An Analysis Pipeline for Identification of RNA Modification, Alternative Splicing and Polyadenylation Using Third Generation Sequencing

Nanopore sequencing based on Oxford Nanopore Technologies (ONT) and Pacific BioSciences (PacBio) single-molecule real-time (SMRT) long-read isoform sequencing (Iso-Seq) have shown great potential in detecting post-transcriptional regulation. Direct RNA sequencing (DRS) has the advantages in capturing RNA modification due to without PCR amplification which is the limitation of the next-generation sequencing (NGS). Here, we provide a comprehensive computational procedure for the quantification of RNA modification in single-base resolution based on DRS data. Moreover, we also provide procedure on the identification of alternative splicing (AS) and alternative polyadenylation (APA) based on both DRS and PacBio Iso-Seq data. The entire step was based on two packages (Nanom6A and PRAPI), which were based on Python language on Linux system.