Primer combination for constructing unknown fusion gene detection library, and use thereof

Abstract

A primer combination for constructing a unknown fusion gene detection library, and the use thereof. A specific primer combination system is designed, which can be effectively used in the construction of an unknown fusion gene detection library. By means of using a random primer, a template-switching primer and a random primer which carries a universal sequence, fixed sequences are respectively added at the 3' end and 5' end of a reverse-transcribed cDNA, so that a target fragment is amplified by means of a 3' / 5' gene-specific primer and a universal primer, and the fusion forms at the 5' and 3' ends of a target gene can be detected simultaneously. The template-switching primer is designed to have the same sequence as the universal sequence, so that linker sequences at two ends of a by-product are complementary during PCR amplification and a loop structure is formed after intramolecular annealing, thereby greatly reducing the proportion of full-length RNA by-products and achieving rapid and comprehensive detection of the target fusion gene.

Description

A primer combination for constructing an unknown fusion gene detection library and its application Technical Field

[0001] The present invention belongs to the technical field of gene detection, and relates to a primer combination for constructing an unknown fusion gene detection library and an application thereof. Background Art

[0002] Fusion genes are a process in which all or part of two genes, at the DNA or RNA level, fuse to form a new gene. This process is a major cause of cancer. Accurate detection of fusion genes is also a crucial basis for targeted cancer treatment.

[0003] High-throughput sequencing (NGS) is one of the main means of detecting unknown fusion genes. However, most of the current library construction methods for detecting unknown fusion genes are time-consuming, difficult to operate, and costly, and need to be further improved.

[0004] Template switch oligo (TSO) is an oligonucleotide that hybridizes with the non-template bases added to the 3' end of the cDNA by reverse transcriptase during reverse transcription. The reverse transcriptase then extends its reverse complementary sequence to the 3' end of the cDNA. This fixed sequence serves as a primer anchor for downstream cDNA amplification. Previous literature has proposed using TSO technology to add a universal sequence to the 3' end of the reverse transcription product, thereby enabling amplification and sequencing of fusion genes corresponding to the target gene (driver gene). For example, CN115074422A discloses a method for detecting unknown fusion genes, wherein the method for constructing an unknown fusion gene detection library includes reverse transcribing the total RNA of a sample in a reverse transcription system or fragmenting the total RNA and then reverse transcribing it in a reverse transcription system to obtain a reverse transcription product; PCR amplifying the obtained reverse transcription product in a first-round amplification system to obtain a first-round PCR amplification product, wherein the first-round amplification system contains a TSO-F primer and a first specific primer; and amplifying the first-round PCR amplification product in a second-round amplification system to obtain an unknown fusion gene detection library, wherein the second-round amplification system contains the TSO-F primer, a second specific primer, and a barcode primer. However, because TSO technology can only add a universal sequence to the 3' end of cDNA, the current library construction method using TSO technology to detect unknown fusion genes can only detect the unknown gene fusion form at the 3' end of the target gene (driver gene) on the RNA sequence, but cannot simultaneously detect the fusion status of the target gene at the 5' end.

[0005] In summary, because different genes have different locations of fusion mutations, some gene fusions occur at the 5' end, and some gene fusions occur at the 3' end. The present invention develops a new method for constructing an unknown fusion gene detection library, which can achieve the simultaneous amplification and sequencing of multiple target genes with gene fusions occurring at different ends, which is of great significance.

[0006] Summary of the Invention

[0007] In response to the deficiencies of the existing technology and actual needs, the present invention provides a primer combination for constructing an unknown fusion gene detection library and its application, in order to achieve rapid and comprehensive detection of target fusion genes, reduce by-products, and have simple operation and low cost.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a primer combination for constructing an unknown fusion gene detection library, characterized in that the primer combination comprises:

[0010] a first random primer, a template displacement primer, a second random primer, a gene-specific primer, a universal primer, and a sequencing adapter primer pair;

[0011] The first random primer includes a first random sequence; the second random primer includes a universal sequence and a second random sequence in sequence from the 5' to the 3' end; the template displacement primer includes the universal sequence; the gene-specific primer includes a linker sequence and a GSP primer in sequence from the 5' to the 3' end, and the GSP primer is reverse complementary to or identical to the driver gene RNA sequence of the gene to be detected;

[0012] The universal primer includes the universal sequence; one primer in the sequencing adapter primer pair includes a sequence at its 3' end that is identical to a portion or all of the adapter sequence in the gene-specific primer, and the other primer in the sequencing adapter primer pair includes a sequence at its 3' end that is identical to a portion or all of the universal primer.

[0013] Preferably, the universal sequence is located at the 5' end of the template displacement primer, so that after reverse transcription and template displacement of the RNA sequence of the gene to be detected, the 3' end of the template-replaced reverse transcription product cDNA of the RNA sequence of the gene to be detected is connected to the reverse complementary sequence of the universal sequence.

[0014] Preferably, the universal sequence is located at the 3' end of the universal primer, so that after reverse transcription of the RNA sequence of the gene to be detected, the 5' end of the reverse transcription product cDNA of the RNA sequence of the gene to be detected can be connected to the universal sequence.

[0015] Preferably, the sequence contained at the 3' end of one primer in the sequencing adapter primer pair is identical to at least a portion of the adapter sequence in the gene-specific primer and is located at the 5' end of the gene-specific primer. Preferably, the sequence contained at the 3' end of the other primer in the sequencing adapter primer pair is identical to at least a portion of the universal primer and is located at the 5' end of the universal primer.

[0016] Preferably, the gene-specific primer includes a 3' gene-specific primer and / or a 5' gene-specific primer, wherein the 3' gene-specific primer includes a linker sequence and a 3'GSP primer in sequence from the 5' to the 3' end, and the 3'GSP primer is reverse complementary to the 3' driver gene RNA sequence of the gene to be detected; the 5' gene-specific primer includes a linker sequence and a 5'GSP primer of the gene to be detected in sequence from the 5' to the 3' end, and the 5'GSP primer sequence is the same as the 5' driver gene RNA sequence.

[0017] In the present invention, a specific primer combination system is designed, which can be effectively applied to the construction of an unknown fusion gene detection library. Random primers, template replacement primers, and random primers with universal sequences are used to add fixed sequences to the 3' and 5' ends of the reverse transcribed cDNA, respectively. Therefore, by amplifying the target fragments with 3' / 5' gene-specific primers and universal primers, the fusion forms at the 3' and 5' ends of the target gene can be simultaneously detected. The sequence of the template replacement primer is designed to have the same sequence as the universal sequence, so that the adapter sequences at both ends of the byproducts are complementary during PCR amplification, and intramolecular annealing forms a loop structure, which greatly reduces the proportion of full-length RNA byproducts and realizes rapid and comprehensive detection of target fusion genes.

[0018] Preferably, the length of the first random sequence and the second random sequence are each independently 6 to 70 nt, including but not limited to 7nt, 8nt, 9nt, 10nt, 11nt, 15nt, 20nt, 25nt, 30nt, 35nt, 40nt, 45nt, 50nt, 55nt, 60nt, 65nt, 66nt, 67nt, 68nt or 69nt.

[0019] Preferably, the length of the universal sequence is 20 to 35 nt, 21 nt, 22 nt, 25 nt, 26 nt, 28 nt, 30 nt, 31 nt, 32 nt, 33 nt or 34 nt.

[0020] Preferably, the length of the sequence at the 3' end of one primer in the sequencing adapter primer pair that is identical to a part or all of the adapter sequence in the gene-specific primer is about 20 nt (e.g., 20-21 nt), preferably about 20 nt (e.g., 20-21 nt) from the 5' end of the adapter sequence in the gene-specific primer.

[0021] Preferably, the length of the sequence at the 3' end of the other primer in the sequencing adapter primer pair that is identical to part or all of the universal primer is about 20 nt (e.g., 20-21 nt), preferably about 20 nt (e.g., 20-21 nt) from the 5' end of the universal primer.

[0022] Preferably, the other primer in the sequencing adapter primer pair contains a sequence at the 3' end that is identical to at least a portion of the universal primer, which is a second adapter sequence. Preferably, the length of the second adapter sequence is 0 to 7 nt, including but not limited to 0nt, 1nt, 2nt, 3nt, 4nt, 5nt, 6nt, and 7nt.

[0023] Preferably, the two primers in the sequencing adapter primer pair are a P5 sequencing adapter primer and a P7 sequencing adapter primer, respectively, the P5 sequencing adapter primer comprises a sequence identical to part or all of the universal primer at its 3' end, and the P7 sequencing adapter primer comprises a sequence identical to part or all of the adapter sequence in the gene-specific primer at its 3' end.

[0024] Preferably, the nucleic acid sequence of the template replacement primer includes the sequence shown in SEQ ID NO.1, and rG is the ribonucleic acid G base.

[0025] Preferably, the nucleic acid sequence of the second random primer includes the sequence shown in SEQ ID NO.2.

[0026] Preferably, the nucleic acid sequence of the linker sequence includes the sequence shown in SEQ ID NO.3.

[0027] Preferably, the nucleic acid sequence of the universal primer includes the sequence shown in SEQ ID NO.4.

[0028] Preferably, the nucleic acid sequence of the sequencing adapter primer includes the sequences shown in SEQ ID NO.5 and SEQ ID NO.6.

[0029] Preferably, the gene to be detected is selected from: FGFR3, ALK, BRAF, NTRK1, NTRK3, RET, ROS1 or MET. Preferably, a 5' GSP primer comprising the following nucleic acid sequence can be used: ACCTGGACCGTGTCCTTArCCGTGA (SEQ ID NO.9); and a 3' GSP primer comprising the following nucleic acid sequence can be used: TCATGATGGTCGAGGTGCrGGAGCT (SEQ ID NO.10), ATGATCTTCATCTGCTGGTCGrGAAGGG (SEQ ID NO.11), TCCACATTTGTTGAGCACAArGGAGCA (SEQ ID NO.12), CACCGGGTCTCCAGATGrUGCTGT (SEQ ID NO.13), TCCGATCTGTCTCTCCTCTTAATGTGCTrGCACAA (SEQ ID NO.14), CCAAGAACCAAGTTCTTCCGAGrGGAATA (SEQ ID NO.15), AAGGAGAAGAGGACAGCGrGCTGCG (SEQ ID NO.16), TGTAACAACCAGAAATATTCCAACTATrAATAGT (SEQ ID NO.17). NO.17) or TCCGATCTTAGTTAGGATGGGGGACATGrUCTGTC (SEQ ID NO.18).

[0030] In a second aspect, the present invention provides use of the primer combination for constructing an unknown fusion gene detection library described in the first aspect in preparing a product for constructing an unknown fusion gene detection library.

[0031] In a third aspect, the present invention provides a product for constructing an unknown fusion gene detection library, which comprises the primer combination for constructing an unknown fusion gene detection library described in the first aspect and optional reverse transcriptase, dNTP, DNA polymerase and reaction buffer.

[0032] Preferably, the product is in the form of a kit for constructing a library for detecting unknown fusion genes.

[0033] Preferably, the product includes the primer combination for constructing the unknown fusion gene detection library described in the first aspect, template-displacing reverse transcriptase, dNTP, DNA polymerase, reaction buffer, and reverse transcription and PCR related reagents.

[0034] In a fourth aspect, the present invention provides use of the primer combination for constructing an unknown fusion gene detection library described in the first aspect in constructing an unknown fusion gene detection library.

[0035] In a fifth aspect, the present invention provides a method for constructing an unknown fusion gene detection library, the method utilizing the primer combination for constructing an unknown fusion gene detection library described in the first aspect, comprising the following steps:

[0036] (1) Sample RNA is divided into two parts as templates, one part is subjected to a template displacement reverse transcription reaction using a first random primer and a template displacement primer to obtain a template displacement reverse transcription product; the other part is subjected to a reverse transcription reaction using a second random primer to obtain a reverse transcription product;

[0037] (2) performing a first amplification reaction using the template-displaced reverse transcription product and the reverse transcription product as a template and using gene-specific primers and universal primers;

[0038] (3) Using sequencing adapter primers, a second amplification reaction is performed on the product of the first amplification reaction to obtain an amplified product for constructing the unknown fusion gene detection library.

[0039] Preferably, step (1) further comprises the step of mixing the template-switched reverse transcription product with the reverse transcription product.

[0040] Preferably, step (1) further comprises the step of purifying the template replacement reverse transcription product and the reverse transcription product.

[0041] Preferably, step (2) further comprises the step of purifying the product of the first amplification reaction.

[0042] In a sixth aspect, the present invention provides a method for detecting an unknown fusion gene, the method comprising constructing a library using the method for constructing an unknown fusion gene detection library described in the fifth aspect, and sequencing and analyzing the library.

[0043] In the present invention, a method for rapid and comprehensive detection of target fusion genes is designed, which has broad application prospects, such as the study of the basic behavior of fusion genes for non-disease diagnosis or treatment purposes, etc.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] The TSO-based method developed by the present invention for detecting unknown fusion genes utilizes random primers with universal sequences, as well as random primers and template-replacement primers to add universal sequences to both ends of the cDNA sequence. This allows for detection of fusions on both sides of the target gene, resulting in more comprehensive results. Furthermore, by designing the template-replacement primer and the second random primer (random primer with a universal sequence), the adapter sequences at both ends of the byproducts in the first PCR amplification reaction are complementary, allowing intramolecular annealing to form a loop structure, effectively inhibiting the amplification of full-length byproducts, especially byproducts represented by rRNA, facilitating subsequent sequencing and analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] FIG1 is a schematic diagram of the technical principle of the i5-i5 primer system in Example 1;

[0047] Figure 2 is a schematic diagram of the technical principle of the i5-i7 primer system in Comparative Example 1;

[0048] FIG3 is a graph showing the detection results of 14 fusion forms using the i5-i5 primer system and the i5-i7 primer system under the same conditions;

[0049] FIG4A is a graph showing the on-target detection rates of the i5-i5 primer system and the i5-i7 primer system;

[0050] Figure 4B shows the percentage of wild-type reads detected by the i5-i5 primer system and the i5-i7 primer system;

[0051] Figure 4C shows the percentage of reads spanning the breakpoints detected by the i5-i5 primer system and the i5-i7 primer system;

[0052] Figure 4D shows the percentage of ribosomal RNA reads detected by the i5-i5 primer system and the i5-i7 primer system. DETAILED DESCRIPTION

[0053] To further illustrate the technical means and effects of the present invention, the present invention is further described below with reference to the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the present invention.

[0054] If specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the field or in the product specifications were used. Reagents or instruments used without manufacturer indication are conventional products available through regular channels. In addition, "reverse transcription" in the claims, description, and examples of the present invention is synonymous with "reverse transcription."

[0055] The present invention develops a method for constructing an unknown fusion gene detection library based on template switch oligo. The technical principle is as follows: 1. A cDNA library with a fixed sequence at the 3' end is generated by reverse transcription reaction using random primers and template replacement primers. At the same time, a cDNA library with a fixed sequence at the 5' end is generated by reverse transcription reaction using random primers with a universal sequence. The fixed sequences at both ends are in a reverse complementary relationship, and the upstream primer is shared in the downstream PCR reaction; 2. Gene-specific primers (GSPs) are designed according to the position of the target gene on the RNA sequence; if the fusion status of the target gene at the 3' end on the RNA sequence is detected, the GSP should carry the reverse complementary sequence of the 3' end region of the target gene on the RNA sequence. If the fusion status of the target gene at the 5' end on the RNA sequence is detected, the GSP should carry the same sequence as the 5' end region of the target gene on the RNA sequence. After effective amplification with the gene-specific primers, unknown fusion events of the target gene can be detected.

[0056] Example 1

[0057] In this example, primers were designed to construct a library for detecting unknown fusion genes.

[0058] The primer sequences are shown in Table 1.

[0059] Table 1

[0060] Where N is a random base, A, G, C, or T; rG is the RNA G base. Primer sequences were synthesized by Integrated DNA Technologies (IDT). The product was delivered as a dry powder. IDTE buffer (IDT) was added according to the manufacturer's recommended volume, and quality control was performed using a NanoDrop spectrophotometer (Thermo Fisher).

[0061] The technical principle of the above primer system (named i5-i5 system) is shown in Figure 1. Driver refers to the driver gene, and Partner refers to the partner gene. Fusion genes include many types. A proto-oncogene fuses with another gene (called a "partner gene"), leading to the occurrence and progression of cancer. ① Reverse transcription step: RNA is divided into two tubes. One tube is reverse transcribed to the 5' end of RNA by random primers, and then non-template bases are added. Finally, a DNA-dependent DNA polymerase reaction is carried out under the guidance of a template-displacement primer to synthesize a cDNA library with a fixed sequence at the 3' end. The other tube uses a random primer with a universal sequence 1 for reverse transcription to generate a cDNA library with a fixed sequence at the 5' end. The two tubes of cDNA libraries are mixed and purified; ② One-round PCR step: Gene-specific primers are designed according to the position of the target gene on the RNA sequence. If a 3' driver gene fusion is detected, the target is amplified by the 3' gene-specific primer and the universal primer 1. If detecting a 5'-driven gene fusion, the target fragment is amplified using 5' gene-specific primer-1 and universal primer 1, and the product is purified. Furthermore, amplification of the same cDNA from both RT tubes can produce byproducts. These byproducts can be any type of RNA, including fusions, wild-type, rRNA, and mRNA. To address this, in this example, the template-replacing primers and random primers with universal sequences are designed to have a common sequence. This ensures that the adapter sequences at both ends of the full-length byproduct are complementary, enabling intramolecular annealing to form a loop structure, inhibiting PCR formation and significantly reducing the proportion of full-length RNA byproducts. Furthermore, the common sequence between the template-replacing primers and the random primers with universal sequences allows the two tubes of cDNA to share the common primers during the first round of PCR after mixing. In addition, the adapter sequences in the 3' gene-specific primer and the 5' gene-specific primer are designed to be identical to the 3' end sequence of one of the sequencing adapter primers, and the universal primer includes a sequence identical to the universal sequence and a sequence identical to the 3' end sequence of another of the sequencing adapter primers, so that the sequencing adapter primers can be used for amplification in the second round of PCR to obtain a sequencing library.

[0062] ③PCR2 step: Amplify the purified PCR1 product using P5 and P7 sequencing adapter primers. After amplification, purify the resulting library (in Figure 1 , the P5 sequencing adapter primer is shown as the P5 universal primer, and the P7 sequencing adapter primer is shown as the P7 universal primer).

[0063] The random primer or the random sequence in the random primer can be a plurality of random bases, for example, 6 to 70 random bases, including but not limited to 7, 8, 9, 10, 11, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 66, 67, 68, 69 bases, etc., for reverse transcribing RNA into cDNA.

[0064] It should be noted that although Table 1 only shows that the P7 sequencing adapter primer contains a sequence identical to a portion of the adapter sequence in the gene-specific primer (3' gene-specific primer or 5' gene-specific primer-1) at the 3' end, the sequence of the identical portion is located at the 5' end of the gene-specific primer, so as to amplify the target and tail the target using the P7 sequencing adapter primer. For this purpose, those skilled in the art can also set the sequence at the 3' end of the P7 sequencing adapter primer (the sequence between the barcode and the 3' end) to be completely identical to the adapter sequence of the gene-specific primer. Although Table 1 only shows that the P5 sequencing adapter primer contains a sequence identical to a portion of the universal primer at the 3' end, the identical sequence is located at the 5' end of the universal primer to achieve the purpose of tailing. For the purpose of amplifying the target and tailing the target using the P5 sequencing adapter primer, those skilled in the art can also set the sequence at the 3' end of the P5 sequencing adapter primer (the sequence between the barcode and the 3' end) to be completely identical to the universal sequence.

[0065] Gene-specific primers (GSP primers) were designed according to the genes to be detected, and an exemplary display is shown in Table 2.

[0066] Table 2

[0067] Example 2

[0068] This example constructs an unknown fusion gene detection library.

[0069] 1. Prepare the TSO reverse transcription system according to Table 3.

[0070] Seracare Fusion RNA Mix v4 (Material No. 0710-0497, Batch No. 10670288) was diluted to 6 copies / ng with a mixture of negative lung cancer clinical RNA samples, for a concentration of 32 ng / μL Seracare mix. 25 ng of each sample (diluted to ~150 copies using EML4-ALK as a standard) was used for template switching (TSO) and reverse transcription (RT) reactions, respectively.

[0071] Table 3

[0072] Prepare the above system, mix by pipetting, centrifuge briefly, and heat the PCR instrument with the lid heated to ≥85°C for 5 minutes at 70°C, then hold at 4°C. Then, add the template switching (TSO) reaction system to continue the reaction. The template switching reaction system is shown in Table 4. In the PCR instrument, perform the reaction at 42°C for 90 minutes, 85°C for 5 minutes, and hold at 4°C to generate template switching reverse transcription products.

[0073] Table 4

[0074] 2. Prepare the reverse transcription system according to Table 5.

[0075] Table 5

[0076] Prepare the above system, mix by pipetting, centrifuge briefly, set the thermal cover of the PCR instrument to ≥85°C, and perform the reaction program of 25°C for 10 min, 42°C for 30 min, 80°C for 20 min, and maintain at 4°C to generate reverse transcription products.

[0077] 3. Product purification.

[0078] Transfer the reverse transcription product to the template-switched reverse transcription product, a total of 20 μL, add 50 μL (2.5×) AMPure Xp Beads and mix well; let it stand at 25°C for 5 minutes, then discard the supernatant on the magnetic stand; then wash twice with 200 μL 80% ethanol, dry at 25°C for 5 minutes, and finally elute the purified product with 20 μL NF-H2O. Recover the supernatant for downstream reactions. It is understandable that the template-switched reverse transcription product can also be transferred to the reverse transcription product. In short, the template-switched reverse transcription product can be mixed with the reverse transcription product. In this way, the template-switched reverse transcription product and the reverse transcription product can be mixed for subsequent amplification process, without the need for separate subsequent amplification, which greatly simplifies the experimental process.

[0079] 4. Preparation of one-round PCR system, as shown in Table 6.

[0080] Table 6

[0081] Prepare the above reaction system and perform the reaction program shown in Table 7 in a PCR instrument.

[0082] Table 7

[0083] 5. Purify and recover the first-round PCR products.

[0084] Add 45 μL (1.5×) AMPure Xp Beads to the first-round PCR product and mix thoroughly. Incubate at 25°C for 5 minutes, then discard the supernatant by aspiration on a magnetic rack. Wash twice with 200 μL 80% ethanol, dry at 25°C for ~5 minutes, and finally elute the purified product with 20 μL NF-H2O. Recycle the supernatant for downstream reactions.

[0085] 6. Preparation of the second-round PCR system is shown in Table 8.

[0086] Table 8

[0087] Prepare the above reaction system and perform the reaction program as shown in Table 9 in a PCR instrument.

[0088] Table 9

[0089] 7. Purification and recovery of the second round of PCR products:

[0090] Add 40 μL (1.0×) AMPure Xp Beads to the second-round PCR product and mix thoroughly. Incubate at 25°C for 5 minutes, then discard the supernatant by aspiration on a magnetic rack. Wash twice with 200 μL of 80% ethanol, dry at 25°C for ~5 minutes, and finally elute the purified product with 20 μL of NF-H2O. Recover the supernatant, which is the library, and prepare it for sequencing.

[0091] Example 3

[0092] Six fusion-positive clinical samples were selected, including two fusion forms, and the detailed information is shown in Table 10.

[0093] Table 10

[0094] The primer system in Example 1 was used and detection was performed according to the method in Example 2. The RNA input was 50 ng each. The supporting reads after sequencing are shown in Table 11.

[0095] Table 11

[0096] It can be seen that this method is also effective for clinical samples. The QC is shown in Table 12. The clinical samples use the QC indicators of the i5-i5 system (including on-target rate%, percentage of wild-type reads, percentage of spanning breakpoint reads, and percentage of ribosomal RNA reads).

[0097] Table 12

[0098] Comparative Example 1

[0099] In this comparative example, a primer system i5-i7 was designed. No template replacement primers and random primers with universal sequences were designed to have a common sequence. That is, the two ends of the PCR byproducts in one round could not be complementary and could not form an intramolecular loop. This was compared with the i5-i5 system in Example 1 to demonstrate that the specially designed primer system of the present invention can reduce the proportion of full-length byproducts.

[0100] The primer sequences of the i5-i7 system are shown in Table 13. The main differences between the i5-i7 system and the i5-i5 system are: 1) random primers with universal sequence 2 replace random primers with universal sequence 1; 2) 5' gene-specific primer-2 replaces 5' gene-specific primer-1, and universal sequence 2 replaces universal sequence 1 to complete the amplification of the 5'-driven fusion form.

[0101] Table 13

[0102] The technical principles of the i5-i7 system are shown in Figure 2. ① Reverse transcription step: RNA is divided into two tubes. One tube undergoes reverse transcription with random primers to the 5' end of the RNA. Non-template bases are then added, and finally, a DNA-dependent DNA polymerase reaction guided by a template-displacing primer is performed to synthesize a cDNA library with a fixed sequence at the 3' end. The other tube undergoes reverse transcription with random primers containing a universal sequence 2 to generate a cDNA library with a fixed sequence at the 5' end. The two cDNA libraries are then mixed and purified. ② PCR1 step: Gene-specific primers are designed based on the location of the target gene in the RNA sequence. If a 3'-driven gene fusion is detected, the target fragment is amplified using a 3' gene-specific primer and universal primer 1. If a 5'-driven gene fusion is detected, the target fragment is amplified using a 5' gene-specific primer-2 and universal primer 2. At the same time, byproducts are generated during amplification of the same cDNA from the two tubes of RT. The byproduct type can be any form contained in RNA, including fusion type / wild type / rRNA / mRNA, etc., and the product is purified; ③PCR2 step: The purified PCR1 product is amplified using P5 and P7 sequencing adapter primers, and the library is purified after amplification (in Figure 2, the P5 sequencing adapter primer is shown as the P5 universal primer, and the P7 sequencing adapter primer is shown as the P7 universal primer in Figure 1).

[0103] The template switching (TSO) reaction and reverse transcription (RT) reaction were carried out as described in Example 2.

[0104] Test Case

[0105] The libraries prepared in Example 1 and Comparative Example 1 were sequenced and analyzed by bioinformatics.

[0106] The detection results of 14 fusion forms under the same conditions for the i5-i5 and i5-i7 systems are shown in Figure 3. It can be seen that the i5-i5 system uses PCR suppression technology to inhibit byproducts, and the average fusion detection rate is increased by 72162 / 4971 = 14.5 times compared with the i5-i7 system. This shows that the special primer system designed in the present invention makes the adapter sequences at both ends of the byproducts of a round of PCR complementary, and intramolecular annealing forms a loop structure, which can reduce the proportion of full-length byproducts.

[0107] Comparative quality control (QC) results for the i5-i5 and i5-i7 systems under identical conditions are shown in Figures 4A, 4B, 4C, and 4D. Considering that rRNA accounts for 80% to 95% of the total RNA content, it is reasonable to assume that 69% of the byproducts in the i5-i7 system are rRNA. In contrast, rRNA only accounts for 5.7% in the i5-i5 system. This demonstrates that the present system effectively reduces byproducts. Furthermore, due to the significant reduction in the rRNA ratio, the on-target rate is significantly increased.

[0108] In summary, the unknown fusion gene detection method developed by the present invention based on TSO technology utilizes random primers with universal sequences, as well as random primers and template-displacing primers to add universal sequences to both ends of the cDNA sequence, thereby enabling detection of fusions on both sides of the target gene. In addition, the present invention designs specific primers that complement the adapter sequences at both ends of the full-length byproducts, enabling intramolecular annealing to form a loop structure and inhibit PCR. The target product is effectively amplified using 5' / 3' gene-specific primers and universal primers, thereby effectively inhibiting the formation of full-length byproducts, especially byproducts represented by rRNA.

[0109] The applicant states that the present invention is intended to illustrate the detailed methods of the present invention through the above-described embodiments, but the present invention is not limited to the above-described detailed methods, that is, it does not mean that the present invention must rely on the above-described detailed methods in order to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions for various raw materials in the products of the present invention, addition of auxiliary ingredients, and selection of specific methods, etc., are all within the scope of protection and disclosure of the present invention.

Claims

1. A primer combination for constructing an unknown fusion gene detection library, characterized in that: The primer combination comprises: a first random primer, a template displacement primer, a second random primer, a gene-specific primer, a universal primer, and a sequencing adapter primer pair; The first random primer includes a first random sequence; the second random primer includes a universal sequence and a second random sequence in sequence from the 5' to the 3' end; the template displacement primer includes the universal sequence; the gene-specific primer includes a linker sequence and a GSP primer in sequence from the 5' to the 3' end, and the GSP primer is reverse complementary to or identical to the driver gene RNA sequence of the gene to be detected; The universal primer includes the universal sequence; one primer in the sequencing adapter primer pair contains a sequence at the 3' end that is identical to part or all of the adapter sequence in the gene-specific primer, and the other primer in the sequencing adapter primer pair contains a sequence at the 3' end that is identical to part or all of the universal primer.

2. The primer combination for constructing an unknown fusion gene detection library according to claim 1, characterized in that: The universal sequence is located at the 5' end of the template displacement primer.

3. The primer combination for constructing an unknown fusion gene detection library according to claim 1, characterized in that: The universal sequence is located at the 3' end of the universal primer.

4. The primer combination for constructing an unknown fusion gene detection library according to claim 1, characterized in that: The sequence contained at the 3' end of one primer in the sequencing adapter primer pair is identical to at least a portion of the adapter sequence in the gene-specific primer and is located at the 5' end of the gene-specific primer.

5. The primer combination for constructing an unknown fusion gene detection library according to claim 1, characterized in that: The other primer in the sequencing adapter primer pair comprises at its 3' end a sequence identical to at least a portion of the universal primer, which is located at the 5' end of the universal primer.

6. The primer combination for constructing an unknown fusion gene detection library according to any one of claims 1 to 5, characterized in that: The lengths of the first random sequence and the second random sequence are independently 6 to 70 nt.

7. The primer combination for constructing an unknown fusion gene detection library according to claims 1-5, characterized in that: The length of the universal sequence is 20 to 35 nt.

8. The primer combination for constructing an unknown fusion gene detection library according to any one of claims 1 to 5, characterized in that: The gene-specific primers include a 3' gene-specific primer and / or a 5' gene-specific primer, wherein the 3' gene-specific primer includes a linker sequence and a 3' GSP primer in sequence from the 5' to the 3' end, and the 3' GSP primer is reverse complementary to the 3' driver gene RNA sequence of the gene to be detected; the 5' gene-specific primer includes a linker sequence and a 5' GSP primer of the gene to be detected in sequence from the 5' to the 3' end, and the 5' GSP primer sequence is the same as the 5' driver gene RNA sequence.

9. The primer combination for constructing an unknown fusion gene detection library according to claim 5, characterized in that: The sequence at the 3' end of the other primer in the sequencing adapter primer pair that is identical to at least a portion of the universal primer is the second adapter sequence of the universal primer. Preferably, the length of the second adapter sequence is 0 to 7 nt.

10. The primer combination for constructing an unknown fusion gene detection library according to any one of claims 1 to 5, characterized in that: The two primers in the sequencing adapter primer pair are respectively a P5 sequencing adapter primer and a P7 sequencing adapter primer, wherein the P5 sequencing adapter primer comprises a sequence identical to a part or all of the universal primer at its 3' end, and the P7 sequencing adapter primer comprises a sequence identical to a part or all of the adapter sequence in the gene-specific primer at its 3' end.

11. The primer combination for constructing an unknown fusion gene detection library according to claim 1, characterized in that: The gene to be detected is selected from: FGFR3, ALK, BRAF, NTRK1, NTRK3, RET, ROS1 or MET.

12. The primer combination for constructing an unknown fusion gene detection library according to claim 1, characterized in that: The nucleic acid sequence of the template replacement primer includes the sequence shown in SEQ ID NO.1; Preferably, the nucleic acid sequence of the second random primer includes the sequence shown in SEQ ID NO.2; Preferably, the nucleic acid sequence of the linker sequence includes the sequence shown in SEQ ID NO.3; Preferably, the nucleic acid sequence of the universal primer includes the sequence shown in SEQ ID NO.4; Preferably, the nucleic acid sequence of the sequencing adapter primer includes the sequences shown in SEQ ID NO.5 and SEQ ID NO.

6.

13. Use of the primer combination for constructing an unknown fusion gene detection library according to any one of claims 1 to 12 in preparing a product for constructing an unknown fusion gene detection library.

14. A product for constructing an unknown fusion gene detection library, the product comprising the primer combination for constructing an unknown fusion gene detection library according to any one of claims 1 to 12 and optional reverse transcriptase, dNTP, DNA polymerase and reaction buffer.

15. Use of the primer combination for constructing an unknown fusion gene detection library according to any one of claims 1 to 12 in constructing an unknown fusion gene detection library.

16. A method for constructing an unknown fusion gene detection library, characterized in that: The method uses the primer combination for constructing an unknown fusion gene detection library according to any one of claims 1 to 12, and comprises the following steps: (1) taking sample RNA and dividing it into two parts as templates, one part is subjected to a template displacement reverse transcription reaction using a first random primer and a template displacement primer to obtain a template displacement reverse transcription product; the other part is subjected to a reverse transcription reaction using a second random primer to obtain a reverse transcription product; (2) using the template-displaced reverse transcription product and the reverse transcription product as a template, using gene-specific primers and universal primers to perform a first amplification reaction; (3) Using sequencing adapter primers, a second amplification reaction is performed on the product of the first amplification reaction to obtain an amplification product for constructing the unknown fusion gene detection library.

17. The method for constructing an unknown fusion gene detection library according to claim 16, characterized in that: Step (1) also includes the step of mixing the template switching reverse transcription product with the reverse transcription product.

18. The method for constructing an unknown fusion gene detection library according to claim 16 or 17, characterized in that: Step (1) also includes the step of purifying the template replacement reverse transcription product and the reverse transcription product.

19. The method for constructing an unknown fusion gene detection library according to any one of claims 16 to 18, characterized in that: Step (2) also includes the step of purifying the product of the first amplification reaction.

20. A method for detecting an unknown fusion gene, characterized in that: The method comprises constructing a library using the method for constructing an unknown fusion gene detection library according to any one of claims 16 to 19, and sequencing and analyzing the library.

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