A comprehensive survey of RNA modifications in a human transcriptome

RNA modifications are critical for transcript function and regulation. Direct RNA nanopore sequencing offers a unique advantage by observing these modifications through characteristic alterations in ionic current signals. Various computational tools have been developed to detect RNA modifications, typically by comparing a sample of interest with a control lacking the target modification, often achieved through enzyme knockdown or knockout. We have developed a robust in vitro transcription protocol to generate a modification-free copy of any input RNA to detect all modifications that alter the raw ionic current signal relative to the canonical nucleotides during nanopore sequencing. We generated an in vitro transcribed sample from K562 cells and used Nanocompore to detect 26,619 modified RNA sites across 2,520 transcript isoforms from 1,742 genes. Of these, 56% are consistent with the well-characterized m6A modification. By inferring the identities of these modifications, we assessed differential usage and correlation patterns, revealing a significant co-occurrence between m6A and m5C modifications within the same transcripts. Additionally, some modifications were non-randomly associated with alternative splicing events. This study provides a comprehensive survey of RNA modifications across the transcriptome, demonstrating the utility of in vitro transcription coupled with direct RNA nanopore sequencing to simultaneously detect multiple modifications without the need for additional independent biochemical assays. The protocol is consistent in the future with more complex experimental designs, for example for differential modification usage between samples.