Sequencing methods

Abstract

The application relates to a sequencing method, which comprises: S1, flowing a first buffer into a chip to clean the chip, the chip being used for performing sequencing by synthesis, the pH value of the first buffer being greater than 8; S2, flowing a first synthesis reagent into the chip to perform an extension reaction on the chip, the first synthesis reagent comprising a first nucleotide, the first nucleotide comprising a cleavable blocking group and an optically detectable label; S3, flowing a developing reagent into the chip to excite the optically detectable label to generate a signal, and collecting the signal; S4, flowing the first buffer into the chip to clean the chip; S5, flowing a cleavage reagent into the chip to remove the blocking group and the optically detectable label; S6, flowing the first buffer into the chip to clean the chip; and S7, flowing a second buffer into the chip to clean the chip; S8, returning to step S1, and repeating steps S1-S7 until the sequencing reaction is completed. The method can effectively improve the efficiency / quality of the sequencing method and reduce the cost.

Description

Technical Field

[0001] This application relates to the field of gene sequencing technology, specifically, to sequencing methods. Background Technology

[0002] Sequencing technology refers to the method of determining the nucleotide sequence of an organism's genetic material (DNA or RNA). This technology is the cornerstone of modern molecular biology and is widely used in genomics, medical diagnostics, forensic medicine, agricultural breeding, and many other fields. Since the first commercially available fully automated sequencer was introduced in the 1980s, sequencing technology, as a key method for determining the nucleotide sequence of DNA or RNA, has undergone nearly 40 years of rapid development. In these decades, we have witnessed several generations of evolution in sequencing technology:

[0003] The first generation of sequencers, based on the Sanger sequencing method, marked the commercialization and automation of sequencing technology, but were limited by throughput and cost.

[0004] Second-generation sequencing platforms: The emergence of high-throughput sequencing technology has made sequencing faster and more economical. Among the competition among many platforms, a dominant technology has gradually emerged, which has greatly promoted genomics research and application.

[0005] Third-generation sequencers: The development of single-molecule sequencing technology, with its long read lengths and real-time sequencing capabilities, has provided new tools for solving complex structures in the genome.

[0006] As sequencing technology moves from the laboratory to wider applications, its application areas are expanding, making sequencing services more accessible. However, with the proliferation of various sequencing applications and intensified market competition, the price of sequencing services is also declining. For sequencer manufacturers, maintaining competitiveness in this fierce market competition by reducing costs and improving efficiency (cost reduction and efficiency improvement) has become an urgent problem to solve.

[0007] Therefore, current sequencing technologies need further improvement. Summary of the Invention

[0008] This application aims to at least partially address one of the technical problems in related technologies. Through in-depth research into sequencing technology, the inventors of this application have developed a variety of sequencing methods that can effectively improve sequencing efficiency / quality and reduce costs.

[0009] One sequencing method provided in this application includes: S1, flowing a first buffer solution into a chip to clean the chip, the chip being used for sequencing-by-synthesis, the pH of the first buffer solution being greater than 8; S2, flowing a first synthetic reagent into the chip to perform an extension reaction on the chip, the first synthetic reagent comprising a first nucleotide, the first nucleotide containing a cleavable blocking group and an optically detectable label; S3, flowing a developing reagent into the chip to excite the optically detectable label to generate a signal, and collecting the signal; S4, flowing the first buffer solution into the chip to clean the chip; S5, flowing a cleavage reagent into the chip to cleave the blocking group and the optically detectable label; S6, flowing the first buffer solution into the chip to clean the chip; and S7, flowing a second buffer solution into the chip to clean the chip; S8, returning to step S1, and repeating steps S1 to S7 until the sequencing reaction is complete.

[0010] According to embodiments of this application, by employing the above-described sequencing method, buffer solutions can be used to isolate reagents that may undergo undesirable reactions or have adverse effects when mixed, thereby avoiding losses due to undesirable reactions or adverse effects from mixing, and thus avoiding increased reagent usage to ensure sequencing efficiency / quality. This operation reduces reagent usage and lowers costs while maintaining sequencing efficiency / quality.

[0011] Specifically, according to the embodiments of this application, by cleaning the chip before step S1 and before step S5 using a first buffer solution, and then cleaning the chip again after step S5 using the first and second buffer solutions sequentially, it is possible to effectively isolate reagents that may react undesirably with each other or reagents that may have adverse effects when mixed. Specifically, the first buffer solution can effectively isolate the second buffer solution from the first synthetic reagent, preventing the second buffer solution from affecting the first synthetic reagent and thus affecting sequencing efficiency / quality; the first buffer solution can also effectively isolate the developing reagent from the cleavage reagent, and the cleavage reagent from the second buffer solution, preventing the developing reagent from contacting or mixing with the cleavage reagent and affecting the cleavage reaction during sequencing, thereby affecting sequencing efficiency / quality, and preventing the second buffer solution from prematurely reacting undesirably with the cleavage reagent and thus affecting sequencing efficiency. In this way, by using the first and second buffer solutions to isolate reagents that may cause undesirable reactions or adverse effects after mixing, unwanted reactions between these reagents or adverse effects after mixing are avoided. This ensures that each reagent maintains its optimal performance during use, thereby improving sequencing efficiency / quality. Furthermore, it eliminates the need to increase reagent usage to maintain sequencing efficiency / quality due to losses caused by unwanted reactions between reagents or adverse effects after mixing, thus reducing reagent usage and lowering costs.

[0012] Another sequencing method provided in this application includes: S10, infusing a first synthetic reagent into the chip to perform an extension reaction on the chip, the first synthetic reagent comprising a first nucleotide, the first nucleotide containing a cleavable blocking group and an optically detectable label; S20, infusing a developing reagent into the chip to excite the optically detectable label to generate a signal, and collecting the signal; S30, infusing a cleaving reagent into the chip to cleave the blocking group and the optically detectable label; S40, infusing a second buffer solution into the chip to clean the chip; S50, returning to step S10, and repeating steps S10 to S40 until the sequencing reaction is complete; wherein the second buffer solution comprises SSC, Tween-20, and cystamine or a salt thereof, the final concentration of cystamine or a salt thereof in the second buffer solution being 10–30 mM.

[0013] According to embodiments of this application, reagent efficiency can be improved by adjusting the reagent formulation, such as increasing the concentration of the active ingredient or changing the pH value and ionic strength, thereby reducing the amount of reagent required. Specifically, according to embodiments of this application, by using a second buffer solution with a higher concentration of cystamine or its salt, the washing efficiency of the second buffer solution can be improved, thereby reducing the amount of second buffer solution used. The second buffer solution can effectively remove the cutting reagents remaining in the chip after the cutting reaction, which is crucial for ensuring the accuracy of the sequencing reaction. The cutting reagents can remove the 3' hydroxyl-terminal blocking group and optically detectable markers of the first nucleotide, restoring the activity of the reaction site on the first nucleotide for the next round of sequencing reaction. If the cutting reagents are not completely removed, it may lead to phase advance, i.e., prephasing, reducing sequencing quality. At the same time, washing the sequencing chip with the second buffer solution after step S30 can also help reduce non-specific binding and background noise during the sequencing process, thereby improving sequencing efficiency / quality and preventing cross-reactions or cross-influences between different reagents, ensuring that each reagent can perform its proper function, which is very important for maintaining the independence and accuracy of each step in the sequencing process.

[0014] Another sequencing method provided in this application includes: S100, allowing a first synthetic reagent to flow into the chip to perform an extension reaction on the chip, the first synthetic reagent comprising a first nucleotide, the first nucleotide containing a cleavable blocking group and an optically detectable label; S200, allowing a developing reagent to flow into the chip to excite the optically detectable label to generate a signal, and collecting the signal; S300, allowing a cleaving reagent to flow into the chip to cleave the blocking group and the optically detectable label; S400, returning to step S100, and repeating steps S100 to S300 until the sequencing reaction is completed; wherein, the method further includes: before or after the first synthetic reagent flows into the chip in step S100, drawing in a predetermined volume of air into the chip.

[0015] According to embodiments of this application, an air column is used to isolate the first synthetic reagent from other reagents, reducing the reduction in effectiveness of the first synthetic reagent due to diffusion. When two reagents enter the chip sequentially, if not isolated, they are prone to cross-diffusion and mutual interference. Therefore, to ensure sequencing efficiency / quality, the dosage needs to be increased to eliminate the diffusion effect, leading to increased reagent usage. However, by using an air column to isolate the first synthetic reagent from other reagents, diffusion between the first synthetic reagent and other reagents can be effectively avoided or reduced. This achieves the goal of reducing the amount of the first synthetic reagent used while maintaining sequencing efficiency / quality, thus lowering costs.

[0016] Another sequencing method provided in this application includes: S1000, allowing a first synthetic reagent to flow into the chip to perform an extension reaction on the chip, the first synthetic reagent comprising a first nucleotide, the first nucleotide containing a cleavable blocking group and an optically detectable tag; S2000, allowing a developing reagent to flow into the chip to excite the optically detectable tag to generate a signal, and collecting the signal; S3000, allowing a cleaving reagent to flow into the chip to cleave the blocking group and the optically detectable tag; S4000, returning to step S1000, and repeating steps S1000 to S3000 until the sequencing reaction is complete; wherein, the method further includes: after step S1000 and before step S2000, recovering the first synthetic reagent after the reaction and / or at least a portion of the first synthetic reagent that did not participate in the reaction; and / or, before step S1000, allowing the recovered first synthetic reagent after the reaction to flow into the chip to clean the chip.

[0017] According to embodiments of this application, the used first synthetic reagent is recovered and reused through a back-tracking method, saving the amount of first synthetic reagent used and reducing costs. Furthermore, using the recovered first synthetic reagent as a cleaning solution to clean the chip can further reduce the amount of cleaning solution used, further lowering costs.

[0018] Another sequencing method provided in this application includes: S10000, inducing a first synthetic reagent into the chip to perform an extension reaction on the chip, the first synthetic reagent comprising a first nucleotide, the first nucleotide containing a cleavable blocking group and an optically detectable label; S20000, inducing a developing reagent into the chip to excite the optically detectable label to generate a signal, and collecting the signal; S30000, inducing a cleavage reagent into the chip to cleave the blocking group and the optically detectable label; S40000, returning to step S10000, and repeating steps S10000 to S30000 until the sequencing reaction is completed; wherein, step S20000, inducing the developing reagent into the chip, includes: pushing the developing reagent into the chip using a first buffer solution; and / or, step S30000, inducing the cleavage reagent into the chip, includes: pushing the cleavage reagent into the chip using a first buffer solution.

[0019] According to embodiments of this application, pre-push developing reagents or cutting reagents into the chip using a first buffer solution can reduce the residue of developing reagents or cutting reagents outside the chip and improve the utilization rate of developing reagents or cutting reagents.

[0020] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0021] Figure 1 A first sequencing method according to an embodiment of this application is shown;

[0022] Figure 2 This illustrates a second sequencing method according to an embodiment of this application;

[0023] Figure 3 This illustrates a third sequencing method according to an embodiment of this application;

[0024] Figure 4 This illustrates a fourth sequencing method according to embodiments of this application;

[0025] Figure 5 This illustrates a fifth sequencing method according to embodiments of this application;

[0026] Figure 6A liquid circuit diagram of the sequencing platform FASTASeq300 according to an embodiment of this application is shown.

[0027] Explanation of reference numerals in the attached figures:

[0028] 1000, Liquid circuit system; 100, Reservoir; 100a, First synthetic reagent reservoir; 100b, Second synthetic reagent reservoir; 200, Valve assembly; 200a, Rotary valve; 200b, Solenoid three-way valve; 300, Pump assembly; 400, Chip; 500, Common pipeline; 600, First pipeline; 700, Second pipeline. Detailed Implementation

[0029] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0030] In this application, the terminology used in the embodiments is for the purpose of describing specific embodiments only and is not intended to limit the application. The term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A alone, A and B simultaneously, or B alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "and" relationship.

[0031] The singular forms “a,” “the,” and “the” 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 used only for descriptive purposes to distinguish objects such as substances, locations, interfaces, messages, requests, and terminals from one another, and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as “first” or “second” may explicitly or implicitly include one or more of that feature.

[0032] In the embodiments of this application, the term "chip" is also referred to as "sequencing chip," "biochip," "flow cell," "reaction cell," "flow cell," "reaction device," etc., and these terms are interchangeable in expression. A chip generally has a sandwich-like structure with three layers (top, middle, and bottom), or a two-layer structure. The top layer is a transparent glass layer, and the middle or bottom layer is a transparent or opaque substrate layer. At least one of the top, middle, or bottom layers has one or more fluid channels arranged in an array. These fluid channels can contain liquids or solutions and provide physical space for the reaction. The chips mentioned in this application include, but are not limited to, chips with the above-described structure.

[0033] The term "cleaning" can refer to removing or replacing another liquid, solution, or other substance with a liquid or solution.

[0034] The term "sequencing," also known as "nucleic acid sequencing" or "gene sequencing," is interchangeable in its terminology. It refers to the determination of the base types and sequence in a nucleic acid sequence, including sequencing by synthesis (SBS).

[0035] Sequencing generally involves multiple rounds to determine the sequence of multiple nucleotides / bases on a nucleic acid template. In this application, each round of "determining the sequence of multiple nucleotides / bases on a nucleic acid template" is referred to as "one round of sequencing." A "sequencing round" (cycle) is also called a "sequencing cycle" and can be defined as one base extension of four nucleotides / bases. In other words, "one round of sequencing" can be defined as determining the base type at any specified position on the nucleic acid template. For sequencing platforms based on polymerization or ligation reactions, one round of sequencing includes the process of binding four nucleotides (including nucleotide analogs) to the so-called nucleic acid template and collecting the corresponding signals. For platforms based on polymerization reactions, the reaction system includes reaction substrate nucleotides or nucleotide analogs, polymerase, and a nucleic acid template. The nucleic acid template has a sequence (sequencing primer) bound to it. Based on the base pairing principle and the polymerization reaction principle, the added reaction substrate nucleotides or nucleotide analogs, under the catalysis of the polymerase, are ligated to the sequencing primers to achieve the binding of the nucleotide or nucleotide analog to a specific position on the nucleic acid template, i.e., base extension. The base extension reaction (or simply extension reaction) is typically involved in a sequencing run. One round of sequencing may include one or more base extension reactions. For example, four nucleotides may be added sequentially to the reaction system, and base extension reactions and corresponding signal acquisition may be performed separately, resulting in four base extension reactions in one round of sequencing. Alternatively, four nucleotides may be added to the reaction system in any combination, such as in pairs or in a one-to-three combination, with each combination performing base extension reactions and acquiring corresponding signal acquisition separately, resulting in two base extension reactions in one round of sequencing. Finally, four nucleotides may be added to the reaction system simultaneously for base extension reactions and signal acquisition, resulting in one base extension reaction in one round of sequencing.

[0036] It should be noted that those skilled in the art will understand that after each base extension reaction, a microscopic imaging system can be used to acquire the corresponding reaction signals and form an image, so as to determine the base type at any specified position on the nucleic acid template based on the image.

[0037] The term "blocking group," also known as a "blocking group," exemplifies a group that can block the reactive site (such as 3'-OH) of a pentose sugar in a nucleotide or nucleotide analog to prevent polymerization at that site, and that group can be removed by chemical or other methods to restore the reactivity of the reactive site. An example of a blocking group is, for instance, an azide group (-N3) attached to the 3' position of a sugar moiety in a nucleotide or nucleotide analog.

[0038] The term "optically detectable label" can refer to any of a variety of detectable labels with optical properties known to those skilled in the art, examples including but not limited to chromophores, fluorophores (or fluorescent groups), luminescent or chemiluminescent molecules, radioactive isotopes, etc. In some cases, preferred labels may include fluorophores. Representative fluorophores include, for example, FAM, (tetramethylrhodamine)Texas. Red™, Green Fluorescent Protein, Blue Fluorescent Protein, Red Fluorescent Protein, Fluorescein, 5-Isothiocyanate Fluorescein (FITC), Cyanide Dyes (Cy3, Cy3.5, Cy5, Cy5.5, Cy7), Bodipy Dyes (Invitrogen) and / or Alexa Fluor Dyes (Invitrogen), Dansyl, Dansyl Chloride (DNS-C1), 5-(Iodoacetamide) Fluorescein (5-IAF), 6-Acryloyl-2-dimethylaminonaphthalene (acrylodan), 7-Nitrobenzo-2-oxo-1,3-diazol-4-ylchloro (NBD-Cl), Ethidium Bromide, Fluorescent Astaxanthin, Rhodamine Dyes (5-Carboxyrhodamine 6G Hydrochloride, Rhodamine B-Sulphocyanate, Rhodamine B-Isothiocyanate (RITC), Rhodamine 800); Tetramethylrhodamine 5-(and 6-)Isothiocyanate (TRITC), Texas RedTM, sulfonyl chloride, naphthylamine sulfonic acid (including but not limited to 1-anilinonaphthalene-8-sulfonic acid (ANS) and 6-(p-toluidine)naphthalene-2-sulfonic acid (TNS)), anthraquinone fatty acids, DPH, octadecanoic acid, TMA-DPH, fluorenyl fatty acids, fluorescein-phosphatidylethanolamine, Texas red-phosphatidylethanolamine, pyrene-phosphatidylcholine, fluorenyl-phosphatidylcholine, bis(2-phosphatidyl)cyanine 540, naphthylstyryl, 3,3'-dipropylthiodicarbonylcyanine (diS-C3-(5)), 4-(p-dipentylaminostyryl)-l-methylpyridinium salt (di-5-ASP), Cy-3-iodoacetamide, Cy-5-N-hydroxysuccinimide, Cy-7-isothiocyanate, IR-125, thiazole orange, azure B, Nile blue, aluminum phthalocyanine, oxazine 1,4',6-diamidinyl-2-phenylindole (DAPI), Hoechst 33342, TOTO, acridine orange, ethidium homodimer, N(ethoxycarbonylmethyl)-6-methoxyquinoline salt (MQAE), Fura-2, calcium green, carboxylated SNARF-6, BAPTA, coumarin, phytofluoresceins, benzene, and metal-ligand complexes. Those skilled in the art will understand that fluorophores can generate fluorescence signals when excited by excitation light of a specific wavelength or wavelength range, and these signals can be acquired using a microscopic imaging system.

[0039] The term "developing reagent" is also known as "imaging reagent." For example, developing reagents contain antioxidants, such as water-soluble vitamin E (Trolox), which can prevent or reduce damage or impact of light on the sample / nucleic acid template during image acquisition.

[0040] The term "cleavage reagent," also known as "excision reagent," is used to remove blocking groups and optically detectable labels from nucleotide analogs. Exemplarily, a cleavage reagent is a reducing agent comprising one or more of THPP (tris(3-hydroxypropyl)phosphine), TCEP (tris(2-chloroethyl) phosphate), DTT (dithiothreitol), or β-mercaptoethanol, used to reduce the blocking group, thereby causing the blocking group to detach from the nucleotide analog.

[0041] Sequencing by Synthesis (SBS) is a high-throughput sequencing technology that determines the sequence of a nucleic acid template by monitoring the addition of nucleotides during the synthesis of the complementary strand. Exemplarily, the main steps of SBS technology include: immobilizing the nucleic acid template on a solid surface, such as a chip or bead surface; adding and binding primers complementary to the nucleic acid template to it; adding four deoxyribonucleotides (dNTPs) with different fluorescent and blocking groups to the reaction system one or more times for a single-base extension reaction; generating a fluorescent signal by exciting the fluorescent group and detecting the signal using a microscopic imaging system; based on the detection results, determining which dNTP attaches to the 3'-OH end of the primer and binds to the nucleic acid template in a base-complementary manner, thereby inferring the corresponding base on the nucleic acid template for determining its sequence; removing the fluorescent and blocking groups; and repeating the single-base extension reaction-signal detection-removal steps until the nucleic acid template sequence is completely read.

[0042] To reduce costs and increase efficiency, the inventors of this application have conducted in-depth research and optimized the sequencing method, hoping to reduce sequencing costs and improve sequencing efficiency by reducing the amount of sequencing reagents used during the sequencing process.

[0043] like Figure 1 As shown, a sequencing method provided in one embodiment of this application includes: S1, allowing a first buffer solution to flow into a chip to clean the chip, the chip being used for sequencing while synthesizing, the pH value of the first buffer solution being greater than 8.

[0044] The first buffer solution contains Trisbase (tris(hydroxymethyl)aminomethane), NaCl (sodium chloride), and Tween. For example, Tween-20.

[0045] Trisbase, as a commonly used buffer, can maintain the stability of the solution's pH value, ensuring that the pH value remains within a suitable range throughout the cleaning process, and avoiding the impact of pH fluctuations on the sequencing primers or nucleic acid templates on the chip surface.

[0046] The addition of NaCl helps to adjust the ionic strength of the solution, promoting the dissolution and removal of residues.

[0047] Tween (e.g., Tween-20), as a nonionic surfactant, can reduce the surface tension of a solution, enhance its permeability and cleaning ability, and help to more effectively remove residues and impurities from the chip surface.

[0048] According to embodiments of this application, the concentration of Trisbase in the first buffer solution is 45-55 mmol / L. Exemplarily, the concentration of Trisbase can be 45 mmol / L, 46 mmol / L, 47 mmol / L, 48 mmol / L, 49 mmol / L, 50 mmol / L, 51 mmol / L, 52 mmol / L, 53 mmol / L, 54 mmol / L, 55 mmol / L, or any two of these values.

[0049] The concentration of NaCl in the first buffer solution is 45-55 mmol / L. For example, the concentration of NaCl can be 45 mmol / L, 46 mmol / L, 47 mmol / L, 48 mmol / L, 49 mmol / L, 50 mmol / L, 51 mmol / L, 52 mmol / L, 53 mmol / L, 54 mmol / L, 55 mmol / L, or any two of these values.

[0050] The volume percentage concentration of Tween (e.g., Tween-20) is 0.01% to 0.1%, for example, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, or a range between any two of these values. For example, the volume percentage concentration of Tween-20 can be 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1%.

[0051] Unless otherwise specified in this document, "Tween volume percentage concentration" refers to the percentage of the Tween volume to the volume of the first buffer solution.

[0052] The pH of the first buffer solution is greater than 8. For example, the pH of the first buffer solution can be 8, 8.5, 9, 9.5, 10, or any two of these values.

[0053] The chip surface is immobilized with sequencing primers and nucleic acid templates that bind to the primers. Before the extension reaction, the chip is washed with a first buffer solution. This replaces any remaining reactants or solutions from the previous step on the chip surface, reducing non-specific binding of these substances to the sequencing primers or nucleic acid templates on the chip surface, thereby improving the signal-to-noise ratio and sequencing quality.

[0054] The sequencing method of this embodiment further includes: S2, allowing a first synthetic reagent to flow into the chip for an extension reaction on the chip, the first synthetic reagent comprising a first nucleotide, the first nucleotide containing a cleavable blocking group and an optically detectable label.

[0055] Specifically, the first nucleotide is one or more deoxyribonucleotides, such as dNTPs. "dNTPs," or deoxyribonucleoside triphosphates, contain a phosphate group, a deoxyribose sugar, and a nitrogenous base (one of A, T, C, and G), typically including dATP, dGTP, dTTP, and dCTP, representing deoxyadenosine triphosphate, deoxyguanosine triphosphate, deoxythymidine triphosphate, and deoxycytidine triphosphate, respectively. After the first synthetic reagent flows into the chip, the first nucleotide in the first synthetic reagent, under the action of DNA polymerase and under conditions suitable for polymerase chain reaction, is ligated to the 3'-OH end of the sequencing primer located on the chip surface and binds to the bases on the nucleic acid template in a base-complementary pairing manner. A cleavable blocking group on the first nucleotide can prevent the polymerization reaction at the reaction site (e.g., 3'-OH) on the sugar of the first nucleotide, thus allowing for a single-base extension reaction. Exemplarily, the DNA polymerase is selected from one or more of rTaq, Canace, Pfu, KOD, Phusion, Primerstar, Tth, BST, and BSU. Suitable conditions for polymerase chain reaction (PCR) include a suitable temperature for PCR. Exemplarily, a suitable temperature for PCR is 55°C-65°C, such as 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, or any two of these values. Preferably, a suitable temperature for PCR is 60°C.

[0056] According to an embodiment of this application, the pH of the first synthetic reagent is greater than 8. Exemplarily, the pH of the first synthetic reagent can be 8, 8.5, 9, 9.5, 10, or any two of these values.

[0057] The sequencing method of this embodiment further includes: S3, allowing the developing reagent to flow into the chip in order to excite the optically detectable marker to generate a signal, and collecting the signal.

[0058] After each base extension reaction, an optically detectable label on the first nucleotide is excited by excitation light to generate an optical signal. For example, when the optically detectable label is a fluorescent group, excitation light of a specific wavelength or wavelength range can generate a fluorescent signal. This fluorescent signal is acquired using a microscopic imaging system to form an image. Based on the image, the type of base attached to the 3'-OH end of the sequencing primer and bound to the nucleic acid template can be determined. Introducing a developing reagent into the chip before or during imaging can avoid or reduce damage or influence on the nucleic acid template caused by excitation light during image acquisition.

[0059] According to an embodiment of this application, the pH of the developing agent is approximately 7. Exemplarily, the pH of the developing agent can be 7, 7.1, 7.2, 7.3, 7.4, or any two values ​​therein.

[0060] The sequencing method in this embodiment further includes: S4, allowing the first buffer solution to flow into the chip to clean the chip.

[0061] In this step, the first buffer solution is allowed to flow into the chip, which can be used to clean the chip again after the development step to remove any residues or byproducts that may have been generated during the development process, in preparation for the next cleavage reaction.

[0062] According to embodiments of this application, the first buffer solution comprises Trisbase (tris(hydroxymethyl)aminomethane), NaCl (sodium chloride), and Tween, such as Tween-20.

[0063] Trisbase, as a commonly used buffer, can maintain the stability of the solution's pH value, ensuring that the pH value remains within a suitable range throughout the cleaning process, and avoiding the impact of pH fluctuations on the sequencing primers or nucleic acid templates on the chip surface.

[0064] The addition of NaCl helps to adjust the ionic strength of the solution, promoting the dissolution and removal of residues.

[0065] Tween (e.g., Tween-20), as a nonionic surfactant, can reduce the surface tension of a solution, enhance its permeability and cleaning ability, and help to more effectively remove residues and impurities from the chip surface.

[0066] According to embodiments of this application, the concentration of Trisbase in the first buffer solution is 45-55 mmol / L, which can be 45 mmol / L, 46 mmol / L, 47 mmol / L, 48 mmol / L, 49 mmol / L, 50 mmol / L, 51 mmol / L, 52 mmol / L, 53 mmol / L, 54 mmol / L, 55 mmol / L, or any two of these values.

[0067] According to the embodiments of this application, the concentration of NaCl in the first buffer solution is 45-55 mmol / L, which can be 45 mmol / L, 46 mmol / L, 47 mmol / L, 48 mmol / L, 49 mmol / L, 50 mmol / L, 51 mmol / L, 52 mmol / L, 53 mmol / L, 54 mmol / L, 55 mmol / L, or any two of these values.

[0068] According to embodiments of this application, the volume percentage concentration of Tween (e.g., Tween-20) is 0.01%-0.1%, and can be 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, or a range between any two of these values.

[0069] According to an embodiment of this application, the pH value of the first buffer solution is greater than 8, and can be 8, 8.5, 9, 9.5, 10, or any two of these values.

[0070] According to embodiments of this application, by cleaning the chip again, residues or byproducts that may be generated during the development process can be removed, ensuring a clean chip surface and providing a favorable environment for subsequent dicing reactions. This helps improve the efficiency and accuracy of the reaction and reduces potential interference and errors.

[0071] The sequencing method of this embodiment further includes: S5, allowing the cutting reagent to flow into the chip to remove the blocking group and the optically detectable marker;

[0072] According to embodiments of this application, in this step, the cleavage reagent is fed into the chip, precisely introduced into the chip to ensure sufficient contact with the first nucleotide. This process requires precise control of the flow rate and velocity of the cleavage reagent to ensure the efficiency and effectiveness of the cleavage reaction. Simultaneously, it is essential to ensure that the cleavage reagent uniformly covers the chip surface to achieve the removal of blocking groups and optically detectable markers on all first nucleotides. Removing the blocking groups and optically detectable markers from the first nucleotides prepares the material for the next round of extension reactions. The presence of the blocking groups on the first nucleotides prevents them from prematurely polymerizing with other nucleotides during the extension reaction. By removing these blocking groups, the 3'-OH activity of the first nucleotide is restored, allowing it to participate in the extension reaction again. Simultaneously, the removal of the optically detectable markers helps reduce background signals, improving the accuracy and sensitivity of subsequent detections. Achieving this objective is crucial for the continuity and accuracy of the sequencing workflow, ensuring that single-base extension reactions can be effectively performed in each cycle.

[0073] According to embodiments of this application, the cleavage reagent typically contains an enzyme or chemical reagent capable of specifically recognizing and cleaving the blocking group. For example, if the blocking group is based on an azide group, the cleavage reagent may contain a reagent capable of reducing the azide group, such as tris(3-hydroxypropyl)phosphine (THPP). The composition and properties of the cleavage reagent determine its ability to specifically recognize and cleave the blocking group. In this embodiment, the enzyme or chemical reagent in the cleavage reagent can accurately recognize and bind to the blocking group, thereby cleaving it through a chemical reaction. Taking the azide group as an example, THPP can effectively reduce the azide group, destroying its structure, thereby achieving the cleavage of the blocking group. This specific recognition and cleavage mechanism ensures the high efficiency and accuracy of the cleavage reaction, avoiding unnecessary influence on other parts. By cleaving the blocking group and the optically detectable label, the 3'-OH activity of the first nucleotide is restored, enabling it to participate in the elongation reaction again. This step is crucial for achieving the cyclical execution of the single-base elongation reaction.

[0074] According to embodiments of this application, the cleavage reaction serves not only to restore the activity of the first nucleotide but also to create conditions for the next round of extension reactions. After the blocking group is removed, the first nucleotide can re-participate as a reactant in the new extension reaction, achieving the sequential addition of single bases. This cyclical process is the core of the sequencing reaction; through continuous extension, detection, and cleavage, a complete nucleic acid template sequence is gradually constructed. Furthermore, removing the optically detectable marker helps reduce background signal and improve the signal-to-noise ratio of subsequent detections, thereby enhancing the quality of sequencing data.

[0075] According to embodiments of this application, the pH of the cutting reagent is greater than 8. Exemplarily, the pH of the cutting reagent can be 8, 8.5, 9, 9.5, 10, or any two of these values.

[0076] According to the embodiments of this application, step S5 is performed after step S4. The first buffer solution effectively isolates the developing reagent and the cutting reagent, preventing the developing reagent from affecting the cutting reagent. For example, mixing the developing reagent and the cutting reagent can cause the pH of the cutting reagent to drop, thus affecting its reaction efficiency. If step S4 is omitted and step S5 is performed directly, the pH of the cutting reagent will drop after the developing reagent comes into contact with or mixes with the cutting reagent. A possible remedy is to increase the amount of cutting reagent used to ensure cutting efficiency, thereby ensuring sequencing efficiency / quality. However, this would result in unnecessary loss of the cutting reagent. The first buffer solution can isolate the developing reagent and the cutting reagent, and its pH range is essentially the same as that of the cutting reagent. In particular, when the pH of the first buffer solution is comparable to that of the cutting reagent, even if the first buffer solution entering the chip first comes into contact with or mixes with the cutting reagent entering the chip later, it will not cause significant fluctuations in the pH of the cutting reagent, thus not affecting its reaction efficiency. Therefore, there is no unnecessary loss of the cutting reagent, and it is not necessary to increase the amount of cutting reagent used to ensure sequencing efficiency / quality, thereby reducing the amount of cutting reagent used and lowering costs.

[0077] According to embodiments of this application, in this step, the flow of the cutting reagent into the chip includes injecting the cutting reagent into the chip in one or multiple portions using a first buffer solution. However, the inventors have discovered that, compared to injecting the cutting reagent into the chip in one go using the first buffer solution, injecting the cutting reagent into the chip in multiple portions using the first buffer solution increases the number of incubation steps in the cutting reaction, thereby improving the efficiency of the cutting reaction. This may be because, as the cutting reaction proceeds, the initially added cutting reagent is continuously consumed, leading to a decrease in the concentration of the cutting reagent in the reaction system and a reduction in efficiency. The subsequently added cutting reagent can replenish this consumption in a timely manner, thus maintaining a high reaction efficiency. Furthermore, injecting the cutting reagent into the chip in multiple portions using the first buffer solution can reduce temperature fluctuations in the reaction system, thereby improving reaction efficiency.

[0078] The sequencing method in this embodiment further includes: S6, allowing the first buffer solution to flow into the chip to clean the chip.

[0079] The first buffer solution is precisely introduced into the chip to ensure complete coverage of the chip surface and full contact with residual cutting reagents and other impurities. According to embodiments of this application, by uniformly flowing the first buffer solution, every area on the chip is ensured to be effectively cleaned.

[0080] According to embodiments of this application, the chip is cleaned again after the cleavage reaction to remove residues and byproducts generated during the cleavage reaction. During the cleavage reaction, some residues may be generated, such as cleaved blocking groups, optically detectable markers, and other possible byproducts. If these residues are not effectively removed, they may affect subsequent sequencing reactions, such as causing nonspecific binding or interfering with signal detection. Cleaning with a first buffer ensures a clean chip surface, providing a favorable environment for the next round of sequencing reactions.

[0081] According to embodiments of this application, cleaning ensures that the chip surface is free of residual cutting reagents or other impurities, providing a clean environment for the next sequencing reaction. This step is crucial for the stability and reliability of the entire sequencing process. Effectively removing residues after the cutting reaction prevents these residues from interfering with subsequent steps such as extension and development, improving sequencing accuracy and efficiency. Simultaneously, keeping the chip surface clean helps extend its lifespan, ensuring good performance throughout multiple sequencing cycles.

[0082] The sequencing method in this embodiment further includes: S7, allowing a second buffer solution to flow into the chip to clean the chip.

[0083] According to embodiments of this application, infusing the second buffer solution into the chip involves flowing the second buffer solution into the chip and pushing the first buffer solution forward, so that the first and second buffer solutions sequentially enter the chip to clean it. In this operation, the first and second buffer solutions are precisely introduced into the chip, ensuring complete coverage of the chip surface and adequate contact with residual reagents and other impurities. The flow rate and velocity of the first and second buffer solutions need to be precisely controlled to ensure effective cleaning while avoiding unnecessary impact on sequencing primers or nucleic acid templates on the chip surface. Uniform flow of the buffer solutions ensures that every area on the chip is effectively cleaned.

[0084] According to embodiments of this application, during the cleavage reaction, some residues may be generated, such as cleaved blocking groups, optically detectable markers, and other possible byproducts. If these residues are not effectively removed, they may affect subsequent sequencing reactions, such as causing nonspecific binding or interfering with signal detection. By sequentially cleaning the chip with a first buffer and a second buffer, the chip surface can be ensured to be clean, providing a good environment for the next sequencing reaction. This step is crucial for the stability and reliability of the entire sequencing process. By effectively removing the residues after the cleavage reaction, interference from these residues in subsequent extension and development reactions can be avoided, improving the accuracy and efficiency of sequencing. At the same time, keeping the chip surface clean helps extend the chip's lifespan, ensuring that it maintains good performance in multiple sequencing cycles.

[0085] According to embodiments of this application, specifically, the chip is first cleaned with a first buffer solution, and then further cleaned with a second buffer solution. This effectively removes residual cleavage reagents from the chip, which is crucial for ensuring the accuracy of the sequencing reaction. The cleavage reagents can remove the 3' hydroxyl-terminal blocking group and optically detectable markers of the first nucleotide, restoring the activity of the reaction site on the first nucleotide for the next round of sequencing. If the cleavage reagents are not completely removed, it may lead to phase advance, i.e., prephasing, reducing sequencing quality. Simultaneously, cleaning the chip with the first buffer solution first, and then further cleaning it with the second buffer solution, can isolate the second buffer solution and the cleavage reagents by the first buffer solution. This prevents undesirable reactions (e.g., the reaction between cystamine in the second buffer solution and THPP in the cleavage reagent) from prematurely occurring after contact or mixing with the cleavage reagents, which could cause the ongoing cleavage reaction to be rapidly terminated, thus reducing cleavage efficiency. Furthermore, cleaning the chip with the first buffer solution first, and then further cleaning it with the second buffer solution, ensures that the cleavage reaction may still be ongoing until the first buffer solution completely replaces the cleavage reagents, thereby improving sequencing efficiency / quality. According to embodiments of this application, the pH of the second buffer solution is approximately 7. For example, the pH of the second buffer can be 7, 7.1, 7.2, 7.3, 7.4, or any two values ​​between them. After cleaning the chip with the first buffer, the chip is further cleaned with the second buffer. The first buffer isolates the second buffer from the cleavage reagent, preventing the pH of the cleavage reagent from decreasing due to contact or mixing, thus avoiding a drop in cleavage efficiency and impacting sequencing efficiency / quality.

[0086] According to embodiments of this application, the second buffer typically comprises SSC, Tween-20, and cystamine or a salt thereof, wherein the final concentration of cystamine or a salt thereof is 10–30 mM, for example, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, or any two values ​​between them. For example, the second buffer may comprise 5X SSC, 0.05% Tween-20, and 20 mM of cystamine dihydrochloride.

[0087] SSC is a commonly used buffer component that provides a stable ionic environment, helping to maintain the structure and stability of DNA. It can modulate the ionic strength of the solution, promoting the dissolution and removal of residues.

[0088] Tween-20, as a nonionic surfactant, can reduce the surface tension of a solution, enhance its permeability and cleaning ability, and help to more effectively remove residues and impurities from the chip surface.

[0089] Cystamine is a compound that can bind to cleavage reagents, further enhancing the cleaning effect and removing residual cleavage reagents and other impurities. In the second buffer, the final concentration of cystamine or its salt is 10–30 mM, which provides sufficient cleaning power while avoiding damage to sequencing primers or nucleic acid templates on the chip surface.

[0090] According to embodiments of this application, the second buffer solution can more effectively remove residues and impurities from the chip surface, ensuring the stability and reliability of the sequencing process. This thorough cleaning helps prevent residues from interfering with subsequent sequencing reactions, improving the quality of sequencing data. Using the second buffer solution for final cleaning ensures a clean chip surface, providing a favorable environment for the next sequencing cycle or the end of the sequencing process. This not only helps improve sequencing accuracy and efficiency but also extends the chip's lifespan, ensuring it maintains good performance across multiple sequencing cycles.

[0091] The sequencing method in this embodiment further includes: S8, returning to step S1, and repeating steps S1 to S7 until the sequencing reaction is complete.

[0092] According to an embodiment of this application, in this operation, the system automatically returns to step S1 and restarts a new sequencing cycle. This includes washing the chip again with the first buffer to prepare for the new extension reaction. The entire process is performed automatically according to a preset program without manual intervention, ensuring the efficiency and stability of the sequencing process. By cyclically executing the above steps, complete sequencing of the nucleic acid template is achieved.

[0093] According to the embodiments of this application, a single round of sequencing can only determine the information of one base on the nucleic acid template. In order to completely determine the sequence of the nucleic acid template, it is necessary to construct the entire sequence step by step through multiple cycles. Each cycle corresponds to a specific base position on the nucleic acid template. By repeating steps S1 to S7, the bases on the nucleic acid template can be determined one by one, and finally the complete sequence information is obtained.

[0094] According to embodiments of this application, this cyclic design enables the sequencing process to be automated and performed at high throughput until complete nucleic acid sequence information is obtained. Automated cycling significantly improves sequencing throughput and efficiency, reducing errors and time costs associated with manual operation. Each cycle strictly follows the same steps and conditions, ensuring the reliability and reproducibility of the sequencing results. Furthermore, this cyclic design can flexibly adapt to nucleic acid templates of different lengths; whether the template is a short or long sequence, sequencing can be completed through an appropriate number of cycles.

[0095] According to embodiments of this application, by employing the above-described sequencing method, buffer solutions can be used to isolate reagents that may undergo undesirable reactions or have adverse effects when mixed, thereby avoiding losses due to undesirable reactions or adverse effects from mixing, and thus avoiding increased reagent usage to ensure sequencing efficiency / quality. This operation reduces reagent usage and lowers costs while maintaining sequencing efficiency / quality.

[0096] Specifically, according to the embodiments of this application, by cleaning the chip before step S1 and before step S5 using a first buffer solution, and then cleaning the chip again after step S5 using the first and second buffer solutions sequentially, it is possible to effectively isolate reagents that may react undesirably with each other or reagents that may have adverse effects when mixed. Specifically, the first buffer solution can effectively isolate the second buffer solution from the first synthetic reagent, preventing the second buffer solution from affecting the first synthetic reagent and thus affecting sequencing efficiency / quality; the first buffer solution can also effectively isolate the developing reagent from the cleavage reagent, and the cleavage reagent from the second buffer solution, preventing the developing reagent from contacting or mixing with the cleavage reagent and affecting the cleavage reaction during sequencing, thereby affecting sequencing efficiency / quality, and preventing the second buffer solution from prematurely reacting undesirably with the cleavage reagent and thus affecting sequencing efficiency. In this way, by using the first and second buffer solutions to isolate reagents that may cause undesirable reactions or adverse effects after mixing, unwanted reactions between these reagents or adverse effects after mixing are avoided. This ensures that each reagent maintains its optimal performance during use, thereby improving sequencing efficiency / quality. Furthermore, it eliminates the need to increase reagent usage to maintain sequencing efficiency / quality due to losses caused by unwanted reactions between reagents or adverse effects after mixing, thus reducing reagent usage and lowering costs.

[0097] According to an embodiment of this application, after step S2 and before step S3, the process further includes: S2-1, allowing a second synthetic reagent to flow into the chip. The second synthetic reagent contains a second nucleotide, which contains a cleavable blocking group but does not contain an optically detectable label. According to an embodiment of this application, the second synthetic reagent is introduced into the chip to ensure sufficient contact with nucleic acid templates that did not participate in the extension reaction in step S2, thereby ensuring that all nucleic acid templates participate in the extension reaction and improving sequencing accuracy. According to an embodiment of this application, in the extension reaction, some nucleic acid templates may not participate in the extension reaction in step S2, resulting in signal loss for these templates in this round of reaction, affecting the integrity and accuracy of the sequencing data. By introducing the second synthetic reagent to complete the reaction, the synchronicity of all nucleic acid template reactions can be ensured without affecting the correct identification of bases in the next round of sequencing. According to an embodiment of this application, the second synthetic reagent contains a second nucleotide, which contains a cleavable blocking group but does not contain an optically detectable label. The second nucleotide, similar to the first nucleotide, also contains a cleavable blocking group but does not contain an optically detectable label. This means that the second nucleotide primarily binds to the nucleic acid template that did not participate in the extension reaction in step S2, without participating in signal detection. This design ensures the efficient execution of the completion reaction while avoiding unnecessary signal interference. The completion reaction ensures that all nucleic acid templates can be accurately determined, enhancing data integrity, reducing sequencing errors caused by asynchrony in the extension reactions of all nucleic acid templates, and improving the quality of sequencing data. Simultaneously, ensuring the accurate determination of all nucleic acid templates helps obtain more complete sequence information, improving the overall performance of sequencing.

[0098] According to embodiments of this application, such as Figure 6 As shown, the chip 400 is detachably disposed in the liquid system 1000, which includes a reservoir 100, a valve assembly 200, and a pump assembly 300. The valve assembly 200 is configured to connect the reservoir 100 and the pump assembly 300, or to connect the reservoir 100 and the chip 400 and the pump assembly 300. The reservoir 100 includes a first synthetic reagent reservoir 100a for storing a first synthetic reagent. Optionally, the method includes: using the pump assembly 300 to drive the first synthetic reagent in the first synthetic reagent reservoir 100a to flow through the valve assembly 200 into the chip 400 for an extension reaction, so that in step S2, the first synthetic reagent flows into the chip 400 to carry out an extension reaction on the chip 400.

[0099] According to an embodiment of this application, the chip 400 is detachably disposed in the liquid circuit system 1000. This design facilitates the replacement and maintenance of the chip 400, and also allows for separate cleaning and processing of the chip 400. The valve assembly 200 includes a rotary valve 200a and a three-way solenoid valve 200b communicatively connected to the rotary valve 200a. The valve assembly 200 connects the reservoir 100 to the pump assembly 300, or connects the reservoir 100 to the chip 400 and the pump assembly 300, using the rotary valve 200a and the three-way solenoid valve 200b.

[0100] According to an embodiment of this application, the liquid circuit system 1000 includes:

[0101] Reservoir 100: Used to store various reagents, such as first synthesis reagent, second synthesis reagent, developing reagent, cutting reagent, first buffer solution, and second buffer solution. The reservoir 100 ensures the orderly storage and supply of reagents.

[0102] Valve assembly 200: Configured to connect reservoir 100 and pump assembly 300, or to connect reservoir 100 and chip 400 and pump assembly 300. The function of valve assembly 200 is to precisely control the flow of reagents, ensuring that the correct reagents are delivered to the designated locations in different sequencing steps.

[0103] Pump Unit 300: Responsible for driving the flow of reagents. It precisely controls the flow rate and volume of reagents according to the needs of the sequencing process, ensuring that the reagents arrive at the designated location on time and in the correct quantity.

[0104] First synthetic reagent reservoir 100a: Specifically used to store the first synthetic reagent, ensuring a stable supply of the first synthetic reagent. The sequencing method includes using a pump assembly 300 to drive the first synthetic reagent in the first synthetic reagent reservoir 100a into the chip 400 via a valve assembly 200 for the extension reaction. This step corresponds to step S2, ensuring that the first synthetic reagent can participate in the extension reaction accurately and in a timely manner.

[0105] According to an embodiment of this application, a pump assembly 300 drives the first synthetic reagent in the first synthetic reagent reservoir 100a to flow into the chip 400 through a valve assembly 200. This ensures that the first synthetic reagent reaches the chip 400 accurately and promptly for the extension reaction. The pump assembly 300 generates sufficient pressure and flow rate according to a preset program and parameters, propelling the first synthetic reagent out of the reservoir 100. Upon receiving a corresponding control signal, the valve assembly 200 opens the corresponding channel, allowing the first synthetic reagent to smoothly pass through the valve assembly 200 and flow into the chip 400. Driven by the pump assembly 300, the first synthetic reagent passes through the valve assembly 200 and finally flows into the chip 400, ready for the extension reaction. According to an embodiment of this application, by precisely controlling the pump assembly 300 and the valve assembly 200, the first synthetic reagent is ensured to participate in the extension reaction accurately and promptly, improving the efficiency and accuracy of sequencing. Simultaneously, this precise control also reduces reagent waste and lowers sequencing costs.

[0106] According to an embodiment of this application, the valve assembly 200 and the chip 400 are connected via a common pipeline 500. The method includes: after step S2 and before step S3, using the pump assembly 300 to drive the first synthetic reagent after the extended reaction in the chip 400 and / or the first synthetic reagent that did not participate in the extended reaction in the common pipeline 500 to flow in a direction toward the first synthetic reagent reservoir 100a, so as to recover at least a portion of the first synthetic reagent after the reaction and the first synthetic reagent that did not participate in the reaction.

[0107] According to an embodiment of this application, after step S2 and before step S3, a pump assembly 300 drives the first synthetic reagent that has undergone the extension reaction within the chip 400 and / or the first synthetic reagent that did not participate in the extension reaction within the common conduit 500 to flow towards the first synthetic reagent reservoir 100a, thereby recovering at least a portion of the reacted first synthetic reagent and the unreacted first synthetic reagent. In this operation, the pump assembly 300 is precisely controlled to generate appropriate positive or reverse flow, causing the first synthetic reagent within the chip 400 and the common conduit 500 to flow towards the first synthetic reagent reservoir 100a. This process requires precise control of the pressure and flow rate of the pump assembly 300 to ensure smooth reagent reflux while avoiding unnecessary impact on the sequencing primers or nucleic acid templates on the surface of the chip 400. In this way, the unreacted first synthetic reagent can be effectively recovered, reducing reagent waste. According to an embodiment of this application, recovering at least a portion of the reacted first synthetic reagent and the unreacted first synthetic reagent can reduce reagent waste and lower sequencing costs. By recovering unreacted reagents from the initial synthesis, they can be reused in subsequent sequencing reactions, improving reagent utilization. This is especially important for expensive reagents, significantly reducing sequencing reagent costs.

[0108] Therefore, by recovering unreacted first-stage synthetic reagents, reagent waste is reduced, sequencing costs are lowered, and reagent utilization is improved. This operation not only helps reduce the direct cost of sequencing but also reduces chemical waste, making it more environmentally friendly. Furthermore, optimizing reagent use simplifies the operational process, improving convenience and accuracy. Through these strategies, the goal of cost reduction and efficiency improvement can be achieved while maintaining or enhancing sequencing quality and efficiency.

[0109] According to an embodiment of this application, the first synthetic reagent reservoir 100a is connected to the valve assembly 200 via a first pipeline 600, wherein the first synthetic reagent after reaction is recovered into the first pipeline 600; and / or, the first synthetic reagent that did not participate in the reaction is recovered into the first pipeline 600 and / or the first synthetic reagent reservoir 100a.

[0110] According to an embodiment of this application, the first synthetic reagent reservoir 100a is connected to the valve assembly 200 via a first conduit 600. This connection provides a physical channel for the recovery of the first synthetic reagent. After the first synthetic reagent undergoes an extension reaction within the chip 400, some of the first synthetic reagent may have participated in the reaction, with the remaining portion being recoverable and reusable. In the common conduit 500, there may be first synthetic reagent that did not participate in the extension reaction; these reagents can also be recovered. The first synthetic reagent after the reaction is recovered into the first conduit 600; and / or, the first synthetic reagent that did not participate in the reaction is recovered into the first conduit 600 and / or the first synthetic reagent reservoir 100a. The first conduit 600 serves as a recovery path, temporarily accommodating the recovered reagent for subsequent processing or reuse. A portion of the first synthetic reagent that did not participate in the reaction can be directly returned to the first synthetic reagent reservoir 100a, mixed with the original reagent, for reuse in subsequent sequencing cycles. According to an embodiment of this application, the pressure difference generated by the pump assembly 300 drives the first synthetic reagent to flow along the first conduit 600 toward the first synthetic reagent reservoir 100a. The pump assembly 300, by precisely controlling the pressure and flow rate, ensures that the reagent can be smoothly recovered from the chip 400 and the common conduit 500 into the first conduit 600 or the first synthetic reagent reservoir 100a. The flow direction of the reagent is opposite to the original delivery direction, from the chip 400 and the common conduit 500 toward the first synthetic reagent reservoir 100a. By precisely controlling the pump assembly 300 and the valve assembly 200, automated management of the recovery process can be achieved, ensuring recovery efficiency and accuracy. Therefore, by recovering the incompletely reacted first synthetic reagent, the utilization rate of the reagent can be significantly improved, and waste can be reduced. The recovery and reuse of reagents directly reduces the cost of sequencing, especially for expensive reagents, where this cost saving is more significant. According to an embodiment of this application, reducing reagent waste and environmental pollution aligns with the concept of sustainable development.

[0111] According to an embodiment of this application, step S1 includes: allowing the recovered first synthetic reagent after the reaction to enter the chip 400 from the first pipeline 600 through the valve group 200 as a first buffer solution to clean the chip 400.

[0112] According to an embodiment of this application, the recovered first synthetic reagent after reaction is introduced into the chip 400 through the first tubing 600 and valve assembly 200, serving as a first buffer solution to clean the chip 400. In this operation, the recovered first synthetic reagent after reaction is reused and delivered into the chip 400 under the control of the first tubing 600 and valve assembly 200. This process requires precise control of the pump assembly 300 and valve assembly 200 to ensure that the recovered reagent can uniformly cover the surface of the chip 400 for effective cleaning. According to an embodiment of this application, the recovered first synthetic reagent after reaction is used as the first buffer solution to clean the chip 400. This aims to further improve reagent utilization and reduce reagent waste and cost. By reusing the recovered reagent in the cleaning step, not only is the cost reduced, but the environmental impact is also reduced. By using the recovered reagent for cleaning, residues and impurities on the surface of the chip 400 can be effectively removed, providing a clean environment for subsequent sequencing reactions. This approach not only helps reduce the direct cost of sequencing but also reduces the generation of chemical waste, making it more environmentally friendly. Meanwhile, by optimizing the use of reagents, the operation process can be simplified, and the convenience and accuracy of the operation can be improved.

[0113] According to an embodiment of this application, the first synthetic reagent recovered into the first pipeline 600 and the first synthetic reagent reservoir 100a is 50% to 80% of the total amount of the first synthetic reagent used, preferably 60% to 65%.

[0114] Therefore, according to the embodiments of this application, by recovering the first synthetic reagent that did not participate in the extension reaction, the total consumption of reagents can be reduced, thereby lowering costs. The recovered reagents can be reused, improving reagent utilization efficiency. Directly discarding reagents that did not participate in the reaction would result in resource waste, while recycling and reuse can reduce this waste. Simultaneously, reducing the waste of chemical reagents is also a form of environmental protection. Furthermore, recycling can reduce the amount of reagent pumped, thereby reducing pumping time and sequencing time while ensuring sequencing quality. According to the embodiments of this application, the amount of reagent returned to the first synthetic reagent reservoir 100a is controlled at 50% to 80% of the total added amount, preferably 60% to 65%. This ratio ensures sufficient reagent participation in the reaction while maximizing recycling, achieving an optimal balance between cost and efficiency.

[0115] According to an embodiment of this application, the reservoir 100 includes a second synthetic reagent reservoir 100b, which is used to store a second synthetic reagent. The process of allowing the second synthetic reagent to flow into the chip 400 includes: using a pump assembly 300 to drive the second synthetic reagent in the second synthetic reagent reservoir 100b through a valve assembly 200 into the chip 400 for a replenishment reaction; and allowing the reacted second synthetic reagent and / or the unreacted second synthetic reagent in the common pipeline 500 to flow towards the second synthetic reagent reservoir 100b to recover the reacted second synthetic reagent and / or the unreacted second synthetic reagent.

[0116] According to an embodiment of this application, a pump assembly 300 drives the second synthetic reagent in the second synthetic reagent reservoir 100b through a valve assembly 200 into the chip 400. In this operation, the pump assembly 300 generates sufficient pressure and flow rate according to a preset program and parameters, pushing the second synthetic reagent out of the reservoir. After receiving a corresponding control signal, the valve assembly 200 opens the corresponding channel, allowing the second synthetic reagent to smoothly pass through the valve assembly 200 and flow into the chip 400. The second synthetic reagent flows into the chip 400 for the completion reaction. The purpose of the completion reaction is to ensure that all nucleic acid templates participate in the extension reaction, thereby improving the accuracy of sequencing. Through this operation, the synchronicity of the reactions of all nucleic acid templates can be ensured, improving the accuracy of sequencing. Thus, by precisely controlling the pump assembly 300 and the valve assembly 200, the second synthetic reagent can be ensured to participate in the completion reaction accurately and in a timely manner, improving the efficiency and accuracy of sequencing. At the same time, this precise control also reduces reagent waste and lowers sequencing costs.

[0117] According to embodiments of this application, the reacted second synthetic reagent and / or unreacted second synthetic reagent in the common tubing 500 are flowed towards the second synthetic reagent reservoir 100b to recover the reacted second synthetic reagent and / or unreacted second synthetic reagent. In this operation, the pump assembly 300 is precisely controlled to generate appropriate positive or reverse flow, causing the second synthetic reagent in the chip 400 and the common tubing 500 to flow towards the second synthetic reagent reservoir 100b. This process requires precise control of the pressure and flow rate of the pump assembly 300 to ensure smooth reagent reflux while avoiding unnecessary impact on the sequencing primers or nucleic acid templates on the surface of the chip 400. At least a portion of the reacted second synthetic reagent and the unreacted second synthetic reagent are recovered. This objective aims to reduce reagent waste and lower sequencing costs. By recovering the incompletely reacted second synthetic reagent, it can be reused in subsequent sequencing reactions, improving reagent utilization. Recovering the incompletely reacted second synthetic reagent reduces reagent waste, lowers sequencing costs, and simultaneously improves reagent utilization. This not only helps reduce the direct cost of sequencing but also reduces the generation of chemical waste, making it more environmentally friendly. According to embodiments of this application, the second synthetic reagent reservoir 100b is connected to the valve assembly 200 via a second conduit 700, and the reacted second synthetic reagent is recovered into the second conduit 700; and / or, unreacted second synthetic reagent is recovered into the second conduit 700 and / or the second synthetic reagent reservoir 100b.

[0118] According to an embodiment of this application, the second synthetic reagent reservoir 100b is connected to the valve assembly 200 via a second conduit 700. This connection provides a physical channel for the recovery of the second synthetic reagent. After the completion reaction is performed within the chip 400, the second synthetic reagent may have partially participated in the reaction, with the remaining portion being recoverable and reusable. Second synthetic reagents that did not participate in the completion reaction may also exist in the common conduit 500, and these reagents can also be recovered. According to an embodiment of this application, the reacted second synthetic reagent is recovered into the second conduit 700; and / or, the second synthetic reagents that did not participate in the reaction are recovered into the second conduit 700 and / or the second synthetic reagent reservoir 100b. The second conduit 700 serves as a recovery path, temporarily accommodating the recovered reagents for subsequent processing or reuse. A portion of the second synthetic reagents that did not participate in the reaction can be directly returned to the second synthetic reagent reservoir 100b, mixed with the original reagents, for reuse in subsequent sequencing cycles.

[0119] According to an embodiment of this application, the pressure difference generated by the pump assembly 300 drives the second synthesis reagent to flow along the second pipeline 700 toward the second synthesis reagent reservoir 100b. The pump assembly 300, by precisely controlling the pressure and flow rate, ensures that the reagent can be smoothly recovered from the chip 400 and the common pipeline 500 into the second pipeline 700 or the reservoir. The flow direction of the reagent is opposite to the original delivery direction, from the chip 400 and the common pipeline 500 toward the second synthesis reagent reservoir 100b. By precisely controlling the pump assembly 300 and the valve assembly 200, automated management of the recovery process can be achieved, ensuring recovery efficiency and accuracy. Therefore, by recovering incompletely reacted second synthesis reagents, reagent utilization can be significantly improved, reducing waste. The recovery and reuse of reagents directly reduces sequencing costs, especially for expensive reagents, where this cost saving is more significant. Reducing reagent waste and environmental pollution aligns with the concept of sustainable development. According to an embodiment of this application, the recovered second synthetic reagent after reaction is introduced from the second conduit 700 through the valve assembly 200 into the chip 400 to replace the solution in the chip 400 after the extended reaction. According to an embodiment of this application, the second synthetic reagent recovered into the second conduit 700 and the second synthetic reagent reservoir 100b is 50% to 80% of the total amount of the second synthetic reagent used, preferably 60% to 65%.

[0120] Therefore, according to the embodiments of this application, by recovering the second synthetic reagents that did not participate in the completion reaction, the total consumption of reagents can be effectively reduced, thereby reducing costs. The recovered reagents can be reused, which not only improves reagent utilization efficiency but also reduces the direct waste of reagents that did not participate in the reaction, avoiding resource waste. Furthermore, reducing the waste of chemical reagents is also a form of environmental protection. Through recycling, the amount of reagents pumped can be reduced, thereby reducing pumping time and sequencing time while ensuring sequencing quality. According to the embodiments of this application, after step S2 and before step S3, at least a portion of the second synthetic reagents that did not participate in the completion reaction are returned to the second synthetic reagent reservoir 100b. According to the embodiments of this application, all the second synthetic reagents that did not enter the chip 400 are returned to the second synthetic reagent reservoir 100b. Optionally, the second synthetic reagents returned to the second synthetic reagent reservoir 100b are 50% to 80% of the total amount of second synthetic reagents added, preferably 55% to 60%. This proportion ensures that sufficient reagents participate in the reaction while maximizing recycling, achieving an optimal balance between cost and efficiency.

[0121] According to an embodiment of this application, allowing the developing reagent to flow into the chip 400 includes: introducing a first buffer solution into a common conduit 500 to push the developing reagent within the common conduit 500 into the chip 400. Thus, by utilizing the flow of the first buffer solution, the developing reagent can be pushed from the common conduit 500 into the chip 400, ensuring that all the developing reagent enters the chip 400 to excite an optically detectable marker to generate a signal and collect that signal. This process not only improves the efficiency of developing reagent utilization but also reduces the residue of developing reagent in the common conduit 500, minimizing waste.

[0122] According to embodiments of this application, allowing the cutting reagent to flow into the chip 400 includes: introducing a first buffer solution into a common conduit 500 to push the cutting reagent within the common conduit 500 into the chip 400. Thus, by utilizing the flow of the first buffer solution to push the cutting reagent from the common conduit 500 into the chip 400, it is ensured that all the cutting reagent enters the chip 400, thereby removing the 3' hydroxyl terminator group and optically detectable marker of the synthetic base, preparing it for the next sequencing reaction. This process not only improves the efficiency of cutting reagent use but also reduces the residue of cutting reagent in the common conduit 500, minimizing waste.

[0123] According to embodiments of this application, the method further includes: drawing a predetermined volume of air into the chip 400 before or after the first synthetic reagent flows into the chip 400. This isolates the first synthetic reagent from other reagents using an air column, reducing the reduction in effectiveness caused by diffusion of the first synthetic reagent. If the two reagents entering the chip 400 sequentially are not isolated, they are prone to mutual diffusion and interference. Therefore, to ensure sequencing efficiency / quality, the dosage needs to be increased to eliminate the diffusion effect, leading to an increase in reagent usage. However, by isolating the first synthetic reagent from other reagents using an air column, diffusion between the first synthetic reagent and other reagents can be effectively avoided or reduced, achieving the goal of reducing the amount of the first synthetic reagent used while ensuring sequencing efficiency / quality, thus reducing costs.

[0124] According to embodiments of this application, sequencing-by-synthesis is performed at 55–65°C, preferably 60°C. Therefore, by maintaining the operating temperature of sequencing-by-synthesis according to embodiments of this application, consistent temperature control throughout the entire process can be ensured, facilitating operation. Furthermore, operating at temperatures such as 60°C can increase the rate of chemical or biological reactions, thereby ensuring that the involved chemical and biological reactions achieve higher efficiency at this temperature. Simultaneously, choosing 60°C as the reaction temperature avoids thermal damage to samples or reagents that might be caused by higher temperatures, protecting the integrity of the samples and the activity of the reagents.

[0125] In a second aspect, this application proposes a sequencing method employing an optimized reagent formulation, as illustrated in the embodiments of this application. Figure 2 As shown, the method includes: S10, allowing a first synthetic reagent to flow into a chip to perform an extension reaction on the chip, the first synthetic reagent comprising a first nucleotide, the first nucleotide containing a cleavable blocking group and an optically detectable label.

[0126] Specifically, the first nucleotide is one or more deoxyribonucleotides, such as dNTPs. "dNTPs," or deoxyribonucleoside triphosphates, contain a phosphate group, a deoxyribose sugar, and a nitrogenous base (one of A, T, C, and G), typically including dATP, dGTP, dTTP, and dCTP, representing deoxyadenosine triphosphate, deoxyguanosine triphosphate, deoxythymidine triphosphate, and deoxycytidine triphosphate, respectively. After the first synthetic reagent flows into the chip, the first nucleotide in the first synthetic reagent, under the action of DNA polymerase and under conditions suitable for polymerase chain reaction, is ligated to the 3'-OH end of the sequencing primer located on the chip surface and binds to the bases on the nucleic acid template in a base-complementary pairing manner. A cleavable blocking group on the first nucleotide can prevent the polymerization reaction at the reaction site (e.g., 3'-OH) on the sugar of the first nucleotide, thus allowing for a single-base extension reaction. Exemplarily, the DNA polymerase is selected from one or more of rTaq, Canace, Pfu, KOD, Phusion, Primerstar, Tth, BST, and BSU. Suitable conditions for polymerase chain reaction (PCR) include a suitable temperature for PCR. Exemplarily, a suitable temperature for PCR is 55°C-65°C, such as 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, or any two of these values. Preferably, a suitable temperature for PCR is 60°C.

[0127] According to an embodiment of this application, the pH of the first synthetic reagent is greater than 8. Exemplarily, the pH of the first synthetic reagent can be 8, 8.5, 9, 9.5, 10, or any two of these values.

[0128] S20, the developing reagent flows into the chip to excite the optically detectable marker to generate a signal and collect the signal.

[0129] After each base extension reaction, an optically detectable label on the first nucleotide is excited by excitation light to generate an optical signal. For example, when the optically detectable label is a fluorescent group, excitation light of a specific wavelength or wavelength range can generate a fluorescent signal. This fluorescent signal is acquired using a microscopic imaging system to form an image. Based on the image, the type of base attached to the 3'-OH end of the sequencing primer and bound to the nucleic acid template can be determined. Introducing a developing reagent into the chip before or during imaging can avoid or reduce damage or influence on the nucleic acid template caused by excitation light during image acquisition.

[0130] According to an embodiment of this application, the pH of the developing agent is approximately 7. Exemplarily, the pH of the developing agent can be 7, 7.1, 7.2, 7.3, 7.4, or any two values ​​therein.

[0131] S30 allows the cutting reagent to flow into the chip to remove the blocking groups and optically detectable markers.

[0132] According to embodiments of this application, in this step, the cleavage reagent is fed into the chip, precisely introduced into the chip to ensure sufficient contact with the first nucleotide. This process requires precise control of the flow rate and velocity of the cleavage reagent to ensure the efficiency and effectiveness of the cleavage reaction. Simultaneously, it is essential to ensure that the cleavage reagent uniformly covers the chip surface to achieve the removal of blocking groups and optically detectable markers on all first nucleotides. Removing the blocking groups and optically detectable markers from the first nucleotides prepares the material for the next round of extension reactions. The presence of the blocking groups on the first nucleotides prevents them from prematurely polymerizing with other nucleotides during the extension reaction. By removing these blocking groups, the 3'-OH activity of the first nucleotide is restored, allowing it to participate in the extension reaction again. Simultaneously, the removal of the optically detectable markers helps reduce background signals, improving the accuracy and sensitivity of subsequent detections. Achieving this objective is crucial for the continuity and accuracy of the sequencing workflow, ensuring that single-base extension reactions can be effectively performed in each cycle.

[0133] According to embodiments of this application, the cleavage reagent typically contains an enzyme or chemical reagent capable of specifically recognizing and cleaving the blocking group. For example, if the blocking group is based on an azide group, the cleavage reagent may contain a reagent capable of reducing the azide group, such as tris(3-hydroxypropyl)phosphine (THPP). The composition and properties of the cleavage reagent determine its ability to specifically recognize and cleave the blocking group. In this embodiment, the enzyme or chemical reagent in the cleavage reagent can accurately recognize and bind to the blocking group, thereby cleaving it through a chemical reaction. Taking the azide group as an example, THPP can effectively reduce the azide group, destroying its structure, thereby achieving the cleavage of the blocking group. This specific recognition and cleavage mechanism ensures the high efficiency and accuracy of the cleavage reaction, avoiding unnecessary influence on other parts. By cleaving the blocking group and the optically detectable label, the 3'-OH activity of the first nucleotide is restored, enabling it to participate in the elongation reaction again. This step is crucial for achieving the cyclical execution of the single-base elongation reaction.

[0134] According to embodiments of this application, the cleavage reaction serves not only to restore the activity of the first nucleotide but also to create conditions for the next round of extension reactions. After the blocking group is removed, the first nucleotide can participate again as a reactant in the new extension reaction, achieving the sequential addition of single bases. This cyclical process is the core of the sequencing reaction, gradually constructing a complete nucleic acid sequence through continuous extension, detection, and cleavage. Furthermore, removing the optically detectable marker helps reduce background signal and improve the signal-to-noise ratio of subsequent detections, thereby enhancing the quality of sequencing data.

[0135] According to embodiments of this application, the pH of the cutting reagent is greater than 8. Exemplarily, the pH of the cutting reagent can be 8, 8.5, 9, 9.5, 10, or any two of these values.

[0136] S40, allowing the second buffer solution to flow into the chip to clean it.

[0137] According to embodiments of this application, in this step, a second buffer solution is precisely introduced into the chip to ensure complete coverage of the chip surface and full contact with residual cutting reagents and other impurities. According to embodiments of this application, by uniformly flowing the buffer solution, every area on the chip is ensured to be effectively cleaned.

[0138] According to embodiments of this application, after the cleavage reaction, the chip is cleaned to remove residues and byproducts generated during the cleavage reaction. During the cleavage reaction, some residues may be generated, such as cleaved blocking groups, optically detectable tags, and other possible byproducts. If these residues are not effectively removed, they may affect subsequent sequencing reactions, such as causing nonspecific binding or interfering with signal detection. Using a second buffer solution for cleaning ensures a clean chip surface, providing a favorable environment for the next round of sequencing reactions.

[0139] According to embodiments of this application, cleaning ensures that the chip surface is free of residual cutting reagents or other impurities, providing a clean environment for the next sequencing reaction. This step is crucial for the stability and reliability of the entire sequencing process. Effectively removing residues after the cutting reaction prevents these residues from interfering with subsequent steps such as extension and development, improving sequencing accuracy and efficiency. Simultaneously, keeping the chip surface clean helps extend its lifespan, ensuring good performance throughout multiple sequencing cycles.

[0140] S50, return to step S10, and repeat steps S10 to S40 until the sequencing reaction is complete.

[0141] According to embodiments of this application, in this operation, the system automatically returns to restart a new sequencing cycle. This includes washing the chip again with the first buffer to prepare for the new extension reaction. The entire process is performed automatically according to a preset program without manual intervention, ensuring the efficiency and stability of the sequencing process. By repeatedly executing the above steps, complete sequencing of the DNA template is achieved.

[0142] According to embodiments of this application, a single round of sequencing can only determine information for one base. To completely determine the sequence of the DNA template, multiple cycles are needed to gradually construct the entire sequence. Each cycle corresponds to a specific base position. By repeating the above steps, the bases on the DNA strand can be determined one by one, ultimately obtaining complete sequence information.

[0143] According to embodiments of this application, this cyclic design enables the sequencing process to be automated and performed at high throughput until complete DNA sequence information is obtained. Automated cycling significantly improves sequencing throughput and efficiency, reducing errors and time costs associated with manual operation. Each cycle strictly follows the same steps and conditions, ensuring the reliability and reproducibility of the sequencing results. Furthermore, this cyclic design can flexibly adapt to DNA templates of different lengths; whether the template is a short or long sequence, sequencing can be completed through an appropriate number of cycles.

[0144] According to an embodiment of this application, the pH of the second buffer solution is approximately 7. Exemplarily, the pH of the second buffer solution can be 7, 7.1, 7.2, 7.3, 7.4, or any two values ​​therein.

[0145] According to embodiments of this application, the second buffer typically comprises SSC, Tween-20, and cystamine or a salt thereof, wherein the final concentration of cystamine or a salt thereof is 10–30 mM, for example, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, or any two values ​​between them. For example, the second buffer may comprise 5X SSC, 0.05% Tween-20, and 20 mM of cystamine dihydrochloride.

[0146] SSC is a commonly used buffer component that provides a stable ionic environment, helping to maintain the structure and stability of DNA. It can modulate the ionic strength of the solution, promoting the dissolution and removal of residues.

[0147] Tween-20, as a nonionic surfactant, can reduce the surface tension of a solution, enhance its permeability and cleaning ability, and help to more effectively remove residues and impurities from the chip surface.

[0148] Cystamine is a compound that can bind to DNA, further enhancing the cleaning effect and removing residual DNA, proteins, and other impurities. In the second buffer, the final concentration of cystamine or its salt is 10–30 mM, which provides sufficient cleaning power while avoiding damage to sequencing primers or nucleic acid templates on the chip surface.

[0149] Therefore, according to embodiments of this application, by adjusting the reagent formulation, such as increasing the concentration of the active ingredient or changing the pH value, ionic strength, etc., the efficiency of the reagent can be improved, thereby reducing the amount of reagent required. Specifically, according to embodiments of this application, by using a second buffer solution with a higher concentration of cystamine or its salt, the washing efficiency of the second buffer solution can be improved. This means that the second buffer solution can effectively perform its function with a lower usage amount, thereby reducing the usage amount. The second buffer solution can effectively remove the cutting reagents remaining in the chip after the cutting reaction, which is crucial for ensuring the accuracy of the sequencing reaction. The cutting reagents can remove the 3' hydroxyl-terminal blocking group and optically detectable markers of the first nucleotide, restoring the activity of the reaction site on the first nucleotide for the next round of sequencing reaction. If the cutting reagents are not completely removed, it may lead to phase advance, i.e., prephasing, reducing sequencing quality. At the same time, washing the sequencing chip with the second buffer solution after step S30 can also help reduce non-specific binding and background noise during the sequencing process, thereby improving the efficiency / quality of sequencing and preventing cross-reactions or cross-influences between different reagents, ensuring that each reagent can perform its proper function. This is very important for maintaining the independence and accuracy of each step in the sequencing process.

[0150] According to an embodiment of this application, after step S10 and before step S20, the process further includes: S10-1, allowing a second synthetic reagent to flow into the chip. The second synthetic reagent contains a second nucleotide, which contains a cleavable blocking group but does not contain an optically detectable label. According to an embodiment of this application, the introduction of the second synthetic reagent into the chip ensures sufficient contact with nucleic acids that did not participate in the extension reaction in step S10, ensuring that all nucleic acid templates participate in the extension reaction, thereby improving sequencing accuracy. According to an embodiment of this application, in the extension reaction, some nucleic acid templates may not participate in the extension reaction in step S10, resulting in signal loss for these templates in this round of reaction, affecting the integrity and accuracy of the sequencing data. By introducing the second synthetic reagent to complete the reaction, the synchronicity of all nucleic acid template reactions can be ensured without affecting the correct identification of bases in the next round of sequencing. According to an embodiment of this application, the second synthetic reagent contains a second nucleotide, which contains a cleavable blocking group but does not contain an optically detectable label. Similar to the first nucleotide, the second nucleotide also contains a cleavable blocking group but does not contain an optically detectable label. This means that the second nucleotide is primarily used to bind to nucleic acid templates that did not participate in the extension reaction in step S10, and does not participate in signal detection. This design ensures the efficient execution of the completion reaction while avoiding unnecessary signal interference. By completing the reaction, all nucleic acid templates are accurately determined, enhancing data integrity, reducing sequencing errors caused by asynchrony in the extension reactions of all nucleic acid templates, and improving the quality of sequencing data. Simultaneously, ensuring the accurate determination of all nucleic acid templates helps obtain more complete sequence information, improving the overall performance of sequencing.

[0151] According to an embodiment of this application, before step S10, a first buffer solution is introduced into the chip to clean the chip, which is used for sequencing-by-synthesis. According to an embodiment of this application, the pH value of the first buffer solution is greater than 8. According to an embodiment of this application, the pH value of the first buffer solution is 9-10.

[0152] The first buffer solution contains Trisbase (tris(hydroxymethyl)aminomethane), NaCl (sodium chloride), and Tween. For example, Tween-20.

[0153] Trisbase, as a commonly used buffer, can maintain the stability of the solution's pH value, ensuring that the pH value remains within a suitable range throughout the cleaning process, and avoiding the impact of pH fluctuations on the sequencing primers or nucleic acid templates on the chip surface.

[0154] The addition of NaCl helps to adjust the ionic strength of the solution, promoting the dissolution and removal of residues.

[0155] Tween (e.g., Tween-20), as a nonionic surfactant, can reduce the surface tension of a solution, enhance its permeability and cleaning ability, and help to more effectively remove residues and impurities from the chip surface.

[0156] According to embodiments of this application, the concentration of Trisbase in the first buffer solution is 45-55 mmol / L. Exemplarily, the concentration of Trisbase can be 45 mmol / L, 46 mmol / L, 47 mmol / L, 48 mmol / L, 49 mmol / L, 50 mmol / L, 51 mmol / L, 52 mmol / L, 53 mmol / L, 54 mmol / L, 55 mmol / L, or any two of these values.

[0157] The concentration of NaCl in the first buffer solution is 45-55 mmol / L. For example, the concentration of NaCl can be 45 mmol / L, 46 mmol / L, 47 mmol / L, 48 mmol / L, 49 mmol / L, 50 mmol / L, 51 mmol / L, 52 mmol / L, 53 mmol / L, 54 mmol / L, 55 mmol / L, or any two of these values.

[0158] The volume percentage concentration of Tween (e.g., Tween-20) is 0.01% to 0.1%, for example, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, or a range between any two of these values. For example, the volume percentage concentration of Tween-20 can be 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1%.

[0159] The pH of the first buffer solution is greater than 8. For example, the pH of the first buffer solution can be 8, 8.5, 9, 9.5, 10, or any two of these values.

[0160] The chip surface is immobilized with sequencing primers and nucleic acid templates that bind to the primers. Before the extension reaction, the chip is washed with a first buffer solution. This replaces any remaining reactants or solutions from the previous step on the chip surface, reducing non-specific binding of these substances to the sequencing primers or nucleic acid templates on the chip surface, thereby improving the signal-to-noise ratio and sequencing quality.

[0161] According to an embodiment of this application, before step S30, a first buffer solution is introduced into the chip to clean it. Specifically, before performing step S30, the first buffer solution can effectively isolate the developing reagent and the cutting reagent, preventing the developing reagent from affecting the cutting reagent. For example, mixing the developing reagent and the cutting reagent can cause the pH of the cutting reagent to drop, thereby affecting the reaction efficiency of the cutting reagent. If this step is omitted and step S30 is performed directly, the pH of the cutting reagent will drop after the developing reagent comes into contact with or mixes with the cutting reagent. A possible remedy is to increase the amount of cutting reagent used to ensure cutting efficiency, thereby ensuring sequencing efficiency / quality. However, this would cause unnecessary loss of the cutting reagent. The first buffer solution can isolate the developing reagent and the cutting reagent, and the pH range of the first buffer solution is basically the same as the pH range of the cutting reagent. In particular, when the pH value of the first buffer solution is comparable to the pH value of the cutting reagent, even if the first buffer solution entering the chip first comes into contact with or mixes with the cutting reagent entering the chip later, it will not cause a large fluctuation in the pH of the cutting reagent, and will not affect the reaction efficiency of the cutting reagent. In this way, there is no unnecessary loss of cutting reagent, and there is no need to increase the amount of cutting reagent used to ensure sequencing efficiency / quality, thereby achieving the goal of reducing the amount of cutting reagent used and reducing costs.

[0162] According to an embodiment of this application, in step S30, the inflow of the cleavage reagent into the chip includes flowing a first buffer solution into the chip and pushing the cleavage reagent into the chip in one or multiple portions to remove the blocking group and optically detectable label on the first nucleotide. In this step, the cleavage reagent can be pushed into the chip in one or multiple portions using the first buffer solution. However, the inventors have found that compared to pushing the cleavage reagent into the chip in one go using the first buffer solution, pushing the cleavage reagent into the chip in multiple portions using the first buffer solution increases the number of incubation steps for the cleavage reaction, thereby improving the efficiency of the cleavage reaction. This may be because as the cleavage reaction proceeds, the initially added cleavage reagent is continuously consumed, leading to a decrease in its concentration and efficiency. The subsequently added cleavage reagent can replenish this consumption in a timely manner, thus maintaining a high reaction efficiency. Furthermore, pushing the cleavage reagent into the chip in multiple portions using the first buffer solution can reduce temperature fluctuations in the reaction system, thereby improving reaction efficiency.

[0163] According to an embodiment of this application, in step S40, the process of allowing the second buffer solution to flow into the chip includes causing the second buffer solution to flow toward the chip and pushing the first buffer solution, so that the first buffer solution and the second buffer solution enter the chip sequentially to clean the chip.

[0164] According to embodiments of this application, in this operation, a first buffer and a second buffer are precisely introduced into the chip to ensure complete coverage of the chip surface and adequate contact with residual reagents and other impurities. The flow rate and velocity of the first and second buffers need to be precisely controlled to ensure effective cleaning while avoiding unnecessary impact on sequencing primers or nucleic acid templates on the chip surface. Uniform flow of the buffers ensures that every area on the chip is effectively cleaned.

[0165] According to embodiments of this application, during the cleavage reaction, some residues may be generated, such as cleaved blocking groups, optically detectable markers, and other possible byproducts. If these residues are not effectively removed, they may affect subsequent sequencing reactions, such as causing nonspecific binding or interfering with signal detection. By sequentially cleaning the chip with a first buffer and a second buffer, the chip surface can be ensured to be clean, providing a good environment for the next sequencing reaction. This step is crucial for the stability and reliability of the entire sequencing process. By effectively removing the residues after the cleavage reaction, interference from these residues in subsequent extension and development reactions can be avoided, improving the accuracy and efficiency of sequencing. At the same time, keeping the chip surface clean helps extend the chip's lifespan, ensuring that it maintains good performance in multiple sequencing cycles.

[0166] According to embodiments of this application, the chip is first cleaned with a first buffer solution, and then further cleaned with a second buffer solution. This effectively removes residual cleavage reagents from the chip, which is crucial for ensuring the accuracy of the sequencing reaction. The cleavage reagents remove the 3' hydroxyl-terminal blocking group and optically detectable markers of the first nucleotide, restoring the activity of the reaction site on the first nucleotide for the next round of sequencing. If the cleavage reagents are not completely removed, it may lead to prephasing, reducing sequencing quality. Furthermore, cleaning the chip with the first buffer solution first, followed by further cleaning with the second buffer solution, isolates the second buffer solution from the cleavage reagents. This prevents unwanted reactions from occurring prematurely after contact or mixing with the cleavage reagents, which could rapidly terminate an ongoing cleavage reaction and reduce cleavage efficiency. Furthermore, cleaning the chip with the first buffer solution first, followed by further cleaning with the second buffer solution, ensures that the cleavage reaction may still be ongoing until the first buffer solution completely replaces the cleavage reagents, thereby improving sequencing efficiency and quality. In addition, the chip is first cleaned with the first buffer and then further cleaned with the second buffer. The first buffer isolates the second buffer from the cleavage reagent, which can also prevent the pH of the cleavage reagent from decreasing after the second buffer comes into contact with or mixes with the cleavage reagent, thus reducing the cleavage efficiency and affecting the sequencing efficiency / quality.

[0167] According to embodiments of this application, such as Figure 6 As shown, chip 400 is detachably disposed in liquid system 1000, which includes reservoir 100, valve assembly 200, and pump assembly 300. Valve assembly 200 is configured to connect reservoir 100 and pump assembly 300, or to connect reservoir 100 with chip 400 and pump assembly 300. Reservoir 100 includes first synthetic reagent reservoir 100a for storing first synthetic reagent. According to an embodiment of this application, the method includes: using pump assembly 300 to drive the first synthetic reagent in first synthetic reagent reservoir 100a through valve assembly 200 into chip 400 for an extension reaction, so that in step S10, the first synthetic reagent flows into chip 400 to carry out an extension reaction on chip 400. According to an embodiment of this application, chip 400 is detachably disposed in liquid system 1000, which facilitates replacement and maintenance of chip 400, and also facilitates separate cleaning and processing of chip 400. Valve assembly 200 includes a rotary valve 200a and a three-way solenoid valve 200b that can communicate with the rotary valve 200a. Valve assembly 200 uses the rotary valve 200a and the three-way solenoid valve 200b to connect the reservoir 100 with the pump assembly 300, or to connect the reservoir 100 with the chip 400 and the pump assembly 300.

[0168] According to an embodiment of this application, the liquid circuit system 1000 includes:

[0169] Reservoir 100: Used to store various reagents, such as first synthesis reagent, second synthesis reagent, developing reagent, cutting reagent, first buffer solution, and second buffer solution. The reservoir 100 ensures the orderly storage and supply of reagents.

[0170] Valve assembly 200: Configured to connect reservoir 100 and pump assembly 300, or to connect reservoir 100 and chip 400 and pump assembly 300. The function of valve assembly 200 is to precisely control the flow of reagents, ensuring that the correct reagents are delivered to the designated locations in different sequencing steps.

[0171] Pump Unit 300: Responsible for driving the flow of reagents. It precisely controls the flow rate and volume of reagents according to the needs of the sequencing process, ensuring that the reagents arrive at the designated location on time and in the correct quantity.

[0172] First synthetic reagent reservoir 100a: Specifically used to store the first synthetic reagent, ensuring a stable supply of the first synthetic reagent. The sequencing method includes using a pump assembly 300 to drive the first synthetic reagent in the first synthetic reagent reservoir 100a into the chip 400 via a valve assembly 200 for the extension reaction. This step corresponds to step S10, ensuring that the first synthetic reagent can participate in the extension reaction accurately and in a timely manner.

[0173] According to an embodiment of this application, a pump assembly 300 drives the first synthetic reagent in the first synthetic reagent reservoir 100a to flow into the chip 400 through a valve assembly 200. This ensures that the first synthetic reagent reaches the chip 400 accurately and promptly for the extension reaction. The pump assembly 300 generates sufficient pressure and flow rate according to a preset program and parameters, propelling the first synthetic reagent out of the reservoir. Upon receiving a corresponding control signal, the valve assembly 200 opens the corresponding channel, allowing the first synthetic reagent to smoothly pass through the valve assembly 200 and flow into the chip 400. Driven by the pump assembly 300, the first synthetic reagent passes through the valve assembly 200 and finally flows into the chip 400, ready for the extension reaction. According to an embodiment of this application, by precisely controlling the pump assembly 300 and the valve assembly 200, the first synthetic reagent is ensured to participate in the extension reaction accurately and promptly, improving the efficiency and accuracy of sequencing. Simultaneously, this precise control also reduces reagent waste and lowers sequencing costs.

[0174] According to an embodiment of this application, the valve assembly 200 and the chip 400 are connected via a common conduit 500. The method includes: after step S10 and before step S20, using a pump assembly 300 to drive the first synthetic reagent that has undergone an extension reaction within the chip 400 and / or the first synthetic reagent that did not participate in the extension reaction within the common conduit 500 to flow towards the first synthetic reagent reservoir 100a, so as to recover at least a portion of the reacted first synthetic reagent and the first synthetic reagent that did not participate in the reaction. According to an embodiment of this application, after step S2 and before step S3, using a pump assembly 300 to drive the first synthetic reagent that has undergone an extension reaction within the chip 400 and / or the first synthetic reagent that did not participate in the extension reaction within the common conduit 500 to flow towards the first synthetic reagent reservoir 100a, so as to recover at least a portion of the reacted first synthetic reagent and the first synthetic reagent that did not participate in the reaction. In this operation, pump assembly 300 is precisely controlled to generate appropriate positive or reverse flow, causing the first synthetic reagent within chip 400 and common tubing 500 to flow toward the first synthetic reagent reservoir 100a. This process requires precise control of the pressure and flow rate of pump assembly 300 to ensure smooth reagent reflux while avoiding unnecessary impact on sequencing primers or nucleic acid templates on the surface of chip 400. In this way, incompletely reacted first synthetic reagents can be effectively recovered, reducing reagent waste. According to embodiments of this application, recovering at least a portion of the reacted first synthetic reagents and those not involved in the reaction can reduce reagent waste and lower sequencing costs. By recovering incompletely reacted first synthetic reagents, they can be reused in subsequent sequencing reactions, improving reagent utilization. This is particularly important for expensive reagents, significantly reducing sequencing reagent costs.

[0175] Therefore, by recovering unreacted first-stage synthetic reagents, reagent waste is reduced, sequencing costs are lowered, and reagent utilization is improved. This operation not only helps reduce the direct cost of sequencing but also reduces chemical waste, making it more environmentally friendly. Furthermore, optimizing reagent use simplifies the operational process, improving convenience and accuracy. Through these strategies, the goal of cost reduction and efficiency improvement can be achieved while maintaining or enhancing sequencing quality and efficiency.

[0176] According to an embodiment of this application, the first synthetic reagent reservoir 100a is connected to the valve assembly 200 via a first conduit 600, wherein the reacted first synthetic reagent is recovered into the first conduit 600; and / or, the first synthetic reagent that did not participate in the reaction is recovered into the first conduit 600 and / or the first synthetic reagent reservoir 100a. According to an embodiment of this application, the first synthetic reagent reservoir 100a is connected to the valve assembly 200 via a first conduit 600. This connection provides a physical channel for the recovery of the first synthetic reagent. After the reacted first synthetic reagent undergoes an extended reaction within the chip 400, the first synthetic reagent may have partially participated in the reaction, and the remaining portion can still be recovered and reused. In the common conduit 500, there may be first synthetic reagent that did not participate in the extended reaction, and these reagents can also be recovered. The reacted first synthetic reagent is recovered into the first conduit 600; and / or, the first synthetic reagent that did not participate in the reaction is recovered into the first conduit 600 and / or the first synthetic reagent reservoir 100a. The first conduit 600 serves as a recovery path, temporarily containing recovered reagents for subsequent processing or reuse. Unreacted first synthetic reagents can be directly returned to the first synthetic reagent reservoir 100a, mixed with the original reagents, for reuse in subsequent sequencing cycles. According to embodiments of this application, the pressure difference generated by the pump assembly 300 drives the first synthetic reagents along the first conduit 600 towards the first synthetic reagent reservoir 100a. The pump assembly 300, through precise control of pressure and flow rate, ensures that reagents can be smoothly recovered from the chip 400 and the common conduit 500 into the first conduit 600 or the first synthetic reagent reservoir 100a. The flow direction of the reagents is opposite to the original delivery direction, from the chip 400 and the common conduit 500 towards the first synthetic reagent reservoir 100a. By precisely controlling the pump assembly 300 and the valve assembly 200, automated management of the recovery process can be achieved, ensuring recovery efficiency and accuracy. Therefore, by recovering incompletely reacted first synthetic reagents, reagent utilization can be significantly improved, and waste reduced. The recycling and reuse of reagents directly reduces the cost of sequencing, especially for expensive reagents, where the cost savings are even more significant. According to embodiments of this application, reducing reagent waste and environmental pollution aligns with the principles of sustainable development.

[0177] According to an embodiment of this application, before step S10: the recovered first synthetic reagent after reaction is introduced from the first pipeline 600 through the valve assembly 200 into the chip 400 to clean the chip 400. According to an embodiment of this application, the first synthetic reagent recovered into the first pipeline 600 and the first synthetic reagent reservoir 100a is 50% to 80% of the total amount of the first synthetic reagent used, preferably 60% to 65%. According to an embodiment of this application, the recovered first synthetic reagent after reaction is introduced from the first pipeline 600 through the valve assembly 200 into the chip 400 as a first buffer solution to clean the chip 400. In this operation, the recovered first synthetic reagent after reaction is reused and delivered into the chip 400 under the control of the first pipeline 600 and the valve assembly 200. This process requires precise control of the pump assembly 300 and the valve assembly 200 to ensure that the recovered reagent can uniformly cover the surface of the chip 400 for effective cleaning. According to an embodiment of this application, the recovered first synthetic reagent after reaction is used as a first buffer solution to clean the chip 400. This objective aims to further improve reagent utilization and reduce waste and costs. Reusing recovered reagents in the cleaning step not only reduces costs but also minimizes environmental impact. Using recovered reagents for cleaning effectively removes residues and impurities from the surface of the chip 400, providing a clean environment for subsequent sequencing reactions. This practice not only helps reduce the direct cost of sequencing but also reduces chemical waste, making it more environmentally friendly. Furthermore, optimizing reagent use simplifies the operational process, improving convenience and accuracy.

[0178] According to an embodiment of this application, the first synthetic reagent recovered into the first pipeline 600 and the first synthetic reagent reservoir 100a is 50% to 80% of the total amount of the first synthetic reagent used, preferably 60% to 65%.

[0179] Therefore, according to the embodiments of this application, by recovering the first synthetic reagent that did not participate in the extension reaction, the total consumption of reagents can be reduced, thereby lowering costs. The recovered reagents can be reused, improving reagent utilization efficiency. Directly discarding reagents that did not participate in the reaction would result in resource waste, while recycling and reuse can reduce this waste. Simultaneously, reducing the waste of chemical reagents is also a form of environmental protection. Furthermore, recycling can reduce the amount of reagent pumped, thereby reducing pumping time and sequencing time while ensuring sequencing quality. According to the embodiments of this application, the amount of reagent returned to the first synthetic reagent reservoir 100a is controlled at 50% to 80% of the total added amount, preferably 60% to 65%. This ratio ensures sufficient reagent participation in the reaction while maximizing recycling, achieving an optimal balance between cost and efficiency.

[0180] According to an embodiment of this application, the reservoir 100 includes a second synthetic reagent reservoir 100b, which is used to store a second synthetic reagent. The process of allowing the second synthetic reagent to flow into the chip 400 includes: using a pump assembly 300 to drive the second synthetic reagent in the second synthetic reagent reservoir 100b into the chip 400 via a valve assembly 200; and allowing the reacted second synthetic reagent and / or the unreacted second synthetic reagent in the common pipeline 500 to flow towards the second synthetic reagent reservoir 100b to recover the reacted second synthetic reagent and / or the unreacted second synthetic reagent.

[0181] According to an embodiment of this application, a pump assembly 300 drives the second synthetic reagent in the second synthetic reagent reservoir 100b through a valve assembly 200 into the chip 400. In this operation, the pump assembly 300 generates sufficient pressure and flow rate according to a preset program and parameters, pushing the second synthetic reagent out of the reservoir. After receiving a corresponding control signal, the valve assembly 200 opens the corresponding channel, allowing the second synthetic reagent to smoothly pass through the valve assembly 200 and flow into the chip 400. The second synthetic reagent flows into the chip 400 to perform the completion reaction. The purpose of the completion reaction is to ensure that all gaps on the DNA template are filled, thereby improving the read length and accuracy of sequencing. Through this operation, it can be ensured that even sequences that are not fully extended in the extension reaction can be accurately determined, enhancing data integrity. Thus, by precisely controlling the pump assembly 300 and the valve assembly 200, the second synthetic reagent can be ensured to participate in the completion reaction accurately and in a timely manner, improving the efficiency and accuracy of sequencing. At the same time, this precise control also reduces reagent waste and lowers sequencing costs.

[0182] According to embodiments of this application, the reacted second synthetic reagent and / or unreacted second synthetic reagent in the common tubing 500 are flowed towards the second synthetic reagent reservoir 100b to recover the reacted second synthetic reagent and / or unreacted second synthetic reagent. In this operation, the pump assembly 300 is precisely controlled to generate appropriate positive or reverse flow, causing the second synthetic reagent in the chip 400 and the common tubing 500 to flow towards the second synthetic reagent reservoir 100b. This process requires precise control of the pressure and flow rate of the pump assembly 300 to ensure smooth reagent reflux while avoiding unnecessary impact on the sequencing primers or nucleic acid templates on the surface of the chip 400. At least a portion of the reacted second synthetic reagent and the unreacted second synthetic reagent are recovered. This objective aims to reduce reagent waste and lower sequencing costs. By recovering the incompletely reacted second synthetic reagent, it can be reused in subsequent sequencing reactions, improving reagent utilization. Recovering the incompletely reacted second synthetic reagent reduces reagent waste, lowers sequencing costs, and simultaneously improves reagent utilization. This not only helps reduce the direct cost of sequencing, but also reduces the generation of chemical waste, making it more environmentally friendly.

[0183] According to an embodiment of this application, the second synthetic reagent reservoir 100b is connected to the valve assembly 200 via a second conduit 700. The reacted second synthetic reagent is recovered into the second conduit 700; and / or, unreacted second synthetic reagent is recovered into the second conduit 700 and / or the second synthetic reagent reservoir 100b. According to an embodiment of this application, the second synthetic reagent reservoir 100b is connected to the valve assembly 200 via a second conduit 700. This connection provides a physical channel for the recovery of the second synthetic reagent. After the completion reaction within the chip 400, the second synthetic reagent may have partially participated in the reaction, and the remaining portion can be recovered and reused. In the common conduit 500, there may be second synthetic reagent that did not participate in the completion reaction; these reagents can also be recovered. According to an embodiment of this application, the reacted second synthetic reagent is recovered into the second conduit 700; and / or, unreacted second synthetic reagent is recovered into the second conduit 700 and / or the second synthetic reagent reservoir 100b. The second line 700 serves as a recovery path, temporarily holding recovered reagents for subsequent processing or reuse. Any unreacted second synthesis reagents can be directly returned to the second synthesis reagent reservoir 100b, mixed with the original reagents, for reuse in subsequent sequencing cycles.

[0184] According to an embodiment of this application, the pressure difference generated by the pump assembly 300 drives the second synthetic reagent to flow along the second conduit 700 toward the second synthetic reagent reservoir 100b. The pump assembly 300, by precisely controlling the pressure and flow rate, ensures that the reagent can be smoothly recovered from the chip 400 and the common conduit 500 into the second conduit 700 or the second synthetic reagent reservoir 100b. The flow direction of the reagent is opposite to the original delivery direction, from the chip 400 and the common conduit 500 toward the second synthetic reagent reservoir 100b. By precisely controlling the pump assembly 300 and the valve assembly 200, automated management of the recovery process can be achieved, ensuring recovery efficiency and accuracy. Therefore, by recovering incompletely reacted second synthetic reagents, reagent utilization can be significantly improved, reducing waste. The recovery and reuse of reagents directly reduces sequencing costs, especially for expensive reagents, where this cost saving is more significant. Reducing reagent waste and environmental pollution aligns with the concept of sustainable development.

[0185] According to an embodiment of this application, the recovered second synthetic reagent is introduced from the second conduit 700 through the valve assembly 200 into the chip 400 to replace the solution in the chip 400 after the extension reaction. According to an embodiment of this application, the second synthetic reagent recovered to the second conduit 700 and the second synthetic reagent reservoir 100b is 50% to 80% of the total amount of the second synthetic reagent used, preferably 60% to 65%. Therefore, according to an embodiment of this application, by recovering the second synthetic reagent that did not participate in the completion reaction, the total consumption of reagents can be effectively reduced, thereby reducing costs. The recovered reagents can be reused, which not only improves reagent utilization efficiency but also reduces the direct waste of reagents that did not participate in the reaction, avoiding resource waste. Furthermore, reducing the waste of chemical reagents is also a form of environmental protection. Through recycling, the amount of reagent pumped can be reduced, thereby reducing pumping time and sequencing time while ensuring sequencing quality. According to an embodiment of this application, after step S10 and before step S20, at least a portion of the second synthetic reagent that did not participate in the completion reaction is returned to the second synthetic reagent reservoir 100b. According to an embodiment of this application, all second synthetic reagents that do not enter the chip 400 are returned to the second synthetic reagent reservoir 100b. Optionally, the amount of second synthetic reagent returned to the second synthetic reagent reservoir 100b is 50% to 80% of the total amount of second synthetic reagents added, preferably 55% to 60%. This proportion ensures that sufficient reagents participate in the reaction while maximizing recycling, achieving an optimal balance between cost and efficiency.

[0186] According to an embodiment of this application, allowing the developing reagent to flow into the chip 400 includes: introducing a first buffer solution into a common conduit 500 to push the developing reagent within the common conduit 500 into the chip 400. Thus, by utilizing the flow of the first buffer solution, the developing reagent can be pushed from the common conduit 500 into the chip 400, ensuring that all the developing reagent enters the chip 400 to excite an optically detectable marker to generate a signal and collect that signal. This process not only improves the efficiency of developing reagent use but also reduces the residue of developing reagent in the conduit, thus reducing reagent waste.

[0187] According to an embodiment of this application, the flow of cleavage reagent into chip 400 includes: introducing a first buffer solution into a common conduit 500 to push the cleavage reagent in the common conduit 500 into chip 400. Thus, by utilizing the flow of the first buffer solution, the cleavage reagent is pushed from the common conduit 500 into chip 400, ensuring that all the cleavage reagent enters chip 400, thereby removing the 3' hydroxyl terminating group and optically detectable label of the synthetic base, preparing for the next round of synthesis reaction. This process not only improves the efficiency of cleavage reagent use but also reduces the residue of cleavage reagent in the conduit, reducing reagent waste.

[0188] According to embodiments of this application, the method further includes: drawing a predetermined volume of air into the chip 400 before or after the first synthetic reagent flows into the chip 400. This isolates the first synthetic reagent from other reagents using an air column, reducing the reduction in effectiveness caused by diffusion of the first synthetic reagent. If the two reagents entering the chip 400 sequentially are not isolated, they are prone to mutual diffusion and interference. Therefore, to ensure sequencing efficiency / quality, the dosage needs to be increased to eliminate the diffusion effect, leading to an increase in reagent usage. However, by isolating the first synthetic reagent from other reagents using an air column, diffusion between the first synthetic reagent and other reagents can be effectively avoided or reduced, achieving the goal of reducing the amount of the first synthetic reagent used while ensuring sequencing efficiency / quality, thus reducing costs.

[0189] According to embodiments of this application, sequencing-by-synthesis is performed at 55–65°C, preferably 60°C. Therefore, by maintaining the operating temperature of sequencing-by-synthesis according to embodiments of this application, consistent temperature control throughout the entire process can be ensured, facilitating operation. Furthermore, operating at temperatures such as 60°C can increase the rate of chemical or biological reactions, thereby ensuring that the involved chemical and biological reactions achieve higher efficiency at this temperature. Simultaneously, choosing 60°C as the reaction temperature avoids thermal damage to samples or reagents that might be caused by higher temperatures, protecting the integrity of the samples and the activity of the reagents.

[0190] In a third aspect, this application proposes a sequencing method employing an air column protocol, as illustrated in the embodiments of this application. Figure 3 As shown, the method includes: S100, allowing a first synthetic reagent to flow into a chip to carry out an extension reaction on the chip, the first synthetic reagent comprising a first nucleotide, the first nucleotide containing a cleavable blocking group and an optically detectable label;

[0191] Specifically, the first nucleotide is one or more deoxyribonucleotides, such as dNTPs. "dNTPs," or deoxyribonucleoside triphosphates, contain a phosphate group, a deoxyribose sugar, and a nitrogenous base (one of A, T, C, and G), typically including dATP, dGTP, dTTP, and dCTP, representing deoxyadenosine triphosphate, deoxyguanosine triphosphate, deoxythymidine triphosphate, and deoxycytidine triphosphate, respectively. After the first synthetic reagent flows into the chip, the first nucleotide in the first synthetic reagent, under the action of DNA polymerase and under conditions suitable for polymerase chain reaction, is ligated to the 3'-OH end of the sequencing primer located on the chip surface and binds to the bases on the nucleic acid template in a base-complementary pairing manner. A cleavable blocking group on the first nucleotide can prevent the polymerization reaction at the reaction site (e.g., 3'-OH) on the sugar of the first nucleotide, thus allowing for a single-base extension reaction. Exemplarily, the DNA polymerase is selected from one or more of rTaq, Canace, Pfu, KOD, Phusion, Primerstar, Tth, BST, and BSU. Suitable conditions for polymerase chain reaction (PCR) include a suitable temperature for PCR. Exemplarily, a suitable temperature for PCR is 55°C-65°C, such as 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, or any two of these values. Preferably, a suitable temperature for PCR is 60°C.

[0192] According to an embodiment of this application, the pH of the first synthetic reagent is greater than 8. Exemplarily, the pH of the first synthetic reagent can be 8, 8.5, 9, 9.5, 10, or any two of these values.

[0193] S200, the developing reagent flows into the chip to excite the optically detectable marker to generate a signal, and the signal is collected;

[0194] After each base extension reaction, an optically detectable label on the first nucleotide is excited by excitation light to generate an optical signal. For example, when the optically detectable label is a fluorescent group, excitation light of a specific wavelength or wavelength range can generate a fluorescent signal. This fluorescent signal is acquired using a microscopic imaging system to form an image. Based on the image, the type of base attached to the 3'-OH end of the sequencing primer and bound to the nucleic acid template can be determined. Introducing a developing reagent into the chip before or during imaging can avoid or reduce damage or influence on the nucleic acid template caused by excitation light during image acquisition.

[0195] According to an embodiment of this application, the pH of the developing agent is approximately 7. Exemplarily, the pH of the developing agent can be 7, 7.1, 7.2, 7.3, 7.4, or any two values ​​therein.

[0196] S300 allows the cutting reagent to flow into the chip to remove the blocking groups and optically detectable markers;

[0197] According to embodiments of this application, in this step, the cleavage reagent is fed into the chip, precisely introduced into the chip to ensure sufficient contact with the first nucleotide. This process requires precise control of the reagent flow rate and velocity to ensure the efficiency and effectiveness of the cleavage reaction. Simultaneously, it is essential to ensure that the cleavage reagent uniformly covers the chip surface to achieve the removal of blocking groups and optically detectable markers on all first nucleotides. Removing the blocking groups and optically detectable markers from the first nucleotides prepares the chip for the next round of extension reactions. The presence of the blocking groups on the first nucleotides prevents them from prematurely polymerizing with other nucleotides during the extension reaction. By removing these blocking groups, the 3'-OH activity of the first nucleotide is restored, allowing it to participate in the extension reaction again. Simultaneously, the removal of the optically detectable markers helps reduce background signals, improving the accuracy and sensitivity of subsequent detections. Achieving this objective is crucial for the continuity and accuracy of the sequencing workflow, ensuring that single-base extension reactions can be effectively performed in each cycle.

[0198] According to embodiments of this application, the cleavage reagent typically contains an enzyme or chemical reagent capable of specifically recognizing and cleaving the blocking group. For example, if the blocking group is based on an azide group, the cleavage reagent may contain a reagent capable of reducing the azide group, such as tris(3-hydroxypropyl)phosphine (THPP). The composition and properties of the cleavage reagent determine its ability to specifically recognize and cleave the blocking group. In this embodiment, the enzyme or chemical reagent in the cleavage reagent can accurately recognize and bind to the blocking group, thereby cleaving it through a chemical reaction. Taking the azide group as an example, THPP can effectively reduce the azide group, destroying its structure, thereby achieving the cleavage of the blocking group. This specific recognition and cleavage mechanism ensures the high efficiency and accuracy of the cleavage reaction, avoiding unnecessary influence on other parts. By cleaving the blocking group and the optically detectable label, the 3'-OH activity of the first nucleotide is restored, enabling it to participate in the elongation reaction again. This step is crucial for achieving the cyclical execution of the single-base elongation reaction.

[0199] According to embodiments of this application, the cleavage reaction not only restores the activity of the first nucleotide but also creates conditions for the next round of extension reactions. After the blocking group is removed, the first nucleotide can participate again as a reactant in the new extension reaction, achieving the sequential addition of single bases. This cyclical process is the core of the sequencing reaction, gradually constructing a complete DNA sequence through continuous extension, detection, and cleavage. Furthermore, removing optically detectable markers helps reduce background signals, improves the signal-to-noise ratio of subsequent detections, and thus enhances the quality of sequencing data.

[0200] S400, return to step S100, and repeat steps S100 to S300 until the sequencing reaction is complete. According to an embodiment of this application, in this operation, the system automatically returns to start a new sequencing cycle. This includes washing the chip again with the first buffer to prepare for the new extension reaction. The entire process is performed automatically according to a preset program without manual intervention, ensuring the efficiency and stability of the sequencing process. By cyclically executing the above steps, complete sequencing of the DNA template is achieved.

[0201] According to embodiments of this application, a single round of sequencing can only determine information for one base. To completely determine the sequence of the DNA template, multiple cycles are needed to gradually construct the entire sequence. Each cycle corresponds to a specific base position. By repeating the above steps, the bases on the DNA strand can be determined one by one, ultimately obtaining complete sequence information.

[0202] According to embodiments of this application, this cyclic design enables the sequencing process to be automated and performed at high throughput until complete DNA sequence information is obtained. Automated cycling significantly improves sequencing throughput and efficiency, reducing errors and time costs associated with manual operation. Each cycle strictly follows the same steps and conditions, ensuring the reliability and reproducibility of the sequencing results. Furthermore, this cyclic design can flexibly adapt to DNA templates of different lengths; whether the template is a short or long sequence, sequencing can be completed through an appropriate number of cycles.

[0203] The methods also include:

[0204] Step S100 involves allowing the first synthetic reagent to flow into the chip before or after it, and then drawing in a preset volume of air into the chip.

[0205] According to embodiments of this application, an air column is used to isolate the first synthetic reagent from other reagents, reducing the reduction in effectiveness of the first synthetic reagent due to diffusion. Two reagents introduced into the chip sequentially, one after the other, are prone to cross-diffusion and mutual interference if not isolated. Therefore, to ensure sequencing efficiency / quality, the dosage needs to be increased to eliminate the diffusion effect, leading to increased reagent usage. Isolating the first synthetic reagent from other reagents with an air column effectively avoids or reduces diffusion between them, achieving the goal of reducing the amount of the first synthetic reagent used while maintaining sequencing efficiency / quality, thus lowering costs. However, air column isolation also has certain drawbacks. First, the surface nucleic acid and primers are bound by hydrogen bonds, which are weak forces. The tension formed by the air column may break these hydrogen bonds, leading to unwinding. Second, introducing an air column involves valve cutting and air pumping, which increases sequencing time. Therefore, using this method to reduce reagent usage requires caution. According to this application, an air column can be introduced before or after valuable reagents, such as before the first synthetic reagent, to further reduce the amount of the first synthetic reagent used.

[0206] According to an embodiment of this application, after step S100 and before step S200, the process further includes: S100-1, allowing a second synthetic reagent to flow into the chip, the second synthetic reagent containing a second nucleotide, the second nucleotide containing a cleavable blocking group but not containing an optically detectable label.

[0207] According to embodiments of this application, a second synthetic reagent is introduced into the chip to ensure sufficient contact with nucleic acids that did not participate in the extension reaction in step S100. This ensures that all nucleic acid templates participate in the extension reaction, thereby improving sequencing accuracy. According to embodiments of this application, some nucleic acid templates may not participate in the extension reaction in step S100, resulting in signal loss for these templates in this round of reaction, affecting the integrity and accuracy of sequencing data. By introducing a second synthetic reagent for a completion reaction, the synchronicity of all nucleic acid template reactions can be ensured without affecting the correct identification of bases in the next round of sequencing. According to embodiments of this application, the second synthetic reagent contains a second nucleotide, which contains a cleavable blocking group but not an optically detectable label. Similar to the first nucleotide, the second nucleotide also contains a cleavable blocking group but not an optically detectable label. This means that the second nucleotide is mainly used to bind to nucleic acid templates that did not participate in the extension reaction in step S100, and does not participate in signal detection. This design ensures efficient completion of the completion reaction while avoiding unnecessary signal interference. By completing the reaction, we ensure that all nucleic acid templates can be accurately determined, enhancing data integrity, reducing sequencing errors caused by asynchrony in the extension reactions of all nucleic acid templates, and improving the quality of sequencing data. At the same time, ensuring that all nucleic acid templates are accurately determined helps to obtain more complete sequence information and improves the overall performance of sequencing.

[0208] According to an embodiment of this application, before step S100, a first buffer solution is introduced into the chip to clean the chip, which is used for sequencing-by-synthesis. According to an embodiment of this application, the pH value of the first buffer solution is greater than 8. According to an embodiment of this application, the pH value of the first buffer solution is 9-10.

[0209] The first buffer solution contains Trisbase (tris(hydroxymethyl)aminomethane), NaCl (sodium chloride), and Tween. For example, Tween-20.

[0210] Trisbase, as a commonly used buffer, can maintain the stability of the solution's pH value, ensuring that the pH value remains within a suitable range throughout the washing process, and avoiding the impact of pH fluctuations on the sequencing primers or nucleic acid templates on the chip surface.

[0211] The addition of NaCl helps to adjust the ionic strength of the solution, promoting the dissolution and removal of residues.

[0212] Tween (e.g., Tween-20), as a nonionic surfactant, can reduce the surface tension of a solution, enhance its permeability and cleaning ability, and help to more effectively remove residues and impurities from the chip surface.

[0213] According to embodiments of this application, the concentration of Trisbase in the first buffer solution is 45-55 mmol / L. Exemplarily, the concentration of Trisbase can be 45 mmol / L, 46 mmol / L, 47 mmol / L, 48 mmol / L, 49 mmol / L, 50 mmol / L, 51 mmol / L, 52 mmol / L, 53 mmol / L, 54 mmol / L, 55 mmol / L, or any two of these values.

[0214] The concentration of NaCl in the first buffer solution is 45-55 mmol / L. For example, the concentration of NaCl can be 45 mmol / L, 46 mmol / L, 47 mmol / L, 48 mmol / L, 49 mmol / L, 50 mmol / L, 51 mmol / L, 52 mmol / L, 53 mmol / L, 54 mmol / L, 55 mmol / L, or any two of these values.

[0215] The volume percentage concentration of Tween (e.g., Tween-20) is 0.01% to 0.1%, for example, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, or a range between any two of these values. For example, the volume percentage concentration of Tween-20 can be 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1%.

[0216] The pH of the first buffer solution is greater than 8. For example, the pH of the first buffer solution can be 8, 8.5, 9, 9.5, 10, or any two of these values.

[0217] The chip surface is immobilized with sequencing primers and nucleic acid templates that bind to the primers. Before the extension reaction, the chip is washed with a first buffer solution. This replaces any remaining reactants or solutions from the previous step on the chip surface, reducing non-specific binding of these substances to the sequencing primers or nucleic acid templates on the chip surface, thereby improving the signal-to-noise ratio and sequencing quality.

[0218] According to an embodiment of this application, before step S300, a first buffer solution is introduced into the chip to clean it. Specifically, before performing step S300, the first buffer solution can effectively isolate the developing reagent and the cutting reagent, preventing the developing reagent from affecting the cutting reagent. For example, mixing the developing reagent and the cutting reagent can cause the pH of the cutting reagent to drop, thereby affecting the reaction efficiency of the cutting reagent. If this step is omitted and step S300 is performed directly, the pH of the cutting reagent will drop after the developing reagent comes into contact with or mixes with the cutting reagent. A possible remedy is to increase the amount of cutting reagent used to ensure cutting efficiency, thereby ensuring sequencing efficiency / quality. However, this would cause unnecessary loss of the cutting reagent. The first buffer solution can isolate the developing reagent and the cutting reagent, and the pH range of the first buffer solution is basically the same as the pH range of the cutting reagent. In particular, when the pH value of the first buffer solution is comparable to the pH value of the cutting reagent, even if the first buffer solution entering the chip first comes into contact with or mixes with the cutting reagent entering the chip later, it will not cause a large fluctuation in the pH of the cutting reagent, and will not affect the reaction efficiency of the cutting reagent. In this way, there is no unnecessary loss of cutting reagent, and there is no need to increase the amount of cutting reagent used to ensure sequencing efficiency / quality, thereby achieving the goal of reducing the amount of cutting reagent used and reducing costs.

[0219] According to embodiments of this application, in step S300, the inflow of the cleavage reagent into the chip includes flowing a first buffer solution into the chip and pushing the cleavage reagent into the chip in one or multiple portions to remove the blocking group and optically detectable label on the first nucleotide. In this step, the cleavage reagent can be pushed into the chip in one or multiple portions using the first buffer solution. However, the inventors have found that, compared to pushing the cleavage reagent into the chip in one go using the first buffer solution, pushing the cleavage reagent into the chip in multiple portions using the first buffer solution increases the number of incubation steps for the cleavage reaction, thereby improving the efficiency of the cleavage reaction. This may be because as the cleavage reaction proceeds, the initially added cleavage reagent is continuously consumed, leading to a decrease in its concentration and efficiency. The subsequently added cleavage reagent can replenish this consumption in a timely manner, thus maintaining a high reaction efficiency. Furthermore, pushing the cleavage reagent into the chip in multiple portions using the first buffer solution can reduce temperature fluctuations in the reaction system, thereby improving reaction efficiency.

[0220] According to an embodiment of this application, after step S300, a second buffer solution is allowed to flow into the chip to clean the chip. This process includes allowing the second buffer solution to flow towards the chip and push the first buffer solution forward, so that the first and second buffer solutions sequentially enter the chip to clean it.

[0221] According to embodiments of this application, in this operation, a first buffer and a second buffer are precisely introduced into the chip to ensure complete coverage of the chip surface and adequate contact with residual reagents and other impurities. The flow rate and velocity of the first and second buffers need to be precisely controlled to ensure effective cleaning while avoiding unnecessary impact on sequencing primers or nucleic acid templates on the chip surface. Uniform flow of the buffers ensures that every area on the chip is effectively cleaned.

[0222] According to embodiments of this application, during the cleavage reaction, some residues may be generated, such as cleaved blocking groups, optically detectable markers, and other possible byproducts. If these residues are not effectively removed, they may affect subsequent sequencing reactions, such as causing nonspecific binding or interfering with signal detection. By sequentially cleaning the chip with a first buffer and a second buffer, the chip surface can be ensured to be clean, providing a good environment for the next sequencing reaction. This step is crucial for the stability and reliability of the entire sequencing process. By effectively removing the residues after the cleavage reaction, interference from these residues in subsequent extension and development reactions can be avoided, improving the accuracy and efficiency of sequencing. At the same time, keeping the chip surface clean helps extend the chip's lifespan, ensuring that it maintains good performance in multiple sequencing cycles.

[0223] According to embodiments of this application, the chip is first cleaned with a first buffer solution, and then further cleaned with a second buffer solution. This effectively removes residual cleavage reagents from the chip, which is crucial for ensuring the accuracy of the sequencing reaction. The cleavage reagents remove the 3' hydroxyl-terminal blocking group and optically detectable markers of the first nucleotide, restoring the activity of the reaction site on the first nucleotide for the next round of sequencing. If the cleavage reagents are not completely removed, it may lead to prephasing, reducing sequencing quality. Furthermore, cleaning the chip with the first buffer solution first, followed by further cleaning with the second buffer solution, isolates the second buffer solution from the cleavage reagents. This prevents unwanted reactions from occurring prematurely after contact or mixing with the cleavage reagents, which could rapidly terminate an ongoing cleavage reaction and reduce cleavage efficiency. Furthermore, cleaning the chip with the first buffer solution first, followed by further cleaning with the second buffer solution, ensures that the cleavage reaction may still be ongoing until the first buffer solution completely replaces the cleavage reagents, thereby improving sequencing efficiency and quality. In addition, the chip is first cleaned with a first buffer solution, and then further cleaned with a second buffer solution. The first buffer solution isolates the second buffer solution from the cleavage reagent, preventing the pH of the cleavage reagent from decreasing due to contact or mixing with the second buffer solution, thus avoiding a drop in cleavage efficiency and impacting sequencing efficiency / quality. According to embodiments of this application, the second buffer solution includes SSC, Tween-20, and cystamine or its salts. Optionally, the final concentration of cystamine or its salts in the second buffer solution is 10–30 mM. According to embodiments of this application, the second buffer typically comprises SSC, Tween-20, and cystamine or a salt thereof, wherein the final concentration of cystamine or a salt thereof is 10–30 mM, for example, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, or any two values ​​between them. For example, the second buffer may comprise 5X SSC, 0.05% Tween-20, and 20 mM of cystamine dihydrochloride.

[0224] SSC is a commonly used buffer component that provides a stable ionic environment, helping to maintain the structure and stability of DNA. It can modulate the ionic strength of the solution, promoting the dissolution and removal of residues.

[0225] Tween-20, as a nonionic surfactant, can reduce the surface tension of a solution, enhance its permeability and cleaning ability, and help to more effectively remove residues and impurities from the chip surface.

[0226] Cystamine is a compound that can bind to DNA, further enhancing the washing effect and removing residual DNA and proteins. In the second buffer, the final concentration of cystamine or its salt is 10–30 mM, which provides sufficient washing power while avoiding damage to sequencing primers or nucleic acid templates on the chip surface.

[0227] Therefore, according to embodiments of this application, by adjusting the reagent formulation, such as increasing the concentration of the active ingredient or changing the pH value, ionic strength, etc., the efficiency of the reagent can be improved, thereby reducing the amount of reagent required. Specifically, according to embodiments of this application, by using a second buffer solution with a higher concentration of cystamine or its salt, the washing efficiency of the second buffer solution can be improved. This means that the second buffer solution can effectively perform its function with a lower usage amount, thereby reducing the usage amount. The second buffer solution can effectively remove the cutting reagents remaining in the chip after the cutting reaction, which is crucial for ensuring the accuracy of the sequencing reaction. The cutting reagents can remove the 3' hydroxyl-terminal blocking group and optically detectable markers of the first nucleotide, restoring the activity of the reaction site on the first nucleotide for the next round of sequencing reaction. If the cutting reagents are not completely removed, it may lead to phase advance, i.e., prephasing, reducing sequencing quality. At the same time, washing the sequencing chip with the second buffer solution after step S30 can also help reduce non-specific binding and background noise during the sequencing process, thereby improving the efficiency / quality of sequencing and preventing cross-reactions or cross-influences between different reagents, ensuring that each reagent can perform its proper function. This is very important for maintaining the independence and accuracy of each step in the sequencing process.

[0228] According to an embodiment of this application, the chip 400 is detachably disposed in a liquid system 1000, the liquid system 1000 including a reservoir 100, a valve assembly 200, and a pump assembly 300; the valve assembly 200 is configured to connect the reservoir 100 and the pump assembly 300, or to connect the reservoir 100 and the chip 400 and the pump assembly 300; the reservoir 100 includes a first synthetic reagent reservoir 100a, the first synthetic reagent reservoir 100a being used to store a first synthetic reagent; the valve assembly 200 and the chip 400 are connected via a common conduit 500, the method including: after step S100 and before step S200, using the pump assembly 300 to drive the first synthetic reagent after the extended reaction in the chip 400 and / or the first synthetic reagent in the common conduit 500 that did not participate in the extended reaction to flow in a direction toward the first synthetic reagent reservoir 100a, so as to recover at least a portion of the first synthetic reagent after the reaction and the first synthetic reagent that did not participate in the reaction.

[0229] According to an embodiment of this application, after step S100 and before step S200, a pump assembly 300 drives the first synthetic reagent that has undergone the extension reaction within the chip 400 and / or the first synthetic reagent that did not participate in the extension reaction within the common conduit 500 to flow towards the first synthetic reagent reservoir 100a, thereby recovering at least a portion of the first synthetic reagent after the reaction and the first synthetic reagent that did not participate in the reaction. In this operation, the pump assembly 300 is precisely controlled to generate appropriate positive or reverse flow, causing the first synthetic reagent within the chip 400 and the common conduit 500 to flow towards the first synthetic reagent reservoir 100a. This process requires precise control of the pressure and flow rate of the pump assembly 300 to ensure smooth reagent reflux while avoiding unnecessary impact on the sequencing primers or nucleic acid templates on the surface of the chip 400. In this way, the incompletely reacted first synthetic reagent can be effectively recovered, reducing reagent waste. According to an embodiment of this application, recovering at least a portion of the first synthetic reagent after the reaction and the first synthetic reagent that did not participate in the reaction can reduce reagent waste and lower sequencing costs. By recovering unreacted reagents from the initial synthesis, they can be reused in subsequent sequencing reactions, improving reagent utilization. This is especially important for expensive reagents, significantly reducing sequencing reagent costs.

[0230] Therefore, by recovering unreacted first-stage synthetic reagents, reagent waste is reduced, sequencing costs are lowered, and reagent utilization is improved. This operation not only helps reduce the direct cost of sequencing but also reduces chemical waste, making it more environmentally friendly. Furthermore, optimizing reagent use simplifies the operational process, improving convenience and accuracy. Through these strategies, the goal of cost reduction and efficiency improvement can be achieved while maintaining or enhancing sequencing quality and efficiency.

[0231] According to an embodiment of this application, the chip 400 is detachably disposed in the liquid circuit system 1000. This design facilitates the replacement and maintenance of the chip 400, and also allows for separate cleaning and processing of the chip 400. The valve assembly 200 includes a rotary valve 200a and a three-way solenoid valve 200b communicatively connected to the rotary valve 200a. The valve assembly 200 connects the reservoir 100 to the pump assembly 300, or connects the reservoir 100 to the chip 400 and the pump assembly 300, using the rotary valve 200a and the three-way solenoid valve 200b.

[0232] According to an embodiment of this application, the liquid circuit system 1000 includes:

[0233] Reservoir 100: Used to store various reagents, such as first synthesis reagent, second synthesis reagent, developing reagent, cutting reagent, first buffer solution, and second buffer solution. The reservoir 100 ensures the orderly storage and supply of reagents.

[0234] Valve assembly 200: Configured to connect reservoir 100 and pump assembly 300, or to connect reservoir 100 and chip 400 and pump assembly 300. The function of valve assembly 200 is to precisely control the flow of reagents, ensuring that the correct reagents are delivered to the designated locations in different sequencing steps.

[0235] Pump Unit 300: Responsible for driving the flow of reagents. It precisely controls the flow rate and volume of reagents according to the needs of the sequencing process, ensuring that the reagents arrive at the designated location on time and in the correct quantity.

[0236] First synthetic reagent reservoir 100a: Specifically used to store the first synthetic reagent, ensuring a stable supply of the first synthetic reagent. The sequencing method includes using a pump assembly 300 to drive the first synthetic reagent in the first synthetic reagent reservoir 100a into the chip 400 via a valve assembly 200 for the extension reaction. This step corresponds to step S100, ensuring that the first synthetic reagent can participate in the extension reaction accurately and in a timely manner.

[0237] According to an embodiment of this application, a pump assembly 300 drives the first synthetic reagent in the first synthetic reagent reservoir 100a to flow into the chip 400 through a valve assembly 200. This ensures that the first synthetic reagent reaches the chip 400 accurately and promptly for the extension reaction. The pump assembly 300 generates sufficient pressure and flow rate according to a preset program and parameters, propelling the first synthetic reagent out of the reservoir. Upon receiving a corresponding control signal, the valve assembly 200 opens the corresponding channel, allowing the first synthetic reagent to smoothly pass through the valve assembly 200 and flow into the chip 400. Driven by the pump assembly 300, the first synthetic reagent passes through the valve assembly 200 and finally flows into the chip 400, ready for the extension reaction. According to an embodiment of this application, by precisely controlling the pump assembly 300 and the valve assembly 200, the first synthetic reagent is ensured to participate in the extension reaction accurately and promptly, improving the efficiency and accuracy of sequencing. Simultaneously, this precise control also reduces reagent waste and lowers sequencing costs.

[0238] According to embodiments of this application, such as Figure 6As shown, the first synthetic reagent reservoir 100a is connected to the valve assembly 200 via a first conduit 600, wherein the reacted first synthetic reagent is recovered into the first conduit 600; and / or, the first synthetic reagent that did not participate in the reaction is recovered into the first conduit 600 and / or the first synthetic reagent reservoir 100a. According to an embodiment of this application, the first synthetic reagent reservoir 100a is connected to the valve assembly 200 via a first conduit 600. This connection provides a physical channel for the recovery of the first synthetic reagent. After the reacted first synthetic reagent undergoes an extended reaction within the chip 400, the first synthetic reagent may have partially participated in the reaction, and the remaining portion can still be recovered and reused. In the common conduit 500, there may be first synthetic reagent that did not participate in the extended reaction, and these reagents can also be recovered. The reacted first synthetic reagent is recovered into the first conduit 600; and / or, the first synthetic reagent that did not participate in the reaction is recovered into the first conduit 600 and / or the first synthetic reagent reservoir 100a. The first conduit 600 serves as a recovery path, temporarily containing recovered reagents for subsequent processing or reuse. Unreacted first synthetic reagents can be directly returned to the first synthetic reagent reservoir 100a, mixed with the original reagents, for reuse in subsequent sequencing cycles. According to embodiments of this application, the pressure difference generated by the pump assembly 300 drives the first synthetic reagents along the first conduit 600 towards the first synthetic reagent reservoir 100a. The pump assembly 300, through precise control of pressure and flow rate, ensures that reagents can be smoothly recovered from the chip 400 and the common conduit 500 to the first conduit 600 or the first synthetic reagent reservoir 100a. The flow direction of the reagents is opposite to the original delivery direction, from the chip 400 and the common conduit 500 towards the first synthetic reagent reservoir 100a. By precisely controlling the pump assembly 300 and the valve assembly 200, automated management of the recovery process can be achieved, ensuring recovery efficiency and accuracy. Therefore, by recovering incompletely reacted first synthetic reagents, reagent utilization can be significantly improved, and waste reduced. The recycling and reuse of reagents directly reduces the cost of sequencing, especially for expensive reagents, where the cost savings are even more significant. According to embodiments of this application, reducing reagent waste and environmental pollution aligns with the principles of sustainable development.

[0239] According to an embodiment of this application, before step S100: the recovered first synthetic reagent after reaction is introduced from the first pipeline 600 through the valve assembly 200 into the chip 400 to clean the chip 400. According to an embodiment of this application, the first synthetic reagent recovered into the first pipeline 600 and the first synthetic reagent reservoir 100a is 50% to 80% of the total amount of the first synthetic reagent used, preferably 60% to 65%.

[0240] According to an embodiment of this application, the recovered first synthetic reagent after reaction is introduced into the chip 400 through the first tubing 600 and valve assembly 200, serving as a first buffer solution to clean the chip 400. In this operation, the recovered first synthetic reagent after reaction is reused and delivered into the chip 400 under the control of the first tubing 600 and valve assembly 200. This process requires precise control of the pump assembly 300 and valve assembly 200 to ensure that the recovered reagent can uniformly cover the surface of the chip 400 for effective cleaning. According to an embodiment of this application, the recovered first synthetic reagent after reaction is used as the first buffer solution to clean the chip 400. This aims to further improve reagent utilization and reduce reagent waste and cost. By reusing the recovered reagent in the cleaning step, not only is the cost reduced, but the environmental impact is also reduced. By using the recovered reagent for cleaning, residues and impurities on the surface of the chip 400 can be effectively removed, providing a clean environment for subsequent sequencing reactions. This approach not only helps reduce the direct cost of sequencing but also reduces the generation of chemical waste, making it more environmentally friendly. Meanwhile, by optimizing the use of reagents, the operation process can be simplified, and the convenience and accuracy of the operation can be improved.

[0241] According to an embodiment of this application, the first synthetic reagent recovered into the first pipeline 600 and the first synthetic reagent reservoir 100a is 50% to 80% of the total amount of the first synthetic reagent used, preferably 60% to 65%.

[0242] Therefore, according to the embodiments of this application, by recovering the first synthetic reagent that did not participate in the extension reaction, the total consumption of reagents can be reduced, thereby lowering costs. The recovered reagents can be reused, improving reagent utilization efficiency. Directly discarding reagents that did not participate in the reaction would result in resource waste, while recycling and reuse can reduce this waste. Simultaneously, reducing the waste of chemical reagents is also a form of environmental protection. Furthermore, recycling can reduce the amount of reagent pumped, thereby reducing pumping time and sequencing time while ensuring sequencing quality. According to the embodiments of this application, the amount of reagent returned to the first synthetic reagent reservoir 100a is controlled at 50% to 80% of the total added amount, preferably 60% to 65%. This ratio ensures sufficient reagent participation in the reaction while maximizing recycling, achieving an optimal balance between cost and efficiency.

[0243] According to an embodiment of this application, the reservoir 100 includes a second synthetic reagent reservoir 100b, which is used to store a second synthetic reagent. The process of allowing the second synthetic reagent to flow into the chip 400 includes: using a pump assembly 300 to drive the second synthetic reagent in the second synthetic reagent reservoir 100b into the chip 400 via a valve assembly 200; and allowing the reacted second synthetic reagent and / or the unreacted second synthetic reagent in the common pipeline 500 to flow towards the second synthetic reagent reservoir 100b to recover the reacted second synthetic reagent and / or the unreacted second synthetic reagent.

[0244] According to an embodiment of this application, a pump assembly 300 drives the second synthetic reagent in the second synthetic reagent reservoir 100b through a valve assembly 200 into the chip 400. In this operation, the pump assembly 300 generates sufficient pressure and flow rate according to a preset program and parameters, pushing the second synthetic reagent out of the reservoir. After receiving a corresponding control signal, the valve assembly 200 opens the corresponding channel, allowing the second synthetic reagent to smoothly pass through the valve assembly 200 and flow into the chip 400. The second synthetic reagent flows into the chip 400 to perform the completion reaction. The purpose of the completion reaction is to ensure that all gaps on the DNA template are filled, thereby improving the read length and accuracy of sequencing. Through this operation, it can be ensured that even sequences that are not fully extended in the extension reaction can be accurately determined, enhancing data integrity. Thus, by precisely controlling the pump assembly 300 and the valve assembly 200, the second synthetic reagent can be ensured to participate in the completion reaction accurately and in a timely manner, improving the efficiency and accuracy of sequencing. At the same time, this precise control also reduces reagent waste and lowers sequencing costs.

[0245] According to embodiments of this application, the reacted second synthetic reagent and / or unreacted second synthetic reagent in the common tubing 500 are flowed towards the second synthetic reagent reservoir 100b to recover the reacted second synthetic reagent and / or unreacted second synthetic reagent. In this operation, the pump assembly 300 is precisely controlled to generate appropriate positive or reverse flow, causing the second synthetic reagent in the chip 400 and the common tubing 500 to flow towards the second synthetic reagent reservoir 100b. This process requires precise control of the pressure and flow rate of the pump assembly 300 to ensure smooth reagent reflux while avoiding unnecessary impact on the sequencing primers or nucleic acid templates on the surface of the chip 400. At least a portion of the reacted second synthetic reagent and the unreacted second synthetic reagent are recovered. This objective aims to reduce reagent waste and lower sequencing costs. By recovering the incompletely reacted second synthetic reagent, it can be reused in subsequent sequencing reactions, improving reagent utilization. Recovering the incompletely reacted second synthetic reagent reduces reagent waste, lowers sequencing costs, and simultaneously improves reagent utilization. This not only helps reduce the direct cost of sequencing, but also reduces the generation of chemical waste, making it more environmentally friendly.

[0246] According to an embodiment of this application, the second synthetic reagent reservoir 100b is connected to the valve assembly 200 via a second conduit 700. The reacted second synthetic reagent is recovered into the second conduit 700; and / or, unreacted second synthetic reagent is recovered into the second conduit 700 and / or the second synthetic reagent reservoir 100b. According to an embodiment of this application, the second synthetic reagent reservoir 100b is connected to the valve assembly 200 via a second conduit 700. This connection provides a physical channel for the recovery of the second synthetic reagent. After the completion reaction within the chip 400, the second synthetic reagent may have partially participated in the reaction, and the remaining portion can be recovered and reused. In the common conduit 500, there may be second synthetic reagent that did not participate in the completion reaction; these reagents can also be recovered. According to an embodiment of this application, the reacted second synthetic reagent is recovered into the second conduit 700; and / or, unreacted second synthetic reagent is recovered into the second conduit 700 and / or the second synthetic reagent reservoir 100b. The second line 700 serves as a recovery path, temporarily holding recovered reagents for subsequent processing or reuse. Any unreacted second synthesis reagents can be directly returned to the second synthesis reagent reservoir 100b, mixed with the original reagents, for reuse in subsequent sequencing cycles.

[0247] According to an embodiment of this application, the pressure difference generated by the pump assembly 300 drives the second synthesis reagent to flow along the second pipeline 700 toward the second synthesis reagent reservoir 100b. The pump assembly 300, by precisely controlling the pressure and flow rate, ensures that the reagent can be smoothly recovered from the chip 400 and the common pipeline 500 into the second pipeline 700 or the reservoir. The flow direction of the reagent is opposite to the original delivery direction, from the chip 400 and the common pipeline 500 toward the second synthesis reagent reservoir 100b. By precisely controlling the pump assembly 300 and the valve assembly 200, automated management of the recovery process can be achieved, ensuring recovery efficiency and accuracy. Therefore, by recovering incompletely reacted second synthesis reagents, reagent utilization can be significantly improved, reducing waste. The recovery and reuse of reagents directly reduces sequencing costs, especially for expensive reagents, where this cost saving is more significant. Reducing reagent waste and environmental pollution aligns with the concept of sustainable development.

[0248] According to an embodiment of this application, the recovered second synthetic reagent after the reaction is introduced from the second conduit 700 through the valve assembly 200 into the chip 400 to replace the solution in the chip 400 after the extension reaction. According to an embodiment of this application, the second synthetic reagent recovered to the second conduit 700 and the second synthetic reagent reservoir 100b is 50% to 80% of the total amount of the second synthetic reagent used, preferably 60% to 65%. Therefore, according to an embodiment of this application, by recovering the second synthetic reagent that did not participate in the completion reaction, the total consumption of reagents can be effectively reduced, thereby reducing costs. The recovered reagents can be reused, which not only improves reagent utilization efficiency but also reduces the direct waste of reagents that did not participate in the reaction, avoiding resource waste. Furthermore, reducing the waste of chemical reagents is also a form of environmental protection. Through recycling, the amount of reagent pumped can be reduced, thereby reducing pumping time and sequencing time while ensuring sequencing quality. According to an embodiment of this application, after step S100 and before step S200, at least a portion of the second synthetic reagent that did not participate in the completion reaction is returned to the second synthetic reagent reservoir 100b. According to an embodiment of this application, all second synthetic reagents that do not enter the chip 400 are returned to the second synthetic reagent reservoir 100b. Optionally, the amount of second synthetic reagent returned to the second synthetic reagent reservoir 100b is 50% to 80% of the total amount of second synthetic reagents added, preferably 55% to 60%. This proportion ensures that sufficient reagents participate in the reaction while maximizing recycling, achieving an optimal balance between cost and efficiency.

[0249] According to an embodiment of this application, allowing the developing reagent to flow into the chip 400 includes: introducing a first buffer solution into a common conduit 500 to push the developing reagent within the common conduit 500 into the chip 400. Thus, by utilizing the flow of the first buffer solution, the developing reagent can be pushed from the common conduit 500 into the chip 400, ensuring that all the developing reagent enters the chip 400 to excite an optically detectable marker to generate a signal and collect that signal. This process not only improves the efficiency of developing reagent use but also reduces the residue of developing reagent in the conduit, thus reducing reagent waste.

[0250] According to an embodiment of this application, the flow of cleavage reagent into chip 400 includes: introducing a first buffer solution into a common conduit 500 to push the cleavage reagent in the common conduit 500 into chip 400. Thus, by utilizing the flow of the first buffer solution, the cleavage reagent is pushed from the common conduit 500 into chip 400, ensuring that all the cleavage reagent enters chip 400, thereby removing the 3' hydroxyl terminating group and optically detectable label of the synthetic base, preparing for the next round of synthesis reaction. This process not only improves the efficiency of cleavage reagent use but also reduces the residue of cleavage reagent in the conduit, reducing reagent waste.

[0251] According to embodiments of this application, the method further includes: drawing a predetermined volume of air into the chip 400 before or after the first synthetic reagent flows into the chip 400. This isolates the first synthetic reagent from other reagents using an air column, reducing the reduction in effectiveness caused by diffusion of the first synthetic reagent. If the two reagents entering the chip 400 sequentially are not isolated, they are prone to mutual diffusion and interference. Therefore, to ensure sequencing efficiency / quality, the dosage needs to be increased to eliminate the diffusion effect, leading to an increase in reagent usage. However, by isolating the first synthetic reagent from other reagents using an air column, diffusion between the first synthetic reagent and other reagents can be effectively avoided or reduced, achieving the goal of reducing the amount of the first synthetic reagent used while ensuring sequencing efficiency / quality, thus reducing costs.

[0252] According to embodiments of this application, sequencing-by-synthesis is performed at 55–65°C, preferably 60°C. Therefore, by maintaining the operating temperature of sequencing-by-synthesis according to embodiments of this application, consistent temperature control throughout the entire process can be ensured, facilitating operation. Furthermore, operating at temperatures such as 60°C can increase the rate of chemical or biological reactions, thereby ensuring that the involved chemical and biological reactions achieve higher efficiency at this temperature. Simultaneously, choosing 60°C as the reaction temperature avoids thermal damage to samples or reagents that might be caused by higher temperatures, protecting the integrity of the samples and the activity of the reagents.

[0253] In a fourth aspect, this application proposes a sequencing method employing a reagent recovery scheme and a reagent volume reduction scheme. According to embodiments of this application, such as... Figure 4 As shown, the method includes:

[0254] S1000, a first synthetic reagent is fed into the chip to carry out an extension reaction on the chip. The first synthetic reagent includes a first nucleotide, which contains a cleavable blocking group and an optically detectable label.

[0255] Specifically, the first nucleotide is one or more deoxyribonucleotides, such as dNTPs. "dNTPs," or deoxyribonucleoside triphosphates, contain a phosphate group, a deoxyribose sugar, and a nitrogenous base (one of A, T, C, and G), typically including dATP, dGTP, dTTP, and dCTP, representing deoxyadenosine triphosphate, deoxyguanosine triphosphate, deoxythymidine triphosphate, and deoxycytidine triphosphate, respectively. After the first synthetic reagent flows into the chip, the first nucleotide in the first synthetic reagent, under the action of DNA polymerase and under conditions suitable for polymerase chain reaction, is ligated to the 3'-OH end of the sequencing primer located on the chip surface and binds to the bases on the nucleic acid template in a base-complementary pairing manner. A cleavable blocking group on the first nucleotide can prevent the polymerization reaction at the reaction site (e.g., 3'-OH) on the sugar of the first nucleotide, thus allowing for a single-base extension reaction. Exemplarily, the DNA polymerase is selected from one or more of rTaq, Canace, Pfu, KOD, Phusion, Primerstar, Tth, BST, and BSU. Suitable conditions for polymerase chain reaction (PCR) include a suitable temperature for PCR. Exemplarily, a suitable temperature for PCR is 55°C-65°C, such as 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, or any two of these values. Preferably, a suitable temperature for PCR is 60°C.

[0256] According to an embodiment of this application, the pH of the first synthetic reagent is greater than 8. Exemplarily, the pH of the first synthetic reagent can be 8, 8.5, 9, 9.5, 10, or any two of these values.

[0257] S2000, the developing reagent flows into the chip to excite the optically detectable marker to generate a signal, and the signal is collected;

[0258] After each base extension reaction, an optically detectable label on the first nucleotide is excited by excitation light to generate an optical signal. For example, when the optically detectable label is a fluorescent group, excitation light of a specific wavelength or wavelength range can generate a fluorescent signal. This fluorescent signal is acquired using a microscopic imaging system to form an image. Based on the image, the type of base attached to the 3'-OH end of the sequencing primer and bound to the nucleic acid template can be determined. Introducing a developing reagent into the chip before or during imaging can avoid or reduce damage or influence on the nucleic acid template caused by excitation light during image acquisition.

[0259] According to an embodiment of this application, the pH of the developing agent is approximately 7. Exemplarily, the pH of the developing agent can be 7, 7.1, 7.2, 7.3, 7.4, or any two values ​​therein.

[0260] S3000 allows cleavage reagent to flow into the chip to remove blocking groups and optically detectable markers;

[0261] According to embodiments of this application, in this step, the cleavage reagent is fed into the chip, precisely introduced into the chip to ensure sufficient contact with the first nucleotide. This process requires precise control of the reagent flow rate and velocity to ensure the efficiency and effectiveness of the cleavage reaction. Simultaneously, it is essential to ensure that the cleavage reagent uniformly covers the chip surface to achieve the removal of blocking groups and optically detectable markers on all first nucleotides. Removing the blocking groups and optically detectable markers from the first nucleotides prepares the chip for the next round of extension reactions. The presence of the blocking groups on the first nucleotides prevents premature polymerization with other nucleotides during the extension reaction. By removing these blocking groups, the 3'-OH activity of the first nucleotide is restored, allowing it to participate in the extension reaction again. Simultaneously, the removal of the optically detectable markers helps reduce background signals, improving the accuracy and sensitivity of subsequent detections. Achieving this objective is crucial for the continuity and accuracy of the sequencing workflow, ensuring that single-base extension reactions can be effectively performed in each cycle.

[0262] According to embodiments of this application, the cleavage reagent typically contains an enzyme or chemical reagent capable of specifically recognizing and cleaving the blocking group. For example, if the blocking group is based on an azide group, the cleavage reagent may contain a reagent capable of reducing the azide group, such as tris(3-hydroxypropyl)phosphine (THPP). The composition and properties of the cleavage reagent determine its ability to specifically recognize and cleave the blocking group. In this embodiment, the enzyme or chemical reagent in the cleavage reagent can accurately recognize and bind to the blocking group, thereby cleaving it through a chemical reaction. Taking the azide group as an example, THPP can effectively reduce the azide group, destroying its structure, thereby achieving the cleavage of the blocking group. This specific recognition and cleavage mechanism ensures the high efficiency and accuracy of the cleavage reaction, avoiding unnecessary influence on other parts. By cleaving the blocking group and the optically detectable label, the 3'-OH activity of the first nucleotide is restored, enabling it to participate in the elongation reaction again. This step is crucial for achieving the cyclical execution of the single-base elongation reaction.

[0263] According to embodiments of this application, the cleavage reaction serves not only to restore the activity of the first nucleotide but also to create conditions for the next round of extension reactions. After the blocking group is removed, the first nucleotide can participate again as a reactant in the new extension reaction, achieving the sequential addition of single bases. This cyclical process is the core of the sequencing reaction, gradually constructing a complete nucleic acid sequence through continuous extension, detection, and cleavage. Furthermore, removing the optically detectable marker helps reduce background signal and improve the signal-to-noise ratio of subsequent detections, thereby enhancing the quality of sequencing data.

[0264] According to embodiments of this application, the pH of the cutting reagent is greater than 8. Exemplarily, the pH of the cutting reagent can be 8, 8.5, 9, 9.5, 10, or any two of these values.

[0265] S4000, return to step S1000, and repeat steps S1000 to S3000 until the sequencing reaction is complete.

[0266] According to embodiments of this application, in this operation, the system automatically returns to restart a new sequencing cycle. This includes washing the chip again with the first buffer to prepare for the new extension reaction. The entire process is performed automatically according to a preset program without manual intervention, ensuring the efficiency and stability of the sequencing process. By repeatedly executing the above steps, complete sequencing of the DNA template is achieved.

[0267] According to embodiments of this application, a single round of sequencing can only determine information for one base. To completely determine the sequence of the DNA template, multiple cycles are needed to gradually construct the entire sequence. Each cycle corresponds to a specific base position. By repeating the above steps, the bases on the DNA strand can be determined one by one, ultimately yielding complete sequence information.

[0268] According to embodiments of this application, this cyclic design enables the sequencing process to be automated and performed at high throughput until complete DNA sequence information is obtained. Automated cycling significantly improves sequencing throughput and efficiency, reducing errors and time costs associated with manual operation. Each cycle strictly follows the same steps and conditions, ensuring the reliability and reproducibility of the sequencing results. Furthermore, this cyclic design can flexibly adapt to DNA templates of different lengths; whether the template is a short or long sequence, sequencing can be completed through an appropriate number of cycles.

[0269] The method further includes: after step S1000 and before step S2000, recovering at least a portion of the first synthetic reagent after the reaction and / or the first synthetic reagent that did not participate in the reaction; and / or, before step S1000, allowing the recovered first synthetic reagent after the reaction to flow into the chip to clean the chip.

[0270] According to embodiments of this application, by recovering the first synthetic reagents that did not participate in the extension reaction, the total consumption of reagents can be reduced, thereby lowering costs. The recovered reagents can be reused, improving reagent utilization efficiency. Directly discarding reagents that did not participate in the reaction would result in resource waste, while recycling and reuse can reduce this waste. Simultaneously, reducing the waste of chemical reagents is also a form of environmental protection. Furthermore, recycling can reduce the amount of reagents pumped, thereby reducing pumping time and sequencing time while ensuring sequencing quality. According to embodiments of this application, the amount of reagent returned to the first synthetic reagent reservoir 100a is controlled at 50% to 80% of the total added amount, preferably 60% to 65%. This ratio ensures sufficient reagents participate in the reaction while maximizing recycling, achieving an optimal balance between cost and efficiency.

[0271] According to embodiments of this application, used reagents can be recovered and reused through a back-pushing method. This mainly applies to reaction reagents; rinsing reagents do not require back-pushing recovery. The reaction process includes three stages: the first stage is the replacement reagent, the second stage is the reaction itself, and the third stage is the main pipeline reagent. The replacement reagent in the first stage is affected by the diffusion of the previous reagent, and its concentration and buffer system are affected, making it unusable. The reaction reagent in the second stage can be used as the replacement reagent for the next round. The reagent in the third stage has not participated in the reaction and its concentration is normal, making it suitable for use as the reaction reagent for the next round. According to embodiments of this application, this scheme is adopted for expensive reagent synthesis reagents 5 and 6. This is mainly because the back-pushing method can effectively reduce the amount of reagents, but certain problems still exist. First, it easily pushes back air bubbles formed in the liquid path, causing bubble accumulation. Second, back-pushing creates positive pressure in the liquid path, requiring control of the back-pushing speed, which increases sequencing time. Third, the positive pressure created by back-pushing increases the risk of liquid trapping, potentially exacerbating reagent residue.

[0272] According to an embodiment of this application, after step S1000 and before step S2000, the method further includes:

[0273] In step S1000-1, a second synthetic reagent is introduced into the chip. This second synthetic reagent contains a second nucleotide, which contains a cleavable blocking group but not an optically detectable label. According to embodiments of this application, the second synthetic reagent is introduced into the chip to ensure sufficient contact with nucleic acids that did not participate in the extension reaction in step S1000, ensuring that all nucleic acid templates participate in the extension reaction, thereby improving sequencing accuracy. According to embodiments of this application, in the extension reaction, some nucleic acid templates may not participate in the extension reaction in step S1000, resulting in signal loss for these templates in this round of reaction, affecting the integrity and accuracy of the sequencing data. By introducing the second synthetic reagent to complete the reaction, the synchronicity of all nucleic acid template reactions can be ensured without affecting the correct identification of bases in the next round of sequencing. According to embodiments of this application, the second synthetic reagent contains a second nucleotide, which contains a cleavable blocking group but not an optically detectable label. Similar to the first nucleotide, the second nucleotide also contains a cleavable blocking group but not an optically detectable label. This means that the second nucleotide is primarily used to bind to nucleic acid templates that did not participate in the extension reaction in step S1000, and does not participate in signal detection. This design ensures the efficient execution of the completion reaction while avoiding unnecessary signal interference. The completion reaction ensures that all nucleic acid templates can be accurately determined, enhancing data integrity, reducing sequencing errors caused by asynchrony in the extension reactions of all nucleic acid templates, and improving the quality of sequencing data. Simultaneously, ensuring the accurate determination of all nucleic acid templates helps obtain more complete sequence information, improving the overall performance of sequencing.

[0274] According to an embodiment of this application, before and after step S3000, a first buffer solution is introduced into the chip to clean it. According to an embodiment of this application, the pH value of the first buffer solution is greater than 8. According to an embodiment of this application, the pH value of the first buffer solution is 9-10.

[0275] The first buffer solution contains Trisbase (tris(hydroxymethyl)aminomethane), NaCl (sodium chloride), and Tween. For example, Tween-20.

[0276] Trisbase, as a commonly used buffer, can maintain the stability of the solution's pH value, ensuring that the pH value remains within a suitable range throughout the cleaning process, and avoiding the impact of pH fluctuations on the sequencing primers or nucleic acid templates on the chip surface.

[0277] The addition of NaCl helps to adjust the ionic strength of the solution, promoting the dissolution and removal of residues.

[0278] Tween (e.g., Tween-20), as a nonionic surfactant, can reduce the surface tension of a solution, enhance its permeability and cleaning ability, and help to more effectively remove residues and impurities from the chip surface.

[0279] According to embodiments of this application, the concentration of Trisbase in the first buffer solution is 45-55 mmol / L. Exemplarily, the concentration of Trisbase can be 45 mmol / L, 46 mmol / L, 47 mmol / L, 48 mmol / L, 49 mmol / L, 50 mmol / L, 51 mmol / L, 52 mmol / L, 53 mmol / L, 54 mmol / L, 55 mmol / L, or any two of these values.

[0280] The concentration of NaCl in the first buffer solution is 45-55 mmol / L. For example, the concentration of NaCl can be 45 mmol / L, 46 mmol / L, 47 mmol / L, 48 mmol / L, 49 mmol / L, 50 mmol / L, 51 mmol / L, 52 mmol / L, 53 mmol / L, 54 mmol / L, 55 mmol / L, or any two of these values.

[0281] The volume percentage concentration of Tween (e.g., Tween-20) is 0.01% to 0.1%, for example, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, or a range between any two of these values. For example, the volume percentage concentration of Tween-20 can be 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1%.

[0282] The pH of the first buffer solution is greater than 8. For example, the pH of the first buffer solution can be 7, 7.5, 8, 8.5, 9, 9.5, 10, or any two of these values.

[0283] The chip surface is immobilized with sequencing primers and nucleic acid templates that bind to the primers. Before the extension reaction, the chip is washed with a first buffer solution. This replaces any remaining reactants or solutions from the previous step on the chip surface, reducing non-specific binding of these substances to the sequencing primers or nucleic acid templates on the chip surface, thereby improving the signal-to-noise ratio and sequencing quality.

[0284] According to an embodiment of this application, before step S3000, a first buffer solution is introduced into the chip to clean it. Specifically, before performing step S3000, the first buffer solution can effectively isolate the developing reagent and the cutting reagent, preventing the developing reagent from affecting the cutting reagent. For example, mixing the developing reagent and the cutting reagent can cause the pH of the cutting reagent to drop, thereby affecting the reaction efficiency of the cutting reagent. If this step is omitted and step S3000 is performed directly, the pH of the cutting reagent will drop after the developing reagent comes into contact with or mixes with the cutting reagent. A possible remedy is to increase the amount of cutting reagent used to ensure cutting efficiency, thereby ensuring sequencing efficiency / quality. However, this would cause unnecessary loss of the cutting reagent. The first buffer solution can isolate the developing reagent and the cutting reagent, and the pH range of the first buffer solution is basically the same as the pH range of the cutting reagent. In particular, when the pH value of the first buffer solution is comparable to the pH value of the cutting reagent, even if the first buffer solution entering the chip first comes into contact with or mixes with the cutting reagent entering the chip later, it will not cause a large fluctuation in the pH of the cutting reagent, and will not affect the reaction efficiency of the cutting reagent. In this way, there is no unnecessary loss of cutting reagent, and there is no need to increase the amount of cutting reagent used to ensure sequencing efficiency / quality, thereby achieving the goal of reducing the amount of cutting reagent used and reducing costs.

[0285] According to embodiments of this application, in step S3000, the inflow of the cleavage reagent into the chip includes flowing a first buffer solution into the chip and pushing the cleavage reagent into the chip in one or multiple portions to remove the blocking group and optically detectable label on the first nucleotide. In this step, the cleavage reagent can be pushed into the chip in one or multiple portions using the first buffer solution. However, the inventors have found that compared to pushing the cleavage reagent into the chip in one go using the first buffer solution, pushing the cleavage reagent into the chip in multiple portions using the first buffer solution increases the number of incubation steps for the cleavage reaction, thereby improving the efficiency of the cleavage reaction. This may be because as the cleavage reaction proceeds, the initially added cleavage reagent is continuously consumed, leading to a decrease in its concentration and efficiency. The subsequently added cleavage reagent can replenish this consumption in a timely manner, thus maintaining a high reaction efficiency. Furthermore, pushing the cleavage reagent into the chip in multiple portions using the first buffer solution can reduce temperature fluctuations in the reaction system, thereby improving reaction efficiency.

[0286] According to an embodiment of this application, after step S3000, a second buffer solution is allowed to flow into the chip to clean the chip. This process includes allowing the second buffer solution to flow toward the chip and push the first buffer solution forward, so that the first and second buffer solutions sequentially enter the chip to clean it.

[0287] According to embodiments of this application, in this operation, a first buffer and a second buffer are precisely introduced into the chip to ensure complete coverage of the chip surface and adequate contact with residual reagents and other impurities. The flow rate and velocity of the first and second buffers need to be precisely controlled to ensure effective cleaning while avoiding unnecessary impact on sequencing primers or nucleic acid templates on the chip surface. Uniform flow of the buffers ensures that every area on the chip is effectively cleaned.

[0288] According to embodiments of this application, during the cleavage reaction, some residues may be generated, such as cleaved blocking groups, optically detectable markers, and other possible byproducts. If these residues are not effectively removed, they may affect subsequent sequencing reactions, such as causing nonspecific binding or interfering with signal detection. By sequentially cleaning the chip with a first buffer and a second buffer, the chip surface can be ensured to be clean, providing a good environment for the next sequencing reaction. This step is crucial for the stability and reliability of the entire sequencing process. By effectively removing the residues after the cleavage reaction, interference from these residues in subsequent extension and development reactions can be avoided, improving the accuracy and efficiency of sequencing. At the same time, keeping the chip surface clean helps extend the chip's lifespan, ensuring that it maintains good performance in multiple sequencing cycles.

[0289] According to embodiments of this application, the chip is first cleaned with a first buffer solution, and then further cleaned with a second buffer solution. This effectively removes residual cleavage reagents from the chip, which is crucial for ensuring the accuracy of the sequencing reaction. The cleavage reagents remove the 3' hydroxyl-terminal blocking group and optically detectable markers of the first nucleotide, restoring the activity of the reaction site on the first nucleotide for the next round of sequencing. If the cleavage reagents are not completely removed, it may lead to prephasing, reducing sequencing quality. Furthermore, cleaning the chip with the first buffer solution first, followed by further cleaning with the second buffer solution, isolates the second buffer solution from the cleavage reagents. This prevents unwanted reactions from occurring prematurely after contact or mixing with the cleavage reagents, which could rapidly terminate an ongoing cleavage reaction and reduce cleavage efficiency. Furthermore, cleaning the chip with the first buffer solution first, followed by further cleaning with the second buffer solution, ensures that the cleavage reaction may still be ongoing until the first buffer solution completely replaces the cleavage reagents, thereby improving sequencing efficiency and quality. In addition, the chip is first cleaned with the first buffer solution and then further cleaned with the second buffer solution. The first buffer solution isolates the second buffer solution from the cleavage reagent, which can also prevent the pH of the cleavage reagent from decreasing after the second buffer solution comes into contact with or mixes with the cleavage reagent, thereby reducing the cleavage efficiency and affecting the sequencing efficiency / quality.

[0290] According to embodiments of this application, the second buffer solution comprises SSC, Tween-20, and cystamine or a salt thereof. Optionally, the final concentration of cystamine or a salt thereof in the second buffer solution is 10–30 mM, for example, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, or any two values ​​between them. According to embodiments of this application, the second buffer solution comprises cystamine dihydrochloride. Optionally, the second buffer solution comprises: 5X SSC; 0.05% Tween-20; and 20 mM of cystamine dihydrochloride. Therefore, according to embodiments of this application, by adjusting the reagent formulation, such as increasing the concentration of the active ingredient or changing the pH value, ionic strength, etc., the efficiency of the reagent can be improved, thereby reducing the amount of reagent required. Specifically, according to embodiments of this application, by using a second buffer solution with a higher concentration of cystamine or its salt, the washing efficiency of the second buffer solution can be improved. This means that the second buffer solution can effectively perform its function with a lower usage amount, thereby reducing the usage amount. The second buffer solution can effectively remove the cutting reagents remaining in the chip after the cutting reaction, which is crucial for ensuring the accuracy of the sequencing reaction. The cutting reagents can remove the 3' hydroxyl-terminal blocking group and optically detectable markers of the first nucleotide, restoring the activity of the reaction site on the first nucleotide for the next round of sequencing reaction. If the cutting reagents are not completely removed, it may lead to phase advance, i.e., prephasing, reducing sequencing quality. At the same time, washing the sequencing chip with the second buffer solution after step S30 can also help reduce non-specific binding and background noise during the sequencing process, thereby improving the efficiency / quality of sequencing and preventing cross-reactions or cross-influences between different reagents, ensuring that each reagent can perform its proper function. This is very important for maintaining the independence and accuracy of each step in the sequencing process.

[0291] According to embodiments of this application, such as Figure 6 As shown, the chip 400 is detachably disposed in the liquid system 1000, which includes a reservoir 100, a valve assembly 200, and a pump assembly 300. The valve assembly 200 is configured to connect the reservoir 100 and the pump assembly 300, or to connect the reservoir 100 with the chip 400 and the pump assembly 300. The reservoir 100 includes a first synthetic reagent reservoir 100a for storing a first synthetic reagent. Optionally, the method includes: using the pump assembly 300 to drive the first synthetic reagent in the first synthetic reagent reservoir 100a through the valve assembly 200 into the chip 400 for an extension reaction, so that in step S1000, the first synthetic reagent flows into the chip 400 to carry out an extension reaction on the chip 400.

[0292] According to an embodiment of this application, the chip 400 is detachably disposed in the liquid circuit system 1000. This design facilitates the replacement and maintenance of the chip 400, and also allows for separate cleaning and processing of the chip 400. The valve assembly 200 includes a rotary valve 200a and a three-way solenoid valve 200b communicatively connected to the rotary valve 200a. The valve assembly 200 connects the reservoir 100 to the pump assembly 300, or connects the reservoir 100 to the chip 400 and the pump assembly 300, using the rotary valve 200a and the three-way solenoid valve 200b.

[0293] According to an embodiment of this application, the liquid system 1000 includes: a reservoir 100 for storing various reagents, such as a first synthetic reagent, a second synthetic reagent, a developing reagent, a cutting reagent, a first buffer solution, and a second buffer solution. The reservoir 100 ensures the orderly storage and supply of reagents.

[0294] Valve assembly 200: Configured to connect reservoir 100 and pump assembly 300, or reservoir 400 and pump assembly 300. The function of valve assembly 200 is to precisely control the flow of reagents, ensuring that the correct reagents are delivered to the designated locations during different sequencing steps.

[0295] Pump Unit 300: Responsible for driving the flow of reagents. It precisely controls the flow rate and volume of reagents according to the needs of the sequencing process, ensuring that the reagents arrive at the designated location on time and in the correct quantity.

[0296] First synthetic reagent reservoir 100a: Specifically used to store the first synthetic reagent, ensuring a stable supply of the first synthetic reagent. The sequencing method includes using a pump assembly 300 to drive the first synthetic reagent in the first synthetic reagent reservoir 100a into the chip 400 via a valve assembly 200 for the extension reaction. This step corresponds to step S1000, ensuring that the first synthetic reagent can participate in the extension reaction accurately and in a timely manner.

[0297] According to an embodiment of this application, a pump assembly 300 drives the first synthetic reagent in the first synthetic reagent reservoir 100a to flow into the chip 400 through a valve assembly 200. This ensures that the first synthetic reagent reaches the chip 400 accurately and promptly for the extension reaction. The pump assembly 300 generates sufficient pressure and flow rate according to a preset program and parameters, propelling the first synthetic reagent out of the reservoir. Upon receiving a corresponding control signal, the valve assembly 200 opens the corresponding channel, allowing the first synthetic reagent to smoothly pass through the valve assembly 200 and flow into the chip 400. Driven by the pump assembly 300, the first synthetic reagent passes through the valve assembly 200 and finally flows into the chip 400, ready for the extension reaction. According to an embodiment of this application, by precisely controlling the pump assembly 300 and the valve assembly 200, the first synthetic reagent is ensured to participate in the extension reaction accurately and promptly, improving the efficiency and accuracy of sequencing. Simultaneously, this precise control also reduces reagent waste and lowers sequencing costs.

[0298] According to an embodiment of this application, the valve assembly 200 and the chip 400 are connected via a common conduit 500. Recovering the first synthetic reagent after the reaction and / or at least a portion of the first synthetic reagent that did not participate in the reaction comprises: using a pump assembly 300 to drive the first synthetic reagent after the extended reaction within the chip 400 and / or the first synthetic reagent that did not participate in the extended reaction within the common conduit 500 to flow towards the first synthetic reagent reservoir 100a, thereby recovering the first synthetic reagent after the reaction and at least a portion of the first synthetic reagent that did not participate in the reaction. According to an embodiment of this application, after step S1000 and before step S2000, the pump assembly 300 drives the first synthetic reagent after the extended reaction within the chip 400 and / or the first synthetic reagent that did not participate in the extended reaction within the common conduit 500 to flow towards the first synthetic reagent reservoir 100a, thereby recovering the first synthetic reagent after the reaction and at least a portion of the first synthetic reagent that did not participate in the reaction. In this operation, pump assembly 300 is precisely controlled to generate appropriate positive or reverse flow, causing the first synthetic reagent within chip 400 and common tubing 500 to flow toward the first synthetic reagent reservoir 100a. This process requires precise control of the pressure and flow rate of pump assembly 300 to ensure smooth reagent reflux while avoiding unnecessary impact on sequencing primers or nucleic acid templates on the surface of chip 400. In this way, incompletely reacted first synthetic reagents can be effectively recovered, reducing reagent waste. According to embodiments of this application, recovering at least a portion of the reacted first synthetic reagents and those not involved in the reaction can reduce reagent waste and lower sequencing costs. By recovering incompletely reacted first synthetic reagents, they can be reused in subsequent sequencing reactions, improving reagent utilization. This is particularly important for expensive reagents, significantly reducing sequencing reagent costs.

[0299] Therefore, by recovering unreacted first-stage synthetic reagents, reagent waste is reduced, sequencing costs are lowered, and reagent utilization is improved. This operation not only helps reduce the direct cost of sequencing but also reduces chemical waste, making it more environmentally friendly. Furthermore, optimizing reagent use simplifies the operational process, improving convenience and accuracy. Through these strategies, the goal of cost reduction and efficiency improvement can be achieved while maintaining or enhancing sequencing quality and efficiency.

[0300] According to an embodiment of this application, the first synthetic reagent reservoir 100a is connected to the valve assembly 200 via a first conduit 600, wherein the reacted first synthetic reagent is recovered into the first conduit 600; and / or, the first synthetic reagent that did not participate in the reaction is recovered into the first conduit 600 and / or the first synthetic reagent reservoir 100a. According to an embodiment of this application, the first synthetic reagent reservoir 100a is connected to the valve assembly 200 via a first conduit 600. This connection provides a physical channel for the recovery of the first synthetic reagent. After the reacted first synthetic reagent undergoes an extended reaction within the chip 400, the first synthetic reagent may have partially participated in the reaction, and the remaining portion can still be recovered and reused. In the common conduit 500, there may be first synthetic reagent that did not participate in the extended reaction, and these reagents can also be recovered. The reacted first synthetic reagent is recovered into the first conduit 600; and / or, the first synthetic reagent that did not participate in the reaction is recovered into the first conduit 600 and / or the first synthetic reagent reservoir 100a. The first tubing 600 serves as a recovery path, temporarily holding recovered reagents for subsequent processing or reuse. Some of the first synthesis reagents that did not participate in the reaction can be directly returned to the first synthesis reagent reservoir 100a, mixed with the original reagents, for reuse in subsequent sequencing cycles.

[0301] According to an embodiment of this application, the pressure difference generated by the pump assembly 300 drives the first synthetic reagent to flow along the first conduit 600 toward the first synthetic reagent reservoir 100a. The pump assembly 300, by precisely controlling the pressure and flow rate, ensures that the reagent can be smoothly recovered from the chip 400 and the common conduit 500 into the first conduit 600 or the first synthetic reagent reservoir 100a. The flow direction of the reagent is opposite to the original delivery direction, from the chip 400 and the common conduit 500 toward the first synthetic reagent reservoir 100a. By precisely controlling the pump assembly 300 and the valve assembly 200, automated management of the recovery process can be achieved, ensuring recovery efficiency and accuracy. Therefore, by recovering the incompletely reacted first synthetic reagent, the utilization rate of the reagent can be significantly improved, and waste can be reduced. The recovery and reuse of reagents directly reduces the cost of sequencing, especially for expensive reagents, where this cost saving is more significant. According to an embodiment of this application, reducing reagent waste and environmental pollution aligns with the concept of sustainable development.

[0302] According to an embodiment of this application, before step S1000: the recovered first synthetic reagent after reaction is introduced from the first pipeline 600 through the valve assembly 200 into the chip 400 to clean the chip 400. According to an embodiment of this application, the first synthetic reagent recovered into the first pipeline 600 and the first synthetic reagent reservoir 100a is 50% to 80% of the total amount of the first synthetic reagent used, preferably 60% to 65%.

[0303] According to an embodiment of this application, the recovered first synthetic reagent after reaction is introduced into the chip 400 through the first tubing 600 and valve assembly 200, serving as a first buffer solution to clean the chip 400. In this operation, the recovered first synthetic reagent after reaction is reused and delivered into the chip 400 under the control of the first tubing 600 and valve assembly 200. This process requires precise control of the pump assembly 300 and valve assembly 200 to ensure that the recovered reagent can uniformly cover the surface of the chip 400 for effective cleaning. According to an embodiment of this application, the recovered first synthetic reagent after reaction is used as the first buffer solution to clean the chip 400. This aims to further improve reagent utilization and reduce reagent waste and cost. By reusing the recovered reagent in the cleaning step, not only is the cost reduced, but the environmental impact is also reduced. By using the recovered reagent for cleaning, residues and impurities on the surface of the chip 400 can be effectively removed, providing a clean environment for subsequent sequencing reactions. This approach not only helps reduce the direct cost of sequencing but also reduces the generation of chemical waste, making it more environmentally friendly. Meanwhile, by optimizing the use of reagents, the operation process can be simplified, and the convenience and accuracy of the operation can be improved.

[0304] According to an embodiment of this application, the first synthetic reagent recovered into the first pipeline 600 and the first synthetic reagent reservoir 100a is 50% to 80% of the total amount of the first synthetic reagent used, preferably 60% to 65%.

[0305] Therefore, according to the embodiments of this application, by recovering the first synthetic reagent that did not participate in the extension reaction, the total consumption of reagents can be reduced, thereby lowering costs. The recovered reagents can be reused, improving reagent utilization efficiency. Directly discarding reagents that did not participate in the reaction would result in resource waste, while recycling and reuse can reduce this waste. Simultaneously, reducing the waste of chemical reagents is also a form of environmental protection. Furthermore, recycling can reduce the amount of reagent pumped, thereby reducing pumping time and sequencing time while ensuring sequencing quality. According to the embodiments of this application, the amount of reagent returned to the first synthetic reagent reservoir 100a is controlled at 50% to 80% of the total added amount, preferably 60% to 65%. This ratio ensures sufficient reagent participation in the reaction while maximizing recycling, achieving an optimal balance between cost and efficiency.

[0306] According to an embodiment of this application, the second synthetic reagent reservoir 100b is connected to the valve assembly 200 via a second conduit 700. The reacted second synthetic reagent is recovered into the second conduit 700; and / or, unreacted second synthetic reagent is recovered into the second conduit 700 and / or the second synthetic reagent reservoir 100b. According to an embodiment of this application, the second synthetic reagent reservoir 100b is connected to the valve assembly 200 via a second conduit 700. This connection provides a physical channel for the recovery of the second synthetic reagent. After the completion reaction within the chip 400, the second synthetic reagent may have partially participated in the reaction, and the remaining portion can be recovered and reused. In the common conduit 500, there may be second synthetic reagent that did not participate in the completion reaction; these reagents can also be recovered. According to an embodiment of this application, the reacted second synthetic reagent is recovered into the second conduit 700; and / or, unreacted second synthetic reagent is recovered into the second conduit 700 and / or the second synthetic reagent reservoir 100b. The second line 700 serves as a recovery path, temporarily holding recovered reagents for subsequent processing or reuse. Any unreacted second synthesis reagents can be directly returned to the second synthesis reagent reservoir 100b, mixed with the original reagents, for reuse in subsequent sequencing cycles.

[0307] According to an embodiment of this application, the pressure difference generated by the pump assembly 300 drives the second synthesis reagent to flow along the second pipeline 700 toward the second synthesis reagent reservoir 100b. The pump assembly 300, by precisely controlling the pressure and flow rate, ensures that the reagent can be smoothly recovered from the chip 400 and the common pipeline 500 into the second pipeline 700 or the reservoir. The flow direction of the reagent is opposite to the original delivery direction, from the chip 400 and the common pipeline 500 toward the second synthesis reagent reservoir 100b. By precisely controlling the pump assembly 300 and the valve assembly 200, automated management of the recovery process can be achieved, ensuring recovery efficiency and accuracy. Therefore, by recovering incompletely reacted second synthesis reagents, reagent utilization can be significantly improved, reducing waste. The recovery and reuse of reagents directly reduces sequencing costs, especially for expensive reagents, where this cost saving is more significant. Reducing reagent waste and environmental pollution aligns with the concept of sustainable development.

[0308] According to an embodiment of this application, the recovered second synthetic reagent after the reaction is introduced from the second conduit 700 through the valve assembly 200 into the chip 400 to replace the solution in the chip 400 after the extension reaction. According to an embodiment of this application, the second synthetic reagent recovered to the second conduit 700 and the second synthetic reagent reservoir 100b is 50% to 80% of the total amount of the second synthetic reagent used, preferably 60% to 65%. Therefore, according to an embodiment of this application, by recovering the second synthetic reagent that did not participate in the completion reaction, the total consumption of reagents can be effectively reduced, thereby reducing costs. The recovered reagents can be reused, which not only improves reagent utilization efficiency but also reduces the direct waste of reagents that did not participate in the reaction, avoiding resource waste. Furthermore, reducing the waste of chemical reagents is also a form of environmental protection. Through recycling, the amount of reagent pumped can be reduced, thereby reducing pumping time and sequencing time while ensuring sequencing quality. According to an embodiment of this application, after step S1000 and before step S2000, at least a portion of the second synthetic reagent that did not participate in the completion reaction is returned to the second synthetic reagent reservoir 100b. According to an embodiment of this application, all second synthetic reagents that do not enter the chip 400 are returned to the second synthetic reagent reservoir 100b. Optionally, the amount of second synthetic reagent returned to the second synthetic reagent reservoir 100b is 50% to 80% of the total amount of second synthetic reagents added, preferably 55% to 60%. This proportion ensures that sufficient reagents participate in the reaction while maximizing recycling, achieving an optimal balance between cost and efficiency.

[0309] According to an embodiment of this application, the flow of developing reagent into chip 400 includes: introducing a first buffer solution into a common conduit 500 to push the developing reagent within the common conduit 500 into chip 400. Thus, by utilizing the flow of the first buffer solution, the developing reagent can be pushed from the common conduit 500 into chip 400, ensuring that the developing reagent accurately and promptly reaches the reaction area on chip 400, thereby exciting an optically detectable marker to generate a signal and collecting that signal. This process not only improves the efficiency of developing reagent use but also reduces the residue of developing reagent in the conduit, thus reducing reagent waste.

[0310] According to an embodiment of this application, the cleavage reagent is fed into the chip 400 by: introducing a first buffer solution into a common conduit 500 to push the cleavage reagent in the common conduit 500 into the chip 400. Thus, by utilizing the flow of the first buffer solution to push the cleavage reagent from the common conduit 500 into the chip 400, it is ensured that the cleavage reagent can accurately and timely reach the reaction region on the chip 400, thereby cleaving the 3' hydroxyl terminating group of the synthetic base and the fluorescence signal, preparing for the next round of synthesis reaction. This process not only improves the efficiency of cleavage reagent use but also reduces the residue of cleavage reagent in the conduit, reducing reagent waste.

[0311] According to embodiments of this application, the method further includes: drawing a predetermined volume of air into the chip 400 before or after the first synthetic reagent flows into the chip 400. This isolates the first synthetic reagent from other reagents using an air column, reducing the reduction in effectiveness caused by diffusion of the first synthetic reagent. If the two reagents entering the chip 400 sequentially are not isolated, they are prone to mutual diffusion and interference. Therefore, to ensure sequencing efficiency / quality, the dosage needs to be increased to eliminate the diffusion effect, leading to an increase in reagent usage. However, by isolating the first synthetic reagent from other reagents using an air column, diffusion between the first synthetic reagent and other reagents can be effectively avoided or reduced, achieving the goal of reducing the amount of the first synthetic reagent used while ensuring sequencing efficiency / quality, thus reducing costs.

[0312] According to embodiments of this application, sequencing-by-synthesis is performed at 55–65°C, preferably 60°C. Therefore, by maintaining the operating temperature of sequencing-by-synthesis according to embodiments of this application, consistent temperature control throughout the entire process can be ensured, facilitating operation. Furthermore, operating at temperatures such as 60°C can increase the rate of chemical or biological reactions, thereby ensuring that the involved chemical and biological reactions achieve higher efficiency at this temperature. Simultaneously, choosing 60°C as the reaction temperature avoids thermal damage to samples or reagents that might be caused by higher temperatures, protecting the integrity of the samples and the activity of the reagents.

[0313] In a fifth aspect, this application proposes a sequencing method employing a reagent pre-application scheme. According to embodiments of this application, such as... Figure 5 As shown, the method includes:

[0314] S10000, a first synthetic reagent is fed into the chip to carry out an extension reaction on the chip. The first synthetic reagent includes a first nucleotide, which contains a cleavable blocking group and an optically detectable label.

[0315] Specifically, the first nucleotide is one or more deoxyribonucleotides, such as dNTPs. "dNTPs," or deoxyribonucleoside triphosphates, contain a phosphate group, a deoxyribose sugar, and a nitrogenous base (one of A, T, C, and G), typically including dATP, dGTP, dTTP, and dCTP, representing deoxyadenosine triphosphate, deoxyguanosine triphosphate, deoxythymidine triphosphate, and deoxycytidine triphosphate, respectively. After the first synthetic reagent flows into the chip, the first nucleotide in the first synthetic reagent, under the action of DNA polymerase and under conditions suitable for polymerase chain reaction, is ligated to the 3'-OH end of the sequencing primer located on the chip surface and binds to the bases on the nucleic acid template in a base-complementary pairing manner. A cleavable blocking group on the first nucleotide can prevent the polymerization reaction at the reaction site (e.g., 3'-OH) on the sugar of the first nucleotide, thus allowing for a single-base extension reaction. Exemplarily, the DNA polymerase is selected from one or more of rTaq, Canace, Pfu, KOD, Phusion, Primerstar, Tth, BST, and BSU. Suitable conditions for polymerase chain reaction (PCR) include a suitable temperature for PCR. Exemplarily, a suitable temperature for PCR is 55°C-65°C, such as 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, or any two of these values. Preferably, a suitable temperature for PCR is 60°C.

[0316] According to an embodiment of this application, the pH of the first synthetic reagent is greater than 8. Exemplarily, the pH of the first synthetic reagent can be 8, 8.5, 9, 9.5, 10, or any two of these values.

[0317] S20000, allows the developing reagent to flow into the chip in order to excite the optically detectable marker to generate a signal and collect the signal;

[0318] After each base extension reaction, an optically detectable label on the first nucleotide is excited by excitation light to generate an optical signal. For example, when the optically detectable label is a fluorescent group, excitation light of a specific wavelength or wavelength range can generate a fluorescent signal. This fluorescent signal is acquired using a microscopic imaging system to form an image. Based on the image, the type of base attached to the 3'-OH end of the sequencing primer and bound to the nucleic acid template can be determined. Introducing a developing reagent into the chip before or during imaging can avoid or reduce damage or influence on the nucleic acid template caused by excitation light during image acquisition.

[0319] According to an embodiment of this application, the pH of the developing agent is approximately 7. Exemplarily, the pH of the developing agent can be 7, 7.1, 7.2, 7.3, 7.4, or any two values ​​therein.

[0320] S30000 allows the cutting reagent to flow into the chip to remove blocking groups and optically detectable markers;

[0321] According to embodiments of this application, in this step, the cleavage reagent is fed into the chip, precisely introduced into the chip to ensure sufficient contact with the first nucleotide. This process requires precise control of the reagent flow rate and velocity to ensure the efficiency and effectiveness of the cleavage reaction. Simultaneously, it is essential to ensure that the cleavage reagent uniformly covers the chip surface to achieve the removal of blocking groups and optically detectable markers on all first nucleotides. Removing the blocking groups and optically detectable markers from the first nucleotides prepares the chip for the next round of extension reactions. The presence of the blocking groups on the first nucleotides prevents them from prematurely polymerizing with other nucleotides during the extension reaction. By removing these blocking groups, the 3'-OH activity of the first nucleotide is restored, allowing it to participate in the extension reaction again. Simultaneously, the removal of the optically detectable markers helps reduce background signals, improving the accuracy and sensitivity of subsequent detections. Achieving this objective is crucial for the continuity and accuracy of the sequencing workflow, ensuring that single-base extension reactions can be effectively performed in each cycle.

[0322] According to embodiments of this application, the cleavage reagent typically contains an enzyme or chemical reagent capable of specifically recognizing and cleaving the blocking group. For example, if the blocking group is based on an azide group, the cleavage reagent may contain a reagent capable of reducing the azide group, such as tris(3-hydroxypropyl)phosphine (THPP). The composition and properties of the cleavage reagent determine its ability to specifically recognize and cleave the blocking group. In this embodiment, the enzyme or chemical reagent in the cleavage reagent can accurately recognize and bind to the blocking group, thereby cleaving it through a chemical reaction. Taking the azide group as an example, THPP can effectively reduce the azide group, destroying its structure, thereby achieving the cleavage of the blocking group. This specific recognition and cleavage mechanism ensures the high efficiency and accuracy of the cleavage reaction, avoiding unnecessary influence on other parts. By cleaving the blocking group and the optically detectable label, the 3'-OH activity of the first nucleotide is restored, enabling it to participate in the elongation reaction again. This step is crucial for achieving the cyclical execution of the single-base elongation reaction.

[0323] According to embodiments of this application, the cleavage reaction not only restores the activity of the first nucleotide but also creates conditions for the next round of extension reactions. After the blocking group is removed, the first nucleotide can participate again as a reactant in the new extension reaction, achieving the sequential addition of single bases. This cyclical process is the core of the sequencing reaction, gradually constructing a complete DNA sequence through continuous extension, detection, and cleavage. Furthermore, removing optically detectable markers helps reduce background signals, improves the signal-to-noise ratio of subsequent detections, and thus enhances the quality of sequencing data.

[0324] S40000, return to step S10000, and repeat steps S10000 to S30000 until the sequencing reaction is complete.

[0325] According to embodiments of this application, in this operation, the system automatically returns to restart a new sequencing cycle. This includes washing the chip again with the first buffer to prepare for the new extension reaction. The entire process is performed automatically according to a preset program without manual intervention, ensuring the efficiency and stability of the sequencing process. By repeatedly executing the above steps, complete sequencing of the DNA template is achieved.

[0326] According to embodiments of this application, a single round of sequencing can only determine information for one base. To completely determine the sequence of the DNA template, multiple cycles are needed to gradually construct the entire sequence. Each cycle corresponds to a specific base position. By repeating the above steps, the bases on the DNA strand can be determined one by one, ultimately obtaining complete sequence information.

[0327] According to embodiments of this application, this cyclic design enables the sequencing process to be automated and performed at high throughput until complete DNA sequence information is obtained. Automated cycling significantly improves sequencing throughput and efficiency, reducing errors and time costs associated with manual operation. Each cycle strictly follows the same steps and conditions, ensuring the reliability and reproducibility of the sequencing results. Furthermore, this cyclic design can flexibly adapt to DNA templates of different lengths; whether the template is a short or long sequence, sequencing can be completed through an appropriate number of cycles.

[0328] Wherein, step S20000, which allows the developing reagent to flow into the chip, includes: pushing the developing reagent into the chip using a first buffer solution; and / or, step S30000, which allows the cutting reagent to flow into the chip, includes:

[0329] The cleavage reagent is pushed into the chip using the first buffer solution.

[0330] According to an embodiment of this application, allowing the developing reagent to flow into the chip includes: introducing a first buffer solution into a common conduit 500 to push the developing reagent within the common conduit 500 into the chip. Thus, by utilizing the flow of the first buffer solution, the developing reagent can be pushed from the common conduit 500 into the chip, ensuring that all the developing reagent enters the chip to excite an optically detectable marker to generate a signal and to collect that signal. This process not only improves the efficiency of developing reagent use but also reduces the residue of developing reagent in the conduit, thus reducing reagent waste.

[0331] According to embodiments of this application, the cleavage reagent is fed into the chip by: introducing a first buffer solution into a common conduit 500 to push the cleavage reagent within the common conduit 500 into the chip. Thus, by utilizing the flow of the first buffer solution to push the cleavage reagent from the common conduit 500 into the chip, it is ensured that all the cleavage reagent enters the chip, thereby removing the 3' hydroxyl terminating group and optically detectable marker of the synthetic base, preparing for the next round of synthesis reaction. This process not only improves the efficiency of cleavage reagent utilization but also reduces the residue of cleavage reagent in the conduit, reducing reagent waste.

[0332] According to embodiments of this application, a portion of the previous reagent in the common conduit 500 can be pre-push into the chip using the next reagent, thereby improving reagent utilization. According to embodiments of this application, reagent pre-push is mainly for relatively inexpensive reagents, such as developing and cutting reagents. This method primarily involves using other reagents to push the reagents in the common conduit 500 into the chip, reducing reagent loss in the main conduit and improving reagent utilization.

[0333] Therefore, according to the embodiments of this application, the raw material cost of reagents can be directly reduced by decreasing the amount of reagents used. Pumping time can be reduced because each round of base synthesis involves pumping reagents into the chip reaction cell; reducing reagent usage effectively reduces pumping time, thereby reducing the total sequencing time. Chemical waste generation can be reduced, resulting in a smaller environmental impact. Optimizing reagent use simplifies the operation process and improves operational convenience and accuracy. Through these strategies, the goal of cost reduction and efficiency improvement can be achieved while maintaining or improving sequencing quality and efficiency.

[0334] According to an embodiment of this application, after step S10000 and before step S20000, the method further includes:

[0335] In step S10000-1, a second synthetic reagent is introduced into the chip. This second synthetic reagent contains a second nucleotide, which contains a cleavable blocking group but not an optically detectable label. According to embodiments of this application, the second synthetic reagent is introduced into the chip to ensure sufficient contact with nucleic acids that did not participate in the extension reaction in step S10000. This ensures that all nucleic acid templates participate in the extension reaction, thereby improving sequencing accuracy. According to embodiments of this application, some nucleic acid templates may not participate in the extension reaction in step S10000, resulting in signal loss for these templates in this round of reaction, affecting the integrity and accuracy of the sequencing data. By introducing the second synthetic reagent to complete the reaction, the synchronicity of all nucleic acid template reactions can be ensured without affecting the correct identification of bases in the next round of sequencing. According to embodiments of this application, the second synthetic reagent contains a second nucleotide, which contains a cleavable blocking group but not an optically detectable label. Similar to the first nucleotide, the second nucleotide also contains a cleavable blocking group but not an optically detectable label. This means that the second nucleotide is primarily used to bind to nucleic acid templates that did not participate in the extension reaction in step S10000, and does not participate in signal detection. This design ensures the efficient execution of the completion reaction while avoiding unnecessary signal interference. By completing the reaction, all nucleic acid templates are accurately determined, enhancing data integrity, reducing sequencing errors caused by asynchrony in the extension reactions of all nucleic acid templates, and improving the quality of sequencing data. Simultaneously, ensuring the accurate determination of all nucleic acid templates helps obtain more complete sequence information, improving the overall performance of sequencing.

[0336] According to an embodiment of this application, before step S10000, a first buffer solution is introduced into the chip to clean it. The chip is used for sequencing-by-synthesis. According to an embodiment of this application, the pH value of the first buffer solution is greater than 8. According to an embodiment of this application, the pH value of the first buffer solution is 9-10.

[0337] The first buffer solution contains Trisbase (tris(hydroxymethyl)aminomethane), NaCl (sodium chloride), and Tween. For example, Tween-20.

[0338] Trisbase, as a commonly used buffer, can maintain the stability of the solution's pH value, ensuring that the pH value remains within a suitable range throughout the washing process, and avoiding the impact of pH fluctuations on the sequencing primers or nucleic acid templates on the chip surface.

[0339] The addition of NaCl helps to adjust the ionic strength of the solution, promoting the dissolution and removal of residues.

[0340] Tween (e.g., Tween-20), as a nonionic surfactant, can reduce the surface tension of a solution, enhance its permeability and cleaning ability, and help to more effectively remove residues and impurities from the chip surface.

[0341] According to embodiments of this application, the concentration of Trisbase in the first buffer solution is 45-55 mmol / L. Exemplarily, the concentration of Trisbase can be 45 mmol / L, 46 mmol / L, 47 mmol / L, 48 mmol / L, 49 mmol / L, 50 mmol / L, 51 mmol / L, 52 mmol / L, 53 mmol / L, 54 mmol / L, 55 mmol / L, or any two of these values.

[0342] The concentration of NaCl in the first buffer solution is 45-55 mmol / L. For example, the concentration of NaCl can be 45 mmol / L, 46 mmol / L, 47 mmol / L, 48 mmol / L, 49 mmol / L, 50 mmol / L, 51 mmol / L, 52 mmol / L, 53 mmol / L, 54 mmol / L, 55 mmol / L, or any two of these values.

[0343] The volume percentage concentration of Tween (e.g., Tween-20) is 0.01% to 0.1%, for example, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, or a range between any two of these values. For example, the volume percentage concentration of Tween-20 can be 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1%.

[0344] The pH of the first buffer solution is greater than 8. For example, the pH of the first buffer solution can be 8, 8.5, 9, 9.5, 10, or any two of these values.

[0345] The chip surface is immobilized with sequencing primers and nucleic acid templates that bind to the primers. Before the extension reaction, the chip is washed with a first buffer solution. This replaces any remaining reactants or solutions from the previous step on the chip surface, reducing non-specific binding of these substances to the sequencing primers or nucleic acid templates on the chip surface, thereby improving the signal-to-noise ratio and sequencing quality.

[0346] According to an embodiment of this application, before step S30000, a first buffer solution is introduced into the chip to clean it. Specifically, before performing step S30000, the first buffer solution can effectively isolate the developing reagent and the cutting reagent, preventing the developing reagent from affecting the cutting reagent. For example, mixing the developing reagent and the cutting reagent can cause the pH of the cutting reagent to drop, thereby affecting the reaction efficiency of the cutting reagent. If this step is omitted and step S30000 is performed directly, the pH of the cutting reagent will drop after the developing reagent comes into contact with or mixes with the cutting reagent. A possible remedy is to increase the amount of cutting reagent used to ensure cutting efficiency, thereby ensuring sequencing efficiency / quality. However, this would cause unnecessary loss of the cutting reagent. The first buffer solution can isolate the developing reagent and the cutting reagent, and the pH range of the first buffer solution is basically the same as the pH range of the cutting reagent. In particular, when the pH value of the first buffer solution is comparable to the pH value of the cutting reagent, even if the first buffer solution entering the chip first comes into contact with or mixes with the cutting reagent entering the chip later, it will not cause a large fluctuation in the pH of the cutting reagent, and will not affect the reaction efficiency of the cutting reagent. In this way, there is no unnecessary loss of cutting reagent, and there is no need to increase the amount of cutting reagent used to ensure sequencing efficiency / quality, thereby achieving the goal of reducing the amount of cutting reagent used and reducing costs.

[0347] According to embodiments of this application, in step S30000, the inflow of the cleavage reagent into the chip includes flowing a first buffer solution into the chip and pushing the cleavage reagent into the chip in one or multiple portions to remove the blocking group and optically detectable label on the first nucleotide. In this step, the first buffer solution can be used to push the cleavage reagent into the chip in one or multiple portions. However, the inventors have found that compared to pushing the cleavage reagent into the chip in one go using the first buffer solution, pushing the cleavage reagent into the chip in multiple portions using the first buffer solution increases the number of incubation steps for the cleavage reaction, thereby improving the efficiency of the cleavage reaction. This may be because as the cleavage reaction proceeds, the initially added cleavage reagent is continuously consumed, leading to a decrease in its concentration and efficiency. The subsequently added cleavage reagent can replenish this consumption in a timely manner, thus maintaining a high reaction efficiency. Furthermore, pushing the cleavage reagent into the chip in multiple portions using the first buffer solution can reduce temperature fluctuations in the reaction system, thereby improving reaction efficiency.

[0348] According to an embodiment of this application, after step S30000, a second buffer solution is allowed to flow into the chip to clean the chip. This process includes allowing the second buffer solution to flow toward the chip and push the first buffer solution forward, so that the first and second buffer solutions sequentially enter the chip to clean it.

[0349] According to embodiments of this application, in this operation, a first buffer and a second buffer are precisely introduced into the chip to ensure complete coverage of the chip surface and adequate contact with residual reagents and other impurities. The flow rate and velocity of the first and second buffers need to be precisely controlled to ensure effective cleaning while avoiding unnecessary impact on sequencing primers or nucleic acid templates on the chip surface. Uniform flow of the buffers ensures that every area on the chip is effectively cleaned.

[0350] According to embodiments of this application, during the cleavage reaction, some residues may be generated, such as cleaved blocking groups, optically detectable markers, and other possible byproducts. If these residues are not effectively removed, they may affect subsequent sequencing reactions, such as causing nonspecific binding or interfering with signal detection. By sequentially cleaning the chip with a first buffer and a second buffer, the chip surface can be ensured to be clean, providing a good environment for the next sequencing reaction. This step is crucial for the stability and reliability of the entire sequencing process. By effectively removing the residues after the cleavage reaction, interference from these residues in subsequent extension and development reactions can be avoided, improving the accuracy and efficiency of sequencing. At the same time, keeping the chip surface clean helps extend the chip's lifespan, ensuring that it maintains good performance in multiple sequencing cycles.

[0351] According to embodiments of this application, the chip is first cleaned with a first buffer solution, and then further cleaned with a second buffer solution. This effectively removes residual cleavage reagents from the chip, which is crucial for ensuring the accuracy of the sequencing reaction. The cleavage reagents remove the 3' hydroxyl-terminal blocking group and optically detectable markers of the first nucleotide, restoring the activity of the reaction site on the first nucleotide for the next round of sequencing. If the cleavage reagents are not completely removed, it may lead to prephasing, reducing sequencing quality. Furthermore, cleaning the chip with the first buffer solution first, followed by further cleaning with the second buffer solution, isolates the second buffer solution from the cleavage reagents. This prevents unwanted reactions from occurring prematurely after contact or mixing with the cleavage reagents, which could rapidly terminate an ongoing cleavage reaction and reduce cleavage efficiency. Furthermore, cleaning the chip with the first buffer solution first, followed by further cleaning with the second buffer solution, ensures that the cleavage reaction may still be ongoing until the first buffer solution completely replaces the cleavage reagents, thereby improving sequencing efficiency and quality. In addition, the chip is first cleaned with the first buffer solution and then further cleaned with the second buffer solution. The first buffer solution isolates the second buffer solution from the cleavage reagent, which can also prevent the pH of the cleavage reagent from decreasing after the second buffer solution comes into contact with or mixes with the cleavage reagent, thereby reducing the cleavage efficiency and affecting the sequencing efficiency / quality.

[0352] According to embodiments of this application, the second buffer solution comprises SSC, Tween-20, and cystamine or a salt thereof. Optionally, the final concentration of cystamine or a salt thereof in the second buffer solution is 10–30 mM, for example, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, or any two values ​​between them. According to embodiments of this application, the second buffer solution comprises cystamine dihydrochloride. Optionally, the second buffer solution comprises: 5X SSC; 0.05% Tween-20; and 20 mM of cystamine dihydrochloride. Therefore, according to embodiments of this application, by adjusting the reagent formulation, such as increasing the concentration of the active ingredient or changing the pH value, ionic strength, etc., the efficiency of the reagent can be improved, thereby reducing the amount of reagent required. Specifically, according to embodiments of this application, by using a second buffer solution with a higher concentration of cystamine or its salt, the cleaning efficiency of the second buffer solution can be improved. This means that the second buffer solution can effectively perform its function with a lower usage amount, thereby reducing the usage amount. The second buffer solution can effectively remove the cutting reagents remaining in the chip after the cutting reaction, which is crucial for ensuring the accuracy of the sequencing reaction. The cutting reagents can remove the 3' hydroxyl-terminal blocking group and optically detectable markers of the first nucleotide, restoring the activity of the reaction site on the first nucleotide for the next round of sequencing reaction. If the cutting reagents are not completely removed, it may lead to phase advance, i.e., prephasing, reducing sequencing quality. At the same time, cleaning the sequencing chip with the second buffer solution after step S30000 can also help reduce non-specific binding and background noise during the sequencing process, thereby improving the efficiency / quality of sequencing and preventing cross-reactions or cross-influences between different reagents, ensuring that each reagent can perform its proper function. This is very important for maintaining the independence and accuracy of each step in the sequencing process.

[0353] According to embodiments of this application, such as Figure 6As shown, the chip 400 is detachably disposed in the liquid system 1000, which includes a reservoir 100, a valve assembly 200, and a pump assembly 300. The valve assembly 200 is configured to connect the reservoir 100 and the pump assembly 300, or to connect the reservoir 100 and the chip 400 and the pump assembly 300. The reservoir 100 includes a first synthetic reagent reservoir 100a for storing a first synthetic reagent. The valve assembly 200 and the chip 400 are connected via a common conduit 500. The method includes: after step S10000 and before step S20000, using the pump assembly 300 to drive the first synthetic reagent that has undergone an extended reaction in the chip 400 and / or the first synthetic reagent that has not participated in the extended reaction in the common conduit 500 to flow toward the first synthetic reagent reservoir 100a, so as to recover at least a portion of the reacted first synthetic reagent and the first synthetic reagent that has not participated in the reaction.

[0354] According to an embodiment of this application, after step S10000 and before step S20000, a pump assembly 300 drives the first synthetic reagent that has undergone the extension reaction within the chip 400 and / or the first synthetic reagent that did not participate in the extension reaction within the common conduit 500 to flow towards the first synthetic reagent reservoir 100a, thereby recovering at least a portion of the reacted first synthetic reagent and the unreacted first synthetic reagent. In this operation, the pump assembly 300 is precisely controlled to generate appropriate positive or reverse flow, causing the first synthetic reagent within the chip 400 and the common conduit 500 to flow towards the first synthetic reagent reservoir 100a. This process requires precise control of the pressure and flow rate of the pump assembly 300 to ensure smooth reagent reflux while avoiding unnecessary impact on the sequencing primers or nucleic acid templates on the surface of the chip 400. In this way, the unreacted first synthetic reagent can be effectively recovered, reducing reagent waste. According to an embodiment of this application, recovering at least a portion of the reacted first synthetic reagent and the unreacted first synthetic reagent can reduce reagent waste and lower sequencing costs. By recovering unreacted reagents from the initial synthesis, they can be reused in subsequent sequencing reactions, improving reagent utilization. This is especially important for expensive reagents, significantly reducing sequencing reagent costs.

[0355] Therefore, by recovering unreacted first-stage synthetic reagents, reagent waste is reduced, sequencing costs are lowered, and reagent utilization is improved. This operation not only helps reduce the direct cost of sequencing but also reduces chemical waste, making it more environmentally friendly. Furthermore, optimizing reagent use simplifies the operational process, improving convenience and accuracy. Through these strategies, the goal of cost reduction and efficiency improvement can be achieved while maintaining or enhancing sequencing quality and efficiency.

[0356] According to an embodiment of this application, the chip 400 is detachably disposed in the liquid circuit system 1000. This design facilitates the replacement and maintenance of the chip 400, and also allows for separate cleaning and processing of the chip 400. The valve assembly 200 includes a rotary valve 200a and a three-way solenoid valve 200b communicatively connected to the rotary valve 200a. The valve assembly 200 connects the reservoir 100 to the pump assembly 300, or connects the reservoir 100 to the chip 400 and the pump assembly 300, using the rotary valve 200a and the three-way solenoid valve 200b.

[0357] According to an embodiment of this application, the liquid circuit system 1000 includes:

[0358] Reservoir 100: Used to store various reagents, such as first synthesis reagent, second synthesis reagent, developing reagent, cutting reagent, first buffer solution, and second buffer solution. The reservoir 100 ensures the orderly storage and supply of reagents.

[0359] Valve assembly 200: Configured to connect reservoir 100 and pump assembly 300, or to connect reservoir 100 and chip 400 and pump assembly 300. The function of valve assembly 200 is to precisely control the flow of reagents, ensuring that the correct reagents are delivered to the designated locations in different sequencing steps.

[0360] Pump Unit 300: Responsible for driving the flow of reagents. It precisely controls the flow rate and volume of reagents according to the needs of the sequencing process, ensuring that the reagents arrive at the designated location on time and in the correct quantity.

[0361] First synthetic reagent reservoir 100a: Specifically used to store the first synthetic reagent, ensuring a stable supply of the first synthetic reagent. The sequencing method includes using a pump assembly 300 to drive the first synthetic reagent in the first synthetic reagent reservoir 100a into the chip 400 via a valve assembly 200 for the extension reaction. This step corresponds to step S10000, ensuring that the first synthetic reagent can participate in the extension reaction accurately and in a timely manner.

[0362] According to an embodiment of this application, a pump assembly 300 drives the first synthetic reagent in the first synthetic reagent reservoir 100a to flow into the chip 400 through a valve assembly 200. This ensures that the first synthetic reagent reaches the chip 400 accurately and promptly for the extension reaction. The pump assembly 300 generates sufficient pressure and flow rate according to a preset program and parameters, propelling the first synthetic reagent out of the reservoir. Upon receiving a corresponding control signal, the valve assembly 200 opens the corresponding channel, allowing the first synthetic reagent to smoothly pass through the valve assembly 200 and flow into the chip 400. Driven by the pump assembly 300, the first synthetic reagent passes through the valve assembly 200 and finally flows into the chip 400, ready for the extension reaction. According to an embodiment of this application, by precisely controlling the pump assembly 300 and the valve assembly 200, the first synthetic reagent is ensured to participate in the extension reaction accurately and promptly, improving the efficiency and accuracy of sequencing. Simultaneously, this precise control also reduces reagent waste and lowers sequencing costs.

[0363] According to an embodiment of this application, the first synthetic reagent reservoir 100a is connected to the valve assembly 200 via a first conduit 600, wherein the reacted first synthetic reagent is recovered into the first conduit 600; and / or, the first synthetic reagent that did not participate in the reaction is recovered into the first conduit 600 and / or the first synthetic reagent reservoir 100a. According to an embodiment of this application, the first synthetic reagent reservoir 100a is connected to the valve assembly 200 via a first conduit 600. This connection provides a physical channel for the recovery of the first synthetic reagent. After the reacted first synthetic reagent undergoes an extended reaction within the chip 400, the first synthetic reagent may have partially participated in the reaction, and the remaining portion can still be recovered and reused. In the common conduit 500, there may be first synthetic reagent that did not participate in the extended reaction, and these reagents can also be recovered. The reacted first synthetic reagent is recovered into the first conduit 600; and / or, the first synthetic reagent that did not participate in the reaction is recovered into the first conduit 600 and / or the first synthetic reagent reservoir 100a. The first tubing 600 serves as a recovery path, temporarily holding recovered reagents for subsequent processing or reuse. Some of the first synthesis reagents that did not participate in the reaction can be directly returned to the first synthesis reagent reservoir 100a, mixed with the original reagents, for reuse in subsequent sequencing cycles.

[0364] According to an embodiment of this application, the pressure difference generated by the pump assembly 300 drives the first synthetic reagent to flow along the first conduit 600 toward the first synthetic reagent reservoir 100a. The pump assembly 300, by precisely controlling the pressure and flow rate, ensures that the reagent can be smoothly recovered from the chip 400 and the common conduit 500 into the first conduit 600 or the first synthetic reagent reservoir 100a. The flow direction of the reagent is opposite to the original delivery direction, from the chip 400 and the common conduit 500 toward the first synthetic reagent reservoir 100a. By precisely controlling the pump assembly 300 and the valve assembly 200, automated management of the recovery process can be achieved, ensuring recovery efficiency and accuracy. Therefore, by recovering the incompletely reacted first synthetic reagent, the utilization rate of the reagent can be significantly improved, and waste can be reduced. The recovery and reuse of reagents directly reduces the cost of sequencing, especially for expensive reagents, where this cost saving is more significant. According to an embodiment of this application, reducing reagent waste and environmental pollution aligns with the concept of sustainable development.

[0365] According to an embodiment of this application, before step S10000: the recovered first synthetic reagent after reaction is introduced from the first pipeline 600 through the valve assembly 200 into the chip 400 to clean the chip 400. According to an embodiment of this application, the first synthetic reagent recovered into the first pipeline 600 and the first synthetic reagent reservoir 100a is 50% to 80% of the total amount of the first synthetic reagent used, preferably 60% to 65%.

[0366] According to an embodiment of this application, the reservoir 100 includes a second synthetic reagent reservoir 100b, which is used to store a second synthetic reagent. The process of allowing the second synthetic reagent to flow into the chip 400 includes: using a pump assembly 300 to drive the second synthetic reagent in the second synthetic reagent reservoir 100b into the chip 400 via a valve assembly 200; and allowing the reacted second synthetic reagent and / or the unreacted second synthetic reagent in the common pipeline 500 to flow towards the second synthetic reagent reservoir 100b to recover the reacted second synthetic reagent and / or the unreacted second synthetic reagent.

[0367] According to an embodiment of this application, a pump assembly 300 drives the second synthetic reagent in the second synthetic reagent reservoir 100b through a valve assembly 200 into the chip 400. In this operation, the pump assembly 300 generates sufficient pressure and flow rate according to a preset program and parameters, pushing the second synthetic reagent out of the reservoir. After receiving a corresponding control signal, the valve assembly 200 opens the corresponding channel, allowing the second synthetic reagent to smoothly pass through the valve assembly 200 and flow into the chip 400. The second synthetic reagent flows into the chip 400 to perform the completion reaction. The purpose of the completion reaction is to ensure that all gaps on the DNA template are filled, thereby improving the read length and accuracy of sequencing. Through this operation, it can be ensured that even sequences that are not fully extended in the extension reaction can be accurately determined, enhancing data integrity. Thus, by precisely controlling the pump assembly 300 and the valve assembly 200, the second synthetic reagent can be ensured to participate in the completion reaction accurately and in a timely manner, improving the efficiency and accuracy of sequencing. At the same time, this precise control also reduces reagent waste and lowers sequencing costs.

[0368] According to embodiments of this application, the reacted second synthetic reagent and / or unreacted second synthetic reagent in the common tubing 500 are flowed towards the second synthetic reagent reservoir 100b to recover the reacted second synthetic reagent and / or unreacted second synthetic reagent. In this operation, the pump assembly 300 is precisely controlled to generate appropriate positive or reverse flow, causing the second synthetic reagent in the chip 400 and the common tubing 500 to flow towards the second synthetic reagent reservoir 100b. This process requires precise control of the pressure and flow rate of the pump assembly 300 to ensure smooth reagent reflux while avoiding unnecessary impact on the sequencing primers or nucleic acid templates on the surface of the chip 400. At least a portion of the reacted second synthetic reagent and the unreacted second synthetic reagent are recovered. This objective aims to reduce reagent waste and lower sequencing costs. By recovering the incompletely reacted second synthetic reagent, it can be reused in subsequent sequencing reactions, improving reagent utilization. Recovering the incompletely reacted second synthetic reagent reduces reagent waste, lowers sequencing costs, and simultaneously improves reagent utilization. This not only helps reduce the direct cost of sequencing, but also reduces the generation of chemical waste, making it more environmentally friendly.

[0369] According to an embodiment of this application, the second synthetic reagent reservoir 100b is connected to the valve assembly 200 via a second conduit 700. The reacted second synthetic reagent is recovered into the second conduit 700; and / or, unreacted second synthetic reagent is recovered into the second conduit 700 and / or the second synthetic reagent reservoir 100b. According to an embodiment of this application, the second synthetic reagent reservoir 100b is connected to the valve assembly 200 via a second conduit 700. This connection provides a physical channel for the recovery of the second synthetic reagent. After the completion reaction within the chip 400, the second synthetic reagent may have partially participated in the reaction, and the remaining portion can be recovered and reused. In the common conduit 500, there may be second synthetic reagent that did not participate in the completion reaction; these reagents can also be recovered. According to an embodiment of this application, the reacted second synthetic reagent is recovered into the second conduit 700; and / or, unreacted second synthetic reagent is recovered into the second conduit 700 and / or the second synthetic reagent reservoir 100b. The second line 700 serves as a recovery path, temporarily holding recovered reagents for subsequent processing or reuse. Any unreacted second synthesis reagents can be directly returned to the second synthesis reagent reservoir 100b, mixed with the original reagents, for reuse in subsequent sequencing cycles.

[0370] According to an embodiment of this application, the pressure difference generated by the pump assembly 300 drives the second synthesis reagent to flow along the second pipeline 700 toward the second synthesis reagent reservoir 100b. The pump assembly 300, by precisely controlling the pressure and flow rate, ensures that the reagent can be smoothly recovered from the chip 400 and the common pipeline 500 into the second pipeline 700 or the reservoir. The flow direction of the reagent is opposite to the original delivery direction, from the chip 400 and the common pipeline 500 toward the second synthesis reagent reservoir 100b. By precisely controlling the pump assembly 300 and the valve assembly 200, automated management of the recovery process can be achieved, ensuring recovery efficiency and accuracy. Therefore, by recovering incompletely reacted second synthesis reagents, reagent utilization can be significantly improved, reducing waste. The recovery and reuse of reagents directly reduces sequencing costs, especially for expensive reagents, where this cost saving is more significant. Reducing reagent waste and environmental pollution aligns with the concept of sustainable development.

[0371] According to an embodiment of this application, the recovered second synthetic reagent is introduced from the second conduit 700 through the valve assembly 200 into the chip 400 to replace the solution in the chip 400 after the extension reaction. According to an embodiment of this application, the amount of second synthetic reagent recovered into the second conduit 700 and the second synthetic reagent reservoir 100b is 50% to 80% of the total amount of second synthetic reagent used, preferably 60% to 65%. Therefore, according to an embodiment of this application, by recovering the second synthetic reagent that did not participate in the completion reaction, the total consumption of reagents can be effectively reduced, thereby reducing costs. The recovered reagents can be reused, which not only improves reagent utilization efficiency but also reduces the direct waste of unreacted reagents, avoiding resource waste. Furthermore, reducing the waste of chemical reagents is also a form of environmental protection. Through recycling, the amount of reagent pumped can be reduced, thereby reducing pumping time and sequencing time while ensuring sequencing quality.

[0372] According to an embodiment of this application, after step S10000 and before step S20000, at least a portion of the second synthetic reagent that did not participate in the replenishment reaction is returned to the second synthetic reagent reservoir 100b. According to an embodiment of this application, all the second synthetic reagent that did not enter the chip 400 is returned to the second synthetic reagent reservoir 100b. Optionally, the second synthetic reagent returned to the second synthetic reagent reservoir 100b is 50% to 80% of the total amount of second synthetic reagent added, preferably 55% to 60%. This proportion ensures sufficient reagent participation in the reaction while maximizing recycling, achieving an optimal balance between cost and efficiency.

[0373] According to an embodiment of this application, the flow of developing reagent into chip 400 includes: introducing a first buffer solution into a common conduit 500 to push the developing reagent within the common conduit 500 into chip 400. Thus, by utilizing the flow of the first buffer solution, the developing reagent can be pushed from the common conduit 500 into chip 400, ensuring that the developing reagent accurately and promptly reaches the reaction area on chip 400, thereby exciting an optically detectable marker to generate a signal and collecting that signal. This process not only improves the efficiency of developing reagent use but also reduces the residue of developing reagent in the conduit, thus reducing reagent waste.

[0374] According to an embodiment of this application, the cleavage reagent is fed into the chip 400 by: introducing a first buffer solution into a common conduit 500 to push the cleavage reagent in the common conduit 500 into the chip 400. Thus, by utilizing the flow of the first buffer solution to push the cleavage reagent from the common conduit 500 into the chip 400, it is ensured that the cleavage reagent can accurately and timely reach the reaction region on the chip 400, thereby cleaving the 3' hydroxyl terminating group of the synthetic base and the fluorescence signal, preparing for the next round of synthesis reaction. This process not only improves the efficiency of cleavage reagent use but also reduces the residue of cleavage reagent in the conduit, reducing reagent waste.

[0375] According to embodiments of this application, the method further includes: drawing a predetermined volume of air into the chip 400 before or after the first synthetic reagent flows into the chip 400. Thus, by using an air column, the two reagents can be isolated, reducing reagent diffusion and thus minimizing reagent efficacy. Without air bubbles to isolate the two reagents drawn one after the other, they can easily diffuse and influence each other, requiring increased dosage to eliminate the diffusion effect. Using an air column effectively reduces the diffusion effect caused by direct contact between the reagents.

[0376] According to embodiments of this application, sequencing-by-synthesis is performed at 55–65°C, preferably 60°C. Therefore, by maintaining the operating temperature of sequencing-by-synthesis according to embodiments of this application, consistent temperature control throughout the entire process can be ensured, facilitating operation. Furthermore, operating at temperatures such as 60°C can increase the rate of chemical or biological reactions, thereby ensuring that the involved chemical and biological reactions achieve higher efficiency at this temperature. Simultaneously, choosing 60°C as the reaction temperature avoids thermal damage to samples or reagents that might be caused by higher temperatures, protecting the integrity of the samples and the activity of the reagents.

[0377] It should be noted that the technical features and effects of this application can be combined with each other, which will not be elaborated here.

[0378] Example

[0379] Note: For ease of command invocation, various reagents are represented by English abbreviations: First Synthetic Reagent (HN), Second Synthetic Reagent (CN), Cleave Reagent / Cleave Reaction (Cleave), Image Reagent (Image), First Buffer (RB_Wash), Second Buffer (WB), Bubble / Air (bubble).

[0380] In the following text, “S0 speed” indicates a controlled fluid flow rate of 7600 μl / min; “S12 speed” indicates a controlled fluid flow rate of 1200 μl / min; “S13 speed” indicates a controlled fluid flow rate of 1350 μl / min; “S15 speed” indicates a controlled fluid flow rate of 850 μl / min; and “S18 speed” indicates a controlled fluid flow rate of 337 μl / min.

[0381] As an example, below are the flow charts for the cleavage and extension reactions before optimization.

[0382] 1. The optimized pre-cutting reaction process is as follows:

[0383] 1) Set the temperature to 60℃, use S13 speed to aspirate RB_Wash 30ul, and let stand for 2 seconds;

[0384] 2) S13 speed aspirate 122ul Cleave, let stand for 2s, then incubate for 16s;

[0385] 3) Draw in 1.25ul of air at speed S18 and let stand for 1 second;

[0386] 4) S13 speed aspirate 23.25ul WB, let stand for 2s, then incubate for 25s;

[0387] 5) S13 speed aspirate 126ul WB, let stand for 2s, then incubate for 0.5s;

[0388] 6) S13 speed aspirate 24.5ul RB_Wash, let stand for 2s, then incubate for 0.5s.

[0389] 2. The extended reaction process before optimization is as follows:

[0390] 1) Set the temperature to 60℃, use speed S13 to aspirate 93ul of RB Wash, and let stand for 2 seconds;

[0391] 2) S13 speed aspirate 188ul of HN, let stand for 2s, then incubate for 15s;

[0392] 3) S18 speed aspirate 24.5ul CN, let stand for 2s, then incubate for 15s;

[0393] 4) S18 speed aspirate 95ul CN, let stand for 2s, then incubate for 23.5s;

[0394] 5) Draw in 1.25ul of air at speed S18 and let stand for 1 second;

[0395] 6) S15 speed aspirate 23.25ul WB, let stand for 2s, then incubate for 40s;

[0396] 7) S15 speed suction 30ul WB, let stand for 2s;

[0397] 8) S15 speed suction of Image 155ul, let stand for 6 seconds;

[0398] 9) S15 speed suction RB_Wash 24.5ul, let stand for 2s.

[0399] As an example, the following is the optimized cleavage and extension reaction flow.

[0400] 1. The optimized cleavage reaction process is as follows:

[0401] 1) Temperature setting: 60℃

[0402] 2) At speed S13, aspirate 51ul cleanly, then aspirate another 10ul, let stand for 4 seconds, then aspirate another 10ul, and let stand for 4 seconds.

[0403] 3) S13 speed suction RB_Wash 17ul, let stand for 2s

[0404] 4) S0 speed draw RB_Wash 17ul (branch), let stand for 2s

[0405] 5) S0 speed suction WB 20ul (branch), let stand for 1s

[0406] 6) S12 speed suction WB 82ul, let stand for 1 second

[0407] 2. The optimized process flow for the extended reaction is as follows:

[0408] 1) Set the temperature to 60℃, use speed S12 to aspirate 44ul of RB Wash, and let stand for 1 second.

[0409] 2) S13 speed absorption HN 190ul, incubate for 15s

[0410] 3) S13 speed back-pull HN 119ul, incubate for 5s

[0411] 4) Add 139 μL of CN at S13 speed, incubate for 15 seconds, then add another 34 μL and incubate for 30 seconds.

[0412] 5) S13 velocity back-pull CN 102ul, incubate for 5s

[0413] 6) S15 speed suction of Image 71ul, let stand for 1 second

[0414] 7) S15 speed suction RB_Wash 24ul

[0415] The present application will be further explained below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0416] Experimental reagents

[0417] Second buffer solution Shenzhen Zhenmai Biotechnology Co., Ltd. Reagent test kit Shenzhen Zhenmai Biotechnology Co., Ltd.

[0418] Experimental instruments

[0419] FASTASeq300 sequencer FASTASeq300 Shenzhen Zhenmai Biotechnology Co., Ltd. Vortex oscillator MX-S SCILOGEX Cubee high-speed mini centrifuge A04307 GeneReachBiotechnology Corp

[0420] Reagent preparation

[0421] Second buffer solution preparation: 1L

[0422]

[0423] Configuration fluid

[0424] 1. Standard fluid

[0425] The sequencing process in a single cycle, or single-round sequencing reaction, is as follows:

[0426] 1) Set the temperature to 60℃, use speed S13 to aspirate RB_Wash 30ul, and let stand for 2 seconds.

[0427] 2) S13 speed aspirate 122ul, let stand for 2 seconds, then incubate for 16 seconds.

[0428] 3) Draw in 1.25ul of air at speed S18 and let stand for 1 second.

[0429] 4) S13 speed aspirate 23.25ul WB, let stand for 2s, then incubate for 25s.

[0430] 5) S13 speed aspirate 126ul WB, let stand for 2s, then incubate for 0.5s.

[0431] 6) S13 speed aspirate 24.5ul RB_Wash, let stand for 2 seconds, then incubate for 0.5 seconds.

[0432] 7) Set the temperature to 60℃, use speed S13 to aspirate RB_Wash 93ul, and let stand for 2 seconds.

[0433] 8) S13 speed aspirate HN 188ul, let stand for 2s, then incubate for 15s.

[0434] 9) S18 speed aspirate 24.5ul CN, let stand for 2s, then incubate for 15s.

[0435] 10) S18 speed aspirate 95ul CN, let stand for 2s, then incubate for 23.5s.

[0436] 11) S18 speed draws in 1.25ul of air, let stand for 1 second

[0437] 12) S15 speed aspirate 23.25ul WB, let stand for 2s, then incubate for 40s.

[0438] 13) S15 speed suction, 30ul WB, let stand for 2 seconds

[0439] 14) S15 speed suction of Image 155ul, let stand for 6 seconds

[0440] 15) S15 speed suction RB_Wash 24.5ul, let stand for 2 seconds

[0441] 2. Optimize the fluid (reduce the amount of reagent in the fluid).

[0442] The sequencing reaction process in a single-round sequencing reaction is as follows:

[0443] 1) Temperature setting: 60℃

[0444] 2) At speed S13, aspirate 51ul cleanly, then aspirate another 10ul, let stand for 4 seconds, then aspirate another 10ul, and let stand for 4 seconds.

[0445] 3) S13 speed suction RB Wash 17ul, let stand for 2 seconds

[0446] 4) S0 speed suction RB Wash 17ul (branch), let stand for 2 seconds

[0447] 5) S0 speed suction WB 20ul (branch), let stand for 1s

[0448] 6) S12 speed suction WB 82ul, let stand for 1 second

[0449] 7) Set the temperature to 60℃, use speed S12 to aspirate RB_Wash 44ul, and let stand for 1 second.

[0450] 8) S13 speed absorption HN 190ul, incubate for 15s

[0451] 9) S13 speed back-pull HN 119ul, incubate for 5s

[0452] 10) S13 speed aspirate 139ul CN, incubate for 15s, then aspirate another 34ul and incubate for 30s.

[0453] 11) S13 velocity back-pull CN 102ul, incubation 5s

[0454] 12) S15 speed suction of Image 71ul, let stand for 1 second

[0455] 13) S15 speed suction RB_Wash 24ul

[0456] Sequencing process

[0457] ① The FASTASeq300 platform chip has four fluid channels (lanes), with two fluid channels belonging to the same unit. Each unit's fluid path system is independent. ② Configure the library according to the FASTASeq300 platform's instruction manual. The recommended library concentration range is 2pM to 4pM, with 3pM being the preferred concentration.

[0458] ③ Place the prepared library in the designated location. The instrument will load the library onto the chip according to the set procedure for single-end (e.g., SE100) sequencing.

[0459] ④ Fluid file configuration

[0460]

[0461] ⑤ Data Analysis

[0462] 1. Basecall Result

[0463]

[0464] The above experiments show that...

[0465] (1) By comparing the sequencing quality of the pre-optimized extension reaction process (Experiment 1) and the post-optimized extension reaction process (Experiment 2), it can be seen that even under the condition of reverse synthesis of reagents (i.e., recycling), the sequencing quality was not affected. This shows that the recycling of synthetic reagents is feasible, and can reduce the amount of reagents used and lower costs without sacrificing sequencing quality.

[0466] (2) By comparing the sequencing quality before and after the cleavage reaction process optimization (Experiment 2 and Experiment 4), it can be clearly seen that the optimized cleavage reaction process significantly improves the removal of cleavage reagent residues, with a significant decrease in prephasing value, a slight decrease in phasing, and a significant increase in Q30. This indicates that by optimizing the cleavage reaction process, the residual cleavage reagents in the cleavage reaction steps can be reduced without sacrificing sequencing quality, thereby improving the accuracy and efficiency of sequencing.

[0467] (3) By comparing the effects of having and not having a three-way solenoid valve in the liquid flow system on sequencing quality (Experiments 3 and 4), it was found that the use of a three-way solenoid valve leads to an increase in residual cutting reagent, resulting in a significant increase in the prephasing value. This indicates that the use of a three-way solenoid valve may have a negative impact on sequencing quality, and therefore should be avoided in optimized sequencing workflows.

[0468] (4) By further comparing the sequencing quality with and without a three-way solenoid valve and by increasing the cystamine concentration in the second buffer after optimizing the cleavage and extension reaction processes (Experiments 3 to 6), it was found that further optimization measures, such as removing the three-way solenoid valve and increasing the cystamine concentration in the liquid system, effectively reduced the prephasing value, indicating that residual cleavage reagent was effectively suppressed. This demonstrates that by optimizing the cleavage and extension reaction processes, removing the three-way solenoid valve in the liquid system, and adjusting the formulation of the second buffer, it is possible to reduce reagent usage and costs while maintaining sequencing quality, and simultaneously improve sequencing accuracy and efficiency.

[0469] The above comparison results support the technical solution of this application, namely, that through meticulous process optimization and reagent management, cost-effectiveness can be achieved while maintaining or improving sequencing quality.

[0470] 2. Analysis of reagent usage

[0471]

[0472] 3. Sequencing time result analysis

[0473] Extended reaction process 177.2s 126.3s 50.9s 28.7％ Cutting reaction process 81.4s 26.9s 54.5s 67.0％ single cycle time 258.6s 153.2s 105.4s 40.8％

[0474] In summary, by improving the sequencing method, including reagent back-pushing and recovery during sequencing, cleaning the chip with the first buffer before and after the cleavage reaction, and using the first buffer to isolate the cleavage reagent from other reagents, the goal of reducing reagent usage was achieved. The overall reagent usage decreased by 50%, and the usage of the most expensive reagent (the first synthesis reagent) decreased by 62%. The reduction in reagent usage not only lowered costs but also reduced liquid throughput time and accelerated sequencing speed, resulting in a 40.8% reduction in single-cycle sequencing time.

[0475] In terms of the fluid path layout of the FASTASeq300 platform, a three-way solenoid valve was added between the rotary valve and the chip. The addition of the solenoid valve resulted in residual cutting reagent, which caused a significant increase in prephasing. Through optimization of the cleaning process after cutting reagent and optimization of the formulation of the second buffer solution, the prephasing returned to normal levels.

[0476] In summary, this application improves the sequencing method by including reagent back-pushing and recovery during sequencing, cleaning the chip with a first buffer before and after the cleavage reaction, and using the first buffer to isolate the cleavage reagent from other reagents. This ensures sequencing quality while reducing reagent consumption by 50% and single-cycle sequencing time by 41%. It not only reduces reagent costs but also improves sequencing efficiency, achieving the goal of cost reduction and efficiency improvement.

[0477] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A sequencing method, characterized in that, include: S1, a first buffer solution is introduced into the chip to clean the chip, which is used for sequencing-while-synthesizing, and the pH value of the first buffer solution is greater than 8. S2, a first synthetic reagent is fed into the chip to carry out an extension reaction on the chip, the first synthetic reagent comprising a first nucleotide containing a cleavable blocking group and an optically detectable label; S3, a developing agent is allowed to flow into the chip to excite the optically detectable marker to generate a signal, and the signal is collected; S4, the first buffer solution is allowed to flow into the chip to clean the chip; S5, the cutting reagent is allowed to flow into the chip to remove the blocking group and the optically detectable marker; S6, the first buffer solution is allowed to flow into the chip to clean the chip; and S7, allow the second buffer solution to flow into the chip to clean the chip; S8, return to step S1, and repeat steps S1 to S7 until the sequencing reaction is complete.

2. The method according to claim 1, characterized in that, The sequencing method satisfies one or more of the following conditions: 1) After step S2 and before step S3, the method further includes: S2-1, a second synthetic reagent is introduced into the chip, the second synthetic reagent containing a second nucleotide, the second nucleotide containing a cleavable blocking group but not an optically detectable label; Optionally, the step of allowing the cutting reagent to flow into the chip includes flowing the cutting reagent into the chip and flowing the first buffer solution into the chip to push the cutting reagent into the chip in one or more portions. Optionally, the step of allowing the second buffer solution to flow into the chip includes flowing the second buffer solution toward the chip and pushing the first buffer solution, so that the first buffer solution and the second buffer solution enter the chip sequentially; 2) The pH value of the first buffer solution is 9-10, and the pH values ​​of the first synthesis reagent and the cleavage reagent are also 9-10; 3) The second buffer solution comprises SSC, Tween-20, and cystamine or a salt thereof. Optionally, the final concentration of cystamine or its salt in the second buffer solution is 10–30 mM; Optionally, the second buffer solution comprises cystamine dihydrochloride. Optionally, the second buffer solution comprises: 5X SSC; 0.05% Tween-20; and 20 mM cystamine dihydrochloride; 4) The chip is detachably disposed in the liquid circuit system, which includes a reservoir, a valve assembly, and a pump assembly; The valve assembly is configured to connect the reservoir to the pump assembly, or to connect the reservoir to the chip and the pump assembly; The reservoir includes a first synthetic reagent reservoir, which is used to store the first synthetic reagent. Optionally, the method includes: using the pump assembly to drive the first synthetic reagent in the first synthetic reagent reservoir through the valve assembly into the chip for an extension reaction, so that in step S2, the first synthetic reagent flows into the chip to carry out an extension reaction on the chip; Optionally, the valve assembly is connected to the chip via a common pipeline, and the method includes: after step S2 and before step S3, using the pump assembly to drive the first synthetic reagent after the extended reaction in the chip and / or the first synthetic reagent in the common pipeline that did not participate in the extended reaction to flow in the direction toward the first synthetic reagent reservoir, so as to recover at least a portion of the first synthetic reagent after the reaction and the first synthetic reagent that did not participate in the reaction; Optionally, the first synthetic reagent reservoir is connected to the valve assembly via a first pipeline, wherein, The first synthetic reagent after the reaction is recovered into the first pipeline; and / or, The first synthetic reagent that did not participate in the reaction was recovered into the first pipeline and / or the first synthetic reagent reservoir; Optionally, step S1 includes: The recovered first synthetic reagent is introduced into the chip through the valve group via the first pipeline to serve as the first buffer solution for cleaning the chip; Optionally, the first synthetic reagent recovered into the first pipeline and the first synthetic reagent reservoir is 50% to 80% of the total amount of the first synthetic reagent used, preferably 60% to 65%. Optionally, the reservoir includes a second synthetic reagent reservoir for storing a second synthetic reagent. The step of allowing the second synthetic reagent to flow into the chip includes: The pump group is used to drive the second synthetic reagent in the second synthetic reagent reservoir into the chip through the valve group, and to make the second synthetic reagent after reaction and / or the second synthetic reagent that has not participated in the reaction in the common pipeline flow towards the second synthetic reagent reservoir, so as to recover the second synthetic reagent after reaction and / or the second synthetic reagent that has not participated in the reaction; Optionally, the second synthetic reagent reservoir is connected to the valve assembly via a second pipeline, and the reacted second synthetic reagent is recovered into the second pipeline; and / or, Unreacted second synthetic reagent is recovered into the second pipeline and / or the second synthetic reagent reservoir; Optionally, the recovered second synthetic reagent after the reaction is introduced from the second pipeline through the valve assembly into the chip to replace the solution in the chip after the extended reaction; Optionally, the amount of the second synthetic reagent recovered into the second pipeline and the second synthetic reagent reservoir is 50% to 80% of the total amount of the second synthetic reagent used, preferably 60% to 65%. Optionally, the process of allowing the developing agent to flow into the chip includes: The first buffer solution is introduced into the common tubing to push the developing reagent in the common tubing into the chip; Optionally, the process of allowing the cutting reagent to flow into the chip includes: The first buffer solution is introduced into the common tubing to push the cutting reagent in the common tubing into the chip; 5) The method further includes: Before or after the first synthetic reagent flows into the chip, a predetermined volume of air is drawn into the chip; 6) The sequencing-by-synthesis process is performed at 55–65 degrees Celsius, preferably 60 degrees Celsius.

3. A sequencing method, characterized in that, include: S10, a first synthetic reagent is fed into the chip to carry out an extension reaction on the chip, the first synthetic reagent comprising a first nucleotide containing a cleavable blocking group and an optically detectable label; S20, a developing agent is allowed to flow into the chip to excite the optically detectable marker to generate a signal, and the signal is collected; S30, the cutting reagent is allowed to flow into the chip to remove the blocking group and the optically detectable marker; S40, the second buffer solution flows into the chip to clean the chip; S50, return to step S10, and repeat steps S10 to S40 until the sequencing reaction is complete. The second buffer solution comprises SSC, Tween-20, and cystamine or its salt, wherein the final concentration of cystamine or its salt in the second buffer solution is 10–30 mM.

4. The method according to claim 3, characterized in that, The sequencing method satisfies one or more of the following conditions: 1) After step S10 and before step S20, the method further includes: S10-1, a second synthetic reagent is introduced into the chip, the second synthetic reagent containing a second nucleotide, the second nucleotide containing a cleavable blocking group but not an optically detectable label; 2) Before step S10, a first buffer solution is introduced into the chip to clean the chip, which is used for sequencing while synthesis; and / or, before step S30, a first buffer solution is introduced into the chip to clean the chip. Optionally, in step S30, the step of allowing the cutting reagent to flow into the chip includes flowing the first buffer solution into the chip and pushing the cutting reagent into the chip in one or more portions; Optionally, in step S40, the step of allowing the second buffer solution to flow into the chip includes causing the second buffer solution to flow toward the chip and pushing the first buffer solution, so that the first buffer solution and the second buffer solution enter the chip sequentially; Optionally, the pH of the first synthetic reagent, the cleavage reagent, and the first buffer solution is greater than 8; Optionally, the pH of the first synthetic reagent, the cleavage reagent, and the first buffer solution is 9 to 10; 3) The second buffer solution includes cystamine dihydrochloride. Optionally, the second buffer solution comprises: 5X SSC; 0.05% Tween-20; and 20 mM cystamine dihydrochloride; 4) The chip is detachably disposed in the liquid circuit system, which includes a reservoir, a valve assembly, and a pump assembly; The valve assembly is configured to connect the reservoir to the pump assembly, or to connect the reservoir to the chip and the pump assembly; The reservoir includes a first synthetic reagent reservoir, which is used to store the first synthetic reagent. Optionally, the method includes: using the pump assembly to drive the first synthetic reagent in the first synthetic reagent reservoir through the valve assembly into the chip for an extension reaction, so that in step S10, the first synthetic reagent flows into the chip to carry out an extension reaction on the chip; Optionally, the valve assembly is connected to the chip via a common pipeline, and the method includes: after step S10 and before step S20, using the pump assembly to drive the first synthetic reagent after the extended reaction in the chip and / or the first synthetic reagent in the common pipeline that did not participate in the extended reaction to flow in the direction toward the first synthetic reagent reservoir, so as to recover at least a portion of the first synthetic reagent after the reaction and the first synthetic reagent that did not participate in the reaction; Optionally, the first synthetic reagent reservoir is connected to the valve assembly via a first pipeline, wherein, The first synthetic reagent after the reaction is recovered into the first pipeline; and / or, The first synthetic reagent that did not participate in the reaction was recovered into the first pipeline and / or the first synthetic reagent reservoir; Optionally, before step S10: The recovered first synthetic reagent is introduced into the chip through the valve group via the first pipeline to clean the chip; Optionally, the first synthetic reagent recovered into the first pipeline and the first synthetic reagent reservoir is 50% to 80% of the total amount of the first synthetic reagent used, preferably 60% to 65%. Optionally, the reservoir includes a second synthetic reagent reservoir for storing a second synthetic reagent. The step of allowing the second synthetic reagent to flow into the chip includes: The pump group is used to drive the second synthetic reagent in the second synthetic reagent reservoir into the chip through the valve group, and to make the second synthetic reagent after reaction and / or the second synthetic reagent that has not participated in the reaction in the common pipeline flow towards the second synthetic reagent reservoir, so as to recover the second synthetic reagent after reaction and / or the second synthetic reagent that has not participated in the reaction; Optionally, the second synthetic reagent reservoir is connected to the valve assembly via a second pipeline, and the reacted second synthetic reagent is recovered into the second pipeline; and / or, Unreacted second synthetic reagent is recovered into the second pipeline and / or the second synthetic reagent reservoir; Optionally, the recovered second synthetic reagent after the reaction is introduced from the second pipeline through the valve assembly into the chip to replace the solution in the chip after the extended reaction; Optionally, the amount of the second synthetic reagent recovered into the second pipeline and the second synthetic reagent reservoir is 50% to 80% of the total amount of the second synthetic reagent used, preferably 60% to 65%. Optionally, the process of allowing the developing agent to flow into the chip includes: The first buffer solution is introduced into the common tubing to push the developing reagent in the common tubing into the chip; Optionally, the process of allowing the cutting reagent to flow into the chip includes: The first buffer solution is introduced into the common tubing to push the cutting reagent in the common tubing into the chip; 5) The method further includes: Before or after the first synthetic reagent flows into the chip, a predetermined volume of air is drawn into the chip; 6) The sequencing-by-synthesis process is performed at 55–65 degrees Celsius, preferably 60 degrees Celsius.

5. A sequencing method, characterized in that, include: S100, a first synthetic reagent is fed into a chip to carry out an extension reaction on the chip, the first synthetic reagent comprising a first nucleotide, the first nucleotide containing a cleavable blocking group and an optically detectable label; S200, a developing agent is flowed into the chip to excite the optically detectable marker to generate a signal, and the signal is collected; S300, a cutting reagent is introduced into the chip to remove the blocking group and the optically detectable marker; S400, return to step S100, and repeat steps S100 to S300 until the sequencing reaction is complete; The method further includes: In step S100, the first synthetic reagent flows into the chip before or after the chip, and a preset volume of air is drawn into the chip.

6. The method according to claim 5, characterized in that, The sequencing method satisfies one or more of the following conditions: 1) After step S100 and before step S200, the method further includes: S100-1, a second synthetic reagent is introduced into the chip, the second synthetic reagent containing a second nucleotide, the second nucleotide containing a cleavable blocking group but not an optically detectable label; 2) Before step S100, a first buffer solution is introduced into the chip to clean the chip, which is used for sequencing while synthesis; and / or, before step S300, a first buffer solution is introduced into the chip to clean the chip. Optionally, in step S300, the process of allowing the cutting reagent to flow into the chip includes flowing the first buffer solution into the chip and pushing the cutting reagent into the chip in one or more portions. Optionally, the pH of the first synthetic reagent, the cleavage reagent, and the first buffer solution is greater than 8; Optionally, the pH of the first synthetic reagent, the cleavage reagent, and the first buffer solution is 9 to 10; Optionally, after step S300, the second buffer solution is allowed to flow into the chip and push the first buffer solution, so that the first buffer solution and the second buffer solution enter the chip in sequence to clean the chip; Optionally, the second buffer solution comprises SSC, Tween-20, and cystamine or a salt thereof. Optionally, the final concentration of cystamine or its salt in the second buffer solution is 10–30 mM; Optionally, the second buffer solution comprises cystamine dihydrochloride. Optionally, the second buffer solution comprises: 5X SSC; 0.05% Tween-20; and 20 mM cystamine dihydrochloride; Optionally, the chip is detachably disposed in the liquid circuit system, which includes a reservoir, a valve assembly, and a pump assembly; The valve assembly is configured to connect the reservoir to the pump assembly, or to connect the reservoir to the chip and the pump assembly; The reservoir includes a first synthetic reagent reservoir, which is used to store a first synthetic reagent. The valve assembly and the chip are connected via a common conduit. The method includes: after step S100 and before step S200, using the pump group to drive the first synthetic reagent after the extension reaction in the chip and / or the first synthetic reagent in the common pipeline that did not participate in the extension reaction to flow in the direction toward the first synthetic reagent reservoir, so as to recover at least a portion of the first synthetic reagent after the reaction and the first synthetic reagent that did not participate in the reaction. Optionally, the first synthetic reagent reservoir is connected to the valve assembly via a first pipeline, wherein, The first synthetic reagent after the reaction is recovered into the first pipeline; and / or, The first synthetic reagent that did not participate in the reaction was recovered into the first pipeline and / or the first synthetic reagent reservoir; Optionally, before step S100: The recovered first synthetic reagent is introduced into the chip through the valve group via the first pipeline to clean the chip; Optionally, the first synthetic reagent recovered into the first pipeline and the first synthetic reagent reservoir is 50% to 80% of the total amount of the first synthetic reagent used, preferably 60% to 65%. Optionally, the reservoir includes a second synthetic reagent reservoir for storing a second synthetic reagent. The step of allowing the second synthetic reagent to flow into the chip includes: The pump group is used to drive the second synthetic reagent in the second synthetic reagent reservoir into the chip through the valve group, and to make the second synthetic reagent after reaction and / or the second synthetic reagent that has not participated in the reaction in the common pipeline flow towards the second synthetic reagent reservoir, so as to recover the second synthetic reagent after reaction and / or the second synthetic reagent that has not participated in the reaction; Optionally, the second synthetic reagent reservoir is connected to the valve assembly via a second pipeline, and the reacted second synthetic reagent is recovered into the second pipeline; and / or, Unreacted second synthetic reagent is recovered into the second pipeline and / or the second synthetic reagent reservoir; Optionally, the recovered second synthetic reagent after the reaction is introduced from the second pipeline through the valve assembly into the chip to replace the solution in the chip after the extended reaction; Optionally, the amount of the second synthetic reagent recovered into the second pipeline and the second synthetic reagent reservoir is 50% to 80% of the total amount of the second synthetic reagent used, preferably 60% to 65%. Optionally, the process of allowing the developing agent to flow into the chip includes: The first buffer solution is introduced into the common tubing to push the developing reagent in the common tubing into the chip; Optionally, the process of allowing the cutting reagent to flow into the chip includes: The first buffer solution is introduced into the common tubing to push the cutting reagent in the common tubing into the chip; 3) The sequencing-by-synthesis process is performed at 55–65 degrees Celsius, preferably 60 degrees Celsius.

7. A sequencing method, characterized in that, include: S1000, a first synthetic reagent is fed into a chip to carry out an extension reaction on the chip, the first synthetic reagent comprising a first nucleotide, the first nucleotide containing a cleavable blocking group and an optically detectable label; S2000, a developing agent is flowed into the chip to excite the optically detectable marker to generate a signal, and the signal is collected; S3000, a cutting reagent is introduced into the chip to remove the blocking group and the optically detectable marker; S4000, return to step S1000, and repeat steps S1000 to S3000 until the sequencing reaction is complete. The method further includes: After step S1000 and before step S2000, at least a portion of the first synthetic reagent after the reaction and / or the first synthetic reagent that did not participate in the reaction is recovered; and / or, Before step S1000, the recovered first synthetic reagent is allowed to flow into the chip to clean the chip.

8. The method according to claim 7, characterized in that, The sequencing method satisfies one or more of the following conditions: 1) After step S1000 and before step S2000, the method further includes: S1000-1, allowing a second synthetic reagent to flow into the chip, the second synthetic reagent containing a second nucleotide, the second nucleotide containing a cleavable blocking group but not containing an optically detectable label; 2) Before step S3000, the first buffer solution is allowed to flow into the chip to clean the chip; Optionally, in step S3000, the process of allowing the cutting reagent to flow into the chip includes flowing the first buffer solution into the chip and pushing the cutting reagent into the chip in one or more portions. Optionally, the pH of the cutting reagent and the first buffer solution is greater than 8; Optionally, the pH of the cutting reagent and the first buffer solution is 9 to 10; Optionally, after step S3000, the second buffer solution is allowed to flow into the chip and push the first buffer solution, so that the first buffer solution and the second buffer solution enter the chip in sequence to clean the chip; Optionally, the second buffer solution comprises SSC, Tween-20, and cystamine or a salt thereof; Optionally, the final concentration of cystamine or its salt in the second buffer solution is 10–30 mM; Optionally, the second buffer solution comprises cystamine dihydrochloride; Optionally, the second buffer solution comprises: 5X SSC; 0.05% Tween-20; and 20 mM cystamine dihydrochloride; 3) The chip is detachably disposed in the liquid circuit system, which includes a reservoir, a valve assembly, and a pump assembly; The valve assembly is configured to connect the reservoir to the pump assembly, or to connect the reservoir to the chip and the pump assembly; The reservoir includes a first synthetic reagent reservoir, which is used to store the first synthetic reagent. Optionally, the method includes: using the pump assembly to drive the first synthetic reagent in the first synthetic reagent reservoir through the valve assembly into the chip for an extension reaction, so that in step S1000, the first synthetic reagent flows into the chip to carry out an extension reaction on the chip; Optionally, the valve assembly and the chip are connected via a common conduit. The recovery of the first synthetic reagent after the reaction and / or at least a portion of the first synthetic reagent that did not participate in the reaction includes: using the pump group to drive the first synthetic reagent after the extended reaction in the chip and / or the first synthetic reagent that did not participate in the extended reaction in the common pipeline to flow in the direction toward the first synthetic reagent reservoir, so as to recover the first synthetic reagent after the reaction and at least a portion of the first synthetic reagent that did not participate in the reaction. Optionally, the first synthetic reagent reservoir is connected to the valve assembly via a first pipeline, wherein, The first synthetic reagent after the reaction is recovered into the first pipeline; and / or, The first synthetic reagent that did not participate in the reaction was recovered into the first pipeline and / or the first synthetic reagent reservoir; Optionally, before step S1000: The recovered first synthetic reagent is introduced from the first pipeline through the valve group into the chip to clean the chip; Optionally, the first synthetic reagent recovered into the first pipeline and the first synthetic reagent reservoir is 50% to 80% of the total amount of the first synthetic reagent used, preferably 60% to 65%. Optionally, the second synthetic reagent reservoir is connected to the valve assembly via a second pipeline, and the reacted second synthetic reagent is recovered into the second pipeline; and / or, Unreacted second synthetic reagent is recovered into the second pipeline and / or the second synthetic reagent reservoir; Optionally, the recovered second synthetic reagent after the reaction is introduced from the second pipeline through the valve assembly into the chip to replace the solution in the chip after the extended reaction; Optionally, the amount of the second synthetic reagent recovered into the second pipeline and the second synthetic reagent reservoir is 50% to 80% of the total amount of the second synthetic reagent used, preferably 60% to 65%. Optionally, the process of allowing the developing agent to flow into the chip includes: The first buffer solution is introduced into the common tubing to push the developing reagent in the common tubing into the chip; Optionally, the process of allowing the cutting reagent to flow into the chip includes: The first buffer solution is introduced into the common tubing to push the cutting reagent in the common tubing into the chip; 4) The method further includes: Before or after the first synthetic reagent flows into the chip, a predetermined volume of air is drawn into the chip; 5) Synthesis-as-sequencing is performed at 55–65 degrees Celsius, preferably 60 degrees Celsius.

9. A sequencing method, characterized in that, include: S10000, a first synthetic reagent is fed into a chip to carry out an extension reaction on the chip, the first synthetic reagent comprising a first nucleotide, the first nucleotide containing a cleavable blocking group and an optically detectable label; S20000, a developing agent is flowed into the chip to excite the optically detectable marker to generate a signal, and the signal is collected; S30000, a cutting reagent is introduced into the chip to remove the blocking group and the optically detectable marker; S40000, return to step S10000, and repeat steps S10000 to S30000 until the sequencing reaction is complete. Specifically, step S20000, which involves allowing the developing reagent to flow into the chip, includes: The developing reagent is pushed into the chip using a first buffer solution; and / or, Step S30000, which involves allowing the cutting reagent to flow into the chip, includes: The cutting reagent is pushed into the chip using the first buffer solution.

10. The method according to claim 9, characterized in that, The sequencing method satisfies one or more of the following conditions: 1) After step S10000 and before step S20000, the method further includes: S10000-1, allowing a second synthetic reagent to flow into the chip, the second synthetic reagent containing a second nucleotide, the second nucleotide containing a cleavable blocking group but not containing an optically detectable label; 2) Before step S10000, the first buffer solution is allowed to flow into the chip to clean the chip, which is used for sequencing while synthesis; and / or, before step S30000, the first buffer solution is allowed to flow into the chip to clean the chip; Optionally, in step S30000, the flow of the cutting reagent into the chip includes flowing the first buffer solution into the chip and pushing the cutting reagent into the chip in one or more portions; Optionally, the pH of the first synthetic reagent, the cleavage reagent, and the first buffer solution is greater than 8; Optionally, the pH of the first synthetic reagent, the cleavage reagent, and the first buffer solution is 9 to 10; Optionally, after step S30000, a second buffer solution is allowed to flow into the chip to clean the chip; Optionally, the step of flowing the second buffer solution into the chip includes flowing the second buffer solution toward the chip and pushing the first buffer solution, so that the first buffer solution and the second buffer solution enter the chip in sequence to clean the chip; Optionally, the second buffer solution comprises SSC, Tween-20, and cystamine or a salt thereof; Optionally, the final concentration of cystamine or its salt in the second buffer solution is 10–30 mM; Optionally, the second buffer solution comprises cystamine dihydrochloride; Optionally, the second buffer solution comprises: 5X SSC; 0.05% Tween-20; and 20 mM cystamine dihydrochloride; 3) The chip is detachably disposed in the liquid circuit system, which includes a reservoir, a valve assembly, and a pump assembly; The valve assembly is configured to connect the reservoir to the pump assembly, or to connect the reservoir to the chip and the pump assembly; The reservoir includes a first synthetic reagent reservoir, which is used to store a first synthetic reagent. The valve assembly and the chip are connected via a common conduit. The method includes: after step S10000 and before step S20000, using the pump group to drive the first synthetic reagent after the extension reaction in the chip and / or the first synthetic reagent in the common pipeline that did not participate in the extension reaction to flow in the direction toward the first synthetic reagent reservoir, so as to recover at least a portion of the first synthetic reagent after the reaction and the first synthetic reagent that did not participate in the reaction. Optionally, the first synthetic reagent reservoir is connected to the valve assembly via a first pipeline, wherein, The first synthetic reagent after the reaction is recovered into the first pipeline; and / or, The first synthetic reagent that did not participate in the reaction was recovered into the first pipeline and / or the first synthetic reagent reservoir; Optionally, before step S10000: The recovered first synthetic reagent is introduced from the first pipeline through the valve group into the chip to clean the chip; Optionally, the first synthetic reagent recovered into the first pipeline and the first synthetic reagent reservoir is 50% to 80% of the total amount of the first synthetic reagent used, preferably 60% to 65%. Optionally, the reservoir includes a second synthetic reagent reservoir for storing a second synthetic reagent. in, The process of allowing the second synthetic reagent to flow into the chip includes: The pump group is used to drive the second synthetic reagent in the second synthetic reagent reservoir into the chip through the valve group, and to make the second synthetic reagent after reaction and / or the second synthetic reagent that has not participated in the reaction in the common pipeline flow towards the second synthetic reagent reservoir, so as to recover the second synthetic reagent after reaction and / or the second synthetic reagent that has not participated in the reaction; Optionally, the second synthetic reagent reservoir is connected to the valve assembly via a second pipeline, and the reacted second synthetic reagent is recovered into the second pipeline; and / or, Unreacted second synthetic reagent is recovered into the second pipeline and / or the second synthetic reagent reservoir; Optionally, the recovered second synthetic reagent after the reaction is introduced from the second pipeline through the valve assembly into the chip to replace the solution in the chip after the extended reaction; Optionally, the amount of the second synthetic reagent recovered into the second pipeline and the second synthetic reagent reservoir is 50% to 80% of the total amount of the second synthetic reagent used, preferably 60% to 65%. Optionally, the step of using the first buffer solution to push the developing reagent into the chip includes: The first buffer solution is introduced into the common tubing to push the developing reagent in the common tubing into the chip; Optionally, the step of pushing the cleavage reagent into the chip using the first buffer solution includes: The first buffer solution is introduced into the common tubing to push the cutting reagent in the common tubing into the chip; 4) The sequencing-by-synthesis process is performed at 55–65 degrees Celsius, preferably 60 degrees Celsius.

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