Patent Description:
Increasingly, biological and medical research is turning to nucleic acid sequencing for enhancing biological studies and medicine. For example, biologists and zoologists are turning to sequencing to study the migration of animals, the evolution of species, and the origins of traits. The medical community is using sequencing for studying the origins of disease, sensitivity to medicines, and the origins of infection. As such, sequencing has wide applicability in many aspects of biology, therapeutics, diagnostics, forensics, and research.

Nevertheless, the use of sequencing can be limited by assay availability, sequencing run time, preparation time, and cost. Additionally, quality sequencing has historically been an expensive process, thus limiting its practice.

In particular, reagent solutions are expensive to transport and store. Further, the composition of reagent solutions can influence amplification of target nucleic acids.

In an example, a rehydration buffer is used to rehydrate nucleotides to form a nucleotide solution. Further components can be added to the nucleotide solution to form an amplification solution useful in performing amplification reactions. Such amplification solutions find particular use in amplifying target nucleotides to form monoclonal populations. In an example, such monoclonal populations can be formed on a substrate or in a microwell.

In an example, a rehydration buffer solution can be an aqueous solution including <NUM>-[<NUM>-(<NUM>-hydroxyethyl)piperazin-<NUM>-yl]ethanesulfonic acid (HEPES), [<NUM>-(methacryloyloxy)ethyl]dimethyl-(<NUM>-sulfopropyl)ammonium hydroxide (DMAPS), and tris(hydroxymethyl)aminomethane (TRIS). Optionally, the rehydration buffer solution can include polyethylene glycol (PEG). In a further example, the rehydration buffer solution can include polyoxyethylene sorbitan monolaurate (TWEEN). Following rehydration of the nucleotides, further solutions can be added to the nucleotide solution to form an amplification solution. In particular, the amplification solution can include enzymes, primers, and stabilizers.

In particular, a reagent solution useful as a rehydration buffer or amplification solution is an aqueous solution including <NUM>-[<NUM>-(<NUM>-hydroxyethyl)piperazin-<NUM>-yl]ethanesulfonic acid, [<NUM>-(methacryloyloxy)ethyl]dimethyl-(<NUM>-sulfopropyl)ammonium hydroxide, and tris(hydroxymethyl)aminomethane. When acting as an amplification solution, the reagent solution can further include nucleotides, primers, and enzymes.

For example, the reagent solution can include <NUM>-[<NUM>-(<NUM>-hydroxyethyl)piperazin-<NUM>-yl]ethanesulfonic acid in an amount of <NUM> to <NUM>, such as an amount of <NUM> to <NUM> or <NUM> to <NUM>, [<NUM>-(methacryloyloxy)ethyl]dimethyl-(<NUM>-sulfopropyl)ammonium hydroxide in an amount of <NUM> to <NUM>, such as <NUM> to <NUM> or <NUM> to <NUM>, and tris(hydroxymethyl)aminomethane in amount of <NUM> to <NUM>, such as <NUM> to <NUM> or <NUM> to <NUM>.

In a further example, the reagent solution can further include polyoxyethylene sorbitan monolaurate (TWEEN). For example, the polyoxyethylene sorbitan monolaurate can be polyoxyethylene (<NUM>) sorbitan monolaurate. In an example, polyoxyethylene sorbitan monolaurate is included in an amount of <NUM> volume percent to <NUM> volume percent, such as an amount of <NUM> volume percent to <NUM> volume percent or <NUM> volume percent to <NUM> volume percent.

In a further example, the reagent solution can further include potassium glutamate. For example, the reagent solution can include potassium glutamate in an amount of <NUM> to <NUM>. For example, potassium glutamate can be included in amount of <NUM> to <NUM>, such as <NUM> to <NUM>.

In an additional example, the reagent solution further includes polyethylene glycol. For example, the reagent solution can include polyethylene glycol in an amount of <NUM> volume percent to <NUM> volume percent. For example, the polyethylene glycol can be included in amount of <NUM> volume percent to <NUM> volume percent, such as <NUM> volume percent to <NUM> volume percent. The polyethylene glycol can have a molecular weight in a range of <NUM> to <NUM>, such as a range of <NUM> to <NUM> or a range of <NUM> to <NUM>. In a particular example, the reagent solution includes polyethylene glycol in different molecular weights. For example, the reagent solution can include a first polyethylene glycol having a molecular weight in a range of <NUM> to <NUM> and a second polyethylene glycol having a molecular weight in a range of <NUM> to <NUM>.

In an additional example, the reagent solution can include a biocide. For example, the biocide can include methyl isothiazolinone or isothiazolinone. In particular, the biocide can be included in amount of <NUM> volume percent to <NUM> volume percent.

In another example, the reagent solution can include acetic acid. For example, the reagent solution can include acetic acid in amount of <NUM> to <NUM>, such as <NUM> to <NUM>.

In a further example, the reagent solution can include dithiothreitol. For example, the reagent solution can include dithiothreitol in amount of <NUM> to <NUM>, such as <NUM> to <NUM> or <NUM> to <NUM>.

In an additional example, the reagent solution can include trehalose. For example, the reagent solution can include trehalose in an amount of <NUM> volume percent to <NUM> volume percent, such as an amount of <NUM> volume percent to <NUM> volume percent or an amount of <NUM> volume percent to <NUM> volume percent.

In another example, the solution can include adenosine triphosphate. For example, the reagent solution can include adenosine triphosphate in amount of <NUM> to <NUM>, such as an amount of <NUM> to <NUM> or <NUM> to <NUM>.

In a further example, the reagent solution can include magnesium acetate. For example, the reagent solution can include magnesium acetate in amount of <NUM> to <NUM>.

Optionally, the reagent solution can also include methylcellulose. For example, the reagent solution can include methylcellulose in an amount of <NUM> volume percent to <NUM> volume percent, such as an amount of <NUM> volume percent to <NUM> volume percent or <NUM> volume percent to <NUM> volume percent.

In an additional example, the reagent solution can include phosphocreatine in amount of <NUM> to <NUM>, such as <NUM> to <NUM> or <NUM> to <NUM>.

When forming an amplification solution, the reagent solution can include nucleotides. For example, the reagent solution can include amounts of each of A, G, C, and T nucleotides or optionally, universal or modified nucleotides. In an example, the nucleotides are found in the reagent solution in an amount of <NUM> to <NUM>. In particular example, the nucleotides are included in amount of <NUM> to <NUM>, such as <NUM> to <NUM>.

Reagent solution can further include primers. For example, such primers may be complementary to positions on target nucleotides. In an example, primers are polynucleotides having between <NUM> and <NUM> bases. Optionally, the primers can include linker moieties. For example, the primer can be coupled to biotin, streptavidin, neutravidin, or avidin, among others.

Further, the reagent solution can include enzymes. Example enzymes include recombinase, polymerase, pyrophosphatase, kinase, or any combination thereof.

Reagent solution can also include stabilizers, such as protein stabilizers.

In a particular example, the reagent solution also includes migration inhibitors. Example migration inhibitors include thickeners or capture molecules.

Such reagent solutions find particular use in rehydrating nucleotides for use in amplification of target polynucleotides. As illustrated in <FIG>, a method <NUM> includes rehydrating nucleotides with a rehydration buffer solution to form a nucleotides solution, as illustrated at block <NUM>. In an example, embodiments of the rehydration buffer solution described above, for example, can include <NUM>-[<NUM>-(<NUM>-hydroxyethyl)piperazin-<NUM>-yl]ethanesulfonic acid, [<NUM>-(methacryloyloxy)ethyl]dimethyl-(<NUM>-sulfopropyl)ammonium hydroxide, and tris(hydroxymethyl)aminomethane. Optionally, the rehydration buffer can include PEG and polyoxyethylene sorbitan monolaurate.

As illustrated at block <NUM>, a first amplification solution can be derived from a portion of the nucleotides solution. Optionally, additive solutions are added to the nucleotide solution. In an example, the first amplification solution includes a primer coupled to a linker moiety.

Optionally, a second application solution can be prepared from another portion of the nucleotides solution, as illustrated at block <NUM>. In particular, a different set of primers or primers free of the linker moieties can be added to the second amplification solution.

In an example, the first amplification of target polynucleotides can be performed using the first amplification solution, as illustrated at block <NUM>. In particular, the application can be performed in accordance with RPA or PCR, as described in more detail below.

Optionally, a second amplification of the target polynucleotides can be performed using the second amplification solution, as illustrated at block <NUM>. Alternatively, the first amplification solution can be used for both the first amplification and the second amplification.

The above reagent solutions and methods find particular use in preparing monoclonal populations for use in sequencing. For example, such monoclonal populations may be formed on a plate and find use in optical sequencing. In another example, such monoclonal populations can be disposed in wells and sequencing can be performed using semiconductor or solid-state sensors. A particular example is illustrated in <FIG>.

In <FIG>, a system <NUM> containing fluidics circuit <NUM> is connected by inlets to at least two reagent reservoirs (<NUM>, <NUM>, <NUM>, <NUM>, or <NUM>), to waste reservoir <NUM>, and to biosensor <NUM> by fluid pathway <NUM> that connects fluidics node <NUM> to inlet <NUM> of biosensor <NUM> for fluidic communication. Reagents from reservoirs (<NUM>, <NUM>, <NUM>, <NUM>, or <NUM>) can be driven to fluidic circuit <NUM> by a variety of methods including pressure, pumps, such as syringe pumps, gravity feed, and the like, and are selected by control of valves <NUM>. Reagents from the fluidics circuit <NUM> can be driven through the valves <NUM> receiving signals from control system <NUM> to waste container <NUM>. Reagents from the fluidics circuit <NUM> can also be driven through the biosensor <NUM> to the waste container <NUM>. The control system <NUM> includes controllers for valves <NUM>, which generate signals for opening and closing via electrical connection <NUM>.

The control system <NUM> also includes controllers for other components of the system, such as wash solution valve <NUM> connected thereto by electrical connection <NUM>, and reference electrode <NUM>. Control system <NUM> can also include control and data acquisition functions for biosensor <NUM>. In one mode of operation, fluidic circuit <NUM> delivers a sequence of selected reagents <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> to biosensor <NUM> under programmed control of control system <NUM>, such that in between selected reagent flows, fluidics circuit <NUM> is primed and washed, and biosensor <NUM> is washed. Fluids entering biosensor <NUM> exit through outlet <NUM> and are deposited in waste container <NUM> via control of pinch valve regulator <NUM>. The valve <NUM> is in fluidic communication with the sensor fluid output <NUM> of the biosensor <NUM>.

The device including the dielectric layer defining the well formed from the first access and second access and exposing a sensor pad finds particular use in detecting chemical reactions and byproducts, such as detecting the release of hydrogen ions in response to nucleotide incorporation, useful in genetic sequencing, among other applications. In a particular embodiment, a sequencing system includes a flow cell in which a sensory array is disposed, includes communication circuitry in electronic communication with the sensory array, and includes containers and fluid controls in fluidic communication with the flow cell. In an example, <FIG> illustrates an expanded and cross-sectional view of a flow cell <NUM> and illustrates a portion of a flow chamber <NUM>. A reagent flow <NUM> flows across a surface of a well array <NUM>, in which the reagent flow <NUM> flows over the open ends of wells of the well array <NUM>. The well array <NUM> and a sensor array <NUM> together may form an integrated unit forming a lower wall (or floor) of flow cell <NUM>. A reference electrode <NUM> may be fluidly coupled to flow chamber <NUM>. Further, a flow cell cover <NUM> encapsulates flow chamber <NUM> to contain reagent flow <NUM> within a confined region.

<FIG> illustrates an expanded view of a well <NUM> and a sensor <NUM>, as illustrated at <NUM> of <FIG>. The volume, shape, aspect ratio (such as base width-to-well depth ratio), and other dimensional characteristics of the wells may be selected based on the nature of the reaction taking place, as well as the reagents, byproducts, or labeling techniques (if any) that are employed. The sensor <NUM> can be a chemical field-effect transistor (chemFET), more specifically an ion-sensitive FET (ISFET), with a floating gate <NUM> having a sensor plate <NUM> optionally separated from the well interior by a material layer <NUM>. The sensor <NUM> can be responsive to (and generate an output signal related to) the amount of a charge <NUM> present on the material layer <NUM> opposite the sensor plate <NUM>. The material layer <NUM> can be a ceramic layer, such as an oxide of zirconium, hafnium, tantalum, aluminum, or titanium, among others, or a nitride of titanium. Alternatively, the material layer <NUM> can be formed of a metal, such as titanium, tungsten, gold, silver, platinum, aluminum, copper, or a combination thereof. In an example, the material layer <NUM> can have a thickness in a range of <NUM> to <NUM>, such as a range of <NUM> to <NUM>, a range of <NUM> to <NUM>, or even a range of <NUM> to <NUM>.

While the material layer <NUM> is illustrated as extending beyond the bounds of the illustrated FET component, the material layer <NUM> can extend along the bottom of the well <NUM> and optionally along the walls of the well <NUM>. The sensor <NUM> can be responsive to (and generate an output signal related to) the amount of a charge <NUM> present on the material layer <NUM> opposite the sensor plate <NUM>. Changes in the charge <NUM> can cause changes in a current between a source <NUM> and a drain <NUM> of the chemFET. In turn, the chemFET can be used directly to provide a current-based output signal or indirectly with additional circuitry to provide a voltage-based output signal. Reactants, wash solutions, and other reagents may move in and out of the wells by a diffusion mechanism <NUM>.

The well <NUM> can be defined by a wall structure, which can be formed of one or more layers of material. In an example, the wall structure can have a thickness extending from the lower surface to the upper surface of the well in a range of <NUM> micrometers to <NUM> micrometers, such as a range of <NUM> micrometers to <NUM> micrometers, a range of <NUM> micrometers to <NUM> micrometers, a range of <NUM> micrometers to <NUM> micrometers, or a range of <NUM> micrometers to <NUM> micrometers. In particular, the thickness can be in a range of <NUM> micrometers to <NUM> micrometer, such as a range of <NUM> micrometers to <NUM> micrometers, or a range of <NUM> micrometers to <NUM> micrometers. The wells <NUM> of array <NUM> can have a characteristic diameter, defined as the square root of <NUM> times the cross-sectional area (A) divided by Pi (e.g., sqrt(<NUM>*A/π)), of not greater than <NUM> micrometers, such as not greater than <NUM> micrometers, not greater than <NUM> micrometers, not greater than <NUM> micrometers, not greater than <NUM> micrometers, not greater than <NUM> micrometers or even not greater than <NUM> micrometers. In an example, the wells <NUM> can have a characteristic diameter of at least <NUM> micrometers. In a further example, the well <NUM> can define a volume in a range of <NUM> fL to <NUM> pL, such as a volume in a range of <NUM> fL to <NUM> pL, a range of <NUM> fL to <NUM> fL, a range of <NUM> fL to <NUM> fL, or even a range of <NUM> fL to <NUM> fL.

In an embodiment, reactions carried out in the well <NUM> can be analytical reactions to identify or determine characteristics or properties of an analyte of interest. Such reactions can generate directly or indirectly byproducts that affect the amount of charge adjacent to the sensor plate <NUM>. If such byproducts are produced in small amounts or rapidly decay or react with other constituents, then multiple copies of the same analyte may be analyzed in the well <NUM> at the same time in order to increase the output signal generated. In an embodiment, multiple copies of an analyte may be attached to a solid phase support <NUM>, either before or after deposition into the well <NUM>. The solid phase support <NUM> may be microparticles, nanoparticles, beads, solid or porous comprising gels, or the like. For simplicity and ease of explanation, solid phase support <NUM> is also referred herein as a particle or bead. For a nucleic acid analyte, multiple, connected copies may be made by rolling circle amplification (RCA), exponential RCA, or like techniques, to produce an amplicon without the need of a solid support.

In particular, the solid phase support, such a bead support, can include copies of polynucleotides. In a particular example illustrated in <FIG>, polymeric particles can be used as a support for polynucleotides during sequencing techniques. For example, such hydrophilic particles can immobilize a polynucleotide for sequencing using fluorescent sequencing techniques. In another example, the hydrophilic particles can immobilize a plurality of copies of a polynucleotide for sequencing using ion-sensing techniques. Alternatively, the above described treatments can improve polymer matrix bonding to a surface of a sensor array. The polymer matrices can capture analytes, such as polynucleotides for sequencing.

A bead support may be composed of organic polymers such as polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy, and polyacrylamide, as well as co-polymers and grafts thereof. A support may also be inorganic, such as glass, silica, controlled-pore-glass (CPG), or reverse-phase silica. The configuration of a support may be in the form of beads, spheres, particles, granules, a gel, or a surface. Supports may be porous or non-porous, and may have swelling or non-swelling characteristics. In some embodiments, a support is an Ion Sphere Particle. Example bead supports are disclosed in <CIT>, titled "Hydrophilic Polymeric Particles and Methods for Making and Using Same," and in <CIT>, titled "Polymer Substrates Formed from Carboxy Functional Acrylamide,".

In some embodiments, the solid support is a "microparticle," "bead," "microbead," etc., (optionally but not necessarily spherical in shape) having a smallest cross-sectional length (e.g., diameter) of <NUM> microns or less, preferably <NUM> microns or less, <NUM> microns or less, approximately <NUM> micron or less, approximately <NUM> microns or less, e.g., approximately <NUM>, <NUM>, <NUM>, or <NUM> microns, or smaller (e.g., under <NUM> nanometer, about <NUM>-<NUM> nanometer, about <NUM>-<NUM> nanometers, or about <NUM>-<NUM> nanometers). In an example, the support is at least <NUM> microns. Microparticles or bead supports may be made of a variety of inorganic or organic materials including, but not limited to, glass (e.g., controlled pore glass), silica, zirconia, cross-linked polystyrene, polyacrylate, polymethylmethacrylate, titanium dioxide, latex, polystyrene, etc. Magnetization can facilitate collection and concentration of the microparticle-attached reagents (e.g., polynucleotides or ligases) after amplification, and can also facilitate additional steps (e.g., washes, reagent removal, etc.). In certain embodiments, a population of microparticles having different shapes sizes or colors is used. The microparticles can optionally be encoded, e.g., with quantum dots such that each microparticle or group of microparticles can be individually or uniquely identified.

Magnetic beads (e.g., Dynabeads from Dynal, Oslo, Norway) can have a size in a range of <NUM> micron to <NUM> microns, such as <NUM> microns to <NUM> microns. The magnetic beads can be formed of inorganic or organic materials including, but not limited to, glass (e.g., controlled pore glass), silica, zirconia, cross-linked polystyrene, polystyrene, or a combination thereof.

In some embodiments, a bead support is functionalized for attaching a population of first primers. In some embodiments, a bead is any size that can fit into a reaction chamber. For example, one bead can fit in a reaction chamber. In some embodiments, more than one bead fit in a reaction chamber. In some embodiments, the smallest cross-sectional length of a bead (e.g., diameter) is about <NUM> microns or less, or about <NUM> microns or less, or about <NUM> microns or less, approximately <NUM> micron or less, approximately <NUM> microns or less, e.g., approximately <NUM>, <NUM>, <NUM>, or <NUM> microns, or smaller (e.g., under <NUM> nanometer, about <NUM>-<NUM> nanometer, about <NUM>-<NUM> nanometers, or about <NUM>-<NUM> nanometers).

In general, the bead support can be treated to include a biomolecule, including nucleosides, nucleotides, nucleic acids (oligonucleotides and polynucleotides), polypeptides, saccharides, polysaccharides, lipids, or derivatives or analogs thereof. For example, a polymeric particle can bind or attach to a biomolecule. A terminal end or any internal portion of a biomolecule can bind or attach to a polymeric particle. A polymeric particle can bind or attach to a biomolecule using linking chemistries. A linking chemistry includes covalent or non-covalent bonds, including an ionic bond, hydrogen bond, affinity bond, dipole-dipole bond, van der Waals bond, and hydrophobic bond. A linking chemistry includes affinity between binding partners, for example between: an avidin moiety and a biotin moiety; an antigenic epitope and an antibody or immunologically reactive fragment thereof; an antibody and a hapten; a digoxigen moiety and an anti-digoxigen antibody; a fluorescein moiety and an anti-fluorescein antibody; an operator and a repressor; a nuclease and a nucleotide; a lectin and a polysaccharide; a steroid and a steroid-binding protein; an active compound and an active compound receptor; a hormone and a hormone receptor; an enzyme and a substrate; an immunoglobulin and protein A; or an oligonucleotide or polynucleotide and its corresponding complement.

As illustrated in <FIG>, a plurality of bead supports <NUM> can be placed in a solution along with a plurality of polynucleotides <NUM> (target or template poylnucleotides). The plurality of bead supports <NUM> can be activated or otherwise prepared to bind with the polynucleotides <NUM>. For example, the bead supports <NUM> can include an oligonucleotide (capture primer) complementary to a portion of a polynucleotide of the plurality of polynucleotides <NUM>. In another example, the bead supports <NUM> can be modified with target polynucleotides <NUM> using techniques such as biotin-streptavidin binding.

In some embodiments, the template nucleic acid molecules (template polynucleotides or target polynucleotides) can be derived from a sample that can be from a natural or non-natural source. The nucleic acid molecules in the sample can be derived from a living organism or a cell. Any nucleic acid molecule can be used, for example, the sample can include genomic DNA covering a portion of or an entire genome, mRNA, or miRNA from the living organism or cell. In other embodiments, the template nucleic acid molecules can be synthetic or recombinant. In some embodiments, the sample contains nucleic acid molecules having substantially identical sequences or having a mixture of different sequences. Illustrative embodiments are typically performed using nucleic acid molecules that were generated within and by a living cell. Such nucleic acid molecules are typically isolated directly from a natural source such as a cell or a bodily fluid without any in vitro amplification. Accordingly, the sample nucleic acid molecules are used directly in subsequent steps. In some embodiments, the nucleic acid molecules in the sample can include two or more nucleic acid molecules with different sequences.

The methods can optionally include a target enrichment step before, during, or after the library preparation and before a pre-seeding reaction. Target nucleic acid molecules, including target loci or regions of interest, can be enriched, for example, through multiplex nucleic acid amplification or hybridization. A variety of methods can be used to perform multiplex nucleic acid amplification to generate amplicons, such as multiplex PCR, and can be used in an embodiment. Enrichment by any method can be followed by a universal amplification reaction before the template nucleic acid molecules are added to a pre-seeding reaction mixture. Any of the embodiments of the present teachings can include enriching a plurality of at least <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>,<NUM>, <NUM>,<NUM>, <NUM>,<NUM>, <NUM>,<NUM>, <NUM>,<NUM>, <NUM>,<NUM>, <NUM>,<NUM>, <NUM>,<NUM>, <NUM>,<NUM>, or <NUM>,<NUM> target nucleic acid molecules, target loci, or regions of interest. In any of the disclosed embodiments, the target loci or regions of interest can be at least <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>,<NUM> nucleotides in length and include a portion of or the entirety of the template nucleic acid molecule. In other embodiments, the target loci or regions of interest can be between about <NUM> and <NUM>,<NUM> nucleotides in length, for example between about <NUM> and <NUM>,<NUM> nucleotides, between about <NUM> and <NUM>,<NUM> nucleotides, or between about <NUM> and <NUM>,<NUM> nucleotides in length. In any of the embodiments of the present teachings, the multiplex nucleic acid amplification can include generating at least <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>,<NUM>, <NUM>,<NUM>, <NUM>,<NUM>, <NUM>,<NUM>, <NUM>,<NUM>, <NUM>,<NUM>, <NUM>,<NUM>, <NUM>,<NUM>, <NUM>,<NUM>, or <NUM>,<NUM> copies of each target nucleic acid molecule, target locus, or region of interest.

In some embodiments, after the library preparation and optional enrichment step, the library of template nucleic acid molecules can be templated onto one or more supports. The one or more supports can be templated in two reactions, a seeding reaction to generate pre-seeded solid supports and a templating reaction using the one or more pre-seeded supports to further amplify the attached template nucleic acid molecules. The pre-seeding reaction is typically an amplification reaction and can be performed using a variety of methods. For example, the pre-seeding reaction can be performed in an RPA reaction, a template walking reaction, or a PCR. In an RPA reaction, template nucleic acid molecules are amplified using a recombinase, polymerase, and optionally a recombinase accessory protein in the presence of primers and nucleotides. The recombinase and optionally the recombinase accessory protein can dissociate at least a portion of a double stranded template nucleic acid molecules to allow primers to hybridize that the polymerase can then bind to initiate replication. In some embodiments, the recombinase accessory protein can be a single-stranded binding protein (SSB) that prevents the re-hybridization of dissociated template nucleic acid molecules. Typically, RPA reactions can be performed at isothermal temperatures. In a template walking reaction, template nucleic acid molecules are amplified using a polymerase in the presence of primers and nucleotides in reaction conditions that allow at least a portion of double-stranded template nucleic acid molecules to dissociate such that primers can hybridize and the polymerase can then bind to initiate replication. In PCR, the double-stranded template nucleic acid molecules are dissociated by thermal cycling. After cooling, primers bind to complementary sequences and can be used for replication by the polymerase. In any of the aspects of the present teachings, the pre-seeding reaction can be performed in a pre-seeding reaction mixture, which is formed with the components necessary for amplification of the template nucleic acid molecules. In any of the disclosed aspects, the pre-seeding reaction mixture can include some or all of the following: a population of template nucleic acid molecules, a polymerase, one or more solid supports with a population of attached first primers, nucleotides, and a cofactor such as a divalent cation. In some embodiments, the pre-seeding reaction mixture can further include a second primer and optionally a diffusion-limiting agent. In some embodiments, the population of template nucleic acid molecules comprise template nucleic acid molecules joined to at least one adaptor sequence which can hybridize to the first or second primers. In some embodiments, the reaction mixture can form an emulsion, as in emulsion RPA or emulsion PCR. In pre-seeding reactions carried out by RPA reactions, the pre-seeding reaction mixture can include a recombinase and optionally a recombinase accessory protein. The various components of the reaction mixture are discussed in further detail herein.

In a particular embodiment of seeding, the hydrophilic particles and polynucleotides are subjected to polymerase chain reaction (PCR) amplification or recombinase polymerase amplification (RPA). In an example, the particles <NUM> include a capture primer complementary to a portion of the template polynucleotide <NUM>. The template polynucleotide can hybridize to the capture primer. The capture primer can be extended to form beads <NUM> that include a target polynucleotide attached thereto. Other beads may remain unattached to a target nucleic acid and other template polynucleotide can be free floating in solution.

In an example, the bead support <NUM> including a target polynucleotide can be attached to a magnetic bead <NUM> to form a bead assembly <NUM>. In particular, the magnetic bead <NUM> is attached to the bead support <NUM> by a double stranded polynucleotide linkage. In an example, a further probe including a linker moiety can hybridize to a portion of the target polynucleotide on the bead support <NUM>. The linker moiety can be attached to a complementary linker moiety on the magnetic bead <NUM>. In another example, the template polynucleotide used to form the target nucleic acid attached to beads <NUM> can include a linker moiety that attaches to the magnetic bead <NUM>. In another example, the template polynucleotide complementary to target polynucleotide attached to the bead support <NUM> can be generated from a primer that is modified with a linker that attaches to the magnetic bead <NUM>.

The linker moiety attached to the polynucleotide and the linker moiety attached to the magnetic bead can be complementary to and attach to each other. In an example, the linker moieties have affinity and can include: an avidin moiety and a biotin moiety; an antigenic epitope and an antibody or immunologically reactive fragment thereof; an antibody and a hapten; a digoxigen moiety and an anti-digoxigen antibody; a fluorescein moiety and an anti-fluorescein antibody; an operator and a repressor; a nuclease and a nucleotide; a lectin and a polysaccharide; a steroid and a steroid-binding protein; an active compound and an active compound receptor; a hormone and a hormone receptor; an enzyme and a substrate; an immunoglobulin and protein A; or an oligonucleotide or polynucleotide and its corresponding complement. In a particular example, the linker moiety attached to the polynucleotide includes biotin and the linker moiety attached to the magnetic bead includes streptavidin.

The bead assemblies <NUM> can be applied over a substrate <NUM> of a sequencing device that includes wells <NUM>. In an example, a magnetic field can be applied to the substrate <NUM> to draw the magnetic beads <NUM> of the bead assembly <NUM> towards the wells <NUM>. The bead support <NUM> enters the well <NUM>. For example, a magnet can be moved in parallel to a surface of the substrate <NUM> resulting in the deposition of the bead support <NUM> in the wells <NUM>.

The bead assembly <NUM> can be denatured to remove the magnetic bead <NUM> leaving the bead support <NUM> in the well <NUM>. For example, hybridized double-stranded DNA of the bead assembly <NUM> can be denatured using thermal cycling or ionic solutions to release the magnetic bead <NUM> and template polynucleotides having a linker moiety attached to the magnetic bead <NUM>. For example, the double-stranded DNA can be treated with low ion-content aqueous solutions, such as deionized water, to denature and separate the strands. In an example, a foam wash can be used to remove the magnetic beads.

Optionally, the target polynucleotides <NUM> can be amplified, referred to herein as templating, while in the well <NUM>, to provide a bead support <NUM> with multiple copies of the target polynucleotides. In particular, the bead <NUM> has a monoclonal population of target polynucleotides. Such an amplification reactions can be performed using polymerase chain reaction (PCR) amplification, recombination polymerase amplification (RPA) or a combination thereof. Alternatively, amplification can be performed prior to depositing the bead support <NUM> in the well.

In a particular embodiment, an enzyme such as a polymerase is present, bound to, or is in close proximity to the particles or beads. In an example, a polymerase is present in solution or in the well to facilitate duplication of the polynucleotide. A variety of nucleic acid polymerase may be used in the methods described herein. In an example embodiment, the polymerase can include an enzyme, fragment, or subunit thereof, which can catalyze duplication of the polynucleotide. In another embodiment, the polymerase can be a naturally occurring polymerase, recombinant polymerase, mutant polymerase, variant polymerase, fusion or otherwise engineered polymerase, chemically modified polymerase, synthetic molecules, or analog, derivative or fragment thereof. Example enzymes, solutions, compositions, and amplification methods can be found in <CIT>, titled "METHODS AND COMPOSITIONS FOR MANIPULATING NUCLEIC ACIDS".

While the polynucleotides of bead support <NUM> are illustrated as being on a surface, the polynucleotides can extend within the bead support <NUM>. Hydrogel and hydrophilic particles having a low concentration of polymer relative to water can include polynucleotide segments on the interior of and throughout the bead support <NUM> or polynucleotides can reside in pores and other openings. In particular, the bead support <NUM> can permit diffusion of enzymes, nucleotides, primers, and reaction products used to monitor the reaction. A high number of polynucleotides per particle produces a better signal.

In an example embodiment, the bead support <NUM> can be utilized in a sequencing device. For example, a sequencing device <NUM> can include an array of wells <NUM>.

In an example, a sequencing primer can be added to the wells <NUM> or the bead support <NUM> can be pre-exposed to the primer prior to placement in the well <NUM>. In particular, the bead support <NUM> can include bound sequencing primer. The sequencing primer and polynucleotide form a nucleic acid duplex including the polynucleotide (e.g., a template nucleic acid) hybridized to the sequencing primer. The nucleic acid duplex is an at least partially double-stranded polynucleotide. Enzymes and nucleotides can be provided to the well <NUM> to facilitate detectible reactions, such as nucleotide incorporation.

Sequencing can be performed by detecting nucleotide addition. Nucleotide addition can be detected using methods such as fluorescent emission methods or ion detection methods. For example, a set of fluorescently labeled nucleotides can be provided to the system <NUM> and can migrate to the well <NUM>. Excitation energy can be also provided to the well <NUM>. When a nucleotide is captured by a polymerase and added to the end of an extending primer, a label of the nucleotide can fluoresce, indicating which type of nucleotide is added.

In an alternative example, solutions including a single type of nucleotide can be fed sequentially. In response to nucleotide addition, the pH within the local environment of the well <NUM> can change. Such a change in pH can be detected by ion sensitive field effect transistors (ISFET). As such, a change in pH can be used to generate a signal indicating the order of nucleotides complementary to the polynucleotide of the particle <NUM>.

In particular, a sequencing system can include a well, or a plurality of wells, disposed over a sensor pad of an ionic sensor, such as a field effect transistor (FET). In embodiments, a system includes one or more polymeric particles loaded into a well which is disposed over a sensor pad of an ionic sensor (e.g., FET), or one or more polymeric particles loaded into a plurality of wells which are disposed over sensor pads of ionic sensors (e.g., FET). In embodiments, an FET can be a chemFET or an ISFET. A "chemFET" or chemical field-effect transistor, includes a type of field effect transistor that acts as a chemical sensor. The chemFET has the structural analog of a MOSFET transistor, where the charge on the gate electrode is applied by a chemical process. An "ISFET" or ion-sensitive field-effect transistor, can be used for measuring ion concentrations in solution; when the ion concentration (such as H+) changes, the current through the transistor changes accordingly.

In embodiments, the FET may be a FET array. As used herein, an "array" is a planar arrangement of elements such as sensors or wells. The array may be one or two dimensional. A one-dimensional array can be an array having one column (or row) of elements in the first dimension and a plurality of columns (or rows) in the second dimension. The number of columns (or rows) in the first and second dimensions may or may not be the same. The FET or array can comprise <NUM><NUM>, <NUM><NUM>, <NUM><NUM>, <NUM><NUM>, <NUM><NUM>, <NUM><NUM> or more FETs.

In embodiments, one or more microfluidic structures can be fabricated above the FET sensor array to provide for containment or confinement of a biological or chemical reaction. For example, in one implementation, the microfluidic structure(s) can be configured as one or more wells (or wells, or reaction chambers, or reaction wells, as the terms are used interchangeably herein) disposed above one or more sensors of the array, such that the one or more sensors over which a given well is disposed detect and measure analyte presence, level, or concentration in the given well. In embodiments, there can be a <NUM>:<NUM> correspondence of FET sensors and reaction wells.

Returning to <FIG>, in another example, a well <NUM> of the array of wells can be operatively connected to measuring devices. For example, for fluorescent emission methods, a well <NUM> can be operatively coupled to a light detection device. In the case of ionic detection, the lower surface of the well <NUM> may be disposed over a sensor pad of an ionic sensor, such as a field effect transistor.

One example system involving sequencing via detection of ionic byproducts of nucleotide incorporation is the Ion Torrent PGM™, Proton™, S5™, or Genexus™ sequencer (Thermo Fisher Scientific), which is an ion-based sequencing system that sequences nucleic acid templates by detecting hydrogen ions produced as a byproduct of nucleotide incorporation. Typically, hydrogen ions are released as byproducts of nucleotide incorporations occurring during template-dependent nucleic acid synthesis by a polymerase. The Ion Torrent PGM™, Proton™, S5™, or Genexus™ sequencer detects the nucleotide incorporations by detecting the hydrogen ion byproducts of the nucleotide incorporations. The Ion Torrent PGM™, Proton™, S5™, or Genexus™ sequencer can include a plurality of template polynucleotides to be sequenced, each template disposed within a respective sequencing reaction well in an array. The wells of the array can each be coupled to at least one ion sensor that can detect the release of H+ ions or changes in solution pH produced as a byproduct of nucleotide incorporation. The ion sensor comprises a field effect transistor (FET) coupled to an ion-sensitive detection layer that can sense the presence of H+ ions or changes in solution pH. The ion sensor can provide output signals indicative of nucleotide incorporation which can be represented as voltage changes whose magnitude correlates with the H+ ion concentration in a respective well or reaction chamber. Different nucleotide types can be flowed serially into the reaction chamber and can be incorporated by the polymerase into an extending primer (or polymerization site) in an order determined by the sequence of the template. Each nucleotide incorporation can be accompanied by the release of H+ ions in the reaction well, along with a concomitant change in the localized pH. The release of H+ ions can be registered by the FET of the sensor, which produces signals indicating the occurrence of the nucleotide incorporation. Nucleotides that are not incorporated during a particular nucleotide flow may not produce signals. The amplitude of the signals from the FET can also be correlated with the number of nucleotides of a particular type incorporated into the extending nucleic acid molecule thereby permitting homopolymer regions to be resolved. Thus, during a run of the sequencer multiple nucleotide flows into the reaction chamber along with incorporation monitoring across a multiplicity of wells or reaction chambers can permit the instrument to resolve the sequence of many nucleic acid templates simultaneously.

The above reagent solutions and methods provide desirable technical and advantages, particularly when used to amplify polynucleotides before monoclonal populations. It is been found that such solutions reduce biasing in amplification of high GC rich regions of target polynucleotides.

Extracted nucleic acid samples are sequenced using a Genexus Sequencing instrument and commercially available Genexus Sequencing Kits and GX5 chip and coupler. Library is prepared using either the commercially available Oncomine™ Myeloid Assay GX (first version) or the Oncomine™ Myeloid Assay GX in which the rehydration buffer is substituted with a test solution. The test solution includes <NUM> vol% PEG <NUM>, <NUM> vol% Neolone M-<NUM>, <NUM> potassium glutamate, <NUM> HEPES, <NUM> DMAPs, <NUM> Tris, and <NUM> vol% TWEEN and has a pH of <NUM>.

Sequencing runs using the test solution, on average, show at least a <NUM>% improvement in % valid reads, longer mean read lengths, and improved raw read accuracy.

Extracted nucleic acid samples are sequenced using a Genexus Sequencing instrument and commercially available Genexus Sequencing Kits and GX5 chip and coupler. Library is prepared using either the commercially available Oncomine™ Myeloid Assay GX (first version) or the Oncomine™ Myeloid Assay GX in which the rehydration buffer is substituted with the test solution described above. The results focus on CEBPA coverage.

Sequencing runs using the test solution, on average, show better CEBPA base coverage and uniformity. The number of CEBPA amplicon reads improved by at least <NUM>%.

In a first embodiment, a reagent solution includes an aqueous solution including <NUM>-[<NUM>-(<NUM>-hydroxyethyl)piperazin-<NUM>-yl]ethanesulfonic acid in an amount of <NUM> to <NUM>; [<NUM>-(methacryloyloxy)ethyl]dimethyl-(<NUM>-sulfopropyl)ammonium hydroxide in an amount of <NUM> to <NUM>; and tris(hydroxymethyl)aminomethane in an amount of <NUM> to <NUM>.

In a second embodiment, a method for amplifying nucleic acids includes rehydrating nucleotides with a rehydration buffer solution to form a nucleotide solution, the rehydration buffer solution including an aqueous solution of <NUM>-[<NUM>-(<NUM>-hydroxyethyl)piperazin-<NUM>-yl]ethanesulfonic acid, [<NUM>-(methacryloyloxy)ethyl]dimethyl-(<NUM>-sulfopropyl)ammonium hydroxide, and tris(hydroxymethyl)aminomethane. The method further includes preparing an amplification solution using the nucleotide solution and adding enzymes and a primer; and performing amplification in the presence of the amplification.

In an example of the first and second embodiments, the <NUM>-[<NUM>-(<NUM>-hydroxyethyl)piperazin-<NUM>-yl]ethanesulfonic acid is included in an amount of <NUM> to <NUM>. For example, the <NUM>-[<NUM>-(<NUM>-hydroxyethyl)piperazin-<NUM>-yl]ethanesulfonic acid is included in an amount of <NUM> to <NUM>.

In another example of the first and second embodiments and the above examples, the [<NUM>-(methacryloyloxy)ethyl]dimethyl-(<NUM>-sulfopropyl)ammonium hydroxide is included in an amount of <NUM> to <NUM>. For example, the [<NUM>-(methacryloyloxy)ethyl]dimethyl-(<NUM>-sulfopropyl)ammonium hydroxide is included in an amount of <NUM> to <NUM>.

In a further example of the first and second embodiments and the above examples, the tris(hydroxymethyl)aminomethane is included in an amount of <NUM> to <NUM>. For example, the tris(hydroxymethyl)aminomethane is included in an amount of <NUM> to <NUM> or an amount of <NUM> to <NUM>.

In an additional example of the first and second embodiments and the above examples, the reagent solution further includes polyoxyethylene sorbitan monolaurate. For example, the polyoxyethylene sorbitan monolaurate is polyoxyethylene (<NUM>) sorbitan monolaurate. In another example, the polyoxyethylene sorbitan monolaurate is included in an amount of <NUM> vol. % to <NUM> vol. %, such as an amount of <NUM> vol. % to <NUM>. % or an amount of <NUM> vol. % to <NUM> vol.

In another example of the first and second embodiments and the above examples, the reagent solution further includes potassium glutamate in an amount of <NUM> to <NUM>. For example, the potassium glutamate is included in an amount of <NUM> to <NUM>, such as an amount of <NUM> to <NUM>.

In a further example of the first and second embodiments and the above examples, the reagent solution further includes polyethylene glycol is included in an amount of <NUM> vol. % to <NUM> vol. For example, the polyethylene glycol is included in an amount of <NUM> vol. % to <NUM> vol. %, such as an amount of <NUM> vol. % to <NUM> vol. In an example, the polyethylene glycol has a molecular weight in a range of <NUM> to <NUM>, such as a range of <NUM> to <NUM> or a range of <NUM> to <NUM>. In a particular example, the polyethylene glycol includes a first polyethylene glycol with a molecular weight in a range of <NUM> to <NUM> and a second polyethylene glycol with a molecular weight in a range of <NUM> to <NUM>.

In an additional example of the first and second embodiments and the above examples, the reagent solution further includes a biocide. For example, the biocide includes methyl isothiazolinone or isothiazolinone. In an example, the biocide is included in an amount of <NUM> vol. % to <NUM> vol.

In a further example of the first and second embodiments and the above examples, the reagent solution further includes nucleotides in an amount of <NUM> to <NUM>. For example, the nucleotides are included in an amount of <NUM> to <NUM>, such as an amount of <NUM> to <NUM>.

In another example of the first and second embodiments and the above examples, the reagent solution further includes acetic acid in an amount of <NUM> to <NUM>. For example, the acetic acid is included in an amount of <NUM> to <NUM>.

In a further example of the first and second embodiments and the above examples, the reagent solution further includes dithiothreitol in an amount of <NUM> to <NUM>. For example, the dithiothreitol is included in an amount of <NUM> to <NUM>, such as an amount of <NUM> to <NUM>.

In an additional example of the first and second embodiments and the above examples, the reagent solution further includes trehalose in an amount of <NUM> vol. % to <NUM> vol. For example, the trehalose is included in an amount of <NUM> vol. % to <NUM> vol. %, such as an amount of <NUM> vol. % to <NUM> vol.

In another example of the first and second embodiments and the above examples, the reagent solution further includes adenosine triphosphate in an amount of <NUM> to <NUM>. For example, the adenosine triphosphate is included in an amount of <NUM> to <NUM>, such as an amount of <NUM> to <NUM>.

In a further example of the first and second embodiments and the above examples, the reagent solution further includes magnesium acetate in an amount of <NUM> to <NUM>, such as an amount of <NUM> to <NUM>.

In an additional example of the first and second embodiments and the above examples, the reagent solution further includes methyl cellulose in an amount of <NUM> vol. % to <NUM> vol. For example, the methyl cellulose is included in an amount of <NUM> vol. % to <NUM> vol. %, such as an amount of <NUM> vol. % to <NUM> vol.

In another example of the first and second embodiments and the above examples, the reagent solution further includes phosphocreatine in an amount of <NUM> to <NUM>. For example, the phosphocreatine is included in an amount of <NUM> to <NUM>, such as an amount of <NUM> to <NUM>.

In a further example of the first and second embodiments and the above examples, the reagent solution further includes enzymes.

In an additional example of the first and second embodiments and the above examples, the reagent solution further includes stabilizers.

In another example of the first and second embodiments and the above examples, the reagent solution further includes primer. In an example, the primer includes a linker moiety.

In a further example of the first and second embodiments and the above examples, the reagent solution further includes migration inhibitors.

In an additional example of the second embodiment and the above examples, the method further includes preparing a second amplification solution using the nucleotide solution and adding enzymes and a second primer.

In the foregoing specification, the concepts have been described with reference to specific embodiments.

For example, a process, method, article, or apparatus that comprises a list of features is not necessarily limited only to those features but may include other features not expressly listed or inherent to such process, method, article, or apparatus.

Also, the use of "a" or "an" are employed to describe elements and components described herein. This description should be read to include one or at least one and the singular also includes the plural unless it is obvious that it is meant otherwise.

Claim 1:
A reagent solution comprising:
an aqueous solution including:
<NUM>-[<NUM>-(<NUM>-hydroxyethyl)piperazin-<NUM>-yl]ethanesulfonic acid in an amount of <NUM> to <NUM>;
[<NUM>-(methacryloyloxy)ethyl]dimethyl-(<NUM>-sulfopropyl)ammonium hydroxide in an amount of <NUM> to <NUM>; and
tris(hydroxymethyl)aminomethane in an amount of <NUM> to <NUM>.