Strategies for sample labelling and library preparation in DNA metabarcoding studies

Research output: Contribution to journalReview articlepeer-review

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  • Kristine Bohmann
    University of Copenhagen
  • Vasco Elbrecht
    ETH Zürich
  • Christian Caroe
    University of Copenhagen
  • Iliana Bista
    Cambridge University
  • Florian Leese
    University of Duisburg-Essen
  • Michael Bunce
    Curtin University, Perth
  • Douglas W. Yu
    Chinese Academy of Sciences
  • Mathew Seymour
    Swedish University of Agricultural Sciences, Uppsala
  • Alex Dumbrell
    University of Essex
  • Simon Creer
Metabarcoding of DNA extracted from environmental or bulk specimen samples is increasingly used to profile biota 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. The latter enables assignment of the sequences back to the samples they originated from. Nucleotide identifiers can be added during the metabarcoding PCR and during “library preparation”, that is, 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, ensuring that data generation is robust and fit for the chosen purpose is critically important for 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; one-step PCR, two-step PCR, and tagged PCR. Further, we distill 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.


  • amplicon sequencing, biodiversity assessment, eDNA, environmental DNA, High-throughout sequencing, Illumina sequencing, library preparation
Original languageEnglish
Pages (from-to)1231-1246
Number of pages16
JournalMolecular Ecology Resources
Issue number4
Early online date22 Sept 2021
Publication statusPublished - May 2022

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