Solution for cleaning sequencing chip, sequencing method and kit

Abstract

The invention provides a solution for cleaning a sequencing chip, a sequencing method and a kit. The solution for cleaning the sequencing chip comprises a basic buffer solution, Na < + > and / or K < + >, a surfactant and a pH regulator, and further comprises at least one of divalent cations, NH4 < + > or polymerase. According to the solution for cleaning the sequencing chip, provided by the invention, at least one of divalent cations, NH4 < + > or polymerase is further added on the basis of the solution containing the basic buffer solution, the Na < + > and / or K < + >, the surfactant and the pH regulator, so that the prepared solution can be used for cleaning the sequencing chip; after the sequencing chip is cleaned by using the solution, the solution left on the sequencing chip can play a buffering role, so that a stable reaction environment is provided for doping nucleotide or nucleotide analogues into a nucleic acid template when the sequencing chip is used for sequencing; therefore, the reaction efficiency of doping the nucleotide or nucleotide analogue into the nucleic acid template is improved, the phase lag is reduced, and the sequencing accuracy is improved.

Description

Technical Field

[0001] This application relates to the technical field of nucleic acid sequencing, and particularly relates to a solution for cleaning a sequencing chip, a sequencing method, and a kit. Background Art

[0002] Next-generation sequencing, also known as high-throughput sequencing or massively parallel sequencing, can determine the nucleic acid sequences of multiple samples in a single sequencing run. A relatively common next-generation sequencing method is Sequencing by Synthesis (SBS). The platform for nucleic acid sequencing based on SBS is based on the base pairing principle and uses DNA polymerase to controllably achieve single-base extension by connecting nucleotides or nucleotide analogs to the 3'-end of a sequencing primer bound to a nucleic acid template. By collecting the signal changes caused by each binding of a nucleotide or nucleotide analog, the base sequence of the nucleic acid template is determined based on the changes in these collected signals. Generally, the SBS method includes the following steps: (1) hybridizing a nucleic acid template to be measured with a probe (or sequencing primer) on the surface of a solid support (such as the substrate surface of a sequencing chip) to connect the nucleic acid template to be measured to the surface of the solid support; (2) under the action of DNA polymerase and under conditions suitable for polymerase chain reaction, incorporating nucleotide analogs with a fluorescent group and a cleavable blocking group into the nucleic acid template to be measured using the probe as a primer, thereby performing single-base extension on the sequencing primer; (3) exciting the fluorescent group on the nucleotide analog to emit light, and then imaging the surface to collect the light emission signal on the surface; (4) using a cleavage reagent to remove the fluorescent group and the cleavable blocking group from the nucleotide analogs bound to the nucleic acid template to be measured; (5) repeating the above steps (2) to (4) to continue extending the sequencing primer to form a complementary strand of the nucleic acid template to be measured. Further, the SBS method further includes step (6): analyzing the optical signals obtained in step (3) to determine the type of nucleotide analog incorporated into the nucleic acid template in each round of extension reaction; reading the types of introduced nucleotide analogs in sequence, and finally obtaining all the nucleotide sequences of the nucleic acid template to be measured. Among them, in step (2), the conditions suitable for polymerase chain reaction include the presence of an extension reagent suitable for polymerase chain reaction. It can be understood that the reaction system for incorporating nucleotide analogs into the nucleic acid template includes this extension reagent, so that the incorporation of nucleotide analogs into the nucleic acid template can be achieved to perform single-base extension on the sequencing primer.

[0003] During the process of incorporating nucleotide analogs into a nucleic acid template to be tested, due to factors such as the reaction efficiency of the introduced nucleotide analogs being incorporated into the nucleic acid template to be tested, phase errors are likely to occur. Generally, phase errors are manifested as phasing (or phase lag) or prephasing (or phase lead). Phase lag refers to the situation where nucleotide analogs that should have reacted and been incorporated into the nucleic acid template to be tested in cycle N are delayed until cycle N + 1 to participate in the reaction. Prephasing refers to the situation where nucleotide analogs that should have reacted and been incorporated into the nucleic acid template to be tested in cycle N participate in the reaction in advance in cycle N - 1, that is, crosstalk occurs between adjacent cycles in the same channel. With the increase in the number of sequencing cycles, this kind of phase error accumulates and becomes stronger. The final result is that the four nucleotides coexist in a single amplification cluster (Cluster) with uniform brightness. In this case, the base recognition algorithm will not be able to recognize the correct sequencing signal in this round, that is, it is impossible to accurately obtain the sequence information of the nucleotide molecules to be tested.

[0004] Therefore, reducing phasing or prephasing during sequencing and improving sequencing accuracy are issues worthy of attention. Summary of the Invention

[0005] This application aims to solve at least one of the technical problems existing in the prior art. For this purpose, this application provides a solution for cleaning a sequencing chip, a sequencing method, and a kit to improve the reaction efficiency of incorporating nucleotides or nucleotide analogs into a nucleic acid template, thereby reducing phase lag and improving sequencing accuracy.

[0006] On the one hand, this application provides a solution for cleaning a sequencing chip, which solution contains a basic buffer, Na + and / or K + , a surfactant, and a pH regulator, and the solution further contains at least one of divalent cations, NH4 + or polymerase.

[0007] The solution for cleaning a sequencing chip provided by this application, based on a solution containing a basic buffer, Na + and / or K + , a surfactant, and a pH regulator, further adds divalent cations, NH4 +or at least one of the polymerase, so that the configured solution can be used for cleaning the sequencing chip. After cleaning the sequencing chip with this solution, the solution remaining on the sequencing chip can play a buffering role, providing a stable reaction environment for incorporating nucleotides or nucleotide analogs into the nucleic acid template during sequencing of the sequencing chip, thereby improving the reaction efficiency of incorporating nucleotides or nucleotide analogs into the nucleic acid template, subsequently reducing phase lag, and improving sequencing accuracy.

[0008] On the other hand, the present application provides a sequencing method, including:

[0009] Introducing a cleaning solution into the sequencing chip to clean the sequencing chip;

[0010] Introducing a first reaction solution into the cleaned sequencing chip to perform a first reaction;

[0011] The sequencing chip includes a solid-phase surface bound with a hybridization complex, and the hybridization complex includes a nucleic acid template and a sequencing primer bound to the nucleic acid template;

[0012] The first reaction solution contains a polymerase, nucleotides or nucleotide analogs;

[0013] The first reaction includes incorporating nucleotides or nucleotide analogs into the nucleic acid template under the action of the polymerase;

[0014] The cleaning solution is the solution for cleaning the sequencing chip provided in the first aspect; or, the cleaning solution is the first reaction solution without nucleotides or nucleotide analogs.

[0015] By cleaning the sequencing chip with this cleaning solution, the solution remaining on the sequencing chip can play a buffering role, providing a stable reaction environment for incorporating nucleotides or nucleotide analogs into the nucleic acid template, thereby improving the reaction efficiency of incorporating nucleotides or nucleotide analogs into the nucleic acid template, subsequently reducing phase lag, and improving the accuracy of sequencing.

[0016] In yet another aspect, the present application provides a kit, including a cleaning solution. The cleaning solution is the solution for cleaning the sequencing chip provided in the first aspect, or the cleaning solution is the first reaction solution without nucleotides or nucleotide analogs. The first reaction solution contains at least one of divalent cations, NH4 + , polymerase, and nucleotides or nucleotide analogs.

[0017] Additional aspects and advantages of the present application will be partially given in the following description, partially become apparent from the following description, or be understood through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a schematic diagram of the sequencing results of the comparative example and the example provided by the present application;

[0019] Figure 2 Schematic diagram of the sequencing results of another embodiment provided for this application. Detailed implementation manners

[0020] In order to make the technical problems to be solved, technical solutions and beneficial effects of this application clearer and more understandable, the following further details this application in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not used to limit this application.

[0021] In this application, the terms used in the embodiments of this application are only for the purpose of describing specific embodiments and are not intended to limit this application. The term "and / or" describes the association relationship of associated objects and indicates that there can be three relationships. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. Among them, A and B can be singular or plural. The character " / " generally represents a "and" relationship between the associated objects before and after.

[0022] The term "at least one" means one or more, and "a plurality" means two or more. "At least one (piece)" or its similar expression refers to any combination of these items, including any combination of single item (piece) or plural items (pieces). For example, "at least one (piece) of a, b, or c", or, "at least one (piece) of a, b, and c" can both represent: a, b, c, a-b (that is, a and b), a-c, b-c, or a-b-c, where a, b, and c can be single item (piece) or multiple items (pieces) respectively.

[0023] The singular forms of "a", "the" and "said" used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise. The terms "first" and "second" are only used for descriptive purposes to distinguish objects such as substances, orientations, interfaces, messages, requests, and terminals from each other, and cannot be understood as indicating or implying relative importance or implicitly indicating the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features.

[0024] In the description of this application, the concentrations of the relevant components mentioned not only can refer to the specific contents of the components, but also can represent the proportional relationship between the contents of the components. Therefore, as long as the contents of the relevant components in the specification of the embodiments of this application are enlarged or reduced in proportion, they are within the scope disclosed in the specification of the embodiments of this application.

[0025] The abbreviations used herein have their conventional meanings in the fields of chemistry and biology. The chemical structures and chemical formulas herein are constructed according to the standard rules of chemical valence known in the chemical field.

[0026] In an embodiment of the present application, the term "sequencing chip", also known as "chip", "biochip", "flow cell", "flow-through cell", "reaction cell", "flow cell", "flow-cell", "flowcell", "reaction device", etc., can be interchanged in expression. The sequencing chip generally has a structure similar to a sandwich with upper, middle and lower layers, or has a structure with upper and lower layers. The upper layer is a transparent glass layer, and the middle or lower layer is a transparent or opaque base layer. One or more fluid channels arranged in an array are provided on at least one of the upper, middle or lower layers. The fluid channels can accommodate liquids or solutions and provide a physical space for reactions. The sequencing chips mentioned in the present application include, but are not limited to, sequencing chips having the above structures.

[0027] The term "washing" can be to remove or replace another liquid, solution or other substance with a liquid or solution, usually without involving substantial processing and / or biochemical reactions.

[0028] The term "sequencing", also known as "nucleic acid sequencing" or "gene sequencing", that is, the three can be interchanged in expression, refers to the determination of the base types and arrangement order in a nucleic acid sequence; includes synthesis sequencing (sequencing by synthesis, SBS) and / or ligation sequencing (sequencing by ligation, SBL), etc., includes DNA sequencing and / or RNA sequencing, includes long fragment sequencing and / or short fragment sequencing. The so-called long fragments and short fragments are relative. For example, nucleic acid molecules longer than 1Kb, 2Kb, 5Kb or 10Kb can be called long fragments, and those shorter than 1Kb or 800bp can be called short fragments; includes paired-end sequencing, single-end sequencing and / or paired-end sequencing, etc. The so-called paired-end sequencing or paired-end sequencing can refer to the readout of any two non-overlapping parts or two parts of the same nucleic acid template; the so-called sequencing includes the process of binding nucleotides or nucleotide analogs to a nucleic acid template and collecting the corresponding signals emitted after the nucleotides or nucleotide analogs are bound to the nucleic acid template.

[0029] Sequencing generally includes multiple rounds of processes to achieve the determination of the sequence of multiple nucleotides / bases on a nucleic acid template. In the embodiments of the present application, each round of the "process to achieve the determination of the sequence of multiple nucleotides / bases on a nucleic acid template" is referred to as "one round of sequencing". "One round of sequencing" (cycle) is also referred to as "sequencing cycle", and can be defined as a single base extension of four nucleotides / bases. In other words, "one round of sequencing" can be defined as completing the determination of the base type at any specified position on the nucleic acid template. For a sequencing platform that achieves sequencing based on polymerization or ligation reactions, one round of sequencing includes the process of enabling the binding of four nucleotides (including nucleotide analogs) to the so-called nucleic acid template and collecting the corresponding signals emitted; for a platform that achieves sequencing based on polymerization reactions, the reaction system includes reaction substrate nucleotides or nucleotide analogs, polymerase, and nucleic acid template. A sequence (sequencing primer) is bound to the nucleic acid template. Based on the principle of base pairing and the principle of polymerization reaction, the added reaction substrate nucleotides or nucleotide analogs are ligated to the sequencing primer under the catalysis of polymerase to achieve the binding of the nucleotide or nucleotide analog to a specific position on the nucleic acid template, that is, incorporating the nucleotide or nucleotide analog into the nucleic acid template; generally, one round of sequencing may include one or multiple base extensions (repeat). For example, the four nucleotides are added to the reaction system in sequence, and base extension and collection of the corresponding reaction signals are carried out respectively. One round of sequencing includes four base extensions; for another example, the four nucleotides are added to the reaction system in any combination, such as in pairs or in combinations of one and three, and the two combinations are respectively subjected to base extension and collection of the corresponding reaction signals. One round of sequencing includes two base extensions; for yet another example, the four nucleotides are added to the reaction system simultaneously for base extension and collection of reaction signals. One round of sequencing includes one base extension.

[0030] The term "nucleic acid template" can refer to a polymeric form of nucleotides of any length and can include ribonucleotides, deoxyribonucleotides, their analogs, or mixtures thereof. The term can refer to single-stranded or double-stranded polynucleotides. Nucleotides in the nucleic acid template can include naturally occurring nucleotides and their functionally interchangeable analogs. Examples of analogs are capable of hybridizing to nucleic acids in a sequence-specific manner or capable of serving as a template for the replication of a specific nucleotide sequence. Naturally occurring nucleotides typically have a backbone containing phosphodiester bonds. The analog structure can have an alternative backbone linkage including any type known in the art. Naturally occurring nucleotides typically have deoxyribose (e.g., found in DNA) or ribose (e.g., found in RNA). The analog structure can have an alternative sugar moiety, including any type known in the art. Nucleotides can include natural bases, and the bases in natural DNA can include one or more of adenine, thymine, cytosine, and / or guanine, and the bases in natural RNA can include one or more of adenine, uracil, cytosine, and / or guanine. Nucleotides can also include any unnatural bases or base analogs, such as locked nucleic acid (LNA) and bridged nucleic acid (BNA).

[0031] The term "sequencing primer" can be an oligonucleotide or nucleic acid molecule that hybridizes to a target sequence of interest. In embodiments, the sequencing primer serves as a substrate onto which nucleotides or nucleotide analogs can be polymerized by a polymerase. For example, the sequencing primer can serve as a starting point for DNA or RNA synthesis. For example, the sequencing primer can hybridize to a nucleic acid template to form a hybridization complex to initiate the synthesis of a new strand complementary to the nucleic acid template. The sequencing primer can include any combination of nucleotides or their analogs. In some instances, the sequencing primer is a single-stranded oligonucleotide or polynucleotide.

[0032] The term "blocking group" includes a group that can block a reactive site (such as 3'-OH) of the pentose sugar in a nucleotide or nucleotide analog to prevent polymerization of the nucleotide or nucleotide analog at that site, and the group can be removed by chemical or other methods to restore the reactivity of the reactive site.

[0033] The term "Q value", namely the sequencing quality score, is used to evaluate the error detection rate of bases, where "Q30" represents an error detection rate of 1 / 1000 and a corresponding detection accuracy of 99.9%. For the definition of the Q value, please refer to the following link:

[0034] https: / / www.illumina.com.cn / science / technology / next-generation- sequencing / pla n-experiments / quality-scores.html 。

[0035] As used herein, "amplification" can be performed on a nucleic acid template in a solid-phase or liquid-phase environment to obtain an amplification product of the nucleic acid template, such as an amplification cluster (Cluster). The amplification method is not limited. For example, it can be achieved by using Taq enzyme or the like for PCR (temperature-variable amplification), or by using Bst or Bsu or recombinase or multi-enzyme systems for isothermal amplification techniques such as RPA (recombinase polymerase amplification), RCA (rolling circle amplification), and SDA (strand displacement amplification). For another example, nucleic acid templates can be amplified on a solid surface by using bridge amplification (bridge PCR) or template walking amplification to form amplicons on the surface; or rolling circle amplification (RCA) can be performed in a liquid phase to obtain an amplification product of the nucleic acid template, and then the amplification product can be loaded onto the surface to form DNA nanoballs or the like on the surface.

[0036] By forming an amplification cluster through the amplification of a nucleic acid template, during the sequencing process, the signal generated when a nucleotide or nucleotide analog is incorporated into the nucleic acid template can be amplified, making it easier for a detection system such as an optical imaging system to detect the signal. However, due to factors such as the reaction efficiency of the introduced nucleotide or nucleotide analog incorporated into the nucleic acid template, phase errors such as phase lag or phase lead are likely to occur, that is, the sequencing reactions between different nucleic acid template molecules in the same amplification cluster gradually lose synchronization. This phase error accumulates and becomes stronger as the number of sequencing cycles increases, ultimately causing serious interference with base recognition, resulting in the inability to correctly perform base recognition, thereby affecting the sequencing read length and the accuracy of sequencing.

[0037] Regarding the phenomenon of phase lag, since one of the main reasons for its occurrence is affected by the reaction efficiency of the introduced nucleotide or nucleotide analog incorporated into the nucleic acid template. Therefore, the prior art usually directly optimizes the formula of the extension reagent to improve the reaction efficiency of incorporating the nucleotide or nucleotide analog into the nucleic acid template, thereby achieving the purpose of reducing phase lag, or directly optimizes the formula of the cleavage reagent to improve the efficiency of removing the fluorescent group and the cleavable blocking group in the nucleotide analog bound to the nucleic acid template, thereby achieving the purpose of reducing phase lag to improve the synchronization of the sequencing reactions between different nucleic acid template molecules in the same amplification cluster. However, due to the limitation of the correlation between the components in the extension reagent and the incorporation reaction principle, it is difficult to further reduce phase lag when the components in the extension reagent and the incorporation reaction principle remain unchanged; or, due to the limitation of the correlation between the components in the cleavage reagent and the excision reaction principle, it is also difficult to further reduce phase lag when the components in the cleavage reagent and the excision reaction principle remain unchanged.

[0038] Based on the above understanding, the inventors of this application have taken a different approach and turned their research and development ideas to the study of solutions used for cleaning sequencing chips, that is, the study of cleaning solutions. As a physical space for containing liquids or solutions and a reaction site for biochemical reactions, the inventors hope to use cleaning solutions to clean the sequencing chips before sequencing to achieve the purpose of reducing phase lag. After repeated studies on the cleaning solution, the inventors found that by optimizing the formula of the cleaning solution, the cleaning solution can be used to clean the sequencing chip before sequencing, so that the cleaning solution remaining on the sequencing chip can play a buffering role after mixing with the reaction system for incorporating nucleotides or nucleotide analogs into the nucleic acid template, which helps to maintain or increase the concentration of the same components as those in the cleaning solution in the above reaction system, and provide a stable reaction environment for incorporating nucleotides or nucleotide analogs into the nucleic acid template, thereby improving the reaction efficiency of incorporating nucleotides or nucleotide analogs into the nucleic acid template, and then reducing the phase lag and improving the sequencing accuracy. Among them, the reaction system for incorporating nucleotides or nucleotide analogs into the nucleic acid template includes an extension reagent, etc. In the following description, the extension reagent is also described as the first reaction solution. Exemplarily, the extension reagent or the first reaction solution comprises divalent cations, NH4 + , polymerase, pH regulator and nucleotides or nucleotide analogs. Wherein, the nucleotides or nucleotide analogs can be, for example, adenine A, cytosine C, guanine G, thymine T, uracil U or their derivatives. The concentration of divalent cations in the first reaction solution is 1-7mmol / L. Exemplarily, the concentration of divalent cations in the first reaction solution can be 1mmol / L, 1.5mmol / L, 2mmol / L, 2.5mmol / L, 3mmol / L, 3.5mmol / L, 4mmol / L, 4.5mmol / L, 5mmol / L, 5.5mmol / L, 6mmol / L, 6.5mmol / L, 7mmol / L or a concentration between any two of them. The divalent cation can be, for example, selected from Mg 2+ , Mn 2+ More specifically, Mg 2+ Mg can be provided for magnesium salts 2+ , and / or Mn 2+ Mn can be provided for manganese salts 2+ NH4 in the first reaction solution + The concentration of NH4 in the first reaction solution is 20-150 mmol / L. +The concentration can be 20 mmol / L, 30 mmol / L, 40 mmol / L, 50 mmol / L, 60 mmol / L, 70 mmol / L, 80 mmol / L, 90 mmol / L, 100 mmol / L, 110 mmol / L, 120 mmol / L, 130 mmol / L, 140 mmol / L, 150 mmol / L or the concentration between any two of them. NH4 + NH4 can be provided by at least one of ammonium salts and ammonia water + . The concentration of the polymerase in the first reaction solution is 0.02 - 0.1 ng / ml. Exemplarily, the concentration of the polymerase in the first reaction solution is 0.02 ng / ml, 0.03 ng / ml, 0.04 ng / ml, 0.05 ng / ml, 0.06 ng / ml, 0.07 ng / ml, 0.08 ng / ml, 0.09 ng / ml, 0.1 ng / ml or the concentration between any two of them. The polymerase can be selected from at least one of 9°N polymerase, Taq polymerase, Canace polymerase, Pfu polymerase, KOD polymerase, Phusion polymerase, Klenow polymerase, Bst polymerase, Phi29 polymerase, PrimerSTAR polymerase, Tth polymerase. The pH regulator in the first reaction solution can be sodium hydroxide, potassium hydroxide, and the pH regulator is used to adjust the pH of the first reaction solution to 8.8 - 9.3.

[0039] An embodiment of the present application provides a solution for cleaning a sequencing chip, which contains a basic buffer, Na + and / or K + , a surfactant and a pH regulator, and the solution further contains at least one of divalent cations, NH4 + or polymerase.

[0040] The solution for cleaning a sequencing chip provided by the embodiment of the present application, on the basis of the solution containing a basic buffer, Na + and / or K + , a surfactant and a pH regulator, further adds divalent cations, NH4 +Or at least one of the polymerases, so that the configured solution can be used for cleaning the sequencing chip, so that after the sequencing chip is cleaned with the solution, the solution remaining on the sequencing chip can play a buffering role after mixing with the reaction system in which nucleotides or nucleotide analogs are incorporated into the nucleic acid template, which helps to maintain or increase the concentration of the components in the above-mentioned reaction system that are the same as the components in the solution, and provide a stable reaction environment for the incorporation of nucleotides or nucleotide analogs into the nucleic acid template. That is, the embodiment of the present application maintains or increases the concentration of the components in the first reaction liquid of the reaction system that are the same as the components in the solution by utilizing the similarity in formula between the solution and the first reaction liquid of the above-mentioned reaction system, thereby improving the reaction efficiency of incorporating nucleotides or nucleotide analogs into the nucleic acid template, thereby reducing the phase lag and improving the sequencing accuracy. In addition, the solution remaining on the sequencing chip can also be used to adjust the above-mentioned reaction system to the desired pH, thereby improving the reaction efficiency of incorporating nucleotides or nucleotide analogs into the nucleic acid template, thereby reducing the phase lag and improving the sequencing accuracy.

[0041] In some embodiments, the basic buffer is selected from at least one of Tris buffer, Hepes buffer, glycine, ethanolamine, tetraethylethylenediamine, tetramethylethylenediamine, N-butyldiethanolamine, diethylaminoethanol, and N,N-dihydroxyethylglycine. Among them, Tris buffer is trishydroxymethylaminomethane buffer; Hepes buffer is a non-ionic amphoteric buffer, and its main component is 2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid. The basic buffer can play a buffering role. When the pH of the above reaction system changes with temperature or as the reaction proceeds, the basic buffer can maintain the relative stability of the pH of the above reaction system, so that the reaction of incorporating nucleotides or nucleotide analogs into the nucleic acid template can be carried out at the desired pH, thereby improving the reaction efficiency, thereby reducing the phase lag and improving the accuracy of sequencing.

[0042] In some embodiments, the concentration of the basic buffer is 50 - 200 mmol / L. Exemplarily, the concentration of the basic buffer can be 50 mmol / L, 55 mmol / L, 60 mmol / L, 65 mmol / L, 70 mmol / L, 75 mmol / L, 80 mmol / L, 85 mmol / L, 90 mmol / L, 95 mmol / L, 105 mmol / L, 110 mmol / L, 115 mmol / L, 120 mmol / L, 125 mmol / L, 130 mmol / L, 135 mmol / L, 140 mmol / L, 145 mmol / L, 150 mmol / L, 155 mmol / L, 160 mmol / L, 165 mmol / L, 170 mmol / L, 175 mmol / L, 180 mmol / L, 185 mmol / L, 190 mmol / L, 195 mmol / L, 200 mmol / L or the concentration between any two of them.

[0043] In some embodiments, the Na in the solution + is provided by at least one of sodium salts, sodium hydroxide, and sodium organic carboxylates. The sodium salts are, for example, at least one of sodium halides and sodium sulfate, the sodium organic carboxylates are, for example, at least one of sodium formate and sodium acetate, and the sodium halides are, for example, at least one of sodium chloride, sodium iodide, and sodium bromide. Preferably, the Na + is provided by sodium hydroxide. While providing Na + , sodium hydroxide can also provide OH + for adjusting the pH of the solution. The K in the solution + is provided by at least one of potassium salts, potassium hydroxide, and potassium organic carboxylates. The potassium salts are, for example, at least one of potassium halides and potassium sulfate, the potassium halides are, for example, at least one of potassium chloride, potassium iodide, and potassium bromide, and the potassium organic carboxylates are, for example, at least one of potassium acetate and potassium oxalate. Preferably, the K - is provided by potassium hydroxide. While providing K + , potassium hydroxide can also provide OH + for adjusting the pH of the solution. + is provided by potassium hydroxide. While providing K + , potassium hydroxide can also provide OH + for adjusting the pH of the solution. -

[0044] In some embodiments, the concentration of Na + and / or K + is 10 - 100 mmol / L. Na + , K + can maintain the activity of the polymerase and the stability of the complementary strand of the synthesized nucleic acid template. Exemplarily, Na +The concentration can be 10 mmol / L, 20 mmol / L, 30 mmol / L, 40 mmol / L, 50 mmol / L, 60 mmol / L, 70 mmol / L, 80 mmol / L, 90 mmol / L, 100 mmol / L or the concentration between any two of them. Exemplarily, K + The concentration can be 10 mmol / L, 20 mmol / L, 30 mmol / L, 40 mmol / L, 50 mmol / L, 60 mmol / L, 70 mmol / L, 80 mmol / L, 90 mmol / L, 100 mmol / L or the concentration between any two of them.

[0045] In some embodiments, the surfactant in the solution can be used to eliminate the bubbles in the solution and increase the stability of the solution system. The surfactant can be selected from at least one of Tween and Triton X-100, for example.

[0046] In some embodiments, the volume percentage content of the surfactant in the solution is 0.01 - 0.1%. Exemplarily, when the surfactant is Tween-20, its volume percentage content can be 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1% or the volume percentage content between any two of them.

[0047] In some embodiments, the concentration of the divalent cation is 1 - 8 mmol / L. Configuring the concentration of the divalent cation in the solution within the range of 1 - 8 mmol / L can make the concentration of the divalent cation in the solution close to or equal to the concentration of the divalent cation in the above reaction system, so as to prevent the solution remaining on the sequencing chip from diluting the above reaction system after using the solution to clean the sequencing chip, resulting in a decrease in the concentration of the same divalent cation in the above reaction system and a reduction in the reaction efficiency, thereby reducing the phase lag and improving the accuracy of sequencing. Exemplarily, the concentration of the divalent cation can be 1 mmol / L, 1.5 mmol / L, 2 mmol / L, 2.5 mmol / L, 3 mmol / L, 3.5 mmol / L, 4 mmol / L, 4.5 mmol / L, 5 mmol / L, 5.5 mmol / L, 6 mmol / L, 6.5 mmol / L, 7 mmol / L, 7.5 mmol / L, 8 mmol / L or the concentration between any two of them.

[0048] In some embodiments, the divalent cation is selected from Mg 2+ 、Mn 2+ and at least one of them. The divalent cation such as Mg 2+ 、Mn2+ As an active auxiliary agent of polymerase, it helps to activate the activity of polymerase, so that the polymerase catalyzes the formation of a phosphodiester bond between the 3'-OH of the sequencing primer bound to the nucleic acid template and the phosphate group of the nucleotide or nucleotide analogue, thereby combining the nucleotide or nucleotide analogue with the sequencing primer, and allowing the nucleotide or nucleotide analogue to bind to the nucleic acid template in a base complementary pairing manner, that is, incorporating the nucleotide or nucleotide analogue into the nucleic acid template.

[0049] In some embodiments, Mg 2+ Mg for magnesium salts 2+ ; and / or, Mn 2+ Mn for manganese salts 2+ That is, in the embodiments of the present application, the solution for cleaning the sequencing chip contains a magnesium salt; and / or the solution for cleaning the sequencing chip contains a manganese salt; or, in the process of preparing the solution for cleaning the sequencing chip, a magnesium salt is added to provide Mg 2+ ; and / or in the process of preparing a solution for cleaning the sequencing chip, adding a manganese salt to provide Mn 2+ .

[0050] In some embodiments, NH4 + The concentration of NH4 is 50-200mmol / L. + The concentration is 50mmol / L, 60mmol / L, 70mmol / L, 80mmol / L, 90mmol / L, 100mmol / L, 110mmol / L, 120mmol / L, 130mmol / L, 140mmol / L, 150mmol / L, 160mmol / L, 170mmol / L, 180mmol / L, 190mmol / L, 200mmol / L or any concentration between them. + The concentration of NH4 in the solution can be set in the range of 50-200mmol / L. + The concentration is close to or equal to the NH4 + concentration, so as to prevent the solution remaining on the sequencing chip from diluting the reaction system after the sequencing chip is cleaned with the solution, thereby causing NH4 + The concentration decreases, which reduces the reaction efficiency, thereby reducing the phase lag and improving the accuracy of sequencing. + It can also increase the reaction activity of polymerase, thereby increasing the reaction efficiency of incorporating nucleotides or nucleotide analogs into nucleic acid templates, thereby reducing phase lag and improving sequencing accuracy. The inventors speculate that NH4 + Improving the polymerase activity may be related to NH4 +is related to being able to change the charge distribution on the surface of the polymerase and / or change the spatial conformation of the polymerase, making the polymerase more likely to bind to the reaction sites of the nucleic acid template and / or sequencing primer, catalyzing the formation of a phosphodiester bond between the 3'-OH of the sequencing primer and the phosphate group of the nucleotide or nucleotide analogue, thereby binding the nucleotide or nucleotide analogue to the sequencing primer and binding the nucleotide or nucleotide analogue to the nucleic acid template in a base complementary pairing manner, that is, incorporating the nucleotide or nucleotide analogue into the nucleic acid template. NH4 + In addition to being able to improve the reaction activity of the polymerase, NH4 + can also weaken the non-specific binding of the hydrogen bond between the nucleotide or nucleotide analogue and the surface of the sequencing chip in the above reaction system. The inventor guesses that this may be related to NH4 + being able to compete for binding with the hydrogen bond on the surface of the sequencing chip. Through NH4 + competing for binding with the hydrogen bond on the surface of the sequencing chip, the non-specific binding of the hydrogen bond between the nucleotide or nucleotide analogue and the surface of the sequencing chip is weakened. In addition, NH4 + can also play a buffering role. When the pH changes with temperature or during the reaction in the above reaction system, NH4 + can maintain the relative stability of the pH of the above reaction system, enabling the reaction of incorporating the nucleotide or nucleotide analogue into the nucleic acid template to proceed at the desired pH, improving the reaction efficiency, thereby reducing phase lag and improving the accuracy of sequencing.

[0051] In some embodiments, NH4 + is provided by at least one of ammonium salts and ammonia water. That is, in the embodiments of the present application, the solution for cleaning the sequencing chip contains at least one of ammonium salts and ammonia water, or during the process of preparing the solution for cleaning the sequencing chip, at least one of ammonium salts and ammonia water is added to provide NH4 + +

[0052] ​​In some embodiments, the concentration of the polymerase is 0.08-0.1 ng / ml. Exemplarily, the concentration of the polymerase is 0.08 ng / ml, 0.085 ng / ml, 0.09 ng / ml, 0.095 ng / ml, 0.1 ng / ml or a concentration between any two of the concentrations. The concentration of the polymerase in the solution is configured in the range of 0.08-0.10 ng / ml, so that the concentration of the polymerase in the solution can be close to or equal to the concentration of the polymerase in the above-mentioned reaction system, so as to prevent the solution remaining on the sequencing chip from diluting the above-mentioned reaction system after the sequencing chip is cleaned with the solution, resulting in a decrease in the concentration of the polymerase in the above-mentioned reaction system and reducing the reaction efficiency. At the same time, in the process of using the solution to clean the sequencing chip, a mounting enzyme can be formed, which increases the enzyme mounting time, thereby improving the reaction efficiency of incorporating nucleotides or nucleotide analogs into the nucleic acid template, thereby reducing the phase lag and improving the accuracy of sequencing.

[0053] In some embodiments, the polymerase is selected from at least one of 9°N polymerase, Taq polymerase, Canace polymerase, Pfu polymerase, KOD polymerase, Phusion polymerase, Klenow polymerase, Bst polymerase, Phi29 polymerase, PrimerSTAR polymerase, and Tth polymerase.

[0054] In some embodiments, the pH of the solution is 8.8-9.7. Exemplarily, the pH of the solution can be 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, etc. The pH of the solution is configured to be in the range of 8.8-9.7, so that when the pH of the above reaction system changes with temperature or as the reaction proceeds (pH decreases), the pH of the above reaction system can be adjusted using the solution remaining on the sequencing chip, and the pH of the above reaction system is adjusted to the desired pH, thereby improving the reaction efficiency of incorporating nucleotides or nucleotide analogs into the nucleic acid template, thereby reducing the phase lag and improving the accuracy of sequencing.

[0055] Since the pH of the reaction system decreases as the temperature changes or as the reaction proceeds, in order to maintain or improve the reaction efficiency, a common practice is to adjust the pH of the extension reagent or the first reaction solution in the reaction system to a higher value in advance, for example, adjusting the pH to 10. However, the inventors found that increasing the pH would cause shelf-life problems. For example, when the pH of the extension reagent or the first reaction solution reaches 10, the activity of the polymerase in the extension reagent or the first reaction solution will be significantly reduced, or nucleotides or nucleotide analogs will be damaged, etc., which is not conducive to the storage of the extension reagent or the first reaction solution. A relatively low pH is more conducive to the storage and stability maintenance of the extension reagent or the first reaction solution. Therefore, in this embodiment, by using the solution remaining on the sequencing chip to adjust the pH of the above reaction system and adjusting the reaction system to the desired pH, the shelf-life problems caused by prematurely increasing the pH of the extension reagent or the first reaction solution can be avoided, which is beneficial to the storage of the extension reagent or the first reaction solution.

[0056] In some embodiments, a pH regulator can be used to adjust the pH of the solution to 8.8 - 9.7. The pH regulator can be selected from at least one of sodium hydroxide and potassium hydroxide. That is to say, in the embodiments of the present application, the solution for cleaning the sequencing chip contains at least one of sodium hydroxide and potassium hydroxide, or at least one of sodium hydroxide and potassium hydroxide is added when preparing the solution for cleaning the sequencing chip.

[0057] In some embodiments, the solution further contains cysteamine dihydrochloride. After using this solution containing cysteamine dihydrochloride to clean the sequencing chip, the cysteamine dihydrochloride remaining on the sequencing chip helps to remove the residual cleavage reagent in the above reaction system.

[0058] In some embodiments, the concentration of cysteamine dihydrochloride is 20 - 200 mmol / L. Exemplarily, the concentration of cysteamine dihydrochloride can be 20 mmol / L, 30 mmol / L, 40 mmol / L, 50 mmol / L, 60 mmol / L, 70 mmol / L, 80 mmol / L, 90 mmol / L, 100 mmol / L, 110 mmol / L, 120 mmol / L, 130 mmol / L, 140 mmol / L, 150 mmol / L, 160 mmol / L, 170 mmol / L, 180 mmol / L, 190 mmol / L, 200 mmol / L or the concentration between any two of them.

[0059] Using the solution in the above embodiments of the present application to clean the sequencing chip before sequencing can significantly reduce the phase lag phenomenon generated during sequencing and improve the sequencing accuracy.

[0060] Another embodiment of the present application provides a sequencing method, including:

[0061] Flush the sequencing chip with a cleaning solution to clean the sequencing chip;

[0062] Flush the cleaned sequencing chip with a first reaction solution to perform a first reaction;

[0063] The sequencing chip includes a solid surface to which a hybridization complex is bound, and the hybridization complex includes a nucleic acid template and a sequencing primer bound to the nucleic acid template;

[0064] The first reaction solution contains a polymerase, nucleotides or nucleotide analogs;

[0065] The first reaction includes incorporating nucleotides or nucleotide analogs into the nucleic acid template under the action of a polymerase;

[0066] The cleaning solution is the above solution for cleaning the sequencing chip.

[0067] The sequencing method of the present application, by cleaning the sequencing chip with the cleaning solution before sequencing, enables the cleaning solution remaining on the sequencing chip to play a buffering role after being mixed with the reaction system for incorporating nucleotides or nucleotide analogs into the nucleic acid template, helps to maintain or increase the concentration of the components in the above reaction system that are the same as the components in the cleaning solution, provides a stable reaction environment for incorporating nucleotides or nucleotide analogs into the nucleic acid template, thereby improving the reaction efficiency of incorporating nucleotides or nucleotide analogs into the nucleic acid template, reducing phase lag, and improving the accuracy of sequencing.

[0068] In some embodiments, the nucleotides or nucleotide analogs contain detectable labels, and the sequencing method further includes: after completing the first reaction, flushing the sequencing chip with an imaging reagent and exciting the detectable labels and collecting signals emitted from the detectable labels. Among them, the "detectable label" includes but is not limited to optically detectable labels such as fluorescent groups CY3, CY5, CY7, ROX, Bodipy (BDP), Comarin, etc. The so-called imaging reagent contains antioxidant components such as water-soluble vitamin E. The imaging reagent can avoid or reduce the damage or influence of light on the sample during image acquisition. After completing the first reaction, the fluorescent group is excited by a laser to generate fluorescence, and the fluorescence signal is collected through a detection system such as an optical imaging system to form an image. Based on the analysis of the image, the type of nucleotide analog incorporated into the nucleic acid template can be determined, and by sequentially reading the types of incorporated nucleotide analogs, the nucleotide sequence of the nucleic acid template can be obtained.

[0069] In some embodiments, after completing the first reaction and before flushing the sequencing chip with the imaging reagent, the sequencing chip is flushed with a cleaning solution to replace the first reaction solution of the previous step and provide a pre-buffering environment for the next round of sequencing reaction.

[0070] In some embodiments, the nucleotide or nucleotide analogue comprises a cleavable blocking group, and the sequencing method further includes: after completing the acquisition of the signal emitted by the detectable label, introducing a cleavage reagent into the sequencing chip to remove the blocking group and the detectable label, so that the next nucleotide or nucleotide analogue can be incorporated into the nucleic acid template. Thus, through multiple rounds of extension reactions, the nucleotide sequence of the nucleic acid template can be obtained. After removing the blocking group and the detectable label, a washing solution is introduced into the sequencing chip to wash the sequencing chip, so that the washing solution replaces the cleavage reagent and provides a pre-buffering environment for the next round of sequencing reaction.

[0071] In some embodiments, before introducing a first reaction solution into the washed sequencing chip to perform a first reaction, a second reaction solution is introduced into the sequencing chip to perform a second reaction. The second reaction includes allowing the nucleic acid template in the sequencing chip to interact with the second reaction solution to achieve amplification of the nucleic acid template, wherein the second reaction solution contains the components required for amplification. By amplifying the nucleic acid template to form an amplification cluster, during the sequencing process, the fluorescence signal generated when a nucleotide or nucleotide analogue is incorporated into the nucleic acid template can be amplified, making it easier for a detection system such as an optical imaging system to detect the fluorescence signal.

[0072] Another embodiment of the present application provides a sequencing method, including:

[0073] Introducing a washing solution into the sequencing chip to wash the sequencing chip;

[0074] Introducing a first reaction solution into the washed sequencing chip to perform a first reaction;

[0075] The sequencing chip includes a solid-phase surface to which a hybridization complex is bound. The hybridization complex includes a nucleic acid template and a sequencing primer bound to the nucleic acid template;

[0076] The first reaction solution contains a polymerase, a nucleotide or a nucleotide analogue;

[0077] The first reaction includes incorporating a nucleotide or a nucleotide analogue into the nucleic acid template under the action of the polymerase;

[0078] Wherein, the washing solution is the first reaction solution without nucleotides or nucleotide analogues.

[0079] Before performing sequencing, the sequencing method of the present application can provide a pre-sequencing buffer environment by washing the sequencing chip with a first reaction solution that does not contain nucleotides or nucleotide analogs. This helps to maintain or increase the concentration of components in the reaction system that are the same as those in the washing solution when incorporating nucleotides or nucleotide analogs into the nucleic acid template, ensuring that the components in the first reaction solution containing nucleotides or nucleotide analogs are not diluted by the washing solution and maintaining a high reaction concentration. This provides a stable reaction environment for the incorporation of nucleotides or nucleotide analogs into the nucleic acid template, thereby improving the reaction efficiency of incorporating nucleotides or nucleotide analogs into the nucleic acid template, reducing phase lag, and improving the accuracy of sequencing.

[0080] In some embodiments, the nucleotide or nucleotide analog contains a detectable label, and the sequencing method further includes: after completing the first reaction, introducing an imaging reagent into the sequencing chip, exciting the detectable label, and collecting the signal emitted from the detectable label. Among them, the "detectable label" includes, but is not limited to, optically detectable labels such as fluorescent groups CY3, CY5, CY7, ROX, Bodipy (BDP), Comarin, etc. The imaging reagent contains antioxidant components such as water-soluble vitamin E. The imaging reagent can avoid or reduce the damage or influence of light on the sample during image acquisition. After completing the first reaction, the fluorescent group is excited by a laser to generate fluorescence, and the fluorescence signal is collected by a detection system such as an optical imaging system to form an image. Based on the analysis of the image, the type of nucleotide analog incorporated into the nucleic acid template can be determined, and by sequentially reading the types of incorporated nucleotide analogs, the nucleotide sequence of the nucleic acid template can be obtained.

[0081] In some embodiments, after completing the first reaction and before introducing the imaging reagent into the sequencing chip, a washing solution is introduced into the sequencing chip to wash the sequencing chip, so that the washing solution replaces the first reaction solution of the previous step and provides a pre-sequencing buffer environment for the next round of sequencing reaction.

[0082] In some embodiments, the nucleotide or nucleotide analog contains a cleavable blocking group, and the sequencing method further includes: after collecting the signal emitted from the detectable label, introducing a cleavage reagent into the sequencing chip to remove the blocking group and the detectable label, so that the next nucleotide or nucleotide analog can be incorporated into the nucleic acid template. Thus, through multiple rounds of extension reactions, the nucleotide sequence of the nucleic acid template can be obtained. After removing the blocking group and the detectable label, a washing solution is introduced into the sequencing chip to wash the sequencing chip, so that the washing solution replaces the cleavage reagent and provides a pre-sequencing buffer environment for the next round of sequencing reaction.

[0083] In some embodiments, before introducing a first reaction solution into the washed sequencing chip to perform a first reaction, a second reaction solution is introduced into the sequencing chip to perform a second reaction. The second reaction includes causing the nucleic acid template in the sequencing chip to interact with the second reaction solution to achieve amplification of the nucleic acid template, wherein the second reaction solution contains the components required for amplification. By amplifying the nucleic acid template to form an amplification cluster, during the sequencing process, the fluorescence signal generated when a nucleotide or nucleotide analog is incorporated into the nucleic acid template can be amplified, making it easier for a detection system, such as an optical imaging system, to detect the fluorescence signal.

[0084] Embodiments of the present application also provide a kit, which includes a cleaning solution. The cleaning solution can be the solution for cleaning the sequencing chip mentioned in the above embodiments of the present application; alternatively, the cleaning solution is a first reaction solution without nucleotides or nucleotide analogs, wherein the first reaction solution contains divalent cations, NH4 + , polymerase, a pH regulator, and nucleotides or nucleotide analogs. Among them, the nucleotides or nucleotide analogs can be, for example, adenine A, cytosine C, guanine G, thymine T, uracil U, or their derivatives.

[0085] In some embodiments, the concentration of divalent cations in the first reaction solution is 1-7 mmol / L. Exemplarily, the concentration of divalent cations in the first reaction solution can be 1 mmol / L, 1.5 mmol / L, 2 mmol / L, 2.5 mmol / L, 3 mmol / L, 3.5 mmol / L, 4 mmol / L, 4.5 mmol / L, 5 mmol / L, 5.5 mmol / L, 6 mmol / L, 6.5 mmol / L, 7 mmol / L, or the concentration between any two of these concentrations.

[0086] In some embodiments, the divalent cations in the first reaction solution can be selected from at least one of Mg 2+ , Mn 2+ . More specifically, Mg 2+ can be Mg 2+ provided by a magnesium salt, and / or Mn 2+ can be Mn 2+ provided by a manganese salt.

[0087] In some embodiments, the concentration of NH4 + in the first reaction solution is 20-150 mmol / L. Exemplarily, the concentration of NH4 +The concentration can be 20 mmol / L, 30 mmol / L, 40 mmol / L, 50 mmol / L, 60 mmol / L, 70 mmol / L, 80 mmol / L, 90 mmol / L, 100 mmol / L, 110 mmol / L, 120 mmol / L, 130 mmol / L, 140 mmol / L, 150 mmol / L or the concentration between any two of them.

[0088] In some embodiments, NH4 in the first reaction solution + can be provided by at least one of ammonium salts and ammonia water. + .

[0089] In some embodiments, the concentration of the polymerase in the first reaction solution is 0.02 - 0.1 ng / ml. Exemplarily, the concentration of the polymerase in the first reaction solution is 0.02 ng / ml, 0.03 ng / ml, 0.04 ng / ml, 0.05 ng / ml, 0.06 ng / ml, 0.07 ng / ml, 0.08 ng / ml, 0.09 ng / ml, 0.1 ng / ml or the concentration between any two of them.

[0090] In some embodiments, the polymerase in the first reaction solution can be selected from at least one of 9°N polymerase, Taq polymerase, Canace polymerase, Pfu polymerase, KOD polymerase, Phusion polymerase, Klenow polymerase, Bst polymerase, Phi29 polymerase, PrimerSTAR polymerase, Tth polymerase.

[0091] In some embodiments, the pH regulator in the first reaction solution can be sodium hydroxide, potassium hydroxide, and the pH regulator is used to adjust the pH of the first reaction solution to 8.8 - 9.3.

[0092] Before sequencing, the sequencing chip is washed by extracting the washing solution from the kit, or the sequencing chip is washed by extracting the washing solution from the kit during sequencing, so that the washing solution remaining on the sequencing chip can play a buffering role after being mixed with the reaction system for incorporating nucleotides or nucleotide analogs into the nucleic acid template, which helps to maintain or increase the concentration of the components in the above reaction system that are the same as the components in the washing solution, provides a stable reaction environment for incorporating nucleotides or nucleotide analogs into the nucleic acid template, thereby improving the reaction efficiency of incorporating nucleotides or nucleotide analogs into the nucleic acid template, reducing the phase lag, and improving the accuracy of sequencing; in addition, the washing solution remaining on the sequencing chip can be used to adjust the above reaction system to the desired pH, so that the above reaction system reacts in the desired pH environment, thereby improving the reaction efficiency of incorporating nucleotides or nucleotide analogs into the nucleic acid template, and then reducing the phase lag and improving the accuracy of sequencing; furthermore, using the first reaction solution without nucleotides or nucleotide analogs to wash the sequencing chip before sequencing can provide a pre-buffering environment for the sequencing reaction, ensuring that the components in the first reaction solution containing nucleotides or nucleotide analogs are not diluted by the washing solution and maintaining a high reaction concentration, thereby improving the reaction efficiency of incorporating nucleotides or nucleotide analogs into the nucleic acid template, reducing the phase lag, and improving the accuracy of sequencing.

[0093] The following will be described in conjunction with specific embodiments. The experimental instruments and their manufacturer information involved in the embodiments of the present application are shown in Table 1:

[0094] Table 1

[0095] Experimental instrument Model Manufacturer FASTASeq300 Sequencer FASTASeq300 Shenzhen GenomiCare Biotech Co., Ltd.

[0096] Example 1:

[0097] Prepare washing solution 1-1 and washing solution 1-2 according to the components shown in Table 2, where both washing solution 1-1 and washing solution 1-2 are adjusted to pH with sodium hydroxide.

[0098] Table 2

[0099] Component Washing Solution 1-1 Washing Solution 1-2 Tris Buffer 50 mM 50 mM Sodium Chloride 50 mM 50 mM Tween-20 0.01％ 0.01％ Cystamine Dihydrochloride 20 mM 0 pH 7.0～7.4 9.2～9.6

[0100] Control group

[0101] Sequencing method:

[0102] According to the requirements of the FASTAseq300 sequencer, prepare the kit, sequencing chip and library;

[0103] Fix the library on the sequencing chip and perform SE100 sequencing;

[0104] During sequencing, the reaction temperature of the sequencing chip was controlled at 60 °C. 75 μl of cleaning solution 1-2 and 122 μl of the first reaction solution (incubated for 15 s) were sequentially aspirated from the reagent kit into the sequencing chip; then 50 μl of cleaning solution 1-2 and 122 μl of imaging reagent were sequentially aspirated into the sequencing chip and photographed; after the photographing was completed, 30 μl of cleaning solution 1-2, 122 μl of cleavage reagent (incubated for 16 s), and 155 μl of cleaning solution 1-1 were sequentially aspirated into the sequencing chip; this cycle was repeated until the sequencing was completed.

[0105] After the sequencing was completed, the sequencing results were counted and analyzed. The sequencing results included the Q30 value, phasing, prephasing, and mismatch ratio (MismatchRatio), as shown in Table A and Figure 1 as follows. Among them, the "mismatch ratio" refers to the ratio of unpaired bases in the nucleotide sequence to the total number of bases.

[0106] Example 2

[0107] Cleaning solutions 2-1, 2-2, 2-3, and 2-4 were prepared according to the components shown in Table 3. The pH values of cleaning solutions 2-1, 2-2, 2-3, and 2-4 were adjusted with sodium hydroxide.

[0108] Table 3

[0109] Component Washing Solution 2-1 Washing Solution 2-2 Washing Solution 2-3 Washing Solution 2-4 Tris Buffer 50 mM 50 mM 50 mM 50 mM Sodium Chloride 50 mM 50 mM 50 mM 50 mM Tween-20 0.01％ 0.01％ 0.01％ 0.01％ Magnesium Sulfate 0 mM 0 mM 5 mM 5 mM Ammonium Sulfate 0 mM 120 mM 0 mM 120 mM pH 8.8 8.8 8.8 8.8

[0110] Experimental group 1-1

[0111] Sequencing method:

[0112] According to the requirements of the FASTAseq300 sequencer, the reagent kit, sequencing chip, and library were prepared;

[0113] The library was fixed on the sequencing chip and SE100 sequencing was performed;

[0114] During sequencing, the reaction temperature of the sequencing chip was controlled at 60 °C. 75 μl of cleaning solution 2-1 and 122 μl of the first reaction solution (incubated for 15 s) were sequentially aspirated from the reagent kit into the sequencing chip; then 50 μl of cleaning solution 2-1 and 122 μl of imaging reagent were aspirated into the sequencing chip and photographed; after the photographing was completed, 30 μl of cleaning solution 2-1, 122 μl of cleavage reagent (incubated for 16 s), and 155 μl of cleaning solution 1-1 were aspirated into the sequencing chip; this cycle was repeated until the sequencing was completed.

[0115] After the sequencing was completed, the sequencing results were counted and analyzed. The sequencing results included the Q30 value, phasing, prephasing, and mismatch ratio (MismatchRatio), as specifically shown in Table A and Figure 1 as follows.

[0116] Experimental Group 1-2

[0117] Sequencing method:

[0118] Prepare the reagent kit, sequencing chip and library according to the requirements of the FASTAseq300 sequencer;

[0119] Fix the library on the sequencing chip and perform SE100 sequencing;

[0120] During sequencing, control the reaction temperature of the sequencing chip at 60°C. Sequentially aspirate 75 μl of Wash Solution 2-2 and 122 μl of the First Reaction Solution (incubate for 15 s) from the reagent kit into the sequencing chip; then aspirate 50 μl of Wash Solution 2-2 and 122 μl of the Imaging Reagent into the sequencing chip and take a photo; after the photo is taken, aspirate 30 μl of Wash Solution 2-2, 122 μl of the Cleavage Reagent (incubate for 16 s) and 155 μl of Wash Solution 1-1 into the sequencing chip; repeat this cycle until sequencing is completed.

[0121] After sequencing is completed, count and analyze the sequencing results. The sequencing results include the Q30 value, phasing, prephasing, and mismatch ratio (MismatchRatio), as shown in Table A and Figure 1 as follows.

[0122] Experimental Group 1-3

[0123] Sequencing method:

[0124] Prepare the reagent kit, sequencing chip and library according to the requirements of the FASTAseq300 sequencer;

[0125] Fix the library on the sequencing chip and perform SE100 sequencing;

[0126] During sequencing, control the reaction temperature of the sequencing chip at 60°C. Sequentially aspirate 75 μl of Wash Solution 2-3 and 122 μl of the First Reaction Solution (incubate for 15 s) from the reagent kit into the sequencing chip; then aspirate 50 μl of Wash Solution 2-3 and 122 μl of the Imaging Reagent into the sequencing chip and take a photo; after the photo is taken, aspirate 30 μl of Wash Solution 2-3, 122 μl of the Cleavage Reagent (incubate for 16 s) and 155 μl of Wash Solution 1-1 into the sequencing chip; repeat this cycle until sequencing is completed.

[0127] After sequencing is completed, count and analyze the sequencing results. The sequencing results include the Q30 value, phasing, prephasing, and mismatch ratio (MismatchRatio), as shown in Table A and Figure 1 as follows.

[0128] Experimental Group 1-4

[0129] Sequencing method:

[0130] Prepare the kit, sequencing chip and library according to the requirements of the FASTAseq300 sequencer;

[0131] Fix the library on the sequencing chip and perform SE100 sequencing;

[0132] During sequencing, control the reaction temperature of the sequencing chip at 60 °C, and sequentially aspirate 75 μl of cleaning solution 2-4 and 122 μl of the first reaction solution (incubate for 15 s) from the kit into the sequencing chip; then aspirate 50 μl of cleaning solution 2-4 and 122 μl of imaging reagent into the sequencing chip and take a photo; after the photo is taken, aspirate 30 μl of cleaning solution 2-4, 122 μl of cleavage reagent (incubate for 16 s) and 155 μl of cleaning solution 1-1 into the sequencing chip; repeat this cycle until sequencing is completed.

[0133] After sequencing is completed, count and analyze the sequencing results. The sequencing results include Q30 value, phasing, prephasing, and mismatch ratio (MismatchRatio), as shown in Table A and Figure 1 as follows.

[0134] Table A

[0135]

[0136] According to Table A and Figure 1 , it can be seen that when Mg 2+ or NH4 + exists in the cleaning solution, it can reduce phasing and base mismatch ratio and increase the Q30 value. When both Mg 2+ and NH4 + exist in the cleaning solution, the decrease in phasing and base mismatch ratio is more obvious, and the increase in Q30 is also more obvious. This shows that after cleaning the sequencing chip with the cleaning solution, the cleaning solution remaining on the sequencing chip can play a buffering role after being mixed with the reaction system for incorporating nucleotides or nucleotide analogs into the nucleic acid template, helping to maintain or increase the concentration of the components in the reaction system that are the same as the components in the cleaning solution, providing a stable reaction environment for incorporating nucleotides or nucleotide analogs into the nucleic acid template, thereby improving the reaction efficiency of incorporating nucleotides or nucleotide analogs into the nucleic acid template, reducing phase lag, and improving the accuracy of sequencing.

[0137] Example 3

[0138] Prepare cleaning solutions 3-1, 3-2, 3-3, and 3-4 according to the components shown in Table 4, where cleaning solutions 3-1, 3-2, 3-3, and 3-4 are all adjusted to pH with sodium hydroxide.

[0139] Table 4

[0140] Component Washing Solution 3-1 Washing Solution 3-2 Washing Solution 3-3 Washing Solution 3-4 Tris Buffer 50 mM 50 mM 50 mM 50 mM Sodium Chloride 50 mM 50 mM 50 mM 50 mM Tween-20 0.01％ 0.01％ 0.01％ 0.01％ Magnesium Sulfate 2 mM 2 mM 2 mM 2 mM Ammonium Sulfate 80 mM 80 mM 80 mM 80 mM pH 8.8 9.1 9.4 9.7

[0141] Sequencing method:

[0142] Experimental group 2-1:

[0143] Prepare the kit, sequencing chip and library according to the requirements of the FASTAseq300 sequencer;

[0144] Fix the library on the sequencing chip and perform SE100 sequencing;

[0145] During sequencing, control the reaction temperature of the sequencing chip at 60°C. Sequentially aspirate 75 μl of cleaning solution 3-1 and 122 μl of the first reaction solution (incubate for 15 s) from the kit into the sequencing chip; then aspirate 50 μl of cleaning solution 3-1 and 122 μl of imaging reagent into the sequencing chip and take a photo; after the photo is taken, aspirate 30 μl of cleaning solution 3-1, 122 μl of cleavage reagent (incubate for 16 s) and 155 μl of cleaning solution 1-1 into the sequencing chip; repeat this cycle until sequencing is completed.

[0146] After sequencing is completed, count and analyze the sequencing results. The sequencing results include Q30 value, phasing, prephasing, and mismatch ratio (MismatchRatio), as shown in Table B and Figure 2 shown below.

[0147] Experimental group 2-2

[0148] Prepare the kit, sequencing chip and library according to the requirements of the FASTAseq300 sequencer;

[0149] Fix the library on the sequencing chip and perform SE100 sequencing;

[0150] During sequencing, control the reaction temperature of the sequencing chip at 60°C. Sequentially aspirate 75 μl of cleaning solution 3-2 and 122 μl of the first reaction solution (incubate for 15 s) from the kit into the sequencing chip; then aspirate 50 μl of cleaning solution 3-2 and 122 μl of imaging reagent into the sequencing chip and take a photo; after the photo is taken, aspirate 30 μl of cleaning solution 3-2, 122 μl of cleavage reagent (incubate for 16 s) and 155 μl of cleaning solution 1-1 into the sequencing chip; repeat this cycle until sequencing is completed.

[0151] After sequencing is completed, count and analyze the sequencing results. The sequencing results include Q30 value, phasing, prephasing, and mismatch ratio (MismatchRatio), as shown in Table B and Figure 2 shown below.

[0152] Experimental Group 2-3

[0153] Prepare the kit, sequencing chip and library according to the requirements of the FASTAseq300 sequencer;

[0154] Fix the library on the sequencing chip and perform SE100 sequencing;

[0155] During sequencing, control the reaction temperature of the sequencing chip at 60 °C. Sequentially aspirate 75 μl of Wash Solution 3-3 and 122 μl of the First Reaction Solution (incubate for 15 s) from the kit into the sequencing chip; then aspirate 50 μl of Wash Solution 3-3 and 122 μl of the Imaging Reagent into the sequencing chip and take a photo; after taking the photo, aspirate 30 μl of Wash Solution 3-3, 122 μl of the Cleavage Reagent (incubate for 16 s) and 155 μl of Wash Solution 1-1 into the sequencing chip; repeat this cycle until sequencing is completed.

[0156] After sequencing is completed, count and analyze the sequencing results. The sequencing results include Q30 value, phasing, prephasing, and mismatch ratio (MismatchRatio), as shown in Table B and Figure 2 as shown below.

[0157] Experimental Group 2-4

[0158] Prepare the kit, sequencing chip and library according to the requirements of the FASTAseq300 sequencer;

[0159] Fix the library on the sequencing chip and perform SE100 sequencing;

[0160] During sequencing, control the reaction temperature of the sequencing chip at 60 °C. Sequentially aspirate 75 μl of Wash Solution 3-4 and 122 μl of the First Reaction Solution (incubate for 15 s) from the kit into the sequencing chip; then aspirate 50 μl of Wash Solution 3-4 and 122 μl of the Imaging Reagent into the sequencing chip and take a photo; after taking the photo, aspirate 30 μl of Wash Solution 3-4, 122 μl of the Cleavage Reagent (incubate for 16 s) and 155 μl of Wash Solution 1-1 into the sequencing chip; repeat this cycle until sequencing is completed.

[0161] After sequencing is completed, count and analyze the sequencing results. The sequencing results include Q30 value, phasing, prephasing, and mismatch ratio (MismatchRatio), as shown in Table B and Figure 2 as shown below.

[0162] Table B

[0163]

[0164] According to Table B and Figure 2As shown in the figure, as the pH of the cleaning solution increases, phasing gradually decreases, and prephasing also gradually decreases. When the pH is 9.4, phasing drops to 0.18%, prephasing drops to 0.15%, and the base error rate is also significantly reduced. This shows that after the sequencing chip is cleaned with the cleaning solution, the cleaning solution remaining on the sequencing chip can adjust the pH of the reaction system for incorporating nucleotides or nucleotide analogs into the nucleic acid template, so that the reaction system can react in the desired pH environment, thereby improving the reaction efficiency of incorporating nucleotides or nucleotide analogs into the nucleic acid template, thereby reducing phase lag and phase advance, and improving the accuracy of sequencing.

[0165] Example 4

[0166] The cleaning solution 4 was prepared according to the components shown in Table 5, wherein the pH of the cleaning solution 4 was adjusted by sodium hydroxide.

[0167] Table 5

[0168] Component Washing Solution 4 Tris Buffer 50 mM Sodium Chloride 50 mM Tween-20 0.01％ Magnesium Sulfate 5 mM Ammonium Sulfate 120 mM Cystamine Dihydrochloride 50 mM pH 9.4

[0169] Sequencing Methods:

[0170] Experimental Group 3

[0171] Prepare the reagent kit, sequencing chip and library according to the requirements of FASTAseq300 sequencer;

[0172] The library was fixed on the sequencing chip and subjected to SE100 sequencing;

[0173] During sequencing, the reaction temperature of the sequencing chip was controlled at 60°C, and 122 μl of the first reaction solution (incubated for 15 s), 50 μl of cleaning solution 3-3, and 122 μl of imaging reagent were sequentially pumped into the sequencing chip from the reagent kit to take pictures; after taking pictures, 30 μl of cleaning solution 3-3, 122 μl of cutting reagent (incubated for 16 s), and 155 μl of cleaning solution 4 were sequentially pumped into the sequencing chip; this cycle was repeated until sequencing was completed.

[0174] After sequencing is completed, the sequencing results are counted and analyzed, and the sequencing results include Q30 value, phasing, prephasing, and mismatch rate (MismatchRatio), as shown in Table C.

[0175] Table C

[0176]

[0177] According to Table C, after the cleavage reagent removes the blocking group and the detectable label, cleaning the sequencing chip with a cleaning solution similar to the first reaction solution formulation can significantly reduce phasing and prephasing, and the absolute value of Q30 increases by 6.7%. This shows that when using this cleaning solution to clean the sequencing chip, the reaction efficiency of incorporating nucleotides or nucleotide analogs into the nucleic acid template is improved, thereby reducing phasing and prephasing and improving the sequencing quality.

Claims

1. A solution for cleaning a sequencing chip, characterized in that: The solution contains a basic buffer, Na + and / or K + , surfactants and pH adjusters; The solution also contains divalent cations, NH4 + or at least one of a polymerase.

2. The solution for cleaning a sequencing chip according to claim 1, characterized in that: The concentration of the divalent cation is 1-8 mmol / L; Optionally, the divalent cation is selected from Mg 2+ , Mn 2+ At least one of; Optionally, the Mg 2+ Mg for magnesium salts 2+ and / or, The Mn 2+ Mn for manganese salts 2+ .

3. The solution for cleaning a sequencing chip according to claim 1 or 2, characterized in that: The NH4 + The concentration is 50-200mmol / L; Optionally, the NH4 + NH4 for ammonium salts and ammonia water + .

4. The solution for cleaning a sequencing chip according to any one of claims 1 to 3, characterized in that: The concentration of the polymerase is 0.08-0.1 ng / ml; Optionally, the polymerase is selected from at least one of 9°N polymerase, Taq polymerase, Canace polymerase, Pfu polymerase, KOD polymerase, Phusion polymerase, Klenow polymerase, Bst polymerase, Phi29 polymerase, PrimerSTAR polymerase, and Tth polymerase.

5. The solution according to claims 1-4, characterized in that The pH of the solution is 8.8-9.7; Optionally, the pH adjuster is selected from at least one of sodium hydroxide and potassium hydroxide.

6. The solution for cleaning a sequencing chip according to claims 1-5, characterized in that: The basic buffer is selected from at least one of Tris buffer, Hepes buffer, glycine, ethanolamine, tetraethylethylenediamine, tetramethylethylenediamine, N-butyldiethanolamine, diethylaminoethanol, and N,N-dihydroxyethylglycine; Optionally, the surfactant is selected from at least one of Tween and TritonX-100; Optionally, the solution further comprises cysteamine hydrochloride.

7. A sequencing method, characterized in that: include: Passing a cleaning solution into the sequencing chip to clean the sequencing chip; Passing a first reaction solution into the cleaned sequencing chip to perform a first reaction; The sequencing chip comprises a solid surface bound to a hybridization complex, wherein the hybridization complex comprises a nucleic acid template and a sequencing primer bound to the nucleic acid template; The first reaction solution comprises a polymerase, nucleotides or nucleotide analogs; The first reaction includes incorporating the nucleotide or nucleotide analog into the nucleic acid template under the action of a polymerase; The cleaning solution is the solution for cleaning a sequencing chip according to any one of claims 1 to 6; or, The cleaning solution is the first reaction solution that does not contain the nucleotide or nucleotide analog.

8. The sequencing method according to claim 7, characterized in that The nucleotide or nucleotide analog comprises a detectable label; The sequencing method further comprises: After the first reaction is completed, an imaging reagent is introduced into the sequencing chip to excite the detectable label and collect the signal emitted by the detectable label; Optionally, the sequencing method further comprises: After the first reaction is completed and before the imaging reagent is introduced into the sequencing chip, the cleaning solution is introduced into the sequencing chip to clean the sequencing chip; Optionally, the nucleotide or nucleotide analog comprises a cleavable blocking group; After completing the acquisition of the signal emitted from the detectable label, introducing a cleavage reagent into the sequencing chip to remove the blocking group and the detectable label; Optionally, before the cleavage reagent is introduced into the sequencing chip, the cleaning solution is introduced into the sequencing chip to clean the sequencing chip; Optionally, after removing the blocking group and the detectable label, the cleaning solution is introduced into the sequencing chip to clean the sequencing chip; Optionally, before the first reaction solution is introduced into the cleaned sequencing chip for the first reaction, a second reaction solution is introduced into the sequencing chip for the second reaction, wherein the second reaction includes allowing the nucleic acid template in the sequencing chip to interact with the second reaction solution to achieve amplification of the nucleic acid template.

9. A kit, characterized in that: The method comprises a cleaning solution, wherein the cleaning solution is the solution for cleaning a sequencing chip according to any one of claims 1 to 6; or the cleaning solution is a first reaction solution that does not contain nucleotides or nucleotide analogs, wherein the first reaction solution contains divalent cations, NH4 + , polymerase and the nucleotide or nucleotide analog.

10. The kit according to claim 9, characterized in that: The concentration of the divalent cation is 1-7 mmol / L; Optionally, the divalent cation is selected from Mg 2+ , Mn 2+ At least one of; Optionally, the Mg 2+ Mg for magnesium salts 2+ ; and / or, the Mn 2+ Mn for manganese salts 2+ ; Optionally, the NH4 + The concentration is 20-150mmol / L; Optionally, the NH4 + NH4 provided by at least one of ammonium salt and ammonia water + ; Optionally, the concentration of the polymerase is 0.02-0.1 ng / ml; Optionally, the polymerase is selected from at least one of 9°N polymerase, Taq polymerase, Canace polymerase, Pfu polymerase, KOD polymerase, Phusion polymerase, Klenow polymerase, Bst polymerase, Phi29 polymerase, PrimerSTAR polymerase, and Tth polymerase; Optionally, the first reaction solution further comprises a pH regulator; Optionally, the pH regulator is selected from at least one of sodium hydroxide and potassium hydroxide; Optionally, the pH of the first reaction solution is 8.8-9.3.

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