Tu1716 Evaluation of the Impact of Different Commercially Available DNA Extraction Kits and Laboratories for Assessing Bacterial Community Structure in Fecal Samples - Implications for IBD Research

Abstract

Introduction Determining faecal sample bacterial community structure through sequence analysis of DNA has become a very important facet of inflammatory bowel disease (IBD) research. The possible impact of different commercially available DNA extraction kits and the influence of different lab environments on the data generated has however received relatively little attention. The study compared bacterial communities in faecal samples extracted using commercial DNA kits in established gastrointestinal research laboratories. Methods Faecal samples from 2 healthy volunteers and 2 IBD patients with relapse were investigated. DNA extraction was undertaken using MoBio and Fastprep DNA extraction kits in two established labs. Two protocols were followed for processing samples using the Fastprep kit. Each DNA sample was then split and an aliquot transferred to the other lab. Pyrosequencing PCR of bacterial 16S rRNA genes was performed in both labs on all samples. Quantitative PCR analysis (q-PCR) to validate sequencing data was also performed. Hierarchical clustering was done using the Jaccard and Theta Yue & Clayton similarity coefficients on the pyrosequencing data. Results DNA extracted using methods FastPrep1, FastPrep2 and the MoBio kit yielded median DNA concentrations of 476 (interquartile range 290–518), 453 (IQR 228–689) and 22 (IQR 9–36) ng/µL respectively. Those obtained with MoBio were significantly lower than FastPrep (p Ruminococcaceae and Bacteroidetes were significantly increased in MoBio extracted samples, while Lachnospiraceae and Enterobacteriaceae were significantly reduced (p Enterobacteriaceae , Bacteroides , Ruminococcaceae and Lachnospiraceae respectively. Conclusion This study demonstrates significant differences in DNA yield and bacterial DNA composition seen when comparing DNA extracted from the same faecal sample with different extraction kits. This highlights the importance of ensuring that all samples to be analysed together are prepared with the same DNA extraction method, and the need for caution when comparing studies that have used different methods. Disclosure of Interest None Declared