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WO2024114696A1 - Technologie de séquençage d'enrichissement de méthylation d'îlots cpg basée sur la digestion d'enzyme de restriction - Google Patents

Technologie de séquençage d'enrichissement de méthylation d'îlots cpg basée sur la digestion d'enzyme de restriction Download PDF

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WO2024114696A1
WO2024114696A1 PCT/CN2023/135179 CN2023135179W WO2024114696A1 WO 2024114696 A1 WO2024114696 A1 WO 2024114696A1 CN 2023135179 W CN2023135179 W CN 2023135179W WO 2024114696 A1 WO2024114696 A1 WO 2024114696A1
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digestion
dna
methylation
sequencing
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姜正文
方欧
王果
林芳斌
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Genesky Technologies Suzhou Inc
SHANGHAI GENESKY BIOTECHNOLOGIES Inc
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Genesky Technologies Suzhou Inc
SHANGHAI GENESKY BIOTECHNOLOGIES Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • the present invention relates to the field of DNA sequencing, and in particular to a CpG island methylation enrichment sequencing technology based on restriction enzyme digestion.
  • DNA methylation modification is crucial to normal gene expression and cell function, and is involved in regulating the most basic life activities such as gene expression and chromatin stability.
  • Gene methylation polymorphism is an important cause of individual phenotypic differences, and gene methylation variation can also lead to individual phenotypic abnormalities.
  • a large number of studies have shown that compared with normal cells, the methylation characteristics of tumor cell genes have undergone extensive and significant changes, and cancer-specific methylation variant genes have also been found in different cancer types. Therefore, gene methylation variation can be used as a pan-cancer biomarker.
  • methylation tumor markers rely on differential methylation gene analysis of genomic DNA in healthy tissues and cancer tissues.
  • WGBS Whole-genome bisulfite sequencing
  • RRBS Reduced representation bisulfite sequencing
  • Methylation detection for specific sites/regions usually uses methods such as MSP (Methylation-specific PCR), BSP (Bisulfite-Sequencing PCR) and MSRE-qPCR.
  • ctDNA (Plasma Cell-free tumor DNA, ctDNA), which is the focus of liquid biopsy, carries methylation information from tumor tissues. Its detection can realize cancer screening, companion diagnosis and prognosis monitoring. Due to the low copy number and high degree of degradation of ctDNA, its detection is not easy, and the weak methylation marker signal will also be masked by background noise. In early applications, taqman probe qPCR technology is usually relied on to detect designated methylation sites or haplotypes. In recent years, whole genome methylation sequencing (WGBS) based on NGS and target region methylation sequencing technology captured by probe hybridization have also been developed for ctDNA detection at the omics level.
  • WGBS whole genome methylation sequencing
  • This type of method can integrate the methylation information of a large number of sites, train mathematical models, and maximize the detection sensitivity and specificity. At the same time, it has the characteristics of tissue tracing, so it has gradually become the mainstream technology for pan-cancer screening.
  • both methods have limitations.
  • WGBS can obtain whole genome methylation information, it is limited by sequencing costs and has a low sequencing depth. For example, the common 90G sequencing volume can only obtain an average sequencing depth of about 20X, and the detection accuracy and sensitivity are poor.
  • the methylation variation regions in the genome that can actually be used to indicate cell carcinogenesis only account for a very small part, and whole genome sequencing is undoubtedly a strategy with low cost performance.
  • Methylation sequencing of target regions based on probe capture certainly makes up for the lack of depth of WGBS sequencing, but due to the complexity of methylation,
  • the haplotype pattern is complex (with the increase of the number of CpG sites in the target region, the number of haplotypes increases exponentially); there are still many technical difficulties in achieving efficient, stable and accurate capture of the target region.
  • the additional steps of probe synthesis and target region hybridization capture also increase the difficulty and cost of the experiment.
  • the purpose of the present invention is to provide a CpG island methylation enrichment sequencing technology based on restriction enzyme cutting.
  • a method for constructing a DNA methylation sequencing library comprising the steps of:
  • the DNA sample to be tested is selected from the following group: genomic DNA (gDNA), cell-free DNA (cfDNA), or a combination thereof.
  • step S1 the DNA sample to be tested is fragmented, preferably fragmented to 100-600 bp, more preferably 200-400 bp.
  • the linker in step S2), is a methylated linker, in which all cytosine Cs are 5-methylated.
  • the linker does not contain AATT or TTAA sequence.
  • the linker comprises a first chain and a second chain, the 3' end of the first chain is partially base complementary to the 5' end of the second chain, and after the two chains are annealed, a single A base of the first chain protrudes.
  • the 5' end of the first chain is modified with a phosphate group.
  • first chain nucleotide sequence is shown as SEQ ID NO.1
  • second chain nucleotide sequence is shown as SEQ ID NO.2.
  • step S2) the steps of end repair and A tailing are also included before connecting the adapter.
  • step S2 after connecting the methylated linker, a sorting and/or purification step is also included.
  • the sorting step includes sorting the length of the DNA insert fragments, preferably 200-400 bp, more preferably 250-350 bp.
  • the purification step includes purifying and recovering DNA fragments with double-ends connected to methylated adapters.
  • the sorting and/or purification includes magnetic bead sorting and/or purification.
  • step S3) the DNA is subjected to one or more rounds of AT enzyme digestion.
  • step S3 the AT enzyme cleavage is performed before CT transformation and/or after CT transformation; or is performed both before CT transformation and after CT transformation.
  • the non-methylated site cleavage is performed before CT conversion.
  • the non-methylated site digestion is performed before AT digestion, or after AT digestion, or simultaneously with AT digestion in the same reaction system.
  • step S3) further includes the step of PCR amplification.
  • step S3) comprises a step selected from the following group:
  • step S3) the AT enzyme cleavage is performed using an enzyme that can recognize and cleave AT-rich sequence DNA.
  • the enzyme is selected from the group consisting of restriction endonucleases, CRISPR gene editing enzymes, ZFNs, TALENs, giant nucleases, or combinations thereof.
  • the AT digestion is performed using a restriction endonuclease whose digestion recognition site contains only A and T bases.
  • the enzyme used for the AT cleavage is selected from the following group: MluCI, MseI, SspI, PsiI, AseI, DraI, PacI, AnaI, AcsI, AgsI, ApoI, AflII, BfrI, BspTI, BstAFI, EcoRI, EcoRV, FaiI, FauNDI, HpaI, KspAI, MfeI, MspCI, MssI, MunI, NdeI, PmeI, PshBI, SaqAI, SmiI, Sse9I, TasI, Tru1I, Tru9I, TspDTI, Tsp509I, VspI, XapI, HindIII, NsiI, NspV, PagI, PciI, SfuI, SnaBI, BfrBI, ClaI, ScaI, SwaI, or a combination thereof.
  • the AT digestion is single digestion with MseI, single digestion with MluCI, or double digestion with MseI and MluCI.
  • the CT conversion is sulfite chemical conversion or APOBEC deaminase conversion, preferably APOBEC deaminase conversion.
  • the CT conversion comprises the steps of:
  • TET2 enzyme oxidatively protects the 5mC base
  • the CT conversion comprises the steps of:
  • step S3 the non-methylated site cleavage is performed using a 5mC methylation-sensitive restriction endonuclease or a combination of endonucleases.
  • the 5mC methylation-sensitive restriction endonuclease or endonuclease combination is selected from the following group: HpaII endonuclease, BstUI endonuclease, FspI endonuclease, or a combination thereof.
  • step S3) the first round of PCR amplification is performed using a high-fidelity DNA amplification enzyme capable of amplifying templates containing uracil (U base).
  • the first round of PCR amplification is performed using adapter sequence-specific primers.
  • the first round of PCR amplification uses a forward primer nucleotide sequence as shown in SEQ ID NO.3, and a reverse primer nucleotide sequence as shown in SEQ ID NO.4.
  • the second PCR amplification uses a forward primer nucleotide sequence as shown in SEQ ID NO.5 and a reverse primer nucleotide sequence as shown in SEQ ID NO.6.
  • each AT digestion and/or non-methylation site digestion step may be followed by a sorting and/or purification step.
  • a DNA methylation sequencing library is provided.
  • the methylation sequencing library is constructed using the method described in the first aspect of the present invention.
  • a method for detecting DNA methylation in a sample comprising the steps of:
  • the sequencing includes using an Illumina sequencing platform or a BGI sequencing platform.
  • a method for diagnosing or predicting diseases related to abnormal DNA methylation comprising the steps of obtaining a DNA sample from a subject to be tested, and detecting methylation of the DNA sample using the method described in the first aspect of the present invention, thereby diagnosing or predicting the disease.
  • the disease is a tumor.
  • the subject is a human or non-human mammal.
  • FIG1 shows the experimental flow chart of the CpG island methylation enrichment sequencing technology based on restriction enzyme digestion.
  • Figure 3 shows the ratio of the sequencing depth of each CpG island after the first round of AT digestion to the depth before digestion in Example 2 when the same amount of sequencing data is used, displayed in a histogram.
  • the part corresponding to the black highlighted horizontal axis [0,1] indicates that after the first round of AT digestion, the sequencing depth is lower than the number of CpG islands without digestion.
  • Figure 4 shows a histogram of sequencing depths of the three libraries treated differently in Example 3 in the commonly detected CpG island regions, with the horizontal axis representing sequencing depths.
  • the top, middle and bottom display the libraries of "no restriction enzyme digestion", “first round AT digestion + non-methylated site digestion” and “first round AT digestion + non-methylated site digestion + second round AT digestion”.
  • the dotted line represents the mean of the sequencing depths of the libraries in these common CpG islands.
  • Figure 5 shows the enrichment effect of enzyme digestion on methylated CpG islands.
  • MseI and MluCI were used for the first round of AT digestion, and HpaII was used for non-methylated site digestion; MseI and MluCI were used for the second round of AT digestion before library construction and sequencing.
  • the results of enrichment sequencing of CpG sites in the promoter region of SEPTIN9 are shown: gray lines represent sequencing reads, NEGATIVE and POSITIVE represent positive and negative strands, respectively; black squares represent methylated CpG sites, and white squares represent non-methylated CpG sites.
  • the inventors After extensive and in-depth research, the inventors have developed a special methylation second-generation sequencing library construction method that introduces a restriction enzyme cutting step during the library construction process. Utilizing the characteristics of CpG islands rich in C and G bases, through the recognition site, the restriction enzyme cutting combination with different characteristics, the enrichment sequencing of CpG islands with methylation modification is realized.
  • the present invention is suitable for the research and application of methylation of CpG islands, not only for conventional genomic DNA, but also for the methylome detection of trace ctDNA, providing a new solution for the field of tumor liquid biopsy that relies on ctDNA methylation. On this basis, the present invention is completed.
  • the present invention comprehensively applies AT digestion, CT conversion and non-methylation site digestion.
  • the AT digestion step the interference of a large number of AT-enriched fragments can be removed, the sequencing efficiency can be improved, and the CpG island can be enriched.
  • non-methylation site digestion the interference of non-methylated CG fragments can be removed, the sequencing efficiency can be improved, and the CpG island containing methylation sites can be enriched.
  • CT conversion step on the one hand, non-methylated cytosine is converted, and only methylated cytosine is retained in the sequence; on the other hand, CT conversion further generates new AT digestion sites in the sequence, and secondary AT digestion can be performed to improve the enrichment efficiency.
  • methylation library and “enzyme digestion sequencing library” have the same meaning, and refer to a double-stranded DNA fragment that is repaired at the end, A is added, and a Y-shaped methylated adapter is connected, followed by one or more steps selected from AT digestion, non-methylated site digestion, CT conversion, or a combination thereof, and finally PCR enrichment amplification to obtain the library.
  • the term "AT digestion” refers to the use of a restriction endonuclease whose recognition sequence contains only A/T to digest the whole genome methylation sequencing library.
  • the restriction endonuclease whose recognition sequence contains only A/T includes any enzyme that can cut AT-rich sequences, such as but not limited to MseI (recognition site is TTAA) and MluCI (recognition site AATT).
  • non-methylated site digestion refers to the use of a 5mC methylation-sensitive restriction endonuclease to digest the whole genome methylation sequencing library.
  • the 5mC methylation-sensitive restriction endonuclease includes but is not limited to HpaII (recognition site is CCGG).
  • CT conversion refers to the deamination of cytosine into uracil or thymine.
  • CT conversion can be performed using an enzyme treatment method, wherein the 5mC base is protected by TET2 enzyme oxidation, and then the unprotected C base (unmethylated C base) is completely deaminated and converted into U base using APOBEC deaminase.
  • Conventional chemical methods can also be used to convert unmethylated C bases into U bases using traditional sulfite treatment. Enzyme treatment is preferred because it is milder and less likely to cause DNA chain breaks.
  • AT digestion can be performed before CT conversion and/or after CT conversion alone; it can also be performed before CT conversion and after CT conversion.
  • Non-methylation site digestion is performed before CT conversion, and can be performed separately or in combination with AT digestion.
  • universal primers at both ends of the library are used to enrich DNA fragments whose insert fragments do not contain the above-mentioned digestion sites.
  • the present invention utilizes the characteristics of CpG islands being rich in C and G bases, utilizes AT digestion and non-methylated site digestion to simplify the sequencing library, retains CpG island information, and can utilize the sequence changes after CT conversion to perform a second round of AT digestion to further improve the simplification efficiency.
  • Non-methylated site digestion further enriches the CpG island information that has undergone methylation.
  • two rounds of restriction enzyme digestion can be performed to remove AT-rich regions (AT digestion) or unmethylated regions (unmethylated site digestion). Performing two rounds of digestion steps can maximize the enrichment of CpG islands that have undergone methylation modification. After CT conversion, the sequence will change, and the new sequence may therefore constitute a new AT digestion site.
  • the method of the present invention can refer to Figure 1, comprising the steps of:
  • DNA fragments are first end-repaired and A-tailed, and methylated adapters are connected to both ends of the DNA fragments;
  • the methylated linker comprises a first strand and a second strand, all cytosine Cs are 5-methylated, wherein the 5' phosphate group of the first strand is modified, the 3' end of the first strand is partially complementary to the 5' end of the second strand, and after the two strands are annealed, a single A base of the first strand protrudes;
  • the type of DNA that can be used for library construction can be genomic DNA (gDNA) or cell-free DNA (cfDNA);
  • a high-fidelity DNA amplification enzyme capable of amplifying templates containing uracil (U base) is used to PCR amplify the fragments of the inserted fragments that do not contain restriction sites to obtain a single-round restriction digestion sequencing library;
  • a second round of PCR amplification is performed using primers matching the universal adapter to enrich the fragments whose inserts still do not contain the digestion site after CT conversion, and obtain a twice-digested sequencing library;
  • the sequencing library is sequenced on a sequencing machine.
  • the sequencing platform is determined based on the connected methylated adapters.
  • the Illumina sequencing platform or the BGI sequencing platform is used.
  • the method of the present invention reduces the sequencing cost while ensuring the acquisition of effective information of CpG islands as much as possible.
  • the CpG island enrichment effect can be as high as 10 times.
  • the method of the present invention eliminates the complex, high-cost and uncertain methylation probe design and capture steps.
  • the method of the present invention is compatible with various types of DNA samples, including genomic DNA and cell-free DNA.
  • This example simulates the enrichment multiple of CpG islands in the human genome sequence (hg19) by performing the first round of AT digestion with MseI and MluCI, respectively, followed by the second round of AT digestion after CT conversion.
  • the genome sequence was divided into N fragments; the number of fragments containing the AATT or TTAA base combination in the sequence was counted, recorded as N1; the number of fragments containing any one of the AATT, TTAA, AACC, AATC, AACT, CCAA, TCAA, and CTAA base combinations in the sequence was counted, recorded as N2;
  • the sequence of human CpG island (data source: UCSC database) is divided into M fragments; the number of fragments containing the base combination of AATT or TTAA in the sequence is counted, recorded as M1; the number of fragments containing any one of the base combinations of AATT, TTAA, AACC, AATC, AACT, CCAA, TCAA, and CTAA in the sequence is counted, recorded as M2;
  • the genome simplification efficiency is positively correlated with the sliding window length regardless of single or double AT digestion.
  • the genome simplification efficiency of single-round AT digestion is more susceptible to the sliding window length, and the genome simplification efficiency of double AT digestion is higher than that of single-round digestion, but is less affected by the sliding window length.
  • the CpG island information retention ratio after single and double digestion is negatively correlated with the sliding window length, and the CpG island information retention ratio of single-round AT digestion is higher, and is less affected by the sliding window length; while the CpG island information retention ratio of double AT digestion is lower than that of single-round digestion, and is more affected by the sliding window length.
  • 5mC-AD-F [ROX]ACACTCTTTCCCTACACGACGCTCTTCCGATCT (SEQ ID NO.1)
  • the primer sequences are:
  • the sequencing results are shown in the following table. The number of bases aligned to the entire genome and the number of bases aligned to the CpG region are counted separately to calculate the average sequencing depth. After one round of AT digestion, the sequencing depth of each G sequencing data in the CpG island is 1.4X compared to 0.4X of the undigested library, and the CpG island enrichment efficiency is about 3.5 times;
  • the sequencing data aligned to the CpG island after one round of AT digestion is mainly divided into the following types: A) all CpG island information is retained, B) CpG island information is partially retained, and C) CpG island information is completely lost ( Figure 2).
  • Figure 3 After calculation, under the same sequencing amount, among the 27,949 CpG islands, the number of sequencing depths after one round of AT digestion is higher than that of sequencing without digestion reached 25,606, accounting for 91.6% ( Figure 3).
  • each CpG site contained in a CpG island usually has a consistent methylation pattern, so After enzyme digestion, even if only part of the information in the CpG island is retained, it can be used to infer the methylation degree of the CpG island.
  • a single round of AT digestion can reduce sequencing costs by more than 70%, and obtain a sequencing depth that is no less than that of whole genome sequencing in more than 90% of CpG island regions.
  • Example 3 CpG island enrichment sequencing after two rounds of AT digestion and non-methylated site digestion of cfDNA
  • Enzymatic Methyl-seq Conversion Module (E7125L, NEB) was used for CT conversion;
  • TET2 Reaction Buffer a. Add 400 ⁇ L TET2 Reaction Buffer to a tube of TET2 Reaction Buffer Supplement and mix well. Mark it as TET2 Reaction Buffer (reconstituted).
  • the primer sequences are the same as in Example 2.
  • the undigested control amplification product is purified, it is directly used as the undigested control sequencing library; after the two digested CT conversion amplification products are purified, one is used as the sequencing library of single-round AT digestion + non-methylated site digestion,
  • the sequencing data were statistically analyzed. When the sequencing data volume was the same as 5G, there were 27949 CpG islands in total without enzyme digestion, of which 21223 CpG islands were sequenced to at least one read; after one round of AT digestion and unmethylated site digestion, at least one read could be detected in 17006 CpG islands, and the sequencing depth was higher than that of the undigested library for 9349 CpG islands; after a second round of AT digestion, at least one read could be detected in 15542 CpG islands, and the sequencing depth was higher than that of the undigested library for 10967 CpG islands.
  • the sequencing results counted the number of bases aligned to the entire genome and the number of bases aligned to the CpG island region, and calculated the average sequencing depth of each CpG island. Due to the differences in genome simplification efficiency of different processing libraries, the CpG islands shared by the sequencing data of the three libraries were counted; when the sequencing volume was 5G, in these effective regions, the average coverage depth was about 1.68X without enzyme digestion; after one round of AT digestion + non-methylated site digestion, the average coverage depth was about 2.99X; after two rounds of AT digestion + one round of non-methylated site digestion, the average coverage depth was 8.25X ( Figure 4).
  • the sequencing data after restriction digestion of non-methylated sites, the sequencing data also enriches CpG islands that have undergone methylation modification. As shown in Figure 5, a CpG island located in the promoter region of the SEPTIN9 gene has 4 reads detected without restriction digestion, all of which are non-methylated. After restriction digestion of non-methylated sites, methylated reads are enriched, and the number of sequenced reads increases with the increase in the number of AT restriction digestions.
  • AT enzyme cleavage can significantly increase the sequencing depth of the CpG island region, and enzyme cleavage at non-methylated sites can enrich the regions where methylation modification occurs.

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Abstract

La présente invention concerne un procédé de construction de bibliothèque de séquençage de méthylation simple et efficace. Une digestion enzymatique combinée d'endonucléase de restriction est ajoutée sur la base d'un procédé de séquençage de méthylation de génome entier classique, permettant ainsi un séquençage d'enrichissement d'îlots CpG, en particulier d'îlots CpG modifiés par méthylation. Le procédé de la présente invention réduit le coût de séquençage et élimine des opérations complexes, étant ainsi approprié pour la recherche et des applications ciblant la méthylation d'îlots CpG.
PCT/CN2023/135179 2022-11-30 2023-11-29 Technologie de séquençage d'enrichissement de méthylation d'îlots cpg basée sur la digestion d'enzyme de restriction Ceased WO2024114696A1 (fr)

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CN113943779A (zh) * 2021-10-15 2022-01-18 厦门万基生物科技有限公司 一种高cg含量dna序列的富集方法及其应用
CN114438184A (zh) * 2022-04-08 2022-05-06 昌平国家实验室 游离dna甲基化测序文库构建方法及应用
CN115976161A (zh) * 2022-11-30 2023-04-18 天昊基因科技(苏州)有限公司 基于限制性酶切的CpG岛甲基化富集测序技术

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