Escherichia phage N4 was isolated in 1966 in Italy and has remained a genomic orphan for a long time. It encodes an extremely large virion-associated RNA polymerase unique for bacterial viruses that became characteristic for this group. In recent years, due to new and relatively inexpensive sequencing techniques the number of publicly available phage genome sequences expanded rapidly. This revealed new members of the N4-like phage group, from 33 members in 2015 to 115 N4-like viruses in 2020. Using new technologies and methods for classification, the Bacterial and Archaeal Viruses Subcommittee of the International Committee on Taxonomy of Viruses (ICTV) has moved the classification and taxonomy of bacterial viruses from mere morphological approaches to genomic and proteomic methods. The analysis of 115 N4-like genomes resulted in a huge reassessment of this group and the proposal of a new family “Schitoviridae”, including eight subfamilies and numerous new genera.

Background: CoreGenes3.5 is a webserver that determines sets of core genes from viral and small bacterial genomes as an automated batch process. Previous versions of CoreGenes have been used to classify bacteriophage genomes and mine data from pathogen genomes.Findings: CoreGenes3.5 accepts as input GenBank accession numbers of genomes and performs iterative BLASTP analyses to output a set of core genes. After completion of the program run, the results can be either displayed in a new window for one pair of reference and query genomes or emailed to the user for multiple pairs of small genomes in tabular format.Conclusions: With the number of genomes sequenced increasing daily and interest in determining phylogenetic relationships, CoreGenes3.5 provides a user-friendly web interface for wet-bench biologists to process multiple small genomes for core gene determinations. CoreGenes3.5 is available athttp://binf.gmu.edu:8080/CoreGenes3.5.

Viruses and small-genome bacteria (~2 megabases and smaller) comprise a considerable population in the biosphere and are of interest to many researchers. These genomes are now sequenced at an unprecedented rate and require complementary computational tools to analyze. "CoreGenesUniqueGenes" (CGUG) is an in silico genome data mining tool that determines a "core" set of genes from two to five organisms with genomes in this size range. Core and unique genes may reflect similar niches and needs, and may be used in classifying organisms. CGUG is available at http://binf.gmu.edu/geneorder.html as a web-based on-the-fly tool that performs iterative BLASTP analyses using a reference genome and up to four query genomes to provide a table of genes common to these genomes. The result is an in silico display of genomes and their proteomes, allowing for further analysis. CGUG can be used for "genome annotation by homology", as demonstrated with Chlamydophila and Francisella genomes. CGUG is used to reanalyze the ICTV-based classifications of bacteriophages, to reconfirm long-standing relationships and to explore new classifications. These genomes have been problematic in the past, due largely to horizontal gene transfers. CGUG is validated as a tool for reannotating small genome bacteria using more up-to-date annotations by similarity or homology. These serve as an entry point for wet-bench experiments to confirm the functions of these "hypothetical" and "unknown" proteins.

Sequence databases are growing exponentially due to 'next generation' DNA analysers and applications of these data. Databases include multiple sequences of previously sequenced organisms, particularly ones of consequence to human health. Applications are limited by tools available to mine them, particularly user-friendly tools that are useful for bench researchers. GeneOrder, CoreGenes and CGUG are web-based 'on-the-fly' tools that examine gene order and synteny, as well as proteomes for comparative genomics and for drug discovery and design targets. CoreGenes (CGUG) now allows analysis of genomes ranging up to 1.9 megabases. Many of these small genome bacteria have impacts on human health.

The determination of core genes in viral and bacterial genomes is crucial for a better understanding of their relatedness and for their classification. CoreGenes5.0 is an updated user-friendly web-based software tool for the identification of core genes in and data mining of viral and bacterial genomes. This tool has been useful in the resolution of several issues arising in the taxonomic analysis of bacteriophages and has incorporated many suggestions from researchers in that community. The webserver displays result in a format that is easy to understand and allows for automated batch processing, without the need for any user-installed bioinformatics software. CoreGenes5.0 uses group protein clustering of genomes with one of three algorithm options to output a table of core genes from the input genomes. Previously annotated “unknown genes” may be identified with homologues in the output. The updated version of CoreGenes is able to handle more genomes, is faster, and is more robust, providing easier analysis of custom or proprietary datasets. CoreGenes5.0 is accessible at coregenes.org, migrating from a previous site.