RNA-Seq Explained: From Reads to Gene Expression

Understanding the Active Transcriptome

While DNA provides the blueprint, the transcriptome reveals active gene expression responding to environmental cues, treatments, or disease states. RNA Sequencing (RNA-Seq) quantifies the abundance of messenger RNA (mRNA) and non-coding RNA molecules across biological samples.

The Step-by-Step RNA-Seq Pipeline

1. Library Preparation & Sequencing: RNA is extracted, reverse-transcribed to cDNA, fragmented, and sequenced on high-throughput instruments.

2. Read Alignment or Pseudoalignment: Splice-aware aligners like STAR align reads across intron-exon junctions. Ultra-fast pseudoalignment algorithms (Salmon/Kallisto) quantify transcript counts directly without full read mapping.

3. Count Normalization: Raw transcript counts are normalized for gene length and total library sequencing depth using algorithms such as TPM (Transcripts Per Million) or DESeq2 median-of-ratios.

4. Differential Expression & GSEA: Statistical frameworks evaluate expression differences between control and treatment groups, yielding fold-changes and false discovery rate (FDR) adjusted p-values.