Library establishment kit and probe for detecting hematologic tumor chemotherapy related gene polymorphism

Abstract

The invention belongs to the technical field of gene sequencing, and particularly relates to a library building kit and a probe for detecting hematologic tumor chemotherapy related gene polymorphism by using a hybrid capture method. According to the present invention, the genotype of the hematologic tumor chemotherapy related gene, the drug treatment effect, the toxicity and the metabolic capability related information can be rapidly obtained through the one-time detection; meanwhile, the library building kit is simple and convenient to operate and short in consumed time, and automatic detection is extremely easy to realize. The detection result completed by using the library building kit is accurate, and the library building kit has important reference significance for treatment and prognosis of hematologic tumor patients needing chemotherapy.

Description

Technical Field

[0001] This invention belongs to the field of gene sequencing technology, specifically relating to a library preparation kit and probe for detecting chemotherapy-related gene polymorphisms in hematological malignancies using hybridization capture method. Background Technology

[0002] Hematologic malignancies are complex diseases involving the interaction of multiple genetic and environmental factors. Chemotherapy remains a cornerstone treatment in the clinical management of hematologic malignancies. However, significant differences in the responsiveness (efficacy and toxicity) of patients with different genetic backgrounds to the same chemotherapy regimen are commonly observed in clinical practice. These individual differences are traditionally unpredictable, often leading to some patients facing the risk of disease relapse or progression due to insufficient drug efficacy, while others experience severe drug toxicity, affecting treatment progress and even endangering their lives. The significant differences in patient responses to chemotherapy drugs make individualized treatment an urgent need.

[0003] Genetic polymorphism (i.e., genetic variations of the same gene in different individuals) plays a crucial role in drug metabolism, efficacy, and adverse reactions. The efficacy and toxicity of chemotherapy drugs (such as irinotecan and cisplatin) are closely related to the genetic polymorphism of patients. With the rise of precision medicine, the application of gene testing technology to guide treatment plans for cancer patients has become a trend. By detecting genetic polymorphisms related to chemotherapy drug metabolism and combining them with current research findings in pharmacogenomics, the response to chemotherapy drugs can be assessed, thereby guiding clinical treatment decisions. For example, for thiopurines (such as 6-mercaptopurine), a core drug for acute lymphoblastic leukemia (ALL), detecting the NUDT15 genotype can accurately identify patients with low enzyme activity. For these patients, clinical guidelines recommend significantly reducing the standard dose to 30-80% of the usual dose (intermediate metabolizer) or 10% of the usual dose (slow metabolizer), thereby effectively preventing fatal myelosuppressive toxicity. Therefore, it is evident that gene testing related to chemotherapy in hematological malignancies is necessary. It can help develop individualized chemotherapy plans for patients, improve the therapeutic effect of chemotherapy drugs, reduce the occurrence of adverse reactions, and improve patients' survival rate and quality of life. Summary of the Invention

[0004] This invention aims to overcome the limitations of existing gene detection technologies in terms of throughput, accuracy, efficiency, or detection range, and provides a novel library preparation kit, its usage method, and probes for detecting gene polymorphisms related to commonly used chemotherapy drugs for hematological malignancies.

[0005] To achieve the above-mentioned objectives, the present invention is implemented through the following technical solution: A probe set for detecting gene polymorphisms related to chemotherapy drugs for hematological malignancies, the probe set comprising multiple probes, the probes being able to specifically hybridize to polymorphic sites of genes related to the metabolism, efficacy or toxicity of chemotherapy drugs for hematological malignancies via hybridization capture method, the base sequences of the probes being selected from at least one of SEQ ID NO. 1 to 64.

[0006] This invention aims to develop a novel gene detection kit for commonly used chemotherapy drugs in hematological malignancies, along with its application method and probes. The goal is to overcome the limitations of existing detection technologies in terms of throughput, accuracy, efficiency, and detection range, providing a more comprehensive, rapid, and accurate solution. This will offer strong technical support for truly personalized chemotherapy in clinical practice, thereby promoting the transformation of hematological malignancy treatment from "empirical medication" to "gene-guided medication," helping physicians predict patient responses to chemotherapy drugs, develop individualized treatment plans, reduce side effects, improve efficacy, and extend patient lifespan.

[0007] Preferably, the probe coverage area contains genes related to chemotherapy drugs for hematologic malignancies, including pharmacogenomics genes related to chemotherapy for hematologic malignancies, and the genes are selected from at least one of the following groups: BCL2L11, ABCB1, CDA, CYP2C9, SERPINE1, ABCC1, ABCC2, CYBA, NCF4, RAC2, CYP2B6, ABCC4, DHFR, MTHFR, SLC01B1, FGF2, NUDT15, SLC7A5, CPA2, NFATC2, SOD2, GNMT, ITPA, TPMT, NOS3, NRP2, ERCC1, XRCC5, CEP72, CYP2E1, SLC22A12, SULT2B1, DCK, NT5C3A, CTH, DROSHA, CTNNB1, GSTA1, RRM1, RRM2, RRM2B, PYGL, GATA3, PNPLA3, FOLH1, GSTP1.

[0008] A library preparation kit for detecting gene polymorphisms related to chemotherapy drugs in hematological malignancies, the library preparation kit comprising the probe set described above.

[0009] This invention discloses a library construction kit for detecting polymorphisms of genes related to chemotherapy drugs for hematological malignancies using a hybridization capture method. Using a very low starting amount of genomic DNA as a template, the kit achieves enzyme digestion, end repair, and A-tailing in a single reaction. Then, an adapter is added, followed by magnetic bead purification. After a brief index amplification, the library is constructed. A personalized probe, a hematological malignancy chemotherapy panel, is then used to rapidly hybridize the library, achieving hybridization efficiency in just 15 minutes that typically requires an overnight reaction. The hybridization products are then captured, enriched, amplified, and purified using streptomycin magnetic beads to obtain the final targeted library. The targeted library is then subjected to high-throughput sequencing. After sequencing data is obtained, bioinformatics analysis and comparison, using relevant bioinformatics analysis software and pharmacogenomics databases, allows for rapid and accurate typing of multiple genes related to chemotherapy drugs for hematological malignancies.

[0010] Preferably, the library construction kit supports the simultaneous construction of libraries with 384 indexes and is compatible with the Illumina sequencing platform.

[0011] Preferably, the library preparation kit further includes a hybridization component comprising FastHybridization Mix and paraffin oil.

[0012] A method for using a library preparation kit for detecting gene polymorphisms related to chemotherapy drugs in hematological malignancies includes the following steps: Using the probe set described above, the sample DNA was hybridized, captured, and sequenced using a hybridization capture method.

[0013] Preferably, the method also includes a library construction step, which includes an integrated reaction of enzyme digestion, end repair, and A-tailing starting with genomic DNA.

[0014] Preferably, the initial amount of genomic DNA is 10-100 ng.

[0015] Preferably, the reaction time for the hybridization capture reaction is 0.25–2 h.

[0016] Preferably, the method also includes the step of capturing and enriching the hybridization product using streptomycin magnetic beads.

[0017] Preferably, the sequencing analysis step includes comparing sequencing data through bioinformatics analysis and outputting genotyping results in conjunction with a pharmacogenomics database to guide the selection of chemotherapy drugs for patients with hematologic malignancies.

[0018] Therefore, the present invention has the following beneficial effects: (1) The library preparation kit manufactured by the present invention can detect the polymorphism of genes related to chemotherapy drugs for hematological malignancies using the hybridization capture method; only a small amount of genomic DNA is needed to construct the DNA library, and the template starting amount can be as low as 10 ng; (2) This invention completes enzyme digestion, end-filling and A-tailing in one reaction system, and then uses the self-designed probe panel for hybridization capture and enrichment of the library, which is time-saving and efficient. (3) The library construction kit of the present invention has up to 384 index pairs, which can realize the simultaneous construction and sequencing of 384 libraries; (4) The rapid hybridization system of the library preparation kit of the present invention can achieve the efficiency of general overnight hybridization in as little as 15 minutes, and finally obtain a library fragment size of about 300~400 bp, which can be compatible with various Illumina sequencing platforms for sequencing; (5) Through a simple and easy-to-use synthesis-on-NGS sequencing solution, rapid and accurate typing of genes that may affect the metabolism, efficacy and toxic side effects of chemotherapy drugs for hematological malignancies can be achieved; (6) A single test can quickly obtain the genotype of chemotherapy drug-related genes and the efficacy and toxicity of various common chemotherapy regimens or single drugs. At the same time, the library preparation kit of this invention is simple to operate, time-saving, and easy to automate. It helps doctors predict patients' responses to chemotherapy drugs, thereby developing individualized medication plans to reduce toxic side effects, improve efficacy, and prolong patients' lifespan. It has good clinical application prospects and is easy to promote and apply in clinical practice. Attached Figure Description

[0019] Figure 1 This is a graph showing the fragment analysis results of the DNA library constructed in Example 1. Detailed Implementation

[0020] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0021] Example 1: [Building a Library] [Library Construction Using Enzyme Digestion] 1.0 DNA fragmentation, end repair, and A-tailing (amplification region) First, turn on the PCR instrument and set the temperature of the PCR instrument's heating cap to 70℃.

[0022] 1.0.1 The initial amount of DNA for library construction is 10~100 ng. After thorough mixing and short-term in vitro incubation, place on ice for later use. 1.0.2 From the reagent preparation area, take a new 1.5 mL centrifuge tube and prepare the enzyme digestion reaction mixture. Thaw 10x Fragmentation Buffer and 5x Fragmentation Enzyme (Twist Bioscience) on ice, prepare the mixture according to Table 1 below, mix well, centrifuge, aliquot the mixture into eight-tube strips, and then transfer them to the amplification area through the transfer window (at the same time, the DNA purification magnetic beads to be used in the next purification step can be taken out of the refrigerator, thoroughly shaken to mix, and then transferred together to the amplification area, allowing to equilibrate at room temperature for at least 30 min). Table 1: reagents Single reaction volume 10x Fragmentation Buffer 5 μL 5x Fragmentation Enzyme 10 μL Total 40 μL .

[0023] 1.0.3 Add 35 μL of each DNA sample to be tested into an eight-tube strip containing the prepared reaction mixture, gently vortex to mix, briefly insulate, and then quickly place the strip into the PCR instrument. Ensure the PCR instrument's heating cap temperature is 70℃, and then follow the procedure shown in Table 2 below: Table 2: temperature time 32℃ 22 min 65℃ 30 min 4℃ HOLD .

[0024] 1.1 Connecting the Universal Adapter 1.1.1 After completing the above steps, add 5 μL of Universal Adapter (Twist Bioscience) to each reaction system. Gently vortex to mix, centrifuge, and place on ice for later use; 1.1.2 Proceed to the reagent preparation area to prepare the ligation reaction mixture. After thawing the DNA Ligation Buffer and DNA Ligation Mix (Twist Bioscience) on ice, prepare the mixture according to Table 3 below, mix well, centrifuge, and transfer to the amplification area on ice for later use; Table 3: reagents Single reaction volume Water 15 μL DNA Ligation Buffer 20 μL DNA Ligation Mix 10 μL Total 45 μL .

[0025] (3) Add 45 μL of the ligation reaction mixture prepared in step 1.1.2 to each sample prepared in step 1.0.3 above. Gently vortex to mix, centrifuge, and then quickly place in a PCR instrument. Incubate at 20°C for 15 minutes (with the hot cap closed). After completion, remove the sample and immediately begin magnetic bead purification.

[0026] 1.2 First step: Magnetic bead purification (amplification region) 1.2.1 Library purification magnetic beads (Vazyme) should be removed from 4°C before use, thoroughly vortexed to mix, and equilibrated at room temperature for at least 30 minutes before use; 1.2.2 Shake the purified magnetic bead suspension (Vazyme) equilibrated at room temperature for 1 min, and aliquot it into 1.5 mL centrifuge tubes at a rate of 80 μL (0.8 times the sample volume) / sample. 1.2.3 Transfer all the bridging products to the corresponding magnetic beads in the centrifuge tube, gently shake to mix, and avoid generating air bubbles; 1.2.4 After letting the centrifuge tubes stand at room temperature for 5 minutes, place them on a magnetic rack for 5 minutes until the supernatant becomes clear. Carefully remove the supernatant. 1.2.5 Keep the centrifuge tubes on the magnetic rack, add 200 μL of freshly prepared 80% ethanol to each tube, let stand at room temperature for 1 min, and discard the supernatant; 1.2.6 Repeat step 1.2.5, and after a brief centrifugation, place the centrifuge tube back into the magnetic rack and use a 10 μL pipette to remove as much residual ethanol solution as possible from the bottom of the tube; 1.2.7 Leave the centrifuge tubes on the magnetic rack at room temperature with the lid off for 5 minutes to allow all the ethanol to evaporate. Do not over-dry. 1.2.8 Add 17 μL of enzyme-free water to each tube and mix thoroughly 10 times using a pipette. Incubate at room temperature for 5 min; 1.2.9 Place each tube on a magnetic rack and let it stand for 5 minutes until the supernatant is clear.

[0027] 1.3 PCR amplification using UDI adapter primers 1.3.1 During step 1.2.9, you can go to the reagent preparation area and prepare eight-tube sets on the ice box according to the number of samples. Take 25 μL of KAPA HiFiHotStartReadyMix (KAPA) and dispense it into each well of the eight-tube set. 1.3.2 Add 10 μL of pre-set UDI primers (Twist Bioscience) at different indices to each well of the eight-tube array. Then transfer the primers through the transfer window to amplify region I. 1.3.3 Following the pre-set position order, add 15 μL of purified supernatant obtained in step 1.2.9 to each well of the pre-allocated PCR reaction mixture, cap the tube, gently shake, and briefly centrifuge to collect the liquid; 1.3.4 Transfer the prepared PCR reaction tubes to the amplification area through the transfer window for PCR reaction. Heat the lid at 105℃. The reaction program settings are shown in Table 4 below. Table 4: .

[0028] 1.4 Second step: Magnetic bead purification (amplification region) 1.4.1 Library purification magnetic beads (Vazyme) should be removed from 4°C before use, thoroughly vortexed to mix, and equilibrated at room temperature for at least 30 minutes before use; 1.4.2 Shake the purified magnetic bead suspension, which has been equilibrated at room temperature, for 1 min, and aliquot it into 1.5 mL centrifuge tubes at a rate of 50 μL (equal volume to the sample). 1.4.3 Remove the PCR reaction product, centrifuge for 30 seconds, transfer all the product to the corresponding magnetic bead in the centrifuge tube, and gently shake to avoid generating air bubbles; 1.4.4 After standing at room temperature for 5 minutes, place on a magnetic separator for 5 minutes until the supernatant is clear. Carefully remove the supernatant; 1.4.5 Place it on a magnetic separator, add 200 μL of freshly prepared 80% ethanol, let it stand at room temperature for 1 min, and discard the supernatant; 1.4.6 Repeat step 1.4.5, and try to remove all residual ethanol solution; 1.4.7 Leave the centrifuge tubes on the magnetic rack at room temperature for 5 minutes to dry, ensuring that all ethanol evaporates completely; 1.4.8 Add 22 μL of enzyme-free water and gently shake. Incubate at room temperature for 5 min; 1.4.9 Place the sample on a magnetic rack for 5 min, then take 20 μL of the supernatant (library) for the next step of library enrichment. Take 1 μL and dilute it 10-fold. Use a Qubit analyzer and a fragment analyzer to determine the library concentration and fragment size, respectively. The average fragment length of the detected library should be around 350 bp.

[0029] [Hybrid Capture] 2.0 Library Hybridization 2.0.1 DNA Mixing Protocol for Library Pools: Library pools typically contain multiple different libraries. The amount of DNA to be added to the library pool is calculated based on the concentration of the library DNA, and the mixtures are then used to obtain the final library pool. The total amount of DNA in the library pool is 1.5–4 μg, and it is preferred to establish libraries pools with samples of equal mass. Refer to Table 5 below for the DNA library usage amounts: Table 5: Mixed sample size Usage per library Total amount of each reaction library 1 1500 ng 1500 ng 2 1000 ng 2000 ng 3 1000 ng 3000 ng 4 800 ng 3200 ng 8 400 ng 3200 ng .

[0030] 2.0.2 Calculate the required amount of each library, mix them in a 1.5 mL centrifuge tube, gently shake, and centrifuge briefly for later use; 2.0.3 Add the prehybridization reagents shown in Table 6 below to the mixed samples, mix well, and centrifuge briefly, trying to avoid generating air bubbles; Table 6: reagents volume HM Panel probes (probe sequences are SEQ ID NO.1~64) 4 μL Universal Blockers (Twist Bioscience) 8 μL Blocking Solution (Twist Bioscience) 5 μL .

[0031] 2.0.4 Open the caps of the prehybridization reagent tubes and place them in a vacuum concentrator. After balancing, dry at 45°C until the solution inside is completely dry. The drying process takes approximately 30 minutes to 1 hour. 2.0.5 When the drying process is almost finished, you can first incubate the Fast Hybridization Mix (Twist Bioscience) at 65°C for 10 min or until all the precipitate is dissolved. Then, set the PCR instrument program according to Table 7 below, with the hot lid at 85°C. Table 7: temperature time 95℃ 5 min 60℃ 15 min ~ 4 h .

[0032] 2.0.6 After drying, remove the centrifuge tube from the desiccator, tear off the sealing film, quickly vortex the Fast Hybridization Mix (Twist Bioscience) and add 20 μL to the tube to resuspend the sample (do not allow the hybridization solution to return to room temperature). Gently tap the tube with your fingertip to mix, avoiding the formation of air bubbles, and let it stand at room temperature for 5 min. Use a pipette to pipette up and down 10 times to completely dissolve the DNA. Transfer all the solution in the tube to a new PCR tube, avoiding the formation of air bubbles. 2.0.7 Add 30 μL of paraffin oil to the tube to cover the surface of the reagent inside, and then centrifuge briefly to remove air bubbles; 2.0.8 Place the PCR tube into a preheated PCR instrument for hybridization and incubate for 15 min to 2 h (during this period, you can go to the reagent preparation area to take out the Streptavidin Binding Beads (Twist Bioscience) used for the next capture step from the refrigerator, shake them thoroughly, and allow them to equilibrate to room temperature for at least 30 min).

[0033] 2.1 Library Capture and Cleaning 2.1.1 In the reagent preparation area, dispense the enrichment reagents (amount of reagent for a single enrichment reaction) into 1.5 mL centrifuge tubes as shown in Table 8. Then, transfer each component and the equilibrated magnetic bead component (Streptavidin Binding Beads) to the amplification area through the transfer window. Table 8: Components Required volume Fast Binding Buffer (Twist Bioscience) 900 μL Fast Wash Buffer 1 (Twist Bioscience) 450 μL Wash Buffer 2 (Twist Bioscience) 700 μL

[0034] 2.1.2 Enter the amplification zone and preheat 450 μL Fast Wash Buffer 1 at 66℃ and 700 μL Wash Buffer 2 at 48℃ in a constant temperature metal bath. 2.1.3 Shake the pre-equilibrated Binding Beads (Twist Bioscience) until completely mixed, then transfer 100 μL of the magnetic beads into a 1.5 mL centrifuge tube; 2.1.4 Add 200 μL of Fast Binding Buffer to the centrifuge tube and mix thoroughly by pipetting. 2.1.5 Place the centrifuge tube on a magnetic rack for 1 minute or until the solution becomes clear, discard the supernatant, and remove the centrifuge tube; 2.1.6 Repeat the washing steps 2.1.4 to 2.1.5 twice, for a total of three times; 2.1.7 After the third wash, add 200 μL of Fast Binding Buffer, and vortex to resuspend and mix thoroughly; 2.1.8 After hybridization in step 2.0.8 above, open the PCR instrument lid and quickly transfer all the hybridization solution to the equilibrated magnetic bead solution. Note: Quickly transferring the reagents from the PCR instrument to the magnetic beads is a critical step. The samples must be loaded directly onto the PCR instrument; do not remove the hybridization tubes to prevent the hybridization solution from cooling down. 2.1.9 After capping the centrifuge tubes, seal the tube openings with sealing film, then place them on a Biocate shaker and mix thoroughly at 1200 rpm for 30 min at room temperature. 2.1.10 Remove the centrifuge tube from the mixer, centrifuge quickly, place it on a magnetic rack for 1 min, discard the supernatant, remove the tube, add 200 μL of preheated Fast Wash Buffer 1 (do not remove it from the metal bath), and gently vortex to mix. 2.1.11 Quickly place the centrifuge tubes incubate at 66°C for 5 min; 2.1.12 After briefly separating the centrifuge tube, place it on a magnetic rack for 1 min, discard the supernatant, remove the tube, add 200 μL of preheated Fast Wash Buffer 1, and gently shake to mix. 2.1.13 Quickly place the centrifuge tubes incubate at 66°C for 5 min; 2.1.14 After briefly separating the centrifuge tube, transfer all the liquid inside to a new 1.5 mL centrifuge tube; place on a magnetic rack for 1 min, then discard the supernatant; 2.1.15 Remove the tube, add 200 μL of preheated Wash Buffer 2 (do not remove it from the metal bath), and gently shake to mix. 2.1.16 Quickly place the centrifuge tubes incubate at 48°C for 5 min; 2.1.17 After briefly separating the centrifuge tubes, place them on a magnetic rack for 1 minute, discard the supernatant, and remove the tubes; 2.1.18 Repeat steps 2.1.15 to 2.1.17 twice, for a total of three times; 2.1.19 After briefly separating the centrifuge tube, place it on a magnetic rack and use a 10 μL pipette tip to remove the washing liquid; 2.1.20 Remove the centrifuge tube from the magnetic rack, add 45 μL of nuclease-free water, gently vortex to mix, and incubate the solution on ice. If PCR amplification is not to be performed temporarily, the magnetic bead mixture can be stored at 20°C.

[0035] 2.2 PCR amplification 2.2.1 Enter the reagent preparation area, take one PCR tube, prepare the mixture, and add (25 μL KAPAHiFiHotStartReadyMix (KAPA) + 2.5 μL Amplification Primers (Twist Bioscience) = 27.5 μL) to the tube. Then transfer the PCR tube to the amplification I area through the transfer window (Note: At this time, DNA PurificationBeads (Vazyme) can be taken out of the refrigerator, thoroughly shaken and mixed, and transferred to the amplification area together with the PCR tube. Allow to equilibrate to room temperature for at least 30 min). 2.2.2 Take 22.5 μL of the magnetic bead suspension obtained in step 2.1.20 and add it to a PCR tube. Gently vortex to mix and then centrifuge briefly. The remaining 22.5 μL can be stored at -20℃ for later use. 2.2.3 Place the PCR tube on the PCR instrument, heat the lid to 105℃, and amplify according to the reaction program shown in Table 9 below: Table 9: .

[0036] 2.3 Magnetic bead purification (amplification region) 2.3.1 Vortex thoroughly to mix the pre-equilibrated DNA purification magnetic beads (Vazyme). Take a clean 1.5 mL centrifuge tube, add 90 μL (1.8*) DNA purification magnetic beads, and after PCR is completed, centrifuge the PCR tube briefly. Then transfer all the solutions in the PCR tube to the 1.5 mL centrifuge tube and vortex thoroughly to mix. 2.3.2 Incubate the centrifuge tubes at room temperature for 5 min; 2.3.3 Place the centrifuge tube on a magnetic rack for 1 minute, and discard the supernatant after the solution has clarified; 2.3.4 Keep the centrifuge tube on the magnetic rack, add 200 μL of freshly prepared 80% ethanol, incubate for 1 min, and discard the supernatant; repeat the 80% ethanol washing once (total 2 times). 2.3.5 After briefly centrifuging the centrifuge tubes, place them on a magnetic rack and carefully remove any residual ethanol with a 10 μL pipette tip. Let them stand at room temperature for 5–10 min or until the magnetic beads are dry, taking care not to let them crack. 2.3.6 Remove the tube from the magnetic rack and add 32 μL of nuclease-free water. Gently shake to mix and incubate at room temperature for 5 min. 2.3.7 Place the centrifuge tube on a magnetic rack for 3 minutes or until the solution becomes clear; 2.3.8 Transfer 30 μL of supernatant to a clean 1.5 mL centrifuge tube, label the tube cap with the library name and construction date, and then transfer it to the library quality control area. Note: If the purified and enriched library cannot be sequenced immediately, it can be stored at -20℃ or below for 30 days. 2.3.9 Take another 1 μL and use the Qubit® dsDNA HS Assay Kit (Vazyme) to determine the library concentration. The library concentration should be greater than 0.5 ng / μL. At the same time, the fragment length distribution of the library can be detected using an Agilent 2100 fragment analyzer. The average fragment length should be between 300 and 400 bp.

[0037] The panel hybridization capture probe sequences for chemotherapy in hematologic malignancies are shown in Table 10 below: Table 10: SEQ ID NO sequence SEQ ID NO 1 CCTTCACAAAGCGGAAGAATGTGTCAGCCTCAAAGAAAAGCTGCGTGATGATGAAATCGGCTCCCGCAGACACCTTCTCCTTCAAGTGCTTCAGGTCAGCCTCAAAGCTTCCTGCTTCGG SEQ ID NO 2 AAATGGGTGAAAATAAAGAAGTTTCTCAGTTATACTGCAGCTTGTTCATGCCTCCTGCCTCGGGATGCCGCAGTGGCTGCCCCAGCCCTGCCCTTTCAGCCTCAGCCCTTCCCTCAGTGAA SEQ ID NO 3 CCTGCATCCTGAAAGCTGCGTACCTGAGAGCCTGCGGTCTGGCTGCAGGGACACACCCAAGGGGAGGAGCTGCAATCGTGTCTGGGGCCCCAGCCCAGGCTGGCCGGAGCTCCTGTTTCC SEQ ID NO 4 AAGGGGAGGAGCTGCAATCGTGTCTGGGGCCCCAGCCCAGGCTGGCCGGAGCTCCTGTTTCCCGCTGCTCTGCTGCCTGCCCGGGGTACCAACATGGCCCAGAAGCGTCCTGCCTGCACCCT SEQ ID NO 5 CCTGCACCCTGAAGCCTGAGTGTGTCCAGCAGCTGCTGGTTTGCTCCCAGGAGGCCAAGAAGTCAGCCTACTGCCCCTACAGTCACTTTCCTGTGGGGGCTGCCCTGCTCACCCAGGAGG SEQ ID NO 6 TATATGTTTAATAACATATTTTTCTTGTGCACTGTTATTATAGCACCCTCCAAGTGGAAGTCACAGCTAGTATTCCAGAGCTGCTATTAGAAGCTGCTTCCTGTGAAGATCAAATCTTCC SEQ ID NO 7 CGCCCGTGCACCCTGTCCCAGCCGTCCTGTCCTGGCTGCTCGCTCTGCTTCGCTGCGCCTCCACTATGCTCTCCCTCCGTGTCCCGCTCGCGCCCATCACGGACCCGCAGCAGCTGCAGC SEQ ID NO 8 GCTTTACTCTCTTCCTTTTATGCTAAAATTGTGACTTCCGAACCTCAGGTGGACCTCTCCAAGGACATTCAGCACTGGGAATCCCTGAAACCCGAGGAGAGATATTTTATATCCCATGTT SEQ ID NO 9 CTGCGTTCGCTTTCTCAAGCGTACCTGGGGCCCCTGCTTGCTCTGCAGGAGTCCTGCGGCCTGTGCCATCCGCACGATTCCCTGGCAGCGAGAAATCTAGGTCCTCCCCTGACCACCTGG SEQ ID NO 10 CAGGCTGAACCTGCAGATATGCGCCCAGAGATATGGATCGCCCAAGAGTTGCGGCGTATTGGAGACGAGTTTAACGCTTACTATGCAAGGAGGGTAATGATGTTTTCTTTACCCGCTTTT SEQ ID NO 11 GCTCCTTTACAATGCGGGAAGTAAACAGTGTCGAGGCCAATATACTCATACAATATGCTCTGTGTAGACACAGAGCGAAGGCTGAGAATTTTCTGTAAGAAACTGCAGAAAAGCAAACTA SEQ ID NO 12 ATGAGTTTCTTAACCCTTTCCAGAGTCCTCCTTTGCCTGATCCTCCAACAGCTGTCACAACTTGTGTTGAGCAAGCAGTAGCATTTGCTTCCTCCCAACAAGCAGCTGGGTTAGGAAAAC SEQ ID NO 13 AGTTGGGTGTATGTAGTTGTTGCCTTCGTTCTCTTCCCTTTTGGAGGGAGCGTTGTCTCCTACTTTGTATCTTCCAGACATCTGTGGTCTTCCCCCCACCCCTCGAGTTTGTGAGTGGTG SEQ ID NO 14 TTTGTGTAATGTTGGAGTTACTTGTTCCTTTTGTAATCTGAAAGTATGCTTTAAAAAAAATTAGTGTACTTTTGAGAATTTTCATTTTGCTTTCTATTCTTCCTTGCTTTGTGCATGTTT SEQ ID NO 15 CTGGGGTCCACCCTTCCTCCCCACCCGACTCCGGAACCTCTTCCCGCGCCCTGCCCGGGCGCCTGGCTGCTTGGGGTAGAGGCCTTCCGCCACACGCGCGGCCCCGCCCCGGCCTTCACG SEQ ID NO 16 GGAGGCGGGCGAGGGCCGAGGGGCAGCTAGGGAGCGCGGCTTGAGGAGGGCGGGGCCGCCCCGCAGGCCCGCCAGTGTCCTCAGCTGCCTCCGCGCGCCAAAGTCAAACCCCGACACCCG SEQ ID NO 17 TCCGATGTCTGAAATGTCACAGCACTTAGTCTTACTCTTCTATGGCCTACTTTCTACTGCTATTTGTGTTACTCATGCTACCCATCTTATCTCCCTCAGTGTGTGAGACGCTGGCATCAG SEQ ID NO 18 GGGTCATGGAGCCATATCTGGGTTTGCAATTTTTGGAAAGTTACGTGACGATCTGCACTCAGGAATGCTGAGTGTTCGGCAGGCCCCATGAACAGTGATCAAATGTGACGCTGTGTGTCC SEQ ID NO 19 AATTTCCTGACTTCTTCTAGTCTCTACTCTATATTCTTATTTTTCCATGCACGCATTAGCACAACTTCTTTTTTTTTTCTTTGAGAAGGAGTCTTGCTCTGTTGCCCAGGCTGGAGTGCA SEQ ID NO 20 GGCAAGGGGTGCGTCTTTTAACCTCCATCCTTTTTTTTTTTCCTTGGTGGTCGAAGAGTTTTACTGATTTTTTAAAAAAGTGCTGGATTGGGTGACTAGAAGAAAGCTGCCCTTAGGTCT SEQ ID NO 21 AAAAACATGTCAGTGTGATTTTATTTTATCTATGTCTCATTTACTTTTCTGTAAGTAGATATAACTTTTCAAAAAGACAGTCAATTCCCCAACTTTTATGTCGTTCTTCAAAAGCATCAA SEQ ID NO 22 GTCCGCGCGCTCACCTGCTATTGGCCAGGTGGGGCTGTCGGCTGCCAGCAGTGCTTATGCTTTAAGTGCGGAGCGGGTGGCTGCGGAGCCAGGCGCGGCGCAGGATGGTGGACAGCGTGT SEQ ID NO 23 GTCCTAATGTATTTATAAGCTCTTTGTTCCTCTCAATAGTTCTCTCCCACTGAAAGAAGAGTCAAGTTAGGGAAAAGCCACTCCCACACATTTCATGGCCAAGGGGCCACCTACTGGATT SEQ ID NO 24 CGTAGTCGTAGGGCAGGTCGGGGAGGCTGTGCTTCTGCCTGGAGCCCAGATACCCCAAAACCGGAGCCAGCTGCCTGCTGGTGCTGAAGACGAGAAAGCACAGCCCGGTCAGTCAGCGCC SEQ ID NO 25 TCCACCATATAAGGGTACTTCTCTTCTACAGTAAGAACAGGATCAACTTCAATAGCGTAATATTTTTCCTTTAGTTGCAATAACTAGGAAGATATGAATAACTTAATCATGAAGATTACT SEQ ID NO 26 CTTACATTAGGCAGTGACTCGATGAAGGCATGTATGTTGGCCTCCTTTGCTGCCCTCACAATCTCTTCCTGTGACACCACCCGGCTGTTGTCTCCATAGGCAATGTTCTCAGCAATGCTG SEQ ID NO 27 TTAGAATACTTTACTCTACTTAATTAATCAATCATATTTAGTTTGACTCACCTTCCCAGAACCTTCTAGTTCTTTCTTATCTTTCAGTGCTTGTCCAGACAACATTTTCATTTCAACAAC SEQ ID NO 28 AGATTAATTGTTGATTAATCATTTATCACTGTACCTTAACTTCTTTTCGAGATGGGTAACTGAAGTGAACATTTCTGAATTCCAAATTTCCCTTAATATTATCTGGTTTGTGCCCACTCT SEQ ID NO 29 CTCTAATAATTTATTTTCTGTATTTTTCCTAGCTCCTCTATTTAGCCCATCTGAGTCCAGATTGAAGGTTCAACATGCTTAAAGAAGGAATGCAACTCACCTCAATGAAGTTAATTGACC SEQ ID NO 30 CTTTCCATTGCTCTAGGATGCAGATGTCTCCAGCCCTCACCTGCCTAGTCCTGGGCCTGGCCCTTGTCTTTGGTGAAGGGTCTGCTGTGCACCATCCCCCATCCTACGTGGCCCACCTGG SEQ ID NO 31 TACCAAGCAATGAAGAACAAATTAAAAATCTGCTACAATTGGAGGCTCAAGAACATCTCCAGGTATTTGATCTAGTGGGAGTTGCTGTTCCCAGAATGAATACAGTTAAACATGATTGAG SEQ ID NO 32 TGGACCCCAGGAAACGGTCGCTTCGACGTGCTGCCCCTGCTGCTGCAGGCCCCAGATGATCCCCCAGAACTCTTCCTTCTGCCCCCCGAGCTGGTCCTTGAGGTGCCCCTGGAGCACCCC SEQ ID NO 33 TGCTTCCTGTGGGTCCACAGACAGACTCAGCCTTGATTCCTGACATTCAGCCAAGAAGCAGAGTGTATACAATGGGAGCTCTTACAAAAAAACAGTTGAACCTGGTTATAACATGACTCA SEQ ID NO 34 TTTTTCAAGAACTGAGAAGAGCCGTTGGGTACTTAGGAAGGCGCCTTTGGCATGCACTGCAGCGTGTTTGTGTTTAATCTCAGGGGTTCTGGGGCACAGCAAAGGCTCTCTGGGGACCCT SEQ ID NO 35 GGCGTTTCTCCCTCATGACGCTGCGGAATTTTGGGATGGGGAAGAGGAGCATTGAGGACCGTGTTCAAGAGGAAGCCCGCTGCCTTGTGGAGGAGTTGAGAAAAACCAAGGGTGGGTGAC SEQ ID NO 36 AGGTTTGCCAGTTATCCGTGCCTTTGAGCACCAGCAGCGATTTCTGAAACACAATGAGGTGAGGATTGACACCAACCAGAAATGTGTCTTTTCCTGGATCACCTCCAACAGGTGAGGCTT SEQ ID NO 37 TTATTTCTTTCTTCCTTGTTTCAGGGTAATGGTCCTAGACAACGGGAAGATTATAGAGTGCGGCAGCCCTGAAGAACTGCTACAAATCCCTGGACCCTTTTACTTTATGGCTAAGGAAGC SEQ ID NO 38 ACAAATGCATGGGGATGTGGCTGGAGTTCCCCGTTGTCTAACCAGTGCCAAAGGGCAGGACGGTACCTCACCCCACGTTCTTAACTATGGGTTGGCAACATGTTCCTGGATGTGTTTGCT SEQ ID NO 39 GAATTTCCTGATCCAGAAAAGTTTAAGCCAGAACACTTCCTGAATGAAAATGGAAAGTTCAAGTACAGTGACTATTTCAAGCCATTTCCACAGGTGAGAAAGATCAGGCAGTACCTT SEQ ID NO 40 CAGCCTCAGTTGTGCTAGGAGGAGTGGGCCCCAGCCATAGGGGAAGGCCTTTCTCATTTATTCCATTCTCGAATCCCTGCTCTTTTTGAGCTGGGGGCCTCATCTTTGCAGGCTGAGAAG SEQ ID NO 41 CTTCTGTCTCTGCTGTCCCAGTTTTGAGGTTTGGTTTCTTGTTTCTGTCCTTGCTTTCGGGCTCCTCCCTCCCACCACTCCCCAACTTCCCCTAGCAGTTGCAGGGAAGATAGGACGAG SEQ ID NO 42 CTTTGCTGGACCCTGTTGCAGGCAAAAGGAGTAATTGATTTAACTGTTAATGATGATAATGATTTTTTTTAAACTCATATTGGGATTTTCACCAAAATGCTTTTGAAAAA SEQ ID NO 43 TTGTAGAATTTGGGTTTTCTTAAATTATGTAATTATATACCTCTTTTGTAGGTTGTGTACCAAGCTGTACATCAGCGGTGTACAATCAACTCTCAGAGTTATTTCCTGAAAAATA SEQ ID NO 44 CAACCTCCTGAGCTCAGCCTCCTCCTCTCCCATCAGTGGAACCTCGTGTGTGACTCTCATGCTCTGAAGCCCATGGCCCAGTCCATCTACCTGGCTGGGATTCTGGTGGGAGCTGCTGCG SEQ ID NO 45 AGGAGGCAGCCCTGGTGGACATGGTGAATGACGGCGTGGAGGACCTCCGCTGCAAATACATCTCCCTCATCTACACCAACTATGTGAGCATCTGCACCAGGGTTGGGCACTGGGGGCTGA SEQ ID NO 46 ACACTCTCTTATCTACATAGGTTGTTTAAAGGAATCTGGGTCATACATGTGGATATATGTGTTCATGGGTAATATGCTTCGTGGAATAGGGGAGACTCCCATAGTACCATTGGGGCTTTC SEQ ID NO 47 CTTCGTGGAATAGGGGAGACTCCCATAGTACCATTGGGGCTTTCTTACATTGATGATTTCGCTAAAGAAGGACATTCTTCTTTGTATTTAGGTAATGTACACAAAATATTAAATTGTATG SEQ ID NO 48 TATTTTCTTCTATTCTGTTATATACGAAGAAACTGTGATTCAAGGATAATAACCAACTTGTCAAAAATCAGAGATAATAGAAAATGGCTAGGATTTGTATGTGAATCTTTTTTGTTTCCA SEQ ID NO 49 GTTGGGAGTGGGTTCCTTGGGAAGAACTACCTCCCCTGGACCAGCTTTTCTGGGGACTGCGTTGTTTAAAAGAACAAGGCTATGATCCATTTAAAGAAGATCTGAACCATCTGGTGGGAT SEQ ID NO 50 ATTTTGGCAGTTAGCTGTGTGTGGTGTGTGCTTTTTAAGGCTTCACTCAATAAAACATCTGTTGCTCTCTGCTGATGAGCAAAAAACTTGCTATACGAAGGACAAATATATGTATACAAA SEQ ID NO 51 GCACTCCAGCCTGCGTAACAGGACGAGACTCCGTCTCAAAAAAAAAAAAAAGTATGATATAAATTTTGAGCAAGTAGACATGGATCACATCACCCTGCCTGGCTATAGGCTACTCCAAGG SEQ ID NO 52 CAGCGTGGCTGAGCCAGGTGTGTTGTGTCGTTTCAGCATCACCTTCTCCATCCCCGAAGGTGCTTTGGTGGCCGTGGTGGGCCAGGTGGGCTGCGGAAAGTCGTCCCTGCTCTCAGCCCT SEQ ID NO 53 CGGGGGGATGACTCTCTGAGGTGCCTCCCTGCCTGATGCCCGTGAACGCTCACCTCTGCGGCAGCCGGCTCTGCTCACCTCCCCTGATGCTGGCATCTTGGTCTCCTGGCCCCAACTCTG SEQ ID NO 54 GGCCCGAACATAGTAATTCCTGGTAAAGGGCCCGAACAGCTTCACCACGGCGGTCATGTACTTCTGTCCCCTGGGGGAGGGAGGAAGGCGAGACGGCGCGGCTGGGCCTCTCCCACTCGG SEQ ID NO 55 AACTCAGCGTCCTCCTCTTCCTTGCACTCCTCACAGGACTCTTGCTACTCCTGGTTCAGCGCCACCCTAACACCCATGACCGCCTCCCACCAGGGCCCCGCCCTCTGCCCCTTTTGGGAA SEQ ID NO 56 GTGTCCTTGACCTGCTGCTTCTTCCTAGGGGCCCTCATGGACCCCACCTTCCTCTTCCAGTCCATTACCGCCAACATCATCTGCTCCATCGTCTTTGGAAAACGATTCCACTACCAAGAT SEQ ID NO 57 TGCTTACATTGGCCACAGTGTGGAGAAGCACCGTGAAACCCTGGACCCCAGCGCCCCCAAGGACCTCATCGACACCTACCTGCTCCACATGGAAAAAGTGGGGTCTGGGAGAGGAAAAAG SEQ ID NO 58 GGAGCCTCTGGGAGTGCTGTTCCCGTCCACCACCAAGAAGAGGAAGAAGCCCAAAGGGAAAGAAACCTTCGAGCCAGAAGACAAGACAGTGAAGCAGGAACAGATTAACACTGAGCCTCT SEQ ID NO 59 TTAAGGGCTGGCTTCGGATGAAGGGCAAAGACAACTTCCTATTTATCACCTACGAGGAGCTGCAGCAGGTGAGTCCCCACCTCCTCCAGGTGCAGCGTCCCCCCCATACCCTCTGCTCAC SEQ ID NO 60 ATAAGTTTCCATGCACTTTGGTGGCACAGAAAATTGACCGTATGTCTCTGTTTTGTTTTATTTTTAAAAGATGGTTGGATTTCTCTGTCTTCCTGTGACCTGACTTTCTGTGTGTCTGTT SEQ ID NO 61 CTACATCCTAATCAATACCCTCTAATAATCCTCCCAATAACCTCCCCTGCCTTGCAAGCACTAACTGCTTTGGAATCACGCTCAAAGACTCCATAACACCGCAAGGAGTATCTCAGCAGC SEQ ID NO 62 GGTGGGGCTTGATGGGCCAGAGGATAAGTGACTCCTCAAGGTCACAAGACACCCTGATGGCTGGGACCCCATCTCTGATGGAGCAAGGCTTCTGATACCCAACTGAGGCTTGGCGCCAAG SEQ ID NO 63 CCCAGCACCCAGGTATCACCTGACCACCACCGAAGCCCACCCCCACGGAGGAAGGATGGTGCATTCAAGGAACCCAGAGACCACAGGGCACGCATGGCTAGTGACTTGGGGGTGAGAAAG SEQ ID NO 64 TCTCGCCTATAACTTCTCTCTCCTTTGCTTTCACAGGCCTTGGTATGTTCCTGCTTCATCCCCTTCTACAGTGGCCTTATCCCTCCTTCCTTCAGAGGCGTGGTAAGTCGGCTTTCTCTG .

[0038] Example 2: Clinical Sample Testing One whole blood sample was collected from each of two volunteers to detect the polymorphism of genes related to hematologic malignancies chemotherapy drugs in these two samples. Genomic DNA was first extracted from the whole blood of four volunteers, and then library construction was performed according to the method described in Example 1, successfully constructing the library (see [link to example]). Figure 1 The library was sequenced on an Illumina high-throughput sequencer with sequencing parameters PE: 2×150. After quality control of the data, bioinformatics analysis was performed using relevant databases such as PharmGKB and CPIC to determine the detection of genes related to chemotherapy drugs for hematological malignancies in each sample. The results were then validated by first-generation sequencing, with a 100% concordance rate.

[0039] The test results for Volunteer 1 are shown in Table 11 below: Table 11: , , , , , .

[0040] The test results for Volunteer 2 are shown in Table 12 below: Table 12: , , , , .

[0041] As can be seen from the detection results in Tables 11-12, the panel hybridization capture probe for hematologic malignancies chemotherapy of the present invention can accurately capture relevant sites of genes related to hematologic malignancies chemotherapy drugs. The library preparation kit of the present invention can detect polymorphisms in genes related to hematologic malignancies chemotherapy drugs, and can accurately genotype these genes, thereby enabling accurate interpretation of chemotherapy drugs based on the genotyping results. Detection of routine samples shows that the probe, library preparation kit, and their usage method of the present invention can detect polymorphisms in genes related to hematologic malignancies chemotherapy drugs.

[0042] The above description is merely a detailed explanation of preferred embodiments and principles of the present invention. For those skilled in the art, there may be changes in specific implementation methods based on the ideas provided by the present invention, and these changes should also be considered within the scope of protection of the present invention.

Claims

1. A probe set for detecting a polymorphism of a blood tumor chemotherapy drug-related gene, characterized by, The probe set comprises a plurality of probes capable of specifically hybridizing to a polymorphic site of a gene related to chemotherapy drug metabolism, efficacy or toxicity of a hematological tumor by hybrid capture method, and the base sequence of the probe is selected from at least one of SEQ ID NO. 1-64.

2. The probe set for detecting polymorphism of blood tumor chemotherapy drug related genes according to claim 1, characterized in that, The probe coverage region comprises a hematological tumor chemotherapy drug related gene, which includes a hematological tumor chemotherapy related pharmacogenomics gene, and the gene is selected from at least one of the following group: BCL2L11, ABCB1, CDA, CYP2C9, SERPINE1, ABCC1, ABCC2, CYBA, NCF4, RAC2, CYP2B6, ABCC4, DHFR, MTHFR, SLC01B1, FGF2, NUDT15, SLC7A5, CPA2, NFATC2, SOD2, GNMT, ITPA, TPMT, NOS3, NRP2, ERCC1, XRCC5, CEP72, CYP2E1, SLC22A12, SULT2B1, DCK, NT5C3A, CTH, DROSHA, CTNNB1, GSTA1, RRM1, RRM2, RRM2B, PYGL, GATA3, PNPLA3, FOLH1, GSTP1.

3. A library kit for detecting a blood tumor chemotherapy drug-related gene polymorphism, characterized by, The library construction kit comprises the probe set according to any one of claims 1-2.

4. The library kit for detecting polymorphism of blood tumor chemotherapy drug related genes according to claim 3, characterized in that, The library construction kit supports simultaneous construction of 384 indexed libraries and is compatible with the Illumina sequencing platform.

5. The library kit for detecting polymorphism of blood tumor chemotherapy drug related genes according to claim 3, characterized in that, The library construction kit further comprises a hybridization assembly comprising a Fast Hybridization Mix and paraffin oil.

6. A method for using a library kit for detecting a blood tumor chemotherapy drug-related gene polymorphism, characterized by, The method comprises the following steps: The sample DNA is subjected to hybridization, capture reaction and sequencing analysis by hybrid capture method using the probe set according to any one of claims 1-2.

7. The method of using the library kit for detecting the polymorphism of blood tumor chemotherapy drug related genes according to claim 6, characterized in that, The library construction step further comprises an integrated reaction of enzyme cutting, end repair and A tailing using genomic DNA as a starting amount.

8. The method for using the library kit for detecting the polymorphism of blood tumor chemotherapy drug related genes according to claim 7, characterized in that, The starting amount of the genomic DNA is 10-100 ng.

9. The method of using the library kit for detecting polymorphism of blood tumor chemotherapy drug related genes according to claim 6, characterized in that, The reaction time of the hybrid capture reaction is 0.25-2 h.

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