autoRNAseq

A bash script and some dependencies to automate RNA-seq analysis. A dummy file structure is provided as an example.
This script has been tested on MacOS Catalina, and should work on Unix-based systems.
Additional Requirements: A sequence for adapater trimming (e.g. TruSeq3-PE.fa) and a reference cDNA sequence (e.g. Homo_sapiens.GRCh38.cdna.all.fa) are required.

Usage

cd container #see the File Structure section of Inputs for more details
chmod 777 PROJECT1.sh #change script permissions to execute
. ./PROJECT1.sh #run script

Inputs

The script depends on a particular file structure and conda.

File Structure

• container
  • PROJECT1.sh
  • PROJECT1
    • PRE
      • sample1_identifier_L002_R1_001.fastq.gz
      • sample1_identifier_L002_R2_001.fastq.gz
      • sample2_identifier_L002_R1_001.fastq.gz
      • sample2_identifier_L002_R2_001.fastq.gz
    • POST
      • sample3_identifier_L002_R1_001.fastq.gz
      • sample3_identifier_L002_R2_001.fastq.gz
      • sample4_identifier_L002_R1_001.fastq.gz
      • sample4_identifier_L002_R2_001.fastq.gz
    • analysis.R
    • studydesign.txt
  • PROJECT2
  • Homo_sapiens.GRCh38.cdna.all.fa
  • TruSeq3-PE.fa

R Analysis

The R analysis script (analysis.R) contains many properties that must be user-defined. analysis.R depends on studydesign.txt

studydesign.txt

studydesign.txt contains a tab-seperated table of pertinent information for each sample. Formatting is as follows (based on the sample heirarchy provided in the File Structure section of Inputs):

sample	group	dir
PREsample1	PRE	sample1_identifier
PREsample2	PRE	sample2_identifier
POSTsample3	POST	sample3_identifier
POSTsample4	POST	sample4_identifier

analysis.R

A barebones R script that outputs basic PCA analysis and a volcano plot of the RNA-seq data into Rplots.pdf. Additionally, outputs LogFC.csv. See Output for more details.

Output

logFC.csv

A comma-seperated-values file of every gene mapped by Kallisto and its corresponding P-val and log fold change from PRE to POST. Genes are sorted by abs(logFC).

geneID	logFC	AveExpr	t	P.Value	adj.P.Value	B
gene1	1.11	2.22	3.33	.444	.555	6.66

Rplots.pdf

Contains a volcano plot of the aforementioned genes as well as plots for sanity checking the data.