Everything you ever wanted to know about working with RNA-Seq data but were afraid to ask

  1. Do you work with RNA-Seq data?
  2. Do you plan to work with RNA-Seq data?
  3. Have you ever heard of RNA-Seq data?

If the answer to any of these questions is 'yes' (or even 'maybe') then you should definitely check out this fantastic online guide to all things RNA-Seq:


The RNA-seqlopedia provides an overview of RNA-seq and of the choices necessary to carry out a successful RNA-seq experiment

Written by Rodger Voelker and Clay Small of the Cresko Lab at the University of Oregon, it is a fantastically detailed, beautifully written resource to walk you through every step of working with RNA-Seq data.

I wish there were more online guides like this! Here's the Table of Contents, with the 'Analysis' section expanded, to give you a feeling for what it covers:

  1. Experimental Design
  2. RNA Preparation
  3. Library Preparation
  4. Sequencing
  5. Analysis
    • Overview
    • Initial Processing
    • Demultiplexing
    • Removing adapters
    • Trimming
    • Kmer Normalization
    • de Novo Assembly
    • de Bruijn Graph assembly
    • Overlap Layout Assembly
    • Aligning reads to a reference
    • Aligning to a ref. genome
    • Aligning to a transcriptome
    • microRNA Aligners
    • Short Read Aligners Output
    • Annotation of transcripts
    • Differential gene expression
    • Normalization
    • Discrete Discrete Models
    • Continuous Discrete Models
    • Nonparametric Models
    • Choice of Analysis Software
  6. References