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

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

Our rationale is that overlapping ORFs with functional benefit will result in negative selection against nucleotide substitutions that introduce stop codons within that gene. Accordingly, the length of ORFs (as measured by the distance between bookend stop codons) is likely to be larger when they are functional than what would be expected by random chance alone where stop codons could be introduced without penalty. Hence, the defining characteristic of our method for the detection of overlapping genes is identifying ORFs larger than expected by chance alone (fig. 1A ). We developed three tests for estimating the distribution of expected ORF lengths. Briefly, in the first test, we estimate the expected length of ORFs by permuting codon positions in the original reading frame and then measuring ORF lengths in other reading frames. This process is repeated to generate an expected distribution of ORF lengths (codon permutation test, fig. 1B ). In the second test, instead of permuting codon positions, the codon order is unchanged and nucleotide substitutions that would introduce synonymous mutations in the original reading frame are randomly generated (synonymous mutation test, fig. 1C ), before measuring ORF lengths in the other reading frames. In the third test, referred to as the combined test, the P values for both the codon permutation test and the synonymous mutation test must fall below some cut-off value.