Principles of Second-Generation Sequencing: A Detailed Comparison of Different Sequencing Technologies

1. Overview of Second-Generation Sequencing (NGS)

Second-generation sequencing (Next-Generation Sequencing, NGS) represents a revolutionary advancement over traditional Sanger sequencing, characterized by high throughput, parallel processing, and low cost, enabling large-scale genomic sequencing. Unlike first-generation sequencing, NGS employs sequencing by synthesis (SBS) or ligation-based sequencing to simultaneously read millions to billions of DNA fragments, generating gigabytes (GB) to terabytes (TB) of data in a single run. The leading NGS platforms include Illumina, Thermo Fisher Ion Torrent, and BGI-MGI, each differing in principle, read length, accuracy, and application scenarios.

2. Principles and Differences Among Major NGS Technologies

1. Illumina Sequencing (Bridge PCR + Sequencing by Synthesis)

Core Technology:

• Library Preparation: DNA fragmentation and adapter ligation, followed by immobilization on a flow cell.
• Bridge PCR Amplification: DNA clusters (~1,000 identical copies per cluster) are formed via bridge amplification.
• Four-Color Fluorescent Labeling: Reversible terminator dNTPs are incorporated one base at a time, with fluorescence indicating the base type.
• Image Capture & Base Calling: A high-resolution camera captures fluorescence, and software interprets the sequence.

Key Features:

• Read Length: Short (50-300 bp), ideal for high-precision sequencing.
• Throughput: Up to terabytes (Tb) per run (e.g., NovaSeq 6000).
• Accuracy: >99.9%, suitable for whole-genome sequencing (WGS), exome sequencing (WES), and RNA-seq.

Limitations:

• Short read lengths limit structural variant detection.
• High equipment costs due to optical systems.

2. Thermo Fisher Ion Torrent (Semiconductor Sequencing)

Core Technology:

• Emulsion PCR: DNA fragments bind to beads and are amplified in water-in-oil droplets, forming monoclonal DNA microspheres.
• Semiconductor Chip Detection: Microspheres are deposited on a chip, where H⁺ ion release (during base incorporation) alters pH, generating electrical signals.
• No Fluorescent Labeling: Direct detection of H⁺ ions eliminates the need for optics.

Key Features:

• Read Length: 200-400 bp, suitable for targeted sequencing (e.g., cancer panels).
• Throughput: Moderate (Gb-level), but fast runtime (2-4 hours).
• Accuracy: >99%, but prone to errors in homopolymer regions (e.g., AAAA).

Limitations:

• Higher error rates in homopolymers.
• Lower throughput, unsuitable for WGS.

3. BGI-MGI (DNB Nanoball Sequencing + cPAS)

Core Technology:

• DNA Nanoballs (DNB): Rolling circle amplification (RCA) generates error-resistant DNA nanoballs.
• Combinatorial Probe-Anchor Synthesis (cPAS): Fluorescent probes and anchored primers enable sequencing.
• High-Density Chip: DNBs are arrayed on a chip for dense sequencing.

Key Features:

• Read Length: 50-200 bp, similar to Illumina but more cost-effective.
• Throughput: Flexible, ideal for small-to-medium projects.
• Localization Advantage: Lower equipment costs, suitable for clinical and research use.

Limitations:

• Shorter read lengths and slightly lower data quality than Illumina.

4. 454 Sequencing (Pyrosequencing, Obsolete)

Core Technology:

• Emulsion PCR: Similar to Ion Torrent but uses pyrosequencing.
• Light Signal Detection: Pyrophosphate release during dNTP incorporation generates light signals.

Key Features:

• Read Length: 700-1000 bp, once preferred for metagenomics.
• Limitations: High cost, low throughput; now replaced by Illumina.

3. NGS Technology Comparison & Selection Guide

Selection Recommendations:

• Whole-genome sequencing (WGS): Illumina (highest accuracy and throughput).
• Targeted sequencing (e.g., cancer panels): Ion Torrent or BGI-MGI (fast and cost-efficient).
• Clinical diagnostics: Ion Torrent (speed) or BGI-MGI (cost-effectiveness).
• Metagenomics: Illumina (short reads) combined with third-gen sequencing (long reads).

4. Future Trends in NGS

While third-generation sequencing (e.g., PacBio, Nanopore) offers long-read advantages, NGS remains dominant due to high accuracy, mature pipelines, and affordability. Future advancements may focus on single-molecule sequencing, higher throughput, and AI-enhanced data analysis.

Conclusion

The diversity of NGS technologies allows researchers to select the optimal platform based on project needs (read length, throughput, cost). Illumina remains the gold standard, but Ion Torrent and BGI-MGI are competitive in niche applications. Understanding these technologies’ principles and differences enables better experimental design and higher-quality data.