WO2016065300A1 - Cartouche microfluidique - Google Patents

Cartouche microfluidique Download PDF

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Publication number
WO2016065300A1
WO2016065300A1 PCT/US2015/057186 US2015057186W WO2016065300A1 WO 2016065300 A1 WO2016065300 A1 WO 2016065300A1 US 2015057186 W US2015057186 W US 2015057186W WO 2016065300 A1 WO2016065300 A1 WO 2016065300A1
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WIPO (PCT)
Prior art keywords
microfluidic cartridge
sample
nucleic acid
component
sequencing
Prior art date
Application number
PCT/US2015/057186
Other languages
English (en)
Inventor
Mark W. Eshoo
Alice T. HANG
Heather L. Smith
Bernadet Meijering
Harma Martine FEITSMA
Martinus Johannes van ZELST
Original Assignee
Eshoo Mark W
Hang Alice T
Smith Heather L
Bernadet Meijering
Feitsma Harma Martine
Van Zelst Martinus Johannes
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Publication date
Application filed by Eshoo Mark W, Hang Alice T, Smith Heather L, Bernadet Meijering, Feitsma Harma Martine, Van Zelst Martinus Johannes filed Critical Eshoo Mark W
Publication of WO2016065300A1 publication Critical patent/WO2016065300A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00351—Means for dispensing and evacuation of reagents
    • B01J2219/00389—Feeding through valves
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00351—Means for dispensing and evacuation of reagents
    • B01J2219/00418—Means for dispensing and evacuation of reagents using pressure
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00479—Means for mixing reactants or products in the reaction vessels
    • B01J2219/00484—Means for mixing reactants or products in the reaction vessels by shaking, vibrating or oscillating of the reaction vessels
    • B01J2219/00486—Means for mixing reactants or products in the reaction vessels by shaking, vibrating or oscillating of the reaction vessels by sonication or ultrasonication
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00495—Means for heating or cooling the reaction vessels
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/005—Beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00585—Parallel processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/0059—Sequential processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00759—Purification of compounds synthesised
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0668—Trapping microscopic beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0684—Venting, avoiding backpressure, avoid gas bubbles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0867—Multiple inlets and one sample wells, e.g. mixing, dilution
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
    • B01L2300/1822—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using Peltier elements
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

Definitions

  • microfluidic cartridges configured to process a biological sample, including but not limited to devices, apparatuses, methods, and systems for generating DNA libraries that are suitable for use in sequencing methods (e.g., next generation sequencing methods) or other nucleic acid analysis technologies.
  • Sequencing of DNA requires large amounts of extracted DNA for use in the preparation of a sequencing library.
  • the process of culturing cells, lysing the cells, extracting DNA, fragmenting the DNA, ligating linkers, and purifying the sequencing template is a multi-step process that can take several days to be performed by a skilled research technician. What is needed are technologies for preparing DNA sequencing libraries that are automated and suitable for integration with nucleic acid sequencing workflows.
  • the microfluidic cartridge accepts as input an aqueous sample (e.g., a biological sample comprising nucleic acids), extracts the nucleic acids, builds a sequencing library, and outputs the sequencing library ready for introduction to an NGS workflow.
  • the sequencing library is delivered directly to an NGS by a fluid connection.
  • the technology is not limited to preparing libraries for any particular NGS workflow or platform and thus provides sequencing libraries to a number of NGS workflows and platforms developed by Pac Bio (e.g., the Pac Bio RS NGS sequencer), Illumina (e.g., the Illumina MiSeq, HiSeq, and NextSeq NGS sequencers), Ion Torrent (e.g., Ion Torrent PGM or Proton sequencers), etc.
  • Pac Bio e.g., the Pac Bio RS NGS sequencer
  • Illumina e.g., the Illumina MiSeq, HiSeq, and NextSeq NGS sequencers
  • Ion Torrent e.g., Ion Torrent PGM or Proton sequencers
  • microfluidic cartridge comprise particular components, e.g., components for thermal control, cell lysis, nucleic acid fragmentation, and/or nucleic acid purification.
  • components for thermal control e.g., components for thermal control, cell lysis, nucleic acid fragmentation, and/or nucleic acid purification.
  • ultrasonic components are used for cell lysis, nucleic acid extraction, and nucleic acid
  • an aqueous sample is lysed, the nucleic acids extracted, and the purified nucleic acids are eluted in a universal reaction buffer.
  • WGA whole genome amplification
  • the amplified DNA is then sheared using a physical method (e.g., by imparting ultrasonic energy into the sample) and end polished to prepare the sample for ligation.
  • the end polished DNA is then ligated with DNA sequencing adapters and cleaned up enzymatically to remove starting material and incomplete reaction products.
  • the sequencing library is then subjected to a final cleanup/size selection process to remove reaction components that are incompatible with downstream sequencing (salts, dNTPs, etc.).
  • a microfluidic cartridge for producing a sequencing library from a biological sample
  • the microfluidic cartridge comprising one or more of a sample processing component comprising a vented headspace to relieve gas pressure in the sample processing component; a waste containment component comprising an absorbent material to absorb waste products; and an output component in fluid communication with a sequencing instrument (e.g., a conduit (e.g., a tube) for delivering a sequencing library directly into a sequencing instrument (e.g., a NGS instrument such as a Solexa, Pac Bio, or Ion Torrent instrument)).
  • a sequencing instrument e.g., a conduit (e.g., a tube) for delivering a sequencing library directly into a sequencing instrument (e.g., a NGS instrument such as a Solexa, Pac Bio, or Ion Torrent instrument)
  • a sequencing instrument e.g., a NGS instrument such as a Solexa, Pac Bio, or Ion Torrent instrument
  • the microfluidic cartridge comprises thermally annealed polymer layers (e.g., annealed at a temperature of approximately 80°C (e.g., 70°C to 90°C, e.g., 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90°C) for approximately 1 hour (e.g., 30 minutes to 90 minutes, e.g., 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 minutes).
  • a temperature of approximately 80°C e.g., 70°C to 90°C, e.g., 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90°C
  • 1 hour
  • the microfluidic cartridge comprises thermally annealed poly(methyl methacrylate) layers (e.g., annealed at a temperature of approximately 80°C (e.g., 70°C to 90°C, e.g., 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90°C) for approximately 1 hour (e.g., 30 minutes to 90 minutes, e.g., 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 minutes).
  • a temperature of approximately 80°C e.g., 70°C to 90°C, e.g., 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90°
  • the sample processing component (e.g., comprising a vented headspace to relieve gas pressure) is a cell lysis component and in some embodiments the sample processing component (e.g., comprising a vented headspace to relieve gas pressure) is a nucleic acid fragmentation component.
  • the microfluidic cartridge comprises one or more sample processing components, e.g., one or more cell lysis components and/or one or more nucleic acid fragmentation components.
  • the sample processing component (e.g., cell lysis and or nucleic acid fragmentation component) is configured to accept a sample into a reaction chamber that has a volume at least 2x the sample volume provided therein, e.g., at least 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, llx, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, or 20x times the sample volume.
  • the reaction chamber has a volume at least 12x the sample volume accepted into the reaction chamber.
  • the vented headspace of the sample processing component (e.g., cell lysis and or nucleic acid fragmentation component) comprises a filter.
  • the vented headspace of the sample processing component (e.g., cell lysis and/or nucleic acid fragmentation component) comprises a HEPA filter.
  • the vented headspace of the sample processing component (e.g., cell lysis and/or nucleic acid fragmentation component) is connected to a pressure release chamber.
  • the vented headspace of the sample processing component (e.g., cell lysis and or nucleic acid fragmentation component) is adapted to lyse cells and/or fragment nucleic acids by an ultrasonic device.
  • the sample processing component e.g., cell lysis and/or nucleic acid fragmentation component
  • the sample processing component is adapted to provide temperature control to (e.g., to heat and/or to cool) a sample in a reaction chamber by a thermoelectric device.
  • the microfluidic cartridge comprises a variety of components, modules, chambers, etc. that are fluidly connected, e.g., with microfluidic and/or macrofluidic channels.
  • a microfluidic cartridge comprises one or more of an amplification component (e.g., an amplification component heated and cooled by a thermoelectric device), an adaptor ligation component, a labeling component, a nucleic acid extraction component, or a nucleic acid size selection component (e.g., a nucleic acid size selection component that accepts an input sample comprising nucleic acids and produces an output sample comprising nucleic acids, wherein the output sample has a higher concentration or amount of nucleic acids longer than a cutoff size that is 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700,
  • the microfluidic cartridge comprises one or more sensors for monitoring sample processing and providing data (e.g., an analog signal and/or a digital signal) as output (e.g., to an apparatus and/or to a computer).
  • the microfluidic cartridge comprises one or more of, e.g., a temperature sensor, a humidity sensor, a pressure sensor, an electrode (e.g., to detect one or more ions), a pH sensor, an imaging sensor (e.g., a charge-coupled device (CCD), an active pixel sensor (e.g., a complementary metal-oxide-semiconductor (CMOS) and/or an N-type metal-oxide- semiconductor (NMOS, Live MOS)), etc.), a motion sensor, an acoustic sensor (e.g., a microphone), a current sensor, a voltage sensor, a magnetometer, a flow sensor, a proximity sensor, a chemical sensor, a gyroscopic sensor, etc.
  • a temperature sensor
  • the microfluidic cartridge is a single-use microfluidic cartridge. In some embodiments, the microfluidic cartridge is a disposable microfluidic cartridge. In some embodiments, the microfluidic cartridge is a sterile microfluidic cartridge. In some embodiments, the microfluidic cartridge further comprises a stabilized and/or a lyophilized reagent. In some embodiments, microfluidic cartridge comprises a stabilized enzyme, buffer, and/or oligonucleotide. In some embodiments, the microfluidic cartridge further comprises a chamber comprising a stabilized reagent. In some embodiments, the microfluidic cartridge further comprises a chamber adapted to comprise a stabilized reagent.
  • a system comprising a microfluidic cartridge as described herein and an apparatus comprising an interface to accept the microfluidic cartridge.
  • the interface provides mechanical, fluidic (e.g., microfluidic, macrofluidic, liquid, gas), electrical (e.g., current, voltage), pneumatic, acoustic (e.g., sonic, ultrasonic), and/or magnetic communication between the microfluidic cartridge and the apparatus.
  • the apparatus comprises an ultrasonic device for lysing cells in a sample contained in the sample processing component of the microfluidic cartridge.
  • a system comprising a microfluidic cartridge as described herein; an apparatus configured to interface with the microfluidic cartridge (e.g., an apparatus comprising an interface to accept the microfluidic cartridge); and a nucleic acid sequencer (e.g., an NGS sequencer).
  • the apparatus interface provides mechanical, fluidic (e.g., microfluidic, macrofluidic, liquid, gas), electrical (e.g., current, voltage), pneumatic, acoustic (e.g., sonic, ultrasonic), and/or magnetic communication between the microfluidic cartridge and the apparatus.
  • the apparatus comprises an ultrasonic device for fragmenting nucleic acids in a sample contained in the sample processing component of the microfluidic cartridge.
  • the apparatus comprises a thermoelectric device for cooling or heating a sample contained in the sample processing component of the microfluidic cartridge.
  • the apparatus comprises a thermoelectric device for cooling or heating a sample contained in an amplification component, adaptor ligation component, labeling component, nucleic acid extraction component, or nucleic acid size selection component.
  • the system comprises a conduit for delivering a sequencing library from an output of the apparatus to an input of the nucleic acid sequencer.
  • a system comprising a computer to accept signals from the apparatus, provide power to the apparatus and/or microfluidic cartridge, and provide nucleic acid sequence data in a user-readable and/or machine-readable format.
  • the microfluidic cartridge is dry coupled to the ultrasonic device of the apparatus (e.g., coupled without use of a liquid, gel, etc. couplant composition).
  • Systems described herein comprise, in some embodiments, one or more sensors, e.g., one or more of a temperature sensor, a humidity sensor, a pressure sensor, an electrode (e.g., to detect one or more ions), a pH sensor, an imaging sensor (e.g., a charge-coupled device (CCD), an active pixel sensor (e.g., a complementary metal-oxide- semiconductor (CMOS) and/or an N-type metal-oxide-semiconductor (NMOS, Live MOS)), etc.), a motion sensor, an acoustic sensor (e.g., a microphone), a current sensor, a voltage sensor, a magnetometer, a flow sensor, a proximity sensor, a chemical sensor, a gyroscopic sensor, a light sensor (e.g., to monitor scattered light, to monitor fluorescence emission, to monitor ultraviolet and/or visible light), etc.
  • a temperature sensor e.g., a humidity sensor, a pressure sensor, an electrode
  • the technology provides a method of producing a sequencing library from a biological sample.
  • some embodiments provide a method comprising providing a microfluidic cartridge as described herein; lysing cells and or fragmenting nucleic acids in one or more sample processing components (e.g., lysing and/or fragmenting by use of an ultrasonic component); and delivering a sequencing library to a sequencing instrument through a direct fluidic connection.
  • Some embodiments provide a step of introducing ultrasonic energy into a sample, e.g., to lyse cells and or to fragment nucleic acids.
  • Some embodiments provide a step of controlling the temperature of a sample in a cell lysis and/or nucleic acid fragmentation component.
  • Some additional method embodiments comprise, e.g., amplifying a nucleic acid in an amplification component, ligating an adaptor to a nucleic acid in an adaptor ligation component, labeling a nucleic acid in a labeling component, extracting nucleic acid in a nucleic acid extraction component, size selecting nucleic acids in a nucleic acid size selection component, and/or interfacing the microfluidic cartridge with an apparatus
  • providing the microfluidic cartridge comprises thermally annealing polymer layers to produce the microfluidic cartridge, e.g., thermally annealing poly(methyl methacrylate) layers to produce the microfluidic cartridge (e.g., at a temperature of approximately 80°C (e.g., 70°C to 90°C, e.g., 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90°C) for approximately 1 hour (e.g., 30 minutes to 90 minutes, e.g., 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 minutes)).
  • 80°C e.g., 70°C to 90°C
  • Some embodiments provide further steps of providing a biological sample (e.g., a human biological sample) comprising a nucleic acid and some embodiments provide further steps of providing a formalin fixed paraffin embedded (FFPE) sample. Additional embodiments will be apparent to persons skilled in the relevant art based on the teachings contained herein.
  • a biological sample e.g., a human biological sample
  • FFPE formalin fixed paraffin embedded
  • Figure 1 is a schematic drawing of an embodiment of the microfluidic cartridge described herein.
  • Figure 1A shows the overall layout of channels and chambers and
  • Figure IB shows the channel layer.
  • Figure 2 is a schematic drawing of an embodiment of an integrated microfluidic cartridge integrated cartridge comprising four sub-circuits ⁇ lysis and extraction; library preparation (e.g., comprising an ultrasonic module); library clean-up; and sequencing set up.
  • library preparation e.g., comprising an ultrasonic module
  • library clean-up e.g., comprising an ultrasonic module
  • Figure 3 is a schematic drawing and photograph of a lysis and extraction sub- circuit of an embodiment of the technology provided herein.
  • Figure 3a shows the design of a lysis and extraction sub-circuit.
  • Light blue lines denote fluidic paths
  • LR denotes liquid reagent reservoirs
  • k denotes a fluidic valve
  • p denotes a fluidic pump
  • US denotes ultrasonic element
  • silica membrane denotes extraction membrane.
  • Figure 3b is a photograph of an embodiment of a lysis and extraction sub-circuit with features denoted.
  • Figure 4 is a schematic drawing and photograph of a library preparation sub- circuit of an embodiment of the technology provided herein.
  • Figure 4a shows the design of a library preparation sub-circuit.
  • Light blue lines denote fluidic paths
  • dr denotes dry reagent reservoirs
  • k denotes a fluidic valve
  • p denotes a fluidic pump
  • US denotes an ultrasonic element
  • Peltier denotes a thermoelectric heating/cooling element.
  • Figure 4b is a photograph of an embodiment of a library preparation sub-circuit with key features denoted.
  • Figure 5 is a schematic drawing and photograph of a library clean-up sub -circuit of an embodiment of the technology provided herein.
  • Figure 5a shows the design of a library clean-up sub-circuit.
  • Light blue lines denote fluidic paths
  • LR denotes liquid reagent reservoirs
  • k denotes a fluidic valve
  • p denotes a fluidic pump
  • magnetic capture denotes electromagnetic element used to capture/actuate microfluidic beads.
  • Figure 5b is a photograph of a library clean-up sub-circuit with key features denoted.
  • Figure 6 is a schematic drawing of a sequencing set-up sub-circuit.
  • Light blue lines denote fluidic paths
  • dr denotes dry reagent reservoirs
  • k denotes a fluidic valve
  • p denotes a fluidic pump
  • Peltier denotes a thermo-electric heating/cooling element
  • sample output denotes a port for fluidic connection to a sequencer.
  • Figure 7 shows labeled photographs of an integrated microfluidic cartridge.
  • Figure 7a shows a cartridge comprising a silica membrane for extraction of nucleic acids, two piezo interfaces (one for lysis prior to extraction and another for DNA fragmentation), two peltier heating/cooling elements (one for library preparation steps and one for sequencing polymerase binding), vacuum/air interfaces for valve control, and an electromagnetic actuation interface for magnetic bead clean-up.
  • Figure 7b shows the location of the lysis and extraction module (Module l); library preparation module (e.g., comprising an ultrasonic module) (Module 2); library clean-up module (Module 3); and sequencing set up module (Module 4).
  • microfluidic cartridges configured to process a biological sample, including but not limited to devices, apparatuses, methods, and systems for generating DNA libraries that are suitable for use in sequencing methods (e.g., next generation sequencing methods) or other nucleic acid analysis technologies.
  • the technology provides embodiments of a technology related to an integrated microfluidics cartridge for preparing libraries for next generation sequencing (NGS).
  • the integrated microfluidic cartridge provides components for cell lysis, nucleic acid extraction, and nucleic acid fragmentation.
  • the technology provides devices and associated methods for extracting nucleic acids from a sample, preparing nucleic acids for construction of a sequencing library, purifying nucleic acids, and constructing a sequencing library.
  • Particular embodiments provide a microfluidic cartridge comprising a combination of one or more poly(methyl methacrylate) (PMMA) layer(s), adhesive layer(s), flexible membrane layer(s), and filter(s).
  • PMMA poly(methyl methacrylate)
  • microfluidic card or “microfluidic cartridge” refers to a device, cartridge, chip, or card with fluidic structures (e.g., channels, chambers, voids, etc.) having microfluidic dimensions, e.g., at least one internal cross-sectional dimension that is less than approximately 500 ⁇ to 1000 ⁇ and typically between approximately 0.1 ⁇ and approximately 500 ⁇ .
  • fluidic structures e.g., channels, chambers, voids, etc.
  • These fluidic structures may include chambers, valves, vents, vias, pumps, inlets, nipples, and detectors and sensors, for example.
  • the microfluidic flow regime is characterized by Poiseuille or "laminar" flow. (See, e.g., Staben et al. 2005. Particle transport in Poiseuille flow in narrow channels. Intl J Multiphase Flow 31:529-47, and references cited therein).
  • Microfluidic devices may be fabricated from various materials using techniques such as laser stenciling, embossing, stamping, injection molding, masking, etching, and three-dimensional soft lithography. Laminated microfluidic devices are further fabricated with adhesive interlayers or by thermal adhesiveless bonding techniques, such as by pressure treatment of oriented polypropylene. The microarchitecture of laminated and molded microfluidic devices can differ. In certain embodiments, the microfluidic cartridges of the present technology are designed to interact or "dock" with a host instrument that provides a control interface and optional temperature and magnetic interfaces.
  • the microfluidic cartridge contains all biological reagents needed to perform the assay and requires only application of a sample or samples (e.g., the microfluidic cartridge comprises one or more stabilized reagent(s); lyophilized reagent(s); stabilized enzyme(s), buffer(s), and/or
  • these microfluidic cartridges are disposable, single-use, and are generally manufactured with sanitary features to minimize the risks of exposure to biohazardous material during use and upon disposal (e.g., in some embodiments, the microfluidic cartridge is a sterile microfluidic cartridge).
  • the term "whole genome amplification” or "WGA” as used herein generally refers to a method for amplification of a nucleic acid (e.g., DNA or RNA) sample in a non ⁇ specific manner (unless targeted WGA is employed) to generate a new sample that is indistinguishable from the original but with a higher nucleic acid concentration.
  • the ideal whole genome amplification technique would amplify a sample up to a microgram level while maintaining the original sequence representation.
  • the nucleic acid of the sample may include one or more entire genomes and/or one or more portions of one or more genomes.
  • DOP oligonucleotide-primed PCR
  • PEP primer extension PCR technique
  • MDA multiple displacement amplification
  • multiple displacement amplification refers to a non-PCR-based isothermal method based on the annealing of random hexamers (or non- random primers in targeted methods) to denatured DNA, followed by strand- displacement synthesis at constant temperature. It has been applied to small genomic DNA samples, leading to the synthesis of high molecular weight DNA with limited sequence representation bias. As DNA is synthesized by strand displacement, a gradually increasing number of priming events occur, forming a network of hyper- branched DNA structures. The reaction can be catalyzed by, for example, the Phi29 DNA polymerase or by the large fragment of the Bst DNA polymerase.
  • solid support or “solid substrate” means a solid material having a surface for attachment of molecules, compounds, cells, or other entities.
  • the surface of a solid support can be flat or not flat.
  • a solid support can be porous or non-porous.
  • a solid support can be a chip or array that comprises a surface and may comprise glass, silicon, nylon, polymers, plastics, ceramics, or metals.
  • a solid support can also be a membrane, such as a nylon, nitrocellulose, or polymeric membrane, or a plate or dish and can be comprised of glass, ceramics, metals, or plastics, such as, for example, polystyrene, polypropylene, polycarbonate, or polyallomer.
  • a solid support can also be a bead, resin, or particle of any shape.
  • Such particles or beads can comprise any suitable material, such as glass or ceramics, and/or one or more polymers, such as, for example, nylon, polytetrafluoroethylene (PTFE), TEFLONTM, polystyrene, polyacrylamide, sepaharose, agarose, cellulose, cellulose derivatives, or dextran, and/or can comprise metals, particularly paramagnetic metals, such as iron.
  • Solid supports may be flexible, for example, a polyethylene terephthalate (PET) film.
  • PET polyethylene terephthalate
  • end polishing or "end finishing” or “end repair” means, as is understood in the art of sequencing, subjecting duplex nucleic acid molecules having staggered single-strand ends to a process by which the ends are made blunted. This can be done enzymatically, such as by using T4 polynucleotide kinase in the presence of the complementary nucleoside triphosphates.
  • sample includes, but is not limited to, biological samples such as, e.g., blood, serum, plasma, buffy coat, saliva, wound exudates, pus, lung and other respiratory aspirates, nasal aspirates and washes, sinus drainage, bronchial lavage fluids, sputum, medial and inner ear aspirates, cyst aspirates, cerebral spinal fluid, stool, diarrheal fluid, urine, tears, mammary secretions, ovarian contents, ascites fluid, mucous, gastric fluid, gastrointestinal contents, urethral discharge, synovial fluid, peritoneal fluid, meconium, vaginal fluid or discharge, amniotic fluid, semen, penile discharge, or the like may be tested.
  • biological samples such as, e.g., blood, serum, plasma, buffy coat, saliva, wound exudates, pus, lung and other respiratory aspirates, nasal aspirates and washes, sinus drainage, bronchial lavage fluids, sputum, medial and inner ear aspir
  • assay a sample from swabs or lavages (e.g., that are representative of mucosal secretions and epithelia), for example, mucosal swabs of the throat, tonsils, gingival, nasal passages, vagina, urethra, rectum, lower colon, and eyes, as are homogenates, lysates, and digests of tissue specimens of all sorts.
  • the sample comprises mammalian cells.
  • the term sample encompasses other samples such as, e.g., samples of water, industrial discharges, food products, milk, air filtrates, etc.
  • test samples are placed directly in the device; in other
  • samples are processed prior to analysis.
  • a pathogen refers to an organism associated with an infection or infectious disease.
  • a pathogenic condition refers to a condition of a host characterized by the absence of health, e.g., a disease, infirmity, morbidity, or a genetic trait associated with potential morbidity.
  • nucleic acid refers to a polymeric form of nucleotides of any length, including but not limited to, ribonucleotides and deoxyribonucleotides. Relatively short (e.g., 10 to 1000 nt or bp) nucleic acid polymers are often used as “primers” or "probes”.
  • nucleic acids from natural sources, e.g., that are methylated or capped, and synthetic forms, e.g., that contain substitute or derivatized nucleobases and that are based on a peptide backbone.
  • Nucleic acids are generally polymers of adenosine, guanine, thymine, and cytosine and their "deoxy" forms, but may also contain other pyrimidines such as uracil and xanthine or spacers and universal bases such as deoxyinosine.
  • Deoxynucleic acids may be single-stranded or double-stranded depending on the presence or absence of complementary sequences and on conditions of pH, salt concentration, temperature, and the presence or absence of certain organic solvents such as formamide, ⁇ , ⁇ -dimethylformamide, dimethylsulfoxide, and n-methylpyrrolidinone.
  • a sample comprising nucleic acids may include, e.g., one or more metagenomes, genomes, chromosomes, genes, portions of genes, regulatory sequences of genes, mRNAs, rRNAs, tRNAs, siRNAs, miRNAs, cDNA, and may be single stranded, double stranded, or triple stranded.
  • Some nucleic acids have polymorphisms, deletions, and alternate splice sequences.
  • target nucleic acid refers to a nucleic acid sequence in a biosample that is to be amplified in an assay by a polymerase and detected.
  • the "target” molecule can be present as a “spike” or as an uncharacterized analyte in a sample, and may consist of DNA, cDNA, gDNA, RNA, mRNA, rRNA, or miRNA, either synthetic or native to an organism.
  • the “organism” is not limited to a mammal.
  • the target nucleic acid sequence is a template for synthesis of a
  • Genomic target sequences are denoted by a listing of the order of the bases, listed by convention from 5' end to 3' end.
  • reporter refers to a biomolecule or modification of a biomolecule that can be detected by physical, chemical,
  • detectable reporters include but are not limited to, radioisotopes, fluorophores, chromophores, mass labels, electron dense particles, magnetic particles, dyed particles, QDots, spin labels, molecules that emit chemiluminescence, electrochemically active molecules, enzymes, cofactors, enzymes linked to nucleic acid probes, and enzyme substrates. Reporters are used in bioassays as reagents, and are often covalently attached to another molecule, adsorbed on a solid phase, or bound by specific affinity binding.
  • immobilized indicates that an entity is present at a defined location or surface, e.g., by physical connection or under effectively irreversible binding (e.g., hybridization) conditions.
  • a "probe” is a nucleic acid capable of binding to a target nucleic acid by complementary base pairing with sufficient complementarity to form a stable double helix at room temperature. Probes may be labeled with reporter groups. Suitable labels that can be attached to probes include, but are not limited to, radioisotopes, fluorophores, chromophores, mass labels, electron dense particles, magnetic particles, spin labels, molecules that emit chemiluminescence, electrochemically active molecules, enzymes, cofactors, and enzyme substrates. Tools for selection of a probe sequence, length, and hybridization conditions are generally familiar to those skilled in the art.
  • amplification refers to a “template-dependent process” that results in an increase in the concentration of a nucleic acid sequence relative to its initial concentration.
  • a “template-dependent process” is a process that involves
  • template-dependent extension of a "primer” molecule.
  • a “template dependent extension” refers to nucleic acid synthesis of RNA or DNA wherein the sequence of the newly synthesized strand of nucleic acid is dictated by the rules of complementary base pairing of the target nucleic acid and the primers.
  • an "amplicon” refers to a double stranded DNA product of amplification and includes double stranded DNA products formed from DNA and RNA templates.
  • a "primer” refers to a single-stranded polynucleotide or polynucleotide conjugate capable of acting as a point of initiation for template-directed DNA synthesis in the presence of a suitable polymerase and cofactors. Primers are generally at least 7 nucleotides long and typically range from 10 to 30 nucleotides in length or longer.
  • the term "primer pair" refers to a set of primers including a 5'
  • primers that hybridizes with the complement of the 5' end of the DNA template to be amplified and a 3' "reverse” or “downstream” primer that hybridizes with the 3' end of the sequence to be amplified. Both primers have 5' and 3' ends and primer extension always occurs in the direction of 5' to 3'. Therefore, chemical conjugation at or near the 5' end does not block primer extension by a suitable polymerase. Primers may be referred to as “first primer” and “second primer”, indicating a primer pair in which the identity of the "forward” and "reverse” primers is
  • Additional primers may be used in nested amplification.
  • Primers can be selected to be "substantially complementary" to the sequence on the template to which it is designed to hybridize and serve as a site for the initiation of synthesis.
  • Polymerases are enzymes defined by their function of incorporating nucleoside triphosphates, or deoxynucleoside triphosphates, to extend a 3' hydroxyl terminus of a primer molecule.
  • polymerases include, but are not limited to, E. coli DNA polymerase I, "Klenow" fragment, Taq-polymerase, T7 polymerase, T4 polymerase, T5 polymerase, and reverse transcriptase.
  • reverse transcriptases examples include HIV-1 reverse transcriptase from the human immunodeficiency virus type 1, telomerase, M-MuLV reverse transcriptase from the Moloney murine leukemia virus, and AMV reverse transcriptase from the avian myeloblastosis virus.
  • reverse transcriptase is used to apply the polymerase chain reaction technique to RNA targets.
  • the classical PCR technique can only be applied directly to DNA, but by using reverse transcriptase to synthesize cDNA from RNA, PCR analysis of RNA targets is possible.
  • the technique is collectively called Reverse Transcription-Polymerase Chain Reaction (RT-PCR).
  • RT-PCR Reverse Transcription-Polymerase Chain Reaction
  • complementary refers to two single- stranded nucleic acid sequences that can hybridize to form a double helix. The matching of base pairs in the double helix of two complementary strands is not necessarily absolute.
  • Selectivity of hybridization is a function of annealing temperature, salt concentration, and solvent, and will generally occur under low stringency when there is as little as 55% identity over a stretch of at least 14 to 25 nucleotides. Stringency can be increased by methods well known in the art. See M. Kanehisa, Nucleic Acids Res. 12:203 (1984).
  • a primer that is "perfectly complementary” has a sequence fully complementary across the entire length of the primer and has no mismatches.
  • a “mismatch” refers to a site at which the base in the primer and the base in the target nucleic acid with which it is aligned are not complementary.
  • particles have at least one dimension, such as apparent diameter or circumference, in the micrometer or nanometer range.
  • particles may be composed of, contain cores of, or contain granular domains of, a paramagnetic or superparamagnetic material, such as iron, cobalt, chromium, nickel, copper, manganese, neodymium, and alloys thereof, e.g., Fe203 and Fe304 (alpha-Fe crystal type); alpha'- FeCo; epsilon-Cobalt; CoPt; CrPt 3 , SmCos, Cu 2 MnAl, alpha-FeZr, Nd 2 Fei 4 B, NoTi.
  • a paramagnetic or superparamagnetic material such as iron, cobalt, chromium, nickel, copper, manganese, neodymium, and alloys thereof, e.g., Fe203 and Fe304 (alpha-Fe crystal type); alpha'- FeCo; epsilon
  • ferrites defined as ferrimagnetic or ceramic compound materials consisting of various mixtures of iron oxides such as hematite (FeiOa) or magnetite
  • These materials as used generally are particles having dimensions smaller than a magnetic domain, and may be formed into particles, beads, or microspheres with binders such as latex polymers (generically), silica, and other materials as is generally well known and inclusive of such materials as are commercially available.
  • binders such as latex polymers (generically), silica, and other materials as is generally well known and inclusive of such materials as are commercially available.
  • the technology comprises use of nanoparticles having diameters in the 50 nn lOO ⁇ range such as those that are commercially available for magnetic bioseparations. These particles are "superparamagnetic", meaning that they are attracted to a magnetic field but retain no residual magnetism after the field is removed. Therefore, suspended superparamagnetic particles tagged to the biomaterial of interest can be removed from a matrix using a magnetic field, but they do not
  • Paramagnetic beads have the property that they align themselves along magnetic flux lines and are attracted from areas of lower magnetic flux density to areas of higher magnetic flux density. Some embodiments provide for the use of magnetic microbeads comprising composite materials. Such beads may further contain other micro- or nanoparticles agglomerated with a binder. Composites with RF-tags, QDots, up-converting
  • a magnetic bead need not be formed entirely of a magnetic material, but may instead comprise both magnetic and non-magnetic materials.
  • Microbeads may themselves be colloidal and have chromogenic properties or may be combined with other colloidal metal particles with chromogenic properties. Mixed suspensions of differently modified microbeads may be used.
  • microbeads are modified with surface active agents such as detergents to control their rheological properties, as in ferrofluids.
  • the surface of microbeads may be modified by adsorption or covalent attachment of bioactive molecules, including immunoaffinity agents, antibodies, enzymes, dyes, fluorescent dyes, fluorescent quenchers, oligomers (e.g., capture probes), peptide nucleomers, and the like, and more specifically by coating with streptavidin or single stranded DNA oligomers, for example.
  • microbeads of interest are comprised of at least one paramagnetic element therein, as would be readily recognized by those skilled in art.
  • Suitable matrices for microbeads include polystyrene, divinylbenzene, polyvinyltoluene, polyester, polyurethane, with optional functional groups selected from S0 3 , COOH, NH 2 , glycidyl (COC), OH, CI, tosyl, aldehyde, and sulfhydryl. Particles often range from 0.3 to 5 ⁇ or larger. Latex particles of 100 nm, and 1, 5, 20, 50, or 100 ⁇ are commercially available in bulk. Silica may be used as a matrix or as a capsule. Derivatized silane includes OH, NH2, COOH and other functional groups. Dextran may also be used as a matrix.
  • Polysaccharide may also be used with silane as silica fortified microbeads of particle size around 250 nm.
  • Agarose and cellulose matrices include particles in the range of 1-10 ⁇ , and may be activated for introduction of functional groups.
  • Protein particles such as of gelatin and albumin, have long been used for magnetic microspheres. These are readily activated for amine, carboxyl, hydroxyl, and sulfhydryl linkages with ligands or tags. Liposomes are somewhat more refractory to chemical derivatization, but have been used to make magnetic particles. Naked iron oxide and other paramagnetic metal particles are also known and may be derivatized by adding sulfhydryl groups or chelators. These particles often have sizes of 5 to 300 nm. Certain types of particle populations are known to be uniform in size; in others the heterogeneity may be controlled or selected.
  • microbeads may be readily prepared.
  • carboxyl-modified microbeads containing approximately 20-60% magnetite are made by dispersing a (magnetite)/ styrene/divinylbenzene ferrofluid mixture in water and emulsion- polymerizing the monomers to trap the magnetite in a polymer matrix of microbeads of approximately 1 ⁇ diameter. The magnetite is thus dispersed throughout the solid beads.
  • Other prior art means for synthesizing and modifying microbeads are commonly known.
  • Suitable microbeads include those available from vendors such as Bangs
  • Dynabeads MyOne Microspheres and the like. Cobalt paramagnetic microbeads are sold as Dynabead's MyOne TALON. BioMag Plus microbeads from Polysciences have an irregular shape and thus more surface area for affinity chemistry.
  • Paramagnetic and superparamagnetic materials e.g., when fabricated as microbeads
  • Paramagnetic and superparamagnetic materials have the property of responding to an external magnetic field when present, but dissipating any residual magnetism immediately upon release of the external magnetic field, and are thus easily resuspended and remain monodisperse, but when placed in proximity to a magnetic field, clump tightly, the process being fully reversible by simply removing the magnetic field.
  • magnetic force field or “magnetic field” refers to a volume defined by the magnetic flux lines between two poles of a magnet or two faces of a coil. Electromagnets and driving circuitry can be used to generate magnetic fields and localized magnetic fields. Permanent magnets may also be used. Preferred permanent magnetic materials include NdFeB (Neodymium-Iron-Boron NdiFewB), Ferrite
  • Magnetic Cobalt (Samarium Cobalt). The magnetic forces within a magnetic force field follow the lines of magnetic flux. Magnetic forces are strongest where magnetic flux is most dense.
  • a moving magnetic force field penetrate most solids and liquids.
  • a moving magnetic force field has two vectors ⁇ one in the direction of travel of the field and the other in the direction of the lines of magnetic flux.
  • the term "reagent" refers broadly to any chemical or biochemical agent used in a reaction, including enzymes.
  • a reagent can include a single agent which itself can be monitored (e.g., a substance that is monitored as it is heated) or a mixture of two or more agents.
  • a reagent may be living (e.g., a cell) or non-living.
  • Exemplary reagents for a nucleic acid amplification reaction include, but are not limited to, buffer, metal ion (for example magnesium salt), chelator, polymerase, primer, template, nucleotide triphosphate, label, dye, nuclease inhibitor, and the like.
  • Reagents for enzyme reactions include, for example, substrates, chromogens, cofactors, coupling enzymes, buffer, metal ions, inhibitors and activators. Not all reagents are reactants.
  • stabilized refers to the protection of a reagents activity, catalytic activity (e.g., for an enzyme reagent), and/or reactive ability by minimizing or preventing the inactivation (e.g., by deterioration, break-down, decomposition, etc.) of the reagent for a period sufficient for typical shipment, storage, and use.
  • detergent refers to anionic, cationic, zwitterionic, and nonionic surfactants.
  • the term "robustness” refers to the relative tolerance of an assay format to variability in execution, to materials substitutions, and to interferences, over a range of assay conditions. Robustness generally increases with the strength of the detection signal generated by a positive result. Robustness negatively correlates with the difficulty and complexity of the assay.
  • the term “specificity” refers to the ability of an assay to reliably differentiate a true positive signal of the target biomarker from any background, erroneous or interfering signals.
  • sensitivity refers to the lower limit of detection of an assay where a negative can no longer be reliably distinguished from a positive.
  • endpoint refers to a "result” from either a qualitative or quantitative assay and may refer to both stable endpoints where a constant plateau or level of reactant is attained and to rate reactions where the rate of appearance or disappearance of a reactant or product as a function of time (e.g., the slope) is the datapoint.
  • universal reaction buffer refers to a single buffer composition in which a series of multiple (e.g., 2 or more) reactions (e.g., enzymatic reactions) or sample manipulations (e.g., physical fragmentation) occurs.
  • a universal buffer is advantageous in that a series of sample manipulations and/or enzymatic reactions does not require buffer replacement between each sample manipulation and/or enzymatic reaction.
  • a preferred universal buffer is one in which all steps of a sample-to-library process (e.g., nucleic acid isolation, amplification (e.g., WGA), nucleic acid fragmentation, end polishing, A-tailing, ligation to sequencing adapters, size selection, and/or final purification of the sequencing library) occur with each sample manipulation and/or enzymatic step having an efficiency that is
  • microfluidic cartridges are fabricated from various materials using techniques such as laser stenciling, embossing, stamping, injection molding, masking, etching, and three-dimensional soft lithography.
  • Laminated microfluidic cartridges are further fabricated with adhesive interlayers or by thermal adhesiveless bonding techniques, such as by pressure treatment of oriented microfluidic cartridges.
  • microarchitecture of laminated and molded microfluidic cartridges can differ.
  • microfluidic channel or “microchannel” refers to a fluid channel having variable length and one dimension in cross-section less than 500 to 1000 ⁇ .
  • Microfluidic fluid flow behavior in a microfluidic channel is highly non-ideal and laminar and may be more dependent on wall wetting properties, roughness, liquid viscosity, adhesion, and cohesion than on pressure drop from end to end or cross- sectional area.
  • the microfluidic flow regime is often associated with the presence of "virtual liquid walls" in the channel.
  • head pressures of 10 psi or more can generate transitional flow regimes bordering on turbulent, as can be important in rinse steps of assays.
  • Microchannels constructed of layers formed by extrusion molding may have more rounded channel profiles and a radius on each "via".
  • the internal channel surfaces of injection molded parts are also somewhat smoother.
  • the flow characteristics of the channels are significant because of the profound surface effects in the microflow regime. Surface tension and viscosity compound surface roughness effects.
  • the most narrow dimension of a channel has the most profound effect on flow. It follows that flow in channels that are based on rectangular or circular cross-sectional profiles is controlled by the diagonal width or diameter, and design is typically varied to take advantage of this behavior. Reduction of taper in the direction of flow leads to a wicking effect for diameters below 200 micrometers. Conversely, flow can be stopped by opening up a channel to form a bulb unless pressure is applied. Vias in a channel can be designed to promote directional flow, e.g., to provide a type of solid state check valve.
  • microfluidic pump include, e.g., bulbs, bellows, diaphragms, or bubbles intended to force movement of fluids, where the substructures of the pump have a thicknesses or other dimension of less than 1 millimeter.
  • Such pumps include the mechanically actuated recirculating pumps described in U.S. Pat. No.
  • Such pumps may be robotically operated or operated by hand. Electroosmotic pumps are also provided. Such pumps can be used in place of external drives to propulse the flow of solubilized reagents and sample in microfluidic device-based assays.
  • bellows pump refers to a device formed as a cavity, often cylindrical in shape, bisected in coronal section by an elastomeric diaphragm to form a first and a second half-chamber that are not fluidically connected.
  • diaphragm is controlled by a pneumatic pulse generator connected to the first half- chamber. Positive pressure above the diaphragm distends it, displacing the contents of the second half-chamber, negative gauge pressure (suction) retracts it, expanding the second half chamber and drawing fluid in.
  • a "check valve” is a one-way valve.
  • via refers to a step in a microfluidic channel that provides a fluid pathway from one substrate layer to another substrate layer above or below, characteristic of laminated devices built from layers.
  • waste chamber or “waste sequestration receptacle” and the like refers to cavity or chamber that serves as a receptacle for sequestering discharged sample, rinse solution, and waste reagents.
  • a waste chamber includes a wicking material (see wick) in some embodiments.
  • Waste chambers may also be sealed under an elastic isolation membrane sealingly attached to the body of the microfluidic device. This inner membrane expands as the bibulous material expands, thus enclosing the waste material. The cavity outside the isolation membrane is vented to atmosphere so that the waste material is contained and isolated. Waste chambers may optionally contain dried or liquid sterilants.
  • a "deformable film”, e.g., lacking elasticity is used in the microfluidic devices of the technology described.
  • a detector or a sensor refers to an apparatus for detecting a signal associated with the endpoint of an assay or for detecting a signal associated with monitoring an assay in real time.
  • a detector or a sensor includes a detection channel.
  • a detector or sensor includes but is not limited to, e.g., a spectrophotometer, fluorometer, luminometer, photomultiplier tube, photodiode, nephlometer, photon counter, voltmeter, ammeter, pH meter, capacitative sensor, radio-frequency transmitter, magnetoresistometer, or Hall-effect device.
  • Magnetic particles, beads, and microspheres having color or impregnated with color or having a higher diffraction index are used in some embodiments to facilitate visual or machine-enhanced detection of an assay endpoint.
  • Magnifying lenses, optical filters, colored fluids, and labeling are used in some embodiments to improve detection and interpretation of assay results.
  • a detector or a sensor may detect a signal produced by a "label” or "tag” such as, but not limited to, dyes such as chromophores and fluorophores, radio frequency tags, plasmon resonance, spintronic, radiolabel, Raman scattering, chemoluminescence, inductive moments, or fluorescence quenching.
  • the technology also comprises use of a variety of substrate and product chromophores associated with enzyme-linked immunoassays that amplify a detection signal to improve the sensitivity of the assay.
  • Detection systems are optionally qualitative, quantitative, or semi ⁇ quantitative. Visual detection is preferred for its simplicity; however, a detector or sensor can comprise visual detection, machine detection, manual detection, or automated detection.
  • a "heating and cooling” includes convective and conductive heating and cooling elements such as electroresistors, hot air, lasers, infrared radiation, Joule heating, thermoelectric or Peltier devices, heat pumps, endothermic reactants, and the like, generally in conjunction with a heat sink for dissipating heat. Heating also includes heating by the motion of magnetic beads driven by a high frequency magnetic field.
  • heating and cooling devices use for cooling
  • thermocycling fall into two categories ⁇ ramped and fixed temperature. Fixed
  • thermoelectric e.g., Peltier
  • heating and cooling means interface with a fluidics member so as to effect heat exchange with the liquid contents, e.g., for PCR and/or other enzymatic reactions.
  • Embodiments of the technology relate to providing a sequencing library compatible with a NGS platform.
  • this process comprises steps such as nucleic acid fragmentation, end polishing, A-tailing, ligation to sequencing adapters, and final purification of the sequencing library.
  • all of these steps are accomplished in the same universal reaction buffer, which reduces the number of reagents, reduces the number of purification steps, and reduces the overall time for preparation of the sequencing library.
  • Embodiments of the technology provide an integrated system for one or more of, e.g., accepting samples (e.g., clinical, biological, and/or environmental samples), lysing cells, extracting nucleic acids, amplifying nucleic acids, (e.g., whole genome amplification in some embodiments), fragmenting nucleic acids, size selecting nucleic acids, labeling nucleic acids, polishing nucleic acid fragment ends, ligating adaptors or linkers to nucleic acids, purifying nucleic acids, sequencing nucleic acids or providing nucleic acids to a sequencer, etc.
  • samples e.g., clinical, biological, and/or environmental samples
  • amplifying nucleic acids e.g., whole genome amplification in some embodiments
  • fragmenting nucleic acids e.g., size selecting nucleic acids, labeling nucleic acids, polishing nucleic acid fragment ends, ligating adaptors or linkers to nucleic acids, purifying nucleic acids, sequencing nucleic
  • Embodiments of the technology provide a microfluidic cartridge comprising one or more components, modules, chambers, etc. associated with these processes.
  • a component, module, chamber may perform two or more of these processes.
  • the cartridge accepts an input sample that is a biological sample and provides as output a nucleic acid sequencing library suitable for NGS.
  • the entire process is integrated into a microfluidic cartridge (e.g., in some embodiments, a single-use and/or disposable microfluidic cartridge) that contains one or more (e.g., in some embodiments, all) reagents needed to perform the process.
  • a microfluidic cartridge e.g., in some embodiments, a single-use and/or disposable microfluidic cartridge
  • the wastes of one or more processes, modules, components, etc. are also contained within the cartridge (e.g., in a waste containment compartment) or, in some embodiments, are removed from the microfluidic cartridge by a component for waste removal.
  • the reagents are stabilized (e.g., in some embodiments in a lyophilized state) so that the cartridges can be stored (e.g., at room temperature) for long periods of time.
  • the cartridges generally contain a port where the resulting output (e.g., a sequencing library) is directly introduced into the workflow of an apparatus that performs DNA sequencing, such as an Illumina sequencing instrument (e.g., a HiSeq, NextSeq, MiSeq, or other instrument based on sequencing-by- synthesis, e.g., Solexa sequencing-by- synthesis), a Life Technologies sequencing instrument (e.g., an Ion Proton, Ion PGM, or other instrument based on the Ion Torrent technology), or a PacBio sequencing instrument (e.g., an RS II, an instrument based on the SMRT technology, or other Pac Bio sequencing instrument).
  • an Illumina sequencing instrument e.g., a HiSeq, NextSeq, MiSeq, or other instrument based on sequencing-by- synthesis, e.g., Solexa sequencing-by- synthesis
  • Life Technologies sequencing instrument e.g., an Ion Proton, Ion PGM, or
  • the technology is suitable for these extant sequencing technologies, platforms, workflows, and instruments, the technology is not limited to providing sequencing libraries that are compatible with these exemplary technologies. Accordingly, the technology is general and provides sequencing libraries to any existing, nascent, or future sequencing technology. Further, in some embodiments, the resulting output nucleic acids are used and/or processed using other nucleic acid reasearch and/or analysis techniques (e.g., microarray, transformation, solution hybridization, phage display, cloning (e.g., shotgun cloning, construction of clone libraries, construction of metagenomic libraries, etc.)
  • nucleic acid reasearch and/or analysis techniques e.g., microarray, transformation, solution hybridization, phage display, cloning (e.g., shotgun cloning, construction of clone libraries, construction of metagenomic libraries, etc.)
  • microfluidic cartridges comprise multiple components, modules, or chambers corresponding to independent processes, tasks, or subprocesses for producing a NGS library.
  • multiple components, modules, or chambers are integrated to provide a single device or two or more interconnected devices. Each component or module in turn comprises microfluidic elements or components.
  • Elements of these components and modules may include, for instance, microfluidic channels, tees (e.g., T-shaped intersections), Y-shaped intersections, chambers, valves, vias, filters, solid phase capture elements, isolation filters, pneumatic manifolds, blister packs (e.g., with reagent pouches), waste sequestration (e.g., waste containment) chambers, sanitary vents, bellows chambers, bellows pumps, optical windows, test pads, and deposits of dehydrated reagents (e.g., including buffers, solubilizers, and passivating agents).
  • tees e.g., T-shaped intersections
  • Y-shaped intersections Y-shaped intersections
  • chambers e.g., valves, vias
  • filters solid phase capture elements
  • isolation filters e.g., pneumatic manifolds
  • blister packs e.g., with reagent pouches
  • waste sequestration e.g.,
  • the microfluidic cartridge e.g., the components, modules, chambers, circuits, subcircuits, etc.
  • the microfluidic cartridge are generally fabricated of an elastomer (e.g., plastic) and are made in some embodiments by lamination, by molding, and/or by lithography (e.g., soft lithography), or by a
  • the cartridges comprise the reagents needed for preparation of an output NGS library from the input sample.
  • the microfluidic devices include RFID (radio-frequency identification) devices, microchips, bar codes, and/or labeling to aid tracking and processing samples and analytical data.
  • RFID radio-frequency identification
  • RFID devices, microchips, bar codes, and/or labeling provide information to an apparatus comprising an interface for cartridge docking, e.g., some embodiments comprise a "smart" instrument or apparatus that obtains information from a
  • microfluidic cartridge and/or communicates sample data and test results to a network.
  • the microfluidic cartridge comprises a pneumatic manifold that serves for control and fluid manipulation, although electronically activated valves find use in some embodiments.
  • air ports are connected to the pneumatic manifold. Air ports are provided in some embodiments with hydrophobic isolation filters (e.g., any liquid-impermeable, gas-permeable filter membrane) where leakage of fluid from within the device is undesirable and unsafe.
  • Some embodiments comprise a flexible membrane layer.
  • the flexible membrane layer provides microfluidic valves and pumps.
  • the flexible membrane layer connects the cartridge to a controller deck where pressure and vacuum valves lie. The manipulation of the valves and pumps on the controller box applies either pressure or vacuum to the flexible membrane and moves the liquid through the channels by pneumatic actuation.
  • reaction chambers are provided on the microfluidic cartridge and can be any suitable shape, such as rectangular chambers, circular chambers, tapered chambers, serpentine channels, and various geometries for performing a reaction.
  • These chambers may have observation windows (e.g., that allow the passage of electromagnetic radiation in the visible, ultraviolet, and/or infrared range of the spectrum), e.g., for examination of the contents (e.g., by a user, by a detector of a visible, ultraviolet, and/or infrared signal, etc.), e.g., to provide one or more detection chambers.
  • Waste chambers are generally provided on the microfluidic cards. Waste chambers are optionally vented with sanitary hydrophobic membranes.
  • the technology comprises non-microfluidic (e.g., macrofluidic) and microfluidic elements.
  • non-microfluidic elements channels, chambers, etc.
  • microfluidic devices e.g., a microfluidic cartridge.
  • the microfluidic cartridge comprises directional control mechanisms, such as valves and pumps, by which fluid is selectively routed between different chambers and along different channels, and by which a single chamber can communicate with a number of other chambers.
  • the instrument uses particulate capture agents that bind a nucleic acid, and that, in various embodiments, are immobilized, suspended, and/or re-suspended in a desired volume of fluid and, in some embodiments, are routed to and from chambers through the microfluidic channels.
  • the amount of capture agent is selected to capture a desired amount of nucleic acid from a sample, for example, to concentrate nucleic acid from a dilute sample, to quantitatively capture all or
  • the instrument comprises a plurality of parallel fluidic circuits by which fluidic operations are performed in parallel.
  • Manipulation of a nucleic acid can include a variety of steps. These can include, for example, preparing the nucleic acid for a reaction (e.g., enzymatic, molecular biological, biochemical, chemical, etc.), mixing with one or more reagents in various sequences, performing a reaction (e.g., enzymatic, molecular biological, biochemical, chemical, etc.) with the nucleic acid, removing wastes, washing, and eluting.
  • a reaction e.g., enzymatic, molecular biological, biochemical, chemical, etc.
  • the technology is used in some embodiments to perform these functions by routing analytes, reagents, wastes, and wash solutions between compartments, modules, chambers, and components.
  • the technology provides a plurality of chambers (e.g., microfluidic and/or non-microfluidic chambers) connected to each other through microfluidic channels. Fluid is moved from one chamber to another by any appropriate motive force, for example, continuous pressure or non-continuous pressure (e.g., positive displacement pumps), electroosmotic flow, and/or by modulating wetting of droplets to the surface (see, e.g., Kim et al. (2013) A Microfluidic DNA Library
  • channels comprise directional control mechanisms to route fluids selectively between chambers as desired. These mechanisms can be valves, such as the diaphragm valves (e.g., as described herein), single use valves such as wax valves, or other valves. By opening and closing valves in a proper sequence, analyte, reagents, and waste are routed into appropriate locations. In this way, the microfluidic cartridge routes fluids between chambers, components, and modules where various functions are performed on the nucleic acids.
  • a chamber, component, or module comprises one or more capture agents to bind a nucleic acid.
  • the capture agents generally comprise a solid substrate and are able to bind nucleic acids specifically or non-specifically.
  • the substrate asserts the binding force or a molecule having binding properties is attached to the substrate, for example, a capture probe complementary to a target nucleic acid.
  • the capture agent is a particulate capture agent.
  • the material in some embodiments, is a chromatographic material. In this case, a sample is passed through the chromatographic material and separated fractions are introduced into the microfluidic cartridge or into one or more chambers, components, or modules of the microfluidic device.
  • the capture agent is a monolith.
  • the capture agent is attached to a surface of the chamber, such as a post, or the chamber surface can be derivatized with a capture molecule.
  • the microfluidic cartridge comprises a filter for extracting nucleic acids from a sample.
  • the filter binds 1 to 5 ⁇ g of nucleic acid (e.g., 1, 2, 3, 4, or 5 ⁇ g; e.g., up to 1, 2, 3, 4, or 5 ⁇ g).
  • the filter binds 1 to 2 ⁇ g (e.g., 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 ⁇ g) of nucleic acids.
  • the filter is a 1-mm to 5-mm (e.g., a 3-mm) filter (from, e.g., Macherey-Nagel).
  • the device is adapted to move particles, such as beads, between one or more chambers, modules, or components.
  • the particles are responsive to magnetic force, electrical forces, or other forces.
  • the particles are paramagnetic or magnetic particles, e.g., to allow manipulation of the particles within the cartridge by application of magnetic fields using fixed or movable magnets including electromagnets.
  • the particles function as a capture agent to capture one or more nucleic acids from a sample.
  • the particles can have specific or non-specific affinity for a nucleic acid.
  • the microfluidic cartridge is used to capture a selected amount of a nucleic acid from a sample and transport the captured nucleic acid.
  • a non-microfluidic volume of a sample is provided to a chamber.
  • a selected amount of the nucleic acid is captured by the capture agent.
  • a small amount of capture agent can be selected so that only a portion of the nucleic acid to be used in a reaction is captured. This allows sampling of a portion of the nucleic in the sample volume, e.g., an amount sufficient or appropriate for a reaction (e.g., enzymatic, molecular biological, biochemical, chemical, etc.).
  • a greater amount of capture agent can be used so that most or substantially all of the nucleic acid present is captured on the capture agent. This effectively concentrates the nucleic acid. Washing the nucleic acid can remove impurities, effectively purifying the nucleic acid from undesirable components of the sample such as inhibitors, proteins, etc.
  • the specificity of the capture can also be controlled by adjusting the chemistry to select broader or narrower ranges of nucleic acids, for example longer or shorter pieces of DNA (e.g., longer or shorter than a defined, specified cutoff as described herein),
  • the cartridge or an instrument adapted to accept a microfluidic cartridge comprises a detector, e.g., an optical detector or other detector as described herein, for detecting nucleic acid molecules at stages of processes performed according to embodiments of the technology provided.
  • reaction sequences are contemplated.
  • a reaction e.g., enzymatic, molecular biological, biochemical, chemical, etc.
  • the reaction product is purified (e.g., wholly or partially purified, e.g., wholly or partially isolated from other components) and subjected to a different reaction (e.g., enzymatic, molecular biological, biochemical, chemical, etc.).
  • a different reaction e.g., enzymatic, molecular biological, biochemical, chemical, etc.
  • this sequence of purifying a reaction product and performing a subsequent different reaction is repeated.
  • this technology contemplates performing a series of enzymatic, molecular biological, biochemical, chemical, etc. reactions separated by purification steps. It is to understood, though, that in some embodiments the output of one reaction is used directly as input for a subsequent reaction without an intervening purification step.
  • the instrument can be used to perform physical actions and/or enzymatic, molecular biological, biochemical, and/or chemical reactions on nucleic acids, such as nucleic acid amplification (e.g., PCR), cycle sequencing, real-time PCR, rolling circle amplification, restriction digestion, nucleic acid fragmentation, protein digestion, ligation, labeling, end polishing, etc.
  • nucleic acid amplification e.g., PCR
  • cycle sequencing e.g., PCR
  • real-time PCR e.g., PCR
  • rolling circle amplification e.g., restriction digestion, nucleic acid fragmentation, protein digestion, ligation, labeling, end polishing, etc.
  • reagents for performing nucleic acid amplification in an appropriate volume are routed through a channel into a chamber configured for thermal cycling (e.g., configured for heating and cooling by a thermoelectric component).
  • the reaction mixture elutes the nucleic acids from particles.
  • cleanup comprises diluting the product to decrease salt concentration and/or binding the nucleic acids to beads, immobilizing them, and washing them.
  • cleanup comprises chromatography, solid phase extraction, and/or other cleanup methods at microfluidic scale or non-microfluidic scale.
  • the microfluidic components route the product to an appropriate location for analysis and/or quantification, e.g., to a capillary tube for capillary electrophoresis, to a fluorimeter, to a UV/visible spectrometer, to a capture probe, etc., or the product is output into an external instrument for analysis such as a commercial capillary array electrophoresis instrument, mass spectrometer, or other analytical instrument.
  • the microfluidic device also can contain a waste confinement area.
  • reaction performed on a nucleic acid is an Eberwine synthesis, in which a first chemical reaction, reverse transcription, is followed by a second reaction, second strand synthesis, which is followed by a third reaction, transcription. Before each subsequent reaction, and typically after the last reaction, the product is removed from contaminants in preparation for the next step.
  • microfluidic devices are monolithic devices.
  • monolithic devices a plurality of components is provided on a single substrate.
  • a monolithic device comprises an elastic layer (e.g., a flexible membrane layer) functioning as a diaphragm for a plurality of valves.
  • one actuation channel operates a plurality of valves on a monolithic device. This allows parallel activation of many fluidic components (e.g., channels).
  • a plurality of actuation channels operates different sets of valves (e.g., a first actuation channel operates a first plurality of valves, a second actuation channel operates a second plurality of valves, etc.).
  • Monolithic devices can have dense arrays of microfluidic components. These components function with high reliability, in part because the components are fabricated simultaneously on a single substrate, rather than being made independently and assembled together.
  • a channel comprises an open fluid conduit and in some embodiments a circuit comprises a closed fluid conduit.
  • the device comprises at least 1 fluidic component per 1000 mm 2 , at least 2 fluidic components per 1000 mm 2 , at least 5 fluidic components per 1000 mm 2 , at least 10 fluidic components per 1000 mm 2 , at least 20 fluidic components per 1000 mm 2 , at least 50 fluidic components per 1000 mm 2 .
  • the device comprises in certain embodiments at least 1 mm of channel length per 10 mm 2 area, at least 5 mm channel length per 10 mm 2 , at least 10 mm of channel length per 10 mm 2 or at least 20 mm channel length per 10 mm 2 .
  • the device in some embodiments comprises valves (either seated or unseated) at a density of at least 1 valve per cm 2 , at least 4 valves per cm 2 , or at least 10 valves per cm 2 .
  • the device comprises elements, features, components, etc., such as channels, reaction chambers, etc., that are no more than 5 mm apart edge-to-edge, no more than 2 mm apart, no more than 1 mm apart, no more than 500 micrometers apart, or no more than 250 micrometers apart.
  • the microfluidic cartridge generally performs a number of preparative and/or analytical reactions on a sample.
  • the device generally comprises a number of discrete reaction, storage, and/or analytical chambers disposed within a single unit or body. The device finds use in sequencing applications, sample identification, and characterization applications (e.g., for taxonomic studies, forensic applications, e.g., criminal investigations, and the like).
  • Embodiments provide that the body of the microfluidic cartridge defines various components and modules (e.g., comprising reaction chambers, valves, fluid passages (e.g., channels), etc.) in which the various operations described herein are performed. Fabrication of the cartridge, and thus the various components (e.g., chambers and channels) disposed within the cartridge, may generally be performed using one or a combination of a variety of well-known manufacturing techniques and materials.
  • the material from which the microfluidic cartridge is fabricated is selected to maximize resistance to the full range of conditions to which the cartridge is exposed, e.g., extremes of temperature, salt, pH, application of electric fields and the like, and is also selected for compatibility with other materials used in the device. Additional components may be later introduced, as necessary, into the cartridge.
  • the cartridge is formed from a plurality of distinct parts that are later assembled or mated (e.g., in some embodiments components or layers are thermally annealed, e.g., at 70 to 90°C (e.g., approximately 80°C)).
  • components or layers are thermally annealed, e.g., at 70 to 90°C (e.g., approximately 80°C)).
  • separate and individual chambers and fluid passages may be assembled to provide the various chambers of the device.
  • the number and size of the reaction chambers included within the microfluidic cartridge vary in various embodiments depending upon the specific application for which the cartridge is to be used.
  • the microfluidic cartridge includes at least two distinct reaction chambers, and in some embodiments, at least three, four, or five distinct reaction chambers, all integrated within a single cartridge.
  • the individual reaction chambers vary in size according to the specific function of the reaction chamber. In general, however, the reaction chambers are from about 0.5 to about 20 mm in width or diameter and about 0.05 to about 5 mm deep.
  • Fluid channels typically range from about 20 to about 1000 ⁇ wide, preferably, 100 to 500 ⁇ wide and about 5 to 100 ⁇ deep.
  • the cartridge is generally fabricated using one or more of a variety of methods and materials suitable for microfabrication techniques.
  • the body of the device comprises a number of planar members that are individually injection molded parts fabricated from a variety of polymeric materials, or that are silicon, glass, or the like.
  • methods for etching, milling, drilling, etc. are used to produce wells and depressions that compose the various reaction chambers and fluid channels within the cartridge.
  • Microfabrication techniques such as those regularly used in the semiconductor and microelectronics industries, are particularly suited to these materials and methods.
  • photolithographic methods of etching substrates are particularly well suited for the microfabrication of these microfluidic cartridges.
  • the first sheet of a substrate may be overlaid with a photoresist.
  • electromagnetic radiation source may then be shined through a photolithographic mask to expose the photoresist in a pattern that reflects the pattern of chambers and/or channels on the surface of the sheet. After removing the exposed photoresist, the exposed substrate may be etched to produce the desired wells and channels.
  • photoresists include those used extensively in the semiconductor industry. Such materials include polymethyl methacrylate (PMMA) and its derivatives, and electron beam resists such as poly(olefin sulfones) and the like (more fully discussed in, e.g., Ghandi, "VLSI Fabrication Principles," Wiley (1983) Chapter 10, incorporated herein by reference in its entirety for all purposes).
  • the wells manufactured into the surface of one planar member make up the various reaction chambers of the microfluidic cartridge.
  • Channels manufactured into the surface of this or another planar member provide fluid channels that are used to fluidly connect the various reaction chambers.
  • Another planar member is then placed over and bonded to the first, whereby the wells in the first planar member define cavities within the body of the microfluidic cartridge. In some embodiments, these cavities are the various reaction chambers of the microfluidic cartridge.
  • fluid channels manufactured in the surface of one planar member, when covered with a second planar member define fluid passages through the body of the microfluidic cartridge. These planar members are bonded together or laminated to produce a fluid- tight body of the microfluidic cartridge.
  • Bonding of the planar members of the device varies depending upon the materials used. For example, adhesives are used in some embodiments to bond the planar members together. For plastic parts, acoustic welding techniques are used in some embodiments. In some embodiments, the technology provides methods of constructing a microfluidic device comprising the thermal
  • annealing e.g., at 70°C to 90°C (e.g., 80°O) of polymer (e.g., PMMA) layers to provide a microfluidic device with improved resistance to temperature changes, solvents, and other exposures that may degrade the stability of the device, e.g., as described hereinbelow.
  • polymer e.g., PMMA
  • the methods provided are used in some embodiments to fabricate individual discrete components of the microfluidic cartridge which are later assembled into the body of the microfluidic cartridge.
  • the microfluidic cartridge comprises a combination of materials and manufacturing techniques.
  • the microfluidic cartridge includes some parts of injection molded plastics, and the like, while other portions of the microfluidic cartridge comprise etched silica or silicon planar members, and the like.
  • the microfluidic cartridge includes some parts formed by photolithography, and the like, while other portions of the microfluidic cartridge comprise glass, etched silica, or silicon planar members, and the like.
  • injection molding techniques are used to form a number of discrete cavities in a planar surface that define the various components, modules, and/or reaction chambers, whereas additional components, e.g., fluid channels, arrays, etc, are fabricated on a planar glass, silica or silicon chip or substrate. Lamination of one set of parts to the other then results in the formation of the various reaction chambers, which are interconnected by the appropriate fluid channels.
  • the microfluidic cartridge is made from at least one injection molded, press molded, or machined polymeric part that has one or more wells or depressions manufactured into its surface to define several of the walls of the reaction chamber or chambers.
  • suitable polymers for injection molding or machining include, e.g., polycarbonate, polystyrene, polypropylene, polyethylene acrylic, and commercial polymers such as Kapton, Valox, Teflon, ABS, Delrin, and the like.
  • a second part that is similarly planar in shape is mated to the surface of the polymeric part to define the remaining wall of the reaction chamber(s).
  • the microfluidic devices are prepared using multilayer soft lithography techniques.
  • microfluidic devices are prepared as multilayer PDMS (e.g., Sylgard 183) devices (e.g., on a solid substrate, e.g., on glass) using multilayer soft lithographic techniques (MSL).
  • MSL multilayer soft lithographic techniques
  • a cartridge comprises a combination of several layers of PMMA (e.g., "ALTUGLASS"), double sided tape (Nitto Denko, 5015P), a flexible layer (Tekniflex, TP68), and a filter (Macherey-Nagel).
  • PMMA e.g., "ALTUGLASS”
  • double sided tape Nito Denko, 5015P
  • Tekniflex, TP68 a flexible layer
  • a filter Macherey-Nagel
  • the thickness of the tape is 100 ⁇ and PMMA having a thickness of both 1 mm and 4 mm were used.
  • the PMMA, tape layers, and the filter were cut by a laser (Coherent
  • the structure (from bottom to top) of the exemplary integrated cartridge comprises the components described herein (e.g., comprising components arranged in Modules 1 to 4).
  • laser-cut holes are provided at the corners (e.g., at two corners) for registration (e.g., alignment) of the layers during assembly.
  • the exemplary cartridge comprises the following layers ⁇
  • This layer is used as membrane for the valves and pumps and does not have any pattern. 2 - Adhesive tape
  • This layer comprises the bases of the reaction chambers (heaters, ultrasonic, magnetic, etc.) and the pump/valve chambers. Also, the input channel to the filter is patterned in this layer.
  • This layer comprises the reaction chambers (heaters, ultrasonic, magnetic), the filter hole, the channels, and the via-holes from the valves to the channels and chambers.
  • the channels are engraved by laser on the top side of the image.
  • the pulse width and focus settings for the channels are 200 ⁇ Z3 and 100 ⁇ Z2 for two types of channels provided in this layer.
  • the width and depth of the engraved channels are respectively 0.82 and 0.28 mm for the first type of channels and 0.7 and 0.14 mm for the second type of channels. 4 - Adhesive tape
  • This layer comprises the reaction chambers (heaters, ultrasonic, magnetic, mix), footprint for the dry reagent and overflow chambers, two via holes to a vent valve, footprint for syringe connection holes (buffers, sample, waste, output). Also two via holes for the output channel from the filter are in this layer.
  • This layer comprises the reaction chambers (heaters, ultrasonic, magnetic, mix), reagent and overflow chambers, two via holes to a vent valve, an output channel from the filter, channels from the dry reagent to the overflow chambers, and the connection holes (buffers, sample, waste, output). Some channels are engraved on the top side of the image and some channels are engraved at the bottom side of the image. The laser settings for these channels are identical to the second and first channel types, respectively, in layer 3. Furthermore, extra alignment holes for assembly of the top cover and ultrasonic chamber are cut in this layer.
  • This layer comprises holes for the overflow chambers, two via holes to a vent valve, two small vent holes for the heater chambers, and a hole for waste.
  • This layer comprises holes for the overflow chambers, a hole for waste, and two engraved (vent) channels at the bottom side of the image. Laser settings are identical to the channels in layer 3. 8— Various parts 1 ' ⁇ magnetic actuation chamber
  • a square plate is used to make the magnetic chamber 2 mm in height. It is a plate of 1 mm PMMA with tape on both sides. Another square 1 mm PMMA plate is used as a cover for the magnetic chamber. 9 - Various parts 2 ' ⁇ vented headspace
  • Vented headspaces two 5-mm thick plates with two 9-mm holes
  • a total height of approximately 14 mm results for the ultrasonic chamber.
  • the second hole forms the overflow chamber, while the small holes are used for alignment during assembly.
  • a "roof is placed (a
  • This rectangular plate is a cover for the ultrasonic chamber and comprises of PMMA with tape on one side and an engraved channel.
  • samples e.g., biological samples comprising cells and/or nucleic acids.
  • samples include various fluid samples.
  • the sample is a bodily fluid sample from the subject.
  • the sample is an aqueous or a gaseous sample.
  • the sample is a gel. In some embodiments, the sample includes one or more fluid component. In some embodiments, solid or semi-solid samples are provided. In some embodiments, the sample comprises tissue collected from a subject. In some embodiments, the sample comprises a bodily fluid, secretion, and/or tissue of a subject. In some embodiments, the sample is a biological sample. In some embodiments, the biological sample is a bodily fluid, a secretion, and/or a tissue sample.
  • biological samples include but are not limited to, blood, serum, saliva, urine, gastric and digestive fluid, tears, stool, semen, vaginal fluid, interstitial fluids derived from tumorous tissue, ocular fluids, sweat, mucus, earwax, oil, glandular secretions, breath, spinal fluid, hair, fingernails, skin cells, plasma, nasal swab or nasopharyngeal wash, spinal fluid, cerebral spinal fluid, tissue, throat swab, biopsy, placental fluid, amniotic fluid, cord blood, emphatic fluids, cavity fluids, sputum, pus, micropiota, meconium, breast milk, and/or other excretions.
  • the sample is provided from a human or an animal, e.g., in some embodiments the sample is provided from a mammal (e.g., a vertebrate) such as a murine, simian, human, farm animal, sport animal, or pet. In some embodiments, the sample is collected from a living subject and in some embodiments the sample is collected from a dead subject.
  • a mammal e.g., a vertebrate
  • the sample is collected from a living subject and in some embodiments the sample is collected from a dead subject.
  • the sample is collected fresh from a subject and in some embodiments the sample has undergone some form of pre-processing, storage, or transport.
  • the sample is a formalin or formaldehyde fixed paraffin embedded (FFPE) sample.
  • FFPE samples e.g., FFPE tissue samples
  • the clinical utility of FFPE samples is substantial, where retrospective analysis of archival tissue enables the correlation of molecular findings with the response to treatment and the clinical outcome.
  • the sample comprises nucleic acids that are amplified, e.g., prior to or after another processing step (e.g., a fragmentation step).
  • the sample is provided to a microfluidic device from a subject without undergoing intervention or much elapsed time.
  • the subject contacts the microfluidic cartridge to provide the sample.
  • a subject provides a sample and/or the sample may be collected from a subject.
  • the subject is a patient, clinical subject, or pre-clinical subject.
  • the subject is undergoing diagnosis, treatment, and/or disease management or lifestyle or preventative care.
  • the subject may or may not be under the care of a health care professional.
  • the sample is collected from the subject by puncturing the skin of the subject; in some embodiments, the sample is collected from the subject without puncturing the skin of the subject. In some embodiments, the sample is collected through an orifice of the subject. In some embodiments, a tissue sample (e.g., an internal or an external tissue sample) is collected from the subject. In some embodiments, the sample is collected from a portion of the subject including, but not limited to, the subject's finger, hand, arm, shoulder, torso, abdomen, leg, foot, neck, ear, or head.
  • a tissue sample e.g., an internal or an external tissue sample
  • the sample is an environmental sample.
  • environmental samples include, but are not limited to, air samples, water samples, soil samples, biofilm samples, or plant samples. Additional samples include food products, beverages, manufacturing materials, textiles, chemicals, biologies, therapies, or any other samples.
  • one type of sample is accepted and/or processed by the microfluidic cartridge.
  • multiple types of samples are accepted and/or processed by the microfluidic cartridge.
  • the microfluidic cartridge is capable of accepting one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, twelve or more, fifteen or more, twenty or more, thirty or more, fifty or more, or one hundred or more types of samples.
  • the microfluidic cartridge is capable of accepting and/or processing any of these numbers of sample types simultaneously and/or at different times from different or the same matrices.
  • the microfluidic cartridge is capable of preparing, assaying, and/or detecting one or multiple types of samples.
  • volume may include, but are not limited to, approximately 10 mL or less, 5 mL or less, 3 mL or less, 1 mL or less, 500 ⁇ or less, 300 ⁇ or less, 250 ⁇ or less, 200 ⁇ or less, 170 ⁇ ⁇ or less, 150 ⁇ ⁇ or less, 125 ⁇ or less, 100 ⁇ or less, 75 ⁇ or less, 50 ⁇ ⁇ or less, 25 ⁇ or less, 20 ⁇ ⁇ or less, 15 ⁇ ⁇ or less, 10 ⁇ or less, 5 ⁇ or less, 3 ⁇ ⁇ or less, 1 ⁇ or less, 500 nL or less, 250 nL or less, 100 nL or less, 50 nL or less, 20 nL or less, 10 nL or less, 5 nL or less, 1 nL or less,
  • the amount of sample may be approximately a drop of a sample.
  • the amount of sample may be approximately 1 to 5 drops of sample, 1 to 3 drops of sample, 1 to 2 drops of sample, or less than a drop of sample.
  • the amount of sample may be the amount collected from a pricked finger or a finger stick. Any volume, including those described herein, is provided to the device in various embodiments.
  • a sample collection unit and/or sample reaction chamber is integral to the microfluidic cartridge. And, in some embodiments the sample collection unit and/or sample reaction chamber is separate from the microfluidic cartridge. In some embodiments, the sample collection unit and/or sample reaction chamber is removable and/or insertable from the microfluidic cartridge or is removable and/or insertable from an apparatus comprising the microfluidic cartridge. In some embodiments, the sample collection unit and/or sample reaction chamber is provided in the microfluidic cartridge; in some embodiments the sample collection unit and/or sample reaction chamber is not provided in the microfluidic cartridge. In some embodiments, the microfluidic cartridge is removable and/or insertable from an apparatus.
  • a sample collection unit and/or sample reaction chamber is configured to receive a sample.
  • the sample collection unit is capable of containing and/or confining the sample.
  • the sample collection unit is capable of conveying the sample to other components, modules, and chambers of the microfluidic cartridge.
  • a microfluidic cartridge is configured to accept a single sample; in some embodiments a microfluidic cartridge is configured to accept multiple samples. In some embodiments, the multiple samples comprise multiple types of samples. For example, in some embodiments a single microfluidic cartridge handles a single sample at a time. For example, in some embodiments a microfluidic cartridge receives a single sample and performs one or more sample processing steps, such as a lysis steps, isolation steps, reaction steps, and/or a fragmentation steps with the sample. In some embodiments, the microfluidic cartridge completes processing a sample before accepting a new sample.
  • a microfluidic cartridge is capable of handling multiple samples simultaneously.
  • a microfluidic cartridge receives multiple samples simultaneously.
  • the multiple samples comprise multiple types of samples.
  • the microfluidic cartridge receives samples in sequence. Samples are provided in some embodiments to the microfluidic cartridge one after another or, in some embodiments, samples are provided to the microfluidic cartridge after any amount of time has passed.
  • a microfluidic cartridge in some embodiments begins sample processing on a first sample, receives a second sample during said sample processing, and processes the second sample in parallel with the first sample. In some embodiments, the first and second samples are not the same type of sample.
  • the microfluidic cartridge processes any number of samples in parallel, including but not limited to more than and/or equal to
  • the microfluidic cartridge processes one, two, or more samples in parallel. The number of samples that are processed in parallel may be determined by the number of available modules, reaction chambers, and/or components in the microfluidic cartridge.
  • embodiments provide that the samples begin and/or end processing at any time. For example, the samples need not begin and/or end processing at the same time.
  • a first sample has completed processing while a second sample is still being processed.
  • the second sample has begun processing after the first sample has begun processing.
  • additional samples are added to the device in some embodiments.
  • the microfluidic cartridge runs continuously with samples being added to the device as various samples have completed processing.
  • multiple samples are provided simultaneously. In some embodiments, multiple samples are not the same type of sample. In some embodiments, multiple sample collection units are provided to a microfluidic cartridge. In some embodiments, the multiple sample collection units receive samples simultaneously and in some embodiments the multiple sample collection units receive samples at different times. In some embodiments, multiples of any of the sample collection mechanisms described herein are used in combination.
  • multiple samples are provided in sequence.
  • multiple sample collection units are used and in some embodiments single sample collection units are used.
  • Embodiments provide any combination of sample collection mechanisms described herein.
  • a microfluidic cartridge accepts one sample at a time, two samples at a time, or more.
  • samples are provided to the microfluidic cartridge after any amount of time has elapsed.
  • the microfluidic cartridge comprises a port, orifice, tube, or other opening or fluid communication for introducing a sample into the microfluidic cartridge, e.g., into a sample collection chamber.
  • sample input is performed by introducing a sample for processing into the device, e.g., directly into a sample collection chamber within the device.
  • a sample is directly injected into the sample collection chamber through a sealable opening, e.g., an injection valve or a septum.
  • sealable valves are preferred to reduce or eliminate potential threat of leakage during or after sample injection.
  • the device may be provided with a hypodermic needle integrated within the device and connected to the sample collection chamber, e.g., for direct acquisition of the sample into the sample chamber. This can substantially reduce the opportunity for contamination of the sample.
  • the sample input accepts a swab, tube, pipette, pipette tip, sub-cartridge, etc. comprising the sample.
  • a solid sample is introduced directly into the cartridge.
  • introducing a sample into the microfluidic device is associated with recording information related to the identification of the sample.
  • the port, orifice, tube, or other opening or fluid communication for introducing a sample into the microfluidic cartridge prevents contamination of the sample by external elements and/or contamination of the environment by the sample.
  • the sample collection portion of the device may also include reagents and/or treatments for neutralization of infectious agents, stabilization of the specimen or sample, pH adjustments, and the like. Stabilization and pH
  • adjustment treatments may include, e.g., introduction of heparin to prevent clotting of blood samples, addition of buffering agents, addition of protease inhibitors or nuclease inhibitors, preservatives, and the like.
  • reagents are stored within the sample collection chamber of the device or are within a separately accessible chamber, wherein the reagents are added to or mixed with the sample upon introduction of the sample into the device. In some embodiments, these reagents are incorporated within the device in either liquid or lyophilized form, depending upon the nature and stability of the particular reagent used.
  • the microfluidic cartridge provides a technology for providing (e.g., outputting) a sequencing library from an input sample comprising nucleic acids (e.g., a biological sample).
  • sample preparation operations are performed upon the sample (e.g., by one or more components or modules of the microfluidic cartridge) to prepare the sequencing library.
  • sample preparation operations include such manipulations as cell lysis, extraction of nucleic acids from whole cell samples (e.g., cell lysates, viruses, and the like), transcription of nucleic acids, amplification of nucleic acids, fragmentation of nucleic acids, labeling, adaptor ligation, quantification, size selection, and/or extension reactions.
  • One or more of these various operations are incorporated into the microfluidic cartridge described herein, e.g., as one or more components or modules of the microfluidic cartridge configured or adapted to perform one or more of these operations.
  • Cell lysis chamber e.g., cell lysis chamber
  • a nucleic acid sequencing library is prepared from an input sample comprising whole cells (e.g., eukaryotic, bacteria, archaea), viruses, environmental samples, and/or tissue. Accordingly, in some embodiments, nucleic acids are obtained from the cells or viruses prior to continuing with other various sample preparation operations. For example, in some embodiments, following sample collection, the collected cells, viral coat, etc., are treated to prepare a crude extract (e.g., a cell lysate), followed by additional treatments to prepare the sample for subsequent operations, e.g., denaturation of contaminating (nucleic acid binding) proteins, purification, filtration, desalting, and the like.
  • a crude extract e.g., a cell lysate
  • Liberation of nucleic acids from the sample cells or viruses may generally be performed by physical or chemical methods.
  • chemical methods generally employ lysing agents (e.g., detergents or solvents) to disrupt the cells and liberate the cellular contents, followed by treatment of the extract with chaotropic salts such as guanidinium isothiocyanate or urea to denature any contaminating and potentially interfering proteins.
  • lysing agents e.g., detergents or solvents
  • chaotropic salts such as guanidinium isothiocyanate or urea
  • the appropriate reagents may be incorporated within the lysis chamber, a separate accessible chamber, or externally introduced.
  • cell lysis and denaturing of contaminating proteins is carried out by applying an alternating electrical current to the sample.
  • the sample of cells is flowed through a channel or multiple channels while an alternating electric current is applied across the fluid flow.
  • a variety of other methods is utilized within the microfluidic cartridge to effect cell lysis/extraction, including, e.g., subjecting cells to ultrasonic agitation or forcing cells through
  • microgeometry apertures thereby subjecting the cells to high shear stress resulting in rupture.
  • the microfluidic cartridge comprises a cell lysis chamber.
  • the cell lysis chamber is configured to accept a sample comprising cells and into a chamber appropriate for lysing cells to release cellular contents, e.g., to release nucleic acids for further isolation and processing.
  • the cell lysis chamber receives a sample introduced into the microfluidic cartridge through the sample port.
  • the cell lysis chamber is configured to accept a sample having a volume of 1 microliter to 10000 microliters, e.g., 10 mL or less, 5 mL or less, 3 mL or less, 1 mL or less, 500 ⁇ ⁇ or less, 300 ⁇ ⁇ or less, 250 ⁇ ⁇ or less, 200 ⁇ ⁇ or less, 170 ⁇ ⁇ or less, 150 ⁇ ⁇ or less, 125 ⁇ ⁇ or less, 100 ⁇ ⁇ or less, 75 ⁇ ⁇ or less, 50 ⁇ ⁇ or less, 25 ⁇ ⁇ or less, 20 ⁇ ⁇ or less, 15 ⁇ ⁇ or less, 10 ⁇ ⁇ or less, 5 ⁇ ⁇ or less, 3 ⁇ ⁇ or less, 2 ⁇ ⁇ or less.
  • a sample having a volume of 1 microliter to 10000 microliters, e.g., 10 mL or less, 5 mL or less, 3 mL or less, 1 mL or less, 500
  • the microfluidic cartridge is docked with or inserted into an apparatus or instrument comprising an ultrasonic device that produces sonic energy to lyse cells.
  • ultrasonic waves are used to treat a sample containing biological material (e.g., cells).
  • the ultrasonic waves are specifically adapted to interact preferentially with supporting matrices in a biological material, such as plant cell walls or
  • extracellular matrices such as bone or collagen, thereby lessening or removing a barrier function of such matrices and, in some embodiments, facilitating lysis of the cells.
  • Other modes of sonic energy can have different effects than disrupting a matrix and can be used either with pre-treatment, with disrupting sonic energy, or by themselves.
  • the conditions to disrupt a matrix can be different from those to permeabilize a cell membrane.
  • the ultrasonic device (and thus the sample in the cell lysis chamber) is cooled by a thermoelectric device in the apparatus or instrument.
  • a thermoelectric device in the apparatus or instrument.
  • Suitable ultrasonic devices and instruments comprising ultrasonic devices are described in U.S. Pat. Appl. Ser. No. 62/068,406 (entitled "Ultrasonics for microfluidic sample
  • the microfluidic cartridge comprises a cell lysis chamber that is designed and configured to be "dry coupled" to an ultrasonic device.
  • a typical ultrasonic "couplant" composition such as a gel, paste, or a liquid is not used to couple the ultrasonic device to the cell lysis chamber.
  • the ultrasonic device is held or pressed tightly to the cell lysis chamber by a spring (e.g., a spring-loaded mechanism) or by pressure (e.g., by applying a vacuum).
  • a piezoelectric component comprising a hole provides ultrasonic energy into the sample.
  • the cell lysis chamber is designed to provide a chamber for cell lysis that also minimizes, eliminates, or otherwise withstands the effects of increased pressure associated with the cell lysis process.
  • the cell lysis chamber has a volume that is larger than the sample volume, e.g., to provide a head space for accepting the extra gas pressure.
  • the cell lysis chamber has a volume of
  • the cell lysis chamber has a volume of approximately 6200 mm 3 (e.g., 5000 to 7500 mm 3 , e.g., 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, or 7500 mm 3 ).
  • 1 mm 3 is equivalent to 0.001 milliliter (l microliter) and thus volumes in mm 3 and milliliters, microliters, picoliters, nanoliters, etc. are readily interconvertible.
  • the cell lysis chamber comprises a vent to release pressure during cell lysis.
  • the cell lysis chamber comprises a vent comprising a filter (e.g., a HEPA filter) to prevent sample components from being released from the cell lysis chamber.
  • a filter e.g., a HEPA filter
  • the top of the cell lysis chamber comprises a vented channel connected to an adjacent chamber for pressure release and to collect aerosols and thus contain the sample from release outside the cell lysis chamber.
  • the vented channel comprises a filter (e.g., an aerosol barrier filter, e.g., a HEPA filter).
  • the cell lysis chamber comprises a particle or bead to increase cavitation and/or mixing of the sample during cell lysis (e.g., a grinding or beating bead).
  • the particle or bead is a glass, ceramic, silica, steel, or ceramic particle or bead.
  • the particle or bead is a yttrium stabilized zircon bead.
  • the beads have a diameter of
  • nucleic acids are extracted from the cells, viruses, and/or cell lysate prior to continuing with other various sample preparation operations.
  • embodiments provide for the separation of nucleic acids from other elements of a crude extract, e.g., denatured proteins, cell membrane particles, and the like, after cell lysis. Removal of particulate matter is generally accomplished by filtration, flocculation, or the like.
  • Various embodiments of the microfluidic cartridge comprise a variety of filter types that are incorporated into the device. Further, in some embodiments in which chemical denaturing methods are used, samples are desalted prior to proceeding to subsequent steps.
  • desalting of the sample and isolation of the nucleic acid are carried out in a single step, e.g., by binding the nucleic acids to a solid phase and washing away the contaminating salts or performing gel filtration chromatography on the sample.
  • Suitable solid supports for nucleic acid binding include, e.g., diatomaceous earth, silica, or the like.
  • Suitable gel exclusion media are also well known in the art and are commercially available from, e.g., Pharmacia and Sigma Chemical.
  • the isolation and/or gel filtration/desalting are carried out in an additional chamber, or alternatively, the particular chromatographic media may be incorporated into a channel or fluid passage leading to a subsequent reaction chamber.
  • the interior surfaces of one or more fluid passages or chambers may themselves be derivatized to provide functional groups appropriate for the desired purification, e.g., charged groups, affinity binding groups, and the like.
  • the nucleic acid is subjected to one or more preparative reactions (e.g., following sample collection, lysis, and/or nucleic acid extraction).
  • preparative reactions e.g., following sample collection, lysis, and/or nucleic acid extraction.
  • exemplary embodiments are associated with preparative reactions that include in vitro transcription, labeling, fragmentation, amplification, and other reactions.
  • Nucleic acid amplification increases the number of copies of the target nucleic acid sequence of interest.
  • a variety of amplification methods are suitable for use in the methods and device described herein, including, e.g., the polymerase chain reaction (PCR), the ligase chain reaction (LCR), self sustained sequence replication (3SR), and nucleic acid based sequence amplification (NASBA).
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • 3SR self sustained sequence replication
  • NASBA nucleic acid based sequence amplification
  • 3SR and NASBA involve isothermal reactions based on isothermal transcription, which produce both single stranded RNA (ssRNA) and double stranded DNA (dsDNA) amplification products in a ratio of approximately 30 or 100 to 1, respectively.
  • ssRNA single stranded RNA
  • dsDNA double stranded DNA
  • amplification comprises PCR techniques. See PCR
  • PCR amplification generally involves the use of one strand of the target nucleic acid sequence as a template for producing a large number of complements to that sequence.
  • two primer sequences complementary to different ends of a segment of the complementary strands of the target sequence hybridize with their respective strands of the target sequence, and in the presence of polymerase enzymes and nucleoside triphosphates, the primers are extended along the target sequence.
  • PCR amplification typically involves repeated cycles of denaturation, hybridization, and extension reactions to produce sufficient amounts of the target nucleic acid.
  • the first step of each cycle of the PCR involves the separation of the nucleic acid duplex formed by the primer extension. Once the strands are separated, the next step in PCR involves hybridizing the separated strands with primers that flank the target sequence. The primers are then extended to form complementary copies of the target strands.
  • the primers are designed so that the position at which each primer hybridizes along a duplex sequence is such that an extension product synthesized from one primer, when separated from the template (complement), serves as a template for the extension of the other primer.
  • the cycle of denaturation, hybridization, and extension is repeated as many times as necessary to obtain the desired amount of amplified nucleic acid.
  • denaturation, hybridization, and extension each occur at a particular reaction temperature.
  • the PCR is heated and cooled (e.g., repeatedly heated and cooled, e.g., "thermocycled") among various temperatures (e.g., in some embodiments approximately 80 to 105°C for denaturation, approximately 40 to 65°C for hybridization, and approximately 60 to 75°C for extension).
  • heating and cooling for PCR thermocycling is provided by a thermoelectric component as described herein or as described in U.S. Pat. Appl. Ser. No.
  • strand separation is normally achieved by heating the reaction to a sufficiently high temperature for a sufficient time to cause the denaturation of the duplex but not to cause an irreversible denaturation of the polymerase enzyme (see U.S.
  • Strand separation can be accomplished by any suitable denaturing method including physical, chemical, or enzymatic processes.
  • Strand separation may be induced by a helicase, for example, or by an enzyme capable of exhibiting helicase activity.
  • the enzyme RecA has helicase activity in the presence of ATP.
  • the reaction conditions suitable for strand separation by helicases are known in the art (see Kuhn Hoffman-Berling, 1978, CSH-Quantitative Biology, 43:63-67; and Radding, 1982, Ann. Rev. Genetics 16:405-436, each of which is incorporated herein by reference).
  • Other embodiments may achieve strand separation by application of electric fields across the sample.
  • PCT Application Nos. WO 92/04470 and WO 95/25177 describe electrochemical methods of denaturing double stranded DNA by application of an electric field to a sample containing the DNA.
  • Structures for carrying out this electrochemical denaturation include a working electrode, counter electrode, and reference electrode arranged in a potentiostat arrangement across a reaction chamber. Such devices may be readily miniaturized for incorporation into the devices of the present invention utilizing the microfabrication techniques described herein.
  • Template-dependent extension of primers in PCR is catalyzed by a polymerizing agent in the presence of adequate amounts of four deoxyribonucleotide triphosphates (typically dATP, dGTP, dCTP, dUTP and dTTP) in a reaction medium that comprises the appropriate salts, metal cations, and pH buffering system.
  • deoxyribonucleotide triphosphates typically dATP, dGTP, dCTP, dUTP and dTTP
  • Suitable polymerizing agents are enzymes known to catalyze template-dependent DNA synthesis.
  • the amplification reaction chamber of the device comprises a sealable opening for the addition of various amplification reagents.
  • the amplification chamber has an effective amount of the various amplification reagents described above predisposed within the amplification chamber or within an associated reagent chamber whereby the reagents are readily transported to the amplification chamber upon initiation of the amplification operation.
  • effective amount refers to a quantity and/or a concentration of reagents required to carry out amplification of a targeted nucleic acid sequence. These amounts are readily determined from known PCR protocols. See, e.g., Sambrook, et al. Molecular Cloning: A Laboratory Manual, (2nd ed.) Vols.
  • reagents are in lyophilized forms to provide maximum shelf life of the overall device. Introduction of the liquid sample to the chamber then reconstitutes the reagents in active form and the particular reactions may be carried out.
  • the polymerase enzyme is present within the polymerase enzyme
  • amplification chamber coupled to a suitable solid support, or immobilized to the walls and surfaces of the amplification chamber.
  • suitable solid supports include those that are well known in the art, e.g., agarose, cellulose, silica, divinylbenzene, polystyrene, etc. Coupling of enzymes to solid supports has been reported to impart stability to the enzyme in question, which allows for storage of days, weeks, or even months without a substantial loss in enzyme activity and without the necessity of lyophilizing the enzyme.
  • the 94 kDa, single subunit DNA polymerase from Thermus aquaticus is particularly suited for the PCR based amplification methods used in the present invention, and is generally commercially available from suppliers such as, e.g., Promega, Inc., Madison, Wisconsin.
  • monoclonal antibodies bind the polymerase enzyme without affecting its polymerase activity. Consequently, covalent attachment of the active polymerase enzyme to a solid support, or the walls of the amplification chamber, can be carried out by using the antibody as a linker between the enzyme and the support.
  • a nucleic acid is an RNA that is reverse transcribed (e.g., by a reverse transcriptase enzyme or by a polymerase comprising reverse transcriptase activity) prior to amplification.
  • amplification is whole-genome amplification of a complex mixture of genomic nucleic acid fragments representing one or more whole, one or more substantially whole, and/or one or more partial genomes that are used as templates for amplification.
  • the amplification is "solid-phase amplification" wherein the nucleic acid amplification reaction is performed on or in association with a solid support (e.g., a bead) such that all or a portion of the amplified products are immobilized on the solid support as they are formed.
  • solid-phase amplification encompasses solid-phase polymerase chain reaction (solid-phase PCR) and solid phase isothermal amplification, which are reactions analogous to standard solution phase amplification, except that one or both of the forward and reverse amplification primers is/are immobilized on the solid support.
  • Solid phase PCR covers systems such as emulsions, wherein one primer is anchored to a bead and the other primer is in free solution, and colony formation in solid phase gel matrices wherein one primer is anchored to the surface and one primer is in free solution.
  • the technology comprises use of whole genome
  • WGA oligo primed PCR
  • CGH comparative genomic hybridization
  • linker-adapter PCR has also been used for whole genome amplification.
  • MDA Multiple displacement amplification
  • cp29 DNA polymerase is a highly processive, strand- displacing polymerase that has a very low error rate (e.g., 1 in 10 6 to 10 7 nucleotides) that is several orders of magnitude better than the error rate for enzymes commonly used in PCR, e.g., Taq polymerase (e.g., error rate of 3 in 10 4 nucleotides) and Pfu polymerase (error rate of 1.6 in 10 6 nucleotides).
  • Taq polymerase e.g., error rate of 3 in 10 4 nucleotides
  • Pfu polymerase error rate of 1.6 in 10 6 nucleotides
  • the MDA reaction is performed under mesothermal conditions (e.g., 15°C to 30°C).
  • mesothermal conditions e.g. 15°C to 30°C.
  • the technology provides a microfluidic cartridge and methods for WGA of trace quantities of nucleic acids using ⁇ 29 DNA polymerase and, in some embodiments, highly specialized random primers. Further, as WGA amplifies all the DNA present in a reaction, some embodiments comprise use of ultra clean (DNA- free) WGA reagents. DNA contaminants in WGA reagents are not significant for the WGA reactions using nanogram quantities of DNA such as required by existing commercial WGA kits, but reagent contaminants become significant for the
  • WGA produces a nucleic acid sample for sequencing from as little as 10 cells from which can be detected single base mutations while maintaining balance and representation.
  • nucleic acids are labeled, e.g., to facilitate subsequent steps and/or to provide for detection of the nucleic acids.
  • labeling is performed during amplification.
  • amplification incorporates a label into the amplified nucleic acid either through the use of labeled primers or the incorporation of labeled dNTPs into the amplified nucleic acid.
  • the nucleic acids are labeled following amplification.
  • Post amplification labeling typically involves the covalent attachment of a particular detectable group to the amplified nucleic acids.
  • Suitable labels or detectable groups include a variety of fluorescent or radioactive labeling groups well known in the art. These labels are coupled in various embodiments to the sequences using methods that are well known in the art. See, e.g., Sambrook and Russell, Molecular Cloning, A Laboratory Manual, third edition.
  • the label comprises, but is not limited to, one or more fluorescent labels (including, but not limited to, FITC, PE, Texas RED, Cyber Green, JOE, FAM, HEX, TAMRA, ROX, Alexa 488, Alexa 532, Alexa 546, Alexa 405, or other fluorescent moieties and dyes), radioactive labels (including, but not limited to, 32 P, 3 H, or 14 C), fluorescent proteins (including, but not limited to, GFP, RFP, or YFP), quantum dots, gold particles, sliver particles, biotin, beads (including but not limited magnetic beads or polystyrene beads).
  • fluorescent labels including, but not limited to, FITC, PE, Texas RED, Cyber Green, JOE, FAM, HEX, TAMRA, ROX, Alexa 488, Alexa 532, Alexa 546, Alexa 405, or other fluorescent moieties and dyes
  • radioactive labels including, but not limited to, 32 P, 3 H, or 14 C
  • fluorescent proteins including, but
  • nucleic acids are subjected to other treatments.
  • embodiments are related to fragmenting nucleic acids prior to subsequent steps.
  • Fragmentation of nucleic acids may generally be carried out by physical, chemical, or enzymatic methods that are known in the art. These treatments may be performed within the amplification chamber, or alternatively, may be carried out in a separate chamber (e.g., a nucleic acid fragmentation chamber or in the cell lysis chamber).
  • physical fragmentation methods include but are not limited to moving the sample containing the nucleic acid over pits or spikes in the surface of a reaction chamber or fluid channel. The motion of the fluid sample, in combination with the surface irregularities, produces a high shear rate that results in fragmentation of the nucleic acids.
  • nucleic acid fragmentation is performed using an ultrasonic device that produces ultrasonic waves in the sample (see, e.g., U.S. Pat. Appl. Ser. No. 62/068,406 (entitled “Ultrasonics for microfluidic sample preparation"), incorporated herein by reference in its entirety).
  • Cavitation and/or streaming within the sample results in substantial shear on the nucleic acids. Similar shear rates may be achieved by forcing the nucleic acid containing fluid sample through restricted size flow passages, e.g., apertures having a cross-sectional dimension in the micron or submicron scale, thereby producing a high shear rate and fragmenting the nucleic acid.
  • the microfluidic cartridge comprises a nucleic acid fragmentation chamber.
  • the nucleic acid fragmentation chamber is configured to accept a sample comprising nucleic acids into a chamber appropriate for fragmenting the nucleic acids, e.g., as a step in the preparation of a nucleic acid library for next- generation sequencing.
  • the nucleic acid fragmentation chamber receives a sample from a component for nucleic acid extraction, purification, and/or isolation.
  • the nucleic acid fragmentation chamber receives a sample from a component for amplification of a nucleic acid (e.g., whole genome amplification, an amplicon panel library, etc.).
  • the nucleic acid fragmentation chamber is configured to accept a sample having a volume of 1 microliter to 1000 microliters, e.g., 1 mL or less, 500 ⁇ ⁇ or less, 300 ⁇ ⁇ or less, 250 ⁇ ⁇ or less, 200 ⁇ ⁇ or less, 170 ⁇ ⁇ or less, 150 ⁇ ⁇ or less, 125 ⁇ ⁇ or less, 100 ⁇ ⁇ or less, 75 ⁇ ⁇ or less, 50 ⁇ ⁇ or less, 25 ⁇ ⁇ or less, 20 ⁇ ⁇ or less, 15 ⁇ ⁇ or less, 10 ⁇ ⁇ or less, 5 ⁇ ⁇ or less, 3 ⁇ ⁇ or less, 2 ⁇ ⁇ or less.
  • 1 microliter to 1000 microliters e.g., 1 mL or less, 500 ⁇ ⁇ or less, 300 ⁇ ⁇ or less, 250 ⁇ ⁇ or less, 200 ⁇ ⁇ or less, 170 ⁇ ⁇ or less, 150 ⁇ ⁇ or less, 125
  • the microfluidic cartridge is docked with or inserted into an apparatus or instrument comprising an ultrasonic device that produces sonic energy to fragment nucleic acids.
  • ultrasonic waves are used to treat a sample containing nucleic acids.
  • the ultrasonic device and thus the sample in the nucleic acid
  • thermoelectric device in the apparatus or instrument.
  • Suitable ultrasonic devices and instruments comprising ultrasonic devices are described in U.S. Pat. Appl. Ser. No. 62/068,406 (entitled “Ultrasonics for microfluidic sample preparation"), which is expressly incorporated herein by reference in its entirety.
  • the microfluidic cartridge comprises a nucleic acid fragmentation chamber that is designed and configured to be "dry coupled" to an ultrasonic device.
  • a typical "couplant" composition such as a gel, paste, or a liquid is not used to couple the ultrasonic device to the nucleic acid fragmentation chamber.
  • the ultrasonic device is held or pressed tightly to the nucleic acid fragmentation chamber by a spring (e.g., a spring-loaded mechanism) or by pressure (e.g., by applying a vacuum). See, e.g., U.S. Pat. Appl. Ser. No. 62/068,406 (entitled "Ultrasonics for microfluidic sample
  • the nucleic acid fragmentation chamber is designed to provide a chamber for fragmenting nucleic acids that also withstands the effects of increased pressure associated with the fragmentation process.
  • the nucleic acid fragmentation chamber has a volume that is larger than the sample volume, e.g., to provide a head space for accepting the extra gas pressure.
  • the nucleic acid fragmentation chamber has a volume of approximately 1000 to 2000 mm 3 (e.g., 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 mm 3 , preferably approximately 1500 mm 3 ).
  • 1 mm 3 is equivalent to 0.001 milliliter (l microliter) and thus volumes in mm 3 and milliliters, microliters, picoliters, nanoliters, etc. are readily interconvertible.
  • the nucleic acid fragmentation chamber comprises a vent to release pressure during cell lysis. In some embodiments, the nucleic acid
  • fragmentation chamber comprises a vent comprising a filter (e.g., a HEPA filter) to prevent sample components from being released from the nucleic acid fragmentation chamber.
  • a filter e.g., a HEPA filter
  • the top of the nucleic acid fragmentation chamber comprises a vented channel connected to an adjacent chamber for pressure release and to collect aerosols and thus contain the sample from release outside the nucleic acid fragmentation chamber.
  • the vented channel comprises a filter (e.g., an aerosol barrier filter, e.g., a HEPA filter).
  • the nucleic acid fragmentation chamber comprises a particle or bead to increase cavitation and/or mixing of the sample during fragmentation of nucleic acids.
  • the particle or bead is a grinding bead such as, e.g., hardened steel beads, stainless steel beads, tungsten carbide beads, agate beads, zirconium oxide beads, PTFE beads, yttrium stabilized zircon beads, glass beads, or silica beads.
  • the particle or bead is a ceramic particle or bead.
  • the particle or bead is a yttrium stabilized zirconia (ZY) bead.
  • the beads have a diameter of approximately 2 mm (e.g., 1 to 5 mm).
  • the nucleic acid fragmentation chamber generates nucleic acid fragments having a size that is approximately 100 bp to 5000 bp (e.g., 100 bp, 150 bp, 200 bp, 250 bp, 300 bp, 350 bp, 400 bp, 450 bp, 500 bp, 550 bp, 600 bp, 650 bp, 700 bp, 750 bp, 800 bp, 850 bp, 900 bp, 950 bp, 1000 bp, 1050 bp, 1100 bp, 1150 bp, 1200 bp, 1250 bp, 1300 bp, 1350 bp, 1400 bp, 1450 bp, 1500 bp, 1550 bp, 1600 bp, 1650 bp, 1700 bp, 1750 bp, 1800 bp, 1850 bp, 1900 bp, 1950 bp,
  • fragmentation chamber generates nucleic acid fragments of 1000 to 3000 base pairs.
  • the nucleic acid fragmentation chamber generates fragments that are in the ranges of 1 kbp to 5 kbp, 10 kbp, 15 kbp, 20 kbp, 25 kbp, 30 kbp, 35 kbp, 40 kbp, 45 kbp, or 50 kbp (e.g., approximately 1000 bp; 2000 bp; 3000 bp; 4000 bp; 5000 bp; 6000 bp; 7000 bp; 8000 bp; 9000 bp; 10,000 bp; 11,000 bp; 12,000 bp; 13,000 bp; 14,000 bp; 15,000 bp; 16,000 bp; 17,000 bp; 18,000 bp; 19,000 bp; or 20,000 bp; 25,000 bp; 30,000 bp; 35,000 bp; 40,000 bp; 45,000 bp; 50,000 bp).
  • the microfluidic cartridge comprises a component for purifying and size selecting nucleic acids such as, e.g., a collection of nucleic acid fragments or a nucleic acid sequencing library (e.g., for NGS, e.g., an NGS library).
  • the component for purifying and size selecting nucleic acids comprises a chamber to hold a nucleic acid sample.
  • a binding buffer and super paramagnetic carboxyl beads are added to the nucleic acid to bind nucleic acids having a specified range of fragment sizes.
  • the binding buffer is formulated to promote the binding of nucleic acids having a specified range of fragment sizes to the beads.
  • Embodiments provide a specific formulation of binding buffer that selects for fragments of nucleic acid having a length of at least approximately 50 to 5000 base pairs, e.g., at least 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, or 5000 base pairs (e.g., to remove small fragments having approximately less than 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, or 5000 base pairs).
  • Particular embodiments provide a specific formulation of binding buffer that selects for fragments of nucleic acid having a length of at least approximately 200 to 1000 base pairs (e.g
  • the binding buffer comprises polyethylene glycol (PEG), sodium chloride, and water (molecular biology grade water, e.g., nuclease free water).
  • the binding buffer comprises between 1% and 10% PEG, e.g., between 4% and 6% w/v PEG (e.g., PEG 8000), e.g., 4.8% PEG. See, e.g., U.S. Pat. App. Ser. No. 62/068,379 (entitled “Systems, compositions and methods for size selective nucleic acid purification"), incorporated herein by reference in its entirety.
  • Some embodiments comprise use of a wash buffer to remove the smaller fragments.
  • the wash buffer comprises approximately 10% Tween- 20, approximately 15% ethanol, and approximately 20 mM MgC . In some embodiments, the wash buffer comprises approximately 10% Tween- 20, approximately 15% ethanol, and approximately 20 mM MgC .
  • the wash buffer does not comprise an alcohol such as ethanol or isopropanol. In some embodiments, the wash buffer comprises approximately 7.5% PEG and approximately 1.4 M NaCl. In some embodiments, the wash buffer comprises 10% PEG and 20 mM MgC . In particular embodiments, the wash buffer is compatible with optical detection based NGS technologies (e.g., in some embodiments the wash buffer does not produce a fluorescence emission that interferes with NGS technologies). See, e.g., U.S. Pat. App. Ser. No. 62/068,379 (entitled “Systems, compositions and methods for size selective nucleic acid purification"), incorporated herein by reference in its entirety.
  • the microfluidic cartridge comprises a component or module for purifying and size selecting nucleic acids.
  • the component or module comprises a chamber to hold a sample and a reservoir comprising paramagnetic beads with a high magnetic moment and a size selecting binding buffer.
  • the component or module comprises a chamber to hold a sample, a reservoir comprising paramagnetic beads with a high magnetic moment, and a reservoir comprising a size selecting binding buffer.
  • the component or module for purifying and size selecting nucleic acids comprises a reservoir comprising a wash buffer that is compatible with microfluidics (e.g., that does not comprise an alcohol, e.g., ethanol or isopropanol, for instance).
  • the reservoirs comprising the beads, binding buffer, and wash buffer are in fluid
  • the chamber for containing the sample is in fluid communication with one or more other components or modules that provide the sample to the component or module for purifying and size selecting nucleic acids and/or that receive the size selected sample from the component or module for purifying and size selecting nucleic acids. See, e.g., U.S. Pat. App. Ser. No.
  • the microfluidic cartridge comprises a component to provide size selective nucleic acid purification using paramagnetic beads with an optimal magnetic moment, a size selecting binding buffer, and a wash buffer compatible with microfluidics.
  • the technology provides a component comprising a magnetic device capable of generating a three-dimensional magnetic field.
  • the microfluidic cartridge is configured to be secured with the magnetic device such that upon securing with the magnetic device the microfluidic cartridge is exposed to a three-dimensional magnetic field.
  • the component to provide size selective nucleic acid purification comprises a binding buffer comprising PEG, wherein the percentage of PEG within the binding buffer is between 10% and 1%, e.g., between 6% and 4%, e.g., the percentage of PEG within the binding buffer is between 5.14% and 4.18%.
  • the binding buffer further comprises nuclease free water and NaCl.
  • the component to provide size selective nucleic acid purification further comprises solid supports comprising super paramagnetic beads coated with carboxylic acid functional groups.
  • the component to provide size selective nucleic acid purification further comprises a washing buffer comprising PEG.
  • the percentage of PEG within the washing buffer is between 5% and 20%. In some embodiments, the percentage of PEG within the washing buffer is between 7.5% and 16%. In some embodiments, the washing buffer further comprises MgC and/or NaCl. See, e.g., U.S. Pat. App. Ser. No. 62/068,379 (entitled “Systems, compositions and methods for size selective nucleic acid purification"), incorporated herein by reference in its entirety. End repair
  • the sample After fragmentation, the sample comprises nucleic acids having a number of different types of ends, e.g., blunt ends, 3' overhangs, 5' overhangs, and incorrect 3' and/or 5' phosphorylation states. Accordingly, for efficient adaptor ligation in
  • nucleic acid ends are converted to a similar end type with 3' overhangs removed, 5' overhangs filled in, and the appropriate
  • an untemplated A is added, e.g., for a T-A ligation scheme.
  • end repair of the nucleic acid fragments is provided by, e.g., a mix of polymerases, polynucleotide kinase (PNK), and adenosine triphosphate (ATP).
  • PNK polynucleotide kinase
  • ATP adenosine triphosphate
  • enzymes present in a WGA reaction are re-used in the end repair reaction to perform the polymerization and the exonuclease activity, PNK, and ATP are added to provide the phosphorylation/dephosphorylation activities.
  • the polymerase activity is provided in the form of a dried (e.g., lyophilized) bead or a mixture of enzymes in one bead.
  • the microfluidic cartridge comprises a component or module for end repair of nucleic acids.
  • the end repair process is generic and common to all NGS platforms and includes in various embodiments, e.g: phosphorylation of 5' ends and desphophorylation of 3' ends; polishing of the ends (e.g., removal of 3' overhang, filling in of 5' overhang); and/or addition of an untemplated A to the 3' end of a blunt nucleic acid (e.g., for A-T mediated adaptor ligation).
  • the microfluidic cartridge comprises a component or module (e.g., a reaction chamber) for ligating adaptors to nucleic acids.
  • a component or module e.g., a reaction chamber
  • the term “adaptor” encompasses both adaptors (e.g., “linkers”) that comprise single stranded nucleotide overhangs at the 5' and/or 3' ends or that do not comprise single stranded nucleotide overhangs at the 5' and/or 3' ends, e.g., a blunt ended adaptor.
  • one or more single stranded overhangs comprise 1, 2, or more nucleotides.
  • adaptors comprise additional nucleic acid sequence for cloning or for the analysis of "inserts.”
  • adaptors comprise labels or affinity tags for analysis or purification of "inserts.”
  • Ligating an adaptor to a nucleic acid fragment is performed using a ligation reaction, which is a biochemical reaction in which an enzyme (e.g., a ligase) forms a chemical link between a nucleic acid fragment and an adaptor.
  • a ligation reaction which is a biochemical reaction in which an enzyme (e.g., a ligase) forms a chemical link between a nucleic acid fragment and an adaptor.
  • Ligation methods are known in the art and utilize standard methods (Sambrook and Russell, Molecular Cloning, A Laboratory Manual, third edition). Such methods utilize ligase enzymes such as DNA ligase to effect or catalyze joining of the ends of two polynucleotide strands by forming a covalent linkage.
  • an adaptor comprises a 5'-phosphate moiety to facilitate ligation to the nucleic acid fragment 3'-OH.
  • a nucleic acid fragment comprises a 5'-phosphate moiety, either residually from the shearing process, or added using an enzymatic treatment step.
  • a nucleic acid fragment has been end repaired and, optionally, extended to produce an overhanging base or bases, to give a 3' ⁇ suitable for ligation.
  • joining means covalent linkage of polynucleotide strands which were not previously covalently linked.
  • joining comprises formation of a phosphodiester linkage between the two polynucleotide strands, but other means of covalent linkage (e.g. non- phosphodiester backbone linkages) may be used.
  • Many ligation methods utilize either a blunt or TA-mediated ligation.
  • the ligase is a T4 ligase, a variant of T4 DNA ligase, an R coli ligase, etc.
  • DNA lig used herein refers to a family of enzymes that catalyze the formation of a covalent phosphodiester bond between two distinct DNA strands, e.g., in a "ligation reaction". While in some embodiments T4 DNA ligase (isolated from the T4 phage) and DNA ligase from R. coli find use in the technology described herein, the technology is not limited by the ligase that is used to perform the ligation reaction. Any enzyme with DNA ligase activity is contemplated by the technology.
  • an oligonucleotide adapter is ligated onto a nucleic acid fragment as a step to prepare a sequencing library for sequencing (e.g., NGS).
  • a sequencing library for sequencing e.g., NGS
  • an end polishing step is performed on nucleic acid fragments to create blunt ends on the nucleic acid fragments.
  • an enzymatic reaction e.g., a PCR, terminal transferase reaction, or Klenow exo minus polymerase reaction adds an untemplated A to the ends of nucleic acids.
  • ligation reactions comprise ligating adaptors with blunt ends and some embodiments of ligation reactions comprise ligating adaptors with overhangs, e.g., with T overhangs that are complementary to the A overhang on the nucleic acid (A-T mediated ligation).
  • the nucleic acids are fragmented nucleic acids and in some embodiments the nucleic acids are size selected nucleic acids.
  • the adaptors comprise an index, barcode, or key that serves to identify the sequencing library after sequencing.
  • the adaptors comprise PCR and/or sequencing priming sites; in some embodiments, the adaptors comprise blunt 3' ends and 5' overhangs; in some embodiments, an adaptor of a pair of adaptors comprises a biotin on the 5' end.
  • each ligated product comprises in various embodiments one or more of (e.g., in various combinations), e.g., a PCR priming site; a sequencing primer site; an index, barcode, or key; a nucleic acid to be sequenced (e.g., a nucleic acid fragment produced from the sample); and a second end comprising a PCR priming site; a sequencing primer site; and an index, barcode, or key.
  • nucleic acid fragments are digested with a restriction enzyme that produces nucleic acid fragments having a 3' or 5' end having a known sequence and/or overhang.
  • an adaptor is used that has an overhang that is complementary to the digested fragments.
  • exonuclease treatment of the ligation reaction removes incomplete ligation products and other incomplete (e.g., off-target or intermediate) reaction components by operating on the free 3' and 5' ends that are not present on insert molecules with an adapter ligated on either side.
  • the exonuclease enzyme mix has both 3' and 5' activity and processes both single stranded and double stranded DNA templates.
  • exonuclease activity is provided in a lyophilized bead, which experiments conducted during the development of embodiments of the technology indicated to have equivalent enzymatic activity to an exonuclease in an aqueous buffer (e.g., in a non-lyophilized state).
  • ligation of the sequencing adaptors is provided by the addition of a ligase and ATP to a reaction mixture comprising the nucleic acid fragments.
  • the ligase using ATP as a cofactor, joins the OH on the 3' ends of the nucleic acids to the phosphate on the 5' end of the nucleic acids to form a phosphodiester bond.
  • ligation of adaptors to either side of the nucleic acids creates a topologically circular, structurally linear nucleic acid that has no free 3' or 5' ends.
  • ligase activity is provided in a lyophilized bead, which experiments conducted during the development of embodiments of the technology indicated to have equivalent enzymatic activity to a ligase in an aqueous buffer (e.g., in a non-lyophilized state).
  • the microfluidic cartridges comprise a waste containment component.
  • the waste containment component accepts various solutions and
  • the waste containment component comprises an absorbent material to eliminate or minimize the release of sample, reagents, waste, etc. into the environment and/or to ease disposal of the used cartridge.
  • the absorbent material solidifies the liquid waste in the waste container to contain the liquid waste created during sample processing.
  • the waste containment component comprises a combination of a filter, a filter paper, an absorbent material, and a storage
  • compartment e.g., to provide an absorbent waste container.
  • the microfluidic cartridge is constructed from a polymer, e.g., a poly(methyl methacrylate) (PMMA) polymer.
  • the microfluidic cartridge comprises multiple layers of a polymer, e.g., multiple layers of PMMA.
  • a microfluidic cartridge comprising layers of PMMA is treated at approximately 80°C (e.g., 70°C to 90°C, e.g., 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90°C) for approximately 1 hour (e.g., 30 minutes to 90 minutes, e.g., 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 minutes).
  • 80°C e.g., 70°C to 90°C, e.g., 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90°C
  • 1 hour e.g., 30 minutes to 90 minutes, e.g., 30, 35,
  • the treatment at approximately 80°C "anneals" the layers of PMMA to one another, e.g., to increase the stability of the microfluidic cartridge (decrease prevalence of cracks and/or strains in the microfluidic cartridge) and to increase the resistance of the microfluidic cartridge to degradation by heat and/or by solvent (e.g., alcohol (e.g., ethanol, propanol) and organic solvents).
  • solvent e.g., alcohol (e.g., ethanol, propanol) and organic solvents.
  • microfluidic devices described herein are fabricated from an elastomeric polymer such as polyisoprene, polybutadiene, polychlorophene, polyisobutylene, poly(styrene-butadiene-styrene), nitriles, the polyurethanes and the polysilicones.
  • GE RTV 615 a vinyl-silane crosslinked (type) silicone elastomer (family), and polydimethysiloxane (PDMS) membrane (e.g., sold as HT-6135 and HT-6240 membranes from Bisco Silicons, Elk Grove, 111.) are useful in selected applications.
  • elastomeric materials that are used in the manufacture of components of the microfluidic devices are described in Unger (2000) Science 288:113-116. Some elastomers of the present devices are used as diaphragms and in addition to their stretch and relax properties, these elastomers are selected for their porosity, impermeability, chemical resistance, and their wetting and passivating characteristics. Other elastomers are selected for their thermal conductivity.
  • microfluidic cartridge One of skill in the art is able to select appropriate material for producing a microfluidic cartridge and/or the different components of a microfluidic cartridge.
  • a variety of plastics and adhesives are available that provide the desired functionalities and/or that can be evaluated for desired functionalities during the production of a microfluidic cartridge.
  • the microfluidic cartridge receives a sample (e.g., an aqueous biological sample), extracts nucleic acids from the sample, amplifies the nucleic acids, prepares the amplified nucleic acids for sequencing (e.g., by fragmenting the nucleic acid, optionally polishing the ends of the nucleic acid fragments, and ligating adaptors to the nucleic acid fragments to provide a NGS library), and delivers the NGS library directly to an NGS workflow, instrument, and/or sequencer for sequencing.
  • a sample e.g., an aqueous biological sample
  • amplifies the nucleic acids e.g., by fragmenting the nucleic acid, optionally polishing the ends of the nucleic acid fragments, and ligating adaptors to the nucleic acid fragments to provide a NGS library
  • sequencing e.g., by fragmenting the nucleic acid, optionally polishing the ends of the nucleic acid fragments, and ligating adaptors to the
  • the microfluidic cartridge produces an NGS library for NGS sequencing and delivers the NGS library to the NGS sequencer without the sample being touched or transported by a person, e.g., which reduces error, contamination, and other problems associated with the transport of samples by a person.
  • the microfluidic cartridge comprises an output component in fluid communication with a sequencing instrument.
  • a tube connection provides a direct fluid conduit from an output of the microfluidic cartridge or from an output of an apparatus comprising the microfluidic cartridge to an input of the sequencing instrument (e.g., NGS sequencing instrument).
  • the microfluidic cartridge and/or the apparatus comprising the microfluidic cartridge is in fluidic communication with a sequencer (e.g., a NGS sequencer).
  • the conduit or tube is made of a polymer, e.g., a thermoplastic polymer such as, e.g., polyether ether ketone (PEEK),
  • PTFE polytetrafluoroethylene
  • PFA perfluoroalkoxy
  • FEP fluorinated ethylene propylene
  • nucleic acid library e.g., a nucleic acid library that is produced as an output of a microfluidic cartridge as described herein.
  • the present technology is not limited by the type of sequencing method employed. Exemplary sequencing methods are described below.
  • nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing.
  • Chain terminator sequencing uses sequence-specific termination of a DNA synthesis reaction using modified nucleotide substrates. Extension is initiated at a specific site on the template DNA by using a short radioactive, or other labeled, oligonucleotide primer complementary to the template at that region.
  • oligonucleotide primer is extended using a DNA polymerase, standard four
  • deoxynucleotide bases and a low concentration of one chain terminating nucleotide, most commonly a di- deoxynucleotide.
  • This reaction is repeated in four separate tubes with each of the bases taking turns as the di- deoxynucleotide.
  • Limited incorporation of the chain terminating nucleotide by the DNA polymerase results in a series of related DNA fragments that are terminated only at positions where that particular di- deoxynucleotide is used.
  • the fragments are size-separated by electrophoresis in a slab polyacrylamide gel or a capillary tube filled with a viscous polymer. The sequence is determined by reading which lane produces a visualized mark from the labeled primer as you scan from the top of the gel to the bottom.
  • Dye terminator sequencing alternatively labels the terminators. Complete sequencing can be performed in a single reaction by labeling each of the di- deoxynucleotide chain-terminators with a separate fluorescent dye, which fluoresces at a different wavelength.
  • next-generation sequencing techniques have emerged as alternatives to Sanger and dye-terminator sequencing methods (Voelkerding et al., Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev. Microbiol., T- 287- 296; each herein incorporated by reference in their entirety).
  • Most current methods describe the use of next-generation sequencing technology for de novo sequencing of whole genomes to determine the primary nucleic acid sequence of an organism.
  • targeted re-sequencing deep sequencing allows for sensitive mutation detection within a population of wild-type sequence.
  • NGS technology produces large amounts of sequencing data points.
  • a typical run can easily generate tens to hundreds of megabases per run, with a potential daily output reaching into the gigabase range. This translates to several orders of magnitude greater than a standard 96-well plate, which can generate several hundred data points in a typical multiplex run.
  • Target amplicons that differ by as little as one nucleotide can easily be distinguished, even when multiple targets from related species are present. This greatly enhances the ability to do accurate genotyping.
  • NGS alignment software programs used to produce consensus sequences can easily identify novel point mutations, which could result in new strains with associated drug resistance.
  • the use of primer bar coding also allows multiplexing of different patient samples within a single sequencing run.
  • NGS methods share the common feature of massively parallel, high-throughput strategies, with the goal of lower costs in comparison to older sequencing methods. NGS methods can be broadly divided into those that require template amplification and those that do not. Amplification-requiring methods include pyrosequencing developed by Solexa and commercialized by Illumina. Non-amplification approaches, also known as single-molecule sequencing, include the Ion Torrent platform commercialized by Life Technologies and the molecule real time sequencing (also known as SMRT) technologies developed by Pacific Biosciences.
  • template DNA is fragmented, end-repaired, ligated to adaptors, and clonally amplified in- situ by capturing single template molecules with beads bearing oligonucleotides complementary to the adaptors.
  • Each bead bearing a single template type is compartmentalized into a water-in-oil microvesicle, and the template is clonally amplified using a technique referred to as emulsion PCR.
  • the emulsion is disrupted after amplification and beads are deposited into individual wells of a picotitre plate functioning as a flow cell during the sequencing reactions. Ordered, iterative introduction of each of the four dNTP reagents occurs in the flow cell in the presence of sequencing enzymes and luminescent reporter such as luciferase. In the event that an appropriate dNTP is added to the 3' end of the sequencing primer, the resulting production of ATP causes a burst of
  • sequencing data are produced in the form of shorter-length reads.
  • single- stranded fragmented DNA is end-repaired to generate 5'-phosphorylated blunt ends, followed by Klenow-mediated addition of a single A base to the 3' end of the fragments.
  • A-addition facilitates addition of T-overhang adaptor oligonucleotides, which are subsequently used to capture the template-adaptor molecules on the surface of a flow cell that is studded with oligonucleotide anchors.
  • the anchor is used as a PCR primer, but because of the length of the template and its proximity to other nearby anchor oligonucleotides, extension by PCR results in the "arching over" of the molecule to hybridize with an adjacent anchor oligonucleotide to form a bridge structure on the surface of the flow cell.
  • These loops of DNA are denatured and cleaved. Forward strands are then sequenced with reversible dye terminators.
  • sequence of incorporated nucleotides is determined by detection of post-incorporation fluorescence, with each fluor and block removed prior to the next cycle of dNTP addition. Sequence read length ranges from 36 nucleotides to over 50 nucleotides, with overall output exceeding 1 billion nucleotide pairs per analytical run.
  • SMRT single molecule real time
  • ZMWs zero-mode waveguides
  • DNA sequencing is performed on SMRT chips, each containing thousands of zero-mode waveguides (ZMWs).
  • ZMW is a hole, tens of nanometers in diameter, fabricated in a 100 nm metal film deposited on a silicon dioxide substrate.
  • Each ZMW becomes a nanophotonic visualization chamber providing a detection volume of just 20 zeptoliters (10 ⁇ 21 liters). At this volume, the activity of a single molecule can be detected amongst a background of thousands of labeled nucleotides.
  • the ZMW provides a window for watching DNA polymerase as it performs sequencing by synthesis.
  • a single DNA polymerase molecule is attached to the bottom surface such that it permanently resides within the detection volume.
  • the engaged fluorophore emits fluorescent light whose color corresponds to the base identity.
  • the polymerase cleaves the bond holding the fluorophore in place and the dye diffuses out of the detection volume. Following incorporation, the signal immediately returns to baseline and the process repeats. Unhampered and uninterrupted, the DNA polymerase continues incorporating bases at a speed of tens per second. In this way, a completely natural long chain of DNA is produced in minutes. Simultaneous and continuous detection occurs across all of the thousands of ZMWs on the SMRT chip in real time.
  • PacBio have demonstrated this approach has the capability to produce reads thousands of nucleotides in length.
  • nanopore sequencing is employed (see, e.g., Astier et al., Am Chem. Soc. 2006 Feb. 8; 128(5): 1705- 10, herein incorporated by reference).
  • the theory behind nanopore sequencing has to do with what occurs when the nanopore is immersed in a conducting fluid and a potential (voltage) is applied across it - under these conditions a slight electric current due to conduction of ions through the nanopore can be observed, and the amount of current is exceedingly sensitive to the size of the nanopore. If DNA molecules pass (or part of the DNA molecule passes) through the nanopore, this can create a change in the magnitude of the current through the nanopore, thereby allowing the sequences of the DNA molecule to be determined.
  • the nanopore may be a solid-state pore fabricated on a metal and/or nonmetal surface, or a protein-based nanopore, such as alpha-hemolysin (Clarke et al., Nat. Nanotech., 4, Feb. 22, 2009: 265-270).
  • HeliScope by Helicos Biosciences (Voelkerding et al., Clinical Chem., 55: 641- 658, 2009; MacLean et al., Nature Rev. Microbiol., 7- 287-296; U.S. Pat. No. 7,169,560; U.S. Pat. No. 7,282,337; U.S. Pat. No. 7,482,120; U.S. Pat. No. 7,501,245; U.S. Pat. No. 6,818,395; U.S. Pat. No. 6,911,345; U.S. Pat. No. 7,501,245; each herein incorporated by reference in their entirety) is the first commercialized single-molecule sequencing platform.
  • Template DNA is fragmented and polyadenylated at the 3' end, with the final adenosine bearing a fluorescent label.
  • Denatured polyadenylated template fragments are ligated to poly(dT) oligonucleotides on the surface of a flow cell.
  • Initial physical locations of captured template molecules are recorded by a CCD camera, and then label is cleaved and washed away.
  • Sequencing is achieved by addition of polymerase and serial addition of fluorescently-labeled dNTP reagents. Incorporation events result in fluor signal corresponding to the dNTP, and signal is captured by a CCD camera before each round of dNTP addition.
  • Sequence read length ranges from 25-50 nucleotides, with overall output exceeding 1 billion nucleotide pairs per analytical run.
  • Other single molecule sequencing methods include real-time sequencing by synthesis using a VisiGen platform (Voelkerding et al., Clinical Chem., 55: 641-658, 2009; U.S. Pat. No. 7,329,492; U.S. patent application Ser. No. 11/671,956; U.S. patent application Ser. No.
  • Some embodiments of the technology are related to systems for processing a biological sample (e.g., a sample comprising cells and/or nucleic acid). Particular embodiments provide systems for producing a sequencing library for input into an NGS workflow. Embodiments of the microfluidic cartridge described herein find use in preparing an NGS library for sequencing.
  • embodiments of the technology provide a system for production of a
  • NGS library from a biological sample comprising a microfluidic cartridge to accept as input a biological sample and produce a NGS sequencing library as output.
  • Some embodiments provide an apparatus that accepts a microfluidic cartridge.
  • Particular embodiments comprise ⁇ l) an apparatus comprising an interface to accept a microfluidic cartridge; and 2) a microfluidic cartridge.
  • the system comprises a piezoelectric component and a thermoelectric component.
  • the system comprises a power supply to provide a voltage and current to the system components.
  • the system comprises a frequency regulator.
  • Some embodiments provide systems for sequencing a nucleic acid (e.g., using NGS).
  • a system comprising an apparatus for producing an NGS library for input to an NGS workflow and/or an NGS sequencer.
  • the NGS sequencer and apparatus are fluidly connected, e.g., by a tube or other conduit for transporting the output NGS library from the apparatus directly to the input of the NGS sequencer.
  • systems for sequencing a nucleic acid comprise ⁇ l) an apparatus comprising an interface to accept a microfluidic cartridge; 2) a microfluidic cartridge; and 3) an NGS sequencer.
  • the methods and systems described herein are associated with a programmable machine designed to perform a sequence of arithmetic or logical operations as provided by the methods described herein. For example, some
  • embodiments of the technology are associated with (e.g., implemented in) computer software and/or computer hardware.
  • the technology relates to a computer comprising a form of memory, an element for performing arithmetic and logical operations, and a processing element (e.g., a processor or a microprocessor) for executing a series of instructions (e.g., a method as provided herein) to read, manipulate, and store data.
  • a processor comprises one or more processors.
  • a processor provides instructions to one or more valves, components, modules,
  • thermoelectric components piezoelectric components, pumps, reagent supplies, etc. in the microfluidic cartridge and/or apparatus.
  • a microprocessor is part of a system comprising one or more of a CPU, a graphics card, a user interface (e.g., comprising an output device such as a display and an input device such as a keyboard), a storage medium, and memory components.
  • Memory components e.g., volatile and/or nonvolatile memory find use in storing instructions (e.g., an embodiment of a process as provided herein) and/or data.
  • Programmable machines associated with the technology comprise conventional extant technologies and technologies in development or yet to be developed (e.g., a quantum computer, a chemical computer, a DNA computer, an optical computer, a spintronics based computer, etc.).
  • Some embodiments provide a computer that includes a computer-readable medium.
  • the embodiment includes a random access memory (RAM) coupled to a processor.
  • the processor executes computer-executable program instructions stored in memory.
  • processors may include a microprocessor, an ASIC, a state machine, or other processor, and can be any of a number of computer processors, such as processors from Intel Corporation of Santa Clara, California and Motorola Corporation of
  • processors include, or may be in communication with, media, for example computer-readable media, which stores instructions that, when executed by the processor, cause the processor to perform the steps described herein.
  • computer-readable media include, but are not limited to, an electronic, optical, magnetic, or other storage or transmission device capable of providing a processor, such as the processor of client, with computer-readable
  • Suitable media include, but are not limited to, a floppy disk, CD-ROM, DVD, magnetic disk, memory chip, ROM, RAM, an ASIC, a configured processor, all optical media, all magnetic tape or other magnetic media, or any other medium from which a computer processor can read instructions.
  • various other forms of computer-readable media may transmit or carry instructions to a computer, including a router, private or public network, or other transmission device or channel, both wired and wireless.
  • the instructions may comprise code from any suitable computer-programming language, including, for example, C, C++, C#, Visual Basic, Java, Python, Perl, Swift, Ruby, Unix, and JavaScript.
  • Computers are connected in some embodiments to a network or, in some embodiments, can be stand-alone machines. Computers may also include a number of external or internal devices such as a mouse, a CD-ROM, DVD, a keyboard, a display, or other input or output devices. Examples of computers are personal computers, digital assistants, personal digital assistants, cellular phones, mobile phones, smart phones, pagers, digital tablets, laptop computers, internet appliances, and other processor-based devices. In general, the computer-related to aspects of the technology provided herein may be any type of processor-based platform that operates on any operating system, such as Microsoft Windows, Linux, UNIX, Mac OS X, etc., capable of supporting one or more programs comprising the technology provided herein. All such components, computers, and systems described herein as associated with the technology may be logical or virtual.
  • sequencing data are produced. Following the production of sequencing data, the sequencing data are reported to a data analysis operation in some embodiments. To facilitate data analysis in some embodiments, the sequencing data are analyzed by a digital computer. In some embodiments, the computer is appropriately programmed for receipt and storage of the sequencing data and for analysis and reporting of the sequencing data gathered, e.g., to provide a nucleic acid sequence in a human or machine readable format. Examples
  • Example 1 Exemplary microfluidic cartridge
  • microfluidic cartridge was designed and tested.
  • an integrated microfluidic cartridge was produced comprising four components (e.g., sub -circuits) ' ⁇ lysis and extraction; library preparation; library clean-up; and sequencing set up ( Figure 2).
  • the lysis and extraction sub-circuit ( Figure 3) comprises the sample input, cell lysis, and nucleic acid extraction components.
  • An exemplary lysis and extraction component provides features and functionalities for tasks such as ⁇
  • Adding an aqueous sample to the cartridge e.g., to the sample input;
  • the library preparation sub-circuit ( Figure 4) comprises components and performs steps to produce a library for a NGS platform.
  • the library preparation sub-circuit produces structurally linear, topologically circular nucleic acid molecules for a Pac Bio sequencing process.
  • An exemplary library preparation sub-circuit provides features and functionalities for tasks such as ⁇
  • the library clean-up module ( Figure 5) comprises components and performs steps to purify the sequencing library, e.g., to remove potential sequencing inhibitors and small circular DNA molecules such as adapter dimers.
  • An exemplary library clean-up module provides features and functionalities for tasks such as ⁇
  • the sequencing set up module ( Figure 6) comprises components and performs steps to prepare the sequencing library for loading onto the sequencer including, e.g., annealing a sequencing primer and binding a sequencing polymerase.
  • An exemplary sequencing set up module provides features and functionalities for tasks such as ⁇
  • experiments were conducted to test the library preparation by the microfluidic cartridge.
  • extracted nucleic acids were added to the library preparation sub-circuit and the entire library preparation process was carried out on the cartridge (e.g., reverse transcription, whole genome amplification, DNA fragmentation, end repair, ligation, and exonuclease treatment).
  • This library product was then purified using a bench top protocol (e.g., Agencourt Ampure bead method) and sequenced on a Pac Bio RS instrument. Based on key sequencing metrics (read quality, total reads, read length, accuracy, and depth of coverage), the on-cartridge library preparation performed comparably to an existing bench top control (Table l).
  • Bioanalyzer gel electrophoresis platform Data collected indicated that the on-cartridge extraction yield was approximately 40% of the benchtop clean-up.
  • tubes made of PEEK and PTFE were attached to the output of a cartridge. Samples were pumped from the cartridge, through the tube, and the sample evaluated at the output of the tube. For both materials, a tube with an inner diameter of 250 ⁇ was selected (e.g., to provide a dead volume of only 24 ⁇ over a tube length of 0.5 m).
  • experiments were conducted to produce a sequencing library in an embodiment of a microfluidic cartridge, output the library to an NGS sequencer, and produce nucleic acid sequence.
  • the technology was tested with varying amounts of Klebsiella pneumoniae (KP) and Staphylococcus aureus (SA) cells as input amounts. Tests were conducted using inputs of approximately 1666, 166, and 16 cells. A number of 1666 cells (or 1666 CFU) corresponds to 8.33 pg of DNA, which was added to 500 ⁇ of buffer; thus the amount of genomic DNA in the samples varied between 1.67 x 10 -5 to 1.67 x 10 -7 ng/ ⁇ .
  • KP Klebsiella pneumoniae
  • SA Staphylococcus aureus
  • a library comprising approximately 2 to 8 ng/ ⁇ of nucleic acid (e.g., 5.61 ⁇ 3.2 ng/ ⁇ ) was outputted by the microfluidic cartridge. Data collected indicated that approximately 3000 to 9000 sequencing reads were obtained from the library. Further, sequence was obtained from a sample comprising approximately 16 bacterial cells as input.

Abstract

La présente invention concerne une technologie associée à des cartouches microfluidiques conçues pour traiter un échantillon biologique, comprenant mais ne se limitant pas à des dispositifs, des appareils, des procédés, et des systèmes permettant de générer des bibliothèques de séquençage d'acide nucléique qui sont appropriées pour être utilisées dans des procédés de séquençage (par exemple, des procédés de séquençage de nouvelle génération) ou d'autres technologies d'analyse d'acide nucléique.
PCT/US2015/057186 2014-10-24 2015-10-23 Cartouche microfluidique WO2016065300A1 (fr)

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US11649275B2 (en) 2018-08-02 2023-05-16 Duke University Dual agonist fusion proteins
CN109576345A (zh) * 2018-10-17 2019-04-05 西人马（厦门）科技有限公司 一种用于dna提取的微流控芯片及其检测方法
US11426097B1 (en) 2018-10-17 2022-08-30 Hound Labs, Inc. Rotary valve assemblies and methods of use for breath sample cartridge systems
US11821821B1 (en) 2019-01-31 2023-11-21 Hound Labs, Inc. Noninvasive point of care biomarker detection from breath samples
US11512314B2 (en) 2019-07-12 2022-11-29 Duke University Amphiphilic polynucleotides
US11643647B2 (en) 2019-08-20 2023-05-09 Seagate Technology Llc Methods of gene assembly and their use in DNA data storage
US11939570B2 (en) 2019-08-20 2024-03-26 Seagate Technology Llc Microfluidic lab-on-a-chip for gene synthesis
US11933731B1 (en) 2020-05-13 2024-03-19 Hound Labs, Inc. Systems and methods using Surface-Enhanced Raman Spectroscopy for detecting tetrahydrocannabinol
WO2022040495A1 (fr) * 2020-08-21 2022-02-24 Duke University Dispositif d'analyse microfluidique
US11806711B1 (en) 2021-01-12 2023-11-07 Hound Labs, Inc. Systems, devices, and methods for fluidic processing of biological or chemical samples using flexible fluidic circuits
WO2022155246A1 (fr) * 2021-01-12 2022-07-21 Definitive Biotechnologies Llc Dispositif et procédé pour détecter des acides nucléiques dans des échantillons biologiques
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