The objective of this renewal proposal is to bring nanopore sequencing technology with MspA to fruition. With funding through this program, our group has engineered the porin MspA specifically for nanopore sequencing. By combining MspA with a processive enzyme technology our team demonstrated the visionary goal of nanopore sequencing first conceived two decades ago. With continued funding, we will take the necessary steps to ready nanopore sequencing for industrial integration. We will also explore improvements and variations that make nanopore sequencing an even more powerful tool.
Our specific aims are: (1) develop the base calling algorithms necessary for our present nanopore sequencing process, (2) further refine the porin MspA through rational - and bold - genetic engineering in order to optimize its sequencing quality, (3) understand and improve DNA translocation dynamics by reducing its stochastic character and (4) demonstrate sequencing of long segments of native genomic DNA including identification of epigenetic modifications. Our team's success to date has enabled us to form partnerships and gain support from many excellent labs whose expertise has assisted us to move nanopore sequencing forward. 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 a technique that can accurately sequence a human genome for well under $1000 and provide sequencing results within the short timeframe required for clinical practice.

Public Health Relevance

The objective of this collaborative proposal is to bring nanopore sequencing to full fruition. With previous support we have engineered the porin MspA for nanopore sequencing and combined it with a processive enzyme to demonstrate nanopore sequencing. We propose to take the next steps necessary to ready the process for industrial integration, but we will also explore improvements and variations that make nanopore sequencing even more powerful.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Research Project (R01)
Project #
5R01HG005115-06
Application #
8728979
Study Section
Special Emphasis Panel (ZHG1-HGR-N (M1))
Program Officer
Schloss, Jeffery
Project Start
2009-09-23
Project End
2017-07-31
Budget Start
2014-08-01
Budget End
2015-07-31
Support Year
6
Fiscal Year
2014
Total Cost
$980,000
Indirect Cost
$214,878
Name
University of Washington
Department
Physics
Type
Schools of Arts and Sciences
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
Maffeo, C; Yoo, J; Comer, J et al. (2014) Close encounters with DNA. J Phys Condens Matter 26:413101
Laszlo, Andrew H; Derrington, Ian M; Ross, Brian C et al. (2014) Decoding long nanopore sequencing reads of natural DNA. Nat Biotechnol 32:829-33
Chaudhry, Jehanzeb Hameed; Comer, Jeffrey; Aksimentiev, Aleksei et al. (2014) A Stabilized Finite Element Method for Modified Poisson-Nernst-Planck Equations to Determine Ion Flow Through a Nanopore. Commun Comput Phys 15:
Ross, Brian C (2014) Mutual information between discrete and continuous data sets. PLoS One 9:e87357
Belkin, Maxim; Maffeo, Christopher; Wells, David B et al. (2013) Stretching and controlled motion of single-stranded DNA in locally heated solid-state nanopores. ACS Nano 7:6816-24
Kowalczyk, Stefan W; Wells, David B; Aksimentiev, Aleksei et al. (2012) Slowing down DNA translocation through a nanopore in lithium chloride. Nano Lett 12:1038-44
Manrao, Elizabeth A; Derrington, Ian M; Laszlo, Andrew H et al. (2012) Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase. Nat Biotechnol 30:349-53
Manrao, Elizabeth A; Derrington, Ian M; Pavlenok, Mikhail et al. (2011) Nucleotide discrimination with DNA immobilized in the MspA nanopore. PLoS One 6:e25723
Langford, Kyle W; Penkov, Boyan; Derrington, Ian M et al. (2011) Unsupported planar lipid membranes formed from mycolic acids of Mycobacterium tuberculosis. J Lipid Res 52:272-7
Bhattacharya, Swati; Muzard, L; Payet, L et al. (2011) Rectification of the current in alpha-hemolysin pore depends on the cation type: the alkali series probed by MD simulations and experiments. J Phys Chem C Nanomater Interfaces 115:4255-4264

Showing the most recent 10 out of 12 publications