Long Amplicon Nanopore Sequencing for Dual-Typing RdRp and VP1 Genes of Norovirus Genogroups I and II in Wastewater

Noroviruses are the leading cause of non-bacterial gastroenteritis across the globe costing US$60.3 billion per annum. Our project involved developing and optimising a nanopore-based method for typing genogroups I (GI) and II (GII) by sequencing partial RNA-dependent RNA polymerase and major structural protein genes from a single fragment of DNA amplified from wastewater. Reverse transcription, PCR and library pooling were optimised for the sample matrix and a consensus-based bioinformatics pipeline was developed. To confirm accuracy of consensus sequences derived from nanopore sequencing data three faecal samples from norovirus positive patients were sequenced using both nanopore and sanger sequencing and compared, to ensure both approaches yeilded equivilient results. After optimisation of experimental, sequencing and bioinformatic protocols the technique was applied to samples which were collected from hundreds of sites in England around October 2021, as part of the SARS-CoV-2 wastewater programme, and pooled into ten samples representing different geographical regions. Within the ten pooled samples 13 GI and 8 GII dual types were identified, highlighting the diversity of norovirus taxa present in wastewater in England at a single point in time.

诺如病毒（Norovirus）是全球范围内引发非细菌性胃肠炎的首要病原体，每年造成的经济损失高达603亿美元。本项目旨在开发并优化一套基于纳米孔测序（nanopore sequencing）的基因分型方法，用于鉴定诺如病毒的基因I型（GI）与基因II型（GII）：该方法通过对从污水样本中扩增得到的单一片段DNA进行测序，靶向覆盖部分RNA依赖的RNA聚合酶（RNA-dependent RNA polymerase）基因与主要结构蛋白基因。针对污水样本基质，我们对逆转录、聚合酶链式反应（PCR）以及文库混合步骤进行了优化，并搭建了基于一致性序列的生物信息学分析流程。为验证基于纳米孔测序数据得到的一致性序列的准确性，我们选取3份诺如病毒阳性患者的粪便样本，分别采用纳米孔测序与桑格测序（Sanger sequencing）进行测序并比对，以确认两种方法的检测结果具有一致性。在优化实验、测序及生物信息学分析流程后，我们将该方法应用于2021年10月前后从英国数百个采样点收集的污水样本：这些样本作为严重急性呼吸综合征冠状病毒2（SARS-CoV-2）污水监测项目的一部分，被混合为10份代表不同地理区域的合并样本。在这10份合并样本中，共鉴定出13例GI型与8例GII型诺如病毒的双重分型，展现了同一时间点英国污水中存在的诺如病毒类群多样性。