Identification of PKD2 mutations in human preimplantation embryos in vitro using a combination of targeted next-generation sequencing and targeted haplotyping.
Identification of PKD2 mutations in human preimplantation embryos in vitro using a combination of targeted next-generation sequencing and targeted haplotyping.
复制标题
结合靶向下一代测序和靶向单倍型分析，在体外鉴定人类植入前胚胎中的 PKD2 突变。
DOI：
10.1038/srep25488
复制
发表时间：
2016-05-06
影响因子：
4.6
通讯作者：
Xu CM
中科院分区：
文献类型：
--
作者：
Chen SC;Xu XL;Zhang JY;Ding GL;Jin L;Liu B;Sun DM;Mei CL;Yang XN;Huang HF;Xu CM
在这里，我们评估了一种新方法的适用性，该方法将靶向下一代测序（NGS）与靶向单倍型分析相结合，在体外鉴定人类植入前胚胎中的 PKD2 基因突变。为了实现这一目标，我们对 PKD2 基因外显子 1 中杂合删除 c.595_595 + 14delGGTAAGAGCGCGCGA 的先证者家族进行了研究。总共分析了 10 个样本，其中包括 7 个胚胎。基于阵列的基因芯片旨在捕获 21 个疾病相关基因（包括 PKD2）的所有外显子。我们进行桑格测序结合靶向单倍型分析来评估这种新方法的可行性。通过NGS从10个样本中总共获得了7.09 G的数据。此外，还鉴定了 24,142 个信息丰富的单核苷酸多态性 (SNP)。我们选择的几个 PKD2 信息丰富的 SNP 的单倍型分析显示，胚胎 3、5 和 6 没有遗传 PKD2 基因的突变单倍型，这一结果 100% 准确，并且与 Sanger 测序一致。我们的结果表明，靶向NGS与靶向单倍型分析相结合可用于体外鉴定人植入前胚胎中的PKD2基因突变，具有高灵敏度、保真度、通量和速度。
Here, we evaluate the applicability of a new method that combines targeted next-generation sequencing (NGS) with targeted haplotyping in identifying PKD2 gene mutations in human preimplantation embryos in vitro. To achieve this goal, a proband family with a heterozygous deletion of c.595_595 + 14delGGTAAGAGCGCGCGA in exon 1 of the PKD2 gene was studied. A total of 10 samples were analyzed, including 7 embryos. An array-based gene chip was designed to capture all of the exons of 21 disease-related genes, including PKD2. We performed Sanger sequencing combined with targeted haplotyping to evaluate the feasibility of this new method. A total of 7.09 G of data were obtained from 10 samples by NGS. In addition, 24,142 informative single-nucleotide polymorphisms (SNPs) were identified. Haplotyping analysis of several informative SNPs of PKD2 that we selected revealed that embryos 3, 5, and 6 did not inherit the mutation haplotypes of the PKD2 gene, a finding that was 100% accurate and was consistent with Sanger sequencing. Our results demonstrate that targeted NGS combined with targeted haplotyping can be used to identify PKD2 gene mutations in human preimplantation embryos in vitro with high sensitivity, fidelity, throughput and speed.