Enrichment of centromeric DNA from human cells

Gamba, Riccardo; Mazzucco, Giulia; Wilhelm, Thérèse; Velikovsky, Leonid; Salinas-Luypaert, Catalina; Chardon, Florian; Picotto, Julien; Bohec, Mylène; Baulande, Sylvain; Doksani, Ylli; Fachinetti, Daniele

doi:10.1371/journal.pgen.1010306

Cited by 3 publications

(3 citation statements)

References 54 publications

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“…Essentially, RPE-1 reads from centromeric chromatin map differently depending on the reference genome used without necessarily reflecting a bona fide biological change. This is noteworthy considering the abundant literature showing epigenetic datasets calling CENP-A localization 30,31,[65][66][67][68][69] , and other centromeric proteins 32,70,71 , on reference genomes. Historically, reference genomes did not contain linear annotation of centromeres.…”

Section: Discussionmentioning

confidence: 86%

“…We show that RPE-1 reads from centromeric chromatin map differently depending on the reference genome used without necessarily reflecting a bona fide biological change. This is noteworthy considering the abundant literature showing epigenetic datasets calling CENP-A localization 42,[60][61][62][65][66][67] , and other centromeric proteins 43,68,69 , previously mapped on reference genomes. Isogenomic mapping using RPE-1 improves all alignments parameters tested, using whole-genome or HDR and either long or short reads compared to the latest references, CHM13 and HG002 maternal and paternal haplotypes 1,2,37 and HG38.…”

Section: Discussionmentioning

confidence: 88%

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The complete diploid reference genome of RPE-1 identifies human phased epigenetic landscapes

Volpe,

Corda,

Di Tommaso

et al. 2023

Preprint

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SUMMARYComparative analysis of recent human genome assemblies highlights profound sequence divergence that peaks within polymorphic loci such as centromeres. This raises the question about the adequacy of relying on human reference genomes to accurately analyze sequencing data derived from experimental cell lines. Here we propose a novel approach, referred to as ‘isogenomic reference’, that leverages a matched reference genome to perform multi-omics analyses. We have generated a new diploid genome assembly for the human retinal epithelial cells (RPE-1), a widely used non-cancer laboratory cell line with a stable diploid karyotype, that presents phased haplotypes and chromosome-level scaffolds that completely span centromeres. Using this assembly, we have characterized haplotype-resolved genomic variation unique to RPE-1, including a stable marker chromosome X with a 73.18 Mb segmental duplication of chromosome 10 translocated onto the microdeleted telomere t(Xq;10q), specific to this cell line. Comparative analyses revealed sequence polymorphism within centromeric regions, including unexpected genetic and epigenetic diversity among haplotypes for all chromosomes. Using our assembly as reference, we re-analyzed both our own and publicly available sequencing, methylation and epigenetic data generated in RPE-1 which had previously been analyzed with non-matched and non-diploid reference genomes. Our results show that the isogenomic reference improves alignments with an increased mapping quality up to 85% while halving mismatches, resulting in significant changes in peaks calling related to centromeres. Our work represents a proof-of-concept, showcasing the use of matched reference genomes for multiomics analyses and, at scale, serves as the foundation for a call to comprehensively assemble experimentally relevant cell lines for a widespread application of isogenomic reference genomes.HighlightsTo improve the standard practice of aligning sequencing reads originating from laboratory human diploid cell lines against a reference, we present a novel approach that leverages the matched, or ‘isogenomic’ reference genome for multi-omics analyses.To achieve this, we have generated a phased human diploid genome assembly of one of the most widely used non-cancer cell lines (RPE-1) with a stable diploid karyotype. We used this genome as a matched reference to analyze sequencing data from RPE-1.We demonstrate how the isogenomic reference genome improves read alignment quality, peak calling accuracy and faithful assignment of sequencing data to each haplotype, uncovering for the first time inter-haplotype variation within the same diploid genome at polymorphic loci such as centromeres.The RPE-1 genome represents a new telomere-to-telomere human diploid reference for the scientific community that will advance genetic and epigenetic research across fields using this cell line.

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Section: Discussionmentioning

confidence: 86%

Section: Discussionmentioning

confidence: 88%

The complete diploid reference genome of RPE-1 identifies human phased epigenetic landscapes

Volpe,

Corda,

Di Tommaso

et al. 2023

Preprint

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“…However, with the advent of long read sequencing where single reads can span large repe99ve regions, complete telomere to telomere assembly has become possible (Nurk et al, 2022). To increase the chance of assembling long repe99ve centromeres, targeted sequencing can be carried out (Gamba et al, 2022).…”

Section: Genome Annotationmentioning

confidence: 99%

A Workflow for Genome Assembly and Annotation of Hāpuku (Polyprion oxygeneios)

Roberts

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<p><strong>Recent advancements in long read DNA sequencing methods have made whole genome sequencing (WGS) and assembly significantly easier for researchers with limited access to resources. In particular, the Oxford Nanopore Technologies (ONT) long read sequencing platform is unique in that both sequencing runs and the instrument itself are relatively inexpensive. Low cost sequencing capabilities mean that non specialists will come to routinely incorporate genome assembly and annotation into a broader range of projects and applications, necessitating the development of better computational workflows. In this thesis, a workflow for genome assembly and annotation using nanopore long reads is presented for the assembly of Hāpuku, a species being developed for aquaculture at NIWAs Northland Aquaculture Centre in New Zealand. The workflow utilises the high accuracy of the latest nanopore base calling models and incorporates the recent duplex and modified base calling models. A tutorial covering installation and details of the commands used is included, details often omitted from published papers. To demonstrate the workflow, nanopore long read sequencing was carried out on a single Hāpuku individual. A total of 158 Gb of reads were produced across two runs, using a GridION sequencer and a PromethION2 sequencer. The final polished assembly, using the Flye assembler and Medaka polisher, had a length of 744 Mb for a coverage of ~212. Genome quality metrics were high with an N50 of 20.7 Mb, an L50 of 15 and a BUSCO completeness of 98.9% using the most recent Actinopterygii gene database. Extremely low heterozygosity of 0.089 was observed in the genome, although the reliability of the long read SNP calling used needs further investigation. Annotation was carried out on the assembly to characterise repetitive regions, tRNAs, rRNAs and protein-coding genes in Hāpuku. A high repeat percentage of 47.8% was found, with the most common repeat type, DNA transposons, making up 17.5% of the genome. Also found were 139 rRNAs, 2,223 standard tRNAs as well as 31,872 putative genes (pre-filtering). A 16,509 bp mitochondrial genome was also obtained and was almost a perfect match to a previous mitochondrial genome assembly for Hāpuku. Distributions of modified bases 5-methylcytosine and 5-hydroxymethylcytosine frequency in repetitive and unique regions are presented, revealing increased methylation in repetitive regions but constant hydroxymethylation between the regions. Finally, synteny analysis of the genome against another Perciformes species suggests all chromosomes have been obtained in the assembly, with many of these dominated by a single scaffold. The workflow generated a high quality reference genome assembly for Hāpuku and permitted a range of annotations, demonstrating that functionally useful whole genome resources can be generated from DNA reads from a single low cost sequencing technology. The assembly contiguity and completeness were high even without additional contiguity information, indicating that long-read sequencing technology is a promising approach for de novo assembly projects. Finally, the genomic resources produced for Hāpuku from this workflow provide important first steps towards a whole-genome informed selective breeding program.</strong></p>

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Genomic technologies to improve variation identification in undiagnosed diseases

Shieh¹

2023

Pediatrics & Neonatology

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Enrichment of centromeric DNA from human cells

Cited by 3 publications

References 54 publications

The complete diploid reference genome of RPE-1 identifies human phased epigenetic landscapes

The complete diploid reference genome of RPE-1 identifies human phased epigenetic landscapes

A Workflow for Genome Assembly and Annotation of Hāpuku (Polyprion oxygeneios)

Genomic technologies to improve variation identification in undiagnosed diseases

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