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

Abstract

The present invention relates to a CYP21A2 and CYP21A1p gene fusion judgment system and method based on Pacbio sequencing data. The present invention constructs a distinguishing site set, generates a sorted and indexed comparison file, obtains the haplotype sequences of CYP21A2 and CYP21A1p, obtains the variation information of each haplotype on the distinguishing site set, and statistically calculates the frequency of occurrence of the distinguishing sites in the obtained distinguishing site set in the CYP21A1p haplotype as the weight of each distinguishing site, and calculates the cumulative score of each distinguishing site. i ; According to Score i The presence and type of fusion can be determined by following the curve trend of the sequence of distinguishing sites. While detecting complex variations in gene fusions, the present invention can accurately locate breakpoints and distinguish fusion directions, thereby improving detection accuracy and resolution.

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 CYP21A2 and CYP21A1p gene fusion based on Pacbio sequencing data. Background Art

[0002] Congenital adrenal hyperplasia (CAH) is an autosomal recessive genetic disorder, most commonly caused by the CYP21A2 gene. The CYP21A2 gene and its pseudogene, CYP21A1p, are closely aligned on chromosome 6p21.3. Their sequences are highly homologous, and recombination events such as unequal crossing over, gene conversion, partial deletion, or fusion often occur, leading to decreased or even loss of 21-hydroxylase activity. Such recombination or fusion variants occur with a certain frequency in CAH patients and are closely associated with disease onset and phenotypic severity.

[0003] Currently, genetic testing for the CYP21A2 / CYP21A1p region primarily relies on traditional techniques such as Sanger sequencing, ARMS-PCR, and MLPA. While these methods offer advantages for detecting single or specific site mutations, due to the high homology between CYP21A2 and CYP21A1p and the structural complexity of this region, they often suffer from the following limitations when large structural rearrangements or fusion variants occur: a. Limited sensitivity: large variants or fusion sites may be missed; b. Specificity is difficult to guarantee: highly homologous sequences can lead to false positives or misinterpretations; and c. Insufficient resolution: the location of breakage (recombination) and the specific fusion pattern cannot be clearly determined.

[0004] Compared to traditional technologies, third-generation sequencing platforms like PacBio, due to their superior read length, can span large repetitive regions or regions of high homology within a single long-read sequence, thereby more accurately reflecting the CYP21A2 / CYP21A1p genomic structure. Combined with bioinformatics analysis pipelines optimized for long-read data, such as precise alignment of repetitive regions and gene fusion detection algorithms, these platforms can better locate recombination breakpoints and determine whether gene fusions have 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 realize the 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 long sequence alignment software, identify the variant sites of CYP21A1p relative to CYP21A2, and obtain a distinguishing site set between CYP21A2 and CYP21A1p;

[0010] The sequencing data alignment module is used to align the high-fidelity sequence of the sample to be tested obtained by PacBio sequencing to the human reference genome, and generate an ordered and indexed alignment 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 at the set of distinguishing sites;

[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 cumulatively 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 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 implementation 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 embodiment, 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 implementation 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 discrimination site set.

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

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

[0023]

[0024] in, is the cumulative score of the i-th distinguishing site in the distinguishing site set; a is the judgment value of whether the distinguishing site exists, which is 1 if a exists and -1 if a does not exist; is the weight of the i-th distinguishing site.

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

[0026] When the As i increases, it shows an overall decreasing trend. 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. 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 increased with the increase of i in the first half and decreased with the increase of i in the second half, and a maximum value appeared in the middle of the distinguishing site set, it was judged to be CYP21A1p-CYP21A2 fusion.

[0030] In another aspect, 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 variant sites of CYP21A1p relative to CYP21A2, and obtain the set of sites that distinguish CYP21A2 from CYP21A1p;

[0032] Align the high-fidelity sequences of the samples to be tested, obtained by PacBio sequencing, to the human reference genome, and generate an ordered and indexed alignment file;

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

[0034] Each obtained CYP21A2 and CYP21A1p haplotype sequence is tested for variation to obtain variation information of each haplotype at the set of distinguishing sites;

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

[0036] Score the variation of all haplotypes in the sample to be tested in the distinguishing site set cumulatively, 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 takes advantage of the high homology, complex structure and easy fusion of CYP21A2 and CYP21A1p, and utilizes the high accuracy and ability to span repeated segments of long-read sequencing such as PacBio to develop a complete set of bioinformatics processes for alignment, haplotype assembly, variant identification and fusion judgment; by calculating the weights and cumulative scoring strategies for distinguishing sites between CYP21A2 and CYP21A1p, it can detect complex gene fusion variations while accurately locating breakpoints and distinguishing fusion directions, greatly improving the accuracy and resolution of 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 and locate recombination breakpoints at one time, 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 While the present invention only provides quantitative clues at the level of large-scale deletions or CNVs, it can provide "qualitative + positioning" with a higher degree of refinement and can be flexibly applied in different demand scenarios such as clinical large-scale 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 CYP21A2 and CYP21A1p gene fusion 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 cumulative 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] Figure 9 Schematic diagram of the cumulative score of sample T3 in Example 2 of the present invention.

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

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

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

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

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

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

[0054] The present invention will be further described in detail below with reference to 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 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 discrimination site set construction module 100, a sequencing data comparison module 200, a haplotype assembly module 300, a haplotype variation identification module 400, a discrimination 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 long sequence alignment software, identify the variant 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, and the sequence is aligned using minimap2 software. The distinguishing site set is constructed based on the SNPs, insertions and / or deletions generated by CYP21A1p relative to CYP21A2, as shown in FIG. Figure 2 shown.

[0060] The sequencing data comparison module 200 is used to 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 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 reference genome, generating a binary alignment bam file. Using the bam processing software samtools, the sequences in the bam file were sorted by alignment position, generating the 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 the 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 several 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, and 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 the variation is recorded at 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 calculate the frequency of occurrence of the distinguishing sites in the obtained distinguishing site set in the CYP21A1p haplotype using the homologous gene assembly results of the normal human control sample set as the weight of each distinguishing site.

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

[0078] For example, using PacBio HiFi data from a set of 40 normal human samples, we performed a human genome alignment and assembled haplotypes for CYP21A2 and CYP21A1p. We then identified variants within these haplotypes and calculated the frequency of occurrence of the resulting discriminatory loci set across all CYP21A1p haplotypes. This frequency was defined as the CYP21A2-CYP21A1p discriminatory loci weight. The discriminatory loci set is shown in Table 1. The larger the locus weight, the higher the discriminatory power of the locus for CYP21A2 and CYP21A1p.

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

[0080]

[0081] Note:

[0082] 5'flk: upstream region of CYP21A2 gene;

[0083] C_e1: CYP21A2 gene exon1 region;

[0084] C_i1: CYP21A2 gene intron1 region.

[0085] The gene fusion judgment module 600 is used to accumulate 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.

[0086] In the gene fusion judgment module 600, the calculation formula for the cumulative score is:

[0087]

[0088] in, is the cumulative score of the i-th distinguishing site in the distinguishing site set; a is the judgment value of whether the distinguishing site exists, which is 1 if a exists and -1 if a does not exist; is the weight of the i-th distinguishing site.

[0089] Determining whether CYP21A2 and CYP21A1p are fused and the type of fusion includes:

[0090] when As i increases, it shows an overall decreasing trend. When the extreme values ​​are located at the two ends of the distinguishing site set, it is judged to be a complete CYP21A2 gene;

[0091] when As i increases, it shows an overall increasing trend. When the extreme values ​​are located at the two ends of the distinguishing site set, it is judged to be a complete CYP21A1p gene;

[0092] when When the value of i 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, the expression of i decreases as i increases, while in the CYP21A1p part, the expression of i increases as i increases. The minimum value occurs in the middle of the site set);

[0093] when When the value of i increases in the first half and decreases 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, the expression of cytochrome P21a1 increases with the increase of i, while in the CYP21A1p part, the expression of cytochrome P21a1 decreases with the increase of i. The maximum value occurs in the middle of the site set).

[0094] For ease of understanding, The curve that changes with i is visualized and plotted, and the final output is the prediction result of whether there is a fusion variation and the fusion type for each haplotype.

[0095] 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:

[0096] S100, obtaining a CYP21A1p reference sequence, aligning it to a CYP21A2 reference sequence using long sequence alignment software, identifying variant sites of CYP21A1p relative to CYP21A2, and obtaining a set of distinguishing sites between CYP21A2 and CYP21A1p;

[0097] S200, aligning the high-fidelity sequence of the sample to be tested obtained by PacBio sequencing to the human reference genome, and generating an ordered and indexed alignment file;

[0098] S300. Based on the alignment file, haplotype assembly software was used to assemble the CYP21A2 and CYP21A1p regions to obtain the haplotype sequences of CYP21A2 and CYP21A1p.

[0099] S400, performing mutation detection on each obtained CYP21A2 and CYP21A1p haplotype sequence to obtain the mutation information of each haplotype at the distinguishing site set;

[0100] S500, using the homologous gene assembly results of the normal human control sample set, the frequency of occurrence of the discriminating sites in the obtained discriminating site set in the CYP21A1p haplotype is counted as the weight of each discriminating site;

[0101] S600, cumulatively 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.

[0102] Example 2

[0103] The results of 6 samples using this method are compared

[0104] Six samples with MLPA verification results were subjected to Pacbio sequencing of the CYP21A2 region long fragment capture. 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 Table 2. Figure 4 and Figure 5 , Paraphase haplotype assembly and cumulative score diagrams 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 Figure 9 , Paraphase haplotype assembly and cumulative score diagrams of sample T4 are shown in Figure 10 and Figure 11 , Paraphase haplotype assembly and cumulative score diagrams of sample T5 are shown in Figure 12 and Figure 13 , Paraphase haplotype assembly and cumulative score diagrams of sample T6 are shown in Figure 14 and Figure 15 .

[0105] Table 2 Comparison of Paraphase assembly haplotypes of CYP21A2 and CYP21A1p

[0106]

[0107] Table 2 shows the comparison of MLPA results of 6 samples and the detection of CYP21A2 and CYP21A1p fusion by the method of the present invention:

[0108] 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.

[0109] 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.

[0110] The method of the present invention can not only confirm the deleted exon, but also identify the specific fusion direction and breakpoint position; while MLPA can only indicate the occurrence of large deletions and cannot accurately locate them.

[0111] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. 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 can 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 long sequence alignment software, identify the variant sites of CYP21A1p relative to CYP21A2, and obtain a distinguishing site set between CYP21A2 and CYP21A1p; The sequencing data alignment module is used to align the high-fidelity sequence of the sample to be tested obtained by PacBio sequencing to the human reference genome, and generate an ordered and indexed alignment 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 at the set of distinguishing sites; 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 cumulatively 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 with the change of the order of the differentiation sites and the type of extreme values ​​appearing at the concentrated end or both ends of the differentiation sites are used to determine whether there is a fusion between CYP21A2 and CYP21A1p and the type of fusion; In the gene fusion judgment module, the calculation formula for the cumulative score is: in, is the cumulative score of the i-th distinguishing site in the distinguishing site set; a is the judgment value of whether the distinguishing site exists, which is 1 if a exists and -1 if a does not exist; is the weight of the i-th distinguishing site; In the gene fusion judgment module, the determination of whether CYP21A2 and CYP21A1p are fused and the type of fusion includes: When the As i increases, it shows an overall decreasing trend. 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. 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 increased with the increase of i in the first half and decreased with the increase of i in the second half, and a maximum value appeared in the middle of the distinguishing site set, it was judged to be CYP21A1p-CYP21A2 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 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 set of distinguishing sites.

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 used to generate sorted and indexed comparison files, CYP21A2 and CYP21A1p haplotype sequences, and variation information of each haplotype at the distinguishing site set. The proportion of mutations that appear or remain consistent at each distinguishing site on all CYP21A1p haplotypes is counted, and this proportion is defined as the weight of the distinguishing site.

7. A method for determining gene fusion between CYP21A2 and CYP21A1p for non-disease diagnosis purposes 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 variant sites of CYP21A1p relative to CYP21A2, and obtain the set of sites that distinguish CYP21A2 from CYP21A1p; Align the high-fidelity sequences of the samples to be tested, obtained by PacBio sequencing, to the human reference genome, and generate an ordered and indexed alignment file; Based on the alignment file, haplotype assembly of the CYP21A2 and CYP21A1p regions was performed using homologous gene haplotype assembly software to obtain haplotype sequences of CYP21A2 and CYP21A1p; Each obtained CYP21A2 and CYP21A1p haplotype sequence is tested for variation to obtain variation information of each haplotype at the set of distinguishing sites; Using the homologous gene assembly results of the normal human control sample set, the frequency of occurrence of the discriminant sites in the obtained discriminant site set in the CYP21A1p haplotype was statistically analyzed as the weight of each discriminant site; Score the variation of all haplotypes in the sample to be tested in the distinguishing site set cumulatively, and calculate the cumulative score of each distinguishing site. i ; According to Score i The trend of the curve with the change of the order of the differentiation sites and the type of extreme values ​​appearing at the concentrated end or both ends of the differentiation sites are used to determine whether there is a fusion between CYP21A2 and CYP21A1p and the type of fusion; The calculation formula for the cumulative score is: in, is the cumulative score of the i-th distinguishing site in the distinguishing site set; a is the judgment value of whether the distinguishing site exists, which is 1 if a exists and -1 if a does not exist; is the weight of the i-th distinguishing site; The determination of whether CYP21A2 and CYP21A1p are fused and the type of fusion comprises: When the As i increases, it shows an overall decreasing trend. 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. 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 increased with the increase of i in the first half and decreased with the increase of i in the second half, and a maximum value appeared in the middle of the distinguishing site set, it was judged to be CYP21A1p-CYP21A2 fusion.

8. The method for determining gene fusion of CYP21A2 and CYP21A1p for non-disease diagnosis purposes based on Pacbio sequencing data according to claim 7, characterized in that: The obtaining of 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.

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