CN115595372B - Methylation detection method of plasma free DNA source, lung cancer diagnosis marker and kit - Google Patents
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Abstract
The invention relates to a methylation detection method of a plasma free DNA source, a lung cancer diagnosis marker and a kit, and belongs to the technical field of gene detection. An optimized MBD-seq technology is used, MBD protein methylation enrichment is carried out on low-entry lung cancer plasma cfDNA, the enriched hypermethylated DNA construction library is subjected to enzymatic conversion, and methylation sequencing is carried out. The optimized MBD enrichment method is higher than the original MBD-seq method in the aspects of enriching methylated DNA and detecting sensitivity and accuracy, reduces nonspecific DNA enrichment, uses cfDNA with lower entry amount, saves sample materials and improves sensitivity. The invention also provides a methylation diagnosis marker analyzed by the detection method, which consists of 20 DMR regions and has better detection accuracy.
Description
Technical Field
The invention relates to a methylation detection method of a plasma free DNA source, a lung cancer diagnosis marker and a kit, and belongs to the technical field of gene detection.
Background
The lung cancer is the most common malignant tumor in the world and is the cancer species with the highest mortality rate in China. Lung cancer can be divided into two broad categories: small cell lung cancer (Small cell lung cancer) and Non-Small cell lung cancer (Non Small cell lung cancer). Non-small cell lung cancer (NSCLC) includes adenocarcinoma, squamous cell carcinoma and large cell carcinoma. Non-small cell lung cancer accounts for approximately 80% of all lung cancers, and adenocarcinoma accounts for approximately 55% of non-small cell lung cancers. Early lung cancer symptoms are not evident, with approximately 75% of patients finding a medium to late stage and a very low 5-year survival rate. Due to the heterogeneity of lung cancer, the prognosis is often not good.
Currently, low dose CT on the chest is the most common method for early screening of lung cancer at present stage, and can detect tumors with a diameter of more than 5 mm. However, this method has problems of radiation exposure, over-diagnosis, insufficient sensitivity, and the like. Liquid biopsy has recently received attention as an in vitro diagnostic technique that detects tumors or tumor cells and tumor DNA fragments that have metastasized to the blood circulation by non-invasive sampling. Has very important significance for screening and detecting early tumors and evaluating the curative effect of the medicament. The most commonly used today is the detection of ctDNA. ctDNA is small piece of DNA released into the human circulation system after tumor cells are ruptured, and may carry genetic information such as tumor mutation, insertion, deletion, copy number variation and the like. Therefore, ctDNA detection based on human blood sources has great significance for early diagnosis of cancer and evaluation of curative effect in cancer treatment process.
DNA methylation is an important component of Epigenetics (Epigenetics). In humans, this occurs frequently in the cytosine of CpG dinucleotides. DNA methylation can cause changes in chromatin structure, DNA conformation, DNA stability, and the way DNA interacts with proteins, thereby controlling gene expression. Numerous studies have shown that there is a confusion of DNA methylation levels and patterns in the development and progression of tumors, including low global genomic methylation levels and hypermethylation of local gene promoter regions. It has been found that methylation abnormality in lung cancer is detected not only in tissues but also in body fluids such as plasma and blood, and that the degree of methylation abnormality increases with the occurrence of lung cancer.
ctDNA methylation studies based on liquid biopsy techniques have become increasingly popular in recent years. Studies have shown that ctDNA methylation can be detected early in tumorigenesis and has good stability, and therefore ctDNA methylation is a valuable marker in tumor fluid biopsies. The methylation detection method includes bisulfite conversion, and a representative method thereof includes WGBS (white-genome bisulfite sequencing). The status is judged, and the MBD-seq (Methylated DNA Binding Domain Sequencing) technology based on protein affinity enrichment is also called protein enrichment whole genome methylation Sequencing. The MBD-seq method utilizes the characteristic that MBD protein families can be specifically combined with methylated DNA so as to enrich CpG high-density methylated DNA fragments on a genome, and combines a high-throughput sequencing technology to sequence the enriched DNA fragments so as to detect the methylation level in the whole genome range. At present, the MBD-seq technology is commonly used for methylated DNA enrichment, the method is based on the fact that high DNA investment is needed, the higher the investment amount is, the better the MBD enrichment effect is, therefore, most commercial kit manufacturers can regulate the input amount of MBD experiments to be more than 1000ng, but the extraction amount of cfDNA from tumor sources is very low, and large-scale methylated DNA enrichment cannot be realized.
Disclosure of Invention
In order to solve the problems of enrichment of cfDNA by MBD-seq and higher detection cost of the traditional WGBS method and the like, the invention optimizes the MBD-seq technology to be used for enrichment methylation detection of cfDNA samples which are from cfDNA of lung cancer plasma and have low input, develops a new cfDNA gene detection technology and assists in early lung cancer detection based on liquid biopsy. The invention also finds the methylation marker with higher lung cancer detection rate, which is obtained by the method.
A methylation detection method of a free DNA source in lung cancer plasma comprises the following steps:
step 3, eluting the magnetic beads obtained by separation in the step 2, wherein the adopted eluent is NaCl solution, the elution process comprises the steps of eluting with a first concentration, then eluting with a second concentration, and collecting all the eluates; the first concentration is 400-600mM, and the second concentration is 800-1200mM;
step 4, adding a sodium acetate solution, absolute ethyl alcohol and glycogen into the eluent to separate out a precipitate, and separating, washing, drying and redissolving to obtain enriched DNA;
and 5, sequencing the enriched DNA obtained in the step 4, and carrying out methylation analysis.
In the step 1, the mixture of magnetic beads and protein is counted by 100. Mu.L, and the mixture contains 5-15. Mu.L of MBD protein and 5-15. Mu.L of streptavidin magnetic beads.
In the step 2, the mixture ratio of the cfDNA to the magnetic bead-protein mixture is as follows: 10-20ng.
In the step 3, the elution times of the first concentration are 1-3 times, and the elution times of the second concentration are 2-4 times.
In the step 4, the volume ratio of the sodium acetate solution to the eluent and the volume ratio of the absolute ethyl alcohol to the eluent are respectively 1:8-12, 1:1-3; the concentration of the sodium acetate solution is 2-4M; the precipitation process is carried out at a temperature below-70 ℃ for at least 2h.
The application of a reagent for detecting a methylation marker derived from plasma free DNA in the preparation of a non-small cell lung cancer diagnostic reagent; the methylation marker is composed of 20 methylation regions on a genome, and the positions of the methylation regions on the genome are shown as follows:
a kit comprising reagents for detecting the above methylated regions.
A methylation detection apparatus for a free DNA source in lung cancer plasma, comprising:
an extraction module: obtaining plasma samples of lung cancer and healthy people, and extracting cfDNA;
MBD-enriched methylated DNA module: for enriching cfDNA by MBD proteins, wherein the enrichment for hypermethylated DNA is performed by elution with an eluent having an optimized ion concentration;
methylation library construction module: constructing a pre-library by connecting the enriched hypermethylated DNA through a joint, and then constructing a methylation library by enzyme conversion and PCR amplification;
a sequencing module: for whole genome methylation high-throughput sequencing of methylation libraries;
a quality control module: the sequence data joint and the low-quality sequence are removed to obtain high-quality data;
a comparison module: for aligning the sequencing data to a reference genome and obtaining the number of reads supporting methylation and the number of unmethylated reads at the CpG sites;
a methylation rate numerical calculation module: for calculating the methylation rate on each methylated region;
enrichment peak identification module: identifying enriched hypermethylated regions for comparing the negative control and the enriched sample;
a difference analysis module: for identifying differentially methylated regions based on enrichment peaks;
lung cancer marker screening module: and (3) establishing classifiers for healthy people and lung cancer patients by utilizing machine learning, screening out specific methylation markers of the lung cancer, and constructing a lung cancer diagnosis regression model.
The methylation rate per methylated region is calculated as the number of methylated reads at all methylated CpG sites in the region divided by the total number of methylated and unmethylated reads.
Identification of enrichment peaks whole genome methylation sequencing samples not enriched for MBD protein can be used.
A computer readable medium bearing a computer program operable to detect the methylation status of lung cancer, the computer program comprising instructions executable to:
step 3, carrying out methylation library building and sequencing on the enriched hypermethylated DNA to obtain sequencing data;
step 4, comparing the sequencing data with a reference genome to obtain CpG sites, and obtaining the number of reads with methylation and the number of reads without methylation on the CpG sites;
step 5, calculating the methylation rate of each methylation region;
step 6, comparing the negative control with the enriched sample to identify the enriched hypermethylation area, and performing gene annotation on the enriched peak;
step 7, performing differential analysis based on the enrichment peaks to identify significant methylation regions in the plasma of healthy people and lung cancer patients;
and 8, establishing classifiers for healthy people and lung cancer patients by using machine learning, screening out specific methylation markers of the lung cancer, and establishing a lung cancer diagnosis regression model.
Has the beneficial effects that: the invention provides a technology for carrying out methylation detection on trace lung cancer plasma cfDNA by using an optimized MBD-seq technology for the first time. The technology can more sensitively enrich methylated DNA, better carry out methylation analysis and save sample materials. The invention obtains the methylation marker of plasma free DNA source, which has higher lung cancer diagnosis accuracy.
Drawings
FIG. 1 shows a flow chart of the present patent;
FIG. 2 shows CpG site methylation profiles before and after optimization of the MBD enrichment method.
Figure 3A shows successful enrichment of the optimized MBD method into hypermethylated regions.
Figure 3B shows the profile of MBD enrichment peaks across the whole genome.
Figure 4 shows a 473 DMR heatmap of plasma free DNA for lung cancer patients and healthy persons.
FIG. 5 shows the best modeling DMR combinatorial screening under the GLM model.
FIG. 6 shows a boxplot of the best 20 methylation markers in lung cancer plasma and normal plasma samples.
Figure 7A shows the area under the receiver operating characteristic curve (ROC) and associated curves (AUC) for the training set for the 20 methylation markers.
Figure 7B shows the area under the receiver operating characteristic curve (ROC) and correlation curve (AUC) for the validation set for 20 methylation markers.
Detailed Description
The optimized MBD enrichment method provided by the invention can better enrich to hypermethylated DNA from low-entry-amount plasma cfDNA, and reduces the nonspecific binding of the DNA and MBD protein in the enrichment process. The optimized enrichment method is mainly realized by adjusting the mass ratio of cfDNA to MBD protein and magnetic beads and selecting an eluent with proper salt ion concentration.
In the following sample enrichment experiments, 2 plasma samples were obtained from patients with early stage non-small cell lung cancer, and plasma was isolated on the day of blood collection. The input amount of the experimental cfDNA for enriching the MBD is 15ng. The test groups were tested using the optimized MBD enrichment method, with the controls using the MBD instructions recommendations for enrichment experiments.
Plasma free DNA was extracted and the QIAamp Circulating Nucleic Acid kit (QIAGEN) was used. This step can be replaced by other kits of the same type that are commercially available.
MBD enrichment of methylated DNA step Using MethylMiner from Invitrogen TM Methylated DNA Enrichment kit.
Comparative example
MBD enrichment experiment, the enrichment process is as follows:
carrying out an experiment according to the requirements of a kit instruction, and comprising the following steps:
1) Taking 10 mu L of streptavidin magnetic beads according to the input amount of 15ng lung cancer cfDNA, putting the streptavidin magnetic beads on a magnetic frame, sucking and removing supernate, adding 100 mu L of 1X Bind/Wash Buffer for washing for 2 times, and finally suspending the magnetic beads by using 100 mu L of 1X Bind/Wash Buffer to prepare magnetic bead suspension;
2) mu.L of MBD protein is taken and added with 1 Xbind/Wash Buffer to make the final volume reach 100. Mu.L. Adding MBD protein into 100 mu L of magnetic bead suspension obtained in the step 1), and performing rotary incubation for 1h at room temperature, wherein the MBD protein and the magnetic beads are combined together to obtain a magnetic bead-protein mixture;
3) Putting the magnetic bead-protein mixture on a magnetic frame, adding 1X Bind/Wash Buffer for 2 times of washing, and finally suspending by using 100 mu L of 1X Bind/Wash Buffer;
4) Adding 15ng of the extracted lung cancer cfDNA into 6 mu L of the suspension obtained in the step 3), supplementing 1X Bind/Wash Buffer to 200 mu L, putting the mixture into a rotary instrument, and incubating the mixture overnight at 4 ℃;
5) Putting the product obtained in the step 4) on a magnetic rack, collecting supernatant, washing for 2 times by using 1X Bind/Wash Buffer, collecting washing liquid for 2 times, and temporarily storing the washing liquid and the supernatant on ice;
6) Eluting the magnetic beads in the step 5) for 2 times by using eluent with the concentration of 250mM NaCl, collecting 250mM eluent, eluting for 2 times by using eluent with the concentration of 500mM salt ions, collecting 500mM eluent, and temporarily storing the eluent on ice;
7) Adding 3M sodium acetate (pH = 5.2) and absolute ethyl alcohol into the recovered supernatant, washing solution and eluent in the steps 5) and 6) according to the proportion of 10. Centrifuging 16000g at 4 deg.C to recover precipitate, and washing the precipitate with 70% ethanol; air drying for 5min, adding 25 μ L enzyme-free water for precipitation and redissolving to obtain high methylated DNA and non/low methylated DNA enriched by MBD, and storing at-20 deg.C.
Example 1
MBD enrichment experiment, the steps are as follows:
1) Taking 5 mu L of streptavidin magnetic beads, putting the streptavidin magnetic beads on a magnetic rack, removing the supernatant by suction, adding 50 mu L of 1X Bind/Wash Buffer for 2 times of Washing, and suspending the magnetic beads by using 50 mu L of 1X Binding Washing Buffer;
2) Mixing 3.5 mu of LMBD protein with magnetic beads, supplementing 1X Bind/Wash Buffer to 100 mu of L, and carrying out rotary incubation for 1h at room temperature to obtain a magnetic bead-protein mixed solution;
3) Putting the magnetic bead-protein mixture on a magnetic frame, adding 1X Bind/Wash Buffer for 2 times of washing, and finally suspending by using 100 mu L of 1X Bind/Wash Buffer;
4) Taking 6 mu L of the magnetic bead-protein mixed solution obtained in the step 3), adding 15ng of the extracted lung cancer cfDNA, supplementing 1X Bind/Wash Buffer to 200 mu L, putting the mixture into a rotary instrument, and incubating overnight at 4 ℃;
5) Putting the product obtained in the step 4) on a magnetic rack, collecting supernatant, washing for 2 times by using 1X Bind/Wash Buffer, collecting washing liquid for 2 times, and temporarily storing the washing liquid and the supernatant on ice;
6) Optimization condition 2: elution was performed using 2 sets of eluents of different salt ion concentrations.
Condition a: eluting the magnetic beads in the step 5) for 2 times by using eluent with the concentration of 250mM NaCl, collecting 250mM eluent, eluting for 2 times by using eluent with the concentration of 500mM NaCl, collecting 500mM eluent, and temporarily storing the eluent on ice to obtain the eluent with the combination of 250+ 500m;
under the condition b, firstly, eluting the magnetic beads in the step 5) for 2 times by using an eluent with the concentration of 500mM salt ions, collecting the 500mM eluent, then eluting for 3 times by using an eluent with the concentration of 1000mM salt ions, collecting the 1000mM eluent, and temporarily storing the eluent on ice to obtain the eluent with the combination of 500+ 1000mM;
7) Adding 3M sodium acetate (pH = 5.2) and absolute ethyl alcohol into the recovered supernatant, washing solution and eluent of the steps 5) and 6) according to the proportion of 10 to 1 to 2, respectively, then adding 1 mu L glycogen (20 mu g/mu L) into the mixture, fully mixing the mixture, standing the mixture at-80 ℃ for at least 2h, and precipitating the enriched DNA. Centrifuging at the temperature of 4 ℃ and 16000g to recover the precipitate, and washing the precipitate by using 70% ethanol; air drying for 5min, adding 25 μ L enzyme-free water for precipitation and redissolving to obtain high methylated DNA and non/low methylated DNA enriched by MBD, and storing at-20 deg.C.
8) Quantification: the enriched hypermethylated DNA under the control condition and the optimized condition is subjected to concentration quantification by using a Qubit 4.0 fluorescence quantifier and a Qubit dsDNA HS assay kit according to the operation requirements of the instruction of the fluorescence quantifier.
Library construction and sequencing procedure:
methylation of the enriched hypermethylated DNA and non/hypomethylated DNA together to create a library was performed using NEBNext from NEB ® Enzymatic Methyl-seq kit and an internal control (a section of unmethylated DNA fragment provided by the kit) was added to the sample. And carrying out methylation-related enzymatic transformation on the constructed library, and carrying out PCR amplification to finally obtain a transformed methylation library. The specific operation steps are shown in the kit instruction.
The plasma free DNA methylation library constructed above was subjected to Genome-wide methylation Sequencing using Novaseq sequencer (WGBS, white Genome Bisulite Sequencing, illumina). After the sequencing is completed and off-line, a fastq file is generated using bcl2 fastq. The data were quality controlled using FastQC software, the adaptor and low quality sequences were removed using Trimmomatic software, and the resulting clearData was aligned to the genome (hg 19) using bismark. Obtaining methylated CpG sites after comparison, determining the methylated reads number of each CpG site and the unmethylated reads number of the site area according to the obtained sites, calculating the methylation rate of each site, and further counting the proportion of the total CpG number of the CpG sites under different levels (figure 2).
Compared with the control group, the optimized MBD condition is obviously enriched to hypermethylated DNA under the condition of 250+500mM salt ion concentration, and the enriched hypermethylated DNA is higher in proportion under the condition of increasing the salt ion concentration of 500+ 1000mM.
Detection of methylated samples and determination of markers:
22 patients with advanced non-small cell lung cancer and 18 healthy human plasma were selected and cfDNA was extracted using QIAamp Circulating Nucleic Acid kit (QIAGEN Co.). Methylated DNA enrichment of 15ng of cfDNA was performed using the MBD enrichment method optimized in example 1 using the salt ion elution conditions of 500+ 1000mM.
Test group:the enriched hypermethylated DNA was all put into methylation for pooling, and NEBNext from NEB was used ® Enzymatic Methyl-seq kit and an internal control (a section of unmethylated DNA fragment provided by the kit) was added to the sample. Performing end repair, adding a base A at the end and adding a connector on the MBD-enriched hypermethylated DNA, and purifying by magnetic beads to obtain a pre-library; carrying out methylation-related enzymatic transformation on the constructed pre-library to obtain a transformed pre-library; and carrying out PCR amplification on the transformed pre-library to obtain an amplified methylated library product. The library is subjected to methylation-related enzymatic transformation, PCR amplification is carried out, and finally a transformed methylation library is obtained.
Control group: methylation pooling was performed directly using 10ng of extracted cfDNA. Adding an internal control into a sample, performing end repairing, adding a base A at the end, adding a joint, and purifying by using magnetic beads to obtain a pre-library; carrying out methylation-related enzymatic transformation on the constructed pre-library to obtain a transformed pre-library; and carrying out PCR amplification on the converted pre-library to obtain an amplified methylation library product.
The specific operation steps are shown in the kit instruction.
The plasma free DNA methylation library constructed above was subjected to Whole Genome methylation Sequencing (WGBS) using Novaseq sequencer from Illumina, a control group was programmed with a throughput of 30G, and a test group was programmed with a throughput of 10G. After the sequencing is completed and off-line, a fastq file is generated using bcl2 fastq. The data were quality controlled using FastQC software, the adaptor and low quality sequences were removed using Trimmomatic software, and the resulting clearData was aligned to the genome (hg 19) using bismark. And after comparison, obtaining methylated CpG sites, and determining the methylated reads number of each CpG site and the unmethylated reads number of the site area according to the obtained sites. The test and control group samples were then compared and the software MACS2 was used to identify enrichment peaks (peaks, fig. 3A and 3B). In FIG. 3A, dark color represents hypermethylation, light color represents hypomethylation, and it can be seen that hypermethylated regions are significantly enriched and peaks are enriched in the whole genome range when comparing the control group and the test group (FIG. 3B).
Further comparison of non-small cell lung cancer patients with healthy human enrichment peaks identified Regions of Differential Methylation (DMRs). One or more CpG sites are contained in a DMR region, and the methylation rate of the DMR is obtained by dividing the sum of the methylation reads of all the CpG sites in the DMR region by the sum of the methylation and the non-methylation total reads of all the CpG sites in the DMR region. By the above sequencing and data processing steps, the methylation rate of each DMR in each cfDNA sample can be obtained.
14 cases and 14 cases are respectively selected from healthy people and non-small cell lung cancer patients as training sets, the remaining 4 cases and 8 cases are used as verification sets, and 473 DMRs with significant differences are screened by comparing enrichment peaks of healthy people and non-small cell lung cancer patients with training set samples (figure 4). Healthy people and non-small cell lung cancer patients can be clustered well, and obvious differential methylation signals can be observed.
A classifier is created by adopting a machine learning method (generalized linear model, glm), the prediction abilities (lung cancer occurrence judgment) of the 473 DMRs obtained by screening are ranked, 10 times of cross validation calculation are performed on a training set, and then candidate DMRs are ranked from front to back according to the total ranking of the 10 times of cross validation importance (fig. 5). The final screening model selected the 20 most accurate DMRs with the highest predictive accuracy for non-small cell lung cancer diagnosis (fig. 6). The genomic positions and base sequences of the 20 most preferred DMRs are shown in the table below.
The 20 DMRs are used as methylation markers for diagnosing the non-small cell lung cancer cfDNA, a diagnostic regression model is established by an H2O method, and diagnostic scoring is carried out on non-small cell lung cancer plasma and healthy human plasma samples of a verification set and a training set. As shown in fig. 7A and 7B, when non-small cell lung cancer and healthy people were distinguished, the training set fruit was stable to 100% and verified to be 100% accurate.
Claims (2)
1. The application of the reagent for detecting the methylation marker derived from plasma free DNA in the preparation of a non-small cell lung cancer diagnostic reagent; the methylation marker is composed of 20 methylation regions on a genome, the genome version number is hg19, and the positions of the methylation regions on the genome are shown as follows:
2. a kit comprising a reagent for detecting the methylated region of claim 1.
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