title: 16S rRNA sequencing of DFU microbiome
design: The bacterial V4 region of the 16S RNA gene, DNA was PCR amplified using 5Prime HotMasterMix and primers 515Fand 806R . The barcodes were attached to the indexed reverse primers. Following amplification of bacterial V4 target sequences, PCR products were purified using the Agencourt AMPure XP clean-up kit. Equimolar amounts of all sample amplicons were pooled into a single multiplexed library and the quality and molarity of the resulting library were assessed using a BioAnalyzer High Sensitivity DNA chip. The samples were sequenced for 250 cycles with custom primers designed for paired-end sequencing.