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All these steps have already been run. We'll be spending time looking at the commands and output. Let's get set up.

Get to the

...

results

Code Block
titleGet set up
cds
cd my_rnaseq_course
cp /corral-repl/utexas/BioITeam/rnaseq_course/cufflinks_exerciseresults .

Step 1:

...

Tophat

We've already gone over how this is done and looked over the results. Lettophat results look here so let's move on to step 2. 

...

Code Block
titleGeneral syntax for cufflinks command
cufflinks [options] <hits.bam>

Some of the important options:
-p/--num-threads 
-G/--GTF
-g/--GTF-guide 
-b/--frag-bias-correct
-u/--multi-read-correct

...

HOW WAS IT RUN?

Expand
How do I look into the file?
How do I look into the file?
#If you've copied it over successfully:
cat run_commands/commands.cufflinks

...

Expand
How do I look into the file?How do I look into the file?
cat $BI
 
#If you don't have a local copy, you can read it from the source: 
cat /corral-repl/utexas/BioITeam/rnaseq_course/cufflinks_exerciseresults/run_commands/commands.cufflinks


HOW DOES THE OUTPUT LOOK?

 Take a minute to look at the output files produced by one cufflinks run.for one of our samples, C1_R1. The important file is transcripts.gtf, which contains Tophat's assembled junctions for C1_R1.

Code Block
titleCufflinks output files
cd $BI#If you have a local copy:
ls -l C1_R1_clout
 
 
#If you don't have a local copy:
ls -l /corral-repl/utexas/BioITeam/rnaseq_course/cufflinks_exercise/results/C1_R1_clout
ls
-l
-rw------- 1 daras G-801020   627673 May 17 16:58 genes.fpkm_tracking
-rw------- 1 daras G-801020  1021025 May 17 16:58 isoforms.fpkm_tracking
-rw------- 1 daras G-801020        0 May 17 16:50 skipped.gtf
-rw------- 1 daras G-801020 14784740 May 17 16:58 transcripts.gtf

Step 3:

...

Cuffmerge

HOW WAS IT RUN?

We first create created a file listing the paths of all per-sample transcripts.gtf files so far, then pass that to cuffmerge:

contents contents
Code Block
titleHow did we do that?
find . -name transcripts.gtf > assembly_list.txt
cuffmerge <assembly_list.txt>
Expand
 
#If you have a local copy:
cat assembly_list.txt


#If you don't have a local copy:
cat /corral-repl/utexas/BioITeam/rnaseq_course/cufflinks_results/assembly_list.txt
Code Block
titlecommands.cuffmerge file
cat
$BI/rnaseq
run_
course/cufflinks_exercise/
commands/commands.cuffmerge

 
     cuffmerge  -g reference/genes.exons.gtf  assembly_list.txt

...

 
HOW DOES THE OUTPUT LOOK?
The most important file is merged.gif, which contains the consensus transcriptome annotations cuffmerge has calculated.
 Code Block
titlecuffmerge output
cd $BI#If you have a local copy:
ls -l merged_asm
 
#If you don't have a local copy:
cat /corral-repl/utexas/BioITeam/rnaseq_course/cufflinks_exerciseresults/merged_asm
ls
-l

-rwxrwxr-x  1 daras G-803889  1571816 Aug 16  2012 genes.fpkm_tracking
-rwxrwxr-x  1 daras G-803889  2281319 Aug 16  2012 isoforms.fpkm_tracking
drwxrwxr-x  2 daras G-803889    32768 Aug 16  2012 logs
-r-xrwxr-x  1 daras G-803889 32090408 Aug 16  2012 merged.gtf
-rwxrwxr-x  1 daras G-803889        0 Aug 16  2012 skipped.gtf
drwxrwxr-x  2 daras G-803889    32768 Aug 16  2012 tmp
-rwxrwxr-x  1 daras G-803889 34844830 Aug 16  2012 transcripts.gtf

Step 4: 
...
 Code Block
cuffdiff [options] <merged.gtf> <sample1_rep1.bam,sample1_rep2.bam> <sample2_rep1.bam,sample2_rep2.bam>

Exercise: What does cuffdiff -b do?
...
-b is for enabling fragment bias correction.
 
...
Cuffdiff
HOW WAS IT RUN?

 
 Code Block
titlecommands.cuffmerge file
#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
titlecuffmerge output
#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

-rw-r--r-- 1 daras G-803889      115 May 18 09:48 cds.diff
-rw-r--r-- 1 daras G-803889      124 May 18 09:48 cds_exp.diff
-rw-r--r-- 1 daras G-803889       91 May 18 09:48 cds.fpkm_tracking
-rw-r--r-- 1 daras G-803889 27309056 May 18 13:42 cuffData.db
-rw-r--r-- 1 daras G-803889  1498907 May 18 09:48 gene_exp.diff
-rw-r--r-- 1 daras G-803889  1619931 May 18 09:48 genes.fpkm_tracking
-rw-r--r-- 1 daras G-803889  2623581 May 18 09:48 isoform_exp.diff
-rw-r--r-- 1 daras G-803889  3238044 May 18 09:48 isoforms.fpkm_tracking
-rw-r--r-- 1 daras G-803889  1168322 May 18 09:48 promoters.diff
-rw-r--r-- 1 daras G-803889  1346411 May 18 09:48 splicing.diff
-rw-r--r-- 1 daras G-803889  1730507 May 18 09:48 tss_group_exp.diff
-rw-r--r-- 1 daras G-803889  1887841 May 18 09:48 tss_groups.fpkm_tracking