Microbial genes: soil microbes
Total DNA from soil samples was extracted using the FastDNA Spin Kit for Soil. The extracted total DNA was used as a template for PCR amplification of the V3-V4 region of the bacterial 16S rRNA gene. The sequence of the PCR primers was 338F (5-ACTCCTACGGGAGGCAGCAG-3) and 806R (5- GGACTACHVGGGTWTCTAAT-3). Sequencing was conducted using Illumina's Miseq PE300/NovaSeq PE250 platforms. The PCR amplification process of the 16S rRNA gene was as follows: Initial denaturation at 95C for 3 minutes, followed by 27 cycles of denaturation at 95C for 30 seconds, annealing at 55C for 30 seconds, extension at 72C for 45 seconds, and a final extension step at 72C for 10 minutes. PCR products are extracted from a 1.5% agarose gel, purified using the AxyPrep DNA Gel Extraction Kit, and quantified using the Quantus Fluorometer. Purified amplicons are pooled together and subjected to paired-end sequencing on Illumina's MiSeq platform. The high-throughput sequencing of the 16S rRNA gene was outsourced to Shanghai Majorbio Bio-Pharm Technology Co., Ltd. The raw FASTQ files were quality-filtered using Trimmomatic to remove operational taxonomic units (OTU) with an abundance of 0.1% and to exclude samples with less than 50 reads. Sequence clustering and chimeric filtering were performed using UPARSE software at a 97% similarity threshold. Species classification annotation was conducted for each sequence using the RDP classifier, with alignment against the Silva 16S rRNA database and a matching threshold set at 73%.