HK1240286B - Method and reagent for constructing nucleic acid double-linker single-strand cyclical library - Google Patents

Description

A method and reagent for constructing a double-linker single-stranded circular library of nucleic acids

Technical Field

The present invention relates to the field of molecular biology technology, and in particular to a method and reagent for constructing a double-linker single-stranded circular library of nucleic acids.

Background Art

Currently, high-throughput sequencing is a key research tool in various fields, including molecular biology and medical diagnostics. Since the advent of high-throughput second-generation sequencing technology, sequencing technology has experienced rapid advancements. Compared to previously expensive sequencing costs, second-generation sequencing has reduced sequencing costs by several orders of magnitude, while also significantly shortening sequencing times. The emergence of third-generation sequencers has intensified competition in the sequencing market, accelerating its development. Therefore, sequencing companies and research teams must reduce sequencing costs, shorten process times, and improve result accuracy to remain competitive.

Among second-generation sequencing platforms, Complete Genomics (CG) has developed a highly accurate and high-throughput platform for whole-genome sequencing of human genomes, achieving data accuracy of up to 99.9998%. This platform provides accurate genomic information for cancer research, detection of low-frequency mutations, and personal genome sequencing. However, the CG platform's library construction process is time-consuming and costly, and the short inserts in the libraries limit subsequent data output and analysis. This not only hinders the development of scientific research projects but also hinders the large-scale and widespread use and development of the CG platform. To maintain its competitive advantage in today's fiercely competitive landscape, the CG platform must optimize its library construction process, shorten construction time, increase library length, and reduce costs. In traditional CG platform library construction, the steps between double-stranded DNA circularization and final single-stranded DNA circularization are quite complex. The library construction process primarily includes 12 steps: enzyme digestion of the circularized DNA, removal of phosphorylation, end repair, 3'-end adapter ligation, 5'-end adapter ligation, nick translation, polymerase chain reaction, single-stranded DNA separation, and circularization. Each enzymatic reaction requires magnetic bead purification. This process is time-consuming and costly, and the library insert fragment generated by enzyme digestion is only 26bp, which neither takes advantage of the large-scale application of the CG platform nor meets the fragment requirements of the CG next-generation sequencing platform.

Summary of the Invention

The present invention provides a method and reagent for constructing a double-linker single-stranded circular library of nucleic acids. The method can increase the length of library insert fragments, simplify the library construction process, shorten the library construction time, and reduce the library construction cost.

According to a first aspect of the present invention, the present invention provides a method for constructing a double-linker single-stranded circular library of nucleic acids, comprising the following steps:

fragmenting the nucleic acid into nucleic acid fragments for library construction;

Connecting a first adapter sequence to both ends of the nucleic acid fragment;

A first product having first adapter sequences at both ends is obtained by a first PCR amplification, wherein the primer sequence used in the first PCR has a U base site and has or does not have a nicking enzyme recognition sequence, and one of the primer sequences has a first affinity tag;

The first product is digested with USER enzyme to generate sticky ends with or without nicks;

Circularizing the first product after enzyme cleavage to produce a circular nucleic acid molecule;

Treating a circular nucleic acid molecule having nicks on both strands with a dephosphorylase, or treating a circular nucleic acid molecule having a nickase recognition sequence on one strand and a nick on the other strand or having a nickase recognition sequence on both strands and no nick with a nickase to generate a nick;

Using a solid support with a second affinity tag to bind to the circular nucleic acid molecule;

Using the circular nucleic acid molecule bound to the solid support as a template, a restriction nick translation reaction is performed starting from the nick and/or gap;

Digestion removes the portion of the circular nucleic acid molecule that has not undergone restriction nick translation reaction to obtain a linear nucleic acid molecule;

Connecting a second linker sequence to both ends of the linear nucleic acid molecule;

A second product having a second linker sequence at both ends is obtained by a second PCR amplification;

The second product is denatured to obtain a single-stranded nucleic acid molecule, and the single-stranded nucleic acid molecule is circularized using a mediating sequence complementary to both ends of one of the single-stranded nucleic acid molecules to obtain a double-linker single-stranded circular library.

As a preferred embodiment of the present invention, the first affinity label is a biotin label; and the second affinity label is a streptavidin label.

As a preferred embodiment of the present invention, the first adapter sequence includes a first 5' adapter sequence and a first 3' L-shaped adapter sequence, which connect the 3' end and 5' end of each chain of the fragment, respectively; the first 5' adapter sequence includes a long chain with a phosphorylated 5' end and a complementary short chain, the 3' end of the short chain is dideoxy modified, and the short chain contains a U base site; the first 3' L-shaped adapter sequence has partial base complementarity with the first 5' adapter sequence in the portion adjacent to the connected fragment;

Connecting the first linker sequence to both ends of the nucleic acid fragment specifically includes:

Dephosphorylation of nucleic acid fragments;

Perform end repair on the dephosphorylated nucleic acid fragments;

Attach a first 5' adapter sequence to the 3' end of each strand of the nucleic acid fragment;

Use USER enzyme to cut the U base site of the short chain of the first 5' linker sequence;

Phosphorylation treatment is performed on the nucleic acid fragments after USER enzyme digestion;

A first 3' L-type linker sequence is connected to the 5' end of each chain of the phosphorylated nucleic acid fragment.

As a preferred embodiment of the present invention, the primer sequences used in the first PCR all have a U base site and a nicking enzyme recognition sequence; after the U base site is cut with the USER enzyme, sticky ends are formed at both ends of the nucleic acid fragment, and the sticky ends are complementary and circularized to produce a circular nucleic acid molecule; then the nicking enzyme recognition sequence is cut with the nicking enzyme to produce a nick.

As a preferred embodiment of the present invention, one of the primer sequences used in the first PCR has two U base sites, and the other primer has one U base site; after the U base site is cleaved by USER enzyme, sticky ends are formed at both ends of the nucleic acid fragment, and the sticky ends complement each other and cyclize to produce a circular nucleic acid molecule.

As a preferred embodiment of the present invention, after cyclizing the first product after enzyme cleavage, the method further comprises: digesting the uncyclized nucleic acid molecule.

As a preferred embodiment of the present invention, in the restriction nick translation reaction, the length of the generated nick translation fragment is controlled by controlling at least one of the molar ratio of dNTP to the nucleic acid molecule as the template, the enzyme reaction temperature and the time.

As a preferred embodiment of the present invention, digestion and removal of the portion of the circular nucleic acid molecule that has not undergone restriction nick translation reaction specifically includes: first using a double-stranded exonuclease to degrade until the nicks at both ends meet; then using a single-stranded exonuclease to degrade the single strand; or using a nuclease endonuclease to directly cut off the portion of the circular nucleic acid molecule that has not undergone restriction nick translation reaction.

As a preferred embodiment of the present invention, the second linker sequence is a bubble linker sequence, which includes two base sequences that are partially complementary at both ends but not complementary in the middle, wherein the middle segment forms a bubble and has a U base site; the 5' end of one chain of the bubble linker sequence has a protruding T base;

Connecting a second linker sequence to both ends of the linear nucleic acid molecule specifically comprises:

Perform end repair and 3' end A addition reaction on linear nucleic acid molecules;

The bubble linker sequence is connected to both ends of the linear nucleic acid molecule through the pairing of T base and A base;

Use USER enzyme to cut the U base site in the middle segment.

As a preferred embodiment of the present invention, after circularizing the single-stranded nucleic acid molecule, the method further comprises: digesting the uncircularized single-stranded nucleic acid molecule.

According to a second aspect of the present invention, the present invention provides a reagent for constructing a double-linker single-stranded circular library of nucleic acids, comprising the following components:

a first adapter sequence, comprising a first 5' adapter sequence and a first 3' L-shaped adapter sequence, connecting the 3' and 5' ends of each chain of the fragments, respectively; the first 5' adapter sequence comprises a long chain phosphorylated at the 5' end and a complementary short chain, the 3' end of the short chain being dideoxy modified and containing a U base site; the first 3' L-shaped adapter sequence has partial base complementarity with the first 5' adapter sequence in the portion adjacent to the connected fragments;

A first PCR primer having a U base site and having or not having a nickase recognition sequence, and one of the primer sequences having a first affinity tag, for obtaining a first product having first adapter sequences at both ends through a first PCR amplification;

USER enzyme, used to cleave the first product, generating sticky ends with or without nicks;

a cyclase for cyclizing the first product after the enzyme cleavage to produce a circular nucleic acid molecule;

a dephosphorylase for dephosphorylating a circular nucleic acid molecule having nicks on both strands; or a nicking enzyme for cleaving a circular nucleic acid molecule having a nicking enzyme recognition sequence on one strand and a nick on the other strand, or having a nicking enzyme recognition sequence on both strands and no nicks, to produce a nick;

a solid support having a second affinity tag for binding to the circular nucleic acid molecule;

Components of a nick translation reaction, used to perform a restriction nick translation reaction starting from the nick and/or nick using a circular nucleic acid molecule bound to a solid support as a template;

A digestive enzyme for digesting and removing the portion of the circular nucleic acid molecule that has not undergone restriction nick translation reaction to obtain a linear nucleic acid molecule;

A second linker sequence, which is a bubble linker sequence, comprising two base sequences that are partially complementary at both ends but not complementary in the middle, wherein the middle segment forms a bubble and has a U base site; the 5' end of one chain of the bubble linker sequence has a protruding T base;

A second PCR primer is used to obtain a second product having a second adapter sequence at both ends through a second PCR amplification;

The mediating sequence is denatured with the second product to obtain a single-stranded nucleic acid molecule, one of which is complementary at both ends and is used to circularize the single-stranded nucleic acid molecule to obtain a double-linker single-stranded circular library.

As a preferred embodiment of the present invention, the first affinity label is a biotin label; and the second affinity label is a streptavidin label.

The method for constructing a double-linker single-stranded circular library of nucleic acids of the present invention combines a restriction nick translation reaction with an enzyme reaction performed on magnetic beads. The restriction nick translation reaction replaces the class III endonuclease site in the traditional method with a new nicking enzyme site, and controllable nucleic acid chain extension is performed starting from the nick or nick, thereby increasing the length of the library insert fragment. In addition, after the circular nucleic acid molecules are bound to the magnetic beads, there is no need to elute the nucleic acid. Instead, an enzyme reaction solution is directly added, and the enzyme reaction is carried out on the magnetic beads until the single-strand elution step. There is no need to bind and elute the magnetic beads multiple times in the middle, which shortens the library construction time and saves the cost of repeatedly adding new magnetic beads.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a flow chart of the process from magnetic bead binding to single-stranded circularization in the method for constructing a double-linker single-stranded circular library of nucleic acids according to one embodiment of the present invention;

FIG2 is a diagram showing the basic principle of CNT incision generation according to one embodiment of the present invention;

FIG3 is a diagram showing the basic principle of CNT gap generation according to an embodiment of the present invention;

FIG4 is a diagram comparing the principles of insert fragment formation and single-strand separation and cyclization using the existing method and the method of the present invention;

FIG5 is a comparison diagram of the existing directional joint connection method and the L-shaped joint connection method of the present invention;

FIG6 is an electrophoresis detection result of the final product of four parallel experiments in one embodiment of the present invention, wherein M represents DNA Marker; 1, 2, 3, and 4 represent the electrophoresis results of four parallel samples C22, D22, E22, and F22, respectively.

7-10 are the test results of the final products of four parallel experiments C22, D22, E22 and F22 in one embodiment of the present invention using a LabChip GX instrument (automatic microfluidic electrophoresis instrument, Caliper).

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.

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 it from other objects.

In the present invention, the first affinity tag and the second affinity tag can be components of commonly used biological binding reactions, such as an antigen or antibody, one strand of a double-stranded DNA fragment, biotin or streptavidin, etc. When the first affinity tag is an antigen, the second affinity tag is an antibody that binds to the antigen, and vice versa. When the first affinity tag is one strand of a double-stranded DNA fragment, the second affinity tag is the other strand that complements the first strand, and vice versa. When the first affinity tag is biotin, the second affinity tag is streptavidin that binds to biotin, and vice versa. In one embodiment of the present invention, the first affinity tag is a biotin tag and the second affinity tag is a streptavidin tag, and the two have strong binding ability.

Please refer to Figure 1. A method for constructing a double-linker single-stranded circular library of nucleic acids according to one embodiment of the present invention includes the following steps: breaking genomic DNA to form nucleic acid fragments for library construction; performing dephosphorylation and end repair reactions; connecting a 5' linker A sequence; USER enzyme digestion and phosphorylation treatment; connecting a 3' L-type linker A sequence; PCR amplification to obtain a product having a 5' linker A sequence and a 3' L-type linker A sequence at both ends, wherein the primer sequence used in the PCR has a U base site and a nicking enzyme recognition sequence, and one primer has a biotin label; using USER enzyme to digest the U base site to generate a sticky end, and circularizing the product after USER enzyme digestion to generate a circular nucleic acid molecule; binding the circular nucleic acid molecule to streptavidin-labeled magnetic beads; using nicking enzyme to cut a nick at the nicking enzyme recognition sequence; and starting a restriction endonuclease reaction (Controlled Nick Translation) from the nick. Translation (CNT); using a nuclease to cut the nucleic acid chain at the nick (or first using a double-stranded exonuclease to degrade until the nicks at both ends meet, and then using a single-stranded exonuclease to degrade the single strand) to obtain a linear nucleic acid molecule; performing end repair and 3'-end A base addition reaction on the linear nucleic acid molecule; connecting the bubble linker sequence; using USER enzyme to cut the U base site on the bubble linker sequence to form an L-shaped linker; PCR amplification to obtain products with different sequences at both ends; denaturation treatment to obtain single-stranded nucleic acid molecules, and circularizing the single-stranded nucleic acid molecules using a mediating sequence that is complementary to both ends of one of the single-stranded nucleic acid molecules to obtain a double-linker single-stranded circular library.

In the present invention, in the method for constructing a double-linker single-stranded circular library shown in Figure 1, a U base site and a nicking enzyme recognition sequence are introduced into the primer sequence used in the first PCR, and a nick is generated using the principle shown in Figure 2, which serves as the starting point of the restriction nick translation reaction. The existing method is to introduce a Class III endonuclease recognition sequence into the linker sequence. After the linker sequence is connected and circularized, the double strands are cleaved with a Class III endonuclease to produce linearized double-stranded DNA; however, the method of the present invention introduces a U base site and a nicking enzyme recognition sequence into the primer sequence used in the first PCR. After PCR amplification, the U base site is cleaved with a USER enzyme to produce a sticky end, and double-stranded circularization is performed. Then, a nicking enzyme (such as Nb.BsrDI, Nb.BsmI, Nt.BbvCI, Nb.Bbv.Nb.BtsI or Nt.BstNBI, etc.) is used to cleave the single strands of the circularized DNA, generating a nick on each single strand to provide an effective starting site for CNT action.

In the present invention, as an alternative, a U base site is introduced into the primer of the first PCR, and a gap is generated by enzyme digestion with USER enzyme, which serves as the starting point of the restriction gap translation reaction. The basic principle of this gap generation is shown in Figure 3: (1) After connecting the 5' adapter A sequence and the 3' L-type adapter A sequence, the adapter A ligation product is amplified using primers with two Us and one U respectively; (2) the U base is digested with USER enzyme, and phosphorylated 3' and 5' ends are formed at the incision; (3) the sticky ends generated by enzyme digestion are used for double-stranded circularization. After circularization, the gap on one chain (Gap 1, formed by USER enzyme digestion) is the phosphorylated 3' end and 5' end, and the gap on the other chain (Gap 2, formed due to the lack of a matching base at this location after circularization) is the dephosphorylated 3' end and phosphorylated 5' end; (4) dephosphorylation treatment is performed to dephosphorylate the 3' end of incision 1 to provide an effective starting site for CNT action.

In the present invention, the reaction initiated from the nick and/or gap is called a "restricted nick translation reaction" because the length of the target fragment generated by the reaction can be controlled within a certain range by controlling factors such as the amount of dNTPs used in the reaction, the amount of nucleic acid molecule used as a template, the enzyme reaction temperature and time, etc. Nucleic acid fragments of a certain length range are suitable for specific sequencing platforms. Generally, the target fragment length in the present invention is preferably controlled within the range of 50-250bp. Such a length is several times greater than the target fragment length obtained by traditional CG sequencing platform library construction schemes. Moreover, the CNT technology of the present invention can control the library insert fragments to a very concentrated range without gel excision and recovery, effectively improving the operability of the nick translation reaction technology.

Please refer to Figure 4 for a comparison between the existing method and the method of the present invention. The existing method utilizes the cleavage characteristics of class III endonucleases to cleave genomic DNA at 25-27 bp on both sides of adapter A to form a target DNA fragment of approximately 104 bp; subsequently, a two-step magnetic bead purification method is used to remove DNA fragments larger than 200 bp without adapter A. At this time, the enzyme-digested products obtained after magnetic bead fragment selection are also mixed with some non-target DNA fragments with a main band of 100-200 bp; after connection with adapter B, a primer with a biotin-labeled base on a primer is used to amplify the DNA fragment connected to adapter B, wherein the single strand amplified by the primer with the biotin-labeled base is a non-target single-stranded nucleic acid; subsequently, the DNA fragment connected to adapter B is enriched by a single streptavidin magnetic bead, and the DNA fragment connected to adapter A is further enriched by a single specific sequence hybridization capture; finally, the double-stranded DNA is melted by alkaline denaturation, the target single-stranded nucleic acid is eluted from the streptavidin magnetic beads, and the target single-stranded nucleic acid is circularized using a mediating sequence. The entire process of the existing method is not only cumbersome and time-consuming, but also consumes expensive reagents (mainly Ampure magnetic beads or streptavidin magnetic beads are required for each step of the reaction). The method of the present invention, on the one hand, uses the biotin label on the first PCR primer to bind the circular nucleic acid molecule to the streptavidin magnetic beads after cyclization. In the subsequent reactions, the target nucleic acid molecule is always bound to the magnetic beads. After each step of the reaction, there is no need to add new magnetic beads for purification. It is only necessary to wash off the reaction solution with a washing reagent before proceeding to the next step of the reaction. This not only reduces the use of magnetic beads, but also saves experimental operation time. On the other hand, the nicking enzyme opens an incision on each of the two chains of the connector A, and then uses the incision translation function of the polymerase in the presence of dNTP to extend the incision from the connector A region to the connector. On both sides of head A, by controlling the molar ratio of dNTP to template DNA, reaction temperature, reaction time and other conditions, the incision extension length can be flexibly controlled, and the size of the main band of the extended fragment can be controlled within the range of 50 to 250bp. Subsequently, a one-step exonuclease digestion reaction is performed to digest the non-target DNA fragment without adapter A. The remaining is the target DNA fragment with adapter A. After being connected with adapter B and PCR amplified with a primer without biotin labeling, the double-stranded DNA is melted by simple high-temperature denaturation, and the target single-stranded DNA is mediated by the mediating sequence to circulize. The target single-stranded DNA can be separated and circulized. It can be seen that the single-stranded circulization method of the present invention only requires thermal denaturation and mediating sequence hybridization to successfully separate and circulize the target single-stranded nucleic acid. It is not only simple in steps and easy to operate, but also does not require the consumption of a large amount of expensive reagents, thereby reducing the cost of library construction.

In a preferred embodiment of the present invention, L-shaped adapter connection is used to replace the traditional adapter connection. Please refer to Figure 5 for a comparison of the existing adapter connection method and the adapter connection method of the present invention. The directional adapter connection method used in the existing method is to minimize the problem of mutual connection between DNA fragments while ensuring the directional connection of the adapter. The method adopts a step-by-step connection method in which the 5' adapter and the 3' adapter are designed separately. Each time an end adapter is added, the adapter sequence, the blocking sequence, and the primer sequence need to work together to complete. The whole process requires dephosphorylation, end repair, addition of the 5' adapter, primer extension, addition of the 3' adapter, nick translation, and connection, which are 6 steps of enzyme reaction and 5 purification operations, before the sequence of adapter A can be directed to both ends of the target DNA. The existing method has complicated steps, high library construction cost (sequence cost, enzyme reaction reagent cost, purification cost), long cycle, and large sample loss, which does not meet the requirements of efficient and simple library construction. The L-shaped adapter connection method of the present invention can improve library construction efficiency and reduce library construction cost while ensuring the directional connection of the adapter. Although the L-shaped adapter connection method also adopts step-by-step connection, the steps are simple relative to the existing method. First, a 5' adapter with a blocking sequence is added. The blocking sequence is approximately 12 bp long and fully complementary to the 5' adapter, forming a partially complementary double-stranded structure that facilitates ligation between the DNA fragment and the 5' adapter. The blocking sequence has a dideoxy modification at its 3' end and a dephosphorylated base at its 5' end. This ensures directional ligation between the 5' adapter and the 3' end of the DNA fragment while preventing the blocking sequence from ligating to the 5' end of the DNA fragment. The blocking sequence contains a U base in the middle. Treatment with USER enzyme degrades the blocking sequence into two single-stranded DNA fragments less than 8 bp in length, which then melts and releases the 5' adapter. Next, an L-shaped single-stranded 3' adapter is added via post-hybridization ligation. Prior to adding the L-shaped adapter, the 5' end of the DNA fragment must be phosphorylated to release the blocking. Experiments have demonstrated that USER enzyme treatment can be performed simultaneously with the phosphorylation reaction. After the reaction, the beads are purified using magnetic beads and then directly resuspended in the ligation buffer for the next step. The clever design of the L-shaped adapter lies in the fact that the last 8 bases at the 3’ end are complementary to the last 8 bases at the 5’ end of the 5’ adapter. This allows direct hybridization to the 5’ adapter, and then the nick is sealed with ligase, allowing the L-shaped 3’ adapter to be ligated to the 5’ end of the DNA fragment. Because some of the bases in the L-shaped adapter are complementary to some of the bases at the 5’ end of the 5’ adapter, while the other bases are not complementary, it appears L-shaped, hence the name L-shaped adapter. After the reaction is completed, an appropriate amount of magnetic bead binding buffer is added to the magnetic beads to purify and recover the ligation product with the adapter added. The entire process only requires five enzymatic reactions: dephosphorylation, end repair, addition of the 5’ adapter, one-step reaction of USER enzyme cleavage and phosphorylation, and addition of the 5’ L-shaped adapter, as well as three purification operations. The sequence of adapter A can be quickly and directionally added to both ends of the target DNA. The steps are simple, the library construction cost is reduced, and the cycle time is shortened.

The unique innovation of the present invention lies mainly in: the enzymatic reaction is carried out on streptavidin magnetic beads, and the nucleic acid does not need to be eluted after binding to the streptavidin magnetic beads before the enzymatic reaction can be carried out; a controllable nick translation reaction is used to generate nucleic acid double strands of specific fragment lengths.

The circular nucleic acid molecules are combined with streptavidin magnetic beads through biotin labeling. In each subsequent enzymatic reaction, the nucleic acid molecules remain bound to the magnetic beads. Only a simple washing step is required to wash away the enzymes, ions, etc. in the reaction, while the double-stranded nucleic acid with biotin labeling will not be washed off. It is not until the second PCR that the double-stranded nucleic acid is denatured into two single strands, and the single strand without biotin labeling is collected.

In the traditional Complete Genomics laboratory, enzyme reactions are performed in solution. After each step, new magnetic beads must be added to bind the target fragment, the enzyme and buffer used in the reaction must be washed away, and the target fragment must be eluted before proceeding to the next step. However, the enzyme reaction of the present invention is performed on streptavidin magnetic beads, requiring only a single addition of the beads. After the double-stranded nucleic acid molecules bind to the magnetic beads, there is no need for multiple elutions and repeated additions of new beads for rebinding. A simple washing step is all that is needed to collect the desired target fragment, eliminating many fragment purification steps. This not only saves experimental time but also reduces the amount of magnetic beads used, thereby saving costs. Furthermore, by avoiding repeated binding and elution between the sample and the magnetic beads, sample loss during the experiment is reduced, and the yield of the target fragment is improved. In one embodiment of the present invention, magnetic beads are used as a solid-phase carrier, but solid-phase carriers are not limited to magnetic beads; other solid-phase carriers, such as microarrays, can also be used. The present invention can be achieved by simply immobilizing streptavidin on the solid-phase carrier.

In the traditional Complete Genomics library construction, circular DNA has a Class III enzyme cleavage site. After the Class III enzyme recognizes the cleavage site, it will enzymatically cut the circular DNA at a distance of 26 bp from the cleavage site, turning the circular DNA into two linear DNA segments. The target fragment is then bound to streptavidin magnetic beads through the biotin label on the DNA to collect the target fragment. The target fragment after enzymatic cleavage in this method is only 26 bp, which limits the fragment size of the library; and the enzyme reaction time is long, requiring 16 hours. The present invention uses the gap generated during circularization, or replaces the recognition site of the Class III enzyme with the recognition site of the nicking enzyme. After the circular DNA is bound to the streptavidin magnetic beads, the enzyme cuts to form a gap or nick on each strand. Then, through the 5'-3' polymerase activity and 3'-5' exonuclease activity of the polymerase, the gap or nick is translated, so that the two chains of the target fragment start from the gap or nick and polymerize and extend in the 5'-3' direction, increasing the length of the library insert fragment and controlling the reaction conditions to control the fragment length. The controlled reaction conditions include the amount of dNTP used, the amount of polymerase, temperature, time, etc. When the dNTP is exhausted, the DNA polymerase will continue to act as an exonuclease and continue cutting along the 3'-5' direction of this chain to produce a sufficiently large gap. Finally, a single-stranded endonuclease is used to cut the other single strand at the gap into double-stranded nucleic acids at both ends. The target fragment that needs to be recovered is biotin-labeled and has already been bound to the streptavidin magnetic beads, and the enzyme reaction can be carried out on the magnetic beads, so only a simple washing step is needed to remove the enzyme, buffer, etc. in the reaction to obtain the target fragment and proceed to the next reaction. The enzyme reaction time in this process is about 2.5 hours. Compared with traditional methods, it not only shortens the time, but also increases the number of inserted fragments in the final library and makes the fragment length controllable.

The present invention is described in detail below by way of examples.

1. Genomic DNA shearing: There are many ways to shear genomic DNA, whether it is physical ultrasound or enzyme reaction, and there are very mature solutions on the market. This example uses the physical ultrasound shearing method.

Take a 96-well PCR plate, insert a piece of Teflon thread, add 1 μg of genomic DNA, and make up to 100 μL with TE buffer or enzyme-free water. Seal the plate and place it on an E220 ultrasonic disruptor for ultrasonic disruption. The disruption conditions are shown in Table 1.

Table 1

parameter Numerical Fill factor twenty one% Pressure (PIP) 500 Pulse coefficient 500 Interruption time 20 seconds, 2 times

2. Selection of fragments: Magnetic bead purification or gel recovery can be used. In this example, magnetic bead purification is used.

Take the sheared DNA, add 45μL Ampure XP magnetic beads, mix well and let it stand for 7-15 minutes; place it on a magnetic rack and collect the supernatant, add 18μL Ampure XP magnetic beads to the supernatant, mix well and let it stand for 7-15 minutes; place it on a magnetic rack and aspirate the supernatant, wash the magnetic beads twice with 75% ethanol; after drying, add 30μL TE buffer solution, mix well and let it stand for 7-15 minutes to dissolve and recover the product.

3. Fragment dephosphorylation reaction: Take the product recovered in the previous step and prepare the system according to Table 2.

Table 2

Reaction components Volume (μL) 10× NEB buffer 2 3.6 Shrimp alkaline phosphatase (1U/μL) 3.6 Total 7.2

Add 7.2 μL of the reaction solution to the recovered product from the previous step, mix well, incubate at 37°C for 45 min, incubate at 65°C for 10 min, and then cool down to 4°C at a rate of 0.1°C per second.

4. Fragment end repair: Prepare the system according to Table 3.

Table 3

Reaction components Volume (μL) Enzyme-free water 7.32 10× NEB buffer 2 1.08 0.1M ATP 0.48 dNTPs (25 mM, Enzymatic) 0.48 Bovine serum albumin (10 mg/ml) 0.24 T4 DNA polymerase (3 U/μL) 1.2 Total 10.8

The system was mixed and then added to the product of the previous step. After mixing, the mixture was incubated at 12°C for 20 minutes. 48 μL of Ampure XP magnetic beads were used for purification, and the product was dissolved and recovered in 40 μL of TE buffer solution.

5. 5' linker A sequence connection: The 5' linker A sequence used in this example is as follows (the sequence in this example is from left to right from the 5' end to the 3' end, "//" indicates a modification group, "phos" indicates phosphorylation, "dd" indicates dideoxy, "bio" indicates biotin, and bold font indicates a tag sequence).

5' linker A sequence:

/5phos/AAGCTGAGGGTACTGTGTCATAAATAGCACGAGACGTTCTCGACT(SEQ ID NO: 1);

5' blocking sequence: TACCCUCAGCT/3ddT/ (SEQ ID NO: 2).

5' linker A mixed solution (10 μM) was prepared according to the formula in Table 4.

Table 4

Reaction components Volume (μL) 5' linker A sequence (100 μM) 12 5' blocking sequence (100 μM) 10 TE buffer 78 Total 100

Add 4.5 μL of the prepared Adapter A mixture (10 μM) to the product from the previous step and mix thoroughly. The ligation reaction system was prepared according to the formula in Table 5 below.

Table 5

Reaction components Volume (μL) Enzyme-free pure water 13.1 2× Ligation Buffer 1 60 T4 DNA ligase (fast) (600 U/μL) 2.4 Total 75.5

The formula of the 2× ligation buffer 1 used in this example is shown in Table 6.

Table 6

The ligation reaction system was mixed with the mixture of the linker and the product, incubated at 25°C for 30 min, incubated at 65°C for 10 min, and then cooled to 4°C.

6. USER enzyme digestion and phosphorylation one-step reaction: Add 1.2 μL USER enzyme (1 U/μL) and 1.2 μL T4 polynucleotide kinase (10 U/μL) to the reaction solution from the previous step, mix well, and incubate at 37°C for 20 minutes. Purify with 108 μL Ampure XP magnetic beads (Agencourt), wash twice with 70% ethanol, aspirate the wash solution, dry at room temperature for 2 minutes, and resuspend the Ampure XP magnetic beads in 48 μL 3'L-type adapter reaction system.

7. 3' L-type linker A sequence ligation: The 3' L-type linker A sequence used in this example is as follows: ACGTTCTCGACUCCTCAGCTT (SEQ ID NO: 3).

Prepare 3' L-type linker reaction system according to Table 7:

Table 7

Reaction components Volume (μL) Enzyme-free pure water 28.98 3× Ligation Buffer 2 16.02 L-type linker sequence (100 μM) 1.8 T4 DNA ligase (fast) (600 U/μL) 1.2 Total 48

The formula of 3× ligation buffer 2 used in this example is shown in Table 8.

Table 8

Reaction components Volume (μL) Polyethylene glycol-8000 (50%) 60 Tris-Cl, pH 7.8 (2 M) 7.5 Adenosine triphosphate (100 mM) 3 Bovine serum albumin (10 mg/mL) 1.5 Magnesium chloride (1M) 3 Dichlorodiphenyltrichloroethane (DDT) (1M) 0.15 Enzyme-free pure water 24.9

Resuspend Ampure XP magnetic beads in 48 μL of 3' L-type linker reaction system and incubate at 300 rpm and 25°C for 30 minutes. After the reaction is complete, add 43.2 μL of Ampure XP magnetic bead binding buffer and incubate at room temperature for 10 minutes. Remove the supernatant, wash twice with 70% ethanol, air-dry at room temperature for 5-10 minutes, and then dissolve and recover the product in 30 μL of TE buffer.

8. Polymerase chain reaction:

The sequence of primer 1 is as follows:

AGTCGAGAACGUCTCG/iBiodT/GCT (SEQ ID NO: 4);

The sequence of primer 2 is as follows:

ACGTTCTCGACUCCTCAGCTT (SEQ ID NO: 5).

Prepare the PCR system according to Table 9.

Table 9

Reaction components Volume (μL) Enzyme-free pure water 186.5 2× PfuTurbo Cx buffer 275 PfuTurbo Cx Hot Start Nucleic Acid Polymerase (2.5U/μL) 11 20 μM Primer 1 13.75 20 μM Primer 2 13.75 Total volume 500

50 μL (180 ng) of the product recovered in the previous step was added to the above system, mixed well, and reacted according to the conditions in Table 10.

Table 10

After the reaction is complete, the product is purified using 550 μL of Ampure XP magnetic beads and recovered by dissolving in 80 μL of TE buffer. 1 μL of the recovered product is collected and its concentration is quantified using the Qubit dsDNA HS Assay Kit (Invitrogen). 2 μg of the product is then used for the next reaction.

9. Removal of uracil: Prepare the reaction solution shown in Table 11 below.

Table 11

Reaction components Volume (μL) Enzyme-free pure water 25.8 10× Taq buffer 11 USER enzyme (1U/μL) 13.2 Total volume 50

The above reaction solution was added to 60 μL (2 μg) of the reaction product from the previous step, mixed well, and incubated at 37°C for 1 h.

10. Double-chain cyclization: Prepare the reaction system 1 shown in Table 12 below.

Table 12

Reaction components Volume (μL) Enzyme-free pure water 1520 10×TA buffer 180 Total volume 1700

Add the reaction product from the previous step to reaction system 1, mix well, divide equally into 4 tubes, and place in a 50°C water bath to react for 15 minutes. After the reaction is completed, place in a room temperature water bath to react for 15 minutes.

The reaction system 2 shown in Table 13 below was prepared.

Table 13

Reaction components Volume (μL) Enzyme-free pure water 98 20×Circ buffer 100 T4 DNA ligase (fast) (600 U/μL) 2 Total volume 200

The formula of 20×Circ buffer used in this example is shown in Table 14.

Table 14

Reaction components concentration Tris-Cl, pH 7.5 66mM Potassium acetate 132mM magnesium acetate 20mM Dichlorodiphenyltrichloroethane (DDT) 1mM ATP 20mM

Add 50 μL of reaction system 2 into the four equally divided tubes of reaction system 1 and incubate at room temperature for 1 h.

To each tube of reaction product (500 μL), add 330 μL Ampure XP magnetic beads, mix well, and let it stand for 7-15 minutes; place it on a magnetic rack, collect the supernatant, add 170 μL Ampure XP magnetic beads to the supernatant, mix well, and let it stand for 7-15 minutes; place it on a magnetic rack, aspirate the supernatant, and wash the magnetic beads twice with 75% ethanol; after drying, add 65 μL TE buffer to dissolve the 4 tubes of purified products.

11. Linear digestion: Prepare the reaction system shown in Table 15 below.

Table 15

The product from the previous step was added to the reaction system, mixed well, and incubated at 37°C for 1 h.

The product was purified using 80 μL of Ampure XP magnetic beads and dissolved in 82 μL of TE buffer. 1 μL of the recovered product was taken and the product concentration was quantified using the Qubit dsDNA HS Assay Kit (Invitrogen). 700 ng of the product was used for the next reaction. The initiation site of the CNT reaction on the double-stranded circularized DNA formed in this example is a nicked type, i.e., both strands are intact double-stranded circular DNA, and the linker A sequence contains a recognition sequence for the nickase.

12. Binding of circular DNA to magnetic beads: 500 ng of circular DNA was added to streptavidin magnetic beads (Life Technologies). Binding was allowed to proceed at room temperature for 1 hour. The biotin label on the circular DNA allowed the DNA to bind to the streptavidin-coated magnetic beads. The beads were then placed on a magnetic rack, the supernatant removed, and the cells were washed once with high-salt wash solution, once with low-salt wash solution, and once with 1× NEB buffer 2. The compositions of the high-salt and low-salt wash solutions are shown in Tables 16 and 17, respectively.

Table 16

Reaction components Volume (μL) Tris-Cl, pH 7.5 (1 M, SIGMA) 5000 Sodium chloride (5M, SIGMA) 10000 Enzyme-free pure water 35,000 Total 50000

Before use, 10% Tween 20 was added to make the final concentration of Tween 20 0.05%.

Table 17

Reaction components Volume (μL) Tris-Cl, pH 7.5 (1 M, SIGMA) 5000 Sodium chloride (5M, SIGMA) 3000 Enzyme-free pure water 42000 Total 50000

Before use, 10% Tween 20 was added to make the final concentration of Tween 20 0.05%.

13. Nicking enzyme digestion reaction: Prepare the system according to the formula in Table 18 below.

Table 18

Reaction components Volume (μL) Enzyme-free water 66.3 10× NEB buffer 2 8 Nt.BvbCI 1.7 Total 80

Add 80 μL of the reaction solution to the magnetic beads in the previous step, mix well, and react at 37°C for 60 min.

After the reaction, the tube was placed on a magnetic rack, the supernatant was removed, and the tube was washed once with high salt solution, once with low salt solution, and once with 1×NEB buffer 2.

14. Restriction nick-shift reaction: Prepare the system according to the formula in Table 19 below.

Table 19

The amounts of dNTPs and DNA polymerase I used are variable and can be adjusted according to the desired length of the target fragment.

Add 60 μL of the reaction solution to the magnetic beads in the previous step, mix well, react at 25°C for 15 min, add 1.2 μL of EDTA (0.5 M, AMBION), and react at 65°C for 15 min.

After the reaction, the tube was placed on a magnetic rack, the supernatant was removed, and the tube was washed once with high salt solution, once with low salt solution, and once with 1×NEB buffer 2.

15. Endonuclease cleavage at the nick: Prepare the system according to the formula in Table 20 below.

Table 20

Reaction components Volume (μL) Enzyme-free pure water 78 10× NEB buffer 2 9 T7 endonuclease I (10 U/μL, NEB) 3 Total 90

Add 90 μL of the reaction solution to the magnetic beads from the previous step, mix thoroughly, and incubate at 25°C for 15 minutes. Add 2 μL of EDTA (0.5 M, AMBION). After the reaction, place the beads on a magnetic rack, remove the supernatant, wash once with high-salt solution, then twice with low-salt solution, and resuspend the beads in 100 μL of low-salt solution.

16. Fill the sticky ends and add A to the 3' end: prepare the system according to the formula in Table 21 below.

Table 21

Reaction components Volume (μL) Enzyme-free pure water 0.8 5×Klex NTA mix 26 Klenow fragment (3′→5′exo-) (5 U/μL, NEB) 3.2 Total 30

Add 30 μL of the reaction solution to the magnetic bead resuspension from the previous step, mix thoroughly, and incubate at 37°C for 60 min. Add 2 μL of EDTA (0.5 M, AMBION). After the reaction, place the beads on a magnetic rack, remove the supernatant, wash three times with low-salt wash solution, and resuspend the beads in 70 μL of low-salt wash solution.

17. Connector B (bubble connector):

Linker B is composed of a complementary pairing of the top chain L and the bottom chain S, and its sequence is as follows:

Top chain L: /phos/AGTCGGAGGCCAAGCGTGCTTAGGACAT (SEQ ID NO: 6);

Bottom strand S: GTCCTAAGCACUGTAGTGTACGATCCGACTT (SEQ ID NO: 7).

The system was prepared according to the formula shown in Table 22 below.

Table 22

Reaction components Volume (μL) Enzyme-free pure water twenty one 3× Ligation Buffer 2 56.8 Linker B (10 μM) 20 T4 ligase (600 U/μL, Enzymatics) 3.2 Total 100

Add 100 μL of the reaction solution to the magnetic bead resuspension solution from the previous step, mix well, and react at room temperature for 30 min, then at 65°C for 10 min.

18. USER enzyme digestion: Add 1 μL of USER enzyme (1 U/μL, NEB), mix well, and incubate at 37°C for 60 min. Then, add 4.5 μL of EDTA (0.5 M, AMBION). After the reaction, place on a magnetic rack, remove the supernatant, and wash three times with low-salt buffer. Separate the unlabeled single strand with 40 μL of 0.1 M sodium hydroxide. Neutralize the resulting product with acidic buffer to a total volume of 60 μL. The biotinylated strand remains bound to the magnetic beads.

19. Polymerase chain reaction:

The sequences of primers F and R used in this example are as follows:

Primer F: /bio/ATGTCCTAAGCACGCTTGGCC (SEQ ID NO: 8);

Primer R: /phos/GTAGTGTACGATCCGACTT (SEQ ID NO: 9).

The system was prepared according to the formula shown in Table 23 below.

Table 23

After mixing, react according to the conditions in Table 24:

Table 24

After the reaction was completed, 400 μL of Ampure XP magnetic beads (Agencourt) were used for purification, and the product was dissolved and recovered in 80 μL of TE buffer solution.

20. Single-stranded cyclization: The nucleic acid single-strand O can be used to connect the two ends of the product from the previous step using the corresponding complementary sequence. The sequence of the nucleic acid single-strand O is as follows:

ATCGTACACTACATGTCCTAAGCA (SEQ ID NO: 10).

Take 100 ng of the PCR product from the previous step, add 10 μL of single-stranded nucleic acid O (10 μM, Sangon), mix well, and incubate at 95°C for 3 minutes; then quickly cool on ice. Prepare the system according to the formula in Table 25 below.

Table 25

Reaction components Volume (μL) Enzyme-free pure water 36.4 10×TA buffer (Epicentre) 12 100 mM ATP (Epicentre) 1.2 T4 ligase (600 U/μL, enzymes) 0.4 Total 50

Add 50 μL of reaction solution to the mixture of PCR product and single-stranded O, mix well and react at 37°C for 60 min.

21. Linear DNA digestion: Prepare the system according to the formula in Table 26 below.

Table 26

Reaction components Volume (μL) Enzyme-free pure water 2 10×TA buffer (Epicentre) 0.8 Exonuclease 1 (20 U/μL, NEB) 3.9 Exonuclease 3 (100 U/μL, NEB) 1.3 Total 8

8 μL of the reaction solution was added to the ligation reaction solution from the previous step, mixed, and reacted at 37°C for 30 min. 6 μL of 0.5 M EDTA was added. The product was then purified and recovered using 170 μL of PEG32 magnetic beads and re-dissolved in 55 μL of TE buffer.

The final product concentration and total amount of the four parallel experiments in this example are shown in Table 27. The electrophoresis results are shown in Figure 6.

Table 27

Sample name Concentration (ng/μL) Total amount (ng) C22 0.33 18.33 D22 0.32 17.87 E22 0.32 17.87 F22 0.31 17.15

As can be seen from the table above, the concentration and total amount of the library are sufficient to meet the library quantity requirements for subsequent sequencing. At the same time, electrophoresis (Figure 6) and test results using the LabChip GX instrument (fully automated microfluidic electrophoresis instrument, Caliper) (Figures 7-10) show that the DNA library bands after polymerase chain reaction are concentrated, with fragment sizes ranging from 200 bp to 300 bp. The electrophoresis bands are concentrated, with a prominent main peak, which meets the fragment range requirements for subsequent sequencing.

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 (11)

1. A method for constructing a single-stranded circular library of nucleic acids with dual adapters, comprising the following steps: Nucleic acid is broken down into nucleic acid fragments for library construction; First adapter sequences are ligated to both ends of the nucleic acid fragment; A first product with the first adapter sequence at both ends is obtained by first PCR amplification, wherein the primer sequences used in the first PCR have U base sites and may or may not have nickase recognition sequences, and one of the primer sequences has a first affinity marker. The first product was digested with USER enzyme to produce sticky ends with or without gaps. The first product after enzyme digestion is cyclized to produce a circular nucleic acid molecule; Circular nucleic acid molecules with nicks on both strands are treated with dephosphorylases, or circular nucleic acid molecules with nicks on one strand having a nicks enzyme recognition sequence and the other strand having a nick, or circular nucleic acid molecules with nicks enzyme recognition sequences on both strands and neither strand having a nick, are treated with nicks enzymes to produce nicks. The circular nucleic acid molecule is bound to a solid-phase carrier with a second affinity label; Using a circular nucleic acid molecule bound to the solid support as a template, a restriction nick translation reaction is initiated from the cut and/or gap. The restriction nick translation reaction refers to a nick translation reaction in which the length of the generated nick translation fragment is controlled by controlling at least one of the following factors: the molar ratio of dNTP to the nucleic acid molecule used as the template, the enzyme reaction temperature, and the time. Digestion removes the portion of the circular nucleic acid molecule that has not undergone the restriction nick translation reaction, yielding a linear nucleic acid molecule; A second adapter sequence is attached to both ends of the linear nucleic acid molecule; A second product with the second adapter sequence at both ends was obtained by a second PCR amplification. The second product is denatured to obtain single-stranded nucleic acid molecules, and the single-stranded nucleic acid molecules are circularized using a mediator sequence that is complementary to both ends of one of the single-stranded nucleic acid molecules to obtain a dual-linker single-stranded circular library. 2. The method according to claim 1, wherein the first affinity label is a biotin label; and the second affinity label is a streptavidin label. 3. The method according to claim 1, wherein the first linker sequence comprises a first 5' linker sequence and a first 3'L-type linker sequence, respectively connecting the 3' end and 5' end of each chain of the fragment; the first 5' linker sequence comprises a long chain with 5' end phosphorylation and a complementary short chain, the short chain having a dideoxy modification at the 3' end, and the short chain containing a U base site; the first 3'L-type linker sequence is partially complementary to the first 5' linker sequence in the portion of the fragment adjacent to the linker sequence; The first adapter sequence is ligated to both ends of the nucleic acid fragment, specifically including: The nucleic acid fragment was dephosphorylated; End repair of dephosphorylated nucleic acid fragments; The first 5' adapter sequence is attached to the 3' end of each strand of the nucleic acid fragment; The U base sites of the short strand of the first 5' linker sequence were cleaved using the USER enzyme. Phosphorylation treatment was performed on the nucleic acid fragments digested by USER enzyme; The first 3' L-type adapter sequence is attached to the 5' end of each strand of the phosphorylated nucleic acid fragment. 4. The method according to claim 1, characterized in that the primer sequences used in the first PCR each have a U base site and a nicking enzyme recognition sequence; after digesting the U base site with USER enzyme, sticky ends are formed at both ends of the nucleic acid fragment, and the sticky ends are complementary and circularized to produce a circular nucleic acid molecule; then the nicking enzyme is used to digest the nicking enzyme recognition sequence to produce a nick. 5. The method according to claim 1, characterized in that, in the primer sequence used in the first PCR, one primer sequence has two U base sites and the other primer has one U base site; after digesting the U base sites with USER enzyme, sticky ends are formed at both ends of the nucleic acid fragment, and the sticky ends are complementary and circularized to produce a circular nucleic acid molecule. 6. The method according to claim 1, characterized in that, after cyclizing the first product after enzyme digestion, it further includes: digesting the uncyclized nucleic acid molecules. 7. The method according to claim 1, wherein the digestion to remove the portion of the circular nucleic acid molecule that has not undergone restriction nick translation reaction specifically includes: firstly, degradation using a double-stranded exonuclease until the nicks at both ends meet; then, degradation of the single strand using a single-stranded exonuclease; or directly cleaving the portion of the circular nucleic acid molecule that has not undergone restriction nick translation reaction using a nuclease. 8. The method according to claim 1, wherein the second linker sequence is a bubble linker sequence, the bubble linker sequence comprising two base sequences that are partially complementary at both ends but not complementary in the middle, wherein the middle section forms a bubble shape and the middle section has a U base site; one strand of the bubble linker sequence has a protruding T base at its 5' end. The second adapter sequences are attached to both ends of the linear nucleic acid molecule, specifically including: The linear nucleic acid molecule undergoes end repair and 3' end A base addition reaction; The bubbling adapter sequence is attached to both ends of the linear nucleic acid molecule by pairing the T base with the A base; The U base sites on the middle segment were cleaved using the USER enzyme. 9. The method according to claim 1, characterized in that, after cyclizing the single-stranded nucleic acid molecule, it further includes: digesting the uncyclized single-stranded nucleic acid molecule. 10. A reagent for constructing a single-stranded circular library of nucleic acids with dual adapters, comprising the following components: The first linker sequence includes a first 5' linker sequence and a first 3'L-type linker sequence, which connect the 3' and 5' ends of each strand of the fragment, respectively; the first 5' linker sequence includes a long chain with 5' phosphorylation and a complementary short chain, the short chain having a dideoxy modification at the 3' end and containing a U base site; the first 3'L-type linker sequence is partially complementary to the first 5' linker sequence in the portion of the fragment adjacent to the linker sequence; The first PCR primer has a U base site and may or may not have a nick enzyme recognition sequence, and one of the primer sequences has a first affinity marker, for obtaining a first product with the first adapter sequence at both ends by the first PCR amplification; USER enzyme is used to cleave the first product, producing sticky ends and with or without nicks; Cycloylase is used to cyclize the first product after enzyme digestion to produce a circular nucleic acid molecule; Dephosphorylase is used to dephosphorylate circular nucleic acid molecules with nicks on both strands; or nicking enzyme is used to digest circular nucleic acid molecules with a nicking enzyme recognition sequence on one strand and a nick on the other strand, or with a nicking enzyme recognition sequence on both strands and no nicks, to create nicks. A solid-phase carrier with a second affinity marker for binding to the circular nucleic acid molecule; The components of the nick translation reaction are used to initiate a restrictive nick translation reaction from the cut and/or nick using a circular nucleic acid molecule bound to the solid support as a template. The restrictive nick translation reaction refers to a nick translation reaction in which the length of the generated nick translation fragment is controlled by controlling at least one of the following factors: the molar ratio of dNTP to the nucleic acid molecule used as a template, the enzyme reaction temperature, and the time. Digestive enzymes are used to digest and remove the portion of the circular nucleic acid molecule that has not undergone the restriction nick translation reaction, to obtain a linear nucleic acid molecule; The second connector sequence is a bubble connector sequence, which includes two base sequences that are partially complementary at both ends but not complementary in the middle, wherein the middle section forms a bubble shape and has a U base site; one strand of the bubble connector sequence has a protruding T base at its 5' end. The second PCR primer is used to amplify the second product with the second adapter sequence at both ends by the second PCR amplification. The mediating sequence is complementary to both ends of one of the single-stranded nucleic acid molecules obtained after denaturation of the second product, and is used to circularize the single-stranded nucleic acid molecule to obtain a dual-linker single-stranded circular library. 11. The reagent according to claim 10, wherein the first affinity label is biotin-labeled; and the second affinity label is streptavidin-labeled.