Increasing the diagnostic PCR and next-generation sequencing capacity for the diagnosis and variant detection of COVID-19 in animals at the North Dakota State University Veterinary Diagnostic Lab

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Summary The primary goal of this project is to increase the capacity for next-generation sequencing (NGS) and SARS-CoV-2 variant detection for the diagnosis of COVID-19 in animals by validating commercially available reagent kits for extraction, PCR, and NGS using a dedicated one-way NGS workflow. This will add the NDSU-VDL to the network of veterinary diagnostic laboratories capable and proficient at testing and sequencing SARS-CoV- 2 in samples from animals. To build this capacity, reagents and dedicated equipment for a one- way workflow are needed. There are three specific aims. 1) Validate the Applied Biosystems™ COVID-19 TaqPath™ Combo Kit for the detection of SARS-CoV-2 and diagnosis of COVID-19 in animal samples. Targeted samples include tissue homogenates and swabs (nasal, oral, fecal) from animals. Extracted product can be used for downstream next generation sequencing on long or short read platforms, such as the MinION or iSeq, respectively. 2) Create a safe, one- way workflow for next generation sequencing that will minimize contamination and increase sequencing capacity by allowing multiple technicians to work on different steps in the process at the same time. 3) Diagnose SARS-CoV-2 variants through next generation sequencing on the MinION platform. This will be accomplished using the NEBNext® ARTIC SARS-CoV-2 Companion Kit and protocol for the MinION for NGS and following the EPI2ME analysis pipeline. The use of the flongle flow cells will be evaluated to reduce sequencing costs. In addition, the amount of time, cost, and ease of use of this NGS library prep kit and analysis pipline will the compared with other laboratories evaluating different kits.