Abstract
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.
Highlights
The analysis of environmental DNA and DNA extracted from bulk specimen samples has experienced an enormous surge in popularity in basic and applied biodiversity studies seeking to detect e.g., animal, plant, algae, fungi, and bacteria (Bálint et al, 2016; Compson et al, 2020; Creer et al, 2016; Jarman et al, 2018; Lindahl et al, 2013; Taberlet et al, 2012)
The applications of metabarcoding range widely; for example, detection of invasive species (e.g., Pochon et al, 2013); assessment of water quality via identification of freshwater invertebrates in bulk specimen samples (e.g., Elbrecht et al, 2017) and environmental samples (e.g., Seymour et al, 2020); identification of plant-pollinator interactions (e.g, Gous et al, 2019; Lucas et al, 2018); detection of vertebrate wildlife via invertebrate “samplers” of vertebrate blood or faeces (e.g., Calvignac-Spencer et al, 2013), and assessment of for example, niche partitioning (e.g., Razgour et al, 2011) and ecosystem services (e.g., Aizpurua et al, 2017) through detection of diet items
Metabarcoding relies on PCR amplification of extracted DNA with primers designed to target a taxonomically informative marker for a selected taxonomic group (Taberlet, Coissac, Pompanon, et al, 2012) (Figure 1)
Summary
Strategies for sample labelling and library preparation in DNA metabarcoding studies. Kristine Bohmann1 | Vasco Elbrecht2 | Christian Carøe1 | Iliana Bista3,4 | Florian Leese5 | Michael Bunce6 | Douglas W. Yu7,8,9 | Mathew Seymour10 | Alex J. Funding information NERC Biomolecular Analysis Facility, Grant/Award Number: NE/M02086X/1, NE/N003756/1, NE/N006216/1, NE/ S000291/1, NE/S005560/1 and NE/ S006958/1; Carlsbergfondet, Grant/ Award Number: CF18-1110; H2020 European Research Council, Grant/ Award Number: 856488; Det Frie Forskningsråd, Grant/Award Number: 5051-00140; Wellcome, Grant/Award Number: WT207492, 104640/Z/14/Z and 092096/Z/10/Z
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