CN109609647B

CN109609647B - Detection Panel for pan-cancer-species targeting, chemotherapy and immune drugs based on next-generation sequencing, detection kit and application thereof

Info

Publication number: CN109609647B
Application number: CN201910074640.XA
Authority: CN
Inventors: 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
Original assignee: Zhenyue Biotechnology Jiangsu Co ltd
Current assignee: Zhenyue Biotechnology Jiangsu Co ltd
Priority date: 2019-01-25
Filing date: 2019-01-25
Publication date: 2022-08-23
Anticipated expiration: 2039-01-25
Also published as: CN109609647A

Abstract

The invention discloses a pan detection kit based on next-generation sequencing for pan-cancer-targeted chemotherapy and immune drugs, 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

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

Technical Field

The invention relates to the technical field of biomedicine, in particular to pan detection kit and application thereof for pan-cancer-targeted chemotherapy and immunization based on next-generation sequencing.

Background

Currently, there are many cancer-related detection techniques, such as high throughput sequencing, target region capture sequencing, fluid biopsy, tumor mutation burden and MSI detection, among others.

Among them, High Throughput Sequencing (also known as Next Generation Sequencing, NGS) is relative to traditional Sanger Sequencing (Sanger Sequencing).

Based on the sequencing method of Sanger et al, fragmented genomic DNA was flanked by adaptors by technical innovation, followed by different methods to generate millions of spatially fixed arrays of PCR clones. Each clone consisted of multiple copies of a single library fragment. Then, primer hybridization and enzyme extension reaction are carried out. Because all clones are on the same plane (flowcell), four different dNTPs are marked by fluorescence with different colors, when DNA polymerase synthesizes a complementary strand, different fluorescence is released when adding one dNTP, DNA sequence extension and imaging detection are repeated continuously, and finally complete DNA sequence information can be obtained through computer analysis. Sequencing hundreds of thousands to millions of nucleic acid molecules at a time is also known as Deep sequencing.

The NGS detection method has high flux, can detect a large number of genes by only constructing and capturing one library for the same sample, meets the requirement of clinical detection, can detect a plurality of genes without a large amount of samples, can detect known mutation sites, can detect unknown mutation sites, can detect various mutation types, and can detect various clinical samples, such as whole blood, tissues, FFPE samples, cfDNA and other sample types.

Target region capture sequencing, applications derived from second generation sequencing in combination with microarray technology, target sequence capture sequencing technology (Targeted sequencing). This technique first uses microarray technology to synthesize a large number of oligonucleotide probes that are capable of binding complementarily to specific regions on the genome, thereby enriching into specific segments, and then sequences these segments using second generation sequencing technology. The sequencing principle is based on the DNA hybridization principle, and the probe customized by the target genome region is utilized to perform chip hybridization or solution hybridization with the genome DNA, enrich the DNA of the target gene region, and then perform sequencing by the NGS technology. The target region selected for sequencing may be a contiguous DNA sequence or may be fragments distributed on different regions of the same chromosome or on different chromosomes.

Liquid biopsy is a method of collecting blood or urine to diagnose diseases such as cancer. Compared to conventional surgical and needle biopsies, liquid biopsies have the advantages: 1) the operation is simple and convenient, the traditional biopsy relies on the discovery of the imaging, the operation or the puncture operation is carried out after the accurate position and the size of the tumor are positioned, the operability is difficult, and the liquid biopsy does not need the support of the imaging; 2) non-invasive, traditional mode because its invasive operation often has complication, the tumor recurrence risk, the liquid biopsy is only to draw a certain amount of blood, the risk is small; 3) the sampling is convenient. Traditional puncture and operation sample taking have great harm to patients, while liquid biopsy generally only needs 5-10 ml of blood, and repeated sampling is supported; 4) effectively cope with tumor heterogeneity. The reason for the conventional sampling of the sampled site may be that the tumor tissue may not be obtained, and the blood may contain information of different mutant tumor cells. 5) The cost is low. Compared with the traditional detection method, the price is lower.

Tumor Mutation Burden (TMB), a completely new biomarker with good prospects for assessing the total number of substitution and insertion/deletion mutations per megabase of exon coding regions of genes, quantitatively estimates the total number of mutations of genomic coding regions in tumor samples. Tumor cells with higher levels of TMB are more easily recognized by the immune system because somatic mutations can be transcribed/expressed at RNA/protein levels, producing new antigens, protein fragments or polypeptide fragments, etc., which are recognized by the autoimmune system as non-self antigens, activating T cells, causing an immune response. Thus, as the number of genetic variations accumulated per megabase increases, many new antigens can be produced, the more easily they can be recognized by immune cells. Thus enabling a stronger immune response to the checkpoint inhibitor, i.e. the higher the TMB, the more beneficial the patient may benefit from immunotherapy, perhaps bear. At present, TMB and tumor neoantigen are proved to be relevant to the curative effect of an immune checkpoint inhibitor in many studies, and an advanced second-generation sequencing technology and an exon sequencing method are utilized to detect the tumor mutation load, so that a solution is provided for clinical personalized treatment.

Microsatellites (microsatellites) are short tandem repeats distributed throughout the human genome, with repeats of single, double or higher nucleotides occurring 10-50 times. Microsatellite length changes in tumor cells as compared to normal cells due to the insertion or deletion of repeat units are called microsatellite instability (MSI). Numerous studies have shown that MSI is caused by a defect in the occurrence of the mismatch repair (MMR) gene and is closely related to tumorigenesis. The MMR system consists of a series of enzymes for specifically repairing DNA base mismatch, and the enzymes can find out and correct mismatched or unmatched bases in the process of DNA replication and damage and ensure the replication accuracy. When MMR gene function loss (Mis-Match Repair specificity, dMMR for short) exists in tumor cells, the marker tumor cells lose the Repair capability of DNA replication errors, a large number of mutations are accumulated in the tumor cells, and microsatellite instability (MSI) characteristics are accompanied, so that abnormal cell proliferation and differentiation and tumor occurrence are caused. MSI is a marker to evaluate the function of the MMR system. Based on MSI instability and extent, it can be classified as microsatellite highly unstable (MSI-H), microsatellite low unstable (MSI-L) and microsatellite stable (MSS). MSI detection can be used to: 1) medication guidance and prognosis prediction for stage II colorectal cancer patients; 2) screening of Lynch (Linche) syndrome; 2) prediction of the benefit of PD-1 immunotherapy. The detection method comprises the following steps: meanwhile, the DNA of normal tissues and tumor tissue samples of the same patient is extracted, a multiplex fluorescence PCR method is adopted to amplify detection sites, amplification products are detected through capillary electrophoresis, and professional software is used to compare and analyze detection results of two tissue sources, so that the MSI state of the patient can be accurately classified.

However, each detection method has different advantages and disadvantages, and products in the current market cannot comprehensively detect samples to be detected.

Disclosure of Invention

The invention aims to provide a pan detection kit based on next-generation sequencing for pan-cancer targeting, chemotherapy and immune drugs, a detection kit and application thereof, so as to solve the technical problem that a sample to be detected cannot be comprehensively detected in the prior art.

To achieve the above objects, according to one aspect of the present invention, there is provided a test panel for pan-cancer species targeting, chemotherapy, and immunization based on next-generation sequencing. The detection panel comprises pan cancer species typing, treatment and prognosis related gene mutation, tumor mutation load calculation related exon region and SNP locus for combined detection.

Further, the exon regions relevant to pan-cancer typing, treatment, prognosis, gene mutation, tumor mutation load calculation include ABCA, ABL, ACDSB, ACOT, ADAMTS, ADRB, ADSS, AGPAT, AK, AKT, ALG, ALK _ fus, ALOX12, ALS2CR, AMER, ANKRA, ANKRD, ANO, APC, APOPT, AR, ARAF, ARAP, ARHGAP, ARID1, ARID4, ARL6IP, ARMC, ARPC, AXASH 1, ASXL, ATM, ATP9, ATR, ATRX, AURKA, AUB, AURKIN, AXL, B2, BAP, BARD, BAT-25, BAT-26, BCL2L, BCOR, YRN, BLM, BRF, BRCA, BRD, BRAD, CALD, CALRD, CDK, CALRD, CDK 5, CDK, CARD, CDK, CARD, CDK, CARD 6, CDK, CARD, CDK, CARD 6, CDK 2, CDK, CARD, CDK, CARD, CDK, CARD, ACOD, CARD, ACRD, CARD, CDK, CARD, CDK, CARD, CDK, CARD, CA, CDKL3, CDKN1A, CDKN1B, CDKN2A, CDKN2B, CDKN2C, CDO1, CEBPA, CEP120, CHD1, CHEK1, CHEK2, CIC, CNKSR3, CNOT8, COSM 6144, COSM1316145, COSM1332498, COSM 76668, COSM3747491, COSM5045664, COX18, CRBBP, CRKL, CRLF2, CSF1R, CSF 382 3R, CTAGE5, CTCF, CTNNB1, CUL3, CXCR4, CYFIP1, CYLD, DADEFLEX 2, DEFU 2, DEFLAFT 2, DIAPH 2, DICER 2, DIS 2, DOCMD 2, DONFET 36149, FODEFGFR 2, FODENFET 2, FODEFUDENFET 2, FODENFET 363636363672, FODENFET 36363636363672, FODENFET 3636363636363672, FODENFET 363636363672, FODENFET 36363636363672, FODENFET 363636363636363672, FODENFET 363672, FODENFET 3636363636363636363636363636363636363672, FODENFET 2, FONFET 363636363636363636363672, FONFET 36363636363636363636363636363636363672, FONFET 2, FONFET 363672, FONFET 2, FONFET 3636363636363636363636363672, FONFET 36363636363636363636363636363672, FONFET 2, FONFET 36363672, FONFET 2, FONFET 363672, FONFET 2, FONFET 363672, FONFET 2, FONFET 363672, FONFET 36363636363672, FONFET 2, FONFET 3636363636363672, FONFET 363636363672, FONFET 363672, FONFET 363636363672, FONFET 2, FONFET 36363636363636363636363636363672, FONFET 2, FONFET 36363636363636363672, FONFET 3636363636363636363636363636363636363636363672, FONFET 3636363672, FONFET 2, FONFET 36363636363636363672, FONFET 363672, FONFET 2, FONFET 36363636363636363672, FONFET 2, FONFET 363636363672, FONFET 2, FONFET 363672, FONFET 2, FONFET 363672, FONFET 2, FONFET 363672, FONFET 36363672, FONFET 2, FONFET 36363636363636, GMEB1, GNA11, GNA13, GNAQ, GNAS, GPM6A, GRIN2A, GSK3B, GSTM1, H3F3A, HAUS2, HCAR2, HEY1_ fus, HGF, HLA-A, HLA-B, HLA-C, HNF 1-1A, HNRNPH1, HRAS, HSPA 11, HSPA1, HYOU1, IARS, ID1, IDH1, IGF 11, IGF1, MDMA TYPK, IK 1, IL7 MYMYMYMYMYMYMYMYMYMYMYMYMY, INHBA, INPP 41, IPO 1, MUAK 1, IRF 1, IRS 1, ITKR, GAL 1, JAK1, KM 1, KM 72K 1, KM 72K 1, KM 72, K1, KM 72, K1, KM 72, K1, KM K1, K1, KM 1, K1, K1, KM K1, K1, K1K 1, K1, K1, K1, K1K 1, K1, K1K 36K K1K 1K 1, K1, K1, K1, K1, K1, K1, K1, K, NAB1, NAB2_ fus, NBN, NCOA6, NDUFS1, NEO1, NF1, NF2, NFE2L2, NFKBIA, NFXL1, NKX 2-RAD 1, NOTCH1, NOTCH2, NOTCH3, NPM1, NR-21, NR-24, NR4A3_ fus, NRAS, NSD1, NT5C2, NTRK1, NT3672 _ fus, NTRK1_ fus, NTNFK 1, NT3672 _ fus, NPP 1, PDCP 1, P1, PALB 1, PAP 1, PAPOLLG, PAQR 1, PARK 1, PARP1, PAX 1, PBRM1, PDCP 1, PDP 1, PSRPP 1, PSRPRAD 1, PSRPP 1, PSADRP 1, PSRPRAD 1, PSADRP 1, PSRPFLK 1, PSRPFLD 1, PSRPFLK 1, PSADRP 1, PSADRF 1, PSADRP 1, PSRPFLD 1, PSADPR 1, PSRPFLD 1, PSADRP 1, PSRPFLK 1, PSRPFLD 1, PSADRF 1, PSRPFLK 1, PSRPFLD 1, PSADRP 1, PSADF 1, PSRPFLD 1, PSADRP 1, PSADF 1, PSADRP 1, PSRPFLD 1, PSADF 1, PSADPR 1, PSADF 1, PSADRP 1, PSADF 1, PSRPFLD 1, PSADF 1, PSADRF 1, PSADF 1, PSADRP 1, PSADF 1, PSADPR 1, PSADRP 1, PSADF 1, PSADRP 1, PSADF 1, PSNFK 1, PSADF 1, PSNFK 1, PSADF 1, PSNFK 1, PSADF, SF3B, SHROOM, SIPA1L, SLC34A _ fus, SMAD, SMARCA, SMARCB, SMO, SNX, SOCS, SOX, SPC, SPEN, SPOP, SRC, SS _ fus, STAG, STAT, STK, STMN, STRBP, SUCLG, SUFU, SUGCT, SYK, TAF, TAGAP, TBC1D8, TBX, TECPR, TERT, TET, TGFBR, TMEM, TMPRSS, TNFAIP, TNRSF, TNSF 13, TNKS, TNRC, TOP, 2, TP, TPH, TRA2, TRIM, TSC, TSHR, TSN, TXNRD, U2AF, UBE2E, UBE3, ULK, UPF, VHY, VHA, XPL, VSIG, WHIG, ZNF, ZVS, ZNF, ZWT and HC 1.

Further, the SNP sites include rs1045642, rs1052555, rs10981694, rs11045585, rs1105525, rs1130214, rs1138272, rs115232898, rs11572080, rs11598702, rs11615, rs12613732, rs12762549, rs13181, rs151264360, rs1517114, rs1570360, rs 697, rs 115723, rs1695, rs1799793, rs1801019, rs 1801131131, rs 1801131133, rs1801265, rs 5018087, rs183484, rs 193494951, rs 2582, rs 207262671, rs2075252, rs2228001, rs2273618, rs 224491075, rs2291767, rs2297595, rs2494752, rs 2503, rs 2584709, rs 27848484709, rs28738963, rs 28321735637321799, rs 227779777946, rs 417977567777777777777777779, rs 417945, rs 42777777777947, rs 4277567945, rs 4277777947, rs 4277567947, rs.

According to another aspect of the invention, a detection kit based on next generation sequencing for pan-cancer species targeting, chemotherapy and immunization is provided. The detection kit comprises a detection probe, wherein the detection probe is used for calculating related exon regions of SNP sites and tumor mutation loads aiming at pan-cancer genotyping, treatment and prognosis related gene mutation.

Further, the detection probe coverage area/target area > is 99%.

Further, pan-cancer typing, treatment, prognosis-related gene mutations and tumor mutation burden calculation-related exon regions include: ABCA, ABL, ACADSB, ACOT, ADAMTS, ADRB, ADSS, AGPAT, AK, AKT, ALG, ALK _ fus, ALOX12, ALS2CR, AMER, ANKRA, ANKRD, ANO, APC, APOPT, AR, ARAF, ARHGAP, ARID1, ARID4, ARL6IP, ARMC, ARPC, ASH1, ASXL, ATM, ATP9, KL, ATRX, AURKA, AURKB, AXIN, AXL, B2, BAP, BARD, BAT-25, BAT-26, BCL2L, BCOR, BCYRN, BLM, BRAF, BRCA, BRIP, BRS, BTF, BTORSM, 6120, 615, CALCD, CALR, CARD, CDK, CARD 766, CDK 2, CDK, CARD, CDK 2, CARD, CDK, CARD 3, CDK, CARD, CDK 2, CDK, CARD, CDK, CARD 3, CDK, CARD, CDK 2, CARD 3, CDK 2, CDK, CARD, CDK 2, CARD 3, CDK 2, CARD, CDK, CARD 3, CDK, CARD, CDK 2, CARD, CDK, CARD, CDK 2, CDK, CARD 3, CDK7, CDK, CARD, CDK 2, CARD, CDK 2, CARD, CA, COX18, CRBBP, CRKL, CRLF2, CSF1R, CSF3R, CTAGE5, CTCF, CTNNB1, CUL3, CXCR4, CYFIP1, CYLD, DAXX, DBT, DDR2, DEPDC5, DIAPH1, DICER1, DIS3, DNMT3 FAST 3A, DOK 11, DOT1L, DROSHA, DSMAM, DXS6804, DXS7132, DXS7423, DYS391, DYS438, DFAT 458, EGFR _ FUS, EIF4G3, EML4_ FUS, EP300, EPHA3, EPHA5, EPHA7, EPHB 7, ERBB 7, ERCC 7, ERG 7, ERI 7, ERGFAK 7, FLF 7, FGFR 7, FLF 7, FLXGFX 7, FLF 7, FLDGF 7, FLEX 7, FLF 7, FLDGF 7, FLX 7, FLDGF 7, FLX 7, FLDE 7, FLX 7, FLDGF 7, FLX 7, FLDE 7, FLX 7, FGFR 7, FLX 7, FGFR 7, FLX 7, FGFR 7, FLX, HLA-C, HNF1A, HNRNPH1, HRAS, HSPA1B, HSPA4, HYOU1, IARS, ID2, ID3, IDH1, IDH2, IGF1R, IGF2, IKBKE, IKZF1, IL7R, IL8, INHBA, INPP 48, IPO 8, IRAK 8, IRF8, IRS 8, ITGAL, JAK 8, JAK 36kc 8, JUN, NONJ 8, KDM 58, KDM 68, KDR, KEAP 8, KIAA 361210, KIAA 2, KIF 58 _ FUS, KIR3 36DX, KIT, KMT 28, KMT2 KM 8, KM 2 KM 72, LON 8, MANF 3, MANF 72, MANFNF 8, MANF 8, MAFN 8, MAMP 8, MANFM 8, MAMP 8, MAFN 8, MANF 8, MAFN 8, MAFNP 8, MAK 8, MAFNP 8, MAFN 8, MAFNP 8, MAFN 8, MAFNP 8, MAFN, NR-24, NR4A3_ fus, NRAS, NSD1, NT5C2, NTRK1, NTRK1_ fus, NTRK2, NTRK2_ fus, NTRK3, NTRK3_ fus, NUP85, NUP93, P2RY8, PALB2, PAPOLG, PAPDG 8, PARK2, PARP1, PAX 1, PBPRNDR 1, PDCD1 LGRADARD 1, PDE 61, PDGFB _ fus, PDGFRA, STAPDGFRPN, PIGF, PIARK 3C 21, PIK3R1, PIK 1, PL3672, PLEKEKHA, PLEK72, PLEK3672, PLEK 1, PSADFH 1, PSORK 1, PSADNFR 1, PSRPRADR 1, PSADRADR 1, PSRT3672, PSADR 1, PSRPRADR 1, PSADR 1, PSADRADR 1, PSRT3672, PSADRADR 1, PSADNFR 1, PSADRADR 1, PSADR 1, PSADRADR 363672, PSADR 1, PSADR 36363672, PSADR 1, PSRPRADR 1, PSADRADR 1, PSADR 1, PSADNFR 1, PSRPRADR 1, PSADNFR 1, PSTFAS 1, PSADNFR 1, PSAD3672, PSADR 1, PSADNFR 1, PSADR 363672, PSADNFR 1, PSADR 1, PSADNFR 1, PSADR 1, PSADNFD 1, PSAD3672, PSADNFR 1, PSADPSADPSADPSADPSADNFD 1, PSADNFR 1, PSTFAS 1, PSADNFR 1, PSNFR 1, PSADNFR 1, PSAD3672, PSADPSADNFR 1, PSAD3672, PSADR 1, PSADNFR 1, PSNFR 1, PSADNFR 1, PSNFR 1, PSADNFR 1, PSNFR 1, PSR 1, PSNFR 1, 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, TPFB 2A, TRIM A, TSC 36HR, TSN, TXD A, TSU 2AF A, UBE2E A, UBE 3A, ULK A, UTF A, VEGFA, VHL, VSVS3672, WHIG A, WHIG 1, WZBN A, ZNF 36ZNF 36711, WZNF A, WZNF 3, 36711, WZNF 3, and WW A.

According to still another aspect of the invention, the second-generation sequencing-based detection panel for pan-cancer-species targeting, chemotherapy and immunization or the second-generation sequencing-based detection kit for pan-cancer-species targeting, chemotherapy and immunization is applied to the preparation of a device for joint detection of gene mutation, SNP (single nucleotide polymorphism) site and exon region related to tumor mutation load calculation related to pan-cancer species typing, treatment and prognosis.

Further, pan-cancerous species include, but are not limited to: lung cancer, intestinal cancer, gastric cancer, liver cancer, breast cancer, endometrial cancer, and the like.

Further, the combined assay includes assays for multiple mutation types, including multiple mutation types: point mutations, fusions, copy number variations, deletions and insertion mutations; preferably, the use comprises concomitant diagnosis of a targeted drug, chemotherapeutic agent or immunological agent.

The design for detecting the panel is not simply adding a plurality of gene regions, but designs a common region for integrating different mutation type information, so that the uniformity of the panel is improved, the overall data volume of the panel is reduced, and the cost is reduced.

The detection panel comprises pan-cancer species typing, treatment and prognosis related genes, exon regions related to tumor mutation load calculation and SNP sites, the included gene information amount is large, combined detection can be better carried out, and more accurate results can be obtained when the panel is applied to the accompanying diagnosis of targeted drugs, chemotherapeutic drugs or immunological drugs.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 is a graph showing the results of the coincidence of the detection panel (P12) and the all-out probe in example 4 with respect to example 1.

Detailed Description

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail below with reference to the accompanying drawings in conjunction with embodiments.

The mechanism of cancer development is very complex, the molecular mechanism and histopathological pathway leading to cancer development are different for different cancers and even patients with the same cancer, even if tumors are in the same part, the treatment effect and method are different from person to person, and the treatment method for different diseases adopted by people and diseases is called 'individualized treatment'. Therefore, only when the individual treatments are carried out according to the different diseases and different from one patient to another in the cancer treatment process, the medicines suitable for the patients can be selected according to different types of patients.

With the continuous and deep research of gene molecule level, more and more tumor cell signal channels are discovered, and a great deal of clinical research shows that the amplification/mutation/expression state of specific genes in the channels is closely related to the effectiveness of targeted and chemotherapeutic drugs. Therefore, the amplification/mutation/expression condition of specific genes in the pathways is clinically detected, and a set of most suitable treatment scheme can be customized for each patient in a targeted manner, so that the treatment effective rate is improved to the maximum extent, the toxic and side effects of the medicines are reduced, and the wrong treatment time due to improper medicine administration is avoided.

The technologies involved in the cancer-related detection products on the market at present have different advantages and disadvantages, and the detection of a sample to be detected cannot be carried out comprehensively.

In view of the above, the present invention provides the following technical solutions: according to an exemplary embodiment of the present invention, a test panel for pan cancer species targeting, chemotherapy, and immunization based on next generation sequencing is provided. The detection panel comprises pan cancer typing, treatment and prognosis related gene mutation (the gene mutation can be also called a mutation gene related to pan cancer typing, treatment and prognosis) for combined detection, an exon region related to tumor mutation load calculation and an SNP locus.

The products in the prior art can not comprehensively carry out the combined detection of multiple tumor variations on tissues and blood samples of pan-cancer species at the same time. The panel and the kit can simultaneously carry out combined detection on gene mutation related to pan-cancer species typing, treatment and prognosis, and exon regions and SNP sites related to tumor mutation load calculation. And the sample type encompasses tissue and blood.

The panel design of the invention is not simple addition, but the type of the detection gene is selected according to the requirement, thereby avoiding panel region redundancy, reducing the output of worthless data, shortening the data analysis period and reducing the detection cost.

By applying the technical scheme of the invention, the detection of panel comprises the genetic mutation related to pan-cancer species typing, treatment and prognosis, and the exon region and the SNP locus related to tumor mutation load calculation, and the gene information content of the panel is large, so that the panel can be better subjected to combined detection, and more accurate results can be obtained when the panel is applied to the concomitant diagnosis of targeted drugs, chemotherapeutic drugs or immunological drugs.

Preferred genetic mutations relevant to pan-cancer typing, therapy and prognosis, exon regions relevant to calculation of tumor mutation load include ABCA13, ABCA8, ABL1, ACADSB, ACOT13, ADAMTS6, ADRB1, ADSS, AGPAT9, AK7, AKT1, AKT2, AKT3, ALG9, ALK _ fus, ALOX12B, ALS2CR11, AMER1, ANKRA2, ANKRD46, ANO KL 1, APC, APOPT1, AR, ARAF, ARHGAP4, ARHGAP6, ARID1A, ARID1B, ARID2, ARID4A, ARL6IP6, ARMC 6, ARPC 6, ASH 16, ASBAT 6, ATM, ATP 369, 6, CALBATBRA 6, CALBC 6, CALBD 6, CABBD 6, CALBC 6, CABBD 6, CALBC 6, CALBD 6, CABBD 6, CANBR 6, CALBC 6, CALBD 6, CALBC 6, CAXB 6, CALBC 6, CANBR 6, CALBD 6, CABBD 6, CALBD 6, CABBD 6, CABB, CDKN1A, CDKN1B, CDKN2A, CDKN2B, CDKN2C, CDO1, CEBPA, CEP120, CHD1, CHEK1, CHEK2, CIC, CNKSR3, CNOT8, COSM1316144, COSM 6145, COSM1332498, COSM3676668, COSM3747491, COSM5045664, COX18, CRKL, CRLF2, CSF1R, CSF3R, CTAGE5, CTCF, CTNNB1, CUL3, CXCR4, FICYP 1, CYLD, DAXX, DBT 2, DEPDC5, DIAPH 5, DIA 5, DIS 5, DOFF 5, DOFFS 363672, DOFFF 5, FANFET 5, FONFET 363672, FONFET 5, FONFET 363636363636363672, FONFET 363636363672, FONFET 363636363636363672, FONFET 3636363672, FONFET 363636363672, FONFET 3636363636363636363636363672, FONFET 363636363636363636363672, FONFET 36363636363636363672, FONFET 363636363672, FONFET 36363636363636363672, FONFET 363636363636363672, FONFET 363636363636363636363636363672, FONFET 363636363636363636363636363636363672, FONFET 5, FONFET 363672, FONFET 5, FONFET 36363672, FONFET 363636363636363636363636363636363636363636363636363636363636363636363636363636363636363636363636363636363636363672, FONFET 36363636363672, FONFET 5, FONFET 363636363672, FONFET 5, FONFET 363672, FONFET 5, FONFET 36363672, FONFET 3636363672, FONFET 5, FONFET 363672, FONFET 5, FONFET 3636363636363672, FONFET 3636363672, FONFET 5, FONFET 3636363636363672, FONFET 5, FONFET 363636363636363636363636363636363672, FONFET 5, FONFET 3636363636363636363636363636363636363636363636363636363636363636363672, FONFET 363672, FONFET 5, FONFET 36363672, FONFET 363672, FONFET 5, FONFET 363636363636363636363636363636363672, FONFET 5, FONFET 3636363636363672, FONFET 5, FONFET 36363636363636363636363636363636363636363636363672, FONFET 5, FONFET 36363672, FONFET 363636363636363672, FONFET 363672, FONFET 36363672, FONFET 5, FONFET 36363672, FONFET 5, FONFET 363636363672, FONFET 5, FONFET 363636363636363636, GNA11, GNA13, GNAQ, GNAS, GPM6A, GRIN2A, GSK3B, GSTM1, H3F3A, HAUS2, HCAR2, HEY1_ fus, HGF, HLA-A, HLA-B, HLA-C, HNF1A, HNRNPH1, HRAS, HSPA1B, HSPA B, HYOU B, IARS, ID B, IDH B, IGF 1B, IGF B, IKBKE B, IKZF B, MDM 7B, MYIL B, INHBA, INPP4B, IPO B, IRAK B, IRF B, JAK B, IRS B, ITGAL B, 3636363672, MYMAMMK B, MAMMK 36K B, MAMMK 36K B, MAPK 36K B, MAMMK B, MAPK B, MAMMAK B, MAPK 36K B, MAPK B, MAMMAK B, MYMAMMK 36K 36363636363672, MYMAMMK 3636K B, MYMAMMK 363672, MYMAMMK 36K 36363636K B, MAMMK 36K B, MYMAMMK 36K B, MAMMK 36363672, MY 36K B, MY B, MAMMK 36K B, MAMMK 36K B, MAMMK 36K 363636K 36K B, MAMMK 36K B, MAMMK 36K B, MAMMK 36K B, MAMMK B, MAPK 36K B, MAMMK 36K B, MAPK 36K B, MAMMK 36K B, MAMMK 36K B, MAMMK 36K B, MAK B, MAMMK 36K B, MAMMK B, MAK B, MAMMK 36K B, MAMMK 36K B, MAMMK B, MAK 36K B, MAK B, MAMMK B, MAK 36K B, MAK B, MAMMK 36K B, MAK 36K B, MAK B, MA, 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, NTRK1, NUP 1, P2RY 1, PALB 1, PAPOPPOQR 1, PARK 1, PARP1, PAX 1, PBRM1, PDCD1, PDF 1, SHROOM, SIPA1L, SLC34A _ fus, SMAD, SMARCA, SMARCB, SMO, SNX, SOCS, SOX, SPC, SPEN, SPOP, SRC, SS _ fus, STAG, STAT, STK, STMN, STRBP, SUCLG, SUFU, SUGCT, SYK, TAF, TAGAP, TBC1D8, TBX, TECPR, TERT, TET, TGFBR, TMEM, TMPRSS, TNFAIP, TNFRSF, TNFSF13, TNKS, TNRC, TOP2, TP, TPH, TRA2, TRIM, TSC, HR, TSN, TXNRD, U2AF, UBE2E, UBE3, ULK, UPF, VEY, VEGFA, VHL, VSIG, WHSC, ZBC, ZNF, ZFH and WW 711. The gene joint detection can enable detection of exon regions related to pan-cancer typing, treatment and prognosis and tumor mutation load calculation to be more accurate.

Preferably, the SNP sites include rs1045642, rs1052555, rs10981694, rs11045585, rs1105525, rs1130214, rs1138272, rs115232898, rs11572080, rs11598702, rs11615, rs12613732, rs12762549, rs13181, rs151264360, rs1517114, rs1570360, rs1650697, rs1650723, rs1695, rs 17793, rs1801019, rs1801131, rs 1801131133, rs 1801801801265, rs 5018087, rs183484, rs 193494951, rs 252582, rs 207262671, rs2075252, rs2228001, rs2273618, rs 22912291798, rs 2276767, rs2297595, rs 24752, rs 250254873, rs 8484709 709, rs 2727777963, rs 2889321799, rs 2273447356377978, rs 4179777946, rs 41797756777777777947, rs 417945, rs 42777947, rs 6777567945, rs 417945, rs 42777947, rs 4277567947, rs. Effective information obtained by detection can be further enriched, and the accuracy of subsequent data application is improved.

According to another aspect of the invention, a detection kit based on next generation sequencing for pan-cancer species targeting, chemotherapy and immunization is provided. The detection kit comprises a detection probe, wherein the detection probe is used for calculating related exon regions aiming at pan-cancer type parting, treatment and prognosis related genes, SNP loci and tumor mutation loads. Preferably, the detection probe coverage area/target area > is 99%.

According to an exemplary embodiment of the present invention, the exon regions related to pan-cancer typing, treatment, prognosis-related gene mutation and tumor mutation burden calculation include: ABCA, ABL, ACADSB, ACOT, ADAMTS, ADRB, ADSS, AGPAT, AK, AKT, ALG, ALK, ALFUS, ALOX12, ALS2CR, AMER, ANKRA, ANKRD, ANO, APC, APOPT, AR, ARAF, ARHGAP, ARID1, ARID4, ARL6IP, ARMC, ARPC, ASH1, ASXL, ATM, ATP9, KL, ATRX, AURKA, AURKB, AXIN, AXL, B2, BAP, BARD, BAT-25, BAT-26, BCL2L, BCOR, BCYRN, BLM, BRAF, BRCA, BRIP, BRS, BTF, BTORSM, 6120, 615, CALCD, CALR, CARD, CDK 2C 766, CDK 2, CARD, CDK 2, CARD 766, CDK 2, CDK, CARD, CDK 2, CARD, CDK, CARD, CDK 2, CDK, CARD, CDK 2, CARD, CDK, CARD, CDK 2, CARD, CDK, CARD, CDK 2, CARD, CDK 2, CARD, CDK, CARD, CDK 2, CARD, CDK 2, CDK, CARD, CDK 2, CARD, CDK 2, CARD, CDK 2, CARD, COSM5045664, COX, CRBBP, CRKL, CRLF, CSF1, CSF3, CTAGE, CTCF, CTNNB, CUL, CXCR, CYFIP, CYLD, DAXX, DBT, DDR, DEPDC, DIAPH, DICER, DIS, DNMT3, DOCK, DOT1, DROSHA, DSCAM, DXS6804, DXS7132, DXS7423, DYS391, DYS438, DYS458, EGFR _ FUS, EIF4G, EML _ FUS, EP300, EPHA, EPHB, ERBB, ERCC, ERG, ERI, ERRFI, ESR, FRAETV, ETV _ FUS, EWSR _ FUS, EXOSC, EZH, EZR _ FUS, F13A, FAM149, FAM, FAN 153, FANCV, FGFR, FG, HLA-C, HNF1A, HNRNPH1, HRAS, HSPA1B, HSPA4, HYOU1, IARS, ID2, ID3, IDH1, IDH2, IGF1R, IGF2, IKBKE, IKZF1, IL7R, IL8, INHBA, INPP 48, IPO 8, IRAK 8, IRF8, IRS 8, ITGAL, JAK 8, JAK 36kc 8, JUN, NONJ 8, KDM 58, KDM 68, KDR, KEAP 8, KIAA 361210, KIAA 2, KIF 58 _ FUS, KIR3 36DX, KIT, KMT 28, KMT2 KM 8, KM 2 KM 72, LON 8, MANF 3, MANF 72, MANFNF 8, MANF 8, MAFN 8, MAMP 8, MANFM 8, MAMP 8, MAFN 8, MANF 8, MAFN 8, MAFNP 8, MAK 8, MAFNP 8, MAFN 8, MAFNP 8, MAFN 8, MAFNP 8, MAFN, NR-24, NR4A3_ fus, NRAS, NSD1, NT5C2, NTRK1, NTRK1_ fus, NTRK2, NTRK2_ fus, NTRK3, NTRK3_ fus, NUP85, NUP93, P2RY8, PALB2, PAPOLG, PAPAK 8, PARK2, PARP1, PAX5, PBPRNDR 5, PDCD1 LGRADARD 5, PDE 65, PDGFB _ fus, PDGFRA, FRPTPTPN, PIGF, PIK3C 25, PIK3R 5, PLEK72, PLEKHA, PLEK72, PLEK 5, PSORK 5, PSORD 36SRRADR 5, PSROR 5, PSRODG 5, PSROR 36363672, PSROR 5, PSROR 363636363672, PSROR 5, PSROR 36, 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, ZZTP B, TPH B, TRA 2B, TRIM B, TSC B, TSHR, NRTXD B, 2AF B, UBE2E B, UBE 3B, UTK B, UPF B, VEY, GFA, VHL, VSIG 4672, WHIG, ZNF B, DHBZNF B, ZNF B, 36ZNF B, UBZ B, WZNF 36711, WW B, WTC B, and WW 36711.

According to a typical embodiment of the present invention, the SNP sites include rs1045642, rs1052555, rs10981694, rs11045585, rs1105525, rs1130214, rs1138272, rs115232898, rs11572080, rs11598702, rs11615, rs12613732, rs12762549, rs13181, rs151264360, rs1517114, rs1570360, rs 157697, rs 723, rs 15195, rs1799793, rs 1801011011019, rs1801131, rs1801133, rs1801265, rs1805087, rs183484, rs1934951, rs2032582, rs 522072671, rs20752, rs 8002221, rs2273618, rs2291075, rs2291767, rs2297595, rs2494752, rs 250752, rs 2503, rs 55552579872, rs 272573872, 282828736011, rs 2273321714332162, 41679, 4167437763, 41679, 41677763, 41679, 41778177817763, 417763, 417980, rs 607763, 41817763, 41798, and 41798.

According to a typical embodiment of the invention, the second-generation sequencing-based pan for pan-cancer-species-targeting, chemotherapy and immunization detection or the second-generation sequencing-based detection kit for pan-cancer-species-targeting, chemotherapy and immunization detection is applied to the preparation of a device for joint detection of gene mutation, SNP (single nucleotide polymorphism) sites and exon regions related to tumor mutation load calculation, wherein the gene mutation is related to pan-cancer species typing, treatment and prognosis. Typically, pan-cancer species include, but are not limited to: lung cancer, intestinal cancer, gastric cancer, liver cancer, breast cancer, endometrial cancer, and the like. The combined assay may include the detection of multiple mutation types, including: point mutations, fusions, copy number variations, deletions and insertion mutations; preferably, the use comprises concomitant diagnosis of a targeted drug, chemotherapeutic agent or immunological agent.

The following examples are provided to further illustrate the advantageous effects of the present invention.

Example 1

The panel of this example includes pan-cancer genotyping, treatment, prognosis related gene mutations, SNP sites and exon regions relevant for tumor mutation burden calculation. Correspondingly, the kit of the present embodiment comprises a detection probe, wherein the detection probe is directed against pan-cancer typing, treatment and prognosis related gene mutation, and exon regions related to calculation of SNP sites and tumor mutation loads, and the detection probe coverage region/target region > is 99%. The detection probes are designed according to conventional design rules in the art.

Wherein, the exon regions related to pan-cancer species typing, treatment and prognosis related gene mutation and tumor mutation load calculation comprise: ABCA, ABL, ACADSB, ACOT, ADAMTS, ADRB, ADSS, AGPAT, AK, AKT, ALG, ALK _ fus, ALOX12, ALS2CR, AMER, ANKRA, ANKRD, ANO, APC, APOPT, AR, ARAF, ARHGAP, ARID1, ARID4, ARL6IP, ARMC, ARPC, ASH1, ASXL, ATM, ATP9, KL, ATRX, AURKA, AURKB, AXIN, AXL, B2, BAP, BARD, BAT-25, BAT-26, BCL2L, BCOR, BCYRN, BLM, BRAF, BRCA, BRIP, BRS, BTF, BTORSM, 6120, 615, CALCD, CALR, CARD, CDK, CARD 766, CDK 2, CDK, CARD, CDK 2, CARD, CDK, CARD 3, CDK, CARD, CDK 2, CDK, CARD, CDK, CARD 3, CDK, CARD, CDK 2, CARD 3, CDK 2, CDK, CARD, CDK 2, CARD 3, CDK 2, CARD, CDK, CARD 3, CDK, CARD, CDK 2, CARD, CDK, CARD, CDK 2, CDK, CARD 3, CDK7, CDK, CARD, CDK 2, CARD, CDK 2, CARD, CA, COX18, CRBBP, CRKL, CRLF2, CSF1R, CSF3R, CTAGE5, CTCF, CTNNB1, CUL3, CXCR4, CYFIP1, CYLD, DAXX, DBT, DDR2, DEPDC5, DIAPH1, DICER1, DIS3, DNMT3A, DOCK11, DOT1L, DROSHA, DSCAM, S6804, DXS7132, DXS7423, DYS391, DYS438, DYS458, EGFR _ FUS, EIF4G3, EML4_ FUS, EP300, EPHA3, EPHA5, EPHA7, EPHB 7, ERBB 7, ERCC 7, ERG 7, ERI 36149, ERGFR 7, EPF 7, FGFR 7, FLF _ 36XFU 7, FLEX 7, FGFR 7, FLF 363636363672, FGFR 7, FGFR 363636363672, FGFR 363636363636363672, FGFR 3636363636363672, FGFR 36363672, FGFR 363636363636363636363636363636363672, FGFR 36363672, FGFR 36363636363636363636363636363672, FGFR 363672, FGFR 7, FGFR 36363636363636363672, FGFR 7, FLF-7, FGFR, FLF-363672, FGFR 3636363672, FGFR 36363636363636363636363636363636363636363636363636363636363672, FGFR 7, FGFR 36363636363636363636363636363672, FGFR 363672, FGFR 36363672, FGFR 363672, FGFR 7, FGFR 3636363636363636363672, FGFR 36363672, FGFR 3636363636363672, FGFR 7, FGFR 363672, FGFR 363636363636363636363636363636363672, FGFR 7, FGFR 7, FGFR 36363672, FGFR 7, FGFR 363636363672, FGFR 36363672, FGFR 7, FGFR 3636363672, FGFR 7, FGFR 7, FGFR 3636363672, FGFR 7, HLA-C, HNF1A, HNRNPH1, HRAS, HSPA1B, HSPA4, HYOU1, IARS, ID2, ID3, IDH1, IDH2, IGF1R, IGF2, IKBKE, IKZF1, IL7R, IL8, INHBA, INPP 48, IPO 8, IRAK 8, IRF8, IRS 8, ITGAL, JAK 8, JAK 36kc 8, JUN, NONJ 8, KDM 58, KDM 68, KDR, KEAP 8, KIAA 361210, KIAA 2, KIF 58 _ FUS, KIR3 36DX, KIT, KMT 28, KMT2 KM 8, KM 2 KM 72, LON 8, MANF 3, MANF 72, MANFNF 8, MANF 8, MAFN 8, MAMP 8, MANFM 8, MAMP 8, MAFN 8, MANF 8, MAFN 8, MAFNP 8, MAK 8, MAFNP 8, MAFN 8, MAFNP 8, MAFN 8, MAFNP 8, MAFN, NR-24, NR4A3_ fus, NRAS, NSD1, NT5C2, NTRK1, NTRK1_ fus, NTRK2, NTRK2_ fus, NTRK3, NTRK3_ fus, NUP85, NUP93, P2RY8, PALB2, PAPOLG, PAPAK 8, PARK2, PARP1, PAX5, PBPRNDR 5, PDCD1 LGRADARD 5, PDE 65, PDGFB _ fus, PDGFRA, FRPTPTPN, PIGF, PIK3C 25, PIK3R 5, PLEK72, PLEKHA, PLEK72, PLEK 5, PSORK 5, PSORD 36SRRADR 5, PSROR 5, PSRODG 5, PSROR 36363672, PSROR 5, PSROR 363636363672, PSROR 5, PSROR 36, 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, ZZTP B, TPH B, TRA 2B, TRIM B, TSC B, TSHR, NRTXD B, 2AF B, UBE2E B, UBE 3B, UTK B, UPF B, VEY, GFA, VHL, VSIG 4672, WHIG, ZNF B, DHBZNF B, ZNF B, 36ZNF B, UBZ B, WZNF 36711, WW B, WTC B, and WW 36711.

The SNP sites comprise rs1045642, rs1052555, rs10981694, rs11045585, rs1105525, rs1130214, rs1138272, rs115232898, rs11572080, rs11598702, rs11615, rs12613732, rs12762549, rs13181, rs 1514360, rs1517114, rs1570360, rs1650697, rs 723, rs1695, rs1799793, rs1801019, rs1801131, rs1801133, rs 1801261265, rs 180505087, rs183484, rs1934951, rs2032582, 2072671, rs2075252, rs2228001, rs 2273075, rs2291075, rs2291767, rs 4497595, rs 24752, rs 25943, rs 203487 487, rs 8484848484848484842763, rs 31288963, rs 31798, rs 32132179321798, rs 4279437762, rs 447777567962, 41777763, 41798, 417763, 567763, 5677567763, 56777763, 5632777763, 56327763, 563277563262, 4156327756327756327763, 4156327763, 417763, 4156327763, 417763, 4156327763, 4156327753, 4156327729, 417729, and 417729, etc.

Example 2

The kit of example 1 was used to detect tissue DNA-mimicking standards.

Firstly, preparing a verification standard product

20 kinds of mutation standard DNA were prepared by mixing tumor cell line DNA containing known mutation and wild type cell line GM12878 DNA in a certain proportion. And the frequency of each mutation in the standard was determined by ddPCR. The tumor cell line DNA is shown in Table 1.

TABLE 1

Secondly, DNA breaking:

DNA was quantified using the Qubit 3.0 and dsDNA HS Assay Kit.

Cutting polytetrafluoroethylene rays to a length of about 1cm by using medical scissors subjected to ultraviolet sterilization, ensuring that the length uniformity of a broken rod is good, placing the broken rod in a clean container, and performing ultraviolet sterilization for 3-4 hours. After completion of sterilization, 1cm of a polytetrafluoroethylene wire was loaded into a 96-well plate using sterilized forceps. And 2 breaking rods are filled into each hole, and then the 96-hole plate is subjected to ultraviolet sterilization for 3-4 hours.

According to the quantitative result of the qubit, 300ng of DNA sample is diluted to 50 μ l by using TE, transferred to a 96-well plate, a tin foil paper film is placed on the 96-well plate, four sides of the film are aligned, the film is sealed for 2 times by using a heat-seal film instrument at 180 ℃ for 5s, and the film is centrifuged by using a microplate centrifuge.

Selecting a preset program Peak Power: 450, Duty Factor: 30, Cycles/Burst: 200, Treatment time: 40s, 3cycles, click "Start position". And running the program at a Run interface point 'Run' button. After this procedure operation was accomplished, take out the sample board, use micropore board centrifuge centrifugation, put the sample board again on the sample frame, select procedure Peak Power: 450, Duty Factor: 30, Cycles/Burst: 200, Treatment time: 40s, 4 cycles. And running the program at a Run interface point 'Run' button. After the procedure was completed, the sample plate was removed and centrifuged using a microplate centrifuge. Taking 1 mul for quality inspection after cutting.

Third, library construction

1. End-repaired and an a-tail added to the 3' -end:

1.1 mu.L of the DNA obtained in the second step was taken, less than 50. mu.L of the DNA was supplemented with nuclease-free water to 50. mu.L, and the DNA was added to the reaction system according to the following Table 2:

TABLE 2

Components	Volume of
		End repair and buffer addition A	7μL
End repair and addition of A enzyme	3μL
		DNA	50μL
Total volume	60μL

1.2 vortex mixing, microcentrifugation, and placing in a PCR instrument, wherein the reaction procedure is as follows in Table 3:

TABLE 3

2. Connecting joints:

2.1 preparation of the linker: linker 2.5. mu.L, diluted to 5. mu.L with 2.5ul water.

2.2 the corresponding reagents were added to the above reaction tubes according to Table 4 below:

TABLE 4

Components	Volume of
		Water (W)	5μL
Ligation buffer	30μL
		DNA ligase	10μL
End repair and addition of A product	60μL
		Total volume	110μL

2.3 vortex, mix, microcentrifuge, place in PCR instrument, the reaction program is as following table 5:

TABLE 5

Step (ii) of	Temperature of	Time
			Connecting joint	20℃	30min
Terminate	20℃	∞

Note that: the temperature of the hot cover is 50 DEG C

3. And (3) purification after connection:

3.1 subpackaging Beckman Agencourt AMPure XP magnetic beads into new eight-connected tubes, wherein each tube contains 88 mu L. And (3) finishing the PCR in the last step, namely finishing 2.3, taking out the sample, carrying out short-time centrifugation, and transferring the sample into a subpackaged 88 mu L magnetic bead centrifuge tube.

3.2 shaking and mixing evenly, and incubating for 15min at room temperature to ensure that the DNA is fully combined with the magnetic beads. Note that: the tube cover is pressed tightly during oscillation. The centrifuge tube was placed on a magnetic rack for clarification of the liquid and the supernatant was discarded (ensuring that the residual volume did not exceed 5. mu.L). Note that: do not attract to the magnetic beads.

3.3 Add 200 u L80% ethanol incubation 30sec after discarding. The 200 μ L80% ethanol wash step was repeated once. Note that: 80% ethanol is prepared in situ.

3.4 suck up the residual ethanol at the bottom of the centrifuge tube with a 10 μ L pipette tip, and dry at room temperature for 3-5min until the ethanol is completely volatilized (the front side is not reflected light, and the back side is dried). Note that: the yield of DNA produced by magnetic beads dried too much is reduced.

3.5 remove the centrifuge tube from the magnetic frame, add 21. mu.L of ultrapure water, shake and mix. Note that: the tube cover is pressed tightly during oscillation. Incubate at room temperature for 5 min.

3.6 centrifuging for a short time, and placing the centrifuge tube on a magnetic frame to clarify the liquid. The remaining 20. mu.L of the supernatant was transferred to a new PCR tube for further amplification.

4. Library amplification:

4.1 the reaction was charged as per Table 6 below:

TABLE 6

Components	Volume of
		Hot start enzyme	25μL
Primer and reaction buffer mixture	5μL
		Joint ligation product	20μL
Total volume	50μL

4.2 vortex, mix, microcentrifuge, place in PCR instrument, the reaction program is as following table 7:

TABLE 7

Obtaining of DNA

5.1 subpackage 25. mu.L of Beckman Agencourt AMPure XP magnetic beads into a new eight-connected tube.

5.2 after the PCR of the previous step (4.2) is finished, taking out a sample.

5.3 short centrifugation, transfer into the subpackaged 25 uL Beckman Agencourt AMPure XP magnetic bead.

5.4 shaking and mixing evenly, and incubating for 15min at room temperature to ensure that the DNA is fully combined with the magnetic beads. Note that the tube cap is pressed down while shaking.

5.5 short-time centrifugation, placing the centrifuge tube on a magnetic frame for clarifying the liquid, and transferring the supernatant to another tube of 25 mu L Beckman Agencourt AMPure XP magnetic beads which are subpackaged. Note that: do not attract to the magnetic beads.

5.6 concussion and mixing evenly, and incubating for 15min at room temperature to ensure that the DNA is fully combined with the magnetic beads, and the tube cover is tightly pressed when concussion is noticed.

5.7 centrifuging for a short time, placing the centrifuge tube on a magnetic frame to clarify the liquid, and discarding the supernatant. Note that: do not attract to the beads.

5.8 Add 200 u L80% ethanol incubation 30sec after discarding. Note that: 80% ethanol is prepared in situ. The 200. mu.L 80% ethanol wash step was repeated once.

5.9 suck up the residual ethanol at the bottom of the centrifuge tube with a 10 μ L pipette tip, and dry at room temperature for 3-5min until the ethanol is completely volatilized (no reflection is seen on the front side, and drying is seen on the back side). Note that: the yield of DNA from beads dried too much is reduced.

5.10 remove the tube from the magnetic frame, add 40. mu.L of ultrapure water, shake and mix.

5.11 incubate for 5min at room temperature to elute DNA.

5.12 short centrifugation, the centrifuge tube is placed on a magnetic rack to clarify the liquid, and the library is transferred to a new centrifuge tube. Store at-20 ℃.

6. Library quality inspection

mu.L of the DNA library was used to determine its concentration using the dsDNA HS Assay Kit.

Library hybrid Capture

The invention is used for detecting panel and hybrid capture reagent to carry out library hybrid capture, and the operation process is carried out according to the product instruction.

1. An equivalent amount of 1. mu.g of library was taken in a 1.5mL centrifuge tube and the volume added for each library was calculated based on the concentration of each library and the number of captured libraries. The volume of library addition was: mu.L (1000 ng/number of captured libraries/concentration of libraries). Add 2.5. mu.L blocking oligonucleotide to the above system, add 5. mu.L COT Human DNA, shake and mix well, centrifuge briefly. Sealing the EP tube with sealing film, and evaporating to dryness (60 deg.C, 20 min-1 hr) in vacuum centrifugal concentrator. Note that at any time it is checked whether it has evaporated to dryness.

2. Hybridization of the library with probes

2.1 adding the reaction system shown in the following table 8 into the dry powder of the library and the like in the previous step, uniformly mixing by vortex, centrifuging for a short time, and heating the mixture at 95 ℃ for denaturation for 10 min.

TABLE 8

Components	Volume of
		Hybridization buffer	7.5μL
Hybridization enhancer	3μL
		Total volume	10.5μL

2.2 take out 4.5ul of probe and centrifuge briefly; and (3) placing the probe in a 47 ℃ PCR instrument after short-time centrifugation, quickly transferring the denatured DNA into a PCR tube containing the probe from 95 ℃, shaking and mixing uniformly, and carrying out short-time centrifugation. Placing in PCR instrument, and hybridizing at 47 deg.C for not less than 16 hr.

3. Library Capture

3.1 preparation of elution solutions, preparation of buffers for capturing the samples according to the following Table, and preparation of buffers according to the number of captures in Table 9 below.

TABLE 9

3.2 preparing capture magnetic beads and elution working solutions 1 and 4, and balancing the capture magnetic beads for 30min at room temperature before use; elution of working solutions 1 and 4 was incubated at 47 ℃ for 2hr before use.

3.3 Each capture was aliquoted into 100. mu.L of capture beads, 100. mu.L of capture beads were placed on a magnetic rack until the liquid was clear, and the supernatant was discarded. Adding 200 mu L of 1X magnetic bead washing liquid, shaking and mixing uniformly. Placing on a magnetic frame until the liquid is clear, and discarding the supernatant. Add 200. mu.L of 1X magnetic bead washing solution, shake and mix. Placing on a magnetic frame until the liquid is clear, and discarding the supernatant. Add 100. mu.L of 1X magnetic bead washing solution, shake and mix. Placing on a magnetic frame until the liquid is clear, and discarding the supernatant. At this point the bead pretreatment was complete and the next run was immediately performed.

3.4 transfer the captured overnight hybridization liquid into the washed magnetic beads, and pipette-blow ten times. Placing in a PCR instrument, incubating at 47 ℃ for 45min (the temperature of a PCR hot cover is set as 57 ℃), and shaking once every 15min to ensure that the magnetic beads are suspended.

3.5, cleaning:

3.5.1 after the incubation was completed, 100. mu.L of 1 × elution working solution 1 preheated at 47 ℃ was added to each tube, and mixed by shaking. Placing on a magnetic frame until the liquid is clear, and discarding the supernatant.

3.5.2 add 200. mu.L of 47 ℃ preheated 1 Xelution working solution 4, blow and beat by a pipette ten times and mix evenly. Incubating at 47 deg.C for 5min, placing on magnetic frame until the liquid is clear, and discarding the supernatant. Care was taken to avoid temperatures below 47 c as much as possible.

3.5.3 mu.L of 47 ℃ preheated 1 Xelution working solution 4 was added, and the mixture was pipetted and mixed ten times. Incubating at 47 deg.C for 5min, placing on magnetic frame until the liquid is clear, and discarding the supernatant. Note that the process was carried out to avoid temperatures below 47 ℃.

3.5.4 adding 200 μ L of 1 × elution solution at room temperature, shaking for 2min, centrifuging for a short time, placing on a magnetic frame until the solution is clear, and discarding the supernatant.

3.5.5 adding 200 μ L of room temperature 1 × elution working solution 2, shaking for 1min, centrifuging for a short time, placing on a magnetic frame until the liquid is clear, and discarding the supernatant.

3.5.6 mu.L of 1 Xelution buffer 3 placed at room temperature was added, shaken for 30sec, centrifuged briefly, placed on a magnetic stand until the solution was clear, and the supernatant was discarded.

3.5.7 adding 18 microliter of ultrapure water into the centrifuge tube for elution, shaking and mixing uniformly, and carrying out the next amplification test.

4. Post-capture PCR:

4.1 the reaction was charged as in Table 10 below.

TABLE 10

Components	Volume of
		Hot start enzyme	50μL
Primer, 5. mu.M	10μL
		DNA eluted in the previous step	40μL
Total volume	100μL

4.2 vortex, mix, microcentrifuge, place in PCR instrument, the reaction program is as following table 11:

TABLE 11

4.3 post amplification purification:

4.3.1 taking out the purified magnetic beads, and balancing for 30min at room temperature for later use.

4.3.2 put 180 μ L of purified magnetic beads into a 1.5mL centrifuge tube, add 100 μ L of amplified capture DNA library, mix well with shaking, incubate at room temperature for 15 min.

4.3.3 place on magnetic frame until the liquid is clear, discard the supernatant.

4.3.4 was incubated with 200. mu.L of 80% ethanol for 30sec and discarded. Note that: 80% ethanol is prepared in situ. The 200 μ L80% ethanol wash step was repeated once.

4.3.5 remove the residual ethanol from the bottom of the centrifuge tube with a 10. mu.L pipette tip and dry at room temperature until the ethanol is completely volatilized (see the beads not reflected light on the front and dry on the back). Note that: the yield of DNA produced by magnetic beads dried too much is reduced.

4.3.6 remove the centrifuge tube from the magnetic rack, add 50. mu.L of ultrapure water, shake and mix. Incubate at room temperature for 2 min.

4.3.7 centrifuging for a short time, placing on a magnetic rack until the liquid is clear, and transferring the capture sample into a new centrifuge tube.

4.3.8 quality control: taking 1 mu L of capture sample for Qubit concentration detection. The capture concentration detection uses the Qubit HS detection kit.

Fifth, testing results

The results are shown in Table 12.

TABLE 12

From the results in Table 12 above, it is clear that the detection of panel in example 1 is superior in the detection of point mutation, indels, fusions and copy number variation in tissue samples, using the ddPCR detection result as the gold standard.

Example 3

The kit of embodiment 1 of the invention is used for constructing and capturing cfDNA samples.

Firstly, preparing a verification standard product

20 kinds of mutation standard DNAs were prepared by mixing tumor cell line DNA containing known mutations and wild type cell line GM12878 DNA in a ratio. And the frequency of each mutation in the standard was determined by ddPCR. The tumor cell line DNA is shown in Table 1. The standard DNA was broken to mimic the fragment length of cfDNA (. about.165 bp).

Library construction

1. End-repaired and a-tailed at the 3' end:

1.1 mu.L of cfDNA was taken and less than 50. mu.L was made up to 50. mu.L with nuclease-free water, and the reaction system was charged as shown in Table 2 of example 2.

1.2 vortex mix, microcentrifuge, place in PCR apparatus and reaction procedure as in Table 3 in example 2.

2. Connecting joints:

2.1 Dilute linker (15. mu.M concentration linker) the volume of linker used was 5. mu.L. The adaptor stock concentration was 15. mu.M, and the adaptor stock concentration was diluted in the following Table 13 based on the initial amount of DNA, and stored in a refrigerator at 4 ℃.

Watch 13

Initial volume of building warehouse (ng)	Joint volume (μ L)	Volume (. mu.L) of nucleic-Free ddH2O
			25-50	5	0
10-25	2.5	2.5
			5-10	1	4

2.2 the reagents were added to the reaction tube as described above in Table 4 of example 2.

2.3 vortex mix, microcentrifuge, place in PCR instrument and the reaction procedure is as shown in Table 5 in example 2.

3. Post-ligation purification (see post-ligation purification in example 2 for specific steps).

4. Library amplification (see examples for specific steps).

Obtaining of DNA (1 × Beads recovery)

5.1 subpackage 50 μ L of Beckman Agencourt AMPure XP magnetic beads into a new eight-connected tube.

5.2 after the PCR of the previous step (4.2) is finished, taking out a sample.

5.3 short centrifugation, transfer into 50 u L Beckman Agencourt AMPure XP magnetic bead.

5.4 shaking and mixing evenly, and incubating for 15min at room temperature to ensure that the DNA is fully combined with the magnetic beads. Note that the tube cap is pressed down during shaking.

5.5 centrifuging for a short time, placing the centrifuge tube on a magnetic frame to clarify the liquid, and discarding the supernatant. Note that: do not attract to the magnetic beads.

5.6 Add 200 u L80% ethanol incubation 30sec after discarding. Note that: 80% ethanol is prepared just before use. The 200 μ L80% ethanol wash step was repeated once.

5.7 suck up the residual ethanol at the bottom of the centrifuge tube with a 10 μ L pipette tip, and dry at room temperature for 3-5min until the ethanol is completely volatilized (no reflection is seen on the front side, and drying is seen on the back side). Note that: the yield of DNA produced by magnetic beads dried too much is reduced.

5.8 remove the tube from the magnetic frame, add 40. mu.L of ultrapure water, shake and mix.

5.9 incubate for 5min at room temperature to elute DNA.

5.10 centrifuge briefly, place the tube on magnetic rack until the liquid is clear, transfer the library to a new tube. Stored at-20 ℃.

6. Library quality inspection

Library hybrid Capture

The procedure was as in example 2.

Fifth, testing the result

The results are shown in Table 14.

TABLE 14

According to the results in the table above, the detection kit of example 1 is superior in detection performance for point mutation, indel, fusion and copy number variation of cfDNA samples, with ddPCR detection results as gold standards.

Example 4

Using the kit of example 1 to detect the identity of tissue TMB and full exon kits

1. Extraction of patient genomic DNA, library preparation and probe hybridization capture

In this embodiment, matched samples of 40 patients are selected for detection, a Tiangen blood cell and tissue extraction kit is used for extracting blood cell and tumor fresh tissue DNA, a Yena FFPE DNA extraction kit is used for extracting tumor FFPE DNA, and the operation process is performed according to the product specification. DNA disruption and library preparation were performed as in example 2, and hybrid capture and on-machine sequencing were performed using the kit and full-length probe of example 1, respectively. Sample information is shown in table 15:

watch 15

The cancer species include: endometrial cancer, low-grade serous adenocarcinoma, colon cancer, rectal cancer, ovarian cancer, gastric cancer, sigmoid colon cancer, breast cancer, cholangiocarcinoma, lung cancer, intestinal cancer, gastric cancer, and glioma.

2. Off-line data analysis

Calculating the formula of TMB: number of mutations 1000000/total number of external bases (depth after deduplication >100X), i.e., total number of substitution and insertion/deletion mutations per megabase

The following case will exclude TMB calculations:

(1) performing germline mutation;

(2) mutations with a mutation frequency of less than 5%;

(3) a synonymous mutation;

(4) the depth of the FFPE sample after de-weighting is lower than 100X;

(5) mutation of the repeat region;

(6) black list mutation;

(7) a driver mutation;

(8) genetic mutation sites in the crowd database.

3. The result of the detection

In this example, 163 patients were tested for paired blood cells and FFPE, and the TMB cut-off was determined, with the results shown in table 16:

TABLE 16

TMB-Moderate	TMB-High	TMB-Extra High
			3.804	8.4278	13.7693

As shown by the results of Table 16, the number of mutations was 13.7693 or more, and the TMB level was judged to be Extra High; the number of mutations was between 8.4278 and 13.7693, and was judged to be High.

In this example, the panel and the exon-full probe of example 1 were used to detect paired blood cells and FFPE of 40 patients, and the consistency between the panel and the exon-full probe of example 1 was determined, and the consistency result is shown in FIG. 1.

As can be seen from the results in FIG. 1, the panel (P12) and the full exon probe tested in example 1 have good consistency, and the consistency R between the two probes ² ＝0.9411。

In the embodiment, the exon regions designed aiming at the tumor mutation load (TMB) simultaneously cover 20 point mutations, 4 insertions and 8 fusions, and the common regions of different mutation type information are integrated, so that the uniformity of the panel is improved, the overall data volume of the panel is reduced, the analysis period is shortened, and the cost is reduced.

Example 5

Detection of Panels in example 1 detection of MSI consistent with a one generation approach

Puncturing tissues, surgical tissues or paraffin sections to extract DNA, taking 200ng of DNA to perform covaris interruption, constructing a library by the interrupted sample, using panel 12 to hybridize and capture the library, and sequencing the captured sample on an Illumina NovaSeq platform. The sequence data is analyzed and processed, and the process aims to accurately detect SNP.

2. Off-line data analysis

MSI QC standard: each MSI marker was deduplicated to a depth > 100X.

24 single nucleotide repeat markers were used in the panel MSI assay of example 1. If the number of markers passed QC for a sample is less than 8, the MSI status is QNS. MSI status is HSI-H if unstable locus/pass locus > is 0.3; if the unstable locus/pass site <0.3, the MSI status is HSI-L/MSS. MSI markers are shown in Table 17.

TABLE 17

3. The result of the detection

The test panel of example 1 was adapted for MSI test, and 140 blood cell samples were used for MSI marker selection. 30 blood cell samples from healthy donors were used for p12 MSI baseline establishment. The 40 samples were subjected to GeneCast Panel 12MSI validation by fluorescence pcr-capillary electrophoresis (MSI Analysis System, Version 1.2, Promega) with known MSI status in 40 clinical tumor tissues. The validation results showed that there were 14 MSI-High, 25 MSI-Low/MSS and 1 QNS. The detailed information is shown in Table 18.

Watch 18

Watch 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

Watch 20

TP	TN	FP	FN	Sensitivity of the probe	Specificity of the drug
						14	25	0	0	100％	100％

The identity of the MSI validation method to the fluorescent PCR-capillary electrophoresis method for detecting panel of example 1 is summarized in tables 19 and 20. Sensitivity was 100%, specificity 98.2%, PPV 94.7%, and accuracy 98.7%.

Example 6

Example 1 detection of panel detection of mutations consistent with the results of a Chamber assessment

1. The information table of the evaluation samples for the room is shown in table 21:

TABLE 21

2. Library preparation and probe hybridization capture of chamber assessment samples

The 23 samples (14 CF and 9 FFPE) of this example were subjected to DNA extraction, fragmentation (FFPE samples are fragmented, CF samples are not fragmented), library preparation, probe hybridization capture and on-machine sequencing as in examples 2 and 3.

3. The results are shown in Table 22.

TABLE 22

Continuation table 22

According to the results in Table 22 above, all mutations were detected and were in agreement with the validation results. It is demonstrated that the detection of panel in example 1 is highly accurate.

Example 7

The kit can detect multiple tumor variations at one time, has accurate detection result, provides the drug selection of three drugs of targeted drugs, chemotherapeutic drugs and immunological drugs for patients, and reduces the cost.

The test panel of example 1 clinical application of panel-concomitant diagnosis of targeted drugs.

1. 3 paired clinical samples of this example DNA extraction, disruption (FFPE or fresh tissue samples with no disruption, CF samples with no disruption), library preparation, probe hybridization capture and on-machine sequencing were performed as in example 2 and example 3.

2. The detection result is shown in

TABLE 23

According to the results in table 23 above, the patient has LOW TMB levels, but detected p.l858r mutation in the EGFR gene in tissue DNA, erlotinib, afatinib, gefitinib, erlotinib, or oxitinib may be suitable for use in the subject. The test panel of example 1 is useful for companion diagnostics of targeted drugs.

Example 8

The results of the test panel clinical application of example 1-immunization are shown in Table 24.

1. Clinical samples for this example DNA extraction, disruption (FFPE or fresh tissue samples with no disruption, CF samples), library preparation, probe hybridization capture and on-machine sequencing were performed as in examples 2 and 3.

2. The results are shown in tables 24 and 25.

Watch 24

According to the results in table 24 above, no sensitive and resistant mutations such as EGFR, KRAS, BRAF and ERBB2 were detected in plasma free DNA and tissue DNA; no increase in MET gene copy number and no sensitive/drug-resistant mutations were detected; fusion of ALK, ROS1 and RET genes and other fusion and sensitive and drug-resistant site mutation are not detected. However, the patient has a TMB level of Extra High and mTORC1/2 inhibitor, ramucirumab, necitumumab, afatinib, and anitinib may be suitable for the subject.

TABLE 25

According to the results of the above table 25, no sensitive and resistant mutations such as EGFR, KRAS, NRAS, BRAF, PIK3CA, KIT, PDGFRA, MTOR genes and the like were detected in plasma free DNA and tissue DNA; fusion and sensitive/drug-resistant mutation of ALK, ROS1 and RET genes are not detected; increased ERBB2, MET gene copy number, and sensitive/resistant mutations were not detected. However, the patient had TMB levels of Extra High.

The results of 2 clinical specimens tested in this example demonstrate that the test panel of example 1 can be used as a guide for immunization.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A detection panel for pan-cancer species targeting, chemotherapy and immunization based on next-generation sequencing, which is characterized in that the detection panel comprises pan-cancer species typing, treatment and prognosis related gene mutation for joint detection, tumor mutation load calculation related exon region and SNP site; the pan cancer genotyping, treatment, prognosis related gene mutations and exon regions related to said tumor mutation burden calculation include ABCA13, ABCA8, ABL1, ACARDSB, ACOT13, ADAMTS6, ADRB1, ADSS, AGPAT9, AK7, AKT1, AKT2, AKT3, ALG9, ALK _ fus, ALOX12B, ALS2CR11, AMER1, ANKRA2, ANKRD46, ANO1, APC, APOPT1, AR, ARAF, ARHGAP4, ARHGAP6, ARID1A, ARID4A, ARL6IP A, ARMC A, ARPC 1 ASH 1A, BAT A, ATM, ATP9, CALSB A, CALSB 7, CALSB 2 ATKN 72, CALSD A, CALSK A, CDK A, CALSK 36OCK A, CALSK 36363672, CALSK A, CALSK 3636363636363672, CALSK A, CALSK 363672, CALSK A, CALSK 363672, CALSK A, CALSK 363672, CALSK 36363672, CALSK A, CALSK 363636363636363672, CALSK A, CA, CDKN2C, CDO1, CEBPA, CEP120, CHD1, CHEK1, CHEK2, CIC, CNKSR3, CNOT8, COSM1316144, COSM1316145, COSM1332498, COSM3676668, COSM 37491, COSM5045664, FOCOX 18, CREBP, CRLF2, CSF1R, CSF3R, CTAGE5, CTCF, CTNNB1, CUL3, CXCR4, CYFIP1, CYLD, DAXX, DBT, 2, DEPDC5, PH 5, DICER 5, DIS 5, DNMT 35, 36K 5, DOT 15, DROSHA, DSCAM, DXS 04, DOCS 7132, DSFLOS 23, DYSOFS 391, DYDENFYS 391, DENFET 391, DEFIS 391, DIS 5, DEFI 5, DEFIFODEFIFODEFIFONFET 5, FLE 5, FLF 5, FLDENFET 5, FLF 5, FLDENFDGF 5, 5-5, 5-5, 5-5, 5-5, 5-5, 5-5, 5-5, 3636363672, 5-5, 36363672, 5, 3636363672, 5, 3636363672, 5, 363636363672, 5, 36363672, 5, 363636363636363636363636363636363672, 5, 36363672, 363636363636363636363636363636363672, 5, 3636363636363672, 5, 363636363672, 3636363672, 5, HCAR2, HEY1_ fus, HGF, HLA-A, HLA-B, HLA-C, HNF 1-1A, HNRNPH1, HRAS, HSPA1B, HSPA4, HYOU1, IARS, ID2, ID3, IDH1, IGF 11, IGF1, IKB KE, IKZF1, IL 71, IL 1, INHBA, INPP 41, IPO 1, IR3672, IRF 1, IRS 1, ITMDM, JAK1, JUN, KCNJ 1, KDM 51, KDM 61, KDM 36AP, KEAP1, KIAA 361432, KIF 361433 _ KIF 1, KI 3_ KM 1, MANPK 1, MANPM 1, MANPMX 1, MANPK 1, MANPM 1, MANPK 1, MANPMX 1, MANF 1, MAMP 1, MANPMANF 1, MANF 1, MAKM 1, MANF 1, MAKM 1, MAMP 1, MANF 1, MAKM 1, MANF 1, MAK 1, MAKM 1, MAPK1, MAKMMAKM 1, MAK 1, MAKMMAKMMAKMMAPK 1, MAPK1, MAK 1, MAPK1, MAMMK 1, MAK 36363672, MAMP 1, MAMP 36363672, MAK 3636363672, MAK 3636363636363636363672, MAPK 363672, MAK 1, MAK 363636363672, MAK 1, MAK 36363636363672, MAK 1, MAK 363636363672, MAK 1, MAKMMAK 1, MAK 36363672, MAK 1, MAK 363672, MAK 3636363672, MAK 363672, MAK 36363672, MAMMK 363672, MAK 1, MAMMK 363672, MAK 36K 1, MAK 363672, MAK 36363636363636363672, MAK 1, MAK 3636363636363672, MAK 1, MAK 36363672, MAK 1, MAK 363672, MAK 1, MAK 36K 1, MAK 36K 36363636363636363636363636K 36K 1, MAK 1, MA, NOTCH3, NPM1, NR-21, NR-24, NR4A3_ fus, NRAS, NSD1, NT5C2, NTRK1, NTRK1_ fus, NTRK2, NTRK2_ fus, NTRK3, NTRK3_ fus, NUP85, NUP93, P2RY8, PALB 8, PAPOLG, PAQR8, PARK 8, PARP 8, PAX 8, PBRARARADR 8, PDCD1 36LG 72, PDE 68, PDGFRPDGFB, PDPN, PIK 8, PIK3C 28, PIK3 RADR 8, PIK 38, PIK 8, PIR 8, PSRAFH 8, PSRAPR 8, PSRADR 8, PSRAPR 8, PSRADR 8, PSRAPR 8, PSR 8, PSRAPR 8, PSR 8, PSRAPR 8, PSR 8, PSRAPR 8, PSR 8, PSRAPR 8, PSR 8, PSRAPR 8, PSR 8, PSRAPR 8, PSR 8, 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, TRZ 2A, TRIM A, TSC HR, TSN, TXD A, TSU 2AF A, UBE2E A, UBE 3A, ULK A, UTF A, VEGFA, VHL, VSVS3672, WHIG A, WHIG 1, ZBF 72, ZNF A, ZNF 36711, WZNF 36Z A, WZNF 36711, WTC A, and WW 36711; the SNP locus comprises rs1045642, rs1052555, rs10981694, rs11045585, rs1105525, rs1130214, rs1138272, rs115232898, rs11572080, rs11598702, rs11615, rs12613732, rs12762549, rs13181, rs151264360, rs1517114, rs1570360, rs1650697, rs 723, rs1695, rs1799793, rs 1801011019, rs1801131, rs1801133, rs 1261801803695, rs1805087, rs183484, rs1934951, rs2032582, rs 207262671, rs2075252, rs2228001, rs2273618, rs 2291221877, rs 22769722595, rs 25094752, rs 254873, rs 8484709, rs 278484709, rs 27288963, rs 3213128899, rs 3219179674762, rs 2979675639, rs 4467777946, rs 41797946, rs 4179797946, rs 4477777947, rs 41797756775648, rs 44777777777947, rs 417947, rs 42775648, rs 42777947, rs 4277817947, rs.

2. Use of the next-generation sequencing-based panel for pan-cancer species targeting, chemotherapy and immunization of claim 1 in the preparation of a device for the joint detection of pan-cancer species-typing, treatment, prognosis-related genetic mutations, SNP sites and exon regions associated with tumor mutation burden calculation.

3. The use of claim 2, wherein the pan-cancerous species comprises: lung cancer, intestinal cancer, gastric cancer, liver cancer, breast cancer and endometrial cancer.

4. The use according to claim 2, wherein said combined detection comprises detection of a plurality of mutation types, said detection comprising: point mutations, fusions, copy number variations, deletions and insertion mutations.

5. Use according to claim 2, wherein the device is a device for the concomitant diagnosis of a targeted drug, a chemotherapeutic drug or an immunological drug.