Barcoded Sleeping Beauty Transposons as Mobile Reporters of Chromatin Architecture.

Transposon integration is influenced by genomic context, but the contribution of three-dimensional genome organization to transposon mobility remains poorly understood. Here, we developed T7-Mediated Unique-barcode Recovery sequencing (TMUR-seq), combining T7-based junction enrichment with uniquely barcoded Sleeping Beauty (SB) transposons to track secondary transposition events and their relationships to individual donor sites. Using this approach, we characterized genome-wide secondary SB transposition in human cells and analyzed the effects of genomic distance, donor-specific directionality, and chromatin organization on target selection. Secondary transposition was strongly dependent on linear genomic distance but showed pronounced donor-specific directional asymmetries, including recurrent mirror-image patterns at neighboring donor sites. Secondary integratiotes also showed enrichment for Hi-C contacts with their donor loci within approximately 50 kb after accounting for genomic distance, consistent with a contribution of local three-dimensional chromatin organization to target selection. An extended chromosome 2 donor cluster further illustrated how donor distribution, chromatin state, domain organization, and local three-dimensional connectivity can coincide with highly asymmetric integration patterns and regions of pronounced integration depletion. Together, these findings indicate that secondary SB transposition is not entirely random and is shaped by the combined effects of genomic distance and local genomic context. TMUR-seq provides a framework for tracking donor-recipient relationships and investigating the genomic determinants of transposon mobility.