2.2 High throughput sequencing and bioinformatics
Soil samples (0.5 g) were used in DNA extraction using Fast DNA Spin Kit (MP Biomedicals, Shanghai, China) according to the manufacturer’s instructions. The primers 515F (5’-GTGCCAGCMGCCGCGGTAA-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’) were used for amplification of the V4 region of the 16S rRNA gene. The amplified PCR products were sequenced on the Illumina Nova 6000 platform (Illumina, San Diego, CA, USA). Subsequently, the native sequences were quality controlled using QIIME2 v2020.11.0 (https://qiime2.org/). DADA2 (https://github.com/benjjneb/dada2) and Deblur (https://github.com/biocore/deblur) were used to clear low-quality sequences and to carry out noise cancellation. Afterward, the remaining sequences were clustered into operational taxonomic units (OTUs) based on 97% sequence similarity and assigned to taxa to the species level with the SILVA Database v132 (Quast et al., 2013) as the reference database. After the deletion of the unassigned OTUs and singleton OTUs, the number of OTUs was counted and an OTU taxonomy information table was computed for further analysis.