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

Area of Science:

• Veterinary Medicine
• Genomics
• Epidemiology

Background:

• Effective wildlife surveillance for pathogens like SARS-CoV-2 is crucial.
• Current methods, such as nucleic acid amplification tests (NAATs), have limitations in detecting co-infections or novel pathogens.
• There is a need for affordable and flexible tools for comprehensive wildlife pathogen detection.

Purpose of the Study:

• To adapt a targeted next-generation sequencing (tNGS) panel for mesocarnivores to the Oxford Nanopore Technologies (ONT) MinION platform.
• To combine this tNGS panel with a SARS-CoV-2 whole-genome sequencing assay for simultaneous detection and variant identification.
• To evaluate the performance of this integrated workflow for wildlife pathogen surveillance.

Main Methods:

• Adapted a validated carnivore tNGS panel from Ion Torrent to the ONT MinION platform, optimizing conditions for multiplexing.
• Integrated SARS-CoV-2 whole-genome sequencing with the tNGS panel before library preparation.
• Assessed analytical sensitivity using contrived samples with known SARS-CoV-2 dilutions and diagnostic performance against NAAT results.

Main Results:

• The MinION tNGS workflow detected SARS-CoV-2 down to Ct 34 and achieved ≥99% genome coverage for Ct ≤24.
• Simultaneous detection of additional pathogens was possible alongside SARS-CoV-2.
• Diagnostic sensitivity was 96.7% and specificity was 100% in tested wildlife samples.

Conclusions:

• The integrated MinION tNGS workflow provides a low-cost, scalable, and efficient method for comprehensive wildlife pathogen surveillance.
• This approach allows for simultaneous detection of SARS-CoV-2, variant identification, and screening for other pathogens.
• The optimized workflow improves turnaround time and reduces costs, making it suitable for low-volume surveillance efforts.