NOP56 gene detection method
Through the ONT platform's long-read, long-term adaptive sampling and sequencing technology, combined with target bed files and IGV analysis, the problem of detection of high GC content and ultra-large repeat amplification sequence length of NOP56 gene was solved, and the accurate diagnosis of SCA36 disease was achieved and the detection was improved. Accuracy.
Patent Information
- Application Number
- CN202411666150.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-20
- Publication Date
- 2025-01-03
AI Technical Summary
Existing gene detection technology is difficult to accurately detect the high GC content and the length of the super-large repeat amplification sequence in the NOP56 gene, resulting in difficulty in diagnosis of SCA36 disease.
Using the ONT platform's long-read long adaptive sampling and sequencing technology, targeted sequencing is carried out by constructing target bed files, including the first intron GGCCTG repeat region of the NOP56 gene, its flanking region and mitochondrial whole genome sequence, targeted sequencing is carried out, combined with Minknow software and IGV graphical analysis to achieve accurate identification of STR dynamic mutations in NOP56 gene.
It improves the accuracy of the complete identification of the length of the six-base repeat sequence of NOP56, enhances the accuracy of SCA36 disease diagnosis, and overcomes the bottleneck of the difficulty of detection of traditional methods.
Smart Images
Figure CN119242776A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of gene detection, and in particular to a method for detecting a NOP56 gene. Background Art
[0002] Spinocerebellar ataxia type 36 (SCA36) (OMIM: 614153) is a spinocerebellar ataxia first described in Japan and Spain, with an autosomal dominant inheritance pattern. SCA36 has a higher incidence in East Asia (Japan and China) and Spain. The main feature of the disease is a late-onset, slowly progressive cerebellar syndrome that usually involves motor neurons or is associated with hearing loss.
[0003] SCA36 is caused by an abnormal expansion of the GGCCTG hexanucleotide sequence in the first intron of the nucleolar protein 56 gene (NOP56). Normal NOP56 alleles contain 3 to 14 GGCCTG hexanucleotide repeats, while abnormally expanded NOP56 alleles usually contain 650 to 2500 hexanucleotide repeats. The large expansion of the GGCCTG hexanucleotide repeat sequence in the intron of the NOP56 gene induces toxic RNA aggregation and sequesters the RNA-binding protein SRSF2, thereby causing disease.
[0004] High GC content and long repeat motifs are typical characteristics of the GGCCTG hexanucleotide repeat expansion of the NOP56 gene. Calculated based on the pathogenic repeat interval of 650 to 2500 times, the length of the pathogenic hexanucleotide repeat expansion sequence can reach 3990 to 15000bp, far exceeding the upper limit of the read length detection of first-generation sequencing and second-generation sequencing (1000bp, 500bp). Therefore, for the hexanucleotide repeat sequence in the NOP56 gene, neither the traditional multiple primer PCR combined with capillary electrophoresis technology / first-generation sequencing technology, nor the STR typing technology based on second-generation sequencing, can fully clarify the specific number of pathogenic repeats, and can only partially indicate the potential pathogenic risk. In addition, due to the high GC content and long fragment length of the repeat sequence region, it is difficult to amplify the repeat sequence region of the NOP56 gene by traditional PCR methods. In summary, conventional detection methods based on first- and second-generation sequencing technologies are difficult to completely solve the detection pain points of the SCA36 repeat sequence region.
[0005] Whole genome sequencing based on third-generation sequencing (Pacbio / ONT) is very suitable for the detection of long-fragment repetitive sequence regions due to its single-molecule, long read length, and PCR-free characteristics, but the cost is still too high at this stage. Targeted capture technology based on third-generation sequencing is regarded as an ideal alternative for detecting long-fragment repetitive sequence regions. However, traditional targeted capture technologies either rely on PCR amplification or on the binding of specific probes to the target region, both of which are susceptible to the GC preference of the target region. Given that the amplification of the GGCCTG hexanucleotide sequence in SCA36 has a significant GC preference, the third-generation sequencing targeted capture technology that relies on PCR amplification and probe hybridization is not completely suitable for the SCA36 detection scenario. Therefore, it is necessary to explore new third-generation sequencing targeted capture technologies suitable for SCA36 detection scenarios.
[0006] The long read adaptive sampling sequencing (LR-AS) technology based on the ONT platform can achieve effective enrichment of target regions across the entire genome with only minor changes to the bioinformatics workflow. This technical method achieves enrichment of specific sequences by reversing the voltage of a single nanopore to reject sequences in or outside the target region. Since this method does not rely on PCR amplification and probe capture to achieve enrichment of target sequences, it is particularly suitable for long-fragment targeted sequencing of high GC content and high repetitive sequence regions.
[0007] The core step of LR-AS technology is to design a target region of appropriate size for uploading to Minknow. If the target region is designed to be too small, too many non-target sequences will pop up frequently, which will affect the activity of the nanopore to a certain extent and affect the sequencing yield of the final target sequence; if the target region is designed to be too large, it will consume a lot of additional computing resources. Therefore, for the human genome, the target region size officially recommended by ONT is 3Mb to 300Mb. In order to match the recommended target region, people often design multiple genomic target regions in the form of panels.
[0008] A recent paper reported the use of LR-AS sequencing to perform targeted sequencing on 110 individuals clinically diagnosed with CA. This method was based on the ONT sequencing platform and designed a target region group for LR-AS sequencing for 334 genomic regions (a total of 111Mb / approximately 3.59% of the hg38 reference genome), including the SCA36 pathogenic gene NOP56. However, this method failed to fully identify the specific repeat expansion size of the GGCCTG hexabase in the SCA36 sample when analyzing STR dynamic mutations using the Straglr (v1.4.1) software (doi: https: / / doi.org / 10.1101 / 2024.07.08.24309939). Therefore, it is necessary to design a new personalized LR-AS detection method for the SCA36 dynamic mutation detection scenario to ensure the complete recognition of the SCA36 hexabase repeat sequence length.
[0009] In view of this, the present invention is proposed. Summary of the invention
[0010] The purpose of the present invention is to provide a method for detecting the NOP56 gene, aiming to solve the bottleneck problem of difficulty in accurately detecting the specific amplified sequence length caused by high GC content and ultra-large repeated amplification in the SCA36 pathogenic gene NOP56.
[0011] In order to achieve the above objectives, the following technical solutions are adopted:
[0012] In a first aspect, the present invention provides a method for detecting the NOP56 gene, comprising the following steps:
[0013] a. Extract DNA from the sample to be tested, and then build an ONT library;
[0014] b. constructing a target bed file, wherein the target bed file includes the GGCCTG repeat sequence region of the first intron of the NOP56 gene, and the whole mitochondrial genome sequence or a partial sequence;
[0015] c. Upload the target bed file constructed in step b in Minknow software, then run the adaptive sampling mode to sequence the library constructed in step a, and then analyze the STR dynamic mutation in the NOP56 gene based on the sequencing results.
[0016] As a further technical solution, the samples to be tested include human blood samples, tissue samples, cell samples, throat swab samples, nasal swab samples and amniotic fluid samples.
[0017] As a further technical solution, in step a, the ligation sequencing kit SQK-LSK110 is used to build the library.
[0018] As a further technical solution, the target bed file includes the GGCCTG repeat sequence region of the first intron of the NOP56 gene and its flanking 20kb regions, as well as the complete mitochondrial genome sequence or partial sequence.
[0019] As a further technical solution, the size of the sequence in the target bed file is 3Mb to 300Mb.
[0020] As a further technical solution, it also includes quality control of sequencing data;
[0021] The quality control includes removal of adapter sequences and low-quality fragments.
[0022] As a further technical solution, the analysis of STR dynamic mutations in the NOP56 gene includes comparing with a reference genome.
[0023] As a further technical solution, the reference genome includes the reference genome hg38.
[0024] As a further technical solution, the analysis of STR dynamic mutations in the NOP56 gene includes analyzing the number of repetitions of the GGCCTG repeat sequence in the first intron region of the NOP56 gene.
[0025] As a further technical solution, the number of repetitions of the GGCCTG repeat sequence in the NOP56 gene is analyzed using bioinformatics software for analyzing STR or based on the IGV diagram.
[0026] Compared with the prior art, the present invention has the following beneficial effects:
[0027] The detection method of the NOP56 gene provided by the present invention uses the mitochondrial genome sequence widely present in cells as the filling sequence of the bed interval, and together with the NOP56 gene target interval, it constitutes the bed file to be uploaded in the Minknow software. Compared with the panel that simultaneously targets multiple genomic regions including NOP56, this strategy enhances the accuracy of STR amplification detection of the NOP56 gene, and can achieve complete recognition of the length of the NOP56 six-base repeat sequence. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0029] Figure 1This is a visualization IGV diagram of the sequence alignment of the reads obtained by sequencing in Example 1. DETAILED DESCRIPTION
[0030] The embodiments of the present invention will be described in detail below in conjunction with the embodiments and examples, but it will be appreciated by those skilled in the art that the following embodiments and examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified, proceed according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.
[0031] Next-generation sequencing: Next-generation sequencing (NGS) is a high-throughput sequencing technology that can sequence tens or millions of DNA molecules in parallel at one time. The read length of next-generation sequencing is relatively short, usually 150bp, 250bp or 300bp, and generally does not exceed 500bp.
[0032] Third-generation sequencing: The third-generation sequencing is a single molecule real-time sequencing technology, also known as long-read sequencing. Different from the traditional second-generation sequencing technology, the third-generation sequencing has a longer read length, which can help solve the problem of difficult assembly of repeated and complex regions in the genome assembly process.
[0033] Targeted Panel Sequencing: Targeted Panel Sequencing refers to targeted enrichment of only the regions of interest in the genome, followed by library construction and sequencing, and comparing the sequencing results with the reference genome to obtain variant screening results for specific genes.
[0034] Whole genome sequencing (WGS): WGS is the sequencing of the entire genome of the subject. In theory, in addition to the coding and non-coding regions of all genes, it also includes intergenic regions.
[0035] In a first aspect, the present invention provides a method for detecting the NOP56 gene, comprising the following steps:
[0036] a. Extract DNA from the sample to be tested, and then build an ONT library;
[0037] b. constructing a target bed file, wherein the target bed file includes the GGCCTG repeat sequence region of the first intron of the NOP56 gene, and the whole mitochondrial genome sequence or a partial sequence;
[0038] c. Upload the target bed file constructed in step b in Minknow software, then run the adaptive sampling mode to sequence the library constructed in step a, and then analyze the STR dynamic mutation in the NOP56 gene based on the sequencing results.
[0039] The detection method of the NOP56 gene provided by the present invention uses the mitochondrial genome sequence widely present in cells as the filling sequence of the bed interval, and together with the NOP56 gene target interval, constitutes the bed file to be uploaded in the Minknow software. Compared with the panel that simultaneously targets multiple genomic regions including NOP56, this strategy enhances the accuracy of STR amplification detection of the NOP56 gene, and can achieve complete recognition of the length of the NOP56 six-base repeat sequence (GGCCTG).
[0040] In some optional embodiments, the samples to be tested include but are not limited to human blood samples, tissue samples, cell samples, throat swab samples, nasal swab samples and amniotic fluid samples.
[0041] In some optional embodiments, the ONT library construction in step a includes a library construction process officially recommended by the ONT, such as selecting a relevant kit for library construction according to the instructions of the ONT manufacturer.
[0042] In some optional embodiments, the library is constructed using the ligation sequencing kit SQK-LSK110.
[0043] In some optional embodiments, the target bed file includes the GGCCTG repeat sequence region in the first intron of the NOP56 gene and its flanking 20 kb regions, as well as the complete or partial mitochondrial genome sequence.
[0044] In some optional embodiments, the size of the sequence in the target bed file is 3Mb to 300Mb.
[0045] In some optional embodiments, the method further comprises performing quality control on the sequencing data;
[0046] The quality control includes removal of adapter sequences and low-quality fragments.
[0047] Among them, quality control includes removing adapter sequences and low-quality fragments to obtain clean sequences.
[0048] In some optional embodiments, the analyzing STR dynamic mutations in the NOP56 gene includes comparing to a reference genome;
[0049] The reference genome includes the reference genome hg38. The clean sequence is aligned with the reference genome hg38 to obtain an aligned bam file.
[0050] Then, the STR dynamic mutations in the NOP56 gene were analyzed based on the bam file.
[0051] In some optional embodiments, the analysis of STR dynamic mutations in the NOP56 gene includes analyzing the number of repetitions of the GGCCTG repeat sequence in the first intron region of the NOP56 gene.
[0052] The number of repetitions of the GGCCTG repeat sequence in the first intron region of the NOP56 gene can be used to determine whether the sample to be tested suffers from the SCA36 disease.
[0053] In some optional embodiments, the number of repetitions of the GGCCTG repeat sequence in the NOP56 gene is analyzed using bioinformatics software for analyzing STR or based on the IGV diagram.
[0054] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.
[0055] Example 1
[0056] 1. Use the blood genomic DNA extraction kit (DP348) to extract the genomic DNA of the sample to be tested (SCA36 patient blood sample).
[0057] 2. Prepare sequencing libraries using the recommended ligation sequencing kit (SQK-LSK110) according to the manufacturer's protocol (ONT).
[0058] 3. The bed interval of the NOP56 gene target region is the GGCCTG repeat sequence region in the first intron of the NOP56 gene and the 20kb buffer region on the flank, i.e., genome chr20:2632733–2672775 (hg38).
[0059] 4. Take 50 fmol of the library and load it into the P2 solo sequencer.
[0060] 5. Upload the target bed file in Minknow software, which includes the bed interval of the target region of NOP56 gene in step 3 and the full-length sequence of mitochondrial genome (NCBI reference sequence: NC_012920.1), and run the adaptive sampling mode for sequencing.
[0061] 6. The flow cell (EXP-WSH004) was washed and the library was loaded onto the flow cell at least twice according to the manufacturer's protocol (ONT).
[0062] 7. The data is QCed to remove the adapter sequences and low-quality fragments to obtain a clean sequence.
[0063] 8. Align the clean sequence with the reference genome hg38 to obtain the aligned bam file.
[0064] 9. Use IGV genome browser software to open the bam file obtained in step 8, and manually identify the specific number of repeats of the GGCCTG repeat sequence region in the first intron region of the NOP56 gene. After calculation, the average number of repeats of the expanded allele is 2037 times (the pathogenic range is 650-2500), reaching the pathogenic threshold, as shown in the attached Figure 1 (In the figure, the red arrow points to the abnormal expansion fragment detected by this scheme, with a base number of 13288.) Compared with the reported method of detecting SCA36 using ONT adaptive sampling, this method improves the accuracy of detection and can ensure the complete recognition of the six-base repeat sequence length of SCA36.
[0065] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements 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 method for detecting NOP56 gene, characterized in that: The following steps are involved: a. Extract DNA from the sample to be tested, and then build an ONT library; b. constructing a target bed file, wherein the target bed file includes the GGCCTG repeat sequence region of the first intron of the NOP56 gene, and the whole mitochondrial genome sequence or a partial sequence; c. Upload the target bed file constructed in step b in Minknow software, then run the adaptive sampling mode to sequence the library constructed in step a, and then analyze the STR dynamic mutation in the NOP56 gene based on the sequencing results.
2. The method for detecting the NOP56 gene according to claim 1, characterized in that: The samples to be tested include human blood samples, tissue samples, cell samples, throat swab samples, nasal swab samples and amniotic fluid samples.
3. The method for detecting the NOP56 gene according to claim 1, characterized in that: In step a, the library was constructed using the ligation sequencing kit SQK-LSK110.
4. The method for detecting the NOP56 gene according to claim 1, characterized in that: The target bed file includes the GGCCTG repeat sequence region of the first intron of the NOP56 gene and the 20 kb regions on its flanking sides, as well as the whole mitochondrial genome sequence or a partial sequence.
5. The method for detecting the NOP56 gene according to claim 1, characterized in that: The size of the sequence in the target bed file is 3Mb to 300Mb.
6. The method for detecting the NOP56 gene according to claim 1, characterized in that: It also includes quality control of sequencing data; The quality control includes removal of adapter sequences and low-quality fragments.
7. The method for detecting the NOP56 gene according to claim 1, characterized in that: The analysis of STR dynamic mutations in the NOP56 gene includes comparison with a reference genome.
8. The method for detecting the NOP56 gene according to claim 7, characterized in that: The reference genome includes the reference genome hg38.
9. The method for detecting the NOP56 gene according to claim 1, characterized in that: The analysis of STR dynamic mutations in the NOP56 gene includes analyzing the number of repetitions of the GGCCTG repeat sequence in the first intron region of the NOP56 gene.
10. The method for detecting the NOP56 gene according to claim 9, characterized in that: The number of repeats of the GGCCTG repeat sequence in the NOP56 gene was analyzed using bioinformatics software for analyzing STR or based on the IGV diagram.