CIRCexplorer
CIRCexplorer is a combined strategy to identify junction reads from back spliced exons and intron lariats.
Version: 1.1.10
Last Modified: 2016-7-14
Authors: Xiao-Ou Zhang (zhangxiaoou@picb.ac.cn), Li Yang (liyang@picb.ac.cn)
Maintainer: Xu-Kai Ma (maxukai@picb.ac.cn)
Download the latest stable version of CIRCexplorer
To see what has changed in recent versions of CIRCexplorer, see the CHANGELOG.
After one year’s tensive development, we have upgraded and extended CIRCexplorer to a new version – CIRCexplorer2, with many improvements and lots of new features. Welcome to use and cite it!
A schematic flow shows the pipeline
Notice
CIRCexplorer is now only a circular RNA annotating tool, and it parses fusion junction information from mapping results of other aligners. The result of circular RNA annotating is directly dependent on the mapping strategy of aligners. Different aligners may have different circular RNA annotations. CIRCexplorer is now only in charge of giving fusion junctions a correct gene annotation. Other functions and supports for more aligners are under tensive developments. Thanks for your supports and understanding!
Prerequisites
Software / Package
TopHat or STAR
- TopHat & TopHat-Fusion
- TopHat 2.0.9
- TopHat-Fusion included in TopHat 2.0.9
- STAR (optional)
- STAR 2.4.0j
Others
- bedtools
- pysam >=0.8.2
- docopt
- interval (If you use the old version of CIRCexplorer, you require to install the interval package from https://github.com/kepbod/interval. For the latest version of CIRCexplorer, you won’t install it by yourself.)
RNA-seq
The poly(A)−/ribo− RNA-seq is recommended. If you want to obtain more circular RNAs, RNase R treatment could be performed.
Aligner
CIRCexplorer was originally developed as a circular RNA analysis toolkit supporting TopHat & TopHat-Fusion. After version 1.1.0, it also supports STAR.
TopHat & TopHat-Fusion
To obtain junction reads for circular RNAs, two-step mapping strategy was exploited:
- Multiple mapping with TopHat
tophat2 -a 6 --microexon-search -m 2 -p 10 -G knownGene.gtf -o tophat hg19_bowtie2_index RNA_seq.fastq
- Convert unmapped reads (using bamToFastq from bedtools)
bamToFastq -i tophat/unmapped.bam -fq tophat/unmapped.fastq
- Unique mapping with TopHat-Fusion
tophat2 -o tophat_fusion -p 15 --fusion-search --keep-fasta-order --bowtie1 --no-coverage-search hg19_bowtie1_index tophat/unmapped.fastq
STAR
To detect fusion junctions with STAR, --chimSegmentMin should be set to a positive value. For more details about STAR, please refer to STAR manual.
Installation
Option 1: using pip
pip install CIRCexplorer
Option 2: via conda
CIRCexplorer is available as conda package with:
conda install circexplorer --channel bioconda
Option 3: from docker container
docker run -it --rm quay.io/mulled/circexplorer:1.1.9--py35_0
Option 4: in galaxy
If you have access to a Galaxy instance, CIRCexplorer is also available from the Galaxy Tool Shed.
Option 5: from source codes
1 Download CIRCexplorer
git clone https://github.com/YangLab/CIRCexplorer.git
cd CIRCexplorer
2 Install required packages
pip install -r requirements.txt
3 Install CIRCexplorer
python setup.py install
Usage
CIRCexplorer.py 1.1.10 -- circular RNA analysis toolkits.
Usage: CIRCexplorer.py [options]
Options:
-h --help Show this screen.
--version Show version.
-f FUSION --fusion=FUSION TopHat-Fusion fusion BAM file. (used in TopHat-Fusion mapping)
-j JUNC --junc=JUNC STAR Chimeric junction file. (used in STAR mapping)
-g GENOME --genome=GENOME Genome FASTA file.
-r REF --ref=REF Gene annotation.
-o PREFIX --output=PREFIX Output prefix [default: CIRCexplorer].
--tmp Keep temporary files.
--no-fix No-fix mode (useful for species with poor gene annotations)
Example
TopHat & TopHat-Fusion
CIRCexplorer.py -f tophat_fusion/accepted_hits.bam -g hg19.fa -r ref.txt
STAR
- convert
Chimeric.out.junctiontofusion_junction.txt(star_parse.pywas modified from STAR filterCirc.awk)
star_parse.py Chimeric.out.junction fusion_junction.txt
- parse
fusion_junction.txt
CIRCexplorer.py -j fusion_junction.txt -g hg19.fa -r ref.txt
Note
- ref.txt is in the format (Gene Predictions and RefSeq Genes with Gene Names) below (see details in the example file)
| Field | Description |
|---|---|
| geneName | Name of gene |
| isoformName | Name of isoform |
| chrom | Reference sequence |
| strand | + or - for strand |
| txStart | Transcription start position |
| txEnd | Transcription end position |
| cdsStart | Coding region start |
| cdsEnd | Coding region end |
| exonCount | Number of exons |
| exonStarts | Exon start positions |
| exonEnds | Exon end positions |
-
hg19.fa is genome sequence in FASTA format.
-
You could use fetch_ucsc.py script to download relevant ref.txt (Known Genes, RefSeq or Ensembl) and the genome fasta file for hg19, hg38 or mm10 from UCSC.
fetch_ucsc.py hg19/hg38/mm10 ref/kg/ens/fa out
Example (download hg19 RefSeq gene annotation file):
fetch_ucsc.py hg19 ref ref.txt
Output
See details in the example file
| Field | Description |
|---|---|
| chrom | Chromosome |
| start | Start of junction |
| end | End of junction |
| name | Circular RNA/Junction reads |
| score | Flag to indicate realignment of fusion junctions |
| strand | + or - for strand |
| thickStart | No meaning |
| thickEnd | No meaning |
| itemRgb | 0,0,0 |
| exonCount | Number of exons |
| exonSizes | Exon sizes |
| exonOffsets | Exon offsets |
| readNumber | Number of junction reads |
| circType | ‘Yes’ for ciRNA, and ‘No’ for circRNA (before 1.1.0); ‘circRNA’ or ‘ciRNA’ (after 1.1.1) |
| geneName | Name of gene |
| isoformName | Name of isoform |
| exonIndex/intronIndex | Index (start from 1) of exon (for circRNA) or intron (for ciRNA) in given isoform (newly added in 1.1.6) |
| flankIntron | Left intron/Right intron |
Note: The first 12 columns are in BED12 format.
Citation
License
Copyright (C) 2014-2016 YangLab. See the LICENSE file for license rights and limitations (MIT).