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: 10158001
• 负责人: MARK T BROWN
• 金额: $ 116.46万
• 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 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10158001
• 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.

总结 克莱蒙生物解决方案有限责任公司（CBIO） 使用第三代测序技术对长片段DNA和RNA进行测序和绘图的能力 技术将改变基因组学领域，使研究人员和临床医生能够获得无与伦比的洞察力 我们基因组和疾病的复杂性。然而，向这些平台的转移提出了新的 长DNA和RNA样品制备中的障碍。最传统的旋转柱样品制备 方法在纯化过程中剪切DNA，虽然琼脂糖塞消化方法可以分离超长的 从样本中提取DNA，该协议可能是困难的，耗时的和高度可变的。而较新 虽然已经出现了允许更快速提取长DNA的技术，但这些试剂盒要么在使用中受到限制， 容易裂解的样品类型（细胞和血液）或需要额外的设备和长的处理时间， 大大增加了成本。在克莱蒙生物解决方案（CBio），我们正在开发一种新的自动化 提取技术将结合裂解、核酸结合和分离。这项技术将允许 用户添加和采样，点击“GO”，并在30分钟内收集DNA或RNA。在目标1中，我们将整合快速 将裂解和分离技术整合到自动化检测盒中，以提供可重现的高质量超长DNA 或RNA提取。在目标2中，我们将使用简单和复杂的方法验证自动化超长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，并证明改进的长读 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RNA提取， 下游天然RNA测序。在目标4中，我们将在非现场alpha和beta阶段进行上市前分析 测试设施，以产生最终用户的反馈和锁定的设计。拟议的系统将简化 工作流程，降低成本，减少可变性。