TempO-Seq Profiling of RNA Epitranscriptomic Modifications

RNA 表观转录组修饰的 TempO-Seq 分析

基本信息

批准号：
9890040
负责人：
BRUCE E. SELIGMANN
金额：
$ 64.29万
依托单位：
BIOSPYDER TECHNOLOGIES, INC.
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-17 至 2022-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9890040
关键词：
Address Antibodies Archives Area Basic Science Benchmarking Bioinformatics Biological Assay Caliber Cell Extracts Cell Line Cell physiology Cells Chicago Clinical Consult DNA Data Data Analyses Databases Development Diagnostic Diagnostic tests Disease Early Diagnosis Etiology Gene Targeting Genes Hela Cells HepG2 Histologic Histology Human Initiator Codon Interferons Knowledge Ligation Literature Malignant Neoplasms Malignant neoplasm of prostate Maps Measures Messenger RNA Methods Methylation Modification Molecular Biology Monitor Morphology National Human Genome Research Institute Performance Phase Play Process Prostate Protocols documentation RNA RNA immunoprecipitation sequencing RNA methylation Recovery Reproducibility Research Research Personnel Resolution Role Sampling Scientist Site Small Business Innovation Research Grant Specificity Speed Terminator Codon Testing Thick Time Tissues Titrations Translating Translations Universities Work assay development base bisulfite bisulfite sequencing commercialization cost demethylation diagnostic assay differential expression disease diagnosis epitranscriptomics experimental study genomic tools high risk instrument interest methylome novel novel therapeutics personalized diagnostics programs sequencing platform success targeted sequencing therapy resistant transcriptome transcriptome sequencing translational medicine

项目摘要

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.

这个快速轨道阶段I-II SBIR解决了NHGRI特殊兴趣主题c：“基因组工具范围从复杂分子生物学试剂盒的新仪器”。最近发现的可逆甲基团甲基化的mRNA和功能作用的早期指示，这些作用在细胞功能和疾病中发挥作用，以及引入RNA免疫沉淀测序（RIP-SEQ）衍生的方法作为突破表面参考分析已使基因内的特定甲基化位点得以识别，请乞求强大，简单且敏感的甲基分析平台，可以提供负担得起的高样品吞吐量不仅细胞的分析，还对单个细胞和临床FFPE组织进行空间分辨率进行分析组织学领域分析数据。我们将展示实施tempo-seq™人类的可行性测量N1-甲基腺苷的mRNA甲基甲基甲基甲基甲基甲基甲基菌素的表面参考方案（M1A，聚集在起始密码子的区域，被发现是对真核mRNA的可逆性表演的修饰，2016年），），和N6-甲基腺苷（M6A，聚集在终止密码子的区域中，可逆，首先映射在在2012年，人类mRNA的表面转录组修饰在2012年）。将针对5-甲基胞嘧啶（M5C）实施第三种甲基组测定方案，优化这三个方案，然后实施和验证节气序列分析测定法，每个甲基团测量约为4,000 内部甲基化的特异性mRNA序列。每种测定的甲基组含量将由我们的联盟专家的工作以及文献和可用数据库，并将得到证实基准M1A-SEQ，M6A-SEQ和BISULFITE-SEQ实验在相同的RNA样品上进行。我们将在提取的细胞RNA，细胞裂解物和FFPE的裂解物上验证TEMPO-SEQ甲基团测定，已建立每个分析测定的敏感性和可重复性，验证其用于分类FACS分类亚群的用途和单细胞，以及直径为130μm的FFPE的焦点区域，证明了与相关的实用性通过分析高级引脚与正常和正常区域，将数据分析到组织的焦点组织学环境前列腺癌组织。然后，我们将它们作为商业产品推出，提供简单而强大的测定使研究人员能够以与Rip-Seq或Bisulfite-Seq相同的成本测试10至20倍的样品，只需1.5个小时即可处理96多个样品，可以完全自动化该次要的周转时间测定高样品吞吐量，进行单细胞分析和直径为130μm的焦点区域的分析存档的FFPE组织，将甲基化合体测定整合到速度 - 季节整体转录组或集中（例如特定于疾病的面板作为单个集成测定法，并通过识别点进行分析不同的甲基化基因而无需生物信息学专家。这意味着任何科学家都可以概述这些甲基组在其研究领域中发挥作用。我们将利用该计划的成功开发所有RNA和DNA种类的甲基化测定，以及诊断测定的开发。