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

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

Abstract
Background
The number of studies using third-generation sequencing using 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.
Results ONT 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.
Conclusions This 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.
Background
Current sequencing-based transcriptomic analyses (RNA-Seq) using the massive throughput of nextgeneration sequencing platforms have enabled us to build a picture of the active transcriptional
patterns within organisms. Among these analyses, short-read RNA-Seq (mainly using Illumina
technology) has been used for over a decade. The numbers of reads output by Illumina sequencers
are sufficient to accurately quantify gene expression. However, because Illumina sequencers are
appropriate only for short read-length sequencing, we must fragment RNA or cDNA during sample
preparation.
Thus, RNA transcript information covering long lengths or distances, including alternative splicing (AS), simple sequence repeats (SSRs), and long non-coding RNA (lncRNA), is lost using this technology.

摘要
背景
在许多不同的研究领域，使用第三代测序技术的Pacific Biosciences(PacBio)和牛津纳米孔技术(ONT)的研究数量正在迅速增加。
其中，植物全长单分子转录组研究多采用PacBio测序，很少使用ONT。
因此，在本研究中，我们考察了植物的ONT RNA测序方法。
我们对PacBio、Nanopore direct cDNA(ONT Dc)和Nanopore PCR cDNA(ONT Pc)的reads序列进行了详细的评价，包括原始数据的特征和转录本的鉴定。
另外，生成匹配的Illumina数据进行比较。
结果ONT Pc显示总体上更好的原始数据质量，而PacBio产生更长的阅读
长度。
在转录组分析中，PacBio和ONT Pc在转录组中表现相似
鉴定，简单序列重复分析，长非编码RNA预测。
PacBio是
在识别可变剪接事件方面表现优异，而ONT Pc可以估计转录本
表达水平。
结论本文对PacBio和纳米孔RNA进行了综合比较
对拟南芥转录组进行测序，结果表明ONT Pc具有更高的性价比
用于生成极长的数据，并能表征转录组和量化
转录表达。
因此，ONT Pc是一种性价比高、值得推广的全长检测新方法
植物单分子转录组分析。
背景
目前基于序列的转录组学分析(RNA-Seq)使用大规模吞吐量的下一代测序平台，使我们能够构建活性转录的图像
模式生物。
在这些分析中，short-read RNA-Seq(主要使用Illumina)
技术)已经被使用了十多年。
Illumina测序仪输出的reads数
足以准确量化基因表达。
然而，因为Illumina测序仪是
只适用于较短的读长测序，我们必须在样本中片段RNA或cDNA
准备。
因此，使用这种技术，包括可变剪接(AS)、简单序列重复序列(SSRs)和长非编码RNA (lncRNA)在内的长长度或距离的RNA转录信息会丢失。

本文标签： comparisonPacBioAnalysisComprehensivenanopore