Figure 1.
ROC curves for aCGH experiments with WGAM amplified single cells.
Cell lines OE19 A) & B) and REH C) & D). An experiment performed according to standard protocol and with gDNA from each corresponding cell line served as a reference array for ROC analysis. A) Digested high molecular DNA vs. single cell amplified DNA used as reference DNA. B) Comparison of different labeling methods (RP = random-primed labeling, MSE = MSE-PCR based labeling 1 or 2, TS = Thermosequenase labeling, ULS = Universal Linkage System™). C) Comparison of aCGH platforms (2×400k, MPS = 5.3 kb, 4×180k, MPS = 13 kb and 8×60k, MPS = 41.4 kb). D) Comparison of different amplification methods (WGAM, WGAN and WGAS).
Figure 2.
Genome wide aCGH profiles of gDNA and associated amplified single cell DNA from healthy controls, REH and OE19.
Peaks upstream the baseline (red area) denote a gain, peaks downstream the baseline (green area) indicate a loss of chromosomal material. A) Healthy control black line = gDNA, red line = female single cell vs. male single cell, blue line = male vs. female single cell. B) REH black line = gDNA, red = single cell. C) OE19 black line = gDNA, red = single cell.
Figure 3.
Effect of custom aberration filter.
Visualization of chromosomal alterations on chromosome 1 in Genomic Workbench using aberration detection algorithm ADM-2. Blue filled areas denote a gain (right) or loss (left) of chromosomal material. A) Analysis of gDNA, WGAM-, WGAN- and WGAS-single cell amplified DNA without aberration filter. B) Analysis of the same samples with aberration filters (≥3 consecutive oligonucleotides, ≥ log2ratio ±0.25).
Figure 4.
Visualization of smallest detected alterations.
Smallest alteration (blue area) in gDNA and single cell amplified DNA generated with three WGA methods (WGAM, WGAN & WGAS from left to right). A) 56 kb deletion of material from chromosome 7p14.1 in cell line REH. Note that the deletion could not be retrieved in the WGAS amplified single cell. B) 115 kb gain of material from chromosome 17q12 in the cell line OE19.
Figure 5.
aCGH experiments with CellSearch® identified and MoFlo™ XDP sorted WGAM single cells from cell line OE19.
A) Screenshot from CellSearch® System from an EpCAM captured CK+/DAPI+/CD45− tumor cell: overlay of DAPI+/CK-PE+ (upper left), CK-PE+ (upper right), DAPI+ (lower left)/CD45-APC− (lower right). B) & C) Dotplot from MoFlo™ XDP, CK+ and CD45+ cells (B) and DAPI+ cells (C). D) Overview of the genomewide profiles from two cells identified with CellSearch®, isolated with MoFlo™ XDP and amplified by WGAM. Black = gDNA, blue = WGAM single cell #1 and red = WGAM single cell #2.
Table 1.
Overview and comparison of non-linear PCR based whole genome amplification methods.