Murrell, C. M.; Sabaawy, A.; Wang, X.; Hashimoto, N.; Ceres, K.; Sun, Y.; Zehr, J. D.; Lehane, A.; Mader, E.; Bailey, N.; Lilly, M. V.; Plimpton, L.; Reboul, G.; Brown, J. J.; Sams, K. L.; Singh, L.; Seiz, E.; Bourgikos, E.; Vogels, C. B. F.; Ciota, A.; Schnurr, V.; Grenier, J. K.; Berthet, X.; Bento, A. I.; Harrington, L. C.; Diuk-Wasser, M.; Olarte-Castillo, X.; Goodman, L. B.

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 (R^2 [≥] 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.