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Circular RNA identification using CIRCexplorer2

1. Example Data

1.1. Working directory

Create a directory for the circular RNA exercises in your home directory and change to that directory

mkdir circ
cd circ

1.2. Get input data

Symbolically link all the CIRCexplorer input files to ‘circ’ folder

ln -s /home/username/Desktop/rnaseq_a/input/circ/* .

1.3. Input file contents

Look at the contents of the input files

head fusion_junction.bed
head ref.txt
head chr21.fa

2. Known CircRNA

a. Run the circexplorer to annotate the known junctions and then identify circular fusion junctions

CIRCexplorer2 annotate -r ref.txt -g chr21.fa /home/username/circ

b. Look at the contents of the output files. Look at the number of annotated and number of circular fusion junctions (use LibreOffice Calc, with Tab space delimiter)

3. denovo CircRNA

3.1. Transcript assembly

Perform transcript assembly using cufflinks for transcript assembly (need tophat output files). Cufflinks output needed for #3.2.

CIRCexplorer2 assemble -r ref.txt /home/username/circ

3.2. Novel CircRNA

Run the  CIRCexplorer2 denovo to identify novel circRNA and to perform alternative splicing of circular RNAs

CIRCexplorer2 denovo -r ref.txt -g chr21.fa /home/username/circ