A capture probe for myeloid hematological tumor detection gene panel and its design method and detection method

Abstract

The present invention provides a capture probe for a myeloid hematological tumor detection gene panel and a design method and a detection method thereof, belonging to the field of gene detection technology. The probe of the present invention includes a panel A DNA capture probe and a panel B cDNA capture probe; the panel A DNA capture probe includes a nucleotide sequence as shown in SEQ ID NO.1-2400, and the panel B cDNA capture probe includes a nucleotide sequence as shown in SEQ ID NO.2401-10559. The probe of the present invention can be used to detect all types of genes for myeloid hematological tumor detection, can solve the detection of myeloid hematological tumor gene mutation, copy number variation, fusion gene, and gene expression in one stop, can be used for detection at all stages of clinical practice, has strong portability, and can be applied to multiple detection platforms.

Description

Technical Field

[0001] The present invention relates to the technical field of gene detection, and in particular to a capture probe for a myeloid hematological tumor detection gene panel and a design method and a detection method thereof. Background Art

[0002] Myeloid hematologic malignancies are a class of malignant tumors that originate from hematopoietic stem cells or early hematopoietic progenitor cells, including acute myeloid leukemia (AML), chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS), and myeloproliferative neoplasms (MPNs). The incidence, prognosis, and treatment options of these tumors vary, but they are all related to genetic abnormalities in myeloid cells.

[0003] According to the International Agency for Research on Cancer (IARC), in 2018, approximately 406,000 people worldwide suffered from myeloid hematologic malignancies, of which AML accounted for 47.6%, CML accounted for 19.9%, MDS accounted for 16.7%, and MPN accounted for 15.8%. The incidence of myeloid hematologic malignancies increases with age, is higher in men than in women, and is higher in developed countries than in developing countries. The five-year survival rate of myeloid hematologic malignancies varies by type and stage, and is generally less than 50%.

[0004] The diagnosis of myeloid hematological tumors mainly relies on clinical manifestations, laboratory tests and molecular biological tests. Laboratory tests include peripheral blood routine tests, bone marrow smears, cell morphology, immunophenotype, cytogenetics and molecular biology. Molecular biological testing refers to the analysis of gene mutations or fusion genes present in myeloid cells to determine the type, stage, prognosis and treatment plan of the tumor. Existing molecular biological detection methods mainly include polymerase chain reaction (PCR), fluorescence in situ hybridization (FISH), chip technology, etc. These methods have the advantages of high sensitivity, strong specificity and simple operation, but they also have limitations, such as only being able to detect known targets, not being able to cover the entire genome, and not being able to detect multiple mutations or low-frequency mutations.

[0005] The occurrence and development of myeloid hematologic malignancies are closely related to genetic abnormalities in myeloid cells, including fusion genes, gene mutations, copy number variations, structural variations, and gene expression. Among them, some gene mutations or fusion genes have diagnostic significance and can help distinguish different types of myeloid hematologic malignancies, such as the BCR-ABL1 fusion gene in CML, the NPM1 mutation in AML, and the SF3B1 mutation in MDS. Other gene mutations or fusion genes have prognostic significance and can reflect the biological behavior and treatment response of tumors, such as the FLT3-ITD mutation in AML, the ABL1 kinetic mutation in CML, and the TP53 mutation in MDS. Some other gene mutations or fusion genes have therapeutic significance and can serve as the basis or monitoring indicators for targeted therapy, such as the BCR-ABL1 fusion gene in CML, the IDH1 / 2 mutation in AML, and the JAK2 V617F mutation in MPN. Therefore, it is very necessary to detect gene mutations and fusion genes in patients with myeloid hematological tumors. It can help doctors develop individualized diagnostic plans, assess patients' prognostic risks, select the most suitable treatment strategy, monitor treatment effects and residual disease, etc.

[0006] Targeted capture sequencing technology is a method that uses specific probes to capture the target gene region and then performs high-throughput sequencing. It can improve the efficiency and sensitivity of sequencing and reduce the cost and time of sequencing. In the diagnosis of myeloid hematological tumors, targeted capture sequencing technology has the following necessity and advantages: Myeloid hematological tumors are a type of highly heterogeneous disease, and their pathogenesis is related to multiple gene mutations. Targeted capture sequencing technology can simultaneously detect the mutations of multiple related genes, provide more comprehensive molecular information, and help with diagnostic typing, prognosis judgment, and treatment guidance. Gene mutations in myeloid hematological tumors are often somatic mutations, and their mutation frequencies and levels vary in different patients and different courses of disease. Targeted capture sequencing technology can improve the sensitivity and accuracy of detection, discover low-frequency or subclonal mutations, and reflect the clonal evolution and heterogeneity of tumors. The treatment effect of myeloid hematological tumors is closely related to the level of minimal residual disease (MRD). Targeted capture sequencing technology can be used as one of the molecular markers of MRD to monitor the elimination of tumor cells after treatment and evaluate the risk of recurrence and prognosis.

[0007] Existing myeloid hematologic malignancies detection has the following defects: 1. The detection content is single, and most of the current sequencing detection technologies only target one of the gene mutations or fusion genes. 2. It is impossible to verify and analyze germline mutations. Germline mutations may be related to hereditary blood tumors or familial blood tumors, which is of great significance for genetic counseling and family screening of patients. 3. Fusion gene detection is mostly targeted at known fusion forms and cannot predict unknown fusion genes. Most fusion gene detection methods can only detect specific fusion genes or fusion partners, and cannot discover new or rare fusion genes, which may ignore some fusion genes with diagnostic, prognostic or therapeutic significance. 4. There is no analysis of gene expression. Gene expression analysis is usually not possible with RNA targeted sequencing technology. Summary of the invention

[0008] The purpose of the present invention is to provide a capture probe for a myeloid hematologic tumor detection gene panel and a design method and detection method thereof. The probe of the present invention can be used to detect all types of genes for myeloid hematologic tumor detection, and can solve the detection of myeloid hematologic tumor gene mutations, copy number variations, fusion genes, and gene expression in one stop, and can be used for detection at all stages of clinical practice, has strong portability, and can be applied to multiple detection platforms.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0010] The present invention provides a capture probe for a myeloid hematological tumor detection gene panel, wherein the capture probe comprises a panel A DNA capture probe and a panel B cDNA capture probe; the panel A DNA capture probe comprises a nucleotide sequence as shown in SEQ ID NO.1-2400, and the panel B cDNA capture probe comprises a nucleotide sequence as shown in SEQ ID NO.2401-10559.

[0011] The present invention also provides an application of the capture probe in preparing a detection reagent or a kit for myeloid hematological tumor genes.

[0012] The present invention also provides a method for designing a capture probe for the above-mentioned myeloid hematological tumor detection gene panel, comprising the following steps:

[0013] (1) Screening of myeloid hematologic malignancies-related genes, divided into panel A and panel B;

[0014] (2) designing and synthesizing probes according to the standard gene names, gene transcript numbers and chromosomal locations of the exons of the relevant genes in the panel A and panel B;

[0015] (3) Performing performance verification on the probe.

[0016] Preferably, the panel A is a myeloid hematological tumor-related mutation gene; the panel B is a myeloid hematological tumor-related fusion gene and gene expression.

[0017] Preferably, the genes in panel A include ABCB1, CCND1, DKC1, GFI1, KMT2C, PDGFRA, RUNX1, TERC, ANKRD26, CDKN2A, DNMT3A, GSKIP, KRAS, PDGFRB, SAMD9, TERT, ASXL1, CEBPA, ELANE, GSTP1, MBD4, PHF6, SAMD9L, TET2, ATG2B, CREBBP, EP300, HAX1, MPL, PIGA, SETBP1, TP53, ATRX, CSF1R, ETNK1, IDH1, MTHFR, PML, S F3B1, TPMT, BCOR, CSF3R, ETV6, IDH2, NF1, PPM1D, SH2B3, U2AF1, BCORL1, CUX1, EZH2, JAK2, NOTCH1, PRPF8, SRP72, WT1, BRAF, CYP2C19, FLT 3. JAK3, NOTCH2, PTEN, SRSF2, ZRSR2, CALR, CYP3A5, GATA1, KDM6A, NPM1, PTPN11, STAG2, NQO1, CBL, DDX41, GATA2, KIT, NRAS, RARα, STAT3;

[0018] The panel Genes in B include ABL1, AFF1, ALK, ANXA3, BAALC, BCR, BMI1, BMP2K, CAT, CBFB, CBL, PROM1, CD34, CPA3, CREBBP , ELL, MLLT1, EP300, ERG, RUNX1T1, ETV6, MECOM, FGFR1, FIP1L1, FLT3, FUS, GATA2, Hes1, HOXA10, HOX A9, JAK2, KAT6A, KIT, MEIS1, KMT2A, MLLT10, MLLT3, AFDN, MN1, MSI2, CBFA2T3, MYH11, NPM1, NT5E, NU P214, NUP98, PDGFRA, PDGFRB, PML, PRAME, SPI1, RARA, RARG, RBM15, ROS1, RPN1, RUNX1, S100A9, WT1.

[0019] Preferably, the probe design method adopts a traversal method based on a machine learning algorithm to exclude probe sequences with a similarity higher than 50% through sequence similarity comparison; the probe specifically binds to the target nucleic acid region, and the single base coupling efficiency is 99.4% to 99.6%. The synthesized probe is quality controlled by electrospray mass spectrometry.

[0020] Preferably, the performance verification includes capture efficiency verification, precision verification, accuracy verification, and analytical sensitivity verification.

[0021] The present invention also provides a method for detecting myeloid hematological tumor-related genes for non-disease diagnosis or treatment purposes, comprising the following steps:

[0022] (1) Extracting sample nucleic acid;

[0023] (2) fragmenting the DNA in the nucleic acid, ligating with adapters, amplifying by PCR, and purifying to obtain a panel A library;

[0024] (3) Shearing the RNA in the nucleic acid, performing reverse transcription, linker ligation, PCR amplification, and purification to obtain a panel B library;

[0025] (4) using the panel A DNA capture probe to perform hybridization capture on the panel A library; using the panel B cDNA capture probe to perform hybridization capture on the panel B library;

[0026] (5) performing second generation sequencing on the panel A library and panel B library after hybridization capture, respectively, and performing data analysis on the sequencing results.

[0027] Preferably, the data analysis of the panel A library includes data filtering, quality control, alignment, mutation detection, copy number analysis, and result annotation; the data analysis of the panel B library includes data filtering, quality control, alignment, fusion detection, expression analysis, and result annotation.

[0028] Preferably, when performing data analysis on the sequencing results of the panel A library after the hybridization capture, it also includes performing germline mutation verification, and the method for verifying the germline mutation is to compare and analyze the low-depth sequencing results of the oral mucosa samples and the bone marrow samples after data analysis.

[0029] The present invention provides a capture probe for a myeloid hematologic tumor detection gene panel, a design method thereof, and a detection method thereof. The gene panel of the present invention includes all types of genes for myeloid hematologic tumor detection. Capture probes are designed for these genes, which can solve the detection of myeloid hematologic tumor gene mutations, copy number variations, fusion genes, and gene expression in a one-stop manner. The detection content has a wide coverage, and can be used for detection at various stages from risk assessment to diagnosis, treatment, and prognosis. It has strong portability and is suitable for sequencing instruments or newly developed detection platforms currently on the market. In addition, the detection method of the present invention includes comparative analysis of oral mucosal samples and bone marrow samples. Through low-depth oral mucosal sample sequencing and high-throughput bone marrow sample sequencing, the annotation of germline mutations and somatic mutations can be achieved without increasing the sequencing cost, solving the problem of germline mutation verification in blood tumor gene mutation detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a diagram of the germline mutation verification results based on 112 patients in Example 2 of the present invention. DETAILED DESCRIPTION

[0031] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0032] Example 1

[0033] This embodiment provides a capture probe for a myeloid hematological tumor gene panel, and the specific design process is as follows:

[0034] 1. Panel design: Select myeloid hematologic malignancies-related genes from published literature, including: high-frequency mutation genes, high-frequency fusion genes, high-frequency expression-related genes in public databases, myeloid hematologic malignancies treatment-related genes in application standards, treatment guidelines, drug labels, and general databases, and genes related to myeloid hematologic malignancies molecular typing, treatment, prognosis, and induction reported in published literature. According to different detection methods, it is divided into Panel A: myeloid hematologic malignancies-related mutation genes (gene types are shown in Table 1), and Panel B: myeloid hematologic malignancies-related fusion and expression genes (gene types are shown in Table 2).

[0035] Table 1 Panel A: Mutation genes associated with myeloid hematological tumors

[0036] ABCB1 CCND1 DKC1 GFI1 KMT2C PDGFRA RUNX1 TERC ANKRD26 CDKN2A DNMT3A GSKIP KRAS PDGFRB SAMD9 TERT ASXL1 CEBPA ELANE GSTP1 MBD4 PHF6 SAMD9L TET2 ATG2B CREBBP EP300 HAX1 MPL PIGA SETBP1 TP53 ATRX CSF1R ETNK1 IDH1 MTHFR PML SF3B1 TPMT BCOR CSF3R ETV6 IDH2 NF1 PPM1D SH2B3 U2AF1 BCORL1 CUX1 EZH2 JAK2 NOTCH1 PRPF8 SRP72 WT1 BRAF CYP2C19 FLT3 JAK3 NOTCH2 PTEN SRSF2 ZRSR2 CALR CYP3A5 GATA1 KDM6A NPM1 PTPN11 STAG2 NQO1 CBL DDX41 GATA2 KIT NRAS RARα STAT3 / / / / /

[0037] Table 2 Panel B: Myeloid hematological neoplasms-related fusion genes and gene expression

[0038]

[0039]

[0040] The above myeloid hematological neoplasms-related genes were merged and removed for redundancy, and the standard gene names, gene transcript numbers and chromosomal locations of the exons of the related genes were determined through the HGNC database (HUGO Gene Nomenclature Committee) for use in probe design and synthesis.

[0041] 2. Probe design and synthesis: According to the chromosomal location of the exons of all transcripts of the extracted relevant genes, the traversal method is used to design probes. Based on the machine learning algorithm, the probe sequences with similarity higher than 50% are excluded through sequence similarity comparison to ensure that the probe covers more than 99% of the target area. The probe can specifically bind to the target nucleic acid region. The probe synthesis requires separate synthesis, and the single base coupling efficiency is between 99.4% and 99.6%. The synthesized probe is quality controlled by electrospray mass spectrometry to ensure the accuracy of the synthesis. The final synthesized Panel A DNA capture probe includes 2400 probes, covering 99.98% of the target area, and the probe sequence is shown in SEQ ID NO.1~2400; Panel B cDNA capture probe includes 8159 probes, covering 99.99% of the target area, and the probe sequence is shown in SEQ ID NO.2401~10559.

[0042] 3. Performance verification: The synthesized probes are verified for precision, accuracy, analytical sensitivity, and capture efficiency.

[0043] 3.1 Sample preparation:

[0044] a) Positive samples: Digital PCR technology is used for testing in the early stage. Clinical samples containing one or more mutant genes / fusion genes in Table 1 / Table 2 are used as positive original samples. Nucleic acid is extracted and the concentration and purity of the obtained nucleic acid are tested. After meeting the quality control requirements, they are mixed in equal proportions according to the nucleic acid concentration, and the mixed positive specimens are diluted with nucleic acid confirmed to be negative to obtain positive samples.

[0045] b) Cell line samples: Cell line culture samples with a known mutation type. Nucleic acids are extracted according to relevant requirements and stored in gradient dilutions.

[0046] c) Negative samples: samples confirmed to be negative (below the detection limit or zero concentration) by digital PCR technology.

[0047] 3.2 Precision Verification

[0048] 3.2.1 Sample preparation

[0049] Panel A: According to the requirements of precision detection, precision detection was performed in the high mutation rate group and the critical mutation rate group respectively. The high mutation rate group selected SNP sites with a mutation frequency of 50% (including three genes, CYP2C19, ABCB1 and CYP3A5), and the low mutation rate group selected mixed cells with a mutation frequency of 1% (mixed with CYP2C19-positive cells and cells without this mutation).

[0050] Panel B: According to the precision detection requirements, precision detection was performed in the high fusion gene frequency group and the low fusion gene frequency group, where the high fusion gene frequency group and the low fusion gene frequency group were obtained by mixing K562 cells (BCR-ABL positive) and normal human peripheral blood cells in proportion. K562 cells accounted for 30% in the high fusion gene frequency group and 5% in the low fusion gene frequency group.

[0051] 3.2.2 Sequencing

[0052] Panel A test: 100 mutation sites of three genes, CYP2C19, ABCB1 and CYP3A5, were selected for the high mutation rate group and divided into 5 batches, with 20 sites in each batch for detection, and the intra-batch and inter-batch SD values ​​and coefficient of variation (CV) values ​​of the high mutation rate group were calculated respectively; 50 mutation sites with a mutation frequency of 1% after mixing of CYP2C19 were selected for the low mutation rate group and divided into 5 batches, with 10 sites in each batch for detection, and the intra-batch and inter-batch SD values ​​and coefficient of variation (CV) values ​​of the low mutation rate group were calculated respectively.

[0053] Panel B test: There were 50 high fusion gene frequency groups, divided into 5 batches, 10 samples in each batch were tested, and the intra-batch and inter-batch SD values ​​and coefficient of variation (CV) values ​​of the high fusion gene frequency group were calculated respectively; there were 50 low fusion gene frequency groups, divided into 5 batches, 10 samples in each batch were tested, and the intra-batch and inter-batch SD values ​​and coefficient of variation (CV) values ​​of the low fusion gene frequency group were calculated respectively.

[0054] 3.2.3 Sequencing results

[0055] Panel A: In the high mutation rate group, the intra-batch average value was 48.05%, the intra-batch coefficient of variation was 6.56%, the inter-batch average value was 48.01%, and the inter-batch coefficient of variation was 7.44%; in the low mutation rate group, the intra-batch average value was 1.36%, the intra-batch coefficient of variation was 13.42%, the inter-batch average value was 1.13%, and the inter-batch coefficient of variation was 23.23%.

[0056] Panel B: In the high fusion gene frequency group, the intra-batch average value was 12.59%, the intra-batch coefficient of variation was 10.93%, the inter-batch average value was 9.45%, and the inter-batch coefficient of variation was 11.41%; in the low fusion gene frequency group, the intra-batch average value was 2.61%, the intra-batch coefficient of variation was 19.04%, the inter-batch average value was 2.36%, and the inter-batch coefficient of variation was 48.28%.

[0057] 3.3 Accuracy Verification

[0058] 3.3.1 Method for evaluating the accuracy of qualitative determination: Digital PCR and fluorescence quantitative PCR are currently among the most sensitive methods for single-site detection. These two methods were selected for accuracy verification together with this method.

[0059] 3.3.2 Reference method

[0060] Panel A DNA capture probe detection reference method: digital PCR

[0061] Panel B cDNA capture probe detection reference method: Fluorescence quantitative PCR

[0062] 3.3.3 Sample preparation: (1) 50 samples with positive gene mutation (IDH2 c.419G>A and DNMT3Ac.1849G>T mutation) and 50 samples with negative gene mutation; (2) 50 samples with positive fusion gene and 50 samples with negative fusion gene.

[0063] 3.3.4 Detection method: The selected samples were tested by two methods at the same time: Panel A was tested by next-generation sequencing (NGS) and digital PCR, respectively, and Panel B was tested by next-generation sequencing (NGS) and fluorescent quantitative PCR, divided into 5 batches, with 10 samples in each batch.

[0064] 3.3.5 Result analysis and judgment

[0065] The test results are shown in Table 3.

[0066] Table 3 Accuracy test results

[0067]

[0068] As can be seen from Table 3, after comparing with the results of digital PCR and fluorescent quantitative PCR, the sensitivity of gene mutation detection by next-generation sequencing (NGS) was 100% and the specificity was 100%; for fusion gene detection, the sensitivity was 100% and the specificity was 100%.

[0069] 3.4 Analytical sensitivity verification

[0070] Analytical sensitivity is usually verified by serially diluting samples containing known mutations, that is, using the same batch of reagents under the same experimental conditions, repeatedly measuring highly diluted samples, and the results are analyzed statistically.

[0071] 3.4.1 Testing process

[0072] Panel A: Known positive samples (IDH2 c.419G>A and DNMT3Ac.1849G>T mutations) were diluted with known negative samples to mutation frequencies of 0.5%, 1%, and 5%. After dilution, samples of each concentration were tested 10 times according to the detection procedure of routine samples and analyzed according to standard analysis procedures.

[0073] Panel B: K562 cells (BCR-ABL positive) and normal human peripheral blood cells were mixed in proportion, with the proportion of K562 cells being 1%, 5%, and 50%, respectively. Samples of different dilution concentrations were tested 10 times at each concentration according to the routine sample testing procedure, and analyzed according to the standard analysis steps.

[0074] 3.4.2 Test results

[0075] The detection sensitivity (lowest detection limit) of Panel A is 1%, and the detection sensitivity (lowest detection limit) of Panel B is 5%.

[0076] 3.5 Probe capture efficiency quality control

[0077] Probe capture capability verification mainly includes capture efficiency and matching rate. Capture efficiency: For target region capture projects, the proportion of reads that can be matched to the target region to all reads. After testing, the capture efficiency of Panel A and B probes is ≥70%. Matching rate: The proportion of reads that can be matched to the target region. After testing, the matching rate of Panel A probe is ≥99%, and the matching rate of Panel B probe is ≥90%.

[0078] Example 2

[0079] This embodiment provides a method for detecting myeloid hematological tumor-related genes using the capture probe in Example 1. The specific process is as follows:

[0080] The instruments used in this embodiment include: Lad-Aid 824s automatic nucleic acid extractor, Xiamen Zhishan Biotechnology Co., Ltd.; ABI-Veriti nucleic acid amplifier, Thermo Fisher Scientific, USA; ABI-Mini-AMP nucleic acid amplifier, Thermo Fisher Scientific, USA; ABI-Qubit 4.0 nucleic acid quantifier, Thermo Fisher Scientific, USA; Illumina-Nextseq-500 high-throughput sequencer, Illumina, USA.

[0081] In the present invention, instruments with the same functions from other manufacturers, such as nucleic acid extractors, amplifiers, and high-throughput sequencers, can be replaced.

[0082] The reagents used in this example include: library construction reagent xGen TM DNA Library Prep EZ Kit, xGen TM RNA Library Prep Kit was purchased from Shanghai Ade Biotechnology Co., Ltd.; hybridization capture reagent xGenHybridization and Wash Kit was purchased from Shanghai Ade Biotechnology Co., Ltd.; sequencing reagent NextSeq500 / 550High Output Kit 2.5 was purchased from Illumina, USA.

[0083] In the present invention, reagents with the same functions from other manufacturers, such as library construction reagents, hybridization capture reagents, and sequencing reagents, can be replaced.

[0084] 1. Extract samples

[0085] Bone marrow samples were collected from 100 patients diagnosed with myeloid hematological diseases (patients who visited the Hematology Department of Henan Cancer Hospital between 2022 and 2023), and DNA and RNA were extracted from the samples using the Lab-Aid 824s / 808 nucleic acid extractor.

[0086] 2. Library Construction

[0087] 2.1 Panel A library construction

[0088] 2.1.1 DNA shearing

[0089] Add the sample DNA extracted in the previous step to a 0.2mL PCR tube, with a DNA input of 300ng, add water to 18.5μL, and add EDTA solution to a total volume of 19.5μL. Prepare the reaction solution shown in Table 4, melt the reagents on ice, and add the prepared reaction solution to each PCR tube to a total volume of 30μL. Then perform DNA shearing treatment on the PCR tube according to the reaction program shown in Table 5.

[0090] Table 4 Reaction solution system

[0091] Reagents Volume (μL) Buffer K1 3 Reagent K2 1.5 Enzyme K3 6 Total volume 10.5

[0092] Table 5 DNA shearing reaction procedure

[0093]

[0094] Note: If the DNA is from oral mucosal samples, the input amount is adjusted to 25ng and the interruption time is 16min.

[0095] 2.1.2 Connection joints

[0096] Prepare the reaction solution as shown in Table 6, and melt the reagents on ice. After preparation, add the reaction solution to the PCR tube after DNA shearing to make the total volume 60 μL.

[0097] Table 6 Reaction solution

[0098] Reagents Volume (μL) Buffer W1 12 Enzyme W3 4 Raegent W5 5 Low EDTA TE 9 Total volume 30

[0099] Note: If the sample is oral mucosa DNA, the amount of Raegent W5 added needs to be diluted as shown in Table 7.

[0100] Table 7 Relationship between the input amount of oral mucosal sample DNA and the dilution factor of Raegent W5

[0101] DNA input amount Raegent W5 dilution factor ≧25ng No dilution 10ng 10 times 1ng 20 times 100pg 30 times

[0102] The mixed PCR tube was subjected to the adapter connection process according to the reaction program shown in Table 8.

[0103] Table 8 Linker ligation reaction procedure

[0104] Temperature(℃) time 20 20min 4 +∞

[0105] 2.1.3 Purification

[0106] Add 48μL magnetic beads to the PCR tube with the connector connected, shake and mix, place at room temperature for 5 minutes, spin off, place on the magnetic stand for 2 minutes until the liquid is clear, and discard the supernatant. Wash twice with 180μL 80% ethanol (the PCR tube needs to be moved back and forth to move the front and back walls of the magnetic beads several times), spin off, use a 10μL dispenser to absorb the remaining residual ethanol, and dry. Add 22μL Low EDTA TE (0.1mM EDTA), shake and mix, place at room temperature for 5 minutes, spin off, and place on the magnetic stand for 2 minutes until the liquid is clear. Transfer 20μL supernatant to a new PCR tube.

[0107] 2.1.4 PCR amplification

[0108] Add 5 μL of indexing primers and 25 μL of PCR Master Mix to the new PCR tube in 2.1.3, shake and mix, and perform PCR amplification according to the reaction program shown in Table 9.

[0109] Table 9 PCR amplification reaction program

[0110]

[0111] Note: For oral mucosa samples, PCR amplification was performed for 8 cycles.

[0112] 2.1.5 Secondary purification

[0113] Add 90μL magnetic beads to the PCR tube after PCR amplification, shake and mix, incubate at room temperature for 5min, spin off, put on the magnetic stand for 2min, until the solution is clear, and discard the supernatant. Wash twice with 180μL 80% ethanol (the PCR tube needs to be moved back and forth to move the front and back walls of the magnetic beads several times), spin off, use a 10μL spray gun to absorb the remaining residual ethanol, and dry. Add 22μL water, shake and mix, place at room temperature for 5min, spin off, put on the magnetic stand for 2min, until the solution is clear, and transfer 20μL supernatant to a new eight-tube.

[0114] 2.1.6 Quantify the library concentration using Qubit.

[0115] 2.2 Panel B library construction

[0116] The sample for Panel B library construction was RNA, and all library construction operations were performed on ice.

[0117] 2.2.1 RNA shearing

[0118] Detect the RIN value of the RNA of the sample to be tested. The RNA input amount is 500ng, add the RNA to a 0.2mL PCR tube, and add water to 5μL. Prepare the reaction solution as shown in Table 10, add the reaction solution to the PCR tube to make the total volume 14μL. Perform RNA shearing treatment according to the reaction procedure shown in Table 11.

[0119] Table 10 Reaction solution

[0120] Reagents Volume (μL) Reagent F1 1 Reagent F2 2 Reagent F3 4 Reagent F4 2 Total volume 9

[0121] Table 11 RNA shearing reaction procedure

[0122] RIN Value Fragment length (bp) Temperature(℃) Time (min) ≥7 200-250 94 15 2-7 200-250 94 10

[0123] After the program is completed, the PCR tubes were immediately placed on ice for 2 min, followed by the addition of the inversion reagent.

[0124] 2.2.2 Reverse transcription

[0125] The reverse reagents were prepared as shown in Table 12, all of which were melted on ice, and added to the PCR tube after RNA shearing (total volume was 20 μL). Reverse transcription was performed according to the reaction procedure shown in Table 13.

[0126] Table 12 Reversal reagents

[0127] Reagents Volume (μL) Enzyme R1 1 Enzyme R2 1 water 4 Total volume 6

[0128] Table 13 Reverse transcription program

[0129] temperature Time (min) 25 10 42 30 70 15 4 hold

[0130] 2.2.3 Purification

[0131] Add 30μL Low EDTATE to the PCR tube after reverse transcription, with a total volume of 50μL. Add 90μL magnetic beads to each PCR tube, shake and mix, place at room temperature for 5min, spin, place on a magnetic rack for 2min until the liquid is clear, and discard the supernatant. Wash twice with 200μL 80% ethanol, spin, discard the residual ethanol, and dry. Add 22μL Low EDTATE, shake and mix, place at room temperature for 5min, spin, place on a magnetic rack for 2min until the liquid is clear. Transfer 20μL supernatant to a new PCR tube without magnetic beads. Add 36μL magnetic beads to each tube, shake and mix, place at room temperature for 5min, spin, place on a magnetic rack for 2min until the liquid is clear, and discard the supernatant. Wash twice with 200μL 80% ethanol, spin, discard the residual ethanol, and dry. Add 12 μL Low EDTA TE, vortex to mix, place at room temperature for 5 min, centrifuge, place on a magnetic rack for 2 min until the liquid is clear, and transfer 10 μL of supernatant to a new PCR tube.

[0132] 2.2.4 Connecting joints

[0133] Preheat the PCR instrument to 95°C in advance. When the temperature reaches 95°C, put the new PCR tube in 2.2.3 into the PCR instrument and incubate for 2 minutes. After the end, quickly transfer the sample to ice and incubate for 2 minutes. After the end, immediately add the reaction solution prepared in advance (as shown in Table 14), shake and mix, put it into the PCR instrument, and connect the adapter according to the reaction program shown in Table 15.

[0134] Table 14 Reaction solution

[0135]

[0136]

[0137] Table 15 Linker ligation reaction procedure

[0138] Temperature(℃) time 37 15min 95 2min 4 hold

[0139] 2.2.5 PCR amplification

[0140] Add 5 μL of indexing primers and 25 μL of PCR Master Mix to the PCR tube after the adapter connection in 2.2.4, shake and mix, and perform PCR amplification according to the reaction program shown in Table 16.

[0141] Table 16 PCR amplification reaction program

[0142]

[0143] 2.2.6 Post-PCR purification

[0144] Add 40.4μL magnetic beads to the PCR tube after PCR amplification, shake and mix, incubate at room temperature for 5min, centrifuge, place on the magnetic stand for 2min, wait until the solution is clear, and discard the supernatant. Wash twice with 200μL 80% ethanol, centrifuge, discard the residual ethanol, and dry. Add 22μL Low EDTATE, shake and mix, place at room temperature for 5min, centrifuge, place on the magnetic stand for 2min, wait until the solution is clear, and transfer 20μL supernatant to a new eight-tube.

[0145] 2.2.7 Quantify the library concentration using Qubit.

[0146] 3. Hybridization capture (Panel A library and Panel B library are operated separately, but the steps are the same)

[0147] 3.1 Library hybridization

[0148] According to the measured library concentration, the volume is calculated based on the required capture mass of the library of 500ng, and the library is pipetted into a low-adsorption 1.5mL centrifuge tube. Prepare the capture reagent as shown in Table 17, and add the capture reagent to the centrifuge tube containing the library. Seal the centrifuge tube with a sealing film, poke a few holes in the sealing film with a gun tip to facilitate liquid evaporation, and place the centrifuge tube in a vacuum centrifugal concentrator to evaporate to dryness (preheated at 60°C). Be sure to check whether it has been evaporated to dryness at any time.

[0149] Table 17 Capture Reagents

[0150] Reagents Volume / μL Human Cot DNA(IDT) 5 Blockers 2

[0151] 3.2 DNA denaturation and hybridization

[0152] Add the Hybridization Master Mix prepared as shown in Table 18 to the evaporated centrifuge tube. Vortex and mix thoroughly, centrifuge briefly, incubate at room temperature for 5 minutes, vortex and mix again, centrifuge briefly, and incubate at room temperature for 5 minutes.

[0153] Table 18 Hybridization Master Mix

[0154] Reagents Volume / μL xGen 2×Hybridization Buffer 8.5 xGen Hybridization Buffer Enhancer 2.7 Panel A / Panel B probe of Example 1 4 water 1.8

[0155] The incubated liquid was transferred to a PCR tube, placed in a PCR instrument, and hybridization reaction was performed according to the program shown in Table 19.

[0156] Table 19 Hybridization reaction program

[0157]

[0158] 3.3 Post-hybridization purification

[0159] 3.3.1 Preparation of Wash Buffer

[0160] The preparation method of the Wash Buffer working solution required during the purification process is shown in Table 20.

[0161] Table 20 Wash Buffer working solution stock solution and preparation method

[0162]

[0163]

[0164] Aliquot the reagents that need to be used at 65°C in the above table and incubate and store at 65°C.

[0165] 3.3.2 Purification

[0166] 3.3.2.1 Cleaning of Streptavidin Magnetic Beads

[0167] Place 50μL Capture beads in the eight-row tube, add 100μL 1×Bead Wash Buffer and shake to mix. Place on the magnetic rack for 1 minute until the liquid is clear, and discard the supernatant. Add 100μL 1×Bead Wash Buffer and shake to mix. Place on the magnetic rack for 1 minute until the liquid is clear, and discard the supernatant. Add 100μL 1×Bead Wash Buffer and shake to mix. Place on the magnetic rack for 1 minute until the liquid is clear, and discard the supernatant. Remove the eight-row tube from the magnetic rack, centrifuge briefly, place on the magnetic rack, and use a 10μL pipette tip to thoroughly discard the residual liquid at the bottom of the centrifuge tube. Add the BeadResuspension Mix prepared according to Table 21 to the washed magnetic beads.

[0168] Table 21 Bead Resuspension Mix

[0169] Reagents Volume / μL xGen 2×Hybridization Buffer 8.5 xGen Hybridization Buffer Enhancer 2.7 Nuclease-Free Water 5.8

[0170] 3.3.2.2 Hot washing

[0171] Transfer the hybridization solution captured overnight to the Bead Resuspension Mix with magnetic beads after incubation at 65℃, and mix well by pipetting. Place in the PCR instrument and incubate at 65℃ for 45min, and use a gun to blow every once in a while to ensure that the magnetic beads are suspended. The time intervals are 11min, 11min, 11min and 12min. The timer needs to be paused during this period. After the incubation is completed, transfer the liquid in the PCR tube to the eight-row tube, add 100μL of 1×Wash Buffer 1 preheated at 65℃, and mix well by pipetting. Place on the magnetic stand for 1min until the liquid is clear, and discard the supernatant. Remove the eight-row tube from the magnetic stand, quickly centrifuge briefly (to prevent the temperature from dropping too much), place it on the magnetic stand, and use a 10μL pipette tip to thoroughly discard the residual liquid at the bottom of the centrifuge tube. Add 150μL of 1× Stringent Wash Buffer preheated at 65℃, mix by pipetting, incubate at 65℃ for 5min, place on the magnetic stand for 1min until the liquid is clear, and discard the supernatant. Add 150μL of 1× Stringent Wash Buffer preheated at 65℃, mix by pipetting, incubate at 65℃ for 5min, place on the magnetic stand for 1min until the liquid is clear, and discard the supernatant. Remove the eight-row tube from the magnetic stand, centrifuge briefly, place on the magnetic stand, and use a 10μL pipette tip to thoroughly discard the residual liquid at the bottom of the centrifuge tube.

[0172] 3.3.2.3 Cleaning at room temperature

[0173] Add 150μL of 1×Wash Buffer 1 at room temperature, oscillate for 30s, let stand for 30s, oscillate again for 30s, let stand for 30s (2min in total), centrifuge briefly, place on a magnetic stand for 1min until the liquid is clear, and discard the supernatant. Add 150μL of 1×Wash Buffer 2 at room temperature, oscillate for 30s, let stand for 30s, oscillate again for 30s, let stand for 30s (2min in total), centrifuge briefly, place on a magnetic stand for 1min until the liquid is clear, and discard the supernatant. Add 150μL of 1×Wash Buffer 3 at room temperature, oscillate for 30s, let stand for 30s, oscillate again for 30s, let stand for 30s (2min in total), centrifuge briefly, place on a magnetic stand for 1min until the liquid is clear, and discard the supernatant. Remove the eight-row tube from the magnetic stand, centrifuge briefly, place on a magnetic stand, and use a 10μL pipette tip to thoroughly discard the residual liquid at the bottom of the centrifuge tube. Add 20 μL of ultrapure water to the centrifuge tube for elution, shake and mix, and proceed to the next amplification test.

[0174] 3.3.3 Post-LM-PCR

[0175] Prepare Post-LM-PCR Mix as shown in Table 22, shake and mix, centrifuge briefly, and dispense into PCR tubes, 30 μL per tube. Place in a PCR instrument and perform PCR reaction according to the program shown in Table 23.

[0176] Table 22 Post-LM-PCR Mix

[0177] Reagents Volume / μL KAPA HiFi HotStart ReadyMix 25 xGen Library Amplification Primer Mix 1.25 water 3.75 DNA eluted in the previous step 20

[0178] Table 23 Reaction procedure

[0179]

[0180] The cycle number is adjusted according to the panel size and the number of samples for hybridization, as shown in Table 24.

[0181] Table 24 Relationship between cycle number, panel size and number of samples for hybridization

[0182] Panel size 1 Miscellaneous 4 Miscellaneous 8 Miscellaneous 12 Miscellaneous Nucleic Acids 10000-100000probes 12cycles 10 cycles 9 cycles 8 cycles RNA 500-10000 13 cycles 11 cycles 10 cycles 10 cycles DNA

[0183] 3.3.4 Post-amplification purification

[0184] Place the amplified captured DNA library on the magnetic rack, take 75μL (1.5×) purified magnetic beads into a new eight-tube strip, add 50μL of the amplified captured DNA library supernatant, shake and mix, and incubate at room temperature for 10min. Centrifuge briefly, place on the magnetic rack for 1min until the liquid is clear, and discard the supernatant. Remove the eight-tube strip from the magnetic rack, centrifuge briefly, place on the magnetic rack, and use a 10μL pipette to completely discard the residual liquid at the bottom of the centrifuge tube. Add 200μL 80% ethanol, let it stand for 30s and discard it, and repeat once. Remove the eight-tube strip from the magnetic rack, centrifuge briefly, place on the magnetic rack, use a 10μL pipette to completely discard the residual liquid at the bottom of the centrifuge tube, and dry at room temperature until the ethanol is completely evaporated. Remove the centrifuge tube from the magnetic rack, add 22μL Low TE, and shake and mix. Incubate at room temperature for 2min. Centrifuge briefly and place on a magnetic stand for 1 min until the liquid is clear. Transfer 20 μL of supernatant to a new 1.5 ml centrifuge tube to obtain the hybridization captured library.

[0185] 3.3.5 Use Qubit to quantify the concentration of the library after hybridization capture.

[0186] 4. Next-generation sequencing (NGS)

[0187] 4.1 Turn on the Illumina Next-seq 500 and wait for a few minutes for the power-on initialization to complete. The system will automatically open the Nextseq500 Control Software. Clean the instrument as required.

[0188] 4.2 Library preparation before loading, library denaturation and dilution

[0189] The libraries were diluted to 1 ng / μL. According to "1×number of samples / concentration=mixed library volume V", the volume of each hybrid library mixed in one tube was determined. Qubit measured several concentrations and took the average value, which was the final library concentration C. Take 5μL of the final library + 5μL0.2M NaOH and mix them, let them stand for 5 minutes for denaturation, add 5μL of ultrapure water and mix them, stop the denaturation, and add 985μLHT1 Buffer to the denaturation tube to make up the volume to 1mL. Prepare a 1.5mL centrifuge tube, marked as 1300, and calculate according to the formula "library sampling volume after denaturation X: X=1.5PM×1300×350×660 / (5×100000×final library concentration C)", draw XμL from the denaturation tube to the EP tube marked 1300, add HT1 Buffer to make up the total volume to 1300μL, shake and mix, and centrifuge to obtain the final library on the machine.

[0190] 4.3 Sequencing

[0191] Follow the instrument instructions and fill in various parameters as required. Click Next to enter the self-test interface and wait for the self-test to complete. Click Star Run to start sequencing.

[0192] 5. Bioinformatics analysis of offline data

[0193] Panel A library sequencing result analysis process: Using the FastQ file from the Illumina sequencing platform as input, the final mutation results and data quality control information are obtained through data filtering, quality control, alignment, mutation detection, copy number analysis, result annotation and other steps.

[0194] Panel B library sequencing result analysis process: Using FastQ file as input, the final fusion gene and gene expression results as well as data quality control information are obtained through data filtering, quality control, alignment, fusion detection, expression analysis, and result annotation.

[0195] The specific process of data filtering is as follows: filter out adapter sequences and low-quality sequences through fastp software, including quality filtering: base quality value ≥15, the percentage of unqualified bases in a single read does not exceed 40%; length filtering: the minimum length of read is 15bp; complexity filtering: read complexity ≥30, complexity is defined as the proportion of bases that are different from one base and its next adjacent base; adapter filtering: automatically identify adapter sequences and remove adapters; quality cutting of each read: use a sliding window to evaluate the average quality value of the 5' and 3' ends of each read. When the average quality value is lower than 15, cut off the window and all bases behind it; base correction: correct the mismatched bases in the overlapping area. If one base has high quality and the other has low quality, replace the low-quality base with a high-quality base; ployG / ployX tail trimming: remove the ployG and ployX tails at the 3' end.

[0196] The specific process of quality control is as follows: the filtered sequences are quality controlled by samtools and bamdst, and the alignment rate, repetition rate, total coverage, Q20\Q30 ratio, and probe capture efficiency are calculated respectively. Among them:

[0197] (1) Alignment rate: Targeted capture library construction alignment rate: indicates the ratio of sequencing data that successfully matches the reference genome. The calculation method is as follows:

[0198] Alignment rate = (number of reads successfully aligned to the reference genome / total sequencing reads) × 100%

[0199] (2) Repeatability: It indicates the proportion of reads with the same sequence in the sequencing data. It is usually considered that this part of the sequence is generated by PCR and should be removed in the analysis. The calculation method is as follows:

[0200] Repeat rate = (number of repeated reads / total number of sequencing reads) × 100%

[0201] Interpretation of duplication rate: What proportion of reads in the sequencing data are caused by technical duplication or PCR amplification.

[0202] (3) Total coverage: refers to the proportion of the target area that can be covered. The calculation method is as follows:

[0203] Total coverage = number of bases detected in the target region / total number of bases in the target region

[0204] Total coverage explained: How comprehensive is the sequencing data for the target region. Higher total coverage generally indicates more comprehensive data.

[0205] (4) Q20 and Q30: Quality score indicators used to measure the quality of each base. The quality score is based on the signal quality measured during the base sequencing process and is usually expressed as a Phred quality score. Q20 represents the percentage of bases with a Phred quality score greater than or equal to 20, representing a base error rate of less than 1%. Q30 represents the percentage of bases with a Phred quality score greater than or equal to 30, representing a base error rate of less than 0.1%. The calculation method is as follows:

[0206] Q20 percentage = (number of bases with a mass score of Q20 or above / total number of bases) × 100%

[0207] Q30 percentage = (number of bases with a mass fraction of Q30 or above / total number of bases) × 100%

[0208] (5) Probe capture efficiency: refers to the ratio of the gene sequences that can be detected to all gene sequences. The calculation method is as follows:

[0209] Probe capture efficiency = (number of reads successfully aligned to the target region / total sequencing reads) × 100%

[0210] Explanation of probe capture efficiency: The higher the capture efficiency, the higher the capture specificity of the probe.

[0211] Panel A uses BWA to align the filtered reads with the human reference genome hg19, and uses sambamba to remove multiple alignments, PCR duplications, and unaligned sequences from the generated bam files. The GATK software package is used for local realignment, base quality score recalibration, and variation detection. The annotation of germline mutations and somatic mutations is achieved through comparative analysis of oral mucosa samples and bone marrow samples. The cnvkit software package is used to perform copy number variation analysis through comparative analysis of oral mucosa samples and bone marrow samples to obtain the copy number value of each target gene, determine the copy number difference of bone marrow samples compared with oral mucosa samples, and obtain the final CNV results; manta is used to detect insertions, deletions, inversions, tandem duplications, and chromosomal translocations.

[0212] Panel B uses SATR to align the filtered reads with the human reference genome hg38. The bam files generated by the alignment are used for fusion gene detection using three softwares: arriba, STAR-Fusion, and Fusioncatcher. The result interpretation principle is:

[0213] Under the premise of qualified QC, in general, if the number of positive reads of the detected fusion gene is ≤3, it is a false positive, and if it is >3, it is a true positive;

[0214] Note: a) When the genes forming the fusion are all in the panel, the number of positive reads = split_reads1 + split_reads2;

[0215] b) When only one of the genes forming the fusion is in the panel, the number of positive reads = split_reads1 or 2 (gene in the panel);

[0216] If more than 2 of the 3 softwares detected the gene and the number of reads was ≥1, or if 1 of the 3 softwares detected the gene and the number of reads was >3, the gene should be verified by fluorescence quantitative PCR.

[0217] When an unknown fusion is detected and the number of detected fusion reads is >3, it needs to be verified by fluorescent quantitative PCR.

[0218] Use featureCounts to extract the counts data of each gene in the comparison results of Panel B, and analyze according to the basic analysis process: count the gene length of each gene, calculate the normalization factor according to the formula = (number of gene counts / gene length) × 1,000,000, and then calculate the expression of each gene TPM = (normalization factor / sum of normalization factors of all genes) × 1,000,000. The high expression threshold of the gene is calculated as the median of the expression of each gene in 300 samples + 3 × standard deviation.

[0219] 6. Test results 6.1

[0221] The test results of 100 samples of panel A are shown in Table 25

[0222] Table 25 Panel A DNA capture probe test results for 100 samples

[0223]

[0224] The test results of 100 samples of panel B are shown in Table 26

[0225] Table 26 Panel B cDNA capture probe test results for 100 samples

[0226]

[0227] 6.2 Germline mutation verification: When performing panel A testing, oral mucosa samples and bone marrow samples are sent for testing at the same time. Through comparison, it can be determined whether the detected mutation site is a germline mutation.

[0228] Panel A testing and result data analysis were performed on 112 patients with myeloid hematologic malignancies who submitted bone marrow samples and oral mucosa for testing between December 2022 and September 2023 (according to steps 1 to 5 above). The results are as follows: Figure 1 As shown, after verification, the part with a system mutation and a VAF value greater than 40% (the green area in the figure) accounts for 14% (62 sites). If there is no oral mucosa comparison, there is a possibility of misjudging it as a germline mutation; after verification, the part with a germline mutation and no RS number (the orange area in the figure) accounts for 42% (169 sites). There are no relevant literature reports and database records for this part of the sites. If there is no oral mucosa comparison, there is a possibility of misjudging it as a system mutation. It can be seen that compared with conventional analysis methods, the germline mutation verification added by panel A can clarify the mutation source of more than 50% of patients.

[0229] 6.3 Detection rate

[0230] According to the above steps 1 to 5, bone marrow samples of 84 patients with acute myeloid leukemia (patients from the Department of Hematology of Henan Cancer Hospital between 2021 and 2023) were tested and analyzed. At the same time, the conventional 56 fusion gene screening methods were used to test and analyze the samples of the above patients. The positive detection rates of the two methods were compared.

[0231] After testing, the positive detection rate of fusion genes by the method of the present invention was 42.9% (36 cases). The positive detection rate of 56 fusion gene screening methods for the same patient samples was 25%. The method of the present invention increased by about 17.9% compared with the conventional method, and detected 12 rare fusions (no reports or less than 10 reports), as shown in Table 27.

[0232] Table 27 Fusion genes not detected by conventional methods

[0233] RUNX1-CUBN KMT2A-ACACA RUNX1-MECOM ETV6-ABL1 SATB1-PDGFRB MSI2-HLF MSI2-SMG6 KMT2A-ACACA RUNX1-EML6 MLLT10-UBE4A ETV6-TBL1XR1 UBE4A-KMT2A

[0234] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A capture probe for a myeloid hematological tumor detection gene panel, characterized in that: The capture probes include panel A DNA capture probes and panel B cDNA capture probes; the sequence of the panel A DNA capture probes is the nucleotide sequence shown in SEQ ID NOs. 1 to 2400, and the sequence of the panel B cDNA capture probes is the nucleotide sequence shown in SEQ ID NOs. 2401 to 10559.

2. Use of the capture probe according to claim 1 in preparing a detection reagent or kit for myeloid hematological tumor genes.

3. A kit comprising a capture probe for the myeloid hematological neoplasm detection gene panel according to claim 1.

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