Enrichment of centromeric DNA from human cells

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. Author summary Centromeres are the portions of the chromosomes required for the correct partitioning of genetic material into the daughter cells. In humans, centromeric DNA is made of highly repetitive DNA sequences that hindered its precise molecular characterization until very recently with the development of pivotal technological advances. However, these approaches require the analysis of the whole human genome, while centromeres only represent less than 5%. For this reason, detailed characterization of human centromeres is still very expensive in terms of cost, timing and data analysis. We propose a method called CenRICH that allows to enrich and purify for human centromeric DNA. We prove that this method provides several advantages: 1) it drastically reduces the cost of centromere sequencing; 2) it can be used to study the epigenetic status of centromeres with high level of resolution; 3) it is suitable for single molecule visualization with advanced microscopy techniques. Therefore, CenRICH is a powerful tool to facilitate many future studies in the ever-expanding field of centromere biology, with potential application in study of genetic disease.