pr2-primers: an 18S rRNA primer database for protists

Abstract

Metabarcoding of microbial eukaryotes (collectively known as protists) has developed tremendously in the last decade, almost uniquely relying on the 18S rRNA gene. As microbial eukaryotes are extremely diverse, many primers and primer pairs have been developed. To cover a relevant and representative fraction of the protist community in a given study system, a wise primer choice is needed as no primer pair can target all protists equally well. As such, a smart primer choice is very difficult even for experts and there are very few on-line resources available to list existing primers. We built a database listing 179 primers and 76 primer pairs that have been used for eukaryotic 18S rRNA metabarcoding. In silico performance of primer pairs was tested against two sequence databases: PR2 for eukaryotes and a subset of Silva for prokaryotes. This allowed to determine the taxonomic specificity of primer pairs, the location of mismatches as well as amplicon size. We developed a R-based web application that allows to browse the database, visualize the taxonomic distribution of the amplified sequences with the number of mismatches, and to test any user-defined primer set (https://app.pr2-primers.org). This tool will provide the basis for guided primer choices that will help a wide range of ecologists to implement protists as part of their investigations.

Oceanic heterotrophic flagellates are dominated by a few widespread taxa

“…Together with Chrysophyceae and Bicosoecida, Diplonemea (and to a lower extent Kinetoplastida) also appeared to be major contributors to deep HF assemblages. This was obvious when analyzing V9 amplicons and metagenomes, but unfortunately these euglenozoans did not appear in the V4‐18S data set, as the combination of longer V4 regions (Pernice et al 2016) and several mismatches with universal primers (Vaulot et al 2021) prevent their correct amplification. Therefore, these euglenozoans do not appear in the catalogue of ASVs, a possibly minor issue for surface assemblages but an important limitation for the deep survey.…”

Newly designed foraminifera primers identify habitat‐specific lineages through metabarcoding analyses

“…This primer set targets an approximately 250–300‐bp‐long region that overlaps with the 43e region of foraminiferal SSU‐rDNA as defined in Pawlowski and Lecroq (2010). An in silico PCR test with the 18F and 19R primers against the PR2 database web version (Vaulot et al, 2020) shows a product ranging from ~200 bp for Tubothalamea to 300–400 bp for other foraminiferal sequences, with virtually no amplification of non‐foraminiferal sequences (Figure ). We deployed these primers on various marine and freshwater ecosystems.…”

18S rRNA gene amplicon sequencing combined with culture‐based surveys of maize rhizosphere protists reveal dominant, plant‐enriched and culturable community members

“…Fifteen unique isolates did not have matching ASVs in any sequenced samples (Table S8): these were identified to five eukaryotic supergroups but predominantly represented the Stramenopiles. These protists may have been absent in the high throughput sequencing data because of their rareness before culturing, or because they were not amplified by the barcoding primers used for the taxonomic sequencing survey (Vaulot et al ., 2020). In summary, the isolation survey captured a substantial proportion of the most conserved and abundant bacteriovore clades in the rhizosphere.…”