CN109609647A - Detection Panel for pan-cancer-species targeting, chemotherapy and immune drugs based on next-generation sequencing, detection kit and application thereof - Google Patents

Detection Panel for pan-cancer-species targeting, chemotherapy and immune drugs based on next-generation sequencing, detection kit and application thereof Download PDF

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CN109609647A
CN109609647A CN201910074640.XA CN201910074640A CN109609647A CN 109609647 A CN109609647 A CN 109609647A CN 201910074640 A CN201910074640 A CN 201910074640A CN 109609647 A CN109609647 A CN 109609647A
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CN109609647B (en
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洪媛媛
夏艳
颜林林
闫慧婷
宋小凤
曾雪霞
郭现超
赵利利
朱伟
何骥
杜波
陈维之
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Zhenyue Biotechnology Jiangsu Co ltd
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Abstract

The invention discloses a pan detection kit for pan-cancer targeting, chemotherapy and immune drugs based on next-generation sequencing, and an application thereof. Wherein, the detection panel comprises pan cancer species typing, treatment and prognosis related gene mutation, tumor mutation load calculation related exon region and microsatellite instability locus. By applying the technical scheme of the invention, the detection panel comprises pan cancer species typing, treatment and prognosis related gene mutation, tumor mutation load calculation related exon regions and microsatellite instability sites, the included gene information is comprehensive, various tumor variations can be directly subjected to combined detection, and the detection panel can be applied to the concomitant diagnosis of targeted drugs, chemotherapeutic drugs or immunological drugs to obtain accurate results.

Description

The detection for the targeting of general cancer kind, chemotherapy and immune medication based on the sequencing of two generations Panel, detection kit and its application
Technical field
The present invention relates to field of biomedicine technology, are used for general cancer kind based on the sequencing of two generations in particular to a kind of Detection panel, detection kit and its application of targeting, chemotherapy and immune medication.
Background technique
Currently, there are many relevant detection technique of cancer, for example, high-flux sequence, target area capture sequencing, liquid biopsy, Tumor mutations load and MSI detection etc..
Wherein, high-flux sequence (High-Throughput Sequencing) also known as next-generation sequencing (Next Generation Sequencing, NGS), it is for (Sanger Sequencing) is sequenced in traditional mulberry lattice.
On the basis of the sequencing approaches such as Sanger, by technological innovation, the genomic DNA two sides of fragmentation is connected and are connect Head then generates the PCR clone's array fixed in millions of a spaces with different methods.Each clone is by single library fragments Multiple copy compositions.Then primer hybridization and enzyme extension are carried out.All in the same plane due to all clones (flowcell), with four kinds of different dNTP of the fluorescent marker of different colours, when archaeal dna polymerase synthesizes complementary strand, every addition A kind of dNTP will release different fluorescence, and DNA sequence dna extends and image checking constantly repeats, and finally analyze by computer It is obtained with complete DNA sequence dna information.Once sequencing is carried out to millions of nucleic acid molecules to hundreds of thousands to be also known as deeply Degree sequencing (Deep sequencing).
NGS detection method flux is high, and same sample only needs to carry out a library construction and capture, so that it may to a large amount of Gene is detected, and meets the needs of clinical detection, and the detection of multiple genes can be carried out by not needing a large amount of sample size, both may be used To be detected to known mutations site, unknown mutation site can also be detected, furthermore NGS detection method can also be to each Kind of mutation type is detected, to Different categories of samples clinically, such as whole blood, tissue, FFPE sample, cfDNA other sample classes Type is able to carry out detection.
The application being derived in conjunction with microarray technology is sequenced in target area capture sequencing, the second generation, and target sequence is caught Obtain sequencing technologies (Targeted Resequencing).This technology synthesizes a large amount of oligonucleotides first with microarray technology Probe, these oligonucleotide probes complementary with the specific region on genome can combine, to be enriched to particular section, then These sections are sequenced with second generation sequencing technologies.It is to utilize target gene group based on DNA hybridization principle that principle, which is sequenced, in it The probe and genomic DNA of region customization carry out chip hybridization or solution hybridization, and target gene regions DNA is enriched with, then passes through NGS technology is sequenced.Selected target area, which is sequenced, can be continuous DNA sequence dna, be also possible to be distributed in same Segment in chromosome different zones or different chromosomes.
Liquid biopsy is exactly to pass through acquisition blood or urine etc. to make diagnosis to diseases such as cancers.It is living with traditional operation Inspection is compared with aspiration biopsy, and the advantage of liquid biopsy is: 1) easy to operate, traditional biopsy depends on the discovery of iconography, fixed Operation or puncture procedure are carried out after the accurate location and size of position tumour, operability is difficult, and liquid biopsy does not need image then It learns and supports；2) non-intrusion type, traditional mode is due to its intrusive mood operation, often with complication, tumor recurrence risk, liquid Biopsy is then only to extract a certain amount of blood, and risk is small；3) sampling is convenient.Tradition punctures and operation samples this and injures to patient It is larger, and liquid biopsy generally only needs 5~10ml blood, supports to be repeated as many times sampling；4) Tumor Heterogeneity is successfully managed.It passes System sampling is likely to occur the problem of taking less than tumor tissues by the reason of sampling point, and may be swollen comprising different mutation in blood The information of oncocyte.5) at low cost.It is cheap compared with traditional detection method.
Tumor mutations load (TMB) is that displacement occurs in the every megabasse of exons coding district for assess gene and is inserted into/lacks The sum for losing mutation, the completely new biomarker with good prospect, TMB can genetic coding region in quantitative predication tumor sample Mutation sum.Tumour cell with higher level TMB is easier to be identified by immune system, the reason is that the mutation of body cell It is transcribed/to be expressed as RNA/ protein level, new antigen, protein fragments or polypeptide segment etc. are generated, these new albumen are by itself Immune system is identified as non-self antigen, activates T cell, causes to be immunoreacted.Therefore, when the gene accumulated in every megabasse becomes When heteromerism mesh increases, so that it may many new antigens are generated, it is easier to be identified by immunocyte.So as to inhibit to checkpoint Agent has stronger immune response, that is to say, that TMB is higher, and patient perhaps benefits in bear immunization therapy more.Currently, very much All confirmed in research TMB and tumor neogenetic antigen to the curative effect of immunologic test point inhibitor be it is relevant, utilize advanced two generation Sequencing technologies, exon sequencing approach detect Tumor mutations load, provide solution for clinic personalized treatment.
Microsatellite (Microsatellite) is the short tandem repeat being dispersed throughout in human genome, there is monokaryon glycosides The repetition of acid, dinucleotide or high-order nucleotide, number of repetition 10~50 times.It is micro- in tumour cell compared with normal cell Satellite due to recurring unit insertion or missing and lead to the change of microsatellite length, be just called microsatellite instability (MSI, microsatellite instability).A large number of studies show that MSI is that defect occurs by mispairing reparation (MMR) gene to cause , it is closely related with the generation of tumour.MMR system is made of a series of enzyme of specific DNA plerosis base mispairings, they can The mispairing occurred in DNA replication dna and damage process or the base that do not match are found and corrected, guarantees the accuracy of duplication.When tumour is thin There are when MMR gene lacks functionality (Mis-Match Repair deficiency, abbreviation dMMR) in born of the same parents, signal tumor cell The repair ability to DNA replication dna mistake is lost, will build up on mass mutation in tumour cell, adjoint microsatellite instability will occur Property (MSI) feature, cause cell proliferation and differentiation abnormal and the generation of tumour.MSI is the label for evaluating MMR system function.According to MSI unstability and degree, can be divided into the highly unstable type of microsatellite (MSI-H), microsatellite minuent instability mode (MSI-L) and Microsatellite stable type (MSS).MSI detection can be used for: 1) II phase colorectal cancer patients medication guide and prognosis prediction；2)Lynch The screening of (Jessica Lynch) syndrome；2) the benefit prediction of PD-1 immunization therapy.Detection method: while extracting normal group of same patient It knits with tumor tissues sample DNA, detection site is expanded using the method for multiple fluorescence PCR, then pass through Capillary Electrophoresis Amplified production is detected, and two kinds of tissue-derived testing results are compared and analyzed using professional software, it can accurately Parting is carried out to patient's MSI state.
But every kind of detection method deposits different advantage and disadvantage, product currently on the market can not be treated comprehensively Test sample is originally detected.
Summary of the invention
The present invention is intended to provide a kind of detection for the targeting of general cancer kind, chemotherapy and immune medication based on the sequencing of two generations Panel, detection kit and its application, with solve in the prior art cannot the technology that is detected of comprehensive sample to be tested ask Topic.
To achieve the goals above, according to an aspect of the invention, there is provided it is a kind of based on the sequencing of two generations for general The detection panel of the targeting of cancer kind, chemotherapy and immune medication.Detection panel includes for carrying out the general cancer kind of joint-detection point Type, treatment, the relevant gene mutation of prognosis, the relevant exon region of Tumor mutations carry calculation and microsatellite instability position Point.
Further, general cancer kind parting, treatment, the relevant gene mutation of prognosis, Tumor mutations carry calculation are relevant outer Aobvious subregion include ABCA13, ABCA8, ABL1, ACADSB, ACOT13, ADAMTS6, ADRB1, ADSS, AGPAT9, AK7, AKT1、AKT2、AKT3、ALG9、ALK、ALK_fus、ALOX12B、ALS2CR11、AMER1、ANKRA2、ANKRD46、ANO1、 APC、APOPT1、AR、ARAF、ARHGAP4、ARHGAP6、ARID1A、ARID1B、ARID2、ARID4A、ARL6IP6、ARMC5、 ARPC2、ASH1L、ASXL1、ATM、ATP9B、ATR、ATRX、AURKA、AURKB、AXIN1、AXL、B2M、BAP1、BARD1、 BAT-25、BAT-26、BCL2、BCL2L1、BCOR、BCYRN1、BLM、BRAF、BRCA1、BRCA2、BRD4、BRIP1、BRS3、 BTF3、BTG1、BTK、C20orf96、C22orf23、C5orf42、C9orf72、CAB39、CALD1、CALM2、CALR、 CARD11、CASP8、CAST,ERAP1、CBFB、CBL、CBR4、CCND1、CCND2、CCND3、CCNE1、CCR4、CD274、 CD40、CD74_fus、CD79A、CD79B、CDC25B、CDC73、CDH1、CDK12、CDK4、CDK6、CDK7、CDK8、CDKL3、 CDKN1A、CDKN1B、CDKN2A、CDKN2B、CDKN2C、CDO1、CEBPA、CEP120、CHD1、CHEK1、CHEK2、CIC、 CNKSR3、CNOT8、COSM1316144、COSM1316145、COSM1332498、COSM3676668、COSM3747491、 COSM5045664、COX18、CREBBP、CRKL、CRLF2、CSF1R、CSF3R、CTAGE5、CTCF、CTNNB1、CUL3、 CXCR4、CYFIP1、CYLD、DAXX、DBT、DDR2、DEPDC5、DIAPH1、DICER1、DIS3、DNMT3A、DOCK11、 DOT1L、DROSHA、DSCAM、DXS6804、DXS7132、DXS7423、DYS391、DYS438、DYS458、EGFR、EGFR_ fus、EIF4G3、EML4_fus、EP300、EPHA3、EPHA5、EPHA7、EPHB1、ERBB2、ERBB3、ERBB4、ERCC4、 ERG、ERI1、ERRFI1、ESR1、ETV1、ETV6、ETV6_fus、EWSR1_fus、EXOSC8、EZH2、EZR_fus、F13A1、 FAM149A、FAM153B、FAM46C、FANCA、FANCC、FANCD2、FANCG、FANCI、FAS、FAT1、FBXW7、FGF16、 FGF19、FGF3、FGF4、FGFR1、FGFR2、FGFR2_fus、FGFR3、FGFR4、FH、FLCN、FLOT1、FLT1、FLT3、 FLT4、FMO1、FOLH1、FOXL2、FOXP1、FRAS1、FUBP1、FUS_fus、GABRP、GANC、GATA1、GATA2、GATA3、 GLI1、GMEB1、GNA11、GNA13、GNAQ、GNAS、GPM6A、GRIN2A、GSK3B、GSTM1、H3F3A、HAUS2、HCAR2、 HEY1_fus、HGF、HLA-A、HLA-B、HLA-C、HNF1A、HNRNPH1、HRAS、HSPA1B、HSPA4、HYOU1、IARS、 ID2、ID3、IDH1、IDH2、IGF1R、IGF2、IKBKE、IKZF1、IL7R、IL8、INHBA、INPP4B、IPO7、IRAK1、 IRF4、IRF6、IRF8、IRS2、ITGAL、JAK1、JAK2、JAK3、JUN、KCNJ2、KDM5A、KDM5C、KDM6A、KDR、 KEAP1、KIAA1210、KIAA1432、KIF5B_fus、KIR3DX1、KIT、KMT2A、KMT2C、KMT2D、KPNA4、KPNB1、 KRAS、LAMA3、LEPR、LMO1、LOC100131626、LONRF3、LRRC34、LYN、MAGOHB、MALT1、MAP2K1、 MAP2K2、MAP2K4、MAP3K1、MAP3K4、MAP4K5、MAPK1、MAPKAP1、MARK2、MCL1、MDM2、MDM4、MED12、 MED19、MEF2B、MEIS1、MEN1、MET、MIR1273H、MITF、MLH1、MMP16、MONO-27、MOV10L1、MPL、 MRE11A、MRPL19、MSH2、MSH3、MSH6、MTF1、MTOR、MTRR、MUTYH、MYADM、MYC、MYCL、MYCN、MYD88、 MYO10、NAB1、NAB2_fus、NBN、NCOA6、NDUFS1、NEO1、NF1、NF2、NFE2L2、NFKBIA、NFXL1、NKX2-1、 NOTCH1、NOTCH2、NOTCH3、NPM1、NR-21、NR-24、NR4A3_fus、NRAS、NSD1、NT5C2、NTRK1、NTRK1_ fus、NTRK2、NTRK2_fus、NTRK3、NTRK3_fus、NUP85、NUP93、P2RY8、PALB2、PAPOLG、PAQR8、 PARK2、PARP1、PAX5、PBRM1、PDCD1、PDCD1LG2、PDE6C、PDGFB_fus、PDGFRA、PDGFRB、PDPN、 PIGF、PIK3C2G、PIK3CA、PIK3CB、PIK3CG、PIK3R1、PIK3R2、PLCG2、PLEKHA1、PLEKHH2、PMS2、 PNO1、POLD1、POLE、PPARG、PPHLN1_fus、PPP2R1A、PRDM1、PREX2、PRKAR1A、PRKCI、PRPF39、 PTCH1、PTEN、PTPN11、PTPRJ、PURA、RABGAP1L、RAC1、RAD21、RAD50、RAD51、RAD51B、RAD51C、 RAD51D、RAD52、RAD54L、RAF1、RARA、RB1、RBM10、RBM27、REL、RET、RET_fus、RHOT1、RICTOR、 RIPK2、RNF19A、RNF43、ROS1、ROS1_fus、RPA4、RPTOR、RRP1B、RUNX1、RYR2、SASH1、SDHA、SDHB、 SDHC、SDHD、SEL1L3、SETD2、SF3B1、SHROOM3、SIPA1L2、SLC34A2_fus、SMAD2、SMAD3、SMAD4、 SMARCA4、SMARCB1、SMO、SNX6、SOCS1、SOX2、SOX9、SPC24、SPEN、SPOP、SRC、SS18_fus、STAG2、 STAT3、STK11、STMN1、STRBP、SUCLG1、SUFU、SUGCT、SYK、TAF15、TAGAP、TBC1D8B、TBX3、 TECPR2、TERT、TET2、TGFBR2、TMEM67、TMPRSS15、TMPRSS2、TNFAIP3、TNFRSF14、TNFSF13B、 TNKS、TNRC18、TOP1、TOP2B、TP53、TPH1、TRA2A、TRIM24、TSC1、TSC2、TSHR、TSN、TXNRD1、 U2AF1、UBE2E3、UBE3C、ULK4、UPF2、UTY、VEGFA、VHL、VSIG10、WHSC1、WHSC1L1、WT1、WWC3、 XPO1, ZBBX, ZDHHC17, ZNF711, ZNF805 and ZZZ3.
Further, microsatellite instability site include rs1045642, rs1052555, rs10981694, rs11045585、rs1105525、rs1130214、rs1138272、rs115232898、rs11572080、rs11598702、 rs11615、rs12613732、rs12762549、rs13181、rs151264360、rs1517114、rs1570360、 rs1650697、rs1650723、rs1695、rs1799793、rs1801019、rs1801131、rs1801133、rs1801265、 rs1805087、rs183484、rs1934951、rs2032582、rs2072671、rs2075252、rs2228001、 rs2273618、rs2291075、rs2291767、rs2297595、rs2494752、rs2501873、rs25487、 rs2784917、rs28738963、rs316019、rs3212986、rs3745274、rs3892097、rs3918290、 rs3957357、rs4148323、rs4244285、rs430397、rs442767、rs4646、rs4673、rs4673993、 rs4728709、rs4880、rs50872、rs55886062、rs60369023、rs67376798、rs699947、rs716274、 Rs7779029, rs7851395, rs8133052, rs8175347, rs873478, rs9679162 and rs9937.
According to another aspect of the present invention, provide it is a kind of based on the sequencing of two generations for the targeting of general cancer kind, chemotherapy and exempt from The detection kit of epidemic disease medication.The detection kit includes detection probe, and detection probe is directed to general cancer kind parting, treatment, prognosis Relevant gene mutation, microsatellite instability site and the relevant exon region of Tumor mutations carry calculation.
Further, detection probe overlay area/target area >=99%.
Further, general cancer kind parting, treatment, the relevant gene mutation of prognosis and Tumor mutations carry calculation are relevant outer Aobvious subregion include: ABCA13, ABCA8, ABL1, ACADSB, ACOT13, ADAMTS6, ADRB1, ADSS, AGPAT9, AK7, AKT1、AKT2、AKT3、ALG9、ALK、ALK_fus、ALOX12B、ALS2CR11、AMER1、ANKRA2、ANKRD46、ANO1、 APC、APOPT1、AR、ARAF、ARHGAP4、ARHGAP6、ARID1A、ARID1B、ARID2、ARID4A、ARL6IP6、ARMC5、 ARPC2、ASH1L、ASXL1、ATM、ATP9B、ATR、ATRX、AURKA、AURKB、AXIN1、AXL、B2M、BAP1、BARD1、 BAT-25、BAT-26、BCL2、BCL2L1、BCOR、BCYRN1、BLM、BRAF、BRCA1、BRCA2、BRD4、BRIP1、BRS3、 BTF3、BTG1、BTK、C20orf96、C22orf23、C5orf42、C9orf72、CAB39、CALD1、CALM2、CALR、 CARD11、CASP8、CAST,ERAP1、CBFB、CBL、CBR4、CCND1、CCND2、CCND3、CCNE1、CCR4、CD274、 CD40、CD74_fus、CD79A、CD79B、CDC25B、CDC73、CDH1、CDK12、CDK4、CDK6、CDK7、CDK8、CDKL3、 CDKN1A、CDKN1B、CDKN2A、CDKN2B、CDKN2C、CDO1、CEBPA、CEP120、CHD1、CHEK1、CHEK2、CIC、 CNKSR3、CNOT8、COSM1316144、COSM1316145、COSM1332498、COSM3676668、COSM3747491、 COSM5045664、COX18、CREBBP、CRKL、CRLF2、CSF1R、CSF3R、CTAGE5、CTCF、CTNNB1、CUL3、 CXCR4、CYFIP1、CYLD、DAXX、DBT、DDR2、DEPDC5、DIAPH1、DICER1、DIS3、DNMT3A、DOCK11、 DOT1L、DROSHA、DSCAM、DXS6804、DXS7132、DXS7423、DYS391、DYS438、DYS458、EGFR、EGFR_ fus、EIF4G3、EML4_fus、EP300、EPHA3、EPHA5、EPHA7、EPHB1、ERBB2、ERBB3、ERBB4、ERCC4、 ERG、ERI1、ERRFI1、ESR1、ETV1、ETV6、ETV6_fus、EWSR1_fus、EXOSC8、EZH2、EZR_fus、F13A1、 FAM149A、FAM153B、FAM46C、FANCA、FANCC、FANCD2、FANCG、FANCI、FAS、FAT1、FBXW7、FGF16、 FGF19、FGF3、FGF4、FGFR1、FGFR2、FGFR2_fus、FGFR3、FGFR4、FH、FLCN、FLOT1、FLT1、FLT3、 FLT4、FMO1、FOLH1、FOXL2、FOXP1、FRAS1、FUBP1、FUS_fus、GABRP、GANC、GATA1、GATA2、GATA3、 GLI1、GMEB1、GNA11、GNA13、GNAQ、GNAS、GPM6A、GRIN2A、GSK3B、GSTM1、H3F3A、HAUS2、HCAR2、 HEY1_fus、HGF、HLA-A、HLA-B、HLA-C、HNF1A、HNRNPH1、HRAS、HSPA1B、HSPA4、HYOU1、IARS、 ID2、ID3、IDH1、IDH2、IGF1R、IGF2、IKBKE、IKZF1、IL7R、IL8、INHBA、INPP4B、IPO7、IRAK1、 IRF4、IRF6、IRF8、IRS2、ITGAL、JAK1、JAK2、JAK3、JUN、KCNJ2、KDM5A、KDM5C、KDM6A、KDR、 KEAP1、KIAA1210、KIAA1432、KIF5B_fus、KIR3DX1、KIT、KMT2A、KMT2C、KMT2D、KPNA4、KPNB1、 KRAS、LAMA3、LEPR、LMO1、LOC100131626、LONRF3、LRRC34、LYN、MAGOHB、MALT1、MAP2K1、 MAP2K2、MAP2K4、MAP3K1、MAP3K4、MAP4K5、MAPK1、MAPKAP1、MARK2、MCL1、MDM2、MDM4、MED12、 MED19、MEF2B、MEIS1、MEN1、MET、MIR1273H、MITF、MLH1、MMP16、MONO-27、MOV10L1、MPL、 MRE11A、MRPL19、MSH2、MSH3、MSH6、MTF1、MTOR、MTRR、MUTYH、MYADM、MYC、MYCL、MYCN、MYD88、 MYO10、NAB1、NAB2_fus、NBN、NCOA6、NDUFS1、NEO1、NF1、NF2、NFE2L2、NFKBIA、NFXL1、NKX2-1、 NOTCH1、NOTCH2、NOTCH3、NPM1、NR-21、NR-24、NR4A3_fus、NRAS、NSD1、NT5C2、NTRK1、NTRK1_ fus、NTRK2、NTRK2_fus、NTRK3、NTRK3_fus、NUP85、NUP93、P2RY8、PALB2、PAPOLG、PAQR8、 PARK2、PARP1、PAX5、PBRM1、PDCD1、PDCD1LG2、PDE6C、PDGFB_fus、PDGFRA、PDGFRB、PDPN、 PIGF、PIK3C2G、PIK3CA、PIK3CB、PIK3CG、PIK3R1、PIK3R2、PLCG2、PLEKHA1、PLEKHH2、PMS2、 PNO1、POLD1、POLE、PPARG、PPHLN1_fus、PPP2R1A、PRDM1、PREX2、PRKAR1A、PRKCI、PRPF39、 PTCH1、PTEN、PTPN11、PTPRJ、PURA、RABGAP1L、RAC1、RAD21、RAD50、RAD51、RAD51B、RAD51C、 RAD51D、RAD52、RAD54L、RAF1、RARA、RB1、RBM10、RBM27、REL、RET、RET_fus、RHOT1、RICTOR、 RIPK2、RNF19A、RNF43、ROS1、ROS1_fus、RPA4、RPTOR、RRP1B、RUNX1、RYR2、SASH1、SDHA、SDHB、 SDHC、SDHD、SEL1L3、SETD2、SF3B1、SHROOM3、SIPA1L2、SLC34A2_fus、SMAD2、SMAD3、SMAD4、 SMARCA4、SMARCB1、SMO、SNX6、SOCS1、SOX2、SOX9、SPC24、SPEN、SPOP、SRC、SS18_fus、STAG2、 STAT3、STK11、STMN1、STRBP、SUCLG1、SUFU、SUGCT、SYK、TAF15、TAGAP、TBC1D8B、TBX3、 TECPR2、TERT、TET2、TGFBR2、TMEM67、TMPRSS15、TMPRSS2、TNFAIP3、TNFRSF14、TNFSF13B、 TNKS、TNRC18、TOP1、TOP2B、TP53、TPH1、TRA2A、TRIM24、TSC1、TSC2、TSHR、TSN、TXNRD1、 U2AF1、UBE2E3、UBE3C、ULK4、UPF2、UTY、VEGFA、VHL、VSIG10、WHSC1、WHSC1L1、WT1、WWC3、 XPO1, ZBBX, ZDHHC17, ZNF711, ZNF805 and ZZZ3.
Further, microsatellite instability site include rs1045642, rs1052555, rs10981694, rs11045585、rs1105525、rs1130214、rs1138272、rs115232898、rs11572080、rs11598702、 rs11615、rs12613732、rs12762549、rs13181、rs151264360、rs1517114、rs1570360、 rs1650697、rs1650723、rs1695、rs1799793、rs1801019、rs1801131、rs1801133、rs1801265、 rs1805087、rs183484、rs1934951、rs2032582、rs2072671、rs2075252、rs2228001、 rs2273618、rs2291075、rs2291767、rs2297595、rs2494752、rs2501873、rs25487、 rs2784917、rs28738963、rs316019、rs3212986、rs3745274、rs3892097、rs3918290、 rs3957357、rs4148323、rs4244285、rs430397、rs442767、rs4646、rs4673、rs4673993、 rs4728709、rs4880、rs50872、rs55886062、rs60369023、rs67376798、rs699947、rs716274、 Rs7779029, rs7851395, rs8133052, rs8175347, rs873478, rs9679162 and rs9937.
In accordance with a further aspect of the present invention, provide it is a kind of it is above-mentioned based on the sequencing of two generations for the targeting of general cancer kind, chemotherapy And the detection for the targeting of general cancer kind, chemotherapy and immune medication that the detection panel or above-mentioned of immune medication is sequenced based on two generations Kit is used for general cancer kind parting, treatment, the relevant gene mutation of prognosis in preparation, and microsatellite instability site and tumour are prominent Varying duty calculates the application in the device of relevant exon region joint-detection.
Further, general cancer kind includes but is not limited to: lung cancer, intestinal cancer, gastric cancer, liver cancer, breast cancer and carcinoma of endometrium etc. Deng.
Further, joint-detection includes the detection of various mutations type, including various mutations type: point mutation, fusion, Copy number variation, missing and insertion mutation；Preferably, using the adjoint diagnosis for including targeting medicine, chemotherapeutic or immune substance.
The design of detection panel of the invention does not instead of simply sum it up multiple gene regions, passes through design integration Panel homogeneity is improved in the shared region of different mutation type information, reduces panel overall amount of data, reduces cost.
Detection panel includes that general cancer kind parting, treatment, the relevant gene of prognosis, Tumor mutations carry calculation are relevant outer Aobvious subregion and microsatellite instability site comprising gene information amount it is big, can preferably carry out joint-detection, apply In targeting medicine, the available more accurate result of adjoint diagnosis of chemotherapeutic or immune substance.
Detailed description of the invention
The accompanying drawings constituting a part of this application is used to provide further understanding of the present invention, and of the invention shows Examples and descriptions thereof are used to explain the present invention for meaning property, does not constitute improper limitations of the present invention.In the accompanying drawings:
Fig. 1 shows the detection consistency knot in embodiment 4 about the detection panel (P12) of embodiment 1 and complete outer probe Fruit figure.
Specific embodiment
It should be noted that in the absence of conflict, the features in the embodiments and the embodiments of the present application can phase Mutually combination.The present invention will be described in detail below with reference to the accompanying drawings and embodiments.
The mechanism that cancer occurs is extremely complex, and various cancers are even suffered from the patient of identical cancer, cancer is caused to be sent out Raw molecular mechanism and histopathology access is all not quite similar, even the tumour at the same position, therapeutic effect and method It should vary with each individual, it is this because people, the various disease treatment method taken by disease are known as " individualized treatment ".Therefore it is controlled in cancer During treatment, only treating the same disease with different methods varies with each individual, and implements individualized treatment, it is suitable could to be directed to different types of patient selection Their drug.
With deepening continuously for gene molecule level research, more and more tumour cell signal paths are found, largely Clinical research shows that amplification/mutation/expression status of the specific gene in access and the validity of targeting, chemotherapeutics are close It is related.Therefore, amplification/mutation/expression of specific gene in these accesses is clinically detected, can be pointedly every trouble Person makes a set of most suitable therapeutic scheme to measure, to farthest improve the effective percentage for the treatment of, the poison for reducing drug is secondary Effect, avoids inappropriate medication from affecting therapic opportunity adversely.
Technology involved in the relevant testing product of cancer currently on the market respectively deposits different advantage and disadvantage, can not Comprehensive sample to be tested is detected.
In view of this, the present invention is the following technical schemes are provided: typically embodiment there is provided one kind for one kind according to the present invention The detection panel for the targeting of general cancer kind, chemotherapy and immune medication based on the sequencing of two generations.Detection panel include for into The general cancer kind parting of row joint-detection, treatment, the relevant gene mutation of prognosis (" gene mutation " herein could also say that with it is general Cancer kind parting, treatment, the relevant mutated gene of prognosis), the relevant exon region of Tumor mutations carry calculation and microsatellite not Stability site.
Product in the prior art comprehensively the tissue to general cancer kind and blood sample cannot carry out kinds of tumors change simultaneously Different joint-detection.Panel and kit of the invention can swell to general cancer kind parting, treatment, the relevant gene mutation of prognosis Tumor mutational load calculates relevant exon region and microsatellite instability site carries out joint-detection simultaneously.And sample type Cover tissue and blood.
Panel design of the invention does not sum it up not instead of simply, is selected according to demand the type of detection gene It selects, avoids the region panel redundancy, reduce the output of priceless Value Data, shorten data analytical cycle, reduce testing cost.
It applying the technical scheme of the present invention, detection panel includes general cancer kind parting, treatment, the relevant gene mutation of prognosis, The relevant exon region of Tumor mutations carry calculation and microsatellite instability site comprising gene information amount it is big, can Preferably to carry out joint-detection, the adjoint diagnosis applied to targeting medicine, chemotherapeutic or immune substance is available more accurate As a result.
Preferably, general cancer kind parting, treatment, the relevant gene mutation of prognosis, Tumor mutations carry calculation are relevant outer aobvious Subregion include ABCA13, ABCA8, ABL1, ACADSB, ACOT13, ADAMTS6, ADRB1, ADSS, AGPAT9, AK7, AKT1, AKT2、AKT3、ALG9、ALK、ALK_fus、ALOX12B、ALS2CR11、AMER1、ANKRA2、ANKRD46、ANO1、APC、 APOPT1、AR、ARAF、ARHGAP4、ARHGAP6、ARID1A、ARID1B、ARID2、ARID4A、ARL6IP6、ARMC5、 ARPC2、ASH1L、ASXL1、ATM、ATP9B、ATR、ATRX、AURKA、AURKB、AXIN1、AXL、B2M、BAP1、BARD1、 BAT-25、BAT-26、BCL2、BCL2L1、BCOR、BCYRN1、BLM、BRAF、BRCA1、BRCA2、BRD4、BRIP1、BRS3、 BTF3、BTG1、BTK、C20orf96、C22orf23、C5orf42、C9orf72、CAB39、CALD1、CALM2、CALR、 CARD11、CASP8、CAST,ERAP1、CBFB、CBL、CBR4、CCND1、CCND2、CCND3、CCNE1、CCR4、CD274、 CD40、CD74_fus、CD79A、CD79B、CDC25B、CDC73、CDH1、CDK12、CDK4、CDK6、CDK7、CDK8、CDKL3、 CDKN1A、CDKN1B、CDKN2A、CDKN2B、CDKN2C、CDO1、CEBPA、CEP120、CHD1、CHEK1、CHEK2、CIC、 CNKSR3、CNOT8、COSM1316144、COSM1316145、COSM1332498、COSM3676668、COSM3747491、 COSM5045664、COX18、CREBBP、CRKL、CRLF2、CSF1R、CSF3R、CTAGE5、CTCF、CTNNB1、CUL3、 CXCR4、CYFIP1、CYLD、DAXX、DBT、DDR2、DEPDC5、DIAPH1、DICER1、DIS3、DNMT3A、DOCK11、 DOT1L、DROSHA、DSCAM、DXS6804、DXS7132、DXS7423、DYS391、DYS438、DYS458、EGFR、EGFR_ fus、EIF4G3、EML4_fus、EP300、EPHA3、EPHA5、EPHA7、EPHB1、ERBB2、ERBB3、ERBB4、ERCC4、 ERG、ERI1、ERRFI1、ESR1、ETV1、ETV6、ETV6_fus、EWSR1_fus、EXOSC8、EZH2、EZR_fus、F13A1、 FAM149A、FAM153B、FAM46C、FANCA、FANCC、FANCD2、FANCG、FANCI、FAS、FAT1、FBXW7、FGF16、 FGF19、FGF3、FGF4、FGFR1、FGFR2、FGFR2_fus、FGFR3、FGFR4、FH、FLCN、FLOT1、FLT1、FLT3、 FLT4、FMO1、FOLH1、FOXL2、FOXP1、FRAS1、FUBP1、FUS_fus、GABRP、GANC、GATA1、GATA2、GATA3、 GLI1、GMEB1、GNA11、GNA13、GNAQ、GNAS、GPM6A、GRIN2A、GSK3B、GSTM1、H3F3A、HAUS2、HCAR2、 HEY1_fus、HGF、HLA-A、HLA-B、HLA-C、HNF1A、HNRNPH1、HRAS、HSPA1B、HSPA4、HYOU1、IARS、 ID2、ID3、IDH1、IDH2、IGF1R、IGF2、IKBKE、IKZF1、IL7R、IL8、INHBA、INPP4B、IPO7、IRAK1、 IRF4、IRF6、IRF8、IRS2、ITGAL、JAK1、JAK2、JAK3、JUN、KCNJ2、KDM5A、KDM5C、KDM6A、KDR、 KEAP1、KIAA1210、KIAA1432、KIF5B_fus、KIR3DX1、KIT、KMT2A、KMT2C、KMT2D、KPNA4、KPNB1、 KRAS、LAMA3、LEPR、LMO1、LOC100131626、LONRF3、LRRC34、LYN、MAGOHB、MALT1、MAP2K1、 MAP2K2、MAP2K4、MAP3K1、MAP3K4、MAP4K5、MAPK1、MAPKAP1、MARK2、MCL1、MDM2、MDM4、MED12、 MED19、MEF2B、MEIS1、MEN1、MET、MIR1273H、MITF、MLH1、MMP16、MONO-27、MOV10L1、MPL、 MRE11A、MRPL19、MSH2、MSH3、MSH6、MTF1、MTOR、MTRR、MUTYH、MYADM、MYC、MYCL、MYCN、MYD88、 MYO10、NAB1、NAB2_fus、NBN、NCOA6、NDUFS1、NEO1、NF1、NF2、NFE2L2、NFKBIA、NFXL1、NKX2-1、 NOTCH1、NOTCH2、NOTCH3、NPM1、NR-21、NR-24、NR4A3_fus、NRAS、NSD1、NT5C2、NTRK1、NTRK1_ fus、NTRK2、NTRK2_fus、NTRK3、NTRK3_fus、NUP85、NUP93、P2RY8、PALB2、PAPOLG、PAQR8、 PARK2、PARP1、PAX5、PBRM1、PDCD1、PDCD1LG2、PDE6C、PDGFB_fus、PDGFRA、PDGFRB、PDPN、 PIGF、PIK3C2G、PIK3CA、PIK3CB、PIK3CG、PIK3R1、PIK3R2、PLCG2、PLEKHA1、PLEKHH2、PMS2、 PNO1、POLD1、POLE、PPARG、PPHLN1_fus、PPP2R1A、PRDM1、PREX2、PRKAR1A、PRKCI、PRPF39、 PTCH1、PTEN、PTPN11、PTPRJ、PURA、RABGAP1L、RAC1、RAD21、RAD50、RAD51、RAD51B、RAD51C、 RAD51D、RAD52、RAD54L、RAF1、RARA、RB1、RBM10、RBM27、REL、RET、RET_fus、RHOT1、RICTOR、 RIPK2、RNF19A、RNF43、ROS1、ROS1_fus、RPA4、RPTOR、RRP1B、RUNX1、RYR2、SASH1、SDHA、SDHB、 SDHC、SDHD、SEL1L3、SETD2、SF3B1、SHROOM3、SIPA1L2、SLC34A2_fus、SMAD2、SMAD3、SMAD4、 SMARCA4、SMARCB1、SMO、SNX6、SOCS1、SOX2、SOX9、SPC24、SPEN、SPOP、SRC、SS18_fus、STAG2、 STAT3、STK11、STMN1、STRBP、SUCLG1、SUFU、SUGCT、SYK、TAF15、TAGAP、TBC1D8B、TBX3、 TECPR2、TERT、TET2、TGFBR2、TMEM67、TMPRSS15、TMPRSS2、TNFAIP3、TNFRSF14、TNFSF13B、 TNKS、TNRC18、TOP1、TOP2B、TP53、TPH1、TRA2A、TRIM24、TSC1、TSC2、TSHR、TSN、TXNRD1、 U2AF1、UBE2E3、UBE3C、ULK4、UPF2、UTY、VEGFA、VHL、VSIG10、WHSC1、WHSC1L1、WT1、WWC3、 XPO1, ZBBX, ZDHHC17, ZNF711, ZNF805 and ZZZ3.Said gene joint-detection can make general cancer kind parting, treatment, The detection of the relevant gene mutation of prognosis, the relevant exon region of Tumor mutations carry calculation is more accurate.
Preferably, microsatellite instability site include rs1045642, rs1052555, rs10981694, rs11045585、rs1105525、rs1130214、rs1138272、rs115232898、rs11572080、rs11598702、 rs11615、rs12613732、rs12762549、rs13181、rs151264360、rs1517114、rs1570360、 rs1650697、rs1650723、rs1695、rs1799793、rs1801019、rs1801131、rs1801133、rs1801265、 rs1805087、rs183484、rs1934951、rs2032582、rs2072671、rs2075252、rs2228001、 rs2273618、rs2291075、rs2291767、rs2297595、rs2494752、rs2501873、rs25487、 rs2784917、rs28738963、rs316019、rs3212986、rs3745274、rs3892097、rs3918290、 rs3957357、rs4148323、rs4244285、rs430397、rs442767、rs4646、rs4673、rs4673993、 rs4728709、rs4880、rs50872、rs55886062、rs60369023、rs67376798、rs699947、rs716274、 Rs7779029, rs7851395, rs8133052, rs8175347, rs873478, rs9679162 and rs9937.It can be further It is abundant to detect obtained effective information, improve the accuracy of follow-up data application.
According to another aspect of the present invention, provide it is a kind of based on the sequencing of two generations for the targeting of general cancer kind, chemotherapy and exempt from The detection kit of epidemic disease medication.The detection kit includes detection probe, and detection probe is directed to general cancer kind parting, treatment, prognosis Relevant gene, microsatellite instability site and the relevant exon region of Tumor mutations carry calculation.Preferably, detection is visited Needle overlay area/target area >=99%.
A kind of typical embodiment according to the present invention, general cancer kind parting, treatment, the relevant gene mutation of prognosis and tumour Mutational load calculate relevant exon region include: ABCA13, ABCA8, ABL1, ACADSB, ACOT13, ADAMTS6, ADRB1、ADSS、AGPAT9、AK7、AKT1、AKT2、AKT3、ALG9、ALK、ALK_fus、ALOX12B、ALS2CR11、AMER1、 ANKRA2、ANKRD46、ANO1、APC、APOPT1、AR、ARAF、ARHGAP4、ARHGAP6、ARID1A、ARID1B、ARID2、 ARID4A、ARL6IP6、ARMC5、ARPC2、ASH1L、ASXL1、ATM、ATP9B、ATR、ATRX、AURKA、AURKB、AXIN1、 AXL、B2M、BAP1、BARD1、BAT-25、BAT-26、BCL2、BCL2L1、BCOR、BCYRN1、BLM、BRAF、BRCA1、 BRCA2、BRD4、BRIP1、BRS3、BTF3、BTG1、BTK、C20orf96、C22orf23、C5orf42、C9orf72、CAB39、 CALD1、CALM2、CALR、CARD11、CASP8、CAST,ERAP1、CBFB、CBL、CBR4、CCND1、CCND2、CCND3、 CCNE1、CCR4、CD274、CD40、CD74_fus、CD79A、CD79B、CDC25B、CDC73、CDH1、CDK12、CDK4、CDK6、 CDK7、CDK8、CDKL3、CDKN1A、CDKN1B、CDKN2A、CDKN2B、CDKN2C、CDO1、CEBPA、CEP120、CHD1、 CHEK1、CHEK2、CIC、CNKSR3、CNOT8、COSM1316144、COSM1316145、COSM1332498、COSM3676668、 COSM3747491、COSM5045664、COX18、CREBBP、CRKL、CRLF2、CSF1R、CSF3R、CTAGE5、CTCF、 CTNNB1、CUL3、CXCR4、CYFIP1、CYLD、DAXX、DBT、DDR2、DEPDC5、DIAPH1、DICER1、DIS3、DNMT3A、 DOCK11、DOT1L、DROSHA、DSCAM、DXS6804、DXS7132、DXS7423、DYS391、DYS438、DYS458、EGFR、 EGFR_fus、EIF4G3、EML4_fus、EP300、EPHA3、EPHA5、EPHA7、EPHB1、ERBB2、ERBB3、ERBB4、 ERCC4、ERG、ERI1、ERRFI1、ESR1、ETV1、ETV6、ETV6_fus、EWSR1_fus、EXOSC8、EZH2、EZR_fus、 F13A1、FAM149A、FAM153B、FAM46C、FANCA、FANCC、FANCD2、FANCG、FANCI、FAS、FAT1、FBXW7、 FGF16、FGF19、FGF3、FGF4、FGFR1、FGFR2、FGFR2_fus、FGFR3、FGFR4、FH、FLCN、FLOT1、FLT1、 FLT3、FLT4、FMO1、FOLH1、FOXL2、FOXP1、FRAS1、FUBP1、FUS_fus、GABRP、GANC、GATA1、GATA2、 GATA3、GLI1、GMEB1、GNA11、GNA13、GNAQ、GNAS、GPM6A、GRIN2A、GSK3B、GSTM1、H3F3A、HAUS2、 HCAR2、HEY1_fus、HGF、HLA-A、HLA-B、HLA-C、HNF1A、HNRNPH1、HRAS、HSPA1B、HSPA4、HYOU1、 IARS、ID2、ID3、IDH1、IDH2、IGF1R、IGF2、IKBKE、IKZF1、IL7R、IL8、INHBA、INPP4B、IPO7、 IRAK1、IRF4、IRF6、IRF8、IRS2、ITGAL、JAK1、JAK2、JAK3、JUN、KCNJ2、KDM5A、KDM5C、KDM6A、 KDR、KEAP1、KIAA1210、KIAA1432、KIF5B_fus、KIR3DX1、KIT、KMT2A、KMT2C、KMT2D、KPNA4、 KPNB1、KRAS、LAMA3、LEPR、LMO1、LOC100131626、LONRF3、LRRC34、LYN、MAGOHB、MALT1、 MAP2K1、MAP2K2、MAP2K4、MAP3K1、MAP3K4、MAP4K5、MAPK1、MAPKAP1、MARK2、MCL1、MDM2、MDM4、 MED12、MED19、MEF2B、MEIS1、MEN1、MET、MIR1273H、MITF、MLH1、MMP16、MONO-27、MOV10L1、 MPL、MRE11A、MRPL19、MSH2、MSH3、MSH6、MTF1、MTOR、MTRR、MUTYH、MYADM、MYC、MYCL、MYCN、 MYD88、MYO10、NAB1、NAB2_fus、NBN、NCOA6、NDUFS1、NEO1、NF1、NF2、NFE2L2、NFKBIA、NFXL1、 NKX2-1、NOTCH1、NOTCH2、NOTCH3、NPM1、NR-21、NR-24、NR4A3_fus、NRAS、NSD1、NT5C2、NTRK1、 NTRK1_fus、NTRK2、NTRK2_fus、NTRK3、NTRK3_fus、NUP85、NUP93、P2RY8、PALB2、PAPOLG、 PAQR8、PARK2、PARP1、PAX5、PBRM1、PDCD1、PDCD1LG2、PDE6C、PDGFB_fus、PDGFRA、PDGFRB、 PDPN、PIGF、PIK3C2G、PIK3CA、PIK3CB、PIK3CG、PIK3R1、PIK3R2、PLCG2、PLEKHA1、PLEKHH2、 PMS2、PNO1、POLD1、POLE、PPARG、PPHLN1_fus、PPP2R1A、PRDM1、PREX2、PRKAR1A、PRKCI、 PRPF39、PTCH1、PTEN、PTPN11、PTPRJ、PURA、RABGAP1L、RAC1、RAD21、RAD50、RAD51、RAD51B、 RAD51C、RAD51D、RAD52、RAD54L、RAF1、RARA、RB1、RBM10、RBM27、REL、RET、RET_fus、RHOT1、 RICTOR、RIPK2、RNF19A、RNF43、ROS1、ROS1_fus、RPA4、RPTOR、RRP1B、RUNX1、RYR2、SASH1、 SDHA、SDHB、SDHC、SDHD、SEL1L3、SETD2、SF3B1、SHROOM3、SIPA1L2、SLC34A2_fus、SMAD2、 SMAD3、SMAD4、SMARCA4、SMARCB1、SMO、SNX6、SOCS1、SOX2、SOX9、SPC24、SPEN、SPOP、SRC、 SS18_fus、STAG2、STAT3、STK11、STMN1、STRBP、SUCLG1、SUFU、SUGCT、SYK、TAF15、TAGAP、 TBC1D8B、TBX3、TECPR2、TERT、TET2、TGFBR2、TMEM67、TMPRSS15、TMPRSS2、TNFAIP3、 TNFRSF14、TNFSF13B、TNKS、TNRC18、TOP1、TOP2B、TP53、TPH1、TRA2A、TRIM24、TSC1、TSC2、 TSHR、TSN、TXNRD1、U2AF1、UBE2E3、UBE3C、ULK4、UPF2、UTY、VEGFA、VHL、VSIG10、WHSC1、 WHSC1L1, WT1, WWC3, XPO1, ZBBX, ZDHHC17, ZNF711, ZNF805 and ZZZ3.
A kind of typical embodiment according to the present invention, microsatellite instability site include rs1045642, rs1052555、rs10981694、rs11045585、rs1105525、rs1130214、rs1138272、rs115232898、 rs11572080、rs11598702、rs11615、rs12613732、rs12762549、rs13181、rs151264360、 rs1517114、rs1570360、rs1650697、rs1650723、rs1695、rs1799793、rs1801019、rs1801131、 rs1801133、rs1801265、rs1805087、rs183484、rs1934951、rs2032582、rs2072671、 rs2075252、rs2228001、rs2273618、rs2291075、rs2291767、rs2297595、rs2494752、 rs2501873、rs25487、rs2784917、rs28738963、rs316019、rs3212986、rs3745274、 rs3892097、rs3918290、rs3957357、rs4148323、rs4244285、rs430397、rs442767、rs4646、 rs4673、rs4673993、rs4728709、rs4880、rs50872、rs55886062、rs60369023、rs67376798、 rs699947、rs716274、rs7779029、rs7851395、rs8133052、rs8175347、rs873478、rs9679162 And rs9937.
It is a kind of according to the present invention typically above-mentioned general cancer kind target to be used for based on the sequencing of two generations embodiment there is provided a kind of To, chemotherapy and the detection panel or above-mentioned of immune medication based on two generations be sequenced for the targeting of general cancer kind, chemotherapy and immune medication Detection kit preparation be used for general cancer kind parting, treatment, the relevant gene mutation of prognosis, microsatellite instability site and Application in the device of the relevant exon region joint-detection of Tumor mutations carry calculation.Typically, general cancer kind includes but not It is limited to: lung cancer, intestinal cancer, gastric cancer, liver cancer, breast cancer and carcinoma of endometrium etc..Joint-detection may include various mutations type Detection, comprising: point mutation, fusion, copy number variation, missing and insertion mutation；Preferably, using including targeting medicine, chemotherapeutic Or the adjoint diagnosis of immune substance.
Beneficial effects of the present invention are further illustrated below in conjunction with embodiment.
Embodiment 1
The panel of the present embodiment includes general cancer kind parting, treatment, the relevant gene mutation of prognosis, microsatellite instability Site and the relevant exon region of Tumor mutations carry calculation.Corresponding, the kit of the present embodiment includes detection probe, inspection Probing needle is directed to general cancer kind parting, treatment, the relevant gene mutation of prognosis, microsatellite instability site and Tumor mutations load Calculate relevant exon region, detection probe overlay area/target area >=99%.Detection probe is according to the normal of this field Rule design rule designs.
Wherein, general cancer kind parting, treatment, the relevant gene mutation of prognosis and the relevant exon of Tumor mutations carry calculation Region include: ABCA13, ABCA8, ABL1, ACADSB, ACOT13, ADAMTS6, ADRB1, ADSS, AGPAT9, AK7, AKT1, AKT2、AKT3、ALG9、ALK、ALK_fus、ALOX12B、ALS2CR11、AMER1、ANKRA2、ANKRD46、ANO1、APC、 APOPT1、AR、ARAF、ARHGAP4、ARHGAP6、ARID1A、ARID1B、ARID2、ARID4A、ARL6IP6、ARMC5、 ARPC2、ASH1L、ASXL1、ATM、ATP9B、ATR、ATRX、AURKA、AURKB、AXIN1、AXL、B2M、BAP1、BARD1、 BAT-25、BAT-26、BCL2、BCL2L1、BCOR、BCYRN1、BLM、BRAF、BRCA1、BRCA2、BRD4、BRIP1、BRS3、 BTF3、BTG1、BTK、C20orf96、C22orf23、C5orf42、C9orf72、CAB39、CALD1、CALM2、CALR、 CARD11、CASP8、CAST,ERAP1、CBFB、CBL、CBR4、CCND1、CCND2、CCND3、CCNE1、CCR4、CD274、 CD40、CD74_fus、CD79A、CD79B、CDC25B、CDC73、CDH1、CDK12、CDK4、CDK6、CDK7、CDK8、CDKL3、 CDKN1A、CDKN1B、CDKN2A、CDKN2B、CDKN2C、CDO1、CEBPA、CEP120、CHD1、CHEK1、CHEK2、CIC、 CNKSR3、CNOT8、COSM1316144、COSM1316145、COSM1332498、COSM3676668、COSM3747491、 COSM5045664、COX18、CREBBP、CRKL、CRLF2、CSF1R、CSF3R、CTAGE5、CTCF、CTNNB1、CUL3、 CXCR4、CYFIP1、CYLD、DAXX、DBT、DDR2、DEPDC5、DIAPH1、DICER1、DIS3、DNMT3A、DOCK11、 DOT1L、DROSHA、DSCAM、DXS6804、DXS7132、DXS7423、DYS391、DYS438、DYS458、EGFR、EGFR_ fus、EIF4G3、EML4_fus、EP300、EPHA3、EPHA5、EPHA7、EPHB1、ERBB2、ERBB3、ERBB4、ERCC4、 ERG、ERI1、ERRFI1、ESR1、ETV1、ETV6、ETV6_fus、EWSR1_fus、EXOSC8、EZH2、EZR_fus、F13A1、 FAM149A、FAM153B、FAM46C、FANCA、FANCC、FANCD2、FANCG、FANCI、FAS、FAT1、FBXW7、FGF16、 FGF19、FGF3、FGF4、FGFR1、FGFR2、FGFR2_fus、FGFR3、FGFR4、FH、FLCN、FLOT1、FLT1、FLT3、 FLT4、FMO1、FOLH1、FOXL2、FOXP1、FRAS1、FUBP1、FUS_fus、GABRP、GANC、GATA1、GATA2、GATA3、 GLI1、GMEB1、GNA11、GNA13、GNAQ、GNAS、GPM6A、GRIN2A、GSK3B、GSTM1、H3F3A、HAUS2、HCAR2、 HEY1_fus、HGF、HLA-A、HLA-B、HLA-C、HNF1A、HNRNPH1、HRAS、HSPA1B、HSPA4、HYOU1、IARS、 ID2、ID3、IDH1、IDH2、IGF1R、IGF2、IKBKE、IKZF1、IL7R、IL8、INHBA、INPP4B、IPO7、IRAK1、 IRF4、IRF6、IRF8、IRS2、ITGAL、JAK1、JAK2、JAK3、JUN、KCNJ2、KDM5A、KDM5C、KDM6A、KDR、 KEAP1、KIAA1210、KIAA1432、KIF5B_fus、KIR3DX1、KIT、KMT2A、KMT2C、KMT2D、KPNA4、KPNB1、 KRAS、LAMA3、LEPR、LMO1、LOC100131626、LONRF3、LRRC34、LYN、MAGOHB、MALT1、MAP2K1、 MAP2K2、MAP2K4、MAP3K1、MAP3K4、MAP4K5、MAPK1、MAPKAP1、MARK2、MCL1、MDM2、MDM4、MED12、 MED19、MEF2B、MEIS1、MEN1、MET、MIR1273H、MITF、MLH1、MMP16、MONO-27、MOV10L1、MPL、 MRE11A、MRPL19、MSH2、MSH3、MSH6、MTF1、MTOR、MTRR、MUTYH、MYADM、MYC、MYCL、MYCN、MYD88、 MYO10、NAB1、NAB2_fus、NBN、NCOA6、NDUFS1、NEO1、NF1、NF2、NFE2L2、NFKBIA、NFXL1、NKX2-1、 NOTCH1、NOTCH2、NOTCH3、NPM1、NR-21、NR-24、NR4A3_fus、NRAS、NSD1、NT5C2、NTRK1、NTRK1_ fus、NTRK2、NTRK2_fus、NTRK3、NTRK3_fus、NUP85、NUP93、P2RY8、PALB2、PAPOLG、PAQR8、 PARK2、PARP1、PAX5、PBRM1、PDCD1、PDCD1LG2、PDE6C、PDGFB_fus、PDGFRA、PDGFRB、PDPN、 PIGF、PIK3C2G、PIK3CA、PIK3CB、PIK3CG、PIK3R1、PIK3R2、PLCG2、PLEKHA1、PLEKHH2、PMS2、 PNO1、POLD1、POLE、PPARG、PPHLN1_fus、PPP2R1A、PRDM1、PREX2、PRKAR1A、PRKCI、PRPF39、 PTCH1、PTEN、PTPN11、PTPRJ、PURA、RABGAP1L、RAC1、RAD21、RAD50、RAD51、RAD51B、RAD51C、 RAD51D、RAD52、RAD54L、RAF1、RARA、RB1、RBM10、RBM27、REL、RET、RET_fus、RHOT1、RICTOR、 RIPK2、RNF19A、RNF43、ROS1、ROS1_fus、RPA4、RPTOR、RRP1B、RUNX1、RYR2、SASH1、SDHA、SDHB、 SDHC、SDHD、SEL1L3、SETD2、SF3B1、SHROOM3、SIPA1L2、SLC34A2_fus、SMAD2、SMAD3、SMAD4、 SMARCA4、SMARCB1、SMO、SNX6、SOCS1、SOX2、SOX9、SPC24、SPEN、SPOP、SRC、SS18_fus、STAG2、 STAT3、STK11、STMN1、STRBP、SUCLG1、SUFU、SUGCT、SYK、TAF15、TAGAP、TBC1D8B、TBX3、 TECPR2、TERT、TET2、TGFBR2、TMEM67、TMPRSS15、TMPRSS2、TNFAIP3、TNFRSF14、TNFSF13B、 TNKS、TNRC18、TOP1、TOP2B、TP53、TPH1、TRA2A、TRIM24、TSC1、TSC2、TSHR、TSN、TXNRD1、 U2AF1、UBE2E3、UBE3C、ULK4、UPF2、UTY、VEGFA、VHL、VSIG10、WHSC1、WHSC1L1、WT1、WWC3、 XPO1, ZBBX, ZDHHC17, ZNF711, ZNF805 and ZZZ3.
Microsatellite instability site include rs1045642, rs1052555, rs10981694, rs11045585, rs1105525、rs1130214、rs1138272、rs115232898、rs11572080、rs11598702、rs11615、 rs12613732、rs12762549、rs13181、rs151264360、rs1517114、rs1570360、rs1650697、 rs1650723、rs1695、rs1799793、rs1801019、rs1801131、rs1801133、rs1801265、rs1805087、 rs183484、rs1934951、rs2032582、rs2072671、rs2075252、rs2228001、rs2273618、 rs2291075、rs2291767、rs2297595、rs2494752、rs2501873、rs25487、rs2784917、 rs28738963、rs316019、rs3212986、rs3745274、rs3892097、rs3918290、rs3957357、 rs4148323、rs4244285、rs430397、rs442767、rs4646、rs4673、rs4673993、rs4728709、 rs4880、rs50872、rs55886062、rs60369023、rs67376798、rs699947、rs716274、rs7779029、 Rs7851395, rs8133052, rs8175347, rs873478, rs9679162 and rs9937.
Embodiment 2
It is detected using standard items of the kit in the present embodiment 1 to simulated tissue DNA.
One, validation criteria product are prepared
By mixing the tumor cell line DNA containing known mutations and wild-type cell system GM12878DNA in proportion, match Produce 20 kinds of mutation standard items DNA.And the frequency of every kind of mutation in standard items is determined by ddPCR.Tumor cell line DNA such as table Shown in 1.
Table 1
Two, DNA is interrupted:
DNA is quantified using Qubit 3.0 and dsDNA HS Assay Kit.
Polytetrafluoroethylene wire is cut with the medical scissors after ultraviolet sterilization to the length of 1cm or so, and guarantees to interrupt stick Length homogeneity it is good, be placed in clean container, ultraviolet sterilization 3~4 hours.After the completion of sterilizing, by the polytetrafluoroethylene (PTFE) of 1cm Line is put into 96 orifice plates with the tweezers after sterilizing.Each hole is packed into 2 and interrupts stick, after the completion again by 96 orifice plate ultraviolet sterilizations 3~ 4 hours.
300ng DNA sample is taken according to qubit quantitative result, 50 μ l is diluted to using TE, is transferred in 96 orifice plates, by tin Foil paper film is placed on 96 orifice plates, and the alignment of four sides using 180 DEG C of heat-sealing film instrument 5s sealer 2 times, is centrifuged using Microplate centrifuge.
Preset program Peak Power:450, Duty Factor:30, Cycles/Burst:200 are selected, Treatment time:40s, 3cycles are clicked " Start position ".In Run interface point " Run " button, run journey Sequence.After the completion of program operation, sample panel is taken out, is centrifuged using Microplate centrifuge, then sample panel is put on specimen holder, Option program Peak Power:450, Duty Factor:30, Cycles/Burst:200, Treatment time:40s, 4cycles.In Run interface point " Run " button, program is run.After the completion of program operation, sample panel is taken out, micropore is used The centrifugation of plate centrifuge.1 μ l is taken to carry out quality inspection after interrupting.
Three, library construction
1. end repairs and adds A tail in 3 ' ends:
1.1 take DNA obtained in 50 μ L above-mentioned steps two, the use nuclease-free water polishing less than 50 μ L to 50 μ L, according to Reaction system is added in the following table 2:
Table 2
Component Volume
Repair and add A buffer in end 7μL
Repair and add A enzyme in end 3μL
DNA 50μL
Total volume 60μL
1.2, which are vortexed, mixes, and micro- centrifugation is placed in PCR instrument, response procedures such as the following table 3:
Table 3
2. jointing:
2.1 connectors prepare: 2.5 μ L of connector adds 2.5ul water to be diluted to 5 μ L.
Corresponding reagent is added into above-mentioned reaction tube in 2.2 accordings to the form below 4:
Table 4
Component Volume
Water 5μL
Connect buffer 30μL
DNA ligase 10μL
Repair and add A product in end 60μL
Total volume 110μL
2.3, which are vortexed, mixes, and micro- centrifugation is placed in PCR instrument, response procedures such as the following table 5:
Table 5
Step Temperature Time
Jointing 20℃ 30min
It terminates 20℃ ∞
Note: hot lid temperature is 50 DEG C
3. being purified after connection:
3.1 packing Beckman Agencourt AMPure XP magnetic beads are into eight new connecting legs, every 88 μ L of pipe.Previous step It is 2.3 end after PCR, takes out sample, of short duration centrifugation is transferred in the 88 μ L magnetic bead centrifuge tubes dispensed.
3.2 concussions mix, and are incubated at room temperature 15min, combine DNA sufficiently with magnetic bead.Note: by tight pipe lid when concussion.It is short Temporarily centrifugation, centrifuge tube is placed on magnetic frame to be clarified to liquid, is discarded supernatant and (is guaranteed that residual volume must not exceed 5 μ L).Note: should not It is drawn onto magnetic bead.
3.3 addition 200 μ L, 80% ethyl alcohol discard after being incubated for 30sec.It is repeated once 200 μ L, 80% ethyl alcohol cleaning step. Note: 80% ethyl alcohol matching while using.
3.4 exhaust the residual ethanol of centrifugation bottom of the tube with 10 μ L pipette tips, and drying at room temperature 3-5min to ethyl alcohol volatilizees (just completely Face sees that the back side, which is seen, have been dried not reflective).Note: magnetic bead overdrying DNA output can be reduced.
3.5 remove centrifuge tube from magnetic frame, and 21 μ L ultrapure waters are added, and concussion mixes.Note: by tight pipe lid when concussion.Room Temperature is incubated for 5min.
3.6 of short duration centrifugations, centrifuge tube is placed on magnetic frame to be clarified to liquid.Remaining 20 μ L clear liquid is transferred to new PCR Pipe carries out amplification test in next step.
4. amplified library:
4.1 are added reaction system according to the following table 6:
Table 6
Component Volume
Thermal starting enzyme 25μL
Primer and reaction buffer mixture 5μL
Connector connection product 20μL
Total volume 50μL
4.2, which are vortexed, mixes, and micro- centrifugation is placed in PCR instrument, response procedures such as the following table 7:
Table 7
The acquisition of 5.DNA
5.1 25 μ L Beckman Agencourt AMPure XP magnetic beads of packing are into eight new connecting legs.
After 5.2 to previous step (4.2) PCR, sample is taken out.
5.3 of short duration centrifugations are transferred in the 25 μ L Beckman Agencourt AMPure XP magnetic beads dispensed.
5.4 concussions mix, and are incubated at room temperature 15min, combine DNA sufficiently with magnetic bead.By tight pipe lid when paying attention to shaking.
5.5 of short duration centrifugations, centrifuge tube is placed on magnetic frame to be clarified to liquid, and supernatant is transferred to 25 that another pipe has dispensed In μ L Beckman Agencourt AMPure XP magnetic bead.Note: not be drawn onto magnetic bead.
5.6 concussions mix, and are incubated at room temperature 15min, combine DNA sufficiently with magnetic bead, by tight pipe lid when paying attention to shaking.
5.7 of short duration centrifugations, centrifuge tube is placed on magnetic frame to be clarified to liquid, abandons supernatant.Note: not be drawn onto magnetic bead.
5.8 addition 200 μ L, 80% ethyl alcohol discard after being incubated for 30sec.Note: 80% ethyl alcohol matching while using.It is repeated once 200 μ L80% ethyl alcohol cleaning steps.
5.9 exhaust the residual ethanol of centrifugation bottom of the tube with 10 μ L pipette tips, and drying at room temperature 3-5min to ethyl alcohol volatilizees (just completely Face sees that the back side, which is seen, have been dried not reflective).Note: magnetic bead overdrying DNA output can be reduced.
5.10 remove centrifuge tube from magnetic frame, and 40 μ L ultrapure waters are added, and oscillation mixes.
5.11 incubation at room temperature 5min eluted dnas.
5.12 of short duration centrifugations, centrifuge tube is placed on magnetic frame to be clarified to liquid, library is transferred in new centrifuge tube.It protects It is stored in -20 DEG C.
6. library quality inspection
1 μ L DNA library is taken to detect its concentration with dsDNA HS Assay Kit.
The capture of four, Library hybridizations
Library hybridization capture is carried out using present invention detection panel and hybrid capture reagent, operating process is said according to product Bright book carries out.
1. taking the equivalent library of 1 μ g of total amount in 1.5mL centrifuge tube, according to the concentration in each library and capture library number Calculate the volume that each library is added.The volume that library is added is: (1000ng/ captures library number/library concentration) μ L.Upwards It states and 2.5 μ L closing oligonucleotide is added in system, 5 μ L COT Human DNA are added, concussion mixes, of short duration centrifugation.With envelope Membrana oralis seals EP pipe, is put into traditional vacuum concentrating instrument and is evaporated (60 DEG C, about 20min~1hr).Pay attention to checking whether to have steamed at any time It is dry.
2. library hybridizes with probe
2.1 walk upwards containing reaction system shown in the following table 8 is added in the dry powder such as library, are vortexed and mix, of short duration centrifugation is placed in 95 DEG C heating module is denaturalized 10min.
Table 8
Component Volume
Hybridization buffer 7.5μL
Hybridization enhancers 3μL
Total volume 10.5μL
2.2 take out 4.5ul probe, of short duration centrifugation；The of short duration centrifugation of probe is placed in 47 DEG C of PCR instruments, rapidly by denaturation DNA is transferred in the PCR pipe containing probe from 95 DEG C, and concussion mixes, of short duration centrifugation.It is placed in PCR instrument, 47 DEG C of hybridization, hybridization Time should be no less than 16hr.
3. library captures
3.1 prepare washing steps liquid, and the preparation method such as following table of a capture required buffer liquid is pressed according to the number of capture The following table 9 prepares buffer.
Table 9
3.2 prepare capture magnetic bead and washing steps liquid 1 and 4, and capture magnetic bead uses preceding palpus equilibrium at room temperature 30min；Elute work Make liquid 1 and 4 and uses 47 DEG C of incubation 2hr of preceding palpus.
3.3 each captures dispense 100 μ L and capture magnetic bead, and 100 μ L capture magnetic bead is placed in magnetic frame and is clarified up to liquid, is abandoned Remove supernatant.200 μ L 1X magnetic bead cleaning solutions are added, concussion mixes.It is placed in magnetic frame to clarify up to liquid, discard supernatant.It is added 200 μ L1X magnetic bead cleaning solutions, concussion mix.It is placed in magnetic frame to clarify up to liquid, discard supernatant.100 μ L 1X magnetic beads are added to wash Liquid is washed, concussion mixes.It is placed in magnetic frame to clarify up to liquid, discard supernatant.Magnetic bead pretreatment at this time is completed, and is carried out immediately next Step test.
3.4 are transferred to overnight hybridization liquid is captured in cleaned magnetic bead, and pipettor is blown and beaten ten times.It is placed in PCR instrument 47 DEG C of incubation 45min (PCR is warm, and lid temperature is set as 57 DEG C) shake primary guarantee magnetic bead every 15min and suspend.
3.5 cleanings:
3.5.1 after the completion of being incubated for, 1 × washing steps liquid 1 of 47 DEG C of 100 μ L preheatings is added in every pipe, and concussion mixes.It is placed in Magnetic frame is clarified up to liquid, is discarded supernatant.
3.5.2 1 × washing steps liquid 4 of 47 DEG C of 200 μ L preheatings is added, pipettor blows and beats ten mixings.47 DEG C of incubations 5min is placed in magnetic frame and clarifies up to liquid, discards supernatant.Notice that operating process avoids temperature lower than 47 DEG C as far as possible.
3.5.3 1 × washing steps liquid 4 of 47 DEG C of 200 μ L preheatings is added, pipettor blows and beats ten mixings.47 DEG C of incubations 5min is placed in magnetic frame and clarifies up to liquid, discards supernatant.Notice that operating process avoids temperature lower than 47 DEG C as far as possible.
3.5.4 1 × washing steps liquid 1 that 200 μ L are placed at room temperature for is added, vibrates 2min, of short duration centrifugation is placed on magnetic frame It clarifies, discards supernatant to liquid.
3.5.5 1 × washing steps liquid 2 that 200 μ L are placed at room temperature for is added, shakes 1min, of short duration centrifugation is placed on magnetic frame It clarifies, discards supernatant to liquid.
3.5.6 1 × washing steps liquid 3 that 200 μ L are placed at room temperature for is added, shakes 30sec, magnetic frame is placed in of short duration centrifugation It clarifies, discards supernatant up to liquid.
3.5.7 the 18 ultrapure water elutions of μ L are added into centrifuge tube, concussion mixes, and carries out amplification test in next step.
4. PCR after capture:
Reaction system is added in 4.1 accordings to the form below 10.
Table 10
Component Volume
Thermal starting enzyme 50μL
Primer, 5 μM 10μL
The DNA of previous step elution 40μL
Total volume 100μL
4.2, which are vortexed, mixes, and micro- centrifugation is placed in PCR instrument, response procedures such as the following table 11:
Table 11
It is purified after 4.3 amplifications:
4.3.1 purifying magnetic bead is taken out, equilibrium at room temperature 30min is spare.
4.3.2 take 180 μ L purifying magnetic bead in 1.5mL centrifuge tube, the capture dna library after 100 μ L amplification is added, oscillation It mixes, is incubated at room temperature 15min.
4.3.3 it is placed in magnetic frame to clarify up to liquid, discard supernatant.
4.3.4 it is added after 200 μ L, 80% ethyl alcohol is incubated for 30sec and discards.Note: 80% ethyl alcohol matching while using.It is repeated once 200 μ L80% ethyl alcohol cleaning steps.
4.3.5 the residual ethanol of centrifugation bottom of the tube is discarded with 10 μ L pipette tips, drying at room temperature to ethyl alcohol is volatilized completely, and (front is seen Magnetic bead is non-reflective, and drying is seen at the back side).Note: magnetic bead overdrying DNA output can be reduced.
4.3.6 centrifuge tube is removed from magnetic frame, 50 μ L ultrapure waters is added, oscillation mixes.It is incubated at room temperature 2min.
4.3.7 of short duration centrifugation is placed in magnetic frame and clarifies up to liquid, capture sample is transferred in new centrifuge tube.
4.3.8 quality inspection: take 1 μ L capture sample for Qubit Concentration Testing.Capture Concentration Testing uses Qubit HS detection kit.
Five, testing results
See Table 1 for details 2 for testing result.
Table 12
According to upper 12 result of table it is found that using ddPCR testing result as goldstandard, the detection panel of embodiment 1 is to tissue sample This point mutation, insertion and deletion, fusion and copy number variation detection performance it is superior.
Embodiment 3
CfDNA sample is carried out using the kit of the embodiment of the present invention 1 and builds library and capture.
One, validation criteria product are prepared
By mixing the tumor cell line DNA containing known mutations and wild-type cell system GM12878DNA in proportion, match Produce 20 kinds of mutation standard items DNA.And the frequency of every kind of mutation in standard items is determined by ddPCR.Tumor cell line DNA such as table Shown in 1.Standard items DNA is interrupted, the fragment length (~165bp) of cfDNA is simulated.
Two, library constructions
1. end repairs and adds A tail in 3 ' ends:
1.1 take 50 μ L cfDNA, the use nuclease-free water polishing less than 50 μ L to 50 μ L, add according to the table 2 in embodiment 2 Enter reaction system.
1.2, which are vortexed, mixes, and micro- centrifugation is placed in PCR instrument, the table 3 in response procedures such as embodiment 2.
2. jointing:
2.1 dilutions connector (connectors that 15 μM of concentration), the use volume of connector is 5 μ L.The preservation concentration of connector is 15 μM, According to the initial amount of DNA, the use concentration of 13 butt joint of according to the form below is diluted, and dilution is placed on 4 DEG C of refrigerators and saves.
Table 13
Build library initial amount (ng) Connector volume (μ L) Nuclease-Free ddH2O volume (μ L)
25-50 5 0
10-25 2.5 2.5
5-10 1 4
2.2 are added corresponding reagent into above-mentioned reaction tube by the table 4 in embodiment 2.
2.3, which are vortexed, mixes, and micro- centrifugation is placed in PCR instrument, and response procedures are as shown in the table 5 in embodiment 2.
3. purifying (specific steps referring to purified after the connection in embodiment 2) after connection.
4. amplified library (specific steps are referring to embodiment).
The acquisition (1 × Beads recycling) of 5.DNA
5.1 50 μ L Beckman Agencourt AMPure XP magnetic beads of packing are into eight new connecting legs.
After 5.2 to previous step (4.2) PCR, sample is taken out.
5.3 of short duration centrifugations are transferred in the 50 μ L Beckman Agencourt AMPure XP magnetic beads dispensed.
5.4 concussions mix, and are incubated at room temperature 15min, combine DNA sufficiently with magnetic bead.By tight pipe lid when paying attention to shaking.
5.5 of short duration centrifugations, centrifuge tube is placed on magnetic frame to be clarified to liquid, is discarded supernatant.Note: not be drawn onto magnetic bead.
5.6 addition 200 μ L, 80% ethyl alcohol discard after being incubated for 30sec.Note: 80% ethyl alcohol matching while using.It is repeated once 200 μ L80% ethyl alcohol cleaning steps.
5.7 exhaust the residual ethanol of centrifugation bottom of the tube with 10 μ L pipette tips, and drying at room temperature 3-5min to ethyl alcohol volatilizees (just completely Face sees that the back side, which is seen, have been dried not reflective).Note: magnetic bead overdrying DNA output can be reduced.
5.8 remove centrifuge tube from magnetic frame, and 40 μ L ultrapure waters are added, and oscillation mixes.
5.9 incubation at room temperature 5min eluted dnas.
5.10 of short duration centrifugations, centrifuge tube is placed on magnetic frame to be clarified to liquid, library is transferred in new centrifuge tube.It protects It is stored in -20 DEG C.
6. library quality inspection
1 μ L DNA library is taken to detect its concentration with dsDNA HS Assay Kit.
The capture of four, Library hybridizations
Concrete operations are the same as embodiment 2.
Five, testing results
See Table 1 for details 4 for testing result.
Table 14
According to upper table as a result, using ddPCR testing result as goldstandard, the detection kit of embodiment 1 is to cfDNA sample Point mutation, insertion and deletion, fusion and copy number variation detection performance it is superior.
Embodiment 4
Using the consistency of kit detection the tissue T MB and full exon kit of embodiment 1
1. the extraction of patient gene's group DNA, library preparation and probe hybrid capture
The paired sample that the present embodiment has chosen 40 patients detects, and uses Tiangeng haemocyte and tissue extraction reagent Box extracts haemocyte, tumour flesh tissue DNA, extracts tumour FFPE DNA, operation using Jena FFPE DNA extraction kit Process is carried out according to product description.It interrupts according to the progress of embodiment 2 DNA and is prepared with library, the reagent of embodiment 1 is respectively adopted Box and complete outer probe carry out hybrid capture and the sequencing of upper machine.Sample information is as shown in Table 15:
Table 15
Cancer kind includes: carcinoma of endometrium, low level serous adenocarcinoma, colon and rectum carcinoma, oophoroma, gastric cancer, second shape knot Intestinal cancer, breast cancer, cholangiocarcinoma, lung cancer, intestinal cancer, gastric cancer and glioma.
2. lower machine data analysis
Calculate TMB formula: in the i.e. every megabasse of the complete outer base number (depth > 100X after duplicate removal) of mutation quantity * 1000000/ The sum of displacement and insertion/deletion mutation occurs
Following situations will exclude TMB and calculate:
(1) germline mutation；
(2) frequency of mutation is lower than 5% mutation；
(3) same sense mutation；
(4) depth is lower than 100X after FFPE sample duplicate removal；
(5) repeat region is mutated；
(6) blacklist is mutated；
(7) driving mutation；
(8) demographic data library genetic mutation site.
3. testing result
The present embodiment has detected the pairing haemocyte and FFPE of 163 patients altogether, it is determined that TMB critical value, critical value knot Fruit is as shown in table 16:
Table 16
TMB-Moderate TMB-High TMB-Extra High
3.804 8.4278 13.7693
By 16 verification result of table it is found that mutation number, which is greater than 13.7693, TMB level, is judged as Extra High；Mutation Number is judged as High between 8.4278-13.7693.
The detection panel and full exon probe of embodiment 1 has been respectively adopted in the present embodiment, has detected 40 patients' altogether Match haemocyte and FFPE, it is determined that the consistency of the detection panel and full exon probe of embodiment 1, consistency result is such as Shown in Fig. 1.
By Fig. 1 result it is found that the detection panel (P12) of embodiment 1 and the consistency of full exon probe are good, the two one Cause property R2=0.9411.
The present embodiment is inserted for 20 point mutation of Tumor mutations load (TMB) exon region designed while covering, 4 Enter and merged with 8, by integrating the shared region of different mutation type information, improves panel homogeneity, it is whole to reduce panel Data volume shortens analytical cycle, reduces cost.
Embodiment 5
The consistency of detection panel the detection MSI and generation method of embodiment 1
1. the extraction of patient gene's group DNA, library preparation and probe hybrid capture
Puncturing tissue, surgical tissue or paraffin section extract DNA, take 200ng DNA to carry out covaris and interrupt, interrupt Sample afterwards constructs library, and using the 12 hybrid capture library panel, the sample after the completion of capturing is in Illumina NovaSeq It is sequenced on platform.Processing sequence data are analyzed, which is intended to accurately detect microsatellite instability (MSI).
2. lower machine data analysis
MSI QC standard: depth > 100X after each MSI marker duplicate removal.
Panel MSI of 24 mononucleotide repeating label objects for embodiment 1 is measured.If a sample passes through QC's Marker quantity is less than 8, then MSI state is QNS.If unstable locus/pass through locus >=0.3, MSI shape State is HSI-H；If unstable locus/pass through site < 0.3, MSI state as HSI-L/MSS.MSI marker is shown in Table 17。
Table 17
3. testing result
The detection panel of embodiment 1 is detected suitable for MSI, and 140 blood cell samples are selected for MSI marker.It comes from 30 blood cell samples of healthy donors are established for p12MSI baseline.Pass through fluorescence pcr- Capillary Electrophoresis (MSIAnalysis System, Version 1.2, Promega), 40 clinical tumor tissue MSI states are it is known that this 40 Sample carries out GeneCast Panel 12MSI verifying.Verification result shows there is 14 MSI-High, 25 MSI-Low/MSS With 1 QNS.Specifying information is shown in Table 18.
Table 18
Table 19
NGS/PCR MSI-H_PCR MSI-L/MSS_PCR
MSI-H_NGS 14 0
MSI-L/MSS_NGS 0 25
QNS_NGS 1 0
Table 20
TP TN FP FN Sensitivity Specificity
14 25 0 0 100% 100%
It is total for the MSI verification method and fluorescent PCR-capillary electrophoresis method consistency of the detection panel of embodiment 1 Knot such as table 19 and table 20.Sensitivity is 100%, and specificity is 98.2%, PPV 94.7%, accuracy 98.7%.
Embodiment 6
The consistency of result is commented in the detection panel detection mutation of embodiment 1 with room interstitial
1. Room interstitial comments sample information table as shown in table 21:
Table 21
2. Room interstitial comments the library of sample to prepare and probe hybrid capture
23 samples (14 CF and 9 FFPE) of the present embodiment carry out DNA extraction according to embodiment 2 and embodiment 3, beat Disconnected (FFPE sample is interrupted, and CF sample does not interrupt), library preparation, probe hybrid capture and the sequencing of upper machine.
3. testing result is shown in Table 22.
Table 22
Continued 22
According to upper table 22 as a result, all mutation have detected, meet verification result.Illustrate that the detection panel of embodiment 1 is quasi- Exactness is high.
Embodiment 7
Kit energy one-time detection kinds of tumors variation of the invention, testing result is accurate, provides patient and targets medicine, chemotherapy The medication of medicine and immune substance three classes drug selects, and reduces expense.
The detection adjoint diagnosis of panel clinical application-targeting medicine of embodiment 1.
1. 3 pairing clinical samples of the present embodiment carry out DNA extraction according to embodiment 2 and embodiment 3, interrupt (FFPE Or flesh tissue sample is interrupted, CF sample does not interrupt), library preparation, probe hybrid capture and upper machine sequencing.
2. testing result is shown in
Table 24
According to upper table 24 as a result, patient's TMB level is LOW, but detect that EGFR gene p.L858R is prominent in tissue DNA Become, Tarceva, Afatinib, Gefitinib, Conmana, difficult to understand wish are likely to be suited for subject for Buddhist nun.Illustrate embodiment 1 Detection panel can be used for targeting the adjoint diagnosis of medicine.
Embodiment 8
Medication, which is immunized, in the detection panel clinical application-of embodiment 1 the results are shown in Table 25.
1. the clinical sample of the present embodiment carries out DNA extraction according to embodiment 2 and embodiment 3, interrupts (FFPE or fresh groups Knit sample to be interrupted, CF sample does not interrupt), library preparation, probe hybrid capture and upper machine sequencing.
2. testing result is shown in Table 25 and table 26.
Table 25
According to upper table 25 as a result, EGFR, KRAS, BRAF and ERBB2 etc. are not detected in plasma DNA and tissue DNA Sensitive and medicament-resistant mutation；The increase of MET gene copy number and sensitivity/medicament-resistant mutation is not detected；ALK, ROS1 and RET is not detected The fusion such as gene and sensitive and drug resistance site mutation.But patient's TMB level is Extra High, mTORC1/2 inhibitor, thunder Lu Dankang, necitumumab, Afatinib, peace sieve are not likely to be suited for subject for Buddhist nun.
Table 26
According to upper table 26 as a result, be not detected in plasma DNA and tissue DNA EGFR, KRAS, NRAS, BRAF, The sensitivity such as PIK3CA, KIT, PDGFRA, MTOR gene and medicament-resistant mutation；The fusion of ALK, ROS1, RET gene and quick is not detected Sense/medicament-resistant mutation；The increase of ERBB2, MET gene copy number and sensitivity/medicament-resistant mutation is not detected.But patient's TMB level is Extra High。
2 clinical sample testing results of the present embodiment prove that the detection panel of embodiment 1 can be used in immune medication Guidance.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.

Claims (10)

1. a kind of detection panel for the targeting of general cancer kind, chemotherapy and immune medication based on the sequencing of two generations, which is characterized in that The detection panel includes for carrying out the general cancer kind parting of joint-detection, treatment, the relevant gene mutation of prognosis, and tumour is prominent Varying duty calculates relevant exon region and microsatellite instability site.
2. detection panel according to claim 1, which is characterized in that the general cancer kind parting, treatment, prognosis are relevant Gene mutation and the relevant exon region of the Tumor mutations carry calculation include ABCA13, ABCA8, ABL1, ACADSB, ACOT13、ADAMTS6、ADRB1、ADSS、AGPAT9、AK7、AKT1、AKT2、AKT3、ALG9、ALK、ALK_fus、ALOX12B、 ALS2CR11、AMER1、ANKRA2、ANKRD46、ANO1、APC、APOPT1、AR、ARAF、ARHGAP4、ARHGAP6、ARID1A、 ARID1B、ARID2、ARID4A、ARL6IP6、ARMC5、ARPC2、ASH1L、ASXL1、ATM、ATP9B、ATR、ATRX、AURKA、 AURKB、AXIN1、AXL、B2M、BAP1、BARD1、BAT-25、BAT-26、BCL2、BCL2L1、BCOR、BCYRN1、BLM、 BRAF、BRCA1、BRCA2、BRD4、BRIP1、BRS3、BTF3、BTG1、BTK、C20orf96、C22orf23、C5orf42、 C9orf72、CAB39、CALD1、CALM2、CALR、CARD11、CASP8、CAST,ERAP1、CBFB、CBL、CBR4、CCND1、 CCND2、CCND3、CCNE1、CCR4、CD274、CD40、CD74_fus、CD79A、CD79B、CDC25B、CDC73、CDH1、 CDK12、CDK4、CDK6、CDK7、CDK8、CDKL3、CDKN1A、CDKN1B、CDKN2A、CDKN2B、CDKN2C、CDO1、 CEBPA、CEP120、CHD1、CHEK1、CHEK2、CIC、CNKSR3、CNOT8、COSM1316144、COSM1316145、 COSM1332498、COSM3676668、COSM3747491、COSM5045664、COX18、CREBBP、CRKL、CRLF2、 CSF1R、CSF3R、CTAGE5、CTCF、CTNNB1、CUL3、CXCR4、CYFIP1、CYLD、DAXX、DBT、DDR2、DEPDC5、 DIAPH1、DICER1、DIS3、DNMT3A、DOCK11、DOT1L、DROSHA、DSCAM、DXS6804、DXS7132、DXS7423、 DYS391、DYS438、DYS458、EGFR、EGFR_fus、EIF4G3、EML4_fus、EP300、EPHA3、EPHA5、EPHA7、 EPHB1、ERBB2、ERBB3、ERBB4、ERCC4、ERG、ERI1、ERRFI1、ESR1、ETV1、ETV6、ETV6_fus、EWSR1_ fus、EXOSC8、EZH2、EZR_fus、F13A1、FAM149A、FAM153B、FAM46C、FANCA、FANCC、FANCD2、 FANCG、FANCI、FAS、FAT1、FBXW7、FGF16、FGF19、FGF3、FGF4、FGFR1、FGFR2、FGFR2_fus、FGFR3、 FGFR4、FH、FLCN、FLOT1、FLT1、FLT3、FLT4、FMO1、FOLH1、FOXL2、FOXP1、FRAS1、FUBP1、FUS_ fus、GABRP、GANC、GATA1、GATA2、GATA3、GLI1、GMEB1、GNA11、GNA13、GNAQ、GNAS、GPM6A、 GRIN2A、GSK3B、GSTM1、H3F3A、HAUS2、HCAR2、HEY1_fus、HGF、HLA-A、HLA-B、HLA-C、HNF1A、 HNRNPH1、HRAS、HSPA1B、HSPA4、HYOU1、IARS、ID2、ID3、IDH1、IDH2、IGF1R、IGF2、IKBKE、 IKZF1、IL7R、IL8、INHBA、INPP4B、IPO7、IRAK1、IRF4、IRF6、IRF8、IRS2、ITGAL、JAK1、JAK2、 JAK3、JUN、KCNJ2、KDM5A、KDM5C、KDM6A、KDR、KEAP1、KIAA1210、KIAA1432、KIF5B_fus、 KIR3DX1、KIT、KMT2A、KMT2C、KMT2D、KPNA4、KPNB1、KRAS、LAMA3、LEPR、LMO1、LOC100131626、 LONRF3、LRRC34、LYN、MAGOHB、MALT1、MAP2K1、MAP2K2、MAP2K4、MAP3K1、MAP3K4、MAP4K5、 MAPK1、MAPKAP1、MARK2、MCL1、MDM2、MDM4、MED12、MED19、MEF2B、MEIS1、MEN1、MET、MIR1273H、 MITF、MLH1、MMP16、MONO-27、MOV10L1、MPL、MRE11A、MRPL19、MSH2、MSH3、MSH6、MTF1、MTOR、 MTRR、MUTYH、MYADM、MYC、MYCL、MYCN、MYD88、MYO10、NAB1、NAB2_fus、NBN、NCOA6、NDUFS1、 NEO1、NF1、NF2、NFE2L2、NFKBIA、NFXL1、NKX2-1、NOTCH1、NOTCH2、NOTCH3、NPM1、NR-21、NR- 24、NR4A3_fus、NRAS、NSD1、NT5C2、NTRK1、NTRK1_fus、NTRK2、NTRK2_fus、NTRK3、NTRK3_fus、 NUP85、NUP93、P2RY8、PALB2、PAPOLG、PAQR8、PARK2、PARP1、PAX5、PBRM1、PDCD1、PDCD1LG2、 PDE6C、PDGFB_fus、PDGFRA、PDGFRB、PDPN、PIGF、PIK3C2G、PIK3CA、PIK3CB、PIK3CG、PIK3R1、 PIK3R2、PLCG2、PLEKHA1、PLEKHH2、PMS2、PNO1、POLD1、POLE、PPARG、PPHLN1_fus、PPP2R1A、 PRDM1、PREX2、PRKAR1A、PRKCI、PRPF39、PTCH1、PTEN、PTPN11、PTPRJ、PURA、RABGAP1L、RAC1、 RAD21、RAD50、RAD51、RAD51B、RAD51C、RAD51D、RAD52、RAD54L、RAF1、RARA、RB1、RBM10、 RBM27、REL、RET、RET_fus、RHOT1、RICTOR、RIPK2、RNF19A、RNF43、ROS1、ROS1_fus、RPA4、 RPTOR、RRP1B、RUNX1、RYR2、SASH1、SDHA、SDHB、SDHC、SDHD、SEL1L3、SETD2、SF3B1、SHROOM3、 SIPA1L2、SLC34A2_fus、SMAD2、SMAD3、SMAD4、SMARCA4、SMARCB1、SMO、SNX6、SOCS1、SOX2、 SOX9、SPC24、SPEN、SPOP、SRC、SS18_fus、STAG2、STAT3、STK11、STMN1、STRBP、SUCLG1、SUFU、 SUGCT、SYK、TAF15、TAGAP、TBC1D8B、TBX3、TECPR2、TERT、TET2、TGFBR2、TMEM67、TMPRSS15、 TMPRSS2、TNFAIP3、TNFRSF14、TNFSF13B、TNKS、TNRC18、TOP1、TOP2B、TP53、TPH1、TRA2A、 TRIM24、TSC1、TSC2、TSHR、TSN、TXNRD1、U2AF1、UBE2E3、UBE3C、ULK4、UPF2、UTY、VEGFA、VHL、 VSIG10, WHSC1, WHSC1L1, WT1, WWC3, XPO1, ZBBX, ZDHHC17, ZNF711, ZNF805 and ZZZ3.
3. detection panel according to claim 1, which is characterized in that the microsatellite instability site includes rs1045642、rs1052555、rs10981694、rs11045585、rs1105525、rs1130214、rs1138272、 rs115232898、rs11572080、rs11598702、rs11615、rs12613732、rs12762549、rs13181、 rs151264360、rs1517114、rs1570360、rs1650697、rs1650723、rs1695、rs1799793、 rs1801019、rs1801131、rs1801133、rs1801265、rs1805087、rs183484、rs1934951、 rs2032582、rs2072671、rs2075252、rs2228001、rs2273618、rs2291075、rs2291767、 rs2297595、rs2494752、rs2501873、rs25487、rs2784917、rs28738963、rs316019、 rs3212986、rs3745274、rs3892097、rs3918290、rs3957357、rs4148323、rs4244285、 rs430397、rs442767、rs4646、rs4673、rs4673993、rs4728709、rs4880、rs50872、 rs55886062、rs60369023、rs67376798、rs699947、rs716274、rs7779029、rs7851395、 Rs8133052, rs8175347, rs873478, rs9679162 and rs9937.
4. a kind of detection kit for the targeting of general cancer kind, chemotherapy and immune medication based on the sequencing of two generations, which is characterized in that The detection kit includes detection probe, and the detection probe is prominent for general cancer kind parting, treatment, the relevant gene of prognosis Become, microsatellite instability site and the relevant exon region of Tumor mutations carry calculation.
5. detection kit according to claim 4, which is characterized in that the detection probe overlay area/target area > =99%.
6. detection kit according to claim 4, which is characterized in that the general cancer kind parting, treatment, prognosis are relevant Gene mutation and the relevant exon region of the Tumor mutations carry calculation include: ABCA13, ABCA8, ABL1, ACADSB, ACOT13、ADAMTS6、ADRB1、ADSS、AGPAT9、AK7、AKT1、AKT2、AKT3、ALG9、ALK、ALK_fus、ALOX12B、 ALS2CR11、AMER1、ANKRA2、ANKRD46、ANO1、APC、APOPT1、AR、ARAF、ARHGAP4、ARHGAP6、ARID1A、 ARID1B、ARID2、ARID4A、ARL6IP6、ARMC5、ARPC2、ASH1L、ASXL1、ATM、ATP9B、ATR、ATRX、AURKA、 AURKB、AXIN1、AXL、B2M、BAP1、BARD1、BAT-25、BAT-26、BCL2、BCL2L1、BCOR、BCYRN1、BLM、 BRAF、BRCA1、BRCA2、BRD4、BRIP1、BRS3、BTF3、BTG1、BTK、C20orf96、C22orf23、C5orf42、 C9orf72、CAB39、CALD1、CALM2、CALR、CARD11、CASP8、CAST,ERAP1、CBFB、CBL、CBR4、CCND1、 CCND2、CCND3、CCNE1、CCR4、CD274、CD40、CD74_fus、CD79A、CD79B、CDC25B、CDC73、CDH1、 CDK12、CDK4、CDK6、CDK7、CDK8、CDKL3、CDKN1A、CDKN1B、CDKN2A、CDKN2B、CDKN2C、CDO1、 CEBPA、CEP120、CHD1、CHEK1、CHEK2、CIC、CNKSR3、CNOT8、COSM1316144、COSM1316145、 COSM1332498、COSM3676668、COSM3747491、COSM5045664、COX18、CREBBP、CRKL、CRLF2、 CSF1R、CSF3R、CTAGE5、CTCF、CTNNB1、CUL3、CXCR4、CYFIP1、CYLD、DAXX、DBT、DDR2、DEPDC5、 DIAPH1、DICER1、DIS3、DNMT3A、DOCK11、DOT1L、DROSHA、DSCAM、DXS6804、DXS7132、DXS7423、 DYS391、DYS438、DYS458、EGFR、EGFR_fus、EIF4G3、EML4_fus、EP300、EPHA3、EPHA5、EPHA7、 EPHB1、ERBB2、ERBB3、ERBB4、ERCC4、ERG、ERI1、ERRFI1、ESR1、ETV1、ETV6、ETV6_fus、EWSR1_ fus、EXOSC8、EZH2、EZR_fus、F13A1、FAM149A、FAM153B、FAM46C、FANCA、FANCC、FANCD2、 FANCG、FANCI、FAS、FAT1、FBXW7、FGF16、FGF19、FGF3、FGF4、FGFR1、FGFR2、FGFR2_fus、FGFR3、 FGFR4、FH、FLCN、FLOT1、FLT1、FLT3、FLT4、FMO1、FOLH1、FOXL2、FOXP1、FRAS1、FUBP1、FUS_ fus、GABRP、GANC、GATA1、GATA2、GATA3、GLI1、GMEB1、GNA11、GNA13、GNAQ、GNAS、GPM6A、 GRIN2A、GSK3B、GSTM1、H3F3A、HAUS2、HCAR2、HEY1_fus、HGF、HLA-A、HLA-B、HLA-C、HNF1A、 HNRNPH1、HRAS、HSPA1B、HSPA4、HYOU1、IARS、ID2、ID3、IDH1、IDH2、IGF1R、IGF2、IKBKE、 IKZF1、IL7R、IL8、INHBA、INPP4B、IPO7、IRAK1、IRF4、IRF6、IRF8、IRS2、ITGAL、JAK1、JAK2、 JAK3、JUN、KCNJ2、KDM5A、KDM5C、KDM6A、KDR、KEAP1、KIAA1210、KIAA1432、KIF5B_fus、 KIR3DX1、KIT、KMT2A、KMT2C、KMT2D、KPNA4、KPNB1、KRAS、LAMA3、LEPR、LMO1、LOC100131626、 LONRF3、LRRC34、LYN、MAGOHB、MALT1、MAP2K1、MAP2K2、MAP2K4、MAP3K1、MAP3K4、MAP4K5、 MAPK1、MAPKAP1、MARK2、MCL1、MDM2、MDM4、MED12、MED19、MEF2B、MEIS1、MEN1、MET、MIR1273H、 MITF、MLH1、MMP16、MONO-27、MOV10L1、MPL、MRE11A、MRPL19、MSH2、MSH3、MSH6、MTF1、MTOR、 MTRR、MUTYH、MYADM、MYC、MYCL、MYCN、MYD88、MYO10、NAB1、NAB2_fus、NBN、NCOA6、NDUFS1、 NEO1、NF1、NF2、NFE2L2、NFKBIA、NFXL1、NKX2-1、NOTCH1、NOTCH2、NOTCH3、NPM1、NR-21、NR- 24、NR4A3_fus、NRAS、NSD1、NT5C2、NTRK1、NTRK1_fus、NTRK2、NTRK2_fus、NTRK3、NTRK3_fus、 NUP85、NUP93、P2RY8、PALB2、PAPOLG、PAQR8、PARK2、PARP1、PAX5、PBRM1、PDCD1、PDCD1LG2、 PDE6C、PDGFB_fus、PDGFRA、PDGFRB、PDPN、PIGF、PIK3C2G、PIK3CA、PIK3CB、PIK3CG、PIK3R1、 PIK3R2、PLCG2、PLEKHA1、PLEKHH2、PMS2、PNO1、POLD1、POLE、PPARG、PPHLN1_fus、PPP2R1A、 PRDM1、PREX2、PRKAR1A、PRKCI、PRPF39、PTCH1、PTEN、PTPN11、PTPRJ、PURA、RABGAP1L、RAC1、 RAD21、RAD50、RAD51、RAD51B、RAD51C、RAD51D、RAD52、RAD54L、RAF1、RARA、RB1、RBM10、 RBM27、REL、RET、RET_fus、RHOT1、RICTOR、RIPK2、RNF19A、RNF43、ROS1、ROS1_fus、RPA4、 RPTOR、RRP1B、RUNX1、RYR2、SASH1、SDHA、SDHB、SDHC、SDHD、SEL1L3、SETD2、SF3B1、SHROOM3、 SIPA1L2、SLC34A2_fus、SMAD2、SMAD3、SMAD4、SMARCA4、SMARCB1、SMO、SNX6、SOCS1、SOX2、 SOX9、SPC24、SPEN、SPOP、SRC、SS18_fus、STAG2、STAT3、STK11、STMN1、STRBP、SUCLG1、SUFU、 SUGCT、SYK、TAF15、TAGAP、TBC1D8B、TBX3、TECPR2、TERT、TET2、TGFBR2、TMEM67、TMPRSS15、 TMPRSS2、TNFAIP3、TNFRSF14、TNFSF13B、TNKS、TNRC18、TOP1、TOP2B、TP53、TPH1、TRA2A、 TRIM24、TSC1、TSC2、TSHR、TSN、TXNRD1、U2AF1、UBE2E3、UBE3C、ULK4、UPF2、UTY、VEGFA、VHL、 VSIG10, WHSC1, WHSC1L1, WT1, WWC3, XPO1, ZBBX, ZDHHC17, ZNF711, ZNF805 and ZZZ3.
7. detection kit according to claim 4, which is characterized in that the microsatellite instability site includes rs1045642、rs1052555、rs10981694、rs11045585、rs1105525、rs1130214、rs1138272、 rs115232898、rs11572080、rs11598702、rs11615、rs12613732、rs12762549、rs13181、 rs151264360、rs1517114、rs1570360、rs1650697、rs1650723、rs1695、rs1799793、 rs1801019、rs1801131、rs1801133、rs1801265、rs1805087、rs183484、rs1934951、 rs2032582、rs2072671、rs2075252、rs2228001、rs2273618、rs2291075、rs2291767、 rs2297595、rs2494752、rs2501873、rs25487、rs2784917、rs28738963、rs316019、 rs3212986、rs3745274、rs3892097、rs3918290、rs3957357、rs4148323、rs4244285、 rs430397、rs442767、rs4646、rs4673、rs4673993、rs4728709、rs4880、rs50872、 rs55886062、rs60369023、rs67376798、rs699947、rs716274、rs7779029、rs7851395、 Rs8133052, rs8175347, rs873478, rs9679162 and rs9937.
8. it is a kind of as claimed any one in claims 1 to 3 based on the sequencing of two generations for the targeting of general cancer kind, chemotherapy and immune The detection panel of medication or the targeting, changing for general cancer kind based on the sequencing of two generations as described in any one of claim 4 to 7 It treats and the detection kit of immune medication is being prepared for general cancer kind parting, treatment, the relevant gene mutation of prognosis, microsatellite not Application in the device of stability site and the relevant exon region joint-detection of Tumor mutations carry calculation.
9. application according to claim 8, which is characterized in that the general cancer kind include: lung cancer, intestinal cancer, gastric cancer, liver cancer, Breast cancer and carcinoma of endometrium.
10. application according to claim 8, which is characterized in that the joint-detection includes the detection of various mutations type, Described includes various mutations type: point mutation, fusion, copy number variation, missing and insertion mutation；
Preferably, the application includes targeting the adjoint diagnosis of medicine, chemotherapeutic or immune substance.
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