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
|
#!/usr/bin/env perl
use strict;
use warnings;
use FindBin;
use lib("$FindBin::RealBin/../../PerlLib");
use Pipeliner;
use File::Basename;
use Cwd;
use Carp;
use Getopt::Long qw(:config no_ignore_case bundling pass_through);
my $HISAT_HOME = $ENV{HISAT_HOME} or die "Error, need env var HISAT_HOME set to the HISAT installation directory.\n\n";
my $usage = <<__EOUSAGE__;
######################################################################
#
# Required:
# --genome <string> target genome to align to
# --reads <string> fastq files. If pairs, indicate both in quotes, ie. "left.fq right.fq"
#
# Optional:
# -N <int> max number of alignments to report. (default: 1)
# -G <string> GTF file for incorporating reference splice site info.
# --CPU <int> number of threads (default: 2)
# --out_prefix <string> output prefix (default: hisat)
# --run_as_single_reads if paired, run as single reads
#
#######################################################################
__EOUSAGE__
;
my ($genome, $reads);
my $CPU = 2;
my $help_flag;
my $out_prefix = "hisat";
my $gtf_file;
my $run_as_single_flag = 0;
my $num_top_hits = 1;
&GetOptions( 'h' => \$help_flag,
'genome=s' => \$genome,
'reads=s' => \$reads,
'CPU=i' => \$CPU,
'out_prefix=s' => \$out_prefix,
'G=s' => \$gtf_file,
'run_as_single_reads' => \$run_as_single_flag,
'N=i' => \$num_top_hits,
);
unless ($genome && $reads) {
die $usage;
}
main: {
my $hisat_index = "$genome.hisat.idx";
if (! -s "$hisat_index.1.bt2") {
## build hisat index
my $cmd = "$HISAT_HOME/hisat-build $genome $hisat_index";
&process_cmd($cmd);
}
my $splice_incl = "";
if ($gtf_file) {
my $gtf_splice = "$gtf_file.hisat.splice";
unless (-s $gtf_splice) {
my $cmd = "$HISAT_HOME/extract_splice_sites.py $gtf_file > $gtf_file.hisat.splice";
&process_cmd($cmd);
}
$splice_incl = " --known-splicesite-infile $gtf_splice ";
}
## run HISAT
$reads = &add_zcat_fifo_and_add_hisat_params($reads);
my $top_hits_count = "";
if ($num_top_hits > 1) {
$top_hits_count = " -k $num_top_hits ";
}
my @tmpfiles;
my $pipeliner = new Pipeliner(-verbose => 1);
my $cmd = "bash -c \"set -o pipefail && $HISAT_HOME/hisat -x $hisat_index -q $reads $splice_incl -p $CPU $top_hits_count @ARGV | gzip -c > $out_prefix.sam.gz\" ";
$pipeliner->add_commands( new Command($cmd, "$out_prefix.sam.gz.ok") );
push (@tmpfiles, "$out_prefix.sam.gz");
$cmd = "bash -c \"set -o pipefail && gunzip -c $out_prefix.sam.gz | samtools view -@ $CPU -F 4 -Sb - > $out_prefix.bam \"";
$pipeliner->add_commands( new Command($cmd, "$out_prefix.bam.ok") );
push (@tmpfiles, "$out_prefix.bam");
$cmd = "samtools sort -@ $CPU $out_prefix.bam $out_prefix.cSorted";
$pipeliner->add_commands( new Command($cmd, "$out_prefix.cSorted.bam.ok") );
$pipeliner->run();
if (-s "$out_prefix.cSorted.bam") {
$cmd = "samtools index $out_prefix.cSorted.bam";
&process_cmd($cmd);
}
unlink(@tmpfiles);
exit(0);
}
####
sub add_zcat_fifo_and_add_hisat_params {
my ($reads) = @_;
$reads =~ s/^\s+|\s+$//g;
my @adj_reads_list;
my $counter = 0;
my @read_files = split(/\s+/, $reads);
my @updated_read_filenames;
foreach my $reads_file (@read_files) {
$counter++;
if ($reads_file =~ /\.gz$/) {
$reads_file = "<(zcat $reads_file)";
}
push (@updated_read_filenames, $reads_file);
# add decoration
$reads_file = (scalar(@read_files) == 2) ? "-$counter $reads_file" : "-U $reads_file";
push (@adj_reads_list, $reads_file);
}
if ($run_as_single_flag) {
return("-U " . join(",", @updated_read_filenames));
}
else {
my $adj_reads = join(" ", @adj_reads_list);
return($adj_reads);
}
}
####
sub process_cmd {
my ($cmd) = @_;
print STDERR "CMD: $cmd\n";
#return;
my $ret = system($cmd);
if ($ret) {
die "Error, cmd: $cmd died with ret ($ret)";
}
return;
}
|
