I've been trying to get Ka/Ks scores of genome-wide pairwise alignments of coding sequences from two strains of the same organism. (i.e. very high similarity)
So far I'm using a custom python script to remove stop codons, Muscle to align the sequences and finally pipe that into KaKs Calculator. I get like 10% of CDS having the coefficient greater than 1, which I find highly unlikely and attribute that to bad alignment.
What alignment programs do you use when trying to align coding sequences (with different sizes and indels) for Ka/Ks analysis?