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

Nelly O Elshafie1, Jobin J Kattoor2, Janetta Kelly3

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Summary
This summary is machine-generated.

Wildlife pathogen surveillance is enhanced with a new, affordable targeted next-generation sequencing (tNGS) method on the Oxford Nanopore MinION platform. This approach enables simultaneous SARS-CoV-2 detection and broader pathogen screening in animals.

Keywords:
MinIONONTOxford Nanopore TechnologiesSARS-CoV-2diagnosticstargeted next-generation sequencing

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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.