Untrimmed ITS2 metabarcode sequences cause artificially reduced abundances of specific fungal taxa.

Advances in DNA metabarcoding have greatly expanded our knowledge of microbial communities in recent years. Pipelines and parameters have been tested extensively for bacterial metabarcoding using the 16S rRNA gene and best practices are largely established. For fungal metabarcoding using the internal transcribed spacer (ITS) gene, however, only a few studies have considered how such pipelines and parameters can affect community prediction. Here, we report a novel bias uncovered during ITS region 2 (ITS2) sequencing of IMPORTANCE: Metabarcode sequencing produces DNA abundance profiles that are presumed to reflect the actual microbial composition of their corresponding input samples. However, this assumption is not always tested, and taxon-specific biases are often not apparent, especially for low-abundance taxa in complex communities. Here, we identified internal transcribed spacer region 2 (ITS2) read quality aberrations that caused dramatic reductions in the relative abundances of specific taxa in multiple data sets characterizing ant fungus gardens. Such taxon-specific biases in read quality may be widespread in other environments and for other fungal taxa, thereby causing incorrect descriptions of these mycobiomes.