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).