Multi-centre extraction and sequencing of IBD mucosal microbiome
The V4 hypervariable region of the 16S rRNA gene was amplified using a universal 515F (GTGCCAGCMGCCGCGGTAA) forward sequencing primer and a uniquely barcoded 806R (GGACTACHVGGGTWTCTAAT) reverse sequencing primer to allow for multiplexing [24]. Amplification reactions were performed using 12.5 L of KAPA2G Robust HotStart ReadyMix (KAPA Biosystems), 1.5 L of 10 M forward and reverse primers, 9.5 L of sterile water, and 1 L of DNA. The V4 region was amplified by cycling the reaction at 95 C for 3 minutes, 27 cycles of 95 C for 15 seconds, 50 C for 15 seconds, and 72 C for 15 seconds, followed by a 5-minute extension at 72 C. All amplification reactions were done in triplicate, visualized on a 1% agarose Tris/Borate/EDTA (TBE) gel, and then pooled to reduce amplification bias. Pooled triplicates were quantified using the Qubit HS DNA kit and combined in equal concentrations. The final library was gel purified on a 2% agarose tris-acetate-EDTA (TAE) gel due to the presence of dual bands after amplification. The gel-extracted library was then purified using 1.8x magnetic Ampure XP beads and quantified. Approximately 7 pM of the purified library pools and PhiX spike-in control DNA (at 5%) were loaded on to the Illumina MiSeq for sequencing, according to manufacturer instructions (Illumina, San Diego, CA). Sequencing was performed on 64 samples in a single run using the V2 chemistry (300 cycles; 2 x 150 bp).