Abstract
BackgroundDNA 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 FindingsIn 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/SignificanceProtocols 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.
Highlights
The microorganisms that colonize various anatomical sites of the human body play important roles in human health and disease [1]
Analysis of variance (ANOVA) showed that the DNA yield varied significantly depending on the DNA extraction method used (p = 0.0017)
For seven of the 11 bacterial species, DNA yields obtained using method 4 were significantly higher than DNA yields obtained using the other five methods that
Summary
The microorganisms that colonize various anatomical sites of the human body play important roles in human health and disease [1]. In recent years there has been increasing interest in knowing more about how differences between individuals, or within individuals over time influence the maintenance of health and risk to disease Such studies require a detailed understanding of the microbial diversity found at various anatomically distinct sites of the human body. The cultivation-dependent methods commonly used in clinical and research laboratories have provided a valuable but incomplete picture of the vast diversity found in the human microbiome because many, if not most human-associated microorganisms have not yet been successfully cultured in the laboratory [8,9,10,11] These methods are limited because most do not lend themselves to the analysis of large numbers of samples because they are labor-intensive and costly. A fundamental challenge in using these approaches has been to isolate DNA that is representative of the microbial community sampled
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