Direct whole-genome sequencing of Plasmodium falciparum specimens from dried erythrocyte spots

Background: Plasmodium falciparum malaria remains a major health burden and genomic research represents one of the necessary approaches for continued progress towards malaria control and elimination. Sample acquisition for this purpose is troublesome, with the majority of malaria-infected individuals living in rural areas, away from main infrastructure and the electrical grid.

The aim of this study was to describe a low-tech procedure to sample P. falciparum specimens for direct whole genome sequencing (WGS), without use of electricity and cold-chain. Methods: Venous blood samples were collected from malaria patients in Bandim, Guinea-Bissau and leukocyte-depleted using Plasmodipur filters, the enriched parasite sample was spotted on Whatman paper and dried.

The samples were stored at ambient temperatures and subsequently used for DNA-extraction. Ratios of parasite:human content of the extracted DNA was assessed by qPCR, and five samples with varying parasitaemia, were sequenced. Sequencing data were used to analyse the sample content, as well as sample coverage and depth as compared to the 3d7 reference genome.

Results: qPCR revealed that 73% of the 199 samples were applicable for WGS, as defined by a minimum ratio of parasite:human DNA of 2:1. WGS revealed an even distribution of sequence data across the 3d7 reference genome, regardless of parasitaemia. The acquired read depths varied from 16 to 99×, and coverage varied from 87.5 to 98.9% of the 3d7 reference genome.

SNP-analysis of six genes, for which amplicon sequencing has been performed previously, confirmed the reliability of the WGS-data. Conclusion: This study describes a simple filter paper based protocol for sampling P. falciparum from malaria patients for subsequent direct WGS, enabling acquisition of samples in remote settings with no access to electricity.