Genome-wide analyses and high-throughput screening was long reserved for biomedical applications and genetic model organisms. With the rapid development of massively parallel sequencing nanotechnology (or next-generation sequencing) and simultaneous maturation of bioinformatic tools, this situation has dramatically changed. Many in the eco-evolutionary sciences will be working with large-scale genomic data sets, and a basic understanding of the concepts and underlying methods is necessary to judge the work of others.
Here, the author briefly introduces next-generation sequencing and then focuses on transcriptome shotgun sequencing (RNA-Seq). This article gives a broad overview and provides practical guidance for the many steps involved in a typical RNA-Seq work flow from sampling, to RNA extraction, library preparation and data analysis. He focuses on principles, presents useful tools where appropriate and point out where caution is needed or progress to be expected. This tutorial is mostly targeted at beginners, but also contains potentially useful reflections for the more experienced.
- Wolf JB. (2013) Principles of transcriptome analysis and gene expression quantification: an RNA-seq tutorial. Mol Ecol Resour [Epub ahead of print]. [article]