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 on transcript abundance and N6-methyladenosine (m6A) modifications, 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 a high diversity of both annotated (31%) and unannotated (61%) transcripts, with only a small proportion expressed across individuals (35% and 7%, respectively). In a genome-wide genetic analysis on transcripts, we identified 105 trQTLs, of which 76 were not detected as eQTLs using a larger published short-read RNAseq dataset (317 samples). A population wide characterization of m6A methylation DRACH motifs identified an average of 40.1 m6A modifications on 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 risk transcripts. Overall, the simultaneous characterization of transcripts and post-transcriptional modifications identified genetic effects on transcription often missed when using other sequencing technologies.