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

对细胞RNA分子进行测序的能力已经成为临床实践中的重要工具， 生物医学研究，因为它提供了基因表达和变异的关键信息。RNA 测序技术是由一种独特的酶：逆转录酶（RT）， 将RNA分子复制成DNA链，然后通过PCR扩增。不幸的是，传统的RT 它们的处理能力并不强，它们只复制RNA模板的短拷贝（短读段），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。在这个过程中，关于远端突变之间的相对流行和联系的信息， 单个转录本内的RNA编辑位点和可变剪接位点丢失。许多重要的细胞和 病毒RNA相当长（>1000 nt），因此需要精确的端到端测序， 对其功能和多样化进行有意义的研究。我们最近发现了一种超进行性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的结构化病毒转录物。我们的目标是优化和提高急诊室。RT生产A 广泛适用于各种生物技术应用的耐用试剂（目标1）。然后我们将利用它来 解决两个主要未满足需求领域。急诊室RT将被纳入下一代测序 (NGS)监测艾滋病毒感染者的病毒演变和耐药性的管道（目标2）。然后它将 优化以从已知但复杂的RNA分子混合物产生全长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 在果蝇中（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RT试剂，从根本上改善NGS，使研究成为可能。 RNA转录物的任何混合物，允许基因组定相和连锁分析。

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.