Studying the gut virome in the metagenomic era: challenges and perspectives

Abstract

The human gut harbors a complex ecosystem of microorganisms, including bacteria and viruses. With the rise of next-generation sequencing technologies, we have seen a quantum leap in the study of human-gut-inhabiting bacteria, yet the viruses that infect these bacteria, known as bacteriophages, remain underexplored. In this review, we focus on what is known about the role of bacteriophages in human health and the technical challenges involved in studying the gut virome, of which they are a major component. Lastly, we discuss what can be learned from studies of bacteriophages in other ecosystems.

Fig. 1

Viruses can be classified based on various characteristics. These terms are used continuously throughout this manuscript. While all characters are important in determining taxonomic relationships, sequence comparisons using both pairwise sequence similarity and phylogenetic relationships have become one of the primary sets of characters used to define and distinguish virus taxa [6]

Fig. 2

Size distributions of genomes and virions of the most prevalent virus families in the gut. Values are given for the prototype virus of each family. Prokaryotic viruses are shown in red, eukaryotic viruses in blue. Structural information as well as genome sizes have been exported from the ICTV Online Report [24]. The prevalence of each family in the human gut has been inferred from the following studies: Inoviridae [20, 25], Circoviridae, Adenoviridae, Microviridae, Podoviridae, Myoviridae, Siphoviridae [26], Anelloviridae [–27], CrAss-like [28, 29]. dsDNA double-stranded DNA. ssDNA single-stranded DNA

Fig. 3

The steps in metagenomic study of the virome. Nucleic acid extraction: the virome can be studied by extraction of nucleic acids from both fractions of the total microbial community which includes bacteria and viruses (left) and purified viral-like particles (VLPs; right), and different types of VLP-enriching techniques might be applied to obtain the latter fraction (see main text for details). Genomic library preparation: the extracted viral genetic material is subjected to sequencing after genomic library preparation. Both the choice of genomic library preparation technique and the sequencing coverage can affect the representation of specific members of the viral community in the sample (see discussion in the main text). Quality control: the raw sequencing reads are further trimmed of sequencing adapters, and low-quality and overrepresented reads are discarded. Virome annotation: there are two main ways of studying viral communities—read-mapping to closed reference databases or de novo assembly of viral genomes with optional, but advised, validation of contigs via reference databases