Library construction method for simultaneously detecting multiple gene mutation types of brain tumors and composition and application thereof

Abstract

The application discloses a library construction method for simultaneously detecting various gene mutation types of brain tumors, and a composition and application thereof. The sequencing library construction method established by the application is a one-step library construction method, which places DNA mutation and RNA fusion in one PCR reaction system, and optimizes pyrophosphate detection MGMT and FISH detection CNV into an amplicon one-step method, so that DNA level mutation and RNA level fusion can be simultaneously detected. Moreover, after the library construction, QPCR quantification is not needed, and only Qubit quantification is needed, and then direct machine sequencing operation is simple. The application can be applied to rapid, simple and accurate detection of brain tumor basic items.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for constructing a library for simultaneously detecting multiple gene mutation types in brain tumors, and the compositions and applications thereof. Background Technology

[0002] Gliomas are the most common primary malignant intracranial tumors, exhibiting high heterogeneity and a poor prognosis. With the development of brain tumor genomics in recent years, molecular pathological testing using gene markers as a prerequisite and foundation for glioma treatment has become a recognized trend in the medical community. Numerous clinical studies have also demonstrated that specific genes are directly related to tumor subtyping and drug efficacy. In 2016, the WHO incorporated molecular pathology into its glioma pathological diagnostic system. Molecular pathology based on tumor genetics can more accurately predict the clinical prognosis of glioma patients and provide a basis for differentiating tumors whose histological diagnosis and grading are difficult to determine.

[0003] Basic brain tumor detection methods include DNA hotspot mutations (such as IDH mutations, TERT promoter mutations, BRAF mutations, ATRX mutations, etc.), RNA fusions, MGMT methylation, 1p / 19q combined deletions, and Chr7 and chr10 copy number variations. Currently, DNA hotspot mutations can be detected using first-generation and second-generation NGS methods. RNA fusions are generally detected using FISH and NGS methods, MGMT methylation is detected using pyrosequencing, and 1p / 19q, Chr7, and chr10 copy number variations are detected using FISH. To detect all these parameters in a single sample, a large initial sample size is required, the methods are complex, and the requirements for sample quality, technical personnel, and laboratory conditions are relatively high. Therefore, there is an urgent need to design a new method for glioma detection to address these issues.

[0004] TNA includes DNA and RNA, and is an abbreviation for the sum of the two. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to quickly, easily and / or accurately detect biomarker genes of glioma or detect basic glioma markers.

[0006] To address the aforementioned technical problems, this invention first provides a method for constructing sequencing libraries. The method may include the following steps: extracting TNA from the sample to be tested; reverse transcribing the TNA to obtain cDNA, and converting the TNA to sulfite to obtain transformed DNA; preparing primer solutions P1, P2, P3, and P4; performing PCR amplification named P1 amplification using the P1 primer solution to obtain a PCR product sub-library named P1; performing PCR amplification named P2 amplification using the P2 primer solution to obtain a PCR product sub-library named P2; performing PCR amplification named P3 amplification using the P3 primer solution to obtain a PCR product sub-library named P3; performing PCR amplification named P4 amplification using the P4 primer solution to obtain a PCR product sub-library named P4; mixing the P1, P2, P3, and P4 sub-libraries to obtain a mixed library; and purifying the mixed library to obtain the sequencing library.

[0007] In the above method, the TNA of the sample to be tested can be divided into 4 parts: 1 part of TNA is used for reverse transcription to obtain cDNA for P1 amplification, 1 part of TNA is used for sulfite conversion to obtain transformed DNA for P3 amplification, and the remaining 2 parts of TNA can be used directly for P2 and P4 amplification without processing.

[0008] In the methods described above, the PCR amplification template for P1 amplification can be the cDNA. The PCR amplification template for P3 amplification can be the sulfite-converted DNA. The PCR amplification templates for P2 and P4 amplification can be the TNA.

[0009] In the method described above, the sequencing library can be used to detect gene mutation types in gliomas. These gene mutation types may include DNA hotspot mutations, RNA fusion mutations, MGMT methylation mutations, 1p / 19q combined deletions, and / or chromosomal copy number variations.

[0010] The DNA hotspot mutations may include IDH1, IDH2, BRAF, H3F3A, HIST1H3B, HIST1H3C, and TERT genes. The RNA fusion mutations may include KIAA1549-BRAF. The methylation mutations may include MGMT. The copy number variations may include 1p / 19q and / or chr7 and chr10 copy number variations.

[0011] In the method described above, the primers in the P1 primer solution are a combination of G13f and G13r. G13f can be a mixture of 11 DNA molecules from sequences 1 to 11 in the sequence listing. G13r can be a mixture of DNA molecules from sequences 15 to 25 in the sequence listing.

[0012] The P2 solution may be a combination of G13f12 and G13r12. G13f12 may be the DNA molecule shown in sequence 12 of the sequence listing. G13r12 may be the DNA molecule shown in sequence 26 of the sequence listing.

[0013] The P3 solution may be a combination of G13f13 and G13r13. G13f13 is the DNA molecule shown in sequence 13 of the sequence listing, and G13r13 is the DNA molecule shown in sequence 27 of the sequence listing.

[0014] The P4 solution may be a combination of G13f14 and G13r14. G13f13 may be the DNA molecule shown in sequence 14 of the sequence listing. G13r13 may be the DNA molecule shown in sequence 28 of the sequence listing.

[0015] In the method described above, G13f, G13f12, G13f13, and G13f14 can be upstream primers. G13r, G13r12, G13r13, and G13r14 can be downstream primers.

[0016] The P1 primer solution also contains TE buffer. The P1 primer solution can be prepared as follows: the 11 DNA molecules shown in sequences 1-11 of the sequence listing are diluted to 100 μM solutions and then mixed in equal volumes to obtain G13f; the DNA molecules shown in sequences 15-25 of the sequence listing are diluted to 100 μM solutions and then mixed in equal volumes to obtain G13r; and the G13f, the G13r, and the TE buffer are mixed in a volume ratio of 1:2:3 to obtain the P1 solution.

[0017] The P2 solution may also contain TE buffer. The P2 solution can be prepared by diluting the DNA molecule shown in sequence 12 of the sequence listing to a 100 μM solution to obtain the G13f12, diluting the DNA molecule shown in sequence 26 of the sequence listing to a 100 μM solution to obtain the G13r12, and mixing the G13f12, the G13r12 and the TE buffer at a volume ratio of 1:2:57 to obtain the P2 solution.

[0018] The P3 solution may also contain TE buffer. The P3 solution can be prepared by: diluting the DNA molecule shown in sequence 13 of the sequence listing to a 100 μM solution to obtain the G13f13, diluting the DNA molecule shown in sequence 27 of the sequence listing to a 100 μM solution to obtain the G13r13, and mixing the G13f13, the G13r13 and the TE buffer at a volume ratio of 1:2:57 to obtain the P3 solution.

[0019] The P4 solution may also contain TE buffer. The P4 solution can be prepared by: diluting the DNA molecule shown in sequence 14 of the sequence listing to a 100 μM solution to obtain the G13f14, diluting the DNA molecule shown in sequence 28 of the sequence listing to a 100 μM solution to obtain the G13r14, and mixing the G13f14, the G13r14 and the TE buffer at a volume ratio of 1:2:57 to obtain the P4 solution.

[0020] In the method described above, the PCR system may further include specific adapters. These specific adapters may be one of the seven DNA molecules represented by sequences 29-35 in the sequence listing.

[0021] The specific adapter may include an upstream universal primer and a downstream universal primer. The upstream universal primer may be a DNA molecule shown in sequences 29-34 of the sequence listing. The downstream universal primer may be a DNA molecule shown in sequence 35 of the sequence listing.

[0022] The upstream universal primer may also contain a first adapter sequence. The 5' end of the DNA molecules represented by sequences 1-11 in the upstream primer G13f described above may contain the first adapter sequence. The DNA molecules represented by sequence 12 in the upstream primer G13f12, sequence 13 in the upstream primer G13f13, and sequence 14 in the upstream primer G13f14 may all contain the first adapter sequence. The first adapter sequence may be nucleotides 1-17 of sequence 1 in the sequence listing. The first adapter sequence can be used to connect the upstream primer and the upstream universal primer.

[0023] The downstream universal primer may contain a second adapter sequence. The 5' end of the DNA molecules represented by sequences 15-25 in G13r described above may contain a second adapter sequence. The DNA molecules represented by sequence 26 in G13r12, sequence 27 in G13r13, and sequence 28 in G13r14 described above may contain a second adapter sequence. The second adapter sequence may be nucleotides 1-19 of sequence 15 in the sequence listing. The second adapter sequence can be used to connect the downstream primer and the downstream universal primer.

[0024] The upstream universal primer also contains a third adapter sequence. This third adapter sequence can be used to add tags and adapters to the sequencing library and the sequencing platform, and is used for the ligation of sequencing primers during the sequencing process.

[0025] The downstream universal primer may also contain a universal sequence. This universal sequence can be used to add a sequencing platform adapter to the downstream universal primer for emulsion PCR during the sequencing process.

[0026] In the method described above, the PCR reaction program can be: denaturation at 95℃ for 2 min; 23 cycles of 95℃ for 30 s, 60℃ for 90 s, and 72℃ for 90 s; termination of the reaction at 72℃ for 10 min. In the method described above, the preparation method of the mixed library can include: diluting the P4 sub-library with 4 times its volume of water to obtain a P4 sub-library diluent; mixing the P1 sub-library, the P2 sub-library, the P3 sub-library, and the P4 sub-library diluent to obtain a mixed library; the volume ratio of the P1, P2, P3, and P4 sub-library diluents is 5:20:20:1.5.

[0027] To address the aforementioned technical problems, the present invention also provides a composition of primers for detecting glioma genes. The composition may contain DNA molecules shown in sequences 1-28 of the sequence listing or DNA molecules shown in sequences 1-35 of the sequence listing.

[0028] To address the aforementioned technical problems, the present invention also provides a method for detecting glioma genes, characterized in that: the method includes the steps of establishing a sequencing library from the sample to be tested using the method described in any one of claims 1-6, sequencing the sequencing library using a sequencer, and analyzing the sequencing data.

[0029] The sequencer mentioned above can be a GENETRON S5 gene sequencer or a DA8600 gene sequencer.

[0030] In the method described above, the sample to be tested can be a paraffin sample.

[0031] The application of the methods and / or compositions described above in the preparation of products for detecting glioma genes and / or brain tumors is also within the scope of protection of this invention.

[0032] The purpose of the above methods can be for disease diagnosis, disease prognosis and / or disease treatment, or it can be for non-disease diagnosis, non-disease prognosis and non-disease treatment purposes; their direct purpose can be to obtain information on intermediate results of disease diagnosis, disease prognosis and / or disease treatment, or their direct purpose can be for non-disease diagnosis, non-disease prognosis and / or non-disease treatment purposes.

[0033] This invention provides a rapid, simple, and accurate method for detecting basic parameters of brain tumors. The sequencing library construction method established in this invention is a one-step library construction method, placing DNA mutations and RNA fusions in a single PCR reaction system; and optimizing pyrosequencing for MGMT and FISH for CNV detection into a one-step amplicon method, capable of simultaneously detecting DNA mutations and RNA fusions. Furthermore, after library construction, qPCR quantification is unnecessary; only qubit quantification is required before direct sequencing, simplifying the process. Specifically, a TNA (Total Nucleic Acid) extraction method is used for one-step library construction, followed by detection on the ION TORRENT platform. An automated bioinformatics workflow analyzes the data and generates results. All four detection parameters are covered using only 100 ng of TNA (Total Nucleic Acid).

[0034] The present invention has the following advantages compared with the prior art due to the adoption of the above technical solutions: (1) The present invention puts DNA mutation and RNA fusion in a PCR reaction system, which can simultaneously detect DNA mutation and RNA fusion, and the operation is simple.

[0035] (2) This invention optimizes the detection of MGMT by pyrosequencing and CNV by FISH (Fluorescence in situ hybridization) into a one-step amplicon method, which is simple and fast.

[0036] (3) After the PCR reaction of the present invention, the library is mixed in proportion, and 4 reaction systems are synthesized into 1 purification system, which is simple to operate.

[0037] (4) After library construction, qPCR quantification is not required. Qubit quantification is sufficient for direct instrumentation, which is simple, convenient and time-saving. Attached Figure Description

[0038] Figure 1This is a distribution diagram of amplified products obtained after the amplicon library construction was completed in Example 1 of this invention. The vertical axis represents the concentration of amplified products, and the horizontal axis represents the length of amplified products. Detailed Implementation

[0039] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0041] Example 1: Library construction method and sequencing result analysis for detecting glioma genes.

[0042] 1. Constructing an amplicon DNA library of the sample to be tested

[0043] The samples to be tested were paraffin (FFPE) samples from 6 brain tumor patients in the Beijing Genecast Sample Bank.

[0044] The specific process for constructing the amplicon library of the sample to be tested is as follows:

[0045] 1.1 Extraction of TNA from Genomic Samples: TNA was extracted from FFPE samples using the RNeasy FFPE Kit (QIAGEN, 73504). The specific steps are as follows:

[0046] 1) Use a scraper to scrape the tissue from the paraffin slide and place it into a 1.5 mL centrifuge tube for step 2; for FFPE rolls, place them directly into a 1.5 mL centrifuge tube for step 2.

[0047] 2) Add 60 μL of dewaxing agent to the centrifuge tube, vortex vigorously for 10 seconds, and then centrifuge briefly.

[0048] 3) Incubate at 56℃ for 3 minutes, then cool to room temperature.

[0049] 4) Add 150uL of Buffer PKD, vortex to mix, and centrifuge at 11000g (10000rpm) for 1 minute.

[0050] 5) Add 10 μL of proteinase K to the bottom colorless solution and mix slowly by pipetting.

[0051] 6) Incubate at 56℃ for 15 minutes, then at 80℃ for 15 minutes.

[0052] 7) Transfer the colorless liquid at the bottom to a new 1.5 mL centrifuge tube.

[0053] 8) Incubate on ice for 3 minutes, then centrifuge at 20000g (13500rpm) for 15 minutes.

[0054] 9) Transfer the supernatant to a new 1.5 mL centrifuge tube, being careful not to aspirate the precipitate.

[0055] 10) Add 320 μL of Buffer RBC, vortex to mix, and then detach.

[0056] 11) Add 720 μL of anhydrous ethanol, mix well by pipetting with a pipette tip, do not centrifuge, and proceed to the next step immediately.

[0057] 12) Transfer 700 μL of sample to an RNA adsorption column (RNeasy MinElute spin column) (place the adsorption column in a 2 mL collection tube), gently cap the column, centrifuge at ≥8000g (≥10000rpm) for 15 seconds, and discard the waste liquid in the collection tube. Repeat step 12 until all the mixture has passed through the adsorption column.

[0058] 13) Add 500 μL of Buffer RPE to the adsorption column, gently cover it, centrifuge at ≥8000g for 15 seconds, and discard the waste liquid in the collection tube.

[0059] 14) Add 500 μL of Buffer RPE to the adsorption column, gently cover it, centrifuge at ≥8000g for 2 minutes, and discard the collection tube.

[0060] 15) Place the adsorption column into a new 2mL collection tube, open the cap and centrifuge at maximum speed for 5 minutes to dry the inner membrane of the adsorption column.

[0061] 16) Place the adsorption column into a new 1.5 mL centrifuge tube, add 30 μL of RNase-free water dropwise onto the inner membrane of the adsorption column, gently cap the tube, incubate at room temperature for 1 min, centrifuge at maximum speed for 1 min, and elute TNA. The TNA was then quantified using Qubit. The extraction concentrations of the six paraffin samples are shown in Table 1.

[0062] Table 1. Sample Extraction Concentration

[0063]

[0064] 1.2. Design and synthesize primers:

[0065] Specific detection items: DNA hotspot mutations, RNA fusions, MGMT methylation, 1p / 19q combined deletions, and Chr7 and chr10 copy number variations.

[0066] The specific detection items correspond to the following target genes: DNA hotspot mutation genes IDH1 / 2, TERT, BRAF, H3F3A, HIST1H3B, HIST1H3C; RNA fusion KIAA1549-BRAF; MGMT methylation; 1p / 19q combined deletion; and Chr7 and chr10 copy number variations (Table 2).

[0067] Primers are designed based on the target gene:

[0068] Upstream primer: First adapter sequence + specific upstream primer sequence;

[0069] Downstream primer: Second adapter sequence + specific downstream primer sequence;

[0070] Upstream universal primer: third adapter sequence + barcode sequence + first adapter sequence;

[0071] Downstream universal primer: universal sequence + second adapter sequence.

[0072] The first adapter sequence in the primer is used for ligation between the upstream universal primer and the upstream primer. The second adapter sequence in the primer is used for ligation between the downstream universal primer and the downstream primer. The third adapter sequence in the primer is the sequencing start sequence.

[0073] The primer combinations used to construct TNA sample amplicon libraries, based on the target genes (common glioma mutant genes) (Table 2), include the following information on specific upstream and downstream primer sequences:

[0074] Table 2 provides the target-specific upstream primer sequences (Special Primer Start, G13f1-G13f14 in Table 2) and the target-specific downstream primer sequences (Special Primer End, G13r1-G13r14 in Table 2). It also provides the sequences of universal upstream and downstream primers: Guf represents optional universal upstream primers (Guf1-Guf6 in Table 2), which are used to add unique tags and sequencing platform adapters to the library for sequencing primer ligation. Gur represents universal downstream primers (Gur, common adapters in Table 2), which are used to add sequencing platform adapters for emulsion PCR during sequencing.

[0075] Table 2. Primer sequence composition

[0076]

[0077]

[0078] 1.3 Sample Preprocessing

[0079] Detection of RNA fusion and MGMT methylation requires first performing reverse transcription and sulfite conversion on the TNA samples extracted in step 1.1. Reverse transcription is performed using the SuperScript kit. TM VILO TM cDNA Synthesis Kit (Invitrogen 11754250), sulfite conversion kit using EZ DNA Methylation-Lightning TM Kit (Zymo, D5031) was used. Following the kit instructions, 20 ng TNA was reverse transcribed and sulfite-converted for each sample, resulting in cDNA and transformed DNA for 6 FFPE samples.

[0080] 1.4 PCR reaction system

[0081] The specific PCR reaction systems are shown in Tables 3-6 below:

[0082] Table 3. P1 PCR reaction system

[0083]

[0084] Table 4. P2 PCR reaction system

[0085]

[0086]

[0087] Table 5. P3 PCR reaction system

[0088]

[0089] Table 6. P4 PCR reaction system

[0090]

[0091] Each PCR reaction system in Table 3-6 is used to construct an amplicon library for a TNA sample. Four sets of PCR product libraries were obtained by PCR amplification using primer mixtures P1, P2, P3, and P4: P1 was used to detect DNA mutations and RNA fusions; P2 was used to detect TERT mutations; P3 was used to detect MGMT methylation; and P4 was used to detect changes in 1p19q and Chr7 / chr10 copy numbers.

[0092] The starting template in the P1 PCR system is the cDNA obtained after pretreatment in step 1.3; the starting template in the P3 PCR system is the sulfite-converted DNA obtained after pretreatment in step 1.3. The starting templates in the P2 and P4 PCR systems are the TNA samples extracted in step 1.1, which do not require pretreatment.

[0093] Preparation of P1 primer solution: Dissolve the dry powder of G13f1-G13f11 (sequences 1-11 in the sequence listing) to 100 μM, and then mix them in equal volumes to prepare G13f; dissolve the dry powder of G13r1-G13r11 (sequences 15-25 in the sequence listing) to 100 μM, and then mix them in equal volumes to prepare G13r; mix G13f:G13r:TE buffer in a volume ratio of 1:2:3 to prepare P1.

[0094] Preparation of P2 primer solution: Dissolve the dry powder of G13f12 (sequence 12 in the sequence listing) and G13r12 (sequence 26 in the sequence listing) to 100 μM, and then mix them together at a volume ratio of G13f12:G13r12:TE buffer = 1:2:57 to prepare P2.

[0095] Preparation of P3 primer solution: Dissolve the dry powder of G13f13 (sequence 13 in the sequence listing) and G13r13 (sequence 27 in the sequence listing) to 100 μM, and then mix them together at a volume ratio of G13f13:G13r13:TE buffer = 1:2:57 to prepare P3.

[0096] Preparation of P4 primer solution: Dissolve the dry powders of G13f14 (sequence 14 in the sequence listing) and G13r14 (sequence 28 in the sequence listing) to 100 μM, and then mix them together in a volume ratio of G13f14:G13r14:TE = 1:2:57 to prepare P4.

[0097] TE buffer (Invitrogen) TM 12090015) is a purchased pre-made reagent that can be used directly.

[0098] 1.5 PCR amplification

[0099] PCR amplification was performed using the four primer solutions P1, P2, P3, and P4 prepared in step 1.4, resulting in four PCR product sub-libraries: P1, P2, P3, and P4 (collectively referred to as Pools). The PCR instrument used was an Applied bio-systems 2720 Thermal Cycler, and the PCR reaction program was as follows:

[0100] Table 7. PCR reaction procedure

[0101]

[0102] 1.6 Purification of PCR Products

[0103] After the PCR reaction, the four PCR product sub-library mixtures obtained in step 1.5 were purified using the Beckman Coulter Agencourt AMGure XP Kit (catalog number A63880). The procedure is as follows:

[0104] The PCR products were diluted according to Table 8 below to obtain 4 sets of sub-library dilutions. The 4 sets of sub-library dilutions were mixed and the resulting libraries were used for purification. The purification steps were performed in accordance with the instructions.

[0105] Table 8. PCR product dilution

[0106]

[0107] After purification using the four pools, the amplicon library was constructed. Figure 1 This is a distribution graph of amplified products obtained from a four-pool mixture purification assay detected by an Agilent 2200 TapeStation System. The horizontal axis represents fragment length, and the vertical axis represents signal intensity (FU). The lower peak is the 25bp marker, and the upper peak is the 1500bp marker. Figure 1 The four PCR product mixtures obtained after PCR amplification, as shown, were concentrated in the 170-300 bp range after purification. Figure 1 This indicates that the experimental results are consistent with the experimental design. Figure 1 It can determine the size and concentration of the constructed library.

[0108] 2. Library sequencing and result analysis

[0109] Follow the instructions for the universal sequencing reaction kit (Beijing Genecast Technology Co., Ltd., RSN006). Complete the template preparation and sequencing process. Use a GENETRON S5 gene sequencer (catalog number: 4000901, hereinafter referred to as the sequencer) manufactured by Chongqing Genecast Biotechnology Co., Ltd. to sequence the PCR amplicon library prepared in step 1.

[0110] After sequencing is completed using the sequencers described above, the sequencers will use their built-in software BaseCaller (Genetron S5: v5.8.10, DA8600: v5.4.11) to identify bases and perform base information statistics. They will also use their built-in TMAP (Genetron S5: v5.8.10, DA8600: v5.4.11) software to align the sequencing results with the human reference genome sequence hg19 (GRCh37). The final output data includes the aligned Bam file and statistical information of all samples in the chip.

[0111] The ghall plugin (built into the GENETRON S5 gene sequencer), developed by Genentech, automatically performs bioinformatics analysis, quality control assessment, variant result interpretation, and annotation on sequencing data.

[0112] After sequencing and data analysis, the actual sample testing results collected in step 1 are as follows: For the 6 paraffin-embedded brain tumor samples, the specific results are shown in Table 9. No tumor-related mutations were detected in the 2 negative samples (S5 and S6 in Table 9). For the 4 paraffin-embedded brain cancer patients (S1-S4 in Table 9), S1 detected a DNA hotspot mutation (TERT mutation) and MGMT methylation. S2 detected a DNA hotspot mutation (IDH1 / TERT mutation), MGMT methylation, and 1p19q combined deletion. S3 detected a DNA hotspot mutation (TERT mutation), chr7 amplification, and chr10 deletion. S4 detected an RNA fusion mutation (KIAA1549e13_BRAFe9 RNA fusion).

[0113] The test results show that the mutation results detected using the amplicon DNA library construction method established in step 1 of this invention fully demonstrate the practical applicability of this invention.

[0114] Table 9. Sample Detection Results

[0115]

[0116] In summary, the sequencing library construction method established in this invention is a one-step library construction method, placing DNA mutations and RNA fusions in a single PCR reaction system; and the pyrosequencing detection of MGMT and FISH detection of CNV are optimized into a one-step amplicon method, which can simultaneously detect DNA mutations and RNA fusions; moreover, after library construction, qPCR quantification is not required, only qubit quantification is needed before direct sequencing, simplifying the operation. It can be applied to the rapid, convenient, and accurate detection of basic brain tumor parameters.

[0117] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for constructing sequencing libraries, characterized in that: The method includes the following steps: extracting TNA from the sample to be tested; reverse transcribing cDNA using RNA in the TNA as a template, and converting the TNA to sulfite to obtain transformed DNA; preparing primer solutions P1, P2, P3, and P4; performing PCR amplification named P1 amplification using the P1 primer solution to obtain a PCR product sub-library named P1; performing PCR amplification named P2 amplification using the P2 primer solution to obtain a PCR product sub-library named P2; performing PCR amplification named P3 amplification using the P3 primer solution to obtain a PCR product sub-library named P3; performing PCR amplification named P4 amplification using the P4 primer solution to obtain a PCR product sub-library named P4; mixing the P1, P2, P3, and P4 sub-libraries to obtain a mixed library; and purifying the mixed library to obtain the sequencing library. The PCR template for P1 amplification is the cDNA; the PCR template for P3 amplification is the sulfite-converted DNA; and the PCR templates for P2 and P4 amplification are the TNA. The primers in the P1 primer solution are a combination of G13f and G13r, wherein G13f is a mixture of 11 DNA molecules whose nucleotide sequences are sequences 1-11 in the sequence listing, and G13r is a mixture of DNA molecules shown in sequences 15-25 in the sequence listing. The P2 primer solution is a combination of G13f12 and G13r12, wherein G13f12 is the DNA molecule shown in sequence 12 of the sequence listing and G13r12 is the DNA molecule shown in sequence 26 of the sequence listing; The P3 primer solution is a combination of G13f13 and G13r13, wherein G13f13 is the DNA molecule shown in sequence 13 of the sequence listing and G13r13 is the DNA molecule shown in sequence 27 of the sequence listing; The P4 primer solution is a combination of G13f14 and G13r14, wherein G13f14 is the DNA molecule shown in sequence 14 of the sequence listing and G13r14 is the DNA molecule shown in sequence 28 of the sequence listing; The PCR system also contains specific adapters; these specific adapters are seven DNA molecules whose nucleotide sequences are sequences 29-35 of the sequence listing. The method for preparing the mixed library includes: diluting the P4 sub-library with 4 times its volume of water to obtain a P4 sub-library diluent; mixing the P1 sub-library, the P2 sub-library, the P3 sub-library, and the P4 sub-library diluent to obtain a mixed library; the volume ratio of the P1 sub-library, the P2 sub-library, the P3 sub-library, and the P4 sub-library diluent is 5:20:20:1.

5.

2. The method according to claim 1, characterized in that: The sequencing library is used to detect gene mutation types in gliomas; the gene mutation types include DNA hotspot mutations, RNA fusion mutations, MGMT methylation mutations, 1p / 19q combined deletions and / or chromosomal copy number variations.

3. The method according to claim 1 or 2, characterized in that: The PCR reaction procedure was as follows: denaturation at 95℃ for 2 min; 23 cycles of denaturation at 95℃ for 30 s, 60℃ for 90 s, and 72℃ for 90 s; and termination of the reaction at 72℃ for 10 min.

4. The method according to claim 1 or 2, characterized in that: The sample to be tested is a paraffin sample.

5. A composition of primers and adapters for detecting glioma genes, characterized in that: The composition consists of DNA molecules represented by sequences 1-35 in the sequence listing.

6. The use of the composition according to claim 5 in the preparation of a product for detecting glioma genes.

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