AssayBLAST v2: major update improving reliability and reporting of the in silico analysis of molecular multi-parameter assays

Introduction Accurate in silico evaluation of primers and probes is essential for the rational design of molecular multi-parameter assays. We present AssayBLAST v2 to automate and simplify this process for extensive assay designs. Results A newly integrated strand and proximity check enables precise validation of corresponding oligonucleotides, ensuring correct orientation and spacing required for amplification. Based on predicted oligonucleotide interactions, AssayBLAST v2 determines the theoretical amplification outcomes, offering a computational benchmark for downstream wet-lab validation and performance correlation. Additionally, the updated software integrates an adaptive BLAST parameter optimization that dynamically scales with database size, thereby improving both analytical sensitivity and computational performance. These improvements are supported by a comparative evaluation against the previous version of AssayBLAST. Conclusions Collectively, these enhancements streamline the assay development workflow, reduce costs associated with suboptimal primer and probe synthesis, and increase the robustness and reliability of molecular diagnostics and research applications.