RNA-seq training PSB 2013

RNA-seq training PSB 2013


Alignment, Differential-expression, Feature-summarisation, Pre-processing, QC


Frederik Coppens (@frcop)


  • Lecture


This lecture gives an overview how to perform an RNA-seq experiment. First, the Illumina sequencing platform is briefly covered, followed by the different file formats in NGS. Next, an RNA-seq workflow is outlined, starting from the raw data up to differential expression. The recommended coverage and sequencing mode is touched upon as well as the effect on your analysis of divergence of your genotype compared to the reference.


The aim is that participants are aware of the general steps in an RNA-seq experiment and are able to communicate with e.g. service providers concerning their needs. The common formats used in NGS and their features are introduced. An overview of the different tools used in an RNA-seq analysis is given, including examples of the command line usage.


  • None

Target audience

Learning objectives

  • list sequencing requirements for your RNA-seq experiment
  • recognise different data formats used in NGS
  • apply and interpret quality control
  • list the different steps in an RNA-seq analysis


  • LaTeX Source.
  • Slides in pdf.


  • not applicable


half a day

Content stability

This content is to date still relevant. There are some new sequencing platforms and new tools, but nothing fundamentally changed. I expect this to stay like this for the foreseeable future.

Technical requirements

  • FastQC
  • FastX toolkit
  • Samtools
  • Picard tools
  • HTseq

Literature references

  • None


  • 2015-08-25: Added links to tools
  • 2015-02-19: Upload to gitlab
  • 2013-04-03: Generation of material


  • I did not check if the use of all figures is allowed or properly acknowledged.
  • A license needs to be added

Keywords: Alignment, Differential-expression, Feature-summarisation, Pre-processing, QC

Additional information

Authors: Frederik Coppens @frcop