CN120866499A - A washing reagent, kit, and usage method for high-throughput sequencing - Google Patents

Abstract

This invention discloses a washing reagent, kit, and method of use for high-throughput sequencing. The washing reagent includes Tris buffer, soluble salt, PCR enhancer, and single-stranded binding protein. The washing reagent containing single-stranded binding protein can greatly improve amplification efficiency while ensuring minimal difference in amplification efficiency between different DNCs, resulting in bright and uniform amplified DNCs within the chip, thus solving the problem of uneven sequencing signals in traditional bridge amplification technology.

Description

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

Technical Field

The invention belongs to the technical field of sequencing reagents, and particularly relates to a flushing reagent for high-throughput sequencing, a kit and a use method thereof.

Background

High throughput sequencers are a technique for determining DNA or RNA sequences that can produce large amounts of different sequence data in a short period of time. It is widely used in many fields including genomics, transcriptomics, polymorphism studies, and the like. These techniques are also used for clinical diagnosis, including rare disease diagnosis, analysis of cancer genomes, personalized medicine, and the like.

The high throughput sequencing technology is implemented in a variety of ways, including the illuminea company sequencing-by-synthesis method, the Hua Dazhi combined probe-anchored polymer sequencing method, and the like. In general, second generation high throughput sequencing techniques can be divided into several steps, sample preparation, library construction, template amplification, sequencing signal acquisition, and base interpretation. The template amplification is to amplify single library fragments into multiple copies of the same sequence which are spatially concentrated together, so that the sequencing signals are amplified, the difficulty and cost of signal detection are reduced, and the sequencing accuracy is improved. Such a single template multiple copy set is a "base signal acquisition unit". Typically, there are about millions of "base signal acquisition units" of different templates per unit area on the detection plane. Based on the method, a large number of base signal acquisition units can be simultaneously subjected to signal acquisition in the sequencing process, so that high-throughput sequencing is realized.

There are also a variety of ways of template amplification, including solid phase surface amplification and liquid phase amplification, and the like, and are typically achieved by isothermal amplification, the most typical of which is bridge amplification. Bridge amplification is the generation of high density DNA clusters (DNCs) on the solid surface where the adaptor primer is immobilized. A DNC is a "base signal acquisition unit" which has hundreds to thousands of copies of the same fragment template, all covalently attached to the solid phase chip surface by linkers. It is one of the most widely used template amplification methods in high throughput sequencing, represented by illuminea corporation. The general steps of bridge amplification are library hybridization, one-strand amplification, DNA denaturation and single-strand hybridization by washing, extension amplification, and then "DNA denaturation and single-strand hybridization by washing, extension amplification" are repeated until the end. For this purpose, reagents used for bridge amplification generally include library hybridization reagents, denaturing reagents, washing reagents, and extension reagents, among others.

However, bridge amplification, particularly where DNCs are randomly distributed on the chip surface, is not controllable as to the number of monoclonal copies within the generated DNCs, and can result in non-uniform DNC size and brightness of the sequencing signal, affecting sequencing quality. Therefore, the reagent used in bridge amplification needs to be optimized, so that the amplification uniformity is ensured as much as possible, the fluctuation of the quantity and density of monoclonal copies in different DNCs is reduced, and the sequencing quality is ensured.

Disclosure of Invention

The invention discloses a flushing reagent for high-throughput sequencing, a kit and a use method thereof, which are used for solving the problem of nonuniform sequencing signals in the prior art.

In a first aspect of the application, a wash reagent for high throughput sequencing is provided comprising Tris buffer, soluble salt, PCR enhancer and single stranded binding protein.

Further, the soluble salt is selected from one or more of sodium salt, potassium salt and magnesium salt.

Further, the PCR enhancer is selected from one or more of BSA, tritonX-100, tween 20, glycerol, formamide, polyethylene glycol, gelatin, tetramethyl ammonium chloride, betaine and DMSO.

Further, the magnesium salt is magnesium sulfate.

In a second aspect of the application, there is provided a kit for high throughput sequencing comprising an extension reagent comprising a polymerase and deoxynucleotides, a wash reagent provided in any of the implementations of the first aspect, and a trimming reagent comprising an ethanolamine buffer, magnesium chloride, an exonuclease and a blocking reagent.

Further, the exonuclease is a single-strand specific 3'-5' exonuclease.

Further, the blocking reagent is selected from one or more of BSA, tween 20, polyethylene glycol, trehalose and dithiothreitol.

In a third aspect of the present application, there is provided a method of using a kit for high throughput sequencing, comprising:

Step a, hybridizing single-stranded library DNA fragments to the adaptor primers on the surface of the chip, wherein the two ends of the library DNA fragments are identical or reversely complementary to the sequences of the adaptor primers;

Step b, amplifying the extension reagent into double chains along the single-stranded library DNA fragment templates from the adaptor primers on the chip;

Step c, using formamide or sodium hydroxide denaturing reagent to break the DNA double strand into single strands;

Step d, washing the denaturing reagent away by using a washing reagent, and leaving the single strand of DNA on the chip, during which time the single strand DNA anneals to other adaptor primers on the chip;

step e, amplifying the extension reagent into double chains along the single-stranded library DNA fragments from the adaptor primer on the chip;

F, repeating the steps c-e for 20-30 times, and treating the chip with a trimming reagent;

and g, repeating the steps c-e for 2-8 times, and finishing amplification.

According to the technical scheme, the flushing reagent, the kit and the using method for high-throughput sequencing are provided, wherein the flushing reagent contains the single-chain binding protein, so that the amplification efficiency can be greatly improved, meanwhile, the amplification efficiency difference between different DNCs is ensured to be smaller, the DNCs amplified in a chip are bright and uniform, and the problem of nonuniform sequencing signals in the traditional bridge amplification technology is solved.

Drawings

FIG. 1 is a fluorescent plot of DNC amplified from different extension reagent formulations;

FIG. 2 is a fluorescent plot of DNC amplified from different wash reagent formulations;

FIG. 3 is a graph showing the effect of the trimming agent treatment;

FIGS. 4 (a) and 4 (b) are graphs of the signal quality Q30 and the signal-to-noise ratio of two DNC chip sequencing before and after the comparison of the optimized wash reagent and the addition of the trimming reagent, respectively.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art without the exercise of inventive faculty, are intended to be within the scope of the invention.

The reagents used for bridge amplification typically comprise library hybridization reagents, denaturing reagents, washing reagents, extension reagents, trimming reagents, and the like. The application mainly optimizes the flushing reagent and the trimming reagent. The library hybridization reagent and the denaturing reagent can be conventional reagents, and in order to better optimize the flushing reagent and the trimming reagent, the concentration of the extending reagent is optimized in series, and the optimization of the extending reagent is described in detail below.

In the prior art, commonly used extension reagents include buffers, soluble salts, PCR enhancers, polymerases, and deoxynucleotides. Wherein the polymerase is Bst polymerase, which is a DNA polymerase having strand displacement activity. The buffer solution is Tris (Tris (hydroxymethyl aminomethane) buffer solution, and the buffer solution is used for providing a proper acid-base environment and providing conditions for amplification. The deoxynucleotides comprise dATP, dGTP, dCTP and dTTP. The soluble salt comprises one or more of sodium salt, potassium salt, ammonium salt, magnesium salt and manganese salt. In one embodiment, the magnesium salt is magnesium sulfate and the manganese salt may be manganese sulfate.

In some embodiments of the 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), tetramethyl ammonium chloride (TMAC), glycerol, dithiothreitol (DTT). These enhancers can increase the efficiency and specificity of PCR in various ways, for example, to help open complex DNA structures, prevent non-specific binding, or improve polymerase activity, etc. In one embodiment, the concentration of polyaspartic acid can be 0.3-0.7%, and the concentration of betaine can be 1M-2.5M, specifically 1M, 1.5M, 2M and 2.5M.

Optimization of the extension reagents is further illustrated by the specific examples below. In the present application, the concentration of each component is the final concentration of that component in the reagent.

Example 1

TABLE 1 extending reagent formulation A (pH 8.8)

TABLE 2 extending reagent formulation B (pH 8.8)

TABLE 3 extending reagent formulation C (pH 8.8)

Table 4 extending reagent formulation D (pH 8.8)

Table 5 comparative example of extending reagent (pH 8.8)

Table 6 flushing reagent formulation (pH 8.8)

Composition of the components	Name of the name	Concentration of
			Buffer solution	Tris-HCl	10mM
Soluble salts	Sodium chloride	50mM
			PCR enhancer	Tween 20	0.02%

Tables 1-4 provide the formulations of four extension reagents, table 5 provides the formulation of a comparative example of an extension reagent, table 6 provides the basic formulation of a rinse reagent, the extension reagent and rinse reagent are used on a sequencing microfluidic chamber chip in combination with a high throughput sequencer, and after completion of one-strand amplification of bridge amplification, the procedure is as follows:

s1, firstly, using formamide denaturing reagent to flow through a chip;

S2, using a flushing reagent to flow through the chip;

S3, using an extension reagent to flow through the chip and incubating for 10S;

S4, repeating the steps 1-3 for 24 times, staining the amplified DNC by using a Qubit dsDNA analysis reagent, and observing the DNC by using a fluorescence microscope, wherein the results are shown in Table 7;

s5, sequencing the chip amplified by the extension reagent D2M betaine and 2.9% DMSO on a high-throughput sequencer, and analyzing the Q30 and the signal to noise ratio, wherein the sequencing quality is still poor as shown by a blue dotted line in FIG. 4. Where Q30 represents the quality value of one base, i.e., the recognition reliability of one base is equal to 99.9%, the error probability is 0.1%, and the signal-to-noise ratio represents the ratio between the sequencing signal and the noise, the lower the signal-to-noise ratio, the less reliable the result.

TABLE 7 Qubit staining Brightness and Brightness coefficient of variation of DNC after amplification with different extension reagents

Comparing the above results, it can be seen that the polyaspartic acid can obtain brighter DNC signal under the conditions of higher salt concentration, dNTPs concentration and enzyme concentration, and especially under the condition of 0.5% polyaspartic acid, DNC is more uniform, and the brightness variation coefficient is the minimum in the conditions. As the concentration of polyaspartic acid used increases, DNC signal decreases, indicating that one of its effects is to inhibit Bst polymerase activity within a certain range, improving uniformity. In contrast, in the comparative example containing a higher concentration of salt component, DNC was low in brightness and poor in uniformity, which was unfavorable for sequencing. In contrast, the addition of multiple PCR enhancers to the amplification system is beneficial to improving brightness and uniformity. FIG. 1 is a fluorescence plot of DNC generated by amplification of four extension reagent formulations, where A in FIG. 1 is a fluorescence plot of DNC generated by extension reagents containing 0.5% polyaspartic acid. FIG. 1B is a fluorescence plot of DNC generated with 0.5% polyaspartic acid and 40mM TMAC extending reagent. FIG. 1C is a fluorescence plot of DNC generated with 2mM manganese sulfate and 1mM DTT extension reagent. FIG. 1D is a fluorescence plot of DNC generated with 2M betaine and 2.9% DMSO extension reagent. As can be seen from FIG. 1, under certain specific salt concentration and enzyme concentration conditions, the use of betaine and DMSO in combination can effectively improve the amplification efficiency, and under the same time, temperature and circulation conditions, the brightest DNC fluorescence signal and better brightness variation coefficient can be obtained, and the result is more preferable than other formulations such as polyaspartic acid, so that the extension reagent formulation D is selected as a formulation for further optimizing the washing reagent and the trimming reagent.

Example 2

The rinsing agent is further described below using example 2.

This example provides a high throughput sequencing wash reagent comprising Tris buffer, soluble salts, PCR enhancers and single stranded binding proteins (SSB, single Strand DNA-binding proteins). In other embodiments, the single-chain binding protein may be a single-chain binding protein of any species origin or corresponding engineering. The concentration of single-stranded binding protein may range from 0.1 to 10ug/mL.

The Tris buffer concentration is preferably in the range of 10-50mM.

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

The PCR enhancer is selected from one or more of BSA, tritonX-100, tween 20, glycerol, formamide, polyethylene glycol (PEG), gelatin, tetramethyl ammonium chloride (TMAC), betaine and DMSO. BSA and gelatin concentration range 0.01-0.1%, tritonX-100 and Tween 20 concentration range 0.01-0.2%, glycerol, formamide and polyethylene glycol concentration range 1-20%, tetramethyl ammonium chloride (TMAC) concentration range 20-100mM, betaine concentration range 1-2.5M, DMSO concentration range 1-5%. In the art, tritonX-100 and Tween 20 are typically equivalent.

Table 8 extending reagent formulation (pH 8.8)

Table 9 flushing reagent formulation A (pH 8.8)

Table 10 flushing reagent formula B (pH 8.8)

Table 8 provides an optimized extension reagent formulation, tables 9-10 provide four rinse reagent formulations, and the extension reagent and rinse reagent provided in example 2 are used on a sequencing microfluidic chamber chip in combination with a high throughput sequencer and after completion of a strand amplification of a bridge amplification, the procedure is as follows:

s1, firstly, using formamide denaturing reagent to flow through a chip;

s2, using a flushing reagent to flow through the chip;

s3, using an extension reagent to flow through the chip and incubating for 10S;

S4, repeating the steps 1-3 for 22 times, staining the amplified DNC with a Qubit dsDNA analysis reagent, and observing with a fluorescence microscope, wherein the analysis results are shown in Table 11.

TABLE 11 Qubit staining Brightness, brightness coefficient of variation and DNC Pixel size of DNC after amplification with different flushing reagents

As can be seen from a comparison of the above results, the inclusion of SSB in the rinse agent provides a significant advantage over other ingredients. FIG. 2 is a fluorescence plot of DNC generated by amplification of four of the rinse reagent formulations, A in FIG. 2 is a-4 generated DNC, B in FIG. 2 is a B-2 generated DNC, C in FIG. 2 is a B-5 (containing 1.5ug/mL SSB, 1mM magnesium sulfate and 10mM sodium chloride rinse reagent), and D in FIG. 2 is a B-6 (containing 1.5ug/mL SSB, 1mM magnesium sulfate and 30mM sodium chloride rinse reagent) generated DNC.

As can be seen from FIG. 2, the SSB contained in the flushing reagent can greatly improve the amplification efficiency and simultaneously ensure that the amplification efficiency difference between different DNCs is small, so that the DNCs amplified in the chip are bright and uniform. However, it can be seen from the examples formulas B-3 to 5 that the SSB increases the DNC amplification efficiency and also increases the DNC size, and that the higher the SSB concentration, the higher the amplification efficiency and the brighter the DNC, and the larger the DNC size. In contrast, the salt ions, such as sodium salt, potassium salt or magnesium salt, in the B-1 to 3 and B-5 to 8 systems inhibit DNC production efficiency, which is not conducive to obtaining uniform amplification results, and the higher the concentration, the poorer the amplification effect. Taken together, salt ions and SSB compete to some extent, with opposite effects on the brightness and uniformity of DNC.

In high throughput sequencing, sequencing throughput is also a very important parameter, and DNC size greatly reduces the chip surface DNC density, which in turn reduces sequencing throughput. Further, by adjusting the ratio of the single-chain binding protein and the salt, we can arbitrarily adjust the size of DNC as needed. Preferably, SSB is used at a concentration of 1.5-10 ug/mL, sodium salt at a concentration of 10-70mM, and magnesium ion at a concentration of 0.2-10mM. Most preferably, 1.5ug/mL SSB, 1mM magnesium sulfate and 30mM sodium chloride wash reagent ensure DNC brightness and uniformity, while DNC size is smaller, ensuring higher sequencing throughput.

Example 3

In order to further limit the DNC size and improve the distinguishable degree of the DNC signal, the amplification process requires trimming of the unused adapter primers on the chip to obtain a high-density, high signal-to-noise ratio DNC signal, improving sequencing accuracy. Thus, the present application also provides a trimming reagent that cooperates with an extension reagent, a rinsing reagent, and a trimming reagent to generate high throughput sequencing clusters. The method of combining the trimming agent and the three agents will be described below.

The trimming reagent comprises ethanolamine buffer, magnesium chloride, exonuclease and a blocking reagent.

The exonuclease is a single-strand specific 3'-5' exonuclease. Preferably, the single strand specific 3'-5' exonuclease is used in an amount ranging from 100 to 1000U/mL.

Preferably, the ethanolamine concentration may range from 10 to 100mM.

Further, the blocking reagent is selected from one or more of BSA, tween 20, polyethylene glycol, trehalose and dithiothreitol. Preferably, the BSA concentration ranges from 0.01 to 0.1%, the Tween 20 concentration ranges from 0.01 to 0.2%, the polyethylene glycol concentration ranges from 1 to 20%, the trehalose is 50 to 200mM, and the dithiothreitol concentration ranges from 0.2 to 5mM.

Table 12 extending reagent formulation (pH 8.8)

Table 13 flushing reagent formulation (pH 8.8)

Table 14 pruning reagent formulation (pH 9.5)

The three reagents are matched with a high-throughput sequencer to be used on a sequencing microfluidic chamber chip, and are used after one-chain amplification of bridge amplification is completed, and the flow is as follows:

s1, firstly, using formamide denaturing reagent to flow through the chip.

In this step, the adaptor primer on the chip has two types of P5 and P7, but is covalently bonded to the chip through the 5' end, and the library is immobilized on the chip by annealing to the library, and amplification on the chip surface is achieved to form double-stranded DNA, and the denaturing agent breaks the double strand into two single-stranded DNAs;

in this example, both ends of the library fragment are identical or complementary to the sequence of the adaptor primer.

S2, using a flushing reagent to flow through the chip.

In this step, the wash reagent contains single-stranded binding protein, allowing the DNA single strand to anneal more efficiently to the adapter primer on the chip, improving bridge amplification efficiency.

In the embodiment, the flushing reagent contains soluble salt, so that the annealing combination efficiency of the DNA single strand and the complementary sequence is improved, the outward diffusion rate of the DNA single strand on the chip surface is reduced, and the DNC size is inhibited;

s3, flowing through the chip by using an extension reagent and incubating for 10S.

S4, repeating the steps 1-3 for 20 times, and using a trimming reagent to flow through the chip and incubating for 10min at 37 ℃.

In this step, the trimming agent comprises exonuclease 1. Exonuclease 1 is a single-strand specific 3'-5' exonuclease. During the treatment, the exonuclease digests off excess linker primers on the chip, but not amplified DNC.

In this example, amplification was continued after the trimming reagent treatment. The amplification of DNC does not continue to spread out, i.e. does not continue to grow large, but may continue to produce more copies.

In this embodiment, the surface of the chip without DNC after the trimming reagent treatment is subjected to blocking treatment by blocking reagents BSA, tween 20, polyethylene glycol, trehalose and the like in the reagent, which is favorable for distinguishing the background from the signal.

S5, using a Qubit dsDNA analysis reagent to dye the amplified DNC, and observing the DNC by using a fluorescence microscope.

S6, repeating steps 1-3 for 7 times again for 27 times total, or repeating steps 1-3 for 4 times again for 24 times total, staining the amplified DNC with the Qubit dsDNA analysis reagent and observing with a fluorescence microscope, as shown in Table 15.

In this step, a fluorescent reagent capable of specifically binding to double-stranded DNA was used as a Qubit dsDNA analysis reagent from the company Sieimer, and observed with a fluorescent microscope. The Qubit dsDNA assay reagent is capable of incorporating fluorescent molecules into double stranded DNA and emitting a fluorescent signal whose intensity is positively correlated with the amount of double stranded DNA within the DNC.

S7, after single-stranded DNC, the sequencing primer is hybridized, 9N enzyme and fluorescent dNTPs are polymerized for one cycle, the DNA is washed by a proper photographing reagent, and the sequencing signal and background ratio is observed by a fluorescent microscope.

S8, sequencing the chip amplified by 27 cycles of the trimming reagent formula E on a high-throughput sequencer, and analyzing the Q30 and the signal-to-noise ratio of the chip, as shown by a yellow solid line in FIG. 4.

Comparative example

For the trimming reagent formulation E-1, the trimming reagent of the 4 th step of the above operation procedure was replaced with a trimming reagent containing no exonuclease, and the other components were compared as in the procedure, and the results are shown in Table 15.

TABLE 15 action of pruning agent

In carrying out example 3 and comparative examples, in order to verify the effect of the trimming reagent, the washing reagent was selected in a configuration that is advantageous in that the amplified DNC size becomes large. From the above results, it can be seen that DNC size of 27 cycles of amplification was large, 9 pixels, and 3 pixels larger than that of 20 cycles of amplification without the trimming agent treatment. And after being treated by the exonuclease trimming reagent containing 230U/mL, the DNC size amplified for 27 cycles is only 5 pixels, and the change of the DNC size is small compared with the DNC size amplified for 20 cycles. It is noted that the use of exonuclease requires careful handling, if the concentration is too high, a large amount of DNA in DNC will be digested, resulting in experimental failure, if the concentration is too low, the effect is not obvious. FIG. 3 is a fluorescent image of DNC obtained by amplification with or without treatment with a pruning reagent (230U/mL exonuclease). In FIG. 3, A is a DNC fluorescent image obtained after 20 cycles of amplification and reagent trimming treatment, B is a DNC fluorescent image obtained after 7 cycles of amplification and reagent trimming treatment, C is a DNC fluorescent image obtained after 20 cycles of amplification and reagent trimming treatment, and D is a DNC fluorescent image obtained after 27 cycles of amplification.

As can be seen from fig. 3, after the trimming reagent treatment, the continued amplification for 7 cycles was only brighter than the continued amplification for 20 cycles of DNC, but of similar size. In Table 15, however, the DNC was brighter and the same size for the 7-cycle further amplification than for the 4-cycle further amplification. That is, DNC size is limited after the trimming reagent treatment, but DNC signal is stronger when the amplification cycle is continued. In addition, after trimming by the trimming reagent, the DNC-free region joint primer on the surface of the chip is digested, and the nonspecific adsorption of fluorescent dNTPs on the surface of the chip is reduced, so that the sequencing background is reduced, and the ratio of the sequencing signal to the background is greatly increased. Preferably, the clipping reagent is treated after 20 cycles of amplification and the amplification is continued for 7 cycles, DNC can be limited to 5 pixels, and better brightness, brightness variation coefficient and signal-to-back ratio can be obtained.

The DNC generated after the optimized washing reagent and trimming reagent and the corresponding amplification procedure was sequenced and the results are shown in FIG. 4. It can be seen that the optimized results are better, such as sequencing accuracy Q30 is better and the signal to noise ratio is higher, than if only the appropriate extension reagent was used. The optimization greatly improves the sequencing quality.

The foregoing is illustrative of the present invention and is not to be construed as limiting thereof, but rather as providing for the use of additional embodiments and advantages of all such modifications, equivalents, improvements and similar to the present invention are intended to be included within the scope of the present invention as defined by the appended claims.

Claims (8)

1. A high-throughput sequencing flushing reagent is characterized by comprising a Tris buffer solution, a soluble salt, a PCR enhancer and a single-chain binding protein.

2. The high throughput sequencing wash reagent of claim 1, wherein said soluble salt is selected from one or more of sodium, potassium, and magnesium salts.

3. The high throughput sequencing washing reagent according to claim 1, wherein the PCR enhancer is selected from one or more of BSA, triton X-100, tween 20, glycerol, formamide, polyethylene glycol, gelatin, tetramethyl ammonium chloride, betaine, and DMSO.

4. The high throughput sequencing wash reagent of claim 2, wherein said magnesium salt is magnesium sulfate.

5. A kit for high throughput sequencing comprising an extension reagent, the wash reagent of any one of claims 1-4, and a trimming reagent, wherein the extension reagent comprises a polymerase and deoxynucleotides, and the trimming reagent comprises an ethanolamine buffer, magnesium chloride, an exonuclease, and a blocking reagent.

6. The high throughput sequencing kit of claim 5, wherein said exonuclease is a single-strand specific 3'-5' exonuclease.

7. The kit for high-throughput sequencing according to claim 5, wherein the blocking reagent is one or more selected from BSA, tween 20, polyethylene glycol, trehalose, dithiothreitol.

8. A method of using a kit for high throughput sequencing comprising:

Step a, hybridizing single-stranded library DNA fragments to the adaptor primers on the surface of the chip, wherein the two ends of the library DNA fragments are identical or reversely complementary to the sequences of the adaptor primers;

Step b, amplifying the extension reagent into double chains along the single-stranded library DNA fragment templates from the adaptor primers on the chip;

Step c, using formamide or sodium hydroxide denaturing reagent to break the DNA double strand into single strands;

Step d, washing the denaturing reagent away by using a washing reagent, and leaving the single strand of DNA on the chip, during which time the single strand DNA anneals to other adaptor primers on the chip;

step e, amplifying the extension reagent into double chains along the single-stranded library DNA fragments from the adaptor primer on the chip;

F, repeating the steps c-e for 20-30 times, and treating the chip with a trimming reagent;

and g, repeating the steps c-e for 2-8 times, and finishing amplification.