Institut Curie, PSL Research University, CNRS, UMR 144, Paris, France.
IFOM, the FIRC Institute of Molecular Oncology, Milan, Italy.
PLoS Genet. 2022 Jul 19;18(7):e1010306. doi: 10.1371/journal.pgen.1010306. eCollection 2022 Jul.
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.
着丝粒是染色体分离的关键元件。经典的着丝粒是由串联重复序列组成的。尽管与其他基因座相比，着丝粒序列非常丰富，但它们仅占人类基因组的 2%到 5%左右，因此研究它们的遗传和表观遗传特征是一个主要挑战。此外，对着丝粒区域进行测序需要高覆盖率才能全面分析长度和序列变异，这可能非常昂贵。为了克服这些问题，我们开发了一种名为 CenRICH 的技术，该技术基于选择性限制性消化和大小分级，从人类细胞中富集着丝粒 DNA。结合在基因组中高频切割的限制酶（除了大多数人类着丝粒之外），以及对 >20kb 片段进行大小选择，可使着丝粒 DNA 富集超过 25 倍。高通量测序显示，富集样本中高达 60%的 DNA 由着丝粒重复序列组成。我们表明，该方法可与长读测序结合使用，以研究某些着丝粒的 DNA 甲基化状态，并且使用特定的酶组合，还可以研究其周围区域（主要是 HSATII）。最后，我们表明 CenRICH 可通过 DNA 梳理促进复制着丝粒纤维的单分子分析。这种方法在降低测序成本和提高效率方面具有很大的潜力，也可用于单个 DNA 分子的研究。
