Isoform Sequencing and State-of-Art Applications for Unravelling Complexity of Plant Transcriptomes

Abstract

Single-molecule real-time (SMRT) sequencing developed by PacBio, also called third-generation sequencing (TGS), offers longer reads than the second-generation sequencing (SGS). Given its ability to obtain full-length transcripts without assembly, isoform sequencing (Iso-Seq) of transcriptomes by PacBio is advantageous for genome annotation, identification of novel genes and isoforms, as well as the discovery of long non-coding RNA (lncRNA). In addition, Iso-Seq gives access to the direct detection of alternative splicing, alternative polyadenylation (APA), gene fusion, and DNA modifications. Such applications of Iso-Seq facilitate the understanding of gene structure, post-transcriptional regulatory networks, and subsequently proteomic diversity. In this review, we summarize its applications in plant transcriptome study, specifically pointing out challenges associated with each step in the experimental design and highlight the development of bioinformatic pipelines. We aim to provide the community with an integrative overview and a comprehensive guidance to Iso-Seq, and thus to promote its applications in plant research.

Keywords: alternative splicing; fusion genes; genome annotation; isoform sequencing; long reads; novel genes; plant; single-molecule real-time sequencing; transcriptomics.

Figure 1

Definition of the polymerase read, subreads, and the read of insert (ROI). DNA template is labeled in blue and adapter in green. SMRT: Single-Molecule Real-Time sequencing.

Figure 2

Schematic workflow of isoform sequencing.

Figure 3

Schematic representation of alternative splicing (AS) and alternative polyadenylation (APA). (a) Shows alternative splicing; (b) shows alternative polyadenylation. The first two APA types—which both are termed as tandem 3′ untranslated region (UTR) APA—generate multiple isoforms that differ in their 3′UTR length without impacting the protein sequence encoded by the gene. The other three APA types potentially affect the coding sequences: alternative terminal exon APA, in which APA generates isoforms that differ in their last exon; intronic APA, which involves cleaving at the cryptic intronic polyA signal (PAS) with an extending terminal exon; and internal exon APA, which involves premature within one coding region with PAS. The filled dark blue boxes denote the retaining exons, and the filled light blue boxes denote the alternative exons. The blue solid lines represent introns. The black dash lines represent AS events. The filled yellow boxes represent 3′ UTR with different length, and the arrows denote PAS.