AOM_DSS_d63_WT
The V5/V6 regions of 16S rRNA genes have been amplified by PCR using Platinum Taq DNA Polymerase (Invitrogen) and an input of 100 ng fecal DNA. The forward core primers have been modified by the addition of a PGM sequencing adaptor (in italic), a GT spacer and unique barcode that allow up to 96 different barcodes: 5-CCATCTCATCCCTGCGTGTCTCCGACTCAG BARCODEATTAGATACCCYGGTAGTCC -3. They were used in combination with a reverse fusion primer 5-CCTCTCTATGGGCAGTCGGTGATACG AGCTGACGACARCCATG-3. The PCR product has a length of 350 bp including adaptor and barcode1,2. Cycling conditions consist of an initial denaturation at 94C for 5 minutes followed by 35 cycles of 1 minute denaturation at 94C, 20 seconds annealing at 46C and 30 seconds elongation at 72C. A final elongation step was performed at 72C for 4 minutes. The PCR products were purified after 1% agarose gel electrophoresis using Gel Extraction Kit (Qiagen) according to manufacturers instructions. Purity and concentration of the amplicon were determined using Qubit 3.0 Fluorometer (Thermo Fisher Scientific) prior to library preparation for sequencing on the IonTorrent PGM system (Life Technologies). Libraries were diluted according to manufacturers instructions and processed using the Ion PGMTM Hi-QTM View OT2 400 kit (Thermo Fischer Scientific) (for up to 400 bp libraries) and the Ion PGMTM Hi-QTM View Sequencing kit (Thermo Fischer Scientific) for sequencing on an Ion 316TM V2 chip (Thermo Fischer Scientific)