The long-term objective is to develop a general utility instrument capable of inexpensive de novo sequencing that can also be used for re-sequencing projects to recognize genome variation in heterozygous genomes. The system being developed will sequentially, and directly, identify the nucleotides in very long fragments of genomic DNA from a base-dependent electronic signal produced by a nanopore articulated with nanotube probes. The final system is intended to provide a relatively high quality sequence from 6.5-fold coverage of a genome using DNA from fewer than 1 million cells, with no amplification and minimal preparative steps.
The specific aims for the initial 5 year period of this project are: 1. Improve nanopore surfaces to reduce nonspecific adsorption, pore clogging, and electrical noise; 2. Fabricate and test a nanopore detector articulated with integrated nanotubes for molecular identification; 3. Investigate and optimize the electronic properties of nanotube-DNA interactions to control DNA translocation, orientation and nucleotide contrast; 4. Develop new enzymatic methods to better control and limit the rate of DNA translocation through articulated nanopores; 5. Develop algorithms for feature detection and identification of signals from articulated nanopores; 6. Demonstrate single base sensitivity and resolution on single-stranded DNA translocating through a nanopore. If, as proposed here, we are able to resolve each base as it passes through a nanopore at the rate of 104 bases/sec, an instrument with an array of 100 such nanopores could produce a high-quality draft sequence of 1 mammalian genome in ~20 hours at a cost of approximately $1,000/mammalian genome. Genomic sequencing at these sharply reduced costs would make vital contributions to improved human health on many fronts, including the understanding, diagnosis, treatment, and prevention of disease; advances in agriculture, environmental science and remediation; and the genetics of human health and disease derived from the understanding of evolution.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Research Project (R01)
Project #
1R01HG003703-01
Application #
6960761
Study Section
Special Emphasis Panel (ZHG1-HGR-N (M1))
Program Officer
Schloss, Jeffery
Project Start
2005-08-01
Project End
2008-05-31
Budget Start
2005-08-01
Budget End
2006-05-31
Support Year
1
Fiscal Year
2005
Total Cost
$1,838,719
Indirect Cost
Name
Harvard University
Department
Engineering (All Types)
Type
Schools of Arts and Sciences
DUNS #
082359691
City
Cambridge
State
MA
Country
United States
Zip Code
02138
Fleming, Stephen J; Lu, Bo; Golovchenko, Jene A (2017) Charge, Diffusion, and Current Fluctuations of Single-Stranded DNA Trapped in an MspA Nanopore. Biophys J 112:368-375
Deamer, David; Akeson, Mark; Branton, Daniel (2016) Three decades of nanopore sequencing. Nat Biotechnol 34:518-24
Levine, Edlyn V; Burns, Michael M; Golovchenko, Jene A (2016) Nanoscale dynamics of Joule heating and bubble nucleation in a solid-state nanopore. Phys Rev E 93:013124
Rollings, Ryan C; Kuan, Aaron T; Golovchenko, Jene A (2016) Ion selectivity of graphene nanopores. Nat Commun 7:11408
Lu, Bo; Fleming, Stephen; Szalay, Tamas et al. (2015) Thermal Motion of DNA in an MspA Pore. Biophys J 109:1439-45
Szalay, Tamas; Golovchenko, Jene A (2015) De novo sequencing and variant calling with nanopores using PoreSeq. Nat Biotechnol 33:1087-91
Nagashima, Gaku; Levine, Edlyn V; Hoogerheide, David P et al. (2014) Superheating and homogeneous single bubble nucleation in a solid-state nanopore. Phys Rev Lett 113:024506
Hoogerheide, David P; Lu, Bo; Golovchenko, Jene A (2014) Pressure-voltage trap for DNA near a solid-state nanopore. ACS Nano 8:7384-91
Hoogerheide, David P; Albertorio, Fernando; Golovchenko, Jene A (2013) Escape of DNA from a weakly biased thin nanopore: experimental evidence for a universal diffusive behavior. Phys Rev Lett 111:248301
Lu, Bo; Hoogerheide, David P; Zhao, Qing et al. (2013) Pressure-controlled motion of single polymers through solid-state nanopores. Nano Lett 13:3048-52

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