Targeted nanopore long read sequencing for efficient validation of the transgenic loci in mice

Transgenic mouse strains are a critical tool for studying an array of disease and genetic mechanisms. Current methods for validating the integrity of transgenic alleles relies on amplification-based techniques, which are time-consuming and labor-intensive. We have developed a CRISPR/Cas9 amplification-free targeted workflow using the Oxford Nanopore sequencing platform and optimized for a wide-range of tissue types, including soft/fibrous tissues and cryopreserved samples. We have applied the protocol to low input material which allows for transgene validation from a single mouse ear notch. This work has provided a rapid and cost-effective workflow to decipher the genomic structures and environment of the transgenic alleles.