DNA Isolation, PCR amplification and 16S rRNA Gene Sequencing
To minimize contamination, DNA extraction was carried out in the laminar flow engine hood using the cell culture scheme provided by the DNasy Blood and Tissue Kit (Qiagen, Germany). The concentration of extracted DNA was determined using a Nanodrop ND-1000 spectrophotometer (Thermo Electron Corporation, USA). The16S rRNA sequence was amplified by PCR using the primer set for V3-V4 region. Extracted negative controls (no urine) and PCR negative controls (no templates) were included to assess the contribution of extraneous DNA from reagents. The Qiaquick PCR purification kit (Qiagen of Valencia,USA) was used to purify the final PCR product from the nucleotides and primers that had not been incorporated. The purified samples were normalized to the same DNA concentration and sequenced by Illumina MiSeq sequencer (Illumina, USA).