Department of Civil and Environmental Engineering, University of California, Berkeley, California 94720, United States.
Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, California 94550, United States.
Environ Sci Technol. 2024 May 14;58(19):8239-8250. doi: 10.1021/acs.est.4c00580. Epub 2024 May 1.
Sequencing human viruses in wastewater is challenging due to their low abundance compared to the total microbial background. This study compared the impact of four virus concentration/extraction methods (Innovaprep, Nanotrap, Promega, and Solids extraction) on probe-capture enrichment for human viruses followed by sequencing. Different concentration/extraction methods yielded distinct virus profiles. Innovaprep ultrafiltration (following solids removal) had the highest sequencing sensitivity and richness, resulting in the successful assembly of several near-complete human virus genomes. However, it was less sensitive in detecting SARS-CoV-2 by digital polymerase chain reaction (dPCR) compared to Promega and Nanotrap. Across all preparation methods, astroviruses and polyomaviruses were the most highly abundant human viruses, and SARS-CoV-2 was rare. These findings suggest that sequencing success can be increased using methods that reduce nontarget nucleic acids in the extract, though the absolute concentration of total extracted nucleic acid, as indicated by Qubit, and targeted viruses, as indicated by dPCR, may not be directly related to targeted sequencing performance. Further, using broadly targeted sequencing panels may capture viral diversity but risks losing signals for specific low-abundance viruses. Overall, this study highlights the importance of aligning wet lab and bioinformatic methods with specific goals when employing probe-capture enrichment for human virus sequencing from wastewater.
由于废水中人类病毒的丰度相对较低，与总微生物背景相比，对其进行测序具有挑战性。本研究比较了四种病毒浓缩/提取方法（Innovaprep、Nanotrap、Promega 和 Solids extraction）对探针捕获富集人类病毒后的测序的影响。不同的浓缩/提取方法产生了不同的病毒谱。Innovaprep 超滤（在去除固体物之后）具有最高的测序灵敏度和丰富度，成功组装了几个接近完整的人类病毒基因组。然而，与 Promega 和 Nanotrap 相比，它在通过数字聚合酶链反应（dPCR）检测 SARS-CoV-2 时的灵敏度较低。在所有的制备方法中，星状病毒和多瘤病毒是丰度最高的人类病毒，而 SARS-CoV-2 则很少见。这些发现表明，使用可以减少提取物中非目标核酸的方法可以提高测序成功率，尽管绝对浓度的总提取核酸（如 Qubit 所示）和靶向病毒（如 dPCR 所示）可能与靶向测序性能没有直接关系。此外，使用广泛靶向的测序面板可能会捕获病毒多样性，但存在失去特定低丰度病毒信号的风险。总体而言，本研究强调了在使用探针捕获富集从废水中对人类病毒进行测序时，将湿实验室和生物信息学方法与特定目标相匹配的重要性。
