circfull: a tool to detect and quantify full-length circRNA isoforms from circFL-seq
Introduction
circfull is a circRNA analysis pipeline to detect and quantity full-length circRNA for circFL-seq data
Availability
circfull is a free software, which can be downloaded from https://github.com/yangence/circfull
Softwares and packages dependencies
- Softwares of minimap2 (>=2.1), bedtools (>=2.29.2), samtools (>=1.6) need to be installed by users.
- porechop(0.2.4) can be downloaded from https://github.com/rrwick/Porechop. We recommand replace adapters.py in porechop directory with circfull/scripts/bin/adapter.py which contain circFL-seq primers
- TideHunter (1.0) and trf (4.09) are included in this program.
- Python 3.x.x and corresponding versions of pysam, numpy, pandas, python-intervals, pyfasta, sklearn, interval, mappy, progressbar, docopt.
Installation
Install latest release from pip
pip install circfull
Install latest release from source codes
git clone https://github.com/yangence/circfull.git
cd circfull/script
pip install -r requirements.txt
python setup.py install
Required files:
Users can prepare the external files under the following instructions:
- Indexed genome fasta file
samtools faidx $genome
- Tabix indexed gene annotation GTF file
grep -v '#' $gtf |sort -k 1,1 -k 4,4n |bgzip >sort.gtf.gz
tabix sort.gtf.gz
- Repetitive elements (optional)
Repetitive elements can be download from https://genome.ucsc.edu/cgi-bin/hgTables
Usage
Usage: circfull <command> [options]
Command:
RG Reference guide detection
DNSC De novo self-correction to get consensus sequence
cRG Use RG algorithem with sequences corrected by DNSC
strand Identify the strand origion of circFL-seq reads
sRG RG algorithem with stranded circFL-seq reads.
mRG Merge circFL results from RG and cRG/sRG.
anno Annotate full-length circRNA.
geneExp Quantify gene expression to evaluate linear RNA residual
var Calling variants in circRNA.
RG
Usage: circfull RG -f fastq -g genome -a anno [-t threads] [-r] [-o output]
Options:
-h --help Show help message.
-v --version Show version.
-f fastq circFL-seq fastq file.
-g genome Fasta file of genome.
-a anno Tabix indexed gtf file of gene annotation.
-t threads Number of threads [default: 20].
-r Filter out false discovery.
-o output Output dir [default: circFL_out].
DNSC
Usage: circfull DNSC -f fastq [-t threads] [-c] [-m mem] [-o output]
Options:
-h --help Show help message.
-v --version Show version.
-f fastq circFL-seq fastq file.
-c Perform cluster of consensus sequence.
-m mem The maximum memory usage, only worked when -c is designated. [default: 100G]
-t threads Number of threads [default: 20].
-o output Output dir [default: circFL_out].
cRG
Usage: circfull cRG -f DNSC -g genome -a anno [-t threads] [-u] [-o output]
Options:
-h --help Show help message.
-v --version Show version.
-f DNSC DNSC directory.
-g genome Fasta file of genome.
-a anno Tabix indexed gtf file of gene annotation.
-t threads Number of threads [default: 20].
-u Stranded reads.
-o output Output dir [default: circFL_out].
strand
Usage: circfull strand -f fastq -F fastq2 -a anno [-r RG_dir] [-l hang_len] [-t threads] [-o output]
Options:
-h --help Show help message.
-v --version Show version.
-f fastq Raw circFL-seq fastq file with adapter remain.
-F fastq2 Trimmed circFL-seq fastq file.
-a anno Tabix indexed gtf file of gene annotation.
-r RG_dir RG output dir. [default: circFL_out]
-l hang_len Length of sequence to find primers in both end. [default: 100]
-t threads Number of threads [default: 20].
-o output Output dir [default: circFL_out].
sRG
Usage: circfull sRG -g genome -a anno [-s strandDir] [-t threads] [-r] [-o output]
Options:
-h --help Show help message.
-v --version Show version.
-g genome Fasta file of genome.
-a anno Tabix indexed gtf file of gene annotation.
-t threads Number of threads [default: 20].
-s dir Strand dir [default: circFL_out].
-r Filter out false discovery.
-o output Output dir [default: circFL_out].
mRG
Usage: circfull mRG -f fastq -g genome [-r RG] [-c cRG] [-s sRG] [-t threads] [-o output]
Options:
-h --help Show help message.
-v --version Show version.
-f fastq circFL-seq fastq file.
-g genome Fasta file of genome.
-r RG RG directory [default: circFL_out].
-c cRG cRG directory [default: circFL_out].
-s sRG sRG directory [default: circFL_out].
-t threads Number of threads [default: 20].
-o output Output dir [default: circFL_out].
anno
Usage: circfull anno -b bed -a anno [-o output]
Options:
-h --help Show help message.
-v --version Show version.
-b bed Full-length circRNA position information in BED format (12 columns).
-a anno Tabix indexed gtf file of gene annotation.
-o output Output file [default: circ_anno.txt].
geneExp
Usage: circfull geneExp -f fastq -r ref [-t threads] [-o output]
Options:
-h --help Show help message.
-v --version Show version.
-f fastq circFL-seq fastq file.
-r ref Gene reference
-t threads Number of threads [default: 20].
-o output Output dir [default: circFL_out].
var
Usage: circfull var -f fastq -g genome [-r RG_dir] [-t threads] [-o output]
Options:
-h --help Show help message.
-v --version Show version.
-f fastq circFL-seq fastq file.
-g genome Fasta file of genome.
-r RG_dir RG output dir [default: circFL_out].
-t threads Number of threads [default: 20].
-o output Output dir [default: circFL_out].
Output files
| File name | Details |
|---|---|
| circFL_Normal.txt | normal circRNA isoforms detected by RG, cRG, sRG |
| circFL_Normal_pass.txt | final circRNAs isoforms from mRG |
| circFL_Fusion.txt | fusion circRNA isoforms detected by RG |
| novoCluster.txt | circRNA isoforms detected by DNSC with -c |
| circ_Var.txt | variants in circRNAs detected by var |
| geneCountDf.txt | gene expression quantified by geneExp |
| strandedFq.fastq | transcript-strand circFL-seq reads in FASTQ format calcuated by strand |
| circ_anno.txt | full-length circRNA annotated by anno |
circFL_Normal.txt and circFL_Normal_pass.txt
| No. | Column name | Details |
|---|---|---|
| 1 | circID | circRNA ID |
| 2 | isoID | isoform ID |
| 3 | chr | chromosome |
| 4 | start | start coordinate of circRNA |
| 5 | end | end coordinate of circRNA |
| 6 | len | length of circRNA |
| 7 | exonNum | number of exons in circRNA |
| 8 | exon_start | start coordinate of exon |
| 9 | exon_end | end coordinate of exon |
| 10 | motif | motif of BS |
| 11 | leftSeq | motif of start junction |
| 12 | rightSeq | motif of end junction |
| 13 | exon_leftSeq | motif of exon start junction |
| 14 | exon_rightSeq | motif of exon end junction |
| 15 | strand | strand direction |
| 16 | geneName | gene name |
| 16 | readCount | Count of reads supporting this circRNA isoform |
| 17 | readID | ID of reads supporting this circRNA isoform |
circFL_Fusion.txt
| No. | Column name | Details |
|---|---|---|
| 1 | circID | circRNA ID |
| 2 | isoID | isoform ID |
| 3 | chr_first | chromosome of one locus |
| 4 | start_first | start coordinate of one locus |
| 5 | end_first | end coordinate of one locus |
| 6 | len_first | length of one locus |
| 7 | exonNum_first | number of exons in one locus |
| 8 | exon_start_first | start coordinate of exon in one locus |
| 9 | exon_end_first | end coordinate of exon in one locus |
| 10 | exon_leftSeq_first | motif of exon start junction of one locus |
| 11 | exon_rightSeq_first | motif of exon end junction of one locus |
| 12 | strand_first | strand direction of one locus |
| 13 | geneName_first | gene name of one locus |
| 14 | chr_second | chromosome of another locus |
| 15 | start_second | start coordinate of another locus |
| 16 | end_second | end coordinate of another locus |
| 17 | len_second | length of another locus |
| 18 | exonNum_second | number of exons in another locus |
| 19 | exon_start_second | start coordinate of exon in another locus |
| 20 | exon_end_second | end coordinate of exon in another locus |
| 21 | exon_leftSeq_second | motif of exon start junction of another locus |
| 22 | exon_rightSeq_second | motif of exon end junction of another locus |
| 23 | strand_second | strand direction of another locus |
| 24 | geneName_second | gene name of another locus |
| 25 | readCount | read counts supporting this circRNA isoform |
| 26 | readID | ID of reads supporting this circRNA isoform |
novoCluster.txt
| No. | Column name | Details |
|---|---|---|
| 1 | ID | read ID |
| 2 | cluster | cluster ID |
| 3 | strand | 1/0 denotes sense/antisense of transcript strand, respectively |
| 4 | readLen | length of read |
| 5 | start | start position of tandem repeats of read |
| 6 | end | end position of tandem repeats of read |
| 7 | consLen | length of consensus sequence |
| 8 | copyNum | number of tandem repeats |
| 9 | usage | proportion of tandem repeats |
| 10 | consensus | consensus sequence |
circ_Var.txt
| No. | Column name | Details |
|---|---|---|
| 1 | isoID | isoform ID |
| 2 | chr | chromosome |
| 3 | pos | coordinate of variants |
| 4 | ref | reference base |
| 5 | alt | alternative base |
| 6 | refCount | read counts supporting reference base |
| 7 | altCount | read counts supporting alternative base |
| 8 | ratio | read counts ratio of alternative base to total base of the variant |
geneCountDf.txt
| No. | Column name | Details |
|---|---|---|
| 1 | geneID | ensembl gene ID |
| 2 | geneName | gene name |
| 3 | num | read counts supporting this gene |
| 4 | ratio | read counts ratio of this gene to all genes |
circ_anno.txt
| No. | Column name | Details |
|---|---|---|
| 1 | chr | chromosome |
| 2 | start | start coordinate of circRNA |
| 4 | end | end coordinate of circRNA |
| 5 | isoID | isoform ID |
| 6 | strand | strand direction |
| 7 | exon_start | start coordinate of exon |
| 8 | exon_end | end coordinate of exon |
| 9 | len | length of circRNA |
| 10 | start_type | annotation for start coordinate of exon |
| 11 | end_type | annotation for end coordinate of exon |
| 12 | geneName | gene name |
| 13 | detail_type | annotation details for isoform |
| 14 | type | annotation for isoform |
Examples
Details in examples.
Reference
Copyright and License Information
Copyright (C) 2021 Zelin Liu (zlliu@bjmu.edu.cn). See the LICENSE file for license rights and limitations.