WO2015070773A1 - 一种全基因组dna甲基化的导向测序技术 - Google Patents

一种全基因组dna甲基化的导向测序技术 Download PDF

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WO2015070773A1
WO2015070773A1 PCT/CN2014/090979 CN2014090979W WO2015070773A1 WO 2015070773 A1 WO2015070773 A1 WO 2015070773A1 CN 2014090979 W CN2014090979 W CN 2014090979W WO 2015070773 A1 WO2015070773 A1 WO 2015070773A1
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methylation
sequence
sequencing
nucleic acid
cytosine
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于文强
李岩
吴飞珍
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复旦大学
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  • the present invention is in the field of molecular biology-epigenetics; more specifically, the present invention relates to directed sequencing techniques for whole genome DNA methylation.
  • DNA methylation is an important component of epigenetics and plays a crucial role in maintaining normal cell function, embryonic development, disease development, and human tumors.
  • the level of DNA methylation, especially the level of methylation in the promoter region of the gene directly affects the transcriptional activity of the gene, controls the expression of the gene, and plays an important role in DNA methylation during life. Therefore, the study of DNA methylation is the most important research in epigenetics and even in the whole life science field.
  • Methylation-sensitive restriction endonucleases are a class of endonucleases that are sensitive to methylation modifications. Such an enzyme contains a methylation base at its cleavage site to block the cleavage of the DNA by the endonuclease and then detect it by Southern Blot or PCR.
  • the most commonly used in this method are the endonucleases of HpaII and MspI. HpaII and MspI have the same recognition sequence but HpaII is sensitive to methylation and MspI is not sensitive to methylation.
  • the advantage of this method is that the operation is simple, and the disadvantage is that Because of the restriction of the endonuclease cleavage site, there are significant limitations on the methylation regions that can be studied.
  • Antibody-based DNA methylation studies use DNA methylcytosine antibodies or methylated binding protein antibodies to study DNA methylation.
  • the principle and operation are similar to ChIP, which involves chip hybridization or sequencing of antibody-affinitive DNA fragments.
  • ChIP which involves chip hybridization or sequencing of antibody-affinitive DNA fragments.
  • the advantage is that the change of DNA methylation can be studied in the genome-wide range, but this method lacks precision, can not know the methylation of a single base, and the method is easily affected by the CG content in different regions of the genome, thus There is a lack of sensitivity to regions of low CG content in the genome.
  • the most widely used method for detecting DNA methylation is a method based on sodium hydrogen sulfite. This has the advantage of accurately detecting single-base resolution methylation changes.
  • the principle of this method is that the DNA passes through the sulfur C (cytosine) not modified by methylation after sodium hydrogen hydride treatment can be converted into U (uracil), but C with methylation modification will not change, after PCR amplification, sequencing, and then Sequence alignment allows for methylation modification of a region of the genome.
  • MethyIC-seq is by far the best method for DNA methylation studies, but its own design flaws and detection biases, as well as problems encountered in bioinformatics analysis, have greatly hampered its application.
  • the positioning concept used in the present invention can completely solve these problems and raise the research on DNA methylation to a new height.
  • a method of assaying nucleic acid methylation, said nucleic acid being double stranded comprising:
  • a polymerase having a 3' ⁇ 5' cleavage function or a 3' ⁇ 5' exonuclease treatment is used to generate 80-200 bases at the 3' end of each of the two strands ( Preferably 100-150 bases are deleted;
  • cytosine (C) is a methylated modified cytosine (5 mC), the deletion of the double-stranded 3' end is filled in and the cytosine therein is methylated modified cytosine;
  • step (3) treating the double strand of step (2), such that the cytosine not modified by methylation is converted into uracil (U), and the methylated modified cytosine is unchanged;
  • telomere sequence used to determine the methylation site; another part of the sequence is identical to the original nucleic acid sequence due to methylation of cytosine, used for sequence localization in data analysis; alignment of the two parts of the sequence (if the sequence of the detected species is known No splicing is required; the species of the unknown sequence can be spliced and then methylated for the alignment, thereby knowing the methylation status of the nucleic acid.
  • the method for determining methylation of a nucleic acid is equally applicable to the detection of a single locus, wherein step (4) comprises: designing a PCR amplification for amplifying a locus of interest Primer, one primer is located at the position of the methylation of cytosine; the other primer is located at the position of the unmethylated cytosine that has been converted to thymine, and PCR amplification is performed to obtain the locus of interest. Sequence and perform methylation analysis.
  • step (3) or step (b) the double chain of step (2) is treated with disulfate, bisulfite, bisulfite or bisulfite.
  • the nucleic acid is a nucleic acid having a length greater than 2 kb (eg, greater than 3 kb, 5 kb, 10 kb) or a whole genome, and prior to step (1), further comprising: interrupting the nucleic acid sequence (preferably Alternatively, by ultrasonic disruption, a double-stranded fragment having a length of 200 to 1000 bp (preferably 400 to 700 bp; more preferably 500 bp) is formed.
  • step (2) after performing the filling, further comprising: adding a sequencing adaptor at both ends of the double strand of step (2) to be applied to high-throughput sequencing.
  • the sequencing linker is ligated by: adding a protruding A) to the 3' end of the double strand, and using a sequencing link (with a prominent T at the 3' end) to be ligated thereto.
  • the sequencing linker is a methyl linker (Methyl Adapter; such as Illumina Methyl Adapter), which is subsequently sequenced using high throughput sequencing methods such as Illumina high seq 2000, Illumina high seq 2500, ABI solid, Roche 454.
  • the methylation modification of the cytosine comprises: CpG methylation, CHG methylation or CHH methylation.
  • the polymerase having a 3' ⁇ 5' directional cleavage function includes, but is not limited to: T4 DNA polymerase, T7 DNA polymerase, Klenow enzyme;
  • the 3' ⁇ 5' exonuclease includes, but is not limited to, exonuclease III; preferably, when step 3 applies a 3' ⁇ 5' exonuclease, step (2) A polymerase is also added; preferably, the polymerase includes, but is not limited to, Taq enzyme, Pfu, reverse transcriptase.
  • the T4 DNA polymerase is treated for a period of from 60 to 140 minutes; preferably from 80 to 120 minutes.
  • the nucleic acid is DNA or RNA.
  • the sequence identical to the original nucleic acid sequence is a read in double-ended sequencing or a part of a read in single-ended sequencing (eg, single-ended sequencing is The first 50 bp is used for localization at 100 bp, and the latter 50 bp is used for methylation detection); preferably, double-end sequencing, in the sequencing result, unmethylated cytosine (C) in one (read1 or read2) nucleic acid sequence Has been converted to thymine (T) for the determination of methylation sites; the other end (read2 or read1) sequence is identical to the original nucleic acid sequence due to methylation of cytosine for sequence localization; The alignment is used to know the methylation status of the nucleic acid.
  • C unmethylated cytosine
  • T thymine
  • an analytical alignment method for a nucleic acid sequence comprising: the sequence is double-end sequencing, one end of the sequence is initially positioned, and the other end of the sequence is searched in the vicinity thereof, the method comprising:
  • the double-ended sequence refers to sequencing of the 5' end and 3' of the nucleic acid sequence, respectively, such as the Illumina Hiseq 200, the Illumina Hiseq 2500, and the Pair-End of the Illumina Analyzer Genome IIx. Sequencing
  • the one-end sequence is initially positioned to a plurality of positions in the genome that are designated to 20 or less than 50 positions (but are not limited thereto).
  • finding in the vicinity of the preliminary positioning refers to 1 to 3 times the size of the sequencing library as the range of the search; the methods of searching include (but are not limited to): character alignment, regular expression retrieval, Sequence retrieval.
  • the optimal alignment position is selected based on whether the total length between the ends is in the text. Within the scope of the library; whether the number of mismatches is the lowest.
  • FIG. 1 A representative read (SEQ ID NO: 1) of clone sequencing of an embodiment of the invention.
  • Fig. 4 shows the results of electrophoresis of the ultrasonically interrupted genome in Example 1 of the present invention.
  • Figure 5 is a graph showing the results of the removal of excess joints by the rubber in Example 5 of the present invention.
  • Fig. 6 shows the results of electrophoresis after PCR in Example 7 of the present invention, and the DNA fragment shown in the figure was 400-700 bp.
  • the present inventors have provided a new method for the determination of methylation of nucleic acids (including DNA and RNA) through research and exploration. Specifically, a "localization" concept for nucleic acid methylation detection is provided, in which part of the sequence is used for localization (same sequence as the genomic sequence) and another part of the sequence is used for methylation detection (after sulfite treatment) The unmethylated cytosine becomes uracil, which completely solves the positioning problem encountered in the bioinformatics analysis of methylation detection. The present invention has been completed on this basis.
  • the present invention utilizes a polymerase having a 3' ⁇ 5' direction cleavage function or a 3' ⁇ 5' exonuclease to treat a double strand of a nucleic acid, resulting in a 3' deletion of the double strand, and then adding a dNTP (wherein cytosine (C) A methylated modified cytosine (5mC), with a polymerase, causes a 5' to 3' extension of the deleted portion of the double strand, a 3' deletion of the nucleic acid strand to fill in and 5mC to the 3' end.
  • C cytosine
  • 5mC A methylated modified cytosine
  • the enzyme which causes the double strand to generate a 3'-end deletion may be a polymerase having a 3' ⁇ 5'-direction cleavage function or a 3' ⁇ 5' exonuclease.
  • the polymerase having a 3' ⁇ 5' direction cutting function includes but is not limited to: T4 DNA polymerase, T7 DNA polymerase, Klenow enzyme.
  • the 3' ⁇ 5' exonuclease includes, but is not limited to, exonuclease III.
  • a polymerase having a 3' ⁇ 5' direction cutting function is applied, so that when the double strand is required to cause a 3' end deletion, it can exert a 3' ⁇ 5' direction cutting function; When the 3' end is extended after the addition of dNTP, it can function as a polymerase.
  • the polymerase having a 3' ⁇ 5' directional cleavage function is T4 DNA polymerase.
  • the bisulfite sequencing PCR (BSP) method is a classical method for detecting gene methylation.
  • the principle is: treatment of genomic DNA with bisulfite modification, all cytosines that have not been methylated (C The cytosine that is converted to uracil (U) and methylated remains unchanged.
  • C cytosine that is converted to uracil
  • U uracil
  • SNP polynucleotide polymorphism
  • the target fragment is amplified.
  • the uracil (U) is completely converted into thymine (T)
  • the PCR product is sequenced to determine whether methylation occurs.
  • the methods of the invention are applicable to nucleic acids of longer length, such as nucleic acids greater than 10 kb in length or whole genome.
  • the methods of the invention can also be applied to short nucleic acids.
  • the length needs to be controlled when the nucleic acid is interrupted, for example by controlling the ultrasound time at which the ultrasound is interrupted.
  • the disruption of the nucleic acid sequence is first performed to facilitate subsequent manipulation.
  • the method of the present invention for interrupting the nucleic acid is not particularly limited, and various methods known in the art can be applied; preferably, a method of ultrasonic disruption is employed.
  • the conditions of the ultrasound mainly depend on the G+C content of the nucleic acid of the ultrasonic device to be applied, the size of the desired fragment, and the like.
  • the non-contact ultrasound system produced by BioRuptor is used for ultrasonic interruption.
  • the G+C content of the genome is about 50%
  • the highest power ultrasound is 6 times
  • each interval of 30 seconds is 30 seconds, which can be about 400-700 bp. Fragment of. Different ultrasound systems are applied. If better ultrasound conditions are to be obtained, the size of the sequence fragment after ultrasonic interruption can be obtained according to the electrophoresis condition.
  • the long nucleic acid or genomic sequence is disrupted to form a double-stranded fragment of 200-1000 bp; preferably 400-700 bp; more preferably 500 bp.
  • the method further comprises: adding the sequencing linker to both ends of the doubled double-stranded Flux sequencing.
  • the sequencing linker refers to a nucleic acid linker involved in some high-throughput sequencing technologies, and those skilled in the art are aware of sequencing linkers applied to specific sequencing technologies, for example, sequencing technology of Illumina Corporation.
  • the method of the present invention is equally applicable to sequencing starting from a single strand of a nucleic acid, by incorporating a cytosine (C) methylation-modified dNTP during synthesis of the second strand, such that subsequent bisulfite treatment, synthesis One of the two strands of the nucleic acid duplex is not transformed by a subsequent base, and the unmethylated modified chain cytosine is converted. Therefore, one sequence is used for localization during sequencing and the other sequence is used for detection of methylation.
  • C cytosine
  • the method of the present invention can not only directly detect the level of methylation of the entire genome, but also can be used in combination with other methods, such as RRBS, thereby improving the map efficiency of the RRBS.
  • the method can also sequence unknown species, use sequence2 to splicing the sequence, and read1 detects the methylation level, so that the genomic sequence and genomic methylation level of the species can be obtained simultaneously.
  • Methylation sequencing of the invention refers to methylation on cytosine, including CpG methylation, CHG methylation, and CHH methylation.
  • the invention overcomes the shortcomings of the existing methylation detection technology, and aims at high-throughput high-resolution, and can truly study the distribution of DNA methylation in a genome-wide range and single-base resolution, and obtain a table. Genomic information can also be obtained from the genome.
  • the following examples are mainly used to detect the whole-genome DNA methylation in tissues and cells.
  • the whole process includes ultrasonic disruption of DNA, T4 DNA polymerase treatment, "A" at the end, linker ligation, gel recovery and de-linker, bisulfite Sodium treatment, PCR amplification and other steps, the main process is shown in Figure 1.
  • the extracted human cell line 293T genomic DNA 3ug was dissolved in 250 ul of ultrapure water, and the DNA was disrupted to a fragment of about 400-700 bp using appropriate ultrasonic conditions, using QIAGEN PCR.
  • the recovery kit recovers the ultrasonic product.
  • the specific procedure is to add 5 volumes (1 ml) of buffer PB to the ultrasonic product, mix well by flicking, transfer to the column, and centrifuge at 13000 rpm for 1 min. Remove the elution liquid. Add 750 ul of buffer PE (alcohol added), centrifuge at 13000 rpm for 1 min, and remove the liquid as above.
  • the ultrasonically interrupted genome was electrophoresed. The results are shown in Fig. 4. It can be seen from the figure that the DNA has been ultrasonically amplified to between 1000 bp and 200 bp at 3 min, and is mainly concentrated at about 500 bp.
  • the flick is fully mixed, transiently (1000g centrifugation for 30 seconds), and digested at 10°C for 100min to remove 100-150 bases from the 3' end; then, add a final concentration of 10mM dNTP (wherein cytosine is methylated) Modified cytosine (5mC), mix well with flick, detach (1000g centrifugation for 30 seconds), 37 °C for 15min.
  • the PCR was recovered (the same procedure as above), and finally eluted with 42 ul of ultrapure water for use.
  • the flick was thoroughly mixed, instantaneously separated, 37 ° C, 1 h, recovered using QIAGEN Mini Elute PCR Recovery Kit, and eluted with 17.5 ul of ultrapure water.
  • Illumina Methyl Adapter is a connector for high-throughput sequencing of methylation produced by Illumina.
  • Fig. 5 The result of the gel recovery to remove the excess linker is shown in Fig. 5.
  • the large fragment of 400 bp or more is the genomic DNA after ultrasound, and the small fragment of about 200 bp is the linker DNA to be removed.
  • the picture on the right shows the cut gel image.
  • the 400-700 bp DNA in the figure has been cut and recovered.
  • the above recovered product was treated with a Zymo-Research methylation kit, and 20 ul of the recovered product was added to 130 ul CT conversion reagent (CT Conversion Reagent), and the transient sample was placed in a PCR instrument, and the reaction procedure was set as follows: 98 ° C , 10 min; 64 ° C, 2.5 h; 4 ° C, ⁇ ; Zymo column (Zymo-Spin (TM) IC Column) into a collection tube (Collection Tube), adding 600ul M-Binding Buffer, Then add the sample after the reaction, cover tightly, invert and mix; centrifuge at 12,000 rpm ( ⁇ 10,000 g) for 30 s; 100ul M-Wash Buffer, centrifuged at 12,000 rpm for 30 s; add 200 ul of M-Desulphonation Buffer, let stand at room temperature (20 ⁇ 30 ° C) for 15-20 min, centrifuge at 12,000 rpm for 30 s; Add 200 ul of M-
  • the PCR product was subjected to electrophoresis, and the electrophoresis results are shown in Fig. 6.
  • the DNA fragment shown in the figure was 400-700 bp, which was consistent with the size of the gel recovery of Example 5.
  • the gel is recovered and quantified for high-throughput sequencing.
  • Figure 2 shows a read of genomic high-throughput sequencing.
  • the shaded area is the region where methylation C is incorporated, that is, the region used for data analysis.
  • the results show that the bases of C and G exist in the shaded region of the 5' end of the sequencing result (equivalent to Qualcomm).
  • the amount is sequenced for the end of the sequence), and at the 3' end of the sequencing result, only C is present and no G is present (equivalent to the end of high-throughput sequencing for methylation detection) (due to cloning and sequencing) It is an anti-chain, the original result should be only G and not C), indicating that the method has reached the requirements of high-throughput sequencing.
  • Figure 3 shows representative results of the alignment of the cloned sequencing results of the present invention in the UCSC genome browser.
  • the upper part of the figure shows that the sequencing results are identical to the original genome (i.e., the portion used for localization), the lower half of the figure. It is shown that all non-CG Gs become A (ie, the part used for methylation detection).
  • the method of the present invention is equally applicable to the detection of a single genetic locus, and the experimental flow chart is shown in FIG.
  • PCR amplification primers for amplifying the gene locus of interest, one primer localizes to the position of the methylation of cytosine; the other primer localizes to the unmethylated cytosine that has been converted to thymine.
  • the position of the sequence is subjected to PCR amplification, and the sequence of the gene locus of interest is obtained and subjected to methylation analysis.

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Abstract

本发明公开了一种全基因组DNA甲基化的导向测序技术。本发明提供了一种新的核酸甲基化的测定方法，具体而言是一种核酸甲基化检测的"定位"理念，即测序时部分序列用来定位，另一部分序列用于甲基化的检测，从而解决全基因组甲基化检测及生物信息学分析时遇到的定位问题。

Description

一种全基因组DNA甲基化的导向测序技术 技术领域
本发明属分子生物学-表观遗传学领域；更具体地，本发明涉及全基因组DNA甲基化的导向测序技术。
背景技术
DNA甲基化是表观遗传学的重要组成部分，其在维持正常细胞功能、胚胎发育、疾病发生以及人类肿瘤中发挥着至关重要的作用。DNA甲基化的水平，尤其是基因启动子区甲基化的水平直接影响着基因的转录活性，控制着基因的表达，在生命过程中DNA甲基化扮演着重要的角色。因此DNA甲基化的研究在表观遗传学，甚至在整个生命科学领域都是研究的重中之重。
DNA甲基化的检测方法很多，但根据其原理主要可以分为以下三类：
1.基于甲基化敏感限制性内切酶的方法
甲基化敏感的限制性内切酶(methylation-sensitive restriction endonucleases,MSREs)是一类对甲基化修饰敏感的内切酶。此类酶在其切割位点中含有一个甲基化碱基就可以阻断该内切酶对DNA的切割，再通过Southern Blot或者PCR来检测。这种方法中最常用的是HpaII和MspI这一对内切酶，HpaII和MspI识别序列相同但HpaII对甲基化敏感而MspI对甲基化不敏感，此法的优点是操作简单，缺点是因为受到内切酶酶切位点的限制，对于可研究的甲基化区域受到很大的限制。
2.基于DNA甲基化抗体的方法
基于抗体的DNA甲基化研究方法是应用甲基胞嘧啶抗体或是甲基化结合蛋白的抗体来研究DNA甲基化。其原理和操作都类似于ChIP，即将抗体亲和的DNA片段进行芯片杂交或者测序。优点是可以在全基因组范围内研究DNA甲基化的变化情况，但此方法缺乏精确性，无法知道单个碱基的甲基化情况，而且该方法容易受到基因组中不同区域CG含量的影响，从而对于基因组中低CG含量的区域缺乏敏感性。
3.基于亚硫酸氢钠的方法
DNA甲基化的检测方法中应用最广的是基于亚硫酸氢钠的方法。其优点在于可以精确检测单碱基分辨率甲基化变化情况。这种方法的原理为DNA经过亚硫 酸氢钠处理后未被甲基化修饰的C(胞嘧啶)可转变为U(尿嘧啶)，然而有甲基化修饰的C则不会发生改变，经过PCR扩增后测序，再与原序列对比就可以得到基因组中某段区域的甲基化修饰情况。
近年来随着人们对DNA甲基化研究的深入，这些传统的方法已经远远不能满足研究的需要。随着高通量测序技术的发展，DNA甲基化检测也由单基因水平逐渐向整个基因组水平发展。这样基于以上三种方法与高通量技术相结合又衍生出了很多方法：如MeDIP，RRBS，HELP等，但其中应用最广，覆盖基因组最多、最精确的方法为MethyIC-seq。其原理为基因组DNA经过亚硫酸氢钠处理后直接进行高通量测序，从理论上讲，对测序结果进行分析后就可以得到整个基因组单碱基分辨率的甲基化修饰情况。但是实际操作中对MethyIC-seq的数据进行分析时却遇到了很大的问题：①由于亚硫酸氢钠处理后基因组中大多数的胞嘧啶(C)都会转变为胸腺嘧啶(T)，造成碱基不平衡，测序得到的结果比对(map)到参考基因组上效率有限，有的位置即使增大测序通量也无法得到DNA甲基化信息，所以到目前为止全世界还没有绘制出任何一张细胞完整甲基化图谱。Felix Krueger等人在Nature Method(Nat Methods.2012 Jan 30；9(2):145-51.)杂志上对于DNA甲基测序数据分析中遇到的问题进行了详细的论述。②该检测方法的设计策略缺陷也使其存在很强的倾向性，容易检测出高甲基化区域，对低甲基化、CG含量少以及一些重复序列缺乏敏感度。
总体来说，到目前为止MethyIC-seq是DNA甲基化研究比较好的方法，但是其本身的设计缺陷和检测偏好性以及生物信息学分析时遇到的问题却极大地阻碍了其应用。而本发明中所使用的定位理念可以彻底解决这些问题，把人们对DNA甲基化的研究提高到了一个新的高度。
发明内容
本发明的目的在于提供一种全基因组DNA甲基化的导向测序技术。
在本发明的第一方面，提供一种核酸甲基化的测定方法，所述的核酸是双链，所述方法包括：
(1)针对核酸双链，利用具有3’→5’方向切割功能的聚合酶或3’→5’核酸外切酶处理，使两条链分别在3’端产生80-200个碱基(较佳地100-150个碱基)缺失；
(2)加入dNTP，其中的胞嘧啶(C)为甲基化修饰的胞嘧啶(5mC)，使双链3’端的缺失被补平且其中的胞嘧啶为甲基化修饰的胞嘧啶；
(3)处理步骤(2)的双链，使未被甲基化修饰的胞嘧啶转变为尿嘧啶(U)、甲基化修饰的胞嘧啶则不变；
(4)PCR扩增(该过程中尿嘧啶转换为胸腺嘧啶(T))，进行甲基化测序，其中一部分序列中未甲基化的胞嘧啶(C)已转变为胸腺嘧啶(T)，用于判断甲基化位点；另一部分序列因胞嘧啶发生甲基化而与原核酸序列相同，用于数据分析时的序列定位；将两部分序列比对(如果检测的物种序列已知则不需要拼接；未知序列的物种可以将用于定位的那一端进行拼接后再进行甲基化比对)，从而得知核酸的甲基化状况。
在本发明的另一方面，所述的核酸甲基化的测定方法同样可用于单个基因位点的检测，其中步骤(4)包括：设计用于扩增感兴趣的基因位点的PCR扩增引物，一条引物定位于胞嘧啶发生甲基化的序列位置；另一引物定位于未甲基化的胞嘧啶已转变为胸腺嘧啶的序列位置，进行PCR扩增，得到感兴趣的基因位点的序列并进行甲基化分析。
在一个优选例中，步骤(3)或步骤(b)中，应用重硫酸盐、重亚硫酸盐、亚硫酸氢盐或重亚硫酸氢盐处理步骤(2)的双链。
在另一优选例中，所述的核酸是长度大于2kb(如大于3kb、5kb、10kb)的核酸或是全基因组，在步骤(1)之前，还包括：将核酸序列进行打断(较佳地，采用超声打断)，形成长度为200-1000bp(较佳地400-700bp；更佳地500bp)的双链片段。
在另一优选例中，步骤(2)中，在进行补平之后，还包括：在步骤(2)的双链的两端加上测序接头，从而应用于高通量测序。
在另一优选例中，所述的测序接头如下连接：在双链的3’末端加上1个突出的A)，应用带有(3’末端含有一个突出的T)的测序接头与之连接；较佳地，所述的测序接头是甲基接头(Methyl Adapter；如Illumina Methyl Adapter)，后续采用高通量测序方法测序，例如Illumina high seq2000，Illumina high seq2500、ABI solid、Roche 454。
在另一优选例中，所述的胞嘧啶的甲基化修饰包括：CpG甲基化，CHG甲基化或CHH甲基化。
在另一优选例中，所述的具有3’→5’方向切割功能的聚合酶包括(但不限于)： T4 DNA聚合酶，T7 DNA聚合酶，Klenow酶；
所述的3’→5’核酸外切酶包括(但不限于)：核酸外切酶III；较佳地，当步骤(1)应用3’→5’核酸外切酶时，步骤(2)中还加入聚合酶；较佳地，所述的聚合酶包括(但不限于)：Taq酶，Pfu，逆转录酶。
在另一优选例中，所述的T4 DNA聚合酶处理的时间为60-140分钟；较佳地为80-120分钟。
在另一优选例中，所述的核酸是DNA或RNA。
在另一优选例中，步骤(4)或步骤(c)中，与原核酸序列相同的序列为双端测序中的一个read，或是单端测序中一个read的一部分(如单端测序为100bp时前50bp用于定位，后50bp用于甲基化检测)；较佳地，进行双端测序，测序结果中，一端(read1或read2)核酸序列中未甲基化的胞嘧啶(C)已转变为胸腺嘧啶(T)，用于判断甲基化位点；另一端(read2或read1)序列因胞嘧啶发生甲基化而与原核酸序列相同，用于序列定位；将双端序列进行比对，从而得知核酸的甲基化状况。
本发明的另一方面，提供一种核酸序列的分析比对方法，包括：序列为双端测序，一端序列作初步定位，另一端序列在其附近检索，所述的方法包括：
(a)将一端序列定位到基因组上，允许每个序列定位到基因组的多个位置；
(b)为已定位到基因组上的序列匹配另一端序列；
(c)根据在基因组中的初步定位，在其附近寻找另一端的位置；
(d)若可获得多个比对位置，选取最佳比对位置；
(e)去除PCR产生的扩增序列；
(f)分析基因组的甲基化水平，统计甲基化率。
在另一优选例中，所述的双端序列是指在核酸序列5’端和3’分别作一定长度的测序，如在Illumina Hiseq200,Illumina Hiseq2500,以及Illumina Analyzer Genome IIx上的Pair-End的测序；
在另一优选例中，所述的一端序列初步定位到基因组的多个位置是指定位到20个或50个以下的位置(但不限于如此)。
在另一优选例中，在初步定位的附近查找，是指根据测序文库的大小的1至3倍作为查找的范围；查找的方法包括(但不限于)：字符比对，正则表达式检索，序列检索。
在另一优选例中，最佳比对位置的选取依据是：两端之间的总长度是否在文 库的范围之内；错配数是否最低。
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
图1、本发明实施方案中所描述的实验流程图。
图2、本发明实施方案克隆测序的一个代表性read(SEQ ID NO:1)。
图3、本发明实施方案克隆测序结果在UCSC genome browser中比对的结果。
图4、本发明实施例1中超声打断的基因组的电泳结果。
图5、本发明实施例5中胶回收去除多余接头的结果。
图6、本发明实施例7中PCR后的电泳结果，图中显示的DNA片段为400-700bp。
图7、应用于单个基因位点的检测的实验流程图。
具体实施方式
本发明人经过研究和探索，提供了一种新的核酸(包括DNA和RNA)甲基化的测定方法。具体地说提供了一种核酸甲基化检测的“定位”理念，即测序时部分序列用来定位(与基因组序列相同)，另一部分序列用于甲基化的检测(经过亚硫酸盐处理后未甲基化的胞嘧啶变为尿嘧啶)，从而彻底解决了甲基化检测生物信息学分析时遇到的定位问题。在此基础上完成了本发明。
本发明是利用具有3’→5’方向切割功能的聚合酶或3’→5’核酸外切酶处理核酸双链，使双链产生3’缺失，然后加入的dNTP(其中胞嘧啶(C)为甲基化修饰的胞嘧啶(5mC))，以聚合酶会使双链上缺失部分产生5’向3’延伸，使核酸链的3’缺失补平并将5mC掺入到3’端。这样经过亚硫酸氢盐(或其类似物)处理后进行双端测序，测序的一端(read1)C就会变为T，而另一端(read2)由于C都为甲基化的C则保持原基因组序列不变，这样read1可以对基因组的甲基化程度进行分析，而read2就可以在基因组中进行map，这样就彻底解决了甲基化测序中数据分析的问题。
使双链产生3’端缺失的酶可以是具有3’→5’方向切割功能的聚合酶或3’→5’核酸外切酶。其中，所述的具有3’→5’方向切割功能的聚合酶包括但不限于：T4 DNA聚合酶，T7 DNA聚合酶，Klenow酶。所述的3’→5’核酸外切酶包括但不限于：核酸外切酶III。
作为本发明的优选方式，应用具有3’→5’方向切割功能的聚合酶，这样，在需要使得双链产生3’端缺失时，其可发挥3’→5’方向切割功能；当需要使得3’端在加入dNTP后延伸时，其可发挥聚合酶的作用。最优选地，所述的具有3’→5’方向切割功能的聚合酶是T4 DNA聚合酶。
当应用3’→5’核酸外切酶使得双链产生3’端缺失时，后续需要进一步加入聚合酶来使得3’端在含有dNTP的情况下延伸。
亚硫酸氢盐处理后测序(bisulfite sequencing PCR，BSP)方法是检测基因甲基化的经典方法，其原理为：用亚硫酸氢盐修饰处理基因组DNA，所有未发生甲基化的胞嘧啶(C)被转化为尿嘧啶(U)而甲基化的胞嘧啶则不变。因而，经过亚硫酸氢盐或重亚硫酸氢盐处理核酸后，甲基化的位点产生类似于一个C/T的多核苷酸多态性(SNP)。基因组DNA经亚硫酸盐处理后，扩增目的片段，此时尿嘧啶(U)全部转化为胸腺嘧啶(T)，最后对PCR产物进行测序就可以判断是否发生甲基化。
本发明的方法适用于长度较长的核酸，例如长度大于10kb的核酸或是全基因组。本发明的方法也可以应用于短的核酸。对于不同长度的核酸，在核酸打断时需要控制长度，例如可通过控制超声打断时的超声时间来控制长度。对于较长的核酸，先进行核酸序列的打断，以有利于后续的操作。本发明对于将核酸打断的方法没有特别的限制，可以应用本领域已知的各种方法；较佳地，采用超声打断的方法。超声的条件主要取决于所应用的超声设备核酸的G+C含量和所需片段的大小等。本实验应用BioRuptor公司生产的非接触式超声仪进行超声打断一般来说如果基因组G+C含量在50％左右，最高功率超声6次，每次30秒间歇30秒，可得到400-700bp左右的片段。应用不同的超声系统，如要获得较佳的超声条件，可以根据电泳情况来获得超声打断后的序列片段的大小。
作为本发明的优选方式，将长的核酸或基因组序列打断，以形成长度为200-1000bp；较佳地400-700bp；更佳地500bp的双链片段。
当核酸序列较长时或针对全基因组序列进行测定时，需要采用高通量测序技术。此时，在使双链3’端的缺失被补平且其中的胞嘧啶为甲基化修饰的胞嘧啶后，还包括：被补平的双链的两端加上测序接头，从而应用于高通量测序。本发明中，所述的测序接头是指配合一些高通量测序技术而涉及的核酸接头，本领域技术人员清楚针对具体的测序技术所应用的测序接头，例如，Illumina公司的测序技术 为广大用户提供了强大的高通量测序方法，其提供商品化的测序接头以便于待测序列与其测序仪器实现对接；Illumina测序技术以外的其它测序技术及其测序接头也是商品化的或是本领域技术人员所了解的。
本发明的方法同样适用于起始于核酸单链的测序，通过在合成第二链时掺入胞嘧啶(C)发生甲基化修饰的dNTP，使得后续的亚硫酸氢盐处理后，合成的核酸双链的两条链中一条链后续碱基不转变，而未甲基化修饰的链胞嘧啶发生转变。因此，测序时一条序列用来定位，另一条序列用于甲基化的检测。
本发明的方法不但可以直接检测整个基因组甲基化的水平，而且还可以与其他方法联合使用，如RRBS，从而提高RRBS的map效率。此外本方法还可以对未知物种进行测序，利用read2进行序列的拼接，而read1检测甲基化水平，这样可以同时得到这个物种的基因组序列和基因组甲基化水平。本发明的甲基化测序是指胞嘧啶上的甲基化，包括CpG甲基化，CHG甲基化和CHH甲基化。
本发明克服了现有甲基化检测技术的缺点，以高通量高分辨率为目的，可以真正做到在全基因组范围内、单碱基分辨率研究DNA甲基化的分布情况，得到表观基因组的同时也可得到基因组的信息。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
以下实施例主要为检测组织及细胞中全基因组DNA甲基化情况，整个过程包括超声打断DNA，T4 DNA聚合酶处理，末端加“A”，接头连接，胶回收去接头，重亚硫酸氢钠处理，PCR扩增等步骤，主要流程如图1。
实施例1、超声打断DNA
将提取好的人类细胞系293T基因组DNA 3ug溶解于250ul超纯水中，使用合适的超声条件将DNA打断至400-700bp左右的片段，使用QIAGEN公司PCR 回收试剂盒回收超声产物，具体的操作步骤为在超声产物中加入5倍体积(1ml)缓冲液PB，轻弹充分混匀，瞬离，转至柱子中，13000rpm离心1min。去除洗脱液体。加入750ul缓冲液PE(已加乙醇)，13000rpm离心1min，同上去除液体。再加入500ul bufferPE，再次洗涤一次，去除液体，盖上盖子，空转2min，打开盖子，静置，晾干。加入170ul超纯水静置1-2min，13000rpm离心1min，洗脱液备用。
超声打断的基因组进行电泳，结果如图4，从图中可以看出超声3min时DNA已经超声到1000bp到200bp之间，且主要集中在500bp左右。
实施例2、T4 DNA聚合酶处理
在回收产物中依次加入下列试剂：
10×NEB缓冲液2    20ul
T4 DNA聚合酶      10ul
轻弹充分混匀，瞬离(1000g离心30秒时间)，12℃酶切100min约切除3’端100-150个碱基；之后，加入终浓度为10mM的dNTP(其中胞嘧啶为甲基化修饰胞嘧啶即5mC)，轻弹充分混匀，瞬离(1000g离心30秒)，37℃15min。PCR回收(过程同上)，最终用42ul超纯水洗脱备用。
实施例3、3’末端加1个“A”
在回收产物中依次加入下列试剂：
10×NEB缓冲液2                   5ul
10mM dATP                        1.5ul
缺失了3’到5’外切酶活性的Klenow 3ul
轻弹充分混匀，瞬离，37℃，1h，使用QIAGEN公司Mini Elute PCR回收试剂盒回收，17.5ul超纯水洗脱备用。
实施例4、接头连接
在回收产物中依次加入下列试剂：
10×T4 DNA连接缓冲液                       2.5ul
Illumina甲基接头(Illumina Methyl Adapter)  5ul
T4 DNA连接酶                               1ul
轻弹充分混匀，瞬离，16℃连接过夜。其中，Illumina Methyl Adapter是illumina公司生产的用于甲基化高通量测序的接头。
实施例5、胶回收去除多余接头
1.配胶
用80-90ml TAE配制2％(重量比)的琼脂糖(Invitrogen)，在微波炉中煮熟沸腾2-3次，待瓶子不烫时，加入EB3ul，充分混匀，倒板。
2.电泳
样品中加入5ul 10×上样缓冲液，准备100bp marker，两边加marker，中间加样品，注意不同样品间隔一个孔，防止污染。150V电泳40 min。
3.割胶
先用纸蘸取TAE清洗紫外凝胶成像载物台，铺上保鲜膜。尽量减少胶在紫外下照射的时间。15ml离心管称重，记录。割取400-700bp的凝胶片段，记录胶在切割前后的照片，将胶放入管子中。称量，记录。
4.胶回收
使用QIAGEN公司Gel Extractionmini elute kit回收凝胶中的DNA，100mg＝100ul体积，加入3倍体积Buffer QG，42℃10min，每隔2-3min混匀一次直至完全溶解。瞬离，加入一倍体积的异丙醇，瞬离，每次取750ul至Elute柱，13000rpm离心1min，加入500ul BufferQG，离心1min。加入750uL Buffer PE，离心1min，再一次加入500ul buffer PE洗涤，空转2min，打开盖子，静置，晾干，加入22ul超纯水，静置1-2min，13000 rpm离心1min，回收备用。
胶回收去除多余接头的结果如图5，左图中400bp以上的大片段为超声后的基因组DNA，200bp左右的小片段为要去除的接头DNA。右图中所示为切较后凝胶图片，图中400-700bp大小的DNA已经切去并进行回收。
实施例6、重亚硫酸氢钠处理DNA
使用Zymo-Research公司甲基化试剂盒处理上述回收产物，将20ul回收产物加入130ul CT转换试剂(CT Conversion Reagent)中，将瞬离后的样品放入PCR仪，设定反应程序如下：98℃，10min；64℃，2.5h；4℃，∞；将Zymo柱(Zymo-Spin(TM)IC Column)放入收集管(Collection Tube)中，加入600ul M-结合液(M-Binding Buffer)，然后加入反应后的样品，盖紧，颠倒混匀；12,000rpm(<10,000g)离心30s；加入 100ul M-清洗液(M-Wash Buffer)，12,000rpm离心30s；加入200ul M-脱磺化液(M-Desulphonation Buffer)，室温(20～30℃)静置15～20min，12,000rpm离心30s；加入200ul M-清洗液(M-Wash Buffer)，12,000rpm离心30s，去除收集管中的废液；重复清洗一次；将柱子移入干净的1.5ml EP管中，在柱子底部加入10ul M-溶解液(M-Elution Buffer)，12,000rpm离心30s，洗脱液备用。
实施例7、PCR扩增
由于亚硫酸氢钠处理后的DNA片段中C转变为U，普通的高保真酶无法识别U从而造成处理后的片段无法扩增，本实验中使用KAPA公司生产的2x KAPAmix进行扩增，可以克服这一问题。
在PCR管中加入下列试剂：
Figure PCTCN2014090979-appb-000001
反应条件：
Figure PCTCN2014090979-appb-000002
PCR产物经过电泳，电泳结果如图6，图中显示的DNA片段为400-700bp，这与实施例5胶回收的大小一致。
电泳后，进行胶回收，定量后可进行高通量测序。
图2显示了基因组高通量测序的一个read。其中阴影区域为甲基化C掺入的区域，即数据分析中用来定位的区域，结果可以看出在测序结果的5’端阴影区域内C和G的碱基都存在(相当于高通量测序用于定位的那一端)，而在测序结果的3’端则只有C存在而没有G的存在(相当于高通量测序用于甲基化检测的那一端)(由于克隆测序测的是反链，原结果应该是只有G而没有C)，说明该方法已经达到了高通量测序的要求。
图3显示了本发明实施方案克隆测序结果在UCSC genome browser中比对的代表性结果，图的上半部分显示测序的结果与原基因组相同(即用于定位的部分)，图的下半部分显示所有非CG的G都变为了A(即用于甲基化检测的部分)。
本发明的方法同样可应用于单个基因位点的检测，其实验流程图如图7。通过设计用于扩增感兴趣的基因位点的PCR扩增引物，一条引物定位于胞嘧啶发生甲基化的序列位置；另一引物定位于未甲基化的胞嘧啶已转变为胸腺嘧啶的序列位置，进行PCR扩增，得到感兴趣的基因位点的序列并进行甲基化分析。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (14)

  1. 一种核酸甲基化的测定方法，其特征在于，所述的核酸是双链，所述方法包括：
    (1)针对核酸双链，利用具有3’→5’方向切割功能的聚合酶或3’→5’核酸外切酶处理，使两条链分别在3’端产生80-200个碱基缺失；
    (2)加入dNTP，其中的胞嘧啶为甲基化修饰的胞嘧啶，使双链3’端的缺失被补平且其中的胞嘧啶为甲基化修饰的胞嘧啶；
    (3)处理步骤(2)的双链，使未被甲基化修饰的胞嘧啶转变为尿嘧啶、甲基化修饰的胞嘧啶则不变；
    (4)PCR扩增，进行甲基化测序，其中一部分序列中未甲基化的胞嘧啶已转变为胸腺嘧啶，用于判断甲基化位点；另一部分序列因胞嘧啶发生甲基化而与原核酸序列相同，用于数据分析时的序列定位；将两部分序列比对，从而得知核酸的甲基化状况。
  2. 如权利要求1所述的方法，其特征在于，步骤(3)中，应用重硫酸盐、重亚硫酸盐、亚硫酸氢盐或重亚硫酸氢盐处理步骤(2)的双链。
  3. 如权利要求1所述的方法，其特征在于，所述的核酸是长度大于2kb的核酸或是全基因组，在步骤(1)之前，还包括：将核酸序列进行打断，形成长度为200-1000bp的双链片段。
  4. 如权利要求1所述的方法，其特征在于，应用于感兴趣的基因位点的检测，其中步骤(4)包括：
    设计用于扩增感兴趣的基因位点的PCR扩增引物，一条引物定位于胞嘧啶发生甲基化的序列位置；另一引物定位于未甲基化的胞嘧啶已转变为胸腺嘧啶的序列位置，进行PCR扩增，得到感兴趣的基因位点的序列并进行甲基化分析。
  5. 如权利要求1所述的方法，其特征在于，步骤(2)中，在进行补平之后，还包括：在步骤(2)的双链的两端加上测序接头，从而应用于高通量测序。
  6. 如权利要求5所述的方法，其特征在于，所述的测序接头如下连接：在双链的3’末端加上1个突出的A，应用带有3’末端含有一个突出的T的测序接头与之连接。
  7. 如权利要求6所述的方法，其特征在于，所述的测序接头是甲基接头，后续采用高通量测序方法测序。
  8. 如权利要求1所述的方法，其特征在于，所述的胞嘧啶的甲基化修饰包括：CpG甲基化，CHG甲基化或CHH甲基化。
  9. 如权利要求1所述的方法，其特征在于，所述的具有3’→5’方向切割功能的聚合酶包括：T4 DNA聚合酶，T7 DNA聚合酶，Klenow酶；
    所述的3’→5’核酸外切酶包括：核酸外切酶III。
  10. 如权利要求9所述的方法，其特征在于，当步骤(1)应用3’→5’核酸外切酶时，步骤(2)中还加入聚合酶。
  11. 如权利要求10所述的方法，其特征在于，所述的聚合酶包括：Taq酶，Pfu，逆转录酶。
  12. 如权利要求1所述的方法，其特征在于，步骤(4)中，与原核酸序列相同的序列为双端测序中的一个read，或是单端测序中一个read的一部分。
  13. 如权利要求12所述的方法，其特征在于，进行双端测序，测序结果中，一端核酸序列中未甲基化的胞嘧啶已转变为胸腺嘧啶，用于判断甲基化位点；另一端序列因胞嘧啶发生甲基化而与原核酸序列相同，用于序列定位；将双端序列进行比对，从而得知核酸的甲基化状况。
  14. 如权利要求1所述的方法，其特征在于，步骤(4)中，核酸序列的分析比对方法为双端测序，一端序列作初步定位，另一端序列在其附近检索，所述的方法包括：
    (a)将一端序列定位到基因组上，允许每个序列定位到基因组的多个位置；
    (b)为已定位到基因组上的序列匹配另一端序列；
    (c)根据在基因组中的初步定位，在其附近寻找另一端的位置；
    (d)若可获得多个比对位置，选取最佳比对位置；
    (e)去除PCR产生的扩增序列；
    (f)分析基因组的甲基化水平，统计甲基化率。
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