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NHGRI Awards $18.4M to 10 Groups Developing New Sequencing Technologies

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By Julia Karow

The National Human Genome Research Institute has awarded $18.4 million in new grants under its "$1,000 Genome" Advanced Sequencing Technology program to 10 research groups developing low-cost DNA-sequencing technologies.

"The new technologies will sequence a person's DNA quickly and cost-effectively so [they] routinely can be used by biomedical researchers and health care workers to improve the prevention, diagnosis and treatment of human disease," NHGRI said in a statement.

Several grantees (see below) are affiliated with companies working on new commercial sequencing technologies, including GnuBio, Caerus Molecular Diagnostics, Oxford Nanopore Technologies, Intelligent Bio-Systems, NobleGen Biosciences, and Halcyon Molecular.

About half the new grants, which range in size between $240,000 and $5.1 million and last between one and four years, went to groups previously funded by NHGRI's program, making up the majority of the funding.

That is not surprising, said Jeff Schloss, NHGRI's program director for technology development, since most awards under the program are made for relatively short periods of time, and researchers can apply for renewed funding if they have shown progress.

A number of grants this year, which account for more than half of the new funding, were awarded for nanopore sequencing-related projects. Schloss said that overall, nanopore and nanogap projects account for about 55 percent of NHGRI's current funding commitments under the program in fiscal year 2010.

'The Big Unknown'

Such a sizeable investment in nanopore-based strategies is justified, he said, given the progress researchers in this field have made over the last year or so. For example, he said, scientists have now shown that two different protein nanopores can distinguish the four types of DNA bases (IS 8/24/2010).

In addition, they have now demonstrated several strategies for controlling the translocation rate of DNA through a nanopore — a "big step in the right direction" — and they are making continuous, slow progress on solid-state nanopores.

"If this works, the payoff could be great in terms of rate of data output and read length," Schloss said. "The big unknown is the data quality."

But NHGRI has also placed a number of bets on other approaches to DNA sequencing, Schloss pointed out. In this funding round, for example, these approaches include microfluidic sequencing in nanodroplets, sequencing by electron microscopy, and sequencing by measuring charges that are added during DNA synthesis.

'Pleasantly Surprised'

The goal of NHGRI's Advanced Sequencing Technology program, now in its seventh year, is to reduce to $1,000 by 2014 the overall cost — including not only reagents but "fully-loaded" costs — to produce a high-quality draft of a mammalian genome.

Over the last year, that cost has dropped from about $100,000 to below $40,000, according to the institute. Schloss said that this assessment is based on "a combination of what vendors claim, claims in the [scientific] literature, and prices offered by companies."

Illumina, for example, now offers a human genome sequence for $19,500 under its individual genome sequencing service (IS 6/8/2010), and Complete Genomics recently said that it will charge $11,000 per human genome for a project led by the National Cancer Institute (IS 9/7/2010). However, it is not known if these prices cover the firms' actual costs.

The bar for new sequencing technologies is thus getting higher, Schloss said. "Many of us are pleasantly surprised how close to the $1,000 goal second-generation technologies are taking us."

He said the institute hopes that its grantees will reach that goal by 2014, but noted that the quality originally specified — that of the assembly of the mouse genome published in 2002 — is "not good enough probably even for research, let alone for the clinic."

The aim is therefore not only to drive the cost of sequencing down, but also to drive the quality up.

The following groups, listed by total funding amount, received new grants this year:

Reza Ghadiri, Scripps Research Institute
Single-Molecule DNA Sequencing with Engineered Nanopores
$5,150,000 over 4 years
Collaborators: Hagan Bayley, University of Oxford; Amit Meller, Boston University

Ghadiri and his collaborators will be working on strand sequencing with the alpha-hemolysin nanopore, with the goal of decoding a human genome in 15 minutes by 2014. Specifically, they plan to engineer alpha-hemolysin to improve its base recognition and make it "fit for real-time sequencing," according to the grant abstract. They have two strategies for controlling the translocation of DNA through the pore: by using rotaxanes or DNA polymerase.

In addition, they plan to use DNA polymerase in "two novel sequencing modes, based on nanopore detection of conformational changes associated with nucleobase incorporation." Finally, they want to develop a chip with up to 1 million alpha-hemolysin pores that will be placed in a silicon nitride film, which would avoid using lipid bilayers.

The new grant continues work that Ghadiri and Bayley began in 2005 under a 5-year, $4.2 million grant under the same program (IS 3/4/2008).

The work will most likely benefit Oxford Nanopore Technologies, which holds a large patent portfolio that includes DNA base identification using a biological nanopore, such as alpha-hemolysin.

Bayley is a co-founder of the company, and Meller is on its technical advisory board. Through Bayley's previous grant with Ghadiri, Oxford Nanopore has also had a close relationship with Ghadiri's group. Meller recently co-founded another company (see below) that wants to sequence DNA using solid-state nanopores and optical detection.

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Amit Meller, Zhiping Weng, Catherine Klapperich, Boston University
Single Molecule Sequencing with Nanopore-induced Photon Emission (SM-SNIPE)
$4.16 million over 4 years
Collaborator: University of Massachusetts Medical School

Under this grant, Amit Meller and his colleagues will continue their work to marry solid-state nanopores with optical detection, which requires the DNA to be converted such that each base is represented by several bases (IS 3/16/2010). The aim is to develop a cost-effective sequencing platform that delivers long reads at high speed and with high accuracy.

Earlier this year, Meller co-founded NobleGen Biosciences, which plans to commercialize the technology (5/25/2010).

The grantees will use the new funding to increase the throughput, speed, and accuracy of the technology by employing arrays of up to 10,000 nanopores, using four instead of two colors for readout, and increasing the signal over background.

In addition, they want to optimize the DNA conversion approach on a "commercially available benchtop system," followed by developing a microfluidic device that can convert an entire human genome. Further, they plan to develop data analysis algorithms for base calling, consensus building, sequence assembly, and error proofing.

Meller previously won an exploratory grant under the program in 2004, and a 3-year, $2.2 million grant in 2006.

Jeremy Edwards, University of New Mexico Health Sciences Center, Albuquerque
Polony Sequencing and the $1,000 Genome
$2.758 million over 3 years
Collaborators: Susan Atlas, Dimiter Petsev, University of New Mexico

Under their new grant, Jeremy Edwards and his collaborators plan to further develop polony sequencing, with the goal of generating raw data for a human genome, including library preparation and sequencing, in a week at a cost of under $1,000.

To do that, they plan to increase the read length of polony sequencing, originally developed by George Church's lab at Harvard Medical School, using a cyclic-ligation strategy that involves enzymatic cleavage; increase the read density through different clonal amplification strategies; and develop software that will allow them to call SNPs from raw sequence data. Their progress to date is "at an advanced stage," according to the grant abstract, and they are "on the verge of" sequencing a human genome to the required quality.

Edwards, a former postdoc in Church's lab, won a 3-year, $900,000 grant under the same program in 2007. However, at the time, his goal was to reduce the cost of sequencing a human genome to under $100,000. Edwards has been an early-access user of the Polonator since 2009, a commercial instrument jointly developed by the Church lab and Dover, a Danaher Motion company (IS 5/5/2009).

Steven Gordon, Intelligent Bio-Systems
Ordered Arrays for Advanced Sequencing Systems
$2.646 million over 2 years

The new grant will help IBS develop an improved version of its sequencing platform, which uses sequencing-by-synthesis chemistry, exclusively licensed from Columbia University, to sequence a human genome for "a few hundred dollars" on an instrument that is "10 to 20 percent of the cost of existing platforms," according to a company statement this week.

The grant abstract states that company researchers plan to combine novel chip fabrication techniques, the firm's sequencing chemistry, and its prototype instrument to create an instrument that will produce sequence data "faster and at a lower cost than any other near-term next-generation sequencing system."

The company said this week that it has several operating prototype systems and has used them to sequence the genomes of "several organisms." It also plans to continue ongoing work with unnamed collaborators in clinical laboratories "to develop cost-effective and precise diagnostic tests using our technology." The improvements will allow it to offer targeted diagnostic tests for about $100 per test "even if only a single test on a single patient is ordered."

Last year, IBS representatives said that the first generation of its instrument, PinPoint I, would cost approximately $250,000 and generate about 8 gigabases of sequence per day (IS 3/24/2009). At the time, the firm was planning a beta-test program by the end of 2009 and a commercial launch "shortly after," but it has not yet released its commercial instrument.

In 2006, the company won a 1-year, $425,000 grant under the program, to develop a platform for the "$100,000 genome."

Stuart Lindsay, Arizona State University
Tunnel Junction for Reading All Four Bases with High Discrimination
$868,000 over 3 years

Lindsay's group has already shown that a pair of functionalized tunneling electrodes can be used to generate distinct signals for the different DNA bases (IS 2/16/2010). They now want to extend those measurements to nucleotides and later oligos in solution.

In particular, they aim to eliminate signals that come from more than one nucleotide in the gap at the same time, and to improve the discrimination of a single read. Eventually, they want to develop criteria for the design of a nanopore sequencing system with tunneling electrodes for readout. The group plans to make reagents they develop available to other researchers working on nanopore sequencers that use electron tunneling.

Lindsay previously received a 1-year $370,000 grant under the program in 2008, as well as a 3-year, $877,000 grant in 2007, and an exploratory grant in 2004.

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Murugappan Muthukumar, University of Massachusetts Amherst
Modeling Macromolecular Transport for Sequencing Technologies
$804,000 over 3 years

A newcomer to the program, Muthukumar and his colleagues aim to understand the behavior of DNA in a nanopore under the influence of electrical and hydrodynamic forces. According to the grant abstract, major challenges include the predictability of capture of the target molecule at the nanopore, efficient threading into the pore, and slowing down the translocating molecule.

The researchers plan to use a combination of statistical mechanics theory, computer simulations, and numerical computation of coupled nonlinear equations to address polymer statistics and dynamics, electrostatics, and hydrodynamics in the phenomena of DNA translocation.

Xiaohua Huang, University of California, San Diego
Direct Real-time Single Molecule DNA Sequencing
$803,000 over 2 years

Huang's group, which is new to the program, wants to sequence DNA by measuring base-specific conformational changes in DNA polymerase. Potentially, this method would allow researchers to sequence very long DNA molecules with high fidelity "in minutes" and a human genome or epigenome "in less than an hour."

Specifically, the researchers plan to engineer the polymerase so it has pairs of Förster resonance energy transfer, or FRET, sensors in strategic places on its surface that can monitor conformational changes in real time that occur when the enzyme incorporates nucleotides into DNA.

These structural changes will "very likely" provide a unique signature for each base type, according to the grant abstract, and the researchers want to investigate initially whether there is such a distinguishable FRET signal.

Javier Farinas, Caerus Molecular Diagnostics
Millikan Sequencing by Label-Free Detection of Nucleotide Incorporation
$500,000 over 2 years

Caerus Molecular Diagnostics, another newcomer to the program, wants to explore label-free sequencing by measuring the increase in charge as nucleotides are added to a DNA template attached to a tethered bead, an approach they call "Millikan sequencing."

Javier Farinas, who founded the Los Altos, Calif.-based company in 2008, is a former director of assay development at Caliper Life Sciences.

Caerus has an apparently unrelated US patent application for highly parallel Sanger sequencing entitled "Methods for Sanger Sequencing Using Particle Associated Clonal Amplicons and Highly Parallel Electrophoretic Size-Based Separation." The application was published in May.

According to the grant abstract, the researchers believe that Millikan sequencing will yield long and accurate reads, and that reagent costs will be "negligible" because the method is label-free.

Also, the instrument will only require "relatively simple" optics and therefore have a low cost. Because the DNA on the bead would require relatively less amplification, methods "other than" emulsion PCR would be possible, "making initial sample preparation easier and cheaper" than ePCR. Ultimately, the method "could be used on single molecules," they write, further reducing sample-preparation costs.

The scientists said they plan to use opposing electrical, hydrodynamic, and entropic forces to measure the bead displacement, a function of the length of DNA attached to the bead. Initially, they want to demonstrate the ability to sequence DNA on a single tethered bead, and follow that by developing large bead arrays for high-throughput sequencing.

Dean Toste, University of California, Berkeley
Base-selective Heavy Atom Labels for Electron Microscopy-based DNA Sequencing
$436,000 over 2 years

Toste's group, another new grantee, will collaborate with Halcyon Molecular to develop reagents for selectively labeling DNA bases with heavy atoms for sequencing by transmission-electron microscopy.

The aim is to sequence a human genome with reads of more than 150 kilobases, high consensus accuracy, and lack of sequence-specific bias. Eventually, it should be possible to sequence a human genome accurately and completely in fewer than 10 minutes at a cost of less than $100, according to the grant abstract.

The researchers said they plan to perform proof-of-concept experiments first, using NMR spectroscopy on individual DNA bases. Then, they want to test their reagents on single DNA strands and sequence them by TEM.

Halcyon, founded in 2003 by Michael and William Andregg, won a $1.29 million grant under the program a year ago through a collaboration with George Church's group at Harvard Medical School.

Earlier this year at NHGRI's Advanced Sequencing Technology Development meeting in Chapel Hill, NC, a company researcher showed that the firm has developed a method for arraying long single-stranded DNA on a substrate and is working on reading its sequence by TEM (IS 3/23/2010).

Adam Abate, GnuBio
Microfluidic DNA Sequencing
$240,000 over 1 year

GnuBio, also a new grant recipient under the program, aims to develop a microfluidics platform to sequence DNA in nanodroplets. The company, founded by David Weitz at Harvard University, former 454 vice president Michael Weiner, and former Helicos business development head John Boyce, presented an outline of the technology at a conference this spring (IS 6/8/2010).

According to its grant abstract, the method requires no enzymes and limits the amount of sequencing reagents to several milliliters. It still allows for the template to be amplified, which would enable researchers "to use relatively inexpensive and robust detection."

The method will detect the hybridization of short probes in microfluidic droplets by a shift in fluorescence polarization that distinguishes between bound and free oligos.

The researchers first plan to "describe" a simple platform and resequencing method, which they plan to scale up in the future to enable human genome sequencing for under $1,000.

GnuBio said earlier this year that it plans to deliver beta instruments to early-access customers by mid-December. The first commercial instrument will cost on the order of $45,000.

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