RNA seq of diabetic foot ulcer microbiome:paired swab and tissue samples
Materials and methods:Extracted DNA from swab and tissue samples was analysed in the Australian Centre for Ecogenomics (ACE) using identical primers in the prokaryotic V4 region (515F and 806wR) for community profiling modified to contain Illumina specific adaptor sequence  515F and 806Rb. PCR products of approx 466bp were amplified with an alteration in polymerase used to substitute NEBNext Ultra ll Q5 Mastermix (New England Biolabs#MO544)  in standard PCR conditions. Resulting amplicons were purified using Agencourt AMPure XP beads. Purified DNA was indexed with unique 8 bp barcodes using the Illumina Nextera XT 384 sample index kit A-D (Illumina FC -131-1002) in standard PCR conditions with the NEBNext Ultra ll Q5 Mastermix. Pooled indexed amplicons in equimolar concentrations were  sequenced (MiSeq sequencing system Illumina) using paired end sequencing with V3 300bp chemistry in ACE according to manufacturers protocol. Control reactions in the amplicon library construction and sequencing were documented by ACE.