Kit and application thereof in sequencing

Abstract

The invention discloses a kit and application thereof in sequencing, the kit comprises a first reagent and a second reagent, the first reagent is selected from a disulfide bond reducing agent; and the second reagent is selected from blocking reagents suitable for blocking disulfide bonds.

Description

Kits and their applications in sequencing Technical Field

[0001] The present invention relates to the biological field, and in particular, to a kit and its application in sequencing. Background Art

[0002] Multiple displacement amplification (MDA) is an isothermal strand displacement amplification method. Under constant temperature, multiple random primers randomly anneal to a template. This is followed by a strand displacement reaction under the action of a DNA polymerase with strand displacement activity. The displaced single strands can then randomly bind to primers, anneal, and extend, ultimately forming branched amplifications. This method is primarily based on the unique strand displacement activity of DNA polymerase. MDA technology has been widely used in sequencing. The main steps include sequencing a single strand. After the first round of sequencing, multiple displacement amplification is used to remove the template strand from the first round and generate a complementary strand for the second round of sequencing. The DNA polymerase with strand displacement activity and the first strand are then removed by elution, and the second round of sequencing of the complementary strand is then performed.

[0003] After MDA is completed, DNA polymerase with strand displacement activity needs to be eluted and removed. However, residual DNA polymerase is prone to remain during the elution process. Residual DNA polymerase will entangle with dNTPs (chemically modified deoxyribonucleotides) at the beginning of complementary chain sequencing. Because the molecular structure of modified dNTPs contains fluorescent dyes, the entanglement of non-specifically bound enzymes and dNTPs will increase the sequencing background signal, resulting in a decrease in the signal-to-noise ratio, thereby affecting sequencing quality. Therefore, it is important to screen for reagents that can effectively remove DNA polymerase with strand displacement activity to prevent the enzyme from affecting complementary chain sequencing.

[0004] Summary of the Invention

[0005] The present invention aims to solve the technical problems existing in the prior art at least to a certain extent.

[0006] It should be noted that the present invention is completed based on the following findings of the inventors:

[0007] The inventors of the present invention have discovered that by disrupting the three-dimensional structure of DNA polymerase, it is possible to prevent it from entangled with modified dNTPs with fluorescent dyes. Since the three-dimensional structure of the enzyme protein is mainly formed by disulfide bonds, during the elution process, the inventors creatively first use a disulfide bond reducing agent to open the disulfide bonds in the enzyme structure, and then use a blocking reagent to block the sulfhydryl groups, thereby releasing the dNTPs and reducing the background signal. Furthermore, the inventors found that the residual DNA polymerase during the elution process is mainly due to the presence of hydrogen bonds between the enzyme and the modified chip. Furthermore, the use of a protonating agent during the elution process can interrupt the hydrogen bonds, help remove the DNA polymerase, prevent its residue from affecting subsequent sequencing, and improve sequencing quality. At the same time, the amount of elution reagent used can be reduced, shortening the elution time and improving sequencing efficiency.

[0008] To this end, in one aspect, the present invention provides a kit. According to an embodiment of the present invention, the kit comprises: a first reagent selected from a disulfide bond reducing agent; and a second reagent selected from a blocking reagent suitable for blocking disulfide bonds.

[0009] In the kit according to an embodiment of the present invention, the disulfide bond reducing agent can open the disulfide bonds in the DNA polymerase, and then the sulfhydryl groups can be blocked under the action of the blocking reagent, thereby preventing the entanglement of the sulfhydryl groups with the modified dNTP with fluorescent dye during the sequencing process, thereby preventing the background signal from increasing and the signal-to-noise ratio from decreasing, thereby improving the sequencing quality.

[0010] According to an embodiment of the present invention, the kit further comprises: a third reagent, wherein the third reagent comprises a protonating agent.

[0011] According to an embodiment of the present invention, the protonating agent is selected from an ammonium salt. The inventors have discovered that ammonium salts are highly effective in disrupting hydrogen bonds between DNA polymerase and the modified chip, facilitating the removal of DNA polymerase. Furthermore, ammonium salts are easily removed by elution and are less likely to adversely affect subsequent sequencing.

[0012] According to an embodiment of the present invention, the protonating agent is selected from at least one of ammonium chloride, ammonium nitrate, and ammonium sulfate. This effectively disrupts hydrogen bonds between the DNA polymerase and the modified chip, facilitating the removal of the DNA polymerase. Furthermore, the protonating agent itself is easily removed by elution and is unlikely to adversely affect subsequent sequencing. Ammonium sulfate is particularly effective.

[0013] In another aspect, the present invention provides the use of the above-mentioned kit in sequencing. Thus, using the kit according to an embodiment of the present invention can effectively ensure sequencing quality, reduce the amount of elution reagents used in the sequencing process, shorten the elution time, and improve sequencing efficiency.

[0014] In another aspect, the present invention provides a sequencing method using the above-mentioned kit. According to an embodiment of the present invention, the method comprises: performing a multiple displacement amplification reaction on a template strand on a chip to generate a complementary strand, wherein the multiple displacement amplification process utilizes a DNA polymerase with strand displacement activity; loading a first reagent onto the chip and eluting and removing the first reagent; loading a second reagent onto the chip, incubating, and eluting and removing the second reagent; and performing a sequencing reaction on the template strand or the complementary strand. Thus, using the method according to an embodiment of the present invention, high-quality sequencing data can be obtained, the amount of elution reagent used during the sequencing reaction can be reduced, the elution time can be shortened, and sequencing efficiency can be improved.

[0015] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments with reference to the following drawings, in which:

[0017] FIG1 shows a schematic flow chart of a sequencing method using a kit according to one embodiment of the present invention;

[0018] FIG2 shows a schematic diagram of Q30 value analysis of a DNA double strand according to one embodiment of the present invention;

[0019] FIG3 shows a schematic diagram of mismatch analysis after comparing DNA sequencing data with a standard gene spectrum according to one embodiment of the present invention. DETAILED DESCRIPTION

[0020] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0021] The present invention provides a kit, an application of the kit in sequencing, and a sequencing method using the kit, which will be described in detail below.

[0022] Reagent test kit

[0023] In one aspect, the present invention provides a kit. According to an embodiment of the present invention, the kit comprises: a first reagent selected from a disulfide bond reducing agent; and a second reagent selected from a blocking reagent suitable for blocking disulfide bonds.

[0024] In the kit according to an embodiment of the present invention, the disulfide bond reducing agent can open the disulfide bonds in the DNA polymerase, and then the sulfhydryl groups can be blocked under the action of the blocking reagent, thereby preventing the entanglement of the sulfhydryl groups with the modified dNTP with fluorescent dye during the sequencing process, thereby preventing the background signal from increasing and the signal-to-noise ratio from decreasing, thereby improving the sequencing quality.

[0025] According to an embodiment of the present invention, the disulfide bond reducing agent is selected from at least one of β-mercaptoethanol, dithiothreitol, and tris(2-carboxyethyl)phosphine. The inventors have developed these preferred disulfide bond reducing agents through extensive experimentation, which can effectively break disulfide bonds. Furthermore, these agents are easily eluted and removed, and are unlikely to adversely affect subsequent sequencing. Among these, dithiothreitol (DTT) is particularly effective.

[0026] According to an embodiment of the present invention, the blocking reagent is selected from a reagent that irreversibly blocks sulfhydryl groups. Selecting an irreversible blocking reagent can prevent the blocked sulfhydryl groups from being reversibly restored to disulfide bonds during the sequencing process, thereby ensuring sequencing quality.

[0027] According to an embodiment of the present invention, the blocking reagent is selected from at least one of N-ethylmaleimide and iodoacetamide. The inventors have obtained the above-mentioned preferred blocking reagent through a large number of experiments, which can block the sulfhydryl group to avoid its influence on the sequencing of the complementary chain. In addition, the above-mentioned reagent is easily eluted and removed, and is not easy to have a negative impact on subsequent sequencing. Because iodoacetamide is easily decomposed by light, its stability is slightly worse, but it can still be used in a light-proof environment. At the same time, N-ethylmaleimide has better stability and is less affected by the environment.

[0028] According to an embodiment of the present invention, the kit further comprises: a third reagent, wherein the third reagent comprises a protonating agent.

[0029] The term "protonating agent" as used in the present invention refers to an agent that can cause atoms, molecules or ions to obtain protons (H). The sequencing chip is pre-modified before sequencing. The inventors have found that the residual DNA polymerase during the elution process is mainly due to the presence of hydrogen bonds between the enzyme and the modified chip. Furthermore, the use of a protonating agent during the elution process can break the hydrogen bonds and help remove the DNA polymerase. The combination of the first reagent, the second reagent and the third reagent of the present invention can work synergistically to avoid the adverse effects of DNA polymerase on complementary chain sequencing and improve sequencing quality.

[0030] According to an embodiment of the present invention, the protonating agent is selected from an ammonium salt. The inventors have discovered that ammonium salts are highly effective in disrupting hydrogen bonds between DNA polymerase and the modified chip, facilitating the removal of DNA polymerase. Furthermore, ammonium salts are easily removed by elution and are less likely to adversely affect subsequent sequencing.

[0031] According to an embodiment of the present invention, the protonating agent is selected from at least one of ammonium chloride, ammonium nitrate, and ammonium sulfate. This effectively disrupts hydrogen bonds between the DNA polymerase and the modified chip, facilitating the removal of the DNA polymerase. Furthermore, the protonating agent itself is easily removed by elution and is unlikely to adversely affect subsequent sequencing. Ammonium sulfate is particularly effective.

[0032] According to an embodiment of the present invention, the third reagent further includes a buffer; the protonating agent concentration in the third reagent is 50-80 mM and the pH is 6-7, thereby effectively removing background. Specifically, the buffer can be WB1 buffer. The source of WB1 buffer is not strictly limited and can be prepared by oneself or purchased commercially.

[0033] According to an embodiment of the present invention, the first reagent and the second reagent are provided in independent forms.

[0034] According to an embodiment of the present invention, the first reagent, the second reagent and the third reagent are provided in independent forms.

[0035] According to an embodiment of the present invention, the kit further includes a DNA polymerase and / or a polymerization reaction reagent. According to an embodiment of the present invention, the DNA polymerase is selected from a DNA polymerase with chain displacement activity, such as phi29 DNA polymerase. The DNA polymerase with chain displacement activity, especially phi29 DNA polymerase, has a disulfide bond. The disulfide bond reducing agent mentioned above can be used to open the disulfide bond in the DNA polymerase, and then the sulfhydryl group can be blocked under the action of a blocking reagent, thereby avoiding the entanglement with the modified dNTP with a fluorescent dye during the sequencing process, thereby causing an increase in the background signal and a decrease in the signal-to-noise ratio. At the same time, the use of the above-mentioned protonating agent during the elution process can break the hydrogen bond, which helps to remove the DNA polymerase. The first reagent, the second reagent and the third reagent of the present invention are used in combination to work synergistically, avoid the adverse effects of DNA polymerase on complementary chain sequencing, and improve the sequencing quality.

[0036] The "polymerization reaction reagent" used in the present invention refers to a reagent that can participate in a polymerization reaction. The present invention does not strictly limit the polymerization reaction reagent, and it can be a reagent commonly used in the art required for multiple displacement amplification reactions, reagents required for sequencing reactions, etc.

[0037] Application of kits in sequencing

[0038] In another aspect of the present invention, the present invention provides the use of the aforementioned kit in sequencing. Thus, the use of the kit according to the embodiment of the present invention can effectively ensure the sequencing quality.

[0039] According to an embodiment of the present invention, the sequencing comprises the step of performing multiple displacement amplification using a DNA polymerase having strand displacement activity.

[0040] It should be noted that the features and advantages described above for the kit are also applicable to this application and will not be repeated here.

[0041] Sequencing method using kit

[0042] In another aspect of the present invention, the present invention provides a sequencing method using the above kit. According to an embodiment of the present invention, referring to FIG1 , the method comprises:

[0043] S100 multiple displacement amplification process

[0044] In this embodiment, a multiple displacement amplification reaction is performed on the template strand on the chip to generate complementary strands, wherein a DNA polymerase having strand displacement activity is used in the multiple displacement amplification process.

[0045] According to an embodiment of the present invention, the DNA polymerase having strand displacement activity is selected from phi29 DNA polymerase. Phy29 DNA polymerase is a mesophilic DNA polymerase cloned from the Bacillus subtilis bacteriophage phi29. This enzyme is characterized by unwinding and displacing the leading strand in the enzyme's forward direction from the original template strand, displacing the strand to obtain a complementary strand. The present invention does not impose strict restrictions on the specific operating steps of the multiple displacement amplification process; conventional techniques in the art can be employed, as long as the phi29 DNA polymerase is used.

[0046] In this example, the chip was eluted.

[0047] S200 loads the first reagent onto the chip and elutes

[0048] In this embodiment, the first reagent is loaded onto the chip and then eluted to remove the first reagent, thereby opening the disulfide bonds of the residual DNA polymerase and facilitating subsequent blocking with a blocking reagent.

[0049] According to an embodiment of the present invention, before loading the first reagent onto the chip, the chip is cleaned after the multiple displacement amplification reaction, and the cleaning process uses the third reagent. Sequencing chips are pre-modified before sequencing. The inventors have discovered that residual DNA polymerase during the cleaning process is primarily due to hydrogen bonding between the enzyme and the modified chip. Furthermore, using a protonating agent during the cleaning process can disrupt hydrogen bonds, facilitating the removal of DNA polymerase.

[0050] According to an embodiment of the present invention, the cleaning process includes: loading the third reagent onto the chip after elution, incubating for 2 to 5 minutes, and then eluting the third reagent with an elution reagent. The incubation process allows the third reagent to fully act on hydrogen bonds, disrupting them, thereby preventing residual DNA polymerase. Specifically, the elution reagent is the same as the elution reagent used in step S200.

[0051] According to an embodiment of the present invention, prior to the cleaning process, the chip after the multiple displacement amplification reaction is eluted. Specifically, the elution reagent used in the elution is derived from the MGISEQ-2000RS sequencer kit. This facilitates the elution of the template strand and DNA polymerase, and the composition of the elution solution does not adversely affect subsequent sequencing.

[0052] S300 loads the second reagent onto the chip and elutes

[0053] In this embodiment, the second reagent is loaded onto the chip, incubated, and eluted to remove the second reagent, thereby sealing the opened disulfide bonds.

[0054] According to an embodiment of the present invention, the incubation time is 20 to 40 minutes, so as to irreversibly block all sulfhydryl groups and avoid adverse effects on subsequent second-strand sequencing.

[0055] S400 performs sequencing reaction on template strand or complementary strand

[0056] In this step, a sequencing reaction is performed on the template strand or the complementary strand.

[0057] It should be noted that the present invention is not strict about the order of steps S100 and S500. Specifically, the timing of the template strand sequencing reaction is not strictly limited, as long as the template strand sequencing can be achieved. For example, the template strand can be subjected to a sequencing reaction first, and then the template strand is subjected to a multiple displacement amplification reaction to generate a complementary strand, and then the chip is processed in steps S200 and S300, and then the complementary strand is subjected to a sequencing reaction; or the template strand on the chip can be subjected to a multiple displacement amplification reaction to generate a complementary strand, and then the chip is processed in steps S200 and S300, and then the template strand and the complementary strand are subjected to a sequencing reaction. According to one embodiment of the present invention, the sequencing method further includes: first performing the sequencing reaction on the template strand fixed on the chip, and then performing the multiple displacement amplification reaction to generate a complementary strand. According to another embodiment of the present invention, the sequencing method further includes: performing the multiple displacement amplification reaction on the template strand fixed on the chip to generate a complementary strand, and then treating the chip with the first reagent and the second reagent, and then performing the sequencing reaction on the template strand and the complementary strand.

[0058] According to an embodiment of the present invention, the sequencing is performed on a DNBSEQ sequencing platform.

[0059] It should be noted that the features and advantages described above for the kit are also applicable to the sequencing method using the kit and will not be described in detail here.

[0060] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0061] Example 1

[0062] 1. Experimental equipment

[0063] MGISEQ-2000RS sequencer, MGIDL-200H loader, MGISEQ-2000RS sequencing slide. The excitation wavelengths of the MGISEQ-2000RS sequencer are 532 nm and 650 nm, respectively.

[0064] 2. Reagents and raw materials used in the experiment

[0065] Ammonium bicarbonate (analytical grade), N-ethylmaleimide (analytical grade), ammonium sulfate (analytical grade), elution reagent (MGISEQ-2000RS sequencer kit), WB1 buffer (from BGI), and ultrapure water.

[0066] The following experiments all used E. coli single-stranded circular DNA as a template (i.e., Standard Library Reagent V3.0) and the CoolMPS High-Throughput Sequencing Reagent Set (MGISEQ-2000RS FCL PE100, containing phi29 DNA polymerase) to prepare DNA nanoballs and load them onto the chip for subsequent sequencing.

[0067] dNTP mixture: contains fluorescently modified dATP, dTTP, dCTP and dGTP.

[0068] 3. Experimental Methods and Procedures

[0069] (a) Preparation of blocking reagent:

[0070] Ammonium bicarbonate is dissolved in ultrapure water to prepare an ammonium bicarbonate aqueous solution, and N-ethylmaleimide (NEM for short) is dissolved in the ammonium bicarbonate aqueous solution to obtain a blocking reagent.

[0071] (b) Preparation of 62.5 mM Slide wash reagent (pH 6.45):

[0072] Accurately weigh ammonium sulfate powder and dissolve it in ultrapure water to prepare a 1 M aqueous solution of ammonium sulfate. Add the 1 M aqueous solution of ammonium sulfate to WB1 buffer to prepare a 62.5 mM Slide Wash reagent. Adjust the pH to 6.45 with concentrated H2SO4.

[0073] (c) DNA sequencing method

[0074] Step 1: Load the DNA nanospheres onto the prepared chip;

[0075] Step 2: Pump the prepared dNTP mixture into the chip and perform single-strand sequencing according to the CoolMPS High-Throughput Sequencing Reagent Set instructions;

[0076] Step 3: After the first strand is sequenced, multiple displacement amplification (MDA) is performed using phi29 DNA polymerase to generate the second strand. After the MDA reaction is complete, the excess phi29 DNA polymerase is eluted using an elution reagent (from the CoolMPS High-Throughput Sequencing Reagent Set).

[0077] Step 4: Set up the following experimental groups and perform the following operations:

[0078] Control group: WB1 buffer (adjusted to pH 6.45 with concentrated H2SO4) was pumped into the chip and reacted for 3 minutes, then washed with WB1 buffer and subjected to second-strand sequencing.

[0079] Experimental group: 62.5 mM Slide wash reagent (pH 6.45) was pumped into the chip and reacted for 3 minutes, followed by elution with 62.5 mM Slide wash reagent (pH 6.45) for double-strand sequencing.

[0080] Experimental Group 1: 62.5 mM Slide wash reagent (pH 6.45) was pumped into the chip for 3 minutes, followed by elution with 62.5 mM Slide wash reagent (pH 6.45). DTT was pumped into the chip to break the disulfide bonds in the enzyme protein, followed by elution with an elution reagent. The blocking reagent was pumped into the chip for 30 minutes of incubation, and the solution was eluted again with an elution reagent for double-strand sequencing.

[0081] Experimental Group 2: 62.5mM Slide Wash Reagent (pH 6.45) was pumped into the chip, reacted for 3 minutes, and then eluted with 62.5mM Slide Wash Reagent (pH 6.45). DTT was pumped into the chip to break the disulfide bonds in the enzyme protein, and then the DTT was eluted with the elution reagent. The blocking reagent was pumped into the chip and incubated for 30 minutes. The solution was eluted again with the elution reagent, and then duplex sequencing was performed. To verify that this treatment method is also applicable to sequencing kits that use FT regeneration reagents, the reagent in well 15 of the sequencing reagent reservoir (FT regeneration reagent) in the DNBSEQ-G99RS High-Throughput Sequencing Reagent Set (G99SM FCL PE150, material code 940-000410-00) produced by MGI was used to replace the reagent in well 9 of the reagent reservoir (regeneration reagent) in Experimental Group 1. A parallel experiment was performed (i.e., the other reagents in the operation kit remained unchanged).

[0082] 4. Experimental results

[0083] As shown in Figure 2, compared with the control group that did not undergo disulfide bond opening and blocking treatment and did not use a protonating agent to treat the chip, the PE100 sequencing results showed that the chip had a higher Q30 and a slower Q30 decrease rate after (NH4)2SO4 was used as a protonating agent to clean the chip, DTT was used to open the disulfide bonds, and NEM was used as a blocking agent to block the sulfhydryl groups. The present invention can be applied to different sequencing and regeneration reagents, that is, it has universality for different sequencing and regeneration reagents and is suitable for wide application.

[0084] As shown in FIG3 , compared with the control group that was not subjected to disulfide bond opening and blocking treatment and the chip was not treated with a protonating agent, the PE100 sequencing results showed a lower error rate when (NH 4 ) 2 SO 4 was used as a protonating agent to clean the chip, DTT was used to open the disulfide bonds, and NEM was used as a blocking agent to block the sulfhydryl groups. The present invention is applicable to different sequencing and regeneration reagents, that is, it is universal for different sequencing and regeneration reagents and is suitable for wide application.

[0085] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0086] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A kit, characterized in that: The kit comprises: a first reagent, wherein the first reagent is selected from a disulfide bond reducing agent; The second reagent is selected from blocking reagents suitable for blocking disulfide bonds.

2. The kit according to claim 1, wherein The disulfide bond reducing agent is selected from at least one of β-mercaptoethanol, dithiothreitol and tris(2-carboxyethyl)phosphine.

3. The kit according to claim 1, wherein The disulfide bond reducing agent is selected from dithiothreitol.

4. The kit according to claim 1, wherein The blocking reagent is selected from reagents that irreversibly block sulfhydryl groups.

5. The kit according to claim 1, wherein The blocking reagent is selected from at least one of N-ethylmaleimide and iodoacetamide.

6. The kit according to claim 1, wherein The blocking reagent is selected from N-ethylmaleimide.

7. The kit according to claim 1, wherein The kit further comprises: A third reagent comprises a protonating agent.

8. The kit according to claim 7, characterized in that The protonating agent is selected from ammonium salts.

9. The kit according to claim 7, characterized in that The protonating agent is selected from at least one of ammonium chloride, ammonium nitrate and ammonium sulfate.

10. The kit according to claim 7, characterized in that The protonating agent is selected from ammonium sulfate.

11. The kit according to claim 7, characterized in that The third reagent further includes a buffer; The concentration of the protonating agent in the third reagent is 50-80 mM, and the pH value is 6-7.

12. The kit according to claim 1, wherein The first reagent and the second reagent are provided in separate forms.

13. The kit according to claim 7, characterized in that The first reagent, the second reagent and the third reagent are provided in independent forms.

14. The kit according to claim 1, wherein The kit further comprises a DNA polymerase and / or a polymerization reaction reagent.

15. The kit according to claim 14, characterized in that The DNA polymerase is selected from DNA polymerases having strand displacement activity.

16. Use of the kit according to any one of claims 1 to 15 in sequencing.

17. The use according to claim 16, characterized in that The sequencing comprises the step of performing multiple displacement amplification using a DNA polymerase having strand displacement activity.

18. A sequencing method using the kit according to any one of claims 1 to 15, characterized in that: include: Performing a multiple displacement amplification reaction on the template strand on the chip to generate a complementary strand, wherein a DNA polymerase having strand displacement activity is used in the multiple displacement amplification process; loading the first reagent onto the chip, and eluting to remove the first reagent; loading the second reagent onto the chip, incubating, and eluting to remove the second reagent; The template strand or the complementary strand is subjected to a sequencing reaction.

19. The method according to claim 18, characterized in that The incubation time is 20 to 40 minutes.

20. The method according to claim 18, wherein Before loading the first reagent onto the chip, the chip after the multiple displacement amplification reaction is cleaned, and the third reagent is used in the cleaning process.

21. The method according to claim 20, characterized in that The cleaning comprises: The third reagent is loaded onto the chip after the multiple displacement amplification reaction, incubated for 2 to 5 minutes, and then eluted with an elution reagent to remove the third reagent.

22. The method according to claim 18, wherein The sequencing method further comprises: first performing a sequencing reaction on the template strand fixed on the chip, and then performing the multiple displacement amplification reaction to generate a complementary strand.

23. The method according to claim 18, wherein The sequencing method further includes: performing the multiple displacement amplification reaction on the template chain fixed on the chip to generate a complementary chain, treating the chip with the first reagent and the second reagent, and then performing the sequencing reaction on the template chain and the complementary chain.

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