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

Environ Microbiol Rep. 2019 Oct;11(5):659-668. doi: 10.1111/1758-2229.12776. Epub 2019 Jul 4.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA Barcoding, Taxonomic / methods*
  • DNA Primers / genetics
  • DNA, Fungal / genetics
  • DNA, Ribosomal Spacer / genetics*
  • Eukaryota / genetics*
  • Fungi / genetics
  • High-Throughput Nucleotide Sequencing
  • Phylogeny
  • Sequence Analysis, DNA

Substances

  • DNA Primers
  • DNA, Fungal
  • DNA, Ribosomal Spacer