16S rRNA amplification
The full length of 16S rRNA was amplified using 50 ng of gDNA with Q5® High-Fidelity 2X Master Mix (New England Biolabs, MA, USA) and the universal primers 27F (5’-AGRGTTYGATYMTGGCTCAG-3’) and 1492R (5’-CGGYTACCTTGTTACGACTT-3’) (Lane, 1991). The polymerase chain reaction (PCR) conditions were 98°C for 30 s, 35 cycles at 98°C for 10 s, 55°C for 30 s, 72°C for 1 min, and 72°C for 10 min. The PCR products were purified and concentrated using FavorPrep GEL/PCR Purification Mini Kit (Medibena, Austria). The purified PCR products were quantified and checked for purity by the NanoDrop machine (Thermo Fisher Scientific, USA). The concentration of purified PCR products was adjusted to 200 ng in 10 µl nuclease-free water.