Identification of DNA-replication domains in single S-phase cells: part 2 whole genome amplified single-cell. Homo sapiens

DNA-replication is a key process in life and can lead to disease when disturbed. Cell-type specific early and late replication domains have been discovered throughout genomes by analysis of DNA from populations of cells. However, cell to cell differences and the association of these differences with other cellular processes remain largely elusive. Here we demonstrate for the first time that consecutive domains of early and late DNA-replication can be detected in single S-phase cells using array comparative genomic hybridization, providing proof-of-concept for a novel tool to investigate DNA-replication genome wide at the single-cell level. Furthermore, methods to profile the genome of a single cell for DNA-copy number aberrations are revolutionizing both basic genome research and clinical genetic diagnosis. It is thus important to apprehend not only technical but also biological reasons for false positive copy number detection. None of the current single-cell copy number calling methods distinguishes between a cell in G1-, S- or G2/M-phase of the cell cycle and mostly use cells isolated randomly from populations. We demonstrate that the oscillating pattern between early and late replicating loci instigates significantly more false-positive DNA copy-number calls in a diploid cell in S-phase cells than in G1- or G2/M-phase, depending on the specific aCGH-signal normalization method used. We propose a work-flow to detect single cells in S-phase and to correct for DNA-replication bias before copy number profiling. Overall design: The genome of 2 S-phase, 1 M-phase and 1 G-phase single cells were amplified using the Sureplex amplification system. These test samples were hybridized comparatively to commercial male reference DNA,

DNA复制（DNA-replication）是生命活动中的关键过程，当其受到干扰时可引发疾病。通过对细胞群体的DNA进行分析，人们已在全基因组范围内发现了细胞类型特异性的早期与晚期复制结构域。然而，细胞间的差异以及这些差异与其他细胞过程的关联，在很大程度上仍未明确。本研究首次证明，可利用阵列比较基因组杂交（array comparative genomic hybridization）技术在单个S期细胞中检测到连续的早期与晚期DNA复制结构域，为在单细胞水平上全基因组范围内研究DNA复制提供了概念验证性的新型工具。此外，针对单细胞基因组进行DNA拷贝数变异分析的方法，正彻底改变基础基因组研究与临床遗传诊断领域。因此，不仅要厘清假阳性拷贝数检测的技术成因，还要明确其生物学根源，这一点至关重要。当前的单细胞拷贝数呼叫方法均无法区分处于细胞周期G1期、S期或G2/M期的细胞，且大多使用从细胞群体中随机分离得到的细胞。我们的研究表明，根据所采用的特定aCGH信号标准化方法，早期与晚期复制位点间的振荡模式，会使S期二倍体细胞产生的假阳性DNA拷贝数呼叫结果，显著多于G1期或G2/M期细胞。我们提出了一套工作流程，可用于检测处于S期的单细胞，并在进行拷贝数分型前校正DNA复制带来的偏差。实验整体设计：采用Sureplex扩增系统对2个S期、1个M期及1个G期的单细胞基因组进行扩增。将这些待测样本与商业化男性参考DNA进行比较杂交。