RNA Seq Splicing Analysis Pipeline

Overview

This splicing analysis pipeline is a novel compilation of computational biology tools that quantifies RNA splicing by providing intron excision ratios and maps sQTLs by performing analysis of SNP statistical significance.

Software Requirements

• Linux
• Python 3.x
  • The Pandas module also should be installed.
• R 3.3.3
• LeafCutter
• Matrix eQTL

Scripts

• pipelineparser.py
  • Extracts the genotype values from a VCF files to a new file in the format necessary for Matrix eQTL. It also creates a table of information regarding the chromosomal location for each SNP.

  •    Input: chrX.vcf.gz 
       Output: SNPGenotypes_chrX, SNPLoc_chrX
    

• leaf-command
  • Calls LeafCutter to generate gene expression and gene location information from .bam files in the form of qqnorm outputs.

  •    Input: /path/to/bamfiles/
       Output: sampleName.gz.qqnorm_chrX
    

• qqnormparser.py
  • Separates the qqnorm files into gene location and gene expression files necessary for input to Matrix eQTL.

  •    Input: sampleName.gz.qqnorm_chrX
       Output: Gene_expression_chrX, Gene_location_chrX 
    

• file_reorder.py
  • Reorders the samples within the Genotype file to match the output from LeafCutter's gene expression files.

  •    Input: SNPGenotypes_chrX, Gene_expression_chrX
       Output: ReorderedSNPGenotypes_chrX
    

• matrix_eQTL_parse.R
  • Tests for association between SNPs and the introns to find which are associated with splicing

  •    Input: /path/to/test_data/ finalSNPGenotypes_chrX SNPLoc_chrX Gene_expression_chrX Gene_location_chrX
       Output: cis_eQTLs_X.txt, trans_eQTLs_X.txt, Cis_trans_hist_X.pdf
    

• AntHill
  • Runs all scripts in correct order for all 23 chromosomes and outputs intron excision ratios and maps sQTLs.

Downloading the project

Open a terminal session and enter the following:

git clone https://github.com/breecoffey/RNA_Seq_Splicing-Analysis-Pipeline.git

Input Files

.vcf.gz,.bam

Make sure your files are in the same directory. You can do this by making a new directory like:

   mkdir anthill_files
   cd anthill_files

And keep all scripts and files together here.

Example

To see a worked out example, head to the test_example directory and follow the worked out example guide there.