Alignment for Circular RNA Fusion Junction Reads

CIRCexplorer2 supports TopHat2/TopHat-Fusion and other aligners (STAR, segemehl, BWA and MapSplice). Although different aligners showed slightly different in circular RNA identification, TopHat2/TopHat-Fusion has a perfect match with Cufflinks. As a result, TopHat2/TopHat-Fusion is recommended in alignment step, especially for circular RNA characterization pipeline.


Because TopHat2 needs gene annotation file for better alignment, you could select one GTF file from hg19_ref.gtf, hg19_kg.gtf and hg19_ens.gtf. In addition, TopHat2 needs genome index files for bowtie2, and TopHat-Fusion require indices for bowtie1, so you could index the genome sequence in advance or let CIRCexplorer2 align to do it from scratch. (See Setup)

  • From index files (bowtie1_index is the prefix for bowtie1 index files, and bowtie2_index is the prefix for bowtie2 index files):
CIRCexplorer2 align -G hg19_kg.gtf -i bowtie1_index -j bowtie2_index RNA_seq.fastq > CIRCexplorer2_align.log
  • Or from genome sequence:
CIRCexplorer2 align -G hg19_kg.gtf -g hg19.fa RNA_seq.fastq > CIRCexplorer2_align.log


  1. Because Cufflinks is well compatible with TopHat2/TopHat-Fusion, it is recommended to use TopHat2/TopHat-Fusion alignment for characterization pipeline.
  2. CIRCexplorer2 align will create a directory circ_out by default, and the BED file fusion_junction.bed under this directory is required for following analysis. You could also check tophat.log and tophat_fusion.log file for detailed logs of Tophat2 and TopHat-Fusion alignment.
  3. See Align for detailed information about CIRCexplorer2 align.
  4. If you have already had alignment results with TopHat2/TopHat-Fusion, you could use CIRCexplorer2 parse to convert their results compatible with CIRCexplorer2. For the alignment parameters of TopHat2/TopHat-Fusion, you could refer to CIRCexplorer manual.
CIRCexplorer2 parse -t TopHat-Fusion tophat_fusion/accepted_hits.bam > CIRCexplorer2_parse.log

Other aligners

1 Align sequencing reads to the reference genome. Commands for different aligners for detecting fusion junction reads are listed below, and you could modify them according to your different requirements.

STAR --chimSegmentMin 10 --runThreadN 10 --genomeDir hg19_STAR_index --readFilesIn RNA_seq.fastq
  • MapSplice (See MapSplice for more information) -p 10 -k 1 --non-canonical --fusion-non-canonical --min-fusion-distance 200 -c hg19_dir -x bowtie1_index --gene-gtf hg19_kg.gtf -1 RNA_seq.fastq
  • BWA (See BWA for more information)
bwa mem -T 19 -t 10 hg19_bwa_index RNA_seq.fastq > RNA_seq_bwa.sam
segemehl.x -q RNA_seq.fastq -d hg19.fa -i hg19_segemehl.idx -S -M 1 -t 10 -o RNA_seq.sam
testrealign.x -d hg19.fa -q RNA_seq.sam -n

2 Use CIRCexplorer2 parse to parse and convert fusion junction information.

  • STAR
CIRCexplorer2 parse -t STAR Chimeric.out.junction > CIRCexplorer2_parse.log
  • MapSplice
CIRCexplorer2 parse -t MapSplice mapsplice_out/fusions_raw.txt > CIRCexplorer2_parse.log
  • BWA
CIRCexplorer2 parse -t BWA RNA_seq_bwa.sam > CIRCexplorer2_parse.log
  • segemehl
CIRCexplorer2 parse -t segemehl splicesites.bed > CIRCexplorer2_parse.log


  1. You could align raw sequencing reads or unmapped reads from TopHat2 alignment (circ_out/tophat/unmapped.fastq).
  2. CIRCexplorer2 parse will create a directory circ_out by default, and the BED file fusion_junction.bed under this directory is required for following analysis.
  3. See Parse for detailed information about CIRCexplorer2 parse.