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.

项目总结