Bisulfite sequencing has been employed to great effect to identify DNA methylation changes between different tissues, through stem cell differentiation, and even in cancer development. However, there is an ever increasing problem of assessing the functional importance of these methylation changes- though work has demonstrated that methylation affects protein-DNA interaction, a clear and comprehensive delineation of the strength of these effects is lacking. We employed a modified chromatin immunoprecipitation method combined with bisulfite sequencing to identify the distribution of methylation patterns present in bound DNA. We used both synthetically generated in vitro samples for an unbiased measurement and samples derived from cell lines for physiologically relevant patterns. Our library preparation methodology uses magnetic bead ChIP (Life Technologies) combined with methylated hairpin adapters (NEB) and low-input bisulfite methods (Zymo Research) with custom modifications. We then sequenced these libraries on an Illumina MiSeq for the long-read length offered (2x300). The results were compared to the input methylome to determine the relative frequency of methylation patterns bound by protein. In our initial work, we examined the methylation patterns of DNA bound by CTCF and MeCP2. The number of methylated locations required to affect protein-binding was measured, as well as the frequency of binding versus a known binding sequence. We specifically examined DNA upstream and downstream of the known binding motifs, to determine non-local effects of methylation on protein-DNA affinity.

It is now possible to manipulate individual molecules using a nanopore to read DNA and proteins, or write DNA by inserting mini-genes into cells. Furthermore, development of these methodologies will kick open the door to new biology and chemistry that has been logistically intractable previously. Nanopore technology will place molecular and sub-molecular analysis within the reach of the typical bench-top scientist or clinical lab-no longer limited to genomics or mass spectrometry specialists. Moreover, the prospects for synthetic biology-using nanopores to program or reprogram cells-are promising as well, but have been examined only at the level of a single cell, so far.