Complex monogenic disease detection and blocking method
Through nanopore methylation sequencing methods, sample gDNA is extracted and repaired and combined ligated, and a library is constructed for sequencing, and pathogenic mutations and methylation information is obtained, which solves the problem of diagnosis and blocking of complex single-gene diseases and achieves efficient diagnosis and blocking effects.
Patent Information
- Application Number
- CN202411848875.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-16
- Publication Date
- 2025-03-28
AI Technical Summary
The prior art is difficult to effectively diagnose and block complex monogenetic diseases, especially due to read length limitations and technical limitations, and traditional methods are difficult to identify methylation information of complex mutation types and gene regions.
Using nanopore methylation sequencing methods, the whole genome or targeted sequencing is achieved by extracting sample gDNA, end repair and A, combining ligands with linkers, library construction and nanopore sequencing, and obtaining pathogenic mutations and methylation information to assist in the construction of haplotypes to achieve diagnosis and blockade.
This method can effectively improve the detection rate of pathogenic mutations of complex single-gene diseases, directly detect nucleic acid methylation modification information, provide more comprehensive and fidelity methylation site information, assist in the construction of haplotypes, and achieve accurate diagnosis and blockade of complex single-gene diseases.
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Figure CN119709976A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of complex single gene detection and blocking technology, and more specifically, to a complex single gene disease detection and blocking method based on nanopore methylation sequencing. Background Art
[0002] Oxford Nanopore's core technology is nanopore sequencing technology. Different from previous sequencing technologies, it does not read the base sequence by detecting the color of light or fluorescent signals, but is a sequencing technology based on electrical signals. Protein nanopores are embedded in synthetic membranes and immersed in electrophysiological solutions, allowing ion currents to pass through the nanopores. When molecules such as DNA or RNA pass through the nanopores, they interfere with the current and cause characteristic changes in the current signal. In this process, the electrical signal is analyzed in real time to determine the base sequence of the DNA or RNA chain passing through the hole, and even the base modification information on the nucleic acid chain can be directly read.
[0003] Long read length and the ability to directly read methylation information without the need for sulfite and enzyme treatment are two major technical advantages of third-generation sequencing. These technical advantages make nanopore sequencing particularly suitable for detecting complex single-gene diseases and blocking them in medical clinics (third-generation test-tube babies, PGT). The complex single-gene diseases mentioned here refer to single-gene genetic diseases with complex mutation types (large fragment structural variations, such as large repeat deletions, dynamic mutations, etc.) or genes in complex genomic regions (highly repeated regions, true and false genes, etc.), such as facioscapulohumeral disease (FSHD), spinal muscular atrophy (SMA), adrenal hyperplasia (CAH), etc. Due to technical limitations such as read length, traditional detection methods are difficult to diagnose and block these complex single-gene diseases. More powerful and effective tools and methods are needed in clinical practice to overcome such difficult problems and provide solutions for patients.
[0004] The present invention is based on a long-read nanopore sequencing method that can simultaneously obtain pathogenic mutations and methylation information of gene regions, accurately identify complex mutation types, and assist in constructing haplotypes through methylation information, thereby simultaneously realizing the diagnosis and blocking (PGT) of complex monogenic diseases. Summary of the invention
[0005] In view of this, in order to solve the above problems, the present invention provides a method for detecting and blocking complex monogenic diseases, comprising the following steps: extracting sample gDNA; repairing the ends of the sample gDNA and adding A to obtain gDNA after adding A; connecting the gDNA after adding A to a connector combination to obtain a connection product; a library construction step, wherein the library construction step includes a whole genome library construction step and a targeted sequencing library construction step; adding the sample sequencing library obtained by the connection product to the Oxford Nanopore sequencing chip and performing whole genome sequencing or targeted sequencing on the machine; bioinformatics analysis, due to the long read length of nanopore sequencing , which can reach more than 100 times the read length of the second-generation sequencing, and can easily detect various types of mutations, including SNV, InDel and SV, effectively improving the detection rate of pathogenic mutations in complex monogenic diseases. Nanopore sequencing does not require enzyme treatment or sulfite treatment, and can directly detect the methylation modification information of nucleic acid molecules. Compared with enzyme or sulfite methods, nanopore sequencing detects methylation site information more comprehensively and faithfully, and has no effect on nucleic acids. The methylation information obtained can be used to assist in the construction of haplotypes. For example, the number of repeating units in FSHD will have a significant effect on the methylation level, which facilitates PGT to achieve blocking of monogenic diseases.
[0006] A method for detecting and blocking complex monogenic diseases based on nanopore methylation sequencing, characterized in that it comprises the following steps:
[0007] 1) Extract sample gDNA;
[0008] 2) repairing the ends of the sample gDNA and adding A to obtain A-added gDNA;
[0009] 3) connecting the gDNA after A addition to the adapter combination to obtain a connection product;
[0010] 4) Adding the sample sequencing library obtained by ligation products to the Oxford Nanopore sequencing chip;
[0011] 5) placing the Oxford Nanopore sequencing chip from step 4) on a machine for whole genome sequencing or targeted sequencing;
[0012] 6) Bioinformatics analysis.
[0013] Furthermore, in step 1), use Genomic kit (Cat#13343) to extract gDNA; or use other long fragment DNA extraction kits to extract gDNA.
[0014] Furthermore, in step 1), the extracted gDNA is obtained from tissues, blood or cells, and the specific steps are: 2-5 ml of peripheral blood is drawn and stored in an EDTA anticoagulant tube.
[0015] Furthermore, in step 3), the Ultra II End Repair / dA-tailing Kit (Cat#E7546) or other similar kits are used for end repair and dA addition, and then the official Oxford Nanopore LSK109, or LSK110 or other similar kits are used to complete the adapter connection.
[0016] Further, in step 4), the Oxford Nanopore sequencing chip model may be R9.4 or R10.1.
[0017] Further, in step 5), the specific method of targeted sequencing comprises the following steps:
[0018] The sequencer was connected to a server running Ubuntu 20.04 and equipped with a GTX3090 GPU; the third-party software readfish (https: / / github.com / LooseLab / readfish) was installed and used. The software connected to the MinKNOW server by calling the official Read Until API of Oxford Nanopore to obtain data in real time and analyze it to achieve targeted sequencing.
[0019] Furthermore, before performing targeted sequencing in step 5), it is necessary to prepare a reference sequence, a readfish configuration file, and a bed file containing the target region. The reference sequence can be downloaded from UCSC hg19 or hg38 human reference genome sequence, http: / / hgdownload.soe.ucsc.edu / goldenPath / hg19 / bigZips; the readfish configuration file can be downloaded from
[0020] https: / / github.com / / LooseLab / readfish / tree / main / docs / _static / example_tomls / hu man_chr_selection.toml is used as the initial template; the bed file containing the target region is the bed of the target region or specified gene obtained using UCSCTable Browser;
[0021] And change the parameter break_reads_after_seconds=1.0 of the corresponding TOML configuration file under / opt / ont / minknow / conf / package / sequencing to break_reads_after_seconds=0.4; then start sequencing on MinKNOW UI and run readfish, turn on the adaptive sampling function, fill in the prepared corresponding files, and start sequencing.
[0022] The command to run Readfish is readfish targets --experiment-name "XT" --deviceMN** --toml human_readfish_XT.toml --log-file XT.log --port 9502 (experiment-name indicates the machine input name of the sequencing experiment, which needs to be consistent with the experiment name on MinKNOW; device indicates the machine serial number of the Oxford Nanopore sequencer, which can be seen on the machine and the MinKNOW software connected to the machine; toml is the configuration file of the experiment, including the reference sequence file for comparison and the bed file of the target region.
[0023] Furthermore, step 6) also includes the steps of: identifying methylation information through SUP mode, filtering out low-quality data using NanoFilt (v2.8.0), aligning sequences using Minimap2 (v2.22), detecting structural variations using Sniffles (v2.0.4) software, detecting upstream and downstream SNPs of mutations and constructing haplotypes using PEPPER-Margin-DeepVariant (r0.7-gpu) software, and assisting in the assembly of haplotypes and error correction using methylation typing results.
[0024] Beneficial effects of the invention: The invention provides a method for detecting and blocking complex monogenic diseases, comprising the following steps: extracting sample gDNA; repairing the ends of the sample gDNA and adding A to obtain gDNA after adding A; connecting the gDNA after adding A to a connector combination to obtain a connection product; a library construction step, wherein the library construction step includes a whole genome library construction step and a targeted sequencing library construction step; adding the sample sequencing library obtained by the connection product to the Oxford Nanopore sequencing chip and performing whole genome sequencing or targeted sequencing on the machine; bioinformatics analysis, due to the long read length of nanopore sequencing, It is more than 100 times longer than the second-generation sequencing read length and can easily detect various types of mutations, including SNV, InDel and SV, effectively improving the detection rate of pathogenic mutations in complex monogenic diseases. Nanopore sequencing does not require enzyme or sulfite treatment and can directly detect the methylation modification information of nucleic acid molecules. Compared with enzyme or sulfite methods, nanopore sequencing can detect methylation site information more comprehensively and accurately and has no effect on nucleic acids. The methylation information obtained can be used to assist in the construction of haplotypes. For example, the number of repeating units in FSHD will have a significant effect on the methylation level, making it easier for PGT to block monogenic diseases. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 This is a schematic diagram of the process of Example 1 of the present invention;
[0026] Figure 2 It is a schematic diagram of the methylation information of the targeted region of the present invention;
[0027] Figure 3 It is a schematic diagram of the process of the complex monogenic disease detection and blocking method of the present invention; DETAILED DESCRIPTION
[0028] Embodiment 1:
[0029] like Figure 1 As shown, for example, XT is a carrier of facioscapulohumeral muscular dystrophy (FSHD). Due to the decreased methylation level of the DUX4 gene region, the expression in skeletal muscle is abnormally increased, causing muscle damage. The D4Z4 repeat sequence located at 4q35 is shortened (normal people>10RUs, FSHD1 1-10RUs), that is, the number of D4Z4 repeats is reduced, the methylation level is reduced, and the clinical phenotype is related to the methylation level. The 4q35 region and its upstream and downstream 10M range are used as the target region to form a bed file,
[0030] like Figure 3 As shown, the next step is to prepare the sequencing sample library. The specific steps are as follows:
[0031] Extract sample gDNA and use Genomic kit (Cat#13343) to extract gDNA; or use other long-fragment DNA extraction kits to extract gDNA, and the extracted gDNA is obtained in the flow product;
[0032] End repair and A addition: The sample gDNA is end repaired and A is added to obtain gDNA after A addition. The reagent usage system is shown in Table 1. Then the mixed reagent is placed in a PCR instrument, incubated at 20°C for 5 minutes and incubated at 65°C for 5 minutes. Then it is purified with AMPure XP magnetic beads;
[0033] Table 1
[0034] Reagents volume DNA 48ul NEBNext FFPE Retrieval Buffer 3.5ul NEBNext FFPE Restoration Mix 2ul NEBNext Ultra II End Repair Reaction Buffer 3.5ul NEBNext Ultra II End Repair Enzyme Mix 3ul Total volume 60ul
[0035] Add barcode: Use the barcode (NB01-96) provided by ONT to add an identity tag to each sample. Use the reagents shown in Table 2 below, then incubate at room temperature for 10 minutes. After incubation, perform magnetic bead purification;
[0036] Table 2
[0037] Reagents volume End-repaired DNA 22.5ul Amplification-free barcode (NB01-96) 2.5ul Blunt / TA Ligase Master Mix 25ul Total volume 50ul
[0038] Ligation of sequencing adapters: Incubate the reaction at room temperature for 10 minutes using the reagents shown in Table 3 below, and purify using magnetic beads;
[0039] Table 3
[0040] Reagents volume DNA sample obtained in the previous step 65ul Ligation Buffer (LNB) 20ul NEBNext Fast T4 DNA Ligase 10ul Sequencing adapter AMXII 5ul
[0041] Add the sample sequencing library of the ligation product to the Oxford Nanopore sequencing chip, the chip model can be R9.4 or R10.1. The following is an explanation using the PromethION 2Solo model. First, perform a quality inspection on the number of chip holes. The sequencing chip can be used for sequencing when the number of holes is not less than 5000. Then open the liquid inlet of the chip, use a pipette to remove the air in the chip flow channel, and then inject the priming mix provided by the official. Prepare the library sample on the machine as shown in Table 4 below, add the configured library sample on the machine to the sequencing chip by pipette, and then close the liquid inlet. Click the button on the interface of the accompanying sequencing software to start sequencing;
[0042] Table 4
[0043] Reagents volume Sequencing Buffer II (SBII) 75ul Loading particles II (LBII) or loading solution (LS) 51ul DNA library 24ul Total volume 150ul
[0044] The targeted sequencing data was statistically analyzed, and the results are shown in Table 1. With about 9.6G data output, about 960M data was sequenced data of the target region, and the enrichment efficiency was 10%. The sequencing depth of the target region reached about 26.22X, the sequencing depth of the non-target region was 2.82X, and the enrichment multiple reached 9.3 times.
[0045] Table 1. Sample targeted sequencing data statistics
[0046]
[0047] like Figure 2 As shown, through bioinformatics analysis, the number of D4Z4 repetitions and methylation information were successfully detected, and the typing of related mutations was also successfully completed.
[0048] The specific steps are as follows: Raw data in FASTQ format were obtained by targeted capture sequencing of electrical signals. In order to use more accurate data, NanoFilt (v2.8.0, https: / / github.com / wdecoster / nanofilt) was used to filter low-quality reads (Qphred≤7) and short reads (<1000bp). In addition, 50bp of sequences containing barcode and index at both ends of the reads were removed. The command was gunzip-c sample.fastq.gz|NanoFilt-q10-l 1000--headcrop 50--tailcrop 50|gzip>highQuality-reads.fastq.gz. Then Minimap2 (https: / / github.com / lh3 / minimap2) was used to accurately align the reads to the reference genome hg19 (GRCh37). The command was minimap2-ax map-ont hg19.fa highQuality-reads.fastq.gz>aln.sam.
[0049] The generated SAM file was then converted into BAM format using samtools (v1.2, https: / / github.com / samtools / samtools) for further processing and analysis.
[0050] For the structural variation part, Sniffles2 was used
[0051] (https: / / github.com / fritzsedlazeck / Sniffles) processes BAM files to detect structural variations (SVs) in genomic data, with commands such as sniffles -i aln.bam -v sniffles.sv.vcf; for detecting single nucleotide variations (SNVs) and insertions and deletions (indels), PEPPER-Margin-DeepVariant (r0.8-gpu, https: / / github.com / kishwarshafin / pepper) is used, and the construction of haplotypes is also completed, here again with BAM files as input.
[0052] For methylation information, dorado software was used to analyze pod5 data to obtain a bam file with methylation information. The command line was dorado basecaller sup,5mCG_5hmCG,6mA pod5s / >calls.bam. Then, by combining the common typing of SNP, SV and methylation information, the haplotype of the target variant can be distinguished for subsequent judgment of embryos carrying the target variant.
[0053] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.
Claims
1. A method for detecting and blocking complex monogenic diseases based on nanopore methylation sequencing, characterized in that: The following steps are involved: 1) Extract sample gDNA; 2) repairing the ends of the sample gDNA and adding A to obtain A-added gDNA; 3) connecting the gDNA after A addition to the adapter combination to obtain a connection product; 4) Adding the sample sequencing library obtained by ligation products to the Oxford Nanopore sequencing chip; 5) placing the Oxford Nanopore sequencing chip from step 4) on a machine for whole genome sequencing or targeted sequencing; 6) Bioinformatics analysis.
2. The method for detecting and blocking complex monogenic diseases based on nanopore methylation sequencing according to claim 1, characterized in that: In step 1), use Genomic kit (Cat#13343) to extract gDNA; Alternatively, use other long fragment DNA extraction kits to extract gDNA.
3. The method for detecting and blocking complex single gene diseases based on nanopore methylation sequencing according to claim 1, characterized in that: In step 1), the extracted gDNA is obtained from tissues, blood or cells. The specific steps are: 2-5 ml of peripheral blood is drawn and stored in an EDTA anticoagulant tube.
4. The method for detecting and blocking complex monogenic diseases based on nanopore methylation sequencing according to claim 1, characterized in that: In step 3), use Ultra II End Repair / dA-tailing Kit (Cat#E7546) or other similar kits for end repair and dA addition, and then use Oxford Nanopore's official LSK109, or LSK110 and other similar kits to complete the adapter connection.
5. The method for detecting and blocking complex monogenic diseases based on nanopore methylation sequencing according to claim 1, characterized in that: In step 4), the Oxford Nanopore sequencing chip model may be R9.4 or R10.
1.
6. The method for detecting and blocking complex monogenic diseases based on nanopore methylation sequencing according to claim 1, characterized in that: In step 5), the specific method of targeted sequencing comprises the following steps: The sequencer was connected to a server running Ubuntu 20.04 and equipped with a GTX3090 GPU; the third-party software readfish (https: / / github.com / LooseLab / readfish) was installed and used. The software connected to the MinKNOW server by calling the official Read Until API of Oxford Nanopore to obtain data in real time and analyze it to achieve targeted sequencing.
7. The method for detecting and blocking complex monogenic diseases based on nanopore methylation sequencing according to claim 1, characterized in that: Before performing targeted sequencing in step 5), you need to prepare the reference sequence, readfish configuration file, and bed file containing the target region. The reference sequence is downloaded from UCSC hg19 or hg38 human reference genome sequence. http: / / hgdownload.soe.ucsc.edu / goldenPath / hg19 / bigZips; readfish configuration file from https: / / github.com / / LooseLab / readfish / tree / main / docs / _static / example_tomls / human_chr_selection.toml is used as the initial template; the bed file containing the target region is the bed of the target region or specified gene obtained using UCSCTable Browser; And change the parameters of the corresponding TOML configuration file under / opt / ont / minknow / conf / package / sequencing break_reads_after_seconds=1.0 is changed to break_reads_after_seconds=0.4; Then start sequencing on MinKNOW UI, run readfish, turn on the adaptive sampling function, fill in the prepared corresponding files, and start sequencing; The command run by Readfish is readfish targets --experiment-name "XT" --deviceMN** --toml human_readfish_XT.toml --log-file XT.log --port 9502 (experiment-name indicates the machine input name of the sequencing experiment, which needs to be consistent with the experiment name on MinKNOW; device indicates the machine serial number of the Oxford Nanopore sequencer, which can be seen on the machine and the MinKNOW software connected to the machine; toml is the configuration file of the experiment, including the reference sequence file for comparison and the bed file of the target region.
8. The method for detecting and blocking complex monogenic diseases based on nanopore methylation sequencing according to claim 1, characterized in that: Step 6) also includes the following steps: identifying methylation information through the SUP mode, filtering out low-quality data using NanoFilt (v2.8.0), aligning sequences using Minimap2 (v2.22), detecting structural variations using Sniffles (v2.0.4) software, detecting upstream and downstream SNPs of mutations and constructing haplotypes using PEPPER-Margin-DeepVariant (r0.7-gpu) software, and assisting in the assembly of haplotypes and error correction using methylation typing results.