Long-read metabarcoding of the eukaryotic rDNA operon to phylogenetically and taxonomically resolve environmental diversity

The small subunit (SSU) RNA gene is a widely-used marker for environmental sequencing to assess eukaryotic biodiversity, and discover new species in various environmental samples. This approach typically employs next generation sequencing technologies such as Illumina, which generates millions of reads through massive parallelisation. One major limitation of this approach is sequence length, which has resulted in an unprecedented wealth of data but for only short (=500bp) regions of the SSU gene. Consequently, the phylogenetic resolution of current data is generally poor. This problem may be overcome by the advent of the high-throughput, long-read sequencing platform PacBio. While PacBio sequencing has successfully been used to amplify the complete SSU in prokaryotes for metabarcoding, a similar approach has not yet been tested for eukaryotes. To address this, we obtained three soil samples and used long-range PCR to amplify a 4000-4500 bp region of the rRNA operon, including 18S, ITS1, 5.8S, ITS2, and 28S. Amplicons were sequenced on three SMRT cells on the new Sequel system, generating a total of ~ 200,000 long reads. Here, we will present our pipeline for cleaning and analysing eukaryotic ribosomal data generated with this method and show its potential to assess eukaryotic diversity using the stronger phylogenetic signal of longer reads. Most notably, this approach allows to reliably infer taxonomic affiliation using phylogenetic trees rather than simpler homology binning, which represents a key advantage to discover novel lineages. It also significantly develops databases of ribosomal genes other than the SSU, increasing taxon sampling and phylogenetic coverage of ITS, 5.8S, and 28S regions.