Table 3.

Bioinformatic tools used to study the gut phageome, including techniques, research questions they may address and name of the software.

Technique	Research questions	Tools	Examples in publication
Annotate genomes	Do the phage genomes encode auxiliary metabolic genes (AMGs)?	Prodigal (Hyatt et al., 2010) or PHANOTATE (McNair et al., 2019) to predict coding sequences.
	Do the phage genomes encode viral taxon-specific genes?	Prokka (Seemann, 2014) or eggNOG (Powell et al., 2012), to functionally annotate draft genomes.
	Are the phages temperate or virulent?	Hmmscan (Eddy, 2011), to align protein sequences to databases (e.g. pVOGs (Grazziotin et al., 2016) and Pfam/vFam (Skewes-Cox et al., 2014; El-Gebali et al., 2019) and Genecatalog (Li et al., 2014).	(Clooney et al., 2019)
	Is there a global phage gene pool?	Phyre2 (Kelley et al., 2015), to find proteins with structural homology .
		DRAM-v to predict putative AMGs associated with metabolism (Shaffer et al., 2020).
Phage prediction	Are the genomes assembled phage genomes?	De-novo prediction of phages using tools such as VirFinder/ DeepVirFinder (Ren et al. 2017; Ren et al., 2002) and VirSorter2 (Guo et al., 2021)
		Confirmation of viral genome completeness with CheckV (Nayfach et al., 2020) and IMG/VR v3 (Roux et al., 2021).
	How diverse are the phages?	Alignment to hallmark taxon-specific marker genes.	(Waller et al., 2014)
	Are any known viral taxa predicted?	BLAST (Camacho et al., 2009), PhymmBL (Brady and Salzberg, 2009) and k-mer approaches (Trifonov and Rabadan, 2010), for taxonomic assignment.	(Vazquez-Castellanos et al., 2014)
		Comparison to gut viral databases, e.g. Gut Virome Database (GVD) (Gregory et al., 2020).
	How similar are they to phages in other ecosystems?	Single protein phylogenies with alignment of core phage genes, e.g. using MAFFT (Katoh and Standley, 2013).
		De-novo classification with vConTACT2 (Zablocki et al., 2019).
Phage-host relationships	Which gut hosts do the phages infect?	Genetic homology of integration sites and CRISPR spacer sequences between phage and host.	(Stern et al., 2012)
	Is phage predation species or strain specific?	Phage genome signature-based recovery.	(Ogilvie et al., 2013)
		Identification of integrated prophage.
		Abundance profiles.	(Dutilh et al., 2014)
		De-novo relationships with WIsH (Galiez et al., 2017)