2.4. 16S rRNA Sequencing

The DNA was extracted from 100 mg samples using DNeasy Power Soil Kit (Cat. 47014, Qiagen, Germany). The DNA concentration was measured with Nanodrop (Thermo scientific). The DNA was diluted to proper concentration for further 16S rRNA gene fragments (V3–V4) amplification. The polymerase used for 16S rRNA gene amplification was Phanta Max Master Mix (Vazyme Biotech Co., Ltd. Nanjing, China) following the manufacturer's procedure. The sequencing was implemented at Novogene Co. (Beijing, China) with a 2 × 250-bp paired-end sequencing strategy.

The V3-V4 regions of microbial 16S rDNA genes were amplified with primers of 341F (5′-CCTAYGGGRBGCASCAG-3′) and 806R (5′GGACTACNNGGGTATCTAAT-3′). The 25 µl PCR amplification mixture contained 25 ng DNA, 1 µl each primer (10 µM), 0.5 µl dNTP (2.5 mM), 12.5 µl Vazyme Phata max buffer, and 0.5 µl vazyme polymerase (Vazyme Biotech). The PCR was performed with an initial denaturation (5 minutes at 95°C), followed by 27 cycles of 15 seconds at 95°C, 15 seconds at 55°C, and 30 seconds at 72°C, and final with one cycle of 5 min at 72°C. The PCR products were purified with the KAPA Pure Beads (Roche) according to the manufacturer's instructions and further sequenced with an Illumina NovaSeq 6000 system (Illumina). The sequences were then clustered into OTUs (operational taxonomic units) at 99% identity thresholds using MOTHUR. Representative sequences of OTUs were selected and compared with ribosomal RNA database to obtain species annotation information. According to species annotation information, OTUs were filtered to obtained valid OTUs.