Laboratory of Pharmaceutical Biotechnology, Ghent University, Ghent, Belgium.
PLoS One. 2018 Apr 26;13(4):e0196334. doi: 10.1371/journal.pone.0196334. eCollection 2018.
Single Gene Disorders (SGD) are still routinely diagnosed using PCR-based assays that need to be developed and validated for each individual disease-specific gene fragment. The TruSight One sequencing panel currently covers 12 Mb of genomic content, including 4813 genes associated with a clinical phenotype. When only a limited number of cells are available, whole genome amplification (WGA) is required prior to DNA target capture techniques such as the TruSight One panel. In this study, we compared 4 different WGA methods in combination with the TruSight One sequencing panel to perform single nucleotide polymorphism (SNP) genotyping starting from 3 micro-manipulated cells. This setting simulates clinical settings such as day-5 blastocyst biopsy for Preimplantation Genetic Testing (PGT), liquid biopsy of circulating tumor cells (CTCs) and cancer-cell profiling. Bulk cell samples were processed alongside these WGA samples to serve as a performance reference. Target coverage, coverage uniformity and SNP calling accuracy obtained using any of the WGA, is inferior to the results obtained on bulk cell samples. However, results after REPLI-g come close. Compared to the other WGA methods, the method using REPLI-g WGA results in a better coverage of the targeted genomic regions with a more uniform read depth. Consequently, this method also results in a more accurate SNP calling and could be considered for clinical genotyping of a limited number of cells.
单基因疾病（SGD）仍然通过基于 PCR 的检测方法进行常规诊断，这些检测方法需要针对每个特定疾病的基因片段进行开发和验证。TruSight One 测序面板目前覆盖 12Mb 的基因组内容，包括与临床表型相关的 4813 个基因。当只有有限数量的细胞可用时，在进行 TruSight One 面板等 DNA 靶标捕获技术之前，需要进行全基因组扩增（WGA）。在这项研究中，我们比较了 4 种不同的 WGA 方法与 TruSight One 测序面板相结合，从 3 个微操作细胞开始进行单核苷酸多态性（SNP）基因分型。这种设置模拟了临床情况，例如用于植入前基因检测（PGT）的第 5 天囊胚活检、循环肿瘤细胞（CTC）的液体活检和癌细胞分析。与这些 WGA 样本一起处理批量细胞样本，作为性能参考。使用任何 WGA 获得的目标覆盖率、覆盖率均匀性和 SNP 调用准确性均劣于批量细胞样本的结果。然而，REPLI-g 的结果接近。与其他 WGA 方法相比，使用 REPLI-g WGA 的方法可以更好地覆盖靶向基因组区域，并具有更均匀的读取深度。因此，这种方法还可以更准确地调用 SNP，并可考虑用于有限数量细胞的临床基因分型。
