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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).
"$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
Golovchenko, Jene
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
Huang, Xiaohua
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
Ju, Jingyue
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
Mishra, Bhubaneswar
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
Kellogg, Greg
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 Reviewed: April 29, 2009
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