Background <p>The soil amoeba <i>Acanthamoeba castellanii</i> is an emerging model organism with which to study a wide range of biomedical, microbiological, and evolutionary phenomena. While transformation systems were established for this organism more than two decades ago, the fate of artificial transgenes has not been well characterized. In this study, artificial transformation experiments were performed to investigate how the <i>A. castellanii</i> genome responds to foreign DNA presented in both circular and linear plasmid form.</p> Results <p>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 <i>A. castellanii</i> is proposed, one in which incoming foreign DNA is tandemly duplicated and telomeres are added to the ends.</p> Conclusions <p>Our results suggest that transformation of <i>A. castellanii</i> with foreign DNA leads to linear molecules that are maintained as telomere-containing, transgene-bearing minichromosomes, which may facilitate chromosomal integration. This process may allow lateral gene transfer by expanding the window of opportunity for exogenous DNA to be taken up and integrated into the <i>A. castellanii</i> genome. Similar mechanisms exist in other eukaryote groups, suggesting this may be a widespread feature of eukaryote genome biology.</p>