A kit for detecting Turner syndrome based on MLPA-NGS method and its use method and application

Abstract

The present invention discloses a kit for detecting Turner syndrome based on the MLPA-NGS method, and a method for using and application thereof, wherein the kit comprises a CNV probe for detecting the copy number of the DNA position to which the probe binds, a SNP probe for giving the SNP genotype of the hybridization position of the probe on the DNA, and a universal primer with an index sequence for amplifying the connected probe; each pair of probes is provided with a characteristic sequence for distinguishing, so as to analyze the number of reads of each probe for the sequencing result, thereby analyzing the SNP typing, the sequencing quality of the sample, the parent-child relationship of the core family, the parental origin of the X chromosome, the relative copy number of the X chromosome, and the parental origin of the X chromosome mosaic. The MLPA-NGS method of the above kit has a large amount of detection information, can achieve the effect of the SNP chip for the copy number of the X chromosome, can achieve the effect of the STR for the analysis of the parental origin of the X chromosome, has high sensitivity, is easy to operate, and is convenient for large-scale promotion.

Description

Technical Field

[0001] The present invention relates to the field of biological gene technology, and in particular to a kit for detecting Turner syndrome based on an MLPA-NGS method, and a use method and application thereof. Background Art

[0002] Turner Syndrome (TS) is a chromosomal disease caused by insufficient gene dosage due to the loss of one or part of the X chromosome, with a female prevalence of about 1 / 2500. The TS chromosome karyotype consists of monosomy, mosaicism, X chromosome structural abnormality, and Y chromosome karyotype. Most patients have clinical phenotypes such as short stature, abnormal skeletal development, kidney and heart malformations, intellectual and cognitive impairment, uterine or ovarian development abnormalities, external genital development different from normal people, and primary amenorrhea. Due to the differences in the composition of the chromosome karyotype of patients, their clinical manifestations vary. Clinically, TS mosaic patients are mostly found to have the disease when they seek medical treatment for infertility due to slow growth and development, amenorrhea in female patients, irregular menstruation, or infertility in adulthood. In general, the phenotype is determined by the loss of the X chromosome. The loss of the terminal X chromosome is usually associated with ovarian dysfunction, and the loss of the proximal Xq (such as Xq13) usually leads to breast dysplasia, primary amenorrhea, and gonadal failure. Both the Xq26-q28 region and the Xq13.3-q21.1 region are thought to contain ovarian determinant cluster genes and are associated with premature ovarian failure.

[0003] Among the TS patients, about 45% of the patients have a 45,X karyotype, and mosaicism accounts for about 20%-30%. The X chromosome comes from the father (Xp) or the mother (Xm), and its clinical phenotype and prognosis are different. At present, many foreign studies have confirmed that the characteristics related to X-linked genes are different in TS patients with two different monomers, Xm and Xp. Van's study pointed out that Xm in TS patients will promote selective visceral fat accumulation and dyslipidemia; Chu found that the prevalence of cardiovascular disease in Xm subjects was relatively higher than that in the Xp group; Bishop found that TS patients with Xp subjects had more significantly impaired visual spatial memory. Different sources of X chromosomes have different treatment effects. For example, Hamelin found in his study that the height increase caused by growth hormone treatment was greater in TS patients with Xm monomers. Previous studies have also found that different sources of X chromosomes have different clinical manifestations. Therefore, the X-STR technology has been improved and combined with karyotype analysis to detect the type and source of chromosomal karyotypes of TS patients. This not only improves the diagnosis rate of the disease and clarifies the cause of the disease, but also provides a scientific basis for guiding the prevention, treatment and prognosis of the disease, which has certain forward-looking significance.

[0004] TS mosaicism is classified according to whether the second cell line contains all or part of the X chromosome. 45,X / 46,XiXq, 45,X / 46,XX, 45,X / 47,XXX, 45,X / 46,X,del(Xp) are the more common types of mosaicism. Different cell lines have tissue-specific expression, and the clinical abnormal phenotype of mosaic patients containing 46,XX, 47,XXX, and 46,XY cell lines may be improved. In TS patients, pure 45,X is more likely to have congenital lymphedema. Spontaneous menstruation and pregnancy are more common in mosaic 45,X / 46,XX and 45,X / 47,XXX. Patients with 45,X / 46,XX are slightly taller than other TS patients. Most TS patients have normal intelligence, but patients with small ring X chromosomes have an increased risk of intellectual disability; due to the inactivation of small ring chromosomes, these patients may have a severe typical S phenotype, and ring chromosomes increase the risk of mental retardation and atypical phenotypes. Most women with TS are infertile due to primary ovarian failure, however, approximately 5% experience spontaneous pregnancies, and most cases of spontaneous pregnancies are mosaic. Many studies have shown that pregnancies in patients with TS are at increased risk of miscarriage, stillbirth, and malformed babies.

[0005] In China, most TS are diagnosed through the aid of chromosome karyotype analysis, and the misdiagnosis rate and missed diagnosis rate are high. Internationally, in addition to karyotype analysis, auxiliary diagnosis such as chip technology, MLPA technology, microsatellite analysis technology, and FISH technology are also used. These diagnostic schemes have improved the diagnosis rate to a certain extent, but there are also various types of defects, and they only play a certain supplementary role to karyotype analysis. Therefore, some new detection methods are tried to develop to accurately provide relevant information about the X chromosome, which can be used as an auxiliary tool to assist doctors in clinical diagnosis. Summary of the invention

[0006] In order to overcome at least one problem existing in the prior art, the present invention develops an MLPA-NGS method, which is based on the principle of MLPA (Multiples ligation-dependent probe amplification) technology to build a library and then use the next-generation sequencing technology (NGS) for sequencing, which can realize accurate analysis of CNV and SNV on genes, and is a high-throughput MLPA method. The above MLPA-NGS method is used to analyze human X chromosomes, and a detection scheme with a wide range, low cost, simple operation and short cycle on X chromosomes is obtained; based on the above MLPA-NGS method, a Turner syndrome detection kit and its analysis method are developed, which can accurately detect Turner syndrome at the molecular level, give the dosage of X chromosomes and each segment of X chromosomes, give the parental origin of X chromosomes and each segment of X chromosomes, accurately understand the duplication (if any) and deletion range of the patient's X chromosome, the proportion of chimeras, and the parental origin, obtain the accurate characteristics of the patient's X chromosome, and provide accurate test data for the patient's Turner syndrome (TS) subtype analysis and treatment.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] The first aspect of the present invention is to provide a kit for detecting Turner syndrome based on the MLPA-NGS method, which includes a SNP probe composition, a CNV probe composition and a primer composition; wherein the SNP probe composition includes SNP probe pairs designed for autosomes, X chromosomes, and Y chromosomes, respectively, and the CNV probe composition includes CNV probe pairs designed for autosomes, X chromosomes, and Y chromosomes, respectively; the primer composition includes a universal primer with an index sequence for amplifying the connected probes, each of the probe pairs includes a left probe and a right probe, and the 5' end of the left probe and the 3' end of the right probe respectively contain a universal sequence, which serves as a binding sequence when the universal primer is amplified. The above index sequence is used to distinguish different samples.

[0009] Further, in the above kit, the SNP probe composition includes 142 probe pairs, which are respectively designed at 66 SNP sites of autosomes, 51 SNP sites of chromosome X, and 25 SNP sites of chromosome Y, wherein each of the SNP sites contains two genotypes: wild type and mutant type; the CNV probe composition includes 84 probe pairs, which are respectively designed at 44 pairs of probes of autosomes, 30 pairs of probes of chromosome X, and 10 pairs of probes of chromosome Y. The above CNV probe composition is used to detect the CNV situation at the location of the probe, and the above SNP probe composition can give the typing of SNP after detection.

[0010] Furthermore, in each of the probe pairs, a phosphate group is added to the 5' end of the right probe for probe connection.

[0011] Furthermore, three SNP sites were selected on each autosome (based on GRCh38 / hg38), for a total of 66 sites, which were located at the head, tail and middle regions. Therefore, the distance between any two selected SNPs on the same chromosome was greater than 1000K, and linkage analysis was not required for parentage testing; the SNPs selected for the X chromosome and the Y chromosome were distributed in a nearly equidistant manner.

[0012] Furthermore, two pairs of CNV probes were designed on each autosome, for a total of 44 pairs, which were located near the two ends of the chromosome; the probes selected on the X chromosome and the Y chromosome were distributed in a nearly equidistant manner.

[0013] Further, in the above kit, in the SNP probe composition, the sequence of each SNP probe pair used for binding to the template DNA is shown as SEQ ID NO.1 to SEQ ID NO.284; in the CNV probe composition, the sequence of each CNV probe pair used for binding to the template DNA is shown as SEQ ID NO.285 to SEQ ID NO.452; the sequence of the universal primer is shown as SEQ ID NO.453 to SEQ ID NO.454, the universal sequence at the 5' end of the left probe is shown as SEQ ID NO.455, and the universal sequence at the 3' end of the right probe is shown as SEQ ID NO.456. Specifically, the overall sequence of the probes involved in the reaction is: left probe: 5'-universal sequence-binding template DNA sequence-3', right probe: 5'-binding template DNA sequence-universal sequence-3'.

[0014] Specifically, the probe pair sequences designed for 66 SNP sites on the autosomes are shown in SEQ ID NO.1 to SEQ ID NO.132, the probe pair sequences designed for 51 SNP sites on the X chromosome are shown in SEQ ID NO.133 to SEQ ID NO.234, and the probe pair sequences designed for 25 SNP sites on the Y chromosome are shown in SEQ ID NO.235 to SEQ ID NO.284; the 44 CNV probe pair sequences designed for the autosomes are shown in SEQ ID NO.285 to SEQ ID NO.372, the 30 CNV probe pair sequences designed for the X chromosome are shown in SEQ ID NO.373 to SEQ ID NO.432, and the 10 CNV probe pair sequences designed for the Y chromosome are shown in SEQ ID NO.433 to SEQ ID NO.452.

[0015] It is understandable that in the selection and design of the above-mentioned SNP probes and CNV probes, based on the same MLPA-NGS method, if the SNP site is replaced or the CNV site is replaced to design a new probe, it should not be regarded as an essential change to the probe described in the present invention if it does not have a fundamental impact on the analysis of Turner syndrome.

[0016] Further, in the above-mentioned kit, the SNP probe composition and the CNV probe composition are mixed to obtain a probe working solution, and the concentration of each probe in the probe working solution is 0.2-20fmol / μL. Specifically, all SNP probes and CNV probes are mixed in appropriate amounts to prepare a mixed solution with a concentration of 0.2-20fmol / μL for each probe, and the concentration is optimized according to the test results to determine the optimal probe working solution. Specifically, the optimal concentration of the probe working solution is 2fmol / μL.

[0017] Furthermore, in the above kit, in the primer composition, each universal primer is prepared at a concentration of 2 to 200 pMol. Specifically, preferably 10 to 50 pMol, more preferably 20 pMol, each primer is prepared separately without mixing, and only when amplifying probe-ligated products of different samples, the upstream and downstream primers are combined differently to obtain products with different indexes that can distinguish samples.

[0018] Furthermore, the kit also includes at least one of MLPA buffer, ligase, ligase buffer, PCR buffer, dNTP, and PCR enzyme.

[0019] Furthermore, the dosage of each component in the kit includes: 5 μL of 50-250 ng DNA sample, 1.5 μL of MLPA buffer, 1.5 μL of probe working solution; 6 μL of ligase buffer, 1 μL of ligase; 5 μL of PCR buffer, 4 μL of dNTP, 0.25 μL of PCR enzyme, and 1 μL each of upstream and downstream universal primers.

[0020] It is understandable that, in the above-mentioned kit, appropriate changes may be made to the reagents used and their concentrations, dosages, etc., which are not regarded as essential changes to the above-mentioned kit.

[0021] Furthermore, in the above-mentioned kit, each CNV probe pair and each SNP probe pair each has 2 to 4 characteristic sequences for Turner syndrome detection, specifically 3 characteristic sequences; each of the probe pairs includes a left probe and a right probe, and the characteristic sequences are base sequences with predetermined lengths respectively intercepted from the left side of the left probe and template DNA binding region, the connection position between the left probe and the right probe, and the right side of the right probe and template DNA binding region; wherein the sequence length of the characteristic sequence is 6 to 12 bases, specifically the sequence length is 8 to 10 bases, and the characteristic sequences are spaced at least one base apart from each other (for example, 2 to 35 bases apart, specifically 3, 4, 5...30 bases apart, etc.).

[0022] Specifically, after using the above kit to test and sequence Turner syndrome patients and their parents using NGS, in order to analyze the number of reads of each CNV and each SNP allele type in the fastQ file, it is necessary to first design the characteristic sequence of the corresponding probe as described above. In a specific embodiment, the sequence length of the characteristic sequence of each CNV probe pair is 8 bases; the sequence length of the characteristic sequence of each SNP probe pair is 10 bases, and each SNP probe pair can detect two alleles of the SNP, each allele contains 3 characteristic sequences, and the characteristic sequences of the two groups of probes of the two alleles have only one base difference in the middle specific sequence, and the base corresponds to the wild type and mutant type respectively. The above constitutes the characteristic sequence of all detection sites. If the probe of a SNP containing degenerate bases is regarded as two groups of probes, then any group of characteristic sequences corresponds one to one with the corresponding probes.

[0023] It is understandable that when designing characteristic sequences, the number, length, position, etc. of the sequences may be appropriately adjusted, provided that the analysis of the amplified products is ensured, and this should not be regarded as an essential change to the characteristic sequences described in the present invention.

[0024] Based on the above-mentioned probe sequence, primer sequence and other adapter sequences derived from Illumina second-generation sequencing, amplification itself is also a library construction process before Illumina is put on the machine. It is understandable that adjustments can also be made to use other suitable high-throughput sequencing platforms; for example, Roche / 454 sequencing platform, ABI SOLiD sequencing platform, Ion Torrent sequencing platform, CG sequencing platform, etc.

[0025] The sequence information involved in the above kit is shown in the following table:

[0026] Table 1 - Probes, primers and characteristic sequence information

[0027]

[0028]

[0029]

[0030]

[0031]

[0032]

[0033]

[0034]

[0035]

[0036]

[0037]

[0038]

[0039] The second aspect of the present invention is to provide a method for using a kit as described in any one of the first aspects of the present invention, which comprises the steps of: denaturing a DNA sample; performing probe hybridization between a SNP probe composition and a CNV probe composition and a DNA sample; connecting the hybridization probes using a ligase and a ligase buffer; performing PCR amplification on the probe connection product and a primer composition; and sequencing the PCR amplification product to obtain a sequencing result.

[0040] Furthermore, the method for using the above kit specifically comprises the steps of:

[0041] Step S1, DNA denaturation and probe hybridization: denaturing the DNA sample; fully mixing the MLPA buffer and the probe working solution, and performing a hybridization reaction. The reaction procedure is: 95°C for 2 min, 65°C to 55°C, incubating for 1 hour at each decrease in temperature, and then maintaining at 54°C for 3-10 hours to obtain a hybridization product;

[0042] Step S2, preparing a ligase master solution containing a ligase buffer, adding the ligase to the ligase master solution and mixing, heating at 54° C. for 1 minute, adding the ligase to the hybridization product at a constant temperature of 54° C. and mixing, continuing to incubate for 25 minutes, heating at 98° C. for 5 minutes, and cooling to 20° C. for pause, to achieve ligation of the hybridization probes and obtain a ligation product;

[0043] Step S3, subjecting the ligation product to PCR amplification reaction with a PCR reaction solution, wherein the PCR reaction solution includes a PCR reaction buffer, dNTPs, upstream and downstream universal primers with indexes, and Taq enzyme, and the reaction conditions are: 95°C for 30s, 60°C for 30s, 72°C for 60s, 35 cycles; incubation at 72°C for 20min, and finally incubation at 15°C to obtain a PCR amplification product;

[0044] Step S4: Take an appropriate amount of sample from each PCR amplification product, mix them evenly, and send them to the NGS sequencer for sequencing to obtain the sequencing results (specifically, fastQ files).

[0045] In a specific embodiment, the method of using the above kit can be as follows: (1) on the first day, DNA denaturation and probe hybridization are performed, wherein 5 microliters of DNA sample (50-250 ng) is added to a PCR tube, denatured at 98°C for 5 minutes, cooled to 25°C and taken out; 1.5 μL of MLPA buffer (from MRC-Holland) is mixed with 1.5 μL of probe working solution, added to the sample tube, and mixed thoroughly; (2) the thermal cycle program is continued: 95°C for 2 minutes, 65°C to 55°C, incubated for 1 hour at each decrease in temperature, and then maintained at 54°C for 3-10 hours; (3) on the second day, the ligase-65 master solution is prepared: each reaction contains 25 μL of each dH2O + 3μL ligase buffer B + 3μL ligase buffer A, then add 1μL ligase-65 enzyme, and gently pipette to mix evenly; buffer A, B and ligase-65 are all from MRC-Holland; place the mixture in a PCR instrument (54°C) and heat it for 1 minute, then add it to the incubating PCR tube at a constant temperature of 54°C, mix evenly, and continue incubating for 25 minutes; (4) Heat the above reaction at 98°C for 5 minutes, cool to 20°C to pause, and remove the PCR tube; (5) When performing PCR amplification, the PCR enzyme used is HS Taq enzyme from Takara; 50μL PCR reaction solution includes the following components: 5μL reaction buffer, 4μL dNTP, 1 μL of upstream and downstream universal primers with index (20 pMol), 10 μL of ligation product, 0.25 μL of enzyme, and water to make up to 50 μL; reaction conditions: 95°C for 30 s, 60°C for 30 s, 72°C for 60 s, 35 cycles; incubate at 72°C for 20 min, and finally incubate at 15°C; (6) Take an appropriate amount of sample from each PCR amplification product, mix well, freeze and store, and send to the NGS sequencer produced by Illumina for sequencing to obtain the fastQ file of the sequencing.

[0046] It is understandable that in the above-mentioned usage method, appropriate adjustments can be made to the reagents, temperature, incubation time, sequencing instruments, etc., which should not be regarded as essential changes to the usage method of the above-mentioned kit without affecting the detection and analysis.

[0047] Furthermore, in the above-mentioned method of use, each probe pair has a characteristic sequence for Turner syndrome detection. The design of the relevant characteristic sequence and its sequence information are detailed in the above-mentioned first aspect of the present invention.

[0048] Furthermore, in the above-mentioned method of use, the step of analyzing the results according to the sequencing results is also included, and at least one of the following analyses is performed: analyzing the number of probe reads, determining SNP typing, judging the sequencing quality of the sample, judging the parent-child relationship of the core family, judging the parental origin of the X chromosome and each segment of the X chromosome, determining the relative copy number of the X chromosome and each segment of the X chromosome, determining the parental origin of the X chromosome mosaic, and determining the presence or absence of the Y chromosome.

[0049] Furthermore, in the above-mentioned method of use, when used to analyze the number of probe reads, it includes the steps of: taking the combination of three characteristic sequences of each pair of probes as the text to be searched, taking each read in the fastQ file as the search object, taking findall of the regular expression in python as the search function, and counting the number of reads containing each text to be searched; wherein, for each SNP, if the sum of the number of reads of the two alleles is less than 20, it is considered to be of unqualified quality and is not analyzed; for the SNPs that pass the quality control, for each sample, the wild-type reads are divided by the total reads of the SNP of the sample to obtain the typing value of the SNP of the sample, and the value range of the typing value is [0,1].

[0050] Furthermore, in the above-mentioned method of use, when used to determine SNP typing, it includes the steps of: using the kit to test a population sample of healthy controls, for each SNP typing value, drawing a scatter plot of the typing value distribution of each SNP of the population sample, and calculating a dividing point for distinguishing each typing of each SNP; based on the dividing point, typing the corresponding SNP of the sample to be tested.

[0051] Furthermore, in the above-mentioned method of use, when used to judge the sequencing quality of a sample, it includes the following steps: after determining the SNP typing, the wild type or homozygous mutant type of the same SNP in multiple normal samples can have a population typing value that can be represented by the average value of the typing value plus or minus the standard deviation, and the population typing value is used as a measurement feature of the SNP to judge the detection quality of the sample; wherein, the larger the average value, the worse the sequencing quality.

[0052] It is understandable that the present invention is not limited to the SNP typing of the Turner syndrome detection kit or its detection method designed based on the MLPA-NGS principle. Other kits or self-built methods designed based on the MLPA-NGS principle, to solve the SNP typing problem, if they follow the same method to establish the judgment framework of SNP analysis and the judgment of sample sequencing quality, are not regarded as essential changes to the above-mentioned analysis method.

[0053] Furthermore, in the above method of use, when used to determine the parent-child relationship of a core family, it includes the steps of: after obtaining the typing results of the autosomal SNPs of the core family, based on the population frequency of each allele type of each SNP found, following the analysis rules of the cumulative paternal index, confirming or denying the parent-child relationship of the core family; wherein each of the core families includes the father, the mother, and the child, and when the SNP sites identified after the test all conform to the family separation rule and the cumulative paternal index is greater than or equal to 10,000, the parent-child relationship of the core family is confirmed. The confirmation of the parent-child relationship of the above core family is a prerequisite for determining the parental origin of the proband's X chromosome.

[0054] It is understandable that the present invention is not limited to the confirmation of the parent-child relationship of the core family of the Turner syndrome detection kit designed based on the MLPA-NGS principle. Any other kits or self-built methods designed based on the MLPA-NGS principle that use SNP typing to solve the problem of confirming or denying the parent-child relationship of the core family are not considered to be essential changes to the above-mentioned analysis method.

[0055] Furthermore, in the above method of use, when used to determine the parental origin of the X chromosome and each segment of the X chromosome, it includes the following steps: after confirming the parent-child relationship of the core family, for a certain X-SNP of the child, if the parents are hemizygous and homozygous respectively and are different from each other (such as the parents are AA and TT respectively), without considering the SNP mutation, it can be determined that the two allele types of the child can only come from the father and the mother respectively, and the father and the mother can only provide their own allele types at the SNP position respectively; and after detecting the typing value of the X-SNP of the child, according to the corresponding relationship between the typing value and the dosage value, the relative dosage of the two alleles of the child is calculated, and the ratio of the X chromosome from the father and the mother (specifically, the approximate ratio) can be determined.

[0056] It is understood that the present invention is not limited to the Turner syndrome detection kit designed based on the MLPA-NGS principle. Any other kits or self-built methods designed based on the MLPA-NGS principle, for determining the parental origin of the X chromosome and each segment of the X chromosome, which use the above method of the present invention, are not regarded as essential changes to the above analysis method.

[0057] It is to be understood that the present invention is not limited to the kit designed based on the MLPA-NGS principle or the self-built method for analyzing the parental origin of the X chromosome and each segment of the X chromosome. For determining the parental origin of other chromosomes, the above method of the present invention is used, which is not regarded as an essential change to the above analysis method.

[0058] Furthermore, in the above method of use, when used to determine the relative copy number of the X chromosome and each segment of the X chromosome, it includes the steps of: for each sample, taking the sum of the reads of the autosomal CNV, and comparing the reads of each CNV including X-CNV with it, to obtain the proportion of the reads of each CNV; taking the mother or father of the proband or other healthy normal person as the reference sample, comparing the proportion of each CNV of the proband with the corresponding CNV proportion of the reference sample, if the comparison object is a female, multiply by 2, if the comparison object is a male, multiply by 1, and the obtained value is the relative copy number of the site. Among them, if the mother's probe contains SNPs that cause the proband's CNV detection to be obviously wrong, the proband's father can be used as the reference sample, and at this time, it is not necessary to multiply by 2 to calculate X-CNV. Generally speaking, the relative copy number within the predicted copy number deviation range of 30% can be considered qualified.

[0059] Furthermore, in the above method of use, when used to determine the parental origin of an X chromosome mosaic, it includes the steps of: after determining the parental origin of the X chromosome and each segment of the X chromosome and the relative copy number of the X chromosome and each segment of the X chromosome, jointly analyzing the two to determine the parental origin of the X chromosome mosaic.

[0060] Furthermore, in the above-mentioned usage method, when used to determine the presence or absence of a Y chromosome, the SNP sites and Y-CNV detection on the Y chromosome can confirm the gender of the proband, and SNP can assist in the kinship identification between the proband's father and son, grandparents and grandchildren, uncles and nephews, and brothers; Y-CNV detection can be used to determine whether the proband has a microdeletion of the Y chromosome.

[0061] The third aspect of the present invention is to provide an application of the kit as described in any one of the first aspects of the present invention, which specifically comprises: using the kit to test the genomic DNA sample of the subject to be tested and performing MLPA-NGS detection.

[0062] Furthermore, in the above application, at least one of the following results is obtained by MLPA-NGS detection: number of probe reads, SNP typing, sequencing quality of samples, parent-child relationship of nuclear family, parental origin of X chromosome and each segment of X chromosome, relative copy number of X chromosome and each segment of X chromosome, parental origin of X chromosome mosaicism, and presence or absence of Y chromosome.

[0063] Furthermore, in the above application, the preparation of the DNA sample includes: collecting peripheral blood and using a blood DNA extraction kit to prepare the DNA sample. It is understandable that other forms of samples may also be used.

[0064] It is understood that the analysis of the above results may be for non-diagnostic purposes, and may be used to obtain intermediate results of relevant analyses.

[0065] In order to verify the accuracy of MLPA-NGS in SNP detection, the present invention uses Sanger sequencing and InfiniumOmni ZhongHua-8 SNP chip for verification; the verification results show that compared with other results, the SNP analysis consistency rate is 99.99%. The accuracy of MLPA-NGS in CNV detection is verified by the karyotype analysis results of most TS patients, and CNV can also be verified by the Infinium Omni ZhongHua-8 chip. The accuracy of MLPA-NGS in detecting the parental origin of the X chromosome is verified by using the X-STR method; the verification method proves that the Turner syndrome detection kit developed based on MLPA-NGS is accurate in CNV detection and X chromosome parental origin detection.

[0066] Compared with the prior art, the present invention adopts the above technical solution to have the following beneficial effects:

[0067] The present invention provides a kit based on the MLPA-NGS principle for detecting Turner syndrome and an analysis method thereof. The kit is composed of a probe working solution, an MLPA buffer, a high temperature resistant connection solution, a universal primer, a PCR reaction solution, etc., and its operation process is similar to the MLPA technology. The fastQ file obtained after sequencing can be automatically analyzed. The analysis content includes: SNP typing, pedigree paternity testing, the number of copies in the X chromosome, the parental source and the source ratio, the presence or absence of the Y chromosome, etc. The kit is detected by the second generation sequencing method, the amount of detection information is large, the number of copies of the X chromosome can achieve the effect of the SNP chip, and the analysis of the parental source of the X chromosome can achieve the effect of the STR, the sensitivity is high, the reagent cost is low, the operation is simple, the skill requirements for the operator are low, and it is convenient for large-scale promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are only used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0069] Figure 1 : is a flow chart of the MLPA-NGS method in one embodiment of the present invention; wherein, A: the designed left and right probes and their binding positions with the target DNA; B: after the template is melted, the probes are hybridized with the template; C: the probes are connected under the action of ligase; D: the linker sequence of the second generation sequencing is used as a primer pair to connect the product amplification; E: amplify the product with the same ends and different in the middle; F: perform NGS sequencing; G: perform data analysis and statistics;

[0070] Figure 2is a schematic diagram of the characteristics of probes used for SNP detection in the MLPA-NGS method in one embodiment of the present invention;

[0071] Figure 3 This is a typical SNP typing value distribution scatter plot in one embodiment of the present invention;

[0072] Figure 4 1 is a schematic diagram of the Sanger sequencing results of rs4608 in one embodiment of the present invention; wherein the five samples used are p2866, p2801, p2864, p2845, and p2806;

[0073] Figure 5 1 is a schematic diagram of the Sanger sequencing results of rs1054480 in one embodiment of the present invention; wherein the five samples used are p2801, p2864, p2851, p2845, and p2806;

[0074] Figure 6 This is a schematic diagram of the results of analyzing the parental origin of autosomes using autosomal SNPs of the sample using HF01 sample as an example in one embodiment of the present invention;

[0075] Figure 7 This is a schematic diagram of the results of analyzing the parental origin of autosomes using autosomal SNPs of a father's bone marrow transplant sample, taking HF53 sample as an example in one embodiment of the present invention;

[0076] Figure 8 This is a schematic diagram of the results of analyzing the parental origin of the X chromosome using a female sample X-SNP, taking the child sample of HF01 as an example in one embodiment of the present invention;

[0077] Fig. 9 This is a schematic diagram of the results of analyzing the parental origin of the X chromosome using a male sample X-SNP, taking the child sample of HF02 as an example in one embodiment of the present invention;

[0078] Fig.10 This is a schematic diagram of the results of analyzing the parental origin of the X chromosome using the T8 sample as an example and the X-SNP of the TS patient sample in one embodiment of the present invention;

[0079] Fig.11 This is a schematic diagram of the results of analyzing the parental origin of the X chromosome using the X-STR of a TS patient sample using the T8 sample as an example in one embodiment of the present invention;

[0080] Fig.12 This is a schematic diagram of the results of analyzing the parental origin of the X chromosome using the T1 sample as an example and the X-SNP of the TS patient sample in one embodiment of the present invention;

[0081] Fig.13This is a schematic diagram of the results of analyzing the parental origin of X chromosomes using X-STR of TS patient samples, taking T1 samples as an example in one embodiment of the present invention;

[0082] Fig.14 This is a schematic diagram of the results of CNV analysis of X chromosomes of two healthy individuals, one female and one male, in one embodiment of the present invention;

[0083] Fig.15 is a schematic diagram of the results of CNV analysis of chromosome X of TS patients using T8 sample as an example in one embodiment of the present invention;

[0084] Fig.16 This is a schematic diagram of the results of SNP chip analysis of chromosome X of TS patients using T11 sample as an example in one embodiment of the present invention;

[0085] Fig.17 This is a schematic diagram of the results of autosomal SNP analysis of TS patients based on MLPA-NGS, taking T11 sample as an example in one embodiment of the present invention;

[0086] Fig.18 This is a schematic diagram of the results of X chromosome SNP analysis of TS patients based on MLPA-NGS, taking T11 sample as an example in one embodiment of the present invention;

[0087] Fig.19 This is a schematic diagram of the results of X chromosome CNV analysis of TS patients based on MLPA-NGS, taking T11 sample as an example in one embodiment of the present invention;

[0088] Fig. 20 This is a schematic diagram of the results of SNP chip analysis of chromosome X of TS patients using sample B1026 as an example in one embodiment of the present invention;

[0089] Fig.21 This is a schematic diagram of the results of autosomal SNP analysis of TS patients based on MLPA-NGS, taking sample B1026 as an example in one embodiment of the present invention;

[0090] Fig. 22 This is a schematic diagram of the results of X chromosome SNP analysis of TS patients based on MLPA-NGS, taking sample B1026 as an example in one embodiment of the present invention;

[0091] Fig.23 This is a schematic diagram of the results of X chromosome CNV analysis of TS patients based on MLPA-NGS, taking sample B1026 as an example in one embodiment of the present invention;

[0092] Fig.24 This is a schematic diagram of the results of SNP chip analysis of chromosome X of TS patients using H53 sample as an example in one embodiment of the present invention;

[0093] Fig.25This is a schematic diagram of the results of autosomal SNP analysis of TS patients based on MLPA-NGS, taking H53 sample as an example in one embodiment of the present invention;

[0094] Fig.26 1 is a schematic diagram of the results of X chromosome SNP analysis of TS patients based on MLPA-NGS, taking H53 sample as an example in one embodiment of the present invention; wherein, Figure 6 to Figure 10 , Fig.12 , Figure 17-18 , Figure 21-22 , Figure 25-26 In the figure, the horizontal axis is the SNP number. A solid five-pointed star on the SNP number means that the site conforms to the genetic law, a hollow five-pointed star means that it does not conform to the genetic law, and a ※ sign means that the SNP has not been measured; the height of the bar graph only represents the proportion of the allele type, not the dosage;

[0095] Fig. 27 This is a schematic diagram of the results of X chromosome CNV analysis based on MLPA-NGS of TS patients taking H53 sample as an example in one embodiment of the present invention. DETAILED DESCRIPTION

[0096] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the scope of protection of the present invention. The experimental methods without specific conditions in the following embodiments are usually measured according to national standards. The experimental materials without the source indicated in the following embodiments are all commercially available raw materials. The equipment used in each step in the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to the general international standards, conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise specified, all parts are weight parts and all percentages are mass percentages. Unless otherwise defined or specified, all professional and scientific terms used in the present invention have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equal to the recorded content can be applied to the method of the present invention.

[0097] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention is further described below in conjunction with the drawings and specific embodiments, but is not intended to be a limitation of the present invention.

[0098] In the following examples, exemplary discussions are given from the perspectives of design ideas, kit preparation methods, sample collection, testing methods, and analysis methods. The samples used include more than 50 healthy core family samples (each core family includes a father, a mother, and a child) and 15 core family samples of TS patients. The following examples will give the statistical results of some samples, specifically taking 3 patients as examples for a more detailed description. The results of the above 3 samples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. The reagents used in the following examples are all conventional products that can be purchased commercially.

[0099] In the following examples, the principle of TS detection based on MLPA-NGS is briefly described as follows:

[0100] (1) Design detection probes based on the MLPA-NGS principle;

[0101] MLPA-NGS technology is a technology that combines MLPA technology with NGS technology. It has the high accuracy of MLPA technology for CNV and expected SNV detection and the high throughput of NGS technology for target fragment detection. It is a high-throughput MLPA technology. The amplification products of the classic MLPA technology are distinguished by capillary electrophoresis due to different lengths. However, the MLPA-NGS technology introduces the universal adapter sequence of NGS into the connection fragment through PCR after the MLPA probe is connected. The fusion product is directly sequenced by high-throughput sequencing, thereby changing the length detection of MLPA to the detection of the sequence itself by MLPA-NGS, breaking the limitation of the limited diversity of the length of the MLPA amplified fragment on the number of detections, and greatly improving the detection throughput. MLPA can detect up to about 60 sites at a time, while MLPA-NGS can detect more than 2,000 sites at a time according to the test. In order to detect TS, the designed detection probes are divided into two types, one is a CNV probe, which is used to detect the copy number of the DNA position bound by each pair of probes, and the other is a SNP probe, which is used to detect the genotype of the SNP at the connection site of the hybridization position of the probe on the DNA. The regions where the two probes are distributed are located on each autosome, chromosome X and chromosome Y, and are most densely distributed on chromosome X. The selected SNP to be detected is generally a single base substitution type SNP containing only two genotypes (these two genotypes are called wild type and mutant type), and two signals of SNP are detected by designing degenerate bases for the two genotypes on the probe.

[0102] (2) Determine SNP genotype using a kit designed based on the MLPA-NGS principle;

[0103] The signals of the two genotypes of each SNP measured are expressed in the number of reads. Then, by calculating the proportion of reads of one genotype (wild type) to the two genotypes, the typing value is obtained (the value interval of this value is obviously within [0,1]), and the genotype is judged to be wild type, heterozygous or homozygous mutant type by the size of the typing value. Before using the typing value to judge the genotype, the genotype cutoff value needs to be set. The method is to present the typing values ​​of a certain SNP of a series of normal samples in the form of a scatter plot. In most cases, these sites can be seen to be scattered in three areas, namely, the near 0 area, the near 1 area and the middle area. Between the three areas, two large or small blank areas are left, and the two midpoints of the two blank areas are taken as the dividing lines of the three distribution areas. In this way, the new typing value to be tested can be divided into one of the three areas. According to whether the typing value of the SNP to be tested falls into the near 0 area, the near 1 area or the middle area, it is judged that the SNP belongs to the homozygous mutant type, wild type or heterozygous type. The accuracy of this method in determining genotypes is verified through two experimental methods. One is the first-generation sequencing method, which randomly selects some SNP sites and some samples for first-generation sequencing, and compares the consistency of first-generation sequencing and MLPA-NGS results; the other is to use SNP chip technology, take some samples for SNP chip detection, and compare the consistency of SNPs covered by the two technologies. The accuracy of CNV probes used for copy number variation detection in CNV detection of the region is compared with karyotype analysis experiments and X-STR experiments. SNP probes and CNV probes are combined to form the main part of the probe working solution for TS detection.

[0104] (3) Use the kit designed based on MLPA-NGS principle to determine the parent-child relationship of the core family;

[0105] The samples of the proband and parents need to be tested at the same time. Before that, it should be ensured that the parents tested are the biological parents of the proband. The proband and parents constitute the core pedigree, and the autosomal SNPs of the core pedigree are analyzed to determine whether they conform to the laws of genetics, and paternity testing can be performed. Specifically, the parent-child relationship needs to be determined by calculating the cumulative paternity index (CPI) of the SNP. For the core pedigree to be tested, if the SNP sites identified after the test conform to the family separation law and the CPI is greater than or equal to 10,000, the parent-child relationship of the core family can be confirmed. A large number of core pedigrees with known relationships are used as the test objects, and MLPA-NGS is used as the test method. The confirmation of the test objects can in turn indicate the reliability of this method for SNP detection.

[0106] (4) Using a kit designed based on the MLPA-NGS principle to determine the parental origin of the proband's X chromosome;

[0107] After the parent-child relationship is confirmed, the parental origin of the proband's X chromosome can be further determined. Usually, the father has one X chromosome, and each X-SNP is hemizygous, and the mother has two X chromosomes, and each X-SNP is wild-type, heterozygous, or homozygous mutant. After all, a child can only have one, two, or more X chromosomes from his parents. For a certain X-SNP, if the mother is not heterozygous and is different from the father (such as AA for the father and GG for the mother), the parental origin of the child's SNP can be clearly determined: if it is the same as the father, it comes from the father; if it is the same as the mother, it comes from the mother; if there are both father and mother signals, it comes from the parents. The ratio of the child's signals from both parents is the ratio of the parents of the X chromosome. Usually, when the signal from one parent is significantly smaller than the other, it indicates the presence of X chromosome mosaicism. The detection of multiple consecutive CNVs on the X chromosome, combined with the ratio of the parental origins of multiple consecutive X-SNPs, can confirm the origin and ratio of both parents in the X chromosome region where the consecutive X-SNPs are located.

[0108] (5) Analyze the quality of test results using a kit designed using the MLPA-NGS principle;

[0109] SNP data on autosomes can also be used to analyze the quality of test results. The analysis found that for samples with better quality, the typing value of the wild type is closer to 1, and the typing value of the homozygous mutant is closer to 0; while for samples with poor quality, or samples that are not tested strictly according to the experimental process, the typing value of the wild type will be farther from 1, and the typing value of the homozygous mutant will be farther from 0. The population typing value of the wild type or homozygous mutant of the same SNP in multiple normal samples can be expressed by the average value of the typing value plus or minus the standard deviation. The population typing value can be regarded as the measurement characteristic of the SNP. The absolute value of the difference between the typing value of each SNP of a new sample and the typing value of the corresponding allele of the corresponding SNP is divided by the standard deviation, named "typing deviation", which can be regarded as the degree to which the SNP meets the measurement characteristics of the SNP. The average value of the typing deviation of all SNPs in a sample, as long as they are not heterozygous, can be used to judge the detection quality of the sample. Obviously, the larger the average value, the worse the detection quality and the less reliable the result.

[0110] The above is the general principle of detecting the X chromosome copy number and parental origin of TS based on the MLPA-NGS method.

[0111] Example 1 - Design and use of kit

[0112] This embodiment designs a kit for detecting Turner syndrome based on the MLPA-NGS method, and describes the operation method of the kit. The above design specifically includes:

[0113] (1) Site selection and probe design;

[0114] SNP probes and CNV probes are designed according to the principle of MLPA-NGS technology. The process of MLPA-NGS technology is as follows: Figure 1 (This figure is a previous study figure, so that the reader can understand it, and it does not play a specific limiting role). When designing SNP probes, 3 SNP sites are selected on each autosome (based on GRCh38 / hg38), with a total of 66 sites, which are located at the head, tail and middle regions respectively. Therefore, any two selected SNPs on the same chromosome have a distance greater than 1000K, and there is no need to perform linkage analysis when used for paternity testing. 51 SNPs were selected on the X chromosome, and 25 SNPs were selected on the Y chromosome. SNPs on X and Y are distributed in a manner close to equidistant. SNP is a single base substitution type, generally only contains two allele types, and the minor allele frequency (MAF) is generally greater than 0.2, but the MAF of SNPs on X and Y chromosomes is not strictly limited. The frequency of each SNP comes from the NCBI 1000 Genome Database. The detection probe for each SNP is composed of two seamlessly adjacent probes that bind to the template, wherein the 3' end of the left probe contains degenerate bases corresponding to the two alleles of the SNP, and the 5' end base of the right probe carries a phosphorylation group for probe connection. The left and right probes constitute a set of probes for detecting one SNP. Take the SNP wild-type site and a 20bp fragment on both sides, and compare them in the human genome. If the fragment is not unique, it will not be selected; take the SNP mutant site and a 20bp fragment on both sides, and compare them in the human genome. If the fragment exists, it will not be selected. In some left probes, a mismatch is introduced in the third base of the 3' end to improve the specificity of binding during connection. For the sequence with the mismatch, a 20bp fragment is also taken for comparison in the genome. If the fragment exists, it will not be selected. The above-mentioned SNP probe sequences are shown in SEQ ID NO.1 to SEQ ID NO.284. When using SNP probes to detect SNPs, the pattern is as follows: Figure 2 As shown, for example: for a SNP whose wild-type and mutant alleles are A and G respectively, the degenerate bases designed on the probe can be successfully connected when they hybridize with the corresponding T and C on the template respectively; if there is no match on the template, it cannot be successfully connected or the connection ratio is low. This principle is used to detect SNP.

[0115] The CNV probe of each site is composed of two seamlessly adjacent probes that bind to the template, which is called a pair of probes. Two pairs of CNV probes are designed on each autosome, with a total of 44 pairs, located near the two ends of the chromosome. 30 pairs of probes are designed on chromosome X and 10 pairs of probes are designed on chromosome Y. The probes on X and Y are distributed in a nearly equidistant manner. The design of the probe follows the probe design guidelines of MRC. A total of 20bp fragments on both sides of the probe connection site are taken as the comparison object to search in the human genome to verify its uniqueness. Probes that are not unique are not used. Try to exclude the presence of SNP sites in the probe binding area, especially those with high MAF, to reduce the interference of SNP. A phosphate group is added to the 5' end of the right probe to prepare for the connection of the probe. The above CNV probe sequences are shown in SEQ ID NO.285 to SEQ ID NO.452.

[0116] In the SNP probe and CNV probe, a sequence is added to the 5' end of the left probe and the 3' end of the right probe respectively as the binding sequence during universal primer amplification, and the corresponding sequences are shown in SEQ ID NO.455 to SEQ ID NO.456. After the SNP probe and CNV probe are synthesized and phosphate groups are added, each probe is mixed in appropriate amounts to prepare a mixed solution with a concentration of 0.2-20fmol / uL for each probe. The concentration is optimized according to the test results (the optimal concentration is 2fmol / uL) as the probe working solution. The binding sequence, primers, etc. are derived from the adapter sequence of Illumina second-generation sequencing, and the amplification itself is also the library construction process before Illumina is put on the machine.

[0117] (2) Design of the analytical sequence of the probe (characteristic sequence)

[0118] After adjacent probes are connected, amplified and sequenced, the sequencing results are obtained. The sequencing results exist in the form of fastQ files, which contain the sequence and related information of each fragment being tested. These sequences are reads. In order to analyze the number of reads after the designed probe is amplified, the characteristic sequence of the probe (i.e., specific sequence) is used to search in the fastQ file. In the fastQ file, all reads containing these specific sequences are classified as the probe and counted. The specific sequence of each pair of CNV probes contains three segments, which are respectively cut from the left side of the left probe and the template binding area, the connection position of the left probe and the right probe, and the right side of the right probe and the template binding area. A sequence with a length of 8 bases is taken as the specific sequence of the pair of probes. The three sequences are at least one base apart from each other. Each group of SNP probes can detect two alleles of the SNP, and each allele contains 3 specific sequences, which are located in the same position as the CNV probe but are 10 bases long. The two small groups of specific sequences of the two alleles have only one base difference in the middle specific sequence, which corresponds to the wild type and mutant type respectively. This constitutes the specific sequence of all detection sites. If the probe of a SNP containing degenerate bases is regarded as two sets of probes, then any set of specific sequences corresponds to the corresponding probes one by one. For the relevant characteristic sequence information, please refer to "Table 1 - Probe, Primer and Characteristic Sequence Information Table".

[0119] In order to attribute the reads of the fastQ file to each pair of probes designed, the conventional analysis method is to align the reads with the template and then calculate the depth of the reads that have been successfully aligned. The analysis method designed in this embodiment is different from the conventional method described above. The conventional method is to align the sequencing reads to the reference sequence, and then extract the differential sites and differential site frequency data, and the result obtained is the same as the method designed in this embodiment. However, the conventional method described above requires bioinformatics professionals to use specific software and analyze on the server, which takes a long time and is complicated to operate. The method designed in this embodiment is to select three sequences for each pair of probes to search in the fastQ file and count the number of occurrences. This method can obtain the same results as the conventional method described above, and the operation and operating conditions are simpler, and no professional bioinformatics personnel and expensive servers are required. The method designed in this embodiment can automatically exclude low-quality sequencing, because low-quality sequencing usually leads to random sequencing errors, and sequences with random errors can be shielded outside the specific sequence search range. The search method utilizes regular expressions.

[0120] (3) Preparation of DNA samples;

[0121] The research of this embodiment has been approved by the ethics committee. This embodiment uses DNA from 53 healthy core families (father, mother, child), 2 core families from hematology children who have undergone bone marrow transplantation, and 15 core families diagnosed with Turner syndrome, and all family members obtain informed consent. There is no blood relationship between each family. Each member collects 2mL of peripheral blood and uses a blood DNA extraction kit (TIANGEN) to prepare DNA. DNA concentration is measured by Nanodrop (Thermo Fisher Scientific).

[0122] (4) MLPA-NGS experimental process;

[0123] On the first day, DNA denaturation and probe hybridization were performed by adding 5 μL of DNA sample (50-250 ng) to a PCR tube, denaturing at 98°C for 5 minutes, cooling to 25°C and removing. 1.5 μL of MLPA buffer (from MRC-Holland) was mixed with 1.5 μL of probe working solution, added to the sample tube, and mixed thoroughly. The thermal cycle program was continued: 95°C for 2 minutes, 65°C to 55°C, incubating for 1 hour at each decrease, and then maintaining at 54°C for 3-10 hours. On the second day, the main solution of ligase-65 was prepared: each reaction contained 25 μL dH2O + 3 μL ligase buffer B + 3 μL ligase buffer A, and then 1 μL ligase-65 enzyme was added and the pipette was gently blown to mix evenly. Buffers A, B and ligase-65 were all from MRC-Holland. Heat the mixture in a PCR instrument (54°C) for 1 minute, then add it to the PCR tube being incubated at 54°C, mix well, and continue incubating for 25 minutes. Heat the above reaction at 98°C for 5 minutes and cool to 20°C to pause. Take out the PCR tube.

[0124] When performing PCR amplification, the PCR enzyme used is HS Taq enzyme from Takara (Cat. No.: R007Q). 50uL PCR reaction solution includes the following components: 5uL reaction buffer, 4uL dNTP, 1uL of upstream and downstream primers (20pMol), 10uL of ligation product, 0.25uL of enzyme, and water is added to 50uL. The sequence of the upstream primer and the sequence of the downstream primer are shown in SEQ ID NO.453 and SEQ ID NO.454, respectively. The poly (N) in the two primers is the index sequence to distinguish the samples. The reaction conditions are: 95℃30s, 60℃30s, 72℃60s, 35 cycles; incubate at 72℃ for 20min, and finally incubate at 15℃. Take an appropriate amount of sample from each PCR amplification product, mix evenly, freeze and store, and send to Nanjing Novogene Biotechnology Co., Ltd. for sequencing. Use Qubit 2.0 to preliminarily quantify the library concentration, use Agilent 2100 to detect the integrity of the library DNA fragment and the size of the insert fragment, and use Illumina high-throughput sequencer (such as HiSeq2500 / HiSeq4000 / HiSeqX / MiSeq) for double-end 150bp sequencing to obtain the sequencing fastQ file.

[0125] Example 2 - Analysis of kit sequencing results

[0126] This example performs corresponding result analysis on the fastQ file obtained in Example 1, which specifically includes:

[0127] (1) Statistical methods of reads and calculation of typing values

[0128] The fastQ file was obtained from a high-throughput sequencer and analyzed using a python program. The combination of the three analysis sequences for each site designed in the "(1) Site selection and probe design and (2) Design of probe analysis sequence" section of Example 1 was used as the text to be searched, each read in the fastQ file was used as the search object, and the findall of regular expressions in python was used as the search method to count the number of reads containing each text to be searched. For each SNP, if the sum of the number of reads of the two alleles was less than 20, it was considered to be of poor quality and was not analyzed.

[0129] For SNPs that have passed quality control, the wild-type reads of each sample are divided by the total reads of this SNP in the sample to obtain the typing value of the SNP. Obviously, the value range of this value is [0,1].

[0130] (2) Create a SNP typing judgment framework to perform SNP typing and SNP typing quality control

[0131] (A) SNP typing;

[0132] In order to accurately determine the typing of each SNP based on the typing value, this example uses the typing values ​​of 73 healthy people selected from healthy core families to produce a distribution scatter plot of the typing value of each SNP to explore the distribution pattern of SNP.

[0133] In most cases, the genotyping value scatter points of each SNP can be divided into three groups: scatter points near 1, scatter points near 0, and scatter points in the middle. The middle scatter points are generally concentrated and have a clear isolation interval from the scatter points near 0 and scatter points near 1. The genotyping value interval [0,1] is stretched 1000 times and placed in the coordinate system to form a baseline containing 1000 units, with an X coordinate interval of [0,1000] and a Y coordinate of 0. After each genotyping value is multiplied by 1000, the corresponding site on the baseline is taken 10 units before and after, and the Y coordinate of the corresponding unit on the baseline is reduced by 1. After each genotyping value of the SNP is processed in this way, three grooves will generally be formed on the baseline, corresponding to the homozygous mutant type, heterozygous type, and wild type, respectively. Between the homozygous mutant type and the heterozygous type, and between the heterozygous type and the wild type, a flat baseline with a Y value of 0 that is either long or short will be retained, and the midpoint of the flat baseline is used as the dividing value for distinguishing the homozygous mutant type from the heterozygous type, and between the heterozygous type and the wild type, and the length of the flat area represents the reliability of the site as a dividing point. For the SNP site on the Y chromosome, there are only two forms, the wild type and the mutant type, and there is no heterozygous type. The typing value of each SNP is divided into three groups or two groups of scattered points by the dividing point, corresponding to three or two genotypes respectively. The dividing point on the baseline divided by 1000 is the dividing point of the typing value, and the typing values ​​of the two or three groups of scattered points obtained are expressed as mean ± standard deviation (x ± s).

[0134] There is a rough correspondence between the typing value and the dosage value. The typing value scatter plot shows the relationship between the typing value and the dosage value when the wild-type dosage is 0, 0.5, and 1, respectively. Outside these three points, a rough judgment is made based on the above correspondence. When the typing value is in an interval close to 0 or close to 1, the typing value corresponding to the dosage of 0 and 1 is used for correction; when the typing value is in the middle area, the typing value corresponding to the dosage value of 0.5 is used for correction. The split values ​​of the typing values ​​in different intervals are set to 0.1 and 0.9 for most SNPs.

[0135] In order to establish the SNP typing framework, 73 samples were used as the analysis objects. According to the above method, a scatter plot was made for the typing value of each SNP, and then the midpoint and length of the two blank areas (boundaries), the average value of the small value (typing value close to 0), the standard deviation of the small value, the average value of the large value, the standard deviation of the large value, etc. were calculated based on the scatter plot. Among them, a typical SNP typing value scatter plot is shown as follows Figure 3As shown, the horizontal axis is the typing value interval [0,1] stretched 1000 times, the vertical axis is the number of samples, the scatter points are the distribution of wild type, heterozygous type, and homozygous mutant type, and the two points on the 0-point line of the vertical axis are the dividing points between homozygous mutant type and heterozygous type, and the dividing point between heterozygous type and wild type. The wild type and mutant bases of this SNP (rs12040811) are C and T, respectively.

[0136] The following table contains the boundary midpoint, small value average, small value standard deviation, large value average, large value standard deviation, etc. for each SNP. Due to space limitations and the fact that the average value and method of the heterozygous type were not used, this table is not included. Figure 3 The data of rs12040811 in have been integrated into the table below.

[0137]

[0138]

[0139]

[0140]

[0141] (B) Quality control of SNP typing;

[0142] In order to analyze the sequencing quality of samples, the concept of average typing deviation is defined. The typing value of a certain SNP of an individual in the above sample is distinguished by the genotype according to the cutoff point. If it is non-heterozygous, it is subtracted from the mean of the typing value of the corresponding genotype of the SNP in the sample. The absolute value of the obtained value is compared with the corresponding standard deviation (set: when the standard deviation is less than 0.003, 0.003 is the standard deviation), which is defined as the typing deviation of the SNP of the individual. The average of all the calculable typing deviations of all the SNPs measured by the individual is taken as the average typing deviation value. A scatter plot of the typing values ​​of all SNPs of each sample is made. It is found that the larger the average typing deviation value, the more diffuse the distribution of the SNP typing values ​​of the sample, and the lower the reliability of its typing. Therefore, the samples with too large average typing deviation values ​​in the healthy samples are eliminated, and the mean and standard deviation of the remaining samples are recalculated, and then the average typing deviation value of each sample is calculated with the new mean and standard deviation.

[0143] (3) Calculate the cumulative paternity index based on the SNPs of the pedigree;

[0144] For each SNP typing value of the subject's sample, the subject is subjected to SNP typing according to the above typing value cutoff point. When the child, father, and mother of a core family undergo the same MLPA-NGS test and SNP typing, the parent-child relationship can be identified by analyzing the situation in which the three conform to the law of allele separation. The likelihood ratio (LR) is used to indicate the credibility of the paternity test results. LR is calculated as the ratio of the likelihood value (L) based on two hypotheses (H0: the subject is the biological father of the child in a given pedigree; H1: the subject is unrelated). The overall LR is determined by calculating the cumulative paternity index (CPI) using the SNP panel on the autosomes.

[0145]

[0146] When calculating CPI, there is no need to perform linkage analysis on adjacent SNPs because the distance between SNPs on each chromosome is far, much greater than 1000K.

[0147] (4) using X-SNP to analyze the parental origin of the X chromosome;

[0148] After determining the biological parents of the child through autosomal SNP, the parental origin and proportion of the X chromosome can be further determined through X-SNP.

[0149] For the two alleles of a child's SNP, there are three situations in determining the parental origin. The first situation is that it cannot be determined, such as the child's parents are (AT, AT, AT), one of the child's alleles can come from either parent, so it cannot be determined. The second situation can be barely determined, such as the child's parents are (AT, AT, TT), because the child's A can only come from the father, so even though the father also has T, it can still be determined that the child's T comes from the mother. The third situation is that the parental origin can be strongly determined, such as the child's parents are (AT, AA, TT), without considering the SNP mutation, it can be determined that the child's A and T can only come from the father and mother respectively, and the father and mother can only provide A and T respectively at the SNP position. In Turner syndrome, since there are many situations for the status of the X chromosome, such as one from the father or the mother, two from each parent or both from the mother, or there is a low proportion of mosaicism, therefore, in order to accurately determine the parental origin of the X chromosome, the third situation is selected as the basis for judgment. The main feature of the third case is that a certain SNP is hemizygous and homozygous in the genes of the parents and the alleles are different. In this case, after detecting the typing value of the child's SNP, the relative dosage of the two alleles of the child is calculated based on the corresponding relationship between the typing value and the dosage value, and the approximate ratio of the X chromosome from the father and the mother can be determined.

[0150] (5) CNV analysis methods;

[0151] The CNV calculation method for the sample to be tested refers to the CNV calculation principle of MRC-Holland. Specifically, for each sample, the reads of the autosomal CNV are summed, and the reads of each CNV, including X-CNV, are compared with it to obtain the percentage value of the reads of each CNV. Taking the mother of the proband as the reference sample, the percentage value of each CNV of the proband is compared with the corresponding CNV percentage value of the reference sample and multiplied by 2. The value obtained is the relative copy number of the site. If the mother's probe contains SNPs that cause the CNV detection of the proband to be obviously incorrect, the father of the proband can be used as the reference sample. At this time, there is no need to multiply by 2 to calculate X-CNV. Generally speaking, relative copy numbers within 30% of the predicted copy number deviation are considered accurate.

[0152] (6) controlled trials;

[0153] In order to verify the accuracy of MLPA-NGS in SNP detection, two control tests were used. One was to randomly select 10 SNP sites, select 5 samples for each site, and the 5 samples contained three genotypes in the MLPA-NGS test. Using the DNA of these samples as a template, the amplification primers of the SNP were designed and amplified, and then Sanger sequencing was performed to compare the consistency of the Sanger sequencing results with the MLPA-NGS analysis results. The second control test was to use Illumina's SNP chip called InfiniumOmni ZhongHua-8 (service company: Shanghai Hezhuo Medical Testing Laboratory Co., Ltd.) to detect chromosome copy number abnormalities across the genome. The above test used three samples, namely T11-proband, B1026-proband, and H53-child. The test was carried out strictly in accordance with quality control standards, including DNA extraction, enzyme digestion, ligation, PCR, purification, fragmentation, labeling, hybridization, elution, scanning and analysis. This method covers more than 1.17 million SNP sites in SNP detection, with a consistency rate of over 99%, and can perform CNV analysis.

[0154] The accuracy of MLPA-NGS in CNV detection is verified by the karyotype analysis results of most TS patients. The karyotype analysis is completed by Shanghai Dian Medical Laboratory, and the number of cells tested for each patient varies from 30, 50, to 100. The Infinium Omni ZhongHua-8 chip can also be used to verify CNV, and its detection accuracy is higher than that of karyotype analysis technology.

[0155] The accuracy of MLPA-NGS in detecting the parental origin of X chromosomes was verified by using the X-STR method. The method is detailed in Ma Hongdu's master's thesis "Establishment of a fluorescent composite amplification system for 9 X-STR loci and their genetic polymorphism".

[0156] Example 3 - Validation and application of the analytical method of the kit

[0157] This embodiment uses certain samples to verify the analysis method in Example 2, and provides relevant specific application examples, which specifically include:

[0158] (1) Statistics of SNP reads;

[0159] After the query program using the analysis method in Example 2 is run, the number of times each specific sequence of each pair of probes appears in the fastQ file and the number of times the three specific sequences of each pair of probes appear together in one read in the fastQ file are summarized in an excel file.

[0160] For example, the reads data of several SNPs in a test of patient T4's family are as follows. The three columns on the right are the reads of the child, father, and mother from left to right.

[0161] rs_number Alleles 1000Genomes_EAS C_reads AF_reads M_reads rs12751100 [G] G＝0.3502 0 10 12 [A] 11 20 25 rs12040811 [C] C＝0.6597 125 243 240 [T] 229 473 521 rs9350 [C] C＝0.5317 15 20 51 [T] 106 254 295 rs3740304 [T] T＝0.7619 300 726 1009 [C] 0 4 3 rs3758490 [G] G＝0.3085 126 256 4 [T] 322 784 2149

[0162] (2) SNP typing, verification and correlation analysis;

[0163] According to the cutoff point of the typing values ​​of the three genotypes of each SNP, each SNP can be typed based on the typing value calculated for each SNP. Substituting the typing results and the population frequency of the allele, the PI value of each SNP can be calculated. If the total number of reads measured for a certain SNP is too low, it is considered to be of unqualified quality and will not be calculated.

[0164] (A) SNP typing value, typing results and PI value;

[0165] The following table shows the typing values, typing results and PI values ​​of some SNPs in a test of patient T4's family. The data are as follows: Columns 3-5, 6-8 from left to right are children, father, and mother. This table corresponds to the previous table of T4 family. Multiplying the PI values ​​of all SNPs in the same core family is the CPI.

[0166]

[0167] The following table shows the typing values, typing results, and PI values ​​of several SNPs in a test of patient B1100. In the PI column of this result, there are multiple results of "Err", indicating that the typing of multiple SNPs between the child and the parents does not conform to the genetic law, and it is judged that the father tested is not the biological father of the child. This result is consistent with the STR test result. For such patients, the subsequent analysis of the parental origin of the X chromosome is inaccurate. In the two cases from the hematology department, the children had undergone bone marrow transplantation, and a large number of "Err" appeared when calculating PI.

[0168]

[0169] (B) The calculation of SNP typing deviation value can be used for experimental quality control;

[0170] The three columns on the right of the table below are the typing deviation values ​​of several SNPs in the T4 family. The average of the typing deviation values ​​of all autosomal SNPs in each sample is the average typing deviation value, which represents the data quality of the sequencing of the sample.

[0171]

[0172] (C) Analysis of SNP exclusion number and average typing deviation value for paternity testing;

[0173] The 53 healthy families, 2 families that had undergone bone marrow transplantation, and 15 TS patient families were analyzed for the paternity test SNP exclusion number and average typing deviation value. The results are as follows:

[0174]

[0175]

[0176] Among these pedigrees, HF53 and HF55 families are families whose children have undergone bone marrow transplantation, and the SNP exclusion numbers for paternity testing are 6 and 17, respectively, which are consistent with expectations. For the B1100 family, the SNP exclusion numbers for paternity testing are 11, respectively, and the father is judged to be not the biological father. The results of B1100 are consistent with the STR test results. In addition, there are 3 healthy pedigrees, each with 1 SNP exclusion number. This embodiment sequenced and verified the three sites, and the results showed that rs2976399 of the HF42 family has another SNP adjacent to its left, which affects the accuracy of rs2976399. The other two pedigrees, after Sanger sequencing, should belong to the second-generation sequencing judgment problem. Therefore, when using this method for paternity testing, if only one or very few SNP sites do not conform to the genetic law, the problem of sequencing errors cannot be completely ruled out. In addition, there is room for improvement in this method.

[0177] (D) Validation test: Sanger sequencing and SNP chip detection;

[0178] Except for the problems of some individual sites, this example conducted verification tests on other sites. Two methods were used for verification, one was Sanger sequencing and the other was SNP chip detection.

[0179] The Sanger sequencing method randomly selects some SNP sites and selectively selects some samples (so as to contain three genotypes). The corresponding relationships and results are shown in the following table and Figure 4-5 See Figure 4 , the Sanger sequencing results of rs4608 locus on 5 samples were consistent with the MLPA-NGS method; see Figure 5 The Sanger sequencing results of rs1054480 on 5 samples were consistent with the MLPA-NGS method. The above results show that the Sanger sequencing results are completely consistent with the MLPA-NGS results.

[0180] SNP sample sample sample sample sample rs2976399 p2837 P4002 P4004 P4005 P4252 rs1652727 p2801 p2806 p2845 p2851 p2864 rs2281974 p2801 p2806 p2845 p2851 p2864 rs8451 p2801 p2845 p2851 p2864 p2863 rs2289759 p2801 p2806 p2845 p2851 p2864 rs4608 p2801 p2806 p2845 p2864 p2866 rs11353 p2801 p2806 p2845 p2851 p2864 rs1054480 p2801 p2806 p2845 p2851 p2864 rs2229137 p2801 p2806 p2845 p2851 p2865 rs2270672 p2806 p2845 p2851 p2864 p2863 SNP result result result result result rs2976399 GA AA GG AA GA rs1652727 CT CC TT CT TT rs2281974 GA GG AA GA GA rs8451 GG GG GG GA AA rs2289759 GG AG GG AG AA rs4608 TT CT CT CT CC rs11353 TC TC TT CC TC rs1054480 CC CC TT CT CT rs2229137 AA CC CC AA AC rs2270672 CC CC TT TT CT

[0181] When the Infinium Omni ZhongHua-8 SNP chip was used to test the three samples of the T11 proband, the B1026 proband, and the H53 child, after comparison, only the sequencing result of the T11 proband sample at rs5744944 was different from the sequencing result. This result has been mentioned before. The other sites, where the results were obtained by both technologies, were completely consistent.

[0182]

[0183]

[0184]

[0185] (3) SNP-based analysis and verification of chromosome parental origin;

[0186] As mentioned above, there are three situations for determining the parental origin of chromosomes based on SNPs in the core family.

[0187] (A) For autosomal SNPs to determine the parental origin of chromosomes, this embodiment uses the second and third situations.

[0188] Taking HF01 as an example, all SNPs that cannot distinguish between parental origins are displayed in blue bar graphs. SNPs that can distinguish between parental origins and calculate the parental origin dosage are represented in orange and gray respectively. After converting to black and white graphs, judgment can be made based on the color of the legend. Each SNP that conforms to the genetic law is represented by a solid five-pointed star, and those that do not conform are represented by a hollow five-pointed star. If the SNP is not measured, it is represented by a ※. Figure 6For SNPs that can distinguish parental origin, the dosage comes from both parents, which is in line with expectations.

[0189] Taking HF53 as an example, the child in this sample underwent a bone marrow transplant, and the bone marrow donor was his father. Figure 7 In the figure, it can be seen that there are a lot of hollow five-pointed stars on the SNP, indicating that it does not conform to the genetic separation law. However, the SNPs that do not conform to the separation law are all orange, that is, they are paternal, suggesting that this method has certain application value even in the judgment of bone marrow transplantation. Therefore, it is effective to judge the parental origin by family SNP on autosomes.

[0190] (B) This method was used to determine the parental origin of the X chromosome, using the third of three cases (parents are non-heterozygous and different for a certain SNP). The children of HF01 and HF02 are female and male, respectively (see Figure 8 , Fig. 9 ). It can be seen that in HF01, all SNP sites that can distinguish the origin of parents have similar dosages on both sides. It can be proved at 9 SNP sites that the X chromosome of the subject comes from both parents, half from each parent, which is consistent with the common sense that women have two X chromosomes, one from each parent. In HF02 samples, all SNP sites that can distinguish the origin of parents have been proved at 3 SNP sites. The X chromosome of the subject comes from the mother, which is consistent with the common sense that the male X chromosome comes from the mother. All 53 healthy nuclear families tested meet the gender characteristics.

[0191] (C) The same method was used to analyze the parental origin of the X chromosome in TS patients;

[0192] Take T8 sample as an example (see Fig.10 ), it was proved at 13 SNP sites that the X chromosome of the subject came from both parents, and the part from the father was higher than the part from the mother. It can be seen that the X chromosome from the father is much larger than the X chromosome from the mother. After calculation, the proportion of X chromosome from the father is 70.2%. The above results are consistent with the X-STR test results. Fig.11 The results of the STR test for the proband T8 and his parents, named GATA172D05, show that the two STR loci of the proband come from the father and mother respectively. The lengths of the STRs carried by the parents are different. The dosage of the STR from the mother of the proband is significantly lower than that of the parents, which indicates that the X chromosome of the proband comes from both parents, and the signal of the X chromosome from the mother is weaker. The result of the karyotype analysis of the proband T8 is: 45,X

[12] / 46,XX

[38] , which is consistent.

[0193] Take the T1 sample as an example (see Fig.12 ) and used the same method to analyze the parental origin of the X chromosome of TS patients. Fig.12It can be seen that the 10 SNP sites proved that the X chromosome of the subject came from both parents, and the part from the father was much higher than that from the mother. The X chromosome from the father was much larger than that from the mother. After calculation, the proportion of X chromosome from the father was 89.7%. The above results are consistent with the results of X-STR test. Fig.13 The STR test results of the T1 proband and his parents, named DXS10146 (the STR peak indicated by the arrow of the proband comes from the mother), show that the X chromosome of the proband comes from the father, but there is a weak X chromosome signal from the mother. This weak signal is sometimes regarded as contamination or non-specific influence. In the analysis method of this embodiment, the mother's signal appears repeatedly in multiple SNP sites, and there is no such signal in the healthy family, which shows that its existence is real. In the karyotype analysis, the T1 proband is 45, X, which shows that the X chromosome from the mother is completely ignored.

[0194] Therefore, the SNP Panel test based on the MLPA-NGS method can determine the parental origin and proportion of the X chromosome, and the results are consistent with the X-STR results and chromosome karyotype analysis results.

[0195] (4) CNV analysis and verification;

[0196] The CNV probe can be used to detect the CNV at the probe location. The number of copies of the X chromosome in a female and a male in a healthy family is 2 and 1, respectively (see Fig.14 , the X chromosome copy number of healthy females is 2, and the X chromosome copy number of healthy males is 1). Among them, the stability of CNV48 and CNV53 probes is slightly poor due to unknown reasons.

[0197] The CNV probe can also detect samples with large CNVs on chromosome X. For example, for sample T8, the copy number of chromosome X can be detected. Fig.15 , indicating that the patient's X chromosome copy number is significantly greater than 1 and less than 2, so it should be a mosaic. Its average copy number is about 1.32, which is basically consistent with the karyotype analysis result of the sample "45, X

[12] / 46, XX

[38] ".

[0198] In this example, three TS positive samples (T11, B1026, H53) were tested by SNP chip. The test results were consistent with the MLPA-NGS results, proving that MLPA-NGS technology is accurate in CNV detection. The results of these three samples are shown in the following "(5) Joint analysis of X chromosome parental origin and CNV".

[0199] (5) Joint analysis of parental origin and CNV of X chromosome;

[0200] This example uses three samples as examples to conduct a joint analysis of the parental origin and CNV of chromosome X. These three samples have all been subjected to karyotype analysis and SNP chip testing, and the accuracy of the MLPA-NGS method is confirmed by mutual comparison.

[0201] 1) Example 1:

[0202] Example 1 is a sample from the T11 family. The karyotype of the T11 proband is: "45,X

[17] / 46,XX

[83] ", which means: 100 cells were analyzed, 17 cells had a karyotype of 45,X, with one less sex chromosome; 83 cells had a karyotype of 46,XX, so it was a 45,X / 46,XX mosaic karyotype.

[0203] The results of the SNP chip analysis of the T11 proband sample named Infinium Omni ZhongHua-8 were: "arr[hg19]Xp22.33q28(60,425-155,236,747)x1~2". The result of the report was explained as follows: a mosaic deletion of chromosome X occurred, with the copy number of 1~2, which was 45,XO / 46,XX mosaicism, and the deletion ratio was about 40%. The results of chromosome X detected by SNP chip can be found in Fig.16 , which shows that the dosage of the two alleles at the heterozygous SNP site on the X chromosome is unequal, and this inequality runs through the entire X chromosome, suggesting the existence of euploid mosaicism.

[0204] In this example, MLPA-NGS was performed on the T11 family. The autosomal SNP analysis profile of the T11 family is shown in Fig.17 , showing that equal amounts of autosomes come from both parents, which is consistent with common sense. Fig.17 It showed that there was a genetic discrepancy at the rs5744944 position of T11 (which had been mentioned before). After SNP chip detection and Sanger sequencing detection, it was proved that there was a detection error at this position in the T11 proband. After eliminating this error, it can be confirmed that the tested parents are the biological parents of the T11 proband. The proportion of autosomes from the parents was calculated by SNP dosage to be 48.2% and 51.8%, respectively, which is consistent with the common sense that the autosomes come from the parents in equal amounts.

[0205] The X chromosome SNP analysis map of the T11 family is shown in Fig.18 , showing that the X chromosome comes from both parents, and the part from the father is more than that from the mother; the proportion of X chromosome from both parents is 64.2% and 35.8%, respectively.

[0206] Assuming that each cell contains an X chromosome from the father and some cells contain chromosomes from the mother, the proportion of cells missing the X chromosome from the mother should be (64.2%-35.8%) / 64.2%=44.2%, which is consistent with the conclusion that the missing proportion of the chip test result is about 40%. The proportion of missing an X chromosome in the karyotype analysis is 17%, which is quite different from the result of MLPA-NGS. Since core analysis generally only analyzes 100 cells, its quantitative error is generally large.

[0207] The X chromosome CNV analysis pattern of the proband in the T11 family is shown in Fig.19 , showing that the copy number of chromosome X in sample T11 is around 1.5, which is basically uniform on each probe, and the average copy number of chromosome X is 1.52.

[0208] If the proband has a 46,XX karyotype, the relative copy number of chromosome X should be 2. According to the current calculation of 1.52, the deletion ratio is 2-1.52=48%, which is consistent with the previous result of 44.2%. Therefore, the MLPA-NGS test results of the T11 family are qualitatively consistent with the karyotype analysis and SNP chip test, and are quantitatively closer to the SNP chip test results.

[0209] 2) Example 2:

[0210] Example 2 is a sample of the B1026 family. The karyotype of the B1026 proband is: "46,X,del(X)(q22)(18) / 45,X(12)". The karyotype description is: After peripheral blood was cultured, chromosome karyotype analysis was performed, and a total of 30 cells were counted. The karyotype of 18 cells was 46,X,del(X)(q22); the karyotype of 12 cells was 45,X, and one sex chromosome was missing.

[0211] The results of the Infinium Omni ZhongHua-8 SNP chip analysis of the B1026 proband sample were: "arr[hg19]Xp22.33(60,425-976,171)x1~2; Xp22.33q13.1(979,904-69,415,867)x1~2; Xq13.1q21.31(69,424,308-91,754,954)x1~2; Xq21.31q28(91,765,739-155,236,747)x1", and the report's interpretation of the karyotype was: a mosaic deletion of one copy of the X chromosome Xp22.33 band, with a length of approximately 916kbp, and a deletion ratio of approximately 35%. The X chromosome Xp22.33q13.1 region has a mosaic deletion of one copy, with a length of about 68.44Mbp, and the deletion ratio is about 70%. The X chromosome Xq13.1q21.31 region has a mosaic deletion of one copy, with a length of about 22.33Mbp, and the deletion ratio is about 35%. The X chromosome Xq21.31q28 region has a mosaic deletion of one copy, with a length of about 63.47Mbp.

[0212] The results of the X chromosome detected by SNP chip are shown in Fig. 20 , showing from the SNP chip, there is only one chromosome copy in the main region below the long arm of the X chromosome, while the X chromosome in the short arm region is uneven.

[0213] In this example, MLPA-NGS was performed on the B1026 family. The autosomal SNP analysis profile of the B1026 family is shown in Fig.21 , showing that equal amounts of autosomes come from both parents, which is consistent with common sense. Fig.21 The results showed that all the autosomal SNPs tested for B1026 were genetically consistent, confirming that the tested parents were the biological parents of the proband B1026. The proportion of autosomes from the parents, calculated by SNP dosage, was 51.3% and 48.7%, respectively, which is consistent with the common sense that the autosomes come from the parents in equal amounts.

[0214] The X chromosome SNP analysis map of the B1026 family is shown in Fig. 22 , showing that there are 5 SNP sites on the left side of the patient, which can distinguish the X chromosome from the parents, with less from the father and more from the mother, and there are four SNP sites on the right side, showing that the X chromosome only comes from the mother. The proportions of X chromosomes from the parents are 16.1% and 83.9%, respectively.

[0215] The X chromosome CNV analysis pattern of the proband in the B1026 family is shown in Fig.23 From the CNV results, the copy number of sites before CNV61 is higher than 1, and the copy number of sites after CNV61 is basically equal to 1; the average copy number of chromosome X is 1.19.

[0216] According to the results of the SNP chip, the above sample is a patient with a complex X chromosome karyotype. Its main feature is that the cells are divided into two types, one with a karyotype of 45,X and the other with a karyotype of 46,X,del(X)(q22), and the two are combined into a chimera. The results of MLPA-NGS are consistent with the results of karyotype analysis and SNP chip.

[0217] 3) Example 3:

[0218] Example 3 is a sample of the H53 family. The karyotype of the H53 proband is: "46, X, del (11.2)", which indicates that a portion of the short arm of one of the X chromosomes is missing.

[0219] The result of the SNP chip analysis of the H53 proband sample named Infinium Omni ZhongHua-8 was: "arr[hg19]Xp22.33p11.21(60,425-57,169,551)x1". The reported result explanation: One copy of the X chromosome Xp22.33p11.21 band is missing, with a length of approximately 57.11Mbp, involving 666 genes including ARSL, STS, ANOS1, MID1, TRAPPC2, SHOX, etc.

[0220] The results of the X chromosome detected by SNP chip are shown in Fig.24 , indicating that most of the short arm part is missing, making the SNP lack heterozygosity in this region.

[0221] In this example, MLPA-NGS was performed on the H53 family. The autosomal SNP analysis map of the H53 family is shown in Fig.25 , showing that equal amounts of autosomes come from both parents, which is consistent with common sense. Fig.25 The results showed that all autosomal SNPs tested for H53 were genetically consistent, confirming that the tested parents were the biological parents of the proband H53. The proportion of autosomes from the parents, calculated by SNP dosage, was 51.0% and 48.9%, respectively, which is consistent with the common sense that the autosomes come from the parents in equal amounts.

[0222] The X chromosome SNP analysis map of the H53 family is shown in Fig.26 , indicating that there are 4 SNP sites on the left side of the patient that can distinguish the origin of the parents, indicating that this section of the X chromosome only comes from the mother; there are 6 SNP sites on the right side that can distinguish the origin of the parents, indicating that the X chromosome is basically from the parents in equal amounts; the proportions of the X chromosome from the parents are 30.1% and 69.9%, respectively.

[0223] The X chromosome CNV analysis map of the proband in the H53 family is shown in Fig. 27From the CNV results, the copy number of CNV56 and the previous sites is 1, and the copy number of the sites after CNV56 is basically equal to 2; the average copy number of chromosome X is 1.56. Therefore, the MLPA-NGS test of the above samples is also consistent with the karyotype analysis and SNP test.

[0224] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A kit for detecting Turner syndrome based on the MLPA-NGS method, characterized in that: The kit comprises a SNP probe composition, a CNV probe composition and a primer composition; wherein the SNP probe composition comprises SNP probe pairs designed for autosomes, chromosome X and chromosome Y, respectively, and the CNV probe composition comprises CNV probe pairs designed for autosomes, chromosome X and chromosome Y, respectively; the primer composition comprises universal primers with index sequences for amplifying the connected probes, each of the probe pairs comprises a left probe and a right probe, the 5' end of the left probe and the 3' end of the right probe respectively comprising a universal sequence, which serves as a binding sequence for the universal primers during amplification; The SNP probe composition includes 142 probe pairs, which are respectively designed at 66 SNP sites on autosomes, 51 SNP sites on chromosome X, and 25 SNP sites on chromosome Y, wherein each SNP site contains two genotypes: wild type and mutant type; the CNV probe composition includes 84 probe pairs, which are respectively designed at 44 pairs of probes on autosomes, 30 pairs of probes on chromosome X, and 10 pairs of probes on chromosome Y; In the SNP probe composition, the sequences of each SNP probe pair used to bind to the template DNA are shown in SEQ ID NO. 1 to SEQ ID NO. 284; in the CNV probe composition, the sequences of each CNV probe pair used to bind to the template DNA are shown in SEQ ID NO. 285 to SEQ ID NO. 452; the sequence of the universal primer is shown in SEQ ID NO. 453 to SEQ ID NO. 454, the universal sequence at the 5' end of the left probe is shown in SEQ ID NO. 455, and the universal sequence at the 3' end of the right probe is shown in SEQ ID NO.

456.

2. The kit according to claim 1, characterized in that The SNP probe composition and the CNV probe composition are mixed to obtain a probe working solution, wherein the concentration of each probe in the probe working solution is 0.2~20 fmol / μL; and / or, in the primer composition, the preparation concentration of each universal primer is 2~200 pMol; and / or, the kit also includes at least one of MLPA buffer, ligase, ligase buffer, PCR buffer, dNTP, and PCR enzyme.

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