Detection reagent and analysis method for copy number variation and gene mutation of embryonic cells

Abstract

The invention relates to a detection reagent and an analysis method for copy number variation and gene mutation of embryonic cells, in particular to a reagent and an analysis method for gene mutation of 4500 genetic related diseases and copy number variation analysis of 23 pairs of chromosomes. According to the method disclosed by the invention, 4500 genetic disease related genes can be analyzed, and sequencing data of 63000 exons of the genes and areas distributed in 23 pairs of chromosomes after adjacent targeted enrichment are analyzed; the copy number variation of embryo biopsy cells and the carrying condition of genetic mutation of 4500 genetic related diseases can be quickly screened through single detection, and the embryos of the epiploid and the embryos without pathogenic genetic mutation are preferentially implanted, so that the innovation and progress of assisted reproduction and prenatal and postnatal breeding technologies are promoted.

Description

Technical Field

[0001] The present invention belongs to the field of gene genetic diagnosis and human assisted reproductive technology, and relates to a detection reagent and analysis method for embryonic cell copy number variation and gene mutation, and specifically to preimplantation genetic testing (PGT), which is a detection reagent and analysis method for identifying whether an embryo carries chromosome copy number variation and pathogenic mutations of 4,500 genetic disease-related genes. Background Art

[0002] For nearly 30 years, single-gene preimplantation genetic testing (PGT-M, which was a category of PGD before 2017, PGD-preimplantation genetic diagnosis) involves identifying gene mutations in embryos through mutation-specific PCR (polymerase chain reaction)-based testing. These tests are usually designed and customized for individual couples, with good customization costs and long testing times. Gene mutations are usually subsequently analyzed by allele-specific PCR, gel electrophoresis of amplified fragments or DNA sequencing, and analysis by capillary-based fragment analyzers. Although practical, the results of typical analyses are prone to errors related to allele dropout (ADO), failure to amplify one of the alleles, or more likely, preferential amplification of one allele (PA), making the other allele undetectable by this classic detection method.

[0003] In order to overcome this phenomenon and avoid or at least reduce the errors in the interpretation of the results, researchers have further designed composite tests that combine mutation detection with linkage (dis)equilibrium between polymorphic markers and mutation sites. This test requires that the polymorphic marker sites preserve information to distinguish between parental alleles and the combined information in the resulting embryo. Although some of the tests developed may be partially or fully applicable to PGT-M testing of unrelated couples with the same genetic disease, it is usually also necessary to construct new personalized marker sets. The time and resource costs required for the first development of genetic disease testing methods are too high, which may cause some couples to delay PGT intervention or even miss the opportunity for PGT intervention. Therefore, the development of universal PGT testing technology and the simplification of the test development cycle may be an urgent clinical need for more convenient and widespread use of PGT.

[0004] The robustness of PGT-M testing can be improved by using linked polymorphic markers in adjacent intervals. This usually involves identifying such markers in a database, designing primers to amplify the markers, optimizing the PCR reaction for each polymorphic site, screening the parental information, and then mixing all the appropriate primer pairs into one reaction that can amplify the selected target from several initial cells. This type of technology requires a long time period and resources, and if these necessary procedures can be reduced, this type of technology will be widely used in PGT laboratories.

[0005] As the understanding of the impact of embryonic chromosome copy number variation on the success rate of embryo implantation gradually deepens, research teams are now trying to combine gene mutation analysis with chromosome analysis in order to screen the best embryos for transplantation. As mentioned above, gene mutation analysis often uses specific targeted PCR, while chromosome copy number variation analysis involves whole genome amplification of DNA from embryo biopsy cells, followed by analysis of the relative balance between chromosomes and within chromosomes by gene chips or sequencing. Analysis of chromosome copy number can be achieved in a variety of ways. The usual method sequences random DNA fragments, then identifies the chromosome position and "bins" the fragments according to the position. These boxes are then statistically compared to infer the relative copy number between chromosomes and between chromosomes, thereby drawing a chromosome map. Euploid embryos are considered to be in a state of equilibrium between chromosomes and sequencing reads within a single box. Another method uses SNPs (single nucleotide polymorphisms) to draw haplotype patterns along chromosomes. This method can easily identify inconsistencies in polymorphic inheritance. If the loss of an entire or partial chromosome extends the homozygous region, for example: in a region of minor allele imbalance at a haplotype or SNP locus, it indicates a trisomy or a segmental deletion / duplication, and this method is often used to identify common copy number variation changes. In addition, haplotype patterns can also be used to determine polymorphic extended regions associated with specific genes to complement any targeted gene analysis. Developing a method that combines both copy number variation screening and single gene mutation detection will attract widespread attention in the PGT field.

[0006] As the first step of the test, most research teams first choose to pre-amplify the whole genome of a single cell, and then perform chromosome copy number variation analysis and gene mutation detection on the amplified samples separately. Simplifying the two analyses and combining them into one method will provide technical advantages for genetic laboratories.

[0007] In recent years, bioinformatics has achieved the combination of chromosome detection and gene mutation detection. An example of this is called WES (Whole Exome Screening), in which DNA from gene exons (~23,000) is enriched and sequenced to identify variants of interest in any gene. This technology can be used for patients with difficult genetic diseases that cannot be directly identified by direct detection methods. WES usually requires 30-40 million sequence reads to achieve an average sequence depth of 30-100 times, which is considered sufficient to reliably identify sequence variations. However, it is clear that most sequencing data resources are used to analyze non-genetic mutations or gene sequences that are insufficiently studied. Since genes are scattered on most chromosomes, WES analysis covers many chromosome regions. The stable exon enrichment process provides the possibility of copy number analysis of chromosome regions. Although not continuous, a large number of genes and more exon targets result in relatively good coverage of most chromosomes. Within chromosomes, there are millions of SNP-type polymorphic sites, most of which are located outside the target gene region. However, many genes also contain such SNP sites, providing the possibility of allele identification. The use of next-generation variant exon screening (ngVES) bioinformatics analysis technology can combine sequence screening, copy number assessment and SNP analysis. This will be of great help to laboratories that want to use PGT-M / A / SR combination analysis.

[0008] For routine testing of embryos, the cost and difficulty of whole exome sequencing (WES) alone are prohibitive. Whole exome sequencing requires a large amount of sequencing resources, which increases the cost of testing. If the scope of analysis can be determined, it can satisfy the analysis of copy number variation and focus on the study of more mature pathogenic gene mutations, which may become a more effective PGT method. Summary of the invention

[0009] In view of the actual needs of PGT-A / -SR / M combined detection in preimplantation embryo diagnosis in the prior art, the present invention provides a detection reagent and analysis method for embryonic cell copy number variation and gene mutation, which is a reagent and analysis method for analyzing 4,500 genetic disease-related gene mutations and copy number variation analysis of 23 pairs of chromosomes. According to the method of the present invention, the analysis of 4,500 genes related to genetic diseases can be analyzed. By analyzing the sequencing data of the regions distributed in 23 pairs of chromosomes after the enrichment of 63,000 exons and adjacent targeted enrichments of the above genes, the copy number variation of embryo biopsy cells and the carrying of genetic mutations of 4,500 genetic-related diseases can be quickly and accurately identified through a single test, thereby improving the success rate of clinical embryo implantation, achieving preferential implantation of embryos with normal chromosomes and no pathogenic genetic mutations, and further blocking the transmission of pathogenic genetic mutations to the next generation. Promote the innovation and progress of assisted reproductive technology.

[0010] The present invention aims to provide a highly sensitive, relatively simple to operate, relatively low-cost and highly accurate detection reagent and method for embryonic cell copy number variation and 4500 genetic mutation-related genes, which is applied to preimplantation embryo diagnosis.

[0011] To achieve the above purpose, the technical solution of the present invention is as follows:

[0012] This is a test kit for copy number variation and gene mutation in (single) embryonic cells, and also a test kit for copy number variation and 4500 genes related to hereditary mutations, prepared by the following steps:

[0013] S1: A total of 4,500 genes related to genetic diseases were screened from public databases;

[0014] S2: Design double-ended capture probes for the upstream and downstream DNA double strands of approximately 63,000 exon regions of all relevant genes;

[0015] S3: For 23 chromosomes, a double-ended capture probe was designed by selecting the upstream and downstream of the forward and reverse strands of the DNA sequence within a 500bp region between each 1Mb region;

[0016] S4: Design probes based on the regions selected by S2 and S3. The sites are extended upstream and downstream respectively, and the preset values ​​of alignment ability and GC content are within the preset range;

[0017] S5: The probe finally designed by S4 is used as a detection reagent for embryonic single-cell aneuploidy and genetic mutations.

[0018] Furthermore, in S3, the preset distance between two adjacent probes is greater than 0.1M and less than 0.5M.

[0019] Furthermore, in S4, the site is extended by 100 bp upstream and downstream respectively.

[0020] Furthermore, in S4, the preset value of the comparison capability is 1, and the preset range of the GC content is greater than or equal to 0.46 and less than or equal to 0.55.

[0021] The present invention also relates to a method for analyzing embryonic cell copy number variation and gene mutation, and also to a system for analyzing embryonic single cell copy number variation and gene mutation, the system comprising software for calculating and analyzing sample data and hardware for loading the software, and comprising the following steps:

[0022] Step 1: Use one of the above-mentioned embryonic cell copy number variation and gene mutation detection reagents to perform targeted enrichment, library preparation and sequencing on the sample after single cell amplification from embryo biopsy; select embryo biopsy samples from the 3rd to 6th day of development, and obtain 3 to 10 cells from the biopsy as embryo biopsy samples;

[0023] Step 2: Use the system to perform bioinformatics analysis on the sequencing results, count the sequencing depth of each site in the capture region of each probe in the detection kit and the sequencing depth of the four bases A / T / C / G, calculate the frequency of the four bases A / T / C / G at each site, and compare with the reference allele and alternative allele in the population frequency database to determine the minor allele frequency;

[0024] Step 3: Use the system to conduct gene mutation correlation analysis, apply analysis tools for comparison and information annotation, set confidence intervals and conduct association analysis in combination with upstream and downstream polymorphic sites. The analysis features are that the pathogenic mutations are annotated and interpreted for all exon coverage areas of the 4,500 screened genes, and upstream and downstream association analysis of polymorphic sites is performed to assist in identifying the parental source of heterozygous mutations, and automatic interpretation is performed to generate analysis reports and visual images;

[0025] Step 4: The system is used to analyze the ploidy of 23 chromosomes, and the analysis tool is used for data comparison. The analysis feature is that the algorithm for calculating the copy number is set to combine the total number of reads within the 500bp range between all chromosome intervals of 1M regions with the number of reads within the analysis window of each 500bp split into 1Mb regions of 4500 gene exons, and the same batch of normal embryo reference samples are compared and calculated, and the confidence interval of the same batch of control samples is set to be greater than 0.95;

[0026] Step 5: According to the analysis results of step 4, draw 23 chromosome analysis graphs, set a standard error of 20% for the analysis, normal embryo (-0.2-+0.2), haploid (-1.8--2.0), triploid (1.8-2.0), and generate corresponding analysis results.

[0027] Furthermore, the embryo biopsy sample described in step 1 is selected from cells derived from embryo blastomere biopsy or blastocyst trophectoderm biopsy.

[0028] Furthermore, the embryo biopsy sample described in step 1, wherein the embryo biopsy sample whole genome amplification method includes: WGA, MDA, MALBAC and other whole genome amplification methods.

[0029] Compared with the prior art, the present invention has the following advantages:

[0030] 1. The present invention provides a detection reagent for embryonic cell copy number variation and gene mutation, which is a detection reagent for embryonic single cell aneuploidy and 4500 genetic mutation-related genes. The probe is designed to cover a total of about 63,000 exon regions including 4500 genetic mutation genes, as well as 1Mb regions upstream and downstream of the region and chromosome sequences of all intervals within 500bp between 1Mb intervals of 23 pairs of chromosomes. The detection reagent method is preferentially applicable to next-generation gene sequencing, and is also applicable to long molecule sequencing platforms.

[0031] 2. The present invention provides an analysis method for copy number variation and gene mutation in embryonic cells, which can detect all possible genetic mutations and de novo mutations in the full-length region of 4500 targeted genes in a single test. The single test analyzes the ploidy of all chromosomes by performing copy number analysis on the target region on the chromosome.

[0032] 3. The present invention provides an analysis method for copy number variation and gene mutation in embryonic cells, identifying linkage polymorphisms within or around target genes to improve the credibility of gene analysis. Identify aberrations in polymorphic loci that may support copy number analysis and interpretation. Identify polymorphic loci that may indicate abnormal fertilization of embryos. Identify polymorphic loci that may indicate abnormal chromosome segregation patterns. It can be applied to most current pedigrees of genetic mutation carriers. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is an analysis chart of the results of 4500 gene pathogenic mutation detection in embryo biopsy samples in an embodiment of the present invention;

[0034] Figure 2 This is a diagram of chromosome analysis results of an embryo biopsy sample in an embodiment of the present invention. DETAILED DESCRIPTION

[0035] The technical scheme in the embodiment of the present invention is described clearly and in detail in conjunction with the following drawings. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0036] The specific embodiments of the present invention are described in detail, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

[0037] Example:

[0038] A reagent for detecting embryonic cell copy number variation and gene mutation is prepared by the following steps:

[0039] 1: Development of test kits

[0040] A total of 4,500 lists of genes related to genetic diseases were screened from public databases, and double-ended capture probes were designed for the upstream and downstream of the double-stranded DNA in the approximately 63,000 exon regions of all related genes; for 23 chromosomes, double-ended capture probes were designed for the upstream and downstream of the forward and reverse double-stranded DNA sequences in a 500bp region between each 1Mb region. The sites were extended 100bp upstream and downstream respectively, and the preset values ​​of the alignment ability were 1 and the GC content were both within the preset range of greater than or equal to 0.46 and less than or equal to 0.55. The preset spacing between two adjacent probes was greater than 0.1M and less than 0.5M;

[0041] 2: Embryo biopsy and whole genome amplification and library preparation

[0042] Five embryos that developed to D5 were selected for cell biopsy, with 5-8 cells. Single-cell amplification was performed using Discover-sc Single Cell WGA Kit, N602, and quantification was performed using Qubit 4.0. 200 ng of each biopsy sample was taken for next-generation sequencing library preparation.

[0043] 3: Targeted enrichment and sequencing

[0044] The above-mentioned example library and the probe designed by the detection kit were subjected to liquid phase hybridization capture, elution and magnetic bead capture, and then PCR amplification and purification were performed before second-generation sequencing. The sequencing platform was Novasek6000 (Illumina), using a 150bp double-end sequencing mode;

[0045] 4: Data Analysis

[0046] 4.1 UMI tag filtering

[0047] The fastq files obtained by sequencing were aligned and filtered for UMI using fgbio and BWA to obtain bam files containing UMI molecular tag information;

[0048] 4.2 Analysis of gene mutations in embryonic cells

[0049] The system is used to conduct gene mutation correlation analysis, and analysis tools are used for comparison and information annotation. Confidence intervals are set to combine upstream and downstream polymorphic sites for association analysis. The analysis features are that pathogenic mutations are annotated and interpreted for all exon coverage areas of 4500 screened genes, and upstream and downstream association analysis of polymorphic sites is performed to assist in identifying the parental source of heterozygous mutations, and automatic interpretation is performed to generate analysis reports and visualization images ( Figure 1 );

[0050] 4.3 Copy number variation analysis of embryonic cells

[0051] The system was used to analyze the ploidy of 23 chromosomes, and the analysis tool was used for data comparison. The analysis features are as follows: the algorithm for calculating the copy number is set, the total number of reads within the 500bp range between all chromosome intervals of 1M regions is combined with the number of reads within the analysis window of each 500bp split into 1Mb regions of 4500 gene exons, and the same batch of normal embryo reference samples are compared and calculated, and the confidence interval of the same batch of control samples is set to be greater than 0.95; 23 chromosome analysis graphs are drawn, and a standard error of 20% is set for the analysis, normal embryos (-0.2-+0.2), haploids (-1.8--2.0), triploids (1.8-2.0), and the corresponding analysis results are generated ( Figure 2 ).

[0052] Results and Discussion:

[0053] Table 1:

[0054] Gene EXON cDNA Protern Class OMIM Inheritance CFTR exon12 c.A1666G p.I556V P 277180,219700 AD KCNJ18 exon3 c.997_999del p.E334del P 613239 AD LRP6 exon2 c.A246T p.K82N P 610947 AD PRRT2 exon2 c.641dupC p.R217Pfs*8 P 605751 AD PRSS1 exon2 c.A86T p.N29I P 167800 AD IFT140 exon31 c.T4196C p.L1399P P 617781,266920 AR|AR KCNV2 exon1 c.G80A p.R27H P 610356 AR

[0055] Example 1: Embryo number: DHX-E1

[0056] The analysis result is: 45, XX, -22. (such as Figure 1 and 2 The red box shows the embryo copy number variation, which is consistent with the clinical PGT-A test results of the embryo. At the same time, 7 pathogenic mutation sites were detected in this example, of which 5 were annotated as autosomal dominant pathogenic mutation sites and 2 were autosomal recessive mutation sites according to the OMIM database (as shown in Table 1).

[0057] Through the patented detection method, the copy number variation of the embryo in the embodiment and the genetically related pathogenic mutations were analyzed simultaneously in one test, which reduced the cost of completing the same test by 3 times and increased the accuracy of the target region analysis carrying the mutation by 3-10 times. Figure 1 ) and chromosome abnormality analysis ( Figure 2 ) combined with the above method improves the accuracy of chromosome abnormality detection and shortens the time of PGT diagnosis and treatment services for patients. The detection range of 4500 genes covered by this embodiment can continue to expand the number of probes based on the original reagent design in the future to meet different PGT diagnosis and treatment service needs.

[0058] The embodiments described above are some embodiments of the present invention, rather than all embodiments. They are not intended to limit the scope of the invention claimed for protection, but merely represent selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Claims

1. A reagent for detecting embryonic cell copy number variation and gene mutation, characterized in that: It is also a test kit for embryonic single-cell aneuploidy and 4,500 genetic mutation-related genes, prepared by the following steps: S1: A total of 4,500 genes related to genetic diseases were screened from public databases; S2: Design double-ended capture probes for the upstream and downstream DNA double strands of 63,000 exon regions of all relevant genes; S3: For 23 chromosomes, a double-ended capture probe was designed by selecting the upstream and downstream of the forward and reverse strands of the DNA sequence within a 500bp region between each 1Mb region; S4: Design probes based on the regions selected by S2 and S3. The sites are extended upstream and downstream respectively, and the preset values ​​of alignment ability and GC content are within the preset range; S5: The probe finally designed by S4 is used as a detection reagent for embryonic single-cell aneuploidy and genetic mutations.

2. A reagent for detecting embryonic cell copy number variation and gene mutation according to claim 1, characterized in that: In S3, the preset distance between two adjacent probes is greater than 0.1M and less than 0.5M.

3. A reagent for detecting embryonic cell copy number variation and gene mutation according to claim 1, characterized in that: In S4, the site is extended by 100 bp upstream and downstream respectively.

4. A reagent for detecting embryonic cell copy number variation and gene mutation according to claim 1, characterized in that: In S4, the preset value of the comparison capability is 1, and the preset range of the GC content is greater than or equal to 0.46 and less than or equal to 0.

55.

5. A method for analyzing embryonic cell copy number variation and gene mutation, characterized in that: It is also a system for analyzing embryonic single cell copy number variation and gene mutation, which includes software for calculating and analyzing sample data and hardware for loading the software, and includes the following steps: Step 1: using the embryonic cell copy number variation and gene mutation detection reagent of claim 1, performing targeted enrichment, library preparation and sequencing on the sample after single cell amplification from embryonic biopsy; selecting embryonic biopsy samples from the 3rd to 6th day of development, and obtaining 1 to 10 cells by biopsy as embryonic biopsy samples; Step 2: Use the system to perform bioinformatics analysis on the sequencing results, count the sequencing depth of each site in the capture region of each probe in the detection kit and the sequencing depth of the four bases A / T / C / G, calculate the frequency of the four bases A / T / C / G at each site, and compare with the reference allele and alternative allele in the population frequency database to determine the minor allele frequency; Step 3: Use the system to conduct gene mutation correlation analysis, apply analysis tools for comparison and information annotation, set confidence intervals and conduct association analysis in combination with upstream and downstream polymorphic sites. The analysis features are that the pathogenic mutations are annotated and interpreted for all exon coverage areas of the 4,500 screened genes, and upstream and downstream association analysis of polymorphic sites is performed to assist in identifying the parental source of heterozygous mutations, and automatic interpretation is performed to generate analysis reports and visual images; Step 4: The system is used to analyze the copy number of 23 pairs of chromosomes, and the analysis tool is used for data comparison. The analysis feature is that the algorithm for calculating the copy number is set to combine the total number of reads within the 500bp range between all chromosome intervals of 1M regions with the number of reads within the analysis window of each 500bp split into each 1Mb region of the 4500 gene exon regions, and the same batch of normal embryo reference samples are compared and calculated, and the confidence interval of the same batch of control samples is set to be greater than 0.95; Step 5: According to the analysis results of step 4, draw 23 chromosome analysis graphs, set a standard error of 20% for the analysis, normal embryo (-0.2-+0.2), haploid (-1.8--2.0), triploid (1.8-2.0), and generate corresponding analysis results.

6. The method for analyzing embryonic cell copy number variation and gene mutation according to claim 5, characterized in that: The embryo biopsy sample described in step 1 is selected from cells derived from embryo blastomere biopsy or blastocyst trophectoderm biopsy.

7. The method for analyzing embryonic cell copy number variation and gene mutation according to claim 5, characterized in that: The embryo biopsy sample described in step 1, wherein the whole genome amplification method of the embryo biopsy sample includes: WGA, MDA, MALBAC and other whole genome amplification methods.

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