An improved primer set and amplification protocol with increased specificity and sensitivity targeting the Symbiodinium ITS2 region

PeerJ
Benjamin C C HumeChristian R Voolstra

Abstract

The Internal Transcribed Spacer 2 (ITS2) rRNA gene is a commonly targeted genetic marker to assess diversity of Symbiodinium, a dinoflagellate genus of algal endosymbionts that is pervasively associated with marine invertebrates, and notably reef-building corals. Here we tested three commonly used ITS2 primer pairs (SYM_VAR_5.8S2/SYM_VAR_REV, ITSintfor2/ITSReverse, and ITS-DINO/ITS2Rev2) with regard to amplification specificity and sensitivity towards Symbiodinium, as well as sub-genera taxonomic bias. We tested these primers over a range of sample types including three coral species, coral surrounding water, reef surface water, and open ocean water to assess their suitability for use in large-scale next generation sequencing projects and to develop a standardised PCR protocol. We found the SYM_VAR_5.8S2/SYM_VAR_REV primers to perform superior to the other tested ITS2 primers. We therefore used this primer pair to develop a standardised PCR protocol. To do this, we tested the effect of PCR-to-PCR variation, annealing temperature, cycle number, and different polymerase systems on the PCR efficacy. The Symbiodinium ITS2 PCR protocol developed here delivers improved specificity and sensitivity towards Symbiodinium with apparent mi...Continue Reading

Associated Datasets

References

Apr 8, 2010·Molecular Phylogenetics and Evolution·Xavier Pochon, Ruth D Gates
Jul 23, 2011·Science·John M PandolfiAnne L Cohen
Oct 25, 2011·PloS One·Laetitia PlaisanceNancy Knowlton
Apr 15, 2015·Trends in Microbiology·Nils RädeckerChristian Wild
Jun 2, 2015·Scientific Data·Stéphane PesantUNKNOWN Tara Oceans Consortium Coordinators
Apr 5, 2016·Proceedings of the National Academy of Sciences of the United States of America·Benjamin C C HumeJörg Wiedenmann
Oct 26, 2016·The ISME Journal·Nadine M BoulotteMadeleine Jh van Oppen
Aug 3, 2016·Annual Review of Microbiology·David G BourneNicole S Webster
Dec 3, 2016·The ISME Journal·Rachel Ashley LevinMadeleine Josephine Henriette van Oppen
Feb 12, 2017·Nature Communications·Maren ZieglerChristian R Voolstra
Feb 12, 2017·Molecular Ecology·D J ThornhillS R Santos
Feb 18, 2017·The ISME Journal·Edward G SmithJohn A Burt
Mar 23, 2017·Frontiers in Microbiology·Raquel S PeixotoDavid G Bourne
Apr 22, 2017·Global Change Biology·Claudia PogoreutzChristian Wild
Jun 2, 2017·Nature·Terry P HughesMarten Scheffer
Aug 2, 2017·Scientific Data·Adriana AlbertiPatrick Wincker
Dec 2, 2017·The ISME Journal·Maren ZieglerChristian R Voolstra

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Citations

Feb 12, 2019·Molecular Ecology Resources·Benjamin C C HumeChristian R Voolstra
Feb 13, 2020·Global Change Biology·Lauren I Howe-KerrAdrienne M S Correa
Sep 29, 2020·Scientific Reports·Quentin CarradecPatrick Wincker
Jul 7, 2020·Global Change Biology·Marcela HerreraManuel Aranda
Sep 17, 2020·The ISME Journal·Marcela HerreraManuel Aranda
Jun 13, 2020·Frontiers in Microbiology·Osama S SaadSenjie Lin
Dec 21, 2019·Scientific Reports·Arjen TilstraChristian Wild
Mar 24, 2021·Animal Microbiome·Carsten G B GrupstraAdrienne M S Correa

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Methods Mentioned

BETA
electrophoresis
PCR
PCRs

Software Mentioned

bowtie
Advantage
REV
split
ITSintfor2
blastn
MiSeq
SYM
fasta
VAR

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