Rapid Method to Enhance and Shape Long-Read Sequencing Read Length Distributions

增强和塑造长读长测序读长分布的快速方法

• 批准号: 10228764
• 负责人: Kelvin Liu
• 金额: --
• 依托单位: CIRCULOMICS, INC.
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-04 至 2021-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10228764
• 关键词: Address; Biological; Biological Sciences; Chemistry; Clinical; Collaborations; Complement; Consensus; DNA; DNA sequencing; Development; Diagnostic tests; Fractionation; Gel; Generations; Genomics; Hazardous Chemicals; Health; Human; Insecta; Length; Libraries; Link; Manuals; Methods; Molecular Weight; Nucleic Acids; Organic solvent product; Performance; Phase; Polymer Chemistry; Precipitation; Preparation; Process; Protocols documentation; RNA; Reaction; Recovery; Sales; Sampling; Science; Shapes; Solubility; Stretching; Structure; Technology; Variant; base; commercialization; cost; data quality; experience; flexibility; improved; instrument; nanopore; next generation sequencing; novel diagnostics; rapid technique; scaffold; sequencing platform; single molecule; targeted sequencing; therapeutic target; transcriptome sequencing; wasting

Project Summary By offering the ability to analyze long stretches of DNA spanning hundreds of kb to Mb in length, 3rd generation and linked-read sequencing technologies from PacBio, Oxford Nanopore, and 10X Genomics have begun to revolutionize an increasing number of genomics applications including de novo assembly, phasing/scaffolding, and structural variant analysis. These capabilities are predicated on the ability to efficiently manipulate high molecular weight (HMW) DNA. Not only must HMW DNA be extracted, but it is equally important that it is not damaged or lost during subsequent library preparation. For optimal sequencing read length, throughput, and quality, precise control of insert lengths is crucial. In Oxford Nanopore sequencing, elimination of short DNA can be used to increase mean read lengths and enhance the fraction of ultra-long reads (>100 kb). In PacBio HiFi sequencing, tight control of insert size allows the generation of high consensus accuracy (>QV20) single molecule reads. Previously, the only method to perform such size selection of DNA in the 10-100 kb range was through manual or, more commonly, automated gel purification. Gel purification instruments have high cutoffs but are slow, expensive, and have low recovery of long DNA. During the course of developing our Nanobind DNA extraction technology, we invented Short Read Eliminator (SRE) size selection technology and quickly brought it to market. In only 9 months of commercial sales, it has become a leading method of size selection for nanopore sequencing due to its high performance, low cost, and ease of use. To achieve this, Circulomics developed proprietary polymer chemistries that enable high cutoffs, high recovery of HMW DNA, and rapid processing. In this Direct to Phase II proposal, we will expand the Short Read Eliminator product portfolio to enable users to further shape read length distributions and address a wider range of sequencing workflows. We will develop new versions of Short Read Eliminator: 1) with higher and sharper cutoffs, 2) for band-pass size selection, 3) for low input samples, and 4) to partition DNA and RNA from the same biological sample. These new versions of the Short Read Eliminator will be validated on both PacBio and Oxford Nanopore using a variety of sample types and applications.

项目概要 通过提供分析长度跨越数百 kb 至 Mb 的长 DNA 片段的能力，第三代 PacBio、Oxford Nanopore 和 10X Genomics 的 Linked-read 测序技术已开始 彻底改变越来越多的基因组学应用，包括从头组装、定相/支架、 和结构变异分析。这些能力取决于有效操纵高 分子量 (HMW) DNA。不仅必须提取 HMW DNA，而且同样重要的是不提取 HMW DNA。 在随后的文库制备过程中损坏或丢失。为了获得最佳的测序读长、通量和 为了确保质量，刀片长度的精确控制至关重要。在牛津纳米孔测序中，短 DNA 的消除可以 用于增加平均读取长度并提高超长读取（> 100 kb）的比例。在 PacBio HiFi 中 测序，严格控制插入片段大小可以生成高一致性精度（>QV20）单 分子读取。此前，在 10-100 kb 范围内进行 DNA 大小选择的唯一方法是 通过手动或更常见的自动凝胶纯化。凝胶纯化仪器具有高截止值 但速度慢、成本高且长 DNA 回收率低。在开发 Nanobind 的过程中 DNA提取技术，我们发明了Short Read Eliminator（SRE）大小选择技术和 很快将其推向市场。商业销售仅 9 个月，已成为尺寸领先的方法 纳米孔测序因其高性能、低成本和易用性而被选择。为了实现这一目标， Circulomics 开发了专有的聚合物化学物质，可实现 HMW DNA 的高截止值和高回收率， 和快速处理。在这个直接进入第二阶段的提案中，我们将扩展 Short Read Eliminator 产品 产品组合，使用户能够进一步塑造读长分布并解决更广泛的问题 测序工作流程。我们将开发新版本的Short Read Eliminator：1）更高、更锐利 截止值，2) 用于带通大小选择，3) 用于低输入样本，以及 4) 从相同样本中分离 DNA 和 RNA 生物样本。这些新版本的 Short Read Eliminator 将在 PacBio 和 Oxford 上进行验证 纳米孔使用多种样品类型和应用。