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

项目摘要 PacBio、牛津纳米孔和10X基因组公司的第三代和链接阅读测序技术 已经开始给一系列基因组学应用带来革命性的变化，包括从头测序、分阶段和 结构变异分析，通过提供对跨越数百个DNA的长片段进行排序的能力 从千基到兆基的长度。然而，这些能力的前提是高效地 操纵高分子量(HMW)DNA。不仅必须提取HMW DNA，而且同样重要的是 在随后的库准备过程中，它不会损坏或丢失。测序文库制备通常 需要3-4个大小选择纯化步骤来清理酶反应，并确保只有正确的 对所需插入大小的制备的文库分子进行测序。在第一阶段，我们演示了Nanobind 大小选择可以增加长阅读测序读取长度，同时显著加快文库准备和 提高图书馆的产量。建议的Nanobind Size Select技术是唯一可以提供 磁性粒子的速度与凝胶纯化的高截止值有关。而不是数百万人 对于微粒子，Nanobind使用实心磁盘(直径1-6 mm)。层叠着高密度的微细和 纳米结构二氧化硅。独特的磁盘格式保护DNA免受剪切损伤，从而导致更多 超长阅读量(&gt；100 kb)。它还实现了比任何磁性材料都宽10倍的截止范围(100 BP-10kb 粒子法或柱法。同时，Nanobind的高净化效率最多可减少19倍 适配器二聚体污染，HMW DNA回收率提高5倍，处理时间加快8.5倍。在第二阶段，我们 将创建一个单一的、统一的试剂盒，能够对高分子量gDNA文库进行大小选择纯化， 片段gDNA文库和聚合酶链式反应扩增子文库。该套件将针对更广泛的第三方 代排序应用，同时还允许将来扩展到短读NGS应用。第一, 我们将为具有更高尺寸选择的HMW gDNA文库开发纳米结合尺寸选择化学 并要求比第一阶段更低的输入浓度，然后，我们将优化化学成分以进行净化 片段DNA和聚合酶链式反应扩增子文库。最后，通过对比实验验证了Nanobind尺寸选择的有效性 现有商业产品采用长读读测序。

Project Summary 3rd generation and linked read sequencing technologies from PacBio, Oxford Nanopore, and 10X Genomics have begun revolutionizing an array of genomics applications, including de novo sequencing, phasing, and structural variant analysis, by offering the ability to sequence long stretches of DNA spanning hundreds of kilobases to megabases in length. However, these capabilities are predicated on the ability to efficiently manipulate high molecular weight (HMW) DNA. Not only must HMW DNA be extracted, it is equally important that it is not damaged or lost during subsequent library preparation. Sequencing library preparation typically requires 3 - 4 size selection purification steps to cleanup enzymatic reactions and to ensure that only properly prepared library molecules of the desired insert size are sequenced. In Phase I, we demonstrated that Nanobind Size Select can enhance long-read sequencing read lengths while dramatically speeding library preparation and improving library yields. The proposed Nanobind Size Select technology is the only method that can offer the speed of magnetic particles with the high cutoffs of gel purification. Rather than millions of individual microparticles, Nanobind uses a solid magnetic disk (1 - 6 mm dia.) layered with a high density of micro- and nanostructured silica. The unique disk format protects DNA from shear damage resulting in greater numbers of very long reads (>100 kb). It also achieved a 10X wider range of cutoffs (100 bp – 10 kb) than any magnetic particle or column method. At the same time, Nanobind’s high purification efficiency results in up to 19X less adapter dimer contamination, 5X higher recovery of HMW DNA, and 8.5X faster process time. In Phase II, we will create a single, unified kit capable of size selection purification of high MW gDNA libraries, fragmented gDNA libraries, and PCR amplicon libraries. This kit will address a broader range of 3rd generation sequencing applications while allowing future extension to short-read NGS applications as well. First, we will develop Nanobind Size Select chemistries for HMW gDNA libraries that have even higher size selection cutoffs and require lower input concentrations than in Phase I. Then, we will optimize the chemistries to purify fragmented DNA and PCR amplicon libraries. Finally, we validate Nanobind Size Select by comparing against existing commercial products using long-read read sequencing.