title: Metabarcoding of fungal ITS2:  Control1_ForestLitter
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).