CLEAR/CIRCexplorer3

A computational pipeline for Circular and Linear RNA Expression Analysis from Ribosomal-RNA depleted (Ribo–) RNA-seq (CLEAR/CIRCexplorer3)

Schema

Installation requirements

• Software
  • CIRCexplorer2 (>=2.3.6)
  • HISAT2 (>=2.0.5)
  • StringTie (>1.3.6)
• Package (python 2.7 +)
  • pysam (>=0.8.4)
  • pybedtools

Installation

git clone https://github.com/YangLab/CLEAR
cd CLEAR
python ./setup.py install

Usage

Start from fastq file:

usage: clear_quant [-h] -1 M1 [-2 M2] -g GENOME -i HISAT -j BOWTIE1 -G GTF
              [-o OUTPUT] [-p THREAD]

optional arguments:
  -h, --help            Show this help message and exit.
  -1 M1                 Comma-separated list of read sequence files in FASTQ
                        format. When running with pair-end read, this should
                        contain #1 mates.
  -2 M2                 Comma-separated list of read sequence files in FASTQ
                        format. -2 is only used when running with pair-end
                        read. This should contain #2 mates.
  -g GENOME, --genome GENOME
                        Genome FASTA file.
  -i HISAT, --hisat HISAT
                        Index files for HISAT2.
  -j BOWTIE1, --bowtie1 BOWTIE1
                        Index files for TopHat-Fusion.
  -G GTF, --gtf GTF     Annotation GTF file.
  -o OUTPUT, --output OUTPUT
                        The output directory.
  -p THREAD, --thread THREAD
                        Running threads. [default: 5]

Start from CIRCexplorer2 output file:

usage: circ_quant [-h] -c CIRC -b BAM -r REF [--threshold THRESHOLD]
                     [--ratio RATIO] [-l] [-t] [-o OUTPUT]

optional arguments:
  -h, --help            Show this help message and exit.
  -c CIRC, --circ CIRC  Input circular RNA file from CIRCexplorer2.
  -b BAM, --bam BAM     Input mapped reads from HISAT2 in BAM format.
  -r REF, --ref REF     The refFlat format gene annotation file.
  --threshold THRESHOLD
                        Threshold of FPB for choose circRNAs to filter linear
                        SJ.[default: 1]
  --ratio RATIO         The ratio is used for adjust comparison between circ
                        and linear.[default: 1]
  -l, --length          Whether to consider all reads' length? [default: False]
  -t, --tmp             Keep tmp dir? [default: False]
  -o OUTPUT, --output OUTPUT
                        Output file. [default: circRNA_quant.txt]

Example

Start from fastq file:

clear_quant -1 mate_1.fastq -2 mate_2.fastq -g hg38.fa -i hg38.hisat_index -j hg38.bowtie_index -G annotation.gtf -o output_dir

Start from CIRCexplorer2 output file:

circ_quant -c CIRCexplorer2_output.txt -b hisat_aligned.bam -t -r annotation.refFlat -o quant.txt

hisat_aligned.bam should not contain unmapped reads.

Output

• output_dir/quant/quant.txt

Field	Description
chrom	Chromosome
start	Start of circular RNA
end	End of circular RNA
name	Circular RNA/Junction reads
score	Flag of fusion junction realignment
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	Type of circular RNA
geneName	Name of gene
isoformName	Name of isoform
index	Index of exon or intron
flankIntron	Left intron/Right intron
FPBcirc	Expression of circRNA
FPBlinear	Expression of cognate linear RNA
CIRCscore	Relative expression of circRNA

Citation

Ma XK*, Wang MR, Liu CX, Dong R, Carmichael GG, Chen LL and Yang L#. A CLEAR pipeline for direct comparison of circular and linear RNA expression. 2019, bioRxiv doi: 10.1101/668657

License

Copyright (C) 2019 YangLab. Licensed GPLv3 for open source use or contact YangLab (yanglab@@picb.ac.cn) for commercial use.