Unbiased Long-Read Whole-Genome Sequencing Enables High-Resolution Mapping of Transgene Concatenation and Off-target Genomic Disruption in a Mouse Model

Abstract Transgenic mouse models are indispensable for dissecting disease mechanisms; yet, their interpretability is frequently compromised by cryptic genomic alterations introduced during transgenesis. Thus, robust quality control strategies are needed to elucidate integration architecture and evaluate model performance when such unintended events occur. Here, we applied unbiased whole-genome long-read sequencing using the PacBio Revio to investigate a mouse model exhibiting unexpected transgene silencing, originally designed to recapitulate autosomal-dominant hereditary macular dystrophy driven by upregulation of a ZZEF1-ALOX15 fusion gene. Long-read sequencing analysis revealed a ≥29-kb head-to-tail concatemer containing more than three copies of the transgene vector. Reconstruction of transgene-genome junctions revealed off-target integration of the concatemer into the calcium-sensing receptor gene (Casr), along with exogenous E. coli DNA, that together defined final transgene architecture. 5-methylcytosine profiling identified hypermethylation of the transgene promoter and additional phenotyping indicated disruption of endogenous Casr function resulting from the rearrangement. Our workflow enabled direct detection of transgene concatenation and off-target mapping. These findings establish long-read sequencing as a powerful and scalable quality control standard for genetically engineered animal models, uniquely capable of uncovering hidden genomic complexity, resolving aberrant phenotypes, and enhancing the reliability of in vivo disease modelling. Competing Interest Statement The authors have declared no competing interest.