Method for detecting ctDNA without obtaining lymphoma tissue sample and application thereof

Abstract

The invention relates to the technical field of biomedicine, in particular to a method for detecting ctDNA without obtaining a lymphoma tissue sample and application. Comprising the following steps: 1, collecting a blood sample; 2, constructing a hybrid library; 3, bioinformatics analysis; the blood sample is derived from peripheral blood. On the basis of a next-generation sequencing technology, a tumor tissue sample does not need to be obtained, the ctDNA content of the sample can be detected and determined only by collecting a peripheral blood sample of a lymphoma patient, and the treatment effect of the patient can be predicted by combining the ctDNA content value and the equivalent value multiple change in the treatment process. Plasma cfDNA and leukocyte genome DNA are collected, and UDI and UMI molecular tags are adopted for NGS sequencing library construction. After sequencing, cfDNA sample sequencing data and leukocyte genome DNA sample sequencing data are combined for filtering analysis, and the ctDNA equivalent of the sample can be accurately calculated.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology (IPC classification number: C12Q), and in particular to a method and application for detecting ctDNA without obtaining lymphoma tissue samples. Background Art

[0002] Lymphoma is one of the most common malignant tumors in China, with approximately 100,000 new cases of lymphoma each year. Lymphoma can originate from any organ and tissue in the body, and is usually divided into two categories: primary lymph nodes and extra-lymph nodes; the most common manifestation is painless progressive lymphadenopathy. On the one hand, for patients with non-superficial lymph node lesions, or patients with unresectable lymphoma lesions, such as central nervous system lymphoma, there is a technical problem that tumor tissue samples are often difficult to obtain after treatment. On the other hand, the most commonly used method for evaluating the efficacy of lymphoma is PET-CT (positron emission computed tomography) imaging evaluation. However, PET-CT is radioactive, and patients cannot undergo frequent PET-CT scans; and the sensitivity of PET-CT for monitoring tiny residual lesions after treatment does not meet the needs.

[0003] The most common circulating tumor DNA (ctDNA) detection method is the tumor-informed assay, which requires first performing next-generation sequencing on tumor tissue to identify gene mutations in tumor cells and filtering germline mutations through oral mucosal samples. These mutation sites of the tumor are then monitored in plasma free DNA samples, and the ctDNA equivalent is calculated. For patients who have difficulty obtaining tumor tissue samples, ctDNA testing based on the tumor-informed assay is difficult to perform. Therefore, a ctDNA detection method that does not rely on lymphoma tumor tissue samples can effectively make up for the shortcomings of the current tumor-informed ctDNA detection technology.

[0004] Existing technologies attempt to use peripheral blood samples for ctDNA detection. However, in plasma cfDNA (free DNA) samples, the proportion of ctDNA from tumor cells is relatively small, which places high demands on the sensitivity of detecting mutation sites in trace ctDNA. Therefore, in the NGS (high-throughput sequencing technology or second-generation sequencing technology) sequencing process of cfDNA samples, adding UMI (molecular tag technology) for library construction and then performing high-depth sequencing is a technical method to ensure high accuracy and high sensitivity in detecting ctDNA mutation sites. At present, the hotspot mutation detection of important genes related to lymphoma generally uses the probe capture method for NGS sequencing library construction, and usually such probe capture panels (detection combinations) are between 100-200 genes. The average number of mutation sites detected in lymphoma samples is between 10 and 20, and some lymphoma samples have even fewer mutation sites.

[0005] Chinese patent CN114446386A discloses a method for detecting blood ctDNA, which determines the genomic regions where benign mutations such as CHV occur frequently by designing a Catcher that can enrich ctDNA mutation signals, and by fitting the blood background distribution of healthy people, and simultaneously captures ctDNA of various malignant tumors including lung cancer and breast cancer, thereby improving the clinical applicability of ctDNA. Chinese patent CN116157539A discloses a multimodal analysis of circulating tumor nucleic acid molecules, which adds at least a portion of a first amount of methylated filler DNA to the sample, and then further denatures the sample, uses a binder that is selective for methylated polynucleotides to capture cell-free methylated DNA, and then sequences and analyzes the captured cell-free methylated DNA to determine the method for detecting ctDNA in cancer cells. However, these prior arts still have defects such as inaccurate detection results, low sensitivity, or complicated operations and difficulties in implementation, and none of them solve the above technical problems.

[0006] Based on the above background, high-sensitivity ctDNA detection based on peripheral blood samples can be used as an effective supplementary means to evaluate the efficacy of lymphoma and deserves in-depth study. Summary of the invention

[0007] The present invention is based on the second-generation sequencing technology. It does not require the acquisition of tumor tissue samples. It can detect and determine the ctDNA content by only collecting peripheral blood samples from lymphoma patients. The effect of patient treatment can be predicted by combining the ctDNA content value and the multiple change of the equivalent value during the treatment process.

[0008] The first aspect of the present invention provides a method for detecting ctDNA without obtaining a lymphoma tissue sample, comprising the following steps:

[0009] 1. Collect blood samples;

[0010] 2. Construction of hybridization library;

[0011] 3. Bioinformatics analysis.

[0012] Preferably, the blood sample is derived from peripheral blood, and the plasma sample and the leukocyte sample are obtained by collecting, centrifuging and separating the peripheral blood.

[0013] Preferably, the peripheral blood includes venous blood, arterial blood, and capillary blood; more preferably, it is venous blood.

[0014] Preferably, the step 1 comprises: collecting a blood sample, centrifuging it at 1000-4000 rpm for 5-15 min, collecting the upper liquid, i.e., the plasma sample; collecting the lower cells, removing the red blood cells, and obtaining the white blood cell sample. (See Figure 1 )

[0015] The core advantage of the method for detecting ctDNA provided by the present invention is that it does not require obtaining tumor tissue samples. It only collects peripheral blood samples from lymphoma patients, centrifuges and separates the blood samples, obtains plasma samples and white blood cell samples respectively, obtains cfDNA information from plasma samples, and obtains germline mutations, CHIP mutations and other base information based on white blood cell gDNA samples. After denoising, the ctDNA equivalent of the sample can be determined; and combined with the ctDNA content value and the multiple change of the equivalent value during the treatment process, the treatment effect can be predicted. Compared with the existing ctDNA detection method, the present invention not only solves the problem that tumor tissue samples are difficult to obtain in actual operation of the traditional tumor-informed method, but also simplifies the sampling operation. The present invention further preferably uses venous blood as the sample, the sample is easy to obtain and the plasma freezing does not affect the experimental results, it is easy and fast, and batch operation is performed, which greatly improves the feasibility of the detection method.

[0016] Preferably, the step 2 comprises: S1. constructing a leukocyte genomic DNA hybridization library; S2. constructing a cfDNA hybridization library; S3. hybridization capture.

[0017] Preferably, in the step S1, the steps of constructing the leukocyte genomic DNA hybridization library include: (1) performing DNA fragmentation, end repair, and dA tailing treatment on the leukocyte sample; (2) linker ligation and purification; (3) double-end labeling with UDI, followed by PCR (polymerase chain reaction) amplification and purification to obtain pre-library one; and (4) quality control of pre-library one.

[0018] dA tailing: refers to the addition of a sequence consisting of adenine (A) nucleotides to the 3' end of a DNA molecule through an enzymatic reaction to form an adenine nucleotide tail (A tail).

[0019] UDI: Unique Dual Index, refers to the adapter with a unique index sequence on both ends; index is a marker sequence used to identify DNA samples.

[0020] Preferably, in the quality control of the pre-library 1, the concentration of the pre-library 1 is ≥50 ng / μL, and the average length of the pre-library 2 is 300 bp-400 bp.

[0021] Preferably, in the step S2, the steps of constructing the cfDNA hybridization library include: (1) cfDNA quantification; (2) end repair and dA tailing of cfDNA; (3) terminal UMI adapter connection and purification; (4) PCR amplification and purification to obtain pre-library 2; and (5) quality control of pre-library 2.

[0022] Preferably, in the quality control of the pre-library 2, the concentration of the pre-library 2 is greater than 25 ng / μL, and the average length of the pre-library 2 is 250 bp-350 bp.

[0023] Preferably, in the S3 step, the hybridization capture step includes: (1) hybridizing pre-library 1 or pre-library 2 with the probe; (2) preparing capture magnetic beads; (3) applying the capture magnetic beads to the hybridization system (1) to capture the target region DNA library; (4) performing PCR amplification after capture to obtain the final library; (5) quality control of the final library; and (6) sequencing the final library on a machine.

[0024] Preferably, in the quality control of the hybrid DNA library, the concentration of the second pre-library is 1-20 ng / μL, and the main peak length of the hybrid DNA library is 270 bp-320 bp.

[0025] Preferably, the probe information is shown in Table 1 below.

[0026] Table 1

[0027]

[0028]

[0029]

[0030]

[0031]

[0032]

[0033]

[0034]

[0035]

[0036]

[0037]

[0038]

[0039] The present invention is based on blood samples, sets a matching library construction method, constructs NGS sequencing libraries through specific UDI and UMI labeling technologies, constructs leukocyte genomic DNA hybridization libraries (prelibrary one) and cfDNA hybridization libraries (prelibrary two), respectively, hybridizes the prelibrary with customized probes, covers high-frequency mutation sites of key driver genes related to lymphoma and high-frequency mutation sites of non-key driver genes, and genes related to BTK inhibitor (Bruton's tyrosine kinase) resistance; captures the target region DNA library under comprehensive coverage; PCR amplifies the target region DNA library to obtain the final library. The detection sensitivity of this method is further improved, and the comparative experiment verifies that the method of the present invention can track more mutation sites, significantly reducing the possibility of missed mutations.

[0040] The present invention further implements strict quality control on pre-library 1, pre-library 2 and final library, screens the test results whose library concentration and library length meet the requirements as the basis for data analysis, reduces experimental errors, and ensures the accuracy of ctDNA detection results.

[0041] Note: The core concept of the present invention is the construction of biological characteristic information such as sequencing libraries. For the implementation of technical means such as extraction methods, extraction methods, library sequencing, bioinformatics analysis processes, etc., the generally available methods in the art can be used to achieve the experimental objectives without any special limitation.

[0042] For example, when constructing a leukocyte genomic DNA hybridization library, a universal library construction kit can be used, such as Aigitaikang, Novezan, Diwin, Twist, etc.

[0043] Preferably, the bioinformatics analysis includes: (1) filtering the sequencing data for background noise and germline variation; (2) calculating the ctDNA equivalent in cfDNA; and (3) a prognostic risk prediction model. The present invention sets specific filtering rules for specific cfDNA samples and leukocyte gDNA samples, thereby minimizing the interference of factors such as background noise and germline variation, thereby ensuring the validity of the test results.

[0044] Preferably, the filtering includes filtering cfDNA, and the filtering principle is set as follows:

[0045] (1) SSCS mutation self-filtering screening: Screening of mutation test data based on single-strand correction mode: The mutation sequencing depth is required to be greater than 100, and the mutation frequency is greater than 1%, the number of supported mutation reads is greater than 1, and the average base quality of the mutation read is at least 39;

[0046] (2) The mutation data can still be obtained in the double-strand correction mode;

[0047] (3) SNP: Data with a population frequency of less than 0.1% were screened based on the ExAC, 1000Genome, and gnomAD databases;

[0048] (4) Non-synonymous mutations: Annovar was used to annotate exon region mutations.

[0049] Preferably, the filtering also includes filtering of germline samples, and the filtering principle is set as follows: if the test results of the leukocyte gDNA sample support the number of mutation reads greater than 1, and the mutation frequency is greater than 0.005, and the sequencing depth of the site is greater than 50, and the average base quality of the mutation reads supported by the site is >30, then this site needs to be excluded in the primary sample.

[0050] Preferably, the filtering also sets the intersection of the mutation sites of the cfDNA tracking samples and the screening sites of the initial samples as the inform set, and performs screening according to the properties of the mutation in the single-strand correction mode, requiring that the sequencing depth of the mutation is greater than 100, and the mutation frequency is greater than 0.1%, supporting the number of mutation reads greater than 1, and the average base quality of the mutation reads is at least 39.

[0051] Preferably, the filtering also sets the non-intersecting part of the mutation sites of the cfDNA tracking samples and the initial sample screening site results as the naive set, and performs screening according to the properties of the mutation in the single-strand correction mode, requiring that the sequencing depth of the mutation is greater than 100, and the mutation frequency is greater than 1%, supporting the number of mutation reads greater than 1, and the average base quality of the mutation read length is at least 39.

[0052] Preferably, the calculation of ctDNA equivalent in the cfDNA includes (1) calculating the ctDNA ratio; (2) calculating the ctDNA concentration; wherein:

[0053]

[0054] The number of cell mutations detected in the cfDNA sequencing results is recorded as j; the mutation read length count at the jth site among the j mutation sites in cfDNA is M j , the sequencing depth is recorded as N j .

[0055] Preferably, when calculating ctDNA concentration

[0056]

[0057] The plasma volume of the i-th blood sample is V i The total amount of cfDNA extracted from the plasma in this blood sample is m i , haploid copy is the haploid copy number.

[0058] The ctDNA equivalent of each blood sample was calculated using the above algorithm.

[0059] Preferably, the prognostic risk prediction model includes: in the scenario of prognostic risk assessment, the change rate of ctDNA concentration in the free samples collected between adjacent time points of the patient is used to predict the patient's efficacy and recurrence possibility. Assuming that the patient's blood was collected at time t0 and t1 for free DNA testing (t0 is earlier than t1), and the ctDNA concentrations detected at the two time points are C0 and C1 respectively, the change rate of ctDNA is calculated as follows

[0060]

[0061] Samples with ctDNA change rate less than a certain threshold (ex-2) are defined as patients with better efficacy, and vice versa as patients with poor efficacy.

[0062] The second aspect of the present invention provides application of the above method in lymphoma detection.

[0063] Beneficial effects:

[0064] The present invention provides a method and application for detecting ctDNA without obtaining lymphoma tissue samples, which has the following advantages:

[0065] (1) The present invention is based on the second-generation sequencing technology. It does not require the acquisition of tumor tissue samples. By simply collecting peripheral blood samples from lymphoma patients, the ctDNA content of the samples can be detected and determined. The effect of patient treatment can be predicted by combining the ctDNA content value and the multiple change of the equivalent value during the treatment process.

[0066] (2) The present invention uses peripheral blood samples, especially venous blood, because the samples are easy to obtain and freezing of plasma does not affect the experimental results. It is easy and quick to operate in batches, and the technical feasibility is strong.

[0067] (3) The ctDNA detection method of the present invention includes collecting plasma cfDNA and leukocyte genomic DNA, and using UDI and UMI molecular tags to construct NGS sequencing libraries. After sequencing, the cfDNA sample sequencing data and the leukocyte genomic DNA sample sequencing data are combined for filtering and analysis, and the ctDNA equivalent of the sample can be accurately calculated.

[0068] (4) The NGS sequencing probe design panel of the present invention covers the high-frequency mutation sites of key driver genes related to lymphoma and the high-frequency mutation sites of non-key driver genes, as well as genes related to BTK inhibitor resistance. Through the detection method of the present invention, more mutation sites are tracked, which helps to improve the detection sensitivity and avoid the situation of missed mutation detection.

[0069] (5) The present invention sets specific filtering rules for specific cfDNA samples and leukocyte gDNA samples, thereby minimizing the interference of factors such as background noise and germline variation, thereby ensuring the validity of the test results.

[0070] (6) The present invention provides a new CAPP-Seq (deep sequencing method for personalized tumor analysis) method, which realizes accurate and efficient detection of ctDNA and provides a new idea for the diagnosis and treatment of lymphoma; it has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 . Schematic diagram of the present invention for collecting blood samples; (where A is a plasma sample, from which cfDNA can be obtained; B is a white blood cell sample, from which white blood cell gDNA (germline mutation, CHIP mutation) can be obtained; and C is a red blood cell)

[0072] Figure 2 . Plasma filtration conditions of the method of the present invention; (wherein plasma filtration condition A: mutation frequency>1%; plasma filtration condition B: inform mutation frequency>0.1% and naive mutation frequency>1%, supplementary retrieval: retrieval of 0.1%-1% sites that appear simultaneously in the initial diagnosis and C2)

[0073] Figure 3 .Comparison chart of the detection results (number of mutation sites, ctDNA equivalents and ctDNA proportion) of fresh blood primary specimens using CAPPseq (the detection method of Example 1 of the present invention) and traditional tumor-informed methods (comparison method: 152 panel detection);

[0074] Figure 4 .Comparison of the results of the fresh blood samples tested again for the number of mutation sites, ctDNA equivalents, and ctDNA proportion after one course of treatment;

[0075] Figure 5 . Comparison of the test results (number of mutation sites, ctDNA equivalents, and ctDNA proportion) of frozen blood primary specimens using CAPPseq (the detection method of Example 1 of the present invention) and traditional tumor-informed methods (comparison method: 152 panel detection);

[0076] Figure 6. A comparison chart of the test results (number of mutation sites, ctDNA equivalents, and ctDNA proportion) of blood specimens after one course of cryopreservation treatment using CAPPseq (the detection method of Example 1 of the present invention) and traditional tumor-informed methods (comparison method: 152 panel detection);

[0077] Figure 7 .Comparison chart of plasma freezing results. DETAILED DESCRIPTION

[0078] Note:

[0079] If not otherwise specified, 1) the reagents and materials in the specific embodiments of the present invention can be obtained commercially; 2) the room temperature in the specific embodiments of the present invention is 25° C.; 3) the solvent of the solution in the specific embodiments of the present invention is water.

[0080] Example 1

[0081] The present invention provides a method for detecting ctDNA without obtaining lymphoma tissue samples, and the specific steps are as follows.

[0082] 1. Collecting blood samples

[0083] 1) Collect blood samples

[0084] Peripheral blood samples from lymphoma patients were collected using cfDNA-specific storage tubes (streck tubes). Specifically, 7.5±2.5 mL of venous blood was collected and centrifuged at 2000 rpm for 10 minutes to separate the upper layer sample (plasma) and the lower layer sample (leukocytes).

[0085] 2) Extraction of plasma cfDNA

[0086] Use a cfDNA-specific storage tube (streck tube) to collect 7.5±2.5mL of venous blood from lymphoma patients, centrifuge at 2000rpm for 10min, separate the upper sample (plasma) for cfDNA extraction (if no subsequent operation is performed immediately, temporarily store the plasma at 4℃ or freeze it at -80℃); use a free DNA extraction kit (supplier: Kangwei Century) and refer to the instructions of the Kangwei Free Nucleic Acid DNA Extraction Kit (Magnetic Bead Method) to extract plasma cfDNA.

[0087] 3) Extraction of leukocyte genomic DNA (gDNA)

[0088] The blood sample white blood cells are collected in step 1), and the white blood cell genomic DNA in the blood sample is selected as a detection control to filter out the noise of germline and clonal hematopoietic related mutation site factors. Specifically, after taking out the plasma sample, the lower layer sample (white blood cells) is taken and subjected to a universal red blood cell removal experimental operation, and then a universal genomic DNA extraction method is used to prepare a white blood cell gDNA sample.

[0089] 2. Construction of hybridization library

[0090] 2.1 Leukocyte genomic DNA hybridization library (genomic DNA sample)

[0091] 2.1.1 Input DNA fragmentation, end repair, dA tailing

[0092] 1. Take out the Frag / AT buffer and Frag / AT enzyme in the kit, thaw and mix thoroughly, centrifuge briefly to collect to the bottom of the tube, and place on ice for later use. All the following steps are performed on ice.

[0093] 2. Prepare the following reaction in a sterile PCR tube:

[0094]

[0095] * The amount of leukocyte genomic DNA added in the input DNA is 100 ng, and the DNA sample is diluted with ddH2O to a volume of 40 μL.

[0096] Note: The Input DNA here refers to the leukocyte gDNA sample collected above.

[0097] 3. Use a pipette to blow or shake to mix, and briefly centrifuge to collect the reaction solution to the bottom of the tube, and immediately place it in a PCR instrument for reaction.

[0098] 4. Place the PCR tube in the PCR instrument and perform the following reactions:

[0099]

[0100] Note: When the thermal cycler program is finished and the sample block returns to 4°C, remove the samples from the template and place them on ice. Proceed immediately to the next step.

[0101] 2.1.2 Sequencing adapter ligation and purification

[0102] This step will ligate adapters to the ends of the fragmented end-repair products from the previous step.

[0103] 1. Take out the universal connector from -20℃, thaw it, mix it thoroughly, centrifuge it briefly to collect it to the bottom of the tube, and place it on ice for later use.

[0104] 2. Prepare the reaction system according to the following table:

[0105]

[0106] 3. Use a pipette to gently pipette to mix (do not oscillate to mix), and briefly centrifuge to collect the reaction solution at the bottom of the tube.

[0107] 4. Place the PCR tube in the PCR instrument and perform the following reactions:

[0108]

[0109] 5. Use purification magnetic beads to purify the reaction products:

[0110] 1) After the magnetic beads are equilibrated to room temperature (30 min), vortex and oscillate to mix and purify the magnetic beads.

[0111] 2) Pipette 60 μL (0.8x) purified magnetic beads into 75 μL Adapter Ligation product and vortex or pipette gently 10 times to mix thoroughly.

[0112] 3) Incubate at room temperature for 5 minutes.

[0113] 4) Briefly centrifuge the PCR tube and place it on a magnetic rack to separate the magnetic beads and liquid. After the solution is clear (5 minutes), carefully remove the supernatant.

[0114] 5) Keep the PCR tube in the magnetic rack, add 180 μL of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 seconds, and carefully remove the supernatant.

[0115] 6) Repeat step 5 for a total of two rinses.

[0116] 7) Keep the PCR tube in the magnetic rack at all times, open the lid and air-dry the magnetic beads for 5-10 minutes until no ethanol remains.

[0117] 8) Remove the PCR tube from the magnetic rack, add 17 μL ddH2O to elute, vortex or use a pipette to gently blow to mix thoroughly, and place at room temperature for 2 minutes. Centrifuge the PCR tube briefly and place it on the magnetic rack to stand. After the solution is clear (5 minutes), carefully transfer 15 μL of supernatant to a new EP tube. Do not touch the magnetic beads.

[0118] 2.1.3 PCR amplification, purification and quality control using UDI primers

[0119] 1. Thaw the UDI primers and Equinox library amplification mixture (2x) in the kit and mix them by inversion. Prepare the following reaction in a sterile PCR tube:

[0120]

[0121] 2. Use a pipette to gently pipette to mix (do not oscillate to mix), and briefly centrifuge to collect the reaction solution at the bottom of the tube.

[0122] 3. Place the PCR tube in the PCR instrument and start the PCR program as follows:

[0123] Heating cover temperature 105℃; 98℃ for 45s; (98℃ for 15s, 60℃ for 30s, 72℃ for 30s) 8 cycles; 72℃ for 1min; 4℃ hold

[0124] 4. Purify the reaction product:

[0125] 1) After the magnetic beads are equilibrated to room temperature, vortex and oscillate to mix and purify the magnetic beads.

[0126] 2) Pipette 50ul (1x) purified magnetic beads into 50μL Library Amplification product and vortex or pipette gently 10 times to mix thoroughly.

[0127] 3) Incubate at room temperature for 5 minutes.

[0128] 4) Briefly centrifuge the PCR tube and place it on a magnetic rack to separate the magnetic beads and liquid. After the solution is clear (5 minutes), carefully remove the supernatant.

[0129] 5) Keep the PCR tube in the magnetic rack, add 180 μL of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 seconds, and carefully remove the supernatant.

[0130] 6) Repeat step 5 for a total of two rinses.

[0131] 7) Keep the PCR tube in the magnetic rack at all times, open the lid and air-dry the magnetic beads for 5-10 minutes until no ethanol remains.

[0132] 8) Remove the PCR tube from the magnetic rack, add 65 μL ddH2O to elute, vortex or use a pipette to gently blow to mix thoroughly, place at room temperature for 2 minutes, centrifuge the PCR tube briefly and place it on the magnetic rack to stand. After the solution is clarified (5 minutes), carefully transfer 65 μL of supernatant to a new EP tube. The supernatant in the tube is the prepared pre-library 1.

[0133] 2.1.4 Library Quantification

[0134] Take 1 μL of pre-library and use Qubit 2.0 Fluorometer (Qubit dsDNA HS Assay Kit) to measure the library concentration and record the library concentration. The concentration range of normal library is above 50 ng / μL, and the library concentration is mainly related to the quality of the template.

[0135] 2.1.5 Library quality detection

[0136] The Qsep100 fully automatic nucleic acid protein analysis system was used to evaluate the length of library fragments. The fragment distribution range of a normal library was between 300bp and 400bp.

[0137] Note: Depending on the integrity of the Input DNA, the library fragments may be slightly different. If the library fragments are too short, the 37°C enzyme digestion time can be shortened to 10 minutes; if the library fragments are too long, the DNA can be re-extracted for re-testing.

[0138] 2.2 Construction of cfDHA hybridization library pre-hybridization library (cfDNA sample)

[0139] 2.2.1 Input cfDNA quantification (qPCR method)

[0140] The cfDNA concentration was measured using a fluorometer Qubit 2.0 Fluorometer (Qubit dsDNA HS Assay Kit) and the cfDNA concentration was recorded.

[0141] The target cfDNA injection volume (cfDNA extracted from plasma samples) determined based on cfDNA concentration is shown in the table below. If the target injection volume is less than 20 ng, all of it will be used in the subsequent amplification step, with 10 cycles of amplification.

[0142]

[0143]

[0144] 2.2.2 Input DNA end repair and dA tailing

[0145] 1. Take out the End Repair & A-Tailing Buffer in the kit from the -20℃ refrigerator in advance, place it on an ice box to melt, vortex briefly to mix, centrifuge it instantaneously, and place it on an ice box for later use. Take out the End Repair & A-Tailing Enzyme Mix from the -20℃ refrigerator, invert and mix, centrifuge it instantaneously, and place it on an ice box for later use. Prepare the reaction system according to the table below. This operation must be performed on an ice box.

[0146]

[0147] 2. Mix by pipetting on ice (avoid violent shaking).

[0148] 3. Set the PCR instrument parameters according to the table below, place the reaction system on the PCR instrument and run the program:

[0149]

[0150] Note: After the program is finished, proceed to the next step of adapter ligation reaction immediately. This step cannot be paused.

[0151] 2.2.3 Unique molecular tag UMI adapter connection and purification

[0152] This step will connect the UMI adapters to the ends of the fragmented and repaired products in the previous step.

[0153] 1. Take out the Adapter from the -20℃ refrigerator in advance and place it on an ice box to thaw. After thawing, vortex briefly to mix and centrifuge instantly, and place it on an ice box for later use.

[0154] 2. Dilute the UMI Adapter (15 μM) to an appropriate concentration in advance according to the amount of DNA used in library construction:

[0155]

[0156] Note: Too high an Adaper input may cause the Adapter to self-ligate; insufficient input will affect the ligation efficiency and reduce the library output. The general injection volume is 20ng, and the UMI Adapter (15μM) is diluted 2 times for use.

[0157] 3. In a PCR tube, prepare the reaction system on ice according to the following table:

[0158]

[0159] Note: If you need to pre-mix the reaction reagents when operating a large amount of samples at one time, please do not pre-mix the Adapter. The best operation method is to mix the Adapter and the sample after the Step 1 reaction first, and then add the pre-mixed reaction reagents, which can effectively reduce the self-ligation of the adapter.

[0160] 4. Mix by gently pipetting 6 times to avoid creating bubbles, then centrifuge briefly.

[0161] 5. Set the PCR instrument parameters according to the table below, close the heated cover, put the PCR tube into the PCR instrument, and run the program.

[0162]

[0163] Note: After the program is finished, proceed to the next step of magnetic bead purification immediately. This step cannot be paused.

[0164] 2.2.4 Post-ligation purification

[0165] 1. Place Agencourt AM Pure XP magnetic beads at room temperature for 30 minutes in advance and shake to mix well for later use.

[0166] 2. After the program is finished, add 0.8 times the volume of magnetic beads (88 μL) to the 110 μL reaction system after 7.2.5, and mix by pipetting or vortexing.

[0167] 3. Let stand at room temperature for 5 minutes and place the centrifuge tube on the magnetic rack for 3 minutes until the solution is clear.

[0168] 4. Keep the centrifuge tube on the magnetic rack, remove the supernatant, add 200 μL 80% ethanol to the centrifuge tube, and let it stand for 30 seconds.

[0169] 5. Remove the supernatant and add 200 μL of 80% ethanol solution to the centrifuge tube. After standing for 30 seconds, completely remove the supernatant and use a 10 μL pipette to remove the residual ethanol solution at the bottom.

[0170] 6. Let stand at room temperature for 5 minutes until the magnetic beads are dry and the residual ethanol evaporates completely.

[0171] 7. Add 22 μL of nuclease-free water, remove the centrifuge tube from the magnetic stand, gently pipette to resuspend the magnetic beads to avoid bubbles, centrifuge briefly, and let stand at room temperature for 2 minutes.

[0172] 8. Place the centrifuge tube on the magnetic rack for 2 minutes until the solution becomes clear.

[0173] 9. Use a pipette to aspirate 20 μL of supernatant and transfer it to a new PCR tube for the next reaction.

[0174] Note: This step can be paused and can be stored in a -20℃ freezer for 1 month.

[0175] 2.2.5 Pre-PCR reaction

[0176] 1. Take out the pre-PCR amplification reaction solution, I5 end adapter primer, and I7 end adapter primer from the kit stored at -20℃ in advance, put them on an ice box to melt, mix them well, and put them on ice for later use.

[0177] 2. Prepare the reaction system according to the table below (this operation is performed on an ice box):

[0178]

[0179] 3. Mix by gently pipetting and centrifuge briefly.

[0180] 4. Set the PCR instrument parameters according to the table below, heat the cover at 105°C, place the above reaction system on the PCR instrument and run the program:

[0181]

[0182] Note: The recommended number of cycles for cfDNA sample amplification is 10 to 12, so that each cfDNA molecule connected to the adapter can be fully amplified, which is conducive to improving the sensitivity of detecting variants. After the program is completed, magnetic bead purification is performed immediately. 2.2.6 Magnetic bead purification after PCR amplification

[0183] 1. Place Agencourt AMPure XP magnetic beads at room temperature 30 minutes in advance, shake and mix thoroughly for later use;

[0184] 2. After the program is finished, transfer 50 μL of the product in the PCR tube to a new 1.5 mL centrifuge tube, add 1 volume of magnetic beads (50 μL), pipette to mix, and centrifuge briefly.

[0185] 3. Let stand at room temperature for 5 minutes, and place the centrifuge tube on the magnetic rack for 3 minutes until the solution is clear.

[0186] 4. Keep the centrifuge tube on the magnetic rack, remove the supernatant, add 200 μL 80% ethanol solution into the centrifuge tube, and let it stand for 30 seconds.

[0187] 5. Remove the supernatant and add 200 μL of 80% ethanol solution to the centrifuge tube. After standing for 30 seconds, completely remove the supernatant and use a 10 μL pipette to remove the residual ethanol solution at the bottom.

[0188] 6. Let stand at room temperature for 5 minutes until the magnetic beads are dry and the residual ethanol evaporates completely.

[0189] 7. Add 50 μL of nuclease-free water, remove the centrifuge tube from the magnetic stand, gently pipette to resuspend the magnetic beads to avoid bubbles, centrifuge briefly, and let stand at room temperature for 2 minutes.

[0190] 8. Place the centrifuge tube on the magnetic rack for 2 minutes until the solution becomes clear.

[0191] 9. Use a pipette to aspirate 48 μL of supernatant and transfer it to a new centrifuge tube. The supernatant in the tube is the prepared pre-library 2, which should be marked.

[0192] 10. Perform subsequent experimental steps (if subsequent operations are not performed immediately, store the library at -20°C).

[0193] 2.2.7 Prelibrary Second Quality Control

[0194] 1. Take 1 μL of pre-library 2 and use Qubit dsDNAHS Assay Kit to measure the library concentration and record the pre-library concentration. The pre-library concentration must be >25 ng / μL.

[0195] 2. Take 1 μL of the pre-library and use Agilent 2100 Bioanalyzer System, Qsep100 (Bioptic) and other nucleic acid analyzers to perform quality inspection of library fragment distribution. The average length of the pre-library 2 is between 250-350 bp, the main peak length is 300 bp, and the concentration is 50 ng / uL.

[0196] 2.3 Hybridization capture

[0197] 2.3.1 Library and probe hybridization

[0198] Use vacuum concentration to concentrate the library. When vacuum concentrating the library, please control the concentration time to avoid excessive drying and sample loss.

[0199] Note: During the hybridization experiment, be sure to confirm that the tube mouth is completely sealed to avoid prolonged evaporation of the sample, which may lead to hybridization failure.

[0200] The specific operations are:

[0201] 1) Take out Hyb Human Block, Adapter Blocker, RNase Block, TargetProbe (from TargetSeq) from the refrigerator in advance. Series Hybridization Capture Kit) was placed on an ice box to melt, and after melting, mixed and placed on ice or at 4°C for use;

[0202] 2) Hybridize the leukocyte and cfDNA libraries, control the amount of each library to be equal and the total amount of library added to the PCR tube to be 1200ng (for example, if it is 2-in-1, add 600ng to each library), and mark it. Place the PCR tube in a vacuum concentrator centrifuge, open the PCR tube cover, start the centrifuge, turn on the vacuum pump switch, and start concentrating to a dry state (the concentration time must be strictly controlled when the library is vacuum concentrated, so before concentration, use the same volume of water to test the concentration time and estimate the time required for concentration to avoid excessive drying due to too long concentration time, resulting in sample loss);

[0203] 3) After the library is concentrated, it is added to the hybridization reaction system as shown in the table below; the information of the lymphoma ctDNA detection probe is shown in Table 1 above, which is from Shanghai Yuanqi.

[0204]

[0205]

[0206] 1) Mix by gentle pipetting and centrifugation.

[0207] 2) Place the PCR tube in a PCR instrument and set the program as follows: hot cover temperature 85°C; 80°C for 5 min; incubate at 50°C overnight.

[0208] 2.3.2 Preparation of capture magnetic beads

[0209] The magnetic beads used for capture must be Cap beads or Dynabeads MyOne Streptavidin T1 magnetic beads. Other types of magnetic beads such as C1, M270, M280, Agencourt AMPure XP, etc. cannot be used as substitutes.

[0210] 1) Take out the capture magnetic beads (T1) from 4°C, equilibrate at room temperature for 30 minutes, and then vortex to resuspend;

[0211] 2) For one hybridization library, take 50 μL of magnetic beads and add them to a new PCR tube. Place it on a magnetic rack for 1 minute to allow the solution to clear, and remove the supernatant.

[0212] 3) Remove the PCR tube from the magnetic stand, add 180 μL Binding Buffer, gently pipette several times to mix, and resuspend the magnetic beads;

[0213] 4) Place on the magnetic stand for 1 min and remove the supernatant;

[0214] 5) Repeat steps 3-4 twice, washing the magnetic beads 3 times in total;

[0215] 6) Remove the PCR tube from the magnetic stand, add 180 μL Binding Buffer, gently pipette and resuspend the magnetic beads for later use. 2.3.3 Capture the DNA library of the target region

[0216] 1) Keep the hybridization product on the PCR instrument, add 180 μL of Cap beads resuspended in the previous step to the hybridization product, mix with a pipette, and place on a rotating mixer at room temperature for 30 minutes;

[0217] 2) Place the PCR tube on a magnetic rack for 2 minutes to clarify the solution and remove the supernatant;

[0218] 3) Add 150 μL of Wash Buffer 1 to the hybridization product, gently pipette to mix, place on a rotary mixer to wash for 15 min, then briefly centrifuge, place the PCR tube on a magnetic rack for 2 min to clarify the solution, and remove the supernatant;

[0219] 4) Add 150 μL 50°C preheated TargetSeq Wash Buffer, gently pipette to mix, then centrifuge briefly, and incubate on a constant temperature shaker at 50°C for 10 min;

[0220] 5) Centrifuge briefly, place the PCR tube on a magnetic rack for 2 minutes to clarify the solution, and remove the supernatant.

[0221] 6) Repeat steps 4-5 twice, washing the magnetic beads 3 times in total;

[0222] 7) Keep the sample on the magnetic rack, add 150 μL 80% ethanol to the PCR tube, let it stand for 30 seconds, then completely remove the ethanol solution (a 10 μL pipette can be used to remove the residue), and dry at room temperature;

[0223] 8) Add 24 μL Nuclease-free Water to the PCR tube, remove the PCR tube from the magnetic stand, and use a pipette to gently resuspend and mix the magnetic beads for later use.

[0224] Operation Note:

[0225] When capturing the target area, add the resuspended capture magnetic beads into the PCR tube and keep the PCR tube on the PCR instrument during the process.

[0226] The probe and magnetic beads were combined and incubated at room temperature for 30 minutes. A mixer was used to prevent the magnetic beads from settling. The rotation speed should not be too high, and it is recommended not to exceed 10 rpm.

[0227] After the capture is completed, be careful not to discard the magnetic beads. The next step is the PCR reaction with the magnetic beads in the system.

[0228] If Wash Buffer 1 stored at room temperature has precipitates, please heat it in a 37°C water bath and use it only after the reagent is completely dissolved.

[0229] Using TargetSeq Preheat the Wash Buffer to 50°C.

[0230] 2.3.4 Post-capture PCR amplification

[0231] 1) Take out Post PCR Master Mix and Post PCR Primer from -20℃ refrigerator, place them on ice box to melt, mix well and place on ice or 4℃ for later use.

[0232] 2) After capture, the DNA library needs to be amplified by PCR. The reaction system is prepared according to the following table:

[0233]

[0234] 3) Adjust the pipette to 40 μL, gently pipette and mix 6 times, and then immediately place on the PCR instrument.

[0235] 4) Place the PCR tube in a PCR instrument and set the program to: 95°C for 1 min; (98°C for 20 s; 60°C for 30 s; 72°C for 30 s) 12 cycles; 72°C for 5 min; 4°C hold

[0236] 5) After PCR, add 55 μL of Agencourt AMPure XP magnetic beads to the sample, vortex or pipette to mix, and let stand at room temperature for 5 minutes;

[0237] 6) Briefly centrifuge and place the PCR tube on a magnetic rack for 3 minutes to allow the solution to clarify;

[0238] 7) Keep the PCR tube on the magnetic rack, remove the supernatant, add 180 μL 80% ethanol solution into the PCR tube, and let it stand for 30 seconds;

[0239] 8) Keep the PCR tube on the magnetic rack, remove the supernatant, add 180 μL 80% ethanol solution to the PCR tube again, let it stand for 30 seconds, and then completely remove the supernatant (a 10 μL pipette can be used to remove the residual ethanol solution at the bottom);

[0240] 9) Let stand at room temperature for 3-5 minutes to allow the residual ethanol to evaporate completely;

[0241] 10) Add 25 μL of Nuclease-free water, remove the PCR tube from the magnetic rack, shake or pipette to mix 10 times, and let stand at room temperature for 2 minutes;

[0242] 11) Briefly centrifuge and place the PCR tube on a magnetic rack for 2 minutes to allow the solution to clarify;

[0243] 12) Use a pipette to transfer 23 μL of supernatant to a 1.5 mL centrifuge tube. The supernatant in the tube is the prepared final library. Mark the sample information.

[0244] 2.3.5 Library Quantification

[0245] Take 1 μL of the final library and use Qubit 2.0 Fluorometer (Qubit dsDNA HS Assay Kit) to measure the library concentration and record the library concentration; the library concentration is 1-20 ng / μL.

[0246] Note: The normal library concentration range is 5ng / μL-40ng / μL. The library concentration is mainly related to the quality of the template.

[0247] 2.3.6 Library quality detection

[0248] The Qsep100 fully automatic nucleic acid protein analysis system was used to evaluate the length of library fragments. The main peak length of a normal library was between 270bp and 320bp.

[0249] 2.4 Library mixing and sequencing

[0250] Dilute each final library with water to 4 ng / μL, and add the corresponding volume of final library to a new 1.5 mL EP tube according to the target sequencing data volume. The sequencing data volume of cfDNA samples is 5G, and the data volume of leukocyte genomic DNA samples is 0.5G. Finally, select the appropriate instrument for sequencing according to the final expected data volume to obtain the original fastq data.

[0251] 3. Bioinformatics Analysis

[0252] 3.1 Bioinformatics Process Analysis

[0253] For the original fastq data of sequencing, use picard FastqToSam to convert the filtered data into bam format, use fgbio ExtractUmisFromBam to extract the UMI information in the bam file, use samtools fastq to convert the bam file into fastq format, then bwa mem is used for alignment, samtools view converts the aligned sam results into bam format, use picard MergeBamAlignment to merge the aligned bam file and the unaligned bam file, sort by coordinates, use fgbio GroupReadsByUmi to split Reads according to the UMI information of different groups, and use fgbioCallMolecularConsensusReads.

[0254] Merge the reads from the same molecule into a consensus read, use samtools fastq to convert consensus.ubam to fastq, then use bwa mem to align the reference genome, and then use samtools view to convert the alignment sam results to bam, use picard MergeBamAlignment to merge the unaligned bam and the aligned bam above, and sort them by coordinates, use fgbio FilterConsensusReads to filter the results of the previous step according to the set conditions, use fgbio ClipBam to clip the mutations of the overlapping parts of the reads from the same template according to the set conditions, use Lianti software to identify the point mutation SNV and insertion and deletion INDEL results, and use Annovar software for gene annotation.

[0255] Background noise and germline (i.e., leukocyte gDNA) variation were filtered out based on the following rules:

[0256] 1. cfDNA filtering rules (see Figure 2 ):

[0257] (1) SSCS mutation self-filtering screening: Screening of mutation test data based on single-strand correction mode: The mutation sequencing depth is required to be greater than 100, and the mutation frequency is greater than 1%, the number of supported mutation reads is greater than 1, and the average base quality of the mutation read is at least 39;

[0258] (2) In double-strand correction mode, mutation data retained by filtering conditions can still be obtained;

[0259] (3) SNP (single nucleotide polymorphism): based on the ExAC, 1000Genome, and gnomAD databases, the population frequency is less than 0.1%; the population frequency is for all populations, including healthy and diseased populations;

[0260] (4) Non-synonymous mutations: Annovar was used to annotate ExonicFunc.refGene;

[0261] (5) Supplementary retrieval: Retrieve 0.1%-1% of the sites that appear simultaneously at the initial diagnosis and C2.

[0262] 2. Filtering rules for germline (ie, white blood cell gDNA) samples:

[0263] If the test results of the leukocyte gDNA sample show that the number of supported mutation reads is greater than 1, the mutation frequency is greater than 0.5%, the sequencing depth of the site is greater than 50, and the average base quality of the supported mutation reads is >30, then this site needs to be excluded in the initial sample.

[0264] 3. Set the intersection of the mutation sites of cfDNA tracking samples and the screening sites of primary samples as the inform set, and screen according to the properties of the mutation in the single-strand correction mode. The mutation sequencing depth is required to be greater than 100, and the mutation frequency is greater than 0.1%. The number of mutation reads supported is greater than 1, and the average base quality of the mutation read length is at least 39.

[0265] 4. Set the non-intersecting part of the cfDNA tracking sample mutation sites and the initial sample screening site results as the naive set (where the cfDNA tracking sample mutation sites correspond to the mutation site results of the plasma cfDNA sample, and the initial sample screening sites correspond to the mutation site results of the white blood cell DNA sample). Screen according to the properties of the mutation in the single-strand correction mode, requiring that the mutation sequencing depth is greater than 100, and the mutation frequency is greater than 1%, supporting the number of mutation reads to be greater than 1, and the average base quality of the mutation read length is at least 39.

[0266] Based on the above filtering conditions, a list of somatic mutations for each tumor tissue sample was obtained.

[0267] 3.2 Calculation of ctDNA equivalent in free DNA

[0268] For a patient, J individual cell mutations were detected. In the free DNA of the patient, the mutation read length s at the jth site is counted as M. j , the sequencing depth is recorded as N j , then the proportion of ctDNA in free DNA is calculated as follows:

[0269]

[0270] The ctDNA concentration in plasma can be assessed by measuring the plasma volume and the total amount of cfDNA extracted from the plasma during the experiment. The ctDNA concentration indicates the number of ctDNA molecules contained in a unit volume of plasma.

[0271] Assume that the plasma volume of the i-th blood sample is V i The total amount of cfDNA extracted from plasma is m i , the ctDNA concentration in plasma is calculated as follows:

[0272]

[0273] The ctDNA concentration (i.e., ctDNA equivalent) of each blood sample is calculated using the above algorithm, which can characterize how many copies of the genome are present in each milliliter of plasma.

[0274] 3.3 Prognostic risk prediction model

[0275] In the scenario of prognostic risk assessment, the change rate of ctDNA concentration in free samples collected between adjacent time points during the treatment of the same patient is used to predict the patient's efficacy and recurrence possibility. Assuming that the patient's blood was collected for free DNA testing at time t0 and t1 (t0 is earlier than t1), and the ctDNA concentrations detected at the two time points are C0 and C1 respectively, the change rate of ctDNA is calculated as follows

[0276]

[0277] Samples with a ctDNA change rate less than a certain threshold (ex-2, referring to + / - 2 log values) are defined as patients with better efficacy, and vice versa as patients with poor efficacy.

[0278] Performance Testing

[0279] 1. Fresh blood sample test

[0280] Compare the detection results of fresh blood samples (the samples come from the remaining blood samples of patients with hemangioma in scientific research cooperation with Ruijin Hospital), CAPPseq (the detection method of Example 1 of the present invention) and traditional tumor-informed methods (comparison method: 152panel detection, for specific probe information, see patent 2023108289926). The number of mutation sites, ctDNA equivalents and ctDNA proportions detected by the two detection methods are compared, and the results are shown in Figure 3 .

[0281] from Figure 3 As can be seen from the data, this detection method adopts a simpler sampling method, and the detected ctDNA proportion and equivalent level are comparable to those of the traditional method; what is even more rare is that the detection method of the present invention can detect more mutation sites, which shows that the CAPP-Seq method of the present invention tracks more sites, which helps to improve detection sensitivity and reduce the possibility of missed mutations.

[0282] Note: Fresh blood specimen refers to the biological sample collected from the experimental subject for the first time.

[0283] 2. Verification of fresh blood test results

[0284] After one course of treatment (before the second course of treatment), the above test is performed again on fresh blood samples. The test results are shown in Figure 4 .from Figure 4 It can be seen that among the samples not detected by CAPP-Seq in the fresh blood samples, one mid-term PET-CT was CR (negative), which was consistent with the test results, and the others did not reach the mid-term. The data results prove that the test results of the present invention are accurate and can be used as a basis for determining the efficacy.

[0285] 3. Testing of frozen blood samples

[0286] Compare the test results of CAPPseq (the detection method of Example 1 of the present invention) and traditional tumor-informed method (comparison method: 152 panel detection) in frozen blood samples (samples were obtained from venous blood of lymphoma patients in Ruijin Hospital; the freezing conditions were to take the initial samples and freeze them at -20°C for 30-90 days, and then take them out for testing). The number of mutation sites, ctDNA equivalents and ctDNA proportions detected by the two detection methods were compared, and the results are shown in Figure 5 , Figure 6 ;in Figure 5 For test results of cryopreserved blood samples, Figure 6 The test results are obtained after freezing blood samples after one course of treatment and at the beginning of the second course of treatment.

[0287] from Figure 5 It can be seen from the data that the test results of frozen blood primary specimens are similar to those of fresh blood specimens. The ctDNA equivalent and ctDNA proportion results detected by the two methods are close, and the number of mutation sites that can be tracked by this method is significantly greater than that of the 152panel detection method.

[0288] from Figure 6 It can be seen from the data that in the test results of blood samples after one course of cryopreservation treatment, the ctDNA equivalent levels and ctDNA proportions detected by the two methods are similar, while the number of mutation sites detected by the present invention is greater.

[0289] The analysis results of 32 samples are shown in Figure 7 ,from Figure 7 It can be seen that plasma freezing does not affect the test results of the present invention for detecting ctDNA (Note: Figure 7 Fresh refers to the first blood sample).

[0290] Note: This invention project was partially funded by scientific research projects, including: Project No. 2023YFC3605700, Research and application of new technologies for rapid auxiliary diagnosis of comorbidities in the elderly.

Claims

1. A method for detecting ctDNA without obtaining lymphoma tissue samples, characterized in that: The steps include:

1. Collect blood samples; 2. Construction of hybridization library; 3. Bioinformatics analysis; The blood sample is derived from peripheral blood, and the plasma sample and the leukocyte sample are obtained by collecting, centrifuging and separating the peripheral blood.

2. The method for detecting ctDNA without obtaining lymphoma tissue samples according to claim 1, characterized in that: The step 1 comprises: collecting a blood sample, centrifuging it at 1000-4000 rpm for 5-15 minutes, collecting the upper layer of liquid, i.e., the plasma sample; collecting the lower layer of cells, removing the red blood cells, and obtaining the white blood cell sample.

3. The method for detecting ctDNA without obtaining lymphoma tissue samples according to claim 1, characterized in that: The step 2 comprises: S1. constructing a leukocyte genomic DNA hybridization library; S2. constructing a cfDNA hybridization library; S3. hybridization capture; Preferably, in step S1, the steps of constructing the leukocyte genomic DNA hybridization library include: (1) performing DNA fragmentation, end repair, and dA tailing treatment on the leukocyte sample; (2) linker ligation and purification; (3) double-end labeling with UDI, followed by PCR amplification and purification to obtain pre-library 1; (4) quality control of pre-library 1; Preferably, in the step S2, the steps of constructing the cfDNA hybridization library include: (1) cfDNA quantification; (2) end repair and dA tailing of cfDNA; (3) terminal UMI adapter connection and purification; (4) PCR amplification and purification to obtain pre-library 2; and (5) quality control of pre-library 2.

4. The method for detecting ctDNA without obtaining lymphoma tissue samples according to claim 3, characterized in that: In the S3 step, the hybridization capture step includes: (1) hybridizing pre-library 1 or pre-library 2 with the probe; (2) preparing capture magnetic beads; (3) applying the capture magnetic beads to the hybridization system (1) to capture the target region DNA library; (4) performing PCR amplification after capture to obtain the final library; (5) quality control of the final library; and (6) sequencing the final library on a machine.

5. The method for detecting ctDNA without obtaining lymphoma tissue samples according to claim 1, characterized in that: The step three includes: (1) filtering the sequencing data for background noise and germline variation; (2) calculating the ctDNA equivalent in cfDNA; and (3) a prognostic risk prediction model.

6. The method for detecting ctDNA without obtaining lymphoma tissue samples according to claim 5, characterized in that: The filtering includes filtering of cfDNA, and the filtering principle is set as follows: (1) SSCS mutation self-filtering screening: Screening of mutation test data based on single-strand correction mode: The mutation sequencing depth is required to be greater than 100, and the mutation frequency is greater than 1%, the number of supported mutation reads is greater than 1, and the average base quality of the mutation read is at least 39; (2) The mutation data can still be obtained in the double-strand correction mode; (3) SNP: Data with a population frequency of less than 0.1% were screened based on the ExAC, 1000Genome, and gnomAD databases; (4) Non-synonymous mutations: Annovar was used to annotate exon region variants.

7. The method for detecting ctDNA without obtaining lymphoma tissue samples according to claim 5, characterized in that: The filtering also includes filtering of germline samples, and the filtering principle is set as follows: if the number of supported mutation reads in the test results of the white blood cell sample is greater than 1, and the mutation frequency is greater than 0.005, and the sequencing depth of the site is greater than 50, and the average base quality of the supported mutation reads of the site is >30, then this site needs to be excluded in the primary sample; Preferably, the filtering further sets the intersection of the mutation sites of the cfDNA tracking samples and the screening sites of the initial samples as the inform set, and performs screening according to the properties of the mutation in the single-strand correction mode, requiring that the sequencing depth of the mutation is greater than 100, and the mutation frequency is greater than 0.1%, supporting the number of mutation reads greater than 1, and the average base quality of the mutation reads is at least 39; preferably, the filtering further sets the non-intersection of the mutation sites of the cfDNA tracking samples and the screening sites of the initial samples as the naive set, and performs screening according to the properties of the mutation in the single-strand correction mode, requiring that the sequencing depth of the mutation is greater than 100, and the mutation frequency is greater than 1%, supporting the number of mutation reads greater than 1, and the average base quality of the mutation reads is at least 39.

8. The method for detecting ctDNA without obtaining lymphoma tissue samples according to claim 5, characterized in that: The calculation of ctDNA equivalent in cfDNA includes (1) calculating the ctDNA ratio; (2) calculating the ctDNA concentration; wherein: The number of cell mutations detected in the cfDNA sequencing results is recorded as j; among the j mutation sites in cfDNA, the mutation read length count at the jth site is Mj, and the sequencing depth is Nj.

9. The method for detecting ctDNA without obtaining lymphoma tissue samples according to claim 8, characterized in that: When calculating ctDNA concentration: The plasma volume of the i-th blood sample is Vi, and the total amount of cfDNA extracted from the plasma in this blood sample is mi.

10. Use of the method according to any one of claims 1 to 9 in lymphoma detection.

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