Rapid-Response Viral Genome Detection using TWIST Capture and Nanopore Flongle Sequencing

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Abstract Background Rapid detection of viral pathogens can be challenging, especially when routine PCR fails. Conventional assays typically detect known viruses which are specifically targeted by the assay, which may result in the failure to identify novel or non-targeted viruses. Broad-range hybrid-capture sequencing enables unbiased detection of viruses, including those that are uncommon or divergent. Methods We combined the TWIST Comprehensive Viral Research Panel (>3,000 virus species) with Oxford Nanopore Flongle sequencing for easy and quick viral genome detection. The workflow includes random-primed cDNA synthesis, dsDNA conversion, TWIST probe enrichment, and Nanopore sequencing. Performance was evaluated using the QCMD 2024 Viral Metagenomics EQA panel and one clinical sample. Results All expected targets of the QCMD 2024 Viral Metagenomics EQA panel were detected; eight of thirteen viruses achieved ≥90% genome coverage. The negative control showed no targeted viral reads. Mixed infections of DNA and RNA viruses were resolved accurately. The workflow from nucleic acid extraction to obtaining sequence data was completed within 3 days. Conclusions Combination of Flongle and hybrid-capture offers a rapid, sensitive sequencing method for detecting diverse viral genomes, suitable for outbreak response, urgent diagnostics, and surveillance of multiple divergent viruses at once.

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