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Short- and long-read metabarcoding of the eukaryotic rRNA operon: evaluation of primers and comparison to shotgun metagenomics sequencing
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  • Meike Anna Christine Latz,
  • Vesna Grujcic,
  • Sonia Brugel,
  • Jenny Lycken,
  • Bengt Karlson,
  • Uwe John,
  • Agneta Andersson,
  • Anders Andersson
Meike Anna Christine Latz
KTH Royal Institute of Technology School of Biotechnology

Corresponding Author:[email protected]

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Vesna Grujcic
KTH Royal Institute of Technology School of Biotechnology
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Sonia Brugel
UmeƄ Universitet
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Jenny Lycken
Swedish Meteorological and Hydrological Institute
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Bengt Karlson
Swedish Meteorological and Hydrological Institute
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Uwe John
Alfred Wegener Institute for Polar and Marine Research
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Agneta Andersson
Umea Universitet Teknisk-Naturvetenskaplig Fakultet
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Anders Andersson
KTH Royal Institute of Technology School of Biotechnology
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High-throughput sequencing for analysis of environmental microbial diversity has evolved vastly over the last decade. Currently the go-to method for microbial eukaryotes is short-read metabarcoding of variable regions of the 18S rRNA gene with <500 bp amplicons. However, there is a growing interest in long-read sequencing of amplicons covering the rRNA operon for improving taxonomic resolution. For both methods, the choice of primers is crucial. It determines if community members are covered, if they can be identified at a satisfactory taxonomic level, and if the obtained community profile is representative. Here, we designed new primers targeting 18S and 28S rRNA based on 177,934 and 21,072 database sequences, respectively. The primers were evaluated in silico along with published primers on reference sequence databases and marine metagenomics datasets. We further evaluated a subset of the primers for short- and long-read sequencing on environmental samples in vitro and compared the obtained community profile with primer-unbiased metagenomic sequencing. Of the short-read pairs, a new V6-V8 pair and the V4_Balzano pair used with a simplified PCR protocol provided good results in silico and in vitro. Fewer differences were observed between the long-read primer pairs. The long-read amplicons and ITS1 alone provided higher taxonomic resolution than V4. Together, our results represent a reference and guide for selection of robust primers for research on and environmental monitoring of microbial eukaryotes.
30 Sep 2021Submitted to Molecular Ecology Resources
07 Oct 2021Submission Checks Completed
07 Oct 2021Assigned to Editor
28 Oct 2021Reviewer(s) Assigned
22 Dec 2021Review(s) Completed, Editorial Evaluation Pending
02 Feb 2022Editorial Decision: Revise Minor
17 Mar 2022Review(s) Completed, Editorial Evaluation Pending
17 Mar 20221st Revision Received
11 Apr 2022Editorial Decision: Accept
Aug 2022Published in Molecular Ecology Resources volume 22 issue 6 on pages 2304-2318. 10.1111/1755-0998.13623