CN115472223A

CN115472223A - Methylation sequencing data analysis method

Info

Publication number: CN115472223A
Application number: CN202210609003.XA
Authority: CN
Inventors: 文路; 黄锦; 汤富酬; 乔杰; 陈依东; 高原
Original assignee: Peking University; Peking University Third Hospital Peking University Third Clinical Medical College
Current assignee: Peking University; Peking University Third Hospital Peking University Third Clinical Medical College
Priority date: 2021-06-11
Filing date: 2022-05-31
Publication date: 2022-12-13

Abstract

The invention relates to a methylation sequencing data analysis method. Specifically, the interference of methylation sequencing data from a parent (such as maternal DNA) on the methylation sequencing data of the blastocyst can be greatly reduced by performing methylation sequencing on free DNA in a blastocyst culture solution and screening reads with the average methylation level of CpG sites in the sequencing data of 0-0.7.

Description

Methylation sequencing data analysis method

Technical Field

The invention relates to the field of sequencing data analysis, in particular to a method for analyzing methylation sequencing data of a biological sample containing embryo free DNA (embryo cell-free DNA). Specifically, the invention utilizes bioinformatics means to process the genome-wide methylation sequencing data of the blastocyst culture solution, and particularly obtains a reading with a certain range of average methylation level through screening, thereby greatly reducing the interference of the methylation sequencing data from a mother body (such as maternal DNA) on the methylation sequencing data of the blastocyst.

Background

Currently, the main procedure for PGT-a involves isolating 1 or more embryonic cells to evaluate 23 copies of chromosomes and sub-chromosomal regions, with 3 major embryonic cell materials being used, the first being the isolation of polar cells for detection, which is a method that is less clinically applicable because of the lack of a parental genome to accurately detect fertilized embryos and because polar bodies are highly degradable. The second method is to isolate one cell in a blastomere for detection. The method can affect the development of embryos, can reduce the implantation potential, and is clinically not used at present because only 1 cell is obtained for detection, misdiagnosis caused by chimerism (multiple copy number conditions exist in one embryo) can exist. The third is to isolate 5-10 cell biopsies in trophectoderm, and this TE biopsy method is the current clinical routine detection method. However, the literature reports that the biopsy method is harmful to embryos and parents, the long-term safety of the offspring is not fully evaluated, and the chimerism risk also exists. All the methods have invasive operations on embryos and have a plurality of disadvantages.

The fact that free DNA exists in the culture solution of the blastula cultured in vitro is discovered by italian scientists in 2013, and the discovery brings hope for non-invasive genetic detection before implantation. Professor Xie Xiaoliang in 2016 developed a novel single cell whole genome amplification method, which includes whole genome sequencing of trace amount of DNA in culture liquid and calculation of chromosome copy number, and found that the coincidence rate of the copy number obtained from the culture liquid and the copy number obtained from the whole embryo is over 85%. And the transferred embryos are guided by the culture solution results, and the final live yield exceeds 70%. An article reported in 2017 that there was contamination of maternal granular cells in the culture broth. Contamination of the granulosa cells has a fatal influence on the evaluation of chromosome copy number. Because the granulosa cells are 2-fold, if there is a copy number abnormality in the embryo, the detection result that euploid appears in the abnormal copy number is masked.

The invention provides a method for analyzing methylation sequencing data of a blastocyst culture solution, which can greatly reduce the interference of the methylation sequencing data of a mother body (such as maternal DNA) on the methylation sequencing data of the blastocyst, thereby more accurately reflecting the DNA methylation condition of the blastocyst.

Disclosure of Invention

The research finds that the interference of methylation sequencing data from a parent (such as maternal DNA) on the methylation sequencing data of the blastocyst can be greatly reduced by performing methylation sequencing on free DNA in a blastocyst culture solution and screening reads with the average methylation level of CpG sites in the sequencing data of 0-0.7.

Thus, in one aspect, the present application provides a method of analyzing methylation sequencing data of a biological sample containing embryo-free DNA, comprising the steps of:

providing the methylation sequencing data;

and screening to obtain reads (reads) with the average methylation level of the CpG sites in the methylation sequencing data of 0-0.7.

The methylation level of a CpG site is the ratio of the number of reads containing methylated cytosines at the CpG site to the total number of reads. For example, a read with a 0 methylation level of a CpG site means that the read contains 1 or more CpG sites, but all CpG sites are unmethylated. It is specifically defined that the CpG sites herein are specifically CpG sites in DNA that have not been methylation sequenced by chemical or enzymatic transformation, etc., and that may be expressed differently in the sequencing data depending on the methylation sequencing method.

In some embodiments, the methylation sequencing data consists of a plurality of reads.

In general, the methylation sequencing data can be obtained by performing methylation sequencing on the biological sample. It will be readily appreciated that the methods of the invention are not limited to a particular methylation sequencing method. Thus, various methylation sequencing methods can be used to obtain the methylation sequencing data described herein. In some embodiments, whole genome methylation sequencing, targeted methylation sequencing, third generation single molecule-long read-based methylation sequencing methods, methods of sequencing,

Methylation sequencing by Enzymatic transformation (EM-seq), TET-assisted pyridine borane sequencing (TAPS) methylation sequencing of the biological samples (A new sequencing method to detect DNA modifications, chunxao Song, et al.Ludwig institute for cancer research. Feb.25,2019; bisutite-free detection of 5-Methyl and 5-hydroxy ethyl sugar base, yiibin Liu, et al.Nature Biotechnology, volume 37, pages 424-429 (2019)).

The whole genome methylation sequencing is a single cell whole genome methylation sequencing technology. Such techniques are well known to those skilled in the art, see, e.g., stuart T, satija r. Integrated single-cell analysis. Nat Rev gene.2019 may;20 (5):257-272. In some embodiments, the single cell genome-wide methylation sequencing technology is selected from the group consisting of the science-seq (single-cell bisulphite sequencing) (see, e.g., smallwood, S.A. et al. Single-cell genome-side sequencing for assessing genetic specificity. Methods 11, 817-2014)), the snmC-seq (single-nuclear methylation sequencing) (see, e.g., luo, C.et al. Single-cell identity genes and sequencing in biological samples of single cell genome-nucleic acids, science, 600-2017), the single cell genome methylation sequencing technology is cited in the genome-genome sequencing, see, e.g., the sample-cell genome sequencing, and the simple-cell genome sequencing, see, e.g., sample-12, sample-cell genome sequencing, and sequencing in biological sample analysis, and sequencing, the detection of single cell genome-nucleotide, science, 7-20126, and the detection of single cell genome-cell, see, sample-gene, 7-12, sample-cell, and the combination of single cell.

In some embodiments, the whole genome methylation sequencing is based on bisulfite sequencing technology. The principle of bisulfite sequencing technology is as follows: following bisulfite treatment, specific primers were designed for the altered DNA sequence and Polymerase Chain Reaction (PCR) was performed. The original unmethylated cytosine sites in the PCR product are replaced by thymine, while the methylated cytosine sites remain unchanged. The PCR product was cloned and then sequenced. By this method, the methylation state of a specific site in each genomic DNA molecule can be obtained.

In some exemplary embodiments, the whole genome methylation sequencing is scBS-seq.

In some embodiments, the screening obtains reads with an average methylation level of 0 at CpG sites in the methylation sequencing data. In some embodiments, the screening obtains reads in the methylation sequencing data having an average methylation level of CpG sites of 0.1 or less. In some embodiments, the screening obtains reads in the methylation sequencing data having an average methylation level of CpG sites of 0.2 or less. In some embodiments, the screening obtains reads in the methylation sequencing data having an average methylation level of CpG sites of 0.3 or less. In some embodiments, the screening obtains reads in the methylation sequencing data having an average methylation level of CpG sites of 0.4 or less. In some embodiments, the screening obtains reads in the methylation sequencing data having an average methylation level of CpG sites of 0.5 or less. In some embodiments, the screening obtains reads in the methylation sequencing data having an average methylation level of CpG sites of 0.6 or less. In some embodiments, the screening obtains reads in the methylation sequencing data having an average methylation level of CpG sites of 0.7 or less.

In some embodiments, the biological sample is a blastocyst broth. The blastocyst is particularly preferably a mammalian blastocyst, especially a human blastocyst. In some embodiments, the biological sample is a culture fluid obtained by culturing a human fertilized egg in vitro for 4 to 7 days, such as a culture fluid obtained on days 4 to 5, a culture fluid obtained on days 4 to 6, or a culture fluid obtained on days 4 to 7. In some embodiments, granulosa cell DNA is present in the culture fluid in addition to embryo-free DNA. In some embodiments, the granulosa cell DNA comprises less than 10%, less than 20%, less than 30%, less than 40%, less than 50%, less than 60%, or less than 70% of the total DNA. In some embodiments, polar DNA is present in the culture broth at the same time. In some embodiments, polar DNA is not present in the culture fluid. In some embodiments, the granulosa cell DNA comprises less than 60% of total DNA in the culture fluid, and polar DNA is absent.

The above-described method for analyzing methylation sequencing data of biological samples containing embryo-free DNA can be implemented using bioinformatics methods. Thus, in a second aspect, the present application provides a computer readable medium encoded with a plurality of instructions for controlling a computing system to perform analysis of methylation sequencing data of a biological sample containing embryo-free DNA, wherein the instructions when executed perform the steps of:

receiving methylation sequencing data of the biological sample;

and screening to obtain reads (reads) with the average methylation level of the CpG sites of 0-0.7 in the data.

After screening the methylation sequencing data, it can be further processed to obtain copy number variations for a chromosomal, sub-chromosomal, and/or localized region of interest on the genome. In a third aspect, the present application provides a method of analyzing a biological sample containing embryo-free DNA, comprising the steps of:

providing the biological sample;

performing methylation sequencing on the biological sample to obtain methylation sequencing data of the biological sample;

screening to obtain a read segment of which the average methylation level of the CpG sites in the methylation sequencing data is 0-0.7;

the data from the screening is further processed to obtain copy number variations for the chromosome, sub-chromosome and/or localized region on the genome of interest.

Methods for detecting Copy Number Variation (CNV) by methylation sequencing techniques are known to those skilled in the art. Because sequence information including methylated and unmethylated genomic regions is obtained while performing methylation sequencing, the principle of detecting chromosome or gene copy number variation is similar to that of genome sequencing. These sequencing data-based copy number variation detection methods typically may include: the sequencing data is aligned to a reference sequence and the change in copy number is determined from the cumulative amount (depth) change or coverage change of reads in the region where the sequencing data matches.

Methods for detecting copy number variation, for example by CNV-seq, are described in detail in McKernan KJ, et al, sequence and structural variation in a human genome uncovered by short-read, mapping parallel ligation sequencing using two-base encoding. Genome Res.2009;19 1527-1541, the entire disclosure of which is incorporated herein by reference.

Methods for detecting copy number variation, for example, by whole genome methylation sequencing are described in detail in Bian, s.et al.single-cell multiomics sequencing and analysis of human color cancer.science.2018nov 30;362 (6418): 1060-1063, the entire contents of which are incorporated herein by reference.

Methods for detecting copy number variation, for example by a semi-targeted methylation sequencing, degenerate representative bisulfite sequencing (RRBS), are described in detail in Hou, y.et al.single-cell triple sequencing genetic, epigenetic, and transcriptional methylation in transcriptional molecular machinery.cell.2016mar; 26 304-19, the entire disclosure of which is incorporated herein by reference.

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

In some embodiments, the reference sequence is human reference genome hg19.

In some embodiments, the chromosome of interest comprises one or more chromosomes selected from the group consisting of:

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.

In some embodiments, the sub-chromosomal region or localized region on the genome refers to a segment of a chromosome with a copy number variation of greater than 10 Mb.

For the method of the third aspect of the present application, the present application further provides a computer readable medium encoded with a plurality of instructions for controlling a computing system to perform an analysis of a biological sample containing embryo-free DNA, wherein the instructions when executed perform the steps of:

receiving the biological sample methylation sequencing data;

screening and obtaining reads (reads) with the average methylation level of the CpG sites of 0-0.7 in the data;

processing the screened data to obtain the copy number of the chromosome, the sub-chromosome and/or the local region on the genome of interest;

diagnosing chromosomal aneuploidy or degree of chimerism thereof, or copy number variation of a sub-chromosomal region or a local region on a genome, based on the copy number of the chromosome, sub-chromosome, and/or local region on the genome of interest; for example, the processed data is matched against a reference sequence, and copy number variation is determined from cumulative (depth) or coverage variations of reads in the matching region.

The degree of chimerism refers to the disparity in chromosomes of a portion of the cells in the embryo, and in particular in the number of chromosomes in the present context.

Yet another aspect of the present application relates to the use of the aforementioned method or computer readable medium for pre-implantation aneuploidy genetic testing.

Yet another aspect of the present application provides a method for determining chromosomal copy number variation, comprising the steps of:

(1) Providing methylation sequencing data of a biological sample containing embryo-free DNA;

(2) Processing said methylation sequencing data according to the method of claim 1;

(3) Matching the processed data with a reference sequence to obtain the accumulated quantity (depth) or coverage of the read of a matching area, and calculating the copy number;

(4) And (4) comparing the copy number obtained by calculation in the step (3) with a reference value, and judging whether the copy number of the chromosome concerned has variation according to the comparison result.

Definition of terms

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, the laboratory procedures of genomics, nucleic acid chemistry, molecular biology, etc. used herein are all conventional procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

As used herein, the terms "nucleic acid", "polynucleotide" refer to deoxyribonucleic acid (DNA) or ribonucleic acid "RNA" in either single-or double-stranded form, and multimers thereof. Unless otherwise specified, the term includes nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences, as well as the sequence explicitly indicated. Specifically, substitution of degenerate codons can be achieved by generating the following sequences: wherein the third position of one or more selected (or all) codons is substituted with mixed base and/or deoxyinosine residues (Batzer et al, nucleic Acid Res.19:5081 (1991); ohtsuka et al, J.biol.Chem.260:2605-2608 (1985); and Rossolini et al, mol.cell.Probes 8 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, small non-coding RNA, microrna (miRNA), piwi-interacting RNA, and short hairpin RNA (shRNA) encoded by a gene or locus.

As used herein, the term "gene" refers to a segment of DNA that is involved in the production of a polypeptide chain. It may include regions preceding and following the coding region (leader and non-transcribed trailer), as well as intervening sequences (introns) between the individual coding segments (exons).

As used herein, the term "DNA methylation" refers to a form of chemical modification of DNA that alters the genetic appearance without altering the DNA sequence. By DNA methylation is meant the covalent attachment of a methyl group at a specific nucleotide position (e.g., the cytosine 5 position of a genomic CpG dinucleotide) under the action of DNA methyltransferase, resulting in a change in chromatin structure, DNA conformation, DNA stability and the manner in which DNA interacts with proteins, thereby controlling gene expression.

As used herein, the term "copy" refers to the number of copies of a chromosome fragment. It may be the same copy of a chromosomal fragment, or different, homologous copies of a chromosomal fragment, where different copies of the chromosomal fragment contain a substantially similar set of genetic loci, where one or more alleles are different. In some cases of aneuploidy, such as M2 copy errors, it is possible that some copies of a given chromosome segment are identical and some copies of the same chromosome segment are different.

As used herein, the term "Copy number variation" refers to Copy Number Variation (CNV), which is an increase or decrease in Copy number of large segments on a genome resulting from rearrangement of the genome, primarily manifested as deletions and duplications at the sub-microscopic level. Are important components of Structural Variation (SV) of the genome. It can be said that it is another important pathogenic mechanism of chromosomal disorders.

As used herein, a "CpG site" (or CG site) is a region of DNA in which, in the linear sequence of nucleotides, in its 5 'to 3' direction, a cytosine nucleotide is followed by a guanine nucleotide. CpG is a shorthand for 5 '-C-phospho-G-3'.

As used herein, the term "chromosomal aneuploidy" refers to a condition in which an incorrect number of chromosomes is present in a cell, including the expression of any genetic defect having an abnormal number of chromosomes, e.g., having more or less chromosomes than the normal number of any one chromosome, and having an excess portion of any one chromosome in addition to the normal pair, or lacking a portion of any one chromosome in the normal pair. In the case of human cells, it may refer to the case where the cells do not contain 22 pairs of autosomes and one pair of sex chromosomes. In the case of human germ cells, it may refer to the case where 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 the situation in which more or less than two X or Y chromosomes are present, or exactly two Y chromosomes are present.

As used herein, the term "degree of chimerism" refers to the disparity in chromosomes of a portion of cells in an embryo as compared to chromosomes of other cells, and specifically in the number of chromosomes herein.

As used herein, the term "granulosa cell" refers to a cell that constitutes the layer of follicular granules.

As used herein, the term "oocyte" refers to an oogonium cell that undergoes meiosis during oogenesis.

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

As used herein, the term "blastocyst" refers to an early stage of embryo development, consisting of a hollow cell sphere enclosing a fluid-filled cavity called the blastomere.

As used herein, the term "blastocyst medium (SBM)" or "embryo medium (SEM)" have the same meaning and are used interchangeably to refer to a medium used during in vitro culture of a blastocyst/embryo prior to implantation, wherein free DNA (cfDNA) released from the embryo is present and may comprise free DNA released from feeder cells (e.g., granulocytes).

Advantageous effects of the invention

The application provides a method for analyzing methylation sequencing data of a biological sample containing embryo free DNA (embryo cell-free DNA), wherein methylation sequencing is carried out on free DNA existing in a blastocyst culture solution, reads with the average methylation level of CpG sites in sequencing data of 0-0.7 are obtained through screening, particularly reads with the methylation level of CpG sites in sequencing data of 0 are obtained through screening, interference of methylation sequencing data from a parent body (such as maternal DNA) on the methylation sequencing data of the blastocyst can be reduced to a great extent, and therefore the DNA methylation condition of the blastocyst can be accurately reflected. This method is particularly advantageous, for example, it can be conveniently used for subsequent analysis to determine true ploidy of embryo chromosomes, and has high application value for screening for aneuploidy before implantation.

Drawings

FIG. 1 shows the results of unsupervised hierarchical cluster analysis of DNA methylation levels in blastocyst broth samples, human preimplantation embryos, germ cells and granulosa cells. GV: GV oocytes; MII: MII oocytes; PN: and (3) pronucleus.

FIGS. 2A-2D show the results of the detection of chromosomal aneuploidy by scBS-seq.

FIG. 2A: CN profiles of HCT116 cells determined by scBS-seq (inner layer) and MALBAC (outer layer).

FIG. 2B: coefficient of Variation (CV) distribution of sequencing results based on unique aligned reads of different data volumes.

FIG. 2C: representative CN profiles of different types of blastocyst broths and corresponding TE biopsy results.

FIG. 2D: total consistency (GCR), false Negative (FNR) and False Positive (FPR) analyses of copy number results from 2 scBS-seq and TE biopsies with varying degrees of particulate cell contamination (no contamination, moderate contamination, severe contamination).

FIG. 3 shows the effect of mean methylation levels of CpG sites on granular cell contamination in blastocyst broth. PBAT _ G _ merge: granulosa cells, PBAT _ S24 and S53: blastocyst culture fluid without maternal contamination, i.e., free DNA within S24 and S53, was derived from embryonic DNA.

FIG. 4 shows the in silico contamination analysis of different ratios of granular cell DNA into blastocyst cultures without maternal contamination and the inclusion of inner cell mass, trophectoderm, and maternal contamination.

FIG. 4A: mixing granular cell DNA with different proportions into the DNA of the inner cell mass and performing decontamination analysis. #1 is an ICM cell with CNV having increased copy number of chromosome 12; #2 is another ICM cell with CNV at chromosome 1 copy number reduction.

FIG. 4B: and mixing the DNA of the trophectoderm cells with the DNA of the granular cells in different proportions and performing decontamination analysis. #1 is a TE cell with CNV having increased copy number of chromosome 12; #2 is another TE cell, with CNV having a reduced copy number of chromosome 5.

FIG. 4C: and mixing granular cell DNA with different proportions into the blastocyst culture solution without maternal pollution and performing decontamination analysis.

FIG. 4D: and (3) a table of the variation coefficients of the CNV after mixing different proportions of granular cells.

FIG. 5 shows the effect of decontamination analysis under different methylation levels.

FIG. 5A: in silico, DNA of ICM cells and granulosa cells of the inner cell mass are mixed according to the proportion of 1/2, the number of CpG contained is preserved > =1, the average methylation level is less than 0.1,0.2,0.3,0.4,0.5,0.6,0.7, and the number of chromosome copies is calculated. #1 is an ICM cell with CNV having an increased copy number of chromosome 12.

FIG. 5B: in silico, DNA of ICM cells and granulosa cells of the inner cell mass are mixed according to the proportion of 1/2, the number of CpG contained is preserved > =1, the average methylation level is less than 0.1,0.2,0.3,0.4,0.5,0.6,0.7, and the number of chromosome copies is calculated. #2 is another ICM cell with CNV of increased chromosome 1 copy number.

FIG. 6 shows chromosome ploidy analysis by blastocyst broth decontamination and TE biopsy.

FIG. 6A: embryo culture fluid sample PBAT _ S28_ B5 decontamination and TE biopsy analysis.

FIG. 6B: and (3) carrying out decontamination and TE biopsy analysis on the embryo culture solution sample PBAT _ S89_ B9.

FIG. 6C: embryo culture fluid sample PBAT _ S214_ B16 decontamination and TE biopsy analysis.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention.

Unless otherwise indicated, the molecular biological experimental methods and immunoassays used in the present invention are essentially described in reference to j.sambrook et al, molecular cloning: a laboratory manual, 2 nd edition, cold spring harbor laboratory Press, 1989, and F.M. Ausubel et al, eds. Molecular biology laboratory Manual, 3 rd edition, john Wiley & Sons, inc., 1995; the use of restriction enzymes is in accordance with the conditions recommended by the product manufacturer. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

1. Human blastocyst Medium Collection

A total of 194 PGT-A (preproplantation genetic testing for embryo) blastocysts and their corresponding culture media were included in this study. In all of these PGT-A cycles, fertilization was performed by intracytoplasmic sperm injection (ICSI) on the day of oocyte retrieval. On day 3, embryos were transferred to blastocyst medium. On day 4, each compacted embryo or mulberry was again carefully stripped of surrounding granular cells, thoroughly washed, and then cultured individually in new dishes (15 μ l of each culture drop). When the embryo reaches the fully enlarged blastocyst stage, it is transferred to a biopsy dish from day 5 to day 7. The culture broth in the foregoing dish was collected by a Polymerase Chain Reaction (PCR) tube and stored at-20 ℃. TE from the corresponding blastocysts was biopsied and each biopsy specimen was individually vitrified frozen. PGT analysis was performed on the biopsy cells using SNP arrays.

2. Whole genome DNA methylation sequencing of blastocyst culture solution

The single cell whole genome methylation sequencing method (scBS-seq) was used to detect the DNA methyl group in blastocyst culture fluid. The culture broth was filled to 20ul volume with nuclease-free, and lysed at 50 ℃ for 1.5h by adding a corresponding volume of lysis buffer (20 mM Tris-EDTA,20 mM potassium chloride, 0.3% Triton X-100 and 1 mg/ml proteinase K) followed by bisulfite treatment with EZ-96DNA Methylation-Direct MagPrep kit. After purification, the first DNA strand was synthesized using random primers P5-N9 (5 '-CTACACGACGCTCTTCCGATCTNNNNNNNN-3', SEQ ID NO: 1) and Klenow polymerase. This step is performed four times. The second DNA strand was synthesized using the P7-N9 primer (5 '-AGACGTGTGCTCTTCCGATCTNNNNNNNN-3', SEQ ID NO: 2). And performing PCR amplification by using the index primers and the Illumina universal PCR primers to obtain a sequencing library, and detecting 5G data of each sample.

3. DNA methylation sequencing data processing

First, we deleted the sequencing adaptors, amplification primers and low quality bases in the original bisulfite sequencing paired end read (reads) data. Then, we discard the R2 reads containing more than 3 unmethylated CHs, as well as the corresponding R1 reads. These clean reads (clean reads) were mapped to the human reference genome (hg 19) in end-to-end alignment mode using BS-Seeker 2. Unaligned reads were re-paired with the hg19 genome in a local alignment, removing the low confidence alignment of the regions of micro-homology. Next, duplicates caused by PCR amplification were deleted using the Picard tool. The ratio of the number of reads methylated to the total reads (methylated and unmethylated) for C is defined as the DNA methylation level; more than 3 reads covered CpG sites for subsequent calculations. Samples with unique aligned reads (unique mapping reads) greater than 100 million were retained for subsequent analysis.

Example 1: granular cell contamination of blastocyst culture solution

We combined analysis of DNA methylation data of preimplantation embryos, germ cells and blastocyst fluid data previously published by our group (p.zhu et al, single-cell DNA methylation sequencing of human transplantation organisms. Nat gene 50,12-19 (2018)), found that whole genome methylation levels of culture fluid free DNA ranged from 13% to 74%, median 36%, significantly higher than methylation levels of ICM and TE (24% and 24% for ICM and TE, respectively). Clustering analysis indicated that some of the broth samples (50 out of 191) were pooled with granulosa cells. The DNA methylation levels of these samples were high (60% on average), close to the DNA methylation level of granulosa cells (71% on average) (fig. 1).

Example 2: single cell whole genome methylation sequencing method (scBS-seq) for detecting aneuploidy

Our previous studies have demonstrated that scBS-seq is able to assess Copy Number (CN) variation (Y.Hou et al, cell Res 26,463 304-319 (2016.; S.H.Bian et al, science 362,1060- + (2018)). We first analyzed HCT116 cells and the results showed that scBS-seq and multiple annealing and loop-based amplification cycles (MALBAC) (c.h.zong, s.j.lu, a.r.chapman, x.s.xie, genome-Wide Detection of Single-Nucleotide and Copy-Number Variations of a Single Human science 338,1622-1626 (2012)) gave the same expected CN spectrum (fig. 2A). To accurately determine the lower sequencing depth limit of the copy number variation result, we randomly sampled the data to reduce the sequencing depth, and the result shows that the Coefficient of Variation (CV) is steadily low at a data size of 2M (fig. 2B). Next, we performed the identification of copy number results on the culture broth samples and found that most SEM samples (182 out of 191) gave clearly informative copy number maps; the remaining 9, which failed to yield a definitive result (more than 6 aneuploid fragments), were defined as "aneuploid confusion" and discarded. Comparing the copy number results obtained from the culture and TE biopsies, embryos were classified into 4 types: 1) broth and TE biopsy euploids (Euploid-Euploid), 2) broth and TE biopsy aneuploidies (Euploid-Aneuploid), 3) broth and TE biopsy euploids (Aneuploid-Euploid), 4) broth and TE biopsy aneuploidies (Aneuploid-Aneuploid). Aneuploidy-aneuploidy samples were further classified as "full-ploidy identity", "partial-ploidy identity (overlap)", "partial-ploidy identity (complement)", and "partial-ploidy identity (non-overlap)". Fig. 2C shows a representative sample of each category. As a result of analyzing the copy number obtained by the 2 methods, the ploidy consistency rate of the chromosomes of the blastocyst culture solution without particle cell pollution is the highest (68/92, 73.9%) and the false negative rate is the lowest (7/51, 13.7%) when the blastocyst culture solution with particle cell pollution is detected by the 2 methods, and the ploidy consistency rate of the chromosomes of the blastocyst culture solution with particle cell pollution is the lowest (46.5%) and the false negative rate is the highest (90.0%). The false positive rates of culture broth without particle cell contamination, moderate contamination and severe contamination were 41.5%, 35.0% and 21.7%, respectively, indicating that particle contamination masked the false positive aneuploidy, which was probably due to chimeras (fig. 2D).

Example 3: reading analysis of blastocyst culture solution free DNA and granular cell methylation data

The results above show that there was severe contamination of the blastocyst culture broth with granulosa cell-derived DNA removed from the culture broth by bioinformatics, leaving only blastocyst-derived DNA.

For DNA sequencing by methylation data processed by the method "DNA sequencing by methylation data processing" of item 3 above, we utilized methylDackel(https://github.com/dpryan79/methyldackel) The perRead command in the tool calculates the total CpG number and average methylation level in each read and counts the distribution of CpG number and average methylation level of all reads for granular cells and blastocyst culture fluid samples without maternal contamination. Further screening the CpG content>Reads with an average methylation level of 0, at which the reads derived from granulosa cells accounted for 7.5% and the reads derived from embryos accounted for 27.1%, enriched embryonic DNA by a factor of 3.6 compared to granulosa cells, at which in all cases there was minimal DNA derived from granulosa cells and most DNA derived from blastocysts (fig. 3).

Example 4: computer simulation of sample decontamination analysis

The computer simulates the pollution of particle cells in different proportions, calculates the chromosome ploidy of the simulated data, removes DNA derived from the particle cells by a bioinformatics method (the concrete method is referred to as example 3), and calculates the chromosome copy number condition.

We mixed 3 different embryonic cell types (inner cell mass ICM, trophectoderm TE, blastocyst broth SEM) and granular cell DNA with copy number variation in 4 different ratios (1/4, 1/3, 1/2, 3/4 granular cell DNA to total DNA). We found that as the DNA proportion of the granular cells increases, it becomes more difficult to see the copy number variation of the chromosome (when the particle cell contamination proportion is greater than 50%, the copy number variation is not seen at all), and the Coefficient of Variation (CV) of the mixed sample becomes smaller. We calculated the chromosome copy number case from the computer-mixed mock data with the proportion of granulocytes accounting for 50% and 75%, retaining only reads (reads) containing CpG number > =1 and an average methylation level of 0. Under the condition, the chromosome copy number condition of the most original embryo data of the simulation data can be obtained.

FIG. 4A shows the contamination analysis with different ratios of granular cell DNA mixed in the inner cell mass DNA.

In this case, #1 is an ICM cell, and CNV is increased in the copy number of chromosome 12. When the DNA of the granular cells accounts for 1/4 and 1/3 of the total DNA, the CNV is more even as a whole, and the copy number of the 12 th chromosome is still increased. When the DNA of the granular cells accounts for 1/2 of the total DNA, the position of the total reading amount (covering amount) of the chromosome 12 is obviously higher than that of other chromosomes, but the copy number is judged to be normal by software. When the number of CpG contained > =1 and the average methylation level is 0 in the case of granular cell DNA accounting for 1/2 of the total DNA, the number of copies of chromosome 12 increases, and the case where there is no maternal contamination is restored.

#2 is another ICM cell with CNV at chromosome 1 copy number reduction. When the DNA of the granular cells accounts for 1/4 and 1/3 of the total DNA, the CNV is more even as a whole, and the copy number of the No. 1 chromosome is still reduced. When the DNA of the granular cells accounts for 1/2 of the total DNA, the position of the total reading amount (covering amount) of the chromosome 1 is obviously lower than that of other chromosomes, but the copy number is judged to be normal by software. When the number of CpG contained was > =1 when the granular cell DNA accounted for 1/2 of the total DNA, and the read (read) with an average methylation level of 0 was retained, the number of chromosome 1 copies decreased, and the case where there was no maternal contamination was restored.

FIG. 4B shows the incorporation of varying proportions of granular cellular DNA into the DNA of trophectoderm cells and the decontamination analysis.

In this case, #1 was a TE cell, and CNV showed an increase in the copy number of chromosome 12. When the DNA of the granular cells accounts for 1/4 and 1/3 of the total DNA, the CNV is more even as a whole, and the copy number of the 12 th chromosome is still increased. When the DNA of the granular cells accounts for 1/2 of the total DNA, the position of the total reading amount (covering amount) of the chromosome 12 is obviously higher than that of other chromosomes, but the copy number is judged to be normal by software. When the number of CpG contained > =1 and the average methylation level is 0 in the case of granular cell DNA accounting for 1/2 of the total DNA, the number of copies of chromosome 12 increases, and the case where there is no maternal contamination is restored.

#2 is another TE cell, with CNV having a reduced copy number of chromosome 5. When the DNA of the granular cells accounts for 1/4 and 1/3 of the total DNA, the CNV is more even as a whole, and the copy number of the chromosome 5 is still reduced. When the DNA of the granular cells accounts for 1/2 of the total DNA, the position of the total number of reads (coverage) on chromosome 5 is obviously lower than that on other chromosomes, but the copy number is judged to be normal by software. When the granular cell DNA accounted for 1/2 of the total DNA, only reads (reads) containing CpG > =1 and the average methylation level was 0 were retained, and the number of chromosome 5 copies decreased, returning to the case of no maternal contamination.

FIG. 4C shows that there is no maternal contamination of blastocyst culture media mixed with different ratios of granular cell DNA and decontaminated for analysis.

CNV #1 showed an increase in copy number of chromosome 22. When the DNA of the granular cells accounts for 1/4 of the total DNA, the CNV is more even as a whole, and the copy number of the 22 th chromosome is still increased. When the DNA of the granular cells accounts for 1/3 and 1/2 of the total DNA, the total number of reads (coverage) on chromosome 22 is obviously higher than that on other chromosomes, but the copy number is judged to be normal by software. When the number of CpG contained > =1 and the number of reads (reads) with an average methylation level of 0 were retained when the granular cell DNA accounted for 1/2 of the total DNA, the number of 22 chromosome copies increased, and the case where there was no maternal contamination was restored.

CNV #2 decreased for chromosome 16 and increased for chromosome 18 copy number. When the DNA of the granular cells accounts for 1/4 and 1/3 of the total DNA, the CNV is more flattened as a whole, the copy number of the 16 chromosome is still reduced, and the copy number of the 18 chromosome is still increased. When the DNA of the granular cells accounts for 1/2 of the total DNA, the position of the total reading amount (covering amount) of the chromosome 16 is obviously lower than that of other chromosomes, the copy number of the chromosome 18 is still increased, but the copy number is judged to be normal by software. When the granular cell DNA accounted for 1/2 of the total DNA, only reads (reads) containing CpG > =1 and the average methylation level was 0 were retained, the number of copies of chromosome 16 decreased, and the number of copies of chromosome 18 increased, and the case where there was no maternal contamination was restored.

Fig. 4D shows a table of the coefficient of variation for CNV after mixing different proportions of granulosa cells. The more granulosa cell DNA that is mixed in, the smaller the coefficient of variation of CNV.

Example 5: computer simulation sample decontamination analysis-exploration decontamination condition

The computer simulates the pollution of granular cells in different proportions, calculates the chromosome ploidy of the simulated data, removes DNA derived from the granular cells by a bioinformatics method (the concrete method is referred to as example 3), and calculates the chromosome copy number condition.

We mixed the DNA of ICM cells and granulosa cells of inner cell mass with copy number variation according to the 1/2 ratio (granulosa cell DNA is 1/2 of the total DNA), and retained the reads (reads) containing CpG number > =1 and average methylation level less than 0.1,0.2,0.3,0.4,0.5,0.6,0.7, respectively, to calculate the chromosome copy number. Under the conditions, the chromosome copy number condition of the most original embryo data of the simulation data can be obtained (shown in figures 5A and 5B).

Example 6: decontamination analysis of actual samples

We retained the CpG-containing number > =1 in the actual sample, and read with an average methylation level of 0, and calculated the chromosome copy number. We found that under these conditions we could obtain a consistent chromosome copy number profile with TE biopsies (FIGS. 6A-6C).

FIG. 6A shows PBAT _ S28_ B5 with TE biopsy result +19,XX. Before the embryo culture fluid sample is decontaminated, the distance between the coverage of the reading of the chromosome 19 and the base line 2 (copy) is 0.28 and is lower than 0.5 cutoff, so that the reading is judged to be a normal copy number, and the calculated karyotype shows that the reading is normally 46,XX. When only reads (reads) containing CpG > =1, with an average methylation level of 0 are retained, the read coverage for chromosome 19 increases by 0.51 from baseline 2, exceeding the reference value of 0.5, and significantly greater than the distance from baseline 2 for the other chromosomes, and the chromosome copy number shows +19,xx, which is in perfect agreement with the TE biopsy results.

FIG. 6B shows PBAT _ S89_ B9, TE biopsy result-22. Before the embryo culture fluid sample is decontaminated, a chromosome copy number graph obtained by calculating the culture fluid shows that the reading total quantity (coverage quantity) position of the chromosome 22 is obviously lower than that of other chromosomes, but the distance from the baseline 2 (copy) is 0.44 and is lower than the reference value 0.5, so that the embryo culture fluid sample is judged to be normal copy number. When only reads (reads) containing CpG > =1, with an average methylation level of 0, were retained, the read coverage change for chromosome 22 increased from baseline 2 by 0.65, exceeding the reference value of 0.5, while the other chromosomes were still normal chromosome copy numbers, which was shown to be-22,xy, which is in complete agreement with the TE biopsy results.

FIG. 6C shows PBAT _ S214_ B16 with TE biopsy results of +21,XX. Before the embryo culture fluid sample is decontaminated, the distance between the reading coverage of the chromosome 21 and the base line 2 (copy) is 0.26, and the chromosome copy number is judged to be normal 46,XX. When only reads (reads) containing CpG > =1, with an average methylation level of 0 were retained, the read coverage change for chromosome 21 increased 0.66 from baseline 2, beyond the reference value of 0.5, and the chromosome copy number was shown to be +21,xx, which is in complete agreement with the TE biopsy results.

Note that in these embryo culture fluids and computer-simulated samples, the read coverage of the genomic regions with copy number variation significantly increased from baseline by decontamination treatment, exceeding the reference value, and thus changed from unrecognizable to correctly recognized; the reference value is set to 0.5 in the present embodiment, but is not limited to 0.5, and may be adjusted to achieve the best copy number judgment effect.

Although specific embodiments of the invention have been described in detail, it will be appreciated by those skilled in the art that, based upon the overall teachings of the disclosure, various modifications and alternatives to those details could be developed and still be encompassed by the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A method of analyzing methylation sequencing data of a biological sample containing embryonic free DNA (embryonic cell-free DNA), comprising the steps of:

providing the methylation sequencing data;

and screening to obtain reads (reads) with the average methylation level of the CpG sites of 0-0.7 in the methylation sequencing data.

2. A computer readable medium encoded with a plurality of instructions for controlling a computing system to perform analysis of methylation sequencing data of a biological sample containing embryo free DNA, wherein the instructions when executed implement the steps of:

receiving methylation sequencing data of the biological sample;

and screening to obtain the reads with the average methylation level of the CpG sites in the data of 0-0.7.

3. A method of analyzing a biological sample containing embryo-free DNA comprising the steps of:

providing the biological sample;

4. A computer readable medium encoded with a plurality of instructions for controlling a computing system to perform an analysis of a biological sample containing embryo-free DNA, wherein the instructions when executed perform the steps of:

receiving the biological sample methylation sequencing data;

screening and obtaining the reads with the average methylation level of the CpG sites in the data of 0-0.7;

diagnosing chromosomal aneuploidy or the degree of chimerism thereof, or copy number variation of a sub-chromosomal region or a local region on the genome, based on the copy number of the chromosome, sub-chromosome, and/or local region on the genome of interest.

5. The method or computer readable medium of any of claims 1-4, wherein the screening obtains reads with an average methylation level of CpG sites in the methylation sequencing data of 0, 0.1 or less, 0.2 or less, 0.3 or less, 0.4 or less, 0.5 or less, 0.6 or less, or 0.7 or less.

6. The method or computer readable medium of any of claims 1-5, wherein the biological sample is a blastocyst broth, such as a mammalian, especially human, blastocyst broth; preferably, the biological sample is a culture solution obtained by culturing human fertilized eggs in vitro on days 4 to 7, such as a culture solution obtained on days 4 to 5, a culture solution obtained on days 4 to 6, or a culture solution obtained on days 4 to 7.

7. The method or computer-readable medium of any of claims 1-6, wherein the biological sample is methylation sequenced using whole genome methylation sequencing or targeted methylation sequencing to obtain the methylation sequencing data.

8. The method of claim 3 or the computer readable medium of claim 4, matching the filtered data to a reference sequence, and determining copy number variation based on cumulative amount (depth) changes or coverage changes of reads of the matching region.

9. The method or computer-readable medium of claim 8, wherein the reference sequence is human reference genome hg19.

10. Use of the method or computer readable medium of any one of claims 1-9 for pre-implantation genetic detection of aneuploidy.

11. A method for determining chromosomal copy number variation, comprising the steps of:

(1) Providing methylation sequencing data of a biological sample containing embryo free DNA;

(3) Matching the processed data with a reference sequence to obtain the accumulated amount (depth) or coverage of the read of a matching area, and calculating the copy number;