Analysis of Transcriptome and Epitranscriptome in Plants Using PacBio Iso-Seq and Nanopore-Based Direct RNA Sequencing.

Liangzhen Zhao,Lianfeng Gu,Hangxiao Zhang,Markus V Kohnen,Kasavajhala V S K Prasad,Anireddy S N Reddy

doi:10.3389/fgene.2019.00253

Abstract

Nanopore sequencing from Oxford Nanopore Technologies (ONT) and Pacific BioSciences (PacBio) single-molecule real-time (SMRT) long-read isoform sequencing (Iso-Seq) are revolutionizing the way transcriptomes are analyzed. These methods offer many advantages over most widely used high-throughput short-read RNA sequencing (RNA-Seq) approaches and allow a comprehensive analysis of transcriptomes in identifying full-length splice isoforms and several other post-transcriptional events. In addition, direct RNA-Seq provides valuable information about RNA modifications, which are lost during the PCR amplification step in other methods. Here, we present a comprehensive summary of important applications of these technologies in plants, including identification of complex alternative splicing (AS), full-length splice variants, fusion transcripts, and alternative polyadenylation (APA) events. Furthermore, we discuss the impact of the newly developed nanopore direct RNA-Seq in advancing epitranscriptome research in plants. Additionally, we summarize computational tools for identifying and quantifying full-length isoforms and other co/post-transcriptional events and discussed some of the limitations with these methods. Sequencing of transcriptomes using these new single-molecule long-read methods will unravel many aspects of transcriptome complexity in unprecedented ways as compared to previous short-read sequencing approaches. Analysis of plant transcriptomes with these new powerful methods that require minimum sample processing is likely to become the norm and is expected to uncover novel co/post-transcriptional gene regulatory mechanisms that control biological outcomes during plant development and in response to various stresses.

Highlights

Analysis of transcriptomes, which represent the activity of genes in the genome, is vital for understanding the relationship between genotype and phenotype
Single-molecule realtime (SMRT) isoform sequencing (Iso-Seq) using Pacific BioSciences (PacBio) platform captures the full length of transcripts (GonzalezGaray, 2016) and thereby presents easier and more accurate ways for different applications, such as gene annotation (Zhao et al, 2018), isoform identification (Abdel-Ghany et al, 2016; Wang T. et al, 2017), identification of fusion transcripts (Weirather et al, 2015), and long non-coding RNA discovery (Li et al, 2016)
We present currently available bioinformatics methods for PacBio and Oxford Nanopore Technologies (ONT) read analysis, including reads-of-interest (ROI) extraction, error correction (Au et al, 2012), mapping (Wu and Watanabe, 2005), isoform clustering (Fu et al, 2012), and identification of multiple transcript isoforms (Abdel-Ghany et al, 2016)

Summary

INTRODUCTION

Analysis of transcriptomes, which represent the activity of genes in the genome, is vital for understanding the relationship between genotype and phenotype. We present currently available bioinformatics methods for PacBio and ONT read analysis, including reads-of-interest (ROI) extraction, error correction (Au et al, 2012), mapping (Wu and Watanabe, 2005), isoform clustering (Fu et al, 2012), and identification of multiple transcript isoforms (Abdel-Ghany et al, 2016). For the analysis of post-transcriptional regulation based on long-read sequencing, TAPIS pipeline (Abdel-Ghany et al, 2016) and PRAPI (Gao et al, 2017) are two main bioinformatics tools that use Iso-Seq reads to identify AS and APA (Figure 1). PRAPI (Gao et al, 2017) can identify several other events/processes, such as ATI, and production of circular RNAs (circRNAs)

IDENTIFICATION OF FUSION TRANSCRIPTS

NATURAL ANTISENSE TRANSCRIPTS IDENTIFICATION

THE APPLICATION OF NANOPORE DIRECT RNA SEQUENCING

Findings

FUTURE DIRECTIONS