1. Files Needed
- Gene annotation GTF
- 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
-o |
Directory to write all results |
-L |
Sample names separated by commas |
-p |
Number of processors to be used |
--no-update-check |
Do not check for new version. If not used, can |
-b |
Correct for fragment bias to improve expression estimates |
-u |
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