A computational workflow for exitron splicing identification
You need Python 3.12 to run ScanExitron.
Install anaconda (python 3.12) firstly, then install dependent packages via conda in bioconda channel.
conda install -c bioconda samtools
conda install -c bioconda bedtools
conda install -c bioconda pyfaidx
conda install -c bioconda regtools=0.5.0
# hg38 genome
wget https://hgdownload.cse.ucsc.edu/goldenpath/hg38/bigZips/hg38.fa.gz
gunzip hg38.fa.gz
# hg19 genome
wget https://hgdownload.cse.ucsc.edu/goldenpath/hg19/bigZips/hg19.fa.gz
gunzip hg19.fa.gz
# hg38 annotation
wget ftp://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_human/release_37/gencode.v37.annotation.gtf.gz
gunzip gencode.v37.annotation.gtf.gz
# hg19 annotation
ftp://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_human/release_19/gencode.v19.annotation.gtf.gz
gunzip gencode.v19.annotation.gtf.gz
# hg38 CDS
cat gencode.v37.annotation.gtf | awk 'OFS="\t" {if ($3=="CDS") {print $1,$4-1,$5,$10,$16,$7}}' | tr -d '";' > gencode.hg38.CDS.bed
# hg19 CDS
cat gencode.v19.annotation.gtf | awk 'BEGIN{OFS="\t"} { if ($3=="CDS") {if ($13=="ccdsid"){print $1,$4-1,$5,$20,$22,$7} else{ print $1,$4-1,$5,$18,$20,$7}}}' | tr -d '";' > gencode.hg19.CDS.bed
[fasta]
# reference genome file in FASTA format (absolute path)
hg38=/abs/path/to/hg38.fa
hg19=/abs/path/to/hg19.fa
[annotation]
# gene annotation file in GTF format (absolute path)
hg38=/abs/path/to/gencode.v21.annotation.gtf
hg19=/abs/path/to/gencode.v19.annotation.gtf
[cds]
# CDS annotation in BED format (absolute path)
hg38=/abs/path/to/gencode.hg38.CDS.bed
hg19=/abs/path/to/gencode.hg19.CDS.bed
ScanExitron.py -i input_rna_seq_bam_file -r [hg38/hg19] -m mapping_quality
-h, --help show this help message and exit
-i INPUT, --input INPUT
RNA-seq alignment file (BAM/CRAM)
-a AO, --ao AO AO cutoff (default: 3)
-p PSO, --pso PSO PSO cutoff (default: 0.05)
-s STRAND, --strand STRAND Strand specificity of RNA library preparation (0 = unstranded, 1 = first-
strand/RF, 2, = second-strand/FR) (default: 1)
--mapq consider reads with MAPQ >= cutoff (default: 50)
-r {hg19,hg38}, --ref {hg19,hg38}
reference genome (default: hg38)
input_bam_file :input RNA-seq BAM/CRAM file. (e.g., rna-seq.bam)
reference_genome :specify reference genome (hg19 or hg38)
exitron_file :Reported exitrons in a TAB-delimited file. (rna-seq.exitron)
Report Columns
| Column Name | Description |
|---|---|
| chrom | The chromosome of this exitron |
| start | The start position of this exitron in the zero-based, half-open coordinate system |
| end | The stop position of this exitron in the zero-based, half-open coordinate system |
| name | Identifier for the junction |
| ao | Observed supporting reads for exitron |
| strand | The strand the exitron is identified |
| gene_symbol | The Gene symbol of the affected gene |
| length | Length of the exitron |
| splice_site | The two basepairs at the donor and acceptor sites separated by a hyphen |
| gene_id | The Ensembl ID of the affected gene |
| pso | The percent spliced out (PSO) index |
| psi | The percent spliced in (PSI) index |
| dp | The average depth of the exitron |
| total_junctions | The total number of junctions in the sample |
We also keep RegTools interim results (rna-seq.janno) for developers.
For a detailed explanation, please refer to The Documentation of RegTools
The project is licensed under the MIT license.
Bug reports or feature requests can be submitted on the ScanExitron Github page.
Please see and cite our papers at Molecular Cell and STAR protocols.