There are couple of ways to do it. If you are interested in using the Clustalw (EBI) then you have to install Bioperl module for your perl to give the multiple sequences as a set and get the output through it. The module within Bioperl package for using it is here: Clustalw. Have a look at the synopsis as it very clearly defined

The other way is by writing a Needleman Wunsch Program as a function in your perl program, providing the sequences one by one and making it compare with the ref seq sequence. Your Needleman Wunsch function is as follows: 


# Needleman-Wunsch  Algorithm 

sub NeedlemanWunsch { 
my ($seq1, $seq2) = @ARGV;

# scoring scheme
my $MATCH    =  1; # +1 for letters that match
my $MISMATCH = -1; # -1 for letters that mismatch
my $GAP      = -1; # -1 for any gap

# initialization
my @matrix;
$matrix[0][0]{score}   = 0;
$matrix[0][0]{pointer} = "none";
for(my $j = 1; $j <= length($seq1); $j++) {
    $matrix[0][$j]{score}   = $GAP * $j;
    $matrix[0][$j]{pointer} = "left";
}
for (my $i = 1; $i <= length($seq2); $i++) {
    $matrix[$i][0]{score}   = $GAP * $i;
    $matrix[$i][0]{pointer} = "up";
}

# fill
for(my $i = 1; $i <= length($seq2); $i++) {
    for(my $j = 1; $j <= length($seq1); $j++) {
        my ($diagonal_score, $left_score, $up_score);

        # calculate match score
        my $letter1 = substr($seq1, $j-1, 1);
        my $letter2 = substr($seq2, $i-1, 1);                         
+   
        if ($letter1 eq $letter2) {
            $diagonal_score = $matrix[$i-1][$j-1]{score} + $MATCH;
        }
        else {
            $diagonal_score = $matrix[$i-1][$j-1]{score} + $MISMATCH;
        }

        # calculate gap scores
        $up_score   = $matrix[$i-1][$j]{score} + $GAP;
        $left_score = $matrix[$i][$j-1]{score} + $GAP;

        # choose best score
        if ($diagonal_score >= $up_score) {
            if ($diagonal_score >= $left_score) {
                $matrix[$i][$j]{score}   = $diagonal_score;
                $matrix[$i][$j]{pointer} = "diagonal";
            }
        else {
                $matrix[$i][$j]{score}   = $left_score;
                $matrix[$i][$j]{pointer} = "left";
            }
        } else {
            if ($up_score >= $left_score) {
                $matrix[$i][$j]{score}   = $up_score;
                $matrix[$i][$j]{pointer} = "up";
            }
            else {
                $matrix[$i][$j]{score}   = $left_score;
                $matrix[$i][$j]{pointer} = "left";
            }
        }
    }
}

# trace-back

my $align1 = "";
my $align2 = "";

# start at last cell of matrix
my $j = length($seq1);
my $i = length($seq2);

while (1) {
    last if $matrix[$i][$j]{pointer} eq "none"; # ends at first cell o
+f matrix

    if ($matrix[$i][$j]{pointer} eq "diagonal") {
        $align1 .= substr($seq1, $j-1, 1);
        $align2 .= substr($seq2, $i-1, 1);
        $i--;
        $j--;
    }
    elsif ($matrix[$i][$j]{pointer} eq "left") {
        $align1 .= substr($seq1, $j-1, 1);
        $align2 .= "-";
        $j--;
    }
    elsif ($matrix[$i][$j]{pointer} eq "up") {
        $align1 .= "-";
        $align2 .= substr($seq2, $i-1, 1);
        $i--;
    }    
 }
$align1 = reverse $align1;
$align2 = reverse $align2;
print "$align1\n";
print "$align2\n";
}

[download]

I would prefer that you should use the Clustalw rather than using a needleman wunsch program as it would answer your questions related to the matrices that you want to use. I hope it works for you. All the best

In reply to Re: how to construct multiple sequence alignment from group of pairwise alignments by snape
in thread how to construct multiple sequence alignment from group of pairwise alignments by gudluck

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