DE202019106695U1 - System for sequencing nucleic acids in fluid foam - Google Patents

Abstract

A flow cell comprising a ternary complex immobilized within a flow cell channel, the ternary complex comprising a polymerase, a primed template nucleic acid and a next correct nucleotide for the template; anda fluid foam in contact with the ternary complex in the flow cell channel, the fluid foam having a plurality of gas bubbles in a liquid, the fluid foam having a volume fraction of bubbles which is at least 25% of the total volume of the fluid foam in the flow cell channel, and wherein the average effective diameter of the gas bubbles is at most 95% of the diameter of the flow cell channel.

Description

This application is based on and takes advantage of the preliminary U.S. Application No. 62 / 930,688 , filed on November 5, 2019, the provisional U.S. Application No. 62 / 883,276 , filed on August 6, 2019, and preliminary U.S. Application No. 62 / 774,998 , filed December 4, 2018, which are hereby incorporated by reference.

BACKGROUND

The present disclosure relates generally to molecular assays and has specific applicability to nucleic acid sequencing methods.

An exact sequence determination of a template nucleic acid strand is important for molecular diagnostics. The identification of a single nucleotide base among alternatives at a known position can serve as the basis for the analysis of single nucleotide polymorphisms (i.e. "SNPs"). An SNP, in turn, can be used to determine a phenotype for the individual, such as susceptibility to disease or the propensity to have a desired trait. Evidence of genetic variations in a patient can indicate the efficacy of certain medications for treating the patient or the risk of side effects when treating the patient with certain medications.

Commercially available nucleic acid sequencing platforms have significantly expanded our knowledge of the genetic basis of implementable traits. The improvements in the sequencing of biochemistry and detection hardware are progressing. However, the cost of the sequencing platforms currently available has hampered sequencing in the clinic despite widespread use in research laboratories. In addition, sequencing platforms are relatively slow to deliver a diagnostic or prognostic response within a timeframe that meets patient and physician expectations. The present disclosure provides fluidic systems and methods that reduce sequencing time, reduce sequencing costs, reduce reagent volume, and also offer related advantages. The systems and methods of the present disclosure can be used for chemical and biological assays beyond nucleic acid sequencing.

SHORT SUMMARY

The present disclosure provides a nucleic acid sequencing system that includes a table, a liquid delivery component, a gas delivery component, a bubble generator component, and an optical detection component, the table configured to receive a flow cell with a detection channel, the optical detection component configured to do this Detect interior of the detection channel, wherein the liquid delivery component is configured to deliver liquid from a plurality of containers / reservoirs to the detection channel, the gas delivery component is configured to deliver gas from one or more sources to the bubble generator component, and the bubble generator component is configured to introduce bubbles from the gas delivery component into liquid from the liquid delivery component at a location that is between the plurality of reservoirs and the flow cell, thereby e feeds a fluid foam to the detection channel, which contains gas bubbles from the gas delivery component.

A flow cell is also provided which contains a ternary complex immobilized within a flow cell channel, the ternary complex containing a polymerase, a primed template nucleic acid and a next correct nucleotide for the template; and
contains a fluid foam that is in contact with the ternary complex in the flow cell channel, the fluid foam containing a plurality of gas bubbles in a liquid, the fluid foam having a volume fraction of bubbles that makes up at least 25% of the total volume of the fluid foam in the flow cell channel, and wherein the average effective diameter of the gas bubbles is at most 95% of the diameter of the flow cell channel.

Figure list

• 1 shows a flow cell with two detection channels, each of the detection channels being fluidly connected to a gas mixing component, the gas mixing component using a T-transition for connecting a liquid channel and a gas channel.
• 2nd shows a flow cell with two detection channels, each of the detection channels being fluidly connected to a gas mixing component, the gas mixing component using a Y transition for connecting a liquid channel and a gas channel.
• 3rd shows a bubble generator with a T-transition and a threaded coupling for a gas line.
• 4th shows a bubble generator with a T-transition and threaded couplings for a gas line and a liquid line.
• 5 shows a bubble generator with a Y junction and a hydrophobic filter membrane, which acts as a gas resistor at the junction.
• 6 shows a fluid circuit for dispensing a fluid foam to a flow cell.
• 7 shows graphs of signal intensity versus sequencing cycle for sequencing runs where the delivery of reagents in liquid ( 7A ) or dispensing reagents in fluid foam ( 7B ) has been used.
• 8th shows a schematic representation of functional components of a nucleic acid sequencing system.
• 9 shows a perspective view of an arrangement of several components of a nucleic acid sequencing system.
• 10th shows a plan view of a distributor which is fluidly connected to sipper arrays.
• 11 shows a bottom view of a manifold which is engaged with rotary valves.
• 12A shows a perspective view of the fluidic connection between a nucleic acid sequencing system and a flow cell; 12B shows the same perspective, but with separate connectors.
• 13 shows the connection between a flow cell and the fluid delivery component of a nucleic acid sequencing system.
• 14A and 14B show exploded views of fluid connectors that contain a bubble generator.
• 15A shows a bubble generator; 15B shows an exploded view of the bubble generator; 15C shows the inside of part of the bubble generator and 15D shows the other part of the bubble generator.

DETAILED DESCRIPTION

Bubbles are known to adversely affect molecular analyzes such as nucleic acid sequencing processes and protein activity assays. Accordingly, avoiding or removing bubbles has previously been a design goal and user requirement for methods and devices used in molecular analysis. Analytical methods that are typically configured to avoid bubbles include, for example, those in which a protein is contacted with analytes of interest under conditions in which the proteins bind to the analytes or in which the proteins catalyze a change in the analytes . The binding or catalysis can generate a signal or other detectable event that indicates the presence, amount, composition, function, or other property of the protein and / or analyte. The analytical methods are typically carried out in liquids which are formulated in such a way that the stability of the reaction components, in particular of the proteins, is retained. Bubbles are believed to damage proteins due to surface denaturation at the gas-liquid interface. See, for example, Clarkson et al., J Colloid Interface Sci. 215 (2): 323-332 (1999), which is hereby expressly incorporated by reference. As a result, bubbles are avoided in molecular analysis, especially in those that use proteins.

Bubbles can also cause interference to the detection devices used in many molecular analyzes such as nucleic acid sequencing processes and protein activity assays. For example, a bubble that enters the beam path of a luminescence detector scatters the light that would otherwise be detected. Many sequencing processes and other analytical assays / tests use solid phase substrates. In these assays, liquid reagents interact with analytes on a surface to produce a detectable product or signal. However, bubbles adhering to the surface can scatter optical signals and, at least temporarily, prevent the liquid reagents from coming into contact with the analytes and can permanently damage the analytes by drying them out.

Bubbles are routinely avoided in molecular analysis such as nucleic acid sequencing processes and protein activity assays because it is believed that problems such as those mentioned above will occur. The present disclosure provides systems that use bubbles for good use in nucleic acid sequencing processes and other analytical methods. Surprisingly, it has been found that bubbles can be introduced into a liquid stream to create a fluid foam that is in turn able to participate in one or more steps of a nucleic acid sequencing reaction. The bubbles can be introduced into the liquid stream under controlled conditions to achieve a variety of desired effects and to avoid undesirable results. For example, a fluid foam can be used to wash a solid support on which a sequencing reaction is taking place, for example, where a previously supplied solution is removed relatively efficiently and replaced with a new solution.

In addition, the flow of a fluid foam through a channel has a different profile than a liquid laminar flow. A laminar flow has a parabolic velocity profile (faster in the middle, slower on the outside). In a flow of fluid foam, the bubbles can be kept in step, for example, by suitable selection of the flow rate and flow together. This helps swap one fluid for another, as the foam maintains a flat front regardless of how far it has spread through the system. Reduced diffusion between proximally flowing fluids and reduced speed shear can help maintain each fluid reagent block as it propagates through a flow cell or other fluid channel. Together, these two effects can reduce the amount of time it takes to reach a certain level of fluid exchange compared to the time it takes for a non-foam fluid to achieve the same level of exchange for the same fluid reagents.

In addition, bubbles can facilitate mixing in a solution to increase the efficiency of reagent transfer between a fluid foam and the surface of a solid support. Rapid convective diffusion near bubbles can increase the rate of reaction of diffusion-limited kinetics near the surface by replenishing (or completely destroying) the depletion area. Another advantage of using bubbles is a reduction in reagent costs due to a reduction in the volume or amount of fluid used. In particular, because gases used to generate bubbles are cheaper than many reagents used for nucleic acid sequencing, and because a relatively large volume of fluid may be required to perform a particular step / steps in a sequencing reaction, this can Adding bubbles to the fluid act as an inert filler to reduce the amount of reagent fluid consumed to effectively perform the particular step (s). The controlled delivery and removal of bubbles, as set forth herein, can allow bubbles to be removed from a flow cell if desired, for example to facilitate optical detection of the interior of the flow cell.

Bubbles can be used for improved heat regulation, for example when solutions are pre-equilibrated to the temperature of a flow cell or other fluid channel. For example, the temperature of a liquid reagent can be raised or lowered by introducing a gas that is heated or cooled. The bubbles in the resulting foam provide a large contact area with the bulk liquid phase and this can allow for a quick and efficient change in the temperature of the reagents in the bulk liquid.

A fluid foam can also be used to remove other bubbles, such as surface bubbles that are otherwise difficult to remove from a surface. In some situations, foams can remove surface bubbles more efficiently than homogeneous liquids, which are similar to the bulk phase of the foams. Bubbles can also be a useful visual aid in determining flow rates in a flow cell.

It is understood that the terms used herein, unless otherwise stated, have their ordinary meaning in the relevant art. Some of the terms used here and their meanings are listed below.

As used herein, the term "array" refers to a population of molecules attached to one or more solid supports so that the molecules in one location can be distinguished from molecules in other locations. An array can contain different molecules, each of which is located on a solid support at different addressable locations. Alternatively, an array may contain separate solid supports, each functioning as a site carrying another molecule, with the different molecules according to the positions of the solid supports on a surface to which the solid supports are attached, or according to the positions of the solid Carriers in a liquid such as a fluid stream can be identified. The molecules of the array can be, for example, nucleotides, nucleic acid primers, nucleic acid matrices, primed nucleic acid matrices or nucleic acid enzymes such as polymerases, ligases, exonucleases or combinations thereof.

As used here, the term "attached / bound" refers to the state of two things that are connected, attached, glued, or tied together. For example, a reaction component, such as a primed template nucleic acid or a polymerase, can be attached / bound to a solid phase component by a covalent or non-covalent bond. A covalent bond is characterized by the sharing of electron pairs between atoms. A non-covalent bond is a chemical bond in which no electron pairs are shared. These include, for example, hydrogen bridges, ion bridges, Van der Waals forces, hydrophilic interactions and hydrophobic interactions.

As used herein, the term "blocking moiety" when used with respect to a nucleotide refers to a portion of the nucleotide that prevents the 3'-oxygen of the nucleotide from covalently binding to a next correct / correct nucleotide during a nucleic acid polymerization reaction, or prevented. The blocking unit of a "reversible terminator" nucleotide can be removed from the nucleotide analog or otherwise modified to allow the 3 'oxygen of the nucleotide to covalently bind to a next correct nucleotide. This process is known as "deblocking" the nucleotide analog. Such a blocking unit is referred to here as a “reversible terminator unit”. Exemplary reversible terminator units are in the U.S. Patent No. 7,427,673 ; 7,414,116 ; 7,057,026 ; 7,544,794 or 8,034,923 ; or PCT publications WO 91/06678 or WO 07/123744 each of which is incorporated herein by reference. A nucleotide that has a blocking moiety or a reversible terminator moiety can be at the 3 'end of a nucleic acid such as a primer, or it can be a monomer that is not covalently linked to a nucleic acid.

The term "bubble" as used here refers to a gas ball in a liquid or solid. A bubble can be observed in a fluid because the gas has a different refractive index than the surrounding liquid. A bubble that is completely surrounded by liquid is called a "bulk bubble". A bubble that adheres to a solid phase surface is called a "surface bubble".

As used herein, the term "catalytic metal ion" refers to a metal ion that facilitates the formation of phosphodiester bonds between the 3'-oxygen of a nucleic acid (e.g., a primer) and the phosphate of an incoming nucleotide by a polymerase. A "divalent catalytic metal cation" is a catalytic metal ion with a valence of two. Catalytic metal ions can be present in concentrations that stabilize the formation of a complex between a polymerase, a nucleotide and a primed template nucleic acid, which are referred to as non-catalytic concentrations of a metal ion, unless a phosphodiester bond is formed. The catalytic concentrations of a metal ion refer to the amount of a metal ion sufficient for polymerases to catalyze the reaction between the 3'-oxygen of a nucleic acid (e.g. a primer) and the phosphate unit of an incoming nucleotide. Exemplary catalytic metal ions include Mg ²⁺ and Mn ²⁺ .

The term “comprising / having” should be open here and should not only include the elements mentioned, but also include any additional elements.

As used herein, the term "everyone" when used in relation to a collection of items is intended to identify a single item in the collection but not necessarily on every item in the collection. Exceptions can occur if the explicit disclosure or context clearly dictates otherwise.

As used herein, "balance" refers to a state of equilibrium due to the same effect of opposing forces. For example, a ternary complex formed between a primed template nucleic acid, polymerase and a related nucleotide is in equilibrium with unbound polymerase and unbound nucleotide when the rate of formation of the ternary complex is balanced by the rate of dissociation. Under this condition, the reversible binding reaction stops changing its net ratio of products (e.g. ternary complex) to reactants (e.g. polymerase, nucleotide and nucleic acid). If the rate of a forward reaction (e.g. formation of a ternary complex) is balanced by the rate of a reverse reaction (e.g. dissociation of the ternary complex), there is no net change in the ratio of products to reactants.

As used herein, the term "exogenous" when used in relation to a unit of a molecule refers to a chemical unit that is not present in a natural analogue of the molecule. For example, an exogenous label of a nucleotide is a label that is not present on a naturally occurring nucleotide. Similarly, an exogenous label present on a polymerase is not found on the polymerase in its original milieu.

As used herein, the term "extension" when used in relation to a nucleic acid refers to a process of adding at least one nucleotide to the 3 'end of the nucleic acid. The term "polymerase extension" when used in relation to a nucleic acid refers to a polymerase-catalyzed process of adding at least one nucleotide to the 3 'end of the nucleic acid. A nucleotide or oligonucleotide that is added to a nucleic acid by extension is said to be incorporated into the nucleic acid. Accordingly, the term "incorporation" can be used to refer to the act of connecting a nucleotide or oligonucleotide to the 3 'end of a nucleic acid by forming a phosphodiester bond.

As used herein, the term "extendable" when used with respect to a nucleotide means that the nucleotide has an oxygen or hydroxyl group at the 3 'position and is capable of covalently linking to a next correct nucleotide to form if and when it is incorporated into a nucleic acid. An extendable nucleotide can be at the 3 'position of a primer or it can be a monomeric nucleotide. A nucleotide that can be extended lacks blocking units, such as reversible terminator units.

As used herein, a "flow cell" is a reaction chamber that includes one or more channels that direct fluid to a detection zone. The detection zone can be functionally coupled to a detector, so that a reaction taking place in the reaction chamber can be observed. For example, a flow cell can contain primed template nucleic acid molecules that are bound to a surface to which nucleotides and auxiliary reagents are iteratively applied and washed off. The flow cell may contain a transparent material that allows imaging of the sample after a desired reaction has occurred. For example, a flow cell may contain a glass or plastic support that contains detection channels through which polymerases, dNTPs and buffers can be pumped. The glass or plastic within the channels can be decorated with one or more primed template nucleic acid molecules to be sequenced. An external imaging system can be positioned to detect the molecules in a detection zone in the detection channel or on a surface in the detection channel. Exemplary flow cells, methods of making them, and methods of using them are described in U.S. Patent Application Publication No. 2010/0111768 A1 or 2012/0270305 AI; or in WO 05/065814 each of which is incorporated herein by reference.

As used herein, the term "fluid" refers to a liquid or gas that is flowable and that changes shape to fill a vessel. In many conditions, a fluid changes shape at a steady rate when it is subjected to a force that tends to change shape.

The term "fluid foam" as used herein refers to a liquid that contains gas bubbles. The liquid acts as a dispersion phase and the bubbles act as a dispersed phase. Exemplary dispersion phase liquids include those that contain reagents or products of a reaction, such as a binding reaction, a nucleic acid sequencing reaction, or one Reaction used in an analytical assay. Aqueous liquids are a particularly useful dispersion phase. Exemplary gases include inert gases such as nitrogen (N ₂ ) or noble gases. Useful noble gases include, for example, helium (He), neon (Ne), argon (Ar), krypton (Kr) and xenon (Xe). Another useful gas is atmospheric air from planet Earth. A fluid foam may include bulk bubbles (ie bubbles surrounded by liquid) and / or surface bubbles (ie bubbles that contact a surface of a solid support such as a surface of a flow cell). In some configurations, a fluid foam can be substantially free of bulk bubbles and / or surface bubbles. A fluid micro foam has bubbles with an average diameter of 1 micrometer or less, whereas a fluid macro foam has bubbles with an average diameter of more than 1 micrometer.

As used herein, the term "fluidly coupled," when used in relation to two things, means that a fluid or a solute in the fluid is able to transition from one thing to another. For example, a reservoir can be fluidly coupled to a flow cell via a pipe through which fluid can flow. In another example, two locations in an array are fluidly coupled when the array is in a liquid so that a solute can diffuse from one location to another.

As used herein, the terms "free" or "unbound" when used with respect to components that can complex in a binding reaction refer to components that are not in a bound state. For example, an equilibrium binding reaction can include a product (e.g., a ternary complex) and reactants that are not bound in the product (e.g., free polymerases, free nucleic acids, or free nucleotides).

As used herein, the term "inhibitory metal ion" refers to a metal ion that, when present in the presence of a polymerase enzyme, inhibits the formation of phosphodiester bonds necessary for the chemical incorporation of a nucleotide into a primer. An inhibitory metal ion can, for example, interact with a polymerase via competitive binding in comparison to catalytic metal ions. A "divalent inhibitory metal ion" is an inhibitory metal ion with a valence of two. Examples of divalent inhibitory metal ions include, but are not limited to, Ca ²⁺ , Zn ²⁺ , Co ²⁺ , Ni ²⁺ , and Sr ²⁺ . The trivalent Eu ³⁺ and Tb ³⁺ ions are inhibitory metal ions with a valence of three.

As used herein, the term "immobilized" when used in relation to a molecule refers to a direct or indirect, covalent or non-covalent bond of the molecule to a solid support. Covalent bonding may be preferred in some configurations, but generally all that is required is that the molecules (e.g., nucleic acids) remain immobilized or bound to the support under the conditions under which the support is to be used, e.g. B. in applications where the immobilization of nucleic acid or polymerase is used at or near a sensor.

As used herein, the term "label" refers to a molecule or entity thereof that provides a detectable property. The detectable property can be, for example, an optical signal, such as absorption of radiation, luminescence or fluorescence emission, luminescence or fluorescence life, luminescence or fluorescence polarization or the like; Rayleigh and / or Mie scattering; Binding affinity for a ligand or receptor; magnetic properties; Electrical Properties; Charge; Dimensions; Radioactivity or the like. Exemplary labels include, without limitation, a fluorophore, a luminophore, a chromophore, a nanoparticle (e.g. gold, silver, carbon nanotubes), a heavy atom, a radioactive isotope, a mass label, a charge label, a spin label, a receptor, a ligand or similar.

As used herein, the term "mixed phase" when used in relation to a fluid means a liquid that contains a suspension of gas bubbles, liquid spheres, or solid particles that are immiscible with the liquid. Exemplary mixed phase fluids include, but are not limited to, a fluid foam (gas bubbles in liquid), a fluid emulsion (beads of a first liquid that are immiscible with a surrounding second liquid), a fluid slurry (solid particles in liquid). Exemplary liquids include those that contain reagents or products of a reaction, such as a binding reaction, a nucleic acid sequencing reaction, a synthesis reaction, or a reaction used in an analytical assay.

As used herein, the term "next correct / correct nucleotide" refers to the type of nucleotide that binds and / or is incorporated at the 3 'end of a primer to complement a base in a template strand to which the primer hybridizes. The base in the template strand is referred to as the "next base" and lies directly 5 'to the base in the template, which is hybridized with the 3' end of the primer. The next correct nucleotide can be called “related” to the next base and vice versa. Related nucleotides that specifically interact with each other in a ternary complex or in a double-stranded nucleic acid are said to "pair" with each other. A nucleotide with a base that is not complementary to the next template base is called a "wrong", "mismatched" or "unrelated" nucleotide.

As used herein, the term "nucleotide" can be used to refer to a native nucleotide or an analog thereof. Examples include, but are not limited to, nucleotide triphosphates (NTPs), such as ribonucleotide triphosphates (rNTPs), deoxyribonucleotide triphosphates (dNTPs), exogenously labeled nucleotides, or non-natural analogues thereof, such as dideoxyribonucleotide triphosphates (ddNtTPsN), or reversibly.

As used herein, the term "polymerase" can be used to refer to a nucleic acid synthesizing enzyme, including, but not limited to, DNA polymerase, RNA polymerase, reverse transcriptase, primase and transferase. Typically, the polymerase has one or more active sites at which nucleotide binding and / or catalysis of nucleotide polymerization can occur. The polymerase can catalyze the polymerization of nucleotides at the 3 'end of the first strand of the double-stranded nucleic acid molecule. For example, a polymerase catalyzes the addition of a next correct nucleotide to the 3'-oxygen group of the first strand of the double-stranded nucleic acid molecule via a phosphodiester bond, as a result of which the nucleotide is covalently incorporated into the first strand of the double-stranded nucleic acid molecule. A polymerase may not be able to incorporate nucleotides under one or more conditions used in a method set forth herein. For example, a mutant polymerase may be able to form a ternary complex, but may not be able to catalyze the incorporation of nucleotides. The amount of polymerase in a fluid can be quantified in units of activity. For example, one unit of a polymerase can be equal to the amount of enzyme that catalyzes the incorporation of 10 nmol dNTP into DNA in 30 min at a certain temperature. For example, thermostable polymerases can be measured at 75 ° C, whereas thermolabile polymerases can be measured at 37 ° C.

As used herein, the term "predefined" when used in relation to a functional property of a system means that the property is a known, predictable, or expected result of manipulation of the system that is intended to produce the property. For example, the fluidity of a fluid is a known and expected result of introducing bubbles into a liquid using a bubble generator, such as a gas-liquid mixing system. Exemplary predefined properties of a system can optionally include the rate at which a dispersed phase is formed in a bulk phase, the amount of dispersed phase that is introduced into a bulk phase, the relative ratio of dispersed phase and bulk phase that is generated by the system, include the size of dispersed phase elements (e.g., bubbles, particles, or beads) generated by the system or the like.

As used herein, the term "primed template nucleic acid" refers to a nucleic acid hybrid with a double-stranded region so that one of the strands has a 3 'end that can be extended by a polymerase. The two strands can be parts of a contiguous nucleic acid molecule (e.g. a hairpin structure), or the two strands can be separable molecules that are not covalently bound to one another.

As used herein, the term "primer" refers to a nucleic acid with a sequence that binds to a nucleic acid sequence at or near a template sequence. In general, the primer binds in a configuration that enables replication of the template, for example via the polymerase extension of the primer. The primer may be a first part of a nucleic acid molecule that binds to a second part of the nucleic acid molecule, the first part being a primer sequence and the second part being a primer binding sequence (e.g. a hairpin primer). Alternatively, the primer can be a first nucleic acid molecule that binds to a second nucleic acid molecule with the template sequence (e.g. a dissociable primer). A primer can consist of DNA, RNA or analogues thereof. A primer can be blocked at the 3 'end or extendable.

As used herein, the term "location" when used in relation to an array means a location in an array where a particular molecule is present. A site can contain only a single molecule, or it can contain a population of several molecules of the same species (ie an ensemble of molecules). Alternatively, a site may include a population of molecules that are different species (e.g., a population of ternary complexes with different template sequences). The locations of an array are usually discrete. The discrete locations can be contiguous or have spaces. For example, an array useful herein may have locations separated by less than 100 microns, 50 microns, 10 microns, 5 microns, 1 micron, or 0.5 microns. Alternatively or additionally, an array may have locations separated by at least 0.5 microns, 1 microns, 5 microns, 10 microns, 50 microns or 100 microns. The locations can each have an area of less than 1 square millimeter, 500 square micrometers, 100 square micrometers, 25 square micrometers, 1 square micrometer or less. The term “feature” when used in relation to an array is intended to be synonymous with the term “job”.

As used herein, the term "solid support" refers to a rigid substrate that is insoluble in an aqueous liquid. The substrate can be non-porous or porous. The substrate may optionally be able to absorb a liquid (e.g. due to porosity), but will typically be sufficiently rigid that the substrate does not swell significantly when the liquid is absorbed and does not contract significantly when the liquid dries Will get removed. A non-porous solid support is generally impervious to liquids or gases. Exemplary solid supports include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylic, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, Teflon ™, cyclic olefins, polyimides, etc.) , Nylon, ceramics, resins, Zeonor ™, silica or silica based materials including silicon and modified silicon, carbon, metals, inorganic glasses, optical fiber bundles and polymers.

As used herein, the term "substantially free of" means that there is no effective or detectable amount of a particular thing or property. For example, a liquid can be without bubbles, with bubbles that are too small or too few to be observed by a particular system, with bubbles that are too small or too few to be observed in a particular method, or with Bubbles that are too small or too few to have a significant impact on a response (e.g., a binding or sequencing reaction) can be characterized as a liquid that is substantially free of bubbles. In another example, a gas may be without molecular oxygen (e.g., disoxygen (O ₂ ), tri-oxygen or ozone (O ₃ ), or singlet oxygen), with a concentration of molecular oxygen that is undetectable in a system or method described herein , or with a concentration of molecular oxygen that has no significant effect on a reaction (e.g., a binding reaction or a sequencing reaction), can be characterized as a gas that is substantially free of molecular oxygen.

The term "surface" as used herein refers to a portion of a solid support that is in contact with a fluid. The fluid can be a gas or liquid. The surface can be essentially flat or flat. Alternatively, the surface can be rounded or contoured. Exemplary contours that can be contained on a surface are bores, depressions, columns, ribs, channels or the like. Exemplary materials that can be used as a solid support include, but are not limited to, glass such as modified or functionalized glass; Plastic such as acrylic, polystyrene or a copolymer of styrene and another material, polypropylene, polyethylene, polybutylene, polyurethane or Teflon ™; Nylon; Nitrocellulose; Resin; Silica or silica-based materials including silicon and modified silicon; Carbon fiber; Metal; inorganic glass; optical fiber bundle or the like.

As used herein, the term "ternary complex" refers to an intermolecular association between a polymerase, a double-stranded nucleic acid and a nucleotide. Typically, the polymerase facilitates the interaction between a next correct nucleotide and a template strand of the primed nucleic acid. A next correct nucleotide can interact with the template strand via a Watson-Crick hydrogen bond. The term "stabilized ternary complex" means a ternary complex with a promoted or extended lifespan or a ternary complex whose destruction has been inhibited. In general, stabilization of the ternary complex prevents covalent incorporation of the nucleotide component of the ternary complex into the primed nucleic acid component of the ternary complex.

As used herein, the term "type" or "species" is used to identify molecules that have the same chemical structure. For example, a mixture of nucleotides can contain several dCTP molecules. It is understood that the dCTP molecules are the same type (or type) of nucleotides but a different type (or type) of nucleotides compared to dATP, dGTP, dTTP etc. It is also understood that individual DNA molecules with the same sequence are nucleotides of the same type (or type) of DNA, whereas DNA molecules with different sequences are different types (or types) of DNA. The term "type" or "species / species" can also identify entities that have the same chemical structure. For example, it should be understood that the cytosine bases in a template nucleic acid have the same base type (or the same base species) regardless of their position in the template sequence.

The embodiments listed below and listed in the claims can be understood in view of the above definitions.

The present disclosure provides a sequencing system that includes a table, a liquid phase delivery component, a second phase delivery component, and a phase mix component, the liquid phase being immiscible with the second phase, the table configured to have one Receive flow cell, wherein the liquid delivery component is configured to deliver liquid from one or more reservoirs to the phase mixing component, the second phase delivery component is configured to deliver the second phase to the phase mixing component, and wherein the phase mixing component is configured to deliver liquids from mix the liquid delivery component with the second phase to deliver a mixed phase fluid to the interior of the flow cell, the mixed phase fluid containing bubbles of the second phase in the liquid.

For example, a system can be configured as a monolithic device that contains all subsystems or components used for a specific purpose in a single housing. Alternatively, one or more of the subsystems or components described here can be located away from other components or can be easily separated from other components. For example, a nucleic acid sequencing system can include a fluidic component and a detection component that are held together in a single housing, whereas a computer processing unit that is operatively connected to the fluidic component and the detection component can be physically housed in a separate housing. Nonetheless, components that are separated from one another or that are located at a distance from one another can be functionally networked via hardware (e.g. fluid lines, optical fibers or electrical lines) or wireless communication.

A system of the present disclosure may be configured to use a flow cell. The flow cell is a device that can include a detection channel in which an analytical reaction of interest can be observed. The analytical reaction can take place in bulk solution within the flow cell. For example, two solutions can be mixed and the product of the mixture can be observed in the detection channel. Alternatively, an analytical reaction can take place on a solid support within the detection channel. For example, a reagent solution can be flowed over a solid support bound to analytes of interest such as nucleic acids and a resulting reaction can be observed on the solid support. A flow cell enables easy fluid manipulation by guiding solutions through an inlet opening into the detection channel and through an outlet opening from the interior. The detection channel also has an observation area or an observation volume, for example an optically transparent window through which optical signals can be observed, an electrical contact through which electronic signals can be observed, or the like. A particularly useful flow cell has a window that is transparent to excitation and emission radiation used for luminescence detection. Exemplary flow cells that can be used for a system or method set forth herein are e.g. B. in U.S. Patent Application Publication No. 2010/0111768 A1, WO 05/065814 or U.S. Patent Application Publication No. 2012/0270305 A1, each of which is hereby incorporated by reference.

A flow cell or similar device can have one or more detection channels. The detection channel (s) can be closed off from the atmosphere (or another surrounding environment, such as, for example, the local environment that immediately surrounds the flow cell) and can form, for example, a tube or a tunnel within the flow cell structure. The detection channel can have a variety of cross-sectional shapes, including, for example, circular, oval, triangular, square, rectangular, polyhedral, or other closed shapes. The Cross-sectional area of the detection channel can be uniform over its length. For example, a detection channel with a circular cross-sectional area that is uniform over the length of the channel has a cylindrical shape, while a detection channel with a circular cross-sectional area that increases or decreases over the length of the channel will have a conical or funnel shape. The cross-sectional area of a detection channel can be at least about 1 µm ² , 10 µm ² , 100 µm ² , 1 mm ² , 10 mm ² , or 100 mm ² or more. Alternatively or additionally, the cross-sectional area of a detection channel can be at most approximately 100 mm ² , 10 mm ² , 1 mm ² , 100 µm ² , 10 µm ² , 1 µm ² , or less. The volume of a detection channel in a flow cell can be at least about 1 nl, 10 nl, 100 nl, 1 µl, 10 µl, 100 µl, 1 ml or more. Alternatively or additionally, the volume of a detection channel in a flow cell can be at most about 1 ml, 100 μl, 10 μl, 1 μl, 100 nl, 10 nl, 1 nl or less.

A flow cell of the present disclosure may have one or more openings for transferring fluids. In certain configurations, a first opening can function as an inlet for the fluids and a second opening as an outlet for the fluids. Alternatively, a flow cell can have a single opening that functions as both an inlet and an outlet. The fluid can be a liquid, gas or mixed phase fluid. The flow cell may also include an area in which analytes are detected. Fluid can flow into the flow cell through the inlet, then through the area and then out of the outlet to exit the flow cell. For example, the area can be examined or detected through a window in the flow cell. For example, an optical detector can observe an inner region of the flow cell through an optically transparent window of the flow cell. The area of the flow cell can be examined or observed by techniques other than optical techniques, including, for example, the detection techniques set forth herein. Accordingly, the flow cell can have a transmission surface which transmits signals from the area of the flow cell to the corresponding detector device. It goes without saying that a flow cell need not be configured to detect analytes. For example, the flow cell can provide a chamber for a reaction to occur, and a reaction product can flow out of the flow cell for subsequent use or subsequent detection. Accordingly, a flow cell need not have an optically transparent window or other surface that is configured to transmit analytical signals.

In some configurations, a flow cell is a fixed component of a fluid system, for example special tools and / or special training are required to remove it. Alternatively, a flow cell can be a removable component of a fluid system. For example, a system of the present disclosure may include a table configured to conveniently place and remove the flow cell. Thus, the flow cell can be a consumable component that is intended for use in a first analytical test and is then removed to be replaced by a second flow cell that is used for a second analytical test. The two flow cells can be configured similarly to one another, for example contain similar analytes, similar samples or sub-fractions of a specific sample.

A table can be configured to detect a flow cell. The table can be fixed or slidable. For example, a slidable table can be moved linearly relative to a detector in one or more directions defined by a Cartesian coordinate system. For example, a flow cell may be shifted in one or more directions (e.g., to scan a strip of a flow cell along the y dimension), a second direction (e.g., to shift the flow cell along the x dimension, to align them for scanning a second strip of the flow cell) and a third direction (e.g. to bring the flow cell into focus of a detector along the z dimension). Examples of sliding tables are in US Patent Nm. 8,951,781 or 10,227,636, each of which is incorporated herein by reference. Examples of fixed tables that can be useful are in U.S. Patent No. 9,650,669 which is hereby expressly incorporated by reference. A fixed table can be useful when scan detection is not in use or when scanning is accomplished by moving the detector instead of the flow cell. A pinned table may also be configured to provide a reference surface that contacts a flow cell to align it with a detector, the flow cell being moved relative to the reference surface by, for example, moving the flow cell while in contact with it the reference surface. Exemplary systems configured with a reference surface and with mechanisms for moving a flow cell along the reference surface are described in US Patent Application Publication No. 2019/0055598 A1 or U.S. Patent Application No. 62 / 807,934, which are hereby expressly incorporated by reference.

Any of a variety of analytes may be present in a flow cell or other vessel described herein. The analytes can be brought into contact with a mixed phase liquid. The mixed phase fluid can flow in contact with the analytes or be static. Exemplary analytes include, but are not limited to, the analytes exemplified herein or in references cited herein. Particularly useful analytes are involved in nucleic acid sequencing processes. Accordingly, a flow cell may contain one or more nucleic acids, polymerases, polymerase inhibitors, polymerase cofactors (e.g. catalytic metal ions), ternary complex stabilizers (e.g. inhibitory metal ions), nucleotides, nucleic acid binding proteins, nucleotide blocking reagents, or the like.

Other analytes that may be present in a flow cell include, for example, biological tissues, biological cells; Organelles; protein-based enzymes; protein-based receptors such as antibodies, lectins or streptavidin; Peptides; RNA molecules; Aptamers or the like. The content of the flow cell can optionally be in contact with a mixed-phase fluid (e.g. a fluid foam). Exemplary protein-based enzymes that can be used include, but are not limited to, polymerase, transposase, ligase, recombinase, kinase, phosphatase, exonuclease, endonuclease, sulfurylase, apyrase, luciferase, green fluorescent protein (GFP), or phycobiliprotein (e.g. B. Phycocyanin, Allophycocyanin, or Phycoerythrin). It is understood that one or more of the analytes set forth in the present disclosure or known in the field of biological or chemical analysis may avoid contact with a mixed phase fluid in one or more steps of a method set forth herein.

In some aspects, a flow cell or other vessel is provided, the flow cell containing a stabilized ternary complex immobilized within the flow cell, the stabilized ternary complex containing a polymerase, a primed template nucleic acid, and a next correct nucleotide for the template; and contains a mixed phase fluid with multiple gas bubbles in a liquid, the mixed phase fluid being in contact with the stabilized ternary complex.

In some configurations of the systems set forth herein, such as some nucleic acid sequencing systems, a mixed-phase fluid (e.g., fluid foam) is used in some steps that use a stabilized ternary complex, but not in other steps that use a stabilized ternary complex. In an exemplary configuration, a mixed phase fluid may optionally be in contact with components that form a ternary complex or have detached from a ternary complex.

A flow cell or other vessel used in a system of the present disclosure may contain a polymerase. The polymerase may be in contact with a mixed phase fluid (e.g. a fluid foam). A variety of polymerases can be used. Reference to a particular polymerase, such as that exemplified in this disclosure, includes functional variants thereof, unless otherwise specified. Particularly useful functions of a polymerase include the formation of a ternary complex, the extension of a primer to introduce a nucleotide (such as a reversibly terminated nucleotide) or the catalysis of the polymerization of a nucleic acid strand using an existing nucleic acid as a template.

Polymerases can be classified into families identified as A, B, C, D, X, Y, and RT based on structural homology, such as the classification of polymerases. DNA polymerases in family A include, for example, T7 DNA polymerase, eukaryotic mitochondrial DNA polymerase γ, E. coli DNA Pol I, Thermus aquaticus Pol I, and Bacillus stearothermophilus Pol I. DNA polymerases in family B include, for example, eukaryotic DNA Polymerases α, δ, and ε; DNA polymerase ζ; T4 DNA polymerase; Phi29 DNA polymerase; and RB69 bacteriophage DNA polymerase. For example, family C includes the E. coli DNA polymerase III alpha subunit. Family B archaeon DNA polymerases include, for example, Vent, Deep Vent, Pfu, and 9 ° N polymerases (e.g., Therminator ™ DNA polymerase from New England BioLabs Inc .; Ipswich, MA). For example, family D includes polymerases derived from Archaea's Euryarchaeota subdomain. DNA polymerases in family X include, for example, eukaryotic polymerases Pol β, pol σ, Pol λ and Pol µ, and S. cerevisiae Pol4. DNA polymerases in family Y include, for example, Pol η, Pol ɩ, Pol κ, E. coli Pol IV (DINB) and E. coli Pol V (UmuD'2C). The RT family (reverse transcriptase) of DNA polymerases includes, for example, reverse transcriptases from retroviruses and eukaryotic telomerases. Exemplary RNA polymerases include, but are not limited to, viral RNA polymerases such as T7 RNA polymerase; eukaryotic RNA polymerases such as RNA polymerase I, RNA polymerase II, RNA polymerase III, RNA polymerase IV, and RNA polymerase V; and Archaea RNA polymerase.

A polymerase with 3'-5 'intrinsic proofreading exonuclease activity may be useful for some applications of the systems set forth herein. Polymerases that have essentially no 3'-5 'proofreading exonuclease activity are also useful in some configurations, for example in most sequencing systems. The lack of exonuclease activity can be a wild-type property or a property mediated by a variant or a constructed polymerase structure.

Polymerases that can be used in a system set forth herein include naturally occurring polymerases and modified variations thereof, including but not limited to mutants, recombinants, fusions, genetic modifications, chemical modifications, synthetics, and analogs. Useful polymerases for the formation and detection of ternary complexes are not limited to polymerases that have the ability to catalyze a polymerization reaction. Optionally, a useful polymerase can catalyze a polymerization reaction in at least one state that is not used during the formation or study of a stabilized ternary complex. Exemplary polymerases that can be used to form a stabilized ternary complex include, for example, wild-type and mutant polymerases described in U.S. Patent Application Publication Nm. 2017/0314072 A1 or 2018/0155698 A1 or U.S. Patent Application No. 16 / 567,476, which are hereby expressly incorporated by reference.

Polymerases that contain an exogenous labeling component (e.g., an exogenous luminophore) that can be used to detect the polymerase may be useful in some embodiments. Optionally, the exogenous labeling component can be chemically bound to the polymerase. An exogenous labeling component can also be bound to a polymerase via protein fusion. Exemplary labeling components that can be bound via protein fusion include, for example, green fluorescent protein (GFP), phycobiliprotein (e.g. phycocyanin and phycoerythrin) or wavelength-shifted variants of GFP or phycobiliprotein.

Polymerases can be present in a mixed phase fluid (e.g., a fluid foam) in a concentration that is at least about 1 nM, 10 nM, 100 nM, 1 µM, 10 µM, 100 µM or more. Alternatively or additionally, the concentration of polymerases in a mixed phase fluid can be at most about 100 μM, 10 μM, 1 μM, 100 nM, 10 nM, 1 nM or less. The polymerase concentration can be determined based on activity units (U). For example, polymerases can be present in a mixed phase fluid in a concentration that is at least about 5 U / ml, 10 U / ml, 25 U / ml, 50 U / ml, 75 U / ml, 100 U / ml or more. Alternatively or additionally, the concentration of polymerases in a mixed phase liquid can be at most about 100 U / ml, 75 U / ml, 50 U / ml, 25 U / ml, 10 U / ml, 5 U / ml or less.

A flow cell or other vessel used in a system or method of the present disclosure may contain a nucleic acid. The nucleic acid can be in contact with a mixed phase fluid (e.g. a fluid foam). In some configurations, a single nucleic acid molecule is to be manipulated or detected. The nucleic acid molecule can be dispensed into a vessel and optionally bound to a surface in the vessel. In some embodiments, the molecule is subjected to detection under conditions in which individual molecules are separated from one another (e.g. single molecule sequencing). Alternatively, multiple copies of the nucleic acid can be made and the resulting ensemble can be detected or sequenced. For example, the nucleic acid ensemble can be attached to a surface (e.g. on the inner wall of a flow cell).

In multiplex embodiments, several different nucleic acid molecules (i.e. a population with a multitude of different sequences) are manipulated or detected. The molecules can optionally be bound to a surface in a flow cell or other vessel. The nucleic acids can be bound at unique locations on the surface and individual nucleic acid molecules that can be spatially distinguished from one another can be detected or sequenced in parallel. Alternatively, nucleic acid ensembles can be spatially distinguishable from one another and modified, detected or sequenced in parallel.

A flow cell or other vessel can contain any nucleic acid product from a variety of nucleic acid amplification techniques. Mixed phase fluids can be in contact with the nucleic acids. Exemplary techniques that can be used include, but are not limited to, polymerase chain reaction (PCR), rolling circle amplification (RCA), multiple displacement amplification (MDA), bridge amplification, or random prime amplification (RPA). Mixed phase fluid may be in contact with primers, matrices, or other amplification reagents such as those exemplified here or in the references cited herein. In certain embodiments, one or more primers used for amplification can be bound to a surface in a flow cell. In such embodiments, extending the surface-bound primers along the template nucleic acids results in copies of the templates being bound to the surface. Accordingly, one or both strands of a double-stranded nucleic acid can be bound to a surface in a flow cell.

Nucleic acid matrices used in a method or composition herein can be DNA such as genomic DNA, synthetic DNA, amplified DNA, complementary DNA (cDNA), or the like. RNA can also be used, such as mRNA, ribosomal RNA, tRNA or the like. Nucleic acid analogs can also be used as templates herein. Primers used herein can be DNA, RNA, or analogs thereof.

A nucleic acid used in a device herein can be linear, for example flanked by a 3 'end and a 5' end. Alternatively, a nucleic acid can be circular, missing a 3 'and 5' end. Whether linear, circular, or in any other conformation, a nucleic acid used herein can have a size that is desirable for a particular use or is the result of manipulations performed on the nucleic acid. For example, a nucleic acid may have a length that is at least 50 bases, 100 bases, 1 × 10 ³ bases, 1 × 10 ⁴ bases, 1 × 10 ⁵ bases, 1 × 10 ⁶ bases long or longer. Alternatively or additionally, the nucleic acid length can be at most 1 × 10 ⁶ bases, 1 × 10 ⁵ bases, 1 × 10 ⁴ bases, 1 × 10 ³ bases, 100 bases, 50 bases long or shorter. If a population of nucleic acids is used, the average length for the population may have a lower and / or an upper limit selected from these ranges.

A flow cell or other vessel used in a system or method of the present disclosure may contain an array of nucleic acids, proteins, or other analytes. In certain configurations, stabilized ternary complexes are present at one or more locations in an array. The array of analytes can be in contact with a mixed phase fluid (e.g., a fluid foam).

Arrays offer the advantage of multiplex processing of analytes, with the many different types of analytes being manipulated or detected in parallel. An array can contain at least 2, 10, 100, 1 × 10 ³ , 1 × 10 ⁴ , 1 × 10 ⁵ , 1 × 10 ⁶ , 1 × 10 ⁹ , or more different analyte sites. Alternatively or additionally, an array can contain at most 1 × 10 ⁹ , 1 × 10 ⁶ , 1 × 10 ⁵ , 1 × 10 ⁴ , 1 × 10 ³ , 100, 10, 2 or fewer different analyte sites.

An array can be attached to an inner surface of a flow cell wall or to a solid support within a flow cell. The flow cell or solid support can be made from any of a variety of materials used in analytical biochemistry. Suitable materials can include glass, polymer materials, silicon, quartz (quartz glass), boron float glass, silicon dioxide, silicon dioxide-based materials, carbon, metals, an optical fiber or a bundle of optical fibers, sapphire or plastic materials. The material can be selected based on properties that are desired for a particular use. For example, materials that are transparent to a desired wavelength of radiation are useful for analytical techniques that use radiation at that wavelength. Conversely, it may be desirable to select a material that does not transmit radiation of a particular wavelength (e.g., opaque, absorbent, or reflective). Other properties of a material that can be exploited are inertness or reactivity to certain reagents used in a downstream process, such as those given here, or ease of use or low manufacturing costs.

A particularly useful solid support for use in a flow cell or other vessel is a particle such as a bead or microsphere. Populations of beads can be used to attach populations of analytes, such as stabilized ternary complexes or components capable of forming the complexes (e.g. polymerases, matrices, primers or nucleotides). In some configurations, each bead has a single type of stabilized ternary complex or a single type of component that is capable of forming the complex or single type of another analyte, as set forth herein or in references cited herein. For example, a single bead can be attached to a single type of ternary complex, one single type of a template allele, a single type of a template locus, a single type of a primer or a single type of a nucleotide. Alternatively, different types of components do not have to be separated bead by bead. As such, a single bead can carry several different types: ternary complexes, template nucleic acids, primers, primed template nucleic acids and / or nucleotides. The composition of a bead can vary, for example, depending on the format, chemistry and / or type of attachment. Exemplary bead compositions include, for example, plastics, ceramics, glass, polystyrene, melamine, methylstyrene, acrylic polymers, paramagnetic materials, thorium sol, carbon graphite, titanium dioxide, glass with controlled pores (CPG), latex or cross-linked dextrans such as Sepharose ™, cellulose, nylon, cross-linked micelles and Teflon ™ and other materials listed in the "Microsphere Detection Guide" by Bangs Laboratories, Fishers Ind., which is hereby expressly incorporated by reference.

Beads may have a symmetrical shape, such as spherical, polyhedral, cylindrical, or the like. Alternatively, beads can have an irregular or non-symmetrical shape. Exemplary bead sizes used herein can have an average diameter that is at least about 10 nm, 100 nm, 1 µm, 5 µm, 10 µm, 100 µm, 1 mm or larger. Alternatively or additionally, the beads used herein can have an average diameter that is at most about 1 mm, 100 µm, 10 µm, 5 µm, 1 µm, 100 nm, 10 nm, 1 nm or smaller. Beads in these size ranges can be used as array features or as particles in a fluid slurry.

Exemplary compositions and techniques that can be used to make an array of beads include, but are not limited to, those used for BeadChip ™ arrays available from Illumina, Inc. (San Diego, CA) ; which in U.S. Patent Nm. 6,266,459; 6,355,431; 6,770,441; 6,859,570; or 7,622,294; or PCT publication no. WO 00/63437 are described, each of which is hereby incorporated by reference. Beads can be placed in discrete locations, such as wells, on a solid support, each location receiving a single bead. Alternatively, discrete locations where beads are located may each contain a plurality of beads, such as in U.S. Patent Application Publication Nm. 2004/0263923 A1, 2004/0233485 A1, 2004/0132205 A1, or 2004/0125424 A1, each of which is incorporated herein by reference.

Other useful arrays include those used in nucleic acid sequencing applications. For example, arrays used to immobilize amplicons of genomic fragments (often referred to as clusters) can be particularly useful. Examples of nucleic acid sequencing arrays that can be used here include those described in US Pat Bentley et al., Nature 456: 53-59 (2008) , PCT release Nm. WO 91/06678 ; WO 04/018497 or WO 07/123744 ; U.S. Patent Nos. 7,057,026; 7,211,414; 7,315,019; 7,329,492 or 7,405,281; or U.S. Patent Application Publication No. 2008/0108082, each of which is hereby incorporated by reference.

Nucleic acids or other analytes can be bound to an array in such a way that they can be detected at the single molecule or ensemble level. For example, a large number of different nucleic acids can be bound to an array in such a way that a single stabilized ternary complex which forms on a nucleic acid molecule in the array can be distinguished from all adjacent ternary complexes which form in the array. Alternatively, an array of the present disclosure may include a plurality of ensembles, an ensemble being a population of analytes of the same type, such as a population of nucleic acids with a common template sequence. An array may have a plurality of ensembles, each of the ensembles being formed using methods known in the art such as bridge amplification, emulsion PCR, or other methods set forth herein.

A flow cell or other vessel used in a system or method of the present disclosure may contain a nucleotide. The nucleotide may be in contact with a mixed phase fluid (e.g. a fluid foam). The nucleotide can be a native nucleotide, a nucleotide analog or a modified nucleotide, as desired, to suit a particular application or configuration of the methods. Such nucleotides can be in a ternary complex or used in a sequencing process.

Optionally, a nucleotide analog has a nitrogen-containing base, a sugar with five carbon atoms and a phosphate group, each unit of the nucleotide being modified, removed and / or replaced in comparison to a native nucleotide. Nucleotide analogs can be non-insertable nucleotides (ie nucleotides that are unable to react with the 3'-oxygen of a primer to form a covalent bond). Such nucleotides that cannot be incorporated include, for example, monophosphate and diphosphate nucleotides, nucleotides that contain modifications to the triphosphate group that make the nucleotide non-insertable, alpha-phosphate-modified nucleotides, alpha-beta nucleotide Analogs, beta-phosphate-modified nucleotides, beta-gamma-nucleotide analogs, gamma-phosphate-modified nucleotides, or caged nucleotides.

Nucleotide analogs used in a device or system herein can include terminators that reversibly prevent subsequent nucleotide incorporation at the 3 'end of the primer after the analog has been incorporated into the primer. For example, describe US 7,544,794 and US 8,034,923 (the disclosures of these patents are hereby expressly incorporated by reference) reversible terminators in which the 3'-OH group is replaced by a 3'-ONH ₂ unit. Another type of reversible terminator is bound to the nitrogen-containing base of a nucleotide, as described, for example, in US 8,808,989 (the disclosure of which is incorporated herein by reference). Other reversible terminators that may similarly be used in connection with the methods described herein include an azidomethyl group at the 3 'position or those described in the references cited elsewhere herein or in US 7,956,171 , US 8,071,755 and US 9,399,798 (the disclosures of these U.S. patents are incorporated herein by reference). In certain embodiments, a reversible terminator unit can be removed from a primer in a process known as "deblocking", which enables the subsequent incorporation of nucleotides.

Alternatively, nucleotide analogs irreversibly prevent the incorporation of nucleotides at the 3 'end of the primer into which they were incorporated. Irreversible nucleotide analogs include 2 ', 3'-dideoxynucleotides (ddNTPs such as ddGTP, ddATP, ddTTP, ddCTP). Dideoxynucleotides lack the 3'-OH group of dNTPs, which would otherwise be involved in the polymerase-mediated primer extension. Thus the 3 'position has a hydrogen residue instead of the native hydroxyl unit.

In certain embodiments, nucleotide analogs used herein, for example, to participate in stabilized ternary complexes, do not include blocking groups (e.g., reversible terminators).

In some embodiments, a nucleotide used herein, for example, to participate in the formation of a stabilized ternary complex, may contain an exogenous label. An exogenously labeled nucleotide may include a reversible or irreversible terminator unit, an exogenously labeled nucleotide may not be insertable, an exogenously labeled nucleotide may not have terminator units, an exogenously labeled nucleotide may be insertable, or an exogenously labeled nucleotide may be both insertable and not - be terminated. Exogenously labeled nucleotides can be particularly useful when used to form a stabilized ternary complex with an unlabeled polymerase.

Alternatively, a nucleotide used here, for example, to participate in the formation of a ternary complex may not have any exogenous labels (i.e. the nucleotide may be "unlabeled"). An unlabeled nucleotide can include a reversible or irreversible terminator unit, an unlabeled nucleotide can be non-insertable, an unlabeled nucleotide cannot have terminator units, an unlabeled nucleotide can be insertable, or an unlabeled nucleotide can be both installable and non-terminated. It is understood that the lack of a unit or function for a nucleotide refers to the nucleotide that has no such function or unit. However, it is also understood that one or more of the functions or units set forth herein for a nucleotide or an analog thereof, or otherwise known in the art for a nucleotide or an analog thereof, specifically in a method or composition as set forth herein can be omitted.

Optionally, a nucleotide (e.g., a native nucleotide or a nucleotide analog) is present in a mixture during or after the formation of a stabilized ternary complex. For example, there may be at least 1, 2, 3, 4 or more types of nucleotides. Alternatively or additionally, a maximum of 4, 3, 2 or 1 nucleotide types can be present. Similarly, one or more existing nucleotide types can be complementary to at least 1, 2, 3 or 4 base types in a template nucleic acid. Alternatively or additionally, one or more existing nucleotide types can be complementary to at most 4, 3, 2 or 1 base types in a template nucleic acid. Different base types can be created by the Presence of different exogenous labels on the different nucleotides should be identifiable. Alternatively, two or more types of nucleotides may have exogenous labels that are indistinguishable.

Nucleotides can be present in a mixed phase fluid (e.g. a fluid foam) in a concentration which is at least about 1 nM, 10 nM, 100 nM, 1 uM, 10 uM, 100 uM or more. Alternatively or additionally, the concentration of nucleotides in a mixed phase fluid can be at most about 100 μM, 10 μM, 1 μM, 100 nM, 10 nM, 1 nM or less. The previous concentrations may apply to the concentration of a single nucleotide type that is present in the fluid or to the total concentration of two or more nucleotide types that are present in the fluid.

Systems of the present disclosure that use optical detectors can also use optically detectable labels on reactants or products that are to be detected. In many cases, the labels are exogenous labels that are added to a reactant or product, such as a polymerase, a nucleic acid, or a nucleotide. Examples of useful exogenous labels include, but are not limited to, radioactive labeling units, luminophore units, fluorophore units, quantum dot units, chromophore units, enzyme units, electromagnetic spin-labeled units, nanoparticle light scattering units, and any of a variety of other signal-generating units known in the art. Suitable enzyme units include, for example, horseradish peroxidase, alkaline phosphatase, beta-galactosidase or acetylcholinesterase. Exemplary fluorophore labels include, but are not limited to, rhodols; Resorufine; Coumarins; Xanthene; Acridines; Fluoresceins; Rhodamine; Erythrins; Cyanines; Phthalaldehydes; Naphthylamine; Fluorescamine; Benzoxadiazoles; Stilbene; Pyrene; Indoles; Borapolyazaindacene; Quinazolinones; Eosin; Erythrosin; Malachite green; CY dyes (GE Biosciences), including Cy3 (and its derivatives), Cy5 (and its derivatives) and Cy7 (and its derivatives); DYOMICS and DYLIGHT dyes (Dyomics) including DY-547, DY-630, DY-631, DY-632, DY-633, DY-634, DY-635, DY-647, DY-649, DY-652, DY-678, DY-680, DY-682, DY-701, DY-734, DY-752, DY-777 and DY-782; Lucifer yellow; CASCADE BLUE; TEXAS RED; BODIPY (Boron dipyrromethene) (Molecular Probes) dyes including BODIPY 630/650 and BODIPY 650/670; ATTO dyes (Atto-Tec) including ATTO 390, ATTO 425, ATTO 465, ATTO 610 611X, ATTO 610, ATTO 635; ALEXA FLUORS including ALEXA FLUOR 633, ALEXA FLUOR 647, ALEXA FLUOR 660, ALEXA FLUOR 700, ALEXA FLUOR 750, and ALEXA FLUOR 680 (Molecular Probes); DDAO (7-hydroxy-9H- (1,3-dichloro-9,9-dimethylacridin-2-one or any derivative thereof) (Molecular Probes); QUASAR dyes (Biosearch); IRDYES dyes (LiCor) including IRDYE 700DX (NHS ester), IRDYE 800RS (NHS ester) and IRDYE 800CW (NHS ester); EVOBLUE dyes (Evotech Biosystems); JODA 4 dyes (Applied Biosystems); HILYTE dyes (AnaSpec); MR121 and MR200 - Dyes (Roche); Hoechst Dyes 33258 and 33242 (Invitrogen); FAIR OAKS RED (Molecular Devices); SUNNYVALE RED (Molecular Devices); LIGHT CYCLER RED (Roche); EPOCH (Glen Research) dyes including EPOCH REDMOND RED, EPOCH YAKIMA YELLOW, EPOCH GIG HARBOR GREEN; Tokyo Green (M. Kamiya, et al., 2005 Angew. Chem. Int. Ed. 44: 5439-5441); and CF dyes including CF. 647 and CF555 (Biotium), and others known in the art, such as those described in Principles of Fluorescence Spectroscopy, Joseph R. Lakowicz (Editor), Plenum Pub Corp., 2nd Edition (July 1999) and the 6th Edition of Molecular Probes Handbook by Richard P. Hoagland.

In alternative embodiments, a reactant or product may lack exogenous labels. For example, a stabilized ternary complex and all components involved in the stabilized ternary complex (e.g., polymerase, template nucleic acid, primer and / or related nucleotide) may lack one, more, or all of the exogenous labels described herein or in the references cited and incorporated herein. In such embodiments, ternary complexes can be detected based on intrinsic properties of the stabilized ternary complex, such as mass, charge, intrinsic optical properties, or the like. Exemplary detection devices for detecting unlabelled ternary complexes are common U.S. Patent Application No. 2017/0022553 A1 PCT application no. PCT / US16 / 68916 , or U.S. Patent Application Nos. 62 / 375,379 or 15 / 677,870, each of which is incorporated herein by reference.

A system of the present disclosure may use a bubble generator component to supply gas to a liquid, thereby forming a fluid foam. The bubble generator component can be configured to deliver a population of bubbles of a desired size (e.g., measured as diameter or volume) and / or number (e.g., measured as concentration or count).

Bubbles can be of a variety of sizes, including, for example, small nanobubbles with an effective diameter between about 1 nm and 100 nm, large nanobubbles with an effective one Diameters of more than 100 nm and less than 500 nm, small microbubbles with an effective diameter between approximately 0.5 µm and 100 µm, large microbubbles with an effective diameter greater than 100 µm and less than 1 mm. Bubbles with an effective diameter of less than 500 µm, 250 µm, 100 µm, 50 µm, 40 µm, 30 µm, 20 µm, 10 µm, 5 µm or 1 µm can be useful, for example, due to their relative stability in liquids. Bubbles that are even smaller and have, for example, an effective diameter of less than 500 nm, 100 nm, 50 nm or 10 nm can be useful because they are more stable and smaller than the wavelengths of light that are typically used for luminescence detection will. Alternatively or in addition to these exemplary upper limits, bubbles can have an effective diameter which is greater than 10 nm, 50 nm, 100 nm, 500 nm, 1 µm, 5 µm, 10 µm, 25 µm, 50 µm, 100 µm, 250 µm or 500 µm, as exemplary lower limits. A population of bubbles can have an average diameter, an average effective diameter, a maximum diameter, or a minimum diameter delimited by one or both of the upper and lower limits exemplified above.

It is understood that the "effective" diameter of a bubble is a measure of the bubble in a spherical state. If the bladder is a disc (e.g. squeezed through the walls of a flow cell) or some other shape, the effective diameter of the disc bladder is a measure of the diameter of the bladder when in a spherical state with the same Volume is converted.

In certain configurations, the bubbles in a fluid foam can have an average diameter, a maximum diameter, or a minimum diameter that is measured relative to the dimensions of a detection channel in a particular detection area in a flow cell. In general, it is preferred that the bubbles be smaller than the detection channel of a flow cell through which the bubbles flow. For example, the average effective diameter, the maximum effective diameter or the minimum diameter of the bubbles can be at most 99%, 95%, 90%, 75%, 50%, 25%, 10%, 5%, 1%, 0.1%, or 0.01% or less of a relevant dimension of the detection channel area. Alternatively or additionally, the average effective diameter, the maximum effective diameter or the minimum effective diameter of the bubbles can be at least 0.01%, 0.1%, 1%, 5%, 10%, 25%, 50%, 75%, 90 %, 95%, 99% or more of the relevant dimension of the detection channel at the detection point. The relevant dimension can be, for example, the width of the channel (e.g. the x dimension, where the y dimension is the direction of the fluid flow and the z dimension is the depth of the channel or the focus dimension for optical detection through the channel) . The relevant dimension can be the depth of the channel in the z dimension or the relevant dimension can be along the y dimension. The relevant dimension can be the cross-sectional area of the channel.

In some configurations, a bladder can form a bolus or plug that occupies the entire cross-sectional area of a flow cell channel in which the bladder is located. A blister plug or bolus can be useful to remove a particular fluid from a flow cell, such as a fluid that contains one or more of the reagents or analytes that occur in a method described herein. As such, various liquids that are delivered to a flow cell can be separated by a bladder or bolus. Although the cross-sectional area of the flow cell limits the maximum cross-sectional area of the bubble bolus or plug, larger bubbles are absorbed by being deformed by the channel. As the volume of the bladder increases, the length of the channel the bladder occupies increases. For example, a blister bolus or plug can have a volume that, in an undeformed state (i.e., spherical shape), has a maximum cross-sectional area that is at least 1x, 2x, 5x, 10x, or 100x larger than the cross-sectional area of the channel in which it is located located. Bubble plugs with larger volumes can cause an increased physical separation of two fluids. This can offer advantages in reducing the accuracy required for detection or fluid delivery steps. Smaller volume bubble plugs can separate fluids while reducing the time the surface of the flow cell is exposed to gas. This reduced exposure can be beneficial when an array or other surface of an active flow cell is sensitive to dehydration or other effects from exposure to gas.

A population of bubbles in a mixed phase fluid can be monodisperse or polydisperse. For example, monodisperse populations have bubbles of uniform size, with the coefficient of resizing (CV) being no more than 10%. Even closer uniformity may be useful in some configurations, including, for example, that the size CV is no more than 1% or 5% of the mean. For example, the size CV for a polydisperse population of bubbles can range from more than 10% to at most about 25%, 50%, 100%, 2-fold, 5-fold, 10-fold, 100-fold or more. In some situations there is a relatively high degree of polydispersity and as such the size can be at least CV for the bubbles about 25%, 50%, 75%, 100%, 2-fold, 5-fold, 10-fold, 100-fold or more. For example, the sizes of the bubbles in a polydisperse population can be within a range given above.

The concentration of gas bubbles in a fluid foam can be selected for a particular application. In some cases, the concentration of gas bubbles in a fluid foam can be controlled by a bubble generator component. For example, the concentration (ie the volume fraction) of the bubbles in a fluid foam can be at least 0.01%, 0.1%, 1%, 5%, 10%, 25%, 50%, 75%, 90%, 95%, 99 % or more of the total volume of the fluid foam in a specific vessel, for example a detection channel of a flow cell. Increasing the volume fraction of bubbles in a fluid can provide an advantage in reducing reagent consumption without necessarily reducing the effective concentration of the reagent in the foam compared to the original liquid reagent. This can lead to a cost reduction, especially if relatively expensive reagents are used. Example 1 below shows the savings in reagents due to the introduction of bubbles into various reagent solutions used for nucleic acid sequencing. Another advantage of increasing the bubble concentration in a fluid foam is the simultaneous reduction in the volume of the liquid to be transported, stored, discarded or otherwise handled.

As an alternative or in addition to the optional lower limits given above, the concentration of bubbles in a fluid foam can be at most 99%, 95%, 90%, 75%, 50%, 25%, 10%, 5%, 1%, 0.1% or 0.01% or less of the total volume of the fluid foam in a particular vessel, such as a flow cell detection channel. Reducing the bubble fraction can allow the delivery of a larger amount of a particular reagent in the same volume of fluid.

A bubble generator can be configured to deliver fluid foams with a variety of bubble sizes, bubble concentrations, or bubble polydispersities, for example in the ranges exemplified above. Alternatively or additionally, a bubble generator component can be configured to deliver a desired number of bubbles. For example, the bubble generator component can generate or deliver bubbles at a rate of at least 1 s ^-1 , 5 s ^-1 , 10 s ^-1 , 50 s ^-1 , 100 s ^-1 , 250 s ^-1 , 500 s ^-1 or more . Alternatively or additionally, the speed of bubble generation or delivery can be at most 500 s ^-1 , 250 s ^-1 , 100 s ^-1 , 50 s ^-1 , 10 s ^-1 , 5 s ^-1 , 1 s ^-1 or less. Again, a bubble generator component of the present disclosure can be configured to generate a population of bubbles with a number of bubbles in an exemplary range and with a bubble size in an exemplary range above.

An exemplary bubble generator component that can be used to produce a fluid foam is shown in FIG 1 shown. Flow cell 100 includes two detection channels 110 and 120 as well as in the flow cell substrate 101 integrated bubble generator components. The detection channels can optionally have a length of a few centimeters to several centimeters (in the direction of flow), a width of a few micrometers to several millimeters and a depth of a few micrometers to several millimeters. For the sake of brevity, the fluid paths through the detection channels 110 and 120 described together, with parentheses referring to the channel 120 is taken. Liquid enters the inlet 115 ( 125 ) and passes through the liquid channel 114 ( 124 ) to a T transition 113 ( 123 ) that deals with the gas channel 116 ( 126 ) forms, with the gas through the inlet 117 ( 127 ) got into the system. The gas channel 116 ( 126 ) has a cross-sectional area that is smaller than the detection channel 110 ( 120 ). The gas channel is optionally circular with a diameter between about 1 micron and 200 microns, but can be larger in other configurations, for example up to 500 microns. The T-junction can have a narrowed point to form a venturi where gas and liquid mix to form a fluid foam. The fluid foam then passes through the channel to pass over the connection 112 ( 122 ) in the detection channel 110 ( 120 ) to occur. The fluid foam then leaves the detection channel 110 ( 120 ) through the outlet 111 ( 121 ).

An alternatively configured flow cell is shown in FIG: 2. The flow cell 200 comprises two detection channels, each of which a fluid foam from one in the flow cell substrate 201 integrated phase mixing component is supplied. The upper detection channel 210 is about a phase mixing component that has a Y transition 212 at the opening of the detection channel 210 has added fluid foam. As such, liquid enters the inlet 215 and flows through the canal 214 to the Y transition 212 where it mixes with gas that goes through the inlet 217 has entered the flow cell and then along the channel 216 flows to in the Y transition 212 to get. The fluid foam then passes through the detection channel 210 and steps over the outlet 211 out. The lower detection channel 220 is fluid foam over a Phase mixing component fed, which has a Y transition 223 upstream of the opening 222 of the detection channel 220 having. As such, liquid enters the inlet 225 and flows through the canal 224 to the Y transition 223 where it mixes with gas that goes through the inlet 227 into the flow cell and along the channel 226 to the Y transition 223 has arrived. The fluid foam then flows through the rest of the fluid channel 224 , into the detection channel inlet 222 , through the detection channel 220 and steps over the outlet 221 out.

The Y transitions in the flow cell 200 are formed from inlet channels that meet at an acute angle. For example, form the fluid channels 214 and 216 at the Y transition 212 an acute angle. The angle is relatively small compared to the acute angle caused by the intersection of the channels 226 and 224 at the Y transition 223 is formed. The angle can be adjusted to produce desired properties for the fluid foam, such as average bubble size and number of bubbles (ie concentration of bubbles).

As by those in the 1 and 2nd Illustrated device shown, a bubble generator may be an integral part of a flow cell or other vessel. In alternative configurations, a bubble generator can be a separate or separable component with respect to a flow cell or other vessel. An example of a bubble generator 300 , which is separable from other fluidic components, is in 3rd shown. The bubble generator 300 includes a body 301 with internal piping on which the liquid inlet 315 , the gas inlet 326 and the foam outlet 311 access. A straight channel 314 connects the liquid inlet 315 with the foam outlet 311 . The gas inlet 326 is across a channel 316 with the channel 314 connected, the two channels making a T transition 323 form. That from the gas inlet 326 flowing gas can at the T transition 323 in from the liquid inlet 315 flowing liquid can be introduced to form a fluid foam that exits the foam outlet 311 emanates. The channel 314 narrows in places 330 and 331 to a narrowed area 310 to build. The T transition 323 occurs in the narrowed area 310 of the straight channel 314 on. A gas source can have a threaded coupling 326 with the bubble generator 300 get connected. The internal thread 325 form a female connector that is configured to receive an externally threaded connection for a gas line. It is understood that when connecting a gas line to the bubble generator 300 opposite connectors (ie male connector on the bubble generator and female connector on the gas pipe) or non-threaded connectors can be used. For example, a pressure connection, a pipe connection, an adhesive-mediated connection, a clamp, or another connection that is known in the field of fluid technology and sanitary engineering can be used for the connection. A liquid line can go through the connector 302 with the inlet 315 get connected. The connector 302 forms, for example, a male connector that can be inserted into a flexible pipe to make a connection. A similar connection can be made for the connector 303 made to foam from the outlet 311 to a flow cell or other fluidic component.

4th shows a bubble generator 400 with a body 401 and an internal T transition 423 which is similar to that in 3rd shown is configured. The T transition 423 connects the channel 414 with the channel 416 so that about the gas inlet 426 introduced gas with that from the liquid inlet 415 introduced liquid can mix to produce a foam that comes out of the foam outlet 411 emanates. The channel 414 narrows in places 430 and 431 to a narrowed area 410 to generate The gas inlet connection 426 and the foam outlet connector 403 are similar to those in 3rd . A gas source can have a threaded coupling 426 with the bubble generator 400 get connected. The internal thread 425 form a female connector that is configured to receive an externally threaded connection for a gas line. The connector 402 for the liquid inlet is a male connector with an external thread. The threaded connection can provide a convenient connection to a fluid line; however, other types of connections such as those related to above can be used 3rd were given as examples.

A bubble generator does not necessarily have to use a T-junction (i.e., when a gas-carrying channel intersects a straight channel at an orthogonal angle, with the straight channel having a fluid inlet at one end and a foam outlet at the other end). Rather, channels can intersect at non-orthogonal angles. The cutting angle can be configured to achieve the desired properties of a foam for a particular use. In a further alternative, a T-junction can be configured such that a fluid-carrying channel intersects a straight channel at an orthogonal angle, the straight channel having a gas inlet at one end and a foam outlet at the other end.

5 shows a bubble generator 500 with a Y transition 518 . Gas flows from the gas inlet 526 to the Y transition 518 where it's through the channel 514 flowing liquid from the liquid inlet 515 contacted. The resulting foam flows through the channel 513 and out of the foam outlet 511 . channels 514 and 513 are curved, but can be straight. The bubble generator 500 includes a connector with an internal thread 525 for the gas inlet 526 , a male connector without thread 502 for the liquid inlet 515 and a male threaded connector 503 for the foam outlet 511 . The bubble generator 500 includes a filter membrane 550 with a lace pattern 551 in a hydrophobic material 552 that acts as a gas diffuser. The membrane 550 can in the cylinder of the gas inlet 526 be arranged.

Für die obige Gleichung 1 ist Δp der Laplace-Druck, die Druckdifferenz über der Fluidgrenzfläche; γ ist die Oberflächenspannung (oder Wandspannung), n^ ist die Einheitennormale, die aus der Oberfläche herauszeigt, H ist die mittlere Krümmung und R₁ und R₂ sind die Hauptkrümmungsradien. Die Membran 550 oder eine ähnliche Vorrichtung kann in anderen hierin beschriebenen Blasengeneratoren verwendet werden, einschließlich beispielsweise solchen, die in den 1 bis 4 gezeigte strukturelle oder funktionelle Elemente verwenden.The membrane 550 , in the 5 is shown is exemplary. The membrane is configured to act as an array of holes with a Young Laplace pressure drop. The Young-Laplace equation is a nonlinear partial differential equation that describes the capillary pressure difference that occurs at the interface between two static liquids, such as a liquid and a gas, due to the surface tension phenomenon. The equation can also describe the capillary pressure difference that occurs at the interface between two static liquids due to the phenomenon of wall tension when the wall is very thin. The Young-Laplace equation relates the pressure difference to the shape of the surface or wall. It is a representation of the normal stress compensation for static liquids that meet at an interface, whereby the interface is treated as a surface (thickness = zero): $$\begin{array}{ll}\Delta p\hfill & =-\gamma \nabla \cdot \widehat{n}\hfill \\ \hfill & =\mathrm{2nd}\gamma H\hfill \\ \hfill & =\gamma \left(\frac{1}{R_1}+\frac{1}{R_{\mathrm{2nd}}}\right)\hfill \end{array}$$

For equation 1 above, Δp is the Laplace pressure, the pressure differential across the fluid interface; γ is the surface tension (or wall tension), n ^ is the unit normal that emerges from the surface, H is the mean curvature and R ₁ and R ₂ are the main radii of curvature. The membrane 550 or a similar device can be used in other bubble generators described herein, including, for example, those described in Figs 1 to 4th use the structural or functional elements shown.

The membrane thickness can be any that achieves the desired properties for the mixed phase fluid. For example, the membrane can have a thickness of at least 100 nm, 500 nm, 1 μm, 10 μm, 100 μm, 500 μm or more. The upper limit of the membrane thickness can be determined based on the desired properties of the mixed phase fluid. For example, as an alternative or supplement to the exemplary lower end of the range specified above, the thickness can be at most 500 μm, 100 μm, 10 μm, 1 μm, 500 nm, 100 nm or less. Of course, using a membrane is an option. In some configurations, the phase mixing component may lack a membrane.

Alternatively or in addition to adjusting the membrane thickness, the number, size and spacing of the holes in the membrane can be adjusted to provide a foam with the desired properties. In general, a hole size between 1 micron and 80 microns can be useful to provide a means of controlling the bubble fraction for differences in gas pressure and oil flow rate, respectively. For example, changing the gas pressure in the range of 15 to 50 psi for gas flowing through a membrane with 1 µm holes enables control of the bubble fraction and polydispersity. If the membrane has 5 µm holes instead, the gas pressure range that allows control of the bubble fraction and polydispersity is 5 to 30 psi. Although a membrane with 80 µm holes can create bubbles, the bubble fraction and polydispersity are less responsive to changes in gas pressure. Accordingly, membranes with holes smaller than about 80 microns are generally preferred, for example in configurations where variable control of foam properties is desired. Larger hole sizes can be used if the variability in foam properties is to be minimized.

The average distance for an accumulation of holes in a membrane can be of a variety of lengths, including, for example, at least 1 µm, 5 µm, 10 µm, 25 µm, 50 µm, 100 µm or more. Alternatively or additionally, the distance for the holes in a particular membrane can be at most 100 µm, 50 µm, 25 µm, 10 µm, 5 µm, 1 µm or less. In general, increasing the distance for the holes can increase the response of the bubble fraction and the polydispersity to the pressure provide the applied gas. A reduction in distance may be desirable if less variability in foam properties is desired.

Other properties of the membrane can also affect the properties of the foam. For example, a hydrophobic material for the membrane is generally preferred. Exemplary materials that can be used for the membrane include, but are not limited to, polycarbonate and polytetrafluoroethylene (PTFE). Another exemplary property is the surface of the membrane. It has been observed that a larger surface area between the gas phase and the liquid phase provides better control of the gas fraction and the bubble polydispersity in a foam generator. The area for a membrane used to produce a foam can be at least about 10 µm ² , 50 µm ² , 100 µm ² , 500 µm ² , or 1 mm ² or more. As an alternative or in addition, the area for a membrane which is used to produce a foam can be at most about 1 mm ² , 500 µm ² , 100 µm ² , 50 µm ² , 10 µm ² or less.

The gas resistant membranes described above are optional and do not have to be used. Whether or not a gas resistant membrane is used. The size of the air inlet that intersects the liquid line at a phase mixture transition can range from a few microns to several hundred microns. For example, the transition for a phase mixing device can have a cross section which lies in the above-mentioned ranges for the surface of the above-mentioned membranes.

A variety of bubble generator systems can be used to generate bubbles, such as those described in Garstecki et al., Bulletin of the Polish Academy of Sciences 53: 361-372 (2005), which is hereby expressly incorporated by reference. In certain configurations, a channel transition can be used to introduce gas bubbles into a liquid. In this geometry, the gas is fed into a main channel that carries the liquid. The liquid preferably wets the walls of the channel and the gas phase breaks up into bubbles as it enters the main channel. Another exemplary bubble generator uses geometries in which liquid is forced through a narrow constriction in the main channel and the gas - which is emitted from a nozzle located close upstream of the constriction - is focused into this opening by the converging flow lines of the liquid. Useful bubble generators include those known in the art as bubble injectors, emulsifiers (e.g., ultrasonic emulsifiers), venturi injectors, and bubble diffusers.

In some configurations, a mixed phase fluid can be formed in a fluid system at a location that is downstream of a fluid reservoir (e.g., a fluid reservoir containing a nucleic acid sequencing reagent) and upstream of a vessel used for analytical detection or synthetic reaction (e.g. flow cell containing an array of nucleic acids to be sequenced). This configuration has the advantage that relatively large reservoirs are avoided to accommodate the mixed phase fluid volume compared to the volume of the liquid phase alone. Introducing a dispersed phase downstream of a reservoir can also reduce damage to a reagent that is sensitive to the material of the dispersed phase. In alternative configurations, bubbles can be introduced into reservoirs that contain various liquids to create fluid foam in the reservoirs before the liquids are conveyed out of the reservoir.

A mixed phase fluid can flow in and out of a flow cell in a single direction. Alternatively, the mixed phase fluid can flow into a flow cell and flow out of it after a change of direction, for example in order to achieve mixing or to remove unwanted materials. Switching can be accomplished by changing the direction of pressure applied to the mixed phase fluid. An example of switching (also called "wiggle") a fluid foam is given in Example 1 below.

A system of the present disclosure may include a heater or cooler configured to control the temperature of a material used to create a dispersed phase in a mixed phase fluid. For example, a heater can be used to raise the temperature of a gas used to create bubbles in a fluid foam. The heated dispersed phase has a large contact area with the bulk phase and as such can heat the bulk phase very efficiently. In other examples, a cooling device can be used to lower the temperature of a gas used to create bubbles in a fluid foam is used. Here too, efficient cooling can be achieved due to the large contact area between the dispersed phase and the bulk phase.

A heater or cooler can be positioned to control the temperature of the materials when present in a fluid reservoir, fluid supply channel, vessel, or other fluid component. In certain configurations, the location of heating or cooling may be downstream of a fluid reservoir and upstream of a phase mixing component. Alternatively or additionally, heating or cooling can occur at the location where phase mixing occurs (i.e., the location of the phase mixing component). As such, a liquid reagent can be stored in a reservoir at a first temperature (e.g., a temperature that provides stability) and the temperature of a sub-fraction of the reagent can later be changed for use in a downstream process.

If the desired temperature for a mixed phase fluid is higher than the temperature of the liquid phase from which it is formed, the gas which forms the dispersed phase can be heated to a temperature which is higher than the desired temperature. For example, the heater may have a set point that is at least about 5 ° C, 10 ° C, 15 ° C or higher than the desired temperature of the fluid in a downstream flow cell. In particular, the heater located upstream of the flow cell may have a setpoint that is higher than the setpoint for a heater or cooler that regulates the temperature of the flow cell. The temperature difference can be selected to account for the cooling that can occur for the mixed phase fluid as it moves to the flow cell. The temperature difference can be selected based on the heat capacity of the mixed phase, the isolation level for the fluidic components, the temperature difference between the ambient temperature and the flow cell, and the time the mixed phase fluid spends between heating and use in the flow cell.

If the desired temperature for a mixed phase fluid is lower than the temperature of the liquid phase from which it is formed, the gas forming the dispersed phase can be cooled to a temperature which is lower than the desired temperature. For example, the cooling device may have a set point that is at least about 15 ° C, 10 ° C, 5 ° C or lower than the desired temperature of the fluid in a downstream flow cell. In particular, the cooling device located upstream of the flow cell may have a setpoint that is lower than the setpoint for a heating device or a cooling device that regulates the temperature of the flow cell. The temperature difference can be selected to account for the heating that can occur for the mixed phase fluid as it moves to the flow cell. The temperature difference can be selected based on properties similar to those exemplified above for the heating.

In some configurations, the liquid phase that forms the bulk phase (e.g., the liquid phase that carries reagents, analytes, or the like) can be heated or cooled. Alternatively or additionally, a mixed phase fluid may be heated or cooled downstream of a phase mixed component, including, for example, in fluid lines that deliver the mixed phase fluid to a flow cell and / or in the passages of a flow cell or other vessel.

Heating or cooling can be accomplished using a variety of heat transfer mechanisms, including but not limited to Joule heating, convection, Peltier control, or radiation. Devices and systems for heating or cooling fluids upstream of a flow cell that can be modified for use with mixed phase fluids are disclosed in U.S. Patent Application No. 62 / 782,565, which is hereby expressly incorporated by reference.

Valves, heaters, coolers, or other elements of a phase mixing component or a bubble generator component can be controlled by a control module. Exemplary configurations for a control module are set out in more detail below. For example, the control module can be programmed to open a valve to allow gas to mix with a dispersion liquid, thereby supplying a mixed phase fluid to the flow cell, and the control module can also be programmed to close the valve to a single phase fluid to deliver to the flow cell.

The inner surfaces of the fluid lines in the bubble generator component and the flow cell channel can be hydrophilic or hydrophobic. The surface can thus be adjusted to reduce or prevent bubbles from adhering to the surfaces. When using an aqueous liquid, it may be advantageous to use hydrophilic components (e.g. flow cells and tubes) Surfaces. The hydrophilicity prevents bubbles from sticking to the surfaces. Surfactants can be included in a foam to further prevent bubbles from adhering to surfaces. Particularly useful surfaces are made from cycloolefin copolymer (COP), which shows a useful hydrophilic character when it comes into contact with aqueous solutions containing surfactants.

Liquids can be fed to a phase mixing component, a bubble generator component and / or a flow cell under pressure. Liquids are typically dispensed under positive pressure, but in some configurations negative pressure can also be used. Suitable pumps include those that induce hydrodynamic fluid pressure, such as those based on mechanical principles (e.g. external syringe pumps, pneumatic diaphragm pumps, vibrating diaphragm pumps, vacuum devices, centrifugal forces, piezoelectric / ultrasonic pumps and acoustic forces); electrical or magnetic principles (e.g. electroosmotic flow, electrokinetic pumps, ferrofluidic plugs, electrohydrodynamic pumps, attraction or repulsion using magnetic forces and magnetohydrodynamic pumps); Gravity; electrostatic forces (e.g. electroosmotic flow); Centrifugal flow (substrate arranged on a CD and rotated); magnetic forces (e.g. oscillating ions cause flux); magnetohydrodynamic forces; and vacuum or pressure differential work.

The fluids used in a system set forth herein may be formulated to contain desired reactive reagents, including, for example, those set forth herein in the context of sequencing processes. The liquid reagents can be stored in reservoirs. For example, one or more reservoirs in a system set forth herein may contain polymerases, nucleotides, nucleic acids, deblocking reagents, polymerase cofactors, polymerase inhibitors, ternary complex stabilizers, or the like.

One or more of the liquids may also contain surfactants, for example to prevent bubbles from joining together when the liquid is used to produce a mixed phase fluid. Surfactants can be anionic, cationic, nonionic or zwitterionic. Exemplary surfactants include, but are not limited to, sodium dodecyl sulfate (SDS), Tween-20 (Polysorbate 20), Tween-80 (Polysorbate 80), cetrimonium bromide, cetyltrimethylammonium bromide, Triton X-100, CHAPS, or NP-40. Surfactants can help stabilize bubbles in a fluid foam. Surfactants can also help stabilize other components of a foam, such as proteins or nucleic acids. A relatively low concentration of surfactant can be useful in a mixed phase fluid. For example, a mixed phase fluid may contain at least 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5% or more surfactant. For some applications, the amount of surfactant is limited, for example to prevent undesirable interactions with other components in a mixed phase fluid. For example, the concentration of the surfactant in a mixed phase fluid can be at most 5%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005% or less surfactant.

Alternatively or in addition to the presence of surfactants, one or more of the liquids can contain solutes which increase or decrease the viscosity. For example, the viscosity can be adjusted by adding polyols or sugars such as glycerin, erythritol, agarose, arabitol, sorbitol, mannitol, xylitol, mannisdomannitol, glucosylglycerol, glucose, fructose, sucrose, trehalose or isofluoroside; or polymers such as dextrans, levane, gelatin or polyethylene glycol can be increased. Nanoparticles can also be useful to stabilize bubbles.

A gas delivery component is typically configured to supply gas to a bubble generator component or flow cell under positive pressure. In some configurations, however, gas can be released under negative pressure. Inert gases such as N ₂ or noble gases are particularly advantageous because they reduce the risk of an adverse interaction with biological components such as enzymes and nucleic acids. In some configurations, a system of the present disclosure, and particularly the gas delivery component, is configured to prevent molecular oxygen from entering a fluid foam. For example, the system and its fluidic components can be configured to exclude atmospheric oxygen. As such, the gas bubbles in a fluid foam can be substantially free of molecular oxygen. For example, the bubbles in a fluid foam may contain less than 20%, 10%, 1%, 0.5%, 0.1%, 100 ppm, 10 ppm or less molecular oxygen. Alternatively, a system or gas delivery component can be configured to deliver molecular oxygen or atmospheric oxygen if desired for a particular application. In some configurations, the gas is atmospheric air, which may or may not be filtered before entering the liquid stream. Filtering air or other gases can remove particles and other contaminants.

An example fluid circuit that includes a bubbler for delivering foam to a flow cell is shown in FIG 6 shown. A liquid reservoir contains a reagent or other liquid and is open to the atmosphere (ATM). The liquid can be fed to a bubble generator under the force of a volumetric pump. Atmospheric air (ATM) is fed to the bubble generator (bubbler) under the force of a regulated gas pressure source. The fluid circuit may include an optional gas resistance (in 6 shown). In some configurations, a membrane located within the bubble generator or in the gas line upstream of the bubble generator serves as a gas resistor. Alternatively or in addition to using a membrane, other types of gas resistors can be placed upstream in the gas line, examples of which include a micro-opening (e.g. available from O'Keefe Controls Co., Monroe CT) or a capillary resistor. A fluid foam is formed by mixing gas and liquid in the bubble generator. The fluid foam flows to the flow cell and then to an intermediate waste reservoir. Gas can be discharged from the intermediate waste reservoir and liquid can be conveyed into a downstream waste reservoir under the force of a pump. The downstream waste reservoir is open to the atmosphere (ATM).

wobei Pi der Einlassdruck ist, P_o der Auslassdruck ist, L die Länge des Rohrs ist, µ die Viskosität ist, R der Radius des Rohrs ist, V das Volumen des Fluids beim Auslassdruck ist und v die Geschwindigkeit des Fluids beim Auslassdruck ist.As in 6 shown, the volume flow for the fluid foam ( Q _f ) by modifying the volume flow of the liquid ( Q _l ) and the volume flow of the gas ( Q _g ) can be set. Flow rates can be adjusted or evaluated using the Poiseuille equation for compressible liquids. For a compressible fluid in a pipe, the volume flow and the linear velocity along the pipe are not constant. The flow is usually expressed as the outlet pressure. When the fluid is compressed or expanding, work is done and the fluid is heated or cooled. This means that the flow rate depends on the heat transfer to and from the fluid. For an ideal gas in the isothermal case, in which the temperature of the fluid can balance itself with its surroundings, and when the pressure difference between the ends of the pipe is small, the volume flow at the pipe outlet is given by equation 2: $$Q=\frac{dV}{dt}=v\pi R^{\mathrm{2nd}}=\frac{\pi R^{\mathrm{4th}}\left(P_i-P_O\right)}{\mathrm{8th}\mu L}\times \frac{P_i+P_O}{\mathrm{2nd}{P}_O}=\frac{\pi R^{\mathrm{4th}}}{16\mu L}\left(\frac{P_i^{\mathrm{2nd}}-P_O^{\mathrm{2nd}}}{P_O}\right)$$

where Pi is the inlet pressure P _o the outlet pressure is L is the length of the tube, µ is the viscosity, R is the radius of the tube, V is the volume of the fluid at the outlet pressure and v is the velocity of the fluid at the outlet pressure.

A system of the present disclosure may optionally include an optical detection component configured to detect the interior of a flow cell, such as the interior of a detection channel in a flow cell. Particularly useful optical detection components include those found in subsystems or components of nucleic acid sequencing systems. Several such detection devices are configured for optical detection, for example for the detection of luminescence signals. Accordingly, an optical detection component can include an excitation system configured to irradiate the interior of a flow cell channel. The optical detection component may further include an emission system configured to detect luminescence from inside the flow cell channel. The detection of the luminescence can be carried out using detection devices known in the prior art which relate to nucleic acid arrays or nucleic acid sequencing. A luminophore can be detected based on a variety of luminescent properties, including, for example, emission wavelength, excitation wavelength, fluorescence resonance energy transfer (FRET) intensity, quenching, anisotropy or lifetime.

Examples of detection devices and their components that can be used in a system or method herein are described, for example, in U.S. Patent Application Publication No. 2010/0111768 A1 or U.S. Patent Nos. 7,329,860; 8,951,781 or 9,193,996, which are hereby expressly incorporated by reference. Other detection devices include those commercially available for nucleic acid sequencing, such as those from Illumina ™, Inc. (e.g. HiSeq ™, MiSeq ™, NextSeq ™ or NovaSeq ™ systems), Life Technologies ™ (e.g. B. ABI PRISM ™ or SOLiD ™ systems), Pacific Biosciences (e.g. systems using SMRT ™ technology such as Sequel ™ or RS II ™ systems) or Qiagen (e.g. genereader ™ system). Other useful detectors are in U.S. Patent Nm. 5,888,737; 6,175,002; 5,695,934; 6,140,489; or 5,863,722; or U.S. Patent Application Publication Nm. 2007/007991 A1, 2009/0247414 A1 or 2010/0111768; or WO2007 / 123744 which are all incorporated herein by reference in their entirety.

Although the system and methods of the present disclosure related to optical detection are shown herein in several exemplary embodiments, it should be understood that other detection modalities may be used in addition or instead. For example, the detector may be an electronic detector that is used to detect protons or pyrophosphate (see, for example, U.S. Patent Application Publication No. Nm. 2009/0026082 A1; 2009/0127589 A1; 2010/0137143 A1; or 2010/0282617 AI, which hereby all of which are incorporated by reference in their entirety, or those commercially available from Ionofent ™ systems from ThermoFisher, Waltham, MA, or as used to detect nanopores such as those commercially available from Oxford Nanopore ™, Oxford UK Systems (e.g. MinlON ™ or PromethION ™ systems) or those in U.S. Patent No. 7,001,792 ; Soni & Meller, Clin. Chem. 53, 1996-2001 (2007); Healy, Nanomed. 2, 459-481 (2007); or Cockroft et al. J. Am. Chem. Soc. 130, 818-820 (2008) systems set forth, all of which are hereby incorporated by reference. An FET detector can be used, such as one or more of those disclosed in U.S. Patent Application Nos. 62 / 767,712; U.S. Patent Application Publication Nm. 2017/0240962 A1, 2018/0051316 AI, 2018/0112265 A1, 2018/0155773 A1 or 2018/0305727 A1; or U.S. Patent Nos. 9,164,053, 9,829,456, 10,036,064 or 10,125,391, all of which are incorporated herein by reference.

The control of system components such as a bubble generator or a phase mixing component can include a general-purpose processor, a digital signal processor (DSP), an application-specific integrated circuit (ASIC), a field programmable gate array (FPGA) or another programmable logic device, discrete gate or transistor logic, use discrete hardware components or any combination thereof designed to perform the functions described herein. A general purpose processor may be a microprocessor, but alternatively the processor may be any conventional processor, controller, microcontroller, or state machine. A processor can also be implemented as a combination of computing devices, e.g. B. a combination of a DSP and a microprocessor, a plurality of microprocessors, one or more microprocessors in conjunction with a DSP core or any other such configuration. For example, a processor or other device may be programmed to actuate a valve or other fluid component that controls the flow of particles, gas or liquid into a phase mixing component, thereby creating a mixed phase fluid with a desired number of bubbles, bubbles of a desired size or composition, or other desirable properties of bubbles. Alternatively or in addition to controlling the flow of material forming the dispersed phase of the mixed phase fluid, a processor or other device may be programmed to actuate a valve or other fluid component that controls the flow of liquid that forms the bulk phase of the mixed phase fluid. The same or a different processor used to control fluids can interact with the system to acquire, store, and process signals (e.g., signals that are detected using a device described herein). In certain embodiments, a processor can be used to determine from the signals the identity of the nucleotide present at a particular location in a template nucleic acid. In some cases, the processor will identify a sequence of nucleotides for the template from the detected signals.

The memory architecture of a processor can include at least one program product that has at least one program module that is implemented as executable instructions that are configured to operate one or more components of a system described here. For example, executable instructions can include an operating system, one or more application programs, other program modules, and program data. In general, program modules can include routines, programs, objects, components, logic, data structures, etc. that perform certain tasks set forth herein, such as: For example, controlling the concentration, number, size, polydispersity, or composition of bubbles delivered to a liquid to produce a mixed phase fluid.

Particularly useful sequencing processes that can be performed in a system or device described herein are cyclic processes that use repeated cycles of reagent delivery. Each cycle can consist of one or more steps. For example, each cycle can include all of the steps required to detect a single nucleotide position in a template nucleic acid. Some sequencing methods use the chemistry of a cyclic reversible terminator (CRT), with each cycle comprising the steps of: (i) adding a single reversibly terminated nucleotide to gradually extend an emerging primer to a nucleotide position to be detected; (ii) detecting the nucleotide at the single nucleotide position and (iii) unblocking the resulting primer to allow return to step (i) to start a subsequent cycle. One or more of these steps can be performed in a mixed phase fluid such as a fluid foam. In some configurations, all three steps are in one Mixed phase fluid executed. A mixed phase fluid can be used to dispense and / or remove reagents or products from one or more of these steps. Examples of reaction steps, reagents and products that can be used in a CRT process using mixed phase fluids are listed below and in the references given below. Alternatively, one or more of the exemplary steps, reagents, or products can be performed in the absence of a mixed phase fluid.

A specific example of a useful CRT nucleic acid sequencing method is a Sequencing By Binding ™ (SBB ™) reaction, as described, for example, in U.S. Patent Application Publication No. Nm. 2017/0022553 A1; 2018/0044727 A1; 2018/0187245 A1; or 2018/0208983 AI, each of which is incorporated herein by reference. In general, SBB ™ methods for determining the sequence of a template nucleic acid molecule can be based on the formation of a stabilized ternary complex (between polymerase, primed nucleic acid and related nucleotide) under certain conditions. The method may include an investigation phase and a nucleotide incorporation phase. One or more sequencing phases can be performed using a mixed phase fluid (e.g., a fluid foam).

The investigation phase of an SBB ™ process can be carried out for at least one template nucleic acid molecule which is primed with a primer, a polymerase and at least one nucleotide type. The interaction of polymerase and a nucleotide with the primed template nucleic acid molecule (s) can be observed under conditions where the nucleotide is not covalently added to the primer (s); and the next base in each template nucleic acid can be identified based on the observed interaction. For example, the nucleotides can contain a detectable label. In some embodiments, the polymerase can be labeled. A variety of conditions and reagents can be useful to stabilize a ternary complex during the study phase. For example, the primer may contain a reversible blocking moiety that prevents nucleotide covalent binding; and / or polymerase cofactors required for extension, such as divalent metal ions, may be absent; and / or inhibitory divalent cations that inhibit polymerase based primer extension may be present; and / or the polymerase present in the test phase can have a chemical modification and / or mutation which inhibits primer extension; and / or the nucleotides can have chemical modifications that inhibit incorporation.

The extension phase can be carried out by creating conditions in which a nucleotide can be added to the primer on each template nucleic acid molecule. In some embodiments, this includes removing reagents that were used in the assay phase and replacing those reagents with reagents that facilitate extension. For example, test reagents can be replaced by a polymerase and one or more reversibly terminated nucleotides.

Another useful CRT sequencing method is sequencing by synthesis (SBS). SBS generally involves the enzymatic extension of an emerging primer by the iterative addition of nucleotides complementary to a template strand to which the primer is hybridized. In short, SBS can be initiated by contacting primed target nucleic acids with one or more labeled nucleotides, DNA polymerase, etc. The primers will incorporate a labeled nucleotide that can be detected. Optionally, the labeled nucleotides can also contain a reversible terminator unit. As such, a single reversibly terminated nucleotide is added to the primer so that subsequent extension cannot take place until a deblocking agent is delivered to remove the reversible terminator unit from the primer. The SBS cycle can be performed n times to extend the primer by n nucleotides, whereby a sequence of length n is detected. Exemplary SBS methods, reagents, and detection components that can be easily adapted for use with a system or device herein are described, for example, in US Pat Bentley et al., Nature 456: 53-59 (2008) , WO 04/018497 ; WO 91/06678 ; WO 07/123744 ; U.S. Patent Nm. 7,057,026; 7,329,492; 7,211,414; 7,315,019 or 7,405,281 and U.S. Patent Application Publication No. 2008/0108082 A1, which are hereby incorporated by reference. Also useful are SBS methods, commercially available from Illumina, Inc. (San Diego, CA).

Some SBS embodiments are cyclic, but do not need to use reversible terminator nucleotides. Such methods can also use mixed phase fluids. A particularly useful method involves the detection of a proton that occurs when a nucleotide is incorporated into an extension product is released. For example, sequencing based on the detection of released protons can use reagents and an electrical detector commercially available from Thermo Fisher (Waltham, MA) or in U.S. Patent Application Publication Nm. 2009/0026082 A1; 2009/0127589 A1; 2010/0137143 A1; or 2010/0282617 A1, each of which is hereby incorporated by reference. Another cyclic sequencing method in which no reversible terminator nucleotides have to be used is pyrosequencing. Pyrosequencing detects the release of inorganic pyrophosphate (PPi) when nucleotides are incorporated into an emerging primer hybridized to a template nucleic acid strand (Ronaghi et al., Analytical Biochemistry 242 (1), 84-9 (1996); Ronaghi, Genome Res. 11 (1), 3-11 (2001), Ronaghi et al., Science 281 (5375), 363 (1998), U.S. Patent Nos. 6,210,891, 6,258,568 and 6,274,320, each of which is incorporated herein by reference).

Sequencing by ligation reactions are also useful, including, for example, those described in Shendure et al. Science 309: 1728-1732 (2005); U.S. Patent No. 5,599,675 ; or U.S. Patent No. 5,750,341 are described, each of which is hereby incorporated by reference. Hybridization sequencing methods can be used as described, for example, in Bains et al., Journal of Theoretical Biology 135 (3), 303-7 (1988); Drmanac et al., Nature Biotechnology 16, 54-58 (1998); Fodor et al., Science 251 (4995), 767-773 (1995); or WO 1989/10977, each of which is hereby incorporated by reference. In both sequencing by ligation and sequencing by hybridization, primers hybridized with nucleic acid matrices are subjected to repeated extension cycles by oligonucleotide ligation, for example using fluorescent-labeled oligonucleotides.

example 1

Nucleic acid sequencing with and without bubbles

This example provides a comparison of nucleic acid sequencing using a reagent delivery fluid or a fluid foam for the reagent delivery. The comparison showed that a fluid foam instead of a liquid could be used to deliver sequencing reagents to an array of nucleic acids during a sequencing run. The results also showed that the use of a fluid foam instead of a liquid delivery medium resulted in a significant reduction in reagent consumption, which resulted in lower reagent costs and a decrease in waste volume. The quality of the sequencing data obtained using fluid foam to deliver sequencing reagents was comparable to the quality of the results obtained using a liquid to deliver the reagents.

Materials and processes

Flow cells containing primed template nucleic acids were made using liquid reagents as follows. Template nucleic acid strands synthesized in 12 PCR reactions were made and then independently bound to beads. This resulted in a population of 12 types of beads, each bead containing a homogeneous collection of one of the 12 template strands. Beads containing immobilized template strands were next attached to the inner surface of a flow cell. The flow cell interior surface was hydrophilized to prevent bubbles from adhering to the flow cell surface. Next, sequencing primers were passed into the flow cell and allowed to hybridize to the immobilized template strands to form immobilized primer-template hybrids.

Sequencing was carried out cyclically, with each cycle comprising the following steps: (i) extension: addition of a reversibly terminated nucleotide to the primers of the immobilized primer-template hybrids; (ii) Investigation: formation and detection of stabilized ternary complexes on the reversibly terminated, immobilized primer-template hybrids, and (iii) activation: cleavage of the reversible terminator from the extended primers. Each cycle resulted in the addition of a single nucleotide and the detection of a subsequent nucleotide position. Thus the number of cycles correlated directly with the length of the sequence read for each bead. Table 1 shows the steps performed for each cycle of a control sequencing run that was in the liquid phase (ie, with no bubbles introduced), along with the time for each step, the flow rate for the fluidic reagents, and the total volume of each step used liquid reagent. TABLE 1: Sequencing cycle for liquid phase sequencing (no bubbles STEP TIME (SEC) LIQUID PHASE FLOW RATE (µL / SEC) LIQUID PHASE TOTAL VOLUME USED (µL) RTS 5 32 160 RTS 30th 1 30th NSB 5 32 160 EXT 5 32 160 BREAK 15 0 0 IMG 7 32 224 DETECT 1 0 0 NSB 5 32 160 EXA 5 32 160 BREAK 15 0 0 IMG 7 32 224 DETECT 1 0 0 NSB 5 32 160 EXC 5 32 160 BREAK 15 0 0 IMG 7 32 224 DETECT 1 0 0 NSB 5 32 160 EXG 5 32 160 BREAK 15 0 0 IMG 7 32 224 DETECT 1 0 0 NSB 5 32 160 CLV 5 32 160 CLV 12th 1 12th IMG 5 32 160

The sequencing cycle was initiated by incorporating reversible terminator nucleotides at the 3 'ends of the primers of the immobilized primer-template hybrids. This was accomplished by the RTS step, in which the flow cell was contacted with unlabeled reversibly terminated nucleotide analogs of dATP, dGTP, dCTP and dTTP in the presence of M15 polymerase (see U.S. Patent Application No. 62 / 732,510, which is hereby expressly incorporated by reference). The reversible terminator nucleotide used in this illustrative method contained a reversible 3'-ONH ₂ terminator unit. A description of this reversible terminator nucleotide can be found in U.S. Patent No. 7,544,794 , which is hereby expressly incorporated by reference.

The NSB step was performed to remove the dNTPs from the RTS solution and wash the flow cell. In particular, for the NSB step, a solution containing isopropanol, Tween-80, hydroxylamine and EDTA was passed through the flow cell. The NSB step retained the M15 polymerase (see U.S. Patent Application No. 16 / 518,321, which is hereby expressly incorporated by reference).

The cycle then continued with an assay subroutine in which each of four different nucleotides was individually delivered to the flow cell (EXT, EXA, EXC and EXG provided Cy5-labeled dTTP, Cy5-labeled dATP, Cy5-labeled dCTP and Cy5-labeled, respectively dGTP). The system stopped fluid flow to allow the formation of a ternary complex. The free nucleotide was removed from the flow cell by delivery of an IMG reagent, and then the flow cell was examined for the formation of a ternary complex on the immobilized primer-template hybrids. The IMG reagent included LiCl, betaine, Tween-80, KCI, ammonium sulfate, hydroxylamine and EDTA, which stabilized the ternary complexes after removal of free nucleotides (see U.S. Patent No. 10,400,272 , which is incorporated here by reference). The flow cell was imaged using fluorescence microscopy to detect ternary complexes that contained a labeled nucleotide that was related to the next correct nucleotide in each of the template nucleic acids. Reversible terminator units on the 3 'nucleotides of the primer strands precluded the incorporation of nucleotides during the ternary complex formation and detection steps.

After the assay subroutine, the NSB wash was performed to free the flow cell of nucleotides from the assay subroutine. Then the sequencing cycle continued with the CLV step in which the reversible terminator units were removed from the primers using sodium acetate and sodium nitrite as in U.S. Patent No. 7,544,794 which is hereby expressly incorporated by reference. The flow cell was then washed in IMG solution. The polymerase from the assay steps was removed by the CLV and IMG steps. The sequencing process then proceeded to the next nucleotide position by returning to the first step of the next cycle.

Table 2 shows the steps performed for each cycle of a sequencing run performed in fluid foam. The table shows the time, flow rate of the liquid reagent, liquid volume consumption and gas pressure used for each step. The liquid phase of each step was the same as that of the same name in the control sequencing run as stated above in connection with Table 1. However, the liquid phase was used for several of the steps using a bubble generator with a Y transition and a gas diffuser as in FIG 5 shown mixed with gas. The bubble generator was placed downstream of reservoirs containing the liquid reagents and upstream of the flow cell. The bubble generator produced a foam when the pressure was above 2 PSI (pounds per square inch). For detection steps, the gas pressure was reduced to 2 PSI in order to omit bubbles. Accordingly, the detection was carried out in the absence of foam in a liquid reagent as was done in the control sequencing run of Table 1. TABLE 2: Sequencing cycle for fluid foam sequencing (+ bubbles) STEP TIME (SEC) LIQUID PHASE FLOW RATE (µL / SEC) LIQUID PHASE TOTAL VOLUME USED (µL) GAS PHASE PRESSURE (PSI) RTS 5 10th 50 15 RTS Jiggle 30th 0 0 2nd NSB 5 10th 50 10th NSB Jiggle 5 0 0 2nd EXT 5 10th 50 20th EXT 1.5 -10 0 2nd BREAK 15 0 0 2nd IMG 5 10th 50 15 IMG 1 50 50 2nd IMG 1.5 -10 0 2nd DETECT 1 0 0 2nd NSB 5 10th 50 10th NSB Jiggle 5 0 0 2nd EXA 5 10th 50 20th EXA 1.5 -10 0 2nd BREAK 15 0 0 2nd IMG 5 10th 50 15 IMG 1 50 50 2nd IMG 1.5 -10 0 2nd DETECT 1 0 0 2nd NSB 5 10th 50 10th NSB Jiggle 5 0 0 2nd EXC 5 10th 50 20th EXC 1.5 -10 0 2nd BREAK 15 0 0 2nd IMG 5 10th 50 15 IMG 1 50 50 2nd IMG 1.5 -10 0 2nd DETECT 1 0 0 2nd NSB 5 10th 50 10th NSB Jiggle 5 0 0 2nd EXG 5 10th 50 20th EXG 1.5 -10 0 2nd BREAK 15 0 0 2nd IMG 5 10th 50 15 IMG 1 50 50 2nd IMG 1.5 -10 0 2nd DETECT 1 0 0 2nd NSB 5 10th 50 10th NSB Jiggle 5 0 0 2nd CLV 3rd 10th 30th 15 CLV 2nd 10th 20th 2nd CLV 12th 1 12th 2nd IMG 5 10th 50 15

Several steps listed in Table 2 included a "wiggle" in which the flow direction for the fluid foam was switched from one direction to the other and back to allow mixing in the flow cell. Some steps also included a continued reversal of the flow direction for the fluid foam or liquid reagent. The reverse flow is indicated by negative values in the flow rate column and zero values for the total volume of the fluid flowing through the flow cell.

Results

7 shows graphical representations of signal intensity versus sequencing cycle for the sequencing protocol, in which the delivery of reagents in liquid form ( 7A ) or dispensing reagents in fluid foam ( 7B ) has been used. Individual curves are displayed for the 'ON' intensity determined for each nucleotide type and for the 'Off' intensity for each nucleotide type. For each bead in each cycle, the nucleotide type that generated the highest signal was identified as the ", AN" signal and the other three nucleotide types were identified as the "off" signal. The 'AN' signals for each nucleotide type were averaged over all bead types detected in a given cycle, and the mean intensity was plotted over all cycles (100 cycles were performed using reagent liquid delivery ( 7A ) and 150 Cycles were performed using the dispensing of reagents with fluid foam ( 7B ) performed) in order to obtain all the AN` signal curves shown in the figure. A Similar averaging of signal intensities across all bead types on a per cycle basis was used to match those in 7 shown to get off 'intensity curves.

wobei I die Signalintensität ist, n die Anzahl von Zyklen ist und τ der Zyklus ist, wenn das Signal ungefähr 37% von I₀ ist (anfängliche Signalintensität). Ein höheres τ zeigt eine verringerte Rate des Signalabfalls an, was im Allgemeinen bevorzugt wird, da es eine erhöhte Leselänge und Sequenziergenauigkeit anzeigt, während eine erhöhte Rate des Signalabfalls durch niedrigere Werte für τ gekennzeichnet ist. Die Anpassungsgüte wurde als Bestimmungskoeffizient R² berechnet. Höhere R²-Werte korrelieren mit einer verringerten Signalintensitätsvarianz während des Sequenzierungslaufs. Die in 7A gezeigten Kurven für die Flüssigkeitsabgabe von Reagenzien hatten einen Durchschnitt τ von 83 und einen Durchschnitt R² von 0,97; und die in 7B gezeigten Kurven für die Abgabe von Fluidschaum von Reagenzien hatten einen Durchschnitt τ von 66 und einen Durchschnitt R² von 0,97 (die Mittelwerte wurden für alle ,An‘-Kurven aller vier Nukleotidtypen ermittelt). Wie durch die τ-Werte angegeben, hat die Verwendung von Fluidschaum-Abgabe von Reagenzien anstelle von Flüssigreagenz-Abgabe einen geringen Einfluss auf die Leselänge, die Verwendung von Fluidschaum erlaubte jedoch immer noch relativ lange Leselängen. Darüber hinaus waren die R²-Werte für mit und ohne Blasen durchgeführte Sequenzierungsläufe vergleichbar, was darauf hinweist, dass die Verwendung von Fluidschaum zur Abgabe von Sequenzierungsreagenzien einen unbedeutenden Einfluss auf die Varianz der Signalintensität hatte.The signal drop for the 'on' curves was assessed by fitting the curves to a curve defined by the following equation: $$\text{I.}={\text{I.}}_0{e}^{-\left(\text{n}/\mathrm{\tau }\right)}$$

where I is the signal intensity, n is the number of cycles and τ is the cycle when the signal is approximately 37% of I ₀ (initial signal intensity). A higher τ indicates a reduced rate of signal decay, which is generally preferred because it indicates increased read length and sequencing accuracy, while an increased rate of signal decay is characterized by lower values for τ. The goodness of fit was calculated as the coefficient of determination R ² . Higher R ² values correlate with a reduced signal intensity variance during the sequencing run. In the 7A curves for liquid delivery of reagents shown had an average τ of 83 and an average R ² of 0.97; and the in 7B The curves shown for the dispensing of fluid foam from reagents had an average τ of 66 and an average R ² of 0.97 (the mean values were determined for all 'An' curves of all four nucleotide types). As indicated by the τ values, the use of fluid foam delivery of reagents instead of liquid reagent delivery has little effect on the reading length, but the use of fluid foam still allowed relatively long reading lengths. In addition, the R ² values for sequencing runs with and without bubbles were comparable, indicating that the use of fluid foam to deliver sequencing reagents had a negligible impact on the variance in signal intensity.

Table 3 shows the postprocessing results for the sequencing run using reagent liquid dispensing as in 7A represented graphically. Table 4 shows the sequencing results for the sequencing run using liquid delivery as in 7B represented graphically. The first column of each table shows the percentage of observed beads that were omitted from the analysis, where "PNN" contains all observed beads, P01 the lowest quality 1% of the data and P02 the lowest quality 2% of the data etc. to P06, the lowest quality omits 6% of the data. For each line, the number of “No Calls”, “Right Calls” and “Wrong Calls” together with the sum of these three columns in the “Total Calls” column displayed. The percentage of errors is displayed along with the relevant Q-Score calculated for each value in the Q% column.

Beispielsweise entspricht ein Q-Score von 20 (Q20) für eine bestimmte Basenbestimmung einer Wahrscheinlichkeit von 1 zu 100, dass die Basenbestimmung falsch ist. Dies bedeutet, dass die Genauigkeit der Basenbestimmung (d. h. die Wahrscheinlichkeit einer korrekten Basenbestimmung) 99,0% beträgt. Eine höhere Basenbestimmungsgenauigkeit von 99,9% wird durch einen Q-Score von Q30 angezeigt und zeigt eine falsche Basenbestimmungswahrscheinlichkeit von 1 zu 1000 an. Q40 zeigt eine Basenbestimmungsgenauigkeit von 99,99% an (d. h. eine falsche Basenbestimmungswahrscheinlichkeit von 1 zu 10.000), Q50 weist auf eine noch höhere Basenbestimmungsgenauigkeit von 99,999% (d. h. eine falsche Basenbestimmungswahrscheinlichkeit von 1 zu 100.000) usw. hin. Derzeit verfügbare Hochdurchsatz-Sequenzierungsplattformen (d. h. Sequenzierungsplattformen der nächsten Generation, wie sie beispielsweise von Illumina, Inc., San Diego, CA erhältlich sind) verwenden normalerweise Q30 als Maßstab für die Qualität. Höhere Q-Scores deuten auf eine höhere Genauigkeit von Variantenbestimmungen hin, was eine höhere Genauigkeit von Schlussfolgerungen und geringere Kosten für Validierungsexperimente zur Folge hat.In nucleic acid sequencing applications, the Q-Score is a property that is logarithmically related to the error probabilities (P) of base determination according to the following equation $$\text{Q}=-\mathrm{10th}\text{log}\left(\text{P}\right)$$

For example, a Q-Score of 20 (Q20) for a particular base determination corresponds to a probability of 1 in 100 that the base determination is wrong. This means that the accuracy of the base determination (ie the probability of a correct base determination) is 99.0%. A higher base determination accuracy of 99.9% is indicated by a Q-Score of Q30 and indicates an incorrect base determination probability of 1 in 1000. Q40 indicates a 99.99% base determination accuracy (ie an incorrect base determination probability of 1 in 10,000), Q50 indicates an even higher base determination accuracy of 99.999% (ie an incorrect base determination probability of 1 in 100,000) etc. Currently available high throughput sequencing platforms (ie, next generation sequencing platforms such as those available from Illumina, Inc., San Diego, CA) typically use Q30 as a measure of quality. Higher Q-scores indicate higher accuracy of variant determinations, which leads to higher accuracy of conclusions and lower costs for validation experiments.

As can be seen from the results of Table 3, when performing sequencing using liquid reagents, a Q score of almost Q50 was obtained without discarding any of the observed data. Omitting the lowest quality 1% of the data from the analysis resulted in a Q-Score of Q70 (ie an incorrect base determination probability of 1 in 10,000,000). In comparison, dispensing the reagents in foam fluid instead of liquid gave a Q value of about 38 without discarding any data. A rating of Q70 was obtained by omitting only 6% of the data. Accordingly, the use of foam fluid to deliver sequencing reagents resulted in highly accurate sequencing. TABLE 3: Sequencing Results (Without Bubbles) Q% NO CALLS RIGHT CALLS WRONG CALLS TOTAL CALLS ERROR PERCENT Q-SCORE PNN 25th 789688 8th 789696 0.001 49.94 P00 0 789688 8th 789696 0.001 49.94 P01 0 781846 0 781846 0 70 P02 0 774056 0 774056 0 70 P03 0 767193 0 767193 0 70 P04 0 767193 0 767193 0 70 P05 0 756686 0 756686 0 70 P06 0 756686 0 756686 0 70 "No Calls": no base determinations; "Right Calls": correct base determinations; "Wrong Calls": wrong base determination; "Total Calls": Total base determinations TABLE 4: Sequencing Results (with Bubbles) Q% NO CALLS RIGHT CALLS WRONG CALLS TOTAL CALLS ERROR PERCENT Q-SCORE PNN 966 1029434 160 1 1029594 0.016 38.09 P00 0 1029434 160 1 1029594 0.016 38.09 P01 0 1019277 21 1 1019298 0.002 46.86 P02 0 1008994 8 1 1009002 0.001 51.01 P03 0 998703 3rd 998706 0 55.22 P04 0 988407 3rd 988410 0 55.18 P05 0 978112 2nd 978114 0 56.89 P06 0 967818 0 967818 0 70 "No Calls": no base determinations; "Right Calls": correct base determinations; "Wrong Calls": wrong base determination; "Total Calls": Total base determinations

Table 5 shows the consumption of liquid reagents per step and sequencing cycle. The foam fluid was generated by adding bubbles to the liquid reagent. Thus, the bubbles take up volume in the fluid foam, which effectively reduces the total amount of reagent in a given volume of fluid foam compared to the same volume of liquid reagent. However, the concentration of the reagent in the liquid phase of the fluid foam is the same as the apparent concentration of the reagent in the liquid reagent experienced by nucleic acids on the surface of the flow cell. Thus, the surface nucleic acids are contacted with sequencing reagents which have the same "apparent" concentration in the fluid foam as in the non-bubbled liquid reagent, although the total amount of reagent consumed per sequencing cycle is reduced when bubbles are added, to create the fluid foam. TABLE 5: Reagent consumption per step and cycle REAGENZ + BUBBLED LIQUID NO BUBBLES USED LIQUID REAGENT SAVING RTS 50 190 74% NSB 250 800 69% EXT 50 160 69% EXA 50 160 69% EXC 50 160 69% EXG 50 160 69% IMG 500 1056 53% CLV 62 172 64% 1062 2858 63%

The results of Table 5 show that by adding bubbles to the sequencing reagents, reagent savings of between 53% and 74% were achieved. The total reagent savings for each cycle was 63%. However, as from the results of 7 and Tables 3 and 4, the use of fluid foam for sequencing resulted in reading lengths and reading qualities comparable to those made using liquid phase reagents in the sequencing platform.

Example 2

Nucleic acid sequencing system using fluid foam

This example describes a nucleic acid sequencing system that includes a table, a liquid delivery component, a gas delivery component, a bubble generator component, and an optical detection component, the table configured to receive a flow cell with a detection channel, the optical detection component configured to inside the Detect the detection channel, the liquid delivery component configured to deliver liquid from a plurality of reservoirs to the detection channel, the gas delivery component configured to deliver gas from one or more sources to the bubble generator component, and the bubble generator component configured to blow bubbles introducing the gas delivery component into liquid from the liquid delivery component at a location that is downstream of the plurality of reservoirs and upstream of the flow cell, to thereby detect the det ection channel to provide a fluid foam that contains bubbles of gas from the gas delivery component.

Some of the components and functions of the nucleic acid sequencing system are set out in more detail below. It goes without saying that the system is exemplary. One or more of the components listed below may be omitted or replaced with other components, as set forth elsewhere herein, for example. Other components set forth herein may be added to the exemplary system without necessarily replacing a component exemplified below.

A block diagram of the nucleic acid sequencing system 1000 is in 8th shown. Fluid connections and the directions in which the fluids are typically in the system 1000 flow are indicated by arrows, solid arrows indicating the flow of liquid or foam and dotted arrows indicating the gas flow. Under certain circumstances, fluids can move in opposite directions, for example when switching / wiggling the fluids. The block diagram shows fluidic connections, but is not intended to necessarily reflect the physical locations of the components, the lengths of the fluidic lines, or the physical structure of the components.

The nucleic acid sequencing system 1000 includes a sipper array 1300 that contains liquids in a variety of reagent reservoirs 1400 contacted. The sipper array 1300 is fluid with the manifold 1500 connected, the liquid from individual sippers of the sipper array 1300 to the bubble generator 1100 or to Bubble generator 1155 (the bubble generators are also referred to as bubble generator components). The bubble generators 1100 and 1155 mix gas with the liquid to form a foam. The bubble generator 1100 is fluid with the inlet 1211 of the detection channel 1201 the flow cell 1200 connected. The bubble generator 1155 is fluid with the inlet 1221 of the detection channel 1202 the flow cell 1200 connected. The gas becomes the bubble generators 1100 and 1155 passed through a gas delivery component that is a source of compressed air 1350 contains the gas through the pneumatic control 1300 , through a specified line in the distributor 1500 , to the bubble generators. The one through the bubble generator 1100 Foam generated is due to displacement forces created by the pump 1050 or 1051 be placed at a location in the fluid delivery component that is between the plurality of reagent reservoirs 1400 and the bubble generators 1100 and 1155 to the detection channel 1201 headed. The outlet 1212 for the detection channel 1201 and the outlet 1222 are fluid with the manifold 1500 connected so that the foam from the detection channels 1201 respectively. 1202 downstream through the fluidic sensor array 1600 or 1601 and then through the downstream selector valve 1700 to the waste tank 1800 can be transferred. The downstream selector valve 1700 allows the fluid systems through the detection channel 1201 or the detection channel 1202 stream, can be selectively opened or closed.

The nucleic acid sequencing system 1000 also includes the detector 1900 . In this example is the detector 1900 an optical detector, which is an excitation system for irradiating the detection channels within the flow cell 1200 , and an emission system for detecting the luminescence from the detection channels inside the flow cell. The flow cell 1200 is through the table 1950 in relation to the fluid components and in relation to the detector 1900 positioned. The nucleic acid sequencing system 1000 further includes a heater 1650 configured to hold the liquids in the liquid delivery component at a location between the plurality of reagent reservoirs 1400 and bubble generator component 1100 to heat / warm up.

9 shows a perspective view of an arrangement of several components of the nucleic acid sequencing system 1000 . The system 1000 is from the base 1009 and the frame 1010 carried. The upper middle area of the frame 1010 is configured so that a user can access the system from the flow cell 1200 on table 1950 to place. Flow cell 1200 is by pretensioner 1951 on the table 1950 pressed. The flow cell 1200 is positioned so that it is from the optical detector 1900 can be detected. The flow cell 1200 is due to translation components 1955 along the table 1950 moved and to a reference surface on the table 1950 held to detect an array of nucleic acids (or other analytical sample) within the flow cell 1200 to enable. Components and functionality of the pretensioner 1951 , the scan system 1955 and the optical detector 1900 are disclosed in U.S. Patent Application Publication No. 2019/0055596 A1, which is hereby expressly incorporated by reference. The system also includes a vent pipe 1920 and a fan 1921 for dissipating the heat generated by the optical detector.

As in the 8th and 9 shown comprises the nucleic acid sequencing system 1000 also peristaltic pumps 1050 and 1051 configured to apply fluid displacement forces (e.g., positive pressure, positive displacement, or the like) at a location in the fluid delivery component that is between the plurality of reservoirs 1400 and bubble generators 1100 and 1155 which causes fluid to flow from the fluid delivery component to the flow cell 1200 is promoted. In the exemplary system 1000 The pumps may be configured to apply fluid displacement forces to the fluid delivery component at a location between the rotary valve and the bubble generator component, thereby delivering fluid from the fluid delivery component to the flow cell. In the view from 9 were fluid connections between the manifold 1500 and the flow cell 1200 removed and are instead in the 12A and 12B shown. The variety of reservoirs 1400 is shown at the time when the reservoirs with the sipper array 1300 are engaged. There are also drawers 1402 and 1403 shown that allow the user to replace liquids in the reservoirs. The reservoirs can be filled with reagents for a nucleic acid sequencing process. Exemplary reagents include, but are not limited to, polymerases, nucleotides, and other reagents listed in Example 2 elsewhere herein or in references cited herein in connection with nucleic acid sequencing. Optionally, the nucleotides and / or polymerases can be labeled exogenously, for example with luminophores as set forth herein or in references cited therein.

10th shows a top view of the distributor 1500 , which is fluid with the sipper distributor 1302 and the sipper distributor 1303 connected is. The sippers are attached to the sipper distributors in such a way that from the Sipper fluid drawn from a reservoir through the sipper manifold through defined channels to the manifold 1500 is directed. For example, the sipper distributor contains 1303 Sipper attachment points 1310 , 1311 , 1313 that with the fluid lines 1314 to 1316 inside the sipper distributor 1303 are fluidly connected. The fluid lines 1314 to 1316 are about connections 1500 with corresponding lines in the distributor 1500 connected to rotary valve 1560 supply the reagents. 10th also shows connections 1510 on the distributor 1500 with corresponding connections on the sipper distributor 1302 are connected so that fluid sucked in by the sipper to the rotary valve 1550 can be directed. Connections are similar 1511 on the distributor 1500 with corresponding connections on the sipper distributor 1303 connected so that fluid sucked in by the sipper to the rotary valve 1560 can be directed. The rotary valves 1550 and 1560 are in 11 visible which is a bottom view of the distributor 1500 shows. 10th also shows an outlet opening 1178 that the outlet of the rotary valve 1550 with the bubble generator 1100 connects a fluid inlet opening 1176 which is the outlet of the flow cell channel 1202 ( 8th ) with waste tank 1800 ( 8th ) connects, and gas outlet opening 1177 , the gas from the compressed air source 1350 ( 8th ) to the bubble generator 1100 ( 8th ) delivers. Similar fluid connections are for the rotary valve 1560 available.

12A shows a perspective view of the fluid connection between the nucleic acid sequencing system 1000 and the flow cell 1200 . 12B shows the same perspective, but with separate and slightly offset connectors. 13 shows the connection between the flow cell 1200 and the fluid delivery components of the nucleic acid sequencing system 1000 .

As in the 12A and 12B shown is the flow cell 1200 fluidly connected to the instrument. The instrument connector 1110 reaches into the instrument connection opening 1172 a. The instrument connection opening 1172 contains a gas outlet 1177 ( 12B ), a fluid inlet 1176 ( 12B ) and a liquid outlet 1178 ( 12B ). The canals 1116 and 1117 of the instrument connector 1110 are through flexible pipes 1191 respectively. 1192 fluid with the flow cell connector 1180 connected. The instrument connector 1110 can with the instrument connection opening 1172 be engaged by hand since the connector 1110 a collapsible hook 1120 ( 12B ), which in a complementary lock on the instrument connection opening 1176 fits.

Also shown in 12A is a peristalic pump 1050 that has a rotor that holds the flexible tube 1193 touched to upstream of the bubble generator 1100 ( 8th ) Apply overpressure. The flexible pipe 1193 runs from the hole 1130 of the instrument connector 1110 via the rotor of the peristalic pump 1050 and then into the hole 1131 of the instrument connector 1110 . The leader 1951 is also in 12A and 12B shown.

In relation to 13 includes the flow cell 1200 a flow cell connector 1180 with the flow cell opening 1190 is engaged. The flow cell connector 1180 contains an inlet 1211 for the detection channel 1201 ( 8th ) and an outlet 1222 for the detection channel 1202 ( 8th ). The instrument connector 1110 is configured to the flexible pipe 1191 with the sewer inlet 1211 fluidly connect and the flexible pipe 1192 with the duct outlet 1222 connect to. Similarly, the flow cell connector 1180 with the flow cell connection opening 1190 be engaged by hand since the connector 1180 collapsible hooks 1181 and 1185 has to the locks 1198 respectively. 1195 fit.

14A shows an exploded view of the left side instrument connector 1110 , the fluidic components of the system 1000 connects to the flow cell. 14B shows an exploded view of the right side instrument connector 1160 , the fluidic components of the system 1000 connects to the flow cell. To simplify the illustration, there are several elements of the instrument connector 1160 with elements with a similar function in the instrument connector 1110 numbered. The instrument connector 1100 contains two plastic parts 1121 and 1122 that can be assembled around the bubble generator 1100 to record. The two parts can snap together and be held in place by compression fittings. The male components of the compression fittings 1113 to 1115 in part 1122 snap into the female screw connections 1133 to 1135 in part 1121 a. The bubble generator 1100 fits in your pocket 1111 on the parts 1121 and a similar pocket on the part 1122 .

If the connector 1100 is assembled, the liquid inlet opening is 1161 of the bubble generator 1100 in fluid communication with the flexible tube 1193 ( 12A and 12B ). The flexible pipe 1193 ( 12A and 12B ) is through the outlet opening 1178 ( 10th ) on the rotary valve 1550 ( 11 ) attached and runs through the channel 1119 of the connector 1110 and then comes out of the hole 1130 ( 12A ) around a loop around a rotor on the peristaltic pump 1050 ( 12A ) to build. The flexible pipe 1193 ( 12A and 12B ) then enters the connector again 1110 through the hole 1131 ( 12A ) and runs through the canal 1118 where the flexible pipe 1193 ( 12A and 12B ) at the liquid inlet opening 1161 on the bubble generator 1100 is attached. The channel 1116 of the connector 1110 is with the flexible tube 1191 ( 13 ) connected in such a way that the foam covering the bubble generator 1100 at the fluid opening 1162 leaves, to the entrance 1211 ( 8th and 13 ) of the detection channel 1201 ( 8th ) is conducted. The instrument connector 1110 also contains a channel 1112 which is the gas delivery component of the nucleic acid sequencing system 1000 with the bubble generator 1100 connects. In particular, gas is released from the gas discharge component from the gas outlet opening 1177 ( 10th ) through the channel 1112 to the gas inlet opening 1163 ( 15A ) on the bubble generator 1100 transfer.

On the instrument connector 1110 is also a channel 1117 included the flexible pipe 1192 ( 13 ) with the instrument fluid inlet opening 1176 ( 10th ) connects. Thus, foam that covers the detection channel 1202 ( 8th ) through the outlet 1222 ( 8th ) leaves through the flexible pipe 1192 ( 13 ), then through the channel 1117 ( 14A ), then through the fluid inlet opening 1176 of the instrument ( 10th ) to the distributor on its way to the fluid sensor 1600 or 1601 ( 8th ), then to the downstream selection valve 1700 ( 8th ) to the waste tank 1800 ( 8th ) hike.

15A shows the bubble generator 1100 . 15B shows an exploded view of the bubble generator 1100 . 15C shows the inside of the part 1169 of the bubble generator 1100 and 15D shows the part 1168 of the bubble generator 1100 . The two outer parts 1168 and 1169 ( 15B ) of the bubble generator 1100 fit via a clamp connection between the web 1170 ( 15B and 15C ) and the gutter 1171 ( 15D ) together. The outer part 1169 includes a gas inlet opening 1163 ( 15B and 15C ). The outer part 1168 includes a liquid inlet port 1161 and a foam outlet 1162 ( 15A , 15B and 15D ). The two assembled parts take seal 1164 ( 15B ) and hydrophobic membrane 1165 ( 15B ) that has an array of holes. The holes in the membrane have diameters between 1 µm and 80 µm and are not visible when the figure is resolved. As in 15B shown the seal 1164 an elongated slot on it when it is between the surface 1166 of the upper part 1168 and the membrane 1165 is arranged, a fluid channel for the flow of liquid from the liquid inlet opening 1161 and the foam outlet 1162 forms. The liquid that flows through the channel formed by the seal picks up bubbles from the membrane 1165 get into the channel, creating the foam.

The gas emerges from the compressed air source 1350 , for example a gas canister containing an inert gas or an inert gas, in the nucleic acid sequencing system 1000 a. Alternatively, the compressed air source 1350 an air compressor configured to the bubble generator 1100 to supply atmospheric air. The compressed air source 1350 generates an overpressure on the gas that is fed to the bubble generator. The Eagle EA is a useful air compressor 2000 with a single-phase motor with 115 volts, 44 dBA and 3.5 amps; and with a 50% duty cycle single stage oil free pump that has a CFM rated power of 90 PSI and a maximum PSI of 125 Has. The gas from the compressed air source 1350 becomes pneumatic control 1300 transmitted, which is configured to regulate and modify the supply of gas from one or more sources into the liquid. The pneumatic control 1300 is configured to vary the pressure of the gas, for example, in a range between 2 and 50 psi.

Example 3

Flow cell that contains fluid foam inside

An exemplary flow cell includes a ternary complex immobilized within a flow cell channel, the ternary complex comprising a polymerase, a primed template nucleic acid, and a next correct nucleotide for the template; and a fluid foam that is in contact with the ternary complex in the flow cell channel, the fluid foam containing a plurality of gas bubbles in a liquid, the fluid foam containing a volume fraction of bubbles that is at least 25% of the total volume of the fluid foam in the flow cell channel, and wherein the average diameter of the gas bubbles is at most 95% of the cross-sectional diameter of the flow cell channel.

The polymerase in the flow cell may be bound to an exogenous luminescent label. Alternatively or additionally, the next correct nucleotide can be bound to an exogenous luminescent label.

Regardless of whether there are labeled components or not, the ternary complex can be present at a site of an array of nucleic acids that is immobilized within the flow cell channel. The array may include at least 1 × 10 ³ sites that have immobilized ternary complexes. Optionally, the average area for each of the locations in the array is less than 25 square microns. Each of the sites can comprise an ensemble of nucleic acids, each ensemble having nucleic acids with a common template sequence.

The ternary complex can be immobilized in the flow cell by attaching the primed template nucleic acid to an inner surface of the flow cell channel. For example, the ternary complex can be immobilized by attaching the primed template nucleic acid to a bead within the flow cell channel. The primed template nucleic acid can be immobilized by linking to the 5 'end of the primer, linking to the 3' end of the template, or linking to the 5 'end of the template.

The fluid foam can flow through the flow cell channel under positive pressure. However, the fluid foam can be static for at least some time during its use. The gas bubbles in the fluid foam can be essentially oxygen-free, for example instead contain an inert gas or noble gas. Alternatively, a fluid foam can be composed of bubbles that contain atmospheric air or oxygen.

In addition to the immobilized components, the flow cell can contain free components that are solvated in the liquid phase of the fluid foam. For example, the fluid foam can contain free nucleotides. The free nucleotides can be present in any desired concentration, for example in a concentration of at least 50 nM. The free nucleotides can have exogenous luminescent labels, blocking units (such as reversible terminator units), both types of units or no type of unit. Regardless of whether the fluid foam contains free nucleotides or not, the fluid foam can contain free polymerases. The polymerases can be present in any desired concentration, for example a concentration of at least 5 units / ml.

If necessary, the cross-sectional area of the detection channel can be at most 100 mm ² . Alternatively or additionally, the cross-sectional area of the detection channel is at least 100 µm ² . The length and cross-sectional area of the detection channel can be adjusted to accommodate a desired volume of fluid foam. For example, the volume of the detection channel can be at most 1 ml. Alternatively or additionally, the volume of the detection channel is at least 1 µl. For example, other channel sizes can be used on a scale known in the art as a microfluidic scale or a nanofluidic scale.

The detection channel of the flow cell can optionally contain an optically transparent window through which the ternary complex is visible. The window can be optically transparent for the light wavelengths used for detection. For example, the window may be transparent to ultraviolet light, visible light or infrared light, or more of them.

The primed template nucleic acid of the ternary complex can comprise a primer that is reversibly terminated or a primer with an extendable 3 'end (i.e. the primer can have a 3'-hydroxyl group). The ternary complex can optionally be a stabilized ternary complex. However, the ternary complex can alternatively be formed during a reaction in which the next correct nucleotide is incorporated into the primer. As such, the ternary complex need not be stabilized or prevented from participating in the polymerase-catalyzed primer extension.

Optionally, the bubbles in the fluid foam can have an average effective diameter that is less than 500 μm. Alternatively or additionally, the bubbles in the fluid foam can have an average effective diameter that is greater than 500 nm. The fluid foam can contain surfactants, viscosity agents, or other chemicals that alter the stability, size, or other properties of bubbles in the foam.

Throughout this application, reference has been made to various publications, patents, and / or patent applications. The disclosures of these documents in their entirety are hereby incorporated by reference into this application.

A number of embodiments have been described. However, it should be understood that various modifications can be made. Accordingly, other embodiments are within the scope of the following claims.

QUOTES INCLUDE IN THE DESCRIPTION

This list of documents listed by the applicant has been generated automatically and is only included for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

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Claims (27)

Flow cell, comprehensive a ternary complex immobilized within a flow cell channel, the ternary complex comprising a polymerase, a primed template nucleic acid and a next correct nucleotide for the template; and a fluid foam that contacts the ternary complex in the flow cell channel, the fluid foam having a plurality of gas bubbles in a liquid, the fluid foam having a volume fraction of bubbles that is at least 25% of the total volume of the fluid foam in the flow cell channel, and wherein the average effective diameter of the gas bubbles is at most 95% of the diameter of the flow cell channel. Flow cell after Claim 1 , wherein the polymerase has an exogenous luminescent label. Flow cell after Claim 1 , the next correct nucleotide having an exogenous luminescent label. Flow cell after Claim 1 , the ternary complex being present at one point in an array. Flow cell after Claim 4 wherein the array has at least 1 × 10 ³ sites that have immobilized ternary complexes. Flow cell after Claim 5 , with the average area for each of the locations being less than 25 square microns. Flow cell after Claim 6 wherein each of the sites has an ensemble of the primed template nucleic acids, the primed template nucleic acids having a common template sequence. Flow cell after Claim 1 wherein the ternary complex is immobilized by attaching the primed template nucleic acid to an inner surface of the flow cell channel. Flow cell after Claim 1 wherein the ternary complex is immobilized by attaching the primed template nucleic acid to a bead within the flow cell channel. Flow cell after Claim 1 , wherein the fluid foam is able to flow through the flow cell channel due to displacement force. Flow cell after Claim 1 , wherein the gas bubbles contain inert gas or noble gas, and wherein the gas bubbles are substantially free of oxygen. Flow cell after Claim 1 , the gas bubbles containing atmospheric air. Flow cell after Claim 1 , wherein the fluid foam comprises free nucleotides in a concentration of at least 50 nM. Flow cell after Claim 13 , wherein the free nucleotides have exogenous luminescent labels. Flow cell after Claim 13 or 14 , wherein the free nucleotides have blocking units. Flow cell after Claim 1 , wherein the fluid foam has free polymerases in a concentration of at least 5 units / ml. Flow cell after Claim 1 , wherein the cross-sectional area of the flow cell channel is at most 100 mm ² . Flow cell after Claim 17 , wherein the cross-sectional area of the flow cell channel is at least 100 µm ² . Flow cell after Claim 1 , the volume of the flow cell channel is at most 1 ml. Flow cell after Claim 19 , the volume of the flow cell channel being at least 1 µl. Flow cell after Claim 1 , wherein the flow cell channel has an optically transparent window through which the ternary complex is visible. Flow cell after Claim 1 , wherein the primed template nucleic acid has a reversibly terminated primer. Flow cell after Claim 1 , wherein the bubbles in the fluid foam have an average diameter that is less than 500 microns. Flow cell after Claim 23 , wherein the bubbles in the fluid foam have an average diameter that is greater than 500 nm. Flow cell after Claim 1 , wherein the ternary complex has a stabilized ternary complex. Flow cell after Claim 1 , wherein the polymerase in the ternary complex catalyzes the covalent addition of the next correct nucleotide to the primed template nucleic acid. Flow cell after Claim 1 , the liquid containing a surfactant.

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