[Bioperl-l] Regarding blast in Bioperl
Roopa Raghuveer
rtbio.2009 at gmail.com
Mon Jan 25 22:35:32 UTC 2010
Hello Russell,
Thank you very much for your reply. My problem is that Remote blast is
getting well executed with my code and I am getting the .out file with
sequences producing significant alignments. But, when I am trying to
retrieve the sequences into an array @seqs, I am able to retrieve all the
sequences except for the first hit. If the number of hits that I get in the
.out file to be 3, I am able to retrieve only 2 hits i.e., I am able to get
only 2 sequences. If there is only one significant hit for my sequence, then
the name and description of the sequence appears in the .out file, but I am
unable to get it into the array,the array count shows 0 and there would not
be any sequence in the array.
I hope that you have got me now.
Here comes my code,
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.";
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 <br>
This page will automatically reload in 30 seconds <br>
</BODY>\n
</HTML>\n";
close(OUTFILE);
@compseqs = blastcode($in{'Inputseq'},$in{'Organism'});
$in{'Inputseq'} =~ s/>.*$//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,"\n";
close(NUC);
my $prog = 'blastn';
my $db = 'refseq_rna';
my $e_val= '1e-10';
my $organism= $organ;
$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 $factory = Bio::Tools::Run::RemoteBlast->new(@params, -ENTREZ_QUERY =>
"$organ\[ORGN]");
#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' => $nuc , '-format' => 'fasta' ,
'-organism' => "$organ\[ORGN]");
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);
open(OUTFILE,'>',$debugfile);
# print OUTFILE $r;
close(OUTFILE);
print STDERR "waiting...." if($v>0);
while ( my @rids = $factory->each_rid ) {
foreach my $rid ( @rids ) {
my $rc = $factory->retrieve_blast($rid);
if( !ref($rc) )
{
if( $rc < 0 )
{
$factory->remove_rid($rid);
}
print STDERR "." if ( $v > 0 );
sleep 5;
}
else {
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 = $serverpath."/blastdata_".time()."\.out";
# open(DEBUGFILE,'>',$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);
while ( my $hit = $result->next_hit ) {
next unless ( $v >= 0);
my $sequ = $gb->get_Seq_by_version($hit->name);
my $dna = $sequ->seq(); # get the sequence as a string
$dummy++;
open(OUTFILE,'>',$debugfile);
open(OUTFILE,'>',$debugfile);
# print OUTFILE $dna;
close(OUTFILE);
push(@seqs,$dna);
}
}
}
}
}
$warum=scalar(@seqs);
open(OUTFILE,'>',$debugfile);
print OUTFILE $warum;
# print OUTFILE @seqs;
close(OUTFILE);
return(@seqs);
}
open(OUTFILE, '>',$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=0;$k<$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;
}
}
Regards,
Roopa.
On Mon, Jan 25, 2010 at 10:14 PM, Smithies, Russell <
Russell.Smithies at agresearch.co.nz> wrote:
> That's a fair mix of incomplete code you've supplied!!
> Did you read the documentation for RemoteBlast? The example there will do
> 99% of what you want.
> http://search.cpan.org/~cjfields/BioPerl-1.6.1/Bio/Tools/Run/RemoteBlast.pm<http://search.cpan.org/%7Ecjfields/BioPerl-1.6.1/Bio/Tools/Run/RemoteBlast.pm>
>
> I'm not entirely sure what you're trying to do (as you've left out a bit of
> your code) but I assume you're trying to retrieve and print the sequence for
> each hit.
>
> Here's something that works, not sure exactly what/why you want to print
> but it should get you a bit further.
>
> --Russell
>
>
> ================================
> #!perl -w
>
> use Bio::Tools::Run::RemoteBlast;
> use Bio::DB::GenBank;
>
> use CGI ':standard';
>
> use strict;
>
> my $q = new CGI;
>
> my @params = (
> -prog => 'blastn',
> -data => 'nr',
> -expect => '1e-30',
> -entrez_query => 'Homo sapiens [ORGN]',
> -readmethod => 'SearchIO'
> );
>
> my $gb = Bio::DB::GenBank->new;
>
> my $factory = Bio::Tools::Run::RemoteBlast->new(@params);
>
> #$v is just to turn on and off the messages
> my $v = 1;
>
> my $str = Bio::SeqIO->new( -file => 'test.faa', -format => "fasta" );
>
> while ( my $input = $str->next_seq() ) {
>
> my $r = $factory->submit_blast($input);
>
> print STDERR "waiting..." if ( $v > 0 );
> while ( my @rids = $factory->each_rid ) {
> foreach my $rid (@rids) {
> my @seqs = ();
> my $rc = $factory->retrieve_blast($rid);
> if ( !ref($rc) ) {
> if ( $rc < 0 ) {
> $factory->remove_rid($rid);
> }
> print STDERR "." if ( $v > 0 );
> sleep 5;
> }
> else {
> my $result = $rc->next_result();
>
> #save the blast output
> my $filename = $result->query_accession . '.out';
> $factory->save_output($filename);
> $factory->remove_rid($rid);
> print "\nQuery Name: ", $result->query_name(), "\n";
> while ( my $hit = $result->next_hit ) {
>
> # store the hit sequences
> push @seqs, $gb->get_Seq_by_version( $hit->name );
>
> next unless ( $v > 0 );
> print "\thit name is ", $hit->name, "\n";
> while ( my $hsp = $hit->next_hsp ) {
> print "\t\tscore is ", $hsp->score, "\n";
> }
> }
>
> ## print the seqs you've retrieved??
> open( OUTFILE, '>', $result->query_accession . '.htm' );
> print OUTFILE $q->start_html('RNAi Result'),
> $q->h1('RNAi Result'),
> $q->h2('Input'),
> $q->pre( toString($input) ),
> $q->h2('Output');
>
> foreach (@seqs) {
>
> #there's probably a better way of printing the seq
> print OUTFILE $q->pre( toString($_) );
> }
> print OUTFILE $q->end_html;
> close OUTFILE;
> }
> }
> }
> }
>
> sub toString {
> my $s = shift;
> return '>' . $s->display_id . " " . $s->desc . "\n" . $s->seq;
> }
>
>
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