title: 16S gut
design: Primers spanning the V3-4 region of 16S ribosomal RNA gene including an Illumina adaptor, an 8-nucleotide barcode and a region-specific primer were utilised (341F (5#-CCTACGGGNGGCWGCAG-3#); 806R (5#-GGACTACHVGGGTWTCTAAT-3#)). Cycling conditions were 95C for 3 minutes followed by 30 cycles of 95 C for 30 s; 54 C for 30 s and 72 C for 1 minute followed by a final extension of 72 C for 10 minutes. PCR products were purified using Agencourt AMPure XP beads as per manufacturers instructions (0.7x, Beckman Coulter). Amplicon quantity and quality was confirmed using the Agilent Tapestation (2200, Agilent) and the NEBNext Library Quant Kit for Illumina (NEB).  Pooled libraries were spiked with 10% PhiX and sequenced using the V2 kit (500-cycles) on the Miseq sequencing platform (Illumina).