Methods for non-invasive preimplantation aneuploidy genetic detection

Abstract

The present invention relates to preimplantation genetic testing for aneuploidy (PGT-A). Specifically, the present invention identifies granulosa cell-specific differentially methylated regions (C-DMRs) and oocyte / polar body cell-specific differentially methylated regions (O-DMRs) for assessing maternal DNA contamination in blastocyst culture fluid, and provides a method for assessing maternal DNA contamination in blastocyst culture fluid based on the above-mentioned differentially methylated regions. Compared with the traditional SNP sequencing method, the method for determining maternal DNA contamination of the present invention is simpler, more economical, and time-saving, and is suitable for large-scale clinical applications. The present invention also provides a method for non-invasive preimplantation aneuploidy genetic testing based on blastocyst culture fluid, which simultaneously detects aneuploidy and maternal contamination rates, thereby achieving improved detection accuracy.

Description

Technical Field

[0001] The present invention relates to preimplantation genetic testing foraneuploidy (PGT-A). Specifically, the present invention identifies granulosa cell-specific differentially methylated regions (C-DMRs) and oocyte / polar body cell-specific differentially methylated regions (O-DMRs) for assessing maternal DNA contamination in blastocyst culture medium, and provides a method for assessing maternal DNA contamination in blastocyst culture medium based on the above-mentioned differentially methylated regions. The present invention also provides a method for non-invasive preimplantation genetic testing foraneuploidy based on blastocyst culture medium. Background Art

[0002] At present, the main process of preimplantation genetic testing for aneuploidy (PGT-A) includes isolating one or more embryonic cells to evaluate the copy number of 23 pairs of chromosomes and subchromosomal regions. There are three main types of embryonic cell materials used. The first is to isolate polar body cells for testing (1). This method cannot accurately detect the embryo after fertilization due to the lack of paternal genome, and because polar bodies are easily degraded, this method is not often used in clinical practice. The second method is to isolate a cell from the blastomere for testing (2,3). This method will affect embryonic development and reduce implantation potential. At the same time, because only one cell is obtained for testing, misdiagnosis may be caused by mosaicism (the presence of multiple copy numbers in one embryo). This method is not commonly used in clinical practice. The third method is to isolate 5-10 cells from the trophectoderm for biopsy. This TE biopsy method is currently a routine detection method in clinical practice (4,5). However, according to literature reports, this biopsy method is harmful to the embryo and the mother, and the long-term safety of the offspring has not been fully evaluated. There is also a risk of mosaicism. All of the above methods involve invasive operations on embryos and have many disadvantages (6).

[0003] In 2013, Italian scientists discovered the presence of free DNA in the culture medium of blastocysts cultured in vitro. This discovery brought hope for non-invasive preimplantation genetic testing (7). In 2016, Professor Xie Xiaoliang and others developed a new single-cell whole genome amplification method, which sequenced the entire genome of a trace amount of DNA in the culture medium and calculated the chromosome copy number. They found that the copy number obtained from the culture medium and the copy number obtained from the whole embryo were consistent with each other by more than 85%. In addition, the embryos transplanted based on the culture medium results had a live birth rate of more than 70% (8). In 2017, an article reported that there was contamination of maternal granulosa cells in the culture medium (9). Granulosa cell contamination has a fatal effect on the assessment of chromosome copy number. Because granulosa cells are diploid, if the embryo has a copy number abnormality, it will mask the abnormal copy number and produce a euploid test result.

[0004] There is currently controversy in this field regarding the origin of free DNA in culture medium. It is unclear whether this free DNA originates from trophectoderm cells, inner cell mass, or cells during early development. In 2018, a paper used single nucleotide polymorphisms (SNPs) to quantify the amount of contaminating granulosa cells and embryonic DNA in culture medium (10). This method requires the extraction of maternal follicular fluid, and the calculations are time-consuming and labor-intensive, making it unsuitable for large-scale clinical application.

[0005] Therefore, the field needs new, cost-effective, and convenient methods to calculate the amount of contamination, thereby improving the accuracy of non-invasive preimplantation genetic testing. Summary of the Invention

[0006] The present invention provides a new approach for performing pre-implantation aneuploidy genetic detection based on detecting free DNA present in blastocyst culture medium. The present invention identifies the source of free DNA in blastocyst culture medium and its components, thereby identifying granulosa cell-specific differentially methylated regions (C-DMRs) and oocyte / polar body cell-specific differentially methylated regions (O-DMRs) for evaluating maternal DNA contamination in blastocyst culture medium, and further provides a method for evaluating maternal DNA contamination in blastocyst culture medium based on the above-mentioned differentially methylated regions. Compared with the traditional SNP sequencing method, the method of determining maternal DNA contamination of the present invention is simpler, more economical, and time-saving, and is suitable for large-scale clinical application. The present invention also provides a method for non-invasive pre-implantation aneuploidy genetic detection based on blastocyst culture medium, which simultaneously detects chromosome copy number and maternal contamination rate, and improves clinical diagnostic accuracy through integrated analysis.

[0007] Differentially methylated regions

[0008] The present invention discovered for the first time that blastocyst culture medium is contaminated not only with granulosa cells but also with polar body cells, and thus further identified granulosa cell-specific differentially methylated regions (C-DMRs) and oocyte / polar body cell-specific differentially methylated regions (O-DMRs) that can be used to assess the proportion of maternal DNA contamination.

[0009] Therefore, in a first aspect, the present invention provides granulosa cell-specific differentially methylated regions (C-DMRs) and oocyte / polar body cell-specific differentially methylated regions (O-DMRs) that can be used to assess the proportion of maternal DNA contamination, wherein the C-DMRs are selected from at least one of Table 1 (e.g., at least 5, at least 10, at least 50, at least 80, at least 100, at least 150, at least 200, or at least 500); and the O-DMRs contain at least one of Table 2 (e.g., at least 5, at least 10, at least 50, at least 80, at least 100, at least 150, at least 200, at least 500, or at least 700).

[0010] In certain embodiments, the C-DMR comprises or at least comprises at least one of the species ranked 1-10 in Table 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or all 10).

[0011] In certain embodiments, the C-DMR comprises or at least comprises at least one selected from Ranks 1-50 of Table 1 (eg, at least 10, at least 20, at least 30, at least 40, at least 45, or all 50).

[0012] In certain embodiments, the C-DMR comprises or at least comprises at least one selected from rankings 1-80 of Table 1 (e.g., at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, or all 80).

[0013] In certain embodiments, the C-DMR comprises or at least comprises at least one of the species ranked 1-100 in Table 1 (e.g., at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or all 100).

[0014] In certain embodiments, the C-DMR comprises or at least comprises at least one of the species ranked 1-150 in Table 1 (e.g., at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, or all 150).

[0015] In certain embodiments, the C-DMR comprises or at least comprises at least one of the species ranked 1-200 in Table 1 (e.g., at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or all 200).

[0016] In certain embodiments, the C-DMR comprises a differentially methylated region of rank 1-10, rank 1-50, rank 1-80, rank 1-100, rank 1-150, rank 1-200, rank 1-300, rank 1-400, or rank 1-500 of Table 1.

[0017] In certain embodiments, the C-DMR comprises all of the differentially methylated regions shown in Table 1.

[0018] In certain embodiments, the O-DMR comprises or comprises at least one of the species ranked 1-10 in Table 2 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or all 10).

[0019] In certain embodiments, the O-DMR comprises or comprises at least one selected from Ranks 1-50 of Table 2 (eg, at least 10, at least 20, at least 30, at least 40, at least 45, or all 50).

[0020] In certain embodiments, the O-DMR comprises or comprises at least one selected from rankings 1-80 of Table 2 (e.g., at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, or all 80).

[0021] In certain embodiments, the O-DMR comprises or comprises at least one of the species ranked 1-100 in Table 2 (e.g., at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or all 100).

[0022] In certain embodiments, the O-DMR comprises or comprises at least one of the species ranked 1-150 in Table 2 (e.g., at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, or all 150).

[0023] In certain embodiments, the O-DMR comprises or comprises at least one of the species ranked 1-200 in Table 2 (e.g., at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or all 200).

[0024] In certain embodiments, the O-DMR comprises a differentially methylated region of rank 1-10, rank 1-50, rank 1-80, rank 1-100, rank 1-150, rank 1-200, rank 1-300, rank 1-400, rank 1-500, rank 1-600, or rank 1-700 of Table 2.

[0025] In certain embodiments, the O-DMR comprises all of the differentially methylated regions shown in Table 2.

[0026] In certain embodiments, the C-DMR comprises or comprises at least one of the species ranked 1-10 in Table 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or all 10), and the O-DMR comprises or comprises at least one of the species ranked 1-10 in Table 2 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or all 10).

[0027] In certain embodiments, the C-DMR comprises or at least comprises at least one of the species ranked 1-10 in Table 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or all 10), and the O-DMR comprises or at least comprises at least one of the species ranked 1-50 in Table 2 (e.g., at least 10, at least 20, at least 30, at least 40, at least 45, or all 50).

[0028] In certain embodiments, the C-DMR comprises or at least comprises at least one of the species ranked 1-10 in Table 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or all 10), and the O-DMR comprises or at least comprises at least one of the species ranked 1-80 in Table 2 (e.g., at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, or all 80).

[0029] In certain embodiments, the C-DMR comprises or at least comprises at least one of the species ranked 1-10 in Table 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or all 10), and the O-DMR comprises or at least comprises at least one of the species ranked 1-100 in Table 2 (e.g., at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or all 100).

[0030] In certain embodiments, the C-DMR comprises or at least comprises a rank 1-10 selected from Table 1, and the O-DMR comprises or at least comprises a rank 1-10 selected from Table 2. In certain embodiments, the C-DMR comprises or at least comprises a rank 1-50 selected from Table 1, and the O-DMR comprises or at least comprises a rank 1-50 selected from Table 2. In certain embodiments, the C-DMR comprises or at least comprises a rank 1-80 selected from Table 1, and the O-DMR comprises or at least comprises a rank 1-80 selected from Table 2. In certain embodiments, the C-DMR comprises or at least comprises a rank 1-100 selected from Table 1, and the O-DMR comprises or at least comprises a rank 1-100 selected from Table 2. In certain embodiments, the C-DMR comprises or at least comprises a rank 1-150 selected from Table 1, and the O-DMR comprises or at least comprises a rank 1-150 selected from Table 2. In certain embodiments, the C-DMR comprises or comprises at least a sequence selected from 1 to 200 in Table 1, and the O-DMR comprises or comprises at least a sequence selected from 1 to 200 in Table 2. In certain embodiments, the C-DMR comprises all of the differentially methylated regions shown in Table 1, and the O-DMR comprises all of the differentially methylated regions shown in Table 2.

[0031] Assessment of maternal DNA contamination rate

[0032] In a second aspect, the present invention provides a method for evaluating the proportion of maternal DNA contamination in a test sample containing embryonic cell-free DNA, comprising: obtaining the methylation levels of granulosa cell-specific differentially methylated regions (C-DMRs) and oocyte / polar body cell-specific differentially methylated regions (O-DMRs) based on the DNA methylation sequencing data of the test sample; wherein the C-DMRs and O-DMRs are as defined in the first aspect of the present invention.

[0033] In certain embodiments, the method further comprises: performing DNA methylation sequencing on the sample to be tested to obtain DNA methylation sequencing data thereof. In certain embodiments, the obtained raw sequencing data is preprocessed to obtain clean data. In certain embodiments, the preprocessing comprises deleting sequencing adapters, amplification primers, and low-quality bases, and optionally deleting R2reads containing more than three unmethylated CHs and corresponding R1reads.

[0034] In certain embodiments, the DNA methylation sequencing is selected from whole-genome methylation sequencing or targeted methylation sequencing.

[0035] In certain embodiments, the whole genome methylation sequencing is a single cell whole genome methylation sequencing technology. Such technology is well known to those skilled in the art, see, for example, Stuart T, Satija R. Integrative single-cell analysis. Nat Rev Genet. 2019 May; 20 (5): 257-272. In certain embodiments, the single-cell whole-genome methylation sequencing technology is selected from scBS-seq (single-cell bisulfite sequencing, single-cell bisulfite sequencing) (see, for example, Smallwood, SA et al. Single-cell genome-wide bisulfite sequencing for assessing epigenetic heterogeneity. Nat. Methods 11, 817–820 (2014).), snmC-seq (single-nucleus methylcytosine sequencing, single-cell nuclear methylcytosine sequencing) (see, for example, Luo, C. et al. Single-cell methylomes identify neuronal subtypes and regulatory elements in mammalian cortex. Science 357, 600–604 (2017).), sci-MET (single-cell combinatorial indexing for methylation analysis, single-cell combinatorial marker methylation detection) (see, for example, Mulqueen, RM et al. Highly scalable generation of DNA methylation profiles in single cells.Nat.Biotechnol.36,428–431(2018).), scRRBS (single-cell reduced representation bifulfite sequencing, single-cell simplified representation bisulfite sequencing) (see, for example, Guo, H. et al.Single-cell methylome landscapes of mouse embryonic stem cells and early embryos analyzed using reduced representation bisulfite sequencing. Genome Res. 23, 2126–2135 (2013).), scTrio-seq (single-cell triple omics sequencing technique) (see, for example, Hou, Y. et al. Single-cell triple omics sequencing reveals genetic, epigenetic, and transcriptomic heterogeneity in hepatocellular carcinomas. Cell Res. 2016 Mar; 26(3): 304-19.), the entire contents of which are incorporated herein by reference.

[0036] In certain embodiments, the whole-genome methylation sequencing is based on bisulfite sequencing technology.

[0037] In certain exemplary embodiments, the whole-genome methylation sequencing is scBS-seq.

[0038] In certain embodiments, the methylation level refers to the ratio of the number of reads containing methylated cytosine to the total number of reads. In certain embodiments, CpG sites covered by more than 3 reads are used to calculate the ratio.

[0039] In certain embodiments, the method further comprises determining the proportion of maternal DNA contamination in the sample based on the obtained methylation levels of C-DMR and O-DMR.

[0040] In certain embodiments, the method further comprises the steps of:

[0041] - The proportion of granulocyte-derived DNA in the sample (P k1 ) and the proportion of polar body cell-derived DNA in the sample (P k2 ):

[0042]

[0043] Among them, k1-k3 represent components, which are granulosa cells, polar body cells, and blastocysts, respectively; C represents C-DMR, O represents O-DMR; P k1、P k2 、P k3 Respectively represent the proportion of DNA derived from granulosa cells, polar body cells, and blastocysts in the samples;

[0044] represents the average methylation level of the measured C-DMR; C / k3 Respectively represent the average methylation level reference values ​​of the C-DMR in granulosa cells, polar body cells, and blastocysts; a C / k1 、a C / k2 、a C / k3 are correction factors, representing the average PCR amplification efficiency of the C-DMR in granulosa cells, polar body cells, and blastocysts, respectively;

[0045] represents the average methylation level of the measured O-DMR; Respectively represent the average methylation level reference values ​​of the O-DMR in granulosa cells, polar body cells, and blastocysts; a O / k1 、a O / k2 、a O / k3 are correction factors, representing the average PCR amplification efficiency of the O-DMR in granulosa cells, polar body cells, and blastocysts, respectively;

[0046] - Calculate the proportion of maternal DNA contamination using the following formula: P 母源污染 =P k1 +P k2 .

[0047] In this article, P k1 +P k2 +P k3 =1 is intended to indicate that the sum of the proportions of the above three components is considered to be 1 when calculating the maternal DNA contamination ratio using the method of the present invention. However, this does not mean that the sample is free of DNA contamination from other sources. For example, the sample may also contain paternal DNA contamination (such as sperm cell-derived DNA contamination). Therefore, the method of the present invention does not exclude the presence of DNA contamination from other sources in the sample. The proportion of DNA contamination from other sources can be combined with the maternal DNA contamination ratio provided by the method of the present invention for non-invasive preimplantation aneuploidy genetic testing.

[0048] In certain embodiments, (i.e., the average methylation level reference value of the C-DMR in granulosa cells) refers to the average methylation level of the C-DMR in the granulosa cell standard. The granulosa cell standard refers to a cell sample consisting of or essentially consisting of granulosa cells, which does not include oocytes, polar body cells, and blastocyst cells. In certain embodiments, The granulocyte standard is obtained by performing the same measurement steps as the sample to be tested.

[0049] In certain embodiments, (i.e., the average methylation level reference value of the C-DMR in polar body cells) refers to the average methylation level of the C-DMR in polar body cell standards. The polar body cell standards refer to cell samples consisting of or essentially consisting of oocytes / polar body cells, which do not include granulosa cells and blastocyst cells. In certain embodiments, The polar cell standard is obtained by performing the same measurement steps as the sample to be tested.

[0050] In certain embodiments, (i.e., the average methylation level reference value of the C-DMR in blastocysts) refers to the average methylation level of the C-DMR in blastocyst standards. The blastocyst standard refers to a cell sample consisting of or essentially consisting of blastocysts or blastocyst cells, which does not include granulosa cells and oocytes / polar body cells. In certain embodiments, The blastocyst standard is obtained by performing the same measurement steps as the sample to be tested.

[0051] In certain embodiments, (i.e., the average methylation level reference value of the O-DMR in granulosa cells) refers to the average methylation level of the O-DMR in the granulosa cell standard. The granulosa cell standard refers to a cell sample consisting of or essentially consisting of granulosa cells, which does not include oocytes, polar body cells, and blastocyst cells. In certain embodiments, The granulocyte standard is obtained by performing the same measurement steps as the sample to be tested.

[0052] In certain embodiments, (i.e., the average methylation level reference value of the O-DMR in polar body cells) refers to the average methylation level of the O-DMR in polar body cell standards. The polar body cell standards refer to cell samples consisting of or essentially consisting of oocytes / polar body cells, which do not include granulosa cells and blastocyst cells. In certain embodiments, The polar cell standard is obtained by performing the same measurement steps as the sample to be tested.

[0053] In certain embodiments, (i.e., the average methylation level reference value of the O-DMR in blastocysts) refers to the average methylation level of the O-DMR in blastocyst standards. The blastocyst standard refers to a cell sample consisting of or essentially consisting of blastocysts or blastocyst cells, which does not include granulosa cells and oocytes / polar body cells. In certain embodiments, The blastocyst standard is obtained by performing the same measurement steps as the sample to be tested.

[0054] In certain embodiments, the C-DMR comprises the differentially methylated regions ranked 1-10 as shown in Table 1, In certain embodiments, the C-DMR comprises the differentially methylated regions ranked 1-50 as shown in Table 1, In certain embodiments, the C-DMR comprises the differentially methylated regions ranked 1-80 as shown in Table 1, In certain embodiments, the C-DMR comprises the differentially methylated regions ranked 1-100 as shown in Table 1, In certain embodiments, the C-DMR comprises the differentially methylated regions ranked 1-150 as shown in Table 1, In certain embodiments, the C-DMR comprises the differentially methylated regions ranked 1-200 as shown in Table 1, In certain embodiments, the C-DMR comprises all differentially methylated regions shown in Table 1. They are 92%, 3% and 4% respectively.

[0055] In certain embodiments, the O-DMR comprises the differentially methylated regions ranked 1-10 as shown in Table 2, In certain embodiments, the O-DMR comprises the differentially methylated regions ranked 1-50 as shown in Table 2, In certain embodiments, the O-DMR comprises the differentially methylated regions ranked 1-80 as shown in Table 2, In certain embodiments, the O-DMR comprises the differentially methylated regions ranked 1-100 as shown in Table 2, In certain embodiments, the O-DMR comprises the differentially methylated regions ranked 1-150 as shown in Table 2, In certain embodiments, the O-DMR comprises the differentially methylated regions ranked 1-200 as shown in Table 2, In certain embodiments, the O-DMR comprises all differentially methylated regions shown in Table 2. They are 19%, 82% and 22% respectively.

[0056] In certain embodiments, the C-DMR comprises the differentially methylated regions ranked 1-10 as shown in Table 1, 93%, 2%, and 4% respectively; the O-DMR includes the differentially methylated regions ranked 1-10 shown in Table 2, They are 22%, 91% and 0% respectively.

[0057] In certain embodiments, the C-DMR comprises the differentially methylated regions ranked 1-50 as shown in Table 1, 90%, 3%, and 4% respectively; the O-DMR includes the differentially methylated regions ranked 1-50 as shown in Table 2, They are 31%, 75% and 15% respectively.

[0058] In certain embodiments, the C-DMR comprises the differentially methylated regions ranked 1-80 as shown in Table 1, 91%, 3%, and 4% respectively; the O-DMR includes the differentially methylated regions ranked 1-80 shown in Table 2, They are 24%, 74% and 13% respectively.

[0059] In certain embodiments, the C-DMR comprises the differentially methylated regions ranked 1-100 as shown in Table 1, 91%, 3%, and 4% respectively; the O-DMR includes the differentially methylated regions ranked 1-100 shown in Table 2, They are 23%, 76% and 13% respectively.

[0060] In certain embodiments, the C-DMR comprises the differentially methylated regions ranked 1-150 as shown in Table 1, 91%, 3%, and 4% respectively; the O-DMR includes the differentially methylated regions ranked 1-150 shown in Table 2, They are 23%, 75% and 13% respectively.

[0061] In certain embodiments, the C-DMR comprises the differentially methylated regions ranked 1-200 as shown in Table 1, 92%, 3%, and 4% respectively; the O-DMR includes the differentially methylated regions ranked 1-200 shown in Table 2, They are 23%, 77% and 12% respectively.

[0062] In certain embodiments, the C-DMR comprises all of the differentially methylated regions shown in Table 1, 92%, 3%, and 4% respectively; the O-DMR includes all differentially methylated regions shown in Table 2, They are 19%, 82% and 22% respectively.

[0063] In certain embodiments, a C / k1 In certain embodiments, a O / k2 In certain embodiments, a C / k2 、a C / k3 、a O / k1 、a O / k3 In certain embodiments, a C / k1 About 0.6, a O / k2 About 0.6, a C / k2 、a C / k3 、a O / k1 、a O / k3 About 1.

[0064] In certain embodiments, the sample to be tested is blastocyst culture fluid.

[0065] Preimplantation genetic testing for aneuploidy

[0066] Once the maternal DNA contamination ratio is determined, those skilled in the art can easily use it in applications for pre-implantation aneuploidy genetic detection.

[0067] Therefore, in the third aspect, the present invention also provides a method for non-invasive pre-implantation aneuploidy genetic detection based on blastocyst culture medium, the method comprising detecting the copy number and maternal DNA contamination ratio of the target region (such as a chromosome, subchromosomal region and / or a local region on the genome), and determining the aneuploidy and its mosaicism degree, or the copy number variation and its mosaicism degree of the subchromosomal region or the local region on the genome by integrating and analyzing the detection data of the two.

[0068] In certain embodiments, the method comprises:

[0069] - Obtain methylation sequencing data of blastocyst culture fluid;

[0070] - obtaining the copy number of a target region based on the sequencing data, wherein the target region is selected from a chromosome, a subchromosome, a local region on the genome, or any combination thereof of interest;

[0071] - using the method described in the second aspect to obtain the maternal DNA contamination ratio in the blastocyst culture medium;

[0072] - using the maternal DNA contamination ratio to correct the obtained copy number and / or mosaicism of the target region;

[0073] - diagnosing chromosomal aneuploidy and / or its mosaicism based on the corrected copy number of the target region, and / or the copy number variation and / or its mosaicism of subchromosomal regions and / or local regions on the genome;

[0074] The obtaining of the copy number of the target region and the obtaining of the maternal DNA contamination ratio can be performed in any order or simultaneously.

[0075] In certain embodiments, the method further comprises: performing methylation sequencing on the blastocyst culture fluid to obtain sequencing data. In certain embodiments, the obtained raw sequencing data is preprocessed to obtain clean data. In certain embodiments, the preprocessing comprises removing sequencing adapters, amplification primers, and low-quality bases, and optionally removing R2 reads containing more than three unmethylated CHs and corresponding R1 reads.

[0076] Methods for detecting copy number variation (CNV) by methylation sequencing technology are known to those skilled in the art. Since methylation sequencing also obtains sequence information including methylated and unmethylated genomic regions, its principle for detecting chromosome or gene copy number variation is similar to genome sequencing. These copy number variation detection methods based on sequencing data typically include: comparing the sequencing data with a reference sequence, and determining the copy number variation based on the cumulative amount (depth) change or coverage change of the read segments (reads) in the matching region of the sequencing data.

[0077] For example, the method of detecting copy number variation by CNV-seq is described in detail in McKernan KJ, et al. Sequence and structural variation in a human genome uncovered by short-read, massively parallel ligation sequencing using two-base encoding. Genome Res. 2009; 19(9): 1527-1541., the entire document of which is incorporated herein by reference.

[0078] For example, a method for detecting copy number variation by whole-genome methylation sequencing is described in detail in Bian, S. et al. Single-cell multiomics sequencing and analyses of human colorectal cancer. Science. 2018 Nov 30; 362(6418): 1060-1063., all of which are incorporated herein by reference.

[0079] For example, a method for detecting copy number variation by semi-targeted methylation sequencing and reduced representation bisulfite sequencing (RRBS) is described in detail in Hou, Y. et al. Single-cell triple omics sequencing reveals genetic, epigenetic, and transcriptomic heterogeneity in hepatocellular carcinomas. Cell Res. 2016 Mar; 26(3): 304-19.; all of which are incorporated herein by reference.

[0080] The various methods described above for detecting copy number variation can be used to determine chromosome copy number. In certain embodiments, the methylation sequencing technology is selected from CNV-seq, whole genome methylation sequencing, and RRBS.

[0081] In certain embodiments, the sequencing data consists of a plurality of reads.

[0082] In certain embodiments, obtaining the copy number of the target region based on the sequencing data includes: aligning the sequencing data with a reference sequence, and determining the change in copy number based on the cumulative amount (depth) or coverage of reads in the matching region of the sequencing data.

[0083] In certain embodiments, the reference sequence is a human genome (eg, human reference genome hg19).

[0084] In certain embodiments, the chromosomes of interest include one or more chromosomes selected from chromosome 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, X, or Y.

[0085] In certain embodiments, the subchromosomal region or local region on the genome refers to a fragment on the chromosome with a copy number variation greater than 1 Mb.

[0086] In certain embodiments, the method for performing pre-implantation aneuploidy genetic testing of the present invention includes a step of correcting the copy number and / or mosaicism of the obtained target region (e.g., chromosome, subchromosome, and / or local region on the genome) according to the maternal DNA contamination ratio. The mosaicism refers to the inconsistency between the chromosomes, subchromosomes, and / or local regions on the genome of a portion of cells in the embryo and the chromosomes, subchromosomes, and / or local regions on the genome of other cells. In certain embodiments, the inconsistency may refer to inconsistencies in terms of numbers. In certain embodiments, the correction process is as follows: if there is maternal DNA contamination in the culture fluid sample, the chromosome copy number tends to be diploid. Based on the normal maternal DNA copy number of two and the maternal DNA contamination ratio, the copy number of the culture fluid sample actually detected can be corrected to restore the actual copy number before the maternal DNA contamination; if there is no maternal DNA contamination in the culture fluid sample, the copy number is a non-integer that is not two, then the ratio of euploid cells to aneuploid cells in the culture fluid sample can be obtained by correcting the mosaicism, thereby knowing the actual copy number of the embryo.

[0087] Electronic Implementation Plan

[0088] The method described in the second or third aspect of the present application can be implemented as software code executed by a processor using any suitable computer language, such as Java, C++, or Perl using conventional or object-oriented techniques. The software code can be stored as a series of instructions or commands on a computer-readable medium for storage and / or transmission, suitable media including random access memory (RAM), read-only memory (ROM), magnetic media such as hard disks or floppy disks, or optical media such as compact disks (CDs) or DVDs (Digital Versatile Discs), flash memory, etc. The computer-readable medium can be any combination of such storage or transmission devices.

[0089] Therefore, on the other hand, the present invention also provides a computer-readable medium or computer program product for performing operations to evaluate the proportion of maternal DNA contamination in a test sample containing embryonic cell-free DNA, which includes multiple instructions for controlling a computing system, and the multiple instructions are used to control the computing system to execute the method described in any embodiment of the second aspect.

[0090] In some embodiments, the plurality of instructions, when executed, implement the following steps:

[0091] -optionally receiving DNA methylation sequencing data of the sample to be tested;

[0092] - Based on the DNA methylation sequencing data of the test sample, obtaining the methylation levels of granulosa cell-specific differentially methylated regions (C-DMRs) and oocyte / polar body cell-specific differentially methylated regions (O-DMRs); wherein the C-DMRs and O-DMRs are as defined in any embodiment of the first aspect;

[0093] - The proportion of granulocyte-derived DNA in the sample (P k1 ) and the proportion of polar body cell-derived DNA in the sample (P k2 ):

[0094]

[0095] Among them, k1-k3 represent components, which are granulosa cells, polar body cells, and blastocysts, respectively; C represents C-DMR, O represents O-DMR; P k1 、P k2 、P k3 Respectively represent the proportion of DNA derived from granulosa cells, polar body cells, and blastocysts in the samples;

[0096] represents the average methylation level of the measured C-DMR; Respectively represent the average methylation level reference values ​​of the C-DMR in granulosa cells, polar body cells, and blastocysts; a C / k1 、a C / k2 、a C / k3 are correction factors, representing the average PCR amplification efficiency of the C-DMR in granulosa cells, polar body cells, and blastocysts, respectively;

[0097] represents the average methylation level of the measured O-DMR; Respectively represent the average methylation level reference values ​​of the O-DMR in granulosa cells, polar body cells, and blastocysts; aO / k1 、a O / k2 、a O / k3 are correction factors, representing the average PCR amplification efficiency of the O-DMR in granulosa cells, polar body cells, and blastocysts, respectively;

[0098] - Calculate the proportion of maternal DNA contamination using the following formula: P 母源污染 =P k1 +P k2 ;

[0099] In certain embodiments, the parameters in the formula are as defined in any embodiment of the second aspect.

[0100] On the other hand, the present invention also provides a computer-readable medium or computer program product for performing non-invasive pre-implantation aneuploidy genetic detection based on blastocyst culture medium, which includes multiple instructions for controlling a computing system, and the multiple instructions are used to control the computing system to execute the method described in any embodiment of the third aspect.

[0101] In some embodiments, the plurality of instructions, when executed, implement the following steps:

[0102] -Receive methylation sequencing data of blastocyst culture fluid;

[0103] - obtaining the copy number of a target region based on the sequencing data, wherein the target region is selected from a chromosome, subchromosome and / or local region on the genome of interest or any combination thereof;

[0104] - using the method described in the second aspect to obtain the maternal DNA contamination ratio in the blastocyst culture medium;

[0105] - using the maternal DNA contamination ratio to correct the obtained copy number and / or mosaicism of the target region;

[0106] - diagnosing chromosomal aneuploidy or its mosaicism, or copy number variation or its mosaicism in subchromosomal regions or local regions on the genome based on the corrected copy number of the target region;

[0107] The obtaining of the copy number of the target region and the obtaining of the maternal DNA contamination ratio can be performed in any order or simultaneously.

[0108] In certain embodiments, the above steps are as defined in any embodiment of the third aspect.

[0109] Definition of terms

[0110] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, laboratory procedures in genomics, nucleic acid chemistry, molecular biology, and other fields used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0111] As used herein, the term "DNA methylation" refers to a form of chemical modification of DNA that can alter genetic expression without changing the DNA sequence. DNA methylation occurs when a methyl group is covalently attached to the 5th carbon position of cytosine in genomic CpG dinucleotides by the action of DNA methyltransferases. This can alter chromatin structure, DNA conformation, DNA stability, and the way DNA interacts with proteins, thereby controlling gene expression.

[0112] As used herein, the term "copy number variation" (CNV) refers to a gene that increases or decreases the number of copies of large segments of the genome due to genomic rearrangements, primarily manifesting as submicroscopic deletions and duplications. It is a key component of genomic structural variation (SV) and can be considered another important pathogenic mechanism of chromosomal diseases. CNV, as defined herein, refers to a copy number increase or decrease of a genomic segment greater than 1 Mb in length.

[0113] As used herein, the term "chromosomal aneuploidy" refers to a state in which the wrong number of chromosomes is present in a cell, including any genetic defect that exhibits an abnormal number of chromosomes, such as having more or less chromosomes than the normal number of any chromosome, and having an extra portion of any chromosome in addition to the normal pair, or lacking a portion of any chromosome in the normal pair. In the case of human cells, it may refer to a situation in which the cell does not contain 22 pairs of autosomes and a pair of sex chromosomes. In the case of human germ cells, it may refer to a situation in which the cell does not contain each of the 23 chromosomes. When referring to a single autosome, it may refer to a situation in which more or less than two homologous chromosomes are present. When referring to sex chromosomes, it may refer to a situation in which more or less than two X or Y chromosomes are present, or just two Y chromosomes are present.

[0114] As used herein, term " mosaicism " refers to that the chromosome of a part of cell, subchromosome and / or the local area on genome and the chromosome of other cells, subchromosome and / or the local area on genome of embryo are inconsistent.In certain embodiments, described inconsistent specifically refers to the inconsistent aspect.

[0115] As used herein, the term "differentially methylated region (DMR)" refers to a region in chromosomal DNA that is differentially methylated in DNA from different sources. For example, in the present invention, granulosa cell-specific differentially methylated regions (C-DMRs) may refer to regions that are highly methylated in granulosa cells, which are hypomethylated or nearly unmethylated in other embryonic cells (e.g., polar body cells). For example, in the present invention, polar body cell-specific differentially methylated regions (O-DMRs) may refer to regions that are highly methylated in oocytes / polar body cells, which are hypomethylated in other cells (e.g., granulosa cells, sperm).

[0116] As used herein, the term "cumulus cell" refers to cells that constitute the granulosa layer of the follicle wall.

[0117] As used herein, the term "oocyte" refers to an oogonia that undergoes meiosis during oogenesis.

[0118] As used herein, the term "polar body" refers to a female reproductive cell that undergoes two meiotic divisions during its formation process to form a large haploid egg cell and 2 to 3 small cells, which are called polar bodies.

[0119] As used herein, the term "blastocyst" refers to an early stage of embryonic development consisting of a hollow ball of cells enclosing a fluid-filled cavity called the cleavage cavity.

[0120] As used herein, the terms "spent blastocyst culture media (SBM)" or "spent embryo culture media (SEM)" have the same meaning and are used interchangeably, and refer to the culture medium used during the in vitro culture of the blastocyst / embryo before implantation, in which free DNA (cfDNA) released by the embryo is present. In certain embodiments, the blastocyst culture medium refers to the culture medium when the embryo cultured in vitro reaches the fully expanded blastocyst stage. In certain embodiments, the blastocyst culture medium is obtained by the following steps: fertilization by intracytoplasmic sperm injection (ICSI) on the day of oocyte recovery; embryos are transferred to blastocyst culture medium (e.g., on day 3); granulosa cells surrounding the embryo or morula are removed, washed thoroughly, and then cultured separately in a new culture dish (e.g., on day 4); when the embryo reaches the fully expanded blastocyst stage (e.g., day 5 to day 7), the culture medium is collected as blastocyst culture medium.

[0121] As used herein, the term "computer-readable medium" refers to any suitable medium for storing, holding, or containing data or information that can be directly read and accessed by a computer. Such media may include, but are not limited to: magnetic storage media such as floppy disks, hard disk storage media, and magnetic tape; optical storage media such as optical disks; electronic storage media such as RAM, ROM, EPROM, EEPROM, etc.; general-purpose hard disks and hybrids of these types such as magnetic / optical storage media.

[0122] Advantageous Effects of the Invention

[0123] The present invention identifies the source and components of free DNA in blastocyst culture medium, thereby identifying granulosa cell-specific differentially methylated regions (C-DMRs) and oocyte / polar body cell-specific differentially methylated regions (O-DMRs) for evaluating maternal DNA contamination in blastocyst culture medium, and further provides a method for evaluating maternal DNA contamination in blastocyst culture medium based on the above-mentioned differentially methylated regions. Compared with the traditional SNP sequencing method, the method of the present invention for determining maternal DNA contamination is simpler, more economical, and time-saving, and is suitable for large-scale clinical application. The present invention also provides a method for non-invasive pre-implantation aneuploidy genetic detection based on blastocyst culture medium, which simultaneously detects chromosome copy number and maternal contamination rate, and improves clinical diagnostic accuracy through integrated analysis.

[0124] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are intended only to illustrate the present invention and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0125] Figure 1 A research flow chart of one embodiment of the present invention is schematically depicted.

[0126] Figure 2 Shown are the results of an assessment of granulosa cell contamination of cell-free DNA in blastocyst culture medium.

[0127] (A) Unsupervised hierarchical cluster analysis of DNA methylation levels in blastocyst culture fluid samples, human preimplantation embryos, germ cells, and granulosa cells. GV: GV oocyte; MII: MII oocyte; PN: pronucleus.

[0128] (B) Heatmap of methylation levels of 769 CpG island differentially methylated regions (C-DMRs), which are specifically hypermethylated in granulosa cells.

[0129] (C) Correlation analysis between the genome-wide DNA methylation level and the C-DMR methylation level in blastocyst culture fluid cfDNA.

[0130] (D) Whole-genome DNA methylation level detection results of three types of blastocyst culture fluid samples: ICM, TE, granulosa cells, and samples with different degrees of granulosa cell contamination (no contamination, moderate contamination, and severe contamination). The degree of granulosa cell contamination was estimated based on C-DMR methylation levels.

[0131] Figures 3A-3D Shown are the results of detecting chromosome aneuploidy by scBS-seq.

[0132] Figure 3A : CN profiles of HCT116 cells determined by scBS-seq (inner panel) and MALBAC (outer panel).

[0133] Figure 3B : Coefficient of variation (CV) distribution of sequencing results based on different amounts of uniquely aligned reads.

[0134] Figure 3C :Representative CN spectra of different types of blastocyst culture media and corresponding TE biopsy results.

[0135] Figure 3D : The global consistency rate (GCR), false negative rate (FNR) and false positive rate (FPR) of copy number results obtained by using scBS-seq and TE biopsy methods to detect blastocyst culture fluid samples with different degrees of granulosa cell contamination (no contamination, moderate contamination, and severe contamination) were analyzed.

[0136] Figures 4A-4E Shown are the results of an assessment of polar body cell contamination of cell-free DNA in blastocyst culture medium.

[0137] Figure 4A : Unsupervised hierarchical clustering analysis of genome-wide DNA methylation in samples without granulosa cell contamination, as well as preimplantation embryos and germ cells. GV: GV oocyte; MII: MII oocyte; PN: pronucleus.

[0138] Figure 4B : Heatmap of methylation levels of 548 oocyte / polar body cell-specific differentially methylated regions (O-DMRs), which are hypermethylated in MII oocytes.

[0139] Figure 4C : Comparison of methylation levels of O-DMRs in three blastocyst culture fluid samples pooled with MII oocytes and maternal pronuclei and other samples.

[0140] Figure 4D : Chromosome CN profiles, TE biopsy results, and C-DMR and O-DMR methylation level determination results of the blastocyst culture medium sample clustered with maternal pronuclei (#S167), the blastocyst culture medium sample clustered with MII oocytes (#S176), and the blastocyst culture medium sample clustered with maternal pronuclei (#S193).

[0141] Figure 4E : Results of correlation analysis between the methylation levels of non-CpG islands and O-DMR methylation levels in culture medium samples.

[0142] Figures 5A-5B Shown are the results of embryonic origin analysis of cell-free DNA in blastocyst culture medium.

[0143] Figure 5A : Principal component analysis results of single-cell DNA methylation data of EPI (n=22) and TE (n=25, all from day 6 embryos), based on the promoter regions of the top 300 differentially expressed genes between EPI and TE. The DNA methylation data of this single-cell multi-omics sequencing were derived from a study previously published by our laboratory (F. Zhou et al., Reconstituting the transcriptome and DNA methylome landscapes of human implantation. 572, 660-664 (2019)).

[0144] Figure 5B : Results of principal component analysis of day 6 blastocyst culture fluid samples (without granulosa cell or polar body cell contamination) and EPI and TE single cells.

[0145] Figure 6 The accuracy verification results of the derived formula are shown.

[0146] (A) Results of simulated DNA incorporation experiments. Computer-generated simulation data were generated for a series of different incorporation ratios of polar bodies (MII oocytes), ICM / TE, and granulosa cells, including 100% incorporation of one of the three components (100% input), 50% incorporation of each of the two components (50% + 1 input), 75% incorporation of one component plus 25% incorporation of the other component (75% + 1 input), 50% incorporation of one component plus 25% incorporation of the other two components (50% + 2 input), and 75% incorporation of one component plus 12.5% ​​incorporation of the other two components (75% + 2 input). The figure shows a comparison of the incorporation percentages with the predicted percentages.

[0147] (B) Correlation analysis between the calculated estimated percentage and the actual incorporation percentage.

[0148] Figures 7A-7E Results of the analysis of the effect of maternal contamination on chromosome copy number are shown.

[0149] Figure 7A : Number and percentage of blastocyst culture medium samples with different granulosa cell contamination ratios (left) and polar body cell contamination ratios (right).

[0150] Figure 7B :Correlation analysis between granulosa cell contamination and polar body cell contamination in blastocyst culture medium.

[0151] Figure 7C : Number and percentage of blastocyst culture medium samples with different proportions of net maternal DNA contamination.

[0152] Figure 7D : Gender discordance rate (GDR) and false negative rate (FNR) of copy number results and TE biopsy results obtained from culture medium samples with different proportions of granulosa cell contamination, polar body cell contamination, or net maternal DNA contamination.

[0153] Figure 7E : The proportion of granulosa cell contamination in the culture medium on days 5, 6, and 7, as well as the gender discordance rate and false negative rate on days 5 and 6.

[0154] Figures 8A-8B : Correlation analysis between the contamination ratios of cumulus and polar bodies calculated using the top 10, top 50, top 80, top 100, top 150, and top 200 C-DMRs and O-DMRs and the actual incorporation percentages. Example

[0155] The invention will now be described with reference to the following examples which are intended to illustrate the invention but not to limit it.

[0156] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention are basically based on the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Ausubel et al., Molecular Biology: A Laboratory Manual, 3rd edition, John Wiley & Sons, Inc., 1995. Restriction endonucleases were used according to the conditions recommended by the product manufacturers. It will be appreciated by those skilled in the art that the examples are provided to illustrate the present invention and are not intended to limit the scope of the invention.

[0157] The experimental methods involved in the following examples are as follows:

[0158] 1. Experimental Design

[0159] We collected 194 blastocyst culture fluid samples and obtained two layers of information through single-cell DNA methylation sequencing: 1) DNA methylation information to infer DNA origin and proportion, and 2) read coverage to infer chromosome copy number. Through integrated analysis, the accuracy of clinical diagnosis can be improved. The experimental process is shown below. Figure 1 shown.

[0160] 2. Collection of human blastocyst culture medium

[0161] A total of 194 PGT-A (preimplantation genetic testing foraneuploidy) blastocysts and their corresponding culture media were included in this study. In all these PGT-A cycles, fertilization was performed by intracytoplasmic sperm injection (ICSI) on the day of oocyte retrieval. On day 3, the embryos were transferred to blastocyst culture medium. On day 4, each compacted embryo or morula was carefully stripped of the surrounding granulosa cells again, thoroughly washed, and then cultured individually in a new culture dish (15 μl per culture drop). From day 5 to day 7, when the embryos reached the fully expanded blastocyst stage, they were transferred to a biopsy dish and the culture medium was collected using a polymerase chain reaction (PCR) tube. The samples were stored at -20°C. The TE of the corresponding blastocyst was biopsied, and each biopsy specimen was individually vitrified. The biopsied cells were analyzed for PGT using a SNP array.

[0162] 3. Whole-genome DNA methylation sequencing of blastocyst culture medium

[0163] Single-cell whole-genome methylation sequencing (scBS-seq) was used to analyze the DNA methylome in blastocyst culture medium. The culture medium was made up to 20 μl with nuclease-free buffer and lysed at 50°C for 1.5 h with the appropriate volume of lysis buffer (20 mM Tris-EDTA, 20 mM KCl, 0.3% Triton X-100, and 1 mg / ml Proteinase K). The cells were then bisulfite-treated using the EZ-96 DNA Methylation-Direct MagPrep Kit. After purification, first-strand DNA was synthesized using random primers P5-N9 (5'-CTACACGACGCTCTTCCGATCTNNNNNNNN-3', SEQ ID NO: 1) and Klenow polymerase. This step was repeated four times. Second-strand DNA was synthesized using primers P7-N9 (5'-AGACGTGTGCTCTTCCGATCTNNNNNNNN-3', SEQ ID NO: 2). PCR amplification was performed using index primers and Illumina universal PCR primers to obtain sequencing libraries, with 5G data measured for each sample.

[0164] 4. DNA methylation sequencing data processing

[0165] First, we removed the sequencing adapters, amplification primers, and low-quality bases from the raw bisulfite sequencing paired-end reads data. Then, we discarded R2reads and corresponding R1reads containing more than 3 unmethylated CHs. These clean reads were mapped to the human reference genome (hg19) using BS-Seeker2 in end-to-end alignment mode. Unaligned reads were re-paired with the hg19 genome in a local alignment manner, and low-confidence alignments in microhomology regions were removed. Next, the Picard tool was used to remove duplicates caused by PCR amplification. The ratio of the number of C-methylated reads to the total reads (methylated and unmethylated) was defined as the DNA methylation level; CpG sites covered by more than 3 reads were used for subsequent calculations. Samples with more than 1 million unique mapping reads were retained for subsequent analysis.

[0166] Example 1: Granulosa cell contamination in blastocyst culture medium

[0167] We combined the DNA methylation data of preimplantation embryos and germ cells (P. Zhu et al., Single-cell DNA methylome sequencing of human preimplantation embryos. Nat Genet 50, 12-19 (2018)) with the blastocyst culture medium data and found that the genome-wide methylation level of culture medium free DNA was 13% to 74%, with a median of 36%, which was significantly higher than the methylation levels of ICM and TE (ICM and TE were 24% and 24%, respectively). Cluster analysis showed that some culture medium samples (50 out of 191) clustered with granulosa cells. The DNA methylation level of these samples was high (average 60%), close to the DNA methylation level of granulosa cells (average 71%) ( Figure 2 A).

[0168] To accurately assess the proportion of granulosa cells in blastocyst culture medium, we identified 769 granulosa cell-specific CpG island differentially methylated regions (C-DMRs). By calculating the methylation differences between these 769 C-DMRs and granulosa cell standards and all other embryonic cells, we ranked them in ascending order based on the p-values ​​obtained by significance calculation. These C-DMRs are shown in the attached Table 1. These differentially methylated regions are highly methylated in granulosa cells and almost unmethylated in other embryonic cells ( Figure 2B). At the same time, the average methylation levels of these C-DMRs were positively correlated with the genome-wide DNA methylation levels, indicating that the genome-wide high methylation levels in the culture medium were mainly caused by contamination from granulosa cells ( Figure 2 C).

[0169] By calculating the methylation levels of these C-DMRs in the culture medium, we determined that approximately half of the culture medium samples (95 of 191) were contaminated with granulosa cells (C-DMR methylation levels higher than 8%, the mean (4%) + 3SD (1.3%) of the C-DMR methylation levels in ICM / TE). Approximately half (50 / 95) of the culture medium samples contaminated with granulosa cells showed moderate contamination (C-DMR methylation levels: 8%-40%), and the other half (45 / 95) showed severe contamination (C-DMR methylation levels: greater than 40%) ( Figure 2 D).

[0170] Example 2: Detection of aneuploidy by single-cell whole-genome methylation sequencing (scBS-seq)

[0171] Our previous studies have demonstrated that scBS-seq can assess copy number (CN) variation (Y. Hou et al., Cell Res 26, 463 304-319 (2016).; SHBian et al., Science 362, 1060-+ (2018).). We first analyzed HCT116 cells and showed that scBS-seq and multiple annealing and loop-based amplification cycles (MALBAC) (CH Zong, SJ Lu, ARChapman, XSXie, Genome-Wide Detection of Single-Nucleotide and Copy-Number Variations of a Single Human Cell. Science 338, 1622-1626 (2012).) gave the same expected CN profile ( Figure 3A In order to accurately determine the lower limit of sequencing depth for copy number variation results, we randomly sampled the data to reduce the sequencing depth. The results showed that when the data volume was 2M, the coefficient of variation (CV) was stable and low ( Figure 3BWe then analyzed the copy number results from the culture medium samples and found that the majority of SEM samples (182 of 191) produced clear and informative copy number maps. The remaining nine samples, for which indeterminate results were obtained (with more than six aneuploid fragments), were defined as "aneuploidy chaos" and discarded. Comparison of copy number results from culture medium and TE biopsy revealed that embryos were classified into four categories: 1) culture medium euploid and TE biopsy euploid (Euploid-Euploid), 2) culture medium euploid and TE biopsy aneuploid (Euploid-Aneuploid), 3) culture medium aneuploid and TE biopsy euploid (Aneuploid-Euploid), and 4) culture medium aneuploid and TE biopsy aneuploid (Aneuploid-Aneuploid). Aneuploid-aneuploid samples were further categorized as "homoploid concordant," "partial ploid concordant (overlapping)," "partial ploid concordant (complementary)," and "partial ploid concordant (non-overlapping)." Figure 3C Representative samples of each category are shown. By analyzing the copy number results obtained by the two methods, it was found that the chromosome ploidy consistency rate detected by the two methods for the blastocyst culture medium without granulosa cell contamination was the highest (68 / 92, 73.9%) and the false negative rate was the lowest (7 / 51, 13.7%), while the chromosome ploidy consistency rate detected by the two methods for the blastocyst culture medium with severe granulosa cell contamination was the lowest (46.5%) and the false negative rate was the highest (90.0%). The false positive rates of the culture medium without granulosa cell contamination, moderate contamination and severe contamination were 41.5%, 35.0% and 21.7%, respectively, indicating that granulosa contamination masks false positive aneuploidy, which may be caused by mosaicism ( Figure 3D ).

[0172] Example 3: Contamination of blastocyst culture medium with polar body cells

[0173] To further explore the cellular origin of free DNA in culture medium, we performed cluster analysis on 96 samples without granulosa cell contamination, as well as preimplantation embryonic cells and germ cells. The results showed that most culture medium samples (92 out of 96) clustered with ICM and TE, while one sample (#S167) and two samples (#S176 and #S193) clustered with MII oocytes and maternal pronuclei, respectively ( Figure 4A Since the genomic DNA and pronucleus of the oocyte should not be released, these cultures are likely to contain components of polar bodies that are produced by the oocyte during meiosis.

[0174] To further quantify polar body contamination, we identified 548 oocyte / polar body cell-specific differentially methylated regions (O-DMRs). By calculating the methylation differences of these 548 O-DMRs in oocyte standards, blastocyst cells, and granulosa cells, we ranked them in ascending order based on the p-values ​​obtained by significance calculation. These O-DMRs are shown in the attached Table 2. These differentially methylated regions are hypermethylated in MII oocytes and hypomethylated in preimplantation embryonic cells, granulosa cells, and sperm ( Figure 4B The three culture medium samples that were clustered with MII oocytes and maternal pronuclei had significantly higher methylation levels at O-DMRs than the other culture medium samples (median methylation levels: 100%, 56%, and 79% for #S167, #S176, and #S193, respectively, compared to a median methylation level of 14% for the other culture medium samples ( Figure 4C ). Observing the copy number of the three culture fluid samples, it was found that all of them were false negative or gender inconsistent: the TE biopsy results of #S176 and #S193 were "46,XY", and the TE biopsy result of #S167 was "-21,XX", but the culture fluid results of the three samples showed "46,XX" ( Figure 4D Calculation of the C-DMR methylation levels for these three samples revealed that they were clearly not contaminated by granulosa cells.

[0175] We calculated the methylation of O-DMRs in culture fluid samples and found that approximately 1 / 3 (27%, 53 / 191) of the culture fluid samples were contaminated with polar bodies (O-DMR methylation levels higher than 31%, the average O-DMR methylation level in ICM / TE (22%) + 3 SD (3%)). We also detected that the methylation levels of non-CpG islands in culture fluid samples were positively correlated with the O-DMR methylation levels ( Figure 4E ).

[0176] Example 4: Embryonic cells in blastocyst culture medium are derived from ICM and TE

[0177] In order to further explore the embryonic origin of free DNA in the blastocyst culture medium, we further conducted an in-depth analysis of the culture medium samples without granulosa cell and polar body cell contamination. We first analyzed the DNA methylation data of the ectoderm (EPI) and trophoblast (TE) samples of single-cell multi-omics sequencing recently published by our group (F. Zhou et al., Reconstituting the transcriptome and DNA methylome landscapes of human implantation. Nature 572, 660-+ (2019).), and found through principal component analysis (PCA) that EPI and TE can be roughly separated based on the DNA methylation map. Then, we added culture medium samples without granulosa cell and polar body contamination on the 6th day (n=61) and performed principal component analysis. The results showed that about 1 / 3 (18 / 61) of the culture medium samples clustered with TE, and about 2 / 3 (43 / 61) clustered with EPI ( Figure 5A ). EPI and TE can be distinguished by dividing the methylation level of EPI differentially expressed genes by the methylation level of TE differentially expressed genes. From this, we inferred that the embryonic origin of free DNA in the culture medium is TE and ICM ( Figure 5B ).

[0178] Example 5: Derivation of maternal DNA contamination rate in culture medium and chromosome aneuploidy analysis

[0179] We sought to quantify the proportion of maternal DNA contamination in culture medium. We established an algorithm to deduce the DNA fractions of granulosa cells and polar cells in culture medium using the methylation levels of 769 C-DMRs and 548 O-DMRs. The sum of the proportions of these two fractions was then used as the net maternal DNA contamination fraction, calculated as follows:

[0180]

[0181] Among them MM i Represents DMR in culture medium free DNA i Methylation level of MC ik Indicates DMR in component k i The methylation level of krepresents the proportional contribution of component k to the free DNA in the culture medium. DMRs are of two types: C-DMRs and O-DMRs. The culture medium contains three components: blastocysts, granulosa cells, and polar bodies. The net sum of the three components is 100%. The DNA methylation levels of DMRs are referred to as: i) C-DMRs, which are 92% in granulosa cells, 4% in blastocysts, and 3% in oocytes / polar bodies; ii) O-DMRs, which are 19% in granulosa cells, 22% in blastocysts, and 82% in oocytes / polar bodies. Correction factor a ik represents the PCR amplification efficiency of DMRi in component k, because PCR amplification of bisulfite-converted DNA tends to favor the unmethylated allele. Our data show that the correction factor a for C-DMRs in granulosa cells is ik The correction factor a for polar somatic O-DMRs is approximately 0.6. ik is about 0.6, and all other values ​​are 1.

[0182] To validate the accuracy of this method, we generated a series of simulated data for ICM / TE and granulosa cells with different incorporation ratios by computer for simulation analysis. The simulated data were obtained as follows: by sampling high-quality methylation sequencing data from MII oocytes (n=33), ICM / TE (ICM, n=9; TE, n=9), and granulosa cells (n=12), polar body, blastocyte, and cumulus cell simulated data were synthesized for simulation analysis. 3,030,303 uniquely aligned reads were randomly sampled from each of the 33 MII oocytes to synthesize "average" MII oocyte simulation data (i.e., polar body simulation data) with 1 million reads. Then, a certain proportion of reads, such as 50% (50,000,000 reads), is randomly selected from the MII oocyte simulation data and mixed with a certain proportion of reads from other cell types, such as 50% (50,000,000 reads) from the blastocyst simulation data, to generate a mixed cell simulation data with approximately one million unique aligned reads. After a series of simulation data with different incorporation ratios of ICM / TE and granulosa cells are generated by computer, the percentages of granulosa cells and polar body cells are calculated using the method of the present invention, and the results are as follows: Figure 6 As shown in A, the calculated percentage has a good linear correlation with the actual incorporation percentage (R = 0.99, Pearson correlation) ( Figure 6 B).

[0183] Next, we calculated the proportion of each component in the actual culture medium samples. Granulocytes in the culture medium caused more serious contamination than polar body cells (the number of culture medium samples with granulocyte contamination > 60%: 39 / 182, 22%; the number of culture medium samples with polar body contamination > 60%: 7 / 182, 4%) ( Figure 7A There was a slight correlation between granulosa cell contamination and polar body cell contamination in the culture medium (R = -0.19, Pearson correlation), which may reflect the situation where low fetal DNA content leads to high maternal contamination ( Figure 7B Approximately one-third (31.3%, 57 / 182) of the culture medium samples showed a net maternal DNA contamination greater than 60%, and one-third (34.1%, 62 / 182) showed a net maternal DNA contamination less than 20% ( Figure 7C ).

[0184] To explore the effect of maternal contamination on chromosome copy number, we calculated the gender discordance rate (GDR) and false negative rate (FNR) of the copy number results obtained from the culture medium and the TE biopsy results. The results of gender discordance rate showed that when the net maternal contamination ratio was less than 20%, the gender discordance rate was zero (0%, 0 in 24). When the granulosa cell contamination ratio was lower than 20%, the gender discordance rate remained at 18% (9 / 49), and when the polar body cell contamination ratio was lower than 20%, the gender discordance rate remained at 42% (24 / 57) ( Figure 7D ).

[0185] The false negative rate results showed that when the net maternal contamination ratio was less than 20%, the false negative rate was still high (16%, 6 / 37) ( Figure 7D ). Careful examination of the chromosome copy number profiles revealed that these false-negative culture fluid samples were mosaic aneuploids with signs of copy number gain or loss, matching or complementing the TE biopsy results in the majority of cases (5 of 6). This suggests that these embryos contained both aneuploid and euploid cells, and that the euploid cells were not sampled by the TE biopsy. Both the sex-inconsistency rate and the false-negative rate increased with increasing granulosa cells, polar body cells, and net maternal contamination rates. When the net maternal contamination ratio was higher than 60%, the sex-inconsistency rate and the false-negative rate increased to 100% (31 / 31) and 75% (6 / 8), respectively ( Figure 7D ).

[0186] We also studied the relationship between sampling time and maternal contamination and found that the proportion of granulosa cell contamination, gender inconsistency rate and false negative rate in the culture medium on day 6 were significantly lower than those on day 5. The proportion of polar body cell contamination in the culture medium was not related to the sampling time, which proved that the release of DNA from polar body cells is a continuous process ( Figure 7E ).

[0187] Example 6: Derivation of maternal DNA contamination rate in culture medium using different numbers of DMRs

[0188] We used the top 10 / 50 / 80 / 100 / 150 / 200 C-DMRs and O-DMRs, respectively, to calculate the contamination rate of maternal DNA in the culture medium. In short, a series of simulated data of polar bodies (MII oocytes), ICM / TE and granulosa cells with different incorporation ratios were generated by computer. The contamination percentages of granulosa cells and polar body cells were calculated using the formula shown in Example 5 based on the top 10 / 50 / 80 / 100 / 150 / 200 DMRs in Tables 1 and 2, and the linear correlation between the estimated percentages and the actual incorporation percentages was compared. The values ​​that need to be substituted into the calculation formula are: The values ​​are shown in the following table.

[0189] Table 3: Calculation using top10 / 50 / 80 / 100 / 150 / 200 DMR value

[0190]

[0191]

[0192] The test results are as follows Figures 8A-8B The results showed that when only the top 10 DMRs (i.e., top 10 C-DMRs and top 10 O-DMRs), top 50 DMRs (i.e., top 50 C-DMRs and top 50 O-DMRs), top 80 DMRs (i.e., top 80 C-DMRs and top 80 O-DMRs), top 100 DMRs (i.e., top 100 C-DMRs and top 100 O-DMRs), top 150 DMRs (i.e., top 150 C-DMRs and top 150 O-DMRs), and top 200 DMRs (i.e., top 200 C-DMRs and top 200 O-DMRs) were measured, the granulosa cell contamination ratio and polar body cell contamination ratio in the culture medium derived from the formula were almost consistent with the actual spike-in ratio. These results demonstrate that accurate detection of maternal DNA contamination rates can be achieved by using some of the C-DMRs shown in Table 1 and the O-DMRs shown in Table 2.

[0193] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

[0194] Table 1: C-DMRs

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212] Note: The chromosome positions shown in Table 1 refer to hg19.

[0213] Table 2: O-DMRs

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226] Note: The chromosome positions shown in Table 2 refer to hg19.

[0227] References

[0228] 1.Montag M, M,Strowitzki T,Toth B.Polar body biopsy.FertilSteril.2013Sep;100(3):603-7.doi:10.1016 / j.fertnstert.2013.05.053.Epub 2013Jun21.PMID:23796365.

[0229] 2.Ray PF, Kaeda JS, Bingham J, Roberts I, Handyside AH. Preimplantation genetic diagnosis of beta-thalassaemia major. Lancet.1996Jun 15;347(9016):1696.doi:10.1016 / s0140-6736(96)91524-x.PMID:8642985.

[0230] 3.Handyside AH,Pattinson JK,Penketh RJ,Delhanty JD,Winston RM,Tuddenham EG.Biopsy of human preimplantation embryos and sexing by DNAamplification.Lancet.1989Feb 18;1(8634):347-9.doi:10.1016 / s0140-6736(89)91723-6.PMID:2464730.

[0231] 4.Kokkali G,Vrettou C,Traeger-Synodinos J,Jones GM,Cram DS,Stavrou D,Trounson AO,Kanavakis E,Pantos K.Birth of a healthy infant followingtrophectoderm biopsy from blastocysts for PGD of beta-thalassaemia major.HumReprod.2005Jul;20(7):1855-9.doi:10.1093 / humrep / deh893.Epub 2005May 5.PMID:15878929.

[0232] 5.Schoolcraft WB,Fragouli E,Stevens J,Munne S,Katz-Jaffe MG,WellsD.Clinical application of comprehensive chromosomal screening at theblastocyst stage.Fertil Steril.2010 Oct;94(5):1700-6.doi:10.1016 / j.fertnstert.2009.10.015.Epub 2009Nov 25.PMID:19939370.

[0233] 6.Cimadomo D,Capalbo A,Ubaldi FM,Scarica C,Palagiano A,Canipari R,Rienzi L.The Impact of Biopsy on Human Embryo Developmental Potential duringPreimplantation Genetic Diagnosis.Biomed Res Int.2016;2016:7193075.doi:10.1155 / 2016 / 7193075.Epub 2016Jan 28.PMID:26942198;PMCID:PMC4749789.

[0234] 7.Stigliani S,Anserini P,Venturini PL,Scaruffi P.Mitochondrial DNAcontent in embryo culture medium is significantly associated with humanembryo fragmentation.Hum Reprod.2013Oct;28(10):2652-60.doi:10.1093 / humrep / det314.Epub 2013 Jul 25.PMID:23887072.

[0235] 8.Xu J,Fang R,Chen L,Chen D,Xiao JP,Yang W,Wang H,Song X,Ma T,Bo S,Shi C,Ren J,Huang L,Cai LY,Yao B,Xie XS,Lu S.Noninvasive chromosome screeningof human embryos by genome sequencing of embryo culture medium for in vitrofertilization.Proc Natl Acad Sci U S A.2016 Oct18;113(42):11907-11912.doi:10.1073 / pnas.1613294113.Epub 2016 Sep 29.PMID:27688762;PMCID:PMC5081593.

[0236] 9.Hammond ER,McGillivray BC,Wicker SM,Peek JC,Shelling AN,Stone P,Chamley LW,Cree LM.Characterizing nuclear and mitochondrial DNA in spentembryo culture media:genetic contamination identified.Fertil Steril.2017 Jan;107(1):220-228.e5.doi:10.1016 / j.fertnstert.2016.10.015.Epub 2016Nov 16.PMID:27865449.

[0237] 10. Vera-Rodriguez M, Diez-Juan A, Jimenez-Almazan J, Martinez S, NavarroR, Peinado V, Mercader A, Meseguer M, Blesa D, Moreno I, Valbuena D, Rubio C, Simon C. Origin and composition of cell-free DNA in spent medium from human embryoculture during preimplantation development. Hum Reprod. 2018 Apr 1;33(4):745-756.doi:10.1093 / humrep / dey028.PMID:29471395. SEQUENCE LISTING <110> Beijing University <120> Methods for non-invasive preimplantation aneuploidy genetic detection <130> IDC210005 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Primer P5-N9 <220> <221> misc_feature <222> (23)..(30) <223> n is a, c, g, or t <400> 1 ctacacgacg ctcttccgat ctnnnnnnnn 30 <210> 2 <211> 29 <212> DNA <213> Artificial sequence <220> <223> P7-N9 primers <220> <221> misc_feature <222> (22)..(29) <223> n is a, c, g, or t <400> 2 agacgtgtgc tcttccgatc tnnnnnnnn 29

Claims

1. A method for assessing the proportion of maternal DNA contamination in a test sample containing embryonic cell-free DNA, comprising: Based on the DNA methylation sequencing data of the sample to be tested, obtaining the methylation levels of granulosa cell-specific differentially methylated regions (C-DMRs) and oocyte / polar body cell-specific differentially methylated regions (O-DMRs); wherein the C-DMRs are selected from Table 1 and at least include the sequences 1-10 selected from Table 1; and the O-DMRs are selected from Table 2 and at least include the sequences 1-10 selected from Table 2; Furthermore, the method further comprises the following steps: - The proportion of granulocyte-derived DNA in the sample (P k1 ) and the proportion of polar body cell-derived DNA in the sample (P k2 ): Among them, k1-k3 represent components, which are granulosa cells, polar body cells, and blastocysts, respectively; C represents C-DMR, O represents O-DMR; P k1 、P k2 、P k3 Respectively represent the proportion of DNA derived from granulosa cells, polar body cells, and blastocysts in the samples; represents the average methylation level of the measured C-DMR; C / k3 represent the average methylation level reference values ​​of the C-DMR in granulosa cells, polar body cells, and blastocysts, respectively; a C / k1 、a C / k2 、a C / k3 are correction factors, representing the average PCR amplification efficiency of the C-DMR in granulosa cells, polar body cells, and blastocysts, respectively; represents the average methylation level of the measured O-DMR; O / k3 represent the average methylation level reference values ​​of the O-DMR in granulosa cells, polar body cells, and blastocysts, respectively; a O / k1 、a O / k2 、a O / k3 are correction factors, representing the average PCR amplification efficiency of the O-DMR in granulosa cells, polar body cells, and blastocysts, respectively; - Calculate the proportion of maternal DNA contamination using the following formula: P 母源污染 =P k1 +P k2 ; Furthermore, the sample to be tested is blastocyst culture medium.

2. The method according to claim 1, wherein The C-DMR comprises a differentially methylated region ranked 1-10, 1-50, 1-80, 1-100, 1-150, 1-200, 1-300, 1-400 or 1-500 of Table 1.

3. The method according to claim 1, wherein The C-DMRs included all differentially methylated regions shown in Table 1.

4. The method according to claim 1, wherein The O-DMR comprises a differentially methylated region of rank 1-10, rank 1-50, rank 1-80, rank 1-100, rank 1-150, rank 1-200, rank 1-300, rank 1-400, rank 1-500, rank 1-600 or rank 1-700 of Table 2.

5. The method according to claim 1, wherein The O-DMRs contained all differentially methylated regions shown in Table 2.

6. The method of claim 1, wherein are the average methylation levels of C-DMR and O-DMR in the granulocyte standard, respectively. are the average methylation levels of C-DMR and O-DMR in polar body cell standards, The methylation levels of C-DMR and O-DMR in blastocyst standards are respectively the average.

7. The method of claim 1, wherein: The C-DMR comprises the differentially methylated regions ranked 1-10 as shown in Table 1, 93%, 2%, and 4% respectively; The C-DMR comprises the differentially methylated regions ranked 1-50 as shown in Table 1, 90%, 3%, and 4% respectively; The C-DMRs comprise the differentially methylated regions ranked 1-80 as shown in Table 1, 91%, 3%, and 4% respectively; The C-DMRs comprise differentially methylated regions ranked 1-100 as shown in Table 1, 91%, 3%, and 4% respectively; The C-DMR comprises the differentially methylated regions ranked 1-150 as shown in Table 1, 91%, 3%, and 4% respectively; The C-DMR comprises the differentially methylated regions ranked 1-200 as shown in Table 1, 92%, 3%, and 4% respectively; or The C-DMR includes all differentially methylated regions shown in Table 1, They are 92%, 3% and 4% respectively.

8. The method of claim 1, wherein: The O-DMR comprises the differentially methylated regions ranked 1-10 as shown in Table 2, 22%, 91%, and 0% respectively; The O-DMRs comprise differentially methylated regions ranked 1-50 as shown in Table 2, 31%, 75%, and 15% respectively; The O-DMRs comprise the differentially methylated regions ranked 1-80 as shown in Table 2, 24%, 74%, and 13% respectively; The O-DMRs comprise the differentially methylated regions ranked 1-100 as shown in Table 2, 23%, 76%, and 13% respectively; The O-DMRs comprise differentially methylated regions ranked 1-150 as shown in Table 2, 23%, 75%, and 13% respectively; The O-DMR comprises the differentially methylated regions ranked 1-200 as shown in Table 2, 23%, 77%, and 12% respectively; or The O-DMRs include all differentially methylated regions shown in Table 2, They are 19%, 82% and 22% respectively.

9. A computer-readable medium comprising a plurality of instructions for controlling a computing system, wherein the plurality of instructions are used to control the computing system to execute the method described in any one of claims 1 to 8 so as to perform an operation for evaluating the proportion of maternal DNA contamination in a test sample containing embryonic free DNA.

10. A computer-readable medium comprising a plurality of instructions for controlling a computing system, wherein the plurality of instructions are used to control the computing system to execute the following method to perform the operation of preimplantation aneuploidy genetic detection based on blastocyst culture medium, wherein the method comprises: - Obtain methylation sequencing data of blastocyst culture fluid; - obtaining the copy number of a target region based on the methylation sequencing data, wherein the target region is selected from a chromosome, a subchromosomal region, a local region on the genome, or any combination thereof; - Obtaining the maternal DNA contamination ratio in the blastocyst culture medium using the method according to any one of claims 1 to 8; wherein obtaining the copy number of the target region and obtaining the maternal DNA contamination ratio can be performed in any order or simultaneously; - using the maternal DNA contamination ratio to correct the obtained copy number and / or mosaicism of the target region; - diagnosing chromosomal aneuploidy and / or its mosaicism, and / or copy number variation and / or its mosaicism in subchromosomal regions and / or local regions on the genome based on the corrected data.

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