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Method Paper
Selective removal of human DNA from metagenomic DNA samples extracted from dental plaque
Article first published online: 7 FEB 2011
DOI: 10.1002/jobm.201000372
Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Additional Information
How to Cite
Hunter, S. J., Easton, S., Booth, V., Henderson, B., Wade, W. G. and Ward, J. M. (2011), Selective removal of human DNA from metagenomic DNA samples extracted from dental plaque. J. Basic Microbiol., 51: 442–446. doi: 10.1002/jobm.201000372
Publication History
- Issue published online: 8 AUG 2011
- Article first published online: 7 FEB 2011
- Manuscript Accepted: 22 OCT 2010
- Manuscript Received: 16 SEP 2010
Funded by
- Wellcome Trust. Grant Number: 078131/Z/05/Z
Keywords:
- Dental plaque;
- Differential centrifugation;
- Selective lysis;
- Metagenomics;
- Real-time PCR
Abstract
Metagenomic techniques are used to analyse bacterial communities allowing both culturable and unculturable species to be represented. However, the screening of oral metagenomic samples can be hindered by high animal host DNA content. This study evaluated methods for the reduction of human DNA concentrations within oral metagenomic samples.
Plaque samples were collected from 27 patients presenting with periodontal disease and treated to remove human DNA using either selective lysis of eukaryotic cells at several buffer concentrations or differential centrifugation after treatment with trypsin and/or detergents. Human and bacterial DNA levels were determined by quantitative polymerase chain reaction (qPCR).
The human DNA content of plaque extracts was significantly reduced by all treatments compared with an untreated control (P < 0.05). However, differential centrifugation simultaneously reduced the bacterial DNA content unless samples were pretreated with a detergent. Observations of Gram stained samples that were processed using differential centrifugation without detergent suggest that many bacteria remain adhered to human cells. An approach that uses differential centrifugation in parallel with selective lysis is recommended to fully represent the oral microbiota in metagenomic samples, including those tightly adhered to human cells and more delicate bacteria such as Mycoplasma. (© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)