Kit suitable for Pacbio platform and application thereof

Abstract

The invention relates to a kit suitable for construction of a Pacbio platform library and application of the kit, and the kit comprises a repair treatment reagent which comprises repair treatment enzyme, NAD < + >, ATP and dNTPs; the connection treatment reagent comprises a connection treatment enzyme, a joint and ATP (adenosine triphosphate); a digestion treatment reagent including a digestion treatment enzyme; and a universal buffer compatible with a repair process, a ligation process, and a digestion process. The kit provided by the embodiment of the invention is simple in composition and convenient to use, reagent and sample loss caused by buffer system replacement is reduced, the library yield is high, the library quality is good, 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 a kit applicable to the Pacbio platform and its applications. Background Art

[0002] At present, sequencing technology has become an essential research means in the field of genes. Although first-generation sequencing has advantages such as long read lengths and high accuracy, it cannot perform large-scale sequencing 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, without 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 applicable to the Pacbio platform, including: a repair treatment reagent, including a repair treatment enzyme, NAD + , ATP, and dNTPs; a ligation treatment reagent, including a ligation treatment enzyme, an adaptor, and ATP; a digestion treatment reagent, including a digestion treatment enzyme; and a universal buffer, which is compatible with repair treatment, ligation treatment, and digestion treatment.

[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 universal buffer includes 1-50 mM Tris buffer, 10-100 mM monovalent cations, 5-20 mM divalent cations, and 50-200 mg / L enzyme stabilizer; preferably, the universal buffer includes: 5-30 mM Tris buffer, 30-60 mM monovalent cations, 8-15 mM divalent cations, and 80-150 mg / L enzyme stabilizer.

[0006] For example, in some embodiments, the pH of the universal buffer is 6-9; preferably, the pH of the universal buffer is 7-9; preferably, the pH of the universal buffer is 7-8.

[0007] For example, in some embodiments, the Tris buffer includes at least one of Tris-hydrochloride buffer and Tris-acetate buffer; and / or, the monovalent cations include Na + , K + and NH 4 +at least one of; and / or, the divalent cation includes Mg 2+ , Mn 2+ , Fe 2+ , Ca 2+ and Zn 2+ at least one of; and / or, the enzyme stabilizer includes at least one of bovine serum albumin and recombinant albumin.

[0008] For example, in some embodiments, the universal buffer includes 1 - 50 mM Tris-Ac, 10 - 100 mM KAc, 5 - 20 mM MgAc2, and 50 - 200 mg / L recombinant albumin; the pH of the universal buffer is 6 - 9; preferably, the universal buffer includes: 5 - 30 mM Tris Tris-Ac, 30 - 60 mM KAc, 8 - 15 mM MgAc2, and 80 - 150 mg / L recombinant albumin; the pH of the universal buffer is 7 - 9; preferably, the universal buffer includes: 10 - 25 mM Tris-Ac, 40 - 55 mM KAc, 9 - 12 mM MgAc2, and 90 - 120 mg / L recombinant albumin; the pH of the universal buffer is 7 - 8.

[0009] For example, in some embodiments, the repair processing enzyme includes at least one of exonuclease, repair enzyme, polymerase, and phosphorylase; preferably, the exonuclease includes EXO VII; the repair enzyme includes PARPs; the polymerase includes Taq DNA polymerase and / or T4 DNA polymerase; the phosphorylase includes T4 PNK; and / or, the ligation processing enzyme includes T4 DNA ligase; and / or, the digestion processing enzyme includes at least one of EXO III, EXO VI, and EXO VII.

[0010] For example, in some embodiments, the content of the linker is 40 - 300 pmol.

[0011] For example, in some embodiments, the enzyme activity concentration of the ligase is 5 - 50 U / μL.

[0012] For example, in some embodiments, it further includes: a fragmentation reagent, including a fragmenting enzyme; and / or, a purification reagent, including magnetic beads; preferably, the purification reagent is used after the digestion reagent.

[0013] In the research, the inventors of the present application found that the library construction cost of the Pacbio sequencing platform is high and cumbersome, which limits its wide application. In the official library construction kits of the current Pacbio platform, it is necessary to configure suitable buffer systems and substrates based on different enzymes. The substrates and ion residues in the previous steps affect the subsequent reaction reagents. The buffer systems between different reactions are difficult to be compatible, reducing the reaction efficiency, affecting the library yield and quality, and also increasing the reagent cost.

[0014] To solve at least the above problems, the kit provided by the present application has a simple composition and is convenient to use. It can use a single universal buffer solution to complete library construction in a single reaction system, reducing the loss of reagents and samples caused by replacing the reaction system. Moreover, the library has a high yield and good quality, significantly reducing the library construction cost and strongly promoting the wide application of third-generation sequencing. Detailed implementation manners

[0015] 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 manners 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.

[0016] It should be noted that the terms "first", "second", etc. in the description and claims of the present application are used to distinguish similar objects, and do not necessarily need to describe a specific order or sequence. It should be understood that such data can be interchanged under appropriate circumstances so that the embodiments of the present application described here can be implemented in an order other than those described here. In addition, the terms "include" and "have" and any variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, system, product or device including 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 pointed out in the following text of the embodiments of the present 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.

[0017] The kit, method and their applications provided by the present application will be described below 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 the present 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.

[0018] At least one embodiment of the present application provides a kit applicable to a library on the Pacbio platform, and this kit can be used to construct a library. For example, the kit includes: a repair treatment reagent, including a repair treatment enzyme, NAD + , ATP, and dNTPs; a ligation treatment reagent, including a ligation treatment enzyme, an adaptor, and ATP; a digestion treatment reagent, including a digestion treatment enzyme; and a universal buffer, which is compatible with repair treatment, ligation treatment, and digestion treatment.

[0019] For example, the repair treatment enzyme in the repair treatment reagent 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 repair treatment enzyme can perform end repair, excision repair, mismatch repair, and double-strand break repair, etc. For example, the repair treatment enzyme can be a combination of one or more enzymes; for example, when the repair treatment enzyme includes multiple enzymes, these enzymes can be individually packaged as independent reagents or can be packaged together.

[0020] For example, when the repair treatment 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 can provide raw materials for the repair reaction.

[0021] For example, the repair treatment enzyme, NAD in the repair treatment reagent + , 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 universal buffer.

[0022] For example, the ligation treatment enzyme in the ligation treatment reagent can ligate the repair treatment product with the adaptor to form a dumbbell-shaped structure required for library construction on the Pacbio platform. For example, the ligation treatment 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 adaptor can be any adaptor suitable for library construction on the Pacbio platform. For example, the adaptor sequence is as shown in Seq IDNo.1. For example, when performing multi-sample library construction, different tags can be added to the adaptor to facilitate data splitting after sequencing. For example, the adaptor sequence is as shown in at least one of Seq ID No.2 to 17.

[0024] For example, the ligation treatment enzyme, adaptor, and ATP in the ligation treatment reagent can be individually packaged as independent reagents.

[0025] For example, at least one of ATP and the adaptor can be incorporated into the universal buffer.

[0026]

[0027]

[0028] For example, the digestion reagent can process (e.g., remove) non-library molecules in the ligation product, such as library molecules that do not form dumbbell structures. For example, the digestion enzyme in the digestion reagent can be a DNA exonuclease. For example, the digestion enzyme includes one or more exonucleases. For example, these enzymes can be individually packaged as separate reagents or packaged together.

[0029] For example, the kit further includes a universal buffer that is compatible with repair, ligation, and digestion. For example, the universal buffer is used to provide a buffer system suitable for repair, ligation, and digestion, enabling the reactions to proceed smoothly. It can be understood that the universal buffer being compatible with repair, ligation, and digestion means that at least one of the above processes can be carried out in the universal buffer; or, the universal buffer can be used for at least one of the above processes. For example, all of the above processes are carried out in the universal buffer. For example, repair and ligation are carried out in the universal buffer. For example, ligation and digestion are carried out in the universal buffer. In the kit provided by the embodiments of the present application, the universal buffer can be compatible with multiple library construction steps, with a simple reagent composition and convenient operation.

[0030] In some embodiments, at least one of the repair reagent, ligation reagent, and digestion reagent further includes a universal buffer. For example, the repair reagent further includes a universal buffer. For example, the repair reagent further includes a universal buffer, and the ligation reagent further includes a universal buffer.

[0031] In some embodiments, the universal buffer can also be an independent reagent, for example, it can be individually packaged and used in at least one of the processes of repair, ligation, and digestion. For example, during repair, the universal buffer and the repair reagent are used. For example, the universal buffer and the repair reagent are used during repair, and the universal buffer and the ligation reagent are used during ligation.

[0032] The inventors of the present application found in their research that when using the kit provided by the embodiments of the present application, there is no need to design different buffer solutions for each processing step during library construction. The uniquely designed universal buffer solution can be compatible with multiple steps of reactions, and multiple steps from repair treatment to digestion treatment can be carried out in the same reaction system, for example, in the same reaction tube. The reagent composition is simple and the operation is convenient. Moreover, the inventors surprisingly found that the kit provided by the embodiments of the present application also effectively improves the library yield, and the library quality is good. 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. For example, the library quality can be evaluated through sequencing data, such as the number of enzyme reads (polymerase_reads), the enzyme read length base (polymerase_base), and the average enzyme read length (Average_polymerase_length), etc.

[0033] Based on at least one of the above embodiments, the universal buffer solution includes 1 - 50 mM Tris buffer solution, 10 - 100 mM monovalent cation, 5 - 20 mM divalent cation, and 50 - 200 mg / L enzyme stabilizer. For example, in some preferred embodiments, the universal buffer solution includes: 5 - 30 mM Tris buffer solution, 30 - 60 mM monovalent cation, 8 - 15 mM divalent cation, and 80 - 150 mg / L enzyme stabilizer. For example, in some preferred embodiments, the universal buffer solution includes: 10 - 25 mM Tris buffer solution, 40 - 55 mM monovalent cation, 9 - 12 mM divalent cation, and 90 - 120 mg / L enzyme stabilizer.

[0034] For example, the pH of the universal buffer solution is 6 - 9; for example, the pH of the universal buffer solution is 7 - 9; for example, the pH of the universal buffer solution is 7.0 - 8.0; for example, the pH of the universal buffer solution can be 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. For example, the pH of the universal buffer solution can be its pH value at room temperature (for example, 20 - 25 °C).

[0035] Based on at least one of the above embodiments, 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 at least one of Na + , K + and NH 4 + ; for example, the divalent cation includes at least one of Mg 2+ , Mn 2+ , Fe 2+ , Ca 2+ and Zn 2+ ; for example, the enzyme stabilizer includes at least one of bovine serum albumin and recombinant albumin.

[0036] For example, the universal buffer includes: 1 - 50 mM Tris-Ac, 10 - 100 mM KAc, 5 - 20 mM MgAc2, and 50 - 200 mg / L recombinant albumin; the pH of the universal buffer is 6 - 9. For example, the universal buffer includes: 5 - 30 mM Tris Tris-Ac, 30 - 60 mM KAc, 8 - 15 mM MgAc2, and 80 - 150 mg / L recombinant albumin; the pH of the universal buffer is 7 - 9. For example, the universal buffer includes: 10 - 25 mM Tris-Ac, 40 - 55 mM KAc, 9 - 12 mM MgAc2, and 90 - 120 mg / L recombinant albumin; the pH of the universal buffer is 7 - 8.

[0037] For example, in the universal buffer, the concentration of Tris-Ac can be 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, or 22 mM. For example, the concentration of KAc can be 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, or 50 mM. For example, the concentration of MgAc2 can be 8 mM, 9 mM, 10 mM, 11 mM, or 12 mM. For example, the concentration of recombinant albumin can be 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 115 mM, or 120 mM.

[0038] For example, the concentration of the universal buffer is the concentration of its 1× component; or, the concentration of the universal buffer can be its working concentration, such as its concentration during use. It can be understood that the universal buffer can also be configured to a higher concentration, such as 2× buffer, 5× buffer, or 10× buffer, etc. For example, the 10× universal buffer correspondingly includes: 10 - 500 mM Tris buffer, 100 - 1000 mM monovalent cation, 50 - 200 mM divalent cation, and 500 - 2000 mg / L enzyme stabilizer. That is, adopting the same composition and / or ratio as the universal buffer provided in the embodiments of the present application, even if the concentration is different due to different concentration multiples, it still belongs to the protection scope of the present application.

[0039] Based on the above at least one embodiment, the universal buffer further includes NAD + , at least one of ATP and dNTPs. For example, the universal buffer further includes NAD + and dNTPs, that is, NAD + and dNTPs required for nucleic acid repair can be incorporated. For example, the universal buffer further includes ATP, for example, to provide energy for the reaction.

[0040] Based on the above at least one embodiment, NAD +, at least one of dNTPs and ATP can also be separately packaged from the universal buffer and added to the universal buffer separately during library construction.

[0041] Based on at least one of the above embodiments, the repair processing enzyme includes at least one of exonuclease, repair enzyme, polymerase, and phosphorylase. For example, the repair processing enzyme consists of exonuclease, repair enzyme, polymerase, and phosphorylase.

[0042] 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 repair processing enzyme includes EXO VII, PARPs, Taq DNA polymerase, and T4 PNK.

[0043] Based on at least one of the above embodiments, the ligation processing enzyme includes T4 DNA ligase. For example, the repair processing product may not have an A tail and may be blunt-ended; T4 DNA ligase can achieve blunt-end ligation and can ligate the repair processing product to the adapter. For example, after ligation to the adapter, a circular nucleic acid molecule that meets the library construction requirements of the Pacbio platform is formed.

[0044] Based on at least one of the above embodiments, the digestion processing enzyme 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.

[0045] Based on at least one of the above embodiments, 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 adding to the reaction system is 5 - 50 U / μL.

[0046] 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.

[0047] Based on the above at least one embodiment, the active concentration of the ligase can be: the enzyme activity per μL of liquid in the reaction system / the volume of the reaction system during the ligation treatment. For example, the active concentration of T4 DNA ligase is 5 - 50 U / μL. For example, for the kit provided in the embodiments of the present application, the active concentration of the ligase can be 5 - 50 U / μL when in use.

[0048] For example, the active concentration of T4 DNA ligase is 10 - 40 U / μL. For example, the active concentration of T4 DNA ligase is 15 - 30 U / μL. For example, the active concentration of T4 DNA ligase is 20 - 30 U / μL. For example, the active 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.

[0049] 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, appropriate amounts of the adapter can be added under different starting amounts, enabling the ligation reaction to proceed fully, and also precisely controlling the usage amount of the adapter reagent and reducing 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.

[0050] 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 also minimize the generation of by-products such as adapter dimers.

[0051] 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 also minimize the generation of by-products such as adapter dimers.

[0052] 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 a 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 requirements for the fragment size in library construction on the Pacbio platform.

[0053] 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.

[0054] For example, the purification treatment reagent can enrich sequences with a preset fragment length in the digestion treatment product. For example, it can enrich fragments with a length of 5 - 25 kbp in the digestion treatment product. For example, it can enrich fragments with a length of 20 - 22 kbp in the digestion treatment product. It can be understood that the sequences in the library outside the preset fragment length include adapter dimers, uncircularized template strands, too long or too short fragments, template single strands, etc.; these sequences cannot be used for subsequent sequencing and will cause waste of sequencing throughput. Therefore, the purification treatment reagent can further improve the library quality.

[0055] In some embodiments, the purification treatment reagent is used after the digestion treatment 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 treatment reagent is greater than that of the digestion treatment reagent. For example, the numbers of the repair treatment reagent, ligation treatment reagent, and digestion treatment kit purification treatment reagent increase in sequence. The kit provided by the embodiments of the present application uses the purification treatment reagent after the digestion treatment reagent, avoiding the purification operation after each treatment process in the existing kit. It can achieve library construction in a single reaction device, with simple operation and improved library yield.

[0056] In some embodiments, the kit provided by the embodiments of the present application does not contain an A - addition reagent. For example, the A - addition reagent includes at least one of an A - Tailing Enzyme and dATP. For example, the repair treatment reagent may not contain an A - addition reagent. For example, the ligation treatment reagent may not contain an A - addition reagent. The kit provided by the embodiments of the present application omits the A - addition reagent, reducing the library construction cost and simplifying the operation process.

[0057] In some embodiments, the kit provided by the embodiments of the present application does not contain an A - addition reagent, and the purification treatment reagent is used after the digestion treatment reagent, avoiding the A - tailing operation of the existing kit and eliminating the need for purification after each treatment process. It can achieve library construction in a single reaction device (such as a reaction tube), with simple reagent composition, simple operation, high library yield, and good library quality.

[0058] Based on the above at least one embodiment, the processing specification of the kit provided by the embodiments of the present application can be 1 to 10,000 ng. For example, the processing specification can be 1,000 to 8,000 ng. For example, the processing specification can be 2,000 to 5,000 ng. For example, the processing specification of the kit can be the amount of the nucleic acid sample to be tested. For example, the amount of the nucleic acid sample to be tested can be regarded as the starting amount during library construction.

[0059] Based on the above at least one embodiment, the usage conditions of the repair treatment reagent include: reaction temperature 20 to 45 °C, reaction time 20 to 90 min. For example, the reaction conditions can 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 40 °C, reaction time 30 min. For example, the reaction conditions of the repair treatment include: reaction temperature 40 °C, reaction time 60 min. For example, the usage conditions of the repair treatment reagent include: reaction temperature 37 °C, reaction time 30 min. For example, the usage conditions of the repair treatment reagent include: reaction temperature 37 °C, reaction time 45 min. For example, the usage conditions of the repair treatment reagent include: reaction temperature 37 °C, reaction time 60 min. Under these conditions, the reaction efficiency of the repair treatment reagent is high and more sufficient.

[0060] Based on the above at least one embodiment, the usage conditions of the ligation treatment reagent include: first reaction temperature 15 to 40 °C, first reaction time 30 to 150 min; second reaction temperature 40 to 80 °C, second reaction time 5 to 30 min. For example, when using the ligation treatment reagent, it includes two reaction stages. The first reaction stage includes: first reaction temperature 15 to 40 °C, first reaction time 30 to 150 min; the second reaction stage includes: second reaction temperature 40 to 80 °C, second reaction time 5 to 30 min. For example, when using the ligation treatment reagent, after completing the reaction of the first stage, 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 can be continuous or discontinuous.

[0061] For example, when using the ligation treatment reagent, a two-temperature-step reaction can be carried out. The reaction can be carried out at a reaction temperature of 15-35°C for a continuous reaction time of 30-60 min, and then continuously at a reaction temperature of 50-70°C for a continuous reaction time of 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.

[0062] 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 these reaction conditions, it can be combined with the universal 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.

[0063] Based on at least one of the above embodiments, 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, it can be at a reaction temperature of 30-50°C for a continuous reaction time of 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 these reaction conditions, the efficiency of the digestion treatment is high and the reaction is more complete.

[0064] For example, the usage conditions of the above reagents can be the reaction conditions of the above reagents in the corresponding treatment steps.

[0065] 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 ligation product; wherein, no purification treatment is included between the repair treatment and the digestion 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, improves the library yield, and has good library quality.

[0066] In some embodiments, the repair treatment, the ligation treatment, and the digestion treatment are all carried out in a universal buffer; for example, the universal buffer can be the universal buffer provided in any of the above embodiments.

[0067] 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.

[0068] 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.

[0069] 1.1 Repair treatment

[0070] 1) Prepare the buffer at room temperature, mix well; add the DNA sample to be tested to the buffer;

[0071] 2) Add NAD + , ATP, dNTPs, and repair treatment enzymes to the bottom of the reaction tube, gently flick the tube 3-5 times with the finger pulp and gently invert the tube 3-5 times to fully mix the solution in the tube, and centrifuge briefly to eliminate air bubbles.

[0072] Reaction conditions: reaction temperature 37 °C, reaction time 60 min.

[0073] The repair treatment enzymes include EXO VII enzyme, DNA repair enzyme, DNA polymerase, and T4 PNK phosphorylase. The buffer added is 10×buffer.

[0074] Table 1 Repair treatment reagents

[0075]

[0076] 1.2 Ligation treatment

[0077] At this time, the reaction tube contains the repair treatment product. Without changing the reaction tube, add the adaptor, ATP, and ligase to the reaction tube, gently flick the tube 3-5 times with the finger pulp and gently invert the tube 3-5 times to fully mix the solution in the tube, and centrifuge briefly to eliminate air bubbles.

[0078] Reaction conditions:

[0079] First reaction stage: The first reaction temperature is 25 °C, and the first reaction time is 120 min;

[0080] Second reaction stage: The second reaction temperature is 65 °C, and the second reaction time is 10 min.

[0081] The ligation enzyme is T4 DNA ligase.

[0082] The adapter sequences are shown in Seq ID No. 1-17.

[0083] The added buffer is 10× buffer.

[0084] Table 2 Ligation reagents

[0085]

[0086] 1.3 Digestion

[0087] At this time, the reaction tube contains the ligation product. Without changing the reaction tube, add the third enzyme to the reaction tube, gently flick it 3-5 times with your finger pulp and gently invert it 3-5 times to fully mix the solution in the tube, and briefly centrifuge to remove air bubbles.

[0088] Reaction conditions:

[0089] The reaction temperature is 37 °C, and the reaction time is 30 min.

[0090] The digestion enzymes are EXO III enzyme and EXO VI enzyme.

[0091] Table 3 Digestion reagents

[0092]

[0093] 1.4 Purification

[0094] After the digestion is completed, use magnetic beads to purify the reaction product. The magnetic beads are purchased from Novoprotein. The purification is carried out according to the following procedure:

[0095] 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;

[0096] 2) Pipette 0.45× volume (the volume after digestion), e.g., 26 μL of the fully mixed magnetic beads into the digestion reaction product, and gently pipette up and down about 15 times with a pipette to fully mix;

[0097] 3) Let it stand at room temperature for 15 min. During this period, the PCR tube can be gently inverted 3-5 times every 5 min to prevent the magnetic beads from settling.

[0098] 4) After briefly centrifuging the reaction tube, place it on a magnetic stand to adsorb the magnetic beads. After about 1 - 5 minutes when the solution becomes clear, carefully remove the supernatant to a new tube for temporary storage, taking care not to aspirate the magnetic beads;

[0099] 5) Keep the reaction tube on the magnetic stand at all times. Add 200 μL of 80% ethanol solution to wash the magnetic beads. Gently pipette up and down about 15 times, then let it stand for 30 - 60 seconds, and carefully remove the supernatant, taking care not to aspirate the magnetic beads;

[0100] 6) Repeat the above operation, that is, wash the magnetic beads twice in total;

[0101] 7) After brief centrifugation, centrifuge the residual ethanol to the bottom of the tube, then place 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;

[0102] 8) Open the lid of the reaction tube and air - dry it at room temperature until the surface of the magnetic beads turns dull and there is no water - stained edge for the magnetic beads close to the tube wall (about 1 - 2 minutes), then take the reaction tube out of the magnetic stand;

[0103] 9) Add 12 μL of TET for elution in the reaction tube. Gently pipette up and down about 15 times to fully mix the magnetic beads, then let it stand at room temperature for 10 - 30 minutes. During this period, the PCR tube can be gently inverted 3 - 5 times every 5 minutes to prevent the magnetic beads from settling. After brief centrifugation, place it on the magnetic stand to adsorb the magnetic beads.

[0104] 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.

[0105] Glossary

[0106] Polymerase Bases: The number of bases in the polymerase read.. Enzyme read length base, that is, all the data volume obtained by sequencing, including adaptors sequences.

[0107] Polymerase Reads: The number of polymerases generating high quality reads.

[0108] Polymerase reads are trimmed to the high quality region and include bases from adaptors, as well as potentially multiple passes around a circular template. The enzyme reads count, namely high-quality sequencing reads, includes adaptors and multiple subreads obtained from multiple runs.

[0109] Average Polymerase Length: The mean trimmed read length of all polymerase reads. The value includes bases from adaptors as well as multiple passes around a circular template. Average enzyme read length, the average length of the sequencing reads, includes adaptors and multiple subreads.

[0110] Polymerase Read N50: 50% of all polymerase reads are longer than this value. Among the sequencing reads, 50% of the reads are longer than the N50 value.

[0111] CCS: Circular Consensus Sequence, a sequence obtained by mutually correcting multiple subreads on a single Polymerase read, which reflects the true library sequence.

[0112] Hifi reads: High fidelity reads, sequencing sequences that combine long read lengths and high accuracy. Hifi reads have

[0113] >99.9% (Q20) accuracy of single molecule reads.

[0114] Yield: The total number of bases.

[0115] Example 1

[0116] On the basis of the above embodiments, the initial amount of the DNA sample to be tested is set to 5000 ng, and the added buffer is 10× buffer. The buffer used is a universal buffer, and its 1× component includes 20 mM Tris-Ac, 50 mM KAc, 10 mM MgAc2 and 100 mg / L recombinant albumin, with a pH of 7.9.

[0117] Detect the library yield of the kit provided in the embodiments of the present application; and perform sequencing and evaluate the sequencing data. The results are shown in Table 4. It can be seen that the kit provided in the embodiments of the present application successfully realizes the third-generation library construction on the Pacbio platform, and has a high library yield and good library quality.

[0118] Table 4 Library Yield and Sequencing Data

[0119]

[0120] Table 5 Sequencing Data of the Library of the Present Application

[0121]

[0122]

[0123] Comparative Example 1

[0124] Compared with the kit provided in Example 1, the difference lies in: adding a purification treatment reagent and using it 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:

[0125] 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 mix well;

[0126] 2) Pipette 0.45× volume (the 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;

[0127] 3) Let it stand at room temperature for 15 min, and during this period, gently invert the PCR tube 3 - 5 times every 5 min to prevent the magnetic beads from settling.

[0128] 4) After briefly centrifuging the reaction tube, place it on a magnetic rack 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;

[0129] 5) Keep the reaction tube on the magnetic stand at all times. Add 200 μL of 80% ethanol solution to rinse the magnetic beads. Gently pipette up and down about 15 times, then let it stand for 30 - 60 sec. Carefully remove the supernatant, taking care not to aspirate the magnetic beads.

[0130] 6) Repeat the above operation, i.e., rinse the magnetic beads twice in total.

[0131] 7) After a brief centrifugation, centrifuge the residual ethanol to the bottom of the tube. Then place 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.

[0132] 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.

[0133] 9) Add 12 μL of TET for elution in the reaction tube. Gently pipette up and down about 15 times to fully mix the magnetic beads. 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 a brief centrifugation, place it on the magnetic stand to adsorb the magnetic beads.

[0134] 10) After the solution becomes clear, aspirate the supernatant into 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 to 11 μL. This is the obtained product.

[0135] The library was constructed using the kit of Comparative Example 1, and its library yield was detected. Moreover, for the libraries produced in Comparative Example 1 and Example 1, after equimolar pooling, they were sequenced on the same cell to detect their library quality. The results are shown in Table 5 and Table 6.

[0136] Comparing the two, for the library constructed using the kit provided in Example 1, its indicators such as yield, polymerase base, and Average_polymerase_length are all better than those of Comparative Example 1.

[0137] Table 5 Library Yield of Comparative Example 1

[0138]

[0139] Table 6 Comparison of Library Construction Data between Comparative Example 1 and the Kit of the Present Application

[0140] Index This application Comparative Example 1 Number of polymerase_reads enzyme reads 454,998 431,064 Polymerase read length base of polymerase_base enzyme 56,721,688,259 52,973,337,859 Average_polymerase_length Average enzyme read length 124,663.60 122,889.73 polymerase_n50 205,145 204,110 ccsreads_reads 222,539 212,724 ccsreads_base 3,088,812,519 2,925,934,017 Average_ccsreads_length 13,879.87 13,754.6

[0141] Comparative Example 2

[0142] Set the initial amount of the DNA sample to be tested at 5000 ng, and construct the library using the official kit of the Pacbio platform. The trade name of the kit is SMRTbell prep kit 3.0. Detect the library yield and library quality, and compare them with Example 1. The results are shown in Table 7 and Table 8.

[0143] 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; and, in terms of sequencing data, it is basically the same as that of Comparative Example 2, indicating that the library quality is good and it can be applied to the Pacbio platform and can replace the existing library construction process and kit.

[0144] 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.

[0145] Table 7 Comparison of library yields between Comparative Example 2 and the present application

[0146] Library yield Comparative Example 2 Example 1 Batch 1 45.00％ 52.80％ Batch 2 43.68％ 57.15％

[0147] Table 8 Comparison of library sequencing data between Comparative Example 2 and the present application

[0148] Comparative Example 2 Comparative Example 2 Example 1 Example 1 Polymerase read length base 482.37 471.49 452.69 437.14 PolymeraseReads 5,706,000 5,511,936 5,271,858 5,554,199 PolymeraseReadLength(mean)(bp) 84,537 85,540 85,868 78,704 HIFIReads(≥Q20) 1,501,680 1,544,642 1,456,839 1,242,745 HIFIreadsMeanLength(bp) 18,402 17,439 16,840 17,568

[0149] It can be seen from at least Example 1, Comparative Example 1 and Comparative Example 2 that:

[0150] 1) The kit provided in the embodiment of the present application has a simple composition and is easy to use. It can complete library construction in a single reaction system, reducing problems such as complex composition, common loss of reagents and samples existing in existing kits;

[0151] 2) For the kit provided in the embodiment of the present application, the library yield reaches 57.15%, which is significantly higher than that of the Pacbio official; and the library quality is good, which can replace the existing kit and has good applicability; in addition, the library construction cost of the embodiment of the present application is only 1 / 9 of the existing technology, with excellent economic applicability;

[0152] 3) The universal buffer used in the kit provided in the embodiment of the present application has good adaptability when applied to library construction, can be compatible with multiple reactions, further simplifies the reagent composition, and can effectively improve the library yield.

[0153] The basic principles of the present invention have been described in conjunction with specific embodiments. However, it should be noted that the advantages, benefits, effects, etc. mentioned in the present invention are merely examples and not limitations, and it cannot be considered that these advantages, benefits, effects, etc. are essential for each embodiment of the present invention. Additionally, the specific details disclosed above are only for illustrative and facilitating understanding purposes, rather than limitations.

Claims

1. A kit suitable for Pacbio platform library construction, characterized in that: include: Repair treatment reagents, including repair treatment enzymes, NAD + , ATP and dNTPs; Ligation processing reagents, including ligation processing enzymes, linkers and ATP; Digestion treatment reagents, including digestion treatment enzymes; as well as A universal buffer that is compatible with repair, ligation, and digestion treatments.

2. The kit according to claim 1, characterized in that The universal buffer comprises 1-50 mM Tris buffer, 10-100 mM monovalent cations, 5-20 mM divalent cations and 50-200 mg / L enzyme stabilizer; Preferably, the universal buffer comprises: 5-30 mM Tris buffer, 30-60 mM monovalent cations, 8-15 mM divalent cations and 80-150 mg / L enzyme stabilizer.

3. The kit according to claim 1 or 2, characterized in that The pH of the universal buffer is 6 to 9; Preferably, the pH of the universal buffer is 7 to 9; Preferably, the pH of the universal buffer is 7-8.

4. The kit according to claim 2 or 3, 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; and / or, The enzyme stabilizer includes at least one of bovine serum albumin and recombinant albumin.

5. The kit according to claim 4, characterized in that The universal buffer comprises 1-50 mM Tris-Ac, 10-100 mM KAc, 5-20 mM MgAc2 and 50-200 mg / L recombinant albumin; the pH of the universal buffer is 6-9; Preferably, the universal buffer comprises: 5-30 mM Tris Tris-Ac, 30-60 mM KAc, 8-15 mM MgAc2 and 80-150 mg / L recombinant albumin; the pH of the universal buffer is 7-9; Preferably, the universal buffer comprises: 10-25 mM Tris-Ac, 40-55 mM KAc, 9-12 mM MgAc2 and 90-120 mg / L recombinant albumin; the pH of the universal buffer is 7-8.

6. The kit according to any one of claims 1 to 5, characterized in that The repair enzyme comprises at least one of an exonuclease, a repair enzyme, a polymerase and a phosphorylase; Preferably, the exonuclease comprises 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 ligation processing enzyme comprises T4 DNA ligase; and / or, The digestion treatment enzyme includes at least one of EXO III, EXO VI and EXO VII.

7. The kit according to any one of claims 1 to 5, characterized in that The content of the linker is 40-300 pmol.

8. The kit according to any one of claims 1 to 7, characterized in that Also includes: fragmentation treatment agents, including fragmentation enzymes; and / or, Purification treatment reagents, including magnetic beads.

9. The kit according to any one of claims 1 to 8, characterized in that The purification treatment reagent is used after the digestion treatment reagent.

10. Use of the kit according to any one of claims 1 to 9 in the field of sequencing.

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