5WBF: a low-cost and straightforward whole blood filtration method suitable for whole-genome sequencing of Plasmodium falciparum clinical isolates
Authors: Coppée Romain , Mama Atikatou , Sarrasin Véronique , Kamaliddin Claire , Adoux Lucie , Palazzo Lawrence , Ndam Nicaise Tuikue , Letourneur Franck , Ariey Frédéric , Houzé Sandrine , Clain Jérôme 
Published: February 2022

AbstractBackgroundWhole-genome sequencing (WGS) is becoming increasingly helpful to assist malaria control programmes. A major drawback of this approach is the large amount of human DNA compared to parasite DNA extracted from unprocessed whole blood. As red blood cells (RBCs) have a diameter of about 7–8 µm and exhibit some deformability, it was hypothesized that cheap and commercially available 5 µm filters might retain leukocytes but much less ofPlasmodium falciparum-infected RBCs. This study aimed to test the hypothesis that such a filtration method, named 5WBF (for5 µmWholeBloodFiltration), may provide highly enriched parasite material suitable forP. falciparumWGS.MethodsWhole blood was collected from five patients experiencing aP. falciparummalaria episode (ring-stage parasitaemia range: 0.04–5.5%) and from mock samples obtained by mixing synchronized, ring-stage culturedP. falciparum3D7 parasites with uninfected human whole blood (final parasitaemia range: 0.02–1.1%). These whole blood samples (50 to 400 µL) were diluted in RPMI 1640 medium or PBS 1× buffer and filtered with a syringe connected to a 5 µm commercial filter. DNA was extracted from 5WBF-treated and unfiltered counterpart blood samples using a commercial kit. The 5WBF method was evaluated on the ratios of parasite:human DNA assessed by qPCR and by sequencing depth and percentages of coverage from WGS data (Illumina NextSeq 500). As a comparison, the popular selective whole-genome amplification (sWGA) method, which does not rely on blood filtration, was applied to the unfiltered counterpart blood samples.ResultsAfter applying 5WBF, qPCR indicated an average of twofold loss in the amount of parasite template DNA (Pf ARN18Sgene) and from 4096- to 65,536-fold loss of human template DNA (humanβ actingene). WGS analyses revealed that > 95% of the parasite nuclear and organellar genomes were all covered at ≥ 10× depth for all samples tested. In sWGA counterparts, the organellar genomes were poorly covered and from 47.7 to 82.1% of the nuclear genome was covered at ≥ 10× depth depending on parasitaemia. Sequence reads were homogeneously distributed across gene sequences for 5WBF-treated samples (n = 5460 genes; mean coverage: 91×; median coverage: 93×; 5th percentile: 70×; 95th percentile: 103×), allowing the identification of gene copy number variations such as forgch1. This later analysis was not possible for sWGA-treated samples, as a much more heterogeneous distribution of reads across gene sequences was observed (mean coverage: 80×; median coverage: 51×; 5th percentile: 7×; 95th percentile: 245×).ConclusionsThe novel 5WBF leucodepletion method is simple to implement and based on commercially available, standardized 5 µm filters which cost from 1.0 to 1.7€ per unit depending on suppliers. 5WBF permits extensive genome-wide analysis ofP. falciparumring-stage isolates from minute amounts of whole blood even with parasitaemias as low as 0.02%.