We have assembled the diploid genome of the human diploid laboratory cell line RPE-1. This telomere-to-telomere (T2T) fully phased de novo assembly can be used as matched reference genome 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. This proof-of-concept calls 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. Here we describe the work to assemble RPE1v1.1 as one of only a handful of chromosome-level diploid human reference genome, and the first of a laboratory cell line, also sequenced, curated and assembled completely in Italy/Europe, first reported on bioRxiv in 2023 (https://pubmed.ncbi.nlm.nih.gov/38168337/), released in 2024 and finally published in 2025 (https://www.nature.com/articles/s41467-025-62428-z). For the related work on how to use this assembly genome as isogenomic reference for experiments done in RPE-1 cell lines, see https://github.com/GiuntaLab/RPE1/commits/main/RPE1v1.1_FunctionalGenomics (Corda et al., 2025).
This is the latest release, incorporating manual curation of gaps present in RPE1v1.0 and the recovery of previously unassigned telomeric sequences.
Assembly, validation and comparison with other genomes are described in the following publication:
Volpe, Colantoni et al. The reference genome of the human diploid cell line RPE-1. Nature Communications, 2025.
Documentation, scripts, files and figures relative to the main figures are available in the RPE1v1.1/ folder.
Supplemental information is available in the Zenodo Repository:
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:
The complete diploid reference genome of RPE-1 identifies human phased epigenetic landscapes
Volpe et al., preprint
Documentation, scripts, files and figures relative to the main figures are available in the RPE1v1.0_biorXiv/ folder.
We generated the near complete phased genome assembly of one of the most widely used non-cancer cell lines (RPE-1) with a stable diploid karyotype. We produced state-of-the-art sequencing data using third generation sequencing: Pacific Biosciences (PacBio) high-fidelity (HiFi) and Oxford Nanopore Technologies (ONT). ONT long and ultra-long (UL, >100 kb) reads were exclusively generated using R10.14 (pore chemistry V14 yielding 99.9% base accuracy). We aimed at above-average reads depth coverage with 46x sequence coverage of HiFi reads, 95x of ONT long, and 30x ONT-UL reads. During DNA extraction, we assessed the length of high molecular weight DNA from hTERT RPE-1 cells using Femto Pulse with a yield of native DNA size distribution around 116 kb (main peak) and up to 1 Mb in length (smaller peaks) with centrifugation below 1000 RPM. For haplotype phasing, we generated 30x Hi-C reads (Arima Genomics).
The HiFi, ONT, and Hi-C sequencing data used to generate the genome have been deposited in the SRA under BioProject PRJNA1193286. Accession numbers are as follows:
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PacBio HiFi raw reads:
SRR33464826, SRR33464827, SRR33464828, SRR33464829 -
ONT reads:
SRR33464817, SRR33464818, SRR33464819, SRR33464820, SRR33464821, SRR33464822, SRR33464823, SRR33464824, SRR33464830, SRR33464831 -
Hi-C Arima reads:
SRR33464825
The first draft of the genome was generated using Verkko v1.4 with HiFi, ONT and Hi-C reads. Verkko was run on the Sapienza TERASTAT2 cluster.
In absence of parental information to support Trio-binning, we obtained fully phased haplotypes for the RPE-1 genome using contact maps. We used the Vertebrate Genome Project (VGP) pipeline. Hi-C raw reads were aligned against the merged assembly composed of both RPE-1 haplotypes and unassigned reads generated from Verkko. The alignment step was performed using the short-read aligner BWA. All reads were retained, including those classified as supplementary, with low mapping quality or having multiple alignments. The final aligned file was converted into a 3D Contact Map viewable through PretextView. PretextView allows the modification of the contact map file by changing contig positions along the diagonal and finding the correct chromatin interaction path. The final diploid Hi-C contact map was based on Nadolina Brajuka RapidCuration2.0. Contigs over 300 kb were assigned to the chromosome merged to the assembly file, yet a remaining 667 contigs <100 kb could not be aligned manually due to short size. In the final contact map, each scaffold was assigned to a specific haplotype Meta Data Tag (Hap 1 and Hap 2). This latest contact map was then converted into an A Golden Path (AGP) file, which was used as input for the subsequent separation of both haplotypes and generating the two fully phased haploid FASTA files after running Curation2.0_pipe.sh.
To address gaps remaining after dual manual curation, we applied two complementary gap-closing strategies:
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Long-read alignment-based gap filling
ONT reads >100 kb were aligned to the assembly using minimap2.
Gaps were identified when reads spanned at least 40 kb on both flanking regions. These gaps were temporarily filled with ONT read sequences.
HiFi reads were then aligned to the patched genome, and final gap-filling was performed using the consensus sequence from these alignments. -
Assembly graph-based gap filling
ONT reads were aligned to the assembly graph using gfalign.
When alignments supported a single unambiguous path through a graph bubble, the corresponding sequence of unitigs was extracted and used to fill the gap.
Additionally, the p-arm telomeres of chromosomes 16 and X, and the q-arm telomere of chromosome 4, were identified among the unassigned sequences generated by Verkko and subsequently recovered through manual curation.
Multi-step pipeline for the manual curation of the RPE-1-specific structural variant identified as 46,XX,dup(10q),t(Xq;10q),del(Xq28):
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Reads alignment
The de novo diploid genome assembly RPE1v1.0 was used as a reference to map HiFi and ONT reads using minimap2. -
Visualization
Long-read alignments were visualized using IGV, revealing an increased read coverage on the long arm of chromosome 10 (10q). A read interruption was observed, with ~100 bp difference in mapping position between chromosome 10 of Hap1 and Hap2. -
Assessment of haplotypic differences in read mapping quality
Chromosome 10 Hap1 displayed only reads with mapping quality 0. In contrast, Hap2 exhibited reads with mapping qualities between 10–60 and supplementary alignments in the telomeric region of chromosome X Hap1, corresponding to the translocation breakpoint. -
Manual curation (translocation)
The sequence of the duplicated and translocated 10q arm from chromosome 10 Hap2 was appended to the telomeric region of chromosome X Hap1. This was achieved by merging the sequences into the existing FASTA file of the RPE-1 assembly. -
Read alignment verification
RPE-1 HiFi and ONT reads were re-aligned to the modified FASTA using minimap2. IGV inspection of the fusion point on chromosome X revealed a 3,603 bp microdeletion in the read alignments, suggesting sequence loss during the X–10 rearrangement. -
Manual curation (deletion)
The deleted bases were manually removed from the FASTA sequence of chromosome X. The modified genome was then aligned again to the HiFi and ONT reads for confirmation. -
Final verification
In the final RPE1v1.0 diploid genome, long reads align fully across the fusion point on chromosome X, confirming successful correction of the structural variant.
The quality of the RPE1v1.1 genome was assessed using a variety of tools:
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NucFreq
Primary alignments of HiFi reads to the RPE1 diploid genome were used to generate a NucFreq plot, which displays the frequencies of the most and second most common bases at each genomic position. TheHetDetection.Rscript was used to identify possible errors in the assembly. -
Merqury
By comparing the k-mers in the HiFi reads to the k-mers found in the assembly, we obtained a quality value (QV) of 64.1 for Hap1 and 61.8 for Hap2. Furthermore, k-mer spectra revealed a multiplicity profile consistent with a near-complete, haplotype-resolved assembly. -
CRAQ
Using HiFi read clipping information, CRAQ detected only ~2 Mb of potential assembly errors, primarily corresponding to regions with unresolved gaps and specific telomeric sequences. The AQI score exceeded 98, underscoring the quality of the assembly. -
Flagger
Based on the alignment of HiFi reads to the RPE1v1.1 assembly, Flagger identified 2.4 Mb as assembly errors and 55 Mb as collapsed regions. The genomic coordinates of these low-confidence regions are provided as BED files and can be used as reference tracks in downstream analyses. -
Compleasm
Compleasm identified 99.71% complete genes (with 3.28% duplicated) in Hap1, and 99.73% (with 0.7% duplicated) in Hap2. The missing genes were 0.21% and 0.09%, while the fragmented genes were 0.08% and 0.09% for Hap1 and Hap2, respectively. -
SecPhase
SecPhase was used to identify HiFi reads aligning to the incorrect haplotype. No read needed to be relocalized, indicating that Hi-C data were sufficient to phase the assembly into two haplotype blocks. -
breakpointR R package
Strand-seq reads from Sanders et al., generated from 80 RPE-1 cells, were aligned using BWA-MEM to the RPE1v1.1 diploid genome.breakpointRwas used to detect strand state patterns in each cell that supported the correct haplotype phasing of the assembly.
RPE1 Hap1 vs. RPE1 Hap2, CHM13 vs. RPE1 Hap1, and CHM13 vs. RPE1 Hap2 comparisons were performed using two approaches:
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SyRI
Based on the genome-to-genome alignments obtained using minimap2, SyRI was used to identify synthenic regions (SYN), inversions (INV), insertions (INS), translocations (TRANS), inverted translocations (INVTR), duplications (DUP), inverted duplications (INVDP), unaligned regions (NOTAL), highly diverged regions (HDR), copy gains (CPG) and SNPs (SNP). -
NUCmer and dnadiff
On average, RPE1 haplotypes aligned over segments of 471 kb with a sequence identity of 99.83%. 393 Mb of Hap1 did not align to Hap2, and 323 Mb of Hap2 did not align to Hap1, reflecting the different length of the two haplotypes.
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RepeatMasker and HumAS-HMMER_for_AnVIL
Used for annotation of repeats, including alpha-satellite monomers. -
StV
Used for HOR Structural Variant (StV) prediction based on HOR-monomer annotation. -
StainedGlass0.5
Generated sequence identity heatmaps.
Liftoff was used to map genes from the human transcriptome annotation of ENSEMBL 112 (GRCh38.p14 genome) to the RPE1v1.1 genome.
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:
- RPE1.0 Hap1
- RPE1.0 Hap2
- CHM13v2.0
- GRCh38.p14
- HG002v1.0 maternal
- HG002v1.0 paternal CENP-A high-confidence peaks were determined with MACS3.
Methylation profiles for 5-methylcytosine (5mc) were generated from the ONT RPE-1 POD5 (3.6 TB) using Dorado v4.2.0 basecalling model and the output processed with Modkit. Following the evaluation of reads coverage values Ncanonical and Nmod, we selected the filter (Nmod / Nvalid_cov) >60 applied to the bedMethyl output.