Use of insertion / deletion polymorphism genetic markers in detecting copy number of human chromosomal subtelomeric regions, and reagent panel and method for detecting copy number of human chromosomal subtelomeric regions

Abstract

Provided in the present invention are the use of insertion / deletion polymorphism genetic markers in detecting the copy number of human chromosomal subtelomeric regions, a reagent panel for detecting the insertion / deletion polymorphisms genetic markers, and a method for detecting the copy number of human chromosomal subtelomeric regions.

Description

Application of insertion / deletion polymorphism genetic marker in detection of copy number of human chromosome sub-telomere region, and reagent set and method for detecting copy number of human chromosome sub-telomere region TECHNICAL FIELD

[0001] The present application relates to the technical field of chromosome sub-telomere region copy number detection, and particularly relates to application of insertion / deletion polymorphism genetic marker in detection of copy number of human chromosome sub-telomere region, and reagent set and method for detecting copy number of human chromosome sub-telomere region. BACKGROUND

[0002] In recent years, with the development of molecular genetics and genomics, people pay more and more attention to chromosomal structural variations, especially the copy number variations (CNVs) of chromosome sub-telomere region. Abnormal copy number of chromosome sub-telomere region, i.e. copy number increase or decrease, is closely related to the occurrence and development of various genetic diseases, including but not limited to nervous system diseases, developmental retardation, mental retardation, etc. Such variations are often difficult to detect by traditional genetic methods, because they are located in complex regions of the genome, and may involve large fragments of DNA duplication or deletion.

[0003] Insertion / deletion polymorphism (InDel) as a common genetic variation form is widely distributed in the human genome, with an average of 7.2 kb of InDel site. InDels not only have high genetic polymorphism, but also have good stability and strong species specificity, which are ideal genetic markers. InDel sites have wide application prospects in multiple fields such as forensic science, medical genetics, disease association studies, etc., and show unique advantages in the detection of chromosomal sub-telomere region copy number variations.

[0004] At present, the detection methods of copy number mainly include real-time quantitative PCR (qPCR), comparative genomic hybridization (CGH), microarray comparative genomic hybridization (array CGH), whole genome sequencing (WGS), etc. Among them, real-time quantitative PCR is widely used in copy number variation detection due to its simple operation, relatively low cost, good specificity, etc. However, real-time quantitative PCR usually needs to design primers for specific sequences, and for the detection of copy number of chromosome sub-telomere region, it is difficult to design specific primers due to the complexity and high repeatability of these regions, and the interpretation of detection results also requires professional knowledge.

[0005] Therefore, developing a reagent set and method based on insertion / deletion polymorphism genetic marker suitable for detecting copy number of human chromosome sub-telomere region will help to improve the accuracy and efficiency of detection, and provide important genetic basis for risk assessment, diagnosis and treatment of genetic diseases. SUMMARY

[0006] The present application aims at the deficiencies in the prior art, and provides an application of an insertion-deletion polymorphism genetic marker in detecting a copy number of a sub-telomere region of a human chromosome, and a reagent set and a method for detecting the copy number of the sub-telomere region of the human chromosome.

[0007] To achieve the above object, the present application adopts the technical solution of:

[0008] The first aspect of the present application provides an application of an insertion-deletion polymorphism genetic marker in detecting a copy number of a sub-telomere region of a human chromosome, which is selected from at least one in Table 1 based on the human genome reference sequence version 38:

[0009] Table 1

[0010] The second aspect of the present application provides a reagent set for detecting a copy number of a sub-telomere region of a human chromosome, comprising: a primer pair for amplifying an insertion-deletion polymorphism genetic marker by polymerase chain reaction; wherein,

[0011] The insertion-deletion polymorphism genetic marker is selected from at least one in Table 1.

[0012] Preferably, the primer pair comprises: an upstream primer, and a downstream primer corresponding to the upstream primer; the upstream primer or / and the downstream primer is labeled with a fluorescein; wherein,

[0013] The upstream primer, the downstream primer, and the fluorescein are shown in the following table:

[0014] Table 2

[0015] Preferably, it comprises a polymerase chain reaction amplification system as shown in Table 3.

[0016] Table 3

[0017] The third aspect of the present application provides a method for detecting a copy number of a sub-telomere region of a human chromosome, comprising the steps of:

[0018] Step one, obtaining a characterization of an allele corresponding to a plurality of insertion-deletion polymorphism genetic markers of a sample to be detected; wherein the plurality of insertion-deletion polymorphism genetic markers are selected from a plurality in Table 1.

[0019] Step two, judging the characterization of an allele corresponding to an insertion-deletion polymorphism genetic marker of a sample to be detected:

[0020] If the allele corresponding to the insertion-deletion polymorphism genetic marker is not characterized, the copy number of the sub-telomere region of the human chromosome corresponding to the insertion-deletion polymorphism genetic marker is the fourth preset copy value, and the process ends.

[0021] If the allele corresponding to the insertion-deletion polymorphism genetic marker is characterized as a single allele, step four is entered.

[0022] If the allele corresponding to the insertion-deletion polymorphism genetic marker is characterized as two alleles, step three is entered.

[0023] In step three, it is determined whether the ratio of the characterization levels of the two alleles corresponding to the insertion-deletion polymorphism genetic marker is in a first preset interval.

[0024] If the ratio of the characterization levels of the two alleles corresponding to the insertion-deletion polymorphism genetic marker is in the first preset interval, the copy number of the sub-telomere region of the human chromosome corresponding to the insertion-deletion polymorphism genetic marker is obtained according to the ratio of the characterization levels of the two alleles corresponding to the insertion-deletion polymorphism genetic marker, and the process ends.

[0025] If the ratio of the characterization levels of the two alleles corresponding to the insertion-deletion polymorphism genetic marker is not in the first preset interval, step four is entered.

[0026] In step four, the copy number of the sub-telomere region of the human chromosome corresponding to the insertion-deletion polymorphism genetic marker is obtained according to the proportion of the characterization of the allele corresponding to the insertion-deletion polymorphism genetic marker in all alleles corresponding to the insertion-deletion polymorphism genetic markers, and the process ends.

[0027] Preferably, in step one, the method for obtaining the characterization of the allele corresponding to the insertion-deletion polymorphism genetic marker of the sample to be detected comprises:

[0028] After the insertion-deletion polymorphism genetic marker is amplified by polymerase chain reaction using primers labeled with fluorescein, the characterization of the allele corresponding to the insertion-deletion polymorphism genetic marker of the sample to be detected is obtained according to the fluorescence intensity of the polymerase chain reaction amplification product.

[0029] Preferably, in step three, the first preset interval includes a first sub-preset interval and a second sub-preset interval.

[0030] When the ratio of the characterization levels of the two alleles corresponding to the insertion-deletion polymorphism genetic marker is in the first sub-preset interval or in the second sub-preset interval, the ratio of the characterization levels of the two alleles corresponding to the insertion-deletion polymorphism genetic marker is in the first preset interval.

[0031] If the ratio of the representation levels of the two alleles corresponding to the insertion-deletion polymorphism genetic marker is in the first sub-pre-set interval, the copy number of the sub-telomere region of the human chromosome corresponding to the insertion-deletion polymorphism genetic marker is the second pre-set copy value, and the process ends.

[0032] If the ratio of the representation levels of the two alleles corresponding to the insertion-deletion polymorphism genetic marker is in the second sub-pre-set interval, the copy number of the sub-telomere region of the human chromosome corresponding to the insertion-deletion polymorphism genetic marker is the third pre-set copy value, and the process ends.

[0033] When the ratio of the representation levels of the two alleles corresponding to the insertion-deletion polymorphism genetic marker is not in the first sub-pre-set interval and not in the second sub-pre-set interval, the ratio of the representation levels of the two alleles corresponding to the insertion-deletion polymorphism genetic marker is not in the first pre-set interval.

[0034] Preferably, the step four comprises:

[0035] The representation proportion of the allele corresponding to the insertion-deletion polymorphism genetic marker in all alleles corresponding to the insertion-deletion polymorphism genetic markers is recorded as a first ratio.

[0036] The representation of the alleles corresponding to the corresponding multiple insertion-deletion polymorphism genetic markers of the control sample is obtained, and the representation proportion of the allele corresponding to the insertion-deletion polymorphism genetic marker of the control sample in all alleles corresponding to the insertion-deletion polymorphism genetic markers of the control sample is recorded as a second ratio.

[0037] It is judged whether the ratio of the first ratio and the second ratio is in a second pre-set interval, and the second pre-set interval comprises a third sub-pre-set interval, a fourth sub-pre-set interval, and a fifth sub-pre-set interval.

[0038] When the ratio of the representation levels of the two alleles corresponding to the insertion-deletion polymorphism genetic marker is in the third sub-pre-set interval or in the fourth sub-pre-set interval or in the fifth sub-pre-set interval, the ratio of the first ratio and the second ratio is in the second pre-set interval, and the process ends.

[0039] If the ratio of the first ratio and the second ratio is in the third sub-pre-set interval, the copy number of the sub-telomere region of the human chromosome corresponding to the insertion-deletion polymorphism genetic marker is the first pre-set copy value, and the process ends.

[0040] If the ratio of the first ratio and the second ratio is in the fourth sub-pre-set interval, the copy number of the sub-telomere region of the human chromosome corresponding to the insertion-deletion polymorphism genetic marker is the second pre-set copy value, and the process ends.

[0041] If the ratio of the first ratio and the second ratio is in the fifth sub-pre-set interval, the copy number of the sub-telomere region of the human chromosome corresponding to the insertion-deletion polymorphism genetic marker is a third pre-set copy value, and the process ends.

[0042] If the ratio of the first ratio and the second ratio is not in the third sub-pre-set interval, not in the fourth sub-pre-set interval, and not in the fifth sub-pre-set interval, and the ratio of the first ratio and the second ratio is not in the second pre-set interval, the copy number of the sub-telomere region of the human chromosome corresponding to the insertion-deletion polymorphism genetic marker is a gray value, and the process ends.

[0043] Compared with the prior art, the application has the following technical effects:

[0044] The application provides a detection scheme for the copy number of the sub-telomere region of the human chromosome, which is efficient, low in cost, simple to operate and accurate, and is expected to have a wide application prospect in clinical diagnosis and genetic disease research.

[0045] Compared with high-throughput technologies such as gene chips, second-generation sequencing, long-read third-generation sequencing or optical genome mapping, the application of the insertion-deletion polymorphism genetic marker in detecting the copy number of the sub-telomere region of the human chromosome has a significantly reduced cost and is more suitable for large-scale screening and clinical application.

[0046] The operation process of the method for detecting the copy number of the sub-telomere region of the human chromosome is simplified, and complex biological information analysis is not needed, so that an experimental personnel can master the method after basic training, thereby greatly reducing the operation threshold and the required professional skill level and being conducive to popularization and use in a laboratory environment with limited resources.

[0047] The method for detecting the copy number of the sub-telomere region of the human chromosome can complete the whole process from sample processing to data analysis within 4 hours, significantly improves the detection efficiency, and is suitable for clinical diagnosis scenarios that require rapid results.

[0048] Through peak analysis of the insertion-deletion polymorphism genetic marker, the copy number of the target region can be accurately judged, and the judgment of 2 copies or 3 copies is particularly convenient. DETAILED DESCRIPTION

[0049] FIG. 1 is a result graph of a first multiplex polymerase chain reaction FAM channel of a sample to be detected in Embodiment 1 of the application;

[0050] FIG. 2 is a result graph of a first multiplex polymerase chain reaction HEX channel of a sample to be detected in Embodiment 1 of the application;

[0051] FIG. 3 is a result graph of a second multiplex polymerase chain reaction TAMRA channel of a sample to be detected in Embodiment 1 of the application;

[0052] Figure 4 is a result chart of the second multiplex polymerase chain reaction ROX channel of the sample to be detected in Example 1 of the present application;

[0053] Figure 5 is a result chart of the third multiplex polymerase chain reaction HEX channel of the sample to be detected in Example 1 of the present application;

[0054] Figure 6 is a result chart of the first multiplex polymerase chain reaction FAM channel of the control sample in Example 1 of the present application;

[0055] Figure 7 is a result chart of the first multiplex polymerase chain reaction HEX channel of the control sample in Example 1 of the present application;

[0056] Figure 8 is a result chart of the second multiplex polymerase chain reaction TAMRA channel of the control sample in Example 1 of the present application;

[0057] Figure 9 is a result chart of the second multiplex polymerase chain reaction ROX channel of the control sample in Example 1 of the present application;

[0058] Figure 10 is a result chart of the third multiplex polymerase chain reaction HEX channel of the control sample in Example 1 of the present application;

[0059] Figure 11 is a result chart of the first multiplex polymerase chain reaction FAM channel of the sample to be detected in the comparative example of the present application;

[0060] Figure 12 is a result chart of the first multiplex polymerase chain reaction HEX channel of the sample to be detected in the comparative example of the present application;

[0061] Figure 13 is a result chart of the second multiplex polymerase chain reaction TAMRA channel of the sample to be detected in the comparative example of the present application;

[0062] Figure 14 is a result chart of the second multiplex polymerase chain reaction ROX channel of the sample to be detected in the comparative example of the present application;

[0063] Figure 15 is a result chart of the third multiplex polymerase chain reaction HEX channel of the sample to be detected in the comparative example of the present application;

[0064] Taking the first fluorescence peak on the left side in Figure 1 as an example, the upper "2P" mark is the name of the insertion-deletion polymorphism genetic marker corresponding to the fluorescence peak, the lower number "1" represents the name of the allele of the insertion-deletion polymorphism genetic marker corresponding to the fluorescence peak, the lower number "93.45" represents the electrophoretic length of the allele of the insertion-deletion polymorphism genetic marker corresponding to the fluorescence peak, and the lower number "5740" represents the peak height of the fluorescence peak;

[0065] Figure 16 is a result chart of the normal distribution curve fitting in the verification example of the present application. DETAILED DESCRIPTION

[0066] The detailed description of the specific embodiments of the present application will be described in detail below.

[0067] Unless otherwise defined, technical and scientific terms used in the claims and specification have their ordinary meaning in the art.

[0068] The term "comprising" or similar terms as used in the specification and claims means that the elements recited are open-ended and do not exclude additional, unrecited elements.

[0069] Numerical values recited in the specification and claims encompass all values from the lower to the upper value by one unit, provided that any lower value is separated from any higher value by at least two units. In this context, a statement that a component quantity or physical quantity is from 1 to 100, 10 to 90, and 20 to 80, is intended to convey that 5 to 95, 14 to 76, 23 to 67, 32 to 58, 41 to 49, and the like are also expressly stated in this specification. For values less than 1, 0.0001, 0.001, 0.01, or 0.1 are considered to be suitable units. The foregoing examples are merely illustrative, and all combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this specification in a similar manner.

[0070] The present application provides an application of an insertion-deletion polymorphic genetic marker in detecting the copy number of the sub-telomere region of human chromosomes, based on the 38th edition of the human genome reference sequence, and the insertion-deletion polymorphic genetic marker is shown in Table 1.

[0071] On this basis, the present application further provides a reagent group for detecting the copy number of the sub-telomere region of human chromosomes, comprising: a first multiplex polymerase chain reaction amplification system for amplifying a first group of insertion-deletion polymorphic genetic markers, a second multiplex polymerase chain reaction amplification system for amplifying a second group of insertion-deletion polymorphic genetic markers, and a third multiplex polymerase chain reaction amplification system for amplifying a third group of insertion-deletion polymorphic genetic markers; wherein,

[0072] The first group of insertion-deletion polymorphic genetic markers comprises: insertion-deletion polymorphic genetic markers with names 1p, 1q, 2p, 2q, 3p, 3q, 4p, 4q, 5p, 5q, 6q, 7p, 7q, 8p, 8q, 9p, 9q, 10q, 11p, 17p, and 19q shown in Table 1;

[0073] The first multiplex polymerase chain reaction amplification system is shown in the following table: The present application provides an application of an insertion-deletion polymorphic genetic marker in detecting the copy number of the sub-telomere region of human chromosomes, based on the 38th edition of the human genome reference sequence, and the insertion-deletion polymorphic genetic marker is shown in Table 1.

[0071] On this basis, the present application further provides a reagent group for detecting the copy number of the sub-telomere region of human chromosomes, comprising: a first multiplex polymerase chain reaction amplification system for amplifying a first group of insertion-deletion polymorphic genetic markers, a second multiplex polymerase chain reaction amplification system for amplifying a second group of insertion-deletion polymorphic genetic markers, and a third multiplex polymerase chain reaction amplification system for amplifying a third group of insertion-deletion polymorphic genetic markers; wherein,

[0072] The first group of insertion-deletion polymorphic genetic markers comprises: insertion-deletion polymorphic genetic markers with names 1p, 1q, 2p, 2q, 3p, 3q, 4p, 4q, 5p, 5q, 6q, 7p, 7q, 8p, 8q, 9p, 9q, 10q, 11p, 17p, and 19q shown in Table 1;

[0073] The first multiplex polymerase chain reaction amplification system is shown in the following table: The present application provides an application of an insertion-deletion polymorphic genetic marker in detecting the copy number of the sub-telomere region of human chromosomes, based on the 38th edition of the human genome reference sequence, and the insertion-deletion polymorphic genetic marker is shown in Table 1.

[0071] On this basis, the present application further provides a reagent group for detecting the copy number of the sub-telomere region of human chromosomes, comprising: a first multiplex polymerase chain reaction amplification system for amplifying a first group of insertion-deletion polymorphic genetic markers, a second multiplex polymerase chain reaction amplification system for amplifying a second group of insertion-deletion polymorphic genetic markers, and a third multiplex polymerase chain reaction amplification system for amplifying a third group of insertion-deletion polymorphic genetic markers; wherein,

[0072] The first group of insertion-deletion polymorphic genetic markers comprises: insertion-deletion polymorphic genetic markers with names 1p, 1q, 2p, 2q, 3p, 3q, 4p, 4q, 5p, 5q, 6q, 7p, 7q, 8p, 8q, 9p, 9q, 10q, 11p, 17p, and 19q shown in Table 1;

[0073] The first multiplex polymerase chain reaction amplification system is shown in the following table:

[0074] The first primer set comprises: the primer pairs with names 1p, 1q, 2p, 2q, 3p, 3q, 4p, 4q, 5p, 5q, 6q, 7p, 7q, 8p, 8q, 9p, 9q, 10q, 11p, 17p and 19q shown in Table 2, and the upstream primer or / and the downstream primer in the primer pairs is labeled with a fluorescein;

[0075] The second set of insertion-deletion polymorphism genetic markers comprises: the insertion-deletion polymorphism genetic markers with names 11q, 12q, 13q, 14q, 16p, 16q, 17q, 18p, 18q, 19p, 20p, 20q, 22q and Xq shown in Table 1;

[0076] The second multiplex polymerase chain reaction amplification system is shown in the following table:

[0077] The second primer set comprises: the primer pairs with names 11q, 12q, 13q, 14q, 16p, 16q, 17q, 18p, 18q, 19p, 20p, 20q, 22q and Xq shown in Table 2, and the upstream primer or / and the downstream primer in the primer pairs is labeled with a fluorescein;

[0078] The third set of insertion-deletion polymorphism genetic markers comprises: the insertion-deletion polymorphism genetic markers with names 6p, 10p, 12p, 15q, 21q and Xp shown in Table 1;

[0079] The third multiplex polymerase chain reaction amplification system is shown in the following table:

[0080] The third primer set comprises: the primer pairs with names 6p, 10p, 12p, 15q, 21q and Xp shown in Table 2, and the upstream primer or / and the downstream primer in the primer pairs is labeled with a fluorescein;

[0081] The multiplex polymerase chain reaction conditions corresponding to the first multiplex polymerase chain reaction amplification system, the second multiplex polymerase chain reaction amplification system and the third multiplex polymerase chain reaction amplification system are shown in the following table:

[0082] On this basis, the application further provides a method for detecting the copy number of the sub-telomere region of human chromosomes, comprising the following steps:

[0083] Step one, after the multiple polymerase chain reaction amplification of each insertion and deletion polymorphism genetic marker by the aforementioned reagent group for detecting the copy number of human chromosome sub-telomere region, the fluorescence intensity (characterization) of the alleles corresponding to the insertion and deletion polymorphism genetic marker of the sample to be tested is obtained by capillary electrophoresis of 1 μL of the multiple polymerase chain reaction amplification product by 3500 genetic analyzer or 3500Dx genetic analyzer or other instruments capable of multiple fluorescence detection;

[0084] Step two, the fluorescence intensity (characterization) of the alleles corresponding to a certain insertion and deletion polymorphism genetic marker of the sample to be tested is determined.

[0085] If the alleles corresponding to the insertion and deletion polymorphism genetic marker do not show a fluorescence peak (not characterized), the copy number of the human chromosome sub-telomere region corresponding to the insertion and deletion polymorphism genetic marker is 0, and the process ends.

[0086] If the alleles corresponding to the insertion and deletion polymorphism genetic marker show a single fluorescence peak (characterizing a single allele), step four is entered.

[0087] If the alleles corresponding to the insertion and deletion polymorphism genetic marker show two fluorescence peaks (characterizing two alleles), step three is entered.

[0088] Step three, the ratio (characterization level ratio) PHR of the fluorescence signal intensity (peak height or peak area) of the two alleles corresponding to the insertion and deletion polymorphism genetic marker is calculated, PHR = H S / H L

[0089] Wherein, H S represents the fluorescence signal intensity of the fluorescence peak of the shorter allele of the two alleles corresponding to the insertion and deletion polymorphism genetic marker, and H L represents the fluorescence signal intensity of the fluorescence peak of the longer allele of the two alleles corresponding to the insertion and deletion polymorphism genetic marker.

[0090] If PHR ∈ [0.80, 1.20], the copy number of the human chromosome sub-telomere region corresponding to the insertion and deletion polymorphism genetic marker is 2, and the process ends.

[0091] If PHR ∈ [0.45, 0.65]∪[1.60, 2.60], the copy number of the human chromosome sub-telomere region corresponding to the insertion and deletion polymorphism genetic marker is 3, and the process ends.

[0092] If Then go to step four;

[0093] Step four, the first part, calculate the ratio of the fluorescence signal intensity of the fluorescence peak of the allele corresponding to the insertion-deletion polymorphism genetic marker to the sum of the fluorescence signal intensity of the fluorescence peak of all alleles corresponding to the insertion-deletion polymorphism genetic marker (the proportion of the characterization level) R S , R S = H X / sumH X H X = H1+H2

[0094] Wherein, H X represents the fluorescence signal intensity of the fluorescence peak of the allele corresponding to the insertion-deletion polymorphism genetic marker, H1 represents the fluorescence signal intensity of the fluorescence peak of the first allele corresponding to the insertion-deletion polymorphism genetic marker, H2 represents the fluorescence signal intensity of the fluorescence peak of the second allele corresponding to the insertion-deletion polymorphism genetic marker, if the allele corresponding to the insertion-deletion polymorphism genetic marker shows only a single fluorescence peak, then H2=0; sumH X represents the sum of the fluorescence signal intensity of the fluorescence peak of all alleles corresponding to the insertion-deletion polymorphism genetic marker, for example, in Figure 1, sumH X , that is, the sum of the fluorescence signal intensity of the fluorescence peak of 18 alleles corresponding to 12 insertion-deletion polymorphism genetic markers;

[0095] Step four, the second part, step one is synchronized to obtain the fluorescence intensity of the allele corresponding to the insertion-deletion polymorphism genetic marker of the control sample (characterization); Step four, the first part is synchronized to calculate the ratio of the fluorescence signal intensity of the fluorescence peak of the allele corresponding to the insertion-deletion polymorphism genetic marker of the control sample to the sum of the fluorescence signal intensity of the fluorescence peak of all alleles corresponding to the insertion-deletion polymorphism genetic marker of the control sample (the proportion of the characterization level) R C ;

[0096] Step four, the third part, calculate the judgment coefficient RR, RR=R S / R C

[0097] Determine RR∈(0,0.7]∪[0.75,1.25]∪[1.3,+∞):

[0098] If RR∈(0,0.7], then the copy number of the human chromosome sub-telomere region corresponding to the insertion-deletion polymorphism genetic marker is 1 copy, and the process ends.

[0099] If RR∈[0.75,1.25], then the copy number of the human chromosome sub-telomere region corresponding to the insertion-deletion polymorphism genetic marker is 2 copies, and the process ends.

[0100] If RR∈[1.3, +∞), the copy number of the human chromosome sub-telomere region corresponding to the insertion-deletion polymorphism genetic marker is 3 copies or above, end.

[0101] If RR∈[0, 1.3), the copy number of the human chromosome sub-telomere region corresponding to the insertion-deletion polymorphism genetic marker is 2 copies, end. If RR∈[0, 1.3), the copy number of the human chromosome sub-telomere region corresponding to the insertion-deletion polymorphism genetic marker is 2 copies, end.

[0102] Example 1

[0103] In this embodiment, the sample to be tested is a normal adult male sample, which is the remaining sample of EDTA anticoagulation blood routine test of Zhengzhou University Third Affiliated Hospital Obstetric Clinic;

[0104] The EDTA anticoagulation blood is used to extract human genomic DNA by using DNA Blood Mini kit, and the genomic DNA concentration is diluted to 2 ng / μL after quantification by using ultraviolet spectrophotometer. In this embodiment, the upstream primer is labeled with fluorescein.

[0105] In the first primer group, the concentration of the primer pair with the name of 1p is 300 nM, the concentration of the primer pair with the name of 1q is 300 nM, the concentration of the primer pair with the name of 2p is 150 nM, the concentration of the primer pair with the name of 2q is 80 nM, the concentration of the primer pair with the name of 3p is 150 nM, the concentration of the primer pair with the name of 3q is 100 nM, the concentration of the primer pair with the name of 4p is 80 nM, the concentration of the primer pair with the name of 4q is 150 nM, the concentration of the primer pair with the name of 5p is 150 nM, the concentration of the primer pair with the name of 5q is 200 nM, the concentration of the primer pair with the name of 6q is 80 nM, the concentration of the primer pair with the name of 7p is 100 nM, the concentration of the primer pair with the name of 7q is 80 nM, the concentration of the primer pair with the name of 8p is 80 nM, the concentration of the primer pair with the name of 8q is 150 nM, the concentration of the primer pair with the name of 9p is 150 nM, the concentration of the primer pair with the name of 9q is 150 nM, the concentration of the primer pair with the name of 10q is 200 nM, the concentration of the primer pair with the name of 11p is 80 nM, the concentration of the primer pair with the name of 17p is 200 nM, and the concentration of the primer pair with the name of 19q is 80 nM.

[0106]

[0107] ​In the second primer group, the concentration of the primer pair with the name 11q is 200nM, the concentration of the primer pair with the name 12q is 800nM, the concentration of the primer pair with the name 13q is 300nM, the concentration of the primer pair with the name 14q is 800nM, the concentration of the primer pair with the name 16p is 600nM, the concentration of the primer pair with the name 16q is 300nM, the concentration of the primer pair with the name 17q is 600nM, the concentration of the primer pair with the name 18p is 400nM, the concentration of the primer pair with the name 18q is 300nM, the concentration of the primer pair with the name 19p is 800nM, the concentration of the primer pair with the name 20p is 400nM, the concentration of the primer pair with the name 20q is 200nM, the concentration of the primer pair with the name 22q is 800nM, and the concentration of the primer pair with the name Xq is 800nM;

[0108] In the third primer group, the concentration of the primer pair with the name 6p is 80nM, the concentration of the primer pair with the name 10p is 100nM, the concentration of the primer pair with the name 12p is 150nM, the concentration of the primer pair with the name 15q is 150nM, the concentration of the primer pair with the name 21q is 80nM, and the concentration of the primer pair with the name Xp is 150nM;

[0109] The number of cycles of the multiplex polymerase chain reaction is 28, the multiplex polymerase chain reaction uses a Veriti Dx PCR instrument, and 1μL of each group of multiplex polymerase chain reaction amplification products is subjected to capillary electrophoresis using a 50cm capillary and POP7 gel;

[0110] The LIZ fluorescently labeled molecular weight marker Gene Scan TM LIZ500 TM used in the foregoing equipment, consumables, and capillary electrophoresis are all products of Thermo Fisher Corporation of the United States;

[0111] In this embodiment, the fluorescence signal intensity is counted by peak height.

[0112] The results of the first multiplex polymerase chain reaction FAM channel are shown in FIG. 1, the results of the first multiplex polymerase chain reaction HEX channel are shown in FIG. 2, the results of the second multiplex polymerase chain reaction TAMRA channel are shown in FIG. 3, the results of the second multiplex polymerase chain reaction ROX channel are shown in FIG. 4, and the results of the third multiplex polymerase chain reaction HEX channel are shown in FIG. 5.

[0113] Taking the insertion and deletion polymorphic genetic markers 1p and 1q in FIG. 1 as an example:

[0114] The insertion-deletion polymorphism genetic marker 1p shows two fluorescence peaks, PHR = 6567 / 6330 ≈ 1.037 ∈ [0.80, 1.20], so the copy number of the subtelomere region of the human chromosome corresponding to the insertion-deletion polymorphism genetic marker 1p is 2 copies;

[0115] The insertion-deletion polymorphism genetic marker 1q shows a single fluorescence peak, R S = 9146 / (5740+5231+6567+6330+9146+8737+9455+7694+3777+3905+4847+4972+3118+3552+4865+4656+15710+11371) ≈ 0.076, and the results of the control sample are shown in FIGS. 6-10, RR ∈ [0.75, 1.25], so the copy number of the subtelomere region of the human chromosome corresponding to the insertion-deletion polymorphism genetic marker 1q is 2 copies;

[0116] In addition to the insertion-deletion polymorphism genetic marker Xq, the copy numbers of the subtelomere regions of the human chromosomes corresponding to the other insertion-deletion polymorphism genetic markers are all 2 copies;

[0117] The copy number of the subtelomere region of the human chromosome corresponding to the insertion-deletion polymorphism genetic marker Xq is 1 copy, which is consistent with the normal adult male having only one X chromosome;

[0118] It should be additionally noted that the insertion-deletion polymorphism genetic marker Xp is the homologous sequence of the Yp chromosome (pseudoautosomal region 1, PAR1), so the copy number of the subtelomere region of the human chromosome corresponding to the insertion-deletion polymorphism genetic marker Xp is 2 copies, which is consistent with the normal adult male having one Xp chromosome and one Yp chromosome.

[0119] Comparative Example

[0120] In the present comparative example, the sample to be tested is a miscarried fetus from a phenotypically normal adult female in the prenatal diagnosis clinic of the Third Affiliated Hospital of Zhengzhou University;

[0121] The karyotype of the peripheral blood of the adult female suggests 46, XX, t(9; 15) (q33; q26.3), i.e., balanced translocation of the distal long arm of chromosome 9 and the distal long arm of chromosome 15, and the karyotype of the peripheral blood of her partner is 46, XY, without abnormalities;

[0122] The CMA test of the miscarried fetus suggests: 9q33.2q34.3 (121,505,760-138,124,196) x3, i.e., the subtelomere region of the distal 9q should be 3 copies; 15q26.3 (100,492,012-101,888,837) x1, i.e., the subtelomere region of the distal 15q should be 1 copy;

[0123] The parameters in the present comparative example are the same as those in Example 1.

[0124] The first multiplex polymerase chain reaction FAM channel results are shown in Figure 11, the first multiplex polymerase chain reaction HEX channel results are shown in Figure 12, the second multiplex polymerase chain reaction TAMRA channel results are shown in Figure 13, the second multiplex polymerase chain reaction ROX channel results are shown in Figure 14, and the third multiplex polymerase chain reaction HEX channel results are shown in Figure 15.

[0125] Referring to Figure 12, the insertion-deletion polymorphic genetic marker 9q shows two fluorescence peaks, PHR = 5637 / 2791 ≈ 2.0197 ∈ [1.60, 2.60], so the copy number of the sub-telomere region of the human chromosome corresponding to the insertion-deletion polymorphic genetic marker 9q is 3 copies;

[0126] Referring to Figure 14, the insertion-deletion polymorphic genetic marker Xq shows a single fluorescence peak for the allele corresponding to the insertion-deletion polymorphic genetic marker Xq, R S = 1256 / (3033+2706+6149+4276+4113+8340+3226+3086+2987+3261+3757+3224+1256) ≈ 0.025, and the results of the control sample are still shown in Figures 6-10, RR ∈ (0, 0.7], so the copy number of the sub-telomere region of the human chromosome corresponding to the insertion-deletion polymorphic genetic marker Xq is 1 copy;

[0127] Referring to Figure 15, the insertion-deletion polymorphic genetic marker 15q shows a single fluorescence peak for the allele corresponding to the insertion-deletion polymorphic genetic marker 15q, R S = 7031 / (7151+7099+6016+6683+9184+7938+9366+7031) ≈ 0.0116, and the results of the control sample are still shown in Figures 6-10, RR ∈ (0, 0.7], so the copy number of the sub-telomere region of the human chromosome corresponding to the insertion-deletion polymorphic genetic marker 15q is 1 copy;

[0128] The copy numbers of the sub-telomere regions of the human chromosomes corresponding to the other insertion-deletion polymorphic genetic markers are all 2 copies.

[0129] Similarly, the copy number of the sub-telomere region of the human chromosome corresponding to the insertion-deletion polymorphic genetic marker Xq is 1 copy, and the copy number of the sub-telomere region of the human chromosome corresponding to the insertion-deletion polymorphic genetic marker Xp is 2 copies, which is consistent with the sex chromosome being XY shown by the sample.

[0130] Example 2

[0131] In the embodiment, the upstream primer in part of the primer pairs is labeled with fluorescein, and the downstream primer in the other primer pairs is labeled with fluorescein;

[0132] In the embodiment, the concentration of each primer in the first primer group is not less than 80nM and not more than 300nM, the concentration of each primer in the second primer group is not less than 200nM and not more than 500nM, and the concentration of each primer in the third primer group is not less than 80nM and not more than 150nM; the number of cycles of the first, second and third multiplex polymerase chain reaction amplification systems is not less than 26 and not more than 28;

[0133] Those skilled in the art can understand that, by obtaining the characterization level of the alleles of the insertion-deletion polymorphic genetic marker of the sample to be detected (and the control sample), the present application can determine the copy number of the sub-telomere region of the human chromosome corresponding to the insertion-deletion polymorphic genetic marker, and therefore the method for obtaining the aforementioned characterization level should not be regarded as a further limitation of the present application;

[0134] On this basis, as long as at least one primer in the primer pair is labeled with fluorescein, those skilled in the art can obtain the aforementioned characterization level by multiplex polymerase chain reaction and capillary electrophoresis; similarly, as long as the aforementioned characterization level is still within the detection range of the instrument and equipment, those skilled in the art can freely adjust the parameters of the multiplex polymerase chain reaction, such as the concentration of the primer, the number of cycles of the multiplex polymerase chain reaction, or the concentration of the DNA template, and the parameters of the capillary electrophoresis, such as the content of the multiplex polymerase chain reaction amplification product, etc.

[0135] Obviously, as long as the sample to be detected and the control sample use the same parameters, the adjustment of the aforementioned parameters will have a global effect on the aforementioned characterization level, and will not affect the final determination result;

[0136] Therefore, the adaptive adjustment of the aforementioned parameters by those skilled in the art to make the obtained characterization level within the detection range of the instrument and equipment, or the adjustment of the method for obtaining the aforementioned characterization level, should be regarded as a routine technical means in the art, and does not exceed the protection scope of the present application.

[0137] The detection scheme for the copy number of the sub-telomere region of the human chromosome provided by the present application uses insertion-deletion polymorphic genetic markers of the sub-telomere region, and when two alleles of a certain insertion-deletion polymorphic genetic marker are detected, the copy number of the human chromosome sub-telomere region corresponding to the insertion-deletion polymorphic genetic marker can be directly determined according to the peak height ratio of the alleles; obviously, the polymorphism or heterozygosity of the population of the insertion-deletion polymorphic genetic markers used is directly related to the convenience of the present application.

[0138] In the verification example, 362 adult individuals with normal phenotype were subjected to genotyping test, Hardy-Weinberg genetic equilibrium test was performed on 39 autosomal insertion-deletion polymorphic genetic markers by using GenAlEx 6.501 software package, and the heterozygosity of the population was obtained;

[0139] The 362 samples with normal phenotype were all the remaining samples of EDTA anticoagulated blood routine test in the obstetric clinic of the Third Affiliated Hospital of Zhengzhou University, and the human genomic DNA was extracted from the EDTA anticoagulated blood, which was the same as in Example 1.

[0140] The observed heterozygosity (Ho), the expected heterozygosity (He), the minimum allele frequency (MAF (East Asia)) of East Asian population in the dbSNP database, and the expected heterozygosity (He (East Asia) or He-east) calculated according to the MAF of the 39 autosomal insertion-deletion polymorphic genetic markers in the 362 samples are shown in the following table:

[0141] According to the average heterozygosity of the 39 sites as the overall rate and using the binomial distribution probability formula, the corresponding probability distribution of the test of any one normal sample with i (I=0, 1, 2, …, 39) markers being 1:1 heterozygous was calculated, and curve fitting was performed by using GraphPad Prism software, and the results are shown in Figure 16; according to the basic properties of binomial distribution, the probability distribution of detecting 1:1 heterozygous genetic markers obtained by taking the average observed heterozygosity (Ho), the average expected heterozygosity (He), and the average expected heterozygosity (He-east) calculated according to the MAF as the overall rate, can be approximately normally distributed, and the normal distribution curves of the three fitted by GraphPad Prism 5 are basically overlapped;

[0142] The detection scheme of human chromosome subtelomere region copy number provided by the application, among the 39 autosomal insertion-deletion polymorphic genetic markers in the subtelomere region, more than 95% of normal individuals will have 12 to 24 showing bimodal type, and these bimodal insertion-deletion polymorphic genetic markers can directly judge the copy number of the corresponding subtelomere region.

[0143] In summary, the application provides a set of efficient, low-cost, simple and accurate human chromosome subtelomere region copy number detection scheme, which is expected to have broad application prospects in clinical diagnosis and genetic disease research, specifically,

[0144] Compared with high-throughput technologies such as gene chips, second-generation sequencing, long-read third-generation sequencing or optical genome mapping, the application of insertion-deletion polymorphism genetic markers in detecting the copy number of the subtelomere region of human chromosomes has a significantly reduced cost and is more suitable for large-scale screening and clinical application;

[0145] The operation process of the method for detecting the copy number of the subtelomere region of human chromosomes is simplified, and complex biological information analysis is not required, so that an experimental personnel can master the method after basic training, thereby greatly reducing the operation threshold and required professional skill level and being conducive to popularization and use in a laboratory environment with limited resources;

[0146] The method for detecting the copy number of the subtelomere region of human chromosomes can complete the whole process from sample processing to data analysis within 4 hours, thereby significantly improving the detection efficiency and being suitable for a clinical diagnosis scenario requiring rapid results;

[0147] Through peak type analysis of the insertion-deletion polymorphism genetic markers, the copy number of the target region can be accurately judged, and the judgment of 2 copies or 3 copies is particularly convenient.

[0148] The above merely describes preferred embodiments of the present application, and does not limit the implementation manners and protection scope of the present application; for those skilled in the art, it should be realized that equivalent replacements and obvious changes made by applying the contents of the present application and drawings should be included in the protection scope of the present application.

Claims

1. Use of an insertion-deletion polymorphic genetic marker in detecting copy number in the subtelomeric region of human chromosomes, characterized in that, Based on human genome reference sequence version 38, the physical location of the insertion-deletion polymorphic genetic marker is selected from at least one of: chr1 : 1014734-1014737, chr1 : 248344885-248344884, chr2: 1853465-1853469, chr2: 241981638-241981643, chr3: 1469652-1469655, chr3: 197081691-197081695, chr4: 1540277-1540279, chr4: 188679385-188679384, chr5: 438737-438739, chr5: 179927343-179927345, chr6: 529405-529413, chr6: 170034015-170034018, chr7: 526246-526250, chr7: 158928777-158928784, chr8: 2001041-2001050, chr8: 143782455-143782461, chr9: 998499-998503, chr9: 137403593-137403597, chr10: 1300362-1300376, chr10: 133184021-133184021, chr11 : 209895-209900, chr11 : 134161942-134161942, chr12: 272923-272927, chr12: 132600305-132600308, chr13: 113406671-113406673, chr14: 106560681-106560686, chr15: 100876312-100876317, chr16: 2009173-2009175, chr16: 89463813-89463815, chr17: 733027-733040, chr17: 82720753-82720755, chr18: 1375182-1375185, chr18: 79914568-79914571, chr19: 294745-294749, chr19: 58358150-58358157, chr20: 1388748-1388754, chr20: 63792424-63792428, chr21 : 46055283-46055288, chr22: 50557738-50557740, chrX: 446148-446161, or chrX: 155332644-155332652.

2. A reagent set for detecting copy number of sub-telomere region of human chromosome, characterized by, comprises: A primer pair for polymerase chain reaction amplification of an insertion-deletion polymorphic genetic marker; wherein, Based on human genome reference sequence version 38, the physical location of the insertion-deletion polymorphic genetic marker is selected from at least one of: chr1 : 1014734-1014737, chr1 :248344885-248344884, chr2: 1853465-1853469, chr2:241981638-241981643, chr3: 1469652-1469655, chr3: 197081691-197081695, chr4: 1540277-1540279, chr4: 188679385-188679384, chr5: 438737-438739, chr5: 179927343-179927345, chr6: 529405-529413, chr6: 170034015-170034018, chr7: 526246-526250, chr7: 158928777-158928784, chr8: 2001041-2001050, chr8: 143782455-143782461, chr9: 998499-998503, chr9: 137403593-137403597, chr10: 1300362-1300376, chr10: 133184021-133184021, chr11 : 209895-209900, chr11 : 134161942-134161942, chr12: 272923-272927, chr12: 132600305-132600308, chr13: 113406671-113406673, chr14: 106560681-106560686, chr15: 100876312-100876317, chr16: 2009173-2009175, chr16: 89463813-89463815, chr17: 733027-733040, chr17: 82720753-82720755, chr18: 1375182-1375185, chr18: 79914568-79914571, chr19: 294745-294749, chr19: 58358150-58358157, chr20: 1388748-1388754, chr20: 63792424-63792428, chr21 : 46055283-46055288, chr22: 50557738-50557740, chrX: 446148-446161, or chrX: 155332644-155332652.

3. The reagent set according to claim 2, characterized by The primer pair comprises an upstream primer and a downstream primer corresponding to the upstream primer; wherein The upstream primer is selected from at least one of the nucleotide sequences shown in SEQ ID NO: 1-41; The upstream primer is selected from at least one of the nucleotide sequences shown in SEQ ID NO: 42-82.

4. The reagent set according to claim 2, characterized by The primer pair comprises an upstream primer and a downstream primer corresponding to the upstream primer; wherein The upstream primer or / and the downstream primer is labeled with a fluorescent substance; The fluorescent substance is selected from at least one of FAM, HEX, TAMRA, or ROX.

5. The reagent set according to claim 2, characterized by Further comprising: Polymerase chain reaction amplification buffer, hot-start deoxyribonucleic acid polymerase, and double deionized water.

6. A method of detecting copy number of subtelomeric regions of human chromosomes, the method comprising the steps of Comprising: Step one, obtaining the characterization of the alleles corresponding to the multiple insertion-deletion polymorphism genetic markers of the sample to be tested;Among them, based on the physical position of human genome reference sequence version 38, the plurality of insertion-deletion polymorphic genetic markers are selected from: chr1:1014734-1014737, chr1:248344885-248344884, chr2:1853465-1853469, chr2:241981638-241981643, chr3:1469652-1469655, chr3:197081691-197081695, chr4:1540277-1540279, chr4:188679385-188679384, chr5:438737-438739, chr5:179927343-179927345, chr6:529405-529413, chr6:170034015-170034018, chr7:526246-526250, chr7:158928777-158928784, chr8:2001041-2001050, chr8:143782455-143782461, chr9:998499-998503, chr9:137403593-137403597, chr10:1300362-1300376, chr10:133184021-133184021, chr11:209895-209900, chr11:134161942-134161942, chr12:272923-272927, chr12:132600305-132600308, chr13:113406671-113406673, chr14:106560681-106560686, chr15:100876312-100876317, chr16:2009173-2009175, chr16:89463813-89463815, chr17:733027-733040, chr17:82720753-82720755, chr18:1375182-1375185, chr18:79914568-79914571, chr19:294745-294749, chr19:58358150-58358157, chr20:1388748-1388754, chr20:63792424-63792428, chr21:46055283-46055288, chr22:50557738-50557740, chrX:446148-446161, or chrX:155332644-155332652. Step two, judging the characterization of the allele corresponding to the insertion-deletion polymorphic genetic marker of the sample to be tested: If the allele corresponding to the insertion-deletion polymorphic genetic marker is not characterized, the copy number of the human chromosome sub-telomere region corresponding to the insertion-deletion polymorphic genetic marker is the fourth preset copy value, and the process ends; If the allele corresponding to the insertion-deletion polymorphic genetic marker is characterized as a single allele, proceed to step four; If the allele corresponding to the insertion-deletion polymorphic genetic marker is characterized as two alleles, proceed to step three; Step three, judging whether the ratio of the characterization levels of the two alleles corresponding to the insertion-deletion polymorphic genetic marker is in a first preset interval: If the ratio of the characterization levels of the two alleles corresponding to the insertion-deletion polymorphic genetic marker is in the first preset interval, the copy number of the human chromosome sub-telomere region corresponding to the insertion-deletion polymorphic genetic marker is obtained according to the ratio of the characterization levels of the two alleles corresponding to the insertion-deletion polymorphic genetic marker, and the process ends; If the ratio of the characterization levels of the two alleles corresponding to the insertion-deletion polymorphic genetic marker is not in the first preset interval, proceed to step four; Step four, obtaining the copy number of the human chromosome sub-telomere region corresponding to the insertion-deletion polymorphic genetic marker according to the proportion of the characterization of the allele corresponding to the insertion-deletion polymorphic genetic marker among all alleles corresponding to insertion-deletion polymorphic genetic markers, and the process ends.

7. The method of claim 6, wherein, In step one, the method for obtaining the characterization of the allele corresponding to the insertion-deletion polymorphic genetic marker of the sample to be tested comprises: After the insertion-deletion polymorphic genetic marker is amplified by polymerase chain reaction using a primer pair labeled with a fluorescent substance, the characterization of the allele corresponding to the insertion-deletion polymorphic genetic marker of the sample to be tested is obtained according to the fluorescence intensity of the polymerase chain reaction amplification product.

8. The method of claim 6, wherein, In step three, the first preset interval comprises a first sub-preset interval and a second sub-preset interval; When the ratio of the characterization levels of the two alleles corresponding to the insertion-deletion polymorphic genetic marker is in the first sub-preset interval or in the second sub-preset interval, the ratio of the characterization levels of the two alleles corresponding to the insertion-deletion polymorphic genetic marker is in the first preset interval, and If the ratio of the representation levels of the two alleles corresponding to the insertion-deletion polymorphism genetic marker is in the first sub-pre-set interval, the copy number of the human chromosome sub-telomere region corresponding to the insertion-deletion polymorphism genetic marker is the second pre-set copy value, and the process ends. If the ratio of the representation levels of the two alleles corresponding to the insertion-deletion polymorphism genetic marker is in the second sub-pre-set interval, the copy number of the human chromosome sub-telomere region corresponding to the insertion-deletion polymorphism genetic marker is the third pre-set copy value, and the process ends. When the ratio of the representation levels of the two alleles corresponding to the insertion-deletion polymorphism genetic marker is not in the first sub-pre-set interval and not in the second sub-pre-set interval, the ratio of the representation levels of the two alleles corresponding to the insertion-deletion polymorphism genetic marker is not in the first pre-set interval.

9. The method of claim 6, wherein, The step four comprises: The representation proportion of the allele corresponding to the insertion-deletion polymorphism genetic marker in all alleles corresponding to the insertion-deletion polymorphism genetic markers is recorded as a first ratio. The representation of the alleles corresponding to the corresponding multiple insertion-deletion polymorphism genetic markers of the control sample is obtained, and the representation proportion of the allele corresponding to the insertion-deletion polymorphism genetic marker of the control sample in all alleles corresponding to the insertion-deletion polymorphism genetic markers of the control sample is recorded as a second ratio. It is judged whether the ratio of the first ratio and the second ratio is in a second pre-set interval, and the second pre-set interval comprises a third sub-pre-set interval, a fourth sub-pre-set interval, and a fifth sub-pre-set interval. When the ratio of the representation levels of the two alleles corresponding to the insertion-deletion polymorphism genetic marker is in the third sub-pre-set interval or in the fourth sub-pre-set interval or in the fifth sub-pre-set interval, the ratio of the first ratio and the second ratio is in the second pre-set interval, and If the ratio of the first ratio and the second ratio is in the third sub-pre-set interval, the copy number of the human chromosome sub-telomere region corresponding to the insertion-deletion polymorphism genetic marker is the first pre-set copy value, and the process ends. If the ratio of the first ratio and the second ratio is in the fourth sub-pre-set interval, the copy number of the human chromosome sub-telomere region corresponding to the insertion-deletion polymorphism genetic marker is the second pre-set copy value, and the process ends. If the ratio of the first ratio and the second ratio is in the fifth sub-pre-set interval, the copy number of the human chromosome sub-telomere region corresponding to the insertion-deletion polymorphism genetic marker is the third pre-set copy value, and the process ends. If the ratio of the first ratio and the second ratio is not in the third sub-pre-set interval, not in the fourth sub-pre-set interval, and not in the fifth sub-pre-set interval, the ratio of the first ratio and the second ratio is not in the second pre-set interval, and the copy number of the human chromosome sub-telomere region corresponding to the insertion-deletion polymorphism genetic marker is a gray value, and the process ends.

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