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#!/usr/bin/env perl
use strict;
use warnings;
use Carp;
use Getopt::Long qw(:config no_ignore_case bundling);
use Cwd;
use File::Basename;
use lib ("/usr/lib/trinityrnaseq/PerlLib");
use Fasta_reader;
use Data::Dumper;
my $usage = <<__EOUSAGE__;
#################################################################################################
#
# Required:
#
# --matrix|m <string> matrix of raw read counts (not normalized!)
#
# --batches_file|b <string> tab-delimited text file indicating biological replicate relationships.
# ex.
# batch_1 cond_A_rep1
# batch_1 cond_B_rep1
#
# batch_2 cond_A_rep2
# batch_2 cond_B_rep2
#
#
################################################################################################
__EOUSAGE__
;
my $matrix_file;
my $batches_file;
my $help_flag;
&GetOptions ( 'h' => \$help_flag,
'matrix|m=s' => \$matrix_file,
'batches_file|b=s' => \$batches_file,
);
if ($help_flag) {
die $usage;
}
unless ($matrix_file
&& $batches_file
) {
die $usage;
}
main: {
&run_edgeR_remove_batch_effect($matrix_file, $batches_file);
exit(0);
}
####
sub run_edgeR_remove_batch_effect {
my ($matrix_file, $batches_file) = @_;
use File::Basename;
my $base = basename($batches_file);
my $Rscript_name = "$base.batch_eff_removal.Rscript";
my $out_matrix_name = basename($matrix_file) . ".batch_eff_removal.matrix";
## write R-script to run edgeR
open (my $ofh, ">$Rscript_name") or die "Error, cannot write to $Rscript_name";
print $ofh "library(edgeR)\n";
print $ofh "\n";
print $ofh "rnaseqMatrix = read.table(\"$matrix_file\", header=T, row.names=1, com='', check.names=F)\n";
print $ofh "batches = read.table(\"$batches_file\", header=F, row.names=2, check.names=F)\n";
print $ofh "batch_factors = as.factor(batches[colnames(rnaseqMatrix),])\n";
print $ofh "exp_study = DGEList(counts=rnaseqMatrix)\n";
print $ofh "exp_study = calcNormFactors(exp_study)\n";
print $ofh "logCPM <- cpm(exp_study,log=TRUE)\n";
print $ofh "logCPM <- removeBatchEffect(logCPM,batch=batch_factors)\n";
print $ofh "millions = colSums(rnaseqMatrix)/1e6\n";
print $ofh "myCPM = 2^logCPM\n";
print $ofh "myRecounts = apply(myCPM, 1, function (x) x*millions)\n";
print $ofh "write.table(t(myRecounts), file=\'$out_matrix_name\', quote=F, sep='\t')\n";
close $ofh;
## Run R-script
my $cmd = "R --no-save --no-restore --no-site-file --no-init-file -q < $Rscript_name";
eval {
&process_cmd($cmd);
};
if ($@) {
print STDERR "$@\n\n";
print STDERR "\n\nWARNING: This EdgeR comparison failed...\n\n";
## if this is due to data paucity, such as in small batch data sets, then ignore for now.
}
return;
}
####
sub process_cmd {
my ($cmd) = @_;
print "CMD: $cmd\n";
my $ret = system($cmd);
if ($ret) {
die "Error, cmd: $cmd died with ret ($ret) ";
}
return;
}
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