Ilumina sequencing of fungal ITS-amplicon library of metagenomic DNA extracts from soil samples
Dual-indexed ITS1 and 2 amplicons were generated via a two-step PCR processing during library preparation, following the operational design described in Guerreiro et al.59. In the first PCR step, four modified version of the forward primer  ITS1F (5-CTTGGTCATTTAGAGGAAGTAA-3) and the reverse ITS4 (5-TCCTCCGCTTATTGATATGC-3), were applied, each containing a unique TAG region and part of the Illumina sequencing primer. The first PCR step was carried out using 5.5 l GoTaq Colourless Master Mix (Promega, Fitchburg, United States), 6.5 l sterile PCR grade dH2O, 1 l of each primer [10 M] and 1 l DNA template (1:100 dilution of the metagenomic DNA extract in PCR grade dH2O), adding to a final volume of 14 l. Thermal cycling was conducted in a Primus 96 Thermal Cycler (MWG-Biotech, Ebersberg, Germany) consisted of an initial heat-activation at 95 C for 3 min, followed by 33 cycles of a denaturation step at 94 C for 27 s, an annealing step at 57 C for 1 min and an elongation step at 72 C for 1:30 min. Each PCR program ended with a final elongation at 72 C for 7 min. The remaining reactants were cleaned up enzymatically during an ExoSAP digestion (Exonuclease I and Shrimp Alkaline Phosphatase, New England Biolabs, Ipswich, Massachusetts, US), at 37 C for 15 min, followed by a heat inactivation of the enzymes. The second PCR step was run under the same conditions as the first, with a higher total reaction volume of 25 l, only 5 cycles and a primer combination consisting of the Illumina sequencing primer (dnatech.genomecenter.ucdavis.edu), an INDEX sequence, for the index-run and P5 and P7 adapter sequences, respectively. Combination of the TAG (PCR1) and INDEX (PCR2) sequences allowed the creation of unique sequence patterns for each environmental sample for the multiplexing of amplicons. Successful amplicon generation was confirmed by agarose gel electrophoresis (0.8% w/v, 0.5x TBE buffer at 75 V) and subsequent imaging of the DNA stained with ethidium bromide under UV light exposure (GelDocTM Station, Invitrogen, Carlsbad, United States). Resulting amplicons were pooled to equimolar concentrations.