Day 1 - RNA-Seq Analysis

Day 1 - RNA-Seq Analysis


Alignment, BAM, FASTA, FASTQ, QC



  • Both


Day 1 starts at the very beginning of a typical RNA-Seq workflow, explaining the sequencing technology and considerations for experimental design, then starts with hands-on application of working with sequencing data fresh off the sequencer.


This day covers an overview of Illumina sequencing technology and proper experimental design, a review of Unix/Linux, common file formats of sequence data, how to do sequencing QC and quality trimming, and an introduction to alignment.


  • Basic UNIX and how to submit jobs to a computing cluster

Target audience

Graduates students/post docs/beginning faculty

Learning objectives

  • Be able to describe the rough basics of Illumina sequencing and how to design an experiment
  • Be able to describe the first steps in a typical RNA-Seq workflow.
  • Be able to follow and modify UNIX scripts for QC and trimming


  • Lecture on Illumina sequencing and experimental design
  • Lecture on file formats, FastQC and trimming
  • Lecture on alignment theory
  • Review of Linux
  • Linux/R comparison sheet
  • Script templates
  • Example sequencing report
  • Explanation of strandedness


  • tarball of yeast genome, gene models, STAR index files and 12 fastq files
  • list of files in tarball
  • md5sum for the tarball


6 hours contact time; practicals intersperced with lectures; easily can fit into 1 day + lunch and breaks

Content stability

Should be stable

Technical requirements

  • UNIX server with FastQC >= 0.11.2, Trimmomatic >= 0.30,

Literature references

Keywords: Alignment, BAM, FASTA, FASTQ, QC

Additional information

Authors: Jenny Drnevich @jenny, Radhika Khetani @radhika, Jessica Kirkpatrick