An introduction to comparative genomics, EukProt, and the reciprocal best hit (RBH) method for bench biologists: Ancestral phosphorylation of Tom22 in eukaryotes as a case study.

Comparative genomics is a useful approach for hypothesis generation for future functional investigations at the bench. However, most bench biologists shy away from computational methods. Here we reintroduce the simple but extremely effective Reciprocal Best Hit method for inferring protein orthologues. Because taxon set delimitation is perhaps the most important step in comparative genomics, we introduce The Comparative Set, a taxonomically representative subset of EukProt, a comprehensive eukaryotic predicted proteome database. After introducing the basic methods, we provide a step-by-step guide, including screen shots, for a case study on collecting Tom22 sequences from diverse eukaryotes. As an example of possible downstream analyses, we show that Tom22 proteins from diverse eukaryotes are likely regulated by conserved kinases at several sites. Though the sites evolve quickly, the processes and functions involved are likely ancestral and conserved across many eukaryotes.