Li Yabing, Bhatt Pankaj, Xagoraraki Irene
Department of Civil and Environmental Engineering, Michigan State University, East Lansing, MI, USA.
Department of Civil and Environmental Engineering, Michigan State University, East Lansing, MI, USA.
Water Res. 2025 Sep 1;283:123803. doi: 10.1016/j.watres.2025.123803. Epub 2025 May 10.
Sequencing approaches may enable monitoring of a broad range of viruses in wastewater, including potential emerging and non-reportable human viruses. Considering the fact that metagenomic sequencing may be non-specific for low-abundance human viruses, integration of viral amplification and enrichment strategies are proposed to enhance the accurate detection of a broad range of human viruses in municipal wastewater. In this study, we focused on the in-depth comparison analysis of three untargeted amplification methods (Multiple Displace Amplification [MDA], Reverse Transcription - MDA [RT-MDA], and a PCR-based random amplification [PCR-based]) and one targeted method (Twist Comprehensive Viral Research Panel [TWIST]) for detecting virus diversity in wastewater. In addition, we included the comparisons of two extraction kits (Qiagen QIAamp VIRAL RNA Mini Kit and ZymoBIOMICSTM DNA/RNA Minipre Kit) and four virus identification tools (Diamond blast, Kraken2, VirSorter2 and geNomad) for a systematic study. Performances of Qiagen and Zymo extraction kits in recovering viruses and human viruses in wastewater were comparable. By the three untargeted methods we detected 12,808 contigs with lengths longer than 10,000 bp. No contig longer than 10,000 bp was detected by the targeted method. Presence of human viruses were analyzed further by comparing the viral contigs against a custom Swiss-Prot human virus database. There were 45 viruses that are potentially associated with human health found in wastewater, 8 of them were unique to the targeted method and 7 of them were unique to the three untargeted methods. Four enteric viruses Mamastrovirus, Norovirus, Rotavirus and Sapovirus were detected with high abundance in samples prepared with the targeted method. Dimensional scaling analysis demonstrated the divergent virus and human virus communities from the untargeted and targeted methods. Patterns of virus and human virus populations identified by Kraken2 and geNomad were similar. Presence of selected viruses (SARS-CoV-2 [N1&N2], SC2, RSV, Norovirus GI and GII) were confirmed with ddPCR. This work indicates integration of untargeted and targeted sequencing methods, and complementary ddPCR can ensure the accurate detection of known and novel viruses using wastewater surveillance.
测序方法或许能够监测废水中的多种病毒，包括潜在的新出现的和未报告的人类病毒。鉴于宏基因组测序可能对低丰度人类病毒缺乏特异性，因此建议整合病毒扩增和富集策略，以提高对城市废水中多种人类病毒的准确检测。在本研究中，我们重点对三种非靶向扩增方法（多重置换扩增[MDA]、逆转录 - MDA[RT - MDA]和基于PCR的随机扩增[基于PCR的]）和一种靶向方法（Twist综合病毒研究面板[TWIST]）进行深入比较分析，以检测废水中的病毒多样性。此外，我们还纳入了两种提取试剂盒（Qiagen QIAamp VIRAL RNA Mini试剂盒和ZymoBIOMICSTM DNA/RNA微量制备试剂盒）以及四种病毒鉴定工具（Diamond blast、Kraken2、VirSorter2和geNomad）的比较，以进行系统研究。Qiagen和Zymo提取试剂盒在回收废水中的病毒和人类病毒方面表现相当。通过三种非靶向方法，我们检测到12,808条长度超过10,000 bp的重叠群。靶向方法未检测到长度超过10,000 bp的重叠群。通过将病毒重叠群与自定义的Swiss - Prot人类病毒数据库进行比较，进一步分析人类病毒的存在情况。在废水中发现了45种可能与人类健康相关的病毒，其中8种是靶向方法独有的，7种是三种非靶向方法独有的。在用靶向方法制备的样本中，检测到四种肠道病毒，即星状病毒、诺如病毒、轮状病毒和札如病毒，丰度较高。维度缩放分析表明，非靶向方法和靶向方法的病毒和人类病毒群落存在差异。Kraken2和geNomad鉴定的病毒和人类病毒种群模式相似。通过数字滴度PCR（ddPCR）确认了所选病毒（严重急性呼吸综合征冠状病毒2[SARS-CoV-2][N1&N2]、SC2、呼吸道合胞病毒、诺如病毒GI和GII）的存在。这项工作表明，非靶向和靶向测序方法的整合以及互补的ddPCR能够确保利用废水监测准确检测已知和新型病毒。
