Li Na, Wang Li, Wang Hui, Ma Minyue, Wang Xiaohong, Li Yi, Zhang Wenke, Zhang Jianguang, Cram David S, Yao Yuanqing
Department of Gynecology and Obstetrics, Reproductive Medicine Center, Tangdu Hospital, Fourth Military Medical University, Xi'an 710038, China; Department of Obstetrics and Gynecology, Affiliated Hospital of Academy of Military Medical Sciences, Beijing 100071, China.
Department of Obstetrics and Gynecology, Chinese PLA General Hospital, Beijing 100853, China.
J Genet Genomics. 2015 Apr 20;42(4):151-9. doi: 10.1016/j.jgg.2015.03.001. Epub 2015 Mar 14.
Reliable and accurate pre-implantation genetic diagnosis (PGD) of patient's embryos by next-generation sequencing (NGS) is dependent on efficient whole genome amplification (WGA) of a representative biopsy sample. However, the performance of the current state of the art WGA methods has not been evaluated for sequencing. Using low template DNA (15 pg) and single cells, we showed that the two PCR-based WGA systems SurePlex and MALBAC are superior to the REPLI-g WGA multiple displacement amplification (MDA) system in terms of consistent and reproducible genome coverage and sequence bias across the 24 chromosomes, allowing better normalization of test to reference sequencing data. When copy number variation sequencing (CNV-Seq) was applied to single cell WGA products derived by either SurePlex or MALBAC amplification, we showed that known disease CNVs in the range of 3-15 Mb could be reliably and accurately detected at the correct genomic positions. These findings indicate that our CNV-Seq pipeline incorporating either SurePlex or MALBAC as the key initial WGA step is a powerful methodology for clinical PGD to identify euploid embryos in a patient's cohort for uterine transplantation.
通过下一代测序（NGS）对患者胚胎进行可靠且准确的植入前基因诊断（PGD），依赖于对具有代表性的活检样本进行高效的全基因组扩增（WGA）。然而，目前最先进的WGA方法在测序方面的性能尚未得到评估。使用低模板DNA（15 pg）和单细胞，我们发现基于PCR的两种WGA系统SurePlex和MALBAC在全基因组覆盖的一致性和可重复性以及24条染色体上的序列偏差方面优于REPLI-g WGA多重置换扩增（MDA）系统，从而能更好地将测试数据与参考测序数据进行标准化。当将拷贝数变异测序（CNV-Seq）应用于通过SurePlex或MALBAC扩增得到的单细胞WGA产物时，我们发现3 - 15 Mb范围内已知的疾病CNV能够在正确的基因组位置被可靠且准确地检测到。这些发现表明，我们以SurePlex或MALBAC作为关键初始WGA步骤的CNV-Seq流程，是一种用于临床PGD以在患者队列中识别整倍体胚胎用于子宫移植的强大方法。
