Computational Tools for the Analysis of Uncultivated Phage Genomes

Abstract

Over a century of bacteriophage research has uncovered a plethora of fundamental aspects of their biology, ecology, and evolution. Furthermore, the introduction of community-level studies through metagenomics has revealed unprecedented insights on the impact that phages have on a range of ecological and physiological processes. It was not until the introduction of viral metagenomics that we began to grasp the astonishing breadth of genetic diversity encompassed by phage genomes. Novel phage genomes have been reported from a diverse range of biomes at an increasing rate, which has prompted the development of computational tools that support the multilevel characterization of these novel phages based solely on their genome sequences. The impact of these technologies has been so large that, together with MAGs (Metagenomic Assembled Genomes), we now have UViGs (Uncultivated Viral Genomes), which are now officially recognized by the International Committee for the Taxonomy of Viruses (ICTV), and new taxonomic groups can now be created based exclusively on genomic sequence information. Even though the available tools have immensely contributed to our knowledge of phage diversity and ecology, the ongoing surge in software programs makes it challenging to keep up with them and the purpose each one is designed for. Therefore, in this review, we describe a comprehensive set of currently available computational tools designed for the characterization of phage genome sequences, focusing on five specific analyses: (i) assembly and identification of phage and prophage sequences, (ii) phage genome annotation, (iii) phage taxonomic classification, (iv) phage-host interaction analysis, and (v) phage microdiversity.

Keywords: computational analysis; microdiversity; phage and prophage identification; phage annotation; phage metagenomics; phage taxonomy; phage-host interaction; uncultivated viruses; viromes.

FIG 1

Proposed workflow for the analysis of phage metagenomic data. Raw metagenomic reads are first filtered for contaminants and then assembled into contigs and binned. While in principle the identification of phage and/or prophage contigs could be omitted if the researcher knows that the reads are enriched for viruses, we suggest performing it as an additional way to filter contaminant nonviral bins. Phage or prophage contigs can then be subjected to genome annotation, taxonomic classification, microdiversity analysis, and host-association analysis. Moreover, while they are different analyses, genome annotation and taxonomic classification are usually done conjointly. While one can carry out microdiversity and/or phage-host analysis without prior annotation and/or taxonomic classification, the former two are usually done before either of the latter.