metabarcoding of soil fungi: Nahuelbuta reference
DNA was extracted from the samples with the FastDNATM Spin Kit for Soil  according to the manufacturers instructions. The target region was amplified by PCR with fusion primers containing partial Illumina sequencing adapters for later addition of indexed adapters. The forward primer (5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCATCGATGAAGAACGCAG-3') was based on ITS3mix1 (Tedersoo et al., 2015) and the reverse primer (5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTCCTSCGCTTATTGATATGC-3') was based on ITS4 (White et al., 1990). PCRs were conducted in 25 l reactions using 0.4 l  Herculase II Fusion Polymerase , 5 l of 5x PCR buffer, 1 l each primer (10 uM), 0.5 l dNTPs (25 mM each), 1.75 l DMSO, 2.5 l MgCl2 (25mM, NEB), 0.3 l BSA (20mg/mL, NEB). After the first PCR the product was purified with magnetic beads cleanup. The DNA was washed in two steps with 70% ethanol. The purified pellet was air dried and resuspended in 25 l of TE. A unique combination of two indexes was added to each sample by a second PCR step. It was conducted in 25 l reactions using 10 l of the product of the first PCR, 0.25 l  Herculase II Fusion Polymerase, 5 l of 5x PCR buffer, 0.625 l each primer, 0.25 l dNTPs (25 mM each), 1 l DMSO. After that two additional magnetic beat cleanup steps were performed. Libraries were sequenced on a Illumina MiSeq (chemistry v3 600). Reads from different samples were de-multiplexed with MiSeq Reporter (version 2.6).