WO2016078095A1 - 鼓泡状接头元件和使用其构建测序文库的方法 - Google Patents

鼓泡状接头元件和使用其构建测序文库的方法 Download PDF

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WO2016078095A1
WO2016078095A1 PCT/CN2014/091952 CN2014091952W WO2016078095A1 WO 2016078095 A1 WO2016078095 A1 WO 2016078095A1 CN 2014091952 W CN2014091952 W CN 2014091952W WO 2016078095 A1 WO2016078095 A1 WO 2016078095A1
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nucleic acid
fragment
sequence
sequencing
acid fragment
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PCT/CN2014/091952
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English (en)
French (fr)
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江媛
赵霞
李巧玲
刘生茂
王博
陈利
章文蔚
蒋慧
徳马纳克拉多杰
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深圳华大基因科技有限公司
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Priority to US15/528,362 priority Critical patent/US10954559B2/en
Priority to PCT/CN2014/091952 priority patent/WO2016078095A1/zh
Priority to CN201480083528.8A priority patent/CN107124888B/zh
Publication of WO2016078095A1 publication Critical patent/WO2016078095A1/zh
Priority to US17/172,927 priority patent/US11827933B2/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • C12Q1/6855—Ligating adaptors

Definitions

  • the present invention relates to the field of biotechnology, and in particular to a bubble-like connector element, a method for constructing a sequencing library using the connector element, a sequenced library constructed, and an application thereof.
  • the Blackbird sequencing platform of Complete Genomics (hereinafter referred to as CG) has higher sequencing accuracy than other platforms (99.9998%), and the sequencing flux has greater advantages over other platforms.
  • the application of research fields has great prospects.
  • the library construction cycle of the CG platform is too long, which seriously hinders the progress of scientific research and projects, and the challenges that various next-generation sequencing technologies are rapidly emerging.
  • the technology optimization optimizes the database construction process and shortens the database construction time. In order to shorten the operating cycle of the CG platform and reduce production costs, it is an urgent and important research and development task.
  • a linker is a specially designed DNA sequence that is ligated to both ends of a DNA fragment by ligation, etc., and can be identified during sequencing and used as a starting site for sequencing, for the instrument to read subsequent sequence information. To ensure that the sequence information read is easy to analyze, two different linker sequences need to be added at both ends (5' and 3') of a DNA fragment.
  • the classic database construction method of CG platform needs to introduce 4 sequencing joints into the sequencing library, which is cumbersome and has a long cycle.
  • CG's dual-join construction method simplifies the number of joints to be added, and the construction period can be shortened by about half.
  • the database construction process mainly includes: genomic DNA interruption, first joint connection and PCR (Polymerase Chain Reaction, Polymerase chain reaction), double-stranded cyclization and restriction enzyme digestion, second joint ligation PCR, single-strand separation and cyclization.
  • the method of adding two joints although cleverly designed by sequence, can minimize the interconnection between DNA fragments while ensuring the directional connection of the joints, but each joint connection requires 6 steps of enzyme reaction to complete.
  • the method of constructing the linker of the library is still too cumbersome, which is reflected in many steps of the enzyme reaction and many purification steps.
  • the disadvantages are low efficiency, high cost and low sample recovery rate. Due to the low sample recovery rate, the database construction process requires two PCR amplifications to meet the total volume of the library, which further increases the cost of building the library.
  • two enrichment screenings are required, which further reduces the efficiency of database construction. Therefore, although the four-joint construction method has been simplified into a two-join construction method, the problem of long process construction, high cost, and high initial demand is still not solved better.
  • the present invention provides a bubble-like joint element, a method of constructing a sequencing library using the same, a sequencing library constructed and an application thereof.
  • the invention focuses on solving the excessive number of joint connection steps and the number of PCR amplifications in the double-link construction method of the CG sequencing platform through the innovative bubbling joint element and its connection reaction technology in solution and on the magnetic beads.
  • the overall library construction time is long, the cost is high, and the initial amount of construction is required.
  • the entire construction cycle and cost are reduced by about half, and the initial amount of database construction can be reduced from 3ug to 1ug.
  • the present invention provides a linker element which is a hybrid formed by a long chain A of a nucleic acid and a short chain B of a nucleic acid, wherein the two ends are complementary in sequence, and the intermediate sequences are not complementary, and are bubbling.
  • the 5' terminal base of the nucleic acid long chain A of the hybrid is phosphorylated, and the 3' end of the nucleic acid short chain B of the hybrid has a prominent base T;
  • the hybrid has a class III restriction endonuclease recognition site; preferably, the recognition site for the class III restriction endonuclease is 0-2 bp from the linker element-target DNA junction.
  • the hybrid is divided into the following three parts according to a configuration:
  • Composition 1 a nucleic acid fragment A1 comprising a 3' end of a long chain A and a 5' end nucleic acid fragment B1 of the short chain B, the nucleic acid fragment A1 partially hybridizing with the nucleic acid fragment B1 to form a complementary double-stranded portion and located in the long a free portion of the 3' end of the strand A;
  • Composition 2 a nucleic acid fragment A2 comprising a middle segment of a long chain A and a nucleic acid fragment B2 of a short chain B; the nucleic acid fragment B2 is not complementary to the nucleic acid fragment A2, thereby forming a bubble structure;
  • Composition 3 a nucleic acid fragment A3 comprising a 5' end of the long chain A and a nucleic acid fragment B3 at the 3' end of the short chain B; a 5' end of the nucleic acid fragment A3 is phosphorylated, and a 3' end of the nucleic acid fragment B3 is a base T; Except for the base T at the 3' end, all bases of the nucleic acid fragment B3 are complementary paired with the base of the nucleic acid fragment A3, thereby forming a complementary double strand of the short chain 3' end protruding by 1 base T;
  • composition 3 a recognition sequence having a class III restriction endonuclease is optionally included.
  • the nucleic acid fragment A1 has a length of 10-20 nt
  • the nucleic acid fragment B1 has a length of 8-16 nt
  • the nucleic acid fragment A2 has a length of 11-36 nt
  • the nucleic acid fragment B2 has a length of 11- 21 nt
  • the nucleic acid fragment A3 is 6-8 nt in length
  • the nucleic acid fragment B3 is 7-9 nt in length.
  • the nucleic acid fragment B1 has an enzyme action site; more preferably, the enzyme action site is U or dU, and the corresponding enzyme is USER enzyme;
  • the nucleic acid fragment A2 contains a tag sequence.
  • the class III restriction enzymes are Acu I, Bpm I, BceA I, Bbv I, BciV I, BpuE I, BseM II, BseR I, Bsg I, BsmF I, BtgZ I, Eci I, EcoP15 I, Eco57M I, Fok I, Hga I, Hph I, Mbo II, Mnl I, SfaN I, TspDT I, TspDW I or Taq II.
  • sequence of the nucleic acid long chain A of the linker element described above is:
  • N indicates a tag sequence
  • /ddC/ indicates dideoxycytidine
  • the tag sequence length Is 6 nt to 10 nt; more preferably, the tag sequence is 5'-TGTCATAAAT-3'; that is, in a more preferred embodiment, the sequence of the nucleic acid long chain A of the linker element is:
  • sequence of nucleic acid short chain B (SEQ ID NO: 2) is:
  • sequence of the nucleic acid long chain A of the linker element is:
  • sequence of nucleic acid short chain B (SEQ ID NO: 4) is:
  • the present invention provides a linker attachment method for ligating a linker element as described in the first aspect to both ends of a DNA fragment to be tested.
  • the linker element is added to both ends of the DNA fragment to be tested by a ligation reaction
  • the step of performing blunt end repair, 5' end phosphorylation and 3' end plus base A of the DNA fragment to be tested is further included.
  • a blunt-end repair is performed using a DNA polymerase, preferably a T4 DNA polymerase; for example, a 5'-end phosphorylation using a nucleotide kinase, preferably a T4 polynucleotide kinase; for example, removal is used
  • a 3' ⁇ 5' exonuclease activity polymerase, such as Klenow exo-, performs the step of adding the base A at the 3' end.
  • the present invention provides a method of constructing a sequencing library using a linker element as described in the first aspect or a linker connection using the linker method as described in the second aspect.
  • the method for constructing the above sequencing library specifically comprises the following steps:
  • the double-stranded DNA fragment is prepared by the following steps:
  • the double-stranded DNA fragment is directly obtained by fragmenting a DNA sample
  • the fragmentation is to randomly interrupt or cut the sample by physical or chemical methods; further preferably, the fragmentation is performed by physical ultrasonic method or enzymatic reaction method;
  • the blunt end repair is performed using T4 DNA polymerase
  • said phosphorylation is carried out using a nucleotide kinase, preferably a T4 polynucleotide kinase;
  • the 3' terminal plus base A is carried out by Klenow polymerase which removes 3' ⁇ 5' exonuclease activity;
  • step 2) by the ligation reaction, the first step of the DNA fragment obtained in step 1) is added to the linker 1;
  • sequence of the nucleic acid long chain A of the linker element 1 is:
  • N indicates a tag sequence
  • /ddC/ indicates dideoxycytidine
  • the tag sequence length is More preferably, the tag sequence is 5'-TGTCATAAAT-3'; that is, in a more preferred embodiment, the sequence of the nucleic acid long chain A of the linker element is:
  • sequence of its short chain B of nucleic acid is:
  • the two primers have an enzyme action site, and a biotin label is introduced into one of the primers;
  • the enzyme action site is U or dU, and the corresponding enzyme is USER enzyme;
  • the primer pair here is:
  • the primer 1 sequence (SEQ ID NO: 5) is as follows:
  • the primer 2 sequence (SEQ ID NO: 6) is as follows:
  • step 4 using the enzyme action site, making a sticky end at both ends of the amplified fragment obtained in step 3), and using the sticky end, the amplified fragment is ligated into a circular nucleic acid double strand;
  • step 5) digesting the circular nucleic acid double strand obtained in step 4) with a class III restriction endonuclease, and then capturing the digested DNA fragment with avidin magnetic beads;
  • step 4) first capture the circular nucleic acid duplex obtained in step 4) with avidin magnetic beads, and then digest the captured circular nucleic acid duplex with a class III restriction endonuclease;
  • the avidin magnetic bead is a streptavidin magnetic bead
  • the sequence of the linker element 2 is different from the sequence of the linker element 1, and the nucleic acid fragment B1 of the linker element 2 contains an enzyme action site; preferably, the enzyme action site is U or dU, and the corresponding enzyme is USER enzyme;
  • the sequence of the nucleic acid long chain A of the linker element 2 for:
  • sequence of its short chain B of nucleic acid is:
  • nucleic acid fragment B1 of the linker element 2 on the DNA fragment obtained in the step 7) is cleaved while phosphorylating the 5' end of the linker element excising the nucleic acid fragment B1;
  • step 9) subjecting the DNA fragment obtained in the step 8) to a denaturation treatment, separating and recovering the non-biotin-labeled nucleic acid single strand; preferably, using an alkali denaturation method or a high temperature denaturation method;
  • the cyclization of the single strand of the nucleic acid is achieved by using a mediated fragment having a corresponding complementary sequence for joining the two ends of the nucleic acid single strand;
  • the sequence of the mediated fragment is: 5'-ATCGTACACTACATGTCCTAAGCA-3', see SEQ ID NO:7;
  • the method further comprises the step of digesting the linear single strand after completion of the single-chain cyclization of the nucleic acid; further preferably, digesting with the exonuclease 1 and/or 3.
  • Step 10 The obtained single-stranded circular nucleic acid product can directly enter a subsequent sequencing step, and after the rolling circle is replicated, a nucleic acid nanosphere (DNB) is formed to perform nucleic acid sequence information reading.
  • NDB nucleic acid nanosphere
  • the invention provides a sequencing library produced by the sequencing library construction method of the third aspect.
  • the prepared sequencing library is a single-stranded circular sequencing library.
  • the present invention provides the use of the sequencing library according to the fourth aspect in genome sequencing, Site selection, application in sequencing of target genomic regions.
  • sequencing is performed using a single-stranded circular library sequencing platform; further preferably, sequencing is performed using a sequencing platform of Complete Genomics.
  • the present invention provides a nucleic acid sequencing method comprising the step of sequencing a sequencing library as described in the fourth aspect;
  • sequencing is performed using a single-stranded circular library sequencing platform; further preferably, sequencing is performed using a sequencing platform of Complete Genomics;
  • the step of assembling and/or splicing the sequencing results is also included.
  • the present invention provides a sequencing library construction kit comprising the linker element of the first aspect.
  • the kit further comprises a nucleotide kinase, preferably a T4 polynucleotide kinase; a DNA polymerase, preferably a T4 DNA polymerase or a polymerase having 3' ⁇ 5' exonuclease activity removed, such as Klenow Exo-; USER enzyme; and class III restriction enzymes, such as Acu I, Bpm I, BceA I, Bbv I, BciV I, BpuE I, BseM II, BseR I, Bsg I, BsmF I, BtgZ I, Eci I, EcoP15 I, Eco57M I, Fok I, Hga I, Hph I, Mbo II, Mnl I, SfaN I, TspDT I, TspDW I or Taq II.
  • a nucleotide kinase preferably a T4 polynucleotide kinase
  • a DNA polymerase
  • the invention adopts a novel bubbling joint element, a simple connection method, and utilizes a method for performing an enzymatic reaction on a magnetic bead.
  • the directional joint connection method adopted by the prior art method can minimize the interconnection between DNA fragments while ensuring the directional connection of the joints, and adopts a method of separately designing and connecting the 5' joint and the 3' joint.
  • Each end of the linker requires a linker sequence, a blocking sequence, and a primer sequence to complete the process.
  • the entire process requires 6 steps of enzyme reaction and 5 steps of purification. The steps are cumbersome and the cost of the library is established (sequence cost, enzyme reagent cost, purification). High cost, long cycle, and large sample loss.
  • the bubbling joint component and the connecting method thereof used in the invention can improve the efficiency of building the library and reduce the construction cost and the starting amount under the premise of ensuring the directional connection of the joint.
  • the 3'T protruding bubbling linker element can be efficiently directionally linked to the 3'A overhanging target DNA.
  • the method adopted by the invention realizes one-step connection of the joint, and only 3-4 steps of enzyme reaction are required to complete the whole process, the 3-step purification operation is simple, the cost of building the library is reduced, and the cycle is shortened.
  • the reduction in the number of purifications reduces the sample loss, and the initial amount of the library can be reduced from the original 3 ⁇ g to 1 ⁇ g.
  • FIG. 3 A comparison of the conventional directional joint joining method with the foaming joint member joining method of the present invention is shown in FIG. It can be seen from Fig. 3 that the directional joint connection method requires dephosphorylation, terminal repair, addition of 5' linker, primer extension, addition of 3' linker, nick translation and ligation of the 6-step enzymatic reaction and 5 purification operations.
  • the sequence orientation of A is added to both ends of the DNA of interest, and the method of ligation of the bubbling linker of the present invention only requires a one-step reaction of phosphorylation and end repair, addition of A, addition of bubbling linker, and USER digestion (may be omitted).
  • the 4-step (or 3-step) enzymatic reaction and three purification operations can be used to rapidly add the sequence of linker A to both ends of the DNA of interest.
  • the traditional enzymatic reaction method in solution the DNA and the reaction solution are mixed together during the enzymatic reaction, and after the reaction is completed, the enzyme and other impurities need to be removed, and thus it is required to be purified by a silicon column (such as Qiagen PCR Purification Kit). Or the purification of DNA by ordinary magnetic bead purification (such as Agencourt AMPure XP beads). These purification methods not only cost high reagents, but also have a long purification operation, and the DNA sample will have a loss of 10-30% after each purification.
  • the invention uniquely adopts a method for performing an enzymatic reaction on a magnetic bead.
  • the target DNA is always bound to the avidin magnetic bead, after the enzyme reaction is completed, only the magnetic bead is used to adsorb and fix the magnetic bead, and then the reaction is discarded. The supernatant is cleaned and the residual impurities on the magnetic beads are washed away with a suitable buffer to purify the DNA. Therefore, the method not only saves the cost of the purification reagent, reduces the loss of the DNA sample (only about 1% loss), but also facilitates the realization of the automation operation and the improvement of the work efficiency.
  • FIG. Clear description A comparison of the method of performing an enzymatic reaction in a solution and performing an enzymatic reaction on a magnetic bead is carried out in FIG. Clear description;
  • Figure 4 shows the approximate steps of the enzymatic reaction from class III endonuclease cleavage to single-strand separation. Specifically, Figure 4 shows that after the class III endonuclease is cleaved, the enzymatic reaction in the solution requires two-step magnetic bead purification to select a fragment that matches the size of the DNA of interest, but the fragment selection method also introduces a partial fragment.
  • the size of the non-target DNA cleavage fragment; and the enzymatic reaction on the magnetic beads can be accurately enriched to the desired DNA cleavage fragment by the specific binding of the labeled biotin on the target DNA fragment A linker to the streptavidin magnetic beads.
  • the existing method requires 6 enzyme reactions and 6 magnetic beads purification to complete; the method used in the present invention can be completed by only 2 enzyme reactions and 2 magnetic beads washing.
  • the existing method of adding the linker element 2 requires PCR amplification to increase the yield of the target DNA, and then performing single-strand separation and one-time specific single-strand capture reaction to obtain the target single-stranded DNA; After completion of the adaptor element 2, PCR amplification is not required, and the non-target linker sequence is cleaved by USER to form a 5' phosphorylated end, and the target single-stranded DNA can be eluted by denaturation.
  • the invention adopts an enzymatic reaction method on the magnetic beads, so that the loss of the DNA sample is greatly reduced, and after the addition of the connector element 2, a sufficient amount of the machine can be obtained without PCR-free.
  • the library not only eliminates the one-step PCR amplification reaction, but also simplifies the single-stranding step of screening, and greatly simplifies the process of building the linker B to the single-strand separation.
  • the library capacity can be improved, the number of repeated read lengths in the library can be reduced, and the sequencing data of the library can be improved.
  • the present invention successfully improves and optimizes the CG double-join library construction process through novel bubbling joint elements, as well as techniques for performing linker ligation reactions in solution and on magnetic beads, with major improvements.
  • the first joint connection is simplified by 6 steps of enzyme reaction, 5 times of magnetic bead purification to 4 steps of enzyme reaction, 3 steps of purification
  • the second joint connection is simplified by 6 times of enzyme reaction and 6 times of magnetic bead purification to 2 times of enzyme. Reaction, 2 times of magnetic bead washing, while eliminating the second PCR amplification and a single-strand enrichment screening step, reducing the entire construction cycle and cost by about half, and the initial amount of database construction can be reduced from 3ug to 1ug. And it is conducive to the realization of high-throughput database automation and library sequencing data efficient.
  • Figure 1 illustrates the sequencing library construction scheme of the present invention: 1 is a fragmented DNA fragment; 2 is a fragment after blunt-end repair, phosphorylation; 3 is a DNA fragment having a 3' end plus a base A; The DNA fragment after the addition of the adaptor element 1; 5 is the non-specific linker sequence (ie, nucleic acid fragment B1) degraded by USER, this step can be omitted; 6 is the DNA fragment of the linker element 1 which is expanded by PCR; a double-stranded DNA cyclized after the formation of a sticky end, wherein the biotin-labeled circular double-strand can be bound to streptavidin magnetic beads; 8 is an end-projection 1-2 formed by cleavage with a class III endonuclease a DNA fragment of a base (N represents any one of A, T, C, and G); 9 is a DNA fragment that has undergone terminal repair, and the A' end is added with A; 10 is a linker element 2 and then subjecte
  • the treated DNA fragment; 11 is a single-stranded DNA eluted from streptavidin magnetic beads; 12 is the final product of the library construction, ie, single-stranded circularized DNA.
  • the biotin marker in the figure indicates biotin labeling.
  • Figure 2 illustrates the step of ligation of the linker element to PCR amplification in the construction of the sequencing library of the present invention
  • 1 shows the 3'T overhanging linker element (composed of compositions 1, 2, 3) and 3'A highlighted DNA
  • the ligation of the fragment 2 shows that the non-target linker sequence on the forward strand is composed by digestion of the linker element with USER, and this step can be omitted; 3 shows that the primer is matched with the forward and reverse strands of the target linker sequence.
  • a DNA fragment carrying the sequence of the linker of interest is amplified.
  • Figure 3 is a comparative illustration of a conventional directional joint joining method and a method of joining the bubble joint member of the present invention.
  • Figure 4 is a comparative representation of the enzymatic reaction in solution and enzymatic reaction on magnetic beads showing the approximate steps of the enzymatic reaction from cleavage of class III endonuclease to single-strand separation.
  • Figure 5 is a result of electrophoresis of a DNA fragment after addition of the adaptor element 1 in the embodiment of the present invention
  • M is D2000 (Tiangen);
  • C is the product of step 2, that is, the DNA fragment before the adaptor element 1;
  • 1L, 2L are steps 5 product 1, 2, that is, the DNA fragment after the addition of the linker element 1;
  • 1P, 2P is the PCR amplification product of the DNA fragment of the step 6, product 1, 2, that is, the linker element 1.
  • M1 is 50 bp (Tiangen); M2 is D2000 (Tiangen); 1-7 is the product of step 13, that is, the joint element 2 is added. PCR amplification of the resulting DNA fragment 1-7.
  • Figure 7 is the result of electrophoresis of the final product;
  • M is RiboRuler High Range RNA Ladder, ready-to-use (Fermentas); 1-7 is the product 1-7 of step 16, ie, single-stranded cyclized DNA product 1-7.
  • the interrupt condition is set as follows:
  • Magnetic bead purification or gel recovery can be employed.
  • This embodiment adopts a magnetic bead purification method, which is specifically as follows:
  • Enzyme-free pure water 9.33ul 10X NEBNext End Repair Buffer 7.00ul T4 deoxyribonucleic acid polymerase (3U/ul) 1.87ul T4 Polynucleotide Kinase (10U/ul) 1.40ul Bovine serum albumin (20mg/ml) 0.40ul Total 20.00ul
  • reaction solution 20 ul of the reaction solution was added to the recovered product of the previous step, mixed, and incubated at 20 ° C for 30 min. After the reaction, purification was carried out with 70 ul of Ampure XP magnetic beads, and 40 ul of TE buffer was dissolved to recover the product.
  • purifying the reaction product such as magnetic bead method, column purification method, gel recovery method, etc. Both can be used for replacement. This embodiment is purified by magnetic beads method unless otherwise specified.
  • the system was mixed and added to the product of the previous step, mixed and incubated at 37 ° C for 30 min. With 60ul Ampure XP beads were purified and 40 ul of TE buffer solution was used to dissolve the recovered product.
  • the sequence of the linker element 1 used in the present scheme is as follows (the sequence in the present embodiment is from the left end to the 5' end to the 3' end, "//” indicates a modifying group, “phos” indicates phosphorylation, and “dd” indicates Dideoxy, "bio” shows biotin, font bolded label sequence):
  • the joint element 1 mixture (25 uM) was formulated as follows:
  • the ligation reaction system was formulated as follows:
  • Tris-hydroxymethane-hydrochloric acid (pH 7.8) 150mM Polyethylene glycol 8000 15% Magnesium chloride 30mM Ribonucleoside triphosphate 3mM
  • reaction mixture was mixed with a mixture of the linker and the product, and incubated at 20 ° C for 30 min. After the reaction was completed, 35 ul of disodium edetate (35 uM) was added and mixed. Purification was carried out with 50 ul of Ampure XP magnetic beads and the recovered product was dissolved in 52 ul of TE buffer.
  • This step completes the attachment of the nucleic acid fragment of interest to the linker element 1.
  • the results of electrophoresis of the PCR amplification products before and after the ligation and after ligation are shown in Fig. 5.
  • the primer 1 sequence (SEQ ID NO: 5) is as follows:
  • the primer 2 sequence (SEQ ID NO: 6) is as follows:
  • the above reaction solution was added to 37 ul (2.4 ug) of the reaction product, and the mixture was incubated at 37 ° C for 1 h.
  • reaction product of the previous step was added to the reaction system 1 and placed in a water bath at 60 ° C for 30 minutes. After the reaction was completed, it was placed in a normal temperature water bath for 20 minutes.
  • the reaction system 2 was added to the above reaction system, and incubated at room temperature for 1 hour.
  • the product of the above step was added to the reaction system, and after mixing, it was incubated at 37 ° C for 1 h.
  • the product of the above step was added to the reaction system, mixed and incubated at 37 ° C for 16 h.
  • the reaction system was added to the above-mentioned magnetic bead resuspension, mixed and incubated at 37 ° C for 1 h.
  • the joint element 2 mixture (10 uM) was formulated as follows:
  • the ligation reaction system was formulated as follows:
  • the ligation reaction system was added to the magnetic bead suspension, mixed, and allowed to stand at room temperature for 30 min. After the reaction, 3.42 ul of disodium edetate (0.5 M) was added, mixed, and allowed to stand for 1 min, and the supernatant was discarded. Wash twice with 1x low salt wash buffer (containing 0.05% Tween 20) and resuspend the beads with 80 ul of 1x low salt wash buffer.
  • This step completes the attachment of the nucleic acid fragment of interest to the linker element 2.
  • the effect of the connection can be obtained by eluting 3 ul of single-stranded nucleic acid in the next step, and after 8 cycles of amplification, 5 uL of electrophoresis is detected, and the electrophoresis results are shown in FIG. 6 .
  • reaction system 1 was prepared in which the mediated fragment has the corresponding complementary sequence for ligating the two ends of the single strand, the sequence of which is: ATCGTACACTACATGTCCTAAGCA (ie SEQ ID NO: 7).
  • the reaction system 1 was added to the single-chain separation product of the above step, and mixed.
  • the reaction system 2 was added to the reaction system 1, mixed, and incubated at 37 ° C for 1.5 h.
  • reaction buffer 20 ul of reaction buffer to 350.5 ul of the reaction product from the previous step, mix and incubate at 37 ° C. Min.
  • the present invention illustrates the detailed process equipment and process flow of the present invention by the above embodiments, but the present invention is not limited to the above detailed process equipment and process flow, that is, does not mean that the present invention must rely on the above detailed process equipment and The process can only be implemented. It should be apparent to those skilled in the art that any modifications of the present invention, equivalent substitution of the various materials of the products of the present invention, addition of auxiliary components, selection of specific means, and the like, are all within the scope of the present invention.

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Abstract

本发明提供了一种鼓泡状接头元件和使用其构建测序文库的方法。所述接头元件是由一条核酸长链A和一条核酸短链B形成的两端序列互补、中间序列不互补而呈鼓泡状的杂交体。

Description

鼓泡状接头元件和使用其构建测序文库的方法 技术领域
本发明涉及生物技术领域，具体地，涉及一种鼓泡状接头元件，使用该接头元件构建测序文库的方法，所构建的测序文库及其应用。
背景技术
上世纪九十年代，自从AB公司推出毛细管电泳测序仪，人类基因组计划启动之后，DNA(Deoxyribonucleic acid，脱氧核糖核酸)测序技术便开始以难以想象的速度发生翻天覆地的变化。第二、第三代测序仪也相继推出，面向市场。
在第二代测序平台中，Complete Genomics公司(以下简称CG)的Blackbird测序平台凭借测序准确性相对其他平台更高(99.9998％)、测序通量相对其他平台更大的优势，在分子诊断等临床研究领域的应用具有巨大前景。然而，CG平台的文库构建周期太长，严重阻滞了科研、项目的开展进度，加上各种新一代测序技术迅速兴起所面临的挑战，通过技术优化来简化建库流程、缩短建库时间，以缩短CG平台的运行周期、降低生产成本是迫在眉睫的重要研发任务。
接头是一段特殊设计的DNA序列，通过连接等方法固定在DNA片段两端后，在测序时能被识别并作为测序的起始位点，供仪器读取其后的序列信息。为保证读取的序列信息易于分析，在一个DNA片段的两端(5’端和3’端)需要加上两种不同的接头序列。
CG平台经典的建库方法需要在测序文库中引入4个测序接头，建库流程繁琐，周期很长。通过改进，CG推出的双接头建库法简化了接头加入个数，建库周期可缩短约一半时间，建库流程主要包括：基因组DNA打断、第一次接头连接及PCR(Polymerase Chain Reaction，聚合酶联反应)、双链环化并酶切、第二次接头连接及 PCR、单链分离环化。其中两次加接头的方法虽然通过巧妙地序列设计，可以在保证接头定向连接的同时，最大程度地降低DNA片段间相互连接问题，但每次接头连接都需要6步酶反应才能完成。相对其他测序平台的文库构建接头连接方法还是过于繁琐，体现在酶反应步骤多、纯化步骤多，这样带来弊端就是建库效率低、成本高、样品回收率低。由于样品回收率低，建库过程需要做两次PCR扩增才能满足上机文库的总量需求，这样又进一步提高了建库成本。另外，在单链分离环节，为了富集到特异的单链DNA，需要进行两次富集筛选，也进一步降低了建库效率。因此，尽管将四接头建库法简化成了两接头建库法，建库流程长、成本高、起始量要求高的问题仍然没有得到更好地解决。
为解决Complete Genomics公司测序平台文库构建中存在的接头连接步骤过多，PCR扩增次数多、整体文库构建时间长、成本高、建库起始量要求高的问题，特提出了本发明。
发明内容
针对上述现有技术的不足，本发明的目的在于提供一种鼓泡状接头元件，使用该接头元件构建测序文库的方法，所构建的测序文库及其应用。本发明通过创新的鼓泡状接头元件及其在溶液中和在磁珠上进行的连接反应技术，着重解决了CG测序平台双接头建库法中存在的接头连接步骤过多、PCR扩增次数多、整体文库构建时间长、成本高、建库起始量要求高的问题。与现有方法相比，整个建库周期和成本降低了约一半，建库起始量从3ug可降低至1ug。
第一方面，本发明提供了一种接头元件，该接头元件是由一条核酸长链A和一条核酸短链B形成的两端序列互补、中间序列不互补而呈鼓泡状的杂交体，所述杂交体的核酸长链A的5’末端碱基经磷酸化修饰，所述杂交体的核酸短链B的3’末端带有突出的碱基T；
任选地，所述杂交体中具有III类限制性内切酶识别位点；优选地，所述III类限制性内切酶的识别位点与接头元件-目标DNA连接处相距0-2bp。
关于上述接头元件，作为优选，所述杂交体按构型分为如下三个部分：
组成1：包括长链A的3’端的核酸片段A1与短链B的5’端的核酸片段B1，所述核酸片段A1与所述核酸片段B1部分互补杂交，形成互补的双链部分和位于长链A 3’末端的游离部分；
组成2：包括长链A中段的核酸片段A2与短链B中段的核酸片段B2；所述核酸片段B2与核酸片段A2不互补，从而形成鼓泡结构；
组成3：包括长链A的5’端的核酸片段A3与短链B的3’端的核酸片段B3；核酸片段A3的5’末端为磷酸化修饰，核酸片段B3的3’末端为碱基T；除了3’末端的碱基T外，核酸片段B3的所有碱基均与核酸片段A3的碱基互补配对，从而形成短链3’末端突出1个碱基T的互补双链；
其中，任选地，所述组成3中具有III类限制性内切酶的识别序列。
进一步优选地，所述核酸片段A1的长度为10-20nt，所述核酸片段B1的长度为8-16nt；所述核酸片段A2的长度为11-36nt，所述核酸片段B2的长度为11-21nt；所述核酸片段A3的长度为6-8nt，所述核酸片段B3的长度为7-9nt。
进一步优选地，所述核酸片段B1中具有酶作用位点；更进一步优选地，所述酶作用位点为U或dU，对应的酶为USER酶；
进一步优选地，所述核酸片段A2中含有标签序列。
进一步优选地，所述III类限制性内切酶为Acu I、Bpm I、BceA I、Bbv I、BciV I、BpuE I、BseM II、BseR I、Bsg I、BsmF I、BtgZ I、Eci I、EcoP15 I、Eco57M I、Fok I、Hga I、Hph I、Mbo II、Mnl I、SfaN I、TspDT I、TspDW I或Taq II。
在一个优选的具体实施方案中，上述接头元件的核酸长链A的序列为：
5’-/Phos/CTGCTGACGTACTG(N)AGCACGAGACGTTCTCGACT/ddC/-3’；其中，/Phos/表示磷酸化修饰，N表示标签序列，/ddC/表示双脱氧胞苷；优选地，所述标签序列长度为6nt-10nt；更优选地，所述标签序列为5’-TGTCATAAAT-3’；即，在一个更优选的具体实施方案中，所述接头元件的核酸长链A的序列为：
5’-/Phos/CTGCTGACGTACTGTGTCATAAATAGCACGAGACGTTCTCGACT/ddC/-3’(见SEQ ID NO：1)；
其核酸短链B的序列(SEQ ID NO：2)为：
5’-GAGAACGUCTCGTGCUACGTTCTCGACTCAGCAGT-3’。
在另一个优选的具体实施方案中，上述接头元件的核酸长链A的序列(SEQ ID NO：3)为：
5’-/Phos/AGTCGGAGGCCAAGCGTGCTTAGGACAT-3’，其中/Phos/表示磷酸化修饰；
其核酸短链B的序列(SEQ ID NO：4)为：
5’-GTCCTAAGCACUGTAGTGTACGATCCGACTT-3’。
第二方面，本发明提供了一种接头连接方法，其为将如第一方面所述的接头元件连接在待测DNA片段两端。
在具体实施方案中，通过连接反应，将所述接头元件加在待测DNA片段的两端；
优选地，在所述接头元件连接前，还包括将待测DNA片段进行平端修复、5’末端磷酸化和3’末端加碱基A的步骤。
在具体实施方案中，例如，使用DNA聚合酶、优选T4 DNA聚合酶进行平端修复；例如，使用核苷酸激酶、优选T4多聚核苷酸激酶进行5’末端磷酸化；例如，使用去除了3’→5’外切酶活性的聚合酶，例如Klenow exo-，进行3’末端加碱基A的步骤。
第三方面，本发明提供了一种测序文库的构建方法，其使用如第一方面所述的接头元件或使用如第二方面所述的接头连接方法进行接头连接。
作为优选，上述测序文库的构建方法具体包括以下步骤：
1)将双链DNA片段进行平端修复、5’末端磷酸化和3’末端加碱基A；
优选地，所述双链DNA片段是通过如下步骤制备的：
1-1)对mRNA样本进行片段化处理，从而获得片段化的mRNA；
1-2)对所述片段化的mRNA进行反转录，从而获得cDNA扩增产物，作为双链DNA片段；
任选地，所述双链DNA片段直接由DNA样本进行片段化处理而得；
优选地，所述片段化为利用物理方法或化学方法，对样本进行随机打断或切断；进一步优选地，利用物理超声法或酶反应法进行所述片段化；
优选地，所述平端修复是利用T4 DNA聚合酶进行的；
优选地，所述磷酸化是利用核苷酸激酶、优选T4多聚核苷酸激酶进行的；
优选地，所述3’末端加碱基A是利用去除3’→5’外切酶活性的Klenow聚合酶进行的；
2)通过连接反应，在步骤1)所得DNA片段两端分别加上接头元件1；
在一个优选的具体实施方案中，所述接头元件1的核酸长链A的序列为：
5’-/Phos/CTGCTGACGTACTG(N)AGCACGAGACGTTCTCGACT/ddC/-3’，其中/Phos/表示磷酸化修饰，N表示标签序列，/ddC/表示双脱氧胞苷；优选地，所述标签序列长度为6nt-10nt；更优选地，所述标签序列为5’-TGTCATAAAT-3’；即，在一个更优选的具体实施方案中，所述接头元件的核酸长链A的序列为：
5’-/Phos/CTGCTGACGTACTGTGTCATAAATAGCACGAGACGTTCTCGACT/ddC/-3’(见SEQ ID NO：1)；
其核酸短链B的序列为：
5’-GAGAACGUCTCGTGCUACGTTCTCGACTCAGCAGT-3’；
3)以步骤2)所得DNA片段为模板，以分别与接头元件1的长链3’端，和短链的核酸片段B2和/或B3中的部分序列互补的核酸单链为引物，进行PCR扩增；
两条引物中部具有酶作用位点，且其中一条引物中引入生物素标记；
优选地，所述酶作用位点为U或dU，对应的酶为USER酶；
当使用上述具体的接头元件1时，此处的引物对为：
引物1序列(SEQ ID NO：5)如下：
AGTCGAGAACGUCTCG/iBiodT/GCT；/iBiodT/表示生物素标记的dT；
引物2序列(SEQ ID NO：6)如下：
ACGTTCTCGACUCAGCAG；
4)利用所述酶作用位点，在步骤3)所得扩增片段两端制造粘性末端，利用粘性末端，将扩增片段连接成环状核酸双链；
5)用III类限制性内切酶酶切消化步骤4)所得环状核酸双链，然后利用亲和素磁珠捕获酶切后的DNA片段；
或者，5’)先利用亲和素磁珠捕获步骤4)所得环状核酸双链，再用III类限制性内切酶酶切消化捕获的环状核酸双链；
优选地，所述亲和素磁珠为链霉亲和素磁珠；
6)对步骤5)或5’)所得酶切后的DNA片段进行平端修复和3’末端加碱基A；
7)通过连接反应，在步骤6)所得DNA片段两端分别加上接头元件2；
所述接头元件2的序列不同于接头元件1的序列，且所述接头元件2的核酸片段B1中含酶作用位点；优选地，所述酶作用位点为U或dU，对应的酶为USER酶；
当使用上述具体的接头元件1时，优选地，所述接头元件2的核酸长链A的序列 为：
5’-/Phos/AGTCGGAGGCCAAGCGTGCTTAGGACAT-3’，其中/Phos/表示磷酸化修饰；
其核酸短链B的序列为：
5’-GTCCTAAGCACUGTAGTGTACGATCCGACTT-3’；
8)利用接头元件2的核酸片段B1中的酶作用位点，将步骤7)所得DNA片段上的接头元件2的核酸片段B1切断，同时使切除核酸片段B1的接头元件5’末端磷酸化；
9)将步骤8)所得DNA片段进行变性处理，分离并回收非生物素标记的核酸单链；优选地，采用碱变性法或高温变性法；
10)将步骤9)所得非生物素标记的核酸单链进行环化，形成单链环状核酸产物，即为测序文库；
优选地，利用介导片段实现所述核酸单链的环化，所述介导片段具有相应互补序列用于连接核酸单链的两端；
当使用上述具体的接头元件2时，优选地，所述介导片段的序列为：5’-ATCGTACACTACATGTCCTAAGCA-3’，见SEQ ID NO：7；
优选地，还包括在核酸单链环化完成后，消化线性单链的步骤；进一步优选地，用核酸外切酶1和/或3进行消化。
步骤10)所得单链环状核酸产物可以直接进入后续的测序步骤，经过滚环复制后形成核酸纳米球(DNB)进行核酸序列信息读取。
第四方面，本发明提供了一种测序文库，由如第三方面所述的测序文库构建方法制得。所制得的测序文库为单链环状测序文库。
第五方面，本发明提供了如第四方面所述的测序文库在基因组测序中的应用，优 选地，在目标基因组区域测序中的应用。
作为优选，使用单链环状文库测序平台进行测序；进一步优选地，使用Complete Genomics公司的测序平台进行测序。
第六方面，本发明提供了一种核酸测序方法，包括将如第四方面所述的测序文库进行测序的步骤；
优选地，使用单链环状文库测序平台进行测序；进一步优选地，使用Complete Genomics公司的测序平台进行测序；
优选地，还包括将测序结果进行组装和/或拼接的步骤。
第七方面，本发明提供了一种测序文库构建试剂盒，其包括如第一方面所述的接头元件。
优选地，所述试剂盒还包括核苷酸激酶，优选T4多聚核苷酸激酶；DNA聚合酶，优选T4 DNA聚合酶或去除了3’→5’外切酶活性的聚合酶，例如Klenow exo-；USER酶；以及III类限制性内切酶，例如Acu I、Bpm I、BceA I、Bbv I、BciV I、BpuE I、BseM II、BseR I、Bsg I、BsmF I、BtgZ I、Eci I、EcoP15 I、Eco57M I、Fok I、Hga I、Hph I、Mbo II、Mnl I、SfaN I、TspDT I、TspDW I或Taq II。
有益效果
相比现有的双接头建库法，本发明采用了新颖的鼓泡状接头元件，简便的连接方法，并利用了在磁珠上进行酶反应的方法。
现有方法采用的定向接头连接法，在保证接头定向连接的同时，最大程度地降低DNA片段间相互连接问题，采用了将5’接头和3’接头分开设计，分步连接的方法。每加一端接头，都需要接头序列、封闭序列、引物序列共同作用来完成，完成整个过程需要6步酶反应、5步纯化操作，步骤繁琐，建库成本(序列成本、酶反应试剂成本、纯化成本)高，周期长，样品损耗大。
而本发明采用的鼓泡状接头元件及其连接方法，能够在保证接头定向连接的前提下，提高建库效率，降低建库成本和起始量。
由于在连接方法设计时，将目的DNA片段进行了3’末端加A处理，因此3’T突出的鼓泡状接头元件可以与3’A突出的目的DNA进行有效地定向连接。相对现有方法的分步连接策略，本发明采用的方法实现了接头的一步连接，完成整个过程只需要3-4步酶反应，3步纯化操作，步骤简单，建库成本降低，周期缩短，另外，纯化次数减少使样品损耗降低，建库起始量可从原来的3μg降低至1μg。
传统的定向接头连接法与本发明的鼓泡状接头元件连接方法的对比详见图3。由图3可以看出，定向接头连接方法需要经过去磷酸化、末端修复、加5’接头、引物延伸、加3’接头、切口平移及连接这6步酶反应及5次纯化操作才能将接头A的序列定向加入到目的DNA两端，而本发明的鼓泡状接头元件连接方法只需要经过磷酸化与末端修复一步反应、加A、加鼓泡状接头、USER酶切(可省略)这4步(或3步)酶反应及3次纯化操作即可较快速地将接头A的序列定向加入到目的DNA两端。
此外，传统的在溶液中的酶反应方法，酶反应过程中DNA和反应液是混合在一起的，反应完成后，需要去除掉酶和其他杂质，因而需要采用硅柱纯化(如Qiagen PCR Purification Kit)或普通磁珠纯化(如Agencourt AMPure XP beads)的方法来纯化DNA，这些纯化方法不仅试剂成本高，纯化操作时间较长，而且每次纯化之后DNA样品会有10-30％的损失。本发明独创地采用在磁珠上进行酶反应的方法，由于目的DNA一直被绑定在亲和素磁珠上，酶反应完之后只需要用磁力架吸附固定住磁珠，然后吸弃反应上清液，再用合适的缓冲液冲洗掉磁珠上残留的杂质，即可达到纯化DNA的目的。因此，本方法不仅节省了纯化试剂成本，降低了DNA样品损失(只有约1％的损失)，还有利于自动化操作的实现和工作效率的提升。
关于在溶液中进行酶反应和在磁珠上进行酶反应的方法对比，在图4中进行了清 晰的说明；图4显示了从III类内切酶切割至单链分离的酶反应大致步骤。具体地，图4显示，III类内切酶切割之后，溶液中的酶反应需要做两步磁珠纯化以选择符合目的DNA大小的酶切片段，但该片段选择方法同时也会引入部分同样片段大小的非目的DNA酶切片段；而磁珠上的酶反应利用目的DNA片段A接头上标记的生物素与链霉亲和素磁珠的特异结合，可准确地富集到目的DNA酶切片段。对于接头元件2的连接，现有方法需要做6次酶反应和6次磁珠纯化才能完成；本发明采用的方法只需要做2次酶反应和2次磁珠洗涤即可完成。
另外，现有方法加完接头元件2需要进行PCR扩增提高目的DNA产量，再进行1次单链分离和1次特异性单链捕获反应，才能得到目的单链DNA；本发明采用的方法加完接头元件2之后无需PCR扩增，通过USER酶切切除非目的接头序列的同时形成5’磷酸化末端，再通过变性的方法，就可将目的单链DNA洗脱下来。这是由于本发明采用在磁珠上的酶反应方法，使得DNA样品的损耗大大降低，在加完接头元件2之后，不需要做PCR扩增(PCR-free)就能得到足量的上机文库，这样不仅省去了一步PCR扩增反应，而且简化了筛选目的单链的步骤，使接头B至单链分离环节的建库流程得到极大地简化。此外，由于减少了文库构建的PCR扩增循环数，可以提高文库的库容，降低上机文库中重复读长的个数，提高文库的测序数据有效率。
总之，本发明通过新颖的鼓泡状接头元件，以及在溶液中和在磁珠上进行接头连接反应的技术，成功对CG双接头建库法文库构建流程的进行了改进和优化，主要改进的地方为第一次接头连接由6步酶反应、5次磁珠纯化简化为4步酶反应、3步纯化，第二次接头连接由6次酶反应、6次磁珠纯化简化为2次酶反应、2次磁珠洗涤，同时省去了第二次PCR扩增和一次单链富集筛选步骤，使整个建库周期和成本降低了约一半，建库起始量从3ug可降低至1ug，并有利于实现高通量建库的自动化和文库测序数据有效率的提高。
附图说明
图1图解说明了本发明的测序文库构建方案：1为打断后的DNA片段；2为经过平端修复、磷酸化作用后的片段；3为3’末端加上碱基A的DNA片段；4为加接头元件1后的DNA片段；5为经过USER酶切降解非特异接头序列(即核酸片段B1)，该步骤可省略；6为PCR扩增加接头元件1的DNA片段；7为经过酶切形成粘性末端后环化的双链DNA，其中带生物素标记的环状双链可以与链霉亲和素磁珠结合；8为经过III类内切酶切割之后形成的末端突出1-2个碱基(N表示A、T、C、G中的任何一种碱基)的DNA片段；9为经过末端修复、3’末端加A的DNA片段；10为加接头元件2后经过USER酶切处理的DNA片段；11为从链霉亲和素磁珠上洗脱下来的单链DNA；12为文库构建最终产物，即单链环化DNA。图中biotin标记，表示经过生物素标记。
图2图解说明了本发明的测序文库构建中，从接头元件连接到PCR扩增的步骤；1显示3’T突出的接头元件(由组成1、2、3组成)与3’A突出的DNA片段的连接；2显示用USER酶酶切消化接头元件组成1正向链上的非目的接头序列，可省略这一步骤；3显示用与目的接头序列正向链和反向链相匹配的引物扩增带有目的接头序列的DNA片段。
图3为传统的定向接头连接法与本发明的鼓泡状接头元件连接方法的对比图示。
图4为在溶液中进行酶反应和在磁珠上进行酶反应的对比图示，其中显示了从III类内切酶切割至单链分离的酶反应大致步骤。
图5为本发明实施例中加接头元件1后的DNA片段的电泳结果；其中，M为D2000(Tiangen)；C为步骤2产物，即加接头元件1前的DNA片段；1L、2L为步骤5产物1、2，即加接头元件1后的DNA片段；1P、2P为步骤6产物1、2，即加接头元件1后的DNA片段的PCR扩增产物。
图6为本发明实施例中加接头元件2后的DNA片段的电泳结果；其中，M1为50bp(Tiangen)；M2为D2000(Tiangen)；1-7为步骤13产物，即加完接头元件2后的DNA片段的PCR扩增产物1-7。
图7为最终产物的电泳结果；M为RiboRulerHigh Range RNA Ladder，ready-to-use(Fermentas)；1-7为步骤16产物1-7，即单链环化DNA产物1-7。
具体实施方式
为便于理解本发明，本发明列举实施例如下。本领域技术人员应该明了，所述实施例仅仅是帮助理解本发明，不应视为对本发明的具体限制。
实施例1本发明的测序文库构建
1、基因组DNA打断：
炎黄1号细胞基因组DNA打断有多种方式，无论是物理超声法还是酶反应法，市场上有非常成熟的方案。本实施例采用的是物理超声打断法。
取96孔PCR板一块，加入一根聚四氟乙烯线，加入基因组DNA 1ug，加入TE缓冲溶液或无酶纯水补齐100ul。将板封膜后至于E220超声打断仪上超声打断。
打断条件设置如下：
填充系数 21％
压力(PIP) 500
脉冲系数 500
打断时间 20s，2次
2、打断片段选择：
可以采用磁珠纯化法或凝胶回收法。本实施例采用磁珠纯化法，具体如下：
取打断后的DNA，加入45ul Ampure XP磁珠，混匀后放置7-15min；置入磁力架后收集上清，在上清中加入18ul Ampure XP磁珠，混匀后放置7-15min；置入磁力 架吸去上清，用75％乙醇洗磁珠两次；晾干后加入50ul TE缓冲溶液，混匀后放置7-15min溶解回收产物。
3、片段磷酸化及末端修复一步反应：
取上步骤回收产物，按下表配制体系：
无酶纯水 9.33ul
10X NEBNext末端修复缓冲液 7.00ul
T4脱氧核糖核酸聚合酶(3U/ul) 1.87ul
T4多聚核苷酸激酶(10U/ul) 1.40ul
牛血清白蛋白(20mg/ml) 0.40ul
总共 20.00ul
将20ul反应液加入前一步的回收产物中，混匀，置于20℃孵育30min。反应完后，用70ul Ampure XP磁珠进行纯化，40ulTE缓冲液溶解回收产物。(反应产物的纯化有多种方式，有磁珠法、柱纯化法、凝胶回收法等等。均可用于替换。本实施例如不做特殊说明，均采用磁珠法纯化。)
4、片段末端加A反应：
按下表配制体系：
Figure PCTCN2014091952-appb-000001
将体系混匀后加入上一步骤产物中，混匀后置于37℃孵育30min。用60ul Ampure  XP磁珠进行纯化，40ul TE缓冲溶液溶解回收产物。
5、接头元件1连接：
本方案中使用的接头元件1序列如下(本实施例中的序列从左到右为5’端至3’端，“//”示修饰基团，“phos”示磷酸化，“dd”示双脱氧，“bio”示生物素，字体加粗示标签序列)：
长链1：
/Phos/CTGCTGACGTACTGTGTCATAAATAGCACGAGACGTTCTCGACT/ddC/
短链1：GAGAACGUCTCGTGCUACGTTCTCGACTCAGCAGT
接头元件1混合液(25uM)按以下配方配制：
长链1(200uM) 12.5ul
短链1(200uM) 12.5ul
氯化钠(5M) 1.2ul
三羟甲基氨基甲烷-盐酸(1M，pH7.8) 1.2ul
乙二胺四乙酸二钠(20mM) 0.5ul
无酶纯水 72.1ul
总共 100.0ul
将6ul配制好的接头元件1混合液(25uM)加入上一步骤产物中，充分混匀。
连接反应体系按以下配方配制：
Figure PCTCN2014091952-appb-000002
Figure PCTCN2014091952-appb-000003
本方案使用的连接缓冲液1配方：
三羟甲基氨基甲烷-盐酸(pH 7.8) 150mM
聚乙二醇8000 15％
氯化镁 30mM
核糖核苷三磷酸 3mM
将连接反应体系与接头和产物的混合液混匀，置于20℃孵育30min。反应完成后，加入35ul乙二胺四乙酸二钠(35uM)，混匀。用50ul Ampure XP磁珠进行纯化，52ulTE缓冲液溶解回收产物。
此步骤完成了目的核酸片段与接头元件1的连接。连接前、后产物及连接后的PCR扩增产物电泳结果如图5所示。
6、聚合酶链式反应：
引物1序列(SEQ ID NO：5)如下：
AGTCGAGAACGUCTCG/iBiodT/GCT
引物2序列(SEQ ID NO：6)如下：
ACGTTCTCGACUCAGCAG
按下表配制反应体系：
Figure PCTCN2014091952-appb-000004
Figure PCTCN2014091952-appb-000005
将上步骤50ul回收产物，加入到以上体系中，混匀后按下表条件进行反应：
Figure PCTCN2014091952-appb-000006
反应完成后，使用450ul Ampure XP磁珠进行纯化，65ul TE缓冲液溶解回收产物。取1ul回收产物，用Qubit dsDNA HS分析试剂盒(invitrogen公司)定量产物浓度。取2.4ug产物进行下一步反应。
7、去尿嘧啶：
配制以下反应液：
10X Taq缓冲液 5ul
USER酶(1U/ul) 8ul
总体积 13ul
将以上反应液加入37ul(2.4ug)上步骤反应产物中，混匀后置于37℃孵育1h。
8、双链环化：
配制以下反应体系1：
Figure PCTCN2014091952-appb-000007
Figure PCTCN2014091952-appb-000008
将上一步骤反应产物加入反应体系1中，置于60℃水浴反应30min。反应完成后置于常温水浴反应20min。
配制以下反应体系2：
Figure PCTCN2014091952-appb-000009
将反应体系2，加入上步反应体系中，置于室温孵育1h。
500ul反应产物中加入330ul Ampure XP磁珠，混匀后放置7-15min；置入磁力架后收集上清，在上清中加入170ul Ampure XP磁珠，混匀后放置7-15min；置入磁力架吸去上清，用75％乙醇洗磁珠两次；晾干后加入68.1ul TE缓冲液溶解纯化产物。
9、线性消化：
配制以下反应体系：
Figure PCTCN2014091952-appb-000010
Figure PCTCN2014091952-appb-000011
将上步骤产物加入反应体系中，混匀后置于37℃孵育1h。
使用80ul Ampure XP磁珠纯化。使用82ul TE缓冲液溶解回收产物。
10、酶切处理：
配制以下反应体系：
无酶纯水 233.2ul
10X NEBuffer3.1 36ul
Ecop15 I内切酶(10U/ul) 10.8ul
总体积 280ul
将上步骤产物加入反应体系中，混匀后置于37℃孵育16h。
11、结合链霉亲和素磁珠：
取90ul MyOne Streptavidin C1磁珠，吸弃上清，用1x磁珠结合缓冲液清洗两次(每次450ul，每次清洗完需要吸弃上清)后重悬于90ul 1x磁珠结合缓冲液中，并加入0.9ul 0.5％吐温20，混匀备用。
向360ul上步骤反应产物中加入72ul氯化钠溶液(3M)，混匀。加入已清洗的90ul MyOne Streptavidin C1磁珠，混匀，室温静置10min。置磁力加上3min，去除上清液。用1x低盐清洗缓冲液(含0.05％吐温20)清洗两次(每次150ul，每次清洗完需要吸弃上清)，用90ul 1x低盐清洗缓冲液重悬磁珠。
12、末端修复及末端加A一步反应：
配制以下反应体系：
Figure PCTCN2014091952-appb-000012
将该反应体系加入上步骤磁珠重悬液中，混匀后置于37℃孵育1h。
反应完成后，加入2.2ul乙二胺四乙酸二钠溶液(0.5M)，混匀，室温静置1min，吸弃上清。用1x低盐清洗缓冲液(含0.05％吐温20)清洗两次，用80ul 1x低盐清洗缓冲液重悬磁珠。
13、接头元件2连接：
长链2：/Phos/AGTCGGAGGCCAAGCGTGCTTAGGACAT
短链2：GTCCTAAGCACUGTAGTGTACGATCCGACTT
接头元件2混合液(10uM)按以下配方配制：
长链2(100uM) 10ul
短链2(100uM) 10ul
氯化钠(1M) 5ul
三羟甲基氨基甲烷-盐酸(0.2M，pH7.8) 5ul
乙二胺四乙酸二钠(2mM) 5ul
无酶纯水 65ul
总共 100ul
将30ul配制好的接头元件2混合液(10uM)加入上一步骤磁珠重悬液中，充分 混匀。
连接反应体系按以下配方配制：
Figure PCTCN2014091952-appb-000013
将连接反应体系加入磁珠重悬液中，混匀，室温静置30min。反应完之后，加入3.42ul乙二胺四乙酸二钠(0.5M)，混匀，静置1min，吸弃上清。用1x低盐清洗缓冲液(含0.05％吐温20)清洗两次，用80ul 1x低盐清洗缓冲液重悬磁珠。
此步骤完成了目的核酸片段与接头元件2的连接。连接效果可通过取下一步洗脱单链核酸3ul，扩增8个循环之后，取5uL电泳检测得知，电泳结果如图6所示。
14、去尿嘧啶及洗脱单链核酸：
将1ul USER酶加入到上步骤磁珠重悬液中，混匀，置于37孵育1h。反应完之后，加入8.1ul乙二胺四乙酸二钠(0.1M)，混匀，静置1min，吸弃上清。用1x低盐清洗缓冲液(含0.05％吐温20)清洗两次，吸弃上清。用75ul氢氧化钠(0.1M)重悬磁珠，室温静置5min，洗取上清。加入37.5ul酸性缓冲液中和获得的单链分离产物，中和后产物总体积112.5ul。
15、单链环化：
配制以下反应体系1，其中介导片段具有相应互补序列用于连接单链两端，其序列为：ATCGTACACTACATGTCCTAAGCA(即SEQ ID NO：7)。
无酶纯水 59ul
介导片段(100uM) 4ul
总共 63ul
将反应体系1加入上步骤的单链分离产物中，混匀。
配制反应体系2：
Figure PCTCN2014091952-appb-000014
将反应体系2加入反应体系1中，混匀，置于37℃孵育1.5h。
16、消化线性单链：
配置以下反应缓冲液：
Figure PCTCN2014091952-appb-000015
将20ul反应缓冲液加入上一步骤的350.5ul反应产物中，混匀，置于37℃孵育30 min。加入15.4ul乙二胺四乙酸(500mM)，混匀。使用500ul PEG32磁珠纯化回收，70ul TE缓冲液溶解产物。
本实施例最终产物浓度和总量情况如下：电泳结果见图7。
  浓度(ng/ul) 总量(ng) 分子量(pmol)
产物1 0.35 24.50 0.44
产物2 0.34 23.80 0.42
产物3 0.35 24.50 0.44
产物4 0.28 19.60 0.35
产物5 0.37 25.90 0.46
产物6 0.39 27.30 0.49
产物7 0.30 21.00 0.37
从结果来看，各产物浓度与总量满足了后续测序要求(分子量≥0.12pmol)，电泳结果也显示片段集中，是质量非常高的文库。证明本方案是完全成功的。
申请人声明，本发明通过上述实施例来说明本发明的详细工艺设备和工艺流程，但本发明并不局限于上述详细工艺设备和工艺流程，即不意味着本发明必须依赖上述详细工艺设备和工艺流程才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。

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  1. 一种接头元件，其特征在于，由一条核酸长链A和一条核酸短链B形成的两端序列互补、中间序列不互补而呈鼓泡状的杂交体，所述杂交体的核酸长链A的5’末端碱基经磷酸化修饰，所述杂交体的核酸短链B的3’末端带有突出的碱基T；
    任选地，所述杂交体中具有III类限制性内切酶识别位点；优选地，所述III类限制性内切酶的识别位点与接头元件-目标DNA连接处相距0-2bp。
  2. 根据权利要求1所述的接头元件，其特征在于，所述杂交体按构型分为如下三个部分：
    组成1：包括长链A的3’端的核酸片段A1与短链B的5’端的核酸片段B1，所述核酸片段A1与所述核酸片段B1部分互补杂交，形成互补的双链部分和位于长链A 3’末端的游离部分；
    组成2：包括长链A中段的核酸片段A2与短链B中段的核酸片段B2；所述核酸片段B2与核酸片段A2不互补，从而形成鼓泡结构；
    组成3：包括长链A的5’端的核酸片段A3与短链B的3’端的核酸片段B3；核酸片段A3的5’末端为磷酸化修饰，核酸片段B3的3’末端为碱基T；除了3’末端的碱基T外，核酸片段B3的所有碱基均与核酸片段A3的碱基互补配对，从而形成短链3’末端突出1个碱基T的互补双链；
    其中，任选地，所述组成3中具有III类限制性内切酶的识别序列；
    优选地，所述核酸片段A1的长度为10-20nt，所述核酸片段B1的长度为8-16nt；所述核酸片段A2的长度为11-36nt，所述核酸片段B2的长度为11-21nt；所述核酸片段A3的长度为6-8nt，所述核酸片段B3的长度为7-9nt；
    优选地，所述核酸片段B1中具有酶作用位点；进一步优选地，所述酶作用位点为U或dU，对应的酶为USER酶；
    优选地，所述核酸片段A2中含有标签序列；
    优选地，所述III类限制性内切酶为Acu I、Bpm I、BceA I、Bbv I、BciV I、BpuE I、BseM II、BseR I、Bsg I、BsmF I、BtgZ I、Eci I、EcoP15 I、Eco57M I、Fok I、Hga I、Hph I、Mbo II、Mnl I、SfaN I、TspDT I、TspDW I或Taq II。
  3. 根据权利要求1或2所述的接头元件，其特征在于，所述核酸长链A的序列为：
    5’-/Phos/CTGCTGACGTACTG(N)AGCACGAGACGTTCTCGACT/ddC/-3’，其中/Phos/表示磷酸化修饰，N表示标签序列，/ddC/表示双脱氧胞苷；优选地，所述标签序列长度为6nt-10nt；更优选地，所述标签序列为5’-TGTCATAAAT-3’；
    所述核酸短链B的序列为：
    5’-GAGAACGUCTCGTGCUACGTTCTCGACTCAGCAGT-3’。
  4. 根据权利要求1或2所述的接头元件，其特征在于，所述核酸长链A的序列为：
    5’-/Phos/AGTCGGAGGCCAAGCGTGCTTAGGACAT-3’，其中/Phos/表示磷酸化修饰；
    所述核酸短链B的序列为：
    5’-GTCCTAAGCACUGTAGTGTACGATCCGACTT-3’。
  5. 一种接头连接方法，其特征在于，将权利要求1-4任一项所述的接头元件连接在待测DNA片段两端；
    优选地，通过连接反应，将所述接头元件加在待测DNA片段的两端；
    优选地，在所述接头元件连接前，还包括将待测DNA片段进行平端修复、5’末端磷酸化和3’末端加碱基A的步骤。
  6. 一种测序文库的构建方法，其特征在于，包括使用如权利要求1-4任一项所述 的接头元件或使用如权利要求5所述的接头连接方法进行接头连接；
    优选地，所述构建方法包括以下步骤：
    1)将双链DNA片段进行平端修复、5’末端磷酸化和3’末端加碱基A；
    优选地，所述双链DNA片段是通过如下步骤制备的：
    1-1)对mRNA样本进行片段化处理，从而获得片段化的mRNA；
    1-2)对所述片段化的mRNA进行反转录，从而获得cDNA扩增产物，作为双链DNA片段；
    任选地，所述双链DNA片段直接由DNA样本进行片段化处理而得；
    优选地，所述片段化为利用物理方法或化学方法，对样本进行随机打断或切断；进一步优选地，利用物理超声法或酶反应法进行所述片段化；
    优选地，所述平端修复是利用T4 DNA聚合酶进行的；
    优选地，所述磷酸化是利用核苷酸激酶、优选T4多聚核苷酸激酶进行的；
    优选地，所述3’末端加碱基A是利用去除3’→5’外切酶活性的Klenow聚合酶进行的；
    2)通过连接反应，在步骤1)所得DNA片段两端分别加上接头元件1；
    优选地，所述接头元件1的核酸长链A的序列为：
    5’-/Phos/CTGCTGACGTACTG(N)AGCACGAGACGTTCTCGACT/ddC/-3’，其中/Phos/表示磷酸化修饰，N表示标签序列，/ddC/表示双脱氧胞苷；优选地，所述标签序列长度为6nt-10nt；更优选地，所述标签序列为5’-TGTCATAAAT-3’；
    其核酸短链B的序列为：
    5’-GAGAACGUCTCGTGCUACGTTCTCGACTCAGCAGT-3’；
    3)以步骤2)所得DNA片段为模板，以分别与接头元件1的长链3’端，和短链的核酸片段B2和/或B3中的部分序列互补的核酸单链为引物，进行PCR扩增；
    两条引物中部具有酶作用位点，且其中一条引物中引入生物素标记；
    优选地，所述酶作用位点为U或dU，对应的酶为USER酶；
    4)利用所述酶作用位点，在步骤3)所得扩增片段两端制造粘性末端，利用粘性末端，将扩增片段连接成环状核酸双链；
    5)用III类限制性内切酶酶切消化步骤4)所得环状核酸双链，然后利用亲和素磁珠捕获酶切后的DNA片段；
    或者，5’)先利用亲和素磁珠捕获步骤4)所得环状核酸双链，再用III类限制性内切酶酶切消化捕获的环状核酸双链；
    优选地，所述亲和素磁珠为链霉亲和素磁珠；
    6)对步骤5)或5’)所得酶切后的DNA片段进行平端修复和3’末端加碱基A；
    7)通过连接反应，在步骤6)所得DNA片段两端分别加上接头元件2；
    所述接头元件2的序列不同于接头元件1的序列，且所述接头元件2的核酸片段B1中含酶作用位点；优选地，所述酶作用位点为U或dU，对应的酶为USER酶；
    进一步优选地，所述接头元件2的核酸长链A的序列为：
    5’-/Phos/AGTCGGAGGCCAAGCGTGCTTAGGACAT-3’，其中/Phos/表示磷酸化修饰；
    其核酸短链B的序列为：
    5’-GTCCTAAGCACUGTAGTGTACGATCCGACTT-3’；
    8)利用接头元件2的核酸片段B1中的酶作用位点，将步骤7)所得DNA片段上的接头元件2的核酸片段B1切断，同时使切除核酸片段B1的接头元件5’末端磷酸化；
    9)将步骤8)所得DNA片段进行变性处理，分离并回收非生物素标记的核酸单链；优选地，采用碱变性法或高温变性法；
    10)将步骤9)所得非生物素标记的核酸单链进行环化，形成单链环状核酸产物，即为测序文库；
    优选地，利用介导片段实现所述核酸单链的环化，所述介导片段具有相应互补序列用于连接核酸单链的两端；进一步优选地，所述介导片段的序列为：5’-ATCGTACACTACATGTCCTAAGCA-3’；
    优选地，还包括在核酸单链环化完成后，消化线性单链的步骤；进一步优选地，用核酸外切酶1和/或3进行消化。
  7. 一种测序文库，其特征在于，由权利要求6所述构建方法制得。
  8. 如权利要求7所述测序文库在基因组测序、优选在目标基因组区域测序中的应用；
    优选地，使用单链环状文库测序平台进行测序；
    进一步优选地，使用Complete Genomics公司的测序平台进行测序。
  9. 一种核酸测序方法，其特征在于，包括将权利要求7所述的测序文库进行测序的步骤；
    优选地，使用单链环状文库测序平台进行测序；进一步优选地，使用Complete Genomics公司的测序平台进行测序；
    优选地，还包括将测序结果进行组装和/或拼接的步骤。
  10. 一种测序文库构建试剂盒，其特征在于，包括权利要求1-4任一项所述的接头元件。
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