Strategies for sample labelling and library preparation in DNA metabarcoding studies

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.

Airborne environmental DNA for terrestrial vertebrate community monitoring

“…This was done for both markers. For each set of four PCR replicates, we used different tag combinations to lower risk of primer cross-contamination and importantly, to allow stringent filtering of sequences across each sample’s PCR replicates 67,71,72 . We employed a conservative approach in which we only retained sequences that were found in min.…”

Best practices in metabarcoding of fungi: From experimental design to results

“…The alternative strategy is to use shorter primers with only sample‐specific indexes, which are c. 30–40% cheaper and easier to amplify but require specific library preparation depending on the sequencing platform. Approaches requiring several PCR steps are also available (Figure 2; Bohmann et al, 2022), but these are more prone to contamination and chimera formation. Although vulnerable to contamination, the use of combinations of Illumina flow cell indices in the second PCR step enables ultra‐high multiplexing of samples without index‐switching bias (Holm et al, 2020).…”

Controlling critical mistag‐associated false discoveries in metagenetic data

“…Within the metagenetic literature, a number of techniques are used to append sample-specific indices during amplicon library preparation and these methods vary with regard to mistagging propensities, as reviewed in Bohmann et al (2021). Critical mistagging can be nearly eliminated through the use of ligation-based library preparation procedures (Carøe & Bohmann, 2020) or by dual-indexing samples using an identity-based matrix of forward and reverse tags, referred to as 'twin tagging' (Schnell et al, 2015;Zepeda-Mendoza et al, 2016).…”