Validation of a universal set of primers to study animal‐associated microeukaryotic communities

Abstract

Summary The application of metabarcoding to study animal‐associated microeukaryotes has been restricted because the universal barcode used to study microeukaryotic ecology and distribution in the environment, the Small Subunit of the Ribosomal RNA gene (18S rRNA), is also present in the host. As a result, when host‐associated microbial eukaryotes are analysed by metabarcoding, the reads tend to be dominated by host sequences. We have done an in silico validation against the SILVA 18S rRNA database of a non‐metazoan primer set (primers that are biased against the metazoan 18S rRNA) that recovers only 2.6% of all the metazoan sequences, while recovering most of the other eukaryotes (80.4%). Among metazoans, the non‐metazoan primers are predicted to amplify 74% of Porifera sequences, 4% of Ctenophora, and 15% of Cnidaria, while amplifying almost no sequences within Bilateria. In vivo, these non‐metazoan primers reduce significantly the animal signal from coral and human samples, and when compared against universal primers provide at worst a 2‐fold decrease in the number of metazoan reads and at best a 2800‐fold decrease. This easy, inexpensive, and near‐universal method for the study of animal‐associated microeukaryotes diversity will contribute to a better understanding of the microbiome.

Single-cell metabarcoding reveals biotic interactions of the Arctic calcifierNeogloboquadrina pachydermawith the eukaryotic pelagic community

“…kit following manufacture instructions including blank extractions to control for (cross-) contamination events. DNA extractions were then amplified in triplicates using the universal Eukaryotic V4 tagged primers TAReuk454FWD1 (5'-CCAGCA(G⁄C)C(C⁄T)GCGG-TAATTCC-3') and TAReukREV3 (5'-ACTTTCGTTCTTGAT(C⁄T)(A⁄G)A-3') (Stoeck et al, 2010) that amplify only eukaryotes and offer a good taxonomic resolution across the entire eukaryote realm (del Campo et al, 2019). Each tagged PCR primer consists of a unique tag sequence of 8 nucleotide appended to the 5'end of the common amplification primer sequence.…”

The eukaryome: Diversity and role of microeukaryotic organisms associated with animal hosts

“…A hybrid approach is to develop primers that work on a wide taxonomic range of eukaryotes but exclude all or most metazoans. One such set of primers developed to look at parasites (Bower et al, ) has recently been shown to work on a wide range of microeukaryotes associated with taxonomically diverse animal hosts (del Campo et al, ). Extensive deployment of these methods will certainly help to identify the main questions to ask about the eukaryome, many of which are currently not even clear.…”

Revealing the Composition of the Eukaryotic Microbiome of Oyster Spat by CRISPR-Cas Selective Amplicon Sequencing (CCSAS)

“…up to 92% in coral, 42% in krill, and 45% in fish) [57,72,63]. This approach also requires designing and optimizing the blocking-primers for each animal host, which remains a challenge [71][72].…”