Assessing fungal endophyte diversity: a comparative study of three automated metabarcoding pipelines

Abstract

High-throughput sequencing approaches have become frequent in the study of endophyte communities allowing the cumulative description of fungal diversity in the last decade. However, they brought new challenges to researchers in terms of programming and developing of informatics tools. Currently, there is no consensus concerning the appropriate bioinformatics to process such sequence data. The aim of this study was to compare the performance of three pipelines of two cost-free toolkits designed to be friendly to non-programmer users, and specifically developed for fungal data: AMPtk and PIPITS. The sapwood-inhabiting fungal assemblages of two Nothofagus species from the Patagonian Forests were assessed through metabarcoding of the internal transcribed spacer (ITS) and compared with an extant sequence dataset obtained from culture prospection in the same study sites and trees. The AMPtk toolkit has performed better concerning community description in terms of precision of taxa clustering, mainly due to the DADA2 algorithm; PIPITS evidenced a higher sensitivity in detecting taxa known to be present, hence it is potentially useful for future specific taxa detection surveys. Because of a current lack of information of the reference databases, both bioinformatic toolkits performed poorly as to taxonomy assignment. It is imperative to continue studying these ecosystems to, concomitantly, improve databases and the explanatory potential of the new technologies.