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Methodology
Analysis and comprehensive comparison of PacBio and nanopore-based RNA sequencing of the Arabidopsis transcriptome
Biao Jin
Yangzhou University
Corresponding Author
ORCiD: https://orcid.org/0000-0002-7330-334X
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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.
Keywords
Plant transcriptome, Third-generation sequencing, PacBio, Nanopore, RNA-Seq, Single-molecule sequencing
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CURRENT STATUS: Under Review
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Received 31 May, 2020
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Received 31 May, 2020
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Invitations sent on 19 May, 2020
Reviewer #1 agreed
On 19 May, 2020
Reviewer #2 agreed
On 19 May, 2020
Reviewer #3 agreed
On 19 May, 2020
Review #1 received
Received 19 May, 2020
Submission checks complete
On 18 May, 2020
Editor invited
On 18 May, 2020
No comments provided
Review #3 received
Received 31 Mar, 2020
Editorial decision: Major revision
On 31 Mar, 2020
Review #1 received
Received 12 Mar, 2020
Reviewer #3 agreed
On 11 Mar, 2020
Review #2 received
Received 07 Mar, 2020
Reviewer #2 agreed
On 28 Feb, 2020
Reviewer #1 agreed
On 24 Feb, 2020
Reviewers invited
Invitations sent on 30 Dec, 2019
Editor assigned
On 18 Dec, 2019
Submission checks complete
On 17 Dec, 2019
Editor invited
On 17 Dec, 2019
First submitted
On 16 Dec, 2019
Metrics
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PDF Downloads: ...
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Subject Areas
Plant Molecular Biology and Genetics
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This preprint is under consideration at Plant Methods. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Methodology
Analysis and comprehensive comparison of PacBio and nanopore-based RNA sequencing of the Arabidopsis transcriptome
Biao Jin
Yangzhou University
Corresponding Author
ORCiD: https://orcid.org/0000-0002-7330-334X
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Review
Version 2
Posted 28 May, 2020
No community comments so far
Review #2 received
Received 31 May, 2020
Review #3 received
Received 31 May, 2020
Editor assigned
On 19 May, 2020
Reviewers invited
Invitations sent on 19 May, 2020
Reviewer #1 agreed
On 19 May, 2020
Reviewer #2 agreed
On 19 May, 2020
Reviewer #3 agreed
On 19 May, 2020
Review #1 received
Received 19 May, 2020
Submission checks complete
On 18 May, 2020
Editor invited
On 18 May, 2020
No comments provided
Review #3 received
Received 31 Mar, 2020
Editorial decision: Major revision
On 31 Mar, 2020
Review #1 received
Received 12 Mar, 2020
Reviewer #3 agreed
On 11 Mar, 2020
Review #2 received
Received 07 Mar, 2020
Reviewer #2 agreed
On 28 Feb, 2020
Reviewer #1 agreed
On 24 Feb, 2020
Reviewers invited
Invitations sent on 30 Dec, 2019
Editor assigned
On 18 Dec, 2019
Submission checks complete
On 17 Dec, 2019
Editor invited
On 17 Dec, 2019
First submitted
On 16 Dec, 2019
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Plant Molecular Biology and Genetics
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7