Overestimation of nonsynonymous/synonymousrate ratio by reverse-translation ofaligned amino acid sequences

Abstract

In the analysis of protein-coding nucleotide sequences, the ratio of the number of nonsynonymous substitutions to that of synonymous substitutions (dN/dS) is used as an indicator for the direction and magnitude of natural selection operating at the amino acid sequence level. The dS and dN values are estimated based on the comparison of homologous codons, which are often identified by converting (reverse-translating) aligned amino acid sequences into codon sequences. In this method, however, homologous codons may be mis-identified when frame-shifts occurred or amino acid sequences were mis-aligned, which may lead to overestimation of the dN/dS ratio. Here the effect of reverse-translating aligned amino acid sequences on the estimation of dN/dS ratio was examined through a large-scale analysis of protein-coding nucleotide sequences from vertebrate species. Apparently, 1-9% of codon sites that were identified as homologous with reverse-translation contained non-homologous codons, where the dN/dS ratio was unduly high. By correcting the dN/dS ratio for these codon sites, it was inferred that the ratio was 5-43% overestimated with reverse-translation. These results suggest that caution should be exerted in the study of natural selection using the dN/dS ratio by reverse-translating aligned amino acid sequences.