Method for constructing nucleic acid library and use thereof

Abstract

Provided is a method for constructing a nucleic acid library. The method comprises: (a) providing a nucleic acid molecule comprising a self-amplifying adapter, the self-amplifying adapter having a palindromic sequence; (b) performing self-amplification on the nucleic acid molecule to obtain the nucleic acid library, wherein the self-amplification is realized by means of one or more cycles of a heating treatment and a cooling treatment, the palindromic sequence of the self-amplifying adapter is adapted for forming a hairpin structure, and the hairpin structure is adapted for serving as a primer for self-amplification.

Description

Method for constructing nucleic acid library and application thereof TECHNICAL FIELD

[0001] The present application belongs to the field of gene sequencing, and in particular, the present application relates to a method for constructing a nucleic acid library and application thereof. More particularly, the present application relates to a method for constructing a nucleic acid library comprising a palindromic sequence, a nucleic acid library, a kit for nucleic acid sequencing, application of the nucleic acid library in sequencing, and a method for sequencing the nucleic acid library. BACKGROUND

[0002] Single-molecule sequencing is a high-throughput sequencing technology that can directly sequence single-stranded nucleic acid molecules, providing more comprehensive and accurate sequence information, and has become an important tool in the field of genomics and biology research, providing more possibilities for scientific research and medical applications. Nanopore sequencing technology is a typical single-molecule level detection technology, which has the advantages of fast sequencing speed, long read length, direct sequencing, high throughput, low cost, small volume, and convenient portability. In the process of nanopore sequencing, a single nanopore is embedded in an insulating impermeable membrane to form a stable ion current channel. Under the action of voltage, a single-stranded nucleic acid molecule passes through the nanopore, thereby reducing the ion current passing through the nanopore. Due to the difference in molecular structure and volume size of different bases on the single-stranded nucleic acid molecule, the current passing through the nanopore exhibits differences corresponding to the base sequence. By using algorithms to analyze the changing current signal, the sequence information of the single-stranded nucleic acid passing through the nanopore can be read in real time. However, the sequencing accuracy of existing single-molecule sequencing technology is still limited, which seriously restricts the application range of single-molecule sequencing technology.

[0003] Therefore, there is an urgent need to improve the accuracy of single-molecule sequencing technology.

[0004] SUMMARY

[0005] The present application aims to at least partially solve at least one of the technical problems existing in the prior art.

[0006] The inventors found that existing single-molecule sequencing technology usually only determines the sequence of a single strand of the target nucleic acid, and the sequencing accuracy is limited. In order to overcome this problem, the inventors perform self-amplification treatment on the target nucleic acid to be tested, and the self-amplification treatment is realized in a cyclic manner of "heating treatment and cooling treatment", thereby obtaining a multiple copy nucleic acid library, and further realizing multiple sequencing of the target nucleic acid to be tested, and improving the sequencing accuracy. Moreover, the number of cycles of "heating treatment and cooling treatment" can be determined according to the number of copies of the nucleic acid library required, and the library construction process is more flexible and controllable. In addition, the library construction process of the method of the present application is simple, low in cost, and high in library yield.

[0007] Based on this, the first aspect of the present application provides a method for constructing a nucleic acid library. According to an embodiment of the present application, the method comprises (a) providing a nucleic acid molecule comprising a self-amplification adaptor, the self-amplification adaptor having a palindromic sequence; (b) performing self-amplification on the nucleic acid molecule to obtain the nucleic acid library, wherein the self-amplification is achieved by cyclically performing "heating treatment and cooling treatment" one or more times, and the palindromic sequence of the self-amplification adaptor is suitable for forming a hairpin structure, and the hairpin structure is suitable for serving as a primer for self-amplification. According to the method of the embodiment of the present application, the library construction process is simple, the cost is low, the library yield is high, the palindromic complementary sequence of ineffective repeated amplification is reduced, and the number of cycles of "heating treatment and cooling treatment" can be determined according to the required copy number of the nucleic acid library, and the library construction process is more flexible and controllable.

[0008] The second aspect of the present application provides a nucleic acid library. According to an embodiment of the present application, the nucleic acid library is constructed by the method of the first aspect of the present application. According to the nucleic acid library obtained by the method of the embodiment of the present application, the cost is low, the library yield is high, the palindromic complementary sequence of ineffective repeated amplification is reduced, and the target copy number of the target nucleic acid is controllable.

[0009] The third aspect of the present application provides a kit for constructing a nucleic acid library. According to an embodiment of the present application, the kit comprises the self-amplification adaptor of the first aspect of the present application, the self-amplification adaptor having the palindromic sequence of the first aspect of the present application; a ligase for connecting the self-amplification adaptor to a nucleic acid molecule; a polymerase having strand displacement activity for self-amplifying the nucleic acid molecule; a buffer suitable for a ligation reaction, a buffer suitable for a self-amplification reaction, and dNTPs. According to the kit of the embodiment of the present application, a nucleic acid library with low cost, high yield, reduced palindromic complementary sequence of ineffective repeated amplification, and target copy number can be obtained.

[0010] The fourth aspect of the present application provides a use of the nucleic acid library constructed by the method of the first aspect of the present application or the nucleic acid library of the second aspect of the present application in sequencing.

[0011] The fifth aspect of the present application provides a method for sequencing a nucleic acid library. According to an embodiment of the present application, the method sequences the nucleic acid library constructed by the method of the first aspect of the present application or the nucleic acid library of the second aspect of the present application to obtain a sequencing result. According to the method of the embodiment of the present application, multiple sequencing of the target nucleic acid to be sequenced can be achieved, and the sequencing accuracy can be improved. BRIEF DESCRIPTION OF DRAWINGS

[0012] FIG. 1 is a schematic diagram of a specific structure of a target nucleic acid to which a first self-amplification adaptor and a second self-amplification adaptor are respectively arranged at two ends according to an embodiment of the present application.

[0013] FIG. 2 is a schematic diagram of a specific structure of a target nucleic acid to which a first self-amplification adaptor is arranged at one end according to an embodiment of the present application.

[0014] FIG. 3 is a schematic diagram of a specific structure of a target nucleic acid to which a second self-amplification adaptor is arranged at one end according to an embodiment of the present application.

[0015] FIG. 4 is a schematic diagram of a specific structure of a self-amplification adaptor according to an embodiment of the present application.

[0016] FIG. 5 is a schematic diagram of a specific structure of a first self-amplification product according to an embodiment of the present application.

[0017] FIG. 6 is a schematic diagram of a specific structure of a second self-amplification product according to an embodiment of the present application.

[0018] FIG. 7 is a schematic diagram of a library construction method of a target nucleic acid to which self-amplification adaptors are connected at two ends according to an embodiment of the present application.

[0019] FIG. 8 is a schematic diagram of a library construction method of a target nucleic acid to which a self-amplification adaptor is connected at one end according to an embodiment of the present application.

[0020] FIG. 9 is a schematic diagram of a method of constructing a nucleic acid library and nanopore sequencing in one embodiment of the present application.

[0021] FIG. 10 is a schematic diagram of a method of constructing a sequencing adaptor complex in Example 1.

[0022] FIG. 11 is an electropherogram of a sequencing adaptor complex in Example 1.

[0023] FIG. 12 is a current signal diagram obtained after nanopore sequencing of a target nucleic acid sequence subjected to multiple self-amplifications in Example 1.

[0024] FIG. 13 is a schematic diagram of a method of constructing a nucleic acid library and nanopore sequencing in another embodiment of the present application. DETAILED DESCRIPTION

[0025] Embodiments of the present application are described in detail below. The embodiments described below are exemplary only and are not to be construed as limiting the present application.

[0026] It should be noted that the terms "first", "second" are used only for descriptive purposes and should not be construed as indicating or implying relative importance or an indicated number of technical features. Thus, features defined with "first", "second" can explicitly or implicitly include one or more of the features. Further, in the description of the present application, the meaning of "a plurality of" is two or more, unless otherwise specified.

[0027] For the purposes of the present invention, certain technical and scientific terms are specifically defined below. Unless specifically defined herein, all other technical and scientific terms used in the present invention have the meanings that are commonly understood by one of ordinary skill in the art in the field of the present invention.

[0028] In the present invention, the term "comprising" or "including" is an open term, which means that it includes the recited elements, but not excluding other elements.

[0029] In the present invention, the term "optionally", "optional" or "optional" generally means that the subsequently described event or circumstance can or can not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0030] In the present invention, the term "self-amplification process" refers to the amplification of the target nucleic acid itself, obtaining a single-stranded self-amplification product, which has a complementary pairing region, and then forming a complementary pairing secondary structure.

[0031] In the present invention, the term "palindrome" refers to a specific nucleotide fragment in a DNA or RNA molecule, which has a sequence read in the 5' to 3' order on one strand identical to the sequence read in the same 5' to 3' order on its complementary strand. The DNA or RNA of the palindrome has a center of symmetry, the bases on both sides of the center of symmetry are symmetrical about the center of symmetry, and can form a complement. Therefore, the palindrome can form a hairpin structure (stem-loop structure).

[0032] The present invention proposes a method for constructing a nucleic acid library, a nucleic acid library, a kit for nucleic acid sequencing, a method for sequencing a nucleic acid library, and uses thereof, which will be described in detail below.

[0033] Method for constructing a nucleic acid library

[0034] In a first aspect of the present invention, the present invention proposes a method for constructing a nucleic acid library. According to an embodiment of the present invention, the method comprises (a) providing a nucleic acid molecule comprising a self-amplification adapter, the self-amplification adapter having a palindrome sequence; (b) performing self-amplification on the nucleic acid molecule to obtain the nucleic acid library, wherein the self-amplification is achieved by cyclically performing "heating treatment and cooling treatment" one or more times, the palindrome sequence of the self-amplification adapter is adapted to form a hairpin structure, and the hairpin structure is adapted to serve as a primer for self-amplification. According to the method of the embodiment of the present invention, the library construction process is simple, the cost is low, the library yield is high, and the palindrome complementary sequence of ineffective repeated amplification is reduced.

[0035] According to an embodiment of the present invention, the nucleic acid library is a multi-copy nucleic acid library.

[0036] According to an embodiment of the present application, the number of cycles of self-amplification is determined according to the copy number of the nucleic acid library.

[0037] According to an embodiment of the present application, the temperature of each heating treatment is 40-80°C.

[0038] According to an embodiment of the present application, the time of each heating treatment is 1-5 min.

[0039] According to an embodiment of the present application, the temperature of each cooling treatment is 20-40°C.

[0040] According to an embodiment of the present application, the time of each cooling treatment is 1-30 min.

[0041] According to an embodiment of the present application, the number of cycles is 1-30.

[0042] According to an embodiment of the present application, the self-amplification procedure is to perform "incubation at 65°C for 2 min, incubation at 37°C for 2 min" n times, wherein n is an integer and 1≤n≤30. n

[0043] According to an embodiment of the present application, the nucleic acid library is a multiple-copy nucleic acid library. For example, a two-copy nucleic acid library, a four-copy nucleic acid library, an eight-copy nucleic acid library, or a sixteen-copy nucleic acid library.

[0044] According to an embodiment of the present application, the number of cycles of self-amplification is determined according to the copy number of the nucleic acid library.

[0045] In this context, the so-called "multiple-copy nucleic acid library" refers to the target double-stranded polynucleotide in the library comprising multiple nucleotide segments for determining the same piece of nucleotide sequence information. For example, the so-called "two-copy nucleic acid library" refers to the target double-stranded polynucleotide in the library comprising two nucleotide segments for determining the same piece of nucleotide sequence information, one of which is located on the first strand and the other on the second strand, the first and second strands are connected together by a linker (such as a self-amplification linker), and the two nucleotide segments are base complementary to each other; similarly, the so-called "four-copy nucleic acid library" refers to the target double-stranded polynucleotide in the library comprising four nucleotide segments for determining the same piece of nucleotide sequence information, two of which are connected in series on the first strand and the other two are connected in series on the second strand, the first and second strands are connected together by a linker (such as a self-amplification linker), and the two nucleotide segments on the first strand are base complementary to the other two nucleotide segments on the second strand; and so on to explain more copy number of nucleic acid library.

[0046] ​The person skilled in the art can understand that the "heating treatment and cooling treatment" is performed one or more times in cycles, and the number of cycles required for self-amplification can be determined according to the number of copies of the nucleic acid library required.

[0047] For example, when a two-copy nucleic acid library is required, the "heating treatment and cooling treatment" is performed only once, thereby obtaining a two-copy nucleic acid library; more specifically, when a two-copy nucleic acid library is required, the "65℃ incubation for 2 minutes, 37℃ incubation for 2 minutes" is performed only once, thereby obtaining a two-copy nucleic acid library. The two-copy nucleic acid library is a single strand of nucleic acid containing a complementary pairing region, and its sequence consists of the following parts: self-amplification adapter sequence-first strand sequence of target nucleic acid (original strand, 1D)-self-amplification adapter sequence-second strand sequence of target nucleic acid (amplification strand amplified for the first time, 2D)-self-amplification adapter sequence.

[0048] For example, when a two-copy nucleic acid library is required, the "heating treatment and cooling treatment" is performed only once, thereby obtaining a two-copy nucleic acid library; more specifically, when a two-copy nucleic acid library is required, the "65℃ incubation for 2 minutes, 37℃ incubation for 2 minutes" is performed only once, thereby obtaining a two-copy nucleic acid library. The two-copy nucleic acid library is a single strand of nucleic acid containing a complementary pairing region, and its sequence consists of the following parts: self-amplification adapter sequence-first strand sequence of target nucleic acid (original strand, 1D)-self-amplification adapter sequence-second strand sequence of target nucleic acid (amplification strand amplified for the first time, 2D)-self-amplification adapter sequence.

[0049] For example, when an eight-copy nucleic acid library is needed, the "heating and cooling treatment" is performed three times, thereby obtaining an eight-copy nucleic acid library; more specifically, when an eight-copy nucleic acid library is needed, the "65°C incubation for 2 minutes, 37°C incubation for 2 minutes" is performed once, then the "65°C incubation for 2 minutes, 37°C incubation for 4 minutes" is performed once, and then the "65°C incubation for 2 minutes, 37°C incubation for 8 minutes" is performed once, thereby obtaining an eight-copy nucleic acid library. Since the nucleic acid library length is doubled after the first self-amplification, the time for the cooling incubation is doubled in the second self-amplification; after the second self-amplification, the nucleic acid library length is quadrupled, so the time for the cooling incubation is quadrupled in the third self-amplification. The eight-copy nucleic acid library is a nucleic acid single strand containing a complementary pairing region, and its sequence is composed of the following parts: self-amplification adapter sequence-target nucleic acid first strand sequence (original strand, 1D)-self-amplification adapter sequence-target nucleic acid second strand sequence (amplification strand after the first amplification, 2D)-self-amplification adapter sequence-target nucleic acid first strand sequence (amplification strand after the second amplification, 3D)-self-amplification adapter sequence-target nucleic acid second strand sequence (amplification strand after the second amplification, 4D)-self-amplification adapter sequence-target nucleic acid first strand sequence (amplification strand after the third amplification, 5D)-self-amplification adapter sequence-target nucleic acid second strand sequence (amplification strand after the third amplification, 6D)-self-amplification adapter sequence-target nucleic acid first strand sequence (amplification strand after the third amplification, 7D)-self-amplification adapter sequence-target nucleic acid second strand sequence (amplification strand after the third amplification, 8D)-self-amplification adapter sequence.

[0050] According to the above method, a nucleic acid library with more copies can be obtained.

[0051] According to an embodiment of the present application, determining the number of cycles of self-amplification according to the copy number of the nucleic acid library comprises determining the number of cycles of self-amplification according to the following formula: y = log2(x)

[0052] wherein y is the number of cycles of self-amplification, x is the copy number of the nucleic acid library, x is an integer, and x≥2.

[0053] According to an embodiment of the present application, 2≤x≤1024, and 1≤y≤10.

[0054] According to an embodiment of the present application, 2≤x≤32, and 1≤y≤5.

[0055] The skilled in the art can understand that the relationship between the copy number of the nucleic acid library and the cycle number of the isothermal self-amplification is exponential at the beginning, and then reaches a plateau due to the inactivation of polymerase and the exhaustion of dNTP, and the amplification curve is similar to the conventional PCR amplification curve. Generally, the copy number of the nucleic acid library is only required to be 2-1024 in practical application, and the cycle number of the self-amplification is 1-10; more commonly, the copy number of the nucleic acid library is 2-32, and the cycle number of the self-amplification is 1-5. In these common cases, the copy number of the nucleic acid library and the cycle number of the isothermal self-amplification are exponentially related.

[0056] In addition, it needs to be further explained that the copy number of the nucleic acid library generated by the "temperature rising-temperature falling" self-amplification is related to the cycle number of the isothermal self-amplification. Therefore, theoretically, the copy number of the library can be controlled by the cycle number of the "temperature rising-temperature falling" self-amplification. However, in the real experimental environment, due to the problem that the amplification efficiency / amplification success rate cannot reach 100%, the nucleic acid library generated by the "temperature rising-temperature falling" self-amplification is not absolutely two copies, four copies, eight copies, sixteen copies, etc., and a small amount of three copies, five copies, six copies, seven copies, etc. non-target copy number may also be formed. According to the needs, these non-target copy number nucleic acid libraries can be removed in the subsequent steps, for example, removed by DNA fragment size screening in nucleic acid library purification, or removed by screening the length of sequencing reads after sequencing, etc. These non-target copy number nucleic acid libraries can also not be removed, which are essentially still multiple copies of the target nucleic acid to be tested, and will not have adverse effects on subsequent sequencing and sequencing read analysis.

[0057] According to an embodiment of the present application, the self-amplification adapter comprises at least one of a first self-amplification adapter and a second self-amplification adapter; wherein the first self-amplification adapter and the second self-amplification adapter are connected to the two ends of the nucleic acid molecule, respectively.

[0058] According to an embodiment of the present application, the self-amplification adapter comprises a first self-amplification adapter and a second self-amplification adapter; wherein the first self-amplification adapter and the second self-amplification adapter are connected to the two ends of the nucleic acid molecule, respectively, and the specific structure schematic diagram is shown in FIG. 1.

[0059] According to an embodiment of the present application, the self-amplification adapter comprises a first self-amplification adapter or a second self-amplification adapter; wherein the self-amplification adapter is connected to one end of the nucleic acid molecule, and the specific construction method schematic diagram is shown in FIG. 2 and FIG. 3.

[0060] According to an embodiment of the present application, the first self-amplification adapter and the second self-amplification adapter are the same.

[0061] According to an embodiment of the present application, the self-amplification linker has a first strand and a second strand, and the first strand and the second strand are at least partially complementary.

[0062] According to an embodiment of the present application, the palindrome sequence is located at one end of the self-amplification linker, and the other end of the self-amplification linker is connected to the nucleic acid molecule.

[0063] According to an embodiment of the present application, the palindrome sequence is located at the 5' end of the first strand and the 3' end of the second strand of the self-amplification linker, and the other end of the self-amplification linker is connected to the nucleic acid molecule. The specific structure is shown in FIG. 4.

[0064] According to an embodiment of the present application, the palindrome sequence has at least one of the sequences of (A) n1 (T) n1 , (T) n2 (A) n2 , (G) n3 (C) n3 , or (C) n4 (G) n4 , wherein n1, n2, n3 and n4 are independently selected from an integer between 5 and 20.

[0065] According to an embodiment of the present application, the palindrome sequence has any one of the nucleotide sequences shown in SEQ ID NO: 1-3.

[0066] According to an embodiment of the present application, the palindrome sequence has the nucleotide sequence shown in SEQ ID NO: 2.

[0067] According to an embodiment of the present application, the self-amplification linker further comprises a non-palindrome sequence. The specific structure is shown in FIG. 4.

[0068] According to an embodiment of the present application, the self-amplification is performed by the following steps, as shown in FIG. 5 and FIG. 6.

[0069] 1) In the first heating process, the double strand of the self-amplification linker is unwound.

[0070] 2) In the first cooling process, the palindrome sequence in the unwound self-amplification linker spontaneously forms a hairpin structure, the 3' end sequence of the hairpin structure is used as a primer, one strand of the nucleic acid molecule is used as a template, and the primer is extended under the action of a polymerase to obtain a first self-amplification product. The first self-amplification product comprises a template strand, a hairpin structure and an extended strand. The specific structure of the first self-amplification product is shown in FIG. 5.

[0071] According to an embodiment of the present application, the self-amplification further comprises:

[0072] 3) in the second heating treatment process, the double strands of the first self-amplification product at both ends are disentangled;

[0073] 4) in the second cooling treatment process, the disentangled first self-amplification product at both ends spontaneously forms a hairpin structure, the 3' end sequence of the hairpin structure is used as a primer, the first self-amplification product is used as a template, and the primer is extended under the action of a polymerase to obtain a second self-amplification product, the second self-amplification product includes a template strand, a hairpin structure, an extension one strand, and an extension two strand. The specific structure of the second self-amplification product is shown in FIG. 6.

[0074] According to an embodiment of the present application, the self-amplification process further comprises: repeating steps "3) and 4)" one or more times to obtain a self-amplification product containing x copies of the nucleic acid molecule, wherein x is an integer and x≥2.

[0075] According to an embodiment of the present application, the self-amplification is performed by a polymerase with strand displacement activity.

[0076] According to an embodiment of the present application, the polymerase is selected from a DNA polymerase or an RNA polymerase.

[0077] According to an embodiment of the present application, the polymerase is a polymerase with strand displacement activity obtained by modification of a polymerase without strand displacement activity.

[0078] According to an embodiment of the present application, the polymerase is selected from at least one of Bst DNA polymerase, SD DNA polymerase, phi29 DNA polymerase, Bsu Large Fragment DNA polymerase, Klenow Fragment DNA polymerase, T4 DNA polymerase, T7 DNA polymerase, DNA Polymerase I, T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, and E. coli RNA polymerase.

[0079] According to an embodiment of the present application, FIG. 7 shows a method flow of performing self-amplification by connecting self-amplification adapters to both ends of a target nucleic acid and performing "heating treatment and cooling treatment" twice in a cycle. Both ends of a double-stranded target nucleic acid molecule are connected to double-stranded self-amplification adapters, the double-stranded self-amplification adapters are opened into single strands after heating, and then cooling is performed. The single-stranded self-amplification adapters spontaneously form a hairpin structure due to the presence of a palindromic sequence, and then the 3' end sequence of the hairpin structure is used as a primer to perform the first amplification to obtain a first self-amplification product. The above-mentioned "heating treatment and cooling treatment" are repeated once, and the first self-amplification product is used as a template to perform the second amplification to obtain a second self-amplification product.

[0080] According to an embodiment of the present application, FIG. 8 shows a method flow of performing self-amplification by connecting a single end of a target nucleic acid to a self-amplification linker and performing "heat treatment and cooling treatment" once. One end of a double-stranded target nucleic acid molecule is connected to a double-stranded self-amplification linker. After heat treatment, the double-stranded self-amplification linker is opened to a single strand. Then, cooling is performed. The single strand of the self-amplification linker spontaneously forms a hairpin structure due to the presence of a palindromic sequence. Then, amplification is performed using the 3' end sequence of the hairpin structure as a primer. A self-amplification product is obtained.

[0081] According to an embodiment of the present application, step (b) further comprises: extending the end of the self-amplification product to obtain an extension product, wherein the extension product is a nucleic acid library.

[0082] According to an embodiment of the present application, the extension is catalyzing the addition of deoxynucleotides to the 3' end of the self-amplification product under the action of a terminal transferase without template dependence to form an extension product containing a 3' overhang sequence.

[0083] According to an embodiment of the present application, the 3' end overhang sequence is poly A, poly T, poly C, or poly G.

[0084] According to an embodiment of the present application, the extension is connecting a Y-type linker to the end of the self-amplification product under the action of a ligase to form an extension product containing a Y-type linker sequence.

[0085] According to an embodiment of the present application, further comprising: performing polymerase amplification using the extension product as a template and a fragment complementary to the 3' end overhang sequence or a fragment complementary to the Y-type linker sequence as a primer to form a double-stranded extension product. At this time, there is a primer only at one end of the template, and the polymerase only performs one round of amplification, i.e., two-chain synthesis of the product, to form a double-stranded extension product after amplification. The product is a nucleic acid library and can be used for subsequent sequencing. Moreover, the nucleic acid library is in a linear double-stranded form. Compared with the single-stranded template containing a base complementary pairing region before amplification (the first strand and the second strand are connected by a self-amplification linker to form a double-stranded form with a hairpin structure at the end), the template is unwound by a helicase and the first strand passes through a nanopore under the control of the helicase during sequencing, especially single-molecule nanopore sequencing based on the helicase. When the helicase unwinds to the hairpin structure at the connection between the first strand and the second strand, the double-stranded structure of the template is completely opened, the second strand forms a new secondary structure, and the sequencing stability of the second strand is reduced (for example, the secondary structure causes the pore to be blocked, and the stability of the helicase is reduced).

[0086] According to an embodiment of the present application, a specific method flow chart for constructing a nucleic acid library when two ends of a target nucleic acid are connected with a first self-amplification adaptor and a second self-amplification adaptor respectively is shown in FIG. 7.

[0087] According to an embodiment of the present application, a specific method flow chart for constructing a nucleic acid library when one end of a target nucleic acid is connected with a self-amplification adaptor is shown in FIG. 8.

[0088] Nucleic acid library

[0089] According to a second aspect of the present application, a nucleic acid library is provided. According to an embodiment of the present application, the nucleic acid library is constructed by the method of the first aspect of the present application. The nucleic acid library obtained by the method of the present application has low cost, high yield, reduced palindrome complementary sequences of invalid repeated amplification, and controllable target copy number of target nucleic acid.

[0090] Kit

[0091] According to a third aspect of the present application, a kit for constructing a nucleic acid library is provided. According to an embodiment of the present application, the kit comprises a self-amplification adaptor of the first aspect of the present application, the self-amplification adaptor having a palindrome sequence of the first aspect of the present application; a ligase for connecting the self-amplification adaptor to a nucleic acid molecule; a polymerase having strand displacement activity for self-amplification of the nucleic acid molecule; a buffer suitable for a ligation reaction, a buffer suitable for a self-amplification reaction, and dNTPs. The kit of the present application can obtain a nucleic acid library with low cost, high yield, reduced palindrome complementary sequences of invalid repeated amplification, and controllable target copy number.

[0092] According to an embodiment of the present application, the polymerase is selected from at least one of Bst DNA polymerase, SD DNA polymerase, phi29 DNA polymerase, Bsu Large Fragment DNA polymerase, Klenow Fragment DNA polymerase, T4 DNA polymerase, T7 DNA polymerase, DNA Polymerase I, T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, and E. coli RNA polymerase.

[0093] According to an embodiment of the present application, the kit further comprises a Y-type adaptor or a terminal transferase. The nucleic acid library is obtained by adding a deoxynucleotide to the 3' end of the self-amplification product through the terminal transferase. Alternatively, the nucleic acid library is obtained by connecting a Y-type adaptor to the 3' end of the self-amplification product through the ligase.

[0094] According to an embodiment of the present application, the kit further comprises a Y-shaped adaptor and a primer having a sequence complementary to the Y-shaped adaptor. The self-amplification product with the Y-shaped adaptor added at the 3' end is used as a template, and the primer complementary to the Y-shaped adaptor is used as a primer to perform polymerase amplification, thereby forming a double-stranded extension product. At this time, the primer only exists at one end of the template, and the polymerase only performs one round of amplification, i.e., the double-strand synthesis of the self-amplification product, thereby forming the double-stranded extension product, which is the nucleic acid library.

[0095] According to an embodiment of the present application, the kit further comprises a terminal transferase and a primer having a poly sequence. The self-amplification product with the plurality of repeated deoxynucleotides added at the 3' end is used as a template, and the primer having a poly sequence is used as a primer to perform polymerase amplification, thereby forming a double-stranded extension product. At this time, the primer only exists at one end of the template, and the polymerase only performs one round of amplification, i.e., the double-strand synthesis of the self-amplification product, thereby forming the double-stranded extension product, which is the nucleic acid library. According to an embodiment of the present application, the primer is poly A, poly T, poly C or poly G. The primer can be at least partially complementary to the self-amplification product with the plurality of repeated deoxynucleotides added at the 3' end, and the polymerase performs the double-strand synthesis of the self-amplification product under the action of the polymerase.

[0096] Use

[0097] In a fourth aspect of the present application, the present application provides a use of the nucleic acid library constructed by the method of the first aspect of the present application or the nucleic acid library of the second aspect of the present application in sequencing.

[0098] According to an embodiment of the present application, the sequencing method is single molecule sequencing.

[0099] According to an embodiment of the present application, the single molecule sequencing is nanopore-based single molecule sequencing or single molecule fluorescent sequencing.

[0100] Method for sequencing nucleic acid library

[0101] In a fifth aspect of the present application, the present application provides a method for sequencing a nucleic acid library. According to an embodiment of the present application, the method sequences the nucleic acid library constructed by the method of the first aspect of the present application or the nucleic acid library of the second aspect of the present application to obtain a sequencing result. According to the method of the embodiment of the present application, multiple sequencing of the target nucleic acid to be measured can be realized, and the sequencing accuracy is improved.

[0102] According to an embodiment of the present application, the sequencing method is single molecule sequencing.

[0103] According to an embodiment of the present application, the single molecule sequencing is nanopore-based single molecule sequencing or single molecule fluorescent sequencing.

[0104] Figure 9 shows the method steps of nucleic acid library construction and nanopore sequencing in one embodiment of the present application. The target nucleic acid molecule is connected with self-amplification adapters at both ends, and the "heating treatment and cooling treatment" is performed multiple times in a cycle to obtain a self-amplification product, a poly sequence is added to the end of the self-amplification product using terminal transferase, and the product is amplified again to form a double-stranded product, which is the nucleic acid library and can be subjected to nanopore sequencing.

[0105] Figure 13 shows the method steps of nucleic acid library construction and nanopore sequencing in another embodiment of the present application. The target nucleic acid molecule is connected with self-amplification adapters at both ends, and the "heating treatment and cooling treatment" is performed multiple times in a cycle to obtain a self-amplification product, a Y-shaped adapter is added to the end of the self-amplification product using ligase, and the product is amplified again to form a double-stranded product, which is the nucleic acid library and can be subjected to nanopore sequencing.

[0106] The protein sequences and nucleotide sequences used in the present application are shown in Table 1.

[0107] Table 1: Protein sequences and nucleotide sequences

[0108] The schemes of the present application will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only used to illustrate the present application and should not be regarded as limiting the scope of the present application. If the specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art or according to the product instructions are used. If the reagents or instruments are not specified by the manufacturer, they are all conventional products that can be obtained commercially.

[0109] Example 1:

[0110] 1. Multiple self-amplification of target nucleic acid sequence

[0111] The experimental steps of multiple self-amplification of nucleotide sequence are shown as follows:

[0112] (1) End repair and 3' end A addition of the target nucleotide to be tested (nucleotide sequence as shown in SEQ ID NO: 7) with characteristic sequence were performed using NEBNext FFPE DNA Repair Mix (NEB, M6630) and NEBNext Ultra II End repair / dA-tailing Module (NEB, E7546) according to the manufacturer's instructions.

[0113] (2) According to the manufacturer's instructions, dissolve the first strand of the self-amplification adapter (nucleotide sequence as shown in SEQ ID NO: 8), the second strand of the self-amplification adapter (nucleotide sequence as shown in SEQ ID NO: 9) with TE buffer (pH = 8), and anneal into self-amplification adapter 1 (5A + 5T). According to the manufacturer's instructions, dissolve the first strand of the self-amplification adapter (nucleotide sequence as shown in SEQ ID NO: 10), the second strand of the self-amplification adapter (nucleotide sequence as shown in SEQ ID NO: 11) with TE buffer (pH = 8), and anneal into self-amplification adapter 2 (10A + 10T). According to the manufacturer's instructions, dissolve the first strand of the self-amplification adapter (nucleotide sequence as shown in SEQ ID NO: 12), the second strand of the self-amplification adapter (nucleotide sequence as shown in SEQ ID NO: 13) with TE buffer (pH = 8), and anneal into self-amplification adapter 3 (15A + 15T). The annealing process is 95°C incubation for 5 minutes, 0.1°C / s cooling to 25°C, and continue incubation for 30 minutes.

[0114] (3) According to the manufacturer's instructions, use NEBNext Quick Ligation Module (NEB, E6056) to perform ligation reaction on the end-repaired and 3-end A-added target nucleotides to be tested and the annealed self-amplification adapters 1, 2, and 3, respectively. The reaction conditions are 25°C incubation for 60 minutes.

[0115] (4) According to the manufacturer's instructions, use AMPure XP beads (Beckman Coulter, A63882) to purify the three groups of ligation products in step (3) respectively.

[0116] (5) Perform self-amplification on the three groups of ligation products respectively, mix the ligation products, BST polymerase, and reaction buffer (ARCTICZYMES, 71502-202), dNTP (NEB, N0447L) uniformly, and perform self-amplification reaction. The self-amplification reaction system is: 500 ng of ligation product, 2 μL of BST polymerase (8 U / μL), 5 μL of BST polymerase 10×Buffer, 5 μL of dNTP (10 mM), and the total volume is 50 μL. The self-amplification process is: 65°C incubation for 2 minutes, 37°C incubation for 2 minutes; 65°C incubation for 2 minutes, 37°C incubation for 4 minutes; 65°C incubation for 2 minutes, 37°C incubation for 8 minutes; 65°C incubation for 2 minutes, 37°C incubation for 16 minutes; 37°C incubation for 30 minutes, and the obtained self-amplification products are named as self-amplification product 1 (corresponding to self-amplification adapter 1 (5A + 5T)), self-amplification product 2 (corresponding to self-amplification adapter 1 (10A + 10T)), and self-amplification product 3 (corresponding to self-amplification adapter 1 (15A + 15T)).

[0117] (6) Purify the three groups of self-amplification products 1, 2 and 3 with AMPure XP beads (Beckman Coulter, A63882) according to the manufacturer's instructions, and obtain 20 μL of purified products, respectively. Determine the concentrations of the three groups of self-amplification products with Qubit dsDNA HS Assay Kit (Thermo, Q32851) according to the manufacturer's instructions. The concentration of self-amplification product 1 is 27.3 ng / μL, that of self-amplification product 2 is 30.9 ng / μL, and that of self-amplification product 3 is 24.8 ng / μL. Since the reaction systems of the three groups of experiments are completely consistent, the yields of the three self-amplification products are all high, among which the yield of self-amplification product 2 is the highest. This embodiment exemplarily shows the results of selecting self-amplification product 2 for subsequent experimental operations.

[0118] The specific process of multiple self-amplification of nucleotide sequences is shown in FIG. 9.

[0119] 2. Double-strand synthesis of self-amplification products

[0120] (1) Continuously add A to the 3' end of self-amplification product 2 with terminal transferase (NEB, M0315) according to the manufacturer's instructions. The reaction system is as follows: 300 ng of self-amplification product 2, 1 μL of terminal transferase (20,000 U / mL), 10 μL of terminal transferase 10x Buffer, 10 μL of CoCl2 (2.5 mM), 10 μL of dATP (1 mM), and water to a total volume of 100 μL. The reaction conditions are 37 °C for 30 minutes and 70 °C for 10 minutes.

[0121] (2) Purify the continuously added A product in step (1) with AMPure XP beads (Beckman Coulter, A63882) according to the manufacturer's instructions.

[0122] (3) Perform double-strand synthesis of the continuously added A product by mixing the continuously added A product, phi29 polymerase and reaction buffer (NEB, M0269S), dNTP and PolyT primer (the nucleotide sequence is shown in SEQ ID NO: 14). The double-strand synthesis reaction system is as follows: 200 ng of continuously added A product, 2 μL of phi29 polymerase (10,000 U / mL), 10 μL of phi29 polymerase 10x Buffer, 10 μL of dNTP (10 mM), 10 μL of PolyT primer (10 μM), and water to a total volume of 100 μL. The double-strand synthesis conditions are 30 °C for 30 minutes. Since the first strand of the self-amplification linker (the nucleotide sequence is shown in SEQ ID NO: 10) contains spacer modification in the middle, one end of the double-strand synthesis product is a specially designed sticky end.

[0123] (4) Purify the double-strand synthesis product with AMPure XP beads (Beckman Coulter, A63882) according to the manufacturer's instructions.

[0124] The specific process of double-strand synthesis of self-amplification product is shown in Figure 9.

[0125] 3. Sequencing adapter complex construction

[0126] The process of sequencing adapter complex construction is shown in Figure 10, and the specific process of constructing the sequencing adapter complex is as follows:

[0127] (1) Dissolve SEQ ID NO: 15 and SEQ ID NO: 16 in TE buffer (pH = 8) according to the manufacturer's instructions. Anneal SEQ ID NO: 15 and SEQ ID NO: 16 into sequencing adapters at a ratio of 1:1. The annealing process is incubated at 95°C for 5 minutes, and then cooled to 25°C at a rate of 0.1°C / s, and continue to incubate for 30 minutes.

[0128] (2) Complete the prokaryotic expression of helicase He (T4 Dda-(AM1)G1 / E94C / C109A / C136A / K194L / A360C, the amino acid sequence is shown in SEQ ID NO: 17) in E. coli, and obtain the target protein after multi-step purification.

[0129] (3) Mix the helicase and the sequencing adapter at a molecular ratio of 9:1, and the final concentration of the reaction buffer is 25mM HEPES, 50mM KCl, 0.5mM EDTA, 2.5mM MgCl2, pH = 8.0, and incubate at room temperature for 30 minutes.

[0130] (4) Add 0.25 volume of 5mM ATP to the incubation product, and incubate at room temperature for 30 minutes.

[0131] (5) Purify the sequencing adapter complex with AMPure XP beads (Beckman Coulter, A63882) according to the manufacturer's instructions to obtain the sequencing adapter complex. The purified sequencing adapter complex is detected by electrophoresis, and the results are shown in Figure 11 to obtain the sequencing adapter complex.

[0132] 4. Nanopore sequencing library construction

[0133] (1) Connect the product of multiple self-amplification and double-strand synthesis of target nucleic acid sequence and the sequencing adapter complex with NEBNext Quick Ligation Module (NEB, E6056) according to the manufacturer's instructions, and incubate at 25°C for 60 minutes.

[0134] (2) Purification with AMPure XP beads (Beckman Coulter, A63882) according to the manufacturer's instructions to obtain sequencing library.

[0135] 5. Nanopore sequencing

[0136] (1) The single-channel nanopore detection system based on patch clamp and signal amplifier was built according to the method disclosed in the literature (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), and the single pore protein was embedded in the hole.

[0137] (2) The sequencing library was mixed with the constraint sequence (nucleotide sequence as shown in SEQ ID NO: 18), and was added to the single-channel nanopore detection system. The change of current signal was observed under the condition of 180 mV, and the sequencing buffer was: 470 mM KCl, 25 mM HEPES, 10 mM MgCl2, 30 mM ATP, pH = 8.10; the sequencing temperature was: 30°C.

[0138] (3) The current signal of the target nucleotide sequence to be detected obtained by multiple nanopore sequencing was obtained, wherein the representative current signal is shown in FIG. 12. According to the linker sequence signal and the characteristic sequence signal, it can be judged that the target sequence is continuously sequenced 8 times. In the figure, 1D, 2D, 3D, 4D, 5D, 6D, 7D and 8D are the current signals of the first, second, third, fourth, fifth, sixth, seventh and eighth sequencing, respectively. From the figure, it can be seen that the method used in this embodiment realizes the multiple nanopore sequencing of the target nucleic acid sequence.

[0139] 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.

[0140] 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 those 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 method of constructing a library of nucleic acids, characterized by, The method comprises: (a) providing a nucleic acid molecule comprising a self-amplification linker, the self-amplification linker having a palindromic sequence; (b) performing self-amplification on the nucleic acid molecule to obtain the nucleic acid library, wherein the self-amplification is achieved by cyclically performing "heating treatment and cooling treatment" one or more times, the palindromic sequence of the self-amplification linker is suitable for forming a hairpin structure, and the hairpin structure is suitable for serving as a primer for self-amplification.

2. The method of claim 1, wherein, The nucleic acid library is a multi-copy nucleic acid library.

3. The method of claim 2, wherein, The number of cycles of self-amplification is determined according to the copy number of the nucleic acid library.

4. The method of claim 3, wherein, Determining the number of cycles of self-amplification according to the copy number of the nucleic acid library comprises determining the number of cycles of self-amplification according to the following formula: y = log2(x) wherein y is the number of cycles of self-amplification, x is the copy number of the nucleic acid library, x is an integer, and x ≥ 2; Preferably, 2 ≤ x ≤ 1024, and 1 ≤ y ≤ 10; More preferably, 2 ≤ x ≤ 32, and 1 ≤ y ≤ 5.

5. The method according to any one of claims 1 to 4, characterized in that, The temperature of each heating treatment is 40-80°C.

6. The method of claim 5, wherein, The time of each heating treatment is 1-5 min.

7. The method according to any one of claims 1 to 4, characterized in that, The temperature of each cooling treatment is 20-40°C.

8. The method of claim 7, wherein, The time of each cooling treatment is 1-30 min.

9. The method according to any one of claims 1 to 4, characterized in that, The number of cycles is 1-30.

10. The method of claim 1, wherein, The self-amplification linker comprises a first self-amplification linker or a second self-amplification linker, and the self-amplification linker is connected to one end of the nucleic acid molecule; or the self-amplification linker comprises a first self-amplification linker and a second self-amplification linker, and the first self-amplification linker and the second self-amplification linker are respectively connected to two ends of the nucleic acid molecule.

11. The method of claim 10, wherein, The first self-amplification linker and the second self-amplification linker are the same.

12. The method of claim 1, wherein, The self-amplification linker has a first strand and a second strand, and the first strand and the second strand are at least partially complementary.

13. The method of claim 1, wherein, The palindromic sequence is located at one end of the self-amplification linker, and the other end of the self-amplification linker is connected to the nucleic acid molecule.

14. The method of claim 1 or 13, wherein, The palindromic sequence has a sequence of at least one of (A) n1 (T) n1 , (T) n2 (A) n2 , (G) n3 (C) n3 , or (C) n4 (G) n4 , wherein each of n1, n2, n3, and n4 is independently selected from an integer between 5 and 20.

15. The method of claim 14, wherein, The palindromic sequence has any one of the nucleotide sequences shown in SEQ ID NO: 1-3.

16. The method of claim 1, wherein, The self-amplification linker further comprises a non-palindromic sequence.

17. The method of claim 1, wherein, The self-amplification is performed by: 1) During the first heating treatment, the double strand of the self-amplification linker is unwound; 2) During the first cooling treatment, the palindromic sequence in the unwound self-amplification linker spontaneously forms a hairpin structure, the 3' end sequence of the hairpin structure is used as a primer, one strand of the nucleic acid molecule is used as a template, and the primer is extended under the action of a polymerase to obtain a first self-amplification product, the first self-amplification product comprises a template strand, a hairpin structure, and an extension one strand.

18. The method of claim 17, wherein, The self-amplification further comprises: 3) During the second heating treatment, the double strand at both ends of the first self-amplification product is unwound; 4) During the second cooling treatment, the two ends of the unwound first self-amplification product spontaneously form a hairpin structure, the 3' end sequence of the hairpin structure is used as a primer, the first self-amplification product is used as a template, and the primer is extended under the action of a polymerase to obtain a second self-amplification product, the second self-amplification product comprises a template strand, a hairpin structure, an extension one strand, and an extension two strand.

19. The method of claim 18, wherein, The self-amplification further comprises: Steps "3) and 4)" are repeated one or more times to obtain a self- amplification product containing x copies of the nucleic acid molecule, wherein x is an integer and x > 2.

20. The method of any one of claims 1, 17, or 18, wherein, The self-amplification is performed by a polymerase having strand displacement activity.

21. The method of claim 20, wherein, The polymerase is selected from a DNA polymerase or a RNA polymerase.

22. The method of claim 20, wherein, The polymerase is a polymerase having strand displacement activity obtained by modification of a polymerase without strand displacement activity.

23. The method of claim 20, wherein, The polymerase is selected from at least one of Bst DNA polymerase, SD DNA polymerase, phi29 DNA polymerase, Bsu Large Fragment DNA polymerase, Klenow Fragment DNA polymerase, T4 DNA polymerase, T7 DNA polymerase, DNA Polymerase I, T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, and E. coli RNA polymerase.

24. The method of claim 1, wherein, Step (b) further comprises extending the ends of the self-amplification product to obtain an extension product, wherein the extension product is a nucleic acid library.

25. The method of claim 24, wherein, The extension is catalyzing the addition of deoxynucleotides to the 3' end of the self-amplification product by a terminal transferase to form an extension product containing a 3' overhang sequence without template dependence. Preferably, the 3' overhang sequence is poly A, poly T, poly C or poly G.

26. The method of claim 24, wherein, The extension is connecting a Y-shaped adaptor to the ends of the self- amplification product by a ligase to form an extension product containing a Y-shaped adaptor sequence.

27. The method of claim 25 or 26, wherein, Further comprising: performing polymerase amplification using the extension product as a template and a fragment complementary to the 3' overhang sequence or a fragment complementary to the Y-shaped adaptor sequence as a primer to form a double-stranded extension product.

28. A nucleic acid library, characterized in that, comprising: constructing using the method of any one of claims 1 to 27.

29. A kit for constructing a library of nucleic acids, characterized in that, comprising: the self-amplification adaptor of any one of claims 1, 10 to 12, 16, wherein the self-amplification adaptor has the palindromic sequence of any one of claims 13 to 15; a ligase for connecting the self-amplification adaptor to a nucleic acid molecule; a polymerase having strand displacement activity for self-amplifying the nucleic acid molecule; a buffer suitable for the ligation reaction, a buffer suitable for the self-amplification reaction, and dNTPs.

30. The kit of claim 29, wherein The polymerase is selected from at least one of Bst DNA polymerase, SD DNA polymerase, phi29 DNA polymerase, Bsu Large Fragment DNA polymerase, Klenow Fragment DNA polymerase, T4 DNA polymerase, T7 DNA polymerase, DNA Polymerase I, T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, and E. coli RNA polymerase.

31. The kit of claim 29 or 30, wherein, Further comprising: i) a Y-shaped adaptor and a primer having a sequence complementary to a portion of the Y-shaped adaptor; or ii) a terminal transferase and a primer, the primer having a poly sequence; preferably, the primer is poly A, poly T, poly C or poly G.

32. Use of a nucleic acid library constructed by the method of any one of claims 1 to 27 or the nucleic acid library of claim 28 in sequencing.

33. Use according to claim 32, characterized in that, The sequencing is single molecule sequencing.

34. Use according to claim 33, characterized in that, The single molecule sequencing is nanopore-based single molecule sequencing or single molecule fluorescent sequencing.

35. A method of sequencing a nucleic acid library, characterized in that, The nucleic acid library constructed by the method of any one of claims 1 to 27 or the nucleic acid library of claim 28 is sequenced to obtain sequencing results.

36. The method of claim 35, wherein, The sequencing is single molecule sequencing.

37. The method of claim 36, wherein, The single molecule sequencing is nanopore-based single molecule sequencing or single molecule fluorescent sequencing.

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