技术领域technical field
本发明涉及生物技术领域，涉及基因组序列分析领域，具体涉及一种鉴定胚胎平衡易位携带状态的方法。The invention relates to the field of biotechnology, to the field of genome sequence analysis, and in particular to a method for identifying the carrying state of an embryo's balanced translocation.
背景技术Background technique
医学遗传学是目前医学中最前沿的学科，也是新兴学科。是生命科学主要研究的课题。医学遗传学主要利用DNA技术来研究疾病与基因的关系。开展新型的诊断技术和治疗方法。可以从分子水平为疾病的早期诊断，预防出生缺陷、以及疑难杂症的诊断和治疗提供更高效的新型医学服务。医学遗传学的发展离不开各种技术的快速进步，尤其是近年来高通量测序技术的发展。Medical genetics is the most cutting-edge discipline in medicine at present, and it is also an emerging discipline. It is the main research subject of life science. Medical genetics mainly uses DNA technology to study the relationship between diseases and genes. Develop new diagnostic techniques and treatment methods. It can provide more efficient new medical services for the early diagnosis of diseases, the prevention of birth defects, and the diagnosis and treatment of intractable diseases from the molecular level. The development of medical genetics is inseparable from the rapid progress of various technologies, especially the development of high-throughput sequencing technology in recent years.
高通量测序技术的诞生可以说是基因组学研究领域一个具有里程碑意义的事件。该技术使得核酸测序的单碱基成本与第一代测序技术相比急剧下降，以人类基因组测序为例，上世纪末进行的人类基因组计划花费30亿美元解码了人类生命密码，而第二代测序使得人类基因组测序已进入万(美)元基因组时代。如此低廉的单碱基测序成本使得我们可以实施更多物种的基因组计划从而解密更多生物物种的基因组遗传密码。同时在已完成基因组序列测序的物种中，对该物种的其他品种进行大规模地全基因组重测序也成为可能。The birth of high-throughput sequencing technology can be said to be a milestone event in the field of genomics research. This technology makes the single-base cost of nucleic acid sequencing drop sharply compared with the first-generation sequencing technology. Taking human genome sequencing as an example, the Human Genome Project at the end of the last century spent $3 billion to decode the code of human life, while the second-generation sequencing The sequencing of the human genome has entered the era of the 10,000 (US) genome. Such a low cost of single-base sequencing allows us to implement genome projects for more species and decipher the genetic codes of genomes of more biological species. At the same time, in the species whose genome sequence has been sequenced, it is also possible to perform large-scale whole genome resequencing of other varieties of the species.
高通量测序技术的发展也响应了精准医疗时代的到来，精准医疗是以个人基因组信息为基础，结合蛋白质组、代谢组等相关内环境信息，为人们量身设计出健康管理和疾病治疗方案，以期达到治疗效果最大化和副作用最小化的定制医疗方式。可以确定的是，精准医学是在医学临床试验的交汇融合应用，是医学科技发展的前沿方向。在国家“精准医学研究”重点专项中强调：要系统加强精准医学研究布局，加快重大疾病防控技术突破，占据未来医学及相关产业发展主导权，重点打造我国生命健康产业发展的新驱动力。The development of high-throughput sequencing technology also responds to the arrival of the era of precision medicine. Precision medicine is based on personal genome information, combined with relevant internal environment information such as proteome and metabolome, to design health management and disease treatment plans for people. , in order to achieve a customized medical approach that maximizes therapeutic effects and minimizes side effects. What is certain is that precision medicine is the convergence and application of medical clinical trials, and it is the frontier direction of medical technology development. In the national key project of "Precision Medicine Research", it is emphasized that it is necessary to systematically strengthen the layout of precision medicine research, accelerate technological breakthroughs in the prevention and control of major diseases, occupy the leading position in the development of future medicine and related industries, and focus on creating new driving forces for the development of my country's life and health industries.
染色体异常又分为结构异常和数目异常，染色体数目异常一般会造成明显的表型，经B超等筛查可以有效检出。而染色体结构异常的患者一般不会有表型，通常在生育下一代时因反复流产或不孕筛查而确诊。染色体结构性异常主要分为：平衡易位、罗氏易位与染色体倒位。染色体平衡易位是指两条染色体发生断裂后相互交换，仅有位置的改变，没有可见的染色体片段的增减，在人群中的发病率为0.1％-0.2％。Chromosomal abnormalities are divided into structural abnormalities and numerical abnormalities. Abnormal chromosome numbers generally cause obvious phenotypes, which can be effectively detected by B-ultrasound and other screening. Patients with abnormal chromosomal structure generally do not have a phenotype and are usually diagnosed by recurrent miscarriage or infertility screening when giving birth to the next generation. Chromosomal structural abnormalities are mainly divided into: balanced translocation, Roche translocation and chromosomal inversion. Chromosomal balanced translocation refers to the exchange of two chromosomes after they break, only the position changes, and there is no visible increase or decrease of chromosome fragments. The incidence rate in the population is 0.1%-0.2%.
辅助生殖胚胎植入前遗传学诊断(PGD)是染色体结构性异常有效的治疗手段。PGD诊疗是染色体结构性异常患者临床治疗较好策略，对于平衡易位的患者，无法检测患者胚胎是否携带了遗传自亲代的平衡易位，这就导致了平衡易位携带者的患者经PGD助孕后子代中仍有平衡易位的携带者，成年后仍将面临反复流产或者原发不孕等问题。我中心PGD随访数据显示：PGD助孕后染色体平衡易位患者出生后代中约10％仍为平衡易位携带者。如何在胚胎植入前分辨患者胚胎是否携带平衡易位，阻断平衡易位垂直传递，是PGD技术面临的重大科学问题。Preimplantation genetic diagnosis (PGD) is an effective treatment for chromosomal structural abnormalities. PGD diagnosis and treatment is a good strategy for the clinical treatment of patients with structural abnormalities of chromosomes. For patients with balanced translocations, it is impossible to detect whether the embryos of the patients carry the balanced translocations inherited from their parents. There are still balanced translocation carriers in the offspring after pregnancy, and they will still face problems such as recurrent miscarriage or primary infertility in adulthood. The PGD follow-up data of our center show that about 10% of the offspring of patients with balanced chromosome translocations after PGD are still balanced translocation carriers. How to distinguish whether a patient's embryo carries a balanced translocation before embryo implantation, and how to block the vertical transmission of a balanced translocation is a major scientific problem faced by PGD technology.
FISH技术应用与胚胎植入前遗传学诊断有效排查了胚胎的不平衡性，提高了染色体结构异常患者的妊娠率，但FISH操作过程比较复杂，检测位点少，只能检测几条染色体的非整倍体；每种平衡易位都需要设计专门的探针来区分平衡易位携带者与正常；该方法检测周期长，无法检测微缺失、微重复，无法进行全基因染色体非整倍性筛查，无法开展单基因遗传病胚胎植入前遗传学诊断等限制在临床的应用，每个平衡易位都需要设计特殊探针、操作方法繁琐以及检测范围受限，多中心随机对照研究发现FISH技术应用于PGS并没有有效提高患者的妊娠率。FISH技术无法对全基因组染色体进行筛查，因此对胚胎进行全基因组筛查就十分必要。The application of FISH technology and preimplantation genetic diagnosis can effectively check the imbalance of embryos and improve the pregnancy rate of patients with abnormal chromosomal structure. Euploid; each type of balanced translocation needs to design special probes to distinguish balanced translocation carriers from normal; this method has a long detection cycle and cannot detect micro-deletions and micro-duplications, and cannot screen for whole-gene chromosome aneuploidy Due to the inability to carry out preimplantation genetic diagnosis of monogenic genetic diseases and other limitations in clinical application, each balanced translocation requires the design of special probes, cumbersome operation methods, and limited detection range. Multi-center randomized controlled studies have found that FISH The application of technology to PGS has not effectively improved the pregnancy rate of patients. FISH technology cannot screen the genome-wide chromosomes, so genome-wide screening of embryos is very necessary.
目前在胚胎植入前遗传学诊断领域内CGH可以对胚胎进行非整倍体筛查，ArrayCGH,SNP array,NGS能够覆盖整个基因组范围、操作流程较为简单，检测通量高在临床广泛采用，但这些高通量筛查方法仅能检测染色体的非平衡易位：即染色体片段的缺失或重复；SNP array的方法较CGH位点多，覆盖度好，可以较好的进行胚胎非整倍体的筛查，亦有少量报道采用SNP array进行连锁分析进行胚胎携带状态诊断，但是无法确定易位位点，在没有易位胚胎不平衡胚胎形成的情况下，无法进行胚胎携带状态的诊断。因此CGH与SNP array临床应用于胚胎易位携带状态筛查具有一定局限性。At present, in the field of preimplantation genetic diagnosis, CGH can screen embryos for aneuploidy. ArrayCGH, SNP array, and NGS can cover the entire genome, the operation process is relatively simple, and the detection throughput is high. It is widely used in clinical practice, but These high-throughput screening methods can only detect unbalanced chromosomal translocations: that is, deletions or duplications of chromosomal segments; SNP array methods have more sites than CGH, and the coverage is better, which can better detect embryonic aneuploidy For screening, there are also a small number of reports using SNP array for linkage analysis to diagnose embryo carrier status, but the translocation site cannot be determined. In the case of unbalanced embryo formation without translocation embryos, the diagnosis of embryo carrier status cannot be performed. Therefore, the clinical application of CGH and SNP array to the screening of embryonic translocation carrier status has certain limitations.
目前有报道对平衡易位携带者进行区分采用的方式为Mate-pair文库的策略来获得断裂点，此方法建库难度相对更高，在获得具体的断裂点位置时还要经过大量的PCR后采用一代测序验证，费用高、耗时长。亦有报道采用显微切割的方式来进行平衡易位断裂点的获得，该方法操作需要显微切割仪器，显微切割的片段还要进行纯化后才可进行后续扩增实验，该法技术难度较大，过程较为繁琐，成功率相对较低。At present, it has been reported that the method used to distinguish balanced translocation carriers is the Mate-pair library strategy to obtain breakpoints. This method is relatively more difficult to build a library, and a large number of PCRs are required to obtain the specific breakpoint position. Verification by next-generation sequencing is expensive and time-consuming. It is also reported that microdissection is used to obtain balanced translocation breakpoints. This method requires microdissection equipment, and the microdissection fragments must be purified before subsequent amplification experiments. This method is technically difficult. Larger, the process is more cumbersome, and the success rate is relatively low.
除上述技术上的缺陷，以上检测技术都不能精准的确定胚胎平衡易位携带/不携带的状态。因此，本领域迫切需要开发一种能够更有效综合鉴定胚胎平衡易位携带/不携带的方法，提高判断平衡易位的精准性。In addition to the above-mentioned technical defects, none of the above-mentioned detection techniques can accurately determine the carrier/non-carrier status of the balanced translocation of the embryo. Therefore, there is an urgent need in this field to develop a method that can more effectively and comprehensively identify embryos carrying/not carrying balanced translocations, and improve the accuracy of judging balanced translocations.
发明内容Contents of the invention
针对现有技术的不足及实际的需求，本发明提供一种鉴定胚胎平衡易位携带状态的系统和方法，所述系统和方法采用的单精子扩增方式与靶向PCR的方式来寻找断裂位点，操作相对简单易行，可以有效降低PCR扩增偏倚，灵敏度高，还可以同时提供胚胎非整倍体筛查与平衡易位携带状态的信息。Aiming at the deficiencies of the prior art and actual needs, the present invention provides a system and method for identifying the carrier status of balanced translocations in embryos. The system and method use single sperm amplification and targeted PCR to find the break site Point, the operation is relatively simple and easy, can effectively reduce PCR amplification bias, has high sensitivity, and can also provide information on embryo aneuploidy screening and balanced translocation carrier status at the same time.
为达此目的，本发明采用以下技术方案：For reaching this purpose, the present invention adopts following technical scheme:
第一方面，本发明提供一种鉴定胚胎平衡易位携带状态的系统，包括如下单元：In a first aspect, the present invention provides a system for identifying the carrying state of an embryo's balanced translocation, comprising the following units:
(1)取样单元：获取胚胎待测样本、父方精液和母方DNA；(1) Sampling unit: obtain the embryo sample to be tested, the father's semen and the mother's DNA;
(2)测序单元：从父方获得的精液中挑选单精子，进行扩增，构建文库后测序；(2) Sequencing unit: select single sperm from the semen obtained from the father, perform amplification, and sequence after constructing a library;
(3)拷贝数分析单元：对测序结果进行染色体拷贝数(CNV)分析，初步确定染色体易位位置；(3) Copy number analysis unit: perform chromosome copy number (CNV) analysis on the sequencing results, and preliminarily determine the position of chromosome translocation;
(4)SNP分析单元：对染色体易位位置及周围位置进行SNP分析，通过多个单精子的比较，获得正常单精子的单倍型；(4) SNP analysis unit: perform SNP analysis on the chromosomal translocation position and surrounding positions, and obtain the haplotype of normal single sperm by comparing multiple single sperm;
(5)对母方的染色体相应的易位位置及周围位置进行SNP分析；(5) Carry out SNP analysis on the corresponding translocation position and surrounding positions of the maternal chromosome;
(6)比对单元：对待测样本的胚胎的染色体相应的易位位置及周围位置进行SNP分析，将分析的结果与正常的单精子单倍型以及母方的SNP信息进行比对，确定胚胎平衡易位携带状态。(6) Comparison unit: SNP analysis is performed on the corresponding translocation position and the surrounding position of the chromosome of the embryo to be tested, and the analysis result is compared with the normal single sperm haplotype and the SNP information of the mother to determine the balance of the embryo Translocation carry state.
根据本发明，步骤(1)所述的待测样本为胚胎的活检细胞，所述活检细胞为胚胎发育到卵裂球时期或囊胚时期取下的外胚层细胞，所述外胚层细胞可以是1个也可以是多个滋养外胚层细胞。According to the present invention, the sample to be tested in step (1) is a biopsy cell of an embryo, and the biopsy cell is an ectoderm cell removed from the blastomere stage or blastocyst stage during embryo development, and the ectoderm cell can be 1 or multiple trophectoderm cells.
根据本发明，所述母方DNA为能够提取DNA的任何人源样本都是可行的，在此不做特殊限定，本领域技术人员可以根据实验需要进行提取，本发明的母方DNA选取来自外周血、淋巴液或口腔黏膜细胞中的任意一种或至少两种的组合。According to the present invention, it is feasible for the maternal DNA to be any human-derived sample from which DNA can be extracted, and there is no special limitation here. Those skilled in the art can extract it according to experimental needs. The maternal DNA of the present invention is selected from peripheral blood, Any one or a combination of at least two of lymph fluid or oral mucosal cells.
根据本发明，步骤(2)所述挑选单精子的数量为15-30个，例如可以是15个、16个、17个、18个、19个、20个、21个、22个、23个、24个、25个、26个、27个、28个、29个或30个，优选为18-25个，以及上述数值之间的具体点值，限于篇幅及出于简明的考虑，本发明不再穷尽列举所述范围包括的具体点值。According to the present invention, the number of selected single sperm in step (2) is 15-30, for example, it can be 15, 16, 17, 18, 19, 20, 21, 22, 23 . The specific point values encompassed by the stated ranges are not intended to be exhaustive.
根据本发明，步骤(2)所述的扩增为单细胞扩增，通过单细胞扩增对活检细胞中的微量核酸进行扩增，以获得更多的核酸用于后续分析。According to the present invention, the amplification described in step (2) is single cell amplification, through which a small amount of nucleic acid in the biopsy cells is amplified to obtain more nucleic acid for subsequent analysis.
根据本发明，所述单细胞扩增为能够进行单细胞扩增的方法都是可行的，在此不做特性限定，本领域技术人员可以根据实验需要进行选择，本发明采用扩增前引物延伸PCR(Primer extension preamplification PCR,PEP-PCR)、退变寡核苷酸引物PCR(Degenerate oligonucleotide primer-PCR,DOP-PCR)、多重置换扩增技术(MultipleDisplacement Amplification,MDA)或多次退火环状循环扩增技术(Multiple Annealingand Looping Based Amplification Cycles,MALBAC)中的任意一种或至少两种的组合，优选为多次退火环状循环扩增技术。According to the present invention, the single-cell amplification is a method capable of single-cell amplification is feasible, and the characteristics are not limited here, and those skilled in the art can choose according to the experimental needs. The present invention uses primer extension before amplification. PCR (Primer extension preamplification PCR, PEP-PCR), degenerate oligonucleotide primer PCR (Degenerate oligonucleotide primer-PCR, DOP-PCR), multiple displacement amplification technique (Multiple Displacement Amplification, MDA) or multiple annealing circular cycles Any one or a combination of at least two of the amplification techniques (Multiple Annealing and Looping Based Amplification Cycles, MALBAC), preferably multiple annealing and looping cycle amplification techniques.
根据本发明，步骤(2)所述的测序将扩增后的样本进行文库构建后，采用高通量测序平台进行测序，所述高通量测序平台为第二代测序平台，本领域的第二代测序平台都是可行的，在此不做特殊限定，本领域技术人员可以根据需要进行选择，本发明可采用Illumina公司的GA、GAII、GAIIx、HiSeq1000/2000/2500/3000/4000、X Ten、X Five、NextSeq500/550、MiSeq、MiSeqDx、MiSeq FGx、MiniSeq、NovaSeq 5000/6000；AppliedBiosystems的SOLiD，Roche的454 FLX，Thermo Fisher Scientific(Life Technologies)的Ion Torrent、Ion PGM、Ion Proton I/II，华大基因的BGISEQ1000、BGISEQ500、BGISEQ100、BGISEQ50，博奥生物集团的BioelectronSeq 4000，中山大学达安基因股份有限公司的DA8600，贝瑞和康的NextSeq CN500，紫鑫药业旗下子公司中科紫鑫的BIGIS，华因康基因HYK-PSTAR-IIA中的任意一种，本发明优选采用Illumina公司的HiSeq2500高通量测序平台。According to the present invention, in the sequencing described in step (2), after the amplified sample is constructed into a library, a high-throughput sequencing platform is used for sequencing. The high-throughput sequencing platform is a second-generation sequencing platform, the first in the art. The next-generation sequencing platforms are all feasible, and there is no special limitation here. Those skilled in the art can choose according to the needs. The present invention can use Illumina's GA, GAII, GAIIx, HiSeq1000/2000/2500/3000/4000, X Ten, X Five, NextSeq500/550, MiSeq, MiSeqDx, MiSeq FGx, MiniSeq, NovaSeq 5000/6000; AppliedBiosystems' SOLiD, Roche's 454 FLX, Thermo Fisher Scientific (Life Technologies)'s Ion Torrent, Ion PGM, Ion Proton I/ II, BGISEQ1000, BGISEQ500, BGISEQ100, BGISEQ50 from BGI, BioelectronSeq 4000 from Boao Bio Group, DA8600 from Sun Yat-Sen University Daan Gene Co., Ltd., NextSeq CN500 from Berry Hekang, Zhongke, a subsidiary of Zixin Pharmaceutical Any one of Zixin's BIGIS, Huayinkang Gene HYK-PSTAR-IIA, the HiSeq2500 high-throughput sequencing platform of Illumina Company is preferably used in the present invention.
优选地，所述测序类型为单端测序和/或双端测序，优选为单端测序。Preferably, the type of sequencing is single-end sequencing and/or paired-end sequencing, preferably single-end sequencing.
根据本发明，所述测序的长度为不小于30bp，例如可以是是30bp、40bp、50bp、80bp、100bp、150bp、300bp、500bp，优选为50bp，以及上述数值之间的具体点值，限于篇幅及出于简明的考虑，本发明不再穷尽列举所述范围包括的具体点值。According to the present invention, the length of the sequencing is not less than 30bp, for example, it can be 30bp, 40bp, 50bp, 80bp, 100bp, 150bp, 300bp, 500bp, preferably 50bp, and the specific points between the above values are limited by the space And for the sake of brevity, the present invention does not exhaustively enumerate the specific points included in the range.
根据本发明，所述测序的深度为不小于基因组的0.1倍，例如可以是是0.1倍、0.5倍、1倍、2倍、5倍、10倍、30倍、50倍、100倍，优选为基因组的0.1倍，以及上述数值之间的具体点值，限于篇幅及出于简明的考虑，本发明不再穷尽列举所述范围包括的具体点值。According to the present invention, the sequencing depth is not less than 0.1 times of the genome, for example, it can be 0.1 times, 0.5 times, 1 times, 2 times, 5 times, 10 times, 30 times, 50 times, 100 times, preferably 0.1 times of the genome, and the specific point values between the above values, due to space limitations and for the sake of simplicity, the present invention will not exhaustively list the specific point values included in the range.
优选地，本发明中测序采用MALBAC单细胞扩增方法，Illumina公司的HiSeq2500高通量测序平台，测序类型为单端测序，测序长度50bp，测序深度为基因组的0.1倍。Preferably, the sequencing in the present invention adopts the MALBAC single-cell amplification method, the HiSeq2500 high-throughput sequencing platform of Illumina Company, the sequencing type is single-end sequencing, the sequencing length is 50 bp, and the sequencing depth is 0.1 times that of the genome.
根据本发明，步骤(4)所述的周围位置为染色体易位位置1-5M内的位点，例如可以是1M、2M、3M、4M或5M，优选为染色体易位位置1-3M内的位点，以及上述数值之间的具体点值，限于篇幅及出于简明的考虑，本发明不再穷尽列举所述范围包括的具体点值。According to the present invention, the surrounding positions described in step (4) are sites within 1-5M of chromosomal translocation positions, such as 1M, 2M, 3M, 4M or 5M, preferably within 1-3M of chromosomal translocation positions Positions, as well as specific point values between the above numerical values, are limited in space and for the sake of brevity, the present invention does not exhaustively list the specific point values included in the range.
优选地，所述周围位置的SNP数量一般检测30个以上，每个胚胎上的可用位点约占1/3，10个以上可用位点就可以确定单倍型连锁关系，本发明中所述SNP的数量为10-500，例如可以是10、20、30、40、50、60、80、100、120、130、150、200、250、300、350、400、450、500，优选为30-100，以及上述数值之间的具体点值，限于篇幅及出于简明的考虑，本发明不再穷尽列举所述范围包括的具体点值。Preferably, the number of SNPs at the surrounding positions is generally more than 30, and the available sites on each embryo account for about 1/3, and the haplotype linkage relationship can be determined with more than 10 available sites, as described in the present invention. The number of SNPs is 10-500, such as 10, 20, 30, 40, 50, 60, 80, 100, 120, 130, 150, 200, 250, 300, 350, 400, 450, 500, preferably 30 -100, and the specific point values between the above numerical values, due to space limitation and for the sake of simplicity, the present invention does not exhaustively list the specific point values included in the range.
根据本发明，所述SNP分析的方法为本领域公知的方法，在此不做特殊限定，本领域技术人员可以根据需要进行选择，本发明采用设计探针芯片捕获测序、设计引物对扩增子进行一代测序或设计引物对扩增子进行二代测序中的任意一种或至少两种的组合。According to the present invention, the method for SNP analysis is a method known in the art, and there is no special limitation here. Those skilled in the art can select according to needs. The present invention adopts the design of probe chip capture sequencing, design of primers to amplicon Either one or a combination of at least two of performing first-generation sequencing or designing primers to perform second-generation sequencing on amplicons.
根据本发明，步骤(4)所述获得正常染色体的单倍型的具体步骤为：将具有染色体重复或缺失的单精子进行比较，发生易位的单精子的SNP减去易位缺失的单精子的SNP，确定正常单精子的单倍型。According to the present invention, the specific step of obtaining the haplotype of the normal chromosome in step (4) is: comparing the single sperm with chromosome duplication or deletion, and subtracting the SNP of the single sperm with translocation SNPs that determine the haplotype of normal single spermatozoa.
根据本发明，步骤(5)母方的染色体相应的易位位置及周围位置的SNP为纯合子。According to the present invention, in step (5), the SNPs at the corresponding translocation position and surrounding positions on the maternal chromosome are homozygous.
根据本发明，步骤(6)所述确定胚胎平衡易位携带状态的具体步骤如下：将待测样本的胚胎的SNP减去母方的SNP后获得的单倍型SNP与正常单精子单倍型进行比较，若一致，为正常胚胎，若不一致，为携带者胚胎。According to the present invention, the specific steps for determining the balanced translocation carrying state of the embryo described in step (6) are as follows: the haplotype SNP obtained after subtracting the SNP of the mother's SNP from the SNP of the embryo of the sample to be tested is compared with the normal single sperm haplotype Comparison, if consistent, it is a normal embryo, if not, it is a carrier embryo.
第二方面，本发明提供一种用于非诊断目的的胚胎平衡易位携带状态的检测方法，采用如第一方面所述的系统，包括如下步骤：In a second aspect, the present invention provides a method for detecting the carrying state of balanced translocations of embryos for non-diagnostic purposes, using the system as described in the first aspect, comprising the following steps:
(1)获取胚胎待测样本、父方精液和母方DNA；(1) Obtain the embryo sample to be tested, the father's semen and the mother's DNA;
(2)从父方获得的精液中挑选单精子，进行扩增，构建文库后测序；(2) Selecting single sperm from the semen obtained from the father, amplifying, constructing a library and then sequencing;
(3)对测序结果进行染色体拷贝数(CNV)分析，初步确定染色体易位位置；(3) Chromosomal copy number (CNV) analysis was performed on the sequencing results to preliminarily determine the position of chromosomal translocation;
(4)对染色体易位位置及周围位置进行SNP分析，通过多个单精子的比较，获得正常单精子的单倍型；(4) Carry out SNP analysis on the chromosomal translocation position and surrounding positions, and obtain the haplotype of normal single sperm by comparing multiple single sperm;
(5)对母方的染色体相应的易位位置及周围位置进行SNP分析；(5) Carry out SNP analysis on the corresponding translocation position and surrounding positions of the maternal chromosome;
(6)对待测样本的胚胎的染色体相应的易位位置及周围位置进行SNP分析，将分析的结果与正常的单精子单倍型以及母方的SNP信息进行比对，确定胚胎平衡易位携带状态。(6) Carry out SNP analysis on the corresponding translocation position and the surrounding position of the chromosome of the embryo to be tested, and compare the analysis result with the normal single sperm haplotype and the SNP information of the mother to determine the balanced translocation carrier state of the embryo .
根据本发明，步骤(1)所述的待测样本为胚胎的活检细胞，所述活检细胞为胚胎发育到卵裂球时期或囊胚时期取下的外胚层细胞，所述外胚层细胞可以是1个也可以是多个滋养外胚层细胞。According to the present invention, the sample to be tested in step (1) is a biopsy cell of an embryo, and the biopsy cell is an ectoderm cell removed from the blastomere stage or blastocyst stage during embryo development, and the ectoderm cell can be 1 or multiple trophectoderm cells.
根据本发明，所述母方DNA为能够提取DNA的任何人源样本都是可行的，在此不做特殊限定，本领域技术人员可以根据实验需要进行提取，本发明的母方DNA选取来自外周血、淋巴液或口腔黏膜细胞中的任意一种或至少两种的组合。According to the present invention, it is feasible for the maternal DNA to be any human-derived sample from which DNA can be extracted, and there is no special limitation here. Those skilled in the art can extract it according to experimental needs. The maternal DNA of the present invention is selected from peripheral blood, Any one or a combination of at least two of lymph fluid or oral mucosal cells.
根据本发明，步骤(2)所述挑选单精子的数量为15-30个，例如可以是15个、16个、17个、18个、19个、20个、21个、22个、23个、24个、25个、26个、27个、28个、29个或30个，优选为18-25个，以及上述数值之间的具体点值，限于篇幅及出于简明的考虑，本发明不再穷尽列举所述范围包括的具体点值。According to the present invention, the number of selected single sperm in step (2) is 15-30, for example, it can be 15, 16, 17, 18, 19, 20, 21, 22, 23 . The specific point values encompassed by the stated ranges are not intended to be exhaustive.
根据本发明，步骤(2)所述的扩增为单细胞扩增，通过单细胞扩增对活检细胞中的微量核酸进行扩增，以获得更多的核酸用于后续分析。According to the present invention, the amplification described in step (2) is single cell amplification, through which a small amount of nucleic acid in the biopsy cells is amplified to obtain more nucleic acid for subsequent analysis.
根据本发明，所述单细胞扩增为能够进行单细胞扩增的方法都是可行的，在此不做特性限定，本领域技术人员可以根据实验需要进行选择，本发明采用扩增前引物延伸PCR(Primer extension preamplification PCR,PEP-PCR)、退变寡核苷酸引物PCR(Degenerate oligonucleotide primer-PCR,DOP-PCR)、多重置换扩增技术(MultipleDisplacement Amplification,MDA)或多次退火环状循环扩增技术(Multiple Annealingand Looping Based Amplification Cycles,MALBAC)中的任意一种或至少两种的组合，优选为多次退火环状循环扩增技术。According to the present invention, the single-cell amplification is a method capable of single-cell amplification is feasible, and the characteristics are not limited here, and those skilled in the art can choose according to the experimental needs. The present invention uses primer extension before amplification. PCR (Primer extension preamplification PCR, PEP-PCR), degenerate oligonucleotide primer PCR (Degenerate oligonucleotide primer-PCR, DOP-PCR), multiple displacement amplification technique (Multiple Displacement Amplification, MDA) or multiple annealing circular cycles Any one or a combination of at least two of the amplification techniques (Multiple Annealing and Looping Based Amplification Cycles, MALBAC), preferably multiple annealing and looping cycle amplification techniques.
根据本发明，步骤(2)所述的测序将扩增后的样本进行文库构建后，采用高通量测序平台进行测序，所述高通量测序平台为第二代测序平台，本领域的第二代测序平台都是可行的，在此不做特殊限定，本领域技术人员可以根据需要进行选择，本发明可采用Illumina公司的GA、GAII、GAIIx、HiSeq1000/2000/2500/3000/4000、X Ten、X Five、NextSeq500/550、MiSeq、MiSeqDx、MiSeq FGx、MiniSeq、NovaSeq 5000/6000；AppliedBiosystems的SOLiD，Roche的454FLX，Thermo Fisher Scientific(Life Technologies)的Ion Torrent、Ion PGM、Ion Proton I/II，华大基因的BGISEQ1000、BGISEQ500、BGISEQ100、BGISEQ50，博奥生物集团的BioelectronSeq 4000，中山大学达安基因股份有限公司的DA8600，贝瑞和康的NextSeq CN500，紫鑫药业旗下子公司中科紫鑫的BIGIS，华因康基因HYK-PSTAR-IIA中的任意一种，本发明优选采用Illumina公司的HiSeq2500高通量测序平台。According to the present invention, in the sequencing described in step (2), after the amplified sample is constructed into a library, a high-throughput sequencing platform is used for sequencing. The high-throughput sequencing platform is a second-generation sequencing platform, the first in the art. The next-generation sequencing platforms are all feasible, and there is no special limitation here. Those skilled in the art can choose according to the needs. The present invention can use Illumina's GA, GAII, GAIIx, HiSeq1000/2000/2500/3000/4000, X Ten, X Five, NextSeq500/550, MiSeq, MiSeqDx, MiSeq FGx, MiniSeq, NovaSeq 5000/6000; SOLiD from AppliedBiosystems, 454FLX from Roche, Ion Torrent, Ion PGM, Ion Proton I/II from Thermo Fisher Scientific (Life Technologies) , BGISEQ1000, BGISEQ500, BGISEQ100, BGISEQ50 from BGI, BioelectronSeq 4000 from Boao Bio Group, DA8600 from Sun Yat-Sen University Da’an Gene Co., Ltd., NextSeq CN500 from Berry Hekang, and Zhongkezi, a subsidiary of Zixin Pharmaceutical Any one of Xin's BIGIS, Huayinkang Gene HYK-PSTAR-IIA, the present invention preferably uses Illumina's HiSeq2500 high-throughput sequencing platform.
优选地，所述测序类型为单端测序和/或双端测序，优选为单端测序。Preferably, the type of sequencing is single-end sequencing and/or paired-end sequencing, preferably single-end sequencing.
根据本发明，所述测序的长度为不小于30bp，例如可以是是30bp、40bp、50bp、80bp、100bp、150bp、300bp、500bp，优选为50bp，以及上述数值之间的具体点值，限于篇幅及出于简明的考虑，本发明不再穷尽列举所述范围包括的具体点值。According to the present invention, the length of the sequencing is not less than 30bp, for example, it can be 30bp, 40bp, 50bp, 80bp, 100bp, 150bp, 300bp, 500bp, preferably 50bp, and the specific points between the above values are limited by the space And for the sake of brevity, the present invention does not exhaustively enumerate the specific points included in the range.
根据本发明，所述测序的深度为不小于基因组的0.1倍，例如可以是是0.1倍、0.5倍、1倍、2倍、5倍、10倍、30倍、50倍、100倍，优选为基因组的0.1倍，以及上述数值之间的具体点值，限于篇幅及出于简明的考虑，本发明不再穷尽列举所述范围包括的具体点值。According to the present invention, the sequencing depth is not less than 0.1 times of the genome, for example, it can be 0.1 times, 0.5 times, 1 times, 2 times, 5 times, 10 times, 30 times, 50 times, 100 times, preferably 0.1 times of the genome, and the specific point values between the above values, due to space limitations and for the sake of simplicity, the present invention will not exhaustively list the specific point values included in the range.
优选地，本发明中测序采用MALBAC单细胞扩增方法，Illumina公司的HiSeq2500高通量测序平台，测序类型为单端测序，测序长度50bp，测序深度为基因组的0.1倍。Preferably, the sequencing in the present invention adopts the MALBAC single-cell amplification method, the HiSeq2500 high-throughput sequencing platform of Illumina Company, the sequencing type is single-end sequencing, the sequencing length is 50 bp, and the sequencing depth is 0.1 times that of the genome.
根据本发明，步骤(4)所述的周围位置为染色体易位位置1-5M内的位点，例如可以是1M、2M、3M、4M或5M，优选为染色体易位位置1-3M内的位点，以及上述数值之间的具体点值，限于篇幅及出于简明的考虑，本发明不再穷尽列举所述范围包括的具体点值。According to the present invention, the surrounding positions described in step (4) are sites within 1-5M of chromosomal translocation positions, such as 1M, 2M, 3M, 4M or 5M, preferably within 1-3M of chromosomal translocation positions Positions, as well as specific point values between the above numerical values, are limited in space and for the sake of brevity, the present invention does not exhaustively list the specific point values included in the range.
优选地，所述周围位置的SNP数量一般检测30个以上，每个胚胎上的可用位点约占1/3,10个以上可用位点就可以确定单倍型连锁关系，本发明中所述SNP的数量为10-500，例如可以是10、20、30、40、50、60、80、100、120、130、150、200、250、300、350、400、450、500，优选为30-100，以及上述数值之间的具体点值，限于篇幅及出于简明的考虑，本发明不再穷尽列举所述范围包括的具体点值。Preferably, the number of SNPs at the surrounding positions is generally more than 30, and the available sites on each embryo account for about 1/3, and the haplotype linkage relationship can be determined with more than 10 available sites, as described in the present invention. The number of SNPs is 10-500, such as 10, 20, 30, 40, 50, 60, 80, 100, 120, 130, 150, 200, 250, 300, 350, 400, 450, 500, preferably 30 -100, and the specific point values between the above numerical values, due to space limitation and for the sake of simplicity, the present invention does not exhaustively list the specific point values included in the range.
根据本发明，所述SNP分析的方法为本领域公知的方法，在此不做特殊限定，本领域技术人员可以根据需要进行选择，本发明采用设计探针芯片捕获测序、设计引物对扩增子进行一代测序或设计引物对扩增子进行二代测序中的任意一种或至少两种的组合。According to the present invention, the method for SNP analysis is a method known in the art, and there is no special limitation here. Those skilled in the art can select according to needs. The present invention adopts the design of probe chip capture sequencing, design of primers to amplicon Either one or a combination of at least two of performing first-generation sequencing or designing primers to perform second-generation sequencing on amplicons.
根据本发明，步骤(4)所述获得正常染色体的单倍型的具体步骤为：将具有染色体重复或缺失的单精子进行比较，发生易位的单精子的SNP减去易位缺失的单精子的SNP，确定正常单精子的单倍型。According to the present invention, the specific step of obtaining the haplotype of the normal chromosome in step (4) is: comparing the single sperm with chromosome duplication or deletion, and subtracting the SNP of the single sperm with translocation SNPs that determine the haplotype of normal single spermatozoa.
根据本发明，步骤(5)母方的染色体相应的易位位置及周围位置的SNP为纯合子。According to the present invention, in step (5), the SNPs at the corresponding translocation position and surrounding positions on the maternal chromosome are homozygous.
根据本发明，步骤(6)所述确定胚胎平衡易位携带状态的具体步骤如下：将待测样本的胚胎的SNP减去母方的SNP后获得的单倍型SNP与正常单精子单倍型进行比较，若一致，为正常胚胎，若不一致，为携带者胚胎。According to the present invention, the specific steps for determining the balanced translocation carrying state of the embryo described in step (6) are as follows: the haplotype SNP obtained after subtracting the SNP of the mother's SNP from the SNP of the embryo of the sample to be tested is compared with the normal single sperm haplotype Comparison, if consistent, it is a normal embryo, if not, it is a carrier embryo.
作为优选技术方案，所述胚胎平衡易位携带状态的检测方法包括如下步骤：As a preferred technical solution, the method for detecting the carrying state of the balanced translocation of the embryo comprises the following steps:
(1)获取胚胎的活检细胞、父方精液和母方DNA；(1) Obtain the biopsy cells of the embryo, the semen of the father and the DNA of the mother;
(2)从父方获得的精液中挑选15-30个单精子，进行多次退火环状循环扩增，构建文库后采用高通量测序平台进行测序；(2) Select 15-30 single sperm from the semen obtained from the father, perform multiple annealing circular cycle amplification, and use a high-throughput sequencing platform for sequencing after constructing the library;
(3)对测序结果进行染色体拷贝数分析，初步确定染色体易位位置：通过染色体拷贝数分析，染色体重复或缺失的位置，初步判断为染色体易位位置；(3) Carry out chromosome copy number analysis on the sequencing results, and preliminarily determine the position of chromosomal translocation: through the analysis of chromosome copy number, the position of chromosome duplication or deletion is preliminarily judged as the position of chromosomal translocation;
(4)对染色体易位位置及周围位置1-5M内进行SNP分析，通过多个单精子的比较，获得正常单精子的单倍型：将具有染色体重复或缺失的单精子进行比较，接受易位的2倍体单精子的SNP减去易位缺失的单精子的SNP，确定正常单精子的单倍型；(4) Perform SNP analysis on the chromosomal translocation position and the surrounding position within 1-5M, and obtain the haplotype of normal single sperm through the comparison of multiple single sperm: compare the single sperm with chromosome duplication or deletion, and accept easy The haplotype of normal single sperm was determined by subtracting the SNP of translocation-deleted single sperm from the SNP of diploid single sperm;
(5)对母方的染色体相应的易位位置及周围位置1-5M内进行SNP分析；(5) Carry out SNP analysis on the corresponding translocation position of the maternal chromosome and the surrounding position within 1-5M;
(6)对待测样本的胚胎的染色体相应的易位位置及周围位置1-5M内进行SNP分析，将分析的结果与正常的单精子单倍型以及母方的SNP信息进行比对，确定胚胎平衡易位携带状态：将待测样本的胚胎的SNP减去母方的SNP后获得的单倍型SNP与正常单精子单倍型进行比较，若一致，为正常胚胎，若不一致，为携带者胚胎。(6) Perform SNP analysis on the corresponding translocation position of the chromosome of the embryo to be tested and within 1-5M of the surrounding position, and compare the analysis results with the normal single sperm haplotype and the SNP information of the mother to determine the balance of the embryo Translocation carrier status: compare the haplotype SNP obtained by subtracting the maternal SNP from the SNP of the embryo to be tested with the normal single sperm haplotype. If they are consistent, they are normal embryos; if they are inconsistent, they are carrier embryos.
与现有技术相比，本发明具有如下有益效果：Compared with the prior art, the present invention has the following beneficial effects:
(1)本发明系统和方法采用的单精子扩增方式与靶向PCR的方式来寻找断裂位点，能够将断裂点位置精确到200kb-500kb，可以有效降低PCR扩增偏倚，灵敏度高；(1) The single sperm amplification method and the targeted PCR method adopted by the system and method of the present invention are used to find the breakpoint, which can accurately locate the breakpoint to 200kb-500kb, can effectively reduce the bias of PCR amplification, and has high sensitivity;
(2)本发明系统和方法可同时做胚胎染色体非整倍体的筛选以及平衡易位断裂位点附近2M区域内SNP位点的挖掘，可以同时提供胚胎非整倍体筛查与平衡易位携带状态的信息，同时确定易位位置和胚胎非整倍性；(2) The system and method of the present invention can simultaneously perform screening of embryonic chromosome aneuploidy and excavation of SNP sites in the 2M region near the break site of balanced translocation, and can simultaneously provide embryonic aneuploidy screening and balanced translocation information on carrying status, simultaneously determining translocation location and embryonic aneuploidy;
(3)本发明系统和方法操作相对简单易行，成本低，易于推广应用。(3) The system and method of the present invention are relatively simple to operate, low in cost, and easy to popularize and apply.
附图说明Description of drawings
图1是本发明实施例1中的样本6_2_MAL_CNV的CNV分析；Fig. 1 is the CNV analysis of the sample 6_2_MAL_CNV in Example 1 of the present invention;
图2是本发明实施例1中的样本12_2_MAL_CNV的CNV分析；Fig. 2 is the CNV analysis of the sample 12_2_MAL_CNV in Example 1 of the present invention;
图3是本发明实施例1中的样本13_2_MAL_CNV的CNV分析；Fig. 3 is the CNV analysis of the sample 13_2_MAL_CNV in Example 1 of the present invention;
图4是本发明实施例1中的样本17_2_MAL_CNV的CNV分析；Fig. 4 is the CNV analysis of the sample 17_2_MAL_CNV in Example 1 of the present invention;
图5是本发明实施例1中的胚胎的CNV分析。Fig. 5 is the CNV analysis of the embryos in Example 1 of the present invention.
具体实施方式detailed description
为更进一步阐述本发明所采取的技术手段及其效果，以下结合附图并通过具体实施方式来进一步说明本发明的技术方案，但本发明并非局限在实施例范围内。In order to further illustrate the technical means and effects adopted by the present invention, the technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and through specific implementation methods, but the present invention is not limited within the scope of the embodiments.
实施例1 鉴定胚胎平衡易位携带状态的系统Example 1 System for Identifying Balanced Translocation Carrier Status of Embryos
所述鉴定胚胎平衡易位携带状态的系统，包括如下单元：The system for identifying the balanced translocation carrying state of an embryo comprises the following units:
(1)取样单元：获取胚胎的活检细胞、父方精液和母方DNA；(1) Sampling unit: obtain the biopsy cells of the embryo, the father's semen and the mother's DNA;
(2)测序单元：从父方获得的精液中挑选15-30个单精子，进行多次退火环状循环扩增，构建文库后采用高通量测序平台进行测序；(2) Sequencing unit: select 15-30 single sperm from the semen obtained from the father, perform multiple annealing circular cycle amplification, and use a high-throughput sequencing platform for sequencing after constructing the library;
(3)拷贝数分析单元：对测序结果进行染色体拷贝数分析，初步确定染色体易位位置：通过染色体拷贝数分析，染色体重复或缺失的位置，初步判断为染色体易位位置；(3) Copy number analysis unit: analyze the chromosome copy number of the sequencing results, and preliminarily determine the position of chromosome translocation: through the analysis of chromosome copy number, the position of chromosome duplication or deletion is preliminarily judged as the position of chromosome translocation;
(4)SNP分析单元：对染色体易位位置及周围位置1-5M内进行SNP分析，通过多个单精子的比较，获得正常单精子的单倍型：将具有染色体重复或缺失的单精子进行比较，接受易位的2倍体单精子的SNP减去易位缺失的单精子的SNP，确定正常单精子的单倍型；(4) SNP analysis unit: conduct SNP analysis on the chromosomal translocation position and the surrounding position within 1-5M, and obtain the haplotype of normal single sperm by comparing multiple single sperm: perform single sperm with chromosomal duplication or deletion For comparison, the SNP of the diploid single sperm that underwent the translocation was subtracted from the SNP of the single sperm that lacked the translocation to determine the haplotype of the normal single sperm;
(5)对母方的染色体相应的易位位置及周围位置1-5M内进行SNP分析；(5) Carry out SNP analysis on the corresponding translocation position of the maternal chromosome and the surrounding position within 1-5M;
(6)比对单元：对待测样本的胚胎的染色体相应的易位位置及周围位置1-5M内进行SNP分析，将分析的结果与正常的单精子单倍型以及母方的SNP信息进行比对，确定胚胎平衡易位携带状态：将待测样本的胚胎的SNP减去母方的SNP后获得的单倍型SNP与正常单精子单倍型进行比较，若一致，为正常胚胎，若不一致，为携带者胚胎。(6) Comparison unit: conduct SNP analysis on the corresponding translocation position of the chromosome of the embryo to be tested and within 1-5M of the surrounding position, and compare the analysis results with the normal single sperm haplotype and the SNP information of the mother , to determine the balanced translocation carrier state of the embryo: compare the haplotype SNP obtained by subtracting the SNP of the mother’s SNP from the SNP of the embryo to be tested with the normal single sperm haplotype, if consistent, it is a normal embryo, if not consistent, it is Carrier embryos.
实施例2 胚胎平衡易位携带状态的检测Example 2 Detection of Balanced Translocation Carrier Status of Embryos
所述鉴定胚胎平衡易位携带状态的方法包括如下步骤：The method for identifying the balanced translocation carrier state of an embryo comprises the following steps:
(1)获取胚胎的活检细胞、父方精液和母方DNA，父方的染色体为：46,XY,t(9,21)(q24,q22.1)；(1) Obtain the biopsy cells of the embryo, the semen of the father and the DNA of the mother. The chromosome of the father is: 46, XY, t(9,21) (q24, q22.1);
(2)从父方获得的精液中挑选15-30个单精子，进行多次退火环状循环扩增，构建文库后采用高通量测序平台进行测序，其中样本6、、12、13和17为四个单精子样本，其结果如图1-4和表1所示：(2) Select 15-30 single sperm from the semen obtained from the father, perform multiple annealing circular cycle amplification, and use a high-throughput sequencing platform for sequencing after constructing the library. Samples 6, 12, 13 and 17 are Four single sperm samples, the results are shown in Figures 1-4 and Table 1:
表1Table 1
(3)对测序结果进行染色体拷贝数分析，初步确定染色体易位位置：通过染色体拷贝数分析，染色体重复或缺失的位置，初步判断为染色体易位位置为chr21，精确度在500kb左右；(3) Carry out chromosome copy number analysis on the sequencing results, and preliminarily determine the position of chromosomal translocation: through the analysis of chromosome copy number, the position of chromosome duplication or deletion is preliminarily judged to be chr21, and the accuracy is about 500kb;
(4)对染色体易位位置及周围位置1-5M内进行SNP分析，结果如表2所示，通过多个单精子的比较，获得正常单精子的单倍型：将具有染色体重复或缺失的单精子进行比较，接受易位的2倍体单精子的SNP减去易位缺失的单精子的SNP，确定正常单精子的单倍型；(4) SNP analysis was performed on the chromosomal translocation position and the surrounding position within 1-5M. The results are shown in Table 2. Through the comparison of multiple single sperm, the haplotype of normal single sperm was obtained: those with chromosome duplication or deletion For single sperm comparison, the SNP of the diploid single sperm that received the translocation was subtracted from the SNP of the single sperm that lost the translocation to determine the haplotype of the normal single sperm;
表2Table 2
(5)对母方的染色体相应的易位位置及周围位置1-5M内进行SNP分析；(5) Carry out SNP analysis on the corresponding translocation position of the maternal chromosome and the surrounding position within 1-5M;
(6)对待测样本的胚胎的染色体相应的易位位置及周围位置1-5M内进行SNP分析，结果如表3所示，将分析的结果与正常的单精子单倍型以及母方的SNP信息进行比对，确定胚胎平衡易位携带状态：将待测样本的胚胎的SNP减去母方的SNP后获得的单倍型SNP与正常单精子单倍型进行比较，其CNV的结果如图5所示，其减去母本的SNP后与正常chr21一致，为正常胚胎。(6) Perform SNP analysis on the corresponding translocation position of the chromosome of the embryo to be tested and the surrounding position within 1-5M, the results are shown in Table 3, and the results of the analysis are compared with the normal single sperm haplotype and the SNP information of the mother Perform comparison to determine the balanced translocation carrier state of the embryo: compare the haplotype SNP obtained after subtracting the maternal SNP from the SNP of the embryo to be tested with the normal single sperm haplotype, and the CNV results are shown in Figure 5 It is shown that it is consistent with normal chr21 after subtracting the mother's SNP, which is a normal embryo.
表3table 3
综上所述，本发明方法可同时做胚胎染色体非整倍体的筛选以及平衡易位断裂位点附近2M区域内SNP位点的挖掘，可以同时提供胚胎非整倍体筛查与平衡易位携带状态诊断的信息，同时确定易位位置和胚胎非整倍性。In summary, the method of the present invention can simultaneously screen embryonic chromosome aneuploidy and excavate SNP sites in the 2M region near the balanced translocation break site, and can simultaneously provide embryonic aneuploidy screening and balanced translocation Carrying information for state diagnostics, simultaneous determination of translocation location and embryonic aneuploidy.
申请人声明，本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。The applicant declares that the present invention illustrates the detailed methods of the present invention through the above-mentioned examples, but the present invention is not limited to the above-mentioned detailed methods, that is, it does not mean that the present invention must rely on the above-mentioned detailed methods to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.
| Application Number | Priority Date | Filing Date | Title |
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| CN201710294366.8ACN106929595B (en) | 2017-04-28 | 2017-04-28 | A system and method for identifying balanced translocation carrier status in embryos |
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| CN201710294366.8ACN106929595B (en) | 2017-04-28 | 2017-04-28 | A system and method for identifying balanced translocation carrier status in embryos |
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| CN201710294366.8AActiveCN106929595B (en) | 2017-04-28 | 2017-04-28 | A system and method for identifying balanced translocation carrier status in embryos |
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