Abstract
Recent technical advances highlight that to understand mammalian development and human disease we need to consider transcriptional and epigenetic cell-to-cell differences within cell populations. This is particularly important in key areas of biomedicine like stem cell differentiation and intratumor heterogeneity. The recently developed nucleosome occupancy and methylome (NOMe) assay facilitates the simultaneous study of DNA methylation and nucleosome positioning on the same DNA strand. NOMe-treated DNA can be sequenced by sanger (NOMe-PCR) or high throughput approaches (NOMe-seq). NOMe-PCR provides information for a single locus at the single molecule while NOMe-seq delivers genome-wide data that is usually interrogated to obtain population-averaged measures. Here, we have developed a bioinformatic tool that allow us to easily obtain locus-specific information at the single molecule using genome-wide NOMe-seq datasets obtained from bulk populations. We have used NOMePlot to study mouse embryonic stem cells and found that polycomb-repressed bivalent gene promoters coexist in two different epigenetic states, as defined by the nucleosome binding pattern detected around their transcriptional start site.
Highlights
Eukaryotic DNA is wrapped around histones octamers to form nucleosomes and chromatin[1]
There is availability of tools to analyse average nucleosome positioning using nucleosome occupancy and methylome (NOMe)-seq[13] and other technics[24], but there is not a software that facilitates the analysis at the single molecule of BS-seq and NOMe-seq datasets generated by high throughput sequencing
We report the development of NOMePlot as an alternative method to carry out single molecule analysis of nucleosome binding and DNA methylation
Summary
Eukaryotic DNA is wrapped around histones octamers to form nucleosomes and chromatin[1]. The initial version of these three methods require thousands of cells as input material and their analysis includes a step to average sequencing reads – averaging out cell-cell differences within the analysed population These protocols have been adapted to allow single cell analysis of chromatin accessibility and nucleosome binding genome-wide[8,9,10,11]. NOMe-treated DNA can be sequenced upon PCR amplification using traditional sanger (NOMe-PCR, to analyse candidate regions)[12] or after library construction using high throughput sequencing (NOMe-seq, genome-wide analysis)[13] In both cases the resulting sequences will retain information of endogenous DNA methylation and nucleosome binding for the same molecule of DNA. As a use example we analysed the epigenome of mouse embryonic stem cells (mESCs)
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