Specificity
Each species/assay combination (26 *42 = 1092 combinations) yielded at least four qPCR data points (i.e. assay in duplicate tested against the target in duplicate). When assessing assay performance, consistency in amplification across all four qPCR reactions was considered. The overall results are shown in Figure 2 . In total, separating Bivalvia and Malacostraca, AMP values were generated for 276 reaction combinations. 12 of these reactions showed minor but positive amplification when the AMP value was below 0.3. 3 out of 55 reactions were positive with AMP values of 0.3-0.4 whilst 0 out of 5 reactions with AMP-values of 0.5 amplified. Of the higher AMP-values,Mytilus edulis assay reacted positively on the obtained M. trossulus specimen from Scotland, however, the obtained oligo based on reference data remained negative resulting in it being considered an artifact. The M. galloprovincialis assay amplified M. edulis target (100000 copies) <10 copies (AMP=0.7). Following removal of the below 0.2 AMP value reactions (as they were considered artifacts), of the 42 assays tested 38 showed satisfactory levels of specificity to the intended target species, whereby they either amplified only the target or produced a distinct melt temperature. The 4 remaining assays amplified other targets with similar melt curves, indicating a lack of specificity. Out of these 4 assays, 2 assaysHamericanus COI, and Oedulis 16S showed substantial amplification in other closely related off targets (signal strength above 10 copies/µL). The other 2 assays (M. galloprovincialis(PAPM), Mytilus 3 (18S) showed positive but weak amplification (signal strength below 10 copies/µL). Both Mytilus andEnsis species complexes could be successfully resolved based on inspecting melt curves and cross-referencing amplification (or lack thereof) of multiple assays. Specifically, the Mytilus markers on the PAPM gene showed a range of melt curves, including 81°C (Mytilus edulis ), 79-80°C (Mytilus trossulus ), and 79°C (Mytilus galloprovincialis ). Similarly, the Ensis species (COI) marker generated distinct melt curves at 75°C (Ensis siliqua ) and 77°C (Ensis ensis ), hence providing some level of resolution within these species complex. Based on the highest AMP-values tested, detected and the in vitro testing 29 out of 42 assays were found to have no risk of false positives (Table 3,supplementary Table S6 ).. A total of 6/42 assays were found to have a risk that could be eliminated by another assay present on the panel. Furthermore, a total of 4/42 assays were considered non-specific and therefore the detection of C. pagarus and C. glaucumcannot be confirmed if the absence of Liocarcinus depurator andParvicardium exiguum is uncertain. The remainder of the target species could be successfully detected by the newly developed HT-qPCR panel (Table 3, supplementary Table S6 ).