Selective whole-genome sequencing ofPlasmodiumparasites directly from blood samples by Nanopore adaptive sampling

Abstract

AbstractBackgroundWhole-genome sequencing (WGS) is becoming an increasingly popular tool to study the population genetics and drug resistance ofPlasmodiumspp. However, the predominance of human DNA in a malaria patient blood sample requires time-consuming lab procedures to filter out human DNA or enrichPlasmodiumDNA. Here, we investigated the potential of adaptive sampling to enrich forPlasmodiumDNA 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. falciparumDNA in human DNA was sequenced. Half of the flowcell channels were run in adaptive sampling mode, enriching for theP. falciparumreference genome, resulting in a 3.2 fold enrichment ofP. falciparumbases on average. Samples with a lower concentration of parasite DNA had a higher enrichment potential. We confirmed these findings by sequencing twoP. falciparumpatient 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 theP. falciparumreference 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-consumingPlasmodium-enrichment protocols and sequence directly from patient blood, given further improvements in cost-efficiency.