[Bioperl-l] Regarding blast in Bioperl

Roopa Raghuveer rtbio.2009 at gmail.com
Fri Jan 8 10:00:21 EST 2010


Hello all,

I was trying Remote blast using Bioperl. My input data is a Trypanosoma
brucei sequence in Fasta format. When I was trying to submit to BLAST using
the step
$r=$factory->submit_blast($input)
It was not returning anything which I checked by debugging the code. It is
not blasting my input sequence even though I mentioned all the parameters.I
would paste the code below.

Please help me in solving put this problem. It is very urgent.

Regards
Roopa.

#!/usr/bin/perl

#path for extra camel module
use lib "/srv/www/htdocs/rain/RNAi/";
use Roopablast;


use Bio::SearchIO;
use Bio::Search::Result::BlastResult;
use Bio::Perl;
use Bio::Tools::Run::RemoteBlast;
use Bio::Seq;
use Bio::SeqIO;
use Bio::DB::GenBank;

$serverpath = "/srv/www/htdocs/rain/RNAi";
$serverurl = "http://141.84.66.66/rain/RNAi";
$outfile = $serverpath."/rnairesult_".time().".html";
$nuc = $serverpath."/nuc".time().".txt";
$debugfile = $serverpath."/debug_".time().".txt";
$blastdebugfile = $serverpath."/blastdebug_".time().".txt";

my $outstring ="";

&parse_form;

print "Content-type: text/html\n\n";
print "<HTML>\n";
print "<head><title>RNAi Result</title>";
print "<META HTTP-EQUIV=\"Refresh\" CONTENT=\"30;
URL=$serverurl/rnairesult_".time().".html\"> \n";
print "</head>\n";
print "<body>\n";
print " Your results will appear <a
href=$serverurl/rnairesult_".time().".html>here</a><br>";
print " Please be patient, runtime can be up to 5 minutes<br>";
print " This page will automatically reload in 30 seconds. Roopa";
print "</BODY>\n";
print "</HTML>\n";

defined(my $pid = fork) or die "Can't fork: $!";
exit if $pid;
open STDIN, '/dev/null' or die "Can't read /dev/null: $!";
open STDOUT, '>/dev/null' or die "Can't write to /dev/null: $!";
open STDERR, '>&STDOUT' or die "Can't dup stdout: $!";



open(OUTFILE, '>',$outfile);

print OUTFILE "<HTML>\n
 <head><title>RNAi Result</title>
 <META HTTP-EQUIV=\"Refresh\" CONTENT=\"30;
URL=$serverurl//rnairesult_".time().".html\"> \n
 <meta http-equiv=\"expires\" content=\"0\">
 </head>\n
 <body>\n
  Your results will appear <a
href=$serverurl/rnairesult_".time().".html>here</a><br>
  Please be patient, runtime can be up to 5 minutes wait wait wait......<br>
 This page will automatically reload in 30 seconds Roopa <br>
 </BODY>\n
 </HTML>\n";

close(OUTFILE);


@compseqs = blastcode($in{'Inputseq'});

$in{'Inputseq'} =~ s/>.*$//m;
$in{'Inputseq'} =~ s/[^TAGC]//gim;
$in{'Inputseq'} =~ tr/actg/ACTG/;

@out = similar($in{'Inputseq'}, \@compseqs, $in{'Windowsize'},
$in{'Threshold'});


sub blastcode
{

$inpu1= $_[0];

#$organ= $_[1];

open(NUC,'>',$nuc);
print NUC $inpu1;
close(NUC);

 my $prog = 'blastn';
 my $db   = 'refseq_rna';
 my $e_val= '1e-10';
 my $organism= 'Trypanosoma Brucei';

$gb = new Bio::DB::GenBank;

 my @params = ( '-prog' => $prog,
         '-data' => $db,
         '-expect' => $e_val,
         '-readmethod' => 'SearchIO',
         '-Organism'   => $organism );

            # open(OUTFILE,'>',$debugfile);
             #  print OUTFILE @params;
             # close(OUTFILE);


 my $factory = Bio::Tools::Run::RemoteBlast->new(@params);

  #change a paramter

 $Bio::Tools::Run::RemoteBlast::HEADER{'ENTREZ_QUERY'} = 'Trypanosoma
Brucei[ORGN]';

#change a paramter
# $Bio::Tools::Run::RemoteBlast::HEADER{'ENTREZ_QUERY'} = '$input2[ORGN]';

  my $v = 1;
  #$v is just to turn on and off the messages

 my $str = Bio::SeqIO->new('-file' => $nuc , '-format' => 'fasta' ,
'-organism' => 'Trypanosoma Brucei' );


while (my $input = $str->next_seq())
{
   #Blast a sequence against a database:
    #Alternatively, you could  pass in a file with many
    #sequences rather than loop through sequence one at a time
    #Remove the loop starting 'while (my $input = $str->next_seq())'
    #and swap the two lines below for an example of that.

             open(OUTFILE,'>',$debugfile);
               print OUTFILE $input;
              close(OUTFILE);


   my $r = $factory->submit_blast($input);    #The program stops here it
does not return any value and it does not enter the While loop,Please help
me in this regard.#
                open(OUTFILE,'>',$debugfile);
                print OUTFILE $r;
                close(OUTFILE);


   print STDERR "waiting...." if($v>0);

  while ( my @rids = $factory->each_rid ) {
      open(OUTFILE,'>',$debugfile);
               print OUTFILE "while entered";
              close(OUTFILE);
     foreach my $rid ( @rids ) {

               open(OUTFILE,'>',$debugfile);
               print OUTFILE "foreach entered";
              close(OUTFILE);

        my $rc = $factory->retrieve_blast($rid);

        if( !ref($rc) )
        {
        if( $rc < 0 )
        {
        $factory->remove_rid($rid);
        }
         open(OUTFILE,'>',$debugfile);
               print OUTFILE "if entered";
              close(OUTFILE);
         print STDERR "." if ( $v > 0 );
         sleep 5;
        }
       else {
              open(OUTFILE,'>',$debugfile);
               print OUTFILE "else entered";
              close(OUTFILE);

          my $result = $rc->next_result();
         #save the output
        $blastdebugfile = $serverpath."/blastdebug_".time().".txt";

          open(BLASTDEBUGFILE,'>',$blastdebugfile);
          print BLASTDEBUGFILE $result->next_hit();
          close(BLASTDEBUGFILE);

        my $filename =
$serverpath."/blastdata_".time().$result->query_name()."\.out";

         # open(DEBUGFILE,'>',$debugfile);
         # open(new,'>',$filename);
         # @arra=<new>;
         # print DEBUGFILE @arra;
         # close(DEBUGFILE);
         # close(new);

         $factory->save_output($filename);

       # open(BLASTDEBUGFILE,'>',$debugfile);
       # print BLASTDEBUGFILE  "Hello $rid";
       # close(BLASTDEBUGFILE);

       $factory->remove_rid($rid);

       open(BLASTDEBUGFILE,'>',$blastdebugfile);
       print BLASTDEBUGFILE  $organism;
        close(BLASTDEBUGFILE);

    # open(OUTFILE,'>',$outfile);
    # print OUTFILE "Test2 $result->database_name()";
    # close(OUTFILE);

#$hit = $result->next_hit;
#open(new,'>',$debugfile);
#print $hit;
#close(new);

   while ( my $hit = $result->next_hit ) {

            next unless ( $v > 0);

          #     open(OUTFILE,'>',$debugfile);
           #    print OUTFILE "$hit in while hits";
            #  close(OUTFILE);

       my $sequ = $gb->get_Seq_by_version($hit->name);
           my $dna = $sequ->seq();        # get the sequence as a string
                  push(@seqs,$dna);
          }
        }
      }
    }
  }

  #open(OUTFILE,'>',$debugfile);
  #print OUTFILE $seqs[0];
  #close(OUTFILE);

return(@seqs);

}

open(OUTFILE, '>',$outfile) || die ;

print OUTFILE "<HTML>\n
<head><title>RNAi Result</title>
<meta http-equiv=\"expires\" content=\"0\"></head>\n
<body>\n
<p><font face=\"Courier, monospace font set\">
Inputsequence: <br>";

for ($i=0; $i<length ($in{'Inputseq'}); $i++) {

    print OUTFILE substr ($in{'Inputseq'}, $i, 1);

    if ( ($i+1)%10==0){
        print OUTFILE " ";
    }
    if ( ($i+1)%60==0){
        print OUTFILE "<br>\n";
    }
}



print OUTFILE "</font> <p>";

$z=@compseqs;

for($k=1;$k<$z;$k++) {
    print OUTFILE "<font face=\"Courier, monospace font set\"><p>Compare
Sequence: <br>";

    for ($i=0; $i<length ($compseqs[$k]); $i++) {

        print OUTFILE substr ($compseqs[$k], $i, 1);

        if ( ($i+1)%10==0){
            print OUTFILE " ";
        }
        if ( ($i+1)%60==0){
            print OUTFILE "<br>\n";
        }
    }
    print OUTFILE "<p></font>";
}

print OUTFILE "<p>
Window: <br>$in{'Windowsize'}
<p>
<p>
Threshold: <br>$in{'Threshold'}
<p>";
my $j=0;

for ($i=0; $i<length ($in{'Inputseq'}); $i++) {

    if ($i<=length ($in{'Inputseq'})-$in{'Windowsize'}){
        if ($out[$i]->{similar}<=$in{'Threshold'}){
            $j=$in{'Windowsize'};
        }
        $height=$out[$i]->{similar}*5;
    }

    if ($j>0) {
        print OUTFILE "<img src=\"$serverurl/blue.gif\" width=\"1\"
height=\"5\">";
        $outstring .= "<font color=\"green\">".substr ($in{'Inputseq'}, $i,
1)."</font>";
        $j--;
    }
    else {
        print OUTFILE "<img src=\"$serverurl/red.gif\" width=\"1\"
height=\"5\">";
        $outstring .= "<font color=\"red\">".substr ($in{'Inputseq'}, $i,
1)."</font>";
    }

    if ( ($i+1)%10==0){
        $outstring .= " ";
    }
    if ( ($i+1)%60==0){
        $outstring .= "<br>\n";

    }
    if ( ($i+1)%800==0){
        print OUTFILE "<br><br>\n";

    }
}

print OUTFILE "<br><br><font face=\"Courier, monospace font
set\">$outstring</font>";

#foreach (@out) {
#print OUTFILE "<p>Sequence: $_->{sequence}: $_->{similar} matchs<p>";
#if ($_->{similar}<=$in{'Threshold'}){

#    }
#}

print OUTFILE "</BODY>\n</HTML>\n";

close OUTFILE;

#nameprint();

sub parse_form {
    local ($buffer, @pairs, $pair, $name, $value);
    # Read in text
    $ENV{'REQUEST_METHOD'} =~ tr/a-z/A-Z/;
    if ($ENV{'REQUEST_METHOD'} eq "POST")
    {
        read(STDIN, $buffer, $ENV{'CONTENT_LENGTH'});
    }
    else
    {
        $buffer = $ENV{'QUERY_STRING'};
    }
    @pairs = split(/&/, $buffer);
    foreach $pair (@pairs)
    {
        ($name, $value) = split(/=/, $pair);
        $value =~ tr/+/ /;
        $value =~ s/%([a-fA-F0-9][a-fA-F0-9])/pack("C", hex($1))/eg;
        $in{$name} = $value;
    }
}


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