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.
Benthic macrofauna is regularly used in monitoring programmes, however the vast majority of benthic eukaryotic biodiversity lies mostly in microscopic organisms, such as meiofauna (invertebrates < 1 mm) and protists, that rapidly responds to environmental change. These communities have traditionally been hard to sample and handle in the laboratory, but DNA sequencing has made such work less time consuming. Compared to DNA sequencing that captures both alive and dead organisms, environmental RNA (eRNA) can be used to better target alive communities. Here, we assessed the biodiversity of three known bioindicator microeukaryote groups (nematodes, foraminifera, and ciliates) in sediment samples collected at seven coastal sites along an organic carbon (OC) gradient. We aimed to investigate if eRNA shotgun sequencing can be used to simultaneously detect differences in 1) biodiversity of multiple microeukaryotic communities, and 2) functional feeding traits of nematodes. Results showed that biodiversity was lower for nematodes and foraminifera in high OC (6.2–6.9 %), when compared to low OC sediments (1.2–2.8 %). The beta diversity for all three groups were different along the OC gradient, as well as the classified feeding type of nematode genera (with more non-selective deposit feeders in high OC sediment). High relative abundant genera included nematode Sabatieria and foraminifera Elphidium in high OC, and Cryptocaryon-like ciliates in low OC sediments. Considering that future sequencing technologies are likely to decrease in cost, the use of eRNA shotgun sequencing to assess biodiversity of living benthic microeukaryotes could be a powerful tool in recurring monitoring programmes.