The fate of artificial transgenes in Acanthamoeba castellanii

In this study, artificial transformation experiments were performed to investigate how the A. castellanii genome responds to foreign DNA presented in both circular and linear plasmid form. Nanopore sequencing was used as a high throughput method to screen for transgene DNA in the resulting transformant cultures, and candidate transgene integrations were identified. Molecular biology experiments were performed to validate the sequence data and provide additional context on the fate of transgenes. A method was devised to estimate the rate of read chimerism in nanopore sequencing runs and accurately account for the effects of read chimerism in identifying putative transgene integrations. Based on the experimental data in hand, a potential mechanism for transgene maintenance in A. castellanii is proposed, one in which incoming foreign DNA is tandemly duplicated and telomeres are added to the ends. This nascent linear molecule is maintained as a transgene-bearing minichromosome, while also allowing for chromosomal integration.