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Fig. 1 | Molecular Neurodegeneration

Fig. 1

From: Long-read sequencing across the C9orf72 ‘GGGGCC’ repeat expansion: implications for clinical use and genetic discovery efforts in human disease

Fig. 1

Schematic diagrams for plasmids used to test PacBio and ONT long-read sequencing technologies. To minimize biases when comparing the PacBio RS II and ONT MinION, we constructed four plasmids, including three repeat-containing plasmids and a non-repeat-containing plasmid. Each plasmid map identifies estimated plasmid size, and the location and size of the repeat within the plasmid. a The first plasmid did not contain a repeat, as a control, but instead included the EGFP gene. The EGFP plasmid was linearized at position 2969 with the AvrII restriction enzyme. b We also constructed a plasmid with 62 repeats of the spinocerebellar ataxia type 36 (SCA36) ‘GGCCTG’ repeat, which was linearized at position 2873 with AvrII. c A third plasmid contained 423 C9orf72 ‘GGGGCC’ repeats, and was linearized at position 6368 with MluI to maximize non-repeat sequence both up and downstream of the plasmid, thus avoiding bias against reads in either direction; allowing the repeat to be too close to either end could compromise sequencing or downstream analyses. d We included an additional plasmid with 774 C9orf72 ‘GGGGCC’ repeats to simulate the expansion size found in ALS- or FTD-affected expansion carriers. While 774 repeats is dramatically smaller than the expansion found in many affected carriers, it was the largest we were able to construct reliably, because these repeats are unstable in bacteria. Additionally, while we targeted the number of specified repeats for each plasmid, most colonies contained fewer than the targeted repeats because repeats are generally unstable in bacteria (Additional file 1: Figure S1). Thus, the targeted number of repeats serves as an estimated maximum number of repeats. Plasmids were visualized using AngularPlasmid

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