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 ?m 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 ?m 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.
Public Health Relevance Statement: This Fast Track Phase I-II project will demonstrate the feasibility of, then optimize and validate, simple and robust TempO-Seq® human epitranscriptomic assays measuring mRNA internally modified with N1-methyladenosine, N6-methyladenosine and 5-methylcytosine at specific known bases, enabling investigators to profile these methylomes across any sample type (cells or FFPE) at less than 1/10th the cost of RNA immunoprecipitation sequencing assays, with single cell sensitivity enabling assay of FACS sorted subpopulations and single cells, with the ability to associate the methylation profile to 130 ?m diameter focal areas of morphology of FFPE sections, with next-day rather than 1 week turn-around, and with automated data analysis. This high risk, high benefit program will be just in time to impact the method, the success, and the speed with which investigators pursue the roles these recently discovered regulated methylations and demethylations of mRNA have in function and disease, accelerating the advancement of this new field from basic research to translational medicine on a platform that is suited to diagnostics, and thus enable RNA epitranscriptomic diagnostic assay development. TempO-Seq methylome assays will not only play a major role in the accelerated understanding of the function of these methylations, but enable those discoveries to be rapidly associated with disease and translated into new therapies and more powerful and precise diagnostic tests and/or enable earlier diagnosis of disease and monitoring for resistance to therapy.
Project Terms: Address; Antibodies; Archives; Area; assay development; base; Basic Science; Benchmarking; Bioinformatics; Biological Assay; bisulfite; bisulfite sequencing; Caliber; Cell Extracts; Cell Line; Cell physiology; Cells; Chicago; Clinical; commercialization; Consult; cost; Data; Data Analyses; Databases; demethylation; Development; Diagnostic; diagnostic assay; Diagnostic tests; differential expression; Disease; disease diagnosis; DNA; Early Diagnosis; epitranscriptomics; Etiology; experimental study; Gene Targeting; Genes; genomic tools; Hela Cells; HepG2; high risk; Histologic; Histology; Human; Initiator Codon; instrument; interest; Interferons; Knowledge; Ligation; Literature; Malignant neoplasm of prostate; Malignant Neoplasms; Maps; Measures; Messenger RNA; Methods; Methylation; methylome; Modification; Molecular Biology; Monitor; Morphology; National Human Genome Research Institute; novel; novel therapeutics; Performance; Phase; Play; Process; programs; Prostate; Protocols documentation; Recovery; Reproducibility; Research; Research Personnel; Resolution; RNA; RNA immunoprecipitation sequencing; RNA methylation; Role; Sampling; Scientist; sequencing platform; Site; Small Business Innovation Research Grant; Specificity; Speed; success; targeted sequencing; Terminator Codon; Testing; therapy resistant; Thick; Time; Tissues; Titrations; transcriptome; transcriptome sequencing; Translating; translational medicine; Translations; Universities; Work
