Next-Generation Sequencing Technologies: An Overview

Abstract

• Evolution of Sequencing: Transition from Sanger sequencing to next-generation sequencing (NGS).
• Classification: NGS divided into short-read (second-generation) and long-read (third-generation) technologies.
• Examples:
  • Short-read: Illumina and Ion Torrent.
  • Long-read: Pacific Biosciences and Oxford Nanopore.
• Advancements: Third-generation technologies improving read length and accuracy.

Introduction

• Historical Context:
  • DNA structure discovered nearly 25 years ago.
  • Sanger sequencing as the first method, involving chain-terminating dideoxynucleotides and gel electrophoresis.
  • Capillary electrophoresis improved throughput, but limitations in sequencing diploid DNA.
  • Human genome project took over a decade, costing $2.7 billion.
• Need for New Technologies:
  • Sanger sequencing reached a ceiling for time and cost.
  • Introduction of NGS between 2004 and 2006, allowing massively parallel sequencing.
  • NGS characterized by high throughput and single-molecule DNA sequencing.

Short-read Sequencing (Second-Generation)

• Features: Massive sequencing of short, clonally amplified DNA molecules (250-800 bp).
• Workflow: Includes library preparation, sequencing, and data analysis.
• Challenges: Reassembly needed for long DNA stretches, handling structural variations and low-complexity regions.

Long-read Sequencing (Third-Generation)

• Advantages: Can generate sequences >10kb from native DNA, overcoming short-read issues.
• Improvements: Increased accuracy over time, particularly with software advancements.
• Applications: Useful for genetic disease diagnosis and large DNA molecule sequencing.

Conclusion and Future Directions

• Technological Shift: From first fully sequenced human genome to current NGS technologies.
• Goals: Serve research and clinical applications with sensitive, specific, and reproducible results.

Declaration of Competing Interest

• Dimitri Monos has certain affiliations with Omixon Inc.; other authors have no conflicts.

Acknowledgment

• Thanks to Jamie L. Duke, Deborah Ferriola, and Timothy L. Mosbruger for their review and suggestions.

References

• Several references included, highlighting key developments and applications in NGS technology.