#1
#!/usr/bin/perl


$ENV{CLUSTALDIR} = 'C:\\\\\\\\\\\\\\\\\\\\\;
use Bio::Tools::Run::Alignment::Clustalw;

my $file = <>; # Get file name (fasta file) from command prompt.

#  Build a clustalw alignment factory
my $factory = Bio::Tools::Run::Alignment::Clustalw->new(-matrix => 'BLOSUM'); 
my $ktuple = 2;
$factory->ktuple($ktuple);

my $inseq = Bio::SeqIO->new(
                    '-file' => "<$file",
                    '-format' => 'fasta'
                   );
my $seq;
my @seq_array;
while ($seq = $inseq->next_seq) {
    push(@seq_array, $seq);
}
# Now we do the actual alignment.
my $seq_array_ref = \@seq_array;
my $aln = $factory->align($seq_array_ref);
##</code><code>##

#2
my $alnio = Bio::AlignIO->new('-file'=> 'out_file','-format' => 'clustalw');

my $dfactory = Bio::Tree::DistanceFactory->new('-method' => 'NJ');

my $stats = Bio::Align::ProteinStatistics->new;

my $treeout = Bio::TreeIO->new('-format' => 'newick');

while( my $aln = $alnio->next_aln ) 
{
 my $mat = $stats->distance('-method' => 'Kimura',
                            '-align'  => $aln);
 my $tree = $dfactory->make_tree($mat);
 $treeout->write_tree($tree);
}