Tetranucleotide usage in mycobacteriophage genomes: alignment-free methods to cluster phage and infer evolutionary relationships

Abstract

Background The genomic sequences of phages isolated on mycobacterial hosts are diverse, mosaic and often share little nucleotide similarity. However, about 30 unique types have been isolated, allowing most phage to be grouped into clusters and further into subclusters [1]. Many tools for the analysis of mycobacteriophage genomes depend on sequence alignment or knowledge of gene content. These methods are computationally expensive, can require significant manual input (for example, gene annotation) and can be ineffective for significantly diverged sequences [2]. We evaluated tetranucleotide usage in mycobacteriophages as an alternative to alignment-based methods for genome analysis. Description We computed tetranucleotide usage deviation, the ratio of observed counts of 4-mers in a genome to the expected count under a null model [3]. Tetranucleotide usage deviation is comparable for members of the same phage subcluster and distinct between subclusters. Neighbor joining phylogenetic trees were constructed on pairwise Euclidean distances between all genomes in the mycobacteriophage database. In almost every case, phage were placed in a monophyletic clade with members of the same subcluster. With few exceptions, trees computed from tetranucleotide usage deviation accurately reconstruct trees based on gene content for a subset of the mycobacteriophage population (Figure 1). We also evaluated the possibility of assigning clusters to unknown phage based on tetranucleotide usage deviation. Under a simple nearest neighbor classifier, cluster assignments were recovered at a frequency greater than 98%. In addition, we looked for evidence of horizontal gene transfer by using tetranucleotide difference index, a measure of the deviation in tetranucleotide usage from the genomic mean in a sliding window across the genome [3]. Tetranucleotide difference index plots showed a strong spike at the end of cluster L mycobacteriophages, which could indicate horizontal gene transfer in the region. Conclusions