Targeted whole- genome recovery of single viral species in a complex environmental sample

• 成果类型: Article
• 署名作者: Chen, Liyin; Chen, Anqi; Zhang, Xinge Diana; Robles, Maria Teresa Saenz; Han, Hee- Sun; Xiao, Yi; Xiao, Gao; Pipas, James M.; Weitz, David A.
• 署名单位: Harvard University; Pennsylvania Commonwealth System of Higher Education (PCSHE); University of Pittsburgh; University of Illinois System; University of Illinois Urbana-Champaign; University of Illinois System; University of Illinois Urbana-Champaign; Harvard University
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-10663
• DOI: 10.1073/pnas.2404727121
• 发表日期: 2024-07-30
• 关键词: amplification; CHALLENGES

摘要

Characterizing unknown viruses is essential for understanding viral ecology and preparing against viral outbreaks. Recovering complete genome sequences from environmental samples remains computationally challenging using metagenomics, especially for low- abundance species with uneven coverage. We present an experimental method for reliably recovering complete viral genomes from complex environmental samples. Individual genomes are encapsulated into droplets and amplified using multiple displacement amplification. A unique gene detection assay, which employs an RNA- based probe and an exonuclease, selectively identifies droplets containing the target viral genome. Labeled droplets are sorted using a microfluidic sorter, and genomes are extracted for sequencing. We demonstrate this method's efficacy by spiking two known viral genomes, Simian virus 40 (SV40, 5,243 bp) and Human Adenovirus 5 (HAd5, 35,938 bp), into a sewage sample with a final abundance in the droplets of around 0.1% and 0.015%, respectively. We achieve 100% recovery of the complete sequence of the spiked- in SV40 genome with uniform coverage distribution. For the larger HAd5 genome, we cover approximately 99.4% of its sequence. Notably, genome recovery is achieved with as few as one sorted droplet, which enables the recovery of any desired genomes in complex environmental samples, regardless of their abundance. This method enables single- genome whole- genome amplification and targeting characterizations of rare viral species and will facilitate our ability to access the mutational profile in single- virus genomes and contribute to an improved understanding of viral ecology.