SBIR-STTR Award

Most next-generation DNA sequencing methods have focused on either 1) template amplification followed by massively-parallel sequencing-by-synthesis, or 2) single molecule detection. The first method is now commercially available but suffers from relatively large volumes of expensive reagent usage. The latter method, although not yet commercially available, will have a disadvantage in signal-to-noise and therefore require more sensitive and expensive instrumentation. To avoid these disadvantages, we will use droplet-based microfluidics to sequence DNA. By using microfluidics we limit the amount of reagent required to sequence DNA to less than several milliliters, while still retaining the ability to amplify the template that thereby enables us to use relatively inexpensive and robust detection. Hybridization of short probes will be detected in microfluidic droplets by a shift in fluorescence polarization that distinguishes between bound and free oligo. This removes the requirement for a separation phase to detect hybridization. The method is simple and does not require enzymes. In Phase I we will describe a simple platform and resequencing method that will be scaled in a future Phase II project to enable human genome sequencing for under $1000. , ,

Public Health Relevance:
The proposed Phase I droplet-based microfluidics instrument will generate, selectively merge and analyze over 10,000 aqueous droplets per second. We want to leverage this capability to implement a DNA sequencing method on a microfluidics platform. We have broken the project up into two phases. Phase I is proof of principle. In Phase I we demonstrate that the biochemical assay works in drops and also that our detector works. In Phase II we will scale the process to build a machine that will be able to sequence a human genome for less than $1000.

Thesaurus Terms:
Analysis, Cost;Assay;Bar Codes;Base Pairing;Binding;Binding (Molecular Function);Bio-Informatics;Bioassay;Biochemical;Bioinformatics;Biologic Assays;Biological Assay;Buffers;Caliber;Cell Communication And Signaling;Cell Signaling;Coloring Agents;Computer Analysis;Cost Analyses;Cost Analysis;Dna;Dna Resequencing;Dna Sequence;Deoxyribonucleic Acid;Detection;Devices;Diameter;Digestion;Disadvantaged;Drops;Dyes;Emulsions;Enzymes;Fluorescence Polarization;Future;Genome;Human Genome;Informatics;Instrumentation, Other;Intracellular Communication And Signaling;Label;Libraries;Life;Measures;Methods;Microfluidic;Microfluidics;Molecular Interaction;Monitor;Noise;Oligo;Oligonucleotides;Phase;Preparation;Process;Quality Control;Reader;Reading;Reagent;Resequencing;Signal Transduction;Signal Transduction Systems;Signaling;Solutions;Speed;Speed (Motion);Stream;System;System, Loinc Axis 4;Temperature;Testing;Time;Variant;Variation;Work;Aqueous;Base;Biological Signal Transduction;Computational Analysis;Cost;Detector;Experiment;Experimental Research;Experimental Study;Genome Sequencing;Instrument;Instrumentation;Member;Milliliter;Next Generation;Novel;Public Health Relevance;Reconstruction;Research Study;Scaffold;Scaffolding;Single Molecule;Virtual

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 to 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.

Public Health Relevance:
In Phase II of this project, we will scale the process we have completed in our Phase I to build a machine that will be able to sequence a human genome for less than $1000. This is a project to develop a user-friendly, desktop instrument that can sequence a person's entire genetic make-up in approximately 6 hours;the entire process will cost less than $1000, including analysis. By understanding a person's genetic make-up, researchers and physicians can use the information to make personalized decisions, thereby changing the healthcare landscape and improve healthcare costs and outcomes. This instrument will greatly enhance medical treatments for tens of millions of people around the world and greatly improve our ability to understand how the body functions, which could lead to major breakthroughs in medicine.