2.4 DNA extraction and Illumina MiSeq sequencing
Soil DNA was extracted from 0.5 g frozen soil using the FastDNA spin kit (DNeasy® Power Soil® Kit (100), QIAGEN, Germany) according to the manufacturer manual. DNA quality was assessed by a NanoDrop ND-1000 Spectrometer (NanoDrop™, Thermo Scientific™, USA) at the wavelength of 260/280 nm. DNA samples were stored at -40°C for further analysis.
The V4-V5 region of the bacterial 16S rRNA gene were amplified by PCR using primers 338F (ACTCCTACGGGAGGCAGCA) and 806R (GGACTACHVGGGTWTCTAAT) (Lee et al., 2012). Triplicate PCR reactions for each sample was executed in 20 μL mixture containing 4 μL of 5 × FastPfu Buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of forward and reverse primer (5 μM), 0.4 μL of FastPfu Polymerase, and 10 ng of template DNA in ABI GeneAmp® 9700. Purified PCR products were pooled in equimolar and paired-end sequenced (2 × 250) on an Illumina MiSeq platform (Majorbio Bio-Pharm Technology Co. Ltd., Shanghai, China) following the standard protocols. The Quantitative Insight into Microbial Ecology (QIIME V1.9.1) pipeline (Caporaso et al., 2010) was used to process the raw sequences. A chimera filtering approach UPARSE was applied to bin the sequences into operational taxonomic units (OTUs) at the 97% sequence identity level (Edgar, 2013). As an even depth of sampling is required for diversity comparison, samples were randomly rarefied according to the lowest number (i.e. 17,113 sequences) among all the samples for downstream analysis. The rarefied OTU was used to calculate alpha diversity (OTU richness: observed number of OTUs, Shannon index) and community dissimilarity (Bray-Curtis dissimilarity distance). The raw reads were deposited into the NCBI Sequence Read Archive (SRA) database with the project accession number PRJNA603198.