TempO-Seq Profiling of RNA Epitranscriptomic Modifications

本快速通道I-II期SBIR涉及NHGRI特别兴趣主题C：“基因组学工具， 从新仪器到复杂的分子生物学试剂盒”。可逆性甲基化的最新发现 甲基化的mRNA和这些在细胞功能和疾病中发挥的功能作用的早期迹象， RNA免疫沉淀测序（RIP-Seq）衍生方法的引入是 表观转录组学分析使基因内甲基化的特定位点得以鉴定， 一个强大、简单和灵敏的甲基化组分析平台，可提供负担得起的高样品通量 不仅对细胞进行分析，而且对单细胞和临床FFPE组织进行分析，其空间分辨率与病灶相关 组织学领域的分析数据。我们将证明实施TempO-Seq™人免疫原性的可行性。 测量N1-甲基腺苷（m1A，聚集在 起始密码子的区域，在2016年被发现为真核mRNA的可逆表位转录组修饰）， 和N6-甲基腺苷（m6 A，聚集在终止密码子区域，可逆，首先定位在 2012年作为人mRNA的表转录组修饰的全转录组水平）。在第二阶段，我们 将对5-甲基胞嘧啶（m5 C）实施第三个甲基化组测定方案，优化所有三个，然后 实施并验证TempO-Seq分析测定，每个甲基化组测定约4，000 内部甲基化的特定mRNA序列。每次测定的甲基化组含量将由我们的 联盟专家从他们的工作和文献和可用的数据库，并将验证 在相同的RNA样品上进行的基准m1A-Seq、m6 A-Seq和亚硫酸氢盐-Seq实验。我们将 验证提取的细胞RNA、细胞裂解物和FFPE裂解物的TempO-Seq甲基化组测定， 每种分析测定灵敏度和再现性验证了它们用于分析FACS分选的亚群的用途 和单细胞，并描绘直径小至130 μm的FFPE焦点区域，证明了与 通过分析高级别PIN与正常区域的关系， 前列腺癌组织然后，我们将推出这些检测作为商业产品，提供简单和强大的 分析使研究人员能够以与RIP-Seq或Bisulite-Seq相同的成本测试10到20倍的样品， 只需1.5小时的动手时间即可在次日完成周转，处理96多个样品，能够完全自动化 分析高样品通量，进行单细胞分析和分析130 μm直径的焦点区域， 存档的FFPE组织，将甲基化组测定整合到TempO-Seq全转录组中或聚焦（例如， 疾病特异性）面板作为一个单一的集成检测，并通过识别点进行分析 差异甲基化基因，而不需要生物信息学专家。这意味着任何科学家都可以 这些甲基化在他们的研究领域发挥的作用。我们将利用这一计划的成功， 开发用于所有种类的RNA和DNA的甲基化组测定，以及开发诊断测定。

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.