[Bioperl-l] Error calling alignment method

[Lorenzo Carretero Paulet]

Hi all,
I'm trying to create the following subroutine but I get the error message:
"Can't call method "alignment" on an undefined value at 
/Users/marioafares/Documents/workspace/PlantEvolGen/test.pl line 80, 
<GEN1> line 2."
which indicates $dna_aln is undefined. However, I manage to print it 
using Dumper, so I guess it is actually defined.
Can anyone see where the problem is?
I attach the two files I'm using in the test.
Thanks in advance,
Lorenzo

    #!/usr/local/bin/perl -w
    use 5.010;
    use strict;
    use Data::Dumper;
    # definition of the environmental variable CLUSTALDIR
    BEGIN {$ENV{CLUSTALDIR} =
    '/Applications/Bioinformatics/clustalw-2.1-macosx/'}
    use Bio::Tools::Run::Alignment::Clustalw;
    use Bio::Align::Utilities qw(aa_to_dna_aln);

    BEGIN {$ENV{PAMLDIR} = '/Applications/Bioinformatics/paml44/bin/'}
    use Bio::Tools::Run::Phylo::PAML::Codeml;

    my $sequencesfilenameAA =
    "/Users/marioafares/Documents/SequenceDatabase/plaza_public_02_Apr27/plaza_public_02/BLAST_Parsed_results/PerSpecies/test_vs_test.par.aa.1.fas";

    my $sequencesfilenameNT =
    "/Users/marioafares/Documents/SequenceDatabase/plaza_public_02_Apr27/plaza_public_02/BLAST_Parsed_results/PerSpecies/test_vs_test.par.nt.1.fas";

    my $format = 'fasta';

    GettingBioperlAlignmentAAtoDNAplusPAMLcalculation
    ($sequencesfilenameAA, $sequencesfilenameNT, $format);


    sub GettingBioperlAlignmentAAtoDNAplusPAMLcalculation
    {
    my ( $sequencesfilenameAA, $sequencesfilenameNT, $format, $ktuple,
    $matrix  ) = @_;
    my %hashNTseqs = ();
    my $likelihood;
    my $Ks;
    my $Ka;
    my $omega;
    my $factory = Bio::Tools::Run::Alignment::Clustalw->new (); #use
    default parameters in here
    my $pep_aln = $factory->align($sequencesfilenameAA);


    my $inseq = Bio::SeqIO->new(-file => "<$sequencesfilenameNT",
                                 -format => $format );
         while (my $seq = $inseq->next_seq)
             {
             my $seq_id = $seq->display_id();
             $hashNTseqs{$seq_id} = $seq;
             }
    my $dna_aln = aa_to_dna_aln($pep_aln, \%hashNTseqs);
    say Dumper \%hashNTseqs;
    say Dumper $dna_aln;
    ###########################################################
    #my $codeml_runs = 5;
    my $ks_factory = new Bio::Tools::Run::Phylo::PAML::Codeml
                           (     -params =>     {
                                               #'verbose' => 0,
                                               #'noisy' => 0,
                                               'runmode' => -2,
                                              'seqtype' => 1,
                                              #'model' => 0,
                                              #'NSsites' => 0,
                                              #'icode' => 0,
                                              #'fix_alpha' => 0,
                                              #'fix_kappa' => 0,
                                              #'RateAncestor' => 0,
                                             'CodonFreq' => 2,
                                             'cleandata' => 1,
                                             'ndata' => 1
                                            },

                           );

    my $kaks_factory->alignment($dna_aln);

    say "\nCalculating Ks-values of current cluster ...";

         # The estimation of the Ks-values is repeated $codeml_runs
    times ...
    #    for(my $k=1;$k <= $codeml_runs;$k++)
    #    {
    #        print "\nCodeml-Run $k:\n\n";

             # Ka and Ks-vlaues are calculated using codeml
             my ($rc,$parser) = $kaks_factory->run();
             #$kaks_factory->cleanup();
             # If the calculation was succsessful ...
    #        if($rc != 0)
    #        {
                 my $result = $parser->next_result;#not sure what it
    does here
                 #my $NGmatrix = $result->get_MLmatrix();
                 my $MLmatrix = $result->get_MLmatrix();
                     $likelihood = $MLmatrix->[0]->[1]->{'lnL'};
                     $Ks = $MLmatrix->[0]->[1]->{'dS'};
                     $Ka = $MLmatrix->[0]->[1]->{'dA'};
                     $omega = $MLmatrix->[0]->[1]->{'omega'};
                     print " likelihood = $likelihood, Ka = $Ka, Ks =
    $Ks, Ka/Ks = $omega\n";
    #        }
    #        else # If an error occured during the Ks-calculation ...
    #        {
    #            return (-1);
    #        }
    #    }
    return ( $likelihood, $Ks, $Ka, $omega );
    }


-- 
*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*
Lorenzo Carretero Paulet
Institute for Plant Molecular and Cell Biology - IBMCP (CSIC-UPV)
Integrative Systems Biology Group
C/ Ingeniero Fausto Elio s/n.
46022 Valencia, Spain

Phone:  +34 963879934
Fax:    +34 963877859
e-mail:locarpau at upvnet.upv.es
*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*

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