README

#Luis Zapata 2017
#Mega tool to detect positively selected and negatively selected genes in cancer exomes.

##Dependencies
bedtools 2.26.0
R-3.3.3
R library tidyr
perl 5
GNU command line tools
variant effect predictor v89

## Important Notes
- earlier versions of bedtools will not work
- tab encoding should be \t (might be a problem for windows/OSX versions)
- genome file is a two column file specifying the fasta id and the length of the sequence

##To install first clone the tool
git clone https://github.com/luisgls/SSB_selection.git

##Install synapse client
pip install synapseclient

##Download zipped data files from synapse syn11681952
synapse get -r syn11681952

##Go to the tool directory and
1.1) Create data folder within the cloned folder
1.2) Unzip data files into data folder
1.3) Open run_negDriver script and specify the location of the genome file (e.g. hg19.genome) and the fasta file (e.g. hg19.fasta) 
1.4) chmod +x run_negDriver

2) run ./run_negDriver to see help

##Input file
The input file is the standard output of variant effect predictor using the following command line (by providing to vep the ensembl default input file format)
perl variant_effect_predictor.pl -i input -o input.annotated --cache --all_refseq --assembly GRCh37 --pick --symbol --no_stats --fasta genome.fasta
If you want to filter putative germline variants use the option --plugin ExAC when running VEP.

Example input files can be found on synapse: ID syn11681983

##Important points before running
a) No header needed for input VEP file
b) VEP annotated first column must be in the format (chr_pos_ref/alt)
c) VEP annotated file must only have chromosomes that are 1,2,3,4...22 or uppercase X,Y
d) After you run vep with the option for ExAC frequencies, it would be necessary to remove all variants present in more than 0.1 percent of the population. You could apply the filter using:
filter_vep -i input.annotated -f "ExAC_AF < 0.1 or not ExAC_AF" --ontology --filter "Consequence is coding_sequence_variant" 

###The UNIX system used should be able to recognize \t as a tab separator
e) Be sure that you are using the GNU command line if you are running in a MacOS (https://www.topbug.net/blog/2013/04/14/install-and-use-gnu-command-line-tools-in-mac-os-x/)

f) Add dependencies to your path for easy running or hardcode the scripts
g) The genome file used in SSB is a two column file that contains the info of the name of the fasta id (column 1) and the length of that sequence (column 2).

NOTES:
This version is a simplified and easy-to-use version of the tool developed in the manuscript "Negative Selection on tumour evolution acts on essential cellular functions and the immunopeptidome" (https://doi.org/10.1186/s13059-018-1434-0). For the reproducibility of the results presented in the manuscript, please contact the authors directly.

The tool to run the analysis of the immunopeptidome is called SOPRANO:

To run the tool with version 38 of the human genome, simply update the path of you GENOME and FASTA file in the run_negdriver script.
Also, use the Data file provided in synapse labeled as Data2.zip. It contains updated transcript information.