CN116426607A - DNA library construction premix - Google Patents

Abstract

The application provides a DNA library construction premix liquid, which belongs to the technical field of biology. The premix liquid provided by the application pre-mixes the reaction buffer and the enzyme mix required by the terminal repair and the joint connection in advance, and only a sample and a joint are needed to be provided to realize one-tube type warehouse establishment. Compared with the traditional full-flow DNA library construction method, the method for constructing the DNA library by using the premix liquid is more convenient and quicker, and can also reduce the risk of uncovering in the operation process and effectively reduce pollution and sample adding errors.

Description

A kind of DNA library construction premix

technical field

The present application relates to the field of biotechnology, in particular to a DNA library preparation premix.

Background technique

In the past 5 years, NGS technology has been widely used in the field of life sciences. At the same time, with the development and progress of sequencing technology, some nucleic acid extraction and library preparation methods have been derived. Library preparation is also the basis of next-generation sequencing, and its basic steps include: (1) Break the DNA molecules to be sequenced into 200-500bp long sequence fragments by ultrasonic waves, or enzymatically break (if free DNA in blood or other This step can be ignored for fragmented DNA); (2) The 3' end of the fragmented genome is protruded and trimmed, and the 5' end is protruded and filled; (3) The 3' end of the fragment after being cut or filled Adding A and 5' ends for phosphorylation; (4) performing adapter ligation on the repaired fragments; (5) performing magnetic bead purification on the adapter ligated fragments; (6) performing PCR amplification on the purified DNA fragments; ( 7) Purifying the amplified library with magnetic beads; (8) Enriching the target region of the amplified and purified DNA library with a probe capture method or a targeted primer method to prepare a sequencing library.

At present, the existing kits on the market adopt a step-by-step process, and the operation is relatively cumbersome. It is inevitable to open the cover frequently during the intermediate process, and there is a risk of introducing contamination. At present, the reagents used in the detection process of mNGS technology include common environmental microorganisms, such as nucleic acid extraction kits and library construction kits. These microorganisms are collectively referred to as "reagent background bacteria". Salter S et al. (Salter S J, Cox M J, et al. Reagent and laboratory contamination can critically impact sequence-based microbiome analyses. [J]. BMC biology, 2014, 12 (1).) found that as simulated samples (input quantitative Enterobacteriaceae) dilution factor increases, the proportion of Salmonella enterica-specific sequences gradually decreases, and the proportion of other microbial sequences (kit genomes) gradually increases. Therefore, minimizing the presence and interference of background bacteria from samples and environmental sources is a major challenge for mNGS technology.

Contents of the invention

The purpose of this application is to provide a DNA library construction premix, which premixes the enzymes and buffers required in the process of end filling, 5' end phosphorylation, and adapter ligation in advance, which reduces the operation steps to a certain extent, and there is no opening in the middle. Cap or just one-step uncap operation, which greatly reduces the risk of background bacteria introduction.

The first aspect of the present application provides a DNA library construction premix, which includes: buffer components, metal salts, dNTP, ATP, DTT (dithiothreitol), polynucleotide kinase, DNA polymerase and DNA ligase.

In some embodiments, the buffer component is, for example, one or more of Tris, Hepes and MES, preferably Tris, more preferably 100-500mM Tris, most preferably 200-400mM Tris, including 210mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280mM, 290mM, 300mM, 310mM, 320mM, 330mM, 340mM, 350mM, 360mM, 370mM, 380mM, 390mM and 400mM.

In some embodiments, the metal salt includes one or more of Mg ²⁺ salt, Mn ²⁺ salt, Ca ²⁺ salt, K ⁺ salt, Na ⁺ salt, Cs ⁺ salt, Ni ²⁺ salt. In some embodiments, the concentration of the metal salt is 100-500mM, preferably 100-300mM, including 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM, 200mM, 210mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280mM, 290mM and 300mM.

In some embodiments, the metal salts include Mg ²⁺ salts, K ⁺ salts and Na ⁺ salts, preferably MgCl ₂ , NaCl and KCl, more preferably, the concentration of MgCl ₂ is 20mM-100mM, including the 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM and 100mM in the above range; the concentration of the NaCl is 50mM-200mM, including the 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM and 200mM in the above range; the concentration of KCl is 20mM-100mM, including the range Within 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM and 100mM.

In some embodiments, the concentration of the dNTP is 0.1 mM-10 mM, including 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, and 10 mM.

In some embodiments, the concentration of ATP is 1-20 mM, preferably 1-10 mM, including 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM and 10 mM within said range.

In some embodiments, the concentration of DTT is 10-50mM, preferably 10-30mM, including 10mM, 12mM, 15mM, 18mM, 20mM, 21mM, 22mM, 23mM, 24mM, 25mM, 26mM, 27mM within said range , 28mM, 29mM and 30mM.

In some embodiments, the polynucleotide kinase is T4 polynucleotide kinase, and the concentration of the T4 polynucleotide kinase is 0.1-10 U/μl, preferably 0.1-5 U/μl, including 0.1U/μl, 0.15U/μl, 0.2U/μl, 0.25U/μl, 0.3U/μl, 0.35U/μl, 0.4U/μl, 0.45U/μl, 0.5U/μl, 0.55 U/μl, 0.6U/μl, 0.65U/μl, 0.7U/μl, 0.8U/μl, 0.9U/μl, 1U/μl, 1.5U/μl, 2U/μl, 2.5U/μl, 3U/μl , 3.5U/μl, 4U/μl and 5U/μl.

In some embodiments, the DNA polymerase includes a DNA polymerase for end filling, for example, one or more selected from T4 DNA polymerase, T3 DNA polymerase, T7 DNA polymerase and Escherichia coli DNA polymerase, Escherichia coli DNA polymerase is preferred, more preferably, the concentration of said Escherichia coli DNA polymerase is 0.1-10U/μl, preferably 0.1-5U/μl, including 0.1U/μl and 0.15U/μl within said range, 0.2U/μl, 0.25U/μl, 0.3U/μl, 0.35U/μl, 0.4U/μl, 0.45U/μl, 0.5U/μl, 0.55U/μl, 0.6U/μl, 0.65U/μl, 0.7U/μl, 0.8U/μl, 0.9U/μl, 1U/μl, 1.5U/μl, 2U/μl, 2.5U/μl, 3U/μl, 3.5U/μl, 4U/μl and 5U/μl.

In some embodiments, the DNA polymerase also includes a DNA polymerase for completing terminal addition of dA (adenine deoxyribonucleotide), such as TaqDNA polymerase, KODDNA polymerase and Klenow DNA polymerase (3'-5 one or more of 'exo-), preferably Klenow DNA polymerase (3'-5'exo-). In some embodiments, the master mix further includes dATP.

In some embodiments, the DNA ligase is, for example, one or more of T4 DNA ligase or Escherichia coli DNA ligase, preferably T4 DNA ligase, and the concentration of the T4 DNA ligase is 20-100U/μl, preferably 30-60U/μl, including 30U/μl, 32U/μl, 35U/μl, 38U/μl, 40U/μl, 41U/μl, 42U/μl, 43U/μl, 44U/μl, 45U/μl μl, 46U/μl, 47U/μl, 48U/μl, 49U/μl, 50U/μl, 51U/μl, 52U/μl, 55U/μl, 56U/μl, 58U/μl and 60U/μl.

In some embodiments, the premix also includes a surfactant, preferably a nonionic surfactant, such as TritonX-100 (polyethylene glycol p-isooctyl phenyl ether), NP10 (nonylphenol polyoxyethylene ether) (10)), NP40 (ethylphenyl polyethylene glycol), Tween 20 (polysorbate 20), Tween 80 (polysorbate 80), PEG2000 (polyethylene glycol 2000), PEG8000 (polyethylene glycol One or more of diol 8000); preferably NP40, more preferably 0.1-10% (volume fraction) NP40, including 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6% in the range , 0.7%, 0.8%, 0.9%, 1%, 1.2%, 1.5%, 1.8%, 2%, 2.2%, 2.5%, 2.8%, 3%, 3.5%, 4%, 4.5%, 5%, 6 %, 7%, 8%, 9% and 10%.

In some embodiments, the master mix includes Tris, Mg ²⁺ , Na ⁺ , K ⁺ , dNTP, ATP, DTT, T4 polynucleotide kinase, E. coli DNA polymerase, and T4 DNA ligase.

In some embodiments, the master mix includes Tris, MgCl ₂ , NaCl, KCl, dNTP, ATP, DTT, T4 polynucleotide kinase, Escherichia coli DNA polymerase and T4 DNA ligase, optionally, the The master mix also includes NP40.

In some embodiments, the master mix includes 100-500mM Tris, 20-100mM _MgCl2 , 50-200mMNaCl, 20-100mMKCl, 0.1-10mMdNTP, 1-20mMATP, 10-50mM DTT, 0.1-10U/μl T4 polynucleoside Acid kinase, 0.1-10 U/μl E. coli DNA polymerase, 20-100 U/μl T4 DNA ligase and optionally 0.1-10% (volume fraction) NP40.

In some embodiments, the master mix includes 200-400mM Tris, 20-80mM _MgCl2 , 50-150mMNaCl, 20-80mMKCl, 0.1-5mMdNTP, 1-10mMATP, 10-30mM DTT, 0.1-5U/μl T4 polynucleotide Kinase, 1-5 U/μl E. coli DNA polymerase, 20-80 U/μl T4 DNA ligase and optionally 0.1-5% (volume fraction) NP40.

In some embodiments, the master mix comprises 200-300mM Tris, 40-60mM MgCl ₂ , 80-100mM NaCl, 30-60mM KCl, 1-3mMdNTP, 3-6mMATP, 20-30mM DTT, 0.1-1U/μl T4 polynucleotide Kinase, 1-3 U/μl E. coli DNA polymerase, 40-60 U/μl T4 DNA ligase and optionally 0.5-2% (volume fraction) NP40.

In some embodiments, the master mix comprises 282mM Tris, 50mMMgCl2, 100mM NaCl, 40mMKCl, 1mMdNTP, 5mMATP, 22mMDTT, 0.56U/μl T4 polynucleotide kinase, 1.78U/μl Escherichia coli DNA polymerase, 44.4U/μl μl T4 DNA ligase.

In some embodiments, the master mix comprises 250mM Tris, 50mMMgCl2, 100mM NaCl, 40mMKCl, 1mMdNTP, 5mMATP, 20mMDTT, 1% NP40, 0.56U/μl T4 polynucleotide kinase, 1.78U/μl Escherichia coli DNA polymerase , 44.4U/μl T4 DNA ligase.

The second aspect of the present application provides a kit, which comprises the master mix described in the first aspect.

In some embodiments, the kit further includes a sequencing adapter.

In some embodiments, the sequencing adapters include, but are not limited to, the sequencing adapters of the Illumina platform, the MGI platform, and the IonTorrent platform known in the art. In some embodiments, the adapter is a Y-adapter or a bubble adapter or a blunt end adapter. In some embodiments, the linker is a sequencing long linker or a short linker of the Illumina platform or a bubble linker of the MGI platform of MGI or a blunt linker of the IonTorrent platform.

In some embodiments, the kit also includes other auxiliary detection reagents such as pure water.

A third aspect of the present application provides a method for constructing a DNA library, the method comprising:

(1) Provide a fragmented DNA sample, add the premix described in the first aspect of the application, react under suitable conditions, and perform end repair on the fragmented DNA sample;

(2) adding a sequencing linker to the reaction product of step (1), performing a linker ligation reaction under suitable conditions to obtain a ligation product;

(3) Optionally, purify or not purify the ligation product;

(4) Optionally, amplifying the ligation product or amplifying the ligation product after purification;

(5) Optionally, purifying the amplified product.

In some embodiments, the fragmented DNA is obtained by fragmenting a DNA sample by, for example, enzymatic digestion, chemical and/or mechanical methods. In some embodiments, the fragmented DNA is, for example, gDNA, cfDNA, or double-stranded cDNA.

In some embodiments, the end repair includes end filling and 5' end phosphorylation, that is, both ends of the DNA fragment after end repair are blunt ends, which can be bluntly connected with the adapter, such as the blunt end adapter of the IonTorrent platform .

In some embodiments, the end repair includes end filling, 5' end phosphorylation and 3' end adding dA, that is, adding adenine deoxyribonucleotide at the 3' end of the DNA fragment after end filling, which can Connect with the adapter through A-T, such as the Y-type adapter of the Illumina platform.

In some embodiments, the purification step adopts magnetic bead method, high salt precipitation method, spin column method or phenol-chloroform extraction method for purification; magnetic bead method is preferred.

In some embodiments, the sequencing adapters include, but are not limited to, the sequencing adapters of the Illumina platform, the MGI platform, and the IonTorrent platform known in the art. In some embodiments, the linker is a blunt end linker, a Y linker, or a bubble linker.

In some embodiments, the reaction conditions of step (1) are, for example, 20-30°C for 10-20 minutes, preferably 25-30°C for 10-15 minutes, more preferably 25°C for 10 minutes.

In some embodiments, the reaction conditions of the step (2) are, for example, 20-30° C. for 5-20 minutes, preferably 25-30° C. for 5-15 minutes, more preferably 25-30° C. for 5-10 minutes, most preferably 25 minutes. ℃ for 5 minutes.

A fourth aspect of the present application provides a method for constructing a DNA library, the method comprising:

(1) Provide a fragmented DNA sample, add the premix and adapter described in the first aspect of the application, react under suitable conditions, and perform end repair and adapter ligation on the fragmented DNA sample;

(2) Optionally, purify or not purify the ligation product;

(3) Optionally, amplifying the ligation product or amplifying the ligation product after purification;

(4) Optionally, purifying the amplified product.

In some embodiments, the fragmented DNA is obtained by fragmenting a DNA sample by, for example, enzymatic digestion, chemical and/or mechanical methods. In some embodiments, the fragmented DNA is, for example, gDNA or cfDNA or double-stranded cDNA.

In some embodiments, the end repair includes end filling and 5' end phosphorylation, that is, both ends of the DNA fragment after end repair are blunt ends, which can be bluntly connected with the adapter, such as the blunt end adapter of the IonTorrent platform .

In some embodiments, the end repair includes end filling, 5' end phosphorylation and 3' end adding dA, that is, adding adenine deoxyribonucleotide at the 3' end of the DNA fragment after end filling, which can Connect with the adapter through A-T, such as the Y-type adapter of the Illumina platform.

In some embodiments, the purification step adopts magnetic bead method, high salt precipitation method, spin column method or phenol-chloroform extraction method for purification; magnetic bead method is preferred.

In some embodiments, the sequencing adapters include, but are not limited to, the sequencing adapters of the Illumina platform, the MGI platform, and the Ion Torrent platform known in the art. In some embodiments, the linker is a blunt end linker, a Y linker, or a bubble linker.

In some embodiments, the reaction conditions of step (1) are, for example, 30-40°C for 10-30min, such as 35-40°C for 20-30min, such as 35-40°C for 25-30min, such as 37°C 25min.

The fifth aspect of the present application provides the application of the master mix described in the first aspect or the kit described in the second aspect in constructing a DNA library.

Description of drawings

Figure 1: The conventional full-process DNA library construction and the library output obtained by using the master mix 1 of the present invention for DNA library construction;

Figure 2: The peak diagram of the conventional full-process DNA library construction and DNA library construction using the master mix 1 of the present invention;

Figure 3: The conventional full-process DNA library construction and the library output obtained by using the master mix 2 of the present invention for DNA library construction;

Figure 4: The peak diagram of the library obtained by the conventional full-process DNA library construction and the DNA library construction using the master mix 2 of the present invention.

Specific embodiments (embodiments)

The technical scheme of the present application will be further described below in conjunction with the accompanying drawings and through specific implementation methods, but the following examples are only simple examples of the present application, and do not represent or limit the protection scope of the application. The protection scope of the application is based on the rights Requirements shall prevail.

In the following examples, unless otherwise specified, the reagents and consumables used were purchased from conventional reagent manufacturers in the field; unless otherwise specified, the experimental methods and technical means used were conventional methods and means in the field.

experimental reagent

End Repair Buffer: Tris 150mM, MgCl ₂ 30mM, NaCl 100mM, KCl 40mM, dNTP 1mM, DTT 20mM, PH8.2;

End repair enzyme mix: E.Coli DNA polymerase (Escherichia coli DNA polymerase) 16U/μl, T4 PNK (T4 polynucleotide kinase) 5U/μl;

Adapter Connection Buffer: Tris 132mM, MgCl ₂ 20mM, DTT 2mM, ATP 5mM, PH7.8;

Ligase: T4 DNALigase (T4 DNA ligase) 400U/μl;

Premix 1: Tris 282mM, MgCl ₂ 50mM, NaCl 100mM, KCl 40mM, dNTP 1mM, DTT 22mM, ATP 5mM, T4 DNA Ligase 44.4U/μl, E.Coli DNA polymerase 1.78U/μl, T4 PNK 0.56U/μl;

Master mix 2: Tris 250mM, MgCl ₂ 50mM, NaCl 100mM, KCl 40mM, dNTP 1mM, DTT 20mM, ATP 5mM, 1% NP40, T4 DNA Ligase 44.4U/μl, E.Coli DNA polymerase 1.78U/μl, T4 PNK0 .56U/μl.

Note: T4 polynucleotide kinase is from Vazyme #N102, T4 DNA ligase is from Vazyme #N103, and other reagents are purchased from conventional reagent manufacturers in the field.

Example 1

100ng of fragmented salmon was used as a sample for whole-process DNA library construction as a control. The specific steps are as follows:

(1) End repair:

Prepare the following reaction system in a sterilized PCR eight-tube tube:

Use a pipette to mix gently by pipetting (do not shake to mix), centrifuge briefly and collect the reaction solution at the bottom of the tube. Place the reaction tube in a PCR machine for the following reactions:

temperature time 105°C hot cover 25°C 10min 4°C hold

(2) Joint connection

Prepare the reaction system (prepared on ice) according to the following table, a total of 100 μl, in which the DNAAdapter is used in a 1:2 dilution:

Place the reaction tube in a PCR machine for the following reactions:

temperature time 105°C hot cover 25°C 5min 4°C hold

(3) Magnetic bead purification

A. Vortex to mix VAHTS DNA Cleanbeads (Vazyme #N411).

B. Pipette 80μl (0.8×) VAHTS DNA Cleanbeads into 100μl ligation product, cover the lid, vortex gently to mix, and centrifuge briefly to collect the reaction solution to the bottom of the tube, then remove the cap.

C. Incubate at room temperature for 5 minutes.

D. Briefly centrifuge the reaction tube and place it in a magnetic stand to separate the magnetic beads and liquid. After the solution cleared (approximately 5 minutes), the supernatant was carefully removed.

E. Keep the EP tube in the magnetic stand all the time, add 200 μl of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 seconds, and carefully remove the supernatant.

F. Repeat step E twice for a total of 2 rinses.

G. Keep the EP tube in the magnetic stand all the time, open the cover and air-dry the magnetic beads for 5 minutes (always pay attention to the change of the state of the magnetic beads, when the surface of the magnetic beads is no longer reflective, it means that the magnetic beads have dried. The magnetic beads are too dry and the surface There will be cracks, which will affect the recovery efficiency; if the ethanol is not volatilized, it will affect the downstream enzymatic reaction).

H. Take the EP tube out of the magnetic stand, add 22 μl sterilized ultrapure water for DNA elution. Put the cap on, vortex to mix, and centrifuge briefly to collect the reaction solution to the bottom of the tube, then remove the cap from the tube.

I. Briefly centrifuge the reaction tube and place it in a magnetic stand to separate the magnetic beads and liquid. After the solution is clear (about 5 minutes), carefully pipette 20 μl of the supernatant into a sterilized PCR tube.

(4) PCR amplification

Prepare the reaction system according to the following table, a total of 50 μl:

Place the prepared reaction system in a PCR instrument for the following reactions:

After amplification, use 1× magnetic beads (Vazyme#N411) to purify, 22.5 μl DDW to elute, take 20 μl, measure the concentration, and calculate the library output as shown in Figure 1. The Agilent 2100 Bioanalyzer peak shape detection is performed on the obtained library products. The result is shown in Figure 2.

100ng of fragmented salmon was used as a sample for one-tube DNA library construction (adapter added alone) as a control, the specific steps are as follows:

(1) End repair:

Prepare the following reaction system in a sterilized PCR eight-tube tube, a total of 100 μl:

components volume Sample (100ng/μl) 1μl Master Mix 1 45μl ddH ₂ O 54μl

Use a pipette to mix gently by pipetting (do not shake to mix), centrifuge briefly and collect the reaction solution at the bottom of the tube. Place the reaction tube in a PCR machine for the following reactions:

temperature time 105°C hot cover 25°C 10min 4°C hold

(2) Joint connection

Prepare the reaction system (prepared on ice) according to the following table, a total of 100 μl, in which the DNAAdapter is used in a 1:2 dilution:

Place the reaction tube in a PCR machine for the following reactions:

temperature time 105°C hot cover 25°C 5min 4°C hold

Subsequent magnetic bead purification and PCR amplification steps are the same as the aforementioned steps (3)-(4) of the whole process of the control group. After the amplification is completed, use 1× magnetic beads (Vazyme#N411) to purify, 22.5 μl DDW for elution, and take 20 μl , measure the concentration, and calculate the library output as shown in Figure 1. The Agilent 2100 Bioanalyzer peak shape detection was performed on the obtained library products, and the results are shown in Figure 2.

Result analysis

As shown in Figure 1-2, using the one-tube DNA library preparation premix of the present invention can build a library normally, the output of the library is normal, and the peak shape at 2100 is normal. Use premix 1, react under certain conditions, add the joint after opening the cap, and the normal connection product can be formed, which shows that the method is feasible and can improve the simplicity of operation to a certain extent.

Example 2

100ng of fragmented salmon was used as a sample for full-process DNA library construction as a control. The specific steps were the same as those of the control group in Example 1. The resulting library output is shown in Figure 3, and the peak shape of the 2100 library is shown in Figure 4.

100ng of fragmented salmon was used as a sample for one-tube DNA library construction (adapter added alone) as a control, the specific steps are as follows:

(1) End repair and adapter connection

Prepare the following reaction system in a sterilized PCR eight-tube tube, in which the DNAAdapter is used in a 1:2 dilution:

Use a pipette to mix gently by pipetting (do not shake to mix), centrifuge briefly and collect the reaction solution at the bottom of the tube. Place the reaction tube in a PCR machine for the following reactions:

temperature time 105°C hot cover 37°C 25min 4°C hold

Subsequent magnetic bead purification and PCR amplification steps are the same as the aforementioned steps (3)-(4) of the whole process of the control group. After the amplification is completed, use 1× magnetic beads (Vazyme#N411) to purify, 22.5 μl DDW for elution, and take 20 μl , measure the concentration, and calculate the library output as shown in Figure 3. The Agilent 2100 Bioanalyzer peak shape detection was performed on the obtained library products, and the results are shown in Figure 4.

Claims (18)

1. A DNA pooling premix comprising: buffer components, metal salts, dNTP, ATP, DTT (dithiothreitol), polynucleotide kinase, DNA polymerase and DNA ligase.

2. Premix according to claim 1, wherein the polynucleotide kinase is a T4 polynucleotide kinase, preferably 0.1-10U/. Mu.l T4 polynucleotide kinase, more preferably 0.1-5U/. Mu.l T4 polynucleotide kinase.

3. Premix according to claim 1, wherein the DNA ligase is one or more selected from T4DNA ligase or e.coli DNA ligase, preferably T4DNA ligase, more preferably 20-100U/. Mu.l T4DNA ligase, most preferably 30-60U/. Mu. l T4DNA ligase.

4. Premix according to claim 1, the DNA polymerase comprising a DNA polymerase for end filling, preferably one or more from the group consisting of T4DNA polymerase, T3DNA polymerase, T7DNA polymerase and e.coli DNA polymerase, more preferably 0.1-10U/. Mu.l e.coli DNA polymerase, most preferably 0.1-5U/. Mu.l e.coli DNA polymerase.

5. Premix according to claim 1, wherein the buffer substance comprises Tris, preferably 100-500mM Tris;

the metal salt comprises Mg ²⁺ Salt, K ⁺ Salts and Na ⁺ Salts, preferably MgCl ₂ KCl and NaCl, more preferably 20-100mM MgCl ₂ 20-100mM KCl and 50-200mM NaCl;

the concentration of dNTPs is 0.1mM-10mM, preferably 1-5mM;

the concentration of ATP is 1-20mM, preferably 1-10mM;

the concentration of DTT is 10-50mM, preferably 10-30mM.

6. Premix according to claim 1, further comprising a surfactant, preferably a nonionic surfactant, more preferably NP40, most preferably 0.1-10% (volume fraction) NP40.

7. The premix of claim 1, comprising Tris, mgCl ₂ NaCl, KCl, dNTP, ATP, DTT, T4, E.coli DNA polymerase and T4DNA ligase, optionally, the premix further comprises NP40;

preferably, the premix comprises 100-500mM Tris, 20-100mM MgCl ₂ 50-200mM NaCl, 20-100mM KCl, 0.1-10mM dNTP, 1-20mM TP, 10-50mM DTT, 0.1-10U/. Mu.l T4 polynucleotide kinase, 0.1-10U/. Mu.l E.coli DNA polymerase, 20-100U/. Mu. l T4DNA ligase and optionally 0.1-10% (volume fraction) NP40;

preferably, the premix comprises 200-400mM Tris, 20-80mM MgCl ₂ 50-150mM NaCl, 20-80mM KCl, 0.1-5mM dNTP, 1-10mM TP, 10-30mM DTT, 0.1-5U/. Mu.l T4 polynucleotide kinase, 1-5U/. Mu.l E.coli DNA polymerase, 20-80U/. Mu. l T4DNA ligase and optionally 0.1-5% (volume fraction) NP40.

Preferably, the premix comprises 200-300mM Tris, 40-60mM MgCl ₂ 80-100mM NaCl, 30-60mM KCl, 1-3mM dNTP, 3-6mM MATP, 20-30mM DTT, 0.1-1U/. Mu. l T4 polynucleotide kinase, 1-3U/. Mu.l E.coli DNA polymerase, 40-60U/. Mu. l T4DNA ligase and optionally 0.5-2% (volume fraction) NP40.

8. A kit comprising the premix of any one of claims 1-7.

9. The kit of claim 8, further comprising a sequencing linker.

10. Use of a premix according to any one of claims 1 to 7 or a kit according to any one of claims 8 to 9 for constructing a DNA library.

11. A method of constructing a DNA library, the method comprising:

(1) Providing a fragmented DNA sample, adding the premix of any one of claims 1-7, reacting under suitable conditions, and performing end repair on the fragmented DNA sample;

(2) Adding a sequencing joint into the reaction product of the step (1), and carrying out joint connection reaction under proper conditions to obtain a connection product;

(3) Optionally, purifying or not purifying the ligation product;

(4) Optionally, amplifying the ligation product or amplifying the purified ligation product;

(5) Optionally, purifying the amplified product.

12. The method of claim 11, wherein the end repair comprises end-filling and 5' end-phosphorylation.

13. The method according to claim 12, the joint being a butt joint, preferably a butt joint of an IonTorrent platform.

14. The process according to claim 11, wherein the reaction conditions of step (1) are, for example, 20-30 ℃ for 10-20min, preferably 25-30 ℃ for 10-15min, more preferably 25 ℃ for 10min;

the reaction conditions of step (2) are, for example, 20-30℃for 5-20min, preferably 25-30℃for 5-15min, more preferably 25-30℃for 5-10min, most preferably 25℃for 5min.

15. A method of constructing a DNA library, the method comprising:

(1) Providing a fragmented DNA sample, adding the premix of any one of claims 1-7 and a linker, reacting under suitable conditions, and performing end repair and linker ligation on the fragmented DNA sample;

(2) Optionally, purifying or not purifying the ligation product;

(3) Optionally, amplifying the ligation product or amplifying the purified ligation product;

(4) Optionally, purifying the amplified product.

16. The method of claim 15, wherein the end repair comprises end-filling and 5' end-phosphorylation.

17. The method of claim 16, wherein the joint is a butt joint, preferably a butt joint of an Ion Torrent platform.

18. The method according to claim 15, wherein the reaction conditions of step (1) are, for example, 30-40 ℃ for 10-30min, such as 35-40 ℃ for 20-30min, such as 35-40 ℃ for 25-30min, such as 37 ℃ for 25min.

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