Analysis of the V4 region of the 16S rRNA gene was done using mothur (version 1.40.1) [35, 36]. Briefly, the standard operating procedure (SOP) at http://www.mothur.org/wiki/MiSeq_SOP was followed to process the MiSeq data. The paired-end reads were assembled into contigs and then aligned to the SILVA 16S rRNA sequence database (release 132) [37, 38] and were classified to the mothur-adapted RDP training set v16 [39] using the Wang method and an 80% bootstrap minimum to the family taxonomic level. All samples with <500 sequences were removed. Chimeric sequences were removed using UCHIME [40]. Sequences were clustered into operational taxonomic units (OTU) using a 3% species-level definition. The OTU data were then filtered to include only those OTU that made up 1% or more of the total sequences. The percentage of relative abundance of bacterial phyla and family members in each sample was calculated. A cutoff of 0.03 (97%) was used to define operational taxonomic units (OTU) and Yue and Clayton dissimilarity metric (θYC) was utilized to assess beta diversity. In addition to NMDS ordination, principle coordinate analysis (PCoA) biplots using Spearman correlation were used to examine difference in microbial community structures between ursodiol treatments and compared to pretreatment. Standard packages in R (http://www.R-project.org) were used to create NMDS ordination on serial fecal samples.

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