Advanced Sequencing Technology Awards 2008

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 2008 awards were announced on August 20, 2008 (See: NHGRI Seeks DNA Sequencing Technologies Fit for Routine Laboratory and Medical Use )

Go to "$100,000 Genome" Grants

"$1,000 Genome" Grants

Electronic Sequencing in Nanopores

Branton, Daniel, and Golovchenko, Jene A.
Harvard College, Cambridge, MA
R01 HG003703
$6.5 million (4 years)

The long-term objective is a nanopore detector chip for a general utility instrument capable of inexpensive de novo sequencing that can also be used for re-sequencing projects. The instrument directly generates base-dependent electronic signals as multi-kilobase length fragments of single stranded genomic DNA is driven sequentially through nanopores articulated with electrically contacted single walled carbon 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 or labeling.

The specific aims are:

Characterize ungapped nanotube articulated nanopore detectors in ionic solution with and without DNA molecules to establish a device model.
Control ssDNA binding, translocation, and sliding on the nanotube surface exposed in ungapped nanotube articulated nanopores.
Study and optimize DNA molecule induced field effect modulation of nanotube electrode conductance in ungapped nanotube articulated nanopores as a function of nanotube bias, gate voltage, and solution properties.
Analyze and optimize tunneling current modulations between gapped nanotube electrodes in the first generation detector.
Design and fabricate a second generation detector with embedded 'T' nanotube geometry and achieve 1 Kb/sec sequencing on Kb length strands of DNA.
Design a third generation nanopore detector for high throughput 10 Kb/sec/nanopore sequencing.If we are able to resolve each base as it passes through a nanopore at the rate of 104 bases/sec as proposed here, 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 reduced costs would make vital contributions to improved human health on many fronts, including the understanding, diagnosis, treatment, and prevention of disease; environmental science and remediation; and the genetics of human health and disease derived from the understanding of evolution.

Nanogap Detector (Arrays) Inside Nanofluidic Channel for Fast Real-Time DNA Sequencing

Chou, Stephen Y.
Princeton University, Princeton, NJ
R21 HG004781
$920,000 (3 years)

The goal of the proposed research is to explore a new single-molecule real-time detector, termed "nanogap detector", and the methods to fabricate this detector, that has the potential to directly sequence single bases of an individual DNA molecule at ultra-high speed and ultra-low cost without labeling or copying, as well as other applications in single DNA analysis and manipulation.

Fundamentally different from nanopore-based designs, the nanogap detector uniquely combines a single nanochannel with a nanogap detector (formed by a pair of electrodes with a gap). The nanochannel stretches a single DNA strand into a linear chain and the nanogap sensor inside the channel measures the electrical signal transverse to the DNA backbone as it moves through the channel.

Recently, we have fabricated these nanogap detectors, and have observed, for the first time, the electrical signal perpendicular to the backbone of a single DNA flowing in a nanochannel, and demonstrated (in preliminary tests) sub-5 nm spatial resolution. The detection resolution can be greatly improved by reducing the device dimensions and operating the device in the tunneling-current regime, which is known to have atomic resolution, thus giving us one of the best chances to achieve single base resolution.

The proposed research has two parallel interconnected focuses: (a) to explore the nanogap detectors for fast, real-time, de novo, DNA analysis and sequencing without copying and (in many cases) without labeling; and (b) to further develop innovative nanofabrication technologies and designs that drastically improve nanogap detector sensitivity and functionalities (to the benefit of other real-time single DNA detectors).

The proposed nanogap detectors offer a wide range of possibilities in revolutionary genome sequencing, from measuring DNA length (label-free) and DNA restriction maps (with markers) to potentially our ultimate goal - high speed single base-pair detection (i.e., from low risk but significant, to high risk and high payoff).

The proposed research is based on our many years of experience in nanofabrication and nanodevices in combination with our experience in molecular biology for DNA detection, and will be carried out by a single group of multidisciplinary researchers with electrical engineering, chemistry, biology, and nanotechnology expertise.

DNA Sequencing Using Nanopore-Nanoelectrode Devices for Sensing and Manipulation

Drndic, Marija
University of Pennsylvania, Philadelphia, PA
R21 HG004767
$820,000 (3 years)

Collaborator: Ken Healy (University of Pennsylvania)

The aim of this research effort is to leverage the unique capabilities of our recently developed sub-nanometer precision transmission electron beam ablation lithography (TEBAL) to demonstrate that the precise integration of solid-state nanopores with nanoelectrodes, nanochannels and other nanostructures will address key obstacles that must be overcome to achieve nanopore-based low-cost high-speed sequencing of chromosomal length DNA molecules, and the resultant medical and scientific benefits enabled by this technology. Fast and low cost full genome DNA sequencing will allow, for example, major improvements in the understanding, diagnosis, treatment and prevention of disease, and significant advances in evolutionary research and the understanding of cellular operation.

This project will build on the remarkable progress towards nanopore-based DNA sequencing over the past decade, and it is planned to continue the tradition of sharing results, techniques and nanopore devices with the research community so that the work carried out will make the maximal contribution to advancing the state of the art. It is expected that the nanopore-nanoelectrode devices produced will seed further work by other groups on a variety of transverse electrode sensing methodologies and nanoelectrode-based single DNA molecule manipulation, which will contribute to the achievement of a nanopore-based "$1000 genome sequencer". This development (R21) project will begin work on the long term goals described above by demonstrating the improvements that can be achieved using nanopore devices with integrated nanoelectrodes and nanochannels. Beyond developing reliable nanopore-nanoelectrode devices, the unique aspects of the proposed work include the integration of geometrically more complex electrode patterns to manipulate DNA motion, and the integration of these devices with microfluidics and a fluorescent microscope setup to allow tracking of DNA molecules, so that they can be actively transported to the nanopore.

The specific tasks are to show that:

DNA molecule length can be measured more accurately by transverse sensing with nanoelectrodes.
The translocation speed of double stranded DNA can be reduced by applying forces via nanoelectrodes.
By constraining DNA molecules, length measurement resolution improves for longer molecules.
Individual DNA molecules can be selected, transported to a nanopore and translocated through it.

These objectives will be accomplished in several steps. The required nanopore-nanoelectrode, nanopore-nanochannel and microfluidics devices will first be fabricated and characterized (some of this has already been achieved). Next, experiments with these devices will be conducted to evaluate their performance and identify problems. Finally, several cycles of device refinement and further experiments will resolve these problems and improve device performance to optimal levels, so that achievement of the objectives can be demonstrated.

DNA Sequencing at a Stretch

Gao, Di
University of Pittsburgh, Pittsburgh, PA
R21 HG004769
$370,000 (2 years)

The objective of the proposed research is to develop a revolutionary DNA sequencing technology that is based on pulling DNA strands off a solid surface when stretched under an electric field. It will replace the electrophoresis-based technologies by a separation-at-a-stretch method to separate DNA strands in different lengths produced from the Sanger reaction. Separation is accomplished by attaching the DNA strands to a solid surface and stretching them under an electric field. The anchor that links the DNA to the surface is designed so that the critical force that is needed to detach the DNA strands from the surface is independent of the DNA length, while the stretching force that is applied to the DNA is proportional to the DNA length. Therefore, as the strength of the electric field gradually increases, longer DNA strands are detached first. Through a fluorescence resonance energy transfer (FRET) mechanism, detachment of the DNA strand can be directly detected from the fluorescence signals, which can be used to determine the sequence of the template DNA. Significantly, this simple and efficient method, in principle, has no upper limit on the length of the DNA strands that can be separated based on their length. We propose to use this technology to increase both the read length and the read speed of the current Sanger cycling sequencing process, while eliminating the need of costly equipments for capillary array electrophoresis, thereby reducing the current cost of DNA sequencing by 3 to 4 orders of magnitude. In addition, we propose to use this technology to separate chromosome-sized DNA molecules whose lengths exceed the current limit of electrophoresis-based technologies, with the goal of developing a promising tool to prepare samples for new DNA sequencing approaches (such as nanopore sequencing and sequencing-by-extension) where very long DNA molecules (more than several hundred million base pairs) are needed. In our preliminary studies, we have been able to achieve single-base resolution on the position of adenine (A) base in a fragment of p53 gene determined by stretching DNA strands (with 410-430 bases) produced from ddATP-terminated reaction. We have also demonstrated efficient separation of lambda dsDNA (48,502 bp) from human genomic dsDNA (100 kbp?1 Mbp) based on their length difference, using the proposed method. Further development of this technology as proposed here includes: (1) determination of the DNA sequence by combining data obtained from stretching DNA strands produced from all four dideoxy (ddATP, ddTTP, ddCTP, and ddGTP)-terminated reactions, (2) evaluation of the phred quality value as a function of base position and the read length of this sequencing technology, (3) investigating how the process parameters affect the phred quality value and the read length, thereby searching for optimized process parameters to improve the performance of the technology, (4) increasing the read speed of this method by programming the voltage source and the fluorescence signal detection equipment, and (5) separation of chromosome-sized DNA molecules (10 million to several billion base pairs) based on their lengths.

Genome Sequencing by Natural DNA Synthesis on Amplified DNA Clones

Huang, Xiaohua
University of California, San Diego, CA
R01 HG004804
$2.5 million (4 years)

Collaborator: Pavel Pevzner (University of California, San Diego)

We propose to combine the best proven aspects of SBS with streamlined methods for DNA amplification and high-speed fluorescence imaging to develop and implement a platform for rapid and inexpensive genome resequencing and de novo sequencing. Our platform is called "Natural Sequencing by Synthesis" (nSBS). Amplified DNA molecular clones will be sequenced in massive parallel by cyclic sequencing by synthesis using DNA polymerases and mostly natural nucleotides. The key is to use a small percentage of a cleavable fluorescently-labeled nucleotide along with the natural nucleotide in the cyclic base-by-base DNA sequencing by synthesis process for sequence detection. Not only will the fluorescently-labeled nucleotide incorporation be sparse but the fluorescent moiety will also be cleaved off after each imaging step. This will minimize the modification of the natural structure of the extending DNA template and ensure that DNA synthesis will not be significantly affected. With this strategy, homopolymer tracts can be sequenced and very long read lengths can be achieved. We present a concept for a new breakthrough technology called natural DNA sequencing by synthesis (nSBS). We also present several other breakthrough innovations: 1) In situ massive parallel amplification of single DNA molecules with micro fabricated arrays and rapid assembly of DNA templates. 2) The usage of an automaton to validate and optimize the new nSBS chemistry for cyclic sequencing by synthesis using DNA polymerases and commercially available nucleotides and nucleotides we will design and synthesize for efficient incorporation; 3) The decoupling of the reaction from detection to make the system scalable to very high-density arrays for whole genome sequencing. Since much higher density arrays can be used and only one enzyme (DNA polymerase) will be used, much less reagent will be needed. This will result in dramatic improvement of throughput and reduction in reagent cost. 4) The implementation of a double barrel paired-end strategy and new algorithms for de novo sequence assembly. In the long run this technology will have a great potential to enable very accurate re-sequencing and de novo sequencing of genomes at high speed and much lower cost for biomedical research and personalized medicine.

DNA Transport and Sequencing Through a Quadrupole Gate

Krstic, Predrag S.
Oak Ridge National Laboratory/UT Battelle, LLC, Oak Ridge, TN
R21 HG004764
$720,000 (2 years)

Collaborator: Mark A. Reed (Yale University)

Direct sequencing using electronic measurements is potentially orders of magnitude faster and proportionally less costly than existing methods. The purpose of this project is to prototype and fully characterize a new device for isolation, trapping, localization and control of the DNA motion, which is envisioned as a central component for a future high speed device for the genome sequencing. The proposed device, nanoscale quadrupole Paul trap, represents an alternative to nanopore sequencing with enhanced control capabilities both in translocation and detection. The DNA will be stabilized by a combined static (DC) and radio-frequency (RF) quadrupole trapping electric fields, which control its translocation through the device, resolving one of the main obstacles in reproducible DNA sequencing in a nanogap or nanophore based on reading electrical characteristics of the bases. This additionally allows for spatial modulation and thus detection modulation spectroscopy further enhancing the detection signal-to-noise ratio. Proof of principle of the successful design, fabrication and functionality of the device in increased control of motion and localization of a ssDNA is the principal research goal of the project. This work builds on important results from preliminary molecular dynamics simulations. The result confirms that a nanoscale quadrupole Paul trap is capable of effectively confining ions in an aqueous environment.

The main advantage of the Paul trap is relaxation of critical dimension control, which simplifies the device fabrication. Critical dimension control becomes very problematic at dimensions below 10nm due to resist resolution and pattern transfer limits of electron-beam lithography. This approach avoids these fabrication difficulties since the electrostatic trapping volume (~nm) is significantly smaller than the fabricated dimensions (20-100 nm range). The fabrication scheme is relatively simple, utilizing a self-aligned approach that positions the Paul-trap electrodes automatically in the center of the physical nanopore through which the ssDNA will translocate. Another advantage of a nanofabricated Paul trap is that a visible pathway for massively parallel sequencing device can be identified by using arrays of Paul traps.

The novel idea of localization of single-strand DNA (ssDNA) molecules within a Paul-type quadrupole trap offers increased electrical detection efficiency for heteropolymers confined within a nanopore regardless of detection scheme. The approach lends itself to numerous detection schemes, to be developed in the follow on research. Our long-term objective, to be realized in the time scale exceeding the duration of the current proposal, is fabrication and calibration of a Paul trap device capable to use both the RF and DC detection techniques for precise DNA sequencing. Specifically, we envision two novel detection schemes to be explored for integration with the Paul trap in the final form of the sequencing device: (1) a radio-frequency single-electron transistor (RF SET), and (2) a nitrogen doped-CNT to measure the resonant tunneling current through the gap. The scope of the present proposal is to prototype and fully characterize a nanoscale quadrupole trap for these applications.

Exploration of Solid-State Nanopore Reading Labeled Linear DNA Sequence

Li, Jiali
University of Arkansas, Fayetteville, AR
R21 HG004776
$830,000 (3 years)

Collaborators: David S. McNabb (University of Arkansas)

In this proposal, we investigate a key component of the nanopore sequencing concept. Although the ultimate goal of the nanopore sequencing concept is to read the linear sequence of nucleotides without copying the DNA and without incorporating of labels, here, we explore of reading the linear sequence of a DNA by incorporating biotin labeled nucleotides. We plan to incorporate biotin labeled nucleotides to better differentiate the signal difference between all four nucleotides and conduct the basic research and technology development that are needed to read the linear sequence of a DNA.

The strategy is to first increase the signal difference between bases in a DNA by incorporating Biotin-Labeled Nucleotides (BLN) into one of the four bases in the presence of the other natural nucleotides. For example, Biotin-Labeled Guanine (BLG) will be incorporated into DNA and then we will detect the locations of BLG by a single base resolution nanopore sensing system. We will investigate the ability to detect the positions of BLN in a single-stranded or double-stranded DNA molecule. One DNA molecule with all the same type of nucleotides substituted by a BLN, all G(xj) for example, will move at a controlled speed through a voltage biased solid state nanopore in an electrolyte solution. The translocating DNA will partially block the ionic current flow in the nanopore, and the current blockage signal generated will distinguish the difference between the biotin-labeled G(xj) and the rest of the bases as an electrical current signal GI(tj), here t is the time. Thus by calibrating the relation between xj and tj, the locations of G(x1), G(x2), ..., G(xj) can be estimated from GI(tj). The locations of the other three types nucleotides A(xj), T(xj), and C(xj) can be determined the same way. Thus, the whole sequence of a DNA molecule could be determined. Our initial goal is to sequence a DNA molecule of ~1000 base pair (bp) whose sequence is known. This will permit us to develop the basic technology necessary for solid-state nanopore-based sequencing.

The specific goals in this proposal are: 1. Fabricate and test single base resolution (1-2 nm in thickness) solid-state nanopores. 2. Develop methods to control the rate of DNA translocation through a nanopore at ~100¼s/base. 3. Optimize our nanopore sensing system and data analysis routines to determine the BLN(xj) in a ~1000 base long DNA. Study how accurate a high resolution solid-state nanopore device can determine the location of BLN(xj) in DNA.

Sequencing by Recognition

Lindsay, Stuart
Arizona State University, Tempe, AZ
R21 HG004770
$370,000 (1 year)

Hydrogen-bond mediated electron tunneling can be used to signal the base composition of single stranded DNA. We seek to combine hydrogen-bond mediated molecular recognition with DNA translocation through a nanopore to present each base to a recognition reader in turn. In its simplest embodiment, two types of recognition molecule are each tethered to a pair of electrodes that span a nano-scale gap. As the ssDNA passes the electrodes via a nanopore, the two recognition molecules bind independent sites on the target DNA to complete a tunneling circuit, signaling the presence of the particular base for which the reagents were designed. So long as both molecular recognition events overlap in time, a significant current will flow, with the consequent charge pulse identifying the target base. The full sequence is assembled by juxtaposing data from four different readers. Such a sequence reader could read genomic DNA at a speed of hundreds to thousands of bases per second, do so with high accuracy with a limited number of repeated reads and read sequence lengths of 100,000 bases (or possibly more) in one continuous read.

A first specific aim of this proposal is to demonstrate interaction of single stranded DNA with at least one of the reading elements by comparing the translocation of DNA through bare and functionalized nanopores to see if, and how, the hydrogen bond interactions with the 'reading head' modify translocation.

A second specific aim is to design and test a 'fixed gap' device for as a basis for the development of a prototype reader at a future date.

In this proposal, we address three key issues: (1) Can we replicate the mechanical flexibility and precision of a scanning tunneling microscope (STM) on a chip, so that we can duplicate the high contrast, high accuracy chemical recognition previously achieved with an STM? (2) Can we align such a reading head with a nanopore so as to read the sequence of translocating DNA? (3) Can we design and synthesize "base-readers" that are better than the native bases at recognizing their molecular targets?

We will also examine other issues, such as the role of secondary structure in translocation, and the juxtaposition of data from different reading heads. We have assembled an exemplary team with skills in nanofabrication, molecular electronics and DNA chemistry. We will: (1) Fabricate tunneling gaps on membranes that permit atomic-scale inspection, optimizing the gap fabrication and chemical functionalization for recognition of nucleoside monophosphates and DNA dimers. (2) Use a novel electrodeposition process to align gaps of optimal geometry with nanopores. (3) Study translocation of long DNA molecules through these gaps using magnetic beads both for manipulation and position read-out. (4) Design and synthesize new types of "base-reader". We will make these base-reading reagents available to the community to facilitate exploration of other types of nanoscale readout. We aim to demonstrate a prototype instrument within five years, laying the groundwork for the design and manufacture of a viable commercial instrument.

"$100,000 Genome" Grants

Near Term Development of Reagents and Enzymes for Genome Sequencing

Benner, Steven A.
Foundation for Applied Molecular Evolution, Inc., Gainesville, FL
R01 HG004831
$1.1 million (3 years)

This proposal, targeted for the National Human Genome Research Institute, has the goal of funding an R01 program to develop near term technologies to lower the cost of DNA sequencing. As illustrated by the history of DNA synthesis, costs will most likely drop through a stream of "small step" innovations in chemistry, enzymology, and instrumentation. This proposal, from a laboratory with a track record of innovation in nucleic acid chemistry, enzymology and bioinformatics, will provide this stream, in part by combining individual innovations that have emerged from the Benner laboratories, including nucleoside analogs and polymerases that accept them.

The innovations are:

Artificially expanded genetic information systems (AEGIS), a DNA-like system that forms duplexes following Watson-Crick rules, but has no interaction with natural nucleic acids [Ben04].
A self-avoiding molecular recognition system (SAMRS), a DNA-like molecule whose components do bind natural DNA, but do not bind other components of the same unnatural SAMRS system [Ben07b].
DNA polymerases and reverse transcriptases that accept both AEGIS and SAMRS components.
Emulsion-based directed evolution (EBDE) to optimize polymerases that incorporate unnatural nucleotides.
Reversible terminators that terminate primer extension, but reversibly, and polymerases that accept them.
SNAP2, which allows priming of DNA synthesis with the specificity of a 16mer, but discrimination of 8mers.
Naturally organized genomic databases that support primer design and help analyze the biological meaning of sequence data collected.

These technologies will be combined to develop (a) primers that incorporate SAMRS components, permitting highly multiplexed priming and PCR with primers having an effective length of 16mers (or longer) without creating PCR artifacts and supporting single nucleotide discrimination, (b) AEGIS tags appended to SAMRS primers that allow binning of the non-repeating elements of a patient's genome on an array without the need for single molecule chemistry, and (c) DNA polymerases that accept as substrates triphosphates that combine a fluorescent tag on their gamma phosphorus units with a 3'-ONH2 reversible terminator.

We will benchmark the new combination reagents and their corresponding enzymes to optimize rates of addition of triphosphates in template-directed polymerization reactions, as well maximize discrimination and minimize mismatching. Throughout, we will use the Luminex bead system as a "beta test " platform to examine combination reagents in the context of small-scale " sequencing-during synthesis " tests. The deliverables will be reagents that meet the "wish lists" of NHGRI with respect to cost, helping meet its $100,000 genome sequence goal, with reduced costs likely as further incremental improvements in the chemistry follow.

DNA Sequencing with Reversible dNTP and Cleavable Fluorescent ddNTP Terminators

Ju, Jingyue
Columbia University, New York, NY
R01 HG004774
$950,000 (2 years)

Collaborator: Nicholas Turro (Columbia University)

DNA sequencing by synthesis (SBS) on a solid surface during polymerase reaction can decipher many sequences in parallel. In this grant application, we will pursue the development of a new DNA sequencing method that is a hybrid between the Sanger dideoxynucleotide terminating reaction and SBS. In this approach, four nucleotides, modified as reversible terminators by capping the 3'-OH with a small reversible moiety so that they are still recognized by DNA polymerase as substrates, are combined with four cleavable fluorescent dideoxynucleotides to perform SBS. The ratio of the two sets of nucleotides is adjusted as the extension cycles proceed. Sequences will be determined by the unique fluorescence emission of each fluorophore on the DNA products terminated by ddNTPs. Upon removing the 3'-OH capping group from the DNA products generated by incorporating the 3'-O-modified dNTPs and the fluorophore from the DNA products terminated with the ddNTPs, the polymerase reaction reinitiates to continue the sequence determination. We have already established the feasibility of this new DNA sequencing approach and will further modify the nucleotides and other elements of the system to achieve longer reads in combination with a "primer reset" walking strategy. We anticipate that up to 100 bp of continuous sequences will be produced by this new sequencing method, which will have wide applications in biomedical research projects cost.

Development of a 10Gb Pyrosequencer

Ronaghi, Mostafa
Illumina, Inc., San Diego, CA
R01 HG003571
$5.1 million (3 years)

Collaborators: Helmy Eltoukhy, Stevan Jovanovich (Microchip Biotechnologies, Inc.)

We propose to develop a low-cost 10-Gb Pyrosequencer for de novo DNA sequencing that would enable any lab to perform high-throughput genome analyses. The platform implements automated sample preparation scheme combined with massive Pyrosequencing, which will potentially enable mammalian genome sequencing in a single run. A sensitive CMOS image sensor has been designed and fabricated specialized for Pyrosequencing chemistry, which is integrated with fluidic platform. Each well in the fluidic platform has a pixel on CMOS for detection of light signal generated from Pyrosequencing. The integrated chip will have a 2-megapixel CMOS, each pixel located on a single well enabling 2 million sequencing on each chip. Sixteen of such chips are set up on a board to develop a 32-million wells Pyrosequencer which potentially enable sequencing of more than 10 gigabase of genomic DNA. The raw data are immediately collected and assembled with our developed algorithms. We envision that the genome sequencing of mammalian genome to be reduced below $100,000 level for large-scale genome sequencing projects.

Last updated: April 04, 2012