CN117904276A - Probe composition, kit and method for detecting Y chromosome microdeletion - Google Patents

Abstract

The application provides a probe composition, a kit and a method for detecting Y chromosome microdeletion. The probe composition comprises at least 26 probes for detecting AZF regions of the Y chromosome. According to the application, a shingled design mode is adopted, the probe density is 3X, probes are respectively designed aiming at SNP loci of AZFa, AZFb and AZFc regions of Y chromosomes, and each SNP locus is separated by 2kb, so that the designed probe composition has good coverage on a Y chromosome microdeletion region, and the accuracy and sensitivity of a detection result are improved. The probe composition designed by the application can be used for detecting the microdeletion condition of each region of AZF on Y chromosome, and has the advantages of high efficiency, high speed, high sensitivity, high accuracy and the like.

Description

Probe composition, kit and method for detecting Y chromosome microdeletion

Technical Field

The application relates to the technical field of molecules, in particular to a probe composition, a kit and a method for detecting Y chromosome microdeletion.

Background

Infertility is a public health problem which afflicts human beings for a long time, and has great influence on social, psychological and economic aspects. According to epidemiological investigation results, the infertility ratio of the couples in the childbearing age period is about 10% -15%, wherein male factors account for 30% -50%. Among the causes of infertility, male insemination disorders are an important factor, where oligospermia, oligospermia are the extrinsic manifestations of Y-chromosome infertility.

Studies have shown that Y chromosome microdeletion (Y-chromosome microdeletions, YCMs) is the second largest genetic factor in male sterility following the korotkoff syndrome. The proximal centromere of the Y chromosome is divided into two parts, namely a long arm (Yq) and a short arm (Yp), wherein a zone 1 (Yq 11) of the long arm 1 contains azoospermia factors (Azoospermia factors, AZF), the region deletion is a main factor causing male spermatogenesis disorder, and the deletion of the gene fragment is called YCMs. Studies prove that 10% -15% of azoospermia and 5% -10% of severe oligospermia patients show YCMs, and the genetic defect is directly transmitted to offspring, so that the screening of Y chromosome microdeletion before assisted reproduction treatment is of great significance.

The AZF region is divided into three non-overlapping sub-segments, AZFa (proximal), AZFb (middle) and AZFc (distal), each of which controls a different stage of spermatogenesis correspondingly. The occurrence rate of microdeletion of AZFa region is relatively low and is about 1% -5% of YCMs%, but the symptoms are most serious after AZFa is deleted, partial deletion is related to spermatogenesis, and complete deletion can block the generation and development of germ cells. The occurrence rate of AZFb deletion is 16% -30%, and the AZFb deletion is mainly expressed as seminal arrest, and spermatogenesis is arrested at the spermatocyte stage. AZFc region deletions are most common, accounting for about 60% of total YCMs, and can result in azoospermia or severe oligospermia. Partial deletions of the AZFc region mainly comprise gr/gr deletion, b2/b3 deletion and the like, and have the highest gr/gr deletion rate and higher occurrence rate in Asia for b2/b3 times; the gr/gr deficiency can lead to the reduction of the number of sperms, the b2/b3 deficiency is related to the spermatogenesis disorder, and the research in China shows that the gr/gr deficiency and the b2/b3 deficiency are related to male infertility.

In clinical practice, YCMs testing plays a critical role in assessing male infertility and sub-fertility. Currently, common YCMs detection methods mainly comprise a CNV-Seq, a multiplex PCR-agarose gel electrophoresis method, an MLPA-capillary electrophoresis method, a real-time fluorescent quantitative PCR method, a multiplex PCR-gene chip method, a fluorescent in situ hybridization technology and the like. The CNV-seq and multiplex PCR are the most commonly used methods, however, due to the limitations of the CNV-seq routine analysis procedure, special design cannot be performed for the AZF region to improve the detection accuracy, so that there may be a risk of false positive or false negative; the primer design of multiplex PCR is complex, the resolution of agarose gel electrophoresis is low, and false positive or false negative results are easy to generate.

Thus, conventional methods for detecting Y chromosome microdeletion are in need of improvement.

Disclosure of Invention

Based on the above, one or more embodiments of the present application provide a probe composition, a kit and a method for detecting Y chromosome microdeletion with high detection speed and high accuracy. The technical proposal comprises:

According to a first aspect of embodiments of the present application there is provided a probe composition for detecting Y chromosome microdeletion, the genomic version of reference being GRCh38, the probe composition comprising probes ：chrY:12318588～12318688、chrY:12330588～12330688、chrY:12354588～12354688、chrY:12640588～12640688、chrY:12940588～12940688、chrY:13196588～13196688、chrY:17807879～17807979、chrY:17857458～17857558、chrY:18029249～18029349、chrY:18320052～18320152、chrY:18752440～18752540、chrY:19047939～19048039、chrY:19987849～19987949、chrY:20503375～20503475、chrY:20896316～20896416、chrY:21197814～21197914、chrY:21840243～21840343、chrY:22356551～22356651、chrY:22746063～22746163、chrY:23104533～23104633、chrY:23824142～23824242、chrY:24670728～24670828、chrY:25043147～25043247、chrY:25769311～25769411、chrY:25826318～25826418 and chrY:26671322 ～ 26671422 for detecting the following regions.

In one embodiment, the probe composition includes probes having nucleotide sequences shown in SEQ ID NO. 1-SEQ ID NO. 26.

According to a second aspect of embodiments of the present application, there is provided a kit for detecting Y chromosome microdeletion, comprising the probe composition described above.

In one embodiment, the kit further comprises hybridization reaction reagents.

In one embodiment, the hybridization reaction reagent comprises one or more of magnetic beads, a blocking solution, and a hybridization buffer.

In one embodiment, the kit further comprises a DNA extraction reagent.

According to a third aspect of embodiments of the present application, there is provided the use of a probe composition as described above for the preparation of a product for detecting microdeletion of the Y chromosome.

According to a fourth aspect of embodiments of the present application, there is provided a method of detecting Y chromosome microdeletion, comprising the steps of:

the probe composition or the kit is adopted to carry out hybridization capturing on a sample to be detected; and

And sequencing the hybridization captured product, and determining the Y chromosome microdeletion condition in the sample to be tested according to the sequencing result.

In one embodiment, the step of performing hybrid capture on the sample to be tested includes:

constructing a whole genome sequencing library by taking genomic DNA of a sample to be detected as a template; and

The whole genome sequencing library is subjected to hybridization capture by using the probe composition or the kit.

In one embodiment, the method of sequencing comprises high throughput sequencing.

Compared with the prior art, the application has the following beneficial effects:

The application adopts a shingled design mode, the probe density is 3X, and compared with 2X probe tiling, the probe density of the design mode is higher, the coverage of a target area is higher, and the target is not easy to miss in the capturing process. According to the application, probes are respectively designed aiming at SNP loci of AZFa, AZFb and AZFc regions of Y chromosomes, and each SNP locus is separated by 2kb, so that the designed probe composition has good coverage on a Y chromosome microdeletion region, and the accuracy and sensitivity of a detection result are improved. The probe composition designed by the application can be used for detecting the microdeletion condition of each region of AZF on Y chromosome, and has the advantages of high efficiency, high speed, high sensitivity, high accuracy and the like.

Drawings

FIG. 1 is a schematic diagram of the probe coverage sites of AZFa region in example 1 of the present application;

FIG. 2 is a schematic diagram of the probe coverage sites of AZFb and AZFc regions in example 1 of the present application;

FIG. 3 is a schematic flow chart of the detection of Y chromosome microdeletion in example 1 of the present application;

FIG. 4 is a scatter diagram of the detection result of sample P01 in example 1 of the present application;

FIG. 5 is a scatter diagram showing the detection result of the sample P02 in example 1 of the present application;

FIG. 6 is a scatter diagram of the detection result of sample P03 in example 1 of the present application;

FIG. 7 is a scatter diagram of the detection result of sample P04 in example 1 of the present application;

FIG. 8 is a scatter diagram of the detection result of sample P05 in example 1 of the present application;

FIG. 9 is a scatter diagram of the detection result of the sample P06 in example 1 of the present application;

FIG. 10 is a scatter diagram showing the detection result of sample P07 in example 1 of the present application;

FIG. 11 is a scatter diagram of the detection result of sample P08 in example 1 of the present application;

FIG. 12 is a scattergram of the detection result of sample P09 in example 1 of the present application;

FIG. 13 is a scatter diagram showing the detection result of the sample P10 in example 1 of the present application;

FIG. 14 is a scattergram of the detection result of sample P11 in example 1 of the present application;

FIG. 15 is a scattergram of the detection result of sample P12 in example 1 of the present application;

FIG. 16 is a scattergram of the detection result of sample P13 in example 1 of the present application;

FIG. 17 is a scattergram of the detection result of sample P14 in example 1 of the present application;

FIG. 18 is a scattergram of the detection result of sample P15 in example 1 of the present application;

FIG. 19 is a scattergram of the detection result of sample P16 in example 1 of the present application;

FIG. 20 is a scattergram of the detection result of sample P17 in example 1 of the present application;

FIG. 21 is a scattergram of the detection result of sample P18 in example 1 of the present application;

FIG. 22 is a scatter diagram showing the detection result of sample P19 in example 1 of the present application;

FIG. 23 is a scatter diagram showing the detection result of the sample P20 in example 1 of the present application;

FIG. 24 is a scattergram of the CNV-seq detection result of sample P01 in example 1 of the present application;

FIG. 25 is a scattergram of the CNV-seq detection result of sample P02 in example 1 of the present application;

FIG. 26 is a scattergram of the CNV-seq detection result of sample P03 in example 1 of the present application;

FIG. 27 is a scattergram of the CNV-seq detection result of sample P04 in example 1 of the present application;

FIG. 28 is a scattergram of the CNV-seq detection result of sample P05 in example 1 of the present application;

FIG. 29 is a scattergram of the CNV-seq detection result of sample P06 in example 1 of the present application;

FIG. 30 is a scattergram of the result of CNV-seq detection of sample P07 in example 1 of the present application;

FIG. 31 is a scattergram of the CNV-seq detection result of sample P08 in example 1 of the present application;

FIG. 32 is a scattergram of the CNV-seq detection result of sample P09 in example 1 of the present application;

FIG. 33 is a scattergram of the CNV-seq detection result of sample P10 in example 1 of the present application;

FIG. 34 is a scattergram of the CNV-seq detection result of sample P11 in example 1 of the present application;

FIG. 35 is a scattergram of the CNV-seq detection result of sample P12 in example 1 of the present application;

FIG. 36 is a scattergram of the CNV-seq detection result of sample P13 in example 1 of the present application;

FIG. 37 is a scattergram of the CNV-seq detection result of sample P14 in example 1 of the present application;

FIG. 38 is a scattergram of the CNV-seq detection result of sample P15 in example 1 of the present application;

FIG. 39 is a scattergram of the CNV-seq detection result of sample P16 in example 1 of the present application;

FIG. 40 is a scattergram of the CNV-seq detection result of sample P17 in example 1 of the present application;

FIG. 41 is a scattergram of the CNV-seq detection result of sample P18 in example 1 of the present application;

FIG. 42 is a scattergram of the CNV-seq detection result of sample P19 in example 1 of the present application;

FIG. 43 is a scattergram of the result of CNV-seq detection of sample P20 in example 1 of the present application.

Detailed Description

The detailed description of the present application will be provided to make the above objects, features and advantages of the present application more obvious and understandable. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. The present application may be embodied in many other forms than described herein and similarly modified by those skilled in the art without departing from the spirit of the application, whereby the application is not limited to the specific embodiments disclosed below.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the description of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present application are commercially available or may be prepared by existing methods.

In the present application, "optional" means optional or not, that is, means any one selected from two parallel schemes of "with" or "without". If multiple "alternatives" occur in a technical solution, if no particular description exists and there is no contradiction or mutual constraint, then each "alternative" is independent.

In the application, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.

In the present application, a numerical range (i.e., a numerical range) is referred to, and optional numerical distributions are considered to be continuous within the numerical range and include two numerical endpoints (i.e., a minimum value and a maximum value) of the numerical range and each numerical value between the two numerical endpoints unless otherwise specified. Where a numerical range merely refers to integers within the numerical range, including both end integers of the numerical range, and each integer between the two ends, unless otherwise indicated, each integer is recited herein as directly, such as where t is an integer selected from 1-10, and where t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Further, when a plurality of range description features or characteristics are provided, these ranges may be combined. In other words, unless otherwise indicated, the ranges disclosed herein are to be understood to include any and all subranges subsumed therein.

CNV-seq is a low-depth whole genome sequencing of DNA samples based on next-generation sequencing technology, comparing the sequencing result with human reference genome base sequences, analyzing whether the sample to be detected has copy number variation of the AZF region by bioinformatics, however, since the CNV-seq conventional analysis flow has limitation on analysis of highly repetitive regions, there may be a risk of false positive or false negative.

The multiplex PCR-agarose gel electrophoresis technology simultaneously amplifies a plurality of targets through one PCR reaction, combines agarose gel electrophoresis, and makes qualitative analysis on the absence or absence of each region of AZF on a Y chromosome according to a band. However, the method has complex primer design; the amplification of each target fragment may interfere with each other, easily causing false positive results; agarose gel electrophoresis has low separation rate and can not distinguish STS sites with relatively close molecular mass; the flux is low, and the detection of a large amount of samples is not facilitated.

The MLPA-capillary electrophoresis technology has more detectable sites and better accuracy and sensitivity, but has high requirements on DNA samples, the MLPA process is easy to be polluted by impurities, the requirement on operators is high, and compared with other detection methods, the MLPA-capillary electrophoresis technology has more time-consuming and more complex analysis methods.

The real-time fluorescent quantitative PCR technology has higher sensitivity, simple and convenient operation and reliable result. However, false positive results may be produced when the fluorescent dye non-specifically binds to double-stranded DNA; the fluorescent probe method needs to design different probes according to different sequences, and has high cost; the single tube reaction has few detectable sites and low detection flux; multi-site detection tends to increase cross contamination rates, while also increasing workload and analysis difficulty.

The multiplex PCR-gene chip technology has higher sensitivity and specificity, compared with other detection methods, has higher flux, but has obvious defects, has the defects of the multiplex PCR technology, has higher design requirement on the gene chip, needs to design a large number of probes, has higher cost, and has more complicated whole detection and analysis.

Compared with the traditional radiolabeled in situ hybridization, the fluorescence in situ hybridization technology has the advantages of rapid operation, strong detection signals and the like, but compared with the methods such as multiplex PCR, Q-PCR, a chip method and the like, the fluorescence in situ hybridization technology has the advantages of longer detection period, complicated steps, low detection sensitivity and specificity and higher requirements on the interpretation of results of operators.

Based on this, in a first aspect of the present application, there is provided a probe composition for detecting Y chromosome microdeletion, the reference genome version being GRCh38, the probe composition comprising probes ：chrY:12318588～12318688、chrY:12330588～12330688、chrY:12354588～12354688、chrY:12640588～12640688、chrY:12940588～12940688、chrY:13196588～13196688、chrY:17807879～17807979、chrY:17857458～17857558、chrY:18029249～18029349、chrY:18320052～18320152、chrY:18752440～18752540、chrY:19047939～19048039、chrY:19987849～19987949、chrY:20503375～20503475、chrY:20896316～20896416、chrY:21197814～21197914、chrY:21840243～21840343、chrY:22356551～22356651、chrY:22746063～22746163、chrY:23104533～23104633、chrY:23824142～23824242、chrY:24670728～24670828、chrY:25043147～25043247、chrY:25769311～25769411、chrY:25826318～25826418 and chrY:26671322 ～ 26671422 for detecting the following regions.

In some embodiments, the probe composition comprises a probe having a nucleotide sequence as shown in SEQ ID NO. 1-SEQ ID NO. 26.

Specifically, the nucleotide sequences shown in SEQ ID NO. 1 to SEQ ID NO. 26 are shown in Table 1.

The application is based on a shingled design mode, the probe density is 3X, and one SNP locus is selected every 2k for the AZFa, AZFb and AZFc regions of a Y chromosome to carry out probe design. For the AZFa region, the probe design range was chrY:12316638 ～ 13122862, and a total of about 350 sites were selected. For AZFb and AZFc regions, the probe design range was chrY:17818205 ～ 26668849, and a total of about 2500 sites were selected.

Based on the interval arrangement mode and the arrangement density, the coverage of the probe composition to the AZF region of the Y chromosome can be improved, and meanwhile, the cost of the probe can be saved, and the probe is not influenced by polymorphic SNP loci.

In a second aspect of the application, there is provided a kit for detecting Y chromosome microdeletion comprising the above probe composition.

In some embodiments, the kit further comprises a hybridization reaction reagent.

In some embodiments, the hybridization reagents described above include one or more of magnetic beads, blocking solutions, RNA inhibitors, and hybridization buffers.

In one example, the magnetic beads are streptavidin magnetic beads. Alternatively, the magnetic beads are Dynabeads streptavidin.

In some embodiments, the blocking fluid comprises Eco Universal Blocking Oligo and RNase block. Alternatively, eco Universal Blocking Oligo and RNase Block are both purchased from Ai Jitai Biotechnology Inc.

In some embodiments, the RNA inhibitor comprises RNase Block.

In some embodiments, the hybridization reagents described above further comprise placental DNA. Alternatively, the placental DNA is Human Cot-1 DNA. Further alternatively, the Human Cot-1 DNA is purchased from Thermo Fisher.

In some embodiments, the kit further comprises a DNA extraction reagent.

In a third aspect of the application, there is provided the use of a probe composition as described above or a kit as described above in the preparation of a product for detecting microdeletion of the Y chromosome.

In a fourth aspect of the present application, there is provided a method for detecting Y chromosome microdeletion, comprising steps S10 to S20.

Step S10: the probe composition or the kit is adopted to carry out hybridization capturing on the sample to be detected.

In some of these embodiments, step S10 includes steps S11 to S12.

Step S11: and constructing a whole genome sequencing library by taking genomic DNA of a sample to be detected as a template.

In some embodiments, in step S11, the step of constructing a whole genome sequencing library comprises: genomic DNA fragmentation, end repair plus a tail, adaptor ligation and PCR amplification.

In some embodiments, the sample to be tested is a blood sample.

Step S12: the whole genome sequencing library is subjected to hybridization capture by using the probe composition or the kit.

In some embodiments, in step S12, the hybridization capture reaction system comprises 180ng to 220ng of whole genome sequencing library, 13 μL to 15 μL hybridization buffer, 2 μL to 5 μ L Eco Universal Blocking Oligo, 2 μL to 5 μL human cot-1 DNA ^TM, 2 μL to 5 μL RNase block, and 0.5 μL to 2.5 μL probe composition.

In step S12, the hybridization captured reaction system included 200ng of whole genome sequencing library, 15. Mu.L of hybridization buffer, 3. Mu. L Eco Universal Blocking Oligo, 3. Mu.L of human cot-1 DNA ^TM, 3. Mu.L of RNase Block, and 0.5. Mu.L of probe composition.

In some embodiments, in step S12, the reaction conditions for hybridization capture include: incubation is carried out at 80 ℃ for 5min, and then incubation is carried out at 50 ℃ for 12-18 h.

Step S20: sequencing the hybridization captured product, and determining the Y chromosome microdeletion condition in the sample to be tested according to the sequencing result.

In some of these embodiments, in step S20, the method of sequencing comprises high throughput sequencing.

In some embodiments, high throughput sequencing includes, but is not limited to, the following: hua Dazhi MGI sequencing, hua Dazhi BGI sequencing and Illumina sequencing. Understandably, when different sequencing platforms are selected for sequencing analysis, the linker sequence of the linker ligation in step S11 is different.

Further, depending on the sequencing platform, the library may be processed accordingly. If the Huada platform requires that the library on the machine is nanosphere (DNB), the prepared target gene library needs to be denatured into single-stranded DNA, the single-stranded DNA is connected into single-stranded annular DNA through a connection reaction, the single-stranded annular DNA is prepared into nanosphere library through rolling circle replication, and finally sequencing is carried out on the Huada platform.

The application relates to a method for detecting Y chromosome microdeletion, which is based on liquid phase probe hybridization capture sequencing to segment human whole genome DNA, repair tail end and A tail, connect and PCR amplification to obtain whole genome library, then the probe composition is used to hybridize a target region, and the target gene library is obtained through capture, cleaning and PCR amplification. And after the quality control of the library is qualified, selecting a second generation platform for sequencing according to the type of the joint, wherein the average depth of sequencing is more than or equal to 200×, and finally analyzing sequencing data through a letter generation algorithm. The method can integrally and efficiently detect the whole or part of the deficiency of the AZF region on the Y chromosome, can be used for detecting male infertility caused by the deficiency of the Y chromosome region, and quickly determines the genetics cause of the patient, thereby providing feasible clinical advice for the patient. Furthermore, the method of the application may be used for diagnostic or non-diagnostic purposes. Non-diagnostic purposes include use in genetic research, ethnic research, human chemical industry, and the like.

The method for detecting Y chromosome microdeletion has at least the following advantages:

(1) The method has the advantages of high efficiency and high flux, can detect tens or even hundreds of samples at a time, is suitable for batch detection, and can also identify unknown new variation.

(2) The detection accuracy and the sensitivity are high, and the false positive rate can be effectively reduced.

(3) Simple and efficient, high automation degree and greatly shortened detection time.

The present application will be further described with reference to specific examples and comparative examples, which should not be construed as limiting the scope of the application. The materials used in the following examples were all commercially available, unless otherwise specified, the equipment used, and the processes involved, unless otherwise specified, were all routinely selected by those skilled in the art.

Example 1:

(1) Probe design

Adopting a shingled design mode, wherein the probe density is 3X, the reference genome version is GRCh38, and RNA probes are designed for AZFa, AZFb and AZFc regions of a Y chromosome; the AZFa region probe coverage is shown in FIG. 1, and the AZFb+AZFc region probe coverage is shown in FIG. 2; the sequence information of a part of the probe is shown in Table 1.

The length of each probe is about 100bp, and the probe sequence can be complementary and paired with the base of the target region, so that the partial or complete deletion of the AZF region of the Y chromosome can be detected integrally, efficiently and comprehensively, and the detection accuracy is improved.

TABLE 1

(2) And (3) detecting 20 samples to be detected (comprising YCMs positive samples and YCMs negative samples) by using the probes designed in the step (1) based on a liquid-phase probe hybridization capture technology and a second-generation sequencing technology. Fig. 3 is a schematic diagram of the detection principle, and the specific steps are as follows.

A) Extraction of genomic DNA and construction of Whole genome library

The peripheral blood genomic DNA of 20 samples to be examined was extracted using a blood genomic DNA extraction kit (Tiangen), and the concentration of the extracted DNA was detected.

Genome pre-library construction is carried out by KAPA Hyperplus kit (Roche), genome DNA fragmentation, end repair and A tail addition, linker connection (double-Barcode linker is formed by annealing two sequences, the linker sequences are shown as SEQ ID NO:27 and SEQ ID NO: 28) and PCR amplification (the PCR amplification primers are shown as SEQ ID NO:29 and SEQ ID NO: 30) are carried out according to the method described in the kit specification, and a whole genome library is obtained. And the library was quantified using the Qubit ^TM dsDNA quantification kit (Thermo Fisher).

SEQ ID NO:27

5’-Phos/AGTCGGAGGCCAAGCGGTCTTAGGAAGACAATCAG-3’

SEQ ID NO:28

5’-TTGTCTTCCTAAGCAACTCCTTGGCTCACAGAACGACATGGCTACGATCCGACTT-3’

SEQ ID NO:29

5’-Phos/CTCTCAGTACGTCAGCAGTT-(Barcode2)-CAACTCCTTGGCTCACAGAAC-3’

SEQ ID NO:30

5’-GCATGGCGACCTTATCAG-(Barcode1)-TTGTCTTCCTAAGACCGCTTGG-3’

B) Post-capture library construction

Hybridization of the probe: hybridization reaction systems (Eco Universal Blocking Oligo and RNase Block from Ai Jitai Biotechnology Co., ltd.; human Cot-1 DNA ^TM from thermo Fisher; hybridization buffer from Bei Kang medical J000099) were prepared according to the systems shown in Table 2, incubated at 80℃for 5 minutes, and then incubated at 50℃for 12 hours to give hybridization products.

TABLE 2

Composition of the components	System of
		The whole genome library prepared in step a)	200ng
Hybridization buffer	15μL
		Eco Universal Blocking Oligo	3μL
Human Cot-1DNATM	3μL
		RNase Block	3μL
Probe with a probe tip	1μL

Library enrichment: to the hybridization product was added 50. Mu.L of streptavidin magnetic beads to enrich the library of interest. The streptavidin magnetic beads were Thermofisher Dynabeads streptavidin.

Collecting a target library: washing the enriched library with 150 μl of wash buffer 2 for 15 min, and discarding the supernatant; then, 150. Mu.L of the washing buffer 3 preheated to 50℃was used for washing 3 times for 10 minutes each, and the supernatant was discarded; finally, 150 mu L of ethanol solution with the volume concentration of 80% is used for cleaning the library, and after standing for 30s at room temperature, the ethanol solution is thoroughly removed and the library is dried at room temperature. 24. Mu.L nuclease-free water was added for elution to obtain a solution containing the objective library. Both wash buffer 2 and wash buffer 3 were from Bei Kang kit model J000099.

Post-capture PCR amplification: PCR amplification was performed using the solution containing the library of interest as a template, and the post-capture amplification enzyme mixture (25. Mu.L) and the post-capture primer mixture (1. Mu.L). The mixed solution of the amplification enzymes after capturing is VAHTS HIFI Amplification Mix of Nanjinouzan biotechnology Co-Ltd; the primer mix after capture was synthesized from Jin Weizhi. The amplification system comprises: 24. Mu.L of target library solution, 1. Mu.L of primer, and amplification enzyme mixture after capture.

The amplification conditions were: pre-denaturation at 95 ℃ for 1 min; denaturation at 98℃for 20 seconds, annealing at 60℃for 30 seconds, extension at 72℃for 30 seconds, and cycling 11 times; extending at 72 ℃ for 5 minutes; preserving at 4 ℃. The amplified products were purified using Agencourt AMPure XP Kit kit to obtain a post-capture library for high throughput sequencing.

Concentration measurement: concentration determination was performed on the library after capture using the Qubit ^TM dsDNA quantification kit.

Sequencing on a machine: a high throughput (combined probe anchored sequencing by polymerization) sequencing platform was selected for on-machine sequencing.

C) Data analysis

The quality control results of the high-throughput sequencing data of 20 samples to be detected are shown in table 3, and the results show that the method of the application shows higher sequencing quality for the samples to be detected, and the data quality control of the samples P01 to P20 all meet the analysis requirement.

The sequence depth scatter diagrams of the samples P01 to P20 are shown in fig. 4, 5,6,7,8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 and 23, respectively (the abscissa "position" in fig. 4 to 23 represents the sequence position, the ordinate "log2 ratio" represents the ratio of the sequence depth of the sample to be tested to the reference library sample to log2, and the "ratio" represents the ratio of the sequence depth of the sample to be tested to the reference library sample).

Specifically, as can be seen from fig. 4, the deletion types of sample P01 include AZFb and AZFc deletions; similarly, the deletion types of samples P02 to P20 can be seen from fig. 5 to 23, and the specific results are shown in table 3. The detection results of the samples P01 to P16 are YCMs positive samples, including AZFb and AZFc complete deletion type, AZFb deletion type, AZFc deletion type, gr/gr deletion and b2/b3 deletion; the detection results of samples P17 to P20 were YCMs negative samples.

TABLE 3 Table 3

The detection result shows that the probe composition can be used for rapidly and accurately detecting the microdeletion of the AZF region on the Y chromosome. Furthermore, the probe composition of the application can accurately detect partial deletions existing in the AZFc region, such as gr/gr and b2/b3, and provides more detailed references for defining the genetic etiology of male infertility and evaluating the risk of male infertility.

D) CNV-seq verification

In order to verify the detection accuracy of the method, the 20 samples to be detected are further detected by adopting CNV-seq analyzed by an optimized flow. The CNV-seq detection results of the samples P01 to P20 are shown in fig. 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 and 43, respectively, and the specific detection results are shown in table 4.

TABLE 4 Table 4

Sample numbering	Deletion type for CNV-seq detection	The deletion type detected by the application
			P01	AZFb and AZFc deletions	AZFb and AZFc deletions
P02	AZFb and AZFc deletions	AZFb and AZFc deletions
			P03	AZFb deletion	AZFb deletion
P04	AZFb deletion	AZFb deletion
			P05	AZFc deletion	AZFc deletion
P06	AZFc deletion	AZFc deletion
			P07	AZFc deletion	AZFc deletion
P08	AZFc deletion	AZFc deletion
			P09	AZFc deletion	AZFc deletion
P10	AZFc deletion	AZFc deletion
			P11	Gr/gr deletion	Gr/gr deletion
P12	Gr/gr deletion	Gr/gr deletion
			P13	Gr/gr deletion	Gr/gr deletion
P14	B2/b3 deletion	B2/b3 deletion
			P15	B2/b3 deletion	B2/b3 deletion
P16	B2/b3 deletion	B2/b3 deletion
			P17	YCMs negative	YCMs negative
P18	YCMs negative	YCMs negative
			P19	YCMs negative	YCMs negative
P20	YCMs negative	YCMs negative

In conclusion, the consistency of the detection result of the Y chromosome microdeletion and the CNV-seq detection result reaches 100%, which shows that the detection method of the application has high accuracy and can be used for detecting the Y chromosome microdeletion.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples illustrate only a few embodiments of the application, which are described in detail and are not to be construed as limiting the scope of the application. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. Accordingly, the scope of protection of the present application is to be determined by the appended claims.

Claims (10)

1. A probe composition for detecting microdeletion of the Y chromosome, wherein the genomic version of the reference is GRCh38, the probe composition comprising a probe ：chrY:12318588～12318688、chrY:12330588～12330688、chrY:12354588～12354688、chrY:12640588～12640688、chrY:12940588～12940688、chrY:13196588～13196688、chrY:17807879～17807979、chrY:17857458～17857558、chrY:18029249～18029349、chrY:18320052～18320152、chrY:18752440～18752540、chrY:19047939～19048039、chrY:19987849～19987949、chrY:20503375～20503475、chrY:20896316～20896416、chrY:21197814～21197914、chrY:21840243～21840343、chrY:22356551～22356651、chrY:22746063～22746163、chrY:23104533～23104633、chrY:23824142～23824242、chrY:24670728～24670828、chrY:25043147～25043247、chrY:25769311～25769411、chrY:25826318～25826418 for detecting the following regions and a chrY:26671322 ～ 26671422.

2. The probe composition for detecting Y chromosome microdeletion according to claim 1, wherein the probe composition comprises a probe having a nucleotide sequence shown in SEQ ID NO.1 to SEQ ID NO. 26.

3. A kit for detecting microdeletion of the Y chromosome, comprising the probe composition of any one of claims 1-2.

4. The kit of claim 3, further comprising hybridization reaction reagents.

5. The kit of claim 4, wherein the hybridization reaction reagents comprise one or more of magnetic beads, blocking solution, and hybridization buffer.

6. The kit of any one of claims 3 to 5, further comprising a DNA extraction reagent.

7. Use of a probe composition according to any one of claims 1-2 for the preparation of a product for detecting microdeletion of the Y chromosome.

8. A method for detecting microdeletion of the Y chromosome comprising the steps of:

Performing hybridization capture on a sample to be tested by using the probe composition according to any one of claims 1 to 2 or the kit according to any one of claims 3 to 6; and

And sequencing the hybridization captured product, and determining the Y chromosome microdeletion condition in the sample to be tested according to the sequencing result.

9. The method for detecting microdeletion of the Y chromosome of claim 8, wherein the step of performing hybrid capture on the sample to be detected comprises:

constructing a whole genome sequencing library by taking genomic DNA of a sample to be detected as a template; and

Hybridization capture of the whole genome sequencing library using the probe composition of any of claims 1-2 or the kit of any of claims 3-6.

10. The method of detecting Y chromosome microdeletion of any of claims 8-9, wherein the method of sequencing comprises high throughput sequencing.