Sensitive and modular amplicon sequencing of Plasmodium falciparum diversity and resistance for research and public health

Sensitive and modular amplicon sequencing of Plasmodium falciparum diversity and resistance for research and public health

Abstract Targeted amplicon sequencing is a powerful and efficient tool for interrogating the Plasmodium falciparum genome, generating actionable data from infections to complement traditional malaria epidemiology. For maximum impact, genomic tools should be multi-purpose, robust, sensitive, and repr...

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Main Authors: Andrés Aranda-Díaz, Eric Neubauer Vickers, Kathryn Murie, Brian Palmer, Nicholas Hathaway, Inna Gerlovina, Simone Boene, Manuel García-Ulloa, Pau Cisteró, Thomas Katairo, Francis Ddumba Semakuba, Bienvenu Nsengimaana, Hazel Gwarinda, Carla García-Fernández, William Louie, Endashaw Esayas, Clemente Da Silva, Debayan Datta, Shahiid Kiyaga, Innocent Wiringilimaana, Sindew Mekasha Feleke, Adam Bennett, Jennifer L. Smith, Endalamaw Gadisa, Jonathan B. Parr, Melissa D. Conrad, Jaishree Raman, Stephen Tukwasibwe, Isaac Ssewanyana, Eduard Rovira-Vallbona, Cristina M. Tato, Jessica Briggs, Alfredo Mayor, Bryan Greenhouse
Format: Article
Language:English
Published: Nature Portfolio 2025-03-01
Series:Scientific Reports
Subjects:
Malaria
Plasmodium falciparum
Targeted amplicon sequencing
Microhaplotype
Antimalarial resistance
Online Access:https://doi.org/10.1038/s41598-025-94716-5
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Summary:Abstract Targeted amplicon sequencing is a powerful and efficient tool for interrogating the Plasmodium falciparum genome, generating 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 MAD4HatTeR, 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. Additionally, we introduce an analytical approach to detect gene duplications and deletions from amplicon sequencing data. Laboratory control and field samples were used to demonstrate the panel’s high sensitivity and robustness. MAD4HatTeR 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. The panel can also detect non-falciparum Plasmodium species. MAD4HatTeR successfully generated data from low-parasite-density dried blood spot and mosquito midgut samples and detected minor alleles at within-sample allele frequencies as low as 1% with high specificity in high-parasite-density dried blood spot samples. Gene deletions and duplications were reliably detected in mono- and polyclonal controls. Data generated by MAD4HatTeR were highly reproducible across multiple laboratories. The successful implementation of MAD4HatTeR in five laboratories, including three in malaria-endemic African countries, showcases its feasibility and reproducibility in diverse settings. MAD4HatTeR is thus a powerful tool for research and a robust resource for malaria public health surveillance and control.
ISSN:2045-2322

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