不同单细胞全基因组扩增方法的比较及MALBAC在辅助生殖中的应用

单细胞全基因组扩增(whole genome amplification, WGA)是指在单细胞水平对全基因组进行扩增的新技术,其原理是将分离的单个细胞的微量全基因组DNA进行扩增,获得高覆盖率的完整的基因组后进行高通量测序,用于揭示细胞异质性。目前,WGA方法主要包括引物延伸预扩增(primer extension preamplification PCR, PEP-PCR)、简并寡核苷酸引物PCR (degenerate oligonucleotide primed PCR, DOP-PCR)、多重置换扩增(multiple displacement amplification, MDA)、多次退火环状循环扩增(multiple annealing and looping-based amplification cycles, MALBAC)等。本文对不同的单细胞WGA方法的原理及应用情况分别进行了阐述,并对其扩增效率进行评价和比较,包括基因组覆盖度、均一性、重现性、SNV (single-nucleotide variants)和CNV (copy number variants)检测力等。综合对比不同单细胞WGA方法后发现,MALBAC的扩增均一性最高、等位基因脱扣率最低、重现性最好,且对于CNV和SNV的检测效果最好。本文还阐述了MALBAC技术在人类单精子减数重组、非整倍体分析以及人类卵细胞基因组研究中的应用。

Comparison of different single cell whole genome amplification methods and MALBAC applications in assisted reproduction

Single-cell whole genome amplification (WGA) is a new technology, which can amplify small amounts of DNA from single cell and obtain the high coverage whole genome DNA template for revealing cell heterogeneity. Single cell WGA methods mainly include primer extension preamplification PCR (PEP-PCR), degenerate oligonucleotide primed PCR (DOP-PCR), multiple displacement amplification (MDA), and multiple annealing and looping-based amplification cycles (MALBAC). In this review, we describe the principles and applications of different single cell genome wide amplification, and we evaluate and compare their amplification efficiency, including the coverage of genome, homogeneity, reproducibility, and detection power of single-nucleotide variants (SNV) and copy number variants (CNV). The results show that MALBAC have the highest amplification homogeneity, the lowest allelic gene knockdown rate, the best reproducibility, and the best detection effect on CNV and SNV. We also describe the applications of MALBAC in human single sperm meiosis, aneuploidy analysis, and human oocyte genome research.