Project Details
Description
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.
Status | Finished |
---|---|
Effective start/end date | 6/1/21 → 5/31/23 |
Funding
- National Human Genome Research Institute: $193,646.00
- National Human Genome Research Institute: $232,500.00
Fingerprint
Explore the research topics touched on by this project. These labels are generated based on the underlying awards/grants. Together they form a unique fingerprint.