The emergence of SARS-CoV-2 variants of concern (VOCs) and variants of interest (VOIs) poses an increased risk to global public health and underlines the need to prioritise monitoring and research to better respond to the COVID-19 pandemic. Wastewater monitoring can be used to monitor SARS-CoV-2 spread and to track SARS-CoV-2 variants. A long read amplicon sequencing approach based on the Oxford Nanopore technology, targeting the spike protein, was applied to detect SARS-CoV-2 variants in sewage samples collected in central Italy on April 2021. Next-generation sequencing was performed on three pooled samples. For variant identification, two approaches–clustering (unsupervised) and classification (supervised)–were implemented, resulting in the detection of two VOCs and one VOI. Key mutations of the Alpha variant (B.1.1.7) were detected in all of the pools, accounting for the vast majority of NGS reads. In two different pools, mutations of the Gamma (P.1) and Eta (B.1.525) variants were also detected, accounting for 22.4%, and 1.3% of total NGS reads of the sample, respectively. Results were in agreement with data on variant circulation in Italy at the time of wastewater sample collection. For each variant, in addition to the signature key spike mutations, other less common mutations were detected, including the amino acid substitutions S98F and E484K in the Alpha cluster (alone and combined), and S151I in the Eta cluster. Results of the present study show that the long-read sequencing nanopore technology can be successfully used to explore SARS-CoV-2 diversity in sewage samples, where multiple variants can be present, and that the approach is sensitive enough to detect variants present at low abundance in wastewater samples. In conclusion, wastewater monitoring can help one discover the spread of variants in a community and early detect the emerging of clinically relevant mutations or variants.

中文翻译：

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基于纳米孔技术的长读长扩增子测序在意大利城市废水中检测到 SARS-CoV-2 的 Alpha、Gamma 和 Eta 变体的关键突变

SARS-CoV-2 关注变体 (VOC) 和关注变体 (VOI) 的出现给全球公共卫生带来了更大的风险，并强调需要优先进行监测和研究，以更好地应对 COVID-19 大流行。废水监测可用于监测 SARS-CoV-2 的传播和追踪 SARS-CoV-2 的变异。2021 年 4 月，在意大利中部收集的污水样本中，采用基于牛津纳米孔技术的长读长扩增子测序方法检测刺突蛋白，检测 SARS-CoV-2 变异。对三个混合样本进行了新一代测序。对于变体识别，实施了聚类（无监督）和分类（监督）两种方法，从而检测到两种 VOC 和一种 VOI。Alpha 变体的关键突变（B.1.1. 7) 在所有池中检测到，占 NGS 读数的绝大多数。在两个不同的池中，还检测到 Gamma (P.1) 和 Eta (B.1.525) 变体的突变，分别占样本总 NGS 读数的 22.4% 和 1.3%。结果与收集废水样品时意大利的变异循环数据一致。对于每个变体，除了特征键尖峰突变外，还检测到其他不太常见的突变，包括 Alpha 簇（单独和组合）中的氨基酸替换 S98F 和 E484K，以及 Eta 簇中的 S151I。本研究结果表明，长读长测序纳米孔技术可成功用于探索污水样本中 SARS-CoV-2 的多样性，其中可能存在多种变异，并且该方法足够灵敏，可以检测废水样品中低丰度存在的变异。总之，废水监测可以帮助人们发现变异在社区中的传播，并及早发现临床相关突变或变异的出现。