Systematic Design of 18S rRNA Gene Primers for Determining Eukaryotic Diversity in Microbial Consortia

• Published in: PloS one Vol. 9; no. 4; p. e95567
• Main Authors: Hugerth, Luisa W., Muller, Emilie E. L., Hu, Yue O. O., Lebrun, Laura A. M., Roume, Hugo, Lundin, Daniel, Wilmes, Paul, Andersson, Anders F.
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 01.04.2014; Public Library of Science (PLoS)
• Subjects: Analysis; Bacterial Communities; Biodiversity; Biodiversity and Ecology; Bioinformatics; Biology and Life Sciences; Biotechnology; Comparative studies; Consortia; Design; Design engineering; DNA Primers; DNA sequencing; Dynamics; Ecology; Ecology and Environmental Sciences; Ecosystems; Ekologi; Environmental Microbiology; Environmental Sciences; Eukaryotes; Eukaryotic Cells - classification; Fragments; Gene sequencing; Generation; Genetic aspects; High-Throughput Nucleotide Sequencing; Human Gut Microbiota; Identification; Laboratories; Life Sciences; Magnitude; Microbial Consortia - genetics; Microorganisms; Molecular Sequence Annotation; Next-generation sequencing; Nucleotide sequencing; Phylogeny; Physiological aspects; Polymerase chain reaction; Primers; Rdna; Reproducibility of Results; Ribonucleic acid; RNA; RNA, Ribosomal, 18S - genetics; rRNA 16S; rRNA 18S; Science; Species Richness; Taxonomy; Water; Sweden; Luxembourg
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0095567

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.