WO2020135650A1 - 一种基因测序文库的构建方法 - Google Patents

一种基因测序文库的构建方法 Download PDF

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WO2020135650A1
WO2020135650A1 PCT/CN2019/128947 CN2019128947W WO2020135650A1 WO 2020135650 A1 WO2020135650 A1 WO 2020135650A1 CN 2019128947 W CN2019128947 W CN 2019128947W WO 2020135650 A1 WO2020135650 A1 WO 2020135650A1
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transposase
complex
sequencing
target dna
sequence
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French (fr)
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樊隆
夏俊秋
刘家栋
蒋浩君
吴政宪
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江苏金斯瑞生物科技有限公司
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    • C12N15/09Recombinant DNA-technology
    • C12N15/10Processes for the isolation, preparation or purification of DNA or RNA
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    • C40BCOMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06Biochemical methods, e.g. using enzymes or whole viable microorganisms

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  • the invention relates to the field of sequencing technology, in particular to a method for constructing a gene sequencing library.
  • NGS Next generation sequencing
  • RNA-seq to discover new transcriptome-level variations, or accurately quantify the expression of mRNA
  • Analyze epigenetic factors such as various methylation of DNA, and the interaction between DNA and protein
  • accurately sequence cancer and find mutation sites for use in precision medicine and individualized treatment of cancer.
  • sequencers such as Miseq, Nextseq, and Hiseq developed by Illumina use sequencing-by-synthesis (SBS) technology to support large-scale parallel sequencing, which is obtained with the advantages of high throughput, low cost, and short cycle time. A wide welcome.
  • SBS sequencing-by-synthesis
  • Sequencing library construction technology based on transposase interruption can simultaneously realize DNA fragmentation and addition of linkers.
  • Such methods have been reported.
  • Chinese patent CN105525357B discloses a library construction using transposase embedding complex The method can greatly reduce the sample processing time.
  • the DNA fragmentation achieved by the transposase is related to the initial amount of target DNA, more target DNA starting amount will cause the library fragments obtained by the transposase after the DNA fragmentation to be larger, which cannot satisfy the subsequent Sequencing requires a range of library fragments; at the same time, different amounts of target DNA can be used to construct a library based on transposase library construction. Therefore, the current library construction based on transposase interruption requires a certain amount of samples, and the final library is accurately quantified for downstream sequencing.
  • the conventional homogenization method estimates the amount of DNA contained by the absorbance value, so as to draw equal or equal proportions of samples to achieve the purpose of homogenization.
  • the method of absorbance value or fluorescence quantification will be subject to other similar absorption specific Spectral effects such as proteins, other types of nucleic acids or qualities, and fluorescence quantification have the disadvantages of high cost, cumbersome and time-consuming operations; the existing homogenization process can be defined as three steps of quantification-calculation-absorption.
  • the operation time for quantifying 96 samples varies from several minutes to 3 hours due to different instrument platforms; in the calculation process, it takes about 1 hour to enter the concentration of each sample and calculate the specific suction sampling volume; adjustment The pipette draws the corresponding calculated amount of samples independently from each sample to achieve the homogenization between the samples and then the downstream library construction process, which takes 1 hour. Therefore, according to the existing technical process, the entire homogenization process takes 5 hours. When constructing a large-volume sample library, this step is time-consuming and cumbersome. Although it is now assisted by automated instruments, the accompanying cost will be further increased.
  • the invention provides a method for constructing a gene sequencing library.
  • the method includes:
  • each transposase embedding complex contains a transposase and also contains A first linker sequence and/or a second linker sequence;
  • the first linker sequence includes a first sequencing linker sequence and a transposase recognition sequence, and the second linker sequence includes a second sequencing linker sequence and a transposase recognition sequence;
  • the magnetic particles in the complex are combined with the transposase through nickel ion (Ni2+)-histidine interaction;
  • the method includes:
  • Magnetic particles and transposase-embedded complex combine in a certain ratio to form a complex
  • the complex includes magnetic particles and a transposase embedded complex;
  • the transposase embedded complex includes a transposase, a transposase recognition sequence, a first sequencing linker sequence, and/or a second sequencing linker Sequence;
  • the PCR primer includes a front primer containing a first sequencing tag sequence and a rear primer containing a second sequencing tag sequence.
  • the method does not include the step of quantifying the target DNA contained in the target DNA sample.
  • the magnetic particles are magnetic beads that chelate divalent metal cations; as a preferred embodiment of the present invention, the magnetic particles chelate the dinitrogen triacetate (NAT) by coupling matching sites Valence metal cation; more preferably, the divalent metal cation is a divalent nickel ion (Ni 2+ ).
  • the transposase embedding complex is unpurified before contact with magnetic particles.
  • the transposase is a transposase with a protein purification tag; as a preferred embodiment of the present invention, the protein tag is a poly-histidine tag (His-tag); preferably The transposase is Tn5 transposase.
  • the method further includes (3) separating the complex from the reaction system of (2) after incubation; and (4) performing PCR amplification using the complex as a template.
  • the PCR uses a front primer comprising a first sequencing tag sequence and a back primer comprising a second sequencing tag sequence
  • the transposase embedding complex is combined with the magnetic particles in a ratio of 60U: 0.5 mg to 2100U: 0.5 mg through the transposase; as a preferred embodiment of the present invention, the ratio is 750U: 0.5 mg.
  • the magnetic particles and the target DNA sample are incubated with shaking at room temperature at a low imidazole concentration; as a preferred embodiment of the present invention, the low imidazole concentration is 15Mm-50Mm; preferably 15Mm.
  • the incubation conditions of the complex and the target DNA sample are shaking speed 700-2000 rpm; preferably 1100 rpm; shaking time 20-40 min; preferably 30 min.
  • the target DNA used in the present invention may be a plasmid, genomic DNA, or amplified DNA, etc.; wherein, the sample source of genomic DNA may be a cell, tissue, or trace DNA sample.
  • the linker sequence and the PCR primer are selected from sequencing linker sequences of the Illumina Nextera sequencing scheme.
  • the tag sequence is a fixed sequence of 6 to 12 bases; as a preferred embodiment of the present invention, the tag sequence is a fixed sequence of 8 bases.
  • the transposase recognition sequence is the 19-bp chimeric end of the transposon recognized by the transposase Tn5.
  • the method of the invention can be used for sample processing of a new generation high-throughput Illumina sequencing platform.
  • the new generation of high-throughput Illumina sequencing platforms include but are not limited to Miseq, Hiseq, and Nextseq sequencing platforms.
  • the first linker sequence is annealed to the complementary sequence of the transposase recognition sequence to form a first linker
  • the second linker sequence is annealed to the complementary sequence of the transposase recognition sequence to form a second linker
  • the sequence complementary sequence has a base sequence shown by a transposase recognition sequence-reverse (ME-R, that is, a transposase recognition sequence complementary sequence)
  • the first linker sequence has a base sequence shown by Adapter-A
  • the second linker sequence has the base sequence shown by Adapter-B.
  • ME-R is 5'-phos-CTGTCTCTTATACACATCT-3' (SEQ ID NO: 1); wherein, phos is a 5'end phosphorylation modification mark.
  • Adapter-A is
  • the underlined part is the transposase recognition sequence.
  • Adapter-B is
  • the PCR forward primer has the base sequence shown by Primer-F
  • the PCR reverse primer has the base sequence shown by Primer-R.
  • Primer-F is
  • NNNNNN is the first tag sequence, and each N can be selected from any one of A, T, C, and G.
  • Primer-R is
  • NNNNNN is the second tag sequence, and each N can be selected from any one of A, T, C, and G.
  • the method for constructing a sequencing library based on the interruption of immobilized transposase was invented on the basis of the combination of magnetic beads and protein, and the existing method for constructing a sequencing library based on the transposase interruption was optimized to make the final
  • the size and quality of the obtained library fragments are basically not affected by the initial amount of target DNA, which effectively solves the problems of uniform library quality and uniform library size for large-scale NGS library construction.
  • the homogenization of conventional DNA libraries requires a process of quantification-calculation-absorption. The above operations on large-scale samples will take longer and cost more.
  • the invention can complete the sequencing library based on transposase interruption within 3.5 hours
  • the construction of a uniform process greatly shortens the time for sample pre-processing and post-building processing, while saving reagents and labor costs.
  • the method for homogenizing NGS libraries based on immobilized transposase interruption provided by the present invention solves the shortcomings such as high cost, time-consuming and cumbersome operation when constructing large-scale NGS libraries, and its design Unique and easy to operate.
  • Figure 1 shows the construction process of a traditional transposase-based DNA library.
  • FIG. 2 is a process for building a homogenized DNA library based on transposase of the present invention.
  • Figure 3 is a sample of the present invention by adjusting the ratio of magnetic beads and transposase embedding complex in the magnetic bead complex, using the magnetic bead complex for library construction, and finally comparing the size of the DNA library fragment with the ratio Figure.
  • FIG. 4 is a comparison diagram of DNA library fragment sizes obtained by using the method of the present invention and a conventional library building method based on transposase interruption while using different initial amounts of RCA samples in the present invention.
  • FIG. 5 shows the comparison of the quality of the DNA library obtained by the method of the present invention and the conventional library building method based on transposase interruption while using different initial amounts of plasmid samples in the present invention.
  • the traditional transposase-based DNA library construction process includes DNA template quantification, transposase embedding adaptor, transposase embedding complex and a certain amount of DNA template for transposition reaction, PCR enrichment , Magnetic beads purification, library quantification and other steps.
  • the transposase embedding complex formed by embedding the sequencing adaptor and transposase in the present invention passes the poly-histidine tag (His-tag) of the transposase and Ni 2+ on the surface of the magnetic bead Combined, by adjusting the ratio of the amount of input between the two, the size of the DNA fragments formed after the target DNA is interrupted by the transposase embedded complex is controlled; at the same time, because the number of transposase embedded complex attached to the magnetic beads For immobilization, by removing the magnetic beads from the solution, a fixed amount of DNA corresponding to the number of transposase embedded complexes can be obtained. In view of these two points, DNA libraries with similar size ranges and the same quality can be finally obtained.
  • His-tag poly-histidine tag
  • transposase (20 U/ ⁇ L) and 10 ⁇ L of the above-mentioned diluted adapter mixture (the concentration of each annealed adapter is 10 ⁇ M) and mix them in equal volume. Incubate on a PCR machine at 25°C for 60 min, then cool to 4°C to form The transposase embeds the complex, which is stored at -20°C until use.
  • washing buffer 100 mM Na 3 PO 4 , 600 mM NaCl, 0.05% Tween 20, 50 mM imidazole, pH 8.0, 25° C.
  • TAPS 200 mM TAPS-NaOH (pH 8.5, 25° C.), 25 mM MgCl 2 and 50% DMF (dimethylformamide).
  • the 10x P2 buffer, dNTP, and P2 polymerase used in the examples are manufactured by Genscript Corporation.
  • This example compares the size of the library fragments obtained by the interruption of the same sample by the magnetic bead complex formed by the combination of different amounts of transposase embedding complex and the magnetic beads.
  • the magnetic bead complex used in this example is as follows:
  • Fig. 3 shows the results of DNA library fragment sizes obtained after the formation of a library of magnetic bead complexes formed by the combination of different amounts of transposase embedding complexes and the same amount of magnetic beads to the target DNA.
  • products of rolling circle replication rolling circle amplification technology, RCA
  • libraries of different starting amounts are constructed using the method of the present invention and the conventional library building method based on transposase interruption.
  • the target DNA used in the test is a sample of the well-known plasmid pUC57.
  • the plasmid has a total length of 2710 bp.
  • test group one and the control group respectively use the method of the present invention (as described above in the "Example Method of the Invention") and the conventional library-based method based on the transposase interruption of library construction as described below.
  • TAPS 200 mM TAPS-NaOH (pH 8.5, 25° C.), 25 mM MgCl 2 and 50% DMF (dimethylformamide).
  • the 10x P2 buffer, dNTP, and P2 polymerase used in the examples are manufactured by Genscript Corporation.
  • Fig. 4 shows the results of the DNA library fragment size of the method of the present invention and the conventional transposase interruption-based library construction method for different starting amounts of target DNA.
  • results in FIG. 4 show that the method of the present invention can be used to effectively input different amounts of target DNA, and the resulting library fragments are similar in size.
  • the same plasmid sample is used, and the library of three different starting amounts of target DNA is constructed by the method of the present invention, and the conventional library construction method based on transposase interruption is used for library construction.
  • the plasmid samples used and the library construction method are the same as in Example 2.
  • Fig. 5 shows the results of the DNA library quality of the method of the present invention and the conventional transposase interruption-based library construction method for different starting amounts of target DNA.
  • the results in FIG. 5 show that, with the method of the present invention, a DNA library with the same library quality can be obtained even when the target DNA starting amount is different.
  • This example compares the total time required for library construction on the same batch of 96 plasmids using the method of the present invention and the conventional library construction method based on transposase interruption. It can be seen that the method of the present invention takes significantly less time.

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Abstract

本申请提供了一种基因测序文库的构建方法,属于基因测序领域,该方法包括将磁性粒子与转座酶包埋复合物结合形成复合体,并用该复合体与待测序的靶DNA样品孵育,产生两端带有接头的DNA文库。

Description

一种基因测序文库的构建方法 技术领域
本发明涉及测序技术领域,尤其涉及一种基因测序文库的构建方法。
背景技术
二代测序技术(Next Generation Sequence,NGS)以高通量、低成本的优势,自出现之日起就倍受欢迎。随着技术的发展,新一代测序技术在许多科学研究和临床检测方面都有应用。
目前很多科学研究与临床应用需要快速对目标的全基因组进行测序,或者对感兴趣的目标区域进行深度测序;利用RNA-seq发现新的转录组水平上的变异,或者精确定量mRNA的表达量;分析表观遗传学因素,例如DNA的各种甲基化、DNA与蛋白之间的相互作用;对癌症进行准确测序,寻找变异位点,以便用于精准医疗,个体化治疗癌症。
测序技术方面,Illumina公司研发的Miseq、Nextseq和Hiseq等测序仪,采用边合成边测序(Sequencing by Synthesis,SBS)技术,支持大规模平行测序,以高通量、低成本、周期短的优势得到了广泛的欢迎。
在实际利用测序的完成过程中,很多时候对时效性要求相当高,需要在基因检测的每一个环节都尽可能缩短时间。
基于转座酶打断的测序文库构建技术,能够同时实现DNA片段化和接头的添加,此类方法己经有报道,比如中国专利CN105525357B公开了一种利用转座酶包埋复合体进行文库构建的方法,能够极大的减少样品处理的时间。但是,由于通过转座酶实现的DNA片段化与靶DNA的起始量有关,更多的靶DNA起始量会造成转座酶在实现DNA片段化后得到的文库片段更大,不能满足后续测序对于文库片段大小范围的要求;同时,不同起始量的靶DNA进行基于转座酶的文库构建后会得到不同量的DNA文库。因此,目前基于转座酶打断的文库构建,需要一定量的样本进行,并且对最终得到的文库进行精确定量,以便下游进行测序。
常规的均一化方法,通过吸光值高低估算含有DNA量的高低,从而来吸取等量或等比例的样本,实现均一化的目的,然而通过吸光值或荧光定量的方法,会受其他同样吸收特定光谱如蛋白、其他类型核酸或质的影响,而荧光定量存在成本高,操作繁琐费时的缺陷;现有的均一化过程可以定义成定量-计算-吸取三个步骤。定量96个样本的操作时间由于各种仪器平台的不同,由几分钟到3个小时不等;计算环节,录入各样本的浓度并计算具体的吸取样本量,需要耗时约1个小时;调整移液器,从每个样本中独立吸取相应计算量的样本,实现样本之间均一化后进行下游文库构建流程,此过程需要1个小时。因此按照现有的技术流程,整个均一化的过程需要5个小时时间。在进行大批量样本文库构建时,该步骤耗时长且繁琐,虽然现在有自动化仪器的辅助,但随之的成本也将进一步提高。
发明内容
本发明提供一种构建基因测序文库的方法,所述方法包括:
(1)将磁性粒子与转座酶包埋复合物接触,使使得磁性粒子与转座酶包埋复合物形成复合体;其中,每个转座酶包埋复合物包含转座酶,还包含第一接头序列和/或第二接头序列;所述第一接头序列包含第一测序接头序列和转座酶识别序列,所述第二接头序列包含第二测序接头序列和转座酶识别序列;
其中,复合体中的磁性粒子与转座酶之间通过镍离子(Ni2+)-组氨酸相互作用结合;
(2)将(1)中的复合体与靶DNA样品孵育,产生两端带有接头的DNA文库。
根据本发明提供的一种基因测序文库的构建方法,所述方法包括:
(1)磁性粒子与转座酶包埋复合物以一定比例结合形成复合体;
(2)将(1)中的复合体与靶基因孵育;
(3)将复合体从(2)中的反应体系中分离出来;
(4)将(3)中的复合体和带有标签序列的接头序列的引物PCR扩增及纯化;
其中,所述复合体包括磁性粒子和转座酶包埋复合物;所述转座酶包埋复合物包括转座酶、转座酶识别序列、第一测序接头序列和/或第二测序接头序列;所述PCR引物包括含有第一测序标签序列的前引物和含有第二测序标签 序列的后引物。
在一些实施方案中,该方法不包括对靶DNA样品中所含的靶DNA定量的步骤。
在一些实施方案中,,所述磁性粒子为螯合二价金属阳离子的磁珠;作为本发明的优选实施方案,所述磁性粒子通过偶联匹配位的氮川三乙酸(NAT)螯合二价金属阳离子;更优选地,所述二价金属阳离子为二价镍离子(Ni 2+)。
在一些实施方案中,所述转座酶包埋复合物在与磁性粒子接触之前是未经纯化的。
在一些实施方案中,,所述转座酶为带有蛋白纯化标签的转座酶;作为本发明的优选实施方案,所述蛋白标签为多聚组氨酸标签(His-tag);优选地,所述转座酶为Tn5转座酶。
在一些实施方案中,所述方法还包括(3)在孵育之后从(2)的反应体系分离复合体;和(4)以复合体作为模板进行PCR扩增。
在一些实施方案中,所述PCR使用包含第一测序标签序列的前引物和包含第二测序标签序列的后引物
在一些实施方案中,转座酶包埋复合物通过转座酶与磁性粒子以60U:0.5mg~2100U:0.5mg的比例相结合;作为本发明的优选实施方案,所述比例为750U:0.5mg。
在一些实施方案中,磁性粒子与靶DNA样品在低咪唑浓度下室温振荡孵育;作为本发明的优选实施方案,所述低咪唑浓度为15Mm-50Mm;优选15Mm。
在一些实施方案中,复合体与靶DNA样品的孵育条件为振荡速度700-2000rpm;优选1100rpm;震荡时间为20-40min;优选30min。
本发明所用的靶DNA可以是质粒、基因组DNA或扩增的DNA等;其中,基因组DNA的样品来源可以是细胞、组织或微量DNA样品等。
作为本发明的优选实施方案,所述接头序列及PCR引物选自Illumina Nextera测序方案的测序接头序列。
作为本发明的优选实施方案,所述标签序列为固定的6~12个碱基的序列;作为本发明的优选实施方案,所述标签序列为8个碱基的固定序列。
作为本发明的优选实施方案,所述转座酶识别序列为转座酶Tn5识别的19bp的嵌合端转座子末端。
本发明的方法可用于新一代高通量Illumina测序平台的样本处理。其中,新一代高通量Illumina测序平台包括并不限于Miseq、Hiseq、Nextseq测序平台。
作为本发明的优选实施方案,第一接头序列与转座酶识别序列互补序列退火形成第一接头,第二接头序列与转座酶识别序列互补序列退火形成第二接头,所述转座酶识别序列互补序列具有转座酶识别序列-反向(ME-R,即转座酶识别序列互补序列)所示的碱基序列;所述第一接头序列具有Adapter-A所示的碱基序列;所述第二接头序列具有Adapter-B所示的碱基序列。
其中,ME-R为5’-phos-CTGTCTCTTATACACATCT-3’(SEQ ID NO:1);其中,phos为5’端磷酸化修饰标志。
其中,Adapter-A为
5’-TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG-3’(SEQ ID NO:2);
其中,下划线部分为转座酶识别序列。
其中,Adapter-B为
5’-GTCTCGTGGGCTCGG AGATGTGTATAAGAGACAG-3’(SEQ ID NO:3);其中,下划线部分为转座酶识别序列。
作为本发明的优选实施方案,所述PCR正向引物具有Primer-F所示的碱基序列,PCR反向引物具有Primer-R所示的碱基序列。
其中,Primer-F为
5’-AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCAGCGTC-3’(SEQ ID NO:4);其中,NNNNNNNN为第一标签序列,每个N可选自A、T、C和G中任一个。
其中,Primer-R为
5’-CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTCTCGTGGGCTCGG-3’(SEQ ID NO:5);其中,NNNNNNNN为第二标签序列,每个N可选自A、T、C和G中任一个。
需要说明的是,本发明中的“第一”和“第二”等概念仅用于区分不同的表述对象,并能理解为有技术含义或有顺序限定的含义。
有益效果
本发明所采用的基于固定化转座酶打断的测序文库构建方法,基于磁珠与蛋白结合的基础上发明,对现有的基于转座酶打断的测序文库构建方法进 行优化,使得最终得到的文库片段大小及文库质量基本不受靶DNA起始量的影响,有效的解决了大规模NGS文库构建的文库质量均一化及文库大小均一化的问题。常规DNA文库均一化需通过定量-计算-吸取的过程,对大规模样本进行上述操作时将耗时较长,成本较贵,本发明可以在3.5小时以内完成基于转座酶打断的测序文库构建均一化过程,大大缩短了样本前处理和建库后处理的时间,同时节约了试剂及人力成本。总体而言,本发明提供的基于固定化转座酶打断的NGS文库均一化的方法,解决了大规模NGS文库构建时样本均一化的成本高,耗时长,操作繁琐等短板,其设计独特,操作简便。
附图说明
图1为传统的基于转座酶的DNA文库构建流程。
图2为本发明的基于转座酶的均一化DNA文库建库流程。
图3为本发明中的样本通过调整磁珠复合体中磁珠与转座酶包埋复合物的比例,使用该磁珠复合体进行文库构建,最终得到的DNA文库片段大小随比例变化的比较图。
图4为本发明中的RCA样本采用不同的起始量,同时进行本发明方法和常规基于转座酶打断的建库方法得到的DNA文库片段大小的比较图。
图5为本发明中的质粒样本采用不同的起始量,同时进行本发明方法和常规基于转座酶打断的建库方法得到的DNA文库质量比较。
具体实施方式
下面通过具体实施方式结合附图对本发明作进一步详细说明。
如图1所示,传统的基于转座酶的DNA文库构建流程包括DNA模板定量、转座酶包埋接头、转座酶包埋复合物与一定量的DNA模板进行转座反应、PCR富集、磁珠纯化、文库定量等步骤。
如图2所示,本发明将测序接头与转座酶包埋形成的转座酶包埋复合物通过转座酶的多聚组氨酸标签(His-tag)与磁珠表面的Ni 2+相结合,通过调整两者之间投入量的比例,控制转座酶包埋复合物进行靶DNA打断后形成的DNA片段大小;同时,因为磁珠上附着的转座酶包埋复合物数量固定,通过将磁珠从溶液中抓取出来,就能够得到固定量的与转座酶包埋复合物数量相应的 DNA量。鉴于此两点,最终能够得到片段大小范围相近、质量相同的DNA文库。
本发明示例方法
1、接头制备:
(1)合成如下接头序列:
ME-R:5’-phos-CTGTCTCTTATACACATCT-3’
Adapter-A:5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3’
Adapter-B:5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3’
(2)用无核酸酶的水将ME-R、Adapter-A、Adapter-B溶解至100μM;
(3)按照下面的体系将对应第一接头序列及第二接头序列相混合:
Figure PCTCN2019128947-appb-000001
(4)将上述混合液放置在PCR仪上,运行以下程序:
温度(℃) 时间(min)
75 15
60 10
50 10
40 10
25 30
4
(5)程序结束后,将Adapter 1和Adapter 2等体积混合成退火接头混合物,并稀释至每个退火接头的浓度为10μM。
2、转座酶包埋:
取30μL转座酶(20U/μL)和10μL上述稀释后得到的接头混合物(每个退火接头的浓度均为10μM)等体积混合,于PCR仪上25℃孵育60min,然后 降温至4℃,形成转座酶包埋复合物,该复合物保存于-20℃备用。
3、磁珠结合:
(1)将Thermo Fishier公司的HisPur Ni-NTA磁珠从冰箱中取出,室温静置30min;
(2)充分振荡混匀HisPur Ni-NTA磁珠,取40μL至一新的1.5mL离心管中,向其中加入160μL结合缓冲液(100mM Na 3PO 4,600mM NaCl,0.05%Tween20,30mM咪唑,pH 8.0,25℃),振荡混匀10s,再置于磁力架上;
(3)待溶液澄清后弃上清,再向其中加入400μL结合缓冲液,振荡混匀10s,置于磁力架上;
(4)待溶液澄清后,弃上清,向磁珠中加入配制的如下结合成分:
成分 体积(μL)
转座酶包埋复合物 50
Tn5保存缓冲液 150
结合缓冲液 200
总计 400
(5)振荡混匀10s,置于涡旋仪上,1100rpm充分振荡混匀30min;
(6)振荡结束后,将离心管置于磁力架上,待溶液澄清后,弃上清;
(7)向磁珠中加入400μL洗涤缓冲液(100mM Na 3PO 4,600mM NaCl,0.05%Tween20,50mM咪唑,pH 8.0,25℃),振荡混匀10s,置于磁力架上,待溶液澄清后,弃上清;
(8)重复上一步;
(9)向磁珠中加入50μL Tn5保存缓冲液,充分振荡混匀10s,形成磁珠复合体,该复合体于4℃保存。
4、转座酶打断:
(1)按照下述体系配制磁珠复合体打断体系:
Figure PCTCN2019128947-appb-000002
Figure PCTCN2019128947-appb-000003
5x TAPS:200mM TAPS-NaOH(pH 8.5,25℃),25mM MgCl 2和50%DMF(二甲基甲酰胺)。
(2)充分吹打混匀,重悬磁珠;
(3)将上述离心管放置PCR仪上,并按照下述程序设置及运行:
温度 时间 循环数
55℃ 10min 1
4℃ 1
5、磁珠清洗:
(1)反应结束后,瞬离,将离心管置于磁力架上;
(2)待溶液澄清后,弃上清;
(3)向磁珠中加入100μL ddH 2O,充分吹打混匀,重悬磁珠;
(4)将离心管置于磁力架上,待溶液澄清后,弃上清;
(5)重复上一步,用小量程枪弃干净上清,保持磁珠置于磁力架上。
6、PCR富集:
(1)合成如下引物:
Primer-F:
5’-AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCAGCGTC-3’
Primer-R:
5’-CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTCTCGTGGGCTCGG-3’
(2)用无核酸酶的水将Primer-F、Primer-R溶解至2μM;
(3)按照下述体系配制PCR反应体系,并充分吹打混匀:
Figure PCTCN2019128947-appb-000004
Figure PCTCN2019128947-appb-000005
注:实例中所用的10x P2缓冲液、dNTP、P2聚合酶为Genscript公司生产。
(4)将磁珠从磁力架上取下,用上述PCR反应体系重悬磁珠,并充分吹打混匀;
(5)将上述PCR管放置在PCR仪上,设置并运行下述程序:
Figure PCTCN2019128947-appb-000006
7、磁珠纯化
(1)将PCR管置于磁力架上,待溶液澄清后,将所有上清转移至一新的离心管中;
(2)向上步离心管中加入30μL纯化磁珠(Yeasen公司生产Hieff NGS DNA分选磁珠),并充分吹打混匀,静置5min;
(3)将离心管置于磁力架上,待溶液澄清后,弃上清;
(4)向磁珠上加入200μL现配的80%乙醇,静置30s后,弃上清;
(5)重复上一步,并用小量程的枪弃干净残留的上清;
(6)将离心管室温静置2~4min,待磁珠稍许干燥后,将其从磁力架上取下,并向其中加入17μL ddH 2O,充分吹打混匀;
(7)室温孵育5min;
(8)将离心管置于磁力架上,待溶液澄清后,取上清16μL置于一新的离 心管中,上清中即构建好的DNA文库。
为了进一步表明本发明所阐述的方法,以下结合附图及实施例对本发明做进一步的阐述。
实施例1
本实施例比较了不同量转座酶包埋复合物与磁珠相结合后形成的磁珠复合体对同样样本的打断建库得到的文库片段大小。
本实例所用的磁珠复合体如下所示:
Figure PCTCN2019128947-appb-000007
图3显示了不同量的转座酶包埋复合物结合相同量的磁珠而形成的磁珠复合体对靶DNA的打断建库后所得DNA文库片段大小的结果。
图3的结果显示,在与磁珠的结合过程中,越多量的转座酶包埋复合物投入,将会形成片段大小更小的DNA文库。
实施例2
本实施例采用靶DNA的滚环复制(滚环扩增技术,RCA)的产物,同时采用本发明方法和常规基于转座酶打断的建库方法进行不同起始量的文库构建。
测试所用的靶DNA为公知的质粒pUC57样本,该质粒全长2710bp,序列如SEQ ID NO:6所示。
测试组一和对照组分别采用本发明方法(如上文“本发明示例方法”所述) 和如下所述的常规基于转座酶打断建库方法创建文库。
Figure PCTCN2019128947-appb-000008
常规基于转座酶打断的建库方法:
1、转座酶打断:
(1)按照下述体系配制转座酶打断体系:
成分 体积(μL)
DNA x
转座酶 1
5x TAPS 2
ddH 2O 7-x
总计 10
5x TAPS:200mM TAPS-NaOH(pH 8.5,25℃),25mM MgCl 2和50%DMF(二甲基甲酰胺)。
(2)充分吹打混匀,短暂离心;
(3)将上述离心管放置PCR仪上,并按照下述程序设置及运行:
温度 时间 循环数
55℃ 10min 1
4℃ 1
2、PCR富集:
(1)合成如下引物:
Primer-F:
5’-AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCAGCGTC-3’
Primer-R:
5’-CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTCTCGTGGGCTCGG-3’
(2)用无核酸酶的水将Primer-F、Primer-R溶解至2μM;
(3)按照下述体系配制PCR反应体系,并充分吹打混匀:
成分 体积(μL)
打断产物 10
10x P2缓冲液 3
dNTP(25μM) 0.75
Primer-F(2μM) 2
Primer-R(2μM) 2
P2聚合酶 1
ddH 2O 11.25
总计 30
注:实例中所用的10x P2缓冲液、dNTP、P2聚合酶为Genscript公司生产。
(4)将上述PCR管放置在PCR仪上,设置并运行下述程序:
Figure PCTCN2019128947-appb-000009
3、磁珠纯化
(1)向上步离心管中加入30μL纯化磁珠(Yeasen公司生产Hieff NGS DNA分选磁珠),并充分吹打混匀,静置5min;
(3)将离心管置于磁力架上,待溶液澄清后,弃上清;
(4)向磁珠上加入200μL现配的80%乙醇,静置30s后,弃上清;
(5)重复上一步,并用小量程的枪弃干净残留的上清;
(6)将离心管室温静置2~4min,待磁珠稍许干燥后,将其从磁力架上取下,并向其中加入17μL ddH 2O,充分吹打混匀;
(7)室温孵育5min;
(8)将离心管置于磁力架上,待溶液澄清后,取上清16μL置于一新的离心管中,上清中即构建好的DNA文库。
图4显示了本发明方法以及常规的基于转座酶打断的建库方法对于不同起始量的靶DNA,最终所得的DNA文库片段大小的结果。
图4的结果显示,采用本发明方法,可以有效的对不同起始量靶DNA的投入,最终得到的文库片段大小相近。
实施例3
本实施例采用同一个质粒样本,采用本发明方法进行三次不同起始量靶DNA的文库构建,并且与之对照的采用了常规基于转座酶打断的建库方法进行文库构建。所用的质粒样本和建库方法均与实施例2相同。
Figure PCTCN2019128947-appb-000010
图5显示了本发明方法以及常规的基于转座酶打断的建库方法对于不同起始量的靶DNA,最终所得的DNA文库质量的结果。
图5的结果显示,采用本发明方法,可以在靶DNA起始量不同的情况下,依然能得到的文库质量相同的DNA文库。
实施例4
本实施例对比了采用本发明方法及采用常规的基于转座酶打断的文库构 建方法对同一批96个质粒进行文库构建所需总耗时。由此可见,本发明方法用时显著更少。
Figure PCTCN2019128947-appb-000011
以上内容是结合具体的实施方式对本发明所作的进一步详细说明,不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干简单推演或替换。

Claims (12)

  1. 一种构建基因测序文库的方法,所述方法包括:
    (1)将磁性粒子与转座酶包埋复合物接触,使得磁性粒子与转座酶包埋复合物形成复合体;
    其中,每个转座酶包埋复合物包含(a)转座酶及(b)第一接头序列和/或第二接头序列;所述第一接头序列包含第一测序接头序列和转座酶识别序列,所述第二接头序列包含第二测序接头序列和转座酶识别序列;
    其中,复合体中的磁性粒子与转座酶之间通过镍离子(Ni 2+)-组氨酸相互作用结合;
    (2)将(1)中得到的复合体与靶DNA样品孵育,产生两端带有接头的DNA文库。
  2. 根据权利要求1所述的方法,其中该方法不包括对靶DNA样品中所含的靶DNA定量的步骤。
  3. 根据权利要求1或2所述的方法,所述磁性粒子为螯合二价镍离子(Ni 2+)的磁珠,优选地,磁性粒子通过偶联匹配位的氮川三乙酸(NAT)螯合二价镍离子。
  4. 根据权利要求1-3中任一项所述的方法,其中,所述转座酶包埋复合物在与磁性粒子接触之前是未经纯化的。
  5. 根据权利要求1-4中任一项所述的方法,其中,所述转座酶带有多聚组氨酸标签;优选地,所述转座酶为Tn5转座酶。
  6. 根据权利要求1所述的方法,所述方法还包括
    (3)在孵育之后从(2)的反应体系分离复合体;和
    (4)以复合体作为模板进行PCR扩增。
  7. 根据权利要求6所述的方法,所述PCR使用包含第一测序标签序列的 前引物和包含第二测序标签序列的后引物。
  8. 根据前述权利要求任一项所述的方法,其中,所述转座酶包埋复合物中所述转座酶与磁性粒子以60U:0.5mg至2100U:0.5mg的比例结合;优选地,所述比例为750U:0.5mg。
  9. 根据权利要求1-8中任一项所述的方法,其中,所述复合体与靶DNA样品的孵育在15~50mM咪唑的存在下进行;优选15mM。
  10. 根据权利要求1-9中任一项所述的方法,所述复合体与靶DNA样品的孵育是在振荡速度为700-2000rpm和震荡时间为20-40min条件下进行;优选的震荡速度是1100rpm;优选的震荡时间是30min。
  11. 根据权利要求1-10中任一项所述的方法,所述靶DNA是质粒、基因组DNA、或DNA扩增产物。
  12. 根据权利要求1-11中任一项所述的方法,所述靶DNA来源于细胞、组织或微量DNA样品。
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