Direct Analysis of HIV mRNA m⁶A Methylation by Nanopore Sequencing

The post-transcriptional processing and chemical modification of HIV RNA are understudied aspects of HIV virology, primarily due to the limited ability to accurately map and quantify RNA modifications. Modification-specific antibodies or modification-sensitive endonucleases coupled with short-read RNA sequencing technologies have allowed for low-resolution or limited mapping of important regulatory modifications of HIV RNA such as N⁶-methyladenosine (m⁶A). However, a high-resolution map of where these sites occur on HIV transcripts is needed for detailed mechanistic understanding. This has recently become possible with new sequencing technologies. Here, we describe the direct RNA sequencing of HIV transcripts using an Oxford Nanopore Technologies sequencer and the use of this technique to map m⁶A at near single nucleotide resolution. This technology also provides the ability to identify splice variants with long RNA reads and thus, can provide high-resolution RNA modification maps that distinguish between overlapping splice variants. The protocols outlined here for m⁶A also provide a powerful paradigm for studying any other RNA modifications that can be detected on the nanopore platform.