@article{fdi:010084322,
  title = {5{WBF} : a low-cost and straightforward whole blood filtration method suitable for whole-genome sequencing of {P}lasmodium falciparum clinical isolates},
  author = {{C}opp{\'e}e, {R}. and {M}ama, {A}. and {S}arrasin, {V}. and {K}amaliddin, {C}. and {A}doux, {L}. and {P}alazzo, {L}. and {T}uikue {N}dam, {N}icaise and {L}etourneur, {F}. and {A}riey, {F}. and {H}ouz{\'e}, {S}. and {C}lain, {J}.},
  editor = {},
  language = {{ENG}},
  abstract = {{B}ackground: {W}hole-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. {A}s red blood cells ({RBC}s) 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 {P}lasmodium falciparum-infected {RBC}s. {T}his study aimed to test the hypothesis that such a filtration method, named 5{WBF} (for 5 mu m {W}hole {B}lood {F}iltration), may provide highly enriched parasite material suitable for {P}. falciparum {WGS}. {M}ethods: {W}hole 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 3{D}7 parasites with uninfected human whole blood (final parasitaemia range: 0.02-1.1%).{T}hese 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 5{WBF}-treated and unfiltered counterpart blood samples using a commercial kit.{T}he 5{WBF} method was evaluated on the ratios of parasite:human {DNA} assessed by q{PCR} and by sequencing depth and percentages of coverage from {WGS} data ({I}llumina {N}ext{S}eq 500). {A}s a comparison, the popular selective whole-genome amplification (s{WGA}) method, which does not rely on blood filtration, was applied to the unfiltered counterpart blood samples. {R}esults: {A}fter applying 5{WBF}, q{PCR} indicated an average of twofold loss in the amount of parasite template {DNA} ({P}f {ARN} 18{S} 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. {I}n s{WGA} 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. {S}equence reads were homogeneously distributed across gene sequences for 5{WBF}-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. {T}his later analysis was not possible for s{WGA}-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). {C}onclusions: {T}he novel 5{WBF} 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. 5{WBF} 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%.},
  keywords = {{M}alaria ; {P}lasmodium falciparum ; {L}eucodepletion ; {F}iltration ; {W}hole-genome sequencing},
  booktitle = {},
  journal = {{M}alaria {J}ournal},
  volume = {21},
  numero = {1},
  pages = {51 [13 ]},
  year = {2022},
  DOI = {10.1186/s12936-022-04073-1},
  URL = {https://www.documentation.ird.fr/hor/fdi:010084322},
}