Usage

Usage

Basic usage

usage: CIRI-long [-h] [-v] {call,collapse} ...

positional arguments:
  {call,collapse}  commands

optional arguments:
  -h, --help       show this help message and exit
  -v, --version    show program's version number and exit

CIRI-long have two main functions, including (1) candidate circRNAs identification and (2) isoform collapsing.

Step1. circRNA identification

Basic options

usage: CIRI-long call [-h] [-i READS] [-o DIR] [-r REF] [-p PREFIX] [-a GTF] [--canonical] [-t INT] [--debug]

optional arguments:
  -h, --help            show this help message and exit
  -i READS, --in READS  Input reads.fq.gz
  -o DIR, --out DIR     Output directory, default: ./
  -r REF, --ref REF     Reference genome FASTA file
  -p PREFIX, --prefix PREFIX
                        Output sample prefix, (default: CIRI-long)
  -a GTF, --anno GTF    Genome reference gtf
  --canonical           Use canonical splice signal (GT/AG) only, default: True)
  -t INT, --threads INT
                        Number of threads
  --debug               Run in debugging mode, (default: False)

NOTE:

• A bwa index for reference genome is required, please use bwa index command to generate bwa index before running CIRI-long.

Example Usage:

Demo dataset can be downloaded from the GitHub release

# Download demo dataset
wget https://github.com/bioinfo-biols/CIRI-long/releases/download/v0.6-alpha/CIRI-long_test_data.tar.gz

# Decompress demo dataset
tar zxvf CIRI-long_test_data.tar.gz
cd test_data

# Build bwa index before running CIRI-long
bwa index -a bwtsw mm10_chr12.fa mm10_chr12.fa

# Run CIRI-long to identify circular reads from sequencing reads
CIRI-long call -i test_reads.fa \
               -o ./test_call \
               -r mm10_chr12.fa \
               -p test \
               -a mm10_chr12.gtf \
               -t 8

Output Files

The output directory should have the following structure:

test_call
├── test.cand_circ.fa
├── test.json
├── test.log
├── test.low_confidence.fa
└── tmp
    ├── ss.idx
    ├── test.ccs.fa
    └── test.raw.fa

1 directory, 7 files

Step2. isoform collapse

Basic Options

usage: CIRI-long collapse [-h] [-i LIST] [-o DIR] [-p PREFIX] [-r REF] [-a GTF] [--canonical] [-t INT] [--debug]

optional arguments:
  -h, --help            show this help message and exit
  -i LIST, --in LIST    Input list of CIRI-long results
  -o DIR, --out DIR     Output directory, default: ./
  -p PREFIX, --prefix PREFIX
                        Output sample prefix, (default: CIRI-long)
  -r REF, --ref REF     Reference genome FASTA file
  -a GTF, --anno GTF    Genome reference gtf
  --canonical           Use canonical splice signal (GT/AG) only, default: True)
  -t INT, --threads INT
                        Number of threads
  --debug               Run in debugging mode, (default: False)

One should provide a text file listing sample name and path to CIRI-long output files *.cand_circ.fa, seperated by space.

sample1_name /path/to/sample1/cand_circ.fa
sample2_name /path/to/sample2/cand_circ.fa

Example Usage

For exmaple, you can create a file name test.lst with the following content:

test ./test_call/test.cand_circ.fa

Then run CIRI-long collapse to aggregate results from one or multiple samples.

 CIRI-long collapse -i ./test.lst \
                    -o ./test_collpase \
                    -p test \
                    -r ./mm10_chr12.fa \
                    -a ./mm10_chr12.gtf \
                    -t 8

Output Files

The output directory should have the following structure:

test_collpase
├── test_collpase.expression
├── test_collpase.info
├── test_collpase.log
├── test_collpase.reads
└── tmp
    ├── ss.idx
    └── test_collpase.corrected.pkl

1 directory, 6 files

Output Format

The main output

The main output of CIRI-long is a GTF file (e.g. test_collpase.info), that contains detailed information 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	CIRI-long
3	type	circRNA
4	start	5' back-spliced junction site
5	end	3' back-spliced junction site
6	score	Number of total supported 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
splice_site	splicing signal of candidate circRNAs and numbers indicating shifted bases of aligned and annotated splice site. (e.g. AG-GT|0-5)
equivalent_seq	equivalent sequence of splice site
circ_type	circRNA types: exon/intron/intergenic
circ_len	length of the major isoform of circRNA
isoform	structure of isoforms, isoforms are seperated by "|" and circular exons are seperated by "," (e.g. 11627815-111627914,111628190-111628302|11627815-111628302)
gene_id	ensemble id of host gene
gene_name	HGNC symbol of host gene
gene_type	type of host gene in the annotation gtf file

Expression matrix

test_collpase.expression contains the summarized expression level of circRNAs in all samples in tsv format.

Using non-canonical splice signals

If you would like to use other splice signals, please modify the dict SPLICE_SIGNAL in align.py in format: {(5'SS, 3'SS): Priority}

Default configuration:

SPLICE_SIGNAL = {
    ('GT', 'AG'): 0,  # U2-type
    ('GC', 'AG'): 1,  # U2-type
    ('AT', 'AC'): 2,  # U12-type
    ('GT', 'AC'): 2,  # U12-type
    ('AT', 'AG'): 2,  # U12-type
}