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.