Abstract
IntroductionDetermining 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.MethodsFecal 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.ResultsDNA 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).ConclusionThis 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.
Highlights
Determining bacterial community structure in fecal samples through DNA sequencing is an important facet of intestinal health research
DNA yields were significantly higher with either method of the FastDNA kit than with the MoBio kit, with median DNA concentrations of 476 ng/mL for FastDNA method 1, 453 ng/mL (IQR 228–689) for FastDNA method 2 and 22 ng/mL (IQR 9-36) for the MoBio method (p,0.001 for both comparisons, Figure 2)
Compositional analysis indicated a higher proportion of Enterobacteriaceae and Sutterellaceae, and lower Ruminococcaceae, in the samples from the two inflammatory bowel disease (IBD) patients compared with the two healthy controls, regardless of the extraction method or laboratory
Summary
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. Determining the bacterial community structure in fecal samples through amplification and sequence analysis of extracted DNA has revolutionized gastrointestinal microbiology research over recent years. These culture-independent techniques for assessing diversity have largely replaced traditional culture based approaches as they are considered to be less biased in terms of defining true diversity and considerably less labor-intensive[10,11]. All methods rely on chemical or mechanical disruption, lysis using detergents, or a combination of these approaches
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