A Simple Method to Detect Candidate Overlapping Genes in Viruses Using Single Genome Sequences

Schlub, Timothy E; Buchmann, Jan P; Holmes, Edward C

We present a simple new method that uses a single genome sequence to detect candidates for overlapping genes. The method assumes that functional ORFs are longer than expected by random chance as they experience selective pressure against mutations that introduce stop codons. We quantify this by using three ways to estimate the null distribution for ORFs lengths within each reading frame of a gene, and use the null to identify those ORFs significantly longer than predicted by random chance. This approach has a number of advantages over current bioinformatics methods to detect overlapping genes. In addition to being simple and quick, it only requires a single genome sequence. This is in contrast to other bioinformatic methods that require multiple sequences to estimate and compare nucleotide or codon diversity. This feature allows the method to be applied much more broadly in both metagenomics projects where genomes of new viruses are frequently only present in a single copy (Bekal et al. 2011; Ballinger et al. 2014; Shi et al. 2016) , and also in screening scenarios such as demonstrated herein. The method is best suited to refine regions of the genome that contain candidates for functional overlapping genes, after which these regions can be further tested for functionality with more resource intensive laboratory methods such as protein isolation, ribosomal profiling (Michel et al. 2012; Ingolia 2016) , and studying the effects of introduced knock out mutations (Chung et al. 2008) .