title: The impact of different DNA extraction kits and laboratories upon the assessment of human gut microbiota composition by 16S rRNA gene sequencing.
abstract: BACKGROUND
  Determining bacterial community structure in fecal samples through DNA sequencing is an important facet of intestinal health research. The impact of different commercially available DNA extraction kits upon bacterial community structures has received relatively little attention. The aim of this study was to analyze bacterial communities in volunteer and inflammatory bowel disease (IBD) patient fecal samples extracted using widely used DNA extraction kits in established gastrointestinal research laboratories.
  
  
  METHODS
  Fecal samples from two healthy volunteers (H3 and H4) and two relapsing IBD patients (I1 and I2) were investigated. DNA extraction was undertaken using MoBio Powersoil and MP Biomedicals FastDNA SPIN Kit for Soil DNA extraction kits. PCR amplification for pyrosequencing of bacterial 16S rRNA genes was performed in both laboratories on all samples. Hierarchical clustering of sequencing data was done using the Yue and Clayton similarity coefficient.
  
  
  RESULTS
  DNA extracted using the FastDNA kit and the MoBio kit gave median DNA concentrations of 475 (interquartile range 228-561) and 22 (IQR 9-36) ng/µL respectively (p<0.0001). Hierarchical clustering of sequence data by Yue and Clayton coefficient revealed four clusters. Samples from individuals H3 and I2 clustered by patient; however, samples from patient I1 extracted with the MoBio kit clustered with samples from patient H4 rather than the other I1 samples. Linear modelling on relative abundance of common bacterial families revealed significant differences between kits; samples extracted with MoBio Powersoil showed significantly increased Bacteroidaceae, Ruminococcaceae and Porphyromonadaceae, and lower Enterobacteriaceae, Lachnospiraceae, Clostridiaceae, and Erysipelotrichaceae (p<0.05).
  
  
  CONCLUSIONS
  This study demonstrates significant differences in DNA yield and bacterial DNA composition when comparing DNA extracted from the same fecal sample with different extraction kits. This highlights the importance of ensuring that samples in a study are prepared with the same method, and the need for caution when cross-comparing studies that use different methods.
authors.affiliation: Gastrointestinal Unit, Centre for Genomic and Experimental Medicine, University of Edinburgh, Western General Hospital, Edinburgh, United Kingdom.
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Kennedy
authors.affiliation: Pathogen Genomics Group, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, United Kingdom.
authors.forename: Alan W
authors.initials: AW
authors.lastname: Walker
authors.affiliation: Gastrointestinal Research Group, Division of Applied Medicine, Aberdeen University, Aberdeen, United Kingdom.
authors.forename: Susan H
authors.initials: SH
authors.lastname: Berry
authors.affiliation: Rowett Institute of Nutrition and Health, Aberdeen University, Aberdeen, United Kingdom.
authors.forename: Sylvia H
authors.initials: SH
authors.lastname: Duncan
authors.affiliation: Rowett Institute of Nutrition and Health, Aberdeen University, Aberdeen, United Kingdom.
authors.forename: Freda M
authors.initials: FM
authors.lastname: Farquarson
authors.affiliation: Rowett Institute of Nutrition and Health, Aberdeen University, Aberdeen, United Kingdom.
authors.forename: Petra
authors.initials: P
authors.lastname: Louis
authors.affiliation: Department of Digestive Disorders, Aberdeen Royal Infirmary, Foresterhill, Aberdeen, United Kingdom.
authors.forename: John M
authors.initials: JM
authors.lastname: Thomson
authors.affiliation: Gastrointestinal Unit, Centre for Genomic and Experimental Medicine, University of Edinburgh, Western General Hospital, Edinburgh, United Kingdom.
authors.forename: Jack
authors.initials: J
authors.lastname: Satsangi
authors.affiliation: Rowett Institute of Nutrition and Health, Aberdeen University, Aberdeen, United Kingdom.
authors.forename: Harry J
authors.initials: HJ
authors.lastname: Flint
authors.affiliation: Pathogen Genomics Group, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, United Kingdom.
authors.forename: Julian
authors.initials: J
authors.lastname: Parkhill
authors.affiliation: Gastrointestinal Unit, Centre for Genomic and Experimental Medicine, University of Edinburgh, Western General Hospital, Edinburgh, United Kingdom.
authors.forename: Charlie W
authors.initials: CW
authors.lastname: Lees
authors.affiliation: Gastrointestinal Research Group, Division of Applied Medicine, Aberdeen University, Aberdeen, United Kingdom.
authors.forename: Georgina L
authors.initials: GL
authors.lastname: Hold
mesh_terms: D004269:DNA, Bacterial / Q000235:genetics*; D041981:Gastrointestinal Tract / Q000382:microbiology*; D006801:Humans; D007753:Laboratories; D064307:Microbiota / Q000235:genetics*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D011933:Reagent Kits, Diagnostic; D017422:Sequence Analysis, DNA / Q000379:methods
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; D011933:Reagent Kits, Diagnostic