Method for improving stability of sequencing reagent

Abstract

The present invention belongs to the field of gene sequencing. Provided is a method for improving the stability of a sequencing reagent. For a component containing a substance having poor thermal stability (for example, a component containing a nucleotide, a component containing a redox-active substance, or a component containing a protein), converting the component into a lyophilized form improves the stability thereof. This allows the sequencing reagent to maintain stability in a normal-temperature or high-temperature environment, thereby solving the problems of existing sequencing reagents (poor thermal stability during transportation in a room-temperature or high-temperature environment, degradation of active ingredients, increase of by-products, and serious impact on the quality of sequencing data), and reducing costs.

Description

Method for improving stability of sequencing reagent TECHNICAL FIELD

[0001] The present application belongs to the field of gene sequencing, and particularly relates to a method for improving stability of sequencing reagent. BACKGROUND

[0002] Gene sequencing technology can predict the possibility of suffering from diseases and lock pathogenic genes through reading of genomes, and is an important tool for genetics and molecular biology.

[0003] In 1975, Sanger et al. initiated the first generation of sequencing technology, and with the help of the first generation of sequencing technology, the first human genome map was completed in 2001. The first generation of sequencing technology has the disadvantages of low throughput and high cost. In 2005, the first second-generation sequencer was launched, and sequencing technology entered the high-throughput era. The update and iteration of technology have continuously reduced the cost of second-generation sequencing, and made it the most mainstream sequencing technology in the field of life science.

[0004] At present, most sequencing reagents are in liquid form, and need to be kept in a low-temperature frozen state of-15 to-25 DEG C during storage and transportation. During the process of transporting the reagent box from the factory to the customer's location, it usually needs to be experienced for several days to several tens of days, and a large amount of dry ice or ice bag needs to be added. The purchase of dry ice or ice bag itself increases the transportation cost, and the volume occupied by dry ice or ice bag further increases the transportation cost.

[0005] If the sequencing reagent can be transported without temperature control, its cost can be greatly reduced. The conversion of unstable components into a freeze-dried form can improve its stability. At present, the freeze-drying technology of drugs, proteins and strains is widely and maturely studied, and the feasibility of freeze-drying liquid reagents to improve stability and reduce cost is verified. The freeze-drying formula and the key components to be protected are closely related, and the freeze-drying formulas of different substances are quite different. In addition, the components of sequencing reagents are more complex and larger in volume. Therefore, the freeze-drying technology of drugs, proteins and strains has limited guiding effect on the freeze-drying of sequencing reagents. For non-freeze-drying components with good thermal stability or components not suitable for freeze-drying, non-temperature-controlled transportation promotes the reproduction of bacteria.

[0006] Therefore, it is necessary to develop a method for improving the stability of sequencing reagent.

[0007] SUMMARY

[0008] The first aspect of the embodiment of the present application aims to provide a composition.

[0009] The second aspect of the embodiment of the present application aims to provide a sequencing reagent box or a sequencing reagent tank.

[0010] The third aspect of the embodiment of the present application aims to provide a method for reconstituting a composition.

[0011] The fourth aspect of the embodiments of the present application aims to provide a sequencing method.

[0012] The fifth aspect of the embodiments of the present application aims to provide a method for improving the stability of a sequencing reagent.

[0013] To achieve the above-mentioned object, the technical solutions adopted by the embodiments of the present application are as follows:

[0014] The first aspect of the embodiments of the present application provides a composition, which is any one of a1) to a3):

[0015] a1) The composition comprises: a lyophilization protective agent and a component containing nucleotides, the nucleotides being natural nucleotides and / or modified nucleotides;

[0016] a2) The composition comprises: a lyophilization protective agent and a component containing a redox active substance;

[0017] a3) The composition comprises: a lyophilization protective agent and a component containing a protein;

[0018] The composition in a1), a2) and a3) is a lyophilized composition.

[0019] In an embodiment, the lyophilization protective agent in a1), a2) and a3) is independently selected from one or more of the following: a polyhydroxyl compound, a sugar, a polymer.

[0020] In an embodiment, the lyophilization protective agent in a1) is any one of b1) to b5): b1) a polymer, a polyhydroxyl compound and a sugar; b2) a polymer and a sugar; b3) a polyhydroxyl compound and a sugar; b4) a polyhydroxyl compound; b5) a sugar.

[0021] In an embodiment, the lyophilization protective agent in a1) is any one of c1) to c14):

[0022] c1) mannitol and sucrose; c2) mannitol and trehalose; c3) polyvinylpyrrolidone K30 and sucrose; c4) polyvinylpyrrolidone K30 and trehalose; c5) polyvinylpyrrolidone K60 and trehalose; c6) polyvinylpyrrolidone K90 and trehalose; c7) (2-hydroxyethyl)-β-cyclodextrin; c8) hydroxypropyl-β-cyclodextrin; c9) polyvinylpyrrolidone K30, trehalose and (2-hydroxyethyl)-β-cyclodextrin; c10) polyvinylpyrrolidone K30, trehalose and hydroxypropyl-β-cyclodextrin; c11) polyvinylpyrrolidone K30, sucrose and (2-hydroxyethyl)-β-cyclodextrin; c12) polyvinylpyrrolidone K30, sucrose and hydroxypropyl-β-cyclodextrin; c13) polyvinylpyrrolidone K30, trehalose and hydroxyethyl starch; c14) polyvinylpyrrolidone K30, sucrose and hydroxyethyl starch.

[0023] In one embodiment, the mass ratio of the mannitol and sucrose in c1) is (3 to 5) : 1.

[0024] In one embodiment, the mass ratio of the mannitol and trehalose in c2) is (2 to 4) : 1.

[0025] In one embodiment, the mass ratio of the polyvinylpyrrolidone K30 and sucrose in c3) is (1 to 5) : 1.

[0026] In one embodiment, the mass ratio of the polyvinylpyrrolidone K30 and trehalose in c4) is (0.1 to 3.5) : 1.

[0027] In one embodiment, the mass ratio of the polyvinylpyrrolidone K60 and trehalose in c5) is (1 to 2) : 1.

[0028] In one embodiment, the mass ratio of the polyvinylpyrrolidone K90 and trehalose in c6) is (1 to 2) : 1.

[0029] In one embodiment, the mass ratio of the polyvinylpyrrolidone K30, trehalose and (2-hydroxyethyl)-β-cyclodextrin in c9) is (1 to 3) : (1 to 3) : 1.

[0030] In one embodiment, the mass ratio of the polyvinylpyrrolidone K30, trehalose and hydroxypropyl-β-cyclodextrin in c10) is (1 to 3) : (1 to 3) : 1.

[0031] In one embodiment, the mass ratio of the polyvinylpyrrolidone K30, sucrose and (2-hydroxyethyl)-β-cyclodextrin in c11) is (1 to 3) : (1 to 3) : 1.

[0032] In an embodiment, the mass ratio of the polyvinylpyrrolidone K30, sucrose and hydroxypropyl-β-cyclodextrin in c12) is (1-3):(1-3):1.

[0033] In an embodiment, the mass ratio of the polyvinylpyrrolidone K30, trehalose and hydroxyethyl starch in c13) is (1-3):(1-3):1.

[0034] In an embodiment, the mass ratio of the polyvinylpyrrolidone K30, sucrose and hydroxyethyl starch in c14) is (1-3):(1-3):1.

[0035] In an embodiment, the lyoprotectant in a2) is polyethylene glycol; further is at least one of PEG2000, PEG4000, PEG6000, PEG8000, PEG10000, PEG20000.

[0036] In an embodiment, the lyoprotectant in a3) is D-mannitol.

[0037] In an embodiment, the mass ratio or mass volume ratio of the lyoprotectant and the components in a1), a2), a3) is (3-20):50.

[0038] In an embodiment, the mass ratio of the lyoprotectant and the redox-active substance in a2) is 1:(0.3-1.5).

[0039] In an embodiment, the mass volume ratio of the lyoprotectant and the protein-containing component in a3) is 1:(5-15).

[0040] In an embodiment, the nucleotide in a1) is a modified nucleotide.

[0041] In an embodiment, the redox-active substance in a2) comprises: at least one of THPP, TCEP.

[0042] In an embodiment, the component in a2) is a regenerating reagent.

[0043] In an embodiment, the protein in a3) is a non-enzyme protein.

[0044] In an embodiment, the protein-containing component in a3) is a protein washing buffer.

[0045] In an embodiment, the lyophilized composition in a1), a2), a3) is a microsphere, a powder cake or a combination thereof.

[0046] In an embodiment, the microsphere is spherical, elliptical or annular.

[0047] In a second aspect, the present application provides a sequencing kit or a sequencing reagent tank comprising the composition of the first aspect.

[0048] In a third aspect, the present application provides a method for reconstituting a composition, wherein the composition is mixed with a reconstitution reagent to obtain a reconstitution solution, and the composition is the composition of a1), a2), or a3) of the first aspect.

[0049] In an embodiment, the reconstitution reagent comprises water, ethanolamine, a buffer, or a combination thereof.

[0050] In an embodiment, the mixing is performed under conditions effective to reconstitute the composition.

[0051] In an embodiment, the reconstitution solution is used in a sequencing process.

[0052] In a fourth aspect, the present application provides a sequencing method, comprising the following steps:

[0053] f1) mixing a composition with a reconstitution reagent to obtain a reconstitution solution;

[0054] f2) using a reagent tank comprising the reconstitution solution to perform sequencing;

[0055] The composition of f1) is the composition of a1), a2), or a3) of the first aspect.

[0056] In an embodiment, the reconstitution reagent comprises water, ethanolamine, a buffer, or a combination thereof.

[0057] In an embodiment, the mixing is performed under conditions effective to reconstitute the composition.

[0058] In a fifth aspect, the present application provides a method for improving the stability of a sequencing reagent,

[0059] The sequencing reagent comprises a component comprising a substance with poor thermal stability.

[0060] The component comprising the substance with poor thermal stability comprises a component comprising a nucleotide, a component comprising a redox-active substance, or a component comprising a protein.

[0061] The nucleotide is a natural nucleotide and / or a modified nucleotide.

[0062] The method comprises the following steps:

[0063] The substance with poor thermal stability, or the component comprising the substance with poor thermal stability, or the sequencing reagent, is mixed with a lyophilization protective agent and is lyophilized.

[0064] In one embodiment, the component containing a nucleotide, the component containing a redox-active substance, or the component containing a protein is the component containing a nucleotide, the component containing a redox-active substance, or the component containing a protein in the first aspect of the embodiment of the present application.

[0065] In one embodiment, the mass ratio or mass to volume ratio of the lyoprotectant to the component containing a substance with poor thermal stability is the mass ratio or mass to volume ratio of the lyoprotectant to the component as described in a1), a2), a3) in the first aspect of the embodiment of the present application.

[0066] In one embodiment, the component containing a substance with poor thermal stability comprises: a component containing a nucleotide, the lyoprotectant is the lyoprotectant in a1) in the first aspect of the embodiment of the present application. In one embodiment, the component containing a substance with poor thermal stability comprises: a component containing a redox-active substance, the lyoprotectant is the lyoprotectant in a2) in the first aspect of the embodiment of the present application.

[0067] In one embodiment, the component containing a substance with poor thermal stability comprises: a component containing a redox-active substance, the mass ratio of the lyoprotectant to the redox-active substance is the mass ratio of the lyoprotectant to the redox-active substance in a2) in the first aspect of the embodiment of the present application.

[0068] In one embodiment, the component containing a substance with poor thermal stability comprises: a component containing a protein, the lyoprotectant is the lyoprotectant in a3) in the first aspect of the embodiment of the present application.

[0069] In one embodiment, the component containing a substance with poor thermal stability comprises: a component containing a protein, the mass to volume ratio of the lyoprotectant to the component containing a protein is the mass to volume ratio of the lyoprotectant to the component containing a protein in a3) in the first aspect of the embodiment of the present application.

[0070] In one embodiment, the nucleotide is a modified nucleotide.

[0071] In one embodiment, the lyophilization results in lyophilized microspheres, lyophilized cake, or a combination thereof.

[0072] In one embodiment, the microspheres are spherical, ellipsoidal, or toroidal.

[0073] In one embodiment, the poorly heat-stable substance is formulated at a high concentration when its concentration in the poorly heat-stable substance-containing component is low. Here, the poorly heat-stable substance is at a low concentration in the poorly heat-stable substance-containing component refers to a concentration lower than the saturation concentration of the poorly heat-stable substance in a reconstitution reagent (such as water, ethanolamine, a buffer, or a combination thereof) containing a lyoprotectant, for example, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% of the saturation concentration. Formulating at a high concentration refers to increasing the concentration to 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 8 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, 60 times, 80 times, 100 times, 150 times, 200 times, 300 times, 400 times, 500 times, or 600 times. It can be understood that the high concentration after the increase is usually not more than the saturation concentration, and in some cases, not more than the supersaturation concentration.

[0074] In one embodiment, when the poorly heat-stable substance-containing component has a large volume (for different sizes of reagent slots, such as 5 mL, 10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 200 mL, 300 mL, 500 mL, 800 mL, 1000 mL, 1500 mL, 2000 mL, 3000 mL, 4000 mL, 5000 mL or more), the poorly heat-stable substance (such as THPP in the regeneration reagent) is lyophilized separately.

[0075] In one embodiment, the lyophilization comprises the following steps: pre-freezing, sublimation drying, and desorption drying.

[0076] In one embodiment, when the poorly heat-stable substance-containing component is a nucleotide-containing component, the pre-freezing condition comprises incubation at -53 to -47°C for 10 to 300 minutes.

[0077] In one embodiment, when the poorly heat-stable substance-containing component is a nucleotide-containing component, the pre-freezing condition further comprises pre-incubation at -3 to 3°C for 10 to 200 minutes before incubation at -53 to -47°C; that is, the pre-freezing condition comprises incubation at -3 to 3°C for 10 to 200 minutes and incubation at -53 to -47°C for 10 to 300 minutes.

[0078] In one embodiment, when the component containing the substance with poor thermal stability is a component containing nucleotides, the sublimation drying conditions include incubation at -33 to -27°C for 10 to 2000 minutes, incubation at -23 to -17°C for 5 to 90 minutes, incubation at -13 to -7°C for 5 to 90 minutes, and incubation at -3 to 3°C for 5 to 90 minutes.

[0079] In one embodiment, when the component containing the substance with poor thermal stability is a component containing nucleotides, the sublimation drying conditions further include pre-incubation at -43 to -27°C for 10 to 2000 minutes before incubation at -33 to -27°C; that is, the sublimation drying conditions include incubation at -43 to -27°C for 10 to 2000 minutes, incubation at -33 to -27°C for 10 to 2000 minutes, incubation at -23 to -17°C for 5 to 90 minutes, incubation at -13 to -7°C for 5 to 90 minutes, and incubation at -3 to 3°C for 5 to 90 minutes.

[0080] In one embodiment, when the component containing the substance with poor thermal stability is a component containing nucleotides, the sublimation drying conditions include incubation at -33 to -27°C for 1000 to 1400 minutes, incubation at -23 to -17°C for 50 to 70 minutes, incubation at -13 to -7°C for 50 to 70 minutes, and incubation at -3 to 3°C for 50 to 70 minutes.

[0081] In one embodiment, when the component containing the substance with poor thermal stability is a component containing nucleotides, the sublimation drying conditions include incubation at -43 to -37°C for 500 to 1500 minutes, incubation at -33 to -27°C for 50 to 70 minutes, incubation at -23 to -17°C for 50 to 70 minutes, incubation at -13 to -7°C for 50 to 70 minutes, and incubation at -3 to 3°C for 50 to 70 minutes.

[0082] In one embodiment, when the component containing the substance with poor thermal stability is a component containing nucleotides, the sublimation drying conditions include incubation at 22 to 28°C for 140 to 460 minutes, for example, 200, 300, 400 minutes.

[0083] In one embodiment, when the component containing the substance with poor thermal stability is a component containing a redox-active substance, or a component containing a protein, the pre-freezing conditions include incubation at -53 to -47°C for 10 to 300 minutes.

[0084] In one embodiment, when the component containing the substance with poor thermal stability is a component containing a redox-active substance or a component containing a protein, the pre-freezing condition comprises: incubation at -3 to 3 DEG C for 10 to 200 min, and incubation at -53 to -47 DEG C for 10 to 300 min.

[0085] In one embodiment, when the component containing the substance with poor thermal stability is a component containing a redox-active substance or a component containing a protein, the pre-freezing condition comprises: incubation at -3 to 3 DEG C for 100 to 140 min, and incubation at -53 to -47 DEG C for 160 to 200 min.

[0086] In one embodiment, when the component containing the substance with poor thermal stability is a component containing a redox-active substance or a component containing a protein, the sublimation drying condition comprises: incubation at -43 to -37 DEG C for 10 to 2000 min, incubation at -33 to -27 DEG C for 5 to 90 min, incubation at -23 to -17 DEG C for 5 to 90 min, incubation at -13 to -7 DEG C for 5 to 90 min, and incubation at -3 to 3 DEG C for 5 to 90 min.

[0087] In one embodiment, when the component containing the substance with poor thermal stability is a component containing a redox-active substance or a component containing a protein, the sublimation drying condition comprises: incubation at -43 to -37 DEG C for 1000 to 1400 min, incubation at -33 to -27 DEG C for 50 to 70 min, incubation at -23 to -17 DEG C for 50 to 70 min, incubation at -13 to -7 DEG C for 50 to 70 min, and incubation at -3 to 3 DEG C for 50 to 70 min.

[0088] In one embodiment, when the component containing the substance with poor thermal stability is a component containing a redox-active substance or a component containing a protein, the desorption drying condition comprises: incubation at 22 to 28 DEG C for 300 to 420 min.

[0089] The embodiment of the present application has the following beneficial effects:

[0090] The embodiment of the present application provides a method for improving the stability of sequencing reagents, which converts a component containing a substance with poor thermal stability (such as a component containing a nucleotide, a component containing a redox-active substance, or a component containing a protein) into a freeze-dried form to improve the stability; so that the sequencing reagents can maintain stability at room temperature or in a high-temperature environment, solving the existing problems of sequencing reagents (poor thermal stability, degradation of effective components, increase of by-products, and serious impact on the quality of sequencing data) and reducing the cost. BRIEF DESCRIPTION OF DRAWINGS

[0091] Figure 1 is a picture of lyophilized powder cake obtained in Example 1 of "1st (15), 3rd (15), 2nd (15), 3rd (15)" (corresponding to Formulation 15, 51, 33, 51 in turn) : color uniform, structure intact without collapse, can be quickly reconstituted by adding water; no obvious change in appearance and no change in reconstitution speed after being placed at 37°C for 2 weeks or at 55°C for 1 week.

[0092] Figure 2 is a picture of lyophilized powder cake obtained in Example 1 of "4th (1)~(5)" (corresponding to Formulation 55~59 in turn).

[0093] Figure 3 is a picture of reconstituted lyophilized powder cake obtained in Example 1 of "4th (1)~(5)" (corresponding to Formulation 55~59 in turn).

[0094] Figure 4 is a picture of lyophilized powder cake obtained in Example 1 of "5th (1)" (corresponding to Formulation 60).

[0095] Figure 5 is a picture of lyophilized microspheres obtained in Example 1 of "6" (corresponding to Formulation 61) : color uniform, structure intact without collapse, can be quickly reconstituted by adding water; no obvious change in appearance and no change in reconstitution speed after being placed at 37°C for 2 weeks or at 55°C for 1 week. DETAILED DESCRIPTION

[0096] In a first aspect of the embodiments of the present application, a composition is provided, which is any one of a1)~a3) :

[0097] a1) the composition comprises: a lyoprotectant, and a component containing nucleotides, the nucleotides being natural nucleotides and / or modified nucleotides;

[0098] a2) the composition comprises: a lyoprotectant, and a component containing redox active substances;

[0099] a3) the composition comprises: a lyoprotectant, and a component containing proteins;

[0100] The composition in a1), a2), a3) is a lyophilized composition.

[0101] In an embodiment, each of the lyoprotectants in a1), a2), a3) is independently selected from one or more of: a polyhydroxyl compound (such as: mannitol, sorbitol, inositol, a thiol, polyethylene glycol, etc.), a sugar (such as: glucose, a-D-mannopyranose, sucrose, lactose, trehalose, cellobiose, mannose, maltose, inositol, cotton sugar, inulin, dextran, dextrin, cyclodextrin, maltodextrin, maltohexaose, soluble starch, hydroxyethyl starch, octasaccharide sucrose, heparin, a cyclodextrin derivative (such as: (2-hydroxyethyl)-β cyclodextrin, hydroxypropyl-β cyclodextrin), etc.), a polymer (such as: polyethylene glycol, polyvinylpyrrolidone (PVP), polysucrose, gelatin, polyethylene imine); further each is independently selected from one or more of: trehalose, sucrose, lactose, dextran, polysucrose, dextrin, cyclodextrin, hydroxyethyl starch, mannitol, sorbitol, polyvinylpyrrolidone, polyethylene glycol, and a derivative of the foregoing; still further each is independently selected from one or more of: trehalose, sucrose, a cyclodextrin derivative, mannitol, polyvinylpyrrolidone, polyethylene glycol, hydroxyethyl starch.

[0102] In an embodiment, the cyclodextrin comprises one or more of: a-cyclodextrin, β-cyclodextrin, γ-cyclodextrin.

[0103] In an embodiment, the cyclodextrin derivative comprises one or more of: (2-hydroxyethyl)-β cyclodextrin, hydroxypropyl-β-cyclodextrin.

[0104] In an embodiment, the polyvinylpyrrolidone comprises at least one of: polyvinylpyrrolidone K30, polyvinylpyrrolidone K60, polyvinylpyrrolidone K90; further comprises any one of: polyvinylpyrrolidone K30, polyvinylpyrrolidone K60, polyvinylpyrrolidone K90.

[0105] In an embodiment, the polyethylene glycol comprises at least one of: PEG2000, PEG4000, PEG6000, PEG8000, PEG10000, PEG20000; further comprises any one of: PEG8000, PEG20000.

[0106] In an embodiment, the mass-volume ratio or mass ratio of the lyoprotectants and the components in a1), a2), a3) is (3-20): 50; further is (5-15): 50; still further is (5-7.5): 50. Wherein the mass-volume ratio of the former material to the latter material referred to in various places in the embodiments of the present application generally refers to the mass of the former material measured in g and the volume of the latter material measured in mL, and the obtained ratio of the mass and volume without the dimension is obtained.

[0107] In one embodiment, the lyoprotectant in a1 ) is any one of b1 ) to b5):

[0108] b1 ) a polymer (such as: polyvinylpyrrolidone), a polyhydroxy compound (such as: mannitol) and a sugar (such as: sucrose, trehalose, (2-hydroxyethyl)-beta cyclodextrin, hydroxypropyl-beta-cyclodextrin, hydroxyethyl starch);

[0109] b2) a polymer (such as: polyvinylpyrrolidone) and a sugar (such as: sucrose, trehalose, (2-hydroxyethyl)-beta cyclodextrin, hydroxypropyl-beta-cyclodextrin, hydroxyethyl starch);

[0110] b3) a polyhydroxy compound (such as: mannitol) and a sugar (such as: sucrose, trehalose, (2-hydroxyethyl)-beta cyclodextrin, hydroxypropyl-beta-cyclodextrin, hydroxyethyl starch);

[0111] b4) a polyhydroxy compound (such as: mannitol);

[0112] b5) a sugar (such as: sucrose, trehalose, (2-hydroxyethyl)-beta cyclodextrin, hydroxypropyl-beta-cyclodextrin, hydroxyethyl starch).

[0113] In one embodiment, the lyoprotectant in a1 ) is any one of c1 ) to c14):

[0114] c1 ) mannitol and sucrose; c2) mannitol and trehalose; c3) polyvinylpyrrolidone K30 and sucrose; c4) polyvinylpyrrolidone K30 and trehalose; c5) polyvinylpyrrolidone K60 and trehalose; c6) polyvinylpyrrolidone K90 and trehalose; c7) (2-hydroxyethyl)-beta-cyclodextrin; c8) hydroxypropyl-beta-cyclodextrin; c9) polyvinylpyrrolidone K30, trehalose and (2-hydroxyethyl)-beta-cyclodextrin; c10) polyvinylpyrrolidone K30, trehalose and hydroxypropyl-beta-cyclodextrin; c11 ) polyvinylpyrrolidone K30, sucrose and (2-hydroxyethyl)-beta-cyclodextrin; c12) polyvinylpyrrolidone K30, sucrose and hydroxypropyl-beta-cyclodextrin; c13) polyvinylpyrrolidone K30, trehalose and hydroxyethyl starch; c14) polyvinylpyrrolidone K30, sucrose and hydroxyethyl starch; further c3), c9), c10), c11 ), or c12); more further c11 ), or c12).

[0115] In one embodiment, the mass to volume ratio of the lyoprotectant in a1 ) and the components is (3 to 20): 50; further (5 to 15): 50; more further (5 to 7.5): 50.

[0116] In one embodiment, the mass ratio of the mannitol and sucrose in c1) is (3-5): 1; further 4: 1.

[0117] In one embodiment, the mass ratio of the mannitol and trehalose in c2) is (2-4): 1; further 3: 1.

[0118] In one embodiment, the mass ratio of the polyvinylpyrrolidone K30 and sucrose in c3) is (1-5): 1; further (1-4): 1; still further (1-2): 1; yet further 1.5: 1.

[0119] In one embodiment, the mass ratio of the polyvinylpyrrolidone K30 and trehalose in c4) is (0.1-3.5): 1; further (0.2-1.5): 1.

[0120] In one embodiment, the mass ratio of the polyvinylpyrrolidone K60 and trehalose in c5) is (1-2): 1; yet further 1.5: 1.

[0121] In one embodiment, the polyvinylpyrrolidone K60 is a polyvinylpyrrolidone K60 solution; further a 45% polyvinylpyrrolidone K60 solution.

[0122] In one embodiment, the mass ratio of the polyvinylpyrrolidone K90 and trehalose in c6) is (1-2): 1; yet further 1.5: 1.

[0123] In one embodiment, the mass ratio of the polyvinylpyrrolidone K30, trehalose and (2-hydroxyethyl)-β-cyclodextrin in c9) is (1-3):(1-3): 1.

[0124] In one embodiment, the mass ratio of the polyvinylpyrrolidone K30, trehalose and hydroxypropyl-β-cyclodextrin in c10) is (1-3):(1-3): 1.

[0125] In one embodiment, the mass ratio of the polyvinylpyrrolidone K30, sucrose and (2-hydroxyethyl)-β-cyclodextrin in c11) is (1-3):(1-3): 1.

[0126] In one embodiment, the mass ratio of the polyvinylpyrrolidone K30, sucrose and hydroxypropyl-β-cyclodextrin in c12) is (1-3):(1-3): 1.

[0127] In one embodiment, the mass ratio of the polyvinylpyrrolidone K30, trehalose and hydroxyethyl starch in c13) is (1-3):(1-3): 1.

[0128] In an embodiment, the mass ratio of the polyvinylpyrrolidone K30, sucrose and hydroxyethyl starch in c14) is (1-3):(1-3):1.

[0129] In an embodiment, the nucleotides in a1) are modified nucleotides.

[0130] In an embodiment, the modified nucleotides comprise a Base selected from a base, a deaza base or a tautomer thereof, and a sugar modified with a blocking group.

[0131] In an embodiment, the base comprises a purine base and / or a pyrimidine base.

[0132] In an embodiment, the Base is selected from adenine, 7-deazaadenine, thymine, uracil, cytosine, guanine, 7-deazaguanine or a tautomer thereof.

[0133] In an embodiment, the sugar comprises ribose and / or deoxyribose; further comprises deoxyribose.

[0134] In an embodiment, the blocking group modifies the 3’-OH of the sugar.

[0135] In an embodiment, the blocking group is a reversible blocking group, which can be any one of the prior art: such as any one of the reversible blocking groups in patent document WO2022083686A1.

[0136] In an embodiment, the modified nucleotide further comprises: a monophosphate group, a diphosphate group, a triphosphate group, or a tetraphosphate group; further comprises a triphosphate group.

[0137] In an embodiment, the modified nucleotide is linked to a detectable label.

[0138] In an embodiment, the detectable label is a fluorescent label, i.e., a fluorophore, which emits radiation of a defined wavelength upon absorption of energy. Many suitable fluorescent labels are known. For example, Welch et al. (Chem. Eur. J. 5(3): 951-960, 1999) disclose dansyl-functionalized fluorescent moieties that can be used in embodiments of the present application. Zhu et al. (Cytometry 28: 206-211, 1997) describe the use of fluorescent labels Cy3 and Cy5, which can also be used in embodiments of the present application. Prober et al. (Science 238: 336-341, 1987), Connell et al. (BioTechniques 5(4): 342-384, 1987), Ansorge et al. (Nucl. Acids Res. 15(11): 4593-4602, 1987), and Smith et al. (Nature 321: 674, 1986) also disclose labels suitable for use. Other commercially available fluorescent labels include, but are not limited to, fluorescein, rhodamine (including TMR, Texas Red, and Rox), alexa, boron-dipyrromethene, acridine, coumarin, pyrene, benzanthracene, and cyanine.

[0139] In an embodiment, the detectable label is introduced via an affinity reagent (such as an antibody, an aptamer, an Affimer, a Knottin), which carries the detectable label and which can specifically recognize and bind to the modified nucleotide.

[0140] In an embodiment, the detectable label is linked to the modified nucleotide via a linking group.

[0141] In an embodiment, the linking group is a cleavable linking group or a non-cleavable linking group.

[0142] In an embodiment, the cleavable linking group is selected from an electrophilic cleavable linking group, a nucleophilic cleavable linking group, a photolyzable linking group, a linking group cleavable under reducing conditions, a linking group cleavable under oxidizing conditions, a safety- handle type linking group, a linking group cleavable via an elimination mechanism, or any combination thereof.

[0143] In an embodiment, the linking group has the structure shown in Formula (B) in patent document WO2022083686A1.

[0144] In an embodiment, the modified nucleotide comprises one or more of Hot dATP-Z1, Hot dCTP-Z1, Hot dGTP-ZB1, Hot dTTP-Z1, Cold dATP, Cold dCTP, Cold dGTP, Cold dTTP.

[0145] In an embodiment, the component in a1) further comprises a buffer (such as: TE buffer).

[0146] In an embodiment, the buffer comprises at least one of: EDTA, Tris-HCl.

[0147] In an embodiment, the component in a1) is a dNTPs mix.

[0148] In an embodiment, the dNTPs mix comprises: dNTPs mix (comprising: Hot dATP-Z1, Hot dCTP-Z1, Hot dGTP-ZB1, Hot dTTP-Z1, Cold dATP, Cold dCTP, Cold dGTP, Cold dTTP, and a buffer), FT dNTPs mix (comprising: Hot dATP-Z1, Hot dCTP-Z1, Hot dGTP-ZB1, Hot dTTP-Z1, Cold dATP, Cold dCTP, Cold dGTP, and Cold dTTP), or dNTPs mix II (Cold dATP, Cold dCTP, Cold dGTP, Cold dTTP, and a buffer).

[0149] In an embodiment, the dNTPs mix is a high concentration dNTPs mix; further is a > 50x dNTPs mix; further is a 147-750x dNTPs mix.

[0150] In an embodiment, the lyoprotectant in a2) is polyethylene glycol; further is at least one of: PEG2000, PEG4000, PEG6000, PEG8000, PEG10000, PEG20000; further comprises: any one of PEG8000, PEG20000.

[0151] In an embodiment, the mass ratio of the lyoprotectant to the redox active material in a2) is 1:(0.3-1.5); further is 1:(0.37-1.11); further is 1:(0.555-1.11); further is 1:(0.555-0.74).

[0152] In an embodiment, the redox active material in a2) comprises: at least one of: THPP, TCEP; further comprises: THPP.

[0153] In an embodiment, the component in a2) can further comprise other materials (such as: sodium chloride, buffer salts, etc.).

[0154] In one embodiment, the component in a2) is a reagent.

[0155] In one embodiment, the lyoprotectant in a3) is D-mannitol.

[0156] In one embodiment, the mass volume ratio of the lyoprotectant to the protein-containing component in a3) is 1:(5-15); further 1:10.

[0157] In one embodiment, the protein in a3) is a non-enzyme protein; further BSA; more further BSA treated by aging.

[0158] In one embodiment, the protein-containing component in a3) is a protein wash buffer; further a BSA-containing buffer.

[0159] In one embodiment, the lyophilized composition in a1), a2), a3) is a microsphere, a cake or a combination thereof.

[0160] In one embodiment, the microsphere is spherical, elliptical or annular.

[0161] In a second aspect, the present application provides a method for preparing the composition of the first aspect, which is any one of d1)-d3):

[0162] d1) mixing the lyoprotectant in a1) and the nucleotide-containing component in the first aspect, and lyophilizing to obtain, wherein the nucleotide is a natural nucleotide and / or a modified nucleotide;

[0163] d2) mixing the lyoprotectant in a2) and the redox-active substance-containing component in the first aspect, and lyophilizing to obtain;

[0164] d3) mixing the lyoprotectant in a3) and the protein-containing component in the first aspect, and lyophilizing to obtain.

[0165] In one embodiment, the lyophilizing in d1), d2), d3) comprises the following steps: pre-freezing, sublimation drying and desorption drying.

[0166] In one embodiment, the pre-freezing conditions for the freeze-drying in d1) comprise incubation at -53 to -47 °C for 10 to 300 min; alternatively, the pre-freezing conditions comprise incubation at -3 to 3 °C for 10 to 200 min and incubation at -53 to -47 °C for 10 to 300 min; alternatively, the pre-freezing conditions comprise incubation at -3 to 3 °C for 100 to 140 min and incubation at -53 to -47 °C for 160 to 200 min; alternatively, the pre-freezing conditions comprise incubation at 0 °C for 120 min and incubation at -50 °C for 180 min.

[0167] In one embodiment, the sublimation drying conditions for the freeze-drying in d1) comprise incubation at -33 to -27 °C for 10 to 2000 min, at -23 to -17 °C for 5 to 90 min, at -13 to -7 °C for 5 to 90 min, and at -3 to 3 °C for 5 to 90 min; alternatively, the sublimation drying conditions comprise incubation at -43 to -37 °C for 10 to 2000 min, at -33 to -27 °C for 10 to 2000 min, at -23 to -17 °C for 5 to 90 min, at -13 to -7 °C for 5 to 90 min, and at -3 to 3 °C for 5 to 90 min; alternatively, the sublimation drying conditions comprise incubation at -33 to -27 °C for 1000 to 1400 min, at -23 to -17 °C for 50 to 70 min, at -13 to -7 °C for 50 to 70 min, and at -3 to 3 °C for 50 to 70 min; further, -30 °C for 1200 min, -20 °C for 60 min, -10 °C for 60 min, and 0 °C for 60 min; alternatively, the sublimation drying conditions comprise incubation at -43 to -37 °C for 500 to 1500 min, at -33 to -27 °C for 50 to 70 min, at -23 to -17 °C for 50 to 70 min, at -13 to -7 °C for 50 to 70 min, and at -3 to 3 °C for 50 to 70 min; further, -40 °C for 1000 min, -30 °C for 60 min, -20 °C for 60 min, -10 °C for 60 min, and 0 °C for 60 min.

[0168] In one embodiment, the desorption drying conditions for the freeze-drying in d1) comprise incubation at 20 to 30 °C for 140 to 460 min; alternatively, the desorption drying conditions comprise incubation at 25 °C for 200, 300, 400 min.

[0169] In an embodiment, the pre-freezing conditions of d2), d3) comprise: incubation at -3 to 3°C for 10 to 200 min, and incubation at -53 to -47°C for 10 to 300 min; further pre-freezing conditions comprise: incubation at -3 to 3°C for 100 to 140 min, and incubation at -53 to -47°C for 160 to 200 min; further pre-freezing conditions comprise: incubation at 0°C for 120 min, and incubation at -50°C for 180 min.

[0170] In an embodiment, the sublimation drying conditions of d2), d3) comprise: incubation at -43 to -37°C for 10 to 2000 min, incubation at -33 to -27°C for 5 to 90 min, incubation at -23 to -17°C for 5 to 90 min, incubation at -13 to -7°C for 5 to 90 min, and incubation at -3 to 3°C for 5 to 90 min; further sublimation drying conditions comprise: incubation at -43 to -37°C for 1000 to 1400 min, incubation at -33 to -27°C for 50 to 70 min, incubation at -23 to -17°C for 50 to 70 min, incubation at -13 to -7°C for 50 to 70 min, and incubation at -3 to 3°C for 50 to 70 min; further sublimation drying conditions comprise: incubation at -40°C for 1200 min, incubation at -30°C for 60 min, incubation at -20°C for 60 min, incubation at -10°C for 60 min, and incubation at 0°C for 60 min.

[0171] In an embodiment, the desiccation conditions of d2), d3) comprise: incubation at 20 to 30°C for 300 to 420 min; further desiccation conditions comprise: incubation at 25°C for 360 min.

[0172] In an embodiment, the sublimation drying and / or desiccation is performed under vacuum.

[0173] In a third aspect of the embodiments of the present application, a sequencing kit or a sequencing reagent tank is provided, comprising the composition of the first aspect of the embodiments of the present application.

[0174] In a fourth aspect of the embodiments of the present application, a method for reconstituting a composition is provided, comprising mixing the composition with a reconstitution reagent to obtain a reconstitution solution, wherein the composition is the composition of a1), a2), a3) of the first aspect of the embodiments of the present application.

[0175] In an embodiment, the reconstitution reagent comprises: water, ethanolamine, a buffer, or a combination thereof.

[0176] In an embodiment, the water is nuclease-free water.

[0177] In an embodiment, the mixing is performed under conditions effective to reconstitute the composition.

[0178] In one embodiment, the reconstitution solution is used in a sequencing process.

[0179] In a fifth aspect, the present application provides a method for sequencing, comprising the steps of:

[0180] f1) mixing the composition with a reconstitution reagent to obtain a reconstitution solution;

[0181] f2) using a reagent tank containing the reconstitution solution for sequencing;

[0182] The composition in f1) is the composition in a1), a2), a3) of the first aspect of the present application.

[0183] In one embodiment, the reconstitution reagent comprises water, ethanolamine, a buffer, or a combination thereof.

[0184] In one embodiment, the mixing is performed under conditions effective to reconstitute the composition.

[0185] In a sixth aspect, the present application provides a method for improving the stability of a sequencing reagent,

[0186] The sequencing reagent comprises a component containing a substance with poor thermal stability.

[0187] The component containing a substance with poor thermal stability comprises a component containing a nucleotide, a component containing a redox-active substance, or a component containing a protein.

[0188] The nucleotide is a natural nucleotide and / or a modified nucleotide.

[0189] The method comprises the steps of:

[0190] Mixing a substance with poor thermal stability, or a component containing a substance with poor thermal stability, or a sequencing reagent, with a lyoprotectant, and lyophilizing.

[0191] In one embodiment, the component containing a nucleotide, the component containing a redox-active substance, or the component containing a protein is the component containing a nucleotide, the component containing a redox-active substance, or the component containing a protein in the first aspect of the present application.

[0192] In one embodiment, the mass ratio or mass-volume ratio of the lyoprotectant to the component containing a substance with poor thermal stability is the mass ratio or mass-volume ratio of the lyoprotectant to the component in a1), a2), a3) of the first aspect of the present application.

[0193] In one embodiment, the component containing a substance with poor thermal stability comprises a component containing a nucleotide, and the lyoprotectant is the lyoprotectant of a1) in the first aspect of the application.

[0194] In one embodiment, the component containing a substance with poor thermal stability comprises a component containing a nucleotide, and the mass to volume ratio of the lyoprotectant to the component containing a nucleotide is the mass to volume ratio of the lyoprotectant to the component of a1) in the first aspect of the application.

[0195] In one embodiment, the component containing a substance with poor thermal stability comprises a component containing a redox active substance, and the lyoprotectant is the lyoprotectant of a2) in the first aspect of the application.

[0196] In one embodiment, the component containing a substance with poor thermal stability comprises a component containing a redox active substance, and the mass ratio of the lyoprotectant to the redox active substance is the mass ratio of the lyoprotectant to the redox active substance of a2) in the first aspect of the application.

[0197] In one embodiment, the component containing a substance with poor thermal stability comprises a component containing a protein, and the lyoprotectant is the lyoprotectant of a3) in the first aspect of the application.

[0198] In one embodiment, the component containing a substance with poor thermal stability comprises a component containing a protein, and the mass to volume ratio of the lyoprotectant to the component containing a protein is the mass to volume ratio of the lyoprotectant to the component containing a protein of a3) in the first aspect of the application.

[0199] In one embodiment, the nucleotide is a modified nucleotide.

[0200] In one embodiment, the lyophilization results in lyophilized microspheres, lyophilized cake or a combination thereof.

[0201] In one embodiment, the microspheres are spherical, ellipsoidal or toroidal.

[0202] In one embodiment, the poorly heat-stable substance is formulated at a high concentration when its concentration in the poorly heat-stable substance-containing component is low. Here, the poorly heat-stable substance is at a low concentration in the poorly heat-stable substance-containing component refers to a concentration lower than the saturation concentration of the poorly heat-stable substance in a reconstitution reagent (such as water, ethanolamine, a buffer, or a combination thereof) containing a lyoprotectant, for example, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% of the saturation concentration. Formulating at a high concentration refers to increasing the concentration to 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 8 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, 60 times, 80 times, 100 times, 150 times, 200 times, 300 times, 400 times, 500 times, or 600 times. It can be understood that the high concentration after the increase is usually not more than the saturation concentration, and in some cases, not more than the supersaturation concentration.

[0203] In one embodiment, when the poorly heat-stable substance-containing component has a large volume (for different sizes of reagent slots, such as 5 mL, 10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 200 mL, 300 mL, 500 mL, 800 mL, 1000 mL, 1500 mL, 2000 mL, 3000 mL, 4000 mL, 5000 mL or more), the poorly heat-stable substance (such as THPP in the regeneration reagent) is lyophilized separately.

[0204] In one embodiment, the lyophilization comprises the following steps: pre-freezing, sublimation drying, and desorption drying.

[0205] In one embodiment, when the poorly heat-stable substance-containing component is a nucleotide-containing component, the pre-freezing conditions comprise: incubation at -53 to -47°C for 10 to 300 min; optionally, the pre-freezing conditions comprise: incubation at -3 to 3°C for 10 to 200 min, and incubation at -53 to -47°C for 10 to 300 min; optionally, the pre-freezing conditions comprise: incubation at -3 to 3°C for 100 to 140 min, and incubation at -53 to -47°C for 160 to 200 min; optionally, the pre-freezing conditions comprise: incubation at 0°C for 120 min, and incubation at -50°C for 180 min.

[0206] In one embodiment, when the component containing the substance with poor thermal stability is a component containing nucleotides, the sublimation drying conditions include: incubation at -33 to -27°C for 10 to 2000 min, incubation at -23 to -17°C for 5 to 90 min, incubation at -13 to -7°C for 5 to 90 min, and incubation at -3 to 3°C for 5 to 90 min; alternatively, the sublimation drying conditions include: incubation at -43 to -37°C for 10 to 2000 min, incubation at -33 to -27°C for 10 to 2000 min, incubation at -23 to -17°C for 5 to 90 min, incubation at -13 to -7°C for 5 to 90 min, and incubation at -3 to 3°C for 5 to 90 min; alternatively, the sublimation drying conditions include: incubation at -33 to -27°C for 1000 to 1400 min, incubation at -23 to -17°C for 50 to 70 min, incubation at -13 to -7°C for 50 to 70 min, and incubation at -3 to 3°C for 50 to 70 min; further, incubation at -30°C for 1200 min, incubation at -20°C for 60 min, incubation at -10°C for 60 min, and incubation at 0°C for 60 min; alternatively, the sublimation drying conditions include: incubation at -43 to -37°C for 500 to 1500 min, incubation at -33 to -27°C for 50 to 70 min, incubation at -23 to -17°C for 50 to 70 min, incubation at -13 to -7°C for 50 to 70 min, and incubation at -3 to 3°C for 50 to 70 min; further, incubation at -40°C for 1000 min, incubation at -30°C for 60 min, incubation at -20°C for 60 min, incubation at -10°C for 60 min, and incubation at 0°C for 60 min.

[0207] In one embodiment, when the component containing the substance with poor thermal stability is a component containing nucleotides, the sublimation drying conditions include: incubation at -33 to -27°C for 10 to 2000 min, incubation at -23 to -17°C for 5 to 90 min, incubation at -13 to -7°C for 5 to 90 min, and incubation at -3 to 3°C for 5 to 90 min; alternatively, the sublimation drying conditions include: incubation at -43 to -37°C for 10 to 2000 min, incubation at -33 to -27°C for 10 to 2000 min, incubation at -23 to -17°C for 5 to 90 min, incubation at -13 to -7°C for 5 to 90 min, and incubation at -3 to 3°C for 5 to 90 min; alternatively, the sublimation drying conditions include: incubation at -33 to -27°C for 1000 to 1400 min, incubation at -23 to -17°C for 50 to 70 min, incubation at -13 to -7°C for 50 to 70 min, and incubation at -3 to 3°C for 50 to 70 min; further, incubation at -30°C for 1200 min, incubation at -20°C for 60 min, incubation at -10°C for 60 min, and incubation at 0°C for 60 min; alternatively, the sublimation drying conditions include: incubation at -43 to -37°C for 500 to 1500 min, incubation at -33 to -27°C for 50 to 70 min, incubation at -23 to -17°C for 50 to 70 min, incubation at -13 to -7°C for 50 to 70 min, and incubation at -3 to 3°C for 50 to 70 min; further, incubation at -40°C for 1000 min, incubation at -30°C for 60 min, incubation at -20°C for 60 min, incubation at -10°C for 60 min, and incubation at 0°C for 60 min.

[0208] In one embodiment, when the component containing the substance with poor thermal stability is a component containing nucleotides, the sublimation drying conditions include: incubation at -33 to -27°C for 10 to 2000 min, incubation at -23 to -17°C for 5 to 90 min, incubation at -13 to -7°C for 5 to 90 min, and incubation at -3 to 3°C for 5 to 90 min; alternatively, the sublimation drying conditions include: incubation at -43 to -37°C for 10 to 2000 min, incubation at -33 to -27°C for 10 to 2000 min, incubation at -23 to -17°C for 5 to 90 min, incubation at -13 to -7°C for 5 to 90 min, and incubation at -3 to 3°C for 5 to 90 min; alternatively, the sublimation drying conditions include: incubation at -33 to -27°C for 1000 to 1400 min, incubation at -23 to -17°C for 50 to 70 min, incubation at -13 to -7°C for 50 to 70 min, and incubation at -3 to 3°C for 50 to 70 min; further, incubation at -30°C for 1200 min, incubation at -20°C for 60 min, incubation at -10°C for 60 min, and incubation at 0°C for 60 min; alternatively, the sublimation drying conditions include: incubation at -43 to -37°C for 500 to 1500 min, incubation at -33 to -27°C for 50 to 70 min, incubation at -23 to -17°C for 50 to 70 min, incubation at -13 to -7°C for 50 to 70 min, and incubation at -3 to 3°C for 50 to 70 min; further, incubation at -40°C for 1000 min, incubation at -30°C for 60 min, incubation at -20°C for 60 min, incubation at -10°C for 60 min, and incubation at 0°C for 60 min.

[0209] In an embodiment, when the component containing the substance with poor thermal stability is a component containing a redox-active substance, or a component containing a protein, the sublimation drying conditions include: incubation at -43 to -37°C for 10 to 2000 min, incubation at -33 to -27°C for 5 to 90 min, incubation at -23 to -17°C for 5 to 90 min, incubation at -13 to -7°C for 5 to 90 min, and incubation at -3 to 3°C for 5 to 90 min; further sublimation drying conditions include: incubation at -43 to -37°C for 1000 to 1400 min, incubation at -33 to -27°C for 50 to 70 min, incubation at -23 to -17°C for 50 to 70 min, incubation at -13 to -7°C for 50 to 70 min, and incubation at -3 to 3°C for 50 to 70 min; further sublimation drying conditions include: incubation at -40°C for 1200 min, incubation at -30°C for 60 min, incubation at -20°C for 60 min, incubation at -10°C for 60 min, and incubation at 0°C for 60 min.

[0210] In an embodiment, when the component containing the substance with poor thermal stability is a component containing a redox-active substance, or a component containing a protein, the desorption drying conditions include: incubation at 20 to 30°C for 300 to 420 min; further desorption drying conditions include: incubation at 25°C for 360 min.

[0211] In an embodiment, the sublimation drying and / or desorption drying is performed under vacuum.

[0212] The content of the embodiments of the present application is further described in detail below through specific examples.

[0213] It should be understood that these examples are only used to illustrate the embodiments of the present application and are not used to limit the scope of the embodiments of the present application.

[0214] The experimental methods not specified in the following examples are generally performed according to conventional conditions, or according to the conditions recommended by the manufacturer. The materials, reagents, etc. used in the present examples are commercially available reagents and materials unless otherwise specified.

[0215] The equipment used in the present examples is as follows: a set of pipettes, a PCR instrument, an MGISEQ-2000 sequencer, an MGISEQ-2000 RS sequencing slide, a DNBSEQ-G99 sequencer, a DNBSEQ-G99 sequencing slide, a Pilot 2-4T freeze dryer from Bomed, and an MS-1R microsphere forming instrument from Erde.

[0216] The reagents used in the present examples are shown in Table 1.

[0217] Table 1: Reagent names and sources

[0218] The formulations of high concentration dNTPs mix (including 150x dNTPs mix, 147x FT dNTPs mix, 150x dNTPs mix II) and THPP aqueous solution used in this example are as follows:

[0219] 150x dNTPs mix (total 10.000 mL), comprising: 0.750 mL Hot dATP-Z1, 1.800 mL Hot dCTP-Z1, 1.800 mL Hot dGTP-ZB1, 0.900 mL Hot dTTP-Z1, 1.125 mL Cold dATP, 0.600 mL Cold dCTP, 0.600 mL Cold dGTP, 0.300 mL Cold dTTP, and 2.125 mL TE buffer (1X, low EDTA, pH 8.0);

[0220] 147x FT dNTPs mix (total 10.200 mL), comprising: 1.800 mL Hot dATP-Z1, 2.400 mL Hot dCTP-Z1, 1.800 mL Hot dGTP-ZB1, 2.400 mL Hot dTTP-Z1, 0.600 mL Cold dATP, 0.300 mL Cold dCTP, 0.600 mL Cold dGTP, 0.300 mL Cold dTTP;

[0221] 150x dNTPs mix II (total 10.000 mL), comprising: 1.500 mL Cold dATP, 1.500 mL Cold dCTP, 1.500 mL Cold dGTP, 1.500 mL Cold dTTP, and 4.000 mL TE buffer (1X, low EDTA, pH 8.0);

[0222] THPP aqueous solution (total 1.000 mL), consisting of: 0.1110 g THPP, and nuclease-free water to volume.

[0223] Example 1 A method for improving the stability of sequencing reagents

[0224] 1. A method for improving the stability of 150x dNTPs mix

[0225] (1) A method for improving the stability of 150x dNTPs mix, comprising the following steps:

[0226] 1) Preparation of the freeze-drying stock solution (Formulation 1, 16% mannitol + 4% sucrose + 150x dNTPs mix): Take 0.0960 g D-mannitol, 0.0240 g sucrose, 0.600 mL 150x dNTPs mix, dissolve, mix well;

[0227] 2) Put the obtained freeze-drying stock solution into a brown 2 mL plastic tube, open the cap of the plastic tube, place the plastic tube on a suitable plastic tube holder, keep the plastic tube in an upright position, and the bottom can contact the freeze-dryer plate layer, put the plastic tube together with the holder into the freeze-dryer (Pilot 2-4T), set the freeze-drying program according to Table 2, close the door, start the freeze-drying program, after the freeze-drying program is completed, backfill the dry air, open the door, take out the plastic tube holder together with the plastic tube, cap tightly and seal.

[0228] Table 2 Freeze-drying program

[0229] (2) A method for improving the stability of 150x dNTPs mix, which is the same as (1), and the only difference is step 1), which is as follows:

[0230] 1) Preparation of the freeze-drying stock solution (Formulation 2, 15% mannitol + 5% trehalose + 150x dNTPs mix): Take 0.0900 g D-mannitol, 0.0300 g D-(+)-trehalose dihydrate, 0.600 mL 150x dNTPs mix, dissolve, mix well.

[0231] (3) A method for improving the stability of 150x dNTPs mix, which is the same as (1), and the only difference is step 1), which is as follows:

[0232] 1) Preparation of the freeze-drying stock solution (Formulation 3, 16% K30 + 4% sucrose + 150x dNTPs mix): Take 0.0960 g polyvinylpyrrolidone K30, 0.0240 g sucrose, 0.600 mL 150x dNTPs mix, dissolve, mix well.

[0233] (4) A method for improving the stability of 150x dNTPs mix, which is the same as (1), and the only difference is step 1), which is as follows:

[0234] 1) Preparation of the freeze-drying stock solution (Formulation 4, 15% K30 + 5% trehalose + 150x dNTPs mix): Take 0.0900 g polyvinylpyrrolidone K30, 0.0300 g D-(+)-trehalose dihydrate, 0.600 mL 150x dNTPs mix, dissolve, mix well.

[0235] (5) A method for improving the stability of 150x dNTPs mix, which is the same as (1), except that step 1) is specifically as follows:

[0236] 1) Preparation of the freeze-drying stock solution (Formula 5, 2% K30 + 10% trehalose + 150x dNTPs mix): Take 0.0120g polyvinylpyrrolidone K30, 0.0600g D-(+)-trehalose dihydrate, 0.600mL 150x dNTPs mix, dissolve and mix evenly.

[0237] (6) A method for improving the stability of 150x dNTPs mix, which is the same as (1), except that step 1) is specifically as follows:

[0238] 1) Preparation of the freeze-drying stock solution (Formula 6, 3% K30 + 9% trehalose + 150x dNTPs mix): Take 0.0180g polyvinylpyrrolidone K30, 0.0540g D-(+)-trehalose dihydrate, 0.600mL 150x dNTPs mix, dissolve and mix evenly.

[0239] (7) A method for improving the stability of 150x dNTPs mix, which is the same as (1), except that step 1) is specifically as follows:

[0240] 1) Preparation of the freeze-drying stock solution (Formula 7, 5% K30 + 5% trehalose + 150x dNTPs mix): Take 0.0300g polyvinylpyrrolidone K30, 0.0300g D-(+)-trehalose dihydrate, 0.600mL 150x dNTPs mix, dissolve and mix evenly.

[0241] (8) A method for improving the stability of 150x dNTPs mix, which is the same as (1), except that step 1) is specifically as follows:

[0242] 1) Preparation of the freeze-drying stock solution (Formula 8, 6% K30 + 4% trehalose + 150x dNTPs mix): Take 0.0360g polyvinylpyrrolidone K30, 0.0240g D-(+)-trehalose dihydrate, 0.600mL 150x dNTPs mix, dissolve and mix evenly.

[0243] (9) A method for improving the stability of 150x dNTPs mix, which is the same as (1), except that step 1) is specifically as follows:

[0244] 1) Formulation of lyophilization stock solution (Formula 9, 6% K60 + 4% trehalose + 150x dNTPs mix): Take 0.0360 g of polyvinylpyrrolidone solution (K60, 45% in H20), 0.0240 g of D-(+)-trehalose dihydrate, 0.600 mL of 150x dNTPs mix, dissolve, mix well.

[0245] (10) A method for improving the stability of 150x dNTPs mix, the same as (1), the difference is only in step 1), as follows:

[0246] 1) Formulation of lyophilization stock solution (Formula 10, 10% HPβCD + 150x dNTPs mix): Take 0.0600 g of hydroxypropyl-β-cyclodextrin, 0.600 mL of 150x dNTPs mix, dissolve, mix well.

[0247] (11) A method for improving the stability of 150x dNTPs mix, the same as (1), the difference is only in step 1), as follows:

[0248] 1) Formulation of lyophilization stock solution (Formula 11, 10% HEβCD + 150x dNTPs mix): Take 0.0600 g of (2-hydroxyethyl)-β-cyclodextrin, 0.600 mL of 150x dNTPs mix, dissolve, mix well.

[0249] (12) A method for improving the stability of 150x dNTPs mix, the same as (1), the difference is only in step 1), as follows:

[0250] 1) Formulation of lyophilization stock solution (Formula 12, 9% K30 + 6% trehalose + 150x dNTPs mix): Take 0.0540 g of polyvinylpyrrolidone K30, 0.0360 g of D-(+)-trehalose dihydrate, 0.600 mL of 150x dNTPs mix, dissolve, mix well.

[0251] (13) A method for improving the stability of 150x dNTPs mix, the same as (1), the difference is only in step 1), as follows:

[0252] 1) Formulation of lyophilization stock solution (Formula 13, 9% K30 + 6% sucrose + 150x dNTPs mix): Take 0.0540 g of polyvinylpyrrolidone K30, 0.0360 g of sucrose, 0.600 mL of 150x dNTPs mix, dissolve, mix well.

[0253] (14) A method for improving the stability of 150x dNTPs mix, the same as (1), except that step 1) is specifically as follows:

[0254] 1) Preparation of the freeze-drying stock solution (Formula 14, 6% K30 + 6% trehalose + 3% HPβCD + 150x dNTPs mix): Take 0.0360 g of polyvinylpyrrolidone K30, 0.0360 g of D-(+)-trehalose dihydrate, 0.0180 g of hydroxypropyl-β-cyclodextrin, 0.600 mL of 150x dNTPs mix, dissolve and mix uniformly.

[0255] (15) A method for improving the stability of 150x dNTPs mix, the same as (1), except that step 1) is specifically as follows:

[0256] 1) Preparation of the freeze-drying stock solution (Formula 15, 6% K30 + 6% sucrose + 3% HPβCD + 150x dNTPs mix): Take 0.0360 g of polyvinylpyrrolidone K30, 0.0360 g of sucrose, 0.0180 g of hydroxypropyl-β-cyclodextrin, 0.600 mL of 150x dNTPs mix, dissolve and mix uniformly.

[0257] (16) A method for improving the stability of 150x dNTPs mix, the same as (1), except that step 1) is specifically as follows:

[0258] 1) Preparation of the freeze-drying stock solution (Formula 16, 6% K30 + 6% sucrose + 3% HEβCD + 150x dNTPs mix): Take 0.0360 g of polyvinylpyrrolidone K30, 0.0360 g of sucrose, 0.0180 g of (2-hydroxyethyl)-β-cyclodextrin, 0.600 mL of 150x dNTPs mix, dissolve and mix uniformly.

[0259] (17) A method for improving the stability of 150x dNTPs mix, the same as (1), except that step 1) is specifically as follows:

[0260] 1) Preparation of the freeze-drying stock solution (Formula 17, 6% K30 + 6% sucrose + 3% hydroxyethyl starch 130 + 150x dNTPs mix): Take 0.0360 g of polyvinylpyrrolidone K30, 0.0360 g of sucrose, 0.0180 g of hydroxyethyl starch (110-150 kDa, degree of substitution 0.37-0.43), 0.600 mL of 150x dNTPs mix, dissolve and mix uniformly.

[0261] (18) A method for improving the stability of 150x dNTPs mix, which is the same as (1), except that step 1) is specifically as follows:

[0262] 1) Preparation of the freeze-drying stock solution (Formulation 18, 6% PVP K30 + 6% sucrose + 3% HES 200 + 150x dNTPs mix): 0.0360 g of PVP K30, 0.0360 g of sucrose, 0.0180 g of HES (170-230 kDa, degree of substitution 0.46-0.54), and 0.600 mL of 150x dNTPs mix were dissolved and uniformly mixed.

[0263] 2. A method for improving the stability of 147x FT dNTPs mix

[0264] (1) A method for improving the stability of 147x FT dNTPs mix, comprising the following steps:

[0265] 1) Preparation of the freeze-drying stock solution (Formulation 19, 16% mannitol + 4% sucrose + 147x FT dNTPs mix, wherein the percentages are all mass / volume ratios, and the same applies hereinafter): 0.0160 g of D-mannitol, 0.0040 g of sucrose, and 0.100 mL of 147x FT dNTPs mix were dissolved and uniformly mixed.

[0266] 2) The obtained freeze-drying stock solution was placed in a brown 2 mL plastic tube, the plastic tube was uncapped, placed on a suitable plastic tube holder, and kept in an upright position with the bottom part in contact with the freeze-dryer plate layer. The plastic tube was placed in a freeze-dryer (Pilot 2-4T) together with the holder, the freeze-drying program was set according to Table 2, the door was closed, and the freeze-drying program was started. After the freeze-drying program was completed, dry air was backfilled, the door was opened, the plastic tube holder together with the plastic tube was taken out, and tightly capped and sealed.

[0267] (2) A method for improving the stability of 147x FT dNTPs mix, which is the same as (1), except that step 1) is specifically as follows:

[0268] 1) Preparation of the freeze-drying stock solution (Formulation 20, 15% mannitol + 5% trehalose + 147x FT dNTPs mix): 0.0150 g of D-mannitol, 0.0050 g of D-(+)-trehalose dihydrate, and 0.100 mL of 147x FT dNTPs mix were dissolved and uniformly mixed.

[0269] (3) A method for improving the stability of 147x FT dNTPs mix, which is the same as (1), except that step 1) is specifically as follows:

[0270] 1) Preparation of the freeze-drying stock solution (Formulation 21, 16% K30 + 4% sucrose + 147x FT dNTPs mix): Take 0.0160 g of polyvinylpyrrolidone K30, 0.0040 g of sucrose, 0.100 mL of 147x FT dNTPs mix, dissolve, and mix well.

[0271] (4) A method for improving the stability of 147x FT dNTPs mix, which is the same as (1), except that step 1) is specifically as follows:

[0272] 1) Preparation of the freeze-drying stock solution (Formulation 22, 15% K30 + 5% trehalose + 147x FT dNTPs mix): Take 0.0150 g of polyvinylpyrrolidone K30, 0.0050 g of D-(+)-trehalose dihydrate, 0.100 mL of 147x FT dNTPs mix, dissolve, and mix well.

[0273] (5) A method for improving the stability of 147x FT dNTPs mix, which is the same as (1), except that step 1) is specifically as follows:

[0274] 1) Preparation of the freeze-drying stock solution (Formulation 23, 2% K30 + 10% trehalose + 147x FT dNTPs mix): Take 0.0020 g of polyvinylpyrrolidone K30, 0.0100 g of D-(+)-trehalose dihydrate, 0.100 mL of 147x FT dNTPs mix, dissolve, and mix well.

[0275] (6) A method for improving the stability of 147x FT dNTPs mix, which is the same as (1), except that step 1) is specifically as follows:

[0276] 1) Preparation of the freeze-drying stock solution (Formulation 24, 3% K30 + 9% trehalose + 147x FT dNTPs mix): Take 0.0030 g of polyvinylpyrrolidone K30, 0.0090 g of D-(+)-trehalose dihydrate, 0.100 mL of 147x FT dNTPs mix, dissolve, and mix well.

[0277] (7) A method for improving the stability of 147x FT dNTPs mix, which is the same as (1), except that step 1) is specifically as follows:

[0278] 1) Formulation of the lyophilization stock solution (Formulation 25, 5% K30 + 5% trehalose + 147x FT dNTPs mix): Take 0.0050 g of polyvinylpyrrolidone K30, 0.0050 g of D-(+)-trehalose dihydrate, 0.100 mL of 147x FT dNTPs mix, dissolve, mix well.

[0279] (8) A method for improving the stability of 147x FT dNTPs mix, the same as (1), the difference is only in step 1), as follows:

[0280] 1) Formulation of the lyophilization stock solution (Formulation 26, 6% K30 + 4% trehalose + 147x FT dNTPs mix): Take 0.0060 g of polyvinylpyrrolidone K30, 0.0040 g of D-(+)-trehalose dihydrate, 0.100 mL of 147x FT dNTPs mix, dissolve, mix well.

[0281] (9) A method for improving the stability of 147x FT dNTPs mix, the same as (1), the difference is only in step 1), as follows:

[0282] 1) Formulation of the lyophilization stock solution (Formulation 27, 6% K60 + 4% trehalose + 147x FT dNTPs mix): Take 0.0060 g of polyvinylpyrrolidone solution (K60, 45% in H2O), 0.0040 g of D-(+)-trehalose dihydrate, 0.100 mL of 147x FT dNTPs mix, dissolve, mix well.

[0283] (10) A method for improving the stability of 147x FT dNTPs mix, the same as (1), the difference is only in step 1), as follows:

[0284] 1) Formulation of the lyophilization stock solution (Formulation 28, 10% HPβCD + 147x FT dNTPs mix): Take 0.0600 g of hydroxypropyl-β-cyclodextrin, 0.600 mL of 147x FT dNTPs mix, dissolve, mix well.

[0285] (11) A method for improving the stability of 147x FT dNTPs mix, the same as (1), the difference is only in step 1), as follows:

[0286] 1) Formulation of the lyophilization stock solution (Formulation 29, 10% HEβCD + 147x FT dNTPs mix): Take 0.0100 g of (2-hydroxyethyl)-β-cyclodextrin, 0.100 mL of 147x FT dNTPs mix, dissolve, mix well.

[0287] (12) A method for improving the stability of 147x FT dNTPs mix, the same as (1), the only difference is that step 1) is specifically as follows:

[0288] 1) Preparation of the freeze-drying stock solution (Formula 30, 9% K30 + 6% trehalose + 147x FT dNTPs mix): take 0.0090g polyvinylpyrrolidone K30, 0.0060g D-(+)-trehalose dihydrate, 0.100mL 147x FT dNTPs mix, dissolve and mix evenly.

[0289] (13) A method for improving the stability of 147x FT dNTPs mix, the same as (1), the only difference is that step 1) is specifically as follows:

[0290] 1) Preparation of the freeze-drying stock solution (Formula 31, 9% K30 + 6% sucrose + 147x FT dNTPs mix): take 0.0090g polyvinylpyrrolidone K30, 0.0060g sucrose, 0.100mL 147x FT dNTPs mix, dissolve and mix evenly.

[0291] (14) A method for improving the stability of 147x FT dNTPs mix, the same as (1), the only difference is that step 1) is specifically as follows:

[0292] 1) Preparation of the freeze-drying stock solution (Formula 32, 6% K30 + 6% trehalose + 3% HPβCD + 147x FT dNTPs mix): take 0.0060g polyvinylpyrrolidone K30, 0.0060g D-(+)-trehalose dihydrate, 0.0030g hydroxypropyl-β-cyclodextrin, 0.100mL 147x FT dNTPs mix, dissolve and mix evenly.

[0293] (15) A method for improving the stability of 147x FT dNTPs mix, the same as (1), the only difference is that step 1) is specifically as follows:

[0294] 1) Preparation of the freeze-drying stock solution (Formula 33, 6% K30 + 6% sucrose + 3% HPβCD + 147x FT dNTPs mix): take 0.0060g polyvinylpyrrolidone K30, 0.0060g sucrose, 0.0030g hydroxypropyl-β-cyclodextrin, 0.100mL 147x FT dNTPs mix, dissolve and mix evenly.

[0295] (16) A method for improving the stability of 147x FT dNTPs mix, the same as (1), the only difference is that step 1) is specifically as follows:

[0296] 1) Preparation of the lyophilization stock solution (Formulation 34, 6% K30 + 6% sucrose + 3% HE -CD + 147x FT dNTPs mix): Take 0.0360 g of polyvinylpyrrolidone K30, 0.0360 g of sucrose, 0.0180 g of (2-hydroxyethyl)- -cyclodextrin, 0.600 mL of 147x FT dNTPs mix, dissolve and mix well.

[0297] (17) A method for improving the stability of 147x FT dNTPs mix, which is the same as (1), except that step 1) is specifically as follows:

[0298] 1) Preparation of the lyophilization stock solution (Formulation 34, 6% K30 + 6% sucrose + 3% HE -CD + 147x FT dNTPs mix): Take 0.0360 g of polyvinylpyrrolidone K30, 0.0360 g of sucrose, 0.0180 g of (2-hydroxyethyl)- -cyclodextrin, 0.600 mL of 147x FT dNTPs mix, dissolve and mix well.

[0299] (18) A method for improving the stability of 147x FT dNTPs mix, which is the same as (1), except that step 1) is specifically as follows:

[0300] 1) Preparation of the lyophilization stock solution (Formulation 34, 6% K30 + 6% sucrose + 3% HE -CD + 147x FT dNTPs mix): Take 0.0360 g of polyvinylpyrrolidone K30, 0.0360 g of sucrose, 0.0180 g of (2-hydroxyethyl)- -cyclodextrin, 0.600 mL of 147x FT dNTPs mix, dissolve and mix well.

[0301] 3. A method for improving the stability of 150x dNTPs mix II

[0302] (1) A method for improving the stability of 150x dNTPs mix II, comprising the following steps:

[0303] 1) Preparation of the lyophilization stock solution (Formulation 34, 6% K30 + 6% sucrose + 3% HE -CD + 147x FT dNTPs mix): Take 0.0360 g of polyvinylpyrrolidone K30, 0.0360 g of sucrose, 0.0180 g of (2-hydroxyethyl)- -cyclodextrin, 0.600 mL of 147x FT dNTPs mix, dissolve and mix well.

[0304] 2) Put the obtained freeze-drying stock solution into a transparent 2 mL plastic tube, open the plastic tube, place it on a suitable plastic tube support, keep the plastic tube in an upright state, and the bottom can contact the freeze-drying machine plate layer, put the plastic tube together with the support into the freeze-drying machine (Pilot 2-4T), set the freeze-drying program according to Table 2, close the door, start the freeze-drying program, after the freeze-drying program is completed, backfill dry air, open the door, take out the plastic tube support together with the plastic tube, cover tightly and seal.

[0305] (2) A method for improving the stability of 150x dNTPs mix II, which is the same as (1), and the only difference is step 1), which is as follows:

[0306] 1) Preparation of freeze-drying stock solution (Formula 38, 15% mannitol + 5% trehalose + 150x dNTPs mix II): take 0.0750 g of D-mannitol, 0.0250 g of D-(+)-trehalose dihydrate, and 0.500 mL of 150x dNTPs mix II, dissolve and mix uniformly.

[0307] (3) A method for improving the stability of 150x dNTPs mix II, which is the same as (1), and the only difference is step 1), which is as follows:

[0308] 1) Preparation of freeze-drying stock solution (Formula 39, 16% K30 + 4% sucrose + 150x dNTPs mix II): take 0.0800 g of polyvinylpyrrolidone K30, 0.0200 g of sucrose, and 0.500 mL of 150x dNTPs mix II, dissolve and mix uniformly.

[0309] (4) A method for improving the stability of 150x dNTPs mix II, which is the same as (1), and the only difference is step 1), which is as follows:

[0310] 1) Preparation of freeze-drying stock solution (Formula 40, 15% K30 + 5% trehalose + 150x dNTPs mix II): take 0.0750 g of polyvinylpyrrolidone K30, 0.0250 g of D-(+)-trehalose dihydrate, and 0.500 mL of 150x dNTPs mix II, dissolve and mix uniformly.

[0311] (5) A method for improving the stability of 150x dNTPs mix II, which is the same as (1), and the only difference is step 1), which is as follows:

[0312] 1) Preparation of the lyophilization stock solution (Formulation 41, 2% K30 + 10% trehalose + 150x dNTPs mix II): Take 0.0100 g of polyvinylpyrrolidone K30, 0.0500 g of D-(+)-trehalose dihydrate, 0.500 mL of 150x dNTPs mix II, dissolve, and mix well.

[0313] (6) A method for improving the stability of 150x dNTPs mix II, the same as (1), the difference is only in step 1), as follows:

[0314] 1) Preparation of the lyophilization stock solution (Formulation 42, 3% K30 + 9% trehalose + 150x dNTPs mix II): Take 0.0150 g of polyvinylpyrrolidone K30, 0.0450 g of D-(+)-trehalose dihydrate, 0.500 mL of 150x dNTPs mix II, dissolve, and mix well.

[0315] (7) A method for improving the stability of 150x dNTPs mix II, the same as (1), the difference is only in step 1), as follows:

[0316] 1) Preparation of the lyophilization stock solution (Formulation 43, 5% K30 + 5% trehalose + 150x dNTPs mix II): Take 0.0250 g of polyvinylpyrrolidone K30, 0.0050 g of D-(+)-trehalose dihydrate, 0.500 mL of 150x dNTPs mix II, dissolve, and mix well.

[0317] (8) A method for improving the stability of 150x dNTPs mix II, the same as (1), the difference is only in step 1), as follows:

[0318] 1) Preparation of the lyophilization stock solution (Formulation 44, 6% K30 + 4% trehalose + 150x dNTPs mix II): Take 0.0300 g of polyvinylpyrrolidone K30, 0.0200 g of D-(+)-trehalose dihydrate, 0.500 mL of 150x dNTPs mix II, dissolve, and mix well.

[0319] (9) A method for improving the stability of 150x dNTPs mix II, the same as (1), the difference is only in step 1), as follows:

[0320] 1) Formulation of the lyophilization stock solution (Formulation 45, 6% K60 + 4% trehalose + 150x dNTPs mix II): Take 0.0300 g of polyvinylpyrrolidone solution (K60, 45% in H20), 0.0200 g of D-(+)-trehalose dihydrate, 0.500 mL of 150x dNTPs mix II, dissolve, mix well.

[0321] (10) A method for improving the stability of 150x dNTPs mix II, the same as (1), the difference is only in step 1), as follows:

[0322] 1) Formulation of the lyophilization stock solution (Formulation 46, 10% HPβCD + 150x dNTPs mix II): Take 0.0600 g of hydroxypropyl-β-cyclodextrin, 0.600 mL of 150x dNTPs mix II, dissolve, mix well.

[0323] (11) A method for improving the stability of 150x dNTPs mix II, the same as (1), the difference is only in step 1), as follows:

[0324] 1) Formulation of the lyophilization stock solution (Formulation 47, 10% HEβCD + 150x dNTPs mix II): Take 0.0500 g of (2-hydroxyethyl)-β-cyclodextrin, 0.500 mL of 150x dNTPs mix II, dissolve, mix well.

[0325] (12) A method for improving the stability of 150x dNTPs mix II, the same as (1), the difference is only in step 1), as follows:

[0326] 1) Formulation of the lyophilization stock solution (Formulation 48, 9% K30 + 6% trehalose + 150x dNTPs mix II): Take 0.0450 g of polyvinylpyrrolidone K30, 0.0300 g of D-(+)-trehalose dihydrate, 0.500 mL of 150x dNTPs mix II, dissolve, mix well.

[0327] (13) A method for improving the stability of 150x dNTPs mix II, the same as (1), the difference is only in step 1), as follows:

[0328] 1) Formulation of the lyophilization stock solution (Formulation 49, 9% K30 + 6% sucrose + 150x dNTPs mix II): Take 0.0450 g of polyvinylpyrrolidone K30, 0.0300 g of sucrose, 0.500 mL of 150x dNTPs mix II, dissolve, mix well.

[0329] (14) A method for improving the stability of 150x dNTPs mix II, which is the same as (1), except that step 1) is specifically as follows:

[0330] 1) Formulate the lyophilization stock solution (Formula 50, 6% K30 + 6% trehalose + 3% HPβCD + 150x dNTPs mix II): take 0.0300g polyvinylpyrrolidone K30, 0.0300g D-(+)-trehalose dihydrate, 0.0150g hydroxypropyl-β-cyclodextrin, 0.500mL 150x dNTPs mix II, dissolve and mix uniformly.

[0331] (15) A method for improving the stability of 150x dNTPs mix II, which is the same as (1), except that step 1) is specifically as follows:

[0332] 1) Formulate the lyophilization stock solution (Formula 51, 6% K30 + 6% sucrose + 3% HPβCD + 150x dNTPs mix II): take 0.0300g polyvinylpyrrolidone K30, 0.0300g sucrose, 0.0150g hydroxypropyl-β-cyclodextrin, 0.500mL 150x dNTPs mix II, dissolve and mix uniformly.

[0333] (16) A method for improving the stability of 150x dNTPs mix II, which is the same as (1), except that step 1) is specifically as follows:

[0334] 1) Formulate the lyophilization stock solution (Formula 52, 6% K30 + 6% sucrose + 3% HEβCD + 150x dNTPs mix II): take 0.0360g polyvinylpyrrolidone K30, 0.0360g sucrose, 0.0180g (2-hydroxyethyl)-β-cyclodextrin, 0.600mL 150x dNTPs mix II, dissolve and mix uniformly.

[0335] (17) A method for improving the stability of 150x dNTPs mix II, which is the same as (1), except that step 1) is specifically as follows:

[0336] 1) Formulate the lyophilization stock solution (Formula 53, 6% K30 + 6% sucrose + 3% hydroxyethyl starch 130 + 150x dNTPs mix II): take 0.0360g polyvinylpyrrolidone K30, 0.0360g sucrose, 0.0180g hydroxyethyl starch (110-150kDa, degree of substitution 0.37-0.43), 0.600mL 150x dNTPs mix II, dissolve and mix uniformly.

[0337] (18) A method for improving the stability of 150x dNTPs mix II, which is the same as (1), except that step 1) is specifically as follows:

[0338] 1) Preparation of the freeze-drying stock solution (Formula 54, 6% K30 + 6% sucrose + 3% hydroxyethyl starch 200 + 150x dNTPs mix II): Take 0.0360 g of polyvinylpyrrolidone K30, 0.0360 g of sucrose, 0.0180 g of hydroxyethyl starch (170-230 kDa, degree of substitution 0.46-0.54), 0.600 mL of 150x dNTPs mix II, dissolve and mix uniformly.

[0339] 4. A method for improving the stability of THPP aqueous solution

[0340] (1) A method for improving the stability of THPP aqueous solution, comprising the following steps:

[0341] 1) Preparation of THPP aqueous solution: weigh a certain mass of THPP stock solution (considered as 100%), add enzyme-free nucleic acid water to make the final concentration 11.1% w / v.

[0342] 2) Preparation of freeze-drying stock solution (Formula 55, 10% PEG8000 + 5.55% THPP, where the percentages are all mass / volume, the same below): take 0.2500 g of PEG 8000, 1.25 mL of THPP aqueous solution, 1.25 mL of enzyme-free water, dissolve and mix uniformly;

[0343] 3) Fill the freeze-drying stock solution into a transparent vial with a capacity of 5 mL at 2.5 mL per vial, place two forked rubber stoppers on top, reserve a vent hole, arrange the vials uniformly on the freeze-drying machine plate layer, start Pilot 2-4T, set the freeze-drying program according to Table 3, close the door, start the freeze-drying program, after the freeze-drying program is completed, press the stopper, backfill dry air, open the door, take out the vial, and seal it by rolling.

[0344] Table 3 Freeze-drying program

[0345] (2) A method for improving the stability of THPP aqueous solution, which is the same as (1), except that step 2) is specifically as follows:

[0346] 2) Preparation of freeze-drying stock solution (Formula 56, 10% PEG20000 + 5.55% THPP): take 0.2500 g of PEG 20000, 1.25 mL of THPP aqueous solution, 1.25 mL of enzyme-free water, dissolve and mix uniformly.

[0347] (3) A method for improving the stability of THPP aqueous solution, which is the same as (1), except that step 2) is specifically as follows:

[0348] 2) Preparation of the freeze-drying stock solution (Formula 57, 15% PEG 20000 + 5.55% THPP): Take 0.3750 g of PEG 20000, 1.25 mL of THPP aqueous solution, and 1.25 mL of nuclease-free water, dissolve and mix evenly.

[0349] (4) A method for improving the stability of THPP aqueous solution, which is the same as (1), except that step 2) is specifically as follows:

[0350] 2) Preparation of the freeze-drying stock solution (Formula 58, 10% PEG 20000 + 11.1% THPP): Take 0.2500 g of PEG 20000, 2.5 mL of THPP aqueous solution, dissolve and mix evenly.

[0351] (5) A method for improving the stability of THPP aqueous solution, which is the same as (1), except that step 2) is specifically as follows:

[0352] 2) Preparation of the freeze-drying stock solution (Formula 59, 15% PEG 20000 + 11.1% THPP): Take 0.3750 g of PEG 20000, 2.5 mL of THPP aqueous solution, dissolve and mix evenly.

[0353] 5. A method for improving the stability of protein wash buffer

[0354] (1) A method for improving the stability of protein wash buffer (Formula 60, 10% D-mannitol + protein wash buffer, where the percentage is mass / volume ratio): Take 0.2000 g of D-mannitol and 2.00 mL of protein wash buffer (BSA 0.04 mg / mL, sucrose 15 wt%), mix evenly and make the D-mannitol completely dissolved;

[0355] 2) The above mixture is one freeze-drying stock solution, and each freeze-drying stock solution is placed in a transparent vial with a capacity of 5 mL, two forked rubber stoppers are placed on top, a vent hole is reserved, the vials are evenly arranged on the freeze-drying machine plate layer, the Pilot 2-4T is started, the freeze-drying program is set according to Table 3, the door is closed, the freeze-drying program is started, after the freeze-drying program is completed, the stopper is pressed, the dry air is backfilled, the door is opened, the vials are taken out, and the cap is sealed.

[0356] 6. A method for improving the stability of 147x FT dNTPs mix, comprising the following steps:

[0357] 1) Formulate the lyophilization stock solution (Formulation 61, 6% K30 + 6% sucrose + 3% HE -CD + 147x FT dNTPs mix): Take 0.0600 g of polyvinylpyrrolidone K30, 0.0600 g of sucrose, 0.0300 g of (2-hydroxyethyl)- -cyclodextrin, 1.00 mL of 147x FT dNTPs mix, dissolve, and mix well.

[0358] 2) Put the obtained lyophilization stock solution into a brown 2 mL plastic tube and place the plastic tube on ice. Fill the drop ball machine liquid nitrogen cup with liquid nitrogen, and the liquid tube needs to be kept inserted below the liquid level of the lyophilization stock solution. Adjust the drop ball machine droplet size to about 35 μL, start the drop ball program; after the end of the drop ball, store the microspheres in sufficient liquid nitrogen. Set the lyophilization program according to Table 4, start the program in advance to pre-freeze the plate layer, and put the microspheres into the lyophilizer tray together with the liquid nitrogen during the pre-freezing process. Close the door, and after the end of the lyophilization program, backfill dry air, open the door, and put the microspheres into a plastic tube in a dry environment, seal and avoid light.

[0359] Table 4 Lyophilization program

[0360] Effect implementation example

[0361] 1. The lyophilized reagents obtained from "1 in (1)-(18)" in Example 1 (corresponding to Formulations 1-18 in turn) and the corresponding stock solutions without lyophilization protectants (non-lyophilization control, stock, i.e. 150x dNTPs mix) were sealed and stored at 37°C for 1 week (lyophilized + 37°C for 1 week, stock + 37°C for 1 week), 2 weeks (lyophilized + 37°C for 2 weeks, stock + 37°C for 2 weeks), and 55°C for 1 day (lyophilized + 55°C for 1 day, stock + 55°C for 1 day), 1 week (lyophilized + 55°C for 1 week, stock + 55°C for 1 week) in the dark, and then the lyophilized reagents were reconstituted: 0.6 mL of nuclease-free water was added to each tube (the lyophilized stock was not treated); at the same time, the lyophilized reagents obtained from "1 in (1)-(18)" in Example 1 were immediately reconstituted (lyophilized): 0.6 mL of nuclease-free water was added to each tube; the reconstituted solutions (lyophilized, lyophilized + 37°C for 1 week, lyophilized + 37°C for 2 weeks, lyophilized + 55°C for 1 day, lyophilized + 55°C for 1 week), the stock solutions without lyophilization protectants under different storage conditions (stock + 37°C for 1 week, stock + 37°C for 2 weeks, stock + 55°C for 1 day, stock + 55°C for 1 week), and the stock solutions without lyophilization protectants freshly prepared (non-lyophilization stock) were sequenced on the MGISEQ-2000 platform to obtain the Runon slope data (the smaller the Runon slope, the smaller the degree of damage to the reversible blocking group on each modified dNTP monomer and the better the thermal stability); the results are shown in Table 5: by comparing the data of the non-lyophilization stock and the lyophilized reagents, it can be seen that lyophilization and / or reconstitution almost do not affect the function of the 150x dNTPs mix; by comparing the data of the lyophilized and stock reagents treated at different temperatures and for different times, it can be seen that the formulations provided by the application can significantly improve the thermal stability, and in particular, the lyophilized reagent prepared using Formulation 15 has the best thermal stability. When evaluating the effect of the lyophilized reagent, the change in the appearance of the lyophilized cake after heating, such as whether it collapses or melts, and the ease of reconstitution after lyophilization and heating also need to be considered; the lyophilized cakes obtained from "1 in (1)-(18)" in Example 1 are uniform in color, have a complete structure without collapse, and can be quickly reconstituted with water without clogging the gun tip after blowing; under the test conditions, the appearance and reconstitution speed do not change significantly after heating (Figure 1).

[0362] Table 5 Runon slope of the lyophilized reagents obtained from "1 in (1)-(18)" in Example 1 and the corresponding lyophilized stock after different treatments

[0363] Note: " / " indicates that the data was not measured, and some formulations show more than one test result.

[0364] Meanwhile, four repeated tests were performed on "1st(15)" (i.e. Formula 15) in Example 1, including four independent liquid preparation and freeze-drying operations. The freeze-dried reagents were sealed in the dark and placed at 37°C for 1 week (freeze-dried + 37°C for 1 week, sample + 37°C for 1 week), 2 weeks (freeze-dried + 37°C for 2 weeks, sample + 37°C for 2 weeks), and 55°C for 1 day (freeze-dried + 55°C for 1 day, sample + 55°C for 1 day), 1 week (freeze-dried + 55°C for 1 week, sample + 55°C for 1 week), and then the freeze-dried reagents were reconstituted: 0.6 mL of nuclease-free water was added to each tube; at the same time, the freeze-dried reagents obtained from "1st(15)" in Example 1 were immediately reconstituted (after freeze-drying): 0.6 mL of nuclease-free water was added to each tube; the reconstituted solutions (after freeze-drying, freeze-dried + 37°C for 1 week, freeze-dried + 37°C for 2 weeks, freeze-dried + 55°C for 1 day, freeze-dried + 55°C for 1 week), and the freshly prepared stock solution without freeze-drying protectants (non-freeze-dried sample) were sequenced on the MGISEQ-2000 platform to obtain the Runon slope data, and the results are shown in Table 6: freeze-drying and / or reconstitution operations have little effect on the function of 150x dNTPs mix, confirming that the thermal stability of freeze-dried reagents is much higher than that of non-freeze-dried controls (non-freeze-dried sample).

[0365] Table 6 Runon slope of freeze-dried reagents obtained from "1st(15)" in Example 1 and corresponding freeze-dried stock solutions after different treatments

[0366] Note: " / " indicates that the data was not measured.

[0367] 2. Prepare a DNBSEQ-G99RS High-Throughput Sequencing Reagent Kit (G99SM FCL PE150) according to the DNBSEQ-G99RS High-Throughput Sequencing Reagent Kit Instruction Manual. Take out the DNBSEQ-G99 Sequencing Slide and place it at room temperature for standby. During the test, the lyophilized reagent (lyophilized G99 hotmix) obtained from “2 (15)” in Example 1 (i.e., Formula 33) is directly reconstituted, or the lyophilized reagent is sealed from light and placed at 25°C for 5-21 days before reconstitution: add 0.1 mL of nuclease-free water to each tube; take 95 μL of the reconstituted solution and inject it from the aluminum foil corresponding to the 3rd hole from the right in the reagent tank with a syringe, replacing the M1 hole in the reagent tank (without pressing the two holes). Use the same finished reagent tank as a control (standard reagent tank ctrl). Refer to the DNBSEQ-G99RS High-Throughput Sequencing Reagent Kit Instruction Manual, and prepare DNA nanoballs with standard library reagent V3. After mixing DNB with DNB loading buffer II uniformly on ice, load it into the MGISEQ-G99 sequencing slide. Select the PE150+10 sequencing scheme on the machine operation interface, insert the reagent tank and sequencing slide into the corresponding positions of the machine, and start sequencing. The results are shown in Tables 7-1 and 7-2: the lyophilized reagent obtained from “2 (15)” in Example 1 (i.e., Formula 33) has little effect on the function of 147x FT dNTPs mix, and the off-machine results meet the QC requirements; the lyophilized reagent obtained from “2 (15)” in Example 1 (i.e., Formula 33) has little effect on the function of 147x FT dNTPs mix after being placed at 25°C for at least 15 days, and the off-machine results meet the QC requirements. “2 (1)-(14), and (16)-(18)” in Example 1 (corresponding to Formulas 19-32, and 34-36, respectively) have similar effects.

[0368] Table 7-1 Sequencing effect of the reconstituted lyophilized reagent obtained from “2 (15)” in Example 1

[0369] Table 7-2 Sequencing effect of the reconstituted lyophilized reagent obtained from “2 (15)” in Example 1 after being placed at 25°C for 5-21 days

[0370] 3. The lyophilized powder cake obtained from “4 (1)-(5)” in Example 1 (i.e., Formulas 55-59) has a complete structure without collapse, and can be quickly reconstituted with water or the corresponding buffer (Figs. 2, 3). There is no obvious change in appearance and reconstitution speed after being placed at 37°C for 21 days or at 55°C for 10 days.

[0371] A DNBSEQ-G99RS high-throughput sequencing reagent kit (G99SM FCL PE150) was prepared according to the DNBSEQ-G99RS high-throughput sequencing reagent kit instruction. The DNBSEQ-G99 sequencing slide was taken out and placed at room temperature for standby. When testing, the freeze-dried reagents obtained in Example 1 in “4th(1), (2), (4), (5)” (i.e. Formulas 55, 56, 58, 59) were sealed in the dark and placed at 37°C for 21 days (corresponding to freeze-dried 5.55% THPP + 10% PEG8K_37°C 21d, freeze-dried 5.55% THPP + 10% PEG2W_37°C 21d, freeze-dried 11.1% THPP + 10% PEG2W_37°C 21d, freeze-dried 11.1% THPP + 15% PEG2W_37°C 21d, respectively), or the freeze-dried reagents obtained in Example 1 in “4th(1), (4), (5)” (i.e. Formulas 55, 58, 59) were sealed in the dark and placed at 55°C for 10 days (corresponding to freeze-dried 5.55% THPP + 10% PEG8K_55°C 10d, freeze-dried 11.1% THPP + 10% PEG2W_55°C 10d, freeze-dried 11.1% THPP + 15% PEG2W_55°C 10d, respectively), and then the freeze-dried reagents were reconstituted: for Formulas 55 and 56, the reconstitution was mixed at a ratio of 1:9; for Formulas 58 and 59, the reconstitution was mixed at a ratio of 1:19; 0.75% w / v PEG2W in RR (0.75% PEG2W in RR, used to study the effect of freeze-dried protectant itself on sequencing, the PEG2W concentration is equivalent to that after mixing with Formula 57 and 59), the above reconstitution solution and the mixture of FT rapid regeneration reagent buffer (without THPP), 5.55% THPP aqueous solution (5.55% THPP aqueous solution-37°C 21d, non-freeze-dried control group) and FT rapid regeneration reagent buffer (without THPP) mixed at a ratio of 1:9 were mixed at a ratio of 1:9, and the mixture was injected into the reagent tank from the aluminum foil corresponding to the 7th hole from the right of the reagent tank (inject and extract the same volume of FT rapid regeneration reagent), and a control (Ctrl) was also prepared using the same finished product kit. According to the DNBSEQ-G99RS high-throughput sequencing reagent kit instruction, DNA nanoballs were prepared using standard library reagent V3. After the DNB was mixed uniformly with DNB loading buffer II on ice, it was loaded into the MGISEQ-G99 sequencing slide.The PE150+10 sequencing program was selected on the machine operation interface, the reagent tank and sequencing slide were inserted into the corresponding positions of the machine, and the sequencing was started. The results are shown in Tables 8-1 and 8-2: a small amount of PEG2W (PEG20000) added during the freeze-drying process had no obvious effect on sequencing; the freeze-dried reagents obtained in Example 1 “4th (1), (2), (4), (5)” (i.e. formulations 55, 56, 58, 59) significantly improved the thermal stability of THPP (especially formulations 55, 56, 59); after the THPP freeze-dried cake stored at 37°C for 21 days was prepared into a reagent tank, the PE150 sequencing quality was significantly better than that of the non-freeze-dried control group under the same storage conditions, and was equivalent to that of the finished standard reagent tank stored at -20°C; after the THPP freeze-dried cake stored at 55°C for 10 days was prepared into a reagent tank, the PE150 sequencing quality was equivalent to that of the finished standard reagent tank stored at -20°C (especially formulation 59).

[0372] Table 8-1 Sequencing effect of freeze-dried reagents obtained in Example 1 “4th (1), (2), (4), (5)” after 37°C heat treatment

[0373] Table 8-2 Sequencing effect of freeze-dried reagents obtained in Example 1 “4th (1), (4), (5)” after 55°C heat treatment

[0374] 4. The freeze-dried cake obtained in Example 1 “5th (1)” (i.e. formulation 60) had complete structure without collapse, and could be quickly reconstituted by adding water (Figure 4). After being stored at 37°C for 10 days, 20 days, or 55°C for 10 days, the appearance did not change significantly, and the reconstitution speed did not change.

[0375] A DNBSEQ-G99RS high-throughput sequencing reagent kit (G99SM FCL PE150) was prepared according to the DNBSEQ-G99RS high-throughput sequencing reagent kit instructions. Take out the DNBSEQ-G99 sequencing slide and place it at room temperature for standby. During the test, the freeze-dried reagent obtained from "5 middle (1)" (i.e. formula 60) in Example 1 was sealed, or sealed and placed at 37°C for 10 days, 20 days, or sealed and placed at 55°C for 10 days (corresponding to freeze-dried 10% D mannitol-PWB, freeze-dried 10% D mannitol-PWB_37℃10d, freeze-dried 10% D mannitol-PWB_37℃20d, freeze-dried 10% D mannitol-PWB_55℃10d, respectively), and then 2 mL of water was added to reconstitute the freeze-dried reagent; the original reagent was removed from the aluminum foil corresponding to the 8th hole from the left of the finished reagent tank with a syringe, 1 mL of the above reconstituted solution was taken, and another new syringe was used to inject the reagent tank from the aluminum foil corresponding to the 8th hole from the left of the above reagent tank; at the same time, the same finished reagent kit was used as a control (Ctrl). According to the DNBSEQ-G99RS high-throughput sequencing reagent kit instructions, DNA nanoballs were prepared from standard library reagent V3. After the DNB was mixed uniformly with DNB loading buffer II on ice, it was loaded into the MGISEQ-G99 sequencing slide. The PE150+10 sequencing scheme was selected on the machine operation interface, the reagent tank and the sequencing slide were inserted into the corresponding positions of the machine, and the sequencing was started. The results are shown in Table 9: the freeze-dried reagent obtained from "5 middle (1)" (i.e. formula 60) in Example 1 significantly improves the thermal stability of the protein washing buffer; after the protein washing buffer freeze-dried cake is prepared into a reagent tank after freeze-drying, or placing at 37°C for 10 days, 20 days, or placing at 55°C for 10 days, the PE150 sequencing quality is comparable to that of the finished standard reagent tank stored at -20°C.

[0376] Table 9 Sequencing effect of freeze-dried reagent obtained from "5 middle (1)" after heat treatment

[0377] The above examples are preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement methods and shall be within the scope of protection of the present application.

Claims

1. A composition, which is any one of a1) to a3): a1) the composition comprises: a lyoprotectant and a component comprising a nucleotide, which is a natural nucleotide and / or a modified nucleotide; a2) the composition comprises: a lyoprotectant and a component comprising a redox active substance; a3) the composition comprises: a lyoprotectant and a component comprising a protein; the composition in a1), a2), a3) is a lyophilized composition.

2. The composition according to claim 1, wherein: the lyoprotectant in a1), a2), a3) is each independently selected from one or more of: a polyhydroxyl compound, a sugar, a polymer; preferably, the lyoprotectant in a1) is any one of b1) to b5): b1) a polymer, a polyhydroxyl compound and a sugar; b2) a polymer and a sugar; b3) a polyhydroxyl compound and a sugar; b4) a polyhydroxyl compound; b5) a sugar; preferably, the lyoprotectant in a2) is a polyethylene glycol; further at least one of PEG 2000, PEG 4000, PEG 6000, PEG 8000, PEG 10000, PEG 20000; preferably, the lyoprotectant in a3) is D-mannitol.

3. The composition according to claim 2, wherein: the lyoprotectant in a1) is any one of c1) to c14): c1) mannitol and sucrose; c2) mannitol and trehalose; c3) polyvinylpyrrolidone K30 and sucrose; c4) polyvinylpyrrolidone K30 and trehalose; c5) polyvinylpyrrolidone K60 and trehalose; c6) polyvinylpyrrolidone K90 and trehalose; c7) (2-hydroxyethyl)-P-cyclodextrin; c8) hydroxypropyl-P-cyclodextrin; c9) polyvinylpyrrolidone K30, trehalose and (2-hydroxyethyl)-P-cyclodextrin; c10) polyvinylpyrrolidone K30, trehalose and hydroxypropyl-P-cyclodextrin; c11) polyvinylpyrrolidone K30, sucrose and (2-hydroxyethyl)-P-cyclodextrin; c12) polyvinylpyrrolidone K30, sucrose and hydroxypropyl-P-cyclodextrin; c13) polyvinylpyrrolidone K30, trehalose and hydroxyethyl starch; c14) polyvinylpyrrolidone K30, sucrose and hydroxyethyl starch.

4. The composition according to claim 3, wherein: the mass ratio of the mannitol and sucrose in c1) is (3 to 5): 1; preferably, the mass ratio of the mannitol and trehalose in c2) is (2 to 4): 1; preferably, the mass ratio of the polyvinylpyrrolidone K30 and sucrose in c3) is (1 to 5): 1; preferably, the mass ratio of the polyvinylpyrrolidone K30 and trehalose in c4) is (0.1 to 3.5): 1; preferably, the mass ratio of the polyvinylpyrrolidone K60 and trehalose in c5) is (1 to 2): 1; preferably, the mass ratio of the polyvinylpyrrolidone K90 and trehalose in c6) is (1 to 2): 1; ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ Preferably, the mass ratio of the polyvinylpyrrolidone K30, trehalose and (2-hydroxyethyl)-β-cyclodextrin in c9) is (1-3):(1-3):1; Preferably, the mass ratio of the polyvinylpyrrolidone K30, trehalose and (2-hydroxyethyl)-β-cyclodextrin in c9) is (1-3):(1-3):1; Preferably, the mass ratio of the polyvinylpyrrolidone K30, trehalose and (2-hydroxyethyl)-β-cyclodextrin in c9) is (1-3):(1-3):1; Preferably, the mass ratio of the polyvinylpyrrolidone K30, trehalose and (2-hydroxyethyl)-β-cyclodextrin in c9) is (1-3):(1-3):1; Preferably, the mass ratio of the polyvinylpyrrolidone K30, trehalose and (2-hydroxyethyl)-β-cyclodextrin in c9) is (1-3):(1-3):1; Preferably, the mass ratio of the polyvinylpyrrolidone K30, trehalose and (2-hydroxyethyl)-β-cyclodextrin in c9) is (1-3):(1-3):

1.

5. The composition of claim 1, wherein: the mass ratio or mass volume ratio of the lyoprotectant and the component in a1), a2), a3) is (3-20):50; Preferably, the mass ratio of the lyoprotectant and the redox active substance in a2) is 1:(0.3-1.5); Preferably, the mass volume ratio of the lyoprotectant and the protein-containing component in a3) is 1:(5-15).

6. The composition of any one of claims 1-5, wherein: the nucleotide in a1) is a modified nucleotide; Preferably, the redox active substance in a2) comprises at least one of THPP, TCEP; Preferably, the component in a2) is a regenerating reagent; Preferably, the protein in a3) is a non-enzyme protein; Preferably, the protein-containing component in a3) is a protein wash buffer; Preferably, the lyophilized composition in a1), a2), a3) is a microsphere, a powder cake or a combination thereof; Preferably, the microsphere is spherical, oval or ring-shaped.

7. A sequencing kit or a sequencing reagent tank, comprising a composition; the composition is any one or more of a1)-a3): a1) the composition comprises a lyoprotectant and a component containing nucleotides, the nucleotides being natural nucleotides and / or modified nucleotides; a2) the composition comprises a lyoprotectant and a component containing redox active substances; a3) the composition comprises a lyoprotectant and a component containing proteins; the composition in a1), a2), a3) is a lyophilized composition.

8. The sequencing kit or the sequencing reagent tank of claim 7, wherein: the lyoprotectant in a1), a2), a3) is independently selected from one or more of a polyhydroxy compound, a sugar, a polymer; Preferably, the lyoprotectant in a1) is any one of b1)-b5): b1) a polymer, a polyhydroxy compound and a sugar; b2) a polymer and a sugar; b3) a polyhydroxy compound and a sugar; b4) a polyhydroxy compound; b5) a sugar; Preferably, the lyoprotector in a2) is polyethylene glycol; further at least one of PEG2000, PEG4000, PEG6000, PEG8000, PEG10000, PEG20000; Preferably, the lyoprotector in a3) is D-mannitol.

9. The sequencing kit or sequencing reagent tank according to claim 8, wherein: the lyoprotector in a1) is any one of c1) to c14): c1) mannitol and sucrose; c2) mannitol and trehalose; c3) polyvinylpyrrolidone K30 and sucrose; c4) polyvinylpyrrolidone K30 and trehalose; c5) polyvinylpyrrolidone K60 and trehalose; c6) polyvinylpyrrolidone K90 and trehalose; c7) (2-hydroxyethyl)-β-cyclodextrin; c8) hydroxypropyl-β-cyclodextrin; c9) polyvinylpyrrolidone K30, trehalose and (2-hydroxyethyl)-β-cyclodextrin; c10) polyvinylpyrrolidone K30, trehalose and hydroxypropyl-β-cyclodextrin; c11) polyvinylpyrrolidone K30, sucrose and (2-hydroxyethyl)-β-cyclodextrin; c12) polyvinylpyrrolidone K30, sucrose and hydroxypropyl-β-cyclodextrin; c13) polyvinylpyrrolidone K30, trehalose and hydroxyethyl starch; c14) polyvinylpyrrolidone K30, sucrose and hydroxyethyl starch; Preferably, the mass ratio or mass volume ratio of the lyoprotector and the components in a1), a2), a3) is (3 to 20): 50; Preferably, the mass ratio of the lyoprotector and the redox active substance in a2) is 1:(0.3 to 1.5); Preferably, the mass volume ratio of the lyoprotector and the protein-containing component in a3) is 1:(5 to 15); Preferably, the mass ratio of the mannitol and sucrose in c1) is (3 to 5): 1; Preferably, the mass ratio of the mannitol and trehalose in c2) is (2 to 4): 1; Preferably, the mass ratio of the polyvinylpyrrolidone K30 and sucrose in c3) is (1 to 5): 1; Preferably, the mass ratio of the polyvinylpyrrolidone K30 and trehalose in c4) is (0.1 to 3.5): 1; Preferably, the mass ratio of the polyvinylpyrrolidone K60 and trehalose in c5) is (1 to 2): 1; Preferably, the mass ratio of the polyvinylpyrrolidone K90 and trehalose in c6) is (1 to 2): 1; Preferably, the mass ratio of the polyvinylpyrrolidone K30, trehalose and (2-hydroxyethyl)-β-cyclodextrin in c9) is (1 to 3):(1 to 3): 1; Preferably, the mass ratio of the polyvinylpyrrolidone K30, trehalose and hydroxypropyl-β-cyclodextrin in c10) is (1 to 3):(1 to 3): 1; Preferably, the mass ratio of the polyvinylpyrrolidone K30, sucrose and (2-hydroxyethyl)-β-cyclodextrin in c11) is (1 to 3):(1 to 3): 1; Preferably, the mass ratio of the polyvinylpyrrolidone K30, sucrose and hydroxypropyl-β-cyclodextrin in c12) is (1-3):(1-3):1; Preferably, the mass ratio of the polyvinylpyrrolidone K30, trehalose and hydroxyethyl starch in c13) is (1-3):(1-3):1; Preferably, the mass ratio of the polyvinylpyrrolidone K30, sucrose and hydroxyethyl starch in c14) is (1-3):(1-3):

1.

10. The sequencing kit or sequencing reagent tank according to any one of claims 7-9, wherein: the nucleotide in a1) is a modified nucleotide; Preferably, the redox active material in a2) comprises at least one of THPP, TCEP; Preferably, the component in a2) is a regenerating reagent; Preferably, the protein in a3) is a non-enzyme protein; Preferably, the protein-containing component in a3) is a protein washing buffer; Preferably, the lyophilized composition in a1), a2), a3) is a microsphere, a powder cake or a combination thereof; Preferably, the microsphere is spherical, oval or ring-shaped.

11. A method for reconstituting a composition, comprising mixing the composition with a reconstitution reagent to obtain a reconstituted solution. The composition is the composition according to any one of claims 1-6.

12. The method according to claim 11, wherein: the reconstitution reagent comprises water, ethanolamine, a buffer or a combination thereof; Preferably, the mixing is performed under conditions effective to reconstitute the composition; Preferably, the reconstituted solution is used in a sequencing process.

13. A method for sequencing, comprising the following steps: f1) mixing a composition with a reconstitution reagent to obtain a reconstituted solution; f2) using a reagent tank comprising the reconstituted solution to perform sequencing; the composition in f1) is the composition according to any one of claims 1-6; Preferably, the reconstitution reagent comprises water, ethanolamine, a buffer or a combination thereof; Preferably, the mixing is performed under conditions effective to reconstitute the composition.

14. A method for improving the stability of a sequencing reagent, comprising: the sequencing reagent comprises a component containing a substance with poor thermal stability; the component containing a substance with poor thermal stability comprises a nucleotide-containing component, a redox active material-containing component or a protein-containing component; the nucleotide is a natural nucleotide and / or a modified nucleotide; the method comprises the following steps: mixing the substance with poor thermal stability, or the component containing a substance with poor thermal stability, or the sequencing reagent, with a lyophilization protective agent and lyophilizing.

15. The method according to claim 14, comprising: the lyophilization protective agent is independently selected from one or more of a polyol, a sugar and a polymer; Preferably, when the component containing a substance with poor thermal stability comprises a nucleotide-containing component, the lyophilization protective agent is any one of b1)-b5): b1) a polymer, a polyol and a sugar; b2) a polymer and a sugar; b3) a polyol and a sugar; b4) a polyol; b5) a sugar. Further preferably, when the component containing a substance with poor heat stability contains a nucleotide-containing component, the lyoprotector is any one of c1) to c14): c1) mannitol and sucrose; c2) mannitol and trehalose; c3) polyvinylpyrrolidone K30 and sucrose; c4) polyvinylpyrrolidone K30 and trehalose; c5) polyvinylpyrrolidone K60 and trehalose; c6) polyvinylpyrrolidone K90 and trehalose; c7) (2-hydroxyethyl)-β-cyclodextrin; c8) hydroxypropyl-β-cyclodextrin; c9) polyvinylpyrrolidone K30, trehalose and (2-hydroxyethyl)-β-cyclodextrin; c10) polyvinylpyrrolidone K30, trehalose and hydroxypropyl-β-cyclodextrin; c11) polyvinylpyrrolidone K30, sucrose and (2-hydroxyethyl)-β-cyclodextrin; c12) polyvinylpyrrolidone K30, sucrose and hydroxypropyl-β-cyclodextrin; c13) polyvinylpyrrolidone K30, trehalose and hydroxyethyl starch; c14) polyvinylpyrrolidone K30, sucrose and hydroxyethyl starch; Preferably, when the component containing a substance with poor heat stability contains a redox-active substance-containing component, the lyoprotector is polyethylene glycol; further, at least one of PEG2000, PEG4000, PEG6000, PEG8000, PEG10000, PEG20000; preferably, when the component containing a substance with poor heat stability contains a protein-containing component, the lyoprotector is D-mannitol.

16. The method according to claim 14, characterized in that: the mass ratio or mass volume ratio of the lyoprotector and the component containing a substance with poor heat stability is (3 to 20) : 50; Preferably, when the component containing a substance with poor heat stability contains a redox-active substance-containing component, the mass ratio of the lyoprotector to the redox-active substance is 1 : (0.3 to 1.5); Preferably, when the component containing a substance with poor heat stability contains a protein-containing component, the mass volume ratio of the lyoprotector to the protein-containing component is 1 : (5 to 15); Preferably, the mass ratio of the mannitol and sucrose in c1) is (3 to 5) : 1; Preferably, the mass ratio of the mannitol and trehalose in c2) is (2 to 4) : 1; Preferably, the mass ratio of the polyvinylpyrrolidone K30 and sucrose in c3) is (1 to 5) : 1; Preferably, the mass ratio of the polyvinylpyrrolidone K30 and trehalose in c4) is (0.1 to 3.5) : 1; Preferably, the mass ratio of the polyvinylpyrrolidone K60 and trehalose in c5) is (1 to 2) : 1; Preferably, the mass ratio of the polyvinylpyrrolidone K90 and trehalose in c6) is (1 to 2) : 1; Preferably, the mass ratio of the polyvinylpyrrolidone K30, trehalose and (2-hydroxyethyl)-β-cyclodextrin in c9) is (1 to 3) : (1 to 3) : 1; Preferably, the mass ratio of the polyvinylpyrrolidone K30, trehalose and hydroxypropyl-β-cyclodextrin in c10) is (1-3):(1-3):1; Preferably, the mass ratio of the polyvinylpyrrolidone K30, sucrose and (2-hydroxyethyl)-β-cyclodextrin in c11) is (1-3):(1-3):1; Preferably, the mass ratio of the polyvinylpyrrolidone K30, sucrose and hydroxypropyl-β-cyclodextrin in c12) is (1-3):(1-3):1; Preferably, the mass ratio of the polyvinylpyrrolidone K30, trehalose and hydroxyethyl starch in c13) is (1-3):(1-3):1; Preferably, the mass ratio of the polyvinylpyrrolidone K30, sucrose and hydroxyethyl starch in c14) is (1-3):(1-3):

1.

17. The method of any one of claims 14-16, wherein: the nucleotide is a modified nucleotide; Preferably, the redox active species comprises at least one of THPP, TCEP; Preferably, the component is a regeneration reagent; Preferably, the protein is a non-enzymatic protein; Preferably, the protein-containing component is a protein wash buffer; Preferably, the lyophilization results in lyophilized microspheres, lyophilized powder cakes, or a combination thereof; Preferably, the microspheres are spherical, ellipsoidal, or toroidal; Preferably, when the concentration of the substance with poor thermal stability in the component containing the substance with poor thermal stability is low, the substance with poor thermal stability is formulated at a high concentration; Preferably, when the volume of the component containing the substance with poor thermal stability is large, the substance with poor thermal stability is lyophilized separately.

18. The method of any one of claims 14-16, wherein: the lyophilization comprises the steps of pre-freezing, sublimation drying, and desorption drying; Preferably, when the component containing the substance with poor thermal stability is a nucleotide-containing component, the pre-freezing conditions comprise incubation at -53 to -47°C for 10 to 300 minutes; Preferably, when the component containing the substance with poor thermal stability is a nucleotide-containing component, the pre-freezing conditions further comprise pre-incubation at -3 to 3°C for 10 to 200 minutes before incubation at -53 to -47°C; Preferably, when the component containing the substance with poor thermal stability is a nucleotide-containing component, the sublimation drying conditions comprise incubation at -33 to -27°C for 10 to 2000 minutes, incubation at -23 to -17°C for 5 to 90 minutes, incubation at -13 to -7°C for 5 to 90 minutes, and incubation at -3 to 3°C for 5 to 90 minutes; Preferably, when the component containing the substance with poor thermal stability is a nucleotide-containing component, the sublimation drying conditions further comprise pre-incubation at -43 to -27°C for 10 to 2000 minutes before incubation at -33 to -27°C; Preferably, when the component containing the substance with poor thermal stability is a nucleotide-containing component, the desorption drying conditions comprise incubation at 22 to 28°C for 140 to 460 minutes; Preferably, when the component containing a substance with poor heat stability is a component containing a redox-active substance or a component containing a protein, the pre-freezing conditions include incubation at -3 to 3°C for 10 to 200 minutes and incubation at -53 to -47°C for 10 to 300 minutes. Preferably, when the component containing a substance with poor heat stability is a component containing a redox-active substance or a component containing a protein, the sublimation drying conditions include incubation at -43 to -37°C for 10 to 2000 minutes, incubation at -33 to -27°C for 5 to 90 minutes, incubation at -23 to -17°C for 5 to 90 minutes, incubation at -13 to -7°C for 5 to 90 minutes, and incubation at -3 to 3°C for 5 to 90 minutes. Preferably, when the component containing a substance with poor heat stability is a component containing a redox-active substance or a component containing a protein, the desorption drying conditions include incubation at 22 to 28°C for 300 to 420 minutes.

Images