WO2023193456A1 - Biological composition, method for preparing same, and use thereof - Google Patents
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- WO2023193456A1 WO2023193456A1 PCT/CN2022/135759 CN2022135759W WO2023193456A1 WO 2023193456 A1 WO2023193456 A1 WO 2023193456A1 CN 2022135759 W CN2022135759 W CN 2022135759W WO 2023193456 A1 WO2023193456 A1 WO 2023193456A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
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- 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
- C12Q1/6844—Nucleic acid amplification reactions
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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
- C12Q1/6869—Methods for sequencing
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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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/91—Cell lines ; Processes using cell lines
Definitions
- the present application relates to the field of biomedicine, specifically to a biological composition and its preparation method, and also includes the application of the biological composition as a standard for methylation detection.
- Circulating DNA (cfDNA, cell free DNA) is a single-stranded or double-stranded DNA molecule that exists in the blood, and its fragment length is approximately 144-166bp.
- DNA methylation plays an important role in the regulation of gene expression and is closely related to the occurrence and development of tumors.
- DNA methylation has a wide range of sites, high sensitivity, and can With the advantages of tissue traceability, combined with the characteristics of liquid biopsy technology such as less trauma and convenient sampling, blood ctDNA methylation detection has become the most potential early screening technology path at present.
- DNA methylation is an epigenetic modification that is catalyzed by DNA methyltransferase (DNMT) to convert S-adenosylmethionine (SAM) as a methyl group.
- DNMT DNA methyltransferase
- SAM S-adenosylmethionine
- the cytosine of the two nucleotides CG of DNA is selectively added with methyl groups, mainly forming 5-methylcytosine (5-mC) (common in the 5'-CG-3' sequence of genes) and small amounts of N6-methylpurine (N6-mA) and 7-methylguanine (7-mG).
- DNA methylation plays an important role in the regulation of gene expression. Abnormal DNA methylation marks have been reported during the development and progression of various diseases, including cancer. As a high-resolution, high-throughput technology, DNA methylation sequencing is increasingly recognized for its role in early cancer screening, diagnosis, and monitoring.
- WGBS Whole Genome Bisulfite Sequencing
- CpG islands are abundant in the genome, and these detection and analysis can be greatly assisted by massively parallel nucleic acid sequencing (also known as “high-throughput sequencing” or “next generation sequencing” (NGS)), allowing prediction of methylation signals The occurrence of cancer and where it occurs becomes possible.
- massively parallel nucleic acid sequencing also known as “high-throughput sequencing” or “next generation sequencing” (NGS)
- NGS next generation sequencing
- methylation negative standards mainly come in the following forms: 1) Unmethylated Lambda DNA sample (isolated from infected E. coli GM119 strain, which lacks dam and dcm methylase activities ), the advantage is that the genome is small and the CpG site signal is close to 0, but the disadvantage is that it does not come from the human genome; 2) The cell line DNA with artificial knockout of methyltransferase cannot remove the methylation signal of this CpG site Clean, there is still a high CpG signal background, and it is expensive; 3) Change the methylation signal of any sample CpG site to 0 through whole-genome amplification.
- methylation positive standards mainly use 1) cancer cell lines, in which methylation sites close to 100% of positive sites can be found; 2) MsssI (methylase) treats puc19 /DNA or human genomic DNA.
- MsssI methylase
- the methylation level of each CpG site of the DNA obtained by the latter method is as high as 97%, but it cannot achieve tissue traceability information.
- This application provides a standard substance for methylation detection and a preparation method thereof.
- the method uses Atlantis dsDNase (a restriction enzyme) or Micrococcal Nuclease (Micrococcal Nuclease) fragmentation enzyme to digest cell lines.
- a cell line standard with a peak shape similar to that of blood is provided to meet the demand for methylation blood detection standards.
- By gradient mixing of cancer-derived cell line DNA and healthy cell line DNA different gradient tumor signal standards can be obtained products, and at the same time, organizational traceability can be achieved.
- adding a certain proportion of fully methylated pUC19 and unmethylated Lambda genome combination or unmethylated pUC19 and fully methylated Lambda genome combination can evaluate the detection accuracy.
- the present application provides a biological composition, including: at least one positive cell line as a positive standard, at least one negative cell line as a negative standard, and at least one positive genome vector as a positive reference, and at least one negative genomic vector serving as a negative reference.
- the number of bases of the positive genome vector is less than the number of bases of the positive cell line; the number of bases of the negative genome vector is less than the number of bases of the negative cell line.
- the number of bases of the positive genome vector is less than 1/100 of the number of bases of the positive cell line.
- the number of bases of the positive genome vector is less than the number of bases of the positive cell line. 1/1000, more preferably, the number of bases of the positive genome vector is less than 1/10000 of the number of bases of the positive cell line;
- the number of bases of the negative genome vector is less than 1/1000 of the number of bases of the negative cell line /100, preferably, the number of bases of the negative genome vector is less than 1/1000 of the number of bases of the negative cell line, more preferably, the number of bases of the negative genome vector is less than 1/1000 of the number of bases of the negative cell line 1/10000;
- the number of bases of the negative genome vector is approximately 1/1000000 to 1/60000 of the number of bases of the negative cell line; or the number of bases of the positive genome vector is approximately 1/1000000 to 1/60000 of the number of bases of the positive cell line. Approximately 1/1,000,000 to 1/60,000 of the number of bases.
- the mass ratio of the negative cell line to the negative genome vector is 1:2 ⁇ 10 -3 to 1:1 ⁇ 10 6 , preferably 1:2 ⁇ 10 -4 to 1:1 ⁇ 10 -5 ;
- the mass ratio of the positive cell line to the positive genome vector is 1:2 ⁇ 10 -3 to 1:1 ⁇ 10 -6 , preferably 1:2 ⁇ 10 -4 to 1:1 ⁇ 10 -5 .
- the mass ratio of the positive cell line to the negative cell line is between 0.1:99.9 and 25:75, preferably between 0.5:99.5 and 12.5:87.5, and more preferably between 1:99 and 25:75. Between 5:95 or: between 99.9:0.1 and 75:25, preferably between 99.5:0.5 and 87.5:12.5, more preferably between 99:1 and 95:5.
- the mass ratio of the positive genome vector to the negative genome vector is between 1:99 and 25:75 or between 99:1 and 95:5, preferably about 1:19 or 19: 1.
- both the positive cell line and the negative cell line undergo fragmentation processing, and the average fragment size is about 140-160 bp.
- the fragmentation treatment may include enzymatic fragmentation and/or physical fragmentation.
- the enzyme cleavage can be through Atlantis dsDNase enzyme or Micrococcal Nuclease fragmented genomic DNA.
- the enzyme cleavage can be interrupted by ultrasound.
- the enzyme digestion of the positive cell line and the negative cell line can be through Atlantis dsDNase enzyme or Micrococcal Nuclease fragmented genomic DNA.
- the positive cell line is selected from a combination of the following cell lines: human colorectal cancer cell line (for example: SW48), human lung adenocarcinoma cell line (for example: H1975), human liver cancer cell line (for example: H1975) For example: HepG2), human esophageal cancer cell line (for example: T.T), human ovarian cancer cell line (for example: CaoV3), human pancreatic cancer cell line (for example: PANC-1, PSN-1).
- human colorectal cancer cell line for example: SW48
- human lung adenocarcinoma cell line for example: H1975)
- human liver cancer cell line for example: H1975)
- human esophageal cancer cell line for example: T.T
- human ovarian cancer cell line for example: CaoV3
- human pancreatic cancer cell line for example: PANC-1, PSN-1).
- only one positive cell line is included.
- the negative cell line is selected from a combination of the following cell lines: human B lymphocytes (for example: GM24385, GM12878, GM24631, NCI.BL1184) or human normal cell lines (for example: Beads-2B and other human normal cell lines).
- human B lymphocytes for example: GM24385, GM12878, GM24631, NCI.BL1184
- human normal cell lines for example: Beads-2B and other human normal cell lines.
- only one negative cell line is included.
- the positive genome vector is pUC19 DNA treated with methyltransferase and/or Lambda treated with methyltransferase, and the methyltransferase is preferably M.Sssl.
- the negative genome vector is unmethylated Lambda and/or unmethylated pUC19 DNA.
- Another aspect of the present application provides an application of the above biological composition, which is used to evaluate the accuracy of cancer gene detection and standards for tissue traceability.
- the biological composition is used to evaluate the accuracy of cancer gene methylation detection and as a standard for tissue traceability.
- the cancer includes: brain cancer, lung cancer, skin cancer, nasopharyngeal cancer, throat cancer, liver cancer, bone cancer, lymphoma, pancreatic cancer, skin cancer, intestinal cancer, rectal cancer, and thyroid cancer , bladder cancer, kidney cancer, oral cancer, gastric cancer, solid tumors, ovarian cancer, esophageal cancer, gallbladder cancer, bile duct cancer, breast cancer, cervical cancer, uterine cancer, prostate cancer, head and neck cancer, sarcoma, thoracic malignant tumors (except lung (External), melanoma, testicular cancer, and leukemia.
- Another aspect of the application provides a method for preparing a biological composition, including the following steps:
- the enzyme digestion is performed by Atlantis DNase enzyme or Micrococcal Nuclease to fragment genomic DNA.
- it also includes: after the enzyme digestion in step (2), fragment screening is performed to remove large gDNA fragments.
- the positive cell line is selected from a combination of the following cell lines: human colon cancer cells (for example: SW48), human lung adenocarcinoma cells (for example: H1975), human liver cancer cells (for example: HepG2) , human esophageal cancer cells (for example: T.T), human ovarian cancer cells (for example: CaoV3), human pancreatic cancer cells (for example: PANC-1, PSN-1).
- human colon cancer cells for example: SW48
- human lung adenocarcinoma cells for example: H1975)
- human liver cancer cells for example: HepG2
- human esophageal cancer cells for example: T.T
- human ovarian cancer cells for example: CaoV3
- human pancreatic cancer cells for example: PANC-1, PSN-1).
- only one positive cell line is included.
- the negative cell line is selected from a combination of the following cell lines: human B lymphocytes (for example: GM24385, GM12878, GM24631, NCI.BL1184).
- only one negative cell line is included.
- the number of bases of the positive genome vector is less than the number of bases of the positive cell line; the number of bases of the negative genome vector is less than the number of bases of the negative cell line.
- the number of bases of the positive genome vector is less than 1/100 of the number of bases of the positive cell line.
- the number of bases of the positive genome vector is less than the number of bases of the positive cell line. 1/1000, more preferably, the number of bases of the positive genome vector is less than 1/10000 of the number of bases of the positive cell line;
- the number of bases of the negative genome vector is less than 1/1000 of the number of bases of the negative cell line /100, preferably, the number of bases of the negative genome vector is less than 1/1000 of the number of bases of the negative cell line, more preferably, the number of bases of the negative genome vector is less than 1/1000 of the number of bases of the negative cell line 1/10000.
- the number of bases of the negative genome vector is approximately 1/1000000 to 1/60000 of the number of bases of the negative cell line; or the number of bases of the positive genome vector is approximately 1/1000000 to 1/60000 of the number of bases of the positive cell line. Approximately 1/1,000,000 to 1/60,000 of the number of bases.
- the mass ratio of the positive cell line to the negative cell line is between 0.1:99.9 and 25:75, preferably between 0.5:99.5 and 12.5:87.5, and more preferably between 1:99 and 25:75. Between 5:95.
- the mass ratio of the positive genome vector to the negative genome vector is between 1:99 and 25:75 or between 99:1 and 95:5, preferably about 1:19 or 19: 1.
- the products of step (3) and step (4) are mixed according to the following mass ratio: 1:5 ⁇ 10 -4 to 1:1.5 ⁇ 10 -5 , preferably 1:2 ⁇ 10 - 4 .
- the disruption is ultrasonic disruption.
- kits comprising the biological composition as described above.
- the present application provides a method for verifying the accuracy of a gene sequencing method.
- the biological composition according to claim 5 is sequenced according to the gene sequencing method.
- the calculation method of the error is: the positive level score of sequencing minus the difference in the mass ratio of the positive cell line/negative cell line, and then divided by the mass ratio of the positive cell line/negative cell line .
- the gene sequencing method is considered accurate.
- the present application provides a method for verifying the accuracy of the gene sequencing method, wherein the above-mentioned biological composition is sequenced according to the gene sequencing method, and the methylation level of the positive genome vector sequencing is compared with the positive genome vector theory. If the error of methylation level (97%) is less than 5%, the gene sequencing method is considered accurate.
- the calculation method of the error is: the difference between the methylation level of the positive genome vector sequencing minus the theoretical methylation level of the positive genome vector, and then divided by the theoretical methylation level of the positive genome vector. level.
- the gene sequencing method is considered accurate.
- the present application provides a method for verifying the accuracy of the gene sequencing method, wherein the above-mentioned biological composition is sequenced according to the gene sequencing method, and the methylation levels of negative genome vector sequencing are compared. If methylation If the level is less than 1%, the gene sequencing method is considered accurate.
- the gene sequencing method is considered accurate; preferably, if the methylation level is less than 0.1%, the gene sequencing method is considered accurate; more preferably, if If the methylation level is less than 0.02%, the gene sequencing method is considered accurate.
- Figure 1 shows a flow chart for the preparation of standards used for methylation detection in this application.
- Figure 2 shows this application taking SW48 as an example.
- the EZ-SW48 and real cfDNA samples treated with Atlantis dsDNase enzyme were used for library construction and sequencing using the WGBS method to detect the distribution of library fragments of the two types of samples.
- the upper half is the peak shape diagram of fragment evaluation using the PE LabChip fragment evaluator, and the lower half is the fragment distribution diagram evaluated by WGBS sequencing.
- Figure 3 shows the repeatability analysis chart comparing the three different enzyme digestion batches (Lot 1, Lot 2, Lot 3) of this application, from left to right: (1) Lot 1 As the horizontal axis, Lot 2 is used as the vertical axis; (2) Lot 1 is used as the horizontal axis, Lot 3 is used as the vertical axis; (3) Lot 2 is used as the horizontal axis, and Lot 3 is used as the vertical axis. Repeatability comparison results.
- Figure 4 shows 6 different cancer cell lines (H1975, SW48, HepG2, CaoV3, PANC-1, T.T), 4 healthy human cell lines (GM24385, GM12878, GM24631, NCI.BL1184) and 24 exemplified in this application.
- GM24385, GM12878, GM24631, NCI.BL1184 4 healthy human cell lines
- 24 exemplified in this application.
- WGBS heat map of WGBS analysis of a real cfDNA sample. The closer the color is to the right half of the color card in the upper left corner (transitioning from light to dark), the higher the detected methylation level.
- Figure 5 shows that in this application, taking SW48 as an example, it was mixed with EZ-GM24385 at different mass ratios (100% SW48, 20% SW48, 12.5% SW48, and pure EZ-GM24385) for sequencing.
- the methylation level results chart in which dark histograms indicate negative methylation results and light histograms indicate positive methylation results. Shown in brackets below each mass ratio on the left are the actual measured methylation levels of the methylation standards prepared according to the method of the present application, displayed as scores.
- next-generation sequencing generally refer to the second-generation high-throughput sequencing technology and higher-throughput sequencing methods developed thereafter.
- Next-generation sequencing platforms include but are not limited to existing sequencing platforms such as Illumina. With the continuous development of sequencing technology, those skilled in the art can understand that other sequencing methods and devices can also be used for this method. For example, two Next-generation gene sequencing can have the advantages of high sensitivity, high throughput, high sequencing depth, or low cost.
- Massively Parallel Signature Sequencing Massively Parallel Sequencing
- MPSS Massively Parallel Sequencing Signature Sequencing
- Polony Sequencing 454 pyro sequencing
- Illumina (Solexa) sequencing Illumina (Solexa) sequencing
- Ion semi conductor sequencing DNA nano-ball sequencing
- Complete Genomics' DNA nanoarray and combined probe anchored ligation sequencing method etc.
- the second-generation gene sequencing can make it possible to conduct a detailed and comprehensive analysis of the transcriptome and genome of a species, so it is also called deep sequencing (deep sequencing).
- deep sequencing deep sequencing
- the method of this application can also be applied to first-generation gene sequencing, second-generation gene sequencing, third-generation gene sequencing or single molecule sequencing (SMS).
- SMS single molecule sequencing
- sample to be tested generally refers to the sample to be tested. For example, it can be detected whether one or more gene regions on the sample to be tested are modified.
- region of complementarity generally refers to a region of complementarity compared to a reference nucleotide sequence.
- complementary nucleic acids can be nucleic acid molecules that optionally have opposite orientations.
- complementary may refer to having the following complementary associations: guanine and cytosine; adenine and thymine; adenine and uracil.
- the term "modification state” generally refers to the modification state possessed by the gene fragment, nucleotide or base thereof in this application.
- the modification state in this application may refer to the modification state of cytosine.
- a gene fragment with a modified state of the present application may have altered gene expression activity.
- the modification state in this application may refer to the methylation modification of a base.
- the modification state in this application may refer to the covalent binding of a methyl group at the 5' carbon position of cytosine in the CpG region of genomic DNA, which may become 5-methylcytosine (5mC), for example.
- the modification state may refer to the presence or absence of 5-methylcytosine ("5-mCyt") within the DNA sequence.
- methylation generally refers to the methylation state possessed by gene fragments, nucleotides or bases thereof in this application.
- the DNA segment in which the gene in this application is located may have methylation on one or more strands.
- the DNA fragment in which the gene in this application is located may have methylation at one site or multiple sites.
- the term "transformation” generally refers to the conversion of one or more structures into another structure.
- the transformations of the present application may be specific.
- cytosine without methylation modification can be transformed into other structures (such as uracil) after transformation, and cytosine with methylation modification can remain basically unchanged after transformation.
- cytosine without methylation modification can be cleaved after conversion, and cytosine with methylation modification can remain basically unchanged after conversion.
- bisulfite or “bisulfite” generally refers to a reagent that can distinguish between regions of DNA that have a modified state and those that do not.
- bisulfite may include bisulfite, or analogs thereof, or combinations thereof.
- bisulfite can deaminate the amino group of unmodified cytosine, distinguishing it from modified cytosine.
- analog generally refers to substances with similar structure and/or function.
- analogs of bisulfite may have a similar structure to bisulfite.
- a bisulfite analog may refer to a reagent that can also differentiate between regions of DNA that have a modified state and those that do not.
- the term "about” generally refers to a variation within the range of 0.5% to 10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
- This application provides a standard for methylation detection and a preparation method thereof.
- the method uses Atlantis dsDNase or Micrococcal Nuclease fragmentation enzyme to digest cell lines to provide a cell line standard with a peak shape similar to that of blood.
- Atlantis dsDNase or Micrococcal Nuclease fragmentation enzyme to digest cell lines to provide a cell line standard with a peak shape similar to that of blood.
- tissue traceability can be achieved at the same time.
- adding a certain proportion of fully methylated pUC19 and unmethylated Lambda genome combination or unmethylated pUC19 and fully methylated Lambda genome combination can evaluate the detection accuracy.
- the methylation standard includes: a healthy cell line as a negative standard, a cancer cell line as a positive standard, a negative minigenome vector, and a positive minigenome vector.
- the healthy cell line can be selected from one or more of the following: human B lymphocytes (for example: GM24385, GM12878, GM24631, NCI.BL1184).
- the cancer cell line can be selected from one or more of the following: human colon cancer cell line (for example: SW48), human lung adenocarcinoma cell line (for example: H1975), human liver cancer cell line (for example: HepG2) , human esophageal cancer cell line (for example: T.T), human ovarian cancer cell line (for example: CaoV3), human pancreatic cancer cell line (for example: PANC-1, PSN-1).
- the negative small genome vector includes: unmethylated Lambda DNA (for example, isolated from infected E. coli GM119 strain, which lacks dam and dcm methylase activities) or unmethylated pUC19.
- the positive small genome vector includes: pUC19 DNA or LambdaDNA treated with M.SssI methyltransferase (for example: pUC19 is a commonly used cloning vector for expressing Amp resistance, modified from the pBR322 cloning vector).
- the methylation standard of this application can be used to verify the accuracy assessment of various methylation detection processes.
- the methylation standard of this application can be used as a reference standard together with the sample to be tested to conduct any complete methylation test. methylation detection process, so as to judge the reliability of the real sample detection results based on the detection results of the methylation standard.
- the methylation detection can be applied to the detection of tumor genes, and more preferably can be applied to early detection or early screening of cancers.
- the cancers include: brain cancer, lung cancer, skin cancer, nasopharyngeal cancer, throat cancer, Liver cancer, bone cancer, lymphoma, pancreatic cancer, skin cancer, bowel cancer, rectal cancer, thyroid cancer, bladder cancer, kidney cancer, oral cancer, stomach cancer, solid tumors, ovarian cancer, esophageal cancer, gallbladder cancer, bile duct cancer, breast Cancer, cervical cancer, uterine cancer, prostate cancer, head and neck cancer, sarcoma, thoracic malignant tumors (except lung), melanoma, and testicular cancer, leukemia.
- methylation negative standards there are many problems with existing methylation standards in this field.
- the disadvantage of unmethylated Lambda DNA is that it does not come from the human genome, and artificial knockout of methyltransferase
- the disadvantage of cell line DNA is that the methylation signal is not completely removed, there is still a high CpG signal background, and it is expensive.
- Another example is methylation positive standards.
- the shortcoming of standards obtained by treating pUC19 or human genomic DNA with MsssI (methylase) is that tissue traceability information cannot be achieved.
- This application provides a reference material with a small amount of exogenous and stable methylation levels mixed into a human standard material. This composition is used in the application of standard materials and can be used in the evaluation of various methylation detection methods. In the process, excellent detection accuracy assessment and standards suitable for tissue traceability are achieved.
- the overall steps of the preparation method of the standard substance may include:
- Healthy cell lines here include but are not Limited to immortalized B lymphocyte cell lines (for example: GM24385, GM12878, GM24631, NCI.BL1184), cancer cell lines include but are not limited to immortalized cancer cell lines (for example: SW48, H1975, HepG2, T.T, CaoV3, PANC-1, PSN-1). These cell line products are examples only and other suitable cancer cell lines may be used.
- the human genome is selected from pure commercial cell lines.
- Cancer type Representative cell line names healthy person GM24385, GM12878, GM24631, NCI.BL1184 lung cancer H1975 colon cancer SW48 liver cancer HepG2 Esophageal cancer T.T ovarian cancer CaoV3 pancreatic cancer PANC-1,PSN-1 gallbladder cancer NOZ,GBC-SD
- fully unmethylated pUC19 can also be mixed with the Lambda genome size treated by M.SssI methyltransferase at a mass ratio of 1:19 and fragmented.
- the mass ratio of the positive cell line to pUC19 is 1:1 ⁇ 10 -4 to 1:1 ⁇ 10 -6 , preferably 1:1 ⁇ 10 -5 . The same effect can be achieved.
- the applicant's ELSA-seq TM library construction method (see Chinese Invention Patent Application Publication Document CN110892097A) was used to complete the library construction, and the methylation signal was detected on the enzyme-digested cell line DNA, and evaluated by evaluating correlation and other indicators. Method reproducibility. Shown below are the methylation signal detection results of three batches (Lot 1, Lot 2, Lot 3) of the EZ-GM24385 cell line and fragments digested with reagents. It can be seen from the results that the signal correlation between each batch reaches more than 0.999, the repeatability is good, and the preparation process is relatively stable.
- WGBS detection was performed on the extracted healthy/cancer cell lines and healthy human cfDNA.
- the obtained data were subjected to bioinformatics cluster analysis. The higher the methylation level of the detection results, the closer the color is to the upper left corner of the color card on the right. Half part (transition from light to dark).
- the methylation signal clustering results of healthy human cell lines GM24385, GM12878, NCI.BL1184, GM24631
- healthy human cfDNA Plasma.1-24
- NFDNA-1, NFDNA-2, and NFDNA-3 respectively represent nucleosome-cleaved DNA derived from GM24385, GM12878, and GM24631 (for example, NFDNA-1 has about 32% EGFR allelic mutations, which can be fixed by Proportional doping ratio is used to evaluate the detection accuracy of VAF).
- Plasma.1, Plasma.2...Plasma.24 respectively represent healthy human cfDNA from different sources.
- the mass ratios of SW48 obtained by gradient mixing nucleosome-digested SW48 and NFDNA-1 are 100%, 12.5%, 5%, 1%, 0.5%, 0.10%, respectively. 0.05%, 0% methylation standard, respectively using ddPCR and the applicant's HS-UMI (the HS sequencing method can be found in the applicant's Chinese invention patent authorization announcement text CN106835291B, on this basis, combined with UMI technology, namely HS-UMI Method) Sequencing method (deep sequencing >100,000x) was used to detect the allele mutation frequency (VAF) of EGFR:p.G719S for each methylation standard.
- VAF allele mutation frequency
- VAF1 and VAF2 the measured VAF were respectively Denote them as VAF1 and VAF2, and find the average VAF of the two repetitions.
- the specific test results are shown in the table below.
- the VAF values measured by ddPCR and HS-UMI are close to the expected VAF of each methylation standard, indicating that the methylation standard gradient prepared by this method has good accuracy.
- VAF is the theoretical allele mutation frequency of the EGFR:p.G719S site corresponding to different methylation standards. NA means not detected.
- the numbers in the brackets on the left represent the actual measured methylation level values. It can be seen that the actual measured methylation levels (95.9%, 23.8%, 10.1%, 0.5%) are different from those measured by mass.
- the preset methylation levels (100%, 20%, 12.5%, 0%) of the prepared standards are basically consistent, indicating that the standards can be used for performance evaluation of cfDNA-based methylation detection methods.
- Samples 1 to 4 were prepared as described in Example 1, and the applicant's ELSA-seq TM library construction method (see Chinese invention patent application publication CN110892097A) was used to perform library construction and sequencing to detect the methylation level of the methylation standard. See the table below, CpG.methyl.hyper refers to the methylation level of pUC19CpG site, CpG.methyl.hypo refers to the methylation level of Lambda CpG site, when performing normal methylation detection (sample 3, sample 4) , CpG.methyl.hyper signal >95% or more.
- the methylation signal of pUC19 is abnormally low; this abnormality can be caused by inappropriate artificial selection of experiments. Conditions arise, for example: the reaction temperature exceeds the standard temperature during the bisulfite process, or the reaction time is longer than the reaction time, or the methylation reaction is abnormal due to insufficient TET2 dosage or insufficient reaction time during the Methyl-seq enzymatic conversion process, etc. . Therefore, the Lambda and pUC19 genomes can be used as internal references for the accuracy of the detection process to monitor the accuracy of the methylation conversion process.
- composition provided in this application is used as a standard and can be used in the entire methylation sequencing process to determine whether experimental conditions meet the requirements of quality control.
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Abstract
Provided are a biological composition, a method for preparing same, and use thereof. The biological composition comprises at least one positive cell line as a positive standard, at least one negative cell line as a negative standard, at least one positive genome vector as a positive reference, and at least one negative genome vector as a negative reference. The biological composition can be used as a standard capable of achieving different gradient positive signals and used to evaluate the accuracy of a methylation detection method.
Description
本申请涉及生物医学领域，具体的涉及一种生物组合物及其制备方法，还包括该生物组合物用于甲基化检测的标准品的应用。The present application relates to the field of biomedicine, specifically to a biological composition and its preparation method, and also includes the application of the biological composition as a standard for methylation detection.
循环DNA(cfDNA，cell free DNA)是一种存在于血液中的单链或双链形式的DNA分子，其片段长度大约144-166bp。DNA甲基化作为一种常见的表观遗传学修饰方式，在基因表达调控中发挥着重要作用，并与肿瘤的发生发展有着密切的联系，DNA甲基化具有位点广泛，灵敏度高，可进行组织溯源等优势，结合液体活检技术创伤小，取样方便等特点，血液ctDNA甲基化检测已成为当下最具潜力的早筛技术路径。Circulating DNA (cfDNA, cell free DNA) is a single-stranded or double-stranded DNA molecule that exists in the blood, and its fragment length is approximately 144-166bp. As a common epigenetic modification, DNA methylation plays an important role in the regulation of gene expression and is closely related to the occurrence and development of tumors. DNA methylation has a wide range of sites, high sensitivity, and can With the advantages of tissue traceability, combined with the characteristics of liquid biopsy technology such as less trauma and convenient sampling, blood ctDNA methylation detection has become the most potential early screening technology path at present.
DNA甲基化(methylation)是一种表观遗传修饰，它是由DNA甲基转移酶(DNA methyl-transferase，DNMT)催化S-腺苷甲硫氨酸(S-adenosylmethionine，SAM)作为甲基供体，将DNA的CG两个核苷酸的胞嘧啶被选择性地添加甲基，主要形成5-甲基胞嘧啶(5-mC)(常见于基因的5'-CG-3'序列)和少量的N6-甲基嘌呤(N6-mA)及7-甲基鸟嘌呤(7-mG)。DNA methylation (methylation) is an epigenetic modification that is catalyzed by DNA methyltransferase (DNMT) to convert S-adenosylmethionine (SAM) as a methyl group. Donor, the cytosine of the two nucleotides CG of DNA is selectively added with methyl groups, mainly forming 5-methylcytosine (5-mC) (common in the 5'-CG-3' sequence of genes) and small amounts of N6-methylpurine (N6-mA) and 7-methylguanine (7-mG).
DNA甲基化在基因表达调控中起着重要的作用。异常的DNA甲基化标记在多种疾病发生发展中过程中都被报道过，包括癌症。DNA甲基化测序作为一种高分辨率，高通量的技术，其作用在癌症早期筛查，诊断，以及监控的作用越来越被认识。DNA methylation plays an important role in the regulation of gene expression. Abnormal DNA methylation marks have been reported during the development and progression of various diseases, including cancer. As a high-resolution, high-throughput technology, DNA methylation sequencing is increasingly recognized for its role in early cancer screening, diagnosis, and monitoring.
全基因组重亚硫酸盐测序(WGBS，Whole Genome Bisulfite Sequencing)是甲基化测序的金标准，但是因为处理过程中对DNA的严重破坏和过高的测序成本，成为临床应用的困难。更重要的是，人类基因组的大部分区域在癌症发生发展过程中并不活跃，癌症相关的变异往往集中在某些特定区域，如CpG岛(CpG island)。CG二核苷酸是最主要的甲基化位点，它在基因组中呈不均匀分布，存在高甲基化、低甲基化和非甲基化的区域，在哺乳动物中mC约占C总量的2-7％。Whole Genome Bisulfite Sequencing (WGBS) is the gold standard for methylation sequencing, but it has become difficult to apply in clinical applications due to the severe damage to DNA during processing and the high cost of sequencing. More importantly, most regions of the human genome are not active in the development of cancer, and cancer-related mutations are often concentrated in certain specific regions, such as CpG islands. CG dinucleotide is the most important methylation site. It is unevenly distributed in the genome. There are hypermethylated, hypomethylated and non-methylated regions. In mammals, mC accounts for about the total amount of C. 2-7%.
CpG岛在基因组中大量存在，通过大规模平行核酸测序(也称为“高通量测序”或者“下一代测序”(NGS))可以极大地辅助这些检测和分析，使得通过甲基化信号预测癌症的发生以及发生的部位成为可能。CpG islands are abundant in the genome, and these detection and analysis can be greatly assisted by massively parallel nucleic acid sequencing (also known as "high-throughput sequencing" or "next generation sequencing" (NGS)), allowing prediction of methylation signals The occurrence of cancer and where it occurs becomes possible.
目前，用于检测甲基化信号的技术包括:焦磷酸测序技术(pyrosequencing)，甲基化特异 PCR(MSP)，高通量测序(NGS)的方法等。基于ctDNA甲基化信号检测的技术日渐成熟。对于上述方法的试剂开发及性能验证、检测过程质量控制以及不同检测方法和平台结果一致性的比较，需要大量稳定的参考品作为标准。因此，甲基化标准品的建立显得尤为重要。Currently, technologies used to detect methylation signals include: pyrosequencing, methylation-specific PCR (MSP), high-throughput sequencing (NGS) methods, etc. Technology based on ctDNA methylation signal detection is becoming increasingly mature. For the reagent development and performance verification of the above methods, the quality control of the detection process, and the comparison of the consistency of results of different detection methods and platforms, a large number of stable reference materials are needed as standards. Therefore, the establishment of methylation standards is particularly important.
目前商业化的甲基化阴性标准品主要有以下几种形式：1)未甲基化的LambdaDNA样本(从感染的大肠杆菌GM119菌株中分离得到的，该菌株缺乏dam及dcm甲基化酶活性)，优点是基因组较小，CpG位点信号接近于0，缺点是非来自于人基因组；2)人工敲除甲基化转移酶的细胞系DNA，此种CpG位点的甲基化信号去除不干净，仍存在较高的CpG信号背景，且价格昂贵；3)通过全基因组扩增将任何样本CpG位点的甲基化信号变为0。Currently, commercially available methylation negative standards mainly come in the following forms: 1) Unmethylated Lambda DNA sample (isolated from infected E. coli GM119 strain, which lacks dam and dcm methylase activities ), the advantage is that the genome is small and the CpG site signal is close to 0, but the disadvantage is that it does not come from the human genome; 2) The cell line DNA with artificial knockout of methyltransferase cannot remove the methylation signal of this CpG site Clean, there is still a high CpG signal background, and it is expensive; 3) Change the methylation signal of any sample CpG site to 0 through whole-genome amplification.
目前商业化的甲基化阳性标准品主要是采用1)癌症细胞系，癌症细胞系中可以找到甲基化位点接近于100％的阳性位点；2)MsssI(甲基化酶)处理puc19/DNA或者人的基因组DNA来获得，后一种方法处理获得的DNA每个CpG位点的甲基化水平高达97％，但是无法实现组织溯源的信息。Currently, commercialized methylation positive standards mainly use 1) cancer cell lines, in which methylation sites close to 100% of positive sites can be found; 2) MsssI (methylase) treats puc19 /DNA or human genomic DNA. The methylation level of each CpG site of the DNA obtained by the latter method is as high as 97%, but it cannot achieve tissue traceability information.
当前大部分的参考标准品，通常采用超声打断获得酶切为片段化的DNA，超声打断会对DNA造成损伤，产生的DNA片段的长度分布与真实cfDNA(细胞游离DNA；cell free DNA)/ctDNA存在较大差异。因此，需要一种稳定的与真实cfDNA片段较为接近的甲基化质控标准品制备方法。Most of the current reference standards usually use ultrasonic fragmentation to obtain enzyme-digested DNA into fragments. Ultrasonic fragmentation will cause damage to the DNA, and the length distribution of the generated DNA fragments is different from that of real cfDNA (cell free DNA). /ctDNA has large differences. Therefore, a stable method for preparing methylation quality control standards that is close to real cfDNA fragments is needed.
发明内容Contents of the invention
本申请提供了一种用于甲基化检测标准品及其制备方法，所述方法通过Atlantis dsDNase(一种限制性内切酶)或者Micrococcal Nuclease(微球菌核酸酶)片段化酶酶切细胞系提供了一种类似于血液峰形的细胞系标准品，实现甲基化血液检测标准品的需求，通过梯度掺比癌症来源的细胞系DNA和健康细胞系DNA，可以获得不同梯度肿瘤信号的标准品，同时可以实现组织溯源。除外，在标准品中添加一定比例的全甲基化的pUC19及未甲基化的Lambda基因组组合或者非甲基化的pUC19及全甲基化的Lambda基因组组合，可以进行检测准确性的评估。This application provides a standard substance for methylation detection and a preparation method thereof. The method uses Atlantis dsDNase (a restriction enzyme) or Micrococcal Nuclease (Micrococcal Nuclease) fragmentation enzyme to digest cell lines. A cell line standard with a peak shape similar to that of blood is provided to meet the demand for methylation blood detection standards. By gradient mixing of cancer-derived cell line DNA and healthy cell line DNA, different gradient tumor signal standards can be obtained products, and at the same time, organizational traceability can be achieved. In addition, adding a certain proportion of fully methylated pUC19 and unmethylated Lambda genome combination or unmethylated pUC19 and fully methylated Lambda genome combination to the standard can evaluate the detection accuracy.
本申请一方面提供了一种生物组合物，包括：至少一种作为阳性标准品的阳性细胞系，至少一种作为阴性标准品的阴性细胞系，至少一种作为阳性参考品的阳性基因组载体，以及至少一种作为阴性参考品的阴性基因组载体。In one aspect, the present application provides a biological composition, including: at least one positive cell line as a positive standard, at least one negative cell line as a negative standard, and at least one positive genome vector as a positive reference, and at least one negative genomic vector serving as a negative reference.
作为一种优选的实施方式，所述阳性基因组载体的碱基数小于阳性细胞系的碱基数；所 述阴性基因组载体的碱基数小于阴性细胞系的碱基数。As a preferred embodiment, the number of bases of the positive genome vector is less than the number of bases of the positive cell line; the number of bases of the negative genome vector is less than the number of bases of the negative cell line.
作为一种优选的实施方式，所述阳性基因组载体的碱基数小于阳性细胞系的碱基数的1/100，优选地，所述阳性基因组载体的碱基数小于阳性细胞系的碱基数的1/1000，更优选地，所述阳性基因组载体的碱基数小于阳性细胞系的碱基数的1/10000；所述阴性基因组载体的碱基数小于阴性细胞系的碱基数的1/100，优选地，所述阴性基因组载体的碱基数小于阴性细胞系的碱基数的1/1000，更优选地，所述阴性基因组载体的碱基数小于阴性细胞系的碱基数的1/10000；As a preferred embodiment, the number of bases of the positive genome vector is less than 1/100 of the number of bases of the positive cell line. Preferably, the number of bases of the positive genome vector is less than the number of bases of the positive cell line. 1/1000, more preferably, the number of bases of the positive genome vector is less than 1/10000 of the number of bases of the positive cell line; the number of bases of the negative genome vector is less than 1/1000 of the number of bases of the negative cell line /100, preferably, the number of bases of the negative genome vector is less than 1/1000 of the number of bases of the negative cell line, more preferably, the number of bases of the negative genome vector is less than 1/1000 of the number of bases of the negative cell line 1/10000;
作为一种优选的实施方式，所述阴性基因组载体的碱基数为阴性细胞系的碱基数的大约1/1000000～1/60000；或者所述阳性基因组载体的碱基数为阳性细胞系的碱基数的大约1/1000000～1/60000。例如，所述阴性细胞系与所述阴性基因组载体的质量比为1:2×10
-3至1:1×10
6，优选为1:2×10
-4至1:1×10
-5；所述阳性细胞系与所述阳性基因组载体的质量比为1:2×10
-3至1:1×10
-6，优选为1:2×10
-4至1:1×10
-5。
As a preferred embodiment, the number of bases of the negative genome vector is approximately 1/1000000 to 1/60000 of the number of bases of the negative cell line; or the number of bases of the positive genome vector is approximately 1/1000000 to 1/60000 of the number of bases of the positive cell line. Approximately 1/1,000,000 to 1/60,000 of the number of bases. For example, the mass ratio of the negative cell line to the negative genome vector is 1:2×10 -3 to 1:1×10 6 , preferably 1:2×10 -4 to 1:1×10 -5 ; The mass ratio of the positive cell line to the positive genome vector is 1:2×10 -3 to 1:1×10 -6 , preferably 1:2×10 -4 to 1:1×10 -5 .
作为一种优选的实施方式，所述阳性细胞系与阴性细胞系的质量比为0.1:99.9至25:75之间，优选为0.5:99.5至12.5:87.5之间，更优选为1:99至5:95之间或者:99.9:0.1至75:25之间，优选为99.5:0.5至87.5:12.5之间，更优选为99:1至95:5之间。As a preferred embodiment, the mass ratio of the positive cell line to the negative cell line is between 0.1:99.9 and 25:75, preferably between 0.5:99.5 and 12.5:87.5, and more preferably between 1:99 and 25:75. Between 5:95 or: between 99.9:0.1 and 75:25, preferably between 99.5:0.5 and 87.5:12.5, more preferably between 99:1 and 95:5.
作为一种优选的实施方式，所述阳性基因组载体与阴性基因组载体的质量比为1:99至25:75之间或者99:1至95:5之间，优选为大约1:19或者19:1。As a preferred embodiment, the mass ratio of the positive genome vector to the negative genome vector is between 1:99 and 25:75 or between 99:1 and 95:5, preferably about 1:19 or 19: 1.
作为一种优选的实施方式，所述阳性细胞系和所述阴性细胞系都经过片段化处理，平均片段大小为约140-160bp。例如，所述片段化处理可以包含生物酶酶切片段化和/或物理打断片段化。例如，所述酶切可以是通过Atlantis dsDNase酶或者Micrococcal Nuclease片段化基因组DNA。例如，例如，所述酶切可以是通过超声打断。例如，阳性细胞系和所述阴性细胞系所述酶切可以是通过Atlantis dsDNase酶或者Micrococcal Nuclease片段化基因组DNA。As a preferred embodiment, both the positive cell line and the negative cell line undergo fragmentation processing, and the average fragment size is about 140-160 bp. For example, the fragmentation treatment may include enzymatic fragmentation and/or physical fragmentation. For example, the enzyme cleavage can be through Atlantis dsDNase enzyme or Micrococcal Nuclease fragmented genomic DNA. For example, the enzyme cleavage can be interrupted by ultrasound. For example, the enzyme digestion of the positive cell line and the negative cell line can be through Atlantis dsDNase enzyme or Micrococcal Nuclease fragmented genomic DNA.
作为一种优选的实施方式，所述阳性细胞系选自如下细胞系的组合：人结直肠癌细胞系(例如：SW48)，人肺腺癌细胞系(例如：H1975)，人肝癌细胞系(例如：HepG2)，人食管癌细胞系(例如：T.T)，人卵巢癌细胞系(例如：CaoV3)，人胰腺癌细胞系(例如：PANC-1，PSN-1)。As a preferred embodiment, the positive cell line is selected from a combination of the following cell lines: human colorectal cancer cell line (for example: SW48), human lung adenocarcinoma cell line (for example: H1975), human liver cancer cell line (for example: H1975) For example: HepG2), human esophageal cancer cell line (for example: T.T), human ovarian cancer cell line (for example: CaoV3), human pancreatic cancer cell line (for example: PANC-1, PSN-1).
作为一种优选的实施方式，只包含一种阳性细胞系。As a preferred embodiment, only one positive cell line is included.
作为一种优选的实施方式，所述阴性细胞系选自如下细胞系的组合：人B淋巴细胞(例如：GM24385、GM12878、GM24631、NCI.BL1184)或者人正常细胞系(例如：Beads-2B及 其他人正常细胞系)。As a preferred embodiment, the negative cell line is selected from a combination of the following cell lines: human B lymphocytes (for example: GM24385, GM12878, GM24631, NCI.BL1184) or human normal cell lines (for example: Beads-2B and other human normal cell lines).
作为一种优选的实施方式，只包含一种阴性细胞系。As a preferred embodiment, only one negative cell line is included.
作为一种优选的实施方式，所述阳性基因组载体为经过甲基化转移酶处理的pUC19 DNA和/或经过甲基化转移酶处理的Lambda，所述甲基化转移酶优选为M.Sssl。As a preferred embodiment, the positive genome vector is pUC19 DNA treated with methyltransferase and/or Lambda treated with methyltransferase, and the methyltransferase is preferably M.Sssl.
作为一种优选的实施方式，所述阴性基因组载体为未甲基化的Lambda和/或未甲基化的pUC19 DNA。As a preferred embodiment, the negative genome vector is unmethylated Lambda and/or unmethylated pUC19 DNA.
本申请另一方面提供了一种上述生物组合物的应用，所述生物组合物用于评估癌症基因检测的准确性和组织溯源的标准品。Another aspect of the present application provides an application of the above biological composition, which is used to evaluate the accuracy of cancer gene detection and standards for tissue traceability.
作为一种优选的实施方式，所述生物组合物用于评估癌症基因甲基化检测的准确性和组织溯源的标准品。As a preferred embodiment, the biological composition is used to evaluate the accuracy of cancer gene methylation detection and as a standard for tissue traceability.
作为一种优选的实施方式，所述癌症包括：脑癌、肺癌、皮肤癌、鼻咽癌、咽喉癌、肝癌、骨癌、淋巴瘤、胰腺癌、皮肤癌、肠癌、直肠癌、甲状腺癌、膀胱癌、肾癌、口腔癌、胃癌、实体瘤、卵巢癌、食管癌、胆囊癌、胆道癌、乳腺癌、***、子宫癌、***癌、头颈癌、肉瘤、胸腔恶性肿瘤(除肺外)、黑色素瘤、和睾丸癌、白血病。As a preferred embodiment, the cancer includes: brain cancer, lung cancer, skin cancer, nasopharyngeal cancer, throat cancer, liver cancer, bone cancer, lymphoma, pancreatic cancer, skin cancer, intestinal cancer, rectal cancer, and thyroid cancer , bladder cancer, kidney cancer, oral cancer, gastric cancer, solid tumors, ovarian cancer, esophageal cancer, gallbladder cancer, bile duct cancer, breast cancer, cervical cancer, uterine cancer, prostate cancer, head and neck cancer, sarcoma, thoracic malignant tumors (except lung (External), melanoma, testicular cancer, and leukemia.
本申请另一方面提供了一种制备生物组合物的方法，包括如下步骤：Another aspect of the application provides a method for preparing a biological composition, including the following steps:
(1)获得至少一种作为阳性标准品的阳性细胞系和至少一种作为阴性标准品的阴性细胞系；(1) Obtain at least one positive cell line as a positive standard and at least one negative cell line as a negative standard;
(2)将所述阳性细胞系和阴性细胞系进行细胞核分离，随后通过酶切将所述阳性细胞系和阴性细胞系转化为阳性片段化DNA和阴性片段化DNA；(2) Separating the nuclei of the positive cell lines and negative cell lines, and then converting the positive cell lines and negative cell lines into positive fragmented DNA and negative fragmented DNA through enzyme digestion;
(3)将所述阳性片段化DNA和所述阴性片段化DNA混合；(3) Mix the positive fragmented DNA and the negative fragmented DNA;
(4)获得至少一种作为阳性参考品的阳性基因组载体和至少一种作为阴性参考品的阴性基因组载体，将所述阳性基因组载体和阴性基因组载体混合，并进行打断；(4) Obtain at least one positive genome vector as a positive reference product and at least one negative genome vector as a negative reference product, mix the positive genome vector and the negative genome vector, and interrupt;
(5)将上述步骤(3)和步骤(4)的产物混合。(5) Mix the products of the above steps (3) and (4).
作为一种优选的实施方式，所述酶切是通过AtlantisdsDNase酶或者Micrococcal Nuclease片段化基因组DNA。As a preferred embodiment, the enzyme digestion is performed by Atlantis DNase enzyme or Micrococcal Nuclease to fragment genomic DNA.
作为一种优选的实施方式，还包括：在步骤(2)的酶切之后，进行片段筛选去除gDNA大片段。As a preferred embodiment, it also includes: after the enzyme digestion in step (2), fragment screening is performed to remove large gDNA fragments.
作为一种优选的实施方式，所述阳性细胞系选自如下细胞系的组合：人结肠癌细胞(例如：SW48)，人肺腺癌细胞(例如：H1975)，人肝癌细胞(例如：HepG2)，人食管癌细胞(例 如：T.T)，人卵巢癌细胞(例如：CaoV3)，人胰腺癌细胞(例如：PANC-1，PSN-1)。As a preferred embodiment, the positive cell line is selected from a combination of the following cell lines: human colon cancer cells (for example: SW48), human lung adenocarcinoma cells (for example: H1975), human liver cancer cells (for example: HepG2) , human esophageal cancer cells (for example: T.T), human ovarian cancer cells (for example: CaoV3), human pancreatic cancer cells (for example: PANC-1, PSN-1).
作为一种优选的实施方式，只包含一种阳性细胞系。As a preferred embodiment, only one positive cell line is included.
作为一种优选的实施方式，所述阴性细胞系选自如下细胞系的组合：人B淋巴细胞(例如：GM24385、GM12878、GM24631、NCI.BL1184)。As a preferred embodiment, the negative cell line is selected from a combination of the following cell lines: human B lymphocytes (for example: GM24385, GM12878, GM24631, NCI.BL1184).
作为一种优选的实施方式，只包含一种阴性细胞系。As a preferred embodiment, only one negative cell line is included.
作为一种优选的实施方式，所述阳性基因组载体的碱基数小于阳性细胞系的碱基数；所述阴性基因组载体的碱基数小于阴性细胞系的碱基数。As a preferred embodiment, the number of bases of the positive genome vector is less than the number of bases of the positive cell line; the number of bases of the negative genome vector is less than the number of bases of the negative cell line.
作为一种优选的实施方式，所述阳性基因组载体的碱基数小于阳性细胞系的碱基数的1/100，优选地，所述阳性基因组载体的碱基数小于阳性细胞系的碱基数的1/1000，更优选地，所述阳性基因组载体的碱基数小于阳性细胞系的碱基数的1/10000；所述阴性基因组载体的碱基数小于阴性细胞系的碱基数的1/100，优选地，所述阴性基因组载体的碱基数小于阴性细胞系的碱基数的1/1000，更优选地，所述阴性基因组载体的碱基数小于阴性细胞系的碱基数的1/10000。As a preferred embodiment, the number of bases of the positive genome vector is less than 1/100 of the number of bases of the positive cell line. Preferably, the number of bases of the positive genome vector is less than the number of bases of the positive cell line. 1/1000, more preferably, the number of bases of the positive genome vector is less than 1/10000 of the number of bases of the positive cell line; the number of bases of the negative genome vector is less than 1/1000 of the number of bases of the negative cell line /100, preferably, the number of bases of the negative genome vector is less than 1/1000 of the number of bases of the negative cell line, more preferably, the number of bases of the negative genome vector is less than 1/1000 of the number of bases of the negative cell line 1/10000.
作为一种优选的实施方式，所述阴性基因组载体的碱基数为阴性细胞系的碱基数的大约1/1000000～1/60000；或者所述阳性基因组载体的碱基数为阳性细胞系的碱基数的大约1/1000000～1/60000。As a preferred embodiment, the number of bases of the negative genome vector is approximately 1/1000000 to 1/60000 of the number of bases of the negative cell line; or the number of bases of the positive genome vector is approximately 1/1000000 to 1/60000 of the number of bases of the positive cell line. Approximately 1/1,000,000 to 1/60,000 of the number of bases.
作为一种优选的实施方式，所述阳性细胞系与阴性细胞系的质量比为0.1:99.9至25:75之间，优选为0.5:99.5至12.5:87.5之间，更优选为1:99至5:95之间。As a preferred embodiment, the mass ratio of the positive cell line to the negative cell line is between 0.1:99.9 and 25:75, preferably between 0.5:99.5 and 12.5:87.5, and more preferably between 1:99 and 25:75. Between 5:95.
作为一种优选的实施方式，所述阳性基因组载体与阴性基因组载体的质量比为1:99至25:75之间或者99:1至95:5之间，优选为大约1:19或者19:1。As a preferred embodiment, the mass ratio of the positive genome vector to the negative genome vector is between 1:99 and 25:75 or between 99:1 and 95:5, preferably about 1:19 or 19: 1.
作为一种优选的实施方式，按照如下质量比例将步骤(3)和步骤(4)的产物混合：1:5×10
-4至1:1.5×10
-5，优选为1:2×10
-4。例如按照如下质量比例将步骤(3)和步骤(4)的产物混合：约1:2×10
-3、约1:1×10
-3、约1:5×10
-4、约1:1×10
-4、约1:5×10
-5、约1:2×10
-5、约1:1.5×10
-
5、或约1:1×10
-5。作为一种优选的实施方式，所述打断为超声打断。
As a preferred embodiment, the products of step (3) and step (4) are mixed according to the following mass ratio: 1:5×10 -4 to 1:1.5×10 -5 , preferably 1:2×10 - 4 . For example, mix the products of step (3) and step (4) according to the following mass ratio: about 1:2×10 -3 , about 1:1×10 -3 , about 1:5×10 -4 , about 1:1 ×10 -4 , about 1:5×10 -5 , about 1:2×10 -5 , about 1:1.5× 10 -5 , or about 1:1×10 -5 . As a preferred embodiment, the disruption is ultrasonic disruption.
本申请另一方面提供了一种试剂盒，包含如上所述的生物组合物。Another aspect of the present application provides a kit comprising the biological composition as described above.
本申请另一方面提供了一种验证基因测序方法准确性的方法，按照所述基因测序方法对如权利要求5所述的生物组合物进行测序，在生物组合物已验证掺比准确性情况下，比较某一区间测序的阳性水平与阳性细胞系/阴性细胞系的质量比的误差，若误差小于30％，则认为该基因测序方法准确。On the other hand, the present application provides a method for verifying the accuracy of a gene sequencing method. The biological composition according to claim 5 is sequenced according to the gene sequencing method. When the biological composition has verified the accuracy of the mixing ratio, , compare the error between the positivity level of sequencing in a certain interval and the mass ratio of positive cell lines/negative cell lines. If the error is less than 30%, the gene sequencing method is considered accurate.
作为一种优选的实施方式，所述误差的计算方法为：测序的阳性水平分数减去阳性细胞系/阴性细胞系的质量比的差值，再除以阳性细胞系/阴性细胞系的质量比。As a preferred embodiment, the calculation method of the error is: the positive level score of sequencing minus the difference in the mass ratio of the positive cell line/negative cell line, and then divided by the mass ratio of the positive cell line/negative cell line .
作为一种优选的实施方式，若误差小于25％，则认为该基因测序方法准确。As a preferred implementation, if the error is less than 25%, the gene sequencing method is considered accurate.
本申请另一方面提供了一种验证基因测序方法准确性的方法，其中，按照该基因测序方法对上述的生物组合物进行测序，并比较阳性基因组载体测序的甲基化水平与阳性基因组载体理论的甲基化水平(97％)的误差，若误差小于5％，则认为该基因测序方法准确。On the other hand, the present application provides a method for verifying the accuracy of the gene sequencing method, wherein the above-mentioned biological composition is sequenced according to the gene sequencing method, and the methylation level of the positive genome vector sequencing is compared with the positive genome vector theory. If the error of methylation level (97%) is less than 5%, the gene sequencing method is considered accurate.
作为一种优选的实施方式，所述误差的计算方法为：阳性基因组载体测序的甲基化水平减去阳性基因组载体理论的甲基化水平的差值，再除以阳性基因组载体理论的甲基化水平。As a preferred embodiment, the calculation method of the error is: the difference between the methylation level of the positive genome vector sequencing minus the theoretical methylation level of the positive genome vector, and then divided by the theoretical methylation level of the positive genome vector. level.
作为一种优选的实施方式，若误差小于3％，则认为该基因测序方法准确。As a preferred implementation, if the error is less than 3%, the gene sequencing method is considered accurate.
本申请另一方面提供了一种验证基因测序方法准确性的方法，其中，按照该基因测序方法对上述的生物组合物进行测序，并比较阴性基因组载体测序的甲基化水平，若甲基化水平小于1％，则认为该基因测序方法准确。On the other hand, the present application provides a method for verifying the accuracy of the gene sequencing method, wherein the above-mentioned biological composition is sequenced according to the gene sequencing method, and the methylation levels of negative genome vector sequencing are compared. If methylation If the level is less than 1%, the gene sequencing method is considered accurate.
作为一种优选的实施方式，若甲基化水平小于0.5％，则认为该基因测序方法准确；优选地，若甲基化水平小于0.1％，则认为该基因测序方法准确；更优选地，若甲基化水平小于0.02％，则认为该基因测序方法准确。As a preferred embodiment, if the methylation level is less than 0.5%, the gene sequencing method is considered accurate; preferably, if the methylation level is less than 0.1%, the gene sequencing method is considered accurate; more preferably, if If the methylation level is less than 0.02%, the gene sequencing method is considered accurate.
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：The specific features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates can be better understood by reference to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:
图1显示的是本申请用于甲基化检测的标准品的制备流程图。Figure 1 shows a flow chart for the preparation of standards used for methylation detection in this application.
图2显示的是本申请的以SW48为例，同时对经Atlantis dsDNase酶处理后的EZ-SW48和真实cfDNA样本使用WGBS方法进行建库测序，检测两种类型样本的文库片段分布情况。上半部是使用PE LabChip片段评估仪进行片段评估的峰形图，下半部是WGBS测序评估出的片段分布图。Figure 2 shows this application taking SW48 as an example. At the same time, the EZ-SW48 and real cfDNA samples treated with Atlantis dsDNase enzyme were used for library construction and sequencing using the WGBS method to detect the distribution of library fragments of the two types of samples. The upper half is the peak shape diagram of fragment evaluation using the PE LabChip fragment evaluator, and the lower half is the fragment distribution diagram evaluated by WGBS sequencing.
图3显示的是针对本申请的三个不同的酶切批次(Lot 1、Lot 2、Lot 3)两两之间对比的重复性分析图，从左至右依次是：(1)Lot 1作为横轴，Lot 2作为纵轴；(2)Lot 1作为横轴，Lot 3作为纵轴；(3)Lot 2作为横轴，Lot 3作为纵轴的重复性比对结果。Figure 3 shows the repeatability analysis chart comparing the three different enzyme digestion batches (Lot 1, Lot 2, Lot 3) of this application, from left to right: (1) Lot 1 As the horizontal axis, Lot 2 is used as the vertical axis; (2) Lot 1 is used as the horizontal axis, Lot 3 is used as the vertical axis; (3) Lot 2 is used as the horizontal axis, and Lot 3 is used as the vertical axis. Repeatability comparison results.
图4显示的是本申请示例的6种不同癌症细胞系(H1975，SW48，HepG2，CaoV3，PANC-1，T.T)，4种健康人细胞系(GM24385，GM12878，GM24631，NCI.BL1184)和24例真实cfDNA样本WGBS分析的热力图，其中颜色越接近左上角颜色卡的右半部分(由浅色向深色过渡)代表检测到的甲基化水平越高。Figure 4 shows 6 different cancer cell lines (H1975, SW48, HepG2, CaoV3, PANC-1, T.T), 4 healthy human cell lines (GM24385, GM12878, GM24631, NCI.BL1184) and 24 exemplified in this application. Here is an example of a heat map of WGBS analysis of a real cfDNA sample. The closer the color is to the right half of the color card in the upper left corner (transitioning from light to dark), the higher the detected methylation level.
图5显示的是本申请中，以SW48为例，与EZ-GM24385以不同质量比混合(100％SW48，20％SW48，12.5％SW48，和纯EZ-GM24385)进行测序的部分位点的甲基化水平结果图，其中深色柱状图表示甲基化阴性结果，浅色柱状图表示甲基化阳性结果。左侧各质量比下方的括号内显示的是根据本申请方法制备的甲基化标准品的实际测得的甲基化水平，以分数显示。Figure 5 shows that in this application, taking SW48 as an example, it was mixed with EZ-GM24385 at different mass ratios (100% SW48, 20% SW48, 12.5% SW48, and pure EZ-GM24385) for sequencing. The methylation level results chart, in which dark histograms indicate negative methylation results and light histograms indicate positive methylation results. Shown in brackets below each mass ratio on the left are the actual measured methylation levels of the methylation standards prepared according to the method of the present application, displayed as scores.
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。The implementation of the invention of the present application will be described below with specific examples. Those familiar with this technology can easily understand other advantages and effects of the invention of the present application from the content disclosed in this specification.
术语定义Definition of Terms
在本申请中，术语“二代基因测序(NGS)”、高通量测序”或“下一代测序”通常是指第二代高通量测序技术及之后发展的更高通量的测序方法。下一代测序平台包括但不限于已有的Illumina等测序平台。随着测序技术的不断发展，本领域技术人员能够理解的是还可以采用其他方法的测序方法和装置用于本方法。例如，二代基因测序可以具有高灵敏度、通量大、测序深度高、或低成本的优势。根据发展历史、影响力、测序原理和技术不同等，主要有以下几种：大规模平行签名测序(Massively Parallel Signature Sequencing，MPSS)、聚合酶克隆(Polony Sequencing)、454焦磷酸测序(454 pyro sequencing)、Illumina(Solexa)sequencing、离子半导体测序(Ion semi conductor sequencing)、DNA纳米球测序(DNA nano-ball sequencing)、Complete Genomics的DNA纳米阵列与组合探针锚定连接测序法等。所述二代基因测序可以使对一个物种的转录组和基因组进行细致全貌的分析成为可能，所以又被称为深度测序(deep sequencing)。例如，本申请的方法同样可以应用于一代基因测序、二代基因测序、三代基因测序或单分子测序(SMS)。In this application, the terms "second-generation gene sequencing (NGS)", high-throughput sequencing" or "next-generation sequencing" generally refer to the second-generation high-throughput sequencing technology and higher-throughput sequencing methods developed thereafter. Next-generation sequencing platforms include but are not limited to existing sequencing platforms such as Illumina. With the continuous development of sequencing technology, those skilled in the art can understand that other sequencing methods and devices can also be used for this method. For example, two Next-generation gene sequencing can have the advantages of high sensitivity, high throughput, high sequencing depth, or low cost. According to development history, influence, sequencing principles and technologies, there are mainly the following types: Massively Parallel Signature Sequencing (Massively Parallel Sequencing) Signature Sequencing (MPSS), Polony Sequencing, 454 pyro sequencing, Illumina (Solexa) sequencing, Ion semi conductor sequencing, DNA nano-ball sequencing ), Complete Genomics' DNA nanoarray and combined probe anchored ligation sequencing method, etc. The second-generation gene sequencing can make it possible to conduct a detailed and comprehensive analysis of the transcriptome and genome of a species, so it is also called deep sequencing (deep sequencing). For example, the method of this application can also be applied to first-generation gene sequencing, second-generation gene sequencing, third-generation gene sequencing or single molecule sequencing (SMS).
在本申请中，术语“待测样本”通常是指需要进行检测的样本。例如，可以检测待测样本上的一个或者多个基因区域是否存在有修饰状态。In this application, the term "sample to be tested" generally refers to the sample to be tested. For example, it can be detected whether one or more gene regions on the sample to be tested are modified.
在本申请中，术语“互补区域”通常是指与参考核苷酸序列相比具有互补的区域。例如，互补核酸可以为任选地具有相反方向的核酸分子。例如，所述互补可以是指具有下面的互补性关联：鸟嘌呤和胞嘧啶；腺嘌呤和胸腺嘧啶；腺嘌呤和尿嘧啶。In this application, the term "region of complementarity" generally refers to a region of complementarity compared to a reference nucleotide sequence. For example, complementary nucleic acids can be nucleic acid molecules that optionally have opposite orientations. For example, complementary may refer to having the following complementary associations: guanine and cytosine; adenine and thymine; adenine and uracil.
在本申请中，术语“修饰状态”通常是指本申请中基因片段、核苷酸或其碱基具有的修饰状态。例如，本申请中的修饰状态可以是指胞嘧啶的修饰状态。例如，本申请的具有修饰状态的基因片段可以具有改变的基因表达活性。例如，本申请的修饰状态可以是指碱基具有的甲基化修饰。例如，本申请的修饰状态可以是指在基因组DNA的CpG区域的胞嘧啶5'碳位共价结合一个甲基基团，例如可以成为5-甲基胞嘧啶(5mC)。例如，修饰状态可以是指DNA序列内存在或不存在5-甲基胞嘧啶(“5-mCyt”)。In this application, the term "modification state" generally refers to the modification state possessed by the gene fragment, nucleotide or base thereof in this application. For example, the modification state in this application may refer to the modification state of cytosine. For example, a gene fragment with a modified state of the present application may have altered gene expression activity. For example, the modification state in this application may refer to the methylation modification of a base. For example, the modification state in this application may refer to the covalent binding of a methyl group at the 5' carbon position of cytosine in the CpG region of genomic DNA, which may become 5-methylcytosine (5mC), for example. For example, the modification state may refer to the presence or absence of 5-methylcytosine ("5-mCyt") within the DNA sequence.
在本申请中，术语“甲基化”通常是指本申请中基因片段、核苷酸或其碱基具有的甲基化状态。例如，本申请中基因所在的DNA片段可以在一条链或多条链上具有甲基化。例如，本申请中基因所在的DNA片段可以在一个位点或多个位点上具有甲基化。In this application, the term "methylation" generally refers to the methylation state possessed by gene fragments, nucleotides or bases thereof in this application. For example, the DNA segment in which the gene in this application is located may have methylation on one or more strands. For example, the DNA fragment in which the gene in this application is located may have methylation at one site or multiple sites.
在本申请中，术语“转化”通常是指将一种或多种结构转变为另一种结构。例如，本申请的转化可以是具有特异性。例如，不具有甲基化修饰的胞嘧啶经过转化可以变为其它结构(例如尿嘧啶)，且具有甲基化修饰的胞嘧啶经过转化可以基本不发生变化。例如，不具有甲基化修饰的胞嘧啶经过转化可以被剪切，且具有甲基化修饰的胞嘧啶经过转化可以基本不发生变化。In this application, the term "transformation" generally refers to the conversion of one or more structures into another structure. For example, the transformations of the present application may be specific. For example, cytosine without methylation modification can be transformed into other structures (such as uracil) after transformation, and cytosine with methylation modification can remain basically unchanged after transformation. For example, cytosine without methylation modification can be cleaved after conversion, and cytosine with methylation modification can remain basically unchanged after conversion.
在本申请中，术语“重亚硫酸盐”，或称为“亚硫酸氢盐”通常是指一种可以区分具有修饰状态和不具有修饰状态的DNA区域的试剂。例如，重亚硫酸盐可以包括重亚硫酸盐、或其类似物或上述的组合。例如，重亚硫酸盐可以使未修饰的胞嘧啶的氨基脱氨基化，以使其与修饰的胞嘧啶区分。在本申请中，术语“类似物”通常是指具有类似结构和/或功能的物质。例如重亚硫酸盐的类似物可以与重亚硫酸盐具有类似的结构。例如，重亚硫酸盐的类似物可以是指一种同样可以区分具有修饰状态和不具有修饰状态的DNA区域的试剂。In this application, the term "bisulfite" or "bisulfite" generally refers to a reagent that can distinguish between regions of DNA that have a modified state and those that do not. For example, bisulfite may include bisulfite, or analogs thereof, or combinations thereof. For example, bisulfite can deaminate the amino group of unmodified cytosine, distinguishing it from modified cytosine. In this application, the term "analog" generally refers to substances with similar structure and/or function. For example, analogs of bisulfite may have a similar structure to bisulfite. For example, a bisulfite analog may refer to a reagent that can also differentiate between regions of DNA that have a modified state and those that do not.
在本申请中，术语“包含”通常是指包括明确指定的特征，但不排除其他要素。In this application, the term "comprising" generally means the inclusion of explicitly specified features, but not the exclusion of other elements.
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。In this application, the term "about" generally refers to a variation within the range of 0.5% to 10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
发明详述Detailed description of the invention
本申请提供了一种用于甲基化检测标准品及其制备方法，所述方法通过Atlantis dsDNase或者Micrococcal Nuclease片段化酶酶切细胞系提供了一种类似于血液峰形的细胞系标准品，实现甲基化血液检测标准品的需求，通过梯度掺比癌症来源的细胞系DNA和健康细胞系DNA，可以获得不同梯度肿瘤信号的标准品，同时可以实现组织溯源。除外，在标准品中添加一定比例的全甲基化的pUC19及未甲基化的Lambda基因组组合或者未甲基化的pUC19及全甲基化的Lambda基因组组合，可以进行检测准确性的评估。This application provides a standard for methylation detection and a preparation method thereof. The method uses Atlantis dsDNase or Micrococcal Nuclease fragmentation enzyme to digest cell lines to provide a cell line standard with a peak shape similar to that of blood. To meet the demand for methylation blood detection standards, by gradient mixing of cancer-derived cell line DNA and healthy cell line DNA, standards for different gradients of tumor signals can be obtained, and tissue traceability can be achieved at the same time. In addition, adding a certain proportion of fully methylated pUC19 and unmethylated Lambda genome combination or unmethylated pUC19 and fully methylated Lambda genome combination to the standard can evaluate the detection accuracy.
所述甲基化标准品包含：作为阴性标准品的健康细胞系、作为阳性标准品的癌症细胞系、阴性小基因组载体、阳性小基因组载体构成。其中，所述健康细胞系可以选自以下的一种或多种：人B淋巴细胞(例如：GM24385、GM12878、GM24631、NCI.BL1184)。其中，所述癌症细胞系可以选自以下的一种或多种：人结肠癌细胞系(例如：SW48)，人肺腺癌细胞系(例如：H1975)，人肝癌细胞系(例如：HepG2)，人食管癌细胞系(例如：T.T)，人卵巢癌细胞系(例如：CaoV3)，人胰腺癌细胞(例如：PANC-1，PSN-1)。所述阴性小基因组载体包括：未甲基化Lambda DNA(例如：从感染的大肠杆菌GM119菌株中分离得到的,该菌株缺乏dam及dcm甲基化酶活性)或者未甲基化的pUC19。所述阳性小基因组载体包括：通过M.SssI甲基化转移酶处理pUC19 DNA或者LambdaDNA(例如：pUC19是表达Amp抗性的常用克隆载体，由pBR322克隆载体修改而来)。The methylation standard includes: a healthy cell line as a negative standard, a cancer cell line as a positive standard, a negative minigenome vector, and a positive minigenome vector. Wherein, the healthy cell line can be selected from one or more of the following: human B lymphocytes (for example: GM24385, GM12878, GM24631, NCI.BL1184). Wherein, the cancer cell line can be selected from one or more of the following: human colon cancer cell line (for example: SW48), human lung adenocarcinoma cell line (for example: H1975), human liver cancer cell line (for example: HepG2) , human esophageal cancer cell line (for example: T.T), human ovarian cancer cell line (for example: CaoV3), human pancreatic cancer cell line (for example: PANC-1, PSN-1). The negative small genome vector includes: unmethylated Lambda DNA (for example, isolated from infected E. coli GM119 strain, which lacks dam and dcm methylase activities) or unmethylated pUC19. The positive small genome vector includes: pUC19 DNA or LambdaDNA treated with M.SssI methyltransferase (for example: pUC19 is a commonly used cloning vector for expressing Amp resistance, modified from the pBR322 cloning vector).
本申请的甲基化标准品，可用于验证各种甲基化检测流程的准确性评估，例如：随同待测样品一起将本申请的甲基化标准品作为参考标准品进行任一完整的甲基化检测流程，从而根据对该甲基化标准品的检测结果来评判真实样本检测结果的可靠性。其中所述甲基化检测可以适用于肿瘤基因的检测，更优选地可以适用于癌症的早期检测或早期筛查，所述癌症包括：脑癌、肺癌、皮肤癌、鼻咽癌、咽喉癌、肝癌、骨癌、淋巴瘤、胰腺癌、皮肤癌、肠癌、直肠癌、甲状腺癌、膀胱癌、肾癌、口腔癌、胃癌、实体瘤、卵巢癌、食管癌、胆囊癌、胆道癌、乳腺癌、***、子宫癌、***癌、头颈癌、肉瘤、胸腔恶性肿瘤(除肺外)、黑色素瘤、和睾丸癌、白血病。The methylation standard of this application can be used to verify the accuracy assessment of various methylation detection processes. For example, the methylation standard of this application can be used as a reference standard together with the sample to be tested to conduct any complete methylation test. methylation detection process, so as to judge the reliability of the real sample detection results based on the detection results of the methylation standard. The methylation detection can be applied to the detection of tumor genes, and more preferably can be applied to early detection or early screening of cancers. The cancers include: brain cancer, lung cancer, skin cancer, nasopharyngeal cancer, throat cancer, Liver cancer, bone cancer, lymphoma, pancreatic cancer, skin cancer, bowel cancer, rectal cancer, thyroid cancer, bladder cancer, kidney cancer, oral cancer, stomach cancer, solid tumors, ovarian cancer, esophageal cancer, gallbladder cancer, bile duct cancer, breast Cancer, cervical cancer, uterine cancer, prostate cancer, head and neck cancer, sarcoma, thoracic malignant tumors (except lung), melanoma, and testicular cancer, leukemia.
本领域已有的甲基化标准品存在着诸多的问题，例如甲基化阴性标准品中，未甲基化的Lambda DNA的缺点是其不来自于人基因组，人工敲除甲基化转移酶的细胞系DNA的缺点是甲基化信号去除不干净，仍存在较高的CpG信号背景，且价格昂贵。又例如甲基化阳性标准品中，MsssI(甲基化酶)处理pUC19或者人的基因组DNA来获得的标准品的缺点是 无法实现组织溯源的信息。本申请提供的一种在人源标准品中掺入少量外源的、甲基化水平稳定的参考品，该种组合物用于标准品的应用，可以在评估各类甲基化检测方法的过程中，实现优异的检测准确性评估、以及适合作组织溯源的标准品。There are many problems with existing methylation standards in this field. For example, among the methylation negative standards, the disadvantage of unmethylated Lambda DNA is that it does not come from the human genome, and artificial knockout of methyltransferase The disadvantage of cell line DNA is that the methylation signal is not completely removed, there is still a high CpG signal background, and it is expensive. Another example is methylation positive standards. The shortcoming of standards obtained by treating pUC19 or human genomic DNA with MsssI (methylase) is that tissue traceability information cannot be achieved. This application provides a reference material with a small amount of exogenous and stable methylation levels mixed into a human standard material. This composition is used in the application of standard materials and can be used in the evaluation of various methylation detection methods. In the process, excellent detection accuracy assessment and standards suitable for tissue traceability are achieved.
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的方法和用途等，而不用于限制本申请发明的范围。Without intending to be limited by any theory, the following examples are only for illustrating the methods and uses of the present application, and are not intended to limit the scope of the present invention.
实施例Example
实施例1Example 1
总体制备步骤参见附图1，所述标准品的制备方法的总体步骤可，包括：Refer to Figure 1 for the overall preparation steps. The overall steps of the preparation method of the standard substance may include:
1)健康细胞系和癌症细胞系培养。1) Culture of healthy cell lines and cancer cell lines.
2)收集细胞，进行细胞核分离，随后使用Atlantis dsDNase或者Micrococcal Nuclease酶片段化基因组DNA，并进行片段筛选去除gDNA大片段。2) Collect cells, separate nuclei, then use Atlantis dsDNase or Micrococcal Nuclease enzyme to fragment genomic DNA, and perform fragment screening to remove large gDNA fragments.
3)将酶切后的癌症细胞系DNA与健康细胞系酶切DNA进行一定比例的混合，获得不同梯度(0％-100％)的阴性和阳性标准品，此处的健康细胞系包括但不限于永生化B淋巴细胞系(例如：GM24385，GM12878，GM24631，NCI.BL1184)，癌症细胞系包括但不限于永生化癌症细胞系(例如：SW48，H1975，HepG2，T.T，CaoV3，PANC-1，PSN-1)。这些细胞系产品仅作为例举，其他合适的癌症细胞系也可以采用。3) Mix the digested DNA of cancer cell lines and the digested DNA of healthy cell lines in a certain proportion to obtain negative and positive standards of different gradients (0%-100%). Healthy cell lines here include but are not Limited to immortalized B lymphocyte cell lines (for example: GM24385, GM12878, GM24631, NCI.BL1184), cancer cell lines include but are not limited to immortalized cancer cell lines (for example: SW48, H1975, HepG2, T.T, CaoV3, PANC-1, PSN-1). These cell line products are examples only and other suitable cancer cell lines may be used.
例如人基因组，选自纯净的商业化细胞系，针对不同癌种有不同的示例性的细胞系可供选择：For example, the human genome is selected from pure commercial cell lines. There are different exemplary cell lines available for different cancer types:
表1：Table 1:
| 癌种Cancer type | 代表性细胞系名称Representative cell line names |
| 健康人healthy person | GM24385、GM12878、GM24631、NCI.BL1184GM24385, GM12878, GM24631, NCI.BL1184 |
| 肺癌lung cancer | H1975H1975 |
| 结肠癌colon cancer | SW48SW48 |
| 肝癌liver cancer | HepG2HepG2 |
| 食管癌Esophageal cancer | T.TT.T |
| 卵巢癌ovarian cancer | CaoV3CaoV3 |
| 胰腺癌pancreatic cancer | PANC-1，PSN-1PANC-1,PSN-1 |
| 胆囊癌gallbladder cancer | NOZ,GBC-SDNOZ,GBC-SD |
| 胃癌stomach cancer | Hs746THs746T |
| 头颈癌Head and neck cancer | HSC-2HSC-2 |
| 乳腺癌breast cancer | BT-549BT-549 |
| 淋巴瘤Lymphoma | BALL-1BALL-1 |
| 肾癌kidney cancer | 769-P769-P |
| 脑癌brain cancer | A-172A-172 |
| 皮肤癌skin cancer | A-431A-431 |
| 人子宫内膜腺癌细胞human endometrial adenocarcinoma cells | AN3-CAAN3-CA |
| 人睾丸癌细胞human testicular cancer cells | NTERA-2 cl.D1NTERA-2 cl.D1 |
| ***癌prostate cancer | DU145DU145 |
| 甲状腺癌Thyroid cancer | 8305C8305C |
| 膀胱癌Bladder Cancer | 56375637 |
| 骨肉瘤Osteosarcoma | 143B143B |
4)通过M.SssI甲基化转移酶处理pUC19 DNA，根据全非甲基化Lambda和pUC19基因组大小按照质量比19:1的比例混合，并进行打断。4) Treat pUC19 DNA with M.SssI methyltransferase, mix the fully unmethylated Lambda and pUC19 genomes at a mass ratio of 19:1, and interrupt.
5)根据人基因组及Lambda基因组大小差异，按照1:2×10
-3至1:2×10
-5，优选为1:2×10
-
4质量比进行混合获得混合基因组标准品。
5) According to the size difference between the human genome and Lambda genome, mix according to the mass ratio of 1:2×10 -3 to 1:2×10 -5 , preferably 1:2× 10 -4 to obtain a mixed genome standard.
进一步地，分别对2×10
-3、2×10
-4和2×10
-5进行过2组对照测试，实际结果中检出的pUC19甲基化信号并无显著差异，从而证明2×10
-3至2×10
-5这个范围内的掺比，都是可以适用的。(参见下表2结果)
Furthermore, two sets of control tests were conducted on 2×10 -3 , 2×10 -4 and 2×10 -5 respectively. There was no significant difference in the pUC19 methylation signal detected in the actual results, thus proving that 2×10 The mixing ratio in the range of -3 to 2×10 -5 is applicable. (See results in Table 2 below)
表2：不同掺比比例的标准品检测结果Table 2: Test results of standard products with different mixing ratios
同理地，也可以采用全非甲基化pUC19，和通过M.SssI甲基化转移酶处理的Lambda基因组大小按照质量比1:19的比例混合，并进行打断。根据人基因组及Lambda基因组大小差异，按照1:2×10
-3至1:2×10
-5，优选为1:2×10
-4质量比进行混合获得混合基因组标准品，也就是说，混合基因组标准品中，阳性细胞系与pUC19质量比为1:1×10
-4至1:1×10
-6，优选为1:1×10
-5。可以达到相同的效果。
Similarly, fully unmethylated pUC19 can also be mixed with the Lambda genome size treated by M.SssI methyltransferase at a mass ratio of 1:19 and fragmented. According to the size difference between the human genome and Lambda genome, mix according to the mass ratio of 1:2×10 -3 to 1:2×10 -5 , preferably 1:2×10 -4 to obtain the mixed genome standard, that is to say, mix Among the genome standards, the mass ratio of the positive cell line to pUC19 is 1:1×10 -4 to 1:1×10 -6 , preferably 1:1×10 -5 . The same effect can be achieved.
实施例2Example 2
核小体酶切片段评估Nucleosome digestion fragment assessment
参见图2，以SW48为例，同时对EZ-SW48和真实cfDNA样本使用WGBS方法进行建库测序，检测两种类型样本的文库片段分布情况(见图2)，上图使用Perkin Elmer的LabChip片段评估仪进行片段评估，图2为WGBS测序评估出的片段大小，图中可以看出，本方法制备的酶切DNA产物(左侧)及其文库的片段大小分布与真实cfDNA(右侧)的非常一致。See Figure 2, taking SW48 as an example, using the WGBS method for library construction and sequencing on EZ-SW48 and real cfDNA samples at the same time, and detecting the library fragment distribution of the two types of samples (see Figure 2). The above picture uses Perkin Elmer’s LabChip fragments The evaluator performs fragment evaluation. Figure 2 shows the fragment size evaluated by WGBS sequencing. It can be seen from the figure that the fragment size distribution of the digested DNA product (left) and its library prepared by this method is the same as that of the real cfDNA (right). Very consistent.
实施例3Example 3
核小体酶切工艺甲基化信号稳定性检测Methylation signal stability detection during nucleosome digestion process
参见图3，使用申请人的ELSA-seq
TM建库方法(参见中国发明专利申请公开文本CN110892097A)完成建库，并对酶切细胞系DNA进行甲基化信号检测，通过评估相关性等指标评估方法重复性。以下所示为3个批次(Lot 1，Lot 2，Lot 3)的EZ-GM24385细胞系及试剂酶切后片段进行甲基化信号检测结果。从结果可以看出，各批次两两之间信号相关性达到0.999以上，重复性好，制备工艺比较稳定。
Referring to Figure 3, the applicant's ELSA-seq TM library construction method (see Chinese Invention Patent Application Publication Document CN110892097A) was used to complete the library construction, and the methylation signal was detected on the enzyme-digested cell line DNA, and evaluated by evaluating correlation and other indicators. Method reproducibility. Shown below are the methylation signal detection results of three batches (Lot 1, Lot 2, Lot 3) of the EZ-GM24385 cell line and fragments digested with reagents. It can be seen from the results that the signal correlation between each batch reaches more than 0.999, the repeatability is good, and the preparation process is relatively stable.
实施例4Example 4
组织溯源信号分析Organization traceability signal analysis
参见图4，对抽提的健康/癌症细胞系和健康人cfDNA进行WGBS检测，得到的数据进行生信聚类分析，检测结果的甲基化水平越高，颜色越接近左上角颜色卡的右半部分(由浅色向深色过渡)。根据该实验结果显示：健康人细胞系(GM24385，GM12878，NCI.BL1184，GM24631)和健康人cfDNA(Plasma.1-24)甲基化信号聚类结果好，可以使用健康细胞系作为健康人标准品，不同的癌症来源的细胞系(H1975，SW48，HepG2，Caov3，PANC-1，T.T)具有不同的甲基化图样，可以用于表明不同来源的癌症细胞系存在不同的甲基化信号，适合 作组织溯源的标准品。图示中NFDNA-1，NFDNA-2，NFDNA-3依次表示来源于GM24385、GM12878、GM24631的核小体酶切DNA(例如，NFDNA-1具有约32％的EGFR等位基因突变，可以通过固定比例掺比用于评估VAF的检测准确性)。Plasma.1、Plasma.2……Plasma.24分别表示不同来源的健康人cfDNA。Refer to Figure 4. WGBS detection was performed on the extracted healthy/cancer cell lines and healthy human cfDNA. The obtained data were subjected to bioinformatics cluster analysis. The higher the methylation level of the detection results, the closer the color is to the upper left corner of the color card on the right. Half part (transition from light to dark). According to the experimental results, the methylation signal clustering results of healthy human cell lines (GM24385, GM12878, NCI.BL1184, GM24631) and healthy human cfDNA (Plasma.1-24) are good, and healthy cell lines can be used as healthy human standards. Products, cell lines derived from different cancers (H1975, SW48, HepG2, Caov3, PANC-1, T.T) have different methylation patterns, which can be used to indicate that different methylation signals exist in cancer cell lines derived from different sources. Suitable for tissue traceability standards. In the illustration, NFDNA-1, NFDNA-2, and NFDNA-3 respectively represent nucleosome-cleaved DNA derived from GM24385, GM12878, and GM24631 (for example, NFDNA-1 has about 32% EGFR allelic mutations, which can be fixed by Proportional doping ratio is used to evaluate the detection accuracy of VAF). Plasma.1, Plasma.2...Plasma.24 respectively represent healthy human cfDNA from different sources.
实施例5Example 5
梯度标准品掺比准确性分析Gradient Standard Mixing Ratio Accuracy Analysis
以SW48为例，通过梯度混合核小体酶切的SW48和NFDNA-1(例如：可选GM24385)获得SW48质量比分别为100％，12.5％，5％，1％，0.5％，0.10％，0.05％，0％的甲基化标准品，分别使用ddPCR和申请人的HS-UMI(HS测序方法可参见申请人的中国发明专利授权公告文本CN106835291B，在此基础上结合UMI技术即HS-UMI方法)测序方法(深度测序>100，000x)检测每种甲基化标准品的EGFR:p.G719S的等位基因突变频率(VAF)，每种方法均进行两次重复，测出的VAF分别记为VAF1和VAF2，并求取两次重复的平均VAF。具体检测结果如下表所示，ddPCR和HS-UMI测得的VAF值与每种甲基化标准品的预期VAF接近，表明本方法制备的甲基化标准品梯度准确性好。Taking SW48 as an example, the mass ratios of SW48 obtained by gradient mixing nucleosome-digested SW48 and NFDNA-1 (for example: optional GM24385) are 100%, 12.5%, 5%, 1%, 0.5%, 0.10%, respectively. 0.05%, 0% methylation standard, respectively using ddPCR and the applicant's HS-UMI (the HS sequencing method can be found in the applicant's Chinese invention patent authorization announcement text CN106835291B, on this basis, combined with UMI technology, namely HS-UMI Method) Sequencing method (deep sequencing >100,000x) was used to detect the allele mutation frequency (VAF) of EGFR:p.G719S for each methylation standard. Each method was repeated twice, and the measured VAF were respectively Denote them as VAF1 and VAF2, and find the average VAF of the two repetitions. The specific test results are shown in the table below. The VAF values measured by ddPCR and HS-UMI are close to the expected VAF of each methylation standard, indicating that the methylation standard gradient prepared by this method has good accuracy.
表3：ddPCR和HS-UMI检测VAF值与预期VAF值比较Table 3: Comparison of VAF values detected by ddPCR and HS-UMI with expected VAF values
注：预期VAF为不同甲基化标准品对应的EGFR:p.G719S位点的理论等位基因突变频率。NA表示未检出。Note: The expected VAF is the theoretical allele mutation frequency of the EGFR:p.G719S site corresponding to different methylation standards. NA means not detected.
参见图5，以SW48为例，通过混合核小体酶切的SW48和NFDNA-1(例如：GM24385)获得质量比分别为100％，20％，12.5％，0％的甲基化标准品，使用申请人的ELSA-seq
TM建 库方法(参见中国发明专利申请公开文本CN110892097A)进行建库测序检测甲基化标准品的甲基化水平，部分位点测序结果如图5所示(位点为Chr1:2,222,156-2,222,797bp；柱状图中的浅色代表甲基化DNA，深色代表非甲基化DNA。)，随着不同甲基化标准品中SW48占比的降低，不同标准品的甲基化水平也逐渐降低，左侧括号中的数字表示实际测得的甲基化水平数值，可见实际测得的甲基化水平(95.9％、23.8％、10.1％、0.5％)与按质量配比的标准品的预设甲基化水平(100％，20％，12.5％，0％)基本一致，表明该标准品可用于基于cfDNA的甲基化检测方法的性能评估。
Refer to Figure 5, taking SW48 as an example, by mixing nucleosome-digested SW48 and NFDNA-1 (for example: GM24385) to obtain methylation standards with mass ratios of 100%, 20%, 12.5%, and 0% respectively. The applicant's ELSA-seq TM library construction method (see Chinese invention patent application publication CN110892097A) was used to perform library construction and sequencing to detect the methylation level of the methylation standard. The partial site sequencing results are shown in Figure 5 (site is Chr1:2,222,156-2,222,797bp; the light color in the histogram represents methylated DNA, and the dark color represents unmethylated DNA.), as the proportion of SW48 in different methylated standards decreases, the Methylation levels also gradually decreased. The numbers in the brackets on the left represent the actual measured methylation level values. It can be seen that the actual measured methylation levels (95.9%, 23.8%, 10.1%, 0.5%) are different from those measured by mass. The preset methylation levels (100%, 20%, 12.5%, 0%) of the prepared standards are basically consistent, indicating that the standards can be used for performance evaluation of cfDNA-based methylation detection methods.
实施例6Example 6
Lambda和pUC19混合基因组的信号评估Signal assessment of lambda and pUC19 hybrid genomes
样本1至样本4参照实施例1进行制备，以及使用申请人的ELSA-seq
TM建库方法(参见中国发明专利申请公开文本CN110892097A)进行建库测序检测甲基化标准品的甲基化水平。参见下方表，CpG.methyl.hyper指pUC19CpG位点的甲基化水平，CpG.methyl.hypo指Lambda CpG位点的甲基化水平，进行正常的甲基化检测时(样本3、样本4)，CpG.methyl.hyper信号>95％以上，当甲基化位点保护出现异常时(样本1、样本2)，pUC19的甲基化信号异常偏低；该异常可以通过人为选择不恰当的实验条件而产生，例如：重亚硫酸盐过程中反应温度超出标准温度，或者反应时长长于反应时间，或者Methyl-seq酶法转化过程中TET2用量不足或者反应时间不足从而导致的甲基化反应异常等。所以Lambda和pUC19基因组可以作为该检测流程的准确性的内部参考，监控甲基化转化过程的准确性。
Samples 1 to 4 were prepared as described in Example 1, and the applicant's ELSA-seq TM library construction method (see Chinese invention patent application publication CN110892097A) was used to perform library construction and sequencing to detect the methylation level of the methylation standard. See the table below, CpG.methyl.hyper refers to the methylation level of pUC19CpG site, CpG.methyl.hypo refers to the methylation level of Lambda CpG site, when performing normal methylation detection (sample 3, sample 4) , CpG.methyl.hyper signal >95% or more. When the methylation site protection is abnormal (sample 1, sample 2), the methylation signal of pUC19 is abnormally low; this abnormality can be caused by inappropriate artificial selection of experiments. Conditions arise, for example: the reaction temperature exceeds the standard temperature during the bisulfite process, or the reaction time is longer than the reaction time, or the methylation reaction is abnormal due to insufficient TET2 dosage or insufficient reaction time during the Methyl-seq enzymatic conversion process, etc. . Therefore, the Lambda and pUC19 genomes can be used as internal references for the accuracy of the detection process to monitor the accuracy of the methylation conversion process.
上述结果显示，本申请提供的组合物用于标准品，可以在整个甲基化测序流程中，用于判断实验条件是否符合质量控制的要求。The above results show that the composition provided in this application is used as a standard and can be used in the entire methylation sequencing process to determine whether experimental conditions meet the requirements of quality control.
表4：puc19和Lambda甲基化信号示例Table 4: Examples of puc19 and Lambda methylation signals
| 样本sample | CpG.methyl.hyperCpG.methyl.hyper | CpG.methyl.hypoCpG.methyl.hypo |
|
样本1 |
0.92400.9240 | 0.00010.0001 |
|
样本2 |
0.92210.9221 | 0.00010.0001 |
|
样本3 |
0.97380.9738 | 0.00010.0001 |
|
样本4 |
0.97640.9764 | 0.00010.0001 |
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附 的权利要求和其等同方案的范围内。The foregoing detailed description is provided by way of explanation and example, and is not intended to limit the scope of the appended claims. Various modifications to the embodiments described herein will be apparent to those of ordinary skill in the art and remain within the scope of the appended claims and their equivalents.
Claims (32)
- 一种生物组合物，其特征在于，包括：至少一种作为阳性标准品的阳性细胞系，至少一种作为阴性标准品的阴性细胞系，至少一种作为阳性参考品的阳性基因组载体，以及至少一种作为阴性参考品的阴性基因组载体。A biological composition, characterized in that it includes: at least one positive cell line as a positive standard, at least one negative cell line as a negative standard, at least one positive genome vector as a positive reference, and at least A negative genome vector that serves as a negative reference.
- 如权利要求1所述的生物组合物，其特征在于，所述阳性基因组载体的碱基数小于阳性细胞系的碱基数；所述阴性基因组载体的碱基数小于阴性细胞系的碱基数。The biological composition according to claim 1, wherein the number of bases of the positive genome vector is less than the number of bases of the positive cell line; the number of bases of the negative genome vector is less than the number of bases of the negative cell line .
- 如权利要求2所述的生物组合物，其特征在于，所述阳性基因组载体的碱基数小于阳性细胞系的碱基数的1/100，优选地，所述阳性基因组载体的碱基数小于阳性细胞系的碱基数的1/1000，更优选地，所述阳性基因组载体的碱基数小于阳性细胞系的碱基数的The biological composition according to claim 2, wherein the number of bases of the positive genome vector is less than 1/100 of the number of bases of the positive cell line. Preferably, the number of bases of the positive genome vector is less than 1/1000 of the number of bases of the positive cell line. More preferably, the number of bases of the positive genome vector is less than 1/1000 of the number of bases of the positive cell line.1/10000；所述阴性基因组载体的碱基数小于阴性细胞系的碱基数的1/100，优选地，所1/10000; the base number of the negative genome vector is less than 1/100 of the base number of the negative cell line, preferably, the述阴性基因组载体的碱基数小于阴性细胞系的碱基数的1/1000，更优选地，所述阴性基因组载体的碱基数小于阴性细胞系的碱基数的1/10000。The number of bases of the negative genome vector is less than 1/1000 of the number of bases of the negative cell line. More preferably, the number of bases of the negative genome vector is less than 1/10000 of the number of bases of the negative cell line.
- 如权利要求3所述的生物组合物，其特征在于，所述阴性细胞系与所述阴性基因组载体的质量比为1:2×10 -3至1:1×10 -6，优选为1:2×10 -4至1：1×10 -5；所述阳性细胞系与所述阳性基因组载体的质量比为1:2×10 -3至1:1×10 -6，优选为1:2×10 -4至1：1×10 -5。 The biological composition according to claim 3, wherein the mass ratio of the negative cell line to the negative genome vector is 1:2×10 -3 to 1:1×10 -6 , preferably 1: 2×10 -4 to 1:1×10 -5 ; the mass ratio of the positive cell line to the positive genome vector is 1:2×10 -3 to 1:1×10 -6 , preferably 1:2 ×10 -4 to 1: 1×10 -5 .
- 如权利要求1所述的生物组合物，其特征在于，所述阳性细胞系与阴性细胞系的质量比为0.1:99.9至25:75之间，优选为0.5:99.5至12.5:87.5之间，更优选为1:99至5:95之The biological composition according to claim 1, wherein the mass ratio of the positive cell line and the negative cell line is between 0.1:99.9 and 25:75, preferably between 0.5:99.5 and 12.5:87.5, More preferably, it is between 1:99 and 5:95间。between.
- 如权利要求1所述的生物组合物，其特征在于，所述阳性基因组载体与阴性基因组载体的质量比为1:99至25:75之间或者99:1至75:25，优选为大约1:19或者19:1。The biological composition according to claim 1, wherein the mass ratio of the positive genome vector to the negative genome vector is between 1:99 and 25:75 or between 99:1 and 75:25, preferably about 1 :19 or 19:1.
- 如权利要求1所述的生物组合物，其特征在于，所述阳性细胞系和所述阴性细胞系都经过片段化处理，平均片段大小为约140-160bp，优选地，所述片段化处理包含生物酶酶切片段化和/或物理打断片段化。The biological composition of claim 1, wherein both the positive cell line and the negative cell line undergo fragmentation processing, with an average fragment size of about 140-160 bp. Preferably, the fragmentation processing includes Fragmentation by biological enzymes and/or physical fragmentation.
- 如权利要求1所述的生物组合物，其特征在于，所述阳性细胞系选自如下细胞系的组The biological composition of claim 1, wherein the positive cell line is selected from the group of cell lines:合：人结直肠癌细胞系(例如：SW48)，人肺腺癌细胞系(例如：H1975)，人肝癌细胞系(例如：HepG2或)，人食管癌细胞系(例如：T.T)，人卵巢癌细胞系(例如：Compatibility: human colorectal cancer cell line (for example: SW48), human lung adenocarcinoma cell line (for example: H1975), human liver cancer cell line (for example: HepG2 or), human esophageal cancer cell line (for example: T.T), human ovarian cancer cell line Cancer cell lines (eg:CaoV3)，人胰腺癌细胞系(例如：PANC-1，PSN-1)。CaoV3), human pancreatic cancer cell lines (eg: PANC-1, PSN-1).
- 如权利要求1所述的生物组合物，其特征在于，所述阴性细胞系选自如下细胞系的组The biological composition of claim 1, wherein the negative cell line is selected from the group of cell lines:合：人B淋巴细胞(例如：GM24385、GM12878、GM24631、NCI.BL1184)或者人正Combined: human B lymphocytes (for example: GM24385, GM12878, GM24631, NCI.BL1184) or human normal常细胞系(例如：Beads-2B及其他人正常细胞系)。Normal cell lines (e.g. Beads-2B and other normal cell lines).
- 如权利要求1所述的生物组合物，其特征在于，所述阳性基因组载体为经过甲基化转移酶处理的pUC19 DNA和/或经过甲基化转移酶处理的Lambda DNA，所述甲基化转移酶 优选为M.Sssl。The biological composition according to claim 1, wherein the positive genome carrier is pUC19 DNA treated with methyltransferase and/or Lambda DNA treated with methyltransferase, and the methylated The transferase is preferably M.Sssl.
- 如权利要求1所述的生物组合物，其特征在于，所述阴性基因组载体为未甲基化的The biological composition of claim 1, wherein the negative genome vector is unmethylatedLambda DNA和/或未甲基化的pUC19 DNA。Lambda DNA and/or unmethylated pUC19 DNA.
- 一种如权利要求8-11中任意一项所述的生物组合物的应用，其特征在于，所述生物组合物用于评估癌症基因检测的准确性和组织溯源的标准品。An application of the biological composition according to any one of claims 8 to 11, characterized in that the biological composition is used to evaluate the accuracy of cancer gene detection and as a standard for tissue traceability.
- 如权利要求12所述的应用，其特征在于，所述癌症包括：脑癌、肺癌、皮肤癌、鼻咽The application according to claim 12, wherein the cancer includes: brain cancer, lung cancer, skin cancer, nasopharyngeal cancer,癌、咽喉癌、肝癌、骨癌、淋巴瘤、胰腺癌、皮肤癌、肠癌、直肠癌、甲状腺癌、膀胱癌、肾癌、口腔癌、胃癌、实体瘤、卵巢癌、食管癌、胆囊癌、胆道癌、乳腺癌、***、子宫癌、***癌、头颈癌、肉瘤、胸腔恶性肿瘤(除肺外)、黑色素瘤、和睾丸Cancer, throat cancer, liver cancer, bone cancer, lymphoma, pancreatic cancer, skin cancer, bowel cancer, rectal cancer, thyroid cancer, bladder cancer, kidney cancer, oral cancer, stomach cancer, solid tumors, ovarian cancer, esophageal cancer, gallbladder cancer , biliary tract cancer, breast cancer, cervical cancer, uterine cancer, prostate cancer, head and neck cancer, sarcoma, thoracic malignant tumors (except lung), melanoma, and testicular cancer癌、白血病。Cancer, leukemia.
- 一种制备生物组合物的方法，其特征在于，包括如下步骤：A method for preparing a biological composition, characterized by comprising the following steps:(1)获得至少一种作为阳性标准品的阳性细胞系和至少一种作为阴性标准品的阴性细胞系；(1) Obtain at least one positive cell line as a positive standard and at least one negative cell line as a negative standard;(2)将所述阳性细胞系和阴性细胞系进行细胞核分离，随后通过酶切将所述阳性细胞系和阴性细胞系转化为阳性片段化DNA和阴性片段化DNA；(2) Separating the nuclei of the positive cell lines and negative cell lines, and then converting the positive cell lines and negative cell lines into positive fragmented DNA and negative fragmented DNA through enzyme digestion;(3)将所述阳性片段化DNA和所述阴性片段化DNA混合；(3) Mix the positive fragmented DNA and the negative fragmented DNA;(4)获得至少一种作为阳性参考品的阳性基因组载体和至少一种作为阴性参考品的阴性基因组载体，将所述阳性基因组载体和阴性基因组载体混合，并进行打断；(4) Obtain at least one positive genome vector as a positive reference product and at least one negative genome vector as a negative reference product, mix the positive genome vector and the negative genome vector, and interrupt;(5)将上述步骤(3)和步骤(4)的产物混合。(5) Mix the products of the above steps (3) and (4).
- 如权利要求14所述的方法，其特征在于，所述酶切是通过限制性内切酶或者微球菌核酸酶片段化基因组DNA。The method of claim 14, wherein the enzyme cutting is to fragment the genomic DNA by restriction endonuclease or micrococcal nuclease.
- 如权利要求14所述的方法，其特征在于，所述阳性细胞系选自如下细胞系的组合：人结肠癌细胞(例如：SW48)，人肺腺癌细胞(例如：H1975)，人肝癌细胞(例如：The method of claim 14, wherein the positive cell line is selected from a combination of the following cell lines: human colon cancer cells (for example: SW48), human lung adenocarcinoma cells (for example: H1975), human liver cancer cells (For example:HepG2)，人食管癌细胞(例如：T.T)，人卵巢癌细胞(例如：CaoV3)，人胰腺癌细胞HepG2), human esophageal cancer cells (for example: T.T), human ovarian cancer cells (for example: CaoV3), human pancreatic cancer cells(例如：PANC-1，PSN-1)。(Example: PANC-1, PSN-1).
- 如权利要求14所述的方法，其特征在于，所述阴性细胞系选自如下细胞系的组合：人B淋巴细胞(例如：GM24385、GM12878、GM24631、NCI.BL1184)，或者人正常细胞系(例如：Beads-2B及其他人正常细胞系)。The method of claim 14, wherein the negative cell line is selected from a combination of the following cell lines: human B lymphocytes (for example: GM24385, GM12878, GM24631, NCI.BL1184), or human normal cell lines ( For example: Beads-2B and other normal cell lines).
- 如权利要求14所述的方法，其特征在于，所述阳性基因组载体的碱基数小于阳性细胞系的碱基数；所述阴性基因组载体的碱基数小于阴性细胞系的碱基数。The method of claim 14, wherein the number of bases of the positive genome vector is less than the number of bases of the positive cell line; and the number of bases of the negative genome vector is less than the number of bases of the negative cell line.
- 如权利要求18所述的方法，其特征在于，所述阳性基因组载体的碱基数小于阳性细胞系 的碱基数的1/100，优选地，所述阳性基因组载体的碱基数小于阳性细胞系的碱基数的The method of claim 18, wherein the number of bases of the positive genome vector is less than 1/100 of the number of bases of the positive cell line. Preferably, the number of bases of the positive genome vector is less than that of the positive cell line. The base number of the system1/1000，更优选地，所述阳性基因组载体的碱基数小于阳性细胞系的碱基数的1/10000；1/1000, more preferably, the base number of the positive genome vector is less than 1/10000 of the base number of the positive cell line;所述阴性基因组载体的碱基数小于阴性细胞系的碱基数的1/100，优选地，所述阴性基因组载体的碱基数小于阴性细胞系的碱基数的1/1000，更优选地，所述阴性基因组载体的碱基数小于阴性细胞系的碱基数的1/10000。The number of bases of the negative genome vector is less than 1/100 of the number of bases of the negative cell line, preferably, the number of bases of the negative genome vector is less than 1/1000 of the number of bases of the negative cell line, more preferably , the base number of the negative genome vector is less than 1/10000 of the base number of the negative cell line.
- 如权利要求19所述的方法，其特征在于，所述阴性基因组载体的碱基数为阴性细胞系的碱基数的大约1/1000000～1/60000；或者所述阳性基因组载体的碱基数为阳性细胞系的碱基数的大约1/1000000～1/60000。The method of claim 19, wherein the number of bases of the negative genome vector is approximately 1/1000000 to 1/60000 of the number of bases of the negative cell line; or the number of bases of the positive genome vector It is approximately 1/1,000,000 to 1/60,000 of the base number of the positive cell line.
- 如权利要求14所述的方法，其特征在于，所述阳性细胞系与阴性细胞系的按照如下质量比混合：0.1:99.9至25:75之间，优选为0.5:99.5至12.5:87.5之间，更优选为1:99至The method of claim 14, wherein the positive cell line and the negative cell line are mixed according to the following mass ratio: between 0.1:99.9 and 25:75, preferably between 0.5:99.5 and 12.5:87.5 , more preferably 1:99 to5:95之间。Between 5:95.
- 如权利要求14所述的方法，其特征在于，所述阳性基因组载体与阴性基因组载体按照如下质量比混合：1:99至25:75之间或者99:1至75:25，优选为大约1:19或者19:1。The method of claim 14, wherein the positive genome vector and the negative genome vector are mixed according to the following mass ratio: between 1:99 and 25:75, or between 99:1 and 75:25, preferably about 1 :19 or 19:1.
- 如权利要求14所述的方法，其特征在于，按照如下质量比例将步骤(3)和步骤(4)的产物混合：1:2×10 -3至1:2×10 -5，优选为1:2×10 -4。 The method according to claim 14, characterized in that the products of step (3) and step (4) are mixed according to the following mass ratio: 1:2×10 -3 to 1:2×10 -5 , preferably 1 :2×10 -4 .
- 一种试剂盒，包含如权利要求1-11中任意一项所述的生物组合物。A kit comprising the biological composition according to any one of claims 1-11.
- 一种验证基因测序方法准确性的方法，其特征在于，按照所述基因测序方法对如权利要求5所述的生物组合物进行测序，在生物组合物已验证掺比准确性情况下，比较某一区间测序的阳性水平与阳性细胞系/阴性细胞系的质量比的误差，若误差小于30％，则认为该基因测序方法准确。A method for verifying the accuracy of a gene sequencing method, characterized in that the biological composition as claimed in claim 5 is sequenced according to the gene sequencing method, and when the accuracy of the mixing ratio of the biological composition has been verified, a certain If the error between the positivity level of an interval sequencing and the mass ratio of positive cell lines/negative cell lines is less than 30%, the gene sequencing method is considered accurate.
- 如权利要求25所述的方法，其特征在于，所述误差的计算方法为：测序的阳性水平分数减去阳性细胞系/阴性细胞系的质量比的差值，再除以阳性细胞系/阴性细胞系的质量比。The method of claim 25, wherein the error is calculated by: subtracting the difference in the mass ratio of the positive cell line/negative cell line from the positive level score of the sequencing, and then dividing by the positive cell line/negative Cell line mass ratio.
- 如权利要求26所述的方法，其特征在于，若误差小于25％，则认为该基因测序方法准The method of claim 26, wherein if the error is less than 25%, the gene sequencing method is considered accurate.确。Indeed.
- 一种验证基因测序方法准确性的方法，其特征在于，按照所述基因测序方法对如权利要求6所述的生物组合物进行测序，并比较阳性基因组载体测序的甲基化水平与阳性基因组载体理论的甲基化水平(97％)的误差，若误差小于5％，则认为该基因测序方法准A method for verifying the accuracy of a gene sequencing method, characterized by sequencing the biological composition according to claim 6 according to the gene sequencing method, and comparing the methylation level of the positive genome vector sequencing with the positive genome vector If the error of the theoretical methylation level (97%) is less than 5%, the gene sequencing method is considered accurate.确。Indeed.
- 如权利要求28所述的方法，其特征在于，所述误差的计算方法为：阳性基因组载体测序的甲基化水平减去阳性基因组载体理论的甲基化水平的差值，再除以阳性基因组载体理论的甲基化水平。The method of claim 28, wherein the error is calculated by: subtracting the difference between the methylation level of positive genome vector sequencing and the theoretical methylation level of positive genome vector, and then dividing by the positive genome vector. Methylation levels of carrier theory.
- 如权利要求29所述的方法，其特征在于，若误差小于3％，则认为该基因测序方法准The method of claim 29, wherein the gene sequencing method is considered accurate if the error is less than 3%.确。Indeed.
- 一种验证基因测序方法准确性的方法，其特征在于，按照所述基因测序方法对如权利要求6所述的生物组合物进行测序，并比较阴性基因组载体测序的甲基化水平，若甲基化水平小于1％，则认为该基因测序方法准确。A method for verifying the accuracy of a gene sequencing method, characterized by sequencing the biological composition as claimed in claim 6 according to the gene sequencing method, and comparing the methylation levels of negative genome vector sequencing, if methyl If the level is less than 1%, the gene sequencing method is considered accurate.
- 如权利要求31所述的方法，其特征在于，若甲基化水平小于0.5％，则认为该基因测序方法准确；优选地，若甲基化水平小于0.1％，则认为该基因测序方法准确；更优选地，若甲基化水平小于0.02％，则认为该基因测序方法准确。The method of claim 31, wherein if the methylation level is less than 0.5%, the gene sequencing method is considered accurate; preferably, if the methylation level is less than 0.1%, the gene sequencing method is considered accurate; More preferably, the gene sequencing method is considered accurate if the methylation level is less than 0.02%.
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| FANG HUAN, ZHONG BIXI, WEI LEI, ZHANG XIANGLIN, ZHANG WEI, WANG XIAOWO: "Practical stability of whole-genome bisulfite sequencing using plasma cell-free DNA", CHINESE JOURNAL OF BIOTECHNOLOGY, ZHONGGUO KEXUEYUAN WEISHENGWU YANJIUSUO, CHINESE ACADEMY OF SCIENCES, INSTITUTE OF MICROBIOLOGY, CN, vol. 35, no. 12, 25 December 2019 (2019-12-25), CN , pages 2284 - 2294, XP093097810, ISSN: 1000-3061, DOI: 10.13345/j.cjb.190281 * |
| MORRISON JACOB, KOEMAN JULIE M., JOHNSON BENJAMIN K., FOY KELLY K., BEDDOWS IAN, ZHOU WANDING, CHESLA DAVID W., ROSSELL LARISSA L.: "Evaluation of whole-genome DNA methylation sequencing library preparation protocols", EPIGENETICS & CHROMATIN, vol. 14, no. 1, 1 December 2021 (2021-12-01), XP093097812, DOI: 10.1186/s13072-021-00401-y * |
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| CN115896027A (en) | 2023-04-04 |
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