Illumina MiSeq of fungal communities: mountain soil
Total genomic DNA was extracted from 1 gram of soil, obtained after pooling and homogenizing the ten sub-replicate soil cores within each of the three single plots, using Nucleo Spin Soil kit Macherey-Nagel (Du_ren, Germany) following manufacturer instructions.The DNAs from the three replicates of each habitat were pooled at equimolar concentration and used as a template for PCR with primers targeting the fungal internal transcribed spacer 2 (Gardes and Bruns, 1993).PCRs were performed in a volume of 50 _l using Phusion High-Fidelity PCR Master Mix (Thermo Fisher Scientific, MA - USA) following manufacturer instructions, 0.2 mM of each deoxynucleoside triphosphate, 0.5 pmol of each primer and 10/20 ng of genomic DNA. PCR conditions used 30 s at 98¡C, followed by 25 cycles of 1 min denaturation at 98¡C, 30 s annealing at 58¡C and 1 min extension at 72¡C, with a final single extra extension step of 7 min at 72¡C. Finally, amplicons were cleaned-up with Agencourt AMPure XP (Beckman, CA - USA) and the sizes were checked with a 2100 Bioanalyzer Instrument (Agilent Technologies, CA - USA). Libraries were prepared with a second stage PCR using Nextera XT Index 1 Primers (N7XX) and Nextera XT Index 2 Primers (S5XX) from the Nextera XT Index kit (FC-131-1001 or FC-131-1002), following 16S Metagenomic Sequencing Library Preparation protocol (http://www.illumina.com/content/dam/illuminasupport/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf). The libraries obtained were quantified by Real Time PCR with KAPA Library Quantification Kits (Kapa Biosystems, Inc. MA - USA) pooled in equimolar proportions and sequenced by MiSeq sequencer (Illumina, CA Ð USA) using reagent kit v3 with paired-end reads of 250 bp.