The impact of different DNA extraction kits and laboratories upon the assessment of human gut microbiota composition by 16S rRNA gene sequencing

William Newman; Nicholas A. Kennedy; Alan W. Walker; Susan H. Berry; Sylvia H. Duncan; Freda M. Farquarson; Petra Louis; John M. Thomson; Jack Satsangi; Harry J. Flint; Julian Parkhill; Charlie W. Lees; Georgina L. Hold

doi:10.1371/journal.pone.0088982

The impact of different DNA extraction kits and laboratories upon the assessment of human gut microbiota composition by 16S rRNA gene sequencing

William Newman, Nicholas A. Kennedy, Alan W. Walker, Susan H. Berry, Sylvia H. Duncan, Freda M. Farquarson, Petra Louis, John M. Thomson, Jack Satsangi, Harry J. Flint, Julian Parkhill, Charlie W. Lees, Georgina L. Hold

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Abstract

Introduction: Determining bacterial community structure in fecal samples through DNA sequencing is an important facet of intestinal health research. The impact of different commercially available DNA extraction kits upon bacterial community structures has received relatively little attention. The aim of this study was to analyze bacterial communities in volunteer and inflammatory bowel disease (IBD) patient fecal samples extracted using widely used DNA extraction kits in established gastrointestinal research laboratories. Methods: Fecal samples from two healthy volunteers (H3 and H4) and two relapsing IBD patients (I1 and I2) were investigated. DNA extraction was undertaken using MoBio Powersoil and MP Biomedicals FastDNA SPIN Kit for Soil DNA extraction kits. PCR amplification for pyrosequencing of bacterial 16S rRNA genes was performed in both laboratories on all samples. Hierarchical clustering of sequencing data was done using the Yue and Clayton similarity coefficient. Results: DNA extracted using the FastDNA kit and the MoBio kit gave median DNA concentrations of 475 (interquartile range 228-561) and 22 (IQR 9-36) ng/μL respectively (p

Original language	English
Article number	e88982
Journal	PLoS ONE
Volume	9
Issue number	2
DOIs	https://doi.org/10.1371/journal.pone.0088982
Publication status	Published - 24 Feb 2014

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Newman, W., Kennedy, N. A., Walker, A. W., Berry, S. H., Duncan, S. H., Farquarson, F. M., Louis, P., Thomson, J. M., Satsangi, J., Flint, H. J., Parkhill, J., Lees, C. W., & Hold, G. L. (2014). The impact of different DNA extraction kits and laboratories upon the assessment of human gut microbiota composition by 16S rRNA gene sequencing. PLoS ONE, 9(2), Article e88982. https://doi.org/10.1371/journal.pone.0088982

@article{3e15c1c7deb643f5a2acb60df4f53e32,

title = "The impact of different DNA extraction kits and laboratories upon the assessment of human gut microbiota composition by 16S rRNA gene sequencing",

abstract = "Introduction: Determining bacterial community structure in fecal samples through DNA sequencing is an important facet of intestinal health research. The impact of different commercially available DNA extraction kits upon bacterial community structures has received relatively little attention. The aim of this study was to analyze bacterial communities in volunteer and inflammatory bowel disease (IBD) patient fecal samples extracted using widely used DNA extraction kits in established gastrointestinal research laboratories. Methods: Fecal samples from two healthy volunteers (H3 and H4) and two relapsing IBD patients (I1 and I2) were investigated. DNA extraction was undertaken using MoBio Powersoil and MP Biomedicals FastDNA SPIN Kit for Soil DNA extraction kits. PCR amplification for pyrosequencing of bacterial 16S rRNA genes was performed in both laboratories on all samples. Hierarchical clustering of sequencing data was done using the Yue and Clayton similarity coefficient. Results: DNA extracted using the FastDNA kit and the MoBio kit gave median DNA concentrations of 475 (interquartile range 228-561) and 22 (IQR 9-36) ng/μL respectively (p",

author = "William Newman and Kennedy, {Nicholas A.} and Walker, {Alan W.} and Berry, {Susan H.} and Duncan, {Sylvia H.} and Farquarson, {Freda M.} and Petra Louis and Thomson, {John M.} and Jack Satsangi and Flint, {Harry J.} and Julian Parkhill and Lees, {Charlie W.} and Hold, {Georgina L.}",

year = "2014",

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doi = "10.1371/journal.pone.0088982",

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Newman, W, Kennedy, NA, Walker, AW, Berry, SH, Duncan, SH, Farquarson, FM, Louis, P, Thomson, JM, Satsangi, J, Flint, HJ, Parkhill, J, Lees, CW & Hold, GL 2014, 'The impact of different DNA extraction kits and laboratories upon the assessment of human gut microbiota composition by 16S rRNA gene sequencing', PLoS ONE, vol. 9, no. 2, e88982. https://doi.org/10.1371/journal.pone.0088982

TY - JOUR

T1 - The impact of different DNA extraction kits and laboratories upon the assessment of human gut microbiota composition by 16S rRNA gene sequencing

AU - Newman, William

AU - Kennedy, Nicholas A.

AU - Walker, Alan W.

AU - Berry, Susan H.

AU - Duncan, Sylvia H.

AU - Farquarson, Freda M.

AU - Louis, Petra

AU - Thomson, John M.

AU - Satsangi, Jack

AU - Flint, Harry J.

AU - Parkhill, Julian

AU - Lees, Charlie W.

AU - Hold, Georgina L.

PY - 2014/2/24

Y1 - 2014/2/24

N2 - Introduction: Determining bacterial community structure in fecal samples through DNA sequencing is an important facet of intestinal health research. The impact of different commercially available DNA extraction kits upon bacterial community structures has received relatively little attention. The aim of this study was to analyze bacterial communities in volunteer and inflammatory bowel disease (IBD) patient fecal samples extracted using widely used DNA extraction kits in established gastrointestinal research laboratories. Methods: Fecal samples from two healthy volunteers (H3 and H4) and two relapsing IBD patients (I1 and I2) were investigated. DNA extraction was undertaken using MoBio Powersoil and MP Biomedicals FastDNA SPIN Kit for Soil DNA extraction kits. PCR amplification for pyrosequencing of bacterial 16S rRNA genes was performed in both laboratories on all samples. Hierarchical clustering of sequencing data was done using the Yue and Clayton similarity coefficient. Results: DNA extracted using the FastDNA kit and the MoBio kit gave median DNA concentrations of 475 (interquartile range 228-561) and 22 (IQR 9-36) ng/μL respectively (p

AB - Introduction: Determining bacterial community structure in fecal samples through DNA sequencing is an important facet of intestinal health research. The impact of different commercially available DNA extraction kits upon bacterial community structures has received relatively little attention. The aim of this study was to analyze bacterial communities in volunteer and inflammatory bowel disease (IBD) patient fecal samples extracted using widely used DNA extraction kits in established gastrointestinal research laboratories. Methods: Fecal samples from two healthy volunteers (H3 and H4) and two relapsing IBD patients (I1 and I2) were investigated. DNA extraction was undertaken using MoBio Powersoil and MP Biomedicals FastDNA SPIN Kit for Soil DNA extraction kits. PCR amplification for pyrosequencing of bacterial 16S rRNA genes was performed in both laboratories on all samples. Hierarchical clustering of sequencing data was done using the Yue and Clayton similarity coefficient. Results: DNA extracted using the FastDNA kit and the MoBio kit gave median DNA concentrations of 475 (interquartile range 228-561) and 22 (IQR 9-36) ng/μL respectively (p

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DO - 10.1371/journal.pone.0088982

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VL - 9

JO - PLoS ONE

JF - PLoS ONE

IS - 2

M1 - e88982

ER -

The impact of different DNA extraction kits and laboratories upon the assessment of human gut microbiota composition by 16S rRNA gene sequencing

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