CN113736878A - Gene panel for detecting nervous system tumor, kit and application thereof - Google Patents

Abstract

The invention belongs to the technical field of multi-gene detection of nervous system tumors, and discloses a gene panel for detecting nervous system tumors. The multi-gene screening list is used as a probe, high-risk genes are detected in a targeted manner through hybridization capture sequencing, gene mutation which has guiding significance on diagnosis and treatment can be detected more efficiently, the gene detection cost of patients and the operation difficulty of experimenters can be reduced, meanwhile, certain tumor patients can be helped to participate in clinical tests, and the multi-gene screening list has important guiding significance on clinical diagnosis and target point discovery of targeted treatment.

Description

Gene panel for detecting nervous system tumor, kit and application thereof

Technical Field

The invention belongs to the technical field of multi-gene detection of nervous system tumors, and relates to a gene panel based on nervous system cancer detection, a kit and application thereof.

Background

The nervous system includes the central nervous system and the peripheral nervous system, and the tumors of the nervous system include primary intracranial tumors and gliomas, most of which are primary intracranial tumors. Symptoms caused by intracranial tumors include local neurological symptoms caused by tumor compression or damage to surrounding brain tissue, and symptoms caused by intracranial space occupying lesions, such as increased intracranial pressure.

With the development of high-throughput sequencing, the deep genetic research, the continuous confirmation of the relationship between cancer and genetic variation, and the important guidance for the design of targeted drugs and new therapeutic means. In order to further explore the relationship between the variation of key genes and cancers, deepen the application of a sequencing technology in clinical treatment and ensure that target sequencing is carried forward, the sequencing time is effectively shortened and the cost is reduced by sequencing the specified genes, so that the method is more suitable for clinical application and realizes precise medical treatment. The determination of the target sequencing detection gene is an important prerequisite, and the preparation of the detection probe is a necessary means for realizing the target sequencing.

The current diagnostic method for nervous system tumor mainly depends on the traditional location and qualitative of clinical symptoms and signs of patients, and special examination items, such as cerebrospinal fluid examination, traditional X-ray examination and CT scanning, and no detection method combined with high-throughput sequencing is reported.

Disclosure of Invention

The invention selects TCGA database, MSKCC-IMPACT related database, foundation one CDx and common genes closely related to mainstream cancer target treatment in the paper, based on the second generation sequencing technology, utilizes a biological probe hybridization method to enrich important exon regions and partial intron regions of 454 genes, and detects and analyzes contents including somatic mutation, germ line mutation, copy number variation, fusion gene and other variations, aiming at achieving accurate detection.

In a first aspect, the invention provides a gene panel for detecting nervous system tumors, wherein the panel comprises a mutant gene, a copy number variation gene and a fusion gene which are relevant to the prognosis of detecting nervous system cancers.

Further, the mutant gene includes the following genes: ABL1, AMER1, ASXL1, BAP1, BMR1A, CARD11, CD274, CDK4, CHD2, CTCF, ABL2, ANKRD11, ASXL 11, BARD 11, BRAF, CASP 11, CD276, CDK 11, CHEK 11, CTLA 11, ACVR 11, APC, ATM, BCL 11, BRCA 11, CBFB, CD 11, CDK 11, CHEK 11, CTNNA 11, ACVR1 11, AR, ATR, BCL2L 11, BRCA 11, CBL, CD 11, CDKN1 11, CIC, CTB 11, ADGRA 11, ARAF, ATRX, ATRL 2L 11, BRD 11, CD 11, CARD3672, CARD11, CARDC 11, CARDCARDC 11, CARDC 11, CARDCARDC 11, CARDC 11, CARDCARDCARDC 11, CARDC 363672, CARDC 11, CARDC 36363636363672, CARDC 11, CARDC 36363672, CARDC 11, CARDC 36363672, CARDC 11, CARDC 363636363636363672, CARDC 363672, CARDC 11, CARDC 36, GATA, GRM, HRAS, DDX3, EIF4A, ERBB, ETV, FANCL, FGF, FLT, GATA, GSK3, HSD3B, DICER, ELF, ERBB, ETV, FAS, FGF, FLT, GATA, H-3, DIS, EP300, ERCC, ETV, FAT, FGF, FOXA, GID, H3C, IDH, DNMT, EPCAM, ERCC, EWSR, FAT, FGFR, FOXL, GL, H3C, IDH, DNMT3, EPHA, ERCC, EZH, FBXW, FGFR, FOXO, GNA, HDAC, IGF1, DNMT3, EPHA, ERCC, FANCA, FGF, FUGR, GNA, HDACA, HDAC, KMBKE, DOT1, EPHA, ERF, FGF, FACNCC, FGFR, NOTTN, NOTTF, NOTCH, ZF, MAPK, NOTCH, MAPK, MTK, MAPK, KMBK, MAF, MTK, KM, MTK, MAF, MTK 2, MTK, MTF, MTK, MTF, MTK, MTF, MTK, MAP2K2, MDM4, MKNK1, MUTYH, NF1, NRAS, P2RY8, IRS1, KDM6A, KRAS, MAP2K4, MECM, MLH1, MYRADC, NF2, NSD1, PAK5, IRS2, KDF, LATS1, MAP3K 2, MED 2, MP, MYRADC, NFE2L2, NSD 2, PALB2, JAK2, KEAP 2, LATS2, MAP3K 2, MEF 22, MRE 2, MYCN, NFKBIA, 5C2, PARP2, JAK2, PRK 2, PRNPFLD 2, PRNPSLCP 2, PRNPPLS 2, PRS 2, PRNPPARP 2, PRS 2, PRNPS 2, PRS 2, PRNPPARP 2, PRNPS 2, PRS 2, PRACK 2, PRAC, SOX, PDK, PIK3R, PPM1, PRSSB, RAC, RAF, RET, SOHA, SMAD, SOX, PECAM, PIM, PPP2R1, PTCH, RAC, RANBP, RICTOR, SDHB, SMAD, SOX, SPEN, STAT, STK, TCF, TET, TNFAIP, TRAF, TYK, VEGFA, ZBTB, SPOP, STAT, SUFU, TCF, TET, TNFRSF, TRRAP, TYRO, VHL, ZFLX, SPRED, STAT5, SYK, TCF7L, TGFBR, TOP2, TSC, U2AF, WT, ZYM, SRC, TSSTATB, TAF, TEK, TIPARP, USP, TSC, YAF, XPO, ZYM, STAG, STK, TBX, TERT, PRHR, TP, TSLA, TSTP, TSF 703 ZNF, XRF, XRAP.

Further, the gene copy number variation includes the following genes: ABL1, BIRC3, CD74, CTLA4, ERBB3, EZH2, GSK3B, KDM5A, MAPK A, NBN, AKT A, BLM, CDK A, CYLD, ERCC A, FANCA, HDAC A, KDR, MAX, NF A, AKT A, BMPR1A, CDK A, EED, ERCC A, FANCD A, HGF, KEAP A, MDM A, NF A, AKT A, BRAF, CDKN2A, EGFR, ERF, FBXW A, HRAS, KIT, MET, NOTCCH A, ALK, BRCA A, CDKN2A, EIF4A A, ESR A, IDH A, KMT A, KMH A, KMCH A, CTAS A, CTLA A, EPT A, EPR A, EPT A, EPR A, EPT A, EPT A, EPR A, EPT A, EPT A, EPT A, EPR A, EPT A, EPR A, EPR A, EPR A, EPR A, EPT A, EPR A, EPR A, EPR A, EPR A, EPR A, EPR A, EPR A, EPR A, EPR A, EPR A, EPR, VEGFA, PIK3R1, PPP2R1A, RAC1, RAD51C, RECQL, RNF43, SDHA, SOS1, TP 53.

Further, the fusion gene includes the following genes: ATF1, ETV1, EWSR1, FGFR3, MET, NPM1, NTRK1, NTRK2, NTRK3, PDGFRA, BRAF, ALK, ROS1, RELA, YAP 1.

In a second aspect, the present invention provides a probe panel for detecting a tumor in the nervous system, wherein the probe panel is a detection probe for the mutant gene, the copy number variant gene and the fusion gene according to any one of claims 1 to 4.

In a third aspect, the present invention also provides a kit for detecting a tumor in the nervous system, which comprises at least one dose of the probe panel described in the second aspect above.

In a fourth aspect, the use of the gene panel described above or the probe panel described above for the preparation of a device for the detection of tumors in the nervous system.

In a fifth aspect, the present invention also provides a device for detecting a tumor in the nervous system, comprising:

the sequencing module is used for extracting DNA of a sample to be tested and carrying out high-throughput sequencing to obtain a sequencing result;

a comparison module for processing the result of the high-throughput sequencing and comparing the data with the gene panel of any one of claims 1 to 4 to obtain mutation information;

and the analysis module is used for analyzing the obtained mutation information to obtain a medication scheme.

In a sixth aspect, the present invention also provides a method for diagnosing and detecting tumors in the nervous system, comprising the steps of,

step S1: obtaining a detection sample DNA (the sample DNA comprises DNA tissue sample DNA and cfDNA of a blood sample), extracting the DNA of a focus tissue and a paracarcinoma or blood tissue of a patient, and optionally extracting paraffin-embedded tissues.

Step S2: constructing a sample DNA library, randomly fragmenting DNA into small fragments of several hundred bases or less, and then preparing the library by adopting a KAPA library construction kit. The method mainly comprises the following steps: DNA breaking, filling in the tail end, adding ' A ' at the 3 ' end, connecting by a joint, purifying, amplifying a library and purifying. After library preparation was complete, it was stored at-20 ℃.

Step S3: constructing a detection probe for detecting the panel, hybridizing with a DNA library, capturing and sequencing; whole genome or whole exome sequencing can also be performed directly.

Step S4: and (4) carrying out biological information analysis on the sequencing result to obtain a sample mutation result.

Wherein the sequencing result is a fastq file, and the sequencing result is analyzed by using an algorithm for acquiring the gene mutation issued by Broad to obtain the gene mutation result and annotate the gene mutation result. The main steps include the quality control of fastq files, genome association, analysis of somatic mutation (somatic mutation) and germ cell mutation (germline mutation), and annotation. The software used respectively had: the fastqc and fastx _ toolkit are used for quality control; bwa, and gatk and mutect2 obtain somatic mutation; obtaining embryo cell mutation by a HaplotpypeCaller method; ANNOVAR is annotated.

Step S5: comparing the sample mutation result with the detection panel to obtain the sample gene mutation result, and combining clinical information to obtain the final diagnosis.

Compared with the prior art, the invention has the beneficial effects that:

1) the invention selects TCGA database, MSKCC-IMPACT related database, foundation one CDx and common genes closely related to mainstream cancer target treatment in the paper, based on the second generation sequencing technology, utilizes a biological probe hybridization method to enrich important exon regions and partial intron regions of 454 genes, and detects and analyzes contents including somatic mutation, germ line mutation, copy number variation, fusion gene and other variations, aiming at achieving accurate detection.

2) The multi-gene screening list is used as a probe, high-risk genes are detected in a targeted manner through hybridization capture sequencing, gene mutation which has guiding significance on diagnosis and treatment can be detected more efficiently, the gene detection cost of patients and the operation difficulty of experimenters can be reduced, meanwhile, certain tumor patients can be helped to participate in clinical tests, and the multi-gene screening list has important guiding significance on clinical diagnosis and target point discovery of targeted treatment.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments will be briefly described below.

FIG. 1 is a diagram showing the results of quality evaluation of DNA sample extraction and sequencing in examples.

FIG. 2 shows the result of detection of gene germline mutation.

FIG. 3 shows the result of somatic mutation (physiological mutation) detection.

FIG. 4 shows the results of the detection of multiple gene copy number variation in tissues.

FIG. 5 shows the gene mutation and reference dosing regimen (partially shown).

FIG. 6 is the result of microsatellite instability (MSI).

FIG. 7 shows the results of detection of DNA mismatch repair (MMR) -associated genes.

FIG. 8 shows the results of Tumor Mutation Burden (TMB) detection.

Detailed Description

The invention will be better understood from the following examples. However, it is easily understood by those skilled in the art that the description of the embodiment is only for illustrating and explaining the present invention and is not for limiting the present invention described in detail in the claims. Unless otherwise specified, reagents, methods and equipment used in the present invention are conventional methods, and test materials used therein are available from commercial companies, unless otherwise specified.

Examples

1. DNA was extracted from a blood sample of one patient and purified using the QIAsymphony automated platform.

2. Fragment screening

2.1, firstly, quantifying the DNA concentration of the sample, if the total amount of 20 mul reaches 30ng and above, the normal process does not reach 30ng, adding more, concentrating (the maximum volume can be 30 mul, and the water is correspondingly decreased) and the like to reach 20-30ng, continuing the experiment, if the concentration is too low, such as about 0.5 of the initial concentration, at least adding more and then ensuring that the experiment has 10ng of DNA.

2.2 taking the AMPure XP magnetic beads out of the refrigerator half an hour in advance and balancing to room temperature; fresh 80% EtOH was prepared (40 ml absolute ethanol was added to 10ml enucleated acid water NFW).

2.3 shaking the AMPure XP magnetic beads which are balanced to room temperature for not less than 1min or completely mixing the AMPure XP magnetic beads uniformly, taking 20-30 mul of prepared sample DNA, adding water to replenish the sample DNA to 30 mul, adding 10.8 mul of magnetic beads, mixing the sample DNA uniformly, taking 40.8 mul of supernatant, adding 50 mul of magnetic beads again, purifying, abandoning 8.4.3 of the supernatant, placing the round bottom PP plate on a magnetic frame for 5min until the magnetic beads are completely adsorbed on the tube wall, and clarifying the solution.

And 2.4, sucking the supernatant by using a liquid transfer device and abandoning the supernatant, so as to ensure that the gun head cannot suck the magnetic beads. Add 200. mu.l of fresh 80% ethanol to round bottom PP plate, let stand for 30 seconds and remove all supernatant to ensure that no beads were aspirated. Repeat 8.15 times with 80% ethanol for a total of two washes. The round bottom PP plate was dried for 5min until the magnetic beads were completely air dried. Adding 52 mu l of deionized water into the round bottom PP plate in the previous step, covering a sealing film, oscillating until the magnetic beads are completely mixed, centrifuging briefly, and incubating at room temperature for 5 min. The round bottom PP plate is put back on the magnetic frame and is kept stand for 5min at room temperature or the solution is completely clear.

2.5 transfer 50. mu.l of supernatant to a new 96-well plate to ensure that no beads are pipetted.

3. End repair and addition of A

3.1 taking out the End Repair & A-Tailing Buffer and the End Repair & A-Tailing Enzyme in advance, placing the mixture on ice for melting, slightly whirling and briefly centrifuging after melting, preparing the End Repair & A-Tailing Mix according to the following proportion, shaking, uniformly mixing

3.2 End Repair & A-Tailing reaction System (this operation is carried out on an ice box):

Component	60μl Reaction
		DNA	50μl
End Repair&A-Tailing Buffer(purplecap)	7μl
		End Repair&A-Tailing Enzyme(purple cap)	3μl

3.3 filling DNA in 9.1.11 to 50 μ l, then adding 10 μ l prepared End Repair & A-labeling Mix to 8.1.10 prepared DNA sample, shaking and mixing evenly, sealing a sealing film and putting into a PCR instrument.

3.4 set up PCR program (volume 60. mu.l without hot lid):

3.5 after the reaction of the PCR instrument is finished, taking out the 96-well plate

4. Add the piecing

4.1 remove the Ligation Buffer in advance, put the idt adapter mix (20. mu.M) on ice to melt, and remove the DNA Ligase after the reaction of the previous step is completed.

4.2 preparation of the Ligation reaction system (this operation was carried out on ice boxes):

Component	60μl Reaction
		Ligation Buffer	30μl
DNA Ligase(yellow cap)	10μl
		adapter Mix(20μM)(brown cap)	4μl
Nuclease free water	6μl
		preparation of the product in the previous step	60μl

4.3 adding 50 mul of prepared LigationMix to 8.2.5DNA sample, shaking and mixing evenly, sealing a sealing film and putting into a PCR instrument.

4.4 set up PCR program (volume 100. mu.l without hot lid):

STEP	TEMP	TIME
			incubation	20℃	15min
Heat preservation	4℃	∞

4.5 after the reaction of the PCR instrument is finished, taking out the 96-well plate and placing the 96-well plate on a4 ℃ or ice box.

5. Magnetic bead purification

5.1 taking the AMPure XP magnetic beads out of the refrigerator half an hour in advance and balancing to room temperature; fresh 80% EtOH was prepared (40 ml absolute ethanol was added to 10ml enucleated acid water NFW).

5.2 shaking the AMPure XP magnetic beads which are balanced to room temperature for not less than 1min or completely mixing the AMPure XP magnetic beads uniformly, taking 95 mul of magnetic beads to a new round bottom PP plate, sucking 95 mul of DNA reaction sample in 8.3.5 out, mixing the DNA reaction sample with the magnetic beads uniformly, and incubating for 5min at room temperature.

5.3 the round bottom PP plate is placed on a magnetic frame for 5min until the magnetic beads are completely adsorbed on the tube wall, and the solution is clarified.

And 5.4, sucking the supernatant by using a liquid transfer device, and abandoning to ensure that the gun head cannot suck the magnetic beads.

5.5 Add 190. mu.l fresh 80% ethanol to round bottom PP plate, let stand for 30 seconds and remove all supernatant to ensure that no beads were aspirated.

5.6 repeat 8.45 times with 80% ethanol and wash twice in total.

5.7 round bottom PP plates were dried for 5min until the beads were completely air dried.

5.8 Add 31. mu.l NFW to the previous round bottom PP plate, cover the sealing membrane, shake until the beads mix well, centrifuge briefly, incubate for 5min at room temperature.

5.9 put the round bottom PP plate back on the magnetic rack and stand for 5min at room temperature or the solution is completely clear.

5.10 transfer 30. mu.l of supernatant to a new 96-well plate, ensure that no beads are pipetted.

6. Pre-hybridization pcr amplification

6.1 in advance primer Mix96 pore plate, 5 x Herc u laser II Reaction Buffer, Herc u laser II Fusion DNA Polymerase (red cap), 100Mm dNTP from-20 ℃ refrigerator, ice melting, slightly vortex mixing, according to the following ratio preparation of Pre-PCR Reaction Mix, oscillation mixing, brief centrifugation.

6.2 mix with Pre-PCR reaction system (this operation was performed on ice box):

Component	50μl Reaction
		nuclease-free water	3.5μl
primer mix	5μl
		100Mm dNTP(green cap)	0.5μl
5×Herculase II Reaction Buffer(clear cap)	10μl
		Herculase II Fusion DNA Polymerase(red cap)	1μl
preparation of the product in the previous step	30μl

6.3 Add 20. mu.l of the sample in the 96-well plate from Pre-Capture PCR Reaction Mix to 8.4.10, pipette up and down, Mix well, cover the sealing film and put into the PCR instrument.

6.4 set up PCR program (using hot lid, volume 50. mu.l):

6.5 after the reaction in the PCR instrument is finished, taking out the 96-well plate, and centrifuging briefly. Tumor sample DNA 14 circulates, blood sample DNA 11 circulates.

7. Magnetic bead purification

7.1 taking the AMPure XP magnetic beads out of the refrigerator half an hour in advance and balancing to room temperature; fresh 80% EtOH was prepared (40 ml absolute ethanol was added to 10ml enucleated acid water NFW).

7.2 shaking the AMPure XP magnetic beads which are balanced to room temperature for not less than 1min or completely mixing the AMPure XP magnetic beads uniformly, taking 50 mul of magnetic beads to a new round bottom PP plate, sucking out a DNA reaction sample in 8.5.5, mixing the DNA reaction sample with the magnetic beads uniformly, and incubating for 5min at room temperature.

7.3 the round bottom PP plate is placed on a magnetic frame for 5min until the magnetic beads are completely adsorbed on the tube wall, and the solution is clarified.

7.4 use the pipettor to absorb the supernatant and abandon, ensure that the rifle head can not absorb the magnetic bead.

7.5 Add 200. mu.l fresh 80% ethanol to round bottom PP plate, let stand for 30 seconds and remove all supernatant to ensure that no beads were aspirated.

7.6 repeat 8.6.5 with 80% ethanol for a total of two washes.

7.7 round bottom PP plates were dried for 5min until the beads were completely air dried.

7.8 tumor sample DNA and blood sample DNA were added to round bottom PP plates of 22. mu.l DI water and 50. mu.l DI water to 8.6.7, respectively, covered with a sealing membrane, shaken until the beads were thoroughly mixed, briefly centrifuged, and placed on the sample plate for incubation at room temperature for 5 min.

7.9 put the round bottom PP plate back to the magnetic rack and stand at room temperature for 5min or the solution is completely clear.

7.10 transfer 20. mu.l of supernatant to a new 96-well plate, ensure that no beads are pipetted.

8. Library quality inspection

Taking 1 mul of library, and detecting the concentration of the library by using a microplate reader, wherein the total amount of the DNA sample of the tumor sample is not less than 500ng, and the total amount of the DNA of the blood sample is not less than 750 ng.

9. Preparation before hybridization

9.1 Each hybridization reaction requires several samples to be mixed in the instrument and the volume required for each sample in each hybridization reaction is calculated from the concentration results measured in the previous step.

9.2 the samples are added into a new 1.5ml centrifugal tube in sequence according to the volume calculated in the previous step, each hybridization system is concentrated to be below 6 mul under the condition of not higher than 30 ℃ by using a vacuum concentrator, the solution is prevented from being completely evaporated, NFW is added to be supplemented to be 6 mul, the mixture is briefly centrifuged for about 30s by oscillation, the samples are completely transferred into a new 96-well plate, a sealing film is sealed, and the new 96-well plate is placed in a refrigerator at the temperature of minus 20 ℃ for storage for later use.

10. Preparation of the hybrid System

10.1 taking out in advance "Probe pen, Universal bottles, Blocking Solution" and placing on an ice box for melting.

10.2 DNA was formulated in such amounts that the total amount of DNA per reaction system exceeded 2000ng but was not more than 4 ug.

The basic amount of hybridization was 400ng of tumor sample DNA, 50ng of paired tissue sample DNA, an appropriate increase of 100ng if the total amount of original DNA was below 20ng, and an appropriate decrease of 100ng if the total amount of DNA was above 50 ng.

10.3, mixing the samples according to calculation, shaking and uniformly mixing, and preparing the hybridization agent for the mixed samples according to the following system. (this operation was performed on an ice box):

Component	8μl Reaction
		probe panel	4μl
Universal Blockers	8μl
		Blocking Solution	5μl

10.4 the pre-hybridization reagent mixed above is dried in a vacuum concentrator (30 ℃).

10.5 Fast Hybridization Mix was removed, incubated at 65 ℃ for 10min or until all pellet dissolved, vortexed rapidly and resuspended in 20. mu.l to 9.8.4 lyophilized sample, gently flicked at the fingertip, avoiding mixing and avoiding air bubbles.

Note that: if the resuspended reagent is needed, it is transferred to another tube for hybridization, gently mixed with fingertips and left to stand for 5min to allow the reagent to be fully resuspended.

10.6 Rapid centrifugation to remove air bubbles, adding 30. mu.l of Hybridizatio Enhancer to the surface of the above reagent, and then placing in a preheated PCR instrument.

STEP	TEMP	TIME
			Incubation	95℃	5min
	60℃	15min-4h (2 h in general)

11. Elution is carried out

11.1 Binding Beads were removed in advance and allowed to equilibrate at room temperature for at least 30 min. The Fast binding Buffer, Fast Wash Buffer1 and Wash Buffer2, if any precipitate, were removed and placed at 48 ℃ until dissolved (450. mu.l of the Fast binding Buffer1 to 66 ℃ C., 700. mu.l of the Wash Buffer2 to 48 ℃ C. were preheated for each reaction).

11.2 vibrate the pre-equilibrated Binding Beads until fully mixed, add 100. mu.l of magnetic Beads to a 1.5ml centrifuge tube.

11.3 Add 200. mu.l Fast binding Buffer and blow-mix with a tip.

11.4 placing the centrifuge tube on a magnetic frame for 1min or until the solution is clear, discarding the supernatant, taking off the centrifuge tube

11.5 repeat the above step 2 times for a total of 3 times.

11.6 after the last wash, 200. mu.l Fast binding Buffer was added and resuspended with shaking to mix well.

11.7 step 9.8.5 after hybridization, the PCR lid was opened and the hybridization solution was quickly transferred to the well-equilibrated magnetic beads.

Note that: the key step of quickly transferring the reagents in the PCR instrument to the magnetic beads is to directly load the PCR instrument without taking out the hybridization tube to prevent the temperature of the hybridization solution from dropping

And 11.8, fully and uniformly mixing the magnetic beads added with the Fast binding Buffer on a Shaker, a rock or a rotator at room temperature for 30 min.

11.9 taking down the centrifugal tube from the blending instrument, quickly centrifuging, placing on a magnetic frame for 1min, and removing the supernatant.

11.10 the tube was removed, 200. mu.l of preheated Fast Wash Buffer1 was added, mixed well and incubated at 66 ℃ for 5min

11.11 placing the centrifuge tube on a magnetic frame for 1min to remove the supernatant, and taking down the tube.

11.12 repeat (steps 9.9.10-9.9.11)2 times for a total of 3 washes.

11.13 adding 200 μ l preheated Fast Wash Buffer2, mixing, incubating at 48 deg.C for 5min, placing the centrifuge tube on magnetic frame for 1min to remove supernatant.

11.14 repeat (step 9.9.13)2 times, a total of 3 washes, and the last wash is blotted with a 10. mu.l tip, 45. mu.l of water is added, and the solution is incubated on ice.

12. Post hybrid Capture PCR amplification

12.1 taking out the amplification primer, KAPA HiFi HotStart ReadyMix from a refrigerator at-20 ℃, melting on ice, slightly whirling and mixing uniformly, preparing a Post-Capture PCR Reaction Mix according to the following proportion, and shaking and mixing uniformly:

12.2 matching Post-PCR Mix reaction system:

Component	50μl Reaction
		KAPA HiFi HotStart ReadyMix	25μl
amplification primers	2.5μl
		Samples comprising magnetic beads	22.5μl

12.3 set up PCR program (using hot lid, volume set 50 ul):

12.4 after the reaction of the PCR instrument is finished, taking out the PCR tube and centrifuging for a short time

Magnetic bead purification

13.1 taking the AMPure XP magnetic beads out of the refrigerator half an hour in advance and balancing to room temperature; preparing fresh 80% EtOH (40 ml absolute ethanol is added with 10ml stoning acid water NFW)

13.2 shaking and balancing AMPure XP magnetic beads to room temperature for not less than 1min or completely mixing, taking 40ul of magnetic beads to a new round bottom PP plate, sucking out DNA reaction samples in 9.10.4, mixing with the magnetic beads uniformly, and incubating for 5min at room temperature

13.3 placing the round bottom PP plate on a magnetic frame for 5min until the magnetic beads are completely adsorbed on the tube wall, and clarifying the solution

13.4 use the pipettor to suck the supernatant and abandon, ensure that the rifle head can not suck the magnetic bead

13.5 Add 200ul fresh 80% ethanol to round bottom PP plates, let stand for 30 sec and remove all supernatant to ensure that no beads were aspirated

13.6 repeat 9.65 with 80% ethanol and wash twice in total

13.7 drying the round bottom PP plate for 5min until the magnetic beads are completely dried

13.8 Add 30ul TE to 9.66.7 round bottom PP plate, cover with sealing film, shake until magnetic beads are mixed well, centrifuge briefly, incubate for 5min at room temperature

13.9 put the round bottom PP plate back to the magnetic rack, stand for 5min at room temperature or completely clarify the solution

13.10 transfer 29ul of supernatant to a new 96 well plate to ensure that beads are not pipetted

14. And (6) analyzing the data.

The original file for analysis is fastq format data. Bwa is used for matching, and a haplotypeCaller method is used for obtaining germline mutation; somatic mutation analysis of tissue samples, using muttec 2, was then annotated using ANNOVAR. Wherein bwa is set by default parameters, mutation analysis and parameters of subsequent steps. The specific contents are as follows:

14.1 mutation analysis

The textual mutation analysis uses MuTect2 in GATK:

VarDict/bin/VarDict-G Homo_sapiens_assembly19.fasta-f$AF_THR-N test\

-b test.consensus.merge.filter.BAM\

-z-c 1-S 2-E 3-g 4-th 4target.bed|\

VarDictJava/VarDict/teststrandbias.R|\

VarDictJava/VarDict/var2vcf_valid.pl-N test-E-f$AF_THR>test.vcf

14.2 Germline mutation analysis HaplotpypeCaller in GATK was used:

java-jar GenomeAnalysisTK.jar\

-R Homo_sapiens_assembly19.fasta\

-T HaplotypeCaller\

-I sample1.bam\

-o output.raw.snps.indels.vcf

14.3 copy number variation analysis using Varscan:

java-jar～/biosoft/VarScan/VarScan.v2.3.9.jar copynumber-sample1.bam--mpileup 1

java-jar～/biosoft/VarScan/VarScan.v2.3.9.jar copyCaller\

ESCC13-T1_recal.copynumber--output-file varScan.T1.cnv.called

14.4 analysis of mutation results output vcf files were annotated by ANNOVAR:

convert2annovar.pl-format vcf4-allsample-withfreq sample1.vcf>

sample1.avinput

table_annovar.pl sample1.avinput～/Biosoft/annovar/humandb/-buildverhg19

-out sample1-remove-protocol

refGene,knownGene,ensGene,cytoBand,genomicSuperDups,avsnp150,avsift,clinvar_20180603,cosmic88_coding,cosmic88_noncoding,esp6500siv2_all,1000g2015aug_all,1000g2015aug_afr,1000g2015aug_eas,1000g2015aug_eur,ljb26_all,exac03-operation g,g,g,r,r,f,f,f,f,f,f,f,f,f,f,f,f-nastring.-tsvout

the database used by ANNOVAR is as follows: refGene, KnowGene, ensGene,

cytoBand,genomicSuperDups,avsnp150,avsift,clinvar_20180603,

cosmic88_coding,

cosmic88_noncoding,esp6500siv2_all,1000g2015aug_all,1000g2015aug_afr,

1000g2015aug_eas,1000g2015aug_eur,ljb26_all,exac03

15. and after the final result of the sample is obtained, the final result is the content presented by the attached drawing of the invention.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are given by way of illustration of the principles of the present invention, and that various changes and modifications may be made without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (8)

1. A gene panel for detecting nervous system tumors, wherein the gene panel comprises a mutant gene, a copy number variant gene and a fusion gene which are related to prognosis of detecting nervous system cancers.

2. The gene panel according to claim 1, wherein the mutated gene comprises the following genes: ABL1, AMER1, ASXL1, BAP1, BMR1A, CARD11, CD274, CDK4, CHD2, CTCF, ABL2, ANKRD11, ASXL 11, BARD 11, BRAF, CASP 11, CD276, CDK 11, CHEK 11, CTLA 11, ACVR 11, APC, ATM, BCL 11, BRCA 11, CBFB, CD 11, CDK 11, CHEK 11, CTNNA 11, ACVR1 11, AR, ATR, BCL2L 11, BRCA 11, CBL, CD 11, CDKN1 11, CIC, CTB 11, ADGRA 11, ARAF, ATRX, ATRL 2L 11, BRD 11, CD 11, CARD3672, CARD11, CARDC 11, CARDCARDC 11, CARDC 11, CARDCARDC 11, CARDC 11, CARDCARDCARDC 11, CARDC 363672, CARDC 11, CARDC 36363636363672, CARDC 11, CARDC 36363672, CARDC 11, CARDC 36363672, CARDC 11, CARDC 363636363636363672, CARDC 363672, CARDC 11, CARDC 36, GATA, GRM, HRAS, DDX3, EIF4A, ERBB, ETV, FANCL, FGF, FLT, GATA, GSK3, HSD3B, DICER, ELF, ERBB, ETV, FAS, FGF, FLT, GATA, H-3, DIS, EP300, ERCC, ETV, FAT, FGF, FOXA, GID, H3C, IDH, DNMT, EPCAM, ERCC, EWSR, FAT, FGFR, FOXL, GL, H3C, IDH, DNMT3, EPHA, ERCC, EZH, FBXW, FGFR, FOXO, GNA, HDAC, IGF1, DNMT3, EPHA, ERCC, FANCA, FGF, FUGR, GNA, HDACA, HDAC, KMBKE, DOT1, EPHA, ERF, FGF, FACNCC, FGFR, NOTTN, NOTTF, NOTCH, ZF, MAPK, NOTCH, MAPK, MTK, MAPK, KMBK, MAF, MTK, KM, MTK, MAF, MTK 2, MTK, MTF, MTK, MTF, MTK, MTF, MTK, MAP2K2, MDM4, MKNK1, MUTYH, NF1, NRAS, P2RY8, IRS1, KDM6A, KRAS, MAP2K4, MECM, MLH1, MYRADC, NF2, NSD1, PAK5, IRS2, KDF, LATS1, MAP3K 2, MED 2, MP, MYRADC, NFE2L2, NSD 2, PALB2, JAK2, KEAP 2, LATS2, MAP3K 2, MEF 22, MRE 2, MYCN, NFKBIA, 5C2, PARP2, JAK2, PRK 2, PRNPFLD 2, PRNPSLCP 2, PRNPPLS 2, PRS 2, PRNPPARP 2, PRS 2, PRNPS 2, PRS 2, PRNPPARP 2, PRNPS 2, PRS 2, PRACK 2, PRAC, SOX, PDK, PIK3R, PPM1, PRSSB, RAC, RAF, RET, SOHA, SMAD, SOX, PECAM, PIM, PPP2R1, PTCH, RAC, RANBP, RICTOR, SDHB, SMAD, SOX, SPEN, STAT, STK, TCF, TET, TNFAIP, TRAF, TYK, VEGFA, ZBTB, SPOP, STAT, SUFU, TCF, TET, TNFRSF, TRRAP, TYRO, VHL, ZFLX, SPRED, STAT5, SYK, TCF7L, TGFBR, TOP2, TSC, U2AF, WT, ZYM, SRC, TSSTATB, TAF, TEK, TIPARP, USP, TSC, YAF, XPO, ZYM, STAG, STK, TBX, TERT, PRHR, TP, TSLA, TSTP, TSF 703 ZNF, XRF, XRAP.

3. The gene panel of claim 1, wherein the gene copy number variation comprises the following genes: ABL1, BIRC3, CD74, CTLA4, ERBB3, EZH2, GSK3B, KDM5A, MAPK A, NBN, AKT A, BLM, CDK A, CYLD, ERCC A, FANCA, HDAC A, KDR, MAX, NF A, AKT A, BMPR1A, CDK A, EED, ERCC A, FANCD A, HGF, KEAP A, MDM A, NF A, AKT A, BRAF, CDKN2A, EGFR, ERF, FBXW A, HRAS, KIT, MET, NOTCCH A, ALK, BRCA A, CDKN2A, EIF4A A, ESR A, IDH A, KMT A, KMH A, KMCH A, CTAS A, CTLA A, EPT A, EPR A, EPT A, EPR A, EPT A, EPT A, EPR A, EPT A, EPT A, EPT A, EPR A, EPT A, EPR A, EPR A, EPR A, EPR A, EPT A, EPR A, EPR A, EPR A, EPR A, EPR A, EPR A, EPR A, EPR A, EPR A, EPR A, EPR, VEGFA, PIK3R1, PPP2R1A, RAC1, RAD51C, RECQL, RNF43, SDHA, SOS1, TP 53.

4. The gene panel according to claim 1, wherein the fusion gene comprises the following genes: ATF1, ETV1, EWSR1, FGFR3, MET, NPM1, NTRK1, NTRK2, NTRK3, PDGFRA, BRAF, ALK, ROS1, RELA, YAP 1.

5. A probe panel for detecting a tumor in the nervous system, wherein the probe panel is a detection probe for the mutant gene, the copy number variant gene and the fusion gene according to any one of claims 1 to 4.

6. A kit for detecting tumors of the nervous system, comprising at least one dose of the probe panel of claim 5.

7. Use of the gene panel according to any one of claims 1 to 4 or of the probe panel according to claim 5 for the production of a device for the detection of tumors of the nervous system.

8. A device for detecting a tumor in the nervous system, comprising:

the sequencing module is used for extracting DNA of a sample to be tested and carrying out high-throughput sequencing to obtain a sequencing result;

a comparison module for processing the result of the high-throughput sequencing and comparing the data with the gene panel of any one of claims 1 to 4 to obtain mutation information;

and the analysis module is used for analyzing the obtained mutation information to obtain a medication scheme.

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