CN117327788A - Acquisition probe of myeloid blood tumor detection gene panel, design method and detection method thereof - Google Patents

Abstract

The invention provides a capture probe of a myeloid blood tumor detection gene panel, a design method and a detection method thereof, belonging to the technical field of gene detection. The probes of the invention include panelADNA capture probes and panb cDNA capture probes; the panel ADNA capture probe comprises a nucleotide sequence shown as SEQ ID NO. 1-2400, and the panel B cDNA capture probe comprises a nucleotide sequence shown as SEQ ID NO. 2401-10559. The probe can be used for detecting all types of genes for detecting marrow blood tumor, can solve the detection of marrow blood tumor gene mutation, copy number variation, fusion gene and gene expression in one step, can be used for detecting all clinical stages, has strong portability, and can be suitable for a plurality of detection platforms.

Description

Acquisition probe of myeloid blood tumor detection gene panel, design method and detection method thereof

Technical Field

The invention relates to the technical field of gene detection, in particular to a capture probe for detecting a myeloid blood tumor detection gene panel, a design method and a detection method thereof.

Background

Myeloid leukemia is a malignant tumor derived from hematopoietic stem cells or early hematopoietic progenitor cells, including Acute Myeloid Leukemia (AML), chronic Myelogenous Leukemia (CML), myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPN), and the like. The incidence, prognosis and treatment regimen of these tumors vary, but are all related to genetic abnormalities in myeloid cells.

According to the international agency for research on cancer (IARC) data, about 40.6 tens of thousands of people had myeloid hematological neoplasms worldwide in 2018, with 47.6% AML, 19.9% CML, 16.7% MDS, and 15.8% MPN. The incidence of medullary hematological tumors increases with age, with men being higher than women and developed countries being higher than developing countries. Five year survival of myeloid hematological tumors varies by type and stage, generally being less than 50%.

Diagnosis of myeloid blood tumors is primarily dependent on clinical manifestations, laboratory examinations and molecular biological tests. Laboratory tests include peripheral blood routine, bone marrow smears, cell morphology, immunophenotyping, cytogenetics, molecular biology, and the like. Molecular biological assays refer to the analysis of gene mutations or fusion genes present in myeloid cells to determine the type, stage, prognosis, and treatment regimen of a tumor. The existing molecular biology detection methods mainly comprise Polymerase Chain Reaction (PCR), fluorescence In Situ Hybridization (FISH), chip technology and the like, and have the advantages of high sensitivity, strong specificity, simple operation and the like, but have limitations, such as being capable of detecting only a known target, being incapable of covering the whole genome range, being incapable of detecting multiple mutations or low-frequency mutations and the like.

The occurrence and development of myeloid blood tumors are closely related to genetic abnormalities in myeloid cells, including fusion genes, gene mutations, copy number variations, structural variations, gene expression, etc. Wherein, some gene mutations or fusion genes have diagnostic significance and can help to distinguish different types of myeloid blood tumors, such as BCR-ABL1 fusion genes in CML, NPM1 mutations in AML, SF3B1 mutations in MDS, and the like. Other gene mutations or fusion genes have prognostic significance and can reflect biological behaviors and therapeutic responses of tumors, such as FLT3-ITD mutation in AML, ABL1 kinetic mutation in CML, TP53 mutation in MDS, and the like. Still other gene mutations or fusion genes have therapeutic significance and can be used as basis or monitoring indicators of targeted therapy, such as BCR-ABL1 fusion gene in CML, IDH1/2 mutation in AML, JAK 2V 617F mutation in MPN, and the like. Therefore, it is necessary to detect gene mutations and fusion genes in patients with medullary hematological tumors, which can help doctors to develop personalized diagnostic protocols, evaluate the prognosis risk of patients, select the most appropriate treatment strategies, monitor the treatment effect and residual disease, etc.

Targeted capture sequencing technology is a method that utilizes specific probes to capture a region of a target gene, followed by high throughput sequencing. The method can improve the efficiency and the sensitivity of sequencing and reduce the cost and the time of sequencing. In the diagnosis of medullary blood tumor, the targeted capture sequencing technology has the following necessity and advantages: medullary hematological neoplasms are a highly heterogeneous group of diseases whose pathogenesis is associated with multiple genetic mutations. The targeted capture sequencing technology can detect mutation conditions of a plurality of related genes simultaneously, provides more comprehensive molecular information, and is beneficial to diagnosis and typing, prognosis judgment and guiding treatment. Often, the genetic mutation of the myeloid blood tumor is a somatic mutation, the frequency and level of which varies from patient to patient and from course to course. The targeted capture sequencing technology can improve the sensitivity and accuracy of detection, discover mutation of low frequency or subclone, and reflect clone evolution and heterogeneity of tumor. The therapeutic effects of medullary hematological tumors are closely related to the level of Minimal Residual Disease (MRD). The targeted capture sequencing technology can be used as one of molecular markers of MRD, and can be used for monitoring elimination of tumor cells after treatment and evaluating recurrence risk and prognosis.

The existing marrow blood tumor detection has the following defects: 1. the detection content is single, and most of the current sequencing detection technologies only aim at one of gene mutation or fusion genes. 2. The verification and analysis of germ line mutation cannot be performed, the germ line mutation can be related to hereditary blood tumor or familial blood tumor, and the germ line mutation has important significance for genetic consultation and family screening of patients. 3. Fusion gene detection is often directed to known fusion formats, and unknown fusion genes cannot be predicted. Most fusion gene detection methods can only detect specific fusion genes or fusion partners, and new or rare fusion genes cannot be found, so that some fusion genes with diagnostic, prognostic or therapeutic significance can be omitted. 4. Analysis of gene expression is not available in RNA-targeted sequencing techniques.

Disclosure of Invention

The invention aims to provide a capture probe of a myeloid blood tumor detection gene panel, a design method and a detection method thereof. The probe can be used for detecting all types of genes for detecting marrow blood tumor, can solve the detection of marrow blood tumor gene mutation, copy number variation, fusion gene and gene expression in one step, can be used for detecting all clinical stages, has strong portability, and can be suitable for a plurality of detection platforms.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a capture probe of a myeloid blood tumor detection gene panel, which comprises a panel A DNA capture probe and a panel B cDNA capture probe; the panel A DNA capture probe comprises a nucleotide sequence shown as SEQ ID NO. 1-2400, and the panel B cDNA capture probe comprises a nucleotide sequence shown as SEQ ID NO. 2401-10559.

The invention also provides application of the capture probe in preparing a detection reagent or a kit for the marrow blood tumor genes.

The invention also provides a design method of the capture probe of the myeloid blood tumor detection gene panel, which comprises the following steps:

(1) Screening marrow blood tumor related genes, and dividing the genes into panel A and panel B;

(2) Designing and synthesizing probes according to the standard gene names, gene transcription numbers and the positions of the exons of the genes in the panel A and the panel B;

(3) And performing performance verification on the probe.

Preferably, the panel A is a myeloid blood tumor-associated mutant gene; the panel B is a marrow blood tumor related fusion gene and gene expression.

Preferably, the genes in panel a include ABCB1, CCND1, DKC1, GFI1, KMT2C, PDGFRA, RUNX1, TERC, ANKRD26, CDKN2A, DNMT3A, GSKIP, KRAS, PDGFRB, SAMD9, TERT, ASXL1, CEBPA, ELANE, GSTP1, MBD4, PHF6, SAMD9L, TET2, ATG2B, CREBBP, EP, HAX1, MPL, PIGA, SETBP1, TP53, ATRX, CSF1R, ETNK1, IDH1, MTHFR, PML, SF B1, TPMT, BCOR, CSF3R, ETV6, IDH2, NF1, PPM1D, SH B3, U2AF1, BCORL1, CUX1, EZH2, JAK2, NOTCH1, PRPF8, SRP72, WT1, BRAF, CYP2C19, FLT3, JAK3, NOTCH2, PTEN, SRSF2, ZRSR2, CALR, CYP3A5, GATA1, KDM6A, NPM, ptg 2, qn 1D, SH B3, cbx 2, ddl 41, DDX 2, d 2;

genes in panel B include ABL1, AFF1, ALK, ANXA3, BAALC, BCR, BMI1, BMP2K, CAT, CBFB, CBL, PROM1, CD34, CPA3, CREBBP, ELL, MLLT1, EP300, ERG, RUNX1T1, ETV6, MECOM, FGFR1, FIP1L1, FLT3, FUS, GATA2, hes1, HOXA10, HOXA9, JAK2, KAT6A, KIT, MEIS1, KMT2A, MLLT10, MLLT3, AFDN, MN1, MSI2, CBFA2T3, MYH11, NPM1, NT5E, NUP214, NUP98, PDGFRA, PDGFRB, PML, PRAME, SPI1, RARA, RARG, RBM, ROS1, RPN1, RUNX1, S100A9, WT1.

Preferably, the design method of the probe adopts a traversal method, and probe sequences with similarity higher than 50% are eliminated through sequence similarity comparison based on a machine learning algorithm; the probe is specifically combined with a target nucleic acid region, the single base coupling efficiency is 99.4% -99.6%, and the synthesized probe is subjected to quality control by an electrospray mass spectrometry.

Preferably, the performance verification includes capture efficiency verification, precision verification, accuracy verification, and analysis sensitivity verification.

The invention also provides a detection method of marrow system blood tumor related genes for non-disease diagnosis or treatment, which comprises the following steps:

(1) Extracting sample nucleic acid;

(2) Breaking DNA in the nucleic acid, connecting by a joint, amplifying by PCR, and purifying to obtain a panel A library;

(3) Breaking RNA in the nucleic acid, and obtaining a panel B library through reverse transcription, linker ligation, PCR amplification and purification;

(4) Performing hybridization capture on the panel A library by using the panel A DNA capture probe; performing hybridization capture on the panel B library by using the panel B cDNA capture probe;

(5) And respectively carrying out second-generation sequencing on the panel A library and the panel B library after hybridization capture, and carrying out data analysis on sequencing results.

Preferably, the data analysis of the panel A library comprises data filtering, quality control, alignment, mutation detection, copy number analysis and result annotation; the data analysis of the panel B library comprises data filtering, quality control, alignment, fusion detection, expression analysis and result annotation.

Preferably, when the sequencing result of the panel A library after hybridization capture is subjected to data analysis, the method further comprises germ line mutation verification, wherein the germ line mutation verification method is to carry out comparison analysis on the low-depth sequencing result of the oral mucosa sample and the bone marrow sample after data analysis.

The invention provides a capture probe of a myeloid blood tumor detection gene panel, a design method and a detection method thereof. The gene panel of the invention contains all types of genes for detecting marrow blood tumor, and the capture probes are designed for the genes, so that the detection of marrow blood tumor gene mutation, copy number variation, fusion gene and gene expression can be solved in one step, the coverage of detection content is wide, the invention can be used for detecting various stages from risk assessment to diagnosis, treatment, prognosis and the like, has strong portability, and is suitable for sequencing instruments in the current market or newly developed detection platforms. In addition, the detection method comprises the comparison analysis of the oral mucosa sample and the bone marrow sample, and the annotation of germ line mutation and somatic mutation can be realized on the premise of not increasing the sequencing cost by the sequencing of the low-depth oral mucosa sample and the sequencing of the high-throughput bone marrow sample, so that the problem of germ line mutation verification in blood tumor gene mutation detection is solved.

Drawings

FIG. 1 is a graph showing the results of germ line mutation verification based on 112 patients in example 2 of the present invention.

Detailed Description

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

The embodiment provides a capturing probe of a myeloid blood tumor gene panel, which is specifically designed as follows:

panel design: selecting myeloid blood tumor-associated genes from published literature data comprising: the high-frequency mutant genes, the high-frequency fusion genes and the high-frequency expression related genes in the public database are applied to standards, treatment guidelines, drug labels and marrow blood tumor treatment related genes in the general database, and genes related to marrow blood tumor molecular typing, treatment, prognosis and induction are reported in the published literature. According to the detection method, the method is divided into Panel A: myeloid blood tumor-associated mutant genes (gene types are shown in table 1), panel B: the marrow blood tumor related fusion and expressed genes (the gene types are shown in Table 2).

Table 1 Panel a: myeloid blood tumor related mutant gene

ABCB1

CCND1

DKC1

GFI1

KMT2C

PDGFRA

RUNX1

TERC

ANKRD26

CDKN2A

DNMT3A

GSKIP

KRAS

PDGFRB

SAMD9

TERT

ASXL1

CEBPA

ELANE

GSTP1

MBD4

PHF6

SAMD9L

TET2

ATG2B

CREBBP

EP300

HAX1

MPL

PIGA

SETBP1

TP53

ATRX

CSF1R

ETNK1

IDH1

MTHFR

PML

SF3B1

TPMT

BCOR

CSF3R

ETV6

IDH2

NF1

PPM1D

SH2B3

U2AF1

BCORL1

CUX1

EZH2

JAK2

NOTCH1

PRPF8

SRP72

WT1

BRAF

CYP2C19

FLT3

JAK3

NOTCH2

PTEN

SRSF2

ZRSR2

CALR

CYP3A5

GATA1

KDM6A

NPM1

PTPN11

STAG2

NQO1

CBL

DDX41

GATA2

KIT

NRAS

RARα

STAT3

/

/

/

/

/

Table 2 Panel B: fusion gene and gene expression related to marrow blood tumor

Combining the marrow blood tumor related genes, removing redundancy, determining the standard gene name, gene transcription number and the chromosome position of the exon of the related genes through an HGNC database (HUGO Gene Nomenclature Committee) for probe design synthesis.

2. Probe design and synthesis: according to the chromosome position of all transcripts of the extracted related genes, a traversing method is adopted to design probes, and based on a machine learning algorithm, probe sequences with similarity higher than 50% are eliminated through sequence similarity comparison, so that the probes are ensured to cover more than 99% of target areas. The probe can be specifically combined with a target nucleic acid region, the synthesis of the probe requires independent synthesis, the single base coupling efficiency is between 99.4% and 99.6%, and the quality of the synthesized probe is controlled by an electrospray mass spectrometry method so as to ensure the accuracy of the synthesis. The finally synthesized Panel A DNA capture probe comprises 2400 probes, wherein 99.98% of a target area is covered by the 2400 probes, and the sequences of the probes are shown as SEQ ID NO. 1-2400; the Panel B cDNA capture probe comprises 8159 probes, the coverage of the target area is 99.99%, and the probe sequences are shown in SEQ ID NO. 2401-10559.

3. And (3) performance verification: and respectively performing precision verification, accuracy verification, analysis sensitivity verification and capture efficiency verification on the synthesized probes.

3.1 sample preparation:

a) Positive samples: the early stage adopts digital PCR technology to detect, clinical samples containing one or more mutant genes/fusion genes in table 1/table 2 are taken as positive original samples, nucleic acid is extracted, the concentration and purity of the obtained nucleic acid are detected, the mixed positive samples are diluted by the nucleic acid which is confirmed to be negative according to the equal proportion of the concentration of the nucleic acid after the concentration and purity of the obtained nucleic acid meet the quality control requirements, and the positive samples are obtained.

b) Cell line samples: samples were cultured for cell lines of known mutation types, nucleic acids were extracted according to the relevant requirements, and stored in gradient dilutions.

c) Negative samples: specimens that proved negative (below the lower limit of detection or zero concentration) were detected using digital PCR techniques.

3.2 precision verification

3.2.1 sample preparation

Panel A: according to the precision detection requirement, respectively performing precision detection under a high mutation rate group and a critical mutation rate group, wherein the high mutation rate group selects SNP loci (comprising three genes CYP2C19, ABCB1 and CYP3A 5) with mutation frequency of 50%, and the low mutation rate group selects mixed cells (obtained by mixing CYP2C19 positive cells and cells without the mutation) with mutation frequency of 1%.

Panel B: according to the precision detection requirement, respectively performing precision detection under a high fusion gene frequency group and a low fusion gene frequency group, wherein the high fusion gene frequency group and the low fusion gene frequency group are respectively obtained by mixing K562 cells (BCR-ABL positive) and normal human peripheral blood cells in proportion. The K562 cells had a 30% duty cycle in the high fusion gene frequency group and a 5% duty cycle in the low fusion gene frequency group.

3.2.2 sequencing assays

Panel A detection: the high mutation rate group selects 100 mutation sites of three genes CYP2C19, ABCB1 and CYP3A5, 5 batches are divided into 20 batches, detection is carried out on each batch, and the intra-batch SD value and the inter-batch SD value and the Coefficient of Variation (CV) value of the high mutation rate group are respectively calculated; the low mutation rate group is selected from 50 mutation sites with the mutation frequency of 1% after CYP2C19 is mixed, 5 batches are divided, 10 of each batch are detected, and the intra-batch and inter-batch SD values and the Coefficient of Variation (CV) values of the low mutation rate group are calculated respectively.

Panel B detection: the number of the high fusion gene frequency groups is 50, 5 batches are divided, 10 batches are detected, and the intra-batch SD value, the inter-batch SD value and the Coefficient of Variation (CV) value of the high fusion gene frequency groups are calculated respectively; the low fusion gene frequency groups were measured in 50 batches of 5, 10 batches each, and the intra-batch and inter-batch SD values and Coefficient of Variation (CV) values of the low fusion gene frequency groups were calculated, respectively.

3.2.3 sequencing results

Panel A: the high mutation rate group had an intra-batch average value of 48.05%, an intra-batch variation coefficient of 6.56%, an inter-batch average value of 48.01% and an inter-batch variation coefficient of 7.44%; the low mutation rate group had an intra-batch average value of 1.36%, an intra-batch variation coefficient of 13.42%, an inter-batch average value of 1.13%, and an inter-batch variation coefficient of 23.23%.

Panel B: high fusion gene frequency group, average value in batch is 12.59%, variation coefficient in batch is 10.93%, average value between batches is 9.45%, variation coefficient between batches is 11.41%; the low fusion gene frequency group has an average value of 2.61%, an intra-batch variation coefficient of 19.04%, an average value of 2.36% and an inter-batch variation coefficient of 48.28%.

3.3 accuracy verification

3.3.1 method for evaluating accuracy of qualitative determination: the digital PCR and the fluorescent quantitative PCR are one of the most sensitive methods for single-point detection at present, and the two methods are selected for accuracy verification with the method.

3.3.2 reference methods

The Panel A DNA capture probe detection reference method comprises the following steps: digital PCR

The Panel B cDNA capture probe detection reference method comprises the following steps: fluorescent quantitative PCR

3.3.3 sample preparation: (1) 50 positive samples for gene mutation (IDH 2 c.319G > A and DNMT3Ac.1849G > T mutation) and 50 negative samples for gene mutation; (2) 50 fusion gene positive samples and 50 fusion gene negative samples.

3.3.4 detection method: the selected samples are detected by two methods simultaneously: panel A is detected by a second generation sequencing (NGS) method and a digital PCR method respectively, and Panel B is detected by a second generation sequencing (NGS) method and a fluorescent quantitative PCR method, and the samples are divided into 5 batches of 10 samples each.

3.3.5 analysis and judgment of results

The test results are shown in Table 3.

TABLE 3 accuracy test results

As can be seen from Table 3, the sensitivity of the second generation sequencing (NGS) gene mutation detection was 100% and the specificity was 100% after comparison with the results of digital PCR and fluorescent quantitative PCR; fusion gene detection, sensitivity is 100%, and specificity is 100%.

3.4 analytical sensitivity verification

The sensitivity of the assay is typically verified by serial dilution of samples containing known mutations, i.e., repeated measurement of samples at high dilutions using the same lot number of reagents under the same experimental conditions, with statistical analysis of the results.

3.4.1 detection flow

Panel A: diluting known positive samples (IDH 2 c.419G > A and DNMT3A c.189G > T mutation) with known negative samples until the mutation frequency is 0.5%, 1% and 5%, repeatedly detecting each concentration of diluted samples for 10 times according to the detection program of the conventional samples, and analyzing according to standard analysis steps.

Panel B: k562 cells (BCR-ABL positive) and normal human peripheral blood cells were mixed in proportion, the proportion of K562 cells was 1%, 5% and 50%, respectively, and samples of different dilution concentrations were repeatedly tested 10 times per concentration according to the conventional sample test procedure, and analyzed according to the standard analysis procedure.

3.4.2 detection results

The Panel A detection sensitivity (lowest detection lower limit) was 1%, and the Panel B detection sensitivity (lowest detection lower limit) was 5%.

3.5 Probe Capture efficiency quality control

The probe capture capability verification mainly comprises capture efficiency and alignment rate. Capture efficiency: for the target area capturing item, the proportion of reads to the target area to all reads can be compared. Through detection, the capturing efficiency of the Panel A and B probes is more than or equal to 70 percent. Comparison rate: the ratio to the target area reads can be compared. Through detection, the comparison rate of the Panel A probe is more than or equal to 99%, and the comparison rate of the Panel B probe is more than or equal to 90%.

Example 2

The embodiment provides a method for detecting marrow blood tumor related genes by using the capture probe in the embodiment 1, which comprises the following specific processes:

the apparatus used in this embodiment includes: lad-Aid 824s nucleic acid automatic extractor, xiamen good Biotechnology Co., ltd; ABI-Veriti nucleic acid amplification apparatus, thermo Fisher, USA; ABI-Mini-AMP nucleic acid amplification apparatus, thermo Fisher, USA; ABI-Qubit 4.0 nucleic acid quantifier, thermo Fisher, inc., USA; illumina-Nextseq-500 high throughput sequencer, illumina company, usa.

In the invention, other instruments with the same functions of manufacturers, such as a nucleic acid extractor, an amplification instrument and a high-throughput sequencer, can be replaced.

The reagents used in this example include: library construction reagent xGen ^TM DNA Library Prep EZ Kit、xGen ^TM RNA Library Prep Kit from Shanghai Ed Biotech Co., ltd; hybrid capture reagent xGen Hybridization and Wash Kit, available from Shanghai Ed Biotech Co., ltd; sequencing run reagent NextSeq 500/550High Output Kit 2.5, available from Illumina, USA.

In the present invention, reagents of the same function as those of other manufacturers, such as library construction reagents, hybridization capture reagents, sequencing machine reagents, can be replaced.

1. Extracting a sample

Bone marrow samples were collected from 100 patients with a definite diagnosis of marrow blood disease (from patients with a clinical visit in oncology hospitals in Henan province in 2022 to 2023), and DNA and RNA in the samples were extracted using a Lab-Aid 824s/808 nucleic acid extractor.

2. Library construction

2.1 Panel A library construction

2.1.1 DNA disruption

The sample DNA extracted in the previous step was added to a 0.2mL PCR tube with a DNA input of 300ng, water was added to 18.5. Mu.L, and EDTA solution was added to make the total volume 19.5. Mu.L. The reaction solutions shown in Table 4 were prepared, and the reagents were melted on ice, and the prepared reaction solutions were added to each PCR tube so that the total volume was 30. Mu.L. The PCR tubes were then subjected to DNA disruption according to the reaction procedure shown in Table 5.

TABLE 4 reaction liquid System

Reagent(s)	Volume (mu L)
		Buffer K1	3
Reagent K2	1.5
		Enzyme K3	6
Total volume of	10.5

TABLE 5 DNA disruption reaction procedure

Note that: if the DNA of the oral mucosa sample is, the input amount is adjusted to 25ng, and the breaking time is 16min.

2.1.2 Joint

The reaction solution was prepared as shown in Table 6, and the reagents were thawed on ice. After completion of the preparation, the reaction solution was added to the PCR tube after completion of DNA cleavage to give a total volume of 60. Mu.L.

TABLE 6 reaction solution

Reagent(s)	Volume (mu L)
		Buffer W1	12
Enzyme W3	4
		Raegent W5	5
Low EDTA TE	9
		Total volume of	30

Note that: if the oral mucosa sample DNA is used, the addition amount of Raegent W5 is diluted as shown in Table 7.

TABLE 7 relationship between the input amount of DNA in oral mucosa samples and the dilution ratio of Raegent W5

DNA input amount	Raegent W5 dilution factor
		≧25ng	Not diluting
10ng	10 times of
		1ng	20 times of
100pg	30 times

The PCR tubes after mixing were subjected to a linker ligation treatment according to the reaction procedure shown in Table 8.

Table 8 Joint ligation reaction procedure

Temperature (. Degree. C.)	Time
		20	20min
4	+∞

2.1.3 purification

Adding 48 mu L of magnetic beads into the PCR tube which is connected with the connector, shaking and mixing uniformly, standing at room temperature for 5min, instantaneously separating, standing for 2min by a magnetic rack until liquid is clear, and discarding the supernatant. Washing with 180 μL 80% ethanol twice (the PCR tube needs to be moved back and forth to move the front and back walls of the magnetic beads several times), instantaneously separating, sucking the residual ethanol with a 10 μL row gun, and air drying. Adding 22 mu L of Low EDTA TE (0.1 mM EDTA), shaking, mixing, standing at room temperature for 5min, instantly separating, and standing with a magnetic rack for 2min until the liquid is clear. Transfer 20 μl of supernatant to new PCR tube.

2.1.4 PCR amplification

To the new PCR tube of 2.1.3, 5. Mu.L of index primers and 25. Mu.L of PCR Master Mix were added, and mixed by shaking, and PCR amplification was performed according to the reaction procedure shown in Table 9.

TABLE 9 PCR amplification reaction procedure

Note that: if the sample is an oral mucosa sample, the PCR amplification is carried out according to 8 cycles.

2.1.5 Secondary purification

Adding 90 mu L of magnetic beads into the PCR tube after PCR amplification, shaking and mixing uniformly, incubating for 5min at room temperature, instantaneously separating, and releasing a magnetic rack for 2min until the solution is clear, and discarding the supernatant. Washing with 180 μL 80% ethanol twice (the PCR tube needs to be moved back and forth to move the front and back walls of the magnetic beads several times), instantaneously separating, sucking the residual ethanol with a 10 μL row gun, and air drying. Adding 22 mu L of water, shaking and mixing uniformly, standing at room temperature for 5min, instantaneously separating, placing a magnetic rack for 2min until the solution is clear, and transferring 20 mu L of supernatant into a new eight-joint tube.

2.1.6 library concentrations were quantified using Qubit.

2.2 Panel B library construction

The Panel B library was pooled in RNA, and all pooling operations were performed on ice.

2.2.1 RNA disruption

And detecting RIN value of the RNA of the sample to be detected. RNA was added to a 0.2mL PCR tube at 500ng, and the mixture was then filled with water to 5. Mu.L. The reaction solution was prepared as shown in Table 10, and the total volume of the reaction solution was set to 14. Mu.L by adding it to a PCR tube. And RNA disruption treatment was performed according to the reaction procedure shown in Table 11.

TABLE 10 reaction solution

Reagent(s)	Volume (mu L)
		Reagent F1	1
Reagent F2	2
		Reagent F3	4
Reagent F4	2
		Total volume of	9

TABLE 11 RNA disruption reaction procedure

RIN value	Fragment Length (bp)	Temperature (. Degree. C.)	Time (min)
				≥7	200-250	94	15
2-7	200-250	94	10

Immediately after the end of the procedure, the PCR tube was left on ice for 2min, immediately followed by the addition of the inversion reagent.

2.2.2 reverse transcription

The inversion reagents were formulated as shown in Table 12, all melted on ice and added to the PCR tube (total volume 20. Mu.L) after RNA disruption. And reverse transcription was performed according to the reaction procedure shown in Table 13.

TABLE 12 reverse reagent

Reagent(s)	Volume (mu L)
		Enzyme R1	1
Enzyme R2	1
		Water and its preparation method	4
Total volume of	6

TABLE 13 reverse transcription procedure

Temperature (temperature)	Time (min)
		25	10
42	30
		70	15
4	hold

2.2.3 purification

To the reverse transcribed PCR tube was added 30. Mu.L of Low EDTATE in a total volume of 50. Mu.L. Adding 90 mu L of magnetic beads into each PCR tube, shaking and mixing, standing at room temperature for 5min, instantaneously separating, standing with a magnetic rack for 2min until the liquid is clear, and discarding the supernatant. 200 mu L of 80% ethanol is washed twice, instantaneously separated, residual ethanol is discarded and dried. Adding 22 mu L of Low EDTATE, shaking and mixing, standing at room temperature for 5min, instantly separating, and standing with a magnetic rack for 2min until the liquid is clear. Transfer 20. Mu.L of supernatant to a new PCR tube, without magnetic beads. Adding 36 mu L of magnetic beads into each tube, shaking and mixing, standing at room temperature for 5min, instantly separating, standing with a magnetic rack for 2min until the liquid is clear, and discarding the supernatant. 200 mu L of 80% ethanol is washed twice, instantaneously separated, residual ethanol is discarded and dried. Adding 12 mu L of Low EDTA TE, shaking and mixing, standing at room temperature for 5min, instantly separating, standing with a magnetic rack for 2min until the liquid is clear, and transferring 10 mu L of supernatant into a new PCR tube.

2.2.4 Joint

The PCR instrument is preheated to 95 ℃ in advance, a new PCR tube of 2.2.3 is placed into the PCR instrument at 95 ℃ after the temperature is reached, the PCR instrument is incubated for 2min, after the incubation is finished, a sample is rapidly transferred to ice, the incubation is carried out for 2min, a reaction solution prepared in advance is immediately added after the incubation is finished (as shown in a table 14), the mixture is uniformly vibrated, and the mixture is placed into the PCR instrument for joint connection according to a reaction procedure shown in a table 15.

TABLE 14 reaction solution

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TABLE 15 linker ligation reaction procedure

Temperature (. Degree. C.)	Time
		37	15min
95	2min
		4	hold

2.2.5 PCR amplification

To the PCR tube after 2.2.4 linker ligation, 5. Mu.L of index primers was added, followed by 25. Mu.L of PCR Master Mix, and mixed by shaking, and PCR amplification was performed according to the reaction procedure shown in Table 16.

TABLE 16 PCR amplification reaction procedure

2.2.6 Post PCR purification

Adding 40.4 mu L of magnetic beads into the PCR tube after PCR amplification, shaking and mixing uniformly, incubating for 5min at room temperature, instantaneously separating, magnetically holding for 2min until the solution is clear, and discarding the supernatant. Washing with 200 μL 80% ethanol twice, instantaneous separating, discarding residual ethanol, and air drying. Adding 22 mu L of Low EDTATE, shaking and mixing, standing at room temperature for 5min, instantly separating, magnetically releasing the rack for 2min until the solution is clear, and transferring 20 mu L of supernatant into a new eight-joint tube.

2.2.7 library concentrations were quantified using Qubit.

3. Hybridization Capture (Panel A library, panel B library, operate separately, but in the same procedure)

3.1 library hybridization

Based on the determined library concentration, the volume was calculated to be 500ng of the desired capture mass of the library, and the library was pipetted into a low adsorption 1.5mL centrifuge tube. Capture reagents were prepared as shown in table 17 and added to the library-containing centrifuge tubes described above. Sealing the centrifuge tube with sealing film, and perforating several holes with gun head to evaporate liquid, and evaporating the centrifuge tube in vacuum centrifugal concentrator (preheated at 60 deg.c). Care was taken to see if it had evaporated to dryness at any time.

TABLE 17 Capture reagent

Reagent(s)	Volume/. Mu.L
		Human Cot DNA(IDT)	5
Blockers	2

3.2 DNA denaturation and hybridization

Hybridization Master Mix formulated according to Table 18 was added to the evaporated centrifuge tube. Mixing by shaking thoroughly, centrifuging briefly, incubating at room temperature for 5min, mixing by shaking again, centrifuging briefly, and incubating at room temperature for 5min.

Table 18 Hybridization Master Mix

Reagent(s)	Volume/. Mu.L
		xGen 2×Hybridization Buffer	8.5
xGen Hybridization Buffer Enhancer	2.7
		Panel A/Panel B Probe of example 1	4
Water and its preparation method	1.8

The incubated liquid was transferred to a PCR tube, placed in a PCR instrument, and hybridized according to the procedure shown in Table 19.

TABLE 19 hybridization reaction procedure

3.3 purification after hybridization

3.3.1 Preparation of Wash Buffer working solution

The preparation method of the Wash Buffer working solution required in the purification process is shown in Table 20.

Table 20 Wash Buffer working solution stock solution and preparation method thereof

The reagents used at 65℃in the above table were dispensed and stored by incubation at 65 ℃.

3.3.2 purification

3.3.2.1 streptavidin magnetic bead cleaning

50. Mu.L Capture beads were placed in eight rows and 100. Mu.L 1 XBead Wash Buffer was added and mixed with shaking. Placing on a magnetic rack for 1min until the liquid is clear, and discarding the supernatant. 100. Mu.L of a 1 XBead Wash Buffer was added thereto and mixed with shaking. Placing on a magnetic rack for 1min until the liquid is clear, and discarding the supernatant. 100. Mu.L of a 1 XBead Wash Buffer was added thereto and mixed with shaking. Placing on a magnetic rack for 1min until the liquid is clear, and discarding the supernatant. The eight rows were removed from the magnetic rack, centrifuged briefly, placed on the magnetic rack, and the residual liquid at the bottom of the centrifuge tube was thoroughly discarded with a 10 μl gun head. Bead Resuspension Mix formulated according to Table 21 was added to the washed beads.

Table 21 Bead Resuspension Mix

Reagent(s)	Volume/. Mu.L
		xGen 2×Hybridization Buffer	8.5
xGen Hybridization Buffer Enhancer	2.7
		Nuclease-Free Water	5.8

3.3.2.2 heat washing

The hybridization solution captured overnight was transferred to Bead Resuspension Mix with magnetic beads after incubation at 65℃and was mixed by pipetting. Placing the mixture in a PCR instrument for incubation at 65 ℃ for 45min, and blowing the mixture with a gun every other section to ensure the suspension of the magnetic beads. The time intervals are 11min, 11min and 12min. During which the timer is to be suspended. After the incubation was completed, the liquid in the PCR tube was transferred to eight rows, and 100. Mu.L of 1 XWash Buffer 1 preheated at 65℃was added and mixed by pipetting. Placing on a magnetic rack for 1min until the liquid is clear, and discarding the supernatant. The eight rows were removed from the magnet holder, centrifuged briefly (to prevent too much temperature drop), placed on the magnet holder and the residual liquid at the bottom of the centrifuge tube was thoroughly discarded with a 10 μl gun head. 150. Mu.L of preheated 1X Stringent Wash Buffer at 65℃was added, the mixture was blown and mixed with a pipette, incubated at 65℃for 5min, placed on a magnetic rack for 1min until the liquid was clear, and the supernatant was discarded. 150. Mu.L of preheated 1X Stringent Wash Buffer at 65℃was added, the mixture was blown and mixed with a pipette, incubated at 65℃for 5min, placed on a magnetic rack for 1min until the liquid was clear, and the supernatant was discarded. The eight rows were removed from the magnetic rack, centrifuged briefly, placed on the magnetic rack, and the residual liquid at the bottom of the centrifuge tube was thoroughly discarded with a 10 μl gun head.

3.3.2.3 cleaning at normal temperature

Adding 150 μl of 1 XWash Buffer 1 placed at room temperature, shaking for 30s, standing for 30s, shaking again for 30s, standing for 30s (total 2 min), centrifuging briefly, standing on a magnetic rack for 1min until the liquid is clear, and discarding the supernatant. Adding 150 μl of 1 XWash Buffer 2 placed at room temperature, shaking for 30s, standing for 30s, shaking again for 30s, standing for 30s (total 2 min), centrifuging briefly, standing on a magnetic rack for 1min until the liquid is clear, and discarding the supernatant. Adding 150 μl of 1 XWash Buffer 3 placed at room temperature, shaking for 30s, standing for 30s, shaking again for 30s, standing for 30s (total 2 min), centrifuging briefly, standing on a magnetic rack for 1min until the liquid is clear, and discarding the supernatant. The eight rows were removed from the magnetic rack, centrifuged briefly, placed on the magnetic rack, and the residual liquid at the bottom of the centrifuge tube was thoroughly discarded with a 10 μl gun head. Adding 20 mu L of ultrapure water into the centrifuge tube for eluting, shaking and mixing uniformly, and carrying out the next amplification test.

3.3.3 Post-LM-PCR

Post-LM-PCR Mix was prepared as shown in Table 22, mixed by shaking, centrifuged briefly, and split into PCR tubes of 30. Mu.L each. The sample was placed on a PCR instrument, and PCR was performed according to the procedure shown in Table 23.

TABLE 22 Post-LM-PCR Mix

Reagent(s)	Volume/. Mu.L
		KAPA HiFi HotStart ReadyMix	25
xGen Library Amplification Primer Mix	1.25
		Water and its preparation method	3.75
DNA eluted in the previous step	20

Table 23 reaction procedure

The number of cycles was adjusted according to the panel size and the number of samples for mixed hybridization, as shown in Table 24.

TABLE 24 relationship between cycle number, panel size and number of samples for hybrid hybridization

Panel size	1 miscellaneous	4 miscellaneous	8 miscellaneous	12 miscellaneous	Nucleic acid
						10000-100000probes	12cycles	10cycles	9cycles	8cycles	RNA
500-10000	13cycles	11cycles	10cycles	10cycles	DNA

3.3.4 post amplification purification

The amplified capture DNA library was placed on a magnetic rack, 75. Mu.L (1.5X) of purified magnetic beads were placed in a new eight-row tube, 50. Mu.L of the amplified capture DNA library supernatant was added, mixed by shaking, and incubated at room temperature for 10min. Instant separation is carried out, the mixture is placed on a magnetic rack for 1min until liquid is clarified, and the supernatant is discarded. The eight rows of tubes were removed from the magnetic rack, centrifuged briefly, placed on the magnetic rack, and the residual liquid at the bottom of the centrifuge tube was thoroughly discarded with a 10 μl gun head. 200. Mu.L of 80% ethanol was added, left to stand for 30 seconds and discarded, and repeated. The eight rows of tubes are taken off from the magnetic rack, centrifuged briefly, placed on the magnetic rack, the residual liquid at the bottom of the centrifuge tube is thoroughly discarded with a 10 mu L gun head, and dried at room temperature until the ethanol is completely volatilized. The centrifuge tube was removed from the magnetic rack, 22. Mu.L of Low TE was added, and mixed by shaking. Incubate for 2min at room temperature. Briefly centrifuged and placed on a magnetic rack for 1min until the liquid is clear, and 20. Mu.L of supernatant is transferred to a new 1.5ml centrifuge tube to obtain a library after hybridization capture.

3.3.5 quantification of library concentrations after hybridization capture using Qubit.

4. Second generation sequencing (NGS)

4.1 turning on the Illumina Next-seq 500 power supply, waiting for several minutes, and starting up to initialize pass. The system will automatically open the Nextseq500 Control Software. And cleaning the instrument according to the instrument requirement.

4.2 preparation of libraries before on-press, library denaturation dilution

The library was diluted to 1 ng/. Mu.L. According to the number/concentration of 1 x samples=volume V of the mixed library, the added volume of each mixed library in one tube is determined, the Qubit is used for measuring a plurality of concentrations, and the average value is taken to obtain the final library concentration C. Mixing 5 mu L of the final library with 5 mu L of 0.2M NaOH, standing for denaturation for 5min, adding 5 mu L of ultrapure water, blowing and mixing uniformly, stopping denaturation, and adding 985 mu L of HT1 Buffer into a denaturation tube to complement 1mL of volume. 1 1.5mL centrifuge tube, labeled 1300, was prepared according to "sample volume of library after denaturation X: X=1.5PM×1300×350×660/(5X1100000×final library concentration C) ", X. Mu.L was drawn from the denaturing tube into EP tubes labeled 1300, HT1 Buffer was used to supplement the total volume 1300. Mu.L, shaking and mixing followed by centrifugation to obtain the final library for the machine.

4.3 on-machine sequencing

And (3) operating according to instrument guidance, filling in each parameter according to the requirement, clicking Next, entering a self-checking interface to wait for self-checking to be completed, clicking Star Run, and starting sequencing.

5. Off-line data letter generation analysis

Panel A library sequencing results analysis procedure: taking a next FastQ file of the illuminea sequencing platform as input, and obtaining a final mutation result and quality control information of the data through the steps of data filtering, quality control, comparison, mutation detection, copy number analysis, result annotation and the like.

Panel B library sequencing results analysis procedure: the FastQ file is taken as input, and the final fusion gene and gene expression result and the quality control information of the data are obtained through the steps of data filtering, quality control, comparison, fusion detection, expression analysis, result annotation and the like.

The specific process of data filtering is as follows: the adaptor sequences, low quality sequences were filtered out by fastp software, including quality filtering: the base matrix value is more than or equal to 15, and the percentage of unqualified bases in single read is not more than 40%; length filtration: the minimum length of the read is 15bp; complexity filtering: the complexity of read is more than or equal to 30, and the complexity is defined as the number ratio of bases different from one base to the next adjacent base; and (3) joint filtration: automatically identifying a linker sequence and removing the linker; mass cut per read: using a sliding window to evaluate the average mass value of the 5 'and 3' ends of each read, and when the average mass value is below 15, cutting off the window and all bases behind the window; base correction: correcting mismatched bases in the overlapping region, if one base matrix is high and the other base is low in mass, replacing the low-mass base with the high-mass base; ployG/ployX tail pruning: the ployG and ployX tails at the 3' end were removed.

The specific process of quality control is as follows: the filtered sequence is subjected to quality control through samtools, bamdst, and the comparison rate, the repetition rate, the total coverage, the Q20/Q30 ratio and the probe capturing efficiency are counted respectively. Wherein:

(1) Comparison rate: targeting capture library building comparison rate: an alignment of successful matches from sequencing data to the reference genome is shown. The calculation method is as follows:

alignment = (number of reads/total sequencing reads aligned to reference genome) ×100% of the total sequencing

(2) Repetition rate: representing the proportion of reads having the same sequence in the sequencing data, this partial sequence is generally considered to be PCR generated and should be removed in the analysis. The calculation method is as follows:

repetition rate = (number of reads repeated/total number of sequencing reads) ×100%

Interpretation of repetition rate: the large proportion of reads in the sequencing data is due to technical replication or PCR amplification.

(3) Total coverage: indicating the proportion of the target area that can be covered. The calculation method is as follows:

total coverage = number of bases detected within target region/total number of bases of target region

Interpretation of total coverage: how comprehensive the sequencing data is for the target region. A higher overall coverage generally represents more comprehensive data.

(4) Q20 and Q30: an indicator of a quality score, typically expressed as a Phred quality score, is used to measure the quality of each base, which is derived based on the quality of the signal measured during base sequencing. Q20 represents a percentage of bases having a Phred mass fraction of 20 or more, representing a base error rate of less than 1%, and Q30 represents a percentage of bases having a Phred mass fraction of 30 or more, representing a base error rate of less than 0.1%. The calculation method is as follows:

q20% = (number of bases per total number of bases of Q20 and above mass fraction) ×100%

Q30% = (number of bases per total number of bases of Q30 and above mass fraction) ×100%

(5) Probe capture efficiency: represents the ratio of the detectable gene sequences to all gene sequences. The calculation method is as follows:

probe capture efficiency = (number of reads successfully aligned to target region/total sequencing reads) ×100%

Interpretation of probe capture efficiency: the higher the capture efficiency, the higher the capture specificity of the probe.

Panel A uses BWA to align the filtered reads to human reference genome hg19 and the bam files generated by the alignment are subjected to multiple alignment, PCR repeat and non-alignment sequences using sambamba. Local heavy comparison, base quality score rechecking and mutation detection are carried out by using a GATK software package, and the annotation of germ line mutation and somatic mutation is realized through the comparative analysis of an oral mucosa sample and a bone marrow sample. Performing copy number variation analysis by using a cnvkit software package through the comparative analysis of the oral mucosa sample and the bone marrow sample to obtain the copy number of each target gene, and determining the copy number difference of the bone marrow sample compared with the oral mucosa sample to obtain a final CNV result; detection of insertions, deletions, inversions, tandem repeats, chromosomal translocations was performed using manta.

Panel B uses SATR to compare the filtered reads with human reference genome hg38, and the bam files generated by comparison are respectively subjected to fusion gene detection by using three kinds of software, namely ariba and STAR-Fusion, fusioncatcher, and the result interpretation principle is as follows:

under the premise of qualified QC, the number of detected fusion gene positive reads is less than or equal to 3 and is false positive, and >3 are true positive;

note that: a) Positive reads number = split_reads1+split_reads2 when both genes forming the fusion are in panel;

b) Positive reads number = split_reads1 or 2 when only one of the genes forming the fusion is in panel (panel internal gene);

more than 2 pieces of 3 pieces of software are detected, the number of reads is more than or equal to 1 piece, or 1 piece of 3 pieces of software is detected, and when the number of reads is more than 3 pieces, verification is needed through a fluorescence quantitative PCR method;

when the unknown fusion is detected, the number of fusion reads is detected to be more than 3, and verification is needed by a fluorescent quantitative PCR method.

Extracting the gene counts data in the Panel B comparison result by using the featurepoints, and analyzing according to a basic analysis flow: the gene length of each gene was counted, and the normalization factor= (number of genes counts/gene length) ×1,000,000 was calculated according to the formula, and then the expression amount tpm= (normalization factor/sum of normalization factors of all genes) ×1,000,000 was calculated for each gene. The calculation mode of the gene high expression threshold is the median +3×standard deviation of the expression quantity of each gene of 300 samples.

6. Detection result

6.1

The test results of the panel A100 samples are shown in Table 25

Table 25 Panel A DNA Capture Probe 100 sample detection results

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The test results of the panel B100 samples are shown in Table 26

Table 26 Panel B cDNA Capture Probe 100 sample detection results

6.2 germ line mutation verification: when the panel A is detected, the oral mucosa sample and the bone marrow sample are simultaneously detected, and the detected mutation site can be judged whether to be germ line mutation or not through comparison.

Performing panel A detection and result data analysis (according to the operations of steps 1-5) on 112 marrow blood tumor patients who are subjected to simultaneous inspection of a marrow sample and oral mucosa during the period of 12 months 2022 to 9 months 2023, wherein the result is shown in a graph 1, and the system mutation is verified and the part (green area in the graph) with the VAF value of more than 40% accounts for 14% (62 positions), if no oral mucosa contrast exists, the system mutation is possibly misjudged; the germ line mutation is verified, no rs number part (orange region in the figure) accounts for 42 percent (169 sites), the sites of the part have no relevant literature report and database record, and if the part has no oral mucosa contrast, the part is misjudged as the system mutation possibly. From this, it can be seen that the germ line mutation verification of panel A increase enables the identification of the mutation source in more than 50% of patients compared to conventional analysis methods.

6.3 detection rate

According to the procedure of 1 to 5 described above, a sample of bone marrow from 84 patients with acute myelogenous leukemia (from hematological patients in the oncology department of Henan province in 2021 to 2023) was examined and analyzed. Meanwhile, the samples of the patients are detected and analyzed by adopting a conventional 56 fusion gene screening method. Positive detection rate of the two methods was compared.

The detection shows that the positive detection rate of the fusion gene of the method is 42.9 percent (36 cases). The same patient sample is detected by adopting 56 fusion gene screening methods, the positive detection rate is 25%, the method is improved by about 17.9% compared with the conventional method, and fewer fusion (less than 10 cases are not reported or reported) 12 cases are detected, as shown in table 27.

Table 27 fusion genes undetected by conventional methods

RUNX1-CUBN	KMT2A-ACACA	RUNX1-MECOM	ETV6-ABL1
				SATB1-PDGFRB	MSI2-HLF	MSI2-SMG6	KMT2A-ACACA
RUNX1-EML6	MLLT10-UBE4A	ETV6-TBL1XR1	UBE4A-KMT2A

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (10)

1. A capture probe of a myeloid blood tumor detection gene panel, which is characterized in that the capture probe comprises a panel ADNA capture probe and a panel B cDNA capture probe; the panel ADNA capture probe comprises a nucleotide sequence shown as SEQ ID NO. 1-2400, and the panel B cDNA capture probe comprises a nucleotide sequence shown as SEQ ID NO. 2401-10559.

2. Use of the capture probe of claim 1 for the preparation of a detection reagent or kit for myeloid blood tumor genes.

3. A method for designing a capture probe for a myeloid blood tumor detection gene panel according to claim 1, comprising the steps of:

(1) Screening marrow blood tumor related genes, and dividing the genes into paneA and paneB;

(2) Designing and synthesizing probes according to the standard gene names, gene transcription numbers and the positions of the exons of related genes in the panel A and panel B;

(3) And performing performance verification on the probe.

4. The method according to claim 3, wherein panea is a myeloid blood tumor-associated mutant gene; the panel B is a marrow blood tumor related fusion gene and gene expression.

5. The design method according to claim 4, wherein the genes in panelA include ABCB1, CCND1, DKC1, GFI1, KMT2C, PDGFRA, RUNX1, TERC, ANKRD26, CDKN2A, DNMT3A, GSKIP, KRAS, PDGFRB, SAMD9, TERT, ASXL1, CEBPA, ELANE, GSTP1, MBD4, PHF6, SAMD9L, TET2, ATG2B, CREBBP, EP300, HAX1, MPL, PIGA, SETBP1, TP53, ATRX, CSF1R, ETNK1, IDH1, MTHFR, PML, SF B1, TPMT, BCOR, CSF3R, ETV6, IDH2, NF1, PPM1D, SH B3, U2AF1, BCORL1, CUX1, EZH2, JAK2, NOTCH1, PRPF8, SRP72, WT1, BRAF, CYP2C19, FLT3, JAK3, NOTCH2, PTEN, SRSF2, ZRSR2, CALR, 3A5, gakdm 6, cbn 1, tsta 6, STAG 1, STAG2, gaq 2, and vata 2;

Genes in panel B include ABL1, AFF1, ALK, ANXA3, BAALC, BCR, BMI1, BMP2K, CAT, CBFB, CBL, PROM1, CD34, CPA3, CREBBP, ELL, MLLT1, EP300, ERG, RUNX1T1, ETV6, MECOM, FGFR1, FIP1L1, FLT3, FUS, GATA2, hes1, HOXA10, HOXA9, JAK2, KAT6A, KIT, MEIS1, KMT2A, MLLT10, MLLT3, AFDN, MN1, MSI2, CBFA2T3, MYH11, NPM1, NT5E, NUP214, NUP98, PDGFRA, PDGFRB, PML, PRAME, SPI1, RARA, RARG, RBM, ROS1, RPN1, RUNX1, S100A9, WT1.

6. The method according to claim 5, wherein the probe is designed by using a traversal method, and excluding probe sequences with similarity higher than 50% by sequence similarity alignment based on a machine learning algorithm; the probe is specifically combined with a target nucleic acid region, the single base coupling efficiency is 99.4% -99.6%, and the synthesized probe is subjected to quality control by an electrospray mass spectrometry.

7. The design method according to claim 6, wherein the performance verification includes a capture efficiency verification, a precision verification, an accuracy verification, an analysis sensitivity verification.

8. A method for detecting a myeloid blood tumor-associated gene for non-disease diagnosis or treatment purposes, comprising the steps of:

(1) Extracting sample nucleic acid;

(2) Breaking DNA in the nucleic acid, connecting by a joint, amplifying by PCR, and purifying to obtain a panel A library;

(3) Breaking RNA in the nucleic acid, and obtaining a panel B library through reverse transcription, linker ligation, PCR amplification and purification;

(4) Performing hybrid capture on the panea library with the panadna capture probe of claim 1; performing hybridization capture on the panel B library with the panel B cDNA capture probe of claim 1;

(5) And respectively carrying out second-generation sequencing on the panea library and paneb library after hybridization capture, and carrying out data analysis on sequencing results.

9. The assay of claim 8, wherein the data analysis of the panea library comprises data filtering, quality control, alignment, mutation detection, copy number analysis, result annotation; the data analysis of the panel B library comprises data filtering, quality control, alignment, fusion detection, expression analysis and result annotation.

10. The method according to claim 9, wherein the data analysis of the sequencing result of the panel a library after hybridization capture further comprises germ line mutation verification, wherein the germ line mutation verification is performed by comparing the sequencing result of the oral mucosa sample and the bone marrow sample after the data analysis.