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

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.