Clinical Performance Evaluation of a Tiling Amplicon Panel for Whole-Genome Sequencing of Respiratory Syncytial Virus

B Ethan Nunley; Amelia Weixler; Hyeong Geon Kim; Hong Xie; Jaydee Sereewit; Pooneh Hajian; Sean Ellis; Margaret G Mills; Ailyn C Pérez-Osorio; Stephanie Goya; Jolene Gov; Rebecca Dewar; Goncalo Fernandes; Kate E Templeton; Daniel M Maloney; Alexander L Greninger; Pavitra Roychoudhury

doi:10.1016/j.jmoldx.2025.05.005

Clinical Performance Evaluation of a Tiling Amplicon Panel for Whole-Genome Sequencing of Respiratory Syncytial Virus

J Mol Diagn. 2025 Jun 19:S1525-1578(25)00139-4. doi: 10.1016/j.jmoldx.2025.05.005. Online ahead of print.

Authors

B Ethan Nunley¹, Amelia Weixler¹, Hyeong Geon Kim¹, Hong Xie¹, Jaydee Sereewit¹, Pooneh Hajian¹, Sean Ellis¹, Margaret G Mills¹, Ailyn C Pérez-Osorio¹, Stephanie Goya¹, Jolene Gov¹, Rebecca Dewar², Goncalo Fernandes², Kate E Templeton², Daniel M Maloney³, Alexander L Greninger⁴, Pavitra Roychoudhury⁵

Affiliations

¹ Department of Laboratory Medicine and Pathology, University of Washington Medical Center, Seattle, Washington.
² Viral Genotyping Reference Laboratory Edinburgh, NHS Lothian, Royal Infirmary of Edinburgh, Edinburgh, United Kingdom.
³ Viral Genotyping Reference Laboratory Edinburgh, NHS Lothian, Royal Infirmary of Edinburgh, Edinburgh, United Kingdom; Institute of Ecology and Evolution, University of Edinburgh, Edinburgh, United Kingdom.
⁴ Department of Laboratory Medicine and Pathology, University of Washington Medical Center, Seattle, Washington; Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Center, Seattle, Washington.
⁵ Department of Laboratory Medicine and Pathology, University of Washington Medical Center, Seattle, Washington; Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Center, Seattle, Washington. Electronic address: proychou@uw.edu.

PMID: 40543557
DOI: 10.1016/j.jmoldx.2025.05.005

Abstract

Accurate genomic characterization of respiratory syncytial virus (RSV) is crucial for studies of epidemiology and viral evolution, including monitoring potential escape from newly authorized vaccines and prophylactic monoclonal antibodies. We adapted a viral genome tiling amplicon panel (UW-ARTIC) and developed a custom bioinformatic pipeline for high-throughput, cost-effective sequencing of both RSV-A and RSV-B subgroups. We established genome acceptability criteria and determined the performance characteristics of the panel, including assay sensitivity, specificity, breadth of genome recovery, accuracy, and precision, using contrived and remnant clinical specimens. High-quality genomes (>95% genome completeness; >500× and >1000× average depth for whole genome and fusion gene, respectively) were recovered from samples with cycle threshold ≤ 30 (approximately 594 and 2004 copies per reaction for RSV-A and RSV-B, respectively). Minor variants were accurately identified at >5% allele frequency. The assay showed high accuracy when compared with Sanger, shotgun metagenomic, and hybridization capture-based sequencing, as well as high repeatability and reproducibility. The UW-ARTIC RSV panel has utility for cost-effective RSV genome recovery in public health, clinical, and research applications. It has been used to generate US Food and Drug Administration-reportable data for clinical trials of RSV antiviral products, with robust performance in global samples from as recently as the 2023/2024 season. Continued genomic surveillance and future updates to primers will be essential for continued recovery of genomes as RSV continues to evolve.