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

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>

Create a project folder and give it a meaningful Project_ID.

Copy the following files into the project folder:

• Snakefile
• Deseq2_final.R
• create_combinations.R
• config.yaml
• Master_file.txt

Inside the project folder, create a sub-folder named 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).

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.

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

#### 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>"

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

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

Type the following command in the terminal:

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