CN116064819A - Method for detecting mutation and methylation of tumor specific gene in ctDNA - Google Patents
Method for detecting mutation and methylation of tumor specific gene in ctDNA Download PDFInfo
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Abstract
The invention discloses a method for detecting mutation and methylation of tumor specific genes in ctDNA, which comprises the following steps: s1, constructing a sequencing library and creating a tumor sample; s2, analyzing the mutation degree of ctDNA, and capturing and sequencing according to a tumor sample and a sequencing library to obtain a FASTQ file, wherein cfDNA in the tumor sample carries a pre-accessed molecular tag; s3, performing a plurality of rounds of nested PCR amplification according to the sequencing library, and sequencing a product through a first round of PCR amplification; s4, taking the product of the first round of PCR as a template, adopting a primer combination B to carry out second round of PCR amplification, sequencing, and analyzing the occurrence of target methylation in the DNA sample according to the result of combining the first round of PCR amplification with the second round of PCR amplification sequencing. The sequencing library prepared by the method can support subsequent detection, the result of each detection can represent the mutation status of all original ctDNA samples and the methylation modification condition of the coverage area of the enzyme cutting site, and the sensitivity and the specificity cannot be reduced.
Description
Technical Field
The invention relates to the technical field of cancer screening, in particular to a method for detecting mutation and methylation of tumor specific genes in ctDNA.
Background
The circulating tumor DNA (ctDNA) is derived from DNA fragments produced by apoptosis, necrosis or secretion of tumor cells, and contains the same genetic variation and apparent modification as the tumor tissue DNA, such as point mutation, gene rearrangement, fusion, copy number variation, methylation modification, etc. The detection of ctDNA can be applied to early cancer screening, diagnosis and stage division, targeted drug administration guidance, efficacy evaluation, recurrence monitoring and other aspects. Combining the information of mutation and methylation of tumor specific genes carried by ctDNA, the method is helpful for improving the sensitivity and specificity of detection, and can find cancer trails earlier, thus having important significance for early screening of tumors.
Currently, ctDNA mutation analysis for early screening generally utilizes a combination of hotspot mutations characteristic of tumors as markers. However, the mutation marker loci are different in each cancer species, and even in liver cancer with relatively concentrated hot spot mutation, detection of hundreds of loci on at least ten genes is required to cover most patients of the cancer species, so that the screening purpose is achieved.
For the detection of the sites, if a common PCR (polymerase chain reaction) method is used, hundreds of milliliters of blood samples are needed, and the feasibility of the detection for a common early screen is low; and the PCR method can detect the mutation with high false positive of technical source and cloning hematopoietic source.
Disclosure of Invention
The invention aims to provide a method for detecting mutation and methylation of a tumor specific gene in ctDNA, so as to solve the problem that the feasibility of the detection of a common early screening body is low in the background art; and the PCR method can detect mutation with high false positive problems of technical sources and clonal hematopoietic sources.
In order to achieve the above purpose, the present invention provides the following technical solutions:
a method for detecting mutations and methylation of tumor specific genes in ctDNA, comprising the steps of:
s1, constructing a sequencing library and creating a tumor sample;
s2, analyzing the mutation degree of ctDNA, and performing capturing and sequencing according to a tumor sample and a sequencing library to obtain a FASTQ file, wherein cfDNA in the tumor sample carries a pre-accessed molecular tag;
s3, performing a plurality of rounds of nested PCR amplification according to the sequencing library, and sequencing a product through a first round of PCR amplification;
s4, taking the product of the first round of PCR as a template, adopting a primer combination B to carry out second round of PCR amplification, sequencing, and analyzing the occurrence of target methylation in the DNA sample according to the result of combining the first round of PCR amplification with the second round of PCR amplification sequencing.
Preferably, the construction of the sequencing library described in step S1 is specifically:
1) Screening for differential ctDNA methylation sites;
2) Screening ctDNA methylation probes;
3) Constructing and verifying a ctDNA methylation diagnosis model of NKTCL;
4) Construction and verification of ctDNA methylation prognosis model of NKTCL.
Preferably, the procedure for screening the different ctDNA methylation sites is as follows:
plasma samples from 400 NKTCL patients and 400 normal individuals were collected;
extracting free DNA by using a cfDNA extraction kit, detecting the methylation level of the whole genome ctDNA by high-throughput whole genome methylation sequencing, and establishing a methylation library with about 300 ten thousand loci;
screening to obtain methylation sites with p < 0.05 and the methylation level difference multiple of 2000 for subsequent queue research through methylation level difference analysis;
corresponding methylation probes are designed according to the 2000 different methylation sites, and methylation sites which can generate positive and specific PCR amplification signals are screened through PCR verification.
Preferably, the step of extracting free DNA using a cfDNA extraction kit comprises:
adding 200 μl of blood into a 1.5ml centrifuge tube, adding 400 μl of cell lysate CL, mixing, centrifuging at 10000rpm for 1min, discarding supernatant, adding 200 μl of buffer GS into the cell nucleus precipitate, and shaking to thoroughly mix;
adding 200 μl of buffer GB and 20 μl of protease K premix solution, mixing completely upside down, standing at 55deg.C for 12min until the solution becomes clear;
after 5-10min at room temperature, 300. Mu.l of buffer BD was added, and the mixture was thoroughly inverted and mixed.
Preferably, the ctDNA mutation degree analysis described in step S2 specifically includes:
respectively extracting molecular tags in paired reads in the FASTQ file and storing the molecular tags as a uBAM file;
comparing the gene sequence of the FASTQ file with a reference genome, removing duplication to obtain a BAM file, and merging the BAM file with the uBAM file to obtain a BAM file containing a molecular tag;
aggregating reads in the BAM file according to the molecular tag and de-duplicating;
obtaining a sample original mutation set in the gene mutation panel region by using a pileup method;
counting the gene mutation parameters in an original mutation set of a tumor sample, wherein the gene mutation parameters comprise: the gene mutation grade, the mutation quantity of each grade of gene and the mutation frequency;
filtering an original mutation set of a tumor sample according to a pre-constructed filtering rule, and counting gene mutation parameters of each sample;
the mutation degree of the plasma sample to be detected is evaluated by using a pre-constructed mutation analysis model aiming at the gene mutation parameters of the tumor sample.
Preferably, the mutation levels include level D, level C, level B and level a, wherein level D includes oncogenes in a preset cancer database, level C includes oncogenes other than level D or other oncogenes whose functionality is determined to be detrimental in a preset cancer database, level B includes exon region genes other than level D and level C, and level a includes genes other than level D, level C and level B;
the gene mutation parameters include: the mutation number of the grade D, the mutation maximum mutation frequency value of the grade D, the mutation number of the grade C, the mutation maximum mutation frequency value of the grade C, the mutation number of the grade B, the mutation maximum mutation frequency value of the grade B, the mutation number of the grade A and the mutation maximum mutation frequency value of the grade A.
Preferably, the filtering the original mutation set of the tumor sample according to the pre-constructed filtering rule includes:
germ line mutations in peripheral blood leukocytes exceeding a given frequency;
blacklist sites repeatedly appearing in a database specific to a specified panel large number of historical samples, and sites in the database where the frequency of people exceeds a set threshold;
background noise baselines were constructed from cfDNA of greater than a given number of healthy human plasma samples under the same sequencing conditions.
Preferably, the step of screening the ctDNA methylation probe comprises the following steps:
collecting a plurality of samples of NKTCL tumor tissues and corresponding plasma samples before treatment, and extracting tumor tissue DNA and plasma ctDNA by using a tissue DNA and plasma ctDNA kit;
detecting methylation levels of candidate methylation sites of tumor tissue DNA and corresponding matched plasma ctDNA by using targeted bisulfite sequencing with the candidate methylation sites as difference sites;
comparing the consistency of tissue specimen DNA and plasma ctDNA methylation level, selecting candidate methylation sites with better consistency and higher detection abundance for subsequent modeling.
In summary, the beneficial effects of the invention are as follows due to the adoption of the technology:
in the invention, the sample size requirement is low, the sequencing library prepared by the method can support subsequent detection, the result of each detection can represent the mutation status of all original ctDNA samples and the methylation modification condition of the coverage area of the enzyme cutting site, and the sensitivity and the specificity cannot be reduced;
in the invention, when the detection is judged based on ctDNA mutation detection positive, the human body tissue can release trace DNA with mutation into plasma, and meanwhile, the instrument can generate technical error to cause false positive, so the sensitivity and the specificity are poor; when the ctDNA CpG point methylation signal in the blood plasma is singly used for detection, the sensitivity and the specificity are low, and different groups of data can be mutually supplemented and corrected, so that the specificity of a model can be improved, the specificity and the prediction effect of the whole diagnosis can be improved, and the diagnosis of a subsequent doctor can be assisted;
in the invention, methylation detection sensitivity and dynamic monitoring value are higher; multiple methylation sites per target gene region can be detected; ctDNA methylation changes from the same type of tumor cells are relatively stable.
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FIG. 1 is a flow chart of a method for detecting mutation and methylation of tumor specific genes in ctDNA according to the present invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, based on the embodiments of the invention, which are apparent to those of ordinary skill in the art without inventive faculty, are intended to be within the scope of the invention. Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, based on the embodiments of the invention, which are apparent to those of ordinary skill in the art without inventive faculty, are intended to be within the scope of the invention.
The invention provides a method for detecting mutation and methylation of tumor specific genes in ctDNA as shown in figure 1, which comprises the following steps:
s1, constructing a sequencing library and creating a tumor sample;
the step S1 of constructing a sequencing library is specifically as follows:
1) Screening for differential ctDNA methylation sites;
2) Screening ctDNA methylation probes; the procedure for screening for differential ctDNA methylation sites was:
collecting plasma samples from 250-350 NKTCL patients and 250-350 normal individuals;
extracting free DNA by using a cfDNA extraction kit, detecting the methylation level of the whole genome ctDNA by high-throughput whole genome methylation sequencing, and establishing a methylation library with about 300 ten thousand loci; the step of extracting free DNA using cfDNA extraction kit includes:
adding 200 μl of blood into a 1.5ml centrifuge tube, adding 400 μl of cell lysate CL, mixing, centrifuging at 10000rpm for 1min, discarding supernatant, adding 200 μl of buffer GS into the cell nucleus precipitate, and shaking to thoroughly mix;
adding 200 μl of buffer GB and 20 μl of protease K premix solution, mixing completely upside down, standing at 55deg.C for 12min until the solution becomes clear;
standing at room temperature for 5-10min, adding 300 μl buffer BD, and mixing completely;
screening to obtain methylation sites with p < 0.05 and the methylation level difference multiple of 2000 for subsequent queue research through methylation level difference analysis;
designing corresponding methylation probes according to the 2000 different methylation sites, and screening methylation sites capable of generating positive and specific PCR amplification signals through PCR verification;
3) Constructing and verifying a ctDNA methylation diagnosis model of NKTCL;
4) Constructing and verifying a ctDNA methylation prognosis model of NKTCL;
the steps of screening ctDNA methylation probes are:
collecting a plurality of samples of NKTCL tumor tissues and corresponding plasma samples before treatment, and extracting tumor tissue DNA and plasma ctDNA by using a tissue DNA and plasma ctDNA kit;
detecting methylation levels of candidate methylation sites of tumor tissue DNA and corresponding matched plasma ctDNA by using targeted bisulfite sequencing with the candidate methylation sites as difference sites; the methylation detection sensitivity and the dynamic monitoring value are higher; multiple methylation sites per target gene region can be detected; ctDNA methylation changes from the same type of tumor cells are relatively stable;
comparing the consistency of the tissue sample DNA and the plasma ctDNA methylation level, and selecting candidate methylation sites with better consistency and higher detection abundance for subsequent modeling;
s2, analyzing the mutation degree of ctDNA, and capturing and sequencing according to a tumor sample and a sequencing library to obtain a FASTQ file, wherein cfDNA in the tumor sample carries a pre-accessed molecular tag;
the ctDNA mutation degree analysis of step S2 specifically includes:
respectively extracting molecular tags in paired reads in the FASTQ file and storing the molecular tags as a uBAM file;
comparing the gene sequence of the FASTQ file with a reference genome, removing duplication to obtain a BAM file, and combining the BAM file with the uBAM file to obtain a BAM file containing molecular tags;
aggregating reads in the BAM file according to the molecular tag and de-duplicating;
obtaining a sample original mutation set in a gene mutation panel region by using a pileup method;
counting the gene mutation parameters in the original mutation set of the tumor sample, wherein the gene mutation parameters comprise: the gene mutation grade, the mutation quantity of each grade of gene and the mutation frequency;
the method comprises the steps of filtering an original mutation set of a tumor sample according to a pre-constructed filtering rule, counting gene mutation parameters of each sample, and filtering the original mutation set of the sample in the process of filtering the original mutation set of the tumor sample according to the pre-constructed filtering rule, wherein the process comprises the following steps:
germ line mutations in peripheral blood leukocytes exceeding a given frequency;
blacklist sites repeatedly appearing in a database specific to a specified panel large number of historical samples, and sites in the database where the frequency of people exceeds a set threshold;
constructing a background noise baseline from cfDNA of greater than a given number of healthy human plasma samples under the same sequencing conditions;
evaluating the mutation degree of the plasma sample to be detected by using a pre-constructed mutation analysis model aiming at the gene mutation parameters of the tumor sample;
further, the gene mutation levels include level D, level C, level B and level a, wherein level D includes oncogenes in a preset cancer database, level C includes oncogenes other than level D in a preset cancer database or other oncogenes whose functionality is judged to be detrimental, level B includes exon region genes other than level D and level C, and level a includes genes other than level D, level C and level B;
the parameters of the gene mutation include: the mutation number of the grade D, the mutation maximum mutation frequency value of the grade D, the mutation number of the grade C, the mutation maximum mutation frequency value of the grade C, the mutation number of the grade B, the mutation maximum mutation frequency value of the grade B, the mutation number of the grade A and the mutation maximum mutation frequency value of the grade A;
s3, performing a plurality of rounds of nested PCR amplification according to the sequencing library, and sequencing a product through a first round of PCR amplification;
s4, taking the product of the first round of PCR as a template, adopting a primer combination B to carry out second round of PCR amplification, sequencing, and analyzing the occurrence of target methylation in the DNA sample according to the result of combining the first round of PCR amplification with the second round of PCR amplification sequencing.
The sequencing library prepared by the method can support subsequent detection, the result of each detection can represent the mutation status of all original ctDNA samples and the methylation modification condition of the coverage area of enzyme cleavage sites, the sensitivity and the specificity are not reduced, and meanwhile, when the detection is judged based on the ctDNA mutation detection positive, the human body tissue can release a trace of DNA with mutation into plasma, and meanwhile, the instrument can generate technical errors to cause false positive, so the sensitivity and the specificity are poor; when the ctDNA CpG point methylation signal in the blood plasma is singly used for detection, the sensitivity and the specificity are low, the different sets of data can be mutually supplemented and corrected, the specificity of the model can be improved, the overall specificity and the prediction effect of diagnosis can be improved, and the diagnosis of a subsequent doctor can be assisted.
The present invention is not limited to the above-mentioned embodiments, and any person skilled in the art, based on the technical solution of the present invention and the inventive concept thereof, can be replaced or changed within the scope of the present invention.
It is noted that relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Claims (8)
1. A method for detecting mutations and methylation of a tumor specific gene in ctDNA comprising the steps of:
s1, constructing a sequencing library and creating a tumor sample;
s2, analyzing the mutation degree of ctDNA, and performing capturing and sequencing according to a tumor sample and a sequencing library to obtain a FASTQ file, wherein cfDNA in the tumor sample carries a pre-accessed molecular tag;
s3, performing a plurality of rounds of nested PCR amplification according to the sequencing library, and sequencing a product through a first round of PCR amplification;
s4, taking the product of the first round of PCR as a template, adopting a primer combination B to carry out second round of PCR amplification, sequencing, and analyzing the occurrence of target methylation in the DNA sample according to the result of combining the first round of PCR amplification with the second round of PCR amplification sequencing.
2. The method for detecting mutation and methylation of tumor specific gene in ctDNA according to claim 1, wherein: the step S1 is characterized by constructing a sequencing library, which comprises the following steps:
1) Screening for differential ctDNA methylation sites;
2) Screening ctDNA methylation probes;
3) Constructing and verifying a ctDNA methylation diagnosis model of NKTCL;
4) Construction and verification of ctDNA methylation prognosis model of NKTCL.
3. A method according to claim 2 for detecting mutations and methylation of tumor specific genes in ctDNA, characterized in that: the process for screening the different ctDNA methylation sites comprises the following steps:
collecting plasma samples from 250-350 NKTCL patients and 250-350 normal individuals;
extracting free DNA by using a cfDNA extraction kit, detecting the methylation level of the whole genome ctDNA by high-throughput whole genome methylation sequencing, and establishing a methylation library with about 300 ten thousand loci;
screening to obtain methylation sites with p < 0.05 and the methylation level difference multiple of 2000 for subsequent queue research through methylation level difference analysis;
corresponding methylation probes are designed according to the 2000 different methylation sites, and methylation sites which can generate positive and specific PCR amplification signals are screened through PCR verification.
4. A method according to claim 3 for detecting mutations and methylation of tumor specific genes in ctDNA, characterized in that: the step of extracting free DNA using the cfDNA extraction kit comprises:
adding 200 μl of blood into a 1.5ml centrifuge tube, adding 400 μl of cell lysate CL, mixing, centrifuging at 10000rpm for 1min, discarding supernatant, adding 200 μl of buffer GS into the cell nucleus precipitate, and shaking to thoroughly mix;
adding 200 μl of buffer GB and 20 μl of protease K premix solution, mixing completely upside down, standing at 55deg.C for 12min until the solution becomes clear;
after 5-10min at room temperature, 300. Mu.l of buffer BD was added, and the mixture was thoroughly inverted and mixed.
5. The method for detecting mutation and methylation of tumor specific gene in ctDNA according to claim 1, wherein: the ctDNA mutation degree analysis described in step S2 specifically includes:
respectively extracting molecular tags in paired reads in the FASTQ file and storing the molecular tags as a uBAM file;
comparing the gene sequence of the FASTQ file with a reference genome, removing duplication to obtain a BAM file, and merging the BAM file with the uBAM file to obtain a BAM file containing a molecular tag;
aggregating reads in the BAM file according to the molecular tag and de-duplicating;
obtaining a sample original mutation set in the gene mutation panel region by using a pileup method;
counting the gene mutation parameters in an original mutation set of a tumor sample, wherein the gene mutation parameters comprise: the gene mutation grade, the mutation quantity of each grade of gene and the mutation frequency;
filtering an original mutation set of a tumor sample according to a pre-constructed filtering rule, and counting gene mutation parameters of each sample;
the mutation degree of the plasma sample to be detected is evaluated by using a pre-constructed mutation analysis model aiming at the gene mutation parameters of the tumor sample.
6. The method for detecting mutation and methylation of tumor specific gene in ctDNA according to claim 5, wherein: the gene mutation grades comprise a grade D, a grade C, a grade B and a grade A, wherein the grade D comprises oncogenes in a preset cancer database, the grade C comprises oncogenes outside the grade D in the preset cancer database or other oncogenes with functions judged to be harmful, the grade B comprises exon region genes outside the grade D and the grade C, and the grade A comprises genes outside the grade D, the grade C and the grade B;
the gene mutation parameters include: the mutation number of the grade D, the mutation maximum mutation frequency value of the grade D, the mutation number of the grade C, the mutation maximum mutation frequency value of the grade C, the mutation number of the grade B, the mutation maximum mutation frequency value of the grade B, the mutation number of the grade A and the mutation maximum mutation frequency value of the grade A.
7. The method for detecting mutation and methylation of tumor specific gene in ctDNA according to claim 5, wherein: the process for filtering the original mutation set of the sample in the process of filtering the original mutation set of the tumor sample according to the pre-constructed filtering rule comprises the following steps:
germ line mutations in peripheral blood leukocytes exceeding a given frequency;
blacklist sites repeatedly appearing in a database specific to a specified panel large number of historical samples, and sites in the database where the frequency of people exceeds a set threshold;
background noise baselines were constructed from cfDNA of greater than a given number of healthy human plasma samples under the same sequencing conditions.
8. A method according to claim 2 for detecting mutations and methylation of tumor specific genes in ctDNA, characterized in that: the screening steps of the ctDNA methylation probes are as follows:
collecting a plurality of samples of NKTCL tumor tissues and corresponding plasma samples before treatment, and extracting tumor tissue DNA and plasma ctDNA by using a tissue DNA and plasma ctDNA kit;
detecting methylation levels of candidate methylation sites of tumor tissue DNA and corresponding matched plasma ctDNA by using targeted bisulfite sequencing with the candidate methylation sites as difference sites;
comparing the consistency of tissue specimen DNA and plasma ctDNA methylation level, selecting candidate methylation sites with better consistency and higher detection abundance for subsequent modeling.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116504318A (en) * | 2023-06-25 | 2023-07-28 | 西安交通大学医学院第一附属医院 | Tumor ctDNA information statistical processing method based on machine learning |
| CN117316289A (en) * | 2023-09-06 | 2023-12-29 | 复旦大学附属华山医院 | Methylation sequencing typing method and system for central nervous system tumor |
| CN117935914A (en) * | 2024-03-22 | 2024-04-26 | 北京求臻医学检验实验室有限公司 | Unknown-meaning clonal hematopoietic recognition and application method thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116504318A (en) * | 2023-06-25 | 2023-07-28 | 西安交通大学医学院第一附属医院 | Tumor ctDNA information statistical processing method based on machine learning |
| CN116504318B (en) * | 2023-06-25 | 2023-08-25 | 西安交通大学医学院第一附属医院 | Tumor ctDNA information statistical processing method based on machine learning |
| CN117316289A (en) * | 2023-09-06 | 2023-12-29 | 复旦大学附属华山医院 | Methylation sequencing typing method and system for central nervous system tumor |
| CN117316289B (en) * | 2023-09-06 | 2024-04-26 | 复旦大学附属华山医院 | Methylation sequencing typing method and system for central nervous system tumor |
| CN117935914A (en) * | 2024-03-22 | 2024-04-26 | 北京求臻医学检验实验室有限公司 | Unknown-meaning clonal hematopoietic recognition and application method thereof |
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