Flushing reagent for high-throughput sequencing, kit and use method

Abstract

The invention discloses a flushing reagent for high-throughput sequencing, a kit and a use method, and the flushing reagent comprises a Tris buffer solution, a soluble salt, a PCR enhancer and a single-chain binding protein. The washing reagent contains the single-chain binding protein, so that the amplification efficiency can be greatly improved, the small difference of the amplification efficiency among different DNCs is ensured, the amplified DNCs in the chip are bright and uniform, and the problem of non-uniform sequencing signals in the traditional bridge amplification technology is solved.

Description

Technical Field

[0001] This invention belongs to the field of sequencing reagent technology, specifically relating to a washing reagent, kit, and method of use for high-throughput sequencing. Background Technology

[0002] High-throughput sequencing is a technology used to determine the sequence of DNA or RNA, capable of generating large amounts of diverse sequence data in a short time. It is widely used in many fields, including genomics, transcriptomics, and polymorphism research. These technologies are also used in clinical diagnostics, including rare disease diagnosis, cancer genome analysis, and personalized medicine.

[0003] High-throughput sequencing technologies are implemented in various ways, including Illumina's sequencing-by-synthesis method and BGI Genomics' combined probe-anchored polymerization sequencing method. Generally, next-generation high-throughput sequencing technology can be divided into several steps: sample preparation, library construction, template amplification, sequencing signal acquisition, and base interpretation. Template amplification involves amplifying a single library fragment into multiple copies of the same sequence that are spatially clustered together. The purpose is to amplify the sequencing signal, reduce the difficulty and cost of signal detection, and improve sequencing accuracy. Such a set of multiple copies of a single template is a "base signal acquisition unit." Typically, there are approximately one million "base signal acquisition units" with different templates per unit area on the detection plane. Based on this, signals from a large number of "base signal acquisition units" can be acquired simultaneously during the sequencing process, achieving high-throughput sequencing.

[0004] There are many template amplification methods, including solid-phase surface amplification and liquid-phase amplification, which are generally achieved through isothermal amplification. The most representative of these is bridge amplification. Bridge amplification generates high-density DNA clusters (DNCs) on a solid-phase surface immobilized with adapter primers. A DNC is a "base signal acquisition unit" containing hundreds or thousands of copies of the same template fragment, all covalently linked to the solid-phase chip surface via adapters. It is one of the most widely used template amplification methods in high-throughput sequencing, with Illumina being a leading example. The general steps of bridge amplification are: library hybridization, one-strand amplification, DNA denaturation and single-stranding, washing to allow adapter primers to hybridize with the single strand, and extension amplification; this cycle of "DNA denaturation and single-stranding, washing to allow adapter primers to hybridize with the single strand, and extension amplification" is repeated until completion. Therefore, the reagents used in bridge amplification generally include library hybridization reagents, denaturation reagents, washing reagents, and extension reagents.

[0005] However, bridging amplification, especially bridging amplification where DNCs are randomly distributed on the chip surface, results in an uncontrollable number of monoclonal copies within the generated DNCs. This leads to uneven DNC size and brightness in the sequencing signal, affecting sequencing quality. Therefore, it is necessary to optimize the reagents used in bridging amplification to ensure amplification uniformity as much as possible, reduce fluctuations in the number and density of monoclonal copies within different DNCs, and guarantee sequencing quality. Summary of the Invention

[0006] This invention discloses a washing reagent, kit, and method of use for high-throughput sequencing, in order to solve the problem of uneven sequencing signals in the prior art.

[0007] A first aspect of this application provides a washing reagent for high-throughput sequencing, comprising: Tris buffer, soluble salt, PCR enhancer, and single-stranded binding protein.

[0008] Furthermore, the soluble salt is selected from one or more of sodium salts, potassium salts, and magnesium salts.

[0009] Furthermore, the PCR enhancer is selected from one or more of BSA, Triton X-100, Tween 20, glycerol, formamide, polyethylene glycol, gelatin, tetramethylammonium chloride, betaine, and DMSO.

[0010] Furthermore, the magnesium salt is magnesium sulfate.

[0011] A second aspect of this application provides a high-throughput sequencing kit, comprising an extension reagent, a washing reagent provided in any implementation of the first aspect, and a trimming reagent, wherein the extension reagent comprises polymerase and deoxynucleotides, and the trimming reagent comprises ethanolamine buffer, magnesium chloride, exonuclease, and blocking reagent.

[0012] Furthermore, the exonuclease is a single-stranded specific 3'-5' exonuclease.

[0013] Furthermore, the blocking reagent is selected from one or more of BSA, Tween 20, polyethylene glycol, trehalose, and dithiothreitol.

[0014] A third aspect of this application provides a method for using a high-throughput sequencing kit, comprising:

[0015] Step a, the single-stranded library DNA fragment is hybridized to the adapter primers on the chip surface, wherein the two ends of the library DNA fragment have the same or reverse complementary sequences as the adapter primers;

[0016] Step b: The extension reagent amplifies the single-stranded library DNA fragment template into a double-stranded form starting from the adapter primers on the chip.

[0017] Step c: Use denaturing agents such as formamide or sodium hydroxide to untangle the DNA double strand into single strands;

[0018] In step d, the denaturing reagent is washed away using a rinsing agent, leaving the single-stranded DNA on the chip. During this process, the single-stranded DNA anneals and binds to other adapter primers on the chip.

[0019] Step e: The extension reagent amplifies the single-stranded library DNA fragment into a double-stranded form starting from the adapter primers on the chip.

[0020] Step f, repeat step ce 20 to 30 times, and treat the chip with the trimming agent;

[0021] Step g, repeat steps 2 to 8 of ce, until the amplification is complete.

[0022] As can be seen from the above technical solutions, this application provides a washing reagent, kit, and method of use for high-throughput sequencing. The washing reagent contains a single-chain binding protein, which can greatly improve the amplification efficiency and at the same time ensure that the difference in amplification efficiency between different DNCs is small, making the amplified DNCs in the chip bright and uniform, thus solving the problem of uneven sequencing signals in traditional bridge amplification technology. Attached Figure Description

[0023] Figure 1 DNC fluorescence patterns generated by amplification with different extension reagent formulations;

[0024] Figure 2 DNC fluorescence images generated by amplification of different washing reagent formulations;

[0025] Figure 3 This is a diagram showing the effect of treatment with pruning reagent;

[0026] Figures 4(a) and 4(b) show the Q30 and signal-to-noise ratio of the sequencing signals from two DNC chips before and after treatment with optimized rinsing reagent and added trimming reagent, respectively. Detailed Implementation

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] The reagents used in bridge amplification generally include library hybridization reagents, denaturing reagents, washing reagents, extension reagents, and trimming reagents. This application mainly optimizes the washing and trimming reagents. Conventional reagents can be used for library hybridization and denaturation. To better optimize the washing and trimming reagents, this application first optimizes the concentration of the extension reagent. The optimization of the extension reagent is described in detail below.

[0029] In existing technologies, commonly used extension reagents include the following components: buffer, soluble salts, PCR enhancers, polymerase, and deoxynucleotides. The polymerase is Bst polymerase, a DNA polymerase with strand displacement activity. The buffer is Tris (tris(hydroxymethyl)aminomethane) buffer, used to provide a suitable acid-base environment for amplification. Deoxynucleotides include dATP, dGTP, dCTP, and dTTP. The soluble salts include one or more of sodium, potassium, ammonium, magnesium, and manganese salts. In one feasible embodiment, the magnesium salt is magnesium sulfate, and the manganese salt can be manganese sulfate.

[0030] In some embodiments of this application, the PCR enhancer comprises one or more of polyaspartic acid, betaine, dimethyl sulfoxide (DMSO), bovine serum albumin (BSA), Triton X-100, Tween 20, formamide, polyethylene glycol (PEG), tetramethylammonium chloride (TMAC), glycerol, and dithiothreitol (DTT). These enhancers can improve the efficiency and specificity of PCR in various ways, such as helping to open complex DNA structures, preventing nonspecific binding, or improving polymerase activity. In one feasible embodiment, the concentration of polyaspartic acid can be 0.3-0.7%; the concentration of betaine ranges from 1M to 2.5M, specifically, it can be 1M, 1.5M, 2M, or 2.5M.

[0031] The optimization of the extended reagent is further illustrated below through specific embodiments. In this application, the concentration of each component refers to the final concentration of that component in the prepared reagent.

[0032] Example 1

[0033] Table 1. Extension reagent formulation A (pH 8.8)

[0034]

[0035]

[0036] Table 2 Extended reagent formulation B (pH 8.8)

[0037]

[0038] Table 3. Extension reagent formulation C (pH 8.8)

[0039]

[0040] Table 4. Extension reagent formulation D (pH 8.8)

[0041]

[0042]

[0043] Table 5. Comparative examples of extension reagents (pH 8.8)

[0044]

[0045] Table 6 Rinse reagent formulation (pH 8.8)

[0046] Element name concentration buffer solution Tris-HCl 10mM soluble salts Sodium chloride 50mM PCR enhancer Twain 20 0.02％

[0047] Tables 1-4 provide formulations for four extension reagents, Table 5 provides a comparative formulation for one extension reagent, and Table 6 provides a basic formulation for one rinsing reagent. The extension and rinsing reagents are used in conjunction with a high-throughput sequencer on a sequencing microfluidic chip, and are applied after the first strand amplification in bridge amplification. The procedure is as follows:

[0048] S1: First, pass the chip with a formamide denaturing agent;

[0049] S2: Rinse the chip with a rinsing agent;

[0050] S3: Flow the extension reagent through the chip and incubate for 10 seconds;

[0051] S4: Repeat steps 1-3 24 times; use Qubit dsDNA analysis reagent to stain the amplified DNC and observe it with a fluorescence microscope. The results are shown in Table 7.

[0052] S5: The chip containing extension reagent D ("2M betaine and 2.9% DMSO amplification") was sequenced on a high-throughput sequencer, and its Q30 and signal-to-noise ratio were analyzed, as shown by the blue dashed line in Figure 4. The sequencing quality remained poor. Q30 represents the quality value of a single base, meaning the recognition reliability of a single base is 99.9%, with an error probability of 0.1%. The signal-to-noise ratio represents the ratio between the sequencing signal and noise; the lower the signal-to-noise ratio, the less reliable the result.

[0053] Table 7. Qubit staining intensity and coefficient of variation of DNC after amplification with different extension reagents.

[0054]

[0055]

[0056] Comparing the above results, it can be seen that polyaspartic acid (PAA) produces brighter DNC signals under higher salt, dNTP, and enzyme concentrations, especially at 0.5% PAA, where the DNC is more uniform and exhibits the lowest coefficient of variation in brightness. As the PAA concentration increases, the DNC signal decreases, indicating that one of its functions is to inhibit Bst polymerase activity within a certain range, thereby improving uniformity. In contrast, the control group with higher salt concentrations exhibits lower DNC brightness and poorer uniformity, which is detrimental to sequencing. Therefore, adding multiple PCR enhancers to the amplification system is beneficial for improving both brightness and uniformity. Figure 1 These are DNC fluorescence images generated by amplification of four of the extension reagent formulations. Figure 1 In Figure A, the DNC fluorescence pattern is generated using a 0.5% polyaspartic acid extension reagent. Figure 1 In section B, the DNC fluorescence pattern was generated using 0.5% polyaspartic acid and 40mM TMAC extension reagent. Figure 1 In the middle, C represents the DNC fluorescence pattern generated with 2 mM manganese sulfate and 1 mM DTT extension reagent. Figure 1 The D-type fluorescence pattern is generated using a 2M betaine and 2.9% DMSO extension reagent. From... Figure 1 It can be seen that under certain specific salt and enzyme concentration conditions, the combination of betaine and DMSO can effectively improve amplification efficiency. Under the same time, temperature and cycling conditions, the brightest DNC ​​fluorescence signal and a better coefficient of variation in brightness can be obtained. Compared with other formulations such as polyaspartic acid, it is a better result. Therefore, this application selects extension reagent formulation D as the formulation for further optimization of the washing and trimming reagents.

[0057] Example 2

[0058] The rinsing reagent will be further explained below using Example 2.

[0059] This embodiment provides a washing reagent for high-throughput sequencing, comprising: Tris buffer, soluble salt, PCR enhancer, and single-stranded DNA-binding protein (SSB). In other embodiments, the single-stranded DNA-binding protein can be of any species origin or a correspondingly modified single-stranded DNA-binding protein. The concentration range of the single-stranded DNA-binding protein can be 0.1-10 μg / mL.

[0060] The preferred concentration range for Tris buffer is 10-50 mM.

[0061] The soluble salt is selected from one or more of sodium, potassium, and magnesium salts. The concentration range of the soluble sodium and potassium salts can be 10-150 mM. In the art, sodium and potassium salts are generally considered equivalent. The magnesium salt is magnesium sulfate, and the concentration range can be 0.2-10 mM.

[0062] The PCR enhancer is selected from one or more of BSA, Triton X-100, Tween 20, glycerol, formamide, polyethylene glycol (PEG), gelatin, tetramethylammonium chloride (TMAC), betaine, and DMSO. The concentration ranges for BSA and gelatin: 0.01-0.1%; for Triton X-100 and Tween 20: 0.01-0.2%; for glycerol, formamide, and polyethylene glycol: 1-20%; for tetramethylammonium chloride (TMAC): 20-100 mM; for betaine: 1-2.5 M; and for DMSO: 1-5%. In the art, Triton X-100 and Tween 20 are generally considered equivalent.

[0063] Table 8. Extension reagent formulation (pH 8.8)

[0064]

[0065] Table 9 Rinse Reagent Formulation A (pH 8.8)

[0066]

[0067]

[0068] Table 10 Rinse Reagent Formulation B (pH 8.8)

[0069]

[0070] Table 8 provides an optimized extension reagent formulation, and Tables 9-10 provide four rinsing reagent formulations. The extension reagent and rinsing reagent provided in Example 2 are used in conjunction with a high-throughput sequencer on a sequencing microfluidic cell chip, and are used after the first strand amplification of the bridge amplification is completed. The procedure is as follows:

[0071] S1. First, pass the chip through a formamide denaturing agent;

[0072] S2. Rinse the chip with a rinsing agent;

[0073] S3. Flow the extension reagent through the chip and incubate for 10 seconds;

[0074] S4. Repeat steps 1-3 22 times; use Qubit dsDNA analysis reagent to stain the amplified DNC and observe it with a fluorescence microscope. The analysis results are shown in Table 11.

[0075] Table 11 Qubit staining brightness, brightness variation coefficient, and DNC pixel size after amplification with different washing reagents.

[0076]

[0077]

[0078] The results above show that, compared with other components, the rinsing reagent containing SSB has a clear advantage. Figure 2 These are DNC fluorescence images generated by amplification of four of the washing reagent formulations. Figure 2 In the image, A represents the DNC fluorescence pattern generated by A-4; Figure 2 In the image, B represents the DNC fluorescence pattern generated by B-2; Figure 2 DNC fluorescence pattern was generated in C for B-5 (containing 1.5ug / mL SSB, 1mM magnesium sulfate and 10mM sodium chloride washing reagent); Figure 2 D is B-6 (containing 1.5ug / mL SSB, 1mM magnesium sulfate and 30mM sodium chloride washing reagent) to generate DNC fluorescence pattern.

[0079] Depend on Figure 2 It is evident that the washing reagent containing SSB can significantly improve amplification efficiency while ensuring minimal difference in amplification efficiency between different DNCs, resulting in bright and uniform DNCs amplified within the chip. However, as seen in formulations B-3 to B-5 of the examples, while SSB improves DNC amplification efficiency, it also increases the size of the DNCs. Furthermore, higher SSB concentrations lead to higher amplification efficiency, brighter DNCs, and larger sizes. Conversely, salt ions, such as sodium, potassium, or magnesium salts, in systems B-1 to B-3 and B-5 to B-8 inhibit DNC ​​formation efficiency, hindering uniform amplification results, and higher concentrations result in poorer amplification. In summary, salt ions and SSB compete to some extent, having opposing effects on the brightness and uniformity of DNCs.

[0080] In high-throughput sequencing, sequencing throughput is also a crucial parameter, and a large DNC size reduces the DNC density on the chip surface, thereby decreasing sequencing throughput. Furthermore, by adjusting the ratio of single-stranded binding protein to salt, the DNC size can be arbitrarily adjusted as needed. Preferably, the SSB concentration can be 1.5-10 μg / mL, the sodium salt concentration 10-70 mM, and the magnesium ion concentration 0.2-10 mM. Most preferably, a washing reagent of 1.5 μg / mL SSB, 1 mM magnesium sulfate, and 30 mM sodium chloride ensures both DNC brightness and uniformity while maintaining a small DNC size, thus guaranteeing high sequencing throughput.

[0081] Example 3

[0082] To further limit the DNC size and improve the discriminability of the DNC signal, the amplification process requires trimming unused adapter primers on the chip to obtain high-density, high signal-to-noise ratio DNC signals and improve sequencing accuracy. Therefore, this application also provides a trimming reagent that combines extension reagent, washing reagent, and trimming reagent to generate high-throughput sequencing clusters. The following describes the trimming reagent and the method of using the three reagents together.

[0083] The trimming reagents include ethanolamine buffer, magnesium chloride, exonuclease, and blocking reagent.

[0084] The exonuclease is a single-stranded specific 3'-5' exonuclease. Preferably, the amount of single-stranded specific 3'-5' exonuclease used ranges from 100 to 1000 U / mL.

[0085] Preferably, the concentration range of ethanolamine can be 10-100 mM.

[0086] Further, the blocking agent is selected from one or more of BSA, Tween 20, polyethylene glycol, trehalose, and dithiothreitol. Preferably, the concentration range of BSA is 0.01-0.1%, the concentration range of Tween 20 is 0.01-0.2%, the concentration range of polyethylene glycol is 1-20%, the concentration range of trehalose is 50-200 mM, and the concentration range of dithiothreitol is 0.2-5 mM.

[0087] Table 12 Extension reagent formulation (pH 8.8)

[0088]

[0089] Table 13 Rinse Reagent Formulation (pH 8.8)

[0090]

[0091] Table 14. Pruning reagent formulation (pH 9.5)

[0092]

[0093] The above three reagents are used in conjunction with a high-throughput sequencer on a sequencing microfluidic cell chip, and are used after the first strand amplification of the bridge amplification is completed. The procedure is as follows:

[0094] S1. First, pass the chip through a formamide denaturing agent.

[0095] In this step, there are two types of adapter primers on the chip, P5 and P7, but they are both covalently linked to the chip through the 5' end. The library is fixed on the chip by annealing with the library, and double-stranded DNA is amplified on the chip surface. Denaturing reagents untangle the double strand into two single-stranded DNAs.

[0096] In this embodiment, the sequences at both ends of the library fragment are the same as or inversely complementary to those of the adapter primer.

[0097] S2. Use rinsing reagent to flow through the chip.

[0098] In this step, the washing reagent contains single-strand binding proteins, which allow DNA single strands to anneal and bind more efficiently to the adapter primers on the chip, thereby improving the efficiency of bridge amplification.

[0099] In this embodiment, the rinsing reagent contains soluble salts, which improve the annealing binding efficiency of DNA single strands and complementary sequences, reduce the rate of DNA single strands diffusing outward on the chip surface, and suppress DNC size.

[0100] S3. Flow the extension reagent through the chip and incubate for 10 seconds.

[0101] S4. Repeat steps 1-3 20 times; use trimming reagent to flow through the chip and incubate at 37°C for 10 min.

[0102] In this step, the trimming reagent contains exonuclease 1. Exonuclease 1 is a single-stranded, specific 3'-5' exonuclease. During processing, the exonuclease digests excess adapter primers on the chip, but does not digest the adapter primers already amplified within the DNC.

[0103] In this embodiment, amplification continues after treatment with the trimming reagent. The amplification of DNC will not continue to spread outward, that is, it will not continue to grow larger, but it can still generate more copies.

[0104] In this embodiment, the surface of the chip without DNC ​​after trimming is sealed by the sealing reagents BSA, Tween 20, polyethylene glycol, trehalose, etc., which is beneficial for distinguishing between background and signal.

[0105] S5. The amplified DNC was stained with Qubit dsDNA analysis reagent and observed with a fluorescence microscope.

[0106] S6. Repeat steps 1-3 seven times, for a total of 27 times; or repeat steps 1-3 four times, for a total of 24 times; stain the amplified DNCs with Qubit dsDNA analysis reagent and observe them with a fluorescence microscope, as shown in Table 15.

[0107] In this step, the fluorescent reagent that specifically binds to double-stranded DNA is Thermo Fisher Scientific's Qubit dsDNA assay reagent, and the results are observed using a fluorescence microscope. The Qubit dsDNA assay reagent incorporates fluorescent molecules into double-stranded DNA and emits a fluorescent signal; the intensity of the fluorescent signal is positively correlated with the amount of double-stranded DNA within the DNC.

[0108] S7. After single-stranding DNC, add sequencing primers, and polymerize with 9N enzyme and fluorescent dNTPs for one cycle. After rinsing with appropriate photographic reagents, observe the "ratio of sequencing signal to background" using a fluorescence microscope.

[0109] S8. The chip amplified by the trimming reagent formulation E for 27 cycles was sequenced on a high-throughput sequencer, and its Q30 and signal-to-noise ratio were analyzed, as shown by the yellow solid line in Figure 4.

[0110] Comparative Example

[0111] For the above-mentioned trimming reagent formulation E-1, the trimming reagent in step 4 of the above operation procedure was replaced with a trimming reagent without exonuclease, while other components and procedures remained the same. Comparative observations were conducted, and the results are shown in Table 15.

[0112] Table 15. Effects of pruning agents

[0113]

[0114] In Example 3 and the comparative examples, to verify the effect of the trimming reagent, a washing solution was selected that favored increasing the size of the amplified DNC. The results show that without the trimming reagent, the DNC size after 27 cycles of amplification was large, measuring 9 pixels, 3 pixels larger than that after 20 cycles. However, after treatment with a trimming reagent containing 230 U / mL exonuclease, the DNC size after 27 cycles of amplification was only 5 pixels, a very small change compared to 20 cycles. It is important to note that the use of exonuclease requires careful handling. If the concentration is too high, a large amount of DNA within the DNC will be digested, leading to experimental failure; if the concentration is too low, the effect will be minimal. Figure 3 This is a comparison of DNC fluorescence images generated by amplification with and without trimming reagent (230 U / mL exonuclease). Figure 3 Image A shows the DNC fluorescence pattern after 20 cycles of amplification and reagent trimming. Figure 3 In the image, B represents the DNC fluorescence pattern generated after 7 cycles of amplification following treatment with the trimming reagent. Figure 3 In the middle, C represents the fluorescence pattern of DNC generated after 20 amplification cycles; Figure 3 The middle D is the fluorescence image of DNC generated after 27 amplification cycles.

[0115] Depend on Figure 3It can be seen that after treatment with the trimming reagent, the DNC after 7 cycles of amplification is only brighter than that after 20 cycles of amplification, but the size is similar. In Table 15, the DNC after 7 cycles of amplification is brighter than that after 4 cycles of amplification, and the size is the same. That is, after treatment with the trimming reagent, the DNC size is limited, but the DNC signal will be stronger with continued amplification cycles. Moreover, after trimming with the trimming reagent, the adapter primers in the DNC-free regions of the chip surface are digested, reducing the non-specific adsorption of fluorescent dNTPs on the chip surface, thus leading to a decrease in sequencing background and a significant increase in the sequencing signal-to-background ratio. Preferably, after 20 cycles of amplification, treatment with the trimming reagent, and then 7 more cycles of amplification, the DNC can be limited to 5 pixels, and better brightness, brightness variation coefficient, and signal-to-background ratio can be obtained.

[0116] The DNCs generated after optimizing the washing and trimming reagents and the corresponding amplification process were sequenced, and the results are shown in Figure 4. It can be seen that compared to simply using appropriate extension reagents, the optimized results are better, such as a higher sequencing accuracy (Q30) and a higher signal-to-noise ratio. This optimization will significantly improve sequencing quality.

[0117] The above are merely embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention are included within the scope of the claims of the present invention pending approval.

Claims

1. A washing reagent for high-throughput sequencing, characterized in that, include: Tris buffer, soluble salts, PCR enhancers, and single-stranded binding proteins.

2. The washing reagent for high-throughput sequencing according to claim 1, characterized in that, The soluble salt is selected from one or more of sodium, potassium, and magnesium salts.

3. The washing reagent for high-throughput sequencing according to claim 1, characterized in that, The PCR enhancer is selected from one or more of BSA, Triton X-100, Tween 20, glycerol, formamide, polyethylene glycol, gelatin, tetramethylammonium chloride, betaine, and DMSO.

4. The washing reagent for high-throughput sequencing according to claim 2, characterized in that, The magnesium salt is magnesium sulfate.

5. A high-throughput sequencing kit, characterized in that, It includes an extension reagent, a rinsing reagent according to any one of claims 1 to 4, and a trimming reagent, wherein the extension reagent includes polymerase and deoxynucleotides, and the trimming reagent includes ethanolamine buffer, magnesium chloride, exonuclease, and blocking reagent.

6. The high-throughput sequencing kit according to claim 5, characterized in that, The exonuclease is a single-stranded specific 3'-5' exonuclease.

7. The high-throughput sequencing kit according to claim 5, characterized in that, The blocking reagent is selected from one or more of BSA, Tween 20, polyethylene glycol, trehalose, and dithiothreitol.

8. A method for using a high-throughput sequencing kit, characterized in that, include: Step a, the single-stranded library DNA fragment is hybridized to the adapter primers on the chip surface, wherein the two ends of the library DNA fragment have the same or reverse complementary sequences as the adapter primers; Step b: The extension reagent amplifies the single-stranded library DNA fragment template into a double-stranded form starting from the adapter primers on the chip. Step c: Use denaturing agents such as formamide or sodium hydroxide to untangle the DNA double strand into single strands; In step d, the denaturing reagent is washed away using a rinsing agent, leaving the single-stranded DNA on the chip. During this process, the single-stranded DNA anneals and binds to other adapter primers on the chip. Step e: The extension reagent amplifies the single-stranded library DNA fragment into a double-stranded form starting from the adapter primers on the chip. Step f, repeat step ce 20 to 30 times, and treat the chip with the trimming agent; Step g, repeat steps 2 to 8 of ce, until the amplification is complete.

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