2.2 DNA extraction and 16S rRNA gene amplicon sequencing
The total DNA of each pooled sample was extracted using the FastDNA SPIN Kit for Soil (MP Biomedicals, USA) following the manufacturer’s protocol. The quality and integrity of the collected DNA were assessed by 1% agarose gel electrophoresis, its concentrations and purities were determined with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, USA).
The DNA was amplified using 16S rRNA gene V3-V4 regions primers 338F (5’-ACTCCTACGGGAGGCAGCAG-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’) (Kumar et al., 2020). The Polymerase Chain Reaction (PCR) amplification containing 4 μL 5 × buffer, 2 μL dNTPs (2.5 mM), 0.8 μL forward primer (5 μM), 0.8 μL reverse primer (5 μM), 0.4 μL DNA polymerase, 10 ng template DNA, and finally ddH2O up to 20 μL. The PCR reaction under the following conditions: initial denaturation at 95 ℃ for 3 min, and 29 cycles of denaturation at 95 ℃ for 30 s, annealing at 53 ℃ for 30 s and extension at 72 ℃ for 45 s, and a final extension at 72 ℃ for 10 min. The PCR product was extracted from 2% agarose gel and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) according to manufacturer’s instructions and quantified using Quantus™ Fluorometer (Promega, USA). Sequencing was carried out on an Illumina MiSeq platform at Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China.