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; 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.

项目摘要 通过提供分析长度跨越数百kb到MB的长长的DNA的能力，第三代 PACBIO，牛津纳米孔和10倍基因组学的链接阅读测序技术已经开始 彻底改变了越来越多的基因组应用，包括从头组装，定相/脚手架， 和结构变体分析。这些功能是基于有效操纵高的能力 分子量（HMW）DNA。不仅必须提取HMW DNA，而且同样重要的是 随后的图书馆准备期间损坏或丢失。用于最佳测序读取长度，吞吐量和 插入长度的质量，精确控制至关重要。在牛津纳米孔测序中，消除短DNA可以 用于增加平均读取长度并增强超长读数的比例（> 100 kb）。在pacbio hifi中 测序，严格控制插入尺寸，可以产生高共识精度（> QV20）单个 分子读取。以前，在10-100 kb范围内执行这种大小DNA选择的唯一方法是 通过手动或更常见的是自动凝胶纯化。凝胶纯化仪器具有很高的截止 但是缓慢，昂贵，并且长DNA的恢复较低。在开发纳米宾的过程中 DNA提取技术，我们发明了简短的读取器（SRE）尺寸选择技术和 很快将其推向市场。在仅9个月的商业销售中，它已成为大小的领先方法 由于其高性能，低成本和易用性而选择纳米孔测序。为此， 循环学开发了专有的聚合物化学分配，可实现高截止，高恢复HMW DNA， 和快速处理。在此直接到II阶段提案中，我们将扩展简短的消除器产品 投资组合使用户能够进一步塑造读取长度分布并解决更广泛的范围 测序工作流程。我们将开发新版本的简短阅读消除器：1）更高和更清晰的版本 截止，2）用于频道尺寸选择，3）用于低输入样品，4）分隔了来自相同的DNA和RNA 生物样品。这些新版本的简短读取器将在PACBIO和OXFORD上得到验证 纳米孔使用各种样本类型和应用。