Method for detecting chromosome variation and device

Abstract

The present application relates to a method for detecting a chromosome variation and a device. The method comprises: carrying out DNA methylation measurement on a target specimen; and on the basis of the measurement result, analyzing chromosome copy number variation information and / or epigenetic modification information of the target specimen.

Description

Method and device for detecting chromosome variation

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 2023110286589, filed on August 15, 2023, entitled “Method and Apparatus for Detecting Chromosome Variations,” the entire text of which is incorporated herein by reference. Background Art

[0003] Chromosomes are important carriers of human genetic genes. Once abnormal, they may cause birth defects such as miscarriage, congenital idiocy, and congenital multiple malformations.

[0004] Spontaneous abortion is the most common pregnancy complication in obstetrics and gynecology. It generally refers to the failure of a pregnancy, primarily including biochemical pregnancy, empty gestational sac, gradual cessation of embryonic development, embryonic or fetal death, and expulsion of the embryo and its appendages. Testing aborted tissue is the primary means of identifying the cause of miscarriage. Currently, clinical investigation primarily involves testing aborted tissue for chromosome copy number variation. However, clinically, 50% of aborted tissue still has normal chromosome multiplicity, making it impossible to determine the cause of miscarriage using existing technologies. This results in a significant proportion of miscarriage patients being unable to receive treatment or intervention.

[0005] Numerous studies in the past have demonstrated that epigenetics plays an important regulatory role in early embryonic development. Errors in epigenetic information during embryonic development can lead to stagnation of embryonic development, pregnancy failure, and birth defects. However, there is currently a lack of effective clinical methods for detecting embryonic epigenetic information. Amniocentesis is a common prenatal screening method. It involves collecting amniotic fluid for testing between 16 and 20 weeks of gestation to determine the health of the fetus and whether it has genetic diseases. Chromosome multiplex screening is one of the main components of amniocentesis and can effectively reduce the incidence of birth defects.

[0006] Currently, there are three main clinical methods for detecting chromosome copy number variations: chromosome karyotype analysis, chromosome microarray analysis (CMA) technology, and copy number variation sequencing (CNVseq) based on next-generation sequencing technology. Among them, chromosome karyotype refers to an image composed of all chromosomes in a somatic cell, which are paired and grouped according to their size and morphological characteristics. For many years, chromosome karyotype analysis technology has been considered the "gold standard" for determining chromosome copy number variations. Chromosome microarray analysis technology (CMA): refers to scanning the entire genome to detect copy number variations (CNVs) of chromosomal imbalances, especially for detecting genomic imbalances such as genomic microdeletions and microduplications. It has outstanding advantages.

[0007] Current clinical testing of amniotic fluid cannot detect epigenetic modifications in the embryo, making it impossible to determine whether the embryo has an epigenetic disease. Furthermore, when CNV testing is performed on amniotic fluid, if a chromosome deletion is detected, it is impossible to determine whether the missing fragment is from the father or the mother, making it difficult for clinicians to determine whether the fetus may have a disease. This presents a significant challenge for amniotic fluid testing and a technical issue that needs to be addressed urgently.

[0008] Therefore, how to obtain more variation information at the same time is a difficulty in detecting chromosomal variations in embryos.

[0009] Summary of the Invention

[0010] In order to solve the above problems and provide more information about chromosome abnormalities, the first object of the present invention is to provide a method for detecting chromosome mutations, comprising:

[0011] Performing DNA methylation testing on a target sample, where the target sample is derived from at least one of embryonic cells, fetal cells and / or tissues, neonatal blood, umbilical cord blood, and abortion tissue;

[0012] Analyze the chromosome copy number variation information and / or epigenetic modification information of the target sample based on the test results.

[0013] This application uses a new sequencing technology principle to construct a whole-genome DNA methylation map, and detects the chromosome copy number information and epigenetic modification information of the sample based on the DNA methylation map, which can provide more chromosome variation information at the same time.

[0014] In one embodiment, analyzing the chromosome copy number variation information and / or epigenetic modification information of the target sample according to the test results includes:

[0015] Obtain DNA methylation data of the target sample based on the test results;

[0016] Analyzing chromosome copy number variation information and / or epigenetic modification information of the target sample based on the DNA methylation data of the target sample;

[0017] Among them, DNA methylation data includes sequencing-based DNA methylation sequencing data and chip-based DNA methylation chip data.

[0018] In one embodiment, analyzing the chromosome copy number variation information and / or epigenetic modification information of the target sample based on the DNA methylation sequencing data of the target sample includes:

[0019] Obtaining chromosome copy number information and / or DNA methylation information of the target sample based on the DNA methylation data of the target sample;

[0020] Determine the parental origin of the chromosome copy number variation of the target sample based on the chromosome copy number information and DNA methylation information of the target sample; and / or

[0021] Determine whether the target sample has epigenetic modification abnormalities based on the DNA methylation information of the target sample.

[0022] In one embodiment, obtaining chromosome copy number information of a target sample based on DNA methylation data of the target sample includes:

[0023] Determine the distribution position of the sequenced reads on the genome of the target sample based on the alignment results of the DNA methylation sequencing data of the target sample with the reference genome;

[0024] The chromosome copy number variation information of the target sample is determined based on the distribution position of all sequencing reads of the target sample in the genome.

[0025] In one embodiment, obtaining DNA methylation information of a target sample based on DNA methylation data of the target sample includes:

[0026] Determine the methylation modification status of each cytosine site on each sequencing read in the target region based on the DNA methylation sequencing data of the target sample;

[0027] The DNA methylation information of the target sample is obtained based on the methylation modification status of each cytosine site on each sequencing read in the target region of the target sample.

[0028] In one embodiment, obtaining DNA methylation information of a target sample based on DNA methylation data of the target sample includes:

[0029] Determine the DNA methylation status of each sequencing read in the target region based on the DNA methylation sequencing data of the target sample. The DNA methylation status includes a high methylation state or a low methylation state.

[0030] The DNA methylation information of the target sample is obtained based on the DNA methylation status of each sequencing read in the target region of the target sample.

[0031] In one embodiment, the target area satisfies at least one of the following characteristics:

[0032] (1) The target region includes the whole genome region;

[0033] (2) The target region includes the promoter region;

[0034] (3) The target area includes the imprint control area;

[0035] (4) Target regions include enhancer regions in the genome that regulate gene expression;

[0036] (5) Target regions include repressor regions in the genome that regulate gene expression.

[0037] In one embodiment, determining the parental origin of the chromosome copy number variation of the target sample based on the chromosome copy number variation information and DNA methylation information of the target sample includes:

[0038] Obtaining DNA methylation information of the target sample's copy number variation region based on the target sample's chromosome copy number variation information and DNA methylation information;

[0039] Determine the parental origin of the target sample chromosome copy number variation based on the DNA methylation information of the target sample copy number variation region;

[0040] Optionally, the copy number variation region comprises an imprinting control region.

[0041] In one embodiment, determining whether a target sample has abnormal epigenetic modification based on DNA methylation information of the target sample includes:

[0042] The DNA methylation status of each sequencing read in the imprinting control region of the target sample is used to determine whether the target sample has abnormal epigenetic modification of the paternal chromosome or the maternal chromosome.

[0043] A second object of the present application is to provide a device for detecting chromosome variation, comprising:

[0044] DNA methylation detection module: used to perform DNA methylation detection on target samples, where the target samples are from at least one of embryonic cells, fetal cells and / or tissues, neonatal blood, umbilical cord blood, and abortion tissue;

[0045] Information analysis module: used to analyze the chromosome copy number variation information and / or epigenetic modification information of the target sample based on the test results.

[0046] The details of one or more embodiments of the present application are set forth in the description below. Other features, objects, and advantages of the present application will become apparent from the description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In order to better describe and illustrate the embodiments and / or illustrations of the inventions disclosed herein, reference may be made to one or more of the accompanying drawings. The additional details or examples used to describe the accompanying drawings should not be considered to limit the scope of the disclosed inventions, the presently described embodiments and / or embodiments, and any of the best modes currently understood for these inventions.

[0048] FIG1 is a flowchart of a method for detecting chromosome variation provided by one or more embodiments of the present application;

[0049] FIG2 is a block diagram of a device for detecting chromosome variation according to one or more embodiments of the present application;

[0050] FIG3 is a diagram showing the internal structure of a computer device provided by one or more embodiments of the present application;

[0051] FIG4 shows the chromosome detection results of the fetal cell sample isolated from amniotic fluid in Example 1 of the present application;

[0052] FIG5 shows the methylation levels of various imprinted genes in the fetal cell sample isolated from amniotic fluid in Example 1 of the present application;

[0053] FIG6 shows the chromosome detection results of peripheral blood samples of patients with imprinted gene diseases in Example 2 of the present application;

[0054] FIG7 shows the methylation level of the imprinted gene SNURF in peripheral blood samples of patients with imprinted gene diseases in Example 2 of the present application;

[0055] FIG8 is the chromosome detection result of the abortion tissue sample in Example 3 of the present application;

[0056] FIG9 is the methylation information of the imprinted gene GNAS-XL in the abortion tissue sample in Example 3 of the present application;

[0057] FIG10 is the chromosome detection result of the abortion tissue sample in Example 4 of the present application;

[0058] FIG11 shows the methylation information of the imprinted gene H19 in the abortion tissue sample in Example 4 of the present application. DETAILED DESCRIPTION

[0059] References to embodiments of the present application will now be provided in detail, one or more examples of which are described below. Each example is provided to illustrate, not to limit, the present application. Indeed, it will be apparent to those skilled in the art that various modifications and variations may be made to the present application without departing from the scope or spirit of the present application. For example, features illustrated or described as part of one embodiment may be used in another embodiment to produce further embodiments.

[0060] Therefore, it is intended that this application covers such modifications and variations that fall within the scope of the appended claims and their equivalents. Other objects, features, and aspects of the present application are disclosed in or are apparent from the following detailed description. Those skilled in the art will appreciate that this discussion is merely a description of exemplary embodiments and is not intended to limit the broader aspects of the present application.

[0061] In order to at least partially solve the above technical problems, the first aspect of the present application provides a method for detecting chromosome variation, as shown in FIG1 , comprising:

[0062] S10: performing DNA methylation detection on a target sample, where the target sample is from at least one of fetal cells and / or tissues, neonatal blood, umbilical cord blood, and abortion tissue;

[0063] S20: analyzing the chromosome copy number variation information and / or epigenetic modification information of the target sample according to the test results;

[0064] In some embodiments, the target samples are all derived from mammals.

[0065] As used herein, the term "mammal" includes any diploid animal including humans, tigers, wolves, mice, deer, mink, monkeys, tapirs, sloths, zebras, dogs, foxes, bears, elephants, leopards, musk deer, cows, lions, pandas, warts, pigs, antelopes, reindeers, koalas, rhinoceroses, lynxes, pangolins, giraffes, pandas, anteaters, gorillas, manatees, otters, civets, dolphins, walruses, platypuses, hedgehogs, Arctic foxes, polar bears, kangaroos, armadillos, hippos, seals, whales, weasels, and rabbits.

[0066] In some embodiments, the target sample is derived from aborted tissue, a fetus, or a newborn of a mammal.

[0067] In some embodiments, the target sample can be a sample of an embryo before implantation in the uterus during assisted reproductive technology, or a fetal cell and / or tissue sample obtained during pregnancy through invasive methods such as chorionic villus sampling, amniocentesis, and percutaneous umbilical vascular puncture, or aborted tissue or peripheral blood of a newborn.

[0068] Thus, in some embodiments, the target sample is selected from in vitro fertilized eggs, preimplantation embryonic cells, fetal cells or tissues in amniotic fluid, umbilical cord blood, abortion tissue, or neonatal blood.

[0069] The present application performs DNA methylation detection on fetal cells or tissues, and analyzes the chromosome copy number variation information and / or epigenetic modification information based on the test results, which can provide more supporting information for the clinical assessment of the risk of miscarriage of the fetus or cells, and intervene more promptly in situations where termination of pregnancy is required. Performing DNA methylation detection on aborted tissues, and analyzing the chromosome copy number variation information and / or epigenetic modification information based on the test results, can provide more detection information for the analysis of the cause of miscarriage, and when the chromosome copy number is normal, analyze the cause of miscarriage from the perspective of epigenetic modification, and then provide the test subject with subsequent more comprehensive pregnancy recommendations. Performing DNA methylation detection on umbilical cord blood or neonatal blood, and analyzing the chromosome copy number variation information and / or epigenetic modification information based on the test results, can timely intervene in abnormal epigenetic modification diseases in newborns to avoid the deterioration of related diseases.

[0070] Specifically, DNA methylation detection includes sequencing-based DNA methylation detection and chip-based DNA methylation detection. In this application, the method of DNA methylation detection of the target sample is not specifically limited. It can be sequencing-based DNA methylation detection or chip-based DNA methylation detection, as long as the chromosome copy number variation information and epigenetic modification information of the target sample can be obtained simultaneously.

[0071] In some specific embodiments, sequencing-based DNA methylation detection refers to converting the DNA in the target sample so that the unmethylated cytosine on the DNA is converted into uracil, and then constructing a library and sequencing the converted DNA to obtain DNA methylation sequencing data of the target sample.

[0072] In some specific embodiments, the sequencing data is next-generation sequencing data.

[0073] For example, sequencing-based DNA methylation detection includes bisulfite conversion sequencing (BS-Seq), whole genome methylation sequencing (WGBS), and third-generation sequencing (SMRT).

[0074] Chip-based DNA methylation detection refers to the detection of DNA methylation information using a methylation chip. The principle is based on signal detection of DNA sequence hybridization after sulfite treatment. The DNA sample is first treated with sulfite to convert non-methylated cytosine into uracil, while the methylated cytosine remains unchanged, so that it can be distinguished after subsequent sequencing or hybridization.

[0075] In particular, for the case of simultaneously obtaining chromosome copy number variation information and epigenetic modification information based on DNA methylation detection results, the present application provides a method for simultaneously detecting chromosome copy number variation information and epigenetic information, comprising:

[0076] Perform DNA methylation testing on the target sample, and analyze the chromosome copy number variation information and epigenetic modification information of the target sample based on the test results.

[0077] Specifically, the DNA methylation detection results based on the target sample can obtain epigenetic modification abnormality information and chromosome copy number variation information. Combining chromosome copy number variation information and epigenetic modification abnormality information can obtain more chromosome copy number variation information, such as the source of chromosome copy number variation.

[0078] Accordingly, analyzing the chromosome copy number variation information and / or epigenetic modification information of the target sample according to the test results includes:

[0079] Obtain DNA methylation data of the target sample based on the test results;

[0080] Analyzing chromosome copy number variation information and / or epigenetic modification information of the target sample based on the DNA methylation data of the target sample;

[0081] Among them, DNA methylation data includes sequencing-based DNA methylation sequencing data and chip-based DNA methylation chip data.

[0082] In some embodiments, analyzing the chromosome copy number variation information and / or epigenetic modification information of the target sample based on the DNA methylation data of the target sample includes:

[0083] Obtaining chromosome copy number information and / or DNA methylation information of the target sample based on the DNA methylation data of the target sample;

[0084] Determine the parental origin of the chromosome copy number variation of the target sample based on the chromosome copy number information and DNA methylation information of the target sample; and / or

[0085] Determine whether the target sample has epigenetic modification abnormalities based on the DNA methylation information of the target sample.

[0086] Specifically, chromosome copy number information includes chromosome copy number variation regions. Specifically, for humans, the human genome contains approximately 3.16 billion DNA base pairs, which make up 46 chromosomes, divided into 23 pairs. Each pair of chromosomes is inherited from the father and the mother. The two chromosomes are also called homologous chromosomes, and their size and genetic composition are basically the same. 22 pairs are autosomes, and the last pair is the sex chromosomes, which are XX for females and XY for males. The normal copy number value of autosomes and the X chromosome in females is 2, and the normal copy number value of the X and Y chromosomes in males is 1. When the copy number detection value is greater than the normal value, it is a duplication, and when it is less than the normal value, it is a deletion.

[0087] In some embodiments, obtaining chromosome copy number information of a target sample based on DNA methylation data of the target sample includes:

[0088] Determine the distribution position of the sequenced reads on the genome of the target sample based on the alignment results of the DNA methylation sequencing data of the target sample with the reference genome;

[0089] The chromosome copy number variation information of the target sample is determined based on the distribution position of all sequencing reads of the target sample in the genome.

[0090] Specifically, when the target sample is human, the reference genome is the human reference genome, more specifically, the human reference genome hg38.

[0091] In some embodiments, in order to obtain alignment data more accurately, before obtaining DNA methylation information of a target sample based on whole-genome methylation sequencing data, the following steps are further included:

[0092] Remove the adapter sequences and low-quality bases in the DNA methylation sequencing data to obtain sequencing data after data quality control processing;

[0093] The DNA methylation sequencing data after data quality control are aligned with the reference genome, and the repeated sequences in the DNA methylation sequencing data are removed based on the alignment results.

[0094] Specifically, epigenetic modification refers to the regulation of gene expression by changing the DNA and proteins on chromosomes through chemical modification. This modification can affect multiple levels of gene transcription, splicing, stability, translation, nucleosome assembly and chromatin structure, thereby affecting the physiological and pathological processes of cells, as well as the phenotype of offspring.

[0095] Among them, abnormal epigenetic modification refers to the inability of genes to change the DNA and proteins on chromosomes through chemical modification to achieve gene expression, and affects the physiological and pathological processes of cells, as well as the phenotype of offspring.

[0096] In order to obtain embryonic epigenetic modification information, the present application proposes for the first time to obtain the chromosome copy number variation information and / or DNA methylation information of the target sample based on the DNA methylation detection results of the above-mentioned target sample, and then analyze whether the epigenetic modification of the target sample is abnormal based on the DNA methylation information of the target sample, or simultaneously analyze whether the chromosome and epigenetic modification of the target sample are abnormal.

[0097] For DNA methylation sequencing data, DNA methylation information can be calculated by calculating the methylation level of each cytosine site through the methylation modification status of each cytosine site on different sequencing reads, and then obtaining the DNA methylation information of the target sample based on the methylation level of each cytosine site. It can also be calculated by calculating the methylation modification status of different cytosine sites on each sequencing read to calculate the methylation status of each sequencing read, and obtain the methylation information of the target sample based on reads with different methylation status.

[0098] Based on the comparison data of DNA methylation sequencing data and the reference genome, the position of the base of each read in the DNA methylation sequencing data on the chromosome can be determined. For a cytosine site at a certain position on the chromosome, the methylation modification status of the cytosine site can be distinguished according to the base type (C or T) of the cytosine site on each sequencing read.

[0099] In some embodiments, obtaining DNA methylation information of a target sample based on DNA methylation data of the target sample includes:

[0100] Determine the methylation modification status of each cytosine site on each sequencing read based on the DNA methylation sequencing data of the target sample;

[0101] The DNA methylation information of the target region of the target sample is obtained based on the methylation modification status of each cytosine site on each sequencing read of the target sample.

[0102] Specifically, for each cytosine site in the target region, the methylation level of each cytosine site is calculated based on the number of C bases and T bases of the cytosine site on all sequencing reads, and then the DNA methylation information of the target region is calculated based on the average value of the methylation levels of each cytosine site in the target region.

[0103] The calculation formula for the methylation level of each cytosine site is as follows:

[0104] Among them, ML i represents the methylation level of the i-th cytosine site in the target gene region;

[0105] C i Indicates the number of cytosine bases covering the i-th CG site in the DNA methylation sequencing data;

[0106] T i Indicates the number of thymine bases covering the i-th CG site in the DNA methylation sequencing data.

[0107] In other embodiments, obtaining DNA methylation information of a target sample based on the DNA methylation data of the target sample includes:

[0108] Determine the DNA methylation status of each sequencing read in the target region based on the DNA methylation sequencing data of the target sample. The DNA methylation status includes a high methylation state or a low methylation state.

[0109] The DNA methylation information of the target gene region of the target sample is obtained according to the DNA methylation status of each sequencing read in the target region of the target sample.

[0110] Specifically, the methylation state of each sequencing read is used to describe the overall methylation level of each sequencing read, including the first methylation state, the second methylation state, and the third methylation state. The first methylation state can represent a high methylation state, the second methylation state can represent a low methylation state, and the third methylation state can represent a moderate methylation state. The definition of reads with different methylation states can be set based on the methylation level of each cytosine site in the sequencing read according to the detection requirements.

[0111] In some specific embodiments, if more than 65% of the cytosine sites in a read are methylated, the read is defined as a read in a high methylation state; if more than 65% of the cytosine sites in a read are not methylated, the read is defined as a read in a low methylation state; if in a read, the cytosine sites that are methylated account for 35%-65% of all cytosine sites in the read, the read is defined as a read in a moderate methylation state.

[0112] In some embodiments, the target region comprises the entire genome.

[0113] In some embodiments, the region of interest includes a promoter region.

[0114] In some embodiments, the target area comprises a print control zone.

[0115] In some embodiments, the target region includes an enhancer region in the genome that regulates gene expression;

[0116] In some embodiments, the target region includes a repressor region in the genome that regulates gene expression.

[0117] Specifically, the whole genome refers to the sequence of the complete genome of an organism. Excessive or low methylation levels in the whole genome may lead to abnormal gene expression.

[0118] A promoter is a sequence on a genome that binds to RNA polymerase and initiates mRNA synthesis. Excessive methylation in the promoter region can lead to inhibition of gene expression. For example, some gene promoter regions are shown in Table 1.

[0119] Table 1

[0120] Genomic imprinting, also known as genomic imprinting, parental imprinting, or gametic imprinting, refers to the phenomenon in which parental alleles or chromosomes undergo specific processing and modification during gamete or zygote development, resulting in the silencing of one parental allele, leading to differential expression of the two parental alleles in offspring somatic cells. Genes with this difference are therefore called imprinted genes. The vast majority of imprinted genes are clustered across large chromosomal regions. The allele-specific repression of imprinted genes is regulated by imprinting control regions (ICRs), typically through methylation of the ICR on one allele.

[0121] Illustratively, some imprinted gene regions are shown in Table 2, where P represents paternal imprinting, i.e., the paternal gene is not expressed, but the maternal allele is expressed; M represents maternal imprinting, i.e., the maternal gene is not expressed, but the paternal allele is expressed.

[0122] Table 2

[0123] In some specific embodiments, the DNA methylation information of the target region can be calculated based on the ratio of the number of cytosines in the region that undergo methylation modification in all reads to all cytosines in these reads.

[0124] In some embodiments, determining the parental origin of the chromosome copy number variation of the target sample based on the chromosome copy number variation information and DNA methylation information of the target sample includes:

[0125] Obtaining DNA methylation information of the target sample's copy number variation region based on the target sample's chromosome copy number variation information and DNA methylation information;

[0126] The parental origin of the chromosome copy number variation of the target sample is determined based on the DNA methylation information of the copy number variation region of the target sample.

[0127] Specifically, for samples with chromosome copy number variation, whether the chromosome variation region of the target sample includes an imprinting control region is determined based on the chromosome copy number variation. If the chromosome variation region of the target sample includes an imprinting control region, the parental source of the chromosome variation of the target sample is determined based on the methylation information of the imprinting control region of the target sample.

[0128] For example, for paternally highly expressed imprinted genes, if the reads in the imprinting control region in the chromosome copy number loss region are almost all in the first methylation state, it can be inferred that the chromosome copy number loss is caused by paternal chromosome copy number variation. For maternally highly expressed imprinted genes, if the reads in the imprinting control region in the chromosome copy number loss region are almost all in the first methylation state, it can be inferred that the chromosome copy number loss is caused by maternal chromosome copy number variation.

[0129] It is understandable that in clinical cases where there are more chromosome copy number variations in fetal samples, based on the methylation level of the imprinting control region in the chromosome copy number loss area, it can be determined whether the chromosome copy number loss is caused by paternal chromosome copy number variation or maternal chromosome copy number variation, thereby providing reference information for the reproductive health of the embryo's parents and their subsequent fertility strategies.

[0130] In some embodiments, determining whether the target sample has abnormal epigenetic modification based on the DNA methylation information of the target sample includes:

[0131] The DNA methylation status of each sequencing read in the imprinting control region of the target sample is used to determine whether the target sample has abnormal epigenetic modification of the paternal chromosome or the maternal chromosome.

[0132] In reagent applications, for example, when testing aborted tissue, DNA methylation testing can simultaneously detect chromosome ploidy information and / or epigenetic modification information in the aborted tissue, greatly improving clinical testing efficiency, reducing the financial burden on patients, and providing more information for subsequent treatment or clinical intervention. Based on the DNA methylation status of each sequencing read in the imprinting control region of the aborted tissue, it is determined whether the target sample has epigenetic modification abnormalities on the paternal or maternal chromosomes.

[0133] In summary, the method for detecting chromosome variation in this application has the following beneficial effects:

[0134] 1. This application uses a new sequencing technology principle to construct a whole-genome DNA methylation map of fetal genetic material (cells and other fetal tissues) isolated from abortion tissue (chorionic villi, placenta, etc.), peripheral blood, and amniotic fluid, and judges the chromosome copy number information and epigenetic modification information of the test sample based on the DNA methylation map;

[0135] 2. This application provides an analytical strategy for determining whether a chromosome copy number variation is caused by a paternal chromosome copy number variation or a maternal chromosome copy number variation based on a DNA methylation profile;

[0136] 3. This application focuses on the detection of imprinted genes and establishes a set of methods and standards for detecting whether imprinted gene diseases exist.

[0137] A second aspect of the present application provides a device for detecting chromosome variation, comprising:

[0138] DNA methylation detection module: used to perform DNA methylation detection on target samples, where the target samples are from at least one of fetal cells and / or tissues, neonatal blood, umbilical cord blood, and abortion tissue;

[0139] Information analysis module: used to analyze the chromosome copy number variation information and / or epigenetic modification information of the target sample based on the test results.

[0140] In some embodiments, the information analysis module includes:

[0141] Acquisition unit: used to obtain DNA methylation data of target samples according to the test results;

[0142] Information analysis unit: analyzing the chromosome copy number variation information and / or epigenetic modification information of the target sample based on the DNA methylation data of the target sample;

[0143] Among them, DNA methylation data includes sequencing-based DNA methylation sequencing data and chip-based DNA methylation chip data.

[0144] In some embodiments, the information analysis unit includes:

[0145] Information acquisition subunit: used to obtain chromosome copy number information and / or DNA methylation information of the target sample based on the DNA methylation data of the target sample;

[0146] A first information analysis subunit: configured to determine the parental origin of the chromosome copy number variation of the target sample based on the chromosome copy number information and DNA methylation information of the target sample; and / or

[0147] The second information analysis subunit: determines whether the target sample has epigenetic modification abnormalities based on the DNA methylation information of the target sample.

[0148] In some embodiments, the information acquisition subunit includes a chromosome copy number information acquisition subunit and a DNA methylation information subunit;

[0149] The chromosome copy number information acquisition subunit is used to: determine the distribution position of the sequenced reads on the genome of the target sample based on the comparison results of the DNA methylation sequencing data of the target sample on the reference genome;

[0150] Determine the chromosome copy number variation information of the target sample based on the distribution position of all sequencing reads of the target sample in the genome;

[0151] The DNA methylation information acquisition subunit is used to:

[0152] Determine the methylation modification status of each cytosine site on each sequencing read in the target region based on the DNA methylation sequencing data of the target sample;

[0153] Obtain the DNA methylation information of the target sample based on the methylation modification status of each cytosine site on each sequencing read in the target region of the target sample;

[0154] Alternatively, the DNA methylation information acquisition subunit is used to:

[0155] Determine the DNA methylation status of each sequencing read in the target region based on the DNA methylation sequencing data of the target sample. The DNA methylation status includes a high methylation state or a low methylation state.

[0156] The DNA methylation information of the target sample is obtained based on the DNA methylation status of each sequencing read in the target region of the target sample.

[0157] In some embodiments, the target area satisfies at least one of the following characteristics:

[0158] (1) The target region includes the whole genome region;

[0159] (2) The target region includes the promoter region;

[0160] (3) The target area includes the imprint control area;

[0161] (4) Target regions include enhancer regions in the genome that regulate gene expression;

[0162] (5) Target regions include repressor regions in the genome that regulate gene expression.

[0163] In some embodiments, the first information analysis subunit is configured to:

[0164] Obtaining DNA methylation information of the target sample's copy number variation region based on the target sample's chromosome copy number variation information and DNA methylation information;

[0165] Determine the parental origin of the target sample chromosome copy number variation based on the DNA methylation information of the target sample copy number variation region;

[0166] Optionally, the copy number variation region comprises an imprinting control region.

[0167] The second information analysis subunit is used for:

[0168] The DNA methylation status of each sequencing read in the imprinting control region of the target sample is used to determine whether the target sample has abnormal epigenetic modification.

[0169] The specific definition of the device for detecting chromosome variation can be found in the definition of the method for detecting chromosome variation above and will not be repeated here. The various modules in the above-mentioned device for detecting chromosome variation can be implemented in whole or in part by software, hardware, or a combination thereof. The above-mentioned modules can be embedded in or independent of the processor in the computer device in hardware form, or can be stored in the memory of the computer device in software form, so that the processor can call and execute the operations corresponding to the above modules.

[0170] In some embodiments, a computer device is provided, which can be a server 104 or a terminal 102, and its internal structure diagram can be shown in Figure 3. The computer device includes a processor, memory, and a communication interface connected via a system bus. When the computer device is a terminal, it also includes a display screen and an input device connected to the system bus. The processor of the computer device is used to provide computing and control capabilities. The memory of the computer device includes a non-volatile storage medium and an internal memory. The non-volatile storage medium stores an operating system and a computer program. The internal memory provides an environment for the operation of the operating system and computer program in the non-volatile storage medium. The network interface of the computer device is used to communicate with an external terminal via a network connection. When the computer program is executed by the processor, a method for detecting chromosome mutations is implemented. The display screen of the computer device can be a liquid crystal display or an electronic ink display screen. The input device of the computer device can be a touch layer covering the display screen, or it can be a button, trackball, or touchpad provided on the computer device housing, or it can be an external keyboard, touchpad, or mouse.

[0171] Those skilled in the art will understand that the structure shown in FIG3 is merely a block diagram of a portion of the structure related to the solution of the present application, and does not constitute a limitation on the computer device to which the solution of the present application is applied. The specific computer device may include more or fewer components than shown in the figure, or combine certain components, or have a different arrangement of components.

[0172] The present application also provides a computer device comprising a memory and a processor, wherein the memory stores a computer program, and the processor implements the steps of the above-mentioned method for detecting chromosome variation when executing the computer program.

[0173] The present application also provides a computer-readable storage medium having a computer program stored thereon, which, when executed by a processor, implements the steps of the above-mentioned method for detecting chromosome variation.

[0174] The present application also provides a computer program product, which includes a computer program that, when executed by a processor, implements the steps of the above-mentioned method for detecting chromosome variation.

[0175] It should be noted that the user information (including but not limited to user device information, user personal information, etc.) and data (including but not limited to data used for analysis, stored data, displayed data, etc.) involved in this application are all information and data authorized by the user or fully authorized by all parties.

[0176] Those skilled in the art will appreciate that all or part of the processes in the above-mentioned embodiment methods can be implemented by instructing the relevant hardware through a computer program, and the computer program can be stored in a non-volatile computer-readable storage medium. When the computer program is executed, it can include the processes of the embodiments of the above-mentioned methods. Among them, any reference to memory, database or other media used in the embodiments provided in this application may include at least one of non-volatile and volatile memory. Non-volatile memory may include read-only memory (ROM), magnetic tape, floppy disk, flash memory, optical memory, high-density embedded non-volatile memory, resistive random access memory (ReRAM), magnetic random access memory (MRAM), ferroelectric random access memory (FRAM), phase change memory (PCM), graphene memory, etc. Volatile memory may include random access memory (RAM) or external cache memory, etc. By way of illustration and not limitation, RAM can be in various forms, such as static random access memory (SRAM) or dynamic random access memory (DRAM). The database involved in the various embodiments provided herein may include at least one of a relational database and a non-relational database. Non-relational databases may include, but are not limited to, distributed databases based on blockchains. The processor involved in the various embodiments provided herein may be, but are not limited to, a general-purpose processor, a central processing unit, a graphics processing unit, a digital signal processor, a programmable logic unit, a data processing logic unit based on quantum computing, and the like.

[0177] The embodiments of the present application will be described in detail below with reference to examples.

[0178] Example 1

[0179] In this example, peripheral blood from a patient diagnosed with an imprinted gene disease, amniotic fluid from a healthy fetus, and two abortion tissues were collected. Genomic DNA was extracted from the collected abortion tissue, peripheral blood, and amniotic fluid samples according to conventional methods, and then genome library construction and sequencing were performed according to the following process.

[0180] The library construction process includes the following steps:

[0181] (1) DNA fragmentation: Place 100 ng of DNA in a PCR tube, vortex to mix, and centrifuge briefly. Place the PCR tube on a DNA shearing ultrasonic disruptor for fragmentation. The fragmentation parameters are set to ultrasonication for 20 seconds, pause for 20 seconds, fragmentation time for 8 minutes, and 40% amplitude, to fragment the DNA into fragments of approximately 300-350 bp.

[0182] (2) End Repair Plus A: Add End Repair Plus A enzyme and End Repair Plus A buffer to the PCR tube containing the cell sample, mix well, and place in a PCR instrument for incubation. PCR program settings: 20°C, 30 min; 65°C, 30 min; 4°C, ∞.

[0183] (3) Adding adapters: Add methylated adapters, ligase mixture and reaction enhancer to the end repair plus A product, mix well and place in a PCR instrument for incubation. PCR program settings: 20℃, 15min; 4℃, ∞. (4) Purification of ligation product:

[0184] Add magnetic beads to the ligation product, mix well and let it stand for 5 minutes;

[0185] Place the PCR tube in a magnetic rack, let it stand for 2 minutes, and discard the liquid;

[0186] Add freshly prepared 80% ethanol to the tube, let it stand for 30 seconds, and discard the liquid;

[0187] Add freshly prepared 80% ethanol to the tube, let it stand for 30 seconds, discard the liquid, and then open the lid to dry for 5 minutes;

[0188] Add eluent to the tube, mix well and let stand for 5 min;

[0189] Place the PCR tube in a magnetic rack, let it stand for 2 minutes, and transfer the liquid to a new PCR tube.

[0190] (5) TET2 oxidation: Add TET2 enzyme, reaction buffer, reaction enhancer and reaction supplement to the purified library on ice, vortex mix, add iron solution, vortex mix again and place in PCR instrument for incubation at 37℃ for 1 hour.

[0191] (6) Oxidation termination: After the reaction is completed, immediately remove the PCR tube and place it on ice. Add the stop solution, mix well, and then place it in a PCR instrument for incubation at 37°C for 30 minutes.

[0192] (7) Purification of oxidation products:

[0193] Add magnetic beads to the oxidation product, mix well and let it stand for 5 minutes;

[0194] Place the PCR tube in a magnetic rack, let it stand for 2 minutes, and discard the liquid;

[0195] Add freshly prepared 80% ethanol to the tube, let it stand for 30 seconds, and discard the liquid;

[0196] Add freshly prepared 80% ethanol to the tube, let it stand for 30 seconds, discard the liquid, and then open the lid to dry for 5 minutes;

[0197] Add eluent to the tube, mix well and let stand for 5 min;

[0198] Place the PCR tube in a magnetic rack, let it stand for 2 minutes, and transfer the liquid to a new PCR tube.

[0199] (8) DNA denaturation: Add 0.1N NaOH to the PCR tube, then place the sample in a PCR instrument preheated at 50°C for 10 minutes and immediately place on ice after the incubation.

[0200] (9) Deamination reaction: Add deaminase, deamination buffer, and reaction supplement to a PCR tube on ice, vortex to mix, and then place in a PCR instrument and incubate at 37°C for 3 h.

[0201] (10) Purification of deamination products:

[0202] Add magnetic beads to the deamination product, mix well and let it stand for 5 minutes;

[0203] Place the PCR tube in a magnetic rack, let it stand for 2 minutes, and discard the liquid;

[0204] Add freshly prepared 80% ethanol to the tube, let it stand for 30 seconds, and discard the liquid;

[0205] Add freshly prepared 80% ethanol to the tube, let it stand for 30 seconds, discard the liquid, and then open the lid to dry for 5 minutes;

[0206] Add eluent to the tube, mix well and let stand for 5 min;

[0207] Place the PCR tube in a magnetic rack, let it stand for 2 minutes, and transfer the liquid to a new PCR tube.

[0208] (11) PCR amplification

[0209] Add amplification primers and PCR enzyme mixture to the PCR tube, mix well and place in the PCR instrument for amplification. The PCR program settings are: 98°C, 45s; (98°C, 15s; 65°C, 30s; 72°C, 30s) x 15; 72°C for 1min; 4°C, ∞.

[0210] (12) Purification of amplified products

[0211] Add magnetic beads to the amplified product, mix well and let it stand for 5 minutes;

[0212] Place the PCR tube in a magnetic rack, let it stand for 2 minutes, and discard the liquid;

[0213] Add freshly prepared 80% ethanol to the tube, let it stand for 30 seconds, and discard the liquid;

[0214] Add freshly prepared 80% ethanol to the tube, let it stand for 30 seconds, discard the liquid, and then open the lid to dry for 5 minutes;

[0215] Add eluent to the tube, mix well and let stand for 5 min;

[0216] ⑥ Place the PCR tube in the magnetic rack, let it stand for 2 minutes, and transfer the liquid to a new centrifuge tube.

[0217] (13) Library quality control: The library was quantified using a qPCR instrument, and the library fragment size was determined using an Agilent 2100;

[0218] (14) Library sequencing: After the library passes the quality inspection, the library is sequenced using the NGS sequencing platform.

[0219] The whole genome methylation sequencing data obtained after sequencing was analyzed according to the following data analysis process. The specific process is as follows:

[0220] The original whole-genome methylation sequencing data were subjected to data quality control, and the adapter sequences and low-quality bases were removed. Then, genome alignment was performed, and the aligned data were merged to remove PCR duplicate sequences.

[0221] Among them, by comparing the whole-genome methylation sequencing data with the genome, the exact position of each base sequence obtained by sequencing on the chromosome is determined, and then the chromosome multiplexity is analyzed using the currently common analysis process or software for detecting chromosome copy number variation;

[0222] In addition, by analyzing the methylation modification status of reads mapped to the imprinted gene region, the source of the reads (from the maternal DNA copy or the paternal DNA copy) can be determined. Then, based on the ratio of reads from different parental sources, it is determined whether the epigenetic modification of the imprinted gene region is normal.

[0223] Similarly, other regions of the genome, including imprinted regions, such as promoter regions, CpG islands, and other CpG dinucleotide-rich regions, can also be used to calculate the DNA methylation levels in the regions to determine whether there are abnormalities in methylation modifications.

[0224] For cytosine sites on the genome, the methylation level (ML) of each site is calculated by counting how many thymine (T) and cytosine (C) the site is covered by. The calculation formula is as follows:

[0225] ML i : methylation level of the ith cytosine site;

[0226] C i : The number of cytosine bases covering the i-th CG site;

[0227] T i : The number of thymine bases covering the i-th CG site;

[0228] The average methylation level of the genome is then calculated based on the methylation levels of all covered cytosine sites.

[0229] For calculation of DNA methylation levels in specific genomic regions, such as promoter regions or imprinted gene control regions, we can count all the cytosine sites in the region and how many thymines (T) and cytosines (C) are covered in total, and use the above formula to calculate the methylation level of each region. We can also evaluate the methylation status of each sequencing read by the methylation level of all cytosine sites on each sequencing read in the region, and evaluate the methylation level of each region based on the methylation status of each sequencing read in each region.

[0230] Example 2

[0231] The sample in this example was derived from fetal cells isolated from amniotic fluid, and the fetus was born healthy. Using the methods described in Example 1, a library was constructed and sequenced from these fetal cells. Chromosome analysis, based on whole-genome methylation sequencing data, revealed normal chromosome copy number in this sample, as shown in Figure 4 ; epigenetic modification information in various imprinted gene regions was also normal, as shown in Figure 5 .

[0232] Example 3

[0233] The sample in this example was tested for copy number variation using the existing CNV-seq method, and the clinical test report showed a 6.28Mb deletion on chromosome 15. In this example, peripheral blood was collected from the patient and sequenced using the method in Example 1. A 6.2Mb deletion was also found at the same location on chromosome 15, as shown in Figure 6.

[0234] At the same time, this example found that this location is the region where the imprinted gene SNURF is located. For the imprinted gene SNURF, the paternal gene is normally expressed, while the maternal gene is inhibited due to the high methylation level. If the paternal gene is missing, it will lead to epigenetic diseases, while the maternal gene will not affect health status.

[0235] Furthermore, this example adopts the bioinformatics analysis strategy in Example 1 to detect the methylation level of the deleted region, and finds that the sequencing reads of the deleted region are all in a high methylation state, and almost all sequencing reads have more than 85% of the cytosine sites modified by methylation, as shown in Figure 7. The numerical value after each sequencing read in Figure 7 represents the degree of methylation. For example, 1.0000 indicates that 100% of the cytosine sites are modified by methylation, and 0.9500 indicates that 95% of the cytosine sites are modified by methylation.

[0236] The above analysis results indicate that all these reads are from the non-deleted copy of the maternal chromosome, confirming that the deletion is caused by the paternal chromosome deletion. The patient's actual clinical symptoms also show symptoms of an epigenetic disease corresponding to the paternal chromosome deletion.

[0237] Example 4

[0238] In this example, a miscarriage tissue sample was collected and methylation library sequencing was performed using the method of Example 1. The test results showed that the chromosome copy number of the tissue was normal, as shown in Figure 8.

[0239] Furthermore, this example performed epigenetic analysis on the sample and found that the imprinted gene GNAS-XL was highly methylated, with more than 90% of the cytosine sites in almost all sequencing reads being methylated, as shown in Figure 9. The numerical value after each sequencing read in Figure 9 represents the degree of methylation. For example, 1.0000 indicates that 100% of the cytosine sites are methylated, and 0.9200 indicates that 92% of the cytosine sites are methylated.

[0240] These results suggest that epigenetic modifications of the imprinted gene GNAS-XL may be erroneous, potentially leading to miscarriage.

[0241] Example 5

[0242] In this example, a miscarriage tissue sample was collected and methylation library construction and sequencing was performed using the method of Example 1. The test results showed that the chromosome copy number of the tissue was normal, as shown in Figure 10.

[0243] Furthermore, this example performed epigenetic analysis on the sample and found that the imprinted gene H19 was highly methylated. In almost all sequencing reads, more than 70% of the cytosine sites were methylated, and in more sequencing reads, 90% of the cytosine sites were methylated, as shown in Figure 11. The numerical value after each sequencing read in Figure 11 represents the degree of methylation. For example, 1.0000 means that 100% of the cytosine sites are methylated, and 0.7500 means that 75% of the cytosine sites are methylated.

[0244] These results suggest that epigenetic modifications of the imprinted gene H19 may be erroneous, potentially leading to miscarriage.

[0245] The technical features of the above embodiments can be combined arbitrarily. To make the description concise, 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.

[0246] The above embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art could make various modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the present patent application shall be determined by the appended claims.

Claims

1. A method for detecting chromosome variation, characterized in that: include: Performing DNA methylation detection on a target sample, wherein the target sample is from at least one of fetal cells and / or tissues, neonatal blood, umbilical cord blood, and abortion tissue; The chromosome copy number variation information and / or epigenetic modification information of the target sample is analyzed based on the test results.

2. The method according to claim 1, characterized in that Analyzing the chromosome copy number variation information and / or epigenetic modification information of the target sample according to the test results includes: Obtain DNA methylation data of the target sample according to the test results; Analyzing chromosome copy number variation information and / or epigenetic modification information of the target sample based on the DNA methylation data of the target sample; The DNA methylation data include DNA methylation sequencing data based on sequencing and DNA methylation chip data based on chip.

3. The method according to claim 2, characterized in that The step of analyzing the chromosome copy number variation information and / or epigenetic modification information of the target sample according to the DNA methylation data of the target sample comprises: Obtaining chromosome copy number information and / or DNA methylation information of the target sample according to the DNA methylation data of the target sample; Determine the parental source of the chromosome copy number variation of the target sample based on the chromosome copy number information and DNA methylation information of the target sample; and / or Determine whether the target sample has abnormal epigenetic modification based on the DNA methylation information of the target sample.

4. The method according to claim 3, characterized in that The step of obtaining the chromosome copy number information of the target sample according to the DNA methylation data of the target sample includes: Determine the distribution position of the sequenced reads on the genome of the target sample based on the comparison result of the DNA methylation sequencing data of the target sample on the reference genome; The chromosome copy number variation information of the target sample is determined based on the distribution position of all sequencing reads of the target sample in the genome.

5. The method according to claim 3, characterized in that: The step of obtaining the DNA methylation information of the target sample according to the DNA methylation data of the target sample comprises: Determine the methylation modification status of each cytosine site on each sequencing read in the target region based on the DNA methylation sequencing data of the target sample; The DNA methylation information of the target sample is obtained based on the methylation modification status of each cytosine site on each sequencing read in the target region of the target sample.

6. The method according to claim 3, characterized in that The step of obtaining the DNA methylation information of the target sample according to the DNA methylation data of the target sample comprises: Determine the DNA methylation state of each sequencing read in the target region according to the DNA methylation sequencing data of the target sample, wherein the DNA methylation state includes a high methylation state or a low methylation state; The DNA methylation information of the target sample is obtained according to the DNA methylation status of each sequencing read in the target region of the target sample.

7. The method according to claim 5 or 6, characterized in that: The target area satisfies at least one of the following characteristics (1) to (5): (1) The target region includes the whole genome region; (2) the target region includes a promoter region; (3) the target area includes a print control area; (4) The target region includes an enhancer region in the genome that regulates gene expression; (5) The target region includes a repressor region in the genome that regulates gene expression.

8. The method according to claim 7, characterized in that Determining the parental source of the chromosome copy number variation of the target sample according to the chromosome copy number variation information and DNA methylation information of the target sample includes: According to the chromosome copy number variation information and DNA methylation information of the target sample, the DNA methylation information of the copy number variation region of the target sample is obtained; Determine the parental source of the target sample chromosome copy number variation based on the DNA methylation information of the target sample copy number variation region; Optionally, the copy number variation region includes an imprinting control region.

9. The method according to claim 7, characterized in that: Determining whether the target sample has abnormal epigenetic modification based on the DNA methylation information of the target sample includes: Based on the DNA methylation status of each sequencing read in the imprinting control region of the target sample, it is determined whether the target sample has abnormal epigenetic modification of the paternal chromosome or the maternal chromosome.

10. A device for detecting chromosome mutation, characterized in that: include: DNA methylation detection module: used to perform DNA methylation detection on a target sample, wherein the target sample is from at least one of fetal cells and / or tissues, neonatal blood, umbilical cord blood, and abortion tissue; Information analysis module: used to analyze the chromosome copy number variation information and / or epigenetic modification information of the target sample based on the test results.

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