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An Improved Rapid and Sensitive Long Amplicon Method for Nanopore-Based RSV Whole-Genome Sequencing

Xiaomin Dong^1,2, Steven Edwards¹, Yi-Mo Deng^1,2,3

¹WHO Collaborating Centre for Reference and Research on Influenza, Royal Melbourne Hospital, at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia.

Influenza and Other Respiratory Viruses

|April 29, 2025

Summary

This study presents a rapid, cost-effective whole-genome sequencing (WGS) method for respiratory syncytial virus (RSV) using Oxford Nanopore Technology (ONT). The optimized protocol enables efficient WGS of clinical RSV samples, aiding in tracking transmission and mutations.

Area of Science:

Virology
Genomics
Molecular Biology

Background:

Whole-genome sequencing (WGS) is crucial for understanding respiratory syncytial virus (RSV) transmission and mutations affecting vaccines and therapeutics.
Current large-scale RSV WGS methods are often costly and time-consuming.
Oxford Nanopore Technology (ONT) offers potential for faster and more affordable next-generation sequencing (NGS).

Purpose of the Study:

To develop and optimize an RSV WGS protocol for the ONT platform.
To assess the efficiency and effectiveness of this new protocol compared to existing methods.

Main Methods:

A modified RSV long amplicon-based WGS protocol was developed for ONT, incorporating a one-step multiplex RT-PCR and rapid barcoding kit.
135 Australian RSV clinical specimens (RSV-A and RSV-B) from 2023 were analyzed.
The ONT workflow was compared against short amplicon-based WGS assays.

Main Results:

A PCR amplicon clean-up step improved WGS for samples with low amplicon yield but was unnecessary for high-yield samples.
An overall WGS success rate of 85.9% was achieved.
The ONT method demonstrated comparable genome coverage and sequencing depth to more complex short amplicon methods.

Conclusions:

The developed ONT workflow is highly successful for generating RSV WGS from clinical samples.
This method significantly improves turnaround times and cost-effectiveness for RSV WGS.
The protocol yields excellent results for RSV clinical samples with cycle threshold values up to 30.

Keywords:

ONT rapid barcoding respiratory syncytial virus whole‐genome sequencing

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