Guidelines for Setting Up a mRNA Sequencing Experiment and Best Practices for Bioinformatic Data Analysis.

Abstract

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.