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

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. A viral genome tiling amplicon panel (UW-ARTIC) was adapted to develop a custom bioinformatic pipeline for high-throughput, cost-effective sequencing of both RSV-A and RSV-B subgroups. Genome acceptability criteria were established and the performance characteristics of the panel were determined, 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.