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 MAD<sup>4</sup>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. 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. MAD<sup>4</sup>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. The panel can also detect non-falciparum Plasmodium species. MAD<sup>4</sup>HatTeR 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 MAD<sup>4</sup>HatTeR were highly reproducible across multiple laboratories. The successful implementation of MAD<sup>4</sup>HatTeR in five laboratories, including three in malaria-endemic African countries, showcases its feasibility and reproducibility in diverse settings. MAD<sup>4</sup>HatTeR is thus a powerful tool for research and a robust resource for malaria public health surveillance and control.
Authors
- Andrés Aranda-Díaz (ORCID: https://orcid.org/0000-0002-0566-4901)
- Manuel García‐Ulloa (ORCID: https://orcid.org/0000-0002-6194-9565)
- Eric Neubauer Vickers
- Melissa D. Conrad (ORCID: https://orcid.org/0000-0002-9984-8030)
- Thomas Katairo (ORCID: https://orcid.org/0000-0002-2002-8911)
- Simone Boene (ORCID: https://orcid.org/0000-0002-8323-9863)
- Jessica Briggs (ORCID: https://orcid.org/0000-0002-8078-3898)
- Kathryn Murie (ORCID: https://orcid.org/0009-0008-9612-2016)
- Endashaw Esayas (ORCID: https://orcid.org/0000-0001-7116-7033)
- Sindew Mekasha Feleke (ORCID: https://orcid.org/0000-0002-5778-653X)
- Debayan Datta (ORCID: https://orcid.org/0000-0002-2347-0860)
- William Louie (ORCID: https://orcid.org/0000-0003-1139-1476)
- Nicholas J. Hathaway (ORCID: https://orcid.org/0000-0001-9639-2894)
- Jaishree Raman (ORCID: https://orcid.org/0000-0003-0728-3093)
- Jonathan B. Parr (ORCID: https://orcid.org/0000-0002-4465-9830)
- Carla García-Fernández (ORCID: https://orcid.org/0000-0003-2738-0587)
- Bryan Greenhouse (ORCID: https://orcid.org/0000-0003-0287-9111)
- Inna Gerlovina (ORCID: https://orcid.org/0000-0002-7772-7473)
- Alfredo Mayor (ORCID: https://orcid.org/0000-0003-3890-2897)
- Adam Bennett (ORCID: https://orcid.org/0000-0002-7028-311X)
- Brian A. Palmer (ORCID: https://orcid.org/0000-0001-5032-4773)
- Hazel Gwarinda (ORCID: https://orcid.org/0000-0002-8605-7870)
- Pau Cisteró (ORCID: https://orcid.org/0000-0001-7225-4331)
- Clemente da Silva (ORCID: https://orcid.org/0000-0003-2083-2579)
- Endalamaw Gadisa (ORCID: https://orcid.org/0000-0002-5179-6278)
- Isaac Ssewanyana (ORCID: https://orcid.org/0000-0001-8605-2001)
- Shahiid Kiyaga (ORCID: https://orcid.org/0000-0002-6380-4714)
- Cristina M. Tato (ORCID: https://orcid.org/0000-0002-0614-756X)
- Eduard Rovira-Vallbona (ORCID: https://orcid.org/0000-0002-4765-1076)
- Jennifer L. Smith (ORCID: https://orcid.org/0000-0002-9244-4412)
- Stephen Tukwasibwe (ORCID: https://orcid.org/0000-0002-6469-6516)
- Francis D Semakuba
- Bienvenu Nsengimaana (ORCID: https://orcid.org/0009-0002-9786-7294)
- Innocent Wiringilimaana
Institutions
- University of North Carolina at Chapel Hill (US)
- Program for Appropriate Technology in Health (US)
- Eduardo Mondlane University (MZ)
- University of California, San Francisco (US)
- University of the Witwatersrand (ZA)
- La Trobe University (AU)
- Departament de Salut (ES)
- Ethiopian Public Health Institute (ET)
- Manhiça Health Research Centre (MZ)
- Infectious Diseases Research Collaboration (UG)
- Chan Zuckerberg Initiative (United States) (US)
- Barcelona Institute for Global Health (ES)
- Armauer Hansen Research Institute (ET)
- National Institute for Communicable Diseases
- University of Pretoria (ZA)
- Universitat de Barcelona (ES)
- Makerere University (UG)
Publication Details
- Journal
- Scientific Reports
- Published
- 2025-03-28
- DOI
- https://doi.org/10.1038/s41598-025-94716-5
- Citations
- 28
- Primary Topic
- Malaria Research and Control
- Type
- article
- Field-Weighted Citation Impact
- 52.60
Funders
- Bill and Melinda Gates Foundation
- Global Fund to Fight AIDS, Tuberculosis and Malaria
- Generalitat de Catalunya
- Ministerio de Ciencia e Innovación
- University of California, San Francisco
- National Institute of Allergy and Infectious Diseases