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

项目摘要 PacBio、Oxford Nanopore和10 X Genomics的第三代和连接读段测序技术 已经开始彻底改变一系列基因组学应用，包括从头测序、定相和 结构变异分析，通过提供对跨越数百个DNA序列的长片段进行测序的能力， 长度从10亿到10亿。然而，这些能力的前提是能够有效地 操纵高分子量（HMW）DNA。不仅要提取HMW DNA， 在随后的文库制备过程中不会损坏或丢失。测序文库制备通常 需要3 - 4个大小选择纯化步骤来净化酶促反应， 对制备的具有所需插入片段大小的文库分子进行测序。在第一阶段，我们证明了Nanobind 尺寸选择可以增强长读段测序读段长度，同时显著加快文库制备和测序。 提高文库产量。提出的纳米结合尺寸选择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。而不是数以百万计的个人 Nanobind使用固体磁盘（1 - 6 mm直径）。分层高密度的微和 纳米结构二氧化硅。独特的磁盘格式可保护DNA免受剪切损伤，从而导致更多的 非常长的读段（>100 kb）。它还实现了10倍宽的截止范围（100 bp - 10 kb）比任何磁性 颗粒或柱方法。与此同时，Nanobind的高净化效率可将 衔接子二聚体污染，HMW DNA回收率提高5倍，处理时间缩短8.5倍。在第二阶段，我们 将创建能够对高MW gDNA文库进行大小选择纯化的单一统一试剂盒， 片段化gDNA文库和PCR扩增子文库。该套件将解决更广泛的第三代 这将有助于第二代测序应用，同时也允许将来扩展到短读NGS应用。第一、 我们将为HMW gDNA文库开发具有更高尺寸选择性的纳米结合尺寸选择化学 截止值，并且需要比阶段I更低的输入浓度。然后我们会优化化学成分 片段化DNA和PCR扩增子文库。最后，我们通过比较 使用长读段测序的现有商业产品。

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.