What is next‑generation sequencing (NGS) and how many types does it have?

What is NGS and How Many Types?

Next-generation sequencing (NGS) is a high-throughput DNA sequencing technology that enables massively parallel sequencing of millions of DNA fragments simultaneously, revolutionizing genomic analysis by providing comprehensive, rapid, and cost-effective sequencing compared to traditional Sanger methods 1.

Definition and Core Technology

NGS represents a transformative advancement over conventional sequencing, allowing an entire human genome to be sequenced within a single day—a process that previously required over a decade using Sanger technology 2. The technology works by fragmenting DNA, adding adaptors to fragments, amplifying them, and then sequencing millions of molecules in parallel 3.

Types of NGS Technologies

There are fundamentally two main categories of NGS technologies, classified by read length:

1. Short-Read (Second-Generation) Sequencing

• Read lengths: Hundreds of bases
• Primary platforms: Illumina (sequencing by synthesis) and Ion Torrent (ion semiconductor sequencing)
• Advantages: Lower per-base cost, high accuracy, well-established protocols
• Limitations: Difficulty resolving structural variants, genome rearrangements, and repetitive sequences 1, 4

2. Long-Read (Third-Generation) Sequencing

• Read lengths: Tens of kilobases
• Primary platforms: Pacific Biosciences and Oxford Nanopore technologies
• Advantages: Can finish microbial genomes for under $1,000, resolves complex genomic regions, structural variants, and interspersed repeats
• Historical limitation: Previously lower accuracy (now improving with emerging developments) 1, 4

Clinical Applications by Sequencing Scope

Beyond platform technology, NGS is categorized by the genomic scope analyzed 5:

Three Main Clinical Application Types:

1. Targeted Gene Panels

   • Sequence specific sets of medically relevant genes
   • Used extensively in precision oncology and inherited disorders
   • Most cost-effective for known disease genes 1

2. Whole-Exome Sequencing (WES)

   • Sequences all protein-coding regions (exons) of known genes
   • Frontline diagnostic tool for inherited disorders
   • Covers ~1-2% of genome but contains ~85% of disease-causing variants 1, 6

3. Whole-Genome Sequencing (WGS)

   • Sequences entire genome including exons and introns
   • Increasingly becoming method of choice due to lower prices and comprehensive data
   • Superior for detecting structural variants and non-coding regulatory regions 1, 7

Technical Workflow Components

The NGS workflow consists of wet-lab and dry-lab processes 3:

Wet-lab steps:

• DNA/RNA extraction and quantification
• Library preparation (fragmentation and adaptor addition)
• Amplification and sequence generation

Dry-lab bioinformatics (three analysis levels):

• Primary analysis: Converting instrument signals to base calls with quality scores
• Secondary analysis: Quality assessment, trimming, filtering, alignment to reference genome or de novo assembly
• Tertiary analysis: Clinical interpretation, variant calling, functional annotation, report generation 3

Key Clinical Distinctions

DNA-based NGS can simultaneously examine point mutations, amplifications, deletions, fusions, microsatellite instability, and tumor mutational burden—particularly useful in oncology 5.

RNA-based NGS (via reverse transcription) detects gene fusion transcripts and expression profiles, essential for confirming functional fusion proteins 5.

The technology has been widely adopted for clinical genetic testing in inherited conditions, cancer genomics, infectious disease diagnosis, pathogen discovery, and public health surveillance 3, 8.