Question

How do next-generation sequencing (NGS) and third. generation sequencing (TGS) differ from Sanger sequencing?

Short answer

Answer: Sanger sequencing relies on the incorporation of chain-terminating dideoxynucleotides and electrophoresis, with the advantage of high accuracy and long-read length; however, it has low throughput and is costly for large-scale projects. NGS uses massively parallel sequencing approaches and offers high throughput and cost-effectiveness for large-scale projects, but has shorter read lengths and higher error rates than Sanger sequencing. TGS allows direct sequencing of single DNA molecules in real-time, offering long-read lengths, lower error rates, and resolving complex genomic regions, but has a higher cost per base compared to NGS and is a rapidly changing field.

Step-by-step solution

Introduction to Sequencing Methods

Before we dive into the differences between NGS, TGS, and Sanger sequencing, it is important to provide a brief introduction to each sequencing method. Sanger sequencing is the first-generation sequencing method developed by Frederick Sanger in 1977. NGS, also known as Second-Generation Sequencing, is a group of high-throughput sequencing technologies that allows for large-scale DNA sequencing. TGS, also referred to as Third-Generation Sequencing, is the latest in sequencing technologies, enabling direct sequencing of single DNA molecules without amplification, long-read sequencing, and lower error rates.

Working Principles of Sequencing Methods

Sanger Sequencing: Sanger sequencing is based on the selective incorporation of chain-terminating dideoxynucleotides (ddNTPs) by DNA polymerase during in vitro DNA replication. It uses electrophoresis to separate DNA fragments according to their sizes. Next-Generation Sequencing (NGS): NGS uses massively parallel sequencing approaches, where millions of DNA fragments are sequenced at once. These sequencing methods rely on different techniques such as sequencing by synthesis (Illumina), pyrosequencing (454 sequencing), and sequencing by ligation (SOLiD). Third-Generation Sequencing (TGS): TGS methods allow for direct sequencing of single DNA molecules in real-time, without the need for DNA amplification. Some notable TGS platforms include Pacific Biosciences (PacBio) Single Molecule, Real-Time (SMRT) Sequencing, and Oxford Nanopore Technologies.

Advantages and Limitations

Sanger Sequencing: Advantages: High accuracy, long-read length (up to 1000 base pairs), and well-established technique. Limitations: Relatively low throughput, costly for large-scale projects, and not suitable for detecting rare variants. Next-Generation Sequencing (NGS): Advantages: High throughput, cost-effective for large-scale projects, and ability to detect rare genetic variants. Limitations: Short-read length (150-600 base pairs), higher error rates compared to Sanger sequencing, and limitations in resolving repeat regions. Third-Generation Sequencing (TGS): Advantages: Long-read length (10,000-100,000 base pairs), ability to resolve complex genomic regions and repeat sequences, single-molecule sequencing, and lower error rates. Limitations: Higher cost per base compared to NGS, and a developing field with rapidly changing technology.

Conclusion

In summary, Sanger sequencing is the first-generation sequencing method that is accurate but limited in throughput and scalability. NGS refers to high-throughput sequencing methods with shorter read lengths but declining cost for large-scale projects. TGS offers long-read sequencing, real-time data analysis, and the ability to resolve complex genomic regions. Each of these sequencing methods has its own advantages and limitations, making them suitable for different applications in genomics research.