2.5 Soil DNA extraction, amplification and pyrosequencing
According to the manufacturer’s instructions of a Power Soil DNA Isolation Kit (MoBio Laboratories Inc., USA), the total genomic DNA was extracted from the fresh soil subsamples collected on early June 2018. The quality and purity of the extracted genomic DNA was quantified using agarose gel electrophoresis and spectrophotometry on a NanoDrop® ND-2000c UV-Vis spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Then the extracted DNA was diluted to about 10 ng μL-1 and stored at -80 ℃ for the following analytical steps.
Bacterial 16S rRNA gene amplicon sequencing and Miseq library construction was performed by Genesky Biotechnologies Inc. (Shanghai, China). The V4-V5 hypervariable region of the 16S rRNA gene was amplified with dual-indexed Illumina fusion primers: V4for 5’-GTGCCAGCMGCCGCGGTAA-3’and V5rev 5’- CCGTCA ATTCMTTTRAGTTT-3’ (Kozich et al., 2013). Polymerase chain reaction (PCR) was performed with GoTaq® Hot Start PCR Master Mix (Promega, Madison, WI, USA) in a 25 μL reaction. Using ABI 2720 Thermal Cycler (Thermo Fisher Scientific, USA), the following thermal cycling scheme was used: initial denaturation step at 94 ℃ for 3 min, followed by 35 cycles of denaturation at 94 ℃ (45 s), annealing at 50 ℃ (30 s), extension at 70 ℃ (90 s), and a final extension at 72 ℃ for 10 min (Rath et al., 2019). The PCR products were purified with AmpureXPbeads (AGENCOURT) to remove the unspecific products. The average molecule length was determined using the Agilent 2100 bioanalyzer instrument (Agilent Technologies, USA). The library was quantified by real-time quantitative PCR (EvaGreenTM). The Qualified libraries are sequenced pair-end on the Illumina MiSeq Benchtop Sequencer (Illumina, San Diego, CA, USA).
All downstream processing of sequences was conducted using the QIIME pipeline v1.7.0 as described in Caporaso et al., (2010). Paired-end reads were assembled using PANDAseq (Masella et al., 2012), and assembled sequences were quality filtered using USEARCH v7 (Edgar, 2010), retaining only sequences > 100 bases in length with expected errors < 2.0. After the filtering step, 4,790,305 sequences were collected with an average length of 376 bases. Sequences were clustered de novo into operational taxonomic units (OTUs) at a 97% similarity level. Taxonomic information was assigned to OTUs and samples were rarefied to 10,000 sequences to correct for differences in sequencing depth. Samples with < 10,000 sequences and OTUs that were observed < 10 times across all samples were excluded in downstream processing, and these criteria resulted in the removal of 5243 out of the total 14601 OTUs.