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After two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no single chromosome has been finished end to end, and hundreds of unresolved gaps persist 1,2 . Here we present a human genome assembly that surpasses the continuity of GRCh38 2 , along with a gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our efforts on the human X chromosome 3 , we reconstructed the centromeric satellite DNA array (approximately 3.1 Mb) and closed the 29 remaining gaps in the current reference, including new sequences from the human pseudoautosomal regions and from cancer-testis ampliconic gene families (CT-X and GAGE). These sequences will be integrated into future human reference genome releases. In addition, the complete chromosome X, combined with the ultra-long nanopore data, allowed us to map methylation patterns across complex tandem repeats and satellite arrays. Our results demonstrate that finishing the entire human genome is now within reach, and the data presented here will facilitate ongoing efforts to complete the other human chromosomes.

Eukaryotic genomes are folded into three-dimensional structures, such as self-associating topological domains, the borders of which are enriched in cohesin and CCCTC-binding factor (CTCF) required for long-range interactions. How local chromatin interactions govern higher-order folding of chromatin fibres and the function of cohesin in this process remain poorly understood. Here we perform genome-wide chromatin conformation capture (Hi-C) analysis to explore the high-resolution organization of the Schizosaccharomyces pombe genome, which despite its small size exhibits fundamental features found in other eukaryotes. Our analyses of wild-type and mutant strains reveal key elements of chromosome architecture and genome organization. On chromosome arms, small regions of chromatin locally interact to form 'globules'. This feature requires a function of cohesin distinct from its role in sister chromatid cohesion. Cohesin is enriched at globule boundaries and its loss causes disruption of local globule structures and global chromosome territories. By contrast, heterochromatin, which loads cohesin at specific sites including pericentromeric and subtelomeric domains, is dispensable for globule formation but nevertheless affects genome organization. We show that heterochromatin mediates chromatin fibre compaction at centromeres and promotes prominent inter-arm interactions within centromere-proximal regions, providing structural constraints crucial for proper genome organization. Loss of heterochromatin relaxes constraints on chromosomes, causing an increase in intra- and inter-chromosomal interactions. Together, our analyses uncover fundamental genome folding principles that drive higher-order chromosome organization crucial for coordinating nuclear functions.