title: Systematic design of 18S rRNA gene primers for determining eukaryotic diversity in microbial consortia.
abstract: 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.
authors.affiliation: KTH Royal Institute of Technology, Science for Life Laboratory, School of Biotechnology, Division of Gene Technology, Stockholm, Sweden.
authors.forename: Luisa W
authors.initials: LW
authors.lastname: Hugerth
authors.affiliation: Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg.
authors.forename: Emilie E L
authors.initials: EE
authors.lastname: Muller
authors.affiliation: KTH Royal Institute of Technology, Science for Life Laboratory, School of Biotechnology, Division of Gene Technology, Stockholm, Sweden.
authors.forename: Yue O O
authors.initials: YO
authors.lastname: Hu
authors.affiliation: Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg.
authors.forename: Laura A M
authors.initials: LA
authors.lastname: Lebrun
authors.affiliation: Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg.
authors.forename: Hugo
authors.initials: H
authors.lastname: Roume
authors.affiliation: KTH Royal Institute of Technology, Science for Life Laboratory, School of Biotechnology, Division of Gene Technology, Stockholm, Sweden.
authors.forename: Daniel
authors.initials: D
authors.lastname: Lundin
authors.affiliation: Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg.
authors.forename: Paul
authors.initials: P
authors.lastname: Wilmes
authors.affiliation: KTH Royal Institute of Technology, Science for Life Laboratory, School of Biotechnology, Division of Gene Technology, Stockholm, Sweden.
authors.forename: Anders F
authors.initials: AF
authors.lastname: Andersson
mesh_terms: D044822:Biodiversity; D017931:DNA Primers; D004783:Environmental Microbiology; D005057:Eukaryotic Cells / Q000145:classification*; D059014:High-Throughput Nucleotide Sequencing; D059013:Microbial Consortia / Q000235:genetics*; D058977:Molecular Sequence Annotation; D010802:Phylogeny; D012337:RNA, Ribosomal, 18S / Q000235:genetics*; D015203:Reproducibility of Results
chemical_list: D017931:DNA Primers; D012337:RNA, Ribosomal, 18S
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