A kit and method for detecting pathogenic genes in X-linked hereditary retinal degenerative diseases.

Abstract

This invention relates to a kit and method for detecting pathogenic genes of X-chromosome-linked hereditary retinal degenerative diseases. The kit includes a library hybridization reagent and a probe set for detecting XLIRD pathogenic genes, including capture probes that can simultaneously and specifically capture the RPGR, RP2, RS1, and CHM pathogenic genes. The capture probes for capturing the RPGR pathogenic gene include probes with sequences as shown in SEQ ID NO. 1-96, and capture probes targeting the high-GC region of the RPGR gene with sequences as shown in SEQ ID NO. 24-55. This invention targets the above four pathogenic genes, comprehensively analyzes their variation profiles and sequence characteristics, optimizes the amplification region range, and designs a high-density capture probe set including exons, introns, UTR regions, and upstream and downstream 20kb regions. Specifically targeting the high-GC content region of exon 15 of the RPGR gene, the probe sequence and capture strategy are adjusted to achieve efficient capture of the high-GC region. During the library PCR enrichment stage, the amplification enzymes in the system are screened and optimized. Combined with the probe set, multiple amplification enzyme systems are used, and finally, the most suitable amplification enzyme combination for this gene system is optimized.

Description

Technical Field

[0001] This invention belongs to the field of gene diagnostic technology, specifically relating to a kit and method for detecting pathogenic genes of X-chromosome-linked hereditary retinal degenerative diseases. Background Technology

[0002] X-linked inherited retinal dystrophy (XLIRD) is a rare degenerative retinal disease caused by pathogenic gene mutations on the X chromosome. The core manifestation of this disease is progressive vision loss, which can eventually lead to legal blindness or even complete blindness. As an important subtype of inherited retinal dystrophies (IRDs), XLIRDs have unique characteristics compared to other autosomal dominant / recessive IRDs. A comprehensive clinical diagnosis obtained through detailed family history information combined with ophthalmological clinical examination is usually more definitive than for other IRDs, significantly narrowing the range of candidate genes. In such cases, further molecular testing is crucial for definitive diagnosis. Currently, gene therapy research for XLIRDs has made groundbreaking progress, with numerous clinical trials conducted both domestically and internationally. Effective treatments will bring hope to many patients, and accurate molecular diagnosis has become a core prerequisite for patient enrollment, efficacy prediction, and treatment plan development. Rapid and accurate detection of the pathogenic genes for XLIRDs will lay a solid foundation for subsequent precision clinical diagnosis and treatment.

[0003] Mutations in the RPGR gene can lead to various XLIRDs, including X-linked retinitis pigmentosa (XLRP), cone dystrophy, cone-rod dystrophy, or syndromic XLRP. Patients with RP gene mutations account for a high proportion of RP patients, exceeding 70% of XLRP cases. Approximately 60% of pathogenic RPGR mutations are located in open reading frame 15 (ORF15), whose alternative splicing products are primarily expressed in the retina and are involved in the transport of proteins essential for maintaining photoreceptor cells. The high GC content in the RPGR gene's ORF15 region results in extremely low coverage of this region by currently used high-throughput sequencing technologies.

[0004] RP2 gene mutation is another cause of XLRP, accounting for 15% to 20% of XLRP patients. Approximately 90% of XLRP cases are caused by mutations in either the RPGR or RP2 genes. The RP2 protein in the retina may act as a molecular chaperone, helping with the folding and transport of other proteins, thereby maintaining the normal function of photoreceptor cells. Mutations in the RP2 gene disrupt this process, ultimately leading to the degeneration and death of retinal cells, resulting in vision loss.

[0005] The RS1 gene is the only known pathogenic gene for X-linked retinoschisis (XLRS) to date. The RS1 gene encodes retinoschisin (RS1), a protein in the retina that is crucial for maintaining retinal structure and function. XLRS can manifest as early as two or three years of age, presenting with varying degrees of visual impairment, including retinal schisis, radial changes caused by macular schisis, and peripheral retinal schisis. It can also be complicated by vitreous hemorrhage and retinal detachment, severely damaging both retinal structure and function.

[0006] The CHM gene is currently considered the sole pathogenic gene for choroideremia (CHM). CHM is a rapidly progressing disease that causes significant visual impairment, potentially leading to blindness in middle age. The CHM gene expresses Rab helper-binding protein (REP)1, which plays a crucial role in intracellular vesicle transport. Pathogenic mutations in the CHM gene can result in the complete absence or non-function of REP1, ultimately causing progressive atrophy of the retinal pigment epithelium, photoreceptor cells, and choroidal capillaries.

[0007] Currently, gene therapy drugs targeting the four genes mentioned above have entered clinical / preclinical trial stages. Clinical trials for gene therapy targeting RPGR and RS1 have already begun in China. A definitive molecular diagnosis is an essential prerequisite for treatment of XLIRD patients. Currently, there is a lack of economical, rapid, targeted, and accurate detection methods that cover all pathogenic variants for the four XLIRD-causing genes. Only through rapid and targeted testing can patients be provided with timely gene therapy or other effective treatments, thereby slowing disease progression and improving their quality of life. Simultaneously, it helps doctors provide timely genetic counseling and reproductive guidance, reducing the risk of transmitting hereditary diseases. Therefore, conducting this test is of great significance for the diagnosis and treatment of XLIRDs.

[0008] In addition, the diagnosis of XLIRDs mainly relies on gene sequencing technology. Preferred molecular genetic tests can include gene exon region targeting (single gene and multiple genes) as well as whole exome sequencing (WES) and whole genome sequencing (WGS).

[0009] For RPGR gene detection, the ORF region, due to its high GC content, is poorly captured by conventional high-throughput sequencing technologies, resulting in incomplete capture of the ORF15 region and false negatives. While first-generation sequencing technology for ORF15 sequence detection has limitations in sequencing length (700-800 bp), directly affecting the detection of rare structural variations, it also suffers from low throughput.

[0010] For the CHM gene, approximately 75% of CHM patients have deletion / insertion mutations, missense mutations, nonsense mutations, and splicing site mutations within the gene, while about 25% of CHM patients have exon or entire gene deletions or duplications. In these cases, WES or WGS cannot detect the full spectrum of mutations in one step, and their accuracy is limited. After routine WES / WGS screening, targeted gene deletion / duplication analysis is needed to infer structural variations in exons or the entire gene. Furthermore, further validation using methods including quantitative polymerase chain reaction (PCR), long-fragment PCR, multiplex ligation probe amplification technology, and gene-targeting microarrays for detecting single exon deletions or duplications is required, but these methods suffer from significant practical problems such as long detection times and low efficiency.

[0011] Both RP2 and RS1 genes have pathogenic variants such as copy number variation and structural variation, and existing detection methods are difficult to meet the needs of one-step detection.

[0012] In summary, it is necessary to design effective reagent kits and corresponding detection methods to solve the following technical problems:

[0013] 1) Difficulty in gene region amplification: Due to the high GC content and numerous repetitive sequences in the ORF15 region of the RPGR gene, traditional amplification methods are prone to problems such as low amplification efficiency and poor library uniformity, resulting in incomplete sequencing data coverage or large deviations, which affects the reliability of the detection results.

[0014] 2) Diverse variant types make it difficult to be compatible: Traditional gene detection methods usually require the use of different technologies to detect point mutations, small fragment insertions / deletions, copy number variations and structural variations. The process is complex and inefficient, increasing costs and time.

[0015] 3) Time consumption for verification of test results: The preliminary results generated by high-throughput sequencing often need to be confirmed by low-throughput verification methods, which prolongs the testing cycle and increases the consumption of samples and reagents, thus restricting the widespread application of the technology in clinical practice.

[0016] 4) Insufficient applicability: Existing technologies lack optimized amplification schemes during library construction, making it difficult to meet the detection needs of both full-length gene coverage and key regions (such as UTR regions and upstream and downstream regulatory regions), which affects the detection rate of certain pathogenic variants. Summary of the Invention

[0017] To address the aforementioned technical problems, the present invention aims to provide a probe set, kit, and detection method for detecting pathogenic genes in X-linked hereditary retinal degenerative diseases. This invention is based on a highly efficient detection system optimized for target gene variant profiles and sequence characteristics, and aims to comprehensively and accurately detect multiple types of variants in four pathogenic genes—RPGR, RP2, RS1, and CHM—of X-linked hereditary retinal degenerative diseases (XLIRDs).

[0018] The objective of this invention is achieved through the following technical solution:

[0019] A probe set for detecting pathogenic genes of X-chromosome-linked hereditary retinal degenerative diseases, the probe set comprising capture probes capable of simultaneously and specifically capturing the pathogenic genes RPGR, RP2, RS1, and CHM, and the probe set comprising high-density capture probes in exons, introns, UTR regions, and upstream and downstream 20kb regions; the upstream and downstream 20kb regions are used to cover structural variations such as large gene deletions and duplications; wherein, the capture probes specifically capturing the RPGR pathogenic gene include probes with sequences as shown in SEQ ID NO. 1-96, and the capture probes specifically capturing the RPGR pathogenic gene include capture probes targeting the high GC regions of the RPGR gene, with sequences as shown in SEQ ID NO. 24-55.

[0020] Capture probes specifically targeting the RS1 pathogenic gene include probes with sequences shown in SEQ ID NO. 97-149; capture probes specifically targeting the RP2 pathogenic gene include probes with sequences shown in SEQ ID NO. 150-193; and capture probes specifically targeting the CHM pathogenic gene include probes with sequences shown in SEQ ID NO. 194-282. For detailed capture probe sequences targeting the RPGR, RP2, RS1, and CHM pathogenic genes, please refer to the probe sequence listing.

[0021] This invention targets four pathogenic genes—RPGR, RP2, RS1, and CHM—and comprehensively analyzes their variation profiles and sequence characteristics to optimize the amplification region. A high-density capture probe encompassing exons, introns, UTR regions, and upstream and downstream 20kb regions was designed. Specifically targeting the high-GC-content region of exon 15 (ORF15 region) of the RPGR gene, a capture strategy employing adjusted probe dosage (probe gradient experiment) and shingled probe encryption achieves highly efficient capture of the high-GC region, overcoming the problems of low detection efficiency and uneven coverage associated with traditional techniques.

[0022] The application of the probe set described herein in the preparation of a kit for detecting pathogenic genes of X-chromosome-linked hereditary retinal degenerative diseases.

[0023] A kit for detecting pathogenic genes of X-chromosome-linked hereditary retinal degenerative diseases, comprising a library hybridization reagent comprising the probe set for detecting pathogenic genes of X-chromosome-linked hereditary retinal degenerative diseases.

[0024] The library hybridization reagents also include hybridization buffer, hybridization enhancer, universal blocking sequence mixture, and NF water (Nuclease-Free Water); the specific composition of the 19 μL hybridization mixture prepared by mixing the various library hybridization reagents is shown in the table below:

[0025]

[0026] The kit further includes amplification reaction reagents for library PCR enrichment after library hybridization capture. The amplification reaction reagents include a hot-start amplification reaction solution and amplification primer premix solution. The hot-start amplification reaction solution contains an amplification enzyme, which is one of KAPA HiFi HotStart Ready Mix (2X) (hereinafter referred to as KAPA amplification enzyme), Watchmaker Equinox Amplification Master Mix (2X) (hereinafter referred to as Watchmaker amplification enzyme), or TIANGEN2x HiFi PCR Master Mix (hereinafter referred to as TIANGEN amplification enzyme).

[0027] The 50 μL PCR reaction system established using the amplification reaction reagents is as follows:

[0028]

[0029] The DNA suspension with magnetic beads was obtained by capturing the library after hybridization.

[0030] Preferably, the amplification enzyme is TIANGEN 2x HiFi PCR Master Mix.

[0031] Through experiments, the inventors verified that not all amplification enzymes are compatible with the probes designed in this invention; that is, not all amplification enzymes are effective. The inventors screened numerous amplification enzymes (the three enzymes mentioned above are only a few listed in this invention; many more amplification enzymes were optimized during the screening and optimization process). Among them, Takara Ex Premier DNA polymerase (Takara amplification enzyme for short) had the lowest pre-library yield under the same number of amplification cycles (7 cycles), making final library enrichment impossible. Among the three amplification enzymes—KAPA, Watchmaker, and TIANGEN—TIANGEN amplification enzyme had the highest amplification efficiency under the same conditions. Therefore, this invention selected TIANGEN amplification enzyme as the amplification enzyme in the amplification reaction.

[0032] This invention screens and optimizes the amplification enzymes used in amplification reaction reagents. Combining the designed capture probes, this invention employs multiple amplification enzyme systems and screens them using quality parameters such as sequencing data coverage, uniformity, and accuracy. Ultimately, the most suitable amplification enzyme for the gene system selected in this invention is optimized, significantly improving amplification efficiency and detection accuracy.

[0033] The kit also includes pre-library construction reagents, which include enzyme digestion and fragmentation, end repair and A-tailing working solution, adapter ligation reagents, and pre-library amplification reagents.

[0034] The enzyme digestion and fragmentation, end repair and A-tailing working solution includes an end repair enzyme and a fragment end repair buffer, and the specific composition of 10 μL of the enzyme digestion and fragmentation, end repair and A-tailing working solution is shown in the table below:

[0035]

[0036] The connector bonding reagent includes a connector premix and a universal short connector;

[0037] The pre-library amplification reagent includes paired-end tagged primers and a mixed reaction solution for library amplification, and the pre-library amplification system established by the pre-library amplification reagent is as follows:

[0038]

[0039] Among them, the purified product after adapter ligation is the product obtained after purifying the DNA sample, digesting and fragmenting it with enzymes, repairing the ends, adding an A tail, ligating the adapter, and purifying it.

[0040] The library amplification mixing solution is the amplification reaction solution, which includes amplification enzymes. This invention illustrates the use of amplification reaction solutions containing four amplification enzymes for pre-library amplification. The four amplification enzymes are: ①KAPAHiFi HotStart Ready Mix (2X) (hereinafter referred to as KAPA amplification enzyme), ②Watchmaker Equinox Amplification Master Mix (2X) (hereinafter referred to as Watchmaker amplification enzyme), and ③Takara Ex Premier DNA Polymerase (hereinafter referred to as Takara).

[0041] The PCR products were selected from two amplification enzymes: ④ TIANGEN 2x HiFi PCR Master Mix (TIANGEN amplification enzyme). Experiments showed that the TIANGEN amplification enzyme produced the highest concentration of PCR products.

[0042] Through experimental verification, the inventors found that not all amplification enzymes are effective. Takara Ex Premier DNA Polymerase (Takara amplification enzyme) produced too low amplification product concentrations under the same conditions, failing to meet the requirements for further library construction and was therefore excluded. TIANGEN amplification enzyme, however, produced the highest PCR product concentrations and demonstrated superior amplification performance compared to KAPA and Watchmaker amplification enzymes. Therefore, this invention's kit uses TIANGEN amplification enzyme as the amplification enzyme in the pre-library amplification reagent.

[0043] The kit described herein is used in the detection of pathogenic genes in X-chromosome-linked hereditary retinal degenerative diseases.

[0044] The application described herein, specifically the method for detecting pathogenic genes of X-chromosome-linked hereditary retinal degenerative diseases using the aforementioned kit, is as follows:

[0045] The extracted genomic DNA of the target sample was fragmented by enzyme digestion, end-modified, ligated with adapters, and amplified by PCR. A paired-end tag sequence was simultaneously introduced for sample identification during parallel testing of multiple samples. Subsequently, a specific capture probe designed for the target region was used to hybridize and capture the pre-library. Based on the principle of complementary base pairing in nucleic acid molecules, the probe specifically binds to the target DNA fragment in the genome. Streptavidin magnetic beads were used to capture the DNA fragment bound to the probe in the target region. The final library was then enriched by PCR amplification and sequencing using a reversible end-termination sequencing method. The sequencing data underwent bioinformatics analysis and genetic interpretation to determine whether clinically significant germline mutations existed in the tested samples.

[0046] The capture probes include probes capable of simultaneously and specifically capturing the pathogenic genes RPGR, RP2, RS1, and CHM, and the probe set contains high-density capture probes for exons, introns, UTR regions, and upstream and downstream 20kb regions; wherein, the capture probes specifically capturing the pathogenic gene RPGR include probes with sequences as shown in SEQ ID NO.1-96, and the capture probes specifically capturing the pathogenic gene RPGR include capture probes targeting the high GC regions of the RPGR gene, with sequences as shown in SEQ ID NO.24-55.

[0047] The method for detecting pathogenic genes of X-chromosome-linked hereditary retinal degenerative diseases using the aforementioned kit includes the following specific steps:

[0048] (1) Extract genomic DNA from the target sample as a DNA sample;

[0049] (2) Pre-library construction: The purified DNA sample was fragmented by enzyme digestion, end repaired, A-tailed, ligated with adapters, purified, and then amplified. The amplified products were then purified and quality-checked to obtain the pre-library.

[0050] (3) Library hybridization: The pre-libraries are mixed to form a hybrid library; the target regions of the hybrid library are hybridized using capture probes;

[0051] (4) Capture after hybridization: The capture magnetic beads are reacted with the hybridization mixture obtained after the hybridization reaction, and then the mixture is washed and eluted to obtain a DNA suspension with magnetic beads; then the library is enriched by PCR using amplification reaction reagents, and then purified and quality checked to form the final library.

[0052] (5) DNA sequencing reaction: The reversible terminal termination sequencing method was used for the sequencing reaction;

[0053] (6) Data analysis.

[0054] Compared with the prior art, the advantages of the present invention are as follows:

[0055] 1. This invention belongs to the field of genetic disease gene detection technology, specifically involving a highly efficient detection system based on the optimization of target gene variant profiles and sequence characteristics. It aims to comprehensively and accurately detect multiple types of variants in four pathogenic genes—RPGR, RP2, RS1, and CHM—of X-linked inherited retinal degenerative diseases (XLIRDs). The specific technical innovations are as follows:

[0056] 1) Precise design targeting the mutation spectrum of target genes: This invention targets four pathogenic genes, RPGR, RP2, RS1 and CHM, and comprehensively analyzes their mutation spectrum and sequence characteristics to optimize the amplification region range and design a high-density capture probe set that includes exons, introns, UTR regions and upstream and downstream 20kb regions.

[0057] 2) Specifically targeting the high GC content region of exon 15 (ORF15 region) of the RPGR gene, the probe sequence and capture strategy were adjusted to achieve efficient capture of the high GC region, overcoming the problems of low detection efficiency and uneven coverage of traditional techniques.

[0058] 3) In the library PCR enrichment stage, the amplification enzymes in the PCR reaction system are screened and optimized: combined with the above probe set, multiple amplification enzyme systems are used. Through screening by quality parameters such as sequencing data coverage, uniformity and accuracy, the most suitable amplification enzyme combination for the gene system is finally optimized, which significantly improves amplification efficiency and detection accuracy.

[0059] 4) One-step accurate detection of multiple variant types: Utilizing optimized kits and high-throughput sequencing technology, it can simultaneously detect point mutations, small fragment insertions / deletions, large fragment copy number variations, and complex structural variations (such as break sites).

[0060] 5) Compared with traditional technologies, this invention achieves high throughput, full coverage and high accuracy in mutation detection, greatly improving detection efficiency, simplifying the process and reducing costs.

[0061] 2. This invention, through in-depth analysis and optimization of the target gene variant spectrum and sequence characteristics, designs an efficient and accurate amplification system. Combined with high-throughput sequencing technology, it achieves comprehensive detection of multiple types of variants in XLIRD pathogenic genes, significantly improving detection efficiency and accuracy. Compared with existing technologies, this invention has the following outstanding advantages:

[0062] 1) Simplify the testing process and reduce experimental costs;

[0063] 2) Improve the mutation detection rate and coverage uniformity, especially achieving breakthroughs in the amplification and detection of high GC regions;

[0064] 3) It possesses flexibility and potential for personalized applications, providing a powerful tool for the accurate diagnosis and personalized treatment of hereditary retinal diseases.

[0065] Specifically, this is reflected in:

[0066] Compared to whole-genome sequencing (WGS): 1) This kit addresses the specific detection needs of XLIRD pathogenic genes, avoiding data redundancy in WGS detection and reducing reagent and sequencing costs; 2) This kit significantly improves the detection rate of point mutations and small variants through high-depth sequencing of specific regions, especially in terms of detection accuracy in high GC regions such as RPGR and ORF15, which is superior to WGS.

[0067] Compared to whole exome sequencing (WES): 1) This kit covers non-coding region variations and structural variations that WES cannot detect, especially the accurate localization of structural variation breakpoints; 2) In the detection of complex variations in XLIRDs, this kit can provide comprehensive gene mutation analysis, filling the blind spots of WES technology.

[0068] Compared to Sanger sequencing: 1) This kit offers advantages of high throughput and high efficiency, enabling simultaneous detection of multiple genes and variant types, thus avoiding the low-throughput limitations of Sanger sequencing. 2) It provides more comprehensive genetic evidence for the diagnosis and differential diagnosis of XLIRDs.

[0069] 3. This invention's kit innovatively integrates a synergistic optimization strategy of amplification enzymes, probes, and library construction processes, successfully overcoming the long-standing technical bottlenecks in the field of ophthalmic disease gene detection, namely low efficiency of long fragment amplification and uneven coverage of complex regions. Ultimately, it achieves highly uniform coverage and high-fidelity amplification of target genes in NGS detection. Furthermore, this kit is a complete system capable of one-step detection of multiple types of variants, and the probe set is a high-density probe set targeting the entire genome (including high-GC regions) and upstream and downstream 20kb regions of four pathogenic genes. Attached Figure Description

[0070] Figure 1 This represents the average sequencing depth corresponding to different probe usage amounts in this invention.

[0071] Figure 2 This is a comparison chart showing the capture and amplification effects of the three amplification enzymes—Watchmaker, KAPA, and TIANGEN—combined with probes in this invention on high GC regions.

[0072] Figure 3 This is a screenshot of IGV detection at a single point of base mutation in Example 1 of the present invention.

[0073] Figure 4 This is a comparison of other NGS detection results in Embodiment 1 of the present invention.

[0074] Figure 5 This is a screenshot of the IGV detection of microdeletion mutations in Example 2-1 of the present invention.

[0075] Figure 6 This is a comparison of the Sanger sequencing detection results in Example 2-1 of the present invention.

[0076] Figure 7 This is a screenshot of the IGV detection of base microdeletion mutations in Example 2-2 of the present invention.

[0077] Figure 8 This is a comparison of the Sange sequencing detection results in Embodiment 2-2 of the present invention.

[0078] Figure 9 This is a screenshot of IGV detection for large fragment deletion mutations in Embodiment 3-1 of the present invention.

[0079] Figure 10 This is a comparison of the detection results of other NGS sequencing methods in Example 3-1 of the present invention.

[0080] Figure 11 This is a screenshot of IGV detection for large fragment deletion mutations in Embodiment 3-2 of the present invention.

[0081] Figure 12 This is a comparison of qPCR detection results in Example 3-2 of the present invention.

[0082] Figure 13 This is a comparison of the capture results of high GC regions by three amplification enzymes, WES and WGS, in Example 4 of this invention. Detailed Implementation

[0083] The present invention will now be described in detail with reference to the accompanying drawings and embodiments:

[0084] The reagents (preferred) and alternative reagents (optional) used in this invention are shown in the following reagent table:

[0085] Reagent Table

[0086]

[0087] This invention provides a probe set for detecting pathogenic genes of X-chromosome-linked hereditary retinal degenerative diseases. The probe set includes capture probes (hereinafter named: XLIRDs gene capture probes) capable of simultaneously and specifically capturing the pathogenic genes RPGR, RP2, RS1, and CHM. The probe set includes high-density capture probes for exons, introns, UTR regions, and upstream and downstream 20kb regions. Among them, the capture probes specifically capturing the RPGR pathogenic gene include probes with sequences as shown in SEQ ID NO.1-96, and the capture probes specifically capturing the RPGR pathogenic gene include capture probes targeting the high GC regions of the RPGR gene, with sequences as shown in SEQ ID NO.24-55.

[0088] Capture probes specifically targeting the RS1 pathogenic gene include probes with sequences shown in SEQ ID NO. 97-149; capture probes specifically targeting the RP2 pathogenic gene include probes with sequences shown in SEQ ID NO. 150-193; and capture probes specifically targeting the CHM pathogenic gene include probes with sequences shown in SEQ ID NO. 194-282. Detailed capture probe sequences for the RPGR, RP2, RS1, and CHM pathogenic genes are shown in the probe sequence listing below.

[0089] probe sequence listing

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099] A kit for detecting pathogenic genes of X-chromosome-linked hereditary retinal degenerative diseases, comprising a pre-library construction reagent, a library hybridization reagent, a post-hybridization capture reagent, and a library PCR enrichment amplification reaction reagent;

[0100] The preliminary library construction reagents include enzyme digestion and fragmentation, end repair and A-tailing working solution, adapter ligation reagents, and preliminary library amplification reagents.

[0101] The enzyme digestion and fragmentation, end repair and A-tailing working solution includes an end repair enzyme and a fragment end repair buffer, and the specific composition of 10 μL of the enzyme digestion and fragmentation, end repair and A-tailing working solution is shown in the table below:

[0102]

[0103] The connector bonding reagent includes a connector premix and a universal short connector;

[0104] The pre-library amplification reagent includes paired-end tagged primers and a mixed reaction solution for library amplification, and the pre-library amplification system established by the pre-library amplification reagent is as follows:

[0105]

[0106] Among them, the purified product after adapter ligation is the product obtained after purifying the DNA sample, digesting and fragmenting it with enzymes, repairing the ends, adding an A tail, ligating the adapter, and purifying it.

[0107] The reagents used for library hybridization include the probe set for detecting pathogenic genes of X-chromosome-linked hereditary retinal degenerative diseases, namely XLIRDs gene capture probes.

[0108] The library hybridization reagents also include hybridization buffer, hybridization enhancer, universal blocking sequence mixture, and NF-water; the specific composition of the 19 μL hybridization mixture prepared by mixing the various library hybridization reagents is shown in the table below:

[0109]

[0110] The amplification reaction reagents include a hot-start amplification reaction solution and a premixed amplification primer solution; wherein, the hot-start amplification reaction solution contains an amplification enzyme, which is one of KAPA HiFi HotStart Ready Mix (2X), Watchmaker Equinox Amplification Master Mix (2X), Takara Ex Premier DNAPolymerase, and TIANGEN 2x HiFi PCR Master Mix. After screening and optimization, the amplification enzyme used in this invention is TIANGEN 2x HiFi PCR Master Mix.

[0111] The 50 μL PCR reaction system established using the amplification reaction reagents is as follows:

[0112]

[0113] The DNA suspension with magnetic beads was obtained by capturing the library after hybridization.

[0114] The method for detecting pathogenic genes of X-chromosome-linked hereditary retinal degenerative diseases using the kit described in this invention includes the following specific steps:

[0115] This kit is designed for use with human EDTA-anticoagulated whole blood. The following methods are recommended for sample collection, genomic DNA extraction, and sample transportation and preservation:

[0116] (1) Sample type: Human EDTA anticoagulated whole blood.

[0117] (2) DNA sample purification requirements: Applicable to library construction of 50ng-200ng human genomic DNA, and targeted capture and enrichment of gene fragments in target regions. For DNA samples with high degradation, it is recommended to use a starting amount of no less than 100ng for library construction. Sample concentration can be accurately quantified using fluorescent dye methods (such as Qubit), and the 260nm absorbance method is not recommended.

[0118]

Test Methods

[0119] 1. Pretext library construction

[0120] 1.1 Preparations before building the pretext library

[0121] 1.1.1 Purify magnetic beads and deionized (NF) water by letting them stand at room temperature for 30 minutes beforehand; mix the DNA and then centrifuge briefly; take the end repair enzyme and end repair buffer reagents and thaw them on ice for later use.

[0122] 1.1.2 Preparation of fresh 80% ethanol.

[0123] 1.2 DNA template purification

[0124] 1.2.1 Arrange the DNA samples according to the library construction task order, and add NF water and 50ng to 200ng DNA sample to the PCR 8-tube in sequence. The total volume of the sample and NF water is 30μL.

[0125] 1.2.2 Purification process:

[0126] 1) Add 54 μL of purified magnetic beads (1.8×) to each sample, seal, vortex to mix, and let stand for 5 min.

[0127] 2) After instantaneous centrifugation, place the solution on a magnetic rack and wait for the solution to clarify. Discard the supernatant and keep the magnetic beads.

[0128] 3) Add 200 μL of freshly prepared 80% ethanol, let stand for 30 seconds, and discard the supernatant.

[0129] 4) Repeat step 3) twice.

[0130] 5) After sealing, centrifuge briefly, place on a magnetic rack, wait for the solution to clarify, and discard the remaining liquid using a small-range pipette tip.

[0131] 6) Let the magnetic beads dry at room temperature (about 5 minutes).

[0132] 7) Add 42 μL of NF water, seal the container, vortex to mix, and let stand at room temperature for 5 minutes.

[0133] 1.2.3 Place the above PCR 8-tube set on a magnetic rack, let it stand until the solution becomes clear, and then transfer 40 μL of supernatant to a new tube.

[0134] 1.3 Enzyme digestion and fragmentation, end repair, and addition of A-tails

[0135] After thawing, mix the end-repair enzyme and end-repair buffer reagents, centrifuge briefly, and place on an ice box. Remove a 1.5 mL EP tube, label it, and place it on ice. Prepare the enzyme digestion fragmentation, end-repair, and A-tailed working solution according to the proportions in the table below, based on the number of samples:

[0136]

[0137] Gently tap or invert the tube 5-10 times to mix the working solution. After brief centrifugation, add 10 μL to the 40 μL purified sample prepared in the previous step, making the total reaction volume 50 μL. Tightly cap the tube, vortex to mix, and briefly centrifuge. Place the tube in a PCR instrument and run the following enzyme digestion, fragmentation, end-repair, and A-tail addition reaction program:

[0138]

[0139] For high-quality genomic DNA, it is recommended to set the enzyme digestion time according to the following table:

[0140]

[0141] 1.4 Connector Connection

[0142] Thaw the ligation premix and universal short connector on an ice pack. After thawing, invert the ligation premix to mix thoroughly. Vortex the universal short connector for 10 seconds, then briefly centrifuge. Add 5 μL of universal short connector to each 50 μL sample (after A-tailing correction), cap the tube, vortex, and briefly centrifuge. Add 20 μL of ligation premix to the above mixture, cap the tube, vortex, and briefly centrifuge. The total reaction volume is 75 μL. Place the tube in a PCR instrument and run the following connector ligation program:

[0143]

[0144] 1.5 Purification after adapter ligation

[0145] Add 60 μL of purified magnetic beads (0.8×) to each 75 μL sample after connecting the connector, seal the sample, shake to mix, and let stand at room temperature for 5 min. Purify according to steps 2)-6) in 1.2.2. After purification, wash the sample with 17 μL of NF water, separate the magnetic beads, and take 15 μL of supernatant into a new tube, which is the purified product.

[0146] 1.6 Preliminary Library Amplification

[0147] The library amplification mixture is the amplification reaction solution, which includes amplification enzymes (i.e., DNA polymerases). This invention describes the use of amplification reaction solutions containing four amplification enzymes for pre-library amplification. The four amplification enzymes are: ①KAPA HiFi HotStart Ready Mix (2X) (hereinafter referred to as KAPA amplification enzyme), ②Watchmaker Equinox Amplification Master Mix (2X) (hereinafter referred to as Watchmaker amplification enzyme), ③Takara Ex Premier DNA Polymerase (hereinafter referred to as Takara amplification enzyme), and ④TIANGEN 2x HiFi PCR Master Mix amplification enzyme (hereinafter referred to as TIANGEN amplification enzyme). Experiments showed that TIANGEN amplification enzyme was preferred, and it produced the highest concentration of PCR products.

[0148] Prepare the following reaction on ice:

[0149]

[0150] Note: Record the tag primer number corresponding to each sample. Only one type of tag primer should be added to each reaction tube. The tag primers used for the same batch of tests should not be repeated.

[0151] After preparing the solution, cap the tubes, vortex to mix, and briefly centrifuge. Place the tubes in a PCR instrument and run the following PCR amplification program:

[0152]

[0153] Recommended table for pre-library construction amplification cycle number:

[0154]

[0155] 1.7 Purification of Amplification Products

[0156] Add 50 μL of purifying magnetic beads (1×) to each tube of amplification product, seal the tube, shake to mix, and let stand at room temperature for 5 min. Purify according to steps 2)-6) in 1.2.2. After purification, elute the sample with 62 μL of NF water and store at 20℃ for 1 month.

[0157] 1.8 Pretext Library Quality Inspection

[0158] 1.8.1 Qubit Quantization

[0159] Use nucleic acid quantification reagents, such as the Qubit dsDNA HS Assay Kit and its accompanying instruments, to determine the concentration of the sample DNA library. The calculated total library volume should be greater than 300 ng. Otherwise, the library preparation sample does not meet the requirements and should be reconstructed.

[0160] 1.8.2 Fragment Quality Inspection

[0161] Use capillary electrophoresis reagents and instruments for fragment quality control. The library fragments should be between 300-500 bp, without obvious small or large fragment peaks. Otherwise, the library construction sample does not meet the requirements and should be reconstructed.

[0162] 1.9 Pre-library amplification results:

[0163] Four amplification enzymes—Takara, KAPA, Watchmaker, and TIANGEN—were used for pre-library amplification. Under the same conditions, the concentration of the amplified product obtained by Takara enzyme was too low to meet the requirements for further library construction. Among the other three amplification enzymes, TIANGEN enzyme produced the highest PCR product concentration (see Tables 1-5), and its amplification effect was superior to the other two enzymes.

[0164] Table 1. Statistical Analysis Results of Pretext Library Concentration

[0165]

[0166] Table 2. Concentrations of PCR products from Takara amplification enzyme used for pre-library construction

[0167]

[0168] Table 3. Concentrations of PCR products from KAPA amplification enzyme used for pre-library construction

[0169]

[0170] Table 4. Concentrations of PCR products from TIANGEN amplification enzyme used for pre-library construction

[0171]

[0172] Table 5. Concentrations of PCR products from Watchmaker amplification enzyme used for preliminary library construction

[0173]

[0174] 2-Library Hybridization

[0175] 2.1 Creating a Mixed Document Library Task Sheet

[0176] When genetic pedigree samples were included, members of the same pedigree were located in the same mixed library, and there were no duplicates in the same mixed library index. The number of hybridization samples was 12. The total amount of pre-library was 3.6 μg and it was mixed with equal mass.

[0177] 2.2 Mixed Library

[0178] The mixing procedure requires double verification. Following the library mixing task sheet, sequentially pipette the pre-libraries into new 1.5mL centrifuge tubes and record the total volume of each mixed library. Thaw the blocking agent beforehand, add 5μL of blocking agent to each mixed library, vortex to mix, and briefly centrifuge, ready for subsequent purification steps.

[0179] 2.3 Preparation of hybridization mixture

[0180] 1) Optimization test of probe dosage: In order to determine the optimal probe dosage, the inventors conducted optimization tests on the probe dosage:

[0181] Sample information: 7 positive, 3 negative, 2 NA12878, totaling 12 samples. Probe gradient testing: 1uL, 2uL, 4uL. Total throughput: 0.5Gb.

[0182] Experimental results show that there is no difference in the final library fragment size with different probe dosages, but the total size increases with increasing probe dosage.

[0183] Sequencing results showed no significant differences in data quality control indicators across different probe dosages. However, there were significant differences in average coverage within the target region (bed) files; that is, higher probe dosage resulted in higher coverage. Figure 1 (Table 6-8)

[0184] Table 6. Sequencing Data Quality Control

[0185]

[0186] Table 7. Tests with different gradient probes:

[0187]

[0188] Table 8. Statistical Analysis Results of Probe Gradient Test

[0189]

[0190] Based on the above optimization test results, the experiment determined that the amount of XLIRDs gene capture probe used should be 4 uL.

[0191] 2) Preparation of hybridization mixture: Take 0.2 mL of PCR tube labeling solution, and prepare the hybridization mixture according to the table below by adding pre-thawed hybridization buffer, hybridization enhancer, universal blocking sequence mixture, and XLIRDs gene capture probe:

[0192]

[0193] Place the prepared hybridization mixture on ice for later use.

[0194] 2.4 Purification before hybridization

[0195] 1) After the purified magnetic beads have equilibrated at room temperature for 30 minutes, take 3 times the volume of the purified magnetic beads and add them to a centrifuge tube containing the mixed library and blocking agent. Vortex to mix and let stand at room temperature for 5 minutes.

[0196] 2) After instantaneous centrifugation, place the solution on a magnetic rack and wait for the solution to clarify. Discard the supernatant and keep the magnetic beads.

[0197] 3) Add 200 μL of freshly prepared 80% ethanol, let stand for 30 seconds, and discard the supernatant.

[0198] 4) Add 200 μL of freshly prepared 80% ethanol for the second time, let stand for 30 seconds, and discard the supernatant.

[0199] 5) Centrifuge briefly, place on a magnetic rack, wait for the solution to clarify, and discard any remaining ethanol.

[0200] 6) Let the magnetic beads dry at room temperature (about 10 minutes).

[0201] 7) Add 19 μL of the hybridization mixture prepared in step 2.3, vortex to mix, and let stand for 5 min.

[0202] 8) Place on a magnetic rack and let stand until the solution becomes clear. Transfer all the supernatant (volume should be no less than 17 μL) to a new 0.2 mL PCR tube.

[0203] 9) Place the PCR tubes on the PCR instrument and prepare to run the hybridization reaction program.

[0204] 2.5 Running the hybridization reaction

[0205] Run the following hybridization reaction program on the PCR amplification instrument:

[0206]

[0207] Note: The hybridization time should generally not exceed 16 hours. Keep the sample on the PCR instrument and maintain the temperature at 65℃ before proceeding with subsequent steps.

[0208] 3. Capture after hybridization

[0209] 3.1 Preparation of washing working solution

[0210] Thaw the magnetic bead elution buffer and wash buffer in advance, and allow the captured magnetic beads to equilibrate to room temperature (30 min). Prepare the working solutions of magnetic bead elution buffer and wash buffer according to the following table based on the number of captured libraries:

[0211]

[0212] Note: Washing buffer 1 can be heated at 65°C if necessary. The prepared washing working solution can be stored at room temperature (15-25°C) for 4 weeks.

[0213] The usage volume and temperature of Wash Buffer 1 working solution and Tough Wash Buffer working solution should be as shown in the table below:

[0214]

[0215] Note: After preparing the working solutions of Washing Buffer 1 and Strict Washing Buffer, they should be preheated to 65°C immediately. The remaining buffer solutions can be left at room temperature.

[0216] 3.2 Target Fragment Capture

[0217] 1) After equilibrating the capture magnetic beads in 3.1, vortex to mix for 15 seconds. Add 80 μL of magnetic beads (per capture library) to each library in a 0.2 mL PCR tube and mix by pipetting.

[0218] 2) After instantaneous centrifugation, place on a magnetic rack until clear (~1 min), remove the supernatant, and be careful to avoid picking up the magnetic beads.

[0219] 3) Add 200 μL of magnetic bead elution buffer working solution, vortex to mix thoroughly for 10 s, centrifuge briefly, place on a magnetic rack until clear (~1 min), remove the supernatant, and be careful to avoid aspirating the magnetic beads.

[0220] 4) Repeat step 3) once.

[0221] 5) Add 100 μL of magnetic bead elution buffer working solution, vortex thoroughly for 10 s, centrifuge briefly, and then place on a magnetic rack until clear (~1 min). Discard the supernatant, taking care to avoid aspirating the magnetic beads. After brief centrifugation, use a small-range pipette tip to completely remove the supernatant.

[0222] 6) Add the hybridization mixture from the overnight hybridization reaction in step 2.5 (try to operate on a PCR instrument at 65℃), and mix by pipetting 10 times.

[0223] 7) Place the 0.2 mL PCR tube containing the capture magnetic beads and hybridization mixture in a 65°C container for 45 min. Vortex the tube every 15 min during the reaction to prevent the magnetic beads from settling or splashing onto the PCR tube cap.

[0224] Note: You must proceed to the next step immediately after completing this step.

[0225] 3.3 Post-capture rinsing and elution

[0226] 1) Add the above reaction products to a 0.2 mL PCR tube containing 100 μL of washing buffer 1 working solution preheated to 65 °C, and mix thoroughly by pipetting (preferably on a metal bath at 65 °C).

[0227] 2) After instantaneous centrifugation, place on a magnetic rack until clear (~1 min), remove the supernatant, and be careful to avoid picking up the magnetic beads.

[0228] 3) Add 200 μL of preheated (65°C) strict washing buffer working solution, mix by pipetting 10 times, and let stand at 65°C for 5 min.

[0229] 4) After instantaneous centrifugation, place on a magnetic rack until clear (~1 min), remove the supernatant, and be careful to avoid picking up the magnetic beads.

[0230] 5) Repeat steps 3) to 4) once.

[0231] 6) Add 200 μL of washing buffer 1 working solution, mix by pipetting 10 times, and shake at 2000 rpm for 2 min at room temperature.

[0232] 7) After a brief centrifugation, place the sample on a magnetic rack until it becomes clear (~1 min), then remove the supernatant, being careful to avoid picking up the magnetic beads.

[0233] 8) Add 200 μL of washing buffer 2 working solution, mix by pipetting 10 times, and shake at 2000 rpm for 1 min at room temperature.

[0234] 9) After brief centrifugation, place on a magnetic rack until clear (~1 min), remove the supernatant, and be careful to avoid picking up the magnetic beads.

[0235] 10) Add 200 μL of washing buffer 3 working solution, mix by pipetting 10 times, and then shake at 2000 rpm for 30 seconds at room temperature.

[0236] 11) After instantaneous centrifugation, place on a magnetic rack until clear (~1 min), remove the supernatant, and be careful to avoid picking up the magnetic beads.

[0237] 12) Remove the centrifuge tube from the magnetic rack, centrifuge briefly, then place it back on the magnetic rack for ~30s. Use a 10μL pipette to remove as much residual liquid as possible.

[0238] 13) Add 20 μL of NF water, vortex to mix thoroughly, centrifuge briefly, and then perform PCR with magnetic beads.

[0239] Note: After washing with working solutions of washing buffer 2 and washing buffer 3, the magnetic beads should be observed to disperse when placed in the magnetic rack. If they do not disperse, it is necessary to review the reagents and procedures for errors. If this phenomenon is not observed, subsequent experiments will likely fail, resulting in low final library yield.

[0240] 3.4 Library PCR Enrichment and Purification

[0241] 3.4.1 Library PCR Enrichment

[0242] To meet the specific needs of gene testing related to ophthalmic diseases, this invention focuses on the full-length detection of four key target genes. These genes exhibit unique sequence characteristics, including GC content bias, repetitive sequences, and complex secondary structures. A typical example is the RPGR gene, which is rich in high-GC regions and long repetitive sequences. However, traditional NGS universal amplification enzymes often exhibit bottlenecks in amplification efficiency and fidelity when dealing with such regions. Therefore, it is urgent to systematically screen and discover specific PCR amplification enzymes suitable for the system of this invention, thereby significantly improving the amplification uniformity and fidelity of target genes, especially for detection applications with a limited number of target genes but significant sequence characteristics. The screening process for different amplification enzymes in this invention is as follows:

[0243] 1) Optimization experiment with different amplification reaction enzymes:

[0244] The amplification reaction solution in Reagent I and Reagent III of this invention are of the same composition. This invention uses KAPA HiFi HotStart Ready Mix (2X) (hereinafter referred to as KAPA amplification enzyme), Watchmaker Equinox Amplification Master Mix (2X) (hereinafter referred to as Watchmaker amplification enzyme), Takara Ex Premier DNA Polymerase (hereinafter referred to as Takara amplification enzyme), and TIANGEN 2x HiFi PCR Master Mix (hereinafter referred to as TIANGEN amplification enzyme) as candidate amplification enzymes in the amplification reaction solution (i.e., the hot-start DNA polymerases listed in the reagent list). Twenty positive samples and four negative samples were selected, and the operation was performed according to the "XLIRDs Gene Mutation Detection Kit (Reversible Termination Sequencing Method) Product Instructions for Use." The reaction systems and amplification conditions for the four enzymes were consistent with the instructions.

[0245] Thaw the hot-start amplification reaction solution, amplification primer premix, and reagents at room temperature. Prepare the PCR reaction system in a 0.2 mL PCR tube according to the table below:

[0246]

[0247] Run the following program on the PCR amplification instrument:

[0248]

[0249] Note: PCR programs require preheating.

[0250] Experimental results showed that different amplification enzymes resulted in significant differences in the total amount of the final library, as shown in Tables 9 and 10. Furthermore, sequencing of the amplification products and analysis of the sequencing data revealed that the data generated by the TIANGEN amplification enzyme was more accurate, and the TIANGEN amplification enzyme exhibited the highest amplification efficiency for regions with high GC content (Tables 11-12).

[0251] Table 9: Final library volume (ng / µl) for different amplification enzymes

[0252]

[0253] Statistical results showed that among the libraries constructed by different amplification enzymes, Takara Ex Premier DNA polymerase had the lowest pre-library yield under the same number of amplification cycles (7 cycles), and could not be used for final library enrichment. Among the three amplification enzymes KAPA, Watchmaker, and TIANGEN, TIANGEN amplification enzyme had the highest amplification efficiency under the same conditions, and showed a significant statistical difference compared with the other two amplification enzymes (P value < 0.05).

[0254] Table 10: Statistical analysis of the total final library size for different amplification enzymes:

[0255]

[0256] Sequencing data results showed no significant differences in QC indicators, but there were significant differences in average sequencing depth and capture of high-GC regions, such as exons 14 and 15 of the RPGR gene. The library constructed using TIANGEN 2x HiFi PCRMaster Mix showed significantly improved coverage in this region, and its average sequencing depth was higher than that of the other two enzymes. Figure 2 Therefore, we have selected TIANGEN amplification enzyme as the amplification enzyme for this kit. Among them, Figure 2 This study compares the capture and amplification effects of three amplification enzymes—Watchmaker, KAPA, and TIANGEN—on high-GC regions using probes.

[0257] Table 11. Comparison of Q30 values ​​for sequencing data from the three amplases under an average sequencing depth of 500×.

[0258]

[0259] Statistical analysis results show that the data generated by the TIANGEN amplification enzyme is more accurate.

[0260] Table 12 shows the comparison of capture and amplification efficiency of the high GC region of exon 15 of the RPGR gene under an average sequencing depth of 500×.

[0261]

[0262] The comparison results show that TIANGEN amplification enzyme has the highest amplification efficiency for regions with high GC content.

[0263] 3.4.2 Library Purification

[0264] 1) Add 50 μL (1×) DNA purification magnetic beads to the PCR reaction product from step 3.4.1, vortex to mix, and let stand at room temperature for 5 min.

[0265] 2) After instantaneous centrifugation, place the solution on a magnetic rack and wait for the solution to clarify. Discard the supernatant and keep the magnetic beads.

[0266] 3) Add 200 μL of freshly prepared 80% ethanol, let stand for 30 seconds, and discard the supernatant.

[0267] 4) Add 200 μL of freshly prepared 80% ethanol for the second time, let stand for 30 seconds, and discard the supernatant.

[0268] 5) Centrifuge briefly, place on a magnetic rack, wait for the solution to clarify, and use a small-range pipette tip to remove any remaining ethanol.

[0269] 6) Let stand until the magnetic beads dry (about 5 minutes).

[0270] 7) Add 32 μL of NF water, vortex to mix, and let stand for 5 min.

[0271] 8) Place on a magnetic rack and let stand until the solution becomes clear. Transfer 30 μL of supernatant to a new 0.2 mL PCR tube, label it, and this is the prepared capture library.

[0272] 3.5 Final Library Quality Inspection

[0273] 3.5.1 Qubit Quantization

[0274] The concentration of the final library should be determined using nucleic acid quantification reagents, such as the Qubit dsDNA HS Assay Kit and its accompanying instruments. The total amount of the final library should be greater than 10 ng; otherwise, it does not meet the requirements and the library should be reconstructed.

[0275] 3.5.2 Segment Quality Inspection

[0276] Use capillary electrophoresis reagents and related instruments, such as Agilent 4150 and Qsep100, for fragment quality control. The library fragments should be between 300-500 bp, with no obvious small or large fragment peaks. Otherwise, the library preparation sample does not meet the requirements and should be reconstructed. If the library is not sequenced in time, it can be stored at -20℃ for one month.

[0277] 4 DNA sequencing reaction

[0278] The amount of data used for each sample should be no less than 0.2 Gb (500X).

[0279] 5. Data Analysis

[0280] This kit is based on high-throughput sequencing technology and uses an open-source bioinformatics analysis workflow. After data splitting, Sentieon-bwa aligns the raw data with the reference genome hg19. Picard is used for quality control, deduplication, and sorting of the aligned BAM files, and Qualimap is used for quality control of the aligned files. Sentieon-GATK is used to analyze SNVs and small fragment indels in the filtered BAM files, and ISoGTools is used for site function annotation. For CNV copy number variation analysis, the CNVexon algorithm is used for exon-based copy number analysis. For pseudogenes and homologous region genes, a self-developed misalignment homologous region optimization algorithm is used for CNV detection.

[0281] The data quality control process consists of the following two parts:

[0282] Step 1: Sample traceability and quality control, including sample gender determination, which is to determine the actual gender of the tested sample by the ratio of X to Y chromosome coverage in high-throughput sequencing data.

[0283] Step 2: Overall quality control of high-throughput sequencing, meeting the following requirements: Q30 of raw sequencing data ≥ 90%, effective depth of target region ≥ 200X, 20X coverage ≥ 99%, capture efficiency ≥ 80%, transversion rate of library samples between different batches, heterozygous to homozygous ratio, and no deviation in SNV / Indel site count.

[0284] 6 Specific Detection Examples

[0285] To demonstrate the effectiveness of the kit described in this invention, the present invention used the above method to test 20 individuals with XLIRD progenitors and carriers. DNA libraries were constructed and sequenced using the above technical solution. Sequencing results analysis showed that this kit can effectively detect gene mutations such as single-point base mutations, small deletions / duplications, and large fragment deletions.

[0286] Example 1. Screenshot of IGV detection using the kit described in this invention for single-point base mutations is shown below. Figure 3 As shown: R_17: RS1: chrx:18660182: NM_000330.4: c.617G>A(p.Trp206*), hemizygous mutation.

[0287] Comparison with WES testing:

[0288] The basic workflow of WES is as follows: Genomic DNA is extracted and library constructed using the Illumina standard protocol. Exome targeting is performed using IDT xGenExome Research Panel v2. After library preparation, paired-end 150bp (PE150) sequencing is performed on the NovaSeq platform (Illumina). The sequenced data undergoes quality control and analysis to ensure 96% of the regions reach a sequencing depth of 20x. The data is then compared with the human reference genome GRCh37 / hg19. WES results are as follows: Figure 4 As shown.

[0289] Comparative Conclusion: This representative example demonstrates that this kit can detect single-point base mutations with high efficiency and accuracy. Based on the sequencing depth being higher than that of ordinary WES, its accuracy is superior to WES. Furthermore, since the detection range of this kit for the target gene includes exons, intron regions, and gene flanking sequences, the overall performance of this kit is superior to WES and Sanger sequencing.

[0290] Example 2-1. Screenshot of IGV detection obtained by the kit described in this invention for detecting base microdeletion mutations is shown below. Figure 5 As shown: R_09: chrx:46696544-46696546:RP2:NM_006915.3:c.14_16del(p.Phe5del) hemizygous variant.

[0291] Comparison with Sanger sequencing

[0292] The basic Sanger sequencing workflow involves designing multiple primer sequences targeting the gene region to ensure full gene coverage. The target DNA fragment is sequenced using the dideoxy chain termination method (Sanger et al., 1977). The sequencing reaction consists of adding purified DNA template, specific sequencing primers (0.5 μM), and BigDye Terminator v3.1 reagent (containing fluorescently labeled ddNTPs, DNA polymerase, and buffer) to a 20 μL reaction system. The amplification program is then executed in an ABI Veriti thermal cycler (96℃ pre-denaturation for 2 min; 25 cycles × 96℃ for 10 s, 50℃ for 5 s, 60℃ for 4 min). After purifying the PCR products, electrophoresis is performed using an ABI 3730xl sequencer, and base signals are automatically identified using Sequencing Analysis v6 software.

[0293] Test results as follows Figure 6 As shown.

[0294] Example 2-2. Screenshot of IGV detection obtained by the kit described in this invention for detecting microdeletion mutations is shown below. Figure 7 As shown:

[0295] R_13:hrx:38145521-8145522:RPGR:NM_001034853.2:c.2730_2731delGG(p.Glu911Glyfs*167) hemizygous variant.

[0296] Compared to Sanger sequencing (the basic procedure is as described above), the results are as follows: Figure 8 As shown.

[0297] Example 2 Comparison Conclusion: This representative example confirms that this kit can efficiently and accurately detect microdeletion mutations, especially small variations in high-GC regions. The optimized amplification enzyme in this kit provides advantages such as high uniformity in enriching high-GC regions, making it superior to WES for mutation detection in high-GC regions. Furthermore, since this kit's detection range for the target gene includes exons, introns, and flanking sequences, its overall performance surpasses WES and Sanger sequencing, achieving full coverage of XLIRD pathogenic gene mutation types.

[0298] Example 3-1. Screenshot of IGV detection using the kit described in this invention for large fragment deletion mutations is shown below. Figure 9 As shown: R_04:chrx:85096185-85161914 is a half-synonymous deletion containing exons 10-15 of the CHM gene.

[0299] Comparison with WES testing

[0300] The WES (Web-based Memory for Detection) procedure for large deletions is as described above: genome library preparation, sequencing, and obtaining raw data are the same. For CNV analysis, ExomeDepth and CODEX tools are used to analyze coverage changes. The filtering threshold for deleted fragments typically requires a coverage decrease of more than 50% for ≥3 consecutive exons, or a continuous low-coverage region spanning >50kb. Results are as follows: Figure 10 As shown.

[0301] Example 3-2. Screenshot of IGV detection using the kit described in this invention for large fragment deletion mutations is shown below. Figure 11 As shown: R_19: chrx: 18670779-18682478 is a half-synonymous deletion containing exons 2-3 of the RS1 gene.

[0302] Comparison of qPCR detection

[0303] The basic qPCR workflow is as follows: Specific primers are designed for the target gene sequence. After obtaining the genomic DNA to be tested, it is simultaneously detected with a template-free negative control and a known normal sample. A 20 μL reaction system is prepared, containing 10 μL of premixed buffer (containing SYBR Green I dye, hot-start DNA polymerase, dNTPs, and optimization buffer), 1 μL of forward and reverse primers (final concentration 0.2 μM), 2 μL of DNA template (approximately 10 ng / μL), and 7 μL of sterile ultrapure water. Amplification is then performed using a real-time quantitative PCR instrument, with the program set as follows: 95℃ pre-denaturation for 30 seconds; cycles of 95℃ for 5 seconds and 60℃ for 30 seconds (40 cycles in total); finally, melting curve analysis (60℃ to 95℃, continuous temperature increase at a rate of 0.1℃ / second) is performed to verify product specificity. Fluorescence signals are acquired in real-time through the SYBR Green channel, and ROX dye is used as an internal control to correct inter-well fluorescence fluctuations. Amplification efficiency is measured using the standard curve method (R...). 2 >0.98) verification: The Ct value difference between the target region and the internal reference gene was compared using the ΔΔCt method. If the Ct value of the target region was significantly higher than that of the control sample (e.g., ΔCt difference ≥1), it indicated that copy number deletion existed in that region. The relative quantification (RQ) was then calculated by comparing it with the normal control. Results are as follows: Figure 12 As shown.

[0304] Example 3 Comparison Conclusion: This representative example confirms that this kit can efficiently and comprehensively detect large deletion mutations in genes, especially breakpoints in intron regions. WES only has high sensitivity at the exon level (CNV), and qPCR is not the preferred method for routine clinical testing. This kit significantly outperforms WES and qPCR sequencing, achieving full coverage of XLIRD pathogenic gene mutation types.

[0305] Example 4 (6 random samples). Results of effective capture and amplification of the high GC region of exon 15 of the RPGR gene obtained by the kit described in this invention.

[0306] The inventors also replaced the TIANGEN amplification enzyme in the kit with KAPA and Watchmaker amplification enzymes for comparative experiments. Simultaneously, they compared the detection method established using this kit with the WES and WGS methods for capturing high-GC regions. The results of capturing high-GC regions using the three amplification enzymes compared to WES and WGS are as follows: Figure 13 As shown.

[0307] Example 4 Comparison Conclusion: Capturing and amplifying high-GC regions is a challenge in high-throughput sequencing. WES has low capture and amplification efficiency in high-GC regions, resulting in poor overall data uniformity. WGS is similarly limited in high-GC regions, exhibiting low accuracy and uniformity. However, the kit using the optimized TIANGEN amplification enzyme and an effective probe set developed in this patent can efficiently amplify and enrich high-GC regions, demonstrating significantly better performance than WES and WGS in detecting mutations in these regions. Figure 13 The sample names in the text represent, in order: library construction and sequencing using TIANGEN, KAPA, and Watchmaker amplases, and capture sequencing results for high GC regions using WES and WGS.

[0308] Table 13: Detection results of 20 positive samples

[0309] Experiment_ID Gender Mutation Gene Chr_Location Results R_01 M Yes RPGR X：38146015-38146016 NM_001034853.2: c.2236_2237delGA(p.Glu746Argfs*23), Hemi R_02 F Yes RPGR X：38146015-38146016 NM_001034853.2:c.2236_2237delGA(p.Glu746Argfs*23),Hete R_03 M Yes CHM X：85218827 NM_000390.4: c.545del(p.Cys182Leufs*15), Hemi R_04 M Yes CHM X：85096185-85161914 chrX: 85096185-85161914del, CHM: exon10-15del, Hemi R_05 M Yes CHM X：85212923 NM_000390.4: c.877C>T(p.Arg293*), Hemi R_06 M Yes RS1 X：18662583 NM_000330.4: c.489del(p.Trp163*), Hemi R_07 M Yes RP2 X：46715956-46723515 chrX: 46715956-46723515del, RP2: exon3del, Hemi, R_08 F Yes RP2 X：46715176-46725075 chrX: 46715176-46725075del, RP2: exon3del, Hete R_09 M Yes RP2 X：46696544-46696546 NM_006915.3:c.14_16del(p.Phe5del),Hemi R_10 F Yes RP2 X：46696544-46696546 NM_006915.3:c.14_16del(p.Phe5del),Hete R_11 M Yes RP2 X：46713155 NM_006915.3: c.347A>G(p.Gln116Arg), Hemi R_12 F Yes RP2 X：46713155 NM_006915.3:c.347A>G(p.Gln116Arg),Hete R_13 M Yes RPGR X：38145521-38145522 NM_001034853.2: c.2730_2731delGG(p.Glu911Glyfs*167), Heml R_14 F Yes RPGR X：38145521-38145522 NM_001034853.2:c.2730_2731delGG(p.Glu911Glyfs*167)Hete R_15 M Yes RPGR X：38164016 NM_001034853.2: c.806G>A(p.Gly269Glu), Hemi R_16 F Yes RPGR X：38164016 NM_001034853.2: c.806G>A(p.Gly269Glu), Hete R_17 M Yes RS1 X：18660182 NM_000330.4: c.617G>A(p.Trp206*), Hemi R_18 F Yes RS1 X：18660182 NM_000330.4:c.617G>A(p.Trp206*),Hete R_19 M Yes RS1 X：18670779-18682478 chrX:18670779-18682478del,RS1:exon2-3del,Hemi R_20 F Yes RS1 X：18670779-18682478 chrX:18670779-18682478del,RS1:exon2-3del,Hete

[0310] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A kit for detecting pathogenic genes of X-chromosome-linked hereditary retinal degenerative diseases, characterized in that: The kit includes a library hybridization reagent, which includes a probe set for detecting pathogenic genes of X-chromosome-linked hereditary retinal degenerative diseases. The probe set includes capture probes that specifically capture the pathogenic genes RPGR, RP2, RS1, and CHM; wherein, the capture probes that specifically capture the pathogenic gene RPGR include probes with sequences as shown in SEQ ID NO.1-96, and the capture probes that specifically capture the pathogenic gene RPGR include capture probes targeting the high GC region of the RPGR gene, with sequences as shown in SEQ ID NO.24-55. The capture probes that specifically capture the RS1 pathogenic gene include probes with sequences as shown in SEQ ID NO. 97-149; the capture probes that specifically capture the RP2 pathogenic gene include probes with sequences as shown in SEQ ID NO. 150-193; and the capture probes that specifically capture the CHM pathogenic gene include probes with sequences as shown in SEQ ID NO. 194-282. The kit includes a pre-library amplification reagent, which includes a library amplification mixing solution, and the library amplification mixing solution includes TIANGEN 2x HiFi PCR Master Mix; The kit also includes amplification reaction reagents for library hybridization capture followed by library PCR enrichment, including TIANGEN 2x HiFi PCR Master Mix.

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