5WBF : a low-cost and straightforward whole blood filtration method suitable for whole-genome sequencing of Plasmodium falciparum clinical isolates

2022

Article référencé dans le Web of Science WOS:000756829200002

Coppée R., Mama A., Sarrasin V., Kamaliddin C., Adoux L., Palazzo L., Tuikue Ndam Nicaise, Letourneur F., Ariey F., Houzé S., Clain J.

Malaria Journal, 2022, 21 (1), 51 [13 p.]

Background: Whole-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 mu m and exhibit some deformability, it was hypothesized that cheap and commercially available 5 mu m 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 mu m Whole Blood Filtration), 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 parasitaemia 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 parasitaemia range: 0.02-1.1%).These whole blood samples (50 to 400 mu L) were diluted in RPMI 1640 medium or PBS 1 x buffer and filtered with a syringe connected to a 5 pm 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. Results: After applying 5WBF, qPCR indicated an average of twofold loss in the amount of parasite template DNA (Pf ARN 18S gene) and from 4096- to 65,536-fold loss of human template DNA (human beta actin gene). WGS analyses revealed that> 95% of the parasite nuclear and organellar genomes were all covered at >= 10x 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 >= 10x depth depending on parasitaemia. Sequence reads were homogeneously distributed across gene sequences for 5WBF-treated samples (n = 5460 genes; mean coverage: 91 x; median coverage: 93x; 5th percentile: 70x; 95th percentile: 103x), allowing the identification of gene copy number variations such as for gch1. 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: 80x; median coverage: 51x; 5th percentile: 7x; 95th percentile: 245x). Conclusions: The novel 5WBF leucodepletion method is simple to implement and based on commercially available, standardized 5 mu m filters which cost from 1.0 to 1.7(sic) per unit depending on suppliers. 5WBF permits extensive genome-wide analysis of P. falciparum ring-stage isolates from minute amounts of whole blood even with parasitaemias as low as 0.02%.

Sciences fondamentales / Techniques d'analyse et de recherche [020] ; Santé : généralités [050] ; Entomologie médicale / Parasitologie / Virologie [052]

Fonds IRD [F B010084322]

fdi:010084322