Quantification of Gut Microbiota Composition

Fecal, cecal and small intestinal content genomic DNA was extracted from the weighted stool samples using a method that was previously described^{31 (link)}, which is based on the Godon DNA extraction method. Quantifications of all bacteria and B. wadsworthia were performed by qPCR using TaqMan Gene Expression Assays (Life technologies) and Takyon SYBR Green PCR kit (Eurogentec). All bacteria was quantified using the following oligonucleotides: (sense) 5′-CGGTGAATACGTTCCCGG-3′ and (antisense) 5′-TACGGCTACCTTGTTACGACTT-3′ and (probe) 5′-CTTGTACACACCGCCCGTC-3′. B. wadsworthia was quantified using specific primers for the tpa gene (accession no. AF269146): (sense) 5′-CGCCGGTATCGAAATCGTGA-3′ and (antisense) 5′-ATTCGCGGAAGGAGCGAGAG-3′. Sulfite-reducing bacteria were quantified using specific primers for the dsra gene (encoding a dissimilatory sulfite reductase alpha subunit) as described by Devkota^{5 (link)}.