Question

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

Short answer

Answer: Sanger sequencing uses the chain termination method with dideoxynucleotides (ddNTPs), while NGS employs massively parallel sequencing and PCR amplification. TGS sequences individual DNA molecules, providing real-time sequencing and longer read lengths. In terms of speed, Sanger sequencing is slowest, NGS is faster due to parallel processing, and TGS is the fastest with real-time sequencing. For accuracy, Sanger sequencing has a high accuracy and low error rate, while NGS generally has high accuracy but can be prone to PCR amplification errors, and TGS methods vary in accuracy depending on the technology used. In terms of cost, Sanger sequencing has the highest per-base cost, whereas both NGS and TGS have significantly reduced sequencing costs with NGS being more cost-effective currently, but TGS is expected to become more cost-effective as the technology matures.

Step-by-step solution

Define Sanger Sequencing

Sanger sequencing, also known as the chain termination method, was developed by Frederick Sanger in the 1970s. It is a method to determine the nucleotide sequence of DNA by using dideoxynucleotides (ddNTPs) as a terminator to stop DNA replication after a single nucleotide is added. The result is a set of fragmented DNA strands, from which the DNA sequence can be read.

Define Next-Generation Sequencing (NGS)

Next-generation sequencing (NGS), also called second-generation sequencing, is a high-throughput sequencing method which allows many DNA fragments to be sequenced simultaneously. This method involves fragmenting the DNA, attaching adapters, and amplifying the fragments using PCR. NGS utilizes a massively parallel approach to detect the sequences of the DNA fragments, making it much faster and more efficient than Sanger sequencing.

Define Third-Generation Sequencing (TGS)

Third-generation sequencing (TGS) is the latest advancement in DNA sequencing technology. TGS methods do not require DNA amplification like NGS, and can sequence individual molecules of DNA in real-time, resulting in longer read lengths. These methods usually involve single-molecule sequencing platforms such as the PacBio or Nanopore Sequencing.

Compare Sequencing Technologies

Sanger sequencing is a widely accepted and reliable method for DNA sequencing, but it has limitations in speed and throughput. NGS revolutionized DNA sequencing by offering massively parallel sequencing, allowing for the rapid processing of multiple samples and producing a much larger amount of data. Both Sanger and NGS methods require PCR amplification, which can introduce errors or biases. TGS offers the advantage of sequencing individual DNA molecules without amplification and can provide longer read lengths.

Compare Sequencing Speed

In terms of sequencing speed, Sanger sequencing is the slowest method, as it sequences DNA one fragment at a time. NGS significantly improved the speed of sequencing by using parallel processing, making it possible to sequence millions of fragments simultaneously. TGS further improved sequencing speed by providing real-time sequencing of individual DNA molecules.

Compare Accuracy

Sanger sequencing has a high accuracy, with a low error rate. NGS methods generally have high accuracy but can be prone to errors introduced during PCR amplification. TGS methods, which do not require amplification, offer a potential reduction in error rates, although the accuracy can vary depending on the specific TGS technology used.

Compare Cost

Sanger sequencing has the highest per-base cost due to its lower throughput, while both NGS and TGS have significantly reduced the cost of DNA sequencing. NGS has become popular for cost-efficient, high-throughput sequencing projects, but TGS is expected to become more cost-effective as the technology matures and sequencing platforms become more widely available.

Key concepts

Sanger Sequencing

Sanger sequencing is a DNA sequencing method developed by Frederick Sanger in the 1970s. It is also known as the chain termination method. This technique identifies the sequence of DNA nucleotides by using special molecules called dideoxynucleotides (ddNTPs) which terminate DNA strand elongation at specific bases. When DNA polymerase incorporates a ddNTP instead of a regular nucleotide during replication, the addition of further nucleotides is halted.

The process results in several DNA fragments of varying lengths, each ending at a ddNTP. These fragments can be separated by size through gel electrophoresis or a similar method, which helps in determining the precise order of nucleotides in the original DNA strand. Sanger sequencing is highly accurate, but the throughput is limited, making it slower and more expensive for large-scale projects.

It is widely used for sequencing small regions of DNA and for clinical diagnosis where precision is necessary. Despite technological advancements, Sanger sequencing remains a gold standard due to its accuracy.

Next-Generation Sequencing (NGS)

Next-generation sequencing (NGS), also known as second-generation sequencing, is a modern method that allows for the simultaneous sequencing of multiple DNA fragments. Unlike Sanger sequencing, which processes one fragment at a time, NGS leverages a massively parallel approach.

This involves breaking the DNA into smaller segments and attaching small indexing adapters to these fragments. The process is then followed by amplification through a method known as polymerase chain reaction (PCR). Unlike traditional Sanger sequencing, NGS can sequence millions of DNA fragments at once, significantly boosting speed and efficiency.

NGS has dramatically reduced the cost and time involved in sequencing projects. It's especially useful for large-scale studies, such as genomics research and cancer studies, providing an unprecedented amount of data. However, the reliance on PCR can potentially introduce errors and biases into the sequenced data. NGS is a breakthrough in efficiency for researchers, enabling the analysis of entire genomes or large sets of genes in a single experiment.

Third-Generation Sequencing (TGS)

Third-generation sequencing (TGS) represents the forefront of DNA sequencing technology. Unlike Sanger sequencing and even NGS, TGS technologies do not rely on DNA amplification, thereby eliminating potential PCR-induced errors. TGS allows for real-time sequencing, sequencing individual DNA molecules as they are naturally found.

With the development of platforms like Pacific Biosciences (PacBio) and Oxford Nanopore Technologies, TGS provides long read lengths beyond what could be achieved with NGS. Longer reads can span complex regions in the genome and offer more comprehensive data, which is especially helpful in identifying structural variations and repetitive sequences.

TGS offers unique capabilities with its real-time data output, providing insights into nucleotide sequences as they are being detected. Although the technology is continuing to develop, making it not yet as widely accessible as NGS due to costs, it is proving to be critical in fields requiring ultra-long read sequencing, such as detailed genome assemblies and complex microbial communities. As the technology advances, TGS is expected to become more affordable and prevalent in research.