2.2 DNA extraction and 16S rRNA gene amplicon sequencing
The
total DNA of each pooled sample was extracted using the
FastDNA
SPIN Kit for Soil (MP Biomedicals, USA) following the manufacturer’s
protocol. The quality and integrity
of the collected DNA were assessed by 1%
agarose
gel electrophoresis,
its
concentrations and purities were determined with a NanoDrop 2000
spectrophotometer (Thermo Fisher Scientific, Wilmington, USA).
The
DNA was amplified using 16S rRNA gene V3-V4 regions primers
338F (5’-ACTCCTACGGGAGGCAGCAG-3’)
and
806R
(5’-GGACTACHVGGGTWTCTAAT-3’) (Kumar et al., 2020). The Polymerase Chain
Reaction (PCR) amplification containing 4 μL
5
×
buffer,
2 μL dNTPs (2.5 mM), 0.8 μL forward primer (5 μM), 0.8 μL reverse primer
(5 μM), 0.4 μL DNA polymerase, 10 ng template DNA, and finally
ddH2O up to 20 μL. The PCR reaction under the following
conditions: initial denaturation at 95 ℃ for 3 min, and
29
cycles of denaturation at 95 ℃ for 30 s, annealing at 53 ℃ for 30 s and
extension at 72 ℃ for 45 s, and a final extension at 72 ℃ for 10 min.
The PCR product was extracted from 2% agarose gel and purified using
the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA,
USA) according to manufacturer’s instructions and quantified using
Quantus™ Fluorometer (Promega, USA). Sequencing was carried out on an
Illumina
MiSeq
platform at Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China.