Capture probe for hemophagocytic syndrome gene detection, kit and application

Abstract

The invention discloses a group of capture probes for hemophagocytic syndrome related genes, a kit, application and a library construction method. The kit manufactured by the invention can be used for detecting 33 primary hemophagocytic syndrome related genes at one time by utilizing a hybrid capture method, the probe coverage is wide, the sequencing depth is high, and the embryonic line variation of all exon regions of the designed genes can be detected. According to the invention, all related diseases of related genes of the primary hemophagocytic syndrome can be reported, and genetic modes corresponding to the diseases can be provided.

Description

Technical Field

[0001] The present invention relates to the technical field of multi-gene detection, and in particular to a capture probe, a kit and an application thereof for detecting genes related to hemophagocytic syndrome. Background Art

[0002] Hemophagocytic syndrome (HPS), also known as hemophagocytic lymphohistiocytosis (HLH), is an excessive inflammatory response syndrome caused by abnormal activation, proliferation and secretion of a large number of inflammatory cytokines of lymphocytes, monocytes and macrophages due to inherited or acquired immune regulation dysfunction. The main clinical features are fever, hematocytopenia, hepatosplenomegaly, and hemophagocytosis in liver, spleen, lymph nodes and bone marrow tissues. HLH is a rapidly progressive and highly lethal disease. The median survival time of untreated HLH patients does not exceed 2 months.

[0003] According to whether there is a clear HLH-related gene abnormality, HLH can be divided into primary and secondary types. Primary HLH is caused by impaired hereditary lymphocyte toxicity or defects in genes related to inflammatory activity, and the inheritance is mainly sex chromosome and (or) autosomal recessive. Currently, there are more than 100 HLH-related genes reported, and there are 17 relatively clear HLH pathogenic genes. According to the characteristics of gene defects, primary HLH can be divided into familial HLH (familialhemophagocytic lymphohistiocytosis, FHL), immunodeficiency syndrome-related HLH, X-linked lymphoproliferative disease (XLP) and Epstein-Barr virus-driven HLH, involving genes such as PRF1, UNC13D, STX11, STXBP2, RAB27A, LYST, AP3B1, SH2D1A, XIAP, MAGT1, ITK, CD27 and CD70.

[0004] Gene sequencing is the gold standard for diagnosing primary HLH. According to the "Guidelines for the Diagnosis and Treatment of Hemophagocytic Syndrome in China", gene sequencing is suitable for patients with clear abnormalities in cytotoxic function tests and protein expression tests of HLH-related genes, patients with a positive family history or onset age <12 years old, patients with HLH for whom no clear cause has been found, and patients with recurrent HLH.

[0005] The existing detection of hemophagocytic syndrome-related genes is mostly based on PCR and first-generation sequencing technology. Such methods can only detect some hot spot mutations of the selected genes, but cannot detect unknown mutations. Therefore, the present invention is based on second-generation sequencing and adopts a hybridization capture method to achieve a one-time detection of the mutation of the whole exon region of 33 genes in the sample to be tested in the same system, and develops a kit for detecting hemophagocytic syndrome-related genes. Summary of the invention

[0006] In response to the above-mentioned problems, the present invention provides a multi-gene panel for detecting hemophagocytic syndrome-related genes, which can detect 33 hemophagocytic syndrome-related genes, has strong specificity, and can realize one-time detection of the variation of the whole exon region of 33 genes in the test sample in the same system, has a wide coverage area, and can detect germline variation in all exon regions.

[0007] The technical solution adopted by the present invention is:

[0008] The present invention provides a group of capture probes, wherein the capture probes include at least one of the base sequences SEQ ID NOs: 1-1096.

[0009] As a further preferred embodiment of the present invention, a capture probe is used for manufacturing a library construction kit for detecting hemophagocytic syndrome-related genes.

[0010] A library construction kit for detecting multiple genes of hemophagocytic syndrome comprises at least one set of capture probes, wherein the capture probes comprise at least one of base sequences SEQ ID NO: 1-1096.

[0011] The kit also includes at least one of the following: genomic DNA extraction reagents (such as Tiangen blood genomic extraction kit), DNA library construction reagents (such as Vazyme's Equinox Library Amp Mix), capture DNA library construction reagents (such as Twist's liquid hybridization capture kit) and instruments or reagents required for sequencing (such as Illumina high-throughput sequencing platform and human genome sequence).

[0012] The present invention also provides the application of the library construction kit in detecting multiple genes of hemophagocytic syndrome. Further, the application is to detect germline variation of genes related to hemophagocytic syndrome, and the germline variation is divided into pathogenic (P), suspected pathogenic (LP), clinical significance unknown (VUS), benign (B), and suspected benign (LB) variation.

[0013] Furthermore, the kit can also be used to provide related diseases and corresponding genetic patterns.

[0014] A library construction method uses the above capture probe or kit to capture hemophagocytic syndrome related genes.

[0015] As a further preference of the present invention, the hemophagocytic syndrome-related genes include one or more of the following genes: AP3B1, ATM, BLM, BTK, CARD11, CD27, CD70, DKC1, DNMT3B, FAS, GATA2, IKZF3, IL7R, IRF8, ITK, JAK3, LYST, MAGT1, NLRC4, PIK3CD, PIK3R1, PLCG2, PRF1, RAB27A, SH2D1A, SLC29A3, STAT3, STX11, STXBP2, UNC13D, WAS, XIAP, and ZAP70.

[0016] The included related genes were derived from multiple databases, relevant guidelines and expert consensus, covering 33 genes related to primary hemophagocytic syndrome.

[0017] Related diseases include: Hermansky-Pudlak syndrome type 2 (OMIM: 608233), breast cancer (OMIM: 114480), Bloom syndrome (OMIM: 210900), X-linked agammaglobulinemia (OMIM: 300755), B-cell hyperplasia with NFKB and T-cell anergy (OMIM: 616452), lymphoproliferative syndrome type 2 (OMIM: 615122), lymphoproliferative syndrome type 3 (OMIM: 618261), cataracts, hearing impairment, nephrotic syndrome and inflammatory bowel type 1 (OMIM: 301108), digenic facioscapulohumeral muscular dystrophy type 4 (OMIM: 619478), Autoimmune Lymphoproliferative Syndrome IA (OMIM:601859), Acute Myeloid Leukemia (OMIM:601626), Immunodeficiency Type 84 (OMIM:619437), Immunodeficiency Type 104 (OMIM:608971), Immunodeficiency Type 32A (OMIM:614893), Lymphoproliferative Syndrome Type 1 (OMIM:613011), Autochromatic Recessive Severe Combined Immunodeficiency with T Cell Negative, B Cell Positive, and NK Cell Negative (OMIM:600802), Chediak-Higashi Syndrome (OMIM:214500), Congenital Disorder of Glycosylation Icc

[0018] (OMIM:301031), familial cold autoinflammatory syndrome type 4 (OMIM:616115), Roifman-Chitayat syndrome

[0019] (OMIM:613328), autosomal recessive agammaglobulinemia type 7 (OMIM:615214), autoinflammatory-antibody deficiency-immune dysregulation (OMIM:614878), aplastic anemia (OMIM:609135), Griscelli syndrome type 2 (OMIM:607624), X-linked lymphoproliferative syndrome type 1 (OMIM:308240), histiocytosis-lymphoedema syndrome (OMIM:602782), multisystem autoimmune disease of infancy type 1 (OMIM:615952), familial hemophagocytic lymphohistiocytosis type 4 (OMIM:603552), familial hemophagocytic lymphoproliferative syndrome type 2 (OMIM:603552), Histiocytosis type 5 with or without microvillous inclusion disease (OMIM:613101), familial hemophagocytic lymphohistiocytosis type 3 (OMIM:608898), X-linked congenital severe neutropenia (OMIM:300299), X-linked lymphoproliferative syndrome type 2 (OMIM:300635), multisystem autoimmune disease of infancy type 2 (OMIM:617006), ataxia telangiectasia (OMIM:208900), isolated growth hormone deficiency type 3 with agammaglobulinemia (OMIM:307200), immunodeficiency type 11A (OMIM:615206), X-linked akeratosis congenita benign disease (OMIM: 305000), immunodeficiency-centromere instability-facial dysmorphism syndrome type 1 (OMIM: 242860), myelodysplastic syndrome (OMIM: 614286), immunodeficiency type 32B (OMIM: 226990), X-linked immunodeficiency with magnesium deficiency, EPSTEIN-BARR virus infection, and tumors (OMIM: 300853), autoinflammation with infantile enterocolitis (OMIM: 616050), autosomal dominant immunodeficiency type 14A (OMIM: 615513), immunodeficiency type 36 (OMIM: 616005), familial cold-induced autoinflammatory syndrome type 3 (OMIM: 6 14468), familial hemophagocytic lymphohistiocytosis type 2 (OMIM:603553), autosomal dominant high IgE recurrent infection syndrome type 1 (OMIM:147060), X-linked thrombocytopenia (OMIM:313900), immunodeficiency type 48 (OMIM:269840), immunodeficiency type 11B with atopic dermatitis (OMIM:617638), Embberger syndrome (OMIM:614038), autosomal recessive immunodeficiency type 14B (OMIM:619281), SHORT syndrome (OMIM:269880), familial non-Hodgkin lymphoma (OMIM:605027).

[0020] Provide inheritance patterns of related diseases: autosomal dominant inheritance, autosomal recessive inheritance, X-linked dominant inheritance, X-linked recessive inheritance, digenic recessive inheritance, digenic dominant inheritance.

[0021] According to the 2015 ACMG guidelines, the clinical significance of germline variants is classified into the following categories: pathogenic (P), suspected pathogenic (LP), of uncertain clinical significance (VUS), benign (B), and suspected benign (LB) variants.

[0022] The present invention also provides a method for detecting and analyzing hemophagocytic syndrome gene mutations based on NGS, which performs hybridization capture and sequencing on samples according to genes and sites related to hemophagocytic syndrome and related diseases, analyzes sample offline data using a data analysis process specifically designed and developed for the experiment, obtains gene mutation information (point mutations, insertions and deletions, copy number variations, etc.) and automatically generates a test report, thereby making a comprehensive assessment and guidance on the mutation status and clinical diagnosis and treatment.

[0023] A method for detecting and analyzing hemophagocytic syndrome gene mutation based on NGS, comprising the following steps:

[0024] S1. Use the probe capture method to construct and sequence the DNA extracted from normal samples and standard mutation samples to obtain the original sample fq data;

[0025] S2, preprocessing the original sample fq data obtained in step S1 by fastp to remove low-quality reads and adapter-contaminated reads;

[0026] S3. Align the sample data obtained in step S2 to the reference genome using bwa mem, deduplicate the original bam file, and then use GATK best practice to recalibrate the base quality of the bam file to obtain a bam file of sample data for variation detection;

[0027] S4. Use Mutect2 tool and LUMPY software to perform somatic mutation detection and fusion gene detection on the sample data obtained in step S3 respectively: in somatic mutation detection, filter meaningless / benign germline mutations by constructing Panel of Normals, and remove background mutations by using normal sample data as baseline in fusion gene detection; finally, use cnvkit software to detect copy number variation, and obtain vcf result files of variation types such as SNV / INDEL / SV / CNV and a list of filtered variations;

[0028] S5. Analyze the variant structure obtained in step S4 and generate a detection report based on the MSI information.

[0029] As a further preference of the present invention, the design of the probe sequence in step S1 includes: determining the target capture area and obtaining a bed file, designing multiple probe sequences using catch software according to the bed file, aligning the designed probe sequences with the reference genome using blastn software, finding out multiple sequences aligned to the reference genome and removing them to obtain the probe sequence.

[0030] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0031] The present invention is based on second-generation sequencing and adopts a hybridization capture method, which can realize one-time detection of the variation of the whole exon region of 33 genes in the sample to be tested by the same system, with a wide coverage area and capable of detecting germline variation in all exon regions.

[0032] The test kit of this project is designed for primary hemophagocytic syndrome. It can detect 33 genes with clear pathogenic effects related to primary hemophagocytic syndrome and some diseases with similar clinical symptoms that need to be differentially diagnosed at one time. It has strong specificity for patients with primary hemophagocytic syndrome. Compared with the detection method of sending tests separately for a single disease, it can reduce costs and increase efficiency, and it complies with the relevant requirements of the "Guidelines for the Diagnosis and Treatment of Hemophagocytic Syndrome in China" for the diagnosis of primary hemophagocytic syndrome.

[0033] The gene variation determination of this project is based on the ACMG guidelines. There is a standard annotation process for variations not included in the clinvar database to effectively avoid missed detections. The disease annotations related to genes come from a wide range of sources, not just relying on a single database. Multiple databases, relevant guidelines, and expert consensus are manually collated to output all related diseases and provide the corresponding genetic patterns of the diseases.

[0034] The capture probes designed for this project cover the entire coding region sequence of the genes involved, with a target region coverage of up to 99% and an average sequencing depth of up to 500x.

[0035] In summary, the present invention has the following beneficial effects:

[0036] The detection and analysis method provided by the present invention covers most of the genes and sites of hemophagocytic syndrome and related diseases. Compared with traditional hemophagocytic syndrome detection methods, it has the advantages of low sample requirements, convenient operation, moderate detection cost and wide detection range (including SNV / INDEL / CNV / SV and other variation types). BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the fragment analysis result of the hybridization capture library in Example 2. DETAILED DESCRIPTION

[0038] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0039] Example 1

[0040] 1. Nucleic Acid Extraction

[0041] Extract genomic DNA from whole blood or bone marrow samples.

[0042] 2. Library Construction

[0043] 2.1 DNA fragmentation, end repair and dA tailing.

[0044] 2.1.1 Set the following program on the PCR instrument, with the heated cover temperature at 70°C:

[0045] temperature time 37℃ 20min 65℃ 30min 4℃ hold

[0046] 2.1.2 Mix and spin off the reagents, prepare the reaction solution (prepare on ice), and dispense into PCR tubes and keep on ice for later use. The reaction solution is configured as follows:

[0047] Reagents Single reaction volume Frag / AT Buffer 4uL Frag / AT Enzyme 6uL Total volume 10uL

[0048] 2.1.3 Determine the concentration of gDNA using the Qubit quantitative analysis kit. Add 40 μL of DNA sample to the above reaction solution, with a total amount of 50-200 ng. Seal the reaction tube or cover it with a lid, gently shake and mix, centrifuge it, and place it in a PCR instrument with a hot cover temperature of 70°C, and run it according to the program in 2.1.1.

[0049] 2.2 Connect the Universal Adapter.

[0050] 2.2.1 Prepare DNA purification magnetic beads during the PCR reaction in 2.1, shake well to mix, and equilibrate at room temperature for at least 30 min; prepare 80% ethanol; thaw Universal adapter (Twist Bioscience) and DNA Ligation Mix (Twist Bioscience) on ice; set the PCR program to 20℃ hold and close the heated lid.

[0051] 2.2.2 Add 5μl of Universal adapter to each well or tube containing DNA fragments with dA tails (prepared in 2.1), mix gently by pipetting, and then place on ice. Add 20μl of Ligation Mix, mix gently by pipetting, seal the reaction tube or cover it, centrifuge briefly, and place it on the PCR instrument set up in the preparation.

[0052] 2.3 First step magnetic bead purification

[0053] 2.3.1 Add 60 μl of mixed DNA purification magnetic beads (0.8x) to each sample obtained in 2.2, vortex to mix, incubate at room temperature for 5 minutes, centrifuge briefly, and place on a magnetic rack for 5 minutes.

[0054] 2.3.2 Aspirate the supernatant, add 200 μl of freshly prepared 80% ethanol, and incubate for one minute. Repeat once.

[0055] 2.3.3 Aspirate the supernatant and carefully remove the remaining ethanol using a 10μl pipette to avoid touching the magnetic beads. Dry the magnetic beads on a magnetic rack until they are not reflective and cracked.

[0056] 2.3.4 Add 18 μl of ddH2O to each sample, mix well by pipetting, and incubate at room temperature for 25 min; centrifuge briefly, place on a magnetic rack, and let stand for 3 min.

[0057] 2.3.5 Transfer 15 μl of supernatant to a PCR tube for the next PCR amplification.

[0058] 2.4UDI Primer PCR Amplification

[0059] 2.4.1 Thaw Equinox Library Amp Mix (2x) and UDI primers (TwistBioscience) on ice; set the program as shown in the following table on the PCR instrument, with the heated lid temperature at 105°C:

[0060]

[0061] 2.4.2 Add 10 μl of UDI primer and 25 μl of Equinox Library Amp Mix (2x) after inversion to each gDNA library prepared in 2.3, and gently pipette to mix. Centrifuge briefly, transfer to a PCR instrument, and run according to the procedure in 2.4.1.

[0062] 2.5 Second step: magnetic bead purification

[0063] 2.5.1 Vortex the pre-equilibrated DNA purification magnetic beads until mixed. Add 50 μl (1x) of pre-equilibrated DNA purification magnetic beads to each ligation sample prepared in 2.4. Vortex to mix and incubate at room temperature for 5 minutes.

[0064] 2.5.2 After centrifugation, place on the magnetic rack for 5 minutes, discard the supernatant, add 200 μl of freshly prepared 80% ethanol, incubate for one minute, and discard the supernatant. Repeat once.

[0065] 2.5.3 Use a 10μl pipette to absorb the remaining ethanol. Dry the magnetic beads on a magnetic rack until they are non-reflective and dry.

[0066] 2.5.4 Add 24 μl of ddH2O to each sample, mix well by pipetting, incubate at room temperature for 5 min, centrifuge briefly, place on a magnetic rack, and let stand for 2 min.

[0067] 2.5.5 Take 2 μl and dilute 10 times, then use Qubit to measure the library concentration and use fragment analyzer to measure the fragment size. The average fragment length should be between 350 and 425 bp.

[0068] 3. Hybridization Capture

[0069] 3.1 Library hybridization

[0070] 3.1.1 Library pool DNA mixing scheme: The library pool often contains multiple different sample libraries. The library pool input amount is calculated according to the library DNA concentration, and the library pool is obtained by mixing. The total amount of library pool DNA is 1.5-4ug.

[0071] 3.1.2 Calculate the amount of different libraries according to the library concentration determined by Qubit, mix the libraries in a 1.5mL centrifuge tube, shake gently, and centrifuge instantly.

[0072] 3.1.3 Add pre-hybridization reagent to the mixed sample according to the table below, gently shake to mix, and centrifuge instantaneously:

[0073] Reagents Single reaction volume Universal Blockers 8uL Blocking Solution 5uL The probe designed by the present invention 4uL

[0074] 3.1.4 Open the cap of the reaction tube and place it in a vacuum concentrator. After balancing, dry it at 45℃ until the solution in the tube is completely dried. The drying process takes about 30min to 60min.

[0075] 3.1.5 Incubate Fast Hybridization Mix (Twist Bioscience) at 65°C for 10 min until the precipitate is completely dissolved; set the PCR instrument program according to the table below and heat the lid at 85°C.

[0076] temperature time 95℃ 5min 60℃ 2h

[0077] 3.1.6 After drying, take out the centrifuge tube from the vacuum concentrator, tear off the sealing film, quickly vortex the preheated Fast Hybridization Mix (Twist Bioscience) and take 20 μl of the resuspended sample, flick to mix, and let stand at room temperature for 5 minutes.

[0078] 3.1.7 Use a pipette to pipette up and down 10 times. After the DNA is completely dissolved, transfer as much of the solution in the tube as possible to a new PCR tube, add 30 μl of paraffin oil to the tube, and centrifuge to remove bubbles.

[0079] 3.1.8 Transfer the PCR tube to the preheated PCR instrument and run the program in 3.1.5.

[0080] 3.2 Library capture and cleaning

[0081] 3.2.1 Prepare DNA-binding magnetic beads Streptavidin Binding Beads (Twist Bioscience) during the PCR reaction in 3.1, shake well to mix, and equilibrate at room temperature for at least 30 min; preheat 450 μl Fast Wash Buffer 1 at 66°C in a thermostatic metal bath; preheat 700 μl Wash Buffer 2 at 48°C; dispense the enrichment reagent into centrifuge tubes according to the table below.

[0082] Reagents Single reaction volume Fast Binding Buffer(Twist Bioscience) 900ul Fast Wash Buffer 1(Twist Bioscience) 450ul Wash Buffer 2(Twist Bioscience) 700ul

[0083] 3.2.2 Shake the pre-equilibrated Binding Beads (Twist Bioscience) until completely mixed, and transfer 100 μl of magnetic beads to a 1.5 mL centrifuge tube.

[0084] 3.2.3 Add 200 μl Fast Binding Buffer to the centrifuge tube and mix well by pipetting with a pipette tip; place the centrifuge tube on a magnetic rack for 1 min or until the solution is clear, discard the supernatant, and remove the centrifuge tube; repeat the above washing steps 2 times, for a total of 3 times.

[0085] 3.2.4 After washing three times, add 200 μl Fast Binding Buffer and resuspend by vortexing to mix thoroughly.

[0086] 3.2.5 After the hybridization reaction in step 2.1 is completed, open the PCR instrument cover and quickly transfer all the hybridization solution to the balanced magnetic bead solution, and immediately mix by pipetting.

[0087] 3.2.6 Close the cap of the centrifuge tube and seal the tube mouth with sealing film, then place it on the Biocate oscillator at 1200 rpm and mix thoroughly at room temperature for 30 minutes.

[0088] 3.2.7 Centrifuge the centrifuge tube quickly, place it on a magnetic rack for 1 minute, and remove the supernatant.

[0089] 3.2.8 Remove the centrifuge tube from the magnetic rack, add 200 μl preheated FW1, mix gently and quickly incubate at 66°C for 5 min. Repeat 3.2.7 to 3.2.8 once.

[0090] 3.2.9 Transfer all the liquid in the tube to a new 1.5mL centrifuge tube, place it on a magnetic rack for 1 minute, and remove the supernatant.

[0091] 3.2.10 Remove the centrifuge tube from the magnetic rack, add 200 μl preheated W2, mix gently and quickly incubate at 48°C for 5 min, place on the magnetic rack for 1 min, remove the supernatant and repeat the washing with W2 twice, for a total of three times.

[0092] 3.2.11 After centrifugation, place on a magnetic rack and use a 10μl pipette tip to absorb all the residual liquid; add 45μl ddH2O, pipette to mix well, and incubate on ice.

[0093] 3.3 PCR amplification

[0094] 3.3.1 Prepare DNA purification magnetic beads, shake and mix thoroughly, and equilibrate at room temperature for at least 30 min; prepare 80% ethanol; thaw Equinox Library Amp Mix (2x) and Amplification Primers (Twist Bioscience) on ice; set the following program on the PCR instrument, with the hot cover temperature at 105°C.

[0095]

[0096]

[0097] 3.3.2 Prepare PCR tubes and add 25μl of Equinox Library Amp Mix (2x) and 2.5μl of Amplification Primers to each tube. Take 22.5μl of the magnetic bead suspension prepared in 3.2 and add it to the PCR tube, gently shake and mix, and centrifuge briefly.

[0098] 3.3.3 Transfer the PCR tube to the preheated PCR instrument and run the above program.

[0099] 3.4 Magnetic bead purification

[0100] 3.4.1 Vortex the pre-equilibrated DNA purification magnetic beads until mixed. Add 90 μl (1.8x) of pre-equilibrated DNA purification magnetic beads to each enriched sample prepared in 3.3. Vortex to mix and incubate at room temperature for 5 minutes.

[0101] 3.4.2 After the instant separation, place the tube on the magnetic rack for 5 minutes and discard the supernatant.

[0102] 3.4.3 Add 200 μl of freshly prepared 80% ethanol, incubate for one minute, and discard the supernatant. Repeat once.

[0103] 3.4.4 Use a 10μl pipette to absorb the remaining ethanol. Dry the magnetic beads on a magnetic rack until they are non-reflective and dry.

[0104] 3.4.5 Add 32 μl of ddH2O to each sample, mix well by pipetting, incubate at room temperature for 5 min, centrifuge briefly, place on a magnetic rack, and let stand for 2 min.

[0105] 3.4.6 Transfer 30 μl of supernatant to a clean 1.5 ml centrifuge tube and take 2 μl for use The library concentration was determined by dsDNA HS Assay Kit (Vazyme), which should be greater than 0.5 ng / μl. The length distribution of library fragments was detected by 2100 fragment analyzer, and the average fragment length should be around 300-400 bp.

[0106] 4. Sequencing and Analysis

[0107] The library constructed by the present invention is compatible with various sequencing platforms of Illumina for sequencing. After sequencing on the machine according to the dilution multiple and sequencing parameters required by the sequencer, the data off the machine is quality controlled. A method for detecting and analyzing hemophagocytic syndrome gene mutations based on NGS specifically comprises the following steps:

[0108] (1) Determine the target capture region based on the target gene and generate the capture region bed file.

[0109] (2) Design the target region probe sequence using the catch software based on the bed file and the human hg38 reference genome (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.);

[0110] Among them, the catch software operation method is as follows:

[0111] nohup design.py-pl 120-l 60-m 5-e 5-o HLH.fasta&&\

[0112] makeblastdb-in HLH.fasta-out blast / HLH.fasta-dbtype nucl&&\

[0113] echo-e"qseqid\tsseqid\tpident\talignment_length\tmismatch\tgapopen\tqstart\tqend\tsstart\tsend\tevalue\tbitscore">blast / HLH.probe.blastn.txt

[0114] blastn-num_threads 10-query probe / HLH.probe.fasta-db blast / HLH.fasta-evalue 1e-50-max_target_seqs 1000-outfmt 6>>blast / HLH.probe.blastn.txt&&\

[0115] perl filter_mulialign_probe.pl blast / HLH.probe.blastn.txt probe / HLH.probe.fasta probe / HLH.uniq.probe.fasta

[0116] The designed exploration sequence was aligned with hg38 using blastn software, and the sequences that were aligned to hg38 multiple times were removed to obtain the probe sequences shown in Table 1.

[0117] (3) The probe capture method was used to construct libraries and sequence the DNA extracted from normal samples and standard mutation samples.

[0118] (4) The obtained original sample fq data is split into normal samples and standard mutation samples.

[0119] (5) All original sample FQ data were preprocessed by fastp to remove low-quality reads and adapter-contaminated reads; the preprocessing method reference is: Chen S, Zhou Y, Chen Y, et al. fastp: an ultra-fast all-in-one FASTQ preprocessor. 2018.

[0120] (6) The sample data obtained in step (5) was aligned to hg38 using bwa mem, the original bam file was deduplicated, and then the bam file was recalibrated for base quality using GATK best practice to obtain a sample data bam file for variation detection.

[0121] (7) Each sample obtained in step (6) is processed as follows:

[0122] a. Use the Mutect2 tool to detect somatic mutations (snv / indel): establish a data set of normal samples as background filtering (PoN, Panel of Normals), analyze and filter meaningless / benign germline mutations;

[0123] b. LUMPY software detection of fusion genes: normal samples are used as the baseline to remove background variations. The specific detection method reference is: Layer RM, Hall IM, Quinlan A R. LUMPY: A probabilistic framework for structural variant discovery [J]. 2012;

[0124] c. Finally, the copy number variation is detected by cnvkit software to obtain the vcf result files of variation types such as SNV / INDEL / SV / CNV and the filtered variation list.

[0125] (8) The MSI status of each sample was detected based on the normal sample data using the MSIsensor-pro software (reference: Jia P, Yang X, Guo L, et al. MSIsensor-pro: Fast, Accurate, and Matched-normal-sample-free Detection of Microsatellite Instability[J]. Journal of Genomics Proteomics and Bioinformatics: English version, 2020(1):7). ​​A result greater than or equal to 10% was MSI-H, and a result less than 10% was MSI-L.

[0126] (9) Using the clinically relevant hemophagocytic syndrome mutation interpretation data set and reporting program accumulated within the laboratory, the mutation results and MSI information in the information analysis steps (7)-(8) are read to automatically generate a clinical test report.

[0127] The analysis process of steps (4)-(9) above has been packaged into an automated process. When running, you only need to provide the fq list of the downloaded files to automatically run the program and output the results. Use BWA / GATK and other related software to perform bioinformatics analysis on the data and annotate related databases such as gnomAD / Clinvar / OMIM. According to relevant international and domestic guidelines, the diagnosis and treatment information of the mutant gene is provided.

[0128] Example 2

[0129] A male patient diagnosed with hemophagocytic syndrome was tested for mutations in the 33 genes described in the present invention using bone marrow samples. The genomic DNA of the bone marrow samples was extracted using a Tianlong automated extractor (model: NP968-C) and tested according to the experimental procedures described in the implementation method. The fragment analysis results of the hybrid capture library are shown in Figure 1 shown.

[0130] The obtained hybridization capture library was sequenced using the Nextseq6000 instrument of the Illumina platform, with sequencing parameters: PE 2x150. The quality control of the library was performed, and the coverage of the target region was 99.9%, and the average depth of the target region was 791.05, which was qualified.

[0131] The sequencing data was aligned with the human reference genome hg38 / GRCh38, and quality data such as coverage of the target region and average sequencing depth were collected. Bioinformatics methods and various professional databases (such as gnomAD, ClinVar, OMIM and other databases) were used to identify, annotate and filter variant sites. The gene names were in accordance with the rules of the Human Genome Organization Gene Nomenclature Committee (HGNC). The naming of variants refers to the nomenclature provided by the Human Genome Variation Society (HGVS). The interpretation of variant sites refers to the ACMG / AMP genetic variation classification standards and guidelines, as well as the updated supplementary recommendations and consensus of ClinGen and others. The obtained variant site information is shown in the following table.

[0132]

[0133] The test results show that the detection kit of the present invention has qualified probe coverage and average depth of the target area, and can accurately detect the mutation of genes related to hemophagocytic syndrome and provide relevant diagnosis and treatment information.

[0134] The hybridization capture probe sequence of the hemophagocytic syndrome panel is as follows:

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[0185] The specific embodiments described herein are merely examples of the spirit of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in similar ways, but they will not deviate from the spirit of the present invention or exceed the scope defined by the appended claims.

Claims

1. A set of capture probes, characterized in that The capture probe comprises at least one of the base sequences SEQ ID NOs: 1-1096.

2. The use of the capture probe according to claim 1, characterized in that The capture probe is used to manufacture a library construction kit for detecting hemophagocytic syndrome related genes.

3. A library construction kit for detecting genes related to hemophagocytic syndrome, characterized in that: It comprises at least one group of capture probes, wherein the capture probes comprise at least one of the base sequences of SEQ ID NOs: 1-1096.

4. Use of the library construction kit as claimed in claim 3 in detecting genes related to hemophagocytic syndrome.

5. A library construction method, characterized in that: The hemophagocytic syndrome-related gene is captured using the capture probe described in claim 1 or the library construction kit described in claim 3.

6. A library construction method according to claim 5, characterized in that, The hemophagocytic syndrome-related genes include one or more of the following genes: AP3B1, ATM, BLM, BTK, CARD11, CD27, CD70, DKC1, DNMT3B, FAS, GATA2, IKZF3, IL7R, IRF8, ITK, JAK3, LYST, MAGT1, NLRC4, PIK3CD, PIK3R1, PLCG2, PRF1, RAB27A, SH2D1A, SLC29A3, STAT3, STX11, STXBP2, UNC13D, WAS, XIAP, and ZAP70.

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