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

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

For each coding region and for each viable alternative reading frame (þ1,þ2,Àc0, Àc1, Àc2) we performed the following analysis (summarized in fig. 1A ). First, the length of ORFs between the stop codons "TGA," "TAG," and "TAA" on that specific alternative reading frame was calculated. Then, 20,000 new coding sequences were created by either randomly permuting the codons in frame þ0 (codon permutation test; fig. 1B ), or for each amino acid in reading frame þ0, randomly choosing a replacement codon (for which the original codon is a possible candidate) that encodes the same amino acid (synonymous mutation test; fig. 1C ). For each of these 20,000 new coding sequences, the length of ORFs between stop codons in that alternative reading frame was calculated again. The lengths of ORFs over all 20,000 randomly generated coding sequences were pooled to calculate a theoretical distribution of the length of ORFs on that specific alternative reading frame. For each ORF length L in the original unpermuted coding sequence, the probability of observing a length as large or larger by random chance alone is calculated using this theoretical distribution as follows: