During the past decade, environmental research has demonstrated that archaea are abundant and widespread in nature and play important ecological roles at a global scale. Currently, however, the majority of archaeal lineages cannot be cultivated under laboratory conditions and are known exclusively or nearly exclusively through metagenomics. A similar trend extends to the archaeal virosphere, where isolated representatives are available for a handful of model archaeal virus-host systems. Viral metagenomics provides an alternative way to circumvent the limitations of culture-based virus discovery and offers insight into the diversity, distribution, and environmental impact of uncultured archaeal viruses. Presently, metagenomics approaches have been successfully applied to explore the viromes associated with various lineages of extremophilic and mesophilic archaea, including Asgard archaea (Asgardarchaeota), ANME-1 archaea (Methanophagales), thaumarchaea (Nitrososphaeria), altiarchaea (Altiarchaeota), and marine group II archaea (Poseidoniales). Here, we provide an overview of methods widely used in archaeal virus metagenomics, covering metavirome preparation, genome annotation, phylogenetic and phylogenomic analyses, and archaeal host assignment. We hope that this summary will contribute to further exploration and characterization of the enigmatic archaeal virome lurking in diverse environments.

BACKGROUND: Clade 5 Clostridioides difficile diverges significantly from the other clades and is therefore, attracting increasing attention due its great heterogeneity. In this study, we used third-generation sequencing techniques to sequence the complete whole genomes of three ST11 C. difficile isolates, RT078 and another two new ribotypes (RTs), obtained from three independent hospitalized elderly patients undergoing antibiotics treatment. Mobile genetic elements (MGEs), antibiotic-resistance, drug resistance genes, and virulent-related genes were analyzed and compared within these three isolates.
RESULTS: Isolates 10,010 and 12,038 carried a distinct deletion in tcdA compared with isolate 21,062. Furthermore, all three isolates had identical deletions and point-mutations in tcdC, which was once thought to be a unique characteristic of RT078. Isolate 21,062 (RT078) had a unique plasmid, different numbers of transposons and genetic organization, and harboring special CRISPR spacers. All three isolates retained high-level sensitivity to 11 drugs and isolate 21,062 (RT078) carried distinct drug-resistance genes and loss of numerous flagellum-related genes.
CONCLUSIONS: We concluded that capillary electrophoresis based PCR-ribotyping is important for confirming RT078. Furthermore, RT078 isolates displayed specific MGEs, indicating an independent evolutionary process. In the further study, we could testify these findings with more RT078 isolates of divergent origins.