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Tick Microbiome Characterization by Next-Generation 16S rRNA Amplicon Sequencing

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Targeted Next-Generation Sequencing for Comprehensive Testing for Selected Vector-Borne Pathogens in Canines.

Jobin J Kattoor¹, Emma Nikolai¹, Barbara Qurollo²

¹Animal Disease Diagnostic Laboratory, Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47907, USA.

Pathogens (Basel, Switzerland)

|September 23, 2022

Summary

A new targeted next-generation sequencing (NGS) assay offers a cost-effective and rapid method for detecting multiple vector-borne pathogens in dogs. This assay shows high agreement with traditional real-time PCR (rtPCR), improving diagnostic efficiency.

Keywords:

diagnostics targeted next-generation sequencing vector-borne pathogens

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Area of Science:

Veterinary Diagnostics
Molecular Biology
Pathogen Detection

Background:

Real-time PCR (rtPCR) is the standard for vector-borne pathogen detection but requires numerous individual tests.
Current methods can be time-consuming and costly for comprehensive diagnosis.

Purpose of the Study:

To develop and validate a targeted next-generation sequencing (NGS) assay for simultaneous detection of vector-borne pathogens.
To assess the diagnostic accuracy and efficiency of the novel NGS assay compared to rtPCR.

Main Methods:

Development of two primer pools for targeted PCR amplification of pathogen regions.
Preparation and sequencing of barcoded DNA libraries using the Ion Torrent S5 system.
Bioinformatic analysis including data assembly (SPAdes) and mapping to a reference database.

Main Results:

The targeted NGS assay demonstrated specificity for 17 different pathogens.
Analytical sensitivity was comparable to rtPCR (Ct value ~35-36).
High diagnostic agreement with rtPCR was observed: 92% positive percent agreement, 88% negative percent agreement, and Cohen's kappa of 0.804.

Conclusions:

The targeted NGS assay is a viable, cost-effective method for detecting vector-borne pathogens in canine whole blood.
The assay offers a reduced turn-around time of 2-3 days compared to conventional methods.
This approach enhances diagnostic capabilities for vector-borne diseases in veterinary medicine.