Fourth-Generation Nanopore-Assisted Sequencing for Advanced Oncological Analyses

Project Summary Nanopore-based sequencing has numerous advantages over sequencing-by-synthesis (SBS, the current gold standard for accuracy), including its ability to detect genomic variations (on a single molecule basis) and provide long-read, wider genomic contexts, and scaffolds. Additionally, the low cost and simple instrumentation mean that nanopore sequencing can enable accessible and routine clinical cancer screening in the future as comprehensive, disease-specific assays are developed. However, in order to supplant SBS, nanopore-based sequencing techniques must see improvements in methodology to increase accuracy, and this is particularly true for genomic repeats, which represent significant but under-studied regions of the genome. In fact, repetitive DNA sequences comprise almost 50% of the human genome but are still quite challenging to characterize because it is impossible confidently align/order small fragments of the repeat; DNA repeats need to be read continuously and completely (end-to-end). SBS technology specifically is limited by a combination of sequence bias, and polymerase/synthesis error or failure that accumulates during the sequencing process and ultimately limit the technique to short read lengths. Thus, because the most accurate sequencing method on the market (SBS) cannot sequence these regions, their roles in disease progression are largely unknown. However, evidence to date shows DNA repeats have a remarkable quantity of sequence variation(s) for normal vs. diseased cells, and as a function of disease progression. Further, these disease-correlated variations have also been found to indicate therapy success. Unfortunately, the methods used for these studies are primarily academic in nature, relying on complex, qualitative methods that do not transfer to clinical settings. To bridge this technology gap, Dr. Eric Peterson will lead Electronic BioSciences, Inc. (EBS), nanopore technology experts, in developing a novel approach to nanopore-controlled sequencing that will yield state-of-the-art accuracy and read lengths capable of assessing DNA repeats. During this program, EBS will demonstrate functionality of the approach and fully assess the workflow/methodology, including accuracy, against current, standard approaches. To initially characterize the feasibility and practicality of the proposed approach, EBS will also show the ability to multiplex the measurement itself on a small scale, which will position the technology for extensive multiplexing during a follow-on effort, a critical consideration for commercial adoptability. The capability to sequence repetitive sequences with high accuracy and precision, and to identify their variations, will signify a major advancement in cancer genomics, paving the way for improved diagnostics, prognostics, and therapeutics, which will lead to better outcomes in cancer care.