ITS2 Soil Fungal amplicons
DNA was isolated from 2 g freeze-dried soil using a CTAB-chloroform extraction protocol followed by a column-based purification using E.Z.N.A Soil DNA Kit following the manufacturers protocol (Omega Bio-tek). Technical samples (replicates and negatives) and mock communities (positive control) were introduced during the PCR amplification step. We amplified the ITS2 region using primers gITS7 (forward) and ITS4 (reverse) (Ihrmark et al., 2012), tagged with unique molecular identifiers. Each PCR consisted of 1 l DNA template, 21 l master mix and 1.5 l 10 M of each tagged forward and reverse primer. Master mix consisted of 14.6 l dH2O, 2.5 l Gold Buffer, 2.5 l Gold MgCl2, 1l 20 mg ml-1 bovine serum albumin, 0.2 l 10nM dNTPs and 0.2 l Taq GOLD polymerase per reaction, mixed for 60 reactions per library. We ran PCR reactions with the following setup: 95 C initial denaturation for 5 min, followed by 32 cycles of 95 C denaturation for 30 s, 55 C primer annealing for 30 s, and elongation at 72 C for 1 min. A final elongation step of 7 minutes at 72 C was run after the 32 cycles were complete, before cooling to 4 C. We controlled for positive amplification using gel electrophoresis on a 1% agarose gel. Samples were further cleaned and purified using ZR-96 DNA Clean & Concentrator-5 kit (Zymo research). DNA concentrations were measured for each sample using Qubit 2 fluorometer dsDNA BR Assay kit (Thermo Fisher Scientific) and pooled to equimolar concentration in 2 pools. Each pool was cleaned and concentrated with DNA Clean & Concentrator-5 (Zymo Research). The two pools were multiplexed and sequenced in one run using Illumina MiSeq 250-bp paired-end with V3 chemistry at Fasteris Switzerland.