Aranda-Díaz Andrés, Vickers Eric Neubauer, Murie Kathryn, Palmer Brian, Hathaway Nicholas, Gerlovina Inna, Boene Simone, Garcia-Ulloa Manuel, Cisteró Pau, Katairo Thomas, Semakuba Francis Ddumba, Nsengimaana Bienvenu, Gwarinda Hazel, García-Fernández Carla, Louie William, Esayas Endashaw, Da Silva Clemente, Datta Debayan, Kiyaga Shahiid, Wiringilimaana Innocent, Fekele Sindew Mekasha, Bennett Adam, Smith Jennifer L, Gadisa Endalamaw, Parr Jonathan B, Conrad Melissa, Raman Jaishree, Tukwasibwe Stephen, Ssewanyana Isaac, Rovira-Vallbona Eduard, Tato Cristina M, Briggs Jessica, Mayor Alfredo, Greenhouse Bryan
EPPIcenter Research Program, Division of HIV, Infectious Diseases, and Global Medicine, Department of Medicine, University of California, San Francisco, California, USA.
Chan Zuckerberg Biohub, San Francisco, California, USA.
bioRxiv. 2025 Feb 19:2024.08.22.609145. doi: 10.1101/2024.08.22.609145.
Targeted amplicon sequencing is a powerful and efficient tool for interrogating the 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 MADHatTeR, 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.
MADHatTeR targets 165 highly diverse loci, focusing on multiallelic microhaplotypes, key markers for drug and diagnostic resistance (including duplications and deletions), and and potential vaccine targets. The panel can also detect non- species. MADHatTeR 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 MADHatTeR were highly reproducible across multiple laboratories.
The successful implementation of MADHatTeR in five laboratories, including three in malaria-endemic African countries, showcases its feasibility and reproducibility in diverse settings. MADHatTeR is thus a powerful tool for research and a robust resource for malaria public health surveillance and control.
靶向扩增子测序是一种强大且高效的基因组检测工具，能从感染中生成可用于指导行动的数据，以补充传统疟疾流行病学研究。为实现最大影响，基因组工具应具备多功能、稳健、灵敏且可重复的特点。
我们开发、表征并实施了MADHatTeR，这是一种基于多重扩增子的靶向重测序的扩增子测序面板，用于药物、诊断、多样性和分化单倍型分析，同时还开发了一套用于数据分析的生物信息学流程。此外，我们引入了一种从扩增子测序数据中检测基因重复和缺失的分析方法。使用实验室对照样本和现场样本展示该面板的高灵敏度和稳健性。
MADHatTeR靶向165个高度多样化的位点，重点关注多等位基因微单倍型、药物和诊断抗性的关键标记（包括重复和缺失）以及潜在的疫苗靶点。该面板还能检测非疟原虫物种。MADHatTeR成功从低寄生虫密度的干血斑和蚊虫中肠样本中生成数据，并在高寄生虫密度的干血斑样本中以高特异性检测到样本内等位基因频率低至1%的次要等位基因。在单克隆和多克隆对照中可靠地检测到了基因缺失和重复。MADHatTeR生成的数据在多个实验室中具有高度可重复性。
MADHatTeR在五个实验室（包括三个疟疾流行的非洲国家的实验室）的成功实施，展示了其在不同环境中的可行性和可重复性。因此，MADHatTeR是一种强大的研究工具，也是疟疾公共卫生监测和控制的有力资源。
