Computational protocol: High-throughput clone library analysis of the mucosa-associated microbiota reveals dysbiosis and differences between inflamed and non-inflamed regions of the intestine in inflammatory bowel disease

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Protocol publication

[…] DNA was extracted from each mucosal biopsy sample using the 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') []. Clone library construction and sequencing were carried out as described previously [].Sequences were aligned using the NAST aligner [] and these alignments were subject to extensive manual curation using the ARB package [] before further analysis. Sequences were tested for chimeras with Mallard [], Bellerophon at Greengenes [] and Pintail [] and any that appeared to be chimeric were removed. 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 []. 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 [] set to the furthest neighbour and 99%-similarity setting. The resulting phylotypes were then assigned similarities to nearest neighbours using MegaBLAST [].To determine the depth of coverage in each of the clone libraries Good's coverage was calculated using the mothur software package []. 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 []. Rarefaction curves for each sample were calculated using mothur [].Community structure comparisons across the whole dataset, incorporating unweighted and weighted UniFrac, Parsimony testing and cluster analysis using the Jaccard coefficient, were performed using mothur and were based on an alignment created in mothur using the reference SILVA-alignment and with the 60% maximal-base filter and Olsen correction applied prior to distance matrix construction in ARB. Cluster dendrograms, with added bar charts showing the microbial composition of each sample, were visualised using the iTOL web package [].Paired (inflamed and non-inflamed) biopsy sample sequences from individual patients were aligned using the NAST aligner and were again extensively corrected in the ARB package [] before further analysis. Olsen-corrected, 60% maximal-base frequency filtered distance matrices were subjected to ∫-LIBSHUFF analysis []. Unaligned paired-sample sequences were used as input for the Library Compare tool at the RDPII website [].Principal coordinates analysis (PCoA) plots were generated using the Fast UniFrac web application [] based upon neighbour joining trees created in ARB, with 60% maximal-base frequency filter and Olsen correction applied, using the sequences aligned to the SILVA reference in mothur as initial input. […]

Pipeline specifications

Software tools DOTUR, BLASTN, mothur, UniFrac, iTOL, Fast Unifrac
Applications Phylogenetics, 16S rRNA-seq analysis
Organisms Homo sapiens
Diseases Colitis, Ulcerative, Crohn Disease, Inflammatory Bowel Diseases