Monitoring Variation in Mixtures of Long RNAs with End-to-End RT Sequencing

通过端到端 RT 测序监测长 RNA 混合物的变化

基本信息

批准号：
9389200
负责人：
Brenton R. Graveley
金额：
$ 89.68万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-01 至 2020-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9389200
关键词：
Address Alternative Splicing Antiviral Agents Area Behavior Benchmarking Biochemical Biomedical Research Biophysics Biotechnology Blood Cell physiology Cells Complementary DNA Complex Complex Mixtures DNA Development Disease Distal Drosophila genus Drug resistance Engineering Enzymes Evolution Foundations Gene Expression Genes Genetic Genomics Goals HIV HIV Genome HIV drug resistance Hand High-Throughput Nucleotide Sequencing Human Human Development Individual Introns Length Light Link Measures Monitor Mutation Patients Pattern Performance Pharmacotherapy Phase Play Prevalence Process Property Protein Isoforms Proteins RNA RNA Editing RNA Sequences RNA Splicing RNA-Directed DNA Polymerase Reaction Reagent Research Reverse Transcription Role Sampling Site Specificity Spliced Genes Structure Technology Testing Thermodynamics Time Tissues Transcript Untranslated RNA Variant Viral Viral Genome Virion Virus base cDNA Library clinical practice design genetic linkage analysis genomic tools human disease immune resistance improved inhibitor/antagonist nanopore next generation sequencing novel novel strategies prevent protein structure real world application reference genome sequencing platform tool transcriptome transcriptome sequencing transcriptomics viral RNA viral fitness

项目摘要

The ability to sequence cellular RNA molecules has become an essential tool in clinical practice and biomedical research because it provides critical information on gene expression and variation. RNA sequencing technology is made possible by a unique type of enzyme: the reverse transcriptase (RT), which copies RNA molecules into DNA strands that can then be amplified by PCR. Unfortunately, conventional RTs are not highly processive and they only make short copies of RNA templates (short reads), which are then computationally stitched together using a reference genome in order to infer the sequence of intact RNA molecules. During this process, information on the relative prevalence and linkage between distal mutations, RNA editing sites and alternative splice sites within individual transcripts is lost. Many important cellular and viral RNAs are quite long (>1000 nts), and therefore accurate end-to-end sequencing will be required to conduct meaningful studies of their function and diversification. We recently discovered an ultra-processive RT from a eubacterial group II intron (the E.r. RT). Even without extensive optimization, the E.r. RT copies highly structured viral transcripts that are >9kb in size. Our goal is to optimize and enhance the E.r. RT to produce a robust reagent that is widely suitable for diverse biotechnology applications (Aim 1). We will then employ it to address two areas of major unmet need. The E.r. RT will be incorporated into Next Generation Sequencing (NGS) pipelines for monitoring viral evolution and drug resistance in HIV-infected patients (Aim 2). It will then be optimized to generate full-length cDNA libraries from known, but complex mixtures of RNA molecules and then utilized for whole-transcriptome sequencing in order to monitor the tissue specificity of alternative splicing in Drosophila (Aim 3). By performing biochemical optimization in parallel with real-world applications of the E.r. RT, we aim to create a powerful new RT reagent that fundamentally improves NGS, making it possible to study any mixture of RNA transcripts, allowing for genomic phasing and linkage analyses.

对细胞 RNA 分子进行测序的能力已成为临床实践和临床实践中的重要工具。生物医学研究，因为它提供了有关基因表达和变异的关键信息。核糖核酸测序技术是通过一种独特的酶实现的：逆转录酶 (RT)，它将 RNA 分子复制到 DNA 链中，然后通过 PCR 进行扩增。不幸的是，传统的 RT 处理能力不强，它们只制作 RNA 模板的短拷贝（短读），然后使用参考基因组通过计算拼接在一起，以推断完整 RNA 的序列分子。在此过程中，有关远端突变之间的相对流行率和联系的信息，单个转录本中的 RNA 编辑位点和选择性剪接位点丢失。许多重要的细胞和病毒RNA相当长（>1000 nts），因此需要准确的端到端测序对它们的功能和多样化进行有意义的研究。我们最近发现了一种超处理 RT 来自真细菌 II 组内含子 (E.r. RT)。即使没有广泛的优化，E.r. RT复制率高大小 >9kb 的结构化病毒转录本。我们的目标是优化和增强 E.r. RT 产生广泛适用于多种生物技术应用的稳健试剂（目标 1）。然后我们将用它来解决两个主要未满足需求领域。急诊室RT将纳入下一代测序用于监测 HIV 感染患者的病毒进化和耐药性的 (NGS) 管道（目标 2）。然后它会进行优化，从已知但复杂的 RNA 分子混合物中生成全长 cDNA 文库然后用于全转录组测序，以监测选择性剪接的组织特异性果蝇（目标 3）。通过与 E.r. 的实际应用并行执行生化优化。 RT，我们的目标是创造一种强大的新型 RT 试剂，从根本上改进 NGS，使研究成为可能 RNA 转录本的任何混合物，可进行基因组定相和连锁分析。