RNA-seq analysis:

This repo contains the scripts used for analysis of the Bulk-RNA sequencing analysis.

PIPELINE FLOWCHART:

STEPS:

• Pre-processing:
  • Quality Control using FASTQC tool
• Alignment:
  • Salmon : pseudo quantification
  • STAR : local alignment
    • feature counts : to count the reads per sample
• Differential Analysis:
  • Filtering low confidence reads
  • Differential gene expression using deseq2
• Visualization :
  • PCA
  • Volcano Plots
  • Heatmap
• Pathway Analysis :
  • msigDB

CONDA ENVIRONMENT:

• Create a new conda environment and installing tools needed for RNA_seq.sh script

conda create -f conda_envs/RNA_seq.yml
conda activate RNA_seq

HOW TO RUN THE PIPELINE:

• INPUT PATHS:

  • FASTQ_DIR =</path/of/the/directory/containing/fastq/files>
  • REF_DIR =</path/of/the/directory/containing/reference/indexes>
  • RESULTS_DIR =</path/of/the/directory/to/store/results>

• PARAMETER:

  • SPECIES = "mm10" or "hg38"
  • ALIGNER = "salmon" or "STAR"
  • READS = "single" or "paired"

• Command:

  • bash RNA_seq <FASTQ_DIR> <REF_DIR> <RESULTS_DIR>
  • Example:

  bash RNA_seq.sh /test/fastq_files /test/mm10_salmon /test/results "mm10" "salmon" "paired"
  

• OUTPUT FOLDER:

  • fastQC : quality control reports for all the fastq files
  • salmon/STAR : quant or bam files

DOWNSTREAM ANALYSIS:

• downstream-analysis.qmd :
  • differential gene expression analysis
  • Visualization plots
  • Pathway analysis