TempO-Seq Profiling of RNA Epitranscriptomic Modifications - 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.
