This Small Grant for Exploratory Research (SGER) will create a large gold standard reference set of newly annotated diverse genes that were previously designated as hypothetical (having unknown function). This set will serve as a baseline for validation and comparative analysis of prospective approaches to functional annotation of uncharacterized genes. This reference set is aimed at overcoming one of the major bottlenecks of microbiology and modern biology in general, and microbial genome sequencing in particular. Hypothetical (uncharacterized) genes constitute approximately one third of all predicted genes and their number is expanding rapidly with each new genome sequencing project. New computational approaches to gene annotation are being developed almost daily to address the hurdle of currently having 70% or less of each genome assigned with known function. <br/><br/>This research will result in a reference set of 200 previously hypothetical genes with high quality functional annotation. This set will serve as a valuable resource for the development and validation of new methods for the function annotation of new and existing genomes. It will also serve as a benchmark for existing annotation methods and databases.