P705. 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
N.A. Kennedy1, A.W. Walker2, UK IBD Microbiota Consortium3, UK IBD Genetics Consortium3, S.H. Berry4, S.H. Duncan5, F. Farquarson5, P. Louis5, J. Satsangi1, H.J. Flint5, C.W. Lees1, G.L. Hold4, 1Western General Hospital, Gastointestinal Unit, Molecular Medicine Centre, Edinburgh, United Kingdom, 2Wellcome Trust Sanger Institute, Pathogen genomics, United Kingdom, 3, United Kingdom, 4Aberdeen University, Gastrointestinal Research Group, Division of Applied Medicine, Aberdeen, United Kingdom, 5Aberdeen University, Rowett Institute of Nutrition and Health, Aberdeen, United Kingdom
Determining the bacterial community structure in faecal samples through amplification and sequence analysis of extracted DNA is now an important facet of IBD research. The possible impact of different DNA extraction kits upon bacterial community structures has received little attention. Limited evidence exists on the possible influence of different laboratory environments on the data generated.
Aim: to analyse bacterial communities in healthy volunteer and IBD patient faecal samples extracted using two DNA extraction kits in two established gastrointestinal research laboratories (AU and RINH).
Faecal samples from 2 healthy volunteers and 2 IBD patients with relapse were investigated. DNA extraction was done using the MoBio kit and two protocols of the Fastprep DNA extraction kit. Each DNA sample was split and an aliquot sent to the other lab. PCR amplification for Next generation sequencing (NGS) of bacterial 16S rRNA genes was performed on all samples. Quantitative PCR analysis (qPCR) was performed to validate NGS data.
DNA extracted using methods FastPrep1, FastPrep2 and MoBio 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 the MoBio kit were significantly lower than with the FastPrep kit (p < 0.0001), while they were similar between the two methods of the FastPrep kit. Hierarchical clustering of NGS data using the Jaccard similarity coefficient revealed four clusters, with samples clustering by patient.
Linear modelling of the effect of patient and kit on relative abundance of common bacterial classes revealed significant differences between the MoBio kit and the FastPrep kit. Ruminococcaceae and Bacteroidetes were significantly increased in samples extracted with the MoBio kit, while Lachnospiraceae and Enterobacteriaceae were significantly reduced (all p < 0.05). qPCR revealed good correlation with the NGS data, with R2 of 0.94, 0.82, 0.69 and 0.57 for Enterobacteriaceae, Bacteroides, Ruminococcaceae and Lachnospiraceae respectively.
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.