The effect of DNA extraction methodology on gut microbiota research applications

Gerasimidis, K. , Bertz, M., Quince, C., Brunner, K., Bruce, A., Combet, E. , Calus, S., Loman, N. and Ijaz, U. (2016) The effect of DNA extraction methodology on gut microbiota research applications. BMC Research Notes, 9, 365. (doi: 10.1186/s13104-016-2171-7) (PMID:27456340) (PMCID:PMC4960752)

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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:Articles
Glasgow Author(s) Enlighten ID:Gerasimidis, Professor Konstantinos and Quince, Dr Christopher and Ijaz, Dr Umer and Combet Aspray, Professor Emilie and Bertz, Mr Martin
Authors: Gerasimidis, K., Bertz, M., Quince, C., Brunner, K., Bruce, A., Combet, E., Calus, S., Loman, N., and Ijaz, U.
College/School:College of Medical Veterinary and Life Sciences > School of Medicine, Dentistry & Nursing
College of Science and Engineering > School of Engineering
College of Science and Engineering > School of Engineering > Infrastructure and Environment
Journal Name:BMC Research Notes
Publisher:BioMed Central
ISSN (Online):1756-0500
Published Online:26 July 2016
Copyright Holders:Copyright © 2016 The Authors
First Published:First published in BMC Research Notes 9:365
Publisher Policy:Reproduced under a Creative Commons License

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
652771Understanding microbial community through in situ environmental 'omic data synthesisUmer IjazNatural Environment Research Council (NERC)NE/L011956/1ENG - ENGINEERING INFRASTRUCTURE & ENVIR