Automated Ultra-Long DNA and RNA Extraction for Long-read Sequencing Applications

用于长读长测序应用的自动化超长 DNA 和 RNA 提取

• 批准号: 10386934
• 负责人: MARK T BROWN
• 金额: $ 86.77万
• 依托单位: CLAREMONT BIOSOLUTIONS, LLC
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-15 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10386934
• 关键词: Adoption; Bacteria; Blood; Blood specimen; Cells; Chloroform; Complex; Consumption; Cytolysis; DNA; Digestion; Disease; Effectiveness; Engineering; Epigenetic Process; Equipment; Feedback; Flowcharts; Generations; Genome; Genomics; Goals; International; Lead; Manuals; Maps; Metagenomics; Methods; Nucleic Acid Binding; Nucleic Acids; Output; Phase; Phenols; Preparation; Principal Investigator; Protocols documentation; RNA; Reproducibility; Research Personnel; Running; Sampling; Sepharose; Site; Solid; Stretching; System; Technology; Testing; Third Generation Sequencing; Time; Tissue Sample; Tissues; Work; Yeasts; clinical application; cost; design; improved; innovation; insight; microbial; nanopore; new technology; next generation sequencing; novel; point of care; space station; transcriptome; transcriptome sequencing; whole genome

Summary Claremont BioSolutions, LLC (CBIO) The ability to sequence and map long stretches of DNA and RNA using third generation sequencing technologies is set to transform the field of genomics and allow researchers and clinicians unparalleled insight into the complexities of our genome and diseases. However, the move to these platforms presents new hurdles in long DNA and RNA sample preparation. Most conventional spin column sample preparation methods shear DNA during purification, and although agarose plug digestion methods can isolate ultra-long DNA from samples, the protocols can be difficult, time consuming and highly variable. While newer technologies have emerged that allow for more rapid extraction of long-DNA, these kits are either limited in use to easy-to-lyse sample types (cells and blood) or require extra equipment and long processing times, which significantly increases the cost. At Claremont BioSolutions (CBio), we are developing a novel automated extraction technology that will incorporate lysis, nucleic acid binding and isolation. The technology will allow users to add and sample, hit “GO” and in 30 minutes collect DNA or RNA. In Aim 1, we will integrate our fast lysis and isolation technology into an automated cartridge to provide reproducible high quality, ultra-long DNA or RNA extraction. In Aim 2, we will validate automated ultra-long DNA extraction using easy and complex sample types, compare our technology to HMW DNA competitor's kits, and demonstrate improved long-read sequencing using two third generation technologies. In Aim 3 we will test automated RNA extraction and downstream native RNA sequencing. In Aim 4 we will perform pre-market analysis at off-site alpha and beta testing facilities to generate end-user feedback and lockdown the design. The proposed system would simplify workflow, lower cost, and reduce variability.

摘要 克莱蒙生物解决方案公司(Claremont BioSolutions，LLC) 使用第三代测序对DNA和RNA的长片段进行测序和绘制地图的能力 技术将改变基因组学领域，并为研究人员和临床医生提供无与伦比的洞察力 我们基因组和疾病的复杂性。然而，向这些平台的转移带来了新的 长DNA和RNA样品制备中的障碍。最常用的旋转柱样品制备 方法在纯化过程中剪切DNA，虽然琼脂糖塞消化法可以分离超长 从样本中提取DNA，这些方案可能会困难、耗时且高度可变。虽然较新 已经出现了能够更快速地提取长DNA的技术，这些试剂盒要么在使用上受到限制 易于分解的样本类型(细胞和血液)或需要额外的设备和较长的处理时间，这是 大大增加了成本。在克莱蒙特生物解决方案公司(Claremont BioSolutions)，我们正在开发一种新型的自动化 集裂解、核酸结合和分离为一体的提取技术。这项技术将使 用户添加和采样，点击“Go”，在30分钟内收集DNA或RNA。在目标1中，我们将整合我们的FAST 裂解和分离技术进入自动盒，提供可重现的高质量超长DNA 或者提取核糖核酸。在目标2中，我们将使用简单和复杂验证自动超长DNA提取 样品类型，将我们的技术与HMW DNA竞争对手的试剂盒进行比较，并展示改进的长篇阅读 使用两种第三代技术进行测序。在目标3中，我们将测试自动提取RNA和 下游天然RNA测序。在目标4中，我们将在非现场阿尔法和贝塔进行上市前分析 测试设施，以生成最终用户反馈并锁定设计。拟议的系统将简化 工作流程，降低成本，减少变化性。