Abstract
The development of multiplex polymerase chain reaction and microarray assays is challenging due to primer dimer formation, unspecific hybridization events, the generation of unspecific by-products, primer depletion, and thus lower amplification efficiencies. We have developed a software workflow with three underlying algorithms that differ in their use case and specificity, allowing the complete in silico evaluation of such assays on user-derived data sets. We experimentally evaluated the method for the prediction of oligonucleotide hybridization events including resulting products and probes, self-dimers, cross-dimers and hairpins at different experimental conditions. The developed method allows explaining the observed artefacts through in silico WGS data and thermodynamic predictions. PRIMEval is available publicly at https://primeval.ait.ac.at.
Highlights
The development of multiplex polymerase chain reaction and microarray assays is challenging due to primer dimer formation, unspecific hybridization events, the generation of unspecific by-products, primer depletion, and lower amplification efficiencies
While classical microbiological methods are commonly used for the identification and characterization of pathogens, cultivation-independent genetic methods such as polymerase chain reaction (PCR) and DNA microarrays are on the rise[3]
We evaluated the three underlying methods for the number of retrieved alignments, hits, corresponding results and for the special case of oligonucleotides with degenerated bases
Summary
The development of multiplex polymerase chain reaction and microarray assays is challenging due to primer dimer formation, unspecific hybridization events, the generation of unspecific by-products, primer depletion, and lower amplification efficiencies. While classical microbiological methods are commonly used for the identification and characterization of pathogens, cultivation-independent genetic methods such as (real-time) polymerase chain reaction (PCR) and DNA microarrays are on the rise[3] These methods are only cost-effective if they are highly multiplexed, which is challenging due to primer dimer formation, the formation of unwanted by-products, the resulting lower amplification efficiencies and lower sensitivity due to primer depletion and accumulation of unspecific DNA4,5. While singleplex (e.g. Primer[3], Primer-BLAST6,7) and multiplex (e.g. oli2go8) primer design tools include specificity and/or primer dimer checks, we are not aware of a software application which performs in silico specificity checks for combined multiplex amplification and detection assays, allows user-uploaded databases, and uses thermodynamic data to predict hybridization events including thermodynamically stable but mismatched oligonucleotides. We present PRIMEval, a software workflow addressing these issues with multiple underlying algorithms accessible through a public web server
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