Representative plots of a sample with evidence and without evidence of somatic instability. The left part represents a stable RFC1 repeat expansion and the right part represents an unstable CNBP repeat expansion. (A) The number of assembled maps at the region of interest in the local-GA data might indicate somatic instability. In this case, the stable repeat had two consensus maps while the unstable repeat had six consensus maps. (B) A gradient of label distance in the molecule pile-up might also indicate mosaicism. The stable repeat had no gradient, while the unstable repeat presented a gradient of label distances based on the large variability in the distance between the red label and black label in each molecule. This variability results in the gradient or “stairway” pattern. (C) The molecule distance script output plots show the repeat expansion size that is detected in each molecule by determining the distance between two specific labels of interest. This bar plot represents the distance between the labels of interest in each molecule ordered from smallest to largest. Molecule distance bar plots with a steep gradient or a stairway distribution of label distances would suggest somatic instability. The stable repeat had no stairway pattern, while the unstable repeat showed a stairway pattern for the expanded allele. The plot for the stable repeat visualizes the separation of the smaller allele and the larger allele around the middle of the plot (molecule number 57). The plot for the unstable repeat visualizes the same separation of the smaller allele and the larger allele (around molecule number 75). (D) The histogram plots outputted by the molecule distance script represent the separation of the two alleles based on the label distances in each molecule. The smaller alleles are indicated with blue peaks and the larger alleles are indicated with orange peaks. A “smear” instead of a real peak in the histogram for one of the alleles might indicate somatic instability. For the stable repeat, no smear was detected, while the unstable repeat presented with a “smear” for the expanded allele. This is due to large variability in molecule label distances and therefore repeat expansion size.
