April 21, 2020  |  

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

Authors: Tedersoo, Leho and Anslan, Sten

Development of high-throughput sequencing techniques have 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 PacBio-based metabarcoding protocols covering the Internal Transcribed Spacer (ITS region) and partial Small Subunit (SSU) 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 pros and cons of long read-based identification of eukaryotes. This article is protected by copyright. All rights reserved.

Journal: Environmental microbiology reports
DOI: 10.1111/1758-2229.12776
Year: 2019

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