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片段的能力， PacBio、Oxford Nanopore和10 X Genomics的连接阅读测序技术已经开始 革命性地改变了越来越多的基因组学应用，包括从头组装，定相/支架， 和结构变体分析。这些能力的前提是能够有效地操纵高 分子量（HMW）DNA。不仅必须提取HMW DNA，而且同样重要的是， 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段长度、通量和 质量，精确控制刀片长度是至关重要的。在牛津纳米孔测序中，消除短DNA可以 用于增加平均读段长度并提高超长读段（>100 kb）的分数。在PacBio HiFi 通过测序，严格控制插入片段大小允许产生高一致性准确度（> QV 20）的单克隆抗体。 分子读数。以前，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 但是缓慢、昂贵，并且长DNA的回收率低。在开发我们的Nanobind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， 很快将其推向市场。在商业销售仅9个月的时间里，它已经成为规模领先的方法 由于其高性能、低成本和易于使用，因此可以选择用于纳米孔测序。为了实现这一点， Circulomics开发了专有的聚合物化学，能够实现高截留率，高HMW DNA回收率， 和快速处理。在此“直接进入第二阶段”提案中，我们将扩展短读消除器产品 组合，使用户能够进一步塑造读长分布，并解决更广泛的 测序工作流程。我们将开发新版本的短读消除器：1）具有更高和更清晰的 截止值，2）用于带通尺寸选择，3）用于低输入样品，以及4）从相同的样品中划分DNA和RNA 生物样本这些新版本的短读消除器将在PacBio和Oxford上进行验证 纳米孔使用的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.