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.

项目摘要/摘要 大多数长阅读的DNA测序方法取决于库大小选择技术来控制 和/或提高阅读长度。在牛津纳米孔和PACBIO测序方案中，样品 负载取决于库分子向读取器表面的扩散（波导表面 用于PACBIO或牛津的膜）。由于小分子的扩散率高于 较大的分子，尺寸选择以消除小图书馆片段可以大大改善库 读取长度。消除小型库元素（通常<6-10kb）的流行方法 长阅读测序是制备琼脂糖凝胶电泳（Bluepippin，sageelf， Sage Science，Inc。的Pippinht和PEG降水（简短读取消除器套件， 循环系统以及家庭冲泡钉方法）。在这些方法中，制备凝胶 电泳在拒绝小的DNA分子方面表现出色，但需要专门 仪器，具有笨重的凝胶柱（长6-10厘米）的易消耗凝胶盒，低 盒式样品吞吐量和长期运行时间。在许多长阅读的测序工作流程中， 尺寸选择是工作流程中最耗时的步骤。 随着长阅读测序方法迅速成为重要的临床研究工具，并且 越来越吸引医学测试实验室的注意力 - 开发尺寸选择很重要 改善尺寸范围的设备和方法，大片段的分辨率提高了 每个运行周期的大小（10KB-200KB），运行速度更快和较高的样本吞吐量。 本应用的目的是开发新的制备凝胶电泳系统 利用大型DNA的非线性响应：1）响应某些脉冲的重新定位时 野外条件，或2）当受到不同的前进和反向电压脉冲时 力量。在这两种情况下，我们都设想了尺寸选择过程，其中针对尺寸的分数 移动很少，并在样品加载位置内（或接近）内回收。如果成功，凝胶 尺寸和运行时间可以大大减少，从而减轻了瓶颈 受益于严格尺寸选择的工作流程。我们的最终目标是一个大小的家庭 使用SBS-Format，自动化友好的凝胶盒48或96井的选择产品 样本容量。