Validation of COI metabarcoding primers for terrestrial arthropods

PeerJ. 2019;7:e7745 DOI 10.7717/peerj.7745

 

Journal Homepage

Journal Title: PeerJ

ISSN: 2167-8359 (Online)

Publisher: PeerJ Inc.

LCC Subject Category: Medicine

Country of publisher: United States

Language of fulltext: English

Full-text formats available: PDF, HTML, XML

 

AUTHORS

Vasco Elbrecht (Centre for Biodiversity Genomics, University of Guelph, Guelph, ON, Canada)
Thomas W.A. Braukmann (Centre for Biodiversity Genomics, University of Guelph, Guelph, ON, Canada)
Natalia V. Ivanova (Centre for Biodiversity Genomics, University of Guelph, Guelph, ON, Canada)
Sean W.J. Prosser (Centre for Biodiversity Genomics, University of Guelph, Guelph, ON, Canada)
Mehrdad Hajibabaei (Centre for Biodiversity Genomics, University of Guelph, Guelph, ON, Canada)
Michael Wright (Centre for Biodiversity Genomics, University of Guelph, Guelph, ON, Canada)
Evgeny V. Zakharov (Centre for Biodiversity Genomics, University of Guelph, Guelph, ON, Canada)
Paul D.N. Hebert (Centre for Biodiversity Genomics, University of Guelph, Guelph, ON, Canada)
Dirk Steinke (Centre for Biodiversity Genomics, University of Guelph, Guelph, ON, Canada)

EDITORIAL INFORMATION

Blind peer review

Editorial Board

Instructions for authors

Time From Submission to Publication: 10 weeks

 

Abstract | Full Text | Full Text

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).