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.

总结 尽管在核酸技术上进行了大量投资，但对大量全长DNA进行测序的能力仍然很有限。 来自复杂样品的具有最高准确性的单个RNA仍然遥不可及。Pheno介绍 大规模下一代DNA测序（NGS）的进展， 从异质混合物中分离单个RNA分子。我们试图证明这些方法可以 实施的规模和精度，以证明翻译成商业上可行的产品和服务。 该提案的目的是解决两个关键步骤，决定规模和准确性，该技术可以 应用，利用核酸酶学的最新发现。目标#1专注于逆转录， 通过体外定向进化，产生了一种高度精确的、校对的逆转录酶， 具有高的持续合成能力，并且缺乏完整的RNA酶H活性和RT伪像的其它来源。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II组内含子的酶也保证准确 甚至最长的RNA病毒基因组的复制。例如，传统的逆转录病毒酶，即使经过修饰， 通过定点诱变，除了简单的碱基阅读外， 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。这是我们 测序化学利用了由测序化学产生的标记cDNA的同源多联体的拓扑优势。 环化单链模板的滚环扩增（RCA）。最低的规模， 技术的应用受到依赖于外源随机DNA的常规RCA方案的限制 引物并生成具有小尺寸样本的伪序列。新协议使用PrimPol 聚合酶直接从模板合成RNA引物，防止从头人工产物， 同时将放大提高约5个数量级。这就提出了令人兴奋的可能性， 将该技术应用于低于单细胞转录组规模的样品。这些研究将 试图为RNA测序建立新的行业标准。这可以帮助加速大范围的精确度 医学，即精确的癌症诊断，免疫治疗;治疗性基因编辑;新药发现和 验证。这项技术可以为对抗传染病提供变革性的进步，包括 HIV/AIDS和SARS-CoV-2介导的COVID 19。