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Article Dans Une Revue PLoS Genetics Année : 2022

Enrichment of centromeric DNA from human cells

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

Résumé

Centromeres are key elements for chromosome segregation. Canonical centromeres are built over long-stretches of tandem repetitive arrays. Despite being quite abundant compared to other loci, centromere sequences overall still represent only 2 to 5% of the human genome, therefore studying their genetic and epigenetic features is a major challenge. Furthermore, sequencing of centromeric regions requires high coverage to fully analyze length and sequence variations, and this can be extremely costly. To bypass these issues, we have developed a technique, named CenRICH, to enrich for centromeric DNA from human cells based on selective restriction digestion and size fractionation. Combining restriction enzymes cutting at high frequency throughout the genome, except within most human centromeres, with size-selection of fragments >20 kb, resulted in over 25-fold enrichment in centromeric DNA. High-throughput sequencing revealed that up to 60% of the DNA in the enriched samples is made of centromeric repeats. We show that this method can be used in combination with long-read sequencing to investigate the DNA methylation status of certain centromeres and, with a specific enzyme combination, also of their surrounding regions (mainly HSATII). Finally, we show that CenRICH facilitates single-molecule analysis of replicating centromeric fibers by DNA combing. This approach has great potential for making sequencing of centromeric DNA more affordable and efficient and for single DNA molecule studies.
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Dates et versions

hal-03874734 , version 1 (28-11-2022)

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Riccardo Gamba, Giulia Mazzucco, Therese Wilhelm, Leonid Velikovsky, Catalina Salinas-Luypaert, et al.. Enrichment of centromeric DNA from human cells. PLoS Genetics, 2022, 18, ⟨10.1371/journal.pgen.1010306⟩. ⟨hal-03874734⟩
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