Long-read RNA sequencing reveals allele-specific N ⁶-methyladenosine modifications

Long-read sequencing technology enables highly accurate detection of allele-specific RNA expression, providing insights into the effects of genetic variation on splicing and RNA abundance. Furthermore, the ability to directly sequence RNA enables the detection of RNA modifications in tandem with ascertaining the allelic origin of each molecule. Here, we leverage these advantages to determine allele-biased patterns of N ⁶-methyladenosine (m⁶A) modifications in native mRNA. We used human and mouse cells with known genetic variants to assign the allelic origin of each mRNA molecule combined with a supervised machine learning model to detect read-level m⁶A modification ratios. Our analyses reveal the importance of sequences adjacent to the DRACH motif in determining m⁶A deposition, in addition to allelic differences that directly alter the motif. Moreover, we discover allele-specific m⁶A modification events with no genetic variants in close proximity to the differentially modified nucleotide, demonstrating the unique advantage of using long-reads and surpassing the capabilities of antibody-based short-read approaches. This technological advance will further our understanding of the role of genetics in determining mRNA modifications.