Composition and library construction method

Abstract

The present invention discloses a composition and a library construction method. The present invention effectively improves the connection efficiency by introducing an enhancer into the connection reaction system, and is applied to the construction of nucleic acid libraries, with low residual adapter dimers, significantly improving the amount of effective sequencing data, reducing data waste, and reducing sequencing costs to a certain extent.

Description

Technical Field

[0001] The present invention relates to the field of molecular amplification technology, and in particular to a composition and a library construction method. Background Art

[0002] Ligation is the connection of the two ends of the nucleic acid molecule DNA or RNA, such as flat end connection or sticky end connection, and the ligation reaction process usually requires the catalysis of ligase. Ligation is widely used in molecular cloning or detection. For example, in the construction of high-throughput sequencing libraries, nucleic acid molecules are connected to sequencing adapters through ligation. Therefore, it is very meaningful to improve the efficiency of ligation and increase the concentration of the library. In addition, the current library construction methods all use excessive adapter sequences and target fragments, and the ratio is usually 100-400 times. Excessive adapter sequences are prone to cause the adapter to connect itself to form adapter dimers. Therefore, reducing the self-connection of the adapter and increasing the amount of effective data are of great benefit.

[0003] In view of this, the present invention is proposed. Summary of the invention

[0004] The present invention unexpectedly discovered that adding polysucrose and DMSO to the ligation reaction can achieve at least one of the following effects: improving ligation efficiency, increasing library concentration, reducing the content of adapter dimers, and increasing the amount of sequencing data.

[0005] The present invention has at least one of the following technical solutions:

[0006] The invention provides a composition for nucleic acid ligation reaction, comprising polysucrose and DMSO.

[0007] In some embodiments, the above composition further comprises propylene glycol.

[0008] In some embodiments, the propylene glycol is selected from at least one of 1,3-propylene glycol and 1,2-propylene glycol.

[0009] In some embodiments, the volume percentage of propylene glycol in the ligation reaction system is 1-5%.

[0010] In some embodiments, the volume percentage of polysucrose in the ligation reaction system is 5-15%.

[0011] In some embodiments, the average molecular weight of polysucrose is 70,000-400,000.

[0012] In some embodiments, the polysucrose is selected from Ficoll400 or Ficoll70.

[0013] In some embodiments, the volume percentage of DMSO in the ligation reaction system is 2-20%.

[0014] In some embodiments, the above-mentioned composition is used in the ligation reaction stage of nucleic acid library construction.

[0015] In some embodiments, the above composition further comprises a ligase.

[0016] In some embodiments, the ligase is a DNA ligase.

[0017] The DNA ligase is selected from the group consisting of T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Taq DNA ligase, DNA ligase, at least one of Escherichia coli DNA ligases.

[0018] The present invention also provides a kit for constructing a nucleic acid library, comprising any of the above compositions.

[0019] In some embodiments, the above-mentioned kit further comprises at least one of a linker sequence and a ligation reaction buffer.

[0020] The present invention also provides a nucleic acid connection method, comprising: mixing the above composition with nucleic acid fragments, and connecting them under conditions that allow connection.

[0021] The present invention also provides a method for constructing a nucleic acid library, comprising the step of performing a linker ligation reaction using the above-mentioned composition, linker sequence and linker reaction buffer.

[0022] Before the adapter ligation reaction, the nucleic acid fragments are also subjected to end repair and A addition steps.

[0023] In some embodiments, the above-mentioned method further includes a PCR enrichment step after the linker ligation reaction.

[0024] In some embodiments, the above method for constructing a nucleic acid library further comprises a purification step after the linker ligation reaction and before PCR enrichment.

[0025] In some embodiments, the purification step comprises a magnetic bead purification step. DETAILED DESCRIPTION

[0026] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0027] The present invention provides a composition for nucleic acid ligation reaction, comprising Ficoll and DMSO (dimethyl sulfoxide). The present invention effectively improves the ligation efficiency by adding the composition to the ligation reaction system; and the content of the linker dimer is low, thereby increasing the amount of effective sequencing data, reducing data waste, and reducing sequencing costs to a certain extent.

[0028] In some embodiments, the composition further comprises propylene glycol. The addition of propylene glycol can further improve the ligation efficiency and the library concentration to a certain extent.

[0029] In some embodiments, the propylene glycol is selected from at least any one of 1,3-propylene glycol and 1,2-propylene glycol.

[0030] In some embodiments, the volume percentage of propylene glycol in the connection reaction system is 1-5%; for example, it can be 1%, 1.5%, 2%, 2.5%, 3%, 3.5, 4%, 4.5% or 5%, but is not limited thereto.

[0031] In some embodiments, the volume percentage of polysucrose in the ligation reaction system is 5-15%, for example, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15%, but is not limited thereto.

[0032] In some embodiments, the average molecular weight of the polysucrose is 70,000-400,000; for example, it may be 70,000, 100,000, 150,000, 200,000, 300,000, or 400,000, but is not limited thereto.

[0033] In some embodiments, the polysucrose is, for example, Ficoll 400 or Ficoll 70. The average molecular weight of Ficoll 400 is 400,000, and the average molecular weight of Ficoll 70 is 70,000.

[0034] In some embodiments, the volume percentage of DMSO in the ligation reaction system is 2-20%; for example, it can be 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18% or 20%, but is not limited thereto.

[0035] It should be noted that the volume concentration of propylene glycol, polysucrose or DMSO in the ligation reaction system is only the amount added in the form of a solution. In other embodiments, the equivalent molar number, mass fraction, and addition ratio in the form of dry powder are all within the scope of protection of the present invention.

[0036] In some embodiments, the composition is used in the ligation reaction stage of nucleic acid library construction.

[0037] In some embodiments, the composition further comprises a ligase.

[0038] In some embodiments, the ligase is a DNA ligase.

[0039] The DNA ligase is selected from the group consisting of T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Taq DNA ligase, DNA ligase, at least one of Escherichia coli DNA ligases.

[0040] The amount of ligase added to the ligation reaction system is 0.006-120 units / μL. In other embodiments, the amount of ligase added can be adjusted according to actual ligation requirements.

[0041] A ligation reaction buffer comprises the composition defined in any of the above embodiments and a buffer component.

[0042] A kit for constructing a nucleic acid library, comprising the composition in any of the above embodiments.

[0043] In some embodiments, the kit further comprises at least any one of the above-mentioned linker sequence and ligation reaction buffer.

[0044] Wherein, the adapter sequence is adapted to the sequencing platform. In some embodiments, such as the Illumina platform, the adapter sequence can be a U-shaped adapter or a Y-shaped adapter.

[0045] In some embodiments, the adapter sequence is added in an amount of 1-50 μM.

[0046] In some embodiments, the ligation reaction buffer includes a buffer component, ATP, magnesium ions, etc. In some embodiments, the ligation reaction buffer uses a 10-fold concentration. In some embodiments, the working concentration of ATP is 1-10mM. In some embodiments, the buffer component can be Tris-HCl, PB, etc., for example, its working concentration can be 10-100mM. In some embodiments, the working concentration of magnesium ions can be 5-20mM. In some embodiments, the ligation reaction buffer can also include DTT, for example, its working concentration can be 5-20mM.

[0047] In other embodiments, the ligation reaction buffer can be a commercially available ligation reaction buffer, such as the ligation reaction buffer selected from the Fapon two-step library construction kit (Cat. No. NK007), the ligation reaction buffer in the Thermo Fisher library construction kit, and the ligation reaction buffer in the NEB kit.

[0048] A method for nucleic acid ligation comprises mixing the composition of any of the above embodiments with nucleic acid fragments and ligating them under conditions that allow ligation.

[0049] The conditions allowing the connection include the connection temperature and the connection reaction time, for example, connection at 4-50° C. for 5-20 min.

[0050] A method for constructing a nucleic acid library, comprising the step of performing an adapter ligation reaction using the composition of any of the above embodiments, an adapter sequence and a ligation reaction buffer.

[0051] In some embodiments, the temperature of the linker ligation reaction is 4-50°C, and the time of the linker ligation reaction is 5-20 min; in some embodiments, the temperature of the above linker ligation reaction is 4-25°C, and the time of the linker ligation reaction is 15-20 min.

[0052] The temperature and time of the adapter ligation reaction depend on the optimal temperature of the ligase activity. For T3 DNA ligase, T4 DNA ligase, T7 DNA ligase and E. coli DNA ligase, preferably, the optimal temperature of the ligase activity is 4-25°C.

[0053] In some embodiments, the step of end repair and A addition to the nucleic acid fragments is further included before the adapter ligation reaction.

[0054] According to different library construction purposes, nucleic acid fragments to be used are obtained, and genome fragmentation is one of the forms of obtaining nucleic acid fragments. In some embodiments, fragmentation can be mechanical crushing, chemical shearing or enzymatic method. Mechanical crushing is achieved by ultrasonic shearing, atomization force, ultrasonic treatment, and fluid dynamic shearing. In one embodiment, the specific median fragment length of DNA can be prepared by ultrasonic shearing according to the manufacturer's instructions, such as by using an instrument through adaptive focusing acoustics (AFA) TM. In some embodiments, fragmentation is enzymatic. Enzymatic fragmentation is achieved by endonuclease digestion of DNA. Preferably, fragmentation can also be carried out by using transposases known to those skilled in the art. When the enzyme is used for the fragmentation reaction, the fragmentation step can be inactivated by heating.

[0055] In some embodiments, a PCR enrichment step is further included after the adapter ligation reaction to obtain a DNA library.

[0056] In some embodiments, the method for constructing a nucleic acid library further comprises a purification step after the adapter ligation reaction and before PCR enrichment.

[0057] In some embodiments, the purification step includes a magnetic bead purification step, such as purification and recovery using AMPure XP magnetic beads.

[0058] The above implementation methods can achieve the following beneficial effects:

[0059] The present invention provides a composition, a kit, a nucleic acid ligation method and a library construction method for nucleic acid ligation reaction. The present invention effectively improves the ligation efficiency by introducing polysucrose and DMSO as enhancers in the ligation reaction system, and is applied to the construction of nucleic acid libraries at the same time, with low residual adapter dimers, significantly improving the amount of effective sequencing data, reducing data waste, and reducing sequencing costs to a certain extent.

[0060] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0061] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0062] Example 1

[0063] This example is based on the Fapon two-step library construction kit (Cat. No. NK007), and uses human genomic DNA as the starting template for library construction to construct a DNA library and perform library quality inspection. The specific experimental steps are as follows:

[0064] (1) Ultrasonic shearing: The human genome was quantified using a Qubit fluorometer. 1 μg of the sample was taken for ultrasonic shearing, and then the sample was broken using an ultrasonic wall-breaking cleaner (KQ-50E ultrasonic cleaner). The shearing conditions were in accordance with Table 1. The expected length of the target fragment obtained in this example was 500 bp.

[0065] After the ultrasound, 2 μl of the broken DNA was taken for agarose gel electrophoresis for verification. The results showed that the fragment size of the broken DNA was between 300-600 bp, which met the size requirements of the target fragment. Then, the broken DNA was purified with AMPure XP magnetic beads and finally dissolved with 40 μl of nuclease-free water.

[0066] Table 1 Ultrasonic interruption parameters.

[0067] Expected fragment water depth Transmit power temperature time 500bp 5.5cm 50W 8-15℃ 120s 300bp 5.5cm 50W 8-15℃ 360s 230bp 5.5cm 50W 8-15℃ 480s

[0068] (2) End repair and A addition: The Fapon two-step library construction kit standard library construction process is used for end repair and A addition. The specific reaction system is shown in Table 2 below:

[0069] Table 2 Data table of system addition amounts for end repair.

[0070] Reagents volume Ultrasonic DNA fragmentation 1μl(50ng) End repair buffer A 10μl End repair plus A enzyme Mix 5μl total Add water to 50 μl

[0071] The reaction procedure was as follows: 20°C for 30 min, 65°C for 30 min, and maintained at 4°C.

[0072] (3) Adapter ligation: The reaction system was prepared according to the system shown in Table 3, and then different concentrations of enhancers were added to the ligation reaction system (the added volume was X). The reaction procedure for adapter ligation was as follows: treatment at 20°C for 15 minutes and maintained at 4°C.

[0073] The enhancer can also be added to the ligation reaction buffer in advance. The adapter sequence used in this embodiment is the Y adapter of Illumina.

[0074] Table 3 Data table of the amount of reaction system added for connector connection.

[0075] Reagents volume Y linker (15μM) 1μl Ligation Reaction Buffer 10μl T4 DNA Ligase 6μl Enhancer Xμl Previous step reaction solution 50μl total Fill to 100 μl

[0076] (4) Purification and recovery of the adapter ligation product using AMPure XP magnetic beads: Add an equal volume of magnetic beads to the adapter ligation product obtained in step (3), mix and purify on a magnetic stand, then dissolve with 22 μl of nuclease-free water to obtain the first purified ligation product;

[0077] (5) PCR amplification: Amplify the ligation library according to the reaction system shown in Table 4.

[0078] Table 4 PCR amplification system.

[0079] Reagents volume 2×HIFI PCR Mix 25μl Adapter ligation library 20μl P5+P7 amplification primers 5μl total Add water to 50 μl

[0080] The amplification reaction procedure is as follows: Step 1: denaturation at 98°C for 30s; Step 2: denaturation at 98°C for 10s; Step 3: annealing at 65°C for 30s; Step 4: extension at 72°C for 30s; Step 5: repeat steps 2-4, 6-9 times; Step 6: extension at 72°C for 1min; Step 7: hold at 4°C.

[0081] (6) Purifying the PCR amplified product: The amplified product was purified using AMPure XP magnetic beads and then dissolved in 22 μl of nuclease-free water to obtain the final purified ligation product.

[0082] (7) Library quality control: The amplified products were quantitatively analyzed using Qubit quantitative detection reagents. The fragment size distribution of the library products was analyzed using the Qsep capillary electrophoresis analysis detector to examine the proportion of insert fragments of different sizes in the entire ligated library.

[0083] Batch 1

[0084] Example 2

[0085] Based on Example 1, specific enhancers and their volume concentrations in the reaction system are provided: 10% Ficoll400 (10 μl added) and 6% DMSO (6 μl added).

[0086] Example 3

[0087] Based on Example 1, a specific enhancer and its volume concentration in the reaction system are provided: 10% Ficoll70 and 6% DMSO.

[0088] Example 4

[0089] Based on Example 1, a specific enhancer and its volume concentration in the reaction system are provided: 10% Ficoll400, 6% DMSO and 2% 1,3-propylene glycol.

[0090] Comparative Example 1

[0091] The difference from Example 1 is that there is no enhancer in step (3), which serves as a control group.

[0092] Comparative Example 2

[0093] Based on Example 1, a specific enhancer and its volume concentration in the reaction system are provided: 10% PEG4000.

[0094] Comparative Example 3

[0095] Based on Example 1, a specific enhancer and its volume concentration in the reaction system are provided: 6% DMSO.

[0096] Comparative Example 4

[0097] Based on Example 1, a specific enhancer and its volume concentration in the reaction system are provided: 2% 1,3-propylene glycol.

[0098] Comparative Example 5

[0099] Based on Example 1, a specific enhancer and its volume concentration in the reaction system are provided: 5% glycerol.

[0100] Comparative Example 6

[0101] Based on Example 1, a specific enhancer and its molar concentration in the reaction system are provided: 1 M betaine.

[0102] Comparative Example 7

[0103] Based on Example 1, a specific enhancer and its volume concentration in the reaction system are provided: 10% Ficoll400.

[0104] Comparative Example 8

[0105] Based on Example 1, a specific enhancer and its volume concentration in the reaction system are provided: 10% Ficoll70.

[0106] Comparative Example 9

[0107] Based on Example 1, a specific enhancer and its volume concentration in the reaction system are provided: 10% Ficoll400 and 2% 1,3-propylene glycol.

[0108] Comparative Example 10

[0109] Based on Example 1, a specific enhancer and its volume concentration in the reaction system are provided: 6% DMSO and 2% 1,3-propylene glycol.

[0110] Comparative Example 11

[0111] Based on Example 1, a specific enhancer and its volume concentration in the reaction system are provided: 10% PEG4000 and 6% DMSO.

[0112] Comparative Example 12

[0113] Based on Example 1, a specific enhancer and its volume concentration in the reaction system are provided: 10% PEG4000+2% 1,3-propylene glycol.

[0114] Comparative Example 13

[0115] Based on Example 1, specific enhancers and their volume concentrations in the reaction system are provided: 10% PEG4000 and 10% Ficoll400.

[0116] Experimental Example 1

[0117] The inventors tested the library concentrations of Examples 2-4 and Comparative Examples 1-13 respectively. The library was constructed using the method of Example 1, and the library concentrations of the comparative examples and examples were qualitatively analyzed using the Qubit dsDNA HS kit. The test results are shown in Table 5.

[0118] Table 5 Library concentrations of different comparative examples and embodiments.

[0119] Group Library concentration ng / μl Comparative Example 1 6.5 Comparative Example 2 37.9 Comparative Example 3 18.3 Comparative Example 4 13.8 Comparative Example 5 6.7 Comparative Example 6 5.4 Comparative Example 7 22.6 Comparative Example 8 20.4 Comparative Example 9 30.1 Comparative Example 10 28.2 Comparative Example 11 42 Comparative Example 12 38 Comparative Example 13 41.5 Example 2 48.3 Example 3 46.5 Example 4 50.3

[0120] As shown in Table 5, PEG has a good effect of promoting ligation on ligase (refer to Comparative Example 2 and Comparative Examples 11-13), and the ligation efficiency of Ficoll (polysucrose) and DMSO is significantly higher than that of the two alone (refer to Examples 2-4), and the ligation efficiency is also better than PEG. In addition, when 1,3-propylene glycol is used in combination with Ficoll (polysucrose) and DMSO (refer to Example 4), the concentration of the library obtained by amplification is the highest, that is, the ligation promotion effect is better.

[0121] Batch 2

[0122] The inventors conducted a second batch of experiments.

[0123] Example 5

[0124] Based on Example 1, a specific enhancer and its volume concentration in the reaction system are provided: 10% Ficoll400 and 2% DMSO.

[0125] Example 6

[0126] Based on Example 1, a specific enhancer and its volume concentration in the reaction system are provided: 10% Ficoll400 and 10% DMSO.

[0127] Example 7

[0128] Based on Example 1, a specific enhancer and its volume concentration in the reaction system are provided: 10% Ficoll400 and 20% DMSO.

[0129] Example 8

[0130] Based on Example 1, a specific enhancer and its volume concentration in the reaction system are provided: 10% Ficoll400, 6% DMSO and 1% 1,3-propylene glycol.

[0131] Example 9

[0132] Based on Example 1, a specific enhancer and its volume concentration in the reaction system are provided: 10% Ficoll400, 6% DMSO and 2% 1,3-propylene glycol.

[0133] Example 10

[0134] Based on Example 1, a specific enhancer and its volume concentration in the reaction system are provided: 10% Ficoll 400, 6% DMSO and 5% 1,3-propylene glycol.

[0135] Comparative Example 14

[0136] The difference from Example 1 is that there is no enhancer in step (3), which serves as a control group.

[0137] Comparative Example 15

[0138] Based on Example 1, a specific enhancer and its volume concentration in the reaction system are provided: 10% PEG4000.

[0139] Comparative Example 16

[0140] Based on Example 1, a specific enhancer and its volume concentration in the reaction system are provided: 5% Ficoll400.

[0141] Comparative Example 17

[0142] Based on Example 1, a specific enhancer and its volume concentration in the reaction system are provided: 10% Ficoll400.

[0143] Comparative Example 18

[0144] Based on Example 1, a specific enhancer and its volume concentration in the reaction system are provided: 15% Ficoll400.

[0145] Experimental Example 2

[0146] In this experimental example, the concentration of amplified products was detected for the libraries constructed in each treatment group in batch 2, and the Qubit dsDNA HS kit was used to analyze the promoting effect of polysucrose, DMSO, and 1,3-propylene glycol at different concentrations on the connection library.

[0147] Table 6 Effects of different concentrations of polysucrose, DMSO, and 1,3-propylene glycol on the concentration of amplified products.

[0148] Group Library concentration Comparative Example 14 4.42 Comparative Example 15 40.1 Comparative Example 16 13.8 Comparative Example 17 19.8 Comparative Example 18 20.6 Example 5 47.9 Example 6 51 Example 7 50 Example 8 51.3 Example 9 52.2 Example 10 52.9

[0149] As shown in Table 6, polysucrose at a volume concentration of 5-15% combined with 2-20% DMSO and 1-5% 1,3-propylene glycol can significantly improve the connection effect of the ligase and is significantly better than PEG.

[0150] Experimental Example 3

[0151] In order to enhance the connection efficiency, the existing connection system often has a higher PEG content (such as 10%), which will cause the linker dimer to be difficult to purify cleanly during the subsequent magnetic bead purification process, resulting in a higher concentration of linker dimer residues in the connection library. For this reason, the method of the present invention further tests the linker dimer content. The linker dimer content in the products of Comparative Examples 14-15 and Examples 2 and 9 was analyzed on a Qsep capillary electrophoresis analysis detector. In this experimental example, the fragment content of 100-150bp was analyzed.

[0152] Table 7 Linker dimer content in products of different treatment groups.

[0153] Group Adaptor dimer content Comparative Example 14 0.09％ Comparative Example 15 0.89％ Example 2 0.06％ Example 9 0.08％

[0154] As can be seen from Table 7, although PEG has excellent ligation enhancement efficiency, PEG will lead to a high content of linker dimers, thereby affecting the sequencing quality and cost (Comparative Example 15); and the solution provided by the present invention can obtain a better enhanced ligation efficiency than PEG, and at the same time the linker dimer content is very low, which has very good application advantages (Example 2 and Example 9).

[0155] Through the above experiments, it can be concluded that the combination of polysucrose and DMSO as enhancers can significantly improve the connection effect of the ligase, and is better than the connection effect of PEG. When 1,3-propylene glycol is used in combination with the above combination, the connection effect of the ligase can be further improved, and the content of the linker dimer can be significantly reduced.

[0156] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A composition for nucleic acid ligation reaction, comprising polysucrose, DMSO and propylene glycol; The propylene glycol is 1,3-propylene glycol; The volume percentage of 1,3-propylene glycol in the connection reaction system is 1-5%, the volume percentage of polysucrose is 5-15%, and the volume percentage of DMSO is 2-20%; The average molecular weight of the polysucrose is 70,000-400,000; The polysucrose is selected from Ficoll400 or Ficoll70.

2. The composition according to claim 1, characterized in that Also included are ligases.

3. The composition according to claim 2, characterized in that The ligase is DNA ligase.

4. The composition according to claim 3, characterized in that The DNA ligase is selected from at least one of T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Taq DNA ligase, Ampligase® DNA ligase, and Escherichia coli DNA ligase.

5. The composition according to any one of claims 1 to 4, characterized in that The composition is used in the ligation reaction stage of nucleic acid library construction.

6. A kit for constructing a nucleic acid library, characterized in that: The invention comprises the composition according to any one of claims 1 to 4.

7. The kit according to claim 6, characterized in that The kit further comprises at least one of a linker sequence and a ligation reaction buffer.

8. A method for nucleic acid ligation, comprising: The composition according to any one of claims 1 to 4 is mixed with nucleic acid fragments and ligated under conditions that allow ligation.

9. A method for constructing a nucleic acid library, characterized in that: The method comprises the step of performing a linker ligation reaction using the composition according to any one of claims 1 to 4, a linker sequence and a linker reaction buffer.

10. The method for constructing a nucleic acid library according to claim 9, characterized in that: Before the adapter ligation reaction, the nucleic acid fragments are also subjected to end repair and A addition steps.

11. The method according to claim 10, characterized in that A PCR enrichment step is also included after the adaptor ligation reaction.

12. The method according to claim 11, characterized in that After the linker ligation reaction and before PCR enrichment, the method for constructing a nucleic acid library further comprises a purification step.

13. The method according to claim 12, characterized in that The purification step includes a magnetic bead purification step.