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.

Design and Evaluation of Illumina MiSeq-Compatible, 18S rRNA Gene-Specific Primers for Improved Characterization of Mixed Phototrophic Communities

“…1A). Conserved regions were targeted as possible primer locations (22,23,41), and combinations of conserved/variable regions (i.e., possible amplicons) were plotted against Shannon entropy (Fig. 1B) in order to determine areas that had the highest entropy and amplicon length suitable for sequencing on the Illumina MiSeq platform.…”

“…The resultant PCR product was processed using gel electrophoresis, band extraction, and purification with a QIAquick gel purification kit (Qiagen, Valencia, CA) before being cloned via a TOPO TA clone kit (Invitrogen, Carlsbad, CA) with a pCR 4-TOPO TA vector, according to the manufacturer's protocols. The cloned PCR products were sequenced using Sanger sequencing and cloned plasmid primers M13For-20, M13Rev-21 (Invitrogen), and 563f (22). Individual Sanger reads were merged to obtain the full-length sequence, and species identification was confirmed using BLAST (63).…”

“…Despite recent studies that have developed broad eukaryotic primers (22,23) using the small subunit (SSU) 18S rRNA gene, there is no widely accepted target gene used to sequence microalgae. Previous studies have targeted various regions of the rrn operon (e.g., 5.8S plus internal transcribed spacer 2 [ITS-2] [24,25], 18S [8,[26][27][28][29], and 23S [30] regions), mitochondrial genes (e.g., cytochrome c oxidase 1 [COI] [31]), and chloroplast genes (e.g., the rbcL gene which encodes the large subunit for ribulose-1,5-bisphosphate carboxylase/oxygenase [RuBisCO] [32][33][34] and the 16S rRNA gene [35]), but these studies have been predominately limited to marine phytoplankton (e.g., references 29,36,37) and occasionally focused on freshwater phytoplankton (e.g., reference 38).…”

“…Within the 18S rRNA gene, multiple studies have used different variable regions for amplification, including the V1-V2 (39), V3 (40), V4 (41)(42)(43), and V9 (37,41,42) regions, with the V4 and V9 regions often being used together (e.g., references 41 and 42). A number of recent studies have compared variable regions along the entire 18S rRNA gene for all eukaryotes (22,23) and eukaryotic plankton (44) and highlighted conserved regions that may be best suited for amplifying hypervariable regions. These studies identified primer combinations using in silico sequence database coverage and taxonomic resolution and confirmed their feasibility with environmental surveys.…”

DNA Sequencing as a Tool to Monitor Marine Ecological Status

“…For example, amplification of a nuclear 16S rRNA gene segment is typical for taxonomic classification of marine prokaryotic assemblages (Klindworth et al, 2013). Amplification of 18S rRNA regions is used for a variety of communities, including phytoplankton and other microeukaryotes (Dunthorn et al, 2012;Hugerth et al, 2014;Johnson and Martiny, 2015;del Campo et al, 2016;Giner et al, 2016), and amplification of the 23S rRNA gene is used to classify organisms such as zooplankton (Hirai et al, 2015a;Bucklin et al, 2016). Amplification of mitochondrial DNA is used to identify an assortment of organisms, with cytochrome oxidase I (COI), cytochrome b, and mitochondrial 16S as examples of popularly employed target regions (Dauble et al, 2012;Pawlowski et al, 2014;Cowart et al, 2015;Guo et al, 2015;Harada et al, 2015;Johnson and Martiny, 2015;Aylagas et al, 2016;Bucklin et al, 2016;Creer et al, 2016;Leray and Knowlton, 2016;Thompson et al, 2016;Trivedi et al, 2016).…”