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Locating and characterizing a transgene integration site by nanopore sequencing

Publication

Date: 5th March 2019 | Source: G3: Genes, Genomes, Genetics

Authors: Peter K. Nicholls, Daniel W. Bellott, Ting-Jan Cho, Tatyana Pyntikova, David C. Page.

Transgenes are foreign DNA experimentally introduced into a genome; often they integrate at random positions in multiple copies and can disrupt genes. Locating the site of DNA incorporation is difficult so the location of most transgenes is unknown. Benefiting from the long reads produced by nanopore sequencing, Nicholls et al. performed whole-genome sequencing on the MinION to successfully identify the integration site of transgene EGFP:Oct4, a widely used reporter gene, in the mouse genome.

The introduction of foreign DNA into cells and organisms has facilitated much of modern biological research, and it promises to become equally important in clinical practice. Locating sites of foreign DNA incorporation in mammalian genomes has proven burdensome, so the genomic location of most transgenes remains unknown. To address this challenge, we applied nanopore sequencing in search of the site of integration of Tg(Pou5f1-EGFP)2Mnm (also known as Oct4:EGFP), a widely used fluorescent reporter in mouse germ line research. Using this nanopore-based approach, we identified the site of Oct4:EGFP transgene integration near the telomere of Chromosome 9. This methodology simultaneously yielded an estimate of transgene copy number, provided direct evidence of transgene inversions, revealed contaminating E. coli genomic DNA within the transgene array, validated the integrity of neighboring genes, and enabled definitive genotyping. We suggest that such an approach provides a rapid, cost-effective method for identifying and analyzing transgene integration sites.

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