RNA‐seq: Applications and Best Practices [chapter]

Michele Araújo Pereira, Eddie Luidy Imada, Rafael Lucas Muniz Guedes
2017 Applications of RNA-Seq and Omics Strategies - From Microorganisms to Human Health  
RNA-sequencing (RNA-seq) is the state-of-the-art technique for transcriptome analysis that takes advantage of high-throughput next-generation sequencing. Although being a powerful approach, RNA-seq imposes major challenges throughout its steps with numerous caveats. There are currently many experimental options available, and a complete comprehension of each step is critical to make right decisions and avoid getting into inconclusive results. A complete workflow consists of: (1) experimental
more » ... ign; (2) sample and library preparation; (3) sequencing; and (4) data analysis. RNA-seq enables a wide range of applications such as the discovery of novel genes, gene/transcript quantification, and differential expression and functional analysis. This chapter will encompass the main aspects from sample preparation to downstream data analysis. It will be discussed how to obtain high-quality samples, replicates amount, library preparation, sequencing platforms and coverage, focusing on best recommended practices based on specialized literature. Basic techniques and well-known algorithms are presented and discussed, guiding both beginners and experienced users in the implementation of reliable experiments. Sanger sequencing [6], but with advances in next-generation sequencing technology (NGS), transcriptomic studies have evolved considerably and RNA-seq [7, 8] became the state-of-art for transcriptome analysis. RNA-seq consists of the direct sequencing of transcripts by NGS. Several NGS platforms [9] [10] [11] are commercially available nowadays. In general, an RNA set of interest is converted to a library of complementary DNA (cDNA) fragments and sequenced in a high-throughput manner. Compared to ESTs, RNA-seq provides better resolution and representativeness, whereas when compared to microarrays, the independence of reference sequences facilitates the discovery of novel genes and isoforms [8] . RNA-seq experiments harbors challenges from the experimental design to data analysis. Since a complete comprehension of each step is critical to make right decision, this chapter will encompass essential principles required for a successful RNA-seq experiment, focusing on best recommended practices based on specialized and recent literature. Basic techniques and well-known algorithms are presented and discussed, guiding both beginners and experienced users in the implementation of reliable experiments. Applications of RNA-Seq and Omics Strategies -From Microorganisms to Human Health RNA-seq: Applications and Best Practices
doi:10.5772/intechopen.69250 fatcat:bczbwcjb6rf2dhsthfrzuvi3gm