Systematic design of 18S rRNA gene primers for determining eukaryotic diversity in microbial consortia

PLoS One. 2014 Apr 22;9(4):e95567. doi: 10.1371/journal.pone.0095567. eCollection 2014.


High-throughput sequencing of ribosomal RNA gene (rDNA) amplicons has opened up the door to large-scale comparative studies of microbial community structures. The short reads currently produced by massively parallel sequencing technologies make the choice of sequencing region crucial for accurate phylogenetic assignments. While for 16S rDNA, relevant regions have been well described, no truly systematic design of 18S rDNA primers aimed at resolving eukaryotic diversity has yet been reported. Here we used 31,862 18S rDNA sequences to design a set of broad-taxonomic range degenerate PCR primers. We simulated the phylogenetic information that each candidate primer pair would retrieve using paired- or single-end reads of various lengths, representing different sequencing technologies. Primer pairs targeting the V4 region performed best, allowing discrimination with paired-end reads as short as 150 bp (with 75% accuracy at genus level). The conditions for PCR amplification were optimised for one of these primer pairs and this was used to amplify 18S rDNA sequences from isolates as well as from a range of environmental samples which were then Illumina sequenced and analysed, revealing good concordance between expected and observed results. In summary, the reported primer sets will allow minimally biased assessment of eukaryotic diversity in different microbial ecosystems.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biodiversity*
  • DNA Primers*
  • Environmental Microbiology
  • Eukaryotic Cells / classification*
  • High-Throughput Nucleotide Sequencing
  • Microbial Consortia / genetics*
  • Molecular Sequence Annotation
  • Phylogeny
  • RNA, Ribosomal, 18S / genetics*
  • Reproducibility of Results


  • DNA Primers
  • RNA, Ribosomal, 18S

Grant support

This work was funded by the Swedish Research Councils VR [grant 2011-5689], FORMAS [grant 2009-1174] and EC BONUS project BLUEPRINT partially funded by FORMAS through grants to A.F.A, the Luxemburg National Research fund [grants ATTRACT/A09/03 and CORE/11/BM/1186762 to P.W., PHD-MARP-04 to H.R. and PRD-2011-1/SR to E.M.] and the Chinese Scholarship Council. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.