Comprehensive viral oligonucleotide probe design using conserved protein regions

Jabado, Omar J.; Liu, Yang; Conlan, Sean; Quan, P. Lan; Hegyi, Hédi; Lussier, Yves; Briese, Thomas; Palacios, Gustavo; Lipkin, W. I.

The thresholds used to design and validate probes were experimentally determined for the Agilent Technologies array platform and the types of clinical samples our Figure 6 . Gibbs free energy model of hybridization signal. The change in Gibbs free energy of probe-West Nile virus hybrids was computed. Aliquots of West Nile virus (New York 1999 strain RNA) at 10 6 copies were spiked into 200 ng of human lung (background) RNA. The fluorescent signal values of replicate arrays were log 2 transformed, normalized, and converted to Z-scores. 95% confidence intervals of the mean for fluorescence versus Gibbs energy is plotted. Probe-virus hybrids with free energy -32.5 kJ had high fluorescence; this value was chosen as the threshold for considering a probe likely to generate a strong signal when the target virus is present (dotted line). laboratory encounters. Probe length can be selected to emphasize efficient coverage of higher order taxa or speciation. The goal of this project is to cover all known viral sequences and optimize potential for detecting related viral sequences. Thus, we designed 60 nt probes because they can better tolerate mismatched templates than 25 nt oligonucleotide probes (45) . Using an empirical approach, appropriate thresholds can be determined for other array platforms, hybridization conditions, and probe lengths. The method of probe design and setcover minimization is flexible and agnostic of platform; application to bead, solution, or surface-based hybridization technology should be straightforward.