WO2020135347A1 - 一种dna甲基化检测的方法、试剂盒、装置和应用 - Google Patents

一种dna甲基化检测的方法、试剂盒、装置和应用 Download PDF

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WO2020135347A1
WO2020135347A1 PCT/CN2019/127537 CN2019127537W WO2020135347A1 WO 2020135347 A1 WO2020135347 A1 WO 2020135347A1 CN 2019127537 W CN2019127537 W CN 2019127537W WO 2020135347 A1 WO2020135347 A1 WO 2020135347A1
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sequencing
dna
library
methylation
double
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French (fr)
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王冀
王欧
章文蔚
陈奥
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BGI Shenzhen Co Ltd
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BGI Shenzhen Co Ltd
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Definitions

  • the present application relates to the field of DNA methylation detection, in particular to a method, kit, device and application for DNA methylation detection.
  • the rapid development of molecular biology has made gene sequencing technology one of the important methods in modern biological research, which is widely used in tumor prevention, screening, diagnosis, treatment and prognosis.
  • Gene sequencing technology can truly reflect all the DNA genetic information on the genome, and then reveal the tumor's occurrence mechanism and development process more comprehensively, so it has a very important position in the scientific research of tumor.
  • the first generation sequencing technology is the dideoxy nucleotide end termination method invented by Sanger et al.
  • the second generation sequencing technology includes 454 technology by Roche, Solexa technology by Illumina, and ABI’s SOLiD technology and BGI's nanosphere sequencing technology, etc.; Helicos and Pacbio's single molecule sequencing technology is called the third generation sequencing technology. Because the third-generation sequencing technology has higher requirements on libraries and the cost of sequencing is higher, the second-generation sequencing technology is currently the most widely used. For example, whole genome sequencing is applied to non-invasive prenatal gene detection, target area capture sequencing is applied to tumor targeted drug gene detection, single cell genome and transcriptome sequencing are applied to tumor tissue heterogeneity and development mechanism research, long fragment sequencing It is applied to the research of noninvasive thalassaemia detection, etc.
  • Second-generation sequencing technology also known as high-throughput sequencing technology, has revolutionized molecular testing in clinical laboratories; therefore, how to construct a higher-quality sequencing library to obtain better sequencing data and become an efficient test And important issues of wide clinical application.
  • Epigenetic research has once again become a hot spot in genetic diagnosis and precision medicine research.
  • Epigenetics refers to changes in heritable gene expression that occur without changing the nucleotide sequence of the gene, such as methylation modification. Sequencing detection for methylation modification is called methylation sequencing.
  • the epigenetic information provided by methylation sequencing has an important role in gene imprinting, embryonic development, cancer prevention and other research fields.
  • the methylation of bases especially the methylation information of cytosine, is the most easily obtained and the most in-depth researched molecular genetic marker of epigenetics.
  • methylation site can be determined by comparing with the known reference genome.
  • DNA degradation is usually severe after bisulfite treatment, and the degradation rate can even reach more than 90%, so many small DNA molecules will be obtained, which will cause difficulties in later genome comparison; Moreover, part of the information will be lost through this process, and it is easy to lose the full-length information.
  • accurate methylation sequencing can be performed on a specific region of the gene, it is more difficult to assemble methylation information of the whole genome.
  • the bisulfite treatment will change the unmethylated DNA sequence information, it is impossible to add a molecular tag (barcode) sequence in advance to build a library, so that it is difficult to trace the molecular source, which is homologous to polyploid organisms.
  • Methylation information extraction brings difficulties and reduces the credibility of data comparison.
  • some sequencing kits can solve the above-mentioned molecular source tracking problem to a certain extent by using a synthetic methylated sequencing adaptor and arranging the addition step before fragmentation; however, methylated sequencing adaptor synthesis
  • the high cost is not conducive to large-scale high-throughput sequencing operations.
  • the existing methylation sequencing the main time cost is reflected in the data analysis after sequencing; for species without a reference genome, it is very difficult to directly perform methylation analysis.
  • the purpose of this application is to provide a new method, kit, device and application for DNA methylation detection.
  • the first aspect of the present application discloses a DNA methylation detection method, including the following steps,
  • the double-strand protection step includes taking a conventional library prepared by a conventional library construction step, recovering magnetic beads, denaturing the DNA into a single strand, and then converting the molecular tag sequence or sequencing linker sequence into a double strand by annealing or extension; wherein,
  • magnetic beads with molecular tag sequences are used to capture DNA fragments, and then the magnetic beads and captured DNA fragments are directly captured.
  • the stLFR library is constructed together to obtain a conventional library; because the library construction process involves PCR amplification and other steps, there are two parts of nucleic acid in the conventional library, one is the DNA that is always bound to the magnetic beads, and the other is amplified by PCR. Increase the free DNA generated; the DNA used for methylation sequencing is the DNA bound to the magnetic beads. Therefore, it is necessary to take a conventional library for magnetic bead recovery; and the free DNA is then subjected to subsequent routine sequencing to obtain normal gene sequencing. result;
  • the bisulfite conversion step includes adding a double-strand protection agent to the product of the double-strand protection step, and adding bisulfite to perform a deamination reaction;
  • the steps of constructing a methylation sequencing library include performing conventional PCR amplification or rolling circle amplification on the products of the deamination reaction to obtain a methylation sequencing library;
  • the methylation sequencing step includes sequencing the methylation sequencing library, and analyzing and obtaining the methylation information of the genes according to the sequencing results.
  • the analysis of methylation information based on the sequencing results specifically refers to comparing the sequencing results of the methylation library with the results of normal gene sequencing to obtain the methylation information of the genes; for species with reference genomes, it is used to The normal gene sequencing result of the comparison is the reference genome; for the species without the reference genome, the normal gene sequencing result used for the comparison is the sequencing result obtained by conventional sequencing of the aforementioned free DNA.
  • the DNA methylation detection method of the present application uses stLFR technology for conventional library construction, and uses the co-barcoding adopted by this technology to obtain methylation information of long fragments, which solves the existing methylation sequencing
  • the problem of short read length at the same time, the method of double-stranded protection is used to protect the molecular tag sequence or sequencing adapter sequence without affecting the bisulfite treatment of the tested fragment, which simply and effectively solves the existing
  • the problem that methylation sequencing cannot add molecular tag sequence to build database in advance can not only effectively track the molecular source, but also conveniently extract the methylation information of homologous chromosomes of polyploid organisms.
  • the DNA methylation detection method of the present application further includes a conventional sequencing step.
  • the conventional sequencing step includes taking a conventional library constructed using the stLFR technology in a conventional library construction step, denaturing the DNA into a single strand, and then performing conventional PCR expansion. After adding or circularizing, rolling circle amplification is performed to obtain a sequencing library, and the sequencing library is subjected to whole genome sequencing to obtain normal gene sequencing results; therefore, the methylation sequencing step also includes the sequencing results of the methylation sequencing library and the conventional library. The obtained normal gene sequencing results are compared to obtain the methylation information of the gene.
  • the sequencing results of the methylation sequencing library can be directly compared with the reference gene to obtain methylation information of the gene.
  • the preferred solution of the present application is based on the principle of co-building the library, and the conventional library obtained by the conventional library construction step is divided into two parts, one part performs whole genome sequencing to obtain normal gene sequencing results; the other part is processed Then perform methylation sequencing; compare and analyze the sequencing results of the methylation sequencing with the normal gene sequencing results of the whole genome sequencing to obtain the methylation information of the genes.
  • the scheme of co-building a library of the present application can perform one-to-one comparison of the libraries before and after processing at the molecular level without reference libraries or reference genomes, thereby obtaining more accurate methylation information.
  • the processing step of the long fragment DNA further includes: capturing the small fragment DNA by using a carrier with a molecular tag sequence, and then using the captured DNA in a conventional library construction step to construct a library of stLFR technology.
  • the carrier is magnetic beads.
  • magnetic bead carrier is a carrier conventionally used in the art, and it is not excluded that other carriers can also be used.
  • the long-length DNA is broken into small-length DNA specifically using Tn5 transposase.
  • Tn5 transposase interruption to obtain small fragments of DNA is a technique conventionally used in the art, and it is not excluded that other physical or chemical means can also be used to interrupt DNA.
  • the extension method specifically includes using the molecular tag sequence or the 3'end sequence of the sequencing linker sequence itself as a primer or adding a foreign short fragment as a primer, extending under the action of DNA polymerase, and extending the molecule
  • the tag sequence or sequencing linker sequence becomes a double-stranded structure.
  • the double-stranded protective agent is at least one of salt ion, polyethylene glycol, enzyme, and organic solvent, and the bisulfite is sodium bisulfite.
  • salt ions such as NaCl
  • polyethylene glycol such as polyethylene glycol 8000.
  • kits for DNA methylation detection which includes a first group of reagents, a second group of reagents, a third group of reagents, a fourth group of reagents, and a fifth group of reagents;
  • the set of reagents includes reagents for breaking long DNA into small fragments of DNA;
  • the second set of reagents includes reagents for library construction with stLFR technology, which is used to prepare conventional libraries;
  • the third set of reagents includes reagents for annealing or
  • the extended method converts the molecular tag sequence or sequencing linker sequence into a double-stranded reagent, which is used to form a double-stranded structure;
  • the fourth group of reagents includes a double-stranded protective agent and bisulfite, which is used for double-stranded protection and removal Amino reaction;
  • the fifth group of reagents includes reagents used for conventional PCR amplification or rolling circle
  • the kit of the present application is actually an organic combination of the core reagents according to the DNA methylation detection method of the present application. On the one hand, it can facilitate the DNA methylation detection method of the present application. On the other hand, it can improve the efficiency of the use of each reagent and reduce waste, thereby reducing the cost of DNA methylation detection of the present application.
  • the kit of the present application further includes a sixth group of reagents, and the sixth group of reagents includes reagents for performing conventional PCR amplification or circularization and rolling circle amplification on conventional libraries, and the reagents are used for preparing conventional sequencing libraries , In order to facilitate the sequencing of the entire gene and obtain the normal gene sequencing results. It can be understood that, in the sixth group of reagents, reagents required for conventional sequencing can also be added according to requirements.
  • the kit of the present application is developed according to the DNA methylation detection method of the present application, therefore, the first group reagent, the second group reagent, the third group reagent, the fourth group reagent and the fifth group Reagents are actually used in sequence for the long DNA processing steps, conventional library construction steps, double-strand protection steps, bisulfite conversion steps, and methylation sequencing library construction steps in the DNA methylation detection method of the present application;
  • the sixth set of reagents is used for routine library construction in routine sequencing steps.
  • the sixth kit of reagents can be selectively added or not added to the kit of the present application, and Alternatively, the sixth group of reagents can be combined into the kit of the present application, and can be selectively used according to needs.
  • sequencing kit is a separate kit product, so it is not included in the kit of the present application, for example, conventional sequencing has an independent conventional sequencing kit, and the methylation sequencing step has a methylation sequencing kit .
  • the first group of reagents further includes a carrier with a molecular tag sequence for capturing DNA fragments.
  • the carrier is magnetic beads
  • the first group of reagents further includes reagents for magnetic bead purification.
  • reagents for magnetic bead purification include, for example, magnetic bead buffer, washing solution, and eluent.
  • the reagent used to break the long DNA into small DNA is Tn5 transposase and its buffer.
  • reagents for converting molecular tag sequences or sequencing linker sequences into double strands by annealing or extension methods specifically including exogenous short nucleotide fragments, DNA polymerases and their buffers, And dNTPs for DNA extension.
  • the exogenous short nucleotide fragment is used as a primer, which can be used according to needs or not added to the third group of kits; for example, in one implementation of this application, the molecular tag sequence can be directly used Or the 3'end sequence of the sequencing linker sequence itself is extended as a primer, therefore, the exogenous short nucleotide fragments can only be put into the third group of reagents as an alternative.
  • the double-stranded protecting agent is at least one of salt ion, polyethylene glycol, enzyme and organic solvent, and the bisulfite salt is sodium bisulfite.
  • the functions of the DNA methylation detection method of the present application may be implemented by hardware, or by a computer program.
  • the program When implemented by a computer program, the program may be stored in a computer-readable storage medium, and the storage medium may include: read-only memory, random access memory, magnetic disk, optical disk, hard disk, etc.
  • the program is executed by a computer to implement the application Methods.
  • the program is stored in the memory of the device, and when the processor executes the program in the memory, the method of the present application can be implemented.
  • the program may also be stored in a storage medium such as a server, another computer, a magnetic disk, an optical disk, a flash disk, or a mobile hard disk, and saved by downloading or copying to In the memory of the local device or the version update of the system of the local device, when the program in the memory is executed by the processor, all or part of the functions of the DNA methylation detection method of the present application can be realized.
  • a storage medium such as a server, another computer, a magnetic disk, an optical disk, a flash disk, or a mobile hard disk
  • a DNA methylation detection device including a long fragment DNA processing module, a conventional library construction module, a conventional sequencing module, a double-stranded protection module, a bisulfite conversion module, methylation Sequencing library construction module and methylation sequencing module;
  • Long-segment DNA processing module including for breaking long-segment DNA into small-segment DNA; the module can use conventional physical, chemical or enzymatic methods to interrupt long-segment DNA; for physical methods, for example, ultrasonic breaking can be used ; Corresponding to the way of chemistry or enzyme, on the one hand, the module can automatically sample or prepare the reaction solution, on the other hand, it can provide temperature and other reaction conditions for the reaction;
  • Conventional sequencing module including the conventional library constructed by using the stLFR technology in the conventional library construction module, denatures the DNA into single strands, and then performs conventional PCR amplification or circularization and rolling circle amplification to obtain the sequencing library.
  • the sequencing library performs whole genome sequencing to obtain normal gene sequencing results; this module is only used without reference genes. Of course, if you want to obtain more accurate results before and after processing, you can also use this module; this module can refer to the existing Automatic library building and sequencing equipment or devices;
  • Double-stranded protection module including the conventional library constructed using stLFR technology in the conventional library construction module, magnetic bead recovery, denaturation of DNA into single strand, and then the molecular tag sequence or sequencing adapter sequence is transformed by annealing or extension method It is double-stranded; on the one hand, the module can refer to automatic sampling or preparation of the reaction solution, on the other hand, it can provide temperature and other reaction condition control for annealing or extension;
  • Bisulfite conversion module including adding double-strand protection agent to the product processed by the double-strand protection module, and adding bisulfite to carry out the deamination reaction;
  • the module can also refer to the existing automatic sampling and reaction Liquid preparation device or equipment, and provide corresponding reaction conditions for deamination reaction;
  • Methylation sequencing library construction module including conventional PCR amplification or circularization and rolling circle amplification of the product of the deamination reaction, to obtain a methylation sequencing library; this module can refer to the existing sequencing module;
  • Methylation sequencing module including for sequencing methylation sequencing libraries, comparing methylation sequencing results with normal gene sequencing results to obtain gene methylation information; this module can refer to existing methylation Sequencing and analysis module or device.
  • the normal gene sequencing result may be a sequencing result obtained by a conventional sequencing module, or it may be an existing reference genome.
  • the DNA methylation detection device of the present application realizes the automatic detection of DNA methylation in each step of the detection method through an automated module, that is, the DNA methylation of the present application
  • the detection device provides a personalized detection system for DNA methylation detection, which can conveniently and effectively perform DNA methylation detection.
  • the DNA methylation detection device of the present application also has various advantages of the DNA methylation detection method of the present application, for example, solves the problem of short read length of the existing methylation sequencing, and solves the existing A Basic sequencing can not add the molecular tag sequence to build the database in advance, and cannot track the molecular source.
  • its long-segment DNA processing module further includes a carrier with a molecular tag sequence to capture small-segment DNA, and then use the captured DNA in a conventional library
  • the construction module adopts stLFR technology for library construction
  • the carrier is magnetic beads.
  • the long-segment DNA is broken into small-segment DNA specifically using Tn5 transposase.
  • the extension method specifically includes using the molecular tag sequence or the 3'end sequence of the sequencing adapter sequence itself as a primer or adding a foreign short fragment as a primer , Extension under the action of DNA polymerase, the molecular tag sequence or sequencing adapter sequence into a double-stranded structure.
  • the double-stranded protective agent is at least one of salt ion, polyethylene glycol, enzyme, and organic solvent, and the bisulfite is Sodium bisulfite.
  • a DNA methylation detection device which includes a memory and a processor; the memory is used to store a program; the processor is used to implement the DNA methylation detection of the application by executing the program stored in the memory method.
  • Another aspect of the present application also discloses a computer-readable storage medium, including a program stored therein, which can be executed by a processor to implement the DNA methylation detection method of the present application.
  • Another aspect of the application also discloses the application of the DNA methylation detection method of the application, the kit of the application or the DNA methylation detection device of the application in gene imprinting detection, embryo development detection or cancer prevention detection.
  • the DNA methylation detection method, kit and device of the present application can obtain long-segment methylation information, which can not only effectively track the molecular source, but also facilitate the homologous chromosomal methylation of polyploid organisms Information extraction; therefore, it can be conveniently applied to the fields of gene imprinting detection, embryonic development detection, cancer prevention detection, etc.
  • the DNA methylation detection method of the present application uses the advantages of stLFR technology to obtain the methylation information of long fragments; the double-stranded protection method is used to protect the molecular tag sequence or sequencing adapter sequence, and the molecular tag sequence can be added in advance
  • the establishment of a library can effectively track the molecular source and conveniently extract the homologous chromosome methylation information of polyploid organisms.
  • FIG. 1 is a schematic flowchart of a DNA methylation detection method in an embodiment of this application
  • FIG. 2 is a schematic structural diagram of a DNA methylation detection device in an embodiment of the present application.
  • FIG. 3 is a schematic diagram of a double-stranded protective molecular tag based on magnetic bead capture in an embodiment of the present application
  • FIG. 5 is a schematic diagram of different forms of double-chain protection in an embodiment of the present application.
  • FIG. 6 is a schematic diagram of different forms of double-chain protection in the embodiments of the present application.
  • FIG. 7 is an agarose condensation electrophoresis diagram of the stLFR conventional library (WGS) and methylation sequencing library (WGBS) constructed in the examples of the present application.
  • the existing methylation sequencing read length is short, and the general conventional methylation sequencing cannot add the molecular tag sequence to build the database in advance, cannot track the molecular source, and it is difficult to achieve homologous chromosome methylation information of polyploid organisms extract.
  • synthetic methylated sequencing adapters can be used to solve the problem of adding molecular tags in advance, synthetic methylated sequencing adapters are costly and difficult to use for large-scale high-throughput sequencing.
  • the present application creatively borrowed the stLFR technology to construct the library to solve the problem of short read length; then, creatively proposed a double-strand protection strategy to protect the molecular tag sequence or sequencing adapter sequence added in advance to avoid its being Bisulfite treatment destroys to solve the problem that the molecular tag sequence cannot be added in advance to build the library; the strategy of using the same library to build the library twice solves the problem of alignment of the methylated library under the condition of no reference genome.
  • this application has developed a method for DNA methylation detection, as shown in Figure 1, including a long DNA processing step 11, a conventional library construction step 12, a double-strand protection step 14, a bisulfite conversion step 15. Methylation sequencing library construction step 16, methylation sequencing step 17; in a preferred solution, based on the principle of co-building the library, the method of the present application further includes a conventional sequencing step 13.
  • the long DNA processing step 11 includes breaking the long DNA into small DNA; including using physical, chemical or enzymatic methods to break the long DNA, for example, in one implementation of this application, specifically using Tn5 Transposase breaks DNA into small pieces.
  • the conventional library construction step 12 includes using the stLFR technology for library construction to obtain a conventional library.
  • it also includes using a carrier with a molecular tag sequence to capture the interrupted DNA fragment, and then performing stLFR technology to build a database.
  • a carrier with a molecular tag sequence that is, a carrier with multiple copies of a specific tag sequence or molecular tag refers to an oligonucleotide sequence with multiple copies of the same sequence on a single carrier.
  • magnetic beads are specifically used as a carrier.
  • the present application is connected by a double-streptavidin protein chimera, and chemically modified DNA is used on the magnetic beads.
  • Chemical group reactions include but are not limited to I-linker, Amino-modified oligo, Thiol-modified oligo, Acrydite-modified oligo, etc. It can be understood that the magnetic beads need to undergo two rounds of PCR amplification in the subsequent reaction. Therefore, the coupling between the DNA and the magnetic beads must be able to withstand acid and alkali, high temperature, and not be easily degraded.
  • Routine sequencing step 13 including taking a conventional library constructed using the stLFR technology in the conventional library construction step, denaturing the DNA into single strands, and then performing conventional PCR amplification or circularization and rolling circle amplification to obtain a sequencing library, and sequencing library Perform sequencing to obtain normal gene sequencing results.
  • This step is a normal DNA library building and sequencing step. Its purpose is to facilitate the species without known reference genes. The methylation sites can be accurately analyzed by comparison before and after methylation treatment. It can be understood that if the species to be tested already has a more accurate reference gene sequence, this step may not be needed.
  • the double-strand protection step 14 includes taking a conventional library constructed using stLFR technology in a conventional library construction module, magnetic bead recovery, denaturing the DNA into single strands, and then using annealing or extension methods to convert the molecular tag sequence or sequencing adapter sequence into Double chain.
  • Double-stranded protection can be achieved by annealing or extension, where extension is achieved, for example, using the molecular tag sequence or the 3'end sequence of the sequencing linker sequence itself as a primer, as shown in Figure 3, Panel A, or, adding The short exogenous fragment serves as a primer, as shown in B of Figure 3, and is extended under the action of DNA polymerase to change the molecular tag sequence or sequencing linker sequence into a double-stranded structure.
  • the double-stranded protection can also have multiple forms of protection according to different vectors or different forms of DNA, as shown in FIGS. 4 to 6.
  • the left column of Fig. 4 is the double-stranded protection of DNA bound to the magnetic beads, and the right column is the double-stranded protection of free DNA; " ⁇ " indicates the magnetic beads, the dark double solid line indicates the complementary DNA double strand, and the light double solid line indicates Non-complementary DNA double-strand; a for single-ended protection, b for double-ended protection, c for bridge protection, d for 3'and/or 5'end ring structure protection, e for 3'and/or 5'end neck Ring bridge structure protection, f is the intramolecular neck ring structure protection.
  • Figure 5 shows the double-stranded protection mechanism of circular DNA.
  • the dark double solid line indicates the complementary DNA double-strand; a is the double-stranded protection introduced with a single-stranded primer, b is the double-stranded protection through the Y-linker primer, c It forms double-strand protection for single-strand self-forming ring.
  • Figure 6 shows the double-stranded protection mechanism of DNA with a solid surface as a carrier.
  • the dark double solid line represents complementary DNA double-strand
  • the light double solid line represents non-complementary DNA double-strand
  • a and b are single-ended at different positions Protection
  • c is double-ended protection
  • d is neck ring type single-ended protection
  • e is bridge structure protection
  • f is 3'or 5'end neck ring bridge structure protection
  • g is intermolecular bridge structure protection.
  • the specific protection mechanism adopted can be determined according to the design of the DNA sequence, or different sequences can be designed according to different vectors to adopt different double-strand protection mechanisms.
  • the bisulfite conversion step 15 includes adding a double-strand protection agent to the product of the double-strand protection step, and adding bisulfite to perform a deamination reaction.
  • Bisulfite such as sodium bisulfite
  • double-stranded and single-stranded libraries exist
  • the double-stranded protection reagents that can be used in the present application include salt ions, polyethylene glycol, enzymes, and organic solvents, etc., which can effectively prevent denaturation of double-stranded DNA at high temperatures, while maintaining single-strand conversion efficiency.
  • Step 16 of constructing a methylation sequencing library includes performing conventional PCR amplification or circularization and rolling circle amplification on the product of the deamination reaction to obtain a methylation sequencing library.
  • the methylation sequencing step 17 includes sequencing the methylation sequencing library, then performing data analysis, and analyzing and obtaining methylation information of the gene according to the sequencing results.
  • the DNA methylation detection method of the present application has the following advantages:
  • the methylation level of different chromosomes in the polyploid genome can be determined
  • DNA methylation detection method of the present application is also applicable to methylation sequencing (5mC) and hydroxymethylation sequencing (5hmC), detailed description is as follows:
  • the library is first oxidized to convert 5hmC to carboxycytosine (5caC), and then to Bisulfite treatment, both unmethylated C and 5caC will be converted to U, while 5mC will not Was transformed to obtain complete 5mC sequencing information.
  • the library is first oxidized to convert 5mC and 5hmC to carboxycytosine (5caC), and then subjected to borane treatment to convert it to U, thereby obtaining 5mC and 5hmC sequencing information.
  • the present application has developed a DNA methylation detection device, as shown in FIG. 2, which includes a long fragment DNA processing module 21, a conventional library construction module 22, a conventional sequencing module 23, a dual Chain protection module 24, bisulfite conversion module 25, methylation sequencing library construction module 26 and methylation sequencing module 27; long-segment DNA processing module 21, including for breaking long-segment DNA into small-segment DNA; Conventional library construction module 22, including for library construction using stLFR technology; conventional sequencing module 23, including for use of conventional library constructed with stLFR technology, denaturing DNA into single strands, and then performing conventional PCR amplification or loop Rolling circle amplification after chemical conversion to obtain a sequencing library, sequencing the sequencing library to obtain normal gene sequencing results; double-stranded protection module 24, including the conventional library used to access part of the library constructed using stLFR technology, magnetic beads recovery, DNA denaturation Is single-stranded, and then annealed or extended to convert
  • the DNA methylation detection method and device of the present application not only have the advantages of stLFR, but also can give accurate methylation information:
  • human genomic DNA sequencing is used as an example to perform DNA methylation detection.
  • the process includes: first extracting a long fragment of genomic DNA, and then using Tn5 transposase to break the DNA into small fragments, using a magnetic tag sequence Beads are captured.
  • the library was constructed using stLFR technology to obtain a conventional library. After obtaining a library for conventional library construction, perform conventional PCR amplification, draw the PCR product supernatant to obtain library L, and perform conventional computer-based sequencing operations to obtain WGS data.
  • the magnetic beads of the conventional library are recovered for methylation sequencing; the DNA molecules on the recovered magnetic beads are fully denatured into single strands, and then the 3'end sequence of the molecules on the magnetic beads is used as a primer or an external The short fragment of the source is used as a primer.
  • the DNA molecular tag sequence is changed into a double-stranded structure, a double-stranded protective agent is added and a sodium bisulfite conversion reaction is performed, and the reaction is completed
  • library M is obtained, and library M is sequenced to obtain WGBS data; finally, WGS and WGBS data are compared one to one to determine the methylation site.
  • the DNA methylation detection in this example is as follows:
  • Linker is a double-stranded DNA molecule, which is annealed together by two single-stranded DNA strands.
  • the annealing conditions of the two single-stranded DNAs are 70°C for 1 minute, and then slowly cooled to 20°C at a rate of 0.1°C/s and react at 20°C 30 minutes.
  • Linker In Linker's double-stranded DNA molecule, the sense strand is Linker-F and the antisense strand is Linker-R; where Linker-F is the sequence shown in SEQ ID NO.1, and the 3'end of Linker-F has Bio modification, Linker- R is the sequence shown in SEQ ID NO.2.
  • SEQ ID NO. 2 5’-CGTAGCCATGTCGTTCTGCC-3’.
  • the magnetic beads with streptavidin used in this example are Dynabeads M-280 streptation (112.06D, Invitrogen).
  • the magnetic bead washing buffer was washed twice, and finally the magnetic beads were resuspended with 12.5 ⁇ L of ligation buffer at a concentration of 1 times.
  • the magnetic bead binding buffer is composed of 50 mM Tirs-HCl, 150 mM NaCl and 0.1 mM EDTA;
  • the low-salt magnetic bead washing buffer is composed of 50 mM Tirs-HCl, 150 mM NaCl and 0.02% Tween-20; normal
  • the prepared ligation buffer is a 10-fold concentration, a 10-fold concentration of ligation buffer, which is composed of polyethylene glycol 8000 30%, Tris-HCl 150mM, ATP 1mM, BSA 0.15mg/mL, MgCl 2 30mM, DTT 1.5mM .
  • the annealing conditions of tag sequence 1 and auxiliary sequence 1 are: 100 ⁇ M of sequence 1 and auxiliary sequence 1 are mixed at a volume ratio of 1:1, placed on a PCR instrument, 70°C for 1 minute, and then slowly cooled to 0.1°C/s 20°C, 20°C for 30 minutes.
  • the tag sequence 1 is the sequence shown in SEQ ID NO. 3
  • the auxiliary sequence 1 is the sequence shown in SEQ ID NO. 4.
  • the 5'end of the tag sequence 1 contains the molecular tag sequence "Barcode”, and the molecular tag sequence "Barcode” is inserted between the 22nd base and the 23rd base of the auxiliary sequence 1.
  • the ligation mixture contains 1 ⁇ L ligase (T4 DNA ligase 600 U/ ⁇ L, Enzymatics), 1.5 ⁇ L ultrapure water and 1 ⁇ L ligation buffer (10 ⁇ T4 DNA ligation buffer, Enzymatics)
  • the ligation reaction was performed with a total reaction volume of 10 ⁇ L per 384-well plate at a reaction temperature of 25°C and a reaction time of 1 hour. Wash once with 100 ⁇ L of high-salt magnetic bead washing buffer and once again with 100 ⁇ L of low-salt magnetic bead washing buffer to remove the ligase in the reaction and the oligonucleotide that has not completely reacted.
  • the high-salt magnetic beads washing buffer is composed of 50 mM Tirs-HCl, 500 mM NaCl, and 0.02% Tween-20;
  • the low-salt magnetic beads washing buffer is composed of 50 mM Tirs-HCl, 150 mM NaCl, and 0.02% Tween-20.
  • step (3) Collect the magnetic beads cleaned in step (3) through a magnetic stand, and then resuspend the magnetic beads with a ligation buffer of 1 times the concentration. After resuspending, the concentration of Linker is 1.6 ⁇ M, and mix using a shaker mixer .
  • the annealing conditions of tag sequence 2 and auxiliary sequence 2 are: 2 ⁇ L of tag sequence 2100 ⁇ M and 2100uM of auxiliary sequence are mixed in a 384-well plate, and then placed on a PCR instrument at 70°C for 1 minute, and then slowly cooled at a rate of 0.1°C/s To 20 °C, 20 °C reaction for 30 minutes.
  • the tag sequence 2 is the sequence shown in SEQ ID NO.5
  • the auxiliary sequence 2 is the sequence shown in SEQ ID NO.6.
  • the molecular tag sequence "Barcode” is inserted between the 47th base and the 48th base of the tag sequence 2, and the 3'end of the auxiliary sequence 2 contains the molecular tag sequence "Barcode”.
  • SEQ ID NO.6 5’-TAAAACGACG[Barcode]-3’.
  • step (6) Dispense the magnetic beads mixed in step (4) into each well of the 384-well plate of step (5) in an amount of 2.5 ⁇ L per well. Then add 3.5 ⁇ L of ligation mixture containing 1 ⁇ L of ligase (T4 DNA ligase 600 U/ ⁇ L, Enzymatics), 1.5 ⁇ L ultrapure water and 1 ⁇ L of ligation buffer (10 ⁇ T4 DNA ligation buffer, Enzymatics) at 25°C React for 1 hour.
  • ligase T4 DNA ligase 600 U/ ⁇ L, Enzymatics
  • the strong alkali denaturation buffer is composed of 1.6M KOH and 1mM EDTA;
  • the hybridization buffer is composed of 50mM Tris-HCl, 1000mM NaCl, 0.05% Tween-20.
  • step (1) Mix 50 ⁇ L of the interrupted DNA solution with 50 ⁇ L of the labeled (Barcode) magnetic beads in step (1), react at 60°C for 1 minute, then let the reaction solution be placed at room temperature, let it cool down naturally, and place it in a vertical mixer. The reaction was mixed at 25°C for 1 hour and labeled as a hybrid capture system.
  • the mixed reaction solution contains: DNA polymerase T4, DNA Polymerase (3U/ ⁇ L, Enzymatics) 6 ⁇ L, ligase T7 DNA, ligase (3000U/ ⁇ L, NEB) 1 ⁇ L, 2 ⁇ T7 DNA ligase buffer (NEB) 25 ⁇ L, 25mM dNTP 0.5 ⁇ L, 10mM ATP and 5 ⁇ L, make up the volume of water to 50 ⁇ L.
  • the 2 ⁇ T7 DNA ligase buffer contains: 132 mM Tris-HCl, 20 mM MgCl 2 , 2 mM ATP, 2 mM DTT, 15% Polyethyleneglycol (polyethylene glycol 6000).
  • the closed sequence 1 is the sequence shown in SEQ ID NO.7.
  • the polymerase reagent mixed solution contains: Polymerase Standard (Taken) Polymerase (5U/ ⁇ L, NEB) 1 ⁇ L, 10 ⁇ thermopol buffer (NEB) 5 ⁇ L, 25 mM dNTP (Enzymatics) 0.8 ⁇ L, total volume 50 ⁇ L. After the reaction, the magnetic beads were adsorbed with a magnetic stand, and the supernatant was recovered.
  • 10 ⁇ thermopol buffer contains: 200mM Tris-HCl, 100mM(NH 4 ) 2 SO 4 , 100mM KCl, 20mM MgSO 4 , 1% X-100.
  • the ligase reagent mixture contains ligase, which consists of 5 ⁇ L of T4 DNA ligase (600 U/ ⁇ L, Enzymatics), 10 ⁇ L of ligation buffer at a three-fold concentration, and 21.5 ⁇ L of 16.7 ⁇ M adapter, with a total volume of 30 ⁇ L.
  • the three-fold concentration of the ligation buffer contains: 30% polyethylene glycol 8000, 150 mM Tris-HCl, 1 mM ATP, 0.15 mg/mL BSA, 30 mM MgCl 2 , and 1.5 mM DTT.
  • the 16.7 ⁇ M linker, linker 2 is formed by annealing the sense linker 2-F and the antisense linker 2-R.
  • the linker 2-F is the sequence shown in SEQ ID NO. 9, the 5'end of the linker 2-F has a phosphorylation modification, and the 3'end is a dideoxy base;
  • the linker 2-R is the sequence shown in SEQ ID NO. 10.
  • the 3'end of the linker 2-R is a dideoxy base.
  • the high-salt magnetic bead washing solution and the low-salt magnetic bead washing buffer are washed once.
  • Primer 2 is the sequence shown in SEQ ID NO.12, the 5'end of primer 2 has a phosphorylation modification; primer 3 is the sequence shown in SEQ ID NO.8, and the 3'end of blocking sequence 1 has a Bio modification.
  • SEQ ID NO. 12 5’-phos-GAGACGTTCTCGACTCAGCAGA-3’.
  • the polymerase and ligase mixed reagents include: polymerase (T4, DNA Polymerase 3U/ ⁇ L, Enzymatics) 6 ⁇ L, ligase (T7, DNA ligase 3000U/ ⁇ L, NEB) 1 ⁇ L, 2 ⁇ T7 DNA ligase buffer (NEB) 15 ⁇ L, 25 mM dNTP 0.5 ⁇ L, 10 mM ATP 5 ⁇ L, make up the volume of water to 30 ⁇ L.
  • the polymerase reagent mixture contains: polymerase (Bst2.0 DNA Polymerase 8U/ ⁇ L, NEB) 1 ⁇ L, 10 ⁇ Isothermal Amplification Buffer (NEB) 5 ⁇ L, 25 mM dNTP (Enzymatics) 0.8 ⁇ L, 100 mM MgSO 4 1.5 ⁇ L, total volume 50 ⁇ L.
  • polymerase Bst2.0 DNA Polymerase 8U/ ⁇ L, NEB
  • 10 ⁇ Isothermal Amplification Buffer (NEB) 5 ⁇ L
  • 25 mM dNTP (Enzymatics) 0.8 ⁇ L, 100 mM MgSO 4 1.5 ⁇ L, total volume 50 ⁇ L.
  • 10 ⁇ Isothermal Amplification Buffer contains: 200mM Tris-HCl, 100mM(NH 4 ) 2 SO 4 , 500mM KCl, 20mM MgSO 4 , 1% 20.
  • the reaction system is as follows:
  • step (4) 50 ⁇ L of the supernatant recovered in step (4), 75 ⁇ L of 2 ⁇ KAPA HiFi Master (Kapa Biosystems), 0.75 ⁇ L of primer 1 (100 ⁇ mol/L), 0.75 ⁇ L of primer 2 (100 ⁇ mol/L), 23.5 ⁇ L of ultrapure water.
  • the reaction conditions are as follows:
  • PCR product obtained by "two, stLFR technology library construction" under magnetic force, carefully absorb the supernatant, and use AMPure XPA beads (Agencourt AMPure XP-Medium, A63882, AGENCOURT) for purification.
  • AMPure XPA beads Amincourt AMPure XP-Medium, A63882, AGENCOURT
  • the recovered product is a short fragment molecule with molecular tags suitable for sequencing.
  • BGIseq-500 was used for sequencing. Therefore, it is necessary to perform a loop formation reaction on the product purified by magnetic beads. For details of the operation, refer to the loop formation step of the BGIseq-500 standard DNA fragment library construction process. Then, BGIseq-500 sequencing was performed on the cyclized products.
  • the magnetic beads of the stLFR library to a 1.5mL centrifuge tube, wash it thoroughly with 500 ⁇ L of 1 ⁇ Buffer D (0.1M NaOH) twice, place the centrifuge tube on a magnetic stand, discard the supernatant, and recover the magnetic beads. Then wash it twice with low-salt magnetic beads washing buffer, discard the supernatant, and recover the magnetic beads.
  • 47 ⁇ L of ultrapure water 50 ⁇ L of 2 ⁇ PfuCx Mix buffer (Agilent), 2 ⁇ L of PfuCx polymerase (Agilent), and 1 ⁇ L of ME primer were added.
  • the ME primer is a complementary sequence corresponding to the 3'end of the barcode in the library
  • the ME primer is the sequence shown in SEQ ID NO.13.
  • the mixture and magnetic beads were mixed well, centrifuged and transferred to a 250 ⁇ L PCR tube, and the following procedures were run on the PCR instrument: 98°C for 3 minutes, 50°C for 1 minute, 72°C for 10 minutes, and kept at room temperature.
  • the reaction was taken out on ice and washed once with high-salt magnetic beads washing solution and twice with low-salt magnetic beads washing buffer, the supernatant was removed, and the magnetic beads were recovered in a 1.5 mL centrifuge tube.
  • PCR Mix includes: 25 ⁇ L of 2 ⁇ PfuCx Mix (Agilent), 0.25 ⁇ L of 100 ⁇ M PCR Forward Forwarder, 0.25 ⁇ L of 100 ⁇ M PCR Reverse Primer, and 0.75 ⁇ L of PfuCx Polymerase (Agilent).
  • PCR Forward is the sequence shown in SEQ ID NO.14
  • PCR Reverse primer is the sequence shown in SEQ ID NO.15.
  • SEQ ID NO. 15 5’-GAGACGTTCTCGACTCAGCAGA-3’.
  • BGIseq-500 was used for sequencing. Therefore, it is necessary to perform a loop formation reaction on the product purified by magnetic beads. For details of the operation, refer to the loop formation step of the BGIseq-500 standard DNA fragment library construction process. Then, BGIseq-500 sequencing was performed on the cyclized products.
  • the results of the preparation of the methylation sequencing library and the corresponding stLFR library were run for 30 minutes.
  • the results are shown in FIG. 7.
  • the first lane is the DNA marker, in this case the Thermofisher 1kb plus DNA ladder
  • the second and third lanes are the S1 and S2 of WGS, that is, two repeats of the stLFR sequencing library for normal gene sequencing
  • fourth And the fifth lane is S1 and S2 of WGBS, that is, two repeats of the methylation sequencing library.
  • Test Clean_reads Clean_bases(bp) Mapped_reads Mapping_rate(%) Test Group 1 (Conventional Library) 156770568 15677056800 149334322 95.26 Test Group Two (Methylated Library) 156868710 15686871000 150202688 95.75 Control group SRR6006942 157796950 23827339450 133582687 84.65
  • the experimental group one is the data obtained by routine sequencing after the stLFR technology is built in this case.
  • the experimental group two is the data obtained by performing methylation library construction and sequencing after the stLFR technology is established. Data obtained by conventional methylation sequencing. Compared with the control group, the mapping efficiency of the conventional sequencing and methylation sequencing results in this case is higher than that of the conventional methylation sequencing control group.
  • this example extracts reads with the same molecular tag sequence and the same length from test group 1 and test group 2 for one-to-one comparison to obtain methylation information. For example, for the known low methylation level LINE gene fragment:
  • test group 1 1320_499_969 sequence is shown in SEQ ID NO.16,
  • SEQ ID NO. 16 and SEQ ID NO. 17 can confirm that the sequence has no methylation site. There is no need to compare with the human genome reference sequence, which is of great significance for species that do not have a reference genome sequence.
  • the DNA fragmentation enzymes tested in the above experiments can also be other enzymes of the Tn transposase family, such as Tn7; or other transposase families, such as the Mu family; even not limited to transposases Or an enzyme preparation, as long as it can fragment DNA while linking a sequence to DNA.
  • the enzymes, buffers, the number of molecular tags used, the carrier, the surface modification of the carrier, the molecular tag structure, the addition of linkers, etc. can be referred to WO2019217452A1.
  • the DNA methylation detection of this application can be used for whole-genome methylation sequencing, methylation sequencing of unknown regions of genes, methylation sequencing of known specific regions, comparison of methylation information of alleles on homologous chromosomes, Haploid whole genome methylation sequencing and assembly. Furthermore, DNA methylation detection of mitochondria, chloroplasts, etc. may be used.

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Abstract

本申请提供了一种DNA甲基化检测的方法，包括长片段DNA处理步骤，将长片段DNA打断；常规文库构建步骤，用stLFR技术建库；双链保护步骤，取回收的stLFR文库，DNA变性，用退火或延伸方法将分子标签或接头序列转化为双链；重亚硫酸盐转化步骤，加入双链保护剂和重亚硫酸盐，进行脱氨基；甲基化测序文库构建步骤，对脱氨基产物进行常规PCR或环化后滚环扩增，得到甲基化测序文库；甲基化测序步骤，对甲基化测序文库测序，根据测序结果分析基因的甲基化信息；本申请还提供了相应试剂盒、装置和应用。本申请方法利用stLFR技术，获得长片段甲基化信息；通过双链保护策略，可事先加入分子标签序列建库，可有效追踪分子来源。

Description

一种DNA甲基化检测的方法、试剂盒、装置和应用 技术领域
本申请涉及DNA甲基化检测领域，特别是涉及一种DNA甲基化检测的方法、试剂盒、装置和应用。
背景技术
分子生物学的快速发展，使基因测序技术成为现代生物学研究中重要的手段之一，广泛应用于肿瘤的预防、筛查、诊断、治疗和预后中。基因测序技术能够真实地反映基因组上全部DNA遗传信息，进而比较全面地揭示肿瘤的发生机制及其发展过程，因而在肿瘤的科学研究中具有十分重要的地位。第一代测序技术是1977年Sanger等发明的双脱氧核苷酸末端终止法和Gilbert等发明的化学降解法；第二代测序技术包括Roche公司的454技术、Illumina公司的Solexa技术、ABI公司的SOLiD技术和华大基因的纳米球测序技术等；Helicos公司和Pacbio的单分子测序技术被称为第三代测序技术。由于第三代测序技术对文库的要求较高，测序成本也较高，因此目前应用最广泛的是第二代测序技术。例如全基因组测序应用于无创产前基因检测、目标区域捕获测序应用于肿瘤靶向用药基因检测、单细胞基因组和转录组测序应用于肿瘤组织的异质性和发生发展机制的研究、长片段测序应用于无创地贫检测的研究等，多种临床检测及基础研究都是在第二代测序技术的平台上开展进行。第二代测序技术，也称高通量测序技术，为临床实验室的分子检测带来革命性的变化；因此，如何构建更高质量的测序文库，以得到更好的测序数据，成为高效检测和广泛临床应用的重要问题。
近年来，表观遗传学研究再次成为基因诊断和精准医疗研究的热点。表观遗传是指在不改变基因的核苷酸序列的情况下，发生的可遗传的基因表达的变化，例如甲基化修饰。针对甲基化修饰的测序检测称为甲基化测序。甲基化测序所提供的表观遗传学信息，在基因印记、胚胎发育、癌症预防等研究领域都有重要作用。在众多表观遗传因子中，碱基的甲基化，特别是胞嘧啶的甲基化信息，是目前实验手段最容易获得，且研究最为深入的表观遗传学分子级标志物。基于重亚硫酸盐的原理，对非甲基化的胞嘧啶进行化学转化，而甲基化的胞嘧啶不会被转化，从而得到基因组的甲基化信息。利用甲基化分析流程，与已知的参考基因组进行比对，即可确定甲基化位点。
目前，经典的全基因组甲基化测序文库的构建都是通过物理方式、酶切或者化学降解的方式将双链DNA长片段随机打断成几百bp的小片段，再将双链DNA变性成单链后，进行重亚硫酸氢钠处理，将没有甲基化的胞嘧啶通过脱氨基作用转化为尿嘧啶，通过引物退火延伸或加接头后延伸得到双链DNA分子，然后进行末端修复、 加“A”、加“接头”、PCR扩增等步骤，最终得到用于测序的文库。在获得测序数据后，因为大部分未甲基化的胞嘧啶转变为了尿嘧啶，因此需要将所得文库与已知的原始基因组进行比对，以确定甲基化位点或获得靶标区域的甲基化水平。
受限于脱氨基反应的反应原理，一般经过重亚硫酸盐处理后DNA降解严重，降解率甚至可以达到90％以上，因此会得到众多小片段DNA分子，为后期的基因组比对带来困难；并且，经过这个过程会损失一部分信息，容易丢失全长信息，虽然可以对基因的特定区域进行精确的甲基化测序，但是为全基因组的甲基化信息组装增加了难度。其次，因为重亚硫酸盐处理会改变非甲基化的DNA序列信息，因此无法事先加入分子标签(barcode)序列进行建库，以至于难以追踪分子来源，这对于多倍体生物的同源染色体甲基化信息提取带来困难，也会降低数据比对的可信度。虽然，部分测序试剂盒通过采用人工合成的已经甲基化的测序接头，将加接头步骤安排在片段化之前，可以一定程度上解决上述分子来源追踪的问题；但是，甲基化的测序接头合成成本高昂，不利于大规模的高通量测序操作。此外，现有的甲基化测序，其主要时间成本体现在测序后的数据分析上；对于没有参考基因组的物种，直接进行甲基化分析会非常困难。
发明内容
本申请的目的是提供一种新的DNA甲基化检测的方法、试剂盒、装置及其应用。
本申请具体采用了以下技术方案：
本申请的第一方面公开了一种DNA甲基化检测的方法，包括以下步骤，
长片段DNA处理步骤，包括将长片段DNA打断成小片段DNA；
常规文库构建步骤，包括采用stLFR技术进行文库构建，获得常规文库；
双链保护步骤，包括取用常规文库构建步骤制备的常规文库，磁珠回收，将DNA变性为单链，然后利用退火或延伸的方法将分子标签序列或测序接头序列转化为双链；其中，对常规文库的磁珠回收，本申请的一种实现方式中，在进行stLFR技术建库时，是采用带有分子标签序列的磁珠对DNA片段进行捕获，然后直接将磁珠和捕获DNA片段一起进行stLFR建库，获得常规文库；由于建库过程中涉及到PCR扩增等步骤，因此，常规文库中有两部分核酸，一部分是始终与磁珠结合的DNA，另一部分则是通过PCR扩增产生的游离DNA；用于甲基化测序的则是其中与磁珠结合的DNA，因此，需要取常规文库进行磁珠回收；而游离DNA则根据需求进行后续的常规测序，获得正常基因测序结果；
重亚硫酸盐转化步骤，包括向双链保护步骤的产物中加入双链保护剂，并加入重亚硫酸盐，进行脱氨基反应；
甲基化测序文库构建步骤，包括对脱氨基反应的产物进行常规PCR扩增或环化后滚环扩增，得到甲基化测序文库；
甲基化测序步骤，包括对甲基化测序文库进行测序，并根据测序结果分析获得基因的甲基化信息。其中，根据测序结果分析甲基化信息，具体的是指，将甲基化文库的测序结果与正常基因测序结果进行比对，获得基因的甲基化信息；对于有参考基因组的物种，用于比对的正常基因测序结果就是参考基因组；对于没有参考基因组的物种，用于比对的正常基因测序结果则是前述游离DNA进行常规测序获得的测序结果。
需要说明的是，本申请的DNA甲基化检测方法，利用stLFR技术进行常规文库构建，通过该技术采用的co-barcoding，获得长片段的甲基化信息，解决了现有的甲基化测序读长偏短的问题；与此同时，采用双链保护的方法对分子标签序列或测序接头序列进行保护，而不影响待测片段的重亚硫酸盐处理，简单而有效的解决了现有的甲基化测序无法事先加入分子标签序列进行建库的问题，不仅可以有效追踪分子来源，也可以方便的对多倍体生物的同源染色体甲基化信息进行提取。
优选的，本申请的DNA甲基化检测方法还包括常规测序步骤，常规测序步骤包括，取用常规文库构建步骤采用stLFR技术构建的常规文库，将DNA变性为单链，然后再进行常规PCR扩增或环化后滚环扩增，获得测序文库，对测序文库进行全基因组测序，获得正常基因测序结果；因此，甲基化测序步骤还包括，将甲基化测序文库的测序结果与常规文库获得的正常基因测序结果比对，从而获得基因的甲基化信息。
需要说明的是，对于有参考基因组的物种，在甲基化测序步骤后，可以直接将甲基化测序文库的测序结果与参考基因进行比对分析，从而获得基因的甲基化信息。而对于没有参考基因组的物种，本申请的优选方案基于共建库的原理，将常规文库构建步骤获得的常规文库分为两部分，一部分进行全基因组测序，获得正常基因测序结果；另一部分经过处理后进行甲基化测序；将甲基化测序的测序结果与全基因组测序的正常基因测序结果进行比对分析，获得基因的甲基化信息。本申请的共建库的方案，可以在无参考文库或无参考基因组的情况下，在分子级别对处理前后的文库进行一对一比对，从而得到更加精确的甲基化信息。
优选的，长片段DNA处理步骤还包括，采用带有分子标签序列的载体对小片段DNA进行捕获，然后再将捕获的DNA用于常规文库构建步骤进行stLFR技术文库构建。
优选的，载体为磁珠。
可以理解，磁珠载体是本领域比较常规使用的载体，不排除还可以使用其它载体。
优选的，长片段DNA处理步骤中，将长片段DNA打断成小片段DNA具体采用Tn5转座酶。
可以理解，Tn5转座酶打断获得小片段DNA是本领域常规使用的技术，不排除还可以采用其它物理或化学手段打断DNA。
优选的，双链保护步骤中，延伸的方法具体包括，利用分子标签序列或测序接头序列自身的3’端序列作为引物或加入外源短片段作为引物，在DNA聚合酶作用下延伸，将分子标签序列或测序接头序列变为双链结构。
优选的，重亚硫酸盐转化步骤中，双链保护剂为盐离子、聚乙二醇、酶和有机溶剂中的至少一种，重亚硫酸盐为重亚硫酸氢钠。其中，盐离子例如NaCl，聚乙二醇例如聚乙二醇8000。
需要说明的是，在重亚硫酸盐进行脱氨基处理的过程，需要保持一定温度，防止单链DNA形成二级结构从而影响反应效率；但是，本申请采用了将分子标签序列或测序接头序列转化为双链的双链保护策略；因此，在重亚硫酸盐的反应温度下，需要保障转化的双链不被破坏，因此，本申请优选的方案中加入了双链保护剂，其作用就是使得已经双链化的分子标签序列或测序接头序列能够在高温下始终保持双链状态，以避免其参与重亚硫酸盐反应，从而起到有效保护的作用。可以理解，凡是能够起到以上作用的双链保护剂都可以用于本申请。
本申请的另一面公开了一种DNA甲基化检测的试剂盒，该试剂盒中包括第一组试剂、第二组试剂、第三组试剂、第四组试剂和第五组试剂；第一组试剂包括用于将长片段DNA打断成小片段DNA的试剂；第二组试剂包括用于stLFR技术建库的试剂，该试剂用于制备常规文库；第三组试剂包括用于通过退火或延伸的方法将分子标签序列或测序接头序列转化为双链的试剂，该试剂用于形成双链结构；第四组试剂包括双链保护剂和重亚硫酸盐，用于进行双链保护和脱氨基反应；第五组试剂包括用于对脱氨基反应的产物进行常规PCR扩增或环化后滚环扩增的试剂，该试剂用于制备甲基化测序文库。
需要说明的是，本申请的试剂盒实际上就是根据本申请的DNA甲基化检测方法，将其中核心的试剂有机的组合在一起，一方面，可以方便本申请的DNA甲基化检测方法的进行；另一方面，可以提高各试剂的使用效率，减少浪费，从而降低本申请的DNA甲基化检测的成本。
可以理解，本申请试剂盒中的各试剂都可以单独通过市售购买，但是，本申请的DNA甲基化检测方法涉及的试剂较多，单独购买一方面是耗费时间、繁琐、使用不便；另一方面，由于各试剂都不是按照本申请检测方法用量配比进行的剂量设计，所以，必然会存在大量的试剂浪费，增加检测成本。因此，本申请以本申请DNA甲基化检测方法为基础，将其使用的主要核心试剂组合为本申请的试剂盒。
优选的，本申请的试剂盒还包括第六组试剂，该第六组试剂包括用于对常规文库进行常规PCR扩增或环化后滚环扩增的试剂，该试剂用于制备常规测序文库，以 便于进行全基因测序，获得正常基因测序结果。可以理解，第六组试剂中，还可以根据需求增加常规测序所需的试剂。
需要说明的是，本申请的试剂盒是根据本申请的DNA甲基化检测方法而研发的，因此，第一组试剂、第二组试剂、第三组试剂、第四组试剂和第五组试剂，实际上就是依序用于本申请DNA甲基化检测方法中的长片段DNA处理步骤、常规文库构建步骤、双链保护步骤、重亚硫酸盐转化步骤和甲基化测序文库构建步骤；第六组试剂用于常规测序步骤的常规文库构建。可以理解，由于常规测序步骤并非必须的步骤，例如已经有参考基因组的情况下，则不需要进行常规测序；所以本申请的试剂盒中也可以选择性的增加或不增加第六组试剂，又或者将第六组试剂组合到本申请的试剂盒中，可以根据需求选择性的使用。
还需要说明的是，测序试剂盒是单独的试剂盒产品，因此没有包含在本申请的试剂盒中，例如常规测序有独立的常规测序试剂盒，甲基化测序步骤有甲基化测序试剂盒。
优选的，第一组试剂还包括带有分子标签序列的载体，用于对DNA片段进行捕获。
优选的，载体为磁珠，第一组试剂中还包括磁珠纯化的试剂。其中，磁珠纯化的试剂，例如磁珠缓冲液、清洗液、洗脱液等。
优选的，第一组试剂中，用于将长片段DNA打断成小片段DNA的试剂为Tn5转座酶及其缓冲液。
优选的，第三组试剂中，用于通过退火或延伸的方法将分子标签序列或测序接头序列转化为双链的试剂，具体包括外源短核苷酸片段、DNA聚合酶及其缓冲液，以及用于DNA延伸的dNTPs。
其中，外源短核苷酸片段是作为引物使用的，可以根据需求选择使用，也可以不添加到第三组试剂盒中；例如，本申请的一种实现方式中，可以直接利用分子标签序列或测序接头序列自身的3’端序列作为引物进行延伸，因此，外源短核苷酸片段可以只是作为备选方案放到第三组试剂中。
优选的，第四组试剂中，双链保护剂为盐离子、聚乙二醇、酶和有机溶剂中的至少一种，重亚硫酸盐为重亚硫酸氢钠。
可以理解，本申请的DNA甲基化检测方法，其全部或部分功能可以通过硬件的方式实现，也可以通过计算机程序的方式实现。当通过计算机程序的方式实现时，该程序可以存储于一计算机可读存储介质中，存储介质可以包括：只读存储器、随机存储器、磁盘、光盘、硬盘等，通过计算机执行该程序以实现本申请的方法。例如，将程序存储在设备的存储器中，当通过处理器执行存储器中程序，即可实现本申请的方法。当本申请的方法中全部或部分功能通过计算机程序的方式实现时，该程序也可以存储在服务器、另一计算机、磁盘、光盘、闪存盘或移动硬盘等存储介 质中，通过下载或复制保存到本地设备的存储器中，或对本地设备的系统进行版本更新，当通过处理器执行存储器中的程序时，即可实现本申请DNA甲基化检测方法的全部或部分功能。
因此，本申请的另一面公开了一种DNA甲基化检测的装置，包括长片段DNA处理模块、常规文库构建模块、常规测序模块、双链保护模块、重亚硫酸盐转化模块、甲基化测序文库构建模块和甲基化测序模块；
长片段DNA处理模块，包括用于将长片段DNA打断成小片段DNA；该模块可以采用常规的物理、化学或酶的方式对长片段DNA进行打断；对于物理方式，例如可借鉴超声波破碎；对应化学或酶的方式，该模块一方面可以自动取样或配制反应液，另一方面可以为反应提供温度等反应条件；
常规文库构建模块，包括用于采用stLFR技术进行文库构建；stLFR技术，即single-tube Long Fragment Read，也就是长片段DNA信息获取技术，可参考专利文件WO2019217452A1；另外，stLFR技术进行文库构建可以采用华大智造发布的MGIEasy stLFR文库制备试剂盒进行，具体参考试剂盒使用说明；
常规测序模块，包括用于取用常规文库构建模块中采用stLFR技术构建的常规文库，将DNA变性为单链，然后再进行常规PCR扩增或环化后滚环扩增，获得测序文库，对测序文库进行全基因组测序，获得正常基因测序结果；该模块仅仅在没有参考基因的情况下使用，当然，如果为了获得更精准的处理前后的结果，也可以采用该模块；该模块可以参考现有的自动建库和测序设备或装置；
双链保护模块，包括用于取用常规文库构建模块中采用stLFR技术构建的常规文库，磁珠回收，将DNA变性为单链，然后利用退火或延伸的方法将分子标签序列或测序接头序列转化为双链；该模块一方面可以参考自动取样或配制反应液，另一方面可以为退火或延伸提供温度等反应条件控制；
重亚硫酸盐转化模块，包括用于向经过双链保护模块处理的产物中加入双链保护剂，并加入重亚硫酸盐，进行脱氨基反应；该模块同样可以参考现有的自动取样和反应液配制装置或设备，并为脱氨基反应提供相应的反应条件；
甲基化测序文库构建模块，包括用于对脱氨基反应的产物进行常规PCR扩增或环化后滚环扩增，得到甲基化测序文库；该模块可以参考现有的测序模块；
甲基化测序模块，包括用于对甲基化测序文库进行测序，将甲基化测序结果与正常基因测序结果比对，获得基因的甲基化信息；该模块可以参考现有的甲基化测序和分析模块或装置。其中，正常基因测序结果可以是常规测序模块获得的测序结果，也可以是已经存在的参考基因组。
本申请的DNA甲基化检测装置，根据本申请DNA甲基化检测方法的需求，将检测方法中的各步骤通过自动化的模块实现DNA甲基化的自动检测，即本申请的DNA甲基化检测装置，为DNA甲基化检测提供了个性化的检测系统，能够方便有效的进 行DNA甲基化检测。并且，本申请的DNA甲基化检测装置同样具有本申请的DNA甲基化检测方法的各项优点，例如解决了现有的甲基化测序读长偏短的问题，解决了现有的甲基化测序无法事先加入分子标签序列进行建库、不能追踪分子来源的问题。
优选的，本申请的DNA甲基化检测装置中，其长片段DNA处理模块，还包括用于采用带有分子标签序列的载体对小片段DNA进行捕获，然后再将捕获的DNA用于常规文库构建模块采用stLFR技术进行文库构建；
优选的，载体为磁珠。
优选的，本申请的DNA甲基化检测装置，其长片段DNA处理模块中，将长片段DNA打断成小片段DNA具体采用Tn5转座酶。
优选的，本申请的DNA甲基化检测装置，其双链保护模块中，延伸的方法具体包括，利用分子标签序列或测序接头序列自身的3’端序列作为引物或加入外源短片段作为引物，在DNA聚合酶作用下延伸，将分子标签序列或测序接头序列变为双链结构。
优选的，本申请的DNA甲基化检测装置，其重亚硫酸盐转化模块中，双链保护剂为盐离子、聚乙二醇、酶和有机溶剂中的至少一种，重亚硫酸盐为重亚硫酸氢钠。
本申请的再一面还公开了一种DNA甲基化检测装置，该装置包括存储器和处理器；存储器用于存储程序；处理器用于通过执行存储器存储的程序以实现本申请的DNA甲基化检测方法。
本申请的再一面还公开了一种计算机可读存储介质，包括存储于其中的程序，该程序能够被处理器执行以实现本申请的DNA甲基化检测的方法。
本申请的再一面还公开了本申请的DNA甲基化检测方法、本申请的试剂盒或本申请的DNA甲基化检测装置在基因印记检测、胚胎发育检测或癌症预防检测中的应用。
可以理解，本申请的DNA甲基化检测方法、试剂盒和装置，能够获得长片段的甲基化信息，不仅可以有效追踪分子来源，也可以方便的对多倍体生物的同源染色体甲基化信息进行提取；因此，能够方便的应用于基因印记检测、胚胎发育检测、癌症预防检测等领域。
本申请的有益效果在于：
本申请的DNA甲基化检测方法，利用stLFR技术的优点，能够获得长片段的甲基化信息；采用双链保护的方法对分子标签序列或测序接头序列进行保护，可以事先加入分子标签序列进行建库，从而可以有效追踪分子来源，并方便的对多倍体生物的同源染色体甲基化信息进行提取。
附图说明
图1是本申请实施例中DNA甲基化检测方法的流程示意图；
图2是本申请实施例中DNA甲基化检测装置的构造示意图；
图3是本申请实施例中基于磁珠捕获的双链保护分子标签的示意图；
图4是本申请实施例中不同形式的双链保护的示意图；
图5是本申请实施例中不同形式的双链保护的示意图；
图6是本申请实施例中不同形式的双链保护的示意图；
图7是本申请实施例中构建的stLFR常规文库(WGS)和甲基化测序文库(WGBS)的琼脂糖凝聚电泳图。
具体实施方式
现有的甲基化测序读长偏短，并且，一般常规的甲基化测序不能事先加入分子标签序列进行建库，无法追踪分子来源，难以实现多倍体生物的同源染色体甲基化信息提取。虽然有研究提出可以采用人工合成的甲基化的测序接头，解决事先加入分子标签的问题，但是，人工合成甲基化测序接头成本高昂，难以用于大规模的高通量测序。
基于以上问题，本申请创造性的借鉴stLFR技术进行文库构建，解决读长偏短的问题；然后，创造性的提出双链保护策略，对事先添加的分子标签序列或测序接头序列进行保护，避免其被重亚硫酸盐处理破坏，以解决不能事先加入分子标签序列进行建库的问题；利用同一文库两次建库的策略，解决了无参考基因组条件下的甲基化文库比对问题。根据以上构思，本申请研发了一种DNA甲基化检测的方法，如图1所示，包括长片段DNA处理步骤11、常规文库构建步骤12、双链保护步骤14、重亚硫酸盐转化步骤15、甲基化测序文库构建步骤16、甲基化测序步骤17；在优选的方案中，基于共建库的原理，本申请的方法还包括常规测序步骤13。
其中，长片段DNA处理步骤11，包括将长片段DNA打断成小片段DNA；包括采用物理、化学或酶的方式将长片段DNA打断，例如本申请的一种实现方式中，具体采用Tn5转座酶将DNA打断成小片段。
常规文库构建步骤12，包括采用stLFR技术进行文库构建，获得常规文库。该步骤可以参考专利文献WO2019217452A1或者采用华大智造发布的MGIEasy stLFR文库制备试剂盒进行常规建库。在本申请的一种实现方式中，在此之前还包括采用带有分子标签序列的载体对打断的DNA片段进行捕获，然后再进行stLFR技术建库。其中，带有分子标签序列的载体即带有多拷贝特定标签序列或分子标签的载体，是指在一个载体上，带有相同序列的多个拷贝的寡聚核苷酸序列。
本申请的一种实现方式中，具体采用磁珠作为载体，为了保障磁珠和DNA的稳固结合，本申请通过双链霉亲和素蛋白嵌合体连接，使用化学修饰的DNA与磁珠上的化学基团反应，这些化学修饰包括但不限于I-linker、Amino-modified oligo、 Thiol-modified oligo、Acrydite-modified oligo等。可以理解，磁珠在后续的反应中需要进行两轮PCR扩增，因此要求DNA与磁珠的偶联必须能够耐酸碱、耐高温且不易降解。
常规测序步骤13，包括取用常规文库构建步骤采用stLFR技术构建的常规文库，将DNA变性为单链，然后再进行常规PCR扩增或环化后滚环扩增，获得测序文库，对测序文库进行测序，获得正常基因测序结果。该步骤即正常的DNA建库和测序步骤，其目的是为了便于没有已知参考基因的物种，可以通过甲基化处理前后对比，准确的分析甲基化位点。可以理解，如果待测物种已经有比较准确的参考基因序列，则可以不用该步骤。
双链保护步骤14，包括取用常规文库构建模块中采用stLFR技术构建的常规文库，磁珠回收，将DNA变性为单链，然后利用退火或延伸的方法将分子标签序列或测序接头序列转化为双链。
双链保护可以采用退火或延伸的方式实现，其中，延伸的方式实现，例如，利用分子标签序列或测序接头序列自身的3’端序列作为引物，如图3的A图所示，或者，加入外源短片段作为引物，如图3的B图所示，在DNA聚合酶作用下延伸，将分子标签序列或测序接头序列变为双链结构。
另外，双链保护根据不同的载体或者DNA的不同形态，还可以有多种保护形式，如图4至图6所示。图4左边一列为与磁珠结合的DNA的双链保护，右边一列为游离DNA的双链保护；“●”表示磁珠，深色双实线表示互补DNA双链，浅色双实线表示非互补DNA双链；a为单端保护、b为双端保护、c为桥式保护、d为3’和/或5’端颈环结构保护、e为3’和/或5’端颈环桥式结构保护、f为分子内颈环结构保护。图5为环状DNA的双链保护机制，同样的，深色双实线表示互补DNA双链；a为引入单链引物的双链保护、b为通过Y接头引物进行的双链保护、c为单链自成环形成双链保护。图6为以固体表面为载体的DNA的双链保护机制，深色双实线表示互补DNA双链，浅色双实线表示非互补DNA双链；其中，a和b是不同位置的单端保护、c为双端保护、d为颈环式单端保护、e为桥式结构保护、f为3’或5’端颈环桥式结构保护、g为分子间桥式结构保护。具体采用怎样的保护机制可以根据DNA序列的设计而定，或者根据不同的载体设计不同的序列，以采取不同的双链保护机制。
重亚硫酸盐转化步骤15，包括向双链保护步骤的产物中加入双链保护剂，并加入重亚硫酸盐，进行脱氨基反应。
在DNA进行重亚硫酸盐，例如重亚硫酸氢钠，处理过程中，需要保持一定温度，防止单链DNA形成二级结构从而影响反应效率，而在本申请中，文库同时存在双链和单链结构，因此需要加入一种DNA的双链保护试剂，使得已经双链化的序列能够在高温下始终保持双链状态，以起到有效的保护作用。本申请可以采用的双链保护 试剂包括盐离子、聚乙二醇、酶和有机溶剂等能够有效的防止高温下双链DNA变性，同时能够保持单链的转化效率。
甲基化测序文库构建步骤16，包括对脱氨基反应的产物进行常规PCR扩增或环化后滚环扩增，得到甲基化测序文库。甲基化测序步骤17，包括对甲基化测序文库进行测序，然后进行数据分析，并根据测序结果分析获得基因的甲基化信息。
本申请的DNA甲基化检测方法，相比现有技术，有以下优点：
1)通过stLFR自身具有的co-barcoding技术，获得长片段的甲基化信息，解决了甲基化测序中读长偏短的问题；
2)可以进行长片段DNA序列检测，利于新物种的从头测序，基因组组装；
3)可以在不需要参考序列的前提下，对某一特定DNA片段进行快速准确的比对；
4)可以对多倍型基因组不同染色体的甲基化水平进行测定；
5)适用于对基于oxBS-seq原理的5mC的测序；
6)适用于去甲基酶Tet和AID/APOBEC参与的5hmC的测序；
7)适用于TAPS-seq原理的甲基化测序(5mC)、羟甲基化测序(5hmC)、羧基胞嘧啶(5caC)的测序。
本申请的DNA甲基化检测方法同样适用于甲基化测序(5mC)和羟甲基化测序(5hmC)，详细说明如下：
1.采用自BGI自产的甲基化测序试剂盒，对DNA样品进行常规的Bisulfite处理，按照专利WO2019217452A1所描述建库方法建库上机，可以获得5mC测序结果。Bisulfite试剂的主要成分是重重亚硫酸氢钠，其可以将非甲基化的胞嘧啶转化为U，而5mC和5hmC则不会被转化，从而获得DNA的甲基化信息。Bisulfite测序无法区分甲基化的类型是5mC还是5hmC，得到的实际上是一个混合的信号。
2.利用oxBS-seq的原理，先将文库进行氧化处理，将5hmC转化为羧基胞嘧啶(5caC)，再进行Bisulfite处理，未甲基化的C和5caC都会转化为U，而5mC则不会被转化，从而得到完整的5mC测序信息。
3.利用TAB-seq或ACE-seq原理，先对5hmC进行糖基化保护，再用Tet酶或A3A酶对DNA进行去甲基化处理，将其他没有糖基化保护的5mC转化为非甲基化的C，再进行Bisulfite处理，从而获得完整的5hmC的测序信息。
4.利用TAPS-seq的原理，先将文库进行氧化处理，将5mC和5hmC转化为羧基胞嘧啶(5caC)，再进行硼烷处理，将其转化为U，从而获得5mC和5hmC的测序信息。
基于本申请的DNA甲基化检测方法，本申请研发了一种DNA甲基化检测装置，如图2所示，包括长片段DNA处理模块21、常规文库构建模块22、常规测序模块23、双链保护模块24、重亚硫酸盐转化模块25、甲基化测序文库构建模块26和甲 基化测序模块27；长片段DNA处理模块21，包括用于将长片段DNA打断成小片段DNA；常规文库构建模块22，包括用于采用stLFR技术进行文库构建；常规测序模块23，包括用于取用采用stLFR技术构建的常规文库，将DNA变性为单链，然后再进行常规PCR扩增或环化后滚环扩增，获得测序文库，对测序文库进行测序，获得正常基因测序结果；双链保护模块24，包括用于取用部分采用stLFR技术构建的常规文库，磁珠回收，将DNA变性为单链，然后利用退火或延伸的方法将分子标签序列或测序接头序列转化为双链；重亚硫酸盐转化模块25，包括用于向经过双链保护模块处理的产物中加入双链保护剂，并加入重亚硫酸盐，进行脱氨基反应；甲基化测序文库构建模块26，包括用于对脱氨基反应的产物进行常规PCR扩增或环化后滚环扩增，得到甲基化测序文库；甲基化测序模块27，包括用于对甲基化测序文库进行测序，并进行数据分析，将甲基化测序结果与正常基因测序结果比对，获得基因的甲基化信息。
本申请的DNA甲基化检测方法和装置，不但具有stLFR的优点，同时还能够给出精确的甲基化信息：
1)在现有的甲基化测序中，因实验原理限制，通常只能得到较短的读长；而本申请通过co-barcoding技术，可以将短读长组装成长片段，给出更加完整的甲基化图谱；
2)基于共建库的原理，对于WGS和WGBS文库进行比对，可以在无参考文库的情况下，在分子级别对处理前后的文库进行一对一比对，得到更加精确的甲基化信息；
3)通过双链保护机制，有效的避免分子标签信息变异，节约时间及合成成本。
下面通过具体实施例对本申请作进一步详细说明。以下实施例仅对本申请进行进一步说明，不应理解为对本申请的限制。
实施例
本例以人基因组DNA测序为例，进行DNA甲基化检测，其过程包括：首先提取长片段基因组DNA，再用Tn5转座酶将DNA打断成小片段，用带有分子标签序列的磁珠进行捕获。采用stLFR技术进行文库构建，获得常规文库。得到常规建库的文库后，进行常规的PCR扩增，吸取PCR产物上清得到文库L，进行常规的上机测序操作得到WGS数据。同时回收常规文库的磁珠用于甲基化测序建库；将回收的磁珠上的DNA分子充分变性为单链，然后利用磁珠上分子中自身的3’端序列作为引物或加入一个外源的短片段作为引物，如图3所示，在DNA聚合酶作用下，将其中的DNA分子标签序列变为双链结构，加入双链保护剂并进行重亚硫酸氢钠转化反应，反应完成后进行常规的PCR扩增得到文库M，对文库M进行上机测序操作得到WGBS数据；最后将WGS和WGBS数据进行一对一比对，确定甲基化位点。本例的DNA甲基化检测详细如下：
一、制备带有多拷贝的分子标签的载体
(1)将接头序列(linker)通过生物素-链霉亲和素作用连接到修饰有链霉亲和素蛋白的磁珠上。Linker为双链DNA分子，由两条单链DNA链退火在一起，两条单链DNA的退火条件为70℃退火1分钟，然后以0.1℃/s的速度缓慢降温至20℃，20℃反应30分钟。
Linker的双链DNA分子中正义链为Linker-F，反义链为Linker-R；其中，Linker-F为SEQ ID NO.1所示序列，Linker-F的3’端具有Bio修饰，Linker-R为SEQ ID NO.2所示序列。
SEQ ID NO.1：
5’-CTTCCGGCAGAACGACATGGCTACGAAAAAAAAAA-3’Bio
SEQ ID NO.2：5’-CGTAGCCATGTCGTTCTGCC-3’。
本例采用的带有链霉亲和素的磁珠为Dynabeads M-280 streptation(112.06D，Invitrogen)。按照Linker(50μM)2μL与M280磁珠30μL的比例混合；磁珠的保存液替换成1倍浓度的磁珠结合缓冲液，25℃，在垂直混匀仪上混合1个小时，然后用低盐磁珠洗涤缓冲液洗涤2次，最后用12.5μL的1倍浓度的连接缓冲液重悬磁珠。
其中，磁珠结合缓冲液由50mM的Tirs-HCl、150mM的NaCl和0.1mM EDTA组成；低盐磁珠洗涤缓冲液由50mM的Tirs-HCl、150mM的NaCl和0.02％的Tween-20组成；正常配制的连接缓冲液为10倍浓度，10倍浓度的连接缓冲液，其由聚乙二醇8000 30％、Tris-HCl 150mM、ATP 1mM、BSA 0.15mg/mL、MgCl 2 30mM、DTT 1.5mM组成。
(2)将不同编号，即1至1536号，的序列1和辅助序列1分装在384孔板的不同孔中进行退火，1:1退火，一共4μL/孔，共计4个384孔板。
标签序列1与辅助序列1的退火条件是：100μM的序列1与辅助序列1按体积比1:1混合后置于PCR仪器上，70℃1分钟，然后以0.1℃/s的速度缓慢降温至20℃，20℃反应30分钟。
其中，标签序列1为SEQ ID NO.3所示序列，辅助序列1为SEQ ID NO.4所示序列。标签序列1的5’端含有分子标签序列“Barcode”，辅助序列1的第22碱基和第23碱基之间插入分子标签序列“Barcode”。
SEQ ID NO.3：5’-[Barcode]ACCCTGACTAGGTCGC-3’
SEQ ID NO.4：
5’-GGAAGGGCGACCTAGTCAGGGT[Barcode]CGCAGA-3’。
(3)使用DNA连接酶将载体上的接头序列与序列1进行连接。该序列包含一段特定的DNA序列，标记为分子标签1。具体步骤为将步骤(1)中带有Linker的M280磁珠平均分配到4块384孔板的1536个孔中，每孔2.5μL。再在每孔补上3.5 μL连接混合液，连接混合液含有1μL连接酶(T4 DNA ligase 600U/μL，Enzymatics)，1.5μL超纯水和1μL连接缓冲液(10×T4 DNA ligation buffer，Enzymatics)，以每个384孔板10μL的总反应体积进行连接反应，反应温度25℃，反应时间1个小时。使用100μL高盐磁珠洗涤缓冲液洗涤一次，再使用100μL低盐磁珠洗涤缓冲液洗涤一次，用于去除反应中的连接酶与未能完全反应的寡聚核苷酸。
其中，高盐磁珠洗涤缓冲液由50mM Tirs-HCl、500mM NaCl、0.02％Tween-20组成；低盐磁珠洗涤缓冲液由50mM Tirs-HCl、150mM NaCl、0.02％Tween-20组成。
(4)将步骤(3)清洗后的磁珠通过磁力架收集，然后用1倍浓度的连接缓冲液重悬磁珠，重悬后Linker的浓度为1.6μM，并使用震荡混匀仪混匀。
(5)将不同编号，即1至1536号，的标签序列2和辅助序列2分装在全新的384孔板的不同孔中退火，共计4个384孔板。
标签序列2与辅助序列2的退火条件是：标签序列2100μM与辅助序列2100uM各2μL混合于384孔板中，然后置于PCR仪器上，70℃ 1分钟，然后以0.1℃/s的速度缓慢降温至20℃，20℃反应30分钟。
其中，标签序列2为SEQ ID NO.5所示序列，辅助序列2为SEQ ID NO.6所示序列。标签序列2的第47碱基和第48碱基之间插入分子标签序列“Barcode”，辅助序列2的3’端含有分子标签序列“Barcode”。
SEQ ID NO.5：
5’-GTACTGAGGGCTGGCGACCTTGAGTGCTTCGACTGGCCGTCGTTTTA[Barcode]TCTGCG-3’
SEQ ID NO.6：5’-TAAAACGACG[Barcode]-3’。
(6)将步骤(4)混匀后的磁珠按每孔2.5μL的量分配到步骤(5)的384孔板的各个孔中。然后加入3.5μL连接混合液，连接混合液含有1μL连接酶(T4 DNA ligase 600U/μL，Enzymatics)，1.5μL超纯水和1μL连接缓冲液(10×T4 DNA ligation buffer，Enzymatics)，在25℃反应1个小时。
(7)使用100μL高盐磁珠洗涤缓冲液洗涤一次，再使用100μL低盐磁珠洗涤缓冲液洗涤一次，用于去除反应中的连接酶与未能完全反应的寡聚核苷酸；最后，用磁力架收集磁珠，再将磁珠重悬于低盐磁珠洗涤缓冲液中，4℃可保存1年。
至此，完成2359296种分子标签磁珠载体的制备，磁珠的浓度为19000000个/μL。
二、stLFR技术建库
(1)准备带有标签的磁珠，取1千万个磁珠，即5.3μL磁珠，用磁力架去除磁珠中的低盐磁珠洗涤液，用50μL低盐磁珠洗涤液洗涤一次，然后用强碱变性缓冲液50μL重悬磁珠，在常温下孵育5分钟，用磁力架吸附磁珠2分钟，去除强碱 变性缓冲液，再加入强碱变性缓冲液50μL洗涤磁珠一次，磁力架吸附磁珠2分钟，去除强碱变性缓冲液，用低盐磁珠洗涤缓冲液50μL洗涤一次，用杂交缓冲液50μL洗涤一次，最后用杂交缓冲液50μL重悬磁珠。
其中，强碱变性缓冲液由1.6M的KOH和1mM的EDTA组成；杂交缓冲液由50mM Tris-HCl、1000mM NaCl、0.05％Tween-20组成。
(2)取20μL长片段DNA(2ng/μL)，缓慢加入27μL超纯水、12μL打断缓冲液(5×TI Buffer，MGIEasy stLFR)和1μL转座复合体(2pmol/μL,MGIEasy stLFR)，共60μL反应液，55℃孵育10分钟后置于冰上。取15μL反应液，加入35μL超纯水，稀释至50μL，标记为打断DNA溶液。
将50μL的打断DNA溶液与步骤(1)中带标签(Barcode)的磁珠50μL混合，60℃反应1分钟，然后让反应液置于室温，让其自然降温并置于垂直混匀仪，25℃混合反应1个小时，标记为杂交捕获体系。
(3)使用转座复合体1时，在1个小时的杂交时间结束后，加入100μM的封闭序列1(Blocker1)2μL到100μL杂交捕获体系中，置于垂直混匀仪上25℃反应0.5个小时。然后用低盐磁珠洗涤液洗涤磁珠两次，加入聚合酶和连接酶的混合反应液重悬磁珠，置于20℃反应0.5小时。
其中，混合反应液中含有：DNA聚合酶T4 DNA Polymerase(3U/μL，Enzymatics)6μL、连接酶T7 DNA ligase(3000U/μL，NEB)1μL、2×T7 DNA ligase buffer(NEB)25μL、25mM dNTP 0.5μL、10mM ATP 5μL，补足体积的水至50μL。
2×T7DNA ligase buffer中含有：132mM Tris-HCl、20mM MgCl 2、2mM ATP、2mM DTT、15％Polyethyleneglycol(聚乙二醇6000)。
封闭序列1为SEQ ID NO.7所示序列。
SEQ ID NO.7：
5’-CCTAGCATGGACTATCGATCCTTGGTGATCATGTCGTCAGTGC-3’Bio
反应结束后加入5μL的0.44％SDS室温下孵育10分钟使转座酶变性从DNA上释放下来，然后用高盐磁珠洗涤液和低盐磁珠洗涤液分别洗涤一次。加入聚合酶试剂混合液重悬磁珠，置于72℃反应10分钟。
其中，聚合酶试剂混合液中含有：聚合酶Standard Taq polymerase(5U/μL，NEB)1μL、10×thermopol buffer(NEB)5μL、25mM dNTP(Enzymatics)0.8μL，总体积50μL。反应结束后用磁力架吸附磁珠，回收上清液。
10×thermopol buffer中含有：200mM Tris-HCl、100mM(NH 4) 2SO 4、100mM KCl、20mM MgSO 4、1％
Figure PCTCN2019127537-appb-000001
X-100。
(4)使用转座复合体2时，在1个小时的杂交时间结束后，加入100μM的封闭序列2(Blocker2)2μL到100μL的杂交捕获体系中，置于垂直混匀仪上25℃反应0.5个小时。加入0.44％SDS 10μL室温下孵育10分钟使转座酶变性从DNA 分子上释放下来。用100μL高盐磁珠洗涤液和低盐磁珠洗涤液各洗涤一次，用18μL的TE缓冲液重悬磁珠，再加入USER酶(1U/μL，NEB)2μL，置于垂直混匀仪上37℃反应0.5个小时，在37℃环境下，用37℃预热的高盐磁珠洗涤液和低盐磁珠洗涤缓冲液各洗涤一次。用连接酶试剂混合液重悬磁珠，置于垂直混匀仪上25℃反应1个小时。连接酶试剂混合液中包含连接酶，其组成为：T4DNA ligase(600U/μL，Enzymatics)5μL、3倍浓度的连接缓冲液10μL和16.7μM接头21.5μL，总体积为30μL。
其中，3倍浓度的连接缓冲液中含有：30％聚乙二醇8000、150mM Tris-HCl、1mM ATP、0.15mg/mL BSA、30mM MgCl 2、1.5mM DTT。16.7μM接头，即接头2，由正义链接头2-F和反义链接头2-R退火形成。接头2-F为SEQ ID NO.9所示序列，接头2-F的5’端具有磷酸化修饰，3’端为双脱氧碱基；接头2-R为SEQ ID NO.10所示序列，接头2-R的3’端为双脱氧碱基。
SEQ ID NO.9：5’-phos-TCTGCTGAGTCGAGAACGTCTddC-3’
SEQ ID NO.10：5’-CTCGACTCAGCAGddA-3’
反应结束后高盐磁珠洗涤液和低盐磁珠洗涤缓冲液各洗涤一次。用2×T7 ligase buffer(NEB)10μL、1μL浓度100μM的引物2、1μL浓度100μM的引物3和8μL水配制成20μL退火混合液，用退火混合液重悬磁珠，70℃1分钟，以0.1℃每秒的速度缓慢降温至20℃，20℃反应30分钟。然后，直接加入聚合酶和连接酶混合液试剂20℃反应0.5个小时。
引物2为SEQ ID NO.12所示序列，引物2的5’端具有磷酸化修饰；引物3为SEQ ID NO.8所示序列，封闭序列1的3’端具有Bio修饰。
SEQ ID NO.8：
5’-CGTAGCCATGTCGTTCTGCCGGAAGGGCGACCTAGTCAGGGT-3’
SEQ ID NO.12：5’-phos-GAGACGTTCTCGACTCAGCAGA-3’。
其中，聚合酶和连接酶混合试剂包含：聚合酶(T4 DNA Polymerase 3U/μL，Enzymatics)6μL、连接酶(T7 DNA ligase 3000U/μL，NEB)1μL、2×T7 DNA ligase buffer(NEB)15μL、25mM dNTP 0.5μL、10mM ATP 5μL，补足体积的水至30μL。
反应结束后用100μL高盐磁珠洗涤液和低盐磁珠洗涤缓冲液各洗涤一次。然后加入有链置换活性的聚合酶试剂混合液重悬磁珠，置于65℃反应5分钟。聚合酶试剂混合液中含有：聚合酶(Bst2.0
Figure PCTCN2019127537-appb-000002
DNA Polymerase 8U/μL，NEB)1μL、10×Isothermal Ampl ification Buffer(NEB)5μL、25mM dNTP(Enzymatics)0.8μL、100mM MgSO 4 1.5μL，总体积50μL。反应结束后用磁力架吸附磁珠，回收上清液。
其中，10×Isothermal Amplification Buffer中含有：200mM Tris-HCl、100mM(NH 4) 2SO 4、500mM KCl、20mM MgSO 4、1％
Figure PCTCN2019127537-appb-000003
20。
(5)使用引物1和引物2进行DNA分子聚合酶链式扩增13个循环。
反应体系如下：
50μL步骤(4)中回收的上清液，75μL 2×KAPA HiFi Master Mix(Kapa Biosystems)，0.75μL引物1(100μmol/L)，0.75μL引物2(100μmol/L)，23.5μL超纯水。
反应条件如下：
I.98℃ 3分钟；II.95℃ 30秒，58℃ 30秒，72℃ 2分钟，重复步骤(II)
13次；III.72℃ 5分钟；IV.4℃保存。
其中，引物1为SEQ ID NO.11所示序列；引物2为SEQ ID NO.12所示序列，引物2的5’端具有磷酸化修饰。
SEQ ID NO.11：5’-CGTAGCCATGTCGTTCTG-3’。
三、常规测序
将“二、stLFR技术建库”得到的PCR产物置于磁力加上，小心吸取上清液，用AMPure XP beads(Agencourt AMPure XP-Medium,A63882,AGENCOURT)进行纯化，纯化方法参考官方提供的标准操作规程，纯化结束后回收产物为带有分子标签的适合测序的短片段分子。
由于本例采用BGIseq-500进行测序，因此，需要对磁珠纯化的产物进行成环反应，操作详情参照BGIseq-500标准DNA小片段建库流程的成环步骤。然后，对环化的产物进行BGIseq-500测序。
四、双链DNA保护
将stLFR文库磁珠转移至1.5mL离心管中，用500μL的1×Buffer D(0.1M NaOH)充分洗涤2次，将离心管置于磁力架上，弃掉上清液，回收磁珠。再用低盐磁珠洗涤缓冲液充分洗涤2次，弃掉上清液，回收磁珠。向回收的磁珠中加入47μL的超纯水、50μL的2×PfuCx Mix buffer(Agilent)，2μL的PfuCx聚合酶(Agilent)，以及1μL的ME引物。其中，ME引物是对应于文库中靠近barcode的3’末端的一段互补序列，ME引物为SEQ ID NO.13所示序列。
SEQ ID NO.13：5’-CTGTCTCTTATACACATCT-3’。
将混合物及磁珠充分混匀，离心并转移至250μL的PCR管中，在PCR仪器上运行如下程序：98℃ 3分钟，50℃ 1分钟，72℃ 10分钟，室温保持。
将反应物取出，至于冰上，并用高盐磁珠洗涤液洗涤1次，低盐磁珠洗涤缓冲液洗涤2次，移去上清，回收磁珠于1.5mL的离心管中。
五、重亚硫酸氢钠转化和甲基化测序文库
按照BGI甲基化测序试剂盒的实验步骤，完成重亚硫酸氢钠转化反应，详细如下：
1.加入20μL双链DNA保护buffer，加入130μL的Bisulfite Buffer(Zymo BioTech)，充分混匀，用锡箔纸覆盖管体，避光。
2.将离心管置于旋转混合仪上，在60℃，孵育1个小时。
3.将离心管置于磁力架上2分钟，弃去上清液，加入400μL的Wash buffer(Zymo BioTech)，前后置换离心管位置，充分洗涤磁珠后，弃去上清液。
4.加入200μL的Desulphonation buffer(Zymo BioTech)，在室温下孵育25-30分钟，不时轻微震荡离心管，防止磁珠沉降。
5.将磁珠置于磁力架上2分钟，弃去上清液，加入400μL的低盐磁珠洗涤缓冲液，前后置换离心管位置，充分洗涤磁珠后，弃去上清液，将磁珠重悬于TE buffer中。
6.取10M磁珠加入PCR Mix并补水至体积为50μL。
其中，PCR Mix包括：25μL的2×PfuCx Mix(Agilent)、0.25μL的浓度为100μM的PCR Forward primer、0.25μL的浓度为100μM的PCR Reverse primer、0.75μL的PfuCx polymerase(Agilent)。
PCR Forward primer为SEQ ID NO.14所示序列，PCR Reverse primer为SEQ ID NO.15所示序列。
SEQ ID NO.14：5’-TGTGAGCCAAGGAGTTG-3’
SEQ ID NO.15：5’-GAGACGTTCTCGACTCAGCAGA-3’。
将PCR管置于PCR仪器上运行如下程序：98℃ 3分钟，然后进入10个循环：98℃ 30秒、58℃ 30秒、72℃ 2分钟，循环结束后，72℃延伸10分钟，然后降温至4℃保存。
7.用0.8×的AMPure XP beads(Agencourt AMPure XP-Medium,A63882,AGENCOURT)对PCR产物进行纯化，并将PCR产物重悬至20μL超纯水中，即得到LFR甲基化测序文库。
六、甲基化测序
由于本例采用BGIseq-500进行测序，因此，需要对磁珠纯化的产物进行成环反应，操作详情参照BGIseq-500标准DNA小片段建库流程的成环步骤。然后，对环化的产物进行BGIseq-500测序。
七、实验结果
1.文库电泳结果
甲基化测序文库及对应的stLFR文库制备的结果，本例用1.5％琼脂糖凝胶140V、30分钟跑电泳，结果如图7所示。图7中，第一泳道为DNA marker，本例采用的是Thermofisher 1kb plus DNA ladder；第二和第三泳道为WGS的S1和S2，即正常基因测序的stLFR测序文库的两个重复；第四和第五泳道为WGBS的S1和S2，即甲基化测序文库的两个重复。
2.甲基化测序结果
本例采用相同的人基因组DNA，按照现有的甲基化测序方法进行测序，作为对比。结果如表1所示。
表1 测序结果比对
试验 Clean_reads Clean_bases(bp) Mapped_reads Mapping_rate(％)
试验组一(常规文库) 156770568 15677056800 149334322 95.26
试验组二(甲基化文库) 156868710 15686871000 150202688 95.75
对照组SRR6006942 157796950 23827339450 133582687 84.65
表1中，试验组一为本例stLFR技术建库后用于常规测序获得的数据，试验组二为本例stLFR技术建库后进行甲基化建库和测序获得的数据，对照组即按照常规的甲基化测序获得的数据。与对照组相比较，本例常规测序和甲基化测序结果的mapping效率都高于常规甲基化测序的对照组。并且，在无参考基因组的前提下，本例从试验组一和试验组二中分别抽取分子标签序列一致且长度一致的reads进行一对一比对，即可获得甲基化信息。例如，对于已知的低甲基化水平LINE基因片段：
试验组一的分子标签序列:1320_499_969序列如SEQ ID NO.16所示，
SEQ ID NO.16：
5’-ATGGAAACTGAACAACCTGCTCCTGAATGACTACTGGGTACATAACGAA-3’
试验组二的分子标签序列:1320_499_969序列如SEQ ID NO.17所示，
SEQ ID NO.17：
5’-ATGGAAA TTGAA TAA TTTG TT TTTGAATGA TTA TTGGGTA TATAA TGAA-3’
直接比较SEQ ID NO.16和SEQ ID NO.17，即可确认该序列没有甲基化位点。而无需与人基因组参考序列进行比对，这对于本身没有参考基因组序列的物种而言，具有重要意义。
需要说明的是，以上试验只是本申请的一种具体实现方式，试验中采用的具体的反应体系、反应条件只是一个常规的参考，在此基础上可以根据具体试验进行改变。以上试验中试验的DNA片段化的酶，除Tn5转座酶以外，还可以是Tn转座酶家族的其他酶，如Tn7；或者其他转座酶家族，如Mu家族；甚至不限于转座酶或者酶制剂，只要其能够使DNA被片段化同时将一段序列连接至DNA上即可。本实例涉及到的酶、缓冲液、使用的分子标签数量、载体、载体表面修饰、分子标签结构、加接头方案等可参考WO2019217452A1。
本申请的DNA甲基化检测可以用于全基因组甲基化测序、基因未知区域甲基化测序、已知特定区域的甲基化测序、同源染色体上等位基因的甲基化信息对比、单 倍体全基因组甲基化测序及组装。并且，可以是线粒体、叶绿体等的DNA甲基化检测。
以上内容是结合具体的实施方式对本申请所作的进一步详细说明，不能认定本申请的具体实施只局限于这些说明。对于本申请所属技术领域的普通技术人员来说，在不脱离本申请构思的前提下，还可以做出若干简单推演或替换。

Claims (23)

  1. 一种DNA甲基化检测的方法，其特征在于：包括以下步骤，
    长片段DNA处理步骤，包括将长片段DNA打断成小片段DNA；
    常规文库构建步骤，包括采用stLFR技术进行文库构建，获得常规文库；
    双链保护步骤，包括取用所述常规文库，磁珠回收，将DNA变性为单链，然后利用退火或延伸的方法将分子标签序列或测序接头序列转化为双链；
    重亚硫酸盐转化步骤，包括向所述双链保护步骤的产物中加入双链保护剂，并加入重亚硫酸盐，进行脱氨基反应；
    甲基化测序文库构建步骤，包括对脱氨基反应的产物进行常规PCR扩增或环化后滚环扩增，得到甲基化测序文库；
    甲基化测序步骤，包括对甲基化测序文库进行测序，并根据测序结果分析获得基因的甲基化信息。
  2. 根据权利要求1所述的方法，其特征在于：还包括常规测序步骤，
    所述常规测序步骤，包括取用所述常规文库构建步骤中采用stLFR技术构建的所述常规文库，将DNA变性为单链，然后再进行常规PCR扩增或环化后滚环扩增，获得测序文库，对测序文库进行全基因组测序，获得正常基因测序结果；
    所述甲基化测序步骤还包括，将甲基化测序文库的测序结果与所述常规文库获得的正常基因测序结果比对，从而获得基因的甲基化信息。
  3. 根据权利要求1或2所述的方法，其特征在于：所述长片段DNA处理步骤还包括，采用带有分子标签序列的载体对DNA片段进行捕获，然后再将捕获的DNA用于所述常规文库构建步骤进行stLFR技术文库构建。
  4. 根据权利要求3所述的方法，其特征在于：所述载体为磁珠。
  5. 根据权利要求1或2所述的方法，其特征在于：所述长片段DNA处理步骤中，将长片段DNA打断成小片段DNA具体采用Tn5转座酶。
  6. 根据权利要求1或2所述的方法，其特征在于：所述双链保护步骤中，延伸的方法具体包括，利用分子标签序列或测序接头序列自身的3’端序列作为引物或加入外源短片段作为引物，在DNA聚合酶作用下延伸，将分子标签序列或测序接头序列变为双链结构。
  7. 根据权利要求1或2所述的方法，其特征在于：所述重亚硫酸盐转化步骤中，所述双链保护剂为盐离子、聚乙二醇、酶和有机溶剂中的至少一种，所述重亚硫酸盐为重亚硫酸氢钠。
  8. 根据权利要求1-7任一项所述的方法在基因印记检测、胚胎发育检测或癌症预防检测中的应用。
  9. 一种DNA甲基化检测的试剂盒，其特征在于：包括第一组试剂、第二组试剂、第三组试剂、第四组试剂和第五组试剂；
    所述第一组试剂包括用于将长片段DNA打断成小片段DNA的试剂；
    所述第二组试剂包括用于stLFR技术建库的试剂，该试剂用于制备常规文库；
    所述第三组试剂包括用于通过退火或延伸的方法将分子标签序列或测序接头序列转化为双链的试剂；
    所述第四组试剂包括双链保护剂和重亚硫酸盐，用于进行双链保护和脱氨基反应；
    所述第五组试剂包括用于对脱氨基反应的产物进行常规PCR扩增或环化后滚环扩增的试剂，该试剂用于制备甲基化测序文库。
  10. 根据权利要求9所述的试剂盒，其特征在于：还包括第六组试剂，所述第六组试剂包括用于对常规文库进行常规PCR扩增或环化后滚环扩增的试剂，该试剂用于制备常规测序文库，以便于进行全基因测序，获得正常基因测序结果。
  11. 根据权利要求9所述的试剂盒，其特征在于：所述第一组试剂还包括带有分子标签序列的载体，用于对DNA片段进行捕获。
  12. 根据权利要求11所述的试剂盒，其特征在于：所述载体为磁珠，第一组试剂中还包括磁珠纯化的试剂。
  13. 根据权利要求9所述的试剂盒，其特征在于：所述第一组试剂中，用于将长片段DNA打断成小片段DNA的试剂为Tn5转座酶及其缓冲液。
  14. 根据权利要求9所述的试剂盒，其特征在于：所述第三组试剂中，用于通过退火或延伸的方法将分子标签序列或测序接头序列转化为双链的试剂，具体包括外源短核苷酸片段、DNA聚合酶及其缓冲液，以及用于DNA延伸的dNTPs。
  15. 根据权利要求9-14任一项所述的试剂盒，其特征在于：所述第四组试剂中，双链保护剂为盐离子、聚乙二醇、酶和有机溶剂中的至少一种，重亚硫酸盐为重亚硫酸氢钠。
  16. 根据权利要求9-15任一项所述的试剂盒在基因印记检测、胚胎发育检测或癌症预防检测中的应用。
  17. 一种DNA甲基化检测的装置，其特征在于：包括长片段DNA处理模块、常规文库构建模块、常规测序模块、双链保护模块、重亚硫酸盐转化模块、甲基化测序文库构建模块和甲基化测序模块，
    长片段DNA处理模块，包括用于将长片段DNA打断成小片段DNA；
    常规文库构建模块，包括用于采用stLFR技术进行文库构建，获得常规文库；
    常规测序模块，包括取用所述常规文库构建模块中采用stLFR技术构建的所述常规文库，将DNA变性为单链，然后再进行常规PCR扩增或环化后滚环扩增，获得测序文库，对测序文库进行全基因组测序，获得正常基因测序结果；
    双链保护模块，包括用于取用所述常规文库构建模块中采用stLFR技术构建的所述常规文库，磁珠回收，将DNA变性为单链，然后利用退火或延伸的方法将分子标签序列或测序接头序列转化为双链；
    重亚硫酸盐转化模块，包括用于向经过所述双链保护模块处理的产物中加入双链保护剂，并加入重亚硫酸盐，进行脱氨基反应；
    甲基化测序文库构建模块，包括用于对脱氨基反应的产物进行常规PCR扩增或环化后滚环扩增，得到甲基化测序文库；
    甲基化测序模块，包括用于对甲基化测序文库进行测序，将甲基化测序结果与正常基因测序结果比对，获得基因的甲基化信息。
  18. 根据权利要求17所述的装置，其特征在于：所述长片段DNA处理模块，还包括用于采用带有分子标签序列的载体对DNA片段进行捕获，然后再将捕获的DNA用于所述常规文库构建模块采用stLFR技术进行文库构建。
  19. 根据权利要求18所述的装置，其特征在于：所述载体为磁珠。
  20. 根据权利要求17所述的装置，其特征在于：所述长片段DNA处理模块中，将长片段DNA打断成小片段DNA具体采用Tn5转座酶。
  21. 根据权利要求17所述的装置，其特征在于：所述双链保护模块中，延伸的方法具体包括，利用分子标签序列或测序接头序列自身的3’端序列作为引物或加入外源短片段作为引物，在DNA聚合酶作用下延伸，将分子标签序列或测序接头序列变为双链结构。
  22. 根据权利要求17-21任一项所述的装置，其特征在于：所述重亚硫酸盐转化模块中，所述双链保护剂为盐离子、聚乙二醇、酶和有机溶剂中的至少一种，所述重亚硫酸盐为重亚硫酸氢钠。
  23. 根据权利要求17-22任一项所述的装置在基因印记检测、胚胎发育检测或癌症预防检测中的应用。
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