CN112662760A

CN112662760A - Cancer gene methylation detection system and cancer in-vitro detection method implemented in cancer gene methylation detection system

Info

Publication number: CN112662760A
Application number: CN202010116119.0A
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: 2020-02-25
Publication date: 2021-04-16
Also published as: WO2021169875A1; CN115176034A

Abstract

Disclosed herein is 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 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.

Description

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

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 number of genes simultaneously. But the method is limited in 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 sequencing cost in the future, the NGS will inevitably replace gene chips and other high-throughput technologies step by step.

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 the 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 method 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. Research shows that the change of gene methylation level is earlier than gene variation, and the change of gene methylation is tracked and detected, so that the generation of 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.

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 tumors and all metastatic foci, overcomes tumor heterogeneity (the current adopted standard tissue biopsy can only reflect the characteristics of a certain part of tumors), realizes real-time monitoring, has higher sensitivity, even can predict the diseased part through genome information, and can effectively prolong the life cycle of a patient. 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 free and extracellular partially degraded endogenous DNA present in circulating blood. 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 of causing, and the like. Kun distant gene also introduced the product "Changle si" in 2018 for detecting methylation level of colorectal cancer.

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 tubular organ tumors such as esophageal cancer, gastric cancer and colorectal cancer, aiming at the deficiencies and technical limitations of the current multi-cancer detection products. The system and the method performed in the system may: 1) the kit is used for early screening of asymptomatic people and prognosis detection of cancer patients in a non-invasive mode, reduces harm caused by invasive detection, 2) increases sequencing depth, enables the detection range of genes to be superior to that of the existing technology and products, has the characteristics of high flux, high detection speed, low detection cost shared by each gene and the like, 3) can realize sampling of all parts and all metastasis foci of tumors, overcomes tumor heterogeneity, and 4) has higher sensitivity and accuracy, can realize real-time monitoring, and even can effectively prolong the life cycle of patients by predicting diseased parts through genome information.

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 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 has a length of 40-60 bp.

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 50 bp.

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.: 66-67, 79, 81-83, 87-90, 97-102, 111, 117, and 118.

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.: any of 119-215, probes Seq ID No.: any of 216-217, and a probe Seq ID No.: 219-220, 233, 235-237, 241-244, 251-257, 266-268, 275-276.

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.: any of 277-373, probe Seq ID No.: 374-375, and a probe Seq ID No. targeting a tissue-specific region: 377-.

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 call module comprising making calls as follows: 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 n 1/n.gtoreq.20%, preferably n 1/n.gtoreq.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, the tissues targeted by m1 probes greater than or equal to the respective threshold are further analyzed and the number of probes greater than or equal to the threshold per tissue is counted, and the tissue of the patient suffering from cancer is judged to be the tissue with the highest number of probes with the methylation level greater than or equal to the threshold.

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 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.

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:

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 50 bp.

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.: any of 119-215, probes Seq ID No.: any of 216-217, and a probe Seq ID No.: 219-220, 233, 235-237, 241-244, 251-257, 266-268, 275-276.

27. The method of item 21, wherein the hypermethylated probe comprises a probe Seq ID No.: any of 277-373, probe Seq ID No.: 374-375, and a probe Seq ID No. targeting a tissue-specific region: 377-.

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, 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 among the n probes targeting the cancer-specific region is equal to or higher than the respective threshold value, and n1/n is equal to or higher than 20%, preferably n1/n is equal to or higher than 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 making the interpretation: 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, the tissues targeted by m1 probes greater than or equal to the respective threshold are further analyzed and the number of probes greater than or equal to the threshold per tissue is counted, and the tissue of the patient suffering from cancer is judged to be the tissue with the highest number of probes with the methylation level greater than or equal to the threshold.

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 a flow chart of the operation 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 changes of 3 tubular cavity organ tumors such as esophageal cancer, gastric cancer, colorectal cancer and the like are 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 system referred to herein above, 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 identification module is used for comparing and identifying the pan-cancer specific region database; 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 identification module which identifies 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: 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 greater than or equal to the respective threshold value, and n1/n ≧ 20%, preferably n1/n ≧ 30%; a tissue-specific interpretation module that performs the following interpretation: if the methylation level of the region targeted by the 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 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.

Specifically, for example, when m1 is 6, it is further judged that, if 5 probes are probes targeting the stomach and 1 probe is a probe targeting the pancreas, it is judged that the patient has cancer in 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 cancer of the stomach and 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, then the patient is judged to have cancer of esophagus, stomach, and colorectal.

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.119, Seq ID No.120 and Seq ID No.121 is a hypomethylated probe targeting the target region shown in Seq ID No.1, and each of Seq ID No.277, Seq ID No.278 and Seq ID No.279 is a hypermethylated probe targeting the target region shown in Seq ID No. 1. Seq ID No.122 and Seq ID No.123 are both hypomethylated probes targeting the target region shown in Seq ID No.2, and Seq ID No.280 and Seq ID No.281 are both hypermethylated probes targeting the target region shown in Seq ID No. 2. Seq ID No.124 and Seq ID No.125 are both hypomethylated probes targeting the target region shown in Seq ID No.3, and Seq ID No.282 and Seq ID No.283 are both hypermethylated probes targeting the target region shown in Seq ID No. 3. Seq ID No.126 is a hypomethylated probe targeting the target region shown in Seq ID No.4, and Seq ID No.284 is a hypermethylated probe targeting the target region shown in Seq ID No. 4. Seq ID No.127 and Seq ID No.128 are both hypomethylated probes targeting the target region shown in Seq ID No.5, and Seq ID No.285 and Seq ID No.286 are both hypermethylated probes targeting the target region shown in Seq ID No. 3. Seq ID No.129 is a hypomethylated probe targeting the target region shown in Seq ID No.6, and Seq ID No.287 is a hypermethylated probe targeting the target region shown in Seq ID No. 6. Seq ID No.130 is a hypomethylated probe targeting the target region shown in Seq ID No.7, and Seq ID No.288 is a hypermethylated probe targeting the target region shown in Seq ID No. 7. Seq ID No.131 and Seq ID No.132 are both hypomethylated probes targeting the target region shown in Seq ID No.8, and Seq ID No.289 and Seq ID No.290 are both hypermethylated probes targeting the target region shown in Seq ID No. 8. Seq ID No.133, Seq ID No.134 and Seq ID No.135 are hypomethylated probes targeting the target region shown in Seq ID No.9, and Seq ID No.291, Seq ID No.292 and Seq ID No.293 are hypermethylated probes targeting the target region shown in Seq ID No. 9. Seq ID No.136 is a hypomethylated probe targeting the target region shown in Seq ID No.10, and Seq ID No.294 is a hypermethylated probe targeting the target region shown in Seq ID No. 10. Seq ID No.137 is a hypomethylated probe that targets the target region shown in Seq ID No.11, and Seq ID No.295 is a hypermethylated probe that targets the target region shown in Seq ID No. 11. Seq ID No.138 is a hypomethylated probe targeting the target region shown in Seq ID No.12, and Seq ID No.296 is a hypermethylated probe targeting the target region shown in Seq ID No. 12. Seq ID No.139 is a hypomethylated probe targeting the target region shown in Seq ID No.13, and Seq ID No.297 is a hypermethylated probe targeting the target region shown in Seq ID No. 13. Seq ID No.140 and Seq ID No.141 are both hypomethylated probes targeting the target region shown in Seq ID No.14, and Seq ID No.298 and Seq ID No.299 are both hypermethylated probes targeting the target region shown in Seq ID No. 14. Seq ID No.142 and Seq ID No.143 are both hypomethylated probes targeting the target region shown in Seq ID No.15, and Seq ID No.300 and Seq ID No.301 are both hypermethylated probes targeting the target region shown in Seq ID No. 15. Seq ID No.144 and Seq ID No.145 are both hypomethylated probes targeting the target region shown in Seq ID No.16, and Seq ID No.302 and Seq ID No.303 are both hypermethylated probes targeting the target region shown in Seq ID No. 16. Seq ID No.146 and Seq ID No.147 are both hypomethylated probes targeting the target region shown in Seq ID No.17, and Seq ID No.304 and Seq ID No.305 are both hypermethylated probes targeting the target region shown in Seq ID No. 17. Seq ID No.148 is a hypomethylated probe targeting the target region shown in Seq ID No.18, and Seq ID No.306 is a hypermethylated probe targeting the target region shown in Seq ID No. 18. Seq ID No.149 and Seq ID No.150 are both hypomethylated probes targeting the target region shown in Seq ID No.19, and Seq ID No.307 and Seq ID No.308 are both hypermethylated probes targeting the target region shown in Seq ID No. 19. Seq ID No.151 is a hypomethylated probe targeting the target region shown in Seq ID No.20, and Seq ID No.309 is a hypermethylated probe targeting the target region shown in Seq ID No. 20. Seq ID No.152 is a hypomethylated probe targeting the target region shown in Seq ID No.21, and Seq ID No.310 is a hypermethylated probe targeting the target region shown in Seq ID No. 21. Seq ID No.153 is a hypomethylated probe targeting the target region shown in Seq ID No.22, and Seq ID No.311 is a hypermethylated probe targeting the target region shown in Seq ID No. 22. Seq ID No.154, Seq ID No.155 and Seq ID No.156 are hypomethylated probes targeting the target region shown in Seq ID No.23, and Seq ID No.312, Seq ID No.313 and Seq ID No.314 are hypermethylated probes targeting the target region shown in Seq ID No. 23. Seq ID No.157 and Seq ID No.158 are both hypomethylated probes targeting the target region shown in Seq ID No.24, and Seq ID No.315 and Seq ID No.316 are both hypermethylated probes targeting the target region shown in Seq ID No. 24. Seq ID No.159 and Seq ID No.160 are both hypomethylated probes targeting the target region shown in Seq ID No.25, and Seq ID No.317 and Seq ID No.318 are both hypermethylated probes targeting the target region shown in Seq ID No. 25. Seq ID No.161 and Seq ID No.162 are both hypomethylated probes targeting the target region shown in Seq ID No.26, and Seq ID No.319 and Seq ID No.320 are both hypermethylated probes targeting the target region shown in Seq ID No. 26. Seq ID No.163 is a hypomethylated probe targeting the target region shown in Seq ID No.27, and Seq ID No.321 is a hypermethylated probe targeting the target region shown in Seq ID No. 27. Seq ID No.164 and Seq ID No.165 are both hypomethylated probes targeting the target region shown in Seq ID No.28, and Seq ID No.322 and Seq ID No.323 are both hypermethylated probes targeting the target region shown in Seq ID No. 28. Seq ID No.166 and Seq ID No.167 are both hypomethylated probes targeting the target region shown in Seq ID No.29, and Seq ID No.324 and Seq ID No.325 are both hypermethylated probes targeting the target region shown in Seq ID No. 29. Seq ID No.168 is a hypomethylated probe that targets the target region shown in Seq ID No.30, and Seq ID No.326 is a hypermethylated probe that targets the target region shown in Seq ID No. 30. Seq ID No.169 and Seq ID No.170 are both hypomethylated probes targeting the target region shown in Seq ID No.31, and Seq ID No.327 and Seq ID No.328 are both hypermethylated probes targeting the target region shown in Seq ID No. 31. Seq ID No.171 is a hypomethylated probe targeting the target region shown in Seq ID No.32, and Seq ID No.329 is a hypermethylated probe targeting the target region shown in Seq ID No. 32. Seq ID No.172 and Seq ID No.173 are both hypomethylated probes targeting the target region shown in Seq ID No.33, and Seq ID No.330 and Seq ID No.331 are both hypermethylated probes targeting the target region shown in Seq ID No. 33. Seq ID No.174 is a hypomethylated probe targeting the target region shown in Seq ID No.34, and Seq ID No.332 is a hypermethylated probe targeting the target region shown in Seq ID No. 34. Seq ID No.175 and Seq ID No.176 are both hypomethylated probes targeting the target region shown in Seq ID No.35, and Seq ID No.333 and Seq ID No.334 are both hypermethylated probes targeting the target region shown in Seq ID No. 35. Seq ID No.177 is a hypomethylated probe that targets the target region shown in Seq ID No.36, and Seq ID No.335 is a hypermethylated probe that targets the target region shown in Seq ID No. 36. Seq ID No.178 and Seq ID No.179 are both hypomethylated probes targeting the target region shown in Seq ID No.37, and Seq ID No.336 and Seq ID No.337 are both hypermethylated probes targeting the target region shown in Seq ID No. 37. Seq ID No.180 and Seq ID No.181 are both hypomethylated probes targeting the target region shown in Seq ID No.38, and Seq ID No.338 and Seq ID No.339 are both hypermethylated probes targeting the target region shown in Seq ID No. 38. Seq ID No.182 is a hypomethylated probe targeting the target region shown in Seq ID No.39, and Seq ID No.340 is a hypermethylated probe targeting the target region shown in Seq ID No. 39. Seq ID No.183 and Seq ID No.184 are hypomethylated probes targeting the target region shown in Seq ID No.40, and Seq ID No.341 and Seq ID No.342 are hypermethylated probes targeting the target region shown in Seq ID No. 40. Seq ID No.185 is a hypomethylated probe targeting the target region shown in Seq ID No.41, and Seq ID No.343 is a hypermethylated probe targeting the target region shown in Seq ID No. 41. Seq ID No.186 is a hypomethylated probe targeting the target region shown in Seq ID No.42, and Seq ID No.344 is a hypermethylated probe targeting the target region shown in Seq ID No. 42. Seq ID No.187 is a hypomethylated probe targeting the target region shown in Seq ID No.43, and Seq ID No.345 is a hypermethylated probe targeting the target region shown in Seq ID No. 43. Seq ID No.188 is a hypomethylated probe targeting the target region shown in Seq ID No.44, and Seq ID No.346 is a hypermethylated probe targeting the target region shown in Seq ID No. 44. Seq ID No.189 and Seq ID No.190 are hypomethylated probes targeting the target region shown in Seq ID No.45, and Seq ID No.347 and Seq ID No.348 are hypermethylated probes targeting the target region shown in Seq ID No. 45. Seq ID No.191 is a hypomethylated probe targeting the target region shown in Seq ID No.46, and Seq ID No.349 is a hypermethylated probe targeting the target region shown in Seq ID No. 46. Seq ID No.192 is a hypomethylated probe targeting the target region shown in Seq ID No.47, and Seq ID No.350 is a hypermethylated probe targeting the target region shown in Seq ID No. 47. Seq ID No.193 is a hypomethylated probe that targets the target region shown in Seq ID No.48, and Seq ID No.351 is a hypermethylated probe that targets the target region shown in Seq ID No. 48. Seq ID No.194 is a hypomethylated probe that targets the target region shown in Seq ID No.49, and Seq ID No.352 is a hypermethylated probe that targets the target region shown in Seq ID No. 49. Seq ID No.195 is a hypomethylated probe targeting the target region shown in Seq ID No.50, and Seq ID No.353 is a hypermethylated probe targeting the target region shown in Seq ID No. 50. Seq ID No.196 and Seq ID No.197 are both hypomethylated probes targeting the target region shown in Seq ID No.51, and Seq ID No.354 and Seq ID No.355 are both hypermethylated probes targeting the target region shown in Seq ID No. 51. Seq ID No.198 is a hypomethylated probe that targets the target region shown in Seq ID No.52, and Seq ID No.356 is a hypermethylated probe that targets the target region shown in Seq ID No. 52. Seq ID No.199 and Seq ID No.200 are both hypomethylated probes targeting the target region shown in Seq ID No.53, and Seq ID No.357 and Seq ID No.358 are both hypermethylated probes targeting the target region shown in Seq ID No. 53. Seq ID No.201 is a hypomethylated probe targeting the target region shown in Seq ID No.54, and Seq ID No.359 is a hypermethylated probe targeting the target region shown in Seq ID No. 54. Seq ID No.202 and Seq ID No.203 are both hypomethylated probes targeting the target region shown in Seq ID No.55, and Seq ID No.360 and Seq ID No.361 are both hypermethylated probes targeting the target region shown in Seq ID No. 55. Seq ID No.204 and Seq ID No.205 are both hypomethylated probes targeting the target region shown in Seq ID No.56, and Seq ID No.362 and Seq ID No.363 are both hypermethylated probes targeting the target region shown in Seq ID No. 56. Seq ID No.206 and Seq ID No.207 are both hypomethylated probes targeting the target region shown in Seq ID No.57, and Seq ID No.364 and Seq ID No.365 are both hypermethylated probes targeting the target region shown in Seq ID No. 57. Seq ID No.208 and Seq ID No.209 are both hypomethylated probes targeting the target region shown in Seq ID No.58, and Seq ID No.366 and Seq ID No.367 are both hypermethylated probes targeting the target region shown in Seq ID No. 58. Seq ID No.210 and Seq ID No.211 are both hypomethylated probes targeting the target region shown in Seq ID No.59, and Seq ID No.368 and Seq ID No.369 are both hypermethylated probes targeting the target region shown in Seq ID No. 59. Seq ID No.212 is a hypomethylated probe targeting the target region shown in Seq ID No.60, and Seq ID No.370 is a hypermethylated probe targeting the target region shown in Seq ID No. 60. Seq ID No.213 and Seq ID No.214 are both hypomethylated probes targeting the target region shown in Seq ID No.61, and Seq ID No.371 and Seq ID No.372 are both hypermethylated probes targeting the target region shown in Seq ID No. 61. Seq ID No.215 is a hypomethylated probe targeting the target region shown in Seq ID No.62, and Seq ID No.373 is a hypermethylated probe targeting the target region shown in Seq ID No. 62. Seq ID No.216 is a hypomethylated probe targeting the target region shown in Seq ID No.63, and Seq ID No.374 is a hypermethylated probe targeting the target region shown in Seq ID No. 63. Seq ID No.217 is a hypomethylated probe targeting the target region shown in Seq ID No.64, and Seq ID No.375 is a hypermethylated probe targeting the target region shown in Seq ID No. 64. Seq ID No.219 is a hypomethylated probe targeting the target region shown in Seq ID No.66, and Seq ID No.377 is a hypermethylated probe targeting the target region shown in Seq ID No. 66. Seq ID No.220 is a hypomethylated probe targeting the target region shown in Seq ID No.67, and Seq ID No.378 is a hypermethylated probe targeting the target region shown in Seq ID No. 67. Seq ID No.222 is a hypomethylated probe targeting the target region shown in Seq ID No.79, and Seq ID No.391 is a hypermethylated probe targeting the target region shown in Seq ID No. 79. Seq ID No.235 is a hypomethylated probe that targets the target region shown in Seq ID No.81, and Seq ID No.393 is a hypermethylated probe that targets the target region shown in Seq ID No. 81. Seq ID No.236 is a hypomethylated probe targeting the target region shown in Seq ID No.82, and Seq ID No.394 is a hypermethylated probe targeting the target region shown in Seq ID No. 82. Seq ID No.237 is a hypomethylated probe targeting the target region shown in Seq ID No.83, and Seq ID No.395 is a hypermethylated probe targeting the target region shown in Seq ID No. 83. Seq ID No.241 is a hypomethylated probe targeting the target region shown in Seq ID No.87, and Seq ID No.399 is a hypermethylated probe targeting the target region shown in Seq ID No. 87. Seq ID No.242 is a hypomethylated probe that targets the target region shown in Seq ID No.88, and Seq ID No.400 is a hypermethylated probe that targets the target region shown in Seq ID No. 88. Seq ID No.243 is a hypomethylated probe targeting the target region shown in Seq ID No.89, and Seq ID No.401 is a hypermethylated probe targeting the target region shown in Seq ID No. 89. Seq ID No.244 is a hypomethylated probe targeting the target region shown in Seq ID No.90, and Seq ID No.402 is a hypermethylated probe targeting the target region shown in Seq ID No. 90. Seq ID No.251 is a hypomethylated probe targeting the target region shown in Seq ID No.97, and Seq ID No.409 is a hypermethylated probe targeting the target region shown in Seq ID No. 97. Seq ID No.252 is a hypomethylated probe targeting the target region shown in Seq ID No.98, and Seq ID No.410 is a hypermethylated probe targeting the target region shown in Seq ID No. 98. Seq ID No.253 is a hypomethylated probe that targets the target region shown in Seq ID No.99, and Seq ID No.411 is a hypermethylated probe that targets the target region shown in Seq ID No. 99. Seq ID No.254 is a hypomethylated probe targeting the target region shown in Seq ID No.100, and Seq ID No.412 is a hypermethylated probe targeting the target region shown in Seq ID No. 100. Seq ID No.255 is a hypomethylated probe targeting the target region shown in Seq ID No.101, and Seq ID No.413 is a hypermethylated probe targeting the target region shown in Seq ID No. 101. Seq ID No.256 and Seq ID No.257 are both hypomethylated probes targeting the target region shown in Seq ID No.102, and Seq ID No.414 and Seq ID No.415 are both hypermethylated probes targeting the target region shown in Seq ID No. 102. Seq ID No.266 and Seq ID No.267 are both hypomethylated probes targeting the target region shown in Seq ID No.111, and Seq ID No.425 and Seq ID No.426 are both hypermethylated probes targeting the target region shown in Seq ID No. 111. Seq ID No.275 is a hypomethylated probe targeting the target region shown in Seq ID No.117, and Seq ID No.433 is a hypermethylated probe targeting the target region shown in Seq ID No. 117. Seq ID No.276 is a hypomethylated probe targeting the target region shown in Seq ID No.118, and Seq ID No.434 is a hypermethylated probe targeting the target region shown in Seq ID No. 118. 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 above or equal to the threshold, when the number of markers above or equal to the threshold (n1) is more than or equal to 20% of the total number of markers (n), the cancer is determined. 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 greater than or equal to the respective threshold, and n 1/n.gtoreq.20%, preferably.gtoreq.30%, the patient is judged to have 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 higher than the respective threshold, the tissues targeted by m1 probes equal to or higher than the respective threshold are further analyzed and the number of probes equal to or higher than the threshold per tissue is counted, and the tissue of the patient suffering from cancer is judged to be the tissue with the highest number of probes with the methylation level equal to or higher than the threshold. The respective thresholds of the tissue-specific regions are shown in table 1, e.g. as Seq ID No.: 79, the threshold value is 0.29, so that when a probe targeting the region is used for detection, if the detection result is greater than or equal to 0.29, the methylation level of the region detected by the probe is greater than or equal to 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 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, 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.: 66-67, 79, 81-83, 87-90, 97-102, 111, 117, and 118.

In the system referred to herein above, in the hybrid 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.: any of 119-215, probes Seq ID No.: any of 216-217, and a probe Seq ID No.: 219-220, 233, 235-237, 241-244, 251-257, 266-268, 275-276. The hypermethylated probes include probe Seq ID No.: any of 277-373, probe Seq ID No.: 374-375, and a probe Seq ID No. targeting a tissue-specific region: 377-.

In the system as referred to herein above, each probe in the probe composition in the hybrid capture module has a length of 40-60 bp. 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, 50 to 56bp can be used. The length of each probe in the probe composition is preferably 50-56 bp, and more preferably 50 bp.

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 the 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 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:

extraction and purification of cfDNA

1.1.1. Plasma sample preparation:

the blood samples were centrifuged at 2000g for 10min at 4 ℃ and the plasma 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 20 s.

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 30 s.

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

1.1.3.9. The tube was left on the magnetic rack and the residual 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 30 s.

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 1.1.3.10-1.1.3.12 steps 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 dried in air 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 the cfDNA in the supernatant is aspirated.

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

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, 250 s.

1.2.2. After the interruption, 1 μ 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-200 bp.

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 linker was diluted in advance to the appropriate concentration with reference to the following table:

TABLE 7

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):

watch 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 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.

1.4.4.4. 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 resuspended magnetic beads to avoid generating bubbles, and standing at room temperature for 2 min.

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 (1-500ng)
			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 15 min. Centrifuging for 1min, and discarding the centrifuged liquid.

1.5.10. Add 500. mu.l buffer BW (note whether absolute ethanol is added), centrifuge for 1min, discard the liquid from the centrifuge, 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. Placing the column in a clean 1.5ml centrifuge tube, adding 20 μ l nuclease-free water to the center of the column membrane, lightly covering the tube cover, standing at room temperature for 1min, and centrifuging for 1 min.

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 1 min.

1.6. Pre-amplification and purification before hybridization:

1.6.1. preparing a reaction system according to the following table, uniformly mixing by blowing, 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 PCR cycle numbers were adjusted depending on the amount of DNA dosed, 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 frame for 3min to clarify the solution.

1.6.6. The supernatant was removed, the PCR tube was 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 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).

1.6.8. 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 the Qubit and library concentration was 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. Melting Hyb buffer solution at room temperature, allowing precipitate to appear after melting, mixing, preheating in a 65 deg.C water bath, dissolving completely (no precipitate and turbid substance), placing 20 μ l Hyb buffer solution in a new 200 μ l PCR tube, covering the tube cover, labeling with A, and incubating in a 65 deg.C water bath for further use.

1.7.5. The following hypomethylated probes were synthesized by the agutazone biotechnology (beijing) limited:

a) probe Seq ID No.: any of 119-215, b) a probe Seq ID No. targeting a pan-cancer specific region: 216, 217, and c) a probe targeting a tissue specific region Seq ID No.: any of the operations 218-276, if any,

and the following hypermethylated probes were synthesized:

d) probe Seq ID No.: any of 277-373, e) probe Seq ID No.: 374-375, and f) a probe that targets a tissue-specific region Seq ID No.: 376 and 434.

And a probe composition was prepared at a ratio of a: b: c: d: e: f: 1:1:1:1: 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, 5 min; 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.

1.7.10.5min later, C was placed on the PCR and incubated, and the lid was closed to the PCR instrument.

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 at 65 ℃ overnight (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 cleaning the magnetic beads 3 times in total.

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 the PCR instrument, adding the prepared 200. mu.L of capture magnetic beads into the hybridization product PCR tube C, pipetting for 6 times, mixing, and placing on a rotary mixer for bonding at room temperature for 30min (preferably, the rotation speed is not more than 10 rpm).

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. Add 200. mu.L of Wash buffer 1 to PCR tube C, gently pipette 6 times and mix, wash on a spin mixer for 15min (preferably not more than 10 rpm), centrifuge briefly, place PCR tube on magnetic stand for 2min to clarify the solution, remove 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 placed on a magnetic stand, 200. mu.l of 80% ethanol was added to the PCR tube, left to stand for 30s, the ethanol solution was removed completely, and the tube was air-dried at room temperature for 2 min.

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

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

1.9.4. Incubate at room temperature for 5min, and place the PCR tube on a magnetic frame 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 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 frame, gently blow and mix the resuspended beads, and stand at room temperature for 2 min.

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

1.9.10. Pipette 23. mu.l of the supernatant into a 1.5ml centrifuge tube 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 area 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.81
CPE	4	166300242	166300291	0.42	Seq ID No.82
						PLXDC2	10	20104497	20104546	0.46	Seq ID No.97
PLXDC2	10	20104758	20104807	0.46	Seq ID No.98
						PLXDC2	10	20104948	20104997	0.53	Seq ID No.99
PLXDC2	10	20105593	20105642	0.49	Seq ID No.100
						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	30131001	30131050	0.57	Seq ID No.84
						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 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 OTX1, SFRP2, CDO1, TRIM15, ALX4 and CCNA1 are all judged to be greater than or equal to the respective thresholds shown in table 1 (as shown in table 19 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 19 having the respective threshold values or higher, it was found that the 6 target regions Seq ID No.81, Seq ID No.82, Seq ID No.97, Seq ID No.98, Seq ID No.99 and Seq ID No.100, which are stomach tissue-specific, were at least the threshold values of the respective methylation levels, and that the methylation of the other tissue-specific markers was not less than the respective threshold values, and therefore the most stomach tissue-specific markers were among the tissue-specific markers having the respective methylation threshold values or higher, and finally, the sample was judged to be a sample having gastric 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 methylation levels, and the results are shown in table 20 below (table 20 shows that target regions equal to or greater than the methylation threshold were 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.87
C6orf155	6	72130553	72130602	0.71	Seq ID No.88
						C6orf155	6	72130641	72130690	0.65	Seq ID No.89
C6orf155	6	72130755	72130804	0.69	Seq ID No.90
						SHISA2	13	26625273	26625397	0.61	Seq ID No.111
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 identification on a detection sample, firstly judging that the methylation levels of 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 and 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.87, Seq ID No.88, Seq ID No.89, and Seq ID No.90, 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 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 and 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 and colorectal cancer); finally, the tissue-specific markers were interpreted, and based on the target regions in table 21 having the respective thresholds or higher, it was found that the 4 target regions Seq ID No.66, Seq ID No.67, Seq ID No.79, and Seq ID No.80, which are specific to esophageal tissue, have the respective methylation levels of the thresholds or higher, and no other tissue-specific markers have the methylation levels of the thresholds or higher, and therefore, the esophageal tissue-specific markers are 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 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.

Sequence listing

<110> Boercheng (Beijing) science and technology Limited

<120> a cancer gene methylation detection system and a cancer in vitro detection method performed in the system

<130> PC00573

<141> 2020-02-25

<160> 338

<170> SIPOSequenceListing 1.0

<210> 1

<211> 132

<212> DNA

<213> Artificial Sequence

<400> 1

ctctgctctc ccctcaaaac aagtttcagg atcttgcagg cctcgcggcg tcgttcttcg 60

ttgtggcggc ctgtggctct ttgaaaaaca cgacgaggcc tgcaaaatgc gtttttcttt 120

ttttccttta cg 132

<210> 2

<211> 63

<212> DNA

<213> Artificial Sequence

<400> 2

atccgacggt ggccgcaaga tctcatcatg gatctgaccc ctgctcagcg cgcgccattt 60

cgt 63

<210> 3

<211> 61

<212> DNA

<213> Artificial Sequence

<400> 3

cgctccaggg gcgaaggact ctggactcac cccgaccacc gggagagctg gcccctaccc 60

a 61

<210> 4

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 4

tcactgcggg attcggcgtt gccgccagcc cagtggggag tgaattagcg 50

<210> 5

<211> 62

<212> DNA

<213> Artificial Sequence

<400> 5

cggcccttcc gacggcacga ggaactcctg tcctgcccca cagaccttcg gcctccgccg 60

ag 62

<210> 6

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 6

agcttaacag gaatattttc cagcagtgag caggggctgt atgggacgcg 50

<210> 7

<211> 53

<212> DNA

<213> Artificial Sequence

<400> 7

gagcgcctga aatatacttg caaggccgca gcaatatact tgcaaggccg cag 53

<210> 8

<211> 81

<212> DNA

<213> Artificial Sequence

<400> 8

cgatcttcca gtcctagtgc cctggtcgag acggttctat ccttttgcaa agaagccgga 60

aagagctggg tcccgggggc g 81

<210> 9

<211> 118

<212> DNA

<213> Artificial Sequence

<400> 9

cgggatgaca gactctgaca atcattaaac cagccgggcc tgatttccca gcactgcctg 60

ctaagatccg ggccaagtgg cactgaatat gcaaatcacc tggggccagg agcccagt 118

<210> 10

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 10

gggcctctgg tgtcccccat ggtgcagggg gatgacaagg tgtttcgccg 50

<210> 11

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 11

cgtggccacc aatgacccgc ggcgcccccg cgtgtccccg cagccactcc 50

<210> 12

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 12

ctggctgggc gcacggcggt gctgagctgg tggggcggcg gcgctgagcg 50

<210> 13

<211> 53

<212> DNA

<213> Artificial Sequence

<400> 13

agaattctaa tttatttaat tattctaaaa attccaatca caatggcgcg gcg 53

<210> 14

<211> 56

<212> DNA

<213> Artificial Sequence

<400> 14

cgcagccggg acaatttcga gacaacttcg agacaatttc gaatggacaa attgcg 56

<210> 15

<211> 74

<212> DNA

<213> Artificial Sequence

<400> 15

ctgaggagag ccgcgcaatg gaaacctggg tgcagggact gtggggcccg aaggcggggc 60

tgggcgcgct ctcg 74

<210> 16

<211> 65

<212> DNA

<213> Artificial Sequence

<400> 16

cggccagtgc ctccgcgccc cggctccggt ccccaccgtc cccgccccag atttccggag 60

gagca 65

<210> 17

<211> 77

<212> DNA

<213> Artificial Sequence

<400> 17

cgctgcagac tcctgacctg ccgactgcgg atcccgagtc cccggatccc ggacccatcc 60

tgtggagccc actcctg 77

<210> 18

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 18

ttgcaggaac tgtatccctg cctgcgacgg gggcgagata gatgattccg 50

<210> 19

<211> 62

<212> DNA

<213> Artificial Sequence

<400> 19

cgcccccaca gtgagcgagc agggcgcggg ctgcgggagt ggggggcacg cagggcaccc 60

cg 62

<210> 20

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 20

cgggcttgtt ttgccccagt ccgaagtttc tgctgggttg ccaggcatga 50

<210> 21

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 21

gttggggggc tgcgtccctg gtagccgcgt gtgccctgtg atggagcccg 50

<210> 22

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 22

cgaatctcca gccaccgttc agcagcctgt cggtgtgctc cccaatgccg 50

<210> 23

<211> 140

<212> DNA

<213> Artificial Sequence

<400> 23

tatgcgtgtc aactgccatc aacttccttg cttgctgggg actggggccg cgagggcata 60

cccccgaggg gtacggggct agggctaggc aggctgtgcg gttgggcggg gccctgtgcc 120

ccactgcgga gtgcgggtcg 140

<210> 24

<211> 61

<212> DNA

<213> Artificial Sequence

<400> 24

gggccccttt taagcgcttg gagtcactag gaatgtacca acggccctcg gagggaggac 60

g 61

<210> 25

<211> 59

<212> DNA

<213> Artificial Sequence

<400> 25

cggctcacgc gcacatcccc ggcttccccg ggctccgcgc cttcccaaga gccccgttg 59

<210> 26

<211> 80

<212> DNA

<213> Artificial Sequence

<400> 26

cgggcctaaa atgcattagc tggtttttac tgaatttacg cttagcagag acctacagaa 60

aaatgagatc cagctcgccg 80

<210> 27

<211> 52

<212> DNA

<213> Artificial Sequence

<400> 27

cgcgaggctg gagtgatttt tttgataatc ctgctagaga cagaatgggt aa 52

<210> 28

<211> 89

<212> DNA

<213> Artificial Sequence

<400> 28

cggggctcca gccaggcgtc accttccaca gcgaacctgc gaaccacagc gtcccctggg 60

ggtctccgtc cgcgtggccg cttcctctt 89

<210> 29

<211> 57

<212> DNA

<213> Artificial Sequence

<400> 29

cgcgccccgc ctgctggacc acttcatctg tgagctgccg gcgttgctca agctggc 57

<210> 30

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 30

cggagacact accgagaacc agatgttcgc cgcccgcgtg gtcatcctgc 50

<210> 31

<211> 68

<212> DNA

<213> Artificial Sequence

<400> 31

aacttactgc gaggagcacg gcgagaagat ctacttcttc tgcgagaacg atgccgagtt 60

cctctgtg 68

<210> 32

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 32

cgaggcattc ccagactgaa ggcagatagg gctccacttg gatgtgtggt 50

<210> 33

<211> 70

<212> DNA

<213> Artificial Sequence

<400> 33

tcggggccag agtttgaagc cgtggatgtg cctgcctggt ggcttgtccg atttgcacgg 60

tgacttgatt 70

<210> 34

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 34

cgaagcgctt tagtgccttc cgtccctaaa ccgccaacag ccagaacggc ttctc 55

<210> 35

<211> 63

<212> DNA

<213> Artificial Sequence

<400> 35

ttccttcctc ctgctggcca gagacacatt ttgcatctgc aaggcatccg gagatcagcc 60

gcg 63

<210> 36

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 36

agaaagcgag gcttggaggg cgcctacacg gggccccatg gcccgctgcg 50

<210> 37

<211> 66

<212> DNA

<213> Artificial Sequence

<400> 37

agagaagctc ctggagcggc cagatacctg ttggctcctg agcagcatcg cccagtgcag 60

cctccg 66

<210> 38

<211> 63

<212> DNA

<213> Artificial Sequence

<400> 38

cgccaccaaa agtcggtagc tgggagcctt caaagcccgc caaacacact ggaagcatcc 60

aca 63

<210> 39

<211> 56

<212> DNA

<213> Artificial Sequence

<400> 39

cgcactcggt acggcagcat ctctggcctc cagccggggt taaccctgac ctgaac 56

<210> 40

<211> 76

<212> DNA

<213> Artificial Sequence

<400> 40

cggtaatctt cgagagctcg aagggccgag ttgggccagg acgatttccg agcagagccc 60

tcggctcgga tgctcg 76

<210> 41

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 41

cgtgttccga ggccagattt cttctggcca gaggaccctc ggcctgctcc 50

<210> 42

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 42

cgagctacgt ggtgctcttg gcagaccttg actaggttct ttttacagcg 50

<210> 43

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 43

catttcaaag ctacacaatg cgggcggtcc cggcgaggcg ggaggggccg 50

<210> 44

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 44

gagcccgcga tggagctttg tgaagatgca ggcgttgggg ctgctcggcg 50

<210> 45

<211> 57

<212> DNA

<213> Artificial Sequence

<400> 45

cccccttgat gaggtcctga ccaaatgcag gaggagcaat tccagcaccg aggggcg 57

<210> 46

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 46

cggaaaccct gcctgtactg gggccgcagc gctgccccca cccatacgta 50

<210> 47

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 47

cgggaaaaac aaactccaag tcgagtttaa cagccgaaac gctccgtgcc 50

<210> 48

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 48

cggcggacaa gaaggagaca gaacagatcc cttggctccc tcgcagcgat 50

<210> 49

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 49

gcagggggct gtattggaag ccgccgggct ggctgcaggc gccaaagtcg 50

<210> 50

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 50

cgagttgagg gaataatcag aaagagagct ccctctggaa gtcgcagtcc 50

<210> 51

<211> 62

<212> DNA

<213> Artificial Sequence

<400> 51

tgtaaagaca gccttgactc aagcatgcgt tagagcacgt gtcagggccg accgtgctgg 60

cg 62

<210> 52

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 52

agcctggcga gtgaggcgcg aaaccggagg ggtcggcgag gatgcgggcg 50

<210> 53

<211> 66

<212> DNA

<213> Artificial Sequence

<400> 53

tggcttcagc ttcaatccca gaaaagtgga tcaaaacgac aggttccacg aacaaacact 60

gcgccg 66

<210> 54

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 54

cgcctctact ttggagaaaa ggaaaagggc gtgaggtttc ggtcattttt 50

<210> 55

<211> 62

<212> DNA

<213> Artificial Sequence

<400> 55

aaaataaagt tcgattattt cacctggctt gtcagtcacc tatgcaggcg tctgagcccc 60

cg 62

<210> 56

<211> 66

<212> DNA

<213> Artificial Sequence

<400> 56

cgcccggccc ccagcccgga gagctgccac cgaccccctc aacgtcccaa gccccagctc 60

tgtcgc 66

<210> 57

<211> 66

<212> DNA

<213> Artificial Sequence

<400> 57

cggagattcg gaaacccgca gagacttctc aagtcagcag gaacttggaa accgctgttc 60

cctcca 66

<210> 58

<211> 63

<212> DNA

<213> Artificial Sequence

<400> 58

cgtccgtttg caccgtctcc ggaacaactg gcggccagca gcactctcca ccccggccgc 60

aac 63

<210> 59

<211> 57

<212> DNA

<213> Artificial Sequence

<400> 59

tgctgaccgc ctcgcagcgc tggccgggct ccgggaggag ggccccggcg ggtggcg 57

<210> 60

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 60

ggcgggggcg gcgggggtgg tggcggaggc ggcgggggcc cagggtcccg 50

<210> 61

<211> 84

<212> DNA

<213> Artificial Sequence

<400> 61

cgggagggag tcggaggcgc cagcccactg gggaggtggc gctgggcgcg cgggatgcgc 60

ggggagcctt ctctgcagga gccg 84

<210> 62

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 62

cggtctgacg ccccctgctc attcgccagg cagccttgat tggcatgacc 50

<210> 63

<211> 53

<212> DNA

<213> Artificial Sequence

<400> 63

gagcgcctga aatatacttg caaggccgca gcaatatact tgcaaggccg cag 53

<210> 64

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 64

ctggctgggc gcacggcggt gctgagctgg tggggcggcg gcgctgagcg 50

<210> 66

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 66

cacagtcagg gatggtgggc tccagtctgg gccttagggg tggggcgtcg 50

<210> 67

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 67

cgcactgagg ccccgccccc acatggcagg gtgtttttgt ttttgttttt 50

<210> 79

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 79

cgtgatgccg tcttcatttg gaacaagggg gggttcatgc caaaattagg 50

<210> 81

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 81

cgagccagca aataacgcca gcagccctcc cagatccacg ccggcccgtc 50

<210> 82

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 82

gaaggtgagg cgagtagagg ctggtgcgga acttgccgcc cccagcagcg 50

<210> 83

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 83

tgacaagtgc ccctgtggga tacacagccc caggcctcat cggtcactcg 50

<210> 84

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 84

cgcctcagag ccagaagttt atggctccca cctgctcaat ctgacaggaa 50

<210> 85

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 85

cggggtgctt ctggagcctg cctcccaggg agcaggctga ggagctggcg 50

<210> 86

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 86

cggggctgtg gtcacagtaa agcaaggcga tcttcgcaca cagcaagtgc 50

<210> 87

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 87

gatgctgtct tggagcgggc accgccgggg gaaaagtctg gactgcctcg 50

<210> 88

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 88

cggtcaagaa tcctggggaa cccgctccgc cccctggctc cagcgccctc 50

<210> 89

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 89

aggtgtcgga aagcccagcc agtccccggg agtgtagcca atagaaggcg 50

<210> 90

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 90

cgatcctgga gcccagatca agccaagtcc ggccaaagct gtgtcgcaaa 50

<210> 97

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 97

cggggaccac gctgcggtgc accaccttcc gccaccctcc cgcgctgggc 50

<210> 98

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 98

cgcagttggc ggagctgccc agggcgtgcg agcaggctgc gaggatggcg 50

<210> 99

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 99

cggcttaatc tgttggccgc gactcctcag ccagccccga ggaaagtgca 50

<210> 100

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 100

cggcgctggc tgtggaatta gatctgtttt gaacccagtg gagcgcatcg 50

<210> 101

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 101

tgagaggccc ctccgtcctt gaggagcaaa cctggggctg cggagaaccg 50

<210> 102

<211> 67

<212> DNA

<213> Artificial Sequence

<400> 102

cgcccagaaa agcctggaaa tggtgtgggc tattgttggg agaacccgcc tcgcaacaga 60

gctgacg 67

<210> 111

<211> 125

<212> DNA

<213> Artificial Sequence

<400> 111

cgggccctag gtccaggagc tcctaagcca tccccgtcct cagccggtgg cccggtcccc 60

ttagggactg ccttatgagt tgcactcgct gatggaaatc tcgggatgca gtcagaaggc 120

ccccg 125

<210> 117

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 117

cggggacact gcgtaccacc cggcgcacag cccctcccgc gcaaacccga 50

<210> 118

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 118

cgccgtccgg gtgggcctag cagtcgctcc atttatcgct tgagatctcc 50

<210> 119

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 119

cataaaaaaa aaaaaaaaaa acacatttta caaacctcat catatttttc 50

<210> 120

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 120

aaaaaaccac aaaccaccac aacaaaaaac aacaccacaa aacctacaaa 50

<210> 121

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 121

caaaacctac aaaacaaaat cctaaaactt attttaaaaa aaaaacaaaa 50

<210> 122

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 122

acaaaataac acacactaaa caaaaatcaa atccataata aaatcttaca 50

<210> 123

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 123

cactaaacaa aaatcaaatc cataataaaa tcttacaacc accatcaaat 50

<210> 124

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 124

taaataaaaa ccaactctcc caataatcaa aataaatcca aaatccttca 50

<210> 125

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 125

caactctccc aataatcaaa ataaatccaa aatccttcac ccctaaaaca 50

<210> 126

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 126

cactaattca ctccccacta aactaacaac aacaccaaat cccacaataa 50

<210> 127

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 127

ctcaacaaaa accaaaaatc tataaaacaa aacaaaaatt cctcatacca 50

<210> 128

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 128

caaaaatcta taaaacaaaa caaaaattcc tcataccatc aaaaaaacca 50

<210> 129

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 129

cacatcccat acaaccccta ctcactacta aaaaatattc ctattaaact 50

<210> 130

<211> 53

<212> DNA

<213> Artificial Sequence

<400> 130

ctacaacctt acaaatatat tactacaacc ttacaaatat atttcaaaca ctc 53

<210> 131

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 131

cacccccaaa acccaactct ttccaacttc tttacaaaaa aataaaacca 50

<210> 132

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 132

ttacaaaaaa ataaaaccat ctcaaccaaa acactaaaac taaaaaatca 50

<210> 133

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 133

actaaactcc taaccccaaa taatttacat attcaatacc acttaaccca 50

<210> 134

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 134

aatcttaaca aacaatacta aaaaatcaaa cccaactaat ttaataatta 50

<210> 135

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 135

taaaaaatca aacccaacta atttaataat tatcaaaatc tatcatccca 50

<210> 136

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 136

caacaaaaca ccttatcatc cccctacacc ataaaaaaca ccaaaaaccc 50

<210> 137

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 137

aaaataacta caaaaacaca caaaaacacc acaaatcatt aataaccaca 50

<210> 138

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 138

cactcaacac caccacccca ccaactcaac accaccatac acccaaccaa 50

<210> 139

<211> 53

<212> DNA

<213> Artificial Sequence

<400> 139

caccacacca ttataattaa aatttttaaa ataattaaat aaattaaaat tct 53

<210> 140

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 140

cacaatttat ccattcaaaa ttatctcaaa attatctcaa aattatccca 50

<210> 141

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 141

ttatccattc aaaattatct caaaattatc tcaaaattat cccaactaca 50

<210> 142

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 142

caaaaacaca cccaacccca ccttcaaacc ccacaatccc tacacccaaa 50

<210> 143

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 143

caaaccccac aatccctaca cccaaatttc cattacacaa ctctcctcaa 50

<210> 144

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 144

tactcctcca aaaatctaaa acaaaaacaa taaaaaccaa aaccaaaaca 50

<210> 145

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 145

ctaaaacaaa aacaataaaa accaaaacca aaacacaaaa acactaacca 50

<210> 146

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 146

caaaaataaa ctccacaaaa taaatccaaa atccaaaaac tcaaaatcca 50

<210> 147

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 147

aaaatccaaa aactcaaaat ccacaatcaa caaatcaaaa atctacaaca 50

<210> 148

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 148

caaaatcatc tatctcaccc ccatcacaaa caaaaataca attcctacaa 50

<210> 149

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 149

caaaataccc tacatacccc ccactcccac aacccacacc ctactcactc 50

<210> 150

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 150

catacccccc actcccacaa cccacaccct actcactcac tataaaaaca 50

<210> 151

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 151

tcatacctaa caacccaaca aaaacttcaa actaaaacaa aacaaaccca 50

<210> 152

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 152

caaactccat cacaaaacac acacaactac caaaaacaca accccccaac 50

<210> 153

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 153

caacattaaa aaacacacca acaaactact aaacaataac taaaaattca 50

<210> 154

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 154

caacccacac tccacaataa aacacaaaac cccacccaac cacacaacct 50

<210> 155

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 155

acctaaccct aaccccatac ccctcaaaaa tataccctca caaccccaat 50

<210> 156

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 156

caaccccaat ccccaacaaa caaaaaaatt aataacaatt aacacacata 50

<210> 157

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 157

catcctccct ccaaaaacca ttaatacatt cctaataact ccaaacactt 50

<210> 158

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 158

caaaaaccat taatacattc ctaataactc caaacactta aaaaaaaccc 50

<210> 159

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 159

caacaaaact cttaaaaaaa cacaaaaccc aaaaaaacca aaaatataca 50

<210> 160

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 160

tcttaaaaaa acacaaaacc caaaaaaacc aaaaatatac acataaacca 50

<210> 161

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 161

caacaaacta aatctcattt ttctataaat ctctactaaa cataaattca 50

<210> 162

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 162

ctctactaaa cataaattca ataaaaacca actaatacat tttaaaccca 50

<210> 163

<211> 52

<212> DNA

<213> Artificial Sequence

<400> 163

ttacccattc tatctctaac aaaattatca aaaaaatcac tccaacctca ca 52

<210> 164

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 164

aaaaaaaaac aaccacacaa acaaaaaccc ccaaaaaaca ctataattca 50

<210> 165

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 165

actataattc acaaattcac tataaaaaat aacacctaac taaaacccca 50

<210> 166

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 166

accaacttaa acaacaccaa caactcacaa ataaaataat ccaacaaaca 50

<210> 167

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 167

taaacaacac caacaactca caaataaaat aatccaacaa acaaaacaca 50

<210> 168

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 168

acaaaataac cacacaaaca acaaacatct aattctcaat aatatctcca 50

<210> 169

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 169

cacaaaaaaa ctcaacatca ttctcacaaa aaaaataaat cttctcacca 50

<210> 170

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 170

cattctcaca aaaaaaataa atcttctcac catactcctc acaataaatt 50

<210> 171

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 171

accacacatc caaataaaac cctatctacc ttcaatctaa aaatacctca 50

<210> 172

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 172

aatcaaatca ccatacaaat caaacaaacc accaaacaaa cacatccaca 50

<210> 173

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 173

caaacaaacc accaaacaaa cacatccaca acttcaaact ctaaccccaa 50

<210> 174

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 174

aaaaaaccat tctaactatt aacaatttaa aaacaaaaaa cactaaaaca cttca 55

<210> 175

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 175

cacaactaat ctccaaatac cttacaaata caaaatatat ctctaaccaa 50

<210> 176

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 176

caaatacctt acaaatacaa aatatatctc taaccaacaa aaaaaaaaaa 50

<210> 177

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 177

cacaacaaac cataaaaccc catataaaca ccctccaaac ctcactttct 50

<210> 178

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 178

caaaaactac actaaacaat actactcaaa aaccaacaaa tatctaacca 50

<210> 179

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 179

caatactact caaaaaccaa caaatatcta accactccaa aaacttctct 50

<210> 180

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 180

tataaatact tccaatatat ttaacaaact ttaaaaactc ccaactacca 50

<210> 181

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 181

aatatattta acaaacttta aaaactccca actaccaact tttaataaca 50

<210> 182

<211> 56

<212> DNA

<213> Artificial Sequence

<400> 182

attcaaatca aaattaaccc caactaaaaa ccaaaaatac taccatacca aataca 56

<210> 183

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 183

caaacatcca aaccaaaaac tctactcaaa aatcatccta acccaactca 50

<210> 184

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 184

caaaaatcat cctaacccaa ctcaaccctt caaactctca aaaattacca 50

<210> 185

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 185

aaaacaaacc aaaaatcctc taaccaaaaa aaatctaacc tcaaaacaca 50

<210> 186

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 186

cactataaaa aaaacctaat caaaatctac caaaaacacc acataactca 50

<210> 187

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 187

caacccctcc cacctcacca aaaccaccca cattatataa ctttaaaata 50

<210> 188

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 188

caccaaacaa ccccaacacc tacatcttca caaaactcca tcacaaactc 50

<210> 189

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 189

cacccctcaa tactaaaatt actcctccta catttaatca aaacctcatc 50

<210> 190

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 190

caatactaaa attactcctc ctacatttaa tcaaaacctc atcaaaaaaa 50

<210> 191

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 191

tacatataaa taaaaacaac actacaaccc caatacaaac aaaatttcca 50

<210> 192

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 192

aacacaaaac atttcaacta ttaaactcaa cttaaaattt atttttccca 50

<210> 193

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 193

atcactacaa aaaaaccaaa aaatctattc tatctccttc ttatccacca 50

<210> 194

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 194

caactttaac acctacaacc aacccaacaa cttccaatac aaccccctac 50

<210> 195

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 195

aaactacaac ttccaaaaaa aactctcttt ctaattattc cctcaactca 50

<210> 196

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 196

caccaacaca atcaacccta acacatactc taacacatac ttaaatcaaa 50

<210> 197

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 197

caaccctaac acatactcta acacatactt aaatcaaaac tatctttaca 50

<210> 198

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 198

cacccacatc ctcaccaacc cctccaattt cacacctcac tcaccaaact 50

<210> 199

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 199

caacacaata tttattcata aaacctatca ttttaatcca cttttctaaa 50

<210> 200

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 200

cataaaacct atcattttaa tccacttttc taaaattaaa actaaaacca 50

<210> 201

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 201

aaaaataacc aaaacctcac acccttttcc ttttctccaa aataaaaaca 50

<210> 202

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 202

caaaaactca aacacctaca taaataacta acaaaccaaa taaaataatc 50

<210> 203

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 203

cacctacata aataactaac aaaccaaata aaataatcaa actttatttt 50

<210> 204

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 204

acaacaaaac taaaacttaa aacattaaaa aaatcaataa caactctcca 50

<210> 205

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 205

ttaaaacatt aaaaaaatca ataacaactc tccaaactaa aaaccaaaca 50

<210> 206

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 206

taaaaaaaac aacaatttcc aaattcctac taacttaaaa aatctctaca 50

<210> 207

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 207

ttccaaattc ctactaactt aaaaaatctc tacaaatttc caaatctcca 50

<210> 208

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 208

attacaacca aaataaaaaa tactactaac caccaattat tccaaaaaca 50

<210> 209

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 209

taaaaaatac tactaaccac caattattcc aaaaacaata caaacaaaca 50

<210> 210

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 210

caccacccac caaaaccctc ctcccaaaac ccaaccaaca ctacaaaaca 50

<210> 211

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 211

caccaaaacc ctcctcccaa aacccaacca acactacaaa acaatcaaca 50

<210> 212

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 212

caaaacccta aacccccacc acctccacca ccacccccac cacccccacc 50

<210> 213

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 213

caactcctac aaaaaaaact ccccacacat cccacacacc caacaccacc 50

<210> 214

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 214

cacacccaac accacctccc caataaacta acacctccaa ctccctccca 50

<210> 215

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 215

aatcatacca atcaaaacta cctaacaaat aaacaaaaaa catcaaacca 50

<210> 216

<211> 53

<212> DNA

<213> Artificial Sequence

<400> 216

ctacaacctt acaaatatat tactacaacc ttacaaatat atttcaaaca ctc 53

<210> 217

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 217

cactcaacac caccacccca ccaactcaac accaccatac acccaaccaa 50

<210> 219

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 219

caacacccca cccctaaaac ccaaactaaa acccaccatc cctaactata 50

<210> 220

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 220

aaaaacaaaa acaaaaacac cctaccatat aaaaacaaaa cctcaataca 50

<210> 233

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 233

cctaatttta acataaaccc ccccttattc caaataaaaa caacatcaca 50

<210> 235

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 235

aacaaaccaa cataaatcta aaaaaactac taacattatt tactaactca 50

<210> 236

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 236

cactactaaa aacaacaaat tccacaccaa cctctactca cctcaccttc 50

<210> 237

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 237

caaataacca ataaaaccta aaactatata tcccacaaaa acacttatca 50

<210> 238

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 238

ttcctatcaa attaaacaaa taaaaaccat aaacttctaa ctctaaaaca 50

<210> 239

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 239

caccaactcc tcaacctact ccctaaaaaa caaactccaa aaacacccca 50

<210> 240

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 240

acacttacta tatacaaaaa tcaccttact ttactataac cacaacccca 50

<210> 241

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 241

caaaacaatc caaacttttc ccccaacaat acccactcca aaacaacatc 50

<210> 242

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 242

aaaaacacta aaaccaaaaa acaaaacaaa ttccccaaaa ttcttaacca 50

<210> 243

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 243

caccttctat taactacact cccaaaaact aactaaactt tccaacacct 50

<210> 244

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 244

tttacaacac aactttaacc aaacttaact taatctaaac tccaaaatca 50

<210> 251

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 251

acccaacaca aaaaaataac aaaaaataat acaccacaac ataatcccca 50

<210> 252

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 252

caccatcctc acaacctact cacacaccct aaacaactcc accaactaca 50

<210> 253

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 253

tacactttcc tcaaaactaa ctaaaaaatc acaaccaaca aattaaacca 50

<210> 254

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 254

caatacactc cactaaattc aaaacaaatc taattccaca accaacacca 50

<210> 255

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 255

caattctcca caaccccaaa tttactcctc aaaaacaaaa aaacctctca 50

<210> 256

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 256

catcaactct attacaaaac aaattctccc aacaataacc cacaccattt 50

<210> 257

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 257

aacaaattct cccaacaata acccacacca tttccaaact tttctaaaca 50

<210> 266

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 266

caaaaacctt ctaactacat cccaaaattt ccatcaacaa atacaactca 50

<210> 267

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 267

taaaacaatc cctaaaaaaa ccaaaccacc aactaaaaac aaaaataact 50

<210> 268

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 268

ccaccaacta aaaacaaaaa taacttaaaa actcctaaac ctaaaaccca 50

<210> 275

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 275

tcaaatttac acaaaaaaaa ctatacacca aataatacac aatatcccca 50

<210> 276

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 276

aaaaatctca aacaataaat aaaacaacta ctaaacccac ccaaacaaca 50

<210> 277

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 277

cgtaaaaaaa aaaaaaaaaa acgcatttta caaacctcgt cgtatttttc 50

<210> 278

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 278

aaaaaaccac aaaccgccac aacgaaaaac gacgccgcga aacctacaaa 50

<210> 279

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 279

cgccgcgaaa cctacaaaat cctaaaactt attttaaaaa aaaaacaaaa 50

<210> 280

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 280

acgaaataac gcgcgctaaa caaaaatcaa atccataata aaatcttacg 50

<210> 281

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 281

cgctaaacaa aaatcaaatc cataataaaa tcttacgacc accgtcgaat 50

<210> 282

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 282

taaataaaaa ccaactctcc cgataatcga aataaatcca aaatccttcg 50

<210> 283

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 283

caactctccc gataatcgaa ataaatccaa aatccttcgc ccctaaaacg 50

<210> 284

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 284

cgctaattca ctccccacta aactaacgac aacgccgaat cccgcaataa 50

<210> 285

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 285

ctcgacgaaa accgaaaatc tataaaacaa aacaaaaatt cctcgtaccg 50

<210> 286

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 286

cgaaaatcta taaaacaaaa caaaaattcc tcgtaccgtc gaaaaaaccg 50

<210> 287

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 287

cgcgtcccat acaaccccta ctcactacta aaaaatattc ctattaaact 50

<210> 288

<211> 53

<212> DNA

<213> Artificial Sequence

<400> 288

ctacgacctt acaaatatat tactacgacc ttacaaatat atttcaaacg ctc 53

<210> 289

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 289

cgcccccgaa acccaactct ttccgacttc tttacaaaaa aataaaaccg 50

<210> 290

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 290

ttacaaaaaa ataaaaccgt ctcgaccaaa acactaaaac taaaaaatcg 50

<210> 291

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 291

actaaactcc taaccccaaa taatttacat attcaatacc acttaacccg 50

<210> 292

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 292

aatcttaaca aacaatacta aaaaatcaaa cccgactaat ttaataatta 50

<210> 293

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 293

taaaaaatca aacccgacta atttaataat tatcaaaatc tatcatcccg 50

<210> 294

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 294

cgacgaaaca ccttatcatc cccctacacc ataaaaaaca ccaaaaaccc 50

<210> 295

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 295

aaaataacta cgaaaacacg cgaaaacgcc gcgaatcatt aataaccacg 50

<210> 296

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 296

cgctcaacgc cgccgcccca ccaactcaac accgccgtac gcccaaccaa 50

<210> 297

<211> 53

<212> DNA

<213> Artificial Sequence

<400> 297

cgccgcgcca ttataattaa aatttttaaa ataattaaat aaattaaaat tct 53

<210> 298

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 298

cgcaatttat ccattcgaaa ttatctcgaa attatctcga aattatcccg 50

<210> 299

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 299

ttatccattc gaaattatct cgaaattatc tcgaaattat cccgactacg 50

<210> 300

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 300

cgaaaacgcg cccaaccccg ccttcgaacc ccacaatccc tacacccaaa 50

<210> 301

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 301

cgaaccccac aatccctaca cccaaatttc cattacgcga ctctcctcaa 50

<210> 302

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 302

tactcctccg aaaatctaaa acgaaaacga taaaaaccga aaccgaaacg 50

<210> 303

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 303

ctaaaacgaa aacgataaaa accgaaaccg aaacgcgaaa acactaaccg 50

<210> 304

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 304

caaaaataaa ctccacaaaa taaatccgaa atccgaaaac tcgaaatccg 50

<210> 305

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 305

gaaatccgaa aactcgaaat ccgcaatcga caaatcaaaa atctacaacg 50

<210> 306

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 306

cgaaatcatc tatctcgccc ccgtcgcaaa caaaaataca attcctacaa 50

<210> 307

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 307

cgaaataccc tacgtacccc ccactcccgc aacccgcgcc ctactcgctc 50

<210> 308

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 308

cgtacccccc actcccgcaa cccgcgccct actcgctcac tataaaaacg 50

<210> 309

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 309

tcatacctaa caacccaaca aaaacttcga actaaaacaa aacaaacccg 50

<210> 310

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 310

cgaactccat cacaaaacac acgcgactac caaaaacgca accccccaac 50

<210> 311

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 311

cgacattaaa aaacacaccg acaaactact aaacgataac taaaaattcg 50

<210> 312

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 312

cgacccgcac tccgcaataa aacacaaaac cccgcccaac cgcacaacct 50

<210> 313

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 313

acctaaccct aaccccgtac ccctcgaaaa tataccctcg cgaccccaat 50

<210> 314

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 314

cgaccccaat ccccaacaaa caaaaaaatt aataacaatt aacacgcata 50

<210> 315

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 315

cgtcctccct ccgaaaaccg ttaatacatt cctaataact ccaaacgctt 50

<210> 316

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 316

cgaaaaccgt taatacattc ctaataactc caaacgctta aaaaaaaccc 50

<210> 317

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 317

caacgaaact cttaaaaaaa cgcgaaaccc gaaaaaaccg aaaatatacg 50

<210> 318

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 318

tcttaaaaaa acgcgaaacc cgaaaaaacc gaaaatatac gcgtaaaccg 50

<210> 319

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 319

cgacgaacta aatctcattt ttctataaat ctctactaaa cgtaaattca 50

<210> 320

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 320

ctctactaaa cgtaaattca ataaaaacca actaatacat tttaaacccg 50

<210> 321

<211> 52

<212> DNA

<213> Artificial Sequence

<400> 321

ttacccattc tatctctaac aaaattatca aaaaaatcac tccaacctcg cg 52

<210> 322

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 322

aaaaaaaaac gaccacgcga acgaaaaccc ccaaaaaacg ctataattcg 50

<210> 323

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 323

gctataattc gcaaattcgc tataaaaaat aacgcctaac taaaaccccg 50

<210> 324

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 324

accaacttaa acaacgccga caactcacaa ataaaataat ccaacaaacg 50

<210> 325

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 325

taaacaacgc cgacaactca caaataaaat aatccaacaa acgaaacgcg 50

<210> 326

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 326

acaaaataac cacgcgaacg acgaacatct aattctcgat aatatctccg 50

<210> 327

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 327

cacaaaaaaa ctcgacatcg ttctcgcaaa aaaaataaat cttctcgccg 50

<210> 328

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 328

cgttctcgca aaaaaaataa atcttctcgc cgtactcctc gcaataaatt 50

<210> 329

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 329

accacacatc caaataaaac cctatctacc ttcaatctaa aaatacctcg 50

<210> 330

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 330

aatcaaatca ccgtacaaat cgaacaaacc accaaacaaa cacatccacg 50

<210> 331

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 331

cgaacaaacc accaaacaaa cacatccacg acttcaaact ctaaccccga 50

<210> 332

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 332

aaaaaaccgt tctaactatt aacgatttaa aaacgaaaaa cactaaaacg cttcg 55

<210> 333

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 333

cgcgactaat ctccgaatac cttacaaata caaaatatat ctctaaccaa 50

<210> 334

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 334

cgaatacctt acaaatacaa aatatatctc taaccaacaa aaaaaaaaaa 50

<210> 335

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 335

cgcaacgaac cataaaaccc cgtataaacg ccctccaaac ctcgctttct 50

<210> 336

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 336

cgaaaactac actaaacgat actactcaaa aaccaacaaa tatctaaccg 50

<210> 337

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 337

cgatactact caaaaaccaa caaatatcta accgctccaa aaacttctct 50

<210> 338

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 338

tataaatact tccaatatat ttaacgaact ttaaaaactc ccaactaccg 50

<210> 339

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 339

aatatattta acgaacttta aaaactccca actaccgact tttaataacg 50

<210> 340

<211> 56

<212> DNA

<213> Artificial Sequence

<400> 340

attcaaatca aaattaaccc cgactaaaaa ccaaaaatac taccgtaccg aatacg 56

<210> 341

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 341

cgaacatccg aaccgaaaac tctactcgaa aatcgtccta acccaactcg 50

<210> 342

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 342

cgaaaatcgt cctaacccaa ctcgaccctt cgaactctcg aaaattaccg 50

<210> 343

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 343

aaaacaaacc gaaaatcctc taaccaaaaa aaatctaacc tcgaaacacg 50

<210> 344

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 344

cgctataaaa aaaacctaat caaaatctac caaaaacacc acgtaactcg 50

<210> 345

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 345

cgacccctcc cgcctcgccg aaaccgcccg cattatataa ctttaaaata 50

<210> 346

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 346

cgccgaacaa ccccaacgcc tacatcttca caaaactcca tcgcgaactc 50

<210> 347

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 347

cgcccctcga tactaaaatt actcctccta catttaatca aaacctcatc 50

<210> 348

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 348

cgatactaaa attactcctc ctacatttaa tcaaaacctc atcaaaaaaa 50

<210> 349

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 349

tacgtataaa taaaaacaac gctacgaccc caatacaaac aaaatttccg 50

<210> 350

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 350

aacacgaaac gtttcgacta ttaaactcga cttaaaattt atttttcccg 50

<210> 351

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 351

atcgctacga aaaaaccaaa aaatctattc tatctccttc ttatccgccg 50

<210> 352

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 352

cgactttaac gcctacaacc aacccgacga cttccaatac aaccccctac 50

<210> 353

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 353

aaactacgac ttccaaaaaa aactctcttt ctaattattc cctcaactcg 50

<210> 354

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 354

cgccaacacg atcgacccta acacgtactc taacgcatac ttaaatcaaa 50

<210> 355

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 355

cgaccctaac acgtactcta acgcatactt aaatcaaaac tatctttaca 50

<210> 356

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 356

cgcccgcatc ctcgccgacc cctccgattt cgcgcctcac tcgccaaact 50

<210> 357

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 357

cgacgcaata tttattcgta aaacctatcg ttttaatcca cttttctaaa 50

<210> 358

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 358

cgtaaaacct atcgttttaa tccacttttc taaaattaaa actaaaacca 50

<210> 359

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 359

aaaaataacc gaaacctcac gcccttttcc ttttctccaa aataaaaacg 50

<210> 360

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 360

cgaaaactca aacgcctaca taaataacta acaaaccaaa taaaataatc 50

<210> 361

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 361

gcctacataa ataactaaca aaccaaataa aataatccga actttatttt 50

<210> 362

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 362

acgacaaaac taaaacttaa aacgttaaaa aaatcgataa caactctccg 50

<210> 363

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 363

ttaaaacgtt aaaaaaatcg ataacaactc tccgaactaa aaaccgaacg 50

<210> 364

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 364

taaaaaaaac aacgatttcc aaattcctac taacttaaaa aatctctacg 50

<210> 365

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 365

ttccaaattc ctactaactt aaaaaatctc tacgaatttc cgaatctccg 50

<210> 366

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 366

attacgaccg aaataaaaaa tactactaac cgccaattat tccgaaaacg 50

<210> 367

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 367

taaaaaatac tactaaccgc caattattcc gaaaacgata caaacgaacg 50

<210> 368

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 368

cgccacccgc cgaaaccctc ctcccgaaac ccgaccaacg ctacgaaacg 50

<210> 369

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 369

cgccgaaacc ctcctcccga aacccgacca acgctacgaa acgatcaaca 50

<210> 370

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 370

cgaaacccta aacccccgcc gcctccgcca ccacccccgc cgcccccgcc 50

<210> 371

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 371

cgactcctac aaaaaaaact ccccgcgcat cccgcgcgcc caacgccacc 50

<210> 372

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 372

cgcgcccaac gccacctccc caataaacta acgcctccga ctccctcccg 50

<210> 373

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 373

aatcatacca atcaaaacta cctaacgaat aaacaaaaaa cgtcaaaccg 50

<210> 374

<211> 53

<212> DNA

<213> Artificial Sequence

<400> 374

ctacgacctt acaaatatat tactacgacc ttacaaatat atttcaaacg ctc 53

<210> 375

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 375

cgctcaacgc cgccgcccca ccaactcaac accgccgtac gcccaaccaa 50

<210> 377

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 377

cgacgcccca cccctaaaac ccaaactaaa acccaccatc cctaactata 50

<210> 378

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 378

aaaaacaaaa acaaaaacac cctaccatat aaaaacgaaa cctcaatacg 50

<210> 391

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 391

cctaatttta acataaaccc ccccttattc caaataaaaa cgacatcacg 50

<210> 393

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 393

aacgaaccga cgtaaatcta aaaaaactac taacgttatt tactaactcg 50

<210> 394

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 394

cgctactaaa aacgacaaat tccgcaccaa cctctactcg cctcaccttc 50

<210> 395

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 395

cgaataaccg ataaaaccta aaactatata tcccacaaaa acacttatca 50

<210> 396

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 396

ttcctatcaa attaaacaaa taaaaaccat aaacttctaa ctctaaaacg 50

<210> 397

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 397

cgccaactcc tcaacctact ccctaaaaaa caaactccaa aaacaccccg 50

<210> 398

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 398

acacttacta tatacgaaaa tcgccttact ttactataac cacaaccccg 50

<210> 399

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 399

cgaaacaatc caaacttttc ccccgacgat acccgctcca aaacaacatc 50

<210> 400

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 400

aaaaacgcta aaaccaaaaa acgaaacgaa ttccccaaaa ttcttaaccg 50

<210> 401

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 401

cgccttctat taactacact cccgaaaact aactaaactt tccgacacct 50

<210> 402

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 402

tttacgacac aactttaacc gaacttaact taatctaaac tccaaaatcg 50

<210> 409

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 409

acccaacgcg aaaaaataac gaaaaataat acaccgcaac gtaatccccg 50

<210> 410

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 410

cgccatcctc gcaacctact cgcacgccct aaacaactcc gccaactacg 50

<210> 411

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 411

tacactttcc tcgaaactaa ctaaaaaatc gcgaccaaca aattaaaccg 50

<210> 412

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 412

cgatacgctc cactaaattc aaaacaaatc taattccaca accaacgccg 50

<210> 413

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 413

cgattctccg caaccccaaa tttactcctc aaaaacgaaa aaacctctca 50

<210> 414

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 414

cgtcaactct attacgaaac gaattctccc aacaataacc cacaccattt 50

<210> 415

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 415

aacgaattct cccaacaata acccacacca tttccaaact tttctaaacg 50

<210> 424

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 424

cgaaaacctt ctaactacat cccgaaattt ccatcaacga atacaactca 50

<210> 425

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 425

taaaacaatc cctaaaaaaa ccgaaccacc gactaaaaac gaaaataact 50

<210> 426

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 426

ccaccgacta aaaacgaaaa taacttaaaa actcctaaac ctaaaacccg 50

<210> 433

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 433

tcgaatttac gcgaaaaaaa ctatacgccg aataatacgc aatatccccg 50

<210> 434

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 434

aaaaatctca aacgataaat aaaacgacta ctaaacccac ccgaacgacg 50

Claims

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;

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

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

2 probes targeting a pan-cancer specific region,

n probes targeting a cancer specific region, and

m probes targeting a tissue specific region.

6. The system of any one of claims 1-5, 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 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, further preferably 50 bp.

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

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

10. The system of claim 5, wherein in the hybrid capture module, the hypomethylated probes comprise probes that target cancer-specific regions Seq ID No.: any of 119-215, probes Seq ID No.: any of 216-217, and a probe Seq ID No.: 219-220, 233, 235-237, 241-244, 251-257, 266-268, 275-276.