Ilumina sequencing of fungal ITS-amplicon library of metagenomic DNA extracts from plant samples
The amplicon library preparation was performed using the ITS1F/ITS4 forward and reverse primers with specific tag sequence combinations (TAG). A two-step PCR was run, the first step PCR added the tag sequence and part of the sequencing primer and went on for 30 cycles. The second step PCR was carried out following a purification step, applying Exonuclease I (New England Biolabs) and Shrimp Alkaline Phosphatase (New England Biolabs) and involved 5 cycles, using the Illumina sequencing primer, combined with a second tag combination (INDEX) and the Illumina P5 or P7 sequencing adapters, respectively. The final amplicons were purified using magnetic beads and ethanol (70%), quantified using gel staining with ethidium bromide in an 0.8 % agarose gel and 100 % UV light under a GelDoc Station and finally pooled over multiple steps.