Coppée R. auteur aut IRD Mama A. auteur aut IRD Sarrasin V. auteur aut IRD Kamaliddin C. auteur aut IRD Adoux L. auteur aut IRD Palazzo L. auteur aut IRD Tuikue Ndam Nicaise auteur aut IRD Letourneur F. auteur aut IRD Ariey F. auteur aut IRD Houzé S. auteur aut IRD Clain J. auteur aut IRD text journalArticle eng text/pdf reformatted digital access 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%. specialized Malaria Plasmodium falciparum Leucodepletion Filtration Whole-genome sequencing 052 050 020 21 1 51 [13 ] 2022 https://www.documentation.ird.fr/hor/fdi:010084322 10.1186/s12936-022-04073-1 [F B010084322] https://www.documentation.ird.fr/hor/fdi:010084322 https://horizon.documentation.ird.fr/exl-doc/pleins_textes/2022-04/010084322.pdf IRD - Base Horizon / Pleins textes 2022-04-06 2025-02-24 fdi:010084322 fre