Direct long-read RNA sequencing uncovers functional variation affecting transcript production and RNA modifications.

The production of multiple transcripts per gene is a process regulated by inherited genetic variants and epitranscriptomic modifications, and plays a prominent role in modulating complex traits and diseases. To simultaneously characterize the effect of genetic variants and N6-methyladenosine (m6A) modifications on transcript abundance, we produced long-read native poly(A) RNA-seq data for 60 genetically different lymphoblastoid cell lines (LCLs) from the 1000 Genomes/Geuvadis project. We identified both annotated (31%) and unannotated (61%) transcripts, and highlight the diversity of transcripts across individuals, with only a small proportion expressed across all samples (35% and 7%, respectively). A genome-wide genetic analysis on transcripts identified 105 trQTLs, of which 76 were not detected as eQTLs using a larger published short-read RNAseq dataset (317 samples). The characterization of m6A methylation DRACH motifs identified an average of 40.1 m6A modifications in 6,222 genes. Genetic association analysis of highly variable modifications from 1,155 genes identified m6A modification quantitative trait loci (m6A-QTLs) for 16 transcripts. Colocalization analysis of trQTL and m6A-QTLs, identified 33 candidate transcripts mediating GWAS traits, with 46.4% of the colocalized trQTLs implicating novel transcripts. Overall, we were able to simultaneously characterize genetic associations with gene expression for their effect on transcript abundance and post-transcriptional modifications, effects often missed when using short-read sequencing technologies.