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. 2012;7(3):e33865.
doi: 10.1371/journal.pone.0033865. Epub 2012 Mar 23.

Evaluation of methods for the extraction and purification of DNA from the human microbiome

Sanqing Yuan  1 , Dora B Cohen, Jacques Ravel, Zaid Abdo, Larry J Forney
Affiliations

Evaluation of methods for the extraction and purification of DNA from the human microbiome

Sanqing Yuan et al. PLoS One. 2012.

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Boxplot of Euclidean distances between observed and expected species proportions.
Euclidean distances between observed and expected proportions were calculated for each of eight replicates of each method.
Figure 2
Figure 2. Boxplot of Euclidean distances between observed and grand proportions.
To calculate grand proportions, the total counts of 16S rRNA gene reads of each species were calculated for eight replicates of each method. Then grand proportions were calculated based on total counts of 16S rRNA gene reads of each species per method. Grand proportions were used to calculate Euclidean distances between observed and grand proportions.
Figure 3
Figure 3. DNA extractions using different enzymatic lysis modes.
The mean concentrations (columns) were calculated based on nine replicated extractions per sample per mode. Pair-wise comparisons of DNA concentrations between modes per sample were performed by using Wilcoxon rank sum test. Bonferroni correction was used for multiple testing. Letters at the top of columns indicate whether there is significantly difference between columns per sample. Means with the same letter are not significantly different.
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