title: Metabarcoding of fungal ITS2: Shrub_Plot14_Humus3
design: Total DNA was extracted from 50 mg freeze-dried material using  the CTAB protocol, and extracts were cleaned with the Wizard DNA clean-up  system (Promega, Madison, WI, USA). Fungal community amplicons were generated  by PCR amplification of ITS2 markers using the forward primer gITS7 and the  reverse primer ITS4 elongated with a unique 8 bp long identification tag for  each sample. PCRs were run in technical triplicates and template  concentrations and number of PCR cycles were optimized for each sample based  on qPCR, aiming for quantitative amplification (Ihrmark et al. 2012).  Amplicons were purified using the Agencourt AMPure kit (Beckman Coulter,  Beverly, MA, USA), concentrations were measured fluorometrically (Qubit  Fluorometer, Invitrogen, Carlsbad, CA, USA) and products mixed in equal  amounts into two sequencing pools. Adaptor ligation and 454-sequencing were  performed by LGC Genomics GmbH (Berlin, Germany) on a GS-FLX Titanium system  (Roche, Basel, Switzerland).