Assay efficiency, specificity, and sensitivity assessment (in vitro)
This step was facilitated by the use of a High Throughput qPCR (HT-qPCR) platform (Biomark HD™, Standard BioTools Inc, USA), which enabled the time- and cost-efficient in vitro assessment of a relatively large number of makers (e.g. compared to conventional qPCR instruments). For this step, template DNA (i.e. extracted from tissue of available specimens) was normalized to 1.5 ng/µl per sample following spectrophotometric quantification with a dsDNA BR (Broad Range) assay kit using a Qubit 3.0 fluorometer (Invitrogen). In addition, to further test specificity of Mytilus and Ensis species complexes, synthetic double stranded DNA (GBLOCKS, Integrated DNA Technologies) of the relevant target genes was obtained for Mytilus galloprovincialis, Mytilus edulis, Mytilus trossulus, Ensis ensis, Ensis siliqua, Ensis leei ( synonym Ensis directus), Ensis magnus (synonym Ensis arcuatus) and standardized to 10,000 copies/µl. Additionally, to act as a universal positive control and generate standard curves for all assays tested, a six points 10-fold dilution series (ranging from 1,000,000 to 10 copies per µl) was created with an oligonucleotide pool containing synthetic double-stranded DNA fragments of all target genes/species combinations (OLIGOPOOLS, Integrated DNA Technologies)( see supplementary material Table S1) .
The HT-qPCR runs were performed using IFC controller MX and HX (for priming and loading Integrated Fluidic Controls, IFCs) and a BIOMARK HD™ real-time PCR system (Standard Bio Tools Inc, USA), following manufacturer’s recommendations, including a pre-amplification step (as per protocol QR 100-5876C2) and the actual qPCR runs using the 48.48 IFC (as per protocol QR 100-9791Rev3) or 96.96 IFC configurations (as per protocol QR 100-9792Rev3)(all protocols available at https://www.standardbio.com/support/instrument-support/biomark-ep1-support). Exceptions included the addition of of 1µg/µl BSA (Thermofisher) in the pre-amplification reaction, and subsequent PCR reaction clean up by addition of exonuclease at 4 U/reaction (New England Biolabs) and a thermocycling profile including 37°C for 30 minutes and 80°C for 15 minutes. Cleaned up products were then diluted 10-fold with TE suspension buffer (Qiagen) prior to performing the actual qPCR run on the relevant IFC controller and Biomark HD. Final thermal cycling followed manufacturer’s protocols and included a melt curve analysis (i.e. “GE Fast 48x48 PCR+Melt v2.pcl” or “GE Fast 96x96 PCR+Melt v2.pcl”). Each configuration enables the simultaneous real-time qPCR amplification (in parallel) of either 2304 (48 samples tested against 48 assays) or 9216 (96 samples tested against 96 assays) reactions. This allowed the screening of all target species’ template DNA in duplicate, the standard dilutions points in six technical replicates for each dilution point per run, including all assays (at least in duplicates) as well as 3 Internal Amplification Controls (IACs) (see supplementary materialAppendix S1 for a more detailed overview of the protocols used). Following each run, qPCR data was inspected using the Real-Time PCR analysis software version 4.5.2 (Standard BioTools Inc, USA). Output data was exported and analysed in R-studio (Posit team, version 2022.12.0+353).
To assess sensitivity of each assay, data generated by the six points 10-fold dilution series was used to estimate Limit Of Detection (LOD) and Limit Of Quantification (LOQ) for each assay following a discrete threshold as well as a curve-fitting modelling approach, as described in Klymus et al. (2020). The discrete threshold approach for LOD identifies the lowest concentration with 95% positive replicates, while for LOQ the lowest standard concentration that could be quantified with a CV value below 0.35 was selected. These CVs were modeled for exponential decay, linear, and polynomial models to select the lowest residual standard error. When possible, a threshold of CV 0.35 was used on the model to estimate the number of copies for modeled LOQ. Assay efficiency was checked according to expected theoretical values (i.e. optimal efficiency range 90-100% and a regression line slope between −3.6 and −3.3), whereas assay specificity was assessed by checking whether positive amplification (i.e. beyond the LOD) was observed in the target species and/or any off-target species, as well as by melt curve peaks comparison with a difference of >1 degree Celsius selected as the threshold for melt-curve specificity.
After calculating the efficiency, LOD and LOQ, two methods were used to quantify the detected DNA Cq-values to copy numbers. The first method which is the more conventional method, is based on the obtained slope and intercept values from the standard curve. A second method was used to recalibrate intercepts based on the efficiency values of all the assays (see supplementary material Appendix S2 for a more detailed explanation). Assuming that efficiency was accurately calculated over 5 similar concentrations for all markers in the HT-qPCR, for the second quantification method, the intercepts were plotted against the efficiency of the assays, which resulted in a significant linear model. To improve the fit of the linear equation, outliers were defined based on the standardised residuals of the linear model. More specifically, any observation with a standardised residual equal to or greater than 2 (in absolute value) was deemed to be an outlier and was removed to improve the fit. The linear model was then used to calculate an additional intercept that was used to convert Cq values into copy numbers. The assays specificity was assessed by verifying the positive reactions, grouped per sample/assay, using the melt curve and AMP values. Results were displayed by creating a heatmap showing average amplification strength in copies per/µl, with the corresponding AMP values of the same PCR reaction. Matrices for the heatmaps were created in R-studio with visualization done in Excel (Microsoft, Version 2208).
2.3. PHASE 3 –FIELD TESTING AND VALIDATION