The diploid reference genome of a human embryonic stem cell line

Ivana Pačar; Matteo Tommaso Ungaro; Ying Chen; Hamza Dallali; Jack A Medico; Prajna Hebbar; Mark Diekhans; Elena Di Tommaso; Margarita Geleta; Patricia P. Chan; Todd M. Lowe; Jennifer Ballaco; Nivesh Jain; Francine Ackerman; Michela Mochi; Alexander G. Ioannidis; Niranjan Sawarkar; Kasandra Diaz; Kotlapati Sudhakar; Joseph E. Powell; Miten Jain; Alessandro Rosa; Gist F. Croft; Andrea Tanzer A; Erich David Jarvis; Giulio Formenti; Sofie R. Salama; Simona Giunta

doi:10.64898/2026.03.26.714432

GenomicsbioRxivMar 30, 2026

Preprint

The diploid reference genome of a human embryonic stem cell line

Ivana Pačar iD, Matteo Tommaso Ungaro iD, Ying Chen iD, Hamza Dallali iD, Jack A Medico iD, Prajna Hebbar iD, Mark Diekhans iD, Elena Di Tommaso iD, Margarita Geleta iD, Patricia P. Chan iD, Todd M. Lowe iD, Jennifer Ballaco, Nivesh Jain iD, Francine Ackerman iD, Michela Mochi iD, Alexander G. Ioannidis iD, Niranjan Sawarkar iD, Kasandra Diaz, Kotlapati Sudhakar, Joseph E. Powell iD, Miten Jain iD, Alessandro Rosa iD, Gist F. Croft iD, Andrea Tanzer A iD, Erich David Jarvis iD, , Simona Giunta iD

Abstract

Advances in DNA sequencing and assembly technologies are spurring a shift from haploid reference genomes to sample-specific diploid assemblies. Here, we generated the first telomere-to-telomere (T2T) diploid reference for the widely used human embryonic stem cell (hESC) line, H9 (WAe009-A). This haplotype-resolved assembly is highly accurate with comprehensive annotation of genes, segmental duplications, methylation, and chromatin conformation. Pangenomic and phased-locus inference point to H9s mixed ancestry with a predominant European component. H9-specific genomic features include near-perfect telomeres [~]1.65-fold longer than other T2T assemblies, consistent with telomerase activity during pluripotency; chromosome 17 inversions that can predispose offspring to neurological syndromes; and expansions of ncRNA clusters, with overall genomic stability maintained despite extensive culturing. Mapping multi-omic datasets to the genome, we demonstrate the power of this resource for allele-specific, high-precision transcriptomic, genetic, and epigenetic analyses, with far-reaching implications for human development and disease.

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