Transcript Assembly with Cufflinks

Progress

• populate this page accordingly https://wikis.utexas.edu/display/bioiteam/Differential+expression+with+splice+variant+analysis+aug2012#Differentialexpressionwithsplicevariantanalysisaug2012-Step2:Runcufflinks
• more on exercises https://wikis.utexas.edu/display/bioiteam/Tuxedo+Suite+For+Splice+Variant+Analysis+and+Identifying+Novel+Transcripts+II
• examining output file https://wikis.utexas.edu/display/bioiteam/Introduction+to+RNA+Seq+Short+Course+Commands *https://wiki.hpcc.msu.edu/display/TEAC/2015-09-21%3A+RNA-Sequencing+Tools?src=search

Issue

• http://seqanswers.com/forums/showthread.php?t=31074
• https://groups.google.com/forum/#!topic/tuxedo-tools-users/CnCmSlvC9Xc
• http://seqanswers.com/forums/showthread.php?t=31074

Cufflinks

Cufflinks assembles transcripts, estimates their abundances, and tests for differential expression and regulation in RNA-Seq samples. It accepts aligned RNA-Seq reads and assembles the alignments into a parsimonious set of transcripts. Cufflinks then estimates the relative abundances of these transcripts based on how many reads support each one, taking into account biases in library preparation protocols.

• Cufflinks manual

Running Cufflinks

Cufflinks command usage:

cufflinks [options] <aligned_reads.(sam/bam)>

Example of basic cufflinks command:

cufflinks -G <gtffile> -o <outputdirectory> accepted_hits.bam

Input:

• accepted_hits.bam : bam file produced by tophat
• gtffile : gtf file with transcript information
• outputdirectory: Directory to place output files in

Output: See link for more details on Cufflinks output files format

Cufflinks produces three output files:

• transcripts.gtf : This GTF file contains Cufflinks’ assembled isoforms.
• isoforms.fpkm_tracking : This file contains the estimated isoform-level expression values in the generic FPKM Tracking Format.
• genes.fpkm_tracking : This file contains the estimated gene-level expression values in the generic FPKM Tracking Format.

Running on MGHPCC

Step 1| Read alignment with tophat2 which we did in previous section.

Step 2| Transcript Assembly with cufflinks

To assemble transcripts for each sample, first create a script, cufflinks_job.sh in the current directory where we have the Tophat2 outputs.

#!/bin/bash

#BSUB -q short
#BSUB -W 5:00
#BSUB -R "rusage[mem=5125]"
#BSUB -J "cufflinks_job"
#BSUB -o cufflinks.out
#BSUB -e cufflinks.err

module load samtools/1.3
module load cufflinks/2.2.1

cufflinks -p 8 -o C1_R1_clout C1_R1_thout/accepted_hits.bam
cufflinks -p 8 -o C1_R2_clout C1_R2_thout/accepted_hits.bam
cufflinks -p 8 -o C1_R3_clout C1_R3_thout/accepted_hits.bam
cufflinks -p 8 -o C2_R1_clout C2_R1_thout/accepted_hits.bam
cufflinks -p 8 -o C2_R2_clout C2_R2_thout/accepted_hits.bam
cufflinks -p 8 -o C2_R3_clout C2_R3_thout/accepted_hits.bam

Again, this has been run for you and the generated output is in your directory Submit the job:

bsub < cufflinks_job.sh

When the job is done, go to one of the output directory, say C1_R1_clout

cd ./C1_R1_clout
ls 
# You should see all the files as listed below:
genes.fpkm_tracking  isoforms.fpkm_tracking  transcripts.gtf
```

### Step 3| Merging assemblies using cuffmerge 
Create a file listing the paths of all per-sample transcripts.gtf files, then pass that to cuffmerge:

`nano assemblies.txt`

Write in the file the following lines and save it.
```
./C1_R1_clout/transcripts.gtf 
./C2_R2_clout/transcripts.gtf 
./C1_R2_clout/transcripts.gtf 
./C2_R1_clout/transcripts.gtf 
./C1_R3_clout/transcripts.gtf 
./C2_R3_clout/transcripts.gtf
```

Then, run Cuffmerge on all your assemblies to create a single merged transcriptome annotation: 
```
cuffmerge -g genes.gtf -s genome.fa -p 8 assemblies.txt
```

Take a look at the output files produced by cuffmerge in `./merged_sam`
```
ls ./merged_sam
# You should see all the files as listed below:
genes.fpkm_tracking
isoforms.fpkm_tracking
logs 
merged.gtf
skipped.gtf
tmp
transcripts.gtf
```

The most important file is merged.gif, which contains the consensus transcriptome annotations cuffmerge has calculated.

Next up, we will identify differentially expressed genes and isoforms using cuffdiff.

---

| [[Previous Section|Read Alignment with TopHat2]] | [[This Section|Transcript Assembly with Cufflinks]] | [[Next Section|Differential Analysis with Cuffdiff]] |
|:------------------------------------:|:--------------------------:|:--------------------------------------------:|
| [[Read Alignment with TopHat2]]| [[Transcript Assembly with Cufflinks]]| [[Differential Analysis with Cuffdiff]]