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Exo-Seq for the single molecule of DNA molecules

Award Number: R43HG010551
ORGANIZATION: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 06/18/2019
PERIOD OF PERFORMANCE END DATE: 12/31/2020

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Exo-Seq for the single molecule of DNA molecules - ABSTRACT The long-term goal of this project is the commercial development of a low-cost Single-Molecule Sequencing (SMS) technology, called Exo-Seq, which employs a sequencing-by-subtraction strategy for high fidelity disassembly of low amounts of unamplified DNAs. In spite of the SMS approach, Exo-Seq possesses superior single-pass base-call accuracy (>95%) using a multi- parameter identification strategy that targets molecular-dependent electrophoretic mobilities and electrical (current transients) signatures to identify individual mononucleotides (dNMPs). In addition, Exo-Seq can generate long reads (>40 kbp) with potentially sequence-independent calling errors as well as reading modified bases without requiring bisulfite conversion. Exo-Seq is comprised of simple hardware with a small instrument footprint, and a novel mixed-scale plastic chip consumable containing nanosensors to accommodate DNA sequencing in parallel for high throughput applications. Exo-Seq requires only minimally pre-processed DNA inputs minimizing extensive hands-on sample preparation by the end-user, while also significantly reducing downstream reagent/consumable costs associated with most commercial sequencing workflows. Exo-Seq consists of a plastic chip with nanofluidic and microfluidic networks, with 3 functional elements: [1] An input/output fluidic network with each channel containing an in-plane pore (~20 nm) to transduce single DNA molecules. [2] Solid-phase bioreactor with an immobilized exonuclease to allow for high fidelity cleavage of single DNA molecules to generate dNMPs. [3] A nanochannel (50 × 50 nm; length <10 µm) with at least 2 in-plane pores (<10 nm effective diameter) positioned a fixed distance apart used to identify them. The use of mixed-scale replication allows for single-step generation of the chip that can allow for low production costs with high manufacturing compliance. Exo-Seq sequencing uses the following steps: (1) Single- stranded DNA molecules are manipulated by electrokinetics in a nanofluidic network and loaded into the nanosensor. (2) The loaded single DNA molecule is captured by an immobilized exonucleases (Exonuclease I and RecJ) that are covalently attached to a support pillar. Using both exonucleases allows for bi-directional sequencing of ssDNA. (3) The DNA that contacts the exonuclease is continuously cleaved one nucleotide at a time in an ordered direction. (3) The released nucleotides electrokinetically enters a nanochannel containing at least 2 in-plane pores positioned at the entrance/exit ends. (4) Each nucleotide base call is deduced from a molecular- dependent mobility and a current amplitude signal obtained from each nanopore providing 2 independent measurements for unprecedented single-molecule identification efficiency.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2021 ( Subtotal = -$342,831 )
2021	2019	DIGITAL NANOGENETICS LLC	4950 RESEARCH PARKWAY	LAWRENCE	KS	66047	DOUGLAS	USA	Human Genome Research	000	1	8/31/2021	NEW	-$342,831
														Subtotal = -$342,831

Issue Date FY: 2019 ( Subtotal = $342,831 )
2019	2019	DIGITAL NANOGENETICS LLC	4950 RESEARCH PARKWAY	LAWRENCE	KS	66047	DOUGLAS	USA	Human Genome Research	000	1	5/20/2019	NEW	$342,831
														Subtotal = $342,831

Grand Total All Awards = $0

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Exo-Seq for the single molecule of DNA molecules

Award Number: R43HG010551

ORGANIZATION: NATIONAL HUMAN GENOME RESEARCH INSTITUTE

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 06/18/2019

PERIOD OF PERFORMANCE END DATE: 12/31/2020

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