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. 2013 Apr 1;41(6):e66.
doi: 10.1093/nar/gks1352. Epub 2013 Jan 7.

Genome-wide Copy Number Profiling of Single Cells in S-phase Reveals DNA-replication Domains

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Free PMC article

Genome-wide Copy Number Profiling of Single Cells in S-phase Reveals DNA-replication Domains

Niels Van der Aa et al. Nucleic Acids Res. .
Free PMC article

Abstract

Single-cell genomics is revolutionizing basic genome research and clinical genetic diagnosis. However, none of the current research or clinical methods for single-cell analysis distinguishes between the analysis of a cell in G1-, S- or G2/M-phase of the cell cycle. Here, we demonstrate by means of array comparative genomic hybridization that charting the DNA copy number landscape of a cell in S-phase requires conceptually different approaches to that of a cell in G1- or G2/M-phase. Remarkably, despite single-cell whole-genome amplification artifacts, the log2 intensity ratios of single S-phase cells oscillate according to early and late replication domains, which in turn leads to the detection of significantly more DNA imbalances when compared with a cell in G1- or G2/M-phase. Although these DNA imbalances may, on the one hand, be falsely interpreted as genuine structural aberrations in the S-phase cell's copy number profile and hence lead to misdiagnosis, on the other hand, the ability to detect replication domains genome wide in one cell has important applications in DNA-replication research. Genome-wide cell-type-specific early and late replicating domains have been identified by analyses of DNA from populations of cells, but cell-to-cell differences in DNA replication may be important in genome stability, disease aetiology and various other cellular processes.

Figures

Figure 1.
Figure 1.
DNA replication is mirrored in single-cell aCGH log2 intensity ratios. (a) The correlation between log2 intensity ratio values and %GC content is different for S-phase single cells (grey lines) when compared with G1- and G2/M-phase cells (black lines). The X-axis depicts the %GC content per probe, the Y-axis the log2 intensity ratios per sample. Each line is a Loess fit using the data of a single-cell sample. (b) Boxplots for single cells (right) and multi-cell controls (left) depicting autosomal log2 intensity ratios that were pooled per cell cycle phase and per early or late DNA-replication domain. The annotation of the DNA-replication domains is described by Ryba et al. (28). Boxplots show the median of the log2 intensity ratios (central line), the quartiles (box and whiskers) and the mean of the log2 intensity ratios (diamonds). Relevant significant differences between G1-, G2/M- and S-phase cells are marked by a star (Student’s t-test; P < 0.05).
Figure 2.
Figure 2.
Log2 intensity ratios in S-phase cells oscillate according to predicted replication timing. For each single-cell and multi-cell control sample, a heat map of the mean log2 intensity ratio per replication domain across all autosomes is depicted in a circosplot. The colour-code legend of the heat map is depicted at the bottom of the figure. In each circosplot, the outermost circle depicts the predicted replication timing pattern as published by Ryba et al. (28) (early replicating domains in green; late replicating domains in red; replication domains covered by five or more microarray probes are marked by a blue bar on the outside). This is followed (from the outside to the inside of the circosplot) by the heat maps representing all single-cell log2 intensity ratio data (one cell per rim) and subsequently by the heat maps reflecting all multi-cell control log2 intensity ratio data (one multi-cell control per rim). (a and b) All S-phase single-cell and multi-cell samples. The top circosplot depicts the chromosomes 1 to 8 (a), bottom circosplot chromosomes 9 to 22 (b). The 14 S-phase single cell samples are shown in the following order (outside to inside): S1.3, S1.2, S3.1, S7.5, S7.6, S1.4, S7.7, S1.1, S7.1, S7.4, S4.1, S4.2, S7.2 and S7.3; followed by S-phase multi-cell control samples. (c and d) All G1- and G2/M-phase single-cell and multi- cell samples. Top circosplot depicts the chromosomes 1 to 8 (c), bottom circosplot chromosomes 9 to 22 (d). The 16 single-cell samples are shown in the following order (outside to inside): G1.1, G1.2, G1.3, G1.4, G3.1, G4.1, G7.1, G7.2, M1.1, M1.2, M1.3, M3.1, M3.2, M4.1, M7.1 and M7.2; followed by G1- and G2/M-phase multi-cell control samples. The oscillating pattern of consecutive positive and negative log2 intensity ratios genome wide orchestrated by early and late DNA replication in S-phase single cells can be clearly observed, and is concordant with the pattern in multi-cell controls. In contrast, the G1-phase and G2/M-phase single cells do not demonstrate such genome wide oscillation of log2 intensity ratios in accordance with the replication timing pattern. Similar observations can be made for the multi-cell controls. Three specific regions for which the log2 intensity plots are shown in Supplementary Figure S5 at high resolution are marked by a black box.
Figure 3.
Figure 3.
PCA of the single-cell mean log2 intensity ratio values per replication domain. The S-phase single-cell data are clearly separated from G1-, G2/M-phase single-cell data for all but one sample (cell S3.1 clusters aberrantly). Apart from an increased standard deviation across autosomal log2 ratios, cell S3.1 did not show the typical log2 intensity ratio behaviour expected following DNA replication as the other S-phase cells did. This suggests that either this cell is a G1- or G2/M-phase cell sorted wrongly into the S-phase cell population, or more likely that the whole-genome amplification product was of bad quality.
Figure 4.
Figure 4.
Single S-phase cells ranked according to their progression in S-phase. (a) Barplots showing per cell the fraction of early and late replicating domains covered by five or more probes (n = 121) for which (i) the single-cell mean log2 intensity ratios surpassed the threshold that was assigned for the expected replication timing (indicated as ‘Match’ in the legend; see also Materials and Methods), (ii) the single-cell mean log2 intensity ratios did not surpass the threshold but were still above or below the zero axis in accordance with the expected replication timing (indicated as ‘Sub threshold’ in the legend) and (iii) the single-cell mean log2 intensity ratios mismatched the expected replication timing (indicated as ‘Mismatch’ in the legend). Fractions of early replicating domains are depicted in green colours, those for late replicating regions in red colours. The left panel shows all S-phase single-cell samples ordered into groups of early-, mid- and late-S-phase cells (left to right), based on the fraction of early and late replication domains having matching mean log2 intensity ratios in the S-phase cells. The right panel shows the data for all single-cell G1- and G2/M-phase samples. (b) Three single-cell S-phase log2 distribution plots of an early-S-phase cell, a mid-S-phase cell and a late-S-phase cell from left to right. The density plot across all log2 intensity ratios is shown in purple. Cell S1.3 (left panel) shows a bimodal distribution of the log2 intensity values with the highest peak left and a lower peak on the right side of the high peak, suggesting the cell is in early S-phase. Cell S7.6 (middle panel) shows a bimodal distribution with two peaks of approximately the same height, suggestive for a cell in mid-S-phase. Cell S1.1 (right panel) shows a low peak to the left of the high peak which is close to zero, suggesting this cell is at a late stage in S-phase.
Figure 5.
Figure 5.
Copy number aberrations called in S-phase samples are concordant with replication domains. Circosplots depict the loci called as DNA copy number gains (green) or losses (red) across the autosomes for different copy number analysis methods. The left circosplots contain all S-phase single-cell and multi-cell samples (panels a, c), the right circosplots contain all G1- and G2/M-phase samples (panels b, d). In each circosplot, the outermost circle depicts the predicted replication timing pattern as published by Ryba et al. (28) (early replicating domains in green; late replicating domains in red). This is followed (from the outside to the inside of the circosplot) by the copy number heat maps of all single cells (one cell per rim, ranked according to S-phase progression as in Figure 4a) and subsequently by the copy number heat maps of all multi-cell control samples (one multi-cell control per rim). Importantly, (i) the type and size of copy number aberrations called in the S-phase single cells are often concordant with one particular replication domain or with a concatenation of (larger) replication domains having similar replication timing. (ii) Cells ranked to be late in S-phase show more DNA losses while cells ranked to be in early S-phase show more DNA-copy number gains (from the outside to the inside of the circosplot: the S-phase cells are ranked from early to mid to late S-phase stage, respecting the ranking proposed in Figure 4a). (a, b) Piecewise constant fitting and integer DNA-copy number transformation of log2 intensity ratios normalized according to their autosomal median value and corrected for GC bias according to a mean Loess regression curve between %GC and G1/G2/M-phase log2 values (techSD). (c, d) CBS segmentation and CGHcall (Methods) after log2 intensity ratios were normalized according to their autosomal median value and corrected for GC bias according to a mean Loess regression curve between %GC and G1/G2/M-phase log2 values (techSD). (a, c) Specifically, the 14 S-phase single cell samples are shown in the following order (outside to inside): S1.3, S1.2, S3.1, S7.5, S7.6, S1.4, S7.7, S1.1, S7.1, S7.4, S4.1, S4.2, S7.2 and S7.3; followed by S-phase multi-cell control samples. (b, d) The 16 G1- and G2/M-phase single-cell samples are shown in the following order (outside to inside): G1.1, G1.2, G1.3, G1.4, G3.1, G4.1, G7.1, G7.2, M1.1, M1.2, M1.3, M3.1, M3.2, M4.1, M7.1 and M7.2; followed by G1- and G2/M-phase multi-cell control samples.

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