CN115176034A

CN115176034A - Cancer gene methylation detection system and cancer in-vitro detection method implemented in same

Info

Publication number: CN115176034A
Application number: CN202180016643.3A
Authority: CN
Inventors: 韩晓亮; 李永君; 吴宁宁; 郭媛媛; 王建铭
Original assignee: Biochain Beijing Science and Technology Inc
Current assignee: Biochain Beijing Science and Technology Inc
Priority date: 2020-02-25
Filing date: 2021-02-20
Publication date: 2022-10-11
Also published as: CN112662760A; WO2021169875A1

Abstract

The present invention provides a system for detecting cancer methylation, comprising: a sample collection module for collecting a subject sample; a DNA extraction module for extracting DNA in the purified sample; a library building module for building a DNA library for sequencing against the purified DNA sample; a transformation module for transforming the constructed DNA library with bisulfite; a pre-PCR amplification module for pre-PCR amplifying the bisulfite converted DNA library; a hybrid capture module for hybrid capture of the pre-PCR amplified sample using a probe composition; a post-PCR amplification module for amplifying the hybridization-captured product using PCR; a sequencing module for performing high-throughput next generation sequencing on the hybridization-captured product after PCR amplification; a data analysis module for analyzing the sequencing data to determine a methylation level of the sample; an interpretation module for interpreting the patient's condition based on the methylation level of the sample.

Description

Cancer gene methylation detection system and cancer in-vitro detection method implemented in same

Technical Field

The application relates to a cancer gene methylation detection system, in particular to a system for detecting changes of free DNA methylation levels of tumors of 3 lumen organs such as esophageal cancer, gastric cancer and colorectal cancer based on high throughput sequencing (NGS) and an in-vitro cancer detection method implemented in the system.

Background

The high-throughput sequencing (NGS) technology, which is a revolutionary innovation in the field of modern genomics research, can simultaneously perform sequence analysis on dozens to millions of DNA molecules, which marks the arrival of the post-genome era. By controlling the sequencing depth, different targets such as de novo sequencing and re-sequencing can be realized, and the sequences of genome, transcriptome and methylation set can be analyzed through different pre-treatment.

The current clinical gene detection technologies mainly include Polymerase Chain Reaction (PCR), fluorescence In Situ Hybridization (FISH) and gene chip technologies. The PCR instrument has low equipment price, high sensitivity, simple and quick operation and high clinical popularization, but is limited by technology and can only detect a few genes simultaneously. FISH sensitivity is high, but the manipulation difficulty is large. The gene chip has higher flux than the former two, and can detect a large amount of genes simultaneously. However, the method has the limitations that only known genes or variations can be detected, the accuracy is low, and the false positive is high. The NGS technology has the characteristics of high flux (simultaneously detecting a large number of known and unknown genes and variations), accurate result (higher accuracy than a gene chip), high detection speed, low detection cost shared by each gene and the like, and is gradually applied to the fields of clinical disease detection, monitoring and the like. With the further reduction of the cost of sequencing in the future, NGS will inevitably replace other high-throughput technologies such as gene chips.

Due to the high overall price of current conventional whole genome sequencing, the final price is overwhelming for the consumer if the sequencing depth is increased in order to detect rare variations. Therefore, the capture sequencing of the target sequence becomes a mainstream choice, and the technology is to design a capture probe aiming at a genomic region of interest according to the detection requirement, enrich the target fragment DNA by the hybridization complementary principle, and perform NGS detection subsequently. The strategy can be flexibly customized according to the purpose of research or detection, only a small number of gene regions are selected, the sequencing depth is increased, the variation condition of the target region can be effectively found, and the strategy has high sensitivity and accuracy.

During the development and progression of cancer, genetic information can undergo a series of changes, including mutations, insertions/deletions of DNA, structural variations of chromosomes, copy number variations, and alterations in epigenetic information. During the progression of cancer, variations in DNA sequence occur randomly, and can lead to the development of malignant tumors only when the variations occur in key growth control genes. Most gene expression abnormalities are due to epigenetic changes, usually changes in the level of DNA methylation. The research shows that the change of the gene methylation level is earlier than the gene variation, and the change of the gene methylation is tracked and detected, so that the generation of the cancer can be predicted earlier. In recent years, with the development of genomics, epigenomes of over 30 cancers have been studied. The results show that although DNA methylation is not predominant in every cancer, it is doubtful that changes in the pattern of gene methylation modification alter the propensity of cells to develop and the phenotype of tumors, thereby having a significant impact on the development of most cancers.

The genomic map of cancer (TCGA) in 2018 early published 27 summarized analyses, which have so far made the most comprehensive pan-cancerous genomic analysis of more than 30 cancer data over a period of ten years. After analysis by integrating various data such as chromosomal variation, DNA methylation, RNA and protein, it was found that 33 anatomical cancers can be divided into 28 subtypes according to molecular characteristics. A certain molecular subtype will include 25 cancers in the classical sense. This result suggests that cancers derived from different organs share common molecular characteristics. Meanwhile, cancers derived from the same organ may have different genomic profiles. Therefore, in the near future, the development of cancer screening and diagnosis markers will certainly introduce more pan-cancer concepts, and the markers can be used for researching cancers not only in anatomical levels, but also in molecular levels, and developing pan-cancer markers capable of covering certain molecular typing.

The liquid biopsy is a mode of in vitro diagnosis, non-invasive blood detection is adopted, circulating Tumor Cells (CTC) or circulating tumor DNA (ctDNA) released from tumors or metastatic foci to blood can be monitored, the technology can effectively reduce the damage caused by invasiveness, can realize sampling of all parts of the tumors and all metastatic foci, overcomes the heterogeneity of the tumors (the standard tissue biopsy adopted at present can only reflect the characteristics of a certain part of the tumors), realizes real-time monitoring, has higher sensitivity, and even can predict the parts with pathological changes through genome information, and can effectively prolong the life cycle of patients. According to the advantages, the liquid biopsy can be used for early diagnosis, auxiliary staging, prognosis and recurrence monitoring of tumors, medication guidance and the like. The most commonly used free DNA for liquid biopsy today.

Free DNA (cfDNA) is partially degraded endogenous DNA present in circulating blood free and extracellular. Research shows that during the development of tumor tissue, after tumor cells are apoptotic, DNA is released into plasma, and after degradation, free tumor DNA (ctDNA) is formed. The molecular genetic characteristics (such as gene mutation, microsatellite instability, tumor suppressor gene promoter methylation and the like) of the CtDNA are consistent with those of tumor tissue DNA. In early screening and detection of multiple cancers, the method is simpler and more convenient to collect peripheral blood than other clinical detection means, is easy to popularize to the basic level, and is easier to be accepted by asymptomatic people due to the non-invasive characteristic. Therefore, the detection of the change of the ctDNA methylation level in the plasma can become one of the important means for the early screening and diagnosis of multiple cancers.

By using a target sequence capture technology in combination with NGS to monitor the variation of cfDNA and the change of methylation level, the application of early tumor screening, susceptibility gene monitoring, companion diagnosis, personalized medicine application, prognosis monitoring and the like can be realized. At present, various companies at home and abroad push out different scales, and some of the panel has obtained approved literature numbers of FDA or CFDA aiming at cancer detection panels of different application scenes. For example, foundation one CDx proposed by Foundation Medicine covers 324 genes, IMPACT proposed by the memorial Schlumberger Katelin cancer research center (MSK) covers 468 cancer-related genes, a "human EGFR/ALK/BRAF/KRAS gene mutation joint detection kit" proposed by stone burning Medicine, a "human EGFR, KRAS, BRAF, PIK3CA, ALK, ROS1 gene mutation detection kit" proposed by Norway origin, and the like. Kun remote gene also introduced the product "Changle" for detecting methylation level of colorectal cancer in 2018.

Disclosure of Invention

In view of the above, the present application provides a system for detecting cancer methylation and an in vitro cancer detection method performed in the system, which can be used for early screening of 3 luminal organ tumors such as esophageal cancer, gastric cancer and colorectal cancer, in view of the shortage of the current multiple cancer detection products and the technical limitation. The system and the method performed in the system may: 1) is used for the early screening of asymptomatic crowd with the non-invasive mode, and cancer patient's prognosis detects, reduced the harm that invasive detection caused, 2) has increaseed the sequencing depth, make the width of detecting the gene superior to current technique and product, have the flux high, detection speed is very fast, share the characteristics such as detection cost low of every gene, 3) can realize the sampling to each part of tumour and all metastases, overcome tumour heterogeneity, and 4) have higher sensitivity and accuracy, can realize real-time supervision, it is even possible to effectively prolong patient's life cycle through the position that genome information prediction took place the pathological change.

Specifically, the present application relates to the following:

1. a system for detecting cancer methylation, comprising:

a sample collection module for collecting a subject sample;

a DNA extraction module for extracting and purifying DNA in the sample;

a library building module for building a DNA library for sequencing against the purified DNA sample;

a transformation module for transforming the constructed DNA library with bisulfite;

a pre-PCR amplification module for pre-PCR amplifying the bisulfite-converted DNA library;

a hybrid capture module for hybrid capture of the pre-PCR amplified sample using a probe composition;

a PCR amplification module for amplifying the hybridization-captured product by using PCR;

a sequencing module for performing high-throughput next generation sequencing on the hybridization-captured product after PCR amplification;

a data analysis module for analyzing the sequencing data to determine a methylation level of the sample;

an interpretation module for interpreting the patient's diseased condition based on the methylation level of the sample.

2. The system of item 1, wherein the subject is suspected of having cancer.

3. The system of item 1or 2, wherein the sample collected from the subject is a plasma sample.

4. The system of any one of claims 1-3, the probe composition used in the hybrid capture module comprising:

2 probes targeting a pan-cancer specific region,

n probes targeting a cancer specific region, and

m probes targeting a tissue specific region.

5. The system of any one of claims 1-4, the probe composition used in the hybrid capture module comprising:

hypomethylated probes that hybridize to bisulfite-converted, CG-methylation-free, pan-cancer-specific, and tissue-specific regions of the cancer, and

hypermethylated probes that hybridize to the cancer-specific, pan-cancer-specific, and tissue-specific regions where bisulfite-converted CG is fully methylated.

6. The system of any one of items 1-5, wherein each probe in the probe composition used in the hybrid capture module is 40-60 bp in length.

7. The system according to any one of items 1 to 6, wherein each probe in the probe composition used in the hybrid capture module has a length of 45 to 56bp, preferably 50 to 56bp, and more preferably 50bp.

8. The system of any one of claims 1-7, wherein n probes in the probe composition used in the hybrid capture module target a cancer specific region,

wherein n is an integer selected from any of 1 to 192;

wherein the cancer specific region is selected from the group consisting of Seq ID No.: 1-62.

9. The system of any one of claims 1-8, wherein m probes in the probe composition used in the hybrid capture module target the tissue-specific region,

wherein m is an integer selected from any of 1 to 44;

wherein the tissue specific region is selected from the group consisting of Seq ID No.: 65-83.

10. The system of item 5, wherein in the hybrid capture module the hypomethylated probes comprise probes that target cancer-specific regions Seq ID No.:84-180, a probe Seq ID No.:181-182, and a probe Seq ID No.: 183-204.

11. The system of item 5, wherein in the hybrid capture module the hypermethylated probes comprise probes that target cancer-specific regions Seq ID No.:205-301, probe Seq ID No.:302-303, and a probe Seq ID No.: 304-325.

12. The system of any of claims 1-11, the interpretation module comprising:

(1) A pan cancer interpretation module for comparing the pan cancer specific region database and performing interpretation to confirm whether the subject has cancer;

(2) A cancer interpretation module for comparing the cancer specific region database and performing interpretation to further confirm the cancer suffered by the subject as one of several suspected cancers; and

(3) And the tissue specificity interpretation module is used for comparing the tissue specificity region database and performing interpretation so as to confirm the cancer-suffering part of the subject.

13. The system of item 12, the pan cancer interpretation module comprising performing the interpretation of: judging the pan-cancer specific region Seq ID No.:63, and judging whether or not the methylation level of the pan cancer-specific region Seq ID No.:64, if Seq ID No.: methylation level of 63 is 55% or more and Seq ID No.:64 greater than or equal to 60%, the patient is read as having cancer.

14. The system of item 12, the cancer interpretation module comprising performing the interpretation of: the patient is judged to have any one of the tissue-specific cancers if the methylation level of the region targeted by n1 probes among the n probes targeting the cancer-specific region is equal to or greater than the respective threshold value, and n1/n is equal to or greater than 20%, preferably n1/n is equal to or greater than 30%.

15. The system of item 12, the tissue-specific interpretation module comprising performing the interpretation of: if the methylation level of the region targeted by m1 probes among the m probes targeting the tissue-specific region is greater than or equal to the respective threshold value, the tissues targeted by the m1 probes greater than or equal to the respective threshold value are further analyzed and the number of probes greater than or equal to the threshold value in each tissue is counted, and the tissue suffering from cancer of the patient is interpreted as the tissue with the highest methylation level of the probes greater than or equal to the threshold value.

16. An in vitro method for detecting cancer in a subject, comprising the steps of:

collecting a subject sample;

extracting and purifying DNA in the sample;

constructing a DNA library for sequencing against the purified DNA sample;

transforming said constructed DNA library with bisulfite;

pre-PCR amplifying the bisulfite-converted DNA library;

performing hybridization capture on the sample subjected to the pre-PCR amplification by using the probe composition;

amplifying the product after hybridization capture by utilizing PCR;

performing high-throughput second-generation sequencing on a product obtained after hybridization and capture after PCR amplification;

analyzing the sequencing data to determine the methylation level of the sample;

interpreting the patient's condition based on the methylation level of the sample.

17. The method of item 16, wherein the subject is suspected of having cancer.

18. The method of claim 16 or 17, wherein the sample from the subject is a plasma sample.

19. The method of any one of claims 16-18, wherein the conversion is treated with bisulfite.

20. The method of any one of claims 16-18, the probe composition comprising:

2 probes targeting a pan-cancer specific region,

n probes targeting a cancer specific region, and

m probes targeting a tissue specific region.

21. The method of any one of claims 16-20, the probe composition comprising:

hypomethylated probes that hybridize to bisulfite-converted, CG-methylation-free, cancer-specific, pan-cancer-specific, and tissue-specific regions of the subject, and

22. The method of any one of claims 16-21, wherein each probe in the probe composition is 40-60 bp in length.

23. The method according to any one of items 16 to 22, wherein each probe in the probe composition has a length of 45 to 56bp, preferably 50 to 56bp, and more preferably 50bp.

24. The method of any one of claims 16-23, wherein n probes in the probe composition target a cancer specific region,

wherein n is an integer selected from any of 1 to 192;

25. The method of any one of claims 16-24, wherein m probes in the probe composition target the tissue-specific region,

wherein m is an integer selected from any of 1 to 44;

26. The method of item 21, wherein the hypomethylated probe comprises a probe Seq ID No.:84-180, a probe Seq ID No. targeting a pan cancer specific region: 181-182, and a probe Seq ID No.: 183-204.

27. The method of item 21, wherein the hypermethylated probe comprises a probe Seq ID No.:205-301, probe Seq ID No.:302-303, and a probe Seq ID No.: 304-325.

28. The method of any one of items 16-27, the interpreting comprising the steps of:

(1) Comparing the pan-cancer specific region database and performing interpretation to confirm whether the subject has cancer;

(2) Comparing the cancer specific region database, and judging to confirm that the cancer suffered by the subject is one of several suspected cancers;

(3) Comparing the tissue specific region database, and performing interpretation to confirm the cancer part of the subject.

29. The method of item 28, wherein step (1) comprises making the interpretation: judging the pan-cancer specific region Seq ID No.:63, and judging whether or not the methylation level of the pan cancer-specific region Seq ID No.:64 is greater than or equal to 60% if Seq ID No.: methylation level of 63 is 55% or more and Seq ID No.:64 greater than or equal to 60%, the patient is read as having cancer.

30. The method of item 28, wherein step (2) comprises making the interpretation: if the methylation level of the region targeted by n1 probes is more than or equal to the respective threshold value in n probes targeting the cancer specific region, and n1/n is more than or equal to 20%, preferably n1/n is more than or equal to 30%, the patient is judged to have any one of the tissue specific cancers, and then the possibility of each cancer is judged according to pattern recognition.

31. The method of item 28, wherein step (3) comprises performing the interpretation: if the methylation level of the region targeted by m1 probes in the m probes targeting the tissue-specific region is greater than or equal to the respective threshold value, the tissues targeted by the m1 probes greater than or equal to the respective threshold value are further analyzed and the number of probes greater than or equal to the threshold value in each tissue is counted, and the tissue of the patient suffering from the cancer is judged to be the tissue with the highest number of probes with the methylation level greater than or equal to the threshold value.

Due to the limitations of existing cancer detection technologies, there is a need to develop a system for detecting cancer methylation and an in vitro cancer detection method performed in the system, which have the following advantages:

the liquid biopsy belongs to noninvasive tumor detection, and is suitable for asymptomatic people and patient groups who cannot obtain tissue samples.

2, the methylation level change of 3 common tubular organ tumors in China can be detected simultaneously, and more than 80 percent of cancer attack population is covered.

3 to increase the accuracy of the detection, the mean sequencing depth was over 5000X for each cancer.

4, the screening of all high-incidence cancers can be completed for the testee at one time, the detection efficiency is improved, and the average price of each marker is lower than that of the detection of the existing single marker in the market.

5 for enterprises, the screening of main cancers can be completed by using one Panel, so that the probe synthesis cost is saved, the experimental process can be simplified, and the operation of experimenters is facilitated.

6 can also be used in principle for cancer monitoring for the prognosis of cancer patients.

Drawings

Fig. 1 is an operation flow of the present application.

Detailed Description

The present application provides a system for cancer gene methylation detection and a method for cancer in vitro detection performed in the system. The free DNA methylation level change of 3 tubular cavity organ tumors such as esophageal cancer, gastric cancer, colorectal cancer and the like is detected based on a high throughput sequencing (NGS) method. The kit can detect the methylation level change of 3 common cancers simultaneously in a non-invasive mode, has high sensitivity and accuracy, deep sequencing depth and low cost, and is suitable for asymptomatic groups and patient groups incapable of obtaining tissue samples and cancer monitoring of cancer patient prognosis.

Definition of

Unless specifically defined elsewhere herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

The probe is single-stranded or double-stranded DNA with the length of tens to hundreds or even thousands of base pairs, and can be combined (hybridized) with complementary non-labeled single-stranded DNA or RNA in a sample to be detected by hydrogen bonds to form a double-stranded complex (hybrid) by utilizing the denaturation and renaturation of molecules and the high accuracy of base complementary pairing. After washing off the probe which is not coupled, the result of hybridization reaction can be detected by a detection system such as autoradiography or enzyme-linked reaction. In the present application, the region to which the probe complementarily binds or hybridizes is the specific target region. Multiple probes are combined into a probe composition.

A cancer specific region is one in which the methylation level of the region is significantly different in a small percentage of cancer species as compared to normal control tissue.

Pan-cancer specific regions refer to regions of significant difference in methylation levels in most cancer species compared to normal control tissues.

A tissue-specific region is one in which the methylation level of the region is significantly different in a particular tissue as compared to other tissues.

DNA methylation refers to the methylation process of the 5 th carbon atom on cytosine in CpG dinucleotide, and is an important epigenetic mechanism which can be inherited to new filial generation DNA along with the DNA replication process under the action of DNA methyltransferase as a stable modification state. Aberrant methylation includes hypermethylation of cancer suppressor genes and DNA repair genes, hypomethylation of repeat DNA, loss of imprinting of certain genes, which is associated with the development of a variety of tumors.

Herein, panel refers to the probe composition used herein.

The technical solution of the present application is described in detail below.

This document relates to a system for detecting cancer methylation comprising: a sample collection module for collecting a sample of a subject; a DNA extraction module for extracting and purifying DNA in the sample; a library building module for building a DNA library for sequencing against the purified DNA sample; a transformation module for transforming the constructed DNA library with bisulfite; a pre-PCR amplification module for pre-PCR amplifying the bisulfite-converted DNA library; a hybrid capture module for hybrid capture of the pre-PCR amplified sample using a probe composition; a post-PCR amplification module for amplifying the hybridization-captured product using PCR; a sequencing module for performing high-throughput next generation sequencing on the hybridization-captured product after PCR amplification; a data analysis module for analyzing the sequencing data to determine a methylation level of the sample; an interpretation module for interpreting the patient's diseased condition based on the methylation level of the sample.

In the above system referred to herein, the sample collection module refers to a module integrated in the system for automatically collecting a sample to be tested, i.e. blood or plasma of a subject, and containing the sample to be tested;

the DNA extraction module is a module in which a sample to be detected enters and DNA is extracted by a known conventional method, for example, the DNA is released by heating and cracking, and then the DNA enters the next module for reaction and detection through filtration and impurity removal;

the library building module is a module for performing end repair and base A addition on a target fragment extracted from the DNA extraction module, connecting the target fragment with a linker to form a ligation product, and performing amplification, separation and purification on the ligation product to form a DNA sequencing library;

the transformation module is a module for transforming the DNA library constructed in the library construction module by using bisulfite by using a known transformation method;

the pre-PCR amplification module refers to a module that amplifies a bisulfite-transformed DNA library in a transformation module to an amount that can be captured by hybridization with a probe composition described herein using known methods;

the hybridization capture module is a module for performing hybridization capture on a sample amplified by pre-PCR by using a probe composition described herein by using a traditional liquid phase hybridization capture system;

the PCR amplification module refers to a module for amplifying a product captured by hybridization by using a known amplification method;

the sequencing module is a module for sequencing a product obtained after PCR amplification and hybridization capture by utilizing a conventional high-throughput second-generation sequencing platform, such as an Illumina platform;

the data analysis module is a module for analyzing the sequencing data according to a multi-cancer methylation analysis database obtained by integrating public data and the existing sequencing data to determine the methylation level of the sample;

the interpretation module is a module which is used for carrying out pattern recognition according to a database by using a computer based on the methylation level data of the sample obtained from the data analysis module, constructing an interpretation whole cancer risk model, and analyzing the cancer risk and the tissue source of a detection object, thereby interpreting the diseased condition of the patient.

The present disclosure also relates to a system for detecting cancer methylation, comprising: a sample collection module for collecting a sample of a subject; a DNA extraction module for extracting and purifying DNA in the sample; a library building module for building a DNA library for sequencing against the purified DNA sample; a transformation module for transforming the constructed DNA library with bisulfite; a pre-PCR amplification module for pre-PCR amplifying the bisulfite-converted DNA library; a hybrid capture module for hybrid capture of the pre-PCR amplified sample using a probe composition; a post-PCR amplification module for amplifying the hybridization-captured product using PCR; a sequencing module for performing high-throughput next generation sequencing on the hybridization-captured product after PCR amplification; a data analysis module for analyzing the sequencing data to determine a methylation level of the sample; the pan cancer interpretation module is used for comparing the pan cancer specific region database and interpreting; the cancer interpretation module is used for comparing the cancer specific region database and interpreting; and the tissue specificity interpretation module is used for comparing and interpreting the tissue specificity region database.

The present disclosure also relates to a system for detecting cancer methylation, comprising: a sample collection module for collecting a sample of a subject; a DNA extraction module for extracting and purifying DNA in the sample; a library building module for building a DNA library for sequencing against the purified DNA sample; a transformation module for transforming the constructed DNA library with bisulfite; a pre-PCR amplification module for pre-PCR amplifying the bisulfite-converted DNA library; a hybrid capture module for hybrid capture of the pre-PCR amplified sample using a probe composition; a post-PCR amplification module for amplifying the hybridization-captured product using PCR; a sequencing module for performing high-throughput next generation sequencing on the hybridization-captured product after PCR amplification; a data analysis module for analyzing the sequencing data to determine a methylation level of the sample; a pan cancer interpretation module which judges the pan cancer specific region Seq ID No. by comparing a pan cancer specific region database: 63, and the pan cancer-specific region Seq ID No.:64, if Seq ID No.: methylation level of 63 is 55% or more and Seq ID No.:64 greater than or equal to 60%, and the patient is read as having cancer; a cancer interpretation module that performs the interpretation of: interpreting that the patient has any one of the tissue-specific cancers if the methylation level of the region targeted by n1 probes among the n probes targeting the cancer-specific region is greater than or equal to the respective threshold value, and n1/n is greater than or equal to 20%, preferably n1/n is greater than or equal to 30%; a tissue-specific interpretation module that performs the following interpretation: if the methylation level of the region targeted by m1 probes is greater than or equal to the respective threshold value in the m probes targeting the tissue-specific region, the tissues targeted by the m1 probes greater than or equal to the respective threshold value are further analyzed, the number of probes greater than or equal to the threshold value in each tissue is counted, and the tissue suffering from cancer of the patient is judged to be the tissue with the greatest number of probes having the methylation level greater than or equal to the threshold value.

Specifically, for example, when m1 is 6, it is further judged that, if 5 of the probes are probes targeting the stomach and 1 of the probes is a probe targeting the pancreas, it is judged that the patient suffering from the cancer is the stomach. If it is further judged that when m1 is 6, 3 of the probes are probes for targeting the stomach and 3 of the probes are probes for targeting the pancreas, it is judged that the patient has the cancer that is the stomach and the pancreas. For example, when m1 is 6, if 2 probes are probes targeting esophageal cancer, 2 probes are probes targeting gastric cancer, and 2 probes are probes targeting colorectal cancer, the patient is judged to have cancer of esophagus, stomach, and colorectal cancer.

See table 1 below, where the methylation thresholds for each of all target regions are listed in table 1.

As shown in Table 1, wherein each of Seq ID No.84, seq ID No.85 and Seq ID No.86 is a hypomethylated probe targeting the target region shown in Seq ID No.1, and each of Seq ID No.205, seq ID No.206 and Seq ID No.207 is a hypermethylated probe targeting the target region shown in Seq ID No. 1. Seq ID No.87 and Seq ID No.88 are both hypomethylated probes targeting the target region shown in Seq ID No.2, and Seq ID No.208 and Seq ID No.209 are both hypermethylated probes targeting the target region shown in Seq ID No. 2. Seq ID No.89 and Seq ID No.90 are both hypomethylated probes targeting the target region shown in Seq ID No.3, and Seq ID No.210 and Seq ID No.211 are both hypermethylated probes targeting the target region shown in Seq ID No. 3. Seq ID No.91 is a hypomethylated probe targeting the target region shown in Seq ID No.4, and Seq ID No.212 is a hypermethylated probe targeting the target region shown in Seq ID No. 4. Seq ID No.92 and Seq ID No.93 are both hypomethylated probes targeting the target region shown in Seq ID No.5, and Seq ID No.213 and Seq ID No.214 are both hypermethylated probes targeting the target region shown in Seq ID No. 3. Seq ID No.94 is a hypomethylated probe targeting the target region shown in Seq ID No.6, and Seq ID No.215 is a hypermethylated probe targeting the target region shown in Seq ID No. 6. Seq ID No.95 is a hypomethylated probe targeting the target region shown in Seq ID No.7, and Seq ID No.216 is a hypermethylated probe targeting the target region shown in Seq ID No. 7. Seq ID No.96 and Seq ID No.97 are both hypomethylated probes targeting the target region shown in Seq ID No.8, and Seq ID No.217 and Seq ID No.218 are both hypermethylated probes targeting the target region shown in Seq ID No. 8. Seq ID No.98, seq ID No.99 and Seq ID No.100 are hypomethylated probes targeting the target region shown in Seq ID No.9, and Seq ID No.219, seq ID No.220 and Seq ID No.221 are hypermethylated probes targeting the target region shown in Seq ID No. 9. Seq ID No.101 is a hypomethylated probe targeting the target region shown in Seq ID No.10, and Seq ID No.222 is a hypermethylated probe targeting the target region shown in Seq ID No. 10. Seq ID No.102 is a hypomethylated probe that targets the target region shown in Seq ID No.11, and Seq ID No.223 is a hypermethylated probe that targets the target region shown in Seq ID No. 11. Seq ID No.103 is a hypomethylated probe targeting the target region shown in Seq ID No.12, and Seq ID No.224 is a hypermethylated probe targeting the target region shown in Seq ID No. 12. Seq ID No.104 is a hypomethylated probe targeting the target region shown in Seq ID No.13, and Seq ID No.225 is a hypermethylated probe targeting the target region shown in Seq ID No. 13. Seq ID No.105 and Seq ID No.106 are both hypomethylated probes targeting the target region shown in Seq ID No.14, and Seq ID No.226 and Seq ID No.227 are both hypermethylated probes targeting the target region shown in Seq ID No. 14. Seq ID No.107 and Seq ID No.108 are both hypomethylated probes targeting the target region shown in Seq ID No.15, and Seq ID No.228 and Seq ID No.229 are both hypermethylated probes targeting the target region shown in Seq ID No. 15. Seq ID No.109 and Seq ID No.110 are both hypomethylated probes targeting the target region shown in Seq ID No.16, and Seq ID No.230 and Seq ID No.231 are both hypermethylated probes targeting the target region shown in Seq ID No. 16. Seq ID No.111 and Seq ID No.112 are both hypomethylated probes targeting the target region shown in Seq ID No.17, and Seq ID No.232 and Seq ID No.233 are both hypermethylated probes targeting the target region shown in Seq ID No. 17. Seq ID No.113 is a hypomethylated probe targeting the target region shown in Seq ID No.18, and Seq ID No.234 is a hypermethylated probe targeting the target region shown in Seq ID No. 18. Seq ID No.114 and Seq ID No.115 are both hypomethylated probes targeting the target region shown in Seq ID No.19, and Seq ID No.235 and Seq ID No.236 are both hypermethylated probes targeting the target region shown in Seq ID No. 19. Seq ID No.116 is a hypomethylated probe targeting the target region shown in Seq ID No.20, and Seq ID No.237 is a hypermethylated probe targeting the target region shown in Seq ID No. 20. Seq ID No.117 is a hypomethylated probe that targets the target region shown in Seq ID No.21, and Seq ID No.238 is a hypermethylated probe that targets the target region shown in Seq ID No. 21. Seq ID No.118 is a hypomethylated probe targeting the target region shown in Seq ID No.22, and Seq ID No.239 is a hypermethylated probe targeting the target region shown in Seq ID No. 22. Seq ID No.119, seq ID No.120 and Seq ID No.121 are hypomethylated probes targeting the target region shown in Seq ID No.23, and Seq ID No.240, seq ID No.241 and Seq ID No.242 are hypermethylated probes targeting the target region shown in Seq ID No. 23. Seq ID No.122 and Seq ID No.123 are both hypomethylated probes targeting the target region shown in Seq ID No.24, and Seq ID No.243 and Seq ID No.244 are both hypermethylated probes targeting the target region shown in Seq ID No. 24. Seq ID No.124 and Seq ID No.125 are both hypomethylated probes targeting the target region shown in Seq ID No.25, and Seq ID No.245 and Seq ID No.246 are both hypermethylated probes targeting the target region shown in Seq ID No. 25. Seq ID No.126 and Seq ID No.127 are both hypomethylated probes targeting the target region shown in Seq ID No.26, and Seq ID No.247 and Seq ID No.248 are both hypermethylated probes targeting the target region shown in Seq ID No. 26. Seq ID No.128 is a hypomethylated probe targeting the target region shown in Seq ID No.27, and Seq ID No.249 is a hypermethylated probe targeting the target region shown in Seq ID No. 27. Seq ID No.129 and Seq ID No.130 are both hypomethylated probes targeting the target region shown in Seq ID No.28, and Seq ID No.250 and Seq ID No.251 are both hypermethylated probes targeting the target region shown in Seq ID No. 28. Seq ID No.131 and Seq ID No.132 are both hypomethylated probes targeting the target region shown in Seq ID No.29, and Seq ID No.252 and Seq ID No.253 are both hypermethylated probes targeting the target region shown in Seq ID No. 29. Seq ID No.133 is a hypomethylated probe targeting the target region shown in Seq ID No.30, and Seq ID No.254 is a hypermethylated probe targeting the target region shown in Seq ID No. 30. Seq ID No.134 and Seq ID No.135 are both hypomethylated probes targeting the target region shown in Seq ID No.31, and Seq ID No.255 and Seq ID No.256 are both hypermethylated probes targeting the target region shown in Seq ID No. 31. Seq ID No.136 is a hypomethylated probe targeting the target region shown in Seq ID No.32, and Seq ID No.257 is a hypermethylated probe targeting the target region shown in Seq ID No. 32. Seq ID No.137 and Seq ID No.138 are both hypomethylated probes that target the target area shown in Seq ID No.33, and Seq ID No.258 and Seq ID No.259 are both hypermethylated probes that target the target area shown in Seq ID No. 33. Seq ID No.139 is a hypomethylated probe that targets the target region shown in Seq ID No.34, and Seq ID No.260 is a hypermethylated probe that targets the target region shown in Seq ID No. 34. Seq ID No.140 and Seq ID No.141 are both hypomethylated probes targeting the target region shown in Seq ID No.35, and Seq ID No.261 and Seq ID No.262 are both hypermethylated probes targeting the target region shown in Seq ID No. 35. Seq ID No.142 is a hypomethylated probe targeting the target region shown in Seq ID No.36, and Seq ID No.263 is a hypermethylated probe targeting the target region shown in Seq ID No. 36. Seq ID No.143 and Seq ID No.144 are both hypomethylated probes targeting the target region shown in Seq ID No.37, and Seq ID No.264 and Seq ID No.265 are both hypermethylated probes targeting the target region shown in Seq ID No. 37. Seq ID No.145 and Seq ID No.146 are both hypomethylated probes targeting the target region shown in Seq ID No.38, and Seq ID No.266 and Seq ID No.267 are both hypermethylated probes targeting the target region shown in Seq ID No. 38. Seq ID No.147 is a hypomethylated probe that targets the target region shown in Seq ID No.39, and Seq ID No.268 is a hypermethylated probe that targets the target region shown in Seq ID No. 39. Seq ID No.148 and Seq ID No.149 are both hypomethylated probes targeting the target region shown in Seq ID No.40, and Seq ID No.269 and Seq ID No.270 are both hypermethylated probes targeting the target region shown in Seq ID No. 40. Seq ID No.150 is a hypomethylated probe targeting the target region shown in Seq ID No.41, and Seq ID No.271 is a hypermethylated probe targeting the target region shown in Seq ID No. 41. Seq ID No.151 is a hypomethylated probe targeting the target region shown in Seq ID No.42, and Seq ID No.272 is a hypermethylated probe targeting the target region shown in Seq ID No. 42. Seq ID No.152 is a hypomethylated probe targeting the target region shown in Seq ID No.43, and Seq ID No.273 is a hypermethylated probe targeting the target region shown in Seq ID No. 43. Seq ID No.153 is a hypomethylated probe targeting the target region shown in Seq ID No.44, and Seq ID No.274 is a hypermethylated probe targeting the target region shown in Seq ID No. 44. Seq ID No.154 and Seq ID No.155 are both hypomethylated probes targeting the target region shown in Seq ID No.45, and Seq ID No.275 and Seq ID No.276 are both hypermethylated probes targeting the target region shown in Seq ID No. 45. Seq ID No.156 is a hypomethylated probe targeting the target region shown in Seq ID No.46, and Seq ID No.277 is a hypermethylated probe targeting the target region shown in Seq ID No. 46. Seq ID No.157 is a hypomethylated probe targeting the target region shown in Seq ID No.47, and Seq ID No.278 is a hypermethylated probe targeting the target region shown in Seq ID No. 47. Seq ID No.158 is a hypomethylated probe that targets the target region shown in Seq ID No.48, and Seq ID No.279 is a hypermethylated probe that targets the target region shown in Seq ID No. 48. Seq ID No.159 is a hypomethylated probe targeting the target region shown in Seq ID No.49, and Seq ID No.280 is a hypermethylated probe targeting the target region shown in Seq ID No. 49. Seq ID No.160 is a hypomethylated probe targeting the target region shown in Seq ID No.50, and Seq ID No.281 is a hypermethylated probe targeting the target region shown in Seq ID No. 50. Seq ID No.161 and Seq ID No.162 are both hypomethylated probes targeting the target region shown in Seq ID No.51, and Seq ID No.282 and Seq ID No.283 are both hypermethylated probes targeting the target region shown in Seq ID No. 51. Seq ID No.163 is a hypomethylated probe targeting the target region shown in Seq ID No.52, and Seq ID No.284 is a hypermethylated probe targeting the target region shown in Seq ID No. 52. Seq ID No.164 and Seq ID No.165 are both hypomethylated probes targeting the target region shown in Seq ID No.53, and Seq ID No.285 and Seq ID No.286 are both hypermethylated probes targeting the target region shown in Seq ID No. 53. Seq ID No.166 is a hypomethylated probe that targets the target region shown in Seq ID No.54, and Seq ID No.287 is a hypermethylated probe that targets the target region shown in Seq ID No. 54. Seq ID No.167 and Seq ID No.168 are both hypomethylated probes targeting the target region shown in Seq ID No.55, and Seq ID No.288 and Seq ID No.289 are both hypermethylated probes targeting the target region shown in Seq ID No. 55. Seq ID No.169 and Seq ID No.170 are both hypomethylated probes targeting the target region shown in Seq ID No.56, and Seq ID No.290 and Seq ID No.291 are both hypermethylated probes targeting the target region shown in Seq ID No. 56. Seq ID No.171 and Seq ID No.172 are hypomethylated probes targeting the target region shown in Seq ID No.57, and Seq ID No.292 and Seq ID No.293 are hypermethylated probes targeting the target region shown in Seq ID No. 57. Seq ID No.173 and Seq ID No.174 are both hypomethylated probes targeting the target region shown in Seq ID No.58, and Seq ID No.294 and Seq ID No.295 are both hypermethylated probes targeting the target region shown in Seq ID No. 58. Seq ID No.175 and Seq ID No.176 are both hypomethylated probes targeting the target region shown in Seq ID No.59, and Seq ID No.296 and Seq ID No.297 are both hypermethylated probes targeting the target region shown in Seq ID No. 59. Seq ID No.177 is a hypomethylated probe targeting the target region shown in Seq ID No.60, and Seq ID No.298 is a hypermethylated probe targeting the target region shown in Seq ID No. 60. Seq ID No.178 and Seq ID No.179 are hypomethylated probes targeting the target region shown in Seq ID No.61, and Seq ID No.299 and Seq ID No.300 are hypermethylated probes targeting the target region shown in Seq ID No. 61. Seq ID No.180 is a hypomethylated probe that targets the target region shown in Seq ID No.62, and Seq ID No.301 is a hypermethylated probe that targets the target region shown in Seq ID No. 62. Seq ID No.181 is a hypomethylated probe targeting the target region shown in Seq ID No.63, and Seq ID No.302 is a hypermethylated probe targeting the target region shown in Seq ID No. 63. Seq ID No.182 is a hypomethylated probe targeting the target region shown in Seq ID No.64, and Seq ID No.303 is a hypermethylated probe targeting the target region shown in Seq ID No. 64. Seq ID No.183 is a hypomethylated probe targeting the target region shown in Seq ID No.65, and Seq ID No.304 is a hypermethylated probe targeting the target region shown in Seq ID No. 65. Seq ID No.184 is a hypomethylated probe targeting the target region shown in Seq ID No.66, and Seq ID No.305 is a hypermethylated probe targeting the target region shown in Seq ID No. 66. Seq ID No.185 is a hypomethylated probe targeting the target region shown in Seq ID No.67, and Seq ID No.306 is a hypermethylated probe targeting the target region shown in Seq ID No. 67. Seq ID No.186 is a hypomethylated probe targeting the target region shown in Seq ID No.68, and Seq ID No.307 is a hypermethylated probe targeting the target region shown in Seq ID No. 68. Seq ID No.187 is a hypomethylated probe targeting the target region shown in Seq ID No.69, and Seq ID No.308 is a hypermethylated probe targeting the target region shown in Seq ID No. 69. Seq ID No.188 is a hypomethylated probe targeting the target region shown in Seq ID No.70, and Seq ID No.309 is a hypermethylated probe targeting the target region shown in Seq ID No. 70. Seq ID No.189 is a hypomethylated probe that targets the target region shown in Seq ID No.71, and Seq ID No.310 is a hypermethylated probe that targets the target region shown in Seq ID No. 71. Seq ID No.190 is a hypomethylated probe targeting the target region shown in Seq ID No.72, and Seq ID No.311 is a hypermethylated probe targeting the target region shown in Seq ID No. 72. Seq ID No.191 is a hypomethylated probe targeting the target region shown in Seq ID No.73, and Seq ID No.312 is a hypermethylated probe targeting the target region shown in Seq ID No. 73. Seq ID No.192 is a hypomethylated probe targeting the target region shown in Seq ID No.74, and Seq ID No.313 is a hypermethylated probe targeting the target region shown in Seq ID No. 74. Seq ID No.193 is a hypomethylated probe that targets the target region shown in Seq ID No.75, and Seq ID No.314 is a hypermethylated probe that targets the target region shown in Seq ID No. 75. Seq ID No.194 is a hypomethylated probe targeting the target region shown in Seq ID No.76, and Seq ID No.315 is a hypermethylated probe targeting the target region shown in Seq ID No. 76. Seq ID No.195 is a hypomethylated probe targeting the target region shown in Seq ID No.77, and Seq ID No.316 is a hypermethylated probe targeting the target region shown in Seq ID No. 77. Seq ID No.196 is a hypomethylated probe targeting the target region shown in Seq ID No.78, and Seq ID No.317 is a hypermethylated probe targeting the target region shown in Seq ID No. 78. Seq ID No.197 is a hypomethylated probe targeting the target region shown in Seq ID No.79, and Seq ID No.318 is a hypermethylated probe targeting the target region shown in Seq ID No. 79. Seq ID No.198 and Seq ID No.199 are both hypomethylated probes targeting the target region shown in Seq ID No.80, and Seq ID No.319 and Seq ID No.320 are both hypermethylated probes targeting the target region shown in Seq ID No. 80. Seq ID No.200, seq ID No.201 and Seq ID No.202 are hypomethylated probes targeting the target region shown in Seq ID No.81, and Seq ID No.321, seq ID No.322 and Seq ID No.323 are hypermethylated probes targeting the target region shown in Seq ID No. 81. Seq ID No.203 is a hypomethylated probe targeting the target region shown in Seq ID No.82, and Seq ID No.324 is a hypermethylated probe targeting the target region shown in Seq ID No. 82. Seq ID No.204 is a hypomethylated probe that targets the target region shown in Seq ID No.83, and Seq ID No.325 is a hypermethylated probe that targets the target region shown in Seq ID No. 83. The target sequences targeted by the probes are given in table 1.

The determination of the methylation level threshold of the two pan-cancer markers means that this indicator is reached or exceeded in more than 50% of the cancer samples, respectively, and is lower in the corresponding normal controls.

By counting the methylation level of the cancer specific region (marker) and analyzing the markers which are greater than or equal to the threshold value, when the number (n 1) of the markers which are greater than or equal to the threshold value is greater than or equal to 20% of the number (n) of the overall markers, the cancer is judged. Indeed, a change in the methylation level of any one cancer specific marker would indicate that the sample is more or less abnormal. In most cancer samples, markers with differential methylation levels account for over 30% of all markers. Considering that most cancer samples were patients at stage II or later in the pathological staging, to increase the sensitivity of the present panel detection, the ratio was adjusted to 20% to improve the detection of early stage cancer.

Possible tissue sources are finally given by counting the methylation levels of all tissue markers and analyzing the tissues pointed to by the tissue markers which are greater than or equal to the threshold value.

In the above system referred to herein, the pan cancer specific region Seq ID No.:63 is 55%, and therefore, when the detection result is 55% or more, the methylation level of the pan-cancer specific region is considered to be equal to or more than the threshold; the pan-cancer specific region Seq ID No.:64 is 60%, and thus when the detection result is 60% or more, the methylation level of the pan-cancer specific region is considered to be equal to or more than the threshold.

In this context, of the n probes targeting the cancer-specific region, the methylation level of the region targeted by n1 probes is equal to or greater than the respective threshold, and n1/n is equal to or greater than 20%, preferably equal to or greater than 30%, and the patient is interpreted as having any one of the tissue-specific cancers. The respective thresholds for the cancer-specific regions are shown in table 1, e.g. as Seq ID No.:48, the threshold value is 0.35, so that if the detection result is 0.35 or more when the probe targeting the region is used for detection, the methylation level of the region detected by the probe is equal to or more than the threshold value. The methylation thresholds for the remaining cancer specific regions can all be seen in table 1.

Herein, if the methylation level of the region targeted by m1 probes among the m probes targeting the tissue-specific region is equal to or greater than the respective threshold value, the tissues targeted by m1 probes equal to or greater than the respective threshold value are further analyzed and the number of probes equal to or greater than the threshold value per tissue is counted, and the tissue of the patient suffering from cancer is interpreted as the tissue with the highest number of probes with the methylation level equal to or greater than the threshold value. The respective thresholds of the tissue-specific regions are shown in table 1, e.g. as Seq ID No.:67, the threshold value is 0.29, so that if the detection result is 0.29 or more using a probe targeting the region, the methylation level of the region detected by the probe is equal to or greater than the threshold value. The methylation thresholds for the remaining tissue-specific regions can all be seen in table 1.

In the system referred to herein above, a sample is collected from a subject. The subject may be a subject suspected of having cancer, or a subject already having cancer. The cancer may be, esophageal cancer, gastric cancer, or colorectal cancer. The sample may be blood, plasma. In the above-described system referred to herein, purified DNA, which may be gDNA, or cfDNA, is extracted from a sample.

In the system described herein above, the bisulfite-converted DNA library is amplified by PCR in a pre-PCR amplification module, using which the amount of bisulfite-converted DNA can be increased to an amount that can undergo hybridization to a probe composition.

In the system referred to herein above, in the hybrid capture module, the sample is hybrid captured using a probe composition comprising, 2 probes targeting the pan-cancer specific region, n probes targeting the cancer specific region, and m probes targeting the tissue specific region. n may be any integer selected from 1 to 192. n can be, for example, 1,2,3,4,5,6,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, … …,192. The cancer specific region may be selected from Seq ID No.: 1-62. m may be any integer selected from 1 to 44. m may be, for example, 1,2,3,4,5,6,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44. The tissue specific region may be selected from Seq ID No.: 65-83.

In the system referred to herein above, in the hybridization capture module, the probe composition comprises: hypomethylated probes that hybridize to the cancer specific, pan-cancer specific, and tissue specific regions that are bisulfite converted without CG methylation, and hypermethylated probes that hybridize to the cancer specific, pan-cancer specific, and tissue specific regions that are bisulfite converted CG all methylation. The hypomethylated probes include probe Seq ID No.:84-180, a probe Seq ID No. targeting a pan cancer specific region: 181-182, and a probe Seq ID No.: 183-204. The hypermethylated probes include probe Seq ID No.:205-301, probe Seq ID No.:302-303, and a probe Seq ID No.: 304-325.

In the system referred to herein above, each probe in the probe composition in the hybrid capture module is 40 to 60bp in length. For example, 41 to 60bp,42 to 60bp,43 to 60bp,44 to 60bp,45 to 59bp,45 to 58bp,45 to 57bp,45 to 56bp,46 to 56bp,47 to 56bp,48 to 56bp,49 to 56bp and 50 to 56bp. The length of each probe in the probe composition is preferably 50 to 56bp, and more preferably 50bp.

In the system referred to herein, the PCR amplification is used to amplify the hybridization-captured product in the post-PCR amplification module, and the amount of the hybridization-captured product can be increased to the initial amount that can be sequenced on the machine using PCR amplification. If PCR amplification is not used for hybridizing the captured product, the amount of the product cannot meet the requirement of on-machine sequencing.

In the system referred to herein above, in the sequencing module, the platform for high-throughput next generation sequencing is the Illumina platform.

In the system referred to herein above, the database may be a multiple cancer methylation analysis database provided by integrating public data with existing sequencing data in the interpretation module. Pattern recognition is carried out according to a database, and the database comprises three types of marker information which are respectively as follows: pan-cancer markers, tissue-specific markers, and markers characteristic of cancer.

In the methods performed in the systems referred to herein above, the detection method enriches cfDNA by means of hybrid capture and detects methylation sites highly associated with cancer using NGS technology. Covers three lumen sex organ malignant tumors (esophageal cancer, gastric cancer and colorectal cancer) with the highest incidence rate in China. Finally, information is provided for early screening and early diagnosis of multiple cancers according to the detection of the gene methylation change level in the cfDNA of the blood plasma.

Examples

Example 1:

as shown in fig. 1, the implementation flow of the present application is specifically as follows:

1.1.cfDNA extraction purification

1.1.1. Plasma sample preparation:

the blood samples were centrifuged at 2000g for 10min at 4 ℃ and the plasma was transferred to a new centrifuge tube. The plasma samples were centrifuged at 16000g for 10min at 4 ℃ and the next step was performed, depending on the type of collection tube used, which was otherwise used in this experiment.

TABLE 2

1.1.2. Cleavage and binding

1.1.2.1. The binding solution/bead mixture was prepared according to the following table and then thoroughly mixed.

TABLE 3

An appropriate volume of plasma sample was added.

1.1.2.2. The plasma sample and the binding solution/bead mixture were thoroughly mixed.

1.1.2.3. Binding was performed on a spin mixer for 10min sufficient to bind cfDNA to the magnetic beads.

1.1.2.4. The binding tube was placed on a magnetic stand for 5min until the solution became clear and the magnetic beads were completely adsorbed on the magnetic stand.

1.1.2.5. The supernatant was carefully discarded with a pipette, the tube was kept on the magnetic rack for several minutes, and the residual supernatant was removed with a pipette.

1.1.3. Washing machine

1.1.3.1. The beads were resuspended in 1ml of wash solution.

1.1.3.2. The resuspension was transferred to a new non-adsorbing 1.5ml centrifuge tube. The bonded tube is retained.

1.1.3.3. The centrifuge tube containing the bead resuspension was placed on a magnetic rack for 20s.

1.1.3.4. The separated supernatant was aspirated to wash the binding tubes, and the washed residual beads were collected again in a resuspension, discarding the lysis/binding tubes.

1.1.3.5. The tube was placed on a magnetic rack for 2min until the solution became clear, the beads were collected in the magnetic rack, and the supernatant was removed with a 1ml pipette.

1.1.3.6. The tube was left on the magnetic rack and the remaining liquid was removed as much as possible with a 200 μ L pipette.

1.1.3.7. The tube was removed from the magnetic stand, 1ml of wash solution was added, and vortexed for 30s.

1.1.3.8. The solution was placed on a magnetic stand for 2min until the solution cleared, the beads were collected on the magnetic stand, and the supernatant was removed with a 1ml pipette.

1.1.3.9. The tube was left on the magnetic stand and the remaining liquid was removed thoroughly with a 200 μ L pipette.

1.1.3.10. The tube was removed from the magnetic stand, 1ml of 80% ethanol was added, and vortexed for 30s.

1.1.3.11. The solution became clear by placing on a magnetic rack for 2min and the supernatant was removed with a 1ml pipette.

1.1.3.12. The tube was left on the magnetic rack and the remaining liquid was removed with a 200 μ L pipette.

1.1.3.13. Repeat the above 1.1.3.10-1.1.3.12 procedure with 80% ethanol once to remove the supernatant as much as possible.

1.1.3.14. The tube was left on the magnetic stand and the beads were air dried for 3-5 minutes.

1.1.4. Elution of cfDNA

1.1.4.1. The eluent was added as in the table below.

TABLE 4

1.1.4.2. Vortex for 5min, place on magnetic rack for 2min, the solution becomes clear, and aspirate cfDNA in the supernatant.

1.1.4.3. The purified cfDNA was used immediately or the supernatant was transferred to a new centrifuge tube and stored at-20 ℃.

1.2.GDNA disruption and purification:

1.2.1. according to the Qubit concentration, 2. Mu.g of gDNA was taken, supplemented to 125. Mu.l with water, added to a covaris 130. Mu.l stoptube, and the program was set: 50w,20%,200 cycles, 250s.

1.2.2. After the interruption, 1 mu l of sample is taken for fragment detection by using Agilent2100, and the main peak of the sample detection after normal interruption is about 150bp-200bp.

For cfDNA samples, agilent2100 performed fragment detection and the qubits were directly quibit for subsequent experiments.

1.3. End repair, 3' end addition of "a":

1.3.1. taking 20ng of the broken gDNA or cfDNA into a PCR tube, supplementing 50 mu l of the broken gDNA or cfDNA with nuclease-free water, adding the following reagents, and mixing by vortex:

TABLE 5

Components	Volume of
gDNA/cfDNA	50μl
Stop repair and A tailing buffer	7μl
Stop repair and A tailing enzyme mixture	3μl
Total volume	60μl

1.3.2. The following program was set up to perform the reaction on a PCR instrument: the hot lid temperature was 85 ℃.

TABLE 6

Temperature of	Time
20℃	30min
65℃	30min
4℃	∞

1.4. Joint connection and purification:

1.4.1. the linkers were diluted to the appropriate concentrations in advance with reference to the following table:

TABLE 7

Fragmented DNA per 50ul ER and AT reactions	Concentration of linker
1μg	10uM
500ng	10uM
250ng	10uM
100ng	10uM
50ng	10uM
25ng	10uM
10ng	3uM
5ng	5uM
2.5ng	2.5uM

1ng

625nM

1.4.2. The following reagents were prepared according to the following table, gently pipetted and mixed, and briefly centrifuged:

TABLE 8

Components	Volume of
End repair, addition of "A" reaction product	60μl
Joint	5μl
Nuclease-free water	5μl
Ligation buffer	30μl
DNA ligase	10μl
Total volume	110μl

1.4.3. The following program was set up to perform the reaction on a PCR instrument: without a heat cover.

TABLE 9

Temperature of	Time
20℃	30min
4℃	∞

1.4.4. Purified magnetic beads were added for the experiment according to the following system (Agencourt AMPure XP beads were brought to room temperature in advance, shaken and mixed well for use):

TABLE 10

Components	Volume of
Joint ligation product	110μl
Agencourt AMPure XP bead	110μl
Total volume	220μl

1.4.4.1. Gently suck and mix for 6 times.

1.4.4.2. And (3) standing and incubating for 5-15min at room temperature, and placing the PCR tube on a magnetic frame for 3min to clarify the solution.

1.4.4.3. The supernatant was removed, the PCR tube was further placed on a magnetic holder, and 200. Mu.l of 80% ethanol solution was added to the PCR tube, followed by standing for 30 seconds.

1.4.4.4. The supernatant was removed, 200. Mu.l of 80% ethanol solution was added to the PCR tube, and the supernatant was removed thoroughly after standing for 30s (it was recommended to remove the residual ethanol solution at the bottom using a 10. Mu.l pipette).

1.4.4.5. Standing at room temperature for 3-5min to completely volatilize residual ethanol.

1.4.4.6. Adding 22. Mu.l of nuclease-free water, taking down the PCR tube from the magnetic frame, gently sucking and beating the heavy suspension magnetic beads to avoid generating bubbles, and standing at room temperature for 2min.

1.4.4.7. The PCR tube was placed on a magnetic stand for 2min to clarify the solution.

1.4.4.8. Pipette 20. Mu.l of the supernatant and transfer to a new PCR tube.

1.5 bisulfite treatment and purification:

1.5.1. the required reagents were taken out beforehand and dissolved. The reagents were added according to the following table:

TABLE 11

Components	High concentration sample (1 ng-2. Mu.g) volume	Volume of low concentration sample (1-500 ng)
Linker ligation of purified products	20μl	40μl
Bisulfite solution	85μl	85μl
DNA protection buffer	35μl	15μl
Total volume	140μl	140μl

1.5.2.DNA protection buffer added to the liquid turned blue. Mix by gentle pipetting and then divide into two tubes and place on the PCR instrument.

1.5.3. The following programs are set and run: hot lid 105 ℃.

TABLE 12

Temperature of	Time
95℃	5min
60℃	10min
95℃	5min
60℃	10min
4℃	∞

1.5.4. Brief centrifugation pooled two identical samples into the same clean 1.5ml centrifuge tube.

1.5.5. Mu.l of buffer BL (sample size less than 100ng with 1. Mu.l of vector RNA (1. Mu.g/. Mu.l)) was added to each sample, vortexed, and briefly centrifuged.

1.5.6. Add 250. Mu.l of absolute ethanol to each sample, vortex and mix for 15s, centrifuge briefly, and add the mixture to the corresponding spin column ready.

1.5.7. Standing for 1min, centrifuging for 1min, transferring the liquid in the collecting tube to the centrifugal column again, centrifuging for 1min, and discarding the liquid in the centrifugal tube.

1.5.8. Add 500. Mu.l buffer BW (note whether absolute ethanol is added or not), centrifuge for 1min, discard waste.

1.5.9. Add 500. Mu.l buffer BD (note whether absolute ethanol was added), cover the tube, and let stand at room temperature for 15min. Centrifuging for 1min, and discarding the centrifuged liquid.

1.5.10. Add 500. Mu.l buffer BW (note whether absolute ethanol is added or not), centrifuge for 1min, discard the separated liquid, repeat once for 2 times.

1.5.11. Add 250. Mu.l of absolute ethanol, centrifuge for 1min, place the column in a new 2ml collection tube and discard all remaining liquid.

1.5.12. The column was placed in a clean 1.5ml centrifuge tube, 20. Mu.l of nuclease-free water was added to the center of the column membrane, the tube cap was gently closed, and the column was left at room temperature for 1min and centrifuged for 1min.

1.5.13. And transferring the liquid in the collecting pipe to a centrifugal column again, standing at room temperature for 1min, and centrifuging for 1min.

1.6. Pre-amplification and purification before hybridization:

1.6.1. preparing a reaction system according to the following table, blowing, beating, mixing uniformly, and centrifuging for a short time:

watch 13

1.6.2. The following program was set up and the PCR program was started: 105 deg.C thermal cover

TABLE 14

1.6.3. The number of PCR cycles was adjusted depending on the amount of DNA put, and the reference data are as follows:

watch 15

1.6.4. And adding 50 mu l of Agencour AMPure XP magnetic beads into the PCR tube after the reaction is finished, and blowing and uniformly mixing the mixture by using a pipettor to avoid generating bubbles (the Agencour AMPure XP is uniformly mixed and balanced at room temperature in advance).

1.6.5. Incubate at room temperature for 5-15min, and place the PCR tube on a magnetic rack for 3min to clarify the solution.

1.6.6. The supernatant was removed, the PCR tube was further placed on a magnetic stand, 200. Mu.l of 80% ethanol solution was added to the PCR tube, and the tube was allowed to stand for 30 seconds.

1.6.7. The supernatant was removed, 200. Mu.l of 80% ethanol solution was added to the PCR tube, and after standing for 30s, the supernatant was removed completely (it was recommended to remove the bottom residual ethanol solution using a 10. Mu.l pipette).

1.6.8. Standing at room temperature for 5min to completely volatilize residual ethanol.

1.6.9. Add 30. Mu.l of nuclease-free water, remove the centrifuge tube from the magnetic rack, and gently pipette and resuspend the magnetic beads.

1.6.10. After standing at room temperature for 2min, 200. Mu.l of PCR tube was placed on a magnetic stand for 2min to clarify the solution.

1.6.11. The supernatant was transferred to a new 200. Mu.l PCR tube (on an ice box) using a pipette, and the reaction tube was labeled with a sample number and ready for the next reaction.

1.6.12. A1. Mu.l sample was taken for library concentration determination using Qubit and library concentrations were recorded.

1.6.13. A1. Mu.l sample was taken and the library fragment length was determined using Agilent2100, with a library length of approximately 270bp to 320 bp.

1.7. Sample and probe hybridization:

1.7.1. the sample library was mixed well with various Hyb blockers, labeled B, according to the following system:

TABLE 16

Components	Volume of
Pre-amplification product	750ng corresponding volume
Hyb human blockers	5μl
Linker blocker	6μl
Reinforcing agent	5μl

1.7.2. And (3) putting the prepared mixture of the sample and the Hyb blocker into a vacuum concentration centrifuge, opening a PCR tube cover, starting the centrifuge, opening a switch of a vacuum pump, and starting concentration.

1.7.3. The drained sample was redissolved in about 9. Mu.l nuclease-free water in a total volume of 10. Mu.l, gently pipetted and mixed, centrifuged briefly and placed on ice for use, labeled B.

1.7.4. And melting the Hyb buffer solution at room temperature, wherein precipitates appear after melting, uniformly mixing, preheating in a 65 ℃ water bath, completely dissolving (without precipitates and turbid substances), putting 20 mu l of Hyb buffer solution in a new 200 mu l PCR tube, covering a tube cover, marking as A, and continuously putting in the 65 ℃ water bath for incubation for later use.

1.7.5. The following hypomethylated probes were synthesized by Ai Jitai kang biotech (beijing) limited:

a) Probe Seq ID No.: any of 84-180, b) a probe Seq ID No.:181-182, and c) a probe Seq ID No.: any of the components 183-204 may be used,

and the following hypermethylated probes were synthesized:

d) Probe Seq ID No.:205-301, e) a probe Seq ID No.:302-303, and f) a probe Seq ID No.: 304-325.

And preparing a probe composition at a ratio of a: b: c: d: e: f = 1.

1.7.6. Mu.l of RNase blocker and 2. Mu.l of probe composition were placed in a 200. Mu.l PCR tube, gently pipetted and mixed, centrifuged briefly and placed on ice until needed, labeled C.

1.7.7. Setting PCR instrument parameters, hot cover 100 deg.C, 95 deg.C, 5min; and keeping at 65 ℃.

1.7.8. Place PCR tube B on the PCR instrument and run the above program.

And 1.7.9. When the temperature of the PCR instrument is reduced to 65 ℃, placing the PCR tube A on the PCR instrument for incubation, and covering a hot cover of the PCR instrument.

After 1.7.10.5 min, place C on PCR for incubation and cover the PCR instrument hot lid.

1.7.11. And (3) placing the PCR tube C into a PCR instrument for 2min, adjusting a pipettor to 13 mu l, sucking 13 mu l of Hyb buffer solution from the PCR tube A, transferring the Hyb buffer solution into the PCR tube C, sucking all samples in the PCR tube B, transferring the samples into the PCR tube C, slightly sucking and beating for 10 times, fully mixing the samples uniformly to avoid generating a large amount of bubbles, sealing a tube cover, covering a hot cover of the PCR instrument, and incubating overnight at 65 ℃ (16-24 h).

1.8. Capture target region DNA library:

1.8.1. preparation of the Capture magnetic beads

1.8.1.1. The magnetic beads (Dynabeads MyOne Streptavidin T1 magnetic beads) were removed from 4 ℃ and resuspended by vortexing.

1.8.1.2. 50 μ l of the magnetic beads were placed in a new PCR tube, placed on a magnetic rack for 1min to clarify the solution, and the supernatant was removed.

1.8.1.3. The PCR tube was removed from the magnetic frame, 200. Mu.L of binding buffer was added and gently pipetted several times to mix well, and the magnetic beads were resuspended.

1.8.1.4. Placing on a magnetic frame for 1min, and removing the supernatant.

1.8.1.5. Repeating the steps 3-4 twice, and washing the magnetic beads 3 times.

1.8.1.6. The PCR tube was removed from the magnetic frame and 200. Mu.L of binding buffer was added and the resuspended beads were gently pipetted 6 times for use.

1.8.2. Capturing a target DNA library

1.8.2.1. Keeping the hybridization product PCR tube C on a PCR instrument, adding the prepared 200 mu L of capture magnetic beads into the hybridization product PCR tube C, sucking and beating for 6 times by using a pipette, uniformly mixing, and placing on a rotary mixer to combine for 30min at room temperature (the rotating speed is preferably not more than 10 r/min).

1.8.2.2. The PCR tube was placed on a magnetic rack for 2min to clarify the solution and the supernatant was removed.

1.8.2.3. Adding 200 μ L of washing buffer 1 into PCR tube C, gently sucking and beating for 6 times, mixing, washing for 15min (preferably no more than 10 rpm) in a rotary mixer, centrifuging for a short time, placing the PCR tube on a magnetic frame for 2min to clarify the solution, and removing the supernatant.

1.8.2.4. Adding 200 μ l of washing buffer solution 2 preheated at 65 deg.C, gently sucking and beating for 6 times, mixing, placing on mixing machine, incubating at 65 deg.C for 10min, and cleaning at 800 rpm.

1.8.2.5. Briefly, centrifuge, place PCR tube on magnetic rack for 2min, remove supernatant. The wash was repeated 2 more times with wash buffer 2 for a total of 3 times. The wash buffer 2 was removed completely for the last time.

1.8.2.6. The PCR tube was continuously placed on a magnetic stand, 200. Mu.l of 80% ethanol was added to the PCR tube, and after standing for 30s, the ethanol solution was completely removed, and air-dried at room temperature for 2min.

1.8.2.7. Add 30. Mu.L nuclease-free water to the PCR tube, remove the PCR tube from the magnetic frame, and gently pipette 6 times of resuspended beads for use.

1.9. Post capture amplification and purification

1.9.1. A reaction system is prepared according to the following table for enriching the capture library, and after the capture library is lightly blown, uniformly mixed, the capture library is centrifuged for a short time:

TABLE 17

1.9.2. The following program was set up, the samples were placed in a PCR instrument, and the program was run: hot lid 105 ℃.

Watch 18

1.9.3 after the PCR is finished, adding 55 mu l of Agencourt AMPure XP magnetic beads into the sample, and gently sucking and mixing the mixture by using a pipettor.

1.9.4. Incubate at room temperature for 5min, and settle the PCR tube on a magnetic rack for 3min to clarify the solution.

1.9.5. The supernatant was removed, the PCR tube was further placed on a magnetic stand, 200. Mu.l of 80% absolute ethanol was added, and the mixture was allowed to stand for 30 seconds.

1.9.6. The supernatant was removed, 200. Mu.l of 80% absolute ethanol was further added to the PCR tube, and the supernatant was completely removed after standing for 30 days.

1.9.7. Standing at room temperature for 5min to completely volatilize residual ethanol.

1.9.8. Add 25. Mu.l nuclease-free water, remove the PCR tube from the magnetic rack, gently blow and mix the tube with the magnetic beads, and leave the tube at room temperature for 2min.

1.9.9. The PCR tube was placed on a magnetic rack for 2min to clarify the solution.

1.9.10. Transfer 23. Mu.l of supernatant to a 1.5ml centrifuge tube by pipette, and label the sample information.

1.9.11. 1 μ l of the library was quantitated using a Qubit and the library concentration was recorded.

1.9.12. A1. Mu.l sample was taken for library fragment length determination using Agilent 2100.

1.9.13. Sequencing was performed using the Illumina high throughput sequencing platform.

1.10. Methylation letter analysis process. Roughly as follows: checking sequencing quality by using quality control software such as trimmatic and the like, removing low-quality reads, comparing clean data after quality control to a reference genome by using comparison software such as a Bismarker and the like, and extracting corresponding methylation sites by using R packets such as methykit and the like. Finally, the methylation ratio of each target region on Panel was calculated.

Example 2

A pathologically characterized gastric cancer sample was collected as peripheral blood using the Panel test of the present application as described in example 1; establishing a library, and sequencing by an Illumina platform; the sequencing data were subjected to the above-described biological information analysis procedure to obtain the methylation level, and the results are shown in the following Table 19 (Table 19 shows that the target regions equal to or greater than the methylation threshold were detected).

Watch 19

Gene	CHR	Initiation of	Terminate	Ratio of methylation	Target region sequence number
TBX15	1	119527108	119527157	0.55	Seq ID No.63
CRYGD	2	208989200	208989249	0.60	Seq ID No.64
CPE	4	166300051	166300291	0.42	Seq ID No.68
CPE	4	166300242	166300291	0.42	Seq ID No.69
PLXDC2	10	20104497	20104546	0.46	Seq ID No.75
PLXDC2	10	20104758	20104807	0.46	Seq ID No.76
PLXDC2	10	20104948	20104997	0.53	Seq ID No.77
PLXDC2	10	20105593	20105642	0.49	Seq ID No.78
OTX1	2	63281139	63281188	0.57	Seq ID No.11
SFRP2	4	154710475	154710536	0.40	Seq ID No.19
SFRP2	4	154710598	154710647	0.37	Seq ID No.20
SFRP2	4	154710702	154710751	0.36	Seq ID No.21
SFRP2	4	154710796	154710845	0.43	Seq ID No.22
CDO1	5	115152372	115152432	0.48	Seq ID No.24
CDO1	5	115152485	115152543	0.48	Seq ID No.25
TRIM15	6	30131701	30131768	0.62	Seq ID No.31
ALX4	11	44330903	44330952	0.49	Seq ID No.47
ALX4	11	44330958	44331007	0.37	Seq ID No.48
CCNA1	13	37004553	37004618	0.42	Seq ID No.53
CCNA1	13	37004620	37004669	0.47	Seq ID No.54
CCNA1	13	37005441	37005502	0.44	Seq ID No.55
CCNA1	13	37005566	37005631	0.39	Seq ID No.56

Performing pattern recognition classification identification on a detection sample, firstly judging that the methylation levels of the pan-cancer specific markers TBX15 and CRYGD genes are more than or equal to 55% and 60%, and then preliminarily judging that the sample is a sample with cancer; secondly, judging that the methylation levels of the cancer specific markers OTX1, SFRP2, CDO1, TRIM15, ALX4 and CCNA1 are all greater than or equal to the respective thresholds shown in the table 1 (as shown in the table 19 above), further judging that the sample is a sample with any of the following 11 cancers (esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer and endometrial cancer); finally, the tissue-specific markers were read, and based on the target regions in table 19 having respective threshold values or higher, it was found that the 6 target regions Seq ID No.68, seq ID No.69, seq ID No.75, seq ID No.76, seq ID No.77, and Seq ID No.78, which are specific to stomach tissue, have respective threshold values of methylation levels or higher, and that no other tissue-specific marker has methylation of a value equal to or higher than its respective threshold value, and therefore the most stomach tissue-specific marker among the tissue-specific markers having respective methylation threshold values or higher, the sample was finally judged to be a sample having stomach cancer.

The patient was bled again 48 hours post-operatively and peripheral blood was collected as in example 1 using the Panel test of the present application; establishing a library, and sequencing by an Illumina platform; the sequencing data are analyzed by the analysis process of the biological information and a mode identification method, and the result shows that the gene methylation level in the table returns to the normal level.

Example 3

A sample of colorectal cancer, peripheral blood was collected as in example 1 using the Panel test of the present application; establishing a library, and sequencing by an Illumina platform; the sequencing data were subjected to the above-described biological information analysis procedure to obtain the methylation level, and the results are shown in table 20 below (table 20 shows that the target region at or above the methylation threshold was detected).

Watch 20

Gene	CHR	Initiation of	Terminate	Ratio of methylation	Target area sequence number
TBX15	1	119527108	119527157	0.55	Seq ID No.63
CRYGD	2	208989200	208989249	0.60	Seq ID No.64
C6orf155	6	72130359	72130408	0.56	Seq ID No.71
C6orf155	6	72130553	72130602	0.71	Seq ID No.72
C6orf155	6	72130641	72130690	0.65	Seq ID No.73
C6orf155	6	72130755	72130804	0.69	Seq ID No.74
SHISA2	13	26625273	26625397	0.61	Seq ID No.81
TRH	3	129693370	129693434	0.66	Seq ID No.16
TRH	3	129693586	129693662	0.53	Seq ID No.17
CDO1	5	115152372	115152432	0.48	Seq ID No.24
CDO1	5	115152485	115152543	0.48	Seq ID No.25
ELMO1	7	37488516	37488578	0.4	Seq ID No.35
GFRA1	10	118032831	118032906	0.33	Seq ID No.40
GFRA1	10	118032948	118032997	0.52	Seq ID No.41
CCNA1	13	37004553	37004618	0.42	Seq ID No.53
CCNA1	13	37004620	37004669	0.45	Seq ID No.54
CCNA1	13	37005441	37005502	0.39	Seq ID No.55
CCNA1	13	37005566	37005631	0.33	Seq ID No.56
SALL1	16	51184379	51184441	0.63	Seq ID No.58

Performing pattern recognition, classification and identification on a detection sample, and judging that the methylation levels of the pan-cancer specific markers TBX15 and CRYGD genes are more than or equal to 55% and 60%, and then preliminarily judging that the sample is a sample with cancer; secondly, when the methylation levels of the cancer specific markers TRH, CDO1, ELMO1, GFRA1, CCNA1, SALL1 are judged to be greater than or equal to the respective thresholds shown in table 1 (as shown in table 20 above), the sample is further judged to be a sample with any of the following 11 cancers (esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, and endometrial cancer); finally, the tissue-specific markers were identified, and based on the target regions in table 20 having the respective thresholds or higher, it was found that the 6 target regions Seq ID No.63, seq ID No.64, seq ID No.71, seq ID No.72, seq ID No.73, and Seq ID No.74, which are colorectal tissue-specific, were at least the threshold of the respective methylation levels, and no other tissue-specific markers were methylated at or above the respective thresholds, and therefore the colorectal tissue-specific markers were the most among the tissue-specific markers having the respective methylation thresholds or higher, and the sample was finally determined to be a sample having colorectal cancer.

After 48 hours of operation, the patient draws blood again, adopts the Panel detection of the application, collects peripheral blood according to the method of the embodiment 1, constructs a library, and sequences; the sequencing data are analyzed by the analysis process of the biological information and a mode identification method, and the result shows that the gene methylation level in the table returns to the normal level.

Example 4

An esophageal cancer sample, using the Panel test of the present application, peripheral blood was collected as in example 1; establishing a library, and sequencing by an Illumina platform; the sequencing data were subjected to the above-described biological information analysis procedure to obtain methylation levels, and the results are shown in table 21 below (table 21 shows that target regions equal to or greater than the methylation threshold were detected).

TABLE 21

Performing pattern recognition classification identification on a detection sample, firstly judging that the methylation levels of the pan-cancer specific markers TBX15 and CRYGD genes are more than or equal to 55% and 60%, and then preliminarily judging that the sample is a sample with cancer; secondly, when the methylation levels of the cancer specific markers CPE, TFAP2E, TRH, C11orf21, EDNRB are judged to be greater than or equal to the respective thresholds shown in table 1 (as shown in table 21 above), the sample is further judged to be a sample with any of the following 3 cancers (esophageal cancer, gastric cancer, colorectal cancer); finally, the tissue-specific markers were interpreted, and based on the target regions in table 21 that were not less than the respective threshold values, it was found that 5 target regions Seq ID No.65, seq ID No.66, seq ID No.67, seq ID No.68, and Seq ID No.69 that are specific to esophageal tissue were not less than the respective threshold values of methylation level, and no other tissue-specific markers were methylated at not less than the respective threshold values thereof, and therefore, the esophageal tissue-specific markers were the most among the tissue-specific markers that were not less than the respective methylation threshold values, and finally the sample was judged to be a sample with esophageal cancer.

Various changes and modifications can be made by those skilled in the art based on the above technical solutions and concepts, and all such changes and modifications should be included in the protection scope of the claims of the present application.

Claims

A system for detecting cancer methylation, comprising:

a sample collection module for collecting a subject sample;

a DNA extraction module for extracting and purifying DNA in the sample;

a library building module for building a DNA library for sequencing against the purified DNA sample;

a transformation module for transforming the constructed DNA library with bisulfite;

a pre-PCR amplification module for pre-PCR amplifying the bisulfite-converted DNA library;

a hybrid capture module for hybrid capture of the pre-PCR amplified sample using a probe composition;

a PCR amplification module for amplifying the hybridization-captured product by using PCR;

a sequencing module for performing high-throughput next generation sequencing on the hybridization-captured product after PCR amplification;

a data analysis module for analyzing the sequencing data to determine a methylation level of the sample;

an interpretation module for interpreting the patient's diseased condition based on the methylation level of the sample.
The system of claim 1, wherein the subject is suspected of having cancer.
The system of claim 1or 2, wherein the sample collected from the subject is a plasma sample.
The system of any one of claims 1-3, the probe composition used in the hybrid capture module comprising:

2 probes targeting a pan-cancer specific region,

n probes targeting a cancer specific region, and

m probes targeting a tissue specific region.
The system of any one of claims 1-4, the probe composition used in the hybrid capture module comprising:

hypomethylated probes that hybridize to bisulfite-converted, CG-methylation-free, pan-cancer-specific, and tissue-specific regions of the cancer, and

hypermethylated probes that hybridize to the cancer-specific, pan-cancer-specific, and tissue-specific regions where bisulfite-converted CG is fully methylated.
The system of any one of claims 1-5, wherein each probe in the probe composition used in the hybrid capture module is 40-60 bp in length.
The system according to any of claims 1-6, wherein each probe in the probe composition used in the hybrid capture module has a length of 45-56 bp, preferably 50-56 bp, more preferably 50bp.
The system of any one of claims 1-7, wherein n probes in the probe composition used in the hybrid capture module target a cancer specific region,

wherein n is an integer selected from any of 1 to 192;

wherein the cancer specific region is selected from the group consisting of Seq ID No.: 1-62.
The system of any one of claims 1-8, wherein m probes in the probe composition used in the hybrid capture module target the tissue-specific region,

wherein m is any integer selected from 1 to 44;

wherein the tissue specific region is selected from the group consisting of Seq ID No.: 65-83.
The system of claim 5, wherein in the hybrid capture module, the hypomethylated probes comprise probes that target cancer-specific regions Seq ID No.:84-180, a probe Seq ID No. targeting a pan cancer specific region: 181-182, and a probe Seq ID No.: 183-204.
The system of claim 5, wherein in the hybrid capture module, the hypermethylated probes comprise probes that target cancer-specific regions Seq ID No.:205-301, probe Seq ID No.:302-303, and a probe Seq ID No.: 304-325.
The system according to any one of claims 1-11, the interpretation module comprising:

(1) A pan cancer interpretation module for comparing the pan cancer specific region database and performing interpretation to confirm whether the subject has cancer;

(2) A cancer interpretation module for comparing the cancer specific region database and performing interpretation to further confirm the cancer suffered by the subject as one of several suspected cancers; and

(3) And the tissue specificity interpretation module is used for comparing the tissue specificity region database and performing interpretation so as to confirm the cancer suffering part of the subject.
The system of claim 12, the pan cancer call module comprising making calls that: judging the pan-cancer specific region Seq ID No.:63, and judging whether or not the methylation level of the pan cancer-specific region Seq ID No.:64, if Seq ID No.: methylation level of 63 is 55% or more and Seq ID No.:64 greater than or equal to 60%, the patient is read as having cancer.
The system of claim 12, the cancer interpretation module comprising performing the interpretation of: the patient is interpreted as having any one of the tissue-specific cancers if, among the n probes targeting the cancer-specific region, the methylation level of the region targeted by n1 probes is equal to or greater than the respective threshold value, and n1/n is equal to or greater than 20%, preferably n1/n is equal to or greater than 30%.
The system of claim 12, the tissue-specific interpretation module comprising performing the interpretation of: if the methylation level of the region targeted by m1 probes among the m probes targeting the tissue-specific region is greater than or equal to the respective threshold value, the tissues targeted by the m1 probes greater than or equal to the respective threshold value are further analyzed and the number of probes greater than or equal to the threshold value in each tissue is counted, and the tissue suffering from cancer of the patient is interpreted as the tissue with the highest methylation level of the probes greater than or equal to the threshold value.
An in vitro method for detecting cancer in a subject, comprising the steps of:

collecting a sample from a subject;

extracting and purifying DNA in the sample;

constructing a DNA library for sequencing against the purified DNA sample;

transforming said constructed DNA library with bisulfite;

pre-PCR amplifying the bisulfite-converted DNA library;

performing hybridization capture on the sample subjected to pre-PCR amplification by using a probe composition;

amplifying the product obtained after hybridization capture by utilizing PCR;

performing high-throughput second-generation sequencing on a product obtained after hybridization and capture after PCR amplification;

analyzing the sequencing data to determine the methylation level of the sample;

interpreting the patient's condition based on the methylation level of the sample.
The method of claim 16, wherein the subject is suspected of having cancer.
The method of claim 16 or 17, wherein the sample taken from the subject is a plasma sample.
The method of any one of claims 16-18, wherein the conversion is treated with bisulfite.
The method of any one of claims 16-18, the probe composition comprising:

2 probes targeting a pan-cancer specific region,

n probes targeting a cancer-specific region, and

m probes targeting a tissue specific region.
The method of any one of claims 16-20, the probe composition comprising:

hypomethylated probes that hybridize to bisulfite-converted, CG-methylation-free, pan-cancer-specific, and tissue-specific regions of the cancer, and

hypermethylated probes that hybridize to the cancer-specific, pan-cancer-specific, and tissue-specific regions where bisulfite-converted CG is fully methylated.
The method of any one of claims 16-21, wherein each probe in the probe composition is 40-60 bp in length.
The method according to any one of claims 16-22, wherein each probe in the probe composition has a length of 45-56 bp, preferably 50-56 bp, more preferably 50bp.
The method of any one of claims 16-23, wherein n probes in the probe composition target a cancer specific region,

wherein n is an integer selected from any of 1 to 192;

wherein the cancer specific region is selected from the group consisting of Seq ID No.: 1-62.
The method of any one of claims 16-24, wherein m probes in the probe composition target the tissue-specific region,

wherein m is an integer selected from any of 1 to 44;

wherein the tissue specific region is selected from the group consisting of Seq ID No.: 65-83.
The method of claim 21, wherein the hypomethylated probe comprises a probe Seq ID No.:84-180, a probe Seq ID No.:181-182, and a probe Seq ID No.: 183-204.
The method of claim 21, wherein the hypermethylated probes comprise probes that target cancer-specific regions Seq ID No.:205-301, probe Seq ID No.:302-303, and a probe Seq ID No.: 304-325.
The method according to any one of claims 16-27, the interpreting comprising the steps of:

(1) Comparing the pan-cancer specific region database and performing interpretation to confirm whether the subject has cancer;

(2) Comparing the cancer specific region database, and judging to confirm that the cancer suffered by the subject is one of several suspected cancers;

(3) Comparing the tissue specific region database, and performing interpretation to confirm the cancer part of the subject.
The method of claim 28, the step (1) comprising performing the interpretation: judging the pan-cancer specific region Seq ID No.:63, and judging whether or not the methylation level of the pan cancer-specific region Seq ID No.:64, if Seq ID No.:63 is at least 55% and Seq ID No.:64 greater than or equal to 60%, the patient is read as having cancer.
The method of claim 28, said step (2) comprising making the following interpretations: if the methylation level of the region targeted by n1 probes is more than or equal to the respective threshold value in n probes targeting the cancer specific region, and n1/n is more than or equal to 20%, preferably n1/n is more than or equal to 30%, the patient is judged to have any one of the tissue specific cancers, and then the possibility of each cancer is judged according to pattern recognition.
The method of claim 28, said step (3) comprising making the interpretation: if the methylation level of the region targeted by m1 probes in the m probes targeting the tissue-specific region is greater than or equal to the respective threshold value, the tissues targeted by the m1 probes greater than or equal to the respective threshold value are further analyzed and the number of probes greater than or equal to the threshold value in each tissue is counted, and the tissue of the patient suffering from the cancer is judged to be the tissue with the highest number of probes with the methylation level greater than or equal to the threshold value.