design: The V4 hypervariable region of the 16S rRNA gene was amplified using dual indexed, Illumina compatible primers. PCR products were batch normalized using an Invitrogen SequalPrep DNA Normalization plate and product recovered from the plates pooled. The pool was concentrated and cleaned up using a QIAquick Spin column and AMPure XP magnetic beads. The pool was QCd and quantified using a combination of Qubit dsDNA HS, Agilent 4200 TapeStation HS DNA1000 and Invitrogen Collibri Illumina Library Quantification qPCR assays.