So very new to phylogenetics!. I've been scrambling and reading so much to get this done!!.
I would like to see if one or more of the sequences I have (from a gene family) are under positive selection using the dN/dS ratio. The conceptual parts are clear to me, however, practically speaking i have found several problems/questions along the way.
My most important question would be: I have four related organisms (insects), and their full sets of genes in a gene family. I have reconstructed trees to delineate their evolutionary story (duplications and losses). What would be the input for the calculation of dN/dS ratios when a single gene has several possible orthologs in the other organisms?, like gene A in species 1 has gene B and C and D in species 2 and E, F, G, H in species 3 etc... Do I need to align only the orthologs for each species? all of them?, or just align all the sequences form the subfamily including all paralogs and orthologs?, wouldnt that overestimate the ratio?
I have tried PAML yn00 package and got a result for a small "test" set of sequences, which were not codon aligned. Results are pairwise, how can I interpret this?, do I want to get "overall" omega values instead of pairwise? what would be the most interpretation on each case?
Second question is how I get codon alignments for these sequences?. I saw a couple of free software on th internet but they wouldn't work.
thanks a lot!