Additive for eluting residues of sequencing reaction, and cleaning agent comprising same

Abstract

Provided is an additive for a cleaning agent used for eluting the residues of a sequencing reaction. The additive comprises: a: a mercapto binder, which comprises a thiol and / or mercapto blocking agent; and b: a disulfide or a derivative thereof.

Description

Additives for eluting sequencing reaction residues and cleaning agents containing them Technical Field

[0001] This application relates to the field of gene sequencing technology, specifically to an additive for eluting sequencing reaction residues and a cleaning agent containing the additive. Background Technology

[0002] Next-generation sequencing (NGS) has been widely used in life science research due to its advantages of high throughput and low cost. NGS mainly uses sequencing by synthesis (SBS) as its basic scheme. Based on the base pairing principle, it achieves single-base extension by adding a single dNTP to the 3' end of the primer strand that binds to the template, and collects the signal changes caused by each nucleotide binding. Then, it determines the base sequence of the template based on these collected signal changes. In SBS, to achieve controllable single-base extension, the 3'-OH site of the dNTPs used is generally modified with a reversible termination group. This blocks the continuous extension caused by the continuous addition of dNTPs, thus ensuring that the resolution of each round of base reading is a single base. After each round of single extension reaction, a cleavage reagent can be introduced into the system to cleave the reversible termination group at the 3' end of the extended chain, thereby restoring the terminal base to an extendable state. Then, a washing agent is introduced to elute the reaction residues generated in this round of extension (including remaining dNTPs, polymerase, cleavage reagent, cleaved reversible termination group, etc.) to make the background of the reaction system relatively pure. Then, the reagents required for the next round of single-base extension can be pumped in to continue the sequential reading of subsequent bases.

[0003] It is worth noting that residual polymerase, cleavage reagents, and free radicals from the sequencing process can severely affect the accuracy and efficiency of sequencing. For example, they may affect the recognition and binding of polymerase to the template strand and damage product molecules (such as DNA nanospheres and DNA clusters). The cleaning agents used in related technologies are generally salt ion buffers, which have limitations in their elution power for removing residues and in protecting product molecules.

[0004] Therefore, there is an urgent need to provide a cleaning agent that can effectively remove reaction residues during sequencing and has minimal impact on product molecules. Summary of the Invention

[0005] The first aspect of this application provides an additive for a cleaning agent used to wash away residues from elution sequencing reactions, comprising: a. a thiol binder, including thiols and / or thiol blocking agents; and b. a disulfide or a derivative thereof.

[0006] In some embodiments, the thiol is selected from one or more of the following: methanethiol, ethanethiol, ethylenedithiol, 1-propanethiol, 1,3-propanedithiol, cysteine, glutathione, homocysteine, and cysteine ​​residues contained in proteins; and the thiol blocking agent is selected from one or more of the following: N-ethylmaleimide and iodoacetamide.

[0007] In some embodiments, the disulfide is selected from one or more of the following: cystamine or a salt thereof, diallyl disulfide, and lipoic acid. In some embodiments, the cystamine salt is cystamine hydrochloride, preferably cystamine dihydrochloride.

[0008] In some embodiments, the thiol binder is cysteine, homocysteine, and cysteine ​​residues contained in proteins, preferably cysteine; and the disulfide is cystamine dihydrochloride.

[0009] In some embodiments, the additive comprises: a thiol binder having a final concentration of 1-10 mM, preferably 2-8 mM, more preferably 2-5 mM; and a disulfide or a derivative thereof having a final concentration of 1-12 mM, preferably 2-10 mM.

[0010] In some embodiments, the additive comprises: a thiol binder at a final concentration of 2-8 mM, preferably 2-5 mM, more preferably 3 mM; and a disulfide or a derivative thereof at a final concentration of 2-10 mM, wherein the thiol binder is cysteine, homocysteine, or cysteine ​​residues contained in proteins, preferably cysteine; and the disulfide is cystamine dihydrochloride.

[0011] A second aspect of this application provides a method for preparing a cleaning agent for eluting sequencing reaction residues, comprising: introducing an additive into a base cleaning agent, wherein the additive comprises: a. a thiol binder, including thiols and / or thiol blocking agents; and b. a disulfide or a derivative thereof.

[0012] In some embodiments, the thiol is selected from one or more of the following: methanethiol, ethanethiol, ethylenedithiol, 1-propanethiol, 1,3-propanedithiol, cysteine, glutathione, homocysteine, and cysteine ​​residues contained in proteins; and the thiol blocking agent is selected from one or more of the following: N-ethylmaleimide and iodoacetamide.

[0013] In some embodiments, the disulfide is selected from one or more of the following: cystamine or a salt thereof, diallyl disulfide, and lipoic acid. In some embodiments, the cystamine salt is cystamine hydrochloride, preferably cystamine dihydrochloride.

[0014] In some embodiments, the thiol binder is cysteine, homocysteine, and cysteine ​​residues contained in proteins, preferably cysteine; and the disulfide is cystamine dihydrochloride.

[0015] In some embodiments, the method further includes introducing the additives into the base cleaning agent at the following concentrations: a thiol binder with a final concentration of 1-10 mM, preferably 2-8 mM, more preferably 2-5 mM; and a disulfide or a derivative thereof with a final concentration of 1-10 mM, preferably 2-10 mM.

[0016] In some embodiments, the method further includes introducing the following additives into the base cleaning agent: a thiol binder at a final concentration of 2-8 mM, preferably 2-5 mM, more preferably 3 mM; and a disulfide or a derivative thereof at a final concentration of 2-10 mM, wherein the thiol binder is cysteine, homocysteine, or cysteine ​​residues contained in proteins, preferably cysteine; and the disulfide is cystamine dihydrochloride.

[0017] In some embodiments, the base cleaning agent comprises one or more of the following: Tris-HCl buffer, sodium citrate buffer, Tween-20, EDTA, and sodium chloride solution. In some embodiments, the base cleaning agent further comprises cystamine, wherein the concentration of cystamine is not greater than 2 mM, preferably not greater than 1 mM.

[0018] A third aspect of this application provides a cleaning agent for eluting sequencing reaction residues, comprising: a base cleaning agent and an additive as described in any embodiment of the first aspect of this application.

[0019] In some embodiments, the base cleaning agent comprises one or more of the following: Tris-HCl buffer, sodium citrate buffer, Tween-20, EDTA, and sodium chloride solution. In some embodiments, the base cleaning agent further comprises cystamine, wherein the concentration of cystamine is not greater than 2 mM, preferably not greater than 1 mM.

[0020] A fourth aspect of this application provides a sequencing kit comprising: a cleaning agent according to any embodiment of a third aspect of this application, and optional amplification reagents, characterization reagents, and cleavage reagents. The amplification reagents include: modified nucleotides, a polymerase, and optional sequencing primers. The characterization reagents include: an enzyme or substrate for characterizing the modified nucleotides. The cleavage reagents include: an enzyme for cleaving modifications contained in the modified nucleotides. In some embodiments, the modified nucleotides contain a reversible termination group modified at a 3'-OH site, wherein the reversible termination group includes allyl, hemiacetal, amino, mercapto, and -O-azidomethoxy.

[0021] In some embodiments, the kit further comprises a solid-phase carrier for sequencing. In some embodiments, the solid-phase carrier is a chip.

[0022] A fifth aspect of this application provides a sequencing method comprising the following steps: i. incorporating a single modified nucleotide into a nucleic acid template under conditions suitable for polymerization, wherein the modified nucleotide contains a reversible termination group; ii. characterizing the single modified nucleotide; iii. removing the reversible termination group from the modified nucleotide; and iv. eluting the reaction residues generated in steps i-iii by introducing a cleaning agent according to any embodiment of the third aspect of this application.

[0023] In some embodiments, prior to characterizing the individual modified nucleotide, the method further includes: introducing a cleaning agent according to any embodiment of the third aspect of this application to elute the reaction residue generated in step i.

[0024] In some embodiments, the sequencing is DNB sequencing or Illumina sequencing, preferably DNB sequencing.

[0025] The sixth aspect of this application proposes the application of the cleaning agent according to any embodiment of the third aspect of this application in sequencing, wherein the sequencing is single-end sequencing or paired-end sequencing, preferably paired-end sequencing.

[0026] In some embodiments, the sequencing is DNB sequencing or Illumina sequencing, preferably DNB sequencing.

[0027] The technical solution of this application achieves the following technical effects:

[0028] The additives and cleaning agents containing them used in the embodiments of this application for eluting sequencing reaction residues can effectively remove reaction residues during the sequencing process, including residual dNTPs, polymerase, cleavage reagents, and cleaved reversible termination groups, thereby providing a clean background for subsequent reactions and improving the specific binding of polymerase, cleavage reagents, etc., to target molecules. Furthermore, the additives and cleaning agents containing them proposed in the embodiments of this application can also eliminate free radicals in the reaction residues and provide antioxidant properties, thereby effectively reducing the damage of reaction residues to product molecules during the sequencing process, and thus improving the accuracy and efficiency of sequencing (especially paired-end sequencing). Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 shows the recovery of the second-strand signal in the sequencing results produced by eluting sequencing process residues using the various cleaning agents according to this embodiment;

[0031] Figure 2 shows the recovery of second-stranded Q30 in sequencing results obtained by eluting sequencing process residues using various cleaning agents according to this embodiment;

[0032] Figure 3 illustrates the decrease in second-stranded Q30 in sequencing results obtained by eluting sequencing process residues using various cleaning agents according to this embodiment;

[0033] Figure 4 illustrates the effect of using various cleaning agents according to this embodiment to elute sequencing process residues on reducing run-on reactions. Detailed Implementation

[0034] The present invention will now be described in further detail with reference to specific embodiments. The embodiments given are merely illustrative of the invention and are not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0035] In this application, the terminology used in the embodiments is for the purpose of describing specific embodiments only and is not intended to limit the application. The term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A alone, A and B simultaneously, or B alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "and" relationship.

[0036] The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0037] In the description of this application, the concentration of the relevant components mentioned can refer not only to the specific content of each component, but also to the proportional relationship between the contents of each component. Therefore, as long as the content of the relevant components is scaled up or down proportionally according to the embodiment of this application, it is within the scope disclosed in the embodiment of this application.

[0038] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0039] The abbreviations used in this article have their conventional meanings in the fields of chemistry and biology. The chemical structures and formulas presented in this article are constructed according to the standard rules of chemical valence known in the field of chemistry.

[0040] In this application embodiment, the term "sequencing" can also be called "nucleic acid sequencing" or "gene sequencing," meaning that the three terms are interchangeable in expression, referring to the determination of the base types and sequence of a nucleic acid sequence; it includes sequencing by synthesis (sequencing-by-synthesis, SBS) and / or sequencing by ligation (sequencing-by-ligation, SBL), including DNA sequencing and / or RNA sequencing, including long fragment sequencing and / or short fragment sequencing. The terms "long fragment" and "short fragment" are relative; for example, nucleic acid molecules longer than 1Kb, 2Kb, 5Kb, or 10Kb can be called long fragments, and those shorter than 1Kb or 800bp can be called short fragments; it includes paired-end sequencing, single-end sequencing, and / or paired-end sequencing, etc. The term "paired-end sequencing" or "paired-end sequencing" can refer to the readout of any two segments or parts of the same nucleic acid molecule that do not completely overlap; the term "sequencing" includes the process of binding nucleotides (including nucleotide analogs) to a template and collecting the corresponding signals emitted by the nucleotides (including analogs).

[0041] Sequencing typically involves multiple rounds to determine the order of multiple nucleotides / bases on a template. In this application, each round of sequencing is referred to as "one round of sequencing." A "sequencing round" can be defined as one base extension of four nucleotides / bases. In other words, a "sequencing round" can be defined as determining the base type at any specified position on the template. For sequencing platforms that achieve sequencing based on polymerization or ligation reactions, a sequencing run includes the process of binding four nucleotides (including nucleotide analogs) to a template and collecting the corresponding signals. For platforms that achieve sequencing based on polymerization reactions, the reaction system includes substrate nucleotides, polymerase, and a template. The template has a sequence (sequencing primer) attached to it. Based on the base pairing principle and the polymerization reaction principle, the added substrate nucleotides are ligated to the sequencing primers under the catalysis of the polymerase, achieving the binding of the nucleotide to a specific position on the template. Typically, a sequencing run may include one or more base extension cycles. For example, four nucleotides are added to the reaction system sequentially, and base extension and corresponding signal collection are performed separately, resulting in four base extension attempts in one sequencing run. Alternatively, four nucleotides can be added to the reaction system in any combination, such as in pairs or in a one-to-three combination, with each combination performing base extension and corresponding signal collection separately, resulting in two base extension attempts in one sequencing run. Finally, four nucleotides can be added to the reaction system simultaneously for base extension and signal collection, resulting in one base extension attempt in one sequencing run.

[0042] Correspondingly, the cleaning agent in the embodiments of this application can be used to elute / clean the residues from the sequencing process in the reaction space after each round of sequencing. In other embodiments, the cleaning agent can also be used for elution / cleaning during each round of sequencing. For example, when a round of sequencing includes multiple base extension attempts, the cleaning agent can be introduced after each base extension attempt to elute / clean the residues; or after acquiring a binding signal, the residues in the reaction space, including the characterization reagents, can be eluted / cleaned; or after removing the reversible termination group of the nucleotide or nucleotide analog, the cleaning agent can be introduced to elute / clean the residues from the cleavage reagents. It is understood that the cleaning agent in the embodiments of this application can also be used for elution / cleaning in other steps of the sequencing process, such as cleaning before loading the sequencing chip, cleaning during library fixation (e.g., cleaning during library fixation using probes), cleaning during library amplification on the chip, and cleaning after amplification (e.g., the process of generating DNA clusters). Furthermore, for each of the above cleaning methods, the cleaning agent in the embodiments of this application can be used for one or more cleanings, and this application does not limit the number of times the cleaning agent is used.

[0043] In this application, the term "run-on" refers to a situation where the reversible termination group is removed prematurely due to the presence of a removal reagent, resulting in the addition of more than one nucleotide in a single sequencing run. Run-on causes doping of the nucleotide signal generated in a single sequencing run, thereby affecting base interpretation and reducing sequencing accuracy.

[0044] In this embodiment, the term "incorporation" refers to the attachment of a nucleotide to the free 3' hydroxyl group of a second nucleotide by forming a phosphodiester bond with the 5' phosphate group of the nucleotide. The second nucleotide to which this nucleotide is attached typically appears at the 3' end of the polynucleotide chain, and said nucleotide can be a natural nucleotide dNTP or a non-natural nucleotide, such as a modified nucleotide.

[0045] In the embodiments of this application, the term "nucleotide" may refer to naturally occurring nucleotides as well as non-natural nucleotides (xeno-nucleic acid, XNA), such as modified nucleotides or non-natural nucleotide aptamers, such as those disclosed in WO2022 / 083686A1. In some embodiments, the modified nucleotide may be a nucleotide modified with a reversible blocking group (also called a "reversible termination group"), wherein the reversible blocking group modification may occur at the 3' sugar ring hydroxyl group and / or base, and the reversible blocking group may be selected from one or more of the following: alkyl, aralkyl, alkenyl, alkynyl, allyl (e.g., 3'-O-allyl), aryl, heteroaryl, heterocyclic, benzyl, hemiacetal, azide group, azide (e.g., 3'-O-azidomethyl), amino, ketone, mercapto, isocyanate group, phosphate group, carbonate group, thio, acyl, cyano, alkoxy, aryloxy, heteroaryloxy, or amide group, etc., which dissociate under aqueous conditions to produce a molecule with a free 3'-OH.

[0046] In the embodiments of this application, "derivative" or "analogue" refers to a compound or molecule whose core structure is the same as or very similar to that of the parent compound but has chemical or physical modifications, such as having different or additional side groups, or 2' and / or 3' protecting groups.

[0047] In this application, a "solid-phase carrier" refers to a solid medium capable of immobilizing a nucleic acid sample to be tested for subsequent sequencing. In some embodiments, the solid-phase carrier has a sequence that can recognize and bind to the nucleic acid sample to be tested, thereby immobilizing the nucleic acid sample to be tested. In other embodiments, the solid-phase carrier has pre-sized grooves to match the size of the nucleic acid sample to be tested for immobilization. In still other embodiments, chemical groups are attached to the solid-phase carrier to connect with the nucleic acid sample to be tested via chemical bonds, van der Waals forces, etc., to immobilize it. In some embodiments, the solid-phase carrier may be a bead, a chip, etc.

[0048] In some embodiments, the solid-phase carrier may be a chip. In some embodiments, the chip is used to immobilize a conventional double-stranded DNA library or a single-stranded DNA library. In some embodiments, the chip is also used to prepare DNA nanospheres (DNBs) based on the double-stranded DNA library or the single-stranded DNA library. When the nucleic acid sample to be tested is DNB, the DNB can be immobilized on the chip using, for example, probe technology, and the sequencing chip can be sequenced based on cPAL (combined probe anchoring ligation) or CPAS (combined probe anchoring polymerization) to obtain the nucleic acid information of the DNA nanospheres. This application also considers the use of a cleaning agent for cleaning / eluting during the library immobilization process.

[0049] In the embodiments of this application, "rebound" refers to the ratio of the signal value of the first cycle of the second chain to the signal value of the first cycle of the first chain. The higher the rebound multiple, the better the quality of the second chain, and the higher the data quality of the second chain will be.

[0050] In this embodiment, "basic cleaning agent" refers to a conventional cleaning agent used for cleaning residues during sequencing, which is generally a salt ion buffer, such as Tris-HCl buffer, Saline-Sodium Citrate buffer (SSC), Tween-20, EDTA, sodium chloride solution, or any combination thereof. In some embodiments, the basic cleaning agent may also contain cystamine, wherein the concentration of cystamine is not greater than 2 mM, preferably not greater than 1 mM.

[0051] In this application embodiment, additives can be introduced into the base cleaning agent to obtain a cleaning agent with improved performance. In some embodiments, the additives comprise: a. thiol binding agents, including thiols and / or thiol blocking agents; and b. disulfides or derivatives thereof. The cleaning agents in this application embodiment can reduce non-specific binding between sequencing enzymes and sequencing template strands and eliminate free radicals remaining during sequencing, thereby effectively removing reaction residues during sequencing and reducing damage to product molecules (e.g., DNB or DNA clusters).

[0052] In this application, the term "thiol binder" refers to a compound containing a thiol group (-SH) and capable of binding with another thiol-containing molecule (such as a cysteine ​​residue in a sequencing enzyme) to form a disulfide bond. In some embodiments, the thiol binder includes thiols. In some embodiments, thiols may include biothiols and inorganic thiols. Biothiols may be, for example, cysteine ​​(Cysteine / Cys, C3H7NO2S), glutathione (GSH), homocysteine ​​(Hcy), and cysteine ​​residues contained in proteins. Inorganic thiols may be, for example, methanethiol, ethanethiol, ethylenedithiol, 1-propanethiol, and 1,3-propanedithiol. In some embodiments, the thiol binder is cysteine.

[0053] In this application embodiment, the thiol binding agent may further include a thiol blocking agent, such as N-ethylmaleimide (NEM, C6H7NO2) and / or iodoacetamide (ICH2CONH2), both of which can react rapidly with another molecule containing a thiol group. The thiol binding agent in the additive proposed in this application embodiment can reduce non-specific binding between the sequencing enzyme and the sequencing template strand, and eliminate residual free radicals during sequencing, thereby effectively removing reaction residues during sequencing and reducing damage to product molecules (e.g., DNB or DNA clusters).

[0054] In this application, the term "disulfide" refers to an organosulfur compound containing a disulfide bond (-SS-) having the general formula RSS-R'. In some embodiments, the disulfide may be selected from one or more of the following: cysteine ​​or its salts, diallyl disulfide (also known as diallyl disulfide, Divinyl Disulfide, DVDS), and lipoic acid. In some embodiments, the cysteine ​​salt is cysteine ​​hydrochloride. In some embodiments, the cysteine ​​salt is cysteine ​​dihydrochloride (NH2CH2CH2SSCH2CH2NH2·2HCl), which is a hydrochloride derivative of cysteine ​​containing two amino groups (NH2) and two sulfur atoms (S) linked together by disulfide bonds (SS) and combined with two hydrochloric acid molecules to form the hydrochloride. The disulfide in the additive proposed in this application works synergistically with the thiol binder to reduce the run-on reaction in sequencing, allowing each cycle to proceed sequentially. This effectively removes reaction residues during sequencing and ensures the quality of sequencing, especially two-strand sequencing.

[0055] In some embodiments, the additive comprises a thiol binder at a final concentration of 1-10 mM. In some embodiments, the additive comprises a thiol binder at a final concentration of 2-8 mM, such as 2-7.5 mM, 2-7 mM, 2-6.5 mM, 2-6 mM, 2-5.5 mM, 2-5 mM, 2-4.5 mM, 2-4 mM, 2-3.5 mM, 2-3 mM, or any value in between (including the extreme values). In some embodiments, the additive comprises a thiol binder at a final concentration of 3 mM, which may optionally be cysteine, homocysteine, or cysteine ​​residues contained in proteins, preferably cysteine. The additives containing specific concentrations of thiol binders provided in the embodiments of this application can reduce non-specific binding between sequencing enzymes and sequencing template strands and eliminate residual free radicals during sequencing, thereby effectively removing reaction residues during sequencing and reducing damage to product molecules (e.g., DNB or DNA clusters).

[0056] In some embodiments, the additive further comprises a disulfide or its derivative at a final concentration of 1-12 mM. In some embodiments, the additive comprises a disulfide or its derivative at a final concentration of 1-10 mM, such as 1.5-10 mM, 2-10 mM, 1-9 mM, 1.5-9.5 mM, 2-9.5 mM, 2-8 mM, 2-7 mM, 2-6 mM, 2-5 mM, 2-4 mM, 2-3 mM, 1-2 mM, or any value in between (including extreme values) of a disulfide or its derivative. In some embodiments, the additive comprises a disulfide or its derivative at a final concentration greater than 2 mM, wherein the disulfide is optionally cystamine dihydrochloride. The additives containing specific concentrations of disulfide or its derivatives provided in the embodiments of this application can synergistically work with the thiol binder to effectively remove reaction residues during sequencing and effectively ensure the sequencing quality of the second strand.

[0057] This application also proposes a cleaning agent comprising the additives described in any of the above embodiments. In some embodiments, the cleaning agent comprises a base cleaning agent, which may be a salt buffer. In some embodiments, the base cleaning agent may comprise one or more of the following: Tris-HCl buffer, SSC, Tween-20, EDTA, and sodium chloride solution, and optionally cystamine. The cleaning agent comprising the additives proposed in this application can bind to some reactants (e.g., sequencing enzymes with cysteine ​​residues, cleavage reagents for cleaving reversible groups) (e.g., via disulfide bonds), thereby removing reaction residues in the reaction system more efficiently through competitive binding; the additives proposed in this application can also eliminate residual free radicals during sequencing, thereby protecting product molecules from damage. In addition, the additives proposed in this application also have antioxidant properties, thereby extending the shelf life of each component in the base cleaning agent. The cleaning agent proposed in this application can effectively remove reaction residues from each step of the sequencing process, thereby improving the accuracy and efficiency of sequencing (especially paired-end sequencing).

[0058] This application also proposes a method for preparing a cleaning agent for eluting sequencing reaction residues, comprising: introducing the additives as described in any of the above embodiments into a basic cleaning agent. It is understood that the additive components in this application embodiment can be added sequentially or simultaneously to the basic cleaning agent; this application does not limit the order of addition.

[0059] This application also proposes a sequencing kit comprising a cleaning agent as described in any of the above embodiments. In some embodiments, the kit may further comprise amplification reagents, characterization reagents, and / or cleavage reagents, wherein the amplification reagents are used for the extension of single nucleotides in each sequencing round and may include modified nucleotides, polymerases, and optional sequencing primers; the characterization reagents are used to characterize the single nucleotides incorporated in each sequencing round as recognizable signals, such as fluorescence signals, electrical signals, chemical signals, etc., thereby obtaining sequence information. In some embodiments, appropriate characterization reagents may be selected according to the type of modified nucleotides used for sequencing, such as enzymes or substrates that react with the modified nucleotides. In some embodiments, the cleavage reagents may include reagents for removing blocking groups of the incorporated single nucleotides, such as cleavage enzymes. It is understood that the kit may also include other alternative components or other components used in the art for sequencing, such as solid-phase carriers (e.g., chips or beads) for sequencing; this application does not limit the specific contents of the kit.

[0060] This application also proposes a sequencing method comprising the following steps: i. incorporating a single modified nucleotide into a nucleic acid template under conditions suitable for polymerization, wherein the modified nucleotide contains a reversible termination group; ii. characterizing the single modified nucleotide; iii. removing the reversible termination group from the modified nucleotide; and iv. eluting the reaction residues generated in steps i-iii by introducing a cleaning agent according to any of the above embodiments.

[0061] In some embodiments, the sequencing method further includes: before characterizing the individual modified nucleotide, introducing the cleaning agent to elute the reaction residue generated in step i.

[0062] Understandably, each round of sequencing can involve multiple reactions, such as amplification and extension, nucleotide characterization, and removal of blocking groups, thereby introducing various reagents (e.g., dNTP mix, primers, polymerases, metal ions such as Mg) into the reaction space in multiple steps. 2+ The cleaning agent proposed in this application (such as buffer, luciferase, cutting enzyme, etc.) can be introduced after each or multiple steps to clean the reaction space before each reaction, thereby reducing the reaction background and improving sequencing accuracy.

[0063] This application also proposes the application of the cleaning agent described in any of the above embodiments in sequencing. In some embodiments, the sequencing is single-end sequencing or paired-end sequencing, preferably paired-end sequencing. In some embodiments, the sequencing can be DNB sequencing or Illumina sequencing, preferably DNB sequencing. In some embodiments, the sequencing can be two-strand sequencing in DNB sequencing. The cleaning agent proposed in this application can improve the sequencing quality of the sequencing process, especially reverse sequencing in paired-end sequencing. Specifically, it can increase the fold increase of the second-strand signal (reverse sequencing signal) in paired-end sequencing (i.e., the ratio of the signal value of the first cycle of the second strand to the signal value of the first cycle of the first strand; a higher fold increase indicates better quality of the second strand, and the data quality of the second strand will also be higher), making the sequencing reaction more stable, thereby improving the sequencing quality of the reverse strand in paired-end sequencing.

[0064] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0065] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0066] Example 1

[0067] This embodiment is based on a basic cleaning agent (9# elution buffer 1V2.0, the specific formula of 1L 9# elution buffer 1V2.0 is: cystamine powder, 0.1126g; 20×SSC, 25mL; 3M HCl, 0.012mL; Tween-20, 0.05mL), with thiol-binding agent cysteine ​​as an additive. The elution effect and sequencing results of cleaning agents containing different concentrations of this additive were verified. The specific steps are as follows.

[0068] Different concentrations of cysteine ​​(0.2 mM, 2 mM, 3 mM, 10 mM, and 40 mM) were added to the basic cleaning solution, and the corresponding cleaning solutions were used for cleaning during the sequencing process, serving as the experimental group. Sequencing was performed on the G800 platform (DNBSEQ-G800 Sequencer) with a read length of PE150. The sequencing library was a standard Ecoli library, and the sequencing process strictly followed the operating instructions of the G800 platform. The sequencing reagents used were the matching DNBSEQ-G800RS CoolMPS high-throughput sequencing reagent kit. Compared to the standard procedure, only the basic cleaning solution was replaced with the cleaning solution containing different concentrations of cysteine ​​in this example. Simultaneously, the standard procedure was followed, using a basic cleaning solution without added cysteine ​​for elution, serving as the control group.

[0069] The sequencing process monitoring output of the sequencer was used to obtain information on the recovery of the second strand signal. Using default parameters, Basecall Lite and LiteViwer were used to analyze the sequencing data to obtain statistical values ​​for each base quality.

[0070] Figure 1 illustrates the signal recovery of the second strand in sequencing results obtained by eluting sequencing residues using various cleaning agents according to this embodiment. The horizontal axis represents each experimental group, and the recovery is represented by the Recovery Value on the vertical axis, which is the ratio of the second strand signal recovery to the first strand signal. A higher value indicates a higher recovery factor for the second strand and better data quality. As shown in Figure 1, compared with the control group, the addition of cysteine ​​to the basic cleaning agent increases the signal recovery, and the recovery ratio gradually increases with the increase of cysteine ​​concentration. This demonstrates that the cleaning agent containing additives proposed in this embodiment can effectively be used to elute reaction residues during the sequencing process, reduce reaction background, and improve the quality of sequencing, especially two-strand sequencing.

[0071] Example 2

[0072] The control and experimental groups were set up using the same method as in Example 1, and the base quality values ​​of the sequencing results were statistically analyzed for those with different concentrations of cysteine ​​(specifically 0.2 mM, 2 mM, 3 mM, 10 mM, and 40 mM) added as a cleaning agent. The results are shown in Figure 2.

[0073] Figure 2 illustrates the Q30 recovery of the second strand in sequencing results obtained by eluting sequencing residues using various cleaning agents according to this embodiment. The horizontal axis represents each experimental group, and the recovery is represented by the recovery value on the vertical axis. This recovery value represents the Q30 recovery value of the second strand, which is the Q30 value of the first cycle of the second strand (R2) in the PE150 read length minus the Q30 value of the first cycle of the first strand. It reflects the quality of DNB after the completion of the MDA reaction (i.e., second strand synthesis), thus reflecting the sequencing quality of the second strand—a higher Q30 recovery value indicates higher sequencing quality of the second strand. As shown in Figure 2, compared with the control group, the addition of cysteine ​​in the basal cleaning agent increased the Q30 recovery value of the second strand, and the recovery ratio gradually increased with the increase of cysteine ​​concentration, with better recovery performance observed in the 0.2mM-10mM group, especially in the 2-3mM group. This application demonstrates that the cleaning agent containing additives proposed in the embodiments can be effectively used to elute reaction residues during the sequencing process, reduce reaction background, and improve the quality of sequencing, especially two-strand sequencing.

[0074] Example 3

[0075] The control and experimental groups were set up using the same method as in Example 1, and the base quality values ​​of the sequencing results were statistically analyzed for those with different concentrations of cysteine ​​(specifically 0.2 mM, 2 mM, 3 mM, 10 mM, and 40 mM) added as a cleaning agent. The results are shown in Figure 3.

[0076] Figure 3 illustrates the decrease in Q30 of the second strand in sequencing results obtained by eluting sequencing residues using various cleaning agents according to this embodiment. The horizontal axis represents each experimental group, and the decrease is expressed by the decrease value on the vertical axis. In contrast to the rebound value in Example 2, the decrease value shown in Figure 3 represents the Q30 decrease of the second strand, which is the Q30 value of the first cycle of the second strand (R2) in the PE150 read length minus the Q30 value of the 150th cycle of the second strand. This reflects the sequencing quality throughout the entire second strand sequencing process—the smaller the Q30 decrease value, the higher and more stable the sequencing quality of the second strand. As can be seen from Figure 3, compared with the control group, the addition of cysteine ​​to the basal cleaning agent reduced the rebound value of the Q30 of the second strand, and the decrease was greater with increasing cysteine ​​concentration. The experimental groups with concentrations above 2 mM, especially the 2-10 mM (e.g., 2-3 mM) groups, showed the best reduction. This application demonstrates that the cleaning agent containing additives proposed in the embodiments can be effectively used to elute reaction residues during the sequencing process, reduce reaction background, and improve the quality of sequencing, especially two-strand sequencing.

[0077] Example 4

[0078] Similar to the method in Example 1, this example uses a basic cleaning agent with thiol-binding cysteine ​​and cystamine dihydrochloride as additives to verify the elution effect and sequencing results of cleaning agents containing different concentrations of these additives.

[0079] Specifically, cysteine ​​at a concentration of 3 mM was used to elute residues from different sequencing cycles using cystamine dihydrochloride. The concentrations of cystamine dihydrochloride added were 0.5 mM, 1 mM, 2 mM, and 10 mM, and their corresponding usage times are shown in the table below. Taking the 0.5 mM group as an example, a washing agent containing this concentration of cystamine dihydrochloride and 3 mM cysteine ​​was used to wash away sequencing residues during cycles 31-60 of one-strand sequencing, and so on. Similarly, a washing agent containing only 3 mM cysteine ​​was used as a control (i.e., 0 mM Cystamine). The remaining methods were the same as in Example 1.

[0080] Figure 4 illustrates the effect of using various cleaning agents according to this embodiment to wash away sequencing process residues on reducing run-on reactions. The horizontal axis represents each experimental group, and the vertical axis represents the statistical value of run-on reaction 1. As shown in Figure 4, compared with the control group, the run-on reaction caused by cysteine ​​addition was significantly reduced with the addition of cystamine dihydrochloride. Furthermore, the cleaning agents containing 1-10 mM cystamine dihydrochloride, especially ≥2 mM cystamine dihydrochloride, exhibited significant run-on reaction inhibition function. This indicates that the cleaning agents containing cysteine ​​and cystamine dihydrochloride as additives proposed in this application embodiment can effectively wash away reaction residues during the sequencing process while ensuring the sequencing reaction proceeds sequentially, reducing reaction background, and improving the quality of sequencing, especially two-strand sequencing.

[0081] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0082] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. An additive for a cleaning agent used to wash away residues from sequencing elution reactions, comprising: a. Thiol-binding agents, including thiols and / or thiol-blocking agents; and b. Disulfides or their derivatives.

2. The additive according to claim 1, wherein the thiol is selected from one or more of the following: methanethiol, ethanethiol, ethylenedithiol, 1-propanethiol, 1,3-propanedithiol, cysteine, glutathione, homocysteine, and cysteine ​​residues contained in proteins; and The thiol blocking agent is selected from one or more of the following: N-ethylmaleimide and iodoacetamide.

3. The additive according to claim 1 or 2, wherein the disulfide is selected from one or more of the following: cystamine or a salt thereof, diallyl disulfide, and lipoic acid. Optionally, the cystamine salt is cystamine hydrochloride, preferably cystamine dihydrochloride.

4. The additive according to any one of claims 1 to 3, wherein the thiol binder is cysteine, homocysteine, or a cysteine ​​residue contained in a protein, preferably cysteine; and The disulfide is cystamine dihydrochloride.

5. The additive according to any one of claims 1 to 4, comprising: A thiol binder with a final concentration of 1-10 mM, preferably 2-8 mM, more preferably 2-5 mM; and The final concentration is 1-12 mM, preferably 2-10 mM, of disulfide or its derivative.

6. The additive according to any one of claims 1 to 5, comprising: A thiol binder with a final concentration of 2-8 mM, preferably 2-5 mM, more preferably 3 mM; and Disulfides or their derivatives with a final concentration of 2-10 mM The thiol binder is cysteine, homocysteine, or cysteine ​​residues contained in proteins, preferably cysteine; and The disulfide is cystamine dihydrochloride.

7. A method for preparing a cleaning agent for eluting sequencing reaction residues, comprising: Additives are introduced into the basic cleaning agent, including: The additive comprises: a. Thiol-binding agents, including thiols and / or thiol-blocking agents; and b. Disulfides or their derivatives.

8. The method of claim 7, wherein the thiol is selected from one or more of the following: methanethiol, ethanethiol, ethylenedithiol, 1-propanethiol, 1,3-propanedithiol, cysteine, glutathione, homocysteine, and cysteine ​​residues contained in proteins; and The thiol blocking agent is selected from one or more of the following: N-ethylmaleimide and iodoacetamide.

9. The method according to claim 7 or 8, wherein the disulfide is selected from one or more of the following: cystamine or a salt thereof, diallyl disulfide, and lipoic acid. Optionally, the cystamine salt is cystamine hydrochloride, preferably cystamine dihydrochloride.

10. The method according to any one of claims 7 to 9, wherein the thiol binder is cysteine, homocysteine, or a cysteine ​​residue contained in a protein, preferably cysteine; and The disulfide is cystamine dihydrochloride.

11. The method according to any one of claims 7 to 10, further comprising: The additive is introduced into the base cleaning agent at the following concentrations: A thiol binder with a final concentration of 1-10 mM, preferably 2-8 mM, more preferably 2-5 mM; and The final concentration is 1-10 mM, preferably 2-10 mM, of disulfide or its derivative.

12. The method according to any one of claims 7 to 11, further comprising: The following additives are introduced into the base cleaning agent: A thiol binder with a final concentration of 2-8 mM, preferably 2-5 mM, more preferably 3 mM; and Disulfides or their derivatives with a final concentration of 2-10 mM The thiol binder is cysteine, homocysteine, or cysteine ​​residues contained in proteins, preferably cysteine; and The disulfide is cystamine dihydrochloride.

13. The method according to any one of claims 7 to 12, wherein the base cleaning agent comprises one or more of the following: Tris-HCl buffer, sodium citrate buffer, Tween-20, EDTA, and sodium chloride solution. Optionally, the basic cleaning agent further contains cystamine, wherein the concentration of cystamine is not greater than 2 mM, preferably not greater than 1 mM.

14. A cleaning agent for eluting sequencing reaction residues, comprising: a base cleaning agent and an additive according to any one of claims 1 to 6.

15. The cleaning agent of claim 14, wherein the base cleaning agent comprises one or more of the following: Tris-HCl buffer, sodium citrate buffer, Tween-20, EDTA, and sodium chloride solution. Optionally, the basic cleaning agent further contains cystamine, wherein the concentration of cystamine is not greater than 2 mM, preferably not greater than 1 mM.

16. A sequencing kit comprising: a cleaning agent according to claim 14 or 15, and optional amplification reagents, characterization reagents, and cleavage reagents. The amplification reagents include: Modified nucleotides, polymerases, and optional sequencing primers; The characterization reagents include: enzymes or substrates used to characterize the modified nucleotides; The cleavage reagent includes an enzyme for cleaving the modifications contained in the modified nucleotide. Optionally, the modified nucleotide includes a reversible termination group at the 3'-OH site, wherein the reversible termination group includes allyl, hemiacetal, amino, mercapto, and -O-azidomethoxy. Optionally, the kit further includes a solid-phase carrier for sequencing, which may be a chip.

17. A sequencing method, comprising the following steps: i. Under conditions suitable for polymerization, a single modified nucleotide is incorporated into a nucleic acid template, wherein the modified nucleotide contains a reversible termination group; ii. Characterize the individual modified nucleotide; iii. Removing the reversible termination group from the modified nucleotide; and iv. Elute the reaction residues generated in steps i-iii by introducing the cleaning agent according to claim 14 or 15.

18. The method of claim 17, wherein prior to ii. characterizing the single modified nucleotide, the method further comprises: The cleaning agent according to claim 14 or 15 is introduced to elute the reaction residue generated in step i.

19. The method according to claim 17 or 18, wherein the sequencing is DNB sequencing or Illumina sequencing, preferably DNB sequencing.

20. The application of the cleaning agent according to claim 14 or 15 in sequencing, wherein the sequencing is single-end sequencing or paired-end sequencing, preferably paired-end sequencing.

21. The application according to claim 20, wherein the sequencing is DNB sequencing or Illumina sequencing, preferably DNB sequencing. Preferably, the sequencing is two-stranded sequencing in DNB sequencing.

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