Hi all, I'm a very new perl/bioperl user, and I'm running into some trouble so I hope I can find some help here. Here's what I need to do: (from a large number of fasta files) Extract an EST (peromyscus) from the FASTA file, find its closest match in mus musclus, and find the dn/ds between the two sequences. Here's what I'm doing (I know this is probably the least efficient way, any suggestions?): 1) Read in pero EST from a FASTA 2) Standaloneblast it to local mus cDNA database, retrieve accession from best result 3) Retrieve complete mus sequence with features from genbank using ID from (2) 4) Make a clustalw simple align object using the mus protein sequence from (3) against the translated pero EST for all 3 frames, and keep the one with the best identity %. --I'm done up to here-- 5) Convert the aln frrom AA to DNA (there is a builtin aa_to_dna_aln but it isn't working for me) 6) Pass the aln through a DN/DS module (is paml the only one?) So I have 2 problems: 7 out of the 20 ESTs return a poor "best alignment" with less than 20% identity (and in the printout they clearly are not aligning). Does this have something to do with gaps? In spite of that, the other 13 are aligning pretty well, with 60-100% alignment. In order to calculate DN/DS I've looked around and it seems I have to use PAML. But before that I think I'm required to have an aln object of two DNA sequences, starting at the correct frame. How can I do that? Thanks, Ed