Flexible and cost-effective genomic surveillance ofP. falciparummalaria with targeted nanopore sequencing

Abstract

Genomic surveillance ofPlasmodium falciparummalaria can provide policy-relevant information about antimalarial drug resistance, rapid diagnostic test failure, and the evolution of vaccine targets. Yet the large and low complexity genome ofP. falciparumlimits the scope of genomic surveillance, as whole-genome sequencing approaches are costly and targeted approaches are challenging to develop. Moreover, the majority of the morbidity and mortality caused byP. falciparumoccurs in sub-Saharan Africa, where resource constraints can make implementing genomic surveillance difficult. Here, we demonstrate a flexible and cost-effective approach for targeted nanopore sequencing ofP. falciparumto enable genomic surveillance of malaria in low-resource settings. We release open-source software that facilitates rapid and flexible design of amplicon sequencing panels forP. falciparum, coupled with a simple and cost-effective protocol that uses dried blood spots as input. We use this software to design two amplicon panels. The first, called NOMADS8, targets seven major antimalarial drug-resistance associated genes as well as the highly polymorphic genemsp2. The second, NOMAD16, incorporates an additional eight genes including the vaccine targetcspand genes coding for the antigens detected in rapid diagnostic tests,hrp2andhrp3. The panels generate reads between 3 to 4kbp that span the entire coding sequence of most target genes. We validate the panels and protocol on mock and field samples, demonstrating robust sequencing coverage across targets, high single-nucleotide polymorphism calling accuracy within coding sequences, and the ability to explore the within-sample diversity of mixedP. falciparuminfections.

Long-Read Sequencing and De Novo Genome Assembly Pipeline of Two Plasmodium falciparum Clones (Pf3D7, PfW2) Using Only the PromethION Sequencer from Oxford Nanopore Technologies without Whole-Genome Amplification

“…Since this implementation, however, few genomic studies have been published on Plasmodium falciparum due to the accessibility and price of sequencers (high cost) but also due to the richness of the genome in (A + T), which leads to a higher error rate [14]. These studies focus on nanopore sequencers, such as the MinION, to explore resistance in identified genes listed as causing antimalarial drug resistance [15][16][17][18].…”

Long-Read Sequencing and De Novo Genome Assembly Pipeline of Two Plasmodium falciparum Clones (P<em>f</em>3D7, P<em>f</em>W2) Using Only the PromethION Sequencer from Oxford Nanopore Technologies without Whole Genome Amplification

“…Since this implementation, moreover, few genomic studies have been published on Plasmodium falciparum due to the accessibility and price of sequencers but also due to the richness of the genome in (A+T) which leads to a higher error rate [14]. These studies focus on nanopore sequencers, such as the MinION, to explore resistance on identified genes, listed as causing antimalarial drug resistance [15][16][17][18].…”

Estimating multiplicity of infection, haplotype frequencies, and linkage disequilibria from multi-allelic markers for molecular disease surveillance

“…Molecular surveillance of infectious diseases allows for retrieving and reconstructing population or evolutionary genetic information such as the spread of drug-resistant pathogenic variants, or detailed epidemiological information such as routes of transmission and transmission intensities [3]. Such information helps to optimize disease control and prevention and becomes increasingly popular for some of the economically most relevant infectious diseases, such as malaria [4,5].…”