The effect of DNA extraction methodology on gut microbiota research applications

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

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

36 Citations (Scopus)

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.

Original languageEnglish
Article number365
JournalBMC Research Notes
Volume9
Issue number1
DOIs
Publication statusPublished - 26 Jul 2016
Externally publishedYes

Bibliographical note

Funding Information:
C. Quince is funded by an MRC fellowship as part of the CLIMB consortium Grant Ref: MR/L015080/1. U.Z. Ijaz is funded by a NERC fellowship NE/ L011956/1. We would like to thank the anonymous reviewer 3 for his suggestion to summarise findings of the study within a Table.

Publisher Copyright:
© 2016 The Author(s).

Keywords

  • 16S rRNA gene
  • Benchmarking
  • Diversity
  • DNA extraction
  • Metagenomics
  • PCR

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