NDLI: The effect of DNA extraction methodology on gut microbiota research applications

Content Provider	Springer Nature : BioMed Central
Author	Gerasimidis, Konstantinos Bertz, Martin Quince, Christopher Brunner, Katja Bruce, Alanna Combet, Emilie Calus, Szymon Loman, Nick Ijaz, Umer Zeeshan
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.
Related Links	https://bmcresnotes.biomedcentral.com/counter/pdf/10.1186/s13104-016-2171-7.pdf
Ending Page	10
Page Count	10
Starting Page	1
File Format	HTM / HTML
ISSN	17560500
DOI	10.1186/s13104-016-2171-7
Journal	BMC Research Notes
Issue Number	1
Volume Number	9
Language	English
Publisher	BioMed Central
Publisher Date	2016-07-26
Access Restriction	Open
Subject Keyword	Biomedicine Medicine Public Health Life Sciences Metagenomics DNA extraction Benchmarking Diversity PCR 16S rRNA gene Medicine/Public Health
Content Type	Text
Resource Type	Article
Subject	Biochemistry, Genetics and Molecular Biology Medicine
Journal Impact Factor	1.6/2023

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