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