Construction method and application of human mitochondrial gene copy number library

Abstract

The invention provides a construction method and application of a human mitochondrial gene copy number library. The method comprises the following steps: selecting 10 nuclear reference genomes with stable copy numbers by utilizing the characteristic of stable copy numbers of nuclear genes, and carrying out probe design on the nuclear reference genes and mitochondrial whole genomes by adopting an imbricated probe design scheme based on a multi-factor algorithm, so as to design specific probes containing 1375 nuclear reference gene sequences and 498 mitochondrial gene sequences; and carrying out enzyme digestion random interruption, template amplification and target area capture, and forming a biotin-labeled mitochondrial gene copy number probe library by utilizing specific sequences of mitochondrial universal joints labeled by biotin of different samples. The method for detecting the mitochondrial gene copy number and constructing the variation library by utilizing a high-throughput technology is realized, so that the detection is quicker, more economical, simpler and more convenient, the popularization and the application are convenient, and the method is suitable for large-scale screening and preventive inspection of people with related diseases caused by mitochondrial copy number abnormality.

Description

Technical Field

[0001] The present invention belongs to the field of life science and biotechnology, and specifically relates to the construction and application of a human mitochondrial gene copy number library, which is a library construction method and its application for determining the human mitochondrial gene copy number by high-throughput sequencing. Background Art

[0002] Mitochondria are an important organelle in human cells. They are the place where sugars, fats and amino acids are finally oxidized to release energy, providing energy for cell activities. In addition to supplying energy to cells, mitochondria are also involved in processes such as cell differentiation, cell information transmission and cell apoptosis, and have the ability to regulate cell growth and cell cycle. Mitochondrial dysfunction is associated with a variety of human diseases, such as mitochondrial encephalomyopathy, mitochondrial myopathy, ataxia complicated with retinitis pigmentosa, maternal inherited diseases (such as mitochondrial deafness, Leber hereditary optic neuropathy, etc.) and complex mitochondrial diseases.

[0003] Mitochondria are semi-autonomous organelles that have their own complete set of equipment for mitochondrial DNA (mtDNA) replication, transcription, and protein translation. There are thousands of mitochondria in each cell, and each mitochondria contains multiple copies of mtDNA molecules. It has been found that abnormalities in the number of mtDNA copies can lead to a variety of human diseases, such as decreased egg quality, laryngeal squamous cell carcinoma, colorectal cancer, bipolar disorder, and some fatal infantile respiratory disorders, lactic acidosis, or liver and kidney failure.

[0004] In recent years, in order to detect mitochondrial copy number, domestically developed detection methods for mitochondrial copy number variation to meet the screening of mitochondrial copy number for related diseases, such as a patent for a method for detecting mitochondrial copy number variation applied in 2023 (patent application number: CN202311784594.5). This invention uses two algorithms, deep control and breakpoint scanning, to analyze the copy number of the mitochondrial genome, extract the nuclear reference genome sequence and the mitochondrial sequence respectively, and generate a comparison file; use the relevant algorithm to analyze and detect the mitochondrial copy number, and provide a method for detecting mitochondrial copy number variation, which has certain application value. However, this method involves whole exon sequencing and mitochondrial whole genome sequencing, and the process is relatively complicated and expensive. Another application for nucleic acid reagents, kits and multiplex fluorescence quantitative PCR detection methods for detecting human mitochondrial DNA copy number (patent application number: CN202310148732.4). The multiplex fluorescence quantitative PCR detection method is used to analyze the copy number of mitochondrial genes. Although this method can achieve the mitochondrial gene copy number in a short period of time, there are primer non-specific amplification bands that cannot be detected, and there is a chance of false positives.

[0005] The technology adopted in this study is to determine the mitochondrial gene copy number based on liquid phase probe capture technology. The total DNA is randomly interrupted by enzyme cutting technology, and liquid phase probe capture technology is used to capture nuclear reference genes and construct mitochondrial whole genome libraries. Sequencing and biological analysis are then performed on the machine to calculate the mitochondrial genome copy number. Summary of the invention

[0006] The purpose of the present invention is to provide a method for constructing a human mitochondrial gene copy number library in view of the shortcomings of the current methods for detecting mitochondrial DNA copy number. The method is a method for capturing the nuclear reference genome and constructing a mitochondrial whole genome library using liquid phase probe capture technology, and it has the characteristics of accuracy, rapidity and high throughput.

[0007] The present invention provides a method for constructing a human mitochondrial gene copy number library, i.e., a method for detecting mtDNA copy number based on high-throughput sequencing technology, which consists of four components: total genome extraction, random enzyme cutting, template amplification, and target region capture. The specific construction steps are as follows:

[0008] (1) Total genome extraction: Collect whole blood or cell samples and extract total genomic DNA. The extracted total DNA includes nuclear genome and mitochondrial genome; (2) Enzyme digestion and random shearing: The genomic DNA is randomly sheared (150-200 bp) by enzyme digestion, the genomic DNA fragments are repaired and A is added to the ends, adapters are connected, purified, the library is amplified, enriched and purified, and a complete second-generation sequencing library is constructed; (3) Template amplification: The target region is divided into nuclear reference genes and mitochondrial whole genes. Ten reference genes with fixed copy numbers, B2M, ACTB, BECN1, NEB, HBS1L, GSDMA, CYGB, TMIE, PIGQ, and SPMIP9, are selected. A total of 1873 probe components are required for capture, including 1375 nuclear reference gene probe components and 498 mitochondrial whole genome probe components. The nuclear reference gene and mitochondrial whole genome probes are hybridized with the library, and the target region library is captured with streptavidin magnetic beads. The nonspecific binding library is eluted and purified to obtain a specific library. The library is enriched to obtain the final library. (4) Target region capture: Perform high-throughput sequencing and bioinformatics analysis on the machine to remove low-quality reads and duplicate values, complete the alignment of the reads with the internal reference genome, and detect mitochondrial genome mutations; use the sequencing depth of the mitochondrial genome and nuclear internal reference genes to calculate the mitochondrial genome copy number.

[0009] In step (3), the nuclear internal reference gene and the mitochondrial whole genome capture are added simultaneously, which ensures the consistency of the experimental process and improves the accuracy of the mitochondrial gene copy number.

[0010] Another object of the present invention is to provide a use of the human mitochondrial gene copy number library in preparing a kit for comprehensively detecting mitochondrial gene copy number variation, which is achieved by the following steps: Step 1: DNA extraction: Take the sample to be tested and use the genome extraction kit to extract the whole genome containing nuclear genes and mitochondrial genes; Step 2: Library construction: Use enzyme digestion to fragment the genomic DNA, repair the ends, add "A" to the 3' end, connect the adapters, purify the products with magnetic beads, enrich and purify the library through amplification, and construct a complete whole genome library; Step 3: The probe is hybridized with the whole genome library. 750 ng of the whole genome library is concentrated to a dry state, and a hybridization reaction solution is prepared, including: 13 μL of hybridization buffer, 5 μL of blocking reagent for human genome repeat sequences, 2 μL of universal blocking oligonucleotide, 5 μL of RNase inhibitor, 3 μL of deionized water, 2 μL of nuclear reference gene probe components (1375, see Sequence Table 1 for details) and 2 μL of mitochondrial whole genome probe components (498, see Sequence Table 2 for details), and a hybridization reaction is performed. The reaction conditions are: a hot lid temperature of 95°C, 85°C for 5 min for 1 cycle, and then a hybridization at 60°C overnight; Step 4: After hybridization, the nuclear reference gene and mitochondrial genome library are captured with streptavidin magnetic beads, and the non-specific binding library is eluted to obtain the specific library; Step 5: enrichment and purification of the post-capture library, PCR amplification reaction solution: 24 μL of post-capture reaction product, 1 μL of PCR primer, 25 μL of PCR buffer, PCR reaction conditions: 1 cycle of denaturation at 95°C for 1 min, 15 cycles of denaturation at 98°C for 20 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s, final extension at 72°C for 5 min, and storage at 4°C; Step 6: Take 1 μL of the constructed library and place it on a nucleic acid analyzer to measure the concentration and record it; Step 7: Take 1 μL of library sample and use the fully automatic nucleic acid protein analysis system to measure the length and purity of the library fragment. The target fragment distribution range of a normal library is between 260bp-400bp.

[0011] The present invention provides a liquid-phase probe capture that can be constructed using a library of mitochondrial copy number and variation using high-throughput sequencing. The capture probe component has a total of 1,873 sequences, including 1,375 nuclear reference gene probe sequences and 498 mitochondrial gene probe sequences, which can make up for the missed diagnosis caused by fewer detection sites, and better facilitate large-sample screening of mitochondrial DNA copy number and variation, with the characteristics of rapid high throughput. It is characterized in that the probe sequence adopts a "shingled" probe design scheme based on a multi-factor algorithm, and multi-layer probe design is performed on the target region of the nuclear reference gene and the entire mitochondrial genome. At the same time, with the self-developed hybridization capture system, efficient and uniform data performance can be obtained.

[0012] The probe design provided by the present invention is based on a 3× shingled design scheme based on thermodynamic stability, with better coverage of the target area, higher effective coverage, high uniformity, and high capture efficiency;

[0013] In the present invention, the copy number and variation detection results of the probe design achieve high accuracy and good repeatability in low-frequency mutation detection (mutation frequency above 1%).

[0014] The present invention extracts genomic DNA, randomly interrupts by enzyme digestion, amplifies templates, captures target regions, and provides related detection kits. The present invention mainly provides: (1) Target gene capture, using liquid phase probe capture technology, is to capture the target region by probe hybridization, comprehensively evaluate the capture efficiency based on multiple factors such as the size of the target region, high GC region, high repetitive region, homologous sequence, sample conservation, etc., based on a multi-factor algorithm, and specially designs difficult-to-capture regions, to provide probe design solutions with the best coverage and capture efficiency; (2) 1873 sequences, including 1375 nuclear reference gene probe sequences and 498 mitochondrial gene probe sequences; (3) Provide a self-developed hybridization capture system, use a single tube operation, only need two rinse reagents, the experiment is more convenient, and efficient and uniform data are obtained; (4) A method and kit for constructing mitochondrial DNA copy number and variant libraries.

[0015] The method of the present invention and the kit for detecting mitochondrial DNA copy number and variation prepared therefrom have the following characteristics: 1. This study uses targeted hybridization capture sequencing technology, which uses probe hybridization to capture the target area, achieves full coverage of the target area, improves sequencing depth, and significantly reduces sequencing costs; 2. At present, there are several methods for detecting mitochondrial copy number at home and abroad, such as direct sequencing and fluorescence quantitative PCR detection. However, due to their own defects, such as time-consuming and labor-intensive, cumbersome operation and high detection costs, they are not easy to promote in clinical practice. Compared with these methods, the high-throughput detection method of mitochondrial copy number and variation provided by the present invention avoids the problems of low throughput, false negatives, false positives, etc. 3. Targeted hybridization capture sequencing technology can capture and sequence multiple gene targets at the same time. It has high throughput and is suitable for studying the variation of multiple genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a schematic diagram of the design of probes for the mitochondrial DNA copy number library shown in the present invention.

[0017] Figure 2 The flowchart of the mitochondrial DNA copy number library construction shown in the present invention.

[0018] Figure 3 This is a quality control diagram of the mitochondrial DNA copy number library construction provided by the present invention, wherein Figure A is the pre-library and Figure B is the final library. DETAILED DESCRIPTION

[0019] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0020] Example 1 A method for constructing a human mitochondrial gene copy number library and its application The method for detecting mitochondrial copy number and variation provided by the present invention uses high-throughput technology to simultaneously perform targeted capture of the nuclear reference genome and the mitochondrial genome, and includes four components: total genome extraction, random enzyme cutting, template amplification, and target region capture. Among them,

[0021] (1) The selected target nuclear internal reference genes are diploid genes that are not easily changed in human body. Ten internal reference genes with fixed copy numbers were selected: B2M, ACTB, BECN1, NEB, HBS1L, GSDMA, CYGB, TMIE, PIGQ, and SPMIP9;

[0022] (2) The selected nuclear reference genes and mitochondrial full genes were designed using a "shingled" probe design scheme based on a multi-factor algorithm (e.g. Figure 1 ), a total of 1873 probe components were obtained, including 1375 nuclear reference gene probe components (see Table 1 for details) and 498 mitochondrial whole genome probe components (see Table 2 for details);

[0023] Table 1. List of 1375 probe capture sequences of nuclear reference genes

[0024] Table 2 List of 498 probe capture sequences of mitochondrial genome

[0025] (3) A hybridization capture system using a single tube operation and requiring only two rinse reagents, including: 13 μL of hybridization buffer, 5 μL of blocking reagent for human genomic repetitive sequences, 2 μL of universal blocking oligonucleotide, 5 μL of RNase inhibitor, 3 μL of deionized water, and a synthesized probe component;

[0026] (4) The amplification primers involved in the post-capture PCR amplification reaction are: forward primer 5'-AATGATACGGCGACCACCGA; reverse primer: 5'-CAAGCAGAAGACGGCATACGA.

[0027] Example 2 Preparation and analysis of mitochondrial copy number library according to Figure 2 The schematic diagram shown in the figure is used to prepare the library before the mitochondrial DNA copy number determination, as follows:

[0028] Step 1: Genomic DNA extraction: Extract genomic DNA from whole blood or cell samples and perform quality control tests;

[0029] Step 2: (1) DNA fragmentation, end repair, and adding "A" to the 3' end: prepare the reaction system as follows: 40 μL sample DNA, 10 μL fragmentation buffer, and 10 μL fragmentase system; use a pipette to mix (avoid vigorous shaking to mix), and centrifuge instantly; set the PCR instrument parameters as follows, place the reaction system on the PCR instrument, and run the program: hot cover temperature 85°C, 4°C 1 min cycle once, 30°C 30 min cycle once, 65°C 20 min cycle once, and store at 4°C;

[0041] Step 3: Connector connection. Prepare the reaction system according to the following table: 60 μL of the reaction product from the above step, 5 μL of the connector, 30 μL of the connector connection buffer, and 5 μL of the connector ligase; use a pipette to mix (avoid vigorous shaking), and centrifuge instantly; set the PCR instrument parameters: 20℃15min for 1 cycle, store at 4℃, place the reaction system on the PCR instrument, and run the program;

[0030] Step 4: Purification of ligation product: Prepare 80% ethanol with anhydrous ethanol and deionized water in advance and place it at room temperature for use. Please try to use freshly prepared 80% ethanol for magnetic bead purification; take out the purified magnetic beads from the 4°C refrigerator in advance, mix them and place them at room temperature for 30 minutes; vortex the purified magnetic beads that have been equilibrated to room temperature for use; add 70μL of magnetic beads to the 100μL reaction system after step 3. Mix by pipetting or vortexing, and let stand at room temperature for 5 minutes; centrifuge instantly, place the PCR tube on the magnetic rack for 3 minutes, and wait for the solution to clarify; keep the PCR tube on the magnetic rack, carefully discard the supernatant, add 200μL of 80% ethanol solution to the PCR tube, and let stand for 30 seconds; keep the PCR tube on the magnetic rack, discard the supernatant, and add 200μL of 80% ethanol solution to the PCR tube again. 80% ethanol solution, let stand for 30s, and discard the supernatant; centrifuge briefly, place the PCR tube on the magnetic rack, and carefully use a 10μL pipette to discard the ethanol remaining at the bottom, taking care not to suck up the magnetic beads; keep the PCR tube on the magnetic rack, let stand at room temperature for 3-5min, dry the magnetic beads, and allow the residual ethanol to evaporate completely; add 22μL of deionized water, remove the PCR tube from the magnetic rack, pipette or vortex to mix, and let stand at room temperature for 2min; centrifuge briefly, place the PCR tube on the magnetic rack for 2min, and wait for the solution to clarify; use a pipette to aspirate 20μL of supernatant, transfer to a new PCR tube, and mark it;

[0031] Step 5: PCR amplification: Add 20 μL of the reaction product from step 4, 25 μL of the PCR mixture, and 5 μL of the primers; use a pipette to mix, and centrifuge briefly; set the PCR instrument parameters as follows, place the reaction system on the PCR instrument, and run the program: hot cover temperature 105°C, denaturation at 98°C for 1 min for 1 cycle, denaturation at 98°C for 20 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s for 5 cycles, final extension at 72°C for 2 min, and store at 4°C;

[0032] Step 6: Purification of PCR amplification products: Prepare 80% ethanol with anhydrous ethanol and Nuclease-Free Water in advance and place it at room temperature for use. Please try to use freshly prepared 80% ethanol for magnetic bead purification; take out the purified magnetic beads from the 4°C refrigerator in advance, mix them and place them at room temperature for equilibration for 30 minutes; vortex the purified magnetic beads that have been equilibrated to room temperature for use; add 1 volume (50μL) of magnetic beads to the 50μL reaction system after step 5, pipette or vortex to mix them, and let them stand at room temperature for 5 minutes; centrifuge instantly, place the PCR tube on the magnetic rack for 3 minutes, and wait for the solution to clarify; keep the PCR tube on the magnetic rack, carefully discard the supernatant, add 200μL 80% ethanol solution to the PCR tube, and let it stand for 30s; keep the PCR tube on the magnetic rack, discard the supernatant, and add 200μL to the PCR tube again 80% ethanol solution, let stand for 30s, and discard the supernatant; centrifuge briefly, place the PCR tube on the magnetic stand, and carefully use a 10μL pipette to discard the ethanol remaining at the bottom, taking care not to suck up the magnetic beads; keep the PCR tube on the magnetic stand, let it stand at room temperature for 3-5min, dry the magnetic beads, and make the residual ethanol evaporate completely; add 30μL deionized water, remove the PCR tube from the magnetic stand, pipette or vortex to mix, and let it stand at room temperature for 2min; centrifuge briefly, place the PCR tube on the magnetic stand for 2min, and wait for the solution to clarify; use a pipette to absorb 28μL of supernatant, transfer it to a new PCR tube, and mark it; take 1μL of the library and use a spectrophotometer to detect the library concentration, and record the library concentration; take 1μ of the library and use a fragment analyzer to detect the library fragment length;

[0033] Step 7: Preliminary preparation for hybridization capture experiment: Take out the hybridization blocking reagent and RNase blocking reagent from the -20℃ refrigerator, place them on an ice box to melt, vortex briefly to mix and centrifuge them after melting, and place them on an ice box for temporary storage; take out the matching blocking oligonucleotide reagent from the -20℃ refrigerator, place them on an ice box to melt, vortex briefly to mix and centrifuge them for instant storage, and place them on an ice box for temporary storage; take out the probes to be used from the -20℃ refrigerator, place them on an ice box to melt, vortex briefly to mix and centrifuge them for instant storage, and place them on an ice box for temporary storage; take out the library to be hybridized and captured from the -20℃ refrigerator, place them on an ice box to melt, vortex briefly to mix and centrifuge them for instant storage, and place them on an ice box for temporary storage; take out the hybridization buffer, melt it at room temperature, vortex briefly to mix and centrifuge it for instant storage. The hybridization buffer needs to be heated in a 37℃ water bath and used only after the reagent is completely dissolved;

[0034] Step 8: Library and probe hybridization: Take 750ng of library and add it to the PCR tube, mark it; put the PCR tube into a vacuum concentrator centrifuge, open the PCR tube cover, and concentrate it to a dry state; after the library is concentrated, prepare the hybridization reaction solution according to the table below: 13μL of cross-linking buffer, 5μL of blocking reagent for human genome repeat sequences, 2μL of universal blocking oligonucleotide, 5μL of RNase inhibitor, 3μL of deionized water, and 2μL of 1873 probe group; add the hybridization reaction solution to the dried library, vortex and oscillate for 30s to ensure that the DNA dried at the bottom of the tube is dissolved, and centrifuge briefly; set the PCR instrument parameters as follows, place the reaction system on the PCR instrument, and run the program: hot cover temperature 95℃, 85℃5min for 1 cycle, and hybridize overnight at 60℃;

[0035] Step 9: Preparation before capture experiment: Take out the magnetic beads from the 4℃ refrigerator in advance, mix them thoroughly and place them at room temperature for 30 minutes to equilibrate; prepare 80% ethanol with anhydrous ethanol and deionized water in advance, store it temporarily at room temperature, and use it in the purification step; take out the eluate, if there is a precipitate, place it in a 37℃ water bath to heat it, and use it after the precipitate is completely dissolved; take out the eluate and place it in a 60℃ water bath to preheat; take 50μL of magnetic beads and add them to a new PCR tube, place it on the magnetic rack for 1 minute, wait for the solution to clarify, and discard the supernatant; remove the PCR tube from the magnetic rack, add 180μL of binding buffer and pipette or vortex to mix, to resuspend the magnetic beads; after instant centrifugation, place the PCR tube on the magnetic rack for 1 minute, wait for the solution to clarify, and discard the supernatant; repeat twice and wash the magnetic beads three times; remove the PCR tube from the magnetic rack, add 180μL of binding buffer and pipette or vortex to mix;

[0036] Step 10: Capture of target nuclear reference genes and mitochondrial genome: Keep the hybridization product of step 8 on the PCR instrument, add 180μL of magnetic beads prepared in step 9 to the hybridization product, and mix by pipetting; cover the tube cap, remove the PCR tube from the PCR instrument, place it on a vertical rotating mixer, the speed does not exceed 10rpm, and combine at room temperature for 30min; remove the PCR tube, centrifuge it instantly, place it on a magnetic stand for 2min, and after the solution is clarified, discard the supernatant; remove the PCR tube from the magnetic stand, add 150μL of elution solution to the PCR tube, gently mix by pipetting to resuspend the magnetic beads, replace the new tube cap, and then place it on a vertical rotating mixer for cleaning at room temperature for 15min, the speed does not exceed 10rpm; remove the PCR tube, centrifuge it instantly, place it on a magnetic stand for 2min, and after the solution is clarified, discard the supernatant; remove the PCR tube from the magnetic stand, add 150μL of elution solution Preheat the elution buffer at 60℃, gently pipette and mix, centrifuge briefly, place on a constant temperature oscillating mixer or metal bath, and incubate at 60℃ for 10min; remove the PCR tube, centrifuge briefly, place on a magnetic rack for 2min, and after the solution is clarified, discard the supernatant; remove the PCR tube from the magnetic rack, add 150μL of preheated elution buffer at 60℃, gently pipette and mix, centrifuge briefly, place on a constant temperature oscillating mixer or metal bath, and incubate at 60℃ for 10min; remove the PCR tube, centrifuge briefly, gently pipette and mix, transfer all the liquid (including magnetic beads) to a new PCR tube, place on a magnetic rack for 2min, and after the solution is clarified, discard the supernatant; keep the PCR tube on the magnetic rack and add 200μL 80% ethanol, let it stand for 30 seconds and then completely discard the ethanol solution, dry the magnetic beads at room temperature to completely evaporate the residual ethanol; add 24μL of deionized water to the PCR tube, remove the PCR tube from the magnetic stand, briefly vortex to resuspend and mix the magnetic beads, and proceed to the next step of amplification reaction;

[0037] Step 11: Post-capture PCR amplification: Prepare PCR reaction solution: 24 μL of the reaction product of step 10 above, 1 μL of PCR primer, and 25 μL of PCR buffer; after preparation, use a pipette to mix, and quickly transfer to the PCR instrument after mixing. Do not use the method of vortexing and then centrifuging to mix; set the PCR instrument program as follows, place the PCR reaction solution on the PCR instrument, and run the program; the hot cover temperature is 105°C, denaturation at 95°C for 1 min for 1 cycle, denaturation at 98°C for 20 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s for 15 cycles, final extension at 72°C for 5 min, and storage at 4°C;

[0038] Step 12: Post-amplification purification: Take out the purified magnetic beads, mix and place at room temperature for 30 minutes; add 1.1 times the volume of magnetic beads (55μL) to the PCR product in step 11, pipette or vortex to mix, and let stand at room temperature for 5 minutes; centrifuge instantly, and place the PCR tube on the magnetic rack for 3 minutes to wait for the solution to clarify; keep the PCR tube on the magnetic rack, discard the supernatant, add 200μL 80% ethanol solution to the PCR tube, and let stand for 30s; keep the PCR tube on the magnetic rack, discard the supernatant, and add 200μL to the PCR tube again 80% ethanol solution, let it stand for 30 seconds and then completely discard the supernatant; ensure that the PCR tube is on the magnetic stand, let it stand at room temperature for 3-5 minutes, dry the magnetic beads to completely evaporate the residual ethanol; add 25μL of deionized water, remove the PCR tube from the magnetic stand, pipette or vortex to mix, and let it stand at room temperature for 2 minutes; centrifuge instantly, place the PCR tube on the magnetic stand for 2 minutes, and wait for the solution to clarify; use a pipette to draw 23μL of supernatant and transfer it to a new PCR tube; store the capture library in a -20℃ refrigerator. The capture library can be stored in a -20℃ refrigerator for one month;

[0039] Step 13: Take 1 μL of the library and use a nucleic acid analyzer to measure the library concentration. Record the library concentration. Figure 3 Show;

[0041] Step 14: Take 1 μL of library sample and use the fully automated nucleic acid protein analysis system to measure the length and purity of the library fragment.

[0042] Bioinformatics analysis: After sequencing is completed, the results of the determination are removed from low-quality reads and duplicates, and the reads are aligned with the reference genome to detect mitochondrial genome mutations. The mitochondrial genome copy number is calculated using the sequencing depth of the mitochondrial genome and nuclear reference genes, as follows: First, the targeted capture sequencing results of mtDNA and nuclear reference genes are aligned to the reference genome GRCh38 / hg38 using BWA, and low-quality reads and duplicates are further removed. After local realignment is completed using GATK, VarScan software is used to analyze mutations. On this basis, the following standards are used to define mtDNA mutations: ① The sequencing depth of the site is ≥4×; ② The minimum allele frequency is ≥15%; ③ The minimum quality value is ≥20.

[0043] 6. Reliability test The high-throughput detection method of the nuclear reference genome and mitochondrial genome library construction of the present invention is used, and after sequencing and comparison on a machine, the detection results are analyzed. At the same time, the fluorescent PCR amplification products of these specimens are further sequenced and analyzed. The sequencing results are consistent with the gene chip results, indicating that the method of the present invention is stable and reliable for detecting mitochondrial DNA copy number.

[0044] Compared with fluorescence quantitative PCR technology, sequencing and enzyme digestion technology, gene chip technology has the following advantages (see Table 3).

[0045] Table 3 Comparison of the present invention with other related technical indicators

[0046] The above embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Any equivalent substitution or change made by a person skilled in the art based on the present invention is within the protection scope of the present invention.

Claims

1. A method for constructing a human mitochondrial gene copy number library, characterized in that: This is accomplished by following these steps: (1) The genomic DNA is randomly fragmented by enzyme digestion, and the fragment length is 150-200 bp; (2) The target regions were selected and divided into nuclear reference genes and mitochondrial whole genome genes. Ten reference genes with fixed copy numbers, namely B2M, ACTB, BECN1, NEB, HBS1L, GSDMA, CYGB, TMIE, PIGQ, and SPMIP9, were used. A "shingled" probe design was adopted based on a multi-factor algorithm to capture a total of 1,873 probe components, including 1,375 nuclear reference gene probe components and 498 mitochondrial whole genome probe components. (3) The probe was hybridized with the nuclear reference gene and mitochondrial genome library, and a hybridization reaction solution was prepared, the components of which included: 13 μL of hybridization buffer, 5 μL of blocking reagent for human genome repetitive sequences, 2 μL of universal blocking oligonucleotide, 5 μL of RNase inhibitor, and 3 μL of deionized water, and liquid phase capture was performed to obtain a labeled whole genome amplification library; (4) After capture, library enrichment and purification are performed, wherein the amplification primers involved in the PCR amplification reaction are: universal adapter forward primer 5'-AATGATACGGCGACCACCGA; Reverse primer: 5′-CAAGCAGAAGACGGCATACGA.

2. The construction method according to claim 1, characterized in that: The nuclear internal reference gene selected in step (2) is a diploid nuclear gene with a stable copy number, which increases its accuracy.

3. The construction method according to claim 1, characterized in that: The probe components in step (2) are calculated using a shingled calculation method, and the overlapping length is 16 to 35 bp. The probes overlap by 60 bp to 80 bp between every two adjacent probe sequences.

4. The construction method according to claim 1, characterized in that: The liquid phase capture probe technology in step (3) ensures the fidelity of amplifying the nuclear reference gene and the entire mitochondrial genome.

5. The construction method according to claim 1, characterized in that: The label in step (3) is a biotin label of water-soluble vitamin B.

6. Use of the human mitochondrial gene copy number library constructed according to the method of claim 1 in preparing a kit for comprehensive detection of mitochondrial gene copy number variation.

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