Sequencing of PCR amplicons of 16S rRNA was conducted with the Illumina MiSeq platform (Illumina, San Diego, CA, USA) targeting the V4 hyper variable regions (forward primer, 515F, 5'-GTGYCAGCMGCCGCGGTAA-3'; reverse primer, 806R, 5'-GGACTACHVGGGTWTCTAAT-3') for 2250-bp paired-end sequencing. PCR amplification was performed using Roche FastStart High Fidelity PCR System in 50ul reactions under the following conditions: initial denaturation at 95C for 2min, followed by 36 cycles of 95C for 30s, 55C for 30s, 72C for 1 min, and a final extension step of 5min at 72C. The PCR products were purified and normalised to 20ng each using the SequalPrep