A versatile 5' RACE-Seq methodology for the accurate identification of the 5' termini of mRNAs

In this work we present a custom 5 RACE-seq methodology, based on the template-switching mechanism and a downstream targeted nanopore sequencing. This approach enables the broad and in-depth study of 5 prime UTRs of any mRNA of interest, by offering a tremendous sequencing depth, while significantly reducing the cost-per reaction compared to commercially available kits. The optimization of the described 5 prime RACE method was carried out using human BCL2L12 as control gene. Finally, the described method was applied for the identification of 5 prime UTRs for many members of the human kallikrein-related peptidases (KLKs), leading not only to the confirmation of annotated 5 prime mRNA termini, but also to the elucidation of novel ones.