Illumina metabarcoding pipeline for fungal ITS

High-throughput metabarcoding studies on fungi and other eukaryotic microorganisms are rapidly becoming more frequent and more complex, requiring researchers to handle ever increasing amounts of raw sequence data. Here we provide a flexible pipeline for pruning and analyzing fungal barcode (ITS rDNA) data generated as paired-end reads on Illumina MiSeq sequencers. The pipeline presented includes specific steps fine-tuned for ITS, that are mostly missing from pipelines developed for prokaryotes. It (i) employs state of the art programs and follows best practices in fungal high-throughput metabarcoding, (ii) consists of modules and scripts easily modifiable by the user to ensure maximum flexibility with regard to specific needs of a project or future methodological developments, (iii) and is straightforward to use, also in classroom settings. We provide detailed descriptions and revision techniques for each step, thus avoiding a black-box approach and giving the user maximum control over data treatment. Employing this pipeline will improve and speed up the tedious and error-prone process of cleaning fungal Illumina metabarcoding data.