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Validation of COI metabarcoding primers for terrestrial arthropods

Metabarcoding can rapidly determine the species composition of bulk samples and thus aids biodiversity and ecosystem assessment. However, it is essential to use primer sets that minimize amplification bias among taxa to maximize species recovery. Despite this fact, the performance of primer sets emp...

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Autores principales: Elbrecht, Vasco, Braukmann, Thomas W.A., Ivanova, Natalia V., Prosser, Sean W.J., Hajibabaei, Mehrdad, Wright, Michael, Zakharov, Evgeny V., Hebert, Paul D.N., Steinke, Dirk
Formato: Online Artículo Texto
Lenguaje:English
Publicado: PeerJ Inc. 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6786254/
https://www.ncbi.nlm.nih.gov/pubmed/31608170
http://dx.doi.org/10.7717/peerj.7745
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author Elbrecht, Vasco
Braukmann, Thomas W.A.
Ivanova, Natalia V.
Prosser, Sean W.J.
Hajibabaei, Mehrdad
Wright, Michael
Zakharov, Evgeny V.
Hebert, Paul D.N.
Steinke, Dirk
author_facet Elbrecht, Vasco
Braukmann, Thomas W.A.
Ivanova, Natalia V.
Prosser, Sean W.J.
Hajibabaei, Mehrdad
Wright, Michael
Zakharov, Evgeny V.
Hebert, Paul D.N.
Steinke, Dirk
author_sort Elbrecht, Vasco
collection PubMed
description Metabarcoding can rapidly determine the species composition of bulk samples and thus aids biodiversity and ecosystem assessment. However, it is essential to use primer sets that minimize amplification bias among taxa to maximize species recovery. Despite this fact, the performance of primer sets employed for metabarcoding terrestrial arthropods has not been sufficiently evaluated. This study tests the performance of 36 primer sets on a mock community containing 374 insect species. Amplification success was assessed with gradient PCRs and the 21 most promising primer sets selected for metabarcoding. These 21 primer sets were also tested by metabarcoding a Malaise trap sample. We identified eight primer sets, mainly those including inosine and/or high degeneracy, that recovered more than 95% of the species in the mock community. Results from the Malaise trap sample were congruent with the mock community, but primer sets generating short amplicons produced potential false positives. Taxon recovery from both mock community and Malaise trap sample metabarcoding were used to select four primer sets for additional evaluation at different annealing temperatures (40–60 °C) using the mock community. The effect of temperature varied by primer pair but overall it only had a minor effect on taxon recovery. This study reveals the weak performance of some primer sets employed in past studies. It also demonstrates that certain primer sets can recover most taxa in a diverse species assemblage. Thus, based our experimental set up, there is no need to employ several primer sets targeting the same gene region. We identify several suitable primer sets for arthropod metabarcoding, and specifically recommend BF3 + BR2, as it is not affected by primer slippage and provides maximal taxonomic resolution. The fwhF2 + fwhR2n primer set amplifies a shorter fragment and is therefore ideal when targeting degraded DNA (e.g., from gut contents).
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spelling pubmed-67862542019-10-11 Validation of COI metabarcoding primers for terrestrial arthropods Elbrecht, Vasco Braukmann, Thomas W.A. Ivanova, Natalia V. Prosser, Sean W.J. Hajibabaei, Mehrdad Wright, Michael Zakharov, Evgeny V. Hebert, Paul D.N. Steinke, Dirk PeerJ Ecology Metabarcoding can rapidly determine the species composition of bulk samples and thus aids biodiversity and ecosystem assessment. However, it is essential to use primer sets that minimize amplification bias among taxa to maximize species recovery. Despite this fact, the performance of primer sets employed for metabarcoding terrestrial arthropods has not been sufficiently evaluated. This study tests the performance of 36 primer sets on a mock community containing 374 insect species. Amplification success was assessed with gradient PCRs and the 21 most promising primer sets selected for metabarcoding. These 21 primer sets were also tested by metabarcoding a Malaise trap sample. We identified eight primer sets, mainly those including inosine and/or high degeneracy, that recovered more than 95% of the species in the mock community. Results from the Malaise trap sample were congruent with the mock community, but primer sets generating short amplicons produced potential false positives. Taxon recovery from both mock community and Malaise trap sample metabarcoding were used to select four primer sets for additional evaluation at different annealing temperatures (40–60 °C) using the mock community. The effect of temperature varied by primer pair but overall it only had a minor effect on taxon recovery. This study reveals the weak performance of some primer sets employed in past studies. It also demonstrates that certain primer sets can recover most taxa in a diverse species assemblage. Thus, based our experimental set up, there is no need to employ several primer sets targeting the same gene region. We identify several suitable primer sets for arthropod metabarcoding, and specifically recommend BF3 + BR2, as it is not affected by primer slippage and provides maximal taxonomic resolution. The fwhF2 + fwhR2n primer set amplifies a shorter fragment and is therefore ideal when targeting degraded DNA (e.g., from gut contents). PeerJ Inc. 2019-10-07 /pmc/articles/PMC6786254/ /pubmed/31608170 http://dx.doi.org/10.7717/peerj.7745 Text en ©2019 Elbrecht et al. https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited.
spellingShingle Ecology
Elbrecht, Vasco
Braukmann, Thomas W.A.
Ivanova, Natalia V.
Prosser, Sean W.J.
Hajibabaei, Mehrdad
Wright, Michael
Zakharov, Evgeny V.
Hebert, Paul D.N.
Steinke, Dirk
Validation of COI metabarcoding primers for terrestrial arthropods
title Validation of COI metabarcoding primers for terrestrial arthropods
title_full Validation of COI metabarcoding primers for terrestrial arthropods
title_fullStr Validation of COI metabarcoding primers for terrestrial arthropods
title_full_unstemmed Validation of COI metabarcoding primers for terrestrial arthropods
title_short Validation of COI metabarcoding primers for terrestrial arthropods
title_sort validation of coi metabarcoding primers for terrestrial arthropods
topic Ecology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6786254/
https://www.ncbi.nlm.nih.gov/pubmed/31608170
http://dx.doi.org/10.7717/peerj.7745
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