HK1239749B - Method using bubble-shaped connector elements to construct sequencing library - Google Patents

Description

Method for constructing sequencing libraries using bubble-shaped adapter elements

Technical Field

The present invention relates to the field of biotechnology, and in particular to a method for constructing a sequencing library using a bubble-shaped linker element, the constructed sequencing library and applications thereof.

Background Art

Whole-genome sequencing is a research method that covers the entire genome of a species and detects all genetic information in an individual's genome. It offers high accuracy, reaching up to 99.99%. With the development of second-generation high-throughput sequencing technology, sequencing has become increasingly faster, more accurate, and less expensive, and whole-genome sequencing has become increasingly widely used in research.

CG sequencing is based on single-stranded circularized DNA and requires the introduction of adapters with known sequences for sequencing. Typically, when constructing whole-genome libraries on the Complete Genomics (CG) sequencing platform, the standard CG whole-genome library construction process requires the addition of two sets of adapters, each with a different sequence, making the loading process cumbersome and time-consuming.

The present invention is proposed to solve the problems of excessive adapter ligation steps and long overall library construction time in the library construction of Complete Genomics' sequencing platform.

Summary of the Invention

In response to the shortcomings of the above-mentioned prior art, the present invention aims to provide a method for constructing a sequencing library using a bubble-shaped adapter element, as well as a sequencing library constructed thereby and its application. By using a bubble-shaped adapter element, the present invention solves the problems of the double-adapter library construction method on the CG sequencing platform, such as the excessive number of adapter ligation steps, the large number of PCR amplifications, the long overall library construction time, and the high cost. It also improves library construction efficiency and achieves optimization of double-adapter library construction.

The present invention achieves the above-mentioned purpose through the following technical solutions:

In a first aspect, the present invention provides a method for constructing a sequencing library, the method comprising the following steps:

1) Fragmenting the double-stranded DNA, and performing blunt-end repair, 5'-end phosphorylation, and 3'-end addition of base A to the resulting DNA fragments;

2) adding linker elements 1 to both ends of the DNA fragment obtained in step 1) through a ligation reaction; the linker element 1 comprises a long nucleic acid chain and a short nucleic acid chain; the long nucleic acid chain and the short nucleic acid chain can anneal to form a bubble-shaped hybrid with complementary sequences at both ends and non-complementary sequences in the middle; the 5' terminal base of the long nucleic acid chain A of the hybrid is phosphorylated, and the 3' terminal of the short nucleic acid chain B of the hybrid has a protruding base T; the linker element 1 has a class III restriction endonuclease recognition site;

3) Using the DNA fragment obtained in step 2) as a template, PCR amplification is performed using two nucleic acid single strands with different sequences targeting partial sequences of the long nucleic acid strand and the short nucleic acid strand of the linker element 1 as primers;

The middle of the two primers has an enzyme action site; preferably, the enzyme action site is U or dU, and the corresponding enzyme is USER enzyme;

4) using the enzyme action site to create sticky ends at both ends of the amplified fragment obtained in step 3), and using the sticky ends to connect the amplified fragments into a circular nucleic acid double strand;

5) digesting the double-stranded circular nucleic acid obtained in step 4) with a class III restriction endonuclease, and recovering the digested DNA fragments;

6) performing blunt-end repair and adding base A to the 3' end of the DNA fragment obtained after enzyme digestion in step 5);

7) adding linker elements 2 to both ends of the DNA fragment obtained in step 6) through a ligation reaction; the linker elements 2 comprise a long nucleic acid chain and a short nucleic acid chain; the long nucleic acid chain and the short nucleic acid chain can anneal to form a bubble-shaped hybrid with complementary sequences at both ends and non-complementary sequences in the middle; the 5' terminal base of the long nucleic acid chain A of the hybrid is phosphorylated, and the 3' terminal of the short nucleic acid chain B of the hybrid has a protruding base T;

The sequence of the linker element 2 is different from the sequence of the linker element 1;

8) Using the DNA fragment obtained in step 7) as a template, PCR amplification is performed using two single-stranded nucleic acids with different sequences targeting partial sequences of the long and short nucleic acid chains of linker element 2, respectively, as primers; one primer has a phosphorylated first base at its 5' end, and the other primer has a biotin-labeled first base at its 5' end;

9) Recovering the PCR product obtained in step 8) using avidin magnetic beads, and performing denaturation treatment to separate and recover the non-biotin-labeled single-stranded nucleic acid;

Preferably, the avidin magnetic beads are streptavidin magnetic beads;

Preferably, an alkaline denaturation method or a high temperature denaturation method is used;

10) Circularizing the non-biotin-labeled single-stranded nucleic acid obtained in step 9) to form a single-stranded circular nucleic acid product, which is the sequencing library;

Preferably, the circularization of the nucleic acid single strand is achieved using a mediating fragment, wherein the mediating fragment has a corresponding complementary sequence for connecting the two ends of the nucleic acid single strand;

Preferably, the method further comprises a step of digesting the linear single strand after the circularization of the nucleic acid single strand is completed; further preferably, the digestion is performed using exonuclease 1 and/or 3.

For the above construction method, preferably, in step 1), the double-stranded DNA fragment is prepared by the following steps:

1-1) fragmenting the mRNA sample to obtain fragmented mRNA;

1-2) reverse transcribing the fragmented mRNA to obtain cDNA amplification products as double-stranded DNA fragments;

Optionally, the double-stranded DNA fragments are directly obtained by fragmentation of a DNA sample;

Preferably, the fragmentation is to randomly interrupt or cut the DNA to be tested by physical or chemical methods; further preferably, the DNA to be tested is fragmented by physical ultrasound or enzyme reaction;

Preferably, the blunt end repair is performed using T4 DNA polymerase;

Preferably, the phosphorylation is performed using a nucleotide kinase, preferably T4 polynucleotide kinase;

Preferably, the addition of base A at the 3' end is performed using Klenow polymerase with 3'→5' exonuclease activity removed.

Preferably, the class III restriction endonuclease in the linker element 1 is Acu I, Bpm I, BceA I, BbvI, BciV I, BpuE I, BseM II, BseR I, Bsg I, BsmF I, BtgZ I, Eci I, EcoP15I, Eco57M I, FokI, Hga I, Hph I, Mbo II, Mnl I, SfaN I, TspDT I, TspDW I or Taq II;

Preferably, the recognition site of the class III restriction endonuclease is 0-2 bp away from the 3' end of the hybrid;

Preferably, the long nucleic acid chain of the linker element 1 comprises a tag sequence; further preferably, the tag sequence is 6-10 nt in length.

In a preferred embodiment, the sequence of the long nucleic acid chain in the linker element 1 is: 5'-/Phos/ACTGCTGAGTCGAGA(N)CTGACAAGGTCGCCAGCCCTGAGTGCTTCGAA-3'; wherein, /Phos/ represents phosphorylation modification, and N is a tag sequence; preferably, the tag sequence is 6 nt to 10 nt in length; more preferably, the tag sequence is 5'-TGTCATAAAT-3'; that is, in a more preferred specific embodiment, the sequence of the long nucleic acid chain in the linker element 1 is:

5’-/Phos/ACTGCTGAGTCGAGATGTCATAAATCTGACAAGGTCGCCAGCCCTGAGTGCTTCGAA-3’ (see SEQ ID NO: 1);

The sequence of its short nucleic acid chain is:

5'-CGAAGCACTCAAGGTCGCCAGCCCTCAGTACGTCAGCAGTT-3', see SEQ ID NO: 2.

When the above-mentioned linker element 1 is used, preferably, in step 3), the primers used for PCR amplification are:

Forward primer: 5'-AGGUCGCCAGCCCUCAGTAC-3' (see SEQ ID NO: 5);

Reverse primer: 5'-AGGGCUGGCGACCUTGTCAG-3' (see SEQ ID NO: 6).

When the above-mentioned linker element 1 is used, preferably, the sequence of the long nucleic acid chain in the linker element 2 is: 5’-/Phos/AGTCGGAGGCCAAGCGTGCTTAGGATGAGTGCTCTCGAA-3’, wherein /Phos/ indicates phosphorylation modification, see SEQ ID NO: 3; the sequence of the short nucleic acid chain is: 5’-CGAGAGCACTCCATGTAGTGTACGATCCGACTT-3’, see SEQ ID NO: 4.

When the above-mentioned linker element 2 is used, preferably, in step 8), the primers used for PCR amplification are:

Forward primer: 5'-TCCTAAGCACGCTTGGCCT-3' (see SEQ ID NO: 7);

Reverse primer: 5'-CATGTAGTGTACGATCCGACTT-3' (see SEQ ID NO: 8);

The first base at the 5' end of the forward primer is biotin-labeled, and the first base at the 5' end of the reverse primer is phosphorylated.

When the above-mentioned linker element 2 is used, preferably, in step 10), the sequence of the mediating fragment is: 5’-GTACACTACATGTCCTAAGCACGC-3’, see SEQ ID NO: 9.

In a second aspect, the present invention provides a sequencing library, which is prepared by the sequencing library construction method as described in the first aspect.

In a third aspect, the present invention provides a use of the sequencing library as described in the second aspect in genome sequencing, preferably in sequencing a target genome region;

Preferably, sequencing is performed using a single-stranded circular library sequencing platform; further preferably, sequencing is performed using a sequencing platform from CompleteGenomics.

In a fourth aspect, the present invention provides a nucleic acid sequencing method, comprising the steps of sequencing the sequencing library as described in the second aspect;

Preferably, sequencing is performed using a single-stranded circular library sequencing platform; further preferably, sequencing is performed using a sequencing platform from CompleteGenomics;

Preferably, the method further comprises the step of assembling and/or splicing the sequencing results.

Beneficial effects

In the existing double-joint library construction, two different joints need to be added each time a joint is added, and multi-step processing is required to perform PCR amplification; the entire library construction process requires the use of four different joints to complete the loading of the joint twice. The sequencing library construction method of the present invention only needs to use one joint to be loaded at the 3' and 5' ends of the DNA fragment at the same time each time a joint is added, and PCR amplification can be performed; the entire library construction process requires the use of two different joints to complete the loading of the joint twice (see Figure 2). In addition, the joint element adopted by the construction method of the present invention passes through the mismatched sequence of the intermediate bubble structure, and during the PCR process, the mismatched chain is replaced, and the matched sequencing sequence is used as a PCR primer to achieve displacement. In this way, by a joint loading and PCR amplification process, it is possible to ensure that the two ends of the DNA fragment are connected to different sequencing sequences, while achieving fragment amplification, saving the time cost and material cost of building a library.

The sequencing library construction method of the present invention reduces the existing library construction steps, shortens the library construction cycle, effectively saves the cost of library construction, improves the library construction efficiency, and realizes the optimization of double-linker library construction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the hybrid structure formed by annealing the linker element 1 in the embodiment;

FIG2 shows the hybrid structure formed by annealing the linker element 2 in the embodiment;

Figure 3 illustrates the sequencing library construction scheme of the present invention; wherein it indicates that the base is phosphorylated; indicates that the base is biotin-labeled; wherein: 1 is a DNA fragment after shearing; 2 is a fragment after blunt-end repair and phosphorylation; 3 is a DNA fragment with base A added to the 3' end; 4 is a DNA fragment after adding linker element 1; 5 is the product after PCR amplification of the DNA fragment with linker element 1; 6 is a DNA fragment with sticky ends after digestion with USER enzyme; 7 is a double-stranded circular DNA formed by circularization of the sticky ends; 8 is a linear DNA fragment formed after digestion with a class III restriction endonuclease; 9 is a DNA fragment after end repair and phosphorylation; 10 is a DNA fragment with base A added to the 3' end; 11 is a DNA fragment after adding linker element 2; 12 is the product after PCR amplification of the DNA fragment with linker element 2; 13 is single-stranded DNA separated by biotin labeling; 14 is the final product of library construction, i.e., single-stranded circular DNA;

FIG4 is the electrophoresis result of the final product of the library construction in the embodiment; the left lane is the single-stranded circular DNA library, and the right lane is the Low Range RNA Ladder (FERMENTAS).

DETAILED DESCRIPTION

For the convenience of understanding the present invention, the present invention is given below with examples. It should be understood by those skilled in the art that the examples are only for the purpose of helping to understand the present invention and should not be regarded as specific limitations of the present invention.

Example 1 Construction of the sequencing library of the present invention

1. Genome disruption

Take 1 μg of genomic DNA from inflammatory cells of human lymphoid tissue, dilute it to 80 μl with TE buffer, add it to the Covaris dedicated shearing plate, and use the Covaris LE220 ultrasonic shearer to shear the DNA to approximately 200 to 400 bp. The parameters are shown in the following table:

Fill factor twenty one% Pressure (PIP) 500 Pulse coefficient 500 Interruption time 20 seconds, 6 times

Transfer the fragmented genome to a new 1.5ml non-stick EP tube, add 64µl of Ampure XP magnetic beads, mix thoroughly, let stand at room temperature for 5 minutes, and then place the tube on a magnetic rack to absorb the beads. After 5 minutes, aspirate the supernatant and place it in another new non-stick EP tube. Add 32µl of Ampure XP magnetic beads, mix thoroughly, and let stand at room temperature for 5 minutes. Place the tube on a magnetic rack to absorb the beads for 5 minutes. Discard the supernatant and wash the beads twice with 75% ethanol. After drying, add 42µl of TE buffer or enzyme-free water, mix thoroughly, and let stand for 5 minutes to dissolve and recover the product.

2. End repair

Prepare the end-repair reaction mix in advance as shown in the following table:

10x Polynucleotide Kinase Buffer 5.5ul 25 mM deoxynucleotide triphosphates 3ul T4 DNA polymerase 3ul T4 polynucleotide kinase 3ul Klenow snippet 1ul Total volume 15.5ul

Add the DNA obtained in the previous step to the premix, mix thoroughly, and incubate at 20°C for 30 minutes. Remove the DNA and add 60 μl of Ampure XP magnetic beads, mix thoroughly, and let stand at room temperature for 5 minutes. Discard the supernatant and wash twice with 75% ethanol. Allow to dry and dissolve in 42 μl of TE buffer. (This is the standard procedure for magnetic bead purification and will be used for all other purifications unless otherwise specified.)

3. Add "A" at the end

Prepare the premix according to the following table:

10xBlue buffer (Enzymatics) 5ul 10mM ATP 1ul Klenow (3`-5`exo-) enzyme 2ul Enzyme-free water 2ul Total volume 10ul

The DNA obtained in the above step was added to the premix, mixed well, incubated at 37°C for 30 minutes, purified with 50ul Ampurexp magnetic beads, dried, and dissolved in 42ul TE buffer.

4. Connecting connector element 1

The sequence structure of the linker element 1 is shown in FIG1 , wherein the tag sequence N is 5’-TGTCATAAAT-3’.

Prepare the premix in advance according to the following table:

10x ligation buffer (Enzymatics) 5ul Connector element 1 (20uM) 2ul Total volume 7ul

Add the premix to the DNA solution from the previous step and mix thoroughly. Then add 2 μl of T4 DNA ligase, mix again, and incubate at 20°C for 1 hour. Purify with Ampure XP magnetic beads and dissolve in 42 μl of TE.

5. PCR amplification

The sequence of primer 1 is: 5′-AGGUCGCCAGCCCUCAGTAC-3′;

The sequence of primer 2 is: 5’-AGGGCUGGCGACCUTGTCAG-3’.

Prepare the reaction premix in advance according to the table below:

Add the premix to the DNA solution obtained in the previous step and mix thoroughly. Place in a PCR instrument and perform the PCR reaction as follows:

After the reaction was completed, 100 μl of Ampure XP magnetic beads were used for purification and dissolved in 82 μl of TE.

6. De-uracil

Prepare the reaction premix in advance according to the table below:

Enzyme-free water 5.8ul 10X Taq buffer (Takara) 11ul USER enzyme (1000U/ml) 13.2ul Total volume 30ul

The prepared reaction premix was added to the reaction product of the previous step, mixed well, and then allowed to react at 37°C for 1 hour.

7. Double-chain cyclization

Prepare the following reaction system 1:

Add the reaction product from the previous step to reaction system 1, mix well, and then react at 60°C for 30 minutes. After the reaction is complete, place the mixture at 24°C.

Prepare the following reaction system 2:

Enzyme-free water 194ul T4 DNA ligase (fast) (600 U/ul) (enzymatics) 6ul Total volume 200ul

Add reaction system 2 into reaction system 1 and react at 24°C for 1 h.

Take the reaction product, add 1320ul Ampure XP magnetic beads, mix well and let it stand for 5 minutes; place it on a magnetic rack and collect the supernatant, add 680ul Ampure XP magnetic beads to the supernatant, mix well and let it stand for 5 minutes; place it on a magnetic rack and aspirate the supernatant, wash the magnetic beads twice with 75% ethanol; after drying, add 65ul TE buffer solution or enzyme-free water, mix well and let it stand for 5 minutes to dissolve and recover the product.

8. Linear digestion

Prepare the reaction premix in advance according to the table below:

Add the prepared reaction premix to the DNA solution obtained in the previous step, mix well, and then react at 37°C for 1 hour.

Purify using 80ul of Ampure XP magnetic beads and dissolve the recovered product in 42ul of TE buffer or enzyme-free water.

9. Ecop15 enzyme digestion

Prepare the reaction mix in advance as shown in the table below:

Enzyme-free water 274ul 10X NEBuffer 3.1 (NEB) 36ul Ecop15I endonuclease (10U/ul) 10ul Total volume 320ul

Add the reaction premix to the DNA solution obtained in the previous step, mix well, and incubate at 37°C for 16 h.

After the reaction is complete, take the reaction product, add 415ul Ampure XP magnetic beads, mix well, and let it stand for 5 minutes; place it on a magnetic rack, collect the supernatant, add 296ul Ampure XP magnetic beads to the supernatant, mix well, and let it stand for 5 minutes; place it on a magnetic rack, aspirate the supernatant, and wash the magnetic beads twice with 75% ethanol; after drying, add 42ul TE buffer solution or enzyme-free water, mix well, and let it stand for 5 minutes to dissolve and recover the product.

10. End repair

Prepare the end-repair reaction mix according to the following table:

10x Polynucleotide Kinase Buffer 5.5ul 25 mM deoxynucleotide triphosphates 3ul T4 DNA polymerase 3ul T4 polynucleotide kinase 3ul Klenow snippet 1ul Total volume 15.5ul

Add the prepared reaction mix to the DNA solution obtained in the previous step, mix thoroughly, and incubate at 20°C for 30 minutes. Complete the purification with 60 μl of Ampure XP magnetic beads. After drying, add 42 μl of TE buffer to dissolve the solution.

11. Add "A" at the end

Prepare the premix in advance as shown in the table below:

10xBlue buffer (Ezymatics) 5ul 10mM ATP 1ul Klenow (3`-5`exo-) enzyme 2ul Enzyme-free water 2ul Total volume 10ul

The DNA solution obtained in the above step was added with the reaction premix prepared above, mixed well, incubated at 37°C for 30 minutes, purified with 50ul Ampure xp magnetic beads, dried, and dissolved in 42ul TE buffer.

12. Connecting connector element 2

The sequence structure of the linker element 2 is shown in FIG2 .

Prepare the premix in advance as shown in the table below:

Add the premix to the DNA solution from the previous step and mix thoroughly. Then add 2 μl of T4 DNA ligase, mix again, and incubate at 20°C for 1 hour. Purify with Ampure XP magnetic beads and dissolve in 42 μl of TE.

13. PCR amplification

The sequence of primer 3 is: 5′-TCCTAAGCACGCTTGGCCT-3′, and the first base T at its 5′ end is biotin-labeled;

The sequence of primer 4 is: 5’-CATGTAGTGTACGATCCGACTT-3’, and the first base C at its 5’ end has a phosphorylation modification.

Prepare the reaction mix in advance as shown in the table below:

Add the premixed solution prepared above to the DNA solution obtained in the above step and mix thoroughly. Place in a PCR instrument to perform the PCR reaction. The PCR procedure is as follows:

After the reaction was completed, 100 μl of Ampure XP magnetic beads was used for purification and dissolved in 62 μl of TE.

14. Single-strand separation and circularization

Streptavidin-coated magnetic beads (MyOne ^™ Streptavidin, Lifetechnologies) were equilibrated at room temperature for half an hour in advance.

Pipette 30ul of the above magnetic beads into a new centrifuge tube, add 120ul of 1x magnetic bead binding buffer, mix thoroughly, place on a magnetic stand and let stand for 5 minutes. After the supernatant becomes clear, aspirate and remove the supernatant. Add 30ul of 1x magnetic bead binding buffer to resuspend the magnetic beads and set aside.

Dilute the PCR product generated in the previous step with 20ul 4x magnetic bead binding buffer, mix well, add to the resuspended magnetic beads, mix well, bind at room temperature for 5 minutes, then place on a magnetic stand and let it stand for 5 minutes. Wait until the supernatant becomes clear and then aspirate the supernatant.

Resuspend the beads in 1 ml of 1x Magnetic Wash Buffer and place on a magnetic rack for 5 minutes to remove the supernatant. Repeat this process. Add 78 μl of 0.1 M sodium hydroxide solution to the tube to resuspend the beads. Let stand for 5 minutes and then place on a magnetic rack. After 5 minutes, remove the supernatant and transfer it to a new centrifuge tube. Add 37.5 μl of MOPS buffer and mix thoroughly.

Prepare the cyclization reaction premix 1 in advance as shown in the following table (wherein the bridge sequence (also referred to as the mediating fragment) is as shown in SEQ ID NO: 9, i.e., 5'-GTACACTACATGTCCTAAGCACGC-3'):

Enzyme-free water 43ul Bridge Sequence 20ul Total 63ul

Add the premix prepared as above to the separated single-stranded DNA solution and mix well.

Prepare cyclization reaction premix 2 as shown in the following table:

Add reaction premix 2 to the single-stranded DNA solution, mix well, and incubate at 37°C for 1.5 hours.

15. Single-strand purification

Prepare the enzyme digestion reaction solution in advance as shown in the following table:

Add 20ul of enzyme digestion solution to the approximately 350ul sample reaction solution from the previous step, mix well, and continue incubating at 37°C for 30 minutes.

After the reaction was completed, 15.4 μl of 500 mM ethylenediaminetetraacetic acid was added to the reaction solution and mixed thoroughly.

Use 800ul Ampure XP magnetic beads to purify and recover, and after drying, add 40ul TE buffer to dissolve the circular DNA library.

The constructed library was detected on 6% urea denaturing PAGE gel. The results are shown in Figure 4.

The electrophoresis results in FIG4 show that the obtained single-stranded circular DNA fragments are concentrated, and the library is of very high quality, which proves that the technical method adopted by the present invention is completely feasible.

The applicant declares that the present invention uses the above-described embodiments to illustrate the detailed process equipment and process flow of the present invention. However, the present invention is not limited to the above-described detailed process equipment and process flow, and does not necessarily rely on the above-described detailed process equipment and process flow in order to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent replacements for the raw materials of the present invention's products, additions of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

SEQUENCE LISTING

<110> Shenzhen BGI Genomics Co., Ltd.

<120> Method for constructing a sequencing library using a bubble-shaped adapter element

<130> PIDC3171299PCN

<160> 9

<170> PatentIn version 3.3

<210> 1

<211> 57

<212> DNA

<213> Artificial sequence

<223> /molecule type="DNA"

/"/Phos/"="Terminal phosphate modification"

<400> 1

/Phos/actgctgagt cgagatgtca taaatctgac aaggtcgcca gccctgagtg cttcgaa 57

<210> 2

<211> 41

<212> DNA

<213> Artificial sequence

<400> 2

cgaagcactc aaggtcgcca gccctcagta cgtcagcagt t 41

<210> 3

<211> 39

<212> DNA

<213> Artificial sequence

<223> /molecule type="DNA"

/"/Phos/"="Terminal phosphate modification"

<400> 3

/Phos/agtcggaggc caagcgtgct taggatgagt gctctcgaa 39

<210> 4

<211> 33

<212> DNA

<213> Artificial sequence

<400> 4

cgagagcact ccatgtagtg tacgatccga ctt 33

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<211> 20

<212> DNA

<213> Artificial sequence

<400> 5

aggucgccag cccucagtac 20

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence

<400> 6

agggcuggcg accutgtcag 20

<210> 7

<211> 19

<212> DNA

<213> Artificial sequence

<400> 7

tcctaagcac gcttggcct 19

<210> 8

<211> 22

<212> DNA

<213> Artificial sequence

<400> 8

catgtagtgt acgatccgac tt 22

<210> 9

<211> 24

<212> DNA

<213> Artificial sequence

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gtacactaca tgtcctaagc acgc 24

Claims (35)

1. A method for constructing a sequencing library, characterized by comprising the following steps: 1) Fragment the double-stranded DNA and perform blunt-end repair, 5' end phosphorylation, and 3' end addition of base A on the resulting DNA fragments; 2) Through a ligation reaction, adapter elements 1 are added to both ends of the DNA fragment obtained in step 1); the adapter element 1 comprises a long nucleic acid chain and a short nucleic acid chain; the long nucleic acid chain and the short nucleic acid chain can be annealed to form a hybrid with complementary sequences at both ends and non-complementary sequences in the middle, forming a bubbly shape; the 5' end base of the long nucleic acid chain A of the hybrid is phosphorylated, and the 3' end of the short nucleic acid chain B of the hybrid has a protruding T base; the adapter element 1 has a class III restriction endonuclease recognition site; 3) Using the DNA fragment obtained in step 2) as a template, and using two different nucleic acid single strands that target partial sequences of the long and short nucleic acid chains of adapter element 1 as primers, PCR amplification is performed; The two primers have enzyme activity sites in the middle; 4) Using the enzyme's active site, sticky ends are created at both ends of the amplified fragment obtained in step 3), and the amplified fragment is ligated into a circular double strand of nucleic acid using the sticky ends; 5) Digest the circular double-stranded nucleic acid obtained in step 4) with a type III restriction endonuclease and recover the digested DNA fragments; 6) Perform blunt-end repair and add base A to the 3' end of the DNA fragment obtained in step 5). 7) By means of a ligation reaction, adapter elements 2 are added to both ends of the DNA fragment obtained in step 6); the adapter element 2 comprises a long nucleic acid chain and a short nucleic acid chain; the long nucleic acid chain and the short nucleic acid chain can be annealed to form a hybrid with complementary sequences at both ends and non-complementary sequences in the middle, forming a bubbly shape; the 5' end base of the long nucleic acid chain A of the hybrid is modified by phosphorylation, and the 3' end of the short nucleic acid chain B of the hybrid has a protruding T base; The sequence of connector element 2 is different from the sequence of connector element 1; 8) Using the DNA fragment obtained in step 7) as a template, PCR amplification is performed using two different single-stranded nucleic acid molecules that target partial sequences of the long and short nucleic acid chains of adapter element 2, respectively; one primer has a phosphorylated modification at the first base of its 5' end, and the other primer has a biotinylated label at the first base of its 5' end. 9) The PCR product obtained in step 8) was recovered using avidin magnetic beads and denatured to separate and recover the non-biotin-labeled single strands of nucleic acid; 10) Circularize the non-biotin-labeled single-stranded nucleic acid obtained in step 9) to form a single-stranded circular nucleic acid product, which is the sequencing library. 2. The construction method according to claim 1, wherein the avidin magnetic beads are streptavidin magnetic beads. 3. The construction method according to claim 1, wherein the denaturation treatment employs an alkaline denaturation method or a high-temperature denaturation method. 4. The construction method according to claim 1, characterized in that the circularization of the nucleic acid single strand is achieved by using a mediating fragment, wherein the mediating fragment has a corresponding complementary sequence for connecting the two ends of the nucleic acid single strand. 5. The construction method according to claim 1, wherein step 10) further includes a step of digesting linear single strands after the nucleic acid single-strand circularization is completed. 6. The construction method according to claim 5, characterized in that the digestion is performed using exonuclease 1 and/or 3. 7. The construction method according to claim 1, characterized in that, in step 1), the double-stranded DNA fragment is prepared by the following steps: 1-1) The mRNA sample is fragmented to obtain fragmented mRNA; 1-2) The fragmented mRNA is reverse transcribed to obtain cDNA amplification products as double-stranded DNA fragments. 8. The construction method according to claim 1, wherein the double-stranded DNA fragment is obtained directly from a DNA sample by fragmentation. 9. The construction method according to claim 8, wherein the fragmentation is performed by randomly breaking or cutting the DNA to be tested using physical or chemical methods. 10. The construction method according to claim 9, characterized in that the DNA to be tested is fragmented using physical sonication or enzymatic reaction. 11. The construction method according to claim 1, wherein the blunt-end repair is performed using T4 DNA polymerase. 12. The construction method according to claim 1, wherein the phosphorylation is performed using a nucleotide kinase. 13. The construction method according to claim 1, wherein the phosphorylation is performed using T4 polynucleotide kinase. 14. The construction method according to claim 1, wherein the addition of base A to the 3' end is performed using Klenow polymerase with 3'→5' exonuclease activity removed. 15. The construction method according to claim 1, wherein the class III restriction endonuclease in the adapter element 1 is Acu I, Bpm I, BceA I, Bbv I, BciVI, BpuE I, BseMII, BseR I, Bsg I, BsmF I, BtgZI, Eci I, EcoP15 I, Eco57M I, Fok I, Hga I, Hph I, Mbo II, Mnl I, SfaN I, TspDT I, TspDWI, or Taq II. 16. The construction method according to claim 1, wherein the recognition site of the class III restriction endonuclease is 0-2 bp away from the 3' end of the hybrid. 17. The construction method according to claim 1, wherein the nucleic acid long chain of the adapter element 1 contains a tag sequence. 18. The construction method according to claim 17, wherein the tag sequence length is 6-10 nt. 19. The construction method according to any one of claims 1-18, characterized in that the sequence of the nucleic acid long chain of the adapter element 1 is: 5’-/Phos/ACTGCTGAGTCGAGA(N)CTGACAAGGTCGCCAGCCCTGAGTGCTTCGAA-3’; where /Phos/ represents phosphorylation modification and N is the tag sequence. 20. The construction method according to claim 19, wherein the tag sequence length is 6nt-10nt. 21. The construction method according to claim 19, wherein the tag sequence is 5’-TGTCATAAAT-3’; Its short nucleic acid chain sequence is as follows: 5’-CGAAGCACTCA AGGTCGCCAGCCCTCAGTACGTCAGCAGTT-3’. 22. The construction method according to claim 1, characterized in that, in step 3), the primers used for PCR amplification are as follows: Forward primer: 5’-AGGUCGCCAGCCCUCAGTAC-3’; Reverse primer: 5’-AGGGCUGGCGACCUTGTCAG-3’. 23. The construction method according to claim 1, wherein the sequence of the nucleic acid long chain of the adapter element 2 is: 5’-/Phos/AGTCGGAGGCCAAGCGTGCTTAGGATGAGTGCTCTCGAA-3’, where /Phos/ indicates phosphorylation modification; The sequence of the short nucleic acid chain is as follows: 5’–CGAGAGCACTCCATGTAGTGTACGATCCGACTT-3’. 24. The construction method according to claim 1, characterized in that, in step 8), the primers used for PCR amplification are as follows: Forward primer: 5’-TCCTAAGCACGCTTGGCCT-3’; Reverse primer: 5’-CATGTAGTGTACGATCCGACTT-3’; The first base at the 5' end of the forward primer is labeled with biotin, and the first base at the 5' end of the reverse primer is phosphorylated. 25. The construction method according to claim 4, wherein in step 10), the sequence of the mediating fragment is: 5’-GTACACTACATGTCCTAAGCACGC-3’. 26. The construction method according to claim 1, wherein the enzyme activity site in the middle of the two primers is U or dU, and the corresponding enzyme is USER enzyme. 27. A sequencing library, characterized in that it is prepared by the construction method according to any one of claims 1-26. 28. The application of the sequencing library as described in claim 27 in genome sequencing. 29. The application according to claim 28, wherein the genome sequencing is target genome region sequencing. 30. The application according to claim 28, characterized in that sequencing is performed using a single-stranded circular library sequencing platform. 31. The application according to claim 28, characterized in that sequencing is performed using a sequencing platform from Complete Genomics. 32. A nucleic acid sequencing method, characterized in that it includes the step of sequencing the sequencing library as described in claim 27. 33. The nucleic acid sequencing method according to claim 32, characterized in that a single-stranded circular library sequencing platform is used for sequencing. 34. The nucleic acid sequencing method according to claim 32, characterized in that sequencing is performed using the sequencing platform of Complete Genomics. 35. The nucleic acid sequencing method according to claim 32, characterized in that it further includes the step of assembling and/or splicing the sequencing results.