Detection method of RNA fusion gene

Abstract

The invention belongs to the technical field of biology, particularly relates to a detection method of an RNA fusion gene, and more particularly relates to a detection method of an RNA fusion gene based on an FFPE sample source. According to the method, the total RNA is used as a template to build the library, and the step of enriching mRNA or removing rRNA is not needed, so that the experiment time is shortened, and the experiment cost is reduced; according to the invention, a quality control index DV200 of sample RNA and grade division of the sample are provided, so that more accurate library construction of subsequent samples with different quality grades is facilitated; the internal reference quality control product is added in the whole experiment, so that the whole experiment process is better monitored, and the experiment accuracy is improved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically, relates to a method for detecting RNA fusion genes, and more specifically, relates to a method for detecting RNA fusion genes based on FFPE samples. Background Art

[0002] FFPE samples refer to samples that have been fixed with formalin and embedded in paraffin. Formalin fixation can easily cause nucleic acids in tissues to degrade to varying degrees and cause cross-linking between molecules. The high-temperature infiltration of paraffin further accelerates the degradation of nucleic acids. The storage time and environment also have a huge impact on the nucleic acids in the samples. This results in the nucleic acid quality in different FFPE samples often being quite different. When constructing a library, different library construction methods need to be used for samples of different qualities in order to improve the success rate of library construction and the quality of the data off the machine. Usually, reasonable and effective quality control of the samples is required before library construction, and only samples that pass the quality control can be used for subsequent library construction.

[0004] Dozens of targeted drugs have been approved for marketing in China for different genetic mutations in cancer patients, and hundreds of drugs are undergoing clinical trials. The diversity of targeted drugs has greatly promoted the transformation of clinical demand from single-gene testing to multi-gene testing. With the development of NGS technology, multi-gene hotspot testing has gradually transitioned to a more comprehensive panoramic gene testing, covering a more comprehensive testing area and testing more abundant biological targets.

[0005] The "Chinese Expert Consensus on Clinical Practice of Fusion Gene Detection in Non-Small Cell Lung Cancer (2023 Edition)" points out that in non-small cell lung cancer (NSCLC), gene fusion, as an important type of molecular variation, is an ideal target for targeted therapy. Therefore, it is crucial to accurately detect gene fusion in NSCLC to screen patients who can benefit from corresponding targeted therapy.

[0006] Enriching RNA-level fusion, variation and gene expression information, without considering the influence of introns, more accurately detecting fusion genes. Guiding targeted medication and chemotherapy for tumor patients, predicting the efficacy of immune drugs, guiding prognosis, and drug development.

[0007] Currently commonly used methods for fusion gene detection:

[0008] qRT-PCR mutation detection: It can detect fusion RNA in NSCLC at the RNA level, with high sensitivity and short detection time, but it is limited to detecting known fusions within the design range of the primers and cannot detect unknown fusions. In addition, the accuracy of the detection results of this method is highly dependent on the quality of the sample RNA.

[0009] FISH detection of fusion genes: It is a molecular detection technology that hybridizes fluorescently labeled probes with DNA target sequences in the cell nucleus and observes and analyzes gene amplification or fusion variation under a fluorescence microscope. It is generally considered the "gold standard" for detecting gene amplification and fusion, but it also has certain limitations. For example, not all detected fusions will produce expressible fusion RNA, separation probes may miss smaller intrachromosomal rearrangements leading to false negative results, and different fusion gene variation subtypes cannot be determined and distinguished.

[0010] IHC detection of fusion genes: It is a technology that uses antigen-antibody reaction and chemical color development principles to detect the expression of specific proteins in tissue or cytological sections. IHC detection has high sensitivity, low cost, short detection time, and is easy to automate, but its accuracy depends on whether there is a verified antibody for the fusion protein being detected. For example, Ventana-D5F3 IHC has high accuracy in detecting ALK fusion in patients with lung adenocarcinoma, while ROS1 and NTRK positive IHC results still need to be verified by other detection methods.

[0011] DNA targeted capture next-generation sequencing to detect fusion genes: This method is based on DNA targeted capture next-generation sequencing to detect fusion genes. For mutant genes with introns, there will be missed detection. The "Chinese Expert Consensus on Clinical Practice of Fusion Gene Detection in Non-Small Cell Lung Cancer (2023 Edition)" points out that rare fusion partners, exon breakpoint fusions, and intergenic fusions detected at the DNA level should be further verified at the RNA or protein level (strongly recommended); NSCLC patients with negative driver genes at the DNA level are recommended to further test for gene fusions at the RNA or protein level, especially young, female, non-smoking, low TMB mucinous / solid lung adenocarcinoma patients (recommended).

[0012] Therefore, there is a need in the art for a method that can enrich RNA-level fusion, variation, and gene expression information, detect fusion genes more accurately without considering the influence of introns, and has important significance for guiding targeted medication, chemotherapy, predicting the efficacy of immune drugs, guiding prognosis, and drug development for tumor patients. Summary of the invention

[0013] In view of this, in a first aspect, the present invention provides a method for detecting an RNA fusion gene, comprising the following steps:

[0014] S1. Extraction of sample RNA;

[0015] S2. Classification of RNA quality, wherein the sample RNA is subjected to quality inspection to obtain DV 200 value, and according to the DV 200 The sample RNA is divided into four quality levels: high, medium, low, and poor.

[0016] S3, construction of a pre-library, wherein different fragmentation operations are performed according to samples of different levels, and a pre-library is constructed;

[0017] S4, construction of a library, wherein the pre-library is hybridized with a probe of the fusion gene to construct a library; and

[0018] S5. Analyze the library and obtain the final results.

[0019] Furthermore, the step S2 comprises:

[0020] DV 200 The value is used as the standard for RNA quality classification (DV 200 : The percentage of RNA fragments longer than 200 nucleotides (nt) in the sample to the total RNA fragments), RNA samples were divided into four levels:

[0021] DV 200 ≥70%, high-quality samples;

[0022] DV 200 50% to 70%, medium quality samples;

[0023] DV 200 30% to 50% of low-quality samples; or

[0024] DV 200 ≤30%, poor quality samples.

[0025] Further, the step S3 includes step S3-1, and the step S3-1 includes performing different fragmentation operations according to the following conditions:

[0026] The fragmentation conditions for high-quality samples are: 90°C to 95°C, 5 min to 7 min;

[0027] The fragmentation conditions of the medium-quality sample and the low-quality sample are: 83° C. to 89° C., 5 min to 7 min; or

[0028] Poor quality samples do not need to be fragmented.

[0029] Further, the sample is a FFPE sample, or a non-FFPE sample. Further, the sample is a FFPE sample.

[0030] Furthermore, the step S3 also includes step S3-2: introducing an internal reference quality control product.

[0031] Furthermore, the step S3-2 includes: introducing internal reference quality control products according to samples of different qualities, wherein:

[0032] For high-quality and medium-quality samples, the internal reference quality control introduced with 100 ng RNA input is the original internal reference quality control diluted 10-15 times; and

[0033] For low-quality and poor-quality samples, the internal reference quality control product introduced with 100 ng RNA input was the original internal reference quality control product diluted 20-25 times.

[0034] Furthermore, the internal reference quality control product is ERCC. That is, ERCC RNA Spike-In Control Mixes (life technologies TM Catalog Number 4456740) is a set of 92 pre-formulated polyadenylated transcripts from the ERCC Plasmid Reference Library. These transcripts are traceable through the manufacturing process to the NIST Plasmid Reference Material.

[0035] Furthermore, the step S3 also includes step S3-3: cDNA synthesis, repair, adapter ligation, adapter purification, PCR enrichment and magnetic bead purification.

[0036] Specifically, S3-3 includes: synthesizing one strand of cDNA or directly combining with random primers to synthesize one strand of cDNA;

[0037] After the synthesis of one strand of cDNA, the synthesis of the second strand is performed, and the ds cDNA end repair and dA-tailing are performed simultaneously with the synthesis of the second strand, and the synthesis end repair and dA-tailing of the second strand are combined into one step;

[0038] After the ds cDNA ends are repaired, adapter ligation and adapter purification are performed, followed by PCR amplification, and a pre-library is obtained after purification.

[0039] Furthermore, in step S4, the fusion gene can be NTRK3, SSX4, GREB1, AR, ATF1, BRCA1, BRCA2, CDK4, DDIT3, FEV, RPS6KB1, MBTD1, ASPSCR1, YAP1, etc.

[0040] In some specific embodiments, the nucleotide sequence of the probe of the fusion gene NTRK3 is shown as SEQ ID NO.1-26.

[0041] In some specific embodiments, the nucleotide sequence of the probe of the fusion gene SSX4 is shown as SEQ ID NOs. 27-50.

[0042] In some specific embodiments, the nucleotide sequence of the probe of the fusion gene GREB1 is shown as SEQ ID NOs. 51-332.

[0043] Furthermore, in step S5, analyzing the library includes, for example, performing sequencing analysis on the library.

[0044] The present invention uses total RNA as a template to build a library, and does not require mRNA enrichment or rRNA removal steps, which shortens the experimental time and reduces the experimental cost. The present invention proposes a quality control index DV for sample RNA. 200 And the grade division of samples, so that more accurate library construction can be carried out for samples of different quality grades in the future; the present invention adds internal reference quality control products in the whole experiment, so as to better monitor the whole experimental process and the accuracy of the experiment.

[0045] In a second aspect, the present invention provides a composition for detecting an RNA fusion gene, comprising:

[0046] The nucleotide sequences of the probes for detecting the fusion gene NTRK3 are shown in SEQ ID NOs. 1 to 26;

[0047] A probe for detecting the fusion gene SSX4 whose nucleotide sequence is shown in SEQ ID NO. 27 to 50; or

[0048] The nucleotide sequences of the probes for detecting the fusion gene GREB1 are shown in SEQ ID NOs. 51-332.

[0049] In some specific embodiments, the fusion gene detection composition includes: a probe for detecting the fusion gene NTRK3 having a nucleotide sequence as shown in SEQ ID NO.1-26.

[0050] In some specific embodiments, the fusion gene detection composition includes: a probe for detecting the fusion gene SSX4 having a nucleotide sequence as shown in SEQ ID NOs. 27 to 50.

[0051] In some specific embodiments, the fusion gene detection composition includes: a probe for detecting the fusion gene GREB1 with a nucleotide sequence as shown in SEQ ID NO.51-332.

[0052] The composition of the present invention captures specific fusion gene sequences based on hybridization selection. The composition can be used to obtain fusion gene DNA fragments that are enriched several thousand times. The enriched fusion gene fragment samples can be selectively applied to various gene detection technologies, especially the next generation sequencing technology can be used to detect gene mutations, deletions, additions, and transversions, so as to obtain efficient and accurate results, and provide meaningful theoretical and clinical guidance for the subsequent treatment of related symptoms.

[0053] In a third aspect, the present invention provides use of the above composition for preparing an RNA fusion gene detection kit.

[0054] In a fourth aspect, the present invention provides an RNA fusion gene detection kit, comprising any of the above-mentioned compositions.

[0055] Furthermore, the kit also includes: at least one of a nucleic acid extraction reagent, a nucleic acid purification reagent, a library construction reagent, or a reagent required for sequencing.

[0056] Furthermore, the kit also includes an internal reference quality control product. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 and 2 are the fragmentation results of different fragmentation conditions of the present invention;

[0058] Figure 3 is a flowchart example of the method of the present invention;

[0059] Figures 4 to 8 The RNA quality inspection results of the samples of the present invention (samples S1 to S5 respectively). DETAILED DESCRIPTION

[0060] The present invention will be specifically described below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are used to illustrate the present invention, but not to limit the present invention. In the examples, if specific techniques or conditions are not indicated, they are carried out according to the techniques or conditions described in the literature in this area or according to the product instructions. If the manufacturer is not indicated for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0061] Embodiment 1, the method used by the present invention

[0062] 1.1 Total RNA extraction from samples

[0063] Nucleic acid extraction: AllPrep DNA / RNA FFPE Kit (Qiagen, 80234) was used to extract total RNA from FFPE samples, and the concentration was determined using the Qubit RNA HS Assay Kits (Thermo Fisher Scientific, Q32852) quantitative kit. The quality of total RNA was tested using the fully automatic nucleic acid protein analysis system Bioptic Qsep400 (Hangzhou Houze Biotechnology Co., Ltd.). Determine the DV of the starting total RNA 200 value.

[0064] 1.2 Construction and quality control of RNA library

[0065] RNA library construction kit (Shengwei Gene) was used to construct a pre-library of total RNA (the library construction process is as follows Figure 1 ), using the Invitrogen Qubit 4 instrument (Thermo Fisher) and Qubit TM The concentration of the pre-library was detected by 1X dsDNA High Sensitivity (HS) and Broad Range (BR) Quantification Kit (Thermo Fisher, Q33231). The fragment size of the pre-library was detected by Qsep 400 Fragment Analyzer (Houze Biotechnology).

[0066] The RNA library construction kit (Shengwei Gene) recommends that the library insert fragment size is 200-300bp. According to the different quality levels of FFPE RNA samples, we designed different fragmentation conditions to detect their effects on the library insert fragment size. The specific data are shown in Table 1 below.

[0067] Table 1

[0068]

[0069] According to the test results, the inserted fragment size of high-quality samples meets 200-300bp under the fragmentation condition of 94℃5min; the inserted fragment size of low- and medium-quality samples meets 200-300bp under the fragmentation condition of 85℃5min; therefore, the fragmentation condition for high-quality samples is 94℃5min, and the fragmentation condition for low- and medium-quality samples is 85℃5min. Figure 1 , Figure 2 Figure 2 shows the insert fragment sizes obtained under different fragmentation conditions.

[0070] Therefore, the method of the present invention can better distinguish different samples, so that the effect of library construction is better.

[0071] The fragmentation conditions for FFPE RNA of different qualities are shown in Table 2:

[0072] Table 2. Fragmentation conditions of FFPE RNA

[0073] <![CDATA[DV 200 ]]> Fragmentation conditions ≥70％ 94℃,5min 30％～70％ 85℃,5min ≤30％ Don't interrupt

[0074] Standards for ERCC sequencing results of internal reference quality control products in FFPE RNA samples of different qualities: The ERCC Panel of the present invention includes 24 types of probe designs in ERCC, so the captured ERCC types are analyzed based on the detection of these 24 types. The specific information of the 24 ERCC types is shown in Table 3 below.

[0075] Table 3

[0076]

[0077] The capture rate of ERCC types is greater than 70%, that is, the capture rate of ERCC types is greater than or equal to 17, and R.squared>0.7. Meeting the above conditions indicates that there is no deviation in ERCC targeted capture. It is recommended that the ERCC data ratio should be within 10% of the total data. Too high a data occupancy rate will cause data waste. According to this standard, we designed different internal reference quality control product inputs for different quality FFPE RNA samples for testing, and analyzed the sequencing data results, as shown in Table 4 below.

[0078] Table 4

[0079]

[0080] According to the ERCC quality control sequencing data test results, the input amounts of FFPE RNA samples of different qualities that meet the ERCC internal quality control sequencing standards are as follows, that is, the input amounts of internal reference quality control products ERCC of FFPE RNA of different qualities are shown in Table 5 below:

[0081] Table 5. ERCC investment

[0082] <![CDATA[DV 200 ]]> ERCC stock solution dilution multiple Input volume (μL) ≥50％ 10 times 1 ＜50％ 20 times 1

[0083] 1.3 Construction and quality control of post-capture libraries

[0084] The DNA library hybridization capture kit (Shengwei Gene) was used to construct the post-capture library (the hybridization capture process is as follows Figure 3 ). The Invitrogen Qubit 4 instrument (Thermo Fisher) and Qubit TM The concentration of the captured library was detected by 1X dsDNA High Sensitivity (HS) and Broad Range (BR) Quantification Kit (Thermo Fisher, Q33231). The fragment size of the captured library was detected by Qsep400 Fragment Analyzer (Houze Biotechnology).

[0085] 1.4 Post-capture library application - next-generation sequencing

[0086] After the double-stranded concentration and fragment size of the captured library are qualified, the second-generation sequencing is performed using the SansureSeq1000 high-throughput sequencer (Shengxiang Bio). The captured library can also be used for SansureSeq series, GeneMind series, and Illumina series high-throughput sequencing platforms.

[0087] Example 2, test results of the method of the present invention

[0088] 2.1 Sample RNA extraction

[0089] The total RNA of FFPE samples was extracted using AllPrep DNA / RNA FFPE Kit (Qiagen, 80234), and the concentration was determined using the Qubit RNA HS Assay Kits (Thermo Fisher Scientific, Q32852). The quality of the total RNA was tested using the fully automatic nucleic acid protein analysis system Bioptic Qsep400 (Hangzhou Houze Biotechnology Co., Ltd.). 200 value.

[0090] The samples were 5 lung cancer FFPE samples. After extraction, the actual extracted RNA concentration was measured using a Qubit fluorometer as follows. The total amount of extracted RNA was above 100 ng. Sample type, sample extracted RNA concentration, and DV 200 As shown in Table 6, Figures 4 to 8 As shown:

[0091] Table 6. FFPE RNA quality test results

[0092]

[0093] 2.2 Construction and quality control of RNA library

[0094] RNA library construction kit (Shengwei Gene) was used to construct pre-libraries for total RNA samples of different quality levels (library construction process as shown in Figure 1 ), the fragmentation time and ERCC input amount of the sample RNA are shown in Table 7:

[0095] Table 7. Fragmentation time of sample RNA and ERCC input amount

[0096]

[0097] The Invitrogen Qubit 4 instrument (Thermo Fisher) and Qubit TM The concentration of the pre-library was detected by 1X dsDNA high sensitivity (HS) and wide range (BR) quantitative kit (Thermo Fisher, Q33231). The fragment size of the pre-library was detected by Qsep400 fragment analyzer (Houze Bio). The quality inspection results of the pre-library are shown in Table 8:

[0098] Table 8. Sample pre-library quality inspection results

[0099]

[0100] 2.3 Construction of hybridization capture library

[0101] 5 pre-libraries were mixed with equal quality, and the hybrid capture library was constructed using the DNA library hybridization capture kit (Shengwei Gene) and RNA panel kit (Shengwei Gene) (the hybridization capture process is as follows Figure 3 ). The Invitrogen Qubit 4 instrument (Thermo Fisher) and Qubit TM The concentration of the captured library was detected by 1X dsDNA High Sensitivity (HS) and Broad Range (BR) Quantification Kit (Thermo Fisher, Q33231). The fragment size of the captured library was detected by Qsep400 Fragment Analyzer (Houze Bio). The captured library passed the quality inspection and was sent to Illumina Novaseq series high-throughput sequencing.

[0102] 2.4 Sequencing data analysis

[0103] 1. The basic information of the sample sequencing data is shown in Table 9:

[0104] Table 9. Basic information of sequencing data analysis

[0105]

[0106] 2. Fusion analysis: According to the comparison results of sequencing data, analyze the specific breakpoint position of fusion and annotate the fusion breakpoint. The examples and explanations of fusion detection results are shown in Table 10;

[0107] Table 10. Fusion detection result examples and descriptions

[0108]

[0109]

[0110] The results of the detection of 5 FFPE RNA samples are as follows. The results are shown in Table 11 using the Sanger sequencing method. The results show that the method for detecting fusion genes of the present invention detected the presence of fusion gene SLC12A6-NTRK3 in sample S1; the presence of fusion gene RRAGB-SSX4 in sample S2; no fusion gene was detected in sample S3; the presence of fusion gene GREB1-ALK in sample S4; and no fusion gene was detected in sample S5. The Sanger detection results of these samples are consistent with the above detection results, indicating that the detection method provided by the present invention can effectively detect the fusion gene of the sample to be tested.

[0111] Table 11. Results of lung cancer related fusion gene detection

[0112] sample Fusion gene Sanger sequencing results S1 SLC12A6-NTRK3 Consistency S2 RRAGB-SSX4 Consistency S3 none Consistency S4 GREB1-ALK Consistency S5 none Consistency

[0113] 3. Sample ERCC internal reference control data

[0114] ERCC standard: capture of ERCC species greater than 70%, R.squared>0.7, meeting the above conditions indicates that there is no deviation in ERCC targeted capture. It is recommended that the ERCC data ratio should be within 10% of the total data. Too high a data occupancy rate will result in data waste. (The ERCC Panel provided by the present invention includes 24 types of probe designs, so the capture of ERCC species is analyzed based on the detection of these 24 types)

[0115] In the ERCC data analysis of the 5 samples, all 24 types of ERCC were detected, and R.squared>0.7, there was no deviation in ERCC targeted capture, and the ERCC reads ratio was within 10%, proving that the entire library construction, hybridization capture and on-machine sequencing process were qualified. The 5 ERCC internal reference quality control data are shown in Table 12.

[0116] Table 12. Basic information of ERCC internal reference quality control data

[0117] sample No_of_detected_ERCC R.squared ERCC_reads_ration% S1 n＝24 0.9754 9.0505 S2 n＝24 0.9667 5.3349 S3 n＝24 0.936 3.4521 S4 n＝24 0.9710 8.6926 S5 n＝24 0.9618 5.5256

Claims

1. A method for detecting RNA fusion genes, comprising the following steps: S1. Extraction of sample RNA; S2. Classification of RNA quality in, The sample RNA is quality tested to obtain DV 200 value, and according to the DV 200 The sample RNA is divided into four quality levels: high, medium, low, and poor. S3, construction of a pre-library, wherein different fragmentation operations are performed according to samples of different levels, and a pre-library is constructed; S4, construction of a library, wherein the pre-library is hybridized with a probe of the fusion gene to construct a library; and S5. Analyze the library and obtain the final results.

2. The detection method according to claim 1, It is characterized in that The step S2 comprises: DV 200 The value is used as the standard for RNA quality classification, and RNA samples are divided into four levels: DV 200 ≥70% are high-quality samples; 50% ≤ DV 200 <70% is a medium quality sample; 30% <DV 200 <50% are low-quality samples; or DV 200 ≤30% is poor quality sample.

3. The detection method according to claim 2, It is characterized in that The step S3 includes step S3-1, and the step S3-1 includes performing different fragmentation operations according to the following conditions: The fragmentation conditions of the high-quality sample are: 90°C to 95°C, 5min-7min; The fragmentation conditions of the medium-quality sample and the low-quality sample are: 83° C. to 89° C., 5 min to 7 min; or The poor quality samples do not need to be fragmented.

4. The detection method according to claim 3, It is characterized in that The step S3 also includes step S3-2: introducing internal reference quality control products according to samples of different qualities, wherein: For high-quality and medium-quality samples, the internal reference quality control introduced with 100 ng RNA input is the original internal reference quality control diluted 10-15 times; or For low-quality and poor-quality samples, the internal reference quality control product introduced with 100 ng RNA input was the original internal reference quality control product diluted 20-25 times.

5. The detection method according to any one of claims 1 to 4, It is characterized in that In step S4, the fusion gene is NTRK3, SSX4, GREB1, AR, ATF1, BRCA1, BRCA2, CDK4, DDIT3, FEV, RPS6KB1, MBTD1, ASPSCR1, and YAP1.

6. The detection method according to claim 5, It is characterized in that The step S5 includes sequencing the library.

7. A composition for detecting RNA fusion genes, include: The nucleotide sequences of the probes for detecting the fusion gene NTRK3 are shown in SEQ ID NOs. 1 to 26; A probe for detecting the fusion gene SSX4 whose nucleotide sequence is shown in SEQ ID NO. 27 to 50; or The nucleotide sequences of the probes for detecting the fusion gene GREB1 are shown in SEQ ID NOs. 51-332.

8. Use of the composition as claimed in claim 7 in preparing an RNA fusion gene detection kit.

9. An RNA fusion gene detection kit, comprising the composition according to claim 7.

10. The kit according to claim 9, It is characterized in that The kit also includes: at least one of a nucleic acid extraction reagent, a nucleic acid purification reagent, a library construction reagent, or a reagent required for sequencing.

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