Scripts required for running the pipeline to find circular RNAs from RNA-seq data, as used in
Jakub O. Westholm, Pedro Miura, Sara Olson, Sol Shenker, Brian Joseph, Piero Sanfilippo, Susan E. Celniker, Brenton R. Graveley, and Eric C. Lai. Genome-wide Analysis of Drosophila Circular RNAs Reveals Their Structural and Sequence Properties and Age-Dependent Neural Accumulation Westholm et al. Cell Reports, 2014.
These scripts have been tested on various Linux distributions. Before they can be run, make sure that the following prerequisites are installed:
- perl
- awk
- STAR (tested on version 2.7.9a)
- samtools
To run the scripts to identify circular RNAs, first run STAR, once for each data set. For paired end data, the command is
./runStar.pl --inFile1 [R1 fastq] --inFile2 [R2 fastq] --genomeDir [path to STAR genome] --maxMismatch [max mismatches realtive to read length, default 0.02] --outPrefix [output directory and prefix]Here --maxMismatch sets the outFilterMismatchNoverReadLmax parameter in STAR.
Next, run the post processing scripts. If there are STAR outputs for many data sets in the same folder, this command will process each of these in turn:
./postProcessStarAlignment.pl --starDir [directory with STAR results] --minLen [minimum length of circular RNAs] --outDir [output directory]For each library the following output files are produced:
a) _filteredJunctions.bed: A bed file with all circular junctions found by the pipeline. The score column indicates the number reads spanning each junction.
b) _s_filteredJunctions.bed: A bed file with those juction in (a) that are flanked by GT-AG splice sites. The score column indicates the number reads spanning each junction.
c) _s_filteredJunctions_fw.bed: A bed file with the same circular junctions as in file (b), but here the score column gives the average number of forward spliced reads at both splice sites around each circular junction.
d) (Sorted and indexed) bam file with all chimeric reads identified by STAR. The circRNA junction spanning reads are a subset of these.