WO2024138320A1 - Method for reducing free linkers in sequencing library - Google Patents

Abstract

Provided are a method for constructing a sequencing library, a sequencing library, a sequencing method, and a kit for constructing a sequencing library. The method for constructing a sequencing library comprises: performing, under the action of an exonuclease, a digestion treatment on a sample to be tested which is linked to a linker, so as to obtain a target sequencing library.

Description

Methods for reducing free adapters in sequencing libraries Technical Field

The present invention relates to the field of biotechnology, and in particular, to a method for constructing a sequencing library and an application thereof, and more particularly, to a method for constructing a sequencing library, a sequencing library, a method for sequencing a target nucleic acid molecule, and a kit.

Background technique

Nucleic acid sequencing has become an indispensable and important research method in the field of life science research. The accompanying genomic technology based on large-scale sequencing data has also played an important role in different research and application directions, such as tracing the causes of complex diseases and dynamically monitoring their development process, targeted breeding of economic crops and animals, and research and protection of different biological genetic resources.

However, the prerequisite for the above research is how to use high-throughput parallel sequencing to obtain high-precision, complete nucleic acid sequences. The sequencing accuracy of the current mainstream synthetic sequencing technology is relatively high, and the original accuracy of most sequenced bases can reach more than 99.9%. However, its read length is short. Using these short read length sequences, whether it is resequencing or de novo assembly, it is difficult to restore the complete target nucleic acid sequence.

Therefore, how to obtain long-distance information in nucleic acid sequences has become one of the hot issues in the current sequencing technology research. Currently, there are two main commercial companies that can provide long-read sequencing solutions. The first is the single-molecule real-time sequencing of Pacific Bioscience (Pacbio) in the United States, and the nanopore sequencing of Oxford Nanopore (ONT) in the United Kingdom. In addition, foreign companies such as Quantum SI and Genia, and domestic companies such as Qi Tan, Jin Shi, and An Xu Yuan have entered this technology field and have released prototypes.

With the advancement of science and technology, current clinical sample variation detection is no longer satisfied with detecting only small-range variations (single nucleotide mutations and small deletion/insertion mutations). Some genetic abnormalities caused by large-scale structural variations are gradually being analyzed. Since structural variation detection is more sensitive to sequencing read length, long-read sequencing technology will gradually shift from the field of scientific and technological services to the field of clinical testing, and the requirements for the accuracy and cost control of sequencing technology will also increase accordingly.

In terms of accuracy, the Hi-Fi sequencing method based on circularization consensus sequencing launched by Pacbio in 2019 (Figure 1) can achieve an average sequencing accuracy of more than 99% by repeating the reading five times, but because it requires multiple repeated sequencing of the same molecule, the cost is relatively high.

In order to save costs, Oxford Nanopore's latest PromethION 48 system can achieve a cost of $2-16 per Gb, which is gradually approaching the cost of synthetic sequencing, which is widely used in the market. Although the accuracy of this system is still insufficient compared with Pacbio's circularization consensus sequencing method, according to the latest data disclosed by Oxford Nanopore, its accuracy can reach 98.4%, which has been significantly improved compared with the early nanopore data.

The current nanopore sequencing solution is low in cost, but there is still room for improvement in sequencing accuracy. Unlike conventional sequencing by synthesis, nanopore sequencing libraries generally have longer inserts, ranging from a few thousand bases to a million bases, so they have fewer double-stranded ends than short insert libraries, and the efficiency of adapter connection is low. Since the nanopore sequencing adapters are generally coupled with the rate-controlling protein required for sequencing, it is impossible to use alcohol-containing cleaning reagents during magnetic bead or column purification after connection, making it difficult to remove the adapters in the purification step, resulting in the detection of non-target fragments during the sequencing process, resulting in a decrease in sequencing accuracy.

Therefore, there is an urgent need in the art to develop a method for removing free adapters in sequencing libraries.

Summary of the invention

The present invention aims to solve at least one of the technical problems existing in the prior art.

In order to avoid the rate-controlling protein denaturation problem caused by the use of alcohol detergents in the column purification process, the inventors found that a nuclease exonuclease can be added during the library construction process to remove residual connectors in the library. During the purification process, no alcohol detergent is used, and only magnetic beads or column purification is used. This avoids the detection of non-target fragments during the sequencing process, improves the sequencing accuracy, increases the sequencing throughput, and obtains high-quality sequencing reads in a short time.

Based on the above findings, in the first aspect of the present invention, the present invention proposes a method for establishing a sequencing library. According to an embodiment of the present invention, the method comprises: digesting the sample to be tested connected with a connector under the action of a nuclease to obtain the sequencing library. According to the method of an embodiment of the present invention, by introducing a nuclease, the fragment to be tested that has not been connected at all, the single-stranded free connector that is not connected to the two ends of the inserted fragment to be tested, and the product of incomplete connector connection (referring to the product with only one end connected to the connector and the other end not connected to the connector) are degraded, thereby reducing the time occupied by the non-desired library in the sequencing channel time, thereby improving the sequencing throughput and increasing the sequencing accuracy.

According to an embodiment of the present invention, the above method may further include at least one of the following additional technical features:

According to an embodiment of the present invention, the method includes exonuclease digestion of unconnected test fragments, free adapters not connected to both ends of the test insert fragment, and products of incomplete adapter connection to obtain digestion products.

According to an embodiment of the present invention, the method further comprises subjecting the obtained digestion product to a detwisting treatment.

According to an embodiment of the present invention, the unwinding process is performed under the action of a helicase.

According to an embodiment of the present invention, the helicase moves along the DNA in a direction from the 5' end to the 3' end, and the exonuclease is a 5'->3' exonuclease.

According to an embodiment of the present invention, the exonuclease recognizes double-stranded DNA and the exonuclease is a 5'->3' exonuclease, and the exonuclease includes at least one selected from T7 exonuclease, Lambda exonuclease, T5 Exonuclease, Exonuclease VI, and Exonuclease VIII (truncated).

According to an embodiment of the present invention, the nuclease is preferably T7 exonuclease.

According to an embodiment of the present invention, the helicase moves along the DNA in a direction from the 3' end to the 5' end, and the exonuclease is a 3'->5' exonuclease.

According to an embodiment of the present invention, the 3’->5’ exonuclease recognizes double-stranded DNA, and the exonuclease includes at least one selected from Exonuclease III, Exonuclease IX, and Exonuclease X.

According to an embodiment of the present invention, the exonuclease recognizes single-stranded DNA.

According to an embodiment of the present invention, the method for establishing a sequencing library may further include performing anti-degradation modification on the 5' end and/or 3' end of the sample to be tested connected to the connector. For digestion of the connection product using some exonucleases that can recognize the 5' end of single-stranded DNA and hydrolyze it (such as: T7 exonuclease, T5 exonuclease, Lambda exonuclease, Exonuclease VI, Exonuclease VIII (truncated)), the 5' end of the single strand, such as the 5' end of the sequence to be tested connected to the Y-type connector, can be chemically modified in advance to resist degradation by nucleases.

According to an embodiment of the present invention, the anti-degradation modification includes at least one selected from phosphate modification, 2'-OH modification (RNA base), 2'-F modification, LNA locked nucleotide modification and PNA peptide nucleic acid modification.

According to an embodiment of the present invention, the digestion treatment is carried out at 37° C. and the ratio of the ligation product to T7 exonuclease is 44:1 for 4 to 6 minutes.

According to an embodiment of the present invention, the connector is a Y-type connector or a non-Y-type connector.

According to an embodiment of the present invention, the non-Y-shaped linker has at least one of the following structures: complete complementary double strand, complementary double strand-non-complementary single strand-complementary double strand, 5' protruding single strand-complementary double strand, 3' protruding single strand-complementary double strand.

According to an embodiment of the present invention, the method for establishing a sequencing library further comprises purifying the digestion product.

According to an embodiment of the present invention, the purification process adopts Ampure XP magnetic beads purification.

According to an embodiment of the present invention, the sample to be tested connected with the connector is obtained by:

(1) Perform end repair and A-addition treatment (adding poly-A tail) on the sample to be tested;

(2) The product treated with A is subjected to a connector connection treatment to obtain the sample to be tested connected with a connector.

According to an embodiment of the present invention, after the end repair and A addition treatment and before the ligation treatment, a first purification treatment is further included for the A addition treatment product.

According to an embodiment of the present invention, after the ligation process, the method further comprises performing a second purification process on the ligation product.

According to an embodiment of the present invention, the sample to be tested is a DNA sample.

According to an embodiment of the present invention, before performing end-repair and A-addition treatment on the sample to be tested, the process further includes performing fragmentation treatment on the sample to be tested.

In a second aspect of the present invention, the present invention also proposes a sequencing library. According to an embodiment of the present invention, the sequencing library is a sequencing library of a nucleic acid sample obtained according to a method for establishing a sequencing library according to an embodiment of the present invention. The inventors found that the sequencing library obtained by the method has significantly reduced the number of fragments to be tested that are not connected, free joints that are not connected to both ends of the inserted fragment to be tested, and products of incomplete joint connection, and is simple to operate, has significantly high sequencing accuracy, good repeatability, low cost, and high sequencing throughput.

In a third aspect of the present invention, the present invention further provides a sequencing method. According to an embodiment of the present invention, the method comprises sequencing the above-mentioned sequencing library to obtain the sequence of the sample to be tested. The inventors have found that the method can efficiently determine the sequence information of the nucleic acid sample, and has high sensitivity, high accuracy, good repeatability and high sequencing throughput.

According to a specific embodiment of the present invention, the sequencing is performed on a nanopore sequencing platform. When the sequencing is performed on a nanopore sequencing platform, the sequencing adapter is generally coupled with a rate-controlling protein necessary for sequencing, so when the magnetic beads or column purification is performed after the connection, a cleaning reagent containing alcohol cannot be used. When the sequencing library constructed by the first aspect of the present invention is used, the fragments to be tested that have not been connected, the free adapters that are not connected to the two ends of the inserted fragment to be tested, and the products of incomplete adapter connection are degraded during the purification of the sequencing library, thereby avoiding the detection of non-target fragments during the sequencing process, and significantly improving the sequencing accuracy.

In a fourth aspect of the present invention, the present invention also provides a kit for constructing a sequencing library. According to an embodiment of the present invention, the kit includes a reagent containing an exonuclease for digesting a sample to be tested connected to a connector. The inventors have found that by using the kit of the present invention, in combination with the above-mentioned method for establishing a sequencing library, a sequencing library, and a sequencing method, a sequencing library can be effectively obtained, and can be effectively applied to a high-throughput sequencing platform, thereby effectively determining the nucleic acid sequence information of the nucleic acid sample, and the information obtained has high accuracy and a large sequencing throughput.

According to an embodiment of the present invention, the kit further comprises at least one of the following additional technical features:

According to an embodiment of the present invention, the reagent further includes a first reagent, and the first reagent is suitable for fragmenting the sample to be tested.

According to an embodiment of the present invention, the reagent further includes a second reagent, and the second reagent is suitable for performing end repair and A addition treatment on the sample to be tested.

According to an embodiment of the present invention, the reagent further includes a third reagent, and the third reagent is suitable for performing a connector connection process on the sample to be tested.

According to an embodiment of the present invention, the kit further comprises a linker carrying a 5' terminal phosphate group.

According to an embodiment of the present invention, the nuclease includes at least one of T7 exonuclease, Lambda exonuclease, T5 Exonuclease, Exonuclease VI, Exonuclease VIII (truncated), Exonuclease III, Exonuclease IX, and Exonuclease X.

Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and/or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

FIG1 is a diagram showing the Pacbio cyclization consensus sequencing principle according to an embodiment of the present invention;

FIG2 is a flowchart of constructing a nanopore library without exonuclease according to an embodiment of the present invention;

FIG3 is a library construction process including an exonuclease step according to an embodiment of the present invention (sequencing direction is 5'->3');

FIG4 is a library construction process including an exonuclease step according to an embodiment of the present invention (sequencing direction is 3'->5');

FIG5 is a protein control result after purification according to an embodiment of the present invention;

FIG6 is a comparison of library bands before and after enzyme digestion according to an embodiment of the present invention;

FIG7 is a comparison of changes in electrical signals of libraries with and without enzyme digestion according to an embodiment of the present invention;

FIG8 is a statistical diagram of the percentage of free adapters in original library sequencing and restriction digestion library sequencing according to an embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

It should be noted that the terms "first", "second", and "third" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first", "second", and "third" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

Methods for establishing sequencing libraries

According to one aspect of the present invention, the present invention provides a method for establishing a sequencing library. According to an embodiment of the present invention, referring to Figures 3 and 4, the method comprises the following steps:

According to an embodiment of the present invention, in the first step, the DNA sequence of the genome is selectively interrupted to obtain fragmented genomic DNA molecules, so as to improve the library capture rate when sequencing on the machine. In the second step, the fragmented genomic DNA molecules are end-repaired and "A" is added. In the third step, the genomic DNA after adding "A" is subjected to product purification treatment, the purpose is to obtain a purer sample, so as to increase the accuracy and sequencing throughput during sequencing. In the fourth step, the sample obtained after the purification treatment is connected to the adapter. According to an embodiment of the present invention, the adapter connection refers to mixing the adapter with T4DNA ligase (NEB, E6057AVIAL) and finally obtaining the sample after the adapter connection. In the fifth step, the adapter connection sample is subjected to product purification, and the sample with higher purity is extremely important for the accuracy and data volume of the sequencing on the machine. In the sixth step, the purified connection product is first digested under the action of a nuclease exonuclease to degrade the fragment to be tested that has not been connected at all, a chain in the free adapter that is not connected to the two ends of the inserted fragment to be tested, and the product of incomplete adapter connection, thereby reducing the time occupied by the undesired library for the sequencing channel time, thereby improving the sequencing throughput. According to an embodiment of the present invention, the exonuclease may be selected from at least one of T7 exonuclease, Lambda exonuclease, T5 Exonuclease, Exonuclease VI, Exonuclease VIII (truncated), Exonuclease III, Exonuclease IX, and Exonuclease X. In the seventh step, the sample treated with the exonuclease is subjected to product purification to obtain a preliminary library sample. In the eighth step, the obtained preliminary library sample is co-incubated with the helicase Dda protein to finally obtain a library that can be used for sequencing. According to an embodiment of the present invention, the direction of movement of the helicase along the DNA is from the 5' end to the 3' end (Figure 3) or from the 3' end to the 5' end (Figure 4).

According to an embodiment of the present invention, the helicase and T7 exonuclease can be added at the same time.

According to the embodiments of the present invention, the method can be used to efficiently prepare sequencing samples, and the obtained sequencing library can be effectively applied to a high-throughput sequencing platform, thereby effectively determining the nucleic acid sequence information of the library sample. In addition, the inventors surprisingly found that the method for preparing a sequencing library of the present invention is simple in process, extremely easy to operate, and the operation process is extremely easy to standardize and promote, and is low in cost, high in sensitivity, high in accuracy, and good in repeatability.

Acquisition of sequencing library

The library of the sample to be tested is obtained by the above method for establishing a sequencing library.

Sequencing methods

According to a specific embodiment of the present invention, the sample of the library to be tested is sequenced on a nanopore sequencing platform. The electrophoresis technology is used to drive individual molecules through the nanopore one by one to achieve sequencing. Since the diameter of the nanopore is very small, only a single nucleic acid polymer is allowed to pass through. Since the charged properties of the individual ATCG bases are different, the type of bases that pass through can be detected by the difference in the electrical signal, thereby achieving sequencing.

Motor proteins

Motor protein (helicase in Example 2 of the present invention) refers to a type of protein distributed inside or on the surface of cells, which is responsible for the macroscopic movement of a part of the substance in the cell or the entire cell.

Primers

The term "primer" used in this article is a general term for oligonucleotides that can complementarily pair with a template and synthesize a DNA chain complementary to the template under the action of DNA polymerase. Primers can be natural RNA, DNA, any form of natural nucleotides, or even non-natural nucleotides such as LNA or ZNA.

iSpC3

iSpC3 is a three-hydrocarbon carbon chain that is often used as a spacer in oligonucleotide chains.

iSp18

iSp18 is an 18-atom-long hexaethylene glycol chain that is commonly used as a spacer in oligonucleotide chains.

The present invention will be further explained below in conjunction with specific examples. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

Example 1: Preparation of Ad3 linker sequences

In this example, the Ad3 linker sequence was obtained by annealing chemically synthesized SEQ ID NO.1 and SEQ ID NO.2.

1. Order SEQ ID NO.1 and SEQ ID NO.2 primer sequences (Sangon Biotech), and dissolve SEQ ID NO.1 and SEQ ID NO.2 primers in TE buffer (pH=8) to a storage solution with a final concentration of 100 μM according to the instructions. Then take 10 μL of the storage solution and add 40 μL of TE buffer (pH=8) to dilute to a working solution with a final concentration of 20 μM.

5’-XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXTTTTTTTTTTYYYYGGTTGTTTCTGTTGGTGCTGATATTGCT-3’(X＝iSpC3；Y＝iSp18；SEQ ID NO.1)

5’pho-GCAATATCAGCACCAACAGAAACAACCTTTGAGGCGAGCGGTCAA-3’(SEQ ID NO.2)

Take 30 μL of the working solution of SEQ ID NO.1 primer and 30 μL of the working solution of SEQ ID NO.2 primer obtained by dilution in the previous step, mix them together, and use a vortex oscillator to fully mix. Heat to 70°C in a thermal cycler and incubate for 10 minutes. Then cool the temperature to 25°C at a rate of 0.1°C/s and continue incubating for half an hour. At this point, a 10 μM linker solution is obtained after annealing. The linker product is named Ad3 and stored in a -20 degree refrigerator.

Example 2. Cloning, expression and purification of helicase Dda

In this example, the helicase Dda (amino acid sequence is shown in SEQ ID NO.4) is prepared by recombinant expression in Escherichia coli, and the helicase is used as a motor protein.

1. Order the full-length cDNA sequence of Dda from Sangon Biotechnology (nucleotide sequence as shown in SEQ ID NO.3), connect it into the PET.28a(+) plasmid, and cut the vector with double restriction enzymes at Nde1 and Xho1 to express the Dda protein with a 6×His tag and a thrombin restriction site at the N-terminus.

2. Transform the cloned PET.28a(+)-Dda plasmid into ArcticExpress(DE3) competent bacteria (Tolo Biotech, 96183-02) or its derivatives. Pick a single colony and transfer it into 5mL LB medium containing kanamycin for propagation, and culture it at 37℃ with shaking overnight. Then transfer it into 1L LB (containing kanamycin), culture it at 37℃ with shaking until OD ₆₀₀ is 0.6-0.8, cool it down to 16℃, and add IPTG at a final concentration of 500μM to induce Dda expression overnight.

3. Prepare five buffer solutions according to the following formula:

Buffer A: 20 mM Tris-HCl pH 7.5, 250 mM NaCl, 20 mM imidazole;

Buffer B: 20 mM Tris-HCl pH 7.5, 250 mM NaCl, 300 mM imidazole;

Buffer C: 20 mM Tris-HCl pH 7.5, 50 mM NaCl;

Buffer D: 20 mM Tris-HCl pH 7.5, 1000 mM NaCl;

Buffer E: 20 mM Tris-HCl pH 7.5, 100 mM NaCl.

4. Collect the Dda bacteria expressed in step 2, resuspend the bacteria with buffer A in step 3, break the bacteria with a cell disruptor, and then centrifuge to obtain the supernatant. Mix the supernatant with the Ni-NTA filler that has been equilibrated with buffer A in advance, and bind for 1 hour. Collect the filler and wash the filler with buffer A in large quantities until no impurities are washed out. Then add buffer B to the filler to elute Dda. The eluted Dda is passed through a desalting column equilibrated with buffer C for buffer exchange. Then add an appropriate amount of thrombin (Yishen Bio, 20402ES05), and then add the mixed sample to the ssDNA cellulose (Sigma, D8273-10G) filler equilibrated with buffer C, and digest and bind overnight at 4℃. Collect the ssDNA cellulose filler, wash it with buffer C 3-4 times, and then elute it with buffer D. The protein concentrate purified from the ssDNA cellulose is passed through the molecular sieve Superdex 200 (Sigma, GE28-9909-44), and the molecular sieve buffer used is buffer E. The target protein was collected, concentrated, and frozen. The concentration of the purified protein was quantified using Nanodrop. The purity of the protein was tested by HPLC and SDS-PAGE electrophoresis at the same time. The results are shown in Figure 5.

Example 3 Sequencing library construction with exonuclease step:

The complete process of the embodiment of the present invention is shown in Figure 3. The sequencing direction is selected as 5'->3'. The main steps of constructing the sequencing library include: initial nucleic acid molecule fragmentation (optional), end repair and A addition, adapter ligation, nuclease treatment, and anchor sequence annealing.

1. DNA fragmentation (optional)

If the initial genome input is less than 12 μg, in order to increase the library capture rate during sequencing, you can choose to fragment the DNA. It is recommended to use Covaris g-TUBE (Covaris, 520079) for fragmentation. For the fragmentation system and steps, refer to the g-TUBE manual.

2. End repair plus "A"

2.1 In a 1.5 mL DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051), calculate the volume of sample required to add 12 μg of total DNA based on the sample concentration, and add Nuclease-Free water (Thermofisher, AM9932) to normalize the sample volume to 288 μL.

2.2 Prepare the required amount of end repair plus "A" reaction mixture in a centrifuge tube according to the ratio in Table 1. This step needs to be prepared on ice. Vortex the prepared end repair plus "A" reaction mixture 3 times, 3 seconds each time, and centrifuge it instantly to collect the reaction solution at the bottom of the tube.

Table 1: End Repair Plus "A" Reaction Mix

Components Single reaction volume FFPE DNA Repair Mix (NEB, M6630LVIAL) 12μL FFPE DNA Repair Buffer (E6622AAVIAL) 21μL End repair enzyme mixture (NEB, E6051AAVIAL) 18μL End repair reaction buffer (NEB, E6052AAVIAL) 21μL total capacity 72μL

2.3 Use a pipette to aspirate 72 μL of the prepared end repair reaction plus "A" mixture and add it to the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) prepared in step 2.1. Use a flared pipette tip to gently pipette and mix, or tap the tube wall by hand to mix. Centrifuge briefly to collect the reaction solution at the bottom of the tube.

2.4 Use a flared pipette tip to dispense 360 μL of the end repair reaction solution into 6 PCR tubes, 60 μL per tube, and centrifuge briefly to collect the reaction solution at the bottom of the tube.

2.5 Place the PCR tube in 2.4 in a PCR instrument and perform the reaction according to the conditions in Table 2.

Table 2: End Repair Plus "A" Reaction Conditions

temperature time Hot cover (105℃) ON 20℃ 10min 65℃ 10min 4℃ Hold

2.6 Centrifuge briefly to collect the reaction solution at the bottom of the tube, and combine the 6 tubes of end-repaired samples with "A" and transfer them to a clean 1.5mL DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051).

3. End repair plus "A" product purification

3.1 Take out Ampure XP magnetic beads (Beckman Coulter, A63882) from the refrigerator at 4 degrees celsius 30 minutes in advance, shake and mix thoroughly, then place at room temperature. Shake and mix thoroughly before use.

3.2 Pipette 360 μL of magnetic beads and add them to the sample in 2.6. Mix by flicking the tube wall gently, or gently blow with a flared pipette tip for at least 6 times until completely mixed. The last time, make sure that all the liquid and magnetic beads in the pipette tip are pumped into the tube.

3.3 Incubate at room temperature on a rotary mixer for 5 min.

3.4 Centrifuge the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) briefly and place it on a magnetic rack. Let it stand for 2 to 5 minutes until the liquid becomes clear. Use a pipette to carefully aspirate the supernatant and discard it.

3.5 Keep the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) on the magnetic rack, add 750 μL of freshly prepared 80% ethanol to rinse the magnetic beads and the tube wall. After standing for 30 seconds, carefully aspirate the supernatant and discard it.

3.6 Repeat step 3.5. Remove the centrifuge tube from the magnetic rack and centrifuge it instantly. After separation on the magnetic rack, use a small-range pipette to absorb the remaining liquid at the bottom of the tube.

3.7 Keep the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) on the magnetic rack, open the tube cover, and dry at room temperature until the surface of the magnetic beads is non-reflective and free of cracks.

3.8 Remove the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) from the magnetic rack, add 392 μL Nuclease-Free water (Thermofisher, AM9932) to elute the DNA, and flick the tube wall to mix. Centrifuge for 3 seconds and collect the liquid in the tube to the bottom of the tube.

3.9 Incubate at room temperature for 5 minutes.

3.10 Centrifuge the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) for a short time and place it on a magnetic rack. Let it stand for 2-5 minutes until the liquid is clear. Transfer 390 μL of the supernatant to a new 1.5 mL DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051). The remaining sample can be used for concentration determination. It is recommended to use Qubit-dsDNA HS Assay Kit (Thermofisher, Q32854) to determine the concentration of the purified end-repair plus "A" product.

4. Connector connection

4.1 Set the metal bath temperature to 25°C for preheating.

4.2 Take out the adapter (Ad3) and T4 DNA ligase (NEB, E6057AVIAL) from the -20 degree refrigerator, flick the tube wall to mix, centrifuge instantly, and place on ice. Thaw the Quick Ligation Reaction Buffer (NEB, E6058AVIAL), pipette to mix, centrifuge instantly, and then place on ice. Prepare the ligation reaction mixture according to Table 3.

Table 3: Ligation Reaction Mix

Components Single reaction volume Rapid Ligation Reaction Buffer (NEB, E6058AVIAL) 120μL T4 DNA ligase (NEB, E6057AVIAL) 60μL total capacity 180μL

4.3 Add 30uL of the Ad3 linker prepared in Example 1 to the 1.5mL DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) containing 390μL of the purified end-repair plus "A" product in 3.10. Mix by gently pipetting 6 times with a widened pipette tip, and centrifuge for a short time to collect the reaction solution at the bottom of the tube.

4.4 Use a pipette to slowly aspirate 180 μL of the prepared linker ligation reaction mixture and add it into the 1.5 mL DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) in step 4.3. Use a flared pipette tip to gently pipette and mix six times. Centrifuge briefly to collect the reaction solution at the bottom of the tube.

4.5 Place the 1.5 mL DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) in step 4.4 into the metal bath preheated at 25°C in step 4.1 for ligation reaction and set the timer to 30 minutes.

4.6 After the reaction is completed, centrifuge the reaction tube instantly and collect the reaction solution at the bottom of the tube.

5. Purification of Ligation Products

5.1 Take out Ampure XP magnetic beads (Beckman Coulter, A63882) from the 4℃ refrigerator 30 minutes in advance, shake and mix thoroughly, then place at room temperature, and shake and mix thoroughly before use.

5.2 Pipette 240 μL of magnetic beads and add them to the sample in 4.6. Mix by flicking the tube wall gently, or gently blow with a flared pipette tip for at least 6 times until completely mixed. The last time, make sure that all the liquid and magnetic beads in the pipette tip are pumped into the tube.

5.3 Incubate at room temperature on a rotary mixer for 5 min.

5.4 Centrifuge the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) briefly and place it on a magnetic rack. Let it stand for 2 to 5 minutes until the liquid becomes clear. Use a pipette to carefully aspirate the supernatant and discard it.

5.5 Keep the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) on the magnetic rack, add 900μL Wash buffer 1 (for short Fragment) or Wash buffer 2 (for long Fragment), remove the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) from the magnetic rack, and gently tap the tube wall to mix the magnetic beads. After mixing, put it back on the magnetic rack and let it stand for 2-5 minutes until all the magnetic beads are against the wall. Carefully aspirate the supernatant and discard it.

5.6 Repeat step 5.5. Remove the centrifuge tube from the magnetic rack and centrifuge it instantly. After separation on the magnetic rack, use a small-range pipette to absorb the remaining liquid at the bottom of the tube.

5.7 Remove the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) from the magnetic rack, add 68 μL of elution buffer to elute the DNA, and flick the tube wall to mix. Centrifuge for 3 seconds and collect the liquid in the tube to the bottom of the tube.

5.8 Incubate at room temperature for 10 min. If the library insert is long, incubate at 37°C for 10 min.

5.9 Centrifuge the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) for a short time and place it on a magnetic rack. Let it stand for 2-5 minutes until the liquid is clear. Transfer 66 μL of the supernatant to a new 1.5 mL DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051). The remaining sample can be used for concentration determination. It is recommended to use Qubit-dsDNA HS Assay Kit (Thermofisher, Q32854) to determine the concentration of the purified ligation product.

6. Enzyme Digestion

6.1 Set the metal bath temperature to 37°C for preheating.

6.2 Take out T7 Exonuclease (T7 exonuclease; NEB, M0263LVIAL) and NE Buffer ^TM 4 (NEB, B7004SVIAL) from the kit, vortex for 3 times, 3 seconds each time, centrifuge for a short time, and place on ice. Prepare the enzyme digestion reaction mixture according to Table 4.

Table 4: Enzyme digestion reaction mixture

Components Single reaction volume NE Buffer ^TM 4 (NEB, B7004SVIAL) 7.5μL T7 Exonuclease (NEB, M0263LVIAL) 1.5μL total capacity 9μL

6.3 Add 9 μL of enzyme digestion reaction mixture into the 1.5 mL DNA LoBind Microcentrifuge Tube containing 66 μL of purified ligation product in 5.9, gently pipette and mix 6 times with a widened pipette tip, and centrifuge briefly to collect the reaction solution at the bottom of the tube.

6.4 Place the 1.5 mL DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) in step 6.3 in the metal bath preheated at 37°C in step 6.1 for ligation reaction and set the timer to 5 minutes.

6.5 After the reaction is completed, centrifuge the reaction tube instantly and collect the reaction solution at the bottom of the tube.

7. Purification of Enzymatic Digestion Products

7.1 Take out Ampure XP magnetic beads (Beckman Coulter, A63882) from the 4℃ refrigerator 30 minutes in advance, shake and mix thoroughly, then place at room temperature. Shake and mix thoroughly before use.

7.2 Pipette 75 μL of magnetic beads and add them to the sample in 6.5. Mix by flicking the tube wall gently, or gently blow with a flared pipette tip for at least 6 times until completely mixed. The last time, make sure that all the liquid and magnetic beads in the pipette tip are pumped into the tube.

7.3 Incubate at room temperature on a rotary mixer for 5 min.

7.4 Centrifuge the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) briefly and place it on a magnetic rack. Let it stand for 2 to 5 minutes until the liquid becomes clear. Use a pipette to carefully aspirate the supernatant and discard it.

7.5 Keep the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) on the magnetic rack, add 200μL Wash buffer 1 (for short fragment), remove the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) from the magnetic rack, and gently tap the tube wall to mix the magnetic beads. After mixing, put it back on the magnetic rack and let it stand for 2-5 minutes until all the magnetic beads are against the wall. Carefully aspirate the supernatant and discard it.

7.6 Repeat step 7.5. Try to drain the liquid in the tube. If there is a small amount of liquid left on the tube wall, centrifuge the tube instantly, separate it on the magnetic rack, and drain the liquid at the bottom of the tube with a small-range pipette.

7.7 Remove the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) from the magnetic rack, add 62 μL of elution buffer to elute the DNA, and flick the tube wall to mix. Centrifuge for 3 seconds and collect the liquid in the tube to the bottom of the tube.

7.8 Incubate at room temperature for 10 minutes. If the library is too long, incubate at 37°C.

7.9 Centrifuge the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) for a short time and place it on a magnetic rack. Let it stand for 2-5 minutes until the liquid is clear. Transfer 60 μL of the supernatant to a new 1.5 mL DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051). The remaining sample can be used for concentration determination. It is recommended to use Qubit-dsDNA HS Assay Kit (Thermofisher, Q32854) to determine the concentration of the purified enzyme digestion product.

7.10 The entire library construction process is now completed and the product can be stored in a 4-degree refrigerator for 72 hours.

7.11 A plasmid digestion product was used as a quality control product to perform the above library construction process. The comparison results before and after digestion showed that there were three forms of molecules after connection, namely the original template without a connector, the library with a single-end connector, and the library with two-end connectors. After exonuclease treatment, the products without a connector and the products with a single-end connector were significantly reduced, and with the increase of digestion time, the products with double-end connectors did not show a significant decrease, indicating that this step of enzyme treatment can effectively enrich the target double-end connector connection products (Figure 6).

Example 4 Construction of sequencing library without exonuclease step

The complete process of the embodiment of the present invention is shown in FIG2 .

1. DNA fragmentation (optional)

If the initial genome input is less than 12 μg, you can choose to fragment the DNA to increase the library capture rate during sequencing. It is recommended to use Covaris g-TUBE (Covaris, 520079) for fragmentation. For the fragmentation system and steps, refer to the g-TUBE manual.

2. End repair plus "A"

89122.1 In a 1.5mL DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051), calculate the volume of sample required to add 12μg of total DNA based on the sample concentration, and add Nuclease-Free water (Thermofisher, AM9932) to normalize the sample volume to 288μL.

2.2 Prepare the required amount of end repair plus "A" reaction mixture in a centrifuge tube according to the ratio in Table 5. This step needs to be prepared on ice. Vortex the prepared end repair plus "A" reaction mixture 3 times, 3 seconds each time, and centrifuge it instantly to collect the reaction solution at the bottom of the tube.

Table 5: End Repair Plus "A" Reaction Mix

Components Single reaction volume FFPE DNA Repair Mix (NEB, M6630LVIAL) 12μL FFPE DNA Repair Buffer (E6622AAVIAL) 21μL End repair enzyme mixture (NEB, E6051AAVIAL) 18μL End repair reaction buffer (NEB, E6052AAVIAL) 21μL total capacity 72μL

2.3 Use a pipette to aspirate 72 μL of the prepared end repair reaction plus "A" mixture and add it to the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) prepared in step 2.1. Use a flared pipette tip to gently pipette and mix, or tap the tube wall by hand to mix. Centrifuge briefly to collect the reaction solution at the bottom of the tube.

2.4 Use a flared pipette tip to dispense 360 μL of the end repair reaction solution into 6 PCR tubes, 60 μL per tube, and centrifuge briefly to collect the reaction solution at the bottom of the tube.

2.5 Place the PCR tube in 2.4 in a PCR instrument and perform the reaction according to the conditions in Table 6.

Table 6: End Repair Plus "A" Reaction Conditions

temperature time Hot cover (105℃) ON 20℃ 10min 65℃ 10min 4℃ Hold

2.6 Centrifuge briefly to collect the reaction solution at the bottom of the tube, and combine the 6 tubes of end-repaired samples with "A" and transfer them to a clean 1.5mL DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051).

3. End repair plus "A" product purification

3.1 Take out Ampure XP magnetic beads (Beckman Coulter, A63882) from the refrigerator at 4 degrees celsius 30 minutes in advance, shake and mix thoroughly, then place at room temperature. Shake and mix thoroughly before use.

3.2 Pipette 360 μL of magnetic beads and add them to the sample in 2.6. Mix by flicking the tube wall gently, or gently blow with a flared pipette tip for at least 6 times until completely mixed. The last time, make sure that all the liquid and magnetic beads in the pipette tip are pumped into the tube.

3.3 Incubate at room temperature on a rotary mixer for 5 min.

3.4 Centrifuge the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) briefly and place it on a magnetic rack. Let it stand for 2 to 5 minutes until the liquid becomes clear. Use a pipette to carefully aspirate the supernatant and discard it.

3.5 Keep the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) on the magnetic rack, add 750 μL of freshly prepared 80% ethanol to rinse the magnetic beads and the tube wall. After standing for 30 seconds, carefully aspirate the supernatant and discard it.

3.6 Repeat step 3.5. Remove the centrifuge tube from the magnetic rack and centrifuge it instantly. After separation on the magnetic rack, use a small-range pipette to absorb the remaining liquid at the bottom of the centrifuge tube.

3.7 Keep the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) on the magnetic rack, open the tube cover, and dry at room temperature until the surface of the magnetic beads is non-reflective and free of cracks.

3.8 Remove the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) from the magnetic rack, add 392 μL Nuclease-Free water (Thermofisher, AM9932) to elute the DNA, and flick the tube wall to mix. Centrifuge for 3 seconds and collect the liquid in the tube to the bottom of the tube.

3.9 Incubate at room temperature for 5 minutes.

3.10 After instant centrifugation, place the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) on a magnetic rack and let stand for 2-5 minutes until the liquid is clear. Transfer 390 μL of supernatant to a new 1.5 mL DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051). The remaining sample can be used for concentration determination. It is recommended to use Qubit-dsDNA HS Assay Kit (Thermofisher, Q32854) to determine the concentration of the purified end-repair plus "A" product.

4. Connector connection

4.1 Set the metal bath to 25°C for preheating.

4.2 Take out the adapter (Ad3) and T4 DNA ligase (NEB, E6057AVIAL) from the -20 degree refrigerator, flick the tube wall to mix, centrifuge instantly, and place on ice. Thaw Quick Ligation Reaction Buffer (NEB, E6058AVIAL), pipette to mix, centrifuge instantly, and then place on ice. Prepare the ligation reaction mixture according to Table 7.

Table 7: Ligation Reaction Mix

Components Single reaction volume Rapid Ligation Reaction Buffer (NEB, E6058AVIAL) 120μL T4 DNA ligase (NEB, E6057AVIAL) 60μL total capacity 180μL

4.3 Add 30uL of the Ad3 linker prepared in Example 1 to the 1.5mL DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) containing 390μL of the purified end-repair plus "A" product in 3.10. Mix by gently pipetting 6 times with a widened pipette tip, and centrifuge briefly to collect the reaction solution at the bottom of the tube.

4.4 Use a pipette to slowly aspirate 180 μL of the prepared linker ligation reaction mixture and add it into the 1.5 mL DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) in step 4.3. Use a flared pipette tip to gently pipette and mix six times. Centrifuge briefly to collect the reaction solution at the bottom of the tube.

4.5 Place the 1.5 mL DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) in step 4.4 into the metal bath preheated at 25°C in step 4.1 for ligation reaction and set the timer to 30 minutes.

4.6 After the reaction is completed, centrifuge the reaction tube instantly and collect the reaction solution at the bottom of the tube.

5. Purification of Ligation Products

5.1 Take out Ampure XP magnetic beads (Beckman Coulter, A63882) from the refrigerator at 4 degrees 30 minutes in advance, shake and mix thoroughly, then place at room temperature. Shake and mix thoroughly before use.

5.2 Pipette 240 μL of magnetic beads and add them to the sample in 4.6. Mix by flicking the tube wall gently, or gently blow with a flared pipette tip for at least 6 times until completely mixed. The last time, make sure that all the liquid and magnetic beads in the pipette tip are pumped into the tube.

5.3 Incubate at room temperature on a rotary mixer for 5 min.

5.4 Centrifuge the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) briefly and place it on a magnetic rack. Let it stand for 2 to 5 minutes until the liquid becomes clear. Use a pipette to carefully aspirate the supernatant and discard it.

5.5 Keep the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) on the magnetic rack, add 900μL Wash buffer 1 (for short Fragment) or Wash buffer 2 (for long Fragment), remove the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) from the magnetic rack, and gently tap the tube wall to mix the magnetic beads. After mixing, put it back on the magnetic rack and let it stand for 2-5 minutes until all the magnetic beads are against the wall. Carefully aspirate the supernatant and discard it.

5.6 Repeat step 5.5. Remove the centrifuge tube from the magnetic rack and centrifuge it instantly. After separation on the magnetic rack, use a small-range pipette to absorb the remaining liquid at the bottom of the tube.

5.7 Remove the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) from the magnetic rack, add 68 μL of elution buffer to elute the DNA, and flick the tube wall to mix. Centrifuge for 3 seconds and collect the liquid in the tube to the bottom of the tube.

5.8 Incubate at room temperature for 10 min. If the library insert is long, incubate at 37°C for 10 min.

5.9 Centrifuge the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) for a short time and place it on a magnetic rack. Let it stand for 2-5 minutes until the liquid is clear. Transfer 66 μL of the supernatant to a new 1.5 mL DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051). The remaining sample can be used for concentration determination. It is recommended to use Qubit-dsDNA HS Assay Kit (Thermofisher, Q32854) to determine the concentration of the purified ligation product.

Example 5: Sequencing library preparation

In this example, the sequencing libraries obtained in Example 3 and Example 4 were incubated with the helicase Dda protein prepared in Example 2, respectively, to prepare the final library for sequencing.

1. Add 100 mL 1 M Tris-HCL PH 7.5 buffer and 100 mL 1 M KCl solution into a volumetric flask, add ultrapure water to make up to 1 L to prepare 2X binding buffer.

2. Prepare a mixed solution of helicase Dda (prepared in Example 2) and the library on ice according to Table 8 below, and then incubate at 30°C for one hour.

Table 8: Motor protein and library binding system

Reagents volume 2x Binding Buffer 50μL Helicase Dda 20μL After purification, the ligation product 20μL water 10μL

3. Use the Qubit DNA HS kit to quantify the library. After clearly marking the concentration, store the product in a 4°C refrigerator for later use.

Example 6: Nanopore sequencing

This example uses a nanopore detection platform based on a patch clamp platform to perform nanopore sequencing on the target sequencing library prepared in Example 6 to verify the advantages of the sequencing library constructed in this application in nanopore sequencing, namely, the increase in the number of molecules that can be sequenced.

1. Referring to the single-channel electrophysiological detection system in Geng Jia and Guo Peixuan (“Application of bacteriophage phi29 DNA packaging motor phospholipid membrane chimera in single-molecule detection and nanomedicine”. Life Science, 2011, 23(11): 1114-1129), a nanopore detection platform based on the patch clamp platform was constructed, and the porin (Sigma-Aldrich, H9395-5mg) was inserted into the phospholipid bilayer membrane to form a single-channel nanopore.

2. The sequencing library obtained in Example 5 was added to the single-channel system, and the current amplitude change was detected and recorded by the patch clamp system.

3. The current changes per unit time for the sequencing of the libraries in Example 3 and Example 4 are shown in Figure 7. It can be seen that per unit time, the number of sequencing sequences of the libraries after enzyme digestion treatment is significantly increased, the free linker sequence signal is reduced, the time occupied by the resting potential is reduced, and the sequencing throughput is significantly improved.

4. The number of sequencing library read sequences and free adapter sequences was detected by the discrimination algorithm of the adapter signal. The comparison results of the free adapter ratio of the original library and the enzyme-digested library showed that after enzyme digestion, the measured free adapter ratio decreased from 41.46% of the original library to 8.82%, and the library molecule ratio increased from the original 58.54% to 91.18% (Figure 8).

In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims (17)

A method for establishing a sequencing library, comprising: The sample to be tested connected with the adapter is digested under the action of nuclease to obtain the sequencing library. The method according to claim 1, further comprising detwisting the digestion product; Optionally, the unwinding process is performed under the action of a helicase. The method according to claim 2, characterized in that the helicase moves along the DNA from the 5' end to the 3' end, and the exonuclease is a 5'->3' exonuclease; Optionally, the 5'->3' exonuclease recognizes double-stranded DNA, and the exonuclease includes at least one selected from T7 exonuclease, Lambda exonuclease, T5 Exonuclease, Exonuclease VI, and Exonuclease VIII (truncated); Preferably, the nuclease is T7 exonuclease. The method according to claim 2, characterized in that the helicase moves along the DNA from the 3' end to the 5' end, and the exonuclease is a 3'->5' exonuclease; Optionally, the 3’->5’ exonuclease recognizes double-stranded DNA, and the exonuclease includes at least one selected from Exonuclease III, Exonuclease IX, and Exonuclease X. The method according to claim 1, characterized in that the nuclease recognizes single-stranded DNA. The method according to claim 1, further comprising performing an anti-degradation modification treatment on the 5' end and/or 3' end of the sample to be tested connected to the connector; Optionally, the anti-degradation modification includes at least one selected from phosphate modification, 2'-OH modification (RNA base), 2'-F modification, LNA locked nucleotide modification and PNA peptide nucleic acid modification. The method according to claim 1 is characterized in that the digestion treatment is carried out at 37° C. and the ratio of the ligation product to T7 exonuclease is 44:1 for 4 to 6 minutes. The method according to claim 1, characterized in that the connector is a Y-type connector or a non-Y-type connector; Optionally, the non-Y-shaped connector has at least one of the following structures: Complete complementary double strands, complementary double strands-non-complementary single strands-complementary double strands, 5’ protruding single strands-complementary double strands, 3’ protruding single strands-complementary double strands. The method according to claim 1 is characterized in that it further includes purifying the digestion product; optionally, the purification process uses Ampure XP magnetic beads for purification. The method according to claim 1, characterized in that the sample to be tested connected with the connector is obtained by: The samples to be tested are subjected to end repair and A addition treatment; The product treated with A is subjected to a connector connection treatment to obtain the sample to be tested connected with a connector; Optionally, after the end repair and A addition treatment and before the ligation treatment, the process further comprises performing a first purification treatment on the A addition treatment product; Optionally, after the ligation treatment, the method further comprises subjecting the ligation product to a second purification treatment. The method according to claim 1, characterized in that the sample to be tested is a DNA sample. The method according to claim 1 is characterized in that before the end repair and A addition treatment of the sample to be tested, it further includes fragmenting the sample to be tested. A sequencing library, characterized in that the sequencing library is obtained by the method according to any one of claims 1 to 12. A sequencing method, characterized in that it comprises: performing sequencing processing on the sequencing library according to claim 13 to obtain the sequence of the sample to be tested; Optionally, the sequencing is performed on a nanopore sequencing platform. A kit for constructing a sequencing library, characterized in that it comprises: reagents, wherein the reagents comprise exonucleases for digesting a sample to be tested connected with a linker; Optionally, further comprising a first reagent, wherein the first reagent is suitable for fragmenting the sample to be tested; Optionally, the method further comprises a second reagent, wherein the second reagent is suitable for performing end repair and A addition treatment on the sample to be tested; Optionally, a third reagent is further included, and the third reagent is suitable for connecting the sample to be tested to a connector. The kit according to claim 15 is characterized in that the kit further comprises a linker carrying a 5' terminal phosphate group. The kit according to claim 16 is characterized in that the nuclease exonuclease includes at least one of T7 exonuclease, Lambda exonuclease, T5 Exonuclease, Exonuclease VI, Exonuclease VIII (truncated), Exonuclease III, Exonuclease IX, and Exonuclease X.

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