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

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Abstract Background 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. Methods 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. 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. Results 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. The panel can also detect non- falciparum Plasmodium species. MAD 4 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 4 HatTeR were highly reproducible across multiple laboratories. Conclusions The successful implementation of MAD 4 HatTeR in five laboratories, including three in malaria-endemic African countries, showcases its feasibility and reproducibility in diverse settings. MAD 4 HatTeR is thus a powerful tool for research and a robust resource for malaria public health surveillance and control.

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