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.

FAQ

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!

NEWS: CIRCpedia is an integrative database, aiming to annotating alternative back-splicing and alternative splicing in circRNAs across different cell lines. Welcome to use!

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 only in charge of annotating circRNA junctions according to the gene annotation. More functions could be found in CIRCexplorer2.

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

bash 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)

bash bamToFastq -i tophat/unmapped.bam -fq tophat/unmapped.fastq

• Unique mapping with TopHat-Fusion

bash 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.

Example 1 (single reads): bash STAR --chimSegmentMin 10 --runThreadN 10 --genomeDir hg19_STAR_index --readFilesIn RNA_seq.fastq

Example 2 (paired-end reads): bash STAR --chimSegmentMin 10 --runThreadN 10 --genomeDir hg19_STAR_index --readFilesIn read_1.fastq read_2.fastq

For more details about STAR, please refer to STAR manual.

Installation

Option 1: using pip

bash pip install CIRCexplorer

Option 2: via conda

CIRCexplorer is available as conda package with:

bash conda install circexplorer --channel bioconda

Option 3: in galaxy

If you have access to a Galaxy instance, CIRCexplorer is also available from the Galaxy Tool Shed.

Option 4: from source codes

1 Download CIRCexplorer bash git clone https://github.com/YangLab/CIRCexplorer.git cd CIRCexplorer

2 Install required packages bash pip install -r requirements.txt

3 Install CIRCexplorer python setup.py install

Usage

```bash 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

bash CIRCexplorer.py -f tophat_fusion/accepted_hits.bam -g hg19.fa -r ref.txt

STAR

• convert Chimeric.out.junction to fusion_junction.txt (star_parse.py was modified from STAR filterCirc.awk)

bash star_parse.py Chimeric.out.junction fusion_junction.txt

• parse fusion_junction.txt

bash 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.

bash fetch_ucsc.py hg19/hg38/mm10 ref/kg/ens/fa out

Example (download hg19 RefSeq gene annotation file):

bash 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

Zhang XO, Wang HB, Zhang Y, Lu X, Chen LL and Yang L. Complementary sequence-mediated exon circularization. Cell, 2014, 159: 134-147

License

Copyright (C) 2014-2017 YangLab. See the LICENSE file for license rights and limitations (MIT).