Prostate cancer HRR gene mutation detection method, probe set, kit and application
By designing a comprehensive HRR gene probe set for prostate cancer and optimizing detection methods, the problems of incomplete gene coverage and incomplete detection standards in existing technologies have been solved, achieving high-precision HRR gene mutation detection and guiding risk assessment and treatment of prostate cancer.
Patent Information
- Application Number
- CN202411069532.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-06
- Publication Date
- 2026-02-06
AI Technical Summary
Existing technologies for HRR gene detection in prostate cancer have problems such as incomplete gene coverage, incomplete detection standards, low accuracy of copy number deletion, inability to accurately distinguish between heterozygous and homozygous deletions, and inability to assess the purity and ploidy of tissue samples.
A set of probes was designed to capture HRR genes in prostate cancer, covering the target regions of 45 HRR genes. The probes were combined with backbone SNPs in large rearrangement regions and intron regions and detected by high-throughput sequencing. A read-depth strategy was used to detect copy number variations, and the method for identifying biallelic mutations was optimized.
It achieves full coverage detection of HRR gene mutation types, improving the accuracy and precision of detection. It can accurately distinguish between heterozygous and homozygous deletions, making it suitable for risk assessment and auxiliary diagnosis of prostate cancer, and guiding PARP inhibitor therapy.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of gene detection, in particular to the detection of prostate cancer related genes, more particularly to the detection of prostate cancer homologous recombination repair (HRR) gene mutations. BACKGROUND
[0002] With the progress of medical technology, there are various treatment methods for prostate cancer, such as radical surgery or radical radiotherapy in the early stage, androgen deprivation therapy for locally advanced and metastatic prostate cancer to prolong the survival period and improve the quality of life; some patients can choose surgical resection, or multiple means of comprehensive treatment based on radiotherapy.
[0003] In 2020, the US FDA approved two PARP inhibitors, Rucaparib and Olaparib (Olaparib), for the treatment of metastatic castration-resistant prostate cancer patients. Both PARP inhibitors have good efficacy for mCRPC patients and require HRR gene BRCA1 and BRCA2 mutation detection. HRR is a complex signaling pathway involving multiple steps, with BRCA1 and BRCA2 as key proteins. If BRCA gene mutation leads to loss of function of BRCA1 and BRCA2 proteins, it will cause HRR dysfunction (HRD). In addition, mutations in other HRR-related genes such as PALB2, CDK12, RAD51, CHEK2, ATM, or methylation of the BRCA1 gene promoter, as well as other unknown reasons, will cause HRD and lead to genomic instability.
[0004] Double allelic mutation refers to mutation of both alleles of a gene in a sample, resulting in inactivation of both alleles. Double allelic mutation is divided into double allelic heterozygous mutation (both alleles are mutated, but the types of mutation are different) and double allelic homozygous mutation (both alleles have the same mutation). The current commonly used double allelic mutation judgment standard is: 1) homozygous deletion; 2) two or more different pathogenic somatic mutations; 3) one pathogenic germline mutation accompanied by wild-type allele heterozygous deletion; 4) one pathogenic somatic mutation accompanied by heterozygous deletion.
[0005] The current market prostate cancer HRR gene next-generation sequencing products generally have the following problems: 1) fewer genes involved, incomplete target regions included, affecting scientific research exploration, and limited function in discovering new targets; 2) incomplete judgment standard for HRR gene double allelic mutation; 3) small precision of HRR gene copy number deletion, unable to accurately detect exon-level deletion, and unable to accurately distinguish between heterozygous deletion and homozygous deletion; 4) unable to accurately assess tumor purity and ploidy of tissue samples. SUMMARY
[0006] One of the technical problems to be solved by the present application is to provide a set of probes for capturing prostate cancer HRR genes, which has a wide gene coverage and can detect all types of HRR gene mutations.
[0007] To solve the above technical problems, the set of probes for capturing prostate cancer HRR genes of the present application captures the target regions of 45 HRR genes shown in Table 1.
[0008] Further, the genomic regions captured by the set of probes can further include large fragment rearrangement regions, which preferably include the 8 intron regions of 5 genes shown in Table 2.
[0009] Further, the genomic regions captured by the set of probes can further include backbone SNPs sites of intron regions, the population frequency of which is 5% to 95%, the upstream and downstream of which do not touch the exon regions, and the interval between the backbone SNPs sites is 50k.
[0010] The second technical problem to be solved by the present application is to provide a kit comprising the above-mentioned set of probes.
[0011] The third technical problem to be solved by the present application is to provide the use of the above-mentioned set of probes and kit.
[0012] The above-mentioned set of probes can be used to prepare a prostate cancer HRR gene mutation detection reagent, which includes one or more of single nucleotide site variation, small fragment insertion and deletion, copy number variation (amplification, homozygous deletion, heterozygous deletion), large fragment rearrangement, and biallelic mutation.
[0013] The above-mentioned set of probes and kit can also be used for high-throughput sequencing of prostate cancer HRR genes, mutation detection of prostate cancer HRR genes, risk assessment of prostate cancer, auxiliary diagnosis of prostate cancer, and auxiliary treatment of prostate cancer.
[0014] The fourth technical problem to be solved by the present application is to provide a method for judging biallelic mutation, which comprises the following steps:
[0015] 1) using the above-mentioned set of probes to hybridize and capture prostate cancer HRR genes, and performing high-throughput sequencing;
[0016] 2) splitting, preprocessing, aligning, and quality control of the sequencing data obtained, to obtain sequencing data passing quality control;
[0017] 3) using the sequencing data obtained in step 2) to detect single nucleotide site variation, small fragment insertion and deletion, and copy number variation;
[0018] 4) According to the detection result of step 3), determine whether it is a double allele mutation.
[0019] The determination condition of the single nucleotide site variation is: site coverage depth ≥ 100X, sequence number supporting mutation ≥ 3, and mutation frequency ≥ 3%;
[0020] The algorithm for copy number variation preferably comprises the following steps: using the Read-Depth strategy, dividing the probe capture region in a sliding window manner, dividing each gene into 20-90 fragment intervals, and the fragment size of each interval is 60-400bp; selecting the genomic DNA of different non-tumor male cell lines to construct a baseline; calculating the average depth of each interval of the baseline and the sample respectively, and performing normalization processing, and simultaneously correcting with GC content to obtain the log2 value of each interval; according to the calculated tumor purity and ploidy value, the threshold value of the sample returning to integer copy number is calculated according to the following formula:
[0021] Threshold value = log2((1-tumor purity) + tumor purity x (copy number + 0.5) / ploidy)
[0022] When the log2 value of the interval is within the threshold interval, the absolute integer copy number of the interval is calculated; the distribution of the absolute integer copy number of all intervals on each exon is counted, and the threshold value is judged, and the value higher than the threshold value is amplified and the value lower than the threshold value is deleted; the SNPs on each exon segment are counted, and whether it is homozygous deletion or heterozygous deletion is judged according to the MAF distribution.
[0023] The determination criteria of the copy number variation are:
[0024] 1) Copy number increase: the absolute copy number of more than 60% of the intervals of the gene is greater than 3, and at least contains two or more consecutive intervals, then it is judged that the gene is amplified;
[0025] 2) Copy number deletion: copy number deletion is observed in more than 5 consecutive exons in the interval, then it is judged that the interval is deleted;
[0026] 3) Homozygous deletion: the absolute copy number of the interval covered by the exon is 0, and the mutation frequency of all SNP sites in the interval does not deviate from 0, 50% or 100%, then it is judged that the interval is homozygous deletion;
[0027] 4) Heterozygous deletion: the absolute copy number of more than 5 consecutive exons in the interval is 1 or the absolute copy number is 0 but the mutation frequency of the SNP site in the interval deviates from 0, 50% or 100%, then it is judged that the interval is heterozygous deletion.
[0028] The determination condition of the double allele mutation is (satisfying one or more of the following) :
[0029] 1) two or more different pathogenic somatic mutations;
[0030] 2) one pathogenic germline mutation with allelic heterozygous deletion;
[0031] 3) one pathogenic somatic mutation with allelic heterozygous deletion;
[0032] 4) two mutations in close proximity, but not on the same sequencing sequence;
[0033] 5) pathogenic germline mutation and pathogenic somatic mutation occur simultaneously;
[0034] 6) two different pathogenic somatic mutations occur simultaneously;
[0035] 7) homozygous deletion.
[0036] The judgment conditions of the pathogenic somatic mutation or pathogenic germline mutation are:
[0037] 1) mutation frequency is greater than 3%;
[0038] 2) the number of sequence coverage of the mutation site is not less than 100;
[0039] 3) at least 3 sequences support the mutation;
[0040] 4) suspected deleterious variation: truncation variation, frame shift variation, nonsense variation, large fragment rearrangement leading to coding region destruction, part of database interpreted missense or non-frame shift variation;
[0041] 5) deleterious variation: the variation meeting the suspected deleterious variation condition and reported in COMSIC database.
[0042] The application designs a new prostate cancer gene set panel, which contains all types of homologous recombination pathway (HRR) genes for prostate cancer detection, has wide gene coverage, can detect all types of HRR gene mutations, has high accuracy, and can be applied to risk assessment of prostate cancer and each stage of occurrence and development of prostate cancer. Compared with the existing prostate cancer related gene detection technology, the prostate cancer HRR gene mutation detection probe set and detection method of the application has the following advantages and beneficial effects:
[0043] 1. The probe set is full coverage for BRCA1 and BRCA2 genes, full coverage for other HRR genes in coding regions, wide gene coverage, and can detect all types of HRR gene mutations; at the same time, the probe set covers meaningful large fragment rearrangement regions, has strong region targeting, avoids omission of important regions and large fragment rearrangement mutations, ensures the accuracy of the detection results, and in addition, since the coverage of unnecessary regions is reduced, it helps to simplify the size of the kit and reduce the cost.
[0044] 2. The probe coverage and algorithm optimization for CNV regions are optimized, the CNV detection accuracy is improved, one exon level deletion detection can be performed, and gene copy number heterozygous deletion and homozygous deletion can be distinguished.
[0045] 3. The judgment method of double allele mutation is optimized, and the double allele mutation load value of the sample can be accurately counted;
[0046] 4. The probe set of the application is used for hybrid capture of prostate cancer HRR genes for NGS sequencing, under the premise of not needing to be paired, 45 prostate cancer HRR related gene systems and germline variations (including single nucleotide site variation, small fragment insertion / deletion, DNA copy number variation, large fragment rearrangement, etc.) can be detected at one time, the detection is comprehensive, economical and practical. DETAILED DESCRIPTION
[0047] In order to have a more specific understanding of the technical content, characteristics and effects of the application, the technical solutions of the application will be further described in detail in combination with specific embodiments.
[0048] Example 1: Design and preparation of hybrid capture probe
[0049] The selected target gene is named according to the HGNC database standard, the mutation is named according to the human genome variation association (HGVS) standard, and the mutation classification is according to the guidelines released by AMP / ASCO / CAP in 2017. Because there are multiple transcripts for a gene, this embodiment uses the most commonly used disease corresponding mutation annotation transcript recommended by the LRG database (see Table 1), and performs full exon coverage on the target gene, with 60 bp extended to the left and right of the exon region, and the exon of the gene with a shorter length (less than 200 bp) and no coverage of adjacent introns is supplemented and extended to ensure that each exon is covered by no less than two probes. According to public data and literature, the meaningful large fragment rearrangement region (all introns of BRCA1 and BRCA2 and 8 intron regions of 5 genes of ATM, BRIP1, CDK12, RAD51D and RAD51B in Table 2) is covered, and the intron region of the gene with a length of less than 600 bp is also fully covered, as shown in Table 1. The shingle probe is designed for the high AT and GC region of the gene exon, and the skeleton SNP point is covered for the longer intron region. The selection criteria of the skeleton site are as follows:
[0050] 1. Based on the 1000Genomes database of the second generation sequencing data set, the SNPs with a frequency (AF) of 5% to 95% in the population are screened;
[0051] 2. Remove sites with large total deviations in different populations to ensure compatibility with multiple populations;
[0052] 3. Preferentially select SNPs with high MAF values and corresponding high heterozygosity in the Chinese population to ensure applicability to the Chinese population;
[0053] 4. The upstream and downstream of the SNP does not touch the exon region to better compatible with other tumor chip products;
[0054] 5. The GC content of the probe region is controlled at 25% to 75% to reduce GC bias and ensure capture efficiency;
[0055] 6. Multiple software comparison and analysis are used to evaluate the uniqueness of the captured sequence to ensure the effectiveness of the capture sequencing analysis;
[0056] 7. With a 50k spacing, ensure that the SNP sites are evenly distributed throughout the genome, and finally remove the sites with linkage disequilibrium to obtain the final sites.
[0057] Table 1 Target genomic region of the probe
[0058]
[0059]
[0060] Table 2 Eight intronic regions of five genes related to large fragment rearrangement
[0061]
[0062]
[0063] Example 2 Detection of bi-allelic mutation
[0064] Clinical selection of 20 cases of metastatic castration-resistant prostate cancer patients FFPE samples, extraction of DNA, hybridization capture of prostate cancer HRR genes with the probe of Example 1, according to the conventional high-throughput sequencing method and data processing, alignment, quality control method, obtain the quality control passed sequencing data, for the following mutation detection.
[0065] I. SNV / INDEL mutation detection
[0066] Using the quality control passed sequencing data, using vardict to detect single nucleotide site variation (SNV) and small fragment insertion and deletion (INDEL), and then using vep and annovar to filter the detected mutations. The filtered mutations (somatic mutations and germline mutations) are annotated, and the following standards are met for pathogenic mutations:
[0067] 1) Mutation frequency is greater than 3%;
[0068] 2) The number of sequence coverage at the mutation site is not less than 100;
[0069] 3) The minimum number of sequences supporting the mutation is 3;
[0070] 4) Suspected deleterious variation: truncation variation (such as splicing variation (±2bp)), frameshift variation, nonsense variation, large fragment rearrangement leading to coding region destruction, and part of the database interpreted missense or non-frameshift variation;
[0071] 5) Deleterious variation: variation that meets the suspected deleterious variation condition and is reported in the COMSIC database.
[0072] Determination of the limit of detection (LOD) of SNP / INDEL: 10 enterprise limit of detection reference products were used, containing 25 5% Snvs-Indels (Snvs: 13, Indels: 12), 25 2.5% Snvs-Indels (Snvs: 13, Indels: 12), after high-throughput sequencing, vardict was used for single nucleotide site variation (SNV) and small fragment insertion and deletion (INDEL) detection, then vep and annovar were used to annotate and filter the detected mutations. The experiment was repeated three times. The results are shown in Table 3, at a mutation frequency of 5% of the mutation sites, SNP / INDEL were all detected positive; at a mutation frequency of 2.5% of the mutation sites, one INDEL was not detected. Therefore, the limit of detection of SNP / INDEL mutation detection of FFPE samples is 5%.
[0073] Table 3 SNP / INDEL detection limit results
[0074]
[0075]
[0076] II. Detection of copy number variation (CNV)
[0077] 1. Optimization of copy number variation algorithm
[0078] In order to improve the detection accuracy of copy number variation of HRR genes, in addition to the optimization of probe coverage, the embodiment also improves the copy number variation algorithm. The common Read-Depth strategy is used, and the capture region is divided into regions in a sliding window manner. The fragment size distribution is between 60-400bp, and the number of fragments of each gene is distributed between 20-90. The genome DNA of different normal male cell lines is selected to construct a baseline, and the average depth of the baseline in each interval is calculated. At the same time, the average depth of the sample in each interval is calculated. The average depth of each interval of the sample is normalized with the average depth of the baseline, and GC content correction is used to obtain the log2 value of each interval. According to the calculated tumor purity (purity) and ploidy (ploidy) values. The threshold value of the sample normalized to integer (0, 1, 2, 3…) copy number (copy_nums) is calculated according to the following formula:
[0079] log2((1-purity)+purity×(copy_nums+0.5) / ploidy)
[0080] When the log2 of the interval is within the threshold interval, the absolute integer copy number of the interval is calculated. The distribution of all interval absolute integer copy numbers on each exon is counted, and the threshold is judged according to the threshold, and the threshold is higher than the threshold, and the threshold is lower than the threshold; and the SNPs on each exon interval are counted, and the MAF (Minor Allele Frequency, the minimum allele frequency) distribution is judged to be homozygous deletion or heterozygous deletion.
[0081] 2. Determination of copy number variation limit of detection (LOD)
[0082] Four clinical samples WSN4, WSN5, WSN6 and WSN7 were selected, and the original sample tumor purity was 50%, 57%, 41% and 42% respectively. Each sample was diluted with normal control sample DNA to obtain samples with tumor purity of 30%, 20% and 10% gradient levels. Using these samples, the probes of Example 1 were used for high-throughput sequencing to obtain quality control passed sequencing data, and CNVKIT and CNV-call software were used to calculate CNV. The results are shown in Table 4.
[0083] Table 4 CNV detection results
[0084] Sample Raw purity Mutation Original purity 30% purity 20% purity 10% purity WSN4 50％ BRCA2 LOSS Positive Positive Positive Positive WSN5 57％ ATM AMP Positive Positive Positive Negative WSN6 41％ ATM LOSS Positive Positive Positive Negative WSN7 42％ CDK12 AMP Positive Positive Positive Positive
[0085] According to the above results, when the tumor purity is greater than or equal to 20%, CNV can be detected. When the tumor purity is less than 20%, the CNV detection results of two samples have false negatives. Therefore, the detection limit of CNV of the present application requires that the tumor purity is not less than 20%.
[0086] 3. CNV homozygous, heterozygous deletion and amplification annotation
[0087] The judgment standard of copy number variation is:
[0088] 1) The copy number increase must be observed in at least 60% of the exons, and at least two or more continuous intervals are included;
[0089] 2) The copy number deletion must be observed in at least 5 or more continuous gene exons to judge that the region is deleted;
[0090] 3) The homozygous deletion must have an absolute copy number of 0 in the segment covered by the exon, and the mutation frequency of all SNP sites in the segment does not deviate from 0 or 50% or 100%, to judge that the interval is homozygous deletion.
[0091] Using the sequencing data passed quality control, the distribution of absolute copy number of all segments on each exon was calculated using CNVKIT and CNV-call. When the absolute copy number of an exon is 0 (the absolute copy number of all segments of the exon is 0), and the mutation frequency of all SNPs in the segment does not deviate from 0, 50% or 100%, the exon is annotated as homozygous deletion; when the absolute copy number of five consecutive exons is 1, or the absolute copy number is 0 but the mutation frequency of SNPs in the segment deviates from 0%, 50% or 100%, the exon segment is judged to be heterozygous deletion. The absolute copy number of all segments of the gene is calculated, and when more than 60% of the segments have an absolute copy number greater than 3, the gene is judged to be amplified.
[0092] IV. Annotation of biallelic mutations
[0093] Based on the above SNV / INDEL mutation detection and copy number variation detection results, it is determined whether the HRR gene of the sample has a biallelic mutation. The judgment conditions are as follows:
[0094] 1) Two or more different pathogenic somatic mutations;
[0095] 2) One pathogenic germline mutation accompanied by allelic heterozygous deletion;
[0096] 3) One pathogenic somatic mutation accompanied by allelic heterozygous deletion;
[0097] 4) Two mutations close to each other, but not on the same sequencing sequence;
[0098] 5) Simultaneous occurrence of pathogenic germline mutation and pathogenic somatic mutation;
[0099] 6) Simultaneous occurrence of two different pathogenic somatic mutations;
[0100] 7) Homozygous deletion.
[0101] If any of the above conditions is met, it is determined that there is a biallelic mutation.
[0102] The results show that among the 20 samples of metastatic castration-resistant prostate cancer patients, 15 samples are positive (9 of which are biallelic mutations, as shown in Table 5), and the remaining 5 samples are negative.
[0103] Table 5: Results of biallelic pathogenic mutation detection
[0104]
[0105] The above 20 patients were followed up, and the patient's medication and progression-free survival (PFS) were recorded. Among them, 6 samples have follow-up results, and the follow-up data are as follows:
[0106] Table 6 6 patients follow-up data results
[0107]
[0108] According to the follow-up data, it is found that the progression-free survival (PFS) of HRR-positive patients is significantly improved, and the HRR gene mutation can assist in guiding the PARP inhibitor treatment of metastatic castration-resistant prostate cancer. The PARP inhibitor treatment effect of patients with double allelic mutation of BRCA2 gene is better than that of patients with single allelic mutation (P2 vs P12, P8 vs P12), and the PARP inhibitor treatment effect of patients with double allelic homozygous mutation is better than that of patients with double allelic heterozygous mutation (P2 vs P8). However, the PFS of the CDK12 gene positive sample (P6) is not significantly improved, and the drug effect is not obvious.
[0109] The above embodiments are only feasible or preferred embodiments of the present application, which are used to illustrate the present application, and are not used to limit the scope of the patent application. Therefore, all equivalent changes and modifications made according to the scope of the patent application of the present application should belong to the scope covered by the present patent.
Claims
1. A probe set for capturing the HRR gene in prostate cancer, characterized in that, The probe set captures genomic regions including target regions of 45 HRR genes as shown in Table 1.
2. The probe assembly according to claim 1, characterized in that, The genomic regions captured by the probe set also include large rearrangement regions.
3. The probe assembly according to claim 2, characterized in that, The large fragment rearrangement regions include eight intron regions of the five genes shown in Table 2.
4. The probe assembly according to claim 3, characterized in that, The genomic regions captured by the probe set also include backbone SNPs in intron regions. The population frequency of these backbone SNPs is 5% to 95%, and they do not reach exon regions upstream or downstream. The spacing between backbone SNPs is 50k.
5. A method for designing a probe set to capture the HRR gene in prostate cancer, characterized in that the steps include... include: For the 45 HRR genes in Table 1, full exon coverage was performed, with 60 bp extensions on both sides of the exon regions. For exons shorter than 200 bp that were not covered by adjacent introns, additional extensions were performed to ensure that each exon was covered by at least two probes. The study covered all introns of the BRCA1 and BRCA2 genes, as well as eight intron regions of the five genes ATM, BRIP1, CDK12, RAD51D, and RAD51B listed in Table 2, and fully covered intron regions of genes with a length of less than 600 bp. Design of shingled probes targeting the high AT and GC regions of gene exons; The backbone SNPs are covered for longer intron regions; the selection method for the backbone SNPs is as follows: (1) Select SNPs with a population frequency of 5% to 95%; (2) Remove SNPs that deviate significantly from the overall range in different populations; (3) Prioritize SNPs with high MAF values and high heterozygosity in the Chinese population; (4) The upstream and downstream regions of SNP sites do not touch the exon regions; (5) The GC content in the probe region is controlled between 25% and 75%; (6) Analyze and evaluate the uniqueness of the captured sequences; (7) Remove linked unbalanced SNPs at intervals of 50k to obtain the final SNPs.
6. A reagent kit, characterized in that, Includes the probe set as described in any one of claims 1-4.
7. The use of the probe set according to any one of claims 1-4 in the preparation of a reagent for detecting HRR gene mutations in prostate cancer, wherein the use is not for the purpose of disease diagnosis and treatment, characterized in that, The prostate cancer HRR gene mutations include one or more of the following: single nucleotide site variation, small fragment insertion / deletion, copy number variation, large fragment rearrangement, and biallelic mutation.
8. The application of the probe set according to any one of claims 1-4 in high-throughput sequencing of the prostate cancer HRR gene, mutation detection of the prostate cancer HRR gene, risk assessment of prostate cancer, auxiliary diagnosis of prostate cancer, and adjuvant treatment of prostate cancer, wherein the application is not for the purpose of diagnosis and treatment of the disease.
9. The application of the kit according to claim 6 in high-throughput sequencing of the HRR gene in prostate cancer, mutation detection of the HRR gene in prostate cancer, risk assessment of prostate cancer, auxiliary diagnosis of prostate cancer, and adjuvant treatment of prostate cancer, wherein the application is not for the purpose of diagnosis and treatment of the disease.
10. A method for determining biallelic mutations, wherein the method is not intended for the diagnosis and treatment of diseases, characterized in that the steps include... include: 1) The HRR gene of prostate cancer is captured by hybridization using the probe set described in any one of claims 1-4, and high-throughput sequencing is performed; 2) The sequencing data is split, preprocessed, aligned, and quality controlled to obtain sequencing data that has passed quality control; 3) Use the sequencing data obtained in step 2) to detect single nucleotide site variations, small fragment insertions / deletions, and copy number variations; 4) Determine whether it is a biallelic mutation based on the test results of step 3).
11. The method for determining biallelic mutations according to claim 10, characterized in that, The copy number variation algorithm includes the following steps: Using a read-depth strategy, the probe capture region is divided into 20-90 fragment intervals with a fragment size of 60-400 bp using a sliding window approach; genomic DNA from different non-tumor male cell lines is selected to construct baselines; the average depth of the baseline and the sample in each interval is calculated and normalized, while GC content correction is used to obtain the log2 value of each interval; based on the calculated tumor purity and ploidy value, the threshold for normalizing the sample to an integer copy number is calculated using the following formula: Threshold = log2((1 - tumor purity) + tumor purity × (copy number + 0.5) / ploidy) When the log2 value of an interval is within the threshold range, calculate the absolute integer copy number of that interval; count the distribution of the absolute integer copy number of all intervals in each exon, and judge according to the threshold: those above the threshold are amplifications, and those below the threshold are deletions; count the SNPs in each exon segment, and determine whether they are homozygous deletions or heterozygous deletions based on their MAF distribution.
12. The method for determining biallelic mutations according to claim 11, characterized in that, The criteria for determining copy number variation are: 1) Increased copy number: If the absolute copy number of more than 60% of the gene regions is greater than 3 and contains at least two consecutive regions, then the gene is considered to be amplified. 2) Copy number missing: If a copy number missing is observed in 5 or more consecutive exons within a range, then the range is considered missing. 3) Homozygous deletion: If the absolute copy number of the exon-covered interval is 0 and the mutation frequency of all SNP sites within the interval does not deviate from 0, 50% or 100%, then the interval is judged to be a homozygous deletion. 4) Heterozygous deletion: If the absolute copy number of 5 or more consecutive exons in the interval is 1, or if the absolute copy number is 0 but the mutation frequency of SNP sites in the interval deviates from 0, 50% or 100%, then the interval is judged as a heterozygous deletion.
13. The method for determining biallelic mutations according to any one of claims 10-12, characterized in that, Step 4), the criteria for determining the biallelic mutation are: 1) Two or more different pathogenic somatic cell mutations; 2) A pathogenic germline mutation accompanied by allele heterozygous deletion; 3) A pathogenic somatic cell mutation is accompanied by the loss of heterozygosity of alleles; 4) Two mutations located very close to each other, but not on the same sequencing sequence; 5) Both pathogenic germline mutations and pathogenic somatic cell mutations occur simultaneously; 6) Simultaneous mutations in two different pathogenic somatic cells; 7) Homozygous absence; If one or more of the above conditions are met, it is judged to be a biallelic mutation.
14. The method for determining biallelic mutations according to claim 13, characterized in that, Mutations that meet the following criteria are considered pathogenic somatic mutations or pathogenic germline mutations: 1) Mutation frequency greater than 3%; 2) The number of sequences covered by the mutation site is not less than 100; 3) There must be at least 3 sequences that support the mutation; 4) Suspected harmful variants: truncated variants, shifted variants, nonsense variants, large-fragment rearrangements that cause damage to the coding region, and some missense or non-shifted variants that have been interpreted by the database. 5) Harmful variants: Variants that meet the criteria for suspected harmful variants and are reported in the COMSIC database.