{$parts[1]}++;
        if (scalar(@parts) > 2) {
            $grp_ids->{$parts[0]} = $parts[2];
            $grps->{$parts[2]}++;
        }
    }

    if (scalar(keys %{$grps}) == 0) {
    $grps->{"1"}++;

        foreach $sample (@{$sample_list}) {
            $grp_ids->{$sample} = "1";
        }
    }

    print STDERR "Parsed population map: ", scalar(@{$sample_list}), " files in ", scalar(keys %{$pops});
    scalar(keys %{$pops}) == 1 ?  print STDERR " population" : print STDERR " populations";
    print STDERR " and ", scalar(keys %{$grps});
    scalar(keys %{$grps}) == 1 ? print STDERR " group.\n" : print STDERR " groups.\n";

    close($fh);
}

sub initialize_samples {
    my ($parents, $progeny, $samples, $sample_list, $pop_ids, $grp_ids) = @_;

    if (scalar(@{$sample_list}) > 0 && scalar(@{$samples}) == 0) {
        my @suffixes = ("fq",    "fastq", "fq.gz",   "fastq.gz", "fa",    "fasta", "fa.gz",   "fasta.gz");
        my @fmts     = ("fastq", "fastq", "gzfastq", "gzfastq",  "fasta", "fasta", "gzfasta", "gzfasta");

        #
        # Read the samples in from the population map.
        #
        my ($i, $extension, $extension_pe);
        my $first = true;
        foreach $sample (@{$sample_list}) {
            if ($first) {
                $first = false;
                my $found = false;
                for ($i = 0; $i < scalar(@suffixes); $i++) {
                    if (!$paired && -e $sample_path . $sample . "." . $suffixes[$i]) {
                        $found = true;
                        $extension = "." . $suffixes[$i];
                        last;
                    } elsif (-e $sample_path . $sample . ".1." . $suffixes[$i]) {
                        $found = true;
                        $extension = ".1." . $suffixes[$i];
                        $extension_pe = ".2." . $suffixes[$i];
                        last;
                    }
                }
                if (!$found) {
                    print STDERR "Error: Failed to find the first reads file '$sample_path$sample(.1).(fq|fastq|fa|fasta)(.gz)'.\n";
                    exit 1;
                }
                if ($i == 2 || $i == 3 || $i == 6 || $i == 7) {
                    $gzip = true;
                }
            }

            my ($path, $path_pe);

            $path    = $sample_path . $sample . $extension;
            $path_pe = ($paired ? $sample_path . $sample . $extension_pe : "");

            die("Error: Failed to open single-end file '$path'.\n") if (! -e $path);
            die("Error: Failed to open paired-end file '$path_pe'.\n") if ($paired && ! -e $path_pe);
            die("Unable to find an entry for '" . $sample . "' in the population map, '$popmap_path'.\n") if (!defined($pop_ids->{$sample}));

            if ($pop_ids->{$sample} eq "parent") {
                push(@{$parents}, {'path'   => $path,
                                       'file'   => $sample,
                                       'suffix' => $suffixes[$i],
                                       'type'   => "parent",
                                       'fmt'    => $fmts[$i],
                                       'path_pe' => $path_pe});
            } elsif ($pop_ids->{$sample} eq "progeny") {
                push(@{$progeny}, {'path'   => $path,
                                       'file'   => $sample,
                                       'suffix' => $suffixes[$i],
                                       'type'   => "progeny",
                                       'fmt'    => $fmts[$i],
                                       'path_pe' => $path_pe});
            } else {
                push(@{$samples}, {'path'   => $path,
                                       'file'   => $sample,
                                       'suffix' => $suffixes[$i],
                                       'type'   => "sample",
                                       'fmt'    => $fmts[$i],
                                       'path_pe' => $path_pe});
            }
        }
    }

    #
    # If a population map was specified, make sure all samples in the list were found (and vice versa) and assign popualtion IDs.
    #
    my %sample_hash;

    foreach $sample (@{$samples}) {
        $sample_hash{$sample->{'file'}}++;

        if (!defined($pop_ids->{$sample->{'file'}})) {
            die("Unable to find an entry for '" . $sample->{'file'} . "' in the population map, '$popmap_path'.\n");
        } else {
            $sample->{'pop_id'} = $pop_ids->{$sample->{'file'}};
        }
        if (!defined($grp_ids->{$sample->{'file'}})) {
            die("Unable to find an entry for '" . $sample->{'file'} . "' in the population map, '$popmap_path'.\n");
        } else {
            $sample->{'grp_id'} = $grp_ids->{$sample->{'file'}};
        }
    }

    foreach $sample (@{$sample_list}) {
        if (!defined($sample_hash{$sample})) {
            die("Unable to find a file corresponding to the population map entry '" . $sample . "' in the population map, '$popmap_path'.\n");
        }
    }

    #
    # Check that no duplicate files were specified.
    #
    my (%files, $file);
    foreach $file (@{$parents}, @{$progeny}, @{$samples}) {
        $files{$file}++;
    }
    foreach $file (keys %files) {
        if ($files{$file} > 1) {
            die("A duplicate file was specified which may create undefined results, '$file'\n");
        }
    }

    print STDERR "Found ", scalar(@{$parents}), " parental file(s).\n\n" if (scalar(@{$parents}) > 0);
    print STDERR "Found ", scalar(@{$progeny}), " progeny file(s).\n\n" if (scalar(@{$progeny}) > 0);
    print STDERR "Found ", scalar(@{$samples}), " sample file(s).\n\n" if (scalar(@{$samples}) > 0);

    if ( scalar(@{$samples}) > 0 && (scalar(@{$parents}) > 0 || scalar(@{$progeny}) > 0) ) {
	die("Both samples and parents/progeny were specified either on the command line (-s/-r/-p) or within the population map. Only one of the other may be specified.\n");
    }
}

sub write_results {
    my ($results, $log_fh) = @_;

    my $line;

    foreach $line (@{$results}) {
        if ($line =~ /\r/) {
            $line =~ s/^.+\r(.*\n)$/\1/;
        }
        print $log_fh $line;
    }
}

sub write_depths_of_cov {
    my ($depths, $log_fh) = @_;

    print STDERR "\nDepths of Coverage for Processed Samples:\n";
    print $log_fh "\nDepths of Coverage for Processed Samples:\n";

    foreach $a (@{$depths}) {
        print STDERR  $a->[0], ": ", $a->[1], "x\n";
        print $log_fh $a->[0], ": ", $a->[1], "x\n";
    }
}

sub parse_command_line {
    my ($arg);

    my $ustacks_mismatch = -1;
    my $cstacks_mismatch = -1;

    while (@ARGV) {
        $_ = shift @ARGV;
        if    ($_ =~ /^-v$/ || $_ =~ /^--version$/) { version(); exit 1; }
        elsif ($_ =~ /^-h$/) { usage(); }
        elsif ($_ =~ /^-d$/ || $_ =~ /^--dry-run$/) { $dry_run   = true; }
        elsif ($_ =~ /^-o$/)        { $out_path  = shift @ARGV; }
        elsif ($_ =~ /^-e$/)        { $exe_path  = shift @ARGV; }
        elsif ($_ =~ /^-m$/)        { $min_cov     = shift @ARGV; }
        elsif ($_ =~ /^-P$/)        { $min_rcov    = shift @ARGV; }
        elsif ($_ =~ /^--paired$/)  { $paired      = true; }
        elsif ($_ =~ /^--samples$/) { $sample_path = shift @ARGV; }
        elsif ($_ =~ /^-O$/ || $_ =~ /^--popmap$/) {
            $popmap_path = shift @ARGV;
            push(@_cstacks,     "-M " . $popmap_path);
            push(@_sstacks,     "-M " . $popmap_path);
            push(@_tsv2bam,     "-M " . $popmap_path);
            push(@_gstacks,     "-M " . $popmap_path);
            push(@_populations, "-M " . $popmap_path);

        } elsif ($_ =~ /^-t$/) {
            push(@_ustacks, "-d ");

        } elsif ($_ =~ /^-T$/) {
            $arg = shift @ARGV;
            push(@_ustacks, "-p " . $arg);
            push(@_cstacks, "-p " . $arg);
            push(@_sstacks, "-p " . $arg);
            push(@_tsv2bam, "-t " . $arg);
            push(@_gstacks, "-t " . $arg);
            push(@_populations, "-t " . $arg);

        } elsif ($_ =~ /^-M$/) {
            $ustacks_mismatch = shift(@ARGV);
            push(@_ustacks,   "-M " . $ustacks_mismatch);

        } elsif ($_ =~ /^-N$/) {
            push(@_ustacks,   "-N " . shift @ARGV);

        } elsif ($_ =~ /^-n$/) {
            $cstacks_mismatch = shift @ARGV;
            push(@_cstacks, "-n " . $cstacks_mismatch);

        } elsif ($_ =~ /^--var-alpha$/) {
            push(@_gstacks, "--var-alpha " . shift @ARGV);

        } elsif ($_ =~ /^--gt-alpha$/) {
            push(@_gstacks, "--gt-alpha " . shift @ARGV);

        } elsif ($_ =~ /^-X$/) {
            #
            # Pass an arbitrary command-line option to a pipeline program.
            #
            # Command line option must be of the form '-X "program:option"'
            #
            $arg = shift @ARGV;
            my ($prog, $opt) = ($arg =~ /^(\w+):(.+)$/);
            if ($prog eq "ustacks") {
            	push(@_ustacks, $opt);
            } elsif ($prog eq "cstacks") {
            	push(@_cstacks, $opt);
            } elsif ($prog eq "sstacks") {
            	push(@_sstacks, $opt);
            } elsif ($prog eq "tsv2bam") {
            	push(@_tsv2bam, $opt);
            } elsif ($prog eq "gstacks") {
            	push(@_gstacks, $opt);
            } elsif ($prog eq "populations") {
            	push(@_populations, $opt);
            } else {
            	print STDERR "Unknown pipeline program, '$arg'\n";
            	usage();
            }
        } elsif ($_ =~ /^--time-components$/) {
            $time = '/usr/bin/time';
            if (! -e $time) {
                die "Error: '$time': No such file or directory.\n";
            }
        } else {
            print STDERR "Unknown command line option: '$_'\n";
            usage();
        }
    }

    $exe_path = $exe_path . "/"          if (substr($exe_path, -1) ne "/");
    $out_path = substr($out_path, 0, -1) if (substr($out_path, -1) eq "/");

    if (scalar(@parents) > 0 && scalar(@samples) > 0) {
        print STDERR "You must specify either parent or sample files, but not both.\n";
        usage();
    }

    if (length($popmap_path) == 0) {
        print STDERR "You must specify a population map that lists your sample names (--popmap).\n";
        usage();
    }

    if (length($sample_path) == 0) {
        print STDERR "You must specify the path to the directory containing the samples (--samples).\n";
        usage();
    }

    if (length($sample_path) > 0) {
        $sample_path .= "/" if (substr($sample_path, -1) ne "/");
    }

    if ($paired == true) {
        if (length($sample_path) == 0) {
            print STDERR "If you want to assemble paired-ends, you must use a population map to specify samples.\n";
            usage();
        } else {
            push(@_tsv2bam, "-R $sample_path");
        }
    }

    #
    # By default, we want ustacks -M to equal cstacks -n.
    #
    if ($cstacks_mismatch == -1 && $ustacks_mismatch > 0) {
        push(@_cstacks, "-n " . $ustacks_mismatch);
    }
}

sub version {
    print STDERR "denovo_map.pl ", stacks_version, "\n";
}

sub usage {
    version();

    print STDERR <<EOQ;
denovo_map.pl --samples dir --popmap path -o dir [--paired] (assembly options) [-X prog:"opts" ...]

  Input/Output files:
    --samples: path to the directory containing the samples reads files.
    --popmap: path to a population map file (format is "<name> TAB <pop>", one sample per line).
    o: path to an output directory.

  General options:
    X: additional options for specific pipeline components, e.g. -X "populations: -p 3 -r 0.50".
    T: the number of threads/CPUs to use (default: 1).
    d: Dry run. Do not actually execute anything, just print the commands that would be executed.

  Stack assembly options:
    M: number of mismatches allowed between stacks within individuals (for ustacks).
    n: number of mismatches allowed between stacks between individuals (for cstacks; default 1; suggested: set to ustacks -M).

  SNP model options:
    --var-alpha: significance level at which to call variant sites (for gstacks; default: 0.05).
    --gt-alpha: significance level at which to call genotypes (for gstacks; default: 0.05).

  Paired-end options:
    --paired: after assembling RAD loci, assemble mini-contigs with paired-end reads.

  Miscellaneous:
    --time-components (for benchmarking)
EOQ

    exit 1;
}