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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\u20138\u00a0\u00b5m and exhibit some deformability, it was hypothesized that cheap and commercially available 5\u00a0\u00b5m filters might retain leukocytes but much less of\n                      <jats:italic>Plasmodium falciparum<\/jats:italic>\n                      -infected RBCs. This study aimed to test the hypothesis that such a filtration method, named 5WBF (for\n                      <jats:underline>5<\/jats:underline>\n                      \u00a0\u00b5m\n                      <jats:underline>W<\/jats:underline>\n                      hole\n                      <jats:underline>B<\/jats:underline>\n                      lood\n                      <jats:underline>F<\/jats:underline>\n                      iltration), may provide highly enriched parasite material suitable for\n                      <jats:italic>P. falciparum<\/jats:italic>\n                      WGS.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      Whole blood was collected from five patients experiencing a\n                      <jats:italic>P. falciparum<\/jats:italic>\n                      malaria episode (ring-stage parasitaemia range: 0.04\u20135.5%) and from mock samples obtained by mixing synchronized, ring-stage cultured\n                      <jats:italic>P. falciparum<\/jats:italic>\n                      3D7 parasites with uninfected human whole blood (final parasitaemia range: 0.02\u20131.1%). These whole blood samples (50 to 400 \u00b5L) were diluted in RPMI 1640 medium or PBS 1\u00d7\u2009buffer and filtered with a syringe connected to a 5\u00a0\u00b5m 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.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      After applying 5WBF, qPCR indicated an average of twofold loss in the amount of parasite template DNA (Pf ARN\n                      <jats:italic>18S<\/jats:italic>\n                      gene) and from 4096- to 65,536-fold loss of human template DNA (human\n                      <jats:italic>\u03b2 actin<\/jats:italic>\n                      gene). WGS analyses revealed that\u2009&gt;\u200995% of the \u00a0parasite\u00a0nuclear and organellar genomes were all\u00a0covered at\u2009\u2265\u200910\u00d7\u2009depth 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\u2009\u2265\u200910\u00d7\u2009depth depending on parasitaemia. Sequence reads were homogeneously distributed across gene sequences for 5WBF-treated samples (n\u2009=\u20095460 genes; mean coverage: 91\u00d7; median coverage: 93\u00d7; 5th percentile: 70\u00d7; 95th percentile: 103\u00d7), allowing the identification of gene copy number variations such as for\n                      <jats:italic>gch1<\/jats:italic>\n                      . 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\u00d7; median coverage: 51\u00d7; 5th percentile: 7\u00d7; 95th percentile: 245\u00d7).\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>\n                      The novel 5WBF leucodepletion method is simple to implement and based on commercially available, standardized 5\u00a0\u00b5m filters which cost from 1.0 to 1.7\u20ac per unit depending on suppliers. 5WBF permits extensive genome-wide analysis of\n                      <jats:italic>P. falciparum<\/jats:italic>\n                      ring-stage isolates from minute amounts of whole blood even with parasitaemias as low as 0.02%.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/s12936-022-04073-1","type":"journal-article","created":{"date-parts":[[2022,2,16]],"date-time":"2022-02-16T06:03:06Z","timestamp":1644991386000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["5WBF: a low-cost and straightforward whole blood filtration method suitable for whole-genome sequencing of Plasmodium falciparum clinical isolates"],"prefix":"10.1186","volume":"21","author":[{"given":"Romain","family":"Copp\u00e9e","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Atikatou","family":"Mama","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"V\u00e9ronique","family":"Sarrasin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Claire","family":"Kamaliddin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lucie","family":"Adoux","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lawrence","family":"Palazzo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nicaise Tuikue","family":"Ndam","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Franck","family":"Letourneur","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fr\u00e9d\u00e9ric","family":"Ariey","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sandrine","family":"Houz\u00e9","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"J\u00e9r\u00f4me","family":"Clain","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2022,2,16]]},"reference":[{"key":"4073_CR1","doi-asserted-by":"publisher","first-page":"215","DOI":"10.1007\/978-1-60327-367-1_12","volume":"628","author":"PC Ng","year":"2010","unstructured":"Ng PC, Kirkness EF. 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Samples received at the French Malaria Reference Centre, (Paris, France) were registered and declared for research purposes as a biobank for both the Assistance Publique des H\u00f4pitaux de Paris and Sant\u00e9 Publique France.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval and consent to participate"}},{"value":"There are no case presentations that require disclosure of respondent\u2019s confidential data\/information in this study.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Consent for publication"}},{"value":"The authors declare that they have no competing interests.","order":4,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"51"}}