Mitochondrial DNA variation pathogenicity evaluation method
Through high-throughput sequencing and multi-database cross-validation methods, a set of variant sites was constructed and dynamic scoring was performed, which solved the accuracy of the pathogenicity assessment of mitochondrial DNA variants and achieved early accurate diagnosis of mitochondrial diseases.
Patent Information
- Application Number
- CN202510492593.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-18
- Publication Date
- 2025-08-12
AI Technical Summary
In the prior art, the assessment of mitochondrial DNA variant pathogenicity lacks a unified and standardized evaluation system, which leads to low accuracy of evaluation results and is prone to misdiagnosis, making it impossible to effectively consider the differences in variation frequency among different populations.
High-throughput sequencing, quality screening and site comparison were used to obtain VCF file data, and through three-level quality screening and multi-database cross-validation, combined with a dynamic scoring mechanism, a set of variant sites was constructed and pathogenicity was evaluated according to the evaluation standard table.
It improves the accuracy of mitochondrial disease diagnosis, avoids misjudgment and misdiagnosis of normal polymorphic variants, realizes early accurate diagnosis, reduces the misdiagnosis rate, and improves the comprehensiveness and accuracy of the evaluation.
Smart Images
Figure CN120472980A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of variation analysis, and in particular to a method for evaluating the pathogenicity of mitochondrial DNA variation. Background Art
[0002] Mitochondria, as the "energy factories" of cells, undertake the key function of oxidative phosphorylation to produce adenosine triphosphate (ATP), providing energy for the normal physiological activities of cells. Mitochondrial DNA, as the genetic material in cells that is independent of the nuclear genome, has unique structure and genetic characteristics. It has a circular double-stranded structure and lacks the protection of histones. In addition, the mitochondrial DNA repair mechanism is relatively weak, which makes mitochondrial DNA more prone to mutation than nuclear DNA. Once mitochondrial DNA mutates, it may interfere with the normal progress of oxidative phosphorylation and lead to insufficient cellular energy supply. The series of diseases caused by mitochondrial DNA mutations are collectively referred to as mitochondrial diseases. Mitochondrial diseases accumulate in multiple organs and systems, and the clinical manifestations are very complex, covering neuromuscular symptoms, cardiovascular problems and metabolic disorders. Therefore, it is very important to evaluate the pathogenicity of mitochondrial DNA mutations.
[0003] However, current methods for assessing the pathogenicity of mitochondrial DNA variations have many shortcomings, and there is a lack of a unified and standardized mitochondrial DNA variation assessment system. Many methods simply ignore the differences in variation frequencies among different populations based on the type and location of mitochondrial DNA variations. For example, mitochondrial DNA variations that appear frequently in some populations may be normal polymorphisms rather than pathogenic variations. If we only consider the variation itself without considering whether the variation is pathogenic, it is easy to lead to misdiagnosis. In addition, different laboratories use different methods, reference standards and data sources when assessing the pathogenicity of mitochondrial DNA variations, resulting in poor comparability of evaluation results and low accuracy of test results. Summary of the Invention
[0004] In order to overcome the defect in the above-mentioned prior art that it is not possible to comprehensively and accurately assess the pathogenicity of mitochondrial DNA variation, the present invention proposes a method for assessing the pathogenicity of mitochondrial DNA variation.
[0005] To achieve the above objectives, the present invention adopts the following technical solution: a method for assessing the pathogenicity of mitochondrial DNA variation, comprising:
[0006] S1: Obtain mitochondrial DNA from patients suspected of mitochondrial disease to be tested;
[0007] S2: Perform high-throughput sequencing, quality screening, and site alignment on the DNA to obtain the first VCF file data;
[0008] S3: Screen the first VCF file data to construct a second variable site set;
[0009] S4: Based on the second set of variant sites and according to the evaluation criteria table, the pathogenicity of mitochondrial DNA variants in patients suspected of mitochondrial disease is assessed.
[0010] Preferably, in step S2, high-throughput sequencing, quality screening, and site alignment are performed on the DNA to obtain first VCF file data, including:
[0011] S21: Perform high-throughput sequencing on the DNA to obtain raw sequencing data, which includes base data, DNA fragment data, and adapter data;
[0012] S22: Perform quality control on the original sequencing data to obtain sequencing data after quality control;
[0013] S23: performing site alignment on the sites in the quality-controlled sequencing data and the standard sites, marking the variant sites in the quality-controlled sequencing data, and generating first VCF file data.
[0014] Preferably, in step S22, the process of performing quality control on the raw sequencing data includes:
[0015] A. Perform base evaluation and screening on the original sequencing data;
[0016] The Q30 base ratio of the original sequencing data is required to be ≥30%, and the base data with a Q30 base ratio of less than 30% in the original sequencing data will be eliminated;
[0017] B. Screen the DNA fragment length of the original sequencing data;
[0018] The DNA fragment length in the original sequencing data is required to be greater than 75 bp. Short fragments with a length of less than or equal to 75 bp due to degradation or abnormal interruption will be eliminated.
[0019] C. Perform adapter contamination control screening on the raw sequencing data;
[0020] The adapter data with residual adapter content ≥5% in the original sequencing data were removed.
[0021] Preferably, in step S3, screening the first VCF file data to construct a second set of variant sites includes:
[0022] S31: Perform variant site screening on the first VCF file data, obtain the second VCF file data, and construct a first variant site set, where each variant site includes several variant types;
[0023] S32: Based on the first set of variant sites, the variation score A of each variant site in the patient suspected of mitochondrial disease is calculated, and the second set of variant sites is obtained by screening.
[0024] Preferably, step S31 includes:
[0025] S311: removing the variation type data of the variation sites with DP values less than 20 in the first VCF file data;
[0026] S312: Remove the variation type data of the variation sites with QUAL less than 30 in the first VCF file data.
[0027] S313: Use the ValidateVariants module of GATK to determine whether the first VCF file data conforms to the output file format specification; if so, generate the second VCF file data, extract the chromosome coordinates, reference bases, variant bases, allele frequencies and functional annotations of the variant sites in the second VCF file, and construct the first variant site set; if not, return to step S23 and regenerate the first VCF file data.
[0028] Preferably, step S32 includes:
[0029] S321: For each variant site in the first variant site set, perform an allele frequency check in the gnomAD database to obtain an allele score AF1 for each variant site;
[0030] When the variant site is checked in the gnomAD database, if the allele frequency of the variant site in the gnomAD database is ≥1%, the allele score AF1 is 1 point; if the allele frequency of the variant site in the gnomAD database is ≥0.5%, the allele score AF1 is 0 point; otherwise, the allele score AF1 is 0 point;
[0031] S322: For each variant site in the first variant site set, perform an allele frequency check in the Helix database to obtain an allele score AF2 for each variant site;
[0032] When a variant site is checked in the Helix database, if the allele frequency of the variant site in the Helix database is ≥1%, the allele score AF2 is 1 point; if the allele frequency of the variant site in the Helix database is ≥0.5%, the allele score AF2 is 0 point; otherwise, the allele score AF2 is 0 point;
[0033] S323: For each variant site in the first variant site set, perform an allele frequency check in the MitoMap database to obtain an allele score AF3 for each variant site;
[0034] When the variant site is checked in the MitoMap database, if the allele frequency of the variant site in the MitoMap database is ≥1%, the allele score AF3 is 1 point; if the allele frequency of the variant site in the MitoMap database is ≥0.5%, the allele score AF3 is 0 point; otherwise, the allele score AF3 is 0 point;
[0035] S324: Based on the allele scores AF1, AF2, and AF3 of each variant site, calculate the mutation score A of each variant site. The calculation formula is:
[0036] A=P×(AF1+AF2+AF3)
[0037] Among them, P is the pathogenicity score;
[0038] S325: Eliminate the variant sites with a mutation score A greater than or equal to a set threshold in the first variant site set to obtain a second variant site set.
[0039] Preferably, the calculation process of the pathogenicity score P is as follows:
[0040] Determine whether the pathogenicity of the variant site is marked as "cfrm" in the MitoMap database or whether the MITOTIP score of the variant site in the MitoMap database is "Pathogenic"; if yes, record P as 0; if no, determine whether the variant site is scored as "likely benign" in the MitoMap database; if yes, record P as 2 points, if no, record P as 1 point.
[0041] Preferably, in step S4, based on the second set of variant sites and according to the evaluation criteria table, the pathogenicity of mitochondrial DNA variants in patients suspected of mitochondrial disease is evaluated, including:
[0042] S41: Based on the second set of variant sites and the evaluation criteria table, calculate the total pathogenicity risk score S for the patient suspected of mitochondrial disease to be tested; the calculation formula is:
[0043] S=w1*I1+w2*I2+...+w i *I i +…+w 12 *I 12 +α
[0044] Where i is the evaluation standard number, w i The weight corresponding to the i-th evaluation criterion, I i is a Boolean variable for the i-th evaluation criterion. When the mitochondrial DNA variation type of the suspected mitochondrial disease patient meets the criterion, the corresponding Boolean variable is 1, otherwise it is 0; α is the combination addition coefficient;
[0045] S42: The pathogenicity of mitochondrial DNA variation is judged based on the total pathogenicity risk score S. The larger the S value, the stronger the pathogenicity.
[0046] Preferably, the calculation process of the combination addition coefficient α is:
[0047] When the mitochondrial DNA variation of a patient suspected of mitochondrial disease meets condition 1 or condition 2, α is 0.5; when both conditions 1 and 2 are met, α is 1; when neither condition 1 nor condition 2 is met, α is 0;
[0048] Condition 1: Meet the core criteria in the evaluation criteria table and any two strong evidence criteria at the same time;
[0049] Condition 2: All three moderate evidence criteria in the evaluation criteria table are met simultaneously;
[0050] Among them, when the weight is 3, the evaluation standard is the core standard, when the weight is 2, the evaluation standard is the strong evidence standard, when the weight is 1, the evaluation standard is the medium evidence standard, and when the weight is 0.5, the evaluation standard is the weak evidence standard.
[0051] A computer program product comprises a computer program / instructions, which, when executed by a processor, implements a method for assessing the pathogenicity of mitochondrial DNA variation.
[0052] The advantages of the present invention are:
[0053] (1) The present invention obtains mitochondrial DNA of a patient suspected of having mitochondrial disease, performs high-throughput sequencing, quality screening, and site alignment on the DNA, obtains first VCF file data, and then screens the first VCF file data to construct a second set of variant sites. Based on the second set of variant sites and according to an evaluation standard table, the pathogenicity of mitochondrial DNA variants in patients suspected of having mitochondrial disease is evaluated. Compared with the traditional method of simply judging based on variant type and position, the present invention fully and comprehensively considers the differences in variant frequencies in different populations, avoids misjudging normal polymorphic variants as pathogenic variants, and reduces the possibility of missing the true pathogenic variants, significantly improving the accuracy of mitochondrial disease diagnosis, helping clinicians to quickly identify key variant sites, achieve early and accurate diagnosis of mitochondrial disease, and gain valuable time for timely treatment.
[0054] (2) The present invention performs three-level quality screening of mitochondrial DNA, multi-database joint filtering, and a dynamic scoring mechanism to make the assessment of the pathogenicity of mitochondrial DNA variation more comprehensive and accurate.
[0055] (3) The present invention effectively eliminates low-quality data through a three-level quality screening system (including Q30 base ratio ≥30%, DNA fragment length >75bp, and adapter contamination <5%) combined with double filtering conditions for variant sites (DP ≥20 and QUAL ≥30), providing a high-confidence variant site set for subsequent analysis.
[0056] (4) The present invention introduces a three-database cross-validation mechanism (gnomAD / Helix / MitoMap) to avoid the population frequency bias of a single database by weighted calculation of the allele scores AF1-AF3 and the dynamic pathogenicity score P. BRIEF DESCRIPTION OF THE DRAWINGS
[0057] Figure 1 The figure is a flow chart of the steps of the method of the present invention. DETAILED DESCRIPTION
[0058] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.
[0059] like Figure 1 As shown, the present invention proposes a method for assessing the pathogenicity of mitochondrial DNA variation, comprising:
[0060] S1: Obtain mitochondrial DNA from patients suspected of mitochondrial disease to be tested;
[0061] S2: Perform high-throughput sequencing, quality screening, and site alignment on the DNA to obtain the first VCF file data, including:
[0062] S21: Perform high-throughput sequencing on DNA to obtain raw sequencing data;
[0063] Raw sequencing data refers to the initial data directly output by the high-throughput sequencing (NGS) platform without quality control, including base data, DNA fragment data, and connector data.
[0064] Base data refers to the nucleotide sequence (A / T / C / G) and its corresponding quality score directly analyzed by the sequencer through optical signals (such as Illumina's fluorescence detection) or electrochemical signals (such as Ion Torrent's pH changes) during high-throughput sequencing.
[0065] DNA fragment data are short sequences (reads) obtained during high-throughput sequencing, including single-end or paired-end reads, which may cover the target genomic region or non-specific amplification products;
[0066] Adapter data is a known artificial sequence introduced during the high-throughput sequencing process (such as Illumina's P5 / P7) for fragment binding to sequencing chips, PCR amplification, or sequencing primer initiation.
[0067] S22: Perform quality control on the raw sequencing data using FastQC v0.11.9 (Babraham Institute) to obtain quality-controlled sequencing data;
[0068] The quality control process includes:
[0069] A. Perform base evaluation and screening on the original sequencing data;
[0070] The Phred33 scoring system was used, requiring that the Q30 base ratio of the original sequencing data be ≥30% (corresponding to a single-base error rate of ≤0.1%). Base data with a Q30 base ratio of less than 30% in the original sequencing data were removed.
[0071] The Phred33 scoring system (also known as Phred+33) is a core quality standard used to quantify the accuracy of base calls in high-throughput sequencing (NGS).
[0072] B. Screen the DNA fragment length of the original sequencing data;
[0073] The DNA fragment length in the original sequencing data is required to be greater than 75bp (Min RL>75bp). Short fragments with a DNA fragment length of less than or equal to 75bp due to degradation or abnormal interruption will be eliminated;
[0074] During high-throughput sequencing, DNA needs to be physically / chemically broken (such as ultrasound, enzyme digestion) or may be degraded due to sample quality issues, resulting in fragments of different lengths.
[0075] C. Perform adapter contamination control screening on the raw sequencing data;
[0076] Eliminate data with residual adapter content (AC) ≥ 5% in the original sequencing data;
[0077] S23: The BWA-MEM v0.7.17 tool was used to align the quality-controlled sequencing data with the revised Cambridge Mitochondrial Reference Genome Standard (rCRS, NC_012920.1), and the variant sites in the quality-controlled sequencing data were marked. The bcftools tool was used to perform variant normalization and generate the first VCF file data. The VCF (Variant Call Format) file is a text-based file used to store gene sequence mutation information.
[0078] S3: Screen the first VCF file data to construct a second set of variant sites, including:
[0079] S31: Perform variant site screening on the first VCF file data, obtain the second VCF file data, and construct a first variant site set. Each variant site includes several variant types, including:
[0080] S311: Remove the variation type data of the variation sites with a DP (Depth of Coverage) value less than 20 in the first VCF file data;
[0081] The above screening method ensures that each site in the first VCF file data has a sequencing coverage depth of at least 20×, that is, each base position is sequenced 20 times on average;
[0082] S312: Remove the variant type data of the variant sites with QUAL (genotype quality score) less than 30 in the first VCF file data, that is, keep the variant type data of the variant sites with a genotype judgment error rate of ≤0.1% in the corresponding Phred quality scoring system;
[0083] S313: Use the ValidateVariants module of GATK to determine whether the first VCF file data conforms to the output file format specification; if yes, generate the second VCF file data, extract the chromosome coordinates (chrM), reference base (REF), variant base (ALT), allele frequency (AF) and functional annotation (ANN field) of the variant site in the second VCF file, and construct the first variant site set; if no, return to step S23 and regenerate the first VCF file data.
[0084] S32: Based on the first set of variant sites, calculate the variation score A of each variant site in the suspected mitochondrial disease patient, and screen to obtain the second set of variant sites; including:
[0085] S321: For each variant site in the first variant site set, perform an allele frequency check in the gnomAD database to obtain an allele score AF1 for each variant site;
[0086] When the variant site is checked in the gnomAD database, if the allele frequency of the variant site in the gnomAD database is ≥1%, the allele score AF1 is 1 point; if the allele frequency of the variant site in the gnomAD database is ≥0.5%, the allele score AF1 is 0 point; otherwise, the allele score AF1 is 0 point;
[0087] Allele frequency refers to the relative frequency of a particular allele (a specific version of a gene) in the mitochondrial DNA of a patient suspected of having a mitochondrial disease.
[0088] S322: For each variant site in the first variant site set, perform an allele frequency check in the Helix database to obtain an allele score AF2 for each variant site;
[0089] When a variant site is checked in the Helix database, if the allele frequency of the variant site in the Helix database is ≥1%, the allele score AF2 is 1 point; if the allele frequency of the variant site in the Helix database is ≥0.5%, the allele score AF2 is 0 point; otherwise, the allele score AF2 is 0 point;
[0090] S323: For each variant site in the first variant site set, perform an allele frequency check in the MitoMap database to obtain an allele score AF3 for each variant site;
[0091] When the variant site is checked in the MitoMap database, if the allele frequency of the variant site in the MitoMap database is ≥1%, the allele score AF3 is 1 point; if the allele frequency of the variant site in the MitoMap database is ≥0.5%, the allele score AF3 is 0 point; otherwise, the allele score AF3 is 0 point;
[0092] S324: Based on the allele scores AF1, AF2, and AF3 of each variant site, calculate the mutation score A of each variant site. The calculation formula is:
[0093] A=P×(AF1+AF2+AF3)
[0094] Among them, P is the pathogenicity score, and the calculation process is as follows:
[0095] Determine whether the pathogenicity of the variant site is marked as "cfrm (confirmed pathogenic variant)" in the MitoMap database or whether the variant site is scored as "Pathogenic (pathogenic variant)" in the MITOTIP score of the MitoMap database; if yes, P is recorded as 0; if no, determine whether the variant site is scored as "likely benign (likely benign variant)" in the MitoMap database; if yes, P is recorded as 2 points, if no, P is recorded as 1 point;
[0096] S325: removing variant sites with a mutation score greater than or equal to 1 from the first variant site set to obtain a second variant site set;
[0097] S4, based on the second set of variant sites, evaluate the pathogenicity of mitochondrial DNA variants in patients suspected of mitochondrial disease according to the evaluation criteria table, including:
[0098] S41: Based on the second set of variant sites and the evaluation criteria table, calculate the total pathogenicity risk score S for the patient suspected of mitochondrial disease to be tested; the calculation formula is:
[0099] S=w1*I1+w2*I2+...+w i *I i +…+w 12 *I 12 +α
[0100] Where i is the evaluation standard number, w i The weight corresponding to the i-th evaluation criterion, I i is a Boolean variable for the i-th evaluation criterion. When the mitochondrial DNA variation type of the patient suspected of mitochondrial disease meets this criterion, the Boolean variable corresponding to the criterion is 1, otherwise it is 0; α is the combination addition coefficient.
[0101] The combination additive coefficient α is used to quantify the synergistic effect when a specific combination of criteria is met. It indicates that when the mitochondrial DNA variant of a patient suspected of mitochondrial disease meets multiple criteria in the evaluation criteria table, the pathogenicity of the variant is significantly enhanced. Specifically, when the mitochondrial DNA variant of a patient suspected of mitochondrial disease meets condition 1 or condition 2, α is 0.5; when both conditions 1 and 2 are met, α is 1; when neither condition 1 nor condition 2 is met, α is 0.
[0102] Condition 1: meet the core criteria and any two strong evidence criteria at the same time;
[0103] Condition 2: All three moderate evidence criteria are met simultaneously.
[0104] Among them, when the weight is 3, the evaluation standard is the core standard, when the weight is 2, the evaluation standard is the strong evidence standard, when the weight is 1, the evaluation standard is the medium evidence standard, and when the weight is 0.5, the evaluation standard is the weak evidence standard.
[0105] The evaluation criteria table is shown in Table 1.
[0106] Table 1 Evaluation criteria
[0107]
[0108]
[0109]
[0110]
[0111] S42: Determine whether the total pathogenicity risk score is less than or equal to 2; if yes, the mitochondrial DNA variation of the suspected mitochondrial disease patient is a "variation of unknown significance or benign significance", and the variation of the suspected mitochondrial disease patient does not need to be presented in the sequencing report, nor does it need to be considered in clinical practice, and it is discarded; if no, determine whether the total pathogenicity risk score is between 3 (inclusive) and 4 (inclusive). If yes, the mitochondrial DNA variation of the suspected mitochondrial disease patient is a "possibly pathogenic variation", indicating that its pathogenicity cannot be determined but it has been proven to have a "tendency to be pathogenic", and genetic experts and clinicians can selectively interpret its impact according to actual conditions to evaluate whether the mitochondrial DNA variation should be retained; if no, the mitochondrial DNA variation of the suspected mitochondrial disease patient is a "pathogenic variation", and its pathogenicity needs to be clarified in the interpretation of the sequencing results, and the impact of the pathogenic variation needs to be considered in clinical practice, and it is retained.
[0112] The following describes the above-mentioned method for evaluating the pathogenicity of mitochondrial DNA variation with reference to specific examples.
[0113] Example 1
[0114] A variant type m.5521G>A was detected in Patient 1 suspected of mitochondrial disease. Based on the methods disclosed herein, a total pathogenicity risk score (S) for the mitochondrial DNA variant m.5521G>A was calculated as follows: Criterion 1: Not applicable; Criterion 2: Not applicable; Criterion 3: Not applicable; Criterion 4: Valid, single-fiber analysis demonstrates functional impairment; Criterion 5: Valid, reported in four unrelated individuals; Criterion 6: Valid, variant frequency in the MITOMAP database is 0.000%; Criterion 7: Not applicable; Criterion 8: Not applicable; Criterion 9: Not applicable; Criterion 10: Not applicable; Criterion 11: Valid, pathogenicity score in the MITOTIP database; Criterion 12: Not applicable. Based on the above, the combined additive coefficient α = 0. Based on the above scoring criteria, the total pathogenicity risk score (S) for the m.5541C>T variant in the patient suspected of mitochondrial disease was 4, and the mitochondrial DNA variant in this patient suspected of mitochondrial disease was assessed as a "likely pathogenic variant." According to the ClinGen evidence base, the pathogenicity assessment of m.5541C>T should be "likely pathogenic variant." This assessment result is consistent with reality.
[0115] Of course, it will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, but also encompasses the same or similar structures that can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims, not the foregoing description, and it is intended that all variations that fall within the meaning and range of equivalents of the claims be encompassed within the present invention. Any reference signs in the claims should not be construed as limiting the claim to which they relate.
[0116] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.
[0117] The technology, shape, and structure not described in detail in the present invention are all well-known technologies.
Claims
1. A method for evaluating the pathogenicity of mitochondrial DNA variation, characterized in that: include: S1: Obtain mitochondrial DNA from patients suspected of mitochondrial disease to be tested; S2: Perform high-throughput sequencing, quality screening, and site alignment on the DNA to obtain the first VCF file data; S3: Screen the first VCF file data to construct a second variable site set; S4: Based on the second set of variant sites and according to the evaluation criteria table, the pathogenicity of mitochondrial DNA variants in patients suspected of mitochondrial disease is assessed.
2. The method for evaluating pathogenicity of mitochondrial DNA variation according to claim 1, wherein: In step S2, high-throughput sequencing, quality screening, and site alignment are performed on the DNA to obtain first VCF file data, including: S21: Perform high-throughput sequencing on the DNA to obtain raw sequencing data, which includes base data, DNA fragment data, and adapter data; S22: Perform quality control on the original sequencing data to obtain sequencing data after quality control; S23: performing site alignment on the sites in the quality-controlled sequencing data and the standard sites, marking the variant sites in the quality-controlled sequencing data, and generating first VCF file data.
3. The method for evaluating pathogenicity of mitochondrial DNA variation according to claim 2, wherein: In step S22, the process of performing quality control on the raw sequencing data includes: A. Perform base evaluation and screening on the original sequencing data; The Q30 base ratio of the original sequencing data is required to be ≥30%, and the base data with a Q30 base ratio of less than 30% in the original sequencing data will be eliminated; B. Screen the DNA fragment length of the original sequencing data; The DNA fragment length in the original sequencing data is required to be greater than 75 bp. Short fragments with a length of less than or equal to 75 bp due to degradation or abnormal interruption will be eliminated. C. Perform adapter contamination control screening on the raw sequencing data; The adapter data with residual adapter content ≥5% in the original sequencing data were removed.
4. The method for evaluating pathogenicity of mitochondrial DNA variation according to claim 1, wherein: In step S3, the first VCF file data is screened to construct a second set of variant sites, including: S31: Perform variant site screening on the first VCF file data, obtain the second VCF file data, and construct a first variant site set, where each variant site includes several variant types; S32: Based on the first set of variant sites, the variation score A of each variant site in the patient suspected of mitochondrial disease is calculated, and the second set of variant sites is obtained by screening.
5. The method for evaluating pathogenicity of mitochondrial DNA variation according to claim 4, wherein: Step S31 includes: S311: removing the variation type data of the variation sites with DP values less than 20 in the first VCF file data; S312: Remove the variation type data of the variation sites with QUAL less than 30 in the first VCF file data. S313: Use the ValidateVariants module of GATK to determine whether the first VCF file data conforms to the output file format specification; if so, generate the second VCF file data, extract the chromosome coordinates, reference bases, variant bases, allele frequencies and functional annotations of the variant sites in the second VCF file, and construct the first variant site set; if not, return to step S23 and regenerate the first VCF file data.
6. The method for evaluating pathogenicity of mitochondrial DNA variation according to claim 4, wherein: Step S32 includes: S321: For each variant site in the first variant site set, perform an allele frequency check in the gnomAD database to obtain an allele score AF1 for each variant site; When the variant site is checked in the gnomAD database, if the allele frequency of the variant site in the gnomAD database is ≥1%, the allele score AF1 is 1 point; if the allele frequency of the variant site in the gnomAD database is ≥0.5%, the allele score AF1 is 0 point; otherwise, the allele score AF1 is 0 point; S322: For each variant site in the first variant site set, perform an allele frequency check in the Helix database to obtain an allele score AF2 for each variant site; When a variant site is checked in the Helix database, if the allele frequency of the variant site in the Helix database is ≥1%, the allele score AF2 is 1 point; if the allele frequency of the variant site in the Helix database is ≥0.5%, the allele score AF2 is 0 point; otherwise, the allele score AF2 is 0 point; S323: For each variant site in the first variant site set, perform an allele frequency check in the MitoMap database to obtain an allele score AF3 for each variant site; When the variant site is checked in the MitoMap database, if the allele frequency of the variant site in the MitoMap database is ≥1%, the allele score AF3 is 1 point; if the allele frequency of the variant site in the MitoMap database is ≥0.5%, the allele score AF3 is 0 point; otherwise, the allele score AF3 is 0 point; S324: Based on the allele scores AF1, AF2, and AF3 of each variant site, calculate the mutation score A of each variant site. The calculation formula is: A=P×(AF1+AF2+AF3) Among them, P is the pathogenicity score; S325: Eliminate the variant sites with a mutation score A greater than or equal to a set threshold in the first variant site set to obtain a second variant site set.
7. The method for evaluating pathogenicity of mitochondrial DNA variation according to claim 6, wherein: The calculation process of the pathogenicity score P is as follows: Determine whether the pathogenicity of the variant site is marked as "cfrm" in the MitoMap database or whether the MITOTIP score of the variant site is "Pathogenic" in the MitoMap database; if yes, P is scored as 0; If no, determine whether the variant site is scored as "likely benign" in the MitoMap database; if yes, score P as 2; if no, score P as 1.
8. The method for evaluating pathogenicity of mitochondrial DNA variation according to claim 5, wherein: In step S4, based on the second set of variant sites and according to the evaluation criteria table, the pathogenicity of the mitochondrial DNA variants in the patient suspected of mitochondrial disease is evaluated, including: S41: Based on the second set of variant sites and the evaluation criteria table, calculate the total pathogenicity risk score S for the patient suspected of mitochondrial disease to be tested; the calculation formula is: S=w1*I1+w2*I2+...+w i *I i +…+w 12 *I 12 +α Where i is the evaluation standard number, w i The weight corresponding to the i-th evaluation criterion, I i is a Boolean variable for the i-th evaluation criterion. When the mitochondrial DNA variation type of the suspected mitochondrial disease patient meets the criterion, the corresponding Boolean variable is 1, otherwise it is 0; α is the combination addition coefficient; S42: The pathogenicity of mitochondrial DNA variation is judged based on the total pathogenicity risk score S. The larger the S value, the stronger the pathogenicity.
9. The method for evaluating pathogenicity of mitochondrial DNA variation according to claim 8, wherein: The calculation process of the combination addition coefficient α is: When the mitochondrial DNA variation of a patient suspected of mitochondrial disease meets condition 1 or condition 2, α is 0.5; when both conditions 1 and 2 are met, α is 1; When both condition 1 and condition 2 are not met, α is 0; Condition 1: Meet the core criteria in the evaluation criteria table and any two strong evidence criteria at the same time; Condition 2: All three moderate evidence criteria in the evaluation criteria table are met simultaneously; Among them, when the weight is 3, the evaluation standard is the core standard, when the weight is 2, the evaluation standard is the strong evidence standard, when the weight is 1, the evaluation standard is the medium evidence standard, and when the weight is 0.5, the evaluation standard is the weak evidence standard.
10. A computer program product, characterized in that It includes a computer program / instruction, which, when executed by a processor, implements a method for evaluating the pathogenicity of mitochondrial DNA variation according to any one of claims 1 to 9.