Dong, Xiaomin

Edwards, Steven

Deng, Yi-Mo

Dapat, Clyde

Hirankitti, Arada

Wordsworth, Rachel

Whitney, Paul

Baird, Robert

Freeman, Kevin

Daley, Andrew J

Barr, Ian G

2025-05-01

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.

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.

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

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.

School of Biomedical Sciences, The University of Western Australia, Perth, Western Australia, Australia.

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

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

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

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.

Royal Darwin Hospital, Tiwi, Northern Territory, Australia.

Royal Darwin Hospital, Tiwi, Northern Territory, Australia.

Department of Microbiology, The Royal Children's Hospital Melbourne, Parkville, Victoria, Australia.

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.

1750-2659

0000-0003-0302-9442

0000-0002-7351-418X

https://pubmed.ncbi.nlm.nih.gov/40296507/?otool=iaurydwlib

https://hdl.handle.net/10137/14263

ONT

rapid barcoding

respiratory syncytial virus

whole‐genome sequencing

Humans

Respiratory Syncytial Virus Infections

Whole Genome Sequencing

Respiratory Syncytial Virus, Human

Genome, Viral

High-Throughput Nucleotide Sequencing

Nanopores

Australia

Nanopore Sequencing

Multiplex Polymerase Chain Reaction

Sensitivity and Specificity

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

Journal Article

Publication