If you can add placeholders [% name %] to your template.config file like this

[module1]

;;path to speedseq package binary directory
$;SPEEDSEQ_BIN_DIR$; = /usr/local/packages/
;;Sequence file 1
$;Seq1File$; = "[% seq1 %]"
;;Sequence file 2
$;Seq2File$; = "[% seq2 %]"
;;Read Group
$;Read_Group$;='@RG\tID:NA12878\tSM:NA12878\tPL:ILLUMINA\tLB:NA12878\t
+PU:NA12878'
;;Reference
$;Reference$; = "[% ref %]"

[module2]

 ;;Output Chromosome
 $;Chromosome$; = "[% chr_list %]"
 ;;use --v for verbose summary
 $;OTHER_ARGS$; = --v
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then you could use a templating module like Template::Toolkit or Template::Tiny

Thanks a lot! This works perfect. But is there any way to populate without changing the input template file? Thanks again!

With some pattern matching perhaps

#!perl
use strict;

# replace with Getopt::Long code as before
my $vars = {
  seq1 =>'USER_INPUT.txt',
  seq2 => 'USER_INPT2.txt',
  ref => 'USER_INPUT_REF.txt',
  chr_list => 'USER_INPUT_CHR.txt',
};
 
my $input = 'template.config';
open IN,'<',$input or die "Could not open $input : $!";
my $output = 'new_'.$input;
open OUT,'>',$output or die "Could not open $output : $!";

while (<IN>){
  chomp;
  # match selected lines
  # and append "value"
  if (/^ *\$;(Seq\d|Ref|Chromo).*\$; =/){
    my $key = ($1 eq 'Chromo') ? 'chr_list' : lc $1;
    $_ .= '"'.$vars->{$key}.'"';
  }
  print OUT $_."\n";  
}
close IN;
close OUT;
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poj