CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S. C. § 119(e) of U.S. Provisional Application No. 62/534,949, entitled "SYSTEMS AND METHODS FOR CONTROLLED FLUID HANDLING," filed July 20, 2017, the entire disclosure of which is incorporated by reference herein in its entirety.

FIELD

[0002] The present disclosure relates to apparatuses and methods for controlling fluid flow and more particularly to controlling a fluid flow to, in, out and/or through a flow cell. The apparatuses and methods disclosed herein are useful in various biological or chemical reagent delivery and/or reaction systems such as a DNA synthesis system.

BACKGROUND

[0003] Conventional biological or chemical delivery systems employ adjustable positive pressure in reagent bottles combined with empirical assessment of flow restriction to enable unidirectional flow from a reagent reservoir (e.g. , a bottle) to a reaction vessel (typically a column) to a waste container. Certain conventional systems use a pump to draw from the reagent bottles and provide unidirectional flow. However, the absence of flow rate control and directional control in conventional systems limits the precision and efficiency of conventional systems.

[0004] For example, one conventional system, the PCR-MATE Model 391 DNA Synthesizer by Applied Biosystems (FIG. 1) is controlled by a positive-pressure chemical delivery system. The system includes a series of valves and manifolds connected to pressurized reagent bottles.

However, this system has the disadvantage that flow control depends on pressure control in the reagent bottle and combined flow resistance of the many valves, connectors, reaction vessel and tubing lengths defining the fluid paths. Delivering a specific dose to the flow cell requires pre- calibration of all possible fluid paths and opening the path for a specific time, which is time consuming and often lacks precision. The flow resistance of a given path can also change with operation time, thus requiring periodic recalibration. Additionally, such systems, due to the reliance on calibrated fluid paths and limited flow control, require a proximal waste container to avoid long distance waste tubing runs. Since the synthesis process produces large volumes of unpleasant chemical waste, for operator health and safety, such waste should be contained in a well-ventilated waste closet, adding to the complexity and cost of system construction and maintenance.

[0005] Another conventional system described in US 2009/0224482, incorporated herein by reference in its entirety, uses an upstream syringe pump with rotary valve to pull reagent from bottles and push the dose to the flow cell. However, such arrangements have the disadvantage that the volume of the syringe limits the size of a continuous dose. That is, continuous regulated flow is impossible. Additionally, the lack of pressurized reagent puts the fluid path, between the pump and reagent, in negative pressure during syringe fill. This makes syringe fill dependent on fluid path resistance and introduces the potential for air leakage (bubbles) into the system. Furthermore, the syringe pump is positioned upstream of the flow cell reaction chamber, which puts the reagent in contact with the pump before it reaches the flow cell reaction site and raises the potential for contamination and reduced reliability.

[0006] A conventional system known as the "gene machine" (FIG. 2) uses an upstream pump to pull reagent and push it through a column. Such configurations are able to sustain continuous flow but otherwise share the disadvantages discussed above. In particular, the lack of pressurized reagent puts the fluid path, between the pump and reagent, in negative pressure during pumping. This makes pumping flow rate dependent on fluid path resistance and introduces the potential for air leakage (bubbles) into the system. Furthermore, the syringe pump is positioned upstream of the reaction chamber. Because the reagent is in contact with the pump before it reaches the flow cell reaction site, the potential for contamination and reduced reliability is present in the system.

[0007] The PolyGen 12 column oligonucleotide synthesizer (FIG. 3) uses a downstream pump to pull reagent from unpressurized reagent containers through a slider block. The sliders (labeled 1-4) are arranged in such a way that they slide past each other. The reagent slider (slider 4) is stationary and contains the reagent feed lines. The column slider (slider 2) contains the carrier for 10 columns. Sliders 1, 2, and 3 can together slide past slider 4. Slider 2 can slide between sliders 1 and 3. Sliders 1 and 3 can only slide together. Thus, the reaction column (slider 2) can be aligned with the bore holes in sliders 1 and 3, thereby establishing a connection via the reagent feed line (slider 4), between the reagent and the metering pump (DMT).

However, similar to the systems described above, this configuration has the disadvantage that it creates negative pressure upstream of the pump, including within the flow cell, and is therefore flow rate limited by the resistance of the fluid path and introduces the potential for air leakage (bubbles) into the system. [0008] Thus, there is a need for improved biological or chemical reagent delivery and/or reaction systems.

SUMMARY

[0009] Provided herein are improved apparatuses, systems and methods for biological or chemical reagent delivery and/or reaction. In various embodiments, the systems and methods of the present disclosure allow for flow rate control and/or directional control and can significantly improve the precision and efficiency of conventional systems.

[0010] In one aspect, an apparatus of the present disclosure can include a positively pressurized reagent container for providing one or more reagents, a rotary valve in fluid communication with the container to direct a flow of the one or more reagents, a flow cell configured to receive the flow of the one or more reagents directed from the rotary valve, and a pump downstream of the flow cell for controlled metering of the one or more reagents.

[0011] In another aspect, an apparatus of the present disclosure includes a reagent container for providing one or more reagents, a degasser adapted to remove or reduce gas from a flow of the one or more reagents, a valve in fluid communication with the reagent container to direct the flow of the one or more reagents, and a flow cell configured to receive the flow of the one or more reagents directed from the valve. In some embodiments, the degasser is positioned upstream of the flow cell, preferably or optionally between the reagent container and the rotary valve.

[0012] In some embodiments, any of the apparatuses disclosed herein can further include a source of compressed gas (e.g. , inert gas such as helium, argon or nitrogen) for pressurizing the reagent container to provide the flow of the one or more reagents to the rotary valve. The one or more reagents can be for use in any biological or chemical reactions. In some embodiments, the reagents are for use in oligonucleotide synthesis, and can be optionally or preferably selected from deprotecting agent, capping agent, oxidizing agent, and/or wash buffer.

[0013] In some embodiments, the rotary valve can be configured to receive a pressurized gas and direct the pressurized gas to the flow cell to displace residual liquids therein following completion of reactions. More than one rotary valve(s) can be included in the apparatus. For example, the apparatus can include a first rotary valve in fluid communication with a first inlet of the flow cell, and a second rotary valve in fluid communication with a second inlet of the flow cell. In some embodiments, when in use, the flow can be directed from the reagent container to the first rotary valve, from the first rotary valve to the first inlet to enter the flow cell, exiting the flow cell through the second inlet to the second rotary valve, and from the second rotary valve to the pump. In certain embodiments, when in use, the flow can be directed from the reagent container to the second rotary valve, from the second rotary valve to the second inlet to enter the flow cell, exiting the flow cell through the first inlet to the first rotary valve, and then from the first rotary valve to the pump.

[0014] The flow cell can be used to receive the flow of the one or more reagents directed from the rotary valve, such that various biological or chemical reactions (or washing) can take place therein, e.g. , on a substrate or solid support located within the flow cell. In certain

embodiments, the flow cell can be in substantially horizontal orientation when loading the substrate. In some embodiments, when in use, the flow cell can be in substantially vertical orientation and optionally or preferably comprises an upper opening and a lower opening. The lower opening may act as inlet for the flow and the upper opening can act as outlet for the flow. In other embodiments, the direction of the flow can be reversed such that the upper opening acts as inlet for the flow and the lower opening acts as outlet for the flow.

[0015] In certain embodiments, the pump can be a rotary positive displacement pump, optionally or preferably a peristaltic pump. The pump can act to control one or more of flow direction, flow rate, flow volume and/or wave front of the flow. Such control can

advantageously be continuous and/or reversible. In some embodiments, the pump acts to draw the flow away from the reagent container and optionally or preferably into a waste container. The pump can also act to push the flow back towards the reagent container. In some

embodiments, the pump may act to agitate the flow in a forward and backward manner, thereby, e.g. , mixing the reagents to improve reaction dynamics and/or improving washing efficiency.

[0016] In some embodiments, the apparatus can further include a degasser positioned, e.g. , upstream of the flow cell, optionally or preferably between the reagent container and the rotary valve, for removing or reducing gas from the flow. The degasser can comprise a hydrophobic membrane optionally or preferably comprising polytetrafluoroethylene, polyvinylidene fluoride, polypropylene, polyethylene, acrylic polymer, or any combination thereof, optionally or more preferably copolymers of tetrafluoroethylene and perfluoro-2,2-dimethyl-l ,3-dioxole.

[0017] Also provided herein are methods for using the apparatuses of the present disclosure, for controlled fluid handling and improved biological or chemical reagent delivery/reactions such as DNA synthesis. For example, the method can include providing one or more reagents from a positively pressurized reagent container, directing a flow of the one or more reagents through a rotary valve in fluid communication with the container, receiving in a flow cell the flow of the one or more reagents directed from the rotary valve, and metering the one or more reagents using a pump placed downstream of the flow cell in a controlled manner. Another method can include providing one or more reagents from a reagent container, removing or reducing gas from a flow of the one or more reagents using a degasser, directing the flow of the one or more reagents through a valve in fluid communication with the reagent container, and receiving in a flow cell the flow of the one or more reagents directed from the valve.

[0018] Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention. This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The presently disclosed embodiments will be further explained with reference to the attached drawings. The drawings shown are not necessarily to scale, with emphasis instead generally being placed upon illustrating the principles of the presently disclosed embodiments.

[0020] FIG. 1 is a schematic diagram illustrating the PCR-MATE Model 391 DNA Synthesizer by Applied Biosystems.

[0021] FIG. 2 is a schematic diagram illustrating the "gene machine."

[0022] FIG. 3 is a schematic diagram illustrating the PolyGen 12 column oligonucleotide synthesizer.

[0023] FIG. 4 is a schematic diagram illustrating a fluid handling system in accordance with some embodiments of the present disclosure.

[0024] FIG. 5 is a schematic diagram illustrating a DNA synthesis system in accordance with some embodiments of the present disclosure.

[0025] While the above-identified drawings set forth present disclosure, other embodiments are also contemplated, as noted in the discussion. This disclosure presents illustrative embodiments by way of representation and not limitation. Numerous other modifications and embodiments can be devised by those skilled in the art which fall within the scope and spirit of the principles of the present disclosure. DETAILED DESCRIPTION

[0026] In one aspect, an improved design for controlled fluid handling and delivery to, from, through and/or within a reaction vessel is provided, particularly a flow cell deployed to accomplish surface reactions such as reactions involved in DNA synthesis. In some

embodiments, the system combines operation at elevated pressure and a positive displacement pump downstream from the reaction vessel to control reagent flow. In certain embodiments, hydrophobic membranes for in-line degassing (e.g. , removing bubbles from the reagent) can be used. Advantageously, the systems and related methods disclosed herein provide maximum control over reagent dose, flow dynamics in the reaction vessel, and reagent utilization, while inhibiting bubble formation and providing means to inhibit valve failure. In particular, the presently disclosed systems and methods allow for control of flow, including slowing down, speeding up or changing direction of the flow, and present significant advantages as it allows for controlled and efficient introduction of fresh reagents to the reaction surface (e.g. , a solid support located within the flow cell) when compared with uncontrolled flow rates. Velocity and direction control of the flow also can facilitate bubble removal in certain embodiments.

Definitions

[0027] For convenience, certain terms employed in the specification, examples, and appended claims are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0028] The articles "a" and "an" are used herein to refer to one or to more than one (i.e. , at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0029] As used herein, the term "about" means within 20%, more preferably within 10% and most preferably within 5%. The term "substantially" means more than 50%, preferably more than 80%, and most preferably more than 90% or 95%.

[0030] As used herein, "a plurality of means more than 1, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, e.g., 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or more, or any integer therebetween.

[0031] The terms "(reaction) vessel", "(reaction) chamber" and "flow cell" are used

interchangeably and refer to a fluidic region capable of enclosing a substrate so that a fluid can be received by and contained within the flow cell to allow a reaction or wash to take place on at least a portion of the substrate.

[0032] The terms "upstream" and "downstream" are relative to each other with reference to the typical direction of flow from reagent container to flow cell to waste.

[0033] A "fluid" can be a liquid or a gas. A "liquid," as used herein, is a substance that is relatively incompressible and has a capacity to flow and to conform to a shape of a container or a channel that holds the substance. A liquid may be aqueous based and include polar molecules exhibiting surface tension that holds the liquid together. A liquid may also include non-polar molecules, such as in an oil-based or non-aqueous substance. It is understood that references to a liquid in the present application may include a liquid that was formed from the combination of two or more liquids. For example, separate reagent solutions may be later combined to conduct designated reactions.

[0034] As used herein, the term "in fluid communication" or "fluidically coupled" (or like term) refers to two spatial regions being connected together such that a liquid or gas may be directed between the two spatial regions. In some cases, the fluidic coupling permits a fluid to be directed back and forth between the two spatial regions. In other cases, the fluidic coupling is uni-directional such that there is only one direction of flow between the two spatial regions. For example, a reaction chamber may be fluidically coupled with a reagent container such that a liquid may be transported into the reaction chamber from the reagent container.

[0035] As used herein, a "reaction" includes a change in at least one of a biological chemical, electrical, physical, or optical property (or quality) of a reactant. The reaction may be a chemical transformation, chemical change, or chemical interaction. The reaction may also be a change in electrical properties. For example, the reaction may be a change in ion concentration within a solution. Exemplary reactions include, but are not limited to, chemical reactions such as reduction, oxidation, addition, elimination, rearrangement, esterification, amidation, etherification, cyclization, or substitution; binding interactions in which a first chemical binds to a second chemical; dissociation reactions in which two or more chemicals detach from each other; fluorescence; luminescence; bioluminescence; chemiluminescence; and biological reactions, such as nucleic acid synthesis, nucleic acid replication, nucleic acid amplification, nucleic acid hybridization, nucleic acid ligation, phosphorylation, enzymatic catalysis, receptor binding, or ligand binding. In particular embodiments, the reaction includes DNA synthesis.

[0036] As used herein, a "reagent" includes any substance that may be used to obtain a desired reaction. For example, reagents can include biological and/or chemical reagents, catalysts such as enzymes, reactants for the reaction, samples, products of the reaction, other biomolecules, salts, metal cofactors, chelating agents, buffer solutions (e.g. , hydrogenation buffer) and wash solutions. The reagent may be delivered, individually in solutions or combined in one or more mixture, to various locations in a fluidic system. For instance, a reagent may be delivered to a reaction chamber.

[0037] As used herein, "nucleic acid," "nucleic acid sequence," "oligonucleotide,"

"polynucleotide," "gene" or other grammatical equivalents as used herein means at least two nucleotides, either deoxyribonucleotides or ribonucleotides, or analogs thereof, covalently linked together. Polynucleotides are polymers of any length, including, e.g. , 10, 20, 50, 100, 200, 300, 500, 1000, etc. As used herein, an "oligonucleotide" may be a nucleic acid molecule comprising at least two covalently bonded nucleotide residues. In some embodiments, an oligonucleotide may be between 10 and 1,000 nucleotides long. For example, an

oligonucleotide may be between 10 and 500 nucleotides long, or between 500 and 1,000 nucleotides long. In some embodiments, an oligonucleotide may be between about 20 and about 800 nucleotides long (e.g. , from about 20 to 400, from about 400 to 800 nucleotides long). In some embodiments, an oligonucleotide may be between about 50 and about 500 nucleotides long (e.g. , from about 50 to 250, from about 250 to 500 nucleotides long). In some embodiments, an oligonucleotide may be between about 100 and about 300 nucleotides long (e.g. , from about 100 to 150, from about 150 to 300 nucleotides long). However, shorter or longer oligonucleotides may be used. An oligonucleotide may be a single- stranded or double- stranded nucleic acid. As used herein the terms "nucleic acid", "polynucleotide" and

"oligonucleotide" are used interchangeably and refer to naturally-occurring or non-naturally occurring, synthetic polymeric forms of nucleotides. In general, the term "nucleic acid" includes both "polynucleotide" and "oligonucleotide" where "polynucleotide" may refer to longer nucleic acid (e.g. , more than 1,000 nucleotides, more than 5,000 nucleotides, more than 10,000 nucleotides, etc.) and "oligonucleotide" may refer to shorter nucleic acid (e.g. , 10-500 nucleotides, 20-400 nucleotides, 40-200 nucleotides, 50- 100 nucleotides, etc.).

[0038] The nucleic acid molecules of the present disclosure may be formed from naturally occurring nucleotides, for example forming deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules. Alternatively, naturally-occurring nucleic acids may include structural modifications to alter their properties, such as in peptide nucleic acids (PNA) or in locked nucleic acids (LNA). The solid phase synthesis of nucleic acid molecules with naturally occurring or artificial bases is well known in the art. The terms should be understood to include equivalents, analogs of either RNA or DNA made from nucleotide analogs and as applicable to the embodiment being described, single- stranded or double- stranded

polynucleotides. Nucleotides useful in the disclosure include, for example, naturally-occurring nucleotides (for example, ribonucleotides or deoxyribonucleotides), or natural or synthetic modifications of nucleotides, or artificial bases. In some embodiments, the sequence of the nucleic acids does not exist in nature (e.g. , a cDNA or complementary DNA sequence, or an artificially designed sequence).

[0039] Usually in a nucleic acid molecule nucleosides are linked by phosphodiester bonds. Whenever a nucleic acid is represented by a sequence of letters, it will be understood that the nucleosides are in the 5' to 3 ' order from left to right. In accordance to the IUPAC notation, "A" denotes deoxyadenosine, "C" denotes deoxycytidine, "G" denotes deoxyguanosine, "T" denotes deoxythymidine, "U" denotes the ribonucleoside, uridine. In addition, there are also letters which are used when more than one kind of nucleotide could occur at that position: "W" (i.e. , weak bonds) represents A or T, "S" (strong bonds) represents G or C, "M" (for amino) represents A or C, "K" (for keto) represents G or T, "R" (for purine) represents A or G, "Y" (for pyrimidine) represents C or T, "B" represents C, G or T, "D" represents A, G or T, "H" represents A, C or T, "V" represents A, C, or G and "N" represents any base A, C, G or T (U). It is understood that nucleic acid sequences are not limited to the four natural deoxynucleotides but can also comprise ribonucleoside and non-natural nucleotides. A "/" in a nucleotide sequence or nucleotides given in brackets refer to alternative nucleotides, such as alternative U in a RNA sequence instead of T in a DNA sequence. Thus, U/T or U(T) indicate one nucleotide position that can either be U or T. Likewise, A/T refers to nucleotides A or T; G/C refers to nucleotides G or C. Due to the functional identity between U and T any reference to U or T herein shall also be seen as a disclosure as the other one of T or U. For example, the reference to the sequence UUCG (on an RNA) shall also be understood as a disclosure of the sequence TTCG (on a corresponding DNA). For simplicity only, only one of these options is described herein. Complementary nucleotides or bases are those capable of base pairing such as A and T (or U); G and C; G and U.

[0040] One or more modified bases (e.g. , a nucleotide analog) can be incorporated in the nucleic acid molecules. Examples of modifications include, but are not limited to, one or more of the following: methylated bases such as cytosine and guanine; universal bases such as nitro indoles, dP and dK, inosine, uracil; halogenated bases such as BrdU; fluorescent labeled bases; nonradioactive labels such as biotin (as a derivative of dT) and digoxigenin (DIG); 2,4- Dinitrophenyl (DNP); radioactive nucleotides; post-coupling modification such as dR-NH2 (deoxyribose-NEb); Acridine (6-chloro-2-methoxiacridine); and spacer phosphoramides which are used during synthesis to add a spacer "arm" into the sequence, such as C3, C8 (octanediol), C9, C 12, HEG (hexaethlene glycol) and C18.

[0041] As used herein, the term "solid support", "support" and "substrate" are used

interchangeably and refers to a porous or non-porous solid (e.g. , solvent insoluble) material on which polymers such as nucleic acids are synthesized or immobilized. As used herein

"porous" means that the material contains pores having substantially uniform diameters (for example in the nm range). Porous materials can include but are not limited to, paper, synthetic filters and the like. In such porous materials, the reaction may take place within the pores. The support can have any one of a number of shapes, such as pin, strip, plate, disk, rod, bends, cylindrical structure, particle, including bead, nanoparticle and the like. In some embodiments, the support is planar (e.g. , a chip). The support can have variable widths. The solid support can be an organized matrix or network of wells, such as a microarray. In some embodiments, the support can include a plurality of beads or particles, optionally positioned in one or more multiwall plates.

[0042] The support can be hydrophilic or capable of being rendered hydrophilic. The support can include inorganic powders such as silica, magnesium sulfate, and alumina; natural polymeric materials, particularly cellulosic materials and materials derived from cellulose, such as fiber containing papers, e.g. , filter paper, chromatographic paper, etc.; synthetic or modified naturally occurring polymers, such as nitrocellulose, cellulose acetate, poly (vinyl chloride), polyacrylamide, cross linked dextran, agarose, polyacrylate, polyethylene,

polypropylene, poly (4-methylbutene), polystyrene, polymethacrylate, poly(ethylene

terephthalate), nylon, poly(vinyl butyrate), polyvinylidene difluoride (PVDF) membrane, glass, controlled pore glass, magnetic controlled pore glass, ceramics, metals, silicon, modified silicon and the like; either used by themselves or in conjunction with other materials.

[0043] As used herein, the term "array" refers to an arrangement of discrete features for storing, routing, amplifying and releasing oligonucleotides or complementary oligonucleotides for further reactions. The array can be planar. In an embodiment, the support or array can be addressable. Addressable supports or arrays enable the direct control of individual isolated volumes such as droplets.

[0044] As used herein, a "feature" refers to a discrete location (or spot) on a solid support, e.g. , a chip, multiwell tray, or microarray. In some embodiments, oligonucleotides can be synthesized on and/or immobilized to the feature. An arrangement of discrete features can be presented on the solid support for storing, routing, amplifying, releasing and otherwise manipulating oligonucleotides or complementary oligonucleotides for further reactions. In some

embodiments, each feature is addressable; that is, each feature is positioned at a particular predetermined, prerecorded location (i.e. , an "address") on the support. Therefore, each oligonucleotide is localized to a known and defined location on the support. The sequence of each oligonucleotide can be determined from its position on the support. The size of the feature can be chosen to allow formation of a microvolume (e.g. , 1-1000 microliters, 1- 1000 nanoliters, 1-1000 picoliters) droplet on the feature, each droplet being kept separate from each other. As used herein, features are typically, but need not be, separated by interfeature spaces to ensure that droplets between two adjacent features do not merge. Interfeatures will typically not carry any oligonucleotide on their surface and will correspond to inert space. In some embodiments, features and interfeatures may differ in their hydrophilicity or hydrophobicity properties.

[0045] As used herein, the term "immobilized" refers to oligonucleotides bound to a solid support that may be attached through their 5' end or 3 ' end. The support-bound oligonucleotides may be immobilized on the chip via a nucleotide sequence (e.g. , degenerate binding sequence) or linker (e.g. , light-activatable linker or chemical linker). It should be appreciated that by 3 ' end, it is meant the sequence downstream to the 5' end and by 5' end it is meant the sequence upstream to the 3 ' end. For example, an oligonucleotide may be immobilized on the chip via a nucleotide sequence or linker that is not involved in subsequent reactions. Certain

immobilization methods are reviewed by Nimse et al., Sensors 2014, 14, 22208-22229, the disclosure of which is incorporated herein by reference in its entirety.

[0046] As used herein, "including," "comprising," "having," "containing," "involving," and variations thereof, are meant to encompass the items listed thereafter and equivalents thereof as well as additional items. "Consisting of shall be understood as a close-ended relating to a limited range of elements or features. "Consisting essentially of limits the scope to the specified elements or steps but does not exclude those that do not materially affect the basic and novel characteristics of the claimed invention.

[0047] Other terms used herein will be generally understood by one of ordinary skill in the applicable arts such as fluidic handling, microfluidics, recombinant nucleic acid technology, synthetic biology, and molecular biology.

Fluid Handling Systems [0048] In one aspect, an apparatus of the present disclosure can include a positively pressurized reagent container for providing one or more reagents, a rotary valve in fluid communication with the container to direct a flow of the one or more reagents, a flow cell configured to receive the flow of the one or more reagents directed from the rotary valve, and a pump downstream of the flow cell for controlled metering of the one or more reagents.

[0049] In another aspect, an apparatus of the present disclosure includes a reagent container for providing one or more reagents, a degasser adapted to remove or reduce gas from a flow of the one or more reagents, a valve in fluid communication with the reagent container to direct the flow of the one or more reagents, and a flow cell configured to receive the flow of the one or more reagents directed from the valve. In some embodiments, the degasser is positioned upstream of the flow cell, optionally or preferably between the reagent container and the rotary valve.

[0050] Referring now to FIG. 4, a fluid handling system 100 is provided in accordance with various embodiments. The system 100 includes a flow path extending from one or more reagent reservoirs lOla-d through flow cell 107 to a waste container 115. As used herein, "upstream" and "downstream" indicate a relative position along the flow path wherein upstream indicates relative proximity to the one or more reagent reservoirs lOla-d and downstream indicates relative proximity to the waste container 115.

[0051] In accordance with various embodiments, the one or more reagent reservoirs lOla-d can be in selective fluid communication with the flow cell 107 via one or more rotary valves 105a-b. Flow of reagent(s) from one or more of the reagent reservoirs lOla-d through the flow cell 107 (e.g. , flowing via first rotary valve 105a from first opening 109 to second opening 111 or flowing via second rotary valve 105b from second opening 111 to first opening 109) to the waste site 115 can be facilitated, e.g. , by pressurizing the reagent reservoirs lOla-d (e.g. , by delivering compressed gas from a gas source 123 at a pressure regulated by pressure regulator 121) and/or operating a downstream pump 113 such that the reagent(s) are expelled from the reagent reservoirs lOla-d and drawn through the flow path to the flow cell 107 and/or the waste site 115.

[0052] The fluid flow, in accordance with some embodiments, can be reversed (e.g. , by reversing the pump 113 and/or reducing pressurization of the reagent reservoir(s) lOla-d).

Furthermore, in accordance with various embodiments, the reagent(s) can be expelled from the flow cell 107 by drawing the compressed gas from the gas source 123 through the flow cell 107. [0053] As explained in greater detail below, by providing both pressurization of the reagent reservoir(s) lOla-d and a reversible downstream pump 113, the system 100 of FIG. 4

advantageously provides control over flow rate, flow velocity, and directionality of the flow while reducing or inhibiting bubble formation and permitting bubble removal. Bubble formation can be further reduced or inhibited, in accordance with certain embodiments, by the inclusion of one or more in-line degassers 103a-d along the flow path (e.g. , upstream of the flow cell 107) to remove unwanted gaseous inclusions. Additionally, in order to alleviate overpressure in the system 100, one or more safety valves 117, 119 can be provided along the fluid path.

[0054] The reagent reservoirs lOla-d can include any suitable containers. For example, in accordance with various embodiments, the reagent reservoirs lOla-d can include beakers, flasks, bottles, tanks, vials, kegs, continuous supply lines, any other suitable reagent source capable of being pressurized, or combinations thereof. As discussed above, pressurization of the reagent reservoirs lOla-d can, in some embodiments, be achieved by delivering compressed gas (e.g. , inert gas such as helium, argon or nitrogen) from a gas source 123 at a pressure regulated by pressure regulator 121. However, it will be apparent in view of the present disclosure that the reagent reservoirs can be pre-pressurized. Any other suitable methods for pressurizing the reagent reservoirs lOla-d (e.g. , via a pump operatively engaged directly with the reagent reservoirs lOla-d) can also be used.

[0055] The reagent reservoirs lOla-d can be used to supply any suitable reagents, depending on the desired reaction. In some embodiments, the reagents are for use in oligonucleotide synthesis, and can be optionally or preferably selected from deprotecting agent, capping agent, oxidizing agent, and/or wash buffer.

[0056] Reagents can be displaced from the reagent reservoirs lOla-d under pressure (e.g. , constant or as needed) supplied from gas source 123. The gas source 123 can be, for example, a continuous gaseous source (e.g. , shop air) or can be a compressed gas cylinder. In various embodiments, the gas provided can be an inert gas such as helium, neon, argon, krypton, xenon, or combinations thereof so as to avoid interference with the reactions. The pressure regulator 121 can be configured to regulate a pressure of the compressed gas exiting the gas source 123 and can include any suitable pressure regulator, many of which are widely known in the art.

[0057] In some embodiments, the apparatus can further include one or more optional degasser(s) 103a-d for removing or reducing gas from the flow of the reagent(s). In-line degassers 103a-d can include, for example, polymeric membranes (e.g., Teflon® AF available from Chemours™), nanoporous membranes, palladium membranes, combinations thereof or any other suitable in- line degasser. In some embodiments, the degasser can comprise a hydrophobic membrane optionally or preferably comprising polytetrafluoroethylene, polyvinylidene fluoride, polyvinylidene difiuoride (PVDF), polypropylene, polyethylene, acrylic polymer, or any combination thereof, optionally or more preferably copolymers of tetrafluoroethylene and perfluoro-2,2-dimethyl- l,3-dioxole. In one example, the degasser is model number 403-0202-1 available from Random Technologies (Loganville, GA).

[0058] The in-line degassers 103a-d can be positioned at a location upstream of the flow cell 107. For example, as shown in FIG. 4, the in-line degassers 103a-d can be positioned downstream of the reagent reservoirs lOla-d but upstream of the rotary valves 105a-b.

However, it will be apparent in view of this disclosure that, in some embodiments, the in-line degassers 103a-d can be positioned downstream of the rotary valves 105a-b and upstream of the flow cell 107. As will be appreciated by one of skill in the art, when pressure is reduced, such as when the reagents exit the pressurized flow path and enter the relatively large, unpressurized flow cell 107 via the first or second opening 109, 111, the solubility of gas (e.g. , air) within the reagents is reduced according to Henry' s Law. Thus, when the pressure is reduced on the reagents as they enter the flow cell 107, any dissolved gas in excess of saturation can effervesce, causing undesirable bubble formation (hence areas unavailable to reagents/reactions due to coverage by the bubbles). Advantageously, by degassing the reagents or other fluid upstream of the flow cell 107, less gas is mixed with the reagents and thus any remaining gas is less likely to effervesce, thereby inhibiting bubble formation within the flow cell 107.

[0059] One or more rotary valves 105a and/or 105b can be included. Rotary valves 105a-b can be any rotary valve suitable for selectively providing fluid communication between at least one of the reagent reservoirs lOla-d, the flow cell 107, the pump 113, and the waste container 115. The rotary valves 105a-b can include, for example, stopcock valves, spool valves, airlocks, any other suitable rotary valve, and combinations thereof. In accordance with various embodiments, the rotary valves 105a-b can include a rotary valve as disclosed in WO 2015/187868, incorporated herein by reference in its entirety.

[0060] In some embodiments, the rotary valve 105a-b can be rotated from a first valve position to a second valve position so as to direct the reagent from the reagent reservoir to the flow cell. In the second valve position, the flow cell 107 may be fluidically coupled to the reagent reservoir lOla-d. At 105a-b, the reagent may be induced to flow (e.g. , by a pump 113) into the flow cell 107. This can be repeated until each of the desired reagents is disposed within the flow cell 107 where more than one reagent is needed. Accordingly, one or more reagents may be directed into the reaction chamber utilizing the rotary valve. In alternative embodiments, the reagent(s) can have a direct channel to the reaction chamber and does not flow through the rotary valve.

[0061] Adjustment of the rotary valves 105a-b provides control over which of the reagent reservoirs lOla-d and/or the gas source 123 are in fluid communication with the flow cell 107. For example, adjustment of the rotary valves 105a-b can permit selection of one or more of the reagent reservoirs lOla-d for fluid communication with the flow cell 107 to commence a reaction or a wash. Similarly, adjustment of the rotary valves 105a-b can permit selection of the gas source 123 for purging the flow cell 107 upon completion of the reaction. Additionally, adjustment of the rotary valves 105a-b can provide some metering of the reagent flow rate. For example, leaving one or both of the rotary valves 105a-b only partially open restricts flow through the valve 105a-b, thereby metering flow to downstream locations such as the flow cell 107.

[0062] In some embodiments, a first rotary valve 105a can be configured to be in fluid communication with a first inlet 109 of the flow cell 107, and a second rotary valve 105b in fluid communication with a second inlet 111 of the flow cell 107. In some embodiments, when in use, the flow can be directed from one or more of the reagent containers lOla-d to the first rotary valve 105a, from the first rotary valve 105a to the first inlet 109 to enter the flow cell 107, exiting the flow cell 107 through the second inlet 111 to the second rotary valve 105b, and from the second rotary valve 105b to the pump 111. In certain embodiments, when in use, the flow can be directed from one or more of the reagent containers lOla-d to the second rotary valve 105b, from the second rotary valve 105b to the second inlet 111 to enter the flow cell 107, exiting the flow cell 107 through the first inlet 109 to the first rotary valve 105a, and then from the first rotary valve 105a to the pump 113.

[0063] In certain embodiments, one or more of the rotary valves 105a-b can be configured to receive a pressurized gas, via port 106, and direct the pressurized gas to the flow cell 107 to displace residual liquids therein following completion of reactions.

[0064] A flow cell 107 can be used to receive the flow of the one or more reagents directed from the rotary valve 105a-b, such that various biological or chemical reactions (or washing) can take place therein, e.g. , on a substrate or solid support located within the flow cell 107. The flow cell 107 (also referred to as a reaction chamber) can include any sealed vessel for containing a reaction substrate and having first and second openings 109, 111 for permitting fluid flow into and out of the vessel. For example, the system 100 can include a flow cell as disclosed in US 2009/0224482, incorporated by reference in its entirety.

[0065] As shown in FIG. 4, the flow cell 107 can be oriented vertically such that the first and second openings 109, 111 are positioned in a vertically stacked arrangement. However, it will be apparent in view of this disclosure that the flow cell can be positioned in any orientation (e.g. , vertical, horizontal, or at an angle). In some embodiments, a rotating mechanism can be include to change the orientation of the flow cell. As shown in FIG. 4 and discussed above, the system 100 can be operated in any direction to flow the reagents either via the first rotary valve 105a from the first opening 109 to the second opening 111 or via the second rotary valve 105b from the second opening 111 to the first opening 109.

[0066] In certain embodiments, the flow cell 107 can be in substantially horizontal orientation when loading the substrate. In some embodiments, when in use (e.g. , after loading the substrate), the flow cell 107 can be in substantially vertical orientation and optionally or preferably comprises an upper opening 111 and a lower opening 109. In vertical configurations, the flow cell 107 has a long and short dimension positioned such that the long dimension is vertical. In some embodiments, this orientation allows for reagents to enter the flow cell 107 from the first (bottom) opening 109 and flow upwards, enabling bubbles in the reagent stream to exit through the second (top) opening 111. In other embodiments, the direction of the flow can be reversed such that the upper opening 111 acts as inlet for the flow and the lower opening 109 acts as outlet for the flow.

[0067] The system 100 can include one or more pump 113. The pump 113 can include any device suitable for moving fluid through the flow path. For example, the pump 113 can include positive displacement pumps, reciprocal pumps, gear pumps, screw pumps, Roots-type pumps, peristaltic pumps, impulse pumps, velocity pumps, radial-flow pumps, axial-flow pumps, any other pump capable of bidirectional operation, and combinations thereof. In certain

embodiments, the pump 113 can be a rotary positive displacement pump, optionally or preferably a peristaltic pump.

[0068] In one example, pump 113 is a positive displacement pump that draws fluid under the force of the gas pressure in response to the actuation of the pump 113. It can include a piston cylinder and a piston, and causes positive displacement of fluid due to its downstream location relative to flow cell 107 and to the positive gas pressure applied on the system 100 by gas source 123. That is, as the piston is withdrawn, it allows fluid to flow due to positive pressure. [0069] The pump 113 can act to control one or more of flow direction, flow rate, flow volume and/or wave front of the flow. Such control can advantageously be continuous and/or reversible. In some embodiments, the pump 113 acts to draw the flow away from the reagent container lOla-d and optionally or preferably into a waste container 115. The pump 113 can also act to push the flow back towards the reagent container lOla-d. In some embodiments, the pump 113 may act to agitate the flow in a forward and backward manner, thereby, e.g. , mixing the reagents to improve reaction dynamics and/or improving washing efficiency within the flow cell 107.

[0070] As shown in FIG. 4, the pump 113 is optionally or preferably positioned downstream of the flow cell 107. In some embodiments, use of a peristaltic positive displacement pump can advantageously provide continuous regulated flow with enough pressure on the exit to push waste to a distant waste container 115. Also advantageously, positioning the pump 113 downstream of the flow cell 107 alleviates pump effects on the reagents. In accordance with some embodiments, the pump 113 provides control over both flow rate and flow directionality by being operable bi-directionally (i.e. , forward or backward) and at variable speeds. In certain embodiments, flow rates produced within the system 100 by the pump 113 and the

pressurization of the reagent reservoirs lOla-d can depend on a volume of the flow cell 107. For example, in a 10 mL flow cell, reagents can flow between 1 and 200 mL/min, although it will be apparent in view of this disclosure that any flow rate achievable by the system 100 and consistent with specific procedure requirements can be used in accordance with various embodiments.

[0071] In some embodiments, the pump 113 can be positioned between the rotary valve and the flow cell, with a dedicated unit for both top and bottom ports. The pump 113 can also be positioned between the reagent delivery line and the rotary valve.

[0072] The waste container 115 can include, for example, a beaker, flask, bottle, tank, vial, keg, a ventilated waste closet, any other suitable container, or combinations thereof. Due to the small volume of reagents required for reactions, the waste container 115 can be placed on-site and close to the flow cell 107. Where large volumes of unpleasant chemical waste are produced, the waste should generally be contained in a remote, dedicated waste container 115. In particular, the waste should optionally or most preferably be stored in a remote, well- ventilated waste closet. Therefore, the ability of the pump 113 to drive waste through extended waste tubes to the waste container 115 advantageously improves operator health and safety during the synthesis process. [0073] Safety valves 117, 119 can be any suitable safety valve, many of which are widely known in the art, suitable for preventing overpressure within the system. In particular, as shown in FIG. 4, a first safety valve 117 is configured to release overpressure caused by the pump 113 and a second safety valve 119 is positioned to release overpressure caused by compressed gas exiting the gas source 123.

[0074] The configuration of the systems disclosed herein advantageously ensures uniform coverage of the synthesis substrate with reagent inside the flow cell. It should be noted that in flow cell systems, flow control has added importance. Some processes, such as liquid to liquid transfer, can occur at high flow, saving production time. Other processes, such as flow cell draining, require a very slow flow to minimize residual liquid on the reaction surface. To address these varied needs, the flow control and multiple directionality provided by the systems described herein enable a wide range of flow through the same overall flow path. Additionally, the configuration of the system disclosed herein permits the pump to deliver accurate flow over a range of fluid paths. For example, by using a downstream positive displacement pump combined with a waste tube connecting the pump to the waste container, the system disclosed herein is able to move fluids both upstream and downstream (i.e. , towards and away from the reagent containers). Thus, flow can be maintained over the flow cell reaction surface or substrate with a limited volume (e.g. , microliters, milliliters) of reagents by reversing the pump direction periodically. Reversing the pump can also push the reagent back towards the reagent reservoirs thus enabling full valves and lines to be purged with gas from the gas source and/or wash solution during or after one or more reaction steps. This way, various system components are protected from degradation by corrosive or sedimenting reagents. Sill further

advantageously, the fluids can be agitated in a forward and backward manner in order to drive reaction kinetics and/or dislodge gas bubbles.

[0075] Another advantage provided by the systems disclosed herein is the capability to precisely control the position of the wavefront of the fluid in the flow cell. As a result, it is possible to predetermine or preselect a particular area on the substrate (e.g. , a third or a half of the substrate or the features thereon) where one or more of the reactions should take place. This way, different zones or features on the same substrate can be designed to have different reactions or products.

Use of the Fluid Handling Systems [0076] The fluidic or fluid handling systems described herein can be used in various biological and/or chemical reagent delivery and/or reaction applications. The systems also have unique capability to move reagent or product from one flow cell to another where a different process may occur within each flow cell such as reactions with different solid supported reagents.

[0077] One example is the highly parallel production of nucleic acid oligonucleotides and genes where oligonucleotide growth reactions occur on the surface of the flow cell or flow cell features. The present disclosure is application to any synthesis chemistry, the reagents being selected, of course, to fit the desired scheme. One example is the conventional beta

cyanoethylphosphoramidite reaction, which uses, for example, iodine for base oxidation and dichloroacetic acid for detritylation. Further detail of the phosphor amidite reaction scheme can be found in Giles and Morrison, "An Economical System for Automated DNA Synthesis", ABL, Vol. 5, p. 16-25 (March/April 1987).

[0078] Typically, oligonucleotide synthesis involves a number of chemical steps that are performed in a cyclic repetitive manner throughout the synthesis with each cycle adding one nucleotide to the growing oligonucleotide chain. The chemical steps involved in a cycle are a deprotection step that liberates a functional group for further chain elongation, a coupling step that incorporates a nucleotide into the oligonucleotide to be synthesized, and other steps as required by the particular chemistry used in the oligonucleotide synthesis, such as an oxidation step required with the phosphor amidite chemistry. Optionally, a capping step that blocks those functional groups which were not elongated in the coupling step can be inserted in the cycle. The nucleotide can be added to the 5'-hydroxyl group of the terminal nucleotide, in the case in which the oligonucleotide synthesis is conducted in a 3 '→5' direction or at the 3 '-hydroxyl group of the terminal nucleotide in the case in which the oligonucleotide synthesis is conducted in a 5'→3' direction.

[0079] Oligonucleotides may be synthesized on solid support using methods known in the art. In some embodiments, pluralities of different single-stranded oligonucleotides are immobilized at different features of a solid support. In some embodiments, the support-bound

oligonucleotides may be attached through their 5' end or their 3 ' end. In some embodiments, the support-bound oligonucleotides may be immobilized on the support via a linker (e.g. , photocleavable linker or chemical linker). It should be appreciated that by 3 ' end, it is meant the sequence downstream to the 5' end and by 5' end it is meant the sequence upstream to the 3 ' end. For example, an oligonucleotide may be immobilized on the support via a nucleotide sequence or linker that is not involved in subsequent reactions. [0080] Certain embodiments of the present disclosure may make use of a solid support comprised of an inert substrate and a porous reaction layer. The porous reaction layer can provide a chemical functionality for the immobilization of pre-synthesized oligonucleotides or for the synthesis of oligonucleotides. In some embodiments, the surface of the array can be treated or coated with a material comprising suitable reactive group for the immobilization or covalent attachment of nucleic acids. Any material known in the art and having suitable reactive groups for the immobilization or in situ synthesis of oligonucleotides can be used. In some embodiments, the porous reaction layer can be treated so as to comprise hydroxyl reactive groups. For example, the porous reaction layer can comprise sucrose.

[0081] According to some aspects of the disclosure, oligonucleotides having terminal 3 ' phosphoryl group oligonucleotides can be synthesized in a 3 '→5' direction on a solid support having a chemical phosphorylation reagent attached to the solid support. In some embodiments, the phosphorylation reagent can be coupled to the porous layer before synthesis of the oligonucleotides. In an exemplary embodiment, the phosphorylation reagent can be coupled to the sucrose. For example, the phosphorylation reagent can be 2-[2-(4,4'-

Dimethoxytrityloxy)ethylsulfonyl]ethyl-(2-cyanoethyl)-(N, N-diisopropyl)-phosphoramidite. In some embodiments, the 3' phosphorylated oligonucleotide can be released from the solid support and undergo subsequent modifications according to the methods described herein. In some embodiments, the 3 ' phosphorylated oligonucleotide can be released from the solid support using ammonium hydroxide.

[0082] In some embodiments, oligonucleotides are synthesized (e.g. , on an array format) as described in U.S. Patent No. 7,563,600, U.S. Patent Application No. 13/592,827, and

International Patent Application No. WO 2014/004393, which are hereby incorporated by reference in their entireties. For example, single-stranded oligonucleotides are synthesized in situ on a common support wherein each oligonucleotide is synthesized on a separate or discrete feature (or spot) on the substrate. In some embodiments, single- stranded oligonucleotides are bound to the surface of the support or feature. As used herein, the term "array" refers to an arrangement of discrete features for storing, routing, amplifying and releasing oligonucleotides or complementary oligonucleotides for further reactions. The array can be planar. In an embodiment, the support or array is addressable: the support includes two or more discrete addressable features at a particular predetermined location (i.e. , an "address") on the support. Therefore, each oligonucleotide molecule of the array is localized to a known and defined location on the support. The sequence of each oligonucleotide can be determined from its position on the support. Moreover, addressable supports or arrays enable the direct control of individual isolated volumes such as droplets. The size of the defined feature can be chosen to allow formation of a microvolume droplet on the feature, each droplet being kept separate from each other. As described herein, features are typically, but need not be, separated by interfeature spaces to ensure that droplets between two adjacent features do not merge. Interfeatures will typically not carry any oligonucleotide on their surface and will correspond to inert space. In some embodiments, features and interfeatures may differ in their hydrophilicity or

hydrophobicity properties.

[0083] In one embodiment, a solid substrate (e.g. , glass or silicon) can be surface treated to have a linker attached, which acts as an anchor for oligonucleotide synthesis. Oligonucleotide synthesis can then be carried out by stepwise addition of nucleotide residues to the 5 '-terminus of the growing chain until the desired sequence is produced. Each addition includes 4 steps: deprotection— coupling— capping— oxidation.

[0084] The deprotection, capping and oxidation steps can take place in bulk reactions in a flow cell where the a predetermined portion or the entire surface of the substrate is flooded with the same reagents. Advantageously, because the system disclosed herein provides the capability to precisely control the position of the wavefront of the fluid in the flow cell, it is possible to predetermine a particular area on the substrate (e.g. , a third or a half of the substrate or the features thereon) where one or more of the reaction steps should take place. This way, different zones or features on the same substrate can have different reactions or products.

[0085] The coupling step can take place in microvolumes where droplets containing the desired phosphor amidite nucleosides are precisely deposited onto preselected feature locations by inkjet from a print head array.

[0086] As shown in FIG. 5, the print head array and the flow cell can be placed in two work areas separated by a partition wall. The substrate is placed on a movable station and is shuffled back and forth between the print head array work area and the flow cell work area as needed. A visualization system can be used to monitor the inkjet process.

[0087] A system controller can be used to control the inkjet, motion of print head array, motion of substrate, and/or retrieving/handling of reagents in system 100 such as the operation of one or more of the gas source 123, reagent container lOla-d, rotary valve 105a-b, or the pump 113. The system controller can be a collection of circuitry modules, and may be implemented utilizing any combination of dedicated hardware boards, digital signaling processors (DSPs), processors, etc. Alternatively, the system controller may be implemented utilizing an off-the- shelf PC with a single processor or multiple processors, with the functional operations distributed between the processors. As a further option, the circuitry modules described below may be implemented utilizing a hybrid configuration in which certain modular functions are performed utilizing dedicated hardware, while the remaining modular functions are performed utilizing an off-the-shelf PC and the like.

[0088] The system controller and/or the circuitry modules may include one or more logic- based devices, including one or more microcontrollers, processors, reduced instruction set computers (RISC), application specific integrated circuits (ASICs), field programmable gate array (FPGAs), logic circuits, and any other circuitry capable of executing functions described herein. In an exemplary embodiment, the system controller and/or the circuitry modules execute a set of instructions that are stored therein in order to perform one or more assay protocols. Storage elements may be in the form of information sources or physical memory elements.

[0089] The set of instructions may include various commands that instruct the system 100 to perform specific operations such as the methods and processes of the various embodiments described herein. The set of instructions may be in the form of a software program. As used herein, the terms "software" and "firmware" are interchangeable, and include any computer program stored in memory for execution by a computer, including RAM memory, ROM memory, EPROM memory, EEPROM memory, and non-volatile RAM (NVRAM) memory. The above memory types are exemplary only, and are thus not limiting as to the types of memory usable for storage of a computer program.

[0090] The software may be in various forms such as system software or application software. Further, the software may be in the form of a collection of separate programs, or a program module within a larger program or a portion of a program module. The software also may include modular programming in the form of object-oriented programming.

[0091] The system controller may be connected to the other components or subsystems of the system 100 via communication links, which may be hardwired or wireless. The system controller may also be communicatively connected to off-site systems or servers. The system controller may receive user inputs or commands, from a user interface. The user interface may include a keyboard, mouse, a touch-screen panel, a mobile device and/or a voice recognition system, and the like. [0092] The system controller may serve to provide processing capabilities, such as storing, interpreting, and/or executing software instructions, as well as controlling the overall operation of the system 100.

[0093] Various aspects of the present disclosure may be used alone, in combination, or in a variety of arrangements not specifically discussed in the embodiments described in the foregoing and is therefore not limited in its application to the details and arrangement of components set forth in the foregoing description or illustrated in the drawings. For example, aspects described in one embodiment may be combined in any manner with aspects described in other embodiments. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

[0094] Use of ordinal terms such as "first," "second," "third," etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for the use of the ordinal term) to distinguish the claim elements.

INCORPORATION BY REFERENCE

[0095] All publications, patents and sequence database entries mentioned herein are hereby incorporated by reference in their entireties as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

[0096] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.