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 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.

Airborne environmental DNA for terrestrial vertebrate community monitoring

“… 27 To enable identification of potential contamination, we included negative controls in all steps of the workflow, including samples from the air in the DNA extraction lab. To reduce risk of PCR-introduced artifacts, we used the so-called tagged PCR approach 28 and only carried out one PCR amplification step prior to sequencing. A crucial step to ensure authenticity was the inclusion of four differently tagged PCR replicates per sample, which allowed stringent filtering of sequences across each sample’s PCR replicates.…”

Using DNA Metabarcoding to Identify Floral Visitation by Pollinators

“…Although each step of the pollen metabarcoding process has a range of different approaches, only certain elements of the entire pollen metabarcoding workflow have been reviewed [118,125,130], leaving a large proportion of the study design to the authors' discretion. Without a standardisation of approaches, comparison of results across multiple studies must be interpreted with caution.…”

“…These unique identifiers allow hundreds or thousands of samples to be pooled for sequencing (multiplexing), significantly increasing the capacity of one sequencing run. Methods for indexing of samples occur either during the initial PCR through nucleotide additions to amplicons or through a secondary PCR along with adapters to allow successful sequencing (library indices) (reviewed in [130]). Each of the methods comes with tradeoffs between many factors, mainly the risk of cross-contamination, efficiency of PCR and overall cost [130].…”

“…Methods for indexing of samples occur either during the initial PCR through nucleotide additions to amplicons or through a secondary PCR along with adapters to allow successful sequencing (library indices) (reviewed in [130]). Each of the methods comes with tradeoffs between many factors, mainly the risk of cross-contamination, efficiency of PCR and overall cost [130]. The two-step PCR approach is most widely used in pollen metabarcoding studies (Supporting Information), allowing a cost-effective approach to sample labelling whilst allowing effective detection of cross-contamination, but comes with the caveat of increased risk of biases due to an additional amplification stage [130].…”

“…Each of the methods comes with tradeoffs between many factors, mainly the risk of cross-contamination, efficiency of PCR and overall cost [130]. The two-step PCR approach is most widely used in pollen metabarcoding studies (Supporting Information), allowing a cost-effective approach to sample labelling whilst allowing effective detection of cross-contamination, but comes with the caveat of increased risk of biases due to an additional amplification stage [130].…”