Unequal-depth HRD and HRR gene hybridization capture probe composition as well as preparation method and application thereof

Abstract

The invention discloses an unequal-depth HRD and HRR gene hybridization capture probe composition as well as a preparation method and application thereof, and belongs to the technical field of molecular biology. The preparation method of the unequal-depth HRD and HRR gene hybridization capture probe composition comprises the following steps: mixing a streptavidin solution with a targeted HRD region probe group, and incubating to obtain a mixed solution; and mixing the mixed solution with a target HRR gene probe group to obtain the unequal-depth HRD and HRR gene hybridization capture probe composition, the ratio of each probe to the streptavidin in the targeted HRD region probe group is 1.6 fmol: (0.1-1) [mu] g. According to the invention, streptavidin is utilized to selectively close part of probes in the target HRD region probe group, and then the probe group is mixed with the target HRR gene probe group, so that the purpose of unequal-depth sequencing of the two regions is achieved without physical separation, and the cost is reduced by 50% compared with that of branched-tube hybridization capture.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biotechnology, and in particular relates to a unequal-depth HRD and HRR gene hybridization capture probe composition, a preparation method and an application thereof. Background Art

[0002] The analysis of the two biomarkers, HRR gene mutation and HRD, has different requirements for the average effective sequencing depth: a higher sequencing depth is required to ensure the sensitivity of HRR gene mutation detection; while HRD is achieved by typing SNP sites across the entire genome (usually selected at a uniform distance), which can detect loss of heterozygosity (LOH), telomeric allelic imbalance (TAI) and large segment migration (LST) events. Using these data, a comprehensive HRD score is calculated through a specific algorithm to determine the HRD status, which only requires a lower sequencing depth.

[0003] In response to the different sequencing depth requirements for HRR genes and HRD regions, existing unequal-depth hybridization capture of HRR genes and HRD regions is mainly carried out in the following two ways: one is to mix probes targeting the HRR gene and HRD region at a certain material ratio. If the sequencing depth of the HRD region is low, the probe material ratio is low, and if the sequencing depth of the HRR gene region is high, the probe material ratio is high. The other is to perform hybridization capture reactions on the probes of the HRR gene and HRD region separately, and then sequence them at different depths. The sequencing depth of the HRR gene region is high and the sequencing depth of the HRD region is low. These two methods each have disadvantages. In the first method, since the HRR gene and HRD region probes are far in excess of the target DNA molecule, reducing the material input ratio of the HRD region probe can reduce the average effective sequencing depth of the HRD region to a certain extent, but it cannot increase the average effective sequencing depth ratio of the HRR gene and HRD region to more than 5 times. Generally, the maximum depth ratio can only reach about 1.5-2 times, which is limited in practical application. The second method, which hybridizes HRD and HRR gene probes separately, can achieve an average effective sequencing depth ratio of more than 5 between the HRR and HRD regions, but this increases reagent and personnel costs exponentially. Therefore, in the process of hybridization capture detection of HRD and HRR genes at different depths, how to meet the sequencing depth requirements of the HRR and HRD regions while simplifying the operation steps and reducing reagent and time costs has become an urgent problem to be solved. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a method for preparing a hybridization capture probe composition of HRD and HRR genes with unequal depths. The prepared probe composition can pass a single hybridization capture sequencing experiment, so that the average effective sequencing depth of the two capture regions for detecting HRR gene mutations and for analyzing HRD after deduplication meets the quality control requirements, and the detection results of HRR gene mutations and HRD status are consistent with the detection results of separate hybridization of the two parts.

[0005] Another object of the present invention is to provide a probe composition prepared by the preparation method.

[0006] Another object of the present invention is to provide a hybridization capture kit for HRD and HRR genes at different depths.

[0007] Another object of the present invention is to provide an application of the preparation method, the probe composition, or the kit in preparing a product for the combined detection of HRR gene mutation and HRD.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a method for preparing a hybridization capture probe composition for HRD and HRR genes of unequal depths. The preparation method comprises: mixing a streptavidin solution with a probe group targeting an HRD region, incubating the mixture, and obtaining a mixed solution; mixing the mixed solution with a probe group targeting an HRR gene to obtain a hybridization capture probe composition for HRD and HRR genes of unequal depths; the mixing ratio of each probe in the probe group targeting the HRD region to streptavidin is 1.6 fmol: 0.1-1 μg.

[0010] Preferably, the probes in the HRR gene targeting probe set and the HRD region targeting probe set are biotin-labeled double-stranded DNA probes.

[0011] Preferably, the concentration of the streptavidin solution is 0.05-0.5 μg / μL; the concentration of each probe in the probe group targeting the HRD region is 0.1-1 fmol / μL; the concentration of each probe in the probe group targeting the HRR gene is 0.1-1 fmol / μL.

[0012] Preferably, the volume ratio of the probe group targeting the HRD region to the streptavidin solution is 4:1-10.

[0013] Preferably, the molar ratio of each probe in the probe group targeting the HRD region and the probe group targeting the HRR gene is 1:1.

[0014] Preferably, the incubation conditions include: temperature 20-30° C., time 0.5-1.5 h.

[0015] The present invention also provides a probe composition prepared by the preparation method.

[0016] The present invention also provides a unequal depth HRD and HRR gene hybridization capture kit, which comprises the probe composition.

[0017] The present invention also provides an application of the preparation method, the probe composition or the kit in preparing a product for jointly detecting HRR gene mutation and HRD.

[0018] Preferably, the method for jointly detecting HRR gene mutation and HRD includes: hybridizing the probe composition with a gene library to obtain a hybridization product; performing PCR amplification using the hybridization product as a template to obtain a sequencing library; sequencing the sequencing library, analyzing the data obtained by sequencing, and obtaining HRR gene mutation and HRD status information.

[0019] Beneficial effects of the present invention:

[0020] The present invention utilizes streptavidin to selectively block some probes in the probe set targeting the HRD region, which are then mixed with the probe set targeting the HRR gene. This achieves the goal of unequal-depth sequencing of the two regions without the need for physical separation, resulting in an average effective sequencing depth of over 1000× for the HRR gene and over 200× for the HRD region. The ratio of the average effective sequencing depth of the HRR region to the HRD region can reach (3.11-7.67):1. The probe composition prepared using the preparation method of the present invention performs hybridization capture sequencing of HRD and HRR genes at unequal depths, and the detection results are consistent with those of separate hybridization of the two regions, while reducing the cost by 50% compared to separate hybridization capture and is compatible with trace samples.

[0021] The present invention achieves deep, differentiated capture of HRR genes and HRD regions by selectively partially blocking the HRD probe with streptavidin in a single tube without the need for physical separation. Compared with separate-tube hybridization capture, the cost is reduced by 50% and the method is compatible with trace samples. The present invention establishes a linear relationship between the degree of HRD probe blocking and sequencing depth, achieving predictable depth control. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Flowchart of the sequencing method in Example 1;

[0023] Figure 2 This is the comparison of the original sequencing depth in Example 1;

[0024] Figure 3 This is a comparison of the effective sequencing depth in Example 1;

[0025] Figure 4 Comparison of the original sequencing depth of the 7 standards in Example 2;

[0026] Figure 5 Comparison of the average effective sequencing depth of the 7 standards in Example 2;

[0027] Figure 6 : HRD score (HRD Score) of the 7 standard products in Example 2. DETAILED DESCRIPTION

[0028] The present invention provides a method for preparing a hybridization capture probe composition for HRD and HRR genes of unequal depths. The preparation method comprises: mixing a streptavidin solution with a probe group targeting an HRD region, incubating the mixture, and obtaining a mixed solution; mixing the mixed solution with a probe group targeting an HRR gene to obtain a hybridization capture probe composition for HRD and HRR genes of unequal depths; the mixing ratio of each probe in the probe group targeting the HRD region to streptavidin is 1.6 fmol: 0.1-1 μg.

[0029] In the present invention, the HRR gene targeting probe group includes probes designed for all exon regions of 58 HRR genes. The 58 HRR genes preferably include ABRAXAS1, ATM, ATR, BARD1, BLM, BRCA1, BRCA2, BRIP1, CDK12, CHEK1, CHEK2, EMSY, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCI, FANCL, FANCM, MDC1, MRE11, NBN, PALB2, PPP2R2A, PTEN, RAD50, RAD51, R AD51B, RAD51C, RAD51D, RAD52, RAD54L, RBBP8, RPA1, SLX4, TP53, WRN, XRCC2, BAP1, RAD54B, POLD1, TP53BP1, MUS81, ERCC4, POLE, AKT1, BACH1, HFM1, PIK3CB, PIK3R1, RPA3, AKT3, CCND1, CDKN1B, CDKN2A, CUL4A, PARP4. In some embodiments, the HRR gene probe set preferably includes 2066 probes.

[0030] In the present invention, the HRD region targeting probe group includes probes designed for SNP sites across the genome. The selection of the SNP sites preferably follows the following principles: the allele frequency of the SNP is greater than 10% in the Caucasian population or greater than 5% in the Asian population; SNPs that deviate severely from the Hardy-Weinberg equilibrium are filtered; SNPs located in homologous regions are filtered; the design of probes covering SNP sites needs to consider GC content and short tandem repeat regions; SNPs are evenly selected across the genome. The number of the SNP sites is preferably 35,000 to 45,000, for example, 35,000, 36,000, 37,000, 38,000, 39,000, 40,000, 41,000, 42,000, 43,000, 44,000 or 45,000. In some embodiments, the number of the SNP sites is preferably 40,000, and the HRD region targeting probe group preferably includes 40,000 probes.

[0031] In the present invention, the probes in the HRR gene targeting probe set and the HRD region targeting probe set are preferably biotin-labeled double-stranded DNA probes. The length of the probes is preferably 110 to 130 bp, such as 110, 115, 120, 125 or 130 bp.

[0032] In the present invention, the ratio of each probe in the HRD region-targeted probe set to streptavidin is preferably 1.6 fmol:0.1-1 μg, for example, 1.6 fmol:0.1 μg, 1.6 fmol:0.25 μg, 1.6 fmol:0.5 μg, or 1.6 fmol:1 μg. At this ratio, streptavidin can selectively block some probes in the HRD region-targeted probe set, thereby achieving deep, differentiated capture of HRR genes and the HRD region.

[0033] In the present invention, the concentration of the streptavidin solution is preferably 0.05 to 0.5 μg / μL, for example, 0.05, 0.1, 0.2, 0.3, 0.4, or 0.5 μg / μL. In some embodiments, the streptavidin stock solution is diluted with IDTE buffer to obtain a streptavidin solution; the concentration of the streptavidin stock solution is preferably 1 to 10 μg / μL, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μg / μL; the IDTE buffer preferably includes 10 mM Tris and 0.1 mM EDTA, and the pH of the IDTE buffer is preferably 7.5.

[0034] In the present invention, the concentration of each probe in the HRD region targeting probe group is preferably 0.1 to 1 fmol / μL, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 fmol / μL; the concentration of each probe in the HRD region targeting probe group is preferably equal. The concentration of each probe in the HRR gene targeting probe group is preferably 0.1 to 1 fmol / μL, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 fmol / μL; the concentration of each probe in the HRR gene targeting probe group is preferably equal. In some embodiments, the concentration of each probe in the HRD region targeting probe group is preferably equal to that in the HRR gene targeting probe group.

[0035] In the present invention, the volume ratio of the probe group targeting the HRD region and the streptavidin solution is preferably 4:1-10, for example, 4:1, 4:2, 4:2.5, 4:3, 4:4, 4:5, 4:6, 4:7, 4:8, 4:9 or 4:10.

[0036] In the present invention, the incubation temperature is preferably 20-30°C, for example, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30°C; the incubation time is preferably 0.5-1.5h, for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4 or 1.5h.

[0037] In the present invention, the amount ratio of each probe in the HRD region targeting probe group and the HRR gene targeting probe group is preferably 1: 1. In some embodiments, when the concentration of each probe in the HRD region targeting probe group and the HRR gene targeting probe group is equal, the volume ratio of the HRD region targeting probe group and the HRR gene targeting probe group is preferably 1: 1.

[0038] The present invention uses streptavidin to selectively block some probes in a probe set targeting the HRD region, which are then mixed with a probe set targeting the HRR gene. This achieves unequal depth sequencing of the two regions without the need for physical separation, resulting in an average effective sequencing depth of over 1000× for the HRR gene and over 200× for the HRD region. The ratio of the average effective sequencing depth of the HRR region to the HRD region can reach (3.11-7.67):1. Using the probe combination of the present invention for hybridization capture sequencing of HRD and HRR genes at unequal depths yields results consistent with those obtained by separate hybridization of the two regions, while reducing the cost by 50% compared to separate hybridization capture and is compatible with trace samples.

[0039] The present invention also provides a probe composition prepared by the preparation method.

[0040] The present invention also provides a unequal depth HRD and HRR gene hybridization capture kit, which comprises the probe composition.

[0041] The present invention also provides an application of the preparation method, the probe composition or the kit in preparing a product for jointly detecting HRR gene mutation and HRD.

[0042] In the present invention, the method for jointly detecting HRR gene mutation and HRD preferably includes: hybridizing the probe composition with a gene library to obtain a hybridization product; performing PCR amplification using the hybridization product as a template to obtain a sequencing library; sequencing the sequencing library, analyzing the data obtained by sequencing, and obtaining HRR gene mutation and HRD status information.

[0043] In the present invention, the gene library is preferably pretreated before hybridization. In some embodiments, the pretreatment method preferably includes: referring to the instructions of the Enzymatics enzyme digestion library preparation kit, performing end repair, A-tailing, adapter ligation, adapter purification, index PCR, and purification and quantification on the gene library in sequence to prepare a prelibrary.

[0044] In the present invention, the hybridization temperature is preferably 55-65°C, for example, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65°C; the hybridization time is preferably 12-18h, for example, 12, 13, 14, 15, 16, 17 or 18h.

[0045] In the present invention, after the hybridization, rinsing is preferably performed; in some embodiments, the rinsing method preferably includes: 68° C., rinsing twice, and then 48° C., rinsing three times.

[0046] In some embodiments of the present invention, the PCR amplification system, based on 50 μL, preferably includes: 22.5 μL of hybridization product, 25 μL of HiFi hotstart ready mix, and 2.5 μL of universal primers. The PCR amplification program preferably includes: 98°C for 1 minute; 98°C for 15 seconds, 60°C for 30 seconds, 72°C for 30 seconds, 16 cycles; 72°C for 3 minutes.

[0047] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0048] In the following examples, unless otherwise specified, all methods are conventional.

[0049] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0050] The HRD standards used in the following examples include HRD gDNA standards GW-FGTM003 (corresponding to experimental number HRD000003T-D), GW-FGTM009 (corresponding to experimental number HRD000009T-D) and GW-FGTM012 (corresponding to experimental number HRD000012T-D) purchased from Jingliang Technology (Shenzhen) Technology Co., Ltd. and HRD gDNA standards HRD-P1, HRD-P3, HRD-P4 and HRD-P13 purchased from Nanjing Kebai Biotechnology Co., Ltd.

[0051] Example 1

[0052] 1. Reagents:

[0053] Targeting 58 HRR genes (ABRAXAS1, ATM, ATR, BARD1, BLM, BRCA1, BRCA2, BRIP1, CDK12, CHEK1, CHEK2, EMSY, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCI, FANCL, FANCM, MDC1, MRE11, NBN, PALB2, PPP2R2A, PTEN, RAD50, RAD51, RAD51B, RAD51C, RAD51D, RAD52, RAD54L, RBBP8, RPA1, SL 2066 probes were designed from all exon regions of HRR gene targeting genes (X4, TP53, WRN, XRCC2, BAP1, RAD54B, POLD1, TP53BP1, MUS81, ERCC4, POLE, AKT1, BACH1, HFM1, PIK3CB, PIK3R1, RPA3, AKT3, CCND1, CDKN1B, CDKN2A, CUL4A, PARP4), forming a probe set targeting HRR gene. The probes are biotin-labeled double-stranded DNA probes with a length of 120 bp. The concentration of each probe in the probe set targeting HRR gene is 0.4 fmol / μL.

[0054] 40,000 probes were designed for 40,000 SNP sites across the genome to form a probe set targeting the HRD region. The probes were biotin-labeled double-stranded DNA probes, 120 bp in length, and the concentration of each probe in the probe set targeting the HRD region was 0.4 fmol / μL.

[0055] The streptavidin stock solution (5 mg / mL) was diluted 50-fold to 0.1 mg / mL with IDTE buffer (10 mM Tris, 0.1 mM EDTA, pH 7.5, manufactured by IDT), with the conversion unit being 0.1 μg / μL, to obtain the streptavidin blocking solution.

[0056] 2. Methods:

[0057] (1) Add 0.5 μL, 1 μL, 2.5 μL, 5 μL, and 10 μL of streptavidin blocking solution to 4 μL of the HRD region probe set, respectively; pipette and mix 20 times, and incubate at 25°C for 1 hour. Add the blocked HRD probes to 4 μL of the HRR gene probe set, mix well, and use them as hybridization capture probes in the experimental group.

[0058] (2) Dilute the probe set targeting the HRD region 10-fold using IDTE buffer, take 4 μL and mix it with 4 μL of the probe set targeting the HRR gene (ratio 1:10) as the control group probe.

[0059] (3) According to the instructions of the Enzymatics enzyme digestion library preparation kit, NGS pre-library was constructed for HRD standard HRD000003T-D. End repair, A-tailing, adapter ligation, adapter purification, index PCR, and purification and quantification were performed in sequence to prepare the pre-library. The pre-library was aliquoted into 18 aliquots (5 blocked gradients × 3 technical replicates in the experimental group + 3 replicates in the control group), each containing an equal amount of DNA (500 ng).

[0060] (4) The above prelibrary was hybridized overnight at 60℃ for 16h using the control group and experimental group probes respectively; the hybridization solution reaction system was 17μL: 4μL of control group or experimental group probe; 8μL of Universal Blockers; 5μL of Blocker Solution; 800-1000ng of prelibrary sample was vacuum concentrated into dry powder and added to the above 17μL hybridization reaction solution for overnight hybridization; 68℃, rinsed twice, and 48℃, rinsed three times. Then, the hybridization product magnetic bead solution was used as a template for post-hybridization amplification. 22.5μL of hybridization product, 25μL of Hifi hotstart ready mix and 2.5μL of universal primer (P7 / phosphorylated P5 universal primer, a working solution obtained by mixing equal amounts of the two primers, concentration 10μM) were prepared into a 50μL reaction system. The PCR reaction was performed with the following program: 98℃ for 1min; 98℃ for 15s, 60℃ for 30s, 72℃ for 30s, 16 cycles; 72℃ for 3min. 1x magnetic bead purification was performed to prepare the sequencing library. Sequencing was performed on the MGISEQ-2000RS high-throughput gene sequencer (MGI) using the PE100 mode. The expected output of each library was 6G data, but the actual data output fluctuated around 6G. Bioinformatics quality control analysis was performed, focusing on the original sequencing depth and effective depth of the two regions. The process is as follows Figure 1 As shown, the results are Figure 2-Figure 3 shown.

[0061] like Figure 2 As shown, the original sequencing depth ratio of the HRD region and the HRR gene region can be adjusted according to the probe blocking conditions of the HRD low-depth sequencing region, and the ratio is between 1:3 and 1:15. The depth ratio can be controlled and adjusted within the range of 1:3 to 1:15; the original sequencing depth ratio of the control group is about 1:1.37. The average effective sequencing depth ratio of the HRD region and the HRR gene region is controllably adjusted between 1:2.5 and 1:8, while the average effective sequencing depth ratio of the control group is about 1:2.2 ( Figure 3 ). This shows that the method of the present invention can not only significantly differ the average effective sequencing depth of the HRR gene and HRD regions, but also meet the expected average effective depth ratio for HRR gene mutation and HRD status analysis, thus making up for the defects of conventional methods.

[0062] Based on the average effective sequencing depth requirements for HRR gene mutation and HRD status analysis, the volume ratio of blocking buffer to the HRD region-targeted probe set was determined to be 5:8 when blocking the HRD probe with 50x streptavidin blocking buffer. This means that 2.5 μL of blocking buffer was mixed with 4 μL of the HRD region-targeted probe set. An equal amount of 4 μL of the HRR gene-targeted probe set was then mixed with this mixture to create a working solution of hybridization capture probes for HRD and HRR genes with varying depths.

[0063] Example 2

[0064] According to the mixed volume ratio of the streptavidin blocking solution and the probe group targeting the HRD region determined in Example 1, the NGS library preparation and sequencing of 7 HRD standards including HRD000003T-D, HRD000009T-D, HRD000012T-D, HRD-P1, HRD-P3, HRD-P4 and HRD-P13 were performed with reference to the experimental process of Example 1, and 6G data output was expected for each library. Then, the obtained data were subjected to bioinformatics quality control analysis, HRR gene variation detection and HRD status analysis (denoted as HRR+HRDpanel). The 7 HRD standards were detected and analyzed using the whole genome sequencing (WGS) method as a control (denoted as WGS). The results are shown in Tables 1-2 and Figure 4-Figure 6 shown.

[0065] Table 1 Detection of pathogenic mutations in HRR genes

[0066]

[0067] Table 2 Consistency of HRD status

[0068]

[0069]

[0070] The original sequencing depth ratio of the HRR gene region and the HRD region before deduplication was between 5.0-9.0 ( Figure 4 ), the average effective sequencing depth ratio of the HRR gene region and HRD region after deduplication is between 4.4-5.9 ( Figure 5 ), and the depths all met the quality control requirements for HRR gene mutation detection and HRD status analysis. HRD positive standards carrying HRR gene pathogenic mutations all detected the corresponding HRR gene pathogenic mutations, with both positive and negative coincidence rates of 100% (Table 1). The actual HRD scores calculated from the 7 HRD standards showed a highly consistent trend with the HRD score standard values ​​obtained using the WGS method ( Figure 6 ), and the positive and negative status of HRD was 100% consistent (Table 2).

[0071] In addition, there are significant differences in the quality of DNA extracted between ovarian cancer FFPE clinical samples. Under the same data output, the average effective sequencing depth will decrease as the degree of DNA integrity decreases. Extremely poor DNA integrity will result in unqualified average effective depth, making it difficult to ensure the sensitivity of HRR gene mutation detection and the needs of HRD status analysis. However, in the case of slight DNA degradation and actual data output of 6G±2G, the unequal depth HRD and HRR gene hybridization probe combination obtained by the present invention is used for detection. The appropriate reduction in the average effective sequencing depth will not affect the detection of HRR gene mutations and the analysis of HRD status.

[0072] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a hybrid capture probe composition for HRD and HRR genes with different depths, characterized in that: The preparation method comprises: mixing a streptavidin solution with a probe group targeting the HRD region, incubating, and obtaining a mixed solution; mixing the mixed solution with a probe group targeting the HRR gene to obtain a hybridization capture probe composition of HRD and HRR genes of varying depths; the mixing ratio of each probe in the probe group targeting the HRD region to streptavidin is 1.6 fmol: 0.1-1 μg.

2. The preparation method according to claim 1, characterized in that The probes in the HRR gene targeting probe set and the HRD region targeting probe set are biotin-labeled double-stranded DNA probes.

3. The preparation method according to claim 1, characterized in that The concentration of the streptavidin solution is 0.05-0.5 μg / μL; the concentration of each probe in the probe group targeting the HRD region is 0.1-1 fmol / μL; the concentration of each probe in the probe group targeting the HRR gene is 0.1-1 fmol / μL.

4. The preparation method according to claim 3, characterized in that The volume ratio of the HRD region targeting probe group to the streptavidin solution is 4:1-10.

5. The preparation method according to claim 3, characterized in that The molar ratio of each probe in the HRD region targeting probe group and the HRR gene targeting probe group is 1:

1.

6. The preparation method according to claim 1, characterized in that The incubation conditions include: temperature 20-30° C., time 0.5-1.5 h.

7. The probe composition prepared by the preparation method according to any one of claims 1 to 6.

8. A hybridization capture kit for HRD and HRR genes with varying depths, characterized in that: The kit comprises the probe composition according to claim 7.

9. Use of the preparation method according to any one of claims 1 to 6, the probe composition according to claim 7, or the kit according to claim 8 in preparing a product for the combined detection of HRR gene mutation and HRD.

10. The use according to claim 9, characterized in that The method for jointly detecting HRR gene mutation and HRD comprises: hybridizing the probe composition according to claim 7 with a gene library to obtain a hybridization product; performing PCR amplification using the hybridization product as a template to obtain a sequencing library; sequencing the sequencing library, analyzing the data obtained by sequencing, and obtaining HRR gene mutation and HRD information.

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