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 Trichoderma-infected ant fungus gardens and confirmed this bias using mock communities. Abnormally low forward read quality caused Trichoderma ITS2 reads to be computationally filtered before and during read pair merging, thus almost entirely eliminating Trichoderma amplicon sequence variants from the resulting fungal community profiles. Sliding window quality trimming before filtering allowed most of these reads to pass filtering and merge successfully, producing community profiles that now correlated with visual signs of Trichoderma infection and matched the composition of the mock communities. Applying such sliding window trimming to a previously generated environmental ITS2 data set increased the detected fungal diversity and again overcame read quality biases against Trichoderma to detect it in nearly every sample instead and often at high relative abundances. This analysis additionally identified a similar, but distinct, bias against a second fungal genus Meyerozyma. The prevalence of such quality biases against other fungal ITS sequences is unknown but may be widespread. We, therefore, advocate for the routine use of sliding window quality trimming as a best practice in ITS2 metabarcoding analysis.

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.