Digital PCR multiplex assay compatibility checker
Evaluate a set of QIAGEN dPCR assays for fluorophore compatibility, risk of heterodimer formation and amplicon conflicts to determine if they will work together in a multiplex analysis.
Catch conflicts in your dPCR multiplex before you order
Add up to 12 dPCR assays, and we'll check their compatibility for multiplexing.
Add your assays to get started
What is the dPCR multiplex assay compatibility checker?
The dPCR multiplex assay compatibility checker screens combinations of QIAGEN catalog dPCR assays for the failure modes that commonly compromise multiplex experiments and outputs a prediction of assay compatibility. The prediction is based on the ratings assigned to each evaluated failure mode, providing an overall assessment of the likelihood that a given assay combination will perform successfully in a multiplex reaction. Enter the target name or GeneGlobe catalog number for each assay and the checker will evaluate:
- Fluorophore channel conflicts: Assays sharing an optical channel produce overlapping signals (spectral crosstalk) that the instrument can’t separate. The checker flags conflicts by assay type. A maximum of two assays of the same color can be combined using amplitude multiplexing; however, this requires assay-specific optimization and validation to ensure they work together reliably.
- Heterodimer formation risk: Primers and probes from different assays can cross-hybridize, reducing the effective primer concentration or generating false positive-signals. The checker evaluates thermodynamic binding efficiency across all primer and probe combinations.
- Amplicon overlap:Oligos from different assays can bind to non-target amplicons and cause cross-priming, potentially generating false-positive partitions. The checker evaluates all assay oligos against the amplicon sequences of every other assay to identify potential cross-priming interactions across all assay combinations.
- Amplicon length: Significant size differences between amplicons can cause shorter targets to amplify more efficiently than longer ones, resulting in lower RFU values for the longer-target assay, especially in partitions where multiple targets are amplified simultaneously. The checker ensures that all amplicons in a multiplex reaction are within 150 bp of each other to help maintain sufficient RFU separation between positive and negative partitions for all assays.
- Portfolio mix: The checker flags when a multiplex set combines assays from more than one QIAGEN dPCR assay product line. In principle, assays from different portfolios can be combined and are expected to work together. However, product portfolios have different optimal cycling conditions, and multiplexing may require optimization to achieve optimal performance. The warning serves as a prompt to confirm that the combination is intentional and that any necessary optimization has been considered.
Supported assay types
Three QIAGEN dPCR assay portfolios are covered:
- Copy number variation analysis: dPCR CNV Probe Assays
- Mutation analysis: dPCR LNA Mutation Assays
- Microbial analysis: dPCR Microbial DNA Detection Assays
How to use the dPCR multiplex compatibility tool
- Click "Add assay" and start typing your target gene, organism or GeneGlobe ID number.
Alternatively, click "Import or paste a list" to add multiple assays by GeneGlobe ID number at the same time. - If needed, narrow the dropdown results by filtering for CNV, microbial or mutation assays.
- Select your assay of interest from the dropdown and repeat for each assay in your multiplex. Add up to 12 assays.
- Click "Check compatibility". Review the overall result – rated Fully Compatible through Not Compatible – and the results for each of the five parameters. Each flagged check shows the reason and how to resolve it.
What the compatibility result means
The checker provides two types of results: an overall compatibility assessment and detailed results for each of the five parameters evaluated.
Overall compatibility summarizes the combined results of all five checks and is reported as one of three categories: Strong Compatibility, Moderate Compatibility, or Weak Compatibility. These categories indicate a high, moderate, or low likelihood that the selected assays will work together successfully in a multiplexing reaction.
Per-check results return one of three states for each parameter:
- Pass: No issues detected for this check
- Warning: A potential issue that may be acceptable depending on your application; review before ordering
- Error: A conflict that must be resolved before this assay combination can be multiplexed
When a check fails, here's what to do:
- Fluorophore error: A channel has more assays than it supports. Change the dye for some of the assays to resolve the conflict.
- Amplicon length error: The size spread across your assays exceeds 150 bp. This does not necessarily indicate incompatibility. However, to minimize the risk of suboptimal signal separation, the outlier assay can be replaced with one whose amplicon size is closer to those of the other assays in the multiplex set.
- Amplicon overlap error: Two or more assays share amplicon sequence regions. Remove or replace the assay with the highest overlap risk.
- Heterodimer error: One or more assay pairs show cross-hybridization risk. Remove or replace the affected assay.
- Portfolio warning: Your panel combines assays from more than one product line. Confirm this is intentional before ordering.
Frequently asked questions
What makes two dPCR assays compatible for multiplexing?
Two dPCR assays are compatible for multiplexing when they use different fluorophore channels, show low heterodimer formation risk and target non-overlapping amplicon regions. QIAGEN dPCR catalog assays are designed with these criteria in mind. Use the checker above to evaluate any combination from the CNV, mutation or microbial assay portfolios before running your experiment.
What fluorophores work with QIAcuity digital PCR?
The QIAcuity instrument detects fluorescence across six standard optical channels: Green, Yellow, Orange, Red, Crimson and Far Red.
The dyes available depend on the assay product line.
dPCR CNV Probe Assays (gene of interest) are available with FAM, Atto 550, Atto 700* and Cy5, while the reference and centromeric reference assays are available with HEX and ROX. These assays support multiplex analysis of up to 12 targets in a reaction using amplitude multiplexing and the QIAcuity High Multiplex Probe PCR Kit.
dPCR Microbial DNA Detection Assays are available with six dyes: FAM, HEX, ROX, TAMRA, Cy5 and Atto 700* and support multiplex analysis of up to 12 targets in a reaction using amplitude multiplexing and the QIAcuity High Multiplex Probe PCR Kit.
dPCR LNA Mutation Assays use a duplex probe design that detects both the mutant and wild-type allele in the same reaction. Three dye combinations are available: FAM + HEX, Atto 550 + ROX, or Cy5 + Atto 700* for multiplexing of up to 3 targets in a reaction.
*Assays with Atto 700 require the use of the QIAcuity High Multiplex Probe PCR Kit.
What is amplitude multiplexing in digital PCR?
Amplitude multiplexing distinguishes two assays running in the same fluorophore channel by signal amplitude rather than emission wavelength (color multiplexing). In a dPCR nanoplate, partitions containing a high-amplitude assay produce a different fluorescence cluster than partitions containing a low-amplitude assay. The QIAcuity Software Suite separates these clusters automatically. QIAGEN uses amplitude multiplexing in the microbial dPCR portfolio to achieve 12-plex from six fluorophores and six optical channels.
How do I check for primer-dimer formation in a multiplex dPCR assay?
Primer-dimer formation in multiplex dPCR occurs when primers from different assays form off-target duplex structures, reducing amplification efficiency and generating false-positive partitions. The dPCR Multiplex Assay Compatibility Checker screens every primer and probe sequence in your assay combination computationally for heterodimer formation risk. For critical applications, confirm the result with a wet-lab titration: run each assay alone, then run the combination and compare positive-partition counts and cluster positions in the QIAcuity Software Suite.