Ten simple rules for implementing deep amplicon sequencing in parasitology

Abstract

Deep amplicon sequencing is transforming parasitology by enabling high-throughput profiling of parasite communities and detection of resistance-associated genetic variants. Despite its growing adoption, many researchers face challenges in implementation, and its full potential is often hindered by challenges in experimental design, including marker selection, data analysis and reproducibility. This article presents ten simple rules for applying deep amplicon sequencing in parasitology, developed through expert consensus at a deep amplicon sequencing symposium during the 2025 World Association for the Advancement of Veterinary Parasitology conference. These rules cover essential aspects from formulating research questions and choosing appropriate markers to managing data workflows and contributing to reference databases. We highlight the importance of integrating deep amplicon sequencing with traditional parasitological methods, ensuring transparent reporting and investing in capacity building. Whether you are new to deep amplicon sequencing or seeking to improve your current practices, these guidelines offer practical advice to enhance the robustness, reproducibility, and impact of your research. By adopting these principles, parasitologists can contribute to, and advance, a more reliable and collaborative scientific landscape.