Thesis title: The Impact of Climate Change on Human DNA: De Novo Genome Assembly to Enable Future Studies on Environmental Agents
Recent advances in long-read sequencing technologies, including PacBio HiFi and Oxford Nanopore Technologies, have enabled the assembly of telomere-to-telomere (T2T) genomes, offering unprecedented insights into the full structure of the human genome. The T2T human reference assembly CHM13 represents a major achievement in this field, providing a complete and highly detailed view of the human genome, including complex regions such as centromeres. Despite its value, CHM13 and other reference genomes have limitations when applied to functional experiments involving commonly used laboratory cell lines, as they may not accurately reflect the genomic features of these specific cell types. This underscores the importance of generating complete, isogenomic reference assemblies for experimentally relevant cell lines, which can serve as accurate models for functional studies.
In this study, we present a telomere-to-telomere diploid genome assembly of the RPE-1 cell line, a widely used non-cancerous laboratory model characterized by its stable karyotype. Leveraging high-coverage long-read sequencing and Hi-C data integration, we produced a high-quality assembly with chromosome-level scaffolds that span entire centromeres across all chromosomes. This assembly also incorporates haplotype-specific phasing, enabling the detailed study of genomic variations between the two haplotypes. Notably, we identified a characteristic translocation between chromosome 10 and the X chromosome (t(X;10)(Xq28;10q21.2)) in RPE-1 cells, highlighting the value of matched-reference genomes for accurately capturing unique genomic features.
The use of this matched-reference genome demonstrates significant improvements in high-precision analyses, particularly for multi-omics studies. By aligning data to a reference genome specifically tailored to the RPE-1 cell line, we achieve superior resolution in mapping and quantifying genomic features, especially in highly variable regions such as centromeres. These regions, which are known to exhibit substantial sequence divergence, often remain poorly resolved when using generic references, thereby limiting the accuracy of genome-wide analyses. The RPE-1 genome overcomes these limitations, enabling precise identification of structural variations, sequence compositions, and other critical features within centromeric regions.
This work emphasizes the broader implications of matched-reference genomes for functional genomics. Compared to non-matched references, the RPE-1 genome allows for more accurate interpretation of sequencing data, improved mapping quality, and enhanced detection of haplotype-specific variations. These advantages are particularly relevant for studies requiring high-confidence genomic and structural insights, including investigations of chromosomal stability, centromere organization, and genome editing applications. The availability of a complete and validated RPE-1 genome assembly thus provides a robust model for advancing multi-omics research, facilitating precise genomic manipulation, and exploring complex genomic regions with unparalleled accuracy.
By establishing a comprehensive reference genome for RPE-1, this study paves the way for broader efforts to assemble T2T genomes for other experimentally relevant cell lines. Such initiatives hold the potential to transform functional genomics by enabling high-precision analyses tailored to specific cellular contexts. Ultimately, the availability of matched-reference genomes will drive progress across a range of scientific disciplines, offering powerful tools for understanding genomic complexity, conducting targeted experiments, and expanding the scope of multi-omics research.