Using CF11 cellulose columns to inexpensively and effectively remove human DNA from Plasmodium falciparum-infected whole blood samples.
Bottom Line: Genome and transcriptome studies of Plasmodium nucleic acids obtained from parasitized whole blood are greatly improved by depletion of human DNA or enrichment of parasite DNA prior to next-generation sequencing and microarray hybridization.The CF11 procedure was compared with the current two-step standard of leukocyte depletion using parasitized red blood cells cultured in vitro and parasitized blood obtained ex vivo from Cambodian patients with malaria.Procedural variations in centrifugation and column size were tested, along with a range of blood volumes and parasite densities.
Affiliation: Howard Hughes Medical Institute, University of Maryland School of Medicine, Baltimore, MD, USA.Show MeSH
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Mentions: Filtration of blood-parasite mixtures using unmodified LP and CF11 methods in parallel successfully depleted human DNA to 1% and 6% of total DNA, respectively (Figure 2A), well below the current Illumina sequencing threshold of < 50%. CF11 was more effective than LP in recovering total DNA from blood-parasite mixtures (mean 1.11 μg vs. 0.37 μg; p = 0.03) (Figure 2B). All 15 CF11-filtered parasitized blood samples and 12 of 14 LP-filtered parasitized blood samples yielded < 50% human DNA contamination (Figure 3A). Human DNA contamination was lower for parasitized blood filtered by CF11 than by LP (mean 2.4% vs. 15.9%; p = 0.03) (Figure 3B). Recovery of total DNA was comparably high in both sets of samples (mean 4.64 μg for CF11 vs. 2.86 μg for LP; p = 0.46) One LP-filtered sample failed to produce a human DNA estimate by qPCR and was omitted from the analysis.
Affiliation: Howard Hughes Medical Institute, University of Maryland School of Medicine, Baltimore, MD, USA.