Full-length sequencing of individual RNAs from heterogeneous samples

对异质样品中的单个 RNA 进行全长测序

• 批准号: 10482321
• 负责人: William S Agnew
• 金额: $ 39.8万
• 依托单位: PHAENO INC
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-15 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10482321
• 关键词: 2019-nCoV; AIDS/HIV problem; Address; Alzheimer' s Disease; Biological; COVID-19; Cancer Diagnostics; Cells; Chemistry; Circular DNA; Communicable Diseases; Complementary DNA; Complex; Coronavirus; Custom; DNA; DNA Library; DNA Primers; DNA Repair; DNA biosynthesis; DNA sequencing; Directed Molecular Evolution; Disease; Enzymatic Biochemistry; Enzymes; Feasibility Studies; Fee-for-Service Plans; Foundations; Genes; Genetic Transcription; Genomics; Goals; HIV; High-Throughput DNA Sequencing; Homo; Human; Immunotherapy; In Vitro; Individual; Industry Standard; Intellectual Property; Introns; Investments; Legal patent; Length; Libraries; Licensing; Malignant Neoplasms; Mediating; Messenger RNA; Methodology; Methods; Morphologic artifacts; Muscular Dystrophies; Nucleic Acids; Performance; Phase; Polymerase; Positioning Attribute; Preparation; Proteins; Protocols documentation; RNA; RNA Splicing; RNA Viruses; RNA primers; RNA replication; RNA-Directed DNA Polymerase; Reading; Reagent; Recovery; Reproducibility; Retrotransposon; Retroviridae; Reverse Transcription; Ribonuclease H; Rights; Sampling; Secure; Services; Single-Stranded DNA; Site-Directed Mutagenesis; Source; Technology; Testing; Translations; Universities; Validation; Variant; Viral Genome; base; comparative; computerized data processing; drug discovery; improved; instrument; next generation; novel; novel strategies; novel therapeutics; personalized diagnostics; phase 2 study; physical separation; precision medicine; prevent; single cell sequencing; single molecule; single-cell RNA sequencing; therapeutic gene; transcriptome; transcriptome sequencing

Summary Despite large investments in nucleic acid technology, the ability to sequence large numbers of full-length individual RNAs, from complex samples, with highest accuracy, has remained out of reach. Pheno introduces an advance for large scale Next Generation DNA Sequencing (NGS) sequencing of effectively unlimited numbers of individual RNA molecules from heterogeneous mixtures. We seek to prove that these methods can be implemented with the scale and precision to justify translation into commercially viable products and services. The aims of this proposal address two key steps that determine scale and accuracy at which the technology can be applied, exploiting recent discoveries in nucleic acid enzymology. Aim #1 focuses on reverse transcription, prompted by creation, with in vitro directed evolution, of a highly accurate, proof-reading reverse transcriptase, with high processivity and devoid of integral RNase H activity and other sources of RT artefacts. This advance over even the best retroviral and enzymes derived from retrotransposons or group-ii introns promises accurate replication of even the longest RNA viral genomes. Conventional retroviral enzymes, for example, even modified by site directed mutagenesis, are prone to at least 11 transcriptional artefacts in addition to simple base-reading errors. Aim #2 exploits a recently discovered class of primer-polymerases used in DNA repair. A key step in our sequencing chemistry exploits topological advantages of homo-concatamers of tagged cDNAs produced by Rolling Circle Amplification (RCA) of circularized single-stranded templates. The lowest scale at which the technology can be applied is limited by conventional RCA protocols that depend on exogenous random DNA primers. and generate artifactual sequences with samples of small size. New protocols use a PrimPol polymerase to synthesize RNA primers directly from the template, preventing de novo artefacts and simultaneously improving amplification by ~ 5 orders of magnitude. This presents the exciting possibility of applying the technology to samples below the scale of a single-cell transcriptome. Collectively these studies will seek to set new industry standards for RNA sequencing. This could help accelerate a wide range of precision medicine, viz. precision cancer diagnostics, immunotherapy; therapeutic gene editing; new drug discovery and validation. The technology could provide transformational advances in battling infectious diseases, including HIV/AIDS and SARS-CoV-2-mediated COVID 19.

摘要 尽管在核酸技术上投入了大量资金，但对大量全长序列进行测序的能力 来自复杂样本的单个RNA具有最高的准确性，仍然遥不可及。菲诺介绍 大规模有效无限数下一代DNA测序的研究进展 从不同的混合物中提取单个RNA分子。我们试图证明这些方法可以 以规模和精确度实施，以证明转换为商业上可行的产品和服务是合理的。 这项提案的目标是解决两个关键步骤，这两个步骤决定了该技术可以达到的规模和精度 被应用，利用核酸酶学的最新发现。目标1专注于逆转录， 在体外定向进化的推动下，创造了一种高度准确的校对逆转录酶， 具有高加工性，不存在完整的RNaseH活性和其他来源的RT伪影。这一进步 即使是最好的逆转录病毒和来自逆转录转座子或第二组内含子的酶也保证了准确性 即使是最长的RNA病毒基因组也可以复制。例如，传统的逆转录病毒酶，甚至经过修饰 通过定点突变，除了简单的碱基阅读外，还容易产生至少11个转录产物 错误。目的#2利用最近发现的一类用于DNA修复的引物聚合酶。我们的关键一步 测序化学利用标记的cDNA的同源链接物的拓扑优势 环状单链模板的滚环扩增(RCA)的最低级别 可应用的技术受到依赖于外源随机DNA的传统RCA协议的限制 底漆。并用小尺寸的样本生成伪像序列。新协议使用PrimPol 聚合酶直接从模板合成RNA引物，防止从头开始的人工制品和 同时将放大能力提高了~5个数量级。这带来了令人兴奋的可能性 将这项技术应用于单细胞转录组规模以下的样本。总的来说，这些研究将 寻求为RNA测序设定新的行业标准。这可以帮助加速大范围的精度 医学，即。癌症精确诊断、免疫治疗、治疗性基因编辑、新药发现和 验证。这项技术可以在抗击传染病方面提供变革性的进步，包括 艾滋病毒/艾滋病和SARS-CoV-2介导的COVID 19.