By Julia Karow
This article, originally published Oct. 8, has been updated with comments from an NHGRI program director and further information from NHGRI and grant recipients.
The National Human Genome Research Institute has awarded 16 research teams a total of $48 million to develop DNA sequencing technologies that aim to reduce the cost of sequencing a human genome at high quality to $1,000 or less.
Ten of the awards, totaling $19 million, were made under the institute's annual "$1,000 Genome" grant program, which has funded projects to develop "revolutionary" low-cost sequencing technologies since 2004.
Seven additional two-year awards for low-cost sequencing tech development — one to a group that also received funding under the regular $1,000 Genome program this year — were made with $28.7 million in stimulus funding under the American Recovery and Reinvestment Act, and NHGRI selected sequencing technology development as its "Recovery Act Signature Project."
In addition, NHGRI used more than $4 million in ARRA funds to award one-year and two-year "administrative supplements" to at least $4 million in supplemental grants to at least 12 research teams with existing grants for sequencing technology development, according to the NIH grant database.
Jeff Schloss, program director for technology development at NHGRI, told In Sequence last week that the criteria for new projects funded with stimulus money differed from those funded under the "regular" program in that they had to be "projects that could make a major advance in two years," either "some substantial progress toward a real device" or a significant demonstration of feasibility for a new idea.
Seven recipients of new grants are corporate research teams, including one firm — Helicos BioSciences — that already has a sequencing platform in the marketplace, and another, Pacific Biosciences, that plans to commercialize an instrument next year.
Also, a group at GE Global Research that won a '$1,000 Genome' grant in 2006 received funding for the second phase of the project this year.
Corporate "newcomers" to NHGRI's program include IT giant IBM, which won $2.6 million over three years to develop a DNA transistor to control the movement of DNA through a nanopore; Ion Torrent Systems, a low-profile Guilford, Conn.-based startup — founded by 454 Life Sciences co-founder Jonathan Rothberg — that is working on a semiconductor sensor to detect DNA synthesis with no use of optics; Electronic Biosciences, a San Diego-based firm that is developing a protein nanopore strand sequencing technology; and Lightspeed Genomics, based in Santa Clara, Calif., which is working on an ultra-high-throughput optical scanner for sequencing.
In addition, George Church at Harvard Medical School, under his grant, is working with Halcyon Molecular, a low-profile California-based startup that he advises, to develop transmission electron microscopy-based sequencing.
Further, a group of Stanford University that received an award to develop clinical human genome resequencing technology appears to be planning to work with Pacific Biosciences' technology, according to the team's grant abstract.
Schloss said that the "$100,000 genome" barrier was broken some time over the last year, although he said it was difficult to say which sequencing project was the first to surpass that milestone. "I think people are still quoting a range of costs, but I think that sometime between a year ago and now, it [became] believable that you can produce a high-quality genomic sequence for $100,000 or less."
Washington University School of Medicine's Genome Center, for example, a group that "has always been quite careful about counting all the factors in analyzing [its] costs," has been quoting less than $100,000 for generating cancer genomes, Schloss said.
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"That doesn't mean it's easy yet, that's for sure," he added. "A good chunk of the cost is still analysis — that would be one thing that I would hope some of these new technologies could circumvent: to produce data that are not quite so difficult to analyze, to call and assemble." Longer reads, he said, would "be one thing that would help a lot" to reach that goal.
Schloss anticipates that the $1,000 Genome program — applications for the next round of funding are due next week — will continue "until we achieve the goal" of reducing the cost of sequencing by several orders of magnitude.
With that goal in mind, the program might not stop at $1,000 per genome. "Clearly, one would like the cost to be much lower than that, and for the technology to be easy to operate, and distributed one way or another," he said. "It should be widely and readily accessible, and as low cost as we can get it, all while maintaining data quality."
"As long as we see that progress is being made, and even if that should go beyond the $1,000 mark, we would like to continue the program," he said, adding that this will depend on NHGRI's future planning.
This year's funding for low-cost sequencing technology development, listed by total funding amount, goes to the following groups:
Geoffrey Barrall, Electronic Biosciences
99% Accuracy Direct DNA Sequencing via the Protein Nanopore Method, $4.3 million (4 years)
San Diego-based Electronic Biosciences, working in collaboration with researchers at the University of Utah, the University of Texas at Arlington, and the University of Calgary, is developing a nanopore strand sequencing method that measures current blockade as DNA passes through alpha-hemolysin, the same protein pore used by Oxford Nanopore Technologies.
Recent data and accurate modeling of DNA motion in the pore "now indicate threshold feasibility for sequencing by the [protein current blockade] method," according to the grant abstract. The researchers have already identified three "quantifiable" sources of sequence errors, caused by "random variance" in the order the bases pass through the pore, which they aim to reduce by modifying the structure and measurement parameters of their current device.
"The final apparatus will be evaluated by sequencing kilobase strands of natural DNA," according to the abstract.
Electronic Biosciences also won an ARRA-funded two-year supplemental award for an existing SBIR grant, providing an additional $198,000 in fiscal year 2009 (see below).
Xiaoliang Sunney Xie, Harvard University
Real-time Single-molecule Nucleic Acid Sequencing with Fluorogenic Nucleotides, $2.0 million (3 years)
Single-Cell Single-Molecule Digital mRNA Profiling with No PCR Amplification, $1.2 million (2 years, ARRA)
Under its three-year award, Xie's group plans to work on a single-molecule sequencing-by-synthesis method that monitors the release of single fluorophores from terminal-phosphate-labeled nucleotide substrates during DNA synthesis. The sequencing reactions take place in sealed sub-femtoliter nanoreactors, each containing a single DNA strand.
The researchers use conventional soft-lithography to fabricate an array of nanoreactors "that allow simultaneous, real-time monitoring of thousands of isolated sequencing reactions with a fluorescence microscope and CCD camera."
They say the approach will offer "low reagent cost, long read lengths, easy sample preparation, and high throughput at several megabases per minute."
They also propose to integrate their sequencer with devices "that process and deliver genetic material from a single cell."
Under their two-year grant, made with ARRA funds, the researchers want to adapt their DNA sequencing technology for direct mRNA sequencing, using a reverse transcriptase that uses fluorescently labeled nucleotide substrates during the synthesis of cDNA.
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Jens Gundlach, Oleksii Aksimentiev, and Michael Niederweis, University of Washington
Nanopore Sequencing of DNA with MspA, $3.0 million (4 years)
The aim of this team of three research groups, with different areas of expertise, is to engineer another protein nanopore other than alpha-hemolysin — MspA — for DNA strand sequencing, providing potential competition to Oxford Nanopore's long-term plans as well as Electronic Biosciences' approach.
The protein's architecture and stability make it "an ideal, inexpensive, and novel nanopore sequencing development platform," according to the grant abstract, and the group says it has already "obtained exciting results that demonstrate the feasibility of our proposal," which are still unpublished. Specifically, mutated MspA can "already nearly resolve" single nucleotides from co-passing current, and the researchers have built a prototype of a fast, low-noise current amplifier for nanosequencing experiments.
Gundlach, who already won a two-year, $650,000 grant under the same program in 2006, published a paper last year showing that he can detect single molecules of DNA passing through MspA (see In Sequence 1/13/2009). For his existing grant, he also won a two-year supplemental award, paid for by stimulus funding, that totals $276,000 in fiscal year 2009 (see below).
John Thompson, Helicos BioSciences
Providing the $1,000 Genome via Improved Single-Molecule Sequencing, $2.9 million (2 years, ARRA)
Helicos BioSciences plans to use its award to improve its existing platform, the Helicos Genetic Analysis system, in order to increase yield and read length and to reduce error rate.
It plans to achieve this by improving the sequencing surface and nucleotides, developing ordered arrays of DNA primers, and developing dyes with better fluorescence yield, according to the grant abstract, which notes that "many other biological applications that are not possible to carry out with other technologies will be enabled with these improvements." For example, Helicos recently published a paper demonstrating that it can sequence RNA molecules directly, which no commercially available sequencing technology is capable of doing to date (see In Sequence 9/29/2009).
Helicos previously won a $2 million $1,000 Genome grant in 2006, which allowed it "to surmount all the critical technical barriers for single-molecule sequencing at the genome-wide scale," according to the grant abstract. "We have taken a very early stage technology and driven it to a commercial instrument," the abstract notes.
The new funding comes as a welcome shot in the arm for the firm, which has been running low on cash. "The next two years are critical for affirming the commercial viability of this platform and technology," according to the abstract.
Ronald Davis, Stanford University
A Strategy for High-Quality Clinical Resequencing of the Human Genome, $2.8 million (2 years, ARRA)
The goal of this project is to develop a strategy for clinical human genome resequencing, applying sequencing "to large clinical populations for disease gene discovery research" and to "a clinical application that has diagnostic potential," according to the grant abstract.
The project will use single-molecule sequencing "as utilized by the Pacific Biosciences platform." The abstract goes on to indicate that the Stanford group will indeed use PacBio's technology: one of the project's goals is to work on an "intermittent segment sequencing approach" that will use "a set of interspersed reads" from a single DNA molecule, sounding very much like PacBio's "strobe sequencing" approach (see In Sequence 3/12/2009). Another goal is to test "in-solution approaches" to "increase substantially" the fold-coverage of DNA regions of high clinical interest, in combination with genome shotgun sequencing.
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Gustavo Stolovitzky, IBM Research
Nanopore-based Electrical Device for DNA Sequencing, $2.6 million (3 years)
IBM's research group proposes to build a nano-electro-mechanical device, which it calls a "DNA transistor," that can control the translocation of a single DNA molecule inside a nanopore "with single nucleotide accuracy," according to the grant abstract.
The design of the DNA transistor "relies on well researched thin film deposition techniques from the semiconductor industry." It consists of a stack of metal and dielectric layers, each a few atoms thin, with a nanopore penetrating the stack. Voltage differences applied to the metal layers trap the DNA inside the nanopore, and by pulsing the voltages, "the controlled translocation of the molecule with single-base resolution can in principle be achieved."
IBM's effort will "rely on in-house industry-leading semiconductor device fabrication facilities," according to the abstract. Complementing the experiments will be modeling and simulations using the company's Blue Gene supercomputing capabilities.
George Church, Harvard Medical School
Development of Electron-Microscopy-Based Nucleic Acid Polymer Sequencing, $2.5 million (2 years, ARRA)
Church and his colleagues plan to work on an "ultra-low-cost, ultra-fast" sequencing technology that is based on single-atom resolution transmission electron microscopy of heavy atom-labeled DNA. The project is a collaboration with Halcyon Molecular, a company he advises, Church told In Sequence last week.
The method images parallel arrays of labeled single-stranded DNA spaced 3 nanometers apart, and will enable read lengths of "at least" 150 kilobases and "potentially as much as" 2 to 4 megabases "or more, with no special difficulties posed by highly repetitive DNA," according to the grant abstract.
Additional work, and "further funding beyond the scope of this proposal" will potentially enable a human genome to be sequenced "at significantly lower cost and with much greater speed and consensus accuracy/completeness than other proposed third-generation sequencing approaches," the group claims.
The goal is to build a prototype TEM sequencing system "with which we hope to demonstrate the approach's potential by delivering a human reference genome assembly" with "unprecedented consensus accuracy and completeness."
At a conference this spring, Church said that Halcyon Molecular uses osmium- and platinum-labeled DNA to image single-stranded stretched-out DNA using TEM.
John Leamon and Jonathan Rothberg, Ion Torrent Systems
Development of a Semiconductor-based Platform for Genomic Sequencing, $2.3 million (2 years)
Ion Torrent Systems, based in Guilford, Conn., with another location in the San Francisco Bay area, proposes to develop "a novel disposable semiconductor sensor and system able to directly and rapidly read gigabases of de novo sequence," according to the grant abstract.
The company has already designed and developed a new type of semiconductor sensor, which it calls "Ion Torrent Chip" 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."
Its system will consist of disposable chips, an integrated chip reader, and fluidics. It will contain a semiconductor sensor with "10s of millions" of separate detectors, "each capable of sequencing long stretches of DNA."
Using high-speed signal processing and special base-calling algorithms, the system "will be able to establish a new gold standard for low-cost, diploid assembled genome sequences."
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Jingyue Ju, Columbia University
Single Molecule DNA Sequencing by Fluorescent Nucleotide Reversible Terminators, $1.9 million (3 years)
Jing Ju's group at Columbia University has received several grants under NHGRI's Advanced Sequencing Technology program before, most recently last year, when it won a two-year $950,000 award, in collaboration with Nicholas Turro, to work on sequencing with reversible dNTP and cleavable fluorescent ddNTP terminators.
Under his new award, Ju's team plans to take its sequencing-by-synthesis strategy "to the next level" by adapting it for single-molecule sequencing, using fluorescent reversible terminators.
The plan is to attach template DNA to a glass surface that is modified with covalently attached 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." The researchers want to use a modified TIRF microscope to detect single molecules during each cycle of SBS.
The technology will likely not provide long reads: "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," the abstract states.
Stuart Lindsay, Arizona State University
Carbon Nanotubes: A New Synthetic Nanopore for Sequencing, $1.7 million (2 years, ARRA)
Lindsay's group, in collaboration with researchers at Oak Ridge National Laboratory and Columbia University, proposes "an entirely new type of nanopore" for translocating DNA, a single walled carbon nanotube, or SWCNT.
SWCNTs are "relatively homogeneous on an atomic scale, easy to manufacture with no special nanofabrication, and can form excellent electrodes, simplifying tunneling readout and opening the possibility of electrochemical readout," according to the grant abstract.
Because DNA can be trapped inside, they open "a new avenue for control of translocation speed."
The researchers have already built devices in which a single SWCNT connects with two fluid reservoirs and have shown that DNA translocates through it.
Lindsay's group previously won a 3-year, $877,000 grant in 2007, and a 1-year, $370,000 grant in 2008 under NHGRI's program to work on electron tunneling approaches to read DNA sequence. Earlier this year, the researchers published several papers, demonstrating that they can read the base composition of DNA using tunneling (see In Sequence 3/31/2009).
For one of his existing NHGRI grants, Lindsay also won a two-year administrative supplement this year, totaling $552,000 in fiscal year 2009 (see below).
John Nelson, GE Global Research
Closed Complex Single Molecule Sequencing, $1.34 million (2 years)
This grant is the second phase of a two-phase research project to develop a new chemistry that "will simplify the overall system requirements for sequencing-by-synthesis, permitting much more flexible systems and enabling significantly longer read length," according to the grant abstract. The first phase, a two-year $900,000 grant, was awarded in 2006.
The method relies on DNA polymerase capturing a single labeled nucleotide, identifying it using a fluorescence scanner, and completing the synthesis cycle by adding a buffer.
Stephen Turner, Pacific Biosciences
Direct Single Base-Pair Real-Time DNA Methylation Sequencing, $1.2 million (2 years, ARRA)
PacBio will use its award to develop the capability of its single-molecule real-time sequencing platform to detect methylated DNA.
The approach, which the company recently presented at a scientific conference (see In Sequence 9/22/2009), relies on altered DNA polymerase kinetics due to the prescence os 5-methylcytosine in the DNA template.
PacBio also won a one-year supplemental award worth $714,000 (see below) to support the development of its sequencing technology under its existing NHGRI grant, which it received in 2005.
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Mark Akeson, University of California Santa Cruz
Controlling Large DNA Fragments During Nanopore Sequencing, $1.1 million (2 years, ARRA)
Akeson and his colleagues want to test how efficiently nanopores can control and process long DNA templates up to 2.5 kilobases in length "as they are catalytically modified by DNA polymerases."
The group's work will focus on T7 DNA polymerase and the Klenow fragment of DNA polymerase 1, coupled to the alpha-hemolysin protein nanopore.
Derek Stein, Brown University
Sequencing by Nanopore Mass Spectrometry, $896,000 (3 years)
The aim of this project is to test the feasibility of a 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," according to the grant abstract.
Stein recently discussed the approach in an interview with In Sequence (5/26/2009).
Joseph Lakowicz, University of Maryland, Baltimore
DNA Sequencing Using Intrinsic Base Fluorescence, $880,000 (3 years)
This group proposes to develop metallic nanostructures that 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," according to the grant abstract.
The effects are possible due to interactions of the bases with electron clouds in the metal, called plasmons.
Jekwan (Josh) Ryu, Lightspeed Genomics
Deep-Submicron Optical Detection for High-Density, High-Throughput Sequencing, $243,000 (1 year)
Santa Clara, Calif.-based Lightspeed Genomics plans to address "the looming optical detection bottleneck in sequencing" by developing an ultra-high-throughput 250 nanometer-scale optical scanner, according to the grant abstract.
The approach is based on a new imaging technique called Synthetic Aperture Optics that allows a high-resolution image to be reconstructed from a series of low-resolution samples.
The company went through a restructuring phase last year and secured new investments earlier this year (see In Sequence 4/14/2009).
In addition to the grants listed above, the following groups won ARRA-funded administrative supplements for existing DNA sequencing technology grants from NHGRI, listed by fiscal year 2009 funding amount:
Jene Golovchenko, Harvard University, Electronic Sequencing in Nanopores, $745,000 (FY 2009), 2 years
Stephen Turner, Pacific Biosciences, Real-time Multiplex Single-molecule DNA Sequencing, $714,000, 1 year
Stuart Lindsay, Arizona State University, Sequencing by Recognition, $552,000 (FY 2009), 2 years
Jingyue Jun, Columbia University, An Integrated System for DNA Sequencing by Synthesis, $405,000, 1 year
Marija Drndic, University of Pennsylvania, DNA Sequencing Using Nanopore-Nanoelectrode Devices for Sensing and Manipulation, $281,000 (FY 2009), 2 years
Predrag Krstic, UT-Batelle, Oak Ridge National Lab, DNA Transport and Sequencing Through a Quadrupole Gate, $279,000 (FY 2009), 2 years
Jens Gundlach, University of Washington, Nanopore Sequencing of DNA with MspA, $276,000 (FY 2009), 2 years
Steven Benner, Foundation for Applied Molecular Evolution, Near Term Development of Reagents and Enzymes for Genome Sequencing, $267,000 (FY 2009), 2 years
Xiaohua Huang, University of California San Diego, Genome Sequencing by Natural DNA Synthesis on Amplified DNA Clones, $253,000 (FY 2009), 2 years
Andrew Hibbs, Electronic Biosciences, New Platform for Ionic Current Measurement with Application to DNA Sequencing, $198,000 (FY 2009), 2 years
David Schwartz, University of Wisconsin Madison, Sequence Acquisition from Mapped Single DNA Molecules, $59,000, 1 year
Robert Riehn, North Carolina State University Raleigh, Sequencing DNA by Transverse Electrical Measurements in Nanochannels, $49,000, 1 year