NEBNext UltraShear® Long Read：用于临床相关样本长读长测序的酶法DNA片段化

Advances in long read sequencing technologies have significantly improved accuracy and throughput, making them useful for clinical applications and personalized medicine. Ultra-long DNA molecules resulting from high quality extractions, if left intact, are generally inefficiently converted into libraries and sequenced on long-read platforms. Therefore, fragmentation is strongly recommended for high-throughput long read sequencing. DNA fragmentation upstream of long read sequencing typically also results in comparable fragment sizes across samples and experiments with higher and consistent N50 lengths as well as improved sequencing yields compared to ultra-long DNA. Current methods for DNA fragmentation include mechanical shearing and enzymatic fragmentation. Mechanical shearing (e.g., Covaris ® g-TUBE and Megaruptor ® ) is the gold standard method for DNA fragmentation upstream of long read library preparation. Drawbacks to these methods include the use of expensive consumables and instruments that are not automation-friendly and result in sample loss. In contrast, enzymatic DNA fragmentation does not require expensive instruments and is automation friendly, however existing fragmentation parameters may not be optimal for long read sequencing. To address these constraints, we developed a novel enzymatic fragmentation solution, NEBNext UltraShear ® Long Read (UltraShear LR), that is quick, tunable, and automation friendly. Enzymatic fragmentation is time-dependent and can be used to generate a wide-range of DNA fragment sizes (2 to 30 kb) suitable for different applications and sequencing platforms. UltraShear LR is robust across a wide range of gDNA input amounts (250 to 5,000 ng) as well as different gDNA samples and species (e.g., animal, plant and human). Here we demonstrate that UltraShear LR fragmentation generates high quality libraries with tunable read lengths by sequencing on Oxford Nanopore™ Technologies and PacBio ® platforms. UltraShear LR libraries retain base modifications (including CpG methylation) and identify more CpGs than mechanically sheared libraries at the same read count. Additionally, we applied the Twist cancer hotspot long read sequencing capture panel using matched normal and tumor samples to identify copy number variations and perform variant calling, demonstrating the applicability of UltraShear LR for clinically relevant samples. The UltraShear LR system generates time-dependent DNA fragment sizes that are reproducible in a robust and cost-effective manner. UltraShear LR overcomes many limitations of mechanical shearing methods by simplifying sample processing, increasing throughput, and preserving base modifications. These advantages collectively improve usability and data quality in long read sequencing library preparation, making the approach well-suited for clinical applications and personalized medicine.