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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2025-03-01
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| author | 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 |
| author_facet | 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 |
| author_sort | Andrés Aranda-Díaz |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-7a7366ca2dfe428aac844b266bed9cbc |
| institution | OA Journals |
| issn | 2045-2322 |
| language | English |
| publishDate | 2025-03-01 |
| publisher | Nature Portfolio |
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| series | Scientific Reports |
| spelling | doaj-art-7a7366ca2dfe428aac844b266bed9cbc2025-08-20T02:10:13ZengNature PortfolioScientific Reports2045-23222025-03-0115111510.1038/s41598-025-94716-5Sensitive and modular amplicon sequencing of Plasmodium falciparum diversity and resistance for research and public healthAndrés Aranda-Díaz0Eric Neubauer Vickers1Kathryn Murie2Brian Palmer3Nicholas Hathaway4Inna Gerlovina5Simone Boene6Manuel García-Ulloa7Pau Cisteró8Thomas Katairo9Francis Ddumba Semakuba10Bienvenu Nsengimaana11Hazel Gwarinda12Carla García-Fernández13William Louie14Endashaw Esayas15Clemente Da Silva16Debayan Datta17Shahiid Kiyaga18Innocent Wiringilimaana19Sindew Mekasha Feleke20Adam Bennett21Jennifer L. Smith22Endalamaw Gadisa23Jonathan B. Parr24Melissa D. Conrad25Jaishree Raman26Stephen Tukwasibwe27Isaac Ssewanyana28Eduard Rovira-Vallbona29Cristina M. Tato30Jessica Briggs31Alfredo Mayor32Bryan Greenhouse33EPPIcenter Research Program, Division of HIV, Infectious Diseases, and Global Medicine, Department of Medicine, University of CaliforniaEPPIcenter Research Program, Division of HIV, Infectious Diseases, and Global Medicine, Department of Medicine, University of CaliforniaEPPIcenter Research Program, Division of HIV, Infectious Diseases, and Global Medicine, Department of Medicine, University of CaliforniaEPPIcenter Research Program, Division of HIV, Infectious Diseases, and Global Medicine, Department of Medicine, University of CaliforniaEPPIcenter Research Program, Division of HIV, Infectious Diseases, and Global Medicine, Department of Medicine, University of CaliforniaEPPIcenter Research Program, Division of HIV, Infectious Diseases, and Global Medicine, Department of Medicine, University of CaliforniaCentro de Investigação em Saúde de ManhiçaISGlobalISGlobalInfectious Diseases Research CollaborationInfectious Diseases Research CollaborationInfectious Diseases Research CollaborationLaboratory for Antimalarial Resistance Monitoring and Malaria Operational Research (ARMMOR), Centre of Emerging Zoonotic and Parasitic Diseases, National Institute for Communicable DiseasesISGlobalEPPIcenter Research Program, Division of HIV, Infectious Diseases, and Global Medicine, Department of Medicine, University of CaliforniaArmauer Hansen Research InstituteCentro de Investigação em Saúde de ManhiçaISGlobalInfectious Diseases Research CollaborationInfectious Diseases Research CollaborationEthiopian Public Health InstitutePATHGlobal Health Group, Malaria Elimination Initiative, University of CaliforniaArmauer Hansen Research InstituteDivision of Infectious Diseases, University of North CarolinaDivision of HIV, Infectious Diseases, and Global Medicine, Department of Medicine, University of CaliforniaLaboratory for Antimalarial Resistance Monitoring and Malaria Operational Research (ARMMOR), Centre of Emerging Zoonotic and Parasitic Diseases, National Institute for Communicable DiseasesInfectious Diseases Research CollaborationInfectious Diseases Research CollaborationISGlobalChan Zuckerberg BiohubEPPIcenter Research Program, Division of HIV, Infectious Diseases, and Global Medicine, Department of Medicine, University of CaliforniaCentro de Investigação em Saúde de ManhiçaEPPIcenter Research Program, Division of HIV, Infectious Diseases, and Global Medicine, Department of Medicine, University of CaliforniaAbstract 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.https://doi.org/10.1038/s41598-025-94716-5MalariaPlasmodium falciparumTargeted amplicon sequencingMalariaMicrohaplotypeAntimalarial resistance |
| spellingShingle | 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 Sensitive and modular amplicon sequencing of Plasmodium falciparum diversity and resistance for research and public health Scientific Reports Malaria Plasmodium falciparum Targeted amplicon sequencing Malaria Microhaplotype Antimalarial resistance |
| title | Sensitive and modular amplicon sequencing of Plasmodium falciparum diversity and resistance for research and public health |
| title_full | Sensitive and modular amplicon sequencing of Plasmodium falciparum diversity and resistance for research and public health |
| title_fullStr | Sensitive and modular amplicon sequencing of Plasmodium falciparum diversity and resistance for research and public health |
| title_full_unstemmed | Sensitive and modular amplicon sequencing of Plasmodium falciparum diversity and resistance for research and public health |
| title_short | Sensitive and modular amplicon sequencing of Plasmodium falciparum diversity and resistance for research and public health |
| title_sort | sensitive and modular amplicon sequencing of plasmodium falciparum diversity and resistance for research and public health |
| topic | Malaria Plasmodium falciparum Targeted amplicon sequencing Malaria Microhaplotype Antimalarial resistance |
| url | https://doi.org/10.1038/s41598-025-94716-5 |
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