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Strategies for sample labelling and library preparation in DNA metabarcoding studies
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  • Kristine Bohmann,
  • Vasco Elbrecht,
  • Christian Carøe,
  • Iliana Bista,
  • Florian Leese,
  • Michael Bunce,
  • Douglas W. Yu,
  • Mathew Seymour,
  • Alex Dumbrell,
  • Simon Creer
Kristine Bohmann
University of Copenhagen Faculty of Health and Medical Sciences
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Vasco Elbrecht
ETH Zurich
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Christian Carøe
University of Copenhagen Faculty of Health and Medical Sciences
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Iliana Bista
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Florian Leese
University of Duisburg-Essen
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Michael Bunce
Curtin University
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Douglas W. Yu
University of East Anglia
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Mathew Seymour
Swedish University of Agricultural Sciences
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Alex Dumbrell
University of Essex
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Simon Creer
Bangor University
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Abstract

Metabarcoding of DNA extracted from environmental or bulk specimen samples is increasingly used to detect plant and animal taxa in basic and applied biodiversity research because of its targeted nature that allows sequencing of genetic markers from many samples in parallel. To achieve this, PCR amplification is carried out with primers designed to target a taxonomically informative marker within a taxonomic group, and sample-specific nucleotide identifiers are added to the amplicons prior to sequencing. This enables assignment of the sequences back to the samples they originated from. Nucleotide identifiers can be added during the metabarcoding PCR and/or during ‘library preparation’, i.e. when amplicons are prepared for sequencing. Different strategies to achieve this labelling exist. All have advantages, challenges and limitations, some of which can lead to misleading results, and in the worst case compromise the fidelity of the metabarcoding data. Given the range of questions addressed using metabarcoding, the importance of ensuring that data generation is robust and fit for purpose should be at the forefront of practitioners seeking to employ metabarcoding for biodiversity assessments. Here, we present an overview of the three main workflows for sample-specific labelling and library preparation in metabarcoding studies on Illumina sequencing platforms. Further, we distil the key considerations for researchers seeking to select an appropriate metabarcoding strategy for their specific study. Ultimately, by gaining insights into the consequences of different metabarcoding workflows, we hope to further consolidate the power of metabarcoding as a tool to assess biodiversity across a range of applications.

Peer review status:IN REVISION

28 Apr 2021Submitted to Molecular Ecology Resources
10 May 2021Assigned to Editor
10 May 2021Submission Checks Completed
19 May 2021Reviewer(s) Assigned
30 Jun 2021Review(s) Completed, Editorial Evaluation Pending
07 Jul 2021Editorial Decision: Revise Minor