Department of Agricultural Sciences, University of Naples Federico II, Portici, Italy.
CREA Research Centre for Vegetable and Ornamental Crops, Pontecagnano Faiano, Italy.
Methods Mol Biol. 2021;2264:137-162. doi: 10.1007/978-1-0716-1201-9_10.
RNA-sequencing, commonly referred to as RNA-seq, is the most recently developed method for the analysis of transcriptomes. It uses high-throughput next-generation sequencing technologies and has revolutionized our understanding of the complexity and dynamics of whole transcriptomes.In this chapter, we recall the key developments in transcriptome analysis and dissect the different steps of the general workflow that can be run by users to design and perform a mRNA-seq experiment as well as to process mRNA-seq data obtained by the Illumina technology. The chapter proposes guidelines for completing a mRNA-seq study properly and makes available recommendations for best practices based on recent literature and on the latest developments in technology and algorithms. We also remark the large number of choices available (especially for bioinformatic data analysis) in front of which the scientist may be in trouble.In the last part of the chapter we discuss the new frontiers of single-cell RNA-seq and isoform sequencing by long read technology.
RNA 测序，通常称为 RNA-seq，是分析转录组的最新发展方法。它使用高通量下一代测序技术，彻底改变了我们对整个转录组复杂性和动态性的理解。在本章中，我们回顾了转录组分析的关键进展，并剖析了用户可以运行的一般工作流程的不同步骤，以设计和执行 mRNA-seq 实验，并处理通过 Illumina 技术获得的 mRNA-seq 数据。本章提出了正确完成 mRNA-seq 研究的指南，并根据最新文献和技术及算法的最新发展提供了最佳实践建议。我们还注意到，科学家在面对大量（特别是生物信息数据分析）可供选择的情况下可能会感到困惑。在本章的最后一部分，我们讨论了单细胞 RNA-seq 和通过长读长技术进行异构体测序的新前沿。
