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.