胚胎跟踪系统(ETS)是否可以提高安全性,基于大规模平行测序的植入前基因检测(PGT)的功效和可扩展性?
应用ETS-PGT,样本切换的机会减少,而可扩展性和疗效可以很容易地大大提高。
尽管基于最新测序的PGT方法在PGT中发生了范式转变,它们仍然需要劳动密集型的库准备步骤,这使得PGT的成本过高,并带来人为错误的风险.为了增加质量保证,效率,基于测序的测定的鲁棒性和吞吐量,条形码化的DNA片段已用于下一代测序(NGS)方法的几个方面。
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。有(n=693)和没有(n=192)ETS,下游PGT程序对大量DNA样本(n=563)和全基因组扩增(WGAed)少数细胞DNA样本(n=322)进行.随后,我们比较了包含ETS或不包含ETS的WGAed和批量DNA样本的全基因组单倍型景观。
我们设计了一个ETS来跟踪全基因组扩增(WGA)后的胚胎到全基因组单倍型。在这项研究中,我们招募了322份来自IVF胚胎的WGAedDNA样本,以及从预期父母外周血中分离的563份大块DNA样本.要确定ETS在基于NGS的PGT工作流程中的可能干扰,在文库制备之前将条形码化的DNA片段添加到DNA样品中,并与没有ETS的样品进行比较。确定了覆盖范围和变体。
当前的PGT方案是质量敏感的并且易于样品切换。为了避免样品转换并通过基于测序的单倍型分析增加PGT的通量,六个控制步骤应手动进行,并由第二个人在临床环境中进行检查。这里,我们开发了一种ETS方法,其中整个PGT程序中只有一个步骤需要四眼主体。我们证明ETS不仅排除了容易出错的手动检查,而且对植入前胚胎的基因组格局没有影响。重要的是,我们的方法提高了最先进的PGT方法的功效和吞吐量.
即使ETS通过避免方案中六个步骤的潜在错误来简化基于测序的PGT,如果初始分配未正确执行,会导致交叉污染.然而,这可以在下游ETS分析后在计算机中检测到。尽管我们展示了一种评估ETS片段纯度的方法,建议使用非人DNA对ETS扩增子进行pre-PGT质量控制测定,使得每个ETS分子的纯度可以在ETS-PGT之前测定。
ETS-PGT方法显著提高了PGT的功效和可扩展性。ETS-PGT具有广泛的应用价值,因为它可以适应任何单细胞和少数细胞测序方法,其中起始标本是稀缺的,与其他需要大量单元格作为输入的方法相反。此外,ETS-PGT可以很容易地适应任何基于测序的诊断方法,包括通过低通测序进行结构重排和非整倍体的PGT以及非侵入性产前检测。
M.Z.E.由EVA(ErfelijkheidVoortplanting&Aanleg)专业计划(批准号:马斯特里赫特大学医学中心(MUMC)的KP111513),和地平线2020创新(ERIN)(授予号欧盟委员会的EU952516)。
不适用。
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.
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.
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.
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.
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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