Comparison of DNA Extraction Methods in Analysis of Salivary Bacterial Communities

Abstract

Culture-independent high-throughput sequencing-based methods are widely used to study bacterial communities. Although these approaches are superior to traditional culture-based methods, they introduce bias at the experimental and bioinformatics levels. We assessed the diversity of the human salivary microbiome by pyrosequencing of the 16S rDNA V1–3 amplicons using metagenomic DNA extracted by two different protocols: a simple proteinase K digestion without a subsequent DNA clean-up step, and a bead-beating mechanical lysis protocol followed by column DNA purification. A high degree of congruence was found between the two extraction methods, most notably in regard to the microbial community composition. The results showed that for a given bioinformatics pipeline, all the taxa with an average proportion >0.12% in samples processed using one extraction method were also detected in samples extracted using the other method. The same taxa tended to be abundant and frequent for both extraction methods. The relative abundance of sequence reads assigned to the phyla Actinobacteria, Spirochaetes, TM7, Synergistetes, and Tenericutes was significantly higher in the mechanically-treated samples than in the enzymatically-treated samples, whereas the phylum Firmicutes showed the opposite pattern. No significant differences in diversity indices were found between the extraction methods, although the mechanical lysis method revealed higher operational taxonomic unit richness. Differences between the extraction procedures outweighed the variations due to the bioinformatics analysis pipelines used.

Assessment of Airway Microbiota and Inflammation in Cystic Fibrosis Using Multiple Sampling Methods

“…These findings are consistent with previous studies of the salivary microbiome in healthy subjects, although Neisseria was less prominent in our findings compared with other reports (37)(38)(39)(40)(41). Streptococcus, Prevotella, Rothia, and Veillonella were also detected in high RA in ES and IS samples, consistent with previous studies of CF airway samples using molecular approaches and anaerobic cultures (7,9,10,13,42).…”

The yield and quality of cellular and bacterial DNA extracts from human oral rinse samples are variably affected by the cell lysis methodology

“…The variations in the resultant microbial profiles in different studies due to methodological disparities make comparisons difficult and not very meaningful (Mai and Draganov, 2009). Although the results of a few studies available to date have shown no significant effects of different DNA extraction methods on the resultant bacterial profiles of biological samples (Peng et al, 2013, Wagner Mackenzie et al, 2015, a number of workers have recognised and called for a consensus and a universal DNA extraction method for microbiome analyses to overcome the challenges and misrepresentation inherent in using disparate techniques (Abusleme et al, 2014, Claassen et al, 2013, Henderson et al, 2013, Kennedy et al, 2014, Lazarevic et al, 2013, Maukonen et al, 2012, Walker et al, 2015, Yuan et al, 2012.…”

Dead or Alive: Deoxyribonuclease I Sensitive Bacteria and Implications for the Sinus Microbiome

“…To determine how much of the DNA extracted from each of our collection techniques originated from bacteria rather than the host, we performed quantitative PCR by using bacterial-or human-specific primer sets. 17,18 We found that 4-5% of the DNA extracted from our brushes and swabs was of bacterial origin, whereas only ϳ2% of the DNA from the tissue samples was bacterial ( Table 2).…”

“…17 Bacterial 16S rRNA primers used were the following: F 5Ј-ACTCCTACGGGAGGCAGCAGT-3Ј, R 5Ј-ATTACCGCGGCTGCTGGC-3Ј; program: 10 minutes at 95°C, followed by 40 cycles of 95°C for 30 seconds and 68°C for 1 minute. 18 All reactions were performed in triplicate by using a Rotogene RG-3000 (Qiagen, Hilden, Germany). Standard curves were generated by using known concentrations of Human Male Genomic (EMD Millipore, Billerica, MA) and Escherichia coli (Affymetrix, Santa Clara, CA) DNA.…”