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.

概括 尽管在核酸技术上进行了大量投资，但对大量全长进行测序的能力 来自复杂样品的单个RNA，具有最高精度的复杂样品仍无法实现。现场介绍 大规模下一代DNA测序（NGS）测序有效的无限数字的进步 来自异质混合物的单个RNA分子。我们试图证明这些方法可以是 以规模和精度实施，以证明翻译成商业上可行的产品和服务的合理性。 该提案的目的涉及确定技术可以的规模和准确性的两个关键步骤 应用，利用核酸酶学的最新发现。 AIM＃1专注于逆转录， 由创建，并在体外定向进化中，具有高度准确的，验证逆转录酶， 具有较高的加工性，没有整体RNase H活性和其他RT人工制品来源。这进展 甚至超过逆转录座或II组内含子的最佳逆转录病毒和酶也有望准确 即使是最长的RNA病毒基因组的复制。例如，常规逆转录病毒酶甚至修饰 按定位的诱变，除了简单的基础阅读外，还容易至少11个转录人工制品 错误。 AIM＃2利用了在DNA修复中使用的最近发现的一类引物聚合酶。我们的关键步骤 测序化学利用了由标记的cDNA的同性恋者的拓扑优势 圆形单链模板的滚动圆扩增（RCA）。最低的规模 技术可以应用于依赖外源随机DNA的常规RCA方案的限制 底漆。并生成具有小尺寸样品的人为序列。新协议使用PRIMPOL 聚合酶直接从模板中合成RNA底漆，防止从头伪像和 同时提高了约5个数量级的扩增。这给出了令人兴奋的可能性 将技术应用于单细胞转录组规模以下的样品。这些研究将 寻求为RNA测序设定新的行业标准。这可能有助于加速多种精确度 医学，即。精确癌症诊断，免疫疗法；治疗基因编辑；新药发现和 验证。该技术可以在与传染病作斗争中提供变革性进步，包括 HIV/AIDS和SARS-COV-2介导的Covid 19。