Multi-centre extraction and sequencing of IBD mucosal microbiome
Purified DNA was used to amplify the hypervariable regions V3 and V4 of the 16S rRNA gene using a two-stage nested PCR approach. Initially the 8f (AGAGTTTGATCCTGGCTCAG) to 926r (CCGTCAATTCCTTTRAGTTT) region of the 16S rRNA gene was amplified using 100 ng of template with 1 unit of Taq, 1x buffer, 1.5 mM MgCl2 (ThermoFisher Taq DNA recombinant kit), 0.4 mg/mL bovine serum albumin (BSA; Sigma), 0.2 mM dNTPs (New England BioLabs), and 10 pM of each primer. The reaction was carried out at 98 C for 5 minutes followed by 15 cycles of 98 C for 30 seconds, 56 C for 30 seconds and 72 C for 60 seconds, with a final extension of 72 C for 10 minutes. The triplicate reaction was then recombined and used as template in the second stage of PCR. A total of 3 L of the first pooled reaction was used as template for PCR using 1 unit of Taq, 1x buffer, 1.5 mM MgCl2, 0.4 mg/mL BSA, 0.2 mM dNTPs, and 5 pM of 341F (CCTACGGGAGGCAGCAG) and 806R (GGACTACHVGGGTWTCTAAT) Illumina adapted primers, as described elsewhere [citation no idea]. The reaction was carried out at 98 C for 5 minutes followed by 25 cycles of 98 C for 30 seconds, 50 C for 30 seconds and 72 C for 30 seconds, with a final extension of 72 C for 10 minutes. The resulting PCR products were visualized on a 1.5% agarose gel. Positive amplicons were normalized using the SequalPrep normalization kit (ThermoFisher) and sequenced with the Illumina MiSeq platform across seven runs. Spike-in was either 5% PhiX or 1% PhiX plus 2.519% bacterial genomic DNA from other experiments.