Sequencing buffer solution and method for nanopore-based capture of target nucleic acid sequence

Abstract

Provided is a sequencing buffer solution and a method for the nanopore-based capture of a target nucleic acid sequence. The sequencing buffer solution comprises a molecular crowding agent. The method employs a nanopore to capture a target nucleic acid sequence from a sequencing buffer solution. The sequencing buffer solution and the method increase the probability that a sequencing library (or other target analyte) is captured by the nanopore protein during the sequencing process, thereby increasing sequencing data output.

Description

Sequencing buffer and method for capturing nucleic acid sequence to be tested by nanopore TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and in particular, the present application relates to a sequencing buffer and a method for capturing nucleic acid sequence to be tested by nanopore, and more particularly, the present application relates to a nanopore sequencing method. BACKGROUND

[0002] Nanopore sequencing (schematic diagram of the principle of nanopore sequencing is shown in Figure 1) is an emerging single-molecule sequencing technology that directly sequences DNA or RNA molecules by using the current signal change in the nanopore. Nanopore is a tiny pore, usually composed of nanoscale pores, which can be realized through nanoscale geometry or protein channels. In nanopore sequencing, DNA or RNA molecules are guided through the nanopore, and when the bases pass through the pore, it will cause a change in the current signal. These changes in signal can be detected and recorded, thereby determining the bases in the sequence.

[0003] Nanopore sequencing often requires a large amount of library input. At present, the MinION sequencer released by Oxford Nanopore Sequencing Company in the United Kingdom is based on the SQK-LSK109 kit for library construction, which requires a target nucleic acid input of 1 μg or more, and the sequencing library input after library construction needs several tens of femtomoles (fmol). The target nucleic acid input required for large-scale parallel sequencing is generally several tens of nanograms, which is much smaller than the target nucleic acid input required for large-scale parallel sequencing. Compared with large-scale parallel sequencing technology, nanopore sequencing requires a larger amount of target nucleic acid input.

[0004] Therefore, it is urgent to improve the probability of capturing sequencing library by nanopore protein in the sequencing process, reduce the input amount of sequencing library, and thereby achieve the reduction of target nucleic acid input.

[0005] SUMMARY

[0006] The present application aims to at least partially solve at least one of the technical problems existing in the prior art.

[0007] The inventors found that the conventional sequencing buffer in the current nanopore sequencing technology generally contains potassium chloride, ATP, MgCl2, buffer components, and the probability of the sequencing library (or other analyte to be detected) being captured by the nanopore protein is low, and more sequencing library (or other analyte to be detected) needs to be put in to increase the capture. In order to overcome this problem, the inventors add a certain proportion of molecular crowding agent to the conventional sequencing buffer. A certain proportion of PEG and other molecular crowding agents can form a crowding effect, increase the probability of the sequencing library (or other analyte to be detected) being captured by the nanopore protein in the sequencing process, thereby improving the output of sequencing data, and in this process, the threading time (i.e. the sequencing speed) of the sequencing library (or other analyte to be detected) will not change due to the crowding effect.

[0008] Based on this, the first aspect of the present application, the present application provides a sequencing buffer. According to an embodiment of the present application, the sequencing buffer comprises: a molecular crowding agent. According to the sequencing buffer of the embodiment of the present application, the probability of the sequencing library (or other analyte to be detected) being captured by the nanopore protein in the sequencing process can be increased, thereby improving the output of sequencing data.

[0009] The second aspect of the present application, the present application provides a method for capturing a nucleic acid sequence to be detected by a nanopore. According to an embodiment of the present application, the nucleic acid sequence to be detected is captured and processed by a nanopore in the sequencing buffer of the first aspect of the present application. According to the method of the embodiment of the present application, the probability of the sequencing library (or other analyte to be detected) being captured by the nanopore protein in the sequencing process can be increased, thereby improving the output of sequencing data.

[0010] The third aspect of the present application, the present application provides a nanopore sequencing method. According to an embodiment of the present application, the method comprises: performing nanopore sequencing processing on the nucleic acid sequence to be detected in the sequencing buffer of the first aspect of the present application. According to the method of the embodiment of the present application, the probability of the sequencing library (or other analyte to be detected) being captured by the nanopore protein in the sequencing process can be increased, thereby improving the output of sequencing data.

[0011] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 is a schematic diagram of the principle of nanopore sequencing and a current amplitude graph. Figure 1A is a sequencing library with a sequencing adaptor connected being captured by a nanopore protein under the action of an electric field force. When one strand (the sequencing strand) passes through the nanopore protein, it produces a change in ion flow, and the pA-level electrical signal change is read through a signal amplifier and a digital-to-analog converter; Figure 1B shows the electrical signal amplitude graph of a sequencing strand being captured and perforated by a nanopore protein, and the perforated part is shown as a.

[0013] Figure 2 is a current amplitude graph for 1 minute in Example 1-Example 4, and Comparative Example 1. Figure 2A is in a conventional sequencing buffer 1, multiple sequencing events occur, and the time interval between the sequencing events is indicated as I; Figure 2B is in a sequencing buffer containing PEG 1K, multiple sequencing events occur, and the event interval between the sequencing events is indicated as II.

[0014] Figure 3 is a statistical column chart of the interval time between two sequencing events in Example 1-Example 4, and Comparative Example 1.

[0015] Figure 4 is a statistical column chart of the number of sequencing events in 1 hour of sequencing in Example 1-Example 4, and Comparative Example 1.

[0016] Figure 5 is a statistical column chart of the sequencing speed in Example 1-Example 4, and Comparative Example 1. DETAILED DESCRIPTION

[0017] The embodiments of the present application are described in detail below. The embodiments described below are exemplary only, and are intended to explain the present application, and are not to be understood as limiting the present application.

[0018] It should be noted that the terms "first", "second" are only for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined with "first", "second" can explicitly or implicitly include one or more of the features. Further, in the description of the present application, unless otherwise specified, the meaning of "a plurality of" is two or more.

[0019] In order to more easily understand the present application, certain technical and scientific terms are defined in detail below. Unless otherwise clearly defined elsewhere in the present application, all other technical and scientific terms used in the present application have the meanings commonly understood by those of ordinary skill in the art to which the present application belongs.

[0020] In the present application, the term "comprising" or "including" is an open-ended expression, i.e. including the content indicated by the present application, but not excluding other aspects.

[0021] In the present invention, the term "optionally", "optional" or "optional" generally refers to the event or condition described subsequently can but does not necessarily occur, and the description includes the case where the event or condition occurs, as well as the case where the event or condition does not occur.

[0022] In the present invention, the term "molecular crowder" generally refers to a compound added to the reaction system to increase the molecular concentration and intermolecular interaction of the solution in the reaction system. The molecular crowder can be a high molecular compound, such as polyethylene glycol (PEG) or other natural or synthetic high molecular polymer. According to an embodiment of the present invention, the molecular crowder is polyethylene glycol (PEG), and the crowder occupies the volume in the reaction system and interacts with other molecules, resulting in limited space and increased interaction between molecules, thereby simulating the crowded environment in cells. The introduction of the molecular crowder can simulate the high molecular concentration conditions in cells, change the physical and chemical properties of the reaction system. It can increase the interaction between reactants, promote the proximity and reaction rate between molecules, while reducing the occurrence of non-specific reactions and side reactions. The molecular crowder can also improve the selectivity and efficiency of the reaction, and improve the yield and purity of the reaction product.

[0023] In the present invention, the term "crowding effect" generally refers to the influence caused by the interaction and spatial limitation between a large number of molecules or particles in the solution at high concentration. In nanopore sequencing, when a DNA or RNA molecule passes through a nanopore, there are a large number of nucleotides, ions and other molecules in the surrounding solution. These molecules will diffuse, move and interact with the molecules to be detected in the nanopore. Due to the mutual repulsion and interaction of molecules in the solution at high concentration, the crowding effect will have an impact on the process of nanopore sequencing. The crowding effect can cause the following effects: limit the movement of molecules: at high concentration, molecules in the solution will collide and interact with each other, resulting in limited movement of molecules, which can cause the passage rate of molecules in the nanopore to slow down or be blocked; affect ion transport: the concentration and interaction of ions in the solution will affect the transport rate and selectivity of ions in the nanopore, and the crowding effect can cause the transport rate of ions to slow down or cause changes in ion selectivity; interfere with signal detection: the crowding effect can cause interference with signal detection in the nanopore sequencing system, such as causing noise signals or changing signal intensity.

[0024] The present invention proposes a sequencing buffer, a method for capturing a nucleic acid sequence to be detected by a nanopore, and a nanopore sequencing method, which will be described in detail below.

[0025] Sequencing buffer

[0026] In a first aspect, the present application provides a sequencing buffer. According to an embodiment of the present application, the sequencing buffer comprises a molecular crowding agent. According to the sequencing buffer of the embodiment of the present application, the probability of the sequencing library (or other analyte to be tested) being captured by the nanopore protein during the sequencing process can be improved, so as to improve the output of the sequencing data.

[0027] According to an embodiment of the present application, the mass-volume ratio of the molecular crowding agent in the sequencing buffer is (0.1-20):100, in g:mL. According to the sequencing buffer of the embodiment of the present application, the probability of the sequencing library (or other analyte to be tested) being captured by the nanopore protein during the sequencing process can be further improved, so as to further improve the output of the sequencing data.

[0028] According to an embodiment of the present application, the mass-volume ratio of the molecular crowding agent in the sequencing buffer is (0.1-20):100, in g:mL. According to the sequencing buffer of the embodiment of the present application, the probability of the sequencing library (or other analyte to be tested) being captured by the nanopore protein during the sequencing process can be further improved, so as to further improve the output of the sequencing data.

[0029] According to an embodiment of the present application, the molecular crowding agent comprises at least one of dextran, sucrose, sorbitol, trimethylamine oxide, betaine, dimethyl sulfoxide, glycerol, triethylene glycol, ethylene glycol, methanol, ethanol, urea, acetamide, and polyethylene glycol.

[0030] According to an embodiment of the present application, the molecular weight of the polyethylene glycol is not less than 200 daltons. According to the sequencing buffer of the embodiment of the present application, the probability of the sequencing library (or other analyte to be tested) being captured by the nanopore protein during the sequencing process can be improved, so as to improve the output of the sequencing data. The sequencing buffer contains a certain proportion of PEG as a molecular crowding agent, which can form a crowding effect to improve the capture probability of the sequencing library (or other analyte to be tested), so as to improve the output of the sequencing data. In this process, the perforation time (i.e., the sequencing speed) of the sequencing library (or other analyte to be tested) will not change due to the crowding effect.

[0031] According to an embodiment of the present application, the molecular weight of the polyethylene glycol is 1000 daltons to 8000 daltons. According to the sequencing buffer of the embodiment of the present application, the probability of the sequencing library (or other analyte to be tested) being captured by the nanopore protein during the sequencing process can be further improved, so as to improve the output of the sequencing data, and the perforation time (i.e., the sequencing speed) of the sequencing library (or other analyte to be tested) will not change due to the crowding effect.

[0032] According to an embodiment of the present application, the polyethylene glycol has a molecular weight of 2000-3350 Dalton. The sequencing buffer according to the embodiment of the present application can further improve the probability of the sequencing library (or other analyte to be tested) being captured by the nanopore protein during the sequencing process, thereby improving the output of sequencing data, and the threading time (i.e. the sequencing speed) of the sequencing library (or other analyte to be tested) will not be changed due to the crowding effect.

[0033] According to an embodiment of the present application, the buffer further comprises metal ions, buffers, and NTPs and / or dNTPs. These components play an important role in the nanopore sequencing process. According to an embodiment of the present application, the metal ions are a component of the buffer, usually in the form of a salt, and the presence of metal ions can provide a stable ionic environment, adjust the enzyme activity and stability of the reaction system, and common metal ions include magnesium ions (Mg 2+ ), which play a key role in the catalytic activity of DNA polymerase and the process of DNA replication; buffers are compounds used to maintain the pH stability of the buffer, which can adjust the acidity and alkalinity of the solution, and provide suitable enzyme activity and reaction conditions. Commonly used buffers include Tris (Tris(hydroxymethyl)aminomethane), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), etc.; NTPs, dNTPs and / or ddNTPs are nucleoside triphosphates, deoxynucleoside triphosphates and / or dideoxynucleoside triphosphates, which are the basic raw materials required for DNA synthesis by DNA polymerase, and NTPs and dNTPs provide the four bases required for DNA synthesis (adenine, thymine, guanine and cytosine), which are paired with complementary bases on the DNA template strand to achieve the synthesis of a new DNA strand.

[0034] According to an embodiment of the present application, the buffer comprises a pH buffer system and / or an ionic buffer system.

[0035] According to an embodiment of the present application, the pH buffer system comprises at least one of a dihydrogen phosphate-hydrogen phosphate buffer system, a carbonic acid-sodium bicarbonate buffer system, a Tris-HCl buffer system, a HEPES buffer system, and a MOPS buffer system.

[0036] According to an embodiment of the present application, the metal ions comprise at least one of monovalent and divalent metal ions.

[0037] According to an embodiment of the present application, the monovalent metal ions comprise at least one of K + and Na + .

[0038] According to an embodiment of the present application, the divalent metal ions include at least one of Mg 2+ , Mo 2+ , Cu 2+ , Fe 2+ , Zn 2+ , Ca 2+ , Pb 2+ and Cd 2+ .

[0039] According to an embodiment of the present application, the metal ion concentration is 0.1-2.5M; the buffer concentration is 1-100mM; and the concentration of NTP, dNTP and / or dNTP is 0.1-100mM. It is to be noted that the metal ion concentration is 0.1-2.5M, for example, it can be 0.1M, 0.3M, 0.5M, 0.7M, 0.9M, 1M, 1.4M, 1.6M, 1.7M, 1.8M, 1.9M, or a range value between any two of them, such as 0.3-1.9M, 0.5-1.8M, 0.7-1.7M, 0.9-1.6M, 1M-1.4M; the buffer concentration is 1-100mM, for example, it can be 1mM, 5mM, 10mM, 20mM, 35mM, 40mM, 50mM, 55mM, 65mM, 80mM, 90mM, 100mM, or a range value between any two of them, such as 5-90mM, 10-80mM, 20-65mM, 35-55mM, or 40-50mM; and the concentration of NTP, dNTP and / or dNTP is 0.1-100mM, for example, it can be 0.1mM, 0.2mM, 1mM, 3mM, 5mM, 10mM, 20mM, 35mM, 40mM, 50mM, 55mM, 65mM, 80mM, 90mM, 100mM, or a range value between any two of them, such as 5-90mM, 10-80mM, 20-65mM, 35-55mM, or 40-50mM.

[0040] Method for capturing a nucleic acid sequence to be tested by nanopore

[0041] According to a second aspect of the present application, a method for capturing a nucleic acid sequence to be tested by nanopore is provided. According to an embodiment of the present application, the method captures the nucleic acid sequence to be tested by nanopore in the sequencing buffer as described in the first aspect of the present application. According to the method of the embodiment of the present application, the probability of capturing the sequencing library (or other analyte to be tested) by nanopore protein during sequencing can be improved, thereby improving the output of sequencing data.

[0042] According to an embodiment of the present application, the nanopore is in the form of a nanopore channel.

[0043] According to an embodiment of the present application, the nanopore channel is formed by inserting the nanopore protein into the membrane.

[0044] Nanopore sequencing method

[0045] According to a third aspect of the present application, a nanopore sequencing method is provided. According to an embodiment of the present application, the method comprises: performing nanopore sequencing on the nucleic acid sequence to be sequenced in the sequencing buffer according to the first aspect of the present application. According to the method of the embodiment of the present application, the probability of capturing the sequencing library (or other analyte to be sequenced) by the nanopore protein during the sequencing process can be improved, thereby improving the output of sequencing data.

[0046] According to an embodiment of the present application, the nanopore sequencing is performed in a sequencing system, and the sequencing system is constructed by inserting the nanopore protein into the membrane to form a nanopore detection channel.

[0047] According to an embodiment of the present application, before the capturing process or the nanopore sequencing process, the nucleic acid sequence to be sequenced is pre-associated with a sequencing adapter complex, wherein the sequencing adapter complex comprises a sequencing adapter and a motor protein, the motor protein is associated with the sequencing adapter; and the associated product is subjected to library construction processing to obtain a sequencing library of the nucleic acid sequence to be sequenced.

[0048] According to an embodiment of the present application, the sequencing adapter comprises a first strand and a second strand, and at least part of the sequences of the first strand and the second strand are complementary to each other.

[0049] According to an embodiment of the present application, the first strand comprises a guide sequence, a motor protein binding sequence, a limiting sequence, and a first complementary sequence; and the second strand comprises a second complementary sequence and a third complementary sequence; wherein at least part of the sequences of the first complementary sequence and the second complementary sequence are complementary to each other.

[0050] According to an embodiment of the present application, the nucleotide sequence of the first strand is shown in SEQ ID NO: 1, and the nucleotide sequence of the second strand is shown in SEQ ID NO: 2.

[0051] According to an embodiment of the present application, the second strand is associated with a cholesterol molecule.

[0052] According to an embodiment of the present application, the sequencing adaptor complex further comprises an oligonucleotide strand, the third complementary sequence is complementary to at least a part of the sequence of the oligonucleotide strand, and the 5' end or 3' end of the oligonucleotide strand is connected to a cholesterol molecule. It needs to be further explained that, according to the unwinding direction of the helicase, if it is unwound from 5' end to 3' end, the 5' end of the oligonucleotide strand is connected to the cholesterol molecule, and if it is unwound from 3' end to 5' end, the 3' end of the oligonucleotide strand is connected to the cholesterol molecule. According to an embodiment of the present application, it needs to be explained that in the present application, the "oligonucleotide strand" is synonymous with "restraint" and "restraint sequence".

[0053] According to an embodiment of the present application, the motor protein is a helicase.

[0054] According to an embodiment of the present application, the helicase is selected from at least one of CsgG helicase, Dda helicase, Pif1 helicase, XPD helicase, T7 Gp41 helicase, and DnaB helicase.

[0055] According to an embodiment of the present application, the helicase is a mutant enzyme. It needs to be explained that, according to an embodiment of the present application, the mutant modification is achieved by mutating the gene of the enzyme, changing the amino acid sequence of the enzyme, and thus changing the structure and function of the enzyme. Such mutant modification can be achieved by genetic engineering techniques, such as point mutation, insertion mutation, deletion mutation, etc. The mutant helicase can have improved performance, such as higher unwinding rate, wider substrate adaptability, stronger thermal stability, etc. By reasonably mutating and screening the amino acid sequence of the helicase, a helicase variant more suitable for specific application requirements can be obtained. Mutant modification can be achieved by various methods, including random mutation and targeted mutation. Random mutation can be achieved by inducing natural mutation, such as radiation or chemical induction mutation, or by randomly introducing mutations, such as error introduction PCR or DNA modification enzyme mediated mutation. Targeted mutation can be achieved by selectively introducing specific mutations, such as structure or sequence based targeted mutation, or by using recombination based methods, such as DNA shuffling or DNA family mutation.

[0056] According to an embodiment of the present application, the nanopore comprises a transmembrane protein pore and / or a solid-state pore.

[0057] According to an embodiment of the present application, the transmembrane protein is selected from at least one of hemolysin, MspA, MspB, MspC, MspD, FraC, ClyA, PA63, CsgG, CsgD, XcpQ, SP1, phi29 connector protein, InvG, and GspD.

[0058] According to an embodiment of the present application, the transmembrane protein is linked to a polypeptide selected from at least one of a tag, a cleavage site, a signal peptide or a leader peptide, and a detectable label.

[0059] According to an embodiment of the present application, the membrane comprises at least one of an amphiphilic membrane and / or a high polymer membrane. According to an embodiment of the present application, these membranes are used as a component of the electrolyte channel in the nanopore sequencing process. Among them, the amphiphilic membrane is a membrane with hydrophilic and hydrophobic regions, which is composed of a thin film arranged by a layer of molecules, the hydrophilic region (usually hydroxyl or carboxyl) is located on the outside of the membrane and contacts with the solution, and the hydrophobic region is located on the inside of the membrane; the high polymer membrane is a thin film composed of high polymer, and these polymers can be natural polymers such as proteins or polyamic acid, or synthetic polymers such as polyvinyl alcohol (PVA) or polyacrylic acid (PAA).

[0060] According to an embodiment of the present application, the membrane comprises at least one of a phospholipid bilayer, a diblock copolymer and a triblock copolymer. It needs to be explained that, according to an embodiment of the present application, the phospholipid bilayer is a thin film arranged by two layers of phospholipid molecules, and the phospholipid molecules have hydrophilic heads and hydrophobic tails, so that they can form a bilayer structure in water, and this phospholipid bilayer is used as a basic material for nanopore inlay in the nanopore sequencing system; the diblock copolymer and the triblock copolymer are polymers composed of two or three different chemical fragments, and these copolymers can be used as a basic material for nanopore inlay in the nanopore sequencing system.

[0061] According to an embodiment of the present application, the sequencing voltage of the nanopore sequencing is not less than 10 mV. According to the method of the embodiment of the present application, the probability of capturing the sequencing library (or other analyte to be tested) by the nanopore protein in the sequencing process can be improved, so as to improve the output of sequencing data.

[0062] According to an embodiment of the present application, the sequencing voltage of the nanopore sequencing is 50 mV-250 mV. According to the method of the embodiment of the present application, the probability of capturing the sequencing library (or other analyte to be tested) by the nanopore protein in the sequencing process can be further improved, so as to improve the output of sequencing data.

[0063] The sequence list of the present application is as follows:

[0064] Sequence list

[0065] In which, X = iSPC3, Y = iSP18.

[0066] The schemes of the present application will be explained below in connection with examples. Those skilled in the art will understand that the examples below are only for illustration of the present application and should not be regarded as limiting the scope of the present application. If no specific technique or condition is specified in the examples, the technique or condition described in the literature in the art or according to the product manual is used. If no manufacturer of the reagent or instrument is specified, it is a conventional product that can be obtained commercially.

[0067] Example 1

[0068] This example is for collecting sequencing data in sequencing buffer 2 containing PEG 1K. The specific experimental procedure is as follows:

[0069] A single nanopore protein CsgG mutant (the specific protein sequence is shown in SEQ ID NO: 3) is inserted into a phospholipid membrane to form a nanopore detection channel, forming a sequencing system. The Escherichia coli (Genbank: CP000946.1) genome is extracted and randomly interrupted to construct a sequencing library. The sequencing library is connected to the nucleic acid fragment to be tested by using T4 DNA ligase or other commonly used methods to connect the sequencing adapter complex combined with the motor protein. The sequencing adapter complex includes a sequencing adapter and a motor protein combined with the sequencing adapter, and the sequencing adapter is formed by annealing a first strand (the specific nucleotide sequence is shown in SEQ ID NO: 1) and a second strand (the specific nucleotide sequence is shown in SEQ ID NO: 2), the first strand includes a guide sequence, a motor protein binding sequence, a limiting sequence, a first complementary sequence, and the second strand includes a second complementary sequence, a third complementary sequence. The sequencing adapter complex further includes an oligonucleotide chain (also known as a confinement sequence or a confiner, the confinement sequence has a portion combined with the phospholipid membrane at one end), the third complementary sequence is complementary to at least a portion of the sequence of the confinement sequence, and the sequencing library is combined with the phospholipid membrane inlaid with the nanopore under the action of the confinement sequence. 200 ng of the sequencing library, 6 μL of 1 μM confinement sequence (the specific nucleotide sequence is shown in SEQ ID NO: 4), and 300 μL of sequencing buffer are mixed uniformly and then added to the sequencing system. The sequencing buffer is sequencing buffer 2 containing PEG 1K. Incubate at 0 V and 30°C for 20 minutes, and sequence at an open voltage of 0.18 V, and collect sequencing data for 1 hour.

[0070] The formula of sequencing buffer 2 containing PEG 1K is shown as follows:

[0071] Sequencing buffer 2 containing PEG 1K: 380 mM KCl, 25 mM HEPES, 15 mM ATP, 25 mM MgCl2, PEG 1K (Polyethylene glycol 1K, TCI, P2828), pH 8.0, wherein the mass volume ratio of PEG 1K to sequencing buffer 2 is 5 g: 100 mL.

[0072] Example 2

[0073] Experimental example 2 is the same as the experimental procedure of example 1, except that the sequencing buffer is sequencing buffer 3 containing PEG 2K.

[0074] The formula of sequencing buffer 3 containing PEG 2K is shown as follows:

[0075] Sequencing buffer 3 containing PEG 2K: 380 mM KCl, 25 mM HEPES, 15 mM ATP, 25 mM MgCl2, PEG 2K (Polyethylene glycol 2K, TCI, P2034), pH 8.0, wherein the mass volume ratio of PEG 2K to sequencing buffer 3 is 5 g: 100 mL.

[0076] Example 3

[0077] Experimental example 3 is the same as the experimental procedure of example 1, except that the sequencing buffer is sequencing buffer 4 containing PEG 3.35K.

[0078] The formula of sequencing buffer 4 containing PEG 3.35K is shown as follows:

[0079] Sequencing buffer 4 containing PEG 3.35K: 380 mM KCl, 25 mM HEPES, 15 mM ATP, 25 mM MgCl2, 5% PEG 3.35K (Polyethylene glycol 3.35K, HEOWNS, P-69710), pH 8.0, wherein the mass volume ratio of PEG 3.35K to sequencing buffer 4 is 5 g: 100 mL.

[0080] Example 4

[0081] Experimental example 4 is the same as the experimental procedure of example 1, except that the sequencing buffer is sequencing buffer 5 containing PEG 8K.

[0082] The formula of sequencing buffer 5 containing PEG 8K is shown as follows:

[0083] Sequencing buffer 5 containing PEG 8K: 380 mM KCl, 25 mM HEPES, 15 mM ATP, 25 mM MgCl2, PEG 8K (Polyethylene glycol 8K, sigma, 89510-250G-F), pH 8.0, wherein the mass volume ratio of PEG 8K to sequencing buffer 5 is 5 g: 100 mL.

[0084] Comparative example 1

[0085] Comparative example 1 and example 1 have the same experimental steps, the difference is that the sequencing buffer is conventional sequencing buffer 1.

[0086] The formula of conventional sequencing buffer 1 is as follows:

[0087] Conventional sequencing buffer 1: 380 mM KCl, 25 mM HEPES, 15 mM ATP, 25 mM MgCl2, pH 8.0.

[0088] The interval time (s) between adjacent two sequencing events, the number of capture events, and the sequencing speed (nt / s) of each sequencing event in examples 1-4 and comparative example 1 were counted, and the results are shown in Table 1. The statistical results of the interval time (s) between adjacent two sequencing events in examples 1-4 and comparative example 1 are shown in FIG. 3, the number of capture events in examples 1-4 and comparative example 1 are shown in FIG. 4, and the sequencing speed (nt / s) of each sequencing event in examples 1-4 and comparative example 1 are shown in FIG. 5.

[0089] Table 1

[0090] Wherein, N is the sample number of each group of sequencing events;

[0091] The formula for calculating the sequencing speed is as follows:

[0092] Compared with comparative example 1, the interval time between two sequencing events in examples 1-4 is shorter, the number of capture events in examples 1-4 is more, and the sequencing speed of each sequencing event in examples 1-4 is not much different from that of comparative example 1, which shows that using sequencing buffer containing a certain proportion of PEG can significantly increase the probability of library being captured by nanopore, and will not cause the increase of library perforation time.

[0093] In the description of the specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are contained in at least one embodiment or example of the present application. In the specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any suitable manner in any one or more embodiments or examples. In addition, the person skilled in the art can combine and combine the different embodiments or examples described in the specification and the features of the different embodiments or examples without contradiction.

[0094] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and the person skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. A sequencing buffer, characterized in that, The application relates to a sequencing buffer for capturing and sequencing a nucleic acid sequence. The molecular crowding agent has a mass-volume ratio of (0.1-20):100 g:mL in the sequencing buffer.

2. The sequencing buffer of claim 1, wherein, The molecular crowding agent has a mass-volume ratio of (0.5-10):100 g:mL in the sequencing buffer.

3. The sequencing buffer of claim 1, wherein, The molecular crowding agent comprises at least one of dextran, sucrose, sorbitol, trimethylamine oxide, betaine, dimethyl sulfoxide, glycerol, triethylene glycol, ethylene glycol, methanol, ethanol, urea, acetamide and polyethylene glycol.

4. The sequencing buffer of claim 1, wherein, The molecular weight of the polyethylene glycol is not less than 200 Dalton.

5. The sequencing buffer of claim 4, wherein, The molecular weight of the polyethylene glycol is 1000-8000 Dalton.

6. The sequencing buffer of claim 4, wherein, The buffer further comprises metal ions, a buffer and NTP, dNTP and / or ddNTP.

7. The sequencing buffer of claim 1, wherein, The buffer comprises a pH buffer system and / or an ionic buffer system.

8. The sequencing buffer of claim 7, wherein, The pH buffer system comprises at least one of a dihydrogen phosphate-dihydrogen phosphate buffer system, a carbonic acid-sodium bicarbonate buffer system, a Tris-HCl buffer system, a HEPES buffer system and a MOPS buffer system.

9. The sequencing buffer of claim 8, wherein, The metal ions comprise at least one of monovalent and divalent metal ions.

10. The sequencing buffer of claim 7, wherein, The concentration of the metal ions is 0.1-2.5 M; the concentration of the buffer is 1-100 mM; and the concentration of the NTP, dNTP and / or dNTP is 0.1-100 mM.

11. The sequencing buffer of claim 10, wherein, The monovalent metal ion includes at least one of K + and Na + .

12. The sequencing buffer of claim 10, wherein, The divalent metal ion includes at least one of Mg 2+ , Mo 2+ , Cu 2+ , Fe 2+ , Zn 2+ , Ca 2+ , Pb 2+ , and Cd 2+ .

13. The sequencing buffer of claim 7, wherein, The nucleic acid sequence to be detected is captured and treated by a nanopore in the sequencing buffer according to any one of claims 1-13.

14. A method of capturing a nucleic acid sequence under test by a nanopore, characterized by, The application relates to a method for capturing and sequencing a nucleic acid sequence.

15. A method of nanopore sequencing, characterized in that, The method comprises the following steps: Before the capturing treatment or the nanopore sequencing treatment, the nucleic acid sequence to be detected is connected to a sequencing adaptor complex, wherein the sequencing adaptor complex comprises a sequencing adaptor and a motor protein, and the motor protein is connected to the sequencing adaptor; and 16. The method of claim 15, wherein, The connected product is subjected to library construction treatment to obtain a sequencing library of the nucleic acid sequence to be detected.

17. The method of claim 14 or 15, wherein, The sequencing adaptor comprises a first strand and a second strand, and at least part of sequences of the first strand and the second strand are complementary to each other. The first strand comprises a guide sequence, a motor protein binding sequence, a limiting sequence and a first complementary sequence.

18. The method of claim 17, wherein, The second strand comprises a second complementary sequence and a third complementary sequence.

19. The method of claim 18, wherein, At least part of sequences of the first complementary sequence and the second complementary sequence are complementary to each other. The nucleotide sequence of the first strand is shown in SEQ ID NO: 1, and the nucleotide sequence of the second strand is shown in SEQ ID NO:

2. ​ 20. The method of claim 18, wherein, ​

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