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

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

Overlapping genes were first detected following the discovery that the cumulative length of protein sequences in bacteriophage u174 exceeded the length of the genome (Barrell et al. 1976 ). Today, the detection of overlapping genes still largely relies on laboratory methods that isolate, sequence, and align individual proteins to reference genomes (Fellner et al. 2015) . These and other potential laboratory methods such as ribosome profiling (Irigoyen et al. 2016) are costly and time intensive, making large scale screening and identification of overlapping genes expensive. Necessarily, these factors have led to the development of bioinformatics and theoretical methods for the analysis of overlapping genes that rely on genome sequence analyses alone. For example, synonymous sites that exhibit a reduced nucleotide substitution rate are indicative of functional overlapping proteins; because these substitutions affect two proteins they are usually expected to be deleterious and hence are observed at a reduced rate (Firth and Brown 2005, 2006; Jagger et al. 2012) . A number of other properties of overlapping genes have been Article ß The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [e-mail] Open Access used as effective bioinformatics makers, such as synonymous codon dissimilarity between newly generated overlapping genes and the remainder of the genome (Pavesi et al. 1997 (Pavesi et al. , 2013 , and the restriction that particular codon sequence orders place on alternative reading frames (Lebre and Gascuel 2017) . For example, the reverse complementary nucleotide sequence for two adjacent tyrosines (TAT/C and TAT/C) will be A/GTA and A/GTA, which always creates a stop codon (either a TAA or TAG after a reading frame shift of 1 nucleotide). Although these properties help in the development of bioinformatics techniques to discover unknown overlapping genes, they are restricted by their requirement for multiple genomic sequences or by their poor sensitivity. With the rapid rise of metagenomics to discover new viruses (Bekal et al. 2011; Ballinger et al. 2014; Shi et al. 2016 Shi et al. , 2018 , efficient and sensitive approaches of identifying overlapping genes that require genome sequence information alone will be essential.