Primer combination for constructing unknown fusion gene detection library and application thereof

Abstract

The invention discloses a primer combination for constructing an unknown fusion gene detection library and application of the primer combination. A specific primer combination system is designed and can be effectively applied to construction of an unknown fusion gene detection library, and a random primer, a template replacement primer and a random primer carrying a universal sequence are utilized; fixed sequences are respectively added at the 3'end and the 5 'end of the eDNA subjected to reverse transcription, so that a target fragment is amplified by a 3' / 5 'gene specific primer and a universal primer, the fusion forms of the 5'end and the 3' end of a target gene can be simultaneously detected, and the same sequence in the sequence of the template replacement primer and the universal sequence is designed; and the linker sequences at the two ends of the by-product are complementary during PCR amplification, and a loop structure is formed by intramolecular annealing, so that the proportion of the RNA full-length by-product is greatly reduced, and rapid and comprehensive detection of the target fusion gene is realized.

Description

Technical Field

[0001] The 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 gene refers to the process in which all or part of the sequences of two genes fuse into a new gene at the DNA or RNA level. This process is an important cause of cancer. At the same time, the accurate detection of fusion genes is also an important basis for targeted tumor 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 base added to the 3' end of cDNA after reverse transcription by reverse transcriptase during the reverse transcription process, and then extends its reverse complementary sequence at the 3' end of cDNA through the action of reverse transcriptase. This fixed sequence can be used as a primer anchor point for downstream c DNA amplification. Existing literature has proposed using TSO technology to add a universal sequence to the 3' end of the reverse transcription product, thereby achieving amplification and sequencing of the corresponding fusion gene of 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 the sample in a reverse transcription system or reverse transcribing the total RNA in a reverse transcription system after fragmentation 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; 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, since the TSO technology can only add a universal sequence to the 3' end of cDNA, the current library construction method for detecting unknown fusion genes using the TSO technology 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 situation at the 5' end of the target gene.

[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. Summary of the invention

[0006] In view of the deficiencies of the prior art 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 the target fusion gene, reduce by-products, and have simple operation and low cost.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] 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:

[0009] 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;

[0010] 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;

[0011] 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.

[0012] Preferably, the universal sequence is located at the 5' end of the template displacement primer, so as to connect the reverse complementary sequence of the universal sequence to the 3' end of the template displacement reverse transcription product cDNA of the RNA sequence of the gene to be detected after reverse transcription and template displacement of the RNA sequence of the gene to be detected.

[0013] Preferably, the universal sequence is located at the 3' end of the universal primer, and is used for connecting the universal sequence to the 5' end of the cDNA of the reverse transcription product of the RNA sequence of the gene to be detected after reverse transcription of the RNA sequence of the gene to be detected.

[0014] Preferably, the sequence identical to at least a portion of the adapter sequence in the gene-specific primer contained at the 3' end of one primer in the sequencing adapter primer pair is located at the 5' end of the gene-specific primer. Preferably, the sequence identical to at least a portion of the universal primer contained at the 3' end of the other primer in the sequencing adapter primer pair is located at the 5' end of the universal primer.

[0015] Preferably, 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.

[0016] In the present invention, a specific primer combination system is designed, which can be effectively applied to construct an unknown fusion gene detection library, and fixed sequences are added to the 3' end and 5' end of the reverse transcribed eDNA by using random primers, template replacement primers and random primers with universal sequences, so that the fusion forms of the 3' and 5' ends of the target gene can be detected simultaneously by amplifying the target fragment by using 3' / 5' gene-specific primers and universal primers. The sequence of the template replacement primer is designed to have the same sequence as that in the universal sequence, so that the adapter sequences at both ends of the by-product are complementary during PCR amplification, and the intramolecular annealing forms a loop structure, which greatly reduces the proportion of the full-length RNA by-product, and realizes rapid and comprehensive detection of the target fusion gene.

[0017] 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 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 15 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt, 55 nt, 60 nt, 65 nt, 66 nt, 67 nt, 68 nt or 69 nt.

[0018] 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.

[0019] 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) starting from the 5' end of the adapter sequence in the gene-specific primer.

[0020] 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 (eg, 20-21 nt), preferably about 20 nt (eg, 20-21 nt) from the 5' end of the universal primer.

[0021] 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 as a second adapter sequence. Preferably, the length of the second adapter sequence is 0 to 7 nt, including but not limited to 0 nt, 1 nt, 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, and 7 nt.

[0022] 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.

[0023] 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.

[0024] Preferably, the nucleic acid sequence of the second random primer includes the sequence shown in SEQ ID NO.2.

[0025] Preferably, the nucleic acid sequence of the linker sequence includes the sequence shown in SEQ ID NO.3.

[0026] Preferably, the nucleic acid sequence of the universal primer includes the sequence shown in SEQ ID NO.4.

[0027] Preferably, the nucleic acid sequence of the sequencing adapter primer includes the sequences shown in SEQ ID NO.5 and SEQ ID NO.6.

[0028] SEQ ID NO. 1: TTCCCTACACGACGCTCTCCGATCTrGrGrG.

[0029] SEQ ID NO. 2: TTCCTACACGACGCTCTCCGATCTNNNNNN.

[0030] SEQ ID NO. 3: GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT.

[0031] SEQ ID NO. 4: ACACTCTTTCCCTACACGACCGCTTCCGATCT.

[0032] SEQ ID NO.5:

[0033] AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGAC.

[0034] SEQ ID NO.6:

[0035] CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTGACTGGAGTTCAGACGTGT.

[0036] 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).

[0037] 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.

[0038] In a third aspect, the present invention provides a product for constructing an unknown fusion gene detection library, the product comprising 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.

[0039] Preferably, the product is in the form of a kit for constructing an unknown fusion gene detection library.

[0040] Preferably, the product comprises the primer combination for constructing an unknown fusion gene detection library described in the first aspect, a template-displacing reverse transcriptase, dNTPs, a DNA polymerase, a reaction buffer, and related reagents for reverse transcription and PCR.

[0041] 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.

[0042] In a fifth aspect, the present invention provides a method for constructing an unknown fusion gene detection library, the method using the primer combination for constructing an unknown fusion gene detection library described in the first aspect, comprising the following steps:

[0043] (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;

[0044] (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;

[0045] (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.

[0046] Preferably, step (1) further comprises the step of mixing the template switching reverse transcription product with the reverse transcription product.

[0047] Preferably, step (1) further comprises the step of purifying the template replacement reverse transcription product and the reverse transcription product.

[0048] Preferably, step (2) also includes the step of purifying the product of the first amplification reaction.

[0049] 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.

[0050] In the present invention, a method for rapidly and comprehensively detecting a target fusion gene 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.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] The unknown fusion gene detection method based on TSO technology developed by the present invention uses random primers with universal sequences and random primers and template replacement primers to add universal sequences at both ends of the cDNA sequence, which can realize the detection of the fusion situation on both sides of the target gene, and the detection result is more comprehensive. In addition, by designing the template replacement primer and the second random primer (random primer with universal sequence), the adapter sequences at both ends of the by-product in the first PCR amplification reaction are complementary, and the intramolecular annealing forms a loop structure, which effectively inhibits the amplification of the full-length by-product, especially the formation of by-products represented by rRNA, which is beneficial to subsequent sequencing and analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Schematic diagram of the technical principle of the i5-i5 primer system in Example 1;

[0054] Figure 2 This is a schematic diagram of the technical principle of the i5-i7 primer system in Comparative Example 1;

[0055] Figure 3 The figure shows the detection results of 14 fusion forms under the same conditions using the i5-i5 primer system and the i5-i7 primer system;

[0056] Figure 4A The on-target detection results of the i5-i5 primer system and the i5-i7 primer system are shown in FIG.

[0057] Figure 4B The result graph of the proportion of wild-type reads detected by the i5-i5 primer system and the i5-i7 primer system;

[0058] Figure 4C The result graph of the proportion of reads spanning the breakpoints detected by the i5-i5 primer system and the i5-i7 primer system;

[0059] Figure 4D The figure shows the percentage of ribosomal RNA reads detected by the i5-i5 primer system and the i5-i7 primer system. DETAILED DESCRIPTION

[0060] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0061] If no specific technology or conditions are specified in the examples, the technology or conditions described in the literature in the field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels. In addition, "reverse transcription" in the claims, description and examples of the present invention is synonymous with "reverse transcription".

[0062] The present invention develops a method for constructing an unknown fusion gene detection library based on template switch oligo, and the technical principle is as follows: 1. using random primers and template replacement primers to generate a cDNA library with a fixed sequence at the 3' end through reverse transcription reaction, and simultaneously using random primers with a universal sequence to generate a cDNA library with a fixed sequence at the 5' end through reverse transcription reaction, and the fixed sequences at both ends are in a reverse complementary relationship, and the upstream primer is shared in the downstream PCR reaction; 2. designing a gene specific primer (GSP) according to the position of the target gene on the RNA sequence; if the fusion situation of the 3' end of the target gene 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 situation of the 5' end of the target gene 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 of the gene specific primer, the unknown fusion event of the target gene can be detected.

[0063] Example 1

[0064] This example designs primers for constructing an unknown fusion gene detection library.

[0065] The primer sequences are shown in Table 1.

[0066] Table 1

[0067]

[0068] Where N is a random base, A, G, C or T; rG is the RNA G base. The primer sequences were synthesized by Integrated DNA Technologies (USA) (IDT). The product was delivered in the form of dry powder, and IDTE buffer (IDT) was added according to the volume recommended by the manufacturer, and the quality control was performed using a NanoDrop spectrophotometer (Thermo Fisher).

[0069] The technical principle of the above primer system (named as i5-i5 system) is as follows Figure 1As shown, 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 undergoes reverse transcription reaction to the 5' end of RNA through 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-displacing 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 reaction 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. Fragment, if 5' driver gene fusion is detected, the target fragment is amplified by 5' gene specific primer-1 and universal primer 1, and the product is purified. In addition, the same cDNA from the two tubes of RT will produce byproducts during amplification. The byproduct type can be any form contained in RNA, including fusion type / wild type / rRNA / mRNA, etc. In this regard, the template replacement primer and the random primer with a universal sequence are designed in this embodiment to have a common sequence, so that the adapter sequences at both ends of the full-length byproduct are complementary, and can anneal intramolecularly to form a loop structure, inhibit PCR formation, and greatly reduce the proportion of RNA full-length byproducts. And the template replacement primer and the random primer with a universal sequence have a common sequence so that the two tubes of cDNA can share the universal primer 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.

[0070] ③PCR2 step: Use P5 and P7 sequencing adapter primers to amplify the purified PCR1 product. After amplification, purify the library (in Figure 1 In the figure, the P5 sequencing adapter primer is shown as the P5 universal primer, and the P7 sequencing adapter primer is shown as the P5 universal primer. Figure 1 P7 universal primer is shown in FIG.

[0071] 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.

[0072] 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 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, so as to achieve the purpose of tailing, so as to amplify the target and tail the target using the P5 sequencing adapter primer. For this purpose, 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 identical to the universal sequence.

[0073] Gene specific primers (GSP primers) are designed according to the genes to be detected, and an exemplary display is shown in Table 2.

[0074] Table 2

[0075]

[0076]

[0077] Example 2

[0078] This example constructs an unknown fusion gene detection library.

[0079] 1. Prepare TSO reverse transcription system according to Table 3.

[0080] Seracare Fusion RNA Mix v4 (Material No 0710-0497, Batch No 10670288) was diluted to 6 copies / ng with a mixture of lung cancer clinical RNA negative samples, with a concentration of 32ng / μL Seracare mix. 25ng (diluted to ~150 copies with EML4-ALK as the standard) was taken as sample RNA for template switching (TSO) reaction and reverse transcription (RT) reaction respectively.

[0081] Table 3

[0082] Components Volume (μL) Dosage Sample RNA Moderate 25ng N6 random primer (6 random bases; 10 μM) 2 2μM dNTP(25mM) 0.4 1mM Nuclease-free Water Make up to 6 μL -

[0083] The above system was prepared, mixed by pipetting, centrifuged instantaneously, and the thermal cover of the PCR instrument was set to ≥85°C. The reaction procedure was performed at 70°C for 5 min and maintained at 4°C. Subsequently, the template switching (TSO) reaction system was added to continue the reaction. The template switching reaction system is shown in Table 4. The reaction procedure was performed in the PCR instrument at 42°C for 90 min, 85°C for 5 min and maintained at 4°C to generate template switching reverse transcription products.

[0084] Table 4

[0085] Reagents Volume (μL) Final concentration 4× Template Replacement Reverse Transcription Buffer 2.5 1 Template displacement primer (75 μM) 0.5 3.75μM 10× Template-displacing Reverse Transcriptase Mix 1 1×

[0086] 2. Preparation of reverse transcription system: Prepare according to Table 5.

[0087] Table 5

[0088] Components Volume (μL) Dosage Sample RNA Moderate 25ng 10× Reverse Transcription Buffer 1 1× Random primers with universal sequence 1 (1 μM) 1.5 0.15μM dNTP(25mM) 0.5 1mM DTT(20mM) 0.5 1mM Reverse transcriptase (200 U / μL) 1 200U Nuclease-free Water Make up to 10 μL -

[0089] Prepare the above system, mix by pipetting, centrifuge instantaneously, set the heat cover of the PCR instrument to ≥85°C, and carry out the reaction program of 25°C, 10 min, 42°C, 30 min, 80°C, 20 min, and maintain at 4°C to generate reverse transcription products.

[0090] 3. Product purification.

[0091] The reverse transcription product was transferred to the template-switched reverse transcription product, a total of 20 μL, and 50 μL (2.5×) AMPureXp Beads was added and mixed; after standing at 25°C for 5 min, the supernatant was discarded on a magnetic rack; then washed twice with 200 μL 80% ethanol, dried at 25°C for 5 min, and finally washed with 20 μL NF-H 2 O to elute the purified product. Recover the supernatant to the downstream reaction. 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 the subsequent amplification process, and there is no need to perform subsequent amplification separately, which greatly simplifies the experimental process.

[0092] 4. Preparation of a round of PCR system, as shown in Table 6.

[0093] Table 6

[0094] Components Volume (μL) Amplification reaction solution 1 (4×) 7.5 Universal primer (25 μM) 0.66 5'Gene-specific Primer-1 (10 μM) 0.3 3' gene-specific primer (10 μM) 1.35 Step 3 Purification product 20 <![CDATA[H 2 The]]> 0.19

[0095] Prepare the above reaction system and carry out the reaction program shown in Table 7 in a PCR instrument.

[0096] Table 7

[0097]

[0098] 5. Purification and recovery of the first round of PCR products.

[0099] Add 45 μL (1.5×) AMPure Xp Beads to the first round of PCR product system and mix well; let stand at 25°C for 5 min and discard the supernatant on a magnetic rack; then wash twice with 200 μL 80% ethanol, dry at 25°C for ~5 min, and finally wash with 20 μL NF-H 2 The purified product was eluted with 0. The supernatant was recovered for downstream reaction.

[0100] 6. Preparation of the second round PCR system is shown in Table 8.

[0101] Table 8

[0102]

[0103] Prepare the above reaction system and carry out the reaction procedure as shown in Table 9 in a PCR instrument.

[0104] Table 9

[0105]

[0106]

[0107] 7. Purification and recovery of the second round of PCR products:

[0108] Add 40 μL (1.0×) AMPure Xp Beads to the second-round PCR product system and mix well; let stand at 25°C for 5 min and discard the supernatant on a magnetic rack; then wash twice with 200 μL 80% ethanol, dry at 25°C for ~5 min, and finally wash with 20 μL NF-H 2 O Elute the purified product. Recover the supernatant, i.e. the library, and sequence it on a sequencing machine.

[0109] Example 3

[0110] Six fusion-positive clinical samples were selected, including two fusion forms, and the detailed information is shown in Table 10.

[0111] Table 10

[0112] Clinical sample number Tumor type Partner Gene Partner Exon Driver Gene Driver exon 143803S01X1 Lung adenocarcinoma CCDC6 1 RET 12 145579S01X4 Lung adenocarcinoma CCDC6 1 RET 12 160079S01X3 Lung adenocarcinoma CCDC6 1 RET 12 166071S01X3 Lung cancer CCDC6 1 RET 12 160725S01X3 Lung adenocarcinoma CCDC6 1 RET 12 45404S01 Lung cancer EML4 13 ALK 20 45820S01 Lung cancer EML4 13 ALK 20

[0113] The primer system in Example 1 was used and the 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.

[0114] Table 11

[0115]

[0116]

[0117] 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 breakpoints, and percentage of ribosomal RNA reads).

[0118] Table 12

[0119]

[0120] Comparative Example 1

[0121] In this comparative example, a primer system i5-i7 was designed in addition. The template replacement primers and random primers with universal sequences were not designed to have a common sequence, that is, the two ends of a round of PCR byproducts could not be complementary and could not form an intramolecular loop. This was compared with the i5-i5 system in Example 1 to prove that the specially designed primer system of the present invention can reduce the proportion of full-length byproducts.

[0122] 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 are used to 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.

[0123] Table 13

[0124]

[0125] The technical principles of the i5-i7 system are as follows Figure 2 As shown, ① reverse transcription step: RNA is divided into two tubes, one tube is reverse transcribed to the 5' end of RNA by random primers, then non-template bases are added, and finally, a DNA-dependent DNA polymerase reaction is carried out under the guidance of a template replacement primer to synthesize a cDNA library with a fixed sequence at the 3' end, and the other tube is reverse transcribed using a random primer with a universal sequence 2 to generate a cDNA library with a fixed sequence at the 5' end. The two tubes of cDNA libraries are mixed and purified; ② PCR1 step: gene-specific primers are designed according to the position of the target gene on the RNA sequence. If 3'-driven gene fusion is detected, the target fragment is amplified by 3' gene-specific primers and universal primer 1. If 5'-driven gene fusion is detected, the target fragment is amplified by 5' gene-specific primer-2 and universal primer 2. At the same time, the same cDNA from the two tubes of RT will produce byproducts during amplification. The byproduct type can be any form contained in RNA, including fusion type / wild type / rRNA / mRNA, etc., and the product is purified; ③ PCR2 step: The purified PCR1 product is amplified using P5 and P7 sequencing adapter primers, and the library is purified after amplification (in Figure 2 In the figure, the P5 sequencing adapter primer is shown as the P5 universal primer, and the P7 sequencing adapter primer is shown as the P5 universal primer. Figure 1 P7 universal primer is shown in FIG.

[0126] The template switching (TSO) reaction and reverse transcription (RT) reaction were carried out as described in Example 2.

[0127] Test Case

[0128] The libraries prepared in Example 1 and Comparative Example 1 were sequenced and analyzed by bioinformatics.

[0129] The detection results of 14 fusion forms under the same conditions of i5-i5 system and i5-i7 system are as follows Figure 3 As shown, the i5-i5 system uses PCR suppression technology to suppress by-products, and the average fusion detection is increased by 72162 / 4971=14.5 times compared with the i5-i7 system, indicating that the special primer system designed in the present invention makes the adapter sequences at both ends of a round of PCR by-products complementary, and the intramolecular annealing forms a loop structure, which can reduce the proportion of full-length by-products.

[0130] The quality control (QC) comparison chart of i5-i5 system and i5-i7 system under the same conditions is as follows Figure 4A , Figure 4B , Figure 4C and Figure 4D As shown, considering that rRNA accounts for 80% to 95% of the total RNA, the proportion of rRNA in the byproducts of i5-i7 accounts for 69%, which is in line with the fact. In contrast, the rRNA of i5-i5 only accounts for 5.7%. The system of the present invention effectively reduces byproducts. In addition, due to the significant reduction in the proportion of rRNA, the on-target rate is greatly increased.

[0131] In summary, the unknown fusion gene detection method based on TSO technology developed by the present invention uses random primers with universal sequences and random primers and template replacement primers to add universal sequences at both ends of the cDNA sequence, so as to realize the detection of the fusion situation on both sides of the target gene; in addition, the present invention designs specific primers, uses the complementary adapter sequences at both ends of the full-length byproducts, can anneal intramolecularly to form a 100p structure, inhibits the occurrence of PCR, and the target product is effectively amplified using 5' / 3' gene-specific primers and universal primers, thereby achieving effective inhibition of the formation of full-length byproducts, especially byproducts represented by rRNA.

[0132] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope 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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