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.CF11 filtration is a cost-effective, scalable, one-step approach to remove human DNA from P. falciparum-infected whole blood samples.
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.