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

Macrobenthos is a good indicator to evaluate the potential effects of human activities on marine benthic ecosystem. In environmental impact assessments (EIAs), macrobenthic species identification typically based morphological characteristics, time-consuming and labor-intensive process for which specific taxonomic knowledge experts are needed. DNA metabarcoding can circumvent most these shortcomings. However, be applicable in EIAs adapted by policy, standardized protocol that allows reproducible reliable results prerequisite. Here, three research questions were investigated as part international Interreg NSR project GEANS: 1) “How many replicates extractions PCR products needed capture sample?”, 2) “Is ‘fixed’ repeatable across different institutes?” 3) “What small changes this alpha diversity?”. These important steps convince stakeholders efficient quick method generates comparable results. First, variation technical was biological from communities Belgian Part North Sea (BPNS) with high, medium low diversity. For each replicate, six taken one used assess between amplification. Three locations high diversity pick up at least 80% present replicates, while four location Variation detected illustrating importance including lab protocol. Second, we conducted ring test where subsamples 12 bulk macrobenthos samples, originating BPNS (differing density diversity), distributed institutes located Belgium, Netherlands, Germany Denmark. Samples processed using same resulting datasets bioinformatically institute. The number ASVs reflected patterns, i.e. highest values highly diverse community, lowest numbers intermediate samples community. patterns identical institutes, showing repeatability when total, 100 through metabarcoding, 60 picked all 0-14 recorded only institutes. Beta also nMDS plot clearly showed clustering communities, independent institute work. Finally, (different extraction kit, fidelity polymerases amplification, reagents clean-up) resulted minor diversity: similar fixed 70% - 75% shared adjusted protocols. This study shows first time offers assessment beta supports suitability monitoring macrobenthos. valuable establish harmonized uniform protocol, institutions Europe implemented new standard