WO2019024341A1 - 一种体液游离dna的文库构建方法及其应用 - Google Patents

一种体液游离dna的文库构建方法及其应用 Download PDF

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WO2019024341A1
WO2019024341A1 PCT/CN2017/113208 CN2017113208W WO2019024341A1 WO 2019024341 A1 WO2019024341 A1 WO 2019024341A1 CN 2017113208 W CN2017113208 W CN 2017113208W WO 2019024341 A1 WO2019024341 A1 WO 2019024341A1
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dna
body fluid
free dna
transposase
library
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王明月
刘龙奇
刘传宇
吴亮
许江山
袁月
商周春
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深圳华大生命科学研究院
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Priority to CN201780097208.1A priority Critical patent/CN111386362A/zh
Priority to US16/766,983 priority patent/US20210317516A1/en
Priority to EP17920120.7A priority patent/EP3719182B1/en
Priority to PCT/CN2017/113208 priority patent/WO2019024341A1/zh
Publication of WO2019024341A1 publication Critical patent/WO2019024341A1/zh

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    • C12Q1/6844—Nucleic acid amplification reactions
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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
    • C12N15/1068—Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C40—COMBINATORIAL TECHNOLOGY
    • 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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    • C12Q2521/00—Reaction characterised by the enzymatic activity
    • C12Q2521/30—Phosphoric diester hydrolysing, i.e. nuclease
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    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
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    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
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    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
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    • C12Q2525/307—Circular oligonucleotides
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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 invention relates to the field of sequencing technology, in particular to a library construction method of body fluid free DNA and its use in prenatal diagnosis and early detection of cancer.
  • T21 chromosomal abnormality Down's syndrome
  • T18 chromosomal abnormality Edward's syndrome
  • T13 chromosomal abnormality Padang's syndrome
  • the human body contains more than 400 cell types, each of which has the same set of genomes, but their gene expression levels have significant cell specificity. This is regulated by a cell-specific epigenome that includes DNA methylation, histone modifications, nucleosome localization, etc., through which information can be directly determined by means of a cell reference epigenome database. source. Recent studies have shown that implicit information in blood free DNA, including methylation and nucleosome localization, can be used for tissue traceability.
  • the methylation of blood free DNA can reflect the methylation level of the source tissue cells to a certain extent, and the degree and location of methylation of different types of tissue cells are different, so it can be used to judge which free DNA comes from Cell type.
  • Lo et al. reported a technique for detecting blood-free DNA methylation: blood-free DNA carries the methylation status of the cells from which it is derived, so it can be processed by bisulfite treatment and sequenced by blood. The basic analysis can be mapped to a specific cell type to achieve tissue traceability (Lo et al., PNAS 112: 5503-5512 (2015)).
  • the study can be used in a variety of diagnoses, including prenatal diagnosis, cancer localization and tumor cell metastasis diagnosis, and immune system rejection in organ transplant patients.
  • this method has the following drawbacks: (1) there are fewer methylation sites on the genome, and the concentration of blood free DNA itself is low, so the detection results are often not accurate; (2) the methylation sequencing step is cumbersome and may be This leads to the loss of a large amount of DNA methylation information, resulting in low sequencing quality and severe noise.
  • nucleosome localization information in cells can be used to distinguish between cell and tissue types.
  • a large number of reports have reported that the length of blood free DNA is mostly concentrated at about 167 bp, which is consistent with the length of a nucleosome DNA, indicating that a large amount of blood free DNA is DNA encapsulated by nucleosomes.
  • Jay Shendure et al. after extracting free DNA from blood, constructed a sequencing library and used deep sequencing strategies to locate nucleosome information of blood free DNA into different types of cells, thereby enabling tissue traceability (Shendure et Al., CELL 164, 57-68 (2016); Speicher et al., Nature Genetics 48, 1273-1278 (2016)).
  • the method directly extracts free DNA in the blood to build a library, and then uses the sequencing data of blood free DNA to analyze nucleosome localization.
  • the naked DNA present in the blood will generate noise, interfere with the localization of normal nucleosome DNA, and ultimately lead to low efficiency of tissue traceability.
  • the method uses deep sequencing strategy, which makes the sequencing cost too high, and it is difficult to form a wide range in commercialization. Applications.
  • the current routine non-invasive prenatal diagnosis and early detection of cancer are completed by first extracting blood free DNA and then performing routine database construction.
  • the present invention aims to provide a method for library construction of bodily fluid free DNA, and its use in prenatal diagnosis and early detection of cancer.
  • the invention directly attacks the body fluid by using the enzyme to fragment the free DNA and add the linker without extracting the free DNA from the body fluid, the steps are simple, the cost of building the library is low, and the micro-banking strategy of the free DNA of the body fluid can be realized. And in the process of building the library, the fragment information of the free nucleosome DNA of the body fluid is retained to the utmost.
  • the present invention achieves the above object by the following technical solutions.
  • the invention provides a method of library construction of free DNA from a body fluid sample, comprising:
  • the body fluid is selected from at least one of blood, urine, and saliva.
  • the body fluid sample there are two kinds of free DNA: one is nucleosome DNA bound to histones, and the other is unbound naked DNA. After the enzyme is applied, the naked DNA is cleaved into fragments of about 50 bp, and the nucleosome DNA may be cleaved into larger fragments due to binding to histones.
  • the enzyme is a transposase or an endonuclease; preferably, the transposase is a Tn5 transposase, and the endonuclease is a MNase or DNase enzyme.
  • both the transposase and the endonuclease can be directly applied to the body fluid sample.
  • the embodiment of the direct humoral challenge using the transposase can directly complete both the DNA fragmentation and the addition of the linker.
  • the embodiment of the Dicer can only be fragmented, and the step of adding a linker needs to be performed separately separately.
  • step (1) comprises: transposing a body fluid sample containing free DNA using a transposase to fragment the free DNA and adding a linker sequence to obtain a linker sequence comprising DNA fragment
  • the step (1) further comprises the step of performing nucleic acid extraction after fragmenting the free DNA and adding a linker sequence.
  • the linker for the transposition reaction is a linker mixture which is prepared by the following steps:
  • the linker mixture is embedded with the transposase to obtain a transposase-embedded complex for use in a transposition reaction.
  • the linker mixture is embedded with a Tagment Enzyme Advanced V5S comprising a transposase; preferably, the volume ratio of the linker mixture to the Tagment Enzyme Advanced V5S is 1:20 to 1:25, preferably It is 1:24.5; preferably, the embedding is carried out at 22-28 ° C, preferably at 25 ° C for 40-80 min, preferably 60 min.
  • the transposase-embedded complex is incubated with the body fluid sample under transposition reaction conditions to effect a transposition reaction.
  • the volume ratio of the transposase-embedded complex to the body fluid sample is 1:50-1:80, preferably 1:62.5;
  • the transposition reaction temperature is 35-40 ° C, preferably 37 ° C;
  • the transposition reaction time is from 55 to 65 min, preferably 60 min.
  • the Tn5 transposase used has the property of randomly cleaving DNA, and the naked free DNA in the plasma (free DNA not entangled in histones) is cleaved by the Tn5 transposase. A small fragment of about 50 bp is formed, and nucleosome DNA (free DNA entangled on histones) remains longer than 50 bp.
  • PE50+10 sequencing DNA fragments of different lengths can be distinguished to select nucleosome DNA sequences, and the apparent information of DNA sequences and regulatory regions such as promoters or enhancers of different genes and transcription can be analyzed. The degree of enrichment of the starting zone. Subsequent use of these apparent information can be used to construct the nucleosome DNA sequence obtained from the library for tissue traceability of blood free DNA.
  • the step (1) comprises: treating a body fluid sample containing free DNA with an endonuclease to fragment the free DNA, and then in the resulting fragmented DNA Adding a linker sequence to both ends to obtain a DNA fragment comprising the linker sequence;
  • the step (1) further comprises the step of performing nucleic acid extraction after fragmenting the free DNA.
  • the amplification in step (2) comprises two amplifications
  • the number N of cycles that need to be supplemented is determined by qPCR as the number of cycles for the second amplification.
  • the method further comprises the step (3): cyclizing and enzymatically cutting the body fluid free DNA library obtained in the step 2);
  • the cyclizing comprises denaturation of the double-stranded DNA in the body fluid free DNA library into single-stranded DNA, and then joining by oligonucleotide complementary pairing with a single-stranded DNA partial region; Further preferably, a mediated fragment: 5'-GCCATGTCGTTCTGTGAGCCAAGG-3' (SEQ ID NO: 4) is used in complementary pairing with the single-stranded DNA to effect cyclization;
  • the digestion is performed using exonuclease I and exonuclease III to remove uncircularized DNA;
  • step (3) further comprises the step of purifying the digested product; preferably, purifying using magnetic beads.
  • the present invention also provides a method of obtaining apparent information of an individual, comprising:
  • the present invention provides a method for constructing a library of free DNA of a body fluid sample according to the first aspect or a method for obtaining an individual's apparent information according to the second aspect, in prenatal diagnosis and early detection of cancer the use of.
  • the present invention also provides a method for prenatal diagnosis or early detection of cancer, which is as first as The method for constructing a library of free DNA of a body fluid sample or the method for obtaining apparent information of an individual as described in the second aspect is achieved.
  • the present invention provides a kit for analyzing bodily fluid free DNA for prenatal diagnosis or early detection of cancer, comprising the reagent, primer, mediated fragment or the method used in the method according to the first aspect A combination of one or more of them.
  • the kit comprises one or more of the following combinations:
  • Primer A 5'-CTGTCTCTTATACACATCT-3' (SEQ ID NO: 1)
  • primer B 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3'
  • primer C 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3' (SEQ ID NO :3);
  • the enzyme and/or reagent required for transposition, PCR amplification, enzymatic cleavage, ligation reaction required for transposition, PCR amplification, enzymatic cleavage, ligation reaction.
  • the invention directly attacks the body fluid by using an enzyme (for example, a transposase or an endonuclease) to fragment the free DNA therein and add a linker, thereby realizing the direct establishment of the free DNA of the body fluid;
  • the method of the invention is not only simple in steps, but also The cost of the library is low, and the loss of information during the sequencing process is also reduced, and the fragment information of the free DNA of the body fluid is retained to the utmost, and the information of the nucleosome DNA is retained. Therefore, the method for constructing a library for free DNA of body fluid provided by the present invention can not only realize a micro-banking strategy for free DNA of body fluid, but also obtain an apparent information of free DNA of body fluid. Organizational traceability can also be achieved by further analyzing and mining the obtained apparent information, especially the nucleosome DNA.
  • an enzyme for example, a transposase or an endonuclease
  • the method provided by the invention provides a new research method for the study of body fluid free DNA, and has a good application prospect in clinical applications such as prenatal diagnosis, early tumor discovery and new disease monitoring.
  • Example 1 is a data diagram of determining the number of cycles of the platform period by qPCR in Example 1;
  • Example 2 is a graph showing a sample amplification curve in Example 1;
  • Example 3 is a graph showing the results of Agilent 2100 detection after partial sample magnetic beads in Example 1;
  • Example 4 is a correlation between data obtained by sampling samples in Example 1;
  • Example 5 is an enrichment diagram of the fragments obtained after sequencing in Example 1 in the transcription initiation region of the housekeeping gene and the silencing gene, respectively;
  • Example 6 is a clustering result of sample sequencing data and tissue sequencing data in Example 1.
  • This embodiment includes a series of steps such as plasma sample preparation, Tn5 direct transposition plasma, transposition DNA amplification, Tn5 database construction, PE50+10 sequencing, and nucleosome fragment screening.
  • the Tn5 transposase was prepared according to the instructions of Vazyme's TruePrep Mini DNA Sample Prep Kit.
  • Filter head filtration method 3 mL of plasma was taken and filtered into a new EP tube (about 0.5 mL) with a 10 mL syringe and a 0.2 ⁇ m filter.
  • 2-part plasma transposition reaction ie, the method of the present invention, directly using plasma for transposition reaction
  • Primer A 5'-CTGTCTCTTATACACATCT-3' (SEQ ID NO: 1)
  • Primer B 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 2)
  • Primer C 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 3)
  • the annealing product 1 and the annealing product 2 were separately vortexed and shaken thoroughly, and the solution was briefly centrifuged to return the solution to the bottom of the tube. Placed in the PCR machine, the following reaction procedure was carried out: 75 ° C for 15 min; 60 ° C for 10 min; 50 ° C 10 min; 40 ° C 10 min; 25 ° C 30 min.
  • the annealed product 1 and the annealed product 2 are mixed in an equal volume and mixed. Name it Adapter Mix and store at -20 °C.
  • Tagment Enzyme Advanced V5S contains 1000 U Tn5 transposase BGI V5S reagent (supplier: BGI, item number: BGE005S).
  • the plasma transposition system (see Table 3) was prepared. After the preparation on ice was completed, the mixture was uniformly mixed, and the transposition reaction was carried out at 37 ° C for 60 min on a constant temperature metal mixer.
  • the DNA solution was the purified DNA solution obtained in the step 3.17 of Example 1, and the DNA solution used in the following Comparative Example 1 was the purified DNA solution obtained in the step 1.4 of Example 1.
  • NNNNNNNNNN is a sequence of 10 random bases, and each sample uses a different tag sequence.
  • the number of cycles corresponding to the plateau fluorescence intensity 1/3 is the number N of cycles that need to be added.
  • N is the number of cycles determined in step 5.2 and the sample amplification curve is shown in Figure 2.
  • the single-strand mediated fragment used in 7.1 is: 5'-GCCATGTCGTTCTGTGAGCCAAGG-3' (SEQ ID NO: 4).
  • EXO I is exonuclease I and EXO III is exonuclease III.
  • the constructed library was prepared as DNB (DNA nanospheres) according to the operating instructions of the BGI-SEQ500 sequencer, wherein a library of 6 ng was used and RCA was reacted for 20 min. Sequencing was then performed on a sequencer using a conventional PE50+10 strategy.
  • sequencing of the blood free DNA library obtained according to the present invention can be achieved by low-depth sequencing.
  • the present comparative example adopts the method of the prior art, that is, the transposition reaction is carried out after directly extracting blood free DNA, as follows:
  • step 1.2 of Example 1 Direct extraction of blood free DNA from the purified plasma in step 1.2 of Example 1 (refer to step 3 of Example 1 for a specific method).
  • step 6.5 dry the ethanol and dry it until the surface of the magnetic beads is not reflective.
  • Example 1 As shown in FIG. 4, a comparative analysis of the results of Example 1 and Comparative Example 1 reveals that it is apparent that the correlation between the samples obtained according to the method (Example 1) of the present invention is higher than that of the existing method (pair The correlation between the samples of ratio 1) is higher.
  • the sample data obtained by the method of the present invention and the data from different tissues of the human body are clustered by a clustering method, indicating that the method of the present invention can capture body fluid free DNA signals from different tissues of the human body. Interest can be further traced to the organization.

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Abstract

一种对体液样本游离DNA进行文库构建的方法，包括利用转座酶或内切酶直接作用于体液样本，将其中游离DNA片段化，然后进行扩增获得文库。还提供了利用该方法进行产前诊断或癌症早期发现的试剂盒。

Description

一种体液游离DNA的文库构建方法及其应用 技术领域
本发明涉及测序技术领域，尤其涉及一种体液游离DNA的文库构建方法及其在产前诊断及癌症早期发现中的用途。
背景技术
1948年，法国科学家曼德尔和麦特斯首次在人体外周血中检测到游离于细胞外的DNA片段。这些DNA主要源于凋亡或坏死细胞被切割后的片段化染色质。
1997年，来自香港中文大学的卢煜明研究团队发现，在怀孕母亲的外周血中存在胎儿的血液游离DNA，由此开启了孕妇外周血基因检测的大门。孕妇外周血的基因检测技术称为无创产前诊断技术，其原理为：怀孕的母亲的外周血中存在胎儿的DNA，可以对怀孕母亲的血液游离DNA进行建库测序，并通过对比与母亲DNA的单核苷酸多态性差异分离出胎儿的DNA，从而进行产前诊断(Lo et al，Am.J.Hum.Genet 64，218-224(1999))。目前这项技术在国内外均比较成熟，主要筛查常见的三大染色体疾病，分别是T21染色体异常(唐氏综合征)，T18染色体异常(爱德华氏综合征)和T13染色体异常(帕陶氏综合征)，检测准确率高达99％以上。
另外，里昂等人确定了癌症病人的血液游离DNA水平与恶性肿瘤的转移程度存在着某种相关性，为癌症的无创诊断提供了理论基础。进而衍生了癌症早期检测技术：在癌症的病理状态下，由于凋亡和坏死细胞数量增多，因此释放到血液中的游离DNA数量明显增加；这些DNA自身携带癌细胞特有的突变，因此能通过检查血液游离DNA的浓度或突变状态，判断癌症类型以及发展进程(Morelli et al.，Ann Oncol 26，731-736(2015))。并且，在癌症患者血液游离DNA上相继检测到了K-ras，EGFR等基因的突变，这些发现能够应用于肺癌及乳腺癌等恶性肿瘤的早期诊断。
应当注意的是，人体包含400多种细胞类型，每种细胞虽然具有相同的一套基因组，但它们的基因表达水平具有显著的细胞特异性。这是由细胞特异性的表观基因组来调控的，表观基因组信息包括DNA甲基化，组蛋白修饰，核小体定位等，通过这些信息并借助细胞参考表观基因组数据库可以直接判断细胞的来源。最新的研究表明，血液游离DNA中隐含表观信息，包括甲基化和核小体定位，因此能够用来进行组织溯源。
其中，血液游离DNA的甲基化能在一定程度上反映来源组织细胞的甲基化水平，而不同类型的组织细胞甲基化程度和位置均不相同，因此可以用于判断游离DNA来自哪种细胞类型。例如，Lo等人报道了血液游离DNA甲基化检测技术：血液游离DNA携带其来源细胞的甲基化状态，因此能够用重亚硫酸盐处理后进行建库测序，通过对血液游离DNA的甲基化分析，可以将其对应到特定的细胞类型上去，从而实现组织溯源(Lo et al.，PNAS 112：5503-5512(2015))。这项研究可用于多种诊断当中，包括产前诊断、癌症定位及肿瘤细胞的转移情况诊断、器官移植患者免疫系统排异性诊断等。但是该方法存在以下弊端：(1)基因组上甲基化位点较少，且血液游离DNA本身浓度就低，因此检测结果往往精确性不高；(2)甲基化测序步骤繁琐，可能会导致大量DNA甲基化信息丢失，产生测序质量低，噪音严重等后果。
此外，细胞中的核小体定位信息可以用来区分细胞和组织类型。大量文献报道，血液游离DNA的长度多集中在167bp左右，这与一个核小体DNA的长度相符合，表明大量血液游离DNA是由核小体包裹的DNA。2016年，Jay Shendure等人通过提取血液中的游离DNA之后，构建测序文库，并采用深度测序策略，将血液游离DNA的核小体信息定位到不同类型的细胞中，进而实现组织溯源(Shendure et al.，CELL 164，57-68(2016)；Speicher et al.，Nature Genetics 48，1273-1278(2016))。但该方法直接提取血液中的游离DNA进行建库，然后使用血液游离DNA的测序数据来分析核小体定位， 而血液中存在的裸露DNA会产生噪音，干扰正常核小体DNA的定位，最终导致组织溯源效率不高；并且，该方法采用深度测序策略，使得测序成本过高，在商业化上难以形成广泛的应用。
另外，还有研究报导了专门针对染色质开放区域的染色质开放性测序技术：通过对细胞进行裂解和Tn5转座酶处理，在染色质中开放区域插入测序接头，然后通过PCR扩增的方法进行全部开放染色质区域的扩增，并进行建库和测序(Chang et al.，Curr.Protoc.Mol.Biol.109：21.29.1-21.29.9)。但是，该技术恰恰是排除了核小体DNA的信息，因此同样不能根据核小体DNA进行组织溯源。
总之，目前常规的无创产前诊断以及癌症早期检测，是通过先提取血液游离DNA、然后进行常规建库来完成，其存在以下问题：(1)血液游离DNA的含量很低，在建库过程中非常容易带来损失，导致建库失败或者检测灵敏度不高；(2)现有的建库方法都是直接提取血液中的游离DNA，再对提取后的DNA进行建库测序。最终只能得到血液游离DNA的序列或甲基化信息，而核小体分布信息可能会在建库的过程中丢失。(3)测序成本过高。因此，亟待开发出一种高效、灵敏的血液游离DNA建库方法，并且能够用这种方法筛选出血液游离DNA的核小体片段，进行组织溯源。
发明内容
针对上述现有技术中存在的缺陷，本发明旨在提供一种对体液游离DNA进行文库构建的方法，及其在产前诊断及癌症早期发现中的用途。本发明使用酶直接攻击体液，以对其中的游离DNA进行片段化并添加接头，而无需从体液中提取游离DNA，其步骤简单，建库成本低，不仅可以实现体液游离DNA的微量建库策略，而且在建库过程中最大限度保留了体液游离核小体DNA的片段信息。
本发明是通过如下技术方案实现上述目标的。
一方面，本发明提供了一种对体液样本游离DNA进行文库构建的方法，其包括：
(1)用酶直接作用于体液样本，以将体液样本中的游离DNA片段化；
(2)对步骤1)所得片段化的DNA进行扩增，获得体液游离DNA文库。
在优选的具体实施方案中，所述体液选自血液、尿液、唾液的至少一种。在所述体液样本中，游离DNA有两种：一种是与组蛋白结合在一起的核小体DNA，一种是无结合的裸露DNA。在酶作用之后，裸露DNA会被切割成大约50bp的片段，而核小体DNA由于与组蛋白结合，其被切割成的片段可能会大一些。
在优选的具体实施方案中，所述酶为转座酶或内切酶；优选地，所述转座酶为Tn5转座酶，所述内切酶为MNase或DNase酶。
请注意，无论是转座酶还是内切酶，都可以直接作用于体液样本，区别在于：使用转座酶直接进行体液攻击的实施方案可以直接完成DNA片段化与加接头两者，而使用内切酶的实施方案仅能完成片段化，添加接头的步骤需要后续单独进行。
在优选的具体实施方案中，步骤(1)包括：利用转座酶，对含有游离DNA的体液样本进行转座反应，以将所述游离DNA进行片段化并添加接头序列，获得包含接头序列的DNA片段；
优选地，步骤(1)还包括：在将所述游离DNA进行片段化并添加接头序列之后，进行核酸提取的步骤。
在进一步优选的实施方案中，用于所述转座反应的接头为接头混合物，其通过以下步骤制备：
1)将引物A：5′-CTGTCTCTTATACACATCT-3′(SEQ ID NO：1)与引物B：5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3′(SEQ ID NO：2)退火，得退火产物1；
2)将引物A：5′-CTGTCTCTTATACACATCT-3′(SEQ ID NO：1)与引物C：5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3′(SEQ ID NO：3)退火，得 退火产物2；
3)将退火产物1和退火产物2混合，得接头混合物。
优选地，所述接头混合物与所述转座酶进行包埋，获得转座酶包埋复合物，以用于转座反应。
进一步优选地，将所述接头混合物与包含转座酶的Tagment Enzyme Advanced V5S进行包埋；优选地，所述接头混合物与所述Tagment Enzyme Advanced V5S的体积比为1∶20-1∶25、优选为1∶24.5；优选地，所述包埋在22-28℃、优选在25℃下进行40-80min、优选60min。
在具体实施方案中，优选地，将所述转座酶包埋复合物与所述体液样本在转座反应条件下进行孵育，以实现转座反应。
对于所述转座反应，优选地，所述转座酶包埋复合物与所述体液样本的体积比为1∶50-1∶80，优选为1∶62.5；
优选地，所述转座反应温度为35-40℃，优选为37℃；
优选地，所述转座反应时间为55-65min，优选为60min。
在采用转座反应的本发明的一个具体实例中，所用Tn5转座酶具有随机切割DNA的特性，血浆中裸露的游离DNA(未缠绕在组蛋白上的游离DNA)会被Tn5转座酶切割成约50bp的小片段，而核小体DNA(缠绕在组蛋白上的游离DNA)会保持长于50bp的长度。通过PE50+10测序等，可以将不同长度特征的DNA片段区分开来，从而筛选出核小体DNA序列，分析得到DNA序列的表观信息以及在不同基因启动子或增强子等调控区以及转录起始区的富集程度。后续可以通过这些表观信息将建库所得核小体DNA序列用于实现血液游离DNA的组织溯源。
在本发明的另一个优选的具体实施方案中，步骤(1)包括：利用内切酶处理含有游离DNA的体液样本，以将所述游离DNA进行片段化，然后在所得片段化DNA的 两端添加接头序列，获得包含接头序列的DNA片段；
优选地，步骤(1)还包括：在将所述游离DNA进行片段化之后，进行核酸提取的步骤。
在优选的具体实施方案中，步骤(2)中所述扩增包括两次扩增；
优选地，在第一次扩增后，通过qPCR确定需要补加的循环数N，作为第二次扩增的循环数。
在优选的具体实施方案中，所述方法还包括步骤(3)：对步骤2)所获得的体液游离DNA文库进行环化和酶切；
优选地，所述环化包括将所述体液游离DNA文库中的双链DNA变性为单链DNA，再通过与单链DNA部分区域互补的寡核苷酸片段、通过碱基互补配对进行连接；进一步优选地，使用介导片段：5′-GCCATGTCGTTCTGTGAGCCAAGG-3′(SEQ ID NO：4)与所述单链DNA互补配对连接以实现环化；
优选地，使用外切核酸酶I和外切核酸酶III进行所述酶切，以去除未环化的DNA；
优选地，步骤(3)还包括：对酶切产物进行纯化的步骤；优选地，使用磁珠进行纯化。
第二方面，本发明还提供了一种获得个体表观信息的方法，其包括：
(1)根据如第一方面所述的方法，获得个体的体液游离DNA文库；
(2)对步骤(1)所得体液游离DNA文库进行测序和分析，以获得个体表观信息。
第三方面，本发明还提供了如第一方面所述的对体液样本游离DNA进行文库构建的方法或如第二方面所述的获得个体表观信息的方法在产前诊断及癌症早期发现中的用途。
第四方面，本发明还提供了一种产前诊断或癌症早期发现的方法，其通过如第一 方面所述的对体液样本游离DNA进行文库构建的方法或如第二方面所述的获得个体表观信息的方法实现。
第五方面，本发明还提供了一种分析体液游离DNA以进行产前诊断或癌症早期发现的试剂盒，其包括如第一方面所述的方法中所使用的试剂、引物、介导片段或其中一项或多项的组合。
优选地，所述试剂盒包括以下一种或多种的组合：
转座酶Tn5或MNase或Dnase酶；
引物A：5′-CTGTCTCTTATACACATCT-3′(SEQ ID NO：1)，引物B：5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3′(SEQ ID NO：2)与引物C：5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3′(SEQ ID NO：3)；
介导片段：5′-GCCATGTCGTTCTGTGAGCCAAGG-3′(SEQ ID NO：4)；以及
所述转座、PCR扩增、酶切、连接反应所需要的酶和/或试剂。
本发明相较于现有技术的优势
如上所述，在现有技术中，通常采用先从体液样本提取游离DNA，再使用Tn5转座酶进行攻击、建库的方法，然而这些方法存在溯源结果精确度不高，且需要深度测序，成本高等缺点。
本发明采用酶(例如转座酶或内切酶)直接攻击体液，以对其中的游离DNA进行片段化并添加接头，实现了体液游离DNA的直接建库；本发明的方法不但步骤简单，建库成本低，而且还降低了测序过程中的信息丢失，最大限度地保留了体液游离DNA的片段信息，尤其是保留了核小体DNA的信息。因此，本发明提供的对体液游离DNA进行文库构建方法，不仅可以实现体液游离DNA的微量建库策略，同时还可获得体液游离DNA的表观信息。通过对所获得的表观信息、尤其是核小体DNA的相关信息进一步分析挖掘，还可实现组织溯源。
本发明提供的方法为体液游离DNA的研究提供了新的研究方法，在产前诊断、肿瘤早期发现和新的疾病监测等临床应用中具有良好的应用前景。
附图说明
图1为实施例1中qPCR确定平台期循环数的数据图；
图2为实施例1中样品扩增曲线图；
图3为实施例1中部分样品磁珠双选后用Agilent 2100检测结果图；
图4为实施例1中样本测序所得数据之间的相关性；
图5为实施例1中测序后所得片段分别在管家基因和沉默基因转录起始区的富集图；
图6为实施例1中样本测序数据与组织测序数据聚类结果。
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 外周血游离DNA的文库构建及表观信息分析
本实施例包括血浆样品准备、Tn5直接转座血浆、转座后DNA扩增、Tn5建库、PE50+10测序、核小体片段筛选等一系列步骤。Tn5转座酶按照Vazyme公司的TruePrep微量DNA样品制备试剂盒(TruePrep Mini DNA Sample Prep Kit)的说明书进行制备。
1血浆样品准备
1.1血浆样品采集
采集健康人全血样本10mL。4℃，1600g离心10min。取上清(即血浆)至新的15mL离心管中。
1.2血浆纯化(可采用以下两种方法中的任意一种)
1.2.1方法一：
滤头过滤法：取3mL血浆，用10mL注射器、0.2μm滤头过滤至新的EP管中(约损失0.5mL)。
1.2.2方法二：
二次离心法：血浆经4℃，16000g离心10min后，取上清至新15mL离心管中。
2部分血浆的转座反应(即本发明的方法，直接采用血浆进行转座反应)
2.1接头混合物(Adapter Mix)的制备
2.1.1参考引物名称及序列：
引物A：5′-CTGTCTCTTATACACATCT-3′(SEQ ID NO：1)
引物B：5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3′(SEQ ID NO：2)
引物C：5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3′(SEQ ID NO：3)
2.1.2使用退火缓冲液溶解引物A、引物B、引物C至100μM。
2.1.3分别配制如下反应体系，见表1：
表1 接头混合物的制备体系
Figure PCTCN2017113208-appb-000001
2.1.4分别将退火产物1和退火产物2涡旋震荡充分混匀，并短暂离心使溶液回到管底。置于PCR仪内，进行如下反应程序：75℃15min；60℃10min； 50℃10min；40℃10min；25℃30min。
2.1.5反应结束后，将退火产物1和退火产物2等体积混合，混匀。命名为接头混合物(Adapter Mix)，-20℃保存。
2.2接头混合物与Tn5转座酶的包埋，以获得转座酶包埋复合物
2.2.1在灭菌PCR管中依次添加各反应组分，见表2；其中，Tagment Enzyme Advanced V5S含1000U Tn5转座酶的BGI V5S试剂(供货商：BGI，货号：BGE005S)。
表2 接头混合物与Tn5转座酶的包埋体系
Figure PCTCN2017113208-appb-000002
2.2.2使用移液器轻轻吹打，充分混匀。
2.2.3将反应置于25℃反应60min。反应产物命名为Tagment Enzyme Advanced Mix V5S，置于-20℃保存。
2.3血液游离DNA片段化
配制血浆转座体系(见表3)，冰上配制完成后，混合均匀，于恒温金属混匀仪上，37℃下进行转座反应60min。
表3 血浆转座反应体系
Figure PCTCN2017113208-appb-000003
Figure PCTCN2017113208-appb-000004
3转座后血浆游离DNA的提取
3.1利用Magen血液游离DNA提取试剂盒(MAGEN MD5432-01)进行提取。
3.2转座反应完成后，在新的1.5mL离心管中，加入25μL蛋白酶K和35μLMagBind磁珠。
3.3转移样品至含蛋白酶K的离心管中。振荡混匀5秒。
3.4加入700μL MLE至样品中，涡旋混匀。55℃振荡温育15分钟。
3.5转移至磁力架上，静置5～10分钟吸附磁珠。
3.6小心吸弃所有溶液。
3.7加入320μL缓冲液MW1，涡旋混匀15秒。
3.8转移至磁力架上，静置3～5分钟吸附磁珠。小心吸弃所有溶液。
3.9加入320μL缓冲液MW2，涡旋混匀15秒。
3.10转移至磁力架上，静置3～5分钟吸附磁珠。小心吸弃所有溶液。
3.11重复步骤3.9-3.10。
3.12短暂离心，收集管壁上的液滴。转移至磁力架上，小心吸弃所有溶液。
3.13空气干燥5-10分钟。
3.14加20μL缓冲液AE，吹打混匀。
3.15室温静置3分钟。
3.16转移至磁力架上，静置3分钟，用同样方法溶解。
3.17转移DNA溶液至新的1.5mL离心管中。
3.18 Qubit检测浓度。
4片段化DNA扩增
4.1按照表4在0.2mLPCR管中配制PCR反应体系。
表4 转座产物第一次扩增反应体系
Figure PCTCN2017113208-appb-000005
*注：其中，DNA溶液为实施例1的步骤3.17所得的纯化后的DNA溶液；而在下述对比例1中所采用的DNA溶液为实施例1的步骤1.4所得的纯化后的DNA溶液。
N5引物：
Pho-GAACGACATGGCTACGATCCGACTTTCGTCGGCAGCGTC(SEQ ID NO：5)；
N7引物：
TGTGAGCCAAGGAGTTGTTGTCTTCNNNNNNNNNNGTCTCGTGGGCTCGG(SEQ ID NO：6)，
其中NNNNNNNNNN为10个随机碱基组成的标签序列，每个样本使用的标签序列均不同。
4.2按照下列参数进行第一次扩增：
Figure PCTCN2017113208-appb-000006
Figure PCTCN2017113208-appb-000007
5 Q-PCR鉴定及二次扩增
5.1配制Q-PCR反应体系(如表5)：
表5 荧光定量PCR鉴定添加循环数反应体系
Figure PCTCN2017113208-appb-000008
5.2按照如下参数进行qPCR：
Figure PCTCN2017113208-appb-000009
如图1，在qPCR线性扩增Rn/Cycle曲线中，平台期荧光强度1/3对应的循环数即为需要补加的循环数N。
5.3按照如下参数进行第二次扩增：
Figure PCTCN2017113208-appb-000010
其中，N为步骤5.2所确定的循环数，样品扩增曲线见图2。
6 XP磁珠双选
6.1检查PCR管中体积，用NF-H2O补充至50μL；
6.2加入40μL磁珠(0.8×)，吹打混匀，室温静置5分钟；
6.3上磁力架2分钟，转移上清至新管中(上清中的DNA片段都小于350bp)；
6.4加入35μL磁珠(0.7×)，混匀，室温静置5min；
6.5上磁力架2分钟，去掉含小DNA片段和RNA的上清；
6.6保持在磁座上，加入150μL预冷的80％乙醇洗两次(30s)；
6.7保持在磁座上5分钟，使得水分蒸发；
6.8为洗脱DNA，加入20μLTE Buffer(AMBION AM9858)，轻轻吹打混匀，室温孵育5min；
6.9上磁力架2分钟，将上清转移到新管中，勿吸到磁珠；
6.10 Qubit dsDNA High sensitivity assay(INVITROGEN Q32854)定量
6.11对样品进行Agilent 2100检测。检测结果见图3。
7文库环化与酶切
7.1所使用的单链介导片段为：5′-GCCATGTCGTTCTGTGAGCCAAGG-3′(SEQ  ID NO：4)。
7.2取324.5ng步骤6纯化所得的DNA溶液，加入5μL单链介导片段(20μM)，用灭菌超纯水补至70μL。
7.3将7.2混合液转移至热循环仪中，95℃反应3min，迅速放置冰上10min。
7.4根据表6配制单链DNA连接反应体系，混合均匀，快速离心3s。
表6 单链DNA连接反应体系
Figure PCTCN2017113208-appb-000011
*注：表6中，DNA混合液为步骤7.3所得溶液。
7.5转移至热循环仪中，37℃反应60min，4℃暂存。
7.6按照表7配制酶切反应体系，混合均匀，快速离心3s。
表7 单链DNA酶切反应体系
Figure PCTCN2017113208-appb-000012
Figure PCTCN2017113208-appb-000013
*注：表7中，EXO I为外切核酸酶I，EXO III为外切核酸酶III。
7.8转移至热循环仪中，37℃反应30min，4℃暂存。
8文库回收
8.1涡旋震荡混匀PEG32磁珠并吸取170μL体积至128μLPCR产物中，使用移液器轻轻吹打10次充分混匀。室温孵育10分钟；
8.2将EP管短暂离心并置于磁力架中分离磁珠和液体。待溶液澄清(约5min)后小心移除上清；
8.3保持EP管始终处于磁力架中，加入200μL新鲜配制的80％乙醇漂洗磁珠。室温孵育30s后小心移除上清；
8.4重复上步，总计漂洗两次；
8.5保持EP管始终处于磁力架中，开盖空气干燥10min；
8.6将EP管从磁力架中取出，加入25μL灭菌超纯水洗脱。使用移液器轻轻吹打充分混匀，室温放置5min。将反应管短暂离心并置于磁力架中分离磁珠和液体。待溶液澄清(约5min)后小心吸取上清至干净EP管中，于-20℃保存；
8.7纯化后产物取1μL测ssDNA浓度。
9上机测序
根据BGI-SEQ500测序仪的操作说明书，将构建好的文库制备成DNB(DNA纳米球)，其中，使用文库6ng，RCA反应20min。然后在测序仪上采用常规PE50+10策略进行测序。
需要说明的是，对按照本发明获得的血液游离DNA文库的测序，进行低深度测序即可实现。
10下机数据分析
10.1将下机Fastq测序数据进行质量过滤和比对，得到数据比对后的Bam文件。
10.2通过测序数据双端片段的配对，得到每条片段全长和位置信息。
10.3通过对片段大小进行过滤，保留大片段(60bp以上)，计算这些片段所在样本间的相关性，以及这些片段在不同基因的启动子和增强子区域的富集情况，结果如图4与5所示。
10.4将这些片段数据与来自ENCODE数据库中人体不同组织的DNase-seq数据进行聚类分析，结果如图6所示。
对比例1
本对比例采用现有技术中的方法，即直接提取血液游离DNA后进行转座反应，具体如下：
1直接提取血液游离DNA后进行转座反应
1.1对实施例1的步骤1.2中纯化后的血浆直接提取血液游离DNA(具体方法参照实施例1的步骤3)。
1.2按照表8配制Tn5转座反应体系，冰上配制完成后，混合均匀，于恒温金属混匀仪上，37℃转座30min。中间轻轻震荡。
表8 血液游离DNA提取后的转座反应体系
Figure PCTCN2017113208-appb-000014
1.3终止反应：加入7.5μL5×NT缓冲液，轻轻吹打20次，室温放置5分钟。
1.41.8×磁珠回收DNA样品
1.4.1XP磁珠(AGENCOURTA63882)从4℃冰箱中取出，混匀，室温放置10min。
1.4.2加入1.8×XP磁珠，吹打10次混匀，室温静置5min。
1.4.3放到磁力架上，静置2min，磁珠被吸附，液体变澄清。
1.4.4去掉上清，可残留5μL，不要吸到磁珠。
1.4.5加入150μL 80％乙醇，静置30s，弃上清。
1.4.6.重复步骤6.5，最后将乙醇吸干静，晾干至磁珠表面不反光。
1.4.7将PCR管从磁力架上拿下，加入24μL NF水溶解，吹打10次混匀，室温静置3min。
1.4.8上磁力架，静置1min，液体澄清。
1.4.9将上清转移至新管中。
1.4.10Qubit dsDNA高灵敏度检测试剂盒(INVITROGEN Q32854)定量。
根据本对比例1的步骤1.4得到DNA样品溶液后，按照实施例1的步骤4-10继续进行实验，以分析在按照本对比例1的方法所构建的文库中，大片段(60bp以上)在不同基因的启动子和增强子区域的富集情况，结果如图4所示。
如图4所示，对实施例1和对比例1的结果进行比较分析可以发现，很明显，依据本发明的方法(实施例1)所得到的样本之间的相关性比现有方法(对比例1)的样本之间相关性更高。
如图5所示，本发明的方法(实施例1)所得到的区域富集效果比现有方法(对比例1)的富集效果显著增强。
如图6所示，用本发明方法所得到的样本数据与来自人体不同组织的数据通过聚类方法聚类到一起，说明本发明方法能捕获到来自人体不同组织的体液游离DNA信 息，可以进一步进行组织溯源。
申请人声明，本申请通过上述实施例来说明本申请的详细方法，但本申请并不局限于上述详细方法，即不意味着本申请必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本申请的任何改进，对本申请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，均落在本申请的保护范围和公开范围之内。

Claims (14)

  1. 一种对体液样本游离DNA进行文库构建的方法，其包括：
    (1)用酶直接作用于体液样本，以将体液样本中的游离DNA片段化；
    (2)对步骤1)所得片段化的DNA进行扩增，获得体液游离DNA文库。
  2. 根据权利要求1所述的方法，其中，步骤(1)中，所述酶为转座酶或内切酶。
  3. 根据权利要求2所述的方法，其中，所述转座酶为Tn5转座酶，所述内切酶为MNase或DNase酶。
  4. 根据权利要求1所述的方法，其中，步骤(1)包括：利用转座酶，对含有游离DNA的体液样本进行转座反应，以将所述游离DNA进行片段化并添加接头序列，获得包含接头序列的DNA片段；
    优选地，步骤(1)还包括：在将所述游离DNA进行片段化并添加接头序列之后，进行核酸提取的步骤。
  5. 根据权利要求1所述的方法，其中，步骤(1)包括：利用内切酶处理含有游离DNA的体液样本，以将所述游离DNA进行片段化，然后在所得片段化DNA的两端添加接头序列，获得包含接头序列的DNA片段；
    优选地，步骤(1)还包括：在将所述游离DNA进行片段化之后，进行核酸提取的步骤。
  6. 根据权利要求4所述的方法，其中，用于所述转座反应的接头为接头混合物，其通过以下步骤制备：
    1)将引物A：5′-CTGTCTCTTATACACATCT-3′与引物B：5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3′退火，得退火产物1；
    2)将引物A：5′-CTGTCTCTTATACACATCT-3′与引物C：5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3′退火，得退火产物2；
    3)将退火产物1和退火产物2混合，得接头混合物；
    优选地，所述接头混合物与所述转座酶进行包埋，获得转座酶包埋复合物，以用于转座反应；
    进一步优选地，将所述接头混合物与包含转座酶的Tagment Enzyme Advanced V5S进行包埋；优选地，所述接头混合物与所述Tagment Enzyme Advanced V5S的体积比为1∶20-1∶25、优选为1∶24.5；优选地，所述包埋在22-28℃、优选在25℃下进行40-80min、优选60min。
  7. 根据权利要求6所述的方法，其中，将所述转座酶包埋复合物与所述体液样本在转座反应条件下进行孵育，以实现转座反应；
    对于所述转座反应，优选地，所述转座酶包埋复合物与所述体液样本的体积比为1∶50-1∶80，优选为1∶62.5；
    优选地，所述转座反应温度为35-40℃，优选为37℃；
    优选地，所述转座反应时间为55-65min，优选为60min。
  8. 根据权利要求1-7任一项所述的方法，其中，步骤(2)中所述扩增包括两次扩增；
    优选地，在第一次扩增后，通过qPCR确定需要补加的循环数N，作为第二次扩增的循环数。
  9. 根据权利要求1-8任一项所述的方法，其还包括：
    步骤(3)：对步骤2)所获得的体液游离DNA文库进行环化和酶切；
    优选地，所述环化包括将所述体液游离DNA文库中的双链DNA变性为单链DNA，再通过与单链DNA部分区域互补的寡核苷酸片段、通过碱基互补配对进行连接；进一步优选地，使用介导片段：5′-GCCATGTCGTTCTGTGAGCCAAGG-3′与所述单链DNA互补配对连接以实现环化；
    优选地，使用外切核酸酶I和外切核酸酶III进行所述酶切，以去除未环化的DNA；
    优选地，步骤(3)还包括：对酶切产物进行纯化的步骤；优选地，使用磁珠进行纯化。
  10. 一种获得个体表观信息的方法，其包括：
    (1)根据权利要求1-9任一项所述的方法，获得个体的体液游离DNA文库；
    (2)对步骤(1)所得体液游离DNA文库进行测序和分析，以获得个体表观信息。
  11. 根据权利要求1-9任一项所述的对体液样本游离DNA进行文库构建的方法或权利要求10所述的获得个体表观信息的方法在产前诊断及癌症早期发现中的用途。
  12. 一种产前诊断或癌症早期发现的方法，其通过根据权利要求1-9任一项所述的对体液样本游离DNA进行文库构建的方法或权利要求10所述的获得个体表观信息的方法实现。
  13. 一种分析体液游离DNA以进行产前诊断或癌症早期发现的试剂盒，其包括权利要求1-9任一项所述的方法中所使用的试剂、引物、介导片段或其中一项或多项的组合。
  14. 根据权利要求13所述的试剂盒，其特征在于，所述试剂盒包括以下一种或多种的组合：
    转座酶Tn5或MNase或Dnase酶；
    引物A：5′-CTGTCTCTTATACACATCT-3′，引物B：5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3′与引物C：5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3′；
    介导片段：5′-GCCATGTCGTTCTGTGAGCCAAGG-3′；以及
    所述转座、PCR扩增、酶切、连接反应所需要的酶和/或试剂。
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