TempO-Seq Profiling of RNA Epitranscriptomic Modifications

本快速通道第一阶段-第二阶段SBIR涉及NHGRI特别感兴趣的主题C： 尖端分子生物学试剂盒的新仪器“。可逆性核糖核酸甲基组的最新发现 甲基化的信使核糖核酸及其在细胞功能和疾病中所起作用的早期迹象，以及 RNA免疫沉淀测序(RIP-SEQ)衍生方法的引入是对 能够识别基因中甲基化的特定位置的表位转录图谱，乞求 一个强大、简单和灵敏的甲基组分析平台，可以提供负担得起的高样本吞吐量 不仅是细胞的轮廓，而且是单个细胞和临床FFPE组织的轮廓与空间分辨率相关的焦点 从组织学领域到侧写数据。我们将论证实施TEMPO-SEQ™人的可行性 表位转录测定N1-甲基腺苷(M1a，聚集在 起始密码子区域，2016年被发现为真核mRNA的可逆表位转录修饰)， 和N6-甲基腺苷(m6A，聚集在终止密码子区域，可逆，首先定位在 转录组水平作为2012年人类mRNA的表观转录修饰)在第一阶段 将为5-甲基胞嘧啶(M5C)实施第三个甲基组分析方案，优化所有三个方案，然后 实施和验证Tempo-Seq简档分析，每个甲基组的分析约为4,000 内部甲基化的特定的mRNA序列。每一次化验的甲基组含量将由我们的 财团专家从他们的工作、文献和现有数据库中获得信息，并将由 在相同的RNA样品上进行了基准M1A-Seq、M6A-Seq和亚硫酸盐-Seq实验。我们会 验证提取的细胞RNA、细胞裂解产物和FFPE裂解产物的TEMPO-Seq甲基组分析，建立 每种图谱分析的灵敏度和重复性，验证了它们在FACS分类亚群图谱中的使用 和单个细胞，并描绘FFPE的焦点区域，直径小至130FPE，展示了关联的有用 通过分析高级别PIN与正常和正常区域的对比，将数据分析到组织的局部组织学背景 前列腺癌组织。然后，我们将以商业产品的形式推出这些检测方法，提供简单而可靠的 化验使调查人员能够以与RIP-SEQ或亚硫酸盐-SEQ相同的成本检测10至20倍的样本， 只需1.5小时的实际操作时间即可完成第二天的周转时间来处理96个样品，能够完全自动化 对于高样品吞吐量的分析，进行130μm直径焦点区域的单细胞图谱和图谱分析 存档的FFPE组织，将甲基组分析整合到Tempo-Seq整个转录组或聚焦(例如 疾病特异性)板作为单一的综合化验，并通过识别的角度执行分析 不需要生物信息学专家的差异甲基化基因。这意味着任何科学家都可以分析 这些甲基组在他们的研究领域扮演着重要的角色。我们将利用这一计划的成功之处 所有种类的RNA和DNA的甲基组检测的发展，以及诊断性检测的发展。

This Fast Track Phase I-II SBIR addresses the NHGRI Special Interest Topic C: “Genomics tools ranging from new instruments to sophisticated molecular biology kits”. The recent discoveries of methylomes of reversibly methylated mRNA and early indications of the functional role these play in cellular function and disease, and the introduction of RNA immunoprecipitation sequencing (RIP-Seq) derived approaches as a breakthrough in epitranscriptomic profiling that has enabled the specific sites of methylation within genes to be identified, beg for a robust, simple, and sensitive methylome profiling platform that can provide affordable high sample throughput profiling of not just cells, but also single cells and clinical FFPE tissue with the spatial resolution to relate focal areas of histology to profiling data. We will demonstrate the feasibility of implementing TempO-Seq™ human epitranscriptomic protocols measuring the mRNA methylomes of N1-methyladenosine (m1A, clustered in the region of the start codon, discovered as a reversible epitranscriptomic modification of eukaryotic mRNA in 2016), and N6-methyladenosine (m6A, clustered in the region of the stop codon, reversible, first mapped at the transcriptome-wide level as epitranscriptomic modifications of human mRNA in 2012) in Phase I. In Phase II we will implement a third methylome assay protocol for 5-methylcytosine (m5C), optimize all three, and then implement and validate TempO-Seq profiling assays, with the assay of each methylome measuring ~4,000 internally methylated specific mRNA sequences. The methylome content for each assay will be selected by our consortium experts from their work and the literature and available databases, and will be validated by benchmark m1A-Seq, m6A-Seq and Bisulfite-Seq experiments performed on the same RNA samples. We will validate the TempO-Seq methylome assays on extracted cell RNA, cell lysates, and lysates of FFPE, establish the sensitivity and reproducibility of each profiling assay, validate their use to profile FACS sorted subpopulations and single cells, and to profile focal areas of FFPE as small as 130 μm diameter, demonstrating utility to relate profiling data to the focal histologic context of the tissue by profiling high grade PIN vs areas of normal and prostate cancer tissue. Then we will launch these assays as commercial products, providing simple and robust assays enabling investigators to test 10 to 20 times more samples for the same cost as RIP-seq or Bisulfite-Seq, have next-day turnaround with just 1.5 hr hands-on time to process 96+ samples, be able to fully automate the assay for high sample throughput, carry out single cell profiling and profiling of 130 μm diameter focal areas of archived FFPE tissue, integrate the methylome assay into the TempO-Seq whole transcriptome or focused (e.g. disease-specific) panels as a single integrated assay, and perform analysis through the point of identifying differentially methylated genes without need of a bioinformatics expert. That means any scientist can profile the role these methylomes play in their area of research. We will leverage the success of this program into development of methylome assays for all species of RNA and DNA, and the development of diagnostic assays.