Gerasimidis, Konstantinos, Bertz, Martin, Quince, Christopher, Brunner, Katja, Bruce, Alanna, Combet, Emilie, Calus, Szymon, Loman, Nick and Ijaz, Umer Zeeshan (2016) The effect of DNA extraction methodology on gut microbiota research applications. BMC Research Notes, 9 (1). 365. doi:10.1186/s13104-016-2171-7 ISSN 1756-0500.

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Abstract

Background:
The effect that traditional and modern DNA extraction methods have on applications to study the role of gut microbiota in health and disease is a topic of current interest. Genomic DNA was extracted from three faecal samples and one probiotic capsule using three popular methods; chaotropic (CHAO) method, phenol/chloroform (PHEC) extraction, proprietary kit (QIAG). The performance of each of these methods on DNA yield and quality, microbiota composition using quantitative PCR, deep sequencing of the 16S rRNA gene, and sequencing analysis pipeline was evaluated.

Results:
The CHAO yielded the highest and the QIAG kit the lowest amount of double-stranded DNA, but the purity of isolated nucleic acids was better for the latter method. The CHAO method yielded a higher concentration of bacterial taxa per mass (g) of faeces. Sequencing coverage was higher in CHAO method but a higher proportion of the initial sequencing reads were retained for assignments to operational taxonomic unit (OTU) in the QIAG kit compared to the other methods. The QIAG kit appeared to have longer trimmed reads and shorter regions of worse quality than the other two methods. A distinct separation of α-diversity indices between different DNA extraction methods was not observed. When compositional dissimilarities between samples were explored, a strong separation was observed according to sample type. The effect of the extraction method was either marginal (Bray–Curtis distance) or none (unweighted Unifrac distance). Taxon membership and abundance in each sample was independent of the DNA extraction method used.

Conclusions:
We have benchmarked several DNA extraction methods commonly used in gut microbiota research and their differences depended on the downstream applications intended for use. Caution should be paid when the intention is to pool and analyse samples or data from studies which have used different DNA extraction methods.

Item Type:	Journal Article
Subjects:	Q Science > QH Natural history Q Science > QP Physiology
Divisions:	Faculty of Science, Engineering and Medicine > Medicine > Warwick Medical School > Biomedical Sciences Faculty of Science, Engineering and Medicine > Medicine > Warwick Medical School > Biomedical Sciences > Microbiology & Infection Faculty of Science, Engineering and Medicine > Medicine > Warwick Medical School
Library of Congress Subject Headings (LCSH):	Metagenomics, DNA
Journal or Publication Title:	BMC Research Notes
Publisher:	BioMed Central Ltd.
ISSN:	1756-0500
Official Date:	26 July 2016
Dates:	Date Event 26 July 2016 Available 19 July 2016 Accepted 12 May 2016 Submitted
Volume:	9
Number:	1
Article Number:	365
DOI:	10.1186/s13104-016-2171-7
Status:	Peer Reviewed
Publication Status:	Published
Date of first compliant deposit:	16 January 2017
Date of first compliant Open Access:	16 January 2017
Funder:	Medical Research Council (Great Britain) (MRC), Natural Environment Research Council (Great Britain) (NERC)
Grant number:	MR/L015080/1(MRC), NE/L011956/1(NERC)

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