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<p>Comparative analyses of newly available human genome assemblies highlight extensive variation&#xa0;that peaks at centromeres. Reliance on a single&#xa0;generic reference genome can thus hinder whole-genome analysis of sequencing data derived from laboratory cell lines and limit their accurate genomic manipulation. Here, we demonstrate that using an “isogenomic” diploid reference genome&#xa0; – specific for the experimental cell line – substantially improves the accuracy of genomic, epigenomic, transcriptomic analyses and genome editing compared to a non-matched reference. Using our recently generated reference genome of the widely used diploid human cell line RPE-1, we uncover haplotype-specific genetic and epigenetic divergence across all centromeres. Mapping quality of RPE-1 data – DNA- and RNA-seq reads, improves both genome-wide and at highly divergent loci when using the matched&#xa0;RPE1v1.1 reference, resolving haplotype-specific enrichment. For genome engineering experiments, centromeric CRISPR guide RNA efficiency and chromosome specificity are best achieved using the RPE-1 reference. Leveraging high-confidence CUT&amp;RUN read mapping using the matched reference, we define the site of the human kinetochore and identify a wide variation in the position, size and structural organization between haplotypes and chromosomes. This work establishes matched-reference genomics as a powerful framework for high-precision cell biology, calling for the systematic assembly of experimentally relevant cell line genomes.</p>

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Cell line-matched reference enables high-precision functional genomics

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2025年11月20日

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  • Luca Corda,
  • Emilia Volpe,
  • Hamza Dallali,
  • Elena Di Tommaso,
  • Alessio Colantoni,
  • Andrea Guarracino,
  • Sai Swaroop Chittoor,
  • Mattia Capulli,
  • Evelyne Tassone,
  • Simona Giunta

摘要

Comparative analyses of newly available human genome assemblies highlight extensive variation that peaks at centromeres. Reliance on a single generic reference genome can thus hinder whole-genome analysis of sequencing data derived from laboratory cell lines and limit their accurate genomic manipulation. Here, we demonstrate that using an “isogenomic” diploid reference genome  – specific for the experimental cell line – substantially improves the accuracy of genomic, epigenomic, transcriptomic analyses and genome editing compared to a non-matched reference. Using our recently generated reference genome of the widely used diploid human cell line RPE-1, we uncover haplotype-specific genetic and epigenetic divergence across all centromeres. Mapping quality of RPE-1 data – DNA- and RNA-seq reads, improves both genome-wide and at highly divergent loci when using the matched RPE1v1.1 reference, resolving haplotype-specific enrichment. For genome engineering experiments, centromeric CRISPR guide RNA efficiency and chromosome specificity are best achieved using the RPE-1 reference. Leveraging high-confidence CUT&RUN read mapping using the matched reference, we define the site of the human kinetochore and identify a wide variation in the position, size and structural organization between haplotypes and chromosomes. This work establishes matched-reference genomics as a powerful framework for high-precision cell biology, calling for the systematic assembly of experimentally relevant cell line genomes.