Background

Segmental duplication (SD) regions are distinct targets for aneuploidy detection owing to the virtual elimination of amplification bias. The difficulty of searching SD sequences for assay design has hampered their applications.

Methods

We developed a computational program, ChAPDes, which integrates SD searching, refinement, and design of specific PCR primer/probe sets in a pipeline to remove most of the manual work. The generated primer/probe sets were first tested in a multiplex multicolour melting curve analysis for the detection of five common aneuploidies. The primer/probe sets were then tested in a digital PCR assay for the detection of trisomy 21. Finally, a digital PCR protocol was established to quantify maternal plasma DNA sequences for the non-invasive prenatal detection of fetal trisomy 21.

Findings

ChAPDes could output 21,772 candidate primer/probe sets for trisomy 13, 18, 21 and sex chromosome aneuploidies within 2 working days. Clinical evaluation of the multiplex multicolour melting curve analysis involving 463 fetal genomic DNA samples revealed a sensitivity of 100% and specificity of 99.64% in comparison with the reference methods. Using the established digital PCR protocol, we correctly identified two trisomy 21 fetuses and thirteen euploid foetuses from the maternal plasma samples.

Interpretation

The combination of ChAPDes with digital PCR detection could facilitate the use of SD as potential biomarkers for the non-invasive prenatal testing of fetal chromosomal aneuploidies.

中文翻译：

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节段重复作为非整倍体非侵入性产前检测的潜在生物标志物

背景

由于实际上消除了扩增偏差，节段重复 (SD) 区域是非整倍性检测的不同目标。为分析设计搜索 SD 序列的困难阻碍了它们的应用。

方法

我们开发了一个计算程序 ChAPDes，它将特定 PCR 引物/探针组的 SD 搜索、细化和设计集成到一个管道中，以消除大部分手动工作。生成的引物/探针组首先在多重多色熔解曲线分析中进行测试，以检测五种常见的非整倍体。然后在数字 PCR 分析中测试引物/探针组以检测 21 三体。最后，建立数字 PCR 方案以量化母体血浆 DNA 序列，以进行胎儿 21 三体的无创产前检测。

发现

ChAPDes 可在 2 个工作日内输出 21,772 个用于 13、18、21 三体和性染色体非整倍体的候选引物/探针组。涉及 463 个胎儿基因组 DNA 样本的多重多色熔解曲线分析的临床评估显示，与参考方法相比，敏感性为 100%，特异性为 99.64%。使用已建立的数字 PCR 协​​议，我们从母体血浆样本中正确识别出两个 21 三体胎儿和十三个整倍体胎儿。

解释

ChAPDes 与数字 PCR 检测的结合有助于将 SD 作为潜在的生物标志物用于胎儿染色体非整倍体的无创产前检测。