The goal of this project is to establish feasibility for diagnostic sequencing of clinically relevant short tandem repeat (STR) DNA markers on the 3rd generation MinION nanopore (NP) sequencing platform, using tailored STR-specific reagents and analytical software. This new and innovative modality has the potential to create a paradigm shift in clinical-research and diagnostics of repeat disorder, as it can ultimately bring the analysis of recalcitrant STR DNA elements up to par with current genetic NGS assay standards. Our approach leverages the platform?s unique long- and direct-read capabilities for STR applications and comprehensively addresses current NP platform limitations and STR-specific challenges through innovative wetware and interpretive software. Successful implementation of Oxford Nanopore?s sequencing technology for STR analysis will allow high resolution and comprehensive long-range genotyping, improve sample- and gene-multiplexing capabilities, streamline genetic testing workflows and reduce turn-around-time, while markedly shrinking laboratory capital costs and instrumentation footprint to a < $1000 pocket-sized device. Proof of principle for STR sequencing will be pursued for the Fragile-X associated CGG repeat region in the FMR1 gene in the context of a much needed high-throughput and comprehensive Fragile X carrier screening application that will provide FMR1 allele specific sizing and AGG analysis across 96 samples in a single reaction and less than 10 hours to results. Fragile X carrier screening is supported by professional testing guidelines and routinely performed, therefore a single assay that can provide streamlined allele-specific CGG repeat length and AGG interruption profiles is positioned to benefit hundreds of thousands of individuals per year.
The long term goal of this project is to develop and commercialize a novel size-sequencing assay for clinically-relevant short tandem repeat (STR) DNA markers. This approach will be broadly applicable in the diagnosis of multiple neurological disorders, including fragile X syndrome, amyotrophic lateral sclerosis (ALS), Alzheimer?s disease, and myotonic dystrophy. Initially, we will focus on applying this technology to the analysis of the clinically important Fragile X-associated FMR1 gene, and the technology will provide a number of benefits over current methods by enhancing analysis resolution, increasing testing throughput, reducing turn-around time for results and eliminating the need for costly and cumbersome laboratory equipment.
