Overestimation of nonsynonymous/synonymous rate ratio by reverse-translation of aligned 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 (d_N/d_S) is used as an indicator for the direction and magnitude of natural selection operating at the amino acid sequence level. The d_S and d_N 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 d_N/d_S ratio. Here the effect of reverse-translating aligned amino acid sequences on the estimation of d_N/d_S 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 d_N/d_S ratio was unduly high. By correcting the d_N/d_S 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 d_N/d_S ratio by reverse-translating aligned amino acid sequences.