Dong Xiaomin, Edwards Steven, Deng Yi-Mo, Dapat Clyde, Hirankitti Arada, Wordsworth Rachel, Whitney Paul, Baird Rob, Freeman Kevin, Daley Andrew J, Barr Ian G
WHO Collaborating Centre for Reference and Research on Influenza, Royal Melbourne Hospital, at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia.
Department of Microbiology and Immunology, University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia.
Influenza Other Respir Viruses. 2025 May;19(5):e70106. doi: 10.1111/irv.70106.
Whole-genome sequencing (WGS) provides critical insights into the respiratory syncytial virus (RSV) transmission and any emerging mutations that could impair the efficacy of monoclonal antibodies or vaccines that have been recently licenced for clinical use worldwide. However, the ability to sequence RSV genomes at large scale is limited by expensive and time-consuming sequencing methods. Oxford Nanopore Technology (ONT) offers significant improvements in next generation sequencing (NGS) both in turnaround time and cost, compared with other platforms for viral WGS.
We have developed and modified an RSV long amplicon-based WGS protocol for the ONT platform using a one-step multiplex RT-PCR assay and the rapid barcoding kit. One hundred thirty-five RSV positive Australian clinical specimens (91 RSV-A and 44 RSV-B) sampled in 2023 with cycle threshold (Ct) values between 14 to 35 were tested in this study. This ONT workflow was compared with other recent RSV WGS amplification assays based on short amplicons.
A PCR amplicon clean-up step prior to library preparation significantly improved WGS result for samples with poor amplicon generation, but it is not necessary or beneficial for ones that generated high concentrations of amplicons. Overall, a success rate of 85.9% was achieved for WGS. This method performed as well as the more complex short amplicon methods in terms of genome coverage and sequencing depth.
The workflow described here was highly successful in generating RSV WGS on ONT platform and had improved turnaround times and excellent results with RSV clinical samples with Ct values up to 30.
全基因组测序（WGS）为呼吸道合胞病毒（RSV）的传播以及可能影响全球近期获批临床使用的单克隆抗体或疫苗效力的任何新出现突变提供了关键见解。然而，大规模测序RSV基因组的能力受到昂贵且耗时的测序方法的限制。与其他用于病毒全基因组测序的平台相比，牛津纳米孔技术（ONT）在周转时间和成本方面为下一代测序（NGS）带来了显著改进。
我们使用一步多重逆转录聚合酶链反应（RT-PCR）检测和快速条形码试剂盒，为ONT平台开发并改进了基于RSV长扩增子的全基因组测序方案。本研究检测了2023年采集的135份RSV阳性澳大利亚临床样本（91份RSV-A和44份RSV-B），其循环阈值（Ct）值在14至35之间。将这种ONT工作流程与其他基于短扩增子的近期RSV全基因组测序扩增检测方法进行了比较。
文库制备前的PCR扩增子纯化步骤显著改善了扩增子生成较差的样本的全基因组测序结果，但对于生成高浓度扩增子的样本来说并非必要或有益。总体而言，全基因组测序的成功率达到了85.9%。在基因组覆盖度和测序深度方面，该方法与更复杂的短扩增子方法表现相当。
本文所述的工作流程在ONT平台上成功生成RSV全基因组测序结果方面非常成功，周转时间有所改善，对于Ct值高达30的RSV临床样本也能获得出色结果。
