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Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons
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Ten simple rules for implementing deep amplicon sequencing in parasitology.

Jan Šlapeta1, Jürgen Krücken2, Alicia Rojas3

  • 1Sydney School of Veterinary Science, Faculty of Science, The University of Sydney, New South Wales, Australia; Sydney Infectious Diseases Institute, The University of Sydney, Sydney, New South Wales, Australia.

International Journal for Parasitology
|November 23, 2025
PubMed
Summary

Deep amplicon sequencing offers powerful tools for parasitology research. This article provides ten essential rules for effective implementation, improving data analysis and reproducibility in parasite studies.

Keywords:
ASVAmplicon profilingGuidelinesMetabarcodingMethodsMicrobiotaOTUSpecimen handling

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Area of Science:

  • Veterinary Parasitology
  • Molecular Biology
  • Bioinformatics

Background:

  • Deep amplicon sequencing (DAS) revolutionizes parasitology by enabling high-throughput parasite community analysis and resistance gene detection.
  • Implementation challenges, including experimental design and data analysis, limit the full potential of DAS in parasitology.

Purpose of the Study:

  • To present ten simple rules for applying deep amplicon sequencing in parasitology.
  • To guide researchers in experimental design, data analysis, and best practices for DAS in veterinary parasitology.

Main Methods:

  • Expert consensus developed through a symposium at the World Association for the Advancement of Veterinary Parasitology conference.
  • Guidelines cover research questions, marker selection, data workflows, and database contributions.

Main Results:

  • Ten practical rules for robust and reproducible deep amplicon sequencing in parasitology.
  • Emphasis on integrating DAS with traditional methods and ensuring transparent reporting.

Conclusions:

  • Adopting these guidelines enhances the reliability and impact of parasitological research using DAS.
  • Promotes a more collaborative and scientifically rigorous landscape in veterinary parasitology.