DOM: Dual Optical Mapping Integrating Sequence‐Specific Barcodes and A/T Density Profiles

ABSTRACT Accurate single‐molecule optical genome mapping remains challenging due to the limited information content of barcode‐only or profile‐only strategies. Here, we present Dual Optical Mapping (DOM), which integrates sequence‐specific barcode markers with AT frequency‐dependent intensity profiles on the same DNA molecules to enhance mapping accuracy. Conventional optical mapping approaches rely either on sequence‐specific barcodes generated by restriction endonucleases, nicking enzymes, or methyltransferases, or on dense profile mapping using DNA‐binding molecules. By combining these complementary sources of information within a unified dual‐channel framework, DOM increases positional specificity and alignment confidence. As a model system, we applied DOM to the E. coli genome (4.6 Mbp), where the combined dual‐channel scoring metric (cc_rg2) ranked 172 of 182 molecules (95%) at the correct genomic locus as the top match, while 179 of 182 molecules (98%) could be validated after quality‐control review. We further extended DOM to the human genome through genome‐wide cross‐correlation scanning coupled with placement score ranking. This quantitative framework enables objective evaluation of alignment distinctiveness across the entire genome, demonstrating scalability to large and complex genomic contexts. These results establish DOM as a scalable dual‐channel framework for high‐accuracy single‐molecule genome mapping across both bacterial and human genomes.