Comprehensive Iso-Seq Data Analysis Workflow

For the analysis of Iso-Seq data, we obtained high-quality reads of insert (ROI) by ConsensusTools script of SMRT Analysis v.2.3.0 software package from Pacific Biosciences. Then we used pbtranscript.py of SMRT Analysis v.2.3.0 software package to obtain full-length non-chimeric (FLNC) reads. For a small part of the chimeric ROI, they were marked as complete ROI by judging the 5′ primer, 3′ primer, presence of poly(A) and corresponding positional relationship. LSC 1.alpha (Au et al., 2012 (link)) was used to correct Iso-Seq. Finally, these corrected Iso-Seq reads were further used as an input file to identify AS events using PRAPI (Gao et al., 2018 (link)) with default parameters to identify the post-transcriptional regulation events including ATI, AS, and APA (Figure 2).