B6-14-6
Amplicon library preparation was performed using an automated platform (Biomek SPRIworks HT) using a custom liquid handling method. For each sample, the V4 region of the bacterial 16S rRNA gene was amplified in duplicate reactions, using EMP primer set 515F/806R. Each unique barcoded amplicon was generated in pairs of 25l reactions with the following reaction conditions: 13l PCR-grade H2O, 10l HotStart MasterMix, 0.5l of forward and reversed barcoded primer (10M) and 1l template DNA. Reactions were run on a C1000 Touch Thermal Cycler (Bio-Rad) with the following cycling conditions: initial denaturing at 94C for 3 min followed by 35 cycles of denaturation at 94C for 45 seconds, annealing at 50C for 1 minute, and extension at 72 C for 90 seconds, with a final extension of 10 min at 72C. Amplicons were quantified using Agilent 2200 TapeStation system and pooled.