Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/14/2023
Publication Date: 8/20/2023
Citation: Bakre, A.A., Kariithi, H.M., Suarez, D.L. 2023. Alternative probe hybridization buffers for target RNA depletion and viral sequence recovery in NGS for poultry samples. Journal of Virological Methods. 321:114793. https://doi.org/10.1016/j.jviromet.2023.114793.
Interpretive Summary: Removal of host derived nucleic acids in field poultry samples can aid in better identification of poultry pathogens. Current protocols to deplete host derived nucleic acids use a commercial hybridization buffer that is both expensive and a special order reagent that delays procurement. In this study we identify and validate an alternative hybridization buffer that works equivalently well yet reduces per sample costs substantially.
Technical Abstract: Non-targeted next generation sequencing (NGS) is widely applied to identify the diversity of pathogens in field samples. However, abundance of host RNA (especially rRNA) and other environmental nucleic acids can reduce the abundance of pathogen specific reads of interest, reduce depth of coverage and increase surveillance costs. We presently deplete chicken- and selected bacterial-specific rRNAs in poultry field RNA samples with complementary DNA probes in a commercially available probe hybridization buffer followed by digestion of the RNA:DNA hybrids with RNase H. Because the current buffer is an expensive special order reagent of proprietary composition, we tested in-house and other commercially available buffers and identified a viable alternative that yields equivalent host rRNA depletion and viral-specific reads in poultry samples as the current special order reagent but at a reduced cost.