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.

项目概要 我们建议开发用于长读长测序的 Nanobind 反应纯化和大小选择方法 文库准备。这些方法使用我们的 Nanobind 磁盘来纯化酶反应并 通过在长读长测序文库制备过程中去除短 DNA 来增强读长。纳米结合磁盘 是直径为 1-5 毫米的小型磁盘，上面覆盖着高密度的微米和纳米结构二氧化硅。 与磁性粒子相比，Nanobind 磁盘非常温和，可以防止 DNA 剪切和 能够高效回收长度超过兆碱基的 DNA。我们目前销售 DNA 提取试剂盒 能够从各种样品中分离超高分子量 (UHMW) 兆碱基大小的 DNA 类型。为了评估 Nanobind 反应纯化的温和程度，我们对它们进行了超长测试 能够生成 100 kb – 1+ Mb 长度读数的测序协议。该方法能够生成 100 – 200 kb 范围内的极高读长 N50，兆碱基读长是以前的 10 倍 超长方法。该补充品将允许在 Nanobind 上进行额外的开发和测序测试 超长测序方案。在 PromethION 上成功验证该方法将产生重大影响 提高数据吞吐量并相应减少成本、时间和人力，从而实现 通过转化为 NHGRI 泛基因组项目等项目产生直接影响，并产生长期影响 通过大幅降低超长测序的成本。