A high-resolution single-molecule sequencing-based Arabidopsis transcriptome using novel methods of Iso-seq analysis

Runxuan Zhang; Richard Kuo; Max Coulter; Cristiane P. G. Calixto; Juan Carlos Entizne; Wenbin Guo; Yamile Marquez; Linda Milne; Stefan Riegler; Akihiro Matsui; Maho Tanaka; Sarah Harvey; Yubang Gao; Theresa Wießner-Kroh; Alejandro Paniagua; Martin Crespi; Katherine Denby; Asa Ben Hur; Enamul Huq; Michael Jantsch; Artur Jarmolowski; Tino Koester; Sascha Laubinger; Qingshun Quinn Li; Lianfeng Gu; Motoaki Seki; Dorothee Staiger; Ramanjulu Sunkar; Zofia Szweykowska-Kulinska; Shih-Long Tu; Andreas Wachter; Robbie Waugh; Liming Xiong; Xiao-Ning Zhang; Ana Conesa; Anireddy S. N. Reddy; Andrea Barta; Maria Kalyna; John W. S. Brown

doi:10.1186/s13059-022-02711-0

A high-resolution single-molecule sequencing-based Arabidopsis transcriptome using novel methods of Iso-seq analysis

Runxuan Zhang (Lead / Corresponding author)
, Richard Kuo
, Max Coulter
, Cristiane P. G. Calixto
, Juan Carlos Entizne
, Wenbin Guo
, Yamile Marquez
, Linda Milne
, Stefan Riegler
, Akihiro Matsui
, Maho Tanaka
, Sarah Harvey
, Yubang Gao
, Theresa Wießner-Kroh
, Alejandro Paniagua
, Martin Crespi
, Katherine Denby
, Asa Ben Hur
, Enamul Huq
, Michael Jantsch

Artur Jarmolowski, Tino Koester, Sascha Laubinger, Qingshun Quinn Li, Lianfeng Gu, Motoaki Seki, Dorothee Staiger, Ramanjulu Sunkar, Zofia Szweykowska-Kulinska, Shih-Long Tu, Andreas Wachter, Robbie Waugh, Liming Xiong, Xiao-Ning Zhang, Ana Conesa, Anireddy S. N. Reddy, Andrea Barta, Maria Kalyna, John W. S. Brown

Research output: Contribution to journal › Article › peer-review

81 Citations (Scopus)

212 Downloads (Pure)

Abstract

Background: Accurate and comprehensive annotation of transcript sequences is essential for transcript quantification and differential gene and transcript expression analysis. Single-molecule long-read sequencing technologies provide improved integrity of transcript structures including alternative splicing, and transcription start and polyadenylation sites. However, accuracy is significantly affected by sequencing errors, mRNA degradation, or incomplete cDNA synthesis.

Results: We present a new and comprehensive Arabidopsis thaliana Reference Transcript Dataset 3 (AtRTD3). AtRTD3 contains over 169,000 transcripts-twice that of the best current Arabidopsis transcriptome and including over 1500 novel genes. Seventy-eight percent of transcripts are from Iso-seq with accurately defined splice junctions and transcription start and end sites. We develop novel methods to determine splice junctions and transcription start and end sites accurately. Mismatch profiles around splice junctions provide a powerful feature to distinguish correct splice junctions and remove false splice junctions. Stratified approaches identify high-confidence transcription start and end sites and remove fragmentary transcripts due to degradation. AtRTD3 is a major improvement over existing transcriptomes as demonstrated by analysis of an Arabidopsis cold response RNA-seq time-series. AtRTD3 provides higher resolution of transcript expression profiling and identifies cold-induced differential transcription start and polyadenylation site usage.

Conclusions: AtRTD3 is the most comprehensive Arabidopsis transcriptome currently. It improves the precision of differential gene and transcript expression, differential alternative splicing, and transcription start/end site usage analysis from RNA-seq data. The novel methods for identifying accurate splice junctions and transcription start/end sites are widely applicable and will improve single-molecule sequencing analysis from any species.

Original language	English
Article number	149
Number of pages	37
Journal	Genome Biology
Volume	23
DOIs	https://doi.org/10.1186/s13059-022-02711-0
Publication status	Published - 7 Jul 2022

Keywords

Arabidopsis
Iso-seq
Reference transcript dataset
Splice junction
Transcription start and end sites
Alternative splicing
Alternative polyadenylation

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10.1186/s13059-022-02711-0Licence: CC BY

Final Published VersionFinal published version, 3 MBLicence: CC BY

Cite this

Zhang, R., Kuo, R., Coulter, M., Calixto, C. P. G., Entizne, J. C., Guo, W., Marquez, Y., Milne, L., Riegler, S., Matsui, A., Tanaka, M., Harvey, S., Gao, Y., Wießner-Kroh, T., Paniagua, A., Crespi, M., Denby, K., Hur, A. B., Huq, E., ... Brown, J. W. S. (2022). A high-resolution single-molecule sequencing-based Arabidopsis transcriptome using novel methods of Iso-seq analysis. Genome Biology, 23, Article 149. https://doi.org/10.1186/s13059-022-02711-0

@article{585f4b2af68a40758a6454fb22a8fc04,

title = "A high-resolution single-molecule sequencing-based Arabidopsis transcriptome using novel methods of Iso-seq analysis",

abstract = "Background: Accurate and comprehensive annotation of transcript sequences is essential for transcript quantification and differential gene and transcript expression analysis. Single-molecule long-read sequencing technologies provide improved integrity of transcript structures including alternative splicing, and transcription start and polyadenylation sites. However, accuracy is significantly affected by sequencing errors, mRNA degradation, or incomplete cDNA synthesis.Results: We present a new and comprehensive Arabidopsis thaliana Reference Transcript Dataset 3 (AtRTD3). AtRTD3 contains over 169,000 transcripts-twice that of the best current Arabidopsis transcriptome and including over 1500 novel genes. Seventy-eight percent of transcripts are from Iso-seq with accurately defined splice junctions and transcription start and end sites. We develop novel methods to determine splice junctions and transcription start and end sites accurately. Mismatch profiles around splice junctions provide a powerful feature to distinguish correct splice junctions and remove false splice junctions. Stratified approaches identify high-confidence transcription start and end sites and remove fragmentary transcripts due to degradation. AtRTD3 is a major improvement over existing transcriptomes as demonstrated by analysis of an Arabidopsis cold response RNA-seq time-series. AtRTD3 provides higher resolution of transcript expression profiling and identifies cold-induced differential transcription start and polyadenylation site usage.Conclusions: AtRTD3 is the most comprehensive Arabidopsis transcriptome currently. It improves the precision of differential gene and transcript expression, differential alternative splicing, and transcription start/end site usage analysis from RNA-seq data. The novel methods for identifying accurate splice junctions and transcription start/end sites are widely applicable and will improve single-molecule sequencing analysis from any species.",

keywords = "Arabidopsis, Iso-seq, Reference transcript dataset, Splice junction, Transcription start and end sites, Alternative splicing, Alternative polyadenylation",

author = "Runxuan Zhang and Richard Kuo and Max Coulter and Calixto, \{Cristiane P. G.\} and Entizne, \{Juan Carlos\} and Wenbin Guo and Yamile Marquez and Linda Milne and Stefan Riegler and Akihiro Matsui and Maho Tanaka and Sarah Harvey and Yubang Gao and Theresa Wie{\ss}ner-Kroh and Alejandro Paniagua and Martin Crespi and Katherine Denby and Hur, \{Asa Ben\} and Enamul Huq and Michael Jantsch and Artur Jarmolowski and Tino Koester and Sascha Laubinger and Li, \{Qingshun Quinn\} and Lianfeng Gu and Motoaki Seki and Dorothee Staiger and Ramanjulu Sunkar and Zofia Szweykowska-Kulinska and Shih-Long Tu and Andreas Wachter and Robbie Waugh and Liming Xiong and Xiao-Ning Zhang and Ana Conesa and Reddy, \{Anireddy S. N.\} and Andrea Barta and Maria Kalyna and Brown, \{John W. S.\}",

note = "Funding Information: This work was jointly supported by funding from the Biotechnology and Biological Sciences Research Council (BBSRC) BB/P009751/1 to JB; BB/R014582/1 to RW and RZ; BB/S020160/1 to RZ; BB/S004610/1 (16 ERA-CAPS BARN) to RW; the Scottish Government Rural and Environment Science and Analytical Services division (RESAS) [to RZ, RW, and JB]; the National Science Foundation (MCB-2014408) and the National Institute of Health (NIH) (GM-114297) to E.H.; S. H. was supported by funding to K.D. from the University of York; the Austrian Science Fund (FWF) SFB F43 to AB and MJ and [P26333] to MK; The French Agence Nationale de la Recherche grant ANR-16-CE12-0032 to MC; the Japan Science and Technology Agency (JST), the Core Research for Evolutionary Science and Technology (CREST; Grant Number JPMJCR13B4) to M.S.; the National Science Foundation (Grant No. DBI1949036 to A.b.H and A.S.N.R, and Grant No. MCB 2014542 to E.H. and A.S.N.R.); and the DOE Office of Science, Office of Biological and Environmental Research (Grant No. DE-SC0010733) to A.S.N.R and A.b.H.; the Deutsche Forschungsgemeinschaft (DFG) STA653/14-1 and STA653/15-1 to DS; the National Science Foundation grant (IOS-154173) to Q.Q.L.; the German Research Foundation (DFG) WA2167/8-1 to AW and SFB1101/C03 to AW and TWK; the Research Grants Council (RGC) of Hong Kong (GRF 12103020) to LX. NSF grant IOS-1849708 and NSF EPSCoR grant 1826836 to RS; the Academia Sinica to S.-L. T. Publisher Copyright: {\textcopyright} 2022, The Author(s).",

year = "2022",

month = jul,

day = "7",

doi = "10.1186/s13059-022-02711-0",

language = "English",

volume = "23",

journal = "Genome Biology",

issn = "1474-7596",

publisher = "BioMed Central",

}

Zhang, R, Kuo, R, Coulter, M, Calixto, CPG, Entizne, JC, Guo, W, Marquez, Y, Milne, L, Riegler, S, Matsui, A, Tanaka, M, Harvey, S, Gao, Y, Wießner-Kroh, T, Paniagua, A, Crespi, M, Denby, K, Hur, AB, Huq, E, Jantsch, M, Jarmolowski, A, Koester, T, Laubinger, S, Li, QQ, Gu, L, Seki, M, Staiger, D, Sunkar, R, Szweykowska-Kulinska, Z, Tu, S-L, Wachter, A, Waugh, R, Xiong, L, Zhang, X-N, Conesa, A, Reddy, ASN, Barta, A, Kalyna, M & Brown, JWS 2022, 'A high-resolution single-molecule sequencing-based Arabidopsis transcriptome using novel methods of Iso-seq analysis', Genome Biology, vol. 23, 149. https://doi.org/10.1186/s13059-022-02711-0

TY - JOUR

T1 - A high-resolution single-molecule sequencing-based Arabidopsis transcriptome using novel methods of Iso-seq analysis

AU - Zhang, Runxuan

AU - Kuo, Richard

AU - Coulter, Max

AU - Calixto, Cristiane P. G.

AU - Entizne, Juan Carlos

AU - Guo, Wenbin

AU - Marquez, Yamile

AU - Milne, Linda

AU - Riegler, Stefan

AU - Matsui, Akihiro

AU - Tanaka, Maho

AU - Harvey, Sarah

AU - Gao, Yubang

AU - Wießner-Kroh, Theresa

AU - Paniagua, Alejandro

AU - Crespi, Martin

AU - Denby, Katherine

AU - Hur, Asa Ben

AU - Huq, Enamul

AU - Jantsch, Michael

AU - Jarmolowski, Artur

AU - Koester, Tino

AU - Laubinger, Sascha

AU - Li, Qingshun Quinn

AU - Gu, Lianfeng

AU - Seki, Motoaki

AU - Staiger, Dorothee

AU - Sunkar, Ramanjulu

AU - Szweykowska-Kulinska, Zofia

AU - Tu, Shih-Long

AU - Wachter, Andreas

AU - Waugh, Robbie

AU - Xiong, Liming

AU - Zhang, Xiao-Ning

AU - Conesa, Ana

AU - Reddy, Anireddy S. N.

AU - Barta, Andrea

AU - Kalyna, Maria

AU - Brown, John W. S.

N1 - Funding Information: This work was jointly supported by funding from the Biotechnology and Biological Sciences Research Council (BBSRC) BB/P009751/1 to JB; BB/R014582/1 to RW and RZ; BB/S020160/1 to RZ; BB/S004610/1 (16 ERA-CAPS BARN) to RW; the Scottish Government Rural and Environment Science and Analytical Services division (RESAS) [to RZ, RW, and JB]; the National Science Foundation (MCB-2014408) and the National Institute of Health (NIH) (GM-114297) to E.H.; S. H. was supported by funding to K.D. from the University of York; the Austrian Science Fund (FWF) SFB F43 to AB and MJ and [P26333] to MK; The French Agence Nationale de la Recherche grant ANR-16-CE12-0032 to MC; the Japan Science and Technology Agency (JST), the Core Research for Evolutionary Science and Technology (CREST; Grant Number JPMJCR13B4) to M.S.; the National Science Foundation (Grant No. DBI1949036 to A.b.H and A.S.N.R, and Grant No. MCB 2014542 to E.H. and A.S.N.R.); and the DOE Office of Science, Office of Biological and Environmental Research (Grant No. DE-SC0010733) to A.S.N.R and A.b.H.; the Deutsche Forschungsgemeinschaft (DFG) STA653/14-1 and STA653/15-1 to DS; the National Science Foundation grant (IOS-154173) to Q.Q.L.; the German Research Foundation (DFG) WA2167/8-1 to AW and SFB1101/C03 to AW and TWK; the Research Grants Council (RGC) of Hong Kong (GRF 12103020) to LX. NSF grant IOS-1849708 and NSF EPSCoR grant 1826836 to RS; the Academia Sinica to S.-L. T. Publisher Copyright: © 2022, The Author(s).

PY - 2022/7/7

Y1 - 2022/7/7

N2 - Background: Accurate and comprehensive annotation of transcript sequences is essential for transcript quantification and differential gene and transcript expression analysis. Single-molecule long-read sequencing technologies provide improved integrity of transcript structures including alternative splicing, and transcription start and polyadenylation sites. However, accuracy is significantly affected by sequencing errors, mRNA degradation, or incomplete cDNA synthesis.Results: We present a new and comprehensive Arabidopsis thaliana Reference Transcript Dataset 3 (AtRTD3). AtRTD3 contains over 169,000 transcripts-twice that of the best current Arabidopsis transcriptome and including over 1500 novel genes. Seventy-eight percent of transcripts are from Iso-seq with accurately defined splice junctions and transcription start and end sites. We develop novel methods to determine splice junctions and transcription start and end sites accurately. Mismatch profiles around splice junctions provide a powerful feature to distinguish correct splice junctions and remove false splice junctions. Stratified approaches identify high-confidence transcription start and end sites and remove fragmentary transcripts due to degradation. AtRTD3 is a major improvement over existing transcriptomes as demonstrated by analysis of an Arabidopsis cold response RNA-seq time-series. AtRTD3 provides higher resolution of transcript expression profiling and identifies cold-induced differential transcription start and polyadenylation site usage.Conclusions: AtRTD3 is the most comprehensive Arabidopsis transcriptome currently. It improves the precision of differential gene and transcript expression, differential alternative splicing, and transcription start/end site usage analysis from RNA-seq data. The novel methods for identifying accurate splice junctions and transcription start/end sites are widely applicable and will improve single-molecule sequencing analysis from any species.

AB - Background: Accurate and comprehensive annotation of transcript sequences is essential for transcript quantification and differential gene and transcript expression analysis. Single-molecule long-read sequencing technologies provide improved integrity of transcript structures including alternative splicing, and transcription start and polyadenylation sites. However, accuracy is significantly affected by sequencing errors, mRNA degradation, or incomplete cDNA synthesis.Results: We present a new and comprehensive Arabidopsis thaliana Reference Transcript Dataset 3 (AtRTD3). AtRTD3 contains over 169,000 transcripts-twice that of the best current Arabidopsis transcriptome and including over 1500 novel genes. Seventy-eight percent of transcripts are from Iso-seq with accurately defined splice junctions and transcription start and end sites. We develop novel methods to determine splice junctions and transcription start and end sites accurately. Mismatch profiles around splice junctions provide a powerful feature to distinguish correct splice junctions and remove false splice junctions. Stratified approaches identify high-confidence transcription start and end sites and remove fragmentary transcripts due to degradation. AtRTD3 is a major improvement over existing transcriptomes as demonstrated by analysis of an Arabidopsis cold response RNA-seq time-series. AtRTD3 provides higher resolution of transcript expression profiling and identifies cold-induced differential transcription start and polyadenylation site usage.Conclusions: AtRTD3 is the most comprehensive Arabidopsis transcriptome currently. It improves the precision of differential gene and transcript expression, differential alternative splicing, and transcription start/end site usage analysis from RNA-seq data. The novel methods for identifying accurate splice junctions and transcription start/end sites are widely applicable and will improve single-molecule sequencing analysis from any species.

KW - Arabidopsis

KW - Iso-seq

KW - Reference transcript dataset

KW - Splice junction

KW - Transcription start and end sites

KW - Alternative splicing

KW - Alternative polyadenylation

UR - https://www.scopus.com/pages/publications/85133563650

U2 - 10.1186/s13059-022-02711-0

DO - 10.1186/s13059-022-02711-0

M3 - Article

C2 - 35799267

SN - 1474-7596

VL - 23

JO - Genome Biology

JF - Genome Biology

M1 - 149

ER -

A high-resolution single-molecule sequencing-based Arabidopsis transcriptome using novel methods of Iso-seq analysis

Abstract

Keywords

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A Reference Transcript Database for Improved Analysis of RNA-seq Data from Barley

16 ERA-CAPS Barley Yield Associated Networks (Joint with IPK Gatersleben as lead and University of Minnesota)

Dynamic Re-programming of the Cold Transcriptome in Arabidopsis (Joint with JHI)