Island4 Fragmented litter2 Fungal metagonome2
"For production of fungal ITS2 amplicons for 454-sequencing, the forward primer gITS7 (Ihrmark et al. 2012, FEMS Microbiology Ecology, DOI: 10.1111/j.1574-6941.2012.01437.x) was used in combination with the ITS4 primer extended with a sample-specific 8bp long identification tag. Analysis by PCR was conducted with an initial denaturation phase of 5 min at 95?C and 24 - 35 cycles of 30 s at 95?C, 30 s at 56?C and 30 s at 72?C, and a final elongation phase of 7 min at 72?C. The PCR mix consisted of 0.2 mM dNTPs, 0.75 mM MgCl2, 0.3 ?M of ITS4 primer, 0.5 ?M of gITS7 primer and 0.5 U of DNA polymerase (Dream Taq, Fermentas) in 50 ?l reactions. The number of cycles was optimized to reach the exponential phase of the PCR reaction for each sample as determined by qPCR results. Samples were run in technical triplicates that were inspected on a 1% agarose gel and pooled. Amplicons were then purified with the AMPure PCR purification kit (Agencourt Bioscience Corporation, Beverly, MA, USA) and DNA concentrations were measured by spectrophotometry (NanoDrop 1000, Thermo Scientific, Wilmington, DE, USA). Aliquots of each sample containing equal amounts of PCR product were pooled, concentrated by freeze-drying, re-suspended and further purified by the GeneJET PCR purification columns (Fermentas). Adaptor and MID ligation and 454-sequencing were performed by LGC Genomics GmbH (Berlin, Germany) on a GL FLX Titanium system (Roche, Basel, Switzerland)."