Project Summary Approaches to complete the human genome will benefit from careful, benchmarked advances that demonstrate the capability to fully assemble and phase diploid chromosomes. The remaining unresolved regions in our high- resolution genomic maps are known to contain long tracts of repeats. The long-term objective of our research is to develop new experimental methods to complete chromosome scale assemblies to study the sequence organization, structural diversity, and disease impact of these novel sequences. In our first aim, we demonstrate the use of new approaches to generate the first telomere-to-telomere phased assembly of a human genome using effectively haploid complete hydatidiform moles (CHMs), and demonstrate the ability to scale these methods to a panel of CHMs. In our second aim we focus on validation methods of repeat assemblies to improve upon the structural and base-level accuracy of our assemblies. In our third aim we harden haplotype phasing method using high coverage ultra long data from diploid genomes to guide phased chromosome assemblies. We propose to optimize a new, cost-effective method of improving high quality reference genomes to reach complete, telomere-to-telomere genome assemblies. This research has the additional benefit that it will add new sequence to the human genome to systematically explore genetic variation of regions frequently overlooked as part of disease association and functional studies.