Strategies for sample labelling and library preparation in DNA metabarcoding studies

Bohmann, Kristine, Elbrecht, Vasco, Carøe, Christian, Bista, Iliana, Leese, Florian, Bunce, Michael, Yu, Douglas W., Seymour, Mathew, Dumbrell, Alex J. and Creer, Simon (2022) Strategies for sample labelling and library preparation in DNA metabarcoding studies. Molecular Ecology Resources, 22 (4). pp. 1231-1246. ISSN 1755-098X

[thumbnail of Published_Version]
PDF (Published_Version) - Published Version
Available under License Creative Commons Attribution.

Download (2MB) | Preview


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.

Item Type: Article
Additional Information: Research Funding: NERC Biomolecular Analysis Facility. Grant Numbers: NE/M02086X/1, NE/N003756/1, NE/N006216/1, NE/S000291/1, NE/S005560/1, NE/S006958/1; Carlsbergfondet. Grant Number: CF18-1110; H2020 European Research Council. Grant Number: 856488; Det Frie Forskningsråd. Grant Number: 5051-00140; Wellcome. Grant Numbers: WT207492, 104640/Z/14/Z, 092096/Z/10/Z
Faculty \ School: Faculty of Science > School of Biological Sciences
Related URLs:
Depositing User: LivePure Connector
Date Deposited: 14 Oct 2021 01:13
Last Modified: 23 Oct 2022 03:07
DOI: 10.1111/1755-0998.13512


Downloads per month over past year

Actions (login required)

View Item View Item