Kit for sequencing library construction and application thereof

Abstract

The invention relates to a kit for constructing a sequencing library, and the kit comprises a repair treatment reagent which comprises a first enzyme with a repair function, NAD < + >, ATP, dNTPs and a first buffer solution; the connection treatment reagent comprises a second enzyme with a connection function, a joint and a second buffer solution; the digestion treatment reagent comprises a third enzyme with a digestion function; the first buffer solution comprises 10 to 50 mM of a Tris buffer solution, 5 to 120 mM of monovalent cations and 0.1 to 10 mM of divalent cations; and the second buffer solution comprises 20 to 100 mM of a Tris buffer solution, 5 to 20 mM of divalent cations and 1 to 10 mM of dithiothreitol. The kit provided by the embodiment of the invention is simple in composition, convenient to use and high in library yield, the library building cost is remarkably reduced, and wide application of three-generation sequencing can be powerfully promoted.

Description

Technical Field

[0001] The present application relates to the field of sequencing, and in particular to a kit for constructing a sequencing library and its application. Background Art

[0002] At present, sequencing technology has become an essential research tool in the field of genes. Although first-generation sequencing has advantages such as long read lengths and high accuracy, large-scale sequencing cannot be performed due to low sequencing throughput. Second-generation sequencing has high throughput and short sequencing time, and can perform genome sequencing quickly and efficiently, but has disadvantages such as too short read lengths, PCR amplification errors, and GC bias. Third-generation sequencing is based on single-molecule sequencing technology and does not require PCR amplification, avoiding amplification errors, and has been applied to multiple research fields such as genome sequencing, methylation research, and mutation identification, represented by the SMRT technology of Pacbio and the nanopore technology of Oxfordnanopore. Summary of the Invention

[0003] In one aspect of the present application, at least one embodiment provides a kit for constructing a sequencing library, including: a repair treatment reagent, including a first enzyme with a repair function, NAD + , ATP, dNTPs, and a first buffer; a ligation treatment reagent, including a second enzyme with a ligation function, an adaptor, and a second buffer; a digestion treatment reagent, including a third enzyme with a digestion function; wherein, the first buffer includes: 10-50 mM Tris buffer, 5-120 mM monovalent cation, and 0.1-10 mM divalent cation; the second buffer includes: 20-100 mM Tris buffer, 5-20 mM divalent cation, and 1-10 mM dithiothreitol.

[0004] In one aspect of the present application, at least one embodiment provides the application of the above kit in the field of sequencing.

[0005] For example, in some embodiments, the first buffer includes: 15-30 mM Tris buffer, 8-80 mM monovalent cation, 1-5 mM divalent cation; and / or, the second buffer includes: 30-80 mM Tris buffer, 8-15 mM divalent cation, and 2-6 mM dithiothreitol.

[0006] For example, in some embodiments, the Tris buffer includes at least one of Tris-HCl buffer and Tris-acetate buffer; and / or, the monovalent cation includes Na + , K + and NH 4 + at least one of them; and / or, the divalent cation includes Mg 2+ , Mn2+ 、 Fe 2+ 、 Ca 2+ and Zn 2+ at least one of them.

[0007] For example, in some embodiments, the first buffer solution includes: 3 - 100 mM NH 4 + and 2 - 20 mM monovalent metal cation; and / or, the first buffer solution further includes: 0.01 - 1 wt% surfactant, and the surfactant is selected from at least one of octylphenoxypolyethoxyethanol, Tween 20, Tween 40 and Tween 60.

[0008] For example, in some embodiments, the first buffer solution includes: 10 - 50 mM Tris-HCl, 1.5 - 50 mM (NH 4 ) 2 SO 4 , 2 - 20 mM KCl and 0.1 - 10 mM MgSO 4 and 0.01 - 1 wt% octylphenoxypolyethoxyethanol; preferably, the first buffer solution includes: 15 - 30 mM Tris-HCl, 2.5 - 30 mM (NH 4 ) 2 SO 4 , 3 - 20 mM KCl, 1 - 5 mM MgSO 4 and 0.05 - 1 wt% octylphenoxypolyethoxyethanol; preferably, the first buffer solution includes: 18 - 25 mM Tris-HCl, 5 - 15 mM (NH 4 ) 2 SO 4 , 5 - 20 mM KCl, 2 - 3 mM MgSO 4 and 0.05 - 0.5 wt% octylphenoxypolyethoxyethanol.

[0009] For example, in some embodiments, the second buffer solution includes: 20 - 100 mM Tris-HCl, 5 - 20 mM MgCl 2 and 1 - 10 mM dithiothreitol; preferably, the second buffer solution includes: 30 - 80 mM Tris-HCl, 8 - 15 mM MgCl 2 and 2 - 6 mM dithiothreitol; preferably, the second buffer solution includes: 40 - 70 mM Tris-HCl, 9 - 12 mM MgCl 2 and 3 - 6 mM dithiothreitol.

[0010] For example, in some embodiments, the pH of the first buffer is 7 to 10; and / or, the pH of the second buffer system is 6 to 9; preferably, the pH of the first buffer system is greater than the pH of the second buffer system.

[0011] For example, in some embodiments, the first enzyme includes at least one of exonuclease, repair enzyme, polymerase, and phosphorylase; preferably, the exonuclease includes at least one of EXO III, EXO VI, and EXO VII; the repair enzyme includes PARPs; the polymerase includes Taq DNA polymerase and / or T4 DNA polymerase; the phosphorylase includes T4PNK; and / or, the second enzyme includes nucleic acid ligase; preferably, the nucleic acid ligase includes T4 DNA ligase; preferably, the enzyme activity concentration of the nucleic acid ligase is 5 to 50 U / μL; and / or, the third enzyme includes exonuclease; preferably, the exonuclease includes at least one of EXO III, EXO VI, and EXO VII.

[0012] For example, in some embodiments, the ratio of the adapter to the nucleic acid sample to be tested is 20 to 1000; and / or, the content of the adapter is 40 to 300 pmol. For example, in some embodiments, it further includes: a fragmentation treatment reagent, including a fragmenting enzyme; and / or, a purification treatment reagent, including magnetic beads; preferably, the purification treatment reagent is used after the digestion treatment reagent.

[0013] The inventors of the present application found in their research that the kit components used for library construction on the Pacbio sequencing platform are complex, costly, and inconvenient to use, which limits its wide application. To at least solve the above problems, the kit provided by the present application uses a combined buffer that is compatible with multi-step reactions to complete library construction. The reagent types are simple and easy to use, reducing the loss of reagents and samples caused by changing the buffer system. The library yield is high, and the library construction cost is significantly reduced, which can effectively promote the wide application of third-generation sequencing. Detailed implementation manners

[0014] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solutions involved in the present application will be clearly and completely described below. Obviously, the specific implementation examples described are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present application.

[0015] It should be noted that the terms "first", "second", etc. in the description and claims of this application are used to distinguish similar objects and do not necessarily describe a specific order or sequence. It should be understood that such data can be interchanged under appropriate circumstances so that the embodiments of this application described here can be implemented in an order other than those described here. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or device that includes a series of steps or units does not necessarily have to be limited to those steps or units clearly listed, but may include other steps or units not clearly listed or inherent to these processes, methods, products, or devices. When the quantity of a component is not specifically indicated in the following text of the embodiments of this application, it means that the component can be one or more, or can be understood as at least one. "At least one" means one or more, and "a plurality" means at least two.

[0016] The following describes the kits, methods, and their applications provided by this application in combination with specific embodiments. It should be noted that the same components or the same operation processes can adopt the same setting methods. All embodiments of this application are applicable to the above-mentioned multiple protection themes. The same or similar content will not be repeated multiple times in each protection theme, and reference can be made to the descriptions in the corresponding embodiments of other protection themes.

[0017] The inventors of this application found in their research that for the official kit used in the Pacbio platform to construct sequencing libraries currently, the reagents used in each step of library construction need to react based on the enzymes used by configuring their respective suitable buffers and substrates, etc. On the one hand, the residues of the reactants in the previous step affect the subsequent reactions; on the other hand, the buffer systems between different reactions are difficult to be compatible, the operation is complex and the reagent cost is high, reducing the library construction efficiency and affecting the library yield.

[0018] To at least solve the above problems, at least one embodiment of this application provides a kit that is at least applicable to the Pacbio platform and can be used to construct libraries. For example, the kit includes: a repair treatment reagent, including a first enzyme with a repair function, NAD + , ATP, dNTPs, and a first buffer; a ligation treatment reagent, including a second enzyme with a ligation function, an adaptor, ATP, and a second buffer; a digestion treatment reagent, including a third enzyme with a digestion function; wherein, the first buffer includes: 10 - 50 mM Tris buffer, 5 - 120 mM monovalent cation, and 0.1 - 10 mM divalent cation; the second buffer includes: 20 - 100 mM Tris buffer, 5 - 20 mM divalent cation, and 1 - 10 mM dithiothreitol.

[0019] For example, the first enzyme in the repair treatment reagent has a repair function and can repair the nucleic acid sample to be tested. For example, it can be a DNA repair treatment enzyme and / or an RNA repair treatment enzyme. For example, the first enzyme can perform excision repair, mismatch repair, double-strand break repair, etc. For example, the first enzyme can be a combination of one or more enzymes. For example, when the first enzyme includes multiple enzymes, these enzymes can be individually packaged as independent reagents or can be packaged together.

[0020] For example, when the first enzyme is a DNA repair treatment enzyme, NAD in the repair treatment reagent + can serve as a substrate for the DNA repair enzyme. For example, ATP in the repair treatment reagent can provide energy for the repair reaction. For example, dNTPs in the repair treatment reagent can provide raw materials for the repair reaction.

[0021] For example, the first enzyme, NAD + , ATP, and dNTPs in the repair treatment reagent can be individually packaged as independent reagents. For example, at least one of NAD + , ATP, and dNTPs can also be incorporated into the first buffer.

[0022] For example, the second enzyme in the ligation treatment reagent has a ligation function and can ligate the repair product to the adapter, for example, to form a dumbbell structure required for library construction on the Pacbio platform. For example, the second enzyme can be a DNA ligase. For example, ATP in the ligation treatment reagent can provide energy for the ligation reaction.

[0023] For example, the adapter can be any adapter suitable for library construction on the Pacbio platform. For example, the adapter sequence is as shown in Seq IDNo.1. For example, when performing multi-sample library construction, different tags can be added to the adapter to facilitate data splitting after sequencing. For example, the adapter sequence is as shown in at least one of Seq ID No.2 to 17.

[0024] For example, the second enzyme, the adapter, and ATP in the ligation treatment reagent can be individually packaged as independent reagents. For example, at least one of ATP and the adapter can be incorporated into the second buffer.

[0025]

[0026]

[0027] For example, the digestion reagent can process (e.g., remove) non-library molecules, such as library molecules that have not formed dumbbell structures. For example, the third enzyme in the digestion reagent, which has a digestion function, can be, for example, a DNA exonuclease. For example, the exonuclease includes one or more exonucleases. For example, these enzymes can be individually packaged as separate reagents from each other or packaged together.

[0028] For example, the kit provided by the embodiments of the present application further includes a first buffer and a second buffer. The first buffer can be used at least for repair processing, and the second buffer can be used at least for ligation processing and digestion processing. For example, the first buffer is used to provide a buffer system suitable for repair processing to enable the reaction to proceed smoothly. For example, the second buffer is used to provide a buffer system suitable for ligation processing and digestion processing to enable the reaction to proceed smoothly. The kit provided by the embodiments of the present application can complete library construction using a simple combination of buffers. Among them, the second buffer can be compatible with ligation processing and digestion processing, with simple reagent composition and convenient operation.

[0029] For example, the second buffer can be used at least for ligation processing and digestion processing. It can be that the second buffer is used during ligation processing, and digestion processing is carried out in the system after the ligation reaction; or, it can also be that the second buffer is used during ligation processing and the second buffer is also used during digestion processing. For example, the second buffer is added to the reaction system after ligation processing, or the ligation reaction product is purified and then the second buffer is added.

[0030] In some embodiments, the repair reagent further includes a first buffer. For example, the ligation reagent further includes a second buffer. For example, the digestion reagent further includes a second buffer.

[0031] In some embodiments, the first buffer and / or the second buffer can also be used as independent reagents. For example, they can be individually packaged and used in at least one of the processes of repair processing, ligation processing, and digestion processing. For example, during repair processing, the first buffer and the repair reagent are used. For example, during ligation processing, the second buffer and the ligation reagent are used. For example, during digestion processing, the second buffer and the digestion reagent are used.

[0032] For example, the first buffer solution includes: 10 - 50 mM Tris buffer solution, 5 - 120 mM monovalent cation, and 0.1 - 10 mM divalent cation. For example, the second buffer solution includes: 20 - 100 mM Tris buffer solution, 5 - 20 mM divalent cation, and 1 - 10 mM dithiothreitol. For example, the concentration of the first buffer solution and / or the second buffer solution is the concentration of its 1× component; alternatively, the concentration of the first buffer solution and / or the second buffer solution can be its working concentration, such as the concentration when in use. It can be understood that the first buffer solution and / or the second buffer solution can also be configured with higher concentrations, such as 2× buffer, 5× buffer, or 10× buffer, etc. For example, the 10× first buffer solution correspondingly includes: 10 - 500 mM Tris buffer solution, 50 - 1200 mM monovalent cation, 1 - 100 mM divalent cation; the 10× second buffer solution correspondingly includes 200 - 1000 mM Tris buffer solution, 50 - 200 mM divalent cation, and 10 - 100 mM dithiothreitol. That is, adopting the same composition and / or ratio as the buffer solution provided in the embodiments of the present application, even if the concentrations are different due to different concentration multiples, they all fall within the protection scope of the present application.

[0033] The inventors of the present application found in the research that when using the kit provided in the embodiments of the present application for library construction, there is no need to design different buffer solutions for each processing step. The uniquely designed combined buffer solution can be compatible with multiple-step reactions while meeting the different characteristics of each reaction step, and the reagent composition is simple; moreover, the inventors surprisingly found that the kit provided in the embodiments of the present application effectively improves the library yield. For example, the library yield can be the ratio of the molecular weight after library construction to the initial amount of the nucleic acid to be detected. Based on the above at least one embodiment, the first buffer solution and / or the second buffer solution further includes at least one of a surfactant, NAD + , ATP, and dNTPs. For example, the first buffer solution further includes NAD + and dNTPs, that is, NAD + and dNTPs required for nucleic acid repair can be incorporated into the first buffer solution. For example, the first buffer solution further includes ATP, for example, to provide energy for the reaction. For example, the second buffer solution further includes ATP, for example, to provide energy for the reaction.

[0034] Based on the above at least one embodiment, the first buffer solution includes: 15 - 30 mM Tris buffer solution, 8 - 80 mM monovalent cation, 1 - 5 mM divalent cation. For example, in some preferred embodiments, the first buffer solution includes 18 - 25 mM Tris buffer solution, 15 - 50 mM monovalent cation, 2 - 3 mM divalent cation.

[0035] Based on the above at least one embodiment, the second buffer solution includes: 30-80 mM Tris buffer solution, 8-15 mM divalent cation, and 2-6 mM dithiothreitol. For example, in some preferred embodiments, the second buffer solution includes 40-70 mM Tris buffer solution, 9-12 mM divalent cation, and 3-6 mM dithiothreitol.

[0036] Based on the above at least one embodiment, the pH of the first buffer solution is 7-10; for example, the pH of the first buffer solution is 8-9; for example, the pH of the second buffer solution is 6-9; for example, the pH of the second buffer solution is 7-8. For example, the pH of the buffer solution can be its pH value at room temperature (e.g., 20-25 °C). For example, in some embodiments, the pH of the first buffer solution is greater than the pH of the second buffer solution. For example, the pH of the first buffer solution is 8-9, and the pH of the second buffer solution is 7-8. For example, the pH of the first buffer solution is 8.5-9, and the pH of the second buffer solution is 7.5-8; for example, the pH of the first buffer solution can be 8.5, 8.6, 8.7, 8.8, 8.9, or 9.0; for example, the pH of the second buffer solution can be 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0; for example, a larger pH is more suitable for repair treatment, and a smaller pH is more suitable for compatible ligation treatment and digestion treatment.

[0037] Based on the above at least one embodiment, for example, the Tris buffer solution includes at least one of Tris-hydrochloride buffer solution and Tris-acetate buffer solution; for example, the monovalent cation includes Na + , K + and NH 4 + at least one of them; for example, the divalent cation includes Mg 2+ , Mn 2+ , Fe 2+ , Ca 2+ and Zn 2+ at least one of them.

[0038] Based on the above at least one embodiment, the monovalent cation of the first buffer solution includes NH 4 + and monovalent metal cation. For example, the monovalent cation of the first buffer solution includes NH 4 + and K + . For example, the monovalent cation of the first buffer solution includes NH 4 + and Na + . For example, NH 4 + can increase the stability of the buffer solution. For example, NH 4 +It can help increase the stability of the buffer; for example, NH 4 + It can help adjust the pH of the buffer, for example, maintaining a relatively high pH value.

[0039] Based on at least one of the above embodiments, the first buffer further includes a surfactant. For example, the surfactant can increase the stability of the buffer; for example, the surfactant can interact with enzymes, substrates, and / or reaction products to regulate enzymatic reactions.

[0040] For example, the surfactant is selected from at least one of polyethylene glycol octylphenyl ether ( X-100), Tween 20, Tween 40, and Tween 60. For example, the addition amount of the surfactant is based on mass fraction. For example, based on the total weight of the buffer, the content of the surfactant is 0.01-1 wt%. For example, it can be 0.01-1 wt%, 0.05-1 wt%, 0.05-0.5 wt%, etc. For example, it can be 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, or 0.5 wt%.

[0041] For example, the first buffer further includes ATP, for example, providing energy for the reaction. For example, the first buffer further includes NAD + and dNTPs, that is, the NAD + and dNTPs required for nucleic acid repair can also be incorporated into the buffer system; for example, NAD + and dNTPs can also be components outside the buffer system and are added to the buffer separately during library construction.

[0042] For example, the second buffer further includes ATP, for example, providing energy for the reaction. For example, the ATP content is 0.1-50 mM; for example, it can be 0.5-4 mM; for example, it can be 0.5-2 mM. For example, the ATP content can be 1 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, or 1.5 mM.

[0043] Based on at least one of the above embodiments, the monovalent cations in the first buffer include 3-100 mM NH 4 + and 2-20 mM monovalent metal cations. For example, the monovalent cations in the first buffer include 5-60 mM NH 4 + and 3-20 mM monovalent metal cations.

[0044] Based on at least one of the above embodiments, for example, the first buffer includes: 10-50 mM Tris-HCl, 1.5-50 mM (NH 4 )2 SO 4 (3 - 100 mM NH 4 + )、2 - 20 mM KCl and 0.1 - 10 mM MgSO 4 ; for example, the first buffer solution comprises 15 - 30 mM Tris buffer solution, 2.5 - 30 mM (NH 4 ) 2 SO 4 (5 - 60 mM NH 4 + )、3 - 20 mM KCl and 1 - 5 mM MgSO 4 。For example, the first buffer solution comprises 18 - 25 mM Tris-HCl, 5 - 15 mM (NH 4 ) 2 SO 4 (10 - 30 mM NH 4 + )、5 - 20 mM KCl and 2 - 3 mM MgSO 4 。

[0045] For example, the first buffer solution comprises: 10 - 50 mM Tris-HCl, 3 - 100 mM NH 4 Cl, 2 - 20 mM KCl and 0.1 - 10 mM MgCl 2 ; for example, the first buffer solution comprises 15 - 30 mM Tris buffer solution, 5 - 60 mM NH 4 Cl, 3 - 20 mM KCl and 1 - 5 mM MgCl 2 。For example, the first buffer solution comprises 18 - 25 mM Tris-HCl, 10 - 30 mM NH 4 Cl, 5 - 20 mM KCl, 2 - 3 mM MgCl 2 。

[0046] For example, in the first buffer solution, the concentration of Tris-HCl can be 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM or 22 mM; for example, the concentration of (NH 4 ) 2 SO 4 can be 5 mM, 10 mM, 11 mM, 12 mM or 15 mM; for example, the concentration of KCl can be 7 mM, 8 mM, 9 mM, 10 mM, 12 mM or 15 mM; for example, the concentration of MgSO4 can be 2 mM, 2.2 mM, 2.5 mM, 2.8 mM or 3 mM.

[0047] For example, the second buffer solution includes: 20 - 100 mM Tris-HCl, 5 - 20 mM MgCl2, and 1 - 10 mM dithiothreitol; for example, the second buffer solution includes 30 - 80 mM Tris-HCl, 8 - 15 mM MgCl 2 and 2 - 6 mM dithiothreitol. For example, the second buffer solution includes 40 - 70 mM Tris-HCl, 9 - 12 mM MgCl 2 and 3 - 6 mM dithiothreitol.

[0048] For example, in the second buffer solution, the concentration of Tris-HCl can be 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, or 70 mM; the concentration of MgCl 2 can be 9 mM, 9.5 mM, 10 mM, 10.5 mM, 11 mM, 11.5 mM, or 12 mM; the concentration of dithiothreitol can be 3 mM, 4 mM, 5 mM, or 6 mM.

[0049] For example, the second buffer solution includes: 20 - 100 mM Tris-HCl, 5 - 20 mM MgSO 4 and 1 - 10 mM dithiothreitol; for example, the second buffer solution includes 30 - 80 mM Tris-HCl, 8 - 15 mM MgSO 4 and 2 - 6 mM dithiothreitol. For example, the second buffer solution includes 40 - 70 mM Tris-HCl, 9 - 12 mM MgSO 4 and 3 - 6 mM dithiothreitol.

[0050] For example, the above buffer solution can be based on water and include the above components. For example, the above water can be at least one of double-distilled water, ultrapure water, and distilled water.

[0051] Based on the above at least one embodiment, the first enzyme includes at least one of exonuclease, repair enzyme, polymerase, and phosphorylase. For example, the first enzyme is composed of exonuclease, repair enzyme, polymerase, and phosphorylase.

[0052] For example, the exonuclease includes EXO VII; for example, the repair enzyme includes PARPs; for example, the polymerase includes Taq DNA polymerase and / or T4 DNA polymerase; for example, the phosphorylase includes T4 PNK. For example, the first enzyme includes EXO VII, PARPs, Taq DNA polymerase, and T4 PNK.

[0053] Based on the above at least one embodiment, the second enzyme includes a nucleic acid ligase. For example, the nucleic acid ligase includes T4 DNA ligase. For example, the repaired processing product may not have an A tail and may be blunt-ended; T4 DNA ligase can achieve the ligation of blunt ends, and can ligate the repaired processing product with an adaptor. For example, after ligation with the adaptor, a circular nucleic acid molecule that meets the library construction requirements of the Pacbio platform is formed. Based on the above at least one embodiment, the third enzyme includes a nuclease. For example, the nuclease includes at least one of EXO III, EXO VI, and EXO VII. For example, the exonuclease includes EXO III and EXO VI. For example, the exonuclease can remove uncircularized linear nucleic acid molecules, damaged circular nucleic acid molecules, and other defective nucleic acid molecules, further improving the library quality.

[0054] Based on the above at least one embodiment, the activity concentration of the enzyme can be: the enzyme activity per μL of liquid in the reaction system / the volume of the reaction system during ligation processing. For example, the activity concentration of T4 DNA ligase is 5 - 50 U / μL. For example, the embodiments of the present application do not limit the concentration of the enzyme stock solution, as long as the activity concentration of the ligase after being added to the reaction system is 5 - 50 U / μL.

[0055] For example, the activity concentration of T4 DNA ligase is 10 - 40 U / μL. For example, the activity concentration of T4 DNA ligase is 15 - 30 U / μL. For example, the activity concentration of T4 DNA ligase is 20 - 25 U / μL.

[0056] Based on the above at least one embodiment, the activity concentration of the ligase can be: the enzyme activity per μL of liquid in the reaction system / the volume of the reaction system during ligation processing. For example, the activity concentration of T4 DNA ligase is 5 - 50 U / μL. For example, the kit provided by the embodiments of the present application enables the activity concentration of the ligase during use to be 5 - 50 U / μL.

[0057] For example, the activity concentration of T4 DNA ligase is 10 - 40 U / μL. For example, the activity concentration of T4 DNA ligase is 15 - 30 U / μL. For example, the activity concentration of T4 DNA ligase is 20 - 30 U / μL. For example, the activity concentration of T4 DNA ligase can be 20 U / μL, 21 U / μL, 22 U / μL, 23 U / μL, 24 U / μL, 25 U / μL, 26 U / μL, 27 U / μL, 28 U / μL, 29 U / μL, or 30 U / μL.

[0058] Based on the above at least one embodiment, the inventor explored the proportional relationship between the adapter and the nucleic acid sample to be detected. Through this proportional relationship, an appropriate amount of the adapter can be added under different starting amounts, enabling the ligation reaction to proceed fully. It can also precisely control the usage amount of the adapter reagent and reduce the reagent cost. For example, the ratio of the adapter to the nucleic acid sample to be detected is 0.001 - 0.05. For example, the ratio of the adapter to the nucleic acid sample to be detected is 20 - 1000. For example, the ratio of the adapter to the nucleic acid sample to be detected is 50 - 500. For example, the ratio of the adapter to the nucleic acid sample to be detected is 100 - 500. For example, the ratio of the adapter to the nucleic acid sample to be detected is 200 - 400. For example, it can be the ratio of the amount of substance (mol) of the adapter to the amount of substance (mol) of the nucleic acid sample to be detected.

[0059] For example, in some embodiments, the concentration of the adapter is 0.1 - 5 pmol / μL. For example, the concentration of the adapter can be: the added amount of the adapter / the volume of the reaction system during the ligation treatment. For example, the concentration of the adapter is 0.1 - 3 pmol / μL. For example, the concentration of the adapter is 0.1 - 2.5 pmol / μL. A suitable adapter concentration can enable the ligation treatment to proceed fully and can also minimize the generation of by-products such as adapter dimers.

[0060] For example, in some embodiments, the content of the adapter is 40 - 300 pmol. For example, the content of the adapter can be 40 - 200 pmol; for example, the amount of the adapter can be 100 - 200 pmol. A suitable adapter concentration can enable the ligation treatment to proceed fully and can also minimize the generation of by-products such as adapter dimers. Based on the above at least one embodiment, the kit further includes: a fragmentation treatment reagent, including a fragmentation enzyme. For example, the fragmentation enzyme can be an endonuclease. For example, the fragmentation reagent includes a fragmentation enzyme and its reaction buffer. The fragmentation treatment reagent can fragment the sample (such as a DNA sample) into sequences with a certain fragment size range, such as sequences of 5 - 20 kbp, which can meet the fragment size requirements for library construction on the Pacbio platform.

[0061] Based on the above at least one embodiment, the kit further includes a purification treatment reagent, including a reagent for purifying the digestion treatment product. For example, the purification treatment reagent includes magnetic beads. For example, the magnetic beads can be carboxyl magnetic beads.

[0062] For example, the purification reagent can enrich sequences with a preset fragment length in the digestion product. For example, it can enrich fragments with a length of 5 - 25 kbp in the digestion product. For example, it can enrich fragments with a length of 20 - 22 kbp in the digestion product. It can be understood that sequences outside the preset fragment length in the library include adapter dimers, uncircularized template strands, fragments that are too long or too short, template single strands, etc.; these sequences cannot be used for subsequent sequencing and will cause waste of sequencing throughput. Therefore, the purification reagent can further improve the library quality.

[0063] In some embodiments, the purification reagent is used after the digestion reagent. For example, the reagents in the kit can be numbered in ascending order according to the order of use, and the number of the purification reagent is greater than that of the digestion reagent. For example, the numbers of the repair reagent, ligation reagent, and digestion kit purification reagent increase in sequence. The kit provided by the embodiments of the present application uses the purification reagent after the digestion reagent, avoiding the purification operation after each processing step in the existing kit. It can construct a library in a single reaction device, with simple operation and improved library yield.

[0064] In some embodiments, the kit provided by the embodiments of the present application does not contain an A-tailing reagent. For example, the A-tailing reagent includes at least one of A-Tailing Enzyme and dATP. For example, the repair reagent may not contain the A-tailing reagent. For example, the ligation reagent may not contain the A-tailing reagent. The kit provided by the embodiments of the present application omits the A-tailing reagent, reducing the library construction cost and simplifying the operation process.

[0065] In some embodiments, the kit provided by the embodiments of the present application does not contain an A-tailing reagent, and the purification reagent is used after the digestion reagent, avoiding the A-tailing operation of the existing kit and eliminating the need for purification after each processing step. It can construct a library in a single reaction device (such as a reaction tube), with a simple reagent composition and easy operation, and without affecting the library yield.

[0066] Based on the above at least one embodiment, the kit provided by the embodiments of the present application may have a processing specification of 1 to 10,000 ng. For example, the processing specification may be 1,000 to 8,000 ng. For example, the processing specification may be 2,000 to 5,000 ng. For example, the processing specification of the kit may be the amount of the nucleic acid sample to be tested. For example, the amount of the nucleic acid sample to be tested may be considered as the starting amount during library construction. Based on the above at least one embodiment, the usage conditions of the repair treatment reagent include: reaction temperature of 20 to 45°C, reaction time of 20 to 90 min. For example, the reaction conditions may be that at a reaction temperature of 20 to 40°C, the reaction time for continuous reaction is 30 to 60 min. For example, the usage conditions of the repair treatment reagent include: reaction temperature of 40°C, reaction time of 30 min. For example, the reaction conditions of the repair treatment include: reaction temperature of 40°C, reaction time of 60 min. For example, the usage conditions of the repair treatment reagent include: reaction temperature of 37°C, reaction time of 30 min. For example, the usage conditions of the repair treatment reagent include: reaction temperature of 37°C, reaction time of 45 min. For example, the usage conditions of the repair treatment reagent include: reaction temperature of 37°C, reaction time of 60 min. Under these conditions, the reaction efficiency of the repair treatment reagent is high and more sufficient.

[0067] Based on the above at least one embodiment, the usage conditions of the ligation treatment reagent include: first reaction temperature of 15 to 40°C, first reaction time of 30 to 150 min; second reaction temperature of 40 to 80°C, second reaction time of 5 to 30 min. For example, when the ligation treatment reagent is used, it includes two reaction stages. The first reaction stage includes: first reaction temperature of 15 to 40°C, first reaction time of 30 to 150 min; the second reaction stage includes: second reaction temperature of 40 to 80°C, second reaction time of 5 to 30 min. For example, when the ligation treatment reagent is used, after the first stage of the reaction is completed, it can be maintained under certain conditions (such as 4°C), and then the second reaction stage is carried out. That is, the two reaction stages of the ligation treatment may be continuous or discontinuous.

[0068] For example, when using the ligation treatment reagent, a two-temperature-step reaction can be carried out. The reaction temperature can be 15 - 35°C, and the reaction can be continuously carried out for 30 - 60 min. Subsequently, the reaction temperature can be continuously 50 - 70°C, and the reaction can be continuously carried out for 5 - 20 min. For example, the usage conditions of the ligation treatment reagent include: the first reaction temperature is 25°C, and the first reaction time is 30 min; the second reaction temperature is 65°C, and the second reaction time is 10 min. For example, the usage conditions of the ligation treatment reagent include: the first reaction temperature is 25°C, and the first reaction time is 60 min; the second reaction temperature is 65°C, and the second reaction time is 10 min. For example, the usage conditions of the ligation treatment reagent include: the first reaction temperature is 25°C, and the first reaction time is 90 min; the second reaction temperature is 65°C, and the second reaction time is 10 min. For example, the reaction conditions of the ligation reaction include: the first reaction temperature is 25°C, and the first reaction time is 120 min; the second reaction temperature is 65°C, and the second reaction time is 10 min. For example, the usage conditions of the ligation treatment reagent include: the first reaction temperature is 20°C, and the first reaction time is 30 min; the second reaction temperature is 70°C, and the second reaction time is 10 min.

[0069] For example, in the first reaction stage, a linker can be added to the repair product (for example, the repaired nucleic acid molecule); for example, in the second reaction stage, the activity of the ligase in the reaction system can be reduced to minimize its impact on the subsequent digestion treatment (for example, the impact on the exonuclease with digestion function). Thus, under this reaction condition, it can be combined with the combined buffer provided in the above embodiments. There is no need to change the reaction system between the ligation treatment and the subsequent digestion treatment. The efficiency of the ligation treatment is high, the impact on the subsequent reaction is small, and the stability is better.

[0070] Based on the above at least one embodiment, the usage conditions of the digestion treatment reagent include: the reaction temperature is 20 - 50°C, and the reaction time is 10 - 60 min. For example, the reaction temperature can be 30 - 50°C, and the reaction can be continuously carried out for 10 - 50 min. For example, the usage conditions of the digestion treatment reagent include: the reaction temperature is 37°C, and the reaction time is 30 min. For example, the usage conditions of the digestion treatment reagent include: the reaction temperature is 37°C, and the reaction time is 45 min. For example, the usage conditions of the digestion treatment reagent include: the reaction temperature is 37°C, and the reaction time is 15 min. For example, the usage conditions of the digestion treatment reagent include: the reaction temperature is 40°C, and the reaction time is 30 min. For example, the usage conditions of the digestion treatment reagent include: the reaction temperature is 40°C, and the reaction time is 45 min. For example, the usage conditions of the digestion treatment reagent include: the reaction temperature is 45°C, and the reaction time is 15 min. Thus, under this reaction condition, the efficiency of the digestion treatment is high, and the reaction is more sufficient.

[0071] For example, the usage conditions of the above reagents can be the reaction conditions of the above reagents in the corresponding treatment steps.

[0072] In one aspect of the present application, at least one embodiment provides a library construction method applicable to the Pacbio platform, including: subjecting a nucleic acid sample to be tested to a repair treatment; ligating the repair treatment product with an adaptor to perform a ligation treatment, and the repair treatment product does not contain an A tail; digesting the adaptor-ligated product; wherein, no purification treatment is included between the repair treatment and the digesting treatment. The method provided by at least one embodiment of the present application simplifies the operation of the library construction process, reduces the library construction cost, and improves the library yield at the same time.

[0073] In some embodiments, the repair treatment is carried out in a first buffer solution, and the ligation treatment and the digesting treatment are carried out in a second buffer solution. For example, the first buffer solution can be the first buffer solution provided in any of the above embodiments; for example, the second buffer solution can be the second buffer solution provided in any of the above embodiments.

[0074] The present application will be described below with reference to specific examples and comparative examples. These examples are merely illustrative and should not be construed as limiting the present invention.

[0075] The nucleic acid sample to be tested extracted is genomic DNA, and the genomic DNA is fragmented to obtain a DNA sample to be tested.

[0076] 1.1 Repair treatment

[0077] 1) Prepare the buffer solution at room temperature, mix well; add the DNA sample to be tested to the buffer solution;

[0078] 2) Add NAD + , ATP, dNTPs, and the first enzyme to the bottom of the reaction tube, gently flick the tube 3 - 5 times with the fingertips and gently invert the tube 3 - 5 times to fully mix the solution in the tube, and briefly centrifuge to remove air bubbles.

[0079] Reaction conditions: reaction temperature 37°C, reaction time 60 min.

[0080] The first enzyme includes EXO VII enzyme, DNA repair enzyme, DNA polymerase, and T4 PNK phosphorylase.

[0081] The buffer solution added is 10×buffer.

[0082] Table 1 Reagents for repair treatment

[0083]

[0084] 1.2 Ligation treatment

[0085] At this time, the product of the repair treatment is in the reaction tube. Without replacing the reaction tube, add the adapter, ATP, and ligase to the reaction tube, gently flick it 3 - 5 times with the finger pulp and gently invert it 3 - 5 times to fully mix the solution in the tube, and centrifuge briefly to eliminate air bubbles.

[0086] Reaction conditions:

[0087] The first reaction stage: the first reaction temperature is 25 °C, and the first reaction time is 120 min;

[0088] The second reaction stage: the second reaction temperature is 65 °C, and the second reaction time is 10 min.

[0089] The second enzyme is T4 DNA ligase.

[0090] The adapter sequence is shown in Seq ID No.1 - 17.

[0091] The added buffer is 10×buffer.

[0092] Table 2 Ligation treatment reagents

[0093]

[0094] 1.3 Digestion treatment

[0095] At this time, the product of the ligation treatment is in the reaction tube. Without replacing the reaction tube, add the third enzyme to the reaction tube, gently flick it 3 - 5 times with the finger pulp and gently invert it 3 - 5 times to fully mix the solution in the tube, and centrifuge briefly to eliminate air bubbles.

[0096] Reaction conditions:

[0097] The reaction temperature is 37 °C, and the reaction time is 30 min.

[0098] The third enzyme is EXO III enzyme and EXO VI enzyme.

[0099] Table 3 Digestion treatment reagents

[0100]

[0101] 1.4 Purification treatment

[0102] After the digestion treatment, use magnetic beads to purify the reaction product. The magnetic beads are purchased from Novoprotein. The purification treatment is carried out according to the following procedure:

[0103] 1) Equilibrate the temperature of the magnetic beads to room temperature (e.g., 25 °C) 30 - 120 min in advance, and vortex the magnetic beads to fully mix them;

[0104] 2) Pipette 0.45 × volume (volume after digestion treatment), e.g., 26 μL of well-mixed magnetic beads, into the digestion reaction product, and gently pipette up and down about 15 times with a pipette to mix well;

[0105] 3) Let it stand at room temperature for 15 min. During this period, gently invert the PCR tube 3 - 5 times every 5 min to prevent the magnetic beads from settling.

[0106] 4) After briefly centrifuging the reaction tube, place it on a magnetic stand to adsorb the magnetic beads. After about 1 - 5 min when the solution becomes clear, carefully remove the supernatant to a new tube for temporary storage, taking care not to aspirate the magnetic beads;

[0107] 5) Keep the reaction tube on the magnetic stand all the time, add 200 μL of 80% ethanol solution to wash the magnetic beads, gently pipette up and down about 15 times with a pipette, then let it stand for 30 - 60 sec, and carefully remove the supernatant, taking care not to aspirate the magnetic beads;

[0108] 6) Repeat the above operation, i.e., wash the magnetic beads twice in total;

[0109] 7) After brief centrifugation, centrifuge the residual ethanol to the bottom of the tube, then put the reaction tube back on the magnetic stand. After the magnetic beads are adsorbed, carefully remove the residual ethanol, taking care not to aspirate the magnetic beads;

[0110] 8) Open the lid of the reaction tube and let it dry at room temperature until the surface of the magnetic beads turns dull and there is no water stain - like edge for the magnetic beads close to the tube wall (about 1 - 2 min), then take the reaction tube out of the magnetic stand;

[0111] 9) Add 12 μL of TET for elution in the reaction tube, gently pipette about 15 times with a pipette to mix the magnetic beads well, then let it stand at room temperature for 10 - 30 min. During this period, gently invert the PCR tube 3 - 5 times every 5 min to prevent the magnetic beads from settling. After brief centrifugation, place it on a magnetic stand to adsorb the magnetic beads.

[0112] 10) After the solution becomes clear, aspirate the supernatant to a new tube, taking care not to aspirate the magnetic beads. If the aspirated supernatant is less than 11 μL, add EB (ethidium bromide solution) to make it up, and this is the obtained product.

[0113] Example 1

[0114] On the basis of the above - mentioned example, set the initial amount of the DNA sample to be tested as 5000 ng, and the buffer used is a combined buffer. Specifically, the first buffer is used in the repair treatment reagent, and the second buffer is used in the ligation treatment reagent; explore the library yield. The 1× components of the combined buffer include:

[0115] The first buffer includes: 20 mM Tris - HCl, 10 mM (NH 4 ) 2 SO 4, 10 mM KCl, 2 mM MgSO 4 and 0.1% wt of X-100, pH 8.8;

[0116] The second buffer includes: 50 mM Tris-HCl, 10 mM MgCl 2 , 5 mM DTT and 1 mM ATP, pH 7.6.

[0117] The library yield of the kit provided in the embodiments of the present application was detected, and the results are shown in Table 4. It can be seen that the third-generation library construction on the Pacbio platform was successfully achieved using the kit provided in the embodiments of the present application, and the library yield was high.

[0118] Table 4 Library yield of the kit using the combined buffer

[0119] Buffer Batch 1 Batch 1 Batch 2 Batch 2 Combined buffer 56.16％ 55.83％ 49.05％ 50.18％

[0120] Example 2

[0121] Based on the above embodiments, the starting amount of the DNA sample to be tested was set to 2000 ng, and the buffer was the combined buffer. Specifically, the first buffer was used in the repair treatment reagent, and the second buffer was used in the ligation treatment reagent. The 1× components of the buffer were:

[0122] The first buffer includes: 20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 10 mM KCl, 2 mM MgSO 4 and 0.1% wt of X-100, pH 8.8, used during repair treatment;

[0123] The second buffer includes: 50 mM Tris-HCl, 10 mM MgCl 2 , 5 mM DTT and 1 mM ATP, pH 7.6, used during ligation treatment.

[0124] The amount of the adapter was changed to explore the ligation efficiency under different adapter amounts and to find the appropriate amount of adapter to add. The ligation efficiency can be the ratio of the molecular weight of the library after ligation to the molecular weight of the library before ligation.

[0125] It can be seen that when the starting amount of DNA remains unchanged, as the amount of adapter used increases, the ligation efficiency does not increase significantly and will decrease after continuing to increase to a certain value. Considering the overall library construction effect and library construction cost, when the starting amount of DNA is 2000 ng, the amount of adapter used is more appropriate at 40 - 80 pmol.

[0126] Comparison of Ligation Efficiencies at Different Adapter Dosages

[0127] Initial DNA amount Adapter dosage Ligation efficiency 2000 ng 40 pmol 44.4％ 2000 ng 40 pmol 45.8％ 2000 ng 80 pmol 44.9％ 2000 ng 80 pmol 40.6％ 2000 ng 120 pmol 41.9％ 2000 ng 120 pmol 45.6％

[0128] Example 3

[0129] On the basis of the above examples, the initial amount of the DNA sample to be tested was set to 5000 ng, and the buffer was a combined buffer. Specifically, the first buffer was used in the repair treatment reagent, and the second buffer was used in the ligation treatment reagent. The components of the 1× buffer were as follows:

[0130] The first buffer included: 20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 10 mM KCl, 2 mM MgSO 4 and 0.1% wt of X-100, with a pH of 8.8, used during the repair treatment;

[0131] The second buffer included: 50 mM Tris-HCl, 10 mM MgCl 2 , 5 mM DTT and 1 mM ATP, with a pH of 7.6, used during the ligation treatment.

[0132] The dosage of the adapter was changed to explore the ligation efficiency at different adapter dosages. The ligation efficiency can be the ratio of the molecular weight of the library after ligation to the molecular weight of the library before ligation.

[0133] It can be seen that when the initial amount of DNA remains unchanged, as the adapter dosage increases, the ligation efficiency does not increase significantly. After continuing to increase to a certain value, it will decrease. Considering the overall library construction effect and library construction cost, when the initial amount of DNA is 5000 ng, the adapter dosage of 100 - 200 pmol is more appropriate.

[0134] The adapter dosage is related to the initial amount of the nucleic acid sample to be tested. From Examples 1 and 2, it can be seen that within a more appropriate adapter dosage range, the molar ratio of the adapter to the nucleic acid sample to be tested is basically unchanged. For example, this ratio is 20 - 1000 (molar ratio). For example, in some preferred embodiments, this ratio is 200 - 400.

[0135] Table 6 Comparison of Ligation Efficiencies at Different Adapter Dosages

[0136] Initial DNA amount Adapter dosage Ligation efficiency 5000 ng 100 pmol 41.7％ 5000 ng 100 pmol 43.8％ 5000 ng 200 pmol 42.2％ 5000 ng 200 pmol 41.7％ 5000 ng 300 pmol 42.9％ 5000 ng 300 pmol 44.1％

[0137] Example 4

[0138] Based on the above embodiments, the starting amount of the DNA sample to be tested is set to 5000 ng, and the amount of linker used is 100 pmol; the buffer is a combined buffer. Specifically, the first buffer is used in the repair treatment reagent, and the second buffer is used in the ligation treatment reagent. The components of the 1× buffer are as follows:

[0139] The first buffer includes: 20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 ₄, 10 mM KCl, 2 mM MgSO 4 ₄ and 0.1% wt of Triton X-100, with a pH of 8.8, used during the repair treatment;

[0140] The second buffer includes: 50 mM Tris-HCl, 10 mM MgCl 2 ₂, 5 mM DTT and 1 mM ATP, with a pH of 7.6, used during the ligation treatment.

[0141] The enzyme activity of the undiluted ligase is 600 U / μL.

[0142] Control the addition amount of the undiluted enzyme to change the enzyme activity concentration of the ligase, and keep the other conditions unchanged to explore the ligation efficiency at different enzyme activity concentrations. The ligation efficiency can be the ratio of the molecular weight of the library after ligation to the molecular weight of the library before ligation.

[0143] It can be seen that when the starting amount of DNA remains unchanged, as the amount of ligase used increases, the ligation efficiency increases to a certain value and then decreases. The amount of ligase used is more appropriate at 2 μL, that is, the enzyme activity concentration at this time is 22 U / μL.

[0144] Table 7 Comparison of ligation efficiencies at different ligase concentrations

[0145] Initial DNA amount Ligase dosage Ligation efficiency 5000 ng 1 μL 44.89％ 5000 ng 1.5 μL 51.71％ 5000 ng 2 μL 60.9％ 5000 ng 2.5 μL 50.14％

[0146] Comparative Example 1

[0147] Compared with the kit provided in Example 1, the difference is that: a purification treatment reagent is added and used before the digestion treatment reagent. Specifically, a purification treatment reagent is added after the repair treatment reagent to purify the repair treatment product. The purification treatment reagent is magnetic beads, and the magnetic beads are purchased from Novoprotein. The purification treatment includes:

[0148] 1) Equilibrate the temperature of the magnetic beads to room temperature (such as 25 °C) 30 - 120 min in advance, and vortex the magnetic beads to mix well;

[0149] 2) Pipette 0.45 × volume (volume after digestion), e.g., 26 μL of well-mixed magnetic beads, into the digestion reaction product, and gently pipette up and down about 15 times with a pipettor to mix well.

[0150] 3) Let it stand at room temperature for 15 min. During this period, gently invert the PCR tube 3 - 5 times every 5 min to prevent the magnetic beads from settling.

[0151] 4) After briefly centrifuging the reaction tube, place it on a magnetic stand to adsorb the magnetic beads. After about 1 - 5 min when the solution becomes clear, carefully remove the supernatant to a new tube for temporary storage, taking care not to aspirate the magnetic beads.

[0152] 5) Keep the reaction tube on the magnetic stand all the time, add 200 μL of 80% ethanol solution to wash the magnetic beads, gently pipette up and down about 15 times with a pipette, let it stand for 30 - 60 sec, and then carefully remove the supernatant, taking care not to aspirate the magnetic beads.

[0153] 6) Repeat the above operation, i.e., wash the magnetic beads twice in total.

[0154] 7) After brief centrifugation, centrifuge the residual ethanol to the bottom of the tube, then put the reaction tube back on the magnetic stand. After the magnetic beads are adsorbed, carefully remove the residual ethanol, taking care not to aspirate the magnetic beads.

[0155] 8) Open the lid of the reaction tube and let it dry at room temperature until the surface of the magnetic beads turns dull and there is no water stain edge on the magnetic beads close to the tube wall (about 1 - 2 min), then take the reaction tube out of the magnetic stand.

[0156] 9) Add 12 μL of TET for elution in the reaction tube, gently pipette about 15 times with a pipettor to mix the magnetic beads well, then let it stand at room temperature for 10 - 30 min. During this period, gently invert the PCR tube 3 - 5 times every 5 min to prevent the magnetic beads from settling. After brief centrifugation, place it on the magnetic stand to adsorb the magnetic beads.

[0157] 10) After the solution becomes clear, aspirate the supernatant to a new tube, taking care not to aspirate the magnetic beads. If the volume of the aspirated supernatant is less than 11 μL, add EB (ethidium bromide solution) to make it up, and this is the obtained product.

[0158] The library construction was carried out using the kit of Comparative Example 1, and its library yield was detected. The results are shown in Table 8. It can be seen that the library yield of Example 1 is significantly better than that of Comparative Example 1.

[0159] Table 8 Library Yield of Comparative Example 1

[0160]

[0161] Comparative Example 2

[0162] Set the initial amount of the DNA sample to be tested at 5000 ng, construct a library using the official kit of the Pacbio platform, the trade name of the kit is SMRTbell prep kit 3.0, and detect the library yield.

[0163] It can be seen that, on the one hand, for the kit provided in the embodiment of the present application, its library yield is significantly better than that of Comparative Example 2; it shows that the kit provided in the embodiment of the present application can be applied to the Pacbio platform and can replace the existing library construction process and kit.

[0164] On the other hand, the library construction cost of the Pacbio official kit is about 600 - 700 yuan / library; while the library construction cost of the kit provided in the embodiment of the present application is 80 - 90 yuan / library, only about 1 / 9 of the library construction cost of the existing technology, greatly reducing the library construction cost.

[0165] Table 9 Library Yield of Comparative Example 2

[0166] Library yield Batch 1 Batch 2 Comparative Example 2 45.00％ 43.68％

[0167] From the results of Examples 1 - 4 and Comparative Examples 1 - 2, it can be seen that:

[0168] 1) The kit provided in the embodiment of the present application has a simple composition and is easy to use. The combined buffer adapts to the different characteristics of each reaction step and is compatible with multiple steps of reactions, reducing problems such as complex composition, general loss of reagents and samples existing in the existing kits;

[0169] 2) The library yield of the kit provided in the embodiment of the present application reaches 56.16%, significantly higher than that of the Pacbio official kit; it can replace the existing kit and has a good library construction effect;

[0170] 3) The combined buffer used in the kit provided in the embodiment of the present application not only adapts to the different characteristics of each reaction step when applied to library construction, but also can be compatible with multiple reactions, further simplifying the reagent composition and effectively improving the library yield;

[0171] 4) The kit provided in the embodiment of the present application carefully designs the composition and content of each reagent, saves the reagent dosage while ensuring smooth library construction, and the library construction cost is only 1 / 9 of the existing technology, having excellent economic applicability.

[0172] The basic principle of the present invention has been described above in combination with specific embodiments. However, it should be noted that the advantages, advantages, effects, etc. mentioned in the present invention are only examples and not limitations, and it cannot be considered that these advantages, advantages, effects, etc. are essential for each embodiment of the present invention. In addition, the above - disclosed specific details are only for the purposes of illustration and easy understanding, rather than limitations.

Claims

1. A kit for constructing a sequencing library, characterized in that: include: Repair treatment reagents, including a first enzyme with repair function, NAD + , ATP, dNTPs and a first buffer; A ligation treatment reagent, comprising a second enzyme with ligation function, a linker and a second buffer; The digestion treatment reagent includes a third enzyme having a digestion function; wherein, The first buffer comprises: 10-50 mM Tris buffer, 5-120 mM monovalent cations and 0.1-10 mM divalent cations; The second buffer comprises: 20-100 mM Tris buffer, 5-20 mM divalent cations and 1-10 mM dithiothreitol.

2. The kit according to claim 1, characterized in that The first buffer comprises: 15-30 mM Tris buffer, 8-80 mM monovalent cations, 1-5 mM divalent cations; and / or, The second buffer comprises: 30-80 mM Tris buffer, 8-15 mM divalent cations and 2-6 mM dithiothreitol.

3. The kit according to claim 1 or 2, characterized in that The Tris buffer comprises at least one of a Tris-hydrochloric acid buffer and a Tris-acetate buffer; and / or, The monovalent cations include Na + , K + and NH4 + At least one of; and / or, The divalent cations include Mg 2+ , Mn 2+ , Fe 2+ , Ca 2+ and Zn 2+ At least one of .

4. The kit according to claim 3, characterized in that The first buffer comprises: 3-100 mM NH4 + and 2-20 mM monovalent metal cations; and / or, The first buffer solution further comprises: 0.01-1 wt % of a surfactant, wherein the surfactant is selected from at least one of polyethylene glycol octylphenyl ether, Tween 20, Tween 40 and Tween 60.

5. The kit according to claim 4, characterized in that The first buffer comprises: 10-50 mM Tris-HCl, 1.5-50 mM (NH4)2SO4, 2-20 mM KCl, 0.1-10 mM MgSO4 and 0.01-1 wt% of polyethylene glycol octylphenyl ether; Preferably, the first buffer comprises: 15-30 mM Tris-HCl, 2.5-30 mM (NH4)2SO4, 3-20 mM KCl, 1-5 mM MgSO4 and 0.05-1 wt% of polyethylene glycol octylphenyl ether; Preferably, the first buffer comprises: 18-25 mM Tris-HCl, 5-15 mM (NH4)2SO4, 5-20 mM KCl, 2-3 mM MgSO4 and 0.05-0.5 wt% of polyethylene glycol octylphenyl ether.

6. The kit according to any one of claims 1 to 5, characterized in that The second buffer comprises: 20-100 mM Tris-HCl, 5-20 mM MgCl2 and 1-10 mM dithiothreitol; Preferably, the second buffer comprises: 30-80 mM Tris-HCl, 8-15 mM MgCl2 and 2-6 mM dithiothreitol; Preferably, the second buffer comprises: 40-70 mM Tris-HCl, 9-12 mM MgCl2 and 3-6 mM dithiothreitol.

7. The kit according to any one of claims 1 to 6, characterized in that The pH of the first buffer is 7 to 10; and / or, The pH of the second buffer system is 6 to 9; Preferably, the pH of the first buffer system is greater than the pH of the second buffer system.

8. The kit according to any one of claims 1 to 7, characterized in that The first enzyme includes at least one of an exonuclease, a repair enzyme, a polymerase, and a phosphorylase; Preferably, the exonuclease comprises at least one of EXO III, EXO VI and EXO VII; the repair enzyme comprises PARPs; the polymerase comprises Taq DNA polymerase and / or T4 DNA polymerase; the phosphorylase comprises T4 PNK; and / or, The second enzyme includes a nucleic acid ligase; Preferably, the nucleic acid ligase comprises T4 DNA ligase; Preferably, the enzyme activity concentration of the nucleic acid ligase is 5 to 50 U / μL; and / or, The third enzyme includes an exonuclease; Preferably, the exonuclease comprises at least one of EXO III, EXO VI and EXO VII.

9. The kit according to any one of claims 1 to 8, characterized in that The ratio of the linker to the nucleic acid sample to be tested is 20 to 1000; and / or, The content of the linker is 40-300 pmol.

10. The kit according to any one of claims 1 to 9, characterized in that Also includes: fragmentation treatment agents, including fragmentation enzymes; and / or, Purification treatment reagents, including magnetic beads; Preferably, the purification treatment reagent is used after the digestion treatment reagent.

11. Use of the kit according to any one of claims 1 to 10 in the field of sequencing.

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