Developmental Genomics Laboratory, Department of Anatomy, School of Biomedical Sciences, University of Otago, Dunedin, New Zealand.
Methods Mol Biol. 2022;2450:599-615. doi: 10.1007/978-1-0716-2172-1_32.
The decrease in sequencing costs and technology improvements has led to the adoption of RNA-sequencing to profile transcriptomes from further non-traditional regeneration model organisms such as the colonial ascidian Botrylloides leachii. The relatively unbiased way in which transcripts are identified and quantified makes this technique suitable to detect large-scale changes in expression, and the identification of novel transcripts and isoforms. Of particular interest to many researchers is the discovery of differentially expressed transcripts across different treatment conditions or stages of regeneration. This protocol describes a workflow starting from processing raw sequencing reads, mapping reads, assembly of transcripts, and measuring their abundance, creating lists of differentially expressed genes and their biological interpretation using gene ontologies. All programs used in this protocol are open-source software tools and freely available.
测序成本的降低和技术的改进使得 RNA 测序技术得以应用，以分析进一步的非传统再生模式生物（如群居海鞘 Botrylloides leachii）的转录组。这种技术可以识别和定量大量的转录本，并且相对无偏倚，因此非常适合检测表达水平的大规模变化，以及新转录本和异构体的鉴定。对于许多研究人员来说，特别感兴趣的是在不同处理条件或再生阶段发现差异表达的转录本。本方案描述了一个从处理原始测序读段、映射读段、转录本组装以及测量其丰度开始的工作流程，创建了差异表达基因列表，并使用基因本体论对其进行生物学解释。本方案中使用的所有程序都是开源软件工具，并且可以免费获得。
