A composition, kit and application for detecting chromosome ploidy abnormalities of hematological tumors based on SNP high-throughput sequencing technology

Abstract

The application provides a composition, a kit and an application for detecting chromosome ploidy abnormalities of hematological tumors based on SNP high-throughput sequencing technology. Specifically, a specific SNP site combination is screened, and a set of methods for detecting chromosome ploidy abnormalities of hematological tumors based on SNP high-throughput sequencing technology is designed based on the combination. The scheme of the application can be used for separate chromosome ploidy detection, and can also be added as a technical module on the basis of a conventional targeted capture-NGS sequencing process, without occupying independent experimental personnel and professional detection instruments. In combination with the existing NGS detection technology, the cost is greatly saved, the accurate detection of chromosome ploidy is realized, the detection is accurate and high, and full coverage of all 23 pairs of chromosomes is achieved. The foregoing chromosome ploidy abnormality detection scheme overcomes various deficiencies in coverage, detection accuracy, technical complexity and price.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a composition, kit, and application for detecting chromosomal ploidy abnormalities in hematologic tumors based on SNP high-throughput sequencing technology. Background Technology

[0002] Hematologic malignancies refer to malignant tumors that occur in the lymphohematopoietic system, including leukemia, lymphoma, multiple myeloma, and other diseases. This group of diseases has a high mortality rate and a heavy disease burden, and there is a high degree of heterogeneity between and within diseases. Diagnostic methods are complex and difficult, and the entire process of diagnosis and treatment requires the use of pathology, genetics, molecular biology, immunology, and other methods to determine the patient's condition.

[0003] Clonal chromosomal abnormalities are common in various hematologic malignancies. These abnormalities can manifest as abnormalities in chromosome number and structure, leading to changes in the quantity, structure, and function of the corresponding transcription / expression products. These processes are considered to be one of the important factors leading to tumor development and progression.

[0004] Because hematologic malignancies are a group of highly heterogeneous diseases, their chromosomal changes also vary greatly between different diseases and between different patients. Taking chromosomal number abnormalities as an example: (1) For lymphocytic tumors, according to the 2022 WHO classification criteria for lymphoid tissue tumors, B-lymphoblastic leukemia / lymphoma can be divided into hyperdiploidy (B-lymphoblastic leukaemia / lymphoma with hyperdiploidy) (51-65 chromosomes) and hypodiploidy (B-lymphoblastic leukaemia / lymphoma with hypodiploidy) based on chromosomal ploidy (i.e., chromosomal number abnormalities). There are two categories of hypodiploidy (<44 chromosomes), and many domestic and foreign guidelines have pointed out that abnormal chromosome ploidy can directly affect the risk stratification and prognosis grouping of B-lymphoblastic leukemia / lymphoma. Therefore, clarifying the chromosome ploidy status of lymphoblastic leukemia / lymphoma patients is of key significance for accurate diagnosis and treatment. At the same time, the changes in the number of different chromosomes also have a key impact on prognosis. The guidelines point out that for B-ALL patients, the increase of chromosomes 4, 10, and 17 has the most positive impact on prognosis; (2) Another hematologic malignancy that often presents with abnormal chromosome ploidy is multiple myeloma. High diploidy can be seen in >50% of multiple myeloma patients. The amplified chromosomes usually occur on odd-numbered chromosomes such as 3, 5, 7, 9, 11, 15, 19, and 21, which are usually considered to be As an early event in MM, these patients usually have a good prognosis. The incidence of subdiploidy is slightly lower, occurring in about 5% of patients, with a poor prognosis. (3) Chromosomal number abnormalities can also be seen in myeloid tumors such as acute myeloid leukemia, myeloproliferative neoplasms, and myelodysplastic syndromes. In AML patients, those with 49-65 chromosomes are usually considered to be high diploid. High diploidy (49-65 chromosomes) or hyperdiploidy (>65 chromosomes) karyotypes are relatively rare in AML. Chromosomes 8, 9, 21, and 22 are the most common chromosomes that can have numerical abnormalities. The prognosis of hyperdiploidy AML patients with >65 chromosomes is slightly better than that of patients with the usual poor prognosis karyotype, reflecting the unique biological characteristics of patients with chromosomal ploidy abnormalities. In summary, based on the important significance of chromosomal number / ploidy abnormalities in the diagnosis and prognostic stratification of various hematologic tumors, accurate and comprehensive detection of chromosomal ploidy abnormalities is of great significance in the precise diagnosis and treatment of hematologic diseases.

[0005] Currently, the commonly used chromosome detection methods in clinical practice include: chromosome karyotype detection technology, chromosome fluorescence in situ hybridization technology, multiplex probe amplification technology, and chromosome microarray technology. Each of these technologies has its own applicable scope: (1) Chromosome karyotype detection technology is the most conventional chromosome examination method. It involves taking samples from target cells and culturing them, and then observing chromosomes in metaphase under a microscope. This method is the most economical, intuitive, and can comprehensively observe the structural and numerical abnormalities of all 23 pairs of chromosomes. However, its disadvantage is that the resolution is low. It is currently believed that only abnormalities >5M can be observed. The detection technology requires culture and amplification, which is highly dependent on the cell state and culture conditions. The experimental cycle is long, and the analysis results are highly dependent on the technical proficiency and experience of the testing personnel. Its clinical application is limited to a certain extent. (2) Chromosome fluorescence in situ hybridization technology (FISH) is based on the principle of using fluorescently labeled probes to hybridize with target bases in chromosomes to qualitatively, locally, and quantitatively detect nucleic acids in cells. This detection does not require cell culture, has a short analysis cycle, and is highly flexible. The sensitivity and specificity are significantly improved compared to traditional chromosome karyotype testing techniques. It can distinguish some small deletions and complex translocations. However, the limitation of FISH is that it is affected by the preset coverage genes, and the detection range and flexibility are limited. It cannot simultaneously meet the detection of all 46 chromosome abnormalities, and the price is relatively expensive. (3) MPLA detection, short for "multiplex ligation-dependent probe amplification technology", is a high-throughput technique for qualitative and quantitative analysis of specific target sequences in the nucleic acid to be tested. Using simple hybridization, ligation and PCR amplification, it can simultaneously detect copy number changes of more than 40 different nucleotide sequences in a single reaction tube, and can detect fragments smaller than 60bp. However, MLPA is used to detect gene deletions or duplications and is not suitable for detecting unknown point mutation types. At the same time, it is limited by the number of target genes that can be detected in a single tube, and cannot achieve full genome coverage, thus affecting the comprehensiveness of the detection method. (4) Chromosome microarray analysis, i.e. CMA detection, is generally carried out on a chip, so CMA is also called "gene chip". It can detect chromosomal abnormalities such as aneuploidy, copy number changes, loss of heterozygosity, uniparental diploidy, triploidy, and polyploidy in a sample. In clinical practice, CMA has a resolution of up to 100kb, and its detection focus is more concentrated on predictable pathogenic CNV (copy number change) regions. It is a commonly used detection method in clinical practice. However, the limitation of this method is that it cannot detect point mutations and small fragment insertions and deletions at the same time, nor can it detect low-proportion chimeric copy number abnormalities. Therefore, in clinical applications, CMA needs to be used in combination with conventional gene sequencing NGS methods, using two sets of detection and analysis processes and two sets of personnel to analyze gene sequence and copy number changes separately, which cannot be managed in a one-stop manner.

[0006] In summary, developing a cost-effective, simple, convenient, comprehensive chromosome ploidy detection solution that can be embedded in conventional NGS testing procedures is an urgent problem to be solved in the field of hematologic malignancy detection. Summary of the Invention

[0007] Due to the complexity of chromosomal ploidy variations in hematologic malignancies and their importance for patient diagnosis and prognosis, in order to overcome the shortcomings of existing technologies and improve detection performance and coverage, this invention provides a composition, kit, and application for detecting chromosomal ploidy abnormalities in hematologic malignancies based on SNP high-throughput sequencing technology.

[0008] In a first aspect of the invention, a combination of SNPs for detecting chromosomal ploidy abnormalities in hematologic malignancies is provided, characterized in that the combination of SNPs comprises SNP loci 1-11943, totaling 11943 SNP loci, wherein the rs numbers of SNP loci 1-11943 are, in order: rs2340527, rs13302969, rs6701114, rs1272959, rs904586, rs28689700, rs601141, rs12744867, rs3107129, rs1532385, rs2477702, rs187759598, rs897628, rs1474562, rs245... 5137, rs4648507, rs12401505, rs10909797, rs35131135, rs10915428, rs34429154, rs4654438, rs6681520, rs7547152, rs9286973, rs1320179, rs36 139907, rs7528859, rs28479388, rs1295100, rs2294713, rs278017, rs10128028, rs11120877, rs1750851, rs3124803, rs488728, rs2120459, rs27086 29. rs1318218, rs6698830, rs4908803, rs2032539, rs10864414, rs115849843, rs4240910, rs6680076, rs34970915, rs10864459, rs284288, rs11586 085, rs6540964, rs2744815, rs2817580, rs9430208, rs1999594, rs562844, rs3397, rs4845908, rs12089791, rs570910916, rs2995600, rs354581, rs4 661455, rs6673156, rs6662664, rs12401417, rs705576, rs761288, rs1622686, rs550003, rs10927798, rs848217, rs146865861, rs221025, rs220917 4. rs6691954, rs2244416, rs3003449, rs2501778, rs773400848, rs1193338, rs620547, rs11261023, rs1832787, rs2244796, rs2789322, 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rs4824993, rs761559, rs5907953, rs1977161, rs5954475, rs6634356, rs5954640, rs 12844538, rs5954889, rs5908535, rs990581, rs12852291, rs237497, rs73244040, rs4144180, rs5 907555, rs1887613, rs5920340, rs909660, rs5966456, rs1174136, rs12164335, rs12862591, rs48 27611, rs5920245, rs1336838, rs2742905, rs5905137, rs5904833, rs2213174, rs5936206, rs6540 393, rs2149730, rs5940682, rs12400999, rs5924975, rs2266843, rs237370, rs4828561, rs45494 598, rs11798865, rs5970299, rs5970362, rs5925230, rs5925285, rs6526157, rs2980049, rs37473 10. rs5945386, rs5986877, rs2664161, rs573189, rs12557310, rs648970, rs6642252, rs3091262, rs1435934, rs2524543, rs13305774, rs9699368, rs233740, rs9786401, rs879044715, rs4609376. ,

[0009] Furthermore, the hematologic malignancies include, but are not limited to, AML, ALL, MDS, MM, CLL, etc.

[0010] In a second aspect of the invention, a probe combination for detecting combinations of SNPs described in the first aspect of the invention is provided.

[0011] Furthermore, the probe array is used to detect chromosomal ploidy abnormalities in hematologic malignancies.

[0012] Furthermore, the hematologic malignancies include, but are not limited to, AML, ALL, MDS, MM, CLL, etc.

[0013] Furthermore, the probe combination is designed and synthesized according to the SNPs combination described in the first aspect of the present invention, for comprehensive detection of the SNPs combination described in the first aspect of the present invention.

[0014] In a third aspect of the invention, a kit for detecting chromosomal ploidy abnormalities in hematologic malignancies is provided, characterized in that it comprises the probe combination described in the second aspect of the invention.

[0015] Furthermore, the hematologic malignancies include, but are not limited to, AML, ALL, MDS, MM, CLL, etc.

[0016] Furthermore, the kit also includes a library construction reagent kit and a hybridization capture reagent kit.

[0017] Furthermore, the library construction reagent kit includes Frag / AT enzymes, Frag / AT buffer, Ligation Master Mix, Equinox Library Amp Mix, DNA purification magnetic beads, Universal adatpers, UDI primers, and Nuclease-Free Water.

[0018] Furthermore, the hybridization capture reagent kit includes universal blockers, blocking solution, streptavidin binding beads, DNA purification beads, hybridization mix, hybridization enhancer, binding buffer, wash buffer 1, wash buffer 2, Equinox Library Amp Mix, amplification primers, and Nuclease-Free Water.

[0019] In a fourth aspect of the invention, the use of the SNP combination described in the first aspect of the invention or the probe combination described in the second aspect of the invention in the preparation of a kit for detecting chromosomal ploidy abnormalities in hematologic malignancies is provided.

[0020] Furthermore, the hematologic malignancies include, but are not limited to, AML, ALL, MDS, MM, CLL, etc.

[0021] Furthermore, the kit also includes a library construction reagent kit and a hybridization capture reagent kit.

[0022] Furthermore, the library construction reagent kit includes Frag / AT enzymes, Frag / AT buffer, Ligation Master Mix, Equinox Library Amp Mix, DNA purification magnetic beads, Universal adatpers, UDI primers, and Nuclease-Free Water.

[0023] Furthermore, the hybridization capture reagent kit includes universal blockers, blocking solution, streptavidin binding beads, DNA purification beads, hybridization mix, hybridization enhancer, binding buffer, wash buffer 1, wash buffer 2, Equinox Library Amp Mix, amplification primers, and Nuclease-Free Water.

[0024] Furthermore, the specific steps for detecting chromosomal ploidy abnormalities in hematologic malignancies include:

[0025] (1) DNA extraction: DNA was extracted from the sample to be tested using a DNA extraction kit;

[0026] (2) Pre-library construction: The extracted DNA was digested and modified with enzymes using library construction reagents and adapter reagents to obtain a library with a high-throughput sequencing structure;

[0027] (3) Library amplification: The above library was amplified using library construction reagents and adapter reagents, and purified using DNA purification magnetic beads to obtain a pre-library that can be used for probe hybridization.

[0028] (4) Probe hybridization: Using the purified pretext library as a template, a probe hybridization reaction is carried out by applying a blocking reagent, a hybridization elution reagent and the probe combination described in the second aspect of the present invention.

[0029] (5) Streptavidin magnetic bead capture: The library that has completed probe hybridization is eluted by heat using hybridization elution reagent, the library obtained by heat elution is further amplified, and finally the reaction impurities are removed by purification washing to obtain a pure final library.

[0030] (6) Sequencing: High-throughput sequencing and bioinformatics analysis were performed on the purified final library to obtain the detection results of copy number variation in the sample.

[0031] More preferably, the concentration of DNA extracted in step (1) is greater than or equal to 20 ng / μl.

[0032] More preferably, the high-throughput sequencing in step (6) is PE150 high-throughput sequencing.

[0033] More preferably, the high-throughput sequencing in step (6) is performed using the Illumina platform.

[0034] More preferably, the high-throughput sequencing in step (6) is performed using the Illumina Novaseq 6000 platform.

[0035] Further, the specific steps of the bioinformatics analysis in step (6) are as follows: 1) Perform quality control analysis on the raw data using Fastp1 (version 0.23.2, https: / / github.com / OpenGene / fastp) to remove adapters and low-quality reads; 2) Align the quality-controlled data to the hg19 reference gene using bwa2 (version 0.7.17, https: / / github.com / lh3 / bwa) software; 3) Detect copy number variations using CNVkit (v0.9.11, https: / / github.com / etal / cnvkit) to obtain the final detection results.

[0036] In a fifth aspect of the invention, the use of the kit described in the third aspect of the invention in the preparation of products for detecting chromosomal ploidy abnormalities in hematologic malignancies is provided.

[0037] Furthermore, the hematologic malignancies include, but are not limited to, AML, ALL, MDS, MM, CLL, etc.

[0038] Furthermore, the specific steps for detecting chromosomal ploidy abnormalities in hematologic malignancies include:

[0039] (1) DNA extraction: DNA was extracted from the sample to be tested using a DNA extraction kit;

[0040] (2) Pre-library construction: The extracted DNA was digested and modified with enzymes using library construction reagents and adapter reagents to obtain a library with a high-throughput sequencing structure;

[0041] (3) Library amplification: The above library was amplified using library construction reagents and adapter reagents, and purified using DNA purification magnetic beads to obtain a pre-library that can be used for probe hybridization.

[0042] (4) Probe hybridization: Using the purified pretext library as a template, a probe hybridization reaction is carried out by applying a blocking reagent, a hybridization elution reagent and the probe combination described in the second aspect of the present invention.

[0043] (5) Streptavidin magnetic bead capture: The library that has completed probe hybridization is eluted by heat using hybridization elution reagent, the library obtained by heat elution is further amplified, and finally the reaction impurities are removed by purification washing to obtain a pure final library.

[0044] (6) Sequencing: High-throughput sequencing and bioinformatics analysis were performed on the purified final library to obtain the detection results of copy number variation in the sample.

[0045] More preferably, the concentration of DNA extracted in step (1) is greater than or equal to 20 ng / μl.

[0046] More preferably, the high-throughput sequencing in step (6) is PE150 high-throughput sequencing.

[0047] More preferably, the high-throughput sequencing in step (6) is performed using the Illumina platform.

[0048] More preferably, the high-throughput sequencing in step (6) is performed using the Illumina Novaseq 6000 platform.

[0049] Further, the specific steps of the bioinformatics analysis in step (6) are as follows: 1) Perform quality control analysis on the raw data using Fastp1 (version 0.23.2, https: / / github.com / OpenGene / fastp) to remove adapters and low-quality reads; 2) Align the quality-controlled data to the hg19 reference gene using bwa2 (version 0.7.17, https: / / github.com / lh3 / bwa) software; 3) Detect copy number variations using CNVkit (v0.9.11, https: / / github.com / etal / cnvkit) to obtain the final detection results.

[0050] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here.

[0051] Compared with the prior art, the present invention has the following advantages and advancements:

[0052] Comprehensive Chromosomal Coverage: This invention provides a method for detecting chromosomal ploidy abnormalities in hematologic malignancies based on SNP high-throughput sequencing technology. It provides comprehensive coverage of chromosomal ploidy abnormalities that may occur in different hematologic malignancies, eliminating the need for disease pre-specification or pre-specification of specific chromosomes. It covers all autosomes and sex chromosomes, improving the applicability of the method and allowing the detection of rare chromosomal ploidy abnormalities. Compared to common FISH methods that require fixed "AML packages," "ALL packages," and "MDS packages," this invention uses a fixed CNV detection method, covering various diseases including AML, ALL, MDS, and MM, offering comprehensive coverage and high flexibility. Performance comparisons show that the chromosomal ploidy abnormalities detected by this invention have a high degree of agreement with conventional chromosome and FISH detection methods, and can additionally detect chromosomal abnormalities not detected by other conventional methods, providing comprehensive coverage.

[0053] High detection accuracy: Copy number abnormalities exceeding 60% of the length of the long / short arm of the chromosome are defined as ploidy abnormalities, resulting in high detection accuracy; all chromosome structural abnormality parameters are reported by the bioinformatics analysis process and manually compounded, reducing errors caused by human experience factors.

[0054] High mobility: The present invention can be implemented independently or as a detection module, and can be integrated into different conventional NGS detection schemes for hematologic malignancies with appropriate wet laboratory and bioinformatics modes without increasing the detection time or process. It does not require the laboratory to carry out additional projects, which facilitates cost control. It also eliminates the need for additional pathological tissue sampling and delivery, reducing the patient's pain during the sampling process.

[0055] In summary, the present invention can be used for standalone chromosome ploidy detection or as a technical module added to the conventional targeted capture-NGS sequencing workflow. It eliminates the need for dedicated laboratory personnel and specialized testing equipment, and its use in conjunction with existing NGS detection technologies significantly reduces costs. It achieves highly accurate detection of chromosome ploidy, providing comprehensive coverage of all 23 pairs of chromosomes. This overcomes the shortcomings of aforementioned chromosome ploidy abnormality detection schemes in terms of comprehensiveness, accuracy, technical complexity, and cost. Attached Figure Description

[0056] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0057] Figure 1 The present invention provides a general flowchart of the SNP site screening and performance testing process.

[0058] Figure 2Special Case 1: Detection results using the method of this invention. The results showed that the patient's chromosome copy number was significantly abnormal, with multiple deletions, suggesting near-haploidy.

[0059] Figure 3 : Chromosomal karyotype analysis results for special case 1. The karyotype analysis result is a normal karyotype.

[0060] Figure 4 FISH verification results for special case 1. Figure A: Normal copy number of chromosome 21 (green signal); copy number deletion of chromosome 8 (red signal); Figure B: One signal each in the EGR1 gene region (red signal) of the short arm (green signal) and long arm of chromosome 5, indicating copy number deletion; Figure C: One signal each in the short arm (green signal) and long arm (red signal) of chromosome 7, indicating copy number deletion; Figure D: Deletion of centromere 8p11.1-q11.1 of chromosome 8; Figure E: Deletion of short arm (red signal) and centromere (green signal) of chromosome 17; Figure F: One signal each in long arm (red signal) and short arm (red signal) of chromosome 20, indicating copy number deletion.

[0061] Figure 5 Special Case 2: Detection results using the method of this invention. The results showed the presence of chromosomes +4; +6; +8; +10; +17, with CN-LOH present on chromosome 9, and amplification on chromosomes 14, 18, and 21, interpreted as high diploidity.

[0062] Figure 6 : Chromosomal karyotype analysis results for special case 2. The karyotype analysis result is a normal karyotype.

[0063] Figure 7 Figure 2: FISH re-examination results of special case 2. Figure A: 3 signals in the 9p11-q11 region (green) of chromosome 9 centromere, 3 signals in the short arm (red) of chromosome 9, suggesting copy number amplification of chromosome 9; Figure B: 2 signals in the long arm (green) of chromosome 22, 3 signals in the long arm (ABL1 gene) of chromosome 9 (red), indicating copy number amplification of chromosome 9; Figure C: 2 signals in chromosome 19 (green), 3 signals visible in chromosome 1 (red), suggesting copy number amplification of chromosome 1; Figure D: 3-4 signals in RUNX1 (red) of chromosome 21 in some cells, 2 signals in the short arm (green) of chromosome 12, suggesting copy number amplification of chromosome 21; Figure E: Using two-color separation of the MYC probe, 3 fusion signals (green + red) are found on chromosome 8 where MYC is located, suggesting copy number amplification of chromosome 8. Detailed Implementation

[0064] This invention provides a composition, kit, and application for detecting chromosomal ploidy abnormalities in hematologic malignancies based on SNP high-throughput sequencing technology. In principle, when screening and detecting SNP sites, this invention selects probe sequences that can basically uniformly cover classic SNP sites on all 22 pairs of autosomes and the XY sex chromosomes. For key sites, special considerations and processing are applied to the probe coverage area, including but not limited to adjusting and selecting probe coverage density and structure based on differences in GC content, differences in unassembled chromosomal regions, differences in SNP density, key genes, and key locations (terminals, centromeres, etc.).

[0065] The specific steps of SNP site screening in this invention ( Figure 1 )as follows:

[0066] 1. Through preliminary research and review of a large number of literatures and public databases such as dbSNP database and gnomAD, more than 40,000 SNP sites were initially selected. Subsequently, based on big data analysis of the distribution distance and allele frequency of SNP sites, SNPs were initially screened to ensure that the interval between adjacent SNPs is evenly distributed between 200Kb (170-300Kb).

[0067] 2. Further consider the GC content near the SNP site to ensure the uniformity of probe coverage performance;

[0068] 3. At the same time, the distribution of SNPs for more important genes or chromosomes (such as 5q, 17p, etc.) was artificially adjusted to ensure that the coverage density was higher than that of the conventional area; and finally, through manual verification, more than 11,000 SNP sites were selected.

[0069] 4. Adjust and confirm the SNP site combination of the probe;

[0070] 5. We commissioned Twist Bioscience to design and synthesize probes based on the provided SNP site combinations.

[0071] After confirming the stability of the SNP probe performance, in order to use this solution as a technical module and conveniently perform it in sync with conventional NGS detection, we explored the specific experimental scheme multiple times and finally confirmed the existing wet experimental parameters and bioinformatics analysis process.

[0072] The present invention will be specifically described below with reference to embodiments to facilitate a further understanding of the invention by those skilled in the art. However, the embodiments described below are only a part of the embodiments of the present invention and should not be regarded as any form of limitation on the present invention. It should be noted that adjustments and improvements made by those skilled in the art based on the concept of the present invention should be considered within the scope of protection of the present invention. Specific technical operation steps and operators not specified in the embodiments are all performed in accordance with the general technical conditions described in the literature or relevant product manuals.

[0073] Example

[0074] Part 1: Reagent Kits and Detection Methods

[0075] 1. Kit Components

[0076] 1.1 Probe kit name: CNV1-PROBE;

[0077] Components: a combination of ploidy abnormality probes designed and synthesized by Twist Company under the SNP site combination described in this invention, and Nuclease-Free Water.

[0078] 1.2 Library Preparation Kit Name: Twist Library Preparation EF Kit (Catalog No. 104207);

[0079] Ingredients: Frag / AT enzymes, Frag / AT buffer, Ligation Master Mix, EquinoxLibrary Amp Mix, Nuclease-Free Water.

[0080] 1.3 Adapter kit name: Twist Universal Adapter System (catalog number 101308);

[0081] Components: Universal adatpers, UDI primers.

[0082] 1.4 Magnetic bead kit name: Twist Binding and Purification Beads (Catalog No. 100984);

[0083] Components: streptavidin binding beads, DNA purification magnetic beads.

[0084] 1.5 Amplification Kit Name: Equinox Library Amp Mix (Catalog No. 104108);

[0085] Ingredients: Equinox Library Amp Mix.

[0086] 1.6 Universal Blocking Solution Reagent Kit Name: Twist Universal Blockers (Catalog No. 100767);

[0087] Components: universal blockers, blocking solution.

[0088] 1.7 Hybridization and Elution Kit Name: Twist Standard Hyb and Wash Kit (Catalog No. 104447);

[0089] Components: wash buffer1, wash buffer2, hybridization mix, hybridizationenhancer, binding buffer, amplification primers.

[0090] Note: The SNP site combination described in this invention is the core of this invention. The probe combination based on the above SNP combination was designed and synthesized by Twist Company using conventional methods. All other reagents are from Twist Company's reagent kit.

[0091] 2. Experimental Procedure

[0092] (1) DNA extraction: DNA was extracted from the sample to be tested using a DNA extraction kit. The concentration of the extracted DNA was greater than or equal to 20 ng / μl.

[0093] (2) Pre-library construction: The extracted DNA was digested and modified using Frag / AT enzymes, Frag / AT buffer, LigationMaster Mix components in the library construction kit and Universal adatpers components in the adapter kit to obtain a library with high-throughput sequencing structure.

[0094] (3) Library amplification: The above library was amplified using Equinox Library Amp Mix from the library construction kit and UDIprimers from the adapter kit, and purified using DNA purification magnetic beads from the magnetic bead kit to obtain a pre-library that can be used for probe hybridization.

[0095] (4) Probe hybridization: Using the purified pre-library as a template, probe hybridization reaction was carried out by applying universal blockers and blocking solution from the universal blocking solution kit, hybridization mix and hybridization enhancer from the hybridization and elution kit, and CNV1-PROBE from the probe kit of the present invention.

[0096] (5) Streptavidin magnetic bead capture: The library that has completed probe hybridization was heat-eluted using wash buffer 1, wash buffer 2, binding buffer reagents in the hybridization and elution kit and streptavidin magnetic beads (binding beads) reagent in the magnetic bead kit. The library obtained by heat elution was re-amplified using the EquinoxLibrary Amp Mix component in the amplification kit and the amplification primers component in the hybridization and elution kit. Finally, the library was washed with DNA purification magnetic beads reagent in the magnetic bead kit to remove reaction impurities and obtain a pure final library.

[0097] (6) Sequencing: High-throughput sequencing and bioinformatics analysis were performed on the purified final library to obtain the detection results of copy number variation in the sample.

[0098] 3. A method for detecting chromosomal ploidy abnormalities in hematologic malignancies using the kit of the present invention based on SNP high-throughput sequencing technology.

[0099] 3.1. DNA extraction: Use a DNA extraction kit to extract whole-genome DNA from peripheral blood or bone marrow samples.

[0100] Nucleic acid extraction reagent: A commercially available nucleic acid extraction kit (Lab-Aid 896 Genomic DNA type) was used. DNA extraction was performed according to the kit's instructions. The extracted DNA solution was initially tested for nucleic acid concentration using NanoDrop (<60 ng / μl is unacceptable) and A260 / A230 purity (A260 / A230 > 1.7 is acceptable). The extracted DNA solution was then tested again for nucleic acid concentration using Qubit (concentration > 20 ng / μl is acceptable). The nucleic acid extraction kit can be used for manual or automated extraction and should be suitable for nucleic acid extraction from whole cell lines in peripheral blood and bone marrow samples, meeting the above quality control requirements.

[0101] 3.2. The extracted DNA was subjected to library digestion, fragmentation, and end repair.

[0102] The enzyme digestion system is shown in Table 1 below.

[0103] Table 1 Enzyme digestion system

[0104] Frag / AT enzymes 6μL Frag / AT buffer 4μL DNA sample <![CDATA[40 μ L]]>

[0105] The library digestion reaction procedure is as follows:

[0106] Step 1: 37℃, 16min;

[0107] Step 2: 65℃, 30min

[0108] Step 3: Store at 4℃.

[0109] 3.3. Ligating the enzyme-digested fragmented library with universal adapters.

[0110] The universal connector system is shown in Table 2 below.

[0111] Table 2 General Connector Connection System

[0112] Ligation Master Mix 20μL Universal adatpers 5μL Previous product <![CDATA[50 μ L]]>

[0113] The general connector connection procedure for the document library is as follows:

[0114] Step 1: 20℃, 15min;

[0115] Step 2: Store at 20℃.

[0116] 3.4. Purify the product from the previous reaction step.

[0117] (1) Add 60 μL of DNA purification magnetic beads, mix by pipetting or vortexing, and let stand at room temperature for 5 min.

[0118] (2) Centrifuge briefly, place the PCR tube on a magnetic rack for 3 minutes, and wait for the solution to become clear.

[0119] (3) Keep the PCR tube on the magnetic rack and completely remove the supernatant. Add 180 μL of 80% ethanol solution to the PCR tube and let it stand for 30 seconds. Keep the PCR tube on the magnetic rack, discard the supernatant, add 180 μL of 80% ethanol solution to the PCR tube again, let it stand for 30 seconds, and discard the supernatant.

[0120] (4) Carefully use a 10μL pipette to remove any residual ethanol at the bottom, being careful not to pick up the magnetic beads.

[0121] (5) Keep the PCR tube on the magnetic rack and let it stand at room temperature for 3-5 minutes to dry the magnetic beads and allow the residual ethanol to evaporate completely.

[0122] (6) Add 17 μL Nuclease-Free Water, remove the PCR tube from the magnetic rack, mix by pipetting or vortexing, and let stand at room temperature for 2 min.

[0123] (7) Centrifuge briefly, place the PCR tube on a magnetic rack for 2 minutes, and wait for the solution to become clear.

[0124] (8) Use a pipette to draw 15 μL of supernatant, transfer it to a new PCR tube, label it, and prepare for the next reaction.

[0125] 3.5. Expand the library using UDIPrimer to complete the construction of the pre-library.

[0126] The library amplification system is shown in Table 3 below.

[0127] Table 3 Library amplification system

[0128] Previous amplification product 15μL UDI primers 10μL Equinox Library Amp Mix 25μL

[0129] The PCR reaction conditions for library amplification are as follows:

[0130] Step 1: 98℃, 45s;

[0131] Step 2 (8 cycles): 98℃, 15s, 60℃, 30s, 72℃, 30s

[0132] Step 3: 72℃, 1 min.

[0133] Part 4: Store at 4℃.

[0134] 3.6. Purify the amplified product.

[0135] (1) Add 50 μL of DNA purification magnetic beads, mix by pipetting or vortexing, and let stand at room temperature for 5 min.

[0136] (2) Centrifuge briefly, place the PCR tube on a magnetic rack for 3 minutes, and wait for the solution to become clear.

[0137] (3) Keep the PCR tube on the magnetic rack and completely remove the supernatant. Add 180 μL of 80% ethanol solution to the PCR tube and let it stand for 30 seconds. Keep the PCR tube on the magnetic rack, discard the supernatant, add 180 μL of 80% ethanol solution to the PCR tube again, let it stand for 30 seconds, and discard the supernatant.

[0138] (4) Carefully use a 10μL pipette to remove any residual ethanol at the bottom, being careful not to pick up the magnetic beads.

[0139] (5) Keep the PCR tube on the magnetic rack and let it stand at room temperature for 3-5 minutes to dry the magnetic beads and allow the residual ethanol to evaporate completely.

[0140] (6) Add 55 μL Nuclease-Free Water, remove the PCR tube from the magnetic rack, mix by pipetting or vortexing, and let stand at room temperature for 2 min.

[0141] (7) Centrifuge briefly, place the PCR tube on a magnetic rack for 2 minutes, and wait for the solution to become clear.

[0142] (8) Use a pipette to draw 53 μL of the supernatant and transfer it to a new PCR tube. Label the tube and prepare for the next reaction. This step produces a pre-library library. Quantify the pre-library library.

[0143] 3.7. Probe hybridization

[0144] (1) Take 1000 ng of the pre-library and place it in a vacuum concentrator to evaporate to dryness at 45°C.

[0145] (2) Add 8 μL of blockers and 5 μL of blocker solution to the pre-library after evaporation for elution.

[0146] (3) Place the Hybridization mix in a 65°C metal bath and incubate for 10 minutes until the precipitate dissolves. Let it stand at room temperature for 5 minutes.

[0147] (4) Take 20 μL of Hybridization mix and 8 μL of LNV1-PROBE and add them to the library tube containing blockers and blockersolution. After mixing, centrifuge quickly to remove air bubbles, add 30 μL of Hybridization Enhancer to the surface and seal with oil. Place the tube in a PCR instrument for hybridization. The PCR instrument program is 70℃ for 16 hours and then keep warm at 70℃.

[0148] 3.8. Streptavidin magnetic bead capture

[0149] (1) When the hybridization reaction has 1 hour remaining, remove the streptavidin magnetic beads and DNA purification magnetic beads from the refrigerator and allow them to equilibrate at room temperature for 30 minutes.

[0150] (2) Place Binding Buffer, Wash Buffer 1 and Wash Buffer 2 on a 48°C constant temperature metal bath and heat until melted.

[0151] (3) Reagent preheating: Dispense Wash Buffer 1 and Wash Buffer 2 according to the actual required amount, and preheat them at 68℃ and 48℃ respectively.

[0152] (4) Mix the streptavidin magnetic beads thoroughly. Take 100 μL into a 1.5 mL centrifuge tube, add 200 μL of binding buffer, mix by pipetting, place on a magnetic rack until clear, and discard the supernatant. Repeat the addition and discarding of supernatant twice, and finally add 200 μL of binding buffer to resuspend the magnetic beads.

[0153] (5) After the hybridization reaction is complete, take the cleaned streptavidin magnetic beads to the side of the hybridization tube, quickly transfer 50 μL of hybridization solution into the magnetic beads, shake to mix, and then place on a shaker to mix thoroughly at room temperature for 30 minutes.

[0154] (6) Add 200 μL of preheated Wash Buffer 1 to the mixture from the previous step, shake to mix, incubate at 68°C for 5 minutes, place on a magnetic rack until the liquid is clear, discard the supernatant, repeat once, transfer the liquid to a new 1.5 mL centrifuge tube, place on a magnetic rack until the liquid is clear, discard the supernatant.

[0155] (7) Add 200 μL of preheated Wash Buffer 2 to the centrifuge tube, shake to mix, incubate at 48°C for 5 minutes, place on a magnetic rack until the liquid is clear, and discard the supernatant; repeat twice, and use a pipette to remove any remaining liquid, then add 45 μL of preheated Wash Buffer 2. μ Mix with L of water and set aside.

[0156] (8) Take 22.5 μL of the hybridization product and add 27.5 μL of Equinox Library Amp Mix to prepare a PCR amplification system with a total volume of 50 μL. PCR

[0157] The program is as follows: 98℃ pre-denaturation for 45 seconds, 98℃ denaturation for 15 seconds, 60℃ annealing for 30 seconds, 72℃ extension for 30 seconds, 9 cycles of amplification, 72℃ full extension for 1 minute, 4℃ holding, and hot capping at 105℃.

[0158] (9) Add 1.8× DNA purification magnetic beads to the amplification product, mix thoroughly, incubate at room temperature for 5 minutes, place on a magnetic rack until the liquid is clear, discard the supernatant, and add 200

[0159] μ Wash with 80% ethanol aqueous solution for 1 minute, discard the supernatant, repeat the washing once, dry the magnetic beads at room temperature for 4 minutes, air dry, and then use 32... μ Resuspend the magnetic beads in L of water, incubate at room temperature for 5 minutes, place on a magnetic rack until the liquid is clear, and transfer 30 μL of the supernatant to obtain the final library.

[0160] 3.9. Sequencing

[0161] The hybridization capture library was quality controlled using a Qubit quantification system to measure its concentration. Dilution and sequencing were performed according to the official Novaseq 6000 program requirements. The Novaseq 6000 program was started, and the library was placed into the library tube of the Illumina Novaseq 6000 sequencing cartridge as indicated on the sequencer screen. Sequencing reagents were added to the sequencer, which was set to PE150 for sequencing. Sequencing was then initiated. After sequencing, the data was obtained and subjected to bioinformatics analysis. Detailed experimental procedures were performed according to the official Novaseq 6000 sequencing platform documentation (https: / / support.illumina.com / content / dam / illumina-support / documents / documentation / system_documentation / novaseq / 100000001935).

[0162] 8_17_novaseq-6000-system-guide.pdf).

[0163] The specific steps of the bioinformatics analysis are as follows: 1) Perform quality control analysis on the raw data using Fastp1 (version 0.23.2, https: / / github.com / OpenGene / fastp) to remove adapters and low-quality reads; 2) Align the quality-controlled data to the hg19 reference gene using bwa2 (version 0.7.17, https: / / github.com / lh3 / bwa) software; 3) Detect copy number variations using CNVkit (v0.9.11, https: / / github.com / etal / cnvkit) to obtain the final detection results.

[0164] Part Two: Test Results and Verification

[0165] 1. Accuracy

[0166] (1) Experimental scheme

[0167] Fifty-one routine test samples were selected, and the results of chromosome and / or FISH tests submitted in the same batch were compared using the method of this invention to analyze the positive concordance rate of the experimental results.

[0168] (2) Sample test results

[0169]

[0170]

[0171]

[0172]

[0173]

[0174] Conclusion: All 51 patients detected by the present invention showed the same or partially the same chromosomal karyotype results through chromosome karyotype or FISH results, with a high concordance rate: 42 cases showed complete concordance, and 9 cases showed partial concordance. The reasons for partial concordance were mainly (1) complex karyotypes leading to high chromosome clone diversity, which was lower than the LOD reported by CNV in this method; (2) the detection accuracy of small chromosomal abnormalities by this method was lower than that of conventional chromosome karyotype detection. However, none of these factors affected the diagnosis of the disease and the stratification of risk for the patients. Among them, the chromosome karyotype results of some patients indicated no mitotic figures or normal ploidy, but the FISH test of the same batch showed chromosomal ploidy abnormalities, which indirectly verified that this method can be used as a supplementary method for the detection of chromosome ploidy in patients with special cases.

[0175] 2. Detailed analysis of the results of special case testing

[0176] Special Case 1:

[0177] The patient, suffering from acute lymphoblastic leukemia, was found to have multiple chromosome copy number deletions using the method described in this invention. Near-haploidy is considered highly probable, and based on the method described in this invention, the chromosome karyotype is likely 27. <n>,X,+X,+6,+10,+18,+21( Figure 2 ).

[0178] The patient's chromosome karyotype test at another hospital indicated a normal karyotype. Figure 3 It cannot be ruled out that during cell culture, tumor cells may not proliferate well, resulting in the presence of mainly normal cells for karyotype analysis, thus failing to analyze tumor cells with chromosomal ploidy abnormalities, leading to significant deviations in the results.

[0179] To confirm the accuracy of the chromosomal ploidy results detected by the method of this invention, we used multiple FISH-related probes to verify the ploidy results of the relevant chromosomes, confirming that the patient had abnormalities of -5, -7, -8, -17, and -20. Figure 4 The results showed that the chromosome karyotypes obtained were inconsistent with those from other hospitals, which indirectly verified the accuracy of the present invention in detecting multiple chromosomal ploidy abnormalities.

[0180] Special Case 2:

[0181] Patients with acute lymphoblastic leukemia were found to have multiple chromosome copy number amplifications using the method described in this invention. Figure 5 Considering the high probability of hyperdiploidy, the proposed scheme considers abnormalities in chromosomes +4, +6, +8, +9, +10, +17, +18, and +21. However, chromosome karyotype testing indicates a normal karyotype. Figure 6 The patient submitted a FISH test kit for B-ALL related fusion gene testing. No B-ALL related fusion genes were found, but amplification was observed on chromosomes 9, 8, and 21. Figure 7 Considering all factors, the patient is hyperdiploid.

[0182] The above description is merely a preferred embodiment of the present invention and does not limit the invention in any way. Modifications and alterations made to the above embodiments by those skilled in the art based on the technical essence of the present invention should all fall within the protection scope of the present invention.< / n>

Claims

1. A probe array for detecting combinations of SNPs used to detect chromosomal ploidy abnormalities in hematologic malignancies, characterized in that, The SNP combination contains SNP loci 1-11943, a total of 11943 SNP loci. The rs numbers of SNP loci 1-11943 are rs2340527, rs13302969, rs6701114, rs1272959, rs904586, rs28689700, rs601141, rs12744867, rs3107129, rs1532385, rs2477702, rs187759598, rs897628, rs1474562, rs2455137, rs4648507, rs12401505, rs10909797, and rs3513113.

5. rs10915428, rs34429154, rs4654438, rs6681520, rs7547152, rs9286973, rs1320179, rs36139907, rs7528859, rs28479388, rs1295100, rs2294713 , rs278017, rs10128028, rs11120877, rs1750851, rs3124803, rs488728, rs2120459, rs2708629, rs1318218, rs6698830, rs4908803, rs2032539, rs10 864414, rs115849843, rs4240910, rs6680076, rs34970915, rs10864459, rs284288, rs11586085, rs6540964, rs2744815, rs2817580, rs9430208, rs19 99594、rs562844、rs3397、rs4845908、rs12089791、rs570910916、rs2995600、rs354581、rs4661455、rs6673156、rs6662664、rs12401417、rs705576、r s761288, rs1622686, rs550003, rs10927798, rs848217, rs146865861, rs221025, rs2209174, rs6691954, rs2244416, rs3003449, rs2501778, rs77340 0848, rs1193338, rs620547, rs11261023, rs1832787, rs2244796, rs2789322, rs12043029, rs1078894, rs4912067, rs6683419, rs10916667, 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rs13318540, rs3732360, rs1154595, rs1259293, rs6787364, rs470537, rs2331525, rs6787064, rs4482697, rs6801693, rs9864290, rs4485669rs3821688、rs4378918、rs6784995、rs6762009、rs820458、rs4485668、rs1 444754、rs12490497、rs333318、rs6438856、rs6438874、rs2001911、rs297 9345, rs7623362, rs9871359, rs7634065, rs7638412, rs9858948, rs891762, rs58451553, rs6762473, rs7652988, rs4857833, rs10934853, rs2840776 4、rs34774315、rs413183、rs10934882、rs2625951、rs729208、rs2717241、 rs6439205、rs9846729、rs6795367、rs11915110、rs218503、rs876519、rs1 225051、rs1357078、rs9828393、rs2305627、rs4337638、rs6439382、rs764 4980、rs11712866、rs9847085、rs11921527、rs9841380、rs4269060、rs9866 896, rs9811000, rs36054, rs1875374, rs4894292, rs9852169, rs518972, rs711975, rs7616204, rs6439688, rs6439701, rs768496, rs201683, rs3773 770, rs774801, rs7626226, rs361061, rs67386737, rs9823043, rs295493, rs12629519, rs970929, rs2350498, rs347320, rs9843100, rs6808549, rs15 11371, rs6763927, rs55813385, rs9869659, rs10935455, rs57177110, rs9866958, rs3844273, rs6440128, rs2119636, rs6789065, rs6779887, rs982 2444、rs7611326、rs6440234、rs1178893、rs6789186、rs1348657、rs38459 61、rs2673862、rs6440402、rs6800434、rs4681322、rs163731、rs67156303、rs9814423、rs9860691、rs57499472、rs4681371、rs28544926、rs11929120 rs4306816、rs2640543、rs34974178、rs4681486、rs9851045、rs9289790、 rs57150193、rs9857853、rs6788804、rs4234379、rs12634699、rs6809546、 rs9817380、rs6798154、rs35998846、rs73166984、rs11915178、rs472976、r s3872620、rs6764808、rs9837387、rs55932663、rs965573、rs12486542、rs 375641446、rs171390、rs4290775、rs11914501、rs9850393、rs7627097、rs 358702, rs4680228, rs7633871, rs9831425, rs7651778, rs9837271, rs1600978, rs67196056, rs7632702, rs9874900, rs1877593, rs7633541, rs82711 8、rs12489496、rs9866134、rs340294、rs7653174、rs9290040、rs9878736、 rs507927、rs2152425、rs62272791、rs6781583、rs1447626、rs9864752、rs 7631479、rs6441391、rs7622055、rs62278717、rs9828074、rs13071745、rs 4059921、rs12486709、rs9851405、rs1946403、rs6810129、rs67672351、rs7 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rs9353374, rs1856419, rs4707320, rs7755883, rs9450723, rs35133466, rs366676, rs6904058, rs1408732, rs9359815, rs1929264, rs109 44439、rs942115、rs12661693、rs9451316、rs4706361、rs6900685、rs4011 47、rs2325328、rs1220507、rs6454838、rs6937560、rs7748368、rs9451856、 rs1353115、rs13211323、rs6905474、rs2787751、rs1486761、rs12661832、 rs6454976、rs1937357、rs1338338、rs426184、rs6929516、rs1183161、rs51 2153、rs9400791、rs13203167、rs67043512、rs4840232、rs975239、rs2971 610、rs1359932、rs1884168、rs9387216、rs62420695、rs9401295、rs250504 1、rs1451347、rs9401969、rs4555920、rs7759571、rs9385694、rs9373169、 rs9403091、rs4840109、rs6937293、rs4840129、rs1875401、rs12215909、rs 9373602, rs2852498, rs673318, rs59398974, rs1413973, rs2153395, rs6571046, rs9404272, rs176424, rs1008965, rs2506724, rs6912929, rs775457 6, rs9373748, rs2744161, rs156130, rs9377657, rs9404579, rs221634, rs576483662, rs1269289, rs1270380, rs9386485, rs6568433, rs3121433, rs9 373914, rs9373927, rs7775235, rs4425630, rs938972, rs9386711, rs2802290, rs6923730, rs409261, rs2222392, rs1885691, rs9384717, rs10499054rs4317449, rs7753212, rs11751416, rs802658, rs9374234, rs383022, rs4947143, rs9372316, rs7752759, rs1342596, rs9374364, rs1547814, rs3851 202、rs9398350、rs6901075、rs717388、rs9400700、rs1997649、rs9398386 rs4483016、rs2807961、rs4946120、rs6932852、rs1114228、rs484498、rs1 321533、rs601200、rs1321365、rs6901720、rs4945582、rs4946258、rs1321 1821、rs9320622、rs9489314、rs6919271、rs7751213、rs7743045、rs494638 2, rs2558, rs1342316, rs2016684, rs2357678, rs2357759, rs1407798, rs9372598, rs960298, rs10782205, rs4945672, rs9482119, rs6937659, rs9490 337、rs2679683、rs34939692、rs4061300、rs1430817、rs9482284、rs93209 19、rs3813328、rs2611427、rs4406249、rs802295、rs1322588、rs584170、rs 9388374、rs573850、rs1270701、rs4897100、rs4897157、rs647444、rs1159 619、rs9388518、rs12664316、rs9375487、rs9482826、rs6919966、rs776753 1, rs4085784, rs9388666, rs9375607, rs902370, rs6569620, rs6939565, rs9398939, rs12210502, rs4897418, rs9388808, rs6900707, rs9492774, rs7 754788、rs942150、rs2807276、rs6569758、rs233906、rs13205427、rs1044 593、rs9402468、rs6941848、rs212773、rs1468054、rs2327426、rs1743961、rs11969046、rs228442、rs6569961、rs9399126、rs6913228、rs7757431、rs 10872447、rs7765567、rs9942517、rs9942519、rs11154886、rs7750251、rs 276550, rs9494708, rs7749298, rs6921233, rs629953, rs7743142, rs4896375, rs4896401, rs6900336, rs9495412, rs3010290, rs9376465, rs949562 0、rs675432、rs6570405、rs58895415、rs9385948、rs9385960、rs9376644、 rs7774877、rs9484615、rs9496392、rs2328370、rs198683、rs55956634、rs 223596, rs6570565, rs7771214, rs9390136, rs9496852, rs587889, rs4574662, rs6906465, rs6939586, rs4896766, rs680108, rs559087, rs11155416 rs9403726 rs9403746 rs2777477 rs6905721 rs4896872 rs1325701 s7775644、rs2818868、rs9390461、rs634753、rs11155524、rs586166、rs94 97930, rs2493920, rs6903119, rs9485313, rs62426096, rs6935330, rs4897111, rs2789503, rs7767622, ​​rs4870080, rs9478330, rs5002108, rs77720 11、rs4075301、rs9478812、rs536100、rs752543、rs12525200、rs4869739、 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66528070、rs7878946、rs34194220、rs213452、rs5922001、rs7060308、rs5 922062, rs5923253, rs68134057, rs5968583, rs5923438, rs1016716, rs9697976, rs5923637, rs4367719, rs5923944, rs6624071, rs6614705, rs70614 30, rs5924228, rs6617585, rs5942437, rs987725, rs5941128, rs3912025, rs4893203, rs1815386, rs5941518, rs537862, rs7049694, rs4537467, rs31 02580, rs5984896, rs2145774, rs3952482, rs79193482, rs1417323, rs6615509, rs5983556, rs1207445, rs5939840, rs808753, rs2799949, rs1285223 6, rs2353627, rs3131391, rs5949614, rs5990464, rs1018379, rs2803385, rs4969679, rs829799, rs5920650, rs6620231, rs716621, rs5920959, rs59 66829、rs2498870、rs5921411、rs5921531、rs7891677、rs6616172、rs5921 681、rs2360670、rs2239461、rs6621108、rs2213369、rs4128745、rs5986852 rs5945747、rs5945776、rs2032469、rs1180761、rs59367175、rs5962434、 rs2227031、rs5917183、rs4826916、rs210560、rs980099、rs67864005、rs78 83179、rs2064722、rs10284196、rs6642760、rs1936494、rs478023、rs5942 614、rs5942935、rs5942672、rs5929568、rs583430、rs6643146、rs2369619、rs4829572、rs5974095、rs5974230、rs5987737、rs5950482、rs5950570、rs6520224、rs1573725、rs4411072、rs5909705、rs2190281、rs5956746、rs12844666、rs5956058、rs1287170、rs28616029、rs57798042、rs2285552、rs1005649、rs2072067、rs5910941、rs5909783、rs6649076、rs77033101、rs6648722、rs1680088、rs2506498、rs4240008、rs28361959、rs1293470、rs3761557、rs5911614、rs6648536、rs1279829、rs5956652、rs2858409、rs5910113、rs28463787、rs140367774、rs5930312、rs5930634、rs4829815、rs5929897、rs1937560、rs5974858、rs204307、rs201644、rs6637268、rs226610、rs2032467、rs12009653、rs5932497、rs6637517、rs2223196、rs5977088、rs3131270、rs209547、rs4829716、rs4415477、rs6637825、rs5932983、rs2144559、rs415335、rs5977749、rs7889796、rs242138、rs6529574、rs5933328、rs2355972、rs11796053、rs2097402、rs5933529、rs6528273、rs781848432、rs7061195、rs6635257、rs1478832、rs5931092、rs2011284、rs1206208、rs5975975、rs2366517、rs5931321、rs1601809、rs5931506、rs6635777、rs2887002、rs2485724、rs56024130、rs5954027、rs6654402、rs4824993、rs761559、rs5907953、rs1977161、rs5954475、rs6634356、rs5954640、rs12844538、rs5954889、rs5908535、rs990581、rs12852291、rs237497、rs73244040、rs4144180、 rs5907555、rs1887613、rs5920340、rs909660、rs5966456、rs1174136、rs12164335、r s12862591, rs4827611, rs5920245, rs1336838, rs2742905, rs5905137, rs5904833, rs2213174, rs5936206, rs6540393, rs2149730, rs5940682, rs12400999, rs5924975 rs2266843, rs237370, rs4828561, rs45494598, rs11798865, rs5970299, rs5970362, rs5925230, rs5925285, rs6526157, rs2980049, rs3747310, rs5945386, rs5986877 rs2664161, rs573189, rs12557310, rs648970, rs6642252, rs3091262, rs1435934, rs2524543, rs13305774, rs9699368, rs233740, rs9786401, rs879044715, rs4609376.

2. A kit for detecting chromosomal ploidy abnormalities in hematologic malignancies, characterized in that, It includes the probe assembly as described in claim 1.

3. The reagent kit according to claim 2, characterized in that, The kit also includes a library construction reagent set and a hybridization capture reagent set.

4. The use of the probe combination according to claim 1 in the preparation of a kit for detecting chromosomal ploidy abnormalities in hematologic malignancies.

5. The application according to claim 4, characterized in that, The specific steps for detecting chromosomal ploidy abnormalities in hematologic malignancies include: DNA extraction: DNA is extracted from the sample to be tested using a DNA extraction kit; Pre-library construction: The extracted DNA was digested and modified using library construction reagents and adapter reagents to obtain a library with a high-throughput sequencing structure; Library amplification: The above library was amplified using library construction reagents and adapter reagents, and then purified using DNA purification magnetic beads to obtain a pre-library that can be used for probe hybridization. Probe hybridization: Using the purified pre-library as a template, a probe hybridization reaction is performed using a blocking reagent, a hybridization elution reagent, and the probe combination described in claim 1. Streptavidin magnetic bead capture: The library that has completed probe hybridization is eluted by heat using hybridization elution reagent, the library obtained by heat elution is re-amplified, and finally the reaction impurities are removed by purification washing to obtain a pure final library. Sequencing: High-throughput sequencing and bioinformatics analysis are performed on the purified final library to obtain the detection results of copy number variations in the sample.

6. The use of the kit according to claim 2 or 3 in the preparation of products for detecting chromosomal ploidy abnormalities in hematologic malignancies.

7. The application according to claim 6, characterized in that, The specific steps for detecting chromosomal ploidy abnormalities in hematologic malignancies include: DNA extraction: DNA is extracted from the sample to be tested using a DNA extraction kit; Pre-library construction: The extracted DNA was digested and modified using library construction reagents and adapter reagents to obtain a library with a high-throughput sequencing structure; Library amplification: The above library was amplified using library construction reagents and adapter reagents, and then purified using DNA purification magnetic beads to obtain a pre-library that can be used for probe hybridization. Probe hybridization: Using the purified pre-library as a template, a probe hybridization reaction is performed using a blocking reagent, a hybridization elution reagent, and the probe combination described in claim 1. Streptavidin magnetic bead capture: The library that has completed probe hybridization is eluted by heat using hybridization elution reagent, the library obtained by heat elution is re-amplified, and finally the reaction impurities are removed by purification washing to obtain a pure final library. Sequencing: High-throughput sequencing and bioinformatics analysis are performed on the purified final library to obtain the detection results of copy number variations in the sample.

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