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

项目摘要 通过提供分析长度从数百kb到Mb的长DNA的能力，第三代 来自PacBio、牛津纳米孔和10X基因组的链接阅读测序技术已经开始 革新越来越多的基因组学应用，包括从头组装、阶段化/脚手架、 结构变异分析。这些能力的前提是能够有效地处理高 相对分子质量(HMW)DNA。不仅必须提取HMW DNA，而且同样重要的是不提取 在后续的磁带库准备过程中损坏或丢失。为了获得最佳的排序读取长度、吞吐量和 为了保证质量，准确控制插入物的长度至关重要。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提高超长读取的比例(&gt；100 kb)。在PacBio HiFi中 测序，严格控制插入大小，允许产生高共识精度(&gt；QV20)单个 分子读数。以前，对10-100kb范围内的DNA进行这种大小选择的唯一方法是 通过人工或更常见的自动凝胶纯化。凝胶纯化仪器具有高截止值 但都是缓慢、昂贵的，而且长DNA的回收率很低。在开发我们的纳米结合剂的过程中 DNA提取技术，我们发明了短读消除器(SRE)大小选择技术和 很快将其推向市场。在短短9个月的商业销售中，它已经成为一种领先的尺寸测量方法 选择纳米孔测序是因为其高性能、低成本和易于使用。为了实现这一目标， 循环组学开发了专有的聚合物化学，使高截留、高回收HMW DNA， 和快速处理。在这份直接到第二阶段的计划中，我们将扩展短读消除器产品 产品组合使用户能够进一步确定阅读长度分布，并解决更广泛的 对工作流进行排序。我们将开发新版本的短读消除器：1)更高、更锐利 阈值，2)带通大小选择，3)低输入样本，以及4)将DNA和RNA从相同的 生物样本。这些新版本的短读消除器将在PacBio和牛津上进行验证 纳米孔采用了各种样品类型和应用。

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.