#!/usr/bin/perl

use strict;

#
#  Program to scan a FASTA-formatted 454 sequence file for usable microsatellites 
#   by Mohamed Noor,  May 7, 2008  ,  modified August 7, 2008
#

print "\n\nProgram to scan a FASTA-formatted 454 sequence file for microsatellites.\n\n";
print "Scans specifically for di- and trinucleotide repeats.\n\n\n";

print "Enter filename from which to scan for repeats: ";
my $inp_file = <>;
print "Enter filename to output: ";
my $out_file = <>;
print "\nEnter minimum number of repeat units: ";
my $repeats = <>;
chomp $repeats;
print "Enter minimum number bases flanking sequence: ";
my $flank = <>;
chomp $flank;


open (SEQDATA, "< $inp_file")  or die ("Input file not found...\n");
open (SEQOUT, "> $out_file");

my $total_seq=0;
my $microhits=0;

my $line_read = <SEQDATA>;
if ($line_read !~ /^>/) {
    print "File does not appear to be FASTA formatted.\n";
    print "It does not start with a >\n";
 } # end if 
my $end_tester = 1;

while ($end_tester==1) {

   # 
   # Data entry phase
   #

   my $header = $line_read;
   my $hit = 0;
   my $sequence = "";
   while ($hit==0) {
      $line_read = <SEQDATA>;
      if (($line_read =~ /^>/) || (eof(SEQDATA))) {
         $hit = 1;
         } else {
         $sequence .= $line_read;
         chomp $sequence;
         $sequence =~ s/\s//g;  # eliminate extra whitespace
        } # end if/ else

    $total_seq++;

     } # end while ($hit==0)

    # 
    # Data entered: scan for microsats phase
    #

    if ( ($sequence =~ /\S{$flank,}[^AN](CA){$repeats,}[^CN]\S{$flank,}/) || ($sequence =~ /\S{$flank,}[^TN](GT){$repeats,}[^GN]\S{$flank,}/)
      || ($sequence =~ /\S{$flank,}[^AN](AA){$repeats,}[^AN]\S{$flank,}/) || ($sequence =~ /\S{$flank,}[^CN](AC){$repeats,}[^AN]\S{$flank,}/)
      || ($sequence =~ /\S{$flank,}[^GN](AG){$repeats,}[^AN]\S{$flank,}/) || ($sequence =~ /\S{$flank,}[^TN](AT){$repeats,}[^AN]\S{$flank,}/)
      || ($sequence =~ /\S{$flank,}[^CN](CC){$repeats,}[^CN]\S{$flank,}/) || ($sequence =~ /\S{$flank,}[^GN](CG){$repeats,}[^CN]\S{$flank,}/)
      || ($sequence =~ /\S{$flank,}[^TN](CT){$repeats,}[^CN]\S{$flank,}/) || ($sequence =~ /\S{$flank,}[^AN](GA){$repeats,}[^GN]\S{$flank,}/)
      || ($sequence =~ /\S{$flank,}[^CN](GC){$repeats,}[^GN]\S{$flank,}/) || ($sequence =~ /\S{$flank,}[^GN](GG){$repeats,}[^GN]\S{$flank,}/)
      || ($sequence =~ /\S{$flank,}[^AN](TA){$repeats,}[^TN]\S{$flank,}/) || ($sequence =~ /\S{$flank,}[^CN](TC){$repeats,}[^TN]\S{$flank,}/)
      || ($sequence =~ /\S{$flank,}[^GN](TG){$repeats,}[^TN]\S{$flank,}/) || ($sequence =~ /\S{$flank,}[^TN](TT){$repeats,}[^TN]\S{$flank,}/)
      || ($sequence =~ /\S{$flank,}[^CN](AAC){$repeats,}[^AN]\S{$flank,}/) || ($sequence =~ /\S{$flank,}[^GN](AAG){$repeats,}[^AN]\S{$flank,}/)
      || ($sequence =~ /\S{$flank,}[^TN](AAT){$repeats,}[^AN]\S{$flank,}/) || ($sequence =~ /\S{$flank,}[^AN](ACA){$repeats,}[^AN]\S{$flank,}/)
      || ($sequence =~ /\S{$flank,}[^CN](ACC){$repeats,}[^AN]\S{$flank,}/) || ($sequence =~ /\S{$flank,}[^GN](ACG){$repeats,}[^AN]\S{$flank,}/)
      || ($sequence =~ /\S{$flank,}[^TN](ACT){$repeats,}[^AN]\S{$flank,}/) || ($sequence =~ /\S{$flank,}[^AN](AGA){$repeats,}[^AN]\S{$flank,}/)
      || ($sequence =~ /\S{$flank,}[^CN](AGC){$repeats,}[^AN]\S{$flank,}/) || ($sequence =~ /\S{$flank,}[^TN](AGT){$repeats,}[^AN]\S{$flank,}/)
      || ($sequence =~ /\S{$flank,}[^GN](AGG){$repeats,}[^AN]\S{$flank,}/) || ($sequence =~ /\S{$flank,}[^AN](ATA){$repeats,}[^AN]\S{$flank,}/)
      || ($sequence =~ /\S{$flank,}[^CN](ATC){$repeats,}[^AN]\S{$flank,}/) || ($sequence =~ /\S{$flank,}[^GN](ATG){$repeats,}[^AN]\S{$flank,}/)
      || ($sequence =~ /\S{$flank,}[^TN](ATT){$repeats,}[^AN]\S{$flank,}/) || ($sequence =~ /\S{$flank,}[^AN](CAA){$repeats,}[^CN]\S{$flank,}/)
      || ($sequence =~ /\S{$flank,}[^CN](CAC){$repeats,}[^CN]\S{$flank,}/) || ($sequence =~ /\S{$flank,}[^GN](CAG){$repeats,}[^CN]\S{$flank,}/)
      || ($sequence =~ /\S{$flank,}[^TN](CAT){$repeats,}[^CN]\S{$flank,}/) || ($sequence =~ /\S{$flank,}[^AN](CCA){$repeats,}[^CN]\S{$flank,}/)
      || ($sequence =~ /\S{$flank,}[^GN](CCG){$repeats,}[^CN]\S{$flank,}/) || ($sequence =~ /\S{$flank,}[^TN](CCT){$repeats,}[^CN]\S{$flank,}/)
      || ($sequence =~ /\S{$flank,}[^AN](CGA){$repeats,}[^CN]\S{$flank,}/) || ($sequence =~ /\S{$flank,}[^CN](CGC){$repeats,}[^CN]\S{$flank,}/)
      || ($sequence =~ /\S{$flank,}[^GN](CGG){$repeats,}[^CN]\S{$flank,}/) || ($sequence =~ /\S{$flank,}[^TN](CGT){$repeats,}[^CN]\S{$flank,}/)
      || ($sequence =~ /\S{$flank,}[^AN](CTA){$repeats,}[^CN]\S{$flank,}/) || ($sequence =~ /\S{$flank,}[^CN](CTC){$repeats,}[^CN]\S{$flank,}/)
      || ($sequence =~ /\S{$flank,}[^GN](CTG){$repeats,}[^CN]\S{$flank,}/) || ($sequence =~ /\S{$flank,}[^TN](CTT){$repeats,}[^CN]\S{$flank,}/)
      || ($sequence =~ /\S{$flank,}[^AN](GAA){$repeats,}[^GN]\S{$flank,}/) || ($sequence =~ /\S{$flank,}[^GN](GAG){$repeats,}[^GN]\S{$flank,}/)
      || ($sequence =~ /\S{$flank,}[^CN](GAC){$repeats,}[^GN]\S{$flank,}/) || ($sequence =~ /\S{$flank,}[^TN](GAT){$repeats,}[^GN]\S{$flank,}/)
      || ($sequence =~ /\S{$flank,}[^AN](GCA){$repeats,}[^GN]\S{$flank,}/) || ($sequence =~ /\S{$flank,}[^CN](GCC){$repeats,}[^GN]\S{$flank,}/)
      || ($sequence =~ /\S{$flank,}[^GN](GCG){$repeats,}[^GN]\S{$flank,}/) || ($sequence =~ /\S{$flank,}[^TN](GCT){$repeats,}[^GN]\S{$flank,}/)
      || ($sequence =~ /\S{$flank,}[^AN](GGA){$repeats,}[^GN]\S{$flank,}/) || ($sequence =~ /\S{$flank,}[^CN](GGC){$repeats,}[^GN]\S{$flank,}/)
      || ($sequence =~ /\S{$flank,}[^TN](GGT){$repeats,}[^GN]\S{$flank,}/) || ($sequence =~ /\S{$flank,}[^AN](GTA){$repeats,}[^GN]\S{$flank,}/)
      || ($sequence =~ /\S{$flank,}[^CN](GTC){$repeats,}[^GN]\S{$flank,}/) || ($sequence =~ /\S{$flank,}[^GN](GTG){$repeats,}[^GN]\S{$flank,}/)
      || ($sequence =~ /\S{$flank,}[^TN](GTT){$repeats,}[^GN]\S{$flank,}/) || ($sequence =~ /\S{$flank,}[^AN](TAA){$repeats,}[^TN]\S{$flank,}/)
      || ($sequence =~ /\S{$flank,}[^CN](TAC){$repeats,}[^TN]\S{$flank,}/) || ($sequence =~ /\S{$flank,}[^GN](TAG){$repeats,}[^TN]\S{$flank,}/)
      || ($sequence =~ /\S{$flank,}[^TN](TAT){$repeats,}[^TN]\S{$flank,}/) || ($sequence =~ /\S{$flank,}[^AN](TCA){$repeats,}[^TN]\S{$flank,}/)
      || ($sequence =~ /\S{$flank,}[^CN](TCC){$repeats,}[^TN]\S{$flank,}/) || ($sequence =~ /\S{$flank,}[^GN](TCG){$repeats,}[^TN]\S{$flank,}/)
      || ($sequence =~ /\S{$flank,}[^TN](TCT){$repeats,}[^TN]\S{$flank,}/) || ($sequence =~ /\S{$flank,}[^AN](TGA){$repeats,}[^TN]\S{$flank,}/)
      || ($sequence =~ /\S{$flank,}[^CN](TGC){$repeats,}[^TN]\S{$flank,}/) || ($sequence =~ /\S{$flank,}[^GN](TGG){$repeats,}[^TN]\S{$flank,}/)
      || ($sequence =~ /\S{$flank,}[^TN](TGT){$repeats,}[^TN]\S{$flank,}/) || ($sequence =~ /\S{$flank,}[^AN](TTA){$repeats,}[^TN]\S{$flank,}/)
      || ($sequence =~ /\S{$flank,}[^CN](TTC){$repeats,}[^TN]\S{$flank,}/) || ($sequence =~ /\S{$flank,}[^GN](TTG){$repeats,}[^TN]\S{$flank,}/)
 
      ){
        print SEQOUT $header;
        print SEQOUT $sequence."\n"; 
        $microhits++;
     } # end if

    if (eof(SEQDATA)) {
       $end_tester=0;
     } # end if

 } # end while ($end_tester==1)

print "\nTotal number of scanned sequences: $total_seq\n";
print "Total number of microsatellites identified: $microhits\n\n\n";

close;

exit;