Precise Length and 5-Methylcytosine Assessments of Repeat Sequences

Project Summary During this program, Electronic BioSciences, Inc. (EBS) will develop and demonstrate a complete, end-to-end, universal methodology for repeat sequence characterization, including precise length and methylation (5- methylcytosine or 5mC) assessments. This technology will be enabled by the development and demonstration of a sequence-targeted sample and library preparation that will enable the isolation and enrichment of any repeat sequence with a known sequential context or loci for disease-specific diagnostics. Specifically, satellite DNA is comprised of sequence repeats that occur in tandem 5 or more times. Among this DNA, microsatellites have a 1–10 nucleotide (nt) repeat pattern and minisatellites have a andgt;10-nt long repeat pattern. A specific type of satellite DNA found in coding regions of the genome are repeat expansions. Increases in repeat sequence length and epigenetic 5mC status have been correlated to a number of diseases, including Myotonic Dystrophy, Huntington’s Disease, Friedreich’s Ataxia, Fragile X, Fragile XE, Amyotrophic Lateral Sclerosis or ALS, etc., and the targeted characterization of these regions would represent a direct approach (relative to whole genome sequencing) towards clinical and point-of-care diagnostics. However, there is no technology presently available that is ideally suited for characterizing these repeat sequences as a result of read length (associated with next generation sequencing approaches) or accuracy (associated with third generation sequencing approaches), which has significantly limited the understanding of the role repeat sequences have in disease states, their diagnostic/prognostic utility, and the development of associated assays. During this Phase I SBIR program, EBS will develop and demonstrate a technology that will allow these regions to be efficiently assessed for disease state diagnostics.Project Narrative This program will develop and demonstrate the feasibility for a complete end-to-end system for targeted repeat sequence characterization, capable of precisely quantifying the repeat length and epigenetic (5mC) status. The proposed approach will implement direct, electronic, nanopore-based, single-molecule, strand sequencing methodology for precision analyses that are not possible with currently available technology. The achievements made during this program will lay the foundation for the development of a multi-disease panel screening platform that will efficiently characterize repeat sequences and the associated disease diagnostics/prognostics, ultimately improving personalized patient care and population health.