Enrichment of centromeric DNA from human cells

PLoS Genet. 2022 Jul 19;18(7):e1010306. doi: 10.1371/journal.pgen.1010306. eCollection 2022 Jul.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Centromere* / genetics
  • Chromosome Segregation
  • DNA* / genetics
  • Humans

Substances

  • DNA

Grants and funding

D.F. receives salary support from the CNRS. D.F. has received support for this project by Labex « CellnScale», the Institut Curie, the program «Investissements d’Avenir» launched by the French Government and implemented by ANR with the references ANR-10-LABX-0038 and ANR-10-IDEX-0001-02 PSL, the Emergence grant 2018 from the city of Paris and the HFSP grant RGY0070/2019. Y.D. lab is supported by the Associazione Italiana per la Ricerca sul Cancro, AIRC, IG 19901. S.B. is supported by the grants ANR-10-EQPX-03 (Equipex) and ANR-10-INBS-09-08 (France Génomique Consortium) from the Agence Nationale de la Recherche ("Investissements d’Avenir" program), by the ITMO-Cancer Aviesan (Plan Cancer III) and by the SiRIC-Curie program (SiRIC Grant INCa-DGOS- 465 and INCa-DGOS-Inserm_12554). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.