ASCOT

Jonathan P. Ling, Chris Wilks, Rone Charles, Ben Langmead, Seth Blackshaw

ASCOT quantifies alternative splicing and gene expression across tens of thousands of bulk and single-cell RNA-Seq datasets in human and mouse. This repository contains the scripts used to generate the PSI/NAUC tables for this resource.

ASCOT uses annotation-free methods to detect exon percent spliced-in (PSI) values within Snaptron, a rapidly queryable database of splice junction counts derived from public RNA-seq data. Gene expression levels are calculated using a normalized "area-under-curve" (NAUC) metric as described in recount2.

For more details, please refer to Nature Communications (2020) and the accompanying ASCOT website. All data tables are available for download.

The github repository for the ASCOT website UI is available at https://github.com/langmead-lab/ascot-ui

Comments and suggestions are always welcome: ascotfeedback@gmail.com

UCSC TrackHubs for data visualization:

We strongly recommend that users cross-validate any splicing results obtained from ASCOT. One way to do so is to visualize the data on the UCSC Genome Browser. We provide TrackHubs (collections of .bigwig files) from each dataset in ASCOT:

>Mouse cell types and tissues from bulk RNA-Seq (MESA) TrackHub link
    http://snaptron.cs.jhu.edu/data/mesa/sums/MESAHub/hub.txt
    
>Mouse single-cell RNA-Seq data (CellTower) TrackHub link
    http://snaptron.cs.jhu.edu/data/ct_m_s/sums/CTMSHub/hub.txt
    
>Human GTEx tissues + eye (GTEx) TrackHub link
    http://snaptron.cs.jhu.edu/data/gtex/sums/GTEXHub/hub.txt
    
>ENCODE shRNA-Seq knockdown data (ENCODE) TrackHub link
    http://snaptron.cs.jhu.edu/data/encode1159/sums/ENCODEHub/hub.txt

Instructions for using TrackHubs are available on the UCSC help page. In brief, navigate to the top menu bar option My Data > Track Hubs, select the My Hubs tab, enter URL from above and select Add Hub.

Usage instructions (we recommend a system with at least 30Gb ram):

git clone https://github.com/jpling/ascot.git
cd ./ascot/software/snaptron
make

All PSI and NAUC data tables can be downloaded here.

To rederive the splicing PSI data tables:

>Mouse cell types and tissues from bulk RNA-Seq (MESA)
    python3 ascot_psi.py --i ./exons/mesa_exons.tsv --a mesaall --c mesalinked --o mesa_psi.tsv

>Mouse single-cell RNA-Seq data (CellTower)
    python3 ascot_psi.py --i ./exons/ctms_exons.tsv --a ctmsall --c ctmslinked --min 10 --f 1 --o ctms_psi.tsv

>Human GTEx tissues + eye (GTEx)
    python3 ascot_psi.py --i ./exons/gtexeye_exons_1.tsv --a gtexeyeall --c gtexeyelinked --o gtexeye_psi_1.tsv
    python3 ascot_psi.py --i ./exons/gtexeye_exons_2.tsv --a gtexeyeall --c gtexeyelinked --o gtexeye_psi_2.tsv
    python3 ascot_psi.py --i ./exons/gtexeye_exons_3.tsv --a gtexeyeall --c gtexeyelinked --o gtexeye_psi_3.tsv

>ENCODE shRNA-Seq knockdown data (ENCODE)
    python3 ascot_psi.py --i ./exons/encode_exons_1.tsv --a encodegtexall --c encodegtexlinked --o encode_psi_1.tsv
    python3 ascot_psi.py --i ./exons/encode_exons_2.tsv --a encodegtexall --c encodegtexlinked --o encode_psi_2.tsv

To rederive the gene expression NAUC data tables:

>Mouse cell types and tissues from bulk RNA-Seq (MESA)
    python3 ascot_nauc.py --a mesaall --c mesalinked --o mesa_nauc.tsv
    
>Mouse single-cell RNA-Seq data (CellTower)
    python3 ascot_nauc.py --a ctmsall --c ctmslinked --o ctms_nauc.tsv
    
>Human GTEx tissues + eye (GTEx)
    python3 ascot_nauc.py --a gtexeyeall --c gtexeyelinked --o gtexeye_nauc.tsv
    
>ENCODE shRNA-Seq knockdown data (ENCODE)
    python3 ascot_nauc.py --a encodegtexall --c encodegtexlinked --o encode_nauc.tsv