Towards PacBio-based pan-eukaryote metabarcoding using full-length ITS sequences

Development of high-throughput sequencing techniques has greatly benefited our understanding about microbial ecology, yet the methods producing short reads suffer from species-level resolution and uncertainty of identification. Here, we optimize Pacific Biosciences-based metabarcoding protocols covering the internal transcribed spacer (ITS region) and partial small subunit of the rRNA gene for species-level identification of all eukaryotes, with a specific focus on Fungi (including Glomeromycota) and Stramenopila (particularly Oomycota). Based on tests on composite soil samples and mock communities, we propose best suitable degenerate primers, ITS9munngs + ITS4ngsUni for eukaryotes and selected groups therein and discuss the pros and cons of long read-based identification of eukaryotes.