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

用于长读长测序文库制备的高通量尺寸选择系统

• 批准号: 10480521
• 负责人: TRUETT C BOLES
• 金额: $ 32.09万
• 依托单位: SAGE SCIENCE, INC.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-03 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10480521
• 关键词: Agar Gel Electrophoresis; Automation; Characteristics; Clinical Research; DNA; DNA sequencing; Devices; Diffusion; Dimensions; Electrophoresis; Elements; Equipment; Family Sizes; Gel; Genomic DNA; Goals; Home; Hour; Length; Libraries; Liquid substance; Membrane; Methods; Output; Phase; Physiologic pulse; Positioning Attribute; Precipitation; Preparation; Process; Protocols documentation; Reader; Recovery; Resolution; Running; Sampling; Science; Specific qualifier value; Surface; System; Techniques; Testing; Time; Width; gel electrophoresis; improved; instrument; medical attention; nanopore; novel; prototype; rapid technique; response; small molecule; tool; voltage; waveguide

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.

项目概要/摘要 大多数长读长 DNA 测序方法依赖于文库大小选择技术来控制 和/或提高阅读长度。在 Oxford Nanopore 和 PacBio 测序方案中，样品 加载取决于文库分子向读取器表面（波导表面 用于 PacBio，或用于 Oxford 的膜）。由于小分子的扩散速率比 较大的分子，通过尺寸选择消除小的文库片段可以大大提高文库质量 读取长度。消除小库元素（通常<6-10kb）的流行方法 长读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，制备凝胶 电泳在拒绝小 DNA 分子方面具有优越性，但需要专门的技术 仪器、带有大体积凝胶柱（6-10 厘米长）和低性能的消耗性凝胶盒 盒式样品吞吐量和运行时间长。在许多长读测序工作流程中， 尺寸选择是工作流程中最耗时的步骤。 随着长读长测序方法迅速成为重要的临床研究工具——并且 越来越受到医学检测实验室的关注——制定尺寸选择很重要 具有改进的尺寸范围、改进的大碎片分辨率的设备和方法 大小 (10kb-200kb)、更快的运行时间以及每个运行周期更高的样品吞吐量。 本申请的目标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）同时响应某些脉冲而重新定向 现场条件，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，我们设想尺寸选择过程，其中目标尺寸分数 移动很少，并在样品装载位置内（或附近）恢复。如果成功，凝胶 大小和运行时间可以显着减少，从而缓解长读取的瓶颈 受益于严格尺寸选择的工作流程。我们的最终目标是家庭规模 采用 SBS 格式、自动化友好的 48 或 96 孔凝胶盒的精选产品 样品容量。