Methods for detecting partner genes

Abstract

The present invention relates to a method for detecting a partner gene, which comprises extracting total RNA of a sample, reverse transcribing to obtain cDNA, then fragmenting into 150-250 pb fragments, repairing and adding A at the ends, connecting sequencing adapters, and obtaining a prelibrary; blocking the prelibrary with a blocking blocker, then hybridizing a specific probe set with the prelibrary to obtain captured fragments, enriching and purifying the fragments by PCR, and then performing quality inspection to obtain a sequencing library; sequencing the sequencing library using second-generation sequencing to obtain gene fusion information. The method of the present invention can still maintain a high capture efficiency while using fewer probes and a lower amount of sequencing data, obtain more positive reads than traditional capture, reduce probe synthesis and sequencing costs, and improve detection sensitivity.

Description

[0001] This application is a divisional application with the application date of August 31, 2020, application number CN202010895988.8, and the invention name is "Targeted sequencing method for detecting gene fusion." Technical Field

[0002] The present invention relates to targeted methods, and more particularly, the present invention relates to methods for detecting partner genes. Background Art

[0003] Cancer cells frequently undergo gene fusion events through chromosomal rearrangements, such as translocations, deletions, and insertions. With increasing awareness of the clinical importance of fusion genes, accurate diagnosis of fusion genes is gaining increasing attention. Currently, a variety of targeted cancer therapies are available to inhibit fusion genes, including imatinib / BCR-ABL1, crizotinib / EML4-ALK, and larotrectinib / NTRK fusions.

[0004] Rapid and accurate diagnosis of fusion genes not only allows for the diagnosis and classification of cancer but also provides essential information for subsequent treatment. Currently, fusion gene diagnosis primarily involves methods such as fluorescence in situ hybridization (FISH) and IHC. However, these assays typically have low throughput, rely on the experience of the examiner, and are only applicable to known fusion subtypes. Reports indicate that the sensitivity of IHC for detecting NTRK3 fusions is reduced, reaching only 79% [Identifying patients with NTRK fusion cancer.].

[0005] Although several emerging fusion gene detection technologies, such as Nanostring (ncounter Vantage 3D™ Assays) and Agena MassArray, avoid subjective judgment by technicians and offer higher throughput and sensitivity, they are unable to detect new fusion subtypes [Overview of Fusion Detection Strategies Using Next-Generation Sequencing]. Furthermore, whole-transcriptome sequencing based on next-generation sequencing is expensive and incompatible with clinical samples, while multiplex amplicon targeted sequencing has a high false-positive rate and requires the establishment of a population baseline and the use of bioinformatics methods to filter low-confidence results. Summary of the Invention

[0006] To address at least some of the technical issues in the prior art, the present invention provides a targeted sequencing method for detecting gene fusions. Compared with traditional probe designs, the probes of the present invention have higher binding efficiency to the target exon region, require fewer probes, cover a smaller region, and are less expensive.

[0007] The targeted sequencing method for detecting gene fusion of the present invention comprises the following steps:

[0008] (1) Preliminary library construction, which includes extracting total RNA from the sample, reverse transcribing to obtain cDNA, then fragmenting into 150-250 pb fragments, repairing the ends, adding A, and ligating sequencing adapters to obtain a prelibrary;

[0009] (2) blocking the prelibrary with a blocking agent, then hybridizing the probe set with the prelibrary to obtain captured fragments, enriching and purifying the fragments by PCR, and then performing quality inspection to obtain a sequencing library;

[0010] (3) sequencing the sequencing library using next-generation sequencing to obtain gene fusion information;

[0011] The probe set consists of a first probe that is complementary to the 5' end and / or 3' end of each exon of gene A or a second probe that is complementary to the 5' end and / or 3' end of each exon of gene B.

[0012] According to the targeted sequencing method of the present invention, preferably, the 5' end refers to the fragment between the 1st base and the 120th base at the 5' end of the exon, and the 3' end refers to the fragment between the last base and the 120th base from the 3' end of the exon.

[0013] According to the targeted sequencing method of the present invention, preferably, the gene A is a 5' partner gene, and the first probe is complementary to the 3' end of each exon of the 5' partner gene; the gene B is a 3' partner gene, and the second probe is complementary to the 5' end of the 3' partner gene.

[0014] According to the targeted sequencing method of the present invention, preferably, the first probe and the second probe are probes modified with biotin at the 5' end.

[0015] According to the targeted sequencing method of the present invention, preferably, the step (1) is to construct the library using total RNA without removing ribosomal RNA.

[0016] According to the targeted sequencing method of the present invention, preferably, in step (1), PCR enrichment and purification steps are further included after connecting the sequencing adapter.

[0017] According to the targeted sequencing method of the present invention, preferably, in the hybridization step of step (2), the pre-library is a plurality of different pre-libraries.

[0018] This invention reduces the number of probes by optimizing probe design, while also improving capture efficiency by optimizing hybridization and probe label structure. This allows for higher capture efficiency and more positive reads than traditional methods, while using fewer probes and requiring less sequencing data. This reduces probe synthesis and sequencing costs, significantly improving detection sensitivity. Furthermore, it eliminates the need for prior knowledge of the fusion gene and is capable of detecting unknown fusion subtypes. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Flowchart of exemplary fusion gene detection of the present invention.

[0020] Figure 2 Schematic diagram comparing an exemplary probe design method of the present invention with a traditional method.

[0021] Figure 3 The effect of different binding lengths between the probe and the target fragment on the binding efficiency.

[0022] Figure 4 Schematic diagram of the probe design for ALK transcripts according to the present invention.

[0023] Figure 5 Schematic diagram of the EML:exon13 / ALK:exon20 fusion probe designed for the present invention. DETAILED DESCRIPTION

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terms described in the present invention are only for describing particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges in the present invention, it should be understood that the upper and lower limits of the ranges and each intermediate value therebetween are specifically disclosed. Each smaller range between any stated value or intermediate value within a stated range and any other stated value or intermediate value within the stated range is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0026] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related thereto. In the event of any conflict with any incorporated document, the present specification shall prevail. Unless otherwise specified, "%" refers to percentages based on weight.

[0027] The present invention provides a targeted sequencing method for detecting gene fusions, sometimes referred to herein as the "targeted sequencing method of the present invention." Gene fusions include fusions between two genes, Gene A and Gene B, as well as fusions between three or more genes, such as three genes. Gene fusions of the present invention preferably occur between exons of different genes, and do not include fusions within exons derived from different genes. Fusion genes may be referred to as partner genes of each other.

[0028] The targeted sequencing method of the present invention generally comprises the following steps:

[0029] (1) Preliminary library construction, which includes extracting total RNA from the sample, reverse transcribing to obtain cDNA, then fragmenting into 150-250 bp fragments, repairing and adding A at the ends, and ligating sequencing adapters to obtain a prelibrary;

[0030] (2) blocking the prelibrary with a blocking agent, then hybridizing the probe set with the prelibrary to obtain captured fragments, enriching and purifying the fragments by PCR, and then performing quality inspection to obtain a sequencing library;

[0031] (3) Sequencing the sequencing library using second-generation sequencing to obtain gene fusion information.

[0032] Step (1) of the present invention is a pre-library construction step. It generally involves total RNA extraction, reverse transcription into cDNA, fragmentation, and sequencing adapter connection. Optionally, an enrichment step is further included after connection of the sequencing adapter. The method designed by the present invention will not be affected by ribosomal RNA. Therefore, when constructing a library using total RNA, it is not necessary to remove ribosomal RNA. The fragmentation of the present invention can be carried out using known methods, such as ion fragmentation or ultrasonic fragmentation, as long as fragments of 150-250 bp can be obtained.

[0033] Step (2) of the present invention is a step of constructing a sequencing library, which includes blocking the pre-library with a blocking agent, then hybridizing the probe set with the pre-library to obtain capture fragments, and then enriching and purifying them by PCR and quality inspection to obtain a sequencing library.

[0034] The blocking blocker of the present invention can use any known blocker. Preferably, a universal blocker designed by the inventor is used. The universal blocker of the present invention is composed of two oligonucleotides containing natural nucleotides and artificial nucleotides, and does not contain an index sequence (index sequence). Preferably, a spacer arm is designed at the 3' end of the universal blocker to prevent the 3' end exonuclease and 3' end polymerase from taking effect. Preferably, the artificial nucleotide is a locked nucleic acid. Locked nucleic acid has a higher Tm value. By controlling the ratio of locked nucleic acid in the universal blocker, the stability of the double-stranded structure formed can be easily controlled.

[0035] In the present invention, the probe set is composed of a first probe that can complement the 5' end and 3' end of each exon of gene A or a second probe that can complement the 5' end and 3' end of each exon of gene B. It should be noted that the "first probe" and "second probe" herein are only used to distinguish whether they are complementary to gene A or gene B, and are not used to indicate the type of probe. In fact, the first probe or the second probe respectively contain the meaning of a combination of multiple different types of probes. Gene A and gene B do not necessarily represent genes that encode a complete full-length protein. In fact, gene A and gene B can also respectively represent genes that encode a certain part of a complete protein. In addition, gene A and gene B can each contain an exon. Preferably, gene A and gene B each contain multiple exons, for example, each contain 2, 3, 5, or even more exons.

[0036] In the present invention, there is no particular limitation on gene A and gene B, and they can be any gene that may be fused. Examples of gene A and gene B include, but are not limited to, ABL1, AKT1, AKT3, ALK, ARHGAP26, AXL, BRAF, BRD3, BRD4, CALCA, CAMTA1, CCNB3, CCND1, CIC, CTNNB1, DDR2, EGFR, EPC1, ERBB2, ERBB4, ERG, ESR1, ESRRA, ETV1, ETV4, ETV5, ETV6, EWSR1, FGFR1, FGFR2, FGFR3, FGR, FOXO1, FUS, GLI1, GNAS, HMGA2, HRAS, IDH1, IDH2, INSR, JAK2, JAZF1, KRAS, KRT20, MAML2, MAP2K1, MA ST1, MAST2, MEAF6, MET, MKL2, MSMB, MUSK, MYB, NCOA2, NOTCH1, NOTCH2, NRAS, NRG1, NTRK1, NTRK2, NTRK3, NUMBL, PDGFB, PDGFRA, PDGFRB, PIK3CA, PKN1, PLAG1, PPARG, PRKCA, PRKCB, PTH, PTK2B, RAF1, RARA, RELA, RET, ROS1, RSPO2, RSPO3, SLC5A5, SS18, STAT6, TAF15, TCF12, TERT, TFE3, TFEB, TFG, THADA, TMPRSS2, TTF1 and YWHAE genes. Preferably, gene A and gene B are selected from ALK, RET, ROS1, NTRK1, NTRK2, NTRK3, BRAF, PIK3CA, MET, FGFR1, FGFR2, FGFR3, PRKCA, PRKCB, KRAS, JAK2, AKT1 and AKT3.

[0037] Specifically, the first probe comprises one or more probes, each of which is capable of complementing the 5' end or 3' end of a certain exon in gene A. The plurality of probes in the first probe enables the 5' end and 3' end of each exon in gene A to bind complementarily to a corresponding probe. Similarly, the second probe comprises one or more probes, each of which is capable of complementing the 5' end or 3' end of a certain exon in gene B. The plurality of probes in the second probe enables the 5' end and 3' end of each exon in gene B to bind complementarily to a corresponding probe.

[0038] In an exemplary embodiment, where gene A comprises a first exon and a second exon, and gene B comprises a third exon and a fourth exon, a fusion event may occur between the 3' end of the first exon and the 5' end of the third exon, between the 3' end of the first exon and the 5' end of the fourth exon, between the 5' end of the first exon and the 3' end of the third exon, between the 5' end of the first exon and the 3' end of the fourth exon, between the 3' end of the second exon and the 5' end of the third exon, between the 3' end of the second exon and the 5' end of the fourth exon, between the 5' end of the second exon and the 3' end of the third exon, and between the 5' end of the second exon and the 3' end of the fourth exon.

[0039] In the present invention, the 5' end of an exon refers to a fragment between the 1st base and the 120th base from its 5' end, preferably a fragment between the 1st base and the 110th base, more preferably a fragment between the 1st base and the 100th base, and further preferably a fragment between the 1st base and the 90th base. The 3' end of an exon refers to a fragment between the last base and the 120th base from its 3' end, preferably a fragment between the last base and the 110th base from its 3' end, more preferably a fragment between the last base and the 100th base from its 3' end, and further preferably a fragment between the last base and the 90th base from its 3' end. In the present invention, the sum of the lengths of the 5' end and the 3' end of the same exon is generally less than the length of the entire exon. Preferably, the sum of the lengths of the 5' end and the 3' end is less than half of the total length of the exon, more preferably less than 2 / 3, or even 3 / 4 of the total length of the exon. In the probe set of the present invention, each probe is directed only to the 5' end and the 3' end of the exon, thereby greatly reducing the number of probes.

[0040] In order to further improve the binding force between the probe and the target sequence, the present invention further optimizes the label of the capture probe. Preferably, each probe (including each probe in the first probe and the second probe) is a probe modified with biotin at the 5' end. More preferably, the biotin molecule is connected to the hydroxyl group at the 5' end of the probe via a spacer arm. Preferably, the spacer arm has the structure of the following formula (I): -NH(CH2) m O-(-C=O-NH-(CH2-CH2O-)4) n , wherein m is an integer of 4 to 8, preferably an integer of 5 to 6, and n is an integer of 1 to 5, preferably an integer of 2 to 3. The present invention has found that by using formula (I) as a spacer arm, the capture efficiency during targeted sequencing can be greatly improved.

[0041] In the present invention, for 100-120 nt probes, hybridization and washing are preferably performed at 60-70°C; for 80-100 nt probes, hybridization and washing are preferably performed at 55-65°C; for 60-80 nt probes, hybridization and washing are preferably performed at 50-60°C.

[0042] In the present invention, in the hybridization step of step (2), the prelibrary can be one or a mixed library of multiple different prelibraries.

[0043] Step (3) of the present invention is to sequence the sequencing library using second-generation sequencing technology to obtain gene fusion information. Second-generation sequencing technology is sometimes also called high-throughput sequencing technology, which is a method of sequencing hundreds of thousands to millions of DNA molecules in parallel at a time, also known as next-generation sequencing technology. The core concept of second-generation sequencing technology is sequencing while synthesis, that is, determining the sequence of DNA by capturing the markers at the newly synthesized ends. Examples of second-generation sequencing technology include Illumina sequencing technology and Life Tech (Thermo Scientific) sequencing technology.

[0044] Example 1

[0045] This example studies the effect of the binding length between the probe and the target fragment on the probe binding force.

[0046] 1. Probe selection:

[0047] In order to detect the effect of the binding length of the probe and the target fragment on the probe binding force, a human genome probe pool with a size of approximately 100Kb was used for capture sequencing, and then 16 probes with no other probes within 1Kb upstream and downstream and a GC content of 47.5-52.5% were selected for analysis.

[0048] Table 1

[0049]

[0050]

[0051] 2. Preliminary library construction and capture sequencing:

[0052] The prelibrary used in this example was constructed using a DNA library construction kit (Rapid DNA Lib Prep Kit, ABclonal) from NA12878 gDNA (Coriell) (insert fragment size: ∼200 bp; PCR cycle number: 7).

[0053] Hybridization capture was performed for 4 hours according to the steps described in AJ.

[0054] A. Library Pre-blocking

[0055] The reagents in Table 2 were added to a 0.2 mL low-absorption centrifuge tube (Eppendorf), and the solution in the centrifuge tube was evaporated to dryness using a vacuum concentrator (Eppendorf) for later use.

[0056] Table 2

[0057]

[0058] B. Hybridization of probes to libraries

[0059] Add 13 μL of hybridization buffer (0.33 M Sodium phosphate buffer pH 7.0, 0.65% SDS (w / v), 1.31 mM EDTA, 1.31X SSC, 2.62X Denhardt's Solution, 20% formamide (v / v)) to the centrifuge tube in the above step, vortex to mix, and incubate at room temperature for 5 minutes.

[0060] Denature at 95°C for 10 minutes, then add 4 μL (3 pmol) of probe pool, vortex mix, and incubate at 65°C for 4 hours.

[0061] C. Cleaning solution preparation

[0062] Prepare the wash buffer as shown in Table 3. Preheat 1X Wash Buffer S and a portion of 1X Wash Buffer I at 65°C for 30 minutes before use.

[0063] Table 3

[0064]

[0065]

[0066] 1X Beads Wash Buffer: 1M NaCl, 10mM Tris-HCl pH 7.5, 1mM EDTA, 0.1% (v / v) Tween-20

[0067] 1X Wash Buffer S: 1X SSC, 0.1% (v / v) Tween-20, pH7.0

[0068] 1X Wash Buffer I: 1X SSC, 0.1% (w / v) SDS, pH7.0

[0069] 1X Wash Buffer II: 0.5X SSC, pH7.0

[0070] 1X Wash Buffer III: 0.2X SSC, pH7.0

[0071] D. Preparation of Streptavidin Magnetic Beads

[0072] Remove streptavidin magnetic beads (Dyna Beads M270, Invitrogen) from the refrigerator (4°C) and allow to return to room temperature (approximately 30 minutes). Vortex for 15 seconds. Add 100 μL of streptavidin magnetic beads to a new 1.5 mL low-binding centrifuge tube. Place the centrifuge tube on a magnetic rack until the solution clears. Aspirate and discard the supernatant without disturbing the beads. Wash the streptavidin magnetic beads as follows:

[0073] (1) Remove the centrifuge tube from the magnetic stand, add 200 μL 1X Beads Wash Buffer, and vortex for 10 seconds.

[0074] (2) Centrifuge the tube briefly and place it on a magnetic rack until the solution becomes clear. Aspirate and discard the supernatant without disturbing the magnetic beads.

[0075] Repeat steps (1) and (2).

[0076] Remove the centrifuge tube from the magnetic stand and add 100 μL of 1X Beads Wash Buffer. Transfer 100 μL of the magnetic bead resuspension from the centrifuge tube to a new 0.2 mL low-binding centrifuge tube (Eppendorf) for later use. Place the centrifuge tube on the magnetic stand until the solution clears. Discard the supernatant without disturbing the magnetic beads and proceed immediately to the next experimental step.

[0077] E. Streptavidin magnetic bead capture

[0078] Add the hybridization mixture to a 0.2 mL low-binding centrifuge tube containing streptavidin magnetic beads. Mix thoroughly by gently pipetting up and down 10 times. Incubate at 65°C for 45 minutes using a PCR instrument (heated lid set to 75°C). Vortex for 3 seconds every 12 minutes to ensure the beads remain suspended.

[0079] F. Post-capture cleaning

[0080] 1.65℃ cleaning steps:

[0081] Add 100 μL of preheated 1X Wash Buffer I to the 0.2 mL low-binding centrifuge tube containing the hybridization mixture. Pipet to mix thoroughly, then transfer the reaction solution containing the streptavidin magnetic beads to a new 1.5 mL low-binding centrifuge tube. Place the tube on a magnetic stand until the solution is clear, then discard the supernatant.

[0082] Continue cleaning as follows:

[0083] (1) Add 200 μL of preheated 1X Wash Buffer S, pipette or vortex to mix, and incubate at 65°C for 5 minutes.

[0084] (2) Centrifuge briefly and place the centrifuge tube on a magnetic rack until the solution becomes clear, then discard the supernatant.

[0085] Repeat steps (1) and (2).

[0086] 2. Clean at room temperature

[0087] Add 200 μL of 1X Wash Buffer I and vortex mix for 2 minutes. Briefly centrifuge the tube and place it on a magnetic rack until the solution is clear, then aspirate the supernatant. Add 200 μL of 1X Wash Buffer II and vortex mix for 1 minute. Briefly centrifuge the tube and place it on a magnetic rack until the solution is clear, then aspirate the supernatant. Add 200 μL of 1X Wash Buffer III and vortex mix for 30 seconds. Briefly centrifuge the tube and place it on a magnetic rack until the solution is clear, then aspirate the supernatant.

[0088] 3. Resuspend magnetic beads

[0089] Immediately add 20 μL of enzyme-free sterile water. Use a pipette to pipette up and down 10 times to resuspend the magnetic beads and proceed to the next experimental step.

[0090] G. PCR amplification

[0091] Configure the PCR reaction system according to Table 4.

[0092] Table 4

[0093]

[0094] Mix by pipetting or vortexing at low speed to keep the beads suspended and immediately proceed to the PCR step. Use a PCR instrument and run the program in Table 5, with the heated lid at 105°C.

[0095] Table 5

[0096]

[0097] H. PCR Product Purification

[0098] Add 75 μL of AMPure XP purification magnetic beads (Beckman) to each PCR tube. Purify the PCR product according to the AMPure XP instruction manual. Elute with 22 μL of Tris-HCl (10 mM, pH 8.5). Transfer 20 μL of the eluate containing the captured library to a new 1.5 mL low-binding centrifuge tube (Eppendorf).

[0099] I. Library Quality Control

[0100] The library concentration was measured using a Qubit Fluorometer 3.0 (ThermoFisher). The library fragment length was measured using an Agilent 2100, and the products were concentrated around 320 bp with no adapter dimerization.

[0101] J. High-throughput sequencing

[0102] Illumina Novaseq sequencer was used for PE150 sequencing.

[0103] 3. Data Analysis

[0104] Trimmomatic was used to remove adapters and low-quality sequences to obtain clean data. Samtools was then used to extract reads of the target region and analyze the size of the probe-DNA binding based on the alignment position.

[0105] The sequencing depths of Cap01 and Cap02 were 12948x and 13271x, respectively. The average number of target fragments captured by the binding length of 110-119 bp was used as a control, and the ratios of the target fragments captured by the binding length of 1-120 bp to the above controls were calculated.

[0106] like Figure 3 As shown, the minimum binding length of the probe to capture a fragment is approximately 40 bp. Its binding capacity gradually increases with binding length, reaching 50% at around 70 bp and saturating around 100 bp, entering a plateau. Therefore, for a 120 nt probe, ensuring that the probe binds to the target fragment for a length exceeding 100 bp will more effectively capture the target sequence. However, for shorter binding lengths (<70 bp), the probe's ability to capture the target fragment will be significantly impacted.

[0107] Example 2

[0108] This example is an example of fusion gene detection.

[0109] 1. Probe design and synthesis:

[0110] The traditional method and the method of the present invention were used to design probes for 90 genes related to tumor gene fusion mutations. Some gene information is shown in Table 6 below; taking ALK as an example, Figure 4 The figure shows the difference between the traditional method and the method of the present invention in probe design for ALK. The two methods use 39 and 29 120nt probes, respectively, covering the intervals of 4614bp and 3235bp. For the above 90 genes, the traditional method and the method of the present invention use 2075 and 1405 120nt probes, respectively. Among them, the specific transcripts and probe information of ALK, RET and ROS1 genes are shown below.

[0111] Table 6 Traditional probe design

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118] Table 7 Probe design of the present invention

[0119]

[0120]

[0121]

[0122]

[0123] In addition, Figure 5 As shown, fusion probes were designed for known fusion subtypes to further improve the sensitivity of fusion detection. The fusion probe sequences involved in ALK, RET and ROS1 are shown in Table 8 below.

[0124] Table 8

[0125]

[0126]

[0127]

[0128] 2. Preliminary library construction:

[0129] An mRNA-seq Lib Prep Kit for Illumina, ABclonal, was used to construct prelibraries of five total RNAs containing ALK, RET, and ROS1 fusion genes (RNA input: 100 ng; insert size: ~200 bp; PCR cycle number: 10).

[0130] The five prelibraries were subjected to hybridization capture using conventionally designed probes, probes designed in the present invention, and probes designed in the present invention with fusion probes, followed by next-generation sequencing and information analysis. The capture sequencing process was the same as in Example 1.

[0131] 3. Data Analysis

[0132] Trimmomatic was used to remove adapters and low-quality sequences to generate clean data. STAR was then used for sequence alignment, and STAR-fusion was used for fusion gene analysis. As shown in Table 9, data analysis demonstrates that the target rate using the probes designed by the present invention is similar to that of traditional methods, with no significant difference. Furthermore, because fewer probes are used and fewer intervals are covered, the present invention achieves sequencing depth similar to that of traditional designs using less data.

[0133] Table 9

[0134]

[0135]

[0136] Further fusion mutation analysis showed that the method of the present invention detected ALK, RET, and ROS1 fusion genes with 1.4-2.1 times the number of positive reads compared to the traditional design, demonstrating higher sensitivity. Furthermore, after the addition of fusion probes, the number of positive reads increased further to 1.5-3.6 times that of the design of the present invention, further enhancing sensitivity (Table 10). These results demonstrate that the probe design of the present invention achieves higher detection sensitivity using fewer probes and lower sequencing data volumes, and the addition of fusion probes allows for more efficient detection of known fusion gene subtypes.

[0137] Table 10

[0138]

[0139]

[0140] In summary, the present invention has the advantages of high throughput, high sensitivity, high capture efficiency and low cost. Compared with traditional technologies such as qPCR that can only detect one fusion subtype at a time, the present invention can detect the "width" of multiple genes at a time, which will greatly reduce the diagnosis time. In addition, the present invention can also discover new fusion subtypes without the need for prior knowledge of fusion genes. At the same time, the addition of fusion probes can further improve the detection sensitivity of known fusion subtypes. It can be foreseen that with the gradual enrichment of tumor-targeted drugs for fusion genes, the number of fusion genes will also increase, and traditional technologies such as qPCR will not be able to meet the detection needs of fusion genes. The present invention has extremely high application value and potential in the rapid and accurate diagnosis of fusion genes.

[0141] Although the present invention has been described with reference to exemplary embodiments, it should be understood that the invention is not limited to the disclosed exemplary embodiments. Various modifications and variations may be made to the exemplary embodiments of the present specification without departing from the scope or spirit of the present invention. The scope of the claims is to be given the broadest interpretation so as to encompass all such modifications and equivalent structures and functions.

Claims

1. A targeted sequencing method for detecting gene fusions, characterized in that: The gene fusion includes fusion between gene A and gene B, and the targeted sequencing method includes the following steps: (1) Preliminary library construction, which includes extracting total RNA from the sample, reverse transcribing to obtain cDNA, then fragmenting into 150-250 bp fragments, repairing and adding A at the ends, and ligating sequencing adapters to obtain a prelibrary; (2) using a blocking blocker to block the repeat region and the adapter sequence of the pre-library, then hybridizing the probe set to the pre-library, capturing the target region fragment bound by the probe by streptavidin magnetic beads, enriching and purifying the fragments by PCR, and then quality checking to obtain a sequencing library, wherein the blocking blocker is a universal blocker composed of two oligonucleotides containing natural nucleotides and artificial nucleotides, which does not contain an index sequence, and a spacer arm is designed at the 3' end of the universal blocker to prevent the 3' end exonuclease and 3' end polymerase from acting, and the artificial nucleotide is a locked nucleic acid; (3) sequencing the sequencing library using second-generation sequencing to obtain gene fusion information; Wherein, the probe group comprises the probes shown in Table 7; The method is not for diagnostic purposes.

2. The targeted sequencing method for detecting gene fusion according to claim 1, characterized in that The probes are respectively probes with 5'-end biotin modified.

3. The targeted sequencing method for detecting gene fusion according to claim 1, characterized in that The probe set further comprises a fusion probe that is complementary to a partial sequence of gene A and a partial sequence of gene B.

4. The targeted sequencing method for detecting gene fusion according to claim 1, characterized in that The step (1) is to construct the library using total RNA without removing ribosomal RNA.

5. The targeted sequencing method for detecting gene fusion according to claim 1, characterized in that: In the step (1), PCR enrichment and purification steps are further included after connecting the sequencing adapter.

6. The targeted sequencing method for detecting gene fusion according to claim 1, characterized in that: In the hybridization step of step (2), the prelibrary is a plurality of different prelibrary.

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