Advanced Sequencing Technology Awards 2007

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 2007 awards were announced on August 1, 2007 (See: New Grants Bolster Efforts to Generate Faster and Cheaper Tools for DNA Sequencing).

Go to "$100,000 Genome" Grants

"$1,000 Genome" Grants

Continuous Sequencing-by-Synthesis Based on a Digital Microfluidic Platform

Fair, Richard B.
Duke University, Durham, N.C.
R01 HG004354
$3,686,000 (3 years)

Collaborators: Vamsee Pamula, Michael Pollack (Advanced Liquid Logic)
Ronald W. Davis, Peter Griffin, Mostafa Ronaghi (Stanford Univ.)

Whereas we have made significant progress in demonstrating the power of digital microfluidics in sequencing by synthesis in our current R21 proposal, some key experimental aspects of the technology as applied to achieving long reads need to be demonstrated. Thus, the overarching aim of Year 1 is to extend the read length of the droplet based sequencing-by-synthesis pyrosequencing reaction chemistry by incorporating the following improvements: 1) integrate solid-phase attachment of DNA compatible with repeated droplet washing and exposure to reagent droplets; 2) demonstrate synthesis reaction chemistry in a droplet format immersed in a silicon oil medium; 3) interfacing of the electrowetting chip with off-chip reagent supply sources to achieve uninterrupted 350 base pair reads. Our specific aims for Years 2 and 3 of this proposal are: 1) determine read length and throughput limitations using adaptive reagent delivery strategies with feedback control based on the detected light signal. Demonstrate that homopolymer regions can be sequenced through with no or negligible degradation of the subsequent sequence accuracy; 2) extend the simulation capability to develop a physically based model to estimate the achievable accuracy and use the insights to increase the read length of droplet-based pyrosequencing; 3) develop a strategy and architecture for parallel reactions and form estimates for the electrowetting chip area, CMOS photodetector array size, and electrode size needed to scale to our 10 year goal of 10,000 parallel reactions; 4) demonstrate that electrowetting technology can be scaled to a picoliter droplet format by scaling system dimensions to achieve highly parallel reactions; 5) experimentally determine read length limitations in droplet-based sequencing by synthesis, and implement software and signal processing strategies to improve read lengths and data quality and throughput, with a goal to demonstrate 1,000 to 10,000 base pair reads by Year 3. The research design is based on verifiable subtasks for each aim that are driven by group leaders. Two key inventions underlie our proposed modified pyrosequencing approach to obtain long reads: 1) decouple the sequencing and detection steps use feedback to add separately extra nucleotides at any DNA site where homopolymer regions are encountered and detected; 2) utilize droplet-based electrowetting to handle the massively complicated fluid handling problem.

Sequencing by Recognition

Lindsay, Stuart
Arizona State University, Tempe
R21 HG004378
$877,000 (3 years)

Collaborators: Otto Sankey, Peiming Zhang (ASU)
Advised by Gregory Timp (University of Illinois - Urbana Champaign)

We will explore a new approach to DNA "sequencing by recognition" in nanopores. It is based on a recent report of chemical recognition of the DNA bases via enhanced electron-tunneling when Watson-Crick hydrogen bonded base pairs form between a base-functionalized probe and a base on the DNA to be read. This mechanism is confirmed by preliminary experiments reported in this application. When combined with a nanopore-DNA translocation system that presents each base sequentially to the electronic sensor, it appears that at least 100 million bases per day could be read with continuous sequence runs of at least 80,000 bases. The single molecule base-calling accuracy might approach 99%, in which case an array of 10,000 nanopores would yield the required 99.99% accuracy. In order to establish the plausibility of this approach, two key issues need to be resolved. (a) Can flexible 'molecular wires' bridge the gap between sensing electrodes and the target attachment sites on the DNA to be sequenced? These wires must reach from one electrode to a phosphate, and from another electrode to a base, be flexible enough to form bonds at the same time as being highly conductive. (b) Is the conductance of the whole assembly (metal-linker-phosphate-sugar-base-base-linker-metal) large enough to produce an acceptable single-molecule base calling accuracy? We propose to design and synthesize a number of 'molecular wires' as candidate linkers and measure their single-molecule conductance, comparing our data to the results of first-principles simulations. Once suitable linkers are found, we propose to measure the conductance of the entire system, and the statistical distribution of these conductances. We will also develop a multi-scale simulation of the entire system, to help us optimize the design of a real instrument. We will collaborate with other groups fabricating nanopores to optimize the reagents we develop for the task of nanopore sequencing.

Hybridization-assisted Nanopore DNA Sequencing

Ling, Xinsheng Sean
Brown University, Providence, R.I.
R21 HG004369
$820,000 (3 years)

The objective of the proposed research program is to develop a low-cost nanopore sequencing technology taking advantage of the latest advances in solid-state nanopore technology and utilizing the recently proposed concept of hybridization-assisted nanopore sequencing (HANS). It has been proposed by the PI and coworkers that by hybridizing short matching strands of oligonucleotides, known as DNA probes, and using nanopore ionic conductance to detect the positions of the probes, one can construct DNA sequences using the ionic current profiles, thus avoiding the classical "repeat problem" in the standard sequencing-by-hybridization (SBH) method, and bypassing the harsh requirement of single-base spatial resolution (0.4nm) in the original nanopore sequencing proposal of Kasianowicz, et al. The proposed HANS sequencing technology has the potential of being fast, low-cost and portable. The proposed research is to test the feasibility of the HANS concept. The specific aims of the project are: (1) Determine the spatial resolution of the ionic conductance method using a combined optical tweezers and nanopore setup. (2) Parallel manipulation of DNA translocations in multiple nanopores. (3) Detect hybridization of single DNA probes on a single-stranded ssDNA using optical tweezers and ionic conductance of the nanopore. (4) Re-sequencing a sample DNA sequence to determine accuracy. (5) Develop non-specific DNA-bead binding technology for de novo sequencing. The proposed research will greatly advance the field of bio-nanotechnology and could directly result in a low-cost DNA sequencing technology, which in turn will have far-reaching benefits to the public health of the US in disease detection and in the studies of the molecular processes underlying diseases. The research program provides excellent training opportunities for students and postdoctoral researchers in preparing them to join the workforce of the emerging bio-nanotechnology economy.

Ribosome-based Single Molecule Method to Acquire Sequence Data from Genomes

Mandecki, Wlodek
UMDNJ-New Jersey Medical School, Newark
R01 HG004364
$1,672,000 (3 years)

Collaborators: Emanuel Goldman, Hieronim Jakubowski (UMDNJ)
Barry S. Cooperman, Yale Goldman (University of Pennsylvania)
Julian Borejdo, Ignacy Gryczynski, Zygmunt Gryczynski (Univ. of North Texas Health Science Center)

A method is presented for acquiring sequence data from single nucleic acid molecules. The approach involves a fluorescence resonance energy transfer (FRET) assay based on molecules involved in protein biosynthesis. The fluorescence signal is acquired from single molecules using a fluorescence correlation spectroscope in several configurations, including measurements in solution and on surfaces. The project's specific aims are to: (i) perform site-directed labeling with a fluorescent dye and quencher; (ii) optimize the FRET assay; (iii) construct a synthetic template and demonstrate the performance of the system on this template; (iv) investigate nanostructures capable of enhancing fluorescence, (v) study the behavior of single molecules in the system; and (vi) demonstrate the capability of the system to acquire high volumes of sequence data. We will also explore the use of nanoparticles as fluorescent tags and quenchers. The method, once fully developed, will allow fast analyses of many types of nucleic acids, including whole genomes, and will lead to ultra-low cost genome sequencing, in accordance with the NIH's $1,000 genome program. The method can be used for nucleic acid diagnostics in vitro and in drug discovery.

Nanopore Array Force Spectroscopy Chip for Rapid Clinical Genotyping

Marziali, Andre
University of British Columbia, Vancouver, Canada
R01 HG003248
$746,000 (3 years)

A major barrier to utilization of genotype information in personalized health care is the complexity and time required by existing genotyping processes. Rapid clinical intervention that is guided by genetic information from individual patients will require determination of tens of mutations per patient in less than one hour. Though methods currently exist that are capable of analyzing a single mutation in 30-60 minutes, this greatly limits the utility of genotype information to diseases and predispositions that depend on a single mutation. Methods for analyzing tens to hundreds of mutations require several hours to carry out, and are too slow for an appropriate clinical response. Likewise, high throughput genotyping technologies that can analyze thousands of patients simultaneously are inappropriate for the single-patient/rapid response required in a clinical setting. There is therefore a pressing need, driven by clinical experts and industry, for simple and rapid genotyping technologies capable of analyzing on the order of a hundred loci in a matter of minutes. Nanometer-sized pores in an insulating membrane represent an important new mode of detection and analysis of biomolecules. Though nanopore-based single-molecule detection schemes are already candidates for high throughput DNA sequencing, genotyping is a nearer-term application that is clinically important and will produce benefits to the DNA sequencing community. Multi-nanopore force spectroscopy promises to be a rapid, sensitive and label-free nucleic acid analysis scheme. In previous work we demonstrated the ability to detect sequence at single base resolution using organic nanopore force spectroscopy. In this continuation of our previous work, we aim at the development of solid-state nanopore-based force spectroscopy for rapid electronic detection of sequence variation. We envision the eventual development of a commercial device based on an array of nanoporous membrane elements, each designed to recognize a particular sequence. In this grant application, we propose the development of a device and methods to serve as a proof-of-concept of one element of such an array. We will construct a prototype element, and iterate on fabrication and methods development while testing its sensitivity, and specificity.

Hybridization-assisted Nanopore Sequencing

Oliver, John S.
NABsys, Inc., Providence, R.I.
R43 HG004433
$480,000 (2 years)

Collaborators: Xinsheng Sean Ling (Brown University & NABsys, Inc.)
Amit Basu (Brown University)

The rationales for the development of technology that will enable extremely cheap, high speed sequencing are well established. Chief among these is the enablement of personalized medicine. There are currently in development several technologies that promise to markedly decrease the cost of sequencing a human genome. It is unclear, however, that any of these will be able to do so drastically enough to allow whole genome sequencing to become a routine clinical tool. Additionally, those technologies which are most promising on this cost parameter look as if they will face difficulties with respect to performance characteristics such as read length. One technology that promises to be cheap and fast and to provide long read lengths is nanopore-based sequencing. To date, however, nanopore sequencing has faced a number of technical challenges. The method of Hybridization-Assisted Nanopore Sequencing (HANS) overcomes these hurdles. HANS utilizes libraries of probes to detect subsequences in the target DNA as in sequencing by hybridization (SBH). HANS differs from SBH, however, in that positional information is also extracted thus completely circumventing, the limitations of SBH and making genome length sequencing feasible. The HANS platform will be capable of sequencing a human genome for substantially less than $1000. Additionally, it promises to do so quickly and accurately. Our specific aims are as follows: 1) Synthesize and test oligonucleotide tags for their ability to enhance the nanopore's capacity to detect the presence and determine the positions of the oligonucleotides on the target DNA. 2) Determine the optimal algorithmic approach for sequence reconstruction and estimate values for the performance characteristics of the sequencing platform.

Sequencing DNA by Transverse Electrical Measurements in Nanochannels

Riehn, Robert
North Carolina State University, Raleigh
R21 HG004383
$439,000 (2 years)

We propose to develop a sequencing method for DNA that is based on transverse electrical measurements through the molecule. Our platform will be built around manipulating stretched and linearized DNA in nanofluidic channels, and detection using nanoelectrodes. The nanochannel platform will enable ultralong read frames of >100 kbp, which will greatly help assembly of whole genomes. Nanochannel handling will also enable multiple reads of the same molecule, and good control over the translocation speed. We expect that the technique will ultimately fulfill the cost and performance demands of the $1000-genome. In this exploratory R21 phase we aim to experimentally establish that the basic principles underlying the proposed method are viable. This will form the basis of the development of a device that meets the defined figures of merit. For this exploratory grant (R21) we aim to demonstrate that we can fabricate functional nanoelectrodes/nanochannel device using proven semiconductor and nanoelectronics fabrication techniques (ebeam lithography, electromigration, nanoplating), that an electrical signal due to the DNA exists, and that the obtained signal is sequence specific. We will investigate the mechanism by which the signal arises, and will in particular examine tunneling, electrochemistry, or polarization of counterions as possible candidates. In order to understand the nature of the signal and its usefulness to sequencing, we will prepare electrode configurations with gaps between sub-5 nm (1nm) and 50 nm. Devices will be evaluated using both single and double stranded DNA. We will assess the resolution and quality of the obtained data by sequencing or GC-content mapping of synthetic block oligomers and genomic DNA samples.

High Throughput Low Cost DNA Sequencing Using Probe Tip Arrays

Wickramasinghe, H. Kumar
University of California, Irvine
R01 HG004431
$2,184,000 (3 years)

Collaborator: Robert Moyzis (University of California, Irvine)

Sequencing of the human genome created a large impact in medicine and biotechnology and opened up the realm of personal therapeutics. A major progress-limiting factor is the inherent cost of current technologies reaching millions of dollars per mammalian genome. Our approach is based on a novel implementation of the proven Sanger method for DNA sequencing. We rely on nanotechnology to greatly increase throughput, reduce reagent volumes and drive costs down by orders of magnitude. In preliminary work, we have demonstrated the feasibility of a new approach for electrophoretic separation of DNA fragments along the surface of an Atomic Force Microscope probe tip. We showed that based on this surface electrophoresis method strands up to at least 100 bases can be separated in size with sample quantities down to only a few molecules and with speeds that are four to five orders of magnitude faster than in conventional capillary electrophoresis . We now propose to evolve this methodology toward a complete DNA sequencing scheme based on the Sanger method. Fluorescently labeled DNA fragments resulting from the Sanger reactions are sorted by size into bands . We detect their passage at the end of the probe tip using a novel optical detection scheme. We envision a massively parallel mode of operation in an array of 100x100 probe tips. We aim to develop technology that can ultimately reach the goal of $1000 per genome by the parallel operation of probe tip sequencers with high analysis throughput and ultimately very small volumes of reagents. The specific aims of this research are the following: Aim 1. We demonstrate a scheme for DNA sequencing based on single probe tip electrophoretic separation of DNA fragments up to 100bp. Aim 2. To demonstrate scalability, we address the parallel operation of DNA sequencers with a linear array of 10 probe tips.

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"$100,000 Genome" Grants

Polony Sequencing the Human Genome

Edwards, Jeremy S.
University of New Mexico School of Medicine, Albuquerque
R21 HG004350
$900,000 (3 years)

We propose to further develop and utilize ultra-high throughput polony genome sequencing with the primary goal of re-sequencing the human genome in under one week at a cost of less than $10,000. Currently, the technology is well advanced, but further progress is needed to meet our goals. As the critical quantitative milestone of the project, we will report the sequence for a human genome with "a target sequence quality equivalent to, or better than, that of the mouse assembly published in December 2002 (Nature 420:520, 2002)". The project is divided into four specific aims, which are basically to (1) to improve the polony sequencing technology by increasing the read length, improve the library construction protocol, and increase bead density in the sequencing reactions, (2) improve the design of the polony genome sequencing microscope, (3) improve the computational tools that we have for assembling the raw sequence reads, and finally, (4) re-sequence a cancer genome and identify the somatic mutations that have occurred during tumor development. Progress towards our goals is at an advanced stage. We are able to routinely sequence bacterial genomes and we are on the verge of sequencing an entire human genome to the required quality level. Additionally, we feel that there are substantial rewards to be gained by pursuing the goals described herein.

3'-O-Modified Nucleotide Reversible Terminators for Pyrosequencing

Ju, Jingyue
Columbia University, New York
R21 HG004404
$644,000 (2 years)

Collaborator: Mostafa Ronaghi (Stanford University)

Pyrosequencing in a miniaturized device has been shown to have wide applications in genome sequencing. However, pyrosequencing with natural nucleotides has inherent difficulty in accurately deciphering the homopolymeric regions of DNA templates. The goal of this proposal is to design and synthesize a library of reversible nucleotide terminators to address this issue. The following three aims will be pursued: (1) Design and synthesis of four 3'-O-allyl-labeled nucleotides corresponding to A, C, G, T, as reversible terminators for pyrosequencing. We have produced small quantity of four 3'-O-allyl-labeled nucleotides in sufficient purity and have demonstrated that they can be used for pyrosequencing to produce accurate sequencing data in homopolymeric regions of DNA. We will further optimize the enzymatic conditions to increase the read length of pyrosequencing using the 3'-O-allyl-labeled nucleotides; (2) Design and synthesis of photocleavable 3'-O-modified nucleotide reversible terminators to compare with the 3'-O-allyl-labeled nucleotides to further optimize the pyrosequencing platform in terms of accuracy and readlength; (3) Evaluation of different micron-sized beads to immobilize the DNA template that are compatible to photocleavage or chemical cleavage conditions to perform pyrosequencing using the optimized set of reversible nucleotide terminators developed above. The molecular tools developed here will facilitate the optimization of pyrosequencing for de novo genome sequencing with unparalleled accuracy for biological and biomedical applications.

An Integrated System for DNA Sequencing by Synthesis

Ju, Jingyue
Columbia University, New York
R01 HG003582
$2,217,000 (2 years)

The objective of the proposed research is to develop a system for DNA sequencing by synthesis (SBS) using cleavable fluorescent nucleotide reversible terminators. We will (1) design and synthesize a library of 2'-deoxynucleotide analogues that each consists of a unique fluorescent dye attached to the base through a cleavable linker, and whose 3'-OH group is modulated as a reversible terminator in polymerase reaction. These nucleotide analogues are designed in such a way that after they are incorporated into the growing strand of DNA in the polymerase reaction, the fluorescent dyes are cleaved by irradiation at 355 nm or by specific chemical reagents in a mild condition that is compatible with DNA, and the 3'-OH can be regenerated to continue the polymerase reaction; (2) develop an "ePCR-bead-on-chip (ePBC)" approach for amplifying and immobilizing DNA products for sequencing by synthesis. The ePBC approach is based on emulsion PCR on beads and then covalently immobilizing the beads, each containing many copies of unique amplified DNA templates, on a chip; (3) use the nucleotide analogues and the ePBC method on a 4-color SBS prototype to sequence templates to increase DNA sequence read length. This new system for genome sequencing and resequencing will have wide applications in biology and biomedical research.

Sequence Acquisition from Mapped Single DNA Molecules

Schwartz, David C.
University Of Wisconsin, Madison
R21 HG004379
$882,000 (3 years)

Collaborator: Michael A. Newton (U of Wisconsin at Madison)

The direct analysis of "raw" genomic DNA offers an unfiltered and relatively unbiased view of any genome obviating need for clone libraries and PCR amplicons. This advantage is greatly potentiated by the analysis of large numbers of single DNA molecules for creation of voluminous data sets. As such, the development of a scheme for acquisition of sequence information is proposed using large genomic DNA molecules that will be optically barcoded and analyzed for sequence content, offering resolution approaching that of resequencing. Because large DNA molecules are analyzed, this sequence acquisition scheme obtains information from heterochromatic regions, pinpoints structural variants in human genomes, and characterizes aberrations associated with cancer genomes. This scheme also offers opportunities for linking sequence data from emerging sequencing platforms.

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Last Reviewed: April 4, 2012