A bench-to-data analysis workflow for respiratory syncytial virus whole-genome sequencing with short and long-read approaches

Respiratory syncytial virus (RSV) is a leading cause of acute respiratory illness, especially in children, immunocompromised patients, and the elderly. Genomic surveillance of RSV enables detecting and monitoring of circulating lineages and the emergence of amino acid substitutions affecting transmission, severity, and treatment, including resistance to monoclonal antibodies (mAb). Most available RSV genomic surveillance protocols target specific regions of the viral genome, such as the G and F genes, or are designed for a particular sequencing technology. Here, we aimed to develop and validate a comprehensive workflow for whole-genome RSV sequencing adapted for short- and long-read sequencing data, covering all steps from sample processing to lineage classification. Viral RNA was collected from 176 nasopharyngeal swab samples from patients in the Canary Islands (Spain) over two seasons between October 2022 and February 2024. Molecular diagnosis was performed with RT-qPCR and genomic libraries were prepared in parallel with tiling amplicon protocols adapted for Illumina and Oxford Nanopore Technologies (ONT) sequencing approaches. An in-house bioinformatics pipeline built on Nextflow was also developed to enable deployment, ensure built-in dependency management, and provide adaptability to both sequencing technologies. We found a high concordance between Illumina and ONT sequencing results in terms of consensus sequence, lineage assignment, and lineage-defining substitutions. We also identified RSV sequences harboring substitutions associated with mAbs resistance and patient coinfections. Taken together, the developed resources and results allowed us to provide for the first time detailed genomic data of RSV transmissions within the epidemiological context in the Canary Islands.