High-throughput size-selection system for long-read sequencing library preparation

项目总结/摘要 大多数长读段DNA测序方法依赖于文库大小选择技术来控制 和/或改进读段长度。在Oxford Nanopore和PacBio测序方案中，样品 加载取决于库分子向读取器表面（波导表面）的扩散 对于PacBio，或对于Oxford的膜）。由于小分子的扩散速率高于 大分子，大小选择消除小文库片段可以大大提高文库 读取长度。常用的消除小库元素（一般<6- 10 kb）的方法， 长读段测序是制备型琼脂糖凝胶电泳（BluePippin，SageELF， 来自Sage Science，Inc.的PippinHT），和PEG沉淀（Short Read Eliminator Kit， 循环组学以及自制PEG方法）。在这些方法中，制备性凝胶 电泳在排斥小DNA分子方面是上级的，但需要专门的 仪器，具有大体积凝胶柱（6- 10 cm长）的耗材凝胶盒， 盒样品通过量和长运行时间。在许多长读序测序工作流程中， 尺寸选择是工作流程中最耗时的步骤。 随着长读段测序方法迅速成为重要的临床研究工具， 越来越引起医学检验实验室的重视--发展规模选择很重要 具有改进的尺寸范围、改进的大片段分辨率的设备和方法 尺寸（10 kb-200 kb）、更快的运行时间和每个运行周期更高的样品通量。 本申请的目的是开发新的制备型凝胶电泳系统， 利用大DNA的非线性响应：1）当响应于某些脉冲而重新定向时， 场条件，或2）当受到不同的正向和反向电压脉冲时， 实力在这两种情况下，我们设想的大小选择过程中，目标大小部分， 移动非常小，并且在样品加载位置内（或附近）恢复。如果成功，凝胶 大小和运行时间可以显著减少，从而缓解长时间读取的瓶颈 从严格的尺寸选择中受益的工作流程。我们的最终目标是一个家庭的规模 选择使用SBS格式、自动化友好型48或96孔凝胶盒的产品 样品容量

Project Summary/Abstract Most long-read DNA sequencing methods depend on library size selection techniques to control and/or improve read lengths. In Oxford Nanopore and PacBio sequencing protocols, sample loading is dependent on diffusion of library molecules to the reader surface (waveguide surface for PacBio, or membrane for Oxford). Since small molecules have a higher rate of diffusion than larger molecules, size selection to eliminate small library fragments can greatly improve library read lengths. Popular methods for elimination of small library elements (generally <6-10kb) in long read sequencing are preparative agarose gel electrophoresis (BluePippin, SageELF, PippinHT from Sage Science, Inc.), and PEG precipitation (Short Read Eliminator Kit, Circulomics, as well as home brew PEG methods). Of these methods, preparative gel electrophoresis is superior in rejecting small DNA molecules, but requires specialized instruments, consumable gel cassettes with bulky gel columns (6-10cm long) and low per- cassette sample throughputs, and long run times. In many long-read sequencing workflows, size selection is the most time-intensive step of the workflow. As long-read sequencing methods are rapidly becoming essential clinical research tools -- and increasingly attracting attention of medical testing labs -- it is important to develop size selection equipment and methods that have improved size range, improved resolution at large fragment sizes (10kb-200kb), faster run times, and higher sample throughput per run cycle. The goal of the present application is develop new preparative gel electrophoresis systems that utilize the nonlinear response of large DNAs: 1) while reorienting in response to certain pulsed field conditions, or 2) when subjected to forward and reverse voltage pulses of different strength. In both cases, we envision size selection processes in which targeted size fractions move very little and are recovered within (or near) the sample loading position. If successful, gel sizes and run times can be decreased dramatically, thereby relieving a bottleneck in long-read workflows that benefit from stringent size selection. Our ultimate goal is a family of size selection products that utilize SBS-format, automation-friendly gel cassettes with 48 or 96 well sample capacity.