Probe library and method for detecting systemic mastocytosis gene mutation

Abstract

The invention belongs to the technical field of gene detection, and particularly relates to a probe library and a method for detecting systemic mastocytosis gene mutation, the probe library completely covers probe combinations of gene sequences corresponding to a target area, and the gene mutation sequences comprise exon probe combinations for detecting ASXL1, ATM, CBL, DNMT3A, ETV6, EZH2, IDH1, IDH2, JAK2, KIT, KRAS, NRAS, RUNX1, SETBP1, SF3B1, SMC3, SRSF2, TET2, TP53 and U2AF1; the probe library can be used for simultaneously detecting the multi-gene multi-site mutation of the systemic mastocytosis, and the detection method can be used for comprehensively screening the mutation of a plurality of genes related to the diagnosis and treatment of the systemic mastocytosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene detection, and in particular relates to a probe library and a method for detecting gene mutation of systemic mastocytosis. Background Art

[0002] Systemic mastocytosis (SM) is the clonal proliferation of invasive mast cells in one or more tissues, organs or systems outside the dermis, which release a large amount of vasoactive mediators and cause multi-organ dysfunction. Common sites of involvement include bone marrow, skin, spleen, lymph nodes, liver and / or mononuclear-macrophage system.

[0003] KIT gain-of-function mutations are present in approximately 90% of SM patients. The proto-oncogene KIT is a mast cell growth factor receptor, a transmembrane tyrosine kinase receptor. KIT mutations lead to KIT phosphorylation and enzyme activation, thereby promoting cell proliferation and resisting apoptosis. KIT p.D816V mutations are found in more than 80% of adult SM patients and more than 90% of indolent SM patients. Other KIT mutations are common in patients with advanced SM, mast cell leukemia, or mast cell sarcoma. These variants are usually present in the juxtamembrane domain (such as KITp.V560G) or the extracellular domain (such as KIT p.Del419). In addition, in addition to KIT gene mutations, SM can also have mutations in TET2, SRSF2, ASXL1, RUNX1, and RAS pathway-related genes, especially in mastocytosis with related hematological tumors (SM-AHN).

[0004] Accurate identification of these gene changes is of great significance for the clinical treatment of SM. The KIT / PDGFRA inhibitor afatinib has been approved by the FDA for the treatment of SM patients. In addition, gene mutations such as SRSF2, ASXL1, RUNX1, EZH2 and SETD2 may affect the prognosis of SM, especially in aggressive SM.

[0005] Because more than 80% of SM patients have KIT mutations, most of the tests for SM on the market are currently for the mutation site detection of the KIT single gene. However, the WHO and NCCN guidelines clearly state that on the basis of the KIT single gene mutation, multi-gene multi-site mutation detection such as SRSF2, ASXL1, RUNX1, EZH2 and SETD2 should also be tested. Therefore, how to provide a product and method for detecting multi-gene mutations related to systemic mastocytosis, which can effectively overcome the shortcomings of only detecting single gene mutations, save time and improve detection sensitivity, has become an urgent problem to be solved. Summary of the invention

[0006] The object of the present invention is to provide a probe library and method for detecting gene mutation of systemic mastocytosis.

[0007] Based on the above purpose, the present invention adopts the following technical solutions:

[0008] A probe library for detecting gene mutations in systemic mastocytosis, wherein the probe library completely covers probe combinations of gene sequences corresponding to target regions, and the gene mutation sequences include exon probe combinations for detecting ASXL1, ATM, CBL, DNMT3A, ETV6, EZH2, IDH1, IDH2, JAK2, KIT, KRAS, NRAS, RUNX1, SETBP1, SF3B1, SMC3, SRSF2, TET2, TP53, and U2AF1;

[0009] A probe combination for detecting the splicing sites of ASXL1, ATM, CBL, DNMT3A, ETV6, EZH2, IDH1, IDH2, JAK2, KIT, KRAS, NRAS, RUNX1, SETBP1, SF3B1, SMC3, SRSF2, TET2, TP53, and U2AF1; the corresponding probe sequences are SEQ ID No. 1-700.

[0010] A method for synthesizing probes in the probe library is composed of the following steps:

[0011] (1) Nucleic acid extraction: extracting DNA from bone marrow samples;

[0012] (2) Prelibrary construction: 900-1100 ng of sample DNA was sheared into fragments with a peak length of 150-250 bp, and then 200-400 ng of DNA fragments were taken for end repair and A addition. The repaired and A-added DNA fragments were then adapter-ligated and purified using magnetic beads. The purified adapter-ligated products were then amplified to obtain a prelibrary.

[0013] (3) Probe synthesis: The probe design for the target gene when detecting DNA mutations is based on the DNA sequence; the probe is mainly synthesized using a solid phase column method, and a biotin label is added to the 5' end, and then the salt is removed by desalting and purification to finally obtain a high-quality probe;

[0014] (4) The probes obtained in step (3) are then subjected to hybridization capture, eluted and purified to obtain a hybridization capture library of the probes.

[0015] (5) Data analysis: Sequence the library captured by hybridization in step (4), and use Fastp software to process the Fastq data after the library sequencing, mainly for data quality control, linker removal and quality screening, and filter the original Fastq data to generate high-quality Fastq;

[0016] Furthermore, in the step (3), the transcript used in the probe synthesis is selected from the transcript used in the ClinVar database, the length of the probe is 100-150 bp, the probe covers the target gene region in a head-to-tail form, and the GC content of the probe sequence is 35% to 70%. The designed probe sequence is synthesized by Nanoda (Nanjing) Biotechnology Co., Ltd.

[0017] Furthermore, the length of the probe is preferably 120 bp.

[0018] A method for detecting gene mutations of systemic mastocytosis using a probe library in use, comprising the following steps: using BWA software to align a quality-controlled probe hybridization capture library with a human genome (version hg19), using gatk software to remove duplicate DNA fragments, using the software Lianti to perform variant site analysis on the BAM obtained by the above alignment and deduplication, and obtaining single base substitution SNVs and short sequence insertions or deletions InDels, respectively, and finally using the software annovar and SnpEff to annotate the SNV / InDel; filtering the analyzed mutations according to a preset mutation frequency and mutation support reads number threshold to obtain a final mutation list;

[0019] A kit for detecting systemic mastocytosis gene mutations using the above-mentioned probe library, the kit comprising a pre-library construction reagent and a hybridization capture reagent, the pre-library construction reagent comprising an end-repair plus A reagent, a linker connection reagent, a pre-library amplification reagent, and purification magnetic beads, and the hybridization capture reagent comprising a blocking agent, a probe composition, a rapid hybridization premix, a hybridization enhancer, an elution buffer, and streptavidin magnetic beads.

[0020] The present invention provides a probe library for detecting gene mutations of systemic mastocytosis, a method for synthesizing probes in the probe library, and a method for detecting gene mutations of systemic mastocytosis using the probe library. The probe library can simultaneously detect multi-gene and multi-site mutations of systemic mastocytosis, and the detection method can perform all-round screening of multiple gene mutations related to the diagnosis and treatment of systemic mastocytosis. The present invention also designs a kit for detecting gene mutations of systemic mastocytosis, which is easy to use and has accurate detection results. Thus, a basis is provided for corresponding clinical treatment, and the method has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The probe sequences corresponding to ASXL1 are SEQ ID No. 1-49;

[0022] Figure 2 The probe sequences corresponding to ATM are SEQ ID No. 50-177;

[0023] Figure 3 The probe sequences corresponding to CBL are SEQ ID No. 178-210;

[0024] Figure 4 The probe sequences corresponding to DNMT3A are SEQ ID No. 211-247;

[0025] Figure 5 The probe sequences corresponding to ETV6 are SEQ ID No. 248-265;

[0026] Figure 6 The probe sequences corresponding to EZH2 are SEQ ID No. 266-299;

[0027] Figure 7 The probe sequences corresponding to IDH1 are SEQ ID No. 300-316;

[0028] Figure 8 The probe sequences corresponding to IDH2 are SEQ ID No. 317-335;

[0029] Fig. 9 The probe sequences corresponding to JAK2 are SEQ ID No. 336-383;

[0030] Fig.10 The probe sequences corresponding to KIT are SEQ ID No. 384-422;

[0031] Fig.11 The probe sequences corresponding to KRAS are SEQ ID No. 423-430;

[0032] Fig.12 The probe sequences corresponding to NRAS are SEQ ID No. 431-438;

[0033] Fig.13 The probe sequences corresponding to RUNX1 are SEQ ID No. 439-457;

[0034] Fig.14 The probe sequences corresponding to SETBP1 are SEQ ID No. 458-500;

[0035] Fig.15The probe sequences corresponding to SF3B1 are SEQ ID No. 501-552;

[0036] Fig.16 The probe sequences corresponding to SMC3 are SEQ ID No. 553-609;

[0037] Fig.17 The probe sequences corresponding to SRSF2 are SEQ ID No. 610-616;

[0038] Fig.18 The probe sequences corresponding to TET2 are SEQ ID No. 617-673;

[0039] Fig.19 The probe sequences corresponding to TP53 are SEQ ID No. 674-690;

[0040] Fig. 20 The probe sequences corresponding to U2AF1 are SEQ ID No. 691-700;

[0041] Fig.21 The flowchart of the method for detecting systemic mastocytosis gene mutation using the probe library of the present invention;

[0042] Fig. 22 Schematic diagram of the gene mutation site IGV. DETAILED DESCRIPTION

[0043] Embodiment 1:

[0044] A probe library for detecting gene mutations in systemic mastocytosis, characterized in that the probe library completely covers probe combinations of gene sequences corresponding to target regions, and the gene mutation sequences include exon probe combinations for detecting ASXL1, ATM, CBL, DNMT3A, ETV6, EZH2, IDH1, IDH2, JAK2, KIT, KRAS, NRAS, RUNX1, SETBP1, SF3B1, SMC3, SRSF2, TET2, TP53, and U2AF1;

[0045] like Figure 1-20As shown, a probe combination for detecting the splicing sites of ASXL1, ATM, CBL, DNMT3A, ETV6, EZH2, IDH1, IDH2, JAK2, KIT, KRAS, NRAS, RUNX1, SETBP1, SF3B1, SMC3, SRSF2, TET2, TP53, and U2AF1; the corresponding probe sequences are SEQ ID No. 1-700. The probe sequences corresponding to ASXL1 are SEQ ID No.1-49; the probe sequences corresponding to ATM are SEQ ID No.50-177; the probe sequences corresponding to CBL are SEQ ID No.178-210; the probe sequences corresponding to DNMT3A are SEQ ID No.211-247; the probe sequences corresponding to ETV6 are SEQ ID No.248-265; the probe sequences corresponding to EZH2 are SEQ ID No.266-299; the probe sequences corresponding to IDH1 are SEQ ID No.300-316; the probe sequences corresponding to IDH2 are SEQ ID No.317-335; the probe sequences corresponding to JAK2 are SEQ ID No.336-383; the probe sequences corresponding to KIT are SEQ ID No.384-422; the probe sequences corresponding to KRAS are SEQ ID No.423-430; the probe sequences corresponding to NRAS are SEQ ID No.431-438; the probe sequences corresponding to RUNX1 are SEQ ID No.439-457; the probe sequence corresponding to SETBP1 is SEQ ID No.458-500; the probe sequence corresponding to SF3B1 is SEQ ID No.501-552; the probe sequence corresponding to SMC3 is SEQ ID No.553-609; the probe sequence corresponding to SRSF2 is SEQ ID No.610-616; the probe sequence corresponding to TET2 is SEQ ID No.617-673; the probe sequence corresponding to TP53 is SEQ ID No.674-690; the probe sequence corresponding to U2AF1 is SEQ ID No.691-700.

[0046] A kit for detecting systemic mastocytosis gene mutations using the probe library.

[0047] The kit includes pre-library construction reagents and hybrid capture reagents. The pre-library construction reagents include end-repair plus A reagents, linker connection reagents, pre-library amplification reagents, and purification magnetic beads. The hybrid capture reagents include blocking agents, probe compositions, rapid hybridization premixes, hybridization enhancers, elution buffers, and streptavidin magnetic beads. The specific components are shown in Table 1 below:

[0048] Table 1

[0049]

[0050] A method for synthesizing the probe in the above-mentioned probe library

[0051] Probe synthesis was performed using bone marrow samples from 10 patients with systemic mastocytosis, including the following steps:

[0052] Nucleic acid extraction:

[0053] 1. DNA: DNA was extracted from bone marrow samples using QIAGEN's QIAamp DNA Micro Kit, and the nucleic acid concentration of the samples was measured using Thermo Life Qubit 4.0. The results are shown in Table 2 below:

[0054] Sample name DNA concentration (ng / μL) 1000ngDNA volume (μL) Sample 1 254.8 3.9 Sample 2 217.0 4.6 Sample 3 231.0 4.3 Sample 4 103.5 9.7 Sample 5 190.5 5.2 Sample 6 153.6 6.5 Sample 7 113.9 8.8 Sample 8 245.0 4.1 Sample 9 321.6 3.1 Sample 10 198.0 5.1

[0055] Prelibrary construction:

[0056] 1. DNA fragmentation: Using a Covaris M220 ultrasonic disruptor, 900-1100 ng of sample DNA was fragmented into fragments with a peak length of 150-250 bp. The ultrasonic system was configured according to Table 3 below, and ultrasonication was performed according to the parameters listed in Table 4;

[0057] Table 3 DNA ultrasonic system

[0058] Components Volume (μL) DNA (1000ng) X (X = 1000 ng / DNA concentration) Nucleic acid-free water 120-X Total 120

[0059] Table 4 DNA fragmentation ultrasound parameters

[0060] parameter condition Peak Incident Power (W) 50 Duty Factor (%) 20 CyclesperBurst(cpb) 200 TreatmentTime(sec) 170

[0061] 2. End repair plus A: Take 40 μL (≥200 ng DNA) of the DNA fragmentation product in step 2, add 10 μL of the end repair mixture, use a pipette to gently blow or shake to mix, and briefly centrifuge the reaction solution to the bottom of the tube. Set the program: 20℃, 30min, 65℃, 30min, maintain at 4℃, and heat the cover at 75℃ for reaction.

[0062] Table 5 End repair plus A system

[0063] Components Volume (μL) Fragmented DNA 40 EndRepair&A-TailingBuffer 6 EndRepair&A-TailingEnzymeMix 4

[0064] 3. Connector connection: Thaw the reagents in Table 6, mix them by inversion, and place them on ice for later use. Prepare the connector connection system according to Table 6, use a pipette to gently blow or shake to mix, and centrifuge briefly to collect the reaction solution to the bottom of the tube. Place the PCR tube in the PCR instrument, close the hot cover, set to 20℃ for 15min, and maintain at 4℃.

[0065] Table 6 Connector connection system

[0066] Components Volume (μL) Step 3 Product 50 LigationBuffer 26 DNALigase 2 NadPrepUMIAdapter(15μM) 2

[0067] 4. Purification of Ligation Products

[0068] (1) Take the magnetic beads out of the 4°C refrigerator in advance, vortex mix, and leave at room temperature for 30 min;

[0069] (2) Mix the magnetic beads by shaking, take 40 μL of the magnetic beads and add them to the above product, use a pipette to gently blow and repeatedly 10 times to mix them thoroughly, and incubate at room temperature for 10 minutes;

[0070] (3) Place the centrifuge tube on the magnetic rack for 5 min. After the solution becomes clear, discard the supernatant and retain the magnetic beads.

[0071] (4) Keep the centrifuge tube on the magnetic rack, add 185 μL of freshly prepared 80% ethanol to the reaction tube, let it stand for 30 seconds, then discard the supernatant and retain the magnetic beads;

[0072] (5) Repeat the above steps once;

[0073] (6) Remove as much residual ethanol as possible from the centrifuge tube and place it on a magnetic rack for 5 min to dry until it turns matte;

[0074] (7) Add 22.6 μL of nuclease-free water to each sample, mix well by pipetting, centrifuge briefly, and incubate at room temperature for 2 min;

[0075] (8) Place the centrifuge tube on a magnetic rack and wait for the solution to clarify;

[0076] (9) Carefully pipette 20 μL of supernatant into a new PCR reaction tube for later use;

[0077] 5. Library amplification: Thaw the reagents in Table 7, mix them by inversion, place them on ice for later use, and prepare the reaction system. Use a pipette to gently blow or oscillate to mix, and centrifuge briefly to collect the reaction solution at the bottom of the tube. Place the PCR tube in the PCR instrument, set the reaction program shown in Table 9, heat the lid at 105°C, and perform library amplification.

[0078] Table 7 Library amplification system

[0079] Components Volume (μL) Step 5 Product 20 2XHiFiPCRMasterMix 25 AmplificationPrimerMixII 5

[0080] Table 8 PCR amplification reaction program

[0081]

[0082] Table 9 Amplification cycle number reference table

[0083] Fragmented DNA Input Amount 1000ng library yield >5ng 7 1-5ng 10-12 <1ng 13-14

[0084] Purification of amplified products: The amplified products were purified using the procedure in step 5, wherein 50 μL of magnetic beads were added.

[0085] Probe synthesis:

[0086] 1. Design probes according to the exon regions of the above genes, select transcripts used in the ClinVar database, the length of the probe is 100-150bp, preferably 120bp; the probe covers the target gene region in a head-to-tail form, the GC content of the probe sequence is 35% to 70%, and is provided to Nanoda (Nanjing) Biotechnology Co., Ltd. for synthesis.

[0087] 2. The probes were synthesized using a solid phase column method and a biotin label was added to the 5' end. Each probe was synthesized individually.

[0088] 3. After the probe is synthesized, desalting and purification are used to effectively remove salt.

[0089] 4. Quantify each individually synthesized probe and perform mass spectrometry detection simultaneously.

[0090] 5. Combine each probe that passes the quality inspection into a tube and concentrate it.

[0091] Hybridization capture library of probes:

[0092] 1. Take 500 ng of the pre-library in the pre-library construction process and mix them. Add 2 μL NadPrep NanoBlockers and 5 μL Human cot DNA to the mixed pre-library and evaporate them to dryness in a vacuum concentrator at 45°C.

[0093] 2. Mix 8.5 μL Hyb#1, 2.7 μL Hyb#2, 1.8 μL Nuclease-Free Water and 4 μL of the probe composition of the present invention, vortex to mix, add to the evaporated sample, flick to mix, and let stand at room temperature for 5 min.

[0094] 3. Transfer the mixed reaction solution to a PCR tube and place it in a PCR instrument for hybridization. The PCR instrument program is set to 105℃ hot cover, 95℃ for 5min, 65℃ for 4-16h, and 65℃ insulation; take out the streptavidin magnetic beads, equilibrate at room temperature for more than 30min, and shake to mix.

[0095] 4. Dissolve Bead Wash Buffer, Wash Buffer 1, Wash Buffer 2, Wash Buffer 3 and Wash Buffer 4 at room temperature, mix well, and centrifuge for standby use;

[0096] 5. Preheating of reagents: Dispense Wash Buffer 1110μL and Wash Buffer 2160μL, and preheat them at 65℃ for at least 15min;

[0097] 6. Mix the streptavidin magnetic beads thoroughly, take 50μL to a 1.5mL centrifuge tube, add 100μL Bead WashBuffer, mix with a pipette tip, place on a magnetic stand until clear, and discard the supernatant; repeat adding and discarding the supernatant twice, and completely remove the supernatant for the third time, and remove the residue with a 10ul pipette tip;

[0098] 7. Mix 8.5 μL Hyb#1, 2.7 μL Hyb#2 and 5.8 μL Nuclease-Free Water, add to the washed magnetic beads, and then incubate at 65°C for 5 min.

[0099] 8. After hybridization, add 17 μL of magnetic bead resuspension to the PCR reaction tube in step 3, gently blow and mix 8 times, keep incubating at 65℃ for 45 minutes, blow and mix once every 10 minutes. After 45 minutes of incubation, remove the PCR tube from the PCR instrument and immediately wash at 65℃;

[0100] 9. Pipette 100ul 65℃ Wash Buffer 1 (WB1) into the PCR reaction tube, pipette and beat 10 times, transfer all the liquid into a new low-adsorption tube, centrifuge for a moment and place on a magnetic rack, remove the supernatant after 1min, and remove the residue with 10ul pipette tip;

[0101] 10. Remove the low adsorption tube, add 150ul of 65℃ Wash Buffer 2 (WB2), vortex to mix, shake gently, and incubate in a 65℃ thermostat for 5min. Centrifuge briefly, place on a magnetic stand for 1min, remove the supernatant, and repeat once;

[0102] 11. Pipette 150ul of room temperature Wash Buffer 1 / 3 / 4 (WB1 / 3 / 4) respectively and add them to the PCR tube, shake vigorously for 30s, let stand for 30s, shake vigorously for 30s again, place on a handheld centrifuge for 30s and shake gently; place on a magnetic stand for 1min and remove the supernatant;

[0103] 12. Pipette 22.5ul of Nuclease-Free Water into the PCR tube, shake thoroughly to mix, and shake gently for later use;

[0104] 13. Take 22.5 μL of the hybridization product and add 27.5 μL of the amplification reagent mixture to prepare a PCR amplification system with a total volume of 50 μL. The PCR reaction program is as shown in Table 10, and the heat cover is 105°C;

[0105] Table 10 PCR amplification reaction program

[0106]

[0107] Table 11 PCR amplification cycle number reference

[0108]

[0109] 14. Add 40 μL of purified magnetic beads that have been incubated at room temperature for 30 minutes to the amplified product, mix thoroughly and incubate at room temperature for 5 minutes, place on a magnetic stand until the liquid is clear, discard the supernatant, add 185 μL of 80% ethanol, discard the supernatant after 1 minute, repeat the wash once, dry the magnetic beads at room temperature for 5 minutes, resuspend the magnetic beads with 22 μL of water after drying, incubate at room temperature for 2 minutes, place on a magnetic stand until the liquid is clear, and transfer 20 μL of supernatant, which is the probe capture library (i.e., the target DNA fragment).

[0110] Data Analysis:

[0111] 1. The probe capture library was subjected to Qubit quantification and 2100 quality control. After passing the quality control, the Illumina sequencing platform sequencer was used for sequencing. The sequencing experiment was performed according to the operating instructions provided by the manufacturer.

[0112] 2. Perform quality control, remove adapters, and screen the data to obtain clean FASTQ format data. Statistical analysis of target mutation gene region coverage uniformity, targeted capture ratio, repetition rate, average sequencing depth, and uniformity; filter the original data (Fastq) to generate high-quality sequences.

[0113] A method for detecting a systemic mastocytosis gene mutation using the above probe library:

[0114] 1. Use the BWA software package to align the quality-controlled sample data with the human genome (version hg19), and use the sambamba software package to convert the alignment results into BAM file format and sort the BAM according to the position on the chromosome; the input file is a fastq file, and the output file is a BAM file;

[0115] 2. Removal of duplicate sequences: For DNA duplicate fragments, MarkDuplicates in the gatk software package is used to remove sequence duplications. Both the input and output files are BAM files;

[0116] 3. Mutation analysis: The software Lianti was used to analyze the variant sites of the BAM obtained by the above alignment and deduplication, and single base substitution (SNV) and short sequence insertion or deletion (InDel) were obtained respectively; the input file was a BAM file, and the output was a vcf file;

[0117] 4. Variant annotation: Use software annovar and SnpEff to annotate SNV / InDel; input is vcf file, output is txt file;

[0118] 5. Mutation filtering: Filter mutations with mutation frequency < 1% and mutation support read number < 3.

[0119] 6. Result report:

[0120] The filtered mutations are summarized in Table 12, and the detected sample mutation results are as follows:

[0121] Table 12 Test results of 10 samples of systemic mastocytosis

[0122]

[0123] As shown in Table 12 above, missense mutations of multiple genes such as KIT, ASXL1, JAK2, TET2, SRSF2, RUNX1, KRAS, EZH2, ETV6 and U2AF1 were detected in 10 samples, and these gene mutations are all changes related to SM. Among them, KIT gene mutation is the most common gene mutation in SM, and about 90% of SM patients have KIT gain-of-function mutations. In addition, gene mutations such as SRSF2, ASXL1, RUNX1, EZH2 and SETD2 may affect the prognosis of SM, especially in invasive SM.

[0124] IGV (Integrative Genomics Viewer) software is a high-performance, easy-to-use interactive tool for visual exploration of genomic data. IGV supports flexible integration of all common types of genomic data and metadata, and supports visualization of a variety of large data sets, including second-generation sequencing, chip sequencing data, and genomic data. In this patent, IGV is mainly used to visualize genomic variation. First, open the IGV software, import the BAM file of the sample to be verified, and then enter the genomic position of the mutation site in the genomic position input box of the software (such as chr4:55599321). At this time, the software interface will display the mutation status of the sample at this genomic position relative to the reference genome sequence. Fig. 22 As shown in the figure, "1" shows the sequence of the reference genome, and "2" shows the actual genome sequence after mutation.

Claims

1. A probe library for detecting gene mutations in systemic mastocytosis, characterized in that: The probe library completely covers the probe combinations of the gene sequences corresponding to the target regions, and the gene mutation sequences include exon probe combinations for detecting ASXL1, ATM, CBL, DNMT3A, ETV6, EZH2, IDH1, IDH2, JAK2, KIT, KRAS, NRAS, RUNX1, SETBP1, SF3B1, SMC3, SRSF2, TET2, TP53, and U2AF1; A probe combination for detecting the splicing sites of ASXL1, ATM, CBL, DNMT3A, ETV6, EZH2, IDH1, IDH2, JAK2, KIT, KRAS, NRAS, RUNX1, SETBP1, SF3B1, SMC3, SRSF2, TET2, TP53, and U2AF1; the corresponding probe sequences are SEQID No.1-700.

2. A method for synthesizing probes in the probe library according to claim 1, characterized in that: It is composed of the following steps: (1) Nucleic acid extraction: extracting DNA from bone marrow samples; (2) Prelibrary construction: 900-1100 ng of sample DNA was sheared into fragments with a peak length of 150-250 bp, and then 200-400 ng of DNA fragments were taken for end repair and A addition. The repaired and A-added DNA fragments were then adapter-ligated and purified using magnetic beads. The purified adapter-ligated products were then amplified to obtain a prelibrary. (3) Probe synthesis: The probe design for the target gene when detecting DNA mutations is based on the DNA sequence; the probe is mainly synthesized using a solid phase column method, and a biotin label is added to the 5' end, and then the salt is removed by desalting and purification to finally obtain a high-quality probe; (4) The probes obtained in step (3) are then subjected to hybridization capture, eluted and purified to obtain a hybridization capture library of the probes. (5) Data analysis: Sequence the library captured by hybridization in step (4), and use Fastp software to process the Fastq data after the library sequencing, mainly for data quality control, linker removal and quality screening, and filter the original Fastq data to generate high-quality Fastq.

3. The method for synthesizing a probe according to claim 2, characterized in that: In the step (3), the transcript used in the probe synthesis is selected from the transcript used in the ClinVar database, the length of the probe is 100-150 bp, the probe covers the target gene region in a head-to-tail form, and the GC content of the probe sequence is 35% to 70%. The designed probe sequence is synthesized by Nanoda (Nanjing) Biotechnology Co., Ltd.

4. The method for synthesizing a probe according to claim 2, characterized in that: The length of the probe is preferably 120 bp.

5. A method for detecting a systemic mastocytosis gene mutation using the probe library of claim 2, characterized in that: The method consists of the following steps: using BWA software to align the quality-controlled probe hybridization capture library with the human genome (version hg19), and using gatk software to remove duplicate DNA fragments; using the software Lianti to analyze the variant sites of the BAM obtained by the above alignment and deduplication, and obtain single-base substitution SNVs and short sequence insertions or deletions InDels respectively; finally, using annovar and SnpEff software to annotate SNV / InDels; filtering the analyzed mutations according to the preset mutation frequency and mutation support reads number threshold to obtain the final mutation list.

6. A kit for detecting systemic mastocytosis gene mutations using the probe library of claim 1, the kit comprising pre-library construction reagents and hybridization capture reagents, the pre-library construction reagents comprising end-repair plus A reagents, linker connection reagents, pre-library amplification reagents, and purification magnetic beads, and the hybridization capture reagents comprising blocking agents, probe compositions, rapid hybridization premixes, hybridization enhancers, elution buffers, and streptavidin magnetic beads.

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