Abstract
The human gut contains dense and diverse microbial communities which have profound influences on human health. Gaining meaningful insights into these communities requires provision of high quality microbial nucleic acids from human fecal samples, as well as an understanding of the sources of variation and their impacts on the experimental model. We present here a systematic analysis of commonly used microbial DNA extraction methods, and identify significant sources of variation. Five extraction methods (Human Microbiome Project protocol, MoBio PowerSoil DNA Isolation Kit, QIAamp DNA Stool Mini Kit, ZR Fecal DNA MiniPrep, phenol:chloroform-based DNA isolation) were evaluated based on the following criteria: DNA yield, quality and integrity, and microbial community structure based on Illumina amplicon sequencing of the V4 region of bacterial and archaeal 16S rRNA genes. Our results indicate that the largest portion of variation within the model was attributed to differences between subjects (biological variation), with a smaller proportion of variation associated with DNA extraction method (technical variation) and intra-subject variation. A comprehensive understanding of the potential impact of technical variation on the human gut microbiota will help limit preventable bias, enabling more accurate diversity estimates.
Highlights
The human gut harbors the most substantial microbial communities within our bodies, with these communities exhibiting considerable inter- and intra-personal variability (Eckburg et al, 2005; Ley et al, 2006; Qin et al, 2010)
The Human Microbiome Project gel results indicated the least amount of shearing of all the methods
A recent meta-analysis examined beta diversity among microbial communities generated from human stool samples across several human microbiome studies (Lozupone et al, 2013)
Summary
The human gut harbors the most substantial microbial communities within our bodies, with these communities exhibiting considerable inter- and intra-personal variability (Eckburg et al, 2005; Ley et al, 2006; Qin et al, 2010). Gut microbial community composition varies less within an individual than among different individuals, suggesting a strong environmental component (Turnbaugh et al, 2006; Qin et al, 2010; Caporaso et al, 2011; The Human Microbiome Project Consortium, 2012; Schloissnig et al, 2013). Differences in lysis efficiency, heterogeneous distribution of microbes across a sample, and adherence of microbes to the stool matrix can result in preferential lysis of certain cell types and misrepresentation of microbial community diversity (Li et al, 2003; Ariefdjohan et al, 2010; Maukonen et al, 2012). Methodological, or technical, variation such as that due to different DNA extraction techniques, sequencing platforms, and/or sequence processing pipelines can influence descriptions of ecological diversity and observed biological variation, including inter-subject variation
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