Table 2. Limitations of viral sequencing compared with bacterial sequencing.

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Feature	Bacteria	Viruses	Challenges
Genome	dsDNA	dsDNA, ssDNA, partially dsDNA, ssRNA or dsRNA	Different extraction protocols for different viruses. RNA viruses require cDNA synthesis and ssDNA second strand synthesis
Gene conservation	Highly conserved, essential genes (for example, 16s rRNA) enabling broad microbiome studies and surveys of taxa	No homologous genes between viruses of different phyla	Lack of conserved homology between viral phyla prevents universal primer-based surveys of viromes
Culture	Often straightforward to culture and obtain pure, highly enriched bacterial DNA and RNA	Challenging to culture, and require a host cell for replication	Cultured viruses are heavily contaminated with host cell nucleic acids, which decreases viral sequencing output
Clinical specimens	Hardy bacterial cells with cell walls can often be separated from human cells in clinical specimens using differential lysis methods or flow cytometry144 prior to extraction	Viruses are intracellular pathogens, and although separation from the host is possible (for example, by filtration or antibody pull-down), viruses cannot easily be separated from clinical samples prior to extraction	Clinical specimens are heavily contaminated with host nucleic acids, which decreases viral sequencing output
Methylation patterns	Bacteria use different methylation patterns from eukaryotes; host DNA can be depleted post-extraction using restriction endonucleases that are directed against CpG methylation145	DNA viruses are often methylated by the host intracellular machinery, and may have similar methylation patterns	DNA digestion according to methylation patterns is less effective as a means of host depletion for viral sequencing

cDNA, complementary DNA; dsDNA, double-stranded DNA; dsRNA, double-stranded RNA; rRNA, ribosomal RNA; ssDNA, single-stranded DNA; ssRNA, single-stranded RNA.