CN117025753A

CN117025753A - Method and device for detecting chromosomal variation

Info

Publication number: CN117025753A
Application number: CN202311028658.9A
Authority: CN
Inventors: 姚雪龙; 刘江; 林戈
Original assignee: Guangzhou Nuwa Life Technology Co ltd
Current assignee: Guangzhou Nuwa Life Technology Co ltd
Priority date: 2023-08-15
Filing date: 2023-08-15
Publication date: 2023-11-10

Abstract

The application relates to a method and a device for detecting chromosome variation, wherein the method comprises the following steps: performing DNA methylation detection on a target sample; and analyzing chromosome copy number variation information and/or epigenetic modification information of the target sample according to the detection result. According to the application, a novel sequencing technology principle is adopted for a target sample to construct a DNA methylation map, and chromosome copy number information and epigenetic modification information of the sample are detected according to the DNA methylation map, so that variation information obtained simultaneously is increased.

Description

Method and device for detecting chromosomal variation

Technical Field

The application relates to the field of reproductive medicine, in particular to a method and a device for detecting chromosome variation.

Background

Chromosome is an important vector of human genetic genes, and once abnormal, it may cause birth defects such as abortion, congenital foolish, congenital multiple deformity, etc.

Spontaneous abortion is the most common pregnancy complication in obstetrics and gynecology, generally refers to the failure of pregnancy process, and mainly comprises biochemical pregnancy, empty gestational sac, gradual stopping of embryo development, death of embryo or fetus, and discharge of embryo and its appendages. The detection of aborted tissue is a main means for finding the cause of abortion. At present, the clinical practice mainly searches for the cause of abortion by detecting chromosome copy number variation of aborted tissues. However, there are still 50% of clinical miscarriage tissue chromosome fold normal, and the cause of miscarriage cannot be clarified by the prior art means, which results in that a significant proportion of miscarriage patients cannot be treated or intervened at present.

Numerous studies in the past have shown that epigenetic events play an important role in the regulation of early embryo development. Errors in epigenetic information during embryo development can lead to embryo development arrest, pregnancy failure and birth defects, however, the current clinic lack of effective technical methods for detecting the epigenetic information of embryos. Amniotic fluid puncture is a relatively common prenatal screening method, and mainly comprises the steps of detecting amniotic fluid by amniocentesis during the period of 16-20 weeks of gestation to determine the health condition of a fetus and whether the fetus has genetic diseases or not. Chromosome fold screening is one of the main contents of amniotic fluid detection, and can effectively reduce the ratio of birth defects.

The current clinical detection method for chromosome copy number variation mainly comprises the following three steps: chromosome karyotyping, chromosome Microarray Analysis (CMA) techniques, and copy number variation sequencing (CNVseq) based on next generation sequencing techniques. The chromosome karyotype is an image formed by sequentially pairing and grouping all chromosomes in a single body cell according to the size and morphological characteristics, and the chromosome karyotype analysis technology has been considered as a 'gold standard' for determining chromosome copy number variation for many years. Chromosome microarray analysis technology (CMA): the method is used for scanning the whole genome and detecting Copy Number Variation (CNV) of chromosome imbalance, and has outstanding advantages in the aspects of detecting genome imbalance abnormality such as genome microdeletion, microreplication and the like.

In the current clinic, in the detection of amniotic fluid, the epigenetic modification information of the embryo cannot be detected, so that whether the embryo has the epigenetic disease cannot be judged in an assisted manner. On the other hand, in CNV detection of amniotic fluid, if a chromosome deletion is detected, it cannot be determined whether a fragment derived from a parent source or a fragment derived from a maternal source is deleted, and thus a clinician cannot determine whether a fetus is likely to suffer from a disease. The method is a great challenge for amniotic fluid detection at present and is also a technical problem to be solved.

Therefore, how to obtain more mutation information at the same time is a difficulty in embryo detection of chromosomal mutation.

Disclosure of Invention

In order to solve the above problems and provide more chromosome abnormality information at the same time, a first object of an embodiment of the present application is to provide a method for detecting chromosome variation, which includes:

performing DNA methylation detection on a target sample, wherein the target sample is at least one of embryo cells, fetal cells and/or tissues, neonatal blood, umbilical cord blood and aborted tissues;

and analyzing chromosome copy number variation information and/or epigenetic modification information of the target sample according to the detection result.

The application adopts a brand-new sequencing technology principle to construct a whole genome DNA methylation map, detects chromosome copy number information and epigenetic modification information of a sample according to the DNA methylation map, and can provide more chromosome variation information at the same time.

In one embodiment, analyzing the chromosomal copy number variation information and/or the epigenetic modification information of the target sample based on the detection result comprises:

obtaining DNA methylation data of a target sample according to the detection result;

analyzing chromosome copy number variation information and/or epigenetic modification information of the target sample according to the DNA methylation data of the target sample;

wherein the DNA methylation data includes sequencing-based DNA methylation sequencing data and chip-based DNA methylation chip data.

In one embodiment, analyzing chromosomal copy number variation information and/or epigenetic modification information of the target sample based on DNA methylation sequencing data of the target sample comprises:

obtaining chromosome copy number information and/or DNA methylation information of a target sample according to DNA methylation data of the target sample;

determining a parent source of chromosome copy number variation of the target sample according to chromosome copy number information and DNA methylation information of the target sample; and/or

Determining whether the target sample has epigenetic modification abnormality according to DNA methylation information of the target sample.

In one embodiment, obtaining chromosomal copy number information of a target sample from DNA methylation data of the target sample comprises:

determining the distribution position of reads obtained by sequencing on the genome of the target sample according to the comparison result of the DNA methylation sequencing data of the target sample on the reference genome;

and determining the chromosome copy number variation information of the target sample according to the distribution position of all sequencing reads of the target sample in the genome.

In one embodiment, obtaining DNA methylation information of a target sample from DNA methylation data of the target sample comprises:

determining the methylation modification state of each cytosine site on each sequencing reads of the target region according to the DNA methylation sequencing data of the target sample;

and obtaining DNA methylation information of the target sample according to the methylation modification state of each cytosine locus on each sequencing reads of the target region of the target sample.

determining the DNA methylation status of each sequencing reads of the target region according to the DNA methylation sequencing data of the target sample, wherein the DNA methylation status comprises a hypermethylation status or a hypomethylation status;

And obtaining DNA methylation information of the target sample according to the DNA methylation state of each sequencing ready of the target region of the target sample.

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

(1) The target region includes a whole genome region;

(2) The target region includes a promoter region;

(3) The target area includes a footprint control area;

(4) The target region comprises an enhancer region in the genome for regulating and controlling gene expression;

(5) The target region includes an inhibitor region in the genome that regulates gene expression.

In one embodiment, determining the parental source of chromosomal copy number variation of the target sample based on chromosomal copy number variation information and DNA methylation information of the target sample comprises:

acquiring DNA methylation information of a copy number variation region of the target sample according to the chromosome copy number variation information and the DNA methylation information of the target sample;

determining a parent source of the chromosome copy number variation of the target sample according to the DNA methylation information of the copy number variation region of the target sample;

optionally, the copy number variation region comprises a print control region.

In one embodiment, determining whether the target sample has an epigenetic modification abnormality based on DNA methylation information of the target sample comprises:

Determining whether the target sample has the apparent genetic modification abnormality of the parent chromosome or the maternal chromosome according to the DNA methylation state of each sequencing reads of the imprinting control region of the target sample.

A second object of the present application is to provide an apparatus for detecting chromosomal variation, comprising:

DNA methylation detection module: for performing DNA methylation detection on a target sample derived from at least one of embryonic cells, fetal cells and/or tissue, neonatal blood, umbilical cord blood, aborted tissue;

and the information analysis module is used for: for analyzing chromosomal copy number variation information and/or epigenetic modification information of the target sample based on the detection result.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present application, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a block flow diagram of a method for detecting chromosomal variations according to an embodiment of the present application;

FIG. 2 is a block diagram of an apparatus for detecting chromosomal variation according to an embodiment of the present application;

FIG. 3 is an internal block diagram of a computer device according to an embodiment of the present application;

FIG. 4 shows the results of chromosome detection of a fetal cell sample isolated from amniotic fluid in example 1 according to the present application;

FIG. 5 shows methylation levels of individual imprinted genes of an isolated fetal cell sample from amniotic fluid in example 1 according to the application;

FIG. 6 shows the results of chromosome detection of a peripheral blood sample of a patient suffering from a imprinted gene disease in example 2 of the application;

FIG. 7 shows methylation levels of the imprinting gene SNURF of a peripheral blood sample of a patient suffering from an imprinting gene disease in example 2 of the present application;

FIG. 8 shows chromosome testing results of a tissue sample produced in accordance with example 3 of the present application;

FIG. 9 shows methylation information of the imprinted gene GNAS-XL of the tissue sample of example 3;

FIG. 10 shows chromosome testing results of a tissue sample produced in accordance with example 4 of the present application;

FIG. 11 shows methylation information of the imprinted gene H19 of the flow tissue sample in example 4 of the application.

Detailed Description

Reference now will be made in detail to embodiments of the application, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the application. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present application without departing from the scope or spirit of the application. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.

Accordingly, it is intended that the present application cover such modifications and variations as fall within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present application will be disclosed in or be apparent from the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present application.

To at least partially solve the above technical problem, a first aspect of the present application provides a method for detecting chromosomal variation, as shown in fig. 1, including:

s10, performing DNA methylation detection on a target sample, wherein the target sample is at least one of fetal cells and/or tissues, neonatal blood, umbilical cord blood and aborted tissues;

s20, analyzing chromosome copy number variation information and/or epigenetic modification information of the target sample according to the detection result;

in some embodiments, the target samples are all derived from a mammal.

As used herein, the term "mammal" includes any one of a diploid animal of humans, tigers, wolves, mice, deer, minks, monkeys, taping, lazy, zebra, dogs, foxes, bears, elephants, leopards, musk, cattle, lions, pandas, warts, pigs, antelopes, reindeer, kola, rhinoceros, lynx, pangolins, giraffes, pandas, formicary, gorilla, sea cows, otter, civet, dolphin, sea elephants, duckbill, hedgehog, arctic fox, polar bear, kangaroo, armadi, river horses, seals, whales, ferrets, rabbits.

In some embodiments, the target sample is from aborted tissue, a fetus, or a neonate of a mammal.

In some embodiments, the target sample may be derived from a pre-uterine embryo sample implanted in assisted reproductive technology, from fetal cells and/or tissue samples obtained during gestation by invasive methods such as, for example, chorionic villus, amniocentesis, and percutaneous umbilical vascular puncture, and from aborted tissue or neonatal peripheral blood.

Thus, in some embodiments, the sample of interest is selected from the group consisting of an in vitro fertilized egg, a pre-implantation embryonic cell, a fetal cell or tissue in amniotic fluid, umbilical cord blood, aborted tissue, or neonatal blood.

The application carries out DNA methylation detection on fetal cells or tissues, analyzes chromosome copy number variation information and/or epigenetic modification information according to detection results, and can provide more support information for clinical evaluation of fetal or cellular abortion risk evaluation and intervene in the condition of stopping pregnancy more timely. DNA methylation detection is carried out on aborted tissues, chromosome copy number variation information and/or epigenetic modification information are analyzed according to detection results, more detection information can be provided for aborted cause analysis, and when the chromosome copy number is normal, aborted causes are analyzed from the epigenetic modification angle, so that follow-up more comprehensive pregnancy suggestions are provided for detected subjects. DNA methylation detection is carried out on umbilical cord blood or neonatal blood, chromosome copy number variation information and/or epigenetic modification information are analyzed according to detection results, and abnormal epigenetic modification diseases of the neonate can be intervened in time, so that related disease deterioration is avoided.

In particular, DNA methylation detection includes sequencing-based DNA methylation detection and chip-based DNA methylation detection. The DNA methylation detection mode of the target sample is not particularly limited, and can be DNA methylation detection based on sequencing or DNA methylation detection based on a chip, so long as chromosome copy number variation information and epigenetic modification information of the target sample can be obtained simultaneously.

In some embodiments, sequencing-based DNA methylation detection refers to the step of, after transformation of DNA in a target sample such that unmethylated modified cytosines on the DNA are converted to uracil, pooling and sequencing the transformed DNA to obtain DNA methylation sequencing data for the target sample.

In some embodiments, the sequencing data is second generation sequencing data.

Illustratively, sequencing-based DNA methylation detection includes bisulfite conversion sequencing (BS-Seq), whole genome methylation sequencing (WGBS), and third generation Sequencing (SMRT), among others.

The chip-based DNA methylation detection is used for detecting DNA methylation information by using a methylation chip, and the principle is that the DNA sample is subjected to sulfite treatment firstly based on signal detection of hybridization of a DNA sequence after sulfite treatment, so that unmethylated cytosine is changed into uracil, and methylated cytosine is kept unchanged, so that the DNA sample is distinguished after subsequent sequencing or hybridization.

In particular, for the case of simultaneously acquiring chromosomal copy number variation information and epigenetic modification information based on a DNA methylation detection result, the present application provides a method for simultaneously detecting chromosomal copy number variation information and epigenetic information, comprising:

and (3) carrying out DNA methylation detection on the target sample, and analyzing chromosome copy number variation information and epigenetic modification information of the target sample according to the detection result.

Specifically, the epigenetic modification abnormality information may be obtained based on the DNA methylation detection result of the target sample, the chromosomal copy number variation information may be obtained, and further chromosomal copy number variation information, such as a chromosomal copy number variation source, may be obtained in combination with the chromosomal copy number variation information and the epigenetic modification abnormality information.

Accordingly, analyzing the chromosomal copy number variation information and/or the epigenetic modification information of the target sample based on the detection result includes:

In some embodiments, analyzing chromosomal copy number variation information and/or epigenetic modification information of the target sample based on DNA methylation data of the target sample comprises:

Specifically, the chromosomal copy number information includes a chromosomal copy number variation region. Specifically, for humans, the human genome contains about 31.6 hundred million DNA base pairs, constituting 46 chromosomes, divided into 23 pairs, one of each pair inherited from the father, one inherited from the mother, 2 chromosomes, also known as homologous chromosomes, of substantially identical size and genetic composition, 22 pairs being autosomes, the last pair being sex chromosomes, XX for females and XY for males. Normal copy number of autosomal and female X chromosome is 2, normal copy number of male X and Y chromosome is 1, duplication is the case when the copy number detection value is greater than normal, and deletion is the case when the copy number detection value is less than normal.

In some embodiments, obtaining chromosomal copy number information of the target sample from DNA methylation data of the target sample comprises:

Specifically, when the target sample is a human, the reference genome is a human reference genome, and more specifically, human reference genome hg38 may be used.

In some embodiments, to obtain more accurate acquisition of alignment data, the acquiring DNA methylation information of the target sample from the whole genome methylation sequencing data is preceded by:

removing the de-adaptor sequence and low-quality base in the DNA methylation sequencing data to obtain sequencing data after data quality control processing;

comparing the DNA methylation sequencing data subjected to data quality control treatment with a reference genome, and removing the repeated sequence in the DNA methylation sequencing data according to the comparison result.

In particular, epigenetic modification refers to the regulation of gene expression by chemical modification of DNA and proteins on chromosomes that can affect multiple levels of gene transcription, splicing, stability, translation, nucleosome assembly, and chromatin structure, thereby affecting physiological and pathological processes of cells, and phenotype of offspring.

Wherein, epigenetic modification abnormalities refer to the inability of a gene to alter DNA and proteins on a chromosome by chemical modification to effect gene expression, and to affect physiological and pathological processes of cells, as well as the phenotype of offspring.

In order to obtain the epigenetic modification information of the embryo, the application firstly provides that the chromosome copy number variation information and/or the DNA methylation information of the target sample are obtained according to the DNA methylation detection result of the target sample, and then whether the epigenetic modification of the target sample is abnormal or not is analyzed according to the DNA methylation information of the target sample, or whether the chromosome and the epigenetic modification of the target sample are abnormal or not is analyzed simultaneously.

For DNA methylation sequencing data, DNA methylation information can be calculated for the methylation level of each cytosine site by calculating the methylation modification state of each cytosine site on different sequencing reads, and further DNA methylation information of a target sample can be obtained based on the methylation level of each cytosine site, or can be calculated for each sequencing reads by calculating the methylation modification state of different cytosine sites on each sequencing reads, and methylation information of a target sample can be obtained from reads of different methylation states.

The position of the base of each ready in the DNA methylation sequencing data on the chromosome can be determined according to the alignment data of the DNA methylation sequencing data and the reference genome, and for a cytosine site at a certain position on the chromosome, the methylation modification state of the cytosine site is distinguished according to the base type (C or T) of the cytosine site on each sequencing ready.

In some embodiments, obtaining DNA methylation information of the target sample from DNA methylation data of the target sample comprises:

determining the methylation modification state of each cytosine site on each sequencing read according to the DNA methylation sequencing data of the target sample;

and obtaining DNA methylation information of a target region of the target sample according to the methylation modification state of each cytosine locus on each sequencing reads of the target sample.

Specifically, for each cytosine position in the target region, the methylation level of each cytosine position is calculated according to the number of C bases and the number of T bases of the cytosine position on all sequencing reads, and then the DNA methylation information of the target region is calculated according to the average value of the methylation levels of the cytosine positions in the target region.

The methylation level of each cytosine site is calculated as follows:

Wherein ML _i Representing the methylation level of the ith cytosine locus of the target gene region;

C _i the number of cytosine in the DNA methylation sequencing data that covers the ith CG locus;

T _i the number of thymine nucleotides covering the ith CG site in the DNA methylation sequencing data is shown.

In other embodiments, obtaining DNA methylation information of a target sample from DNA methylation data of the target sample comprises:

and obtaining DNA methylation information of the target gene region of the target sample according to the DNA methylation state of each sequencing reads of the target region of the target sample.

In particular, the methylation state of each sequencing reads is used to describe the overall methylation level of each sequencing read, including a first methylation state, a second methylation state, and a third methylation state, wherein the first methylation state can represent a hypermethylation state, the second methylation state can represent a hypomethylation state, and the third methylation state can represent a medium methylation state. The definition of different methylation status reads allows the methylation level of each cytosine site of the sequencing reads to be set according to the detection requirements.

In some embodiments, reads are defined as reads in a hypermethylated state if more than 65% of the cytosine sites in the reads are methylation modified, reads in a hypomethylated state if more than 65% of the cytosine sites in the reads are not methylation modified, and reads in a medium methylation state if the methylation modified cytosine sites account for 35% -65% of all the cytosine sites in the reads.

In some embodiments, the region of interest comprises a whole genome.

In some embodiments, the target region comprises a promoter region.

In some embodiments, the target area comprises an imprinted control region.

In some embodiments, the region of interest comprises an enhancer region in the genome that regulates expression of a gene;

in some embodiments, the region of interest comprises an inhibitor region in the genome that regulates gene expression.

Specifically, whole genome refers to the sequence of the complete genome of an organism, and methylation levels of the whole genome are too high or too low, which may lead to abnormalities in gene expression.

The promoter (promoter) refers to a sequence that binds to RNA polymerase on the genome and initiates mRNA synthesis. Too high a methylation level of the promoter region may result in inhibition of gene expression. Illustratively, some of the gene promoter regions are shown in table 1.

TABLE 1

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Genomic imprinting, also known as genomic imprinting, parental imprinting or gametophyte imprinting, refers to the phenomenon in which alleles or chromosomes from a parent undergo specific processing modifications during development, resulting in silencing of one allele of the parent, and thus in the presence of different expression activities in the two parent-derived alleles in the offspring somatic cell. Accordingly, a gene having such a difference is called a imprinted gene. Most imprinted genes are clustered in large chromosomal regions, and inhibition of allele-specific expression of imprinted genes is regulated by the Imprinted Control Region (ICR), usually methylation of ICR on the allele side.

Illustratively, some imprinted gene regions are shown in table 2, wherein P represents the male parent imprinting, i.e., the male parent's gene is not expressed and the female parent's allele is expressed; m represents maternal imprint, i.e., the gene of the maternal plant is not expressed, the allele of the paternal plant is expressed.

TABLE 2

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In some embodiments, the DNA methylation information for a region of interest can be calculated based on the ratio of the number of methylation-modified cytosines in that region, i.e., in all reads, to the number of cytosines in those reads.

In some embodiments, determining the parental source of chromosomal copy number variation in the target sample based on the chromosomal copy number variation information and the DNA methylation information of the target sample comprises:

and determining the parent source of the chromosome copy number variation of the target sample according to the DNA methylation information of the copy number variation region of the target sample.

Specifically, for a sample with chromosome copy number variation, determining whether a chromosome variation region of the target sample includes a print control region according to the chromosome copy number variation, if the chromosome variation region of the target sample includes the print control region, determining a parent source of the chromosome variation of the target sample according to methylation information of the print control region of the target sample.

For example, for a imprinted gene highly expressed by a parent, reads of the imprinted control region in the chromosome copy number deletion region are almost all in the first methylation state, and it can be inferred that the chromosome copy number deletion is due to the chromosome copy number variation of the parent. For imprinted genes with high maternal expression, reads of imprinted control regions in the chromosome copy number deletion region are almost all in the first methylation state, it can be inferred that the chromosome copy number deletion is due to maternal chromosome copy number variation.

It will be appreciated that in the case of more chromosomal copy number variations in a clinical fetal sample, it may be determined whether the chromosomal copy number loss is due to a parental or maternal chromosomal copy number variation based on the methylation level of the imprinted control region in the chromosomal copy number loss region, thereby providing reference information for reproductive health of the embryo parents and their subsequent fertility strategies.

In some embodiments, determining whether the target sample has an epigenetic modification abnormality based on DNA methylation information of the target sample comprises:

In reagent application, for example, when detecting the abortion tissue, the chromosome fold information and/or the epigenetic modification information of the abortion tissue can be detected simultaneously according to DNA methylation detection, so that the clinical detection efficiency is greatly improved, the economic burden of a patient is reduced, and more information is provided for subsequent treatment or clinical intervention. Determining whether the target sample has the apparent genetic modification abnormality of the parent chromosome or the maternal chromosome according to the DNA methylation state of each sequencing reads of the abortive tissue print control region.

In summary, the method for detecting chromosomal variation of the present application has the following beneficial effects:

1. the application adopts a brand-new sequencing technology principle to realize the construction of a whole genome DNA methylation map of genetic materials (cells and other tissues from fetuses) of fetuses separated from the fluid tissues (villus, placenta and the like), peripheral blood and amniotic fluid, and judges and detects chromosome copy number information and epigenetic modification information of a sample according to the DNA methylation map;

2. the application provides an analysis strategy for judging whether the chromosome copy number variation is caused by the parent chromosome copy number variation or the parent chromosome copy number variation according to the DNA methylation map;

3. the application establishes a set of methods and standards for detecting whether the imprinting gene disease exists or not aiming at the detection of imprinting genes.

In a second aspect, the application provides an apparatus for detecting chromosomal variations, comprising:

DNA methylation detection module: for DNA methylation detection of a target sample derived from at least one of fetal cells and/or tissue, neonatal blood, umbilical cord blood, aborted tissue;

In some embodiments, the information analysis module comprises:

an acquisition unit: the DNA methylation data are used for obtaining a target sample according to the detection result;

information analysis unit: analyzing chromosome copy number variation information and/or epigenetic modification information of the target sample according to the DNA methylation data of the target sample;

In some embodiments, the information analysis unit comprises:

an information acquisition subunit: the method comprises the steps of obtaining chromosome copy number information and/or DNA methylation information of a target sample according to DNA methylation data of the target sample;

a first information analysis subunit: a parent source for determining a chromosomal copy number variation of the target sample based on the chromosomal copy number information and the DNA methylation information of the target sample; and/or

A second information analysis subunit: determining whether the target sample has epigenetic modification abnormality according to DNA methylation information of the target sample.

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

the chromosome copy number information acquisition subunit is configured to: determining the distribution position of reads obtained by sequencing on the genome of the target sample according to the comparison result of the DNA methylation sequencing data of the target sample on the reference genome;

Determining chromosome copy number variation information of the target sample according to the distribution positions of all sequencing reads of the target sample in a genome;

the DNA methylation information acquisition subunit is for:

acquiring DNA methylation information of a target sample according to the methylation modification state of each cytosine locus on each sequencing reads of a target region of the target sample;

alternatively, the DNA methylation information acquisition subunit is configured to:

In some embodiments, the target region satisfies at least one of the following characteristics:

(1) The target region includes a whole genome region;

(2) The target region includes a promoter region;

(3) The target area includes a footprint control area;

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

optionally, the copy number variation region comprises a print control region.

The second information analysis subunit is configured to:

determining whether the target sample has the epigenetic modification abnormality according to the DNA methylation state of each sequencing reads of the imprinting control area of the target sample.

Specific limitations regarding the means for detecting chromosomal variations may be found in the above definitions of methods for detecting chromosomal variations, and are not described in detail herein. The respective modules in the above-described apparatus for detecting chromosomal variations may be implemented in whole or in part by software, hardware, and combinations thereof. The above modules may be embedded in hardware or may be independent of a processor in the computer device, or may be stored in software in a memory in the computer device, so that the processor may call and execute operations corresponding to the above modules.

In some embodiments, a computer device is provided, which may be the server 104 or the terminal 102, and the internal structure diagram thereof may be as shown in fig. 3. The computer device includes a processor, a memory, and a communication interface connected by a system bus. When the computer equipment is a terminal, the system also comprises a display screen and an input device which are connected with the system bus. Wherein the processor of the computer device is configured 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 programs in the non-volatile storage media. The network interface of the computer device is used for communicating with an external terminal through a network connection. The computer program is executed by a processor to implement a method of detecting chromosomal variations. The display screen of the computer equipment can be a liquid crystal display screen or an electronic ink display screen, and the input device of the computer equipment can be a touch layer covered on the display screen, can also be keys, a track ball or a touch pad arranged on the shell of the computer equipment, and can also be an external keyboard, a touch pad or a mouse and the like.

It will be appreciated by those skilled in the art that the structure shown in FIG. 3 is merely a block diagram of some of the structures associated with the present inventive arrangements and is not limiting of the computer device to which the present inventive arrangements may be applied, and that a particular computer device may include more or fewer components than shown, or may combine some of the components, or have a different arrangement of components.

The application also provides computer equipment. The computer device comprises a memory in which a computer program is stored and a processor which, when executing the computer program, carries out the steps of the above-described method of detecting chromosomal variations.

The application also provides a computer readable storage medium. A computer readable storage medium having stored thereon a computer program which, when executed by a processor, performs the steps of the method of detecting chromosomal variations described above.

The application also provides a computer program product. Computer program product comprising a computer program which, when executed by a processor, implements the steps of the above-described method of detecting chromosomal variations.

The user information (including but not limited to user equipment information, user personal information, etc.) and the data (including but not limited to data for analysis, stored data, presented data, etc.) related to the present application are information and data authorized by the user or sufficiently authorized by each party.

Those skilled in the art will appreciate that implementing all or part of the above-described methods may be accomplished by way of a computer program, which may be stored on a non-transitory computer readable storage medium, that when executed may comprise the steps of the embodiments of the methods described above. Any reference to memory, database, or other medium used in embodiments provided herein may include at least one of non-volatile and volatile memory. The nonvolatile memory may include Read-only memory (ROM), magnetic tape, floppy disk, flash memory, optical memory, high density embedded nonvolatile memory, resistive random access memory (ReRAM), magnetic random access memory (Magnetoresistive Random Access Memory, MRAM), ferroelectric memory (Ferroelectric Random Access Memory, FRAM), phase change memory (Phase Change Memory, PCM), graphene memory, and the like. Volatile memory can include random access memory (Random Access Memory, RAM) or external cache memory, and the like. By way of illustration, and not limitation, RAM can be in the form of a variety of forms, such as static random access memory (Static Random Access Memory, SRAM) or dynamic random access memory (Dynamic RandomAccess Memory, DRAM), and the like. The databases referred to in the embodiments provided herein may include at least one of a relational database and a non-relational database. The non-relational database may include, but is not limited to, a blockchain-based distributed database, and the like. The processor referred to in the embodiments provided in the present application may be a general-purpose processor, a central processing unit, a graphics processor, a digital signal processor, a programmable logic unit, a data processing logic unit based on quantum computing, or the like, but is not limited thereto.

Embodiments of the present application will be described in detail below with reference to examples.

Example 1

In this example, peripheral blood of a patient who had been diagnosed with a imprinted gene disease, amniotic fluid of a fetus produced in a healthy state, and two aborted tissues were collected, and genomic DNA was extracted from the aborted tissues, peripheral blood, and amniotic fluid samples collected by a conventional method, and then subjected to genomic library sequencing according to the following procedures.

The library construction procedure included the following steps:

(1) DNA fragmentation: 100ng of DNA was placed in a PCR tube, vortexed, and centrifuged transiently. The PCR sample tube was placed on a DNA shear sonicator for disruption. The breaking parameter is set to ultrasonic 20s, stagnation 20s, breaking time is 8min,40% amplitude, breaking DNA into about 300-350 bp fragments.

(2) End repair plus a: adding end repair and A enzyme and end repair and A buffer solution into a PCR tube filled with a cell sample, uniformly mixing, and then placing into a PCR instrument for incubation. PCR program settings: 20 ℃ for 30min;65 ℃ for 30min;4 ℃ and infinity.

(3) And (3) adding a joint: adding a methylation joint, a ligase mixed solution and a reaction enhancer into the end repair and addition A product, uniformly mixing, and then placing the mixture into a PCR instrument for incubation. PCR program settings: 20 ℃ for 15min;4 ℃ and infinity. (4) purification of ligation product:

Adding magnetic beads into the connection product, uniformly mixing, and standing for 5min;

placing the PCR tube into a magnetic rack, standing for 2min, and discarding the liquid;

adding 80% ethanol into the tube, standing for 30s, and discarding the liquid;

adding 80% ethanol into the tube, standing for 30s, discarding the liquid, uncovering, and air drying for 5min;

adding eluent into the tube, uniformly mixing, and standing for 5min;

the PCR tube was placed in a magnetic rack, left to stand for 2min, and the liquid was transferred to a new PCR tube.

(5) TET2 oxidation: adding TET2 enzyme, reaction buffer, reaction enhancement solution and reaction supplement into the purified library on ice, adding an iron solution after vortex mixing, and placing the mixture into a PCR instrument for incubation at 37 ℃ for 1h after vortex mixing again.

(6) Oxidation termination: immediately taking out the PCR tube after the reaction is finished, putting the PCR tube on ice, adding a stop solution, mixing uniformly, and then putting the mixture into a PCR instrument for incubation at 37 ℃ for 30min.

(7) Purification of oxidation products:

adding magnetic beads into the oxidation product, uniformly mixing, and standing for 5min;

adding 80% ethanol into the tube, standing for 30s, and discarding the liquid;

Adding eluent into the tube, uniformly mixing, and standing for 5min;

(8) DNA denaturation: naOH of 0.1N is added into a PCR tube, then a sample is placed into a PCR instrument preheated at 50 ℃ in advance for incubation for 10min, and the sample is placed on ice immediately after incubation is finished.

(9) Deamination reaction: deaminase, deamination buffer and reaction supplement are added into the PCR tube on ice, after vortex mixing, the mixture is put into a PCR instrument for incubation for 3h at 37 ℃.

(10) Purification of deamination product:

adding magnetic beads into the deamination product, uniformly mixing and standing for 5min;

adding 80% ethanol into the tube, standing for 30s, and discarding the liquid;

adding eluent into the tube, uniformly mixing, and standing for 5min;

(11) PCR amplification

Adding the mixed solution of the amplification primer and the PCR enzyme into a PCR tube, uniformly mixing, and then placing into a PCR instrument for amplification, wherein the PCR program is set as follows: 98 ℃ for 45s; (98 ℃,15s;65 ℃,30s;72 ℃,30 s) x15;72 ℃ for 1min;4 ℃ and infinity.

(12) Amplification product purification

Adding magnetic beads into the amplified product, uniformly mixing, and standing for 5min;

adding 80% ethanol into the tube, standing for 30s, and discarding the liquid;

adding eluent into the tube, uniformly mixing, and standing for 5min;

(6) the PCR tube was placed in a magnetic rack and allowed to stand for 2min, and the liquid was transferred to a new centrifuge tube.

(13) Library quality inspection: quantitative determination of the library using qPCR instrument and determination of library fragment size using agilent 2100;

(14) Library sequencing: and after the library quality is checked to be qualified, sequencing the library by adopting an NGS sequencing platform.

The whole genome methylation sequencing data obtained after sequencing is analyzed according to the following data analysis flow, and the specific process is as follows:

and (3) performing data quality control on the original whole genome methylation sequencing data, removing the linker sequence and the low-quality base, performing genome comparison, merging the compared data, and removing the PCR repeated sequence.

Determining the exact position of each base sequence obtained by sequencing on a chromosome by comparing genome-wide methylation sequencing data with genome, and then analyzing the chromosome fold by using the current general analysis flow or software for detecting chromosome copy number variation;

In addition, the origin of reads (either a copy of DNA from the parent source or a copy of DNA from the parent source) was determined by analyzing the methylation modification status of reads aligned to the imprinted gene region. And then judging whether the epigenetic modification of the imprinted gene region is normal or not according to the ratios of reads from different parent sources.

Similarly, other regions of the genome, including the imprinted region, such as promoter regions, cpG islands, and other CpG dinucleotide rich regions, can also be used to determine if the region is abnormal in methylation modification by calculating the DNA methylation level of the region.

For cytosine sites on the genome, the methylation level (Methylation Level, ML) of each site was calculated by counting how much thymine (T) and cytosine (C) are covered by the site, respectively, as follows:

MLi: methylation level of the ith cytosine site;

ci: the bases covering the ith CG site are the number of cytosines;

ti: the base covering the ith CG site is the number of thymines;

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

For the calculation of the DNA methylation level of a specific region of the genome, such as a promoter region or a imprinted gene control region, it is possible to count all cytosine sites in that region, how much thymine (T) and cytosine (C) are covered in total, and calculate the methylation level of each region by the above formula; methylation status of each sequencing reads can also be assessed by methylation levels of all cytosine sites on each sequencing read within the region, with methylation levels of each region being assessed based on methylation status of each sequencing read of each region.

Example 2

The sample of this example is derived from foetal cells isolated in amniotic fluid, and the foetus is now healthy to birth. This example uses the method of example 1 to perform pooling sequencing of the fetal cells. Chromosome detection results obtained according to genome-wide methylation sequencing data show that the chromosome copy number result of the sample is normal, and the chromosome copy number result is shown in FIG. 4; the epigenetic modification information of each imprinted gene region was also normal, as shown in fig. 5.

Example 3

The sample of this example used the existing CNV-seq method to detect copy number variation, and clinical detection reports showed that chromosome 15 had a 6.28Mb deletion. In this example, peripheral blood of the patient was collected, and library sequencing was performed by the method of example 1, and it was found that a 6.2Mb deletion was also present at the same position as that of chromosome 15, as shown in FIG. 6.

Meanwhile, the embodiment finds that the position is the region where the imprinting gene SNURF is located, for the imprinting gene SNURF, the gene of the parent source is normally expressed, the gene of the parent source is inhibited due to higher expression of methylation level, and if the gene of the parent source is deleted, the epigenetic disease is caused, and the health condition is not influenced due to the deletion of the parent source.

Further, in this example, the methylation level of the deleted region was detected using the bioinformatic analysis strategy of example 1, and it was found that all of the sequencing reads of the deleted region were highly methylated, and that almost all of the sequencing reads had more than 85% of cytosine sites were methylated, as shown in FIG. 7, and the numerical value after each sequencing read in FIG. 7 indicates the degree of methylation, for example, 1.0000 indicates 100% of cytosine sites were methylated, and 0.9500 indicates 95% of cytosine sites were methylated.

The above analysis shows that these reads are all derived from copies of the parent chromosome that are not deleted, i.e., it is clear that the deletion is due to the deletion of the parent chromosome. The actual clinical symptoms of the patient also show symptoms of epigenetic disease corresponding to the loss of the parent chromosome.

Example 4

In this example, methylation library sequencing was performed on a sample of aborted tissue collected by the method of example 1, and the detection result shows that the chromosome copy number of the tissue is normal, as shown in fig. 8.

Further, the sample was subjected to epigenetic analysis in this example, and it was found that the imprinted gene GNAS-XL was highly methylated, and that almost all of the sequencing reads had more than 90% of the cytosine sites were methylation-modified, as shown in fig. 9, and the numerical value after each sequencing read in fig. 9 indicates the degree of methylation, for example, 1.0000 indicates 100% of the cytosine sites were methylation-modified, and 0.9200 indicates 92% of the cytosine sites were methylation-modified.

The analysis results indicated that the imprint gene GNAS-XL had an epigenetic modification error. Thus, errors in the epigenetic modification of GNAS-XL may be responsible for abortions.

Example 5

In this example, methylation library sequencing was performed on a sample of aborted tissue collected by the method of example 1, and the detection result shows that the chromosome copy number of the tissue is normal, as shown in fig. 10.

Further, the sample is subjected to epigenetic analysis in this example, and it is found that the imprinting gene H19 is highly methylated, more than 70% of cytosine sites are methylation-modified in almost all sequencing reads, more than 90% of cytosine sites are methylation-modified in more sequencing reads, and specifically, as shown in FIG. 11, the value after each sequencing read in FIG. 11 indicates the degree of methylation, for example, 1.0000 indicates 100% of cytosine sites are methylation-modified, and 0.7500 indicates 75% of cytosine sites are methylation-modified.

The above analysis shows that the imprinting gene H19 has an epigenetic modification error. Thus, errors in the epigenetic modification of H19 may be responsible for abortion.

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

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

Claims

1. A method for detecting chromosomal variations, comprising:

performing DNA methylation detection on a target sample, wherein the target sample is derived from at least one of fetal cells and/or tissue, neonatal blood, umbilical cord blood, aborted tissue;

2. The method of claim 1, wherein analyzing the chromosomal copy number variation information and/or the epigenetic modification information of the target sample based on the detection result comprises:

3. The method of claim 2, wherein analyzing the chromosomal copy number variation information and/or the epigenetic modification information of the target sample based on the DNA methylation data of the target sample comprises:

4. The method of claim 3, wherein the obtaining chromosomal copy number information of the target sample from the DNA methylation data of the target sample comprises:

5. The method of claim 3, wherein the obtaining DNA methylation information of the target sample from the DNA methylation data of the target sample comprises:

6. The method of claim 3, wherein the obtaining DNA methylation information of the target sample from the DNA methylation data of the target sample comprises:

7. The method according to claim 5 or 6, wherein the target area satisfies at least one of the following features (1) to (5):

(1) The target region comprises a whole genome region;

(2) The target region comprises a promoter region;

(3) The target area includes an imprinting control area;

8. The method of claim 7, wherein determining the parental source of chromosomal copy number variation in the target sample based on chromosomal copy number variation information and DNA methylation information of the target sample comprises:

optionally, the copy number variation region comprises a print control region.

9. The method of claim 7, wherein determining whether the target sample has an epigenetic modification abnormality based on DNA methylation information of the target sample comprises:

10. An apparatus for detecting chromosomal variations, comprising:

DNA methylation detection module: for performing DNA methylation detection on a target sample derived from at least one of fetal cells and/or tissue, neonatal blood, umbilical cord blood, aborted tissue;