Long-read transcriptome-wide RNA structure maps using DMS-FIRST-seq

ABSTRACT RNA modifications induce reverse transcription (RT) errors in an enzyme- and context-dependent manner, enabling transcriptome-wide mapping and RNA structure probing. We present FIRST-seq, a flexible, cost-effective nanopore cDNA method that avoids second-strand synthesis and PCR, making it compatible with any RT enzyme and enabling single-nucleotide resolution RT signature analysis. Benchmarking multiple RT enzymes and buffers identified conditions that reduce premature termination and enhance error detection. Coupled with DMS probing, FIRST-seq accurately detects m1A and m3C at unpaired sites, recapitulating known RNA structures in vitro and in vivo. FIRST-seq offers a versatile platform for profiling chemical-induced and natural RNA modifications using long-read sequencing. Competing Interest Statement EMN is a member of the Scientific Advisory Board of IMMAGINA Biotech. JSM is a member of the Scientific Advisory Board of NextRNA Therapeutics Inc. OB, GD and EMN have received travel bursaries from ONT to present their work at conferences. Footnotes Manuscript was updated in order to answer to the comments from reviewers.