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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 Data::Dumper;
my $usage = <<__EOUSAGE__;
#################################################################################################
#
# Required:
#
# --matrix <string> matrix of raw read counts (not normalized!)
#
################################################################################################
__EOUSAGE__
;
my $matrix_file;
my $help_flag;
&GetOptions ( 'h' => \$help_flag,
'matrix=s' => \$matrix_file,
);
if ($help_flag) {
die $usage;
}
unless ($matrix_file) {
die $usage;
}
main: {
my $tmm_info_file = &run_TMM($matrix_file);
&write_normalized_file($matrix_file, $tmm_info_file);
exit(0);
}
####
sub run_TMM {
my ($counts_matrix_file) = @_;
my $tmm_norm_script = "$counts_matrix_file.runTMM.R";
open (my $ofh, ">$tmm_norm_script") or die "Error, cannot write to $tmm_norm_script";
print $ofh "library(edgeR)\n\n";
print $ofh "rnaseqMatrix = read.table(\"$counts_matrix_file\", header=T, row.names=1, com='', check.names=F)\n";
print $ofh "rnaseqMatrix = as.matrix(rnaseqMatrix)\n";
print $ofh "rnaseqMatrix = round(rnaseqMatrix)\n";
print $ofh "exp_study = DGEList(counts=rnaseqMatrix, group=factor(colnames(rnaseqMatrix)))\n";
print $ofh "exp_study = calcNormFactors(exp_study)\n";
print $ofh "exp_study\$samples\$eff.lib.size = exp_study\$samples\$lib.size * exp_study\$samples\$norm.factors\n";
print $ofh "write.table(exp_study\$samples, file=\"$counts_matrix_file.TMM_info.txt\", quote=F, sep=\"\\t\", row.names=F)\n";
close $ofh;
&process_cmd("R --no-save --no-restore --no-site-file --no-init-file -q < $tmm_norm_script 1>&2 ");
my $tmm_matrix = "$counts_matrix_file.TMM_info.txt";
unless (-s $tmm_matrix) {
confess "Error, TMM matrix $tmm_matrix was not generated. Be sure edgeR is installed and see additional error messages above for other helpful info";
}
return($tmm_matrix);
}
####
sub process_cmd {
my ($cmd) = @_;
print STDERR "CMD: $cmd\n";
my $ret = system($cmd);
if ($ret) {
die "Error, cmd: $cmd died with ret ($ret) ";
}
return;
}
####
sub write_normalized_file {
my ($matrix_file, $tmm_info_file) = @_;
my %col_to_eff_lib_size;
my %col_to_norm_factor;
open (my $fh, $tmm_info_file) or die "Error, cannot open file $tmm_info_file";
my %bloom_to_col;
my $header = <$fh>;
while (<$fh>) {
chomp;
my @x = split(/\t/);
my ($col, $norm_factor, $eff_lib_size) = ($x[0], $x[2], $x[3]);
$col =~ s/\"//g;
my $bloom = $col;
$bloom =~ s/\W/$;/g;
$col_to_eff_lib_size{$col} = $eff_lib_size;
$col_to_norm_factor{$col} = $norm_factor;
if ($bloom ne $col) {
if (exists $bloom_to_col{$bloom}) {
die "Error, already stored $bloom_to_col{$bloom} for $bloom, but trying to also store $col here... Ensure column names are unique according to non-word characters.";
}
$col_to_eff_lib_size{$bloom} = $eff_lib_size;
$col_to_norm_factor{$bloom} = $norm_factor;
}
}
close $fh;
open ($fh, $matrix_file);
$header = <$fh>;
chomp $header;
my @pos_to_col = split(/\t/, $header);
my $check_column_ordering_flag = 0;
while (<$fh>) {
chomp;
my @x = split(/\t/);
unless ($check_column_ordering_flag) {
if (scalar(@x) == scalar(@pos_to_col) + 1) {
## header is offset, as is acceptable by R
## not acceptable here. fix it:
unshift (@pos_to_col, "");
}
$check_column_ordering_flag = 1;
print join("\t", @pos_to_col) . "\n";
}
my $gene = $x[0];
print $gene;
for (my $i = 1; $i <= $#x; $i++) {
my $col = $pos_to_col[$i];
$col =~ s/\"//g;
my $adj_col = $col;
$adj_col =~ s/-/\./g;
my $bloom = $col;
$bloom =~ s/\W/$;/g;
my $eff_lib_size = $col_to_eff_lib_size{$col}
|| $col_to_eff_lib_size{$bloom}
|| $col_to_eff_lib_size{$adj_col}
|| $col_to_eff_lib_size{"X$col"}
|| die "Error, no eff lib size for [$col] or [$bloom] or [$adj_col] or [\"X$col\"]" . Dumper(\%col_to_eff_lib_size);
my $norm_factor = $col_to_norm_factor{$col}
|| $col_to_norm_factor{$bloom}
|| $col_to_norm_factor{$adj_col}
|| $col_to_norm_factor{"X$col"}
|| die "Error, no normalization scaling factor for $col" . Dumper(\%col_to_norm_factor);
my $read_count = $x[$i];
my $converted_val = sprintf("%.3f", $read_count * 1/$norm_factor);
print "\t$converted_val";
}
print "\n";
}
return;
}
|
