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

项目摘要 我们建议开发用于长阅读测序的纳米结合反应、纯化和大小选择方法 图书馆准备工作。这些方法使用我们的纳米结合磁盘来提纯酶反应和 在长阅读测序文库准备过程中，通过去除短DNA来增加阅读长度。纳米粘结盘 是直径1-5毫米的小磁盘，上面覆盖着高密度的微米和纳米结构的二氧化硅。 与磁性颗粒相比，纳米结合盘非常温和，可以防止DNA剪切和 实现DNA的高效回收，长度超过百万碱基。我们目前销售DNA提取试剂盒 这使得能够从各种各样样本中分离超高分子量(UHMW)百万碱基大小的DNA 类型。为了评估纳米结合反应纯化的温和程度，我们在超长时间内对它们进行了测试 能够产生100 kb-1+Mb长度读取的测序协议。该方法能够生成 100-200 kb范围内极高的读取长度N50，并且读取的兆数是以前的10倍 超长的方法。这一补充将允许对Nanobind进行额外的开发和测序测试 超长测序协议。该方法在生产上的成功验证将导致显著的 数据吞吐量的增强以及相应的成本、时间和人力的减少，使 通过转化为NHGRI泛基因组项目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。

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.