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bioRxiv
BIORXIV
bioRxiv
2692-8205
Cold Spring Harbor Laboratory

10.1101/2024.08.22.609145
preprint
3
Article
Sensitive and modular amplicon sequencing of Plasmodium falciparum diversity and resistance for research and public health
Aranda-Díaz Andrés http://orcid.org/0000-0002-0566-4901

Vickers Eric Neubauer
Murie Kathryn
Palmer Brian
Hathaway Nicholas
Gerlovina Inna
Boene Simone
Garcia-Ulloa Manuel
Cisteró Pau
Katairo Thomas
Semakuba Francis Ddumba
Nsengimaana Bienvenu
Gwarinda Hazel
García-Fernández Carla
Da Silva Clemente
Datta Debayan
Kiyaga Shahiid
Wiringilimaana Innocent
Fekele Sindew Mekasha
Parr Jonathan B.
Conrad Melissa
Raman Jaishree
Tukwasibwe Stephen
Ssewanyana Isaac
Rovira-Vallbona Eduard
Tato Cristina M.
Briggs Jessica
Mayor Alfredo http://orcid.org/0000-0003-3890-2897

Greenhouse Bryan
25 8 2024
2024.08.22.609145https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License, which allows reusers to distribute, remix, adapt, and build upon the material in any medium or format for noncommercial purposes only, and only so long as attribution is given to the creator.
http://biorxiv.org/lookup/doi/10.1101/2024.08.22.609145
nihpp-2024.08.22.609145.pdf
Abstract

Targeted amplicon sequencing is a powerful and efficient tool to interrogate the P . falciparum genome and generate actionable data from infections to complement traditional malaria epidemiology. For maximum impact, genomic tools should be multi-purpose, robust, sensitive and reproducible. We developed, characterized, and implemented MAD 4 HatTeR, an amplicon sequencing panel based on Multiplex Amplicons for Drug, Diagnostic, Diversity, and Differentiation Haplotypes using Targeted Resequencing, along with a bioinformatic pipeline for data analysis. MAD 4 HatTeR targets 165 highly diverse loci, focusing on multiallelic microhaplotypes; key markers for drug and diagnostic resistance, including duplications and deletions; and csp and potential vaccine targets. In addition, it can detect non- falciparum Plasmodium species. We used laboratory control and field sample data to demonstrate the high sensitivity and robustness of the panel. The successful implementation of this method in five laboratories, including three in malaria-endemic African countries, showcases its feasibility in generating reproducible data across laboratories. Finally, we introduce an analytical approach to detect gene duplications and deletions from amplicon sequencing data. MAD 4 HatTeR is thus a powerful research tool and a robust resource for malaria public health surveillance and control.
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