Nanostructured Magnetic Disks for Wide Range Size Selection in NGS Library Preparation and PCR Purification

用于 NGS 文库制备和 PCR 纯化中宽范围尺寸选择的纳米结构磁盘

• 批准号: 10212466
• 负责人: Kelvin Liu
• 金额: $ 41.07万
• 依托单位: CIRCULOMICS, INC.
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-18 至 2021-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10212466
• 关键词: Biochemical Reaction; Caliber; DNA; Data; Development; Diagnostic tests; Disease; Genetic; Genome; Human Resources; Length; Libraries; Magnetism; Methods; Molecular Weight; Nanostructures; National Human Genome Research Institute; Preparation; Protocols documentation; Reaction; Recovery; Sampling; Silicon Dioxide; Structure; Testing; Time; Translations; Validation; cost; density; improved; novel diagnostics; pan-genome; particle; prevent; reference genome; therapeutic target

Project Summary We proposed to develop Nanobind reaction purification and size selection methods for long-read sequencing library preparation. These methods use our Nanobind magnetic disks to purify enzymatic reactions and to enhance read lengths by removing short DNA during long-read sequencing library preparation. Nanobind disks are small 1-5 mm diameter magnetic disks covered with a high density of micro- and nanostructured silica. Compared to magnetic particles, Nanobind disks are extraordinarily gentle, preventing DNA shearing and enabling high efficiency recovery of DNA surpassing megabase in length. We currently sell DNA extraction kits that enable isolation of ultra high molecular weight (UHMW) megabase-sized DNA from a wide variety of sample types. To evaluate how gentle the Nanobind reaction purifications are, we tested them in an ultra-long sequencing protocol capable of generating 100 kb – 1+ Mb length reads. This method is capable of generating extremely high read length N50 in the 100 – 200 kb range and 10 times more megabase reads than previous ultra long methods. This supplement will allow additional development and sequencing tests on the Nanobind ultra long sequencing protocols. Successful validation of the method on PromethION will lead to a significant enhancement in data throughput and corresponding in reduction of cost, time, and manpower, enabling an immediate impact via translation into projects such as the NHGRI pan-genome project and a long-term impact by dramatically reducing the cost of ultra long sequencing.

项目摘要 我们建议开发用于长读序测序的Nanobind反应纯化和大小选择方法 图书馆准备。这些方法使用我们的Nanobind磁盘来纯化酶促反应， 通过在长读序测序文库制备期间去除短DNA来增强读序长度。纳米结合盘 是直径1-5 mm的小型磁盘，覆盖有高密度的微米和纳米结构二氧化硅。 与磁性颗粒相比，Nanobind磁盘非常温和，可以防止DNA剪切， 能够高效回收长度超过百万碱基的DNA。我们目前出售DNA提取试剂盒 其能够从多种样品中分离超高分子量（UHMW）兆碱基大小DNA 类型为了评估Nanobind反应纯化的温和程度，我们在一个超长的 测序方案能够产生100 kb - 1+ Mb长度的读数。该方法能够生成 极高的读取长度N50在100 - 200 kb范围内，兆碱基读取比以前多10倍 超长方法该补充将允许对Nanobind进行额外的开发和测序测试 超长测序方案。该方法在异丙嗪上的成功验证将导致显著的 数据吞吐量的提高以及相应的成本、时间和人力的减少， 通过转化为NHGRI泛基因组计划等项目产生的直接影响和长期影响 通过大幅降低超长测序的成本。