Next-generation sequencing (NGS) encompasses a set of modern sequencing technologies that can sequence millions of DNA fragments in parallel, making it possible to sequence entire genomes quickly and affordably. Platforms such as Illumina, Ion Torrent, and PacBio dominate the field.
How NGS Works (Illumina Platform)
- Library Preparation
The DNA is fragmented into small pieces, typically 200–600 base pairs. Adapters—short known sequences—are ligated to both ends of each fragment. These adapters allow the fragments to bind to the flow cell and serve as priming sites for sequencing.
- Cluster Generation
The library is loaded onto a flow cell whose surface is coated with complementary oligonucleotides. Each fragment binds to the flow cell and undergoes bridge amplification: the fragment bends over, a new strand is synthesized, and the process repeats, creating a cluster of thousands of identical copies of each fragment.
- Sequencing by Synthesis
Fluorescently labeled nucleotides are flowed over the flow cell one at a time. Each nucleotide has a reversible terminator that allows only one base to be added per cycle. After each cycle, the fluorescence is imaged to identify which base was added to each cluster.
- Data Analysis
Millions of sequencing reads are generated. These reads are aligned to a reference genome or assembled de novo. Bioinformatics tools identify genetic variants, gene expression levels, or epigenetic modifications depending on the application.
- Applications
NGS is used for whole-genome sequencing, targeted gene panels, RNA sequencing, epigenetic analysis, and metagenomics, among many other applications.
