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

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.

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Publication Details

Journal
UNC Libraries
Published
2025-04-08
DOI
https://doi.org/10.17615/pz14-wx86
Primary Topic
Malaria Research and Control
Type
article
Field-Weighted Citation Impact
0.00
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article

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

Andrés Aranda-Díaz, Manuel García‐Ulloa, Eric Neubauer Vickers, Melissa D. Conrad et al.
UNC Libraries
Malaria Research and Control
article

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

Andrés Aranda-Díaz, Manuel García‐Ulloa, Eric Neubauer Vickers, Melissa D. Conrad, Thomas Katairo, Simone Boene, Jessica Briggs, Kathryn Murie, Endashaw Esayas, Sindew Mekasha Feleke, Debayan Datta, William Louie, Nicholas J. Hathaway, Jaishree Raman, Jonathan B. Parr, Carla García-Fernández, Bryan Greenhouse, Inna Gerlovina, Alfredo Mayor, Adam Bennett, Brian A. Palmer, Hazel Gwarinda, Pau Cisteró, Clemente da Silva, Endalamaw Gadisa, Isaac Ssewanyana, Shahiid Kiyaga, Cristina M. Tato, Eduard Rovira-Vallbona, Jennifer L. Smith, Stephen Tukwasibwe, Francis D Semakuba, Bienvenu Nsengimaana, Innocent Wiringilimaana
article en

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.

UNC Libraries
University of North Carolina at Chapel Hill (US)
Openalex Percentile: Top 61%
Malaria Research and Control
DOIOpen Access PDFOpenAlex
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