Advanced Sequencing Technology Awards 2009

Advanced Sequencing Technology Awards 2009

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 2009 awards were announced online on October 8, 2009 (See: NHGRI Uses Recovery Act Funds to Accelerate Genome Research to Improve Human Health)

Award Abstracts

99.99% Accuracy Direct DNA Sequencing via the Protein Nanopore Method
Barrall, Geoffrey
Electronic Biosciences, LLC, San Diego, Calif.
R01 HG005095
$4,322,000 (4 years)

Collaborators: Cynthia Burrows and Henry White, University of Utah
Xiyun Guan, University of Texas at Arlington
Sergei Nosokov, University of Calgary

Our aim is to develop a DNA sequencing device based upon the blockade of ionic current in the transmembrane protein pore alpha-hemolysin (aHL). Compared to other proposed nanopore-based approaches, the protein pore current blockade (PPCB) method is arguably the simplest, both conceptually and technologically, but recently has been passed over in favor of more complex methods. Recent electronic readout and bilayer lipid membrane advances by the proposers have greatly alleviated prior signal-to-noise ratio and robustness issues. New experimental data and an accurate Stochastic Model for DNA Motion (SMDM) within aHL now indicate threshold feasibility for sequencing by the PPCB method. Inspection of calculated SMDM responses to known input DNA sequences shows that the principal issue for sequencing via the PPCB method is random variance in the order the bases pass through the pore, leading to three quantifiable sources of error. This program will make specific structural and measurement parameter modifications to the present apparatus to reduce each type of error and to produce a minimal overall sequencing error. The final apparatus will be evaluated by sequencing kilobase strands of natural DNA.

Nanopore Sequencing of DNA with MspA
Gundlach, Jens; Aksimentiev, Oleksii; Niederweis, Michael
University of Washington, Seattle
R01 HG005115
$2,988,000 (4 years)

Additional performance sites: University of Illinois Urbana-Champaign, University of Alabama Birmingham

The objective of this project is to engineer a new protein pore, MspA, for nanopore DNA sequencing. MspA's short and narrow constriction, its extreme stability against denaturation and its tolerance to mutations make this protein an ideal, inexpensive and novel nanopore sequencing development platform. We have obtained exciting results that demonstrate the feasibility of our proposal. We designed and made MspA mutants that pass DNA. Importantly, mutated MspA can already nearly resolve single nucleotides using co-passing current alone. Molecular dynamics simulation of MspA agrees excellently with experiment. A prototype fast, low-noise current amplifier was built specifically for nanopore sequencing experiments.

Our specific aims are to (i) rationally design, produce and test MspA mutants to improve DNA base recognition and reduce translocation speed; (ii) use molecular dynamics simulation to understand how DNA interacts with MspA and to optimize MspA for nanopore sequencing; (iii) construct a single chain protein to further improve DNA base sensitivity and control of DNA motion in an asymmetric MspA pore; (iv) construct a highly sensitive electronic amplifier and a practical bilayer apparatus.

We have formed a team of three outstanding labs with complementary expertise in protein science, protein simulation, single-channel experiments, molecular biology, and instrumentation to realize these aims. It is our goal to develop a system that can sequence a human genome for under $1000.

Single Molecule DNA Sequencing by Fluorescent Nucleotide Reversible Terminators
Ju, Jingyue
Columbia University, New York
R01 HG005109
$1,859,000 (3 years)

The ability to sequence a human genome with high accuracy and speed, and at low cost, is critical to the emerging field of personalized medicine. In response to this demand, our research team developed the novel method of DNA sequencing-by-synthesis (SBS) on a solid surface, which has been recognized as a successful new paradigm for deciphering DNA sequences. In this grant application, we will use molecular engineering approaches to take our successful SBS strategy to the next level by adapting it for single molecule sequencing using fluorescent reversible terminators. Template DNA molecules will be attached to a glass surface modified by covalent attachment of PEG-primers under conditions where as many as 1 billion clearly separated single molecules are attached to the slide and their location registered by the presence of a cleavable fluorescent moiety. SBS will then be conducted using reversible blocked nucleotides with an appropriate set of cleavable fluorophores. We will modify a TIRF microscope to create a device with an enhanced microfluidic flow cell platform to permit large-scale detection of single molecules during each cycle of SBS. With a billion DNA templates immobilized on a chip at single molecule resolution, even 30 to 50 base reads will cover the entire human genome at good coverage on a single chip.

DNA Sequencing Using Intrinsic Base Fluorescence
Lakowicz, Joseph R.
University of Maryland, Baltimore
R21 HG005090
$880,000 (3 years)

There are extensive ongoing efforts to develop high-throughput low-cost DNA sequencing with the eventual goal of $1000 for an individual genome. Some approaches use physical properties to identify the bases, but most methods use fluorescent probes as extrinsic labels. Such extrinsic probes are needed because of the low quantum yield of the DNA bases. We propose to develop metallic nanostructures which will increase the brightness of intrinsic nucleotide emission, decrease the background, and efficiently direct the emission toward a detector. Additionally, these structures will provide spectral separation for base calling. These effects are possible due to through-space near-field interactions of the bases with electron clouds in the metal, which are called plasmons. To accomplish single-molecule intrinsic emission base calling we propose:

Specific Aim 1. Use theoretical modeling, primarily the finite-difference time-domain (FDTD) method, to design geometries which enhance base fluorescence, provide directional emission, and which are practical for high throughput sequencing.

Specific Aim 2. Measure the photophysical properties of DNA nucleotides near metal particles which increase the quantum yield.

Specific Aim 3. Determine the detectability and maximum count rates for nucleotides in or near nanoholes in metal films.

Specific Aim 4. Fabricate and test metallic structures which provide directional emission and spectral separation for base calling. We will determine the detection efficiency and accuracy of the base calling.

Development of a Semiconductor-based Platform for Genomic Sequencing
Leamon, John H.; Rothberg, Jonathan Marc
Ion Torrent Systems, Inc. Guilford, Conn.
R01 HG005094
$2,255,000 (2 years)

We propose to develop a novel disposable semiconductor sensor and system able to directly and rapidly read gigabases of de novo sequence. The system is comprehensive, and includes simplified and robust sample preparation technology, and produces data fully compatible with current standards. A new type of semiconductor sensor - an "Ion Torrent Chip" - has been designed and developed to directly detect polymerization of DNA without the need for ANY intermediate enzymatic reactions, chemiluminescence, fluorescence, optics, optical imaging, or other constraints of having to detect light or use unnatural reagents. The system consisting of disposable "Ion Torrent Chips," an integrated chip reader and fluidics, and can be produced at extremely low costs and generate high quality assembled human genome sequence at less then $1,000. At the heart of the system is a semiconductor sensor, with 10's of millions of separate detectors, each capable of sequencing long stretches of DNA. Along with high-speed signal processing, and base-calling algorithms, the system will be able to establish a new gold standard for low cost, diploid assembled genome sequences. Because the heart of the system is a novel sensor built and assembled using standard semiconductor fabrication methodologies, able to sequence without the need for intermediate enzymes, or the constraints of having to image using light, the cost of genome sequencing will continue to fall with each successive generation of denser chips according to Moore's law.

Closed Complex Single Molecule Sequencing
Nelson, John
GE Global Research, Niskayuna, N.Y.
R33 HG004155
$1,344,000 (2 years)

Through almost the entire time that the first Human Genome was being mapped and sequenced, what is now GE Healthcare actively developed and provided essential enzymes, dyes, methods, and instruments to the effort. Now that this effort is complete, NHGRI has recognized that a promising new demand for sequence information will emerge once the cost of obtaining that information is decreased by 1000-fold or more. While a number of promising approaches to collect sequence information quickly and inexpensively are under investigation, GE proposes to use enzyme and dye-tagged nucleotide resources in a new way that will simplify the fundamental, front-end chemistry of massively parallel sequencing-by-synthesis.

GE's proposed method uses the natural catalytic cycle of DNA polymerase to capture just a single nucleotide on an immobilized primer/template. The nucleotide is captured prior to the chemical reaction step of the polymerization cycle, and it can be tagged with a dye attached to the pyrophosphate, a part that eventually is discarded. However, prior to completing the reaction cycle, the tagged nucleotide is identified using a fluorescence scanner that will scan hundreds of thousands of similar molecules at one time. When scanning is complete, the synthesis cycle is finished by simply adding a buffer containing divalent metal ion. The cycle is repeated over and over to generate DNA sequence data for each DNA molecule. This new chemistry will simplify the overall system requirements for sequencing-by-synthesis, permitting much more flexible systems and enabling significantly longer read-lengths.

Deep-Submicron Optical Detection for High-Density, High-Throughput DNA Sequencing
Ryu, Jekwan (Josh)
Lightspeed Genomics, Inc., Santa Clara, Calif.
R44 HG005127
$243,000 (6 months)

This project addresses the looming optical detection bottleneck in sequencing by developing an ultra high-throughput 250nm-scale optical scanner. In addition to a 50X gain in throughput, the approach also supports a 4X feature-size reduction path that can help drive down reagent usage by a factor of 16X. Together, these throughput and density gains provide the needed instrument-side support in the push to break the $1,000 genome barrier. The approach is based on a novel imaging technique called Synthetic Aperture Optics (SAO) that allows a high-resolution image to be reconstructed from a series of low-resolution samples. In this manner, a SAO scanner trades off expensive, time-intensive stage movements with relatively fast imaging samples. Another advantage of SAO is that the number of samples can be dynamically adjusted as the demand for resolution increases or decreases. This fundamental property of SAO allows superior resolution over conventional light microscopy, as well as significantly higher scanning throughput. This project intends to develop a commercially viable prototype SAO scanner and verify its accuracy and throughput by sequencing an ePCR-amplified genomic reference library on 250nm-scale beads. The scanner can potentially be integrated with a range of chemistries and can be used both for de novo sequencing and for re-sequencing. The success of this project will enable the industry to fully exploit gains in sequencing chemistries to drive reagent, instrument, and operation costs down below the $1,000 genome target.

Sequencing by Nanopore Mass Spectrometry
Stein, Derek
Brown University, Providence, R.I.
R21 HG005100
$896,000 (3 years)

The goal of this project is to test the feasibility of a new single-molecule DNA sequencing strategy that combines solid-state nanopores with mass spectrometry. The idea is to sequentially cleave each nucleotide or base from a DNA molecule as it transits a nanopore, then identify each one by determining its mass-to-charge ratio in a mass spectrometer. Identifying the bases of a translocating DNA molecule by mass spectrometry is appealing because: 1) It is an extremely sensitive technique that can easily distinguish the four DNA bases from their significantly different masses; 2) Modern ion detectors can detect the impact of single ions with a quantum efficiency approaching unity; 3) Those same ion detectors register ions as ~ 20 ns electrical pulses, offering a high detection bandwidth that may obviate any need to control the DNA translocation speed; 4) The sequence of DNA is revealed by the order in which ions of different mass impact the detector, and is not affected by variations of translocation speed; 5) Mass measurements are expected to be insensitive to the orientation of a base in the nanopore, which is difficult to control. The success of our strategy hinges on whether ionized bases or nucleotides can be controllably cleaved from the leading end of a DNA molecule as it translocates the nanopore, and transferred into a mass spectrometer that is housed in a vacuum chamber. This project consequently focuses on assembling a nanopore mass spectrometry instrument, and using it to understand and control ionization and molecular fragmentation processes at the liquid-vacuum interface. The specific aims are to: 1) Detect DNA mononucleotides in a quadrupole mass spectrometer coupled to a ?m-scale, chip-based pore; 2) Demonstrate mass spectrometry of single DNA bases ejected from a nanopore; 3) Identify efficient DNA fragmentation and ionization mechanisms; 4) Sequence short DNA homopolymers. Obtaining high quality sequence information (e.g. Q20 bases or better) from DNA homopolymers will demonstrate the viability of the nanopore mass spectrometry technique. This would justify developing a second-generation system, capable of sequencing long, heterogeneous DNA molecules. The $1000 per genome objective would be well within reach.

Nanopore-based Electrical Device for DNA Sequencing
Stolovitzky, Gustavo Alejandro
IBM Corp., Yorktown Heights, N.Y.
R01 HG005110
$2,547,000 (3 years)

The technologies that make sequencing DNA fast, cheap and widely available have the potential to revolutionize bio-medical research and herald the era of personalized medicine. Being able to sequence human genomes for $1000 will enable comparative studies of variations between individuals in both sickness and health. Ultimately it can improve the quality of medical care by identifying patients who will gain the greatest benefit from a particular medicine, and those who are most at risk of adverse reactions. Nanopore-based sequencing technologies attempt to thread a long DNA molecule through a few nanometer wide nanopore and use physical differences between the four base types to read the sequence of bases in DNA. The two major potential benefits of nanopore sequencing are the high speed and the low price. Nanopore sequencing does not need slow and expensive chemistry, therefore electrical-only sequence readout can proceed at highest rates achievable by modern electronics. At present, the nanopore sequencing is still a promise - no single nucleotide resolution has as yet been achieved experimentally. It is very likely that the ability to localize a DNA molecule inside a nanopore with a single base resolution would provide a sufficient time for read-out electronics to determine the base type. We propose a nano-electro-mechanical device (DNA Transistor) capable of controlling the translocation of a single DNA molecule inside a nanopore with single nucleotide accuracy. This function is based on interaction of discrete charges, localized on phosphate groups along the backbone of a DNA molecule, with the externally controlled electric field confined inside the nanopore. The design of the DNA Transistor relies on well researched thin film deposition techniques from the semiconductor industry. The device is a stack of metal and dielectric layers, each a few atoms thin, with a nanopore penetrating through the stack. Voltage differences applied to the metal layers create a trap for the DNA molecule inside the nanopore. By pulsing these voltages, the controlled translocation of the molecule with single base resolution can in principle be achieved. IBM Research is uniquely positioned to implement the proposed idea. Our experimental effort will rely on in-house industry leading semiconductor device fabrication facilities. The experimental component of the effort will be complemented by a modeling and simulation component that will rely on in-house Blue Gene supercomputing capabilities. Our goal is to fabricate the DNA transistor and demonstrate its capability to translocate DNA molecules through the nanopore with single base resolution.

Real-time Single-molecule Nucleic Acid Sequencing with Fluorogenic Nucleotides
Xie, Xiaoliang Sunney
Harvard University, Cambridge, Mass.
R01 HG005097
$2,019,000 (3 years)

Capitalizing on our group's experience on single molecule enzymology, we propose a novel method for multiplex sequencing of individual nucleic acid molecules using a sequencing-by-synthesis approach that employs fluorogenic nucleotide substrates. Upon incorporation of a non-fluorescent, terminal phosphate-labeled nucleotide substrate by a polymerase, a fluorogenic polyphosphate molecule is released, and subject to fast enzymatic digestion, yielding a single fluorophore, the color of which is dependent on the identity of the incorporated nucleotide. To facilitate single molecule fluorescence detection, an individual nucleic acid molecule is confined in a sealed sub-femtoliter nanoreactor, in which the sequencing reaction takes place continuously. Using conventional soft lithography, we fabricate an array of nanoreactors that allow simultaneous, real-time monitoring of thousands of isolated sequencing reactions with a fluorescence microscope and CCD camera. Our new approach offers low reagent cost, long read lengths, easy sample preparation, and high throughput at several megabases per minute. We also propose the integration of a massively parallel single molecule fluorogenic sequencer with microfluidic devices that process and deliver genetic material from a single cell.