Selective whole-genome sequencing of Plasmodium parasites directly from blood samples by Nanopore adaptive sampling

BackgroundWhole-genome sequencing (WGS) is becoming an increasingly popular tool to study the population genetics and drug resistance of Plasmodium spp. However, the predominance of human DNA in a malaria patient blood sample requires time-consuming lab procedures to filter out human DNA or enrich Plasmodium DNA. Here, we investigated the potential of adaptive sampling to enrich for Plasmodium DNA while sequencing unenriched patient blood samples on a minION device. ResultsTo compare adaptive sampling versus regular sequencing, a dilution series consisting of 0% up to 100% P. falciparum DNA in human DNA was sequenced. Half of the flowcell channels were run in adaptive sampling mode, enriching for the P. falciparum reference genome, resulting in a 3.2 fold enrichment of P. falciparum bases on average. Samples with a lower concentration of parasite DNA had a higher enrichment potential. We confirmed these findings by sequencing two P. falciparum patient blood samples with common levels of parasitaemia (0.1% and 0.2%). The estimated enrichment was 3.9 and 5.8, which was sufficient to cover at least 97% of the P. falciparum reference genome at a median depth of 20 (highest parasitaemia) or 5 (lowest parasitaemia). A comparison of 38 drug resistance variants (WHO) obtained via adaptive sequencing or Sanger sequencing showed a high concordance between the two methods, suggesting that the obtained sequencing data is of sufficient quality to address common clinical research questions for patients with parasitaemias of 0.1% and higher. ConclusionsOur results demonstrate that adaptive Nanopore sequencing has the potential to replace more time-consuming Plasmodium-enrichment protocols and sequence directly from patient blood, given further improvements in cost-efficiency.