MinION Adapted tNGS Panel for Carnivore Pathogens Including SARS-CoV-2

Affordable, flexible surveillance tools are needed to detect SARS-CoV-2 and other pathogens in wildlife. Standard nucleic acid amplification tests (NAATs) are reliable but restricted to predefined targets, limiting their ability to detect co-infections or emerging pathogens. To address this, we adapted a targeted next-generation sequencing (tNGS) panel for mesocarnivores to the Oxford Nanopore Technologies (ONT) MinION platform and combined it with a SARS-CoV-2 whole-genome sequencing assay. Merging both assays before library preparation enables simultaneous SARS-CoV-2 detection, variant identification, and broader pathogen screening. The MinION platform also improves turnaround time because sequencing can begin immediately on small numbers of samples, reducing costs in low-volume workflows. We converted our validated carnivore tNGS panel from the Ion Torrent system to MinION, optimizing amplification conditions, primer pools, and barcoding for multiplexing. Analytical sensitivity was measured using contrived wildlife samples spiked with serial dilutions of SARS-CoV-2 and tested in parallel with a commercial NAAT. Diagnostic sensitivity was assessed using contrived positives, and specificity was evaluated using NAAT-negative wildlife samples and in silico analyses. All 161 wildlife samples were NAAT-negative. MinION tNGS detected SARS-CoV-2 down to Ct 34 and produced ≥ 99% genome coverage for Ct ≤ 24 while simultaneously identifying additional pathogens. Diagnostic sensitivity and specificity were 96.7% and 100%. This workflow offers a low-cost, scalable approach for comprehensive wildlife pathogen surveillance.