2.4. Illumina Sequencing of 16S rRNA

Total DNA of each sample was extracted using a FastDNA Spin Kit for Soil (MP Biomedicals, Solin, OH, USA) following the manufacturer's instructions. The V4 hypervariable region of the bacterial 16S rRNA was amplified using the primers 520F and 802R (5′-AYTGGGYDTAAAGNG-3′ and 5′-TACNVGGGTATCTAATCC-3′) described before [5]. A total of 25 μL of reaction was consisted of 5.0 μL of 5 × PCR buffer, 2.0 μL of dNTP (2.5 mM), 5.0 μL of 5 × PCR GC-high enhancer, 1.0 μL of each primer (10 μM), 2.0 μL of template genomic DNA (~200 ng/μL), 0.25 μL of TaKaRa polymerase (5 U/μL), and 8.75 μL of sterilized water. Triplicate PCR amplifications of each sample were performed with an initial denaturation at 98°C for 3 min; a total of 27 cycles of 98°C for 30 s, 50°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 5 min. Pooled triplicate reactions for each sample were purified using an AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and then checked for quality and quantified using a QuantiFluor™-ST fluorometer (Promega, Madison, WI, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). DNA libraries were constructed and evaluated using a 500-cycle (2 × 250 paired ends) kit on an Illumina MiSeq platform at a sequencing company (Personalbio Technology Co., Ltd., Shanghai, China).