Hi, In Bio::EnsEMBL::Variation::Utils::Sequence, it reads: my %unambig = qw(M AC V ACG N ACGT H ACT R AG D AGT W AT S CG B CGT Y CT K GT C CC A AA T TT G GG - --); Any reason for AA, TT, GG, CC for A, T, G and C, respectively? Maybe easier for just a single letter? Cheers, Sung