Development and application of methodology in the study of the gastrointestinal virome

Emerging metagenomic methods have provided the possibility to maximally restore the virome from the gut. This development opens new avenues for studying the unexplored components of the gut virome “dark matter” and its potential applications. Despite these advancements, there is a paucity of data on the effects of storage conditions (buffers, temperatures, and time) on virome samples and their impact on the viral metagenomic dataset. It is still a challenge to capture the gut virome due to the bias created by current sequencing library preparations.Moreover, the archaea virus is still under-exploration.

In Study 1, our findings reveal that phage recovery rates significantly vary depending on the preservation buffer. Specifically, over 90% of phages, particularly T4, were rapidly inactivated by StayRNA and RNAlater after spiking. Conversely, the DNA/RNA Shield buffer exhibited immediate detrimental effects across all the tested conditions, whereas SM buffer effectively maintained phage infectivity, especially at 4°C. Long-term preservation using CANVAX buffer was sufficient in maintaining phage genomes, but it induced the obvious alteration of viral diversity with prolonged storage. Short-term preservation at room temperature has minimal impact on phage infectivity or genomic integrity, as long as samples are promptly stabilized in appropriate buffers. This study highlights the critical role of buffer selection and storage conditions in preserving phage infectivity and maintaining viral community diversity within gut virome study.

To circumvent the limitations associated with existing library preparations, Study 2 introduces a rapid, straightforward, and efficient ligation-based single-stranded library (SSLR) preparation method. This method facilitates the generation of complex libraries within three hours, specifically for gut virome studies. The SSLR method has exceptional efficiency in the quality and quantity of the DNA genomes (dsDNA and ssDNA) and outperforms currentdouble-stranded (Nextera XT) and single-stranded (xGen) library preparation techniques, minimizing amplification bias and achieving nearly complete phage genome assembly.Furthermore, the versatility of SSLR not only allows for the simultaneous preparation of DNA and RNA genome libraries but also enables the identification of the modified viruses in the gut.We believe this innovated SSLR library preparation tool enhances gut virome studies and applies to various environmental samples, potentially revolutionizing virome research with its efficiency and comprehensive capabilities.

Study 3 addresses the current issue of global warming, with enteric methane production by ruminants has been identified as a significant contributor to greenhouse gas emissions. We investigated ruminal virome transfer as a novel microbiome modulation approach to reduce methane emissions in ruminants. By analyzing the microbiome and virome diversities between high and low methane-emitting cows and conducting in vitro virome transfers, we revealed the complexity of the rumen ecosystem. These results demonstrated a distinct microbial and viral community composition linked to methane emission levels, suggesting that virome transfer could be a promising strategy for mitigating methane production. However, the effectiveness of this approach is tempered by individual variations among cows, highlighting the challenges of applying virome transfer universally.

In Study 4, the highly expressed HMG-CoA reductase gene cassette that detoxified the inhibition of simvastatin is used as a selection marker for constructing a Cas6-deficient mutant in Methanobrevibacter smithii (SimS) through homologous recombination. Transformants were seen in half of the experimental trials using the natural transformation method, which was simple and quick to implement. The mutants that grown and demonstrated resistance to simvastatin (SimR), which was verified through subculturing the transformants. While SimR phenotype was clearly visible, there was only partial evidence for the homologous recombination, as the PCR was unable to amplify the entire recombined area at the targeted locations. These findings suggest that more research is required.

In summary, to deal with the study of gut virome lagged substantially behind and explore its potential application, this Ph.D. study delves into the critical aspects of gut virome storage conditions, introduces an innovative library preparation method for in-depth virome exploration, evaluates the potential of virome transfer for mitigating methane emissions from ruminants, and develop a Cas6 gene knockout system based on simvastatinresistance and natural transformation for Methanobrevibacter smithii. Theseinvestigations collectively advance our understanding of the diversity and complexity of gut virome and its future application. The insights obtained highlight the necessity for appropriate storage conditions, minimization of bias, and the usage of innovative methodologies in gut virome study, paving the way for future investigations aimed at exploiting the gut virome for clinical or environmental interventions.