Evaluation of Oral Cavity DNA Extraction Methods on Bacterial and Fungal Microbiota

Jennifer Rosenbaum,Lorna E Thorpe,Mykhaylo Usyk,Levi Waldron,Heidi E Jones,Christine P Zolnik,Zigui Chen,Jennifer B Dowd,Robert D Burk

doi:10.1038/s41598-018-38049-6

Abstract

The objective of this study was to evaluate the most effective method of DNA extraction of oral mouthwash samples for use in microbiome studies that utilize next generation sequencing (NGS). Eight enzymatic and mechanical DNA extraction methods were tested. Extracted DNA was amplified using barcoded primers targeting the V6 variable region of the bacterial 16S rRNA gene and the ITS1 region of the fungal ribosomal gene cluster and sequenced using the Illumina NGS platform. Sequenced reads were analyzed using QIIME and R. The eight methods yielded significantly different quantities of DNA (p < 0.001), with the phenol-chloroform extraction method producing the highest total yield. There were no significant differences in observed bacterial or fungal Shannon diversity (p = 0.64, p = 0.93 respectively) by extraction method. Bray-Curtis beta-diversity did not demonstrate statistically significant differences between the eight extraction methods based on bacterial (R2 = 0.086, p = 1.00) and fungal (R2 = 0.039, p = 1.00) assays. No differences were seen between methods with or without bead-beating. These data indicate that choice of DNA extraction method affect total DNA recovery without significantly affecting the observed microbiome.

Highlights

The human oral cavity hosts a diverse microbial community comprised of bacteria, fungi, protozoa, archaea, and viruses[1]
Raw sequences were processed for quality control and chimera removal, resulting in a total of 373,840 bacterial reads, and 363,881 fungal sequence reads
Our analysis revealed that DNA yield was significantly different among the eight DNA extraction methods with DNA recovery greatest after phenol-chloroform extraction (Fig. 1)

Summary

Introduction

The human oral cavity hosts a diverse microbial community comprised of bacteria, fungi, protozoa, archaea, and viruses[1]. The vast bacterial biota includes pathogenic bacteria that are responsible for local and systemic diseases[2]. For example bacteria have been shown to be responsible for oral ailments such as dental caries[3], and periodontal diseases[4]. The scope of bacteria causing oral ailments is vast with conditions such as mild gum disease and gingivitis affecting over 90% of adults[5] at some point in their lives. The oral cavity hosts a variety of fungal species[15]. The fungal constituents of the oral microbiome have far been understudied when compared to bacteria, but are emerging as being important in human disease. Studies indicate that fungi operate together with bacteria in oral infections[18]

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Conclusion