Cuffdiff

The syntax is below:

     cuffdiff [options] <gene_counts.gff> <sample1_rep1.bam,sample1_rep2.bam> <sample2_rep1.bam,sample2_rep2.bam>

Exercise: Explore some options. What does cuffdiff -b do?

Solution

-b is for enabling fragment bias correction.

We will be inspecting the cuffdiff results further in a little bit.

Examine the differential expression analysis results

cds
cp -r $BI/ngs_course/tophat_cufflinks/diff_out .
ls diff_out

Take a minute to look at the output files produced by cuffdiff.

cuffdiff output

cds
cd diff_out
ls -l

-rwxr-x--- 1 daras G-801020  2691192 Aug 21 12:20 isoform_exp.diff  : Differential expression testing for transcripts
-rwxr-x--- 1 daras G-801020  1483520 Aug 21 12:20 gene_exp.diff     : Differential expression testing for genes
-rwxr-x--- 1 daras G-801020  1729831 Aug 21 12:20 tss_group_exp.diff: Differential expression testing for primary transcripts
-rwxr-x--- 1 daras G-801020  1369451 Aug 21 12:20 cds_exp.diff      : Differential expression testing for coding sequences

-rwxr-x--- 1 daras G-801020  3277177 Aug 21 12:20 isoforms.fpkm_tracking
-rwxr-x--- 1 daras G-801020  1628659 Aug 21 12:20 genes.fpkm_tracking
-rwxr-x--- 1 daras G-801020  1885773 Aug 21 12:20 tss_groups.fpkm_tracking
-rwxr-x--- 1 daras G-801020  1477492 Aug 21 12:20 cds.fpkm_tracking

-rwxr-x--- 1 daras G-801020  1349574 Aug 21 12:20 splicing.diff  : Differential splicing tests
-rwxr-x--- 1 daras G-801020  1158560 Aug 21 12:20 promoters.diff : Differential promoter usage
-rwxr-x--- 1 daras G-801020   919690 Aug 21 12:20 cds.diff       : Differential coding output.

Here is a basic command useful for parsing/sorting the gene_exp.diff or isoform_exp.diff files:

Linux one-liner for sorting cuffdiff output by log2 fold-change values

cat isoform_exp.diff | awk '{print $10 "\t" $4}' | sort -n -r | head

Exercise 1: Find the 10 most up-regulated genes, by fold change that are classified as significantly changed. Look at one example of a up-regulated gene, regucalcin, on IGV.

Solution

Top 10 upregulated genes
scf
CG8979
CG4389
crc
KdelR
Vha68-2
CG3835
Df31
by
Dhpr

Hint

One-line command to get 10 most up regulated genes

cat gene_exp.diff |grep 'yes'|sort -k10nr,10|head

One-line command to get 10 most down regulated genes

cat gene_exp.diff |grep 'yes'|sort -k10n,10|head

Exercise 2: Find the 10 most up-regulated isoforms, by fold change that are classified as significantly changed. What genes do they belong to?

Solution

simj
CG2177
sPLA2
Nipsnap
Pde8
by
CG15814
Dhpr
eIF-4E
spir

Hint

One-line command to get 10 most up-regulated isoforms

cat isoform_exp.diff |grep 'yes'|sort -k10nr,10|head