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WO2020135347A1 - Procédé de détection de méthylation d'adn, kit d'analyse, dispositif et application - Google Patents

Procédé de détection de méthylation d'adn, kit d'analyse, dispositif et application Download PDF

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Publication number
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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Chinese (zh)
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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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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00

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

La présente invention concerne un procédé de détection de méthylation d'ADN, comprenant : une étape de traitement d'ADN long consistant à casser de longs ADN; une étape de construction de bibliothèque générale consistant à utiliser une technologie stLFR pour construire une bibliothèque; une étape de protection double brin consistant à prendre la bibliothèque stLFR obtenue, à dénaturer l'ADN, et à convertir un marqueur moléculaire ou une séquence de liaison en double brin par recuit ou extension; une étape de conversion au bisulfite consistant à ajouter un agent de protection double brin et du bisulfite pour effectuer une désamination; une étape de construction de bibliothèque de séquençage de méthylation consistant à effectuer une amplification par PCR ou circulaire classique sur un produit désaminé pour obtenir une bibliothèque de séquençage de méthylation; et une étape de séquençage de méthylation consistant à séquencer la bibliothèque de séquençage de méthylation, et à analyser des informations de méthylation de gènes selon un résultat de séquençage. La présente invention concerne également un kit d'analyse correspondant, un dispositif et une application. Le procédé selon la présente invention utilise la technologie stLFR pour obtenir des informations de méthylation à long fragment. Au moyen d'une stratégie de protection double brin, la séquence de marqueur moléculaire peut être ajoutée à l'avance pour construire une bibliothèque, pouvant suivre efficacement une source moléculaire.
PCT/CN2019/127537 2018-12-29 2019-12-23 Procédé de détection de méthylation d'adn, kit d'analyse, dispositif et application Ceased WO2020135347A1 (fr)

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CN118389655A (zh) * 2024-03-11 2024-07-26 青岛大学 一种dna中8-氧-2’脱氧鸟嘌呤修饰单碱基分辨率定位分析方法
CN119979688A (zh) * 2025-01-14 2025-05-13 中国科学院遗传与发育生物学研究所 一种高分辨率低成本的组织空间dna甲基化组学测序方法

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