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).