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

项目摘要/摘要 大多数长阅读DNA测序方法依赖于文库大小选择技术来控制 和/或改善读取长度。在牛津纳米孔和PacBio测序方案中，样本 加载依赖于库分子向读取器表面(波导面)的扩散 用于PacBio，或用于牛津的膜)。因为小分子的扩散速度比 选择较大分子、大小的文库片段消除小片段可以大大提高文库 阅读长度。中消除小文库元素(通常为&lt；6-10kb)的流行方法 长阅读测序是制备性琼脂糖凝胶电泳法(BluePippin，SageELF， 来自Sage Science，Inc.的PippinHT)和聚乙二醇沉淀(短读消除器工具包， 循环组学，以及国产BREW聚乙二醇法)。在这些方法中，制备凝胶 电泳法在排除小DNA分子方面具有优势，但需要专门的 仪器、消耗性凝胶盒，凝胶柱体积大(6-10厘米长)，密度低. 磁带样本吞吐量，运行时间长。在许多长时间阅读的排序工作流中， 尺寸选择是工作流程中最耗时的步骤。 随着长阅读测序方法迅速成为必要的临床研究工具--以及 医学检测实验室日益受到重视--开展规模选择工作十分重要 具有改进的大小范围、提高大碎片的分辨率的设备和方法 大小(10kb-200kb)、更快的运行时间和更高的每个运行周期的样本吞吐量。 本申请的目标是开发新的制备凝胶电泳系统，该系统 利用大DNA的非线性响应：1)在响应特定脉冲时进行重定向 场条件，或2)当受到不同的正向和反向电压脉冲时 力量。在这两种情况下，我们设想了针对尺寸分数的尺寸选择过程 移动很少，并在样品装载位置(或附近)回收。如果成功，凝胶 可以显著减少大小和运行时间，从而缓解长期阅读中的瓶颈 受益于严格的大小选择的工作流。我们的最终目标是拥有一个大家庭 选择采用SBS格式、自动化友好的48孔或96孔凝胶盒的产品 样本容量。

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.