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# SpliceFisher

Multiple Fisher's exact tests for differential alternative splicing detection using RNA-seq data


## Method

![](SpliceFisher.method.png)

1. Counting reads from BAM files
   * Exon skipping
     * *a*, *b* exon-junction reads
     * *c* exon-skipping reads
     * *d* exon-mapping reads
     * *e* gene-mapping reads
   * Intron retention
     * *a*, *b* exon-intron reads
     * *c* exon-exon reads
     * *d* intron-mapping reads
     * *e* gene-mapping reads
2. Multiple Fisher's exact tests
   * Head: (control *a* / control *c*) / (test *a* / test *c*)
   * Tail: (control *b* / control *c*) / (test *b* / test *c*)
   * Body: (control *d* / control *e*) / (test *d* / test *e*)
3. Adjustment of p-values by false discovery rate (FDR) method

4. Alternative splice sites (A5SS and A3SS) and Mutually exclusive exons (MXE)
![](SpliceFisher.method.ASS.png)
![](SpliceFisher.method.MXE.png)


## Requirements

1. Perl - https://www.perl.org
2. Perl module "Bio::DB::Sam" - http://search.cpan.org/~lds/Bio-SamTools-1.43/lib/Bio/DB/Sam.pm
3. R (Rscript) - https://www.r-project.org
4. Common linux commands: bash, cut, sort, uniq, ...


## Install

If you already have Git (https://git-scm.com) installed, you can get the latest development version using Git.
```
git clone https://github.com/jiwoongbio/SpliceFisher.git
```


## Usages

1. Preparation of exon/intron regions
   ```
   ./prepare.sh <gene.gtf>
   ```
   * Example: Before running the following command, download "Homo_sapiens_UCSC_hg19.tar.gz" from http://support.illumina.com/sequencing/sequencing_software/igenome.html and unzip the file.
   ```
   ./prepare.sh Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf
   ```
   * The repository already contains prebuilt exon/intron regions in hg19 (exon.unique.txt, intron.unique.txt).

2. Detection of differential alternative splicing
   ```
   ./SpliceFisher.sh <outputPrefix> <alpha> <control.sorted.bam> <test.sorted.bam>
   ```
   * Example: It uses SRA data of https://www.ncbi.nlm.nih.gov/pubmed/24840202. The reads mapped to 11p13 were extracted to generate the input BAM files.
   ```
   ./SpliceFisher.sh hnRNPM_KD 0.05 Control_rep1.bam,Control_rep2.bam hnRNPM_KD_rep1.bam,hnRNPM_KD_rep2.bam
   ```
   * Output: exon skipping and intron retention of CD44 (hnRNPM_KD.exon.filtered.txt, hnRNPM_KD.intron.filtered.txt)

![hnRNPM_KD.CD44.png](hnRNPM_KD.CD44.png)


## Citation

Han T, Goralski M, Gaskill N, Capota E, Kim J, Ting TC, Xie Y, Williams NS, Nijhawan D.
"Anticancer sulfonamides target splicing by inducing RBM39 degradation via recruitment to DCAF15"
Science. 2017 Mar 16. pii: eaal3755.
PMID: [28302793](https://www.ncbi.nlm.nih.gov/pubmed/28302793)