I've no idea about the Bio stuff but here's a way to compare the alignments by XOR'ing them. Identical characters XOR'ed together result in NULL (\x00) so look for non-NULLs to spot where they differ. I use a regex match with a zero-width look-ahead so that pos finds that the regex pointer is looking at the mis-aligned character, not the one behind it. Offsets in strings are zero-based in Perl and note that your last mis-alignment is at character 13, not 12.

$ perl -Mstrict -Mwarnings -E '
my $ref = q{AATTTGGGCTACT};
my $qry = q{AAATTCGGCTACA};

my $xor = $ref ^ $qry;

my @offsets;
push @offsets, pos $xor while $xor =~ m{(?=[^\x00])}g;

say join q{ }, map { ( $_ + 1 ) . substr $qry, $_, 1 } @offsets;'
3A 6C 13A
$
[download]

I hope this is helpful