Usage 1: circRNA quantifcation

Usage 1: circRNA quantifcation

Basic options

Usage:
  CIRIquant [options] --config <config> -1 <m1> -2 <m2>

  <config>          Config file
  <m1>              Input mate1 reads (for paired-end data)
  <m2>              Input mate2 reads (for paired-end data)


Options (defaults in parentheses):

  -v                Run in verbose mode
  -o, --out          Output directory (default: current directory)
  -e, --log         Specific log file (default: sample_prefix.log)
  -p, --prefix      Output sample prefix (default: input sample name)
  -t, --threads     Number of CPU threads to use (defualt: 4)
  -a, --anchor      Minimum anchor length for junction alignment (default: 5)
  -l, --libary-type Library type, 0: unstranded, 1: read1 match the sense strand, 2: read1 match the antisense strand (default: 0)

  --bed             User provided Back-Spliced Junction Site in BED format
  --circ            circRNA prediction results from other tools
  --tool            Tool name, required when --circ is specified ([CIRI2/CIRCexplorer2/DCC/KNIFE/MapSplice/UROBORUS/circRNA_finder/find_circ])

  --RNaseR          CIRIquant output file of RNase R data (required for RNase R correction)
  --bam             Specific hisat2 alignment bam file against reference genome
  --no-gene         Skip StringTie estimation of gene abundance

NOTE:

• For now, --circ and --tool options support results from CIRI2 / CIRCexplorer2 / DCC / KNIFE / MapSplice / UROBORUS / circRNA_finder / find_circ
• For tools like DCC and circRNA_finder, please manually remove duplicated circRNAs with same junction postion but have opposite strands.
• Gene expression values are needed for normalization, do not use --no-gene if you need to run DE analysis afterwards.

Example config

A YAML-formated config file is needed for CIRIquant to find software and reference needed. A valid example of config file is demonstrated below.

// Example of config file
name: hg19
tools:
  bwa: /home/zhangjy/bin/bwa
  hisat2: /home/zhangjy/bin/hisat2
  stringtie: /home/zhangjy/bin/stringtie
  samtools: /home/zhangjy/bin/samtools

reference:
  fasta: /home/zhangjy/Data/database/hg19.fa
  gtf: /home/zhangjy/Data/database/gencode.v19.annotation.gtf
  bwa_index: /home/zhangjy/Data/database/hg19/_BWAtmp/hg19
  hisat_index: /home/zhangjy/Data/database/hg19/_HISATtmp/hg19

Key	Description
name	the name of config file
bwa	the path of bwa
hisat2	the path of hisat2
stringtie	the path of stringite
samtools	the path of samtools, samtools version below 1.3.1 is not supported
fasta	reference genome fasta, a fai index by samtools faidx is also needed under the same directory
gtf	annotation file of reference genome in GTF/GFF3 format
bwa_index	prefix of BWA index for reference genome
hisat_index	prefix of HISAT2 index for reference genome

For quantification of user-provided circRNAs, a list of junction sites in bed format is required, for example:

chr1    10000   10099   chr1:10000|10099    .   +
chr1    31000   31200   chr1:31000|31200    .   -

Example Usage

Recommended: Predict circRNAs using CIRI2 (packaged in CIRIquant)

CIRIquant -t 4 \
          -1 ./test_1.fq.gz \
          -2 ./test_2.fq.gz \
          --config ./chr1.yml \
          -o ./test \
          -p test

Quantify circRNAs using provided BED format input

CIRIquant -t 4 \
          -1 ./test_1.fq.gz \
          -2 ./test_2.fq.gz \
          --config ./chr1.yml \
          -o ./test \
          -p test \
          --bed your_circRNAs.bed

Quantify circRNAs using results from other tools

For example, if you have find_circ results of predicted circRNAs.

CIRIquant -t 4 \
          -1 ./test_1.fq.gz \
          -2 ./test_2.fq.gz \
          --config ./chr1.yml \
          -o ./test \
          -p test \
          --circ find_circ_results.txt \
          --tool find_circ

Output format

The main output of CIRIquant is a GTF file, that contains detailed information of BSJ and FSJ reads of circRNAs and annotation of circRNA back-spliced regions in the attribute columns

Description of each columns's value

column	name	description
1	chrom	chromosome / contig name
2	source	CIRIquant
3	type	circRNA
4	start	5' back-spliced junction site
5	end	3' back-spliced junction site
6	score	CPM of circRNAs (#BSJ / #Mapped reads)
7	strand	strand information
8	.	.
9	attributes	attributes seperated by semicolon

The attributes containing several pre-defined keys and values:

key	description
circ_id	name of circRNA
circ_type	circRNA types: exon / intron / intergenic
bsj	number of bsj reads
fsj	number of fsj reads
junc_ratio	circular to linear ratio: 2 * bsj / ( 2 * bsj + fsj)
rnaser_bsj	number of bsj reads in RNase R data (only when --RNaseR is specificed)
rnaser_fsj	number of fsj reads in RNase R data (only when --RNaseR is specificed)
gene_id	ensemble id of host gene
gene_name	HGNC symbol of host gene
gene_type	type of host gene in gtf file