RNA_seq-analysis

This workflow is for differential gene expression study for the samples with replicates

FOR STAND ALONE RUN (INDIVIDUAL COMMANDS)

1. Perform quality check on fastq file using FASTQC/MULTIQC

FASTQC:

fastqc *.fastq -o <output_directory>

-o : Directory to save output files (file must be created) "*.fastq" represents to select all files with the ".fastq" extension in the working directory

MULTIQC:

multiqc <fastqc_results_directory>/

2. Quality control using Trimgalore

trim_galore -q 20 --paired --fastqc --cores <number_of_threads> <input_R1_fq.gz> <input_R2.fq.gz> -o <output_directory>

3. Indexing reference file

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

4. Alignment using STAR

STAR --genomeDir <index_dir_name> --runThreadN <number_of_threads> --outSAMtype BAM SortedByCoordinate --readFilesCommand zcat --readFilesIn <input_R1.fastq.gz> <input_R2.fastq.gz>  --outFileNamePrefix <output_filename>

5. Read Quantification using Feature count

featureCounts -p -T <number_of_threads> --verbose -t exon -g gene_id -a <path to ".gtf" file> -o <out_count_file_name> <List of BAM files as Input files>

FOR SNAKEMAKE RUN

Step 1: Make a Project Folder with Project_ID

Create a project folder and give it a meaningful Project_ID.

Step 2: Copy Files into Project Folder

Copy the following files into the project folder:

• Snakefile
• Deseq2_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. If in case you want to use characters in sample name make sure to use underscores () instead of hyphens (-) in file names. For example, replace '-' with '' (e.g., Tumor-1_R1.fq.gz --> 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 (Example file)

#### Enter organism name (Scientific name)
org: "Homo sapiens"

#### Enter Kegg organism code
org_code: "hsa"

#### Specify Number of threads
threads: "40"

#### Specify Combinations using "+" between combinations
combinations: "control_Tumor + Tumor_control"

#### Path to indexed reference folder (Reference 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