A significant obstacle for scientific progress in biotechnology and medicine is the current cost of sequencing a mammalian sized genome, estimated to be in the range of 10-50 MUSD, and the time required for genome sequencing. Many efforts have therefore been recently undertaken in order to develop cheaper and faster alternatives to the current paradigm of DNA sequencing, and it has been identified that techniques based on single molecule sequencing (SMS) may provide the ultimate cost reduction and speed. It has however, proven difficult to scale down DNA analysis to the single molecule level, mainly due to the relatively small differences between the four nucleotides constituting DNA, and due to the inherent noise in single molecule probing. To this end we have undertaken a radical new approach, designed to circumvent these problems and to provide the means for low-cost, high-throughput DNA sequencing performed at the single molecule level. Our approach consists of two steps: (1) As part of the initial preparation step DNA molecules are converted into longer and periodically structured DNA molecule, which we name """"""""Design Polymers"""""""". (2) The Design Polymers are read in a highly parallel way using optical detection of Nanopore arrays. Based on our preliminary experiments and computer simulations, we project that our method will yield an exceptionally high readout rate of >5 M bases per second, using a 100x100 nanopore array.
The specific aims of the current application are threefold: First, we will fabricate a two-color TIRF system for the high-throughput detection of design polymers in 5x5, 10x10, 25x25 and eventually 100x100 nanopore arrays. In parallel, we will develop computer algorithms for the quasi real-time analysis and error correction of the nanopore data. Second, we will improve and extend the biochemical conversion method used to produce design polymers. Our focus will be on cost reduction, extending read length to 80-mers and massive parallelism (converting thousands of different DNA targets in a single tube). Third, we will harness our methodology to real-life studies, during the lifetime of this award: (a) we will re-sequence a viral genome (such as Lambda phage) as a benchmark test for speed, cost, and accuracy, (b) Detect Polymorphism in the human mitochondria genome, (c) Map regulatory regions in the human genome enriched by Chromatin Immunoprecipitation.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Research Project (R01)
Project #
5R01HG004128-03
Application #
7492135
Study Section
Special Emphasis Panel (ZHG1-HGR-N (O1))
Program Officer
Schloss, Jeffery
Project Start
2006-09-29
Project End
2011-02-28
Budget Start
2009-09-01
Budget End
2011-02-28
Support Year
3
Fiscal Year
2009
Total Cost
$931,269
Indirect Cost
Name
Boston University
Department
Engineering (All Types)
Type
Schools of Engineering
DUNS #
049435266
City
Boston
State
MA
Country
United States
Zip Code
02215
Singer, Alon; McNally, Ben; Torre, Ruby Dela et al. (2012) DNA sequencing by nanopore-induced photon emission. Methods Mol Biol 870:99-114
Soni, Gautam V; Singer, Alon; Yu, Zhiliang et al. (2010) Synchronous optical and electrical detection of biomolecules traversing through solid-state nanopores. Rev Sci Instrum 81:014301
Singer, Alon; Kuhn, Heiko; Frank-Kamenetskii, Maxim et al. (2010) Detection of urea-induced internal denaturation of dsDNA using solid-state nanopores. J Phys Condens Matter 22:454111
Singer, Alon; Wanunu, Meni; Morrison, Will et al. (2010) Nanopore based sequence specific detection of duplex DNA for genomic profiling. Nano Lett 10:738-42
Dudko, Olga K; Mathe, Jerome; Meller, Amit (2010) Nanopore force spectroscopy tools for analyzing single biomolecular complexes. Methods Enzymol 475:565-89
Wanunu, Meni; Morrison, Will; Rabin, Yitzhak et al. (2010) Electrostatic focusing of unlabelled DNA into nanoscale pores using a salt gradient. Nat Nanotechnol 5:160-5
McNally, Ben; Singer, Alon; Yu, Zhiliang et al. (2010) Optical recognition of converted DNA nucleotides for single-molecule DNA sequencing using nanopore arrays. Nano Lett 10:2237-44
Wanunu, Meni; Sutin, Jason; Meller, Amit (2009) DNA profiling using solid-state nanopores: detection of DNA-binding molecules. Nano Lett 9:3498-502
McNally, Ben; Wanunu, Meni; Meller, Amit (2008) Electromechanical unzipping of individual DNA molecules using synthetic sub-2 nm pores. Nano Lett 8:3418-22
Wanunu, Meni; Sutin, Jason; McNally, Ben et al. (2008) DNA translocation governed by interactions with solid-state nanopores. Biophys J 95:4716-25

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