Single-Platform Nanopore Sequencing Enables Diploid Telomere-to-Telomere Genome Assembly and Haplotype-Resolved 3D Chromatin Maps

Telomere-to-telomere (T2T) genome assembly has transformed human genomics by resolving centromeres, segmental duplications, and other previously inaccessible regions. However, most diploid T2T assemblies rely on the combination of multi-platform sequencing strategies including short read genome sequencing, PacBio HiFi, Oxford Nanopore ultra-long reads, and chromatin conformation capture data (Hi-C), limiting both scalability and accessibility. Here, we present a streamlined Nanopore-only workflow for diploid human T2T assembly using three ultra-long and one Pore-C PromethION flow cell per individual. Across 23 genetically diverse individuals, we generated 360 gapless chromosomes and 446 near-complete T2T scaffolds, achieving median consensus accuracy of QV50 without Duplex sequencing or hybrid polishing. Assembly continuity, gene completeness, and structural variant detection were comparable to multi-platform Human Pangenome Reference Consortium assemblies. Pore-C data enabled chromosome-scale haplotype phasing without parental information and supported generation of haplotype-resolved chromatin contact maps. Integrated methylation and 3D genome analyses revealed allele-specific chromatin organization at imprinted loci and clear signatures of X-chromosome inactivation. Our openly accessible dataset expands public T2T resources and demonstrates that reference-grade diploid assemblies, phased methylomes, and 3D genome maps can be derived from a single sequencing platform. This approach reduces technical barriers and supports scalable population and functional genomics in the T2T era.