Nanopore sequencing unveils the complexity of the cold-activated murine brown adipose tissue transcriptome

Christoph Andreas Engelhard; Sajjad Khani; Sophia Derdak; Martin Bilban; Jan-Wilhelm Kornfeld

doi:10.1016/j.isci.2023.107190

Nanopore sequencing unveils the complexity of the cold-activated murine brown adipose tissue transcriptome

iScience. 2023 Jun 23;26(8):107190. doi: 10.1016/j.isci.2023.107190. eCollection 2023 Aug 18.

Authors

Christoph Andreas Engelhard¹, Sajjad Khani^{2

3}, Sophia Derdak⁴, Martin Bilban⁵, Jan-Wilhelm Kornfeld¹

Affiliations

¹ Department for Biochemistry and Molecular Biology (BMB), University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark.
² Max Planck Institute for Metabolism Research, Gleueler Strasse 50, 50931 Cologne, Germany.
³ Cologne Excellence Cluster on Cellular Stress Responses in Ageing-Associated Diseases (CECAD), University of Cologne, Cologne, Germany.
⁴ Core Facilities, Medical University of Vienna, Lazarettgasse 14, 1090 Vienna, Austria.
⁵ Department of Laboratory Medicine & Core Facilities, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria.

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.

Keywords: Molecular biology; Omics; Sequence analysis; Transcriptomics.