Evaluation of Methods for the Extraction and Purification of DNA from the Human Microbiome

Abstract

Background DNA extraction is an essential step in all cultivation-independent approaches to characterize microbial diversity, including that associated with the human body. A fundamental challenge in using these approaches has been to isolate DNA that is representative of the microbial community sampled. Methodology/Principal Findings In this study, we statistically evaluated six commonly used DNA extraction procedures using eleven human-associated bacterial species and a mock community that contained equal numbers of those eleven species. These methods were compared on the basis of DNA yield, DNA shearing, reproducibility, and most importantly representation of microbial diversity. The analysis of 16S rRNA gene sequences from a mock community showed that the observed species abundances were significantly different from the expected species abundances for all six DNA extraction methods used. Conclusions/Significance Protocols that included bead beating and/or mutanolysin produced significantly better bacterial community structure representation than methods without both of them. The reproducibility of all six methods was similar, and results from different experimenters and different times were in good agreement. Based on the evaluations done it appears that DNA extraction procedures for bacterial community analysis of human associated samples should include bead beating and/or mutanolysin to effectively lyse cells.

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

“…A number of workers have compared different commercial kits and non-commercial protocols using either human samples or a laboratory created bacterial community (Claassen et al, 2013, Kennedy et al, 2014, McOrist et al, 2002, Wagner Mackenzie et al, 2015, Yuan et al, 2012. In contrast, in the current study we attempted to evaluate the qualitative and quantitative changes of the extracted DNA consequential to various cell lysis methods described in the literature.…”

“…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.…”

An Improved Methodology to Overcome Key Issues in Human Fecal Metagenomic DNA Extraction

“…A number of efforts to optimize a methodology for metagenomic DNA isolation from feces have been undertaken [11], [13], [15], [20]. Although progress has been made in this regard, problems of limited applicability ( e.g.…”

“…In summary, current methodology has been successfully used to isolate the metagenomic DNA from one-month-old frozen fecal sample stored at −86 °C with a minimal effect on yield and quality, which was a challenge as per reported metagenomic DNA isolation studies [13], [14], [20], [21], [22].…”

“…The success of these culture-independent studies depends primarily on the quality and quantity of metagenomic DNA isolated from the given samples [11], [12], [13]. Therefore, isolation of metagenomic DNA with a good quality from a heterogeneous source like human feces has been a challenging task.…”