Laboratory for Cytogenetics and Genome Research, Department of Human Genetics, KU Leuven, Leuven, Belgium.
Department of Clinical Genetics, Maastricht University Medical Center, Maastricht, The Netherlands.
Hum Reprod. 2019 Aug 1;34(8):1608-1619. doi: 10.1093/humrep/dez106.
Can reduced representation genome sequencing offer an alternative to single nucleotide polymorphism (SNP) arrays as a generic and genome-wide approach for comprehensive preimplantation genetic testing for monogenic disorders (PGT-M), aneuploidy (PGT-A) and structural rearrangements (PGT-SR) in human embryo biopsy samples?
Reduced representation genome sequencing, with OnePGT, offers a generic, next-generation sequencing-based approach for automated haplotyping and copy-number assessment, both combined or independently, in human single blastomere and trophectoderm samples.
Genome-wide haplotyping strategies, such as karyomapping and haplarithmisis, have paved the way for comprehensive PGT, i.e. leveraging PGT-M, PGT-A and PGT-SR in a single workflow. These methods are based upon SNP array technology.
STUDY DESIGN, SIZE, DURATION: This multi-centre verification study evaluated the concordance of PGT results for a total of 225 embryos, including 189 originally tested for a monogenic disorder and 36 tested for a translocation. Concordance for whole chromosome aneuploidies was also evaluated where whole genome copy-number reference data were available. Data analysts were kept blind to the results from the reference PGT method.
PARTICIPANTS/MATERIALS, SETTING, METHODS: Leftover blastomere/trophectoderm whole genome amplified (WGA) material was used, or secondary trophectoderm biopsies were WGA. A reduced representation library from WGA DNA together with bulk DNA from phasing references was processed across two study sites with the Agilent OnePGT solution. Libraries were sequenced on an Illumina NextSeq500 system, and data were analysed with Agilent Alissa OnePGT software. The embedded PGT-M pipeline utilises the principles of haplarithmisis to deduce haplotype inheritance whereas both the PGT-A and PGT-SR pipelines are based upon read-count analysis in order to evaluate embryonic ploidy. Concordance analysis was performed for both analysis strategies against the reference PGT method.
PGT-M analysis was performed on 189 samples. For nine samples, the data quality was too poor to analyse further, and for 20 samples, no result could be obtained mainly due to biological limitations of the haplotyping approach, such as co-localisation of meiotic crossover events and nullisomy for the chromosome of interest. For the remaining 160 samples, 100% concordance was obtained between OnePGT and the reference PGT-M method. Equally for PGT-SR, 100% concordance for all 36 embryos tested was demonstrated. Moreover, with embryos originally analysed for PGT-M or PGT-SR for which genome-wide copy-number reference data were available, 100% concordance was shown for whole chromosome copy-number calls (PGT-A).
LIMITATIONS, REASONS FOR CAUTION: Inherent to haplotyping methodologies, processing of additional family members is still required. Biological limitations caused inconclusive results in 10% of cases.
Employment of OnePGT for PGT-M, PGT-SR, PGT-A or combined as comprehensive PGT offers a scalable platform, which is inherently generic and thereby, eliminates the need for family-specific design and optimisation. It can be considered as both an improvement and complement to the current methodologies for PGT.
STUDY FUNDING/COMPETING INTEREST(S): Agilent Technologies, the KU Leuven (C1/018 to J.R.V. and T.V.) and the Horizon 2020 WIDENLIFE (692065 to J.R.V. and T.V). H.M. is supported by the Research Foundation Flanders (FWO, 11A7119N). M.Z.E, J.R.V. and T.V. are co-inventors on patent applications: ZL910050-PCT/EP2011/060211- WO/2011/157846 'Methods for haplotyping single cells' and ZL913096-PCT/EP2014/068315 'Haplotyping and copy-number typing using polymorphic variant allelic frequencies'. T.V. and J.R.V. are co-inventors on patent application: ZL912076-PCT/EP2013/070858 'High-throughput genotyping by sequencing'. Haplarithmisis ('Haplotyping and copy-number typing using polymorphic variant allelic frequencies') has been licensed to Agilent Technologies. The following patents are pending for OnePGT: US2016275239, AU2014345516, CA2928013, CN105874081, EP3066213 and WO2015067796. OnePGT is a registered trademark. D.L., J.T. and R.L.R. report personal fees during the conduct of the study and outside the submitted work from Agilent Technologies. S.H. and K.O.F. report personal fees and other during the conduct of the study and outside the submitted work from Agilent Technologies. J.A. reports personal fees and other during the conduct of the study from Agilent Technologies and personal fees from Agilent Technologies and UZ Leuven outside the submitted work. B.D. reports grants from IWT/VLAIO, personal fees during the conduct of the study from Agilent Technologies and personal fees and other outside the submitted work from Agilent Technologies. In addition, B.D. has a patent 20160275239 - Genetic Analysis Method pending. The remaining authors have no conflicts of interest.
减少代表性基因组测序是否可以替代单核苷酸多态性 (SNP) 阵列，作为人类胚胎活检样本中单基因疾病 (PGT-M)、非整倍体 (PGT-A) 和结构重排 (PGT-SR) 的通用和全基因组方法？
使用 OnePGT 的减少代表性基因组测序提供了一种通用的、基于下一代测序的方法，用于在单个卵裂球和滋养外胚层样本中自动进行单体型分析和拷贝数评估，两者都可以单独或组合使用。
染色体作图和 haplarithmisis 等全基因组单倍型策略为全面 PGT 铺平了道路，即在单个工作流程中利用 PGT-M、PGT-A 和 PGT-SR。这些方法基于 SNP 阵列技术。
研究设计、规模、持续时间： 这项多中心验证研究评估了总共 225 个胚胎的 PGT 结果的一致性，其中 189 个最初测试了单基因疾病，36 个测试了易位。在有全基因组拷贝数参考数据的情况下，还评估了全染色体非整倍体的一致性。数据分析员对参考 PGT 方法的结果保持盲态。
参与者/材料、设置、方法： 使用剩余的卵裂球/滋养外胚层全基因组扩增 (WGA) 材料，或对二级滋养外胚层活检进行 WGA。来自 WGA DNA 的减少代表性文库与来自相体参考的 bulk DNA 一起在两个研究地点使用 Agilent OnePGT 解决方案进行处理。文库在 Illumina NextSeq500 系统上测序，Agilent Alissa OnePGT 软件用于数据分析。嵌入式 PGT-M 管道利用 haplarithmisis 的原理来推断单体型遗传，而 PGT-A 和 PGT-SR 管道都基于读取计数分析来评估胚胎的倍性。对两种分析策略与参考 PGT 方法进行了一致性分析。
对 189 个样本进行了 PGT-M 分析。对于 9 个样本，由于数据质量太差无法进一步分析，对于 20 个样本，由于单倍型分析方法的生物学限制，例如同源重组事件的局部化和所关注染色体的单体性，主要无法获得结果。对于其余 160 个样本，OnePGT 与参考 PGT-M 方法之间获得了 100%的一致性。同样对于 PGT-SR，测试的所有 36 个胚胎均显示出 100%的一致性。此外，对于最初分析 PGT-M 或 PGT-SR 的胚胎，并且有全基因组拷贝数参考数据，全染色体拷贝数调用 (PGT-A) 显示出 100%的一致性。
局限性、谨慎的原因： 由于单倍型分析方法的内在原因，仍然需要处理额外的家族成员。生物学限制导致 10%的病例结果不确定。
使用 OnePGT 进行 PGT-M、PGT-SR、PGT-A 或综合全面 PGT 提供了一个可扩展的平台，该平台本质上是通用的，因此无需针对特定家族进行设计和优化。它可以被认为是对当前 PGT 方法的改进和补充。
研究资金/利益冲突： 安捷伦科技公司、鲁汶大学 (C1/018 至 J.R.V. 和 T.V.) 和 Horizon 2020 WIDENLIFE (692065 至 J.R.V. 和 T.V.)。H.M. 得到了佛兰德斯研究基金会 (FWO, 11A7119N) 的支持。M.Z.E、J.R.V. 和 T.V. 是专利申请 ZL910050-PCT/EP2011/060211-WO/2011/157846“单细胞单倍型分析和拷贝数评估的方法”和 ZL913096-PCT/EP2014/068315“使用多态性变体等位基因频率进行单倍型分析和拷贝数分型”的共同发明人。T.V. 和 J.R.V. 是专利申请 ZL912076-PCT/EP2013/070858“基于测序的高通量基因分型”的共同发明人。Haplarithmisis（“使用多态性变体等位基因频率进行单倍型分析和拷贝数分型”）已授权给安捷伦科技公司。OnePGT 正在申请以下专利：美国 2016275239、澳大利亚 2014345516、CA2928013、CN105874081、EP3066213 和 WO2015067796。OnePGT 是一个注册商标。D.L.、J.T. 和 R.L.R. 在研究期间和提交工作之外报告了来自安捷伦科技公司的个人酬金。S.H. 和 K.O.F. 在研究期间和提交工作之外报告了来自安捷伦科技公司的个人酬金和其他酬金。J.A. 在研究期间报告了来自安捷伦科技公司的个人酬金和其他酬金以及来自安捷伦科技公司和鲁汶大学的个人酬金。B.D. 在研究期间报告了来自 IWT/VLAIO 的拨款，在研究期间和提交工作之外报告了来自安捷伦科技公司的个人酬金和其他酬金。此外，B.D. 拥有专利 20160275239-遗传分析方法待批。其余作者没有利益冲突。
