Detection method for variation type of haemophilia virulence gene

Abstract

The invention relates to the technical field of gene detection, in particular to a hemophilia virulence gene variation type detection method, which comprises the following steps: comparing sequence reading of third-generation long-reading long sequencing with a reference genome, comparing a part of sequence reading to an F9 gene region and a VWF gene region to obtain a first comparison result, and comparing the first comparison result with a second comparison result to obtain a second comparison result; performing first variation analysis according to the first comparison result, outputting a first analysis result, performing haplotype typing on the F8 gene region to obtain a differential base sequence of an unconventional gene and a highly homologous sequence of the unconventional gene, and comparing the differential base sequence with the residual sequence reading to obtain a second comparison result; and performing second variation analysis according to the second comparison result, and outputting a second analysis result. According to the application, multiple variation types of the hemophilia virulence gene can be screened at the same time, and variation analysis of the F8 gene with homologous gene sequence interference can be solved.

Description

Technical Field

[0001] The present application relates to the technical field of gene detection, and in particular to a method for detecting the variant type of a hemophilia-causing gene. Background Art

[0002] Hemophilia is a common X-linked recessive genetic disease, in which the types lacking coagulation factor VIII and coagulation factor IX are called hemophilia A and hemophilia B respectively, which together with von Willebrand disease (VWD) constitute 95% to 97% of hereditary bleeding diseases. Patients with this type of disease often have a lifelong bleeding tendency, which affects their social function and quality of life, and may experience severe or even fatal bleeding during trauma, surgery, menstruation or childbirth.

[0003] There are many types of genetic mutations that cause hemophilia, which can be divided into the following categories according to the type of disease: (1) Hemophilia A: caused by mutations in the F8 gene, which encodes coagulation factor VIII. Mutations include intron 22 inversion, point mutations, deletions, or abnormal gene insertions. (2) Hemophilia B: caused by mutations in the F9 gene, which encodes coagulation factor IX. It is known that the F9 gene has a variety of mutation forms, including deletions, insertions, and point mutations. (3) Von Willebrand Disease (VWD): caused by mutations in the VWF gene, which encodes von Willebrand factor (vWF). The VWF gene is located at the end of the short arm of chromosome 12 (12p13.3) and contains 52 exons. Mutations may include a reduction in quantity or abnormal quality. There are many specific mutation types, including missense mutations, nonsense mutations, splice site mutations, deletions, and insertions. Among the above-mentioned hemophilia-causing genes, the structural variation of the F8 gene is mainly that int22h-1 located in intron 22 of the F8 gene is prone to recombination with two sets of homologous sequences int22h-2 and int22h-3 on its telomeric side, forming intron 22 inversion (including Inv22 Type I and Inv22 Type II). Other int22h-related recombination such as deletion (Del22) and duplication (Dup22) have also been reported. In addition, the variation types of the F8 gene also include intron 1 inversion and other large-scale structural variations. Most of the variation types detected in the F9 gene and VWF gene are point mutations, but there are also large-scale duplications, insertions, deletions and other complex structural variations.

[0004] In recent years, the development of personalized treatments such as gene therapy and targeted therapy has provided more options for patients with such genetic diseases. In addition, with the advancement of technologies such as prenatal diagnosis and preimplantation genetic testing, the diagnosis and treatment of genetic diseases is constantly moving forward. However, the implementation of these technologies requires accurate diagnosis of gene defects as a prerequisite.

[0005] At present, there are still challenges in the accurate detection of hemophilia genes. Taking the genetic detection of hemophilia A as an example, point mutations and small structural variations are mainly screened through Sanger sequencing and next-generation sequencing (NGS). However, nearly half of severe hemophilia A cases are caused by inversion of intron 22 and other types of large structural variations, which require long-distance PCR, reverse PCR, multiple ligation probe amplification (MLPA) and other technologies to identify. However, these technologies are difficult to operate and lack accuracy, so they have not been widely used in clinical practice. In order to thoroughly clarify the type of mutation, a set of detection processes combining multiple methods is required. The expensive detection fee is also one of the reasons for the low coverage of genetic testing.

[0006] In actual clinical cases, there are also cases where the clinical phenotype results of patients are inconsistent with the genotype test results. For example, the clinical test results of female samples were found to be F8 protease deficiency, but the inversion results and abnormal mutations were not found through long-distance PCR and NGS testing, and the int22h1-h2 heterozygous deletion type was found through CNV testing. The limitations of detection technology have extremely high requirements for clinical practitioners to judge the test results. It is necessary to combine multiple test results to molecularly type the patient's phenotype, which brings great difficulties to clinical diagnosis.

[0007] Therefore, there is an urgent need to develop a detection method for hemophilia pathogenic genes that can highly accurately identify point mutations and large-fragment complex structural mutations and other types of mutations in one go, so as to address the shortcomings of traditional detection methods, such as low efficiency, high cost and inaccurate judgment of test results in the combined application of multiple methods. Summary of the invention

[0008] Based on this, one or more embodiments of the present application provide a method, detection device, computer equipment, computer readable storage medium and computer program product for detecting variant types of hemophilia pathogenic genes. The detection method of the present application is applicable to people with normal phenotype or phenotype, and can simultaneously perform carrier screening and diagnosis on multiple variant types of multiple genes of hemophilia, and can simultaneously detect point mutations, insertions and deletions, copy number variations and structural variations in the same reaction system, with low detection cost and accurate judgment results.

[0009] The technical solution of this application includes the following contents:

[0010] A method for detecting the variant type of a hemophilia-causing gene comprises the following steps:

[0011] S100, obtaining genomic DNA from the sample to be tested;

[0012] S200, the genomic DNA is fragmented and adapter-added to obtain a pre-capture library;

[0013] S300, targetedly capturing the target region in the pre-capture library to obtain a post-capture library; the target region includes the F8 gene, the F9 gene, the VWF gene and the SRY gene region;

[0014] S400, constructing a sequencing library using the post-capture library and performing third-generation long-read sequencing to obtain sequence reads;

[0015] S500, aligning the sequence reads with a reference genome, aligning a portion of the sequence reads to the F9 gene and VWF gene regions to obtain a first alignment result, performing a first variation analysis based on the first alignment result, outputting a first analysis result, performing haplotype typing on the F8 gene region, obtaining a differential base sequence between the unconventional gene and a highly homologous sequence thereof, aligning the differential base sequence with the remaining portion of the sequence reads to obtain a second alignment result, performing a second variation analysis based on the second alignment result, and outputting a second analysis result; analyzing the gender of the sample to be tested based on the alignment result of the sequence reads with the SRY gene region in the reference genome;

[0016] S600, summarizing the first analysis result and the second analysis result, and outputting a gene mutation analysis result;

[0017] S700, comparing the gene mutation analysis result, the gender of the sample to be tested and the clinical information of the sample to be tested to achieve typing of hemophilia;

[0018] Among them, the conventional gene refers to a gene without a highly homologous sequence, the unconventional gene refers to a gene with a highly homologous sequence, and the gene with a highly homologous sequence means that the gene has a homologous sequence with a similar sequence structure within the genome, and the similarity between the gene sequence and the homologous sequence is ≥90%;

[0019] The first analysis result and the second analysis result each independently include at least one of the following gene mutation results: single nucleotide polymorphism, small-scale insertion and deletion, structural variation and complex genome rearrangement.

[0020] In some embodiments of the present application, the genomic DNA is fragmented and treated with adapters to obtain a pre-capture library, comprising:

[0021] The genomic DNA is fragmented by a transposase method or a physical shearing method and a sequencing adapter is introduced to obtain a DNA fragment with an adapter;

[0022] The fragments with a length of less than 5 kb in the DNA fragments with adapters are removed to obtain the pre-capture library.

[0023] In some embodiments of the present application, the method for targeted capture of the target region in the pre-capture library comprises:

[0024] (1) using the probe composition to perform a probe hybridization capture method on the target region;

[0025] (2) Performing multiplex PCR amplification on the target region using the primer combination.

[0026] In some embodiments of the present application, performing a first variation analysis according to the first comparison result includes:

[0027] Analyzing first variation information of the conventional gene according to the first comparison result, wherein the first variation information includes single nucleotide polymorphism, small-scale insertion and deletion, and structural variation;

[0028] Furthermore, the structural variation includes copy number variation.

[0029] In some embodiments of the present application, performing a first variation analysis according to the first comparison result and outputting a first analysis result further includes:

[0030] The conventional gene is subjected to haplotype typing according to the information of the heterozygous SNP in the first comparison result.

[0031] In some embodiments of the present application, performing a second variation analysis according to the second comparison result includes:

[0032] analyzing second variation information of the unconventional gene according to the second comparison result, wherein the second variation information includes at least one of single nucleotide polymorphism, small-scale insertion and deletion, structural variation and complex genome rearrangement;

[0033] Furthermore, the structural variation includes at least one of copy number variation, gene fusion, and large-segment gene conversion and inversion;

[0034] Furthermore, the complex genome rearrangement refers to the occurrence of multiple types of structural variations within the same gene region.

[0035] In some embodiments of the present application, the gene mutation analysis result, the gender of the sample to be tested and the clinical information of the sample to be tested are compared to achieve the type typing of hemophilia, including:

[0036] By analyzing the pathogenic variants of VWF, F8 and F9 genes and comparing the activity levels of vWF antigen, FVIII and FIX factors with clinical information, the type of hemophilia suffered by the organism from which the sample was tested was confirmed.

[0037] A detection device, comprising:

[0038] The alignment module is used to align the sequence reads of the third-generation long-read sequencing with the reference genome;

[0039] An analysis module is used to analyze the comparison results of the sequence reads of the third-generation long-read sequencing with the reference genome: a part of the sequence reads is compared to the F9 gene and VWF gene regions to obtain a first comparison result, a first variation analysis is performed based on the first comparison result, and the first analysis result is output, and the F8 gene region is subjected to haplotype typing to obtain the differential base sequence between the unconventional gene and its highly homologous sequence, and the differential base sequence is compared with the remaining part of the sequence reads to obtain a second comparison result, a second variation analysis is performed based on the second comparison result, and a second analysis result is output; according to the comparison result of the sequence reads with the SRY gene region in the reference genome, the gender of the sample to be tested is analyzed;

[0040] An output module is used to summarize the first analysis result and the second analysis result and output a gene mutation analysis result; compare the gene mutation analysis result, the gender of the sample to be tested and the clinical information of the sample to be tested to achieve the type typing of hemophilia;

[0041] Among them, the conventional gene refers to a gene without a highly homologous sequence, the unconventional gene refers to a gene with a highly homologous sequence, and the gene with a highly homologous sequence means that the gene has a homologous sequence with a similar sequence structure within the genome, and the similarity between the gene sequence and the homologous sequence is ≥90%;

[0042] The first analysis result and the second analysis result each independently include at least one of the following gene mutation results: single nucleotide polymorphism, small-scale insertion and deletion, structural variation and complex genome rearrangement;

[0043] The clinical information of the sample to be tested includes at least one of the test results of vWF antigen, FVIII and FIX active factors.

[0044] A computer device comprises a memory and a processor, wherein the memory stores a computer program, and wherein the processor implements the steps of the detection method described above when executing the computer program.

[0045] A computer-readable storage medium stores a computer program, wherein the computer program implements the steps of the detection method described above when executed by a processor.

[0046] A computer program product comprises a computer program, wherein the computer program implements the steps of the detection method described above when executed by a processor.

[0047] The detection method of this application is based on the targeted detection technology of the third-generation long-length sequencing. Through a set of experimental processes, it takes into account the accurate identification of point mutations and various large-fragment structural variations, achieving the same effect as combining multiple traditional detection methods, and at the same time can provide more potential information for clinical diagnosis and disease typing. Compared with the combination of other detection methods, it has the advantages of simplifying experimental operations, increasing sequencing throughput, and reducing detection costs.

[0048] The detection method of the present application captures the target region through long-fragment targeted capture technology, and then constructs a third-generation sequencing library from the captured library to obtain third-generation long-read sequencing data, thereby realizing the detection of hemophilia-related genes F8, F9 and VWF gene. Based on the high-precision and long-length characteristics of third-generation sequencing, the detection method can analyze the highly homologous regions of the F8 gene. In view of the difference in the results of hemophilia in gender, the detection method includes the detection of the SRY gene to distinguish between genders.

[0049] After the third-generation sequencing platform is used for targeted sequencing, the detection method of the present application uses a long-fragment genome assembly to construct a haplotype for the pathogenic variation and clarify the cis and trans conditions of the variation. This can solve the variation analysis of the F8 gene with homologous gene sequence interference, including accurate identification of point mutations, insertions and deletions, copy number variations, structural variations, etc.; at the same time, the copy number is determined according to the haplotype to solve the problem of identification accuracy of the third-generation sequencing copy number variation. The detection method, detection device, computer equipment, computer-readable storage medium and computer program product of the present application can compare the genotype results of the subject obtained by the test with the clinical indicator information, accurately distinguish the molecular subtype information of the subject, assist clinical diagnosis, determine the pathogenic gene, and provide a basis for carrier detection and prenatal diagnosis in the same family. 。 BRIEF DESCRIPTION OF THE DRAWINGS

[0050] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0051] Figure 1 IGV spectrum of sample A4 in Example 1 of the present application;

[0052] Figure 2 IGV spectrum of sample A4-2 of Example 1 of the present application;

[0053] Figure 3 IGV spectrum of sample A5 in Example 1 of the present application;

[0054] Figure 4 IGV spectrum of sample A9 in Example 1 of the present application;

[0055] Figure 5 IGV spectrum of sample B1 in Example 1 of the present application;

[0056] Figure 6 IGV spectra of samples B2 and B2-2 of Example 1 of the present application;

[0057] Figure 7 is the IGV spectrum of sample X1 in Example 1 of the present application;

[0058] Figure 8 This is a gel electrophoresis diagram of sample A4 of comparative example 1 of the present application detected by gel electrophoresis long fragment PCR method. DETAILED DESCRIPTION

[0059] The present application will be further described below in conjunction with the embodiments and examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. In addition, it should be understood that after reading the content taught in the present application, those skilled in the art may make various changes or modifications to the present application, and these equivalent forms also fall within the protection scope of the claims attached to the present application.

[0060] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0061] the term

[0062] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0063] The term "and / or" used in this article includes any one of two or more related listed items, and also includes any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B.

[0064] Herein, "preferred", "preferable", "better", etc. are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of this application.

[0065] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0066] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0067] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0068] The hemophilia-causing genes of the present application include F8 gene, F9 gene and VWF gene.

[0069] In order to obtain a detection method for screening for hemophilia pathogenic gene mutations with higher detection accuracy, better efficiency and simpler operation, the researchers of this application conducted a large amount of literature research.

[0070] Studies have found that high-throughput sequencing has been widely used in the detection of mutations in hemophilia pathogenic genes such as the F8 gene. In order to improve throughput and sequencing depth, it is necessary to first target enrichment of the target gene. At present, the targeted enrichment technology for short-read sequencing platforms is relatively mature, and some teams are also exploring the targeted enrichment of long-fragment DNA. Using nucleic acid probes to capture target genes is a simple and efficient method, and there are precedents for long-fragment sequencing platforms. However, due to the short length of the prepared library DNA fragments, it is not enough to completely span the 9.5kb int22h homologous sequence, and conventional gene alignment software cannot cope with these highly homologous DNA fragments. In the process of constructing the library, the use of pulsed field electrophoresis and other technologies to screen long-fragment DNA may be a potential solution, but it is difficult to promote and apply it to high-throughput detection due to the long detection time and economic cost. In addition, there are reports on the use of multiplex PCR amplification to obtain target long-fragment DNA. At the same time, since the position of PCR primers is fixed in hot spot mutation regions such as exons, this may miss the opportunity to discover new mutation sites. As a popular technology in the field of gene editing, the CRISPR-Cas9 system can also be used to target and obtain long DNA fragments. The library fragments prepared by this method retain the epigenetic information of the DNA, but currently it has no advantages in enrichment efficiency and target number.

[0071] In addition, carrier screening based on next-generation sequencing and probe targeted capture is limited by the short read length of NGS, and cannot detect complex mutations in hemophilia pathogenic gene mutations, such as inversion of intron 22 of F8 and other types of large-fragment structural mutations. Due to the interference of highly homologous sequences F8A2 and F8A3, the mutations in the corresponding regions cannot be directly detected. For highly homologous sequences or pseudogenes, due to the influence of the short read length of NGS, it is impossible to determine whether the sequence carrying the mutant base comes from the functional gene or its homologous sequence. Therefore, in the test results for the above genes, false negatives or false positives often occur, which need to be verified and supplemented by other detection technologies. In addition, for a gene mutation with two or more mutations, it is particularly important to determine the cis and trans conditions of the mutation in order to assess the pathogenicity of the individual. However, NGS detection technology is limited by the read length and cannot determine the above situation. For the verification of such cases, PCR amplification primers need to be designed for verification, which consumes a lot of manpower and cannot achieve high-throughput quantification.

[0072] At present, the hemophilia screening products on the market cannot simultaneously detect point mutations and various large-fragment structural variations and other types of mutations in the same experimental system. Usually, multiple methods are required in combination, such as NGS+MLPA+LD-PCR and other multi-method combined detection.

[0073] The study also found that for the screening and diagnosis of hemophilia, since the pathogenic gene is located on the X chromosome, it is necessary to distinguish between males and females. For the MLPA and LD-PCR methods, additional detection of gender-related genes is required. At the same time, the test results of males and females are also different for the relationship between hemophilia-related phenotypes, clinical indicators and genotypes. For example, male carriers of inversion of intron 22 of the F8 gene have a phenotype (diagnosed as severe hemophilia A), while female carriers of heterozygous inversions will not have a phenotype. In clinical practice, we found that if a female has a heterozygous CNV deletion of F8, there will be abnormal activity levels of the clinical indicator FVIII factor. Therefore, it is necessary to associate the one-time test results with the patient's phenotype to obtain the results of molecular typing.

[0074] This application aims to overcome the existing technical difficulties of long-fragment sequencing of the targeted F8 gene, while also covering the screening of F9 and VWF, optimizing the experimental process and bioinformatics analysis, and providing a comprehensive solution for accurate genetic testing for the clinical diagnosis of hemophilia and VWD. In view of the difference in the results of hemophilia in terms of gender, this application also aims to simultaneously test the SRY gene for gender differentiation.

[0075] One or more embodiments of the present application provide a method for targeted detection based on high-precision, long-read third-generation genome sequencing technology. The method is suitable for people with normal phenotypes or those with phenotypes, and can simultaneously perform carrier screening and diagnosis of multiple variant types of multiple genes for hemophilia. It can achieve simultaneous detection of point mutations, insertions and deletions, copy number variations, structural variations and other variations in the same reaction system.

[0076] A method for detecting a variant type of a hemophilia-causing gene according to an embodiment of the present application comprises the following steps:

[0077] S100, obtaining genomic DNA from the sample to be tested;

[0078] S200, genomic DNA was fragmented and adapter-added to obtain the pre-capture library;

[0079] S300, targeted capture of the target region in the pre-capture library to obtain the post-capture library; the target region includes the F8 gene, F9 gene, VWF gene and SRY gene regions;

[0080] S400, constructing a sequencing library using the post-capture library and sequencing the library to obtain sequence reads;

[0081] S500, aligning the sequence reads with the reference genome, aligning a portion of the sequence reads to the F9 gene and VWF gene regions to obtain a first alignment result, performing a first variation analysis based on the first alignment result, outputting a first analysis result, performing haplotype typing on the F8 gene region, obtaining a differential base sequence between the unconventional gene and its highly homologous sequence, aligning the differential base sequence with the remaining portion of the sequence reads to obtain a second alignment result, performing a second variation analysis based on the second alignment result, and outputting a second analysis result; analyzing the gender of the sample to be tested based on the alignment result of the sequence reads with the SRY gene region in the reference genome;

[0082] S600, summarizing the first analysis result and the second analysis result, and outputting a gene mutation analysis result;

[0083] S700, comparing the gene mutation analysis result, the gender of the sample to be tested and the clinical information of the sample to be tested to achieve typing of hemophilia;

[0084] Among them, conventional genes refer to genes without highly homologous sequences, unconventional genes refer to genes with highly homologous sequences, and genes with highly homologous sequences refer to genes with homologous sequences with similar sequence structures within the genome, and the similarity between the gene sequence and the homologous sequence is ≥90%;

[0085] The first analysis result and the second analysis result each independently include at least one of the following gene mutation results: single nucleotide polymorphism, small-scale insertion and deletion, structural variation and complex genome rearrangement;

[0086] The clinical information of the sample to be tested includes at least one of the test results of vWF antigen, FVIII and FIX active factors.

[0087] Hemophilia type typing results, based on the results of gene pathogenic variant testing and clinical information, classify the disease into severe, moderate or mild.

[0088] Optionally, methods for obtaining genomic DNA from the sample to be tested include methods compatible with magnetic bead method, column method for extracting DNA, and automated DNA extraction method, etc.

[0089] Optionally, the third-generation sequencing platform includes a single-molecule real-time sequencing technology platform or a single-molecule nanopore sequencing technology platform.

[0090] Optionally, the genomic DNA is fragmented and adapter-added to obtain a pre-capture library, comprising:

[0091] The genomic DNA is fragmented by a transposase method or a physical shearing method and a sequencing adapter is introduced to obtain a DNA fragment with an adapter;

[0092] The fragments with a length of less than 5 kb were removed from the DNA fragments with adapters to obtain the pre-capture library.

[0093] Optionally, the method of targeted capture of a target region in a pre-capture library comprises:

[0094] (1) using the probe composition to perform a probe hybridization capture method on the target area;

[0095] (2) Multiplex PCR amplification of the target region using the primer combination.

[0096] The analytical steps in the detection method of this application are:

[0097] In step S500, the sequence reads are compared with the reference genome, a portion of the sequence reads are compared to the F9 gene and VWF gene regions to obtain a first comparison result, a first variation analysis is performed based on the first comparison result, the first analysis result is output, and the F8 gene region is haplotyped to obtain a differential base sequence between the unconventional gene and its highly homologous sequence, the differential base sequence is compared with the remaining portion of the sequence reads to obtain a second comparison result, a second variation analysis is performed based on the second comparison result, and the second analysis result is output.

[0098] In some embodiments of the present application, performing a first variation analysis according to the first comparison result includes:

[0099] Analyzing first variation information of conventional genes according to the first comparison result, the first variation information including single nucleotide polymorphisms, small-scale insertions and deletions, and structural variations;

[0100] Furthermore, structural variation includes copy number variation.

[0101] Further, performing a first variation analysis according to the first comparison result and outputting the first analysis result also includes:

[0102] The conventional gene is haplotyped based on the information of the heterozygous SNP in the first alignment result.

[0103] In some embodiments of the present application, performing a second variation analysis according to the second comparison result includes:

[0104] analyzing second variation information of the unconventional gene according to the second comparison result, the second variation information comprising at least one of single nucleotide polymorphism, small-scale insertion and deletion, structural variation and complex genome rearrangement;

[0105] Further, the structural variation includes at least one of copy number variation, gene fusion, and large segment gene conversion and inversion;

[0106] Furthermore, complex genomic rearrangements refer to situations in which multiple types of structural variations occur within the same gene region.

[0107] In some embodiments of the present application, in step S500, the F8 gene is haplotyped to obtain a differential base sequence between the F8 gene and its highly homologous sequence, and the differential base sequence is compared with the remaining sequence reads to obtain a second comparison result, and a second variation analysis is performed based on the second comparison result to output a second analysis result; the highly homologous sequence regions of the F8 gene include F8int22h1, F8int22h2 and F8int22h3 regions.

[0108] Specifically, the method for performing haplotype typing on the F8 gene comprises the following steps:

[0109] S541, aligning the sequence reads with the reference group genome, and extracting the sequencing fragment sequence set S1 aligned to F8int22h1, F8int22h2, and F8int22h3;

[0110] S542, establish the reference genome index of F8int22h2;

[0111] S543, compare the differences among the reference sequences F8int22h1, F8int22h2, and F8int22h3, record the difference position set B (B1 ... BN) of the difference base sequences of F8int22h2, F8int22h1, and F8int22h3, for position B1, record the base information set BN (BN1-1, BN1-2 ... BN1-N) corresponding to B1, and the reference genome number C (e.g., F8int22h1 / F8int22h2 / F8int22h3) corresponding to the set BN, and complete the recording of all base information sets in the set B and the corresponding reference genome numbers;

[0112] S544, according to the set S1 obtained in step S541, align it to the reference genome corresponding to F8int22h to obtain a sequence alignment result;

[0113] S545. According to the result of step S544, for the sequence aligned to the reference genome F8int22h, based on the set B recorded in S544 and the corresponding base set BN and the corresponding reference genome number C, all positions of the set B are traversed and the reference genome number is marked for the sequence aligned in step S544;

[0114] S546. According to the result of step S545, when all positions of set B on the aligned sequence are marked as reference genome F8int22h, the sequence is recorded as F8int22h haplotype; when it is marked as reference genome F8int22h1, the sequence is recorded as F8int22h1 haplotype; when it is marked as reference genome F8int22h3, the sequence is recorded as F8int22h3 haplotype, and the haplotype marking of all aligned sequences is completed in sequence; when there is an aligned sequence, the set B starting at the 5' end is marked as F8int22h1, and the set B site starting at the 3' end is compared as F8int22h2 or F8int22h3, it is recorded as a fusion haplotype type set F (F1...FN);

[0115] S547, performing cluster analysis on the sequences of all haplotype types in step S546, for all sequences of the F8int22h1 haplotype, when two or more haplotypes of different sequences are clustered, the haplotype number set is recorded as F8int22h1-Hap (Hap1..HapN) and the corresponding average sequence number set D (D1...DN); when only one haplotype of sequence is clustered, the haplotype number is recorded as F8int22h1-Hap1; and so on, completing the clustering of other haplotype types (F8int22h2, F8int22h3 and fusion haplotype F), and recording the haplotype number set and the corresponding average sequence number set;

[0116] S548, analyzing the mutation type according to the haplotype number set obtained in step S547:

[0117] In the case where only one or multiple non-consecutive positions in set B were marked as non-F8int22h1, the haplotype type was considered to still be F8int22h1, with the presence of a point mutation or indel;

[0118] There is a fusion gene haplotype type set F, which is considered to have undergone structural variation, among which:

[0119] When the set B starting from the 5' end of the sequence corresponding to the haplotype set F is marked as F8int22h1, the 3' end starting set B site is compared as F8int22h2, and there is no copy number loss in the region from F8int22h1 to F8int22h2 of the reference genome, it is determined to be F8int22 type II inversion. Otherwise, if there is a copy number loss, it is determined to be a F8int22h1 to F8int22h2 loss.

[0120] When the sequence corresponding to the haplotype set F, the set B starting from the 5' end is marked as F8int22h1, and the site of the set B starting from the 3' end is compared as F8int22h3, and there is no copy number loss in the region from F8int22h1 to F8int22h3 of the reference genome, it is determined to be F8int22 type I inversion. Otherwise, if there is a copy number loss, it is determined to be a loss from F8int22h1 to F8int22h3;

[0121] According to the number of haplotype numbers in F8int22h1: if the number of haplotype numbers in male samples is greater than 1 or greater than 2 in female samples, it is considered that the F8int22h1 region of the gene has a copy number duplication; if the number of haplotype numbers in male samples is less than 1 or less than 2 in female samples, it is considered that the F8int22h1 region of the gene has a copy number deletion;

[0122] When the value of the average sequence number DN corresponding to a haplotype number in set B is 1.5-2.5 times the value of D of other numbers, it is considered that there are two identical copies of the haplotype number, and the above copy number variation results are updated.

[0123] The result output steps in the detection method of this application are:

[0124] In step S600, the first analysis result and the second analysis result are summarized and a gene mutation analysis result is output.

[0125] The first analysis result and the second analysis result each independently include at least one of the following gene mutation results: single nucleotide polymorphism, small-scale insertion and deletion, structural variation and complex genome rearrangement.

[0126] In the diagnosis of hemophilia, after the sample has been genetically tested, it is still necessary to further compare the gender and phenotype to determine the subtype. This is because: genetic testing can determine the pathogenic gene, but the phenotype (i.e., the level of coagulation factor activity) is the key to determining the subtype of hemophilia. In the detection of hemophilia, hemophilia is divided into mild, intermediate and severe types according to the level of coagulation factor activity. Mild patients generally seldom bleed, and only bleed after injury or surgery. Severe patients may have spontaneous bleeding since childhood. The severity of bleeding in intermediate patients is between mild and severe. As mentioned above, since hemophilia is an X-linked recessive genetic disease, men have only one X chromosome, so if there is a coagulation factor defect, they will show symptoms; while women have two X chromosomes, even if one X chromosome has a coagulation factor defect, the other normal X chromosome can usually produce enough coagulation factors to prevent bleeding symptoms, so women are usually asymptomatic carriers. Therefore, for samples of different genders, further clinical information is needed to determine the specific disease subtype.

[0127] Therefore, in step S700, the gene mutation analysis result, the gender of the sample to be tested and the clinical information of the sample to be tested are also compared to achieve the type classification of hemophilia.

[0128] The clinical information of the sample in the embodiment of the present application includes at least one of the detection results of vWF antigen, FVIII and FIX active factors.

[0129] vWF antigen (von Willebrand Factor Antigen, vWF:Ag for short) is the antigenic form of von Willebrand Factor (VWF for short). VWF is a large multimeric glycoprotein synthesized by vascular endothelial cells and megakaryocytes. Its main functions include promoting platelet adhesion at vascular injuries and acting as a carrier of coagulation factor VIII (FVIII) to protect it from degradation, thus playing an important role in the hemostasis process. The determination of vWF antigen is mainly used for the diagnosis and differential diagnosis of von Willebrand disease (VWD), and its increased or decreased levels can reflect different pathological states.

[0130] FVIII active factor, namely coagulation factor VIII activity (FVIII:C), refers to the coagulation activity of coagulation factor VIII. FVIII is a macromolecular complex composed of two parts: von Willebrand factor (vWF) and factor VIII coagulation activity (VIII:C). As a cofactor of coagulation factor IXa, FVIII participates in the activation of coagulation factor X and is an important coagulation factor in the intrinsic coagulation pathway. The determination of FVIII activity is very important for the diagnosis and treatment monitoring of hemophilia A, because hemophilia A is caused by the hereditary deficiency of FVIII.

[0131] FIX active factor, namely coagulation factor IX activity (FIX:C), refers to the coagulation activity of coagulation factor IX. FIX is one of the vitamin K-dependent coagulation factors, mainly synthesized in the liver, and participates in the extrinsic coagulation pathway. FIX is activated to FIXa during the coagulation process, and then participates in the activation of coagulation factor X, thereby promoting the conversion of prothrombin to thrombin. The determination of FIX activity is very important for the diagnosis and treatment monitoring of hemophilia B, because hemophilia B is caused by the hereditary deficiency of FIX.

[0132] In some embodiments, in step S700, the type of hemophilia suffered by the organism from which the sample is derived is confirmed by analyzing the pathogenic variants of the VWF, F8 and F9 genes and comparing the activity levels of the vWF antigen, FVIII and FIX factors in the clinical information, specifically:

[0133] When there are FVIII factor activity test results, the following analysis can be performed in combination with the FVIII factor activity test results:

[0134] Severe deficiency of FVIII activity:

[0135] If F8 int22 type I or type II inversion is detected (hemizygous inversion for male samples and homozygous inversion for female samples), it is confirmed as severe hemophilia A;

[0136] If other pathogenic mutations of the F8 gene are detected (such as point mutations, insertions and deletions, etc., hemizygous mutations for males and homozygous mutations for females), it is confirmed as severe hemophilia A;

[0137] If the female sample has heterozygous inversions and other pathogenic mutations, and is determined to be a trans carrier based on the haplotype, it is confirmed to be severe female hemophilia;

[0138] Moderate or mild deficiency of FVIII factor activity:

[0139] If the pathogenic mutation (e.g., point mutation, insertion / deletion, etc.) of the F8 gene is detected as a hemizygous variation in males, it is confirmed as moderate or mild hemophilia A;

[0140] If the F8 gene mutation is not detected, the patient is then checked for pathogenic mutations in the VWF gene. If a pathogenic mutation in the VWF gene is present, the patient is confirmed to have VWD type 2 combined with FVIII factor activity deficiency.

[0141] When there are FIX factor activity test results, the following can be combined for analysis:

[0142] FIX factor activity test results are severe deficiency:

[0143] If hemizygous deletion of the F9 gene is detected in males or homozygous deletion in females, as well as complete inactivation of gene function caused by point mutations or insertions and deletions, it is confirmed as severe hemophilia B;

[0144] FVIII activity is moderately or mildly deficient:

[0145] If the F9 gene point mutation or hemizygous insertion / deletion mutation is detected, resulting in gene function damage, it is confirmed as moderate or mild hemophilia B;

[0146] If testing of the F9 gene does not reveal any pathogenic mutations, the diagnosis of hemophilia B is ruled out.

[0147] When there are activity test results of vWF antigen, the activity test results of vWF antigen can be combined for analysis:

[0148] When a pathogenic variant occurs in the VWF gene and the result of vWF antigen activity is lower than the clinical standard, it can be diagnosed as reduced vWF antigen activity caused by the pathogenic variant of the VWF gene.

[0149] The present application also provides a detection device, comprising:

[0150] The alignment module is used to align the sequence reads of the third-generation long-read sequencing with the reference genome;

[0151] An analysis module is used to analyze the comparison results of the sequence reads of the third-generation long-read sequencing with the reference genome: a part of the sequence reads is compared to the F9 gene and VWF gene regions to obtain a first comparison result, a first variation analysis is performed based on the first comparison result, and the first analysis result is output, and the F8 gene region is subjected to haplotype typing to obtain the differential base sequence between the unconventional gene and its highly homologous sequence, and the differential base sequence is compared with the remaining part of the sequence reads to obtain a second comparison result, a second variation analysis is performed based on the second comparison result, and a second analysis result is output; the gender of the sample to be tested is analyzed based on the comparison result of the sequence reads with the SRY gene region in the reference genome;

[0152] An output module is used to summarize the first analysis result and the second analysis result, and output the gene mutation analysis result; compare the gene mutation analysis result, the gender of the sample to be tested, and the clinical information of the sample to be tested to achieve the type typing of hemophilia;

[0153] Among them, conventional genes refer to genes without highly homologous sequences, unconventional genes refer to genes with highly homologous sequences, and genes with highly homologous sequences refer to genes with homologous sequences with similar sequence structures within the genome, and the similarity between the gene sequence and the homologous sequence is ≥90%;

[0154] The first analysis result and the second analysis result each independently include at least one of the following gene mutation results: single nucleotide polymorphism, small-scale insertion and deletion, structural variation and complex genome rearrangement;

[0155] The clinical information of the sample to be tested includes at least one of the test results of vWF antigen, FVIII and FIX active factors.

[0156] The present application also provides a computer device, including a memory and a processor, wherein the memory stores a computer program, and wherein the processor implements the steps of the above detection method when executing the computer program.

[0157] The present application also provides a computer-readable storage medium on which a computer program is stored, wherein the computer program implements the steps of the above detection method when executed by a processor.

[0158] The present application also provides a computer program product, including a computer program, characterized in that when the computer program is executed by a processor, the steps of the above detection method are implemented.

[0159] The following are some specific embodiments.

[0160] For experimental parameters not specified in the following specific embodiments, reference is made to the instructions given in the present application document, and reference may also be made to experimental manuals in the art or other experimental methods known in the art, or to experimental conditions recommended by manufacturers.

[0161] The raw materials and reagents involved in the following specific examples can be obtained from commercial sources, or can be prepared by those skilled in the art according to known methods.

[0162] Example 1

[0163] This embodiment provides clinical sample testing of hemophilia A and hemophilia B patients and their families, and before the gene test, the samples to be tested have completed the examination of conventional biochemical indicators related to hemophilia, and the results of abnormal activity values ​​of FVIII factor and FIX factor are determined to classify hemophilia A and hemophilia B patients. The sample information table is shown in Table 1.

[0164] Table 1

[0165]

[0166] Testing steps:

[0167] S100, obtaining genomic DNA from a peripheral blood DNA sample of a subject to be examined;

[0168] S200, fragmenting the DNA by transposase method, introducing sequencing adapters, and screening the fragments to remove libraries below 5 kb as much as possible to obtain the pre-capture library;

[0169] S300, using a probe hybridization capture method of the target region to capture the target region and obtain a post-capture library;

[0170] S400, constructing a sequencing library using the post-capture library and performing third-generation long-length sequencing to obtain sequence reads;

[0171] S500, alignment of sequence reads to reference genome and data analysis, including:

[0172] S510, removing sequence tags from the offline data, including the third-generation sequencing library tag, the post-capture library tag, the sample marker tag, and the transposon tag. After completion, the original offline data file is split into sequence data files (fastq format) of each sample;

[0173] S520, aligning the split sequence data files to the reference genome (hg19 / hg38) and creating an index;

[0174] S530, analyzing the point mutations, insertion and deletion information of F8, F9 and VWF genes; further, analyzing the copy number variation and structural variation results;

[0175] Based on the information of heterozygous SNPs, the F8, F9, and VWF gene sequences in the targeted amplification region were assembled to generate haplotype typing results;

[0176] S540, for the regions of highly homologous sequences of F8 (F8int22h1, F8int22h2 and F8int22h3), sequence assembly is performed based on the differential base sequences between the highly homologous genes, thereby distinguishing the haplotypes of F8int22h1, F8int22h2 and F8int22h3;

[0177] Based on the results of the haplotypes of F8int22h1, F8int22h2, and F8int22h3, the point mutations, indels, and copy number variation results of the regions of the F8 highly homologous sequences (F8int22h1, F8int22h2, and F8int22h3) were analyzed;

[0178] According to the haplotype results of F8int22h1, F8int22h2 and F8int22h3, when a certain haplotype has block fusion, the fusion gene is identified and the structural variation results are confirmed.

[0179] S600, molecular typing results and result output

[0180] For patients with hemophilia A, in addition to the F8 gene, the VWF gene is checked for mutations, and the molecular typing results of the patients are completed and the test results are output. The test results are shown in Table 2.

[0181] Table 2

[0182]

[0183] The test results of each sample in Table 2 are analyzed:

[0184] (1) Sample A4

[0185] Mutation detection results: F8 int22 inversion type I, IGV map see Figure 1 .

[0186] Disease type: Hemophilia A severe

[0187] Molecular typing results: severe hemophilia A caused by F8 int22 inversion type I leading to inactivation of FVIII factor.

[0188] The detection method of the present application realizes haplotype typing for F8int22h1 / F8int22h2 / F8int22h3, wherein the F8int22h3 haplotype is a fusion gene haplotype, the 5' sequence of the region is F8int22h1, and the 3' sequence of the region is F8int22h3, so the fusion sequence is a structural variation caused by F8 int22 type I inversion.

[0189] (2) Sample A4-2

[0190] Mutation detection results: F8 int22 inversion type I carrier.

[0191] IGV map see Figure 2 .

[0192] The detection method of the present application realizes haplotype typing for F8int22h1 / F8int22h2 / F8int22h3 (two copies of each), wherein the F8int22h3 haplotype hap2 is a fusion gene haplotype, the 5' sequence of the region is F8int22h1, and the 3' sequence of the region is F8int22h3, so the fusion sequence is a structural variation caused by F8 int22 type I inversion.

[0193] (3) Sample A5

[0194] Mutation detection results: F8 hemizygous mutation: NM_000132.4:c.6496C>T.

[0195] IGV map see Figure 3 .

[0196] Disease type: Hemophilia A severe

[0197] Molecular typing results: severe hemophilia A caused by inactivation of FVIII factor due to F8 hemizygous mutation.

[0198] (4) Sample A9:

[0199] Mutation detection results: F8 int22 type II inversion.

[0200] IGV map see Figure 4 .

[0201] Disease type: Hemophilia A severe

[0202] Molecular typing results: severe hemophilia A with inactivation of FVIII factor due to F8 int22 type II inversion.

[0203] The detection method of the present application realizes haplotype typing for F8int22h1 / F8int22h2 / F8int22h3, wherein the int22 h2 haplotype is a fusion gene haplotype.

[0204] The sequence of the 5' region is F8int22h1, and the sequence of the 3' region is F8int22h2, so the fusion sequence is a structural variation caused by F8int22 type II inversion.

[0205] (5) Sample B1:

[0206] Mutation detection results: F9 mutation NM_000133.4:c.205T>G.

[0207] IGV map see Figure 5 .

[0208] Disease Type: Hemophilia B Moderate

[0209] Molecular typing results: moderate hemophilia B with reduced FIX factor activity due to F9 mutation.

[0210] (6) Samples B2 and B2-2:

[0211] Mutation detection result: F9 hemizygous deletion.

[0212] IGV map see Figure 6 .according to Figure 6 It can be seen intuitively that the upper one is the B2 sample and the lower one is the B2-2 sample. The B2 sample has a hemizygous deletion of F9, and the B2-2 sample has a heterozygous deletion of F9.

[0213] Disease type: Hemophilia B severe

[0214] Molecular typing results: severe hemophilia B with reduced FIX factor activity due to hemizygous deletion of F9.

[0215] (7) Sample X1

[0216] Mutation detection result: F8 int22h1-h2 heterozygous deletion.

[0217] IGV map see Figure 7 .

[0218] Disease type: None, low FVIII factor activity

[0219] Molecular typing results: severe hemophilia A caused by inactivation of FVIII factor due to heterozygous deletion of F8 int22h1-h2.

[0220] The detection method of the present application realizes haplotype typing for F8int22h1 / F8int22h2 / F8int22h3, wherein the int22 h2 haplotype is a fusion gene haplotype, and thus the fusion sequence is a structural variation caused by the deletion of F8 int22h1-h2.

[0221] Comparative Example 1

[0222] This comparative example uses gel electrophoresis long fragment PCR method to detect clinical samples of hemophilia patients. The test results are as follows Figure 8 .

[0223] according to Figure 8 It can be seen that A4 is a F8int22 type I inversion; A4-2 is a F8int22 type I inversion carrier; A9 is a F8int22 type II inversion; and X1 is a F8int22h1-h2 deletion.

[0224] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0225] The technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. To make the description concise, not all possible combinations of the technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0226] The above-described embodiments only express several implementation methods of the present application, but they cannot be understood as limiting the scope of the patent application. It should be pointed out that, for those of ordinary skill in the art, without departing from the concept of the present application, several variations and improvements can also be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by those skilled in the art on the basis of the technical solutions provided in the present application through logical analysis, reasoning or limited experiments are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. A method for detecting the variant type of a hemophilia-causing gene, characterized in that: The steps include: S100, obtaining genomic DNA from the sample to be tested; S200, the genomic DNA is fragmented and adapter-added to obtain a pre-capture library; S300, targetedly capturing the target region in the pre-capture library to obtain a post-capture library; the target region includes the F8 gene, the F9 gene, the VWF gene and the SRY gene region; S400, constructing a sequencing library using the post-capture library and performing third-generation long-read sequencing to obtain sequence reads; S500, aligning the sequence reads with a reference genome, aligning a portion of the sequence reads to the F9 gene and VWF gene regions to obtain a first alignment result, performing a first variation analysis based on the first alignment result, outputting a first analysis result, performing haplotype typing on the F8 gene region, obtaining a differential base sequence between the unconventional gene and a highly homologous sequence thereof, aligning the differential base sequence with the remaining portion of the sequence reads to obtain a second alignment result, performing a second variation analysis based on the second alignment result, and outputting a second analysis result; analyzing the gender of the sample to be tested based on the alignment result of the sequence reads with the SRY gene region in the reference genome; S600, summarizing the first analysis result and the second analysis result, and outputting a gene mutation analysis result; S700, comparing the gene mutation analysis result, the gender of the sample to be tested and the clinical information of the sample to be tested to achieve typing of hemophilia; Among them, the conventional gene refers to a gene without a highly homologous sequence, the unconventional gene refers to a gene with a highly homologous sequence, and the gene with a highly homologous sequence means that the gene has a homologous sequence with a similar sequence structure within the genome, and the similarity between the gene sequence and the homologous sequence is ≥90%; The first analysis result and the second analysis result each independently include at least one of the following gene mutation results: single nucleotide polymorphism, small-scale insertion and deletion, structural variation and complex genome rearrangement; The clinical information of the sample to be tested includes at least one of the test results of vWF antigen, FVIII and FIX active factors.

2. The detection method according to claim 1, characterized in that: The genomic DNA is fragmented and treated with adapters to obtain a pre-capture library, comprising: The genomic DNA is fragmented by a transposase method or a physical shearing method and a sequencing adapter is introduced to obtain a DNA fragment with an adapter; The fragments with a length of less than 5 kb in the DNA fragments with adapters are removed to obtain the pre-capture library.

3. The detection method according to claim 1, characterized in that: The method for targeted capture of the target region in the pre-capture library comprises: (1) using the probe composition to perform a probe hybridization capture method on the target region; (2) Performing multiplex PCR amplification on the target region using the primer combination.

4. The detection method according to claim 1, characterized in that: Performing a first variation analysis according to the first comparison result includes: Analyzing first variation information of the conventional gene according to the first comparison result, wherein the first variation information includes single nucleotide polymorphism, small-scale insertion and deletion, and structural variation; Furthermore, the structural variation includes copy number variation.

5. The detection method according to claim 4, characterized in that: The method further comprises: performing a first variation analysis according to the first comparison result and outputting a first analysis result. The conventional gene is subjected to haplotype typing according to the information of the heterozygous SNP in the first comparison result.

6. The detection method according to claim 1, characterized in that: Performing a second variation analysis according to the second comparison result includes: analyzing second variation information of the unconventional gene according to the second comparison result, wherein the second variation information includes at least one of single nucleotide polymorphism, small-scale insertion and deletion, structural variation and complex genome rearrangement; Furthermore, the structural variation includes at least one of copy number variation, gene fusion, and large-segment gene conversion and inversion; Furthermore, the complex genome rearrangement refers to the occurrence of multiple types of structural variations within the same gene region.

7. The detection method according to claim 1, characterized in that: Comparing the gene mutation analysis result, the gender of the sample to be tested and the clinical information of the sample to be tested to achieve the type classification of hemophilia, including: By analyzing the pathogenic variants of VWF, F8 and F9 genes and comparing the activity levels of vWF antigen, FVIII and FIX factors with clinical information, the type of hemophilia suffered by the organism from which the sample was tested was confirmed.

8. A detection device, characterized in that: The detection device comprises: The alignment module is used to align the sequence reads of the third-generation long-read sequencing with the reference genome; An analysis module is used to analyze the comparison results of the sequence reads of the third-generation long-read sequencing with the reference genome: a part of the sequence reads is compared to the F9 gene and VWF gene regions to obtain a first comparison result, a first variation analysis is performed based on the first comparison result, and the first analysis result is output, and the F8 gene region is subjected to haplotype typing to obtain the differential base sequence between the unconventional gene and its highly homologous sequence, and the differential base sequence is compared with the remaining part of the sequence reads to obtain a second comparison result, a second variation analysis is performed based on the second comparison result, and a second analysis result is output; according to the comparison result of the sequence reads with the SRY gene region in the reference genome, the gender of the sample to be tested is analyzed; An output module is used to summarize the first analysis result and the second analysis result and output a gene mutation analysis result; compare the gene mutation analysis result, the gender of the sample to be tested and the clinical information of the sample to be tested to achieve the type typing of hemophilia; Among them, the conventional gene refers to a gene without a highly homologous sequence, the unconventional gene refers to a gene with a highly homologous sequence, and the gene with a highly homologous sequence means that the gene has a homologous sequence with a similar sequence structure within the genome, and the similarity between the gene sequence and the homologous sequence is ≥90%; The first analysis result and the second analysis result each independently include at least one of the following gene mutation results: single nucleotide polymorphism, small-scale insertion and deletion, structural variation and complex genome rearrangement; The clinical information of the sample to be tested includes at least one of the test results of vWF antigen, FVIII and FIX active factors.

9. A computer device comprising a memory and a processor, wherein the memory stores a computer program, wherein: When the processor executes the computer program, the steps of the detection method according to any one of claims 1 to 8 are implemented.

10. A computer-readable storage medium having a computer program stored thereon, characterized in that: When the computer program is executed by a processor, the steps of the detection method according to any one of claims 1 to 8 are implemented.

11. A computer program product, comprising a computer program, characterized in that When the computer program is executed by a processor, the steps of the detection method according to any one of claims 1 to 8 are implemented.

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