Characterization of the 18S rRNA Gene for Designing Universal Eukaryote Specific Primers

Abstract

High throughput sequencing technology has great promise for biodiversity studies. However, an underlying assumption is that the primers used in these studies are universal for the prokaryotic or eukaryotic groups of interest. Full primer universality is difficult or impossible to achieve and studies using different primer sets make biodiversity comparisons problematic. The aim of this study was to design and optimize universal eukaryotic primers that could be used as a standard in future biodiversity studies. Using the alignment of all eukaryotic sequences from the publicly available SILVA database, we generated a full characterization of variable versus conserved regions in the 18S rRNA gene. All variable regions within this gene were analyzed and our results suggested that the V2, V4 and V9 regions were best suited for biodiversity assessments. Previously published universal eukaryotic primers as well as a number of self-designed primers were mapped to the alignment. Primer selection will depend on sequencing technology used, and this study focused on the 454 pyrosequencing GS FLX Titanium platform. The results generated a primer pair yielding theoretical matches to 80% of the eukaryotic and 0% of the prokaryotic sequences in the SILVA database. An empirical test of marine sediments using the AmpliconNoise pipeline for analysis of the high throughput sequencing data yielded amplification of sequences for 71% of all eukaryotic phyla with no isolation of prokaryotic sequences. To our knowledge this is the first characterization of the complete 18S rRNA gene using all eukaryotes present in the SILVA database, providing a robust test for universal eukaryotic primers. Since both in silico and empirical tests using high throughput sequencing retained high inclusion of eukaryotic phyla and exclusion of prokaryotes, we conclude that these primers are well suited for assessing eukaryote diversity, and can be used as a standard in biodiversity studies.

Under the canopy: Community-wide effects of invasive algae in Marine Protected Areas revealed by metabarcoding

“…18S in general is known to underestimate the diversity present at the species level in metabarcoding studies (in metazoans in particular, Tang et al, 2012;Leray and Knowlton, 2016), and several species or even several genera can share the same sequence for the v7 region amplified (Guardiola et al, 2016). In addition, several fragments of 18S have been commonly used in metabarcoding, each with its own characteristics (Hadziavdic et al, 2014;Tanabe et al, 2016). The fragment of COI sequenced here, on the other hand, is longer and more variable, thus allowing the delimitation of a much higher number of MOTUs.…”

The influence of vent systems on pelagic eukaryotic micro-organism composition in the Nordic Seas

“…Extraction was carried out according to the manufacturer's instructions. PCR was conducted using the Takara Ex HS taq (Takara BIO, Inc., Japan) with the universal eukaryotic 18S rRNA gene-targeted primers Univ-1131F-7 (5 0 -AAA CTT AAA GRA ATT GAC GG-3 0 ) and Univ-1428R (5 0 -CTA AGG GCA TCA CAG ACC-3 0 ) bp (Troedsson et al 2008;Hadziavdic et al 2014) generating a *315-bp product. The amplification conditions included an initial denaturation cycle (3 min at 94°C), followed by 30 amplification cycles (30 s at 94°C, 30 s at 57.4°C and 1 min at 72°C).…”

“…Caron et al 2009). Indeed, previous studies have explored different cut-off levels ranging from 90 to 100 % (Stoeck et al 2007;Edgcomb et al 2011;Orsi et al 2011;Hadziavdic et al 2014), which is necessary for any new primer set used to amplify a fragment of the 18S rRNA gene. However, to our knowledge, this is the first study using the hierarchical rank approach with environmental DNA data.…”

Novel Diversity of Deeply Branching Holomycota and Unicellular Holozoans Revealed by Metabarcoding in Middle Paraná River, Argentina

“…Both V4 and V9 are considered suitable regions for biodiversity studies given their great variability and the combination of high and low entropy in these sections, which allows forgood primer binding (Behnke et al, 2011;Hadziavdic et al, 2014;Tanabe et al, 2016). They also have been widely used in molecular diversity studies, so the research community knows the advantages and limitations of both regions (AmaralZettler et al, 2009;Stoeck et al, 2010;Massana et al, 2015).…”