Abstract
Single-molecule sequencing (SMS), a long-read DNA sequencing technology, plays a crucial role in genomics research. However, traditional ultrasonic shearing techniques struggle to efficiently produce DNA fragments ≥10 kbp, limiting the efficiency of SMS library preparation. Here, we developed a zirconia bead-mediated ultrasonic shearing method that enables precise DNA fragmentation through zirconia bead mechanical agitation induced by sonication cavitation. By optimizing parameters such as zirconia bead size, quantity, ultrasonic probe distance, ultrasonic time, water bath temperature, DNA sample volume, and DNA concentration, we obtained target fragments in the 10-20 kbp range. The results demonstrated that this method sheared purified λDNA (48.5 kbp) into fragments averaging 15 kbp within 20 seconds, achieving performance comparable to commercial g-TUBE methods. The method was also successfully applied to human genomic DNA. This simple, rapid and reliable DNA fragmentation method provides an effective solution for SMS library preparation with great potential for molecular detection and diagnostic applications.