A Software Tool for Reagent Design to Expand Access to Single-Nucleotide Variant Detection by the Oligonucleotide...

Area of Science:

• Molecular Biology
• Bioinformatics
• Genetics

Background:

• Single-nucleotide variants (SNVs) are crucial biomarkers for diseases and drug resistance.
• Oligonucleotide ligation assays (OLAs) offer high specificity for SNV detection but have complex design processes.
• Existing methods for SNV detection require specialized expertise and are time-consuming.

Purpose of the Study:

• To develop an automated, open-source tool (OLAgen) to streamline the design of oligonucleotide ligation assays (OLAs) coupled with PCR.
• To reduce the complexity and uncertainty associated with OLA assay development.
• To enhance the accessibility and application of OLA technology in clinical and research settings.

Main Methods:

• Development of OLAgen, an open-source software tool for automating OLA and PCR assay design.
• Sequence alignment around SNVs and generation of ligation probes with dimerization potential screening.
• Validation of OLAgen using known SNV targets (HIV-1, SARS-CoV-2, KRAS) and development of new assays for drug resistance and genetic variants.

Main Results:

• OLAgen successfully generated validated ligation probes for HIV-1, SARS-CoV-2, and KRAS.
• New assays designed by OLAgen for Mycobacterium tuberculosis drug resistance and human gene variants (JAK2, BRAF, factor V) showed 100% sensitivity and specificity.
• Proof-of-concept studies demonstrated OLAgen's ability to detect mutant frequencies as low as 1%-5% in wild-type backgrounds.

Conclusions:

• OLAgen significantly simplifies and automates the design of OLA-based assays for SNV detection.
• The tool enhances the reliability and accessibility of OLA technology for diverse applications.
• OLAgen promotes broader adoption of SNV detection methods in clinical diagnostics and biological research.