2.4. Sample Preparation and Data Analysis
MP
The collected samples were transported to Cell Biotech, Co., Ltd. (Gyeonggi-do, Korea) and immediately frozen at −80 °C. A total of 204 samples were used for microbiome analysis. Microbial DNA was extracted using the FastDNA SPIN Kit for Soil (MP Biochemicals, Santa Ana, CA, USA) according to the manufacturer’s instructions. The extracted microbial DNA was purified using DNeasy PowerClean Cleanup Kit (Qiagen, Hilden, Germany) and then DNA quality was measured using NanoDrop (Thermo Fisher Scientific, Carlsbad, CA, USA). The purified DNA was measured for DNA concentration using the Qubit™ dsDNA BR Assay kit (Thermo Fisher Scientific). A sequencing library was prepared according to the Illumina 16S Metagenomic Sequencing Library Preparation Guide (Illumina, San Diego, CA, USA). The V4-V5 region of the bacterial 16S rRNA gene was amplified for 16S rRNA gene sequencing. The forward primer in the v4 region (CCA GCM GCC GCG GTA ATW C) and the reverse primer in the v5 region (CC GTC AAT TYY TTT RAG TTT) were used for PCR amplification in this study. The amplified sequencing library was purified with Agencourt