Single-molecule Real-time (SMRT) Isoform Sequencing (Iso-Seq) in Plants: The Status of the Bioinformatics Tools to Unravel the Transcriptome Complexity

Abstract

Background: The advent of the Single-Molecule Real-time (SMRT) Isoform Sequencing (Iso-Seq) has paved the way to obtain longer full-length transcripts. This method was found to be much superior in identifying full-length splice variants and other post-transcriptional events as compared to the Next Generation Sequencing (NGS)-based short read sequencing (RNA-Seq). Several different bioinformatics tools to analyze the Iso-Seq data have been developed and some of them are still being refined to address different aspects of transcriptome complexity. However, a comprehensive summary of the available tools and their utility is still lacking. Objective: Here, we summarized the existing Iso-Seq analysis tools and presented an integrated bioinformatics pipeline for Iso-Seq analysis, which overcomes the limitations of NGS and generates long contiguous Full-Length Non-Chimeric (FLNC) reads for the analysis of posttranscriptional events. Results: In this review, we summarized recent applications of Iso-Seq in plants, which include improved genome annotations, identification of novel genes and lncRNAs, identification of fulllength splice isoforms, detection of novel Alternative Splicing (AS) and Alternative Polyadenylation (APA) events. In addition, we also discussed the bioinformatics pipeline for comprehensive Iso-Seq data analysis, including how to reduce the error rate in the reads and how to identify and quantify post-transcriptional events. Furthermore, the visualization approach of Iso-Seq was discussed as well. Finally, we discussed methods to combine Iso-Seq data with RNA-Seq for transcriptome quantification. Conclusion: Overall, this review demonstrates that the Iso-Seq is pivotal for analyzing transcriptome complexity and this new method offers unprecedented opportunities to comprehensively understand transcripts diversity.

Chromosome-level genome assembly of largemouth bass (Micropterus salmoides) using PacBio and Hi-C technologies

“…The whole genome information is the basis for studying the nature of organisms, including advantages during biological invasions and adaptation to extreme environments such as hypoxia 10 – 12 , climate change 13 , 14 , temperature 15 , 16 and salinity 17 , 18 . With the development of sequencing technology, genome research has been studied more deeply and accurately 19 . More and more fish genomes have been decoded, such as yellow perch ( Perca flavescens ) 20 , golden pompano ( Trachinotus ovatus ) 21 and dark sleeper ( Odontobutis potamophila ) 22 , etc.…”

PSDX: A Comprehensive Multi-Omics Association Database of Populus trichocarpa With a Focus on the Secondary Growth in Response to Stresses

“…Single-molecule real-time (SMRT) Isoform Sequencing (Iso-Seq) presents a great advantage in the identification of post-transcriptional regulation based on full-length splicing isoforms (Gao et al, 2019;Zhang et al, 2019;Zhao et al, 2019). Using Iso-Seq data we identified a total of 58,000 AS events in 9,199 genes which included 22,882 IR, 4,175 ES, 17,031 AltA, and 13,912 AltD, respectively (Supplementary Table 7).…”

Transcriptome sequencing of cochleae from constant-frequency and frequency-modulated echolocating bats

“…In these earlier studies, the reference used for quantification of gene expression was from a de novo assembly based on the short RNA-seq reads which may contain many artificial transcripts 11 . The PacBio single-molecule real-time isoform sequencing (Iso-seq) can generate full-length (FL) sequences of all transcripts without the need for assembly 12 , which has been integrated with RNA-seq for transcriptome quantification in multiple studies 12,13 . PacBio Iso-seq is also used to detect alternative splicing events without the help of a reference genome sequence 14 and to identify previously unannotated transcripts 15 .…”

“…They can be used as the reference to reanalyze the RNA-seq datasets of cochlea in previous comparative transcriptomic studies 8 – 10 . Quantification of transcript expression by mapping reads to the FL transcriptome will help to improve the accuracy of identifying differentially expressed transcripts 12 . Moreover, by comparing with transcripts expressed in non-echolocating mammals, the current FL transcriptomes from echolocating bats will help to test whether alternative splicing plays an important role in the origin of novel phenotype (ultrahigh frequency hearing).…”