Testing repeatability, testing repeatability, testing repeatability: harmonization of the DNA metabarcoding protocol for macrobenthos across Europe

Environmental impact assessments (EIAs) are performed to evaluate the potential effects of human activities on marine benthic ecosystems. Traditionally, taxonomic experts identify macrobenthic species based on morphological characteristics, a time-consuming and labor-intensive process for which specific taxonomic knowledge is needed. DNA-based monitoring methods can circumvent most of these shortcomings. However, a standardized protocol creating reproducible and reliable DNA metabarcoding results is a prerequisite for use of DNA metabarcoding in EIAs. Specific changes in the lab protocol, such as the choice of the DNA extraction kit, primer pair or PCR conditions, can influence diversity estimates. So far, no study has investigated whether a certain ‘fixed’ DNA metabarcoding protocol is repeatable across different institutes, while this is important to convince stakeholders that the new method generates reliable and comparable results regardless of who is conducting the work.
To this end, we conducted a ring test where subsamples of the same bulk samples (12 in total), originating from four macrobenthic communities (three biological replicates) in the Belgian Part of the North Sea, were distributed to four different institutes located in Belgium, Germany, the Netherlands and Denmark. Subsamples were processed by each institute following the same laboratory protocol. Each institute extracted DNA from the subsample and a portion of the mitochondrial cytochrome c oxidase subunit I (COI) gene was amplified and sequenced using Illumina Miseq. The resulting sequences were processed by the DADA2 pipeline to generate amplicon sequence variants (ASVs) and a custom-made reference database was used to assign taxonomy to these ASVs. Alpha and beta diversity patterns were compared between institutes. The number of ASVs and the number of species reflected the morphological diversity patterns, with highest values in the samples of the high diversity location, lowest numbers in the samples of the low diversity location and intermediate values in the samples of the locations with medium diversity. These patterns were identical between the four datasets, showing high repeatability between institutes. Of the 100 macrobenthic species that were detected in total, 60 species were picked up by all four institutes, while 0-14 species were only found by one institute. Next to similar alpha diversity, the nMDS plot clearly showed clustering based on the different macrobenthic communities, independent of which institute conducted the work.
This study shows for the first time that DNA metabarcoding offers a highly repeatable assessment of alpha and beta diversity patterns for macrobenthic communities which supports the use of DNA metabarcoding for monitoring marine macrobenthos. In a next step, we have used the same 12 samples to test the robustness of DNA metabarcoding by changing some steps in the lab protocol as well as the impact of different bioinformatics pipelines for macrobenthos diversity estimates. Together, our results have high value to harmonize the DNA metabarcoding method across different institutes in Europe and to implement the DNA metabarcoding method for EIAs.