Targeted sequencing method for simultaneous detection of multiple molecular defects associated with imprinting disorders and kit for performing the same

Abstract

The application discloses a targeted sequencing method for synchronously detecting various molecular defects related to imprinting diseases and a kit for implementing the method. The targeted sequencing method comprises the following steps: designing a capture probe capable of specifically capturing one or more imprinting-related genomic target regions, the target regions covering known imprinting control regions and related imprinting genes; hybridizing the probe to genomic DNA in a sample to be detected to generate DNA products and dividing the products into two parts, the first part being used for constructing a conventional sequencing library and the second part being used for constructing a methylation sequencing library after being subjected to bisulfite conversion; performing high-throughput sequencing on the conventional sequencing library and the methylation sequencing library respectively to generate sequencing data; and analyzing the sequencing data by bioinformatics to generate a detection report. Advantages of the targeted sequencing method at least include that DNA methylation detection and genomic variation detection can be performed on imprinting regions in a large amount or a small amount of DNA, and disease diagnosis can be performed in combination with clinical manifestations.

Description

Technical Field

[0001] This application relates to the field of sequencing detection technology, specifically to targeted sequencing methods for the simultaneous detection of multiple molecular defects associated with imprinted diseases, and kits for implementing such methods. Background Technology

[0002] Genomic imprinting is an epigenetic mechanism that regulates gene expression, causing hundreds of mammalian genes to express only the alleles from one parent, with this expression dependent on the parental origin of that allele. In its classic model, imprinting arises from the differential establishment and maintenance of DNA methylation on maternal and paternal autosomes. Paternal and maternal imprinted genes may provide counterbalancing growth effects in embryonic development; paternal imprinted genes tend to promote growth and nutrient acquisition, while maternal imprinted genes tend to suppress growth to reduce pregnancy burden. Currently, over 100 and 150 imprinted genes have been identified in humans and mice, respectively (https: / / www.geneimprint.com). These imprinted genes are crucial for embryonic development, intrauterine development, pregnancy maintenance, neonatal transition, and growth and development, and function in brain behavior, energy metabolism, respiration, and reproduction. Disorders of imprinted gene expression can lead to congenital imprinting disorders (ImpDis), manifesting as abnormalities in growth, metabolism, endocrine function, and neurobehavioral processes. Currently, 13 imprinted diseases have been identified, and the molecular mechanisms causing abnormal imprinted gene expression include uniparental disomy (UPD) involving the imprinted region, microdeletions or microduplications within the imprinted region, mutations in pathogenic imprinted genes, and epigenetic mutations leading to abnormal methylation of disease-associated differentially methylated regions (DMRs). Figure 1 In recent years, chimeric multi-locus imprinting disturbance (MLID) has been observed in some patients with imprinting diseases, characterized by the loss of methylation of one or more DMRs in addition to those typically associated with the disease. Although the clinical significance of MLID is still under investigation, its biological importance has become apparent: several MLID cases present with atypical clinical features, including a high risk of relapse and maternal reproductive problems.

[0003] Early symptoms of imprinted diseases often appear before birth or in infancy; however, the timing and specific manifestations of the same disease can vary drastically among different patients. This often leads to difficulties for clinicians in making diagnoses and selecting appropriate genetic testing, resulting in many patients remaining without a clear diagnosis for extended periods and even entering a cycle of repeated medical visits. Therefore, there is an urgent need to develop next-generation molecular diagnostic tools for imprinted diseases and to increase evidence-based medicine from various aspects to improve diagnostic efficiency and pinpoint the cause as early as possible. Most imprinted diseases currently identified are closely related to chromosomal structural abnormalities, such as uniparental disodiasis (UPD) and chromosomal microdeletions / microduplications. These abnormalities can be identified through routine genomic testing (such as chromosomal microarray analysis and karyotype analysis). However, there is a more subtle type of imprinted disease in clinical practice—simple epigenetic modification abnormalities, where changes in DNA methylation patterns are not accompanied by genomic sequence variations. These diseases require targeted methylation detection technologies (such as methylation-specific multiplexed probe amplification (MS-MLPA), pyrosequencing, or whole-genome methylation analysis) for diagnosis. In addition, for imprinted diseases caused by imprinted gene mutations, Sanger sequencing, whole genome or whole exome sequencing are required for detection.

[0004] Previous detection methods for imprinted diseases could not screen all four mechanisms using a single approach (see...). Figure 1 For imprinted diseases caused by [unspecified factors], conventional single-method testing may result in false negatives, preventing timely diagnosis. Comprehensive etiological screening often requires a combination or sequential testing of several methods, which is not only costly but also time-consuming. Furthermore, many tests (such as Sanger sequencing and DNA methylation testing) target specific regions and cannot identify multi-site imprinted crosstalk (MLID), while whole-genome-based analyses (whole-genome sequencing and whole-genome methylation sequencing) are costly and cause data redundancy. Summary of the Invention

[0005] Based on this, this application provides at least one targeted sequencing method for simultaneously detecting multiple molecular defects associated with imprinted diseases, as well as a kit for implementing it.

[0006] This application describes the design of a PANEL for imprinted disease detection, which covers the imprinted region and genes related to the imprinted region. With a single test, the methylation status and variations of known imprinted regions can be detected, enabling efficient screening and diagnosis of imprinted diseases.

[0007] In a first aspect of this application, a targeted sequencing method for simultaneously detecting multiple molecular defects associated with imprinted diseases is provided, comprising the following steps:

[0008] Design capture probes capable of specifically capturing one or more imprinted genomic target regions, which cover known imprinting control regions and associated imprinted genes;

[0009] The probe is used to hybridize the genomic DNA in the sample to be tested, generating DNA products;

[0010] The DNA product was divided into two parts. The first part was used to construct a conventional sequencing library, and the second part was converted with bisulfite and used to construct a methylated sequencing library.

[0011] High-throughput sequencing was performed on the conventional sequencing library and the methylation sequencing library to generate sequencing data;

[0012] The sequencing data is analyzed using bioinformatics to generate a test report, which includes one or more of the following data:

[0013] (i) Data on heterozygosity loss patterns based on single nucleotide polymorphisms (SNPs);

[0014] (ii) Copy number variation (CNV) data for the target region;

[0015] (iii) Sequence variation data of imprinted genes; and

[0016] (iv) DNA methylation level data at CpG sites within the target imprinting control region.

[0017] In a second aspect of this application, a kit is provided for implementing a targeted sequencing method as described in the first aspect for the simultaneous detection of multiple molecular defects associated with imprinted diseases, comprising:

[0018] (a) A capture probe capable of specifically capturing one or more imprinted genomic target regions, said target regions covering known imprinting control regions and associated imprinted genes;

[0019] (b) A library construction reagent module for constructing conventional sequencing libraries; and,

[0020] (c) Library preparation reagent module for constructing methylated sequencing libraries, which includes bisulfite conversion reagent.

[0021] The method involved in this application can detect DNA methylation and genomic variation in imprinted regions of large or small amounts of DNA, and combine this with clinical manifestations for disease diagnosis. The targeted capture detection method has lower costs, less data redundancy, and can detect all possible molecular mechanisms of all imprinted diseases in a single test, saving time and helping to make molecular diagnoses of suspected imprinted disease patients in clinical practice. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without creative effort. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in illustrating this application.

[0023] Figure 1 The genetic etiology of the imprinted disease in one embodiment of this application; UPD, uniparental disomy; P, paternal allele; M, maternal allele; DMR(s), differentially methylated region(s) [PMID: 40033833].

[0024] Figure 2 This is the imprint capture experimental procedure in one embodiment of this application.

[0025] Figure 3 This document shows the detection status of test samples in one embodiment of this application; and the methylation status of the captured SNURF imprinted regions. S11 is a normal control (moderate SNURF methylation), S12 is a paternally deleted PWS positive sample (highly methylated SNURF), and S11665, S13823, and S9731 are all maternally deleted AS positive samples (lowly methylated SNURF). Red indicates highly methylated (>80%) reads, blue indicates lowly methylated (<20%) reads, and white indicates moderately methylated (20%-80%) reads.

[0026] Figures 4A to 4D This invention shows the detection results of imprint defects in an example BWS family according to one embodiment of the present application, wherein KvDMR1(IC2) is a region of abnormal methylation; Figure 4A : Family tree, the arrow points to the proband of BWS (who has already had an abortion); Figure 4B Methylation levels in imprinted regions detected using the WGBS platform (displayed by the number of reads with low / medium / high methylation levels). Figure 4C Analysis was performed using next-generation sequencing following targeted amplification of KvDMR1(IC2), and the results show gel images, alignment rates, and CpG site methylation levels. Figure 4DThe methylation level of each imprinted region detected by the capture method of this kit is shown by the number of reads with low / medium / high methylation levels; where low methylation reads are defined as methylation levels <20% and high methylation reads are defined as methylation levels >80%.

[0027] Figure 5A and Figure 5B This application displays information about an exemplary probe in one embodiment. Detailed Implementation

[0028] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings. Preferred embodiments of this application are shown in the drawings. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this application.

[0029] Unless otherwise defined, 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 is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0030] In this application, unless otherwise specified, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and other instances that otherwise indicate "one or more" shall be understood in the same way unless otherwise specified.

[0031] The terms “combinations thereof,” “any combination thereof,” and “any combination thereof” as used in this application include all suitable combinations of any two or more of the listed items.

[0032] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method" etc., shall be defined as being able to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0033] In this application, terms such as "further," "even more," "particularly," "for example," "like," "example," and "exemplary" are used for descriptive purposes to indicate that different technical solutions preceding and following each other are related in terms of their coverage, but should not be construed as limiting the preceding technical solution or restricting the scope of protection of this application. In this application, unless otherwise specified, A (e.g., B) indicates that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0034] The terms “containing,” “comprising,” and “including” as used in this application are synonyms and are inclusive or open-ended, not excluding additional, uncited members or features. Members or features include, for example, materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions under which actions occur, timing, states, etc.

[0035] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0036] In this application, the exemplary descriptions such as "in some implementations (or embodiments)" and "in one implementation (or embodiment)" may cover, but are not limited to, the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0037] In this application, the terms "first aspect," "second aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first aspect," "second aspect," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0038] In this application, when numerical intervals (i.e., numerical ranges) are involved, unless otherwise specified, the distribution of selectable numerical values ​​within the numerical interval is considered continuous, and includes the two endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. The term "numerical interval" can be broadly included to include numerical interval types such as percentage intervals, ratio intervals, and proportion intervals.

[0039] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0040] Imprinted disorders are a class of genetic diseases caused by abnormal genomic imprinting function. Common imprinted disorders include Angelman syndrome (AS), Prader-Willi syndrome (PWS), and Beckwell syndrome (BWS).

[0041] Genomic imprinting: This is a special epigenetic regulatory mechanism. After fertilization, certain genes from both parents are not expressed equally. For certain specific genes, only the allele from the father (or mother) is active, while the allele from the other parent is "silenced" (not expressed) in somatic cells. This is like tagging genes with "paternal" or "maternal" labels, determining whether they function. This "tag" is mainly achieved through epigenetic modifications such as DNA methylation. These regions marked by DNA methylation are called imprinted regions.

[0042] Beckwith-Wiedemann Syndrome (BWS): BWS is a congenital disorder characterized by three main features: macroglossia, omphalocele, and hyperplasia. It is also accompanied by excessive organ growth, visceral enlargement (primarily of the liver, kidneys, and spleen), hypoglycemia at birth, and unilateral hypertrophy (excessive growth on one side of the body). It is typically caused by factors such as abnormal methylation in the 11p15.5 region, gene mutations, chromosomal rearrangements, and paternal UPD.

[0043] DNA methylation is a form of DNA chemical modification that can alter genetic expression without changing the DNA sequence. It is generally believed that DNA methylation leads to gene silencing.

[0044] CpG: CpG is an abbreviation for cytosine (C)-phosphate (p)-guanine (G). CpG islands are mainly located in the promoter and exon regions of genes and are regions rich in CpG dinucleotides. The C in CpG is easily methylated, so imprinted regions in the genome are usually rich in CpG.

[0045] Reads: refers to sequence fragments obtained through sequencing;

[0046] Uniparental disomy (UPD): refers to an individual's chromosome region / segment being replaced by a homologous portion from the other parent, or an individual having two homologous chromosomes that both originate from the same parent.

[0047] Karyotype: Karyotype refers to the phenotype of a chromosome set during metaphase of mitosis. It is the sum of chromosome number, size, and morphological characteristics. When describing a karyotype using a karyotype formula, the first item is the total number of chromosomes (including sex chromosomes), the second item is a comma, and the third item is the sex chromosomes.

[0048] Copy number variation (CNV) refers to the increase or decrease of large segments (over 1 kb) in the genome, involving various structural variations such as deletions and duplications.

[0049] Methylation-specific MLPA (MS-MLPA) is a derivative of MLPA technology that can be used for semi-quantitative analysis of methylation in multiple targets simultaneously. Like conventional MLPA probes, MS-MLPA probes consist of a left-hand probe and a right-hand probe. In addition to the hybridization sequence, universal primer recognition sequence, and filler sequence of conventional MLPA probes, MS-MLPA probes also contain methylation-sensitive Hha I endonuclease cleavage sites for methylation analysis. MS-MLPA can detect aberrant methylation in target regions. However, MLPA is highly sensitive to impurities, requires sophisticated sample preparation and technical manipulation, and may experience reduced probe signal due to rare polymorphisms or mutations. Furthermore, MLPA is expensive, and the need for simultaneous detection of multiple imprinted regions in clinical practice can double the time and cost. It cannot detect imprinted diseases caused by gene mutations.

[0050] Whole-genome bisulfite sequencing (WGBS) is considered the "gold standard" for DNA methylation research. Combining bisulfite treatment and high-throughput sequencing technology, it enables the analysis of methylation of individual cytosine bases across the entire genome, making it suitable for constructing fine methylation maps of the entire genome. However, WGBS is costly, involves numerous steps, has a long data acquisition cycle, generates redundant data, and its cost is directly proportional to sequencing depth. It cannot accurately reflect the methylation modification status of imprinted regions, requiring higher detection depths for these regions, which inevitably leads to higher costs. Furthermore, it cannot detect imprinted diseases caused by gene mutations.

[0051] SNP-arrays (single nucleotide polymorphism chips) detect the genotypes (homozygous AA / TT / CC / GG or heterozygous AT / CG) of numerous single nucleotide polymorphism (SNP) sites in the genome through probe hybridization. Combined with parental information (parental samples are required for control) or self-genotype pattern analysis, they can diagnose UPD. While detecting UPD, they can simultaneously analyze copy number variations (deletions / duplications) across the entire genome, but the resolution for copy number variations is relatively low. A single sample cannot detect uniparental heterodizygosity (derived from two different chromosome copies from the same parent), nor can it detect imprinting defects caused by abnormal DNA methylation and gene mutations.

[0052] CNV-seq is a genome-wide copy number analysis technology based on next-generation sequencing (NGS). It randomly fragments and sequences genomic DNA, calculates the sequencing depth (number of reads) for each genomic window, and compares it with a normal reference genome to determine the copy number of target regions (normal diploid, deletion, duplication, etc.). Since the core characteristic of UPD (Ultra-Polydilation Disorder) is "normal copy number (2 copies) but abnormal parental origin," and CNV-seq can only detect "copy number changes" and cannot directly analyze SNP genotypes or parental information, CNV-seq cannot directly detect UPD. Furthermore, CNV-seq cannot detect imprinting defects caused by abnormal DNA methylation and gene mutations.

[0053] WES (whole exome sequencing) only sequences the coding regions of the genome (approximately 1-2%), focusing on detecting SNVs / Indels, small CNVs, and LOH (loss of heterozygosity) patterns in exons and nearby splice sites. WGS (whole genome sequencing) sequences the entire genome (coding and non-coding regions), enabling comprehensive detection of SNVs, Indels, CNVs, UPDs, structural variations (SVs), and deep intron variations. However, both methods require high sequencing depth, resulting in high data redundancy and the following drawbacks: they only read DNA sequence information and cannot directly identify abnormal methylation states in imprinted regulatory regions. Approximately 20-30% of imprinted diseases are caused by isolated methylation defects, and these patients may be completely missed. They also have insufficient efficiency in detecting UPD (low sensitivity and the need for special bioinformatics processes). Furthermore, they have limited ability to detect structural variations. WES cannot detect structural variations in uncaptured regions and has poor sensitivity in detecting microdeletions / microduplications in captured regions. Although WGS is superior to WES due to uneven probe capture efficiency and the influence of GC content, it still has insufficient coverage in highly repetitive regions (such as fragment repetitions in imprinted gene clusters), making it difficult to accurately determine breakpoints.

[0054] The customized probe capture combined with dual-path library construction and sequencing method designed in this application has the core advantage of achieving, for the first time, integrated and simultaneous detection of genetic variations and epigenetic methylation status in imprinted diseases. This method utilizes probes designed for known imprinted regions for highly efficient targeted capture (capture efficiency >50%). On the one hand, conventional sequencing can be performed to comprehensively analyze genomic variations such as CNVs, SNVs, and UPDs; on the other hand, after bisulfite conversion and library construction, the methylation level of key differentially methylated regions (DMRs) can be directly and accurately detected. This innovative design fundamentally overcomes the limitations of traditional techniques (such as SNP-array, WES, and MS-MLPA, which do not detect methylation, and do not detect sequence variations), allowing for the acquisition of complete molecular typing information in a single experiment without the need for multi-platform validation.

[0055] The high homogeneity of this method (>85% good regional coverage) ensures the sensitivity and reliability of the detection, making it particularly suitable for prenatal diagnosis with limited sample sizes and for the analysis of mosaicism and low-proportion variants. It provides an efficient and comprehensive solution for the accurate diagnosis, genetic counseling and clinical management of imprinted diseases.

[0056] Based on the next-generation sequencing platform, the signal is amplified, and the accuracy of chromosome structural variations, mutations, and regional DNA methylation levels is high; targeted capture reduces data redundancy, and the data depth and effective utilization rate are high.

[0057] Dual-path library construction can save sample and time costs, while avoiding the need for patch testing due to a negative test result, and reducing the psychological burden on patients due to waiting.

[0058] The streamlined operation process is suitable for establishing standardized testing procedures and is easy to promote.

[0059] It greatly reduces testing costs and has strong applicability.

[0060] This method is applicable to the following samples: peripheral blood, amniotic fluid cells before and after culture, other tissue cells, semen, oral mucosal cells, and other trace cellular DNA.

[0061] This method is applicable to platforms compatible with Illumima Miseq and MGI 150 / 200 platforms, etc.

[0062] For basic technical procedures, please refer to Figure 2 .

[0063] A single experiment can simultaneously obtain the methylation status of the imprinted region and gene sequence mutations, avoiding the sample consumption, technical bias, and data integration difficulties of separate testing. It is especially suitable for the comprehensive diagnosis of imprinted diseases (such as Beckwith-Wiedemann syndrome and Silver-Russell syndrome), and can accurately detect imprinted diseases caused by four types of molecular defects. It is also more suitable for clinical diagnosis of induced labor.

[0064] Best clinical applicability and cost-effectiveness:

[0065] ① Compared to WGBS, it has lower cost, smaller data volume, deeper target region coverage, and avoids data redundancy. In addition, it can obtain information on genomic variations (WGBS cannot accurately identify such single-base variations because C bases change to T after methylation conversion), which makes the analysis faster and more suitable for clinical laboratories; ② Compared to MS-MLPA, it has fewer operational limitations, controllable cost, provides higher detection accuracy (single-base resolution) and a wider coverage (customizable expansion), and can detect mutations; ③ Compared to single-region targeted methylation detection, it enables simultaneous analysis of multiple sites, improving detection efficiency and systematicity; ④ Compared to CNV-seq or SNP-array, it overcomes the inability of CNV-seq or SNP-array to detect DNA methylation and the false negatives caused by low resolution (only above 100kb), and can detect simple DNA methylation abnormalities without chromosomal copy number variations or uniparental disomy. WGS also causes data redundancy due to the high data volume required; ⑤ Compared with WES and other technologies that can only detect mutations, it fully demonstrates its superiority in DNA methylation detection.

[0066] One aspect of this application provides a targeted sequencing method for the simultaneous detection of multiple molecular defects associated with imprinted diseases, comprising the following steps:

[0067] Design capture probes capable of specifically capturing one or more imprinted genomic target regions, which cover known imprinting control regions and associated imprinted genes;

[0068] The probe is used to hybridize the genomic DNA in the sample to be tested, generating DNA products;

[0069] The DNA product was divided into two parts. The first part was used to construct a conventional sequencing library, and the second part was converted with bisulfite and used to construct a methylated sequencing library.

[0070] High-throughput sequencing was performed on the conventional sequencing library and the methylation sequencing library to generate sequencing data;

[0071] The sequencing data is analyzed using bioinformatics to generate a test report, which includes one or more of the following data:

[0072] (i) Data on heterozygosity loss patterns based on single nucleotide polymorphisms (SNPs);

[0073] (ii) Copy number variation (CNV) data for the target region;

[0074] (iii) Sequence variation data of imprinted genes; and

[0075] (iv) DNA methylation level data at CpG sites within the target imprinting control region.

[0076] For example, the design principles of the capture probe include, but are not limited to:

[0077] 1) Sequence specificity: After being shielded by RepeatMasker, the probe sequence avoids high copy repeat regions such as LINE, SINE, and satellite DNA; the uniqueness of the probe sequence in the reference genome is ensured by genome alignment; for targets with pseudogenes or highly homologous family genes, the probe is designed in the differential region.

[0078] 2) Homogenization of physicochemical properties: The probe length is controlled between 60 nt and 120 nt; the melting temperature (Tm) of all probes in the probe pool is controlled between 75℃ and 85℃, with a maximum temperature difference of no more than 5℃; the GC content of the probe is controlled between 30% and 70%, and the capture efficiency is compensated by increasing the probe density in high GC regions.

[0079] 3) Probe layout and coverage: A tiling design is adopted, with the probe spacing being 1 / 2 or 1 / 3 of the probe length, ensuring that each base in the target region is covered by 2 to 3 probes; the probe coverage extends 50 bp to 200 bp to both sides of the target region to eliminate the impact of decreased capture efficiency in the edge region after library disruption.

[0080] 4) Secondary structure constraints: Avoid forming stable hairpin structures or dimers when designing probes; for regions with stable secondary structures such as G-quadruplexes, optimize by increasing hybridization temperature or increasing probe density.

[0081] 5) Modification and labeling: Biotin is modified at the 5' end of each probe to bind with streptavidin magnetic beads to achieve the separation of the captured product.

[0082] 6) Polymorphism site treatment: For probes located in regions with high frequency single nucleotide polymorphisms (SNPs), degenerate base (mixed base) synthesis or probe position adjustment is used to avoid a decrease in capture efficiency due to allele deactivation.

[0083] Furthermore, the design principles of the capture probe include, but are not limited to:

[0084] 1. Ensure sequence specificity

[0085] a. Avoid repetitive sequences: Probe sequences must avoid high-copy-repetitive regions such as LINE, SINE, and satellite DNA. If the target region contains repetitive sequences, mask them using RepeatMasker before designing the probe.

[0086] b. Genomic uniqueness: BLAST alignment ensures the probe is unique within the reference genome. If the target gene contains pseudogenes or highly homologous family genes, the probe should be designed to target the differentially expressed region.

[0087] 2. Physicochemical properties (Tm and GC content)

[0088] a. Length: Strictly controlled between 60 nt and 120 nt. Short probes (~60 nt) have higher specificity, while long probes (~120 nt) have higher capture efficiency.

[0089] b. Tm value uniformity: The melting temperature of all probes in the probe cell should be controlled between 75°C and 85°C, and the maximum temperature difference (ΔTm) should not exceed 5°C to ensure hybridization uniformity.

[0090] c. GC content: Must be controlled within the range of 30% to 70%. In the high GC (>70%) region, hybridization efficiency decreases significantly, and it is recommended to increase probe density (reduce spacing) to compensate. In the low GC (<30%) region, specificity is poor and non-specific background is easily generated, so the hybridization temperature needs to be appropriately increased.

[0091] 3. Probe layout and coverage

[0092] a. Immovable layering: Using a density with an interval of 1 / 2 or 1 / 3 of the probe length, ensure that each base in the target region is covered by 2 to 3 probes.

[0093] b. Edge extension: The probe coverage must extend 50 bp to 200 bp to both sides of the target region (such as exons) to eliminate the problem of a sharp drop in capture efficiency in the edge region after library interruption.

[0094] 4. Secondary structure limitations

[0095] a. Avoid stable structures: The probe itself should not form a stable hairpin structure or dimer (ΔG value should not be too low). If a stable secondary structure (such as G-quadruplex) exists in the target region, the DNA probe will have difficulty penetrating, which needs to be compensated for by increasing the hybridization temperature or increasing the probe density.

[0096] 5. Modification and Marking

[0097] a. Biotin modification: Biotin (for streptavidin magnetic bead binding) must be modified at the 5' end of the probe. Modification must not be placed in the internal or critical 3' end region that would affect hybridization efficiency.

[0098] 6. Handling Polymorphism

[0099] To prevent allele decoupling: If the design site happens to be located in a region with a high density of high-frequency SNPs, the binding affinity between the DNA probe and the mismatched template will decrease. Use degenerate bases (mixed bases) to synthesize probes, or adjust the probe position appropriately to avoid key mutation sites.

[0100] In some implementations, the genomic target region includes one or more of the following loci or imprinted control regions:

[0101] The SNURF (SNRPN) imprinting control region located on chromosome 15q11.2-q13, the KvDMR1 (IC2) imprinting control region and / or the H19 / IGF2:IG-DMR (IC1) imprinting control region located on chromosome 11p15.5, the UBE3A gene, the MAGEL2 gene, the KCNQ1OT1 gene, the CDKN1C gene, the IGF2 gene; and the H19 gene.

[0102] In some implementations, the process of constructing a conventional sequencing library includes: end repair of DNA fragments, addition of an "A" to the 3' end, ligation of universal sequencing adapters, and library amplification.

[0103] In some implementations, the process of constructing a methylation sequencing library includes:

[0104] DNA was subjected to bisulfite conversion, purification of the conversion product, end repair and adapter ligation of the converted fragments, and library amplification using DNA polymerase.

[0105] In some implementations, the bioinformatics analysis includes:

[0106] Data from conventional sequencing libraries are aligned to a reference genome for sequence alignment, variant identification, and annotation to generate the sequence variant data and copy number variant data. Genotypes of SNP sites are analyzed to generate the heterozygous loss pattern data; and / or,

[0107] Data from methylation sequencing libraries are compared using an alignment tool adapted for bisulfite conversion data to identify target CpG sites and calculate their methylation levels, thereby generating the DNA methylation level data.

[0108] In some implementations, the high-throughput sequencing is performed on an Illumina sequencing platform or an MGI sequencing platform.

[0109] In some implementations, the sample to be tested is derived from peripheral blood, amniotic fluid, villi, oral swabs, or paraffin-embedded tissue.

[0110] In some embodiments, the imprinted disease includes Angelman syndrome (AS), Prader-Willi syndrome (PWS), Beckwith-Wiedemann syndrome (BWS), Silver-Russell syndrome (SRS), or Schaaf-Yang syndrome (SYS).

[0111] Another aspect of this application also provides a kit for implementing the targeted sequencing method described above for the simultaneous detection of multiple molecular defects associated with imprinted diseases, comprising:

[0112] (a) A capture probe capable of specifically capturing one or more imprinted genomic target regions, said target regions covering known imprinting control regions and associated imprinted genes;

[0113] (b) A library construction reagent module for constructing conventional sequencing libraries; and,

[0114] (c) Library preparation reagent module for constructing methylated sequencing libraries, which includes bisulfite conversion reagent.

[0115] In some embodiments, the library preparation reagent module for constructing conventional sequencing libraries includes end repair and A-tailing reagents, DNA ligase, universal sequencing adapters, and DNA polymerase.

[0116] In some embodiments, the library preparation reagent module for constructing methylated sequencing libraries includes bisulfite conversion reagent, DNA purification reagent, end repair and A-tailing reagent, methylated sequencing adapter, and DNA polymerase.

[0117] In some implementations, hybridization buffer for library capture and streptomycin-labeled magnetic beads are also included.

[0118] The following are some examples.

[0119] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, reference should be made to the guidelines given in this application, or to experimental manuals or conventional conditions in the art, or to the conditions recommended by the manufacturer, or to experimental methods known in the art.

[0120] Example 1

[0121] 1.1 DNA Fragmentation - End Repair - dA Tail Addition

[0122] (1) Five samples were selected for testing, including a normal control (S11), a positive sample of paternal deletion type PWS syndrome (S12, SNURF hypermethylation), and a positive sample of maternal deletion type AS syndrome (S11665, S13823 and S9731; SNURF hypomethylation). The DNA samples were broken down to 150-250bp using sonication.

[0123] (2) End repair / dA tail addition: After thawing each reagent in Table 1, invert and mix well, keep on ice for later use, and prepare the reaction system in a sterile PCR tube according to the ratio; use a pipette to gently blow or shake to mix well, and briefly centrifuge to the bottom of the tube.

[0124] Table 1. PCR reaction system with end repair / dA tail addition

[0125]

[0126] (3) Place the PCR tubes in the PCR instrument and set the reaction program according to Table 2. Heat the lid to 105°C.

[0127] Table 2. PCR reaction program for end repair / dA tail addition

[0128]

[0129] 1.2 Connector Connection

[0130] (1) Dilute the Adapter to the appropriate concentration and volume according to the amount of DNA input. Dissolve the reagents required in Table 3 naturally, mix well, and keep on ice for later use. Prepare the reaction system according to Table 3, mix well, and collect to the bottom of the tube by short-circuiting. Perform the reaction on the PCR instrument according to Table 4.

[0131] Table 3 Adapter Ligation PCR System

[0132]

[0133] Table 4 Adapter Ligation PCR Reaction Procedure

[0134]

[0135] 1.3 Product Purification

[0136] (1) Take the Clean magnetic beads out of the refrigerator in advance, allow them to equilibrate at room temperature for 10 minutes, and prepare 80% ethanol;

[0137] (2) Vortex or thoroughly invert the magnetic beads, add 66 μl of Clean magnetic beads to the 110 μl system after the connection reaction, shake, and incubate at room temperature for 5 minutes;

[0138] (3) Briefly centrifuge the PCR tube and place it in a magnetic rack to separate the magnetic beads and liquid. Discard the supernatant, add 200 μl of 80% ethanol to rinse the magnetic beads for 30 seconds, remove the supernatant, and repeat the rinsing once.

[0139] (4) The PCR tubes should always be placed on a magnetic rack and left open to dry until they crack (3-5 minutes).

[0140] (5) Add 25 μl H2O, vortex and mix well, and let stand at room temperature for 5 minutes.

[0141] 1.4 Hybrid Capture

[0142] (1) Take Human Cot-1 DNA and Un-Blocker out of the refrigerator and let them thaw naturally on ice at room temperature. Prepare the reaction system according to Table 5.

[0143] Table 5 Closed System Before Capture

[0144]

[0145] (2) Vortex, momentarily remove the reaction liquid to the bottom of the tube, and place it in a vacuum rotary evaporator to evaporate the reaction liquid to dryness;

[0146] (3) Take out the following reagents and let them thaw naturally on ice at room temperature: HYB-Buffer, Enhancer, ZXXY_METHY-Probe (the sequences of exemplary probes are shown in SEQ ID NO: 1~100).

[0147] (4) Add the following reagents to the evaporated PCR tube (Table 6).

[0148] Table 6 Capture System

[0149]

[0150] (5) Mix the reaction solution from the previous step thoroughly, let it stand at room temperature for 5-10 minutes, mix again, and incubate briefly; place it in a pre-set PCR instrument for incubation at 95°C, with the PCR hot cover set at 105°C.

[0151] (6) Take out the PCR tube, shake to mix, briefly separate, and immediately place it on the pre-set PCR instrument for incubation at 65°C (>30 minutes), with the PCR hot cap at 75°C.

[0152] (7) Elution: Remove the following buffer reagents from the refrigerator and thaw at room temperature: 2x BWB, 10x WI, 10x WII, 10x SW.

[0153] Table 7. Preparation of Buffer for a Single Hybridization Reaction (1 rxns) (Theoretical)

[0154]

[0155] The working solution should be preheated at 65°C for at least 15 minutes on the PCR instrument before use.

[0156] (8) Preparation of streptomycin affinity magnetic beads (SA magnetic beads): Remove the SA magnetic beads from the refrigerator, let them stand at room temperature for 10 minutes, equilibrate the system, vortex for 15 seconds to mix, and take 50 μl of SA magnetic bead solution for each hybridization reaction. Place 50 μl of the required reaction solution into a 1.5 ml centrifuge tube, place it on a magnetic rack, and discard the supernatant; add N. 100 μl of 1X BWB, vortex oscillation, placed on a magnetic rack, supernatant discarded, repeated once; then take N Mix 50 μl of 1X BWB well, and pipette 50 μl into a new PCR tube for each hybridization reaction.

[0157] (9) Magnetic bead binding: Place the SA magnetic beads obtained in the previous step on a magnetic rack, discard the supernatant, and immediately place the PCR tube containing the SA magnetic beads on a PCR instrument at 65°C (heated lid 75°C). Also transfer 16.5 μl of the hybridization reaction to the PCR tube containing the SA magnetic beads and mix well.

[0158] (10) React on the PCR instrument for 30 minutes. Every 10 minutes, take out the PCR tube, shake for 5 seconds, and immediately put it back into the PCR instrument until the total reaction time of 30 minutes is met.

[0159] (11) Hot washing: Except for the use of a magnetic rack, all other steps were performed on a PCR instrument at 65℃. Add 120 μl of preheated 1XWI, mix well by pipetting, incubate for 10-20 s, place on a magnetic rack, and discard the supernatant; then add 150 μl of preheated 1X SW solution, fully suspend the SA magnetic beads, put back into the PCR instrument and incubate for 5 minutes, place on a magnetic rack, discard the supernatant, and repeat the 1X SW washing once;

[0160] (12) Elution at room temperature: Add 150 μl of 1X WI, vortex for 2 minutes, suspend the SA magnetic beads, place on a magnetic rack, and discard the supernatant; add 150 μl of 1X WI, vortex for 1 minute, suspend the SA magnetic beads, place on a magnetic rack, and discard the supernatant; add 150 μl of 1X WI, mix by pipetting, place on a magnetic rack, and discard the supernatant;

[0161] (13) Resuspending magnetic beads: Add 25 μl of enzyme-free water, shake to mix, take 5 μl for unmethylated library construction, and 20 μl for methylated library construction.

[0162] 1.5 Methylation treatment

[0163] (1) Prepare the reaction system according to Table 8, mix well, briefly centrifuge to the bottom of the tube, and carry out the conversion according to Table 9.

[0164] Table 8 Methylation Conversion Reaction System

[0165]

[0166] Table 9 Methylation Conversion Reaction Procedure

[0167]

[0168] (2) Purification of the transformation product:

[0169] Add 10 μl of METH magnetic beads and the conversion product from the previous step to a 1.5 ml centrifuge tube, add 500 μl of binding buffer, mix thoroughly, incubate at room temperature for 10 minutes, then place on a magnetic rack and remove the supernatant; add 400 μl of washing buffer, mix thoroughly, place on a magnetic rack and remove the supernatant.

[0170] Add 200 μl of desulfurization buffer, mix thoroughly, let stand at room temperature for 10 minutes, then incubate briefly and transfer the supernatant to a magnetic rack; then add 400 μl of washing buffer, mix thoroughly, incubate briefly and transfer the supernatant to a magnetic rack, repeat once;

[0171] Dry at room temperature, remove residual liquid but do not crack the surface of the magnetic beads, add 20 μl of ddH2O, fully resuspend the magnetic beads, incubate at room temperature for 5 minutes, briefly separate and place on a magnetic rack, and after the solution is clear, transfer the supernatant to a new PCR tube.

[0172] 1.6 POST-PCR Amplification

[0173] (1) Prepare the unmethylated and methylated reaction systems in PCR tubes according to the volumes in Table 10 below. The reagents need to be thawed naturally at room temperature.

[0174] Table 10 Methylation Conversion Reaction System

[0175]

[0176] (2) Swirl and shake off the magnetic beads to suspend them. Set the PCR program according to Table 11 below for PCR amplification.

[0177] Table 11 POST-PCR reaction procedure

[0178]

[0179] (3) Take 1 μl of the amplification product and measure its concentration using Qubit.

[0180] 1.7 Product Purification

[0181] (1) Add 50 μl of selected magnetic beads to the PCR product, vortex to mix, let stand at room temperature for 5 minutes, place on a magnetic rack, and discard the supernatant.

[0182] (2) Add 100 μl of 80% ethanol to each PCR tube, place the PCR tube on a magnetic rack and adsorb it back and forth 1-2 times, wash it thoroughly, then place it on a magnetic rack and discard the supernatant;

[0183] (3) Dry the magnetic beads at room temperature, add 25 μl ddH2O or 0.1X TE Buffer to fully suspend the magnetic beads, let stand for 5 minutes, place on a magnetic rack, transfer the supernatant to a new EP tube, mark it, and store at 4℃ overnight.

[0184] 1.8 Document Quality Inspection

[0185] The obtained library was quantified using Qubit, and the peak shape of the library was checked using an Agilent 2100 analyzer. After the library passed the quality check, it could be arranged for sequencing or stored at -20℃.

[0186] 1.9 Sequencing

[0187] (1) Construct the library according to the standard library construction process of the Illumima or MGI sequencing platform, add tags to different samples, and use them to distinguish different samples in subsequent analysis. This allows for simultaneous sequencing of multiple samples in mixed tubes.

[0188] (2) Sequencing was performed using an Illumina or MGI sequencer.

[0189] 1.10 Bioinformatics Analysis: First, data quality control was performed, including Raw_Reads, Raw_Bases, Raw_Q30, Adapter_Rate, Clean_Reads, Clean_Bases, Clean_Q30, Clean_Rate, Uniq_Mapping_Reads, Mapping_Rate, Dup_Rate, and Mean_Depth. The quality control data was visualized, and the data was filtered to remove low-quality data and adapters. The data was then aligned with the hg19 genome sequence to obtain the position of the reads on the reference genome. Further filtering using relevant software and alignment files was performed, including checking for duplicates and multiple alignments.

[0190] For methylation data analysis: Alignment tools adapted for methylation sequencing (such as BSMAP and Bismark) are used to align clean data to the reference genome. During alignment, specific parameters need to be set to distinguish between transformed unmethylated cytosine (converted to U, identified as T during sequencing) and untransformed methylated cytosine (still C). Multiple alignments and low-quality alignment reads (alignment quality value < 30) are filtered out to generate a sorted BAM format file. Picard Tools are used to mark and remove PCR duplicate reads to avoid interference from repetitive sequences in methylation quantification. Based on the aligned BAM file, tools such as MethylDackel and Bismark methylation extractor are used to identify methylation sites across the entire genome (mainly including CG, CHG, and CHH types, where H represents A / T / C). The methylation level (methylation rate = number of methylated reads / total number of covered reads × 100%) was calculated by statistically analyzing the number of methylated reads at each cytosine site and the total number of covered reads. A BedGraph or BED format file containing site location and methylation rate was generated, while sites with a coverage depth < 5× were filtered to ensure quantitative reliability. Tools such as annotatePeaks.pl were used to perform genomic functional annotation on the detected methylated sites, associating them with their respective genomic regions (e.g., promoters, exons, introns, CpG islands, repetitive sequences, etc.). A methylation imprinting region pipeline was used to analyze high / medium / low methylated reads, target region methylation levels, target region coverage, and depth.

[0191] For genomic data analysis: The HaplotypeCaller module of the Genome Analysis Toolkit (GATK) was used to detect variants in the processed BAM files, identifying single-nucleotide variants (SNVs) and small insertions / deletions (InDels), generating a raw variant call format (VCF) file. The GATK VariantFiltration module was used to rigorously screen the raw variants, using criteria including QUAL > 30, QD > 2.0, FS < 60.0, and MQ > 40.0, to eliminate technical errors and low-confidence variants. After variant screening, high-confidence variants are comprehensively annotated using ANNOVAR or Variant Effect Predictor (VEP) tools. The annotation includes: 1) basic genetic information (such as chromosome location, reference / alternate alleles, gene symbols, transcript IDs); 2) variant functional impact prediction (such as missense variants, nonsense variants, frameshift variants, splice site variants, and pathogenicity scores from tools such as Polyphen-2, SIFT, and CADD); 3) population frequency data from public databases (such as gnomAD, ExAC, and the 1000 Genomes Project database); and 4) clinical relevance annotation (such as association information with hereditary diseases in the OMIM and ClinVar databases).

[0192] 2. Exemplary probe information can be found in SEQ ID NO: 1~100 (details can be found in [link to documentation]). Figure 5A and Figure 5B ).

[0193] 3. Test Results:

[0194] The success rate of library construction for the 5 samples was 100%. The detection results for the 1 normal control and 4 positive samples were in line with expectations: S11 was a normal control (moderate SNURF methylation), S12 was a paternal deletion type PWS positive sample (SNURF hypermethylation), and S11665, S13823 and S9731 were maternal deletion type AS positive samples (SNURF hypomethylation).

[0195] Table 12. Capture efficiency and quality control parameters of test samples

[0196]

[0197] Note: Unf10 represents the proportion of 0.1 times the average depth.

[0198] Example 2: Eleven cases and ten controls with previous gene diagnoses of imprinted diseases were selected. There were four types of diseases, involving molecular defects of all four imprinted diseases, including seven different regions. The above kit was used for detection, and the positive detection rate was 100% and the false positive rate was 0%.

[0199] All regions achieved 100% coverage. Overall capture efficiency and depth were related to the amount of data. Among the 11 positive samples, 68.8% coverage was achieved with the lowest data volume (603,846 reads), and the target region depth was above 40X. The kit accurately detected molecular defects in the imprinted region, fully demonstrating its accuracy and sensitivity. No false positives were observed in the normal control samples.

[0200] Table 13 shows the methylation information of the samples.

[0201] Table 13

[0202]

[0203] Notes: AS, Angelman syndrome; PWS, Prader–Willi syndrome; BWS, Beckwith–Wiedemann syndrome; SYS, Schaaf–Yang syndrome; SNV, Single Nucleotide Variant; LOM, loss of methylation; GOM, gain of methylation.

[0204] Table 14 shows the quality control parameters corresponding to sample testing.

[0205] Table 14

[0206]

[0207] Example 3: PANEL screening of a fetus prenatally diagnosed as suspected BWS (compared with whole-genome methylation sequencing and targeted methylation results)

[0208] Prenatal ultrasound in a case of intrauterine pregnancy at 18 weeks gestation indicated omphalocele. Figure 4AFetal karyotype and CNV abnormalities have been ruled out (no loss of heterozygosity was observed, suggesting non-UPD), and whole-exome sequencing of fetal amniotic fluid cells and peripheral blood DNA from both parents showed no phenotypic abnormalities. Whole-genome methylation sequencing (WGBS) was performed separately. Figure 4B ), targeted methylation ( Figure 4C ) and this kit ( Figure 4D Detection was performed using [method name missing], and all results showed BWS-related KvDMR1(IC2) hypomethylation. WGBS, using 10G of data (a high-cost sequencing method), yielded only 27 sequencing reads in the KvDMR1(IC2) target region, while this kit used only 0.5G of sequencing data, achieving a sequencing depth of 3224 reads in the KvDMR1(IC2) target region, significantly improving accuracy. The methylation levels detected by WGBS, targeted methylation, and this kit were 4.3%, 6.5%, and 7.1%, respectively. Targeted methylation, as a near-gold standard for measuring regional methylation levels, indicates that this kit has higher detection accuracy.

[0209] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0210] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A targeted sequencing method for simultaneous detection of multiple molecular defects associated with imprinted diseases, characterized in that, Includes the following steps: Design capture probes capable of specifically capturing one or more imprinted genomic target regions, which cover known imprinting control regions and associated imprinted genes; The probe is used to hybridize the genomic DNA in the sample to be tested, generating DNA products; The DNA product was divided into two parts. The first part was used to construct a conventional sequencing library, and the second part was converted with bisulfite and used to construct a methylated sequencing library. High-throughput sequencing was performed on the conventional sequencing library and the methylation sequencing library to generate sequencing data; The sequencing data is analyzed using bioinformatics to generate a test report, which includes one or more of the following data: (i) Data on heterozygosity loss patterns based on single nucleotide polymorphisms (SNPs); (ii) Copy number variation (CNV) data for the target region; (iii) Sequence variation data of imprinted genes; and (iv) DNA methylation level data at CpG sites within the target imprinting control region.

2. The targeted sequencing method for simultaneous detection of multiple molecular defects related to imprinted diseases as described in claim 1, characterized in that, The genomic target region includes one or more of the following loci or imprinted control regions: The SNURF (SNRPN) imprinting control region located on chromosome 15q11.2-q13, the KvDMR1 (IC2) imprinting control region located on chromosome 11p15.5 and / or the H19 / IGF2:IG-DMR (IC1) imprinting control region, the UBE3A gene, the MAGEL2 gene, the KCNQ1OT1 gene, the CDKN1C gene, and the IGF2 gene; And the H19 gene.

3. The targeted sequencing method for simultaneous detection of multiple molecular defects related to imprinted diseases as described in claim 1, characterized in that, The process of constructing a conventional sequencing library includes: end repair of DNA fragments, addition of an "A" to the 3' end, ligation of universal sequencing adapters, and library amplification.

4. The targeted sequencing method for simultaneous detection of multiple molecular defects related to imprinted diseases as described in claim 1, characterized in that, The process of constructing a methylation sequencing library includes: DNA was subjected to bisulfite conversion, purification of the conversion product, end repair and adapter ligation of the converted fragments, and library amplification using DNA polymerase.

5. The targeted sequencing method for simultaneous detection of multiple molecular defects associated with imprinted diseases as described in claim 1, characterized in that, The bioinformatics analysis includes: Data from conventional sequencing libraries are aligned to a reference genome for sequence alignment, variant identification, and annotation to generate the sequence variant data and copy number variant data. Genotypes of SNP sites are analyzed to generate the heterozygous loss pattern data; and / or, Data from methylation sequencing libraries are compared using an alignment tool adapted for bisulfite conversion data to identify target CpG sites and calculate their methylation levels, thereby generating the DNA methylation level data.

6. The targeted sequencing method for simultaneous detection of multiple molecular defects associated with imprinted diseases as described in claim 1, characterized in that, The high-throughput sequencing was performed on the Illumina sequencing platform or the MGI sequencing platform.

7. The targeted sequencing method for simultaneous detection of multiple molecular defects associated with imprinted diseases as described in claim 1, characterized in that, The test samples are derived from peripheral blood, amniotic fluid, villi, oral swabs, or paraffin-embedded tissue.

8. The targeted sequencing method for simultaneous detection of multiple molecular defects associated with imprinted diseases as described in any one of claims 1 to 7, characterized in that, The imprinted disorders include Angelman syndrome (AS), Prader-Willi syndrome (PWS), Beckwith-Wiedemann syndrome (BWS), Silver-Russell syndrome (SRS), or Schaaf-Yang syndrome (SYS).

9. A kit for implementing the targeted sequencing method for simultaneously detecting multiple molecular defects related to imprinted diseases as described in any one of claims 1 to 8, characterized in that, include: (a) A capture probe capable of specifically capturing one or more imprinted genomic target regions, said target regions covering known imprinting control regions and associated imprinted genes; (b) A library construction reagent module for constructing conventional sequencing libraries; and, (c) Library preparation reagent module for constructing methylated sequencing libraries, which includes bisulfite conversion reagent.

10. The kit according to claim 9, characterized in that, It meets one or more of the following conditions: (1) The library preparation reagent module for constructing conventional sequencing libraries includes end repair and A-tailing reagents, DNA ligase, universal sequencing adapters and DNA polymerase; (2) The library construction reagent module for constructing methylated sequencing libraries includes bisulfite conversion reagent, DNA purification reagent, end repair and A-tailing reagent, methylated sequencing adapter and DNA polymerase; (3) It also includes hybridization buffer for library capture and streptomycin-avidin-labeled magnetic beads.

Images