Tumor early screening and diagnosis probe library, kit and detection method based on co-detection of ctDNA methylation and mutation

Abstract

The invention discloses a tumor early screening and diagnosis probe library, a kit and a detection method based on co-detection of ctDNA methylation and mutation. The probe library comprises a specific probe aiming at 120 cg site methylation biomarkers and a specific probe aiming at 158 mutation sites of 14 tumor related genes. The detection method comprises the following steps: extracting free DNA; library preparation; carrying out enzyme digestion on a part of the DNA library by using methylation specific incision enzyme; respectively carrying out PCR amplification on the DNA libraries before and after enzyme digestion, and hybridizing and capturing by using a specific probe library; and carrying out high-throughput sequencing on the captured fragment to identify methylation and mutation states of a target region of the captured fragment. According to the probe library, the kit and the detection method, methylation and mutation conditions of free DNA of plasma can be detected with high sensitivity and high specificity so as to identify and quantify circulating tumor DNA, and the probe library, the kit and the detection method have huge clinical application prospects in the fields of tumor early screening, auxiliary diagnosis, relapse monitoring, curative effect evaluation and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a tumor early screening, diagnostic probe library, kit and detection method based on the co-detection of ctDNA methylation and mutation. Background Art

[0002] Tumors cause a large number of deaths every year. The global number of cancer death cases is close to 10 million. The key to affecting the efficacy of tumors lies in early detection and early treatment. Research shows that the cure rate of early-stage tumors is very high, and many can be cured. The average five-year survival rate of early-stage tumor patients is 91%. However, once the tumor develops to the advanced stage, the cure rate is greatly reduced, and the average five-year survival rate of advanced tumor patients is only 26%. People have realized the importance of early detection of tumors, and various early screening technologies have been developed and applied. Traditional detections include tumor markers, endoscopic examinations, imaging examinations, etc. There are many tumor markers, but the false positive rate is relatively high; the time to detect tumor lesions by imaging is too late; endoscopic examinations are only applicable to some cancer types such as gastric cancer and colorectal cancer, and the patient compliance is low.

[0003] Tumor early screening technologies based on liquid biopsy have attracted much attention in clinical applications due to their advantages such as non-invasive and non-radiative. Currently, detection technologies based on liquid biopsy, such as ctDNA mutation detection and DNA methylation detection technologies, have made important progress and shown good clinical application prospects. However, there are still obstacles such as high false positive rate and low specificity and sensitivity. High false positive detection results will lead to unnecessary subsequent examinations and anxiety for the tested subjects, while low sensitivity may pose a risk of missed diagnosis.

[0004] The occurrence and development of tumors are closely related to abnormal methylation and gene mutations of tumor DNA. Circulating tumor DNA (ctDNA) is a DNA fragment released by tumor cells into the blood circulation, which carries tumor-specific methylation and mutation information and can be used as an important biomarker for early tumor diagnosis. However, the content of ctDNA in the blood of early-stage tumor patients is extremely low, and there is high heterogeneity. Therefore, it is of great significance to develop high-sensitivity and high-specificity ctDNA detection methods.

[0005] Currently, ctDNA detection methods mainly include PCR-based methods and high-throughput sequencing-based methods. Existing ctDNA methylation detection technologies mainly include methylation-specific PCR (MSP), bisulfite sequencing (BS-seq), and methylation-sensitive high-resolution melting curve analysis (MS-HRM). The MSP technology has high sensitivity, but its specificity and quantification ability are limited, and it is difficult to detect multiple methylation sites. The bisulfite sequencing (BS-seq) technology can provide methylation information across the whole genome, but it is costly, has complex data analysis, and has insufficient detection sensitivity for low-abundance ctDNA. Although the MS-HRM technology is easy to operate, its resolution and sensitivity are relatively low, making it difficult to meet the requirements of early tumor detection. In addition, most of the existing ctDNA detection methods only focus on single indicators such as methylation or mutation, and it is difficult to meet the requirements of high sensitivity and high specificity for early tumor screening.

[0006] In addition, there are other technical challenges in using ctDNA methylation analysis for early tumor detection, including: lack of suitable methylation markers. At present, not enough ctDNA methylation sites have been found as biomarkers to distinguish tumors from healthy people. The existing markers are limited in number and not highly specific, resulting in tumor samples often being misjudged as negative (false negatives), or healthy samples being misjudged as positive (false positives), thus causing unnecessary anxiety and over-detection. Existing methylation detection technologies often involve a trade-off between sensitivity and specificity. For example, methylation-specific PCR (MSP) has high sensitivity but insufficient specificity; while bisulfite sequencing (BS-seq) can provide whole-genome methylation information, but its detection sensitivity for low-abundance ctDNA is limited.

[0007] Gene mutation detection has high specificity. Especially when detecting known tumor driver gene mutations (such as KRAS G12D, EGFRL858R, etc.), it can effectively distinguish tumor patients from healthy people. However, the sensitivity of gene mutation detection is low, mainly because not every patient carries gene mutations that can be used for detection. Especially in the early tumor stage, the mutation ratio in ctDNA is low (generally less than 0.1%); and the selectable mutation sites are very limited, making it difficult to achieve early screening through single or a few mutation detections. If combined mutation and methylation detections are used, the detection sensitivity and specificity can be greatly improved. Currently, most technologies cannot perform ctDNA methylation and mutation detections simultaneously. The main reason is that methylation detection and mutation detection usually require different sample processing procedures and detection platforms, and it is difficult to achieve in the same reaction system. For example, methylation detection requires bisulfite treatment of DNA, and this process may damage the DNA sequence, affecting the accuracy of mutation detection. Summary of the Invention

[0008] In view of this, the object of the present invention is to provide a tumor early screening, diagnostic probe library, kit and detection method based on the co-detection of ctDNA methylation and mutation. The probe library, kit and detection method can detect the methylation and mutation of cell-free tumor DNA in a sample with high sensitivity and high specificity, and have great clinical application prospects in the fields of tumor early screening, auxiliary diagnosis, recurrence monitoring, efficacy evaluation, etc.

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] The present invention provides a detection method for co-detection of ctDNA methylation and mutation, the method comprising:

[0011] Extracting cell-free DNA (cfDNA) from a biological sample, the biological sample being selected from blood, sputum, urine, feces, cerebrospinal fluid, pleural effusion, ascites, etc.;

[0012] Performing end repair, A-tailing and adapter ligation on the extracted cfDNA to prepare a DNA library;

[0013] Dividing the DNA library into two parts, wherein, one part is digested with a methylation-sensitive endonuclease;

[0014] Performing PCR amplification on the digested DNA library and the undigested DNA library respectively using primers with different sample indexes;

[0015] Hybridizing and capturing the PCR amplification product with a specific probe library, the probe library comprising specific probes for 120 cg site methylation biomarkers and specific probes for 158 mutation site biomarkers of 14 tumor-related genes;

[0016] Performing high-throughput sequencing on the hybridized and captured DNA fragments to obtain sequencing data;

[0017] Analyzing the sequencing data to identify the methylation and mutation status of the DNA target region in the sample to be tested.

[0018] Preferably, the methylation-sensitive endonuclease is selected from HpaII, BSTUI, AcII, HpyCH41V and their multi-enzyme digestion combinations.

[0019] Preferably, the probes in the probe library are biotinylated single-stranded DNA probes, which are 70-150 nucleotides in length and have high specificity with the target sequence.

[0020] Preferably, the high-throughput sequencing platform is selected from Illumina NovaSeq6000, MiSeq, NextSeq500, BGI MGISEQ-2000, DNBSEQ-G99, DNBSEQ-T7, Element Biosciences (AVITI), UltimaGenomics (UG 100), GenoCare 1600, or Thermo Fisher Ion GeneStudio S5 series, etc.

[0021] Preferably, the data analysis includes the following steps:

[0022] Data preprocessing: Use FastQC software to evaluate the quality of the raw sequencing data, and use adapter removal software for quality filtering and adapter removal;

[0023] Sequence alignment: Use alignment software such as BWA-MEM to align the filtered sequencing data to the human reference genome hg19;

[0024] Methylation analysis: Identify and quantify the number of sequencing reads of the restriction enzyme sites contained in the target region, such as sites of CCGG and CGCG;

[0025] Mutation analysis: Use software such as GATK to detect somatic mutations, and annotate the gene information and functional impacts of the mutation sites.

[0026] The present invention also provides a tumor early screening and diagnosis probe library based on the co-detection of ctDNA methylation and mutation. The probe library contains specific probes for 120 cg-site methylation biomarkers and specific probes for 158 mutation-site biomarkers of 14 tumor-related genes. Among them, the positions of the 120 biomarker cg-sites in the human genome hg19 are shown as follows:

[0027] chr1:3568566, chr1:156406599, chr12:114841155, chr16:86600277, chr2:68546553, chr3:27765348, chr5:1875863, chr7:27196189, chr7:139168349, chr1:4714033, chr1:158150985, chr11:125036498, chr13:37005997, chr17:35165640, chr2:73518649, chr3:50378122, chr7:27204728, chr7:152622582, chr1:13910245, chr11:133938851, chr14:37136251, chr17:46656093, chr2:105459166, chr3:62356223, chr5:42994093, chr7:27205159, chr7:153445808, chr1:14925876, chr1:223302841, chr12:25056207, chr14:60978096, chr17:48042533, chr2:111876726, chr3:139653774, chr5:78365599, chr7:27283528, chr7:156797121, chr1:33219657, chr1:248020908, chr12:25101965, chr14:61110649, chr18:55104979, chr2:121412136, chr4:8863338, chr7:29603279, chr7:156810972, chr1:50882021, chr10:17271503, chr12:50297721, chr14:102247968, chr2:154335110, chr4:13526852, chr5:115152494, chr7:157478340, chr1:63785841, chr14:102248105, chr2:175199881, chr4:24801842, chr5:178421646, chr7:38671009, chr8:17271027, chr1:77333903, chr10:105036745, chr12:63544103, chr15:48937573, chr20:21086198, chr4:141677849, chr6:28411301chr8:41167045, chr12:95942193, chr16:1030641, chr19:30715705, chr20:21685481, chr4:147560279, chr6:32119691, chr7:70596939, chr8:57359311, chr1:119526933, chr10:131770969, chr12:108169051, chr16:25704534, chr20:45142329, chr4:168154865, chr6:137814788, chr8:70981935, chr1:145562905, chr10:133110829, chr12:113917518, chr16:51190145, chr22:33197647, chr4:176987040, chr7:1272935, chr7:100318589, chr8:70982082, chr1:151811370, chr11:31837607, chr12:113917635, chr16:66613195, chr19:46997242, chr4:176987240, chr7:129425828, chr8:97505843, chr1:119532849, chr1:170630712, chr1:63786015, chr1:63795374, chr10:118892493, chr14:57278227, chr17:37321626, chr2:19556813, chr2:223161815, chr2:45231868, chr2:71116118, chr20:21492828, chr20:25062309, chr3:157812320, chr14:38725015, chr6:10417655, chr8:55370844, chr8:99986879;,

[0028] Among them, the mutation sites of 158 biomarkers are shown as follows:

[0029] AKT1 (p.E17K), APC (p.R223X, p.R226X, p.R242X, p.R509X, p.R564X, p.R574X, p.Q793X, p.R815X, p.K885X, p.R886X, p.E951X, p.W1059X, p.K1192X, p.R1124X, p.S1210Ffs, p.Q1313X, p.E1316X, p.E1319X, p.Q1377X, p.R1460X, p.L1498Ffs*25, p.V1424Efs*5, p.H1500Lfs*17, p.E1389X, p.I1557*, p.T1566Nfs*), BRAF (p.G466, p.G469, p.Y472, p.D594, p.G596, p.L597, p.V600), CDKN2A (p.D84Y, p.D153Y, c.151-1G>A, p.E10X, p.H83D, p.H83Y, p.G52Vfs*77, p.C100X, p.R58X, p.S12X, p.H166Y, p.F90Cfs*50, p.E88X, p.Y44Lfs*76), CTNNB1 (p.S37P / A / Y / C / F, p.T41A / I, p.S45F / C / Y / A / P), EGFR (p.E709K / A, p.G719S / C / A, p.E746_A750del, p.E746_A751del, p.E746_T751>A, p.E746_S752>V, p.E746_S752>D, p.L747_A750>P, p.L747_A749del, p.L747_S752del, p.L747_T751del, p.D761Y, p.S768I, p.V769_D770insASV, p.V769_D770insASV, p.D770_N771insG, p.H773_V774insH, p.T790M, p.L858Q, p.L858M, p.L858R, p.L861Q, p.G863D), ERBB2 (p.G309E / A, p.S310Y / F, p.A775_G776insYVMA), FBXW7 (p.R278X, p.R441Q, p.R465C, p.G477S, p.R479Q / X, p.R505 / L, p.C506X, p.Y545C / X, p.H580Y, p.R658X, p.R689W, c.1122+1G>C), KRAS (p.G12S / R / C / D / V / A, p.G13S / R / C / D / A / V, p.Q61K / E / R / L / H, p.K117N / T / V), NRAS (p.Q61K / P / R / L / H, p.K117N, p.A146T), PIK3CA (p.E542K / Q / V, p.E545K, p.E545G / D / A, p.Q546K / E / P / R / Q, p.D549N / H, p.M1043I / V / T, p.N1044K / S / Y / D, p.H1047R / L / Y / Q, p.G1049G / R / S), PTEN (p.R130G / Q / P / L / X, p.R173C / H, p.R233X, p.R233fs), TP53 (p.Q100X, p.R110Ifs, p.S127F, p.H168R, p.E171X, p.R175H, p.C176W, p.H179R, p.V197L, p.R213X, p.Y220C, p.E224D, p.Y234C, p.Y236S, p.Y236H, p.M237I, p.C238Y, p.G245S, p.G245V, p.M246V, p.R248W, p.R248Q, p.R249S, p.I255S, p.R273L, p.R273P, p.C275G, p.P278L, p.T284P, p.E285X, p.E294X, p.R306X, p.Q317, p.F338Lfs*7, c.375+1G>A), SMAD4 (G30X, Q116X, Y195*fs*, W302X, Y353S, R361G, R361H, R361P, G386D, D424A, D493Mfs*, D493Y, G510E, G510V, R531Q).

[0030] In the present invention, the 120 methylated biomarker cg sites are obtained by analyzing and screening a large number of samples and optimized by algorithms, and can cover as many methylated positive tumor patients as possible with the least number of sites. The mutation sites of the 158 biomarkers are hot regions with a high occurrence frequency in tumors and have high specificity, and can effectively distinguish tumor-related gene mutations.

[0031] Preferably, the probe library further includes probes for reference genes.

[0032] Preferably, the probes in the probe library are biotinylated single-stranded DNA probes, with a length of 70-150 nucleotides and high specificity for the target sequence.

[0033] Preferably, the probe library contains specific probes for 120 cg-site methylation biomarkers, the nucleotide sequences of which are shown in SEQ ID No. 1-120; specific probes for 158 mutation-site biomarkers of 14 tumor-related genes, the nucleotide sequences of which are shown in SEQ ID No. 121-342; and 10 probes for reference genes, the nucleotide sequences of which are shown in SEQ ID No. 343-352.

[0034] The tumor early screening and diagnostic probe library provided by the present invention is mainly screened from samples of lung cancer and colorectal cancer, and is mainly used for the detection of lung cancer and colorectal cancer. The present invention has also tested a small number of samples of other cancer types, including liver cancer, gastric cancer, pancreatic cancer, and esophageal cancer, and has proven that these cancer types can also be detected. Therefore, the application is not limited to lung cancer and colorectal cancer, and other cancer types, including liver cancer, gastric cancer, pancreatic cancer, and esophageal cancer, are also applicable to this probe library.

[0035] The present invention also provides a tumor early screening and diagnostic kit based on the co-detection of ctDNA methylation and mutation, and the kit contains the tumor early screening and diagnostic probe library based on the co-detection of ctDNA methylation and mutation described in any one of the above.

[0036] Preferably, the kit further contains:

[0037] cfDNA library construction reagent with molecular tags (UMI): including DNA end repair, A-tailing, and UMI sequencing adapter ligation reaction solutions for end repair, A-tailing, and adapter ligation of DNA;

[0038] Methylation-sensitive endonucleases for methylation-specific digestion of cfDNA, including but not limited to HpaII, BSTU1, AcII, or HpyCH41V methylation-sensitive endonucleases and their multi-enzyme digestion combinations;

[0039] Hybridization capture reagent for hybridizing and capturing the amplification product of cfDNA with the specific probe library;

[0040] PCR reaction solution, including DNA polymerase, dNTPs, PCR reaction buffer, sample index primer, and universal primer required for PCR.

[0041] The tumor early screening and diagnosis kit provided by the present invention utilizes the characteristic that methylation-sensitive endonucleases cannot cleave methylation sites. The methylation-sensitive endonucleases are used to degrade cfDNA from non-tumor sources, and specifically amplify ctDNA from tumor sources, effectively realizing the early screening of lung cancer. Specifically, in a specific embodiment of the kit for tumor early screening provided by the present invention, the methylation-sensitive endonucleases include BSTU1 methylation-sensitive endonuclease, HpaII methylation-sensitive endonuclease, AcII methylation-sensitive endonuclease, and HpyCH41V methylation-sensitive endonuclease. Degrading the test sample with the above methylation-sensitive endonucleases results in very little loss of DNA template molecules during the enzymatic digestion process compared to the traditional bisulfite treatment, which converts unmethylated cytosine into uracil and causes a loss of approximately 50-90% of DNA template molecules. Moreover, the sequence remains intact, enabling the co-detection of methylation and gene mutations. Therefore, the detection sensitivity and specificity can be greatly improved.

[0042] The present invention provides a methylation detection site combination with high signal-to-noise ratio. This site combination comes from 120 CpG sites screened from a large-scale sample, covering high signal-to-noise ratio methylation regions of various tumor types, and can significantly improve the detection sensitivity and specificity.

[0043] In addition, based on the experience accumulated by the inventors in their work, the mutation information of thousands of tumor patients (mainly lung cancer and colorectal cancer) was integrated and analyzed, and 158 mutation hotspots of 14 genes with high-frequency gene mutations were designed into the capture probe pool. The co-detection of methylation and mutations significantly improves the detection sensitivity and specificity. By simultaneously detecting methylation and mutation information and integrating multi-dimensional data, the sensitivity and specificity of tumor early screening are significantly improved.

[0044] Beneficial effects: The tumor early screening, diagnostic probe library, kit, and detection method based on the co-detection of ctDNA methylation and mutations provided by the present invention can detect the methylation and mutation conditions of tumor ctDNA in plasma samples with high sensitivity and high specificity, and have great clinical application prospects in the fields of tumor early screening, auxiliary diagnosis, recurrence monitoring, and efficacy evaluation.

[0045] The tumor early screening, diagnostic probe library, kit, and detection method based on the co-detection of ctDNA methylation and mutations provided by the present invention have the following advantages:

[0046] 1. A methylation site combination with high signal-to-noise ratio. The best methylation site combination screened from a large number of samples covers approximately 98% of tumor patients, significantly improving the detection sensitivity and specificity;

[0047] 2. It has extremely high discrimination ability for tumor and healthy human samples, and the AUC value is as high as 0.96;

[0048] 3. The sensitivity to early lung cancer samples reaches 0.933;

[0049] 4. The combined detection of ctDNA methylation and mutation, by simultaneously detecting two indicators, significantly improves the sensitivity and specificity of early tumor screening. Among them, the overall detection sensitivity of tumors reaches 97%, the specificity reaches 96%, and the sensitivity to early lung cancer reaches 93.3%.

[0050] 5. The detection sites are optimized, the number of sites is moderate, and the cost is low, which is suitable for early tumor screening, tumor auxiliary diagnosis, recurrence monitoring, efficacy evaluation, etc. of large-scale populations. Description of the Drawings

[0051] Figure 1 It is the result of methylation detection of 100 tumor plasma free DNA samples and 100 healthy individual plasma free DNA samples. The results show that there are obvious methylation signals in the plasma DNA of tumor patients, while the methylation signals of the healthy population are relatively low;

[0052] Figure 2 It is to analyze the sensitivity and specificity of the ctDNA methylation site combination described in the present invention as a biomarker for tumor detection by using the ROC (Receiver Operating Characteristic) curve. The ROC curve (AUC = 0.96) of the methylation site combination of the kit described in the present invention shows that it has extremely high sensitivity and specificity for tumor detection.

[0053] Figure 3 It is the result of co-detection of mutations in the plasma of tumor patients, showing that gene mutations can be detected in most plasma DNA. Detailed Embodiments

[0054] The technical solutions of the present invention will be further described in detail below in conjunction with specific implementation examples. It should be understood that the following examples are only for illustrative explanation of the present invention and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention. It should be noted that the experimental materials not specified in the experimental examples of the present invention can be commercially obtained, and the experimental methods not specified in the experimental examples of the present invention are usually carried out according to the conventional experimental methods in the art or according to the methods recommended by the experimental material manufacturers.

[0055] Example 1

[0056] Sample collection: Plasma samples from at least 100 cancer patients and 100 healthy individuals were collected. The cancer patient samples covered a variety of common cancer types (such as lung cancer, colorectal cancer). The healthy individual samples were strictly screened to ensure no history of cancer or other major diseases.

[0057] Sequencing library construction:

[0058] 1. Library construction: Use a commercial plasma-free DNA extraction kit (such as QIAamp Circulating Nucleic Acid Kit) to extract cfDNA from plasma samples. Perform end repair, A-tailing, and adapter ligation on the extracted cfDNA (the adapter uses the Illumina sequencing adapter), and use a DNA library preparation kit (such as NEBNext Ultra II DNA Library Prep Kit) to complete library construction. Purify the library using DNA purification magnetic beads (such as AMPure XP beads) to remove unligated adapters and reaction reagents.

[0059] 2. Library pre-amplification: Divide the DNA library (cfDNA ligation product) into two parts. When using a methylation-sensitive endonuclease to digest one part of the DNA library, the composition of the digestion reaction solution is: 1× digestion buffer, 10U HpaII, and 10U BSTU1 methylation-sensitive endonuclease, 15 - 60 ng cfDNA. The other part is not digested. Then perform PCR amplification. Among them, the pre-amplification PCR reaction solution includes the PCR reaction solution, the DNA library after digestion or the DNA library without digestion, and amplification primers with sample index sequences (the sequences are as shown on the Illumina official website). The reaction system is as follows:

[0060]

[0061] PCR conditions: Pre-denaturation at 98°C for 2 minutes, denaturation at 98°C for 15 seconds, annealing at 60°C for 20 seconds, extension at 72°C for 20 seconds, a total of 7 - 14 cycles, and finally extension at 72°C for 5 minutes.

[0062] 3. Probe hybridization capture enrichment. Hybridize and capture the PCR amplification product of the DNA library with a capture probe library (including a probe combination targeting methylation sites and mutation sites). The hybridization conditions are as follows:

[0063]

[0064] Note: * is the IDT NGS second-generation sequencing blocking reagent / blocking oligonucleotide, here targeting the Illumina sequencing adapter.

[0065] Mix the hybridization reaction solution evenly and place it in a PCR amplifier for reaction. The conditions are 95°C for 2 minutes. Immediately place it on ice and keep it for 2 minutes after the reaction is completed. Then place it in a 60°C constant temperature hybridization instrument for hybridization, and the hybridization time is 6 - 48 hours. Use DynabeadsTM M-280 Streptavidin (Invitrogen) streptavidin magnetic beads or streptavidin magnetic beads from other commercial companies for probe capture. Take 50 μl of magnetic beads and wash them once with Beads Wash Buffer (1M NaCl, 10 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, 0.1% Triton X-100). Adsorb the supernatant with a magnet, then suspend it in 50 μl of Beads Wash Buffer for standby. Add the above-mentioned obtained magnetic bead suspension to the hybridization mixture that has been hybridized for 8 hours, and rotate it at 60°C in a constant temperature hybridization instrument for 45 minutes to capture DNA. Remove the supernatant under the action of a magnetic field. Wash the magnetic beads captured with DNA with 1000 μl of Beads Wash Buffer at 37°C for 15 minutes, and adsorb the supernatant with a magnet; repeat the washing 2 times, and then adsorb the supernatant with a magnet. The captured DNA is resuspended with 50 μl of nuclease-free water to resuspend the magnetic beads captured with DNA. The suspension is reacted at 95°C on a PCR instrument for 4 minutes. Immediately adsorb the magnetic beads at high temperature and collect the supernatant. At this time, the supernatant contains the captured DNA fragments.

[0066] Further PCR amplify the enriched DNA library to prepare for loading on a sequencing instrument. The PCR reaction system is as follows:

[0067]

[0068] PCR conditions: Pre-denaturation at 98°C for 2 minutes, denaturation at 98°C for 15 seconds, annealing at 60°C for 20 seconds, extension at 72°C for 20 seconds, a total of 20 - 22 cycles, and finally extension at 72°C for 5 minutes.

[0069] 4. Sequencing: After the PCR product is purified by XP beads, use a high-throughput sequencing platform (such as Illumina NovaSeq 6000) to sequence the DNA fragments after hybridization capture to obtain paired-end 150bp sequencing data.

[0070] 5. Data analysis:

[0071] Data preprocessing: Include using the FastQC software to evaluate the quality of the original sequencing data, and using the Trimmomatic software for quality filtering and adapter removal.

[0072] Sequence alignment: Include using the BWA-MEM software to align the filtered sequencing data to the human reference genome (hg19).

[0073] Methylation analysis: It includes quantifying CpG reads at restriction enzyme sites and calculating the number of methylated molecules at these CpG sites. By comparing the methylation differences between cfDNA of healthy individuals and cancer patients, the z-score value of each site is calculated, and a threshold for distinguishing healthy individuals from cancer patients is set.

[0074] Mutation analysis: It includes detecting somatic mutations using GATK software and annotating the gene information and functional impacts of the mutation sites.

[0075] Test result display:

[0076] Positive methylation results: Among 100 cancer patient samples, 96 were detected as methylation positive ( Figure 1 ). Among the 100 cancer patient samples, 30 were plasma samples from stage I lung cancer patients, and 27 of them were detected as methylation positive. False positive results in healthy individual samples: Among 100 healthy individual samples, 4 were misjudged as methylation positive. The overall sensitivity of methylation detection was 96% ( Figure 2 ). The sensitivity for early-stage lung cancer was 90%. The overall specificity was 96%.

[0077] Mutation detection results: Among 100 cancer patients, 49 mutations were detected, and the sensitivity was 49% ( Figure 3 ). No mutations were detected in 100 healthy individual samples, and the specificity was 100%. Among 30 early-stage samples, 5 mutations were detected. Among these 5 cases, 1 was detected as methylation negative but mutation positive.

[0078] Combined methylation and mutation detection results: Among 100 cancer patient samples, 97 were detected as positive, 4 were misjudged as positive, the sensitivity was 97%, and the specificity was 96%. The sensitivity for early-stage lung cancer was 93.3%.

[0079] Conclusion:

[0080] The methods and kits provided by the present invention showed excellent performance in the validation experiments, and the sensitivity, specificity, and AUC values all reached relatively high levels; they can effectively distinguish cancer patients from healthy populations. Compared with single methylation or mutation detection, the combined detection of ctDNA methylation and mutation provided by the present invention can significantly improve the detection rate of early-stage cancer samples. This method has important clinical application value in early cancer detection and is suitable for early cancer screening of large-scale populations.

[0081] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention.

Claims

1. A method for detecting ctDNA methylation and mutation, characterized in that: The method comprises: Extracting free cfDNA from a biological sample, wherein the biological sample is selected from blood, sputum, urine, feces, cerebrospinal fluid, pleural effusion, and ascites; The extracted cfDNA was end-repaired, A-tailed, and adapter-ligated to prepare a DNA library; The DNA library is divided into two parts, one of which is digested with a methylation-sensitive endonuclease; Use primers with different sample indexes to perform PCR amplification on the digested DNA library and the undigested DNA library respectively; The PCR amplification product is hybridized and captured with a specific probe library, wherein the probe library contains specific probes for 120 cg site methylation biomarkers and specific probes for 158 mutation site biomarkers of 14 tumor-related genes; Perform high-throughput sequencing on the DNA fragments captured by hybridization to obtain sequencing data; The sequencing data is analyzed to identify the methylation and mutation status of the DNA target region in the sample to be tested.

2. The detection method according to claim 1, characterized in that: The methylation-sensitive endonuclease includes, but is not limited to, HpaII, BSTU1, AcII or HpyCH41V methylation-sensitive endonuclease and multiple enzyme cutting combinations thereof.

3. The detection method according to claim 1, characterized in that: The high-throughput sequencing platform includes but is not limited to Illumina NovaSeq6000, MiSeq, NextSeq500, BGISEQ-2000, DNBSEQ-G99, DNBSEQ-T7Element Biosciences (AVITI), Ultima Genomics (UG 100), GenoCare 1600, or ThermoFisher Ion GeneStudioS5 series.

4. The detection method according to claim 1, characterized in that: The data analysis includes the following steps: Data preprocessing: Use FastQC and other quality control software to assess the quality of raw sequencing data, and use adapter removal software for quality filtering and adapter removal; Sequence alignment: The filtered sequencing data were aligned to the human reference genome using BWA-MEM alignment software; Methylation analysis: Identify the restriction sites in the target region and quantify the number of sequencing reads; Mutation analysis: GATK analysis software was used to detect somatic mutations and annotate the genetic information and functional impact of the mutation sites.

5. A tumor early screening and diagnosis probe library based on ctDNA methylation and mutation co-detection, the probe library comprising specific probes for 120 cg site methylation biomarkers and specific probes for 158 mutation site biomarkers of 14 tumor-related genes, wherein: The positions of 120 biomarker cg sites in human genome hg19 are as follows: chr1:3568566,chr1:156406599,chr12:114841155,chr16:86600277,chr2:68546553,chr3:27765348,chr5:1875863,chr7:27196189,chr7:139168349,chr1:4714033,chr1:158150985,chr11:125036498,chr13:37005997,chr17:35165640,chr2:73518649,chr3:50378122,chr7:27204728,chr7:152622582,chr1:13910245,chr11:133938851,chr14:37136251,chr17:46656093,chr2:105459166,chr3:62356223,chr5:42994093,chr7:27205159,chr7:153445808,chr1:14925876,chr1:223302841,chr12:25056207,chr14:60978096,chr17:48042533,chr2:111876726,chr3:139653774,chr5:78365599,chr7:27283528,chr7:156797121,chr1:33219657,chr1:248020908,chr12:25101965,chr14:61110649,chr18:55104979,chr2:121412136,chr4:8863338,chr7:29603279,chr7:156810972,chr1:50882021,chr10:17271503,chr12:50297721,chr14:102247968,chr2:154335110,chr4:13526852,chr5:115152494,chr7:157478340,chr1:63785841,chr14:102248105,chr2:175199881,chr4:24801842,chr5:178421646,chr7:38671009,chr8:17271027,chr1:77333903,chr10:105036745,chr12:63544103,chr15:48937573,chr20:21086198,chr4:141677849,chr6:28411301,chr8:41167045,chr12:95942193,chr16:1030641,chr19:30715705,chr20:21685481,chr4:147560279,chr6:32119691,chr7:70596939,chr8:57359311,chr1:119526933,chr10:131770969,chr12:108169051,chr16:25704534,chr20:45142329,chr4:168154865,chr6:137814788,chr8:70981935,chr1:145562905,chr10:133110829,chr12:113917518,chr16:51190145,chr22:33197647,chr4:176987040,chr7:1272935,chr7:100318589,chr8:70982082,chr1:151811370,chr11:31837607,chr12:113917635,chr16:66613195,chr19:46997242,chr4:176987240,chr7:129425828,chr8:97505843,chr1:119532849,chr1:170630712,chr1:63786015,chr1:63795374,chr10:118892493,chr14:57278227,chr17:37321626,chr2:19556813,chr2:223161815,chr2:45231868,chr2:71116118,chr20:21492828,chr20:25062309,chr3:157812320,chr14:38725015,chr6:10417655,chr8:55370844,chr8:99986879;, Among them, the mutation sites of 158 biomarkers are as follows: AKT1(p.E17K),APC(p.R223X,p.R226X,p.R242X,p.R509X,p.R564X,p.R574X,p.Q793X,p.R815X,p.K885X,p.R886X,p.E951X,p.W1059X,p.K1192X,p.R1124X,p.S1210Ffs,p.Q1313X,p.E1316X,p.E1319X,p.Q1377X,p.R1460X,p.L1498Ffs*25,p.V1424Efs*5,p.H1500Lfs*17,p.E1389X,p.I1557*,p.T1566Nfs*),BRAF(p.G466,p.G469,p.Y472,p.D594,p.G596,p.L597,p.V600),CDKN2A(p.D84Y,p.D153Y,c.151-1G>A,p.E10X,p.H83D,p.H83Y,p.G52Vfs*77,p.C100X,p.R58X,p.S12X,p.H166Y,p.F90Cfs*50,p.E88X,p.Y44Lfs*76),CTNNB1(p.S37P / A / Y / C / F,p.T41A / I,p.S45F / C / Y / A / P),EGFR(p.E709K / A,p.G719S / C / A,p.E746_A750del,p.E746_A751del,p.E746_T751>A,p.E746_S752>V,p.E746_S752>D,p.L747_A750>P,p.L747_A749del,p.L747_S752del,p.L747_T751del,p.D761Y,p.S768I,p.V769_D770insASV,p.V769_D770insASV,p.D770_N771insG,p.H773_V774insH,p.T790M,p.L858Q,p.L858M,p.L858R,p.L861Q,p.G863D),ERBB2(p.G309E / A,p.S310Y / F,p.A775_G776insYVMA),FBXW7(p.R278X,p.R441Q,p.R465C,p.G477S,p.R479Q / X,p.R505 / L,p.C506X,p.Y545C / X,p.H580Y,p.R658X,p.R689W,c.1122+1G>C),KRAS(p.G12S / R / C / D / V / A,p.G13S / R / C / D / A / V,p.Q61K / E / R / L / H,p.K117N / T / V),NRAS(p.Q61K / P / R / L / H,p.K117N,p.A146T),PIK3CA(p.E542K / Q / V,p.E545K,p.E545G / D / A,p.Q546K / E / P / R / Q,p.D549N / H,p.M1043I / V / T,p.N1044K / S / Y / D,p.H1047R / L / Y / Q,p.G1049G / R / S),PTEN(p.R130G / Q / P / L / X,p.R173C / H,p.R233X,p.R233fs),TP53(p.Q100X,p.R110Ifs,p.S127F,p.H168R,p.E171X,p.R175H,p.C176W,p.H179R,p.V197L,p.R213X,p.Y220C,p.E224D,p.Y234C,p.Y236S,p.Y236H,p.M237I,p.C238Y,p.G245S,p.G245V,p.M246V,p.R248W,p.R248Q,p.R249S,p.I255S,p.R273L,p.R273P,p.C275G,p.P278L,p.T284P,p.E285X,p.E294X,p.R306X,p.Q317,p.F338Lfs*7,c.375+1G>A),SMAD4(G30X,Q116X,Y195*fs*,W302X,Y353S,R361G,R361H,R361P,G386D,D424A,D493Mfs*,D493Y,G510E,G510V,R531Q)。.

6. The probe library according to claim 5, characterized in that The probe library also contains probes targeting internal reference genes.

7. The probe library according to any one of claims 5 to 6, characterized in that: The probes in the probe library are biotin-modified single-stranded DNA probes with a length of 70-150 nucleotides and high specificity to the target sequence.

8. The probe library according to claim 7, characterized in that The probe library comprises specific probes for 120 cg site methylation biomarkers, whose nucleotide sequences are shown in SEQ ID No.1-120; specific probes for 158 mutation site biomarkers of 14 tumor-related genes, whose nucleotide sequences are shown in SEQ ID No.121-342; and 10 probes for internal reference genes, whose nucleotide sequences are shown in SEQ ID No.343-352.

9. A tumor early screening and diagnosis kit based on ctDNA methylation and mutation co-detection, characterized in that: The kit comprises the tumor early screening and diagnosis probe library based on ctDNA methylation and mutation co-detection according to any one of claims 5 to 8.

10. The kit according to claim 9, characterized in that The kit also contains: cfDNA library construction reagents with molecular indices (UMI): including DNA end repair, A-tailing and UMI sequencing adapter ligation reaction solution, used for DNA end repair, A-tailing and adapter ligation; Methylation-sensitive endonucleases, used for methylation-specific digestion of cfDNA libraries, including but not limited to HpaII, BSTU1, AcII or HpyCH41V methylation-sensitive endonucleases and multi-enzyme digestion combinations thereof; A hybridization capture reagent, used to hybridize and capture the amplified product of cfDNA with a specific probe library; PCR reaction solution, including DNA polymerase, dNTPs, PCR reaction buffer, sample index primer, and universal primer required for PCR.

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