Error: readline on closed filehandle using Perl - file

Question

!/usr/bin/perl
use Cwd;
use warnings;
open($fh,'<', "clinical.txt");
$line = <$fh>;
while($line = <$fh>)
{
my #fields2 =split(" ",$line);
push(#id,$fields2[1]);
push(#status,$fields2[3]);
}
#flow = grep { -d } glob "*";
$arrSize = #flow;
for ($p = 0; $p < $arrSize; $p++)
{
#files=<$flow[$p]/*.maf>;
print $flow[$p],"\n";
foreach $file(#files)
{
print $file,"\n";
open(x,$file);
%hash={};
%tested={};
%Mut_Count={};
$hyper=0;
$line = <x>;
$line = <x>;
$line = <x>;
$line = <x>;
$line = <x>;
$line = <x>;
while($line = <x>)
{
#temp=split("\t",$line);
$key=$temp[4]."_".$temp[5]."_".$temp[6]."_".$temp[10]."_".$temp[11]."_".$temp[12]."_".$temp[15];
push #{$hash{$key}}, "0";
push #{$Mut_Count{$temp[15]}}, "0";
}
#nm=split(/\./,$file);
open(x,$file);
open(Out1,">Results/".$nm[1]."_Hyper.txt");
$line = <x>;
$line = <x>;
$line = <x>;
$line = <x>;
$line = <x>;
#temp=split(" ",$line);
#temp2=split(",",$temp[1]);
print Out1 "Gene\tMutation\tType\tdbSNP\tStatus\tPolyphen\tSift";
for($j=0;$j<scalar(#id);$j++){
my #M=split('-', $id[$j]);
for($i=0;$i<scalar(#temp2);$i++)
{
my #N=split('-', $temp2[$i]);
if(scalar(#{$Mut_Count{$temp2[$i]}})>499 && $M[0] eq $N[0] && $M[1] eq $N[1] && $M[2] eq $N[2] && $status[$j] eq 'MSS')
{
print Out1 "\t",$temp2[$i];
$hyper++;
}
}
}
$line = <x>;
while($line = <x>)
{
$hy=0;
#temp=split("\t",$line);
$key=$temp[4]."_".$temp[5]."_".$temp[6]."_".$temp[10]."_".$temp[11]."_".$temp[12];
if(!exists $tested{$key})
{
push #{$tested{$key}}, "0";
print Out1 "\n",$temp[0],"\t",$key,"\t",$temp[8],"\t",$temp[13],"\t",$temp[25],"\t",$temp[72],"\t",$temp[73];
for($i=0;$i<scalar(#temp2);$i++)
{
$key=$temp[4]."_".$temp[5]."_".$temp[6]."_".$temp[10]."_".$temp[11]."_".$temp[12]."_".$temp2[$i];
my #L=split('-', $temp2[$i]);
for($j=0;$j<scalar(#id);$j++){
my #O=split('-', $id[$j]);
if(scalar(#{$Mut_Count{$temp2[$i]}})>499 && $L[0] eq $O[0] && $L[1] eq $O[1] && $L[2] eq $O[2] && $status[$j] eq 'MSS')
{
if(exists $hash{$key})
{
print Out1 "\t1";
$hy++;
}
else
{
print Out1 "\t0";
}
}
}
}
}
}
open(Out3,">Results/".$nm[1]."_Summary.txt");
print Out3 "Hypermutated\t$hyper\n";
}
}
$line = <$fh>; while($line = <$fh>)
these lines are showing readline() on closed filehandle $fh
%hash={}; %tested={}; %Mut_Count={};
on these three lines it says Reference found where even-sized list expected
.maf are basically GDC downloaded files with bit modified header according to our need with unique TCGA IDs.
Whereas, Clinical Info is file contaning TCGA IDs, its source and MSI_Status that tells us whether it is MSI-L, MSI-H or MSS.
I'm reading multiple .maf files and comparing it with clinical_info file
and if the if condition is satisfied that I want it to generate mastertable(write a file)
I'm doing it in windows. Kindly, help me resolve this, Thanks in anticipation.

Answer 1

Answer to
%hash={}; %tested={}; %Mut_Count={}; on these three lines it says Reference found where even-sized list expected
You can get rid of the errors by getting rid of these three lines, though if you accept (please!) the use strict; recommendation you will need to replace them with my %hash; and so forth.
Under Perl, hashes (and arrays) do not need to be initialized. If you wish to initialize one, you assign it another array, or a list, or another hash. The length of the initializer must be even, since it is interpreted as key/value pairs. Your code supplied a hash reference, which will not be expanded by Perl to an empty hash. Hence the error.
If it makes you nervous not to initialize the hash, you can say my %hash = ();.
On the other hand, if you intend to use a scalar as a hash reference, you will need to initialize it. This is where you use the curly brackets, which (among other things) are a hash constructor which returns a reference to the constructed hash. So:
my $hash = {};
$hash->{$key} = $value;
...