Which method are you talking about, with respect to those 5 seconds?  The XOR method outlined above takes just ~0.05 secs on my 4-year-oldish machine, for 10,000 comparisons against a common 40-char target (with ~3-5 deviations per sequence):

my @set = qw(A T C G);
my $target = join "", map $set[rand @set], 1..40;
my @tests ;
for (1..10000) {
    my $test = $target;
    for (1..5) {
        substr($test, rand(length($target)), 1) = $set[rand @set];
    }
    push @tests, $test;
}


use Time::HiRes qw(time);
my $start = time();

my %change;  # reverse lookup table
for my $t1 (@set) {
    my $t = $t1;
    $t =~ tr/ATCG/HRDZ/;
    for my $t2 (@set) {
        $change{ $t ^ $t2 } = "$t1->$t2";
    }
}

$target =~ tr/ATCG/HRDZ/;

for my $test (@tests) {
    my $diff = $target ^ $test;
    while ($diff =~ /([^\x09\x06\x07\x1d])/g) {
        my $pos    = pos($diff);
        my $change = $change{$1};
        # do something with them...
    }
}

printf "%.3f secs\n", time() - $start;

__END__
0.052 secs
[download]

Storing away the results somewhere or doing something else with them will presumably take considerably longer than computing them...

ok, the XOR method presented by Eliya is fast, though i haven't timed it. the substr methods presented previously take about 5 seconds to run. i don't know about the method that BrowserUk is talking about. bear in mind, i am doing additional things to actually generate the target sequences, but that is a cost that i think i have to deal with due to my dependence on BioPerl.

Character by character processing of strings is the single biggest weakness in perl's arsenal. As you've already used PDl, you probably have a compiler, so dropping into Inline::C should not be a problem for you.

The two routines below differ in the way the return the results. dnacmp() returns a list of strings like this:"123:A:C", whereas dnacmp2() concatenates all those into a single string for return:

#! perl -slw
use strict;
use Inline C => Config => BUILD_NOISY => 1;
use Inline C => <<'END_C',  NAME => 'dnacmp', CLEAN_AFTER_BUILD => 0;

void dnacmp( char *a, char *b ) {
    int n = 0;
    Inline_Stack_Vars;

    Inline_Stack_Reset;
    while( *a && *b ) {
        if( *a != *b ) {
            Inline_Stack_Push( sv_2mortal( newSVpvf( "%d:%c:%c", n, *a
+, *b ) ) );
        }
        a++; b++; n++;
    }
    Inline_Stack_Done;
    return;
}

SV *dnacmp2( char *a, char *b ) {
    SV *res = newSVpv( "", 0 );
    int n = 0;

    while( *a && *b ) {
        if( *a != *b )
            sv_catpvf( res, "%d:%c:%c ", n, *a, *b );
        ++a, ++b, ++n;
    }
    return res;
}

END_C
use Data::Dump qw[ pp ];
use Time::HiRes qw[ time ];

sub rndStr{ join'', @_[ map{ rand @_ } 1 .. shift ] }

print for dnacmp( 'ATTCCGGG', 'ATACCGC' );
print dnacmp2( 'ATTCGGG', 'ATACCGGC' );

our $N //= 10e3;
our $L //= 8;

my @seqs; $#seqs = $N-1;
$seqs[ $_ ] = rndStr( $L, qw[ A C G T ] ) for 0 .. $#seqs;

my $start = time;
for my $i ( 0 .. $#seqs ) {
    for my $j ( $i+1 .. $#seqs ) {
        print "@seqs[ $i, $j ] : ", dnacmp2( @seqs[ $i, $j ] );
    }
}
printf STDERR "Took %f seconds to cross compare $N sequences (%d compa
+risons)\n",
    time() - $start, ($N**2-$N)/2;

__END__
C:\test>dnacmp -N=142 -L=40
2:T:A
6:G:C
2:T:A 4:G:C
Took 0.233414 seconds to cross compare 142 sequences (10011 comparison
+s)

C:\test>head out.dat
2:T:A
6:G:C
2:T:A 4:G:C
CTTTACGGTCGCCAGTGCGCTAGCGGTGCTGGGTATGTCC TTTCGGTCAGGAATGGCGGCCAGCATATA
+ATCGGAGCCGT : 0:C:T 3:T:C 4:A:G 5:C:G 6:G:T 7:G:C 8:T:A 9:C:G 11:C:A 
+12:C:A 13:A:T 15:T:G 16:G:C 17:C:G 20:T:C 24:G:A 25:G:T 26:T:A 27:G:T
+ 28:C:A 29:T:A 30:G:T 31:G:C 33:T:G 35:T:G 36:G:C 37:T:C 38:C:G 39:C:
+T
CTTTACGGTCGCCAGTGCGCTAGCGGTGCTGGGTATGTCC CCGGGAACTCATTGACTCATCTCTGCACG
+CGGAAAGAGAC : 1:T:C 2:T:G 3:T:G 4:A:G 5:C:A 6:G:A 7:G:C 10:G:A 11:C:T
+ 12:C:T 13:A:G 14:G:A 15:T:C 16:G:T 18:G:A 19:C:T 20:T:C 21:A:T 22:G:
+C 23:C:T 25:G:C 26:T:A 27:G:C 28:C:G 29:T:C 32:G:A 33:T:A 35:T:G 36:G
+:A 37:T:G 38:C:A
CTTTACGGTCGCCAGTGCGCTAGCGGTGCTGGGTATGTCC ACACCGTTACAGGAGCTTACGATAACGAC
+CTGACCAATAC : 0:C:A 1:T:C 2:T:A 3:T:C 4:A:C 5:C:G 6:G:T 7:G:T 8:T:A 1
+0:G:A 11:C:G 12:C:G 15:T:C 16:G:T 17:C:T 18:G:A 20:T:G 22:G:T 23:C:A 
+24:G:A 25:G:C 26:T:G 27:G:A 29:T:C 30:G:T 32:G:A 33:T:C 34:A:C 35:T:A
+ 36:G:A 38:C:A
CTTTACGGTCGCCAGTGCGCTAGCGGTGCTGGGTATGTCC TTTATGCTGTAAACTAGACTAACTCCCTT
+AGCTACTGACG : 0:C:T 3:T:A 4:A:T 5:C:G 6:G:C 7:G:T 8:T:G 9:C:T 10:G:A 
+11:C:A 12:C:A 13:A:C 14:G:T 15:T:A 17:C:A 18:G:C 19:C:T 20:T:A 22:G:C
+ 23:C:T 24:G:C 25:G:C 26:T:C 27:G:T 28:C:T 29:T:A 31:G:C 32:G:T 33:T:
+A 34:A:C 37:T:A 39:C:G
CTTTACGGTCGCCAGTGCGCTAGCGGTGCTGGGTATGTCC CTCGTTCGGTATATGGCTGACGGAATTCG
+CTACGGGCACG : 2:T:C 3:T:G 4:A:T 5:C:T 6:G:C 8:T:G 9:C:T 10:G:A 11:C:T
+ 12:C:A 13:A:T 15:T:G 16:G:C 17:C:T 19:C:A 20:T:C 21:A:G 23:C:A 24:G:
+A 25:G:T 27:G:C 28:C:G 29:T:C 30:G:T 31:G:A 32:G:C 33:T:G 34:A:G 35:T
+:G 36:G:C 37:T:A 39:C:G
CTTTACGGTCGCCAGTGCGCTAGCGGTGCTGGGTATGTCC AAAAGGTGCGGCACTAGCGTAAACTGCAC
+GAGCGTACACC : 0:C:A 1:T:A 2:T:A 3:T:A 4:A:G 5:C:G 6:G:T 8:T:C 9:C:G 1
+2:C:A 13:A:C 14:G:T 15:T:A 19:C:T 20:T:A 22:G:A 24:G:T 26:T:C 27:G:A 
+29:T:G 30:G:A 32:G:C 33:T:G 34:A:T 35:T:A 36:G:C 37:T:A
CTTTACGGTCGCCAGTGCGCTAGCGGTGCTGGGTATGTCC TCAAGGGCGGCGGTAAAACAATGATGTAC
+ACCTCGTCGCG : 0:C:T 1:T:C 2:T:A 3:T:A 4:A:G 5:C:G 7:G:C 8:T:G 9:C:G 1
+0:G:C 11:C:G 12:C:G 13:A:T 14:G:A 15:T:A 16:G:A 17:C:A 18:G:C 19:C:A 
+20:T:A 21:A:T 23:C:A 24:G:T 27:G:A 29:T:A 30:G:C 31:G:C 32:G:T 33:T:C
+ 34:A:G 36:G:C 37:T:G 39:C:G

C:\test>tail out.dat
TACATGCCCTATCCAGGCAACGCATTAGGCACTGCGTGTA ACAATGCGACGGCTCCAGACTTGATACCT
+GTATCAAACTA : 0:T:A 1:A:C 2:C:A 7:C:G 8:C:A 9:T:C 10:A:G 11:T:G 13:C:
+T 14:A:C 15:G:C 16:G:A 17:C:G 19:A:C 20:C:T 21:G:T 22:C:G 25:T:A 26:A
+:C 27:G:C 28:G:T 29:C:G 30:A:T 31:C:A 33:G:C 34:C:A 35:G:A 36:T:A 37:
+G:C
TACATGCCCTATCCAGGCAACGCATTAGGCACTGCGTGTA GGTGGAAACGGCGTGTGTAGAAATTTATT
+CACGGTACTGT : 0:T:G 1:A:G 2:C:T 3:A:G 4:T:G 5:G:A 6:C:A 7:C:A 9:T:G 1
+0:A:G 11:T:C 12:C:G 13:C:T 14:A:G 15:G:T 17:C:T 19:A:G 20:C:A 21:G:A 
+22:C:A 23:A:T 27:G:T 28:G:T 32:T:G 34:C:T 35:G:A 36:T:C 37:G:T 38:T:G
+ 39:A:T
TACATGCCCTATCCAGGCAACGCATTAGGCACTGCGTGTA GATAGTTCCAAGCCACCTCCCACCCTGCT
+CAACATCGCTA : 0:T:G 2:C:T 4:T:G 5:G:T 6:C:T 9:T:A 11:T:G 15:G:C 16:G:
+C 17:C:T 18:A:C 19:A:C 21:G:A 23:A:C 24:T:C 26:A:G 27:G:C 28:G:T 31:C
+:A 32:T:C 33:G:A 34:C:T 35:G:C 36:T:G 37:G:C
TACATGCCCTATCCAGGCAACGCATTAGGCACTGCGTGTA AAGGTTGGTACTTACTAACCGGTTTGAGC
+CGTTCAACGAA : 0:T:A 2:C:G 3:A:G 5:G:T 6:C:G 7:C:G 8:C:T 9:T:A 10:A:C 
+12:C:T 13:C:A 14:A:C 15:G:T 16:G:A 17:C:A 18:A:C 19:A:C 20:C:G 22:C:T
+ 23:A:T 25:T:G 28:G:C 30:A:G 31:C:T 33:G:C 34:C:A 35:G:A 36:T:C 38:T:
+A
ACAATGCGACGGCTCCAGACTTGATACCTGTATCAAACTA GGTGGAAACGGCGTGTGTAGAAATTTATT
+CACGGTACTGT : 0:A:G 1:C:G 2:A:T 3:A:G 4:T:G 5:G:A 6:C:A 7:G:A 8:A:C 9
+:C:G 11:G:C 12:C:G 14:C:G 15:C:T 16:A:G 17:G:T 19:C:G 20:T:A 21:T:A 2
+2:G:A 23:A:T 25:A:T 26:C:A 27:C:T 29:G:C 30:T:A 31:A:C 32:T:G 33:C:G 
+34:A:T 36:A:C 37:C:T 38:T:G 39:A:T
ACAATGCGACGGCTCCAGACTTGATACCTGTATCAAACTA GATAGTTCCAAGCCACCTCCCACCCTGCT
+CAACATCGCTA : 0:A:G 1:C:A 2:A:T 4:T:G 5:G:T 6:C:T 7:G:C 8:A:C 9:C:A 1
+0:G:A 13:T:C 14:C:A 16:A:C 17:G:T 18:A:C 20:T:C 21:T:A 22:G:C 23:A:C 
+24:T:C 25:A:T 26:C:G 29:G:C 30:T:A 32:T:C 33:C:A 34:A:T 35:A:C 36:A:G
ACAATGCGACGGCTCCAGACTTGATACCTGTATCAAACTA AAGGTTGGTACTTACTAACCGGTTTGAGC
+CGTTCAACGAA : 1:C:A 2:A:G 3:A:G 5:G:T 6:C:G 8:A:T 9:C:A 10:G:C 11:G:T
+ 12:C:T 13:T:A 15:C:T 17:G:A 18:A:C 20:T:G 21:T:G 22:G:T 23:A:T 25:A:
+G 26:C:A 27:C:G 28:T:C 29:G:C 30:T:G 31:A:T 36:A:C 37:C:G 38:T:A
GGTGGAAACGGCGTGTGTAGAAATTTATTCACGGTACTGT GATAGTTCCAAGCCACCTCCCACCCTGCT
+CAACATCGCTA : 1:G:A 3:G:A 5:A:T 6:A:T 7:A:C 9:G:A 10:G:A 11:C:G 12:G:
+C 13:T:C 14:G:A 15:T:C 16:G:C 18:A:C 19:G:C 20:A:C 22:A:C 23:T:C 24:T
+:C 26:A:G 27:T:C 31:C:A 32:G:C 33:G:A 35:A:C 36:C:G 37:T:C 38:G:T 39:
+T:A
GGTGGAAACGGCGTGTGTAGAAATTTATTCACGGTACTGT AAGGTTGGTACTTACTAACCGGTTTGAGC
+CGTTCAACGAA : 0:G:A 1:G:A 2:T:G 4:G:T 5:A:T 6:A:G 7:A:G 8:C:T 9:G:A 1
+0:G:C 11:C:T 12:G:T 13:T:A 14:G:C 16:G:A 17:T:A 18:A:C 19:G:C 20:A:G 
+21:A:G 22:A:T 25:T:G 27:T:G 28:T:C 30:A:G 31:C:T 32:G:T 33:G:C 34:T:A
+ 37:T:G 38:G:A 39:T:A
GATAGTTCCAAGCCACCTCCCACCCTGCTCAACATCGCTA AAGGTTGGTACTTACTAACCGGTTTGAGC
+CGTTCAACGAA : 0:G:A 2:T:G 3:A:G 4:G:T 6:T:G 7:C:G 8:C:T 10:A:C 11:G:T
+ 12:C:T 13:C:A 14:A:C 15:C:T 16:C:A 17:T:A 20:C:G 21:A:G 22:C:T 23:C:
+T 24:C:T 25:T:G 26:G:A 27:C:G 28:T:C 30:A:G 31:A:T 32:C:T 33:A:C 34:T
+:A 35:C:A 36:G:C 37:C:G 38:T:A
[download]

ok, i think inline::c is a little beyond me. i'm trying it out with the following code:

# V5: Inline::C
#!/opt/local/bin/perl
use strict;
use Inline C => Config => BUILD_NOISY => 1;
use Inline C => <<'END_C', NAME => 'dnacmp', CLEAN_AFTER_BUILD => 0;

void dnacmp( char *a, char *b ) {
    int n = 0;
    Inline_Stack_Vars;

    Inline_Stack_Reset;
    while( *a && *b ) {
        if( *a != *b ) {
            Inline_Stack_Push( sv_2mortal( newSVpvf( "%d:%c:%c", n, *a
+, *b ) ) );
        }
        a++; b++; n++;
    }
    Inline_Stack_Done;
    return;
}

SV *dnacmp2( char *a, char *b ) {
    SV *res = newSVpv( "", 0 );
    int n = 0;

    while( *a && *b ) {
        if( *a != *b )
            sv_catpvf( res, "%d:%c:%c ", n, *a, *b );
        ++a, ++b, ++n;
    }
    return res;
}
END_C

use Bio::Seq;
use Bio::DB::Sam;

my $bamfile = Bio::DB::Sam->new( -bam => "eg1.bam" );
my @allReads = $bamfile->features( -type => 'match' );
for my $read (@allReads) {
    ( $ref, $matches, $query ) = $read->padded_alignment;
    print $read->qname, "\n", dnacmp2('$ref', '$query');
}
[download]

first, i extract some information from the .bam file. then, i create the target and the test sequence on the fly in the subsequent for loop. $ref represents the target and $query represents the test (both are just strings). i then try to feed these two variables into dnacmp2 and print a few more things before it ($read->qname represents a sequence identifier), but it throws the following error:

Global symbol "$query" requires explicit package name at mutations.pl 
+line 133.
Execution of mutations.pl aborted due to compilation errors.
Pradeep-Bandarus-MacBook-Pro:test pradeepbandaru$ perl mutations.pl
Global symbol "$ref" requires explicit package name at mutations.pl li
+ne 133.
Global symbol "$matches" requires explicit package name at mutations.p
+l line 133.
Global symbol "$query" requires explicit package name at mutations.pl 
+line 133.
Execution of mutations.pl aborted due to compilation errors.
[download]

sorry if this is a rookie question, but what's going on with this?