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J Oral Microbiol. 2014 Apr 23;6. doi: 10.3402/jom.v6.23990. eCollection 2014.

Influence of DNA extraction on oral microbial profiles obtained via 16S rRNA gene sequencing.

Author information

1
Division of Periodontology, Department of Oral Health and Diagnostic Sciences, The University of Connecticut Health Center, Farmington, CT, USA ; Laboratory of Oral Microbiology, Faculty of Dentistry, University of Chile, Santiago, Chile.
2
Division of Periodontology, Department of Oral Health and Diagnostic Sciences, The University of Connecticut Health Center, Farmington, CT, USA.
3
Department of Molecular and Cell Biology, The Center for Applied Genetics and Technologies, The University of Connecticut, Storrs, CT, USA.

Abstract

BACKGROUND AND OBJECTIVE:

The advent of next-generation sequencing has significantly facilitated characterization of the oral microbiome. Despite great efforts in streamlining the processes of sequencing and data curation, upstream steps required for amplicon library generation could still influence 16S rRNA gene-based microbial profiles. Among upstream processes, DNA extraction is a critical step that could represent a great source of bias. Accounting for bias introduced by extraction procedures is important when comparing studies that use different methods. Identifying the method that best portrays communities is also desirable. Accordingly, the aim of this study was to evaluate bias introduced by different DNA extraction procedures on oral microbiome profiles.

DESIGN:

Four DNA extraction methods were tested on mock communities consisting of seven representative oral bacteria. Additionally, supragingival plaque samples were collected from seven individuals and divided equally to test two commonly used DNA extraction procedures. Amplicon libraries of the 16S rRNA gene were generated and sequenced via 454-pyrosequencing.

RESULTS:

Evaluation of mock communities revealed that DNA yield and bacterial species representation varied with DNA extraction methods. Despite producing the lowest yield of DNA, a method that included bead beating was the only protocol capable of detecting all seven species in the mock community. Comparison of the performance of two commonly used methods (crude lysis and a chemical/enzymatic lysis+column-based DNA isolation) on plaque samples showed no effect of extraction protocols on taxa prevalence but global community structure and relative abundance of individual taxa were affected. At the phylum level, the latter method improved the recovery of Actinobacteria, Bacteroidetes, and Spirochaetes over crude lysis.

CONCLUSION:

DNA extraction distorts microbial profiles in simulated and clinical oral samples, reinforcing the importance of careful selection of a DNA extraction protocol to improve species recovery and facilitate data comparison across oral microbiology studies.

KEYWORDS:

DNA extraction; bias; oral microbiome

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