Adapting the Illumina COVIDSeq for Whole Genome Sequencing of Other Respiratory Viruses in Multiple Workflows and a Single Rapid Workflow

Acute respiratory infections (ARIs) continue to pose a major global health threat, particularly among vulnerable populations. These infections often present with similar clinical symptoms, complicating accurate diagnosis and facilitating unmonitored transmissions. Genomic surveillance has emerged as an invaluable tool for pathogen identification and monitoring of such infectious pathogens; however, its implementation is frequently limited by high costs. The widespread use of high-throughput sequencing during the COVID-19 pandemic has created an opportunity to repurpose existing genomic platforms for broader respiratory virus surveillance. In this study, we evaluated the feasibility of adapting the Illumina COVIDSeq assay—initially designed for SARS-CoV-2 whole-genome sequencing—for use with Influenza A/B, Respiratory Syncytial Virus (RSV), and Rhinovirus. Positive control samples were processed using two approaches for library preparation: four virus-specific multiple workflows and a combined rapid workflow. Both workflows incorporated pathogen-specific primers for amplification and followed the Illumina COVIDSeq protocol for library preparation and sequencing. Sequencing quality metrics were analysed, including Phred scores, read length distribution, and coverage depth. The study did not identify significant differences in genome coverage and genetic diversity metrics between workflows. Genome Detective consistently identified the correct species across both methods. The findings of this study demonstrate that the COVIDSeq assay can be effectively adapted for multi-pathogen genomic surveillance and that the combined rapid workflow can offer a cost- and labour-efficient alternative with minimal compromise to data quality.