title: The effect of DNA extraction methodology on gut microbiota research applications.
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.
authors.affiliation: Human Nutrition, School of Medicine, College of Medical, Veterinary and Life Sciences, Glasgow Royal Infirmary, University of Glasgow, Glasgow, UK.
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Gerasimidis
authors.affiliation: Human Nutrition, School of Medicine, College of Medical, Veterinary and Life Sciences, Glasgow Royal Infirmary, University of Glasgow, Glasgow, UK.
authors.forename: Martin
authors.initials: M
authors.lastname: Bertz
authors.affiliation: Warwick Medical School, University of Warwick, Warwick, UK.
authors.forename: Christopher
authors.initials: C
authors.lastname: Quince
authors.affiliation: Human Nutrition, School of Medicine, College of Medical, Veterinary and Life Sciences, Glasgow Royal Infirmary, University of Glasgow, Glasgow, UK.
authors.forename: Katja
authors.initials: K
authors.lastname: Brunner
authors.affiliation: Human Nutrition, School of Medicine, College of Medical, Veterinary and Life Sciences, Glasgow Royal Infirmary, University of Glasgow, Glasgow, UK.
authors.forename: Alanna
authors.initials: A
authors.lastname: Bruce
authors.affiliation: Human Nutrition, School of Medicine, College of Medical, Veterinary and Life Sciences, Glasgow Royal Infirmary, University of Glasgow, Glasgow, UK.
authors.forename: Emilie
authors.initials: E
authors.lastname: Combet
authors.affiliation: School of Engineering, University of Glasgow, Oakfield Avenue, Glasgow, G12 8LT, UK.
authors.forename: Szymon
authors.initials: S
authors.lastname: Calus
authors.affiliation: Institute of Microbiology and Infection, University of Birmingham, Birmingham, B15 2TT, UK.
authors.forename: Nick
authors.initials: N
authors.lastname: Loman
authors.affiliation: School of Engineering, University of Glasgow, Oakfield Avenue, Glasgow, G12 8LT, UK. Umer.Ijaz@glasgow.ac.uk.
authors.forename: Umer Zeeshan
authors.initials: UZ
authors.lastname: Ijaz
mesh_terms: D019985:Benchmarking; D002725:Chloroform / Q000737:chemistry; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D058893:DNA Barcoding, Taxonomic / Q000592:standards*; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D005243:Feces / Q000382:microbiology; D000069196:Gastrointestinal Microbiome / Q000187:drug effects* / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D059627:Liquid Phase Microextraction / Q000379:methods; D019800:Phenol / Q000737:chemistry; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; D019800:Phenol; D002725:Chloroform; D004247:DNA
keywords: 16S rRNA gene; Benchmarking; DNA extraction; Diversity; Metagenomics; PCR