Advanced Sequencing Technology Awards 2005
The National Human Genome Research Institute (NHGRI) has continued its coordinated effort to support the development of technologies to dramatically reduce the cost of DNA sequencing, a move aimed at broadening the applications of genomic information in medical research and health care. The 2005 awards were announced on August 8, 2005 (NHGRI Expands Effort to Revolutionize Sequencing).
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"$1,000 Genome" Grants
The National Human Genome Research Institute's (NHGRI) Revolutionary Genome Sequencing Technologies grants have as their goal the development of breakthrough technologies that will enable a human-sized genome to be sequenced for $1,000 or less.
Grant recipients and their approximate total funding are:
Droplet-Based Digital Microfluidic Genome Sequencing
Fair, Richard B.
Duke University, Durham, North Carolina
R21 HG003706-01 approx $510,000 (2 years)
The overall goal of the pilot phase (R21) is to demonstrate how existing droplet-based microfluidic electrowetting technology can be modified to perform sequencing by synthesis reaction chemistry based on the introduction of DNA cloned on microbeads into a droplet that is subjected to repeated cycles of nucleotide addition and washing. The aims are to: 1) adapt electrowetting technology to demonstrate synthesis reaction chemistry in a bead-based droplet format, including bead washing, bead retention, and transport of enzymatic by-products to remote detection sites; 2) demonstrate a quantitative model for the diffusion reaction equations that describe sequencing on microbeads in droplets and the subsequent amount of light generated; and 3) demonstrate through simulation that a single-sided assembly strategy will provide an assembly equivalent to the mouse genome standards. The goal of subsequent research would be to develop an integrated detection strategy for the first genome sequencing demonstration. The major aims of that later phase of the work would be to: 1) adapt electrowetting technology to provide experimental, integrated platforms to support research in synthesis reaction chemistry in a bead-based droplet format, including bead washing, retention, on-chip dispensing, and transport of enzymatic by-products to a remote array with an integrated CMOS photosensor array; 2) demonstrate that electrowetting technology can be scaled to a picoliter droplet format, including on-chip dispensing with ± 2% volume control, reproducible numbers of beads per dispensed droplet, 100% bead retention during washing operations, controlled merging and splitting of bead droplets and wash droplets, and good reproducibility and control of sequencing-by-synthesis processes; 3) experimentally determine read length limitations in droplet-based sequencing-by synthesis, and implement software and signal processing strategies and assembly methodologies to improve read lengths and data quality, with a goal to demonstrate 1,000 to 10,000 base pair reads and to sequence a small genome.
Single-Molecule DNA Sequencing with Engineered Nanopores
Ghadiri, M. Reza
Scripps Research Institute, La Jolla, Calif.
R01 HG003709-01 approx funding $4.2 million (5 years)
The proposed research program is an integrated collaborative effort between two laboratories experienced in nanopore research, protein engineering, and molecular recognition (laboratories of the PI and Hagan Bayley, University of Oxford). The proposal addresses experimentally the most fundamental and critical issues in the field of single-molecule DNA sequencing by the protein nanopore approach, namely the nanopore itself, nucleobase recognition, and the moderation of single stranded (ss)-DNA transit times through the nanopore. The proposed work will establish protein nanopore technology for short (< 1000 base) reads at a considerable price reduction. It is an important step on the path to accurate high-speed genome sequencing at greatly reduced cost. The specific aims of the proposed research program are: (1) Genetically engineered a-hemolysin pores for base recognition. A constriction will be formed within the pore at which a single base confronts a ring of amino acid side chains generated by mutagenesis. The interaction restricts the current flow through the pore and the residual current differs for each base. (2) Chemically modified pores for base recognition. Natural nucleobases and unnatural analogues will be attached at specific sites within the pore. The modifications will provide base recognition and act as molecular brakes to slow the DNA transit time. (3) Attached enzymes to control translocation. Unidirectional DNA transit through the pore will be controlled by DNA polymerases so that sequence determination by amplitude-based recognition can be optimized. (4) Additional improvements to the nanopore through protein engineering. We will examine: (i) Control of the orientation of DNA within the pore with a molecular slide, (ii) Polymer-filled pores to slow DNA transit; (iii) Engineered pores other than a-hemolysin; (iv) Molecular adapters for base recognition. (5) Multipass reading with rotaxanes. DNA trapped as a supramolecular rotaxane can be moved back and forth in the pore by switching the applied potential, allowing multipass sequencing of DNA strands with reduced error rates. (6) Manipulation of the physical conditions. Nucleic acids contain secondary structure. The threading of ss-DNA at high temperatures or from denaturants will improve reads and prevent permanent blockades of the pore.
Electronic Sequencing in Nanopores
Harvard University, Cambridge, Mass.
R01 HG003703-01 approx funding $5.2 million (3 years)
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 10^4 bases/sec, an instrument with an array of 100 such nanopores could produce a high-quality draft sequence of one 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. This collaboration involves the laboratories of the PI along with Daniel Branton (Harvard), and David Deamer, Mark Akeson and Stephen Winters-Hilt (UC Santa Cruz).
Real-Time DNA Sequencing
Hardin, Susan H.
VisiGen Biotechnologies, Inc., Houston, Tex.
R01 HG003580-01 approx funding $4.2 million (3 years)
VisiGen Biotechnologies, Inc. is developing a sequencing system in which polymerase and nucleotides act together as direct molecular sensors of DNA base identity. More specifically, the technology detects the interaction between a modified nucleotide and a fluorescently-modified polymerase. As a nucleotide is incorporated into the growing DNA polymer, energy transfers from an excited donor fluorophore within the polymerase to an acceptor fluorophore within the nucleotide, stimulating the emission of a base-specific incorporation signature that is directly detected in real-time. Cutting-edge technologies, including single molecule detection, fluorescent molecule chemistry, computational biochemistry, and biomolecule engineering, are combined to create this novel sequencing system. DNA samples will be processed in massively parallel arrays that will enable large genomes to be sequenced in less than a day for approximately $1000 and permit sufficient oversampling to produce redundant data that will minimize errors. The system is being developed to identify pathogens (or variations thereof) and to enable comprehensive genome analysis. Proof of principle for VisiGen's technology is demonstrated: sequence information has been detected at the single molecule level in real-time using an immobilized, donor-labeled polymerase and 2 different acceptor-labeled dNTPs. The specific aims of the proposed research are to refine the sequencing chemistry and to design, build and test the next generation single-molecule DNA sequencing instrument. This instrument, Alpha-1, will be placed at VisiGen and accelerate technology development. This instrument will be used for beta testing by researchers at the Baylor College of Medicine, Human Genome Sequencing Center (Houston, TX) and SeqWright, Inc. (Houston, TX). Feedback from personnel involved in these beta tests will be incorporated into the next phase of technology development.
Massively Parallel Cloning and Sequencing of DNA
University of California, San Diego, La Jolla
R01 HG003587-01 approx $750,000 (2 years)
The objective of our research is to develop two innovative technologies: massively parallel whole genome amplification and DNA sequencing by denaturation (SBD). The proposed research will address two technological issues critical for the development of the next-generation sequencing technologies: 1) the development of methods for the parallel clonal amplification of individual DNA molecules from whole genomes; and 2) the development of an ultra high throughput sequencing strategy that can integrate genome-scale sample amplification and processing into the sequencing workflow in an integrated miniaturized device. We have demonstrated that hundreds of millions of single circular DNA molecules can be separated and cloned in massive parallel on solid supports using a powerful isothermal DNA amplification technique called rolling circle amplification (RCA). We have also developed a conceptual framework for the "sequencing by denaturation" technology for rapid and accurate DNA sequencing. We propose to demonstrate the feasibility of separating and cloning individual shot-gun DNA fragments from a whole mammalian-size genome in a small area on a single chip using the rolling circle amplification technology. We also will demonstrate the proof-of-principle of the novel "sequencing by denaturation" method for high throughput DNA sequencing. Accomplishing the proposed milestones will lay down a technological framework for an integrated system that will enable whole genome amplification and sequencing to be carried out in a single miniaturized device.
Modulating Nucleotide Size in DNA for Detection by Nanopore
Columbia University, New York
R21 HG003718-01 approx $970,000 (3 years)
The goal of the proposal is to design and synthesize modified nucleotides to increase their size difference for single molecule DNA analysis by nanopores. We will pursue the following aims to study the feasibility of this approach: (1) Use solid phase synthesis to prepare single stranded DNA consisting of nucleotides carrying different sized modification groups and test these modified DNAs using nanopores to evaluate the parameters that are required to generate distinct blockade signals from each nucleotide in the DNA; (2) With knowledge gained in aim 1, design and synthesize modified nucleotides carrying different size groups for synthesis of modified DNAs in polymerase reaction. The single stranded DNA will then be detected using nanopores to search for condition to guide the design and modification of the nucleotides to achieve distinct blockade signals; (3) Design and synthesize nucleotides carrying small functional groups as hooks for DNA polymerase reaction to generate hook-labeled DNA products. Due to the small size of the hook, these nucleotides are expected to be good substrates for commonly used DNA polymerase to produce DNA products carrying the hook. The single stranded DNA products carrying the hook will then be isolated and selectively reacted with several different large functional groups to increase the size difference among the nucleotides in DNA. This DNA strand with the modified nucleotides will then be detected distinctly by nanopores to produce sequence data. The molecular tools developed here will facilitate achieving the long-term goal of single molecule sequencing by nanopores at single base resolution.
Haplotype Sequencing via Single Molecule Hybridization
New York University, New York
R21 HG003714-01 approx funding $585,000 (2 years)
The goal of this project is to develop, within 10 years, a technology to sequence a human size genome of about 6 gigabases including both haplotypes. We aim to accomplish these goals by successfully integrating three different component technologies: (1) Optical mapping to create ordered restriction maps with respect to an enzyme, (2) Hybridization of a pool of oligonucleotide probes (LNA probes) with single genomic DNAs on surface, and (3) Algorithms to solve "localized versions" of PSBH (positional sequencing by hybridization) problems over the whole genome. The project supports the pilot phase of a two-stage project: (1) Pilot study to assess scientific soundness [R21] and (2) Large-scale system engineering [R33]. The R21 phase aims to demonstrate first the soundness of whole-genome mapping of LNA probe hybridization sites, and then algorithmic feasibility of combining these maps into haplotype sequences. The potential for success of these two aims may be inferred from our preliminary work on (1) haplotype mapping of T. pseudonana and a segment of human chromosome 4; (2) fluorescent imaging of DNA and its validation by AFM technology; and (3) existing body of work on optical mapping by our investigators. Subsequent research would aim to engineer the final system by constructing in succession: (1) high throughput optical system, (2) preliminary validation by sequencing 100bp segment of P. falciparum genome (small-size), (3) more complex validation by sequencing 100bp segment of H. sapiens genome (large-size), (4) final system engineering and validation by sequencing the entire H. sapiens genome.
Sequencing a DNA Molecule using a Synthetic Nanopore
Timp, Gregory L.
University of Illinois, Urbana-Champaign
R01 HG003713-01 approx funding $2.1 million (3 years)
We plan to explore the feasibility of sequencing a DNA molecule using a revolutionary type of silicon integrated circuit that incorporates a nanopore mechanism with a molecular trap. The essential component is a single, nanometer-diameter pore in a robust, nanometer-thick membrane formed from a Metal Oxide Semiconductor (MOS) capacitor. To sequence the molecule, the voltage induced by the dipole moment associated with each base is measured using the electrodes on the capacitor as the DNA translocates through the pore. The 1 nm diameter of the pore is a key specification since it forces the unique dipole moment associated with each base to be nearly transverse to electrodes during a translocation, while minimizing thermal fluctuations and excluding most of the water. Another crucial specification is the thickness of the SiO2 insulator separating the electrodes forming the capacitor. The spatial resolution for sequencing is essentially determined by the SiO2 thickness. With a 1 nm diameter pore and a 0.7nm thick oxide, we expect to be able to measure the electrical signal associated with a single base spanning the insulator during a translocation. To facilitate signal recovery, we intend to trap the molecule during the translocation through the pore, forcing it to oscillate back-and-forth between the electrodes. The oscillation in the position of the DNA allows for narrow-band synchronous detection (lock-in techniques) to be used to improve the electrical signal-to-noise level without compromising the throughput and effectively averages out the noise associated with conformational changes in the DNA and the ion distribution. While we plan to fabricate and test an integrated circuit incorporating a nanopore-capacitor mechanism with a molecular trap and optimize it for sequencing a single molecule of DNA, at the same time we also plan to simulate the performance and test the theoretical resolution of the mechanism using molecular dynamics in conjunction with a self-consistent 3D Poisson solver. This collaboration involves the UIUC laboratories of the PI, Aleksei Aksimentiev, Jean-Pierre Leburton, Klaus Schulten and Stephen Sligar.
Real-time Multiplex Single-Molecule DNA Sequencing
Turner, Stephen W.
Nanofluidics, Menlo Park, Calif.
R01 HG003710-01 approx funding $6.6 million (3 years)
Genome sequencing has revolutionized biology and medicine. A five-fold decrease in sequencing cost over the past ten years has fueled an explosive growth in the availability of genome sequence data for numerous organisms. Despite these advances, the vast majority of the value from sequence data has yet to be realized, as the cost of routine sequencing is prohibitive. Current sequencing technologies based on capillary electrophoresis will likely not allow order-of-magnitude decreases in cost. Alternative sequencing technologies are required. Here we propose to use DNA polymerase enzyme as a fast and frugal sequencing engine by monitoring DNA polymerization in real-time. Nanofluidics, Inc. was established as a spin-out from Cornell University explicitly to leverage two technological advances that enable real-time single-molecule sequencing system. The first is an optical confinement technology, the zero-mode waveguide (ZMW), which allows detection of single nucleotide incorporation in real-time during processive DNA polymerization. The second, terminal-phosphate fluorescent labeling, is a method of attaching fluorophores to nucleotides such that they are automatically removed from the DNA strand after incorporation. By leaving the DNA structure un-hindered with fluorophores, this method allows highly processive incorporation even using 100% replacement with labeled nucleotides. The combination of these technologies eliminates the need for slow and expensive washing of the reaction or un-blocking of the polymerase. Because the polymerase is free-running, the sequence read can proceed as long as the polymerase continues synthesizing, which can be as long as hundreds of thousands of bases. Both the ZMW and the polymerase are small, and the system has no fluidics or moving parts, making the technology amenable to high degrees of multiplexing. The goal of this program is to deploy these technologies in a four-color, real-time, multiplex single-molecule DNA sequencing system that will enable sequencing of a mammalian genome for $50,000 by 2008, and $1000 by 2010. This will be achieve through collaboration with laboratories at Cornell, Stanford, Childrens┐ Hospital Oakland Research Institute, University of Washington, Oak Ridge National Laboratories, the University of Texas, Austin, and Washington University.
"$100,000 Genome" Grants
NHGRI's "Near-Term Development for Genome Sequencing" grants will support research aimed at sequencing a human-sized genome at 100 times lower cost than is possible today. There is strong potential that, five years from now, some of these technologies will be at or near commercial availability.Grant recipients and their approximate total funding are:
Bead-Based Polony Sequencing
Costa, Gina L.
Agencourt Bioscience Corp., Beverly, Mass.
R01 HG003570-01 (Supplement) approx funding $1.2 million (2 years)
The goals of this project are to develop a robust sequencing by synthesis methodology for de novo and resequencing applications using the bead-based polony technology. Our overall R & D focus is to address key aspects of the technology that need to be refined to enable robust, high quality polony sequencing. Our experience in large-scale genome sequencing will serve well to ensure that the key issues involved in optimizing the technology against current industry standards, data processing, management, and analysis are effectively addressed in a time- and cost-efficient manner. The specific aims are to: 1) Develop effective procedures for production of paired-end PCR libraries with virtual insert sizes (distance between read pairs) in the range of 2 to 50 kilobases; 2) Develop methods for effective solid-phase template amplification on derivatized microspheres and for enrichment of beads containing amplified templates; 3) Develop methods for robust array preparation; 4) Develop procedures for fluorescent in situ sequencing by ligation and cleavage; 5) Develop an automated data acquisition system including optics for four-color signal detection, CCD camera, movable stage, peltier flow cell, fluidics system and control software; 6) Develop and implement image analysis, sequence acquisition, data management and assembly software; 7) Develop and produce cleavable ligation substrates; and 8) Develop modified ligase enzymes with improved performance.
Ultra High Throughput DNA Sequencing System Based on Two-Dimensional Monolith Multi-Capillary Arrays and Nanoliter Reaction Volume
Gorfinkel, Vera B.
SUNY Stony Brook, NY
R21 HG003717-01 approx funding $1.5 million (2 years)
The is a feasibility project for the development, implementation and testing of a highly efficient method and an instrument for DNA sequencing capable of both automated re-sequencing and de-novo sequencing of mammalian size genomes at 100-fold reduced cost. The proposed system will be based on highly parallel CE separation and detection of fluorescently labeled dideoxynucleotide-terminated DNA extension product generated by gel matrix-immobilized colonies of single template molecules (polonies) in two dimensional monolith multi-capillary arrays (2D-MMCA). The cost reduction will come from both using nanoliter volume reactions and employing 2D-MMCAs which increase the throughput of the CE separation and detection by at least two orders of magnitude compared to commonly used high-throughput DNA machines. We will perform pilot studies toward the following goals: Development of the technology platform, building of a pilot 55x55 lane automated DNA sequencer capable of sequencing 1,000 bp/second with 450bp Q20 read length at cost of $0.007/kbp, and demonstration of the production scale re-sequencing and automated generation of a genome sequence of the quality of the mouse draft genome at less than $100,000 per mammalian size genome; Further development of the proposed system and introducing the technology and the instrumentation changes enabling de-novo sequencing; Optimization of polonies cultivation technology and obtaining an efficient amplification and cycle sequencing of 3-5kb DNA fragments; Building a pilot 100x300 lane DNA sequencer capable of automated generation of raw sequencing data at 7500 bp/second with 800 bp Q20 read length at cost of $0.002/kbp; Demonstration of the system's potential for de-novo sequencing of human size genome at 10 fold coverage for $100,000 - $200,000.
$100,000 Genome Using Integrated Microfluidic CE
Network Biosystems, Woburn, Mass.
R01 HG003704-01 approx funding $4.5 million (3 years)
The objective of this project is to develop a commercial system for DNA sequencing using microfabricated devices that would enable whole-genome mammalian sequencing for about $100,000. "Sequencing by separation" has been the de facto method of DNA analysis since the 1970s; however current commercial systems that implement this using capillary electrophoresis are unlikely to be developed beyond the point where whole-genome sequencing will cost about $5,000,000. This project will support the development of commercial systems that will advance the price-performance of DNA separation using current, proven genome sequencing methodologies (i.e., PCR and Sanger sequencing) beyond that achievable with capillary systems. Current "assembly line" sequencing requires expensive automation and robotics, as well as the preparation of several hundred times the amount of expensive sample and reagent needed for electrophoresis. This project will eliminate the need for this by using large scale microfabrication to integrate the various component steps in a large scale biochip device. Specific microfabricated systems and sub-systems will be developed that can be deployed as direct replacements of existing components in high and medium-throughput sequencing facilities. Substantial cost reductions are expected from the integration of microscale fluid processes, leading to significant reductions in reagent consumption, and the replacement of expensive liquid-handling automation by dedicated microfluidics-based liquid-handling.
Last Updated: October 3, 2011