Selective removal of human DNA from metagenomic DNA samples extracted from dental plaque

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.

A catalog of microbial genes from the bovine rumen unveils a specialized and diverse biomass-degrading environment

“…Briefly, the pellet was suspended in water (Millipore Waters Milli Q purification unit) and incubated for 1 h at room temperature followed by DNase treatment (Benzonase, Novagen) as described [40]. The suspension was filtered through a 10 µm monofilament textile, collected by centrifugation as before, suspended in an appropriate volume of PBS and stored at -80 °C until DNA extraction.…”

Evaluation of human and microbial DNA content in subgingival plaque samples collected by paper points or curette

“…Incorporation of human DNA in samples may be overcome by adding enzymatic or centrifugation steps during nucleic acid (NA) extraction. However, as these approaches may affect bacterial DNA and cause misinterpretation of the microbial diversity (Horz et al, 2008;Hunter et al, 2011), it is relevant to determine the subgingival plaque sampling method able to carry the lowest content of host cells. Therefore, this study compared the levels of content of host DNA in subgingival plaque collected by paper points (PP) and curette.…”

Dna From Human Ancient Bacteria: A Novel Source of Genetic Evidence From Archaeological Dental Calculus

“…1995), due to the incorporation of human cells (epithelial cells, leucocytes and macrophages) in calculus (Xie et al . 2010; Hunter et al . 2011).…”