Gene sequencing method

Abstract

The invention discloses a gene sequencing method, which is characterized in that two groups of sequencing signals are obtained by successively carrying out two extension reactions and imaging reactions in a single sequencing cycle, and the sequencing signal of the substrate nucleotide in each extension reaction has distinguishability. The combination of the sequencing signals corresponding to different substrate nucleotides of the two extension reactions also has distinguishability, the combination of the sequencing signals obtained by the two extension reactions is utilized to distinguish the types of the extended nucleotides, and then the sequence of the template polynucleotide to be detected is determined according to complementary base pairing. Therefore, monochromatic sequencing reaction of template polynucleotide is realized, crosstalk between optical channels is avoided, and the sequencing precision is high.

Description

Technical Field

[0001] The present invention relates to a gene sequencing method, and belongs to the field of gene sequencing. Background Art

[0002] At present, high-throughput sequencing technology has developed rapidly and has become a commonly used analytical method for life research. It has the characteristics of high throughput, fast detection speed, flexibility and versatility, and low cost. This technology usually requires the detection of fluorescent signals from the reaction to detect the types of bases incorporated in the sequencing reaction, thereby obtaining sequence information. However, in existing four-color or two-color sequencing platforms, due to signal crosstalk between different wavelengths, when the optical channel of a specific base is photographed, the dyes of other bases will produce a certain amount of fluorescent signals. This is because the different optical channels cannot be completely separated, and the excitation wavelengths and emission wavelengths of different dyes overlap each other. This crosstalk phenomenon causes the sequencing accuracy to be adversely affected. In addition, four-color or two-color imaging also makes the optical system of the sequencer complicated and significantly increases the hardware cost of the sequencer. Therefore, in order to overcome these problems, it is necessary to develop new sequencing methods to improve the accuracy of sequencing and reduce costs. Summary of the Invention

[0003] This application claims priority to the following Chinese patent application: Chinese patent application with application number 202410171020.9 filed on February 6, 2024, and titled “Nucleic Acid Sequencing Method and Sequencing Kit”, the entire contents of which are incorporated herein by reference in their entirety.

[0004] The present invention discloses a gene sequencing method, which obtains two sets of sequencing signals by successively performing two extension reactions and imaging reactions in a single sequencing cycle. The sequencing signals of the substrate nucleotides of each extension reaction are distinguishable, and the combination of sequencing signals corresponding to different substrate nucleotides of the two extension reactions is also distinguishable. The combination of sequencing signals obtained by the two extension reactions is used to distinguish the type of extended nucleotides, thereby realizing a single-color sequencing reaction for the template polynucleotide, and at least partially solving the aforementioned technical problems.

[0005] Specifically, the present invention discloses a gene sequencing method, characterized by comprising:

[0006] Sequencing a template polynucleotide immobilized at a reaction site in a flow cell, providing two sequencing reaction solutions, and sequentially performing nucleotide extension reactions using the two sequencing reaction solutions in each sequencing cycle. Each sequencing cycle incorporates only a single nucleotide, and a single imaging event is performed after each extension reaction to collect fluorescence emission signals.

[0007] Distinguishing the type of incorporated nucleotide monomers based on a combination of fluorescence emission signals obtained after two extension reactions, thereby obtaining the sequence of the template polynucleotide;

[0008] The two sequencing reaction solutions contain a total of four types of nucleotide monomers: A, G, C, and T (or U). One sequencing reaction solution contains at least one type of nucleotide monomer, and the other sequencing reaction solution contains the remaining types of nucleotide monomers. The two sequencing reaction solutions do not contain common types of nucleotide monomers. The relative signal intensities of different types of nucleotide monomers contained in each sequencing reaction solution are different, and at least three of the four types of nucleotide monomers contain nucleotides labeled with a detection label, and at least two types of nucleotide monomers contain nucleotides not labeled with a detection label.

[0009] In a specific embodiment, the relative signal intensity of each nucleotide monomer ranges from [0, 1], which is achieved by mixing nucleotides labeled with detection tags and nucleotides not labeled with detection tags.

[0010] In a specific embodiment, in each sequencing cycle, the fluorescence generated by the first extension reaction is removed before performing the second extension reaction. Preferably, the nucleotides labeled with the detection tag are nucleotides labeled with a 5' terminal phosphate fluorescent label.

[0011] In a preferred embodiment, the structure of the nucleotide labeled with a detection tag is formula (I):

[0012]

[0013] Wherein, Y is O or S, B is a heterocyclic base, and n is an integer of 0-6; R is selected from azidomethyl, amino, allyl, substituted disulfanyl, substituted methoxymethyl, o-nitrobenzyl, coumarins, nitroethyl phosphate, trimethylsilyl, tetrahydropyranyl ether, azido, alkylhydroxylamino, thiophosphate, malonyl, benzyl, acetal, thiocarbamate, and vinyl; and the fluorogenic dye is selected from anthracenes, phenoxazines, acridines, or coumarins having fluorescence switching properties, wherein the anthracenes having fluorescence switching properties include one or more of xanthenes-fluoresceins, carbonanthenes-Beijing oranges, siliconanthenes, germaniumanthenes, phosphoroxanthenes, and thioanthenes.

[0014] In a preferred embodiment, the fluorescence switching property refers to a significant enhancement of the fluorescence signal after sequencing compared to before the sequencing reaction.

[0015] In a preferred embodiment, the structure of the nucleotide without a detection tag is formula (II):

[0016] Wherein Y is O or S; X is O or a group that cannot be degraded by phosphatase, wherein G D is OH or any non-fluorescent organic group; n is an integer from 0 to 6, B is a heterocyclic base, and Cap1 is a reversible termination group;

[0017] Preferably, X is O, G D is selected from optionally substituted C1-10 alkyl, C1-10 alkyloxy, amino, mono- or di-substituted amine, C6-14 aryl, C6-14 aryloxy, C6-14 arylamino, or C3-12 cycloalkyl, C3-12 cycloalkyloxy, C3-12 cycloalkylamino; C2-13 heteroaryl or C2-13 heterocyclyl or a combination thereof; preferably, G D is selected from C1-4 alkyl, C1-4 alkoxy, C1-4 alkylamino or di(C1-4 alkyl)amino, or amino, or phenyl, phenoxy, anilino or a combination thereof;

[0018] Preferably, X is CH2, CF2 or NH, G D is selected from OH, C1-10 alkyl, C1-10 alkyloxy, mono- or di-substituted amine, C6-14 aryl, C6-14 aryloxy, C6-14 arylamino, or C3-12 cycloalkyl, C3-12 cycloalkyloxy, C3-12 cycloalkylamino; C2-13 heteroaryl or C2-13 heterocyclyl or a combination thereof; preferably, G D For OH.

[0019] In a preferred embodiment, the reaction site includes a plurality of reaction volumes created by a plurality of reaction chambers arranged on an array, and the template polynucleotide is immobilized in the reaction volumes; after the sequencing reaction solution is delivered to each reaction volume, each reaction volume can be sealed and / or separated from other reaction volumes on the array; then, the fluorescence emission signal from the detection label can be detected and / or recorded by each reaction volume.

[0020] In a preferred embodiment, one of the two sequencing reaction solutions contains two types of nucleotide monomers, and the other sequencing reaction solution contains another two types of nucleotide monomers, and at most one type of nucleotide monomer contains only nucleotides without detection tag labeling.

[0021] In a preferred embodiment, the relative signal intensities of the two nucleotide monomers in one sequencing reaction solution are (0-0.3, 0.7-1), and the relative signal intensities of the two nucleotide monomers in another sequencing reaction solution are (0.3-0.5, 0.7-1).

[0022] In a preferred embodiment, the relative signal intensities of the two nucleotide monomers in one sequencing reaction solution are (0-0.1, 0.9-1), respectively, and the relative signal intensities of the two nucleotide monomers in another sequencing reaction solution are (0.4-0.5, 0.9-1), respectively; preferably, the relative signal intensities of the two nucleotide monomers in one sequencing reaction solution are (0, 1), respectively, and the relative signal intensities of the two nucleotide monomers in another sequencing reaction solution are (0.4-0.45, 1), respectively.

[0023] In a specific embodiment, the 3' ends of the four types of nucleotide monomers contain a reversible termination group, and the reversible termination group at the 3' end of the incorporated nucleotide is removed before the next sequencing cycle, so that the incorporated nucleotide becomes extendable.

[0024] The monochrome gene sequencing method disclosed in the present invention has the following advantages compared with the existing technology: it has low requirements for the optical system of the sequencer, only requires a single wavelength of excitation light and receives a single wavelength of fluorescence signal. Compared with the current mainstream four-color and two-color sequencers, the imaging system is simple and the hardware cost is greatly reduced; since only monochrome imaging is required, there is no crosstalk between optical channels, and the sequencing accuracy is higher. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 .Shows a schematic diagram of the principles of some embodiments of the gene sequencing method of the present invention.

[0026] Figure 2 . Shows the statistical results of the sequencing error rate corresponding to each sequencing cycle of Example 2 of the present invention.

[0027] Figure 3 . Shows the statistical results of the sequencing error rate corresponding to each sequencing cycle of Example 3 of the present invention.

[0028] Figure 4 . A comparison chart showing the statistical results of the sequencing error rate corresponding to each sequencing cycle of Example 4 of the present invention and the sequencing error rate of Example 3 is shown. DETAILED DESCRIPTION

[0029] Explanation of terms

[0030] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0031] The use of the term "comprising" is not limiting. The use of the term "comprising" is not limiting. As used in this specification, whether in a transitional phrase or in the body of the claims, the terms "comprising" and "including" are to be interpreted as having an open-ended meaning. That is, the above terms are to be interpreted synonymously with the phrases "having at least" or "including at least." For example, when used in the context of a process, the term "comprising" means that the process includes at least the recited steps, but may also include additional steps. When used in the context of a compound, composition, or device, the term "comprising" means that the compound, composition, or device includes at least the recited features or components, but may also include additional features or components.

[0032] The term "or" means one or all of the listed elements, or a combination of any two or more of the listed elements, unless the context indicates otherwise.

[0033] The words "preferred" and "preferably" refer to embodiments of the invention that may offer certain benefits under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention.

[0034] Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxyl orientation.

[0035] Unless otherwise indicated, all headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading. The headings provided herein are not limitations of the various aspects or embodiments of the invention, which can be obtained by reference to the entire specification. Therefore, the terms defined below are more fully defined by reference to the entire specification.

[0036] As used herein, "polymerase" refers to an enzyme that catalyzes the polymerization of nucleotides (i.e., polymerase activity). Typically, the enzyme will initiate synthesis at the 3' end of a primer annealed to a polynucleotide template sequence and proceed toward the 5' end of the template strand. "DNA polymerase" catalyzes the polymerization of deoxyribonucleotides. Deoxyribonucleotides can be natural nucleotides or modified or labeled nucleotides.

[0037] As used in the present invention, " nucleotide " comprises nitrogenous heterocyclic base, ribose and one or more phosphate groups. They are the monomeric units of nucleic acid sequences. In RNA, sugar is ribose, and in DNA it is deoxyribose, that is, lacking the oxygen of the hydroxyl group present in the 2' position of ribose. Nitrogenous heterocyclic base can be purine, deazapurine or pyrimidine base. Purine base includes adenine (A) and guanine (G) and their modified derivatives or analogs, such as 7-deazaadenine or 7-deazaguanine. Pyrimidine base includes cytosine (C), thymine (T) and uracil (U) and their modified derivatives or analogs. The C-1 atom of deoxyribose is bonded to the N-1 of pyrimidine or the N-9 of purine. In some cases, the term " nucleotide " can also encompass nucleotide modifications or conjugates.

[0038] As used in the present invention, the template polynucleotide is a single-stranded nucleotide or nucleotide cluster of any origin and of a length suitable for sequencing by synthesis; preferably, the template polynucleotide is immobilized on the surface of a carrier.

[0039] As used herein, "extending" (or "incorporating") into an oligonucleotide or polynucleotide means forming a covalent bond between a nucleotide and an oligonucleotide or polynucleotide. In some such embodiments, a phosphodiester bond is formed between the 3' hydroxyl group of the oligonucleotide or polynucleotide and the 5' phosphate group of the nucleotide.

[0040] reversible terminator nucleotides

[0041] The nucleic acid sequencing method of the present invention requires that only one base can be extended per sequencing cycle. Therefore, the sequencing method of the present invention is first based on the use of reversible terminator nucleotides. Reversible terminator nucleotides are well known in the field of sequencing and nucleic acid synthesis, such as conventionally used 3'-modified reversible terminator nucleotides (such as oxyamino groups). Those skilled in the art will understand how to connect a suitable blocking group to the 3' position of ribose to block the interaction between the polymerase and the 3'-OH. The aforementioned blocking group can be directly connected to the 3' position of ribose. The blocking group connected to the 3' position can be cleaved (or removed) and turned into a 3'-OH that can be extended again. As used herein, the term "blocking" or "blocking" or "termination" refers to the use of a specific group to form a protection for the 3'-OH of a nucleoside or nucleotide so that the polymerization of a possible polymerase (such as DNA polymerase) is terminated. The group used for blocking is referred to as a "blocking group" or "blocking group" or "termination group" or "protecting group". The blocking group can be removed so that the nucleotide can be turned into an extendable state again. Such blocking is referred to as "reversible blocking" or "reversible blocking" or "reversible termination". The group used for reversible termination is called a "reversible termination group", and the process of removing the reversible termination group and converting the 3' position of the ribose to a 3'-OH group that can be extended can be called "deprotection". Regarding the reversible termination group and the corresponding deprotection reagent: The reversible termination group (Cap1) used in this article can be selected from the following groups: 3'-O-azidomethyl, 3'-O-amino, 3'-O-allyl, 3'-O-substituted disulfanyl, 3'-O-substituted methoxymethyl, 3'-O-o-nitrobenzyl, 3'-O-coumarin, 3'-O-phosphonitrileethyl ester, 3'-O-trimethylsilyl, 3'-O-THP, 3'-O-azido, 3'-O-alkylhydroxylamino, 3'-phosphorothioate, 3'-O-malonyl, 3'-O-benzyl, 3'-O-acetal, 3'-O-thiocarbamate, 3'-vinyl, etc. Other available reversible termination groups also include, for example, those disclosed in international applications WO2014139596A1 and CN201780039312.5. The present invention does not particularly limit the types of reversible termination groups. According to the principles of the present invention, any 3'-OH group used for reversible blocking in the art can be used as a reversible termination group in the present invention. For example, 3'O reversible termination groups that can be cleaved by a reducing agent (e.g., phosphine) include but are not limited to azidomethyl. 3'-O reversible termination groups that can be cleaved by ultraviolet light include but are not limited to nitrobenzyl. 3'-O reversible termination groups that can be cleaved by contact with a Pd aqueous solution include but are not limited to allyl. 3'-O reversible termination groups that can be cleaved by acid include but are not limited to methoxymethyl.3'-O reversible termination groups that can be cleaved by contact with sodium nitrite buffered aqueous solution (pH = 5.5) include, but are not limited to, aminoalkoxy groups. Correspondingly, commonly used deprotection reagents can be selected from: for example, for 3'-O-thiocarbamates, they can be removed under a variety of conditions, such as, non-limiting exemplary conditions include NaIO4 and potassium peroxymonosulfate; for 3'-O-acetals, they can be cleaved by palladium catalysts; for 3'-O-azido or 3'-O-azidomethyl, the azide group can be converted into an amino group by contact with a phosphine, such as, non-limiting exemplary conditions include phosphines, such as trialkylphosphines, non-limiting examples of trialkylphosphines include tris(hydroxymethyl)phosphine (THP), tris(2-carboxyethyl)phosphine (TCEP), tris(hydroxymethyl)phosphine (THMP) or tris(hydroxyethyl)phosphine (THEP), etc. For 3'-vinyl groups, a variety of tetrazine substances can be used to deprotect the 3' vinyl protecting group. The deprotection mechanism of the 3'-vinyl protecting group is likely similar to that of simple tetrazines. For example, substituted tetrazines, including v-tetrazine, as-tetrazine, and / or s-tetrazine, can be used to deprotect the 3'-vinyl terminus. For 3'-O-allyl groups, the blocking group is removed via a palladium-catalyzed deallylation reaction using palladium and triphenylphosphine tris-metasulfonic acid trisodium salt as a ligand. For 3'-O-THP, the THP protecting group is stable under alkaline conditions but unstable under mild acidic conditions. It can be cleaved using, for example, HOAc / THF / H₂O (4:2:1) with slight heating.

[0042] 5'-terminal fluorescently labeled reversible terminating nucleotides

[0043] In some preferred embodiments of the present invention, the realization of the nucleic acid sequencing method relies on reversible termination nucleotides labeled with 5'-terminal phosphate fluorescence. For 5'-terminal phosphate fluorescence-labeled nucleotides, see CN104910229A. Hydroxanthenes, coumarins, and resorufin fluorescent molecules containing hydroxyl groups are introduced into the 5' end of the nucleotide through polyphosphate as detection labels. The nucleotides of this structure can be used as DNA or RNA polymerase substrates, recognized by DNA or RNA polymerase and incorporated into the DNA or RNA chain, and release polyphosphate molecules containing fluorescent groups or self-cleavage molecules containing fluorescent groups. The polyphosphate molecules labeled with terminal phosphate fluorescence can be further decomposed as substrates for enzymes (e.g., alkaline phosphatase) until all phosphate groups are detached from the fluorescent molecules, thereby generating fluorescent signals. The aforementioned fluorescent molecules are fluorophores with fluorescence switching properties. In a preferred embodiment, the sequencing method may include using an enzyme to release the fluorophore with fluorescence switching properties, optionally including first using a DNA polymerase to release the fluorophore substituted with polyphosphate, and then using a phosphatase to remove the substituted polyphosphate, thereby releasing the fluorophore. The dyes that can be used in the embodiments of the present application include anthracenes, phenoxazines, acridines, and coumarins with fluorescence switching properties. The aforementioned anthracenes with fluorescence switching properties include: xanthenes-fluoresceins, carbon anthracenes-Beijing oranges, silicon anthracenes, germanium anthracenes, phosphorus xanthenes, thioanthenes, etc. Exemplary fluorophores include phenolic dyes, such as fluorescein (e.g., methoxyfluorescein), phenoxazines (e.g., resorufin), acridines (e.g., DDAO) and coumarins (e.g., 7-hydroxycoumarin, 3-carboxy-7-hydroxycoumarin, 4-acetic acid-7-hydroxycoumarin, 7-hydroxy-4-trifluoromethylcoumarin), Beijing Orange (Peking Orange, PO), Tokyo Green (TG), Tokyo Magenta (see Table 1 for example structures of the above fluorophores). The chemical reaction of fluorescent nucleic acid substrates based on phenolic dyes is relatively simple because the phenolic oxygen is esterified to a phosphate group. For dyes containing amines (e.g., rhodamine and its derivatives, cresyl violet, etc.), nucleotide substrates can be introduced by introducing self-cleaving groups. Once DNA polymerase incorporates the labeled nucleotide substrate and cleaves between the α- and β-phosphate groups of the nucleotide, the released fluorophore will emit fluorescence directly after cleavage from the nucleotide or after further enzymatic action of other enzymes (e.g., alkaline phosphatase). These new fluorescent molecules are then detected using standard fluorescence detection techniques (e.g., total internal reflection fluorescence, epifluorescence, or confocal microscopy). The fluorescence switching property refers to the fact that the fluorescence signal after sequencing is significantly changed compared to the situation before the sequencing reaction. Commonly, the fluorescence signal after sequencing is significantly enhanced (or increased) compared to the situation before the sequencing reaction.

[0044] The 5'-terminal fluorescently labeled reversible terminator nucleotide used in a preferred embodiment of the present invention has a structure shown in formula (I):

[0045]

[0046] Wherein, Y is O or S, B is a heterocyclic base, and n is an integer of 0-6; R is selected from azidomethyl, amino, allyl, substituted disulfanyl, substituted methoxymethyl, o-nitrobenzyl, coumarins, nitroethyl phosphate, trimethylsilyl, tetrahydropyranyl ether, azido, alkylhydroxylamino, thiophosphate, malonyl, benzyl, acetal, thiocarbamate, and vinyl; and fluorogenic dye is selected from the aforementioned fluorophores having fluorescence switching properties.

[0047] Table 1 summarizes exemplary optional dyes. In order to reduce photodamage during sequencing, preferably, dyes with an excitation wavelength greater than 400 nm can be selected from them.

[0048] Table 1

[0049]

[0050]

[0051] The reversible terminator nucleotides without detection label label (abbreviated as dark nucleotides) in the present invention refer to the situation where no specific signal is generated in the detection event, for example, the nucleotide substrate itself lacks a fluorescent label, and these nucleotide substrates are referred to as dark nucleotides in the present invention. The present invention designs dark nucleotides with a unique structure, which can be used as ordinary non-luminescent substrates and matched with fluorescently labeled substrates to achieve monochrome sequencing reactions. In order to most conveniently achieve sequencing reactions, the reversible terminator group contained in the dark nucleotide is the same as the reversible terminator group contained in the reversible terminator nucleotide labeled with 5′-terminal phosphate fluorescent label, or at least can be deprotected in the same manner.

[0052] In the present invention, single-color sequencing (or single-channel sequencing) refers to a sequencing method that uses a single excitation light source and a single optical channel for detection to identify four nucleotides. The four types of nucleotide analogs can contain the same type of fluorophore or different fluorophores with the same or similar excitation / emission spectra. The aforementioned use of a single excitation light source and a single optical channel for detection is referred to as performing a single imaging event in the present invention. As can be seen, this single imaging event is independent of the number of images captured.

[0053] In the present invention, "relative signal intensity" refers to the ratio of the fluorescence signal intensity of each substrate nucleotide to the fluorescence signal intensity corresponding to the fluorescence signal intensity when each substrate nucleotide molecule in the substrate nucleotide generates a fluorescence signal upon extension (the value range of this value is [0, 1], that is, greater than or equal to 0 and less than or equal to 1), which is achieved by mixing nucleotides labeled with detection tags and nucleotides not labeled with detection tags. For example, if 100% of the A substrate nucleotides are nucleotides labeled with detection tags, that is, each substrate nucleotide molecule generates a fluorescence signal upon extension, then the relative signal intensity of A is 1; if a portion of the T substrate nucleotides are nucleotides labeled with detection tags and the remaining portion are nucleotides not labeled with detection tags, mixing the above two nucleotides and adjusting the ratio between the two can make the relative signal intensity of T a value greater than 0 and less than 1; the C substrate nucleotides can be entirely nucleotides not labeled with detection tags, then the relative signal intensity of C is 0. Detailed Description of the Invention

[0055] In existing high-throughput sequencing platforms, most use four-color or two-color imaging to distinguish four bases. There is signal crosstalk between different wavelengths, which affects the accuracy of sequencing. In addition, four-color or two-color imaging also makes the optical system of the sequencer very complicated and greatly increases the hardware cost of the sequencer. To address these problems, patent CN202080003541.3 proposes a monochrome imaging sequencing method, which uses nucleotides with light-switchable labels. These labels can undergo a reversible change from an “on” state to an “off” state under irradiation with light of a specific wavelength, allowing the fluorescent state of the label to be controlled by light during the sequencing process, thereby achieving monochrome sequencing. However, this monochrome sequencing method must strictly rely on this “light-switchable label”, and ordinary fluorescent labels cannot be applied. In addition, the development and synthesis of light-switchable labels may involve complex chemical processes, which may increase production costs and difficulty. Therefore, the scope of application of this monochrome sequencing method is narrow and has not been widely used. It is necessary to develop a monochrome sequencing method that is both highly versatile and low-cost.

[0056] The present invention provides a single-color gene sequencing method. For example, the schematic diagram of the principle of some embodiments of the single-color sequencing method can be found in Figure 1In a single sequencing cycle, two extension reactions and imaging reactions are performed sequentially, generating two sets of sequencing signals. The signals for different substrate nucleotides in each extension reaction are distinguishable. In this example, the relative signal intensities for substrate nucleotides A and C in the first extension are (1, 0), respectively; and the relative signal intensities for substrate nucleotides G and T in the second extension are (0.5, 1), respectively. The combination of sequencing signals corresponding to different substrate nucleotides in the two extension reactions is also distinguishable. The combination of the two sets of sequencing signals obtained from the two extension reactions is used to distinguish the type of nucleotide extended in that sequencing cycle. Four sequencing cycles are performed: in this example, if the signal combination for the two extension reactions is (0, 1), the extended nucleotide is T; if the signal combination is (1, 0), the extended nucleotide is A; if the signal combination is (0, 0.5), the extended nucleotide is G; and if the signal combination is (0, 0), the extended nucleotide is C. Finally, the sequence of the template polynucleotide to be tested is determined based on complementary base pairing. Since only single-color imaging is required, there is no crosstalk between optical channels, resulting in higher sequencing accuracy.

[0057] Specifically, the present invention discloses a gene sequencing method, comprising: sequencing a template polynucleotide immobilized at a reaction site in a flow cell, providing two sequencing reaction solutions, performing a nucleotide extension reaction in sequence using the two sequencing reaction solutions in each sequencing cycle, incorporating only a single nucleotide in each sequencing cycle, performing a single imaging event after each extension reaction, and collecting a fluorescence emission signal;

[0058] Distinguishing the type of incorporated nucleotide monomers based on a combination of fluorescence emission signals obtained after two extension reactions, thereby obtaining the sequence of the template polynucleotide;

[0059] The two sequencing reaction solutions contain a total of four types of nucleotide monomers: A, G, C, and T (or U). One sequencing reaction solution contains at least one type of nucleotide monomer, and the other sequencing reaction solution contains the remaining types of nucleotide monomers. The two sequencing reaction solutions do not contain common types of nucleotide monomers. The relative signal intensities of different types of nucleotide monomers contained in each sequencing reaction solution are different, and at least three of the four types of nucleotide monomers contain nucleotides labeled with a detection label, and at least two types of nucleotide monomers contain nucleotides not labeled with a detection label.

[0060] In a specific embodiment, one sequencing reaction solution contains one nucleotide monomer (for example, A), and another sequencing reaction solution contains the remaining three nucleotide monomers (which can be C, G, and T), and the two sequencing reaction solutions do not contain a common type of nucleotide monomer; in the sequencing reaction solution containing the three types of nucleotide monomers, the relative signal intensities of the three types of nucleotide monomers are different. In other words, the sequencing signals of the three nucleotide monomers can be distinguished from each other. For example, the relative signal intensities of C, G, and T are (0, 0.5, and 1), respectively. Different relative signal intensities are achieved by mixing nucleotides labeled with a detection tag and nucleotides not labeled with a detection tag: the nucleotide (C) with a relative signal intensity of 0 only contains nucleotides not labeled with a detection tag and does not contain nucleotides labeled with a detection tag, the nucleotide (G) with a relative signal intensity of 0.5 contains nucleotides labeled with a detection tag and nucleotides not labeled with a detection tag, and the nucleotide (T) with a relative signal intensity of 1 only includes nucleotides labeled with a detection tag. For a sequencing reaction solution containing only one type of nucleotide monomer, the relative signal intensity of that nucleotide monomer (A) cannot be 0, but can be a value greater than 0 and less than or equal to 1, for example, it can be equal to 1. In each sequencing cycle, a reversibly blocked substrate nucleotide is used, and only one nucleotide is extended in each cycle. The nucleotide extension reaction is performed sequentially using the two aforementioned sequencing reaction solutions. After each extension reaction, a single imaging event is performed and the fluorescence emission signal is collected. For example, the first extension reaction is a sequencing reaction solution containing A, and imaging is performed after extension. The second extension reaction is a sequencing reaction solution containing C, G, and T, and imaging is performed after extension. The type of nucleotide monomer incorporated in the sequencing cycle is then distinguished based on the combination of fluorescence emission signals obtained after the two extension reactions. A total of four sequencing cycles are performed, i.e., cycle 1-cycle 4. Afterwards, the sequence of the template polynucleotide to be tested can be obtained according to the base complementary pairing rules. The specific process is shown in Table 2. If the sequence of the extended nucleotide obtained in the above sequencing cycle is ATGC, then the sequence of the template polynucleotide to be tested is GCAT.

[0061] Table 2

[0062]

[0063] In a specific embodiment, one sequencing reaction solution contains two nucleotide monomers (for example, A and T), and another sequencing reaction solution contains the remaining two nucleotide monomers (which can be C and G); in the aforementioned sequencing reaction solutions, the relative signal intensities of the two types of nucleotide monomers contained in each reaction solution are different. In other words, the sequencing signals of the two nucleotide monomers can be distinguished from each other. For example, the relative signal intensities of A and T are (0, 0.5), respectively, and the relative signal intensities of C and G are (0.5, 1); the nucleotide (A) with a relative signal intensity of 0 only contains nucleotides without a detection tag and does not contain nucleotides labeled with a detection tag; the nucleotide (T, C) with a relative signal intensity of 0.5 contains nucleotides labeled with a detection tag and nucleotides without a detection tag; the nucleotide (G) with a relative signal intensity of 1 only contains nucleotides labeled with a detection tag. In each sequencing cycle, a reversibly blocked substrate nucleotide is used, and only one nucleotide is extended in each cycle. Nucleotide extension reactions are performed sequentially using the two aforementioned sequencing reaction solutions. After each extension reaction, a single imaging event is performed and the fluorescence emission signal is collected. For example, the first extension reaction is a sequencing reaction solution containing A and T, followed by imaging. The second extension reaction is a sequencing reaction solution containing C and G, followed by imaging. The type of nucleotide monomer incorporated in the sequencing cycle is then distinguished based on the combination of emission signals obtained after the two extension reactions. A total of four sequencing cycles, cycle 1 to cycle 4, are performed. Afterwards, the sequence of the template polynucleotide to be tested can be obtained based on the base complementary pairing rules. The specific process is shown in Table 3. If the sequence of the extended nucleotide obtained in the above sequencing cycle is CGTA, then the sequence of the template polynucleotide to be tested is TACG.

[0064] Table 3

[0065]

[0066] In a preferred embodiment, of the two sequencing reaction solutions provided, one sequencing reaction solution contains two types of nucleotides, and the relative signal intensities of the two nucleotides are (0-0.3, 0.7-1), respectively. The other sequencing reaction solution contains two other types of nucleotides, and the relative signal intensities of the two nucleotides are (0.3-0.5, 0.7-1), respectively. This is easy to understand. In the sequencing method of the present invention, each sequencing cycle relies on the distinguishability of the combination of sequencing signals obtained from two extension reactions. The higher the distinguishability of the signal combination, the higher the sequencing accuracy. For each extension reaction, the sequencing signals of different substrate nucleotides should also have as high a distinction as possible. Taking the aforementioned combination of two nucleotides containing the detection tag as A and T as an example, if the relative signal intensity of A is close to 0, then the relative signal intensity of T should be as close to 1 as possible. Otherwise, as the sequencing reaction proceeds to the later stage, A and T will become increasingly difficult to distinguish. Therefore, the signal distinction between A and T is required to be as high as possible. For example, the relative signal intensity of A is 0-0.3, and the relative signal intensity of T is 0.7-1. For another sequencing reaction solution, this extension reaction needs to distinguish three signal values: 0 and two other signal values. The signal value range obtained by mixing a fluorescent substrate with a non-fluorescent substrate is limited to 0 to 1. Therefore, when one of the other two signal values is close to 1 and the other is close to 0.5, the three signals are best distinguished, i.e., the highest discrimination is achieved. Preferably, the relative signal intensities of the two nucleotide monomers in one sequencing reaction solution are (0-0.1, 0.9-1), respectively, and the relative signal intensities of the two nucleotide monomers in the other sequencing reaction solution are (0.4-0.5, 0.9-1), respectively. Preferably, the relative signal intensities of the two nucleotide monomers in one sequencing reaction solution are (0, 1), respectively, and the relative signal intensities of the two nucleotide monomers in the other sequencing reaction solution are (0.4-0.45, 1), respectively. The higher the distinguishability of the signal combination, the higher the sequencing accuracy.

[0067] In specific embodiments, the fluorescence signal generated by the first extension reaction must not interfere with the collection of the fluorescence signal generated by the second extension reaction. Therefore, the fluorescence generated by the first extension reaction must be removed before the second extension reaction is performed. For example, if the detection tag of the substrate nucleotide is attached to the base, a specific reagent must be introduced after the imaging event corresponding to the first extension reaction to remove the detection tag group. This reagent must not destroy the reversible blocking group of the substrate nucleotide to ensure that only one nucleotide is extended per sequencing cycle.

[0068] In a preferred embodiment, the nucleotides with detection label mark are 5' terminal phosphate fluorescent-labeled nucleotides, which can be used as DNA or RNA polymerase substrates, are recognized by DNA or RNA polymerase and incorporated into DNA or RNA chains, and release polyphosphate molecules comprising fluorescent groups. The polyphosphate molecules with terminal phosphate fluorescent-labeled can be further decomposed as substrates of enzymes (e.g., alkaline phosphatase) until all phosphate groups are disengaged from the fluorescent molecules, thereby generating fluorescent signals, that is to say, the fluorescent molecules generated are free, and therefore, after the single imaging event corresponding to the first extension reaction, cleaning reagents are introduced into the chip to remove the above-mentioned fluorescent groups, preventing them from interfering with the sequencing signals of the second extension reaction. The removal of the fluorescent groups of this kind of substrate nucleotides does not require the use of specific chemical reagents or specific deblocking conditions, has no effect on the structure of the substrate nucleotides, is simple to operate, and will not form molecular scars, which is conducive to the increase of sequencing read length.

[0069] In a preferred embodiment, the structure of the nucleotide labeled with the detection tag is formula (I):

[0070]

[0071] Wherein, Y is O or S, B is a heterocyclic base, and n is an integer from 0 to 6; R is selected from azidomethyl, amino, allyl, substituted disulfanyl, substituted methoxymethyl, o-nitrobenzyl, coumarin, nitroethyl phosphate, trimethylsilyl, tetrahydropyranyl ether, azido, alkylhydroxylamino, thiophosphate, malonyl, benzyl, acetal, thiocarbamate, and vinyl; and the fluorogenic dye is selected from anthracenes, phenoxazines, acridines, and coumarins with fluorescence-switching properties. The anthracenes with fluorescence-switching properties include xanthenes-fluoresceins, carboanthracenes-Beijing oranges, silanthenes, germananthenes, phosphoxanthenes, and thioanthenes. The substrate nucleotide of this structure can serve as a substrate for DNA or RNA polymerase, is recognized by the DNA or RNA polymerase, and is incorporated into the DNA or RNA chain, releasing a polyphosphate molecule containing a fluorescent group. The polyphosphate molecule with terminal phosphate fluorescent labeling can be further decomposed as a substrate of an enzyme (e.g., alkaline phosphatase) until all phosphate groups are detached from the fluorescent molecule, thereby generating a fluorescent signal, that is, the generated fluorescent signal is free, therefore, after the imaging event corresponding to the first extension reaction, the fluorescent group can be removed by introducing a cleaning reagent into the chip to prevent it from interfering with the sequencing signal of the second extension reaction. In addition, a monochrome sequencing reaction is performed using the reversible terminator nucleotide with 5′-terminal phosphate fluorescent labeling disclosed in this embodiment. Since the fluorescent group is connected to the terminal phosphate and the fluorescent group can be released by enzymatic reaction cleavage, no group remains, thus avoiding the formation of molecular scars, and the synthesized polynucleic acid chain can maintain a natural conformation, which is beneficial to the increase in sequencing read length. And since the fluorescent group is connected to the terminal phosphate, substrate synthesis is cheaper.

[0072] For the single-color sequencing reaction disclosed in the present application, nucleotides that are in a "dark state" (that is, nucleotides without fluorescent labeling) are necessary. However, for the aforementioned sequencing using reversible termination nucleotides with 5'-terminal phosphate fluorescent labels, since a fluorescence activation reagent such as alkaline phosphatase is required, non-fluorescently labeled nucleotide monomers (for example, cold nucleotides commonly used in the art, with a structure as shown in formula (III), the terminal phosphate is in an open state, and Cap1 is a reversible termination group) will be hydrolyzed by alkaline phosphatase and cannot be incorporated into the newly synthesized chain by the polymerase. For nucleotides containing detection labels (such as A, C), this will result in all nucleotide molecules incorporated into the newly synthesized chain being able to emit fluorescence, and different nucleotide types will have the same relative signal intensity (that is, 1). For nucleotides that do not contain detection labels (such as G, T), they are directly hydrolyzed by alkaline phosphatase and cannot be incorporated into the newly synthesized chain at all. This will result in only some types of nucleotides containing detection labels (A, C) being able to be incorporated, while the rest of the nucleotides (G, T) cannot be incorporated, so single-color sequencing cannot be achieved at all.

[0073]

[0074] To address this difficulty, the inventors of this application designed a unique structure of unlabeled nucleotides (referred to as dark nucleotides) to achieve single-color sequencing based on reversible termination nucleotides with 5'-terminal fluorescent phosphate labels. In a specific embodiment of the present invention, the structure of the aforementioned dark nucleotide is formula (II):

[0075]

[0076] Wherein Y is O or S; X is O or a group that cannot be degraded by phosphatase, wherein G D is OH or any non-fluorescent organic group; n is an integer from 0 to 6, B is a heterocyclic base, and Cap1 is a reversible termination group;

[0077] Preferably, X is O, G D is selected from optionally substituted C1-10 alkyl, C1-10 alkyloxy, amino, mono- or di-substituted amine, C6-14 aryl, C6-14 aryloxy, C6-14 arylamino, or C3-12 cycloalkyl, C3-12 cycloalkyloxy, C3-12 cycloalkylamino; C2-13 heteroaryl or C2-13 heterocyclyl or a combination thereof; preferably, G D is selected from C1-4 alkyl, C1-4 alkoxy, C1-4 alkylamino or di(C1-4 alkyl)amino, or amino, or phenyl, phenoxy, anilino or a combination thereof;

[0078] Preferably, X is CH2, CF2 or NH, G D is selected from OH, C1-10 alkyl, C1-10 alkyloxy, mono- or di-substituted amine, C6-14 aryl, C6-14 aryloxy, C6-14 arylamino, or C3-12 cycloalkyl, C3-12 cycloalkyloxy, C3-12 cycloalkylamino; C2-13 heteroaryl or C2-13 heterocyclyl or a combination thereof; more preferably, G D For OH.

[0079] In a specific embodiment, the 3' ends of the four types of nucleotide monomers in the sequencing reaction all contain reversible termination groups as described above, and the reversible termination groups at the 3' ends of the incorporated nucleotides are removed before the next sequencing cycle, making the nucleotides extendable.

[0080] In another embodiment, the sequencing reaction of template polynucleotide can be carried out in array, such as on chip.Array can comprise a plurality of reaction volumes such as created by a plurality of reaction chambers arranged on array.Template polynucleotide sequence or its fragment can be fixed in the reaction volume, such as by adsorption or specific binding on the capture molecule on the solid carrier in each reaction volume.After providing reaction solution and being delivered to each reaction volume in reaction mixture, each reaction volume can be sealed and / or separated with other reaction volumes on array.Then, can detect and / or record the signal such as fluorescence information by each reaction volume.Aforementioned sealing (seal) refers to, because the fluorophore of the reversible termination nucleotide of 5 ' terminal phosphoric acid fluorescent labeling in the sequencing reaction can be released by enzymatic reaction fracture, therefore, need it to be limited in microreaction chamber, to prevent or reduce fluorophore diffusion and solution evaporation etc. For example, the micro-reaction chamber can be sealed with a water-immiscible fluid, such as an oil or a water-immiscible gas. Examples of such oils include mineral oil, silicone oil, fluorinated oils (such as perfluorocarbons and HFE-7500, 2-trifluoromethyl-3-ethoxydodecafluorohexane, or Fluorinert), or hydrocarbon oils (such as isoparaffins); examples of such gases include air, nitrogen, oxygen, saturated water vapor, or a rare gas. In a specific embodiment, the micro-reaction chamber can be sealed with fluorinated oil. First, the desired sequencing aqueous phase liquid is introduced into the reaction chamber (flowcell), and then the fluorinated oil is introduced into the reaction chamber to seal the sequencing aqueous phase liquid within the micro-reaction chamber and cover the aqueous phase liquid, thereby preventing the components within the micro-reaction chamber from diffusing or evaporating.

[0081] In a specific embodiment of the present invention, each round of sequencing comprises at least 50 cycles, preferably, comprises at least 75, or at least 100, or at least 150, or at least 200, or at least 250, or at least 300, or at least 350, or at least 400, or at least 450, or at least 500, or at least 600, or at least 700, or at least 800, or at least 900, or at least 1000 cycles.

[0082] Example 1

[0083] This example describes the synthesis of 5'-end-labeled 3'-azidomethyl-2'-deoxynucleoside derivatives that serve as polymerase substrates for extension reactions during sequencing. It should be understood that this example merely illustrates a general synthetic pathway for these derivatives and does not necessarily limit the method presented.

[0084] Part I: Synthesis of 5'-terminally phosphate-labeled 3'-azidomethyl-2'-deoxynucleoside tetraphosphates To synthesize the labeling monophosphate, the hydroxyl group of the labeled molecule is first phosphorylated. The specific method is described as follows:

[0085]

[0086] Where,

[0087] R4 is: H, Me, MeO, COOH, SO3H, F, Cl;

[0088] R5 is: H, Me, MeO, COOH, SO3H, F, Cl;

[0089] R6 is: Me, Et, MeO, EtO.

[0090] The labeled molecule (Ar-OH, 5 mmole) was added to 200 mL of anhydrous acetonitrile. The solid suspension was placed under argon protection and placed in an ice-water bath for 5 min. Phosphorus oxychloride (5 eq) was added to the solution under rapid stirring and stirred rapidly for 5 min. Tributylamine solution (10 eq) was continued to be added to the solution and the reaction was kept stirring in an ice-water bath for 4 hours. The disappearance of the reaction starting material was monitored by TLC. After the starting material was substantially eliminated, 100 mL of 100 mM TEAA buffer solution (pH = 8.0) was added to the solution. The reaction was stirred in an ice-water bath for 1 hour. The resulting mixed solution was concentrated to approximately 1 / 4 of the total volume by rotary evaporation. The resulting solution was filtered through a 0.22 μm aqueous membrane filter and purified by medium-pressure preparative chromatography and a C18 column (0-50% B gradient, where A = 50 mM TEAA pH = 6.9, B = acetonitrile). The target fractions were collected and concentrated to near dryness by rotary evaporation. A small amount of purified water was added to the fractions, and the pH of the solution was adjusted to slightly alkaline (pH = 8) with tetrabutylammonium hydroxide (40% W / V) solution. The corresponding ammonium monophosphate solid product was obtained after freeze-drying. Some representative molecular information is as follows:

[0091]

[0092] 2) Synthesis of 3'-azidomethyl-2'-deoxynucleoside triphosphate ammonium salt: Commercial 3'-azidomethyl-2'-deoxynucleoside monophosphate is activated and reacted with pyrophosphate to obtain the corresponding 3'-azidomethyl-2'-deoxynucleoside triphosphate:

[0093]

[0094] Preparation of 3'-azidomethyl-2'-deoxynucleoside monophosphate ammonium salt: Commercial 3'-azidomethyl-2'-deoxynucleoside monophosphate (5 mmole) (if it is in the form of sodium phosphate, the sodium nucleoside monophosphate needs to be ion-exchanged and replaced with the phosphate form before starting this step) is mixed with 200 mL of purified water, and the pH of the solution is adjusted to a slightly alkaline pH of 8 with tetrabutylammonium hydroxide (40% W / V) solution. At this time, the solution changes from turbidity to clarity. The adjusted solution is transferred to a 1L round-bottom flask and freeze-dried to obtain 3'-azidomethyl-2'-deoxynucleoside monophosphate tetrabutylammonium salt as a white solid. The ammonium salt described in this patent is not limited to tetrabutylammonium salt, and tributylammonium salt and triethylammonium salt are both suitable options.

[0095] Preparation of 3'-azidomethyl-2'-deoxynucleoside triphosphates: Dissolve 3'-azidomethyl-2'-deoxynucleoside monophosphate ammonium salt (5 mmole) in 100 mL of dry acetonitrile. Remove the solvent using a rotary evaporator and vacuum dry. Repeat the process twice with the addition of the same volume of acetonitrile and rotary evaporation. Vacuum dry for 2 h. Add 250 mL of dry, anhydrous acetonitrile to the flask. Cool in an ice-water bath. Add 2 eq of imidazolium benzenesulfonate activator and 3 eq of diisopropylethylamine solution. Stir and react for 30 min in an ice-water bath. Add ammonium pyrophosphate (5 eq) and anhydrous magnesium chloride (5 eq) to the activated nucleoside monophosphate solution. Allow to warm to room temperature with stirring for 24 h. The reaction solution was placed on a rotary evaporator to remove most of the acetonitrile. 100 mL of 100 mM TEAA buffer solution (pH = 8.0) was added to the remaining solution and stirred in an ice-water bath for 1 hour. The resulting mixed solution was extracted twice with dichloromethane using a separatory funnel (100 mL x 2). The organic phase was separated and the resulting aqueous phase was filtered through a 0.22 μm aqueous membrane filter and purified by high-pressure preparative chromatography and a C18 column (0-30% B 20 min, where A = 50 mM TEAA pH = 8.0, B = acetonitrile). The target fraction was collected and concentrated to near dryness on a rotary evaporator. The fraction was then co-evaporated three times with dry acetonitrile on the rotary evaporator (50 mL acetonitrile / time) to effectively remove residual water. The resulting triphosphate product was dissolved in 200 mL of dry acetonitrile and used directly in the next step after the concentration was determined by HPLC. (Yield 45%)

[0096] To the above dissolved 3'-azidomethyl-2'-deoxynucleoside triphosphate acetonitrile solution, add CDI activator (5 eq) and diisopropylethylamine (3 eq) and react at room temperature with stirring for 3 hours to generate the activated trimetaphosphate nucleoside derivative intermediate. Methanol (5 eq) is added to the reaction solution using a microsyringe. After stirring for 30 minutes, the reaction solution is cooled in an ice-water bath. The solid marker monophosphate (3 eq) and anhydrous magnesium bromide (5 eq, added at low temperature, exothermic) synthesized in the previous step are added. The temperature is allowed to rise to room temperature and react for 40 hours. 100 mL of 100 mM TEAA buffer solution (pH = 8.0) is added to the reaction solution and stirred in an ice-water bath for 1 hour. The resulting mixed solution is extracted twice with dichloromethane using a separatory funnel (100 mL x 2). The organic phase was separated and removed, and the obtained aqueous phase was filtered through a 0.22 μm aqueous filter membrane and purified by high-pressure preparative chromatography and a C18 column (0-20% B 20 min, 20-70% B 15 min, where A is 50 mM TEAA solution, pH = 8.0, B = acetonitrile). The target fractions with a purity of ≥99% were analyzed and collected (the impure fraction was collected and concentrated and then purified again). The fractions meeting the purity requirements were pooled and concentrated on a rotary evaporator, redissolved in 100 mL of purified water, and freeze-dried to obtain the target product.

[0097] Part II: Synthesis of 5'-terminally phosphate-labeled 3'-azidomethyl-2'-deoxynucleoside pentaphosphates

[0098] 1) Preparation of ammonium trimetaphosphate: 20 g of commercial sodium trimetaphosphate was dissolved in water and exchanged with Dowex 50W X8(H) ion exchange resin to form a trimetaphosphoric acid solution. The pH of the solution was adjusted to slightly alkaline (pH = 8.0) with tetrabutylammonium hydroxide (40% w / v) solution. The adjusted solution was transferred to a 1 L round-bottom flask and freeze-dried to obtain tetrabutylammonium trimetaphosphate as a white solid.

[0099] 2) Preparation of the marker monophosphate ammonium salt: The marker monophosphate obtained as described in the first part of Example 1 was added to 200 mL of anhydrous acetonitrile and the marker molecule (Ar-OH, 5 mmole). The solid suspension was placed under argon protection in an ice-water bath for 5 minutes. Phosphorus oxychloride (5 eq) was added to the solution with rapid stirring and stirred rapidly for 5 minutes. Tributylamine solution (10 eq) was then added to the solution, and the reaction was stirred in an ice-water bath for 4 hours. The disappearance of the reaction starting material was monitored by TLC. After the starting material was substantially disappeared, 100 mL of 100 mM TEAA buffer solution (pH = 8.0) was added to the solution, and the reaction was stirred in an ice-water bath for 1 hour. The resulting mixed solution was concentrated to approximately 1 / 4 of the total volume by rotary evaporation. The resulting solution was filtered through a 0.22 μm aqueous membrane filter and purified by medium-pressure preparative chromatography and a C18 column (0-50% B gradient, where A = 50 mM TEAA pH = 6.9, B = acetonitrile). The target fractions were collected and concentrated to near dryness by rotary evaporation. A small amount of purified water was added thereto, and the pH of the solution was adjusted to slightly alkaline (pH = 8.0) with tetrabutylammonium hydroxide (40% W / V) solution. The corresponding ammonium monophosphate solid product was obtained after freeze-drying.

[0100] 3) Synthesis of 5'-terminal phosphate-labeled 3'-azidomethyl-2'-deoxynucleoside pentaphosphate:

[0101] Place solid ammonium trimetaphosphate (2 mmole) in a 500 mL round-bottom flask, add 50 mL of dry acetonitrile, remove the solvent using a rotary evaporator, and drain using a vacuum pump. Repeat the process twice with the same volume of acetonitrile and rotary evaporation, then drain using a vacuum pump for 2 hours. Add 250 mL of dry acetonitrile to this flask, cool in an ice-water bath, then add imidazolium benzenesulfonate activator (1.5 eq) and diisopropylethylamine solution (3 eq). Stir and react in an ice-water bath for 30 minutes. Add dried marker monophosphate (1 eq) to this activated trimetaphosphate solution, and continue cooling in an ice-water bath for 3 hours. Then, add dried 3'-azidomethyl-2'-deoxynucleoside monophosphate (1 eq) and dry magnesium chloride (5 eq). Allow to warm to room temperature with stirring for 24 hours. The reaction solution was placed on a rotary evaporator to remove most of the acetonitrile, and 100 mL of 100 mM TEAA buffer solution (pH = 8.0) was added to the remaining solution, and stirred in an ice-water bath for 1 hour. The resulting mixed solution was extracted twice with dichloromethane using a separatory funnel (100 mL * 2). The organic phase was separated and the obtained aqueous phase was filtered through a 0.22 μm aqueous phase filter membrane and purified by high-pressure preparative chromatography and a C18 column (0-30% B20 min, where A = 50 mM TEA pH = 8.0, B = acetonitrile). The target fractions were collected, analyzed, and determined to be ≥99% (the impure portion was collected and concentrated and then purified again). The fractions that met the purity requirements were concentrated by rotary evaporation, redissolved in 100 mL of purified water, and freeze-dried to obtain the target product.

[0102] Part III: Synthesis of 5'-terminal phosphoryl fluorescently labeled 3'-azidomethyl-2'-deoxynucleoside hexaphosphate

[0103] 1) Preparation of ammonium trimetaphosphate: 20 g of commercial sodium trimetaphosphate was dissolved in water and exchanged with Dowex 50W X8(H) ion exchange resin to form a trimetaphosphoric acid solution. The pH of the solution was adjusted to slightly alkaline (pH = 8.0) with tetrabutylammonium hydroxide (40% w / v) solution. The adjusted solution was transferred to a 1 L round-bottom flask and freeze-dried to obtain tetrabutylammonium trimetaphosphate as a white solid.

[0104] 2) Preparation of 3'-azidomethyl-2'-deoxynucleoside triphosphate ammonium salt: 3'-azidomethyl-2'-deoxynucleoside triphosphate ammonium salt was obtained as described in the first part of Example 1.

[0105] 3) Synthesis of 5'-terminally phosphate-labeled 3'-azidomethyl-2'-deoxynucleoside hexaphosphate: Place solid ammonium trimetaphosphate (2 mmole) in a 500 mL round-bottom flask, add 50 mL of dry acetonitrile, remove the solvent using a rotary evaporator, and drain using a vacuum pump. Repeat the process twice with the same volume of acetonitrile and rotary evaporation, then drain using a vacuum pump for 2 h. Add 250 mL of dry acetonitrile to the flask, cool in an ice-water bath, then add imidazolium benzenesulfonate activator (1.5 eq) and diisopropylethylamine solution (3 eq), and stir in an ice-water bath for 30 min. Add dried labeling agent ammonium triphosphate (1 eq) to the activated trimetaphosphate solution, and continue cooling in an ice-water bath for 3 h. Add dried 3'-azidomethyl-2'-deoxynucleoside ammonium triphosphate (1 eq) and dry magnesium chloride (5 eq) to this solution. Allow to warm to room temperature with stirring for 24 h. The reaction solution was placed on a rotary evaporator to remove most of the acetonitrile. 100 mL of 100 mM TEAA buffer solution (pH = 8.0) was added to the remaining solution and stirred in an ice-water bath for 1 hour. The resulting mixed solution was extracted twice with dichloromethane using a separatory funnel (100 mL * 2). The organic phase was separated and the obtained aqueous phase was filtered through a 0.22 μm aqueous phase filter membrane and purified by high-pressure preparative chromatography and a C18 column (0-30% B 20 min, where A = 50 mM TEAA pH = 8.0, B = acetonitrile). The target fractions were collected, analyzed, and determined to be ≥99% (the impure portion was collected and concentrated and then purified again). The fractions that met the purity requirements were concentrated by rotary evaporation, redissolved in 100 mL of purified water, and freeze-dried to obtain the target product solid.

[0106] Part IV: Synthesis of 5'-nonphosphatase-hydrolyzed 3'-azidomethyl-2'-deoxynucleoside triphosphates

[0107] 1) Preparation of 3'-azidomethyl-2'-deoxynucleoside monophosphate ammonium salt:

[0108] Commercially available 3'-azidomethyl-2'-deoxynucleoside monophosphate (5 mmole) (if in Na salt form, the sodium nucleoside monophosphate salt must be ion-exchanged to H form before this step) was mixed with 200 mL of purified water. The pH of the solution was adjusted to slightly alkaline (pH = 8.0) using tetrabutylammonium hydroxide (40% W / V) solution, at which point the solution turned from turbid to clear. The adjusted solution was transferred to a 1 L round-bottom flask and freeze-dried to obtain 3'-azidomethyl-2'-deoxynucleoside monophosphate tetrabutylammonium salt as a white solid.

[0109] 2) Preparation of ammonium methylene diphosphate: (The same procedure applies to difluoromethylene diphosphoric acid or aminodiphosphoric acid) Commercial methylene diphosphoric acid (2 mmole) was mixed with 100 mL of purified water. The pH of the solution was adjusted to slightly alkaline (pH = 8) using tetrabutylammonium hydroxide (40% w / v) solution, at which point the solution turned from turbid to clear. The adjusted solution was transferred to a 500 mL round-bottom flask and freeze-dried to obtain solid tetrabutylammonium methylene diphosphate.

[0110] 3) Preparation of 3'-azidomethyl-2'-deoxynucleoside substituted triphosphate derivatives

[0111] Dissolve 2 mmole of 3'-azidomethyl-2'-deoxynucleoside monophosphate in 100 mL of dry acetonitrile. Remove the solvent using a rotary evaporator and drain using a vacuum pump. Repeat the process twice with the addition of the same volume of acetonitrile and rotary evaporation. Drain using a vacuum pump for 2 h. Add 100 mL of dry, anhydrous acetonitrile to this flask, cool in an ice-water bath, then add 2 eq of imidazolium benzenesulfonate activator and 3 eq of diisopropylethylamine solution. Stir and react for 30 min in an ice-water bath. Add methylene diphosphate ammonium salt (5 eq) and anhydrous magnesium chloride (5 eq) to this activated nucleoside monophosphate solution. Allow to warm to room temperature while stirring for 24 h. Remove the majority of the acetonitrile using a rotary evaporator. Add 100 mL of 100 mM TEAA buffer (pH 8.0) to the remaining solution, stir in an ice-water bath for 1 h, and extract the resulting mixture once with 100 mL of dichloromethane using a separatory funnel. The organic phase was separated and removed, and the resulting aqueous phase was filtered through a 0.22 μm aqueous membrane filter and purified by high-pressure preparative chromatography and a C18 column (0-20% B for 30 min, where A = 50 mM TEAA, pH = 8.0, and B = acetonitrile). Fractions with ≥98% of the target product were collected and analyzed (the impure fraction was collected and concentrated and then purified again). Fractions meeting the purity requirements were pooled and concentrated on a rotary evaporator, redissolved in 50 mL of purified water, and freeze-dried to obtain the target product in a yield of 15%. The corresponding nucleoside triphosphorus derivatives of difluoromethylene diphosphonic acid or aminodiphosphonic acid were synthesized in the same manner.

[0112] Representative compound structures and mass spectrometry data are as follows:

[0113]

[0114] Part V: Synthesis of 5'-nonphosphatase-hydrolyzed 3'-azidomethyl-2'-deoxynucleoside tetraphosphates

[0115] 1) Preparation of ammonium trimetaphosphate: 20 g of commercial sodium trimetaphosphate was dissolved in water and filtered through Dowex 50W X8

[0116] (H) Ion exchange resin was exchanged with trimetaphosphoric acid solution and tetrabutylammonium hydroxide (40% W / V)

[0117] Adjust the pH of the solution to a slightly alkaline pH of 8.0 and transfer the adjusted solution to a 1L

[0118] The round-bottom flask was freeze-dried to obtain tetrabutylammonium trimetaphosphate as a white solid.

[0119] 2) Synthesis of 5'-terminal phosphate-labeled 3'-azidomethyl-2'-deoxynucleoside tetraphosphate:

[0120] Place solid ammonium trimetaphosphate (2 mmole) in a 500 mL round-bottom flask, add 50 mL of dry acetonitrile, remove the solvent using a rotary evaporator, and drain using a vacuum pump. Repeat the process twice with the same volume of acetonitrile and rotary evaporation, then drain using a vacuum pump for 2 h. Add 250 mL of dry acetonitrile to this flask, cool in an ice-water bath, then add imidazolium benzenesulfonate activator (2 eq) and diisopropylethylamine solution (6 eq). Stir and react in an ice-water bath for 30 min. Add solid ammonium 3'-azidomethyl-2'-deoxynucleoside monophosphate (2 mmole) and dry magnesium chloride (5 eq). Keep cooling in an ice-water bath for 2 h. Add dimethylamine solution (5 eq). Allow to warm to room temperature with stirring for 24 h. The reaction solution was placed on a rotary evaporator to remove most of the acetonitrile, and 100 mL of 100 mM TEAA buffer solution (pH = 8.0) was added to the remaining solution, and stirred in an ice-water bath for 1 hour. The resulting mixed solution was extracted twice with dichloromethane using a separatory funnel (100 mL * 2). The organic phase was separated and the obtained aqueous phase was filtered through a 0.22 μm aqueous phase filter membrane and purified by high-pressure preparative chromatography and a C18 column (0-30% B 20 min, where A = 50 mM TEA pH = 8.0, B = acetonitrile). The target fractions were collected, analyzed, and determined to be ≥99% (the impure portion was collected and concentrated and then purified again). The fractions that met the purity requirements were concentrated by rotary evaporation, redissolved in 100 mL of purified water, and freeze-dried to obtain the target product.

[0121]

[0122] Part VI: Synthesis of 5'-nonphosphatase-hydrolyzed 3'-azidomethyl-2'-deoxynucleoside pentaphosphate

[0123] Preparation of 3'-azidomethyl-2'-deoxynucleoside triphosphate ammonium salt: 3'-azidomethyl-2'-deoxynucleoside triphosphate ammonium salt was obtained as described in the first part of Example 1.

[0124] 3'-Azidomethyl-2'-deoxynucleoside triphosphate ammonium salt (1 mmole) was placed in a 100ml round-bottom flask. 30ml of dry acetonitrile was added. The solvent was removed using a rotary evaporator and vacuumed to dryness using an oil pump. The same volume of acetonitrile was added twice and the mixture was rotary evaporated twice, followed by vacuuming to dryness using an oil pump for 2 hours. 50ml of dry acetonitrile was added to the round-bottom flask, and a magnetic bar was added to dissolve the mixture thoroughly. CDI activator (5 eq) and diisopropylethylamine (3 eq) were added to the solution with stirring. The mixture was stirred at room temperature for 3 hours to generate the activated trimetaphosphate nucleoside derivative intermediate. Methanol (5 eq) was added to the reaction solution using a microsyringe. The mixture was stirred for 30 minutes and then cooled in an ice-water bath. The diphosphate derivative ammonium salt (5 eq) and anhydrous dry magnesium chloride (5 eq) were added to the activated nucleoside monophosphate solution. The mixture was allowed to warm to room temperature with stirring for 24 hours. The reaction solution was placed on a rotary evaporator to remove most of the acetonitrile, and 100 mL of 100 mM TEAA buffer solution (pH = 8.0) was added to the remaining solution, and stirred in an ice-water bath for 1 hour. The resulting mixed solution was extracted once with a separatory funnel and 100 mL of dichloromethane. The organic phase was separated and the obtained aqueous phase was filtered through a 0.22 μm aqueous phase filter membrane and purified by high-pressure preparative chromatography and a C18 column (0-20% B 30 min, where A = 50 mM TEAA pH = 8.0, B = acetonitrile). The target fractions were collected, analyzed, and determined to be ≥98% (the impure portion was collected and concentrated and then purified again). The fractions that met the purity requirements were concentrated by rotary evaporation, redissolved in 50 mL of purified water, and freeze-dried to obtain the target product.

[0125] Example 2

[0126] Prepare the following solution:

[0127] The first sequencing reaction solution: 20 mM Tris-HCl pH = 8.3; 10 mM (NH4)2SO4; 50 mM KCl; 1 mM MnCl2; 100 mM glycine; 0.1% Tween, 9°N polymerase, CIP (alkaline phosphatase, calf intestine), PO4 fluorescently labeled dC4Ps. The structural formula of PO4 fluorescently labeled dC4Ps is as follows: The base is simplified as B, and the specific structure of PO can be found in Table 1;

[0128] Second sequencing reaction solution: 20 mM Tris-HCl pH = 8.3; 10 mM (NH4)2SO4; 50 mM KCl; 1 mM MnCl2; 100 mM glycine; 0.1% Tween, 9°N polymerase, CIP (alkaline phosphatase, calf intestine), PO fluorescently labeled dG4Ps and non-fluorescently labeled dG4Ps, the ratio of these two nucleotides being adjusted so that the relative signal intensity of the sequenced dG4Ps is 0.5, PO fluorescently labeled dT4Ps, and non-fluorescently labeled dA4Ps. The structural formulas of PO fluorescently labeled dG4Ps and dT4Ps are as follows: The base is simplified as B, and the specific structure of PO can be found in Table 1; the structural formulas of dG4Ps and dA4Ps without fluorescent labeling are as follows The base is simplified as B;

[0129] Deblocking reaction solution: 20 mM tris (3-hydroxypropyl) phosphine (THPP), 0.5 M NaCl, 50 mM Tris-HCl, pH = 8.6, 0.05% Tween-20;

[0130] Washing solution: 20 mM Tris-HCl pH=8.3; 10 mM (NH4)2SO4; 50 mM KCl; 0.1 mM EDTA and 0.1% Tween 20.

[0131] Oil seal fluid: Novec TM 7500 electronic fluoride liquid (purchased from 3M TM ).

[0132] After constructing the lambda phage genomic DNA library, it is fixed to a sequencing chip, which is then placed in a sequencer to construct nucleic acid clusters through amplification. The following steps are performed:

[0133] 1) Hybridize the sequencing primers onto the prepared DNA array;

[0134] 2) Control the chip temperature to 4°C and introduce the first sequencing reaction solution into the chip;

[0135] 3) Pour 100-200 μL of oil sealant into the chip;

[0136] 4) Raise the temperature of the chip to 55°C to carry out nucleotide polymerization reaction, so that nucleotides are incorporated into the growing nucleic acid chain.

[0137] 3′ end and terminate the continued growth; extension 60s;

[0138] 5) Take a picture, detect the fluorescence signal using a 540 nm light source as the excitation wavelength, take a picture, and store the image;

[0139] 6) First, clean the oil sealant, then pass 400uL of cleaning solution into the chip and clean it three times;

[0140] 7) Control the chip temperature to 4°C and introduce the second sequencing reaction solution into the chip;

[0141] 8) Pour 100-200 μL of oil sealant into the chip;

[0142] 9) Raise the temperature of the chip to 55°C to carry out nucleotide polymerization reaction, so that nucleotides are incorporated into the growing nucleic acid chain.

[0143] 3′ end and terminate the continued growth; extension 60s;

[0144] 10) Take a picture, detect the fluorescence signal using a 540 nm light source as the excitation wavelength, take a picture, and store the image;

[0145] 11) First clean the oil seal liquid, add 200μL of the sealing reaction solution into the chip, and set the chip temperature to

[0146] 55°C for 120 s to remove the blocking group azidomethyl;

[0147] 12) Cleaning: Pour 400 μL of cleaning solution into the chip and clean it three times;

[0148] 13) Repeat steps 2) to 12) (this is one sequencing cycle) to proceed to the next sequencing cycle, for a total of 150 sequencing cycles.

[0149] 14) Sequencing data processing, analysis of sequencing results, the first extension photo is taken to determine the type of extended base based on whether there is a fluorescent signal. If there is fluorescence, it is C; if there is no fluorescence, it is A, G, or T.

[0150] Combined with the relative fluorescence signal intensity obtained from the second extension photo, specific base information can be obtained:

[0151] When there is no signal in the first extension, if the relative fluorescence signal intensity of the second extension is 1, the extended nucleotide is T, and if the relative fluorescence signal intensity of the second extension is 0.5, the extended nucleotide is

[0152] G, if there is no fluorescence, the extended nucleotide is A, by using the above automatic analysis program for sequencing, the sequencing results are obtained and a report is output. The above analysis process can also be simplified as follows: the combination of sequencing signals after two extensions is used as the judgment basis, if the signal combination is (1, 0), the extended nucleotide is C, if the signal combination is (0, 1), the extended nucleotide is T, if the signal combination is

[0153] If the signal combination is (0, 0.5), the extended nucleotide is G. If the signal combination is (0, 0), the extended nucleotide is A. The sequencing results are as follows Figure 2 As shown, a total of 150 cycles were sequenced, with an average error rate of approximately 0.048, which can meet the requirements of single-color sequencing.

[0154] Example 3

[0155] Prepare the following solution:

[0156] The first sequencing reaction solution includes: 20 mM Tris-HCl (pH 8.3); 10 mM (NH4)2SO4; 50 mM KCl; 1 mM MnCl2; 100 mM glycine; 0.1% Tween; 9°N polymerase; CIP (alkaline phosphatase, calf intestine); PO fluorescently labeled dA4Ps and non-fluorescently labeled dA4Ps, the ratio of these two nucleotides being adjusted so that the relative signal intensity of the sequenced dA4Ps is 0.5; and PO fluorescently labeled dC4Ps. The structural formulas of PO fluorescently labeled dA4Ps and dC4Ps are as follows: The base is simplified as B, and the structural formula of dA4Ps without fluorescent label is as follows The base is simplified as B, and the specific structure of PO can be found in Table 1;

[0157] Second sequencing reaction solution: 20 mM Tris-HCl pH = 8.3; 10 mM (NH4)2SO4; 50 mM KCl; 1 mM MnCl2; 100 mM glycine; 0.1% Tween, 9°N polymerase, CIP (alkaline phosphatase, calf intestine), PO fluorescently labeled dG4Ps and non-fluorescently labeled dG4Ps, the ratio of these two nucleotides being adjusted so that the relative signal intensity of the sequenced dG4Ps is 0.5, and PO fluorescently labeled dT4Ps. The structural formulas of PO fluorescently labeled dG4Ps and dT4Ps are as follows: The base is simplified as B, and the specific structure of PO can be found in Table 1; the structural formula of dG4Ps without fluorescent label is as follows The base is simplified as B

[0158] Deblocking reaction solution: 20 mM tris (3-hydroxypropyl) phosphine (THPP), 0.5 M NaCl, 50 mM Tris-HCl, pH = 8.6, 0.05% Tween-20;

[0159] Washing solution: 20 mM Tris-HCl pH=8.3; 10 mM (NH4)2SO4; 50 mM KCl; 0.1 mM EDTA and 0.1% Tween 20.

[0160] Oil seal fluid: Novec TM 7500 electronic fluoride liquid (purchased from 3M TM ).

[0161] After constructing the lambda phage genomic DNA library, it is fixed to a sequencing chip, which is then placed in a sequencer to construct nucleic acid clusters through amplification. The following steps are performed:

[0162] 1) Hybridize the sequencing primers onto the prepared DNA array;

[0163] 2) Control the chip temperature to 4°C and introduce the first sequencing reaction solution into the chip;

[0164] 3) Pour 100-200 μL of oil sealant into the chip;

[0165] 4) Raise the chip temperature to 55°C to allow nucleotide polymerization to occur, allowing nucleotides to be incorporated into the 3′ end of the growing nucleic acid chain and terminate further growth; extend for 60 seconds;

[0166] 5) Take a picture, detect the fluorescence signal using a 540 nm light source as the excitation wavelength, take a picture, and store the image;

[0167] 6) First, clean the oil sealant, then pass 400uL of cleaning solution into the chip and clean it three times;

[0168] 7) Control the chip temperature to 4°C and introduce the second sequencing reaction solution into the chip;

[0169] 8) Pour 100-200 μL of oil sealant into the chip;

[0170] 9) Raise the chip temperature to 55°C to allow nucleotide polymerization to occur, allowing nucleotides to be incorporated into the 3′ end of the growing nucleic acid chain and terminate further growth; extend for 60 seconds;

[0171] 10) Take a picture, detect the fluorescence signal using a 540 nm light source as the excitation wavelength, take a picture, and store the image;

[0172] 11) First, wash off the oil sealant and add 200 μL of deblocking reaction solution to the chip. Set the chip temperature to 55°C and react for 120 seconds to remove the blocking group azidomethyl.

[0173] 12) Cleaning: Pour 400 μL of cleaning solution into the chip and clean it three times;

[0174] 13) Repeat steps 2)-12) to perform the next cycle of sequencing, for a total of 150 cycles of sequencing.

[0175] 14) Sequencing data processing and analysis of sequencing results. The type of extended base is determined based on the relative fluorescence signal intensity during the first extension photograph. If the relative fluorescence signal intensity is 1, the extended nucleotide is C. If the relative fluorescence signal intensity is 0.5, the extended nucleotide is A. If there is no fluorescence, it is G or T. Combined with the relative fluorescence signal intensity obtained from the second extension photograph, the specific base information can be obtained:

[0176] When there is no signal in the first extension, if the relative fluorescence signal intensity of the second extension is 1, the extended nucleotide is T, and if the relative fluorescence signal intensity of the second extension is 0.5, the extended nucleotide is

[0177] G, by using the above automatic analysis program for sequencing, the sequencing results are obtained and a report is output. The above analysis process can also be simplified as follows: the combination of sequencing signals after two extensions is used as the basis for judgment. If the signal combination is (1, 0), the extended nucleotide is C; if the signal combination is (0.5,

[0178] 0), the extended nucleotide is A, if the signal combination is (0, 1), the extended nucleotide is T, if the signal combination is (0, 0.5), the extended nucleotide is G. The sequencing results are as follows Figure 3 As shown, a total of 150 cycles were sequenced, with an average error rate of approximately 0.036, which is significantly lower than the average error rate in Example 2, indicating that each sequencing reaction solution contains a combination of two substrate nucleotides that is more suitable for implementing the monochrome sequencing method of the present application.

[0179] Example 4

[0180] Prepare the following solution:

[0181] The first sequencing reaction solution includes: 20 mM Tris-HCl (pH 8.3); 10 mM (NH4)2SO4; 50 mM KCl; 1 mM MnCl2; 100 mM glycine; 0.1% Tween, 9°N polymerase, CIP (alkaline phosphatase, calf intestine), PO fluorescently labeled dA4Ps and non-fluorescently labeled dA4Ps, the ratio of these two nucleotides being adjusted so that the relative signal intensity of the sequenced dA4Ps is 0.45, and PO fluorescently labeled dC4Ps. The structural formulas of PO fluorescently labeled dA4Ps and dC4Ps are as follows: The base is simplified as B, and the structural formula of dA4Ps without fluorescent label is as follows The base is simplified as B, and the specific structure of PO can be found in Table 1;

[0182] Second sequencing reaction solution: 20 mM Tris-HCl pH = 8.3; 10 mM (NH4)2SO4; 50 mM KCl; 1 mM MnCl2; 100 mM glycine; 0.1% Tween, 9°N polymerase, CIP (alkaline phosphatase, calf intestine), non-fluorescently labeled dG4Ps, PO fluorescently labeled dT4Ps, wherein the structural formula of PO fluorescently labeled dT4Ps is as follows: The base is simplified as B, and the specific structure of PO can be found in Table 1; the structural formula of dG4Ps without fluorescent label is as follows The base is simplified as B

[0183] Deblocking reaction solution: 20 mM tris (3-hydroxypropyl) phosphine (THPP), 0.5 M NaCl, 50 mM Tris-HCl, pH = 8.6, 0.05% Tween-20;

[0184] Washing solution: 20 mM Tris-HCl pH=8.3; 10 mM (NH4)2SO4; 50 mM KCl; 0.1 mM EDTA and 0.1% Tween 20.

[0185] Oil seal fluid: Novec TM 7500 electronic fluoride liquid (purchased from 3M TM ).

[0186] The remaining experimental conditions and experimental steps 1) to 13) are the same as those in Example 3 and will not be repeated here.

[0187] 14) Sequencing data processing and analysis of sequencing results. The first extension photograph determines the type of extended base based on the relative fluorescence signal intensity. If the relative fluorescence signal intensity is 1, the extended nucleotide is C; if the relative fluorescence signal intensity is 0.45, the extended nucleotide is A; if there is no fluorescence, it is G or T. Combined with the relative fluorescence signal intensity obtained from the second extension photograph, specific base information can be obtained: when there is no signal in the first extension, if the relative fluorescence signal intensity of the second extension is 1, the extended nucleotide is T; if there is no fluorescence, the extended nucleotide is G. By using the above automatic analysis program for sequencing, the sequencing results are obtained and a report is output. The above analysis process can also be simplified as follows, using the combination of sequencing signals after the two extensions as the judgment basis: if the signal combination is (1, 0), the extended nucleotide is C; if the signal combination is (0.45, 0), the extended nucleotide is A; if the signal combination is (0, 1), the extended nucleotide is T; if the signal combination is (0, 0), the extended nucleotide is G. A total of 150 cycles were sequenced, with an average error rate of approximately 0.029, which is lower than the error rate in Example 3.

[0188] 15) The data from this experiment were processed and compared with the sequencing data from Example 3. The sequencing results were as follows: Figure 4 As shown, the orange line represents the sequencing error rate per bp in Example 3, and the green line represents the sequencing error rate per bp in Example 4. It can be seen that the error rates of the two are basically the same in the early stage of sequencing (about the first 100 bp). However, as the sequencing reaction proceeds to the later stage, the sequencing error rate of Example 4 is significantly lower than that of Example 3. This shows that under the same experimental conditions, the greater the signal differentiation of the two nucleotides in each extension reaction, the lower the sequencing error rate, which is conducive to achieving a sequencing reaction with a longer read length.

[0189] The above detailed description and examples are given only for a clear understanding of the present invention. They should not be construed as unnecessary limitations. The present invention is not limited to the exact details shown and described, as variations obvious to those skilled in the art are intended to be included within the invention defined by the claims.

Claims

1. A gene sequencing method, characterized in that: include: Sequencing a template polynucleotide immobilized at a reaction site in a flow cell, providing two sequencing reaction solutions, and sequentially performing nucleotide extension reactions using the two sequencing reaction solutions in each sequencing cycle. Each sequencing cycle incorporates only a single nucleotide, and a single imaging event is performed after each extension reaction to collect fluorescence emission signals. Distinguishing the type of incorporated nucleotide monomers based on a combination of fluorescence emission signals obtained after two extension reactions, thereby obtaining the sequence of the template polynucleotide; The two sequencing reaction solutions contain a total of four types of nucleotide monomers, namely A, G, C, T or U. One sequencing reaction solution contains at least one type of nucleotide monomer, and the other sequencing reaction solution contains the remaining types of nucleotide monomers. The two sequencing reaction solutions do not contain a common type of nucleotide monomer; the relative signal intensities of the different types of nucleotide monomers contained in each sequencing reaction solution are different, and at least three of the four types of nucleotide monomers contain nucleotides labeled with a detection label, and at least two nucleotide monomers contain nucleotides not labeled with a detection label.

2. The gene sequencing method according to claim 1, characterized in that The relative signal intensity of each nucleotide monomer ranges from [0,1] and is achieved by mixing nucleotides labeled with detection tags and nucleotides without detection tags.

3. The gene sequencing method according to claim 2, characterized in that In each sequencing cycle, the fluorescence generated by the first extension reaction is removed before the second extension reaction is performed. Preferably, the nucleotides labeled with the detection tag are nucleotides labeled with a 5' terminal phosphate fluorescence.

4. The gene sequencing method according to claim 3, characterized in that The structure of the nucleotide labeled with the detection tag is formula (I): Wherein, Y is O or S, B is a heterocyclic base, and n is an integer of 0-6; R is selected from azidomethyl, amino, allyl, substituted disulfanyl, substituted methoxymethyl, o-nitrobenzyl, coumarins, nitroethyl phosphate, trimethylsilyl, tetrahydropyranyl ether, azido, alkylhydroxylamino, thiophosphate, malonyl, benzyl, acetal, thiocarbamate, and vinyl; and the fluorogenic dye is selected from anthracenes, phenoxazines, acridines, or coumarins having fluorescence switching properties, wherein the anthracenes having fluorescence switching properties include one or more of xanthenes-fluoresceins, carbonanthenes-Beijing oranges, siliconanthenes, germaniumanthenes, phosphoroxanthenes, and thioanthenes.

5. The gene sequencing method according to claim 4, characterized in that The structure of the nucleotide without a detection tag is formula (II): Wherein Y is O or S; X is O or a group that cannot be degraded by phosphatase, wherein G D is OH or any non-fluorescent organic group; n is an integer from 0 to 6, B is a heterocyclic base, and Cap1 is a reversible termination group; Preferably, X is O, G D is selected from optionally substituted C1-10 alkyl, C1-10 alkyloxy, amino, mono- or di-substituted amine, C6-14 aryl, C6-14 aryloxy, C6-14 arylamino, or C3-12 cycloalkyl, C3-12 cycloalkyloxy, C3-12 cycloalkylamino; C2-13 heteroaryl or C2-13 heterocyclyl or a combination thereof; preferably, G D is selected from C1-4 alkyl, C1-4 alkoxy, C1-4 alkylamino or di(C1-4 alkyl)amino, or amino, or phenyl, phenoxy, anilino or a combination thereof; Preferably, X is CH2, CF2 or NH, G D is selected from OH, C1-10 alkyl, C1-10 alkyloxy, mono- or di-substituted amine, C6-14 aryl, C6-14 aryloxy, C6-14 arylamino, or C3-12 cycloalkyl, C3-12 cycloalkyloxy, C3-12 cycloalkylamino; C2-13 heteroaryl or C2-13 heterocyclyl or a combination thereof; preferably, G D For OH.

6. The gene sequencing method according to any one of claims 1 to 5, characterized in that: The reaction site includes a plurality of reaction volumes created by a plurality of reaction chambers arranged on the array, and the template polynucleotide is immobilized in the reaction volume; after the sequencing reaction solution is delivered to each reaction volume, each reaction volume can be sealed and / or separated from other reaction volumes on the array; The fluorescent emission signal from the detection label can then be detected and / or recorded from each reaction volume.

7. The gene sequencing method according to claim 6, characterized in that Of the two sequencing reaction solutions, one sequencing reaction solution contains two types of nucleotide monomers, and the other sequencing reaction solution contains another two types of nucleotide monomers, and at most one type of nucleotide monomer contains only nucleotides without detection tag labels.

8. The gene sequencing method according to claim 7, characterized in that: The relative signal intensities of the two nucleotide monomers in one sequencing reaction solution are (0-0.3, 0.7-1), and the relative signal intensities of the two nucleotide monomers in another sequencing reaction solution are (0.3-0.5, 0.7-1).

9. The gene sequencing method according to claim 8, characterized in that The relative signal intensities of the two nucleotide monomers in one sequencing reaction solution are (0-0.1, 0.9-1), respectively, and the relative signal intensities of the two nucleotides in another sequencing reaction solution are (0.4-0.5, 0.9-1), respectively; preferably, the relative signal intensities of the two nucleotide monomers in one sequencing reaction solution are (0, 1), respectively, and the relative signal intensities of the two nucleotide monomers in the other sequencing reaction solution are (0.4-0.45, 1), respectively.

10. The gene sequencing method according to any one of claims 1 to 9, characterized in that: The 3' ends of the four types of nucleotide monomers contain reversible termination groups, and the reversible termination groups at the 3' ends of the incorporated nucleotides are removed before the next sequencing cycle, so that the incorporated nucleotides become extendable.

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