Abstract
Alternative transcription increases transcriptome complexity by expression of multiple transcripts per gene. Annotation and quantification of transcripts using short-read sequencing is non-trivial. Long-read sequencing aims at overcoming these problems by sequencing full-length transcripts. Activation of brown adipose tissue (BAT) thermogenesis involves major transcriptomic remodeling and positively affects metabolism via increased energy expenditure. We benchmark Oxford Nanopore Technology (ONT) long-read sequencing protocols to Illumina short-read sequencing assessing alignment characteristics, gene and transcript detection and quantification, differential gene and transcript expression, transcriptome reannotation, and differential transcript usage (DTU). We find ONT sequencing is superior to Illumina for transcriptome reassembly, reducing the risk of false-positive events by unambiguously mapping reads to transcripts. We identified novel isoforms of genes undergoing DTU in cold-activated BAT including Cars2, Adtrp, Acsl5, Scp2, Aldoa, and Pde4d, validated by real-time PCR. The reannotated murine BAT transcriptome established here provides a framework for future investigations into the regulation of BAT.
| Originalsprog | Engelsk |
|---|---|
| Artikelnummer | 107190 |
| Tidsskrift | iScience |
| Vol/bind | 26 |
| Udgave nummer | 8 |
| Antal sider | 24 |
| ISSN | 2589-0042 |
| DOI | |
| Status | Udgivet - 18. aug. 2023 |
Bibliografisk note
Funding Information:This work was supported by a European Research Council starting grant (TransGen RNA, 675014), a Novo Nordisk Foundation grant (Adiposign, NNF18OC0033444) and a research grant from the Danish Diabetes Academy, which is funded by the Novo Nordisk Foundation (NNF12SA1016522). ONT sequencing was performed at the Core Facilities of the Medical University of Vienna, a member of VLSI. We thank Markus Jeitler (Core Facilities, Medical University of Vienna) for help with ONT library preparation and sequencing, and Bjørk Ditlev Marcher Larsen and Ajeetha Josephrajan (Department for Biochemistry and Molecular Biology, University of Southern Denmark) for generating and visualizing the Cars2 structure predictions, respectively. The wt1-SAM brown preadipocyte cell line as well as the empty sgRNA vector have been generated as described in Lundh et al.65 and were kind gifts of Brice Emanuelli. Conceptualization, C.A.E. M.B. J.-W.K.; Software, C.A.E. S.D. Validation, C.A.E. M.B. J.-W.K.; Formal Analysis, C.A.E.; Investigation, C.A.E. S.K.; Data Curation, C.A.E. S.D.; Visualization, C.A.E.; Writing—Original Draft Preparation, C.A.E.; Writing—Review and Editing, C.A.E. M.B. J.-W.K.; Supervision, M.B. J.-W.K.; Resources, M.B. J.-W.K.; Project Administration, M.B. J.-W.K.; Funding Acquisition, M.B. J.-W.K. J.-W.K supervised and provided resources for the generation of in vitro and in vivo samples and the short-read sequencing, M.B. supervised and provided resources for the long-read sequencing. Both contributed equally in the supervision of the data analysis and the preparation of the manuscript. All authors have read and agreed to the published version of the manuscript. The authors declare no competing interest. We support inclusive, diverse, and equitable conduct of research.
Funding Information:
This work was supported by a European Research Council starting grant (TransGen RNA, 675014 ), a Novo Nordisk Foundation grant (Adiposign, NNF18OC0033444 ) and a research grant from the Danish Diabetes Academy , which is funded by the Novo Nordisk Foundation ( NNF12SA1016522 ).
Publisher Copyright:
© 2023 The Author(s)