Optimization and evaluation of viral metagenomic amplification and sequencing procedures toward a genome-level resolution of the human fecal DNA virome

作者：Guangyang Wang, Shenghui Li, Qiulong Yan, Ruochun Guo, Yue Zhang, Fang Chen, Xiangge Tian, Qingbo Lv, Hao Jin, Xiaochi Ma, Yufang Ma · 发表于：Journal of Advanced Research · 年份：2022 · DOI：10.1016/j.jare.2022.08.011 · 被引用次数：45 · 研究领域：Bacteriophages and microbial interactions、Plant and Fungal Interactions Research、Genomics and Phylogenetic Studies

INTRODUCTION: Viruses in the human gut have been linked to health and disease. Deciphering the gut virome is dependent on metagenomic sequencing of the virus-like particles (VLPs) purified from the fecal specimens. A major limitation of conventional viral metagenomic sequencing is the low recoverability of viral genomes from the metagenomic dataset. OBJECTIVES: To develop an optimal method for viral amplification and metagenomic sequencing for maximizing the recovery of viral genomes. METHODS: We performed parallel virus enrichment and DNA extraction to generate ∼ 30 viral DNA samples from each of 5 fresh fecal specimens and conducted the experiments including 1) optimizing the cycle number for high-fidelity enzyme-based PCR amplification, 2) evaluating the reproducibility of the optimally whole viral metagenomic experimental process, 3) evaluating the reliability of multiple displacement amplification (MDA), 4) testing the capability of long-read sequencing for improving viral metagenomic assembly, and 5) comparing the differences between viral metagenomic and bulk metagenomic approaches. RESULTS: Our results revealed that the optimal cycle number for PCR amplification is 15. We verified the reliability of MDA and the effectiveness of long-read sequencing. Based on our optimized results, we generated 151 high-quality viruses using the dataset combined from short-read and long-read sequencing. Genomic analysis of these viruses found that most (60.3%) of them were previously u...