CN111154851A - Embryo implantation pre-chromosome aneuploidy detection reference product based on high-throughput sequencing and preparation method thereof - Google Patents
Embryo implantation pre-chromosome aneuploidy detection reference product based on high-throughput sequencing and preparation method thereof Download PDFInfo
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Abstract
The invention provides a high-throughput sequencing-based embryo implantation pre-chromosome aneuploidy detection reference product, which comprises a positive reference product, a negative reference product, a chimera reference product and a data quality control reference product; meanwhile, the invention also provides a preparation method of the embryo implantation pre-chromosome aneuploid detection reference substance based on high-throughput sequencing. The reference product is the most comprehensive embryo implantation pre-chromosome aneuploidy detection reference product library in China at present, 89 cases of embryo implantation pre-chromosome aneuploidy detection reference products are prepared in total, and most of clinical chromosome abnormality types are covered; the kit can be used for verifying the accuracy of different sequencing platforms and clinical application of the kit based on the different sequencing platforms; the method can also be widely applied to indoor quality control and indoor quality assessment of clinical detection projects, and the accuracy and reliability of detection results are verified.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a high-throughput sequencing-based embryo implantation pre-chromosome aneuploidy detection reference product and a preparation method thereof.
Background
In vitro fertilization-embryo transfer (IVF-ET) technique refers to taking out ovum and sperm, performing in vitro fertilization under manual operation, culturing into embryo, and transplanting the embryo back into mother body. This technique can help couples who want children but do not have the corresponding fertility for physical reasons. The first test tube baby worldwide was born in the uk in 1978 by louis bronsted. The 2010 nobel prize on physiology and medicine is awarded to the father Edwards of "test-tube babies", which marks the success of the human assisted reproduction technology, and by 2012, the number of test-tube babies born by the in vitro fertilization-embryo transfer technology all over 500 ten thousand have been reached all over the world.
Despite the increasing efficiency of IVF technology, the success rate of tube infants is still relatively low. Chromosome number and structure abnormalities are the main causes of embryo implantation failure and abortion. With the increasing age of the puerpera, the number of ova is reduced, the quality is reduced, the aneuploidy of the embryo is gradually increased, the abortion rate is gradually increased, and the live birth rate is obviously reduced. However, spontaneous abortion in women, whether or not there is a history of recurrent abortion, is caused by chromosomal abnormalities in the fetus in more than half of the cases. Therefore, the detection of chromosomal abnormalities in embryos is of great importance in elderly women and couples with recurrent abortion.
For the people who carry out IVF treatment, normal and healthy embryos can be selected for transplantation through morphological evaluation and/or embryo genetic material detection before the embryos are implanted into the uterus, so that the clinical pregnancy rate of IVF treatment is improved, and the pain of pregnant women caused by pregnancy termination is avoided. However, the traditional embryo morphology evaluation system cannot reflect the genetic reality of the embryo. It is reported that only 42% of embryos are completely normal in chromosomes among morphologically good embryos. On the basis of the first and second generation tube baby technologies, the third generation tube baby technology for detecting embryo genetic materials is developed along with the progress of detection technologies such as fluorescence in situ hybridization and comparative genome hybridization. The embryo implantation pre-chromosome abnormality detection means that chromosome abnormality analysis is carried out on gametes or embryos before being transplanted into a uterine cavity by using a microscopic technology and a DNA analysis technology, embryos with normal chromosome karyotypes are screened for transplantation, and abortion of pregnant women caused by implantation of abnormal embryos or offspring suffering from serious diseases are avoided.
The utility of this technique has increased with the first report in 1995 of cases where successful pregnancy was obtained after Preimplantation Genetic Screening (PGS). The European association Of Human Reproduction and embryo (ESHRE) assistance group reported a total Of 27,630 in vitro fertilization-embryo transfer and preimplantation genetic screening and diagnosis cycles (IVF-PGD) during 1997-2007 with 57 IVF centers involved, 61% Of which were used for preneoplastic chromosomal aneuploidy testing. The combination of the genetic detection technology before embryo implantation and the adoption of the Assisted Reproduction Technology (ART) can obviously improve the implantation success rate and pregnancy rate of the embryo and reduce the abortion rate.
The traditional PGS method is fluorescence in situ hybridization FISH, but the detection range is limited to only 9-11 common chromosomes, and the detection precision is limited, so the method is gradually replaced. Emerging microarray technologies (including arrayCGH and SNParray) can expand the detection range to all chromosomes, the detection precision can be accurate to the kb level, but the sample size of one-time detection is limited, and the early construction cost and the investment are large. The whole genome sequencing method based on high-throughput sequencing has the advantages of high stability, high specificity, high throughput and the like, and has good clinical application value in PGS.
The current PGS detection methods for clinical application mainly comprise: FISH techniques, SNParray and arrayCGH techniques. The single cell-based FISH technology uses a fluorescence labeling probe to combine with a specific chromosome region to determine the chromosome state, is mainly used for detecting chromosome abnormalities such as aneuploidy or structural aberration, and can also be used for sex selection of X-linked diseases. The number of probes used in each round of hybridization in FISH is limited and all 23 pairs of chromosomes cannot be detected. The SNParray and arrayCGH technologies can be used for diagnosing 23 chromosomes simultaneously and detecting chromosome unbalanced translocation and chromosome microdeletion micro-duplication. The high throughput sequencing technique is: when the embryo after in vitro fertilization develops to 3-5 days, one blastomere cell at 8 cell stage or 3-8 trophoblast cells at the blastocyst stage are taken, and the normal development of the embryo is not influenced. However, single cell or several cells obtained during embryo biopsy have extremely low DNA content, which is not enough for subsequent genetic analysis, so that single cell whole genome amplification is required. The taken out cells can obtain enough embryo genome DNA through whole genome amplification, thereby completing whole genome low coverage sequencing and subsequent information analysis.
Clinical applications for PGS have become increasingly widespread, and preliminary clinical studies have demonstrated high sensitivity and specificity of detection means. On the other hand, uniformly approved reference products and operation specifications are lacked in clinical detection in China and outside, and with the popularization of a high-throughput sequencing platform, how to effectively guide the technical standardization development while expanding the application is a problem needing to be considered together. With the popularization and application of the technology in a wider range and the standard use, the evaluation of detection methods of different sequencing platforms is particularly important.
In order to promote the clinical application of the PGS technology, a set of PGS reference products is developed for carrying out quality evaluation on the detection result of the gene detection before implantation based on high-throughput sequencing, carrying out comparison of the detection results among different sequencing platforms, different detection processes and different sampling modes, determining the difference and the technical level among different platforms, determining the influence of the sampling mode on the detection result and the like, determining key influence factors, and formulating the technical standard for embryo detection before implantation based on high-throughput sequencing.
Disclosure of Invention
The invention provides a high-throughput sequencing-based embryo implantation pre-chromosome aneuploidy detection reference product and a preparation method thereof, which are used for performing quality evaluation on embryo implantation pre-chromosome aneuploidy detection results based on high-throughput sequencing and performance evaluation of an embryo implantation pre-chromosome aneuploidy detection kit. The difference and the technical level between different platforms are determined through the application of a reference substance, and the influence of a sampling mode on a detection result is determined. The technical scheme adopted by the invention is as follows:
the method comprises the following steps of (1) detecting an embryo preimplantation chromosome aneuploidy detection reference product based on high-throughput sequencing, wherein the reference product comprises a positive reference product, a negative reference product, a chimera reference product and a data quality control reference product; the positive reference comprises different chromosome CNV sizes, a three-body cell line sample and a three-body embryonic stem cell sample; the negative reference comprises a human oral epithelial cell sample; the chimeric reference comprises a chimeric sample mixed by a mutant cell line with a larger CNV and a mutant cell line with a smaller CNV; the data quality control reference comprises a YH cell line sample;
preferably, the positive reference comprises 66 different chromosome CNV size and three body cell line samples and 6 three body embryonic stem cell samples;
preferably, the negative reference comprises 10 oral epithelial cell samples of normal people of different sources;
preferably, the chimeric reference substance is 30% by weight;
preferably, the chimeric reference comprises 1 chimeric sample of 6 different mutant cell lines with larger CNV mixed with 6 different mutant cell lines with smaller CNV;
preferably, the chimeric reference substance is prepared by mixing 6 positive cell lines with larger CNV and 6 positive cell lines with smaller CNV according to the proportion of 1: 2;
the invention also provides a preparation method of the embryo implantation pre-chromosome aneuploidy detection reference substance based on high-throughput sequencing, which comprises the following steps:
1) harvesting cells after culturing said cell line;
2) washing, resuspending, counting, diluting, and performing subpackaging by micromanipulation;
3) picking cells from a culture dish and placing the cells into a PCR reaction tube;
4) respectively mixing the chimeric samples with the mutant cells with larger CNV and the mutant cells with smaller CNV, picking the samples and putting the samples into a PCR reaction tube;
5) putting the PCR reaction tube with the sample into a freezing tube, and labeling;
6) freezing and storing the prepared reference product;
preferably, the number of the cells in the step 3) is 3-5;
preferably, the PCR reaction tube in the step 4) is a 0.2ml PCR reaction tube;
preferably, the temperature for cryopreservation in the step 6) is-80 ℃;
the invention provides an application of the reference substance or the reference substance prepared by the method in quality evaluation of embryo implantation pre-chromosome aneuploid detection results based on high-throughput sequencing;
the invention also provides an application of the reference substance or the reference substance prepared by the method in performance evaluation of the embryo implantation pre-chromosome aneuploidy detection kit.
The invention discloses a set of embryo implantation pre-chromosome aneuploidy detection reference products based on high-throughput sequencing and a preparation method thereof, wherein the set of reference products comprises 89 reference products, wherein 72 positive reference products, 10 negative reference products, 6 chimera reference products and 1 data quality control reference product. The reference product is used for carrying out quality evaluation on embryo implantation pre-chromosome aneuploidy detection results based on high-throughput sequencing and performance evaluation of embryo implantation pre-chromosome aneuploidy detection kits. The difference and the technical level between different platforms are determined through the application of a reference substance, the influence of a sampling mode on a detection result is determined, and the like, so that the high-throughput sequencing-based embryo pre-implantation detection technical standard can be formulated.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
1. the reference product is the most comprehensive embryo implantation pre-chromosome aneuploidy detection reference product library in China at present, 89 cases of embryo implantation pre-chromosome aneuploidy detection reference products are prepared in total, and most of clinical chromosome abnormality types are covered. Meanwhile, the most comprehensive embryo sample reference database in China is constructed.
2. The reference substance can be used for verifying the accuracy of different sequencing platforms and the clinical application of the kit based on the different sequencing platforms.
3. The reference substance can be widely applied to indoor quality control and time-lapse evaluation of clinical detection items, and the accuracy of detection results is ensured.
Drawings
FIG. 1 is a flow chart of the use of high throughput sequencing based embryo preimplantation chromosome aneuploidy detection reference.
Detailed Description
In order that the invention may be more clearly expressed, the invention will now be further described by way of specific examples. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
EXAMPLE 1 cell line sample preparation
The reference samples were obtained from the European Coriell Institute for Medical Research, and 6 embryonic stem cell lines were provided in Zhongxiangya genetic and reproductive specialty Hospital, and 6 30% chimeric reference samples were prepared from 6 positive cell lines with larger CNV and 6 positive cell lines with smaller CNV at a ratio of 1:2, wherein 66 positive cell line samples of the reference samples (66 positive cell line samples and 1 positive yellow cell line sample) were obtained from oral epithelial cells of normal population.
The results of the reference karyotype are shown in the following table: basically, each sample is labeled according to the form of 'sequence number + abnormal type + abnormal size', for example, '1-del (3) -4.5M' represents that the karyotype of the No. 1 sample is a reference substance of a No. 3 chromosome deletion 4.5M fragment. The following table shows sample information of specific reference substances.
TABLE 1 reference information
Example 2 reference preparation
Reference sample including the following components was prepared, and reference 1 tube of each type was prepared, and 89 tubes were used in total
1) The relevant cell lines in table 1 were cultured under the respective culture conditions and then harvested.
2) And washing, resuspending, counting, diluting, and performing subpackaging by micromanipulation.
3) 3-5 cells were picked from the culture dish and placed in each 0.2ml PCR reaction tube.
4) The chimera sample is mixed with 6 kinds of CNV smaller mutant cells in the ratio of 1 to 2 and set in 0.2ml PCR reaction tube.
5) And (3) putting the 0.2ml PCR reaction tube filled with the sample into a 2ml cryopreservation tube, and attaching a corresponding label.
6) The prepared reference was stored at-80 ℃.
EXAMPLE 3 use of reference
The reference product is suitable for the requirements of all high-throughput sequencing platforms for embryo pre-implantation detection, and the detection method generally comprises the following steps according to the operation flow of products of each platform (such as NextSeq CN500, DA8600 and BGISEQ-500): whole genome amplification, DNA fragmentation, library preparation and sequencing.
Wherein, the product after the primary whole genome amplification is stored at the temperature of-20 ℃ or-80 ℃ after DNA fragmentation by using one part. The constructed library sample can be used for multiple sequencing quality control, and after the library sample is used up, the stored whole genome amplification product can be taken out for continuous fragmentation and library construction. The detailed flow chart is shown in figure 1.
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (12)
1. The embryo implantation pre-chromosome aneuploidy detection reference product based on high-throughput sequencing is characterized by comprising a positive reference product, a negative reference product, a chimera reference product and a data quality control reference product; the positive reference comprises different chromosome CNV sizes, a three-body cell line sample and a three-body embryonic stem cell sample; the negative reference comprises a human oral epithelial cell sample; the chimeric reference comprises a chimeric sample mixed by a mutant cell line with a larger CNV and a mutant cell line with a smaller CNV; the data quality control reference comprises a YH cell line sample.
2. The high throughput sequencing-based embryonic preimplantation chromosome aneuploidy detection reference of claim 1, wherein the positive reference comprises 66 different chromosome CNV size and trisomy cell line samples and 6 trisomy embryonic stem cell samples.
3. The high throughput sequencing-based embryo preimplantation chromosome aneuploidy detection reference product of claim 1, wherein the negative reference product comprises 10 oral epithelial cell samples of normal human of different origins.
4. The high throughput sequencing-based embryonic preimplantation chromosome aneuploidy detection reference of claim 1, wherein the chimeric reference is 30% by weight.
5. The high-throughput sequencing-based embryo preimplantation chromosome aneuploidy detection reference substance of claim 1, wherein the chimeric reference substance comprises 1 each chimeric sample of 6 different mutant cell lines with larger CNV mixed with 6 different mutant cell lines with smaller CNV.
6. The high throughput sequencing-based embryo preimplantation chromosome aneuploidy detection reference substance of claim 1, wherein the chimeric reference substance is prepared by mixing a positive cell line with a larger CNV and a positive cell line with a smaller CNV in a ratio of 1: 2.
7. The method for preparing the reference substance for detecting the embryo implantation pre-chromosome aneuploidy based on the high-throughput sequencing in any one of claims 1 to 6, which comprises the following steps:
1) harvesting cells after culturing said cell line;
2) washing, resuspending, counting, diluting, and performing subpackaging by micromanipulation;
3) picking cells from a culture dish and placing the cells into a PCR reaction tube;
4) respectively mixing the chimeric samples with the mutant cells with larger CNV and the mutant cells with smaller CNV, picking the samples and putting the samples into a PCR reaction tube;
5) putting the PCR reaction tube with the sample into a freezing tube, and labeling;
6) and (5) freezing and storing the prepared reference product.
8. The method for preparing the reference substance for detecting the embryo implantation pre-chromosome aneuploidy based on the high-throughput sequencing of claim 7, wherein the number of the cells in the step 3) is 3-5.
9. The method for preparing the reference substance for detecting the embryo implantation pre-chromosome aneuploidy based on the high-throughput sequencing in claim 7, wherein the PCR reaction tube in the step 4) is a 0.2ml PCR reaction tube.
10. The method for preparing the reference substance for detecting the embryo implantation pre-chromosome aneuploidy based on high-throughput sequencing according to claim 7, wherein the temperature for cryopreservation in the step 6) is-80 ℃.
11. Use of a reference according to any one of claims 1 to 6 or a reference prepared according to any one of claims 7 to 10 for quality assessment based on the results of high throughput sequencing based embryo preimplantation chromosomal aneuploidy detection.
12. Use of a reference according to any one of claims 1 to 6 or a reference prepared by a method according to any one of claims 7 to 10 in the performance evaluation of a kit for the detection of chromosomal aneuploidy prior to embryo implantation.
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| CN114990106A (en) * | 2022-06-10 | 2022-09-02 | 序康医疗科技（苏州）有限公司 | Sample preservation liquid for embryo preimplantation aneuploid genetic detection and preparation method and application thereof |
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| WO2014116881A1 (en) * | 2013-01-23 | 2014-07-31 | Reproductive Genetics And Technology Solutions, Llc | Compositions and methods for genetic analysis of embryos |
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|---|---|---|---|---|
| CN112582022A (en) * | 2020-07-21 | 2021-03-30 | 序康医疗科技（苏州）有限公司 | System and method for non-invasive embryo transfer priority rating |
| CN114990106A (en) * | 2022-06-10 | 2022-09-02 | 序康医疗科技（苏州）有限公司 | Sample preservation liquid for embryo preimplantation aneuploid genetic detection and preparation method and application thereof |
| CN114990106B (en) * | 2022-06-10 | 2024-05-24 | 序康医疗科技（苏州）有限公司 | Sample preservation solution for genetic detection of aneuploidy before embryo implantation and preparation method and application thereof |
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