5WBF: A low-cost and straightforward whole blood filtration method suitable for whole-genome sequencing of <i>Plasmodium falciparum</i> clinical isolates

ABSTRACT Background Whole-genome sequencing (WGS) is becoming increasingly helpful to assist malaria control programs. 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, we hypothesized that cheap and commercially available 5 μm filters might retain leukocytes but much less of Plasmodium falciparum -infected RBCs. This study aimed to test the hypothesis that such a filtration method, named 5WBF (for 5 μm W hole B lood F iltration), may provide highly enriched parasite material suitable for P. falciparum WGS. Methods Whole blood was collected from five patients experiencing a P. falciparum malaria episode (ring-stage parasitemia range: 0.04-5.5%) and from mock samples obtained by mixing synchronized, ring-stage cultured P. falciparum 3D7 parasites with uninfected human whole blood (final parasitemia range: 0.02-1.1%). These whole blood samples (50 to 400 μL) were diluted in RPMI 1640 medium or PBS 1X buffer and filtered with syringes connected to a 5 μm commercial filter. DNA was extracted from filtered 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, we also applied to the same unprocessed whole blood samples the selective whole-genome amplification (sWGA) method which does not rely on blood filtration. Results After applying 5WBF, qPCR indicated an average of 2-fold loss in the amount of parasite template DNA (Pf ARN 18S gene) and from 4,096- to 65,536-fold loss of human template DNA (human β actin gene). WGS analyses revealed that > 95% of the nuclear genome and the entire whole organellar genomes were covered at ≥ 10× depth for all samples tested. In sWGA counterparts, none of the organellar genomes were covered, and from 47.7 to 82.1% of the nuclear genome was covered at ≥ 10× depth depending on parasitemia. Sequence reads were homogeneously distributed across gene sequences for 5WBF-treated samples (n = 5,460 genes; mean coverage: 91×; median coverage: 93×; 5 th percentile: 70×; 95 th percentile: 103×), allowing the identification of gene copy number variations such as for gch1 . This later analysis was not possible for sWGA-treated samples, as we observed a much more heterogeneous distribution of reads among gene sequences (mean coverage: 80×; median coverage: 51×; 5 th percentile: 7×; 95 th percentile: 245×). Conclusions The 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 of P. falciparum DNA from minute amounts of whole blood even with parasitemias as low as 0.02%.

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