6.3 Transcriptome reconstruction and expression using cufflinks

There are a number of tools that perform reconstruction of the transcriptome and for this workshop you are going to use cufflinks, which can do transcriptome assembly with and without a reference annotation. It also quantifies the isoform expression in FPKMs. Let’s import the cufflinks module and check its (rather long) list of parameters:

$ module load cufflinks/2.1.1
$ cufflinks --help

You are going to use cufflinks with the UCSC annotation file for zebrafish limiting the transcriptome reconstruction strictly to the available annotations. You could also use the annotations as a guide but letting the algorithm look also for transcribed loci not included in the annotations. In the first case cufflinks will only report isoforms that are included in the annotation, while in the latter case it will report novel isoforms as well. First, copy the annotation file from UCSC for chromosome 12 of Danio rerio:

$ cp –r /ibers/repository/public/courses/Rna-Seq/annotations .  

Then let’s create a folder for the cufflinks output storage:

$ mkdir cuff_out

Now you are ready to run cufflinks. The general format of the cufflinks command is:

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

where the input is the aligned reads (either in SAM or BAM format). Prepare a script file to launch cufflinks, by opening it with a text editor (you can call it for example cufflinks_2cells.sh). Copy the same header for the scheduler that you used previously, use the commands to load the required modules, and point the script to your working space. Your script should look something like this:

#$ -S /bin/sh
#$ -cwd
#$ -q amd.q,large.q,intel.q
#$ -l h_vmem=24G
#$ -e cff_12.e
#$ -N cff
#$ -o cff_12.o
module load cufflinks/2.2.1
cd /ibers/ernie/home/vpl/zebrafish

Then you can write (all in one line) the command:

cufflinks -o cuff_out_chr12/2cells -G annotations/Zebrafish_refGene_chr12.gtf \
-b /ibers/ernie/home/vpl/zebrafish/bt2Index/chr12.fa \
-u --library-type fr-unstranded /ibers/ernie/scratch/vpl/zebra_fish/tophat_out_chr12/accepted_hits.bam