A Computational Workflow for Estimation of Short RNA Polyadenylation using Direct RNA Nanopore Sequencing with Polyuridylation

Polyadenylation or poly(A) tail at the 3’-end of RNA is a type of post-transcriptional modification that regulates RNA biological functions and stability. While poly(A) tails in eukaryotes are 80–200 nucleotides long and generally stabilize the RNAs, the poly(A) tails in prokaryotes are significantly shorter and usually destabilize the RNAs. Although Oxford Nanopore Technologies (ONT) direct RNA sequencing provides long-read sequencing that allows poly(A) tail length estimation in recent years, the standard nanopore RNA library preparation, which requires sequencing oligo(dT) adapter ligation, could not capture short poly(A) tail RNAs, especially RNAs in prokaryotes. Hence, we introduced in vitro uridylation and developed a computational processing workflow to dissect poly(A) tails and estimate their length. Using synthetic RNAs as standard, this study provides the workflow for short poly(A) tail length estimation by reassigning underestimated poly(A) tail length using base-called sequencing data and re-segmenting misassigned poly(A) tail. This workflow could be beneficial to obtain poly(A) tail length in different organisms and potentially provide insights into their gene expression, regulation, and modification.