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The effect of DNA extraction methodology on gut microbiota research applications.
Gerasimidis, Konstantinos; Bertz, Martin; Quince, Christopher; Brunner, Katja; Bruce, Alanna; Combet, Emilie; Calus, Szymon; Loman, Nick; Ijaz, Umer Zeeshan.
Affiliation
  • Gerasimidis K; Human Nutrition, School of Medicine, College of Medical, Veterinary and Life Sciences, Glasgow Royal Infirmary, University of Glasgow, Glasgow, UK.
  • Bertz M; Human Nutrition, School of Medicine, College of Medical, Veterinary and Life Sciences, Glasgow Royal Infirmary, University of Glasgow, Glasgow, UK.
  • Quince C; Warwick Medical School, University of Warwick, Warwick, UK.
  • Brunner K; Human Nutrition, School of Medicine, College of Medical, Veterinary and Life Sciences, Glasgow Royal Infirmary, University of Glasgow, Glasgow, UK.
  • Bruce A; Human Nutrition, School of Medicine, College of Medical, Veterinary and Life Sciences, Glasgow Royal Infirmary, University of Glasgow, Glasgow, UK.
  • Combet E; Human Nutrition, School of Medicine, College of Medical, Veterinary and Life Sciences, Glasgow Royal Infirmary, University of Glasgow, Glasgow, UK.
  • Calus S; School of Engineering, University of Glasgow, Oakfield Avenue, Glasgow, G12 8LT, UK.
  • Loman N; Institute of Microbiology and Infection, University of Birmingham, Birmingham, B15 2TT, UK.
  • Ijaz UZ; School of Engineering, University of Glasgow, Oakfield Avenue, Glasgow, G12 8LT, UK. Umer.Ijaz@glasgow.ac.uk.
BMC Res Notes ; 9: 365, 2016 Jul 26.
Article de En | MEDLINE | ID: mdl-27456340
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.
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Texte intégral: 1 Collection: 01-internacional Base de données: MEDLINE Sujet principal: ADN / ADN bactérien / ARN ribosomique 16S / Codage à barres de l'ADN pour la taxonomie / Microbiome gastro-intestinal Limites: Humans Langue: En Journal: BMC Res Notes Année: 2016 Type de document: Article Pays d'affiliation: Royaume-Uni

Texte intégral: 1 Collection: 01-internacional Base de données: MEDLINE Sujet principal: ADN / ADN bactérien / ARN ribosomique 16S / Codage à barres de l'ADN pour la taxonomie / Microbiome gastro-intestinal Limites: Humans Langue: En Journal: BMC Res Notes Année: 2016 Type de document: Article Pays d'affiliation: Royaume-Uni