Methods and Tools for MeRIP-Seq and Nanopore Detection of m6A(m) Changes.

Previous studies have reported that various stimuli, including viral infection, cancer, and heat shock, lead to changes to m6A methylation of mRNAs from global increases or decreases in m6A to shifts in the distribution of methylation over transcripts or changes at specific loci. Reported estimates of m6A gains and losses range from zero to thousands of sites, depending on the stimulus and analysis methods. Most studies map m6A using methylated RNA immunoprecipitation sequencing (MeRIP-seq). Methylated regions are identified through peaks in transcript coverage from the immunoprecipitated fraction compared to an input fraction. However, for any two given studies looking at similar conditions, we found peak overlap varied from ∼30 to 60%, independent of the cell lines studied. Although we verified appropriate statistical methods to detect changes in peaks using positive and negative controls, we were unable to validate consistent changes across studies of similar experimental interventions. Eventually, single-molecule methods may help in these challenges, and as such, we also present evidence of m6A within nature RNA nanopore molecules with a new tool (EpinNano).

Duke Scholars

Author Stacy M. Horner Integrative Immunobiology