zhengcq / igtex

GTEx Isoform Quantification via the XAEM algorithm

iGTEx

This project describes an automated pipeline to quantify isoform expressions for the GTEx V8 RNA sequencing data. The protocol uses XAEM, a powerful method for isoform expression estimation across multiple samples. You also can find the detailed information about XAEM website and in the published paper in Bioinformatics.

Prerequisites

R (recommended version >= 3.5.1)
Python (recommended version >= 3.7)

Installation

Step 1: Setup XAEM

Install the XAEM tool for this protocol via the bash command:

git https://github.com/ZhengCQ/iGTEx.git

or

wget https://github.com/ZhengCQ/iGTEx/archive/refs/tags/iGTEx_XAEM_v0.1.2.zip
unzip iGTEx_XAEM_v0.1.2.zip
ln -fs iGTEx-iGTEx_XAEM_v0.1.2 iGTEx_XAEM

Step 2: Setup R dependencies

In R, install the R dependencies via:

install.packages("foreach")
install.packages("doParallel")

Step 3: Download the annotation reference

Run the following commands to download the reference annotating the transcripts:

cd /path/to/iGTEx_XAEM
python down_ref.py

(Optional) To down a particular reference with gencode hg38, use:

python down_ref.py -db gencode_38

Example

An example is prepared in the project Example folder, executable as

cd /path/to/iGTEx_XAEM/Example
sh run_example.sh 

Isoform Estimation using GTEx data

XAEM performs better when multiple samples of similar data type are considered. In the GTEx V8 data, for each tissue, we create a project directory:

mkdir -p /path/to/Tissue1
cd /path/to/Tissue1

Input files

In /path/to/Tissue1, create a file /path/to/Tissue1/infastq_lst.tsv listing the FASTQ input files. The file is a tab-delimited text file with 4 columns: Sample name, Source name, FASTQ file name for paired-end read 1, and FASTQ file name for paired-end read 2. Source name indicates the batch or sequencing library of the sample, so that the same sample may correspond to more than one sources. A standard example, where each sample has only a single batch or multiple batches, is given as /path/to/iGTEx_XAEM/Example/infastq_lst.tsv:

single batch

sample4 S0007   S0007_1.fg.gz   S0007_2.fg.gz
sample5 S0008   S0008_1.fg.gz   S0008_2.fg.gz

multiple batches

sample1 S0001   S0001_1.fg.gz   S0001_2.fg.gz
sample1 S0002   S0002_1.fg.gz   S0002_2.fg.gz
sample2 S0003   S0003_1.fg.gz   S0003_2.fg.gz
sample2 S0004   S0004_1.fg.gz   S0004_2.fg.gz
sample3 S0005   S0005_1.fg.gz   S0005_2.fg.gz
sample3 S0006   S0006_1.fg.gz   S0006_2.fg.gz

Run XAEM

XAEM can be easily run with:

python /path/to/iGTEx_XAEM/run_xaem.py -i /path/to/Tissue1/infastq_lst.tsv

(Optional) To specify a particular reference with gencode hg38, use:

--ref gencode_38

(Optional) To specify a particular output directory, use:

-o /path/to/Tissue1_output_directory

(Optional) Further customized configuration of XAEM can be setup by:

-c /path/to/Tissue1_config.ini

An example of the config.ini file can be found in /path/to/iGTEx_XAEM/.

Calculate isoform ratio

Isoform ratio can be easily run with

python /path/to/iGTEx_XAEM/exp2ratio.py -i /path/to/XAEM_isoform_expression.RData

(Optional) To specify a particular reference with gencode hg38, use:

--ref gencode_38

(Optional) The default output directory as same as input isoform file. To specify a particular output directory, use:

-o /path/to/Tissue1_output_directory

(Optional) To specify a covariates file for linear regression:

--covariates /path/to/covariates_file

(For GTEx) For GTEx v8, we have prepared the covariates information for calculation, you can specify a tissue name:

--tissue Brain_Amygdala 

(For GTEx) For GTEx v8 demo:

python /path/to/iGTEx_XAEM/exp2ratio.py -i /path/to/Brain_Amygdala_XAEM_isoform_expression.RData --ref gencode_38 --tissue Brain_Amygdala

sQTL THISTLE

BOD file

/path/to/osca --efile /path/to/TissueName/isoform_splice_ratio.tsv --gene-expression --make-bod --no-fid --out TissueName 
/path/to/osca --befile TissueName --update-opi /path/to/iGTEx_XAEM/ref/gencode_38/anno_gene_info.opi

sQTL

/path/to/osca --sqtl --bfile /path/to/Genotype/BED_All/TissueName_Genotype --befile TissueName --maf 0.05 --call 0.85 --cis-wind 1000 --thread-num 10 --task-num 1 --task-num 1 --task-id 1 --to-smr --bed /path/to/iGTEx_XAEM/ref/gencode_38/anno_gene_info.bed --out sQTL_results/TissueName