A 'polyORFomic' analysis of prokaryote genomes using disabled-homology filtering reveals conserved but undiscovered short ORFs

Prokaryote gene annotation is complicated by large numbers of short open reading frames (ORFs) that arise naturally from genetic code design. Historically, many hypothetical ORFs have been annotated as genes in microbes, usually with an arbitrary length threshold (e.g. greater than 100 codons). Given the use of such thresholds, what is the extent of genuine undiscovered short genes in the current sampling of prokaryote genomes? To assess rigorously the potential under-annotation of short ORFs with homology, we exhaustively compared the polyORFome--all possible ORFs in 64 prokaryotes (53 bacteria and 11 archaea) plus budding yeast--to itself and to all known proteins. The novelty of our analysis is that, firstly, sequence comparisons to/between both annotated and un-annotated ORFs are considered, and secondly a two-step disabled-homology filter is applied to set aside putative pseudogenes and spurious ORFs. We find that un-annotated homologous short ORFs (uhORFs) correspond to a small but non-negligible fraction of the annotated prokaryote proteomes (0.5-3.8%, depending on selection criteria). Moreover, the disabled-homology filter indicates that about a third of uhORFs correspond to putative pseudogenes or spurious ORFs. Our analysis shows that the use of annotation length thresholds is unnecessary, as there are manageable numbers of short ORF homologies conserved (without disablements) across microbial genomes. Data on uhORFs are available from http://pseudogene.org/polyo