RPE1 genome for Functional Genomics

This folder contains documentation, scripts and files relative to the main figures of the following manuscript:

Cell line-matched reference enables high-precision functional genomics. Nature Communications, 2025 (in press)

In this new work, we demonstrate the improvements when using the diploid reference genome of the human laboratory cell line RPE-1, RPE1v1.1 (The reference genome of the human diploid cell line RPE-1. Nature Communications, 2025; https://github.com/GiuntaLab/RPE1), including the two fully phased haplotypes, for sequencing and omics data analyses.
RPE1v1.1 has been used as matched reference genomes to analyze sequencing data generated from the same cell line, an approach we refer to as isogenomic reference genome. The improvement in alignment quality using matched reads-reference enables high-precision mapping for profiling phased epigenome and methylome.

The two publications were originally shared as preprints in one manuscript: Preprint: The complete diploid reference genome of RPE-1 identifies human phased epigenetic landscapes, 2023.
Documentation, scripts, files and figures relative to the main figures are available in the https://github.com/GiuntaLab/RPE1/tree/main/RPE1v1.1_FunctionalGenomics folder.

Altogether, the improvements in haplotype-resolved mapping, guide RNA (gRNA) design for genome engineering, transcriptome analysis and alignment of sequencing reads serve as a proof-of-concept calling for a comprehensive catalog of complete assemblies for commonly used cells for a widespread application of isogenomic reference genomes to enable high-precision multi-omics analyses.

Human Diplopid Reference for high-precision Functional Genomics, Genome Engineering, Transcriptomic, Phased Epigenetic & beyond

Functional Genomics using the diploid reference genome matched to the model system

Here, we analysed sequencing, omics data and other genomic information through an isogenomic approach where the reads and the reference are matched. In this case, we used experimental data from RPE-1 cells and used the RPE1.v1.1 (see below) to map and interpret the data. Documentation, scripts, files and figures relative to the main figures are available in the https://github.com/GiuntaLab/RPE1/tree/main/RPE1v1.1 folder. Supplemental information is available in the Zenodo repository: https://zenodo.org/records/17608523 Scripts for the analyses, codes and data for each figure can be found in the respective folders (Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6), including supplementary information pertaning to each figure.

RPE1v1.1

The reference genome of the human diploid cell line RPE-1. Nature Communications, 2025

As described in a separate publication, our laboratory performed sequencing, de novo assembly and complete haplotypes phasing of the reference genome for the RPE-1 cell line. Documentation, scripts, files and figures relative to the main figures are available in the https://github.com/GiuntaLab/RPE1/tree/main/RPE1v1.1 folder. Supplemental information is available in the Zenodo repository: https://doi.org/10.5281/zenodo.15789913.

Genome availability

The RPE1v1.1 genome has been deposited in NCBI GenBank under the accession numbers JBJQNK000000000 (Hap1) and JBJQNL000000000 (Hap2), with links to BioProject accession numbers PRJNA1193286 (Hap1) and PRJNA1193302 (Hap2), both under the umbrella BioProject accession number PRJNA1195024.

To download the diploid RPE1v1.1 genome with the chromosome names used in the study, please use the following link, fill in the form, access the link that will be emailed to you and download the GIUNTAlab_RPE1V1.1.gz file: https://forms.gle/7TPggHvT1Na2G3oX7

RPE1v1.1 genome is also available in the UCSC Genome Browser:

• Hap1: https://genome.ucsc.edu/h/GCA_050656345.1
• Hap2: https://genome.ucsc.edu/h/GCA_050656315.1

Sequencing data availability

PacBio HiFi and Illumina raw reads generated and used for this study are available under the BioProject accession number PRJNA1359433.

• PacBio HiFi raw reads used for genome assembly and the NucPlot analysis: SRR33464826, SRR33464827, SRR33464828, and SRR33464829
• ONT raw reads used for genome assembly: SRR33464817, SRR33464818, SRR33464819, SRR33464820, SRR33464821, SRR33464822, SRR33464823, SRR33464824, SRR33464830, and SRR33464831
• Hi-C raw reads used for genome assembly: SRR33464825
• PacBio HiFi raw reads from RPE-1 cells expressing an inducible Cas9 and a PAN-centromeric sgRNA used for the NM and MAPQ score analysis: SRR35988140 SMRT-Link was used to obtain Circular Consensus Sequencing (CCS) reads from subreads generated using the Sequel IIe platform with SMRT Cells 8M (PacBio 101-389-001).
• Illumina raw reads from RPE-1 cells expressing an inducible Cas9 and a non-targeting sgRNA used for the NM and MAPQ score analysis: SRR35988139 HMW DNA from 1.5 x 10^6 cells was used to generate short read sequencing libraries with the Nextera XT DNA Library Preparation kit (Illumina FC-131-1024). Sequencing was performed on the Illumina Nova-Seq 6000.
• Methylation The 5-methylcytosine (5mC) profile of the RPE1v1.1 genome was calculated from the Oxford Nanopore Technologies long reads used to assemble the genome, employing Dorado v0.7.3 (https://github.com/nanoporetech/dorado) with the simplex basecalling model. The DNA methylation profile of the CHM13 genome was downloaded using the following link https://s3-us-west-2.amazonaws.com/human-pangenomics/T2T/CHM13/assemblies/annotation/regulation/chm13v2.0_CHM13_CpG_ont_guppy3.6.0_nanopolish0.13.2.bw.
• CRISPR guide RNA 76 centromere-targeting single guide RNAs (sgRNAs) designed on the CHM13 genome and validated on the hg38 genome were collected from Bosco, N. et al. 2023 (https://www.cell.com/cell/fulltext/S0092-8674(23)00326-4).
• RNA-seq RNA-seq data from three biological replicates of RPE-1 cells were obtained from Papathanasiou et al., 2023 (https://www.nature.com/articles/s41586-023-06157-7), using the following SRA accession numbers: SRR23924195, SRR23924196, SRR23924197.
• ChIP-seq Publicly available CUT&RUN raw reads generated from hTERT RPE-1 cells (NCBI BioProject: PRJNA546288) and ChIP-seq raw reads generated from CHM13hTERT cells (NCBI BioProject: PRJNA559484) were downloaded to perform the epigenetic analysis of centromeres. Specifically, we downloaded datasets with the following SRA accession numbers: SRR9201843 (CENP-A CUT&RUN, RPE-1), SRR9201837, SRR9201839 and SRR9201844 (CENP-B CUT&RUN, RPE-1), SRR9201840, SRR9201841 and SRR9201845 (CENP-C CUT&RUN, RPE-1), SRR9201837, SRR9201839 and SRR9201844 (CENP-B CUT&RUN, RPE-1), SRR10164102 (CENP-T CUT&RUN, RPE-1), SRR9201838, SRR9201842, SRR9201846 and SRR9201847 (whole-genome sequencing, RPE-1), SRR13278683 and SRR13278684 (CENP-A ChIP-seq, CHM13), SRR13278681 and SRR13278682 (Input, CHM13).

Gene annotation

Liftoff was used to map genes from the human transcriptome annotation of ENSEMBL 112 (GRCh38.p14 genome) to the RPE1v1.1 genome.

Epigenetic analysis

Centromere chromatin phased landscapes were obtained from RPE-1 CUT&RUN CENP-A dataset (GSE132193). Short reads were aligned using Bowtie to the following reference genomes: • RPE1.1 Hap1 • RPE1.1 Hap2 • CHM13v2.0 • GRCh38.p14 • HG002v1.0 maternal • HG002v1.0 paternal CENP-A, CENP-C, CENP-B and CENP-T high-confidence peaks were determined with MACS3. Our data show diversity between haplotypes and chromosomes in the size and peak enrichment in CENP-A, CENP-B and other constitutive centromere associated network (CCAN) factors, identifying through isogenomic mapping of reads from RPE-1 to its own matched reference genome the human kinetochore site at base pair resolution.

All the analyses were performed in the Giunta Lab HPC or using the University server at HPC TeraStat2.