Sequencing linker, sequencing linker compound and method for multiple nanopore sequencing of target nucleic acid sequence

Abstract

The invention provides a sequencing linker, a sequencing linker compound and a method for multiple nanopore sequencing of a target nucleic acid sequence. Wherein the sequencing linker comprises a first chain and a second chain, the first chain comprises a sequencing guide sequence, a helicase binding sequence, a limiting structure, a first complementary sequence and a first primer sequence from the 5'end to the 3 'end in sequence, the limiting structure prevents helicase from moving, and the first chain is provided with a 3' free end; the second chain comprises a second complementary sequence and a restraint sequence or a sequence complementary with the nucleic acid sequence of the restraint sequence from the 5'end to the 3 'end in sequence, and the restraint sequence comprises a nucleic acid sequence of which the tail end is connected with a hydrophobic molecule; the first complementary sequence and the second complementary sequence are reversely complementary, and the sequencing linker is formed by annealing the first strand and the second strand. The sequencing linker and the sequencing linker compound are simple in construction process and high in yield.

Description

Sequencing adapter, sequencing adapter complex, and method for multiple nanopore sequencing of target nucleic acid sequences Technical Field

[0001] The present invention relates to the field of sequencing technology, and in particular to a sequencing adapter, a sequencing adapter complex, and a method for multiple nanopore sequencing of a target nucleic acid sequence. Background Art

[0002] Nanopore sequencing technology is a single-molecule level detection technology with the advantages of fast sequencing speed, long read length, direct sequencing, high throughput, low cost, small size, and portability. During the nanopore sequencing process, a single nanopore is embedded in an insulating impermeable membrane to form a stable ion current channel. Under the action of voltage, a single-stranded nucleic acid molecule passes through the nanopore, thereby reducing the ion current passing through the nanopore. Due to the different molecular structures and sizes of different bases on single-stranded nucleic acid molecules, the current passing through the nanopore shows differences corresponding to the base sequence. By using algorithms to analyze the current change signal, the perforated single-stranded nucleic acid sequence can be read in real time. However, the accuracy of existing nanopore sequencing technology is still limited, which will seriously restrict the scope of application of nanopore sequencing technology.

[0003] For example, Oxford Nanopore Technologies (ONT) has developed a four-step nanopore sequencing technology for target nucleic acid sequences, which includes: (1) connecting different sequencing adapters at both ends of the double-stranded nucleic acid to be tested, one end of which is a Y-shaped adapter that combines a helicase and a polymerase, and the other end of which is a hairpin structure adapter. The top chain of the Y-shaped adapter consists of a guide sequence + a helicase binding site + a spacer sequence + a sequence complementary to the bottom chain, and the bottom chain of the adapter consists of a sequence complementary to the top chain + a sequence containing a hairpin structure. (2) The helicase binds between the guide sequence and the spacer sequence on the top chain of the Y-shaped adapter and is blocked by the spacer sequence; the polymerase binds to the hairpin position of the Y-shaped adapter and uses dNTP for amplification, so that both the forward and reverse chains of the nucleic acid to be tested are amplified once. (3) The amplified product is added to the sequencing system embedded with a nanopore. After the guide sequence is captured by the nanopore, the helicase passes through the spacer sequence under the action of the electric field force and controls the nanopore sequencing, thereby completing four sequencing of the target nucleic acid sequence.

[0004] The disadvantages of the four-pass nanopore sequencing technology developed by ONT are as follows: (1) Connecting different sequencing adapters at both ends of the double-stranded nucleic acid to be tested is a relatively complex problem, which requires complex preliminary operations (such as using PCR and connecting adapters at both ends to introduce restriction endonuclease sites, obtaining different sticky ends through enzyme cutting, and then connecting with adapters with different sticky ends; or adding different adapters at the same time during the TA connection process, and then screening to obtain the target product with different adapters at both ends). This process is complex to operate and has low product acquisition efficiency. (2) This method can only sequence the target nucleic acid sequence up to four times, and the number of sequencing times of the target nucleic acid fragment is limited. (3) When this method is used for four-pass sequencing, the helicase controls the sense chain, antisense chain, sense chain (amplification), and antisense chain (amplification) to pass through the nanopore in sequence to complete sequencing. However, the sequencing speed of different chains in such a sequencing process is significantly different. The reason is that the speed of the same helicase controlling double-strand sequencing and single-strand sequencing is different. This four-pass sequencing method inevitably requires controlling both double-strand and single-strand sequencing, which will increase the difficulty of base recognition when analyzing the sequencing results and ultimately affect the sequencing accuracy.

[0005] Summary of the Invention

[0006] The present invention aims to provide a sequencing adapter, a sequencing adapter complex, and a method for multiple nanopore sequencing of a target nucleic acid sequence to improve sequencing efficiency and accuracy.

[0007] To achieve the above-mentioned object, according to one aspect of the present invention, a sequencing adapter is provided. The sequencing adapter comprises a first strand and a second strand, wherein: the first strand comprises a sequencing guide sequence, a helicase binding sequence, a limiting structure, a first complementary sequence, a structure for preventing displacement of the first primer sequence by a polymerase, and the first primer sequence, connected sequentially from the 5' end to the 3' end, the limiting structure preventing the helicase from moving, and the first strand having a 3' free end; the second strand comprises a second complementary sequence, and a restraining sequence or a sequence complementary to the nucleic acid sequence of the restraining sequence, connected sequentially from the 5' end to the 3' end, the restraining sequence comprising a nucleic acid sequence with a hydrophobic molecule attached to its end; the first complementary sequence is reversely complementary to the second complementary sequence, and the sequencing adapter is formed by annealing the first strand and the second strand.

[0008] Furthermore, the structure that prevents the first primer sequence from being replaced by the polymerase is a GC-rich motif, an artificially modified nucleotide sequence, or a normal nucleic acid coupled with other inhibitory molecules (such as a polymerase inhibitor, etc.), or any combination thereof; preferably, the structure that prevents the first primer sequence from being replaced by the polymerase is an artificially modified nucleotide sequence.

[0009] Furthermore, the artificially modified nucleotides in the artificially modified nucleic acid sequence include at least one of LNA, PNA and BNA; further, the artificially modified nucleic acid sequence includes 2 to 10 artificially modified nucleotides.

[0010] Furthermore, the limiting structure includes a spacer region, the spacer region includes a spacer, and the spacer is selected from at least one of iSpC18, iSpC9, iSpC3, iSpC6 and iSpC12; the limiting structure includes 2 to 8 spacers;

[0011] Preferably, the sequencing guide sequence comprises 10 to 50 nucleotides or iSpC3;

[0012] Preferably, the helicase binding sequence comprises 5 to 40 nucleotides, more preferably, the helicase binding sequence comprises 5 to 40 thymine nucleotides;

[0013] Preferably, the first complementary sequence or the second complementary sequence comprises 5 to 80 nucleotides;

[0014] Preferably, the nucleic acid sequence of the constraint sequence comprises 10 to 50 nucleotides.

[0015] Furthermore, the hydrophobic molecules are selected from any one or more of the following: lipids, fatty acids, sterols, carbon nanotubes, polypeptides, proteins, amino acids, such as cholesterol, palmitate or tocopherol.

[0016] Furthermore, the first primer sequence includes a specific primer or a random primer, and the first primer sequence can bind to the circular library; preferably, the first primer sequence includes 5 to 80 nucleotides;

[0017] Optionally, the circular library is a single-stranded circular library or a double-stranded circular library;

[0018] Optionally, the single-stranded circular library contains an introduced known sequence, which is complementary to the first primer sequence;

[0019] Optionally, the double-stranded circular library contains an introduced known sequence, and a bubble or a gap exists at the complementary binding site between the known sequence and the first primer sequence;

[0020] Optionally, there is no known sequence in the circular library, the first primer sequence is a degenerate primer sequence, and any sequence in the circular library is complementary to the degenerate primer sequence.

[0021] According to another aspect of the present invention, a sequencing adapter complex is provided. The sequencing adapter complex comprises the aforementioned sequencing adapter and a helicase, wherein the helicase is bound to the helicase binding sequence and unwinds in a 5'-3' direction. Preferably, the helicase is selected from any one or more of the following: Dda helicase, Pif 1 helicase, XPD helicase, T7 Gp41 helicase, and DnaB helicase. The helicase is modified by mutation, wherein the modified mutation primarily refers to a mutation that improves sequencing performance.

[0022] According to another aspect of the present invention, a kit for nanopore sequencing of a target nucleic acid sequence is provided, comprising any of the aforementioned sequencing adapters, a polymerase with strand displacement activity, a reaction buffer, and a helicase.

[0023] According to another aspect of the present invention, a kit for nanopore sequencing of a target nucleic acid sequence is provided, comprising any one of the above-mentioned sequencing adapter complexes; further comprising a polymerase with strand displacement activity and a reaction buffer.

[0024] Furthermore, the kit further comprises one or more of dNTP or NTP, and reagents related to circular library construction.

[0025] According to another aspect of the present invention, a method for multiple nanopore sequencing of a target nucleic acid sequence is provided, comprising the following steps: S1, combining a sequencing adapter complex, a circular library containing the target nucleic acid sequence, and a polymerase with strand displacement activity to obtain a sequencing adapter amplification complex; S2, the polymerase using dNTPs or NTPs to perform a polymerization reaction to extend the first strand of the sequencing adapter, and when the polymerase amplifies the sequencing chain along the circular library once and encounters a sequence in the first strand that can inhibit the strand displacement activity of the polymerase, the polymerization reaction stops, and an amplified sequencing adapter complex is obtained; S3, the amplified sequencing adapter complex is bound to the membrane of the nanopore sequencer via a constraint sequence, and the amplified sequencing adapter complex is sequenced under the control of a helicase to achieve multiple sequencing of the target nucleic acid sequence.

[0026] Furthermore, S1 includes: the first chain of the sequencing adapter in the sequencing adapter complex is combined with the circular library through base complementary pairing, and the polymerase uses the circular library as a template and the first chain as a primer to form an amplification complex at the junction of the primer and the template; in S2, the helicase bound to the sequencing adapter cannot use the dNTP in the sequencing buffer to unwind the double strand due to the presence of the restriction sequence; S3 includes: the second chain of the sequencing adapter is combined with the constraint sequence, and the amplified sequencing adapter complex is combined with the membrane of the nanopore sequencer, or the constraint sequence on the second chain of the sequencing adapter directly combines the amplified sequencing adapter complex with the membrane of the nanopore sequencer; after applying the sequencing voltage, the sequencing adapter is combined with the binding force of the electric field. The guide sequence is captured by the nanopore, and the guide sequence passes through the nanopore. The electric field force pushes the helicase through the limiting sequence and the sequence that can inhibit the polymerase chain displacement activity, and normal unwinding is achieved to achieve stable sequencing; at the same time, because the sequence that can inhibit the polymerase chain displacement activity passes through the nanopore, the polymerase is able to continue circular amplification and continuously extend the sequencing chain; the polymerase continuously extends the sequencing chain, and under the action of the electric field force, the helicase alone, or the helicase and polymerase jointly control the sequencing speed to achieve multiple sequencing; the sequence length of the constraint sequence is 10 to 50 nucleotides; the end of the constraint sequence is connected to a hydrophobic molecule, and the hydrophobic molecule is selected from any one or more of the following: lipids, fatty acids, sterols, carbon nanotubes, polypeptides, proteins, amino acids, such as cholesterol, palmitate or tocopherol.

[0027] Furthermore, the polymerase having strand displacement activity is selected from DNA polymerase or RNA polymerase;

[0028] Optionally, the polymerase is a polymerase with strand displacement activity obtained by modifying a polymerase without strand displacement activity;

[0029] Preferably, the polymerase is selected from Bst DNA polymerase, SD DNA polymerase, phi29 DNA polymerase, Bsu Large Fragment DNA polymerase, Klenow Fragment DNA polymerase, T4 DNA polymerase, T7 DNA polymerase, DNA Polymerase I, T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, E. coli RNA polymerase or any combination thereof.

[0030] Furthermore, the sequencing voltage is above 10 mV, preferably 50 mV-250 mV.

[0031] Furthermore, the nanopore of the nanopore sequencer is a transmembrane protein pore or a solid-state pore;

[0032] Preferably, the transmembrane protein pore is derived from a transmembrane protein selected from hemolysin, MspA, MspB, MspC, MspD, FraC, ClyA, PA63, CsgG, CsgD, XcpQ, SP1, phi29 connector protein, InvG, GspD, or any combination thereof;

[0033] Optionally, the transmembrane protein is further linked to another polypeptide, the other polypeptide being selected from a tag, an enzyme cleavage site, a signal peptide or a leader peptide, a detectable marker, or any combination thereof;

[0034] Optionally, the membrane of the nanopore sequencer is an amphiphilic membrane, a high molecular polymer membrane or any combination thereof, and the membrane of the nanopore sequencer is a phospholipid bilayer, a diblock copolymer, or a triblock copolymer.

[0035] Furthermore, the sequencing buffer used in sequencing contains a pH buffer system. Preferably, the pH buffer system is a dihydrogen phosphate-hydrogen phosphate buffer system, a carbonic acid-sodium bicarbonate buffer system, a Tris-HCl buffer system, a HEPES buffer system, a MOPS buffer system, or any combination thereof;

[0036] Preferably, the sequencing buffer contains NTP, dNTP, ddNTP, or any combination thereof;

[0037] Preferably, the sequencing buffer contains K + 、Na 2+ , or any combination thereof;

[0038] Preferably, the sequencing buffer contains Mg 2+ 、Mo 2+ 、Cu 2+ 、Fe 2+ 、Zn 2+ , Ca 2+ , Pb 2+ 、Cd 2+ , or any combination thereof;

[0039] Preferably, the reaction buffer or sequencing buffer contains additives or auxiliary reagents that enhance the polymerase extension reaction, and the additives or auxiliary reagents are dimethyl sulfoxide, glycerol, formamide, bovine serum albumin, ammonium sulfate, polyethylene glycol, gelatin, non-ionic detergent, N,N,N-trimethylglycine, single-stranded nucleic acid binding protein or any combination thereof.

[0040] The sequencing adapter of the present invention contains a sequence complementary to the circular library. A polymerase with strand displacement activity amplifies a sequencing chain using the circular library as a template and the sequencing adapter as a primer. The sequencing adapter contains an artificially modified nucleic acid sequence that inhibits the strand displacement activity of the polymerase. This sequence is also bound to the circular library through base complementarity. When the polymerase amplifies the sequencing chain along the circular library, it encounters the sequence that inhibits the strand displacement activity of the polymerase, thereby inhibiting the activity of the polymerase. When the guide sequence on the sequencing adapter is captured by the nanopore, the electric field force pushes the helicase on the sequencing adapter complex through the limiter sequence and the sequence that inhibits the strand displacement activity of the polymerase, allowing normal unwinding to achieve stable sequencing. At the same time, because the sequence that inhibits the strand displacement activity passes through the nanopore, the polymerase can continue circular amplification to continuously extend the sequencing chain, ultimately achieving multiple sequencing of the target nucleic acid.

[0041] The application of the technical solution of the present invention has at least the following beneficial effects: 1) the sequencing adapter used in the present invention has a simple structure, and annealing of two chains to the target configuration is relatively easy and has a high yield; 2) the sequencing library construction process used in the present invention is simple and has a high yield; 3) the present invention combines the amplification process with the sequencing process, eliminating the need for a long amplification process before sequencing, significantly saving experimental time; 4) the present invention can sequence multiple times, and the sequencing speed of different times is relatively consistent, reducing the difficulty of data analysis; 5) in the sequencing method adopted by the present invention, the synthesis of new chains of DNA will not continue before the sequencing library is captured, thereby generating complex secondary structures and preventing normal sequencing. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0043] FIG1 shows a schematic diagram of a method for multiple nanopore sequencing of a target nucleic acid sequence according to one embodiment of the present application;

[0044] FIG2 shows a schematic diagram of the process for constructing a sequencing adapter complex in Example 1;

[0045] FIG3 shows the electrophoresis detection diagram of the sequencing adapter complex constructed in Example 1, wherein A is the electrophoresis diagram of the sequencing adapter annealing, and B is the electrophoresis diagram of the sequencing adapter complex;

[0046] Figure 4 shows the current signal graphs of the sequencing adapter complexes in Example 2 subjected to nanopore detection, wherein A shows the current signal graph of the sequencing adapter complex He+Ad-2LNA subjected to nanopore sequencing. B shows the current signal graph of the sequencing adapter complex He+Ad-4LNA subjected to nanopore sequencing. C shows the current signal graph of the sequencing adapter complex He+Ad-8LNA subjected to nanopore sequencing.

[0047] FIG5 shows the single molecule permeation time of the sequencing adapter complex for nanopore detection in Example 2;

[0048] FIG6 shows a schematic diagram of constructing a single-chain circular library in Example 3;

[0049] Figure 7 shows the electrophoresis detection diagram of the single-stranded circular library constructed in Example 3, wherein A is the annealing electrophoresis diagram of the sequence containing the hairpin structure (SEQ ID NO. 8), and B is the electrophoresis diagram of the single-stranded circular library;

[0050] FIG8 shows a current signal diagram of multiple sequencing runs on a single nanopore in Example 4;

[0051] FIG9 shows an enlarged view of the characteristic sequence current signal in FIG8 ;

[0052] FIG10 shows an amplified diagram of the current signal of the card issuing sequence in FIG8 ;

[0053] FIG11 shows a schematic diagram of a sequencing library (double-stranded circular library) according to another embodiment of the present application, wherein the binding site for the first strand of the sequencing adapter complex has a bubble or a gap;

[0054] FIG12 shows a schematic diagram of a method for multiple nanopore sequencing of a target nucleic acid sequence according to another embodiment of the present application (double-stranded circular library). DETAILED DESCRIPTION

[0055] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments in this application can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0056] As mentioned in the background, existing nanopore sequencing technology still faces a series of technical issues, including limited accuracy, which restricts its application. Performing multiple nanopore sequencing runs on a target nucleic acid sequence could significantly improve sequencing accuracy. To address this, this application provides the following technical solutions.

[0057] According to a typical embodiment of the present application, a sequencing adapter is provided. The sequencing adapter comprises a first strand and a second strand, wherein: the first strand comprises a sequencing guide sequence, a helicase binding sequence, a restricting structure, a first complementary sequence, and a first primer sequence, sequentially connected from the 5' end to the 3' end, the restricting structure preventing the movement of the helicase, and the first strand having a 3' free end; the second strand comprises a second complementary sequence and a restraining sequence or a sequence complementary to the nucleic acid sequence of the restraining sequence, sequentially connected from the 5' end to the 3' end, the restraining sequence comprising a nucleic acid sequence with a hydrophobic molecule attached to its end; the first complementary sequence is reversely complementary to the second complementary sequence, and the sequencing adapter is formed by annealing the first and second strands. The sequencing adapter of the present application is simple in design, and annealing of the two strands to the desired configuration is relatively easy, resulting in high yield.

[0058] It is understood that if the constraint sequence is on the second strand, the 3' end of the second strand is connected to a hydrophobic molecule; if the sequence complementary to the nucleic acid sequence of the constraint sequence is on the second strand, the 5' end of the constraint sequence is connected to a hydrophobic molecule.

[0059] According to another typical embodiment of the present application, a sequencing adapter complex is provided. The sequencing adapter complex comprises the aforementioned sequencing adapter of the present application and a helicase, wherein the helicase binds to the helicase binding sequence of the first strand. The helicase unwinds in a 5'-3' direction; preferably, the helicase is selected from any one or more of the following: Dda helicase, Pif 1 helicase, XPD helicase, T7 Gp41 helicase, and DnaB helicase; and the helicase is modified by mutation.

[0060] Based on the above-mentioned sequencing adapter complex provided by the present application, the applicant further invented a method for achieving multiple sequencing of a target nucleic acid. The sequencing adapter of the present invention contains a sequence complementary to the circular library, and a polymerase with chain displacement activity amplifies a sequencing chain using the circular library as a template and the complex sequencing adapter as a primer; the sequencing adapter contains an artificially modified nucleic acid sequence that can inhibit the polymerase chain displacement activity, and this sequence is also bound to the circular library through base complementarity. When the polymerase amplifies the sequencing chain along the circular library, it encounters this sequence that can inhibit the polymerase chain displacement activity, so that the activity of the polymerase is inhibited; when the guide sequence on the sequencing adapter is captured by the nanopore, the electric field force drives the helicase on the sequencing adapter complex to pass through the limiter sequence and the sequence that can inhibit the polymerase chain displacement activity, and normal unwinding is achieved to achieve stable sequencing. At the same time, because the sequence that inhibits the polymerase chain displacement activity passes through the nanopore, the polymerase is able to continue circular amplification and continuously extend the sequencing chain, ultimately achieving multiple sequencing of the target nucleic acid.

[0061] According to a preferred embodiment of the present application, the specific process of the method for multiple nanopore sequencing of target nucleic acid sequences can be referred to Figure 1: In process (1), the components required for sequencing are prepared: a sequencing adapter complex, a single-stranded ring library and a polymerase with chain displacement activity. The construction process of the sequencing adapter complex: First, the first chain and the second chain are annealed to form a sequencing adapter. The first chain is composed of a guide sequence (30 iSpC3, represented by a gray dotted line), a helicase binding sequence (10 T, represented by a black solid line), a limiter sequence (4 iSp18, represented by a gray dotted line), a sequence complementary to the second chain (represented by a black solid line), and a sequence complementary to the single-stranded ring library (including an artificially modified nucleic acid sequence that can inhibit the chain displacement activity of the polymerase, such as LNA, PNA or BNA, represented by a bold black solid line; the rest of the unmodified nucleic acid sequence is represented by a black solid line). The second chain is composed of a sequence complementary to the first chain and a sequence complementary to the constraint sequence (both represented by black solid lines) from 5' to 3'. Next, the sequencing adapters are incubated with helicase (light gray notched circles, unwinding from 5' to 3'). A reaction buffer containing ATP is then added. Helicase bound to the helicase binding sequence is prevented from displacing due to the restriction sequence and remains bound to the first strand. Helicase bound to the remaining sequences displaces and detaches. Finally, after magnetic bead purification, a sequencing adapter complex with a 1:1 ratio of helicase to sequencing adapter is obtained. Single-stranded circle library construction: Hairpin-containing adapters are ligated to both ends of the target nucleotide double strand to create a single-stranded circle library. Alternatively, a single-stranded circle library is created from the target nucleotide double strand using the MGI MGIEasy PCR-Free DNA Library Preparation Kit (MGI, 1000013456), or other similar methods. During the preparation of the single-stranded circle library, a sequence complementary to the first strand of the sequencing adapter is introduced (bold black solid line). The polymerase used is a polymerase with strand displacement activity (gray oval). In process (2), the sequencing adapter complex, the single-stranded circular library, and the polymerase with chain displacement activity are incubated together, and the first chain of the sequencing adapter is bound to the single-stranded circular library through base complementary pairing (including an artificially modified nucleic acid sequence that can inhibit the polymerase chain displacement activity). The polymerase uses the circular library as a template and the first chain as a primer, and binds to the junction of the primer and template (primer-template junction), and the three form a complex. In process (3), the polymerase uses the dNTP or NTP in the sequencing buffer to perform a polymerization reaction, extending the first chain to obtain a sequencing chain. When the polymerase amplifies the sequencing chain along the circular library, it will encounter the artificially modified nucleic acid sequence in the first chain that can inhibit the polymerase chain displacement activity, and the polymerization reaction stops, thereby obtaining an amplified sequencing adapter complex. At the same time, the helicase bound to the sequencing adapter cannot use the dNTP or NTP in the sequencing buffer to unwind the double strand due to the presence of the limiter sequence.In process (4), the added constraint sequence (3' end is a dideoxynucleotide to prevent amplification by polymerase that has not formed a complex; 5' end is cholesterol modified, represented by a black circle) binds to the second chain of the sequencing adapter, and the amplified sequencing adapter complex is bound to the membrane material near the nanopore (the gray square represents the membrane, and the white channel represents the nanopore). After the sequencing voltage is applied, the guide sequence of the sequencing adapter is captured by the nanopore under the action of the electric field force (indicated by the black arrow). In process (5), the guide sequence passes through the nanopore, and the electric field force (indicated by the black arrow) drives the helicase to pass through the limiting sequence and the sequence that can inhibit the polymerase chain displacement activity, and normal unwinding is achieved to achieve stable sequencing. At the same time, because the sequence that can inhibit the polymerase chain displacement activity passes through the nanopore, the polymerase is able to continue circular amplification and continuously extend the sequencing chain. In process (6), the polymerase continuously extends the sequencing chain. Under the action of the electric field force (indicated by the black arrow), the helicase alone, or the helicase and polymerase together, controls the sequencing speed to achieve multiple sequencing.

[0062] The advantages of this design are: ① When the sequencing chain is not captured by the nanopore, the amplification of long single chains is avoided, forming complex secondary structures, which affects nanopore sequencing, but at the same time, multiple sequencing of the target polynucleotide sequence to be tested can be achieved; ② The sequencing process is always a double-stranded sequencing process controlled by helicase, and the duration and sequencing speed of different sequencing times are relatively consistent; ③ The circular library construction process is simple and can be preserved for a long time; ④ The sequencing adapter complex construction process is simple to operate and has high yield.

[0063] Based on the inventive concept of the present application, some technical features in the above preferred embodiments are variable or replaceable. For example, in certain embodiments, preferably, the guide sequence is 10 to 50 nucleotides or iSpC3; preferably, the helicase binding sequence of the first chain is 5 to 40 nucleotides, more preferably, the helicase binding sequence of the first chain is 5 to 40 thymine nucleotides (T); preferably, the limiting sequence of the first chain is 2 to 8 spacer modifications, such as iSp18, iSp9, iSpC3, iSpC6, iSpC12, or any combination thereof; preferably, the length of the sequence complementary to the first chain and the second chain is 5 to 80 nucleotides; preferably, the length of the sequence complementary to the first chain and the circular library is 5 to 80 nucleotides; preferably, the length of the sequence complementary to the first chain and the second chain is 5 to 80 nucleotides; preferably, the length of the sequence complementary to the second chain and the first chain is 5 to 80 nucleotides; preferably, the length of the sequence complementary to the second chain and the constraint sequence is 10 to 50 nucleotides.

[0064] In certain embodiments, the technical solution of the present application may use a double-stranded circular library, in which a bubble or a gap exists at the complementary binding site with the first primer sequence of the first chain of the sequencing adapter complex. For details on how to achieve multiple nanopore sequencing of the target nucleic acid sequence, see Figures 11 and 12.

[0065] In certain embodiments, the technical solution of the present application may use a circular library, in which there is no known sequence, the first primer sequence of the first chain is a degenerate primer sequence, and any sequence in the circular library is complementary to the degenerate primer sequence.

[0066] In certain embodiments, the sequence capable of inhibiting polymerase chain displacement activity is a GC-rich motif with stronger binding ability, an artificially modified nucleotide sequence, or any combination thereof; in certain embodiments, the artificially modified nucleic acid sequence capable of inhibiting polymerase chain displacement activity located at the 5' end of the second chain is LNA, PNA, BNA, or any combination thereof; the number of sequences capable of inhibiting polymerase chain displacement activity is 2 to 10.

[0067] For the convenience of operation and product production, in certain embodiments, a separate constraint sequence may not be required, and the 3' end of the second strand of the sequencing adapter can be directly modified with a hydrophobic molecule, wherein the hydrophobic molecule is selected from any one or more of the following: lipids, fatty acids, sterols, carbon nanotubes, polypeptides, proteins, amino acids, such as cholesterol, palmitate, or tocopherol.

[0068] In certain embodiments, the helicase unwinds in a 5'-3' direction, and the helicase is selected from any one or more of the following: Dda helicase, Pif 1 helicase, XPD helicase, T7Gp41 helicase, and DnaB helicase. The selected helicase may be modified by mutation.

[0069] According to a typical embodiment of the present application, a method for multiple nanopore sequencing of a target nucleic acid sequence is also provided. The method comprises the following steps: S1, combining a sequencing adapter complex, a circular library containing a target nucleic acid sequence, and a polymerase with strand displacement activity to obtain a sequencing adapter amplification complex; S2, the polymerase using dNTPs or NTPs to perform a polymerization reaction to extend the first strand of the sequencing adapter. When the polymerase amplifies the sequencing chain along the circular library once and encounters a sequence in the first strand that inhibits the polymerase's strand displacement activity, the polymerization reaction stops, thereby obtaining an amplified sequencing adapter complex; S3, binding the amplified sequencing adapter complex to the membrane of the nanopore sequencer via a restraining sequence. The amplified sequencing adapter complex is sequenced under the control of a helicase, thereby achieving multiple sequencing of the target nucleic acid sequence. The circular library can be a single-stranded circular library or a double-stranded circular library. In certain embodiments, the sequence that inhibits the polymerase's strand displacement activity is a GC-rich motif with stronger binding affinity, an artificially modified nucleotide sequence, or any combination thereof. In certain embodiments, the artificially modified nucleotide is LNA, PNA, BNA, or any combination thereof. The number of artificially modified nucleotides can be 1, 2, 3, 4, 5, 6, 7, 8 or even more, preferably 1 to 8.

[0070] In the present application, typically, S1 includes: the first chain of the sequencing adapter in the sequencing adapter complex is bound to the circular library through base complementary pairing, and the polymerase uses the circular library as a template and the first chain as a primer to form an amplification complex at the junction of the primer and the template; in S2: the helicase bound to the sequencing adapter cannot use the dNTP in the sequencing buffer to unwind the double strand due to the presence of the restriction sequence; S3 includes: the second chain of the sequencing adapter is bound to the constraint sequence, and the amplified sequencing adapter complex is bound to the membrane of the nanopore sequencer, or the constraint sequence on the second chain of the sequencing adapter is directly The amplified sequencing adapter complex is bound to the membrane of the nanopore sequencer; after applying the sequencing voltage, the guide sequence of the sequencing adapter is captured by the nanopore under the action of the electric field force, and the guide sequence passes through the nanopore. The electric field force drives the helicase to pass through the limiting sequence and the sequence that can inhibit the polymerase chain displacement activity, and normal unwinding is achieved to achieve stable sequencing; at the same time, because the sequence that can inhibit the polymerase chain displacement activity passes through the nanopore, the polymerase is able to continue circular amplification and continuously extend the sequencing chain; the polymerase continuously extends the sequencing chain, and under the action of the electric field force, the helicase alone, or the helicase and the polymerase jointly control the sequencing speed to achieve multiple sequencing. Multiple nanopore sequencing of the target nucleic acid sequence will significantly improve the sequencing accuracy. Among them, the sequence length of the constraint sequence is 10 to 50 nucleotides; the end of the constraint sequence is connected to a hydrophobic molecule, and the hydrophobic molecule is selected from any one or more of the following: lipids, fatty acids, sterols, carbon nanotubes, polypeptides, proteins, amino acids, such as cholesterol, palmitate or tocopherol.

[0071] In some embodiments, preferably, the sequencing voltage is above 10 mV, preferably 50 mV-250 mV. For ease of operation and product production, in some embodiments, a separate constraint sequence may not be required, and cholesterol modification can be performed directly on the 3' end of the second strand of the sequencing adapter.

[0072] In certain embodiments, the polymerase having strand displacement activity is selected from a DNA polymerase or an RNA polymerase. The selected polymerase may also be a polymerase having strand displacement activity that is modified from a polymerase without strand displacement activity, such as Bst DNA polymerase, SD DNA polymerase, phi29 DNA polymerase, Bsu Large Fragment DNA polymerase, Klenow Fragment DNA polymerase, T4 DNA polymerase, T7 DNA polymerase, DNA Polymerase I, T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, E. coli RNA polymerase, or any combination thereof.

[0073] In certain embodiments, the nanopore is a transmembrane protein pore or a solid-state pore. In certain embodiments, the transmembrane protein pore is derived from a transmembrane protein selected from hemolysin, MspA, MspB, MspC, MspD, FraC, ClyA, PA63, CsgG, CsgD, XcpQ, SP1, phi29 connector protein, InvG, GspD, or any combination thereof.

[0074] In certain embodiments, the transmembrane pore is further linked to an additional polypeptide, wherein the additional polypeptide is selected from a tag, an enzyme cleavage site, a signal peptide or leader peptide, a detectable label, or any combination thereof.

[0075] In certain embodiments, the membrane of the nanopore sequencer is an amphiphilic membrane, a high molecular polymer membrane, or any combination thereof, and the membrane of the nanopore sequencer is a phospholipid bilayer, a diblock copolymer, or a triblock copolymer.

[0076] In certain embodiments, the sequencing buffer used in sequencing contains a pH buffer system. Preferably, the pH buffer system is a dihydrogen phosphate-hydrogen phosphate buffer system, a carbonic acid-sodium bicarbonate buffer system, a Tris-HCl buffer system, a HEPES buffer system, a MOPS buffer system, or any combination thereof.

[0077] In certain embodiments, the sequencing buffer contains NTPs, dNTPs, ddNTPs, or any combination thereof.

[0078] In certain embodiments, the sequencing buffer contains K + , Na 2+ , or any combination thereof.

[0079] In certain embodiments, the sequencing buffer contains Mg 2+ , Mo 2+ , Cu 2+ , Fe 2+ , Zn 2+ , Ca 2+ , Pb 2+ , Cd 2+ , or any combination thereof.

[0080] In certain embodiments, the sequencing buffer contains additives or auxiliary reagents commonly used to enhance polymerase extension reactions, such as dimethyl sulfoxide (DMSO), glycerol, formamide, bovine serum albumin (BSA), ammonium sulfate [(NH4)2SO4], polyethylene glycol (PEG), gelatin, non-ionic detergents (such as Tween 20, Triton X-100), N,N,N-trimethylglycine (betaine), single-stranded nucleic acid binding proteins, or any combination thereof.

[0081] The beneficial effects of the present application will be further explained in detail below with reference to specific embodiments.

[0082] Example 1: Construction of Sequencing Adapter Complex

[0083] The construction process of the sequencing adapter complex is shown in Figure 2, which is divided into three processes: sequencing adapter annealing, sequencing adapter and helicase incubation to form a complex, and sequencing adapter complex purification. In process (1), the synthesized first chain (A, SEQ ID NO.2-4) and the second chain (B, SEQ ID NO.1) are annealed to form a sequencing adapter at a ratio of 1:1. The first chain, from 5' to 3', is composed of a guide sequence (30 iSpC3, represented by gray dashed lines), a helicase binding sequence (10 T, represented by black solid lines), a limiter sequence (4 iSp18, represented by gray dashed lines), a sequence complementary to the second chain (represented by black solid lines), and a sequence complementary to the single-stranded circular library (including an artificially modified nucleic acid sequence that can inhibit polymerase chain displacement activity, represented by a bold black solid line; the remaining unmodified nucleic acid sequences are represented by black solid lines). The second chain, from 5' to 3', is composed of a sequence complementary to the first chain and a sequence complementary to the constraint sequence (both represented by black solid lines). In process (2), the sequencing adapter and helicase (light gray notched circle, unwinding direction 5' to 3', SEQ ID NO. 5) are incubated to form a complex. In process (3), a buffer containing ATP is added. Due to the action of the limiter sequence, only one motor protein bound between the guide sequence and the limiter sequence remains. The sequencing adapter complex with a 1:1 ratio of helicase to sequencing adapter is obtained by magnetic bead purification.

[0084] The specific steps are as follows:

[0085] Sequencing adapter annealing

[0086] 1. Dissolve SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 4 in TE buffer (pH = 8) according to the manufacturer's instructions.

[0087] 2. Sequence ID NO. 2, Sequence ID NO. 3, and Sequence ID NO. 4 were annealed to Sequence ID NO. 1 at a ratio of 1:1 to form sequencing adapters, named Ad-2LNA, Ad-4LNA, and Ad-8LNA. The annealing process was as follows: incubate at 95°C for 5 minutes, cool to 25°C at a rate of 0.1°C / s, and continue incubating for 30 minutes. After annealing, electrophoresis was performed. The results are shown in Figure 3. A large number of successfully annealed Ad-2LNA and Ad-4LNA sequencing adapters were obtained, as well as a small number of successfully annealed Ad-8LNA sequencing adapters. Figure 3 A is an electrophoretic diagram of sequencing adapter annealing. Lane 1 is the second strand (B, SEQ ID NO. 1), lanes 2-4 are the first strands containing 2, 4, and 8 LNA-modified nucleotides (A-2LNA / -4LNA / -8LNA, SEQ ID NOs. 2-4), lanes 5-7 are the annealed sequencing adapters (Ad-2LNA / -4LNA / -8LNA), and lane 8 is the DNA molecular weight standard (M). FIG3B is an electrophoresis diagram of the sequencing adapter complex. Lanes 1-2 are the sequencing adapter Ad-2LNA and the sequencing adapter complex He+Ad-2LNA, lanes 3-4 are the sequencing adapter Ad-4LNA and the sequencing adapter complex He+Ad-4LNA, and lanes 5-6 are the sequencing adapter Ad-8LNA and the sequencing adapter complex He+Ad-8LNA. Arrows indicate the sequencing adapter complex with a 1:1 ratio of helicase to sequencing adapter and the sequencing adapter, respectively.

[0088] Sequencing adapters and helicase are incubated as a complex

[0089] 1. The helicase He (T4Dda-(ΔM1)G1 / E94C / C109A / C136A / K194L / A360C, SEQ ID NO. 5) was expressed in Escherichia coli and the target protein was obtained through multiple purification steps.

[0090] 2. Mix helicase He with sequencing adapters Ad-2LNA, Ad-4LNA, and Ad-8LNA at a molecular ratio of 9:1. The final concentration of the reaction buffer is 25mM HEPES, 50mM KCl, 0.5mM EDTA, 2.5mM MgCl2, pH = 8.0. Incubate at room temperature for 30 minutes.

[0091] Sequencing adapter complex purification

[0092] 1. Add 0.25 volume of 5 mM ATP to the incubation product and incubate at room temperature for 30 minutes.

[0093] 2. Sequencing adapter complexes were purified using AMPure XP beads (Beckman Coulter, A63882) according to the manufacturer's instructions to obtain He+Ad-2LNA, He+Ad-4LNA, and He+Ad-8LNA sequencing adapter complexes. The purified sequencing adapter complexes were examined by electrophoresis. The results, shown in Figure 3, showed a large number of 1:1 He+Ad-2LNA and He+Ad-4LNA sequencing adapter complexes, and a small number of 1:1 He+Ad-8LNA sequencing adapter complexes.

[0094] Example 2: Nanopore detection of sequencing adapter complexes

[0095] 1. Based on the reference (Ji Z, Guo P. Channel from bacterial virus T7 DNA packaging motor for the differentiation of peptides composed of a mixture of acidic and basic amino acids. Biomaterials. 2019 May 21;214:119-222), a nanopore detection system was constructed based on patch clamp and signal amplifier to complete the embedding of a single porin.

[0096] 2. Sequencing adapter complexes (He+Ad-2LNA, He+Ad-4LNA, and He+Ad-8LNA) were mixed with the constraint sequence (SEQ ID NO. 6) and added to the single-channel system. Current signal changes were observed at 100 mV, 120 mV, and 150 mV, respectively. The sequencing buffer consisted of 0.30 M KCl, 50 mM Tris-HCl, 10 mM MgCl2, 0.5 mM dNTPs, pH 8.10. The sequencing temperature was 30°C.

[0097] 3. The three sequencing adapter complexes He+Ad-2LNA, He+Ad-4LNA, and He+Ad-8LNA all obtained clear sequencing current signals under 100mV, 120mV, and 150mV conditions. Figure 4 shows the current signal graphs of the three sequencing adapter complexes under nanopore detection under 120mV conditions, where A shows the current signal graph of the sequencing adapter complex He+Ad-2LNA for nanopore sequencing. B shows the current signal graph of the sequencing adapter complex He+Ad-4LNA for nanopore sequencing. C shows the current signal graph of the sequencing adapter complex He+Ad-8LNA for nanopore sequencing.

[0098] 4. During nanopore detection, we discovered that the permeation time for individual molecules differed between the three sequencing adapter complexes. Figure 5 shows the statistical analysis of permeation times for individual molecules: higher sequencing voltages resulted in shorter permeation times for the sequencing adapter complexes; and higher numbers of LNAs in the sequencing adapter complexes resulted in longer permeation times.

[0099] Example 3: Construction of single-stranded circular library

[0100] 1. Construction of a single-stranded circular library: Various known methods can be used, such as ligating hairpin-containing linkers to both ends of the target nucleotide double strand to prepare a single-stranded circular library, or other similar methods to prepare the target nucleotide as a single-stranded circular library. In this example, a single-stranded circular library was prepared by ligating hairpin-containing linkers to both ends of the target nucleotide double strand. The construction process is shown in Figure 6. The target polynucleotide to be tested (SEQ ID NO. 7) with a characteristic sequence (bold black solid line) was end-repaired and A-added, and then ligated with hairpin-containing linkers (SEQ ID NO. 8) at both ends. The single-stranded circular library was purified, and the arrows indicate the characteristic sequence.

[0101] 2. Perform end repair and dA-tailing on the target polynucleotide with the characteristic sequence (SEQ ID NO. 7) using NEBNext FFPE DNA Repair Mix (NEB, M6630) and NEBNext Ultra II End Repair / dA-tailing Module (NEB, E7546) according to the manufacturer's instructions.

[0102] 3. Dissolve the hairpin sequence (SEQ ID NO.8) in TE buffer (pH = 8) according to the manufacturer's instructions and anneal to form a hairpin structure. The annealing process is to incubate at 95°C for 5 minutes, cool down to 25°C at a rate of 0.1°C / s, and continue incubating for 30 minutes. After the annealing is completed, electrophoresis detection is performed. The results are shown in Figure 7. After annealing, SEQ ID NO.8 forms a hairpin structure. Among them, A in Figure 7 shows the annealing electrophoresis diagram of the sequence containing the hairpin structure (SEQ ID NO.8). Lane 1 is a DNA molecular weight standard (M), and lanes 2-3 are the sequences containing the hairpin structure before and after annealing. B is the electrophoresis diagram of the single-stranded circular library: lane 1 is a DNA molecular weight standard (M), lane 2 is a target polynucleotide to be tested with a characteristic sequence (SEQ ID NO. 7), and lane 3 is a single-stranded circular library (target polynucleotide to be tested + a linker containing a hairpin structure) obtained by ligation and purification. The arrows indicate the target polynucleotide to be tested, the linker containing a hairpin structure to be connected at one end of the target polynucleotide to be tested, and the linker containing a hairpin structure to be connected at both ends of the target polynucleotide to be tested.

[0103] 4. Ligation reaction was performed on the target polynucleotide after end repair and A addition and the annealed hairpin-containing adapter using NEBNext Quick Ligation Module (NEB, E6056) according to the manufacturer's instructions. The reaction conditions were incubation at 25°C for 10 minutes.

[0104] 5. Purify the single-stranded circular library using AMPure XP beads (Beckman Coulter, A63882) according to the manufacturer's instructions and perform electrophoresis detection. The results are shown in Figure 2-7, and a large number of single-stranded circular libraries with hairpin structures connected at both ends of the target sequence are obtained.

[0105] Example 4: Nanopore sequencing

[0106] 1. Mix the sequencing adapter complex He+Ad-4LNA obtained in Example 1, the single-stranded circular library obtained in Example 3, and Klenow Fragment DNA Polymerase (NEB, M0212M) with strand displacement activity. Incubate at 30°C for 30 minutes. The incubation system is shown in Table 1. The final concentration of the reaction buffer is 37.5 mM KCl, 12.5 mM Tris-HCl, 2.5 mM MgCl2, pH = 8.10. After the incubation, add 0.1 volume of 10 mM dNTPs to the product and continue incubating at 30°C for another 30 minutes to obtain the sequencing adapter amplification complex.

[0107] Table 1 Incubation system formula

[0108]

[0109] 2. Build a single-channel nanopore detection system based on patch clamp and signal amplifier to complete the embedding of a single porin.

[0110] 3. The amplified sequencing adapter complex was mixed with the constraint sequence (SEQ ID NO. 6) and added to the single-channel system. The current signal change was observed and obtained at 180 mV (sequencing temperature: 30°C). The sequencing buffer was 0.30 M KCl, 50 mM Tris-HCl, 10 mM MgCl2, 1.25 mM dNTP, pH = 8.10.

[0111] 4. A sequencing signal is captured, and a clear sequencing signal current amplitude change graph is obtained. As shown in Figure 8, based on the characteristic sequence signal and hairpin sequence signal of the reads, it can be judged that the target sequence is sequenced 4 times in a row, and 1D, 2D, 3D, and 4D are respectively the current signals of the first, second, third, and fourth sequencing, of which only a part of the fourth sequencing is completed. From the sequencing signal current amplitude change graph, it can be found that the duration and sequencing speed of different sequencing times (first, second, and third) are relatively consistent; the dotted rectangular box indicates the hairpin sequence signal, and the arrow indicates the characteristic sequence signal. Figure 9 shows an enlarged view of the current signal; the dotted rectangular box indicates the hairpin sequence signal, and Figure 10 shows an enlarged view of the current signal.

[0112] Polynucleotide sequences used in the examples

[0113]

[0114]

[0115]

[0116] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects:

[0117] (1) The library construction process of the present invention is simple and has a high yield. For example, by directly connecting a connector containing a hairpin structure at both ends of the double-stranded nucleic acid to be tested, a single-stranded circular library can be constructed. (2) The present invention can achieve multiple sequencing. (3) During the sequencing process of the present invention, the helicase always controls the double-stranded sequencing process, and the sequencing speed of different times is relatively consistent. (4) The sequencing connector of the present invention is simple in design, and it is relatively easy to anneal the two chains to the target configuration, and the yield is high. (5) The sequencing connector of the present invention only needs to be combined with a helicase, and the construction process is simple to operate and has a high yield. (6) During the sequencing process of the present invention, no long single chain will be generated, thereby forming a complex secondary structure, which will lead to the problem of abnormal sequencing. (7) The sequencing process of the present invention does not require a long amplification process. (8) The present invention does not have the problem of amplifying to obtain multiple copies and then connecting the sequencing connector, and there is no need to consider the connection efficiency, and the utilization efficiency of the amplified product is high.

[0118] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A sequencing adapter, characterized in that It includes a first chain and a second chain, wherein: The first strand comprises a sequencing guide sequence, a helicase binding sequence, a limiting structure, a first complementary sequence, a structure for preventing the first primer sequence from being displaced by a polymerase, and the first primer sequence, which are sequentially connected from the 5' end to the 3' end, the limiting structure prevents the helicase from moving, and the first strand has a 3' free end; The second strand includes a second complementary sequence and a restraining sequence or a sequence complementary to the nucleic acid sequence of the restraining sequence, which are sequentially connected from the 5' end to the 3' end, and the restraining sequence includes a nucleic acid sequence with a hydrophobic molecule connected to the end; The first complementary sequence is reverse complementary to the second complementary sequence, and the sequencing adapter is formed by annealing the first strand and the second strand.

2. The sequencing adapter according to claim 1, characterized in that The structure that prevents the first primer sequence from being displaced by the polymerase is a GC-rich motif, an artificially modified nucleotide sequence, or a normal nucleic acid coupled with other inhibitory molecules, or any combination thereof; Preferably, the structure that prevents the first primer sequence from being displaced by a polymerase is an artificially modified nucleotide sequence.

3. The sequencing adapter according to claim 2, characterized in that The artificially modified nucleotides in the artificially modified nucleic acid sequence include at least one of LNA, PNA and BNA; Furthermore, the artificially modified nucleic acid sequence includes 2 to 10 artificially modified nucleotides.

4. The sequencing adapter according to claim 1, characterized in that The limiting structure includes a spacer region, the spacer region includes a spacer, and the spacer is selected from at least one of iSp18, iSp9, iSpC3, iSpC6 and iSpC12; the limiting structure includes 2 to 8 spacers; Preferably, the sequencing guide sequence comprises 10 to 50 nucleotides or iSpC3; Preferably, the helicase binding sequence comprises 5 to 40 nucleotides, more preferably, the helicase binding sequence comprises 5 to 40 thymine nucleotides; Preferably, the first complementary sequence or the second complementary sequence comprises 5 to 80 nucleotides; Preferably, the nucleic acid sequence of the constraint sequence comprises 10 to 50 nucleotides.

5. The sequencing adapter according to claim 1, characterized in that The hydrophobic molecules are selected from any one or more of the following: lipids, fatty acids, sterols, carbon nanotubes, polypeptides, proteins, and amino acids; Preferably, the hydrophobic molecule is cholesterol, palmitate or tocopherol.

6. The sequencing adapter according to claim 1, characterized in that The first primer sequence includes a specific primer or a random primer, and the first primer sequence can bind to a circular library; Preferably, the first primer sequence comprises 5 to 80 nucleotides; Optionally, the circular library is a single-stranded circular library or a double-stranded circular library; Optionally, the single-stranded circular library contains an introduced known sequence, and the known sequence is complementary to the first primer sequence; Optionally, the double-stranded circular library contains an introduced known sequence, and a bubble or a gap exists at the complementary binding site between the known sequence and the first primer sequence; Optionally, there is no known sequence in the circular library, the first primer sequence is a degenerate primer sequence, and any sequence in the circular library is complementary to the degenerate primer sequence.

7. A sequencing adapter complex, characterized in that The method comprises a sequencing adapter and a helicase according to any one of claims 1 to 6, wherein the helicase is bound to a helicase binding sequence, and the helicase unwinds in a 5'-3' direction; preferably, the helicase is selected from any one or more of the following: Dda helicase, Pif 1 helicase, XPD helicase, T7 Gp41 helicase and DnaB helicase; and the helicase is modified by mutation.

8. A kit for nanopore sequencing of a target nucleic acid sequence, characterized in that: Comprising a sequencing adapter as described in any one of claims 1 to 6; Furthermore, it also includes a polymerase with strand displacement activity, a reaction buffer and a helicase.

9. A kit for nanopore sequencing of a target nucleic acid sequence, characterized in that: comprising the sequencing adapter complex as claimed in claim 7; Furthermore, it also includes a polymerase with strand displacement activity and a reaction buffer.

10. The kit according to claim 8 or 9, characterized in that The kit further comprises one or more of dNTP or NTP, and reagents related to circular library construction.

11. A method for multiple nanopore sequencing of a target nucleic acid sequence, characterized in that: The following steps are involved: S1, combining the sequencing adapter complex as claimed in claim 7, a circular library containing a target nucleic acid sequence and a polymerase having strand displacement activity to obtain a sequencing adapter amplification complex; S2, the polymerase uses dNTP or NTP to perform a polymerization reaction to extend the first chain of the sequencing adapter. When the polymerase amplifies the sequencing chain along the circular library, it encounters a sequence in the first chain that can inhibit the polymerase chain displacement activity, and the polymerization reaction stops, thereby obtaining an amplified sequencing adapter complex; S3, the amplified sequencing adapter complex is bound to the membrane of the nanopore sequencer through a restraining sequence, and the amplified sequencing adapter complex is sequenced under the control of a helicase to achieve multiple sequencing of the target nucleic acid sequence.

12. The method according to claim 11, characterized in that The S1 comprises: the first strand of the sequencing adapter in the sequencing adapter complex is combined with the circular library through base complementary pairing, the polymerase uses the circular library as a template and the first strand as a primer to form an amplification complex at the junction of the primer and the template; and / or, The S3 includes: The second chain of the sequencing adapter is combined with the restraining sequence, and the amplified sequencing adapter complex is combined with the membrane of the nanopore sequencer, or the restraining sequence on the second chain of the sequencing adapter directly combines the amplified sequencing adapter complex with the membrane of the nanopore sequencer; after applying the sequencing voltage, the guide sequence of the sequencing adapter is captured by the nanopore under the action of the electric field force, and the guide sequence passes through the nanopore. The electric field force drives the helicase to pass through the limiting sequence and the sequence that can inhibit the polymerase chain displacement activity, and the normal unwinding is realized to achieve stable sequencing; at the same time, the sequence that can inhibit the polymerase chain displacement activity passes through the nanopore, and the polymerase continues to perform circular amplification to continuously extend the sequencing chain; the polymerase continuously extends the sequencing chain, and the helicase alone, or the helicase and the polymerase jointly control the sequencing speed under the action of the electric field force, to achieve multiple sequencing; The sequence length of the restraint sequence is 10 to 50 nucleotides; the end of the restraint sequence is connected to a hydrophobic molecule, and the hydrophobic molecule is selected from any one or more of the following: lipids, fatty acids, sterols, carbon nanotubes, polypeptides, proteins, amino acids, such as cholesterol, palmitate or tocopherol.

13. The method according to claim 11 or 12, characterized in that: The polymerase having strand displacement activity is selected from DNA polymerase or RNA polymerase; Optionally, the polymerase is a polymerase with strand displacement activity obtained by modifying a polymerase without strand displacement activity; Preferably, the polymerase is selected from Bst DNA polymerase, SD DNA polymerase, phi29 DNA polymerase, Bsu Large Fragment DNA polymerase, Klenow Fragment DNA polymerase, T4 DNA polymerase, T7 DNA polymerase, DNA Polymerase I, T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, E. coli RNA polymerase, or any combination thereof.

14. The method according to claim 11 or 12, characterized in that: The sequencing voltage is above 10 mV, preferably 50 mV-250 mV.

15. The method according to claim 11 or 12, characterized in that: The nanopore of the nanopore sequencer is a transmembrane protein pore or a solid-state pore; Preferably, the transmembrane protein pore is derived from a transmembrane protein selected from hemolysin, MspA, MspB, MspC, MspD, FracCe, ClyA, PA63, CsgG, CsgD, XcpQ, SP1, phi29 connector protein, InvG, GspD, or any combination thereof; Optionally, the transmembrane protein is further connected to another polypeptide, and the other polypeptide is selected from a tag, an enzyme cleavage site, a signal peptide or a leader peptide, a detectable marker, or any combination thereof; Optionally, the membrane of the nanopore sequencer is an amphiphilic membrane, a high molecular polymer membrane or any combination thereof, and the membrane of the nanopore sequencer is a phospholipid bilayer, a diblock copolymer, or a triblock copolymer.

16. The method according to any one of claims 11 to 15, characterized in that The sequencing buffer used in the sequencing contains a pH buffer system. Preferably, the pH buffer system is a dihydrogen phosphate-hydrogen phosphate buffer system, a carbonic acid-sodium bicarbonate buffer system, a Tris-HCl buffer system, a HEPES buffer system, a MOPS buffer system, or any combination thereof; Preferably, the sequencing buffer contains NTP, dNTP, ddNTP, or any combination thereof; Preferably, the sequencing buffer contains K + 、Na 2+ , or any combination thereof; Preferably, the sequencing buffer contains Mg 2+ 、Mo 2+ , Cu 2+ , Fe 2+ 、Zn 2+ , Ca 2+ , Pb 2+ 、Cd 2+ , or any combination thereof; Preferably, the reaction buffer or the sequencing buffer contains additives or auxiliary reagents for enhancing the polymerase extension reaction, and the additives or auxiliary reagents are dimethyl sulfoxide, glycerol, formamide, bovine serum albumin, ammonium sulfate, polyethylene glycol, gelatin, non-ionic detergent, N,N,N-trimethylglycine, single-stranded nucleic acid binding protein or any combination thereof.

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