Optimization and Ev a Luation of Viral Metagenomic Amplification and Sequencing Methods Toward a Genome -le Vel Resolution of the Human Fecal DNA Virome

Abstract

Background: Viruses in the human gut have been linked to health and disease. Deciphering of the gut virome is dependent on metagenomic sequencing of the virus-like particles purified from the fecal specimens. A major limitation of conventional viral metagenomic sequencing is the low recoverability of viral genomes from the metagenomic dataset. Results: Herein, we developed an optimal method for viral amplification and metagenomic sequencing to maximize the recovery of viral genomes. Using 5 fecal specimens with multiple repetitions, we revealed the optimal number of PCR cycles of high-fidelity enzyme-based amplification and the reliability of multiple displacement amplification in virome DNA preparation, verified the reproducibility of the optimally whole viral metagenomic experimental process, and tested the capability of long-read sequencing for improving viral metagenomic assembly. Based on our optimized results, we generated 151 high-quality viruses using the data combined from short-read (15 cycles for PCR amplification) and long-read sequencing. Genomic analysis of these viruses found that most (60.3%) of them were previously unknown and showed a remarkable diversity of viral functions, especially the existence of 206 viral auxiliary metabolic genes. Finally, we compared the viral metagenomic and bulk metagenomic sequencing approaches and revealed significant differences in the efficiency and coverage of viral identification between them. Conclusions: Our study demonstrates the potential of optimized experiment and sequencing strategies in uncovering viral genomes from fecal specimens, which will facilitate future research about genome-level characterization of complex viral communities.

Cataloguing and profiling of the gut virome in Chinese populations uncover extensive viral signatures across common diseases

“…4b). The substantial higher level of Microviridae viruses in the viral samples may because that the VLP technology is more inclined to target free viral particles, agreeing with the previous studies 6, 35 .…”