ITS_taxonomy

Six oak bark pieces (~1g) from four oak trees (three pieces from oak 1 and one piece from each of the other trees) in Northern Germany were collected. In addition an infusion (20g oak bark in a sterile tea bag in 300 mL water for 24 hours) for each tree was prepared. DNA was extracted from all samples using the Soil DNA Kit from Omega Bio-Tek. The resulting DNA was sent to LGC Genomics (GmbH, Berlin, Germany) for amplification of the fungal ITS (ITS1, 5.8S and ITS2) sequences using ITS1f (CTTGGTCATTTAGAGGAAGTAA) and ITS4 (TCCTCCGCTTATTGATATGC) primers using the 454 GS FLX+ Titanium Sequencer (Roche).
ITS sequences were processed with mothur and subsampled to 4000 sequences per sample. For taxonomic classification the mothur implementation of naive Bayesian classification was used with a threshold bootstrap value of 70% for each taxonomic level. The used fungal database was the “dynamic” mothur release from 08.12.2013 of the UNITE database.