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.
Highlights
The accurate identification of diversity is a key challenge in microbial ecology research
Recent developments in high-throughput sequencing make deep sequencing of hundreds of samples achievable at affordable costs. This is in turn transforming microbial ecology into a quantitative research field in which, for example, models of spatio-temporal patterns of microbial diversity can be built and tested [5,6,7,8,9,10,11], and links between microbial community composition and host genotype [12] as well as phenotype [13,14,15,16] can be established
Used 18S annealing primers from the literature were included, and these retain the name used in the original work [17,18]. They generally display good taxonomic coverage, Rev3 and EukB were excluded from the analysis since they target the distal 39 end of the 18S rDNA, which has a much sparser representation in the SILVA database (Table S1)
Summary
The accurate identification of diversity is a key challenge in microbial ecology research. Recent developments in high-throughput sequencing make deep sequencing of hundreds of samples achievable at affordable costs. This is in turn transforming microbial ecology into a quantitative research field in which, for example, models of spatio-temporal patterns of microbial diversity can be built and tested [5,6,7,8,9,10,11], and links between microbial community composition and host genotype [12] as well as phenotype [13,14,15,16] can be established. 454 pyrosequencing has been conducted on 18S rDNA amplicons and revealed an unprecedented diversity of eukaryotes in a range of environments [17,18,19,20,21,22,23,24,25,26]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
