...
Step 4: Cuffdiff
HOW WAS IT RUN?
| Code Block | ||
|---|---|---|
| ||
#If you have a local copy: cat run_commands/commands.cuffdiff cuffdiff -o diff_out -b reference/genome.fa -p 8 -L C1,C2 -u merged_asm/merged.gtf C1_R1_thout/accepted_hits.bam,C1_R2_thout/accepted_hits.bam,C1_R3_thout/accepted_hits.bam C2_R1_thout/accepted_hits.bam,C2_R2_thout/accepted_hits.bam,C2_R3_thout/accepted_hits.bam #If you don't have a local copy: cat /corral-repl/utexas/BioITeam/rnaseq_course/cufflinks_results/commands.cuffdiff |
| Code Block | ||
|---|---|---|
| ||
#If you have a local copy:
ls -l diff_out
#If you don't have a local copy:
cat /corral-repl/utexas/BioITeam/rnaseq_course/cufflinks_results/diff_out
-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
|
PARSING CUFFDIFF OUTPUT
Here is a basic command useful for parsing/sorting the gene_exp.diff or isoform_exp.diff files:
| Code Block | ||
|---|---|---|
| ||
cat diff_out/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.
| Code Block | ||
|---|---|---|
| ||
cat diff_out/gene_exp.diff |grep 'yes'|sort -k10nr,10|head
#Lets pull out just gene names
cat diff_out/gene_exp.diff |grep 'yes'|sort -k10nr,10|head|cut -f 3 |
| Expand | ||||
|---|---|---|---|---|
| ||||
Top 10 upregulated genes scf crc KdelR Nacalpha CG8979 CG4389 Vha68-2 regucalcin CG3835 CG1746 |
Exercise 1: Find the 10 most down-regulated genes, by fold change that are classified as significantly changed.
| Code Block | ||
|---|---|---|
| ||
cat diff_out/gene_exp.diff |grep 'yes'|sort -k10n,10|head|cut -f 3
|
| Expand | ||||
|---|---|---|---|---|
| ||||
Top 10 downregulated genes CG17065 Git CG18519 del CG13319 RNaseX25 msb1l achi CG15611 CG11309 |
Exercise 2: Find the 10 most up-regulated isoforms, by fold change that are classified as significantly changed. What genes do they belong to?
| Code Block | ||
|---|---|---|
| ||
cat diff_out/isoform_exp.diff |grep 'yes'|sort -k10nr,10|head
#To pull out gene names:
cat diff_out/isoform_exp.diff |grep 'yes'|sort -k10nr,10|head |
| Expand | ||||
|---|---|---|---|---|
| ||||
sick mub CG5021 akirin CG4587 c(3)G Ank2 CG1674 CrebB-17A stai |