TITAN-RNA: A hybrid-capture sequencing panel detects known and unknown Flaviviridae for diagnostics and vector...

Clinical testing and public health surveillance can be significantly improved by incorporating sequencing-based molecular detection and subtyping for real-time monitoring of virus evolution. With phylogenetic analysis used for speciation and variant subtyping, target analyte specificity can be relaxed well beyond typical parameters acceptable in PCR-based diagnostics. Hybrid capture is a promising way to enrich large numbers of sequences with maximal flexibility, using standard molecular biology laboratory equipment and small benchtop sequencers. Here, we report the development and bench validation of a hybrid capture based next-generation sequencing diagnostic panel for all known viral tick-borne pathogens, TITAN-RNA. Based on systematic testing with simulated novel viruses and field samples, we determined a 10% tolerance for evenly distributed mutations or 27% tolerance for naturally occurring viral divergence. The TITAN-RNA extrapolated limit of detection in blood is 19.1 genome copies by complementary log-log analysis, and linearity performance (R² ≥ 0.99) is amenable for its use as a quantitative assay. As proof of principle for public health surveillance and evolutionary studies, we report two putatively novel segmented Flavi-like viruses in New York State, USA, identified from the invasive Haemaphysalis longicornis tick.