Decoding fungal communities: ITS extraction matters more than ASVs vs. OTUs

Abstract. To capture the complexity of fungal communities, microbial ecologists must often resort to amplicon sequencing. Amplicon sequences are obtained from the nuclear ribosomal internal transcribed spacer (ITS), which is currently used as a marker for fungal taxonomic identity. Raw amplicon sequences are analysed using either amplicon sequence variants (ASVs) or operational taxonomic units (OTUs). However, these two approaches are conceptually different, and there is disagreement over which is more suitable for fungal sequence data. In order to address this problem, we used published fungal genomes to simulate amplicon sequencing of the ITS1 and ITS2 regions of fungal communities with diversity spanning 50 to 800 strains. These data were then analysed via six pipelines with differences at key steps in read processing and ASV/OTU retrieval. In addition, we confirmed the results with sequencing data from a previously published 189-strain mock community. Our results show that the choice between ASV and OTU has only a minor effect on the end result and that the most important step in the analysis pipeline is the extraction of the ITS region from the short-read sequences. Moreover, we recommend sequencing with as large a library size as possible, joining the reads to perform ITS extraction and then clustering the sequences into OTUs. We hope that this will help microbial ecologists choose the most appropriate approach to answer their specific questions.