A pipeline that correlates somatic mutation in tumor/normal WGS paired samples with gene-level allele-specific expression in an effort to systematically uncover cis-regulatory variants involved in tumorigenesis.
#Introduction
It is well known that some DNA mutations can induce uncontrolled cell division and ultimately lead to cancer. Advances in sequencing over the last five years have enabled researchers to scan all of the DNA within a tumor to identify these mutations, and indeed we are finding thousands, leaving us with the daunting task of distinguishing the potentially few causal changes from the vast majority of “passengers”. Researchers are focusing on protein-coding regions where functional effects can be readily predicted. Unfortunately, this approach misses the expansive oncogenic effects caused by changes in gene regulation (turning genes up/down), which are much more difficult to identify. We have pioneered a new bioinformatic approach to probe high-throughput RNA sequencing (RNA-Seq) data, generated from the molecules that carry out gene expression, to identify the truly causal regulatory mutations. We applied our method to all suitable breast cancer samples in The Cancer Genome Atlas (TCGA) and mapped over a hundred genes whose regulation is perturbed by regulatory mutations in breast tumors.
The pipeline performs ASE analysis with using RNA-Seq, affymetrix genotyping array, whole genome sequence, copy number variant, and methylation data from TCGA. It is divided into 5 parts that have 2 or more Perl scripts for each part. They are used to download, run imputation, perform ASE analysis as well as perform WGS analysis. These scripts are easy to use and provide options and usages. To speed up the analysis, the perl module MCE (Multiple Core Engine) was implemented into the pipeline. Additionally, this pipeline uses other software to process data, including samtools, impute2, shapeit, plink, overlapSelect and VarScan with links found below for each software.
#Install
This pipeline was coded on a Centos 7 x86_64 system and some of the software listed below are for an x86_64 linux distribution and some of the software may not be compatible with i686 or other versions. The commands below may also be different based on what distribution is being used.
####Firewall and Other
Make sure that the firewall allows for incoming and outgoing of traffic for HTTPS/HTTP and FTP. Also make sure that MySQL and WGET are installed.
####Packages
This pipeline also requires some packages to be installed as the other software it uses within it needs them.
openssl - sudo yum install openssl
openssl-devel - sudo yum install openssl-devel
EPEL repository - sudo yum install epel-release
yaml - sudo yum install yaml-cpp.x86_64 yaml-cpp-devel.x86_64
gcc - sudo yum install gcc
gcc-c++ - sudo yum install gcc-c++.x86_64
ncurses-devel - sudo yum install ncurses-devel.x86_64
Make sure you have these perl modules installed before you run these scripts.
####In order to install these modules, you will need to use cpan or any other utility that installs perl modules.
If you are using cpan and it is not installed, install it and run it.
Before installing these modules, it is a good idea to check if they are not already installed:
If the module is installed, then nothing will happen but if it is not, it will display an error saying that it could not be found.
also install YAML as some of the other modules rely on it to install
cpan YAML
FindBin - perl -e 'use FindBin;'
File::Copy - perl -e 'use File::Copy;'
File::Basename - perl -e 'use File::Basename;'
FileHandle - perl -e 'use FileHandle;'
MCE - perl -e 'use MCE;'
Cwd - perl -e 'use Cwd;'
Math::CDF - perl -e 'use Math::CDF;'
Getopt::Long - perl -e 'use Getopt::Long;'
LWP::Simple - perl -e 'use LWP::Simple;'
autodie - perl -e 'use autodie;'
lib - perl -e 'use lib;'
strict - perl -e 'use strict'
warnings - perl -e 'use warnings;'
####You will need to install the latest version of aria2 since this pipeline uses it to download RNA-Seq, Genotyping array and WGS data.
aria2 - https://aria2.github.io/
####The commands to install aria2 are as follows
./configure --with-openssl
sudo make
sudo make install
####This pipeline also uses curl to get data from GDC, so make sure you have curl installed on your system.
sudo yum install curl
#NOTE
There is a version of the Dwnld_WGS_RNA.pm module that only uses curl and is available outside of the scripts on the github repository for this pipeline. If there is no way to download and/or install aria2 then put this script in the TCGA_ASE_Analysis_Pipeline/TCGA_Lib directory replacing the other file in there.
###You will need to install the software below as well ####Create a bin directory in your home directory and install the software below to it
cd ~
mkdir bin
####NOTE: The URLs provided below may change or the page they direct to may no longer be available, so if that happens the software will need to be aquired elsewhere. Also, the commands below are optional other than the software that requires configure and make to be run.
####If you don't have root permissions to install the software, contact you system admin to have the software installed.
####samtools
download link - https://sourceforge.net/projects/samtools/files/latest/download?source=files
Place the compressed archive in the bin directory and decompress it
####Installing samtools
tar -jxvf samtools-1.3.1.tar.bz2(version number may be different)
Change the name of the directory to samtools
remove or backup the archive
cd samtools
./configure
make
sudo make install
####shapeit(v2.12 static was used for this pipeline)
download link - https://mathgen.stats.ox.ac.uk/genetics_software/shapeit/shapeit.html
Place the compressed archive in the bin directory and decompress it
####Installing shapeit
tar -zxvf shapeit.v2.r837.GLIBCv2.12.Linux.static.tgz
remove or backup the archive
mkdir shapeit
mv bin example LICENCE shapeit
####impute2(the static verison was used for this pipeline)
download link - https://mathgen.stats.ox.ac.uk/impute/impute_v2.html#download
Place the compressed archive in the bin directory and decompress it
####Installing impute2
tar -zxvf impute_v2.3.2_x86_64_static.tgz
rename the directory impute2
remove or backup the archive
####plink1.9 or later (Developement)
download link - https://www.cog-genomics.org/plink2
Place the compressed archive in the bin directory and decompress it
####Installing plink
unzip plink_linux_x86_64.zip
mkdir plink1(temporary name)
mv plink prettify LICENSE toy.map toy.ped plink1
mv plink1 plink
####overlapSelect
download link - http://hgdownload.cse.ucsc.edu/admin/exe/linux.x86_64.v287/overlapSelect
place the downloaded software in the bin directory and chand the permissions so that it can be executed.
chmod a+x overlapSelect
####VarScan(need java installed in order to run it)
download link - https://sourceforge.net/projects/varscan/files/
place the downloaded software in the bin directory
You will need to add the software to your path in order for the pipeline to be able to run them. VarScan is the ony exception to this as you will need to put the full path to it in order for it to run.
cd ~
vi .bash_profile
####press i to insert into the file
PATH=$PATH:$HOME/bin:$HOME/bin/samtools/:$HOME/bin/shapeit/bin/:$HOME/bin/impute2/:/usr/bin/:$HOME/bin/plink
####press the Esc key
:wq
source ~/.bash_profile
This pipeline also uses many different files which are all in the same directory. The directory with the files can be downloaded from the link below.
#After Download
####1. Copy it to a directory and extract it. ####2. Enter the directory and copy the TCGA_ASE_Analysis_Pipeline dirctory to a new location.
####Database directory download link
https://mega.nz/#!bZN3hSob!B5ybrcH_4frJfFv24sMns2XEzHPO5aQsqyrUq1MpnKc
####Once downloaded place the directory in the TCGA_ASE_Analysis_Pipeline directory and decompress it.
####GDC Key Required
You will also need to have a GDC account to get the key for downloading as it does expire over a certain period of time. Just go to https://gdc-portal.nci.nih.gov/
####1. Go to login. ####2. Click on your account name in the top right to open the drop down and click download token. ####3. Rename the downloaded token to gdc.key ####4. Place it in the Database directory of the pipeline. When running the download scripts, the path to the key will need to be entered.
This pipeline can be used right away in any part of the system once all of the above has been done. You can also add the path to the sctipts if you wish so you don't have to be in the directory or specify the path to them in order to execute them.
#Scripts
Below are the scripts that are included in this pipeline:
0_Download_SNPArray_From_GDC.pl – Downloads SNP-Array data from gdc. The data types that are used in this pipeline are Genotypes and Copy number estimate.
0_Download_SNPArray_Table_From_GDC.pl - Downloads the SNP-Array data table of a specified cancer type from GDC. Only run this script if the table file is missing from the tables directory of the cancer type.
1.0_Prep_SNPs_for_Imputation.pl – This prepares the files that will be used in the imputation process.
1.1_Birdseed_to_ped_and_maps.pl – Takes the birdseed files that were downloaded from GDC and makes the map and ped files that will be used for imputation.
1.2_Shapeit_and_Imputation.pl – Runs shapeit processes as well as run imputation on the files made in the previous script.
1.3_Make_het_cds.pl – Keeps het SNPS and makes cds sorted files.
2.0_Prep_For_Bad_SNPs_CNVs.pl – Performs lookups on the cds sorted files and gets the normal samples out of them.
2.1_Get_Bad_SNPs_and_CNVs.pl – Gets the bad SNPs and CNVs of the cancer type.
3.0_Download_RNASeq_WGS_and_do_Mpileup.pl – Downloads either RNA-Seq or WGS bams for a cancer type. By default this script is set to download a certain number of bams unless the user specifies the amount that they want to download in the command line, deletes them afterwards and downloads more when done.
3.0_Dwnld_Table_4_RNASeq_WGS_From_GDC.pl - Downloads the RNA-Seq or WGS table of specified cancer types from GDC. Only run this script if the table file is missing from the tables directory of the cancer type.
3.1_ASE_Analysis.pl – Performs gene level ASE Analysis on the samples that had mpileups done on them.
3.2_Export_ASE_Data.pl – Performs the final touches by compressing what will be needed for further analysis and moving them into a specific directory.
4.0_Somatic_Variants.pl – Processes WGS data to get somatic variants.
4.1_Upstream_Downstream_Analysis.pl – Performs upstream and downstream analysis and gets the data ready to be analyzed.
#Modules
Modules that are incorporated in the scripts are listed below:
Parsing_Routines.pm - All scripts use this module.
Dwnld_WGS_RNA.pm - 0_Download_SNPArray_From_GDC.pl, 0_Download_SNPArray_Table_From_GDC.pl, 3.0_Download_RNASeq_WGS_and_do_Mpileup.pl, 3.0_Dwnld_Table_4_RNASeq_WGS_From_GDC.pl
Imputation_Plink.pm - 1.0_Prep_TCGA_for_Imputation_and_Plink.pl, 1.1_Birdseed_to_ped_and_maps.pl, 1.2_Shapeit_and_Imputation.pl, 1.3_Make_het_cds.pl
Bad_SNPS_and_CNVS.pm - 2.0_Lookups_for_normal.pl, 2.1_Get_Bad_SNPs_and_CNVs.pl
TCGA_ASE_Analysis.pm - 3.1_ASE_Analysis_for_other_Bams.pl, 3.2_Export_ASE_data.pl
WGS_Analysis.pm - 4.0_Somatic_Variants.pl, 4.1_Upstream_Downstream_Analysis.pl