The effect of DNA extraction methodology on gut microbiota research applications.

Konstantinos Gerasimidis,Katja Brunner,Szymon Calus,Martin Bertz,Christopher Quince,Emilie Combet,Alanna Bruce,Umer Zeeshan Ijaz,Nick Loman

doi:10.1186/s13104-016-2171-7

Konstantinos Gerasimidis, Katja Brunner + Show 7 more

Open Access

https://doi.org/10.1186/s13104-016-2171-7

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Abstract

BackgroundThe 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.ResultsThe 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.ConclusionsWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-016-2171-7) contains supplementary material, which is available to authorized users.

Highlights

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
We have performed a benchmarking study and explored the effect of three popular faecal DNA extraction methods on yield and quality of isolated DNA, as well as microbiota composition determined by a typical sequencing analysis pipeline, using traditional molecular microbiology techniques and high-throughput generation sequencing
Effect on yield and purity of isolated DNA Beginning with the same faecal material mass, the chaotropic method (CHAO) produced the highest and the Qiagen kit (QIAG) the lowest yield of double stranded DNA (Fig. 1a)

Summary

Introduction

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. 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. Isolation and purification of bacterial genomic DNA from gut mucosal and luminal contents is a crucial initial step to Gerasimidis et al BMC Res Notes (2016) 9:365 extraction methods may have on traditional downstream methods (e.g. quantitative PCR) compared to modern generation sequencing approaches have not been extensively explored [1, 2]. We have performed a benchmarking study and explored the effect of three popular faecal DNA extraction methods on yield and quality of isolated DNA, as well as microbiota composition determined by a typical sequencing analysis pipeline, using traditional molecular microbiology techniques and high-throughput generation sequencing

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