Analysis and comprehensive comparison of PacBio and nanopore-based RNA sequencing of the Arabidopsis transcriptome

Jiawen Cui,Biao Jin,Yuyao Wang,Nan Shen,Guolu Xu,Zhaogeng Lu

doi:10.1186/s13007-020-00629-x

Jiawen Cui, Biao Jin + Show 4 more

Open Access

https://doi.org/10.1186/s13007-020-00629-x

Copy DOI

Journal: Plant methods	Publication Date: Jun 12, 2020
Citations: 74	License type: open-access

Affiliation: Yangzhou University, Biomarker Technologies (China)

Abstract

BackgroundThe number of studies using third-generation sequencing utilising Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) is rapidly increasing in many different research areas. Among them, plant full-length single-molecule transcriptome studies have mostly used PacBio sequencing, whereas ONT is rarely used. Therefore, in this study, we examined ONT RNA sequencing methods in plants. We performed a detailed evaluation of reads from PacBio, Nanopore direct cDNA (ONT Dc), and Nanopore PCR cDNA (ONT Pc) sequencing including characteristics of raw data and identification of transcripts. In addition, matched Illumina data were generated for comparison.ResultsONT Pc showed overall better raw data quality, whereas PacBio generated longer read lengths. In the transcriptome analysis, PacBio and ONT Pc performed similarly in transcript identification, simple sequence repeat analysis, and long non-coding RNA prediction. PacBio was superior in identifying alternative splicing events, whereas ONT Pc could estimate transcript expression levels.ConclusionsThis paper made a comprehensive comparison of PacBio and nanopore-based RNA sequencing of the Arabidopsis transcriptome, the results indicate that ONT Pc is more cost-effective for generating extremely long reads and can characterise the transcriptome as well as quantify transcript expression. Therefore, ONT Pc is a new cost-effective and worthwhile method for full-length single-molecule transcriptome analysis in plants.

Highlights

The number of studies using third-generation sequencing utilising Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) is rapidly increasing in many different research areas
Overview of Illumina, PacBio and ONT sequencing To compare the performance of the RNA sequencing methods, we sequenced cDNA libraries from Arabidopsis on Illumina NovaSeq, PacBio Sequel, Nanopore instruments
PCR and Sanger sequencing validated four of eight and five of eight lncRNAs from the PacBio and ONT Pc data, respectively. These results suggest that both ONT Pc and PacBio methods are suitable for identification of lncRNAs

Summary

Introduction

The number of studies using third-generation sequencing utilising Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) is rapidly increasing in many different research areas. Current sequencing-based transcriptomic analyses (RNA-Seq) using the massive throughput of next-generation sequencing platforms have enabled us to build a picture of the active transcriptional patterns within organisms. Among these analyses, short-read RNASeq (mainly using Illumina technology) has been used for over a decade. With the rapid development of sequencing technology, long-read sequencing platforms, including Pacific Biosciences (PacBio) [1] and Oxford Nanopore Technologies (ONT) [2], which have the capability to sequence entire

Objectives

Methods

Results

Discussion

Conclusion