Illumina MiSeq Amplicon Sequencing and Data Analysis

The community compositions of both bacteria and fungi in each plot were identified by the Centre for Genetic and Genomic Analysis, GENESKY Biotechnologies Inc. (Shanghai, China). The DNA of each soil sample was used as a template for PCR amplification. Moreover, the bacterial 16S rRNA V4-V5 and fungal ITS1 were amplified using primer pairs 515F/907R and ITSI/ITS2, respectively. Each sample was amplified in triplicates. At the same time, standard genomic DNA of the bacteria and fungi was used as a positive control. The PCR products were checked in a 1.5% agarose gel electrophoresis to assess their specificity. These were then purified using Agencourt AMpure XP PCR Purification Beads (Sangon Biotech Co., China). Purified amplicons were pooled in equimolar and paired-end sequenced (2 × 250 bp) using Illumina Miseq amplicon sequencing.

The raw sequencing data were processed and trimmed using Quantitative Insights Into Microbial Ecology (QIIME) software to remove the low-quality sequences (quality score < 20), primers, barcodes, adaptors (Caporaso et al., 2010), and chimeras were detected and removed using the UCHIME algorithm (Edgar et al., 2011). The remaining high-quality sequences were clustered into Operational Taxonomic Units (OTUs), with a 97% similarity cutoff. The representative sequences for each OTU were aligned using the Python Nearest Alignment Space Termination (PyNAST) (DeSantis et al., 2006; Yao et al., 2017a). The taxonomy of each depty phylotype was assigned using a BLAST comparison against sequences within the GenBank database. Rarefaction curves and alpha diversity indices were calculated using QIIME software based on the obtained OTUs. All sequences have been deposited in the National Center for Biotechnology Information (NCBI) Short Reads Archive database SRP307320.