Reaction reagents for high-throughput sequencing library construction, library construction method

Abstract

The present invention relates to the field of high-throughput sequencing technology, and specifically relates to reaction reagents for constructing high-throughput sequencing libraries and a library construction method. By creatively adjusting the pH of the reaction system to ≤7.2 and adjusting the components and concentrations of the reaction system, the reaction reagents of the present invention can be prepared as single-tube reagents with the mixed enzymes and reaction buffer system, and precipitation is not likely to occur during storage; at the same time, the reaction reagents of the present invention have good compatibility, have no inhibitory effect on the subsequent processes of library preparation, and improve the efficiency and stability of library construction; specifically, divalent cations, monovalent cations, PEG8000, polyhydroxy compounds, and trimethylamine N-oxide in the reaction system of the present invention serve as activity stabilizers and promoters for the mixed enzymes, and synergistically enhance each other to achieve efficient library construction for samples with low starting concentrations. The starting concentration of DNA fragments in the sample can be as low as 100 pg, meeting the application project requirements for high substrate conversion efficiency.

Description

Technical Field

[0001] The present invention relates to the technical field of high-throughput sequencing, and specifically relates to reaction reagents for constructing high-throughput sequencing libraries and a library construction method. Background Art

[0002] Second-generation sequencing technology is also known as high-throughput sequencing or massively parallel sequencing. It is a new-generation sequencing method based on the sequencing-by-synthesis technology after the first-generation de novo sequencing technology of the Sanger method. It can sequence millions or even billions of DNA molecules simultaneously, thereby achieving goals that cannot be reached by first-generation sequencing technologies such as large-scale and high-throughput.

[0003] The operation process of high-throughput sequencing technology is as follows: 1) Construction of a sequencing library; 2) Hybridization of the library with chip adapter primers; 3) Amplification into clusters; 4) Extension sequencing; 5) Data analysis, etc. Among them, for the construction of a sequencing library, the genome is first prepared, and the DNA is randomly fragmented into small fragments of several hundred bases or shorter, or cfDNA does not require fragmentation treatment, and specific adapters are added to both ends. If it is transcriptome sequencing, the construction of the library is relatively more troublesome. After the RNA is fragmented, it needs to be reverse-transcribed into cDNA, and then an adapter is added, or the RNA is first reversed into cDNA, and then fragmented and an adapter is added. Adding adapters to both ends of the fragmented nucleic acid template to be tested can be achieved by various methods known to those skilled in the art. Usually, synthetic adapters are added through the following steps: (1) Physically or enzymatically shear the DNA sample of interest; (2) Blunt and phosphorylate the obtained DNA fragments; (3) Extend the 3' end of the blunt DNA fragment by one nucleotide, preferably by dATP; (4) Connect the obtained DNA fragment to an adapter with a dTTP extension at the 3' end. The dA tailing of the target DNA fragment prevents their intra- or intermolecular ligation with other DNA fragments, and thus increases the yield of fragments with the expected structure.

[0004] In order to simplify the library construction process and improve the efficiency of library construction, the end repair / blunting and dA tailing reactions of DNA fragments can be completed in one tube. The core principle is to use room-temperature enzymes for end repair and heat-resistant enzymes for dA tailing treatment, and adopt a two-temperature-step continuous reaction (performing DNA blunting and phosphorylation reactions at a lower temperature, and then performing tailing on the blunt DNA fragment at a higher temperature), so that end repair and dA tailing can be continuously carried out without intermediate purification treatment. The main products on the market currently are: Ultra TM End Repair / dA-Tailing Module, Ultra TMII End Repair / dA-Tailing Module, TIANSeq End Repair / dA-Tailing Module, For kits such as Fast Pace End Repair / dA Tailing Module, the main enzyme components include: T4 DNA Polymerase, Klenow Fragment (exo-), and T4 PNK for blunt-ending / phosphorylating end repair, and Taq DNA Polymerase for A-tailing reaction at 65 - 70°C and inactivating the end repair enzymes. There are two specific product forms. One is that all the enzymes required for DNA end repair and dA-tailing reaction are combined by the user into a single mixture (enzyme mixture), and the reaction buffer is provided separately without being premixed with the enzymes into a ready-to-use master mixture. The other is that all components for DNA end repair and dA-tailing reaction are in a single vial. To enable automated pipetting and increase the convenience of operation, most products tend to be developed in the form where all components are in a single vial.

[0005] However, putting all components for DNA end repair and dA-tailing reaction in a single vial requires storing multiple enzymes in the same buffer system, including at least the room-temperature polymerase and phosphorylase for end repair, and the heat-resistant polymerase for dA-tailing. The optimal (high-stability) storage conditions, reaction conditions, etc. of these different enzyme raw materials vary. How to balance the performance of each enzyme and confirm the reaction efficiency of the reagent, especially the reaction efficiency of dA-tailing (which directly determines the overall library construction efficiency), while ensuring the stability of the reagent, has become a technical problem to be solved for such products. Summary of the Invention

[0006] To achieve the above object, the object of the present invention is to provide a reaction reagent for high-throughput sequencing library construction, which realizes a general formula with high stability, maintains high reaction efficiency, simplifies the library construction process, and is conducive to industrialization.

[0007] Meanwhile, the present invention also aims to provide a library construction method, which simplifies and improves the efficiency of library construction by using the reaction reagent provided by the present invention.

[0008] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] A reaction reagent for constructing a high-throughput sequencing library, which is prepared by mixing a mixed enzyme and a reaction buffer system; wherein the mixed enzyme includes a room-temperature DNA polymerase, a polynucleotide kinase, and a heat-resistant DNA polymerase; the reaction buffer system includes 400-550 mM Tris-HCl, 60-120 mM divalent cations, 300-500 mM monovalent cations, 60-100 mM polyhydroxy compound, 5-10 mM PEG 8000, 10-20 mM trimethylamine N-oxide, 80-150 mM DTT, 6-15 mM ATP, 1.0-2.0 mM dNTPs, 5 mM-10 mM dATP; the pH of the reaction reagent ≤ 7.2.

[0010] Further preferably, the pH of the reaction reagent is 6.9-7.2.

[0011] As an example, the polyhydroxy compound used in the examples of the present invention is melezitose.

[0012] Optionally, the divalent cation is Mg 2+ ; the monovalent cation is Na + and K + wherein the ratio of Na + and K + is 2:1.

[0013] Optionally, the reaction buffer system further includes 20-30 mM non-ionic surfactant; the non-ionic surfactant is selected from one or a mixture of two of Triton-x-100, Tween 20, and NP-40.

[0014] Optionally, the non-ionic surfactant is a mixture of Triton-x-100 and Tween 20 in a mass ratio of 2:1.

[0015] Optionally, the room-temperature DNA polymerase is T4 DNA polymerase with a concentration of 3 U / μL; the polynucleotide kinase is T4 PNK with a concentration of 10 U / μL; the heat-resistant DNA polymerase is Taq DNA polymerase with a concentration of 5 U / μL.

[0016] Optionally, the reaction reagent is composed of the following components in the following concentration contents: 3 U / μL T4 DNA polymerase, 10 U / μL T4 PNK, 5 U / μL Taq DNA polymerase, 450 mM Tris-HCl, 80 mM MgCl 2, 200 mM NaCl, 100 mM KCl, 80 mM melezitose, 8 mM PEG 8000, 15 mM trimethylamine N-oxide, 110 mM DTT, 10 mM ATP, 1.3 mM dNTPs, 5 mM dATP, 18 mM Triton-x-100, 9 mM Tween 20.

[0017] A high-throughput sequencing library construction method includes the following operating steps: 1) Take a sample containing DNA fragments and add the above reaction reagents, react at 30-40 °C for 20-35 min, raise the temperature to 70-75 °C, react for 15-25 min, perform end repair and dA tailing on the DNA fragments to obtain a first product;

[0018] 2) Add adapters, ligase and its reaction buffer to the first product, and perform a ligation reaction after mixing.

[0019] The reaction reagent for high-throughput sequencing library construction of the present invention is applied to end repair and dA tailing of second-generation sequencing library construction, realizing that end repair and dA tailing are carried out continuously in a single-tube reaction system without intermediate purification, with simple operation and being suitable for industrial application. The present invention has the following beneficial effects compared with the prior art and commercially available similar products:

[0020] It is known in the art that each enzyme has different optimal storage and reaction conditions. For example, the additives required for one enzyme may be harmful to either the storage and / or catalytic activity of another enzyme in the component blend, such as salts and their concentrations, buffer systems, the pH of the mixture, etc. To achieve the stable blend provided by the present invention that can effectively inactivate, phosphorylate, and add dA tails to DNA fragments in a single step and within a single container, a large number of experiments and creative formulation adjustments are required to achieve the compatibility of different enzymes in the same reaction system. Specifically, in terms of storage stability, the mixed enzymes contained in the reaction reagent provided by the present invention generally include T4 DNA polymerase, T4 PNK, and Taq DNA polymerase. The efficiency of adding A is crucial in library construction. Generally, to ensure the A-addition efficiency of Taq DNA polymerase, the pH of the reaction system is usually set to the optimal pH for Taq DNA polymerase activity, usually pH > 7.6, such as mostly 8 - 10. However, this pH condition is close to the isoelectric point of T4 PNK, and T4 PNK precipitation and inactivation are likely to occur, resulting in poor storage stability. To solve this technical problem, the reaction reagent of the present invention creatively adjusts the pH of the reaction system ≤ 7.2 and adjusts the components and concentrations of the reaction system to improve the stability of the reaction reagent, enabling the preparation of the mixed enzyme and the reaction buffer system as a single-tube reagent that is not prone to precipitation and enzyme activity inactivation; at the same time, the reaction reagent of the present invention has good compatibility, has no inhibitory effect on the subsequent processes of library preparation, and improves the efficiency and stability of library construction;

[0021] Specifically, the divalent cations, monovalent cations, PEG8000, polyhydroxy compounds, and trimethylamine N-oxide in the reaction system of the present invention serve as the activity stabilizers and promoters of the mixed enzyme, and they play a synergistic effect with each other to achieve efficient library construction for samples with low starting concentrations. The starting concentration of DNA fragments in the sample can be as low as 100 pg, meeting the application project requirements for high substrate conversion efficiency;

[0022] Furthermore, the present invention optimizes the dosage ratio of divalent cations and monovalent cations, preferably selects appropriate polyhydroxy compounds, and adds non-ionic surfactants such as Triton-x-100, Tween 20, and NP-40 (and preferably the components and dosage ratios), further improving the library construction efficiency. Description of the Drawings

[0023] Figure 1 Schematic diagram of the detection results of the storage stability test of different reaction reagents;

[0024] Figure 2 Schematic diagram of the analysis results of the precipitated components of the reaction reagent in Comparative Example 1;

[0025] Figure 3Schematic diagram of the detection results of the library construction efficiency of different reaction reagents;

[0026] Figure 4 Schematic diagram of the detection results of the stability of different reaction reagents after 7 days of thermal accelerated storage;

[0027] Figure 5 Schematic diagram of the detection results of the change in reaction activity during the continuous thermal accelerated storage of the reaction reagent in Example 1 for 10 days;

[0028] Figure 6 Schematic diagram of the detection results of the library construction efficiency of the reaction reagent in Example 1 for samples with different concentrations. Detailed implementation mode

[0029] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention pertains.

[0030] Next, in combination with the detailed implementation mode, the present invention will be further described, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field. Unless otherwise specified, the reagents and materials used in the following embodiments are all commercially available.

[0031] Among them, T4 DNA polymerase and T4 PNK are purchased from NEB; Taq DNA polymerase is purchased from Enzymatic; the adapters used in library construction are purchased from illumina; T4 DNA Ligase and T4 DNA Ligation Buffer are purchased from NEB.

[0032] Example 1

[0033] This example provides a reaction reagent for high-throughput sequencing library construction, which is used for end modification and dA tailing of fragmented DNA during library construction, and consists of the following components with the following concentration contents: 1.5 mg / ml T4 DNA polymerase, 0.33 mg / ml T4 PNK, 0.08 mg / ml Taq DNA polymerase, 450 mM Tris-HCl, 80 mM MgCl 2 2, 200 mM NaCl, 100 mM KCl, 80 mM melezitose, 8 mM PEG 8000, 15 mM trimethylamine N-oxide, 110 mM DTT, 10 mM ATP, 1.3 mM dNTPs, 5 mM dATP, 18 mM Triton-x-100, 9 mM Tween 20; the pH of the reaction reagent is 7.0.

[0034] The preparation process of the above reaction reagent is the same as the configuration process of the liquid reagent system known in the art. Each component is taken in an equivalent amount according to the formulated concentration and uniformly mixed to prepare it.

[0035] The present invention also provides other examples and comparative examples for verifying the performance of the reaction reagent provided by the present invention in terms of storage stability and library construction. Among them, the concentrations of T4 DNA polymerase, T4 PNK, and Taq DNA polymerase in the reaction reagents of other examples and comparative examples are the same, and the other components, concentration contents, and pH are shown in Table 1 below:

[0036] Table 1

[0037]

[0038]

[0039] Performance comparison and evaluation of different reaction reagents in test examples

[0040] 1. Evaluation indicators:

[0041] (1) Storage stability: The storage stability of the reaction reagent is evaluated by whether precipitation occurs; the occurrence of precipitation indicates poor storage stability; SDS-PAGE is used to analyze the protein components of the precipitate;

[0042] (2) Library construction efficiency: The library construction efficiency is evaluated by the library conversion rate after the library construction is completed. The higher the conversion rate, the higher the library construction efficiency;

[0043] 2. Test methods:

[0044] (1) Storage stability:

[0045] The reaction reagents provided in Examples 1 to 3 and Comparative Examples 1 to 9 are aliquoted into centrifuge tubes and sealed with a sealing film; placed in a 37 °C biological incubator, and the placement time is recorded; taken out after 12 hours of placement, and centrifuged to observe whether protein precipitation occurs;

[0046] (2) Library construction efficiency:

[0047] 1) The process of library construction is as follows:

[0048] ①: Sample preparation:

[0049] FFPE sample nucleic acid extraction and fragmentation:

[0050] Use QIAamp DNA FFPE Tissue Kit (purchased from Qiagen, catalog number 56404) to extract FFPE DNA, and the extracted DNA is used with Qubit TMThe concentration was determined using the 1X dsDNA High Sensitivity (HS) and Broad Range (BR) Quantification Kit (purchased from Thermo, catalog number Q33231).

[0051] Nucleic acid extraction and fragmentation from whole blood samples:

[0052] gDNA in plasma was extracted using the Blood&Cell Culture DNA Mini Kit (purchased from Qiagen, catalog number 13323). The extracted DNA was quantified using Qubit TM The concentration was determined using the 1X dsDNA High Sensitivity (HS) and Broad Range (BR) Quantification Kit (purchased from Thermo, catalog number Q33231).

[0053] gDNA and FFPE DNA were fragmented using a Bioruptor Pico non-contact ultrasonic disruptor. The DNA fragmentation conditions for whole blood were Time ON 30s, Time OFF 30s, 8 Cycles; for FFPE DNA, the conditions were Time ON 30s, Time OFF 30s, 6 Cycles;

[0054] Extraction of cfDNA from plasma:

[0055] Plasma DNA was extracted using the QIAamp Circulating Nucleic Acid Kit (purchased from Qiagen, catalog number 55114). The extracted DNA was quantified using Qubit TM The concentration was determined using the 1X dsDNA High Sensitivity (HS) and Broad Range (BR) Quantification Kit (purchased from Thermo, catalog number Q33231);

[0056] After the preparation of the above three types of samples, the DNA concentration was quantified;

[0057] ② End repair and A addition: The sample input was 50 - 100 ng. Add 15 μL of end modification and A addition reaction reagent, and make up the system to 65 μL with nuclease-free water. Reaction program: 20°C for 20 min, 65°C for 20 min;

[0058] ③ Adapter ligation: Add 5 μL of 15 μM adapter, 5 μL of T4 DNA Ligase (NEB), and 25 μL of T4 DNA Ligation Buffer (NEB) to the system after the reaction in step ② is completed. Reaction program: 20°C for 20 min;

[0059] ④Purification after joint connection: Purify the ligation product in step ③ and VAHTS DNA Clean Beads (purchased from Novoprotein, product number N411-01) at a volume ratio of 5:4, with an elution volume of 20 μL;

[0060] ⑤Amplification: Amplify the library according to the following system: 25 μL of KAPA HiFi Hotstart Ready Mix (purchased from Roche, product number: KK2602), 5 μL of Primer P5 and P7 (purchased from illumina), and 20 μL of the eluted ligation product; Reaction program: 98 °C for 45 s, 98 °C for 15 s, 60 °C for 30 s, 72 °C for 30 s, 7 cycles; 72 °C for 1 min;

[0061] ⑥Purification of amplification product: Purify the amplification product and VAHTS DNA Clean Beads (purchased from Novoprotein, product number N411-01) at a ratio of 1:1, with an elution volume of 25 μL;

[0062] ⑦Determination of library concentration: Use Qubit TM 1X dsDNA High Sensitivity (HS) and Broad Range (BR) Quantification Kit (purchased from Thermo, product number Q33231) to determine the concentration of the library;

[0063] Library conversion amount = library concentration (ng / μL) × volume (μL);

[0064] Library conversion rate = library conversion amount ÷ sample input amount (ng) ÷ 2 ^ number of cycles.

[0065] (3) Thermal accelerated stability:

[0066] Keep the reaction reagent to be verified at 37 °C for 7 days or 10 days, and detect the stability of the reaction reagent after thermal accelerated storage according to the detection method of the above library construction efficiency;

[0067] (4) Sample starting concentration:

[0068] Set different concentration gradients of sample starting concentration, and detect the library construction efficiency of the reaction reagent under different sample starting concentration conditions according to the detection method of the above non-library construction efficiency.

[0069] 3. Test results:

[0070] (1) The results of the storage stability test showed that no precipitation occurred in Examples 1 to 3 and Comparative Examples 4 to 9, while different degrees of precipitation occurred in Comparative Example 1, Comparative Example 2, and Comparative Example 3, as Figure 1 shown;

[0071] (2) The reaction reagents of Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3 were continuously placed at 37 °C for 7 days. It was observed that no precipitation occurred in the reaction reagent of Example 1, while different degrees of precipitation occurred in Comparative Example 1, Comparative Example 2, and Comparative Example 3, and the precipitation was more than that after 12 h of placement. The reaction reagent of Comparative Example 1 with precipitation was centrifuged, and the lower precipitate and the upper supernatant were subjected to SDS-PAGE analysis. As Figure 2 shown, lane 1 was the reaction reagent of Comparative Example 1 at 0 d as the control group, lane 2 was the centrifuged supernatant, and lane 3 was the centrifuged precipitate. By comparing lanes 1, 2, and 3, it was found that T4 PNK and T4 DNA Polymerase were in the precipitate, indicating that T4 PNK and T4 DNA Polymerase were unstable and prone to precipitation.

[0072] In the present invention, by optimizing the pH of the reaction reagent ≤ 7.2, protein precipitation was avoided, and the stability of the reaction reagent was improved;

[0073] (3) Library construction efficiency: The library construction efficiencies of the reaction reagents of Example 1, Comparative Examples 4-9 were verified according to the above library construction method, and the results were as Figure 3 shown. The library construction efficiencies of different reaction reagents were in the order of Example 1 > Comparative Example 4 > Comparative Example 5 > Comparative Example 7 > Comparative Example 9 > Comparative Example 6 > Comparative Example 8, indicating that divalent cations, monovalent cations, PEG8000, the polyhydroxy compound melezitose, trimethylamine N-oxide, and surfactants, as activity stabilizers and promoters of the mixed enzyme, played a synergistic effect within a specific dosage ratio range, improving the library construction efficiency of the reaction reagent in a neutral environment;

[0074] (4) Thermal acceleration stability: The reaction reagents of Example 1, Comparative Examples 1-5 were continuously placed at 37 °C for 7 days, and the library construction efficiencies of different reaction reagents were detected. The results were as Figure 4 shown. The pH of the reaction reagents of Comparative Examples 1-3 was > 7.2, and they basically lost their reaction activity after thermal acceleration storage, indicating that in the present invention, by optimizing the pH of the reaction reagent to be near neutral, specifically pH, 7.2, enzyme precipitation and inactivation were avoided, and the stability of the reaction reagent was improved;

[0075] At the same time, the reaction reagent of Example 1 was continuously placed at 37 °C for 10 days, and the change in the library construction efficiency during the thermal acceleration storage of the reaction reagent of Example 1 was detected by comparison. As Figure 5 shown, the reaction reagent of Example 1 was continuously placed for 10 days, and its reaction activity did not decrease, indicating that the reaction reagent provided by the present invention had excellent stability;

[0076] (5) Sample starting concentration: Different concentration gradients of FFPE samples and gDNA samples were set, and the library construction efficiencies of the reaction reagent of Example 1 for different concentration sample starting concentrations were detected. The results were as Figure 6As shown, it indicates that the reaction reagent provided in Embodiment 1 of the present invention can be applied to library construction with a sample starting concentration as low as 0.1 ng, meeting different application requirements.

[0077] Conclusion:

[0078] Adjust the components of the reaction system. When pH < 7.20, the mixed enzyme and the reaction buffer system are made into a single-tube reagent for preservation, and precipitation is not likely to occur. The divalent cations, monovalent cations, PEG8000, polyhydroxy compounds, and trimethylamine N-oxide in the reaction system act as the activity stabilizers and promoters of the mixed enzyme, exerting a synergistic effect on each other to achieve efficient library construction for samples with a low starting concentration. The starting concentration of DNA fragments in the sample can be as low as 100 pg, meeting the application project requirements for high substrate conversion efficiency.

[0079] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A reaction reagent for high-throughput sequencing library construction, characterized in that, it is prepared by mixing a mixed enzyme and a reaction buffer system; the mixed enzyme includes a room temperature DNA polymerase, a polynucleotide kinase, and a heat-resistant DNA polymerase; the reaction buffer system includes 400-550 mM Tris-HCl, 60-120 mM divalent cations, 300-500 mM monovalent cations, 60-100 mM polyhydroxy compound, 5-10 mM PEG 8000, 10-20 mM trimethylamine N-oxide, 80-150 mM DTT, 6-15 mM ATP, 1.0-2.0 mM dNTPs, 5 mM-10 mM dATP; the pH of the reaction reagent is 6.9-7.2; the room temperature DNA polymerase is T4 DNA polymerase; the polynucleotide kinase is T4 PNK; the heat-resistant DNA polymerase is Taq DNA polymerase; The polyhydroxy compound is melezitose; the divalent cation is Mg 2+ ; the monovalent cation is Na + and K + , wherein the ratio of Na + and K + is 2:1; the reaction buffer system further includes 20-30 mM non-ionic surfactant; the non-ionic surfactant is a mixture of Triton-x-100 and Tween 20 in a mass ratio of 2:

1.

2. The reaction reagent for high-throughput sequencing library construction according to claim 1, characterized in that, the room temperature DNA polymerase is T4 DNA polymerase with a concentration of 3 U / μL; the polynucleotide kinase is T4 PNK with a concentration of 10 U / μL; the heat-resistant DNA polymerase is Taq DNA polymerase with a concentration of 5 U / μL.

3. The reaction reagent for high-throughput sequencing library construction according to claim 2, characterized in that, The reaction reagent consists of the following components with the following concentration contents: 3 U / μL T4 DNA polymerase, 10 U / μL T4 PNK, 5 U / μL Taq DNA polymerase, 450 mM Tris-HCl, 80 mM MgCl 2 , 200 mM NaCl, 100 mM KCl, 80 mM melezitose, 8 mM PEG 8000, 15 mM trimethylamine N-oxide, 110 mM DTT, 10 mM ATP, 1.3 mM dNTPs, 5 mM dATP, 18 mM Triton-x-100, 9 mM Tween 20.

4. A method for high-throughput sequencing library construction, characterized in that, it includes the following operation steps: 1) Take a sample containing DNA fragments and add the reaction reagent according to any one of claims 1-3, react at 30-40 °C for 20-35 min, raise the temperature to 70-75 °C, and react for 15-25 min to perform end repair and dA tailing treatment on the DNA fragments to obtain a first product; 2) Add an adaptor, a ligase and its reaction buffer to the first product, and perform a ligation reaction after mixing.

5. The method for high-throughput sequencing library construction according to claim 4, characterized in that, the DNA content in the sample containing DNA fragments is 100 pg-60 ng.

Images