Quantitative profiling of N6-methyladenosine at single-base resolution in stem-differentiating xylem of Populus trichocarpa using Nanopore direct RNA sequencing

There are no comprehensive methods to identify N⁶-methyladenosine (m⁶A) at single-base resolution for every single transcript, which is necessary for the estimation of m⁶A abundance. We develop a new pipeline called Nanom6A for the identification and quantification of m⁶A modification at single-base resolution using Nanopore direct RNA sequencing based on an XGBoost model. We validate our method using methylated RNA immunoprecipitation sequencing (MeRIP-Seq) and m⁶A-sensitive RNA-endoribonuclease-facilitated sequencing (m6A-REF-seq), confirming high accuracy. Using this method, we provide a transcriptome-wide quantification of m⁶A modification in stem-differentiating xylem and reveal that different alternative polyadenylation (APA) usage shows a different ratio of m⁶A.