ProtoGene.pl 60.4 KB
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#!/usr/bin/env perl

########################################################
#                                                      #
# ProtoGene : Bona-Fide Back Translation of Protein    #
#             Multiple Sequence Alignments             #
#                                                      #
########################################################

use strict;
use warnings;
use diagnostics;
use Env qw(HOME);                          # Use only environmental (shell) HOME variable
use File::Basename;
use File::Copy qw(move);                   # Avoid external 'mv' command usage
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use LWP::Simple;                           # To test gigablaster availability
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use lib '/mnt/common/share/ProtoGene/';    # Local path for ProtoGene's own perl modules
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use lib '/mnt/local/lib/tcoffee_perl/';
use Getopt::Long;                          # For options specifications
use File::Which qw(which);                 # Locate external executable programs in the PATH
#use Time::Format qw(%time);               # Use local time for part of pseudo-uniq temp file name
use Mail::Send;                            # Send warnings and errors files by e-mail ==> only if the $userEMail variable is defined

use loci_from_Exonerate;                   # Exonerate parser
#use output_checker;                       # Check output for cds consistancy with query



############## Specific to our server
#L'installer doit creer $HOME/.PACMAN/ and $HOME/.PACMAN/.perllib/
#et mettre les differents modules dans $HOME/.PACMAN/.perllib/
$ENV{'PATH'} .= ':/mnt/local/bin/'; # additional path for executable on the server
##############



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my $Version  = '3.2.3';
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my $uct      = 15;                                     # UpdateCacheThreshold: number of days before update
my $cachedir = '/scratch/frt/tcoffee/ProtoGene_Cache'; # Cache directory
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##### User settings ####################################
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my $userEMail    = 'moretti.sebastien@gmail.com';      # To receive an e-mail with encountered problems; leave blank to inactive this option
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my $webblast_exe = which('webblast.pl')   || '';       #
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my $blast_prog   = 'blastall';                         # Or wu-blastall for Wu-BLAST; for local blast usage
my $exonerate    = which('exonerate-1.0') || '';       #
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########################################################
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my $blast_exe = which($blast_prog) || '';
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my $doc       = which('perldoc')   || '';
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#my $date = $time{'yy-mm-dd_hh\hmm-ss.mmm'};   #Used to get a unique file
my $date  = `/usr/bin/env date +%y-%m-%d_%T`; #Used to get a unique file
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chomp($date);
$date =~ s/\:/\-/g;

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&checkProgramsPresence($webblast_exe, $exonerate); # Check external programs presence in the PATH
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$blast_exe =~ s/${blast_prog}$//;

&checkCacheAccessibility($cachedir); # Check cache directory accessibility



##Options management
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my ($msa, $species, $db, $athome, $revtrans, $pep, $hideBOJ)   = ('', 'All_organisms', 'refseq_protein', 0, 0, 0, 0);
my ($version, $help, $run_name, $Cache, $template, $lim, $tmp) = (0, 0, '', 'update', '', 0, 0);
my $giga = 0;
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GetOptions("msa|in=s"       => \$msa,        #Input sequences
           "orgm|species=s" => \$species,    #Organism(s) to blast against
           "db|database=s"  => \$db,         #Database to blast against
           "local"          => \$athome,     #Use to specify a local db query, definied in $local_db
           "giga"           => \$giga,       #Use GigaBlaster server
           "revtrans"       => \$revtrans,   #Use to reverse-translate sequences with no match
           "pep"            => \$pep,        #Add the original peptide query beneath the linked nt seq
           "version|v"      => \$version,    #Print version information
           "help|h"         => \$help,       #Print full help message
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           "run_name=s"     => \$run_name, #Use another name, instead of input seq name, for result files
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           "cache=s"        => \$Cache,      #Specify what cache parameter
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           "hideBOJ"        => \$hideBOJ,
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           "template=s"     => \$template,   #Use a template file
           "lim=i"          => \$lim,        #Limite number of input query sequences
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           "tmp"            => \$tmp,        #To keep traces of fake intermediate files like fake xml from NCBI, fake aln, ...
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           );
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$athome = 2 if ( $giga==1 );
if ( $version==1 ){
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    print "\n\tPROTOGENE : Bona-Fide Back Translation of Protein Multiple Sequence Alignments\n\tversion   : $Version\n\n";
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    exit 0;
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}
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if ( $help==1 ){
    if ( $doc ne '' ){
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        system("$doc $0");
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    }
    else{
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        print {*STDERR} "\n\tperldoc command doesn't seem to be available\n\tto see the documentation in pod format\n\n";
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    }
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    exit(1);
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}
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if ( $blast_exe eq '' && $version==0 && $help==0 ){
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    print {*STDERR} "\n\t$blast_prog program is not reachable\n\tIt could not be in your PATH or not installed\n\tIt is required only for local blast searches\n\n";
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}

#Cache management
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my $cache = &cacheManagement($Cache) || '.';
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#Open and check template file
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my ($templG, $templP, $templSeq);
($templG, $templP, $templSeq) = &checkTemplate($template) if ($template);
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#Short help message
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if ( $msa eq '' || $species eq '' || $db eq '' || $cache eq '' ){
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    my $appli = basename($0);
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    print {*STDERR} "\n\tCannot open the MSA file in fasta format
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\tTry:  $appli --msa=path_of_the_fasta_msa_file [Options]

\tOptions: --orgm=All_organisms, Bacteria, Viruses, Vertebrata,
\t                Eukaryota, Mammalia, Primates, Homo_sapiens,
\t                Gallus_gallus, Bos_taurus, Escherichia_coli,
\t                Arabidopsis_thaliana, Mus_musculus,
\t                Drosophila_Melanogaster, ...
\t                default is 'All_organisms'
\t         --db=nr, pdb, swissprot, refseq_protein
\t                default is 'refseq_protein'
\t         --local to execute a local BLAST query with
\t                --db=path_for_a_local_db_blast_formated
\t         --template to provide your own nucleotidic sequences
\t                following the cds file format
\t         --revtrans to reverse-translate sequences with no
\t                blast hit, in IUB (IUPAC) depiction code
\t                They are removed from the alignement by default
\t         --pep  to add the original peptide query beneath the
\t                back-translated sequence
\t         --cache=none, update, use, own_PATH_directory, old, empty
\t                to select the cache mode
\t                default is 'update'
\t         --version to print version information
\t         --help to print a full help message\n\n";
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    exit(1);
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}



##Open and Check the fasta file
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my $originalMSA   = $msa;
my $change        = 0;
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($msa, $change)   = &checkFastaFile($msa, $change);
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my $fasta_checker = -1;
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my (@input_order, @original_names, @original_seq); #Use 3 lists in parallel : original_names, seq, order(1->n)
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open( my $MSA, '<', "$msa" ) if ( -e "$msa" );
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while(<$MSA>){
    if ( $_ =~ /^>/ ){
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        $fasta_checker++;
        my $name = $_;
        $name    =~ s/\r\n//g; #Remove return lines from windows OS '^M'
        $name    =~ s/^>[ \t]+/>/;
        chomp($name);
        @original_names = (@original_names, $name);
        @input_order    = (@input_order, $fasta_checker);
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    }
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    elsif ( $_ !~ /^>/ ){
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        my $seqq = $_;
        $seqq    =~ s/\r\n//g;
        $seqq    =~ s/\./-/g; #for msa with '.' as gap
        $seqq    =~ s/[^A-Za-z\-\*\n\r]//g; #Remove all the non-gap or non-alphabetic characters from the seq
        chomp($seqq);
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        #fasta sequence on 1 line
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        if ( !exists($original_seq[$fasta_checker]) ){
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            @original_seq = (@original_seq, $seqq);
        }
        else {
            $original_seq[$fasta_checker] = $original_seq[$fasta_checker].$seqq;
        }
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    }
}
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close $MSA;
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if ( $fasta_checker == -1 ){
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    &failure;
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    print {*STDERR} "\tThe MSA file does NOT seem to be a protein fasta format\n\n";
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    exit(1);
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}
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elsif ( $lim >0 && $fasta_checker > $lim ){
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    &failure;
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    print {*STDERR} "\tThe fasta file is too large, try with less than $lim sequences\n\n";
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    exit(1);
}
elsif ( exists( $original_seq[0] ) && $original_seq[0] =~ /[acgtu]/i){
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    my $first_seq = $original_seq[0];
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    #Check if sequences are amino acids and not nucleotides
    my ( $a, $c, $g, $t, $non ) = ( 0, 0, 0, 0, 0 );
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    $a   = ($first_seq =~ s/[aA]//g);
    $c   = ($first_seq =~ s/[cC]//g);
    $g   = ($first_seq =~ s/[gG]//g);
    $t   = ($first_seq =~ s/[tTuU]//g);
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    $non = ($first_seq =~ s/[^aAcCgGtTuUNn-]//g);
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    if ( ($a+$c+$g+$t) >= (($a+$c+$g+$t+$non)*80/100) ){
        &failure();
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        print {*STDERR} "\tYour sequences seem already to be nucleotides\n\tthis program purpose is to turn AMINO ACID alignments into CDS nucleotide alignments\n\n";
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        exit(1);
    }
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}
undef $fasta_checker;


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#Check GigaBlaster status if used
if ( $giga==1 ){
    if ( head('http://www.igs.cnrs-mrs.fr/Giga2/~database/remoteblast.cgi') ){
    }
    else{
        $athome = 0;
    }
}

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#Start main program with version # of programs and list original queries
print "\n\t   Protogene\t$Version\t$date\n\n";
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open(my $EXONERATEISHERE, "$exonerate --version |");
my $IsExonerateHere = <$EXONERATEISHERE>;
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close $EXONERATEISHERE;
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print "\t   $IsExonerateHere\n";
undef $IsExonerateHere;
undef $Version;
for(my $m=0; $m<=$#original_names; $m++){
    print "\t$original_names[$m]\n";
}
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$date .= "_$$"; #Add PID
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# run_name implementation for web server
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$originalMSA = $run_name if ( $run_name ne '' );
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unlink("${originalMSA}.cds");
unlink("${originalMSA}.cdsP");
unlink("${originalMSA}.out");
unlink("${originalMSA}.boj");



# Remove old files from the cache directory
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&cacheManagement('old', '1'); #everytime ProtoGene is running
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##Build the sequences, from the alignment, to perform webblast
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my $quiet = '';
EACH_SEQ:
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for(my $r=0; $r<=$#input_order; $r++){
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    my $right_name = $original_names[$r];
    my $templ_name = $original_names[$r];
    $right_name    =~ s/^>//;
    $templ_name    =~ s/^([^\s\t]+).*$/$1/;
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    open(my $READY2BLAST, '>', "$cache/${date}_seq2blast.fas${r}");
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    my $noGap = $original_seq[$r];
    $noGap    =~ s/[\.-]//g; #remove gaps
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    print {$READY2BLAST} ">$input_order[$r]\n$noGap\n";
    close $READY2BLAST;
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    undef $noGap;


    my @multipleequalblasthits;
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    if ( exists($templG->{$templ_name}) && $templG->{$templ_name} ne '' ){
        @multipleequalblasthits = (@multipleequalblasthits, 'My_own_seq'); #To change for ?
        print "\n\n$r done\n\nNucleotidic template for $r:\t$templG->{$templ_name}\n\n**********************************************************************\n\n";
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    }
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    elsif ( exists($templP->{$templ_name}) && $templP->{$templ_name} ne '' ){
        @multipleequalblasthits = (@multipleequalblasthits, $templP->{$templ_name});
        print "\n\n$r done\n\nBlastP template for $r:\t$templP->{$templ_name}\n\n**********************************************************************\n\n";
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    }
    else{
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    ##   --> send them to webblast
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        $quiet = '-quiet=on ' if ( $r>0 ); #Show webblast parameters only for the first webblast run
        &launchWebblast("$cache/${date}_seq2blast.fas${r}", $quiet, $athome, $db, $species, $blast_exe, "$cache/${date}.blastp${r}");
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    ##Get the BLASTp hit(s)  acc number
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        open(my $BLASTP, '<', "${cache}/${date}.blastp${r}");
        while(<$BLASTP>){
            @multipleequalblasthits = (@multipleequalblasthits, $1) if ( $_ =~ /^>\s*\d+@?\w?\w?\w?__([^\s\|\.]+).*$/ ); #Warning: double '_'
        }
        close $BLASTP;
        unlink("$cache/${date}.blastp${r}", "${cache}/${date}_seq2blast.fas${r}") if ( $tmp == 0 || exists($multipleequalblasthits[0]) );
        if ( !exists($multipleequalblasthits[0]) ){
            print "\n$input_order[$r] ...\tNo blast result found above filter thresholds for $right_name\n\n";
            &buildFailureOutputFiles($r, 'No_BLASTp_Result', 'Unavailable', '');
            undef $original_seq[$r];
            undef $original_names[$r];
            undef $input_order[$r];
            next EACH_SEQ;
        }
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    }



    #When multiple equal blast hits  ==> use, and add, every hits
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    my ($resultPOS, $resultBOJ) = ('', '');
    my @failureStatus = ('');
    my @intronStatus  = ('');
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    for(my $qq=0; $qq<=$#multipleequalblasthits; $qq++){
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        print "\n${right_name}  -->  [${multipleequalblasthits[$qq]}]\n";


        my @nt_GIs;
        my $intronStep = 0;
        if ( exists($templG->{$templ_name}) && $templG->{$templ_name} ne '' && $templG->{$templ_name} !~ /^My_Seq$/i ){
            &downloadSeq($cache, $date, '', '', $templG->{$templ_name});
            @nt_GIs = $templG->{$templ_name};
        }
        elsif ( exists($templG->{$templ_name}) && $templG->{$templ_name} ne '' && $templG->{$templ_name} =~ /^My_Seq$/i ){
            open(my $MYSEQ, '>', "$cache/My_Seq-$$.fas");
            print {$MYSEQ} ">My_Seq-$$ for $templ_name\n".$templSeq->{$templ_name}."\n";
            close $MYSEQ;
            @nt_GIs = "My_Seq-$$";
        }
        else{
        #Prot ACC -> PUID
            my $protGI = &blastPAcc2PGI($multipleequalblasthits[$qq]);
            if ( $protGI eq '' ){
                print "\tNo protein (prot GI) link found for ${multipleequalblasthits[$qq]} in $right_name\n\n";
                @failureStatus = ('PUI_unavailable', ${multipleequalblasthits[$qq]});
                next;
            }
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#           print "\t$protGI\n";

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        #PUID -> nt GIs & GeneID
            my ($ntGIs, $geneID) = &protGI2NTGIs($protGI, $multipleequalblasthits[$qq]);
            if ( $ntGIs eq '' && $geneID eq '' ){
                print "\tNo nucleotide (nt GIs) link found for ${multipleequalblasthits[$qq]} in $right_name\n\n";
                @failureStatus = ('No_nt_link', ${multipleequalblasthits[$qq]});
                next;
            }
            print " linked with [$ntGIs $geneID]\n";


        #Get transcript and contig seq
            @nt_GIs = split(/,/, $ntGIs);
            &downloadSeqFromGIs($cache, $date, '', '', @nt_GIs);
        #GeneID -> Chr
            my @geneIDs = split(/,/, $geneID);
            while(<@geneIDs>){
                my $geneID = $_;
                my ($chr, $amont, $aval) = &geneID2Chr($geneID, $multipleequalblasthits[$qq]);
                print "\n\tNo gene locus found for ${multipleequalblasthits[$qq]} with $geneID in $right_name\n\n" if ($chr eq '' and $geneID ne '');

                if ( $chr ne '' ){
            #Get Chr seq
                    &downloadSeq($cache, $date, $amont, $aval, $chr);
                    $intronStep = 1;

                    @nt_GIs = ("$chr:$amont-$aval", @nt_GIs);
                }
            }
        }


    #Align Nt seq with our protein query seq
        my ($POS, $BOJ) = &runExonerate($exonerate, $cache, $date, ${input_order[$r]}, ${original_seq[$r]}, $right_name, $multipleequalblasthits[$qq], $r, @nt_GIs);

        my ($POSresult, $bestNucleotide) = &prepareResults4CDS($POS, $multipleequalblasthits[$qq], ${original_seq[$r]}, $right_name);
        $resultPOS .= $POSresult if ( $bestNucleotide ne '' && $POSresult ne '' && $resultPOS !~ /_G_$bestNucleotide/ );

        @intronStatus = ($POS->{0}, ${multipleequalblasthits[$qq]}, '')              if ( $intronStep==1 && exists($POS->{0}) && $POS->{0} ne '' && $POS->{0} =~ /\:/ );
        @intronStatus = ($POS->{0}, ${multipleequalblasthits[$qq]}, $bestNucleotide) if ( $intronStep==0 && ($bestNucleotide =~ /^N[CTWZG]_/ || $bestNucleotide =~ /^AC_/) );

        my ($intronLess, $nameLess) = ('', '');
        $intronLess = $POS->{0}       if ( %$BOJ eq 0 && exists($POS->{0}) && $POS->{0} ne '' );
        $nameLess   = $bestNucleotide if ( %$BOJ eq 0 && $intronStep==0 && ($bestNucleotide =~ /^N[CTWZG]_/ || $bestNucleotide =~ /^AC_/) );
        my ($BOJresult, $bestGenomic) = &prepareResults4BOJ($BOJ, $multipleequalblasthits[$qq], ${original_seq[$r]}, $right_name, $intronLess, $nameLess);
        $resultBOJ .= $BOJresult      if ( $bestGenomic ne '' && $BOJresult ne '' && $resultBOJ !~ /_G_$bestGenomic[ \-]/ );
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#    print " $intronStep $POS->{0} [@intronStatus] {$BOJresult} $bestNucleotide - $bestGenomic \t $BOJ->{0}\n";
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    }


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    if ( $resultPOS eq '' ){
        &buildFailureOutputFiles($r, $failureStatus[1], $failureStatus[0], '')     if ( $failureStatus[0] ne '' );
        &buildFailureOutputFiles($r, $multipleequalblasthits[0], 'Failed_Aln', '') if ( $failureStatus[0] eq '' );
        next;
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    }
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    elsif ( $resultBOJ eq '' ){
        if ( $failureStatus[0] eq 'PUI_unavailable' || $failureStatus[0] eq 'No_nt_link' ){
            &buildFailureBOJOutputFile($failureStatus[1], $failureStatus[0], $right_name, ${original_seq[$r]});
        }
        elsif ( $failureStatus[0] eq '' && $intronStatus[0] ne '' ){
            &buildIntronlessBOJOutputFile($intronStatus[1], $intronStatus[0], $intronStatus[2], $right_name, ${original_seq[$r]});
        }
        else{
            &buildFailureBOJOutputFile($multipleequalblasthits[0], 'Unavailable', $right_name, ${original_seq[$r]});
        }
        &createCDSOutputFile($resultPOS, ${original_seq[$r]}, $right_name);
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    }
    else {
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        &createCDSOutputFile($resultPOS, ${original_seq[$r]}, $right_name);
        &createBOJOutputFile($resultBOJ, ${original_seq[$r]});
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    }


    undef $original_seq[$r];
    undef $original_names[$r];
    undef $input_order[$r];
}


&checkAndCleanStderrFiles("$cache/${date}_ExonerateError") if ( -e "$cache/${date}_ExonerateError" );
&checkOutputFiles();
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unlink("$msa") if ( $change==1 );
my @tmpFiles = glob($cache."/${date}*");
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if ( exists($tmpFiles[0]) ){
    mkdir("$cache/${date}_TMP");
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    move("$_", "$cache/${date}_TMP") while(<@tmpFiles>);
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}


#Ouput Checker


#STDOUT Log output for our server
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if ( -s "${originalMSA}.cds" || -s "${originalMSA}.out"){
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    print "\n\nTERMINATION STATUS: SUCCESS\n";
    print "OUTPUT RESULTS\n";
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    print "    #### File Type=        MSA Format= fasta_CDS Name= ${originalMSA}.cds\n" if ( -s "${originalMSA}.cds");
    print "    #### File Type=        MSA Format= fasta_CDS+Query Name= ${originalMSA}.cdsP\n" if ( -s "${originalMSA}.cdsP");
    print "    #### File Type=        MSA Format= Rejected_seq Name= ${originalMSA}.out\n" if ( -s "${originalMSA}.out");
    print "    #### File Type=        MSA Format= fasta_Exon-Boundaries Name= ${originalMSA}.boj\n" if ( -s "${originalMSA}.boj" && -s "${originalMSA}.cds" && $hideBOJ==0);
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}
else{
    &failure;
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    exit(1);
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}

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exit(0);
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=head1 NAME

ProtoGene.pl - Converts a peptidic alignment to a nucleotidic alignment through database search

=head1 SYNOPSIS

B<ProtoGene.pl> --msa=path_of_the_fasta_msa_file [options]

with these options available:

=over 4

=item I<--orgm>=targeted species in the database search

=item All_organisms [default], Bacteria, Viruses, Vertebrata, Eukaryota, Mammalia, Primates, Homo_sapiens, Gallus_gallus, Bos_taurus, Escherichia_coli, Arabidopsis_thaliana, Mus_musculus, Drosophila_Melanogaster, ...

=item I<--db>=targeted database in the database search

=item nr, pdb, swissprot, refseq_protein [default]

=item I<--local> to execute a local BLAST query with  --db=path_for_a_local_db_blast_formated

=item I<--template> to provide your own nucleotidic sequences following the cds file format

=item I<--revtrans> to reverse-translate sequences with no blast hit, in IUB (IUPAC) depiction code

=item They are removed from the alignement by default

=item I<--pep> to add the original peptide query beneath the back-translated sequence

=item I<--cache>=none, update, use, own_path_directory, old, empty

=item none: no cache usage, none temporary files are stored

=item update: use cache but update old files [default]

=item use: force use cache, whatever the age of files

=item own_path_directory: use my own directory, and its files

=item old: remove the old files in the cache directory

=item empty: empty the whole cache directory $HOME/.ProtoGene/

=item I<--version> to print version information

=item I<--help> to print this help message

=back

=head1 DESCRIPTION

PROTOGENE : Bona-Fide Back Translation of Protein Multiple Sequence Alignments
It converts a peptidic alignment to a nucleotidic alignment through database search.
Blast queries allow to get nucleotidic sequences from where peptidic sequences come from.
Exonerate aligns nucleotidic and peptidic sequences together.
PROTOGENE re-builds the original alignment with nucleotidic information it has gotten.

=head1 REQUIREMENT

=over 4

=item B<Perl 5.6 or better> is required !

=item Standard Perl modules B<lib>, B<strict>,  B<warnings>,  B<diagnostics>, B<Env> are required

=item and some other current ones : B<Getopt::Long>,  B<File::Which>,  B<File::Copy>, B<Mail::Send>

=item -

=item I<exonerate> from http://www.ebi.ac.uk/~guy/exonerate/

=item I<blast> from http://www.ncbi.nlm.nih.gov/BLAST/download.shtml or http://blast.wustl.edu/

=back

=head1 VERSION

=over 8

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=item version 3.2.3
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=item on Nov 1st, 2007
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=back

=head1 AUTHORS

=over 8

=item Sebastien MORETTI

=item Sebastien.Moretti@igs.cnrs-mrs.fr

=item Frederic REINIER

=item Frederic.Reinier@igs.cnrs-mrs.fr

=item Lab. Information Genomique et Structurale - IGS

=item CNRS - Life Sciences

=item Marseille, France

=item http://www.igs.cnrs-mrs.fr/

=back

=cut


######################  Management  ######################

#Failure declaration
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sub failure{
    print {*STDERR} "\n\nFATAL\n\nTERMINATION STATUS: FAILURE\n\n";
    return;
}
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#Check external programs presence in the PATH
sub checkProgramsPresence{
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    my ($webblast_exe, $exonerate) = @_;
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    if ( $webblast_exe eq '' ){
        &failure;
        print {*STDERR} "\twebblast.pl program is not reachable\n\tIt could not be in your PATH or not installed\n\n";
        exit(1);
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    }
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    if ( $exonerate eq '' ){
        &failure;
        print {*STDERR} "\texonerate program is not reachable\n\tIt could not be in your PATH or not installed\n\n";
        exit(1);
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    }
}

#Check cache directory accessibility: Test and change rights, if needs, of the $HOME/.ProtoGene/ cache directory
sub checkCacheAccessibility{
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    my ($cachedir) = @_;
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    if ( -d "$cachedir/" && -w "$cachedir/" && -x "$cachedir/" && -r "$cachedir/" ){
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    }
    elsif ( -d "$cachedir/" ){
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        if ( -o "$cachedir/" ){
            chmod 0754, "$cachedir/";
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        }
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        else{
            print {*STDERR} "\n\t** Warning ** :\n\tPermissions do NOT seem to be valide for the current cache directory\n\n";
        }
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    }
    else{
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        mkdir "$cachedir/";
        if ( -o "$cachedir/" ){
            chmod 0754, "$cachedir/"; #0754 est en octal, donc 754 ou '0754' ne sont pas bons !
        }
        else{
            print {*STDERR} "\n\t** Warning ** :\n\tPermissions do NOT seem to be valide for the current cache directory\n\n";
        }
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    }
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    return;
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}

#Cache Management
sub cacheManagement{
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    my ($Cache, $inScript) = @_;
    $inScript = 0 if ( !defined $inScript );

    if ( $Cache eq 'empty' ){
        my @temp_files = glob($cachedir.'/*.fas');
        if ( exists ($temp_files[0]) ){
            unlink $_ while(<@temp_files>);
        }
        @temp_files = glob($cachedir.'/*_ExonerateError');
        if ( exists ($temp_files[0]) ){
            unlink $_ while(<@temp_files>);
        }
        unlink("$cachedir/webblast.log", "$cachedir/web_tempo.result", "$cachedir/debug.tempo");
        print "\n\tcache directory [$cachedir] is empty\n\n";
        exit 0;
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    }
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    elsif ( $Cache eq 'old' ){
        my @temp_files = glob($cachedir.'/*.fas');
        my @tempFiles  = grep { -M $_ > $uct } @temp_files; #Sort list only with $uct days old files
        if ( exists ($tempFiles[0]) ){
            unlink $_ while(<@tempFiles>);
        }
        print "\n\tcache directory [$cachedir] is empty of its old files\n\n" if ($inScript==0);
        exit 0 if ( $inScript==0 );
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    }
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    elsif ( $Cache eq 'update' || $Cache eq 'use' ){
        $cache = $cachedir; #Limit cache files to less than $uct days old ones for 'update'
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    }
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    elsif ( $Cache eq 'none' ){
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    }
    elsif ( -d $Cache ){
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        die "\n\tPACMAN cannot access to your directory\n\n"  if ( ! -x $Cache );
        die "\n\tPACMAN cannot write into your directory\n\n" if ( ! -w $Cache );
        die "\n\tPACMAN cannot read into your directory\n\n"  if ( ! -r $Cache );
        $cache = $Cache;
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    }
    else {
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        die "\n\tWrong cache argument, or your cache folder doesn't seem to exist\n\n";
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    }

    return($cache);
}

##########################################################


######################    Fasta    ######################

#Check input aln
sub checkFastaFile{
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    my ($msa, $change) = @_;
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    if ( -e "$msa" ){
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    #Convert no fasta format to fasta with seq_reformat if available
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        my $seqreformat = which('t_coffeeX') || ''; #desactive tant que t_coffee seq_reformat pose prob
        if ( $seqreformat ne '' ){
            my @formatType = `$seqreformat -other_pg seq_reformat -in $msa -print_format`;
            my $format     = '';
            while(<@formatType>){
                if ( $_ =~ /FORMAT:/ ){
                    $format = $_;
                    chomp($format);
                    last;
                }
            }
            if ( $format ne '' && $format !~ /FORMAT:fasta_/ ){
                system("$seqreformat -other_pg seq_reformat -in $msa -output fasta_aln -out=${msa}.007") if ($format =~ /_aln *$/);
                system("$seqreformat -other_pg seq_reformat -in $msa -output fasta_seq -out=${msa}.007") if ($format =~ /_seq *$/);
                $msa    .= '.007';
                $change  = 1;
            }
        }
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    }
    else {
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        &failure;
        print {*STDERR} "\tThe file \'$msa\' doesn't not seem to be reachable\n\n";
        exit(1);
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    }

    return($msa,$change);
}

#Prepare Fasta Header of the query name to incorporate our annotations
sub fastaHeaders4ProtoGene{
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    my ($QueryName) = @_;
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    $QueryName =~ s/  +/ /g;
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    my ($cqacc, $cqdesc) = ($QueryName, $QueryName);
    $cqacc  =~ s/^ *([^ ]*) .*$/$1/;
    $cqacc  =~ s/[\s\t]*$//g;
    $cqacc  =  $cqacc.'_G_@@';
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    $cqdesc =~ s/^ *[^ ]* *(.*)$/$1/;
    $cqdesc =~ s/^[\| \.]*//;
    $cqdesc =~ s/[\|\. ]*$//;

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    $QueryName = $cqacc.$cqdesc;
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    return($QueryName);
}

#Prepare Fasta Header to put it on the fasta header of the result outputs
sub getFastaHeaderAnnot{
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    my ($bestTarget) = @_;
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    #Get annotation from nucleotide file header
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    open(my $BEST, '<', "$cache/${bestTarget}.fas");
    my $annot=<$BEST> || '';
    close $BEST;
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    chomp($annot);
    $annot =~ s/^ *[^ ]* //;
    $annot =~ s/[\. ]*$//;
    $annot =~ s/^ *//;
    $annot =~ s/  +/ /g;

    return($annot);
}

#########################################################


###################### Translation ######################

#Direct Reverse translation
sub reverse_trad{
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    my ($aa) = @_;
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    my %reverse_code=('A' => 'GCN', 'F' => 'TTy', 'K' => 'AAr', 'P' => 'CCN', 'T' => 'ACN',
                      'C' => 'TGy', 'G' => 'GGN', 'L' => 'yTN', 'Q' => 'CAr', 'V' => 'GTN',
                      'D' => 'GAy', 'H' => 'CAy', 'M' => 'ATG', 'R' => 'mGN', 'W' => 'TGG',
                      'E' => 'GAr', 'I' => 'ATh', 'N' => 'AAy', 'S' => 'wsN', 'Y' => 'TAy',
                      'X' => 'NNN', '-' => '---', '*' => 'Trr'); # '*' is for stop codon

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    my $triplet = 'nnn';
    while( my ($x,$y) =each(%reverse_code) ){
        if ( uc($aa) eq $x ){
            $triplet = $y;
            last;
        }
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    }

    return($triplet);
}

#########################################################


####################### webblast #######################
sub launchWebblast{
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    my ($infile, $quiet, $whatBlast, $db, $species, $blast_exe, $outfile) = @_;
    my $blast_at = '';
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    $blast_at    = "-database=$db"                         if ( $whatBlast==0 );
    $blast_at    = "-database=$db -blast_dir=${blast_exe}" if ( $whatBlast==1 );
    $blast_at    = "-database=$db -gigablast=yes"          if ( $whatBlast==2 );
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#    print "             Evalue treshold : 0.05
#             Matrix : BLOSUM62
#             Filter : F
#             **********************
#             For RefSeq Protein db:
#             Blast_identity_threshold : 100
#             Cover threshold : 100
#             **********************
#             For NR Protein db:
#             Blast_identity_threshold : 95
#             Cover threshold : 95";

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    system("$webblast_exe -program=blastp ${blast_at} -infile=$infile -matrix=BLOSUM62 -evalue=0.05 -method=geneid -filter=Off -organism=$species -identity=100 -cover=100 -hits=10 $quiet -outfile=$outfile");
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    if ( -z "$outfile" ){ #Another blastP query if no hit with refseq and db was not nr !
        $quiet = '-quiet=on';
        print {*STDERR} "run BLAST.. against NR because there was no acceptable hit against $db\n"
            if ( $whatBlast != 1 && $db ne 'nr' );
        print {*STDERR} "run BLAST.. again, with decreased thresholds, because there was no acceptable hit against $db\n"
            if ( $whatBlast == 1 || $db eq 'nr' );
        $blast_at =~ s/-database=[^\s]*/-database=nr/ if ( $whatBlast != 1 );
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        system("$webblast_exe -program=blastp ${blast_at} -infile=$infile -matrix=BLOSUM62 -evalue=0.05 -method=geneid -filter=Off -organism=$species -identity=95 -cover=95 -hits=10 $quiet -outfile=$outfile");
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    }

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    move('webblast.log',     "$cache"); #Move temporary webblast.pl files
    move('web_tempo.result', "$cache");
    move('debug.tempo',      "$cache");

    unlink("$cache/webblast.log")     if ( -z "$cache/webblast.log" );
    unlink("$cache/web_tempo.result") if ( -z "$cache/web_tempo.result" );
    unlink("$cache/debug.tempo")      if ( -z "$cache/debug.tempo" );
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    return;
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}


########################################################


##################### NCBI requests #####################

#Prot ACC -> PUID
sub blastPAcc2PGI{
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    my ($blastHit) = @_;
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    my $protGI = '';
    my $count  = 0;
    GET_PUI:
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    for(my $rep=0;$rep <= 4; $rep++){
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        $count++;
        system("wget -q -O $cache/${date}_${blastHit}protGi.tmp 'http://eutils.ncbi.nlm.nih.gov/entrez/eutils/esearch.fcgi?db=protein&term=${blastHit}[pacc]&retmode=xml'");
        open(my $GIP, '<', "$cache/${date}_${blastHit}protGi.tmp");
        PROT_GI:
        while(<$GIP>){
            if ( $_ =~ /^.*\<Id\>(\d+)\<\/Id\>.*$/ ){
                $protGI = $1;
                last PROT_GI; #OK if only one PUID
            }
        }
        close $GIP;
        $rep = $rep-15 if ( -z "$cache/${date}_${blastHit}protGi.tmp" && $count==1 );
        last GET_PUI if ( $protGI =~ /^\d+$/ );
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    }
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    unlink("$cache/${date}_${blastHit}protGi.tmp") if ( $tmp == 0 || $protGI ne '' );
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    return($protGI);
}

#PUID -> nt GIs
sub protGI2NTGIs{
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    my ($protGI, $blastHit) = @_;
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    my $ntGIs  = '';
    my $geneID = '';
    my $count  = 0;
    GET_NTUI:
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    for(my $rep=0;$rep <= 4; $rep++){
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        $count++;
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        #nt GIs
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        system("wget -q -O $cache/${date}_${blastHit}nucleo.tmp 'http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=protein&db=nuccore,nucleotide,gene&id=$protGI&retmode=xml'");
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        open(my $GIN, '<', "$cache/${date}_${blastHit}nucleo.tmp");
        my $flag = 0;
        NT_GI:
        while(<$GIN>){
            if ( $_   =~ /<LinkName>protein_nuc[a-z]+<\/LinkName>/ ){ #for nuccleotide and nuccore
                $flag = 1;
            }
            elsif ( $_ =~ /\<LinkName\>protein_gene\<\/LinkName\>/ ){
                $flag  = 2;
            }
            elsif ( $flag==1 && $_ =~ /^.*\<Id\>(\d+)\<\/Id\>.*$/ ){
                $ntGIs .= ",$1" if ( $ntGIs ne '' );
                $ntGIs  = $1    if ( $ntGIs eq '' );
            }
            elsif ( $flag==2 && $_ =~ /^.*\<Id\>(\d+)\<\/Id\>.*$/ ){
                $geneID .= ",$1" if ( $geneID ne '' );
                $geneID  =$1     if ( $geneID eq '' );
            }
        }
        close $GIN;
        $rep = $rep-15 if ( -z "$cache/${date}_${blastHit}nucleo.tmp" && $count==1 );
        last GET_NTUI if ( $ntGIs ne '' || $geneID ne '' );
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    }
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    unlink("$cache/${date}_${blastHit}nucleo.tmp") if ( $tmp==0 || ($ntGIs ne '' || $geneID ne '') );
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    return($ntGIs, $geneID);
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}

sub geneID2Chr{
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    my ($geneID, $blastHit) = @_;
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    my $chr   = '';
    my ($amont, $aval) = ('', '');
    my $count = 0;
    GET_CHR:
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    for(my $rep=0;$rep <= 8; $rep++){
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        $count++;
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        #Gene Acc
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        system("wget -q -O $cache/${date}_${blastHit}gene.tmp 'http://eutils.ncbi.nlm.nih.gov/entrez/eutils/efetch.fcgi?db=gene&id=$geneID&retmode=xml'");
        open(my $GACC, '<', "$cache/${date}_${blastHit}gene.tmp");
        my $flag = 0;
        GENE_CHR:
        while(<$GACC>){
            if ( $_  =~ /\<ERROR\>Empty id list \- nothing todo\<\/ERROR\>/ && $flag==0 ){
                $rep = $rep-15 if ( $count==1 );
            }
            if ( $_   =~ /\<Entrezgene_locus\>/ && $flag==0 ){
                $flag = 1;
            }
            elsif ( $_ =~ /\<Gene-commentary_type value=\"genomic\"\>/ && $flag==1 ){
                $flag  = 2;
            }
            elsif ( $_ =~ /\<Gene-commentary_type value=/ && $flag==2 ){
                last GENE_CHR;
            }
            elsif ( $_ =~ /\<Gene-commentary_accession\>([\w\_\-\.]+)\<\/Gene-commentary_accession\>/ && $flag==2 ){
                $chr   = $1;
                $flag  = 3;
            }
            elsif ( $_ =~ /\<Seq-interval_from\>(\d+)\<\/Seq-interval_from\>/ && $flag==3 ){
                $amont = $1;
                $flag  = 4;
            }
            elsif ( $_ =~ /\<Seq-interval_to\>(\d+)\<\/Seq-interval_to\>/ && $flag==4 ){
                $aval  = $1;
                last GENE_CHR;
            }
        }
        close $GACC;
        $rep = $rep-15 if ( -z "$cache/${date}_${blastHit}gene.tmp" && $count==1 );
        last GET_CHR   if ( $chr ne '' && $amont =~ /^\d+$/ && $aval =~ /^\d+$/ );
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    }
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    unlink("$cache/${date}_${blastHit}gene.tmp") if ( $tmp == 0 || $chr ne '' );
    if ( $amont eq '' || $aval eq '' ){
        $amont = '';
        $aval = '';
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    }
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    elsif ( $amont ne '' && $aval ne '' ){
        if ( $amont > $aval ){
            $amont = $amont+5000;
            $aval  = $aval-5000;
        }
        else{
            $amont = $amont-5000;
            $aval  = $aval+5000;
        }
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    }
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    $amont = 1 if ( $amont ne '' && $amont <= 0 );
    $aval  = 1 if ( $aval ne ''  && $aval <= 0 );
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    return($chr, $amont, $aval);
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}

sub downloadSeqFromGIs{
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    my ($cache, $date, $amont, $aval, @acc) = @_;
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    GET_SEQ:
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    for(my $a=0; $a<=$#acc; $a++){
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        my $whatNumber = 0;
        if ( $amont =~ /^\d+$/ && $aval =~ /^\d+$/ ){
            GET_CHR_SEQ:
            for(my $rep=0;$rep <= 4; $rep++){
                system("wget -q -O $cache/${acc[$a]}-${amont}.fas 'http://eutils.ncbi.nlm.nih.gov/entrez/eutils/efetch.fcgi?db=nucleotide&id=${acc[$a]}&rettype=fasta&retmode=text&from=$amont&to=$aval'") if ( !-e "$cache/${acc[$a]}-${amont}.fas" || -z "$cache/${acc[$a]}-${amont}.fas" || $Cache eq 'none' || ($Cache eq 'update' && -M "$cache/${acc[$a]}-${amont}.fas" > $uct ) );
                open(my $CIBLE, '<', "$cache/${acc[$a]}-${amont}.fas");
                my $counter = 0;
                my $lines   = 0;
                CHECK_CHR_SEQ:
                while(<$CIBLE>){
                    $lines++              if ( $_ !~ /^>/ );
                    $counter = $counter+2 if ( $counter==1 && $_ !~ /^\w/ && $lines==1 && $_ !~ /^>/ );
                    $counter++            if ( $_ =~ /^>/ );
                    $counter = $counter+2 if ( $_ !~ /^>/ && ($_ =~ /Error:/ || $_ =~ /[<>]/) );
#                   last CHECK_CHR_SEQ    if ( $_ !~ /^>/ && $counter==1 && $_ =~ /^\w/ );
                }
                close $CIBLE;
                $whatNumber++;
                last GET_CHR_SEQ if ( $whatNumber==20 );
                if ( $counter != 1 ){
                    $rep = $rep-1;
                    unlink("$cache/${acc[$a]}-${amont}.fas");
                }
            }
        }
        else{
            GET_OTHER_SEQ:
            for(my $rep=0;$rep <= 4; $rep++){
                system("wget -q -O $cache/${acc[$a]}.fas 'http://eutils.ncbi.nlm.nih.gov/entrez/eutils/efetch.fcgi?db=nucleotide&id=${acc[$a]}&rettype=fasta&retmode=text'") if ( !-e "$cache/${acc[$a]}.fas" || -z "$cache/${acc[$a]}.fas" || $Cache eq 'none' || ($Cache eq 'update' && -M "$cache/${acc[$a]}.fas" > $uct ) );
                open(my $CIBLE, '<', "$cache/${acc[$a]}.fas");
                my $counter = 0;
                my $lines   = 0;
                CHECK_OTHER_SEQ:
                while(<$CIBLE>){
                    $lines++              if ( $_ !~ /^>/ );
                    $counter = $counter+2 if ( $counter==1 && $_ !~ /^\w/ && $lines==1 && $_ !~ /^>/ );
                    $counter++            if ( $_ =~ /^>/ );
                    $counter = $counter+2 if ( $_ !~ /^>/ && ($_ =~ /Error:/ || $_ =~ /[<>]/) );
                }
                close $CIBLE;
                $whatNumber++;
                last GET_OTHER_SEQ if ( $whatNumber==20 );
                if ( $counter != 1 ){
                    $rep=$rep-1;
                    unlink("$cache/${acc[$a]}.fas");
                }
            }
        }
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    }
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    return;
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}

sub downloadSeq{
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    my ($cache, $date, $amont, $aval, @acc) = @_;
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    my $cp   = 0;
    my $from = $amont;
    my $to   = $aval;
    DOWNL_SEQ:
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    for(my $a=0; $a<=$#acc; $a++){
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        my $pacc2puid = $acc[$a];
        if ( $pacc2puid !~ /^[NAX][CGTWZM]_/ ){
        #pacc = primary acc NOT prot acc !   #265666 -> S55551
            system("wget -q -O $cache/${date}_${acc[$a]}.gui 'http://eutils.ncbi.nlm.nih.gov/entrez/eutils/esearch.fcgi?&db=nucleotide&term=${pacc2puid}[pacc]'");
            open(my $GUI, '<', "$cache/${date}_${acc[$a]}.gui");
            DOWNL:
            while(<$GUI>){
                if ( $_ =~ /\<Id\>(\d+)\<\/Id\>/ ){
                    $pacc2puid = $1;
                    last DOWNL;
                }
            }
            close $GUI;