CN114480660A - Gene Panel for detecting pan-cancer species, probe and application - Google Patents

Abstract

The invention discloses a gene Panel for detecting pan-cancer species, a probe and application. The gene Panel comprises tumor pathway related genes, tumor genetic susceptibility genes, tumor high-frequency mutation genes, tumor targeting drug related genes, tumor driving genes, immunity curative effect related genes, key DDR pathway related genes and other genes playing important roles in the occurrence and development of cancers. The gene Panel can be used for detecting all biomarkers corresponding to solid tumor targeted drugs approved to be on the market by the national drug administration (NMPA)/American Food and Drug Administration (FDA) at one time, so that accurate treatment of tumors can be guided fully. The prognosis of the examined person can be evaluated, the relapse risk of the examined person is layered, and postoperative adjuvant therapy is guided; in addition, the method can also be used for evaluating the hereditary tumor of the examined person to prompt the family genetic risk, so as to realize early discovery, early diagnosis and early treatment.

Description

Gene Panel for detecting pan-cancer species, probe and application

Technical Field

The invention relates to the technical field of tumor polygene detection, in particular to a gene Panel, a probe and application for detecting pan-cancer species.

Background

According to statistics of national cancer centers, the number of new cancer cases in 2015 in China is 186.39/10 ten thousand, and the death rate is 105.84/10 ten thousand. Clinical studies have classified tumors as a gene disorder, mainly due to activation of proto-oncogenes or inactivation of suppressor genes. With the great progress of high-throughput sequencing (NGS) and biotechnology, a plurality of driver genes related to the occurrence and development of cancer and biomarkers for guiding tumor treatment are discovered, and a plurality of drugs are developed aiming at the driver genes and the biomarkers, so that precise treatment can be performed according to the detected driver gene mutation or biomarker in clinical treatment, and the treatment mode of tumors is changed from traditional treatment to precise treatment.

Currently, genetic testing techniques for precision therapy include Sanger sequencing (first-generation sequencing), RT-PCR (polymerase chain reaction), IHC (immunohistochemistry), FISH (fluorescence in situ hybridization), and high throughput sequencing (NGS) techniques, among others. Wherein, Sanger sequencing is a gold standard for sequencing, but one reaction of the sequencing technology can only obtain one sequence, so the sequencing flux is low; although individual reactions are inexpensive, the cost of obtaining large quantities of sequencing is high; the detection sensitivity is low, and generally, only mutations with mutation abundance of more than 20% can be detected, and the possibility of missing detection exists for some low-frequency mutations. RT-PCR can only detect known sites, and can not find unknown sites. IHC is mainly used for detecting protein expression, and cannot detect point mutation (SNV) of a gene, small fragment insertion deletion (Indel), and the like, and therefore, detection requiring medication guidance according to the gene SNV/Indel cannot be achieved by IHC. The FISH assay is a gold standard for identifying gene Fusion (Fusion) and amplification (CNV), but the SNV/Indel assay is not applicable. The high throughput sequencing (NGS) technique has the following advantages: high flux (several genes to hundreds of genes and even whole exome are detected at one time), high sensitivity, lower detection limit and stronger exploration capacity, can find unknown mutation, can detect multiple mutation types at one time (SNV/Indel/Fusion/CNV) and can also detect genome biomarkers (tumor mutation load/microsatellite instability). Comprehensive guidance on precise treatment can be achieved by only one test.

Disclosure of Invention

In order to solve the problems in Sanger sequencing (first generation sequencing), RT-PCR (polymerase chain reaction), IHC (immunohistochemistry) and FISH (fluorescence in situ hybridization) technologies, the invention selects a high throughput sequencing (NGS) technology. The invention provides the following technical scheme.

The invention provides a gene Panel for detecting pan-cancer species, which comprises tumor pathway related genes, tumor genetic susceptibility genes, tumor high-frequency mutation genes, tumor targeting drug related genes, tumor driving genes, immunity curative effect related genes, key DDR pathway related genes and other genes playing important roles in the occurrence and development of cancers; the key DDR pathway related genes comprise: ATM, ATR, BARD1, BLM, BRCA1, BRCA2, BRIP1, CHEK1, CHEK2, ERCC2, ERCC3, ERCC4, ERCC5, FAM175A, FANCA, FANCC, FANCD2, FANCL, MDC1, MLH1, MRE11A, MSH2, MSH3, MSH6, NBN, PALB2, PARP1, PMS1, PMS2, POLE, PRKDC, PTEN, RAD50, RAD51, RAD51B, RAD51C, RAD51D, RAD52, RAD54L, 53, TP53BP1, XRCC 2.

Preferably, the tumor pathway-associated genes include: AKT1, AKT2, AKT3, ALK, APC, AR, ARAF, ASXL1, ATM, ATR, ATRX, AURKA, AURKB, AXIN1, BCL2, BRAF, BRCA1, BRCA2, BRIP 2, BTK, CASP 2, CBL, CCND2, CCNE 2, CD79, CDK 2, KN 12, KN 22, MDM2, CDKN 22, CHEK2, CRKL 2, CRLF2, CSCSF 12, CTNNA 2, CTNNB 2, MDM2, 685MT 3, DOT 2, 685 2, 685 2, 685 2, 685, 2, 685 2, 685, 2, 685 2, 685 2, 685, 685 2, 685 2, 685 2, 685 2, 685 2, 685 2, 685, 2, 685 2, 685, 2, 685 2, 685 2, 685, 685, 685, 685 2, 685, 685, 685 2, 685, 685, 2, 685 2, 685, 685, 685, 685, 685, 685, 685, 685, 685, 685, 685, 685, PDGFRA, PDGFRB, PGR, PIK3CA, PIK3CG, PIK3R1, PIK3R2, PTCH1, PTEN, PTPN11, RAD50, RAD51, RB1, RET, RICTOR, RNF43, ROS1, RPTOR, SETD2, SMAD2, SMAD3, SMAD4, SMO, SOCS1, SRC, STAG2, STAT3, STAT4, STAT5A, STAT5B, SUFU, TET2, TGFBR1, TGFBR2, TNFAIP3, TP53, TSC1, TSC2, TSHR, WISP3, WT 1.

Preferably, the tumor genetic susceptibility gene comprises: ALK, APC, ATM, AXIN, BMPR1, BRCA, BRIP, CDH, CDKN2, CHEK, FH, FLCN, GREM, KIT, MEN, MET, MLH, MSH, MUTYH, NBN, NF, PALB, PMS, POLD, POLE, PTEN, RAD51, RB, SMAD, STK, TP, TSC, VHL, SDHD, TSC, PDGFRA, BARD, CDK, RET, SMARCA, SMARCB, SDHA, PRKAR1, HOXB, BRAF, PTCH, SDHAF.

Preferably, the tumor high-frequency mutant gene comprises: ACVR1B, AKT1, AMER1, APC, ARID 11, ARID1, ASXL1, ATM, ATRX, AXIN1, BAP1, BCL2L1, BCOR, BRA 1, BRCA1, BTG1, CASP 1, CBFB, CCND1, CCNE1, CD274, CDH1, CDK1, KN 11, MDM 11, CDKN K2 1, CHD 1, CREBB 1, CTNNA1, CTB 1, EGFR, ELF 1, EP300, ERBB4, ERBB 1, ERCC 1, ERBG 1, ERBBD 1, KM 1, 1, STK11, TBX3, TCF7L2, TERC, TERT, TGFBR2, TMPRSS2, TNFAIP3, TP53, TSC1, TSC2, U2AF1, VEGFA, VHL, WHSC1L 1.

Preferably, the tumor-targeted drug-associated gene comprises: ABL1, ABL2, ACVR1, AKT1, AKT2, AKT3, ALK, AR, ARAF, ARID1A, ATM, ATR, ATRX, AXL, BAP1, BARD1, BCL1, BLM, BRAF, BRCA1, BRD 1, BRIP1, BTK, C11orf 1, CCND1, CCNE1, CDK1, MDM 1, CDKN 11, CDKN 21, 685 1, 685K 1, KM K, 1, 685K 1, 685K 1, 685D, 1, 685D, 685D, 685D, 685D, 685D, 685D, 685D, 685D, 685D, 685D, 685D, 685D, 685D 685, NOTCH2, NPM1, NRAS, NTRK1, NTRK2, NTRK3, PALB2, PDGFRA, PDGFRB, PGR, PIK3CA, PIK3CB, PIK3CD, PIK3CG, PIK3R1, PMS2, POLD1, POLE, PTCH1, PTEN, RAB35, RAD50, RAD51, RAD52, RAF1, RARA, RB1, RET, RHEB, RICTOR, ROS1, SLX4, SMARCA4, SMARC 1, SMO, SRC, STK11, SYK, TMPRSS2, TOP2A, 53, TSC1, TSC2, VHL, XRCC 2.

Preferably, the tumor driver genes include: ABL1, ACVR1, ACVR1B, AKT1, ALK, AMER1, APC, AR, ARAF, ARID 11, ARID 51, ASXL1, ATM, ATRX, AXIN1, B2 1, BAP1, BCL2L1, BCOR, BRAF, BRCA1, BTG1, CARD1, CASP 1, CBFB, CCND1, CD79 1, CDH1, CDK1, KMK 1, KM K1, KM 1, 1, NCOR, NF, NFE2L, NOTCH, NPM, NRAS, NSD, NUP, PAX, PBRM, PDGFRA, PGR, PIK3R, PIM, PMS, POLE, PPM1, PPP2R1, PPP6, PRKAR1, PTCH, PTEN, PTPN, PTPRD, RAC, RAD, RAF, RARA, RASA, RB, RBM, RET, RHEB, RHOA, RIT, RNF, RRAS, RUNX, RXRRA, SETD, SF3B, SMAD, SMARCA, SOS, SOX, SPOP, SPTA, SF, STAG, SRK, TAF, TBEB, TCEB, TCF7L, TET, TGTGTGTP, VHTP, TSC, AIP, TSC, HAF, TSC, TMAF, TMAS, PTN, PTPN, PTPR, RTR, TSC, and TSC.

Preferably, the immunotherapy effect-related gene includes: TSC2, ATM, B2M, BRCA1, BRCA2, CCND1, DNMT3A, EGFR, FGF19, FGF3, FGF4, JAK1, JAK2, KRAS, MDM2, MDM4, MLH1, MSH2, MYC, PBRM1, PMS2, POLD1, POLE, PTEN, STK11, VHL, NF1, TP 53.

Preferably, the other genes that play an important role in the development of cancer include: DUSP, ETV, FOXP, GID, GRM, HIST1H3, HIST2H3, ICOSLG, IL, IRS, KNSTRN, MAGI, MAPKAP, MSI, NT5C, PAK, PMAIP, PPP4R, RFWD, RRAGC, SOX, TNFRSF, ZNF703, ANKRD, BABAM, E2F, EPCAM, ETV, FGF, FRS, HIST1H3, HIST3H, ID, IRS, MALT, MAX, MSI, NTHL, PAK, RAC, RANBP, RRAS, SGK, GSTM, TOP, TYRO, WWTR, BCORL, BTG, EED, HIST1H3, INHA, DNMST, MYOD, NKX-1, PRDM, PTP4A, SDHB, SH2B, SOX, TEST, SPEAP, SPE, SHCK 4, SHCK, SACK, SHCK 1H, SHCK, SACK, SHCK, SACK, SA, CD22, CENPA, EIF4A2, EPHB4, EZR, HIST1H3H, LTK, MAP3K13, NEGR1, NOTCH4, PARP3, PHOX2B, PIK3R3, PPARG, PRKCI, PTPRS, RECQL, SESN2, SLC34A2, SMYD3, SPRED1, STK19, TCF3, TIPARP, TRAF2, VTCN1, YES1, BCL2L1, CD74, CHD2, CSF3R, DNMT3B, PRRY F4E, HIST1H3I, INSR, MAP3K14, MEF2B, MYB, NUTM1, PIK3C2B, PRKD 685K 1, PRK 1, 6855, 1, 6855, 1, 6855, 1, 6855, 1, 6855, 1, 6855, 1, 6855, 1, 6855, 1, 6855, SACK 2K, 1, 6855, 1, 6855, 1, 6855, 1, SACK 2K, 1, 685.

The second aspect of the invention provides a probe Panel for detecting pan-cancer species, wherein the probe Panel is a detection probe for the tumor pathway related genes, the tumor genetic susceptibility genes, the tumor high-frequency mutation genes, the tumor targeting drug related genes, the tumor driving genes, the immune curative effect related genes, the key DDR pathway related genes and other genes playing an important role in the occurrence and development of cancer.

The third aspect of the invention provides the application of the gene Panel of the first aspect or the probe Panel of the second aspect in preparing a pan-cancer species detection device.

The invention has the beneficial effects that: the invention relates to Panel containing 561 gene, which can detect all biomarkers corresponding to solid tumor targeting drugs approved to be on the market by the national drug administration (NMPA)/American Food and Drug Administration (FDA) at one time. The biomarker for guiding accurate treatment is found by collecting tumor tissue samples and whole blood samples of the examined person, and performing tumor content evaluation, DNA extraction, library establishment, capture, sequencing, bioinformatics analysis and medical interpretation. Secondly, the prognosis of the patient can be predicted, and the relapse risk of the patient is layered by combining the clinical and pathological characteristics of the patient, so as to guide the adjuvant therapy. Finally, the genetic tumor of the detected person can be evaluated for prompting the family genetic risk, and early discovery, early diagnosis and early treatment can be realized.

Detailed Description

In order to better understand the above technical solutions, the above technical solutions are described in detail by specific embodiments below.

The invention aims to provide a detection Panel for pan-cancer species targeting, immunization, chemotherapy medication, molecular typing and genetic risk assessment. The invention utilizes the target region capture next-generation sequencing technology to analyze 561 all exon regions of genes related to tumor personalized medicine and genetic risk, hot spot regions of partial genes and chemotherapy drug related SNP sites. Therefore, biomarkers such as gene point mutation (SNV), small fragment insertion deletion (Indel), Copy Number Variation (CNV), gene Fusion (Fusion), tumor mutation load (TMB), microsatellite instability (MSI) and the like which are related to pan-cancer species (including but not limited to lung cancer, stomach cancer, intestinal cancer, endometrial cancer, ovarian cancer, breast cancer, prostate cancer, pancreatic cancer and the like) and have definite clinical significance are detected, and accurate treatment of the tumor is further guided. Meanwhile, the gene mutation detected by a tumor patient can be used for assisting the grouping of corresponding clinical tests. Provides guiding significance for clinical treatment, diagnosis and discovery of new targets.

The gene Panel provided by the embodiment of the invention mainly comprises the following genes:

genes related to the therapeutic effect of targeted drugs for solid tumors, which have been approved or approved by the National Cancer Complex Network (NCCN) guidelines and are to be approved or under investigation by the national drug administration (NMPA) or the U.S. Food and Drug Administration (FDA);

immunodetection point inhibitor therapeutic effect related genes, including genes positively correlated with therapeutic effect and genes correlated with drug resistance and risk;

core genes of DNA Damage Repair (DDR) pathways selected according to the National Cancer Complex Network (NCCN) guidelines, the american Society for Gynecological Oncology (SGO), etc.;

core genes of other important pathways during the development of cancer;

important driver genes for solid tumors;

frequently mutated genes of cancer species.

The gene Panel provided by the invention is a Panel product containing 561 genes, and comprises all biomarkers corresponding to solid tumor targeting drugs and immune drugs which are on the market at present. The gene Panel of the invention can be used for detecting all biomarkers corresponding to solid tumor targeted drugs which are approved to be on the market by the national drug administration (NMPA)/American Food and Drug Administration (FDA) at one time, thereby fully guiding the accurate treatment of tumors. The prognosis of the examined person can be evaluated, the relapse risk of the examined person is layered, and the postoperative adjuvant therapy is guided.

The gene Panel provided by the invention also comprises a gene related to pan-cancer hereditary tumor, so that the risk of hereditary tumor can be fully evaluated. When the subject has pathogenicity or possible pathogenicity heredity susceptibility gene mutation, healthy people who do not suffer from the disease in the family are recommended to carry out genetic evaluation, and the risk of the corresponding tumor is known in advance.

In addition, the gene Panel provided by the invention also comprises genes related to pan-carcinogenesis and development mechanisms, and the practicability is higher than that of whole exon sequencing. Valuable information can be provided both in clinical medication guidance and in subsequent biomarker detection.

In the specific application process, the biomarker for guiding accurate treatment can be found by collecting tumor tissue samples and whole blood samples of the detected persons, and performing tumor content evaluation, DNA extraction, library building, capturing, sequencing, bioinformatics analysis and medical interpretation. Secondly, the prognosis of the patient can be predicted, and the relapse risk of the patient is layered by combining the clinical and pathological characteristics of the patient, so as to guide the adjuvant therapy. Finally, the genetic tumor of the detected person can be evaluated for prompting the family genetic risk, and early discovery, early diagnosis and early treatment can be realized.

In the embodiment of the present invention, the following method may be specifically adopted for detection:

extracting DNA of a detection sample;

constructing a detection sample DNA library;

constructing a detection probe of the gene Panel as described above;

hybridizing the detection probe with the DNA library for capturing, eluting, purifying and sequencing;

performing bioinformatics analysis on the sequencing result to obtain mutation information of the detection sample;

and judging the pathogenicity of the mutation information.

Wherein the sample DNA comprises blood sample DNA and tumor tissue sample DNA. The following steps are mainly adopted for constructing the DNA library: DNA breaking, end repairing, joint connection, library amplification and purification.

In addition, in the embodiment of the present invention, some parameters that may affect the detection result are verified and evaluated, which specifically include:

1. initial volume assessment of reservoir building

The effect of different starting amounts on the test results was evaluated. 3 samples were taken and 30ng, 50ng and 100ng of starting pools were used, respectively, with three replicates per sample. And (4) comparing the influence of the initial amount on the detection result. Thereby evaluating whether the quality control of the sample passes and whether the target variation is detected under different initial quantities. The results showed that the sample quality control passed under the condition of 30ng and all the objective mutations were detected, so that the minimum input amount was 30 ng.

2. Positive match rate assessment

22 samples were tested, the types of mutations included Single Nucleotide Variation (SNV), small fragment insertion deletion (Indel) and Copy Number Variation (CNV), and the samples were obtained from clinical specimens. All mutations were verified by digital PCR or first-generation sequencing, QPCR, etc. The results showed that all the target variations were detected in total, i.e., the positive site coincidence rate was 100%.

3. Negative match rate evaluation

And (3) detecting 20 negative samples, wherein the sample source is a clinical sample, and evaluating whether a target positive variation is detected. The results showed that all the target variants were detected, i.e., the negative match rate was 100%.

4. Minimum detection limit evaluation

4 samples were selected, the types of variation including Single Nucleotide Variation (SNV), small fragment insertion deletion (Indel), Copy Number Variation (CNV) and Fusion (Fusion), and the sample sources were standard and clinical samples. Each mutation frequency was performed in 3 replicates. Setting mutation frequency: the expected frequencies for SNV and Indel settings were 1%, 2%, 5%, CNV settings were 2.5, 3, 3.5 copies, and Fusion settings were 1%, 2%, 5%. Finally, the lowest expected frequency of all the mutation sites is used as the lowest detection limit.

The final results show that:

1) the SNV mutation site frequency is detected in all sites of 2% or more, so the minimum detection limit of SNV is 2%.

2) The Indel mutation sites are detected in all sites with a frequency of 5% or more, and thus the minimum detection limit of Indel is 5%.

3) The CNV variant copy number was all detected at3 or more, and therefore the CNV minimum detection limit was 3.

4) Fusion mutation sites were detected at a frequency of 5% or more, and thus the minimum detection limit of Fusion was 5%.

5. Repeatability verification

6 clinical specimens were selected and tested for 3 intra-and 3 inter-batch replicates. The results show that the repeatability measurements within and between batches of the same sample are consistent.

6. Interferent analysis

6 samples were taken and the interfering substances bilirubin (concentration 342. mu.M/L), triglyceride (concentration 37 mM/L), hemoglobin (concentration 2 g/L) and 80% ethanol were added, 3 replicates per sample per treatment. The results show that: bilirubin (concentration 342. mu.M/L), triglyceride (concentration 342 mM/L), hemoglobin (concentration 2 g/L) and 80% ethanol all had no effect on the assay results.

7. DNA quality assessment

19 samples of different quality grades were selected and 100ng of DNA was used for the construction of the library. Thereby evaluating the effect of samples of different masses on mutation detection. The mutation sites of the samples with DNA main bands more than 500bp can be detected.

8. Tumor cell proportion evaluation

The verified tissue samples have 20% -90% of tumor content, and all positive sites are detected. Namely, the Panel can detect samples with the tumor content of more than or equal to 20 percent.

9. Sample stability assessment

The tissue samples used in the verification are collected from 3 months to 2021 months in 2019, and all positive sites are detected. Namely, the Panel can detect tissue samples with the storage time less than 2 years.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The invention is exemplified by the enriched 561 gene segment used for mutation detection of cancer patients based on next generation sequencing technology to guide the patients to be treated accurately.

The detection process mainly comprises the following steps: preparing a DNA sample library, constructing the DNA library, hybridizing a detection probe with the DNA library, detecting based on a next generation sequencing technology, analyzing and identifying mutation, interpreting the mutation and giving a report according to the interpretation result.

The method comprises the following steps: and extracting detection sample DNA (the sample DNA comprises blood sample DNA and tumor tissue sample DNA).

Step two: and (5) constructing a DNA library of the test sample. The method mainly comprises the following steps: DNA breaking, end repairing, joint connection, library amplification and purification. The method can be implemented according to the following steps:

1. sample preparation

For paraffin-embedded Tissue samples, QIAamp DNA FFPE Tissue Kit from QIAGEN was used, and for Blood samples, QIAamp DNA Blood Mini Kit was used, and the procedures were strictly followed. The DNA needs to be quantitatively detected, the extracted DNA is detected by using the Qubit dsDNA HS Assay Kit and a matched instrument, and the total extraction amount is not less than 50 ng.

2. DNA library construction

2.1 sample interruption

1) A breaking system as shown in table 1 was used:

2) FFPE DNA was interrupted using Covaris and blood cell genomic DNA was interrupted using Covaris or Bioruptor.

The Covaris interruption operation is as follows:

a. preparing a Covaris broken tube, marking the serial number of the DNA sample on the tube cover

b. Adding TE Buffer pH 8.0 and sample into the interrupt tube

c. Samples were interrupted and the interruption instrument interruption parameters are shown in table 2:

② Bioruptor interruption operation as follows:

a. prepare Bioruptor-cut tube, label DNA sample number on tube cap

b. TE Buffer pH 8.0 and sample were added to the disrupted tube

c. Samples were interrupted and the interruption instrument interruption parameters are shown in table 3:

d. the broken DNA is subjected to quality inspection by 2% agarose gel electrophoresis, 50bp DNA Ladder and D2000 DNA Ladder are used, the voltage is 150V, and the electrophoresis band is qualified within 200bp-300 bp.

2.2 end repair

1) End Repair Mix was prepared as per table 4.

2) The End Repair reaction program was set with reference to table 5, and the hot lid temperature was adjusted to 40 ℃.

3) The sample was placed in a PCR instrument and the End Repair reaction program was run.

4) The used reagents were returned to the original kit and stored at-20 ℃.

2.3 preparation of Ligation1 Mix

Ligation1 Mix was prepared as in table 6.

2.4 purification after repair of the termini

1) The AMPure XP magnetic beads are taken out of a refrigerator at4 ℃, and the temperature is balanced for 30 min.

2) 147.5. mu.L (2.5X) of AMPure XP magnetic beads were added to the end-repair sample and mixed well.

3) Standing at room temperature for 10min, transferring to magnetic rack, standing for 5min until the liquid is completely clarified, discarding the supernatant, and paying attention to avoid sucking magnetic beads.

4) 160 μ L of 80% ethanol was added slowly along the tube side walls, left to stand for 30s, and the supernatant was removed using a pipette.

5) Repeating the step 4) once.

6) The residual ethanol was removed by pipetting with a 10. mu.L pipette, and the mixture was left to dry at room temperature for 3 min.

7) Add 30. mu.L Ligation1 Mix to the sample tube, shake and Mix, and centrifuge instantaneously.

2.5、Ligation 1

1) The Ligation1 reaction program was set up with reference to Table 7, and the hot lid temperature was adjusted to 70 ℃.

Note: after the end of the process, the sample can be stored at4 ℃ for no more than 2 h!

2) The Ligation1 reaction procedure was run.

2.6、Ligation 2

1) Ligation 2 Mix was prepared according to table 8.

2) The PCR reaction tube of Ligation1 was removed from the PCR apparatus, subjected to instantaneous centrifugation, and placed on ice. Each reaction tube is subpackaged with 10 mu L of Ligation 2 Mix, evenly mixed by oscillation and instantaneously centrifuged.

3) The Ligation 2 reaction program was set up with reference to table 9, and the hot lid temperature was adjusted to 70 ℃.

4) The sample was placed in a PCR instrument and the Ligation 2 reaction program was run.

2.7 purification after Adaptor ligation

1) Add 100. mu.L (2.5X) PEG/NaCl to the reacted sample after Ligation 2 and mix well.

2) Standing at room temperature for 10min, transferring to magnetic rack, standing for 5min until the liquid is completely clarified, discarding the supernatant, and paying attention to avoid sucking magnetic beads.

3) 160 μ L of 80% ethanol was added slowly along the tube side walls, left to stand for 30s, and the supernatant was removed using a pipette.

4) Repeating the step 3) once.

5) The residual ethanol was removed by pipetting with a 10. mu.L pipette, and the mixture was left to dry at room temperature for 3 min.

6) Add 20. mu.L of nucleic-Free Water to the sample tube, mix well with shaking, and centrifuge instantaneously.

2.8 PCR amplification

1) PCR reaction systems were prepared in PCR tubes according to the system in Table 10.

2) Shaking and mixing evenly, and carrying out instantaneous centrifugation to ensure that all reaction liquid is placed at the bottom of the PCR tube.

PCR amplification procedure is as in table 11:

2.9 library purification and quantification

1) Taking out AMPure XP magnetic beads from a refrigerator at4 ℃, and balancing for 30min at room temperature.

2) The PCR product was removed from the PCR instrument, centrifuged instantaneously, placed on a magnetic stand and allowed to stand for 5min, and the supernatant was transferred to a new tube.

3) Add 65. mu.L (1.3X) of AMPure XP magnetic beads to the PCR product, pipette (range 65. mu.L) down to 20, mix well.

4) Standing at room temperature for 10min, transferring to magnetic rack, standing for 5min until the liquid is completely clarified, discarding the supernatant, and paying attention to avoid sucking magnetic beads.

5) 160 μ L of 80% ethanol was added slowly along the tube side walls, left to stand for 30s, and the supernatant was removed using a pipette.

Note: if the magnetic beads are absorbed when the waste liquid is discarded, standing for 2min, and discarding the supernatant after the magnetic beads are absorbed.

6) Repeating the step 5) once.

7) The residual ethanol was removed by pipetting with a 10. mu.L pipette, and the mixture was left to dry at room temperature for 3 min.

8) Add 32. mu.L TE Buffer PH 8.0 to the sample tube, mix well with shaking, centrifuge instantaneously, incubate for 8min at room temperature.

9) The sample tube was placed on a magnetic rack for 5min until the liquid was completely clear, and 30 μ Ι _ of supernatant was carefully transferred to a new 1.5ml centrifuge tube using a pipettor.

10) The library, Qubit, was quantified using Qubit 4.0.

2.10 library quality testing, the results are shown in Table 12.

2.11, storing the library, and storing the library in a refrigerator at-20 ℃ in an amplification area.

Step three: constructing a detection probe for detecting Panel, hybridizing with a DNA library, capturing, eluting, purifying and sequencing. Specifically, in the embodiment of the present invention, 30465 IDT probes were designed and manufactured by using the Target Capture Probe Design & Ordering Tool of the website of Integrated DNA Technologies IDT corporation.

Specifically, the method can be carried out as follows.

Library hybridization procedures were as follows:

1. shaking and uniformly mixing the Human Cot DNA and xGen Universal blocks-TS Mix, and instantaneously centrifuging.

2. Hybridization reaction Mix was prepared as shown in Table 13.

3. Add 7. mu.L Mix and 500ng-1ug DNA library to the centrifuge tube, Mix well with shaking, and centrifuge instantaneously. And drying in a vacuum concentrator for later use.

4. XGen according to Table 14 ^®2x Hybridization Buffer and xGen ^®And (3) oscillating and uniformly mixing the 2x Hyb Buffer Enhancer, and performing instantaneous centrifugation to obtain a hybridization reaction solution.

5. And adding the hybridization reaction solution and 4 mu L of probe into the evaporated sample, and incubating for 10min at 25 ℃ on a constant-temperature mixing machine.

6. And (5) putting the sample into a PCR instrument, clicking the program and starting to operate. PCR procedure as in table 15:

the library elution procedure was as follows:

1. streptavidin magnetic bead cleaning

1) Will xGen^®2X Hybridization Buffer and xGen 2X Hyb Buffer Enhancer are shaken, mixed evenly and centrifuged instantly.

2) The magnetic bead suspension Mix was prepared as in table 16.

3) Dynabeads M-270 Streptavidin was shaken and mixed well. The amount of Dynabeads M-270 Streptavidin used per library was 50. mu.L.

4) The using amount of each library 1XBead Wash Buffer (nuclear-Free Water and xGen 2X Bead Wash Buffer prepared according to the proportion of 1: 1) is 100 muL, 1X Bead Wash Buffer with the corresponding volume is added into a tube, the mixture is oscillated and mixed evenly (n X100 muL), the mixture is subjected to instantaneous centrifugation and placed in a magnetic frame for 1min, and after the liquid is completely clarified, the supernatant is sucked and discarded by using a pipette.

5) Repeating the step 4) twice.

6) The residual liquid was aspirated with a pipette.

7) The amount of each library bead suspension was 17. mu.L, and a corresponding volume of bead suspension was added to the tube for suspension.

8) A corresponding number of PCR tubes were prepared, and 17. mu.L of a mixture of magnetic beads and a suspension was dispensed into each of the PCR tubes.

9) The PCR tube containing the magnetic bead suspension was placed in a PCR instrument and incubated for 5 min.

2. Streptavidin magnetic bead capture

1) After 4-16h of hybridization reaction, the resuspended streptavidin magnetic beads are added into the hybridization system twice after being mixed evenly. Transfer the sample tube to the running PCR instrument and click next.

2) Incubate at 65 ℃ for 45min, shake and mix once every 15min, ensure the magnetic beads are completely resuspended.

3. Thermal elution

1) Turn on PCR Instrument setup program according to Table 17

2) The filled containers will contain 1X WashBuffer I (nucleic-Free Water and xGen)^®10 × Wash Buffer I prepared in a ratio of 1: 9) and 1X Stringent Wash Buffer (Nuclear-Free Water and xGen)^®10X Stringent Wash Buffer in a 1:9 ratio) was placed in a PCR apparatus and preheated.

3) After incubation at 65 ℃ for 45min, transferring the hybridization sample captured by the streptavidin magnetic beads into a PCR tube filled with 1X Wash Buffer I and mixing uniformly. The PCR tube was placed on a magnetic stand for 1min, and after the liquid was completely clarified, the supernatant was removed thoroughly with a pipette.

4) Transferring the 1X Stringent Wash Buffer into a PCR tube filled with sample magnetic beads, mixing uniformly, covering the tube cap tightly, and putting the tube cap into a PCR instrument to incubate for 5min at 65 ℃. After the time, the PCR tube with the sample is placed on a magnetic rack until clarified, and the supernatant is removed thoroughly with a pipette.

5) Repeating the step 4) once.

4. Eluting at normal temperature

1) The PCR tube containing the sample was removed from the magnetic stand, and 150ul of 1 XWash Buffer II (nucleic-Free Water and xGen) was pipetted from the dispensing tube^®10x Wash Buffer II in a ratio of 1: 9) into a PCR tube filled with magnetic beads, incubating at room temperature for 2min, uniformly mixing for 30s in a constant-temperature mixer, standing for 30s, and alternately mixing to ensure sufficient mixing. The PCR tube was centrifuged instantaneously and then allowed to stand on a magnetic stand for 1min, and the supernatant was removed thoroughly with a pipette.

2) The PCR tube containing the sample was removed from the magnetic rack, subjected to instantaneous centrifugation, and then placed on the magnetic rack, and a small amount of residual Buffer was removed by a pipette.

3) Add 21. mu.L of Nuclear-Free Water to the tube, suck and mix.

4) KAPA HiFi HotStart ReadyMix, primers FCF (10. mu.M) and FCR (10. mu.M) were thawed 15-20min in advance at4 ℃.

5) The reaction system was prepared in PCR tubes or centrifuge tubes placed on ice-boxes as per Table 18.

6) And (3) subpackaging the configured MIX into a PCR tube with a labeled sample serial number.

7) Transfer 20. mu.L of the product with magnetic beads to the corresponding PCR tube with a pipette, and mix well.

8) The procedure shown in table 19 was started on a PCR instrument:

library purification steps were as follows:

1) and (3) oscillating and uniformly mixing the AMPure XP magnetic beads, sucking 60 mu L of the mixture and adding the mixture into a labeled PCR tube.

2) After PCR amplification, the PCR tube was taken out and placed in a magnetic rack for 2min, and after the liquid was completely clarified, the supernatant was transferred to the PCR tube in the magnetic rack.

3) Shaking and mixing, and incubating at room temperature for 10 min.

4) The PCR tube was centrifuged instantaneously and placed on a magnetic stand for 5min, after the liquid was completely clarified, the supernatant was removed (5-10. mu.L of liquid was left).

5) 200 μ L of 80% ethanol was added slowly along the side walls of the PCR tube, left to stand for 30s, and the supernatant was removed using a pipette.

6) Repeating the step 5) once.

7) The PCR tube was removed from the magnetic rack, placed on the magnetic rack after transient centrifugation, and a small amount of residual ethanol was removed using a pipette, taking care not to attract the magnetic beads.

8) And opening a PCR tube cover, and placing the magnetic beads at room temperature for drying, wherein the surfaces of the magnetic beads have no liquid to reflect light, and the magnetic beads cannot be dried excessively.

9) Add 31. mu.L of TE Buffer pH 8.0 to the PCR tube, mix well with shaking, incubate for 5min at room temperature.

10) The PCR tube was centrifuged briefly and placed on a magnetic rack for 1-2min until the liquid was clear, and 30. mu.L of the supernatant was carefully transferred to a new 1.5mL centrifuge tube with a pipette, taking care not to attract the beads.

11) Performing library quality inspection, and performing machine sequencing after the library quality inspection is qualified.

Step four: and (4) performing bioinformatics analysis on the sequencing result to obtain a mutation result of the detection sample. The method can be specifically implemented by the following steps:

and removing the adaptor sequence and the low-quality base sequence introduced in the experiment and sequencing links by using fastp (v0.19.4) software to obtain high-quality sequencing data. Wherein, the required data quality meets Q30 more than or equal to 80%, otherwise, the quality control is judged not to pass.

The above-described data satisfying the requirements were aligned to hg19 (GRCh 37) reference genome using bwa (0.7.17) sequence alignment software, generating BAM format files recording the alignment results. BAM files were then sorted, de-duplicated, and base quality corrected using Samtools (v 1.9) and genoanalysis tk (v 4.1.0) to obtain the final BAM file, and subsequent mutation analysis was performed based on this file.

Wherein, the comparison rate of the reference genome is more than or equal to 90 percent, the average sequencing depth of the target area of the control sample is more than or equal to 100 times, the average sequencing depth of the target area of the tumor sample is more than or equal to 500 times, the proportion of sites with the depth in the target area larger than the average depth multiplied by 0.2 is more than or equal to 90 percent, and the matching consistency of the tumor and the control sample is more than or equal to 90 percent. If any of the conditions is not met, the quality control is judged to fail, and the experiment needs to be carried out again.

Analyzing point mutation and insertion deletion mutation in a sample by using a variation identification module of genomeAnalystk (v4.1.0); analyzing the gene fusion event in the sample by using a fusion analysis module; copy number variation analysis modules (including the cnvkit (v 0.9.6) software and filter modules) were used to analyze copy number variation in samples. Resulting in mutations that can eventually enter the annotation process.

The resulting mutations were annotated as follows: the point mutation, small fragment insertion deletion, gene fusion and copy number variation are annotated by using a variation annotation module built based on Annovar (v2018.04.16), and databases used for annotation comprise clinvar, cosmic, 1000 genes and the like.

In addition, TMB (tumor mutation burden) values were calculated and samples were analyzed for microsatellite instability (MSI) using a microsatellite instability analysis module.

Step five: and (5) reading mutation results, and judging the pathogenicity of the mutation. Based on the annotated results of the variation in step four, the variation was classified for detected non-benign/likely-benign variations according to the classification criteria of variation as specified in ACMG genetic variation classification criteria and Guidelines and the Cancer variation Interpretation Guidelines (Standards and Guidelines for the Interpretation and Reporting of Sequence Variants in Cancer) jointly issued by the american association of pathology (AMP)/american clinical oncology society of oncology (ACSO)/american society of pathologists (CAP). As an example, such as: in the following colorectal cancer samples, KRAS c.35g > ap.g12d variation was detected in exon 2 of KRAS gene, and patients with this variation could not be treated with cetuximab and panitumumab according to the national cancer complex Network for Colorectal Cancer (NCCN) guidelines and the chinese clinical oncology society of cancer (CSCO) guidelines for colorectal cancer diagnosis and treatment. Finally classifying the variation as a type I variation according to the Cancer variation Interpretation guideline (Standards and Guidelines for the Interpretation and Reporting of Sequence Variants in Cancer) jointly issued by the American Association of Pathology Association (AMP)/American society of clinical Oncology (ACSO)/American society of pathologists (CAP), thereby providing a report and suggesting that the patient is resistant to cetuximab and panitumumab.

In practical application, the above steps are adopted to detect a sample of a patient with endometrial cancer, and the result is: microsatellite instability was high (MSI-H) and tumor mutational load (TMB) was 298.520 mut/Mb, which is attributed to high TMB. Solid tumor patients with high TMB or high microsatellite instability may benefit from curitant (pappaluzumab) according to approval by the united states Food and Drug Administration (FDA). The patients were treated by selecting kreota in 2018 at3 months, and the injection was performed once every 21 days, as the results of which the CA125 level decreased to within the normal range after two treatment courses and remained within the normal range thereafter. 2018-3-30, 2018-5-9 and 2019-3-11, and the results of abdominal ultrasound contrast in three stages show that the lesion is clearly seen to shrink earlier and then disappear.

The above steps are adopted to detect samples of patients with ovarian cancer, and the result is as follows: the tumor mutational load (TMB) was 15.162mut/Mb, which is high in TMB. TMB high solid tumor patients may benefit from curitant (pappalizumab) according to united states Food and Drug Administration (FDA) approval. Patients were treated with kreota as suggested by the test results, and CA125 returned to normal levels (from 179.2U/ml to 18.7U/ml) during treatment, with a significant reduction in metastatic foci (by more than 30%) and Partial Remission (PR).

The sample of the colorectal cancer patient is detected by adopting the steps, and the result is as follows: the variation KRAS c.35G > A p.G12D is detected, and the variation abundance is 17.92%. According to the National Cancer Complex Network (NCCN) guidelines for colorectal cancer and the chinese clinical oncology institute of technology (CSCO) guidelines for colorectal cancer diagnosis and treatment, patients with this variation cannot be treated with cetuximab and panitumumab. The patient may be treated in consideration of the remaining modalities, such as conventional chemotherapy.

Therefore, the technical scheme provided by the invention can be used for detecting pan-cancer species, and the detection result has a positive guiding effect on accurate treatment.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention. It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims (9)

1. A gene Panel for detecting pan-cancer species is characterized by comprising tumor pathway related genes, tumor genetic susceptibility genes, tumor high-frequency mutation genes, tumor targeting drug related genes, tumor driving genes, immunity curative effect related genes, key DDR pathway related genes and other genes playing important roles in the occurrence and development of cancer; the key DDR pathway related genes comprise: ATM, ATR, BARD1, BLM, BRCA1, BRCA2, BRIP1, CHEK1, CHEK2, ERCC2, ERCC3, ERCC4, ERCC5, FAM175A, FANCA, FANCC, FANCD2, FANCL, MDC1, MLH1, MRE11A, MSH2, MSH3, MSH6, NBN, PALB2, PARP1, PMS1, PMS2, POLE, PRKDC, PTEN, RAD50, RAD51, RAD51B, RAD51C, RAD51D, RAD52, RAD54L, 53, TP53BP1, XRCC 2; such other genes that play an important role in the development of cancer include: DUSP, ETV, FOXP, GID, GRM, HIST1H3, HIST2H3, ICOSLG, IL, IRS, KNSTRN, MAGI, MAPKAP, MSI, NT5C, PAK, PMAIP, PPP4R, RFWD, RRAGC, SOX, TNFRSF, ZNF703, ANKRD, BABAM, E2F, EPCAM, ETV, FGF, FRS, HIST1H3, HIST3H, ID, IRS, MALT, MAX, MSI, NTHL, PAK, RAC, RANBP, RRAS, SGK, GSTM, TOP, TYRO, WWTR, BCORL, BTG, EED, HIST1H3, INHA, MST, MYOD, NKX-1, PRDM, PTP4A, SDHB, SH2B, SOX, TET, SPEAP, SPEP, SHCK 1H, SHCK 1H3, SHCK, SACK, SADDS, SA, CENPA, EIF4A, EPHB, EZR, HIST1H3, LTK, MAP3K, NEGR, NOTCH, PARP, PHOX2, PIK3R, PPARG, PRKCI, PTPRS, RECQL, SESN, SLC34A, SMYD, SPRED, STK, TCF, TIPARP, TRAF, VTCN, YES, BCL2L, CD, CHD, CSF3, DNMT3, EIF4, HIST1H3, INSR, GRIN MAP3K, MEF2, MYB, NUTM, PIK3C2, PRKD, PTPRT, RECQL, RPS6KA, SESN, SLIT, CAIP, STK, TMEM127, TRAIFF, ZBTB, FOKML, HIST1H2, HIST1H3, P2, PIRY 3C2, PRXB 3, SDCK 6, SUBCK 2, PRXO, SLC 2, FORCH 2, PRXB, PRXC, PRXK, SLC 6, SLC 2, FORCH 2, FORCK, FORCH 3, FORCH 2, FORCK, FORCH 2, FORCK, FORCH, FORCK, FORCH 2, FORCH 3, FORCK, FORCH 2, FORCK, FORCH 2, FORCH, FORCK, FORCH 2, FORCH 2, FORCH 2, FORCH, FORC.

2. The gene Panel for detecting pan-cancer species of claim 1 wherein the tumor pathway associated gene comprises: AKT1, AKT2, AKT3, ALK, APC, AR, ARAF, ASXL1, ATM, ATR, ATRX, AURKA, AURKB, AXIN1, BCL2, BRAF, BRCA1, BRCA2, BRIP 2, BTK, CASP 2, CBL, CCND2, CCNE 2, CD79, CDK 2, KN 12, KN 22, MDM2, CDKN 22, CHEK2, CRKL 2, CRLF2, CSCSF 12, CTNNA 2, CTNNB 2, MDM2, 685MT 3, DOT 2, 685 2, 685 2, 685 2, 685, 2, 685 2, 685, 2, 685 2, 685 2, 685, 685 2, 685 2, 685 2, 685 2, 685 2, 685 2, 685, 2, 685 2, 685, 2, 685 2, 685 2, 685, 685, 685, 685 2, 685, 685, 685 2, 685, 685, 2, 685 2, 685, 685, 685, 685, 685, 685, 685, 685, 685, 685, 685, 685, PDGFRA, PDGFRB, PGR, PIK3CA, PIK3CG, PIK3R1, PIK3R2, PTCH1, PTEN, PTPN11, RAD50, RAD51, RB1, RET, RICTOR, RNF43, ROS1, RPTOR, SETD2, SMAD2, SMAD3, SMAD4, SMO, SOCS1, SRC, STAG2, STAT3, STAT4, STAT5A, STAT5B, SUFU, TET2, TGFBR1, TGFBR2, TNFAIP3, TP53, TSC1, TSC2, TSHR, WISP3, WT 1.

3. The gene Panel for detecting pan-cancer species of claim 1, wherein said tumor genetic susceptibility gene comprises: ALK, APC, ATM, AXIN, BMPR1, BRCA, BRIP, CDH, CDKN2, CHEK, FH, FLCN, GREM, KIT, MEN, MET, MLH, MSH, MUTYH, NBN, NF, PALB, PMS, POLD, POLE, PTEN, RAD51, RB, SMAD, STK, TP, TSC, VHL, SDHD, TSC, PDGFRA, BARD, CDK, RET, SMARCA, SMARCB, SDHA, PRKAR1, HOXB, BRAF, PTCH, SDHAF.

4. The gene Panel for detecting pan-cancer species according to claim 1, wherein said tumor high-frequency mutant gene comprises: ACVR1B, AKT1, AMER1, APC, ARID 11, ARID1, ASXL1, ATM, ATRX, AXIN1, BAP1, BCL2L1, BCOR, BRA 1, BRCA1, BTG1, CASP 1, CBFB, CCND1, CCNE1, CD274, CDH1, CDK1, KN 11, MDM 11, CDKN K2 1, CHD 1, CREBB 1, CTNNA1, CTB 1, EGFR, ELF 1, EP300, ERBB4, ERBB 1, ERCC 1, ERBG 1, ERBBD 1, KM 1, 1, STK11, TBX3, TCF7L2, TERC, TERT, TGFBR2, TMPRSS2, TNFAIP3, TP53, TSC1, TSC2, U2AF1, VEGFA, VHL, WHSC1L 1.

5. The gene Panel for detecting pan-cancer species of claim 1 wherein the tumor-targeting drug-associated gene comprises: ABL1, ABL2, ACVR1, AKT1, AKT2, AKT3, ALK, AR, ARAF, ARID1A, ATM, ATR, ATRX, AXL, BAP1, BARD1, BCL1, BLM, BRAF, BRCA1, BRD 1, BRIP1, BTK, C11orf 1, CCND1, CCNE1, CDK1, MDM 1, CDKN 11, CDKN 21, 685 1, 685K 1, KM K, 1, 685K 1, 685K 1, 685D, 1, 685D, 685D, 685D, 685D, 685D, 685D, 685D, 685D, 685D, 685D, 685D, 685D, 685D 685, NOTCH2, NPM1, NRAS, NTRK1, NTRK2, NTRK3, PALB2, PDGFRA, PDGFRB, PGR, PIK3CA, PIK3CB, PIK3CD, PIK3CG, PIK3R1, PMS2, POLD1, POLE, PTCH1, PTEN, RAB35, RAD50, RAD51, RAD52, RAF1, RARA, RB1, RET, RHEB, RICTOR, ROS1, SLX4, SMARCA4, SMARC 1, SMO, SRC, STK11, SYK, TMPRSS2, TOP2A, 53, TSC1, TSC2, VHL, XRCC 2.

6. The gene Panel for detecting pan-cancer species of claim 1, wherein the tumor driver gene comprises: ABL1, ACVR1, ACVR1B, AKT1, ALK, AMER1, APC, AR, ARAF, ARID 11, ARID 51, ASXL1, ATM, ATRX, AXIN1, B2 1, BAP1, BCL2L1, BCOR, BRAF, BRCA1, BTG1, CARD1, CASP 1, CBFB, CCND1, CD79 1, CDH1, CDK1, KMK 1, KM K1, KM 1, 1, NCOR, NF, NFE2L, NOTCH, NPM, NRAS, NSD, NUP, PAX, PBRM, PDGFRA, PGR, PIK3R, PIM, PMS, POLE, PPM1, PPP2R1, PPP6, PRKAR1, PTCH, PTEN, PTPN, PTPRD, RAC, RAD, RAF, RARA, RASA, RB, RBM, RET, RHEB, RHOA, RIT, RNF, RRAS, RUNX, RXRRA, SETD, SF3B, SMAD, SMARCA, SOS, SOX, SPOP, SPTA, SF, STAG, SRK, TAF, TBEB, TCEB, TCF7L, TET, TGTGTGTP, VHTP, TSC, AIP, TSC, HAF, TSC, TMAF, TMAS, PTN, PTPN, PTPR, RTR, TSC, and TSC.

7. The gene Panel for detecting pan-cancer species according to claim 1, wherein said immunotherapy efficacy-related gene comprises: TSC2, ATM, B2M, BRCA1, BRCA2, CCND1, DNMT3A, EGFR, FGF19, FGF3, FGF4, JAK1, JAK2, KRAS, MDM2, MDM4, MLH1, MSH2, MYC, PBRM1, PMS2, POLD1, POLE, PTEN, STK11, VHL, NF1, TP 53.

8. A probe Panel for detecting pan-cancer species, wherein the probe Panel is a detection probe for the tumor pathway-associated gene, the tumor genetic susceptibility gene, the tumor high-frequency mutation gene, the tumor-targeted drug-associated gene, the tumor driver gene, the immune efficacy-associated gene, the key DDR pathway-associated gene, and other genes playing important roles in the development of cancer according to any one of claims 1 to 7.

9. Use of the gene Panel according to any one of claims 1 to 7 or the probe Panel according to claim 8 for the preparation of a pan-cancer species detection device.