CN114317741A - Soft tissue sarcoma gene detection kit and system - Google Patents
Soft tissue sarcoma gene detection kit and system Download PDFInfo
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Abstract
The invention provides a soft tissue sarcoma gene detection kit and a system. The detection kit comprises 71 primer compositions for detecting the fusion gene and amplification primers of the following reference genes: MYC, YEATS4, CFHR5, FOSL1, VGLL3, MTTP, PLAG1, FRS2, CSF1, MDM2, HMGA2, and MATN 1. The kit and the detection system provided by the invention can uniformly amplify a target sequence by the modified multiple amplification primers, add 10 internal reference genes for quality control, detect by an ion torrent high-throughput sequencing platform, detect 233 fusion forms altogether, can comprehensively detect fusion genes possibly existing in a patient body at one time, help to comprehensively and rapidly diagnose diseases clinically, and can also be used for prompting targeted medication.
Description
Technical Field
The invention relates to the technical field of biomedical detection, in particular to a soft tissue sarcoma gene detection kit and a system.
Background
Soft tissue sarcoma often undergoes gene fusion, and detection of gene fusion is clinically needed to aid soft tissue sarcoma gene detection. The most widely used detection method at present is Fluorescence In Situ Hybridization (FISH), and the fusion condition of a certain gene is judged by a gene fusion probe or a gene disruption probe. Immunohistochemical (IHC) markers can also be used to detect molecular changes in soft tissue sarcomas, based primarily on the principles of antigen-antibody reactions and chemochromic development. Some soft tissue sarcomas have definite sequence variation and have guiding significance for the prognosis and medication of patients, and the conventional method for clinically detecting gene mutation at present is first-generation sequencing (Sanger sequencing), and the change condition of a base group in a sequence is analyzed by sequencing a PCR product.
Ideally, fusion probes (fusion probes) are adopted to help determine specific gene fusion types, but some tumors have a plurality of chaperone genes, the occurrence frequency of part of the chaperone genes is low, if the fusion probes are adopted for detection, the cost is high, the conventional development is not suitable, the problem of a plurality of fusion partners can be solved by adopting a fragmentation probe, but the problem that the flux is low, and only one fusion fragmentation mode can be detected at one time still exists. Antibodies developed by immunohistochemical markers according to molecular changes or gene expression profiles in soft tissue sarcomas have a certain auxiliary diagnostic value, but some antibodies are not specific, such as Fli1, TLE1, MDM2, SATB2, BCOR and the like, and can only detect the protein expression level, and the expression level needs to be judged by subjective observation of testers, so that different testers may have different results, and need to pay attention to dialectical analysis. The method is limited by the content and proportion of tumor cells in a specimen, and has the problems of low sensitivity and low detection flux. With the continuous emergence of new molecular anomaly reports, the detection of gene mutation by sequencing of one generation is increasingly unable to meet clinical requirements.
In addition, soft tissue sarcoma relates to many types of diseases, the types of related fusion genes are complex, and with the rapid development of gene detection technology, more and more fusion genes related to soft tissue sarcoma which are not reported before are reported, but the fusion genes recorded in a public database are far behind the leading-edge research at present, and the fusion genes and fusion fracture types recorded in the public database are only relied on, so that the increasing requirements of clinic are difficult to meet, and how to cover the fusion gene types and fusion fracture modes as many as possible is the key for improving the detection performance and detection significance of the kit. Therefore, there is a need for a product and method for comprehensive and rapid assessment of genetic variation in soft tissue sarcoma patients.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a soft tissue sarcoma gene detection kit and a system.
In order to achieve the purpose, the invention adopts the following technical scheme:
the first aspect of the invention provides a soft tissue sarcoma gene detection kit, which comprises a primer composition for detecting the following fusion gene pairs: ACTB-GLI, AHRR-NCOA, ASPSCR-TFE, ATIC-ALK, BCOR-CCNB, C11 orf-MKL, CARS-ALK, CIC-, CLTC-ALK, COL 1A-PDGFB, COL 3A-PLAG, COLA-CSF, ETV-NTRK, EWSR-ATF, EWSR-CREB, EWSR-DDIT, EWSR-ERG, EWSR-ETV, EWSR-FEV, EWSR-FLI, EWSR-NFATC, EWSR-NR 4A, EWSR-PATZ, EWSR-PBX, EWSR-POU 5F, EWSR-SMARCA, EJAR-SP, EWSR-WT, EWSR-ZNF 444, FUS-ATF, FUS-CREB3L, FUS-CREB3, FUS-CREB3, FUS-SARG, HSP-FEV, HSP-SAGA-2-ABGA, HSP-NFAT, EWSR-NFATF, EW-NFATZ, ESA, ESR-NFAT, ESR-FES-SARG-SAG, ESR-SARG-SALPS-SAG, SARG-SALPS-SAG, SAG-SALPS-SAG, SALPS-SARG, SALPS-SAG-SALPS-SAG, SALPS-SAL, SALPS-SAC, SALPS-SAG, SALPS-SAL, SALPA, SAG, SALPS-SALPA, SAL, SAC, SALTS-SALPS-SALPA, SALTS-SALTA, SALTS-SAL, SALTS-SALTA, SALTS-SALTS, SALTS-SALTS, SALTS-SALTS, SALTS-SALTS, SALT, NAB2-STAT6, NTRK3-ETV6, PAX3-FOXO1, PAX 1-NCOA 1, PAX 1-FOXO 1, PPFIBP1-ALK, RAB2 1-PLAG1, RANBP 1-ALK, SEC31 1-ALK, SRF-NCOA 1, SS18L1-SSX1, SS 1-SSX1, TAF 1-NR4A 1, TCF 1-NR4A 1, TEAD1-NCOA 1, TFG-NR4A 1, YAAE 1-ALK, TPM 1-ALK, VG 1-CITED 1, VG-NCLL 1, WHTFE-NCTFE 72, WH 1-TCTFE 1-NCOA 1, FAYTFE 1-WTYTR 1, FAYTFM 1-1, FAYTFYTFM 1-1, FAYTFYAAE 1-1, FAYTFYA 1-1, FAYTFM 1, FAYTFYA 1-1, FAYTFA 1, FAYTFX 1, and FAYTFX 1.
Further, the primer composition of the fusion gene pair comprises a sequence with a nucleotide sequence of SEQ ID No. 1-198.
Furthermore, the kit also comprises an amplification primer of the reference gene; the reference genes include MYC, YEATS4, CFHR5, FOSL1, VGLL3, MTTP, PLAG1, FRS2, CSF1, MDM2, HMGA2 and MATN 1.
The second aspect of the present invention provides a soft tissue sarcoma gene detection system using the above kit, comprising:
a detection unit: performing high-throughput sequencing by using the kit;
a data analysis unit: establishing an analysis flow on an ion torrent platform IR server, and analyzing offline data;
a quality control unit: and checking through a preset quality control program, outputting a detection result if the checking is passed, and displaying that the sample is invalid if the checking is not passed.
Further, in the above detection unit, high throughput sequencing is performed by:
step one, sample processing;
step two, RNA extraction: extracting RNA in a sample;
step three, constructing an Ampliseq library: performing reverse transcription on the RNA to form cDNA, respectively amplifying a cDNA target region and a DNA target region by using a primer composition in the kit, mixing amplification products, performing digestion primer, performing ligation and adaptor, purifying the ligation products, and finally performing library enrichment and purification;
and step four, quantifying the library Qubit and sequencing on a computer.
Further, in the above data analysis unit, the data analysis is performed by:
checking DNA sequencing data QC condition on a TS server;
secondly, initially analyzing off-line data to obtain QC information and an original BAM file;
and step three, uploading the data to IR for analysis to obtain an RNA library fusion result.
Further, the quality control unit performs verification by the following procedure: and (3) checking the quality control and/or the comparison rate of the reference genes.
Further, the quality control standard of the quality control unit is as follows: the number of reads matched with the sample is more than 20000, and the contrast ratio is more than 85%.
By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:
the kit and the detection system provided by the invention can uniformly amplify a target sequence by the modified multiple amplification primers, add 10 internal reference genes for quality control, detect by an ion torrent high-throughput sequencing platform, detect 233 fusion forms altogether, can comprehensively detect fusion genes possibly existing in a patient body at one time, help to comprehensively and rapidly diagnose diseases clinically, and can also be used for prompting targeted medication.
Drawings
FIG. 1 is a schematic diagram of the working procedure of a soft tissue sarcoma gene detection system according to an embodiment of the present invention.
Detailed Description
Soft tissue sarcoma relates to many types of diseases, and relates to complex types of fusion genes, along with the rapid development of gene detection technology, more and more fusion genes related to soft tissue sarcoma which are not reported before are reported, but the fusion genes recorded in a public database are far behind the leading-edge research at present, and the fusion genes and fusion fracture types recorded in the public database are only relied on, so that the increasing requirements of clinic are difficult to meet, and how to cover the fusion gene types and the fusion fracture modes as much as possible is the key for improving the detection performance and the detection significance of the kit. Based on the above, the invention provides a soft tissue sarcoma gene detection kit and system, which can detect 71 fusion genes (233 fusion fracture modes related) related to sarcoma.
The present invention will be described in detail and specifically with reference to the following examples and drawings so as to provide a better understanding of the invention, but the following examples do not limit the scope of the invention.
In the examples, the conventional methods were used unless otherwise specified, and reagents used were those conventionally commercially available or formulated according to the conventional methods without specifically specified.
Example 1
This example provides a soft tissue sarcoma gene assay kit, which can detect 233 fusion forms. The kit includes primer compositions (table 3) for detecting fusion genes shown in table 1 below, and the clinical significance of these fusion genes is shown in table 2 below.
TABLE 1 fusion genes
TABLE 2 correspondence of fusion genes to disease detection
TABLE 3 primer composition for fusion genes
The kit also comprises the following amplification primers of the reference genes: MYC, YEATS4, CFHR5, FOSL1, VGLL3, MTTP, PLAG1, FRS2, CSF1, MDM2, HMGA2, and MATN 1.
Example 2
The embodiment provides a soft tissue sarcoma gene detection system, which comprises the following module units:
1. a detection unit: high throughput sequencing was performed using the kit of example 1.
2. A data analysis unit: and (4) establishing an analysis process on an ion torrent platform IR server, and analyzing the offline data.
3. A quality control unit: and checking through a preset quality control program, outputting a detection result if the checking is passed, and displaying that the sample is invalid if the checking is not passed.
Referring to fig. 1, the working flow of the soft tissue sarcoma gene detection system is as follows:
first, detecting
1.1 sample treatment: and (3) extracting nucleic acid from the tissue specimen by using an RNA extraction kit. The extraction kit comprises: magen paraffin RNA extraction kit.
1.2 Paraffin removal:
1.2.1 the specimens on the sections were scraped with a clean blade and immediately transferred to a 1.5ml centrifuge tube.
1.2.2 Paraffin removal with xylene.
1.2.3 Add 1ml xylene to the tube containing the sample, vortex vigorously for 10-30s and then place in a metal bath at 56 ℃ for 10 min.
1.2.414,000 Xg, centrifuging for 3 min; carefully discard the supernatant without sucking the pellet.
1.2.5 adding 1ml of absolute ethyl alcohol, and uniformly mixing for 10-30s by vortex; centrifuge at 14,000 Xg for 5 min.
1.2.6 carefully discard the supernatant thoroughly without sucking the pellet.
1.2.7 open the tube lid and place the centrifuge tube in a metal bath at 56 deg.C for 10min to completely remove the residual ethanol (note: it is important to dry sufficiently to remove the ethanol! the residual ethanol affects the efficiency of RNA extraction).
1.2.8 and cracking and digesting.
1.2.9 Add 200. mu.l Buffer FRL and 20. mu.l protease K to the sample and vortex to resuspend the sample for 10 s.
1.2.10 the samples were placed in a metal bath at 56 ℃ for 10min and then on ice for 3 min.
Centrifuge at 20,000 Xg for 10min at 1.2.11 RT.
1.2.12 prepare a new 1.5ml centrifuge tube and write the sample number + R on the tube cap.
1.2.13 the supernatant was carefully transferred to the centrifuge tube described above for RNA extraction.
1.3RNA extraction
1.3.1 the samples were placed in a 80 ℃ metal bath for 15 min.
1.3.2 short centrifugation, add 200. mu.l Buffer RL and 600. mu.l absolute ethanol, vortex mix for 20s, short centrifugation.
1.3.3 HiPure RNA Mini ColumnI was loaded into a 2ml collection tube and the sample number + R was written on the tube cap.
1.3.4 transfer half of the volume of the mixture to the column and centrifuge at 8,000 Xg for 30 s.
1.3.5 pour out the effluent, pack the column in a collection tube, transfer the remaining mixture to the column, centrifuge for 30s at 8,000 Xg.
1.3.6 pour out the effluent, the column is mounted in a collection tube, 500. mu.l Buffer RW2 (diluted with ethanol) is added to the column, and centrifugation is carried out for 30s at 8,000 Xg.
1.3.7 repeat step 1.3.6.
1.3.8 pour out the effluent, pack the column in a collection tube, centrifuge the empty column at 13,000 Xg for 3min to spin dry the matrix of the column.
1.3.9 prepare a new 1.5ml centrifuge tube, and write the sample number + R, patient name, sample number, and extraction date on the tube cap.
1.3.10 transfer the column to the 1.5ml centrifuge tube described above. Add 30. mu.l RNase-Free Water to the center of the membrane of the column and let stand for 1min without touching the filter. Centrifuge at 13,000 Xg for 1 min.
1.3.11 discarding HiPure RNA Mini Column I, covering the tube cap, confirming whether the number on the tube cap is consistent with that on the adsorption Column, vortexing and mixing for 10s, and centrifuging for a short time.
1.3.12 quant quantification: quantification of samples Using the Qubit dsRNA HS Assay Kit with reference to the Qubit Quantification Protocol Guide
1.4Ampliseq library construction
1.4.1RNA library construction.
1.4.2 reverse transcription of RNA into cDNA.
1.4.2.1 take out the ReverTra Ace kit (TOYOBO) from the freezer of the refrigerator, check the production lot number, the production date and the expiration date, and disassemble the kit after checking.
1.4.2.2 and balancing at room temperature for 10-20 min (when the reagent is completely melted), shaking and mixing on a vortex mixer for 3-5 s, and centrifuging in a micro-centrifuge for 5 s.
1.4.2.3 reverse transcription reaction solution (1 part by person) was prepared according to the following composition of Table 4:
TABLE 4 reverse transcription reaction solution
| Name of reagent | Dosage (mu l) |
| Template RNA | 10 |
| Primer mix | 1 |
Note that: the optimal total amount of RNA is 1000ng, and if the concentration of the extracted RNA is more than 100 ng/. mu.l, the extracted RNA is diluted to 100 ng/. mu.l.
The mixture was placed in a general PCR apparatus (Bori), and the reaction program was set as follows:
TABLE 5 reaction procedure
1.4.2.4 at the end of the procedure, the reagents shown in Table 6 below were added continuously:
TABLE 6 preparation of reaction solution
| Name of reagent | Dosage (mu l) |
| 5×PCR Buffer | 4 |
| ReverTra Ace | 1 |
| ddH2O | 4 |
The mixture was placed in a general PCR apparatus (Bori), and the reaction program was set as follows:
TABLE 7 reaction procedure
1.4.2.5 after the procedure is completed, the reverse transcription product is removed and subjected to the next experiment, if not used immediately, and stored in a refrigerator at 4 ℃.
1.4.3 amplification of cDNA target regions
1.4.3.1 after brief centrifugation of the PCR product, the reagents were slowly added by pipette in the order shown in Table 8 below, where the PCR enzyme mixture and cDNA amplification primer mixture were thoroughly thawed, shaken, mixed and centrifuged before use, suggesting the formulation of the reaction system on ice:
TABLE 8 composition of the reaction System
| Components | Single sample volume |
| HIFI MIX | 2μL |
| 5×STS RNA Research panel | 2μL |
| cDNA | 6μl |
| Total volume | 10μL |
1.4.3.2 after the preparation, the PCR tube cover is covered tightly, the mixture is mixed thoroughly by vortex oscillation, the mixture is separated instantly to collect the residual solution on the tube wall and the tube cover, and the PCR tube is placed on ice for standby.
1.4.3.3 setting the PCR program according to the following Table 9, after selecting "start", setting the volume to 5 μ L, ensuring that the octant tube cover is covered tightly, selecting "start" to start running, and the PCR running time is about 3 h; the hot lid temperature was 105 ℃ (programmed in advance each time).
TABLE 9PCR procedure
1.4.4 amplification of DNA target regions
1.4.4.1PCR mixture (see Table 10 below):
TABLE 10 composition of PCR mixture
| Components | PCR mix of 2 pool for a single sample |
| 5×HIFI MIX | 2μL |
| DNA | 40ng |
| Water (W) | Mixing with DNA to 6.5. mu.l |
| Total volume | 8.5μl |
1.4.4.2 were aliquoted (2 pool, table 11 below):
TABLE 11 composition of the individual dispensing tubes
| Components | Pool1 | Poo12 |
| 1.4.4.1PCR mixture prepared in step | 4μl | 4μl |
| 5×STS DNAResearch panel | 1μl | 1μl |
| Total volume | 5μl | 5μl |
1.4.4.3 after the PCR tube is assembled, the tube is covered tightly, vortexed and shaken thoroughly, and then separated instantly to collect the residual solution on the tube wall and the tube cover, and the PCR tube is placed on ice for use.
1.4.4.4 the PCR program was set up according to Table 12 below, and after "start" was chosen, the volume was set to 5. mu.L, ensuring that the octant caps were tightly capped, and "start" was chosen to start the run, with a PCR run time of about 2.5 h; the hot lid temperature was 105 ℃ (programmed in advance each time). This step may be carried out at 10 ℃ overnight.
TABLE 12 PCR procedure
1.4.4.5 after PCR, the DNA PCR amplification products pool1 and pool2 were combined into 1 tube with a total volume of 10. mu.l.
1.4.4.6 the digestive enzymes in the library construction kit were removed from the freezer at-20 deg.C, placed on ice, shaken well and centrifuged before use.
1.4.4.7 the combined DNA PCR amplification product (total volume 10. mu.l) and cDNA amplification product (10. mu.l) were flash-separated, 1. mu.l of digesting enzyme was added to each tube of amplification product, the PCR cap was closed, vortexed, mixed well, and centrifuged briefly to collect the residual solution on the tube wall and tube cap.
1.4.4.8 the PCR program was set up according to Table 13 below, and after "start" was chosen, the volume was set at 12. mu.L, ensuring that the PCR lid was closed, and "start" was chosen to start the run:
TABLE 13 PCR reaction procedure
| Temperature of | Time |
| 50℃ | 10 minutes |
| 55℃ | 10 minutes |
| 60℃ | 20 minutes |
| 10℃ | Hold (1 hour at most) |
1.4.4.9 after the PCR instrument is started, the next step of "adaptor ligation of amplicons" should be prepared in advance.
1.4.5 adaptor ligation of amplicons
1.4.5.1 the pre-prepared label was placed on ice to melt thoroughly from a-20 ℃ freezer. Fully oscillating, uniformly mixing, centrifuging and placing on an ice box for later use. Where there are 96 tags (1 to 96), typically one unique linker is used per library.
TABLE 14 linker ligation reaction solution
| Components | Volume of |
| Digested PCR product | 11μl |
| Label (R) | 1μl |
| DNA Ligase(blue cap) | 1μl |
| Switch Solution(yellow cap) | 2μl |
| Total volume | 15μl |
1.4.5.2 mix well with shaking, set up the PCR reaction program (as in Table 15 below):
watch 15 joint connection procedure
| Temperature of | Time |
| 22℃ | 30 minutes |
| 68℃ | 5 minutes |
| 72℃ | 5 minutes |
| 10℃ | Hold |
1.4.6 ligation purification
1.4.6.1 standing the magnetic beads at room temperature for 30min, and mixing by vortexing;
1.4.6.2 adding 45 μ L (1.5 times volume) of magnetic beads into each sample after PCR reaction in step 1.4.4.3, mixing by air-blowing, and incubating at room temperature for 5 min;
1.4.6.3 placing on a magnetic frame for 2min until the supernatant becomes clear, and discarding the supernatant;
1.4.6.4 adding 200 μ L of 70% ethanol, rotating the centrifuge tube to clean the magnetic beads, and discarding the supernatant;
1.4.6.5 repeat step 1.4.6.4 once;
1.4.6.6 keeping the centrifuge tube on magnetic rack, air drying for 5min, but not drying too much;
1.4.6.7 mu.L of nuclease-free water (Life: 50. mu.L of 1 Xenzyme solution Black cap) was added to the tube, the tube was removed from the magnetic frame, and the magnetic beads were vortexed thoroughly to mix them well.
1.4.6.8 was incubated at room temperature for 3 min.
1.4.6.9 the centrifuge tubes were placed on a magnetic rack and left at room temperature for 2min or until the solution was clear.
1.4.7 library enrichment
1.4.7.1 mu.L of primer mix and 25. mu.L of high fidelity reaction mix were added to the PCR tube, and 23. mu.L of supernatant from step 1.4.6.9 (self-developed kit).
1.4.7.2 mu.L of 1 Xeluted product and 2. mu.L of enzyme (red cap) were added to the PCR tube.
1.4.7.3 the following procedure was performed on a PCR instrument:
1.4.8 library purification
1.4.8.1 standing the magnetic beads at room temperature for 30min, and mixing by vortexing;
1.4.8.2 adding 25 μ L (0.5 times volume) of magnetic beads, mixing, and incubating at room temperature for 5 min;
1.4.8.3 placing on a magnetic frame for 5min until the supernatant becomes clear, transferring the supernatant to a new centrifuge tube;
1.4.8.4 adding 60 μ L (1.2 times volume) of magnetic beads, mixing, and incubating at room temperature for 5 min;
1.4.8.5 placing on a magnetic frame for 3min until the supernatant becomes clear, and discarding the supernatant;
1.4.8.6 adding 200 μ L of 70% ethanol, rotating the centrifuge tube to clean the magnetic beads, and discarding the supernatant;
1.4.8.7 repeat step 1.4.8.6 once;
1.4.8.8 keeping the centrifuge tube on magnetic rack, air drying for 5min, but not drying too much;
1.4.8.9 taking the centrifugal tube off the magnetic frame, adding 50 μ L nuclease-free water, and mixing by vortex;
1.4.8.10 were placed on a magnetic rack for 2min until the supernatant became clear, and 45. mu.L of the supernatant was aspirated from the centrifuge tube and transferred to a new 1.5ml centrifuge tube.
1.5 quantification of Qubit from library
1.5.1 configure the Qubit tuning solution (for each sample): take 199. mu.l of QubitTMssDNA buffer was added to a 500. mu.l centrifuge tube, followed by 1. mu.l of the Qubit ssDNAreagent, and vortexed to mix.
1.5.2 calibration of standards: take 10. mu.l of QubitTMAdding ssDNA Standard 1 into 190 μ l of the Qubit ionization solution, and mixing by vortex; take 10. mu.l of QubitTMAdding ssDNA Standard 2 into 190 μ l of the Qubit ionization solution, mixing uniformly by vortex, and standing for 2min at room temperature; and (4) selecting a standard substance according to the prompt of the instrument, and reading. Under the condition that the instrument is not powered off, the calibration time of the standard substance is calibrated at least once a week, and the standard substance needs to be reused for calibration once every time the instrument is restarted.
1.5.3 take 1. mu.l of DNA sample and add to 199. mu.l of the Qubit learning solution, vortex and mix well.
1.5.4 DNA samples were selected according to the instrument instructions and then read. Subsequent experiments were performed with library concentrations >0.2 ng/. mu.l. If the concentration of the library is <0.2 ng/. mu.l for which analysis is required, the library is reconstructed.
1.6 machine sequencing.
Second, data analysis
2.1 DNA sequencing data QC status was checked on the TS server.
2.1.1 uploading BED file, setting basic analysis parameter (BED file is design area information file generated when the panel is designed).
2.1.2 open TS Server first (192.168.10.54), find Reference Sequences selection in the interface, enter database storage page.
2.1.3 select Target Regions option, then select right Add Target Regions option, import the detection region bed file of soft tissue sarcoma panel into the system.
2.1.4 the upload is completed, and the uploaded bed file can be seen by selecting the Target registers option again.
2.1.5 selecting Data, clicking to computer Data on the corresponding date, and checking the corresponding specific Data condition.
2.2, performing initial analysis on off-line data to obtain QC information and an original BAM file.
2.2.1 click the Plugins button on the get-ahead data overview page, select Plugins to run selection, click in. The size distribution of the amplified fragments can be viewed at this interface, focusing on the designed amplicon size region.
2.2.2 selection of the coverageAnalysis analysis (generated by Thermo Fisher Scientific autowrite QC script, current version v5.12.0.0).
2.2.3 select library types, find soft tissue sarcoma panel red file just uploaded to the server, click to determine, begin analysis.
2.2.4 the analysis was complete and information on target rate, sequencing depth and data homogeneity was reviewed, where target rate > 90%, sequencing depth >500X and homogeneity > 85% were required.
2.3 upload data to IR for analysis
2.3.1 download the raw data bam file locally.
2.3.2 log in the IR server, click define Sample option, upload local Sample.
2.3.3 begin Analysis using Launch Analysis in the IR server (this flow uses platform self-written script, applying BaseCaller, tmap, variable caller, etc. software).
2.3.4 analysis gave the RNA library fusion results.
Quality control
And checking through a preset quality control program, outputting a detection result if the checking is passed, and displaying that the sample is invalid if the checking is not passed.
Quality control standard: the number of mapping reads of the sample is more than 20000, and the mapping rate is more than 85%.
Verification example 1
In this example, the kit and the corresponding detection method provided in example 2 were validated for methodology, and the specific validation process and results are as follows:
1. accuracy and specificity
The study used clinically diagnosed sarcoma specimens from 35 cases (basic information as in table 16) and was validated in parallel by one generation sequencing and FISH with specificity up to 100% (results as in table 17 below).
TABLE 1635 basic information of sarcoma patients
TABLE 1735 test results for sarcoma specimens
2. Sensitivity and lower limit of detection
The RNA of 5 samples is selected for dilution detection, 5ng, 10ng and 15ng are respectively selected for gradient experiments, as a result, one sample with 5ng of dilution concentration is not detected, and other samples with concentration are successfully detected and fused (see table 18 below), so that the lowest input quantity of the sample is positioned at 10ng, and the lowest detected reads number is defined as 20 according to the credit generation result.
TABLE 185 test results of samples
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. It will be appreciated by those skilled in the art that any equivalent modifications and substitutions are within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.
Sequence listing
<110> Shanghai Tung Tree medical inspection laboratory Co., Ltd
<120> soft tissue sarcoma gene detection kit and system
<160> 198
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
catcactgat ctacgtaaac aa 22
<210> 2
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gcaggatgtg gacatagcaa gtca 24
<210> 3
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gaagctgccc gactccttct tc 22
<210> 4
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
cgccggcagc aggtgaaaca gt 22
<210> 5
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
agcagctact ctcctagctg ta 22
<210> 6
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
tgactctcac agccgaactc tgcg 24
<210> 7
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gagagagcac ctacttgtcc tc 22
<210> 8
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ttgcagcaga aggcgtcgga g 21
<210> 9
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
gatcctacag ccaagctcca ag 22
<210> 10
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
caggagcttg gcaaattgct c 21
<210> 11
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
ctcatcgttg gcccacagag aa 22
<210> 12
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
ctcagggctc tgcagctcca tc 22
<210> 13
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
ccagttcctg ttgcagactg tg 22
<210> 14
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
cgagcggctt cactcagacc ctg 23
<210> 15
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
cctgacccgc tgcttcatct gc 22
<210> 16
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
attcagtggc ttccccatc 19
<210> 17
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
aggagatccg caaatacagc at 22
<210> 18
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
ctcgtctccc acgaccgcgt gg 22
<210> 19
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
agtgagcgaa tggacaggt 19
<210> 20
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
gatggactac gacggcagcc gg 22
<210> 21
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
tggtgcctga cactggcaga 20
<210> 22
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
cgctggagag cgaggtggct tc 22
<210> 23
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
tccagcctat gggcagcagc ca 22
<210> 24
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
tctgatgcat gttgtgatg 19
<210> 25
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
ctcagggctc tgcagct 17
<210> 26
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
gatatagacc aacacagcct gg 22
<210> 27
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
gagtaactac agttatcccc ag 22
<210> 28
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
ccgtgtgccc tggcactgag cg 22
<210> 29
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
actcttcgtg gaagagagtg g 21
<210> 30
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
gatttgatcg tggaggcatg ag 22
<210> 31
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
ggaatgaacc cacatcagac cc 22
<210> 32
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
actaaggtgg gccttgagca cc 22
<210> 33
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
ctcccaggtc aagccag 17
<210> 34
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
ctcctgaggg caaaacccag ag 22
<210> 35
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
ggtgactctc acagccga 18
<210> 36
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
cagttcagcg atgaccccaa gg 22
<210> 37
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
tctttctttt cagattgtgt ac 22
<210> 38
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
gtgacccagc tcacccgcga gg 22
<210> 39
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
gtggccacca gcttcagg 18
<210> 40
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
cctgacagca tcatctggga ca 22
<210> 41
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
ttgtcctcct tgaagggctc ca 22
<210> 42
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
ccaccattct tcagagggca at 22
<210> 43
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
catgttcatc tctaatt 17
<210> 44
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
cctatgtacc cccataccgc cc 22
<210> 45
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
tccacggtca tcttgatgg 19
<210> 46
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
tgggagttgt taaacctggc 20
<210> 47
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
gatgagcttt gtgcaaaagg gg 22
<210> 48
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
tctgaatcat atgaacctcc ac 22
<210> 49
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
tctgaatcat atgaacctcc ac 22
<210> 50
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
agctgctgtt cccattgct 19
<210> 51
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
actctgctgg gctcctctgt ag 22
<210> 52
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
ttctccataa tgtttggtac c 21
<210> 53
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
tctgaatcat atgaacctcc ac 22
<210> 54
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
cctcatcact cggagactca 20
<210> 55
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
gaggagcgcg ctcctgaggg ca 22
<210> 56
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
ggtcagcttc ccacatctcc ag 22
<210> 57
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
ctcagggctc tgcagctcca 20
<210> 58
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
ccctaccttc aggtgttatc ag 22
<210> 59
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
tttgtgcctc cgtcggggcc ct 22
<210> 60
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
gcctcgttgt gaaccttgat 20
<210> 61
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
gtcaatatag ccaacagagc ag 22
<210> 62
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
ctgtatgtct cctttggtgt 20
<210> 63
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
agcaggccta ccccatccag cc 22
<210> 64
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
ggtgactctc acagccgaac t 21
<210> 65
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
gggtacaggc agttgcaatc a 21
<210> 66
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
ggtgccttcc caggtgat 18
<210> 67
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 67
ggttacaacc gcagcagtgg tg 22
<210> 68
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 68
gatgcagctg gagttggagc t 21
<210> 69
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 69
ccctcgggac caaggatcac gt 22
<210> 70
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 70
cagagacccg tgctgagttt gc 22
<210> 71
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 71
ctcagggctc tgcagctc 18
<210> 72
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 72
ctccggcatg tgcaaggccg gc 22
<210> 73
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 73
ccttctacaa tgagctgcgt gt 22
<210> 74
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 74
gggccctttt tggtgattca 20
<210> 75
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 75
cacttgccaa ccagcatgtc c 21
<210> 76
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 76
cagattcagg ctcacgcttc 20
<210> 77
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 77
ctgagcattt catcattgt 19
<210> 78
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 78
tccaagccaa agaagtccaa gt 22
<210> 79
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 79
gctgacagtg atggctggt 19
<210> 80
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 80
tcgccaggag ctcatccagc 20
<210> 81
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 81
ggtgccaagg gagatgctgg tc 22
<210> 82
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 82
ggcgttggag atcatcaa 18
<210> 83
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 83
tgatggtgct actggtgctg cc 22
<210> 84
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 84
gctggccctg ctggaaaccc tg 22
<210> 85
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 85
gccctcctgg tcccaagggt aa 22
<210> 86
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 86
cccaaaggat ctcctggtga ag 22
<210> 87
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 87
gtggtcaggc tggtgtgatg gg 22
<210> 88
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 88
tcggtcctgc tggcaaagat gg 22
<210> 89
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 89
ctccaggtga agcaggcaaa cc 22
<210> 90
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 90
gagcgtggtg tgcaaggtcc cc 22
<210> 91
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 91
cttcagggaa tgcctggtga ac 22
<210> 92
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 92
gcgtccgtgg tctgactggc cc 22
<210> 93
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 93
cctggtgaca agggtgaaag tg 22
<210> 94
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 94
ggcgaacctg gtgatgctgg tg 22
<210> 95
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 95
cctggcccca ttggtaatgt tg 22
<210> 96
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 96
tggtccccct ggtgctactg gt 22
<210> 97
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 97
acgtcctggt gaagttggtc cc 22
<210> 98
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 98
ttgctggaca gcgtggtgtg gt 22
<210> 99
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 99
cccctggatt ggctggaccc cc 22
<210> 100
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 100
ctgctggcaa gagtggtgat cg 22
<210> 101
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 101
ggtgacaagg gtgagacagg cg 22
<210> 102
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 102
tcctggctct cctggtgaac aa 22
<210> 103
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 103
caaagatgga ctcaacggtc tc 22
<210> 104
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 104
cacctgccgt gacctcaaga tg 22
<210> 105
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 105
ctggttcggc gagagcatga cc 22
<210> 106
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 106
gagatggcat ccctggacag cc 22
<210> 107
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 107
ctttgctccc cagctgtctt at 22
<210> 108
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 108
ggtcctcgtg gtctccctgg cc 22
<210> 109
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 109
ccccctggaa agaatggaga tg 22
<210> 110
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 110
ctcagggtgc tcgaggattg cc 22
<210> 111
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 111
cggttttcca ggcatttca 19
<210> 112
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 112
cctgtacccc atcggtacca t 21
<210> 113
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 113
tgcagttgga tcagtctg 18
<210> 114
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 114
gatttgcaag gcggctactt 20
<210> 115
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 115
ggtgccttcc caggtgatg 19
<210> 116
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 116
cggtcacggc caccccacgc 20
<210> 117
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 117
ccctctgtgt ggaggtacat 20
<210> 118
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 118
gtccgctgcc tttctgaacc 20
<210> 119
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 119
cgggcccagg atctgatacg ga 22
<210> 120
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 120
gtgaactttc cctgaggtaa ct 22
<210> 121
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 121
tgacactcaa tcgtgagga 19
<210> 122
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 122
gcaccgctaa gggtccag 18
<210> 123
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 123
gtaagggttg ctgagatggg aa 22
<210> 124
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 124
ccattgatgc cattctcata c 21
<210> 125
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 125
cagttacaaa caaaagggcg tt 22
<210> 126
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 126
agaagtgaca gcagcagaa 19
<210> 127
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 127
aggttggcct tccttgg 17
<210> 128
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 128
gtaagggttg ctgagatggg a 21
<210> 129
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 129
ccatagactt taccaca 17
<210> 130
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 130
ctcctgcttg taggggcgca c 21
<210> 131
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 131
gttattgccc caagctcctc tt 22
<210> 132
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 132
ctggtggacc catggatgaa gg 22
<210> 133
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 133
tctgcgccgc ataactggaa c 21
<210> 134
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 134
gatgccacag tgtcctatga ag 22
<210> 135
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 135
gcaccgctaa gggtccagg 19
<210> 136
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 136
ggaagaagcc tccaatgaac ag 22
<210> 137
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 137
tgcagttgga tcagtctgct 20
<210> 138
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 138
gggtgagaat gttacaattg ag 22
<210> 139
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 139
gagccgtcgc tgtcactgc 19
<210> 140
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 140
cttgcaggtc ctcataccag g 21
<210> 141
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 141
ctcgccgggc agactttaat cg 22
<210> 142
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 142
ccagtgtaag gcccctaaac ca 22
<210> 143
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 143
cagagttaca gtggttatag cc 22
<210> 144
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 144
cagcaaagct ataatccccc tc 22
<210> 145
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 145
tctgaagtcc ctgtactcca 20
<210> 146
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 146
atgaggagcc gtgaggagag 20
<210> 147
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 147
ctaaagcagc tcaaaagaaa gc 22
<210> 148
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 148
ggtgctcaag ctcatcctct g 21
<210> 149
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 149
gtggctcgga gaatccgctc 20
<210> 150
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 150
agacatgtaa gtaagagttc 20
<210> 151
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 151
cgaccttgcc aacgtaccag ct 22
<210> 152
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 152
ggaatctgat cgcctgtt 18
<210> 153
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 153
gagcatccac caagaactt 19
<210> 154
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 154
gtctcacata agcctggcaa 20
<210> 155
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 155
tggcctcgtg tctgggaaaa g 21
<210> 156
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 156
gatcttcgac agtgcctgaa ca 22
<210> 157
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 157
acctccacaa cagggatatc ca 22
<210> 158
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 158
cttctgacac tcccttc 17
<210> 159
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 159
gattttctcc gaggatttca 20
<210> 160
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 160
cccatcattt tgtgggccag 20
<210> 161
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 161
cagggtcgct gatctcttca t 21
<210> 162
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 162
ctgacactcc cttcgaatca 20
<210> 163
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 163
cagcagcatg attcctatag tc 22
<210> 164
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 164
tgacagtcga ttaggagccc at 22
<210> 165
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 165
cggctatggt gcacagcagc cg 22
<210> 166
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 166
ctgacaaact gaaagaggct ga 22
<210> 167
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 167
ggactctaca cttatcatgc ag 22
<210> 168
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 168
tgtggtgaag ggcagagccg 20
<210> 169
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 169
ccttcccagg tgatgcagct 20
<210> 170
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 170
gttagtgaaa tggacagtga ga 22
<210> 171
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 171
gttagtgaaa tggacagtga ga 22
<210> 172
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 172
tcatcgggaa gacctggctt ac 22
<210> 173
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 173
agttctcgct tcagcacgat g 21
<210> 174
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 174
tgcaaggcgg ctacttgttg g 21
<210> 175
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 175
gcttctctga aaggctctcc tt 22
<210> 176
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 176
tacggctgca ccgagtcgta 20
<210> 177
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 177
cttcttccgt gggacaatat gt 22
<210> 178
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 178
agtgtgccca tcttcttctc t 21
<210> 179
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 179
gcactcagtc tgtataaacg gg 22
<210> 180
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 180
gaacagctac tttttctcca tc 22
<210> 181
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 181
cgggcatgtt ccgagacttc gg 22
<210> 182
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 182
ctgactgcca ctcatggtgt t 21
<210> 183
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 183
ctggagcctt tcatggaaca gt 22
<210> 184
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 184
ccagtgggcc cttggattt 19
<210> 185
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 185
tgtgtcagaa tgaaggctgc aa 22
<210> 186
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 186
gacttgggat gccttctgga a 21
<210> 187
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 187
ggccctacaa gtgcatacaa ca 22
<210> 188
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 188
gattcttcag atgatctttc cg 22
<210> 189
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 189
gttgtagctg tccagataaa ga 22
<210> 190
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 190
agtcacgttt gcgggtgtat 20
<210> 191
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 191
gctggagaag gtcaagaatg tc 22
<210> 192
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 192
gtacaggcag ttgcaatca 19
<210> 193
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 193
tggttggatc aggattcagt tt 22
<210> 194
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 194
tgcctgatac acagtaactt g 21
<210> 195
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 195
cacaacaagt tgttcagaag aa 22
<210> 196
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 196
tgcagtgtct tctcccttca 20
<210> 197
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 197
gagggtcctc tctggcacta gc 22
<210> 198
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 198
agagctgtcg acaacaaaca 20
Claims (8)
1. A soft tissue sarcoma gene detection kit, comprising a primer composition for detecting the following fusion gene pairs: ACTB-GLI, AHRR-NCOA, ASPSCR-TFE, ATIC-ALK, BCOR-CCNB, C11 orf-MKL, CARS-ALK, CIC-, CLTC-ALK, COL 1A-PDGFB, COL 3A-PLAG, COLA-CSF, ETV-NTRK, EWSR-ATF, EWSR-CREB, EWSR-DDIT, EWSR-ERG, EWSR-ETV, EWSR-FEV, EWSR-FLI, EWSR-NFATC, EWSR-NR 4A, EWSR-PATZ, EWSR-PBX, EWSR-POU 5F, EWSR-SMARCA, EJAR-SP, EWSR-WT, EWSR-ZNF 444, FUS-ATF, FUS-CREB3L, FUS-CREB3, FUS-CREB3, FUS-SARG, HSP-FEV, HSP-SAGA-2-ABGA, HSP-NFAT, EWSR-NFATF, EW-NFATZ, ESA, ESR-NFAT, ESR-FES-SARG-SAG, ESR-SARG-SALPS-SAG, SARG-SALPS-SAG, SAG-SALPS-SAG, SALPS-SARG, SALPS-SAG-SALPS-SAG, SALPS-SAL, SALPS-SAC, SALPS-SAG, SALPS-SAL, SALPA, SAG, SALPS-SALPA, SAL, SAC, SALTS-SALPS-SALPA, SALTS-SALTA, SALTS-SAL, SALTS-SALTA, SALTS-SALTS, SALTS-SALTS, SALTS-SALTS, SALTS-SALTS, SALT, NAB2-STAT6, NTRK3-ETV6, PAX3-FOXO1, PAX 1-NCOA 1, PAX 1-FOXO 1, PPFIBP1-ALK, RAB2 1-PLAG1, RANBP 1-ALK, SEC31 1-ALK, SRF-NCOA 1, SS18L1-SSX1, SS 1-SSX1, TAF 1-NR4A 1, TCF 1-NR4A 1, TEAD1-NCOA 1, TFG-NR4A 1, YAAE 1-ALK, TPM 1-ALK, VG 1-CITED 1, VG-NCLL 1, WHTFE-NCTFE 72, WH 1-TCTFE 1-NCOA 1, FAYTFE 1-WTYTR 1, FAYTFM 1-1, FAYTFYTFM 1-1, FAYTFYAAE 1-1, FAYTFYA 1-1, FAYTFM 1, FAYTFYA 1-1, FAYTFA 1, FAYTFX 1, and FAYTFX 1.
2. The detection kit according to claim 1, wherein the primer composition of the fusion gene pair comprises a sequence having a nucleotide sequence of SEQ ID No. 1-198.
3. The detection kit according to claim 1, further comprising an amplification primer for an internal reference gene; the reference genes include MYC, YEATS4, CFHR5, FOSL1, VGLL3, MTTP, PLAG1, FRS2, CSF1, MDM2, HMGA2 and MATN 1.
4. A soft tissue sarcoma gene detection system using the kit of any one of claims 1-3, comprising: a detection unit: performing high-throughput sequencing by using the kit;
a data analysis unit: establishing an analysis flow on an ion torrent platform IR server, and analyzing offline data;
a quality control unit: and checking through a preset quality control program, outputting a detection result if the checking is passed, and displaying that the sample is invalid if the checking is not passed.
5. The soft tissue sarcoma gene detection system of claim 4, wherein in the detection unit, high-throughput sequencing is performed by:
step one, sample processing;
step two, RNA extraction: extracting RNA in a sample;
step three, constructing an Ampliseq library: performing reverse transcription on the RNA to form cDNA, respectively amplifying a cDNA target region and a DNA target region by using a primer composition in the kit, mixing amplification products, performing digestion primer, performing ligation and adaptor, purifying the ligation products, and finally performing library enrichment and purification;
and step four, quantifying the library Qubit and sequencing on a computer.
6. The soft tissue sarcoma gene detection system of claim 4, wherein, in the data analysis unit, the data analysis is performed by:
checking DNA sequencing data QC condition on a TS server;
secondly, initially analyzing off-line data to obtain QC information and an original BAM file;
and step three, uploading the data to IR for analysis to obtain an RNA library fusion result.
7. The soft tissue sarcoma gene detection system of claim 4, wherein the quality control unit is verified by the following procedure: and (3) checking the quality control and/or the comparison rate of the reference genes.
8. The soft tissue sarcoma gene detection system of claim 4, wherein the quality control unit has quality control criteria of: the number of reads matched with the sample is more than 20000, and the contrast ratio is more than 85%.
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| CN109811055A (en) * | 2019-01-08 | 2019-05-28 | 广州金域医学检验中心有限公司 | Sarcoma fusion detection kit and system |
| CN112094915A (en) * | 2020-11-17 | 2020-12-18 | 苏州科贝生物技术有限公司 | Sarcoma fusion gene and/or mutation joint detection primer group and kit |
| WO2021078910A1 (en) * | 2019-10-22 | 2021-04-29 | Institut Curie | Immunotherapy targeting tumor neoantigenic peptides |
| CN112921097A (en) * | 2021-04-20 | 2021-06-08 | 四川大学华西医院 | Gene detection kit for fibroblast/myofibroblast tumors and application thereof |
| CN113005200A (en) * | 2021-04-14 | 2021-06-22 | 深圳乐土生物科技有限公司 | Primer composition and kit for detecting sarcoma fusion gene mutation and application of primer composition and kit |
| CN113462763A (en) * | 2021-04-21 | 2021-10-01 | 四川大学华西医院 | Kit for designing gene panel for targeted detection of soft tissue tumor small round cell tumor fusion |
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2021
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| CN109811055A (en) * | 2019-01-08 | 2019-05-28 | 广州金域医学检验中心有限公司 | Sarcoma fusion detection kit and system |
| WO2021078910A1 (en) * | 2019-10-22 | 2021-04-29 | Institut Curie | Immunotherapy targeting tumor neoantigenic peptides |
| CN112094915A (en) * | 2020-11-17 | 2020-12-18 | 苏州科贝生物技术有限公司 | Sarcoma fusion gene and/or mutation joint detection primer group and kit |
| CN113005200A (en) * | 2021-04-14 | 2021-06-22 | 深圳乐土生物科技有限公司 | Primer composition and kit for detecting sarcoma fusion gene mutation and application of primer composition and kit |
| CN112921097A (en) * | 2021-04-20 | 2021-06-08 | 四川大学华西医院 | Gene detection kit for fibroblast/myofibroblast tumors and application thereof |
| CN113462763A (en) * | 2021-04-21 | 2021-10-01 | 四川大学华西医院 | Kit for designing gene panel for targeted detection of soft tissue tumor small round cell tumor fusion |
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