Method and probe set for detecting skipping of 14 # exon of MET gene at RNA level

Abstract

The invention discloses a method and a probe set for detecting skipping of a 14 # exon of an MET gene on the basis of RNA (Ribonucleic Acid) level. The invention further provides a kit for detecting the skipping of the 14 # exon of the MET gene at the RNA level and application of the kit. The method for detecting the skipping of the 14 # exon of the MET gene based on the RNA level, provided by the invention, can be used as supplement and verification of DNA level detection, so that a patient is benefited from gene detection and targeted therapy to a greater extent.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of gene detection, and particularly relates to a method for detecting skipping of exon 14 of MET gene at RNA level and a probe set. BACKGROUND

[0002] The human MET gene, also known as c-MET, is a proto-oncogene, and the full-length encoded MET protein is a potential targeted oncogenic driver. The MET protein can bind to a growth factor (HGF) and activate a plurality of downstream signaling pathways. Degradation inactivation of the MET gene leads to abnormal activation of the HGF / MET pathway, which is considered to be related to the occurrence, invasion and metastasis of a plurality of tumors. Skipping of exon 14 of the MET gene is the main cause of MET degradation inactivation. MET exon 14 skipping is due to abnormal mutation of the key recognition region of MET gene transcription regulation, which causes mRNA to skip exon 14 during MET transcription, resulting in loss of exon 14 and direct connection of exon 13 and exon 15, and inhibition of ubiquitination and degradation of MET protein. About 3% of non-small cell lung cancer cases have MET exon 14 skipping. MET exon 14 skipping occurs in about 14% of secondary glioblastoma cases, and the prognosis is significantly poorer.

[0003] Next-generation sequencing (NGS) can achieve the purpose of large-scale and high-throughput sequencing, can be applied to tumor driver gene sequencing, and is an important part of precise diagnosis and treatment of tumors. Whole genome or exome NGS sequencing has a wide genomic coverage, high related cost and large data complexity, and is not suitable for the needs of clinical scenarios. Targeted sequencing selects genes or regions of interest on the genome as a target detection region for sequencing, has the advantages of high specificity, high coverage depth and reduced sequencing cost, and is therefore widely used in clinical applications. The technical principles of common targeted sequencing are divided into probe hybridization capture and multiplex PCR capture. Multiplex PCR capture simultaneously amplifies multiple target sequences in one reaction by designing multiple pairs of primers, and has the advantages of low initial amount requirement, simple and rapid detection process. However, multiplex PCR primer design is difficult, and it is difficult to supplement and update sites subsequently, and the detection region coverage is small. In addition, there may be amplification bias in the PCR amplification process, affecting the detection sensitivity. The probe hybridization capture method can capture thousands of different target regions in a larger range at the same time, has lower probe design difficulty, high flexibility of target updating, and higher detection sensitivity.

[0004] Currently, the detection of MET exon 14 skipping based on DNA level targeted capture sequencing technology is mainly achieved by identifying the variation of four functional regions around exon 14, i.e. splice donor region, splice acceptor region, branch point and polypyrimidine tract. The variation mainly occurs in the regions of intron 13, exon 14 and intron 14, and the variation types include point mutation and small fragment deletion. Since the variation site range is large, the variation types are complex and diverse, and the intron region is involved. Therefore, the primer design for detecting the complex variation of MET exon 14 skipping at DNA level by multiplex PCR targeted sequencing technology is difficult, and the detection sensitivity is low. Although the probe hybridization capture technology has low probe design difficulty, the number of probes is large, which increases the sequencing data volume and thus leads to an increase in detection cost, and in addition, the imperfect annotation of complex variation types can lead to missed detection.

[0005] MET exon 14 skipping at RNA level is relatively fixedly expressed as the direct connection of exon 13 and exon 15, so the detection of MET exon 14 skipping at RNA level has low technical difficulty, the detection fusion mode is relatively fixed, and the positive detection rate is higher. Currently, the multiplex PCR capture technology identifies MET exon 14 skipping by amplifying the fragment of the fusion of exon 13 and exon 15. However, the target region coverage of multiplex PCR capture is small, which is only suitable for the detection of fusion of known breakpoints, cannot capture the fusion with breakpoint position beyond the coverage range and unknown fusion, and is easily affected by RNA quality, and has poor fusion detection ability for low-quality RNA samples with severe degradation. In comparison, the probe hybridization capture technology has large target coverage, can capture the fusion with variable breakpoint position and unknown fusion, and is less affected by RNA quality. SUMMARY

[0006] In order to make up for the shortcomings of the prior art, the purpose of the present application is to provide a method for detecting MET gene exon 14 skipping at RNA level and a probe set.

[0007] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: The present application provides a probe set for detecting MET gene exon 14 skipping at RNA level.

[0008] Further, the probe set comprises any one probe with a nucleotide sequence as shown in SEQ ID NO. 1-6, or a probe with the same function.

[0009] Further, the probe with the same function refers to a probe with one or several nucleotides substituted and / or deleted and / or added to any one of SEQ ID NO. 1-6 and with the same hybridization and capture function.

[0010] Preferably, the probe with the same function has 80% or more of the same bases as the original probe, more preferably 90% or more of the same bases, and even more preferably 95% or more of the same bases.

[0011] In the present application, the term "exon skipping" refers to the phenomenon that a certain exon is not present in the mature mRNA because it is spliced together with the introns on both sides during transcription, which is a type of alternative splicing. In the present application, the "exon skipping of gene 14" refers to the case where the 14th exon in gene expression is skipped. MET The exon skipping of gene 14 refers to the case where the 14th exon in gene expression is skipped. MET The exon skipping of gene 14 refers to the case where the 14th exon in gene expression is skipped.

[0012] In the present application, the probe with 80% or more of the same bases as the original probe refers to a probe with 80% or more homology to the original probe. "Homology" and "identity" are used interchangeably and refer to the degree to which two nucleotide sequences have identical residues at the same positions in alignment, and is usually expressed as a percentage. Preferably, the identity is determined over the entire length of the sequences being compared. Thus, two copies of the same sequence have 100% identity. Those skilled in the art will recognize that several algorithms can be used to determine sequence identity using standard parameters, such as Blast, Blast2, Smith-Waterman, and ClustalW.

[0013] In some embodiments, the probe can be labeled with a labeling substance. The labeling substance includes but is not limited to biotin, a fluorescent substance, a radioisotope, or an enzyme. Among them, the fluorescent substance includes but is not limited to TAMRAT TM , Alexa555, Alexa647, Cy3, Cy5 of cyanine dye series, and fluorescein. The radioisotope includes but is not limited to 32 P, 33 P, 35 S. The enzyme includes but is not limited to alkaline phosphatase and horseradish peroxidase.

[0014] The second aspect of the present application provides a kit for detecting the exon skipping of gene 14 at the RNA level. MET The second aspect of the present application provides a kit for detecting the exon skipping of gene 14 at the RNA level.

[0015] Further, the kit includes the probe set according to the first aspect of the present application.

[0016] Further, the kit further comprises any one or a combination of at least two of the following: RNA fragmentation reagent, cDNA first strand synthesis reagent, cDNA second strand synthesis primer, cDNA second strand synthesis reagent, adaptor ligation reagent, hybridization capture reagent, hybridization product amplification reagent, or streptavidin affinity magnetic beads.

[0017] Preferably, the kit further comprises an instruction manual.

[0018] In the present application, one or more probes in a suitable amount are provided in one or more containers, or immobilized on a substrate. The probes can be provided in suspension in an aqueous solution, or for example as a freeze-dried or lyophilized powder. Among them, the container provided with nucleic acids can be any conventional container capable of containing the provided form, such as a microcentrifuge tube, an ampoule or a bottle. The kit can contain labeled or unlabeled, for detecting MET a probe for detecting exon skipping of gene 14.

[0019] In the present application, the streptavidin-labeled magnetic beads are separated from the system by the magnetic field force through the binding of streptavidin to biotin on the capture probe, and the target region RNA sequence targetedly combined with the capture probe.

[0020] In the present application, the kit preferably further comprises RNA fragmentation reagent, reverse transcription reaction reagent, cDNA first strand synthesis reagent, cDNA second strand synthesis reaction reagent, adaptor ligation reagent, purification reagent, streptavidin affinity magnetic beads, PCR pre-reaction reagent, and post-capture PCR amplification reaction reagent. The PCR pre-reaction reagent preferably comprises at least one of the following: PCR Master Mix containing UDG enzyme and UDI primer. The RNA fragmentation reagent, reverse transcription reaction reagent, cDNA second strand synthesis reaction reagent, adaptor ligation reagent, purification reagent, PCR pre-reaction reagent are used for constructing a sample RNA library. The present application does not have special restrictions on the reagents for constructing the RNA library, and reagents well known in the art can be used. The post-capture PCR amplification reaction reagent preferably comprises at least one of the following: PCR primer and post-capture PCR reaction premix. The post-capture PCR amplification reaction reagent is used to amplify the captured target region RNA sequence to obtain a large amount of target region RNA sequence to meet the sequencing requirements.

[0021] The third aspect of the present application provides a method for detecting exon skipping of gene 14 at the RNA level. MET The third aspect of the present application provides a method for detecting exon skipping of gene 14 at the RNA level.

[0022] Further, the method comprises using the probe set of the first aspect of the present application to capture the probe hybridization of the RNA intermediate library obtained from the sample to be tested.

[0023] Furthermore, the sample to be tested is selected from one or more of tumor cells, blood, plasma, exosome solution, pleural effusion, peritoneal effusion, saliva, urine, and tissue.

[0024] Preferably, the tumor cell is a central nervous system tumor, pharyngeal cancer, adrenal tumor, AIDS-related cancer, alveolar soft tissue sarcoma, astrocytoma, bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumor, breast cancer, carotid body tumor, cervical cancer, chondrosarcoma, chordoma, renal chromophobe cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, desmoplastic small round cell tumor, ependymoma, Ewing tumor, extraskeletal myxoid chondrosarcoma, fibrous dysplasia, fibrous dysplasia, gallbladder or bile duct cancer, gastric cancer, primary liver cancer, malignant teratocarcinoma, ovarian embryonal tumor, viral hepatitis, gestational trophoblastic disease with cirrhosis, germ cell tumor, head and neck cancer, hepatocellular carcinoma, One or more of pancreatic islet cell tumor, Kaposi sarcoma, kidney cancer, leukemia, liposarcoma / malignant lipomatous tumor, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, biliary cell carcinoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer, papillary thyroid cancer, parathyroid cancer, pediatric cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, melanoma, metastatic kidney cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, synovial sarcoma, testicular cancer, thymic cancer, thymoma, metastatic thyroid cancer, or uterine cancer.

[0025] More preferably, the tumor cells are non-small cell lung cancer cells or brain glioma cells.

[0026] Furthermore, the step of obtaining an RNA intermediate library from the sample to be tested includes extracting RNA from the sample, fragmenting the sample RNA, and sequentially performing cDNA single-strand synthesis, cDNA second-strand synthesis, end-removal and A addition, and adapter ligation on the fragmented RNA. After amplification and purification, the RNA intermediate library is obtained after Qubit quantification and 2100 quality control detection.

[0027] Furthermore, the method also includes library amplification and purification after probe hybridization capture, sequencing and analysis of sequencing results.

[0028] Preferably, the sequencing is performed using the Illumina platform in PE150 mode.

[0029] Preferably, the step of analyzing the sequencing results includes aligning the captured RNA molecule sequencing data to the hg19 version of the human reference genome to obtain alignment information of all sequencing reads; merging the alignment positions of the two reads R1 and R2 obtained by sequencing the same RNA molecule fragment from both ends into a complete read; and classifying according to the alignment information of the merged reads: 1) If the read segment is correctly aligned to MET At the end of exon 13 of the gene, this read is denoted as d; 2) If a gap occurs in the read segment, one end of the gap can be correctly aligned to MET The end of exon 13 of the gene, and the other end can be correctly aligned to MET The start of exon 15 of the gene, this read segment is denoted as k; 3) The total number of sequenced reads is recorded as n; The variant support reads were then normalized to the variant frequency and the variant support reads per megabyte of sequencing reads: a) Mutation frequency = k / d; b) Number of variant support reads per megabyte of sequencing reads = (k*1000000) / n; If the number of variant support reads per megabyte of sequencing reads is greater than or equal to 0.4, or the variant frequency is greater than or equal to 3%, or the number of variant support reads (k) is greater than or equal to 4, the sample is judged as positive, otherwise it is judged as negative.

[0030] In the present invention, RNA level detection MET The method for skipping exon 14 of a gene is preferably to obtain an intermediate library of an RNA sample by fragmentation, reverse transcription and library construction of the total RNA of the sample. The intermediate library is hybridized with the probe group described in the first aspect of the present invention, and the library fragments containing the target sequence are complementary paired with the probes. The probes are captured by streptavidin magnetic beads, and the uncaptured fragments are washed away; then, the hybridization capture of the library containing the target sequence is completed by PCR amplification and magnetic bead sorting and purification. The sequencing data is obtained by high-throughput sequencing, and then analyzed by the corresponding bioinformatics analysis software to obtain MET 14 exon skipping variant information.

[0031] The present invention has no particular limitations on the reagents for constructing the RNA library, the reagents for the post-capture PCR amplification reaction, and the reagents for sequencing, and any reagents well known in the art may be used.

[0032] In the present invention, considering the support MET 14 The number of reads that jump will be affected by METThe influence of gene expression amount and total sequencing data amount, even for the same sample, the number of supporting reads obtained by sequencing different data amounts is different. In order to reduce the bias caused by different sequencing depths, improve the accuracy and reliability of data analysis, on the basis of considering the variation supporting reads number as the basis for judging positive results, the variation supporting reads number is normalized to obtain the variation supporting reads number per meg sequencing read.

[0033] The fourth aspect of the application provides an RNA level detection MET The device for skipping the exon 14 of the gene.

[0034] Further, the device comprises: (1) RNA intermediate library preparation module: the obtained sample RNA is fragmented, and the fragmented RNA is subjected to cDNA single-strand synthesis, cDNA double-strand synthesis, A tailing, adapter ligation, amplification and purification, and then subjected to Qubit quantification and 2100 quality control detection to obtain the RNA intermediate library; (2) probe hybridization capture module: the probe set of the first aspect of the application is added for hybridization capture; (3) amplification and sequencing module: the hybridization capture product is subjected to amplification, purification, quality control detection, and then subjected to sequencing, and the sequencing is performed by the illumina platform in the PE150 mode; (4) analysis module: the captured RNA molecule sequencing data is compared to the human reference genome in the hg19 version to obtain the alignment information of all sequencing reads; the alignment positions of R1 and R2 two reads obtained by sequencing the same RNA molecule fragment from two ends are combined into a complete read; and the combined read alignment information is classified according to the combined read alignment information: 1) if the read is correctly aligned to the end of the exon 13 of the gene, the read is recorded as d; MET 2) if the read has a gap, one end of the gap can be correctly aligned to the end of the exon 13 of the gene, and the other end can be correctly aligned to the start of the exon 15 of the gene, and the read is recorded as k; MET MET 3) the total number of sequencing reads is n; After that, the variation supporting reads number is normalized, and the normalized values are the variation frequency and the variation supporting reads number per meg sequencing read: a) variation frequency = k / d; b) variation supporting reads number per meg sequencing read = (k*1000000) / n; ​​​(5) Result output module: If the number of variant support reads per megabyte of sequencing reads of the sample is greater than or equal to 0.4, or the variant frequency is greater than or equal to 3%, or the number of variant support reads (k) is greater than or equal to 4, it is judged as positive, otherwise it is judged as negative.

[0035] Furthermore, the device also includes a sample nucleic acid extraction module for extracting RNA from the sample.

[0036] The fifth aspect of the present invention provides any of the following applications.

[0037] Furthermore, the application includes: 1) The probe set described in the first aspect of the present invention is prepared for detection MET Application in products that skip exon 14 of a gene; 2) The probe set described in the first aspect of the present invention or the kit described in the second aspect of the present invention is used for MET Application in gene exon 14 skipping sequencing.

[0038] Advantages and benefits of the present invention: 1. Research shows that the current common second-generation sequencing technology based on DNA level is MET 14 Exon skipping detection often leads to missed detection. The present invention establishes a method for detecting RNA levels based on the probe-targeted capture principle. MET The probe set and analysis method for exon 14 skipping can serve as a supplement and verification of DNA-level detection, improving detection capabilities. In addition, the target region does not need to cover introns, the probe design is simple, the sequencing data volume requirement is low, and the detection cost is low.

[0039] 2. Compared with multiplex PCR capture technology detection MET 14 Exon skipping method. The detection method developed by the present invention based on the probe targeted capture principle is less affected by low-quality RNA, can capture fusions of other patterns, and can also be flexibly added to other mixed pools of the same type of probes to complete multi-target synchronous detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Schematic diagram of the design of the primer set provided by the present invention. DETAILED DESCRIPTION

[0041] The technical solutions of the present application will be described clearly and completely in combination with the embodiments. Obviously, the described embodiments are only some of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of the present application. The specific conditions not mentioned in the embodiments are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments not mentioned by the manufacturer are conventional products that can be purchased in the market.

[0042] The present application aims to establish a probe target capture technology for detecting RNA level MET 14 exon skipping probe set and analysis method, as a supplement and verification of DNA level detection, to improve MET The detection rate and accuracy of 14 exon skipping. The present application first designs a capture probe for the region of 13 exon and 15 exon fusion. The specific probe design method is as shown in Figure 1 The 13 exon and 15 exon are close to the fusion breakpoint position 2X probe coverage, and a cross-breakpoint fusion probe sequence containing a 60 bp sequence close to the 3' end of the 13 exon and a 60 bp sequence at the 5' end of the 15 exon is designed. Finally, a total of 6 probe combinations containing 120 nt are designed. Then the total RNA sample is fragmented, reverse transcribed and library constructed to obtain the intermediate library of the RNA sample. The intermediate library is hybridized with the probe set, the library fragments containing the target sequence are complementary to the probe, and the probe is captured by streptavidin magnetic beads, and the fragments not captured are washed away; then the hybridization capture of the library containing the target sequence is completed by PCR amplification and magnetic bead sorting purification. The sequencing data is obtained by high-throughput sequencing, and the 14 exon skipping variation information can be obtained after analysis by the corresponding bioinformatics analysis software. MET

[0043] Embodiment 1 MET 14 Exon skipping positive standard detection I. Step 1-RNA library construction and probe hybridization capture 1. Experimental materials Sample: purchased MET 14 Exon skipping RNA positive standard, manufacturer: Nanjing Kebai Biotechnology Co., Ltd., product number: CBP20171R; ddPCR detection: MicroDrop-100B microdroplet digital PCR system is used to detect RNA fusion frequency, the primers and probes used in the experiment are provided by Nanjing Kebai, and the ddPCR reagents and consumables are provided by Yongnuo; ​Library construction reagent: a commercial RNA library construction reagent kit developed for Illumina high-throughput sequencing platform; double-end UDI adapter: a commercial double-end adapter containing 8 nt unique sample tag (Index) suitable for Illumina high-throughput sequencing platform; Hybrid capture reagent: a commercial library hybrid capture reagent kit for Illumina® sequencing platform; Hybrid capture probe: a biotin-labeled single-stranded DNA probe designed and synthesized for RNA level MET 14 Exon skipping target region design and synthesis of biotin-labeled single-stranded DNA probe, a total of six, primer specific information as shown in Table 1;

[0044] Table 1 Primer information

[0045]

[0046] 2. Experimental method

[0047] (1) Preparation of MET14 exon skipping RNA positive sample

[0048] Take MET14 exon skipping RNA positive standard and known negative clinical sample RNA to prepare different copy number gradient of simulated positive samples, and detect the copy number by ddPCR, the specific sample information is shown in Table 2.

[0049] Table 2 Sample information

[0050]

[0051] (2) RNA library construction

[0052] Step 1. RNA fragmentation

[0053] Take 200 ng of prepared RNA positive sample with RNA-free gun head into a 0.2 mL PCR tube, add nuclease-free water to 10 μL, and place on ice; add 10 μL of 2×fragmentation buffer to each sample tube, vortex to mix, and instantaneously separate; place the fragmentation reaction system in a PCR instrument, and run the program according to Table 3.

[0054] Table 3 RNA fragmentation PCR running program

[0055]

[0056] Step 2. First-strand cDNA synthesis

[0057] Add 8.5 μL of single-strand synthesis reagent to the fragmentation product, vortex to mix, and instantly separate. Place the single-strand synthesis reaction system in a PCR instrument, and run the program in Table 4.

[0058] Table 4. PCR running program for first-strand cDNA synthesis

[0059]

[0060] Step 3. Second-strand cDNA synthesis

[0061] Add 25 μL of second-strand synthesis enzyme and 5 μL of random primer to the single-strand synthesis product in sequence, vortex to mix, and instantly separate. Place the second-strand synthesis reaction system in a PCR instrument, and run the program in Table 5.

[0062] Table 5. PCR running program for second-strand cDNA synthesis

[0063]

[0064] Step 4. cDNA magnetic bead purification

[0065] After the second-strand synthesis reaction is completed, add 105 μL (1.8 ×) of AMPure XP (manufacturer: Beckman Coulter, product number: A63882) magnetic beads to the reaction system for purification, wash twice with 180 μL of 80% ethanol, discard the supernatant, and resuspend in 52 μL of nuclease-free water after drying at room temperature. After magnetic separation, take 50 μL of the supernatant to a new 0.2 mL PCR tube.

[0066] Step 5. End repair and A tailing

[0067] Vortex to mix the double-strand cDNA product according to Table 6, and instantly separate. Place the reaction system in a set PCR instrument, and run the program in Table 7.

[0068] Table 6. End repair and A tailing system

[0069]

[0070] Table 7. PCR reaction program for end repair and A tailing

[0071]

[0072] Step 6. Preparation of adapter ligation reaction system

[0073] Prepare the ligation reaction system premix according to Table 8, vortex to mix, and instantly separate. Place at room temperature for 30-45 min.

[0074] Table 8. Ligation reaction system

[0075]

[0076] Step 7. Ligation

[0077] After end repair and A tailing, 25 μL of ligation reaction system premix was added to the product, vortexed to mix and instantaneously separated; 5 μL of RNA adapter was added, vortexed to mix and instantaneously separated. The ligation reaction system was placed in the set PCR instrument, and the program in Table 9 was run.

[0078] Table 9. Ligation PCR reaction program

[0079]

[0080] Step 8. Magnetic bead purification after ligation

[0081] 80 μL (0.8x) of AMPure XP magnetic beads were added to the ligation product for purification, washed twice with 180 μL of 80% ethanol, and the supernatant was discarded; after drying at room temperature, 35 μL of nuclease-free water was added to resuspend, and after magnetic separation, 34 μL of supernatant was taken to a new 0.2 mL PCR tube.

[0082] Step 9. Library pre-amplification

[0083] After purification, the ligation product was added with PCR reagents according to Table 10, shaken to mix and instantaneously separated; 5 μL of the corresponding double-end tag primer pair was added, shaken to mix and instantaneously separated; the reaction system was placed in the PCR instrument, and the program in Table 11 was run.

[0084] Table 10. Library pre-amplification PCR reaction system

[0085]

[0086] Table 11. Library pre-amplification PCR reaction program

[0087]

[0088] Step 10. Magnetic bead purification after amplification

[0089] 50 μL (1.0x) of AMPure XP magnetic beads were added to the PCR reaction system for purification, washed twice with 180 μL of 80% ethanol, and the supernatant was discarded; after drying at room temperature, 22 μL of nuclease-free water was added to resuspend, and after magnetic separation, 20 μL of supernatant was taken to a new 0.2 mL PCR tube, which was the intermediate library in the RNA sample.

[0090] Step 11. Quality inspection of intermediate library

[0091] The intermediate library concentration was determined using a Qubit 3.0 fluorometer, and the library fragment distribution was detected using an Agilent 2100. The total amount of the library was required to be ≥ 500 ng, and the main peak of the library fragment distribution was required to be in the range of 250-500 bp.

[0092] (3) Probe hybridization capture

[0093] Step 1. Mixing and concentration of intermediate library

[0094] 500 ng of each intermediate library was taken into a 1.5 mL centrifuge tube, mixed, and 10 μL of Cot DNA and 2 μL of universal blocking sequence were added and mixed well and then centrifuged. The mixed system was concentrated to dry powder at 60°C in a concentrator.

[0095] Step 2. Hybridization reaction

[0096] The hybridization reagent was added to the concentrated sample according to Table 12, mixed well, and transferred to a new 0.2 ml PCR tube, and incubated at room temperature for 10 min. After incubation, it was placed in a PCR instrument, incubated at 95°C for 5 min, and then incubated at 65°C for 2 h.

[0097] Table 12 Hybridization reaction system

[0098]

[0099] Step 3. Streptavidin magnetic bead cleaning

[0100] 50 μL of streptavidin magnetic beads were taken and washed twice with 100 μL of 1× magnetic bead cleaning solution, and the supernatant was discarded. Finally, 50 μL of 1× magnetic bead cleaning solution was added to resuspend the beads, and the mixture was transferred to a new 0.2 ml PCR tube.

[0101] Step 4. Capture reaction

[0102] 16 μL of the hybridization product was transferred to the PCR tube containing the capture magnetic beads, mixed well, and incubated at 65°C for 30 min for capture reaction.

[0103] Step 5. Post-capture cleaning

[0104] 65°C cleaning: 120 μL of 65°C preheated 1× hybridization cleaning solution 1 was added to each tube, and after magnetic separation, the supernatant was discarded. Then, 150 μL of 65°C preheated 1× hybridization cleaning solution 4 was added, and after magnetic separation, the supernatant was discarded. The above step was repeated once.

[0105] Room temperature washing: add 150 μL of room temperature 1x hybridization wash 1, wash, and then discard the supernatant by magnetic absorption; add 150 μL of room temperature 1x hybridization wash 2, wash, and then discard the supernatant by magnetic absorption; add 150 μL of room temperature 1x hybridization wash 3, transfer the system to a new PCR tube with the corresponding number, and then discard the supernatant by magnetic absorption; and add 23 μL of nuclease-free water for resuspension.

[0106] Step 6. Library amplification after capture

[0107] Add the PCR reaction reagents in Table 13 to the sample after capture, and mix well by vortexing. Place the sample in a PCR instrument, and run the program in Table 14.

[0108] Table 13. PCR reaction system after capture

[0109]

[0110] Table 14. PCR reaction program for library amplification after capture

[0111]

[0112] Step 7. Magnetic bead purification after library amplification Add 50 μL (1.0x) of AMPure XP magnetic beads to the PCR reaction product for purification, wash twice with 180 μL of 80% ethanol, and discard the supernatant; after drying at room temperature, resuspend with 25 μL of nuclease-free water, and then separate by magnetic separation, and then transfer 23 μL of the supernatant to a new 1.5 mL centrifuge tube, which is the library after capture.

[0113] Step 8. Quality inspection of the library after capture Use the Qubit 3.0 fluorescence quantifier to determine the concentration of the library after capture, and use the Agilent 2100 to detect the fragment distribution of the library. The library concentration is required to be ≥ 3 nM, and the main peak of the library fragment distribution is in the range of 250-500 bp.

[0114] 3. Experimental results The total amount of the intermediate library constructed from each sample is > 500 ng, the main peak of the library fragment distribution is in the range of 250-500 bp, the concentration of the library after capture is > 3 nM, and the main peak of the library fragment distribution is in the range of 250-500 bp.

[0115] II. Step 2 - Library sequencing and data analysis 1. Experimental method The captured library was sequenced using the Illumina platform using the PE150 protocol. After the sequencing data was downloaded, the captured RNA molecule sequencing data was first aligned to the hg19 human reference genome using Sentieon STAR software, generating a bam file containing the alignment information for all sequencing reads. Because the PE150 sequencing strategy was used, the same RNA molecule fragment was sequenced from both ends to generate two reads, R1 and R2. These two reads were merged into a single complete read based on their alignment positions. The merged reads were then classified based on their alignment information: (1) If the read segment is correctly aligned to the end of exon 13, the read segment is recorded as d; (2) If a gap occurs in the read segment, one end of the gap can be correctly aligned to the end of exon 13, and the other end can be correctly aligned to the start of exon 15. This read segment is recorded as k; (3) The total number of sequenced reads is denoted as n.

[0116] Considering support MET 14 The number of reads that jump will be affected by MET The influence of gene expression and total sequencing data volume means that even for the same sample, the number of supporting reads obtained from sequencing different data volumes will be different. In order to reduce the bias caused by different sequencing depths and improve the accuracy and reliability of data analysis, the number of supporting reads for variants is normalized based on the consideration of the number of supporting reads for variants as the basis for judging positive results, and the number of supporting reads for variants is obtained per megabyte of sequencing reads: (1) Mutation frequency = k / d, (number of variant support reads / coverage MET Number of reads for exon 13); (2) Number of variant-supporting reads per megabyte of sequencing reads = (k*1000000) / n ((number of variant-supporting reads*1000000) / total number of sequencing reads).

[0117] If the number of variant support reads per megabyte of sequencing reads in the sample is greater than or equal to 0.4, or the variant frequency is greater than or equal to 3%, or the number of variant support reads is greater than or equal to 4, then it is judged as positive, otherwise it is judged as negative.

[0118] 2. Experimental results The sequencing analysis results are shown in Table 15. In this experiment, the prepared simulation samples were all detected by this detection method. MET 14 Exon skipping was positive, but not in known negative clinical samples MET 14 Exon skipping supported reads detection, indicating the accuracy of the detection results of the present invention.

[0119] Table 15 MET14 exon skipping positive standard sequencing data analysis results

[0120]

[0121] Example 2 Detection of brain glioma clinical samples

[0122] For 3 clinical paraffin samples from high-grade astrocytoma (WHO grade 4) and 3 from other types of central nervous system tumors, the library construction and sequencing data analysis were performed according to the method in the foregoing Example 1.

[0123] 6 clinical samples were subjected to library construction according to the method in Example 1, the intermediate library yield was >500 ng, and the main peak of the library fragment distribution was at 250-500 bp; after capture, the library concentration was >3 nM, and the main peak of the fragment distribution was at 250-500 bp.

[0124] The sequencing data analysis results are shown in Table 16. The results show that the MET14 exon skipping positive occurring in high-grade glioma can be detected by the present application, indicating that the present application has practicality.

[0125] Table 16 Sequencing data analysis results of brain glioma clinical samples

[0126]

[0127] Obviously, the above-described embodiments are only a part of the embodiments of the present application, rather than all the embodiments, and the preferred embodiments of the present application are given in the drawings, but do not limit the patent scope of the present application. The present application can be realized in many different forms, and on the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions recorded in the foregoing specific embodiments, or equivalently replace some technical features thereof. Any equivalent structure made by using the content of the present application specification and drawings, directly or indirectly applied to other related technical fields, is also within the patent protection scope of the present application.

Claims

1. RNA level detection MET The probe set for skipping exon 14 of a gene is characterized in that: The probe group comprises any one of the probes having nucleotide sequences as shown in SEQ ID NOs. 1 to 6, or a probe having the same function as the probe.

2. The probe set according to claim 1, characterized in that The probe with the same function refers to any one of the probes of SEQ ID NO. 1 to 6, which has one or more nucleotides replaced and / or deleted and / or added and has the same hybridization capture function; Preferably, the probe having the same function has more than 80% identical bases with the original probe, more preferably more than 90% identical bases, and even more preferably more than 95% identical bases.

3. RNA level detection MET A kit for skipping exon 14 of a gene, characterized in that: The kit comprises the probe set according to any one of claims 1 to 2; Preferably, the kit further comprises any one or a combination of at least two of an RNA fragmentation reagent, a cDNA single-strand synthesis reagent, a cDNA double-strand synthesis primer, a cDNA double-strand synthesis reagent, a linker ligation reagent, a hybridization capture reagent, a hybridization product amplification reagent or streptavidin affinity magnetic beads.

4. RNA level detection MET A method for skipping exon 14 of a gene, characterized in that: The method comprises performing probe hybridization capture on an RNA intermediate library obtained from a sample to be tested using the probe set according to any one of claims 1 to 2.

5. The method according to claim 4, characterized in that The sample to be tested is selected from one or more of tumor cells, blood, plasma, exosome solution, pleural effusion, peritoneal effusion, saliva, urine, and tissue; Preferably, the tumor cell is a central nervous system tumor, pharyngeal cancer, adrenal tumor, AIDS-related cancer, alveolar soft tissue sarcoma, astrocytoma, bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumor, breast cancer, carotid body tumor, cervical cancer, chondrosarcoma, chordoma, renal chromophobe cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, desmoplastic small round cell tumor, ependymoma, Ewing tumor, extraskeletal myxoid chondrosarcoma, fibrous dysplasia, fibrous dysplasia, gallbladder or bile duct cancer, gastric cancer, primary liver cancer, malignant teratocarcinoma, ovarian embryonal tumor, viral hepatitis, gestational trophoblastic disease with cirrhosis, germ cell tumor, head and neck cancer, hepatocellular carcinoma, one or more of pancreatic islet cell tumor, Kaposi's sarcoma, kidney cancer, leukemia, liposarcoma / malignant lipomatous tumor, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, biliary cell carcinoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer, papillary thyroid cancer, parathyroid cancer, pediatric cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, melanoma, metastatic kidney cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, synovial sarcoma, testicular cancer, thymic cancer, thymoma, metastatic thyroid cancer, or uterine cancer; More preferably, the tumor cells are non-small cell lung cancer cells or brain glioma cells.

6. The method according to claim 4, characterized in that The step of obtaining an RNA intermediate library from a sample to be tested includes extracting RNA from the sample, fragmenting the sample RNA, sequentially subjecting the fragmented RNA to cDNA single-strand synthesis, cDNA second-strand synthesis, end-removal and A addition, and adapter ligation, amplifying and purifying, and then performing Qubit quantification and 2100 quality control testing to obtain the RNA intermediate library.

7. The method according to claim 4, characterized in that The method also includes library amplification and purification after probe hybridization capture, sequencing and analysis of sequencing results; Preferably, the sequencing is performed using the Illumina platform in PE150 mode; Preferably, the step of analyzing the sequencing results includes aligning the captured RNA molecule sequencing data to the hg19 version of the human reference genome to obtain alignment information of all sequencing reads; merging the alignment positions of the two reads R1 and R2 obtained by sequencing the same RNA molecule fragment from both ends into a complete read; and classifying according to the alignment information of the merged reads: 1) If the read segment is correctly aligned to MET At the end of exon 13 of the gene, this read is denoted as d; 2) If a gap occurs in the read segment, one end of the gap can be correctly aligned to MET The end of exon 13 of the gene, and the other end can be correctly aligned to MET The start of exon 15 of the gene, this read segment is denoted as k; 3) The total number of sequenced reads is recorded as n; The variant support reads were then normalized to the variant frequency and the variant support reads per megabyte of sequencing reads: a) Mutation frequency = k / d; b) Number of variant support reads per megabyte of sequencing reads = (k*1000000) / n; If the number of variant support reads per megabyte of sequencing reads is greater than or equal to 0.4, or the variant frequency is greater than or equal to 3%, or the number of variant support reads (k) is greater than or equal to 4, the sample is judged as positive, otherwise it is judged as negative.

8. A RNA level detection MET A device for skipping exon 14 of a gene, characterized in that: The device comprises: (1) RNA intermediate library preparation module: The obtained sample RNA is fragmented, and the fragmented RNA is subjected to cDNA single-strand synthesis, cDNA second-strand synthesis, end-removal A addition, and adapter ligation. After amplification and purification, the RNA intermediate library is obtained after Qubit quantification and 2100 quality control detection; (2) Probe hybridization capture module: adding the probe group described in any one of claims 1-2 for hybridization capture; (3) Amplification and sequencing module: The hybridization capture product is amplified, purified, and quality controlled before sequencing using the Illumina platform in PE150 mode. (4) Analysis module: Align the captured RNA molecule sequencing data to the hg19 version of the human reference genome to obtain the alignment information of all sequencing reads; merge the alignment positions of the two reads R1 and R2 obtained from sequencing the same RNA molecule fragment from both ends into a complete read; classify according to the alignment information of the merged reads: 1) If the read segment is correctly aligned to MET At the end of exon 13 of the gene, this read is denoted as d; 2) If a gap occurs in the read segment, one end of the gap can be correctly aligned to MET The end of exon 13 of the gene, and the other end can be correctly aligned to MET The start of exon 15 of the gene, this read segment is denoted as k; 3) The total number of sequenced reads is recorded as n; The variant support reads were then normalized to the variant frequency and the variant support reads per megabyte of sequencing reads: a) Mutation frequency = k / d; b) Number of variant support reads per megabyte of sequencing reads = (k*1000000) / n; (5) Result output module: If the number of variant support reads per megabyte of sequencing reads of the sample is greater than or equal to 0.4, or the variant frequency is greater than or equal to 3%, or the number of variant support reads (k) is greater than or equal to 4, it is judged as positive, otherwise it is judged as negative.

9. The device according to claim 8, characterized in that The device also includes a sample nucleic acid extraction module for extracting RNA from the sample.

10. Any of the following applications, characterized in that: The applications include: 1) The probe set according to any one of claims 1-2 is used in the preparation of a detection MET Application in products that skip exon 14 of a gene; 2) The probe set according to any one of claims 1 to 2 or the kit according to claim 3 is used for MET Application in gene exon 14 skipping sequencing.

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