Advanced Sequencing Technology Awards 2012
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 2012 awards were announced on September 14,2012 (See: New NIH grants to harness nanoscale technologies to cut DNA sequencing costs)
Low Cost Sequencing with Re-usable Magnetic Arrays and Nanoelectronic Sensors
$3,332,000 (3 years)
Sequencing methods allowing cost effective and accurate de novo and re-sequencing genomes are critical to provide needed insights for human health, disease management and diagnostics at the individual level. Costs still remain too high with inadequate quality, to make sequencing technologies affordable for the routine use of genomics in individual health care. The "Gene Electronic Nano-Integrated Ultra-Sensitive" (GENIUS) platform is based on innovative technologies to provide significant improvements in cost, accuracy, read length, throughput and ease of use relative to current state-of-art systems.
The overall goal is to develop a sequencing system using nano-magnetic-electronic platforms. We are proposing a system that integrates sample preparation and enrichment steps with the sequencing module. After sample preparation, beads are then transferred and held on a nano-magnetic-electronic platforms for direct detection of extension reactions. In lieu of a reverse emulsion we are developing an easy to use, chip-based approach that will combine multiple sample processing steps in a single device. The efficient capture of beads, concentration and confinement of DNA and amplification products, may permit the elimination of whole genome amplification with its inherent bias. The device will eliminate the variability in reaction volumes and double-Poisson distribution inherent in emulsion PCR. The proposed chip provides high-sensitivity detection, efficient and uniform reagent delivery and washing, combined with the high efficiency bead capture and permits longer reads and higher accuracy by minimizing de-phasing and providing uniformity of reaction, and also reduces reagent consumption and cost. In summary, the simplified automatable workflow with low reagent consumption, label-free electronic detection using unmodified nucleotides and polymerase results in significant cost savings and improved accuracy.
Our goal is to develop a platform to sequence a genome with consumable costs of ~$50, average read length of up to 1000 bases, and pre-assembly accuracy of > 99.7% with similar cost reduction and simplification for sample preparation.
Electronic Sequencing in Graphene Nanopores
Golovchenko, Jene A., Daniel Branton
$3,600,000 (3 years)
Our research has shown that a single layer of graphene is an ideal membrane in which to fabricate high resolution nanopore detectors to sense the presence of single DNA molecules and their nucleobases. Our objective is to develop the tools and procedures needed to realize a scalable nanopore sequencing device which will significantly reduce future de novo sequencing costs by directly identifying the nucleobases on single stranded genomic DNA molecules that are driven sequentially through an array of precisely dimensioned graphene nanopores. The final system is intended to provide a relatively high quality sequence from ?6.5-fold coverage of a genome using DNA from fewer than 1 million cells, with no amplification or labeling. The specific aims are to: a) implement a graphene edge-sputtering process to facilitate high precision fabrication of nanopore arrays; and b) optimize discrimination between the four nucleotides of DNA using ionic current blockades. Successful completion of these aims will provide the key building blocks of a nanopore sequencing device that can accurately sequence an entire human genome at a cost of less than $1,000. The ability to inexpensively and accurately sequence complete genomes has the potential of remarkably improving many facets of human life and society, including the understanding, diagnosis, treatment and prevention of disease.
Microfluidic DNA Sequencing
$4,471,000 (3 years)
Collaborator: David Weitz, Harvard University
This is a Phase II SBIR proposal to increase the throughput of our DNA sequencing instrument to enable whole genome sequencing with high efficiency and accuracy for a cost of under $1000 per genome. In Phase I, we developed a working sequencing assay, a microfluidic platform and camera system, and analysis methodology that enable genome alignment and variant calling. We have demonstrated sequencing using both synthetic DNA and genomic DNA amplicons with read lengths up over 600bp and accuracy higher than 99.9% per base.
Using internal funds, we are upgrading the Phase I device to become a field ready beta test device for placement into several sites. This will be a single channel device that is capable of inline selection of 250 sequencing targets (average of 200BP long), sequence at 40X coverage, and variant calling in less than 2.5 hours of total run time.
The Specific Aim of Phase II is to increase the throughput of this beta test single channel instrument to enable whole genome sequencing with high efficiency and accuracy for a cost of under $1000 per genome. To accomplish this Specific Aim, we will carry out seven Tasks:
- Task 1: Develop a full-genome sequencing assay protocol for the preparation of a genomic DNA library for sequencing.
- Task 2: Develop a sensor configuration that can scale to 100s of channels.
- Task 3: Develop a microfluidic device that can scale to 100s of channels.
- Task 4: Develop a method to read droplet sizes of ~10um.
- Task 5: Develop a sorting assay that sorts amplified DNA from empty drops.
- Task 6: Extend current informatics pipeline for resequencing a whole genome.
- Task 7: Extend current barcode clustering algorithms.
At the conclusion of Phase II, we will have a breadboard sequencing system that will be able demonstrate that sequencing a whole genome in about six hours, including data analysis, genome alignment and variant calling, is fully achievable. The system will support reagent volumes that will meet the per run costs target <$1000. Following Phase II, we expect to use internal funds to have a market ready product within one year.
Integrated, Mutiplexed High-Frequency Electronic Analysis of DNA in Nanopores
Shepard, Kenneth L
$1,472,000 (3 years)
Collaborator: Marija Drndic, University of Pennsylvania
There is strong demand for third-generation DNA sequencing systems to be single-molecule, massively-parallel, and real-time. For single-molecule optical techniques, however, the signal from a single fluorophore is typically < 2500 photons/sec (equivalent to electrical current levels on the order of 50 fA). This leads to complex optics to try to collect every photon emitted and makes scaling of the platforms difficult. Additionally, synthesis reactions must be intentionally slowed to 1 Hz (or slower) to allow sufficient imaging times for these weak, noisy optical signals.
The limitations of single-molecule optical techniques highlight key advantages of electrochemical detection approaches, which have significantly higher signal levels (typically three orders of magnitude higher), allowing for the possibility for high-bandwidth detection with the appropriate co-design of transducer, detector, and amplifier. Significant effort has been directed toward the development of nanopore technology as one potential bioelectronic transduction mechanism. Nanopores, however, have proved to be extremely limited by the relatively short time biomolecules spend in the charge-sensitive region of the pore. Restricted by the use of off-the-shelf electronics, the noise-limited bandwidth of nanopore measurements is typically less than 100 kHz, limiting the available sensing and actuation strategies and defying multiplexed integration which would be required for any sequencing application.
In this four-year effort, we focus on improving significantly the noise-limited bandwidth of the detection electronics for nanopores allowing their full potential to be realized through close integration of the electronics and the pore while simultaneously supporting high levels of parallelism with multiple nanopores on the same detection substrate. We consider techniques for integrating both solid-state (Specific Aim 1) and biological pores (Specific Aim 3) onto these measurement substrates in a massively parallel manner (Specific Aim 2). The techniques we propose for leveraging commodity CMOS technology and co-integrating detection electronics are completely general and have significance to all other single-molecule bioelectronic transduction approaches. These high-bandwidth integrated electronics will also enable "closed-loop" sensing and actuation (Specific Aim 4), allowing dynamic manipulation of capture and translocation dynamics at microsecond (or better) timescales.
Single-Molecule, Real-Time Electronic Sequencing
Varma, Madoo, Oguz H. Elibol, Xing Su, Serge Guy Lemay, Kenneth L Shepard, Stephen W Turner
Intel Corporation, University of Twente, Columbia University, Pacific Biosciences
$4,996,000 (4 years)
Third-generation sequencing approaches are largely focusing on single-molecule strategies with the ability to achieve long read lengths. Single-molecule approaches require little or no sample preparation, saving time and reagent costs. They are more accurate since there is less chance of errors as no amplification is needed and there is no bias in molecular quantification. In addition, single-molecule techniques allow direct sequencing of mRNA, allowing understanding of post-transcription editing variations and copy-number studies. Ideally, single-molecule SBS can be massively-parallel and real-time, operating at synthesis rates as high as 1 msec for DNA polymerase, however complex optics required to collect photons efficiently make scaling of the platforms to high densities difficult.
A promising route for overcoming the challenges to optical techniques is bioelectronic detection. The direct, real-time detection of this reaction product by electrical means represents a two-fold challenge. First, the minute amount of charge involved falls well below the noise floor for solid-state detection. Second, the presence of a high concentration of screening ions in physiological buffers greatly reduces the range and strength of electrostatic interactions. As a result, conventional electrical detection strategies, including impedance spectroscopy, field-effect detection and Faradaic reactions, lack sufficient sensitivity to detect single molecules.
In this four-year effort, we develop a real-time, single-molecule sequencing approach based on the electrical detection of specifically engineered electrochemical tags that are attached to each of the four nucleotides. A base-specific electrochemical tag is released during the nucleotide incorporation; this tag is then activated through a phosphatase reaction to become redox active and is subsequently collected into a single molecule fingerprinting region (composed of four nanogap transducers). Redox cycling is used to produce an amplified signal for detection in the fingerprinting region. This approach to signal amplification is the electrical analog of fluorescent labels which see repeated excitation and emission under constant illumination to achieve detection gain. These nanogap transducers are integrated onto a CMOS integrated circuit in a highly multiplexed, parallel format. The proposed approach combines the advantages of single-molecule real time sequencing with a CMOS-compatible single molecule signal transduction platform and its attendant scalability benefits
Picogram-Level DNA Sequencing using Nanopores and Zero-Mode Waveguides
Wanunu, Meni, Jonas Korlach
Northeastern University, Pacific Biosciences
$825,000 (3 years)
Single-molecule array-based approaches to genome sequencing have the potential to deliver a dramatic reduction in sequencing costs over bulk methods by reducing reagent costs, increasing read lengths, and substantially decreasing the input DNA requirements. The latter advantage is critical when information retrieved from the sequencing technique sheds light on epigenetic modifications and damaged DNA bases, which cannot be amplified. Pacific Biosciences has developed and commercialized a single-molecule real-time (SMRT) sequencing instrument that utilizes as substrates an array of zero-mode waveguides (ZMWs), or nanoscale holes through an opaque metallic film. The ZMW array enables multiplexed DNA sequencing by employing single DNA polymerases for sequencing by synthesis, and detection is achieved using 4-color fluorescence microscopy and dye-labeled nucleotides. However, two major challenges currently limit the cost reduction and the capabilities of this technology. The first is the low yield of functional ZMWs that contain exactly one DNA polymerase molecule, as required for sequencing. The second is the reliance on diffusion of polymerase/template complexes into ZMWs, which results in sub-optimal loading into the array and increases the DNA input requirement. Despite the demonstrated ability of SMRT sequencing to identify DNA base modifications, high DNA inputs required preclude the gathering of epigenetic sequence data from native DNA, important for understanding the role of DNA modifications in aging and various diseases. In this proposal we present an innovative approach for reducing the cost of SMRT DNA sequencing, as well as enabling genomic and epigenomic sequencing from picogram levels of DNA without prior amplification. Our approach is to replace the glass bottom of each ZMW with a thin membrane that contains a single nanopore. The nanopore will serve two major purposes: 1) Precise positioning of individual DNA polymerases in ZMW arrays with greatly improved yields and controlled stoichiometry; 2) Orders of magnitude enhancement of the loading rates of input DNA that is to be sequenced into the ZMWs. To achieve our goals we have assembled a multidisciplinary team of investigators with expertise in cutting edge fabrication techniques, nanoscale science, and molecular biology. We divide our goals into aims that include the fabrication of the ZMW-pore devices, anchoring of individual DNA polymerases in ZMW-pore arrays, and the reduction of input DNA requirements by active DNA loading into the ZMW-pore devices. Finally, we will demonstrate using our ZMW-pore arrays sequencing of control DNA libraries, as well as direct epigenomic sequencing from picogram levels of human brain mitochondrial DNA. Our approach, if successful, will dramatically reduce the cost of DNA sequencing and pave way to massive high-throughput genomic and epigenomic sequence data.
Posted: September 14, 2012