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 to interrogate the P . falciparum genome and generate 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 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. 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. In addition, it can detect non- falciparum Plasmodium species. We used laboratory control and field sample data to demonstrate the high sensitivity and robustness of the panel. The successful implementation of this method in five laboratories, including three in malaria-endemic African countries, showcases its feasibility in generating reproducible data across laboratories. Finally, we introduce an analytical approach to detect gene duplications and deletions from amplicon sequencing data. MAD 4 HatTeR is thus a powerful research tool and a robust resource for malaria public health surveillance and control.

Lineage-informative microhaplotypes for recurrence classification and spatio-temporal surveillance of Plasmodium vivax malaria parasites.

Challenges in classifying recurrent Plasmodium vivax infections constrain surveillance of antimalarial efficacy and transmission. Recurrent infections may arise from activation of dormant liver stages (relapse), blood-stage treatment failure (recrudescence) or reinfection. Molecular inference of familial relatedness (identity-by-descent or IBD) can help resolve the probable origin of recurrences. As whole genome sequencing of P. vivax remains challenging, targeted genotyping methods are needed for scalability. We describe a P. vivax marker discovery framework to identify and select panels of microhaplotypes (multi-allelic markers within small, amplifiable segments of the genome) that can accurately capture IBD. We evaluate panels of 50-250 microhaplotypes discovered in a global set of 615 P. vivax genomes. A candidate global 100-microhaplotype panel exhibits high marker diversity in the Asia-Pacific, Latin America and horn of Africa (median HE = 0.70-0.81) and identifies 89% of the polyclonal infections detected with genome-wide datasets. Data simulations reveal lower error in estimating pairwise IBD using microhaplotypes relative to traditional biallelic SNP barcodes. The candidate global panel also exhibits high accuracy in predicting geographic origin and captures local infection outbreak and bottlenecking events. Our framework is open-source enabling customised microhaplotype discovery and selection, with potential for porting to other species or data resources.

Lineage-informative microhaplotypes for spatio-temporal surveillance of Plasmodium vivax malaria parasites.

Challenges in understanding the origin of recurrent Plasmodium vivax infections constrains the surveillance of antimalarial efficacy and transmission of this neglected parasite. Recurrent infections within an individual may arise from activation of dormant liver stages (relapse), blood-stage treatment failure (recrudescence) or new inoculations (reinfection). Molecular inference of familial relatedness (identity-by-descent or IBD) based on whole genome sequence data, together with analysis of the intervals between parasitaemic episodes ("time-to-event" analysis), can help resolve the probable origin of recurrences. Whole genome sequencing of predominantly low-density P. vivax infections is challenging, so an accurate and scalable genotyping method to determine the origins of recurrent parasitaemia would be of significant benefit. We have developed a P. vivax genome-wide informatics pipeline to select specific microhaplotype panels that can capture IBD within small, amplifiable segments of the genome. Using a global set of 615 P. vivax genomes, we derived a panel of 100 microhaplotypes, each comprising 3-10 high frequency SNPs within <200 bp sequence windows. This panel exhibits high diversity in regions of the Asia-Pacific, Latin America and the horn of Africa (median HE = 0.70-0.81) and it captured 89% (273/307) of the polyclonal infections detected with genome-wide datasets. Using data simulations, we demonstrate lower error in estimating pairwise IBD using microhaplotypes, relative to traditional biallelic SNP barcodes. Our panel exhibited high accuracy in predicting the country of origin (median Matthew's correlation coefficient >0.9 in 90% countries tested) and it also captured local infection outbreak and bottlenecking events. The informatics pipeline is available open-source and yields microhaplotypes that can be readily transferred to high-throughput amplicon sequencing assays for surveillance in malaria-endemic regions.