DNA extraction, PCR amplification and high throughput sequencing
Total genomic DNA was extracted from 250 mg of soil per sample using the
PowerSoil DNA Isolation Minikit (Qiagen, Hilden, Germany) according to
the manufacturer’s instructions. DNA integrity was checked on 1.0%
(w/v) agarose gel using 1X TAE (Tris acetate EDTA) as running buffer.
The quantification of isolated DNA was carried out by
QubitTM dsDNA BR assay kit (Thermo Fisher Scientific
MA, USA).
Identification of bacterial and fungal microbiota was accomplished by
amplicon sequencing of the 16S rRNA and ITS2 genes, respectively. 20
nanograms (ng) of DNA was used to amplify hyper variable (V3-V4) region
of the prokaryotic small-subunit (16S) rRNA and the internal transcribed
spacer region 2 (ITS2) of the fungal organisms using KAPA HiFi HotStart
Ready Mix PCR Kit (Kapa Biosystems, Wilmington, MA, USA). The reaction
mixture included KAPA HiFi Hot Start Ready Mix (2X), 100 nm
concentration of primers and 20 ng of DNA. The PCR involved an initial
denaturation of 95°C for 5 min followed by 25 cycles of 95°C for 30s,
55°C for 45s and 72°C for 30s and a final extension at 72°C for 7 min
and holding at 4°C. After amplification, the PCR products were cleaned
up, using 0.8X AMPure XP beads (Beckmann-Coulter, CA, USA) to remove
unused primers and eluted in 15 µls of 0.1X TE buffer. 5 µLs of the
purified PCR product from each sample was taken for performing P7 and P5
barcoding by additional 8 cycles of PCR. The final PCR products were
again purified using 1X AMPure XP beads and the final library was eluted
in 20 µLs of 0.1X TE buffer and the sequencing was performed on Illumina
MiSeq, using MiSeq Reagent Kit v3 (600-cycle) according to the
manufacturer’s instructions (Illumina, Inc., San Diego, CA, United
States).