Comprehensive preimplantation genetic testing by massively parallel sequencing
Comprehensive preimplantation genetic testing by massively parallel sequencing
复制标题
通过大规模并行测序进行全面的植入前基因检测
DOI：
10.1093/humrep/deaa269
复制
发表时间：
2021-01-01
影响因子：
6.1
通讯作者：
Xu, Chenming
中科院分区：
文献类型：
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作者：
Chen, Songchang;Yin, Xuyang;Xu, Chenming
研究问题：全基因组测序(WGS)能否为单基因疾病(PGT-M)、非整倍体(PGT-A)和结构重排(PGT-SR)的胚胎基因组单倍型和植入前遗传学测试(PGT-M)提供一种相对经济有效的方法？总结答案：可靠的全基因组单倍型、PGT-M、PGT-A和PGT-SR可以由WGS进行，其亲代深度为10倍，胚胎测序数据深度为4倍。WHAT已知：基于全基因组测序单倍体解决方案的简化代表基因组测序已被验证为自动化单倍型和综合PGT的通用方法。几种基于低深度大规模并行测序(MPS)的单倍型和综合PGT方法已经被开发出来。研究设计、大小、持续时间：在本研究中，接受过传统体外受精-胚胎移植的10个家系和53个胚胎，包括来自两个PGT-SR家系的13个胚胎和来自8个PGT-M家系的40个胚胎，用于评估基于WGS的方法。共24个卵裂球和29个囊胚。所有胚胎均用于PGT-A。核测图验证了WGS的结果。对10个家系的临床结果进行评估。对照/材料，背景，方法：从胚泡中取一个或几个滋养外胚层细胞进行活检，并进行多重置换扩增(MDA)。文库构建采用家系成员的丙二醛DNA和散体DNA。对文库进行测序，并使用内部管道进行数据分析，包括PGT-M和PGT-SR的单倍型遗传推断和PGT-A的读取计数分析。有先证者的单倍型和没有额外家庭成员的只有父母的单倍型被用来评估WGS方法学。对WGS结果和传统的PGT方法进行一致性分析。MAIN结果和机会的作用：对于40个PGT M和53个PGT A胚胎，无论是否包括额外的家族成员或先证者进行PGT-M单倍型，WGS和单核苷酸多态(SNP)阵列结果都观察到100%的一致性。对于来自两个PGT-SR家族的13个胚胎，检测到了胚胎平衡易位，WGS和MicroSeq与PCR-Seq的一致性为100%。限制，谨慎的理由：本研究的样本数量有限。在某些情况下，由于直接基因分型失败，无法获得仅用于PGT M或PGT SR亲本单倍型的参考胚胎。这些发现的广泛意义：基于WGS的PGT-A、PGT-M和PGT-SR提供了一种综合的PGT单倍型方法，而不需要额外的家庭成员。它提供了一种改进的方法来补充PGT方法，如基于低深度MPS和SNP阵列的方法。
STUDY QUESTION: Can whole genome sequencing (WGS) offer a relatively cost-effective approach for embryonic genome-wide haplotyping and preimplantation genetic testing (PGT) for monogenic disorders (PGT-M), aneuploidy (PGT-A) and structural rearrangements (PGT-SR)?SUMMARY ANSWER: Reliable genome-wide haplotyping, PGT-M, PGT-A and PGT-SR could be performed by WGS with 10 x depth of parental and 4x depth of embryonic sequencing data.WHAT IS KNOWN ALREADY: Reduced representation genome sequencing with a genome-wide next-generation sequencing haplarithmisis-based solution has been verified as a generic approach for automated haplotyping and comprehensive PGT. Several low-depth massively parallel sequencing (MPS)-based methods for haplotyping and comprehensive PGT have been developed. However, an additional family member, such as a sibling, or a proband, is required for PGT-M haplotyping using low-depth MPS methods.STUDY DESIGN, SIZE, DURATION: In this study, 10 families that had undergone traditional IVF-PGT and 53 embryos, including 13 embryos from two PGT-SR families and 40 embryos from eight PGT-M families, were included to evaluate a WGS-based method. There were 24 blastomeres and 29 blastocysts in total. All embryos were used for PGT-A. Karyomapping validated the WGS results. Clinical outcomes of the 10 families were evaluated.PARTICIPANTS/MATERIALS, SETTING, METHODS: A blastomere or a few trophectoderm cells from the blastocyst were biopsied, and multiple displacement amplification (MDA) was performed. MDA DNA and bulk DNA of family members were used for library construction. Libraries were sequenced, and data analysis, including haplotype inheritance deduction for PGT-M and PGT-SR and read-count analysis for PGT-A, was performed using an in-house pipeline. Haplotyping with a proband and parent-only haplotyping without additional family members were performed to assess the WGS methodology. Concordance analysis between the WGS results and traditional PGT methods was performed.MAIN RESULTS AND THE ROLE OF CHANCE: For the 40 PGT M and 53 PGT A embryos, 100% concordance between the WGS and single nucleotide polymorphism (SNP) array results was observed, regardless of whether additional family members or a proband was included for PGT-M haplotyping. For the 13 embryos from the two PGT-SR families, the embryonic balanced translocation was detected and 100% concordance between WGS and MicroSeq with PCR-seq was demonstrated.LIMITATIONS, REASONS FOR CAUTION: The number of samples in this study was limited. In some cases, the reference embryo for PGT M or PGT SR parent only haplotyping was not available owing to failed direct genotyping.WIDER IMPLICATIONS OF THE FINDINGS: WGS-based PGT-A, PGT-M and PGT-SR offered a comprehensive PGT approach for haplotyping without the requirement for additional family members. It provided an improved complementary method to PGT methodologies, such as low-depth MPS- and SNP array-based methods.