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#!/usr/bin/env perl
# -*-CPerl-*-
# Last changed Time-stamp: <2015-06-01 16:40:16 mtw>
#
# Compute normalized expression data in TPM or RPKM from (raw) read
# counts in multicov.
# TPM reference: Wagner et al, Theory Biosci. 131(4), pp 281-85 (2012)
#
# usage: normalize_multicov.pl -i file.multicov -r 50 -o /output/path
#
# ***********************************************************************
# * Copyright notice
# *
# * Copyright 2015 Michael T. Wolfinger <michael@wolfinger.eu>
# * All rights reserved
# *
# * 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/>.
# *
# * This copyright notice MUST APPEAR in all copies of the script!
# ***********************************************************************
use strict;
use warnings;
use Getopt::Long;
use Data::Dumper;
use File::Basename;
use Pod::Usage;
use Cwd;
use Path::Class;
use Bio::ViennaNGS::Expression;
#^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^#
#^^^^^^^^^^ Variables ^^^^^^^^^^^#
#^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^#
my ($infile,$FC,$FC_sample,$conds,$totreads,$i,$sample,$basename);
my ($dir,$ext,$cwd);
my $readlength = 100;
my $dest = getcwd();
my $type = "TPM";
#^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^#
#^^^^^^^^^^^^^^ Main ^^^^^^^^^^^^^#
#^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^#
Getopt::Long::config('no_ignore_case');
pod2usage(-verbose => 1) unless GetOptions("i=s" => \$infile,
"readlength|r=s" => \$readlength,
"out|o=s" => \$dest,
"type|t=s" => \$type,
"man" => sub{pod2usage(-verbose => 2)},
"help|h" => sub{pod2usage(1)}
);
unless(-f $infile){
warn "Could not find input multicov file provided via -i option";
pod2usage(-verbose => 0);
}
unless ($type eq "TPM" || $type eq "RPKM"){
warn "\"$type\" is an unknown normalization type, please specify \"TPM\" or \"RPKM\"";
pod2usage(-verbose => 0);
}
$cwd = getcwd();
unless ($infile =~ /^\// || $infile =~ /^\.\//){$infile = file($cwd,$infile);}
unless ($dest =~ /\/$/){$dest .= "/";}
unless (-d $dest){mkdir $dest;}
($basename,$dir,$ext) = fileparse($infile,qr/\.[^.]*/);
# parse multicov file
my $exp = Bio::ViennaNGS::Expression->new();
$exp->parse_readcounts_bed12("$infile"); # stringified Path::Class object
#print Dumper($exp->nr_features);
#print Dumper($exp->data);
# compute TPM values for all genes in each condition
for ($i=0;$i<$exp->conds;$i++){
if($type eq "TPM"){
$exp->computeTPM($i, $readlength);
}
else{
$exp->computeRPKM($i);
}
}
# write extended BED12 file for each condition past column 12
if($type eq "TPM"){
$exp->write_expression_bed12("TPM", $dest, $basename);
}
else {
$exp->write_expression_bed12("RPKM", $dest, $basename);
}
# compute RPKM from TPM for debugging purposes
#for ($i=0;$i<$exp->conds;$i++){
# my $avRPKM = $exp->computeRPKMfromTPM($i,$readlength);
# warn "RPKMfromTPM (sample $i): avg RPKM $avRPKM";
#}
#$exp->write_expression_bed12("RPKMfromTPM", $dest, $basename);
__END__
=head1 NAME
normalize_multicov.pl - Compute normalized expression data from read
counts
=head1 SYNOPSIS
normalize_multicov.pl [-i I<FILE>] [-t RPKM|TPM] [--readlength I<INT>] [options]
=head1 DESCRIPTION
This program computes normalized expression values in 'Transcript per
Million' (TPM) or 'Reads per Megabase per Million' (RPKM) from read
counts. The latter must be provided in the format produced by the
'bedtools multicov' utility, i.e. an extended BED12 file where each
colum past the 12th lists read counts for one sample/condition.
=head1 OPTIONS
=over
=item B<-i>
Input file in 'bedtools multicov' output format, i.e. an extended BED12
file where each colum past the 12th lists the read counts for one
sample/condition.
=item B<--readlength -r>
Read length of the RNA-seq experiment.
=item B<--out -o>
Output folder.
=item B<--type -t>
Normalization method, either 'TPM' (default) or 'RPKM'.
=item B<--help -h>
Print short help
=item B<--man>
Prints the manual page and exits
=back
=head1 SEE ALSO
TPM reference: Wagner et al, Theory Biosci. 131(4), pp 281-85 (2012)
=head1 AUTHOR
Michael T. Wolfinger E<lt>michael@wolfinger.euE<gt>
=cut