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.
Highlights
Transcriptomics is the study of the transcriptome including gene structure, expression, and regulation
ROI: read of insert; FLNC reads: full-length non-chimeric reads; NA means the data which are not presented in the literature; 1 denotes reads corrected by iterative clustering for error correction (ICE)-Quiver; 2 denotes mapped reads obtained by two full-passes full-length non-chimeric read of insert data; NS means the detail numbers are not shown the analysis has been done
Recent advances in short and long read sequencing technology have led to the dramatic increase of sequenced plant genomes
Summary
Transcriptomics is the study of the transcriptome including gene structure, expression, and regulation. The replication processes in all ZMWs of a SMRT cell are recorded by a ‘movie’ of light pulses, and the pulses corresponding to each ZMW can be interpreted to be a sequence of bases or a polymerase read. 1–2 in most genomes (for example, in Arabidopsis kb in rice [6], kb in [7]), long read can the same transcript multiple times. In this case,case, the maize [7]),the thepolymerase long polymerase readcover can cover the same transcript multiple times.
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