TY - JOUR T1 - Combining the 5.8S and ITS2 gene regions to improve classification of fungi JF - bioRxiv DO - 10.1101/532358 SP - 532358 AU - Felix Heeger AU - Christian Wurzbacher AU - Elizabeth C. Bourne AU - Camila J. Mazzoni AU - Michael T. Monaghan Y1 - 2019/01/01 UR - http://biorxiv.org/content/early/2019/02/05/532358.abstract N2 - The internal transcribed spacer (ITS) is used in DNA metabarcoding of fungi. One disadvantage of its high variability may be a failure to classify OTUs when no similar reference sequence exists. We tested whether the 5.8S region, often sequenced with ITS2 but discarded before analysis, could provide OUT classifications when ITS fails.We used in silico evaluation to compare classification success of 5.8S and ITS from the UNITE database when reference sequences of the same species, genus, or family were removed. We then developed an automated pipeline for a combined 5.8S - ITS2 analysis and applied it to mixed environmental samples containing many lineages that are underrepresented in databases.ITS was clearlysuperior for species-level classifications with a complete reference database, but 5.8S outperformed ITS at higher level classifications with an incomplete database. Our combined 5.8S-ITS2 pipeline classified 3x more fungal OTUs compared to ITS2 alone, particularly within Chytridiomycota (10x) and Rozellamycota (3x).Missing reference sequences led to the failure of ITS to classify many fungal OTUs at all, and to a significant underestimation of environmental fungal diversity. Using 5.8S to complement ITS classification will likely provide better estimates of diversity in lineages for which database coverage is poor. ER -