Principal Investigator/Program director (Last, first, middle): Gundlach, Jens, H. High Accuracy Nanopore Sequencing PROJECT SUMMARY The objective of this proposal is to enable nanopore sequencing technology to reach its fullest potential. With NHGRI funding, our group has played a pivotal role in developing nanopore sequencing. We paved the way to nanopore sequencing by first engineering the highly sensitive pore MspA to provide single-nucleotide resolution and then by controlling the DNA speed through MspA with an enzyme to give the first proof of concept of nanopore sequencing. In this application we outline techniques that will overcome the last hurdle to nanopore sequencing, low base calling accuracy. We request funding through this application to increase the single-passage base calling accuracy of nanopore sequencing by more than an order of magnitude to better than 99%. This goal is a transformative step forward for nanopore sequencing since it opens up ubiquitous applications of this technology in health care and beyond. Additionally, we will explore other improvements and variations that make nanopore sequencing less expensive and more useful.
Our specific aims are: (1) implementing a hybrid-voltage-enzyme control of DNA that dithers DNA back and forth, thereby allowing identification and correction of enzyme error modes, while maximizing the information obtainable from the ion current readings, (2) improving the fundamental components of the nanopore sequencer by (i) improving the pore used for sequencing and (ii) teaming up with helicase experts to develop DNA-controlling enzymes specifically engineered to optimize nanopore sequencing quality, (3) developing techniques that permit high integration density of nanopores to increase sequencing throughput and to minimize DNA input quantity. Our team's success to date has enabled us to form partnerships with prominent collaborators and to gain support from many excellent labs in academia and industry, whose expertise assists us in bringing nanopore sequencing into health care. We will work with our partner labs to complete the aims outlined in this proposal. It is our goal to improve the quality of human life by delivering revolutionary sequencing technology that is accurate, fast and inexpensive. Project Summary

Public Health Relevance

The objective of this proposal is to bring nanopore sequencing technology to its fullest potential. With funding through this program, our group has developed core technologies that are now involved in industrial nanopore sequencing implementation. To be valuable in health care applications the base calling accuracy of nanopore sequencing must be improved. We propose implementation of technology that will increase the base calling accuracy of nanopore sequencing by more than an order of magnitude to better than 99%. This goal is a transformative step forward for nanopore sequencing since it opens up ubiquitous applications of this technology in health care and beyond.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Research Project (R01)
Project #
5R01HG005115-12
Application #
9980937
Study Section
Special Emphasis Panel (ZHG1)
Program Officer
Smith, Michael
Project Start
2009-09-30
Project End
2021-07-31
Budget Start
2020-08-01
Budget End
2021-07-31
Support Year
12
Fiscal Year
2020
Total Cost
Indirect Cost
Name
University of Washington
Department
Physics
Type
Schools of Arts and Sciences
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
Craig, Jonathan M; Laszlo, Andrew H; Brinkerhoff, Henry et al. (2017) Revealing dynamics of helicase translocation on single-stranded DNA using high-resolution nanopore tweezers. Proc Natl Acad Sci U S A 114:11932-11937
Laszlo, A H; Derrrington, I M; Gundlach, J H (2017) Subangstrom Measurements of Enzyme Function Using a Biological Nanopore, SPRNT. Methods Enzymol 582:387-414
Nova, Ian C; Derrington, Ian M; Craig, Jonathan M et al. (2017) Investigating asymmetric salt profiles for nanopore DNA sequencing with biological porin MspA. PLoS One 12:e0181599
Pavlenok, Mikhail; Niederweis, Michael (2016) Hetero-oligomeric MspA pores in Mycobacterium smegmatis. FEMS Microbiol Lett 363:
Comer, Jeffrey; Aksimentiev, Aleksei (2016) DNA sequence-dependent ionic currents in ultra-small solid-state nanopores. Nanoscale 8:9600-13
Laszlo, Andrew H; Derrington, Ian M; Gundlach, Jens H (2016) MspA nanopore as a single-molecule tool: From sequencing to SPRNT. Methods 105:75-89
Bhattacharya, Swati; Yoo, Jejoong; Aksimentiev, Aleksei (2016) Water Mediates Recognition of DNA Sequence via Ionic Current Blockade in a Biological Nanopore. ACS Nano 10:4644-51
Craig, Jonathan M; Laszlo, Andrew H; Derrington, Ian M et al. (2015) Direct Detection of Unnatural DNA Nucleotides dNaM and d5SICS using the MspA Nanopore. PLoS One 10:e0143253
Derrington, Ian M; Craig, Jonathan M; Stava, Eric et al. (2015) Subangstrom single-molecule measurements of motor proteins using a nanopore. Nat Biotechnol 33:1073-5
Morton, Danielle; Mortezaei, Shahab; Yemenicioglu, Sukru et al. (2015) Tailored Polymeric Membranes for Mycobacterium Smegmatis Porin A (MspA) Based Biosensors. J Mater Chem B 3:5080-5086

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