Development and Testing of Species-specific Quantitative PCR Assays for Environmental DNA Applications

1. Generation of a sequence database of mitochondrial DNA sequences from target and non-target species of interest

1. Define the question, targets and system being addressed. Identify the target species for eDNA detection. Identify the geographic system in which the assay will be used. Make a list of species of interest, including the target species, sympatric (co-occurring) species within the same taxa (usually order or family level), and closely related allopatric species, those that may not be in the same geographic location as the target (Figure 1).
   NOTE: Here, the Clinch River populations of the species A. ligamentina were targeted.
2. Search and download sequences from multiple gene regions for species on the list from Step 1. Sequence databases such as NCBI (National Center for Biotechnology Information), BOLD (Barcode of Life Database), EMBL (European Molecular Biology Laboratory) and DDBJ (DNA Data Bank of Japan) can be used. NCBI, EMBL and DDBJ all share sequence information.
   1. Using NCBI’s Nucleotide Database, search for the target organism (e.g., Actinonaias ligamentina) and gene region (e.g., cytochrome c oxidase I (COI) or NADH-dehydrogenase 1 (ND1); Example search string: Actinonaias ligamentina AND ND1)
   2. Next, select all sequences that match the specifications and select Send to. Choose Complete record, File and download format as either GenBank or FASTA and then Create File. These sequences are now saved to the computer.
   3. Repeat these steps for all the species on the list defined in Step 1. Keep sequences for each gene region in a separate file as these will be analyzed separately.
      1. Download all relevant sequences (or a large, representative proportion of sequences) for the target species identified in Step 1. Include geographic variants if possible.
      2. Repeat the search and download sequences for related and sympatric non-target species of the same taxonomic group that were identified in Step 1 (e.g., if the target species is the mucket (A. ligamentina) download sequences for all other freshwater mussel species in the Family Unionidae that occur in the system of interest).
      3. Repeat the search and download for closely related but allopatric (geographically separate) species listed in Step 1.1.
         NOTE: Not all species (targets and non-targets) will be available in the public databases. Increase the local reference database by amplifying and sequencing taxonomically verified specimens of species of interest in-house. If working with a species that has high within-species genetic diversity or working in a geographically large area where geographic variants may be expected, gather sequences from across the range.

2. Assay design

1. Align sequences from each gene region separately using alignment software that can be found in various genetic sequence editing and bioinformatic programs. Do this alignment for each of the different gene regions.
   1. For instance, using Geneious Prime software (https://www.geneious.com) import the downloaded sequence files into the program.
   2. Create separate folders for each gene region.
   3. Within a folder that contains sequences from one gene region, select all the sequences.
   4. Use the Multiple alignment tool to create a nucleotide alignment of the selected sequences. There may be several options for the type of alignment, using the Geneious or MUSCLE alignments and default parameters works well.
2. Choose promising regions for assay design through the visualization of aligned sequence data. A region that has a lot of sequence data available for the species of interest, is highly divergent among species, and shows low within-species variation is a good candidate. This will increase the likelihood that primers and probes designed will be able to discriminate the target from non-target species, while also ensuring intraspecific variants will amplify with the assay.
3. Design of assay primers and probe.
   1. Use qPCR assay design software and follow instructions. IDT’s PrimerQuest Tool (https://www.idtdna.com/) to design 5 sets of qPCR assays was used here.
   2. Paste the sequence selected in step 2.2 into the Sequence entry box. If the alignment created spaces, delete those from the sequence.
   3. Select qPCR 2 Primers + Probe in the Choose Your Design option.
   4. Download the recommended assays.
   5. Copy the sequences from the forward primer of the first assay, and search for this primer sequence in the alignment created in step 2.1.4. If using Geneious Prime, use the Annotate and Predict tool to add the primer region to the alignment. Do this for all the primer and probe combinations (Figure 2).
   6. Inspect these regions of the alignment for variation within the target species as well as within the co-occurring species.
      1. If there is intraspecific genetic variation, search for assays where the primers and probe do not fall within these regions.
      2. To prevent non-target species amplification, search for mismatches with non-target species. Choose assays with the most mismatches for further validation. Currier et al. (2018) suggest choosing sets with at least two of the three regions (the two primers, or a primer and a probe) having at least two mismatches with all the non-target species. However, keep in mind that mismatches at the probe contribute less to specificity¹⁰.
         NOTE: Differences within 3 base pairs of the 3’ end of each primer increase specificity better than differences at the 5’ end of the primers¹⁰.
   7. Consider the following important parameters in assay design.
      1. Determine the melting and annealing temperatures of the primers and probe. Ideally the melting temperature (Tm) of primers should be between 60-64 °C and within 2 °C of each other, and the Tm of the probe should be 6-8 degrees higher than the Tm of the primers. Set the annealing temperature (Ta) of the qPCR reaction 5 °C below the melting temperature, around 55-60 °C¹¹.
      2. Examine the GC content. Choose between 35 – 65% GC content, and avoid regions with 4 or more consecutive Gs. Having 1 or 2 Gs or Cs in the 5 last bases of the 3’ end of the primer (GC clamp) might increase specificity as it would help the primer to make a stronger bond¹².
      3. Search for hairpin and dimer structures. Test primers and probe for predicted hairpin structures and dimers using an oligonucleotide analyzing program (e.g., OligoAnalyzer -IDT¹³; OligoCalculator¹⁴). These structures can cause non-target amplification and lower efficiency. Avoid assays which are predicted to form these structures.
      4. Determine the primer length. Aim for primers between 18-25 bases in length and probe length between 20 –25 bases. Longer primers and probes may have lower amplification efficiency.
      5. Determine the amplicon length. It should be between approximately 100 and 250 base pairs. This range is generally short enough for high PCR efficiency, but long enough for ease of verification by Sanger sequencing^4,15.
      6. Design probes. Make sure the probes do not have a G base at the 5’ end, because it could dampen the signal from green and yellow dyes¹¹. We designed double quenched probes, with IDT 3IABkFQ and ZEN quenchers and FAM or HEX fluorophores.
         NOTE: Determine the MGB probes: TaqMan MGB (minor groove binder) probes are often used for eDNA studies. However, because these probes are very short, they can bind to non-targets even with a 2 or 3 base pair mismatch¹⁰.
      7. Determine the probe Tm. Melting temperature of the probe should be 6-8°C higher than the primers. Lower temperatures decrease the binding success of the probe.
      8. Determine the probe length and location. The probe should be between 20 and 25 bp in length and ideally located close to the primer binding site on the same strand without overlapping it.

3. Assay screening and optimization

1. In silico assay development and testing. Before ordering primer-probe sets, assess specificity (potential non-target amplification) by testing primer amplification in silico.
   1. Test primers through NCBI’s Primer-Blast¹⁶ or similar programs that can identify potential non-targets in the NCBI nt/nr database that might amplify with the assay. If using Primer-Blast paste primers on the Use my own primer box under Primer parameters. In the Primer Pair Specificity Checking Parameters options, select nr as the Database and type the Order of the organism of interest (e.g., “Unionida” or “Unionoida”) in the Organism box.
   2. Continue to assess primer/probe sets visually on aligned sequence data.
      1. In order to assess primers and probes at the same time in silico, create a text string of the forward primer, 12 N’s, the probe, 12 N’s, and the reverse complement of the reverse primer. If the probe sequence is within 12 base pairs of one of the primers, use the number of N’s corresponding to the number of base pairs between the primer and probe.
      2. Use NCBI’s Nucleotide Blast search (Blastn) to search against the nr database¹⁷. Use the Taxonomy tab to look for non-target species with few mismatches; these should be tested in the laboratory during assay optimization.
         NOTE: In silico testing helps rule out non-specific assays, but potentially specific assays must be tested empirically (in vitro), as not all species have sequences in the genetic databases and primer and probes can still bind to non-targets even if deemed unlikely by the software.
2. Choose three to five primer/probe combinations to test in the laboratory.
3. Order primers, probes and a synthetic DNA standard as well as additional M13-tailed primers for amplicon sequencing.
   1. Order synthetic oligonucleotide primers and probes from a company that makes oligos. Probes are labelled with a fluorescent dye and a quencher. Different fluorophores should be selected for assays that need to be multiplexed. Check your qPCR instrument for a list of which fluorophores the instrument can detect.
   2. Design and order M13-tailed primers for verification of qPCR detections with Sanger sequencing by adding the M13 Forward (-20) sequence, GTA AAA CGA CGG CCA GT, to the 5’ end of the forward primer, and the M13 Reverse (-27) sequence, CAG GAA ACA GCT ATG AC, to the 5’ end of the reverse primer.
   3. The synthetic DNA standard contains the target sequence (including the primer regions) at a known concentration in copies/µL. Quantify unknown samples based on a curve made by known concentrations of this standard (i.e., the standard curve). Acquire the synthetic standard from the same company that manufactures the primers and probe. Follow manufacturer recommendations for resuspension and storage. Dilute standards in TE buffer with a tRNA carrier using low-retention plasticware to reduce hydrolysis and binding to surfaces.
      NOTE: If the standard curve does not perform well (poor PCR efficiency, see step 3.4.2), try re-suspending the standard in water or Tris-HCl.
   4. Suspend primers and probes in nuclease free water, Tris-HCl, or TE buffer at convenient concentrations for assay use. Generally, dilute working stocks 20-fold in the master mix to achieve the optimized final assay concentration. Store suspended oligos at a constant -20 °C when not in use.
4. In vitro (in the laboratory) assay optimization and testing. Reject assays that have poor efficiency, cross-react with co-occurring species, or have poor sensitivity¹⁸. Include the use of an internal positive control (IPC) during assay development as well as when running actual samples.
   1. First, find the optimal temperature and primer/probe concentration values for the assay. Once these parameters have been optimized for PCR efficiency (Step 3.4.2), cross-reactivity (Step 3.4.3) and sensitivity (Step 3.4.4), proceed to test the assay with a multiplexed IPC (Step 3.4.5).
      1. Test optimum annealing temperature (Ta) for primer and probes using a PCR temperature gradient centered 5° C below the predicted average primer Tm.
      2. Test optimum primer and probe concentrations. Typically, 200 nM, 400 nM, and 800 nM primer concentrations and 75 nM, 125 nM, and 200 nM probe concentrations are tested.
   2. Create a standard curve and determine efficiency and linear range. Test at least six 10-fold dilutions of a synthetic DNA standard containing the target sequence, at approximately 10⁰ copies/reaction to 10⁵ copies/reaction (Figure 3A).
      1. Use the qPCR software to plot the Cq value (threshold for cycle at quantification) of each standard on the y-axis and the log base 10 of the initial standard concentration in copies/reaction on the x-axis. The qPCR software should automatically run a linear regression (Figure 3B).
      2. Calculate the efficiency from the slope of the regression, E = -1 + 10^(-1/slope). For example, if the slope is -3.4, E= -1 + 10^(0.29) = 0.97 or 97%. Also check the r² values which indicate how well the standard replicates fit on the curve. The qPCR software should automatically calculate this as well (Figure 3B). Aim for efficiency values of 100% (±10%) and r² values of ≥0.98^{9,15,19,20,21,22}.
      3. Visually inspect the standard curve for bias, that is, deviations from the regression in a consistent direction or for poor standard curve performance as measured by efficiency and r² values (Figure 3C and 3D).
   3. Specificity: Assess cross-reactivity with non-target species to decrease the chance of false positives. Where eDNA detections may result in costly management decisions, verify positive detections by amplicon sequencing.
      1. Non-targets: Run the assay against genomic DNA extractions of taxonomically verified specimens of related species and of geographically co-occurring species; with highest priority being to test against closely related, co-occurring species. Use similar total DNA concentrations for both target and non-target samples. The concentration chosen should yield amplification from target species samples near the middle of the linear range of the standard curve. Amplification should be observed only with the target species.
      2. If non-target amplification is observed, clean and sequence the product to verify its identity. It is not uncommon to observe contamination from the target species in tissue samples of non-target species, thus all amplifications at this stage should be verified by sequencing. Reamplify cleaned amplicons from specificity tests using the M13 tailed primers and sequence with M13 primers.
         1. In the post-PCR laboratory, transfer the qPCR products to be sequenced to fresh tubes. Remove residual primers and reaction components with a clean-up kit (e.g., MinElute PCR Purification Kit).
         2. Make 1:100 dilutions of the elutions and amplify 1 µL of each for 30 cycles in a 50 µL PCR reaction with the M13 tailed primers and a high-fidelity polymerase (e.g., Phusion High-Fidelity DNA Polymerase).
         3. Run 10 µL of each reaction on a 1% agarose gel to check for a single band of the expected size. If no band is observed, increase the number of cycles or the amount of sample. If multiple bands are observed, gel purify the band of the expected size.
         4. Remove residual primers and reaction components with a clean-up kit as above and measure the DNA concentrations of the elutions.
         5. Set up sequencing reactions with the M13 primers according to the instructions of the sequencing facility.
            NOTE: Never open amplified samples in the qPCR laboratory. Prepare samples for sequencing in a laboratory dedicated to post-PCR samples.
   4. Sensitivity: Sensitivity affects the chance of false negatives, or failures to detect the target species DNA when it is present. Assess the limit of detection (LOD) and limit of quantification (LOQ) for each assay. Finally, include an internal positive control (IPC) to assess PCR inhibition of samples. Multiplex and test this IPC assay with the designed assay to ensure the two assays do not interfere with one another.
      1. LOD: Make six 4-fold serial dilutions of the synthetic DNA standard, with 8-24 replicates per standard dilution (Figure 4). Calculate the lowest initial concentration with 95% detection. LOD and LOQ plots can be generated with an LOD/LOQ calculator R script⁵.
         NOTE: Data below the LOD should not be censored. Because of the specificity of PCR, there is no lower limit for true positives. The LOD is the highest concentration below which false negatives may be expected to occur.
      2. LOQ: From the same dilution series, calculate the lowest initial DNA standard concentration quantifiable with a coefficient of variation (CV) below 35%.
         NOTE: LOD and LOQ should be reported in copies/reaction. When using a validated assay and field samples amplify below the LOQ, results should be reported as % detections rather than eDNA concentrations, because the exact concentration cannot be measured with confidence⁵.
   5. Use an internal positive control (IPC) to test for PCR inhibition. Inhibition can lead to a decrease in sensitivity and false negatives. Test the ability of the IPC assay to be multiplexed with the target assay.
      1. An IPC assay can be multiplexed with the target assay using a probe with a different reporter dye than the target assay. This IPC assay consists of a short synthetic DNA sequence from a species unrelated to the target taxa, incorporated into the qPCR master mix at a low concentration of approximately 10² copies/reaction, along with primer and probes that detect it. This lower concentration is necessary to avoid competition with the target sequence for polymerase and nucleotides²³.
      2. Compare the Cq value of the sample’s IPC template to that of the IPC template in the no template control. In this no template control (NTC), the only DNA input is that of the IPC template. The IPC template in this reaction should amplify as expected. If the IPC template in a sample amplifies at 2 or more cycles different from that of the IPC template in the NTC, the eDNA sample is inhibited. Samples that do show inhibition can be diluted 1:10 and re-tested. If a sample remains inhibited, that sample should be removed from analysis.
5. In situ assay development and testing
   1. In the laboratory: If access to the organism in the laboratory as well as sympatric species is available; take water samples from enclosures with these species, process the samples and test the assay against these eDNA samples. Sequence products as above to verify amplification of the intended target using the M13 tailed primers.
   2. In the field:
      1. Identify sites where the target organism is known to occur and known not to occur. It is preferable to have some measure of abundance at each site where the target species occurs.
      2. Decide what sample volumes and sample collection methods (e.g., filtration, centrifugation, etc.) will be used.
      3. Include a field blank or negative control at each site, this is clean water that has been brought to the field site and then collected and prepared with the same field equipment and protocols used for eDNA sampling²⁴. The purpose of the field blank is to detect contamination of the sampling equipment and field gear brought to the site. Take the field blank before processing field water samples.
      4. Take multiple water samples per site, preferably 3 samples per site.
      5. Back in the laboratory, process and extract samples.
      6. Run the assay using a plate set up similar to Figure 5A and compare eDNA concentration and detection frequency with known site differences in occurrence and abundance. Confirm all detections by sequencing^24,25.
         NOTE: The above would validate an assay through Level 4 of Thalinger et al.'s (2020) scale⁶ (optimization of the assay’s technical performance) and begin to gather data supporting Level 5 assay validation. Level 5 incorporates probability modeling and use of the assay for eDNA ecology studies. We feel this is beyond the scope of basic assay development, but we encourage these applications of laboratory and field vetted assays to improve assay design and data interpretation.