8B
Primers were used to prepare the 16S metagenomic libraries by amplifying the V3-V4 region of the 16S gene, with Illumina adaptors incorporated as described in the Illumina 16S Metagenomic Library preparation guide with the following exceptions: the first PCR reaction was performed in a total volume of 50l instead of 25l and the number of cycles used in the first PCR was increased from 25 cycles to 30. The volume of AMPure XP beads (NEB) used in the initial clean-up was scaled up accordingly. Following index PCR and purification, Qubit high sensitivity DNA kit (Life technologies) was used to quantify the products. The PCR products were pooled equimolarly. The pooled libraries were assessed by Agilent high sensitivity DNA kit and quantified by qPCR using the Kapa Quantification kit for Illumina (Kapa Biosystems) according to the manufacturers guidelines. Libraries were diluted and denatured following Illumina guidelines and sequenced on the Illumina MiSeq using the V3 600 cycle kit according to Illumina sequencing protocols at the Teagasc sequencing facility, Teagasc Food Research Centre, Fermoy, County Cork, Ireland.