Design and implementation of multiplexed amplicon sequencing panels to serve genomic epidemiology of infectious disease: A malaria case study

LaVerriere, Emily; Schwabl, Philipp; Carrasquilla, Manuela; Taylor, Aimee R; Johnson, Zachary M; Shieh, Meg; Panchal, Ruchit; Straub, Timothy J.; Kuzma, Rebecca; Watson, Sean; Buckee, Caroline O.; Andrade, Carolina M.; Portugal, Sílvia; Crompton, Peter D.; Traoré, Boubacar; Rayner, Julian C.; Corredor, Vladimir; James, Kashana; Cox, Horace; Early, Angela M.; MacInnis, Bronwyn; Neafsey, Daniel E.

doi:10.1111/1755-0998.13622

Cited by 26 publications

(34 citation statements)

References 118 publications

(143 reference statements)

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“…Recent efforts in the malaria field have borne several amplicon panels with wide geographic breadth that can quickly and affordably genotype hundreds or thousands of samples [ 23 , 64 , 65 ]. While whole-genome sequencing will remain the bedrock of selection analyses, these methodological advances in targeted sequencing will facilitate the use of IBD analysis with confidence intervals for describing local population structure (as we do here) [ 66 , 67 ], measuring connectivity between populations [ 68 ], identifying likely importation events [ 69 ], and tracking changes in transmission [ 70 ].…”

Section: Discussionmentioning

confidence: 99%

Resolving drug selection and migration in an inbred South American Plasmodium falciparum population with identity-by-descent analysis

et al. 2022

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The human malaria parasite Plasmodium falciparum is globally widespread, but its prevalence varies significantly between and even within countries. Most population genetic studies in P. falciparum focus on regions of high transmission where parasite populations are large and genetically diverse, such as sub-Saharan Africa. Understanding population dynamics in low transmission settings, however, is of particular importance as these are often where drug resistance first evolves. Here, we use the Pacific Coast of Colombia and Ecuador as a model for understanding the population structure and evolution of Plasmodium parasites in small populations harboring less genetic diversity. The combination of low transmission and a high proportion of monoclonal infections means there are few outcrossing events and clonal lineages persist for long periods of time. Yet despite this, the population is evolutionarily labile and has successfully adapted to changes in drug regime. Using newly sequenced whole genomes, we measure relatedness between 166 parasites, calculated as identity by descent (IBD), and find 17 distinct but highly related clonal lineages, six of which have persisted in the region for at least a decade. This inbred population structure is captured in more detail with IBD than other common population structure analyses like PCA, ADMIXTURE, and distance-based trees. We additionally use patterns of intra-chromosomal IBD and an analysis of haplotypic variation to explore past selection events in the region. Two genes associated with chloroquine resistance, crt and aat1, show evidence of hard selective sweeps, while selection appears soft and/or incomplete at three other key resistance loci (dhps, mdr1, and dhfr). Overall, this work highlights the strength of IBD analyses for studying parasite population structure and resistance evolution in regions of low transmission, and emphasizes that drug resistance can evolve and spread in small populations, as will occur in any region nearing malaria elimination.

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Section: Discussionmentioning

confidence: 99%

Resolving drug selection and migration in an inbred South American Plasmodium falciparum population with identity-by-descent analysis

et al. 2022

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“…Sequencing workflows that can be implemented in endemic settings are essential to drive the decentralisation of genomics, to support its integration into clinical and public health applications, and to push for a more equitable distribution of global genomics capacity. Amplicon sequencing can be a pragmatic approach to malaria molecular surveillance and generate actionable data on parasite populations, including workflows based in endemic LMICs [11,12,[94][95][96][97][98][99][100][101][102][103][104][105][106]. Moreover, by developing and investing in sequencing capacity, the technical skills, experience and technology can potentially be applied to multiple high priority pathogens and emerging infection threats, maximising the impact of genomics in public health and strengthening global pathogen surveillance and health security [107].…”

Section: Discussionmentioning

confidence: 99%

Nanopore sequencing for real-time genomic surveillance ofPlasmodium falciparum

Girgis

Adika

Nenyewodey

et al. 2022

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Malaria is a global public health priority causing over 600,000 deaths annually, mostly young children living in Sub-Saharan Africa. Molecular surveillance can provide key information for malaria control, such as the prevalence and distribution of antimalarial drug resistance. However, genome sequencing capacity in endemic countries can be limited. Here, we have implemented an end-to-end workflow for Plasmodium falciparum genomic surveillance in Ghana using Oxford Nanopore Technologies, targeting antimalarial resistance markers and the leading vaccine antigen circumsporozoite protein (csp). The workflow was rapid, robust, accurate, affordable and straightforward to implement. We found that P. falciparum parasites in Ghana had become largely susceptible to chloroquine, with persistent sulfadoxine-pyrimethamine (SP) resistance, and no evidence of artemisinin resistance. Multiple Single Nucleotide Polymorphism (SNP) differences from the vaccine csp sequence were identified, though their significance is uncertain. This study demonstrates the potential utility and feasibility of malaria genomic surveillance in endemic settings using Nanopore sequencing.

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“…To date, most studies utilizing this method have only targeted one or a few loci to gain information on diversity of infections, drug resistance, or selection 29,[32][33][34] . Increasing the number of loci can provide a higher resolution comparison of infections at the population level as well as pairwise relatedness inference at the individual level 15,35,36 .…”

Section: Introductionmentioning

confidence: 99%

Highly multiplexed ddPCR-amplicon sequencing reveals strongPlasmodium falciparumpopulation structure and isolated populations amenable to local elimination efforts in Zanzibar

Holzschuh

Lerch

Gerlovina

et al. 2023

Preprint

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Over the past 15 years, Zanzibar has made great strides toward malaria elimination; yet progress has stalled. Parasite genetic data of Plasmodium falciparum may inform strategies for malaria elimination by helping to identify contributory factors to parasite persistence. Here we elucidate fine-scale parasite population structure and infer relatedness and connectivity of infections using an identity-by-descent (IBD) approach. We sequenced 518 P. falciparum samples from 5 districts covering both main islands using a novel, highly multiplexed droplet digital PCR (ddPCR)-based amplicon deep sequencing method targeting 35 microhaplotypes and drug-resistance loci. Despite high genetic diversity, we observe strong fine-scale spatial and temporal structure of local parasite populations, including isolated populations on Pemba Island and genetically admixed populations on Unguja Island, providing evidence of ongoing local transmission. We observe a high proportion of highly related parasites in individuals living closer together, including between clinical index cases and the mostly asymptomatic cases surrounding them, consistent with isolation-by-distance. We identify a substantial fraction (2.9%) of related parasite pairs between Zanzibar, and mainland Tanzania and Kenya, consistent with recent importation. We identify haplotypes known to confer resistance to known antimalarials in all districts, including multidrug-resistant parasites, but most parasites remain sensitive to current first-line treatments. Our study provides a high-resolution view of parasite genetic structure across the Zanzibar archipelago and reveals actionable patterns, including isolated parasite populations, which may be prioritized for malaria elimination.

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Design and implementation of multiplexed amplicon sequencing panels to serve genomic epidemiology of infectious disease: A malaria case study

Cited by 26 publications

References 118 publications

Resolving drug selection and migration in an inbred South American Plasmodium falciparum population with identity-by-descent analysis

Resolving drug selection and migration in an inbred South American Plasmodium falciparum population with identity-by-descent analysis

Nanopore sequencing for real-time genomic surveillance ofPlasmodium falciparum

Highly multiplexed ddPCR-amplicon sequencing reveals strongPlasmodium falciparumpopulation structure and isolated populations amenable to local elimination efforts in Zanzibar

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