CYP21A2 and CYP21A1p gene fusion judgment system and method based on Pacbio sequencing data

Abstract

The invention relates to a CYP21A2 and CYP21A1p gene fusion judgment system and a CYP21A2 and CYP21A1p gene fusion judgment method based on Pacbio sequencing data. The method comprises the following steps: constructing a distinguishing site set, generating a sequence, establishing an indexed comparison file, obtaining a haplotype sequence of CYP21A2 and CYP21A1p, obtaining variation information of each haplotype on the distinguishing site set, carrying out statistics on the occurrence frequency of distinguishing sites in the obtained distinguishing site set in the CYP21A1p haplotype, and taking the occurrence frequency as the weight of each distinguishing site, so as to obtain the CYP21A2 and CYP21A1p haplotype sequence of the CYP21A1p haplotype sequence of the CYP21A2 and the CYP21A1p haplotype sequence of the CYP21A1p haplotype sequence. Calculating an accumulated score Score of each distinguishing site; according to the curve trend of Scorei changing along with the sequence of the distinguishing sites, whether fusion exists or not and the fusion type exist is judged; according to the method, while complex variation of gene fusion can be detected, breakpoints are accurately positioned, fusion directions are distinguished, and the detection accuracy and resolution are improved.

Description

Technical Field

[0001] The present invention relates to the field of bioinformatics analysis and genomics technology, and in particular to a system and method for determining the fusion of CYP21A2 and CYP21A1p genes based on Pacbio sequencing data. Background Art

[0002] Congenital adrenal hyperplasia (CAH) is an autosomal recessive genetic disease, and the most common pathogenic gene is CYP21A2. The CYP21A2 gene and its pseudogene CYP21A1p are closely arranged in the chromosome 6p21.3 region. The sequences of the two are highly homologous, and recombination events such as unequal exchange, gene conversion, partial deletion or fusion often occur, resulting in decreased or lost 21-hydroxylase activity. This type of recombination or fusion variation has a certain incidence in CAH patients and is closely related to the occurrence of the disease and the severity of the phenotype.

[0003] At present, genetic testing for the CYP21A2 / CYP21A1p region mainly relies on traditional technologies such as Sanger sequencing, ARMS-PCR, and MLPA. Although these methods have certain advantages in detecting single-point or specific site mutations, due to the high homology between CYP21A2 and CYP21A1p and the complexity of the structure of this region, when large structural rearrangements or fusion mutations occur, they often have the following shortcomings: a. Limited detection sensitivity: large fragment mutations or fusion sites may be missed; b. Specificity is difficult to ensure: high homology sequences lead to false positives or misjudgments; c. Insufficient resolution: the break (recombination) position and specific fusion pattern cannot be clearly determined.

[0004] Compared with traditional technologies, third-generation sequencing platforms such as PacBio can span large repetitive regions or highly homologous regions in a single long read sequence due to their read length advantage, thereby more accurately reflecting the CYP21A2 / CYP21A1p genome structure. If combined with bioinformatics analysis processes optimized for long read data, such as accurate alignment of repetitive segments and gene fusion detection algorithms, it is possible to better locate recombination breakpoints and determine whether gene fusion has occurred.

[0005] Therefore, there is an urgent need for a bioinformatics analysis method to accurately assemble, align, and identify variants in the CYP21A2 and CYP21A1p regions from long-read sequencing data (PacBio or other platforms), so as to accurately identify possible gene fusion events. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the present invention aims to provide a system and method for determining the fusion of CYP21A2 and CYP21A1p genes based on Pacbio sequencing data, so as to achieve determination of the fusion of CYP21A2 and CYP21A1p genes.

[0007] In order to solve the above problems, the present invention adopts the following technical solutions:

[0008] On the one hand, the present invention provides a CYP21A2 and CYP21A1p gene fusion judgment system based on Pacbio sequencing data, including a distinguishing site set construction module, a sequencing data comparison module, a haplotype assembly module, a haplotype variation identification module, a distinguishing site weight calculation module and a gene fusion judgment module;

[0009] The distinguishing site set construction module is used to obtain a CYP21A1p reference sequence, align it to a CYP21A2 reference sequence using a long sequence alignment software, identify the variable sites of CYP21A1p relative to CYP21A2, and obtain a distinguishing site set between CYP21A2 and CYP21A1p;

[0010] The sequencing data comparison module is used to compare the high-fidelity sequence of the sample to be tested obtained by PacBio sequencing to the human reference genome, and generate a sorted and indexed comparison file;

[0011] The haplotype assembly module is used to perform haplotype assembly on the CYP21A2 and CYP21A1p regions based on the alignment file using homologous gene haplotype assembly software to obtain haplotype sequences of CYP21A2 and CYP21A1p;

[0012] The haplotype variation identification module is used to perform variation detection on each obtained CYP21A2 and CYP21A1p haplotype sequence, respectively, to obtain variation information of each haplotype on the distinguishing site set;

[0013] The distinguishing site weight calculation module is used to use the homologous gene assembly results of the normal human control sample set to count the occurrence frequency of the distinguishing sites in the obtained distinguishing site set in the CYP21A1p haplotype as the weight of each distinguishing site;

[0014] The gene fusion judgment module is used to accumulate and score the variation of all haplotypes in the sample to be tested in the distinguishing site set, and calculate the cumulative score of each distinguishing site. i ; According to Score i The trend of the curve changes with the order of the differentiation sites, and the type of extreme values ​​appearing at the concentrated end or both ends of the differentiation sites can be used to determine whether CYP21A2 and CYP21A1p are fused and the type of fusion.

[0015] As an implementable embodiment, in the distinguishing site set construction module, minimap2 software is used for sequence alignment, and the distinguishing site set is constructed based on the SNPs, insertions and / or deletions generated by CYP21A1p relative to CYP21A2.

[0016] As an implementable method, in the sequencing data alignment module, minimap2 is used to align the sequencing reads with the human reference genome in map-hifi mode;

[0017] Use samtools to convert the generated SAM files into BAM format and sort and index the BAM files;

[0018] The alignment quality was evaluated through sequencing depth statistics and coverage analysis, low-quality alignment results were filtered out, and alignment files were generated.

[0019] As an implementable method, in the haplotype assembly module, the homologous gene haplotype assembly software Paraphase is used to cluster and iteratively assemble the reads of the CYP21A2 and CYP21A1p regions, and output a number of corresponding haplotype sequences.

[0020] As an implementable method, in the haplotype variation identification module, DeepVariant variation detection software is used to perform SNP and InDel detection on each haplotype sequence of CYP21A2 and CYP21A1p, and the presence or absence of variation is recorded on the distinguishing site set.

[0021] As an implementable embodiment, in the distinguishing site weight calculation module, a comparison file of sorted and indexed comparison of multiple normal human control samples, haplotype sequences of CYP21A2 and CYP21A1p, and variation information of each haplotype on the distinguishing site set are generated, and the proportion of mutations that appear or remain consistent at each distinguishing site on all CYP21A1p haplotypes is counted, and the proportion is defined as the weight of the distinguishing site.

[0022] As an implementable method, in the gene fusion judgment module, the calculation formula of the cumulative score is:

[0023]

[0024] in, is the cumulative score of the ith distinguishing site in the distinguishing site set; a is the judgment value of whether the distinguishing site exists, if a exists, it is 1, if not, it is -1; is the weight of the ith distinguishing site.

[0025] As an implementable embodiment, in the gene fusion judgment module, the judgment of whether CYP21A2 and CYP21A1p are fused and the fusion type includes:

[0026] When the As i increases, it shows an overall decreasing trend, and when the extreme values ​​are located at the two ends of the distinguishing site set, it is judged to be a complete CYP21A2 gene;

[0027] When the As i increases, it shows an overall increasing trend, and when the extreme values ​​are located at the two ends of the distinguishing site set, it is judged to be a complete CYP21A1p gene;

[0028] When the When the value decreased with the increase of i in the first half and increased with the increase of i in the second half, and a minimum value appeared in the middle of the distinguishing site set, it was judged to be CYP21A2-CYP21A1p fusion;

[0029] When the When the value increases with the increase of i in the first half and decreases with the increase of i in the second half, and a maximum value appears in the middle of the distinguishing site set, it is judged to be CYP21A1p-CYP21A2 fusion.

[0030] On the other hand, the present invention provides a method for determining CYP21A2 and CYP21A1p gene fusion based on Pacbio sequencing data, comprising:

[0031] Obtain the CYP21A1p reference sequence, align it to the CYP21A2 reference sequence using long sequence alignment software, identify the variable sites of CYP21A1p relative to CYP21A2, and obtain the distinguishing site set between CYP21A2 and CYP21A1p;

[0032] Align the high-fidelity sequence of the sample to be tested obtained by PacBio sequencing to the human reference genome, and generate a sorted and indexed alignment file;

[0033] Based on the alignment file, haplotype assembly of CYP21A2 and CYP21A1p regions was performed using homologous gene haplotype assembly software to obtain haplotype sequences of CYP21A2 and CYP21A1p;

[0034] Performing variation detection on each obtained CYP21A2 and CYP21A1p haplotype sequence, respectively, to obtain variation information of each haplotype at the distinguishing site set;

[0035] Using the homologous gene assembly results of the normal human control sample set, the frequency of occurrence of the distinguishing sites in the obtained distinguishing site set in the CYP21A1p haplotype was statistically analyzed as the weight of each distinguishing site;

[0036] Accumulate the scores for the variation of all haplotypes in the sample to be tested in the distinguishing site set, and calculate the cumulative score of each distinguishing site. i ; According to Score i The trend of the curve changes with the order of the differentiation sites, and the type of extreme values ​​appearing at the concentrated end or both ends of the differentiation sites can be used to determine whether CYP21A2 and CYP21A1p are fused and the type of fusion.

[0037] As an embodiment, obtaining the distinguishing site set between CYP21A2 and CYP21A1p includes performing sequence alignment using minimap2 software, and constructing the distinguishing site set based on SNPs, insertions and / or deletions generated by CYP21A1p relative to CYP21A2.

[0038] The beneficial effects of the present invention are as follows: the present invention aims at the characteristics of CYP21A2 and CYP21A1p being highly homologous, complex in structure and easy to fuse, and utilizes the high accuracy of long-read sequencing such as PacBio and the advantage of being able to cross repeated segments, to develop a complete set of bioinformatics processes for alignment, haplotype assembly, variation identification, and fusion judgment; by calculating the weights of the sites distinguishing CYP21A2 and CYP21A1p and using a cumulative scoring strategy, the present invention can accurately locate breakpoints and distinguish fusion directions while detecting complex gene fusion variations, thereby greatly improving the accuracy and resolution of the detection; compared with traditional detection methods for single-site mutations such as Sanger sequencing and ARMS-PCR, the present invention can scan a wider range of variations at one time and locate recombination breakpoints, significantly reducing missed detections or misjudgments caused by insufficient local detection; compared with MLPA, the present invention has the same sensitivity in detecting large fragment deletions, and can further obtain fusion direction and specific breakpoint information; MLPA The present invention only provides quantitative hints at large-scale deletions or CNV levels, while it can "qualitatively + positionally" with a higher degree of refinement, and can be flexibly applied in different demand scenarios such as large-scale clinical screening, rapid diagnosis and precise breakpoint positioning. The present invention can not only provide more comprehensive variation information for the molecular diagnosis of clinical congenital adrenal hyperplasia, but also provide more reliable data support for related genetic research and the formulation of individualized treatment strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Schematic diagram of a CYP21A2 and CYP21A1p gene fusion determination system based on Pacbio sequencing data in Example 1 of the present invention.

[0040] Figure 2 Schematic diagram of the distinguishing site set obtained based on the human reference genome hg19 CYP21A1p region sequence in Example 1 of the present invention.

[0041] Figure 3 This is a flow chart of a method for determining gene fusion of CYP21A2 and CYP21A1p based on Pacbio sequencing data in Example 1 of the present invention.

[0042] Figure 4 This is the Paraphase haplotype assembly diagram of sample T1 in Example 2 of the present invention.

[0043] Figure 5 Schematic diagram of the accumulated score of sample T1 in Example 2 of the present invention.

[0044] Figure 6 This is the Paraphase haplotype assembly diagram of sample T2 in Example 2 of the present invention.

[0045] Figure 7 Schematic diagram of the cumulative score of sample T2 in Example 2 of the present invention.

[0046] Figure 8 This is the Paraphase haplotype assembly diagram of sample T3 in Example 2 of the present invention.

[0047] Fig. 9 Schematic diagram of the cumulative score of sample T3 in Example 2 of the present invention.

[0048] Fig.10 This is the Paraphase haplotype assembly diagram of sample T4 in Example 2 of the present invention.

[0049] Fig.11 Schematic diagram of the cumulative score of sample T4 in Example 2 of the present invention.

[0050] Fig.12 This is the Paraphase haplotype assembly diagram of sample T5 in Example 2 of the present invention.

[0051] Fig.13 Schematic diagram of the cumulative score of sample T5 in Example 2 of the present invention.

[0052] Fig.14 This is the Paraphase haplotype assembly diagram of sample T6 in Example 2 of the present invention.

[0053] Fig.15 Schematic diagram of the cumulative score of sample T6 in Example 2 of the present invention. DETAILED DESCRIPTION

[0054] The present invention is further described in detail below in conjunction with specific embodiments.

[0055] It should be noted that these embodiments are only used to illustrate the present invention rather than to limit the present invention. Simple improvements to the method based on the concept of the present invention all fall within the scope of protection claimed by the present invention.

[0056] Example 1

[0057] See also Figure 1 , which is a CYP21A2 and CYP21A1p gene fusion judgment system based on Pacbio sequencing data, including a distinguishing site set construction module 100, a sequencing data comparison module 200, a haplotype assembly module 300, a haplotype variation identification module 400, a distinguishing site weight calculation module 500 and a gene fusion judgment module 600.

[0058] The distinguishing site set construction module 100 is used to obtain a CYP21A1p reference sequence, align it to a CYP21A2 reference sequence using a long sequence alignment software, identify the variable sites of CYP21A1p relative to CYP21A2, and obtain a distinguishing site set between CYP21A2 and CYP21A1p.

[0059] In the distinguishing site set construction module 100, the human reference genome hg19 CYP21A1p (chr: - ) sequence is obtained, the sequence is aligned using minimap2 software, and a distinguishing site set is constructed based on the SNPs, insertions and / or deletions generated by CYP21A1p relative to CYP21A2, such as Figure 2 shown.

[0060] The sequencing data comparison module 200 is used to compare the high-fidelity sequence of the sample to be tested obtained by PacBio sequencing to the human reference genome, and generate a sorted and indexed comparison file.

[0061] In the sequencing data alignment module 200 , the sequencing reads are aligned with the human reference genome using minimap2 in map-hifi mode;

[0062] Use samtools to convert the generated SAM files into BAM format and sort and index the BAM files;

[0063] The alignment quality was evaluated through sequencing depth statistics and coverage analysis, low-quality alignment results were filtered out, and alignment files were generated.

[0064] Specifically, this embodiment provides a specific implementation process of a sequencing data comparison module 200:

[0065] Using the long sequence alignment software minimap2, based on the map-hifi mode, the PacBio high-fidelity HIFI sequences were aligned to the human hg19 version of the reference genome, generating a binary alignment bam format file. Using the bam processing software samtools, the sequences in the bam file were sorted by alignment position, generating a sorted alignment file sorted.bam:

[0066] minimap2 -t 8 -ax map-hifi hg19.mmi pacbio_hifi_reads.fq > hifi_map2hg19.sam

[0067] samtools view -Sb hifi_map2hg19.sam > hifi_map2hg19.bam

[0068] samtools sort hifi_map2hg19.bam > sorted.bam

[0069] samtools index sorted.bam.

[0070] The haplotype assembly module 300 is used to perform haplotype assembly on the CYP21A2 and CYP21A1p regions based on the alignment file using homologous gene haplotype assembly software to obtain haplotype sequences of CYP21A2 and CYP21A1p.

[0071] In the haplotype assembly module 300, the homologous gene haplotype assembly software Paraphase is used to cluster and iteratively assemble the reads of the CYP21A2 and CYP21A1p regions, and output a number of corresponding haplotype sequences:

[0072] paraphase -b sorted.bam -r hg19_genome.fa --samtools samtools --minimap2 minimap2 -o . / -g rccx --genome 19.

[0073] The haplotype variation identification module 400 is used to perform variation detection on each obtained CYP21A2 and CYP21A1p haplotype sequence, respectively, to obtain variation information of each haplotype at the distinguishing site set.

[0074] In the haplotype variation identification module 400, DeepVariant variation detection software is used to perform SNP and InDel detection on each haplotype sequence of CYP21A2 and CYP21A1p, and the presence or absence of variation is recorded on the distinguishing site set:

[0075] deepvariant --model_type=PACBIO --ref= hg19_genome.fa --reads=hg19.sorted.hapN.bam --output_vcf= hg19.sorted.hapN.vcf.

[0076] The distinguishing site weight calculation module 500 is used to use the homologous gene assembly results of the normal human control sample set to count the occurrence frequency of the distinguishing sites in the obtained distinguishing site set in the CYP21A1p haplotype as the weight of each distinguishing site.

[0077] In the distinguishing site weight calculation module 500, a comparison file of sorted and indexed comparison of multiple normal human control samples, haplotype sequences of CYP21A2 and CYP21A1p, and variation information of each haplotype on the distinguishing site set are generated, and the proportion of mutations that appear or remain consistent at each distinguishing site on all CYP21A1p haplotypes is counted, and the proportion is defined as the weight of the distinguishing site.

[0078] For example, the PacBio HiFi data of 40 normal human sample sets were used for human genome alignment and haplotype assembly of CYP21A2 and CYP21A1p, and haplotype variation identification was performed. The frequency of occurrence of the obtained distinguishing site set in the CYP21A1p haplotype of all samples was statistically calculated, and the frequency was defined as the distinguishing site weight of CYP21A2 and CYP21A1p. The distinguishing site set is shown in Table 1. The larger the site weight value, the higher the discriminative ability of the site for CYP21A2 and CYP21A1p.

[0079] Table 1. Site set distinguishing CYP21A2 from CYP21A1p

[0080] Note:

[0081] 5'flk: upstream region of CYP21A2 gene;

[0082] C_e1: CYP21A2 gene exon1 region;

[0083] C_i1: CYP21A2 gene intron1 region.

[0084] The gene fusion judgment module 600 is used to accumulate and score the variation of all haplotypes in the sample to be tested in the distinguishing site set, and calculate the cumulative score of each distinguishing site. i ; According to Score i The trend of the curve changes with the order of the differentiation sites, and the type of extreme values ​​appearing at the concentrated end or both ends of the differentiation sites can be used to determine whether CYP21A2 and CYP21A1p are fused and the type of fusion.

[0085] In the gene fusion judgment module 600, the calculation formula of the cumulative score is:

[0086]

[0087] in, is the cumulative score of the ith distinguishing site in the distinguishing site set; a is the judgment value of whether the distinguishing site exists, if a exists, it is 1, if not, it is -1; is the weight of the ith distinguishing site.

[0088] Determine whether fusion exists between CYP21A2 and CYP21A1p and the type of fusion includes:

[0089] when As i increases, it shows an overall decreasing trend, and when the extreme values ​​are located at the two ends of the distinguishing site set, it is judged to be a complete CYP21A2 gene;

[0090] when As i increases, it shows an overall increasing trend, and when the extreme values ​​are located at the two ends of the distinguishing site set, it is judged to be a complete CYP21A1p gene;

[0091] when When the value decreases with the increase of i in the first half and increases with the increase of i in the second half, and the minimum value appears in the middle of the distinguishing site set, it is judged to be CYP21A2-CYP21A1p fusion ( In the CYP21A2 part, it decreases with the increase of i, while in the CYP21A1p part, it increases with the increase of i. The minimum value occurs in the middle of the site set);

[0092] when When the value increases with the increase of i in the first half and decreases with the increase of i in the second half, and the maximum value appears in the middle of the distinguishing site set, it is judged to be CYP21A1p-CYP21A2 fusion ( In the CYP21A1p part, it increases with increasing i, while in the CYP21A1p part, it decreases with increasing i. The maximum value occurs in the middle of the site set).

[0093] For ease of understanding, The curve that changes with i is visualized and plotted, and finally the prediction results of whether there is a fusion variation and the fusion type for each haplotype are output.

[0094] See also Figure 3 Based on the above judgment system, a CYP21A2 and CYP21A1p gene fusion judgment method based on Pacbio sequencing data is provided (applied to gene fusion judgment, not disease diagnosis method), including:

[0095] S100, obtaining a CYP21A1p reference sequence, aligning it to a CYP21A2 reference sequence using a long sequence alignment software, identifying the variable sites of CYP21A1p relative to CYP21A2, and obtaining a set of distinguishing sites between CYP21A2 and CYP21A1p;

[0096] S200, aligning the high-fidelity sequence of the sample to be tested obtained by PacBio sequencing to the human reference genome, and generating an alignment file for sorting and indexing;

[0097] S300, based on the alignment file, the haplotypes of CYP21A2 and CYP21A1p regions were assembled using homologous gene haplotype assembly software to obtain the haplotype sequences of CYP21A2 and CYP21A1p;

[0098] S400, performing variation detection on each obtained CYP21A2 and CYP21A1p haplotype sequence, respectively, to obtain variation information of each haplotype at the distinguishing site set;

[0099] S500, using the homologous gene assembly results of the normal human control sample set, the frequency of occurrence of the distinguishing sites in the obtained distinguishing site set in the CYP21A1p haplotype is counted as the weight of each distinguishing site;

[0100] S600: Accumulate and score the variation of all haplotypes in the sample to be tested in the distinguishing site set, and calculate the cumulative score of each distinguishing site. i ; According to Score i The trend of the curve changes with the order of the differentiation sites, and the type of extreme values ​​appearing at the concentrated end or both ends of the differentiation sites can be used to determine whether CYP21A2 and CYP21A1p are fused and the type of fusion.

[0101] Example 2

[0102] Results and comparison of 6 samples using this method

[0103] Six samples with MLPA verification results were subjected to Pacbio sequencing of long fragment capture in the CYP21A2 region. The generated HiFi sequence data were used to determine the fusion results of CYP21A2 and CYP21A1p using the judgment system or method provided in Example 1, and the results were compared, as shown in Table 2. The schematic diagrams of the Paraphase haplotype assembly and cumulative score of sample T1 are shown in Figure 4 and Figure 5 The schematic diagrams of Paraphase haplotype assembly and cumulative scores of sample T2 are shown in Figure 6 and Figure 7 , Paraphase haplotype assembly and cumulative score diagrams of sample T3 are shown in Figure 8 and Fig. 9 , Paraphase haplotype assembly and cumulative score diagrams of sample T4 are shown in Fig.10 and Fig.11 , Paraphase haplotype assembly and cumulative score diagrams of sample T5 are shown in Fig.12 and Fig.13 , Paraphase haplotype assembly and cumulative score diagrams of sample T6 are shown in Fig.14 and Fig.15 .

[0104] Table 2 Comparison of haplotypes of CYP21A2 and CYP21A1p assembled by Paraphase

[0105] Table 2 shows the comparison of MLPA results of 6 samples with the detection of CYP21A2 and CYP21A1p fusion by the method of the present invention:

[0106] MLPA of samples T1, T2, T3, and T4 all indicated exon deletion; the method of the present invention further clarified that it was a fusion of CYP21A1p and CYP21A2 or a fusion of CYP21A2 and CYP21A1p, and pointed out the precise range of the deletion (exon1-exon8 or exon10, etc.), and the results were highly consistent.

[0107] The MLPA results of samples T5 and T6 were normal, and the method of the present invention also detected no fusion, and the results were consistent.

[0108] The method of the present invention can identify the specific fusion direction and breakpoint position while confirming the missing exon; while MLPA can only indicate the occurrence of large-segment deletions and cannot accurately locate them.

[0109] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described with reference to the preferred embodiments of the present invention, it should be understood by those skilled in the art that various changes may be made in form and details without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. A CYP21A2 and CYP21A1p gene fusion determination system based on Pacbio sequencing data, characterized in that: It includes a distinguishing site set construction module, a sequencing data comparison module, a haplotype assembly module, a haplotype variation identification module, a distinguishing site weight calculation module and a gene fusion judgment module; The distinguishing site set construction module is used to obtain a CYP21A1p reference sequence, align it to a CYP21A2 reference sequence using a long sequence alignment software, identify the variable sites of CYP21A1p relative to CYP21A2, and obtain a distinguishing site set between CYP21A2 and CYP21A1p; The sequencing data comparison module is used to compare the high-fidelity sequence of the sample to be tested obtained by PacBio sequencing to the human reference genome, and generate a sorted and indexed comparison file; The haplotype assembly module is used to perform haplotype assembly on the CYP21A2 and CYP21A1p regions based on the alignment file using homologous gene haplotype assembly software to obtain haplotype sequences of CYP21A2 and CYP21A1p; The haplotype variation identification module is used to perform variation detection on each obtained CYP21A2 and CYP21A1p haplotype sequence, respectively, to obtain variation information of each haplotype on the distinguishing site set; The distinguishing site weight calculation module is used to use the homologous gene assembly results of the normal human control sample set to count the occurrence frequency of the distinguishing sites in the obtained distinguishing site set in the CYP21A1p haplotype as the weight of each distinguishing site; The gene fusion judgment module is used to accumulate and score the variation of all haplotypes in the sample to be tested in the distinguishing site set, and calculate the cumulative score of each distinguishing site. i ; According to Score i The trend of the curve changes with the order of the differentiation sites, and the type of extreme values ​​appearing at the concentrated end or both ends of the differentiation sites can be used to determine whether CYP21A2 and CYP21A1p are fused and the type of fusion.

2. The CYP21A2 and CYP21A1p gene fusion determination system based on Pacbio sequencing data according to claim 1, characterized in that: In the distinguishing site set construction module, minimap2 software is used for sequence alignment, and the distinguishing site set is constructed based on the SNPs, insertions and / or deletions generated by CYP21A1p relative to CYP21A2.

3. The CYP21A2 and CYP21A1p gene fusion determination system based on Pacbio sequencing data according to claim 1, characterized in that: In the sequencing data alignment module, minimap2 is used to align the sequencing reads with the human reference genome in map-hifi mode; Use samtools to convert the generated SAM files into BAM format and sort and index the BAM files; The alignment quality was evaluated through sequencing depth statistics and coverage analysis, low-quality alignment results were filtered out, and alignment files were generated.

4. The CYP21A2 and CYP21A1p gene fusion determination system based on Pacbio sequencing data according to claim 1, characterized in that: In the haplotype assembly module, the homologous gene haplotype assembly software Paraphase is used to cluster and iteratively assemble the reads of the CYP21A2 and CYP21A1p regions, and output a number of corresponding haplotype sequences.

5. The CYP21A2 and CYP21A1p gene fusion determination system based on Pacbio sequencing data according to claim 1, characterized in that: In the haplotype variation identification module, DeepVariant variation detection software is used to perform SNP and InDel detection on each haplotype sequence of CYP21A2 and CYP21A1p, and the presence or absence of variation is recorded on the distinguishing site set.

6. The CYP21A2 and CYP21A1p gene fusion determination system based on Pacbio sequencing data according to claim 1, characterized in that: In the distinguishing site weight calculation module, a plurality of normal human control samples are sorted and indexed to generate a comparison file, the haplotype sequences of CYP21A2 and CYP21A1p, and the variation information of each haplotype on the distinguishing site set, and the proportion of each distinguishing site that appears or remains consistent on all CYP21A1p haplotypes is counted, and the proportion is defined as the weight of the distinguishing site.

7. The CYP21A2 and CYP21A1p gene fusion determination system based on Pacbio sequencing data according to claim 6, characterized in that: In the gene fusion judgment module, the calculation formula of the cumulative score is: in, is the cumulative score of the ith distinguishing site in the distinguishing site set; a is the judgment value of whether the distinguishing site exists, if a exists, it is 1, if not, it is -1; is the weight of the ith distinguishing site.

8. The CYP21A2 and CYP21A1p gene fusion determination system based on Pacbio sequencing data according to claim 7, characterized in that: In the gene fusion judgment module, the judgment of whether CYP21A2 and CYP21A1p are fused and the type of fusion includes: When the As i increases, it shows an overall decreasing trend, and when the extreme values ​​are located at the two ends of the distinguishing site set, it is judged to be a complete CYP21A2 gene; When the As i increases, it shows an overall increasing trend, and when the extreme values ​​are located at the two ends of the distinguishing site set, it is judged to be a complete CYP21A1p gene; When the When the value decreased with the increase of i in the first half and increased with the increase of i in the second half, and a minimum value appeared in the middle of the distinguishing site set, it was judged to be CYP21A2-CYP21A1p fusion; When the When the value increases with the increase of i in the first half and decreases with the increase of i in the second half, and a maximum value appears in the middle of the distinguishing site set, it is judged to be CYP21A1p-CYP21A2 fusion.

9. A method for determining gene fusion of CYP21A2 and CYP21A1p based on Pacbio sequencing data, characterized in that: include: Obtain the CYP21A1p reference sequence, align it to the CYP21A2 reference sequence using long sequence alignment software, identify the variable sites of CYP21A1p relative to CYP21A2, and obtain the distinguishing site set between CYP21A2 and CYP21A1p; Align the high-fidelity sequence of the sample to be tested obtained by PacBio sequencing to the human reference genome, and generate a sorted and indexed alignment file; Based on the alignment file, haplotype assembly of CYP21A2 and CYP21A1p regions was performed using homologous gene haplotype assembly software to obtain haplotype sequences of CYP21A2 and CYP21A1p; Performing variation detection on each obtained CYP21A2 and CYP21A1p haplotype sequence, respectively, to obtain variation information of each haplotype at the distinguishing site set; Using the homologous gene assembly results of the normal human control sample set, the frequency of occurrence of the distinguishing sites in the obtained distinguishing site set in the CYP21A1p haplotype was statistically analyzed as the weight of each distinguishing site; Accumulate the scores for the variation of all haplotypes in the sample to be tested in the distinguishing site set, and calculate the cumulative score of each distinguishing site. i ; According to Score i The trend of the curve changes with the order of the differentiation sites, and the type of extreme values ​​appearing at the concentrated end or both ends of the differentiation sites can be used to determine whether CYP21A2 and CYP21A1p are fused and the type of fusion.

10. The method for determining the fusion of CYP21A2 and CYP21A1p genes based on Pacbio sequencing data according to claim 9, characterized in that: The method of obtaining the distinguishing site set between CYP21A2 and CYP21A1p includes performing sequence alignment using minimap2 software, and constructing the distinguishing site set according to the SNP, insertion and / or deletion generated by CYP21A1p relative to CYP21A2.

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