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

Journal Article · · Genome Biology (Online)
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  1. James Hutton Institute, Dundee (Scotland); OSTI
  2. Mocean Energy, Edinburgh (United Kingdom)
  3. University of Dundee (Scotland). James Hutton Institute
  4. James Hutton Institute, Dundee (Scotland)
  5. Centre for Genomic Regulation, Barcelona (Spain)
  6. University of Natural Resources and Life Sciences (BOKU), Vienna (Austria)
  7. RIKEN Center for Sustainable Resource Science, Yokohama (Japan)
  8. University of York Wentworth Way, York (United Kingdom)
  9. Fujian Agriculture and Forestry University, Fuzhou (China)
  10. University of Tubingen (Germany)
  11. Spanish National Research Council, Paterna (Spain)
  12. University Paris-Saclay, Gif-sur-Yvette (France)
  13. Colorado State University, Fort Collins, CO (United States)
  14. University of Texas, Austin, TX (United States)
  15. Medical University of Vienna (Austria)
  16. Adam Mickiewicz University, Poznań (Poland)
  17. Bielefeld University (Germany)
  18. Carl von Ossietzky Universität Oldenburg (Germany); Martin Luther University Halle-Wittenberg, Halle (Germany)
  19. Western University of Health Sciences, Pomona, CA (United States); Xiamen University (China)
  20. Oklahoma State University, Stillwater, OK (United States)
  21. Institute of Plant and Microbial Biology, Taipei (Taiwan)
  22. Hong Kong Baptist University, Hong Kong (China)
  23. St. Bonaventure University, NY (United States)
+ Show Author Affiliations

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. 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. 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.

Research Organization:
Colorado State University, Fort Collins, CO (United States)
Sponsoring Organization:
USDOE Office of Science (SC), Biological and Environmental Research (BER); Biological Sciences Research Council (BBSRC); Scottish Government Rural and Environment Science and Analytical Services division (RESAS); National Science Foundation; National Institute of Health (NIH); Austrian Science Fund (FWF); French Agence Nationale de la Recherche; Japan Science and Technology Agency (JST); Core Research for Evolutionary Science and Technology (CREST); German Research Foundation (DFG); Research Grants Council (RGC) of Hong Kong
Grant/Contract Number:
SC0010733
OSTI ID:
1904476
Journal Information:
Genome Biology (Online), Journal Name: Genome Biology (Online) Journal Issue: 1 Vol. 23; ISSN 1474-760X
Publisher:
BioMed CentralCopyright Statement
Country of Publication:
United States
Language:
English

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Related Subjects

transcription start and end sites
59 BASIC BIOLOGICAL SCIENCES
Arabidopsis
Iso-seq
alternative polyadenylation
alternative splicing
reference transcript dataset
splice junction