深度测序对单细胞21-三体诊断的可行性研究

发布时间：2018-04-18 23:27

本文选题：深度测序 + 单细胞　；参考：《中国人民解放军医学院》2013年硕士论文

【摘要】：【背景】非整倍体是最常见的染色体异常，可导致胚胎停育与自发流产，是限制试管婴儿成功率的重要原因之一。胚胎植入前遗传学筛查（preimplatationgenetic screening，，PGS）是筛选染色体数目和结构正常的胚胎进行移植，以此期望提高移植成功率与妊娠率。目前PGS技术主要包括:原位荧光杂交技术、比较基因组杂交以及微阵列比较基因组杂交和单核苷酸多态性芯片技术等，但各项技术均存在缺点。为了寻找一种更快速、全面检测非整倍体的方法，我们以二代测序技术为基础，尝试一种新方法完成对单细胞21-三体的诊断，从而建立该技术平台为后续研究奠定基础。【目的】初步建立深度测序技术在单细胞水平诊断21三体的技术平台【方法】采用深度测序技术对正常女性外周血DNA进行检测，评估该方法检测的稳定性。对不同浓度DNA的21-三体样本应用多重置换扩增方法进行全基因组扩增，对其产物进行深度测序，评估该方法对21-三体诊断的可行性。利用深度测序技术对单细胞21-三体样本进行检测，初步建立深度测序方法检测单细胞-21三体的技术平台。【结果】1.采用深度测序方法对14例样本进行检测，各染色体标准差均处于低水平，其中最大标准差为0.078。21-三体样本21号染色体拷贝数是正常样本21号染色体拷贝数的1.5倍，其余染色体与正常样本相比未发现拷贝数的明显异常。3. cut off值0.5、1、1.5分别代表单倍体、二倍体和三倍体，21-三体样本测序结果显示21号染色体cut off均值为1.586，其99%可信区间为[1.537,1.635]。采用深度测序方法检测单细胞21-三体具有可行性。【结论】1.采用深度测序方法检测正常女性DNA具有稳定性。2.通过对不同浓度DNA模板扩增产物进行深度测序，证实该检测方法对诊断21-三体具有可行性。3.深度测序方法检测单细胞-21三体具有可行性。
[Abstract]:Background: aneuploidy is one of the most common chromosomal abnormalities, which can lead to aborted embryo and spontaneous abortion, which is one of the important reasons to limit the success rate of IVF.Preimplantation genetic screening (PGSs) is a method of screening embryos with normal chromosome number and structure in order to improve the success rate and pregnancy rate.At present, PGS technology mainly includes: in situ fluorescence hybridization, comparative genomic hybridization, microarray comparative genomic hybridization and single nucleotide polymorphism chip technology.In order to find a more rapid and comprehensive method for the detection of aneuploidy, we try a new method to complete the diagnosis of single-cell 21-trisomy based on the second-generation sequencing technique, and establish the platform for further research.[objective] to establish a technique platform for the diagnosis of trisomy 21 at single cell level by deep sequencing.[methods] the DNA in peripheral blood of normal women was detected by deep sequencing and the stability of this method was evaluated.The whole genome of 21- trisomy samples with different concentrations of DNA was amplified by multiplex replacement amplification method, and its products were deeply sequenced to evaluate the feasibility of this method in the diagnosis of 21-trisomy.The technique platform of detecting single cell trisomy 21 was established by using the deep sequencing technique to detect the single cell 21 trisomy.[result] 1.14 samples were detected by deep sequencing. The standard deviation of each chromosome was low, and the maximum standard deviation was 0.078.21- chromosome 21 copy number of trisomy sample was 1.5 times that of normal sample.There was no obvious abnormality of copy number in the other chromosomes. The cut off value of 0.5 ~ 1. 5 represented haploid, diploid and triploid trisomy 21, respectively. The results of sequencing showed that the mean value of cut off on chromosome 21 was 1.586, and its 99% confidence interval was [1.537 卤1.635].It is feasible to detect single cell 21-trisomy by deep sequencing.[conclusion] 1.The method of deep sequencing was used to detect the stability of DNA in normal women. 2. 2.By deep sequencing of DNA template amplification products with different concentrations, it is proved that this method is feasible for the diagnosis of 21 trisomy. 3.It is feasible to detect trisomy-21 by deep sequencing.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R394;R3416

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