OLGenie: Estimating Natural Selection to Predict Functional Overlapping Genes

Purifying (negative) natural selection is a hallmark of functional biological sequences, and can be detected in protein-coding genes using the ratio of nonsynonymous to synonymous substitutions per site (d(N)/d(S)). However, when two genes overlap the same nucleotide sites in different frames, synonymous changes in one gene may be nonsynonymous in the other, perturbing d(N)/d(S). Thus, scalable methods are needed to estimate functional constraint specifically for overlapping genes (OLGs). We propose OLGenie, which implements a modification of the Wei-Zhang method. Assessment with simulations and controls from viral genomes (58 OLGs and 176 non-OLGs) demonstrates low false-positive rates and good discriminatory ability in differentiating true OLGs from non-OLGs. We also apply OLGenie to the unresolved case of HIV-1's putative antisense protein gene, showing significant purifying selection. OLGenie can be used to study known OLGs and to predict new OLGs in genome annotation.