ABSTRACT
Genomic surveillance of Plasmodium falciparum malaria 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 of P. falciparum limits 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 by P. falciparum occurs 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 of P. falciparum to enable genomic surveillance of malaria in low-resource settings. We release open-source software that facilitates rapid and flexible design of amplicon sequencing panels for P. 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 gene msp2. The second, NOMAD16, incorporates an additional eight genes including the vaccine target csp and genes coding for the antigens detected in rapid diagnostic tests, hrp2 and hrp3. 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 mixed P. falciparum infections.
Competing Interest Statement
The authors have declared no competing interest.
