Capture probe set, kit and detection analysis method for follicular lymphoma multi-gene detection

Abstract

The present application relates to the technical field of multi-gene detection, and in particular to a set of capture probes for follicular lymphoma multi-gene detection, a kit and a detection analysis method ARID1A, ARID1B, ATM, ATP6V1B2, B2M, BCL10, BCL2, BCL6, BCL7A, BTG1, BTG2, BTK, CARD11, CCND3, CD58, CD70, CD79A, CD79B, CDK4, CDKN2A The capture probe set comprises capture probes for specifically binding to 74 follicular lymphoma related genes, and the capture probe set preferably comprises nucleotides with sequences as shown in SEQ ID NO. 1-937. The probe set of the present application can realize one-time detection of variation in the full exon region of 74 follicular lymphoma related genes in the sample to be detected in the same system, has high specificity, high average sequencing depth and wide coverage area.

Description

Technical Field

[0001] This invention relates to the field of multi-gene detection technology, and in particular to capture probe sets, kits, and detection and analysis methods for multi-gene detection of follicular lymphoma. Background Technology

[0002] Follicular lymphoma (FL) is a type of non-Hodgkin lymphoma (NHL) originating from B cells in the center of follicles, with a typical immunophenotype of CD5. － CD10 + CD19 + It is accompanied by t(14;18)(q32;q21) and exhibits high clinical heterogeneity.

[0003] According to the 2022 International Clinical Advisory Committee classification of mature lymphocytic tumors, FL (follicular lymphoma) is graded into grades 1, 2, 3A, and 3B based on the number of central blasts in the follicular region. The 5th edition of the WHO Classification of Hematopoietic and Lymphocytic Tumors (2022) introduced new recommendations for FL grading, no longer requiring strict adherence to grades 1-3B. Instead, it classifies FL into classic FL (cFL), follicular large B-cell lymphoma (FLBL), and FL with uncommon features (uFL). FLBL replaces the original FL 3B designation to emphasize the distinct biological characteristics of this group of FLs compared to cFL. uFL includes FL predominantly characterized by "blastocystic" or "large centrocellular" cell morphology and diffuse growth. The former more frequently exhibits immunophenotypic variations and genotypic diversity, and has a poorer prognosis; the latter often presents as a large mass in the groin area, frequently accompanied by CD23 expression, STAT6 gene mutations, and 1p36 deletion or TNFRSF14 gene mutations, but without BCL2 rearrangements. Based on these advances in pathological diagnosis and treatment, the latest treatment guidelines suggest that institutions with the necessary resources should consider next-generation sequencing to provide a basis for precision diagnosis and treatment of FL.

[0004] Current gene testing for follicular lymphoma is mostly based on PCR and first-generation sequencing technologies. These methods can only detect some hotspot variants of selected genes and cannot detect unknown variants. Furthermore, related second-generation sequencing tests are usually for B-cell lymphoma rather than for detecting gene variants specific to follicular lymphoma, making it difficult to provide relevant clinical significance for follicular lymphoma. Summary of the Invention

[0005] (a) Technical problems to be solved

[0006] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a set of capture probes, kits and detection and analysis methods for multi-gene detection of follicular lymphoma; the capture probes can be used to detect genes related to follicular lymphoma, with high specificity and wide coverage, and can realize the simultaneous detection of the variation of the whole exon region of 74 genes in the sample under test in the same system.

[0007] (II) Technical Solution

[0008] In a first aspect, the present invention provides a set of capture probes for multi-gene detection of follicular lymphoma, comprising capture probes that specifically bind to nucleic acid sequences selected from the following group of genes:

[0009] ARID1A, ARID1B, ATM, ATP6V1B2, B2M, BCL10, BCL2, BCL6, BCL7A, BTG1, BTG2, BTK, CARD11, CCND3, CD58, CD70, CD79A, CD79B, CDK4, CDKN2A, CIITA, CREBBP, CXCR4, DDX3X, DTX1, DUSP2, EBF1, EP300, EPHA7, EZH2, FAS, FOXO1, GNA13, GNAI2, IKZF3, IRF4, IRF8, ITPKB, KLF2, KLHL6, KM T2C, KMT2D, MAP2K1, MEF2B, MYC, MYD88, NOTCH1, NOTCH2, OSBPL10, P2RY8, PAX5, PIM1, PLCG2, PRDM1, PTEN, PTPRD, RB1, REL, RHOA, RRAGC, SETD1B, SGK1, SMARCA4, SOCS1, SPEN, STAT3, STAT6, TBL1XR1, TET2, TMEM30A, TNFAIP3, TNFRSF14, TP53, XPO1; wherein the capture probe set is capable of capturing at least one target sequence of the gene.

[0010] According to a preferred embodiment of the present invention, the capture probe set comprises oligonucleotides with sequences as shown in SEQ ID NO.1-SEQ ID NO.937.

[0011] Secondly, the present invention also provides a kit for detecting multiple genes in follicular lymphoma, the kit comprising the above-mentioned capture probe set for multi-gene detection of follicular lymphoma.

[0012] Preferably, the kit includes a genomic DNA extraction reagent module, a targeted capture enhancement reagent module, an NGS library quality control reagent module, and a sequencing expansion reagent module; the targeted capture enhancement reagent module contains the capture probe set for the multi-gene detection of follicular lymphoma.

[0013] Preferably, the genomic DNA extraction reagent module includes lysis reagents (such as cell lysis buffer, red blood cell lysis buffer), binding / washing reagents (such as DNA binding magnetic beads, binding buffer, washing buffer), purification reagents (such as nucleic acid purification column, DNase / RNase removal solution), and elution / preservation reagents (such as TE buffer, DNA stabilizer).

[0014] The NGS library quality control reagents include fragment screening reagents (such as SPRI magnetic beads, PEG / NaCl solution), quantitative detection reagents (Qubit dsDNA HS detection reagent, qPCR quantitative premix solution containing targeted adapter sequence primers), fragment analysis reagents (such as Agilent Bioanalyzer DNA high-sensitivity chip, sample loading dye), and contaminant removal reagents (such as exonuclease I, phosphatase).

[0015] The targeted capture enhancement reagent module includes a biotinylated DNA probe library, a hybridization system (such as hybridization buffer and blocking reagent), a capture vector (such as streptavidin magnetic beads), a washing system (such as high-strength washing solution and deionized formamide solution), and a molecular tag (such as UMI adapter); wherein, the biotinylated DNA probe library consists of probes from the capture probe group for the multi-gene detection of follicular lymphoma with biotin linked at the 3' end.

[0016] The sequencing expansion reagents include library amplification reagents (such as PCR premix, Illumina P5 / P7 universal primers), index systems (such as 96 combinations of dual index primers), reversible termination dNTPs labeled with four colors of fluorescence, DNA polymerase, regeneration reagents (TCEP cutting buffer, 3' end regeneration solution) and cluster generation reagents (such as Flowcell flow cell, bridge PCR reagents).

[0017] Thirdly, the present invention provides a method for detecting and analyzing gene mutations in follicular lymphoma based on NGS. The method uses the capture probe set or kit described in the above embodiments to sequence the in vitro sample to be tested, analyze the sample data, obtain gene mutation information related to follicular lymphoma, and generate a report.

[0018] Preferably, the gene mutation information includes point mutations, insertions / deletions, copy number variations, etc. Based on the analysis results, a test report is generated to provide medical personnel with a comprehensive assessment and guidance on mutation status and clinical diagnosis and treatment.

[0019] According to a preferred embodiment of the present invention, the detection and analysis method includes the following steps:

[0020] S1, Library Construction and Sequencing

[0021] The capture probe set for multi-gene detection of follicular lymphoma in the above embodiment is used to construct a library and complete sequencing of genomic DNA extracted from in vitro samples to obtain raw sample FastQ format sequencing data;

[0022] S2, Sequencing data preprocessing

[0023] The raw fq data obtained from S1 was quality controlled and preprocessed using the FASTP software. Specific parameter settings included: removing low-quality bases with a base quality value of Q≤20, removing short fragments with a read length of ≤50bp, and removing reads containing adapter contamination.

[0024] S3, Reference Genome Alignment and Data Deduplication

[0025] The clean fq data preprocessed in step S2 were aligned to the human reference genome hg38 using the bwa mem algorithm to generate the original BAM format file. The original BAM file was then subjected to PCR repetitive sequence removal using software, while retaining unique alignment reads. Finally, the deduplicated BAM file was sorted and indexed using software to obtain a standardized BAM file that can be used for variant detection.

[0026] S4. Multi-type variant detection and filtering, including:

[0027] Somatic mutation SNV / INDEL detection: The self-built variant detection tool Parsebam was used to detect SNV and INDEL in S3 standardized BAM files. At the same time, a PoN database was constructed to filter meaningless variants and benign germline variants, while retaining potentially pathogenic somatic variants.

[0028] Fusion gene SV detection: Structural variation detection was performed on the S3 normalized BAM file using LUMPY software to identify fusion gene-related variations. Background variations were removed by using normal sample data as a baseline, while tumor-specific fusion gene variations were retained.

[0029] Copy number variation (CNV) detection: CNVkit software was used to detect CNVs in S3 normalized BAM files. The copy number change was calculated by the ratio of sequencing depth of the target region to the reference region. The CNV detection thresholds were set as copy number gain ≥ 3 copies and copy number loss ≤ 1 copy.

[0030] Finally, the detection results of SNV / INDEL, SV, and CNV were uniformly converted into VCF standard format files, and functional annotations were performed using ANNOVAR software to generate a filtered high-quality variant list.

[0031] S5. Analyze the variant structures and MSI information obtained in S4, and generate a detection report.

[0032] Preferably, in S1, the detection sample is a tumor tissue sample from a patient with follicular lymphoma or a cell-free DNA sample from peripheral blood; during library construction, the initial DNA amount is ≥100ng and the fragmentation length is 150-200bp;

[0033] S2 also includes: filtering reads with N base content ≥5%, and verifying data quality using FastQC software after preprocessing to ensure Q30 ≥ 85%;

[0034] In S3, the human reference genome hg38 uses the latest version released by Ensembl or UCSC; PicardTools software is used to remove PCR repetitive sequences from the original BAM file while retaining unique alignment reads, with an alignment quality value MAPQ ≥ 30.

[0035] Preferably, in S4, when performing SNV and INDEL detection on the S3 standardized BAM file, the minimum allele frequency MAF is set to ≥0.02, the minimum coverage depth is set to ≥20×, and the number of reads supported by the variant site is set to ≥5; the constructed PoN database also contains sequencing data of ≥50 peripheral blood samples from healthy individuals;

[0036] Structural variation detection was performed on the S3 standardized BAM file to identify fusion gene-related variations, including chromosomal translocations or inversions. During the structural variation detection process, the minimum breakpoint support ≥3 reads and the number of reads across breakpoints ≥2. Data from at least 3 healthy normal samples analyzed using the same process were used as a baseline to remove background variations.

[0037] The detection process for copy number variations (CNVs) requires the introduction of sex-matched normal control samples for correction to exclude normal copy number differences in sex-related chromosomes.

[0038] The resulting high-quality variant list includes information such as variant location, variant type, functional impact, allele frequency, and clinical significance.

[0039] This invention is used to confirm gene mutations / genotypes in ex vivo samples from already diagnosed patients.

[0040] The reference gene version for this application is GRCh38 / hg38.

[0041] (III) Beneficial Effects

[0042] Compared to methods for detecting and analyzing follicular lymphoma based on first-generation sequencing technology, this invention leverages NGS technology to achieve multi-dimensional upgrades, with the following specific advantages:

[0043] 1. Detection range and coverage

[0044] Gene and Region Coverage: Overcoming the limitations of traditional methods that only detect partial hotspot variants, this approach employs a hybridization capture method to simultaneously detect the entire exon regions of 74 genes within a single system. This covers all exon regions, including unknown variants, avoiding missed detections due to limited detection range. The accompanying capture probe set covers the entire coding region sequence of the target genes, achieving 99% target region coverage and an average sequencing depth exceeding 1000X, reaching up to 1500X, significantly higher than traditional methods, thus ensuring the detection of low-abundance variants.

[0045] 2. Sensitivity and Detection Limit

[0046] Traditional methods typically have a minimum reportable mutation abundance (AF) of no less than 10%. This invention, through technological optimization, reduces the minimum reportable mutation abundance (AF) to 1%, enabling accurate detection of low proportions of somatic mutations and improving the detection accuracy of early or trace residual lesion samples.

[0047] 3. To leverage technological advantages, this invention has developed a capture probe set and a dedicated reagent kit for the detection of follicular lymphoma. The core design and performance are as follows:

[0048] The probes provided by this invention have a fixed length of 120 bp, a GC content strictly controlled between 40% and 60%, and a stable Tm value (melting temperature) of 65 ± 2℃, ensuring the probe's hybridization specificity and stability. Through sequence screening, probes containing highly repetitive sequences (such as microsatellite sequences) or secondary structures (such as hairpin structures) are completely avoided, reducing non-specific binding interference. More than three probes are designed for each target region, and multiple coverage further improves capture efficiency, ensuring effective detection of the target region.

[0049] The kit provided by this invention focuses on follicular lymphoma and also covers genes related to "some diseases with similar clinical symptoms to follicular lymphoma that require differential diagnosis," containing a total of 74 genes. All selected genes possess clear pathogenic effects, are relevant drug targets, or have prognostic value, making it more targeted than "general detection and analysis methods for mature B-cell lymphoma." The detection content and result interpretation fully comply with the relevant requirements for follicular lymphoma classification and prognostic grading in the 5th edition of the WHO Classification of Hematopoietic and Lymphomas, ensuring standardized clinical application.

[0050] 4. This invention establishes a rigorous system for determining and annotating gene variations, avoiding missed detections and misjudgments. The specific process is as follows:

[0051] (1) Criteria and tools for determining variation

[0052] Gene mutation determination strictly follows the "Guidelines for Interpreting Next-Generation Sequencing Clinical Reports" to ensure that the determination logic is consistent with clinical needs. Dedicated detection tool: Parsebam, a self-developed mutation detection tool based on the above guidelines, can accurately identify mutation types such as SNV / INDEL and is suitable for the detection scenarios of this invention.

[0053] (2) Handling of unrecorded variants: For variants not included in the ClinVar database, a standard annotation process is designed to clarify the significance of variants through multi-dimensional verification (such as evolutionary conservation analysis, protein function prediction, etc.) to effectively avoid missed detection.

[0054] (3) Multi-source information integration: Disease annotation of genes does not rely on a single database, but integrates multiple authoritative databases (such as dbSNP, ExAC, gnomAD, etc.), clinical guidelines and expert consensus to ensure the comprehensiveness and accuracy of annotation information and provide support for subsequent diagnosis and evaluation.

[0055] 5. In summary, the detection and analysis method of the present invention has significant advantages in clinical applications, specifically reflected in:

[0056] (1) Operational and cost advantages

[0057] It has low sample requirements, meeting testing needs without requiring a large number of samples; the operation process is convenient, reducing the complexity of laboratory operations; and the testing cost is moderate, balancing technical performance and clinical accessibility.

[0058] (2) Clinical auxiliary value

[0059] Differential diagnosis: By covering "genes related to follicular lymphoma and similar diseases" and combining standardized annotations, clinical differential diagnosis of follicular lymphoma and similar diseases can be assisted.

[0060] Treatment and prognostic guidance: For the detected variants, the corresponding targeted drug information (if any) and prognostic assessment results (if any) will be reported simultaneously to provide direct reference for the selection of clinical treatment plans and the judgment of patient prognosis.

[0061] (3) Comprehensiveness of detection

[0062] In addition to covering the whole exon regions of 74 genes, it can also detect various variant types such as SNV / INDEL, encompassing "most of the genes and loci included in authoritative guidelines or databases for follicular lymphoma and related diseases", with a detection range far exceeding that of traditional methods. Attached Figure Description

[0063] Figure 1 These are the fragment analysis results from the hybridization capture library of Example 2. Detailed Implementation

[0064] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0065] Example 1

[0066] This embodiment provides a method for detecting and analyzing gene mutations in follicular lymphoma based on NGS, and the analysis process is as follows:

[0067] I. Nucleic acid extraction from ex vivo samples

[0068] Genomic DNA was extracted from paraffin blocks or white flake samples.

[0069] II. Library Construction

[0070] 1. DNA fragmentation, end repair, and A-tailing

[0071] (1) PCR instrument program settings

[0072] First, set the program on the PCR instrument, fixing the hot lid temperature at 70℃. Then, set a three-stage reaction program: the first step is to maintain the temperature at 37℃ for 10 minutes, the second step is to raise the temperature to 65℃ and maintain it for 30 minutes, and the third step is to lower the temperature to 4℃ and maintain it at a constant temperature. Once the program is set, it is ready for use.

[0073] (2) Preparation of reaction solution

[0074] First, thoroughly mix the required reagents, then briefly centrifuge to ensure homogeneity and no residue. Next, prepare the reaction solution on ice. After preparation, aliquot the reaction solution into PCR tubes. Throughout the process, keep the PCR tubes on ice to prevent temperature-related effects on reagent activity until subsequent steps. The reagent volumes for a single reaction system are as follows: 4 μL of Frag / AT Buffer and 6 μL of Frag / AT Enzyme, for a total reaction volume of 10 μL.

[0075] (3) Sample addition and reaction operation

[0076] The concentration of genomic DNA (gDNA) was determined using the Qubit quantitative analysis kit. Based on the results, 40 μL of DNA sample was added to the reaction solution prepared in step (2), ensuring that the total amount of DNA sample added was within the range of 50-200 ng. The PCR tube was then capped, and the tube was gently shaken to thoroughly mix the reaction solution and DNA sample. After mixing, the tube was briefly centrifuged to remove air bubbles and ensure the liquid was concentrated at the bottom. Finally, the PCR tube was placed in a PCR instrument with the program pre-set (i.e., the program set in step "(1)", with the hot cap temperature maintained at 70°C), and the instrument was started to run the reaction program.

[0077] 2. Connect the Universal Adapter

[0078] (1) Preliminary preparations

[0079] During the PCR reaction in Part "1", the following preparations should be carried out simultaneously: First, remove the DNA purification magnetic beads, shake them thoroughly to mix, and then place them at room temperature for equilibration for at least 30 minutes; at the same time, prepare 80% ethanol in advance for later use; then remove the Universal Adapter (purchased from Twist Bioscience) and DNA Ligation Mix (purchased from Twist Bioscience) from the low-temperature environment and place them on ice for slow thawing to avoid repeated freeze-thaw cycles affecting reagent activity; finally, set the PCR instrument program to set the reaction temperature to 20°C and maintain a constant temperature (hold), while closing the PCR instrument's hot cap to complete the preliminary preparations.

[0080] (2) Adapter connection and reactive execution

[0081] After the PCR reaction in Part "1" is completed, immediately remove the reaction product from the PCR instrument and briefly centrifuge the product tube to ensure that the liquid in the tube is concentrated at the bottom and there is no residue. Then, open the cap of the product tube and add the samples in a fixed order: first add 5 μL of Universal Adapter, then add 20 μL of DNA Ligation Mix. After adding the samples, close the cap and briefly centrifuge the product tube for 10 seconds to remove any air bubbles that may be present. Then, place the product tube on a shaker and vortex at low speed to mix the liquid in the tube, ensuring that the reagents and products are in full contact. Perform another brief centrifugation to ensure that the liquid in the tube is completely concentrated at the bottom. Immediately place the product tube into the PCR instrument that has been pre-programmed (20°C hold, hot cap closed) and start the timer. The reaction will continue for 15 minutes.

[0082] After the reaction was complete, the product tube was immediately removed from the PCR instrument, and the subsequent magnetic bead purification steps were carried out.

[0083] 3. First magnetic bead purification

[0084] (1) Combination of magnetic beads with products

[0085] To each reaction product obtained in step “2”, add 60 μL of pre-mixed DNA purification magnetic beads (the volume ratio of magnetic beads to product is 0.8:1). Then, use a vortex mixer to thoroughly mix the mixture and incubate at room temperature for 5 minutes to allow the DNA to fully bind to the magnetic beads. After incubation, perform a short-term centrifugation to remove air bubbles from the tube. Then, place the centrifuge tube on a magnetic rack and let it stand for 5 minutes until the magnetic beads are completely adsorbed to one side of the tube wall. Stop the standing time after that.

[0086] (2) Washing with ethanol

[0087] Keep the centrifuge tube in an adsorbed state on the magnetic rack, and carefully aspirate the supernatant in the tube with a pipette, being careful to avoid touching the adsorbed magnetic beads; then add 200 μL of freshly prepared 80% ethanol to the tube, ensuring that the magnetic beads are completely immersed in the ethanol, and incubate at room temperature for 1 minute to complete the first wash; then aspirate the ethanol supernatant again, and repeat the above steps of adding ethanol and incubation to perform a second wash to further remove impurities.

[0088] (3) Dry the magnetic beads

[0089] After the second ethanol wash, thoroughly aspirate the ethanol supernatant from the tube. Then, using a pipette with a volume of 10 μL, carefully aspirate the remaining ethanol droplets at the bottom of the tube, keeping the magnetic beads untouched throughout the process. Continue to leave the centrifuge tube on the magnetic rack to air dry the magnetic beads at room temperature until no reflection is observed on the surface of the magnetic beads (usually 3-5 minutes).

[0090] (4) DNA elution

[0091] After the magnetic beads have dried, remove the centrifuge tubes from the magnetic rack and add 18 μL of enzyme-free pure water (ddH2O) to each sample tube. Gently pipette the mixture to suspend the magnetic beads completely and ensure they are in full contact with the pure water. Incubate at room temperature for 5 minutes to allow the DNA to be fully eluted from the magnetic beads. After incubation, perform a short-term centrifugation and then return the centrifuge tubes to the magnetic rack. Let them stand for 5 minutes to allow the magnetic beads to re-adhere to the tube wall.

[0092] (5) Elution buffer transfer

[0093] Keep the centrifuge tube on the magnetic rack and carefully pipette 15 μL of the supernatant (i.e., the eluted DNA solution) into a new PCR tube. Label the sample for subsequent PCR amplification steps.

[0094] 4. UDI primer PCR amplification

[0095] (1) Reagent preparation and PCR instrument program settings

[0096] First, thaw two reagents on ice: Equinox Library Amp Mix (2x concentration) and UDI primers (purchased from Twist Bioscience). Avoid repeated freeze-thaw cycles during thawing. Once the reagents are completely thawed, set them for use. Simultaneously, program the PCR instrument, fixing the hot-cover temperature at 105°C. Then, set the reaction program according to the following parameters: Step 1: Hold at 98°C for 45 seconds, this step is repeated once. Step 2: Enter the cycling phase, first hold at 98°C for 15 seconds, then increase to 60°C and hold for 30 seconds, and finally increase to 72°C and hold for 30 seconds. This cycling phase is repeated 8 times. After the cycling is complete, proceed to Step 3: Hold at 72°C for 1 minute, this step is repeated once. Finally, cool down to 4°C and hold at a constant temperature (hold). There is no requirement for the number of cycles. After the program is set, wait for subsequent samples to be added.

[0097] (2) Sample addition and PCR reaction operation

[0098] Take the purified supernatant obtained in step “3”, add 10 μL of UDI primer and 25 μL of Equinox Library Amp Mix (2x) after inverting and mixing to each supernatant; after the sample is added, gently blow the mixture with a pipette to ensure that the reagent and the purified supernatant are fully mixed and to avoid generating bubbles; then perform instantaneous centrifugation on the mixture to concentrate the liquid at the bottom of the tube, and then transfer the PCR tube to the PCR instrument with the program set in advance (i.e. the program set in step (1), the hot cap temperature is 105℃), start the instrument to run the reaction program until the program ends automatically.

[0099] 5. Second magnetic bead purification

[0100] (1) Binding of magnetic beads to PCR products

[0101] Take out the DNA purification magnetic beads that have been pre-equilibrated at room temperature and vortex them to mix them completely. Add 50 μL of 1× DNA purification magnetic beads to the PCR product taken out after PCR amplification in step "4" and vortex again to mix the magnetic beads and PCR product thoroughly. Incubate the mixture at room temperature for 5 minutes to ensure that the DNA binds effectively to the magnetic beads.

[0102] (2) Magnetic bead washing (ethanol washing)

[0103] Centrifuge the incubated mixture briefly, then place the centrifuge tube on a magnetic rack and let it stand for 5 minutes until the magnetic beads are completely adsorbed to one side of the tube wall and the solution is clear. Carefully aspirate the supernatant with a pipette. Add 200 μL of freshly prepared 80% ethanol to the centrifuge tube, incubate at room temperature for 1 minute, and then aspirate the ethanol supernatant. Repeat the above "add ethanol-incubate-discard supernatant" operation to complete two ethanol washes.

[0104] (3) Dry the magnetic beads

[0105] After the second ethanol wash, use a pipette with a volume of 10 μL to carefully remove the remaining ethanol droplets from the bottom of the centrifuge tube, avoiding touching the magnetic beads during the operation; keep the centrifuge tube on the magnetic rack and continue to dry the magnetic beads at room temperature until the surface of the magnetic beads is no longer reflective.

[0106] (4) DNA elution and supernatant transfer

[0107] Add 24 μL of enzyme-free pure water (ddH2O) to each sample tube containing dried magnetic beads. Gently pipette the magnetic beads to ensure they are completely suspended and in full contact with the pure water. Incubate at room temperature for 5 minutes, then perform a short-term centrifugation. Place the centrifuge tube back on the magnetic rack and let it stand for 2 minutes until the solution is clear. Then, aspirate 20 μL of the supernatant (i.e., the purified library) and transfer it to a new container for the next step of library enrichment.

[0108] (5) Document quality inspection

[0109] Take 2 μL of the purified library and dilute it 10 times with enzyme-free pure water. Use the Qubit quantitative analysis kit to determine the library concentration. At the same time, use a fragment analyzer to detect the fragment size of the library. The average fragment length of a qualified library should be in the range of 350-425 bp.

[0110] III. Hybrid Capture

[0111] 1. Library hybridization

[0112] (1) Library pool preparation scheme

[0113] A library pool typically contains libraries from multiple different samples. The amount of each library to be added to the library pool needs to be calculated based on its DNA concentration to ensure that the total amount of DNA in the pool after mixing is within the range of 1.5-4 micrograms, in order to meet the needs of subsequent hybridization.

[0114] (2) Mixed operation of documents

[0115] Based on the concentration of each library determined by Qubit, calculate the required amount of each library and add the corresponding volume of each library to a 1.5 ml (mL) centrifuge tube; gently shake the centrifuge tube to mix the libraries thoroughly, and then perform a short-term centrifugation to ensure that the liquid in the tube is concentrated at the bottom of the tube and that no residue adheres to the tube wall.

[0116] (3) Addition of prehybridization reagent

[0117] Add the following prehybridization reagents sequentially to the mixed library sample: 8 μL Universal Blockers, 5 μL Blocking Solution, and 4 μL probe set (see nucleotide sequences shown in SEQ ID NO.1-SEQ ID NO.937); after adding the reagents, gently shake the centrifuge tube to ensure thorough mixing of the reagents and the library, and then perform a brief centrifugation to avoid liquid residue.

[0118] (4) Vacuum drying of samples

[0119] Open the centrifuge tube cap and place it into the vacuum concentrator. At the same time, place an empty centrifuge tube for balancing to ensure the instrument is balanced. Set the vacuum concentrator temperature to 45℃ and start the instrument to dry until the solution in the tube is completely dry (drying time is usually 30-60 minutes, adjusted according to the solution volume).

[0120] (5) Hybridization reagent pretreatment and PCR instrument settings

[0121] Take out the Fast Hybridization Mix (purchased from Twist Bioscience) and incubate it at 65°C for 10 minutes until the precipitate in the reagent is completely dissolved. At the same time, set the program on the PCR instrument: first keep it at 95°C for 5 minutes, then keep it at 60°C for 2 hours, and finally close the PCR instrument's hot cover to complete the program setting.

[0122] (6) Sample resuspension and mixing

[0123] After drying, immediately remove the centrifuge tube from the vacuum concentrator and quickly cap it; quickly vortex the preheated FastHybridization Mix (be careful not to let the hybridization solution return to room temperature to avoid affecting the activity), add 20 μL to the centrifuge tube, gently tap the tube wall to ensure the solution is in full contact with the dried sample, and let it stand at room temperature for 5 minutes.

[0124] (7) Solution transfer and paraffin oil addition

[0125] Use a pipette to blow the solution in the centrifuge tube up and down 10 times to ensure that the DNA is completely dissolved; transfer all the solution in the tube to a new PCR tube, add 30 μL of paraffin oil to the PCR tube, and centrifuge briefly to remove air bubbles and prevent liquid evaporation during hybridization.

[0126] (8) Hybridization reaction operation

[0127] Transfer the PCR tube with added paraffin oil to a preheated and programmed PCR instrument, start the instrument to run the hybridization program set in step (5) of this step, until the program ends automatically.

[0128] 2. Library capture and cleaning

[0129] (1) Preliminary preparations

[0130] While the hybridization reaction in step “1” of this procedure is underway, perform the following preparations: Take out the DNA-binding magnetic beads (Streptavidin Binding Beads, purchased from Twist Bioscience), shake them thoroughly to mix, and equilibrate at room temperature for at least 30 minutes; preheat Fast Wash Buffer 1 to 66°C and Wash Buffer 2 to 48°C in a constant temperature metal bath; aliquot the enrichment reagents into centrifuge tubes, with the following single-reaction volumes: 900 μL Fast Binding Buffer (Twist Bioscience), 450 μL Fast Wash Buffer 1 (Twist Bioscience), and 700 μL Wash Buffer 2 (Twist Bioscience).

[0131] (2) Measuring magnetic beads

[0132] Shake the pre-equilibrated Streptavidin Binding Beads until the beads are completely mixed, then add 100 μL of beads to a 1.5 mL centrifuge tube.

[0133] (3) Magnetic bead washing (Fast Binding Buffer)

[0134] Add 200 μL of Fast Binding Buffer to the centrifuge tube containing the magnetic beads and gently mix with a pipette tip. Place the centrifuge tube in a magnetic rack and wait for the solution to clarify before discarding the supernatant and removing the centrifuge tube. Repeat the above steps of "adding buffer - mixing - magnetic rack adsorption - discarding supernatant" for a total of 3 washes to ensure the magnetic beads are clean.

[0135] (4) Magnetic bead resuspension

[0136] After three washes, add 200 μL of Fast Binding Buffer to the centrifuge tube and shake to fully resuspend the magnetic beads.

[0137] (5) Hybridization solution combined with magnetic beads

[0138] After the hybridization reaction in step “1” of this procedure is completed, immediately open the PCR instrument lid and transfer all the hybridization solution in the PCR tube to the cleaned magnetic bead suspension. After the transfer is completed, immediately use a pipette to slowly mix the mixture to ensure that the hybridization solution and the magnetic beads are in full contact.

[0139] (6) Constant temperature mixing and bonding

[0140] Cover the centrifuge tubes and place them on a vertical mixer. Set the speed to 1200 rpm and mix thoroughly at room temperature for 30 minutes to allow the hybridization complex to effectively bind with the magnetic beads.

[0141] (7) First wash (Fast Wash Buffer 1)

[0142] Centrifuge the mixed centrifuge tubes quickly, then place them on a magnetic rack and let them stand for 1 minute until the solution becomes clear. Discard the supernatant. Remove the centrifuge tubes from the magnetic rack, add 200 μL of Fast Wash Buffer 1 preheated to 66°C, mix gently, and then quickly incubate at 66°C for 5 minutes. After incubation, repeat the "let the solution stand on the magnetic rack for 1 minute - discard the supernatant" process to complete one Fast Wash Buffer 1 wash.

[0143] (8) Magnetic bead transfer and second washing

[0144] Transfer all liquid from the centrifuge tube to a new 1.5 ml centrifuge tube. Place the new centrifuge tube on a magnetic rack and let it stand for 1 minute. Discard the supernatant to complete the second Fast Wash Buffer 1 wash.

[0145] (9) Third wash (Wash Buffer 2)

[0146] Remove the centrifuge tubes from the magnetic rack, add 200 μL of Wash Buffer 2 preheated to 48°C, mix gently, and then quickly incubate at 48°C for 5 minutes. After incubation, place the centrifuge tubes on the magnetic rack and let them stand for 1 minute, then discard the supernatant. Repeat the above steps of "add Wash Buffer 2 - incubation - magnetic rack adsorption - discard supernatant" for a total of 3 washes.

[0147] (10) Residual liquid removal and magnetic bead resuspension

[0148] After three washes with Wash Buffer 2, the centrifuge tubes were briefly centrifuged and then placed on a magnetic rack. The remaining liquid in the tubes was aspirated using a 10 μL pipette. 45 μL of enzyme-free pure water (ddH2O) was added to the centrifuge tubes, and the mixture was stirred by pipetting. The centrifuge tubes were then placed on ice for incubation.

[0149] 3. PCR amplification

[0150] (1) Preliminary preparation and PCR instrument setup

[0151] Remove the DNA purification beads, vortex thoroughly, and equilibrate at room temperature for at least 30 minutes; prepare 80% ethanol for later use; remove the Equinox Library Amp Mix (2× concentration) and Amplification Primers (purchased from Twist Bioscience) from the low-temperature environment and thaw them on ice; set the program on the PCR instrument: hot lid temperature 105℃, program steps are: 98℃ for 45 seconds (1 cycle), then 98℃ for 15 seconds, 60℃ for 30 seconds, 72℃ for 30 seconds (12 cycles in total), and finally 72℃ for 1 minute (1 cycle), and hold at 4℃.

[0152] (2) Preparation of the reaction system

[0153] Take a PCR tube and add 25 μL of Equinox Library Amp Mix (2×) and 2.5 μL of Amplification Primers to each tube; add 22.5 μL of the magnetic bead suspension prepared in "Step 2" to the PCR tube, gently shake to mix the reaction system thoroughly, and then perform a short-term centrifugation to ensure that the liquid is concentrated at the bottom of the tube.

[0154] (3) PCR reaction run

[0155] Transfer the prepared reaction system to the PCR tubes and start the amplification program in the instrument until the program ends.

[0156] 4. Magnetic bead purification

[0157] (1) Binding of magnetic beads to amplification products

[0158] Remove the pre-equilibrated DNA purification magnetic beads and vortex to mix them completely. Add 90 μL of 1.8× pre-mixed DNA purification magnetic beads to each enriched sample obtained in step “3” of this step, vortex to mix, and incubate at room temperature for 5 minutes to allow the DNA to bind to the magnetic beads.

[0159] (2) Supernatant removal

[0160] After incubation, the mixture was briefly centrifuged. The centrifuge tube was placed on a magnetic rack and left to stand for 5 minutes until the magnetic beads were adsorbed and the solution became clear. The supernatant was then discarded.

[0161] (3) Washing with ethanol

[0162] Add 200 μL of freshly prepared 80% ethanol to the centrifuge tube, incubate at room temperature for 1 minute, and then discard the supernatant. Repeat the above ethanol washing operation twice.

[0163] (4) Dry the magnetic beads.

[0164] Use a 10 μL pipette to remove any residual ethanol from the bottom of the tube. Keep the centrifuge tube on the magnetic rack and allow the magnetic beads to air dry at room temperature until the surface is no longer reflective.

[0165] (5) DNA elution

[0166] Add 34 μL of enzyme-free pure water (ddH2O) to each sample tube, mix by pipetting, and incubate at room temperature for 5 minutes; after brief centrifugation, place the centrifuge tube back on the magnetic rack and let it stand for 2 minutes.

[0167] (6) Document quality inspection

[0168] Transfer 32 μL of supernatant to a clean 1.5 mL centrifuge tube; take 2 μL of supernatant and use the Qubit® dsDNA HS Assay Kit (purchased from Vazyme) to determine the library concentration. The acceptable library concentration should be greater than 0.5 ng / µL; use a 2100 fragment analyzer to detect the fragment length distribution of the library. The average fragment length of an acceptable library should be around 300-400 bp.

[0169] IV. Sequencing and Analysis

[0170] The library constructed according to the aforementioned steps is compatible with all models of Illumina sequencing platforms. Adjust the library concentration according to the dilution factor required by the corresponding sequencer, set standard sequencing parameters (such as read length, sequencing depth, etc.), complete the sequencing on the instrument, and perform quality control on the raw data after sequencing.

[0171] This invention provides a method for detecting and analyzing gene mutations in follicular lymphoma based on NGS, specifically including the following steps:

[0172] (1) Target acquisition area determination

[0173] Based on the target genes associated with follicular lymphoma, the target capture region to be detected is identified, and a BED format file of the region is generated as the basis for probe design and subsequent analysis.

[0174] (2) Probe sequence design

[0175] Based on the .bed file generated in step (1) and the human hg38 reference genome, probe sequences for the target region were designed using CaTCh software (Reference: Metsky HC, Siddle KJ, Gladden-Young A, et al. Capturing sequence diversity in metagenomes with comprehensive and scalable probe design [J]. Nat Biotechnol, 2019:160-168.).

[0176] After design, synthesis, alignment, filtering of repetitive sequences, and removal of non-specific sequences that can be aligned to the hg38 genome in multiple places, the final specific probe sequences are shown as nucleotide sequences in SEQ ID NO.1 to SEQ ID NO.937.

[0177] (3) Sample library construction and sequencing

[0178] Genomic DNA was extracted from normal samples and standard mutant samples respectively. The probe capture method obtained in step (2) was used to construct libraries, and then the libraries were sequenced to obtain raw sequencing data.

[0179] (4) Splitting the raw data

[0180] Based on the sample source (normal sample, standard mutant sample), the raw sample data obtained after processing is split into fq format to ensure that the data of different types of samples are independent, which facilitates subsequent comparative analysis.

[0181] (5) Data preprocessing

[0182] All raw FQ data were preprocessed using the FASTP software (Reference: Chen S, Zhou Y, Chen Y, et al. FASTP: an ultra-fast all-in-one FASTQ preprocessor. 2018). Specifically, reads with low base quality values ​​(low-quality reads) and reads containing adapter contamination were removed to obtain clean data.

[0183] (6) Reference genome alignment and data correction

[0184] The clean data obtained in step (5) is aligned to the hg38 reference genome using the bwa mem algorithm to generate the original BAM format file. The original BAM file is then subjected to PCR repetitive sequence removal, and the base quality of the BAM file is recalibrated using the GATK best practice workflow to finally obtain a standardized BAM file that can be used for variant detection.

[0185] (7) Multi-type variant detection and filtering

[0186] a. Somatic mutation (SNV / INDEL) detection: The self-built variant detection tool Parsebam was used to detect SNV (single nucleotide variant) and INDEL (insertion / deletion variant) in the standardized BAM file; at the same time, a normal sample dataset was constructed as a background filter library (Panel of Normals, PoN). The PoN library was used to analyze and filter meaningless variants and benign germline variants, while retaining potentially pathogenic somatic variants.

[0187] b. Fusion gene (SV) detection: Structural variants (SVs) were detected using LUMPY software, with a focus on identifying fusion gene-related variants (Reference: Layer RM, Hall IM, Quinlan AR. LUMPY: A probabilistic framework for structural variant discovery [J]. 2012); normal sample data were used as a baseline, background noise variants were removed, and tumor-specific fusion gene variants were retained.

[0188] c. Copy number variant (CNV) detection: Copy number variants are detected using cnvkit software. The results of the above SNV / INDEL, SV, and CNV detections are integrated to generate VCF format result files for each variant type and a filtered list of high-quality variants.

[0189] (8) MSI Status Detection

[0190] Using MSIsensor-pro software, with normal sample data as a reference, the microsatellite instability (MSI) status of each sample was detected (Reference: Jia P, Yang X, Guo L, et al. MSIsensor-pro: Fast, Accurate, and Matched-normal-sample-free Detection of MicrosatelliteInstability [J]. Journal of Genomics, Proteomics and Bioinformatics: English Edition, 2020 (1):7); the judgment criteria were: MSI value (proportion of unstable sites) ≥10% was considered microsatellite instability-high (MSI-H), and MSI value <10% was considered high.

[0191] (9) By using the dataset and reporting program for interpreting clinically relevant mutations of follicular lymphoma accumulated in the laboratory, the mutation results and MSI information in the information analysis steps (7)-(8) are read and the clinical test report is automatically generated.

[0192] The above steps (4)-(9) have been encapsulated into an automated process. During runtime, only the fq list of the download file is required for the program to run automatically and output results. Bioinformatics analysis of the data is performed using software such as BWA / GATK, and relevant databases such as gnomAD / Clinvar / OMIM are annotated. Diagnostic and treatment information for mutated genes is provided based on relevant international and domestic guidelines.

[0193] Example 2

[0194] This embodiment uses the gene mutation detection and analysis method for follicular lymphoma constructed in Example 1 to detect gene mutations in a male patient diagnosed with follicular lymphoma. The sample type is a tissue smear, and mutations in 74 follicular lymphoma-related genes are detected. The method is as follows:

[0195] 1. Genomic DNA was extracted from the white sample using a Tianlong automated extraction instrument (model: NP968-C), and analyzed according to the method and procedure of Example 1. The fragment analysis results of the hybridization capture library are as follows: Figure 1 As shown.

[0196] 2. The obtained hybridization capture library was sequenced using an Illumina Nextseq 6000 instrument with sequencing parameters of PE 2x150. Quality control was performed on the sequenced library; the target region coverage was 99.9%, and the average depth of the target region was 1151.05, indicating that the quality control was satisfactory.

[0197] 3. The sequencing data were compared with the human reference genome hg38 / GRCh38, and quality data such as the coverage and average sequencing depth of the target region were collected.

[0198] 4. The corrected BAM file was analyzed using a bioinformatics workflow to identify variant sites, and annotations were performed using multiple professional databases and standards. Specific guidelines are as follows:

[0199] Database support: The annotation of variant sites references authoritative databases such as gnomAD (population frequency database), ClinVar (clinical variant database), and OMIM (online human Mendelian genetics database) to clarify the population frequency, clinical significance, and disease association information of the variants.

[0200] Gene and variant naming rules: Gene names are strictly named in accordance with the rules established by the Human Genome Organization's Committee on Gene Nomenclature (HGNC) to avoid interpretation bias caused by inconsistent naming; the naming of variant sites refers to the standard nomenclature provided by the Human Genome Variation Society (HGVS) to ensure the accuracy and standardization of variant descriptions.

[0201] Variant grading criteria: The interpretation of variant sites is based on the ACMG / AMP genetic variant classification criteria and guidelines, combined with updated supplementary recommendations and consensus from institutions such as ClinGen, and variants are classified into different grades (such as pathogenic, possibly pathogenic, of unknown significance, possibly benign, and benign). In this case, the focus is on pathogenic and possibly pathogenic variants associated with follicular lymphoma.

[0202] Finally, the information on the variant sites obtained from the analysis is shown in Table 1.

[0203] Table 1:

[0204]

[0205] As shown in Table 1, through the above analysis process, two clinically significant mutation sites were detected in the 74 target genes of the male patient with follicular lymphoma: (1) KMT2D gene mutation: The mutation site was named NM_003482.3 (exon34):c.9964C>T according to HGVS, and the corresponding amino acid change was p.Gln3322*. The mutation type was nonsense mutation. The mutation frequency (allele frequency) of this mutation was 24.85%. According to the ACMG / AMP standard and clinical database annotation, the mutation classification was IA (pathogenic mutation). (2) FOXO1 gene mutation: The mutation site was named NM_002015.4(exon1):c.504_510del according to HGVS, and the corresponding amino acid change was p.Ile169fs. The mutation type was frameshift mutation. The mutation frequency of this mutation was 13.61%. The mutation classification was also IA (pathogenic mutation).

[0206] Example 3

[0207] This embodiment uses the gene mutation detection and analysis method for follicular lymphoma constructed in Example 1 to detect gene mutations in a patient whose follicular lymphoma progressed to diffuse large B-cell lymphoma. The sample type is tissue smear, and the mutation status of 74 follicular lymphoma-related genes is detected.

[0208] The analysis method is described in Example 2, and the information on the variant sites obtained from the analysis is shown in Table 2.

[0209] Table 2:

[0210]

[0211] As shown in Table 2, through the above analysis process, 20 clinically significant variant sites of 14 variant genes were detected among the 74 target genes in this male patient with follicular lymphoma.

[0212] The test results show that the coverage and average depth of the target region of the follicular lymphoma panel hybridization capture probe set (see SEQ ID NO.1~SEQ ID NO.937) provided by the present invention are qualified, and can accurately detect the variation of follicular lymphoma-related genes.

[0213] The core characteristics of the 937 capture probes provided by this invention are high synergy and precise coverage, which together support efficient and comprehensive detection within the same system.

[0214] 1. High degree of collaboration

[0215] Supporting simultaneous detection within the same system is the foundation for achieving simultaneous detection of multiple genes, specifically reflected in two aspects: (1) Strong compatibility: All 937 probes in the same reaction system (such as the same test tube, the same buffer solution and temperature conditions) will not interfere with the detection due to interaction and can work stably at the same time. (2) Efficiency matching: The binding efficiency and reaction kinetic parameters (such as annealing temperature and binding rate) of each probe have been optimized, and the target region can be captured simultaneously in the same detection process (such as one PCR amplification and one sequencing reaction) without the need for batch operations.

[0216] 2. Precise coverage: Achieving coverage of the "full exon regions of 74 genes" determines the comprehensiveness and accuracy of the detection. The core features are: (1) Complete regional coverage: The design of 937 probes covers all exon regions of 74 target genes, with no key regions missing, and the coverage rate reaches 99.9%, ensuring that all possible variations (such as point mutations, insertions and deletions) on the exons can be detected. (2) Precise targeting: Each probe can specifically bind to the corresponding gene exon sequence and does not bind to non-target regions (such as introns, other irrelevant genes), avoiding false positives or data interference caused by non-specific capture.

[0217] In summary, the non-obviousness of the detection of 937 capture probes in the same reaction system provided by this invention lies in the need to simultaneously solve the problems of non-interference synergistic work among multiple probes, the problem of precise targeting design covering all exons of 74 genes, and the problem of process optimization to balance the reaction efficiency of all probes in a single process. These all require breakthroughs in multi-dimensional technical bottlenecks rather than simply stacking the number of probes.

[0218] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features. These modifications or substitutions, or combinations of technical features in the above embodiments that do not conflict with each other, can be made in accordance with the manner described in the embodiments. These modifications, substitutions or combinations do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A set of capture probes for multi-gene detection of follicular lymphoma, characterized in that, Capture probes containing nucleic acid sequences that specifically bind to genes selected from the following group: ARID1A, ARID1B, ATM, ATP6V1B2, B2M, BCL10, BCL2, BCL6, BCL7A, BTG1, BTG2, BTK, CARD11, CCND3, CD58, CD70, CD79A, CD79B, CDK4, CDKN2A, CIITA, CREBBP, CXCR4, DDX3X, DTX1, DUSP2, EBF1, EP300, EPHA7, EZH2, FAS, FOXO1, GNA13, GNAI2, IKZF3, IRF4, IRF8, ITPKB, KLF2, KLHL6, KMT2C, KMT2D, MAP2K1, MEF2B, MYC, MYD88, NOTCH1, NOTCH2, OSBPL10, P2RY8, PAX5, PIM1, PLCG2, PRDM1, PTEN, PTPRD, RB1, REL, RHOA, RRAGC, SETD1B, SGK1, SMARCA4, SOCS1, SPEN, STAT3, STAT6, TBL1XR1, TET2, TMEM30A, TNFAIP3, TNFRSF14, TP53, XPO1 The capture probe set is capable of capturing at least one target sequence of the gene.

2. The capture probe set for multi-gene detection of follicular lymphoma according to claim 1, characterized in that, Oligonucleotides comprising sequences as shown in SEQ ID NO.1-SEQ ID NO.

937.

3. A kit for detecting multiple genes in follicular lymphoma, characterized in that, The kit contains the capture probe set for multigene detection of follicular lymphoma as described in claim 1 or 2.

4. The reagent kit according to claim 3, characterized in that, The kit includes a genomic DNA extraction reagent module, an NGS library quality control reagent module, a targeted capture enhancement reagent module, and a sequencing expansion reagent module; the targeted capture enhancement reagent module contains a set of capture probes for the multi-gene detection of follicular lymphoma.

5. The reagent kit according to claim 4, characterized in that, The genomic DNA extraction reagent module includes lysis reagents, binding / washing reagents, purification reagents, and elution / preservation reagents; The NGS library quality control reagents include fragment screening reagents, quantitative detection reagents, fragment analysis reagents, and contaminant removal reagents; The targeted capture enhancement reagent module includes a biotinylated DNA probe library, a hybridization system, a capture vector, a washing system, and a molecular tag; wherein, the biotinylated DNA probe library consists of probes from the capture probe group for multi-gene detection of follicular lymphoma with biotin linked at the 3' end; The sequencing expansion reagent includes library amplification reagent, Index system, four-color fluorescently labeled reversible termination dNTPs, DNA polymerase, regeneration reagent, and cluster generation reagent.

6. A method for detecting and analyzing gene mutations in follicular lymphoma based on NGS, characterized in that, Using the capture probe set as described in claim 1 or 2 or the kit as described in any one of claims 3-5, the in vitro sample to be tested is sequenced, the sample data is analyzed, gene mutation information related to follicular lymphoma is obtained, and a report is generated.

7. The method for detecting and analyzing gene mutations in follicular lymphoma based on NGS according to claim 6, characterized in that, The detection and analysis method includes the following steps: S1, Library Construction and Sequencing Using the capture probe set as described in claim 1 or 2 or the kit as described in any one of claims 3-5, a library is constructed and sequencing is completed on the genomic DNA extracted from the in vitro sample to obtain the original sample FastQ format sequencing data; S2, Sequencing data preprocessing The raw fq data obtained from S1 was quality controlled and preprocessed using the FASTP software. Specific parameter settings included: removing low-quality bases with a base quality value of Q≤20, removing short fragments with a read length of ≤50bp, and removing reads containing adapter contamination. S3, Reference Genome Alignment and Data Deduplication The clean fq data preprocessed in step S2 were aligned to the human reference genome hg38 using the bwa mem algorithm to generate the original BAM format file. The original BAM file was then subjected to PCR repetitive sequence removal using software, while retaining unique alignment reads. Finally, the deduplicated BAM file was sorted and indexed using software to obtain a standardized BAM file that can be used for variant detection. S4. Multi-type variant detection and filtering, including: Somatic mutation SNV / INDEL detection: The self-built variant detection tool Parsebam was used to detect SNV and INDEL in S3 standardized BAM files. At the same time, a PoN database was constructed to filter meaningless variants and benign germline variants, while retaining potentially pathogenic somatic variants. Fusion gene SV detection: Structural variation detection was performed on the S3 normalized BAM file using LUMPY software to identify fusion gene-related variations. Background variations were removed by using normal sample data as a baseline, while tumor-specific fusion gene variations were retained. Copy number variation (CNV) detection: CNVkit software was used to detect CNVs in S3 normalized BAM files. The copy number change was calculated by the ratio of sequencing depth of the target region to the reference region. The CNV detection thresholds were set as copy number gain ≥ 3 copies and copy number loss ≤ 1 copy. Finally, the detection results of SNV / INDEL, SV, and CNV were uniformly converted into VCF standard format files, and functional annotations were performed using ANNOVAR software to generate a filtered high-quality variant list. S5. Analyze the variant structures and MSI information obtained in S4, and generate a detection report.

8. The method for detecting and analyzing gene mutations in follicular lymphoma based on NGS according to claim 7, characterized in that, In S1, the test samples are tumor tissue samples from patients with follicular lymphoma or cell-free DNA samples from peripheral blood; During library construction, the initial DNA amount should be ≥100ng and the fragmentation length should be 150-200bp; S2 also includes: filtering reads with N base content ≥5%, and verifying data quality using FastQC software after preprocessing to ensure Q30 ≥ 85%; In S3, the human reference genome hg38 uses the latest version released by Ensembl or UCSC; PicardTools software is used to remove PCR repetitive sequences from the original BAM file while retaining unique alignment reads, with an alignment quality value MAPQ ≥ 30.

9. The method for detecting and analyzing gene mutations in follicular lymphoma based on NGS according to claim 7, characterized in that, In S4, when performing SNV and INDEL detection on the S3 standardized BAM file, the minimum allele frequency (MAF) was set to be ≥0.02, the minimum coverage depth to be ≥20×, and the number of reads supported by the variant site to be ≥5. The constructed PoN database also contains sequencing data from ≥50 peripheral blood samples from healthy individuals. Structural variation detection was performed on the S3 standardized BAM file to identify fusion gene-related variations, including chromosomal translocations or inversions. During the structural variation detection process, the minimum breakpoint support ≥3 reads and the number of reads across breakpoints ≥2. Data from at least 3 healthy normal samples analyzed using the same process were used as a baseline to remove background variations. The detection process for copy number variations (CNVs) requires the introduction of sex-matched normal control samples for correction to exclude normal copy number differences in sex-related chromosomes. The resulting high-quality variant list includes information on variant location, variant type, functional impact, allele frequency, and clinical significance.

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