High-sensitivity whole-genome recovery of single viral species in environmental samples.
High-sensitivity whole-genome recovery of single viral species in environmental samples.
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环境样本中单一病毒物种的高灵敏度全基因组恢复。
DOI：
10.1101/2023.11.13.566948
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Weitz,DavidA
中科院分区：
文献类型：
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作者：
Chen,Liyin;Chen,Anqi;Zhang,XingeDiana;Robles,MariaSaenzT;Han,Hee-Sun;Xiao,Yi;Xiao,Gao;Pipas,JamesM;Weitz,DavidA
描述未知病毒的特征对于了解病毒生态和预防病毒爆发至关重要。使用宏基因组学从环境样品中恢复完整的基因组序列在计算上仍然具有挑战性，特别是对于覆盖不均匀的低丰度物种。这项工作提出了一种从复杂的环境样品中可靠地回收完整病毒基因组的方法。将单个基因组封装到液滴中并使用多重置换扩增进行扩增。一种新的基因检测方法，它采用了基于RNA的探针和核酸外切酶，选择性地识别含有靶病毒基因组的液滴。使用微流体分选仪对标记的液滴进行分选，并提取基因组进行测序。验证实验使用污水样品与两种已知的病毒证明了该方法的有效性。我们实现了具有均匀覆盖分布的加标SV40（猿猴病毒40，5243 bp）基因组序列的100%回收率，以及较大HAd5基因组（人腺病毒5，35938 bp）的约99.4%回收率。值得注意的是，用少至一个分选的液滴实现基因组回收，这使得能够在复杂的环境样品中回收任何期望的基因组，而不管它们的丰度。该方法能够对罕见病毒物种进行靶向表征，并对单个基因组进行全基因组扩增，以获得单个病毒基因组中的突变谱，有助于提高对病毒生态学的理解。
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. This work presents a method for reliably recovering complete viral genomes from complex environmental samples. Individual genomes are encapsulated into droplets and amplified using multiple displacement amplification. A novel 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. Validation experiments using a sewage sample spiked with two known viruses demonstrate the method’s efficacy. We achieve 100% recovery of the spiked-in SV40 (Simian virus 40, 5243bp) genome sequence with uniform coverage distribution, and approximately 99.4% for the larger HAd5 genome (Human Adenovirus 5, 35938bp). 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 targeted characterizations of rare viral species and whole-genome amplification of single genomes for accessing the mutational profile in single virus genomes, contributing to an improved understanding of viral ecology.