Department of Clinical Genetics, Maastricht University Medical Center+ (MUMC+), Maastricht, The Netherlands.
Department of Genetics and Cell Biology, GROW School for Oncology and Reproduction, Maastricht University, Maastricht, The Netherlands.
Hum Reprod. 2022 Oct 31;37(11):2700-2708. doi: 10.1093/humrep/deac208.
Can the embryo tracking system (ETS) increase safety, efficacy and scalability of massively parallel sequencing-based preimplantation genetic testing (PGT)?
Applying ETS-PGT, the chance of sample switching is decreased, while scalability and efficacy could easily be increased substantially.
Although state-of-the-art sequencing-based PGT methods made a paradigm shift in PGT, they still require labor intensive library preparation steps that makes PGT cost prohibitive and poses risks of human errors. To increase the quality assurance, efficiency, robustness and throughput of the sequencing-based assays, barcoded DNA fragments have been used in several aspects of next-generation sequencing (NGS) approach.
STUDY DESIGN, SIZE, DURATION: We developed an ETS that substantially alleviates the complexity of the current sequencing-based PGT. With (n = 693) and without (n = 192) ETS, the downstream PGT procedure was performed on both bulk DNA samples (n = 563) and whole-genome amplified (WGAed) few-cell DNA samples (n = 322). Subsequently, we compared full genome haplotype landscapes of both WGAed and bulk DNA samples containing ETS or no ETS.
PARTICIPANTS/MATERIALS, SETTING, METHODS: We have devised an ETS to track embryos right after whole-genome amplification (WGA) to full genome haplotype profiles. In this study, we recruited 322 WGAed DNA samples derived from IVF embryos as well as 563 bulk DNA isolated from peripheral blood of prospective parents. To determine possible interference of the ETS in the NGS-based PGT workflow, barcoded DNA fragments were added to DNA samples prior to library preparation and compared to samples without ETS. Coverages and variants were determined.
Current PGT protocols are quality sensitive and prone to sample switching. To avoid sample switching and increase throughput of PGT by sequencing-based haplotyping, six control steps should be carried out manually and checked by a second person in a clinical setting. Here, we developed an ETS approach in which one step only in the entire PGT procedure needs the four-eyes principal. We demonstrate that ETS not only precludes error-prone manual checks but also has no effect on the genomic landscape of preimplantation embryos. Importantly, our approach increases efficacy and throughput of the state-of-the-art PGT methods.
LIMITATIONS, REASONS FOR CAUTION: Even though the ETS simplified sequencing-based PGT by avoiding potential errors in six steps in the protocol, if the initial assignment is not performed correctly, it could lead to cross-contamination. However, this can be detected in silico following downstream ETS analysis. Although we demonstrated an approach to evaluate purity of the ETS fragment, it is recommended to perform a pre-PGT quality control assay of the ETS amplicons with non-human DNA, such that the purity of each ETS molecule can be determined prior to ETS-PGT.
The ETS-PGT approach notably increases efficacy and scalability of PGT. ETS-PGT has broad applicative value, as it can be tailored to any single- and few-cell sequencing approach where the starting specimen is scarce, as opposed to other methods that require a large number of cells as the input. Moreover, ETS-PGT could easily be adapted to any sequencing-based diagnostic method, including PGT for structural rearrangements and aneuploidies by low-pass sequencing as well as non-invasive prenatal testing.
STUDY FUNDING/COMPETING INTEREST(S): M.Z.E. is supported by the EVA (Erfelijkheid Voortplanting & Aanleg) specialty program (grant no. KP111513) of Maastricht University Medical Centre (MUMC+), and the Horizon 2020 innovation (ERIN) (grant no. EU952516) of the European Commission.
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胚胎跟踪系统（ETS）能否提高基于大规模并行测序的植入前遗传学检测（PGT）的安全性、有效性和可扩展性？
应用 ETS-PGT 可以降低样本切换的可能性，同时可以显著提高可扩展性和有效性。
尽管基于最新技术的测序 PGT 方法在 PGT 方面带来了重大转变，但它们仍然需要劳动密集型的文库制备步骤，这使得 PGT 的成本过高，并存在人为错误的风险。为了提高基于测序的检测的质量保证、效率、稳健性和通量，在下一代测序（NGS）方法的多个方面都使用了带有条形码的 DNA 片段。
研究设计、规模、持续时间：我们开发了一种 ETS，它大大减轻了当前基于测序的 PGT 的复杂性。在有（n=693）和没有（n=192）ETS 的情况下，下游 PGT 程序分别在批量 DNA 样本（n=563）和全基因组扩增（WGAed）的少量细胞 DNA 样本（n=322）上进行。随后，我们比较了含有 ETS 或不含 ETS 的 WGAed 和批量 DNA 样本的全基因组单倍型景观。
参与者/材料、设置、方法：我们设计了一种 ETS 来跟踪 WGA 后全基因组扩增（WGA）的胚胎，直至全基因组单倍型图谱。在这项研究中，我们招募了 322 个来自 IVF 胚胎的 WGAed DNA 样本，以及 563 个来自潜在父母外周血的批量 DNA 样本。为了确定 ETS 在基于 NGS 的 PGT 工作流程中的可能干扰，在文库制备之前向 DNA 样本中添加了带有条形码的 DNA 片段，并将其与没有 ETS 的样本进行比较。确定了覆盖率和变体。
当前的 PGT 协议质量敏感，容易发生样本切换。为了避免样本切换并通过基于测序的单体型分析提高 PGT 的通量，在临床环境中需要进行六步质量检查，并由第二人进行检查。在这里，我们开发了一种 ETS 方法，其中整个 PGT 过程中只需要进行一个步骤，需要四人同时进行。我们证明，ETS 不仅可以防止易出错的手动检查，而且对植入前胚胎的基因组景观没有影响。重要的是，我们的方法提高了最先进的 PGT 方法的有效性和通量。
局限性、谨慎的原因：尽管 ETS 通过避免协议中的六个步骤中的潜在错误简化了基于测序的 PGT，但如果初始分配不正确，可能会导致交叉污染。然而，这可以在下游 ETS 分析中通过计算机进行检测。虽然我们展示了一种评估 ETS 片段纯度的方法，但建议对 ETS 扩增子进行 PGT 前质量控制检测，以便在进行 ETS-PGT 之前确定每个 ETS 分子的纯度。
ETS-PGT 方法显著提高了 PGT 的有效性和可扩展性。ETS-PGT 具有广泛的应用价值，因为它可以针对任何需要稀缺起始标本的单细胞和少量细胞测序方法进行定制，而不是其他需要大量细胞作为输入的方法。此外，ETS-PGT 可以很容易地适应任何基于测序的诊断方法，包括基于低通测序的 PGT 用于结构重排和非整倍性，以及非侵入性产前检测。
研究资金/利益冲突：M.Z.E. 得到了 Maastricht 大学医学中心（MUMC+）遗传生殖与发育（EVA）专业计划（赠款号 KP111513）和欧洲委员会的地平线 2020 创新（ERIN）（赠款号 EU952516）的支持。
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