RNA Seq Pipeline - iffatAGheyas/bioinformatics-tutorial-wiki GitHub Wiki

Under RNA-Seq Pipeline we’ll cover each major processing step from raw FASTQ to a gene-level count matrix:

RNA-Seq Pipeline

1. Experimental Design & Metadata
   – Sample grouping, replicates, batch considerations
2. Data Organization & Download
   – Directory structure, SRA/ENA fetch (e.g. fasterq-dump)
3. Raw Read Quality Control
   – FastQC reports
   – Aggregation with MultiQC
4. Adapter & Quality Trimming
   – Cutadapt or Trimmomatic usage
5. Read Alignment or Pseudo-alignment
   • Genome-based: STAR or HISAT2
   • Alignment-free: Salmon or Kallisto
6. Post-Alignment QC
   – RSeQC (e.g. gene-body coverage, inner distance)
   – Qualimap RNA-seq module
7. Quantification & Count Matrix Generation
   – featureCounts or HTSeq-count for aligned BAMs
   – tximport for transcript‐to‐gene summarization
8. Normalization & Exploratory Analysis
   – TPM/CPM calculation
   – PCA, hierarchical clustering
9. Workflow Management & Reproducibility
   – Snakemake or Nextflow pipeline templates
   – Containerization (Docker/Singularity)
   This roadmap will guide our hands-on examples, code snippets, and recommended tools for each step.