WO2016078096A1 - 使用鼓泡状接头元件构建测序文库的方法 - Google Patents

使用鼓泡状接头元件构建测序文库的方法 Download PDF

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WO2016078096A1
WO2016078096A1 PCT/CN2014/091953 CN2014091953W WO2016078096A1 WO 2016078096 A1 WO2016078096 A1 WO 2016078096A1 CN 2014091953 W CN2014091953 W CN 2014091953W WO 2016078096 A1 WO2016078096 A1 WO 2016078096A1
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nucleic acid
sequencing
sequence
linker element
long chain
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PCT/CN2014/091953
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French (fr)
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江媛
田凯
赵霞
章文蔚
徐怀前
蒋慧
徳马纳克拉多杰
耿春雨
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深圳华大基因科技有限公司
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Priority to CN201480083529.2A priority Critical patent/CN107075508B/zh
Priority to US15/528,353 priority patent/US10316356B1/en
Priority to PCT/CN2014/091953 priority patent/WO2016078096A1/zh
Publication of WO2016078096A1 publication Critical patent/WO2016078096A1/zh

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    • 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
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    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
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    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
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    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/532—Closed or circular
    • C—CHEMISTRY; METALLURGY
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    • 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
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    • C12Q2535/00—Reactions characterised by the assay type for determining the identity of a nucleotide base or a sequence of oligonucleotides
    • C12Q2535/122—Massive parallel sequencing

Definitions

  • the present invention relates to the field of biotechnology, and in particular to a method for constructing a sequencing library using a bubbling connector element, a constructed sequencing library and an application thereof.
  • Whole genome sequencing technology is a research method that covers the whole genome base sequence of a species and detects all genetic information in an individual's genome. Its accuracy is high and its accuracy is as high as 99.99%. With the development of second-generation high-throughput sequencing technology, sequencing has become more and more rapid, accurate, and low-cost. Whole-genome sequencing has been widely used in research.
  • CG sequencing is based on single-stranded circularized DNA and requires the introduction of a linker of known sequence for sequencing.
  • CG Complete Genomics
  • the present invention provides a method for constructing a sequencing library using a bubbling connector element, a constructed sequencing library and an application thereof.
  • the invention solves the problems of excessive joint connection steps, multiple PCR amplification times, long overall library construction time and high cost in the double joint construction method of the CG sequencing platform by using a bubbling joint element, and improves the problem.
  • the present invention provides a method of constructing a sequencing library, the method comprising the steps of:
  • the linker element 1 is respectively added to the two ends of the DNA fragment obtained in the step 1);
  • the linker element 1 comprises a long chain of nucleic acid and a short chain of nucleic acid; the long chain of the nucleic acid and the short chain of the nucleic acid can be annealed a hybrid in which the sequences at both ends are complementary and the intermediate sequences are not complementary to be bubbling; the 5' terminal base of the long chain A of the nucleic acid of the hybrid is phosphorylated, and the 3' of the nucleic acid short chain B of the hybrid a terminal with a prominent base T; the linker element 1 has a class III restriction endonuclease recognition site;
  • 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;
  • 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 double-stranded nucleic acid strand obtained in step 4) with a class III restriction endonuclease, and recovering the digested DNA fragment;
  • the two ends of the DNA fragment obtained in step 6) are respectively added with a linker element 2;
  • the linker element 2 comprises a long chain of nucleic acid and a short strand of nucleic acid;
  • the long chain of the nucleic acid and the short strand of the nucleic acid can be annealed a hybrid in which the sequences at both ends are complementary and the intermediate sequences are not complementary to be bubbling;
  • the 5' terminal base of the long chain A of the nucleic acid of the hybrid is phosphorylated, and the 3' of the nucleic acid short chain B of the hybrid a terminal with a prominent base T;
  • the sequence of the linker element 2 is different from the sequence of the linker element 1;
  • the avidin magnetic bead is a simian avidin magnetic bead
  • an alkali denaturation method or a high temperature denaturation method is employed;
  • 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 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.
  • 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 performed by using a physical method or a chemical method, and the DNA to be tested is randomly interrupted or cut; further preferably, the DNA fragmentation to be tested 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.
  • the class III restriction enzymes in the linker element 1 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;
  • the recognition site of the class III restriction endonuclease is 0-2 bp from the 3' end of the hybrid;
  • the linker element 1 comprises a tag sequence in a long chain of nucleic acids; further preferably, the tag sequence is 6-10 nt in length.
  • the sequence of the nucleic acid long chain in the linker element 1 is: 5'-/Phos/ACTGCTGAGTCGAGA(N)CTGACAAGGTCGCCAGCCCTGAGTGCTTCGAA-3'; wherein /Phos/ indicates phosphorylation modification, and N is a tag
  • the tag sequence is 6 nt to 10 nt in length; more preferably, the tag sequence is 5'-TGTCATAAAT-3'; ie, in a more preferred embodiment, the linker element 1
  • the sequence of the long chain of nucleic acids is:
  • sequence of its short strand of nucleic acid is:
  • the primers used for PCR amplification are:
  • Reverse primer 5'AGGGCUGGCGACCUTGTCAG-3' (see SEQ ID NO: 6).
  • the sequence of the long chain of the nucleic acid 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 strand of the nucleic acid is: 5'-CGAGAGCACTCCATGTAGTGTACGATCCGACTT-3', see SEQ ID NO: 4.
  • the primers used for PCR amplification are:
  • Reverse primer 5'-CATGTAGTGTACGATCCGACTT-3' (see SEQ ID NO: 8);
  • first base at the 5' end of the forward primer carries a biotin label
  • first base at the 5' end of the reverse primer has a phosphorylation modification
  • the sequence of the mediated fragment is: 5'-GTACACTACATGTCCTAAGCACGC-3', see SEQ ID NO: 9.
  • the invention provides a sequencing library produced by the method of constructing a sequencing library as described in the first aspect.
  • the present invention provides the use of a sequencing library according to the second aspect in genome sequencing, preferably in sequencing of a target genomic region;
  • 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, the sequencing method comprising the step of sequencing a sequencing library as described in the second 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 method for constructing the sequencing library of the invention reduces the existing building procedure, shortens the database construction period, effectively saves the cost of building the library, improves the efficiency of the database construction, and realizes the optimization of the double joint database construction.
  • Figure 1 shows the hybrid structure formed by annealing the joint member 1 in the embodiment
  • Figure 2 shows the hybrid structure formed by annealing the tab element 2 in the embodiment
  • Figure 3 illustrates a sequencing library construction scheme of the present invention
  • Indicates that the base is labeled with biotin wherein: 1 is the fragmented DNA fragment; 2 is the fragment after blunt-end repair and phosphorylation; 3 is the DNA fragment of the 3' end plus base A; a DNA fragment after the adaptor element 1; 5 is a product obtained by PCR amplification of the DNA fragment of the adaptor element 1; 6 is a DNA fragment having a sticky end after digestion with the USER enzyme; 7 is formed by viscous terminal cyclization Double-stranded circular DNA; 8 is a linear DNA fragment formed by cleavage by a class III restriction endonuclease; 9 is a DNA fragment after terminal repair and phosphorylation; 10 is a 3' end plus base A a DNA fragment; 11 is a DNA fragment after addition of the linker element 2; 12 is a product obtained by PCR amplification of the DNA fragment of the linker element 2; 13 is a
  • the left lane is a single-stranded circular DNA library
  • the right lane is a Low Range RNA Ladder (FERMENTAS).
  • the genomic DNA of the inflammatory yellow blood cells derived from human lymphoid tissue was taken 1 ug, diluted to 80 ul with TE buffer, and the special disrupted plate of Covaris was added.
  • the Covaris LE220 ultrasonic interrupter was used to break the DNA to about 200 bp to 400 bp. The parameters are as shown in the following table:
  • the interrupted genome was inhaled into a new 1.5 ml non-stick EP tube, 64 ul of Ampure xp magnetic beads were added, mixed, and allowed to stand at room temperature for 5 minutes, and then placed in a magnetic stand to adsorb magnetic beads. After 5 minutes, the supernatant was placed in another new non-stick EP tube, and 32 ul of Ampure xp magnetic beads were added, mixed, and allowed to stand at room temperature for 5 minutes. Place the magnetic frame to adsorb the magnetic beads for 5 minutes, discard the supernatant, and wash the magnetic beads twice with 75% ethanol; after drying, add 42 ul of TE buffer solution or no enzyme water, mix and let stand for 5 minutes to dissolve the recovered product.
  • the DNA obtained in the above step was added to the premix, mixed, and incubated at 20 ° C for 30 minutes. After taking out, 60 ul of Ampure xp magnetic beads were added, mixed, and allowed to stand at room temperature for 5 minutes. Discard the supernatant and wash twice with 75% alcohol. Dry and add 42 ul of TE buffer to dissolve (this is the standard procedure for purification of magnetic beads. If no special instructions are used, this method is used for magnetic bead purification, the same below, no further details).
  • the DNA obtained in the above procedure was added to the premix, mixed, incubated at 37 ° C for 30 minutes, purified with 50 ul of Ampure xp magnetic beads, air-dried and dissolved in 42 ul of TE buffer.
  • the sequence structure of the joint member 1 is as shown in Fig. 1, wherein the label sequence N is 5'-TGTCATAAAT-3'.
  • connection buffer Enzymatics
  • Connector component 1 (20uM)
  • the above premix was added to the DNA solution obtained in the previous step, and after mixing, 2 ul of T4 DNA ligase was added, mixed again, and then incubated at 20 ° C for 1 hour. After purification with Ampure XP magnetic beads, it was dissolved in 42 ul of TE.
  • primer 1 The sequence of primer 1 is: 5'-AGGUCGCCAGCCCUCAGTAC-3';
  • primer 2 The sequence of primer 2 was: 5'-AGGGCUGGCGACCUTGTCAG-3'.
  • the prepared reaction premix was added to the reaction product of the above step, mixed, and then reacted at 37 ° C for 1 h.
  • Enzyme free water 1520ul 10 ⁇ TA buffer (epicentre company) 180ul total capacity 1700ul
  • the reaction product of the previous step was added to the reaction system 1, and after mixing, it was subjected to a reaction at 60 ° C for 30 minutes. After the reaction was completed, it was placed at 24 °C.
  • the reaction system 2 was added to the reaction system 1, and the reaction was carried out at 24 ° C for 1 h.
  • the prepared reaction mixture was added to the DNA solution obtained in the previous step, mixed, and then reacted at 37 ° C for 1 h.
  • Enzyme free water 274ul 10X NEBuffer3.1 (NEB) 36ul Ecop15 I endonuclease (10U/ul) 10ul total capacity 320ul
  • reaction premix was added to the DNA solution obtained in the previous step, mixed, and placed at 37 ° C for 16 h.
  • the reaction product was taken, 415 ul of Ampure XP magnetic beads were added, and the mixture was allowed to stand for 5 min. After placing the magnetic stand, the supernatant was collected, and 296 ul of Ampure XP magnetic beads were added to the supernatant, mixed and placed for 5 min; The supernatant was removed from the rack, and the magnetic beads were washed twice with 75% ethanol; after drying, 42 ul of TE buffer solution or no enzyme water was added, and the mixture was allowed to stand for 5 minutes to dissolve the recovered product.
  • the prepared reaction mixture was added to the DNA solution obtained in the above step, mixed, and incubated at 20 ° C for 30 minutes. Purification was accomplished with 60 ul of Ampure xp magnetic beads. After drying, add 42 ul of TE buffer to dissolve.
  • the reaction mixture prepared as above was added to the DNA solution obtained in the above step, mixed, incubated at 37 ° C for 30 minutes, purified with 50 ul of Ampure xp magnetic beads, air-dried and dissolved in 42 ul of TE buffer.
  • the sequence structure of the joint member 2 is as shown in Fig. 2.
  • the above premix was added to the DNA solution obtained in the previous step, and after mixing, 2 ul of T4 DNA ligase was added, mixed again, and then incubated at 20 ° C for 1 hour. After purification with Ampure XP magnetic beads, it was dissolved in 42 ul of TE.
  • primer 3 5'-TCCTAAGCACGCTTGGCCT-3', and the first base T at the 5' end carries a biotin label;
  • the sequence of primer 4 is: 5'-CATGTAGTGTACGATCCGACTT-3', and the first base C at the 5' end has a phosphorylation modification.
  • the premix prepared as above was added to the DNA solution obtained in the above step, and mixed. Place the PCR reaction in the PCR machine.
  • the PCR procedure is as follows:
  • the PCR product produced in the previous step was diluted with 20 ul of 4x magnetic bead binding buffer, mixed, added to the resuspended magnetic beads, mixed, allowed to stand at room temperature for 5 minutes, and placed in a magnetic stand for 5 minutes. After clearing the clarification, the supernatant is aspirated.
  • the magnetic beads were resuspended in 1 ml of 1x magnetic bead wash buffer and placed in a magnetic stand for 5 minutes to aspirate the supernatant. repeat.
  • the magnetic beads were resuspended by adding 78 ul of 0.1 M sodium hydroxide solution to the tube, left to stand for 5 minutes, and then placed in a magnetic stand. After 5 minutes, the supernatant was aspirated into a new centrifuge tube, and 37.5 ul of MOPS buffer was added thereto. Mix well.
  • the cyclization reaction premix 1 is prepared in advance as shown in the following table (wherein the bridge sequence (also referred to as a mediated fragment) is as shown in SEQ ID NO: 9, 5'-GTACACTACATGTCCTAAGCACGC-3'):
  • the premix prepared above was added to the above isolated DNA single-strand solution and mixed.
  • the cyclization reaction premix 2 was prepared as shown in the following table:
  • reaction premix 2 was added to a DNA single-strand solution, mixed, and incubated at 37 ° C for 1.5 hours.
  • the constructed library was tested at 6% urea-denatured PAGE gel. The results are shown in Figure 4.
  • 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

一种使用鼓泡状接头元件构建测序文库的方法，采用两个序列不同的鼓泡状接头元件，该鼓泡状接头元件的中间鼓泡结构为不匹配序列，以匹配的测序序列作为PCR引物，通过PCR过程，实现不匹配链的置换；该方法通过一次接头加载及PCR扩增过程就可以保证DNA片段两端连上不同的测序序列，同时实现片段扩增。利用该方法构建测序文库，可进行核酸测序。

Description

使用鼓泡状接头元件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，涉及一种使用鼓泡状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
全基因组测序技术，是一种广泛覆盖物种全基因组碱基序列，检测个体基因组中全部遗传信息的研究手段。其准确性高，准确率可高达99.99％。随着二代高通量测序技术的发展，测序变得越来越快速、准确、低成本，全基因组测序在研究中得到了越来越广泛的应用。
CG测序是一种基于单链环化DNA的，需要引入已知序列的接头才能进行测序。通常，在构建基于Complete Genomics(CG)测序平台的全基因组文库过程中，标准的CG全基因建库流程，需要分别加两组接头才能完成建库，且每组接头序列不同，加载步骤繁琐，用时较长。
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，整体文库构建时间长等问题，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使用鼓泡状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。本发明通过使用一种鼓泡状接头元件，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、成本高的问题，并且提高了建库效率，实现了双接头建库的优化。
本发明通过以下技术方案实现上述目的：
第一方面，本发明提供了一种测序文库的构建方法，该构建方法包括以下步骤：
1)将双链DNA进行片段化，并对所得DNA片段进行平端修复、5’末端磷酸化和3’末端加碱基A；
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；所述接头元件1包含一条核酸长链和一条核酸短链；所述核酸长链和核酸短链可退火形成两端序列互补、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；所述接头元件1中具有III类限制性内切酶识别位点；
3)以步骤2)所得DNA片段为模板，以分别针对接头元件1的核酸长链和核酸短链的部分序列的两条序列不同的核酸单链为引物，进行PCR扩增；
所述两条引物的中部具有酶作用位点；优选地，所述酶作用位点为U或dU，对应的酶为USER酶；
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，回收酶切后的DNA片段；
6)对步骤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包含一条核酸长链和一条核酸短链；所述核酸长链和核酸短链可退火形成两端序列互补、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；
所述接头元件2的序列不同于接头元件1的序列；
8)以步骤7)所得DNA片段为模板，以分别针对接头元件2的核酸长链和核酸短链的部分序列的两条序列不同的核酸单链为引物，进行PCR扩增；其中一条引物的 5’端第一个碱基带有磷酸化修饰，另一条引物的5’端第一个碱基带有生物素标记；
9)利用亲和素磁珠回收步骤8)所得PCR产物，并进行变性处理，分离回收非生物素标记的核酸单链；
优选地，所述亲和素磁珠为连霉亲和素磁珠；
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；
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。
对于上述构建方法，作为优选，步骤1)中，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，对待测DNA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待测DNA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。
作为优选，所述接头元件1中的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；
优选地，所述III类限制性内切酶的识别位点距离所述杂交体的3’末端0-2bp；
优选地，所述接头元件1的核酸长链中包含标签序列；进一步优选地，所述标签序列长度为6-10nt。
在一个优选的实施方案中，所述接头元件1中的核酸长链的序列为：5’-/Phos/ACTGCTGAGTCGAGA(N)CTGACAAGGTCGCCAGCCCTGAGTGCTTCGAA-3’；其中，/Phos/表示磷酸化修饰，N为标签序列；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1中的核酸长链的序列为：
5’-/Phos/ACTGCTGAGTCGAGATGTCATAAATCTGACAAGGTCGCCAGCCCTGAGTGCTTCGAA-3’(见SEQ ID NO：1)；
其核酸短链的序列为：
5’-CGAAGCACTCAAGGTCGCCAGCCCTCAGTACGTCAGCAGTT-3’，见SEQ ID NO：2。
当采用上述接头元件1时，优选地，步骤3)中，PCR扩增所用引物分别为：
正向引物：5’-AGGUCGCCAGCCCUCAGTAC-3’(见SEQ ID NO：5)；
反向引物：5’AGGGCUGGCGACCUTGTCAG-3’(见SEQ ID NO：6)。
当采用上述接头元件1时，优选地，所述接头元件2中的核酸长链的序列为：5’-/Phos/AGTCGGAGGCCAAGCGTGCTTAGGATGAGTGCTCTCGAA-3’，其中/Phos/表示磷酸化修饰，见SEQ ID NO：3；其核酸短链的序列为：5’-CGAGAGCACTCCATGTAGTGTACGATCCGACTT-3’，见SEQ ID NO：4。
当采用上述接头元件2时，优选地，步骤8)中，PCR扩增所用引物分别为：
正向引物：5’-TCCTAAGCACGCTTGGCCT-3’(见SEQ ID NO：7)；
反向引物：5’-CATGTAGTGTACGATCCGACTT-3’(见SEQ ID NO：8)；
其中，所述正向引物的5’端第一个碱基带有生物素标记，所述反向引物的5’端第一个碱基具有磷酸化修饰。
当采用上述接头元件2时，优选地，步骤10)中，所述介导片段的序列为：5’-GTACACTACATGTCCTAAGCACGC-3’，见SEQ ID NO：9。
第二方面，本发明提供了一种测序文库，其是由如第一方面所述的测序文库的构建方法制得。
第三方面，本发明提供了如第二方面所述测序文库在基因组测序、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。
第四方面，本发明提供了一种核酸测序方法，该测序方法包括将如第二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。
有益效果
现有双接头建库，每次加接头需要加两种不同的接头，需要多步处理才能进行PCR扩增；整个建库过程需要使用四种不同的接头完成两次接头的加载。而本发明的测序文库构建方法，每次添加接头只需要用一种接头同时加载在DNA片段的3’及5’末端，就可以进行PCR扩增；整个建库过程需要使用两种不同的接头完成两次接头的加载 (见附图2)。此外，本发明构建方法所采用的接头元件通过中间鼓泡结构的不匹配序列，在PCR的过程中，不匹配链被替换，匹配的测序序列作为PCR引物而实现置换。这样，通过一次接头加载及PCR扩增过程，就可以保证DNA片段两端连上不同的测序序列，同时实现了片段扩增，节约了建库的时间成本和物料成本。
本发明的测序文库的构建方法缩减了现有建库步骤，缩短了建库周期，有效节约了建库的成本，并且提高了建库效率，实现了双接头建库的优化。
附图说明
图1显示实施例中接头元件1退火形成的杂交体结构；
图2显示实施例中接头元件2退火形成的杂交体结构；
图3图解说明了本发明的测序文库构建方案；其中
Figure PCTCN2014091953-appb-000001
表示碱基被磷酸化；
Figure PCTCN2014091953-appb-000002
表示碱基加有生物素标记；其中：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为加接头元件1的DNA片段进行PCR扩增后的产物；6为经过USER酶酶切后的具有粘性末端的DNA片段；7为利用粘性末端环化所形成的双链环状DNA；8为经过III类限制性内切酶切割之后形成的线状DNA片段；9为经过末端修复、磷酸化处理后的DNA片段；10为3’末端加碱基A的DNA片段；11为加接头元件2后的DNA片段；12为加接头元件2的DNA片段进行PCR扩增后的产物；13为利用生物素标记分离的单链DNA；14为文库构建最终产物，即单链环化DNA。
图4为实施例中文库构建最终产物的电泳结果；左边泳道为单链环状DNA文库，右边泳道为Low Range RNA Ladder(FERMENTAS)。
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、基因组打断
取来源于人淋巴组织的炎黄细胞基因组DNA 1ug，用TE缓冲液稀释至80ul，加入Covaris专用打断平板，选用Covaris LE220超声打断仪，将DNA打断至约200bp到400bp之间。参数如下表所示：
填充系数 21％
压力(PIP) 500
脉冲系数 500
打断时间 20s，6次
将打断完成的基因组吸入新的1.5ml不粘EP管，加入64ul的Ampure xp磁珠，混匀，室温静置5分钟后，置入磁力架吸附磁珠。5分钟后，吸取上清置于另一个新的不粘EP管，再加入32ul的Ampure xp磁珠，混匀，室温放置5分钟。置入磁力架吸附磁珠5分钟，弃去上清，用75％乙醇洗磁珠两次；晾干后加入42ul TE缓冲溶液或无酶水，混匀后放置5min溶解回收产物。
2、末端修复
预先配制末端修复的反应预混液，如下表所示：
10x多核苷酸激酶缓冲液 5.5ul
25mM三磷酸脱氧核苷酸 3ul
T4 DNA聚合酶 3ul
T4多核苷酸激酶 3ul
Klenow片段 1ul
总体积 15.5ul
在上述步骤得到的DNA加入预混液，混匀，置于20℃孵育30分钟。取出后，加入60ulAmpure xp磁珠，混匀，室温静置5分钟。弃去上清，用75％酒精清洗两次。晾干，加入42ul TE缓冲液溶解(此为磁珠纯化标准步骤，如不做特殊说明，都用的是这种方式做磁珠纯化，下同，不再赘述)。
3、末端加“A”
按下表配制预混液：
10xBlue缓冲液(Enzymatics) 5ul
10mM ATP 1ul
Klenow(3`-5`exo-)酶 2ul
无酶水 2ul
总体积 10ul
在上述步骤得到的DNA加入预混液，混匀，置于37℃孵育30分钟，用50ul Ampure xp磁珠纯化，晾干后溶解于42ul TE缓冲液。
4、连接接头元件1
所述接头元件1的序列结构如附图1所示，其中标签序列N为5’-TGTCATAAAT-3’。
按下表提前配制预混液：
10x连接缓冲液(Enzymatics) 5ul
接头元件1(20uM) 2ul
总体积 7ul
将上述预混液加入上一步所得DNA溶液，混匀后，加入2ul T4 DNA连接酶，再次混匀后置于20℃孵育1小时。用Ampure XP磁珠纯化后，溶于42ul TE。
5、PCR扩增
引物1的序列为：5’-AGGUCGCCAGCCCUCAGTAC-3’；
引物2的序列为：5’-AGGGCUGGCGACCUTGTCAG-3’。
按下表提前配制反应预混液：
Figure PCTCN2014091953-appb-000003
在上述步骤得到的DNA溶液中加入预混液，混匀。置于PCR仪中进行PCR反应，程序如下：
Figure PCTCN2014091953-appb-000004
反应完成后使用100ul Ampure XP磁珠进行纯化，溶于82ul TE。
6、去尿嘧啶
按下表提前配制反应预混液：
无酶水 5.8ul
10X Taq缓冲液(Takara) 11ul
USER酶(1000U/ml) 13.2ul
总体积 30ul
将所配制的反应预混液加入上步骤反应产物中，混匀后置于37℃反应1h。
7、双链环化
配制以下反应体系1：
无酶水 1520ul
10×TA缓冲液(epicentre公司) 180ul
总体积 1700ul
将上一步骤反应产物加入反应体系1中，混匀后置于60℃反应30min。反应完成后置于24℃。
配制以下反应体系2：
无酶水 194ul
T4 DNA连接酶(快速)(600U/ul)(enzymatics公司) 6ul
总体积 200ul
将反应体系2加入反应体系1中，置于24℃反应1h。
取反应产物，加入1320ul Ampure XP磁珠，混匀后放置5分钟；置入磁力架后收集上清，在上清中加入680ul Ampure XP磁珠，混匀后放置5分钟；置入磁力架吸去上清，用75％乙醇洗磁珠两次；晾干后加入65ul TE缓冲溶液或无酶水，混匀后放置5分钟溶解回收产物。
8、线性消化
按下表提前配制反应预混液：
Figure PCTCN2014091953-appb-000005
将所制备的反应预混液加入上一步所得到的DNA溶液中，混匀后置于37℃反应1h。
使用80ul Ampure XP磁珠纯化。使用42ul TE缓冲液或无酶水溶解回收产物。
9、Ecop15酶切处理
按下表所示提前配制反应预混液：
无酶水 274ul
10X NEBuffer3.1(NEB) 36ul
Ecop15 I内切酶(10U/ul) 10ul
总体积 320ul
将反应预混液加入上一步所得到的DNA溶液，混匀，置于37℃反应16h。
反应完成后，取反应产物，加入415ul Ampure XP磁珠，混匀后放置5min；置入磁力架后收集上清，在上清中加入296ul Ampure XP磁珠，混匀后放置5min；置入磁力架上吸去上清，用75％乙醇洗磁珠两次；晾干后加入42ul TE缓冲溶液或无酶水，混匀后放置5min溶解回收产物。
10、末端修复
按下表预先配制末端修复的反应预混液：
10x多核苷酸激酶缓冲液 5.5ul
25mM三磷酸脱氧核苷酸 3ul
T4 DNA聚合酶 3ul
T4多核苷酸激酶 3ul
Klenow片段 1ul
总体积 15.5ul
在上述步骤得到的DNA溶液中加入所配制的反应预混液，混匀，置于20℃孵育30分钟。完成用60ul的Ampure xp磁珠纯化。晾干后，加入42ul TE缓冲液溶解。
11、末端加“A”
按下表所示提前配制预混液：
10xBlue缓冲液(Ezymatics) 5ul
10mM ATP 1ul
Klenow(3`-5`exo-)酶 2ul
无酶水 2ul
总体积 10ul
在上述步骤得到的DNA溶液中加入如上配制的反应预混液，混匀，置于37℃孵育30分钟，用50ul Ampure xp磁珠纯化，晾干后溶解于42ul TE缓冲液。
12、连接接头元件2
接头元件2的序列结构如附图2所示。
按下表所示提前配制预混液：
Figure PCTCN2014091953-appb-000006
Figure PCTCN2014091953-appb-000007
将上述预混液加入上一步所得DNA溶液，混匀后，加入2ul T4 DNA连接酶，再次混匀后置于20℃孵育1小时。用Ampure XP磁珠纯化后，溶于42ul TE。
13、PCR扩增
引物3的序列为：5’-TCCTAAGCACGCTTGGCCT-3’，其5’端第一个碱基T带有生物素标记；
引物4的序列为：5’-CATGTAGTGTACGATCCGACTT-3’，其5’端第一个碱基C具有磷酸化修饰。
按下表所示提前配制反应预混液：
Figure PCTCN2014091953-appb-000008
在上述步骤得到的DNA溶液中加入如上配制的预混液，混匀。置于PCR仪中进行PCR反应，PCR程序如下：
Figure PCTCN2014091953-appb-000009
Figure PCTCN2014091953-appb-000010
反应完成后，使用100ul Ampure XP磁珠进行纯化，溶于62ul TE。
14、单链分离与环化
提前将链霉亲和素包裹的磁珠
Figure PCTCN2014091953-appb-000011
MyOneTMStreptavidin，Life technologies)室温平衡半个小时。
吸取30ul上述磁珠加入一新的离心管中，加入120ul 1x磁珠结合缓冲液，混匀，置入磁力架静置5分钟，待上清变澄清后吸去上清。再加入30ul 1x磁珠结合缓冲液重悬磁珠备用。
将上一步骤产生的PCR产物用20ul 4x磁珠结合缓冲液稀释，混匀，加入到重悬后的磁珠，混匀，室温结合5分钟，再置入磁力架静置5分钟，待上清变澄清后吸去上清。
用1ml 1x磁珠洗涤缓冲液重悬磁珠，再置入磁力架静置5分钟以吸去上清。重复一次。向管中加入78ul 0.1M的氢氧化钠溶液重悬磁珠，静置5分钟后置入磁力架，5分钟后吸取上清加入新的离心管中，再在其中加入37.5ul MOPS缓冲液，混匀。
如下表所示提前配制环化反应预混液1(其中桥序列(又可称为介导片段)如SEQ ID NO：9所示，即5’-GTACACTACATGTCCTAAGCACGC-3’)：
无酶水 43ul
桥序列 20ul
总共 63ul
将如上配制的预混液加入上述分离出来的DNA单链溶液中，混匀。
如下表所示配制环化反应预混液2：
Figure PCTCN2014091953-appb-000012
将反应预混液2加入到DNA单链溶液中，混匀，置于37℃孵育1.5小时。
15、单链纯化
如下表所示提前配制酶切反应液：
Figure PCTCN2014091953-appb-000013
向上一步的约350ul的样品反应液中加入20ul的酶切反应液，混匀，继续于37℃孵育30分钟。
反应完成后，向反应液中加入15.4ul的500mM的乙二胺四乙酸，混匀。
使用800ul Ampure XP磁珠纯化回收，待晾干后加入40ul的TE缓冲液溶解环状DNA文库。
在6％的尿素变性PAGE胶检测构建好的文库，结果见附图4。
图4电泳结果显示：所得单链环状DNA片段集中，是质量非常高的文库；证明本发明所采用的技术方法是完全可行的。
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。

Claims (10)

  1. 一种测序文库的构建方法，其特征在于，包括以下步骤：
    1)将双链DNA进行片段化，并对所得DNA片段进行平端修复、5’末端磷酸化和3’末端加碱基A；
    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；所述接头元件1包含一条核酸长链和一条核酸短链；所述核酸长链和核酸短链可退火形成两端序列互补、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；所述接头元件1中具有III类限制性内切酶识别位点；
    3)以步骤2)所得DNA片段为模板，以分别针对接头元件1的核酸长链和核酸短链的部分序列的两条序列不同的核酸单链为引物，进行PCR扩增；
    所述两条引物的中部具有酶作用位点；优选地，所述酶作用位点为U或dU，对应的酶为USER酶；
    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，回收酶切后的DNA片段；
    6)对步骤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包含一条核酸长链和一条核酸短链；所述核酸长链和核酸短链可退火形成两端序列互补、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；
    所述接头元件2的序列不同于接头元件1的序列；
    8)以步骤7)所得DNA片段为模板，以分别针对接头元件2的核酸长链和核酸 短链的部分序列的两条序列不同的核酸单链为引物，进行PCR扩增；其中一条引物的5’端第一个碱基带有磷酸化修饰，另一条引物的5’端第一个碱基带有生物素标记；
    9)利用亲和素磁珠回收步骤8)所得PCR产物，并进行变性处理，分离回收非生物素标记的核酸单链；
    优选地，所述亲和素磁珠为连霉亲和素磁珠；
    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；
    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。
  2. 根据权利要求1所述的构建方法，其特征在于，步骤1)中，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，对待测DNA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待测DNA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。
  3. 根据权利要求1或2所述的构建方法，其特征在于，所述接头元件1中的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；
    优选地，所述III类限制性内切酶的识别位点距离所述杂交体的3’末端0-2 bp；
    优选地，所述接头元件1的核酸长链中包含标签序列；进一步优选地，所述标签序列长度为6-10nt。
  4. 根据权利要求1-3任一项所述的构建方法，其特征在于，所述接头元件1的核酸长链的序列为：
    5’-/Phos/ACTGCTGAGTCGAGA(N)CTGACAAGGTCGCCAGCCCTGAGTGCTTCGAA-3’；其中/Phos/表示磷酸化修饰，N为标签序列；优选地，所述标签序列长度为6nt-10nt；进一步优选地，所述标签序列为5’-TGTCATAAAT-3’；
    其核酸短链的序列为：
    5’-CGAAGCACTCA AGGTCGCCAGCCCTCAGTACGTCAGCAGTT-3’。
  5. 根据权利要求4所述的构建方法，其特征在于，步骤3)中，PCR扩增所用引物分别为：
    正向引物：5’-AGGUCGCCAGCCCUCAGTAC-3’；
    反向引物：5’-AGGGCUGGCGACCUTGTCAG-3’。
  6. 根据权利要求1-5任一项所述的构建方法，其特征在于，所述接头元件2的核酸长链的序列为：
    5’-/Phos/AGTCGGAGGCCAAGCGTGCTTAGGATGAGTGCTCTCGAA-3’，其中/Phos/表示磷酸化修饰；
    所述核酸短链的序列为：
    5’-CGAGAGCACTCCATGTAGTGTACGATCCGACTT-3’。
  7. 根据权利要求6所述的构建方法，其特征在于，步骤8)中，PCR扩增所用引物分别为：
    正向引物：5’-TCCTAAGCACGCTTGGCCT-3’；
    反向引物：5’-CATGTAGTGTACGATCCGACTT-3’；
    所述正向引物的5’端第一个碱基带有生物素标记，所述反向引物的5’端第一个碱基具有磷酸化修饰；
    优选地，步骤10)中，所述介导片段的序列为：5’-GTACACTACATGTCCTAAGCACGC-3’。
  8. 一种测序文库，其特征在于，由权利要求1-7任一项所述构建方法制得。
  9. 如权利要求8所述测序文库在基因组测序、优选地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。
  10. 一种核酸测序方法，其特征在于，包括将权利要求8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。
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