Advanced Sequencing Technology Awards 2013

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 2013 awards were announced on September 6, 2013 (See: New NIH awards focus on nanopore technology for DNA sequencing)

Award Abstracts

Plasmonic Nanopores for Trapping, Controlled Motion and Sequencing of DNA
Aksimentiev, Oleksii
University of Illinois Urbana-Champaign
R01 HG007406
$680,000 FY13, $2,473,000 (4 years)

Collaborator:  Cees Dekker, Delft University of Technology, The Netherlands

This research project aims to combine the unique and powerful capabilities of two exciting, rapidly evolving fields, plasmonics and nanopores, for the analysis of single DNA molecules.  More specifically, recent advances in nanoplasmonics will be utilized to enable label-free, single-molecule trapping and sequencing of DNA using nanopores.  A novel type of synthetic nanostructure will be developed to strongly focus light to very high intensity in a nanometer-dimension spot where a solid-state nanopore is created.  Through that spot, a DNA molecule will be translocated in a controlled way, allowing the detection of the sequence of the DNA fragments that are sequentially exposed to the intense optical fields of the plasmonic hot spot.  The unique aspect of the program is the use of plasmonic tweezers to control DNA in solid-state nanopores.  This novel approach to advancing DNA through the nanopore simultaneously enables DNA sequence detection through surface-enhanced Raman spectroscopy.  Because locally confined plasmonic fields enhance Raman scattering many orders of magnitude and because of the direct relationship of Raman spectra to the underlying molecular structure, sequence detection will be possible directly, without any labeling.  The project's team is a synergetic combination of experts in biomolecular modeling (UIUC), nanopore experiments (TU Delft) and plasmonic sensing (TU Delft).  The specific aims of the projects are to (i) use a plasmonic field to trap DNA in solid-state nanopores, (ii) develop a method to transport DNA through plasmonic nanopores in discrete, ultimately single-nucleotide steps, and (iii) detect the nucleotide sequence of trapped and moving DNA molecules by means of Raman spectroscopy.

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Haplotype Resolved Sequencing Technology
Edwards, Jeremy S.
University of New Mexico Health Sciences Center, Albuquerque
R01 HG006876
$450,000 FY13, $1,350,000 (3 years)

The focus of this proposal is to develop innovative library preparation tools and techniques to enable haplotype resolved whole human genome sequencing.  Recent advances in Next-Gen sequencing technology, along with the development of robust analysis methods, have given researchers the ability to identify sequence variants.  However, the ultimate goal of relating sequence variants to human diseases is still quite difficult and likely not possible except for very simple single-gene diseases.  Furthermore, even for single gene diseases, when large patient cohorts are studied typically less that 50% of the cases can be linked directly to a genetic variant.  It is my hypothesis that improved sequencing methods are needed to elucidate the complex genotype/phenotype relation.  Namely, new methods are needed to understand the long-range sequence contiguity of human genome and resolve the phase of the sequence variants.  I also hypothesize that long-range genome interactions play a critical role in many diseases, and the cis/trans relation between sequence variants is essential for understanding the genetic basis of disease.  However, all high throughput (Next-Gen) sequencing technologies today generate very short reads, and these short reads are insufficient for phasing sequence variants.  Typically, these sequencing technologies produce results that are limited to finding polymorphisms, and the importance of haplotypes (or the cis/trans phasing of variants) has been largely neglected.  In order to truly understand the genetic makeup of a specific disease there is a need to develop methods to identify the specific chromosome of all polymorphisms, and this is the focus of this proposal.

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Nanopore Sequencing of DNA with MspA
Gundlach, Jens
University of Washington, Seattle
R01 HG005115
$829,500 FY13,   $3,829,500 (4 years)

Collaborator:  Michael Niederweis, University of Alabama, Birmingham

The objective of this renewal proposal is to bring nanopore sequencing technology with MspA to fruition.  With funding through this program, our group has engineered the porin MspA specifically for nanopore sequencing.  By combining MspA with a processive enzyme technology our team demonstrated the visionary goal of nanopore sequencing first conceived two decades ago.With continued funding, we will take the necessary steps to ready nanopore sequencing for industrial integration.  We will also explore improvements and variations that make nanopore sequencing an even more powerful tool. Our specific aims are:  (1) develop the base calling algorithms necessary for our present nanopore sequencing process, (2) further refine the porin MspA through rational - and bold - genetic engineering in order to optimize its sequencing quality, (3) understand and improve DNA translocation dynamics by reducing its stochastic character and (4) demonstrate sequencing of long segments of native genomic DNA including identification of epigenetic modifications. Our team's success to date has enabled us to form partnerships and gain support from many excellent labs whose expertise has assisted us to move nanopore sequencing forward.  We will work with our partner labs to complete the aims outlined in this proposal. It is our goal to improve the quality of human life by delivering a technique that can accurately sequence a human genome for well under $1000 and provide sequencing results within the short timeframe required for clinical practice.

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An Integrated System for Single Molecule Electronic Sequencing by Synthesis
Ju, Jingyue; Church, George M.; Russo, James John
Columbia University, New York, Morningside
R01 HG007415
$1,750,000 FY13, $5,250,000 (3 years)

Additional sites and collaborators:  Harvard Medical School
John Kasianowicz, National Institute of Standards and Technology
Randall Davis, Genia Technologies, Inc.
Ken Shepard, Columbia University

There is a great need to reduce the cost of DNA sequencing to achieve the goal of the $1000 genome.  We recently developed a new nanopore-based sequencing by synthesis (Nano-SBS) approach.  In this project, we will pursue the development of the Nano-SBS approach into a high throughput real-time single-molecule sequencing platform.  In the Nano-SBS method, a polymer tag of distinct size and charge is attached to the terminal phosphate of each of the four nucleotides.  When the complementary nucleotide analog enters a template-primer-polymerase complex that is attached to the nanopore during the polymerase reaction, the tag specific for that nucleotide is captured in the voltage gradient within the nanopore and results in a current blockade unique to each tag for sequence determination.  The polymerase is covalently attached to the nanopore by a short linker so the polymeric tag will have sufficient time to enter the vestibule and constriction of the nanopore prior to its release ensuring that its current blockade signal is recorded by the nanopore.  The extended DNA strand bears only natural nucleotides, enabling long reads.  We have carried out the key proof-of-principle experiments to demonstrate the feasibility of this approach.  Here our strong team of nucleic acid chemists, genomic scientists, electrical engineers, and nanofabrication experts will further develop the Nano-SBS as a high throughput genomic sequencing system.  We will develop robust methodology to attach polymerase to the ?-hemolysin (AHL) nanopore and synthesize nano-tags with unique chemical properties resulting in AHL current blockades distinct from each other and nucleotide precursors.  We will test these elements in single pores as well as in new nanopore array chips with separate sensors and circuits for each pore.  We will produce mutant AHL and polymerase constructs and link them to each other, selecting for the combination that assures accurate DNA extension reactions, and rapid capture and detection of tags in nanopores.  The nanopore chips will be enhanced and expanded from the current 260 nanopores to over 125,000 using advanced nanofabrication techniques.  We will conduct real-time single molecule Nano-SBS on DNA templates with known sequences to test and optimize the overall system.  These research and development efforts will lay the foundation for the production of a commercial single molecule electronic DNA sequencing platform, which will enable routine use of sequencing for medical diagnostics and personalized medicine.

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Accurate, Long Read Length, Whole Human Genome Sequencing Under $100
Kotseroglou, Theofilos
Eve Biomedical, Inc., Mountain View, California
R43 HG007386
$250,000 FY13, $493,000 (2 years)

The long-term goal of the proposed research is the design and construction of a DNA sequencing system that can sequence the whole human genome under $100 including sample preparation and with the cost of goods of the system well under $10,000.  The system developed under the proposed research is at least an order of magnitude in cost better than the target of the solicitation while maintaining all performance metrics.  In that respect it will break the barrier towards genomic medicine.  The overall system is based on optically sequencing DNA on a consumer cell-phone camera chip by means of a transducer that relates the nanometer scale dynamics of single base of a DNA to a macro-motion that is easy to image without the use of fluorescence, but only with a single common white-light LED.  Preliminary data shows that the proposed method can already achieve DNA readlength of 1,600 bases and accurate sequencing alignment of 100 base sections.  While this is a great start, Phase I work will continue to investigate the accuracy and readlength and throughput limits of this approach.  To achieve that, software tools such as image processing and bioinformatics need to be constructed at this first phase, while at same time we will need to improve the biology assays.  If successful, the main goal is to completely sequence a microbial DNA (E. coli) at the end of Phase I, and thus completely benchmark the proposed architecture.  Beyond Phase I, a low cost benchtop system will be constructed using commercial cell-phone camera chips and will be used to perform sequencing of Whole Human Genome with the target cost defined as goal of this grant solicitation.

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Modeling Macromolecular Transport Through Protein and Solid-State Nanopores
Muthukumar, Murugappan
University of Massachusetts Amherst
R01 HG002776
$269,000 FY13, $1,072,000 (4 years)

The urgent need to develop low-cost and high-quality revolutionary technologies for sequencing mammalian-sized genomes has inspired many experimental strategies.  Chief among these is the nanopore-based electrophoresis.  While excellent progress is continuously being made with this technique, there are many challenges in reaching the goals of very high quality sequencing and fabricating massively parallel sequencing devices.  These challenges stem from the physics of nanopore-based electrophoresis of DNA which needs to be understood from a fundamental scientific point of view.  The proposed research deals with fundamental understanding of the behavior of DNA in nanopore environments under the influence of electric and hydrodynamic forces, and ratcheting forces from enzymes.  We will investigate the challenges underlying several key system components in the goal of reducing the cost, increasing the speed, and increasing the accuracy of sequencing mammalian-sized genomes.  The major challenges deal with slowing down DNA through nanopore, effects of specific ions, conformational fluctuations of DNA, effects of flow fields arising from hydrodynamics, salt concentration gradients, and electroosmotic flow, and fluctuations in the processivity of enzymes.  We will use a combination of concepts from polymer physics, statistical mechanics theory, computer simulations, and numerical computation of coupled nonlinear equations to address polyelectrolyte statistics and dynamics, electrostatics, and hydrodynamics in the phenomena of DNA translocation.  The proposed research, while being generally relevant to all nanopore-based experiments, will be hinged specifically on:  (a) slowing down DNA and fundamental understanding of translocation, mediated by voltage, temperature, identity and amount of electrolyte, salt concentration gradient, and patterns on pore surface, (b) controlling the stochasticity in enzyme-ratcheted translocation and fundamental understanding of coupling among fluctuations in enzyme processivity, DNA conformational fluctuations, and electrophoretic drift-diffusion, and (c) designing optimum configuration of compact arrays of thousands of nanopores for massively parallel DNA sequencing without crosstalk between the units.

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Nanofluidic Platforms for High Resolution Mapping of Genomic DNA
Ramsey, John Michael
The University of North Carolina at Chapel Hill
R01 HG007407
$534,000 FY13, $2,050,000 (4 years)

We propose to develop a nanofluidic platform for the high-throughput restriction mapping of complete genomes.  This platform consists of monolithically integrated fluidics for the extraction of chromosomal DNA from cells, its digestion by restriction endonucleases in a long nanochannel, and the high-resolution sizing of ordered restriction fragments.  In contrast to other nanochannel-based approaches, we do not rely on the imaging of highly confined and elongated DNA molecules.  Rather, fluorescently labeled DNA is digested by restriction endonucleases in a relatively large diameter (300 - 500 nm) channel in which nanochannel confinement and electrostatic forces prevent diffusive mixing of adjacent fragments.  After digestion, the ordered fragments are electrophoretically driven to an injection point where the channel diameter decreases to ~100 nm.  As each fragment reaches this point, it is accelerated by the higher electric field, creating separation from its trailing neighbors.  These separated fragments migrate through a focused laser spot and the duration and integrated intensity of each fluorescence pulse is detected and analyzed to determine fragment size.

We anticipate several advantages to our approach; it relies on nanochannels greater than 100 nm, ensuring that, after prototyping, devices can be fabricated using low-cost, high-throughput methods.  The ability to fabricate very long nanochannels with diameters of 300 - 500 nm enables us to confine long genomic DNA in the reaction nanochannel, greatly reducing the need for map assembly from smaller DNA molecules.  Integration of DNA extraction on chip increases the probability of interrogating intact chromosomal DNA, eliminating map assembly entirely and providing truly global coverage.  Single-point detection obviates the need for image storage and analysis.  In addition, many unique opportunities for pre- or post-mapping functionality are possible.

We have conducted preliminary studies demonstrating the ordered injection of fragments from a reaction nanochannel into a smaller detection nanochannel and the ability of this approach to resolve neighboring fragments.  Together with other elements developed by the Ramsey group and others, we believe that we would be generating restriction maps during the first half of the proposed project.  Consequently, our team includes members with expertise in next generation sequencing (NGS) and bioinformatics.  We will validate our restriction maps against reference sequences and physical maps generated using other methods.  We will also demonstrate the utility of our restriction maps as scaffolds for de novo assembly of NGS data.  Any errors or biases determined from these assessments will direct platform improvements.

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Towards Viable Nanopore Sequencing by Slowing DNA Translocation
Schibel, Anna
Electronic Biosciences, Inc., San Diego, California
R43 HG006878
$239,000 FY13,  (1 year)

The aim of this project is to investigate a simple method for slowing down the rate of electrophoretic translocation of DNA through a nanopore.  Unique modulations of current across nanopores produced by individual bases during such translocation forms the basis of DNA nanopore sequencing.  The nanopore method requires very simple sample preparation with potentially no amplification steps and can achieve long read sequences with inexpensive hardware and biochemical reagents.  Hence, nanopore technology holds a great deal of promise in the effort to achieve rapid, low cost sequencing.  However, current high bandwidth methods are limited by the inability to accurately distinguish various bases due to the extremely rapid nature of freely translocating DNA.  Methods proposed here maintain the simplicity of nanopore sequencing through biological and electronic innovations.  Progress made through this proposal will potentially lead to an inexpensive and rapid benchtop DNA sequencing solution that will contribute significantly towards the goal of the $1000 genome.

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Posted: September 6, 2013