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.

中文翻译：

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对复杂环境样本中的单个病毒物种进行靶向全基因组回收


表征未知病毒对于了解病毒生态学和准备应对病毒爆发至关重要。使用宏基因组学从环境样本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。我们提出了一种从复杂环境样本中可靠地恢复完整病毒基因组的实验方法。将单个基因组封装成液滴，并使用多重置换扩增进行扩增。一种独特的基因检测检测方法采用基于 RNA 的探针和核酸外切酶，可选择性地识别含有目标病毒基因组的液滴。使用微流控分选仪对标记的液滴进行分类，并提取基因组进行测序。我们通过将两个已知的病毒基因组，即猿猴病毒 40 （SV40,5,243 bp） 和人腺病毒 5 （HAd5,35,938 bp） 加标到污水样品中来证明该方法的有效性，液滴中的最终丰度分别为约 0.1% 和 0.015%。我们实现了加标 SV40 基因组完整序列的 100% 回收率，覆盖分布均匀。对于较大的 HAd5 基因组，我们覆盖了大约 99.4% 的序列。值得注意的是，只需一个分选的液滴即可实现基因组恢复，从而能够在复杂的环境样本中回收任何所需的基因组，无论其丰度如何。这种方法能够实现单基因组全基因组扩增和稀有病毒物种的靶向表征，并将有助于我们获取单病毒基因组中的突变谱，并有助于更好地了解病毒生态学。