John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138.
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260.
Proc Natl Acad Sci U S A. 2024 Jul 30;121(31):e2404727121. doi: 10.1073/pnas.2404727121. Epub 2024 Jul 25.
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.
对未知病毒进行特征分析对于了解病毒生态学和防范病毒爆发至关重要。使用宏基因组学从环境样本中恢复完整的基因组序列仍然具有计算挑战性，尤其是对于丰度低且覆盖不均匀的物种。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。将单个基因组包裹在液滴中，并使用多次置换扩增进行扩增。一种独特的基因检测方法，采用基于 RNA 的探针和外切酶，选择性地识别包含目标病毒基因组的液滴。使用微流控分选器对标记的液滴进行分选，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，猴病毒 40（SV40，5243bp）和人腺病毒 5（HAd5，35938bp），分别以大约 0.1%和 0.015%的最终丰度掺入到污水样本中，证明了该方法的有效性。我们实现了掺入的 SV40 基因组完整序列的 100%回收，且具有均匀的覆盖分布。对于更大的 HAd5 基因组，我们覆盖了其序列的大约 99.4%。值得注意的是，仅使用一个分选液滴即可实现基因组的回收，这使得能够从复杂的环境样本中回收任何所需的基因组，而不论其丰度如何。该方法可实现单基因组全基因组扩增和靶向稀有病毒物种的特征分析，有助于我们获取单病毒基因组中的突变特征，并提高对病毒生态学的理解。
