2.4 | DNA extraction, PCR amplification, and
sequencing
For each sample, DNA was extracted from 0.25 g thawed soil by a DNeasy®
PowerSoil® DNA Isolation Kit (Qiagen, Germany). The quality of isolated
DNA was determined by electrophoresis on 1% agarose gel. The purity was
examined using NanoDrop. In total, 12 rhizosphere and 12 non-rhizosphere
DNA samples were prepared, respectively.
The 16S rRNA gene was amplified to estimate the relative abundance of
soil bacteria while internal transcribed spacer (ITS) region for the
fungi. The paired primers, 341F and 806R, were used to target the
hypervariable V3+V4 region of the 16S rRNA gene; while the paired
primers, ITS5-1737F and ITS2-2043R, for the ITS1 regions of fungi.
The
primers in each sample were tagged with unique
barcodes.
All PCRs were carried out in 30-µL reactions with10 ng of template DNA,
0.2 µM of forward and reverse primers, and 15 µL of Phusion®
High-Fidelity PCR Master Mix (New England Biolabs). For thermal cycles,
the initial denaturation was at 98 °C for 1 min, followed by 30 cycles
of denaturation at 98 °C for 10 s. Then annealing was at 55 °C for 30 s,
and elongation at 72 °C for 30 s. Finally, an extension was at 72 °C for
5 min.
The
PCR products of each sample and 1X loading buffer (with SYBR green) were
mixed with equal volumes and then electrophoresed on a 2% agarose gel
to examine the successful amplification of target DNA fragments. The PCR
products for each sample were quantified, and mixed with 80ng of DNA in
equal proportions. The resultant mixed PCR products were electrophoresed
by 2% agarose gel, and the fragments of DNA ranging from 400 - 450bp
were excised and then purified with a GeneJETTM Gel
Extraction Kit (Thermo Scientific).
Following the manufacturer’s recommendation, Ion Plus Fragment Library
Kit 48 rxns (Thermo Scientific) was used for sequencing libraries
preparation. Qubit@ 2.0 fluorometer (Thermo Scientific) and Agilent
Bioanalyser 2100 system (Agilent, USA) were used to evaluate the library
quality. The libraries were sequenced on IonS5TMXL
platform, generating 400-bp/600-bp single-end reads.