16 S rRNA gene sequencing: Phase4
Genomic DNA extraction was conducted using the FastDNA spin kit for soil (MP Biomedicals, USA). The forward primer Univ341F (5-CCTACGGGNGGCWGCAG-3), the reverse primer Univ806R (5-GGACTACHVGGGTWTCTAAT-3), and Premix Ex Taq Hot Start Version were utilized for PCR amplification. This process was carried out on MiniAmp thermal cyclers (Thermo Fisher Scientific Inc., USA) with the following program: an initial cycle at 95 C for 180 seconds; 30 cycles at 95, 55, and 72 C for 10, 20, and 30 seconds respectively; followed by a final cycle at 72 C for 10 minutes. PCR products were verified via electrophoresis and purified using a QIAquick PCR purification kit (QIAGEN, Germany). The 16S rRNA gene sequencing was performed with an iSeq 100 i1 reagent and iSeq 100 system (Illumina Inc., USA).