Nanopore sequencing method and kit

Abstract

A nanopore sequencing method and a kit are provided. The nanopore sequencing method comprises: performing a first mixed incubation of particles with a nucleic acid library, and obtaining a first library containing the particles; and performing nanopore sequencing on the first library containing the particles. By sticking or connecting the nucleic acid library to the particles and then sequencing the nucleic acid library, the average read length of sequencing is increased compared to a control group without particles. In addition, the described method also has the advantages of improving sequencing read length and reducing the proportion of sequencing adapters.

Description

Nanopore sequencing methods and kits Technical Field

[0001] This invention relates to the field of sequencing, and more specifically, to a nanopore sequencing method and kit. Background Technology

[0002] Long-read sequencing technology plays an important role in genomics, metagenomics, transcriptomics, and other fields. Especially in microbial metagenomics research, longer sequencing reads can span repetitive and complex genomic regions, and even cover the entire genome, which helps to improve the fragmentation problem in short-read assembly, thereby better assembling the complete genome sequence of microorganisms.

[0003] Nanopore sequencing is a long-read sequencing technology. During nanopore sequencing, individual nanopores are embedded in an insulating, impermeable membrane, forming a stable ion current channel. Under voltage, single-stranded nucleic acid molecules pass through the nanopore, thus reducing the ion current flowing through it. Because different bases on the single-stranded nucleic acid molecule have different molecular structures and sizes, the current flowing through the nanopore exhibits differences corresponding to the base sequence. By analyzing the current change signal using algorithms, the sequence of the perforated single-stranded nucleic acid can be read in real time. The read length of nanopore sequencing is closely and positively correlated with the fragment length of the nucleic acid being tested. Various methods can be used to increase the average length of the nucleic acid fragments, such as sorting using magnetic beads, electrophoresis, and agarose gel to obtain long nucleic acid fragments. During library construction, wide-mouth pipette tips are used for gentle and slow aspiration to minimize mechanical breakage of nucleic acid fragments.

[0004] Patent application CN112391380A proposes a suitable purification reagent for the efficient recovery of ultra-long nucleic acids, ensuring that the length of the recovered nucleic acids is not destroyed. Patent application CN115151640A proposes a precipitation buffer containing salt and polyvinyl pyrrolidone (PVP) and / or sucrose to separate nucleic acids within the desired size range, thereby obtaining ultra-high molecular weight nucleic acid fragments. Patent CN213570409U proposes a kit for separating high molecular weight nucleic acids using beads, which can rapidly separate large quantities of high molecular weight nucleic acids. Currently, the most common commercial products for obtaining long nucleic acid fragments are magnetic beads (Beckman Coulter AMPure XP Beads) and pulsed field gel electrophoresis systems. The above-mentioned technologies may have the following drawbacks or limitations: the size cutoff value for nucleic acid length selection is too low (Beckman Coulter AMPure XP Beads); the sorting time is too long, which may damage the nucleic acid (Pulsed Field Gel Electrophoresis Systems); the amount of nucleic acid sorted is limited (patent applications CN112391380A, CN115151640A), etc.

[0005] Current technologies are all geared towards pre-library construction, i.e., sorting nucleic acid fragments to obtain long nucleic acid fragments before library construction. These technologies are limited by their application scenarios and ignore the impact and limitations of the library construction and sequencing processes on library length. For example, in nanopore sequencing, long nucleic acid fragments may still be broken down during library construction, resulting in the presence of some short nucleic acid fragments in the obtained nucleic acid library. Furthermore, because nucleic acid sorting schemes easily inactivate motor proteins, most current nucleic acid sorting schemes are not suitable for nucleic acid libraries containing motor proteins. In nanopore sequencing, short-fragment libraries are more easily captured and sequenced by nanopores, thus reducing the average read length. Therefore, developing a simple, safe, and rapid method to improve the average read length is crucial for advancing this technology.

[0006] Summary of the Invention

[0007] The main objective of this invention is to provide a nanopore sequencing method and kit to solve the problem of short average read length in existing sequencing technologies.

[0008] To achieve the above objectives, according to a first aspect of the present invention, a nanopore sequencing method is provided, the method comprising: performing a first mixing and incubation of particles with a nucleic acid library to obtain a first library containing particles; and performing nanopore sequencing on the first library containing particles.

[0009] To achieve the above objectives, according to a second aspect of the present invention, a nanopore sequencing method is provided, the method comprising: firstly mixing and incubating particles with a nucleic acid library to obtain a first library containing particles; centrifuging the first library containing particles to obtain a second library without particles and a third library containing particles; performing nanopore sequencing on the second library without particles, and / or resuspending the third library containing particles using sequencing buffer, and performing nanopore sequencing on the resuspended third library.

[0010] To achieve the above objectives, according to a third aspect of the present invention, a nanopore sequencing method is provided, the method comprising: firstly mixing and incubating particles with a nucleic acid library to obtain a first library containing particles; centrifuging the first library containing particles to obtain a second library without particles and a third library containing particles; secondly mixing and incubating the particles and the second library without particles to obtain a fourth library containing particles; and performing nanopore sequencing on the fourth library containing particles.

[0011] Furthermore, the time for the first mixed incubation is ≤24h, and the temperature is 25~30℃.

[0012] Furthermore, the second mixed incubation time is ≤24h, and the temperature is 25~30℃.

[0013] Furthermore, the above-mentioned nanopore sequencing is performed in a sequencing system, and the sequencing system includes sequencing reagents and sequencing devices, wherein the sequencing reagents include sequencing buffers and optional restraint reagents; preferably, the sequencing buffers include KCl, HEPES, ATP and MgCl2; preferably, the pH of the sequencing buffers is 7.5 to 8.5; preferably, the restraint reagents include restraint sequences and corresponding buffer reagents.

[0014] Furthermore, the sequencing device includes: an electrolytic cell containing the sequencing buffer; a nanopore sensor located in the center of the electrolytic cell, dividing the electrolytic cell and the sequencing buffer into a positive electrolyte region and a negative electrolyte region; a first electrode and a second electrode, the first electrode and the second electrode being respectively disposed in the positive electrolyte region and the negative electrolyte region, and the first electrode and the second electrode being connected to a signal processing chip.

[0015] Further, the aforementioned nanopore sensor comprises: a membrane and a pore inserted into the membrane to form a channel; preferably, the membrane is selected from any one or more of the following: a lipid layer, a synthetic polymer membrane, and a solid film; preferably, the pore is a biological protein pore or a solid nanopore; preferably, the biological protein pore is selected from any one or more of the following or its homologs or mutants: hemolysin, leukocidin, Mycobacterium smegmatis porin A, MspB, MspC, MspD, α-Haemolysin, CsgG, Aerolysin, cytolysin, outer membrane porin F, outer membrane porin G, outer membrane phospholipase A, Neisseria spp. autotransporter lipoprotein, WZA, GspD, BCP34, or BCP58.

[0016] Furthermore, prior to performing the nanopore sequencing, the method further includes: attaching the library to the membrane using the restraint sequence; wherein the library includes: the first library containing particles, the second library without particles, the third library after resuspension, or the fourth library containing particles.

[0017] Preferably, the library comprises: the first library containing particles, the third library after resuspension, or the fourth library containing particles; the restraint sequence is mixed and incubated a third time with the sequencing buffer in the sequencing system, and the library is incubated a fourth time with the sequencing buffer in the sequencing system, thereby attaching the library to the membrane.

[0018] Preferably, the library is the second library that does not contain particles; the restraint sequence is mixed and incubated with the sequencing buffer in the sequencing system for the third time, and the library is incubated with the sequencing buffer in the sequencing system for the fourth time, thereby attaching the library to the membrane; or the restraint sequence is mixed with the library and then mixed and incubated with the sequencing buffer in the sequencing system for the fifth time, thereby attaching the library to the membrane.

[0019] More preferably, the sequence of the above-mentioned restraint sequence is: 5'-YYYY-SEQ ID NO: 1-3'; wherein, the 5' end of the restraint sequence is modified with cholesterol, and Y represents iSp18; more preferably, the temperature for performing the above-mentioned nanopore sequencing is 28-32℃, and the sequencing voltage is 0.18-0.23V; even more preferably, the time for the third mixing incubation is ≤30min, and the temperature is the sequencing temperature, which is 28-32℃; even more preferably, the time for the fourth mixing incubation is ≤10min, and the temperature is the sequencing temperature, which is 28-32℃; even more preferably, the time for the fifth mixing incubation is ≤10min, and the temperature is the sequencing temperature, which is 28-32℃.

[0020] Furthermore, the aforementioned particles include a matrix and surface modifications, wherein the matrix of the particles includes hydrophobic, hydrophilic, or amphiphilic substances.

[0021] Furthermore, the surface modification of the above particles contains any one of the following groups: -HN + (C2H5)2 or -N + (CH3)3.

[0022] Furthermore, the hydrophobic material is selected from any one or more of the following: glass, ceramics, iron oxide, silicon dioxide, polymers or metals, cholesterol, lipids, fatty acids, carbon nanotubes, amino acids or hydrophobic polymers; preferably, the hydrophobic polymer is polymerized from any two or more of the following substances: polylactic acid, polystyrene or polymethacrylates.

[0023] Furthermore, the aforementioned hydrophilic material includes a cross-linked hydrophilic material, wherein the cross-linked hydrophilic material is selected from any one or more of the following: hydrogel, cross-linked protein particles, cross-linked amino acids, or cross-linked hydrophilic polymers; preferably, the aforementioned cross-linked hydrophilic polymer is polymerized from any two or more of the following substances: sodium polyacrylate, gelatin, or gelatin-sodium gelatate copolymer.

[0024] Further, the aforementioned amphiphilic substances include amphiphilic polymers; preferably, the aforementioned amphiphilic polymers are polymerized from any two or more of the following substances: polysaccharides, amino acids, proteins, acrylic acid, polyethylene glycol, polyvinyl alcohol, polyethylene glycol methyl ether, polyvinylpyrrolidone, polyoxyethylene, polyethylene oxide, acrylates, polyethylene, polystyrene, polyvinyl chloride, polytetrafluoroethylene, polydimethylsiloxane, polyester, or polyurethane.

[0025] Furthermore, the average length of the aforementioned libraries is 1kb-50kb.

[0026] Furthermore, the diameter of the aforementioned particles is ≤900μm; preferably, the diameter of the aforementioned particles is 1-200μm.

[0027] To achieve the above objectives, according to a fourth aspect of the present invention, a nanopore sequencing kit is provided, the kit comprising particles and sequencing reagents; wherein the sequencing reagents comprise a sequencing buffer and optionally a restraint reagent; preferably, the sequencing buffer comprises KCl, HEPES, ATP and MgCl2; preferably, the pH of the sequencing buffer is 7.5 to 8.5; preferably, the restraint reagent comprises a restraint sequence and a corresponding buffer reagent.

[0028] Furthermore, the aforementioned particles include a matrix and surface modification, wherein the matrix of the particles includes a hydrophobic substance, a hydrophilic substance, or an amphiphilic substance; preferably, the surface modification of the aforementioned particles contains any one of the following groups: -HN + (C2H5)2 or -N + (CH3)3. Preferably, the hydrophobic material is selected from any one or more of the following: glass, ceramics, iron oxide, silicon dioxide, polymers or metals, cholesterol, lipids, fatty acids, carbon nanotubes, amino acids or hydrophobic polymers; more preferably, the hydrophobic polymer is polymerized from any two or more of the following substances: polylactic acid, polystyrene or polymethacrylates.

[0029] Preferably, the hydrophilic substance includes a cross-linked hydrophilic substance, wherein the cross-linked hydrophilic substance is selected from any one or more of the following: hydrogel, cross-linked protein particles, cross-linked amino acids or cross-linked hydrophilic polymers; more preferably, the cross-linked hydrophilic polymer is polymerized from any two or more of the following substances: sodium polyacrylate, gelatin or gelatin-sodium gelatate copolymer.

[0030] Preferably, the amphiphilic substance includes an amphiphilic polymer; more preferably, the amphiphilic polymer is polymerized from any two or more of the following substances: polysaccharides, amino acids, proteins, acrylic acid, polyethylene glycol, polyvinyl alcohol, polyethylene glycol methyl ether, polyvinylpyrrolidone, polyoxyethylene, polyethylene oxide, acrylates, polyethylene, polystyrene, polyvinyl chloride, polytetrafluoroethylene, polydimethylsiloxane, polyester, or polyurethane.

[0031] Furthermore, the diameter of the aforementioned particles is ≤900μm; preferably, the diameter of the aforementioned particles is 1-200μm.

[0032] Furthermore, the above-mentioned kit also includes: a membrane, and pores inserted into the membrane to form channels; preferably, the membrane is selected from any one or more of the following: lipid layer, artificial polymer membrane, and solid film; preferably, the pores are biological protein pores or solid nanopores; preferably, the biological protein pores are selected from any one or more of the following or their homologs or mutants: hemolysin, leukocidin, Mycobacterium smegmatis porin A, MspB, MspC, MspD, α-Haemolysin, CsgG, Aerolysin, cytolysin, outer membrane porin F, outer membrane porin G, outer membrane phospholipase A, Neisseria spp. autotransporter lipoprotein, WZA, GspD, BCP34, and BCP58.

[0033] By applying the technical solution of this invention, nucleic acid libraries are sequenced after adsorption or ligation using particles. The average read length is improved compared to the control group (without particles). Furthermore, this invention also offers advantages such as increasing read length and reducing the proportion of sequencing adapters. The research and application of this invention are of great significance to the development and improvement of sequencing technology. Attached Figure Description

[0034] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0035] Figure 1 shows a schematic diagram of the change of E. coli library sequencing results over time using operation scheme 1 with particle A in Embodiment 2 of the present invention, where A represents the average sequencing read length, B represents the sequencing read length N50, and C represents the adapter percentage.

[0036] Figure 2 shows a schematic diagram of the change of sequencing results of NA12878 library using particle A according to operation scheme 1 in embodiment 3 of the present invention over time, where A represents the average sequencing read length, B represents the sequencing read length N50, and C represents the adapter percentage.

[0037] Figure 3 shows a schematic diagram of the change in average read length over time when using particle A to sequence an E. coli library according to operation scheme 2 in Embodiment 4 of the present invention.

[0038] Figure 4 shows a schematic diagram illustrating the change in average read length over time when E. coli library sequencing was performed using particle A according to operation scheme 4 in Embodiment 5 of the present invention.

[0039] Figure 5 shows a schematic diagram illustrating the change in average read length over time when E. coli library sequencing was performed using particle A according to operation scheme 5 in Embodiment 6 of the present invention.

[0040] Figure 6 shows a schematic diagram of the changes in E. coli library sequencing results over time using particle B according to operation scheme 1 in Embodiment 7 of the present invention, where A represents the average sequencing read length, B represents the sequencing read length N50, and C represents the adapter percentage.

[0041] Figure 7 shows a schematic diagram illustrating the change over time of the adsorption or linkage amount of E. coli library by particle A in Embodiment 8 of the present invention.

[0042] Figure 8 shows the pulsed-field gel electrophoresis detection of the supernatant after adsorption or ligation of E. coli library by particle A in Embodiment 8 of the present invention. Detailed Implementation

[0043] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0044] Terminology Explanation:

[0045] Average read length: This refers to the average length of all sequencing reads obtained during genome sequencing or transcriptome sequencing. This metric can be used to measure the quality and reliability of sequencing data; generally, the longer the read length, the better the quality and interpretation capability of the sequencing data.

[0046] Sequencing read N50: This refers to the shortest read in the sequencing data that, after all reads are sorted from longest to shortest, accumulates to 50% of the total length. In other words, N50 is a metric for sequencing data quality, representing the distribution of read lengths in the sequencing data. A higher N50 value generally indicates higher quality sequencing data.

[0047] Adapter ratio: This refers to the proportion of the cumulative number of adapter reads in the sequencing data to the total number of sequencing reads (the sum of adapter reads and library reads). A low adapter ratio indicates high sequencing quality, while a high adapter ratio indicates low sequencing quality.

[0048] As mentioned in the background section, existing sequencing technologies suffer from short average read lengths. While existing technologies offer a method to improve the average read length by selecting long fragments, this method suffers from problems such as a low size selection cutoff, excessively long sorting time, limited sorting quantity, and potential damage to nucleic acids during sorting. In contrast, the present invention employs a method of sequencing nucleic acid libraries after particle adsorption or ligation, eliminating the need for sorting and allowing direct sequencing. This significantly improves the average read length and read length N50, while reducing the proportion of sequencing adapters. Therefore, based on these improvements, the inventors have proposed a series of protection schemes for this invention.

[0049] In a first typical embodiment of the present invention, a nanopore sequencing method is provided, the method comprising: mixing and incubating particles with a nucleic acid library for the first time to obtain a first library containing particles; and performing nanopore sequencing on the first library containing particles.

[0050] After incubating particles with nucleic acid libraries, libraries containing particles can be obtained. Since particles are diverse, the interaction between particles and nucleic acid libraries can include physical adsorption or chemical linkage.

[0051] The role of the particles in this invention may be to adsorb or connect nucleic acid libraries (including long-fragment and short-fragment libraries). Libraries containing particles may be preferentially captured for sequencing. After centrifugation, the libraries containing particles settle near the nanopore membrane due to particle sedimentation or under the influence of an electric field, effectively enriching the libraries near the nanopores and thus promoting their capture by the nanopores. Furthermore, since there is a certain distance between the particles and the pores, long nucleic acid fragments may be more easily captured by the nanopores due to their chain length.

[0052] Prior to nanopore sequencing, the method further includes attaching a first library containing particles to the membrane using a confinement sequence. The confinement sequence is then mixed and incubated a third time with the sequencing buffer in the sequencing system, and the first library containing particles is incubated a fourth time with the sequencing buffer in the sequencing system, thereby attaching the first library containing particles to the membrane.

[0053] In a second typical embodiment of the present invention, a nanopore sequencing method is provided. The method includes: firstly mixing and incubating particles with a nucleic acid library to obtain a first library containing particles; centrifuging the first library containing particles to obtain a second library without particles and a third library containing particles; performing nanopore sequencing on the second library without particles; and / or resuspending the third library containing particles using sequencing buffer, and performing nanopore sequencing on the resuspended third library.

[0054] It should be noted that after centrifugation of the first library containing particles, a particle-free supernatant and a particle-containing precipitate are obtained. The particle-free supernatant is the particle-free second library, which can be directly sequenced using nanopore sequencing. However, as those skilled in the art know, the particle-containing precipitate needs to be resuspended and mixed with sequencing buffer before nanopore sequencing to obtain a particle-containing third library suitable for sequencing. Furthermore, nanopore sequencing of the particle-free second library, compared to conventional libraries without particle incubation, has the beneficial effects of increasing the average sequencing length and N50, and reducing the adapter ratio. It is speculated that this may be because the particles adsorb or ligate small nucleic acid fragments, thereby enriching the long nucleic acids in the second library.

[0055] In a third typical embodiment of the present invention, a nanopore sequencing method is provided. The method includes: firstly mixing and incubating particles with a nucleic acid library to obtain a first library containing particles; centrifuging the first library containing particles to obtain a second library without particles and a third library containing particles; secondly mixing and incubating the particles and the second library without particles to obtain a fourth library containing particles; and performing nanopore sequencing on the fourth library containing particles.

[0056] Multiple experimental studies have shown that the library construction method of this application, which uses particle adsorption and ligation for sequencing, can obtain libraries with an average length of 1kb-50kb (including the first library containing particles, the second library without particles, the resuspended third library, and the fourth library containing particles), which is longer than the length without particles.

[0057] Before performing nanopore sequencing, the above method also includes attaching a first library containing particles, a second library without particles, a resuspended third library, and a fourth library containing particles to the membrane by binding sequences.

[0058] When attaching the first library containing particles, the resuspended third library, and the fourth library containing particles, the aforementioned restraint sequence is first mixed and incubated for the third time with the sequencing buffer in the sequencing system. Then, the first library containing particles, the resuspended third library, and the fourth library containing particles are incubated for the fourth time with the sequencing buffer after the third mixing and incubation, thereby attaching the first library, the second library, and the fourth library to the membrane.

[0059] It is important to note that when the library contains particles, the restraint sequences should not be mixed with the library before incubation with the sequencing buffer. This is because particles in the library may adsorb the restraint sequences, thus affecting the experimental results. Therefore, the restraint sequences should be mixed with the sequencing buffer first, and then the library should be mixed with the incubated sequencing buffer containing the restraint sequences before sequencing. This allows the library to be captured and bound near the nanopores by the restraint sequences without causing a large amount of restraint sequences to be adsorbed by particles.

[0060] When attaching a particle-free second library, the restraint sequence is first mixed and incubated with the sequencing buffer in the sequencing system for the third time, and then the library is incubated with the sequencing buffer after the third mixing and incubation for the fourth time, thereby attaching the library to the membrane; or the restraint sequence is first mixed with the library and then mixed and incubated with the sequencing buffer in the sequencing system for the fifth time, thereby attaching the library to the membrane. Since the second library does not contain particles, the incubation of the restraint sequence and the library is not affected by particles. The restraint sequence can be incubated with the sequencing buffer first and then mixed with the library for incubation; or the restraint sequence can be mixed with the library first and then incubated with the sequencing buffer.

[0061] It is important to note that when using a large quantity of the restrained sequence and the particle-free secondary library, they can be incubated separately with the sequencing buffer in the sequencing system, or the restrained sequence and the particle-free secondary library can be mixed together before incubating together with the sequencing buffer. However, when using a small quantity of the restrained sequence and the particle-free secondary library, it is not suitable to incubate them separately with the sequencing buffer. It is better to mix them first and then incubate them together with the sequencing buffer, as this helps improve the attachment efficiency of nucleic acid fragments.

[0062] More preferably, the sequence of the above-mentioned restraint sequence is: 5'-YYYY-SEQ ID NO: 1-3'; wherein, the 5' end of the restraint sequence is modified with cholesterol, and Y represents iSp18; more preferably, the temperature for performing the above-mentioned nanopore sequencing is 28-32℃, and the sequencing voltage is 0.18-0.23V; even more preferably, the time for the third mixing incubation is ≤30min, and the temperature is the sequencing temperature, which is 28-32℃; even more preferably, the time for the fourth mixing incubation is ≤10min, and the temperature is the sequencing temperature, which is 28-32℃; even more preferably, the time for the fifth mixing incubation is ≤10min, and the temperature is the sequencing temperature, which is 28-32℃.

[0063] The first incubation period was ≤24 hours at a temperature of 25–30°C; the second incubation period was ≤24 hours at a temperature of 25–30°C. The purpose of both incubations was to ensure sufficient adsorption or linkage between the particles and the library, resulting in a larger library containing particles.

[0064] The third mixing incubation serves to ensure adequate adhesion of the restraint sequences to the membrane. During the fourth mixing incubation, the restraint sequences and library connectors fully pair, drawing the library closer to the membrane. The fifth mixing incubation serves both to ensure adequate adhesion of the restraint sequences to the membrane and to draw the library closer to the membrane.

[0065] However, for libraries containing particles, the fourth and fifth mixing incubations also serve to ensure that the libraries containing particles settle sufficiently near the membrane.

[0066] The above-mentioned nanopore sequencing is performed in a sequencing system, and the sequencing system includes sequencing reagents and sequencing devices. The sequencing reagents include sequencing buffers and optional restraint reagents. Preferably, the sequencing buffers include KCl, HEPES, ATP and MgCl2. Preferably, the pH of the sequencing buffers is 7.5 to 8.5. Preferably, the restraint reagents include restraint sequences and their corresponding buffer reagents.

[0067] The sequencing device includes: an electrolytic cell containing sequencing buffer; a nanopore sensor located in the center of the electrolytic cell, dividing the electrolytic cell and sequencing buffer into a positive electrolyte region and a negative electrolyte region; a first electrode and a second electrode, which are respectively disposed in the positive electrolyte region and the negative electrolyte region, and are connected to a signal processing chip.

[0068] The nanopore sensor includes a membrane and a pore inserted into the membrane to form a channel; preferably, the membrane is selected from any one or more of the following: lipid layer, artificial polymer membrane and solid film.

[0069] Preferably, the pores are biological protein pores or solid nanopores; preferably, the biological protein pores are selected from any one or more of the following or their homologs or mutants: hemolysin, leukocidin, Mycobacterium smegmatis porin A (MspA), MspB, MspC, MspD, α-Haemolysin, CsgG, Aerolysin, cytolysin, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A, Neisseria spp. autotransporter lipoprotein (NalP), WZA, GspD, BCP34 and BCP58.

[0070] Using the above sequencing system for nanopore sequencing has the beneficial effect of ensuring successful sequencing.

[0071] The diameter of the particles can affect the amount of nucleic acid adsorbed or linked, as well as the capture of nucleic acids by the nanopores. Larger particles can adsorb or link more nucleic acids on their surface, but if the particles are too large, the distance between the nucleic acids and the nanopores increases, reducing the amount of nucleic acids that can be captured by the nanopores. In this invention, the diameter of the particles is ≤900 μm. In a preferred embodiment of this invention, the diameter of the particles is 1-200 μm. Multiple tests have shown that particles with this diameter have the beneficial effects of a moderate amount of nucleic acid that can be adsorbed or linked, and that nucleic acids are easily captured by the nanopores.

[0072] The aforementioned particles include a matrix and surface modifications, wherein the matrix of the particles includes hydrophobic, hydrophilic, or amphiphilic substances.

[0073] Preferably, the surface modification of the above particles contains any one of the following groups: -HN + (C2H5)2 or -N + (CH3)3 (unlike commonly used magnetic beads with surface modifications such as carboxyl, hydroxyl, Oligo(dT), streptavidin, or biotin modifications). Surface modifications include -HN. + (C2H5)2 or -N +(CH3)3 particles adsorb or ligate the library, which has the beneficial effects of increasing the average length and N50 of the sequencing library and reducing the proportion of adapters.

[0074] Preferably, the hydrophobic material is selected from any one or more of the following: glass, ceramics, iron oxide, silicon dioxide, polymers or metals, cholesterol, lipids, fatty acids, carbon nanotubes, amino acids or hydrophobic polymers; preferably, the hydrophobic polymer is polymerized from any two or more of the following substances: polylactic acid, polystyrene or polymethacrylates.

[0075] Hydrophilic substances include cross-linked hydrophilic substances, wherein the cross-linked hydrophilic substances are selected from any one or more of the following: hydrogels, cross-linked protein particles, cross-linked amino acids, or cross-linked hydrophilic polymers; preferably, the cross-linked hydrophilic polymers are polymerized from any two or more of the following substances: sodium polyacrylate, gelatin, or a gelatin-sodium gelatate copolymer.

[0076] Amphiphilic substances include amphiphilic polymers; preferably, the amphiphilic polymers are polymerized from any two or more of the following substances: polysaccharides, amino acids, proteins, acrylic acid, polyethylene glycol, polyvinyl alcohol, polyethylene glycol methyl ether, polyvinylpyrrolidone, polyoxyethylene, polyethylene oxide, acrylates, polyethylene, polystyrene, polyvinyl chloride, polytetrafluoroethylene, polydimethylsiloxane, polyester, or polyurethane.

[0077] In a fourth typical embodiment of the present invention, a nanopore sequencing kit is provided. The kit includes particles and sequencing reagents, wherein the sequencing reagents include sequencing buffer and optional restraint reagents; preferably, the sequencing buffer includes KCl, HEPES, ATP, and MgCl2; preferably, the pH of the sequencing buffer is 7.5–8.5; preferably, the restraint reagent includes a restraint sequence and a corresponding buffer reagent. The sequencing buffer provides an environment that maintains the stability of the nanopore and membrane layer, and the metal ions contained therein give the sequencing buffer good conductivity.

[0078] The aforementioned particles include a matrix and surface modifications, wherein the matrix of the aforementioned particles includes hydrophobic, hydrophilic, or amphiphilic substances.

[0079] Preferably, the surface modification of the above particles contains any one of the following groups: -HN + (C2H5)2 or -N + (CH3)3.

[0080] Preferably, the hydrophobic material is selected from any one or more of the following: glass, ceramics, iron oxide, silicon dioxide, polymers or metals, cholesterol, lipids, fatty acids, carbon nanotubes, amino acids or hydrophobic polymers; more preferably, the hydrophobic polymer is polymerized from any two or more of the following substances: polylactic acid, polystyrene or polymethacrylates.

[0081] Preferably, the hydrophilic substance includes a cross-linked hydrophilic substance, wherein the cross-linked hydrophilic substance is selected from any one or more of the following: hydrogel, cross-linked protein particles, cross-linked amino acids or cross-linked hydrophilic polymers; more preferably, the cross-linked hydrophilic polymer is polymerized from any two or more of the following substances: sodium polyacrylate, gelatin or gelatin-sodium gelatate copolymer.

[0082] Preferably, the amphiphilic substance includes an amphiphilic polymer; more preferably, the amphiphilic polymer is polymerized from any two or more of the following substances: polysaccharides, amino acids, proteins, acrylic acid, polyethylene glycol, polyvinyl alcohol, polyethylene glycol methyl ether, polyvinylpyrrolidone, polyoxyethylene, polyethylene oxide, acrylates, polyethylene, polystyrene, polyvinyl chloride, polytetrafluoroethylene, polydimethylsiloxane, polyester, or polyurethane.

[0083] The diameter of the particles is ≤900μm; preferably, the diameter of the particles is 1-200μm.

[0084] The kit also includes: a membrane and a pore inserted into the membrane to form a channel; preferably, the membrane is selected from any one or more of the following: lipid layer, artificial polymer membrane and solid film; preferably, the pore is a biological protein pore or a solid nanopore.

[0085] Preferably, the biological protein pores are selected from any one or more of the following, or their homologs or mutants: hemolysin, leukocidin, Mycobacterium smegmatis porin A, MspB, MspC, MspD, α-Haemolysin, CsgG, Aerolysin, cytolysin, outer membrane porin F, outer membrane porin G, outer membrane phospholipase A, Neisseria spp. autotransport lipoprotein, WZA, GspD, BCP34, and BCP58.

[0086] Using the above kit for nanopore sequencing can improve the capture rate of long nucleic acids in the sequencing library by the nanopore, thereby increasing the average sequencing read length and N50, and reducing the proportion of adapters.

[0087] The beneficial effects of the present invention will be explained in more detail below with reference to specific embodiments.

[0088] Example 1 Sequencing Method

[0089] Following 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; 214:119222), a single-channel nanopore detection system was built based on patch clamp and signal amplifier to complete the embedding of a single pore protein.

[0090] Libraries were prepared using the H940-000013CycloneSEQ universal library preparation kit (24RXN). The nucleic acids to be tested were Escherichia coli genomic DNA and NA12878 human cell line genomic DNA, resulting in E. coli nucleic acid libraries (50 ng / μL) and NA12878 nucleic acid libraries (50 ng / μL).

[0091] The tether sequence is 5'-YYYY-SEQ ID NO: 1-3', where the sequence of SEQ ID NO: 1 is TTGACCGCTCGCCTC. The 5' end of the tether sequence is modified with cholesterol, and Y represents iSp18. It was synthesized by Sangon Biotech (Shanghai) Co., Ltd. and dissolved in TE buffer (pH=8) at 10 μM.

[0092] Particle A: Agarois-Q FF (its own material is agarose, and its functional group is -N) + (CH3)3), particle size 45μm, Suzhou Saifen Technology Co., Ltd.), Particle B: Monomix HC60-DEAE (hydrophilic polymethyl methacrylate, functional group -N(C2H5)2), particle size 60μm, Suzhou Saifen Technology Co., Ltd.).

[0093] The sequencing buffer consisted of 420 mM KCl, 23 mM HEPES, 27 mM ATP, and 23 mM MgCl2, with a pH of 8.0. The sequencing temperature was 30 °C, and the sequencing voltage was 0.18 V.

[0094] (1) Scheme 1: Using particles to increase the average read length of sequencing

[0095] a) Add sequencing buffer and restraint reagent to EP tube ①, mix well, and then add to the sequencing buffer of the sequencing system. Incubate for 30 minutes.

[0096] b) Add sequencing buffer, nucleic acid library and particle reagent to EP tube ②, mix well and then add to the sequencing buffer of the sequencing system, incubate for 10 min.

[0097] c) Start sequencing at an on voltage of 0.18V.

[0098] (2) Scheme 2: Using particles to increase the average read length of sequencing

[0099] a) Add sequencing buffer, nucleic acid library and particle reagent to EP tube ①, mix well and incubate for 24 hours.

[0100] b) Centrifuge at low speed to separate the particles from the supernatant, and take the supernatant into EP tube ②.

[0101] c) Add sequencing buffer and restraint reagent to EP tube ③, mix well, and then add to the sequencing buffer of the sequencing system. Incubate for 30 minutes.

[0102] d) Add the supernatant from EP tube ② to the sequencing buffer of the sequencing system and incubate for 10 min.

[0103] e) Start sequencing at an on-state voltage of 0.18V.

[0104] (3) Scheme 3: Using particles to increase the average read length of sequencing

[0105] a) Add sequencing buffer, nucleic acid library and particle reagent to EP tube ①, mix well and incubate for 24 hours.

[0106] b) Centrifuge at low speed to separate the particles from the supernatant, and take the supernatant into EP tube ②.

[0107] c) Add the restraint reagent to EP tube ②, mix well, and then add it to the sequencing buffer of the sequencing system. Incubate for 10 min.

[0108] d) Start sequencing at an on voltage of 0.18V.

[0109] (4) Scheme 4: Using particles to increase the average read length of sequencing

[0110] a) Add sequencing buffer, nucleic acid library and particle reagent to EP tube ①, mix well and incubate for 24 hours.

[0111] b) Centrifuge at low speed to separate the particles from the supernatant, discard the supernatant and retain the particles.

[0112] c) Add sequencing buffer and restraint reagent to EP tube ②, mix well, and then add to the sequencing buffer of the sequencing system. Incubate for 30 minutes.

[0113] d) Add fresh sequencing buffer to the EP tube ① containing the retained particles, mix well, and then add it to the sequencing buffer of the sequencing system. Incubate for 10 min.

[0114] e) Start sequencing at an on-state voltage of 0.18V.

[0115] (5) Scheme 5: Using particles to enhance the average read length of sequencing

[0116] a) Add sequencing buffer, nucleic acid library and particle reagent to EP tube ①, mix well and incubate for 24 hours.

[0117] b) Add sequencing buffer and restraint reagent to EP tube ②, mix well, and then add to the sequencing buffer of the sequencing system. Incubate for 30 minutes.

[0118] c) After the reagent incubation in EP tube ① is complete, mix it well and add it to the sequencing buffer of the sequencing system, and incubate for 10 min.

[0119] d) Start sequencing at an on voltage of 0.18V.

[0120] (6) Scheme 6: Using particles to enhance the average read length of sequencing

[0121] a) Add sequencing buffer, nucleic acid library and particle reagent to EP tube ①, mix well and incubate for 24 hours.

[0122] b) Centrifuge at low speed to separate the particles from the supernatant, and take the supernatant into EP tube ②.

[0123] c) Add sequencing buffer and restraint reagent to EP tube ③, mix well, and then add to the sequencing buffer of the sequencing system. Incubate for 30 minutes.

[0124] d) Add particulate reagent to the EP tube ② containing the supernatant, mix well, and then add it to the sequencing buffer of the sequencing system. Incubate for 10 min.

[0125] e) Start sequencing at an on-state voltage of 0.18V.

[0126] The above schemes 1-6 are briefly shown in Table 1.

[0127] Table 1. Brief steps of the six schemes

[0128] Example 2: E. coli library-particle A-scheme 1 sequencing

[0129] Sequencing conditions: The E. coli library was sequenced using particle A according to operation protocol 1.

[0130] Experimental group: Add 394 μL sequencing buffer and 6 μL restraint reagent to EP tube ①, mix well, and add to the sequencing buffer of the sequencing system. Incubate for 30 min. Then, add 394 μL sequencing buffer, 2 μg library, and 1 μL or 2 μL particle A to EP tube ②, mix well, and add to the sequencing buffer of the sequencing system containing the above buffer and restraint reagent. Incubate for 10 min. Turn on the voltage to 0.18V and start sequencing.

[0131] Control group: Add 394 μL sequencing buffer and 6 μL restraint reagent to EP tube ①, mix well, and add to the sequencing buffer of the sequencing system. Incubate for 30 min. Then, add 394 μL sequencing buffer and 2 μg library (without particles) to EP tube ②, mix well, and add to the sequencing buffer of the sequencing system containing the buffer and restraint reagent. Incubate for 10 min. Turn on the voltage to 0.18V and proceed with sequencing.

[0132] The sequencing results are shown in Figure 1. Figures A, B, and C in Figure 1 represent the changes over time in the average read length, read length N50, and adapter ratio obtained using particle A according to operation protocol 1, respectively. The results indicate that particle A can simultaneously improve the average read length and read length N50 of the E. coli library during the sequencing process, while also reducing the adapter ratio. The effect of increasing the amount of particle added is even more pronounced in improving the average read length and read length N50, and reducing the adapter ratio.

[0133] Example 3: Sequencing of NA12878 library-particle A-scheme 1

[0134] Sequencing conditions: The NA12878 library was sequenced using particle A according to operation protocol 1.

[0135] Experimental group: Add 394 μL sequencing buffer and 6 μL restraint reagent to EP tube ①, mix well, and add to the sequencing buffer of the sequencing system. Incubate for 30 min. Then, add 394 μL sequencing buffer, 1 μg library, and 1 μL or 2 μL particle A to EP tube ②, mix well, and add to the sequencing buffer of the sequencing system containing the buffer and restraint reagent. Incubate for 10 min. Turn on the voltage to 0.18V and start sequencing.

[0136] Control group: Add 394 μL sequencing buffer and 6 μL restraint reagent to EP tube ①, mix well, and add to the sequencing buffer of the sequencing system. Incubate for 30 min. Then, add 394 μL sequencing buffer and 1 μg library to EP tube ②, mix well, and add to the sequencing buffer of the sequencing system containing the buffer and restraint reagent. Incubate for 10 min. Turn on the voltage to 0.18V and proceed with sequencing.

[0137] The sequencing results are shown in Figure 2. Figures A, B, and C in Figure 2 represent the changes over time in the average read length, read length N50, and adapter ratio obtained using particle A according to operation plan 1, respectively. The results indicate that particle A can simultaneously improve the average read length and read length N50 of the NA12878 library and reduce the adapter ratio during the sequencing process. The effect of increasing the amount of particle added is even more pronounced in improving the average read length and read length N50 and reducing the adapter ratio.

[0138] Example 4: E. coli library-particle A-scheme 2 sequencing

[0139] Sequencing conditions: The E. coli library was sequenced using particle A according to operation protocol 2.

[0140] Experimental group: Add 394 μL sequencing buffer, 2 μg library, and 2 μL particle A to EP tube ①, mix well, and incubate for 24 hours. Centrifuge at low speed for 30 seconds to separate the particles from the supernatant, and transfer the supernatant to EP tube ②. Then, add 394 μL sequencing buffer and 6 μL restraint reagent to EP tube ③, mix well, and then add to the sequencing buffer of the sequencing system, incubating for 30 minutes. Finally, add the supernatant from EP tube ② to the sequencing buffer of the sequencing system containing the above buffer and restraint reagent, and incubate for 10 minutes. Turn on the voltage to 0.18V and start sequencing.

[0141] Control group: Add 394 μL sequencing buffer and 2 μg library to EP tube ①, mix well, and incubate for 24 hours. Then, add 394 μL sequencing buffer and 6 μL restraint reagent to EP tube ②, mix well, and then add to the sequencing buffer of the sequencing system, incubating for 30 minutes. Finally, add the solution from EP tube ① to the sequencing buffer of the sequencing system containing the buffer and restraint reagent, and incubate for 10 minutes. Turn on the voltage at 0.18V and proceed with sequencing.

[0142] The sequencing results are shown in Figure 3. Figure 3 shows the change in average read length over time obtained by sequencing the E. coli library using particle A according to operation plan 2. The results indicate that treating the nucleic acid library with particle A can improve the average read length of the E. coli library.

[0143] Example 5: E. coli library-particle A-scheme 4 sequencing

[0144] Sequencing conditions: The E. coli library was sequenced using particle A according to operation protocol 4.

[0145] Experimental group: Add 394 μL sequencing buffer, 2 μg library, and 2 μL particle A to EP tube ①, mix well, and incubate for 24 hours. Centrifuge at low speed for 30 seconds to separate the particles from the supernatant, discard the supernatant, and retain the particles. Then, add 394 μL sequencing buffer and 6 μL restraint reagent to EP tube ②, mix well, and add to the sequencing buffer of the sequencing system, incubate for 30 minutes. Finally, add 394 μL of fresh sequencing buffer to EP tube ①, mix well, and add to the sequencing buffer of the sequencing system containing the buffer and restraint reagent, incubate for 10 minutes. Turn on the voltage to 0.18V and start sequencing.

[0146] Control group: Add 394 μL sequencing buffer and 2 μg library to EP tube ①, mix well, and incubate for 24 hours. Then, add 394 μL sequencing buffer and 6 μL restraint reagent to EP tube ②, mix well, and then add to the sequencing buffer of the sequencing system, incubating for 30 minutes. Finally, add the solution from EP tube ① to the sequencing buffer of the sequencing system containing the buffer and restraint reagent, and incubate for 10 minutes. Turn on the voltage at 0.18V and proceed with sequencing.

[0147] The sequencing results are shown in Figure 4. Figure 4 shows the change in average read length over time obtained by sequencing the E. coli library using particle A according to operation protocol 4. The results indicate that treating the nucleic acid library with particle A can improve the average read length of the E. coli library.

[0148] Example 6: E. coli library-particle A-scheme 5 sequencing

[0149] Sequencing conditions: The E. coli library was sequenced using particle A according to operation protocol 5.

[0150] Experimental group: Add 394 μL sequencing buffer, 2 μg library, and 2 μL particle A to EP tube ①, mix well, and incubate for 24 hours. Then, add 394 μL sequencing buffer and 6 μL restraint reagent to EP tube ②, mix well, and then add to the sequencing buffer of the sequencing system, incubating for 30 minutes. Finally, mix the reagent (including particles) from EP tube ① and add it to the sequencing buffer of the sequencing system containing the buffer and restraint reagent, incubating for 10 minutes. Turn on the voltage to 0.18V and start sequencing.

[0151] Control group: Add 394 μL sequencing buffer and 2 μg library to EP tube ①, mix well, and incubate for 24 hours. Then, add 394 μL sequencing buffer and 6 μL restraint reagent to EP tube ②, mix well, and then add to the sequencing buffer of the sequencing system, incubating for 30 minutes. Finally, add the solution from EP tube ① to the sequencing buffer of the sequencing system containing the buffer and restraint reagent, and incubate for 10 minutes. Turn on the voltage at 0.18V and proceed with sequencing.

[0152] The sequencing results are shown in Figure 5. Figure 5 shows the change in average read length over time obtained by sequencing the E. coli library using particle A according to operation protocol 5. The results indicate that treating the nucleic acid library with particle A can improve the average read length of the E. coli library.

[0153] Example 7: E. coli library-particle B-scheme 1 sequencing

[0154] Sequencing conditions: The E. coli library was sequenced using particle B according to procedure 1. Polyethylene glycol 2000 (average molecular weight: 1900-2200, Sigma-Aldrich) was added to the sequencing buffer to increase the stability of particle B. A sequencing buffer containing 9% PEG was prepared.

[0155] Experimental group: Add 394 μL sequencing buffer and 6 μL restraint reagent to EP tube ①, mix well, and add to the sequencing buffer of the sequencing system. Incubate for 30 min. Then, add 394 μL sequencing buffer, 1 μg library, and 1 μL particle B to EP tube ②, mix well, and add to the sequencing buffer of the sequencing system containing the buffer and restraint reagent. Incubate for 10 min. Turn on the voltage to 0.18V and start sequencing.

[0156] Control group: Add 394 μL sequencing buffer and 6 μL restraint reagent to EP tube ①, mix well, and add to the sequencing buffer of the sequencing system. Incubate for 30 min. Then, add 394 μL sequencing buffer and 1 μg library to EP tube ②, mix well, and add to the sequencing buffer of the sequencing system containing the buffer and restraint reagent. Incubate for 10 min. Turn on the voltage to 0.18V and proceed with sequencing.

[0157] The sequencing results are shown in Figure 6. Figures A, B, and C in Figure 6 represent the changes over time in the average read length, read length N50, and adapter ratio obtained by sequencing the E. coli library using particle B according to operation plan 1. The results indicate that particle B can simultaneously improve the average read length and read length N50 of the E. coli library during the sequencing process, while also reducing the adapter ratio. The effect of increasing the particle amount on improving the average read length and read length N50, and reducing the adapter ratio, is even more pronounced.

[0158] Example 8: Ligation or Adsorption of Particle A to E. coli Library - Qubit Detection

[0159] Objective: To investigate the change in the amount of E. coli library adsorbed or linked by particle A over time.

[0160] Experimental group: 394 μL sequencing buffer, 2 μg library, and 2 μL particle A were mixed and incubated for different times (5 min, 2 h, 6 h, 24 h, 48 h, 72 h). The supernatant containing the library was separated by low-speed centrifugation and analyzed using a Qubit (Thermo Fisher). TM The dsDNA quantitative kit (catalog number Q32851) is used to detect the concentration of DNA in the supernatant.

[0161] Control group: 394 μL sequencing buffer and 2 μg library were mixed and incubated for different times (5 min, 2 h, 6 h, 24 h, 48 h, 72 h), and the concentration of DNA in the solution was detected by Qubit.

[0162] The percentage of library linkage or adsorption by particle A is calculated as follows: (Initial library concentration × Initial library volume - Library concentration in supernatant × Supernatant volume) / Initial library concentration × Initial library volume × 100%. The results are shown in Figure 7. The adsorption capacity of particle A increases with incubation time. After incubation for 5 min, 2 h, 6 h, 24 h, 48 h, and 72 h, the adsorbed library amounts by particle A were 4.5%, 17.2%, 21.0%, 41.1%, 54.8%, and 60.6%, respectively.

[0163] Example 9: Ligation or Adsorption of Particle A to an E. coli Library - Agarose Gel Pulsed Field Electrophoresis

[0164] Objective: To investigate the changes in library length distribution in the supernatant after adsorption or ligation of particle A with E. coli library with incubation time.

[0165] 394 μL of sequencing buffer, 0.4 μg of library, and 0.4 μL of particle A were mixed and incubated for different times (0 h, 0.25 h, 6 h, 24 h). The supernatant containing the library was separated by low-speed centrifugation and detected by 1% agarose gel pulsed-field gel electrophoresis. The results are shown in Figure 8. As the incubation time increased, the gray value in the supernatant decreased, indicating a decrease in the amount of library. This suggests that particle A has a binding or adsorption effect on the library, and the amount of library adsorbed by particle A increases with increasing incubation time.

[0166] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects: The nanopore sequencing method of the present invention includes incubating particles with a nucleic acid library to obtain a test library, and then performing nanopore sequencing on the test library. Compared with nucleic acid libraries without particles, nucleic acid libraries with particles have longer average read lengths, higher N50 read lengths, or lower adapter ratios. In summary, the present invention helps to improve the average read length, increase N50 read lengths, or reduce the adapter ratio, thereby further improving the sequencing quality of nanopore sequencing, which is of great significance to the development of sequencing technology.

[0167] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A nanopore sequencing method, characterized in that, The method includes: The particles and the nucleic acid library were mixed and incubated for the first time to obtain the first library containing the particles. Nanopore sequencing was performed on the first library containing particles.

2. A nanopore sequencing method, characterized in that, The method includes: The particles and the nucleic acid library were mixed and incubated for the first time to obtain the first library containing the particles. The first library containing particles was centrifuged to obtain a second library without particles and a third library containing particles. The second library, which does not contain particles, is subjected to nanopore sequencing, and / or the third library containing particles is resuspended in sequencing buffer, and the resuspended third library is subjected to nanopore sequencing.

3. A nanopore sequencing method, characterized in that, The method includes: The particles and the nucleic acid library were mixed and incubated for the first time to obtain the first library containing the particles. The first library containing particles was centrifuged to obtain a second library without particles and a third library containing particles. The particles and the second library without particles are mixed and incubated for a second time to obtain a fourth library containing particles; The nanopore sequencing was performed on the fourth library containing particles.

4. The method according to any one of claims 1-3, characterized in that, The first mixed incubation period is ≤24 hours, and the temperature is 25~30℃.

5. The method according to claim 3, characterized in that, The second mixing and incubation period is ≤24 hours, and the temperature is 25~30℃.

6. The method according to any one of claims 1-3, characterized in that, The nanopore sequencing is performed in a sequencing system, which includes sequencing reagents and sequencing devices, wherein the sequencing reagents include sequencing buffers and optional restraint reagents; Preferably, the sequencing buffer comprises: KCl, HEPES, ATP, and MgCl2; Preferably, the pH of the sequencing buffer is 7.5–8.5; Preferably, the restraint agent includes a restraint sequence and a corresponding buffering agent.

7. The method according to claim 6, characterized in that, The sequencing device includes: An electrolytic cell containing the sequencing buffer; A nanopore sensor is located in the center of the electrolytic cell, dividing the electrolytic cell and the sequencing buffer into a positive electrolyte region and a negative electrolyte region. The first electrode and the second electrode are respectively disposed in the positive electrolyte region and the negative electrolyte region, and the first electrode and the second electrode are connected to the signal processing chip.

8. The method according to claim 7, characterized in that, The nanopore sensor includes: a membrane and a pore inserted into the membrane to form a channel; Preferably, the membrane is selected from any one or more of the following: lipid layer, artificial polymer membrane, and solid film; Preferably, the pores are biological protein pores or solid nanopores; Preferably, the biological protein pores are selected from any one or more of the following, or their homologs or mutants: hemolysin, leukocidin, Mycobacterium smegmatis porin A, MspB, MspC, MspD, α-Haemolysin, CsgG, Aerolysin, cytolysin, outer membrane porin F, outer membrane porin G, outer membrane phospholipase A, Neisseria spp. autotransporter lipoprotein, WZA, GspD, BCP34 or BCP58.

9. The method according to claim 8, characterized in that, Prior to performing the nanopore sequencing, the method further includes: attaching a library to the membrane using the restraint sequence; The library includes: the first library containing particles, the second library without particles, the resuspended third library, or the fourth library containing particles; Preferably, the library comprises: the first library containing particles, the resuspended third library, or the fourth library containing particles, and the restraint sequence is first mixed and incubated for a third time with the sequencing buffer in the sequencing system, and then the library is incubated for a fourth time with the sequencing buffer after the third mixing and incubation, thereby attaching the library to the membrane; Preferably, the library is the particle-free second library, and the restraint sequence is first mixed and incubated a third time with the sequencing buffer in the sequencing system, and then the library is incubated a fourth time with the sequencing buffer after the third mixing and incubation, thereby attaching the library to the membrane; or The library is the second library without particles, and the restraint sequence is mixed with the library and then mixed and incubated for the fifth time with the sequencing buffer in the sequencing system, thereby attaching the library to the membrane; More preferably, the restraint sequence is: 5'-YYYY-SEQ ID NO: 1-3'; wherein the 5' end of the restraint sequence is modified with cholesterol, and Y represents iSp18; More preferably, the temperature for performing the nanopore sequencing is 28–32°C, and the sequencing voltage is 0.18–0.23V; More preferably, the third mixing incubation time is ≤30 min, and the temperature is the sequencing temperature, which is 28–32 °C; More preferably, the fourth mixing incubation time is ≤10 min, and the temperature is the sequencing temperature. The temperature is 28–32℃; More preferably, the fifth mixing incubation time is ≤10 min, and the temperature is the sequencing temperature, which is 28-32℃.

10. The method according to any one of claims 1-9, characterized in that, The particles include a matrix and surface modifications, wherein the matrix of the particles includes a hydrophobic substance, a hydrophilic substance, or an amphiphilic substance. Preferably, the surface modification of the particles contains any one of the following groups: -HN + (C2H5)2 or -N + (CH3)3.

11. The method according to claim 10, characterized in that, The hydrophobic material is selected from any one or more of the following: glass, ceramics, iron oxide, silicon dioxide, polymers or metals, cholesterol, lipids, fatty acids, carbon nanotubes, amino acids or hydrophobic polymers; Preferably, the hydrophobic polymer is polymerized from any two or more of the following substances: polylactic acid, polystyrene, or polymethacrylates.

12. The method according to claim 10, characterized in that, The hydrophilic material includes a cross-linked hydrophilic material, wherein the cross-linked hydrophilic material is selected from any one or more of the following: hydrogel, cross-linked protein particles, cross-linked amino acids or cross-linked hydrophilic polymers. Preferably, the crosslinked hydrophilic polymer is polymerized from any two or more of the following substances: sodium polyacrylate, gelatin, or a gelatin-sodium gelatate copolymer.

13. The method according to claim 10, characterized in that, The amphiphilic substances include amphiphilic polymers; Preferably, the amphiphilic polymer is polymerized from any two or more of the following substances: polysaccharides, amino acids, proteins, acrylic acid, polyethylene glycol, polyvinyl alcohol, polyethylene glycol methyl ether, polyvinylpyrrolidone, polyoxyethylene, polyethylene oxide, acrylates, polyethylene, polystyrene, polyvinyl chloride, polytetrafluoroethylene, polydimethylsiloxane, polyester, or polyurethane.

14. The method according to any one of claims 1-3, characterized in that, The average length of the library is 1kb-50kb.

15. The method according to any one of claims 1-9, characterized in that, The diameter of the particles is ≤900μm; Preferably, the diameter of the particles is 1-200 μm.

16. A nanopore sequencing kit, characterized in that, The kit includes particles and sequencing reagents; wherein the sequencing reagents include sequencing buffer and optional restraint reagents; Preferably, the sequencing buffer comprises: KCl, HEPES, ATP, and MgCl2; Preferably, the pH of the sequencing buffer is 7.5–8.5; Preferably, the restraint agent includes a restraint sequence and a corresponding buffering agent.

17. The kit according to claim 16, characterized in that, The particles include a matrix and surface modifications, wherein the matrix of the particles includes a hydrophobic substance, a hydrophilic substance, or an amphiphilic substance. Preferably, the surface modification of the particles contains any one of the following groups: -HN + (C2H5)2 or -N + (CH3)3; Preferably, the hydrophobic material is selected from any one or more of the following: glass, ceramics, iron oxide, silicon dioxide, polymers or metals, cholesterol, lipids, fatty acids, carbon nanotubes, amino acids or hydrophobic polymers; More preferably, the hydrophobic polymer is polymerized from any two or more of the following substances: polylactic acid, polystyrene, or polymethacrylates; Preferably, the hydrophilic material includes a cross-linked hydrophilic material, wherein the cross-linked hydrophilic material is selected from any one or more of the following: hydrogels, cross-linked protein particles, cross-linked amino acids, or cross-linked hydrophilic polymers; More preferably, the crosslinked hydrophilic polymer is polymerized from any two or more of the following substances: sodium polyacrylate, gelatin, or a gelatin-sodium gelatate copolymer; Preferably, the amphiphilic substance comprises an amphiphilic polymer; More preferably, the amphiphilic polymer is polymerized from any two or more of the following substances: polysaccharides, amino acids, proteins, acrylic acid, polyethylene glycol, polyvinyl alcohol, polyethylene glycol methyl ether, polyvinylpyrrolidone, polyoxyethylene, polyethylene oxide, acrylates, polyethylene, polystyrene, polyvinyl chloride, polytetrafluoroethylene, polydimethylsiloxane, polyester, or polyurethane.

18. The reagent kit according to claim 16, characterized in that, The diameter of the particles is ≤900μm; Preferably, the diameter of the particles is 1-200 μm.

19. The kit according to claim 16, characterized in that, The kit also includes: a membrane, and a pore inserted into the membrane to form a channel; Preferably, the membrane is selected from any one or more of the following: lipid layer, artificial polymer membrane, and solid film; Preferably, the pores are biological protein pores or solid nanopores; Preferably, the biological protein pores are selected from any one or more of the following, or their homologs or mutants: hemolysin, leukocidin, Mycobacterium smegmatis porin A, MspB, MspC, MspD, α-Haemolysin, CsgG, Aerolysin, cytolysin, outer membrane porin F, outer membrane porin G, outer membrane phospholipase A, Neisseria spp. autotransport lipoprotein, WZA, GspD, BCP34, and BCP58.

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