Single-cell direct RNA sequencing using electrical zero-mode waveguides and engineered reverse transcriptases

项目摘要/摘要 在过去的几十年里，基因组技术的进步大大降低了DNA的成本 测序和阅读长度的大幅增加，后者是由新的单分子的开发提供的 测序技术。这些进展使人们能够探测基因组中被认为是 “暗物质”直到最近，以及组装新的高质量的参考基因组。除了……之外 基因组测序，这些单分子方法为探测化学物质开辟了新的应用领域 DNA的修饰，通过探索利用光波导合成测序的动力学，或者 通过使用纳米孔对修饰的碱基进行电区分。目前，正在努力创造强大的 直接RNA测序的方法，以便关于RNA序列、表观遗传修饰和 量，可以得到。在单个人类细胞中，只有几个皮克的RNA和DNA可用，并且 由于这些核酸的表观遗传修饰不能倍增，一个公认的未来目标 测序技术是为了减少可以在皮卡水平上分析的基因组材料的数量。 我们最近开发了一种将皮克级DNA和RNA加载到零模波导中的方法 (ZMWs)，并展示了通过制造多孔体实现的长DNA片段的DNA测序 ZMWs(PZMWs)，在ZMWs底部嵌入了多孔材料。然而，挑战与 多孔材料的化学成分和寿命限制了该系统的处理能力。在这项提案中，我们 将开发一种全新的直接RNA测序方法，使定量转录组分析成为可能 和RNA碱基修饰信息，仅需要皮卡级别的输入RNA。首先，我们开发了一种 一种新型的ZMW，它的下面嵌入了一个金属盘电极。在两端施加电压 ZMWs产生一个电场，帮助捕获DNA和RNA。这些新设备极大地允许 与上一代PZMW相比，提高了吞吐量，并大幅提高了 获得的数据。其次，对于排序引擎，我们将使用马拉松RT，这是一种超过程反向 一种转录酶，通过酶复制将RNA分子转化为互补DNA(CDNA)分子 稳健和准确，比目前用于RNA测序的酶更准确。第三，我们将聘用 先进的单细胞RNA提取和金标准RNA定量方法。有广泛的支持 初步数据，我们将整合马拉松RT作为引擎，PtZMWs作为敏感的序列阅读器和 先进的单细胞分选和RNA提取工具，首次开发出定量RNA表达 来自真正的单细胞材料的图谱(即没有放大)。此外，使用我们的能力遵循 通过马拉松RT复制动力学，我们将探测保存在这些RNA分子中的化学修饰， 如甲基腺嘌呤和假尿嘧啶。这一独特方法的成功将使转录组发生革命性变化 通过以前所未有的灵敏度提供表观/转录切割工作流程，从单细胞材料进行分析。

Project Summary / Abstract Progress in genome technologies over the past few decades has delivered a dramatic cost reduction in DNA sequencing and vast increases in read lengths, the latter afforded by development of new single-molecule sequencing technologies. These advances enabled probing regions of the genome that were considered as “dark matter” up until recently, as well as the assembly of new high-quality reference genomes. In addition to genome sequencing, these single-molecule methods have opened up new applications for probing chemical modifications in DNA, by either probing the kinetics of sequencing-by-synthesis using optical waveguides, or by electrically distinguishing modified bases using nanopores. Currently, efforts are made to create robust methods for direct RNA sequencing, so that information about RNA sequence, epigenetic modifications, and quantity, can be obtained. In a single human cell, only a few picograms of RNA and DNA are available, and since epigenetic modifications in these nucleic acids cannot be multiplied, a recognized goal of future sequencing technologies is to reduce the amount of genomic material that can be analyzed at picogram levels. We have recently developed a method for loading picogram-level DNA and RNA into zero-mode waveguides (ZMWs), and have demonstrated DNA sequencing of a long DNA fragment, achieved by fabricating porous ZMWs (PZMWs) in which a porous material was embedded at the ZMW bottoms. However, challenges with the chemistry and longevity of porous materials have limited the throughput of this system. In this proposal, we will develop an entirely new method for direct RNA sequencing that enables quantitative transcriptome analysis and RNA base modification information, requiring only picogram-level input RNA. First, we have developed a new type of ZMW that contains a metal-disk electrode embedded underneath it. Applying voltage across the ZMWs produces an electric field that assists with DNA and RNA capture. These new devices allow vastly increased throughput over the previous generation PZMWs, as well as substantial quality improvements to the data obtained. Second, for the sequencing engine we will employ MarathonRT, an ultra-processive reverse transcriptase that converts RNA molecules to complementary DNA (cDNA) molecules by enzymatic replication robustly and accurately, more so than currently used enzymes used for RNA sequencing. Third, we will employ advanced single-cell RNA extraction and gold-standard RNA quantification methods. Backed by extensive preliminary data, we will integrate MarathonRT as the engine, PtZMWs as the sensitive sequence readers and advanced single-cell sorting and RNA extraction tools, to develop for the first time quantitative RNA expression profiles from truly single-cell material (i.e., no amplification). Additionally, using our ability to follow the replication kinetics by MarathonRT, we will probe chemical modifications preserved in these RNA molecules, such as methyladenine and pseudouridine. Success in this unique approach will revolutionize transcriptome analysis from single-cell material by providing a workflow for epi/transcriptomics at unprecedented sensitivity.