DOI: https://doi.org/10.21203/rs.3.rs-2893323/v1
Targeted next-generation sequencing (tNGS) offers a cost-effective option for pathogen diagnostics and therapeutics. Nevertheless, the design of multiplex primers constrains its wide-spectrum target coverage. Herein, we present multiPrime, which enables a more extensive coverage of pathogen spectra while maintaining high multiplexing compatibility. Our study demonstrates that multiPrime outperforms DegePrime with fewer primers and greater coverage on 8 viruses. Furthermore, multiPrime-designed primers successfully amplified all target amplicons across these viruses. We utilized multiPrime to design primers for a tNGS panel that covers 30 viruses, validating the panel's performance in 80 clinical specimens, achieving 94% sensitivity and 89% specificity.