RNA-Seq Work Flows

Martin Morgan, Sonali Arora
February 3, 2015

7-step work flow

1. Experimental design

Keep it simple


Avoid confounding experimental factors with other factors

Record co-variates

Be aware of batch effects

2. Wet-lab

Confounding factors

Artifacts of your particular protocols

3. Sequencing

Axes of variation

Application-specific, e.g.,

4. Alignment

Alignment strategies

Splice-aware aligners (and Bioconductor wrappers)

(5a. Bowtie2 / tophat / Cufflinks / Cuffdiff)

5. Reduction to 'count tables'

Step 6. Analysis



Appropriate error model


Borrowing information

Step 7. Comprehension

Placing differentially expressed regions in context


The lab is based on a modified version of the RNA-seq work flow developed by Michael Love, Simon Anders, Wolfgang Huber.