Differential Gene Expression Analysis for Samples with No Replicates

This guide provides steps to run an RNA_SEQ Snakemake file for performing differential gene expression analysis when samples have no replicates.

Steps to Run RNA_SEQ Snakemake File

Step 1: Make a Project Folder with Project_ID

Create a project folder and assign it a meaningful Project_ID.

Step 2: Copy Files into Project Folder

Copy the following files into the project folder:

• Snakefile
• DEGSeq_no_replicate_final.R
• create_combinations.R
• config.yaml
• Master_file.txt

Step 3: Create a Sub-folder "1_Data"

Inside the project folder, create a sub-folder named 1_Data.

Step 4: Copy Sample Files to 1_Data

Copy the sample files into the 1_Data folder. Ensure that you replace hyphens (-) with underscores (_) in file names. For example, Tumor-1_R1.fq.gz should be renamed to Tumor_1_R1.fq.gz.

Step 5: Create "Master_file.txt"

Create a file named Master_file.txt in the project folder. This file should specify the combinations and replicates. Refer to the example file provided for better clarity.

Step 6: Use Config File to Add Additional Information

Utilize the config.yaml file to add any additional information required for the workflow.

Config.yaml Content for RNA_SEQ Snakemake Workflow

# Enter organism name (Scientific name)
org: "Mus musculus"

# Enter Kegg organism code
org_code: "mmu"

# Specify Number of threads
threads: "15"

# Specify Combinations using "+" between combinations
combinations: "Tumor_Lung + Tumor_Liver + Lung_Liver"

# Reference Assembly version (Indexing command provided below)
reference: "<path/to/indexed/reference/folder>"

Genome indexing using STAR

STAR --runMode genomeGenerate --genomeDir {index_dir_name} --genomeFastaFiles {path to ".fasta" file} --sjdbGTFfile {path to ".gtf" file} --sjdbOverhang 100 --runThreadN 10

Step 7: Open Terminal in Project Folder

Navigate to the project folder in your terminal/command prompt.

Step 8: Run Snakemake

Type the following command in the terminal:

snakemake --configfile=config.yaml --cores 5