In a new study published in Nature Communications titled, “The reference genome of the human diploid cell line RPE-1,” researchers from University of Rome La Sapienza have produced the first reference-quality genome assembly of an experimentally relevant human cell line, retinal pigment epithelial line RPE-1.
Recent advances in sequencing technology have enabled telomere-to-telomere (T2T) genomes, such as the current CHM13 human reference. Yet these assemblies do not reflect the precise genomes of the cell lines used in experimental practice. This mismatch has constrained the accuracy of experimental studies, particularly in highly variable and structurally complex regions such as centromeres.
Led by Simona Giunta, PhD, associate professor of human genomics at University of Rome La Sapienza, the research team assembled RPE1v1.1, a near-complete diploid genome of the hTERT RPE-1 cell line. By integrating high-coverage long-read sequencing with Hi-C chromosome conformation capture, the researchers generated and validated a de novo assembly at reference quality. Notably, assembly resolves the centromeres of RPE-1 chromosomes, offering a haplotype-resolved human reference with maternal and paternal genomes represented.
“Cell lines are the workhorses of modern biology, yet their genome assemblies have lagged behind in accuracy and completeness compared to the human reference,” said Giunta. “By producing a reference-quality assembly of RPE-1 with resolved centromeres, we provide the community with a tool that will drastically improve the precision of genomic and epigenomic studies in this system.”
The RPE-1 line, derived from retinal pigment epithelial cells, has long served as a model system due to its diploid karyotype and stability under culture conditions. Scientists studying RPE-1 have historically relied on the human reference genome, which does not fully capture the structural variation or unique genomic features of this experimental model. This mismatch has limited the accuracy of downstream multi-omics analysis, particularly in repetitive and polymorphic regions.
The new assembly reaches reference quality, with the advance in resolving the centromeres of RPE-1 chromosomes, regions that remain fragmented in the current human reference genome. This achievement enables unprecedented mapping of regulatory and structural features across the genome, including kinetochore assembly, the essential process that governs faithful chromosome segregation.
The work demonstrates that aligning experimental data directly to the RPE-1 reference, an approach named isogenomic mapping, reduces alignment errors, improves haplotype resolution, and enables accurate analysis of structural and regulatory variation. This strategy highlights the importance of matched references for interpreting experimental data and lays the groundwork for a larger initiative to assemble high-quality genomes for other commonly used human cell lines.
The study opens a broader vision of building a Human Pangenome of Experimental Cell Lines. This resource would systematically integrate reference-quality assemblies of widely used laboratory models into graph-based frameworks, ensuring that functional genomic studies are grounded in the genomic architecture of the systems in which they are performed. Comparing the RPE-1 reference genome to the human pangenome shows that the cell line largely retain human like qualities, with the cell line closely associated to the population of origin.