MultiPrime: A reliable and efficient tool for targeted next-generation sequencing

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%. This study introduces multiPrime, a novel tool tailored for the wide-ranging detection of target sequences through targeted next-generation sequencing (tNGS). By integrating degenerate primer design principles with effective mismatch handling, multiPrime demonstrates enhanced precision and specificity in the identification of diverse sequence types. This breakthrough presents a prospective pathway for optimizing the development of tNGS. In performance comparison, multiPrime excelled over conventional tools in terms of execution time, primer coverage, and the number of candidate primers. It offers a streamlined and versatile solution for the rapid and cost-effective detection of diverse microbiomes.Highlightsimage MultiPrime is a user-friendly and one-step tool for designing targeted next-generation sequencing primer sets.It integrates degenerate primer design theory with mismatch handling, resulting in improved accuracy and specificity in detecting broad-spectrum sequences.It outperformed conventional programs in terms of run time, primer number, and primer coverage.The versatility and potential of multiPrime are highlighted by its potential application in detecting single or multiple genes, exons, antisense strands, RNA, or other specific DNA segments.