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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：
10220107
负责人：
BRUCE E. SELIGMANN
金额：
$ 47.67万
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 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10220107
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.

快速通道I-II期SBIR解决了NHGRI特别兴趣主题C：“基因组学工具范围从