Viral Phylogenomics Using an Alignment-Free Method: A Three-Step Approach to Determine Optimal Length of k-mer

The development of rapid, economical genome sequencing has shed new light on the classification of viruses. As of October 2016, the National Center for Biotechnology Information (NCBI) database contained > 2 million viral genome sequences and a reference set of similar to 4000 viral genome sequences that cover a wide range of known viral families. Whole-genome sequences can be used to improve viral classification and provide insight into the viral " tree of life". However, due to the lack of evolutionary conservation amongst diverse viruses, it is not feasible to build a viral tree of life using traditional phylogenetic methods based on conserved proteins. In this study, we used an alignment-free method that uses k-mers as genomic features for a large-scale comparison of complete viral genomes available in RefSeq. To determine the optimal feature length, k (an essential step in constructing a meaningful dendrogram), we designed a comprehensive strategy that combines three approaches: (1) cumulative relative entropy, (2) average number of common features among genomes, and (3) the Shannon diversity index. This strategy was used to determine k for all 3,905 complete viral genomes in RefSeq. The resulting dendrogram shows consistency with the viral taxonomy of the ICTV and the Baltimore classification of viruses.