Gnomes are small magical creatures that mine for treasures, so you might wonder if a nanoNOMe is an even tinier being. Well actually it’s a new molecular technique, but its applications are nothing short of magical. Several methods have been developed to probe DNA methylation, transcription factors, and chromatin state simultaneously. Many are based on the principle of NOMe-seq, which uses a GpC methyltransferase to label open chromatin, allowing bisulfite sequencing to identify both these sites and DNA methylation at CpG sites. The major advantages of these approaches are the single-molecule data output, meaning that DNA methylation and nucleosome occupancy data come from the same DNA strand. A challenge of these methods has been that most sequencing technologies have short reads, meaning this valuable single-strand information is very limited in scale.
The lab of Winston Timp at Johns Hopkins University wanted to expand the read length of these combinatory methods. To do this, they used nanopore sequencing. Nanopore is unique among sequencing technologies for its very long reads (>10kb) of unamplified DNA. In previous work, Dr. Timp’s lab has shown that nanopore sequencing can accurately call DNA methylation using there software Nanopolish. This is a distinct challenge when compared to bisfulfite sequencing, since in this case the DNA is unamplified and thus the nanopore sequencer directly detects the 5mC nucleotide itself. In this new study, they combined nanopore sequencing with GpC methylation in a method they call nanopore sequencing of nucleosome occupancy and methylome (nanoNOMe). They applied this approach in four human cells lines. Here’s what they found on their mining expedition:
- Long reads allow interrogation of repetitive elements, which is difficult with other methods
- Of all repetitive elements, only Alu elements show increased methylation, and chromatin accessibility is reduced at all repetitive elements, especially in LINE and LTR regions
- nanoNOMe can perform nucleosome footprinting, which can also be integrated with CTCF motif and binding data at single-allele resolution
- Promoters show increased accessibility and decreased DNAm with higher gene expression levels, as well as subnucleosomal footprints that represent protein binding events
- Using phased X-chromosome analysis, the authors were also able to identify accessible chromatin on the active X and inaccessible, methylated chromatin at the same loci on the inactive X
- This approach could also be used to explore epigenetic effects on mutated vs. wild type alleles
At its core, nanoNOMe adds an exogenous layer of information to the DNA itself, which can be used to store information about nucleosome occupancy in the cell and generate a fully phased human epigenome. This is then read out along long single molecules using nanopore sequencing. We could imagine many uses for this approach across basic science and disease models. So, if you are mining the epigeNome looking for your own treasures, consider going on a nanoNOme expedition.
Check out all the treasures in this twitter thread and Nature Methods, December 2020