@article{ff065a02787044da8d383de5ffa158fb,
title = "A novel metabarcoding primer pair for environmental DNA analysis of Cephalopoda (Mollusca) targeting the nuclear 18S rRNA region",
abstract = "Cephalopods are pivotal components of marine food webs, but biodiversity studies are hampered by challenges to sample these agile marine molluscs. Metabarcoding of environmental DNA (eDNA) is a potentially powerful technique to study oceanic cephalopod biodiversity and distribution but has not been applied thus far. We present a novel universal primer pair for metabarcoding cephalopods from eDNA, Ceph18S (Forward: 5′-CGC GGC GCT ACA TAT TAG AC-3′, Reverse: 5′-GCA CTT AAC CGA CCG TCG AC-3′). The primer pair targets the hypervariable region V2 of the nuclear 18S rRNA gene and amplifies a relatively short target sequence of approximately 200 bp in order to allow the amplification of degraded DNA. In silico tests on a reference database and empirical tests on DNA extracts from cephalopod tissue estimate that 44-66% of cephalopod species, corresponding to about 310-460 species, can be amplified and identified with this primer pair. A multi-marker approach with the novel Ceph18S and two previously published cephalopod mitochondrial 16S rRNA primer sets targeting the same region (Jarman et al. 2006 Mol. Ecol. Notes. 6, 268-271; Peters et al. 2015 Mar. Ecol. 36, 1428-1439) is estimated to amplify and identify 89% of all cephalopod species, of which an estimated 19% can only be identified by Ceph18S. All sequences obtained with Ceph18S were submitted to GenBank, resulting in new 18S rRNA sequences for 13 cephalopod taxa.",
keywords = "Cephalopoda, environmental DNA, metabarcoding, universal primer",
author = "{De Jonge}, {Dani{\"e}lle S.W.} and V{\'e}ronique Merten and Till Bayer and Oscar Puebla and Reusch, {Thorsten B.H.} and Hoving, {Henk Jan T.}",
note = "Funding Information: Ethics. Cabo Verde has not ratified the Nagoya protocol. To fulfil the national ABS regulations of Cabo Verde, we obtained the required permit for the publication of results based on samples collected in Cabo Verde waters from the Direccao Nacional do Ambiente (National Directorate for the Environment of Cabo Verdes). Permission for fieldwork and publication of results was granted by Minist{\'e}rio da Agricultura e Ambiente of Cape Verde and Ag{\^e}ncia Mar{\'i}tima e Portu{\'a}ria of Cape Verde. Data accessibility. All barcoded sequences are accessible via GenBank accession numbers MT680727–MT680790 and MT680792–MT680800. The electronic supplementary material includes the Supplementary Data and Code zip-file, which contains the code and data used for data analysis and the creation of figures 1, 3 and 4, and Supplementary Table S1, which contains sample station information. Authors{\textquoteright} contributions. D.S.W.d.J. carried out the molecular laboratory work, performed the bioinformatics and data analysis and drafted the manuscript; V.M. participated in the design of the study, collected field data, helped with the laboratory work and participated in data analysis. T.B. helped with the laboratory work and data analysis. O.P. participated in conceiving and designing the study. T.B.H.R. participated in conceiving and designing the study. H.-J.T.H. conceived and designed the study, collected field data, performed morphologies species identifications and coordinated the study. All authors critically revised the manuscript, gave final approval for publication and agree to be held accountable for the work performed therein. Competing interests. We declare we have no competing interests. Funding. This research is funded by the Deutsche Forschungsgemeinschaft (DFG) under grant HO 5569/2-1 (Emmy Noether Junior Research Group) awarded to H.-J.T.H., and by GEOMAR{\textquoteright}s Programme Oriented Funding III OCEANS programme. Acknowledgements. We thank the Institute of Clinical Molecular Biology in Kiel for providing Sanger sequencing as supported in part by the DFG Clusters of Excellence {\textquoteleft}Precision Medicine in Chronic Inflammation{\textquoteright} and {\textquoteleft}ROOTS{\textquoteright}. We thank T. Naujoks, Dr D. Langfeldt and Dr B. L{\"o}scher for technical support. We thank Dr Heino Fock (chief scientist of WH383) and Dr Stephanie Czudaj for the collaboration and opportunity to collect cephalopods in the eastern Atlantic which were used in this study. Publisher Copyright: {\textcopyright} 2021 The Authors.",
year = "2021",
month = feb,
day = "28",
doi = "10.1098/rsos.201388",
language = "English",
volume = "8",
journal = "Royal Society Open Science",
issn = "2054-5703",
publisher = "The Royal Society",
number = "2",
}