Multiplex PCR primer structure for detecting solid tumor gene fusion and method for constructing NGS library

Abstract

The invention relates to the technical field of gene detection, and discloses a multiple PCR primer structure for detecting solid tumor gene fusion and a method for constructing an NGS library, the multiple PCR primer structure for detecting solid tumor gene fusion and the method for constructing the NGS library comprise the following steps: S1, linker connection; s2, purification after connection; s3, carrying out specific amplification; s4, purifying after specific amplification: purifying a PCR amplification product obtained in S3 to obtain a purified NGS library; s5, sequencing on a machine and analyzing a result. According to the multiple PCR primer structure for detecting solid tumor gene fusion and the method for constructing the NGS library, an omega type target primer is designed for a main gene exon participating in fusion, so that all gene sequences fused by the exon can be amplified without difference while the specificity is greatly improved, and the purpose of detecting unknown gene fusion of partners is achieved; the method is simple to operate, short in time consumption, low in cost, high in specificity and capable of simultaneously detecting fusion of various genes.

Description

Technical Field

[0001] This invention relates to the field of gene detection technology, specifically to a multiplex PCR primer structure and an NGS library construction method for detecting gene fusions in solid tumors, used to detect the presence of one or more known or unknown gene fusions in tissue samples. Background Technology

[0002] A fusion gene is a process in which all or part of the sequences of two genes are fused together to form a new gene. This can be caused by chromosomal translocation, intermediate deletion, or chromosomal inversion. Currently, the main detection methods include fluorescence in situ hybridization (FISH), reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and next-generation sequencing (NGS), which can detect fusion genes at the DNA, RNA, and protein levels.

[0003] Among these methods, FISH offers high specificity and visually intuitive results. However, FISH is complex and time-consuming; it cannot identify the genes involved in the fusion or distinguish between different fusion types, potentially missing fusion genes with low incidence or complex rearrangements; conventional FISH methods require calculations of at least 50 cells, making it unsuitable for specimens with small tissue volumes (e.g., biopsy samples); the sequences of the two genes involved in the fusion must be known to design probes; the two genes involved in the fusion must be located close to each other on the chromosomes; and it cannot simultaneously detect the fusion of different genes with a known gene.

[0004] RT-PCR requires different primers to be designed for different fusion methods. It is highly operable and sensitive, but it can only detect a single fusion gene and cannot identify new fusion gene partners or resolve complex structural recombinations. It is also relatively expensive, which makes it difficult to detect fusion genes and reduces the detection rate.

[0005] Although the IHC method is simple and convenient, it cannot directly detect fusion genes, and the interpretation of the results is highly subjective.

[0006] High-throughput sequencing technology is expensive, time-consuming, and has strict requirements for nucleic acid integrity, and can usually only detect fusion genes with known partners;

[0007] In summary, current methods cannot effectively utilize Omega primers for multiplex single-end anchored PCR library construction to detect gene fusions of known and unknown partner genes, and cannot significantly simplify experimental procedures, improve experimental efficiency, or reduce costs. Therefore, we propose a multiplex PCR primer structure and NGS library construction method for detecting gene fusions in solid tumors. Summary of the Invention

[0008] The purpose of this invention is to provide a multiplex PCR primer structure for detecting gene fusions in solid tumors and a method for constructing NGS libraries, so as to solve the problems mentioned in the background art.

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] A method for constructing an NGS library to detect gene fusions in solid tumors includes the following steps:

[0011] S1. Adapter ligation: Mix total cDNA from the sample with the ligation system and perform adapter ligation reaction to obtain ligation product. The ligation system contains ligase and short Y adapter.

[0012] S2. Post-ligation purification: The ligation product obtained in S1 is purified to obtain a purified ligation product.

[0013] S3. Specific amplification: The purified ligation product obtained in S2 is mixed with the amplification system and subjected to PCR amplification to obtain a gene fusion detection library. The amplification system contains universal primers, specific primers and index primers. The specific primers are Omega structure primers designed for the near fusion site region of the main gene, and are used to target and amplify fragments at the fusion site of the main gene and its fusion partner.

[0014] S4. Purification after specific amplification: The PCR amplification product obtained in S3 is purified to obtain a purified NGS library.

[0015] S5. Sequencing and analysis of results.

[0016] In a further embodiment, the short Y-linker comprises a first oligonucleotide and a second oligonucleotide, the first oligonucleotide having a nucleotide sequence as shown in SEQ ID NO:1, and the second oligonucleotide having a nucleotide sequence as shown in SEQ ID NO:2; wherein the nucleotide sequence of SEQ ID NO:1 is: 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3', and the nucleotide sequence of SEQ ID NO:2 is: P-5'-GATCGGAAGAGC-3'.

[0017] In a further embodiment, the universal primer comprises a nucleotide sequence as shown in SEQ ID NO:3, wherein the nucleotide sequence of SEQ ID NO:3 is 5'-ACACTCTTTCCCTACACGAC.

[0018] In a further embodiment, the specific primer is a single-stranded DNA molecule that includes, from the 5' end to the 3' end, a first specific sequence, a universal sequence, and a second specific sequence; the universal sequence is 5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3'; the first specific sequence and the second specific sequence are complementary to the near-fusion site region of the main gene.

[0019] In a further embodiment, the index primer comprises, from the 5' end to the 3' end, a linker sequence 2, an index-specific sequence, and a universal sequence 2. The index-specific sequence is used to distinguish different samples, overcoming the limitation of traditional detection methods (such as FISH and RT-PCR) that can only detect known partner gene fusions. It can indiscriminately amplify all gene sequences fused to the target exon of the main gene. It can not only detect known fusion types such as ROS1-CD74 and ROS1-SLC34A2, but also detect unknown partner genes fused to the same exon, covering more potential fusion types, avoiding the omission of rearranged fusion genes with low incidence or complex structures, and improving the comprehensiveness and completeness of gene fusion detection in solid tumors.

[0020] In a further embodiment, the purification in steps S2 and S4 uses magnetic bead purification, which includes binding, washing, and elution steps.

[0021] In a further embodiment, the method uses ROS1-CD74 and ROS1-SLC34A2 fusion positive samples for detection.

[0022] In a further embodiment, the master gene is selected from at least one of ROS1, ALK, and RET.

[0023] In a further embodiment, the length of the targeted amplified fragment in step S3 is 370 bp, to avoid low amplification efficiency or interference from non-specific fragments due to excessively long fragments.

[0024] A multiplex PCR primer structure for detecting gene fusions in solid tumors, wherein the primer is an Omega-structure primer, comprising, from the 5' end to the 3' end, a first specific sequence, a universal sequence, and a second specific sequence; the first specific sequence and the second specific sequence are designed for the region near the fusion site of the main gene, and are used to target and amplify the fragment at the fusion site of the main gene and its fusion partner, wherein the universal sequence is 5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3'.

[0025] Compared with existing technologies, this invention provides a multiplex PCR primer structure for detecting gene fusions in solid tumors and a method for constructing NGS libraries, which has the following beneficial effects:

[0026] 1. This method for constructing multiplex PCR primers and NGS libraries for detecting gene fusions in solid tumors aims to comprehensively detect fusions of known and unknown partner genes. By designing Omega-structured specific primers and targeting the fusion site region of the main gene, it covers more fusion types and improves detection integrity. The specific primers are designed with an Omega structure, containing a first specific sequence, a universal sequence, and a second specific sequence from the 5' end to the 3' end. The first and second specific sequences are complementary to the fusion site region of the main gene (at least one of ROS1, ALK, and RET), which can accurately target the fusion site fragments of the main gene and its fusion partner.

[0027] Simultaneously, primers designed for specific exons of the main gene eliminate the need to predetermine the fusion partner gene sequence, thus overcoming the limitation of traditional detection methods that can only detect fusions of known partner genes. This allows for the indiscriminate amplification of all gene sequences fused to the target exon of the main gene. It can detect not only known fusion types such as ROS1-CD74 and ROS1-SLC34A2, but also unknown partner genes fused to the exon, covering more potential fusion types and avoiding the omission of rearranged fusion genes with low incidence or complex structures, thereby improving the comprehensiveness and completeness of gene fusion detection in solid tumors.

[0028] 2. This method for constructing NGS libraries and using multiplex PCR primers for detecting gene fusions in solid tumors aims to simplify experimental procedures, shorten detection time, and reduce costs. By replacing the hybridization capture process with ultra-multiplex single-end PCR and using standardized magnetic bead purification, experimental efficiency is optimized and detection costs are controlled. When constructing NGS libraries, the core process is "adapter ligation (S1) - purification after ligation (S2) - specific amplification (S3) - purification after amplification (S4) - sequencing and analysis of results (S5)". Compared with the hybridization capture process, ultra-multiplex single-end PCR does not require complex probe hybridization and elution steps, which greatly simplifies the experimental operation.

[0029] The magnetic bead purification method is standardized, time-saving, and does not require expensive hybridization probe reagents, thus reducing reagent costs. At the same time, the overall process does not require the calculation of no less than 50 cells as FISH, making it applicable to specimens with small tissue volumes (such as puncture biopsy samples), further broadening the range of applicable samples. Ultimately, it achieves the comprehensive effects of simplified experimental procedures, shortened detection time (avoiding the problem of long procedures in traditional methods), and reduced detection costs.

[0030] 3. This method for constructing multiplex PCR primers and NGS libraries for detecting gene fusions in solid tumors aims to improve detection specificity and accuracy. It reduces non-specific interference and ensures detection reliability by precisely controlling the amplified fragment length, optimizing primer and adapter design, and incorporating a sample differentiation mechanism. Specifically, in the S3 specific amplification step, the targeted design of Omega-structured primers precisely controls the amplified fragment length to 370 bp, avoiding low amplification efficiency or non-specific fragment interference caused by excessively long fragments. This achieves efficient ligation of total cDNA from the sample and reduces adapter dimer interference.

[0031] Meanwhile, the index primers contain adapter sequence 2, index-specific sequence, and universal sequence 2 from the 5' to 3' ends. The index-specific sequence is used to distinguish different samples, avoiding cross-contamination between samples. Thus, the 370bp targeted amplification fragment length can ensure amplification efficiency and specificity, reducing the amplification of non-target fragments. The specific sequence design of the short Y adapter improves ligation efficiency and reduces the impact of adapter-related impurities on subsequent amplification. The sample differentiation function of the index primers can accurately trace the detection results of each sample, avoiding misjudgments caused by cross-contamination. Overall, this improves the specificity of gene fusion detection, reduces false positive and false negative results, ensures the accuracy and reliability of detection results, and provides a more accurate basis for the diagnosis of gene fusion in solid tumors. Attached Figure Description

[0032] Figure 1 This is a flowchart of the overall method of the present invention;

[0033] Figure 2 This is a diagram showing the results of the reaction solution being placed on a PCR instrument according to the present invention.

[0034] Figure 3 This is a schematic diagram of the sequencing and analysis results in step S5 of the present invention. Figure 1 ;

[0035] Figure 4 This is a schematic diagram of the sequencing and analysis results in step S5 of the present invention. Figure 2 ;

[0036] Figure 5 This is a schematic diagram of the sequencing and analysis results in step S5 of the present invention. Figure 3 . Detailed Implementation

[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] Please see Figures 1-5 The present invention provides a technical solution:

[0039] A method for constructing NGS libraries to detect gene fusions in solid tumors.

[0040] Includes the following steps:

[0041] The method uses ROS1-CD74 and ROS1-SLC34A2 fusion positive samples for detection;

[0042] S1. Adapter ligation: The total cDNA from the sample is mixed with the ligation system, and an adapter ligation reaction is performed to obtain the ligation product. The ligation system contains ligase and a short Y-adaptor. Further, the short Y-adaptor contains a first oligonucleotide and a second oligonucleotide. The first oligonucleotide has a nucleotide sequence as shown in SEQ ID NO:1, and the second oligonucleotide has a nucleotide sequence as shown in SEQ ID NO:2. The nucleotide sequence of SEQ ID NO:1 is 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3', and the nucleotide sequence of SEQ ID NO:2 is P-5'-GATCGGAAGAGC-3'.

[0043] S2. Post-ligation purification: The ligation product obtained in S1 is purified to obtain a purified ligation product.

[0044] S3. Specific amplification: The purified ligation product obtained in S2 is mixed with the amplification system and subjected to PCR amplification to obtain a gene fusion detection library. The amplification system contains universal primers, specific primers, and index primers. The specific primers are Omega-structure primers designed for the near-fusion site region of the main gene, used to target and amplify fragments at the fusion site of the main gene and its fusion partner. Furthermore, the universal primers contain the nucleotide sequence shown in SEQ ID NO:3, where SEQ ID NO:3... NO:3 nucleotide sequence is 5'-ACACTCTTTCCCTACACGAC; further, the specific primer is a single-stranded DNA molecule, which includes, from the 5' end to the 3' end, the following sequences: a first specific sequence, a universal sequence, and a second specific sequence; the universal sequence is 5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3'; the first and second specific sequences are complementary to the near-fusion site region of the main gene; further, the index primer includes, from the 5' end to the 3' end, the following sequences: adapter sequence 2, index-specific sequence, and universal sequence 2; the index-specific sequence is used to distinguish different samples; further, the length of the fragment targeted for amplification in step S3 is 370 bp;

[0045] S4. Purification after specific amplification: The PCR amplification product obtained in S3 is purified to obtain a purified NGS library.

[0046] S5. Sequencing and analysis of results.

[0047] Furthermore, the purification in steps S2 and S4 uses magnetic bead purification, which includes binding, washing and elution steps; furthermore, the main gene is selected from at least one of ROS1, ALK and RET.

[0048] A multiplex PCR primer structure for detecting gene fusions in solid tumors is disclosed. The primers are Omega-structured primers, which include, from the 5' end to the 3' end, a first specific sequence, a universal sequence, and a second specific sequence. The first and second specific sequences are designed for the region near the fusion site of the main gene, and are used to target and amplify the fragment at the fusion site of the main gene and its fusion partner. The universal sequence is 5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3'.

[0049] In one embodiment of the present invention, the S1 connector connection specifically includes the following steps:

[0050] S1.1 After thawing the reagent, invert and mix well, then place on ice for later use;

[0051] S1.2, Prepare the reactions shown in Table 1 in the PCR tube:

[0052] Components Volume (ul) Previous product 50 RapidLigationbuffer 25 Rapid DNA Ligase 5 ddH2O 10 Custom DNAAdapter (added last) 10 total Total system 100

[0053] Table 1. Components and Volumes in PCR Tubes

[0054] S1.3. Use a pipette to gently mix the liquid (do not shake to mix), and briefly centrifuge to collect the reaction solution to the bottom of the tube;

[0055] S1.4 Place the PCR tube in the PCR instrument and perform the reactions shown in Table 2 below:

[0056]

[0057] Table 2. Temperature and timing for placing PCR tubes in the PCR instrument

[0058] In one embodiment of the present invention, purification after S2 ligation specifically includes the following steps:

[0059] S2.1. Take 60 μL of magnetic beads into an eight-tube bundle, add all the Step 2 product, mix by suction or vortex, and let stand at room temperature for 5 min.

[0060] S2.2 Instantaneous centrifugation: Place the sample on a magnetic rack and let it stand for 3 minutes. Once the liquid is clear, discard the supernatant and avoid contact with the magnetic beads.

[0061] S2.3 Add 180 μL of freshly prepared 80% ethanol, let stand for 30 seconds, and discard the supernatant after the solution becomes clear;

[0062] S2.4 Repeat the previous step, washing twice in total;

[0063] S2.5. Cap the tube and centrifuge briefly to remove residual ethanol to the bottom of the tube. Place the eight-tube strip on a magnetic rack and use a 10μL pipette to remove the residual ethanol. Let it stand at room temperature for 3 minutes to ensure that the ethanol evaporates completely, but do not over-dry.

[0064] S2.6 Remove the eight-tube strip from the magnetic rack, add 32μL of Nuclease-Free Water, mix by suction or vortexing, and let stand at room temperature for 2 minutes.

[0065] S2.7 Place the eight-tube strip on the magnetic rack for 2 minutes until the solution becomes clear;

[0066] S2.8. Transfer 30 μL of supernatant to a new PCR tube or eight-tube strip, label it, and prepare for Step 4 reaction. (Store at 4℃ for 7 days)

[0067] In one embodiment of the present invention, S3-specific amplification specifically includes the following steps:

[0068] S3.1 After thawing the reagents, invert and mix thoroughly, then prepare the PCR solution in sterile PCR tubes as shown in Table 3 below:

[0069]

[0070] Table 3. Components and volumes of reagents mixed in sterile PCR tubes

[0071] S3.2. Use a pipette to mix (avoid vigorous shaking), then centrifuge briefly.

[0072] S3.3. Set the PCR instrument program as follows: Place the reaction solution on the PCR instrument and run the program as follows. Figure 2 As shown.

[0073] In one embodiment of the present invention, purification after S4 specific amplification specifically includes the following steps:

[0074] S4.1. Take 45 μL of magnetic beads into an eight-tube, add all the product from Step 4, mix by suction or vortex, and let stand at room temperature for 5 minutes.

[0075] S4.2. Centrifuge briefly, place on a magnetic rack, let stand for 3 minutes, and discard the supernatant after the liquid becomes clear, avoiding contact with the magnetic beads;

[0076] S4.3 Add 180 μL of freshly prepared 80% ethanol, let stand for 30 seconds, and discard the supernatant after the solution becomes clear.

[0077] S4.4 Repeat the previous step, washing twice in total;

[0078] S4.5. Use a 10μL pipette to remove the residual ethanol, let it stand at room temperature for 3 minutes to ensure that the ethanol evaporates completely, but do not over-dry.

[0079] S4.6 Add 35μL Nuclease-FreeWater, remove the eight-tube strip from the magnetic rack, mix by suction or vortex, and let stand at room temperature for 2 minutes.

[0080] S4.7. Instant centrifugation: Place the eight-tube tube on a magnetic rack for 2 minutes until the solution becomes clear.

[0081] S4.8. Use a pipette to draw 32 μL of supernatant, transfer it to a new 1.5 mL tube, label it, and measure and record the library concentration and fragment size.

[0082] S5 sequencing and analysis results are as follows: Figure 2 , Figure 3 and Figure 4 As shown.

[0083] In summary, the specific primers are designed with an Omega structure, containing a first specific sequence, a universal sequence (5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3'), and a second specific sequence from the 5' end to the 3' end. The first and second specific sequences are complementary to the near-fusion site region of the main gene (at least one of ROS1, ALK, and RET), enabling precise targeting of the fusion site fragments of the main gene and its fusion partner. Furthermore, the primers are designed for specific exons of the main gene, eliminating the need to predetermine the fusion partner gene sequence.

[0084] Results Achieved: Overcoming the limitations of traditional detection methods (such as FISH and RT-PCR) that can only detect known partner gene fusions, this method can indiscriminately amplify all gene sequences fused to the target exon of the main gene. It can detect not only known fusion types such as ROS1-CD74 and ROS1-SLC34A2, but also unknown partner genes fused to the same exon, covering more potential fusion types and avoiding the omission of rearranged fusion genes with low incidence or complex structures, thus improving the comprehensiveness and completeness of gene fusion detection in solid tumors.

[0085] When constructing the NGS library, the core workflow is "adapter ligation (S1) - post-ligation purification (S2) - specific amplification (S3) - post-amplification purification (S4) - sequencing and analysis of results (S5)". In step S3, ultra-multiplex single-end PCR technology is used. The amplification system composed of universal primers (containing SEQ ID NO:3 sequence 5'-ACACTCTTTCCCTACACGAC), Omega structure-specific primers and index primers completes the amplification of the target fragment in one step, replacing the hybridization capture process that traditional high-throughput sequencing relies on. At the same time, steps S2 and S4 uniformly adopt the magnetic bead purification method and strictly follow the standardized operation of "binding-washing-elution" (e.g., in S2, 60 μL of magnetic beads are mixed with the ligation product, incubated at room temperature for 5 min, washed twice with 80% ethanol and then eluted with 32 μL of enzyme-free water; in S4, 45 μL of magnetic beads are used to treat the amplification product).

[0086] Results: Compared to hybridization capture, ultra-multiplex single-end PCR eliminates the need for complex probe hybridization and elution steps, significantly simplifying experimental procedures. Magnetic bead purification is standardized, time-efficient, and eliminates the need for expensive hybridization probe reagents, reducing reagent costs. Furthermore, unlike FISH, the overall process does not require calculating at least 50 cells, making it suitable for specimens with small sample volumes (such as biopsy samples), further broadening the range of applicable samples. Ultimately, this achieves a comprehensive effect of simplified experimental procedures, reduced detection time (avoiding the problem of long procedures in traditional methods), and lower detection costs.

[0087] In the S3 specific amplification step, the length of the amplified fragment is precisely controlled at 370 bp through targeted design of Omega structure primers, avoiding low amplification efficiency or interference from non-specific fragments due to excessively long fragments. A short Y adapter (containing the first and second oligonucleotides) is designed to achieve efficient ligation of total cDNA from the sample and reduce adapter dimer interference. At the same time, the index primer contains adapter sequence 2, index-specific sequence and universal sequence 2 from the 5' end to the 3' end. The index-specific sequence is used to distinguish different samples and avoid cross-contamination between samples.

[0088] Results Achieved: The 370bp targeted amplification fragment length ensures amplification efficiency and specificity, reducing the amplification of non-target fragments; the specific sequence design of the short Y-linker improves ligation efficiency and reduces the impact of linker-related impurities on subsequent amplification; the sample differentiation function of the index primers can accurately trace the test results of each sample, avoiding misjudgments caused by cross-contamination; comprehensively improving the specificity of gene fusion detection, reducing false positive and false negative results, ensuring the accuracy and reliability of test results, and providing a more accurate basis for the diagnosis of gene fusion in solid tumors.

[0089] The present invention has been described in detail above. However, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, any modifications or improvements that do not depart from the spirit of the present invention are within the scope of protection of the present invention.

Claims

1. A method for constructing an NGS library to detect gene fusions in solid tumors, characterized in that, Includes the following steps: S1. Adapter ligation: Mix total cDNA from the sample with the ligation system and perform adapter ligation reaction to obtain ligation product. The ligation system contains ligase and short Y adapter. S2. Post-ligation purification: The ligation product obtained in S1 is purified to obtain a purified ligation product. S3. Specific amplification: The purified ligation product obtained in S2 is mixed with the amplification system and subjected to PCR amplification to obtain a gene fusion detection library. The amplification system contains universal primers, specific primers and index primers. The specific primers are Omega structure primers designed for the near fusion site region of the main gene, and are used to target and amplify fragments at the fusion site of the main gene and its fusion partner. S4. Purification after specific amplification: The PCR amplification product obtained in S3 is purified to obtain a purified NGS library. S5. Sequencing and analysis of results.

2. The method for constructing an NGS library for detecting gene fusions in solid tumors according to claim 1, characterized in that: The short Y-linker comprises a first oligonucleotide and a second oligonucleotide, the first oligonucleotide having a nucleotide sequence as shown in SEQ ID NO:1, and the second oligonucleotide having a nucleotide sequence as shown in SEQ ID NO:2; wherein the nucleotide sequence of SEQ ID NO:1 is: 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3', and the nucleotide sequence of SEQ ID NO:2 is: P-5'-GATCGGAAGAGC-3'.

3. The method for constructing an NGS library for detecting gene fusions in solid tumors according to claim 1, characterized in that: The universal primer contains a nucleotide sequence as shown in SEQ ID NO:3, wherein the nucleotide sequence of SEQ ID NO:3 is 5'-ACACTCTTTCCCTACACGAC.

4. The method for constructing an NGS library for detecting gene fusions in solid tumors according to claim 1, characterized in that: The specific primer is a single-stranded DNA molecule, which includes, from the 5' end to the 3' end, a first specific sequence, a universal sequence and a second specific sequence; the universal sequence is 5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3'; the first specific sequence and the second specific sequence are complementary to the near-fusion site region of the main gene.

5. The method for constructing an NGS library for detecting gene fusions in solid tumors according to claim 1, characterized in that: The index primer comprises, from the 5' end to the 3' end, a linker sequence 2, an index-specific sequence, and a universal sequence 2, wherein the index-specific sequence is used to distinguish different samples.

6. The method for constructing an NGS library for detecting gene fusions in solid tumors according to claim 1, characterized in that: The purification in steps S2 and S4 uses magnetic bead purification, which includes binding, washing, and elution steps.

7. The method for constructing an NGS library for detecting gene fusions in solid tumors according to claim 1, characterized in that: The method uses ROS1-CD74 and ROS1-SLC34A2 fusion positive samples for detection.

8. The method for constructing an NGS library for detecting gene fusions in solid tumors according to claim 1, characterized in that: The main gene is selected from at least one of ROS1, ALK, and RET.

9. The method for constructing an NGS library for detecting gene fusions in solid tumors according to claim 1, characterized in that: The fragment length targeted for amplification in step S3 is 370 bp.

10. A multiplex PCR primer structure for detecting gene fusions in solid tumors, characterized in that, The primers are Omega-structure primers, which include, from the 5' end to the 3' end, a first specific sequence, a universal sequence, and a second specific sequence. The first specific sequence and the second specific sequence are designed for the near-fusion site region of the main gene and are used to target and amplify the fragment at the fusion site of the main gene and its fusion partner. The universal sequence is 5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3'.

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