Dysregulation of epigenetic mechanisms is known to play important roles in the development and progression several disease states including cancer, autoimmune, neurological and inflammatory disorders. In cancer alone, high frequency mutations in sixty epigenetic regulatory proteins have been identified. Like genome sequencing, epigenetic information can be elucidated across the entire genome and profiling can be used to find epigenetic abnormalities that cause or correlate with disease. Computational integration of genome-wide genetic and epigenetic profiles (epi-genotyping) has enabled identification of novel disease subgroups, with pediatric neuro-oncology being a notable example. Developing a systems solution that facilitates integration of epigenetic data sets is key to enabling personalized medicine solutions for pathologies with epigenetic dependencies. Chromatin immunoprecipitation (ChIP) is the primary method for genome-wide mapping of DNA associated proteins. In contrast to the tremendous advances in DNA sequencing technologies, which are approaching the $1,000 genome, ChIP has only had incremental improvements in work flow, reduction of background and sample size requirements. This SBIR Fast Track proposal intends to develop Epi-Seq, a high throughput multiplexing ChIP-Seq platform to map functional alterations in the epigenome on a scale and time frame suitable for clinical and drug screening applications. With automation compatibility in mind, Epi-Seq combines sample preparation and solubilization of fixed chromatin using PIXUL, a 96-well plate sonicator capable of shearing 96 chromatin samples in less than 30 minutes, with Transposase- Assisted Multiplex ChIP-Seq (TAM-ChIP) to functionally annotate the entire genome in a high throughput format. Multiplexing is achieved with a panel of recombinant AbFlex® ChIP-qualified antibodies targeting various hallmark features of the epigenetic landscape, each of which has been conjugated to a transposase- compatible barcoded oligonucleotide via site-directed conjugation. Phase I feasibility will use PIXUL from human and mouse tissues in Aim1 and compare TAM-ChIP enabled AbFlex® antibodies for key DNA binding proteins and histone PTMs in traditional and TAM-ChIP-seq methods. Phase II product development efforts will optimize TAM-ChIP target multiplexing in Aim 3 and Aim 4 efforts will adapt current workflows for automation compatibility such that a hands-free sample-to-sequence workflow is achieved. Phase II efforts will conclude with alpha-testing by independent laboratories whose feedback will be integrated into subsequent commercialization efforts by Active Motif. Epi-Seq is an enabling technology with potential accelerate development of epigenetic diagnostics, prognostic and therapeutics for diverse human pathologies and will be offered both as a contract service and as a product, sold independently or bundled with the PIXUL instrument.
Public Health Relevance Statement: ChIP (chromatin immunoprecipitation) is the keystone method for epigenetics, defined as regulatory mechanisms which affect heritable changes in cellular phenotypes independent of DNA sequence. This SBIR Fast Track proposal describes development of Epi-seq, an automation compatible systems solution which streamlines ChIP, enabling simultaneous interrogation of multiple targets and provides for integration of complex bioinformatic datasets. This technology will simplify epigenetics, and will accelerate its adoption in drug discovery and personalized medicine.
Project Terms: Adipose tissue; Adoption; Affect; Algorithms; Antibodies; Autoimmune Process; Automation; Benchmarking; Bioinformatics; Biological Assay; Biopsy; Cell Line; Cells; Characteristics; Childhood; Chromatin; chromatin immunoprecipitation; Clinical; commercialization; Complex; Contract Services; Coupled; Cultured Cells; Data; Data Set; Dependence; Development; Diagnostic; Disease; disorder subtype; DNA mapping; DNA Polymerase II; DNA Sequence; DNA sequencing; DNA-Binding Proteins; drug discovery; Drug Screening; Epigenetic Process; epigenome; epigenomics; Evaluation; Feedback; Freezing; Frequencies; genetic profiling; genetic regulatory protein; Genetic Transcription; Genome; genome sequencing; genome-wide; Genomics; Genotype; Hand; Heart; HepG2; Heritability; histone modification; Histones; Human; Human Pathology; human tissue; Individual; Inflammatory; innovation; instrument; instrumentation; K-562; Kidney; Laboratories; Libraries; Liquid substance; Malignant Neoplasms; Maps; Measures; Mediating; Methods; Mind; Morphologic artifacts; Mus; Mutation; neuro-oncology; Neurologic; next generation; next generation sequencing; novel; novel diagnostics; novel therapeutics; Oligonucleotides; Pathology; personalized medicine; Phase; Phenotype; Play; Preparation; Procedures; product development; prognostic; Prosencephalon; Proteins; Protocols documentation; Reagent; Recombinants; Recommendation; Resolution; RNA; Role; Sample Size; Sampling; Site; Small Business Innovation Research Grant; Sonication; success; System; Systems Integration; Technology; Teleconferences; Testing; Therapeutic; Time; Tissue Sample; Tissues; Transposase; whole genome; Work
