hybseek: Pathogen primer design tool for diagnostic multi-analyte assays

Due to recent advances in genome sequencing, the detection of pathogens by DNA signatures, i.e. by oligonucleotide sequences that uniquely identify a specific genome, is becoming increasingly popular in modern clinical diagnostics. However, currently available screening methods, such as PCR and microarrays, lack multiplexing and sensitivity, respectively. Solid-phase amplification (SPA) is an emerging approach with the potential to overcome these limitations. SPA-based diagnostic assays require both pathogen-specific and compatible primer pairs for many, often closely related pathogens. Currently, none of the available tools supports an automated design of such primer sets, making it an iterative, labor-intensive, and often difficult procedure. Here we describe hybseek, a Web interface for efficient design of both pathogen-specific and compatible primer pairs for DNA-based diagnostic multi-analyte assays. hybseek achieves pathogen-specificity by selecting only candidates with unique 3^′ subsequence, and the degree of this uniqueness is quantitatively expressed by a specificity score. qPCR experimental data confirm the feasibility of our design strategy. The service is freely available at https://www.hybseek.com.