Differential Expression Analysis Using CuffDiff

1. Files Needed

  1. Gene annotation GTF
  2. Aligned and sorted BAM files from tophat2

2. General Format

cuffdiff options genes.gtf condition1.bam condition2.bam

3. Run cuffdiff

cd ~/mydemo
cuffdiff --no-update-check -o cuffdiff_out –L wt,deletion mm9_chr1.gtf tophat_wt/accepted_hits.bam tophat_deletion/accepted_hits.bam

4. Parameters



Directory to write all results


Sample names separated by commas


Number of processors to be used


Do not check for new version. If not used, can 
cause cuffdiff to hang/freeze.


Correct for fragment bias to improve expression estimates


Correct for multi-mapping reads


5. cuffdiff output

a. Cuffdiff creates ~20 output files
b. Log files are named *.info
run.info – command used to run cuffdiff
Normalized expression values are found in *.fpkm_tracking files
Gene level → genes.fpkm_tracking
Isoform level → isoforms.fpkm_tracking
Tab-delimited files can be opened in excel

c. Expression level differences are found in *.diff files
Gene level  → gene_exp.diff
Isoform level → isoform_exp.diff

d. gene_exp.diff - Important columns

q-value: p-value adjusted for multiple testing
Log2(fold change): fold-difference between condition 1 & 2