Deafness gene therapy related gene capture probe group, detection method and application

Abstract

The invention belongs to the technical field of hereditary deafness molecular diagnosis, and particularly relates to a deafness gene therapy related gene capture probe set, a detection method and application, the capture probe set comprises any one nucleotide sequence in SEQ ID NO.1-SEQ ID NO.404; the detection method comprises the following steps: carrying out hybrid capture on a nucleic acid sample to be detected by using the capture probe group; constructing a library for the captured target sequence, and performing high-throughput double-end sequencing; comparing the sequencing result with a reference genome, and identifying mutation of genes related to deafness gene therapy; the invention also discloses application of the capture probe group for the deafness gene therapy related genes in preparation of detection products for deafness genetic screening, deafness cause diagnosis, deafness genetic typing or gene therapy decision. Compared with the prior art, the gene capture probe set for deafness related to gene therapy is designed, the problem of non-uniform coverage of conventional sequencing in such areas is solved, and the whole genome capture uniformity and efficiency are improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular diagnosis of genetic deafness, and particularly relates to a capture probe set for deafness gene therapy related genes, a detection method and application. BACKGROUND

[0002] Deafness, as the most common sensory impairment disease in humans, has a profound impact on the hearing function, speech communication and quality of life of hundreds of millions of people worldwide. According to the report of the World Health Organization, about 466 million people worldwide suffer from disabling hearing loss, among which genetic factors play a dominant role in the pathogenesis of deafness, and about 60% of congenital deafness cases are caused by genetic factors. In China, the total number of hearing impaired people is more than 70 million, among which about 137,000 are hearing impaired children aged 0-6 years old, and about 35,000 are newly added every year.

[0003] So far, more than 200 genes have been confirmed to be associated with genetic deafness. Chinese population has a unique mutation spectrum, GJB2 Gene mutation is the most common deafness-causing factor, and 21.6% of deaf patients in China carry GJB2 gene mutations; SLC26A4 Gene mutation is the second largest deafness-causing factor, accounting for 19.4%; drug-induced deafness caused by mitochondrial m.1555A>G and m.1494C>T mutations accounts for about 1.87%; USH2A Gene accounts for 3%-6% of congenital deafness population; MPZL2 is a common gene associated with mild to moderate genetic hearing loss. However, the detection of 4 genes and about 20 common mutations can only solve the etiological diagnosis of about 30-36% of genetic deafness patients, and a large number of cases still cannot be clearly diagnosed due to un-covered mutations or genes.

[0004] In recent years, the emergence of gene therapy has brought hope for radical intervention for genetic deafness. OTOF DFNB9 caused by gene mutation accounts for 2%-8% of genetic deafness. Professor Shu Yilai's team of the Eye, Ear, Nose and Throat Hospital of Fudan University carried out the world's first OTOF gene clinical treatment in December 2022, and significantly improved the hearing and speech ability of the children. The gene therapy research targeting USH2A exon 13 mutation also brings hope to patients. In addition, GJB2, MPZL2 gene therapy research in animal models has achieved good results. These breakthroughs mark that genetic deafness treatment has officially entered the gene level era, and gene detection is the key prerequisite for identifying patients suitable for these innovative therapies. In recent years, the development of the second-generation high-throughput sequencing (NGS) technology has provided a new means for genetic deafness gene detection.

[0005] However, the existing detection methods still cannot meet the needs of rapid screening and genetic typing of gene therapy population. Therefore, it is of great significance to develop a capture probe set and its detection method which can comprehensively cover the deafness gene therapy related genes and balance the detection sensitivity and specificity, so as to improve the clinical diagnosis level and promote the deafness gene therapy. SUMMARY

[0006] (1) Technical problems to be solved The purpose of the present application is to solve at least one of the above problems by providing a capture probe set for deafness gene therapy related genes, a detection method and application, so as to solve the problems in the prior art that it is difficult to detect the genes related to deafness gene therapy at a high cost performance and comprehensively, there is a lack of specific probe combination for common deafness genes in Chinese population, and the existing detection methods have the problems of complex operation and high cost in clinical application, so as to achieve the effect of improving the coverage range and accuracy of genetic deafness gene therapy related gene detection.

[0007] (2) Technical solutions The purpose of the present application is achieved by the following technical solutions: One of the technical solutions of the present application is a capture probe set for deafness gene therapy related genes, which comprises any one of the nucleic acid sequences in SEQ ID NO. 1-SEQ ID NO. 404.

[0008] Further, the capture probe set comprises probes for capturing genes related to genetic deafness and drug-induced deafness; The genes include OTOF, GJB2, SLC26A4, MPZL2 , USH2A and mitochondrial genes MT-RNR1 .

[0009] The genes include OTOF, GJB2, SLC26A4, MPZL2 , USH2A and mitochondrial genes MT-RNR1 . The combination of these probes covers the deafness genes which have carried out clinical trials of gene therapy or have potential value of gene therapy and the mutation spectrum unique to Chinese population, and also includes the gene therapy target points which have entered the clinical trial stage, so as to provide molecular diagnostic basis for clinical decision.

[0010] Further, the probes are continuously tiled along the sense strand region of the target gene, covering the entire exon region and part of the intron region near the splicing site of the corresponding gene. This design eliminates the detection blind area by the continuous tiling strategy, completely captures the exon region and potential splicing mutation site, and ensures the detection ability of gene structure variation and splicing site mutation.

[0011] Further, the probe set comprises the following characteristics: (1) The length of each probe is 120 bp; (2) the probe sequence has homology less than 80% with the non-target region after alignment with the human whole genome sequence; (3) the coverage density of the probe is 1.5 times or more than that of the high GC region in the region with GC content less than 45%.

[0012] During the construction of the library, the probe of this length can form a stable double-stranded structure with the target DNA fragment, ensuring the accuracy of subsequent PCR amplification and sequencing. The low GC region has low base pairing stability, and the binding efficiency of the probe to the target sequence is reduced, resulting in reduced capture efficiency. By increasing the coverage density of the low GC region probe to 1.5 times or more than that of the high GC region, the number of effective binding probes in the unit area can be increased to compensate for the lack of single probe binding force. In the design of the probe continuously tiled along the sense strand of the target gene, the 120bp probe can effectively cover the exon region and the adjacent intron region, while the coverage density adjustment strategy for different GC content regions forms a complement.

[0013] The second technical scheme of the present application is a method for detecting deafness gene therapy related genes using a capture probe set for non-diagnostic purposes, comprising the following steps: (1) hybridization capture of the nucleic acid sample to be tested using the capture probe set as described above; (2) constructing a library of the captured target sequence and performing high-throughput double-end sequencing; (3) aligning the sequencing results with the reference genome to identify mutations related to deafness gene therapy.

[0014] The probe set contains the nucleic acid sequences of SEQ ID NO. 1 to SEQ ID NO. 404, and the 120bp length design balances the hybridization efficiency and specificity. The arrangement of the probe in the target region continuously tiled with the differential coverage density strategy effectively overcomes the influence of GC content variation on capture efficiency. In the sequencing stage, the double-end mode is adopted, and the single base resolution accuracy is improved through the complementary verification of the two end reads. This method optimizes the capture probe design, sequencing strategy and analysis standard, and systematically improves the detection rate of pathogenic mutations and the reliability of clinical interpretation.

[0015] Further, the high-throughput double-end sequencing adopts surfseq 5000 platform for double-end sequencing.

[0016] Further, the mutation identification is interpreted according to the HGVS naming standard and ACMG pathogenicity classification guidelines.

[0017] Further, the method as described above is applied to detect the sample of the subject, if a known pathogenic mutation or a possible pathogenic mutation is detected in the gene or site, it is suggested that the subject has or has a high risk of suffering from deafness.

[0018] Further, the method as described above is applied to detect the sample of the subject, if a known pathogenic mutation or a possible pathogenic mutation is detected in the gene or site, it is suggested that the subject has or has a high risk of suffering from deafness. MT-RNR1 Further, the method as described above is applied to detect the sample of the subject, if a known pathogenic mutation or a possible pathogenic mutation is detected in the gene or site, it is suggested that the subject has or has a high risk of suffering from deafness.

[0019] Further, the method as described above is applied to detect the sample of the subject, if a known pathogenic mutation or a possible pathogenic mutation is detected in the gene or site, it is suggested that the subject has or has a high risk of suffering from deafness. OTOF Further, the method as described above is applied to detect the sample of the subject, if a known pathogenic mutation or a possible pathogenic mutation is detected in the gene or site, it is suggested that the subject has or has a high risk of suffering from deafness. OTOF Further, the method as described above is applied to detect the sample of the subject, if a known pathogenic mutation or a possible pathogenic mutation is detected in the gene or site, it is suggested that the subject has or has a high risk of suffering from deafness. OTOF Further, the method as described above is applied to detect the sample of the subject, if a known pathogenic mutation or a possible pathogenic mutation is detected in the gene or site, it is suggested that the subject has or has a high risk of suffering from deafness.

[0020] The third technical solution of the present application is the application of a deafness gene therapy related gene capture probe set as described above, which can be prepared into a detection product for deafness genetic screening, deafness genetic typing, molecular diagnosis or gene therapy candidate screening of precise treatment drugs or potential drugs.

[0021] Further, the capture probe set can prepare a deafness gene detection kit.

[0022] Further, the kit further comprises one or more of the following components: reagents for DNA fragmentation, reagents for end repair, reagents for linker ligation, reagents for PCR amplification, streptavidin magnetic beads, hybridization solution and elution buffer.

[0023] (Three) beneficial effects Compared with the prior art, the present application has the following advantages: (1) The core of the present application is to independently design a set of gene capture probes for deafness: the probe set accurately covers the five nuclear genes (GJB2, GJB3, GJB6, TCF20 and SLC26A4) most closely related to deafness in Chinese population and the key drug-induced deafness mitochondrial gene (12SrRNA), and the 120bp long probes are continuously laid along the target region to ensure comprehensive coverage. For low complexity regions with GC content less than 45% in the genome, the probe density is increased to more than 1.5 times that of high GC regions, overcoming the uneven coverage problem of conventional sequencing in such regions and improving the uniformity and efficiency of whole genome capture. OTOF, GJB2, SLC26A4, MPZL2, USH2A (1) The core of the present application is to independently design a set of gene capture probes for deafness: the probe set accurately covers the five nuclear genes (GJB2, GJB3, GJB6, TCF20 and SLC26A4) most closely related to deafness in Chinese population and the key drug-induced deafness mitochondrial gene (12SrRNA), and the 120bp long probes are continuously laid along the target region to ensure comprehensive coverage. For low complexity regions with GC content less than 45% in the genome, the probe density is increased to more than 1.5 times that of high GC regions, overcoming the uneven coverage problem of conventional sequencing in such regions and improving the uniformity and efficiency of whole genome capture. MT-RNR1

[0024] ​(2) In the first cohort of 30 patients with a clear deafness phenotype, using this probe set and detection method, clear pathogenic or suspected pathogenic gene mutations were successfully detected in 16 patients, with a positive detection rate of up to 53.3%. This data is significantly better than the traditional detection methods for single genes or high-frequency sites of common deafness genes, which fully demonstrates the comprehensiveness and scientific nature of the gene set selected in this invention, as well as the high sensitivity and reliability of the entire detection process (from capture to bioinformatics analysis), providing a powerful tool for the clinical molecular diagnosis of deafness.

[0025] (3) This application achieves full exon coverage of deafness-related genes and simultaneous detection of mitochondrial drug-sensitive sites, effectively solving the problem of missed diagnoses caused by incomplete gene coverage in traditional screening methods. By integrating nuclear gene and mitochondrial variation detection, this scheme can simultaneously identify the genetic causes of congenital deafness and the susceptibility risk to drug-induced deafness, providing clinical diagnosis with complete diagnostic evidence including genotype-phenotype associations. The detection products prepared by this probe set can support the precise screening of gene therapy indications, for example, by detecting... OTOF or USH2A Biallelic pathogenic mutations were identified to screen for patient groups eligible for gene replacement therapy. Attached Figure Description

[0026] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 For patient S1 in Example 1 GJB2 Sanger sequencing validation results for the c.235delC gene mutation; Figure 2 For patient S1 in Example 1 GJB2 Sanger sequencing validation results for the c.109G>A gene mutation; Figure 3 For patient S2 in Example 1 SLC26A4 Sanger sequencing validation results for the c.1336C>T mutation in the gene; Figure 4 For patient S2 in Example 1 SLC26A4 Sanger sequencing validation results for the c.1975G>C mutation in the gene; Figure 5 For patient S3 in Example 1 GJB2 Sanger sequencing validation results for the c.235del gene mutation; Figure 6 Sanger sequencing validation results for the gene c. 109G>A mutation for patient S4 in Example 1; GJB2 Sanger sequencing validation results for the gene c. 109G>A mutation for patient S4 in Example 1; Figure 7 Sanger sequencing validation results for the gene c. 109G>A mutation for patient S4 in Example 1; GJB2 Sanger sequencing validation results for the gene c. 109G>A mutation for patient S4 in Example 1; Figure 8 Sanger sequencing validation results for the gene c. 1645dupA mutation for patient S5 in Example 1; SLC26A4 Sanger sequencing validation results for the gene c. 1645dupA mutation for patient S5 in Example 1; Figure 9 Sanger sequencing validation results for the gene c. 1645dupA mutation for patient S5 in Example 1; SLC26A4 Sanger sequencing validation results for the gene c. 1645dupA mutation for patient S5 in Example 1; Figure 10 Sanger sequencing validation results for the mitochondrial gene m.1555A>G mutation for patient S6 in Example 1; MT-RNR1 Sanger sequencing validation results for the mitochondrial gene m.1555A>G mutation for patient S6 in Example 1; Figure 11 Sanger sequencing validation results for the mitochondrial gene m.1555A>G mutation for patient S7 in Example 1; MT-RNR1 Sanger sequencing validation results for the mitochondrial gene m.1555A>G mutation for patient S7 in Example 1; Figure 12 Sanger sequencing validation results for the gene c. 235del mutation for patient S8 in Example 1; GJB2 Sanger sequencing validation results for the gene c. 235del mutation for patient S8 in Example 1; Figure 13 Sanger sequencing validation results for the gene c. 235del mutation for patient S9 in Example 1; GJB2 Sanger sequencing validation results for the gene c. 235del mutation for patient S9 in Example 1; Figure 14 Sanger sequencing validation results for the gene c. 235del mutation for patient S9 in Example 1; GJB2 Sanger sequencing validation results for the gene c. 235del mutation for patient S9 in Example 1; Figure 15 Sanger sequencing validation results for the gene c. 589G>A mutation for patient S10 in Example 1; SLC26A4 Sanger sequencing validation results for the gene c. 589G>A mutation for patient S10 in Example 1; Figure 16 Sanger sequencing validation results for the gene c. 589G>A mutation for patient S10 in Example 1; SLC26A4 Sanger sequencing validation results for the gene c. 589G>A mutation for patient S10 in Example 1; Figure 17 Sanger sequencing validation results for the gene c. 5192+1G>C mutation for patient S11 in Example 1; OTOF Sanger sequencing validation results for the gene c. 5192+1G>C mutation for patient S11 in Example 1; Figure 18 Sanger sequencing validation results for the gene c. 5192+1G>C mutation for patient S11 in Example 1; OTOF Sanger sequencing validation results for the gene c. 5192+1G>C mutation for patient S11 in Example 1; Figure 19 For patient S12 in Example 1 MPZL2 Sanger sequencing validation results for the c.463delG gene mutation; Figure 20 For patient S12 in Example 1 MPZL2 Sanger sequencing validation results for the c.220C>T mutation in the gene; Figure 21 For patient S13 in Example 1 USH2A Sanger sequencing validation results for the c.9298G>T mutation in the gene; Figure 22 For patient S13 in Example 1 USH2A Sanger sequencing validation results for the c.5581G>A mutation in the gene; Figure 23 For patient S14 in Example 1 GJB2 Sanger sequencing validation results for the c.235delC mutation in the gene; Figure 24 For patient S14 in Example 1 GJB2 Sanger sequencing validation results for the c.109G>A gene mutation; Figure 25 For patient S15 in Example 1 GJB2 Sanger sequencing validation results for the c.235delC mutation in the gene; Figure 26 For patient S15 in Example 1 GJB2 Sanger sequencing validation results for the c.35dup gene mutation; Figure 27 For patient S16 in Example 1 SLC26A4 Sanger sequencing validation results for the c.919-2A>G gene mutation. Detailed Implementation

[0028] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0029] Following, the embodiments of the present application are described through specific examples, and other advantages and effects of the present application can be easily understood by those skilled in the art from the disclosure. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. The present application can also be implemented or applied through other different specific embodiments, and various modifications or changes can be made to the details in the specification without departing from the spirit of the present application. It should be noted that the following embodiments and features in the embodiments can be combined with each other without conflict. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0030] It should be noted that the various aspects of the embodiments described below are within the scope of the appended claims. It should be apparent that the aspects described herein can be embodied in a wide variety of forms and that any specific structure and / or function described herein is merely illustrative. Based on the teachings provided herein one skilled in the art will appreciate that one aspect described herein can be implemented independently of any other aspects and that the various aspects described herein can be combined in various ways. For example, an apparatus can be implemented or a method can be practiced using any number of the aspects set forth herein. In addition, such an apparatus can be implemented or such a method can be practiced using other structure and / or functionality in addition to or other than one or more of the aspects set forth herein.

[0031] In addition, in the following description, specific details are provided to facilitate thorough understanding of examples. However, one skilled in the art will understand that the examples can be practiced without these specific details.

[0032] The raw materials or devices used in the following examples are commercially available raw materials or conventional experimental devices unless otherwise specified.

[0033] The technical solutions provided by the embodiments of the present application are described below in combination with the drawings and examples. Example 1

[0034] The present embodiment discloses a capture probe set for deafness gene therapy related genes, including nucleic acid probes for capturing 5 nuclear genes and 2 mitochondrial sites, the probe set includes the following features: (1) the length of each probe is 120 bp; (2) the probes are continuously tiled along the sense strand region of the target gene, covering the corresponding gene exons and part of the intron region; (3) after aligning the probe sequence with the human whole genome sequence, the homology in the non-target region is less than 80%; (4) In the region where the GC content is less than 45%, the coverage density of the probe is 1.5 times or more of that of the high GC region.

[0035] The genes captured by the capture probe set and the chromosome coordinate information of the genes are shown in the following table: Table 1 Gene captured by the capture probe set and chromosome coordinate information

[0036] The probe set comprises any one of the nucleic acid sequences in SEQ ID NO. 1 to SEQ ID NO. 404 as shown in Table 2: Table 2 Genetic deafness gene therapy related gene capture probe set Example 2

[0037] The present embodiment provides a complete deafness gene detection method, comprising the following steps: (1) Hybridization capture of the nucleic acid sample to be tested by using the capture probe set of the deafness gene therapy related gene; (2) Constructing a library of the captured target sequence and performing double-end sequencing by using the surfseq 5000 platform; (3) Aligning the sequencing results with the reference genome, identifying the gene mutations related to deafness gene therapy, and interpreting the mutations according to the HGVS naming standard and the ACMG pathogenicity classification guidelines.

[0038] Specifically comprising the following steps: (I) Library construction The library construction method is used to prepare a DNA library suitable for double-end sequencing by the Surfseq 5000 platform, and the specific steps are as follows: 1. DNA fragmentation and end repair: Take the sample of genomic DNA to be tested, centrifuge and place on ice, add 5 μL FERA Buffer and 5 μL FERA Enzyme, vortex and centrifuge, and run the mixture on a PCR instrument with the following program: 25°C for 30 min, 65°C for 30 min, 10°C Hold, (FERA Enzyme is a DNA fragmentation and end repair enzyme mixture).

[0039] 2. Adapter ligation and purification: centrifuge the product of the previous step, place on ice, add 26 μL Ligation Buffer and 2 μL DNA Ligase, and finally add 2 μL M-Adapter (DI) along the wall of the tube, mix thoroughly, and centrifuge briefly to place the entire reaction at the bottom of the PCR tube, and start the reaction program on the PCR instrument: 20°C for 15 min, 4°C ∞; add 40 μL SP Beads (magnetic beads) to the ligation product, incubate at 25°C for 5 min, place the PCR tube on a magnetic stand for 5 min, discard the supernatant, slowly add 150 μL 80% ethanol along the side wall of the PCR tube, stand for 30 seconds, then repeat the addition of 80% ethanol and aspiration, centrifuge briefly and aspirate the residual ethanol, stand at room temperature for 2-3 min until the ethanol evaporates, and add 20 μL Nuclease Free Water.

[0040] 3. Pre-library amplification and purification: transfer the resuspended solution with magnetic beads to a new tube, add 25 μL 2X HiFi PCR Master Mix and 5 μL M-Index Primer Mix (MDI), mix well, and run the PCR program: 98°C for 2 min; (98°C for 15 s, 60°C for 30 s, 72°C for 30 s) for 4 cycles; 72°C for 2 min, 4°C ∞, volume set to 50 μL, hot lid 105°C; after amplification, add 50 μL SP Beads magnetic beads to the product, mix well, incubate at 25°C for 5 min, centrifuge the PCR tube briefly and place it on a magnetic stand for 5 min until the liquid is completely clear, slowly add 150 μL 80% ethanol along the side wall of the PCR tube, stand for 30 seconds, then repeat the addition of 80% ethanol and aspiration, centrifuge briefly and aspirate the residual ethanol, stand at room temperature for 2-3 min until the ethanol evaporates, and add 20 μL TE Solution to elute the final sequencing library.

[0041] 4. Library quantification: use Qubit fluorometer or quantitative PCR to quantify the library, and use Agilent Bioanalyzer or similar equipment to detect the fragment size distribution of the library.

[0042] (B) Hybrid capture and sequencing 5、Library hybridization: Take an appropriate amount of pre-library (single hybridization 1000 ng, multiple hybridization 750 ng / library) for pooling, add Human Cot DNA, and then use SP Beads to purify and concentrate the mixed library to remove impurities and adjust the volume. Add hybridization mixture (containing 9.5 μL Hyb #1, 3 μL Hyb #2, 2 μL Blockers, and 4.5 μL WES V2.0) to the purified magnetic beads, mix well, and incubate at room temperature for 5 min. Separate the centrifuge tube on the magnetic stand, and transfer 17 μL of clear supernatant to a new PCR tube. Start the hybridization program on the PCR instrument: 95°C for 30 s; 65°C for 16 h, 65°C for ∞, and hot lid at 100°C.

[0043] 6、Sub-packaging hybridization capture cleaning reagents and configuring magnetic bead suspension: Hybridization capture cleaning reagents include Wash Buffer 1, Wash Buffer 2, and Wash Buffer 3; magnetic bead suspension includes HYB #1, HYB #2, and Nuclease-Free Water.

[0044] 7、Magnetic bead pretreatment and capture: Take 50 μL Streptavidin Beads, wash three times with Bead Wash Buffer to remove impurities in the storage solution; resuspend the washed magnetic beads in the prepared suspension, and preheat at 65°C for 5 min. Add the preheated magnetic beads to the reaction system after hybridization, and incubate at 65°C for 45 min to allow the magnetic beads to fully bind to the biotin-labeled probe-target DNA complex.

[0045] 9、Thermal elution and purification: Use preheated Wash Buffer 1 and Wash Buffer 2 to wash the magnetic bead complex at 65°C twice to completely remove non-specifically bound DNA. Then, at room temperature, use Wash Buffer 1, Wash Buffer 3, etc. to wash at room temperature to remove residual impurities such as salt ions. Finally, discard all eluate, and resuspend the captured target DNA-magnetic bead complex in 22.5 μL Nuclease-Free Water.

[0046] (Three) Amplification and quality control of the library after capture 10、PCR amplification: Directly use the above-mentioned 22.5 μL capture product containing magnetic beads as a template, add PCR master mix and primers for post-capture amplification (10 cycles) to enrich the final sequencing library. The PCR instrument is set as follows: 98°C for 45 s; (98°C for 15 s, 60°C for 30 s, and 72°C for 30 s) for 10 cycles, 72°C for 1 min, and 4°C for ∞, with a volume of 50 μL and a hot lid at 105°C.

[0047] 11. Library purification: standard purification of PCR amplification products using SP Beads to remove primer dimers and reaction system impurities, and finally dissolve the library in 21 μL buffer.

[0048] 12. Library quality control: use Qubit to accurately quantify the final library after purification, and use Bioanalyzer or Qsep100 to detect the fragment size distribution of the library. The final library that passes the quality control is used for double-end sequencing on the Surfseq 5000 platform.

[0049] (Four) bioinformatics analysis 1. The original FASTQ data is subjected to quality control, adapter removal and low-quality data filtering using fastp (version 0.23.3).

[0050] 2. The filtered FASTQ is aligned to the human reference genome (build hg19) using bwa-mem (version 0.7.17-r1188) to generate an alignment file.

[0051] 3. The alignment file is processed and quality controlled using samblaster (version 0.1.26), samtools (version 1.18), and mosdepth (version 0.3.3).

[0052] 4. The processed alignment file is subjected to variant detection using gatk (version 4.2.6.1).

[0053] 5. The variant detection results are filtered and quality controlled using bcftools (version 1.18).

[0054] 6. The variant detection results are annotated using snpeff (version v5.1f) and annovar (version 2020-06-08).

[0055] Detection verification 30 cases of deafness phenotype related patients were detected for deafness gene therapy related genes, and the mutation sites screened in this study were named and pathogenicity rated according to the naming principles of Human Genome Variation Society (HGVS) and ACMG guidelines, and Sanger sequencing verification was performed, and the results are as follows. The analysis results found that 16 cases of deafness phenotype in 30 patients were detected to have clear pathogenic mutations, including OTOF, GJB2, SLC26A4, MPZL2, USH2A and mitochondrial MT-RNR1The detection rate was 53.3% (16 / 30). In the 16 patients, 7 patients were detected GJB2 Variation, the detection rate was 43.75%, 4 patients were detected SLC26A4 Variation, the detection rate was 25%; 2 patients were detected mitochondrial MT-RNR1 Variation, the detection rate was 12.5%; 1 patient was detected OTOF Variation, the detection rate was 6.25%; 1 patient was detected MPZL2 Variation, the detection rate was 6.25%; 1 patient was detected USH2A Variation, the detection rate was 6.25%. The specific results are shown in the following table: Table 3 Detection results of deafness gene treatment related genes of 30 patients with deafness phenotype related diseases

[0056] The 16 patients in the above table 3 were subjected to sanger verification, and the results are shown in the following table: Figures 1-27 The peak chart of sanger sequencing clearly shows homozygous mutation (S3 patient in the middle, Figure 5 S8 patient in the middle and S16 patient in the middle) or heterozygous mutation (, Figure 12 , Figure 27 and Figures 1-4 , Figures 6-11 and Figures 13-26 ) on the mutation site detected by NGS, which confirms that the sensitivity and specificity of detecting the site are very high, and the variation indeed exists in the sample DNA, which is not a false positive error caused by NGS library construction, amplification or bioinformatics analysis software. The above detection verification results show that the above capture probe set provided by the present application can effectively capture the deafness gene treatment related genes, can reduce the detection cost of related genes, and improve the detection efficiency.

[0057] Example 3

[0058] The present embodiment provides a scheme for preparing a kit for detecting deafness genes by using a set of capture probes. The kit prepared in the present embodiment comprises the following reagents: AngTruth-seqTM EZ DNA Library Preparation Module (for MGI), 96 rxn, FERA Buffer, FERA Enzyme, Enhancer Buffer, Ligation Buffer, DNA Ligase, 2X HiFi PCR Master Mix, Nuclease Free Water, TE Solution, AngTruth-seqTM SP Beads, AngTruth-seqTM Target Module (for MGI), 96 rxn, Hyb #1, Hyb #2, Human Cot DNA, Blockers (for MGI), Bead Wash Buffer, Wash Buffer 1, Wash Buffer 2, Wash Buffer 3, Wash Buffer 4, 2X HiFi PCR Master Mix, M-Amplification Primer Mix (for MGI, DI), SP Beads, Streptavidin Beads.

[0059] The same or similar parts among the various embodiments in the present specification can be referred to each other, and each embodiment focuses on the difference from other embodiments.

[0060] The above merely illustrates the specific embodiments of the present application, but the protection scope of the present application is not limited to this. Any person skilled in the art can easily think of the changes or replacements within the technical scope disclosed in the present application, which should be covered in the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A set of capture probes for genes related to deafness gene therapy, characterized in that, The capture probe set includes any nucleic acid sequence from SEQ ID NO.1 to SEQ ID NO.

404.

2. The capture probe set for genes related to deafness gene therapy according to claim 1, characterized in that, The capture probe set includes probes for capturing genes associated with hereditary deafness and drug-induced deafness; The genes include OTOF, GJB2, SLC26A4, MPZL2 , USH2A and mitochondrial genes MT-RNR1 .

3. The capture probe set for genes related to deafness gene therapy according to claim 2, characterized in that, The probe is continuously laid out along the positive strand region of the target gene, covering all exons of the corresponding gene and some intron regions near the splice site.

4. The capture probe set for genes related to deafness gene therapy according to claim 2, characterized in that, The probe group includes the following features: (1) Each probe is 120 bp in length; (2) After the probe sequence is compared with the human whole genome sequence, its homology in non-target regions is less than 80%; (3) In regions where the GC content is less than 45%, the coverage density of the probe is more than 1.5 times that of regions with high GC content.

5. A method for detecting deafness gene therapy-related genes using the capture probe set of claim 1 for non-diagnostic purposes, characterized in that, Includes the following steps: (1) Using the capture probe set as described in claim 1 to perform hybridization capture on the nucleic acid sample to be tested; (2) Construct a library from the captured target sequence and perform high-throughput paired-end sequencing; (3) The sequencing results are compared with the reference genome to identify mutations in genes related to deafness gene therapy.

6. The method for detecting genes related to deafness gene therapy using a capture probe set according to claim 5, characterized in that, The high-throughput paired-end sequencing was performed using the surfseq 5000 platform.

7. The method for detecting genes related to deafness gene therapy using a capture probe set according to claim 5, characterized in that, The mutation identification was interpreted according to the HGVS nomenclature standard and the ACMG pathogenicity classification guidelines.

8. The application of a capture probe set for genes related to deafness gene therapy as described in claim 1, characterized in that, The capture probe set can be used to prepare detection products for genetic screening of deafness, genetic typing of deafness, molecular diagnosis, or screening of gene therapy candidates.

9. The application of the capture probe set of genes related to deafness gene therapy according to claim 8, characterized in that, The captured probe set can be used to prepare a deafness gene detection kit.

10. The application of the capture probe set of genes related to deafness gene therapy according to claim 9, characterized in that, The kit also contains one or more of the following components: reagents for DNA fragmentation, reagents for end repair, reagents for adapter ligation, reagents for PCR amplification, streptavidin beads, hybridization solution, and elution buffer.

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