A probe combination, a kit and a detection method for detecting a human polynucleotide repeat expansion mutation STR site

Abstract

The present application relates to a kind of probe combination, kit and detection method for detecting human polynucleotide repeat expansion mutation STR site.The probes in the probe combination meet the following requirements: ①the length of probe is 120bp, and GC content is 40%-60%;②according to the GC content of the probe hybridization capture region of the related gene of the human polynucleotide repeat expansion mutation STR site to be detected, the probe density of the corresponding region is matched;wherein, when the GC content of the hybridization capture region is 25%-75%, the probe density of the hybridization capture region is 0.20-0.24;when the GC content of the hybridization capture region is less than 25% or more than 75%, the probe density of the hybridization capture region is 0.44-0.48.The present application realizes the comprehensive and effective detection of human polynucleotide repeat expansion mutation STR site, improves the detection ability of polynucleotide repeat sequence, and can provide more accurate polynucleotide repeat number of polynucleotide repeat sequence, which can guide molecular typing, genetic risk assessment, etc.

Description

Technical Field

[0001] This invention relates to the field of gene detection technology, and in particular to a probe combination, kit, and detection method for detecting human polynucleotide repeat extension mutation (STR) sites. Background Technology

[0002] Human polynucleotide repeat expansion mutation diseases, also known as dynamic mutation diseases, mainly include typical genetic diseases such as Huntington's disease, spinocerebellar ataxia, and Fragile X syndrome. The pathogenesis of these diseases involves pathological abnormal expansion of polynucleotide repeat units such as CAG, CTG, GAA, GGC, GGCCTG, and GGGGCC in the human genome. In normal individuals, the copy number of these polynucleotide repeat sequences in the genome remains within a specific threshold range. When the copy number exceeds the normal threshold and pathological expansion occurs, the corresponding genetic disease can be induced. Currently, nearly 70 human polynucleotide repeat expansion mutation diseases have been scientifically and clinically confirmed, covering a wide range of disease types including ataxia, myopathy, myoclonic epilepsy, chorea, Fragile X syndrome, and congenital developmental disorders, demonstrating a broad clinical coverage.

[0003] Epidemiological data show that the overall incidence of polynucleotide repeat expansion mutation diseases can reach 1 / 3000, falling into the category of highly prevalent hereditary diseases. However, due to limitations in current testing technologies, the clinical diagnosis rate of these diseases is low, and the false negative rate is high. Most patients cannot achieve early and accurate diagnosis, severely impacting clinical diagnosis and intervention. Currently, mainstream testing technologies all have significant technical shortcomings, making it difficult to meet the needs of large-scale, accurate clinical testing. Traditional Southern blotting technology can only detect single STR loci one by one, resulting in low throughput and poor overall efficiency, failing to meet the needs of multi-locus batch screening. While PCR combined with capillary electrophoresis can detect some conventional STR loci, it suffers from amplification failure and poor specificity for polynucleotide expansion loci with high repeat counts, as well as STR loci corresponding to high GC content repeat units such as GGC, CCG, and GGGGCC. This makes it impossible to accurately determine the polynucleotide repeat copy number, severely limiting the accuracy and applicability of the detection. This is currently the core technical challenge hindering the accurate detection of dynamic mutation diseases. While third-generation whole-genome long-read sequencing technology can overcome the technical bottlenecks of traditional detection methods and effectively identify various polynucleotide repeat expansion mutation sites, whole-genome sequencing is extremely expensive, the detection process is complex, and the cost-effectiveness and accessibility are extremely poor, making it difficult to apply on a large scale to clinical screening, batch sample testing, and scientific research screening scenarios.

[0004] In summary, there is currently a lack of a low-cost, highly versatile, highly accurate, and efficient technical solution for detecting human polynucleotide repeat extension mutation (STR) sites in the clinical and research fields. Therefore, developing a combination of STR site detection probes and a supporting detection system that is compatible with multiple types of STR sites, provides accurate detection, and is cost-effective has significant clinical value and research significance. Summary of the Invention

[0005] The purpose of this invention is to provide a probe combination, kit, and detection method for detecting human polynucleotide repeat expansion mutation (STR) sites, solving the problems of missed detection, amplification failure, high cost, and low detection efficiency in the prior art, and providing a stable, low-cost, and highly versatile set of detection tools and processes for scientific research screening and sample typing of genetic diseases related to polynucleotide repeat expansion mutations.

[0006] A first aspect of the present invention provides a probe array for detecting human polynucleotide repeat extension mutation (STR) sites, characterized in that each probe in the probe array meets the following requirements: ① The probe length is 120 bp, and the GC content is 40% to 60%; ②Based on the GC content of the probe hybridization capture region of the gene related to the human polynucleotide repeat extended mutation STR site to be detected, the probe density of the corresponding region is set accordingly; Specifically, when the GC content of the hybridization capture region is 25% to 75%, the probe density of the hybridization capture region is 0.20 to 0.24; when the GC content of the hybridization capture region is less than 25% or greater than 75%, the probe density of the hybridization capture region is 0.44 to 0.48. When the GC content is too low or too high, a better effect can be achieved by increasing the probe density.

[0007] In this invention, probe density = probe length / fragment length containing probe hybridization capture region.

[0008] Specifically, the probe length is 120 bp, the GC content of the probe hybridization capture region is 25%–75%, and the probe density is 0.24. Therefore, two probes are designed for every 500 bp of the probe hybridization capture region, i.e., 0.24 = 120 bp / 500 bp. If, during the design, the probe hybridization capture region is less than 500 bp, or if a fragment is less than 500 bp due to non-divisibility by 500, then, according to common knowledge in the field, sufficient bases are selected upstream or downstream of this region / fragment to make up the 500 bp.

[0009] Preferably, when selecting upstream or downstream bases, the principle is to select those with lower GC content in the resulting sequence.

[0010] The present invention preferably designs capture probes with a probe density of 0.24 when the GC content in the probe hybridization capture region is 25% to 75%. If the GC content of the capture probe does not meet the requirement of 40% to 60% when designed with this density, the probe density can be appropriately adjusted to obtain a capture probe that meets the requirements.

[0011] The area to be detected is relatively small. In order to achieve the desired capture effect, the capture area is extended. Preferably, the probe hybridization capture area includes the area related to the human polynucleotide repeat extended mutation STR site to be detected, extended 3000bp towards the 5' end and 3000bp towards the 3' end. 3000bp is sufficient to cover the area to be detected, and the cost is moderate. Combined with third-generation sequencing technology, it can not only greatly improve the detection capability of polynucleotide repeat sequences, but also provide accurate polynucleotide repeat counts.

[0012] Preferably, the human polynucleotide repeat extended mutation STR site-related genes include: ABCD3, AFF2, AFF3, AR, ARX, ATN1, ATXN1, ATXN2, ATXN3, ATXN7, ATXN8OS, ATXN10, BEAN1, C9orf72, C11orf80, CACNA1A, CBL, CNBP, COMP, CSTB, DAB1, DIP2B, DMPK, EIF4A3, FGF14, FMR1, FOXL2, FRA10AC1, FXN, GIPC1, GLS, HOXA13, HOXD13, HTT, JPH3, KCNN3, LRP12, MARCHF6, NAXE, N One or more of the following: IPA1, NOP56, NOTCH2NLC, NUTM2B-AS1, PABPN1, PHOX2B, PLIN4, POLG, PPP2R2B, PRDM12, PRNP, RAI1, RAPGEF2, RFC1, RILPL1, RUNX2, SAMD12, SOX3, STARD7, STRTS / MIR7-2, TAF1, TBP, TBX1, TCF4, THAP11, TMEM185A, TNRC6A, TYMS, VWA1, WDR7, XYLT1, YEATS2, ZFHX3, ZIC2, ZIC3, ZNF713, TBC1D7, FAM193B, and HSF1.

[0013] Preferably, the sex-related genes RPS4Y1 and AMELY are added. These two sex-specific Y chromosome genes can simultaneously achieve precise sex tracing of samples while completing the detection of pathogenic sites, balancing the comprehensiveness of detection with the accuracy of data tracing.

[0014] Preferably, the probe assembly includes probes as shown in Table 1.

[0015]

[0016] A second aspect of the present invention provides the application of the probe combination for detecting human polynucleotide repeat expansion mutation (STR) sites in the preparation of human polynucleotide repeat expansion mutation (STR) site detection products.

[0017] Preferably, the detection products include reagents, reagent kits, chips, software, detection systems, etc.

[0018] A third aspect of the present invention provides a kit for detecting human polynucleotide repeat extension mutation (STR) sites, comprising the probe combination for detecting human polynucleotide repeat extension mutation (STR) sites.

[0019] Preferably, the kit may further include one or more of the following: library preparation reagent, hybridization reagent, and capture reagent.

[0020] Optionally, the kit is not limited to simultaneously detecting the genes related to the above 78 human polynucleotide repeat expansion mutation STR sites, but can also detect human polynucleotide repeat expansion mutation STR sites by combining one or more genes.

[0021] A fourth aspect of the present invention provides a method for detecting human polynucleotide repeat expansion mutation (STR) sites for non-disease diagnosis and treatment purposes, comprising the following steps: S1. Extract genomic DNA from the sample to be tested; S2. The genomic DNA is fragmented to obtain fragmented DNA with a length of 6k to 10k. S3. Construct a long fragment library using the fragmented DNA; S4. The long fragment library is subjected to hybridization capture processing using the probe combination described above; through library construction in step S3 and hybridization capture in step S4, high GC content sequences can be effectively enriched. S5. Based on the captured library products, construct a PacBio sequencing library, then perform long-read sequencing and bioinformatics analysis to complete STR site detection.

[0022] Preferably, in step S1, genomic DNA is extracted from the sample using the Magnetic Universal Genomic DNA Kit.

[0023] Preferably, in step S1, the sample to be tested is a blood sample or a tissue sample.

[0024] Preferably, in step S2, 1-3 μg of genomic DNA is fragmented using a g-Tube, with a fragment length of about 10 kJ; more preferably, 1.5 μg of genomic DNA is fragmented using a g-Tube.

[0025] Preferably, step S3 specifically includes: S31. Perform end repair and deoxyadenine modification on the fragmented DNA to obtain repaired DNA; S32. Connect the repaired DNA to the sequencing adapter to obtain the DNA ligation product; S33. The DNA ligation product is amplified to prepare a long fragment library.

[0026] Preferably, in step S31, the NEBNext® Ultra™ II End Repair / dA-TailingModule is used to perform end repair on the fragmented DNA and add deoxyadenine to the 3′ end.

[0027] Preferably, in step S32, the NEBNext® Ultra™ II Ligation Module is used to connect the repaired DNA to the Barcode-Adapter.

[0028] In this invention, there are no specific restrictions on the Barcode-Adapter sequence. PacBio products can be selected or self-designed. Those skilled in the art can choose according to their needs. For example, the Barcode-Adapter sequence can be selected from any set in Table 2, and different Barcode-Adapter sequences can be used to distinguish different samples.

[0029] Table 2

[0030] Preferably, in step S33, the DNA ligation product is purified with 0.4× magnetic beads, pre-amplified with PCR polymerase, and the amplified product is purified with 0.4× magnetic beads before being used in subsequent steps.

[0031] Preferably, in step S33, the long fragment library (pre-library) is amplified using the DeCodi-Fi High-Fidelity PCR Kit.

[0032] Preferably, in step S33, the concentration of the purified product is determined using Qubit 4.0.

[0033] Preferably, in step S33, the fragment size of the purified product is determined using an Agilent 2100.

[0034] Preferably, step S4 specifically includes: S41. Hybridization and capture are performed using the probe combination and long fragment library. After hybridization and capture are completed, the target region DNA is captured from the hybridization product. S42. Amplify the captured target region DNA to obtain a capture library product that can be used for sequencing.

[0035] Preferably, in step S41, after the hybridization capture is completed, capture magnetic beads and rinsing buffer are used to capture the target region DNA of the hybridization product.

[0036] Preferably, in step S42, the captured library product is purified with 0.4× magnetic beads, amplified with PCR polymerase, and the amplified product is purified with 0.4× magnetic beads before being used in subsequent steps.

[0037] Preferably, in step S42, the long fragment library (pre-library) is amplified using the DeCodi-Fi High-Fidelity PCR Kit.

[0038] Preferably, in step S42, the concentration of the purified product is determined using Qubit 4.0.

[0039] Preferably, in step S42, the fragment size of the purified product is determined using an Agilent 2100.

[0040] Preferably, in step S5, the purified capture library product is used to construct a PacBio library using the SMRTbell® prep kit 3.0 from Pacific Biosciences. The constructed PacBio library is then sequenced using the PacBioRevio platform for long-fragment DNA. The sequencing results of the long-fragment library undergo quality control analysis, including but not limited to GC content, coverage, and average sequencing depth. Alignment to the reference genome is performed using minimap2 (v2.27-r1207-dirty), and dynamic mutations in repeat units of different lengths are analyzed using RepeatHMM and adVNTR (v1.5.0). Simultaneously, haplotype verification of dynamic mutation detection results is analyzed, and the results from various software programs are integrated to indicate the number of polynucleotide repeats at polynucleotide repeat extension mutation STR sites.

[0041] A fifth aspect of the present invention provides a system for detecting human polynucleotide repeat expansion mutation (STR) sites, comprising: Genomic DNA extraction module, used to extract genomic DNA from the sample to be tested; The DNA fragmentation module is used to fragment the extracted genomic DNA. The long fragment library construction module is used to construct long fragment libraries based on fragmented DNA. A targeted hybridization capture module is used to perform target region hybridization capture on a long fragment library using the probe combination described in any one of claims 1-4; The sequencing and data analysis module is used to perform long-read sequencing and bioinformatics analysis on the captured target region DNA to determine the STR site mutation status.

[0042] The present invention has at least the following beneficial effects: (1) This invention strictly limits the probe length to 120bp and the GC content to 40% to 60% to ensure the stability and specificity of probe hybridization. At the same time, it innovatively matches the probe density according to the difference in GC content in the hybridization capture region. Low-density probe layout is adopted in the conventional 25% to 75% GC region, and the probe arrangement is encrypted in the extremely high GC and extremely low GC regions. This completely overcomes the technical bottleneck of traditional capture technology in the poor enrichment effect and difficulty in amplification of high GC repeated expansion STR sites such as GGC, CCG, and GGGGCC. It achieves full coverage, uniform and efficient capture of STR sites with different base characteristics, and greatly reduces the probability of site missed detection.

[0043] (2) This invention covers 78 core genes related to human polynucleotide repeat expansion mutation diseases in one go, including STR sites related to various dynamic mutation diseases such as ataxia, myopathy, chorea, fragile X syndrome, and congenital developmental disorders. It breaks through the limitations of traditional technology that can only detect one or a few sites at a time and has extremely low detection throughput.

[0044] (3) This invention innovatively extends the hybridization capture region of each STR detection site by 3000bp upstream and downstream, and combines 6k-10k ultra-long genomic DNA fragmentation and long fragment library construction technology to completely preserve the complete genomic sequence upstream and downstream of the STR site, effectively avoiding the problems of repetitive sequence breakage and information loss caused by short fragment library construction. Combined with PacBio long read sequencing technology, it can accurately read the true copy number of ultra-long polynucleotide repetitive sequences, solve the defects of traditional PCR and capillary electrophoresis technology that cannot identify high repetition number STR sites, and realize accurate quantitative typing of repetition number.

[0045] (4) Compared with the disadvantages of high cost and inability to be applied in batches in third-generation whole genome sequencing, this invention adopts a scheme of targeted probe hybridization capture combined with long read sequencing, which only enriches the target pathogenic STR region for sequencing. While retaining the advantages of accurate typing of long read sequencing, it greatly reduces the amount of sequencing data and detection cost, and significantly improves the cost-effectiveness of detection. It can be widely applied to scenarios such as scientific research cohort screening, family pathogen verification, and sample source tracing analysis, breaking through the bottleneck of existing high-precision detection technology being difficult to industrialize and popularize.

[0046] (5) This invention simultaneously protects the core probe combination, detection kit, detection method and detection system, forming a standardized complete process from sample processing, library construction, targeted capture, sequencing analysis to result interpretation. The system has strong stability and repeatability. At the same time, it is compatible with a variety of test samples such as blood and tissue, and has a wide range of applicable scenarios. It can provide accurate, stable and efficient technical support for the study of the mechanism of human polynucleotide repeat expansion mutation diseases, molecular subtyping, genetic risk assessment and related drug development. It has extremely high scientific research value and clinical application prospects. Attached Figure Description

[0047] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0048] Figure 1 This is a schematic diagram of the method for detecting human polynucleotide repeat expansion mutation STR sites for non-disease diagnosis and treatment purposes provided by the present invention.

[0049] Figure 2 The images shown are agarose gel electrophoresis results of fragmented DNA in blood samples provided by this invention. From left to right, they are: Marker, blood sample 1#, blood sample 2#, and blood sample 3#.

[0050] Figure 3 The DNA pre-text library detection images of blood samples provided by this invention are shown in the following order from left to right: Marker, blood sample 1#, blood sample 2#, and blood sample 3#.

[0051] Figure 4 Bam diagram of blood sample 1# provided by the present invention.

[0052] Figure 5 Bam diagram of blood sample 2# provided by the present invention.

[0053] Figure 6 Bam diagram of blood sample 3# provided by this invention. Detailed Implementation

[0054] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0055] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form includes the plural form unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0056] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0057] Example 1 This embodiment utilizes a targeted capture probe based on a combination of 78 marker genes to perform gene detection on STR loci associated with human polynucleotide repeat expansion mutations in blood samples from three patients with high GC content that are difficult to detect. The 78 marker gene combinations include: ABCD3, AFF2, AFF3, AR, ARX, ATN1, ATXN1, ATXN2, ATXN3, ATXN7, ATXN8OS, ATXN10, BEAN1, C9orf72, C11orf80, CACNA1A, CBL, CNBP, COMP, CSTB, DAB1, DIP2B, DMPK, EIF4A3, FGF14, FMR1, FOXL2, FRA10AC1, FXN, GIPC1, GLS, HOXA13, HOXD13, HTT, J PH3, KCNN3, LRP12, MARCHF6, NAXE, NIPA1, NOP56, NOTCH2NLC, NUTM2B-AS1, PABPN1, PHOX2B, PLIN4, POLG, PPP2R2B, PRDM12, PRNP, RAI1, RAPGEF2, RFC1, RILPL1, RUNX2, SAMD12, SOX3, STARD7, STRTS / MIR7-2, TAF1, TBP, TBX1, TCF4, THAP11, TMEM185A, TNRC6A, TYMS, VWA1, WDR7, XYLT1, YEATS2, ZFHX3, ZIC2, ZIC3, ZNF713, TBC1D7, FAM193B, HSF1. Additionally, the sex-related genes RPS4Y1 and AMELY were added. The gene detection coverage area includes the STR locus-related region for diseases associated with human polynucleotide repeat extension mutations, extending 3000 bp upstream of the 5' end and 3000 bp downstream of the 3' end. The detection area is shown in Table 3, including the detected gene, chromosome (number), chromosome start position, and chromosome termination position (referencing the GRCh38 version of the genome).

[0058] Table 380 shows the detection areas and specific locations covered by the marker genes.

[0059] The flowchart of the testing steps is as follows Figure 1 As shown, it includes the following steps: 1. Extract genomic DNA from the sample to be tested. Genomic DNA was extracted from the sample according to the instructions of the Magnetic Universal Genomic DNA Kit; the specific operating procedures were strictly followed according to the instructions of the corresponding kit.

[0060] 2. Genomic DNA fragmentation 1.5 μg of genomic DNA was fragmented using a g-Tube tube, centrifuged at 7000 rpm for 1 min, then inverted the g-Tube tube and centrifuged at 7000 rpm for 1 min. The DNA fragment length was approximately 10 kb. The fragmented DNA was detected by agarose gel electrophoresis as shown in the image. Figure 2 As shown. Fragmented DNA was purified using 0.4× magnetic beads, and 27 μl of ddH2O was added to elute the fragmented DNA. The concentration of the purified product was determined using a Qubit 4.0 analyzer.

[0061] 3. Construction of long-fragment libraries 3.1 Genomic DNA end repair and deoxyadenine addition Use the NEBNext® Ultra™ II End Repair / dA-Tailing Module to perform end repair and add deoxyadenine to the 3′ end of fragmented DNA.

[0062] Configure the terminal repair and deoxyadenine-added reaction system according to Table 4 below: Table 4

[0063] Mix using a pipette (avoid vigorous shaking), then centrifuge briefly.

[0064] Place the reaction tubes in the PCR instrument and perform the following procedure: open the PCR instrument heating cap and incubate at 75°C for 30 minutes at 20°C, then at 65°C for 30 minutes, and finally at 4°C.

[0065] 3.2 Connecting connectors The NEBNext® Ultra™ II Ligation Module was used to ligate the repaired DNA into the Barcode-Adapter shown in Table 2.

[0066] Configure the connector connection reaction system according to Table 5 below: Table 5

[0067] Mix using a pipette (avoid vigorous shaking), then centrifuge briefly.

[0068] Place the reaction tubes in the PCR instrument and perform the following procedure: close the PCR instrument's hot cap, incubate at 20°C for 15 minutes, then maintain at 4°C.

[0069] 3.3 Preliminary Library Augmentation After purification with 0.4× magnetic beads, the ligation product was eluted with 22 μL of ddH2O. The ligation product was then used to amplify a pre-library using the DeCodi-FiHigh-Fidelity PCR Kit.

[0070] Configure the pre-library amplification reaction system according to Table 6 below: Table 6

[0071] Mix using a pipette (avoid vigorous shaking), then centrifuge briefly.

[0072] Place the PCR reaction tubes in the PCR instrument and perform the following procedure: open the PCR instrument heating cap and cycle at 105°C for 1 minute at 98°C; (98°C for 15 seconds; 62°C for 15 seconds; 72°C for 10 minutes) for 7 cycles; cycle at 72°C for 5 minutes; and hold at 4°C.

[0073] After purification with 0.4× magnetic beads, the pre-library amplification product was eluted with 30 μL ddH2O.

[0074] Take 1 µl of the pre-library amplification product and determine its concentration using Qubit 4.0.

[0075] Take 1 µl of the pre-library amplification product and determine the fragment size using an Agilent 2100. The results are as follows: Figure 3 As shown.

[0076] 4. Targeted capture of long-fragment libraries (pre-libraries) 4.1 Pre-library capture probe hybridization capture Take 1.5 μg of pretext library, 5 μL of DNA blocking reagent, and 10 μL of adapter blocking reagent and place them in a 1.5 mL centrifuge tube. Mix well and then evaporate to dryness in a concentrator. Add hybridization reagent (as shown in Table 7 below), mix well, and denature at 95 °C for 10 minutes.

[0077] Table 7

[0078] Then, add 4 µL of capture probe (the capture probe containing the above 80 genes shown in Table 1), mix well, and immediately perform hybridization at 65°C for 12 hours.

[0079] Furthermore, after hybridization, the target region DNA was captured using capture magnetic beads and rinsing buffer. The DNA pre-library agarose gel electrophoresis detection image is shown below. Figure 3 As shown.

[0080] 4.2 Capture Library Amplification After purification with 0.4× magnetic beads, the captured product was amplified into a pre-library using the DeCodi-Fi High-Fidelity PCR Kit.

[0081] Configure the pre-library amplification reaction system according to Table 8 below: Table 8

[0082] Mix using a pipette (avoid vigorous shaking), then centrifuge briefly.

[0083] Place the PCR reaction tubes in the PCR instrument and perform the following procedure: open the PCR instrument's hot cap and cycle at 105°C for 1 minute at 98°C; (98°C for 15 seconds; 62°C for 15 seconds; 72°C for 10 minutes) for 15 cycles; cycle at 72°C for 5 minutes; and hold at 4°C.

[0084] After purification with 0.4× magnetic beads, the capture library amplification products were eluted with 22 μL ddH2O.

[0085] Take 1 µl of the capture library amplification product and determine its concentration using Qubit 4.0.

[0086] Take 1 µl of the capture library amplification product and determine the fragment size using an Agilent 2100. The results are as follows: Figure 4 As shown.

[0087] 5. PB library construction and sequencing The purified capture library product was used to construct a PacBio library using the SMRTbell® prep kit 3.0 from Pacific Biosciences (PacBio, Cat#102-141-700). The constructed PacBio library was then sequenced using the PacBio Sequel II platform for long-fragment DNA sequencing. The sequencing results of the long-fragment library underwent quality control analysis, including but not limited to coverage and sequencing depth. The quality control criteria were: data volume ≥ 0.15G; average sequencing depth ≥ 150X. The final detection results of this embodiment are shown in Table 9 below.

[0088] Table 9

[0089] Minimap2 (v2.27-r1207-dirty) was used for alignment to the reference genome, and RepeatHMM and adVNTR (v1.5.0) were used to analyze dynamic mutations in repeat units of different lengths. Simultaneously, haplotype verification of dynamic mutation detection results was analyzed, and the results from various software programs were integrated to indicate the number of polynucleotide repeats at STR sites associated with polynucleotide repeat expansion mutations. The detection results for samples 1#, 2#, and 3# are as follows: Figure 4-6 And as shown in Table 10 below.

[0090] Table 10

[0091] Samples 1#, 2#, and 3# were tested by capillary electrophoresis, and the results were all positive. However, the number of repetitions of the corresponding GGC trinucleotide, GGGGCC hexanucleotide, and GGC trinucleotide could not be determined.

[0092] In summary, this invention targets and enriches a set of 78 marker genes associated with human polynucleotide repeat (PNR) extension mutation (STR) sites, and combines this with third-generation high-throughput sequencing (NGS) technology to identify PNRs at related gene STR sites, overcoming the limitations of commonly used NGS platforms in detecting PNRs at STR sites. Furthermore, the library construction and hybridization capture methods in this invention effectively enrich high-GC-content sequences, and can still effectively amplify STR sites with high-GC-content PNR units such as GGC, GCC, and GGGGCC. This allows for more comprehensive and accurate PNR information, guiding molecular subtyping and genetic risk assessment of related diseases, providing clearer personalized diagnostic and treatment guidelines for clinicians, and also applicable to drug development.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A probe array for detecting human polynucleotide repeat extension mutation (STR) sites, characterized in that, Each probe in the probe assembly meets the following requirements: ① The probe length is 120 bp, and the GC content is 40% to 60%; ②Based on the GC content of the probe hybridization capture region of the gene related to the human polynucleotide repeat extended mutation STR site to be detected, the probe density of the corresponding region is set accordingly; Wherein, when the GC content of the hybridization capture region is 25% to 75%, the probe density of the hybridization capture region is 0.20 to 0.24; when the GC content of the hybridization capture region is less than 25% or greater than 75%, the probe density of the hybridization capture region is 0.44 to 0.

48.

2. The probe combination for detecting human polynucleotide repeat extension mutation (STR) sites according to claim 1, characterized in that, The probe hybridization capture region includes the region obtained by extending 3000bp from the 5' end and 3000bp from the 3' end of the region related to the human polynucleotide repeat extended mutation STR site to be detected.

3. The probe combination for detecting human polynucleotide repeat extension mutation (STR) sites according to claim 1, characterized in that, The genes associated with the human polynucleotide repeat expansion mutation STR sites include: ABCD3, AFF2, AFF3, AR, ARX, ATN1, ATXN1, ATXN2, ATXN3, ATXN7, ATXN8OS, ATXN10, BEAN1, C9orf72, C11orf80, CACNA1A, CBL, CNBP, COMP, CSTB, DAB1, DIP2B, DMPK, EIF4A3, FGF14, FMR1, FOXL2, FRA10AC1, FXN, GIPC1, GLS, HOXA13, HOXD13, HTT, JPH3, KCNN3, LRP12, MARCHF6, NAXE, NIP One or more of the following: A1, NOP56, NOTCH2NLC, NUTM2B-AS1, PABPN1, PHOX2B, PLIN4, POLG, PPP2R2B, PRDM12, PRNP, RAI1, RAPGEF2, RFC1, RILPL1, RUNX2, SAMD12, SOX3, STARD7, STRTS / MIR7-2, TAF1, TBP, TBX1, TCF4, THAP11, TMEM185A, TNRC6A, TYMS, VWA1, WDR7, XYLT1, YEATS2, ZFHX3, ZIC2, ZIC3, ZNF713, TBC1D7, FAM193B, HSF1.

4. The probe combination for detecting human polynucleotide repeat extension mutation (STR) sites according to claim 1, characterized in that, The probe assembly includes probes as shown in Table 1.

5. The use of the probe combination for detecting human polynucleotide repeat expansion mutation STR sites as described in any one of claims 1-4 in the preparation of human polynucleotide repeat expansion mutation STR site detection products.

6. A kit for detecting human polynucleotide repeat extension mutation (STR) sites, characterized in that, Includes the probe combination for detecting human polynucleotide repeat extension mutation STR sites as described in any one of claims 1-4.

7. A method for detecting human polynucleotide repeat expansion mutation STR sites for non-disease diagnosis and treatment purposes, characterized in that, Includes the following steps: S1. Extract genomic DNA from the sample to be tested; S2. The genomic DNA is fragmented to obtain fragmented DNA with a length of 6k to 10k. S3. Construct a long fragment library using the fragmented DNA; S4. Perform hybridization capture processing on the long fragment library using the probe combination described in any one of claims 1-4; S5. Based on the captured library products, construct a PacBio sequencing library, then perform long-read sequencing and bioinformatics analysis to complete STR site detection.

8. The method for detecting human polynucleotide repeat expansion mutation STR sites for non-disease diagnosis and treatment purposes according to claim 7, characterized in that, Step S3 specifically includes: S31. Perform end repair and deoxyadenine modification on the fragmented DNA to obtain repaired DNA; S32. Connect the repaired DNA to the sequencing adapter to obtain the DNA ligation product; S33. The DNA ligation product is amplified to prepare a long fragment library.

9. The method for detecting human polynucleotide repeat expansion mutation STR sites for non-disease diagnosis and treatment purposes according to claim 7, characterized in that, Step S4 specifically includes: S41. Hybridization capture is performed using the probe combination described in any one of claims 1-4 and a long fragment library. After the hybridization capture is completed, the target region DNA is captured from the hybridization product. S42. Amplify the captured target region DNA to obtain a capture library product that can be used for sequencing.

10. A system for detecting human polynucleotide repeat extension mutation STR sites, characterized in that, include: Genomic DNA extraction module, used to extract genomic DNA from the sample to be tested; The DNA fragmentation module is used to fragment the extracted genomic DNA. The long fragment library construction module is used to construct long fragment libraries based on fragmented DNA. A targeted hybridization capture module is used to perform target region hybridization capture on a long fragment library using the probe combination described in any one of claims 1-4; The sequencing and data analysis module is used to perform long-read sequencing and bioinformatics analysis on the captured target region DNA to determine the STR site mutation status.

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