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.

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BSG abstracts Introduction The human intestinal tract is a complex microbial ecosystem unique to every individual and contributing sufficient metabolic equivalents to be considered an organ in its own right. Dysbiosis is thought to contribute to the chronic inflammation which is involved in colorectal cancer (CRC) development. We have shown that the premalignant adenoma stage of CRC is underpinned by chronic inflammation and hypothesise that the colonic microbiota is an important driver of neoplastic progression. The aim of the study was to compare bacterial diversity in adenomas and paired adjacent normal colonic tissue. Methods 72 patients undergoing colonoscopy as part of the national bowel cancer screening programme were recruited. All patients underwent polypectomy for large adenomas (> 1cm). Denaturing gradient gel electrophoresis (DGGE on all subjects) and pyrosequencing (18 subjects) were used to compare the bacterial diversity present between the adenoma and adjacent normal paired tissue samples. Results 46% of patients showed differences in microbial diversity by DGGE analysis between adenoma and adjacent normal tissue. 21% had only limited differences whilst 79% showed dramatically altered diversity profiles. Phylum-level comparisons across the study cohort, based on sequencing, revealed no statistically significant differences for the Bacteroidetes, Firmicutes and Proteobacteria phyla. When samples were split based on DGGE analysis (i.e. similar or different DGGE profiles between paired polyp and normal mucosa), eight of the possible 10 phyla showed statistically significant differences. Collectively samples with similar profiles between polyp and normal tissue (DGGE 'same' group) had higher levels of Bacteroidetes and lower numbers of Actinobacteria, Firmicutes and Proteobacteria than the DGGE 'different' group. However when genus-level comparisons were assessed the DGGE 'different' sample pairs demonstrated a much greater level of dysbiosis. The levels of normal commensal genera were reduced and there was evidence of large numbers of potentially pathogenic bacteria including Shigella, Enterobacter and Fusobacteria spp. Conclusion Differences in microbial diversity harboured by a significant number of polyps compared to their immediately adjacent normal mucosa point to an important role for these bacteria in CRC progression. We hypothesise that the disturbed microbial homeostasis in these polyps, in genetically predisposed individuals and taking into account multiple environmental factors, underpins the pathogenesis of colorectal neoplasia. The identification of a "harmful" pro-inflammatory microbiota that is associated with precancerous stages (i.e. adenomas) offers the potential for manipulations of this microbiota with probiotic, prebiotic and indeed antibiotic means. Disclosure of Interest None Declared.