Engelhard Christoph Andreas, Khani Sajjad, Derdak Sophia, Bilban Martin, Kornfeld Jan-Wilhelm
Department for Biochemistry and Molecular Biology (BMB), University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark.
Max Planck Institute for Metabolism Research, Gleueler Strasse 50, 50931 Cologne, Germany.
iScience. 2023 Jun 23;26(8):107190. doi: 10.1016/j.isci.2023.107190. eCollection 2023 Aug 18.
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 , , , , , and , validated by real-time PCR. The reannotated murine BAT transcriptome established here provides a framework for future investigations into the regulation of BAT.
可变转录通过每个基因表达多个转录本增加了转录组的复杂性。使用短读长测序对转录本进行注释和定量并非易事。长读长测序旨在通过对全长转录本进行测序来克服这些问题。棕色脂肪组织（BAT）产热的激活涉及主要的转录组重塑，并通过增加能量消耗对代谢产生积极影响。我们将牛津纳米孔技术（ONT）长读长测序方案与Illumina短读长测序进行了基准测试，评估了比对特征、基因和转录本检测与定量、差异基因和转录本表达、转录组重新注释以及差异转录本使用（DTU）。我们发现ONT测序在转录组重新组装方面优于Illumina，通过将 reads 明确地映射到转录本降低了假阳性事件的风险。我们鉴定了在冷激活的BAT中经历DTU的基因的新异构体，包括 、 、 、 、 和 ，通过实时PCR验证。这里建立的重新注释的小鼠BAT转录组为未来对BAT调节的研究提供了一个框架。
