Tunable, narrow molecular weight distribution DNA for nanopore sequencing

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Summary Nanopore sequencing is at the cutting-edge of the DNA sequencing revolution, providing long reads that greatly facilitate genome assembly and identify structural variations. The platform’s flexibility and low barrier to entry make it attractive for remote locations, for rapid analysis during disease outbreaks, and for routine sequencing in laboratories that do not have easy access to, or the need for, large-scale centralized sequencing resources. However, nanopore sequencing is biased towards short DNA owing to transport limitations in the device. The polydispersity of the initial molecular weight distribution of the double-stranded DNA (dsDNA) becomes crucial and typically determines the read lengths. Achieving long read lengths requires controlled breakage of megabase genomic dsDNA into smaller fragments with narrow size distributions with a high average molecular weight. The preferred approach is flow-based scission, which yields sequence-independent break points with low DNA damage. The state-of-the-art, developed 20 years ago, pumps the dsDNA many times through a contraction, with commercial devices producing 90% of the molecules within a factor of 2x of the target weight. Other commonly used approaches include multiple passes through a syringe needle, which results in poorly controlled in molecular weight distributions, or centrifugation in a g-tube, which only accesses lower molecular weights. There is considerable room for improvement on the state-of-the-art for dsDNA scission for nanopore sequencing sample preparation, both in terms of the target molecular weights and, more importantly, the distribution about that target weight. This exploratory R21 project will address the unmet need in nanopore sequencing for DNA samples with a narrow distribution about a tunable target molecular weight. Meeting this need would allow users to balance their relative desire for throughput versus read length, while achieving read-length reproducibility between sequencing runs. The goal is to produce a prototype device and protocols that target molecular weights of 30 kilobases (for standard nanopore sequencing), 70 kilobases (for long-read sequencing), and 100 kilobases (for ultra-long read sequencing), with at least 95% of the molecules within 1.5x of the target weight and < 10% variation between runs. The successful completion of this project will establish the feasibility of using flow to provide a relatively simple, inexpensive device with unprecedented tunability of the target DNA molecular weight and an exceptionally narrow size distribution compared to the state-of-the-art. This project is significant because it will provide a new tool in the nanopore sequencing pipeline, complementing ongoing improvements in the sequencing technique itself by addressing a critical need in sample preparation. The project is innovative in its leveraging of concepts in polymer physics and non-Newtonian rheology to improve genomics.