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.