HK1240277B - One-stop treatment method for breaking nucleic acid by means of transposase, and reagent - Google Patents

Description

A one-stop treatment method and reagent for nucleic acid transposase disruption

Technical Field

The present invention relates to the technical field of molecular biology, and in particular to a one-stop processing method and reagent for transposase disruption of nucleic acid.

Background Art

Since Roche invented the pyrosequencing method, which pioneered next-generation sequencing (NGS), NGS has experienced rapid growth. However, with the development of high-throughput sequencing, high-throughput and low-cost sample preparation has become a key consideration in the sequencing field. Various sample processing methods and automated devices have been developed, primarily encompassing sample fragmentation, nucleic acid end-processing, adapter ligation, and ultimately, library release.

Sample fragmentation is primarily achieved through physical methods (such as ultrasonic shearing) or enzymatic methods (i.e., non-specific endonuclease treatment). Physical methods primarily utilize Covaris, based on patented Adaptive Focused Acoustic (AFA) technology. Under isothermal conditions, this method utilizes geometrically focused acoustic energy, focusing a 1mm wavelength onto the sample via a spherical solid-state ultrasonic transducer operating at >400kHz. This method preserves the integrity of nucleic acid samples and achieves high recovery rates. Covaris instruments include the economical M series, the single-tube, full-power S series, and the higher-throughput E and L series. While fragmentation based on physical methods offers high randomness, throughput relies on a large number of Covaris fragmentation instruments, requiring subsequent end-treatment, linker addition, PCR, and various purification steps. Enzymatic methods include NEB Next dsDNA Fragmentase, launched by NEB. This reagent first creates random nick sites in double-stranded DNA, then a second enzyme recognizes the nick sites and cleaves the complementary DNA strand, achieving fragmentation. This reagent can be used for genomic DNA, whole-genome amplification products, and PCR products, and has good randomness. However, it can produce some artificial short insertions and deletions, and inevitably requires subsequent end-treatment, adapter addition, PCR, and corresponding purification operations. Transposase fragmentation kits, led by the Nextera kit from Epicentra (acquired by Illumina), utilize transposase to simultaneously fragment DNA and add adapters, thereby reducing sample processing time.

From the perspective of the simplicity of various operations, the transposase shearing method undoubtedly far surpasses other methods in terms of throughput and ease of operation. However, this shearing method also has its own disadvantages: the transposase is embedded in the target sequence and will inhibit subsequent enzymatic reactions. Although the relevant transposase kit manufacturers provide some reagents for transposase treatment of low-input samples (such as 5ng of starting genomic DNA), thus achieving the goal of eliminating the need for purification after shearing. However, for methods that require a higher amount of transposase, such as 50ng starting amount, the transposase needs to be removed by column or magnetic bead purification, which undoubtedly increases the cost and process of experimental operation (Figure 1A).

Summary of the Invention

The present invention provides a one-stop treatment method and reagent for transposase fragmentation of nucleic acids. The method and reagent can achieve one-stop processing from transposase fragmentation of nucleic acids to downstream PCR amplification reaction, without the need for column or magnetic bead purification, thereby simplifying the experimental operation process and reducing experimental costs.

According to a first aspect of the present invention, the present invention provides a one-stop method for transposase fragmentation of nucleic acids, comprising the following steps:

Randomly fragmenting the nucleic acid using a transposase-embedded complex, wherein the transposase-embedded complex comprises a transposase and a first adapter comprising a transposase recognition sequence;

adding a first reagent for treatment to break the adsorption of the transposase to the target sequence of the nucleic acid;

Adding a second reagent for treatment reduces the effect of the first reagent on the subsequent enzymatic reaction;

A PCR reaction is performed using the product treated with the second reagent as a template component to obtain a PCR product with adapters connected to both ends of the interrupted nucleic acid fragment.

In the method of the present invention, a first reagent is used to treat the reaction product after the transposase breaks the nucleic acid to break the adsorption of the transposase to the target sequence of the nucleic acid, replacing the traditional complex and costly column or magnetic bead purification step. Then, a second reagent is used to reduce the effect of the first reagent on the subsequent enzymatic reaction, ensuring smooth downstream PCR amplification.

As a preferred embodiment of the present invention, the first reagent comprises one or more of a protease solution, a sodium dodecyl sulfate (SDS) solution, and a NT buffer. These solutions degrade or denature the transposase, freeing it from the target nucleic acid sequence. It should be noted that the first reagent can be one or more of the above solutions, wherein the plurality can be two or three, such as a protease solution and an SDS solution, an SDS solution and a NT buffer, a protease solution and a NT buffer, or a protease solution, an SDS solution, and a NT buffer. The NT buffer can be the NT buffer provided with the Truprep kit S5 series.

As a preferred embodiment of the present invention, if the first reagent contains a protease solution, then after the first reagent is added for treatment, an ethylenediaminetetraacetic acid (EDTA) solution is further added for treatment. EDTA inhibits the activity of the protease, preventing the protease from degrading the enzyme in the subsequent PCR reaction.

As a preferred embodiment of the present invention, the second reagent comprises a Triton-X100 solution. Triton-X100, also known as Triton X-100 and chemically known as octylphenyl polyoxyethylene ether, is a nonionic surfactant used in the present invention to reduce the effect of the first reagent on the subsequent enzymatic reaction.

As a preferred embodiment of the present invention, if the first reagent contains an SDS solution, the second reagent also contains a Tween-20 solution. The addition of Tween-20 can further reduce the effect of SDS on subsequent enzymatic reactions and improve the PCR effect. It should be noted that Tween-20 can be used as a second reagent in the form of a mixture with Triton-X100; it can also be provided separately in the form of separation from Triton-X100. In this case, the second reagent refers to Triton-X100 solution and Tween-20 solution. It should be understood that the first reagent and the second reagent in the present invention are not limited to a single object, but may also refer to a combination of multiple objects. Moreover, in the present invention, the concepts of "first" and "second" used in any case should not be understood as having sequential and technical meanings, and their function is only to distinguish them from other objects.

As a preferred embodiment of the present invention, after adding the second reagent for treatment and before conducting the PCR reaction, the method further includes: ligating a second adapter at the gap using a ligase, wherein the gap is a 9-bp base deletion formed after ligating the first adapter at both ends of the fragmented nucleic acid. The addition of the second adapter enables PCR using primers that target the first adapter and primers that target the second adapter, respectively, to produce PCR products with different adapter sequences at both ends. This eliminates the need for the fragmented nucleic acid fragments to be used due to the shared transposase recognition sequence on both sides. The sequence of the second adapter is not limited and can be any sequence.

As a preferred embodiment of the present invention, the 3' end of the second linker is a dideoxynucleotide to prevent the second linker from interconnecting with the first linker.

According to a second aspect of the present invention, the present invention provides a one-stop treatment reagent for transposase fragmentation of nucleic acids, comprising the following components:

A first reagent comprising one or more of a protease solution, an SDS solution, and an NT buffer, for breaking the adsorption of the transposase to the target sequence of the nucleic acid;

The second reagent, comprising Triton-X100 solution, is used to reduce the effect of the first reagent on the subsequent enzymatic reaction.

It should be noted that the first reagent can be one or more of a protease solution, an SDS solution, and a NT buffer. The multiple reagents can be two or three, such as a protease solution and an SDS solution, an SDS solution and a NT buffer, a protease solution and a NT buffer, or a protease solution, an SDS solution, and a NT buffer. The NT buffer can be the NT buffer provided with the Truprep kit S5 series.

As a preferred embodiment of the present invention, if the first reagent includes a protease solution, the first reagent also includes an additional reagent containing EDTA. EDTA inhibits the activity of the protease, preventing the protease from degrading the enzyme in the subsequent PCR reaction. The additional reagent containing EDTA is provided separately from the protease solution as a component of the first reagent.

As a preferred embodiment of the present invention, if the first reagent includes an SDS solution, the second reagent further includes a Tween-20 solution. Tween-20 can be provided as the second reagent in a mixed form with Triton-X100, or can be provided separately from Triton-X100.

As a preferred embodiment of the present invention, the reagent further comprises: a transposase and a first linker containing a transposase recognition sequence, which is used to form a transposase-embedded complex to randomly fragment the nucleic acid.

As a preferred embodiment of the present invention, the reagent further comprises a PCR component for performing a PCR reaction using the product treated with the second reagent as a template component. PCR components generally include DNA polymerase, PCR buffer, dNTPs, Mg2 ⁺ solution, and primers.

As a preferred embodiment of the present invention, the reagent further comprises a second linker component for connecting to the gap formed after the two ends of the interrupted nucleic acid are connected to the first linker.

As a preferred embodiment of the present invention, the reagent further comprises a ligase component for connecting a second linker to the gap formed after the two ends of the broken nucleic acid are connected to the first linker.

In the present invention, the nucleic acid to be interrupted can be genomic DNA, whole genome amplification product or PCR product, can be DNA or cDNA, and there is no limitation on the source of the nucleic acid, which can be a nucleic acid sample of animal, plant or microbial origin.

In the present invention, the working concentrations of the first and second reagents can be determined empirically by those skilled in the art. Generally, in the first reagent, the working concentration of protease is preferably 50-5000 mAU/mL, preferably 75-3750 mAU/mL, and most preferably 1500 mAU/mL; the working concentration of EDTA is preferably 1-50 mmol/L, preferably 14 mmol/L; the working concentration of SDS is preferably 0.01%-1.5% (volume ratio), preferably 1% (volume ratio); and the final concentration of NT buffer can be 1x. In the second reagent, the working concentration of Triton-X100 is preferably 0.1%-2% (volume ratio), preferably 1% (volume ratio); and the working concentration of Tween-20 is preferably 0.1%-2% (volume ratio), preferably 0.5% (volume ratio).

The method of the present invention uses a first reagent and a second reagent to process the product of nucleic acid fragmentation by transposase, replacing traditional column or magnetic bead purification, thereby achieving a one-stop processing from transposase fragmentation of nucleic acid to downstream PCR amplification reaction. The entire process is carried out in a single tube, which simplifies the experimental operation process, reduces experimental costs, shortens the processing cycle, and thus makes high-throughput sample processing possible.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram of the operational flow of a conventional transposase kit from fragmentation to PCR reaction (A), a schematic diagram of the operational flow of the purification-free one-stop reaction of the present invention (B), and a schematic diagram of the operational flow of the purification-free one-stop reaction optimized for the second adapter introduction scheme of the present invention (C).

Figure 2 shows the electrophoresis detection results of the PCR products of the No. 1 adapter double adapter transposase complex of Example 1 of the present invention, wherein D2000 is the DNA Ladder lane; lane 1 is the purification result of 1×PBI and 1.3×Ampure XP beads; lane 2 is the result of treatment with 2 μL protease + 14 mM EDTA + 1% Triton-X100; lane 3 is the result of treatment with 1% SDS + 1% Triton-X100 + 0.5% Tween-20; lane 4 is the result of treatment with NT buffer + 1% Triton-X100; and lane 5 is the result of treatment with 2 μL protease + 1% Triton-X100.

Figure 3 shows the electrophoresis detection results of the PCR products of the No. 1 adapter single adapter transposase complex of Example 2 of the present invention, wherein D2000 is the DNA ladder lane; lane 1 shows the results of treatment with 2 μL protease + 1% Triton-X100; lane 2 shows the results of treatment with NT buffer + 1% Triton-X100; lane 3 shows the results of treatment with 1% SDS + 1% Triton-X100 + 0.5% Tween-20; lane 4 shows the results of treatment with 2 μL protease + 14 mM EDTA + 1% Triton-X100; lane 5 shows the results of treatment with 1×PBI, 1.3×Ampure XP beads + 1% Triton-X100; and lane 6 shows the results of the negative control (no template added).

DETAILED DESCRIPTION

The present invention is further described in detail below by means of specific examples. Unless otherwise specified, the techniques used in the following examples are conventional techniques known to those skilled in the art; the instruments, equipment, and reagents used are all available to those skilled in the art through public channels, such as commercial purchases.

Referring to FIG1B , the operational flow of the one-stop reaction without purification of the present invention mainly includes: (1) using a No. 1 linker (referred to as the first linker elsewhere in the present invention) embedded with a specific modification sequence (including a transposase recognition sequence) of a transposase to randomly interrupt a nucleic acid sequence such as a genomic sequence, a whole genome amplification sequence or a PCR product sequence; (2) treating the No. 1 reagent (referred to as the first reagent elsewhere in the present invention) with a protease or other specific ratio to break the adsorption effect between the transposase and the target sequence to achieve a purification-free operation; (3) adding a No. 2 reagent (referred to as the second reagent elsewhere in the present invention) to the product of the previous step reaction to reduce the effect of the No. 1 reagent on the subsequent enzymatic reaction; (4) directly performing a PCR reaction mediated by a special primer (the primer targets the No. 1 linker, and the No. 1 linker includes two linker parts, each of which contains a common transposase recognition sequence and other different sequences) without purification, thereby obtaining a PCR product in which the two ends of the target sequence are connected to completely different sequences except the transposase recognition sequence, and this product can be used in subsequent molecular biology experiments.

1C , the operational flow of the one-stop reaction without purification corresponding to the scheme for introducing the second adapter of the present invention mainly includes: (1) using the first adapter embedded with a specific modified sequence of transposase to randomly interrupt nucleic acid sequences such as genomic sequences, whole genome amplification sequences and PCR product sequences; (2) breaking the adsorption of the transposase and the target sequence by treating with protease or other specific proportions of the first reagent to achieve a purification-free operation; (3) adding the second reagent to reduce the effect of the first reagent on the subsequent enzymatic reaction; (4) introducing the second adapter (referred to as the second adapter or gap adapter elsewhere in the present invention) by connecting the second adapter at the 9nt gap, changing the adapter base sequence adjacent to the fragmented target sequence, so that the sequences on both sides of the target sequence are completely different, one of which retains the first adapter sequence containing the transposase recognition sequence, and the other is a completely arbitrarily designed second adapter sequence; (5) directly performing a PCR reaction mediated by special primers (primers targeting the first adapter and the second adapter respectively) to obtain a PCR product with completely different sequences connected at both ends of the target sequence, which can be used in subsequent molecular biology experiments.

During the transposase embedding stage, the present invention simultaneously selects two embedding methods: the first is that the transposase embeds the first adapter with a double adapter to form a transposase embedding complex (Figure 1B), in which both adapters contain a special 19bp transposase recognition sequence, but each also contains different other sequences; the second is that the transposase embeds the first adapter with a single adapter to form a complex (Figure 1C). Through special modification of the embedded adapter, a segment of the embedded first adapter sequence contains a special 19bp transposase recognition sequence. At the same time, to prevent the unembedded first adapter from connecting with the second adapter and affecting PCR, the adapter is modified at the 3' end with a dideoxy nucleotide in the present invention.

The present invention uses a domestically produced transposase kit (S50 series of Truprep kit produced by Nanjing Novozymes) for experiments. The kit contains two amounts of 5ng genomic DNA and 50ng genomic DNA. This embodiment uses 50ng genomic DNA for experiments.

Example 1

In this example, 50 ng of high-quality genomic DNA was first fragmented using the embedded transposase complex. The DNA was then treated with protease, SDS, NT, or a combination of protease and EDTA to remove the transposase protein bound to the DNA. PCR primers were then used for direct amplification, and a certain concentration of Triton X-100 was added to the PCR reaction system.

1. Design and order three primer sequences with a 19 bp transposase recognition sequence, sequence A, sequence B, and sequence C, for preparing the No. 1 adapter double adapter (referred to as No. 1 adapter in this invention) for embedding. Sequence A + sequence B constitute the No. 1 adapter double adapter 5 end; sequence A + sequence C constitute the No. 1 adapter double adapter 3 end:

Linker No. 1 double linker sequence A: CTGTCTCTTATACACATCT (SEQ ID NO: 1);

Linker No. 1 double linker sequence B: TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG (SEQ ID NO: 2);

Linker No. 1 double linker sequence C: GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG (SEQ ID NO: 3).

2. Sequence A, sequence B, and sequence C were diluted to 100 μM, respectively, and sequence A + sequence B and sequence A + sequence C were combined. After thorough mixing, centrifugation was performed and annealed in a PCR instrument according to the following procedure (Table 1) to obtain linker No. 1 (stored at -20°C) for preparation of the embedding complex.

Table 1

After the reaction, the two sets of annealed adapters were mixed in equal volumes and used to embed the transposase complex.

3. According to the following system (Table 2), embed the first adapter and transposase into a transposase-embedded complex. Gently pipette 20 times to mix. Incubate at 30°C for 1 hour to complete the complex embedding. Store the complex at -20°C.

Table 2

Components content Transposase S50 reagent 47μL Connector No. 1 2μL Coupling buffer 47μL total 96μL

4. Mix 50 ng of high-quality genome and transposase complex according to the following system (Table 3), gently pipette 20 times to mix, incubate at 55°C for 10 minutes, and then cool to 4°C to complete genome fragmentation.

Table 3

Components content water 5μL 5× disruption buffer 2μL gDNA (50 ng/μL) 1 μL Disrupt enzyme complex 2μL total 10 μL

5. There are several options for sample processing after shearing. Method 1: Add 1 volume of PBI (a commercial reagent in the Qiagen PCR purification kit) and mix well, then purify with 1.3 times of Ampure XP beads, and re-melt with pure water; Method 2: Add 0.1-5μL protease (750mAU/mL) for treatment, and then add EDTA with a final concentration of 1-50mM. In this example, 2μL protease and a final concentration of 14mM EDTA are preferred, and 0.1μL protease plus 1mM EDTA and 5μL protease plus 50mM EDTA are also used. EDTA combination was tested; Method 3: adding 0.01%-1.5% (volume ratio) of SDS, preferably 1% (volume ratio) of SDS in this embodiment, and the concentrations of 0.01% (volume ratio) and 1.5% (volume ratio) were tested respectively; Method 4: adding a final concentration of commercial 1×NT buffer (matching reagent in the Truprep kit S5 series); Method 5: adding 0.1-5 μL of protease for treatment, preferably 2 μL of protease in this embodiment, and the amounts of 0.1 μL and 5 μL of protease were tested respectively.

6. After the above treatment, 0.1%-2% (volume ratio) of Triton-X100 was added to the product. In this embodiment, 1% (volume ratio) was preferred. Triton-X100 at dosages of 0.1% (volume ratio) and 2% (volume ratio) was tested.

7. PCR amplification was performed according to the following PCR reaction system (Table 4) and reaction conditions (Table 5). For the experimental group in which SDS was added, a specific concentration of Tween-20 was also added to the PCR system to partially improve PCR efficiency. The working concentration of Tween-20 can be adjusted to various levels, such as 0.1% to 2% (volume ratio), with 0.5% (volume ratio) being preferred in this example. Both 0.1% (volume ratio) and 2% (volume ratio) working concentrations of Tween-20 were tested.

Table 4

Components content Processed DNA samples 30μL 10 μL 10mM dNTP 1 μL Primer 1 (10 μM) 2μL Primer 2 (10 μM) 2μL PCR enzyme (DNA polymerase) 1 μL pure water 4μL Total 50μL

Note: No. 1 adapter double adapter primer 1: AATGATACGGCGACCACCGA (SEQ ID NO: 4); No. 1 adapter double adapter primer 2: CAAGCAGAAGACGGCATACGA (SEQ ID NO: 5).

Table 5

8. Detection of PCR products of the No. 1 adapter double adapter transposase complex is shown in Figure 2, and the results of PCR product concentration determination are shown in Table 6:

Table 6

Example 2

In this example, 50 ng of high-quality genomic DNA was first fragmented using the embedded transposase complex. The DNA was then treated with protease, SDS, NT, or a combination of protease and EDTA to remove the transposase protein bound to the DNA. After ligation via gap linkers, the DNA was directly amplified using PCR primers. A certain concentration of Triton X-100 was added to the PCR reaction system.

1. Design and order a pair of primer sequences with a 19bp transposase recognition sequence, sequence A and sequence B, for preparing a single adapter No. 1 for embedding (referred to as adapter No. 1 in the present invention):

Linker No. 1 single linker sequence A: TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG (SEQ ID NO: 6);

Linker No. 1 single linker sequence B: CTGTCTCTTATACACATC ddT (SEQ ID NO: 7, dd indicates dideoxy modification).

2. Dilute sequence A and sequence B to 100 μM, mix thoroughly, and centrifuge. Anneal in a PCR instrument according to the following procedure (Table 7) to obtain adapter No. 1 (stored at -20°C) for preparation of the embedding complex.

Table 7

3. Embed the first adapter and transposase into a transposase complex according to the following system (Table 8). Gently pipette 20 times to mix. Incubate at 30°C for 1 hour to complete the complex embedding. Store the complex at -20°C.

Table 8

Components content transposase 85μL Connector No. 1 30μL Coupling buffer 85μL total 200 μL

4. Mix 50 ng of high-quality genome and transposase complex according to the following system (Table 9), gently pipette 20 times to mix, incubate at 55°C for 10 minutes, and then cool to 4°C to complete genome fragmentation.

Table 9

Components content water 5μL 5× disruption buffer 2μL gDNA (50 ng/μL) 1 μL Disrupt enzyme complex 2μL total 10 μL

5. There are several options for processing the sample after shearing. Method 1: Add 0.1-5 μL protease (750 mAU/mL) for treatment. In this embodiment, 2 μL protease is preferred. At the same time, 0.1 μL and 5 μL protease dosages are tested respectively. Method 2: Add a final concentration of commercial 1× NT buffer (supporting reagents in the Truprep kit S5 series); Method 3: Add 0.01%-1.5% (volume ratio) SDS. In this embodiment, 1% (volume ratio) SDS is preferred. At the same time, 0.01% (volume ratio) and 1.5% (volume ratio) concentrations are tested respectively. Method 4: Add 0.1-5 μL protease for treatment, and then add EDTA at a final concentration of 1-50 mM. In this embodiment, 2 μL protease and a final concentration of 14 mM EDTA are preferred. At the same time, the combinations of 0.1 μL protease plus 1 mM EDTA and 5 μL protease plus 50 mM EDTA are tested; Method 5: Add 1 volume of PBI (Qiagen The resulting product was purified using Ampure XP beads at a 1.3x dilution ratio and then reconstituted with pure water.

6. After the above treatment, 0.1%-2% (volume ratio) of Triton-X100 was added to the product. In this embodiment, 1% (volume ratio) was preferred. Triton-X100 at dosages of 0.1% (volume ratio) and 2% (volume ratio) was tested.

7. The Triton-X100-treated product was annealed and connected to the gap adapter (Adapter No. 2) according to the following system (Table 10), and the adapter connection was completed by incubation at 25°C for 60 minutes.

Table 10

Components content water 8μL 3× connection buffer 20 μL Adapter No. 2 (5μM) 10 μL Ligase 2μL DNA 20 μL total 30μL

Note: Linker sequence A: 5’-pAAGTCGGAGGCCAAGCGGTCGT ddC-3’ (SEQ ID NO: 8); Linker sequence B: 5’-TTGGCCTCCGACT ddT-3’ (SEQ ID NO: 9) (p indicates phosphorylation modification; dd indicates dideoxy modification).

8. PCR amplification was performed according to the following PCR reaction system (Table 11) and reaction conditions (Table 12). For the experimental group in which SDS was added, a specific concentration of Tween-20 was also added to the PCR system to partially improve PCR efficiency. The working concentration of Tween-20 can be adjusted to various levels, such as 0.1% to 2% (volume ratio), with 0.5% being preferred in this example. Both 0.1% (volume ratio) and 2% (volume ratio) working concentrations of Tween-20 were tested.

Table 11

Note: Adapter single adapter primer 1:

AGACAAGCTCGAGCTCGAGCGATCGGGATCTACACGACTCACTGATCGTCGGCAGCGTC (SEQ ID NO: 10); Adapter single adapter primer 2: TCCTAAGACCGCTTGGCCTCCGACT (SEQ ID NO: 11).

Table 12

9. The results of PCR product detection after single-linker embedded complex fragmentation and gap linker ligation are shown in Figure 3, and the results of PCR product concentration determination are shown in Table 13.

Table 13

The above is a further detailed description of the present invention in conjunction with specific embodiments, and the specific implementation of the present invention cannot be considered to be limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention.

Claims (5)

1. A one-stop method for transposase-mediated disruption of nucleic acids, comprising the following steps: The nucleic acid is randomly fragmented using a transposase embedding complex, wherein the transposase embedding complex comprises a transposase and a first adapter containing a transposase recognition sequence. The first reagent is added to break the adsorption of the transposase to the target sequence of the nucleic acid; A second reagent is added to reduce the effect of the first reagent on the subsequent enzymatic reaction. Using the product treated with the second reagent as a template, a PCR reaction was performed to obtain a PCR product with adapters attached to both ends of the broken nucleic acid fragments. The first reagent contains an SDS solution; The second reagent contains a Triton-X100 solution and also contains a Tween-20 solution. 2. The method according to claim 1, characterized in that, after treatment with the second reagent and before performing the PCR reaction, it further comprises: ligating a second adapter at the gap using a ligase, wherein the gap is a 9bp deletion formed after the first adapter is ligated to both ends of the broken nucleic acid. 3. The method according to claim 2, wherein the 3' end of the second connector is a dideoxynucleotide to prevent the second connector from interconnecting with the first connector. 4. A one-stop reagent for transposase disruption of nucleic acids, comprising the following components: The first reagent contains an SDS solution, used to break the adsorption of the transposase to the target sequence of the nucleic acid; The second reagent, comprising Triton-X100 solution and Tween-20 solution, is used to reduce the effect of the first reagent on subsequent enzymatic reactions; Also includes: Transposase and a first linker containing a transposase recognition sequence are used to form a transposase embedding complex to randomly break down nucleic acids; Also includes: PCR components are used to perform PCR reactions using the product of the second reagent as a template. 5. The reagent according to claim 4, characterized in that the reagent further comprises: The second adapter component is used to connect to the gap formed after the two ends of the broken nucleic acid are connected to the first adapter: The ligase component is used to attach a second adapter to the gap formed after the first adapter is attached to both ends of the broken nucleic acid.