Nanopore sequencing method using closer

Abstract

Provided in the present invention is a nanopore sequencing method using a closer. The method of the present invention enables a sequencing library to only bind within the nanopore capture range, ensuring that the library is efficiently captured by the nanopores during sequencing, thus improving the utilization efficiency of the library.

Description

Method for nanopore sequencing using blockers Technical Field

[0001] The present invention belongs to the field of nanopore sequencing, and in particular relates to a method for performing nanopore sequencing using a blocker. Background Art

[0002] Nanopore sequencing is a single-molecule detection technology with advantages such as fast sequencing speed, long read length, direct sequencing, high throughput, low cost, small size, and portability. During nanopore sequencing, a single nanopore is embedded in an insulating, impermeable membrane to form a stable ion current channel. The sequencing library is bound to the insulating, impermeable membrane by the action of the restraining sequence. Under the action of voltage, the sequencing library within the capture range of the nanopore passes through the nanopore in the form of single-stranded nucleic acid molecules under the control of the motor protein, thereby reducing the ion current passing through the nanopore. Due to the different molecular structures and sizes of different bases on the single-stranded nucleic acid molecules, the current passing through the nanopore shows differences corresponding to the base sequence. By using an algorithm to analyze the current change signal, the perforated single-stranded nucleic acid sequence can be read in real time.

[0003] The library to be tested developed by Oxford Nanopore Technologies (ONT) is bound to an insulating impermeable membrane under the action of a restraining sequence (Figure 1). The specific process is as follows: (1) The library to be tested binds to the restraining sequence; (2) After incubation, the library to be tested binds to the insulating impermeable membrane embedded with nanopores under the action of the restraining sequence; (3) After applying an electric field, the library to be tested that is bound to the insulating impermeable membrane embedded with nanopores is captured and sequenced. Patent CN103733063B can be found. However, the sequencing library is bound to the insulating impermeable membrane embedded with nanopores under the action of the restraining sequence, and it will also bind to the insulating impermeable membrane that has not been successfully embedded with nanopores, and even to the membrane material remaining in the sequencing system. However, only the small portion of the sequencing library that is bound to the nanopore capture range can be captured and sequenced by the nanopore, resulting in a large amount of library that cannot be sequenced and is wasted. Developing a method to concentrate the sequencing library within the nanopore capture range can ensure that the library is efficiently captured by the nanopore during sequencing, thereby improving the utilization efficiency of the library.

[0004] Figure 2 shows the method developed by ONT to concentrate tethered complexes in an amphiphilic membrane region. The amphiphilic membrane is composed of multiple amphiphilic molecules and can be chemically and / or physically divided into a first region and a second region. A detector, such as a nanopore, is embedded in the first region. The tethered complex includes one or more hydrophilic components connected by a hydrophobic linker and preferentially localizes to the first region. Sequencing libraries can specifically bind to the hydrophilic components of the tethered complex, thereby binding to the first region. (See patent CN115004030A.) Beijing Qitan Technology Co., Ltd. has also developed a similar method; see patent CN113999291B.

[0005] The disadvantages of the method developed by ONT to concentrate tethered complexes in the amphiphilic membrane region include: (1) the amphiphilic membrane is chemically and / or physically divided into a first region and a second region, and the tethered complex is preferentially located in the first region, but the first region and the capture range of the nanopore do not necessarily overlap absolutely, and some of the sequencing libraries bound to the tethered complexes cannot be sequenced and are wasted; (2) in the large-scale array sequencing system developed by ONT, not all amphiphilic membranes are embedded with nanopores that can be sequenced normally, or the nanopores embedded with nanopores that can be sequenced normally are lost after a period of sequencing, but the tethered complexes are still located in the first region of this part of the amphiphilic membrane, resulting in the sequencing libraries bound to these tethered complexes cannot be sequenced and are wasted; (3) the tethered complex is composed of a hydrophobic linker connected to one or more hydrophilic components, and has a complex structure. It is difficult and costly to synthesize and purify the tethered complex; (4) a large number of hydrophobic linkers of the tethered complex pass through the amphiphilic membrane, which will cause the stability of the amphiphilic membrane to deteriorate, affecting the service life of the sequencing system; (5) the amphiphilic membrane and the tethered complex of this method must match each other, and the versatility is poor. A similar method developed by Beijing Qi Tan Technology Co., Ltd. also has similar shortcomings.

[0006] Therefore, there is an urgent need in the art for a method to concentrate the library to be tested within the capture range of a nanopore.

[0007] Summary of the Invention

[0008] As mentioned above, there is an urgent need in the art for a method to concentrate the library to be tested within the capture range of a nanopore.

[0009] In a first aspect, the present invention provides a nanopore sequencing method, comprising the following steps:

[0010] 1) Incubating a blocker with a membrane embedded with a nanopore so that the blocker binds to the membrane; the blocker comprises a first strand, a second strand, and a constraining sequence, the constraining sequence comprising a portion that binds to the membrane, the first strand comprising a first sequence, a second sequence at least partially complementary to the second strand, the second strand comprising a third sequence at least partially complementary to the first strand, and a fourth sequence at least partially complementary to the constraining sequence; wherein the third sequence is configured to bind to a sequencing library by being complementary to a sequencing adapter;

[0011] 2) applying an electric field force to the membrane in step 1), causing the first chain of the blocker located within the capture range of the nanopore to pass through the nanopore, thereby exposing the third sequence in the second chain of the blocker;

[0012] 3) The third sequence is bound to the sequencing library via a sequencing adapter, so that the sequencing library is bound to the capture range of the nanopore and then captured by the nanopore to sequence the strand to be sequenced connected to the sequencing adapter.

[0013] In a second aspect, the present invention provides a nanopore sequencing kit, comprising a blocker, a sequencing buffer, a sequencing adapter, and a second nucleic acid control protein, the blocker comprising a first strand, a second strand, and a restraining sequence, the restraining sequence comprising a portion that binds to a membrane being sequenced, the first strand comprising a first sequence and a second sequence at least partially complementary to the second strand, the second strand comprising a third sequence at least partially complementary to the first strand and a fourth sequence at least partially complementary to the restraining sequence;

[0014] The sequencing adapter and the second nucleic acid control protein form a sequencing adapter complex, which is used to capture the chain to be sequenced, thereby forming a sequencing library. The third sequence is used to bind complementary to the sequencing adapter, so that the sequencing library is bound to the nanopore capture range and then captured by the nanopore to achieve sequencing.

[0015] The beneficial effects of the present invention include: (1) The present invention cleverly designs a blocker that is complementary to the connector of the sequencing library, thereby allowing the sequencing library to be bound only within the capture range of the nanopore, ensuring that the library is efficiently captured by the nanopore during the sequencing process, thereby improving the utilization efficiency of the library; (2) The scheme of the present invention allows the library that has been added to the sequencing system but not sequenced to be suspended in the sequencing buffer after a single sequencing is completed, and the sequencing buffer can be directly drawn out from the sequencing system to recover and reuse almost all of the unsequenced library; (3) The blocker designed by the present invention is simple in structure and low in cost; (4) The blocker designed by the present invention has little effect on the stability of the membrane; (5) The blocker designed by the present invention has strong versatility for different types of insulating and impermeable membranes; (6) The permeation time of a single blocker is on the order of milliseconds, which will not affect the capture of the sequencing library by the nanopore. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the background technology of the present invention and the technical solutions in the specific embodiments, the following is a brief introduction to the drawings.

[0017] Figure 1 shows a schematic diagram of a test library developed by Oxford Nanopore Technologies bound to an insulating, impermeable membrane embedded with nanopores. In Figure A, the direction of helicase action is from 5' to 3', and in Figure B, the direction of helicase action is from 3' to 5'. Figure C is a schematic diagram of the membrane structure.

[0018] FIG2 shows a method developed by Oxford Nanopore Technologies to concentrate tethered complexes in an area of ​​an amphiphilic membrane. FIG2 shows a top view of an amphiphilic membrane and FIG2 shows a cross-sectional view of the amphiphilic membrane. ...

[0019] FIG3 is a schematic diagram showing a method of using a blocker complex to concentrate the test library within the capture range of a nanopore according to the present invention.

[0020] FIG4 is a schematic diagram showing a method of using a blocker to concentrate the test library within the capture range of a nanopore according to the present invention.

[0021] FIG5 shows a schematic diagram of the construction of the blocker complex in Example 1 of the present invention.

[0022] FIG6 shows the electrophoresis diagram of the blocker annealing (Figure A) and the electrophoresis diagram of the blocker complex (Figure B) in Example 1 of the present invention.

[0023] FIG7 shows a schematic diagram of the construction of the sequencing adapter complex in Example 1 of the present invention.

[0024] FIG8 shows the electrophoresis diagram of sequencing adapter annealing (Figure A) and the electrophoresis diagram of sequencing adapter complex (Figure B) in Example 1 of the present invention.

[0025] FIG9 shows the through-hole current signal diagrams of three closed sub-complexes in Example 1 of the present invention, where (A), (B), and (C) correspond to closed-10iSpC3, closed-20iSpC3, and closed-30iSpC3 closed sub-complexes, respectively.

[0026] FIG10 shows a current signal diagram of sequencing the test library-1 using the blocking-30iSpC3 blocking subcomplex and the constraint sequence-1 in Example 1 of the present invention.

[0027] FIG11 shows a schematic diagram of the construction of the blocker in Example 2 of the present invention.

[0028] FIG12 shows the electrophoresis diagram of the blocker in Example 2 of the present invention.

[0029] FIG13 shows a schematic diagram of the construction of the sequencing adapter complex in Example 2 of the present invention.

[0030] FIG14 shows the electrophoresis diagram of sequencing adapter annealing (Figure A) and the electrophoresis diagram of sequencing adapter complex (Figure B) in Example 2 of the present invention.

[0031] FIG15 shows a diagram of via current signals of three blockers in Example 2 of the present invention, where (A), (B), and (C) correspond to blockers 10, 20, and 30, respectively.

[0032] FIG. 16 shows a current signal diagram of sequencing the test library-2 using the blocker-20 blocker and the constraint sequence-2 in Example 2 of the present invention.

[0033] Figure 17 shows a schematic diagram of the experimental design of the experimental group (using closure-20 and restraint sequence-2), control group 1 (using restraint sequence-1), and control group 2 (using restraint sequence-2) in Example 3 of the present invention, (A), (B), and (C) correspond to the experimental group, control group 1, and control group 2, respectively.

[0034] FIG18 shows the read capture results of sequencing 50 ng of test library-2 by the experimental group, control group 1, and control group 2 in two replicates in Example 3 of the present invention, (A) and (B) correspond to the two replicates, respectively.

[0035] FIG19 shows the read capture results of sequencing 5 ng of test library-2 by the experimental group, control group 1, and control group 2 in two replicates in Example 3 of the present invention, (A) and (B) correspond to the two replicates, respectively. DETAILED DESCRIPTION

[0036] Hereinafter, the present invention will be described in detail with reference to the accompanying drawings. It should be understood that the following description is merely illustrative of the present invention and is not intended to limit the scope of the present invention. The scope of protection of the present invention shall be subject to the appended claims. Furthermore, those skilled in the art will understand that the technical solutions of the present invention may be modified without departing from the spirit and purpose of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of the present invention belongs. Before describing the present invention in detail, the following definitions are provided for a better understanding of the present invention.

[0038] Where a numerical range is provided, such as a concentration range, a percentage range, or a ratio range, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of the range and any other stated or intervening values ​​in the stated range are encompassed within the subject matter unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and such embodiments are also encompassed within the subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed within the subject matter.

[0039] In the context of the present invention, many embodiments use the expressions "comprising", "including" or "consisting essentially / mainly of..." The expressions "comprising", "including" or "consisting essentially / mainly of..." can generally be understood as open-ended expressions, indicating that in addition to the elements, components, assemblies, method steps, etc. specifically listed after the expression, other elements, components, assemblies, method steps, etc. are also included. In addition, in this document, the expressions "comprising", "including" or "consisting essentially / mainly of..." can also be understood as closed-ended expressions in some cases, indicating that only the elements, components, assemblies, method steps specifically listed after the expression are included, and no other elements, components, assemblies, method steps are included. In this case, the expression is equivalent to the expression "consisting of..."

[0040] For a better understanding of the present teachings and without limiting the scope of the present teachings, all numbers and other numerical values ​​expressing quantities, percentages or ratios used in the specification and claims should be understood as being modified in all cases by the term "about", unless otherwise indicated. Therefore, unless indicated to the contrary, the numerical parameters set forth in the following specification and the appended claims are approximate values ​​that may vary depending on the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0041] As used herein, the term "nucleotide" refers to ribonucleotides, deoxynucleotides, or modified forms of either type of nucleotides, and combinations thereof. Nucleotides are the basic structural units of nucleic acids.

[0042] As used herein, the term "nucleic acid" refers to a single-stranded or double-stranded covalently linked nucleotide sequence in which the 3' and 5' ends of each nucleotide are connected by a phosphodiester bond. The nucleic acid can be composed of deoxyribonucleotide bases or ribonucleotide bases. The nucleic acid can include DNA and RNA and can be synthesized in vitro or isolated from natural resources. The nucleic acid can further include modified DNA or RNA, such as methylated DNA or RNA, or RNA modified after translation, such as 5'-capping with 7-methylguanosine, 3'-end processing, such as cleavage and polyadenylation, and splicing. The nucleic acid can also include synthetic nucleic acids (XNA), such as hexitol nucleic acids (HNA), cyclohexene nucleic acids (CeNA), threose nucleic acids (TNA), glycerol nucleic acids (GNA), bridge nucleic acids (BNA), locked nucleic acids (LNA) and peptide nucleic acids (PNA). The length of the nucleic acid is usually expressed in base pairs (bp) for double-stranded polynucleotides, or in the case of single-stranded polynucleotides, in terms of the number of nucleotides (nt).

[0043] As used herein, the term "nanopore" refers to a structure having a nanoscale passageway through which an ionic current can flow. The inner diameter of a nanopore can vary greatly depending on the intended use of the device.

[0044] As used herein, the term "nucleic acid control protein" refers to a class of proteins that can bind to single-stranded nucleotides in a nucleotide chain, enabling polymerization, unwinding, or ligation of the nucleotide chain. For example, a nucleic acid control protein can be a helicase, topoisomerase, ligase, or polymerase. Typically, a nucleic acid control protein can bind to the nucleic acid control protein binding sequence of a blocker or to the adapter sequence of a sequencing library.

[0045] In this article, the term "limiting sequence" refers to a class of compound molecules that can prevent nucleic acid control proteins from performing functions such as polymerization, unwinding or connection of nucleotide chains during nanopore sequencing. For example, when the blocker complex is not captured by the nanopore, the helicase cannot unwind the complementary sequence on the blocker due to the presence of the limiting sequence; when the blocker complex is captured by the nanopore, under the action of the electric field force, the helicase moves and passes through the limiting sequence, opening the unwinding of the complementary sequence on the blocker. The molecular structure of the limiting sequence is not particularly limited. For example, the limiting sequence can be iSp18, iSp9, iSpC3, iSpC6, iSpC12 or a modified oligonucleotide. Limiting sequences can usually be purchased through commercial channels, for example, through Sangon Biotech (Shanghai) Co., Ltd. (https: / / www.sangon.com / conventional_primer_synthesis.html).

[0046] As used herein, the term "constraint sequence" refers to a polymer sequence, such as a nucleic acid sequence, with a hydrophobic molecule modified at one end. The constrained sequence is bound to the membrane at one end by the hydrophobic molecule, and the other end is complementary to the sequencing adapter, thereby pulling the library connected to the sequencing adapter near the membrane. The length of the constrained sequence is not particularly limited and can be, for example, 10-50 nucleotides. Part of the sequence in the constrained sequence can bind to the fourth sequence of the blocker.

[0047] Herein, the term "guide sequence" refers to the first sequence in the first strand (i.e., top strand) of the blocker, which is used to capture the blocker by the nanopore, thereby allowing the first strand (i.e., top strand) of the blocker to pass through the nanopore.

[0048] In this article, the term "exposed" refers to the state of the third sequence of the blocker's second strand during the sequencing process. Before the blocker is perforated, the third sequence of the blocker's second strand is in a complementary binding state with the second sequence of the first strand. After the blocker's first strand is captured and passes through the nanopore, the third sequence of its second strand is "exposed" and becomes single-stranded, capable of binding to the sequencing adapter.

[0049] In this document, relational terms such as first and second, etc. are used merely to distinguish one entity or operation from another entity or operation, but do not necessarily require or imply any actual relationship or order between these entities or operations.

[0050] As mentioned above, the present invention aims to provide a method for concentrating the binding of a library to be tested within the capture range of a nanopore.

[0051] Therefore, in a first aspect, the present invention provides a nanopore sequencing method, comprising the following steps:

[0052] 1) incubating a blocker with a membrane embedded with a nanopore so that the blocker is bound to the membrane; the blocker comprises a first strand (also referred to as a top strand), a second strand (also referred to as a bottom strand), and a constraining sequence, wherein the constraining sequence comprises a portion that binds to the membrane, the first strand comprises a first sequence (also referred to as a guide sequence), a second sequence that is at least partially complementary to the second strand, the second strand comprises a third sequence that is at least partially complementary to the first strand, and a fourth sequence that is at least partially complementary to the constraining sequence; wherein the third sequence is used to bind to a sequencing library by being complementary to a sequencing adapter;

[0053] 2) applying an electric field force to the membrane in step 1), causing the first chain of the blocker located within the capture range of the nanopore to pass through the nanopore, thereby exposing the third sequence in the second chain of the blocker;

[0054] 3) The third sequence is bound to the sequencing library via a sequencing adapter, so that the sequencing library is bound to the capture range of the nanopore and then captured by the nanopore to sequence the strand to be sequenced connected to the sequencing adapter.

[0055] In the present invention, the sequencing library is captured within the nanopore capture range via complementary binding of a sequencing adapter sequence to a blocker sequence, and then sequenced after capture by the nanopore. Specifically, in one embodiment of the present invention, a DNA ligase is used to connect the strand to be sequenced to a sequencing adapter complex to construct a sequencing library. The sequencing adapter complex comprises a sequencing adapter and a second nucleic acid control protein bound to the sequencing adapter. The structure of the sequencing adapter is similar to that of the blocker, comprising a top strand and a bottom strand. The top strand comprises a guide sequence, a nucleic acid control protein binding sequence, a limiter sequence, and a sequence complementary to the bottom strand. The bottom strand comprises a sequence complementary to the top strand. Furthermore, a portion of the top or bottom strand can complementarily bind to a third sequence in the blocker.

[0056] In one embodiment, the portion of the restraining sequence that binds to the membrane is a hydrophobic molecule. In a preferred embodiment, the hydrophobic molecule is selected from any one or more of the following: lipids, fatty acids, sterols, carbon nanotubes, polypeptides, proteins, and / or amino acids. In a further preferred embodiment, the hydrophobic molecule is selected from any one or more of the following: cholesterol, palmitate, or tocopherol.

[0057] In one embodiment, the first sequence consists of 10-50 nucleotides, iSpC3, iSp18, or a combination thereof.

[0058] In one embodiment, the first strand further comprises a nucleic acid control protein binding sequence, and in step 1), a first nucleic acid control protein is added such that the first nucleic acid control protein binds to the nucleic acid control protein binding sequence. The first nucleic acid control protein is selected from any one of the following: a helicase, a topoisomerase, a ligase, and a polymerase. The first nucleic acid control protein acts in a 3'-5' direction or a 5'-3' direction.

[0059] In a further embodiment, the first strand further comprises a restriction sequence, the restriction sequence being located between the second sequence and the nucleic acid control protein binding sequence. The restriction sequence is composed of at least one of iSp18, iSp9, iSpC3, iSpC6, and iSpC12. The restriction sequence is at least 1 bp in length.

[0060] In one embodiment, the length of the second sequence, the third sequence, and the fourth sequence is at least 2 bp, respectively. In a preferred embodiment, the length of the second sequence, the third sequence, and the fourth sequence is 5-30 bp, respectively.

[0061] In one embodiment, the complementary length of the third sequence to the sequencing adapter of the sequencing library is at least 2 bp.

[0062] In one embodiment, the blocker is composed of DNA, RNA, LNA, PNA, BNA, or any combination thereof.

[0063] In one embodiment, the nanopore is a transmembrane protein pore or a solid-state pore. In a preferred embodiment, the transmembrane protein pore is selected from hemolysin, MspA, MspB, MspC, MspD, FraC, ClyA, PA63, CsgG, CsgD, XcpQ, SP1, phi29 connector protein (phi29 connector), InvG, GspD, or any combination thereof.

[0064] In one embodiment, the membrane is an amphiphilic membrane, a high molecular polymer membrane, or any combination thereof.

[0065] In one embodiment, the voltage during the sequencing process is above 10 mV. In a preferred embodiment, the voltage is 50 mV-250 mV.

[0066] In one embodiment, the sequencing library is added to the reaction system together with the blocker, added to the reaction system before the blocker, or added to the reaction system after the blocker.

[0067] In one embodiment, the sequencing adapter is composed of two nucleic acid chains, one end of which is complementary to the other end, and the binding mode of the blocker to the sequencing adapter includes:

[0068] 1) The sequencing adapter has a Y-shaped structure, with a non-complementary single-stranded portion at one end and a complementary double-stranded portion at the other end. The third sequence of the blocker is complementary to the single-stranded portion of the sequencing adapter (as shown in Figures 3B and 3C);

[0069] 2) One end of the sequencing adapter is a non-complementary single-stranded portion, and the other end is a complementary double-stranded portion, and the double-stranded portion has an RNA fragment. After the RNA fragment is degraded by RNase, the third sequence of the blocker complementarily binds to the complementary sequence of the RNA fragment (as shown in Figures 3A and 3D).

[0070] In one embodiment, the sequencing adapter is combined with a second nucleic acid control protein to form a sequencing adapter complex, and the second nucleic acid control protein is selected from any one of the following: a helicase, a topoisomerase, a ligase, and a polymerase. In a preferred embodiment, the second nucleic acid control protein is a helicase.

[0071] In one embodiment, in step 3), the third sequence of the blocker is complementary to the sequencing adapter of the sequencing library, thereby achieving connection between the blocker and the sequencing library; under the action of the electric field force, the chain to be sequenced of the sequencing library passes through the nanopore, and the change in the current signal generated by the perforation is detected, thereby obtaining the sequence information of the chain to be sequenced.

[0072] In a second aspect, the present invention provides a nanopore sequencing kit, comprising a blocker, a sequencing buffer, a sequencing adapter, and a second nucleic acid control protein, the blocker comprising a first strand, a second strand, and a restraining sequence, the restraining sequence comprising a portion that binds to a membrane being sequenced, the first strand comprising a first sequence and a second sequence at least partially complementary to the second strand, the second strand comprising a third sequence at least partially complementary to the first strand and a fourth sequence at least partially complementary to the restraining sequence;

[0073] The sequencing adapter and the second nucleic acid control protein form a sequencing adapter complex, which is used to capture the chain to be sequenced, thereby forming a sequencing library. The third sequence is used to bind complementary to the sequencing adapter, so that the sequencing library is bound to the nanopore capture range and then captured by the nanopore to achieve sequencing.

[0074] In one embodiment, the portion of the restraining sequence that binds to the membrane is a hydrophobic molecule. In a preferred embodiment, the hydrophobic molecule is selected from any one or more of the following: lipids, fatty acids, sterols, carbon nanotubes, polypeptides, proteins, and / or amino acids. In a further preferred embodiment, the hydrophobic molecule is selected from any one or more of the following: cholesterol, palmitate, or tocopherol.

[0075] In one embodiment, the first sequence consists of 10-50 nucleotides, iSpC3, iSp18, or a combination thereof.

[0076] In one embodiment, the first strand further comprises a nucleic acid control protein binding sequence, and the kit further comprises a first nucleic acid control protein, such that the first nucleic acid control protein binds to the nucleic acid control protein binding sequence. The first nucleic acid control protein is selected from any one of the following: a helicase, a topoisomerase, a ligase, and a polymerase. In one embodiment, the first nucleic acid control protein acts in a 3'-5' direction or a 5'-3' direction.

[0077] In one embodiment, the first strand further comprises a restriction sequence, the restriction sequence being located between the second sequence and the nucleic acid control protein binding sequence. The restriction sequence is composed of at least one of iSp18, iSp9, iSpC3, iSpC6, and iSpC12. The restriction sequence is at least 1 bp in length.

[0078] In one embodiment, the length of the second sequence, the third sequence, and the fourth sequence is at least 2 bp, respectively. In a preferred embodiment, the length of the second sequence, the third sequence, and the fourth sequence is 5-30 bp, respectively.

[0079] In one embodiment, the complementary length of the third sequence to the sequencing adapter of the sequencing library is at least 2 bp.

[0080] In one embodiment, the blocker is composed of DNA, RNA, LNA, PNA, BNA, or any combination thereof.

[0081] Figures 3 and 4 respectively illustrate schematic diagrams of the present invention's method for using a blocker complex (i.e., blocker + nucleic acid control protein) and a blocker to centrally bind a test library within the nanopore capture area. The method is explained in detail below:

[0082] Figure 3 shows the process of nanopore sequencing using blocker complexes in the following four cases: A: blocker complex 1 composed of 5'→3' helicase + 5'→3' constraint sequence + library bound to 5'→3' helicase; B: blocker complex 1 composed of 5'→3' helicase + 5'→3' constraint sequence + library bound to 3'→5' helicase; C: blocker complex 2 composed of 3'→5' helicase + 3'→5' constraint sequence + library bound to 5'→3' helicase; D: blocker complex 2 composed of 3'→5' helicase + 3'→5' constraint sequence + library bound to 3'→5' helicase. Taking Figure A as an example to illustrate the specific process: Step (1): Prepare a blocker complex, wherein the blocker complex includes a top chain, a bottom chain, a constraint sequence and a helicase. The top chain is composed of a guide sequence (represented by a gray dotted line), a helicase binding sequence (represented by a black solid line), a limiter sequence (represented by a gray dotted line) and a sequence complementary to the bottom chain (represented by a black solid line) from 5' to 3'. The bottom chain is composed of a sequence complementary to the top chain and a sequence complementary to the constraint sequence (both represented by black solid lines). The successfully annealed blocker is incubated with the helicase (light gray circle with a gap, the unwinding direction is from 5' to 3') and purified into a blocker complex; Step (2): After incubation, under the action of the constraint sequence, the blocker complex is bound to the insulating impermeable membrane; Step (3): After applying an electric field force, the blocker complex bound to the capture range of the nanopore is captured, the top chain of the blocker complex passes through the nanopore, and the bottom chain is still bound to the constraint sequence and exposes the sequence bound to the sequencing library (indicated by the arrow). At the same time, since the top chain of the blocker is relatively short, the time it takes for a single blocker complex to pass through the nanopore is on the order of 100 milliseconds, which will not affect the capture of the sequencing library by the nanopore; Step (4): Add the sequencing library, and the sequencing library can only bind to the bottom chain of the blocker within the capture range of the nanopore, exposing the sequence bound to the sequencing library (indicated by the arrow), so that the library to be tested is concentratedly bound within the capture range of the nanopore, thereby ensuring that the library is efficiently captured by the nanopore during the sequencing process and improving the utilization efficiency of the library.

[0083] Figure 4 shows the process of nanopore sequencing using blockers in the following four cases: A: blocker 1 + 5'→3' constraint sequence + library bound to 5'→3' helicase; B: blocker 1 + 5'→3' constraint sequence + library bound to 3'→5' helicase; C: blocker 2 + 3'→5' constraint sequence + library bound to 5'→3' helicase; D: blocker 2 + 3'→5' constraint sequence + library bound to 3'→5' helicase. Take Figure C as an example to illustrate the specific process: Step (1): Prepare a blocker, wherein the blocker includes a top chain, a bottom chain, and a constraint sequence, wherein the top chain is composed of a sequence complementary to the bottom chain (indicated by a black solid line) and a guide sequence (indicated by a gray dotted line) from 5' to 3', and a sequence complementary to the constraint sequence and a sequence complementary to the top chain (both represented by black solid lines) from 5' to 3'; Step (2): After incubation, under the action of the constraint sequence, the blocker is bound to the insulating impermeable membrane; Step (3): After applying an electric field force, the blocker bound to the capture range of the nanopore is captured, the top chain of the blocker passes through the nanopore, and the bottom chain is still bound to the constraint sequence and exposes the sequence bound to the sequencing library (indicated by the arrow). At the same time, since the top chain of the blocker is relatively short, the time it takes for a single blocker to pass through the nanopore is on the order of milliseconds, which will not affect the capture of the sequencing library by the nanopore; Step (4): Add the sequencing library, and the sequencing library can only bind to the bottom chain of the blocker within the capture range of the nanopore, exposing the sequence bound to the sequencing library (indicated by the arrow), so that the library to be tested is concentratedly bound within the capture range of the nanopore, thereby ensuring that the library is efficiently captured by the nanopore during the sequencing process and improving the utilization efficiency of the library.

[0084] The above sequencing process uses the necessary equipment and reagents for sequencing, such as a nanopore sequencer and sequencing buffer. The membrane of the nanopore sequencer is a phospholipid bilayer, a diblock copolymer, or a triblock copolymer;

[0085] The pH buffer system contained in the sequencing buffer 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;

[0086] The sequencing buffer contains NTP, dNTP, ddNTP, or any combination thereof;

[0087] The sequencing buffer contains K + 、Na 2+ , or any combination thereof;

[0088] The sequencing buffer contains Mg 2+ 、Mo 2+ 、Cu 2+ 、Fe 2+ 、Zn2+ , Ca 2+ , Pb 2+ 、Cd 2+ , or any combination thereof.

[0089] Example

[0090] It should be noted that the terms used in the description of the present invention are intended only to describe specific embodiments and are not intended to limit the present invention. The above summary of the invention and the detailed description below are intended only to illustrate the present invention and are not intended to limit the present invention in any way. Without departing from the spirit and purpose of the present invention, the scope of the present invention is determined by the appended claims.

[0091] Example 1

[0092] This embodiment uses the blocker complex shown in FIG3A of the present invention to implement nanopore sequencing, specifically comprising the following steps:

[0093] 1. Preparation of Blocker Complex

[0094] The sequences 5'-XXXXXXXXXXTTTTTTTTTTYYYYGGTTGTTTCTGTTGG-3'(X=iSpC3,Y=iSp18)(SEQ ID NO:1), 5'-XXXXXXXXXXXXXXXXXXXXTTTTTTTTTTYYYYGGTTGTTTCTGTTGG-3'(X=iSpC3,Y=iSp18)(SEQ ID NO:2), 5'-XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXTTTTTTTTTTYYYYGGTTGTTTCTGTTGG-3'(X=iSpC3,Y=iSp18)(SEQ ID NO:3), and 5'-CCAACAGAAACAACCTTTGAGGCGAGCGGTCAA-3'(SEQ ID NO:4) were dissolved in TE buffer (pH=8), respectively.

[0095] Blockers were prepared by annealing the top strands SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 with the bottom strand SEQ ID NO: 4 at a 1:1 ratio. The resulting blockers were named Block-10iSP3, Block-20iSP3, and Block-30iSP3, respectively. The annealing process consisted of incubation at 95°C for 5 minutes, followed by a cooling rate of 0.1°C / s to 25°C, and continued incubation for 30 minutes. The top and bottom chain structures of the blocker are shown in Figure 5: the top chain (A) consists of, from 5' to 3', a guide sequence (10 iSpC3, 20 iSpC3, 30 iSpC3, represented by gray dotted lines), a helicase binding sequence (10 Ts, represented by black solid lines), a limiter sequence (4 iSp18s, represented by gray dotted lines), and a sequence complementary to the bottom chain (represented by black solid lines); the bottom chain (B) consists of, from 5' to 3', a sequence complementary to the top chain and a sequence complementary to the constraint sequence-1 (5'-Cholesterol-YYYYTTGACCGCTCGCCTC-3' (Y = iSp18), SEQ ID NO: 5)) (all represented by black solid lines). After annealing, electrophoresis detection was performed. The results are shown in Figure 6(A), showing successfully annealed blockers. Lanes 1-3 are top strands (A-10iSpC3, A-20iSpC3, A-30iSpC3, SEQ ID NOs: 1-3), lane 4 is the bottom strand (B, SEQ ID NO: 4), lanes 5-7 are annealed blocked-10iSpC3, blocked-20iSpC3, and blocked-30iSpC3, and lane 8 is a DNA molecular weight standard (M).

[0096] The prokaryotic expression of the helicase He (T4Dda-(ΔM1)G1 / E94C / C109A / C136A / K194L / A360C, SEQ ID NO: 6) was completed in Escherichia coli, and the target protein was obtained after multiple purification steps. The amino acid sequence of the helicase is

[0097] The blocker and the helicase (the light gray notched circle in Figure 5, with the unwinding direction from 5' to 3') were incubated to form a complex. Specifically, the helicase He was mixed with the blocked-10iSpC3, blocked-20iSpC3, and blocked-30iSpC3 blockers at a molar ratio of 9:1, respectively. The final concentration of the reaction buffer was 25mM HEPES, 50mM KCl, 0.5mM EDTA, 2.5mM MgCl2, pH = 8.0, and incubated at room temperature for 30 minutes.

[0098] 0.25 times the volume of 5 mM ATP was added to the incubation product and incubated at room temperature for 30 minutes. Due to the effect of the restriction sequence, only one helicase He bound between the guide sequence and the restriction sequence was retained.

[0099] The blocker complex was purified using AMPure XP beads (Beckman Coulter, A63882) to obtain the blocker complex. The purified blocker complex was detected by electrophoresis. The results are shown in Figure 6(B), showing a large amount of blocker complexes with a 1:1 blocker to helicase ratio. Lane 1 represents a DNA molecular weight standard (M), lanes 2-4 represent the complexes of blocker-10iSpC3 and helicase incubated with blocker-10iSpC3 before and after purification, lanes 5-7 represent the complexes of blocker-20iSpC3 and helicase incubated with blocker-20iSpC3 before and after purification, and lanes 8-10 represent the complexes of blocker-30iSpC3 and helicase incubated with blocker-30iSpC3 before and after purification.

[0100] 2. Prepare Sequencing Library

[0101] The sequences 5'-XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXTTTTTTTTTTYYYYGGTTGTTTCTGTTGGTGCTGATATTGCT-3' (X=iSpC3, Y=iSp18) (SEQ ID NO: 7), 5'-Phosphorylation-GCAATATCA-3' (SEQ ID NO: 8), and 5'-CCAACAGAAACAACC-3' (SEQ ID NO: 9, RNA) were dissolved in TE buffer (pH=8) according to the manufacturer's instructions.

[0102] Sequencing adapters were prepared by annealing SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9 at a ratio of 1:1:1. The annealing process was incubation at 95°C for 5 minutes, followed by a cooling rate of 0.1°C / s to 25°C, and continued incubation for 30 minutes. SEQ ID NO:7 and SEQ ID NO:8 were annealed at a ratio of 1:1, and SEQ ID NO:7 and SEQ ID NO:9 were annealed at a ratio of 1:1 as a control. The structure of the sequencing adapter is shown in Figure 7: From 5' to 3' on the top strand, the guide sequence (30 iSpC3s, represented by a gray dashed line), the helicase binding sequence (10 Ts, represented by a black solid line), the limiter sequence (4 iSp18s, represented by a gray dashed line), and the sequence complementary to the bottom strand (represented by a black solid line); the bottom strand-DNA is the sequence complementary to the top strand (represented by a black solid line); the bottom strand-RNA is adjacent to the bottom strand-DNA and complementary to the top strand (represented by a gray solid line).

[0103] After annealing, electrophoresis detection was performed, and the results are shown in Figure 8(A), indicating that SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 were successfully annealed at a ratio of 1:1:1 to obtain sequencing adapters. Lane 1 is a sequencing adapter in which the top strand (SEQ ID NO: 7), bottom strand-DNA (SEQ ID NO: 8), and bottom strand-RNA (SEQ ID NO: 9) were annealed at a ratio of 1:1:1, lane 2 is a control in which the top strand and bottom strand-DNA were annealed at a ratio of 1:1, lane 3 is a control in which the top strand and bottom strand-RNA were annealed at a ratio of 1:1, lane 4 is the top strand, lane 5 is the bottom strand-DNA, and lane 6 is the bottom strand-RNA.

[0104] The prokaryotic expression of helicase He (T4Dda-(ΔM1)G1 / E94C / C109A / C136A / K194L / A360C, SEQ ID NO: 6) was completed in Escherichia coli, and the target protein was obtained through multiple purification steps.

[0105] Helicase He and sequencing adapter were mixed at a molar ratio of 9:1. The final concentration of the reaction buffer was 25 mM HEPES, 50 mM KCl, 0.5 mM EDTA, 2.5 mM MgCl2, pH = 8.0, and incubated at room temperature for 30 minutes.

[0106] 0.25 times the volume of 5 mM ATP was added to the incubation product and incubated at room temperature for 30 minutes. Due to the effect of the restriction sequence, only one helicase He bound between the guide sequence and the restriction sequence was retained.

[0107] The sequencing adapter complex was purified using AMPure XP beads (Beckman Coulter, A63882) to obtain a sequencing adapter complex (Figure 7, the helicase is a light gray notched circle, and the unwinding direction is 5' to 3'). The purified sequencing adapter complex was detected by electrophoresis. The results are shown in Figure 8(B), showing a large amount of sequencing adapter complexes with a 1:1 adapter to helicase ratio. Lanes 1-3 show the incubation of the adapter, helicase, and adapter complex before and after purification, respectively.

[0108] The sequencing adapter complex was ligated to the randomly sheared Bacillus subtilis genomic DNA fragment using T4 DNA Ligase (NEB, M0202T). The RNA on the sequencing adapter was then degraded using RNase H (NEB, M0297L). Finally, the RNA was purified using AMPure XP beads to obtain the test library-1.

[0109] 3. Blocker Complex Nanopore Sequencing

[0110] According to 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 single-channel nanopore detection system was built based on patch clamp and signal amplifier to complete the single porin pore.

[0111] The blocked-10iSpC3, blocked-20iSpC3, and blocked-30iSpC3 blocker complexes obtained in step 1, "Preparing Blocker Complexes," were added to the system along with the restraining sequence-1. The blocker's pore-passing current signals were observed at 180 mV. The pore-passing time for a single blocker complex was on the order of 100 milliseconds. The buffer solution consisted of 470 mM KCl, 25 mM HEPES, 10 mM MgCl2, 30 mM ATP, pH 8.10, and the temperature was 30°C. Figure 9 shows the pore-passing current signals for the three blocker complexes.

[0112] 4. Nanopore sequencing of the test library using the blocker complex and the restraining sequence

[0113] According to 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 single-channel nanopore detection system was built based on patch clamp and signal amplifier to complete the single porin pore.

[0114] The closed-30iSpC3 closed subcomplex obtained in step 1 "Preparation of closed subcomplex", the restraining sequence-1, and the library to be tested-1 were mixed and added to the single-pore system. Sequencing was performed under 180 mV conditions, and the nanopore sequencing current signal diagram of the library was observed and obtained (Figure 10), indicating that nanopore sequencing can be performed using the closed subcomplex and the restraining sequence scheme.

[0115] Example 2

[0116] This embodiment uses the blocker shown in FIG4C of the present invention to implement nanopore sequencing, specifically comprising the following steps:

[0117] 1. Preparation of blockers

[0118] According to the manufacturer's instructions, the sequences 5'-GAGGCGAGCGGTCAATTTTTTTTTTXXXXXXXXXX-3' (X = iSpC3) (SEQ ID NO: 10), 5'-GAGGCGAGCGGTCAATTTTTTTTTTXXXXXXXXXXXXXXXXXXXXXXX-3' (X = iSpC3) (SEQ ID NO: 11), 5'-GAGGCGAGCGGTCAATTTTTTTTTTXXXXXXXXXXXXXXXXXXXXXXXXXXX-3' (X = iSpC3) (SEQ ID NO: 12), and 5'-AATGCCTAAGTACTGAAATTGACCGCTCGCCTC-3' (SEQ ID NO: 13) were dissolved in TE buffer (pH = 8).

[0119] Blockers were prepared by annealing top strands SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12 with bottom strand SEQ ID NO:13 at a 1:1 ratio and named Blocker-10, Blocker-20, and Blocker-30, respectively. The annealing process consisted of incubation at 95°C for 5 minutes, followed by a cooling rate of 0.1°C / s to 25°C, and continued incubation for 30 minutes. The top and bottom strand structures of the blockers are shown in Figure 11: The top strand (A) consists, from 5' to 3', of a sequence complementary to the bottom strand (indicated by a black solid line) and a guide sequence (indicated by a gray dashed line); the bottom strand (B) consists, from 5' to 3', of a sequence complementary to the constraint sequence-2 (5'-CAGTACTTAGGCATTYYYY-Cholesterol 3' (Y = iSp18), SEQ ID NO:14) and a sequence complementary to the top strand (both indicated by black solid lines). After annealing, electrophoresis detection was performed, and the results are shown in Figure 12, where successfully annealed blockers were obtained. Lanes 1-3 are top chains A-10, A-20, and A-30 (containing 10 iSpC3, 20 iSpC3, and 30 iSpC3, respectively), lane 4 is bottom chain B, lanes 5-7 are block-10, block-20, and block-30 after annealing, and lane 8 is a DNA molecular weight standard (M).

[0120] 2. Prepare Sequencing Library

[0121] The sequences 5'-XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXTTTTTTTTTTYYYYGGTTGTTTCTGTTGGTGCTGATATTGCT-3' (X=iSpC3, Y=iSp18) (SEQ ID NO: 7) and 5'-Phosphorylation-GCAATATCAGCACCAACAGAAACAACCTTTGAGGCGAGCGGTCAA-3' (SEQ ID NO: 15) were dissolved in TE buffer (pH=8) according to the manufacturer's instructions.

[0122] SEQ ID NO: 7 and SEQ ID NO: 15 were annealed in a ratio of 1: 1 to prepare sequencing adapters. The annealing process was incubated at 95 ° C for 5 minutes, cooled to 25 ° C at a rate of 0.1 ° C / s, and continued to incubate for 30 minutes. The structure of the sequencing adapter is shown in Figure 13: the top chain from 5' to 3' is the guide sequence (30 iSpC3, represented by gray dotted lines), the helicase binding sequence (10 T, represented by black solid lines), the limiter sequence (4 iSp18, represented by gray dotted lines), and the bottom chain complementary sequence (represented by black solid lines). The bottom chain from 5' to 3' is the complementary sequence to the top chain and the complementary sequence to the blocker (both represented by black solid lines). After the annealing is completed, electrophoresis detection is performed. The results are shown in Figure 14 (A), and a successfully annealed sequencing adapter is obtained, wherein lane 1 is the top chain (SEQ ID NO: 7), lane 2 is the bottom chain (SEQ ID NO: 15), and lane 3 is the sequencing adapter after annealing.

[0123] The prokaryotic expression of helicase He (T4Dda-(ΔM1)G1 / E94C / C109A / C136A / K194L / A360C, SEQ ID NO: 6) was completed in Escherichia coli, and the target protein was obtained through multiple purification steps.

[0124] Helicase He and sequencing adapter were mixed at a molar ratio of 9:1. The final concentration of the reaction buffer was 25 mM HEPES, 50 mM KCl, 0.5 mM EDTA, 2.5 mM MgCl2, pH = 8.0, and incubated at room temperature for 30 minutes.

[0125] 0.25 times the volume of 5 mM ATP was added to the incubation product and incubated at room temperature for 30 minutes. Due to the effect of the restriction sequence, only one helicase He bound between the guide sequence and the restriction sequence was retained.

[0126] The sequencing adapter complex was purified using AMPure XP beads (Beckman Coulter, A63882) to obtain a sequencing adapter complex (Figure 13; the helicase is a light gray, notched circle with a 5' to 3' unwinding direction). The purified sequencing adapter complex was examined by electrophoresis. The results are shown in Figure 14(B), showing a large amount of sequencing adapter complexes with a 1:1 adapter to helicase ratio. Lanes 1 and 2 show the helicase and sequencing adapter complexes before and after purification, respectively.

[0127] The sequencing adapter complex was ligated to the randomly sheared Bacillus subtilis genomic DNA fragment using T4 DNA Ligase (NEB, M0202T) and purified using AMPure XP beads to obtain the test library-2.

[0128] 3. Closer Nanopore Sequencing

[0129] According to 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 single-channel nanopore detection system was built based on patch clamp and signal amplifier to complete the single porin pore.

[0130] Blocker-10, Blocker-20, and Blocker-30, obtained in step 1 ("Preparing Blockers"), were added to the system along with constraint sequence-2 (SEQ ID NO: 14). The pore current signals of the blockers were observed at 180 mV, with the pore duration of a single blocker measured in milliseconds. The buffer solution consisted of 470 mM KCl, 25 mM HEPES, 10 mM MgCl2, and 30 mM ATP, pH 8.10, and the temperature was 30°C. Figure 15 shows the pore current signals of the three blockers.

[0131] 4. Nanopore sequencing of the test library using blockers and restrainer sequences

[0132] According to 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 single-channel nanopore detection system was built based on patch clamp and signal amplifier to complete the single porin pore.

[0133] Blocker-20, constraint sequence-2 (SEQ ID NO: 14), and test library-2 obtained in step 1, "Preparing the Blocker," were mixed and added to the single-pore system. Sequencing was performed at 180 mV, and the nanopore sequencing current signal of the library was observed and obtained ( FIG16 ), demonstrating that nanopore sequencing can be performed using the blocker and constraint sequence scheme.

[0134] Example 3

[0135] This example compares the read capture rates of nanopore sequencing using "blocked-20 and constrained sequence-2," "constrained sequence-1," and "constrained sequence-2," respectively. FIG17 shows a schematic diagram of the experimental design of this example.

[0136] According to 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 single-channel nanopore detection system was built based on patch clamp and signal amplifier to complete the single porin pore.

[0137] Experimental Group: Blocking-20 and Constrained Sequence-2 obtained in Example 2 were mixed, added to a single-well system, and sequenced at 180 mV for 4 hours. 50 ng of Test Library-2 was added and sequenced at 180 mV for 24 hours. After the addition of the sequencing library, the number of reads captured was counted every hour. The results showed that as the sequencing time increased, the number of sequencing reads captured per hour first increased and then gradually decreased.

[0138] Control group 1 (existing conventional technology): The constraint sequence-1 in Example 1 was added to the single-well system, sequenced at 180 mV for 4 hours, 50 ng of the test library-2 was added and sequenced at 180 mV for 24 hours. After the addition of the sequencing library, the reads captured were counted every hour. The results showed that: as the sequencing time increased, the sequencing reads captured per hour decreased significantly, and the rate of decrease was higher than that of the experimental group; the sequencing reads captured in the experimental group were lower than those in the control group 1 in the first 4 hours of sequencing, and then the sequencing reads captured in the experimental group were higher than those in the control group 1.

[0139] Control group 2: The constraint sequence-2 from Example 2 was added to the single-well system and sequenced at 180 mV for 4 hours. 50 ng of test library-2 was added and sequenced at 180 mV for 24 hours. The number of reads captured was counted every hour after the addition of the sequencing library. The results showed that only a few reads were captured during the sequencing process, and essentially no sequencing reads were captured 12 hours after sequencing.

[0140] The same experimental operation as above was repeated once, and the results were consistent ( FIG18 ).

[0141] The amount of test library 2 was reduced to 5 ng, and the above experimental procedures were repeated to complete sequencing of the experimental group, control group 1, and control group 2 (sequencing was performed 12 hours after adding the sequencing library). The experimental results were consistent with those of the sequencing library added with 50 ng. The same experimental procedures were repeated once, and the results were consistent (Figure 19).

[0142] The sequencing buffer in the above experiment was: 470 mM KCl, 25 mM HEPES, 10 mM MgCl2, 30 mM ATP, pH = 8.10; the temperature was 30°C.

[0143] Comparison of the changes in sequencing read capture over time in the experimental group, control group 1, and control group 2 showed that the use of blockers can concentrate the test library within the nanopore capture range, thereby ensuring that the library is captured by the nanopore during sequencing and improving the utilization efficiency of the library, indicating that the present invention has great application prospects.

[0144] The above describes in detail the method provided by the present invention for concentrating the test library within the capture range of the nanopore using a blocker. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above examples is only intended to help understand the method of the present invention and its core concept. At the same time, for those skilled in the art, based on the concept of the present invention, there may be changes in the specific implementation methods and application scopes. In summary, the contents of this specification should not be understood as limiting the present invention.

Claims

1. A nanopore sequencing method, characterized in that: The method comprises the following steps: 1) Incubating a blocker with a membrane embedded with a nanopore so that the blocker is bound to the membrane; the blocker comprises a first chain, a second chain and a restraining sequence, the restraining sequence comprises a portion that binds to the membrane, the first chain comprises a first sequence, a second sequence that is at least partially complementary to the second chain, the second chain comprises a third sequence that is at least partially complementary to the first chain, and a fourth sequence that is at least partially complementary to the restraining sequence; wherein the third sequence is used to bind to a sequencing library by being complementary to a sequencing adapter; 2) applying an electric field force to the membrane in step 1), so that the first chain of the blocker located within the capture range of the nanopore passes through the nanopore, thereby exposing the third sequence in the second chain of the blocker; 3) The third sequence is bound to the sequencing library via a sequencing adapter, so that the sequencing library is bound within the capture range of the nanopore and then captured by the nanopore to sequence the strand to be sequenced connected to the sequencing adapter.

2. The method according to claim 1, characterized in that The portion of the binding sequence that binds to the membrane is a hydrophobic molecule; preferably, the hydrophobic molecule is selected from any one or more of the following: lipids, fatty acids, sterols, carbon nanotubes, polypeptides, proteins and / or amino acids; more preferably, the hydrophobic molecule is selected from any one or more of the following: cholesterol, palmitate or tocopherol.

3. The method according to claim 1 or 2, characterized in that: The first sequence consists of 10-50 nucleotides, iSpC3, iSp18 or a combination thereof.

4. The method according to any one of claims 1 to 3, characterized in that The first chain further comprises a nucleic acid control protein binding sequence, and in step 1), a first nucleic acid control protein is added.

5. The method according to claim 4, characterized in that The first nucleic acid control protein is selected from any one of the following: helicase, topoisomerase, ligase, polymerase.

6. The method according to claim 4 or 5, characterized in that: The first nucleic acid controls the protein to act in a 3'-5' direction or a 5'-3' direction.

7. The method according to any one of claims 4 to 6, characterized in that: The first strand further comprises a restriction sequence, and the restriction sequence is located between the second sequence and the nucleic acid control protein binding sequence.

8. The method according to claim 7, characterized in that The restriction sequence is composed of at least one of iSp18, iSp9, iSpC3, iSpC6, and iSpC12.

9. The method according to claim 7 or 8, characterized in that: The length of the restriction sequence is at least 1 bp.

10. The method according to any one of claims 1 to 9, characterized in that The lengths of the second sequence, the third sequence and the fourth sequence are respectively at least 2 bp; preferably, the lengths of the second sequence, the third sequence and the fourth sequence are respectively 5-30 bp.

11. The method according to any one of claims 1 to 10, characterized in that The complementary length between the third sequence and the sequencing adapter of the sequencing library is at least 2 bp.

12. The method according to any one of claims 1 to 11, characterized in that The blocker is composed of DNA, RNA, LNA, PNA, BNA, or any combination thereof.

13. The method according to any one of claims 1 to 12, characterized in that The nanopore is a transmembrane protein pore or a solid-state pore; preferably, the transmembrane protein pore is selected from hemolysin, MspA, MspB, MspC, MspD, FraC, ClyA, PA63, CsgG, CsgD, XcpQ, SP1, phi29 connector protein, InvG, GspD, or any combination thereof.

14. The method according to any one of claims 1 to 13, characterized in that The membrane is an amphiphilic membrane, a high molecular polymer membrane, or any combination thereof.

15. The method according to any one of claims 1 to 14, characterized in that The voltage during the sequencing process is above 10 mV; preferably, the voltage is 50 mV-250 mV.

16. The method according to any one of claims 1 to 15, characterized in that The sequencing library is added to the reaction system together with the blocker, added to the reaction system before the blocker, or added to the reaction system after the blocker.

17. The method according to any one of claims 1 to 16, characterized in that The sequencing adapter is composed of two nucleic acid chains, one end of the two nucleic acid chains is complementary to the other end, and the combination mode of the blocker and the sequencing adapter includes: 1) The sequencing adapter is a Y-shaped structure, one end of which is a non-complementary single-stranded portion, and the other end is a complementary double-stranded portion, and the third sequence of the blocker is complementary to the single-stranded portion of the sequencing adapter; 2) One end of the sequencing adapter is a non-complementary single-stranded part, and the other end is a complementary double-stranded part, and the double-stranded part has an RNA fragment. After the RNA fragment is degraded by RNase, the third sequence of the blocker complementarily binds to the complementary sequence of the RNA fragment.

18. The method according to any one of claims 1 to 17, characterized in that The sequencing adapter combines with the second nucleic acid control protein to form a sequencing adapter complex; preferably, the second nucleic acid control protein is a helicase.

19. The method according to any one of claims 1 to 18, characterized in that In step 3), the third sequence of the blocker is complementary to the sequencing connector of the sequencing library, thereby achieving connection between the blocker and the sequencing library; under the action of the electric field force, the chain to be sequenced of the sequencing library passes through the nanopore, and the change in the current signal generated by the perforation is detected, thereby obtaining the sequence information of the chain to be sequenced.

20. A nanopore sequencing kit, characterized in that: The kit comprises a blocker, a sequencing buffer, a sequencing adapter and a second nucleic acid control protein, the blocker comprises a first strand, a second strand and a restraining sequence, the restraining sequence comprises a portion that binds to a membrane during sequencing, the first strand comprises a first sequence, a second sequence that is at least partially complementary to the second strand, the second strand comprises a third sequence that is at least partially complementary to the first strand, and a fourth sequence that is at least partially complementary to the restraining sequence; The sequencing adapter and the second nucleic acid control protein form a sequencing adapter complex, and the sequencing adapter complex is used to capture the chain to be sequenced, thereby forming a sequencing library. The third sequence is used to complementarily bind to the sequencing adapter so that the sequencing library is bound to the nanopore capture range and then captured by the nanopore to achieve sequencing.

21. The nanopore sequencing kit according to claim 20, characterized in that The portion of the binding sequence that binds to the membrane is a hydrophobic molecule; preferably, the hydrophobic molecule is selected from any one or more of the following: lipids, fatty acids, sterols, carbon nanotubes, polypeptides, proteins and / or amino acids; more preferably, the hydrophobic molecule is selected from any one or more of the following: cholesterol, palmitate or tocopherol.

22. The nanopore sequencing kit according to claim 20 or 21, characterized in that The first sequence consists of 10-50 nucleotides, iSpC3, iSp18 or a combination thereof.

23. The nanopore sequencing kit according to any one of claims 20 to 22, characterized in that: The first strand further comprises a nucleic acid control protein binding sequence, and the kit further comprises a first nucleic acid control protein.

24. The nanopore sequencing kit according to claim 23, characterized in that The first nucleic acid control protein is selected from any one of the following: helicase, topoisomerase, ligase, polymerase.

25. The nanopore sequencing kit according to claim 23 or 24, characterized in that The first nucleic acid controls the protein to act in a 3'-5' direction or a 5'-3' direction.

26. The nanopore sequencing kit according to any one of claims 20 to 25, characterized in that The first strand further comprises a restriction sequence, and the restriction sequence is located between the second sequence and the nucleic acid control protein binding sequence.

27. The nanopore sequencing kit according to claim 26, characterized in that The restriction sequence is composed of at least one of iSp18, iSp9, iSpC3, iSpC6, and iSpC12.

28. The nanopore sequencing kit according to claim 26 or 27, characterized in that The length of the restriction sequence is at least 1 bp.

29. The nanopore sequencing kit according to any one of claims 20 to 28, characterized in that The lengths of the second sequence, the third sequence and the fourth sequence are respectively at least 2 bp; preferably, the lengths of the second sequence, the third sequence and the fourth sequence are respectively 5-30 bp.

30. The nanopore sequencing kit according to any one of claims 20 to 29, characterized in that The complementary length between the third sequence and the sequencing adapter of the sequencing library is at least 2 bp.

31. The nanopore sequencing kit according to any one of claims 20 to 30, characterized in that The blocker is composed of DNA, RNA, LNA, PNA, BNA, or any combination thereof.

Images