Abstract We present multiPrime, a novel tool that automatically designs minimal primer sets for targeted next‐generation sequencing, tailored to specific microbiomes or genes. MultiPrime enhances primer coverage by designing primers with mismatch tolerance and ensures both high compatibility and specificity. We evaluated the performance of multiPrime using a data set of 43,016 sequences from eight viruses. Our results demonstrated that multiPrime outperformed conventional tools, and the primer set designed by multiPrime successfully amplified the target amplicons. Furthermore, we expanded the application of multiPrime to 30 types of viruses and validated the work efficacy of multiPrime‐designed primers in 80 clinical specimens. The subsequent sequencing outcomes from these primers indicated a sensitivity of 94% and a specificity of 89%.
