Inclusion of database outgroups reduces false positives in fungal metabarcoding taxonomic assignments.

Metabarcoding studies of fungal communities rely on curated databases for assigning taxonomy. Any host or other nonfungal environmental sequences that are amplified during polymerase chain reaction (PCR) are inherently assigned taxonomy by these same databases, possibly leading to ambiguous nonfungal amplicons being assigned to fungal taxa. Here, we investigated the effects of including nonfungal outgroups in a fungal taxonomic database to aid in detecting and removing these nontarget amplicons. We processed 15 publicly available fungal metabarcode data sets and discovered that roughly 40% of the reads from these studies were not fungal, although they were assigned as Fungus sp. when using a database without nonfungal outgroups. We discuss implications for metabarcoding studies and recommend assigning taxonomy using a database with outgroups to better detect these nonfungal amplicons.