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PRIMEval: Optimization and screening of multiplex oligonucleotide assays
Rick Conzemius1, Michaela Hendling2, Stephan Pabinger2
1Center for Health and Bioresources, AIT Austrian Institute of Technology, Giefinggasse 4, A-1210, Vienna, Austria. rick.conzemius.fl@ait.ac.at.
Scientific Reports
|December 19, 2019
Summary
We created PRIMEval, a software tool that predicts issues in multiplex PCR and microarray assays. This in silico evaluation helps explain and prevent experimental artifacts, improving assay development.
Area of Science:
- Molecular Biology
- Bioinformatics
- Assay Development
Background:
- Multiplex polymerase chain reaction (PCR) and microarray assays face challenges like primer dimer formation, unspecific hybridization, and by-product generation.
- These issues lead to primer depletion and reduced amplification efficiencies, complicating assay development and data interpretation.
- Accurate prediction of these artifacts is crucial for optimizing assay design and experimental conditions.
Purpose of the Study:
- To develop a computational workflow for the in silico evaluation of multiplex PCR and microarray assays.
- To predict and explain experimental artifacts such as primer dimers, unspecific hybridization, and hairpins.
- To provide a tool that aids in the optimization of oligonucleotide-based assay design.
Main Methods:
- Developed a software workflow comprising three algorithms with varying specificity for assay evaluation.
- Utilized in silico whole-genome sequencing (WGS) data and thermodynamic predictions for artifact analysis.
- Experimentally validated the prediction of oligonucleotide hybridization events, including self-dimers, cross-dimers, and hairpins under diverse conditions.
Main Results:
- The software workflow successfully performed in silico evaluation of multiplex assays on user-defined datasets.
- Experimental validation confirmed the method's ability to predict hybridization events and resulting artifacts.
- The developed method effectively explains observed experimental artifacts by integrating in silico WGS data and thermodynamic principles.
Conclusions:
- The PRIMEval software provides a comprehensive in silico solution for evaluating multiplex PCR and microarray assays.
- This tool aids researchers in identifying potential issues and understanding experimental artifacts before or during assay development.
- PRIMEval is publicly available, facilitating improved assay design and reliability in molecular diagnostics and research.
