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Using CF11 cellulose columns to inexpensively and effectively remove human DNA from Plasmodium falciparum-infected whole blood samples.

Venkatesan M, Amaratunga C, Campino S, Auburn S, Koch O, Lim P, Uk S, Socheat D, Kwiatkowski DP, Fairhurst RM, Plowe CV - Malar. J. (2012)

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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Howard Hughes Medical Institute, University of Maryland School of Medicine, Baltimore, MD, USA.

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Related in: MedlinePlus

Percent human DNA (A) and total DNA (B) as estimated by qPCR after leukocyte depletion of Plasmodium falciparum-infected blood samples that vary in parasite density. Light gray shading indicates samples with < 50,000 parasites/μl, medium gray shading indicates samples with 50,000-100,000 parasites/μl, and dark gray shading indicates samples with parasite density > 100,000 parasites/μl. All sample volumes were 4 ml. n = sample size for each method within each range of parasite density. Dashed lines indicate current criteria for Illumina sequencing: < 50% human DNA and > 500 ng total DNA. LP = Lymphoprep + Plasmodipur, CF11 = filtration using a 5.5-ml CF11 column. Box plots show median and interquartile range. Whiskers span the range of data points within 1.5 times the interquartile range of the lower and upper quartiles. Outliers are shown as points.
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Figure 3: Percent human DNA (A) and total DNA (B) as estimated by qPCR after leukocyte depletion of Plasmodium falciparum-infected blood samples that vary in parasite density. Light gray shading indicates samples with < 50,000 parasites/μl, medium gray shading indicates samples with 50,000-100,000 parasites/μl, and dark gray shading indicates samples with parasite density > 100,000 parasites/μl. All sample volumes were 4 ml. n = sample size for each method within each range of parasite density. Dashed lines indicate current criteria for Illumina sequencing: < 50% human DNA and > 500 ng total DNA. LP = Lymphoprep + Plasmodipur, CF11 = filtration using a 5.5-ml CF11 column. Box plots show median and interquartile range. Whiskers span the range of data points within 1.5 times the interquartile range of the lower and upper quartiles. Outliers are shown as points.

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.


Using CF11 cellulose columns to inexpensively and effectively remove human DNA from Plasmodium falciparum-infected whole blood samples.

Venkatesan M, Amaratunga C, Campino S, Auburn S, Koch O, Lim P, Uk S, Socheat D, Kwiatkowski DP, Fairhurst RM, Plowe CV - Malar. J. (2012)

Percent human DNA (A) and total DNA (B) as estimated by qPCR after leukocyte depletion of Plasmodium falciparum-infected blood samples that vary in parasite density. Light gray shading indicates samples with < 50,000 parasites/μl, medium gray shading indicates samples with 50,000-100,000 parasites/μl, and dark gray shading indicates samples with parasite density > 100,000 parasites/μl. All sample volumes were 4 ml. n = sample size for each method within each range of parasite density. Dashed lines indicate current criteria for Illumina sequencing: < 50% human DNA and > 500 ng total DNA. LP = Lymphoprep + Plasmodipur, CF11 = filtration using a 5.5-ml CF11 column. Box plots show median and interquartile range. Whiskers span the range of data points within 1.5 times the interquartile range of the lower and upper quartiles. Outliers are shown as points.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3295709&req=5

Figure 3: Percent human DNA (A) and total DNA (B) as estimated by qPCR after leukocyte depletion of Plasmodium falciparum-infected blood samples that vary in parasite density. Light gray shading indicates samples with < 50,000 parasites/μl, medium gray shading indicates samples with 50,000-100,000 parasites/μl, and dark gray shading indicates samples with parasite density > 100,000 parasites/μl. All sample volumes were 4 ml. n = sample size for each method within each range of parasite density. Dashed lines indicate current criteria for Illumina sequencing: < 50% human DNA and > 500 ng total DNA. LP = Lymphoprep + Plasmodipur, CF11 = filtration using a 5.5-ml CF11 column. Box plots show median and interquartile range. Whiskers span the range of data points within 1.5 times the interquartile range of the lower and upper quartiles. Outliers are shown as points.
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.

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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Howard Hughes Medical Institute, University of Maryland School of Medicine, Baltimore, MD, USA.

Show MeSH
Related in: MedlinePlus