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ATLAS Deliverable 3.5: Potential and limits of metabarcoding of eDNA and qPCR

Carlsson, J

Environmental (e)DNA methods (quantitative PCR and metabarcoding) are non-invasive, rapid and cost-efficient tools for detecting single species and monitoring biodiversity with considerable potential for informing aquatic conservation and management. Methods for implementing eDNA are constantly developing and these tools have received significant interest from industry. There have been substantial efforts to develop best practice approaches, standardisation and workflows (c.f. COST-action DNAqua-Net CA15219) that might ultimately complement or replace existing methods and develop new metrics for the implementation of the European Water Framework Directive. These eDNA based methods also have the potential to contribute to the implementation of the Marine Strategy Framework Directive. While the use of eDNA in freshwater has received by far the most attention, there is great potential for using eDNA in the marine environment to address a wide range of questions using non-invasive sampling; ranging from spatial and temporal biodiversity assessments, to assessing distribution patterns and range expansions/contractions of single species. In the ATLAS project, WP3 focused on evaluating the accuracy and sensitivity of meta-barcoding and qPCR methods with the objective of selecting a set of primers amplifying distinct DNA fragments to optimise metabarcoding across the Tree of Life, covering a maximum number of lineages, and developing species-specific probes for PCR detection of VME indicator species, fishery targets and bycatch species.
The emergence of eDNA tools to assess marine biodiversity and detect specific target marine species has generated great hopes to describe biodiversity of ecosystems that have been difficult to access (e.g. deep-sea habitats); as sampling of water and sediments is relatively simple as compared to traditional methods requiring specialised equipment (ROV, camera sledges, fishery gear, etc.). Nevertheless, few examples of such applications existed and even fewer, if any, in the deep sea. The great challenges for using eDNA techniques to assess deep-sea biodiversity are to obtain DNA from more or less blindly collected, low biomass taxa and subsequently low DNA concentration seawater or sediment samples. University College Dublin and IFREMER were tasked with evaluating the performance of next-generation genomic tools (metabarcoding of eDNA) for assessing biodiversity and quantitative qPCR (plankton samples) as a sensitive tool to detect and quantify biomass of target species. The accuracy and sensitivity of metabarcoding and qPCR will be validated on samples assessed using classical taxonomy in selected Case Studies. In respect of the development of qPCR assays, six target species were selected for assay development. Quantitative (q)PCR assays successfully detected and semi-quantified five target species showing that despite extremely low DNA concentration and the large volumes of water in which these species are found, eDNA is a very sensitive tool offering a promising method for detection of target species in the marine environment, including the deep sea. However, it was not possible to develop an assay for Lophelia pertusa due to low polymorphism usually encountered at mitochondrial DNA for scleractinian corals, and the lack of existing sequence data from closely related species. The metabarcoding efforts by IFREMER resulted in the development of six complementary sets of primers capable of assessing biodiversity from deep-sea sediments across the entire Tree of Life. In general, metabarcoding protocols were capable of characterising biodiversity of low biomass deep-sea sediments; even for understudied deep-sea metazoan taxa. These protocols were applied to 350 samples collected during the ATLAS-MEDWAVES cruise, demonstrating the sensitivity of metabarcoding in deep-sea habitats. The development of eDNA species-specific assays and metabarcoding methods demonstrate the utility of eDNA-based methods for assessing and managing deep-sea biodiversity. Further, in line with the successful deployment of these tools in freshwater and in marine waters as demonstrated in this WP, these approaches could also be used to supplement or replace traditional methods such as morphology-based biodiversity used in the marine environment, including in the deep sea where specimens can be extremely small and difficult to identify (c.f. Danovaro et al. 2016). Similarly, future and current applications of eDNA include biodiversity assessments and baselines for Environmental Impact Studies of deep-sea industry operations such as mineral extraction (Boschen et al. 2016). We have demonstrated the usefulness of eDNA methods in the deep sea despite the great challenges they represent in terms of accessing samples and often low concentration of biomass. 

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