Geochemistry and microbiology of tropical serpentine soils in the Santa Elena Ophiolite, a landscape-geographical approach.
Sequencing of PCR amplicons of the ITS2 region of nuclear ribosomal DNA was conducted with the Illumina MiSeq platform (Illumina, San Diego, CA, USA) targeting the ITS2 internal transcribed spacer region between the large subunit (LSU) and the 5.8S ribosomal genes (forward primer, ITS4F, 5?- AGCCTCCGCTTATTGATATGCTTAART -3?, reverse primer, 5.8SR, 5?- AACTTTYRRCAAYGGATCWCT -3?;)1 for 2??300-bp paired-end sequencing (Illumina)2, 3. PCR amplification was performed using Roche FastStart High Fidelity PCR System (Roche Diagnostics Ltd, Burgess Hill, UK) in 50?l reactions under the following conditions: initial denaturation at 95C for 2?min, followed by 36 cycles of 95C for 30?s, 56C for 45?s, 72C for 2 min, and a final extension step of 5?min at 72C. The PCR products were purified and normalised to ~20ng each using the SequalPrep Normalization Kit (Fisher Scientific, Loughborough, UK). The PCR amplicons from all samples were pooled in equimolar ratios. The run was performed using a 10pM sample library spiked with 10pM PhiX to a final concentration of 10% following the method of Schloss and Kozich.