Table 2.

Summary of molecular biology techniques used to study the gut phageome, including the major advantages and limitations of each technique.

Technique	Example gut discoveries	Advantages	Limitations
Electrophoresis	SDS-polyacrylamide gel electrophoresis on a CsCl fraction of human faeces isolated CrAss-like phages for subsequent mass spectrometry (Guerin et al., 2018).	Multiple viral populations can be separated from multispecies samples due to their differing capsid sizes.	No taxonomic information can be determined by electrophoresis alone.
Recombinant protein gene expression	Toxin-antitoxin systems were identified within prophages of Lactobacillus johnsonii (Denou et al., 2008).	Proteins can be overexpressed to obtain high titres for subsequent analysis.	Cloning of viral genes into heterologous host systems can be difficult given that viral genomes often encode modified nucleotides (Warren, 1980) and lethal genes (Wang et al., 2010).
			Expressed proteins may not be functional due to misfolding and incorrect modifications in expression host.
Microarrays	The new-born infant gut viral community was found to be dynamic (Breitbart et al., 2008).	High-throughput.	Incompatible with novel viral genomes as a priori sequence information is required to design probes.
	50% of the strain-specific DNA in Lactobacillus johnsonii was found to derive from prophages (Ventura et al., 2003).		Viral DNA amplification steps prior to microarray analysis can introduce bias, making relative abundances no longer reflect that of the sample studied.
Single-cell DNA sequencing	Sequencing of commensal gut bacteria can facilitate the identification of integrated prophages (Ventura et al., 2003; Denou et al., 2008).	Facilitates taxonomic investigation.	Requires isolation and cultivation of lytic phages and hosts of temperate phages.
		Can assemble viral genomes for viruses excluded from metagenomic approaches (Martinez-Hernandez et al., 2017).	No community-wide view.
qPCR	77% of faecal samples contained phages carrying at least one antibiotic resistance gene (Quiros et al., 2014).	Detection and quantification of specific genes in real time.	Sequence of target gene is required a priori.
	Longitudinal tracking of phage and bacterial hosts in the faeces of a mouse model system facilitated the study of predator-prey dynamics (Hsu et al., 2019).		Nonspecific binding of template can lead to amplification of off-target genes.
Viral tagging & flow cytometry	363 unique phage-host pairings were predicted, including many uncharacterised phages (Džunková et al., 2019).	Infer phage-host relationships.	Direct evidence of successful phage infection is not provided by attachment of phage and host cell.
		Culture-independent.	Different assay conditions can bias the phage-host pairings observed from the community.
		Flow cytometry has the facility to sort individual phage-host pairs for downstream sequencing etc.