Once taken, biopsy samples (approximately 1 × 2 mm) were placed in a cryovial without preservative, immediately snap frozen in liquid nitrogen, and stored at -70°C until analysis. Additional biopsy samples from the same area were also sent for histological analysis. These biopsies were scored independently for presence of ulceration, acute and chronic inflammation by a single gastrointestinal pathologist. Prior diagnosis of active CD or UC was determined by standard clinical, radiological, endoscopic and histopathological criteria. A modified Baron

score with a range from 0-5, where a score of 5 represents the most severe disease, was used to grade the endoscopic severity of inflammation at the site of each biopsy used in the study [76]. DNA extraction and sequence analysis DNA was extracted from each mucosal biopsy sample using the

Selleck BMN-673 QIAamp® DNA Mini-Kit (Qiagen, UK) and the eluted DNA was stored at -20°C. 16S rRNA genes were amplified using the broad-range bacterial primers Bact-8F (5′-AGAGTTTGATCCTGGCTCAG-3′) and Bact-1391R (5′-GACGGGCGGTGTGTRCA-3′) [34]. Clone library construction and sequencing were carried out as described previously [72]. Sequences were aligned using the NAST aligner [77] and these alignments were C646 mw subject to extensive manual curation using the ARB package [78] before further analysis. Sequences were tested for chimeras with Mallard [79], Bellerophon at Greengenes [77] and Pintail [80] and any that appeared to be chimeric were removed. Rutecarpine The sequences (deposited in GenBank under accession numbers FJ503060-FJ513069) were

initially given a broad classification to the phylum and family levels using the Classifier tool at the RDPII website [41]. To obtain more detailed taxonomic information the sequences were then divided into phylotypes. Distance matrices were generated in ARB with the Olsen correction and a 60% maximal-base frequency filter applied. This filter removed many ambiguously-aligned columns but was not so stringent that distinct species were commonly merged into single phylotypes. Distance matrices were then entered into the DOTUR program [81] set to the furthest selleck chemical neighbour and 99%-similarity setting. The resulting phylotypes were then assigned similarities to nearest neighbours using MegaBLAST [82]. To determine the depth of coverage in each of the clone libraries Good’s coverage was calculated using the mothur software package [40]. Using this estimator the median coverage across all samples was found to be 94.35% (range of 83.73-97.3%). Shannon diversity indices were calculated for each library by entering distance matrices generated in ARB, with the Olsen correction and a 60% maximal base-frequency filter applied, into DOTUR [81]. Rarefaction curves for each sample were calculated using mothur [40].