What is Next Generation DNA Sequencing?

In contrast to microarray methods, sequence-based approaches directly determine the nucleic acid sequence of a given DNA or cDNA molecule.

The first major foray into DNA sequencing was the Human Genome Project. This project, which used first-generation sequencing, known as Sanger sequencing (the chain-termination method), took 13 years, cost $3 billion and was completed in 2003.

Around the same time, the first paper describing ‘massively parallel sequencing’ applied to gene expression studies was published; this marked the beginning of a new sequencing era, called Next generation sequencing (NGS). Also known as high throughput sequencing, NGS is the catch-all term used to describe a number of different modern sequencing technologies. These technologies allow for sequencing of DNA and RNA much more quickly and cheaply than the previously used Sanger sequencing, based on capillary electrophoreses. They also provide orders of magnitude more data, revolutionising the study of genomics and molecular biology.

Advantages of NGS

NGS can be used to analyse DNA and RNA samples and is a popular tool in functional genomics. In contrast to microarray methods, NGS-based approaches have several advantages including:

• a priori knowledge of the genome or genomic features is not required
• it offers single-nucleotide resolution, making it possible to detect related genes (or features), alternatively spliced transcripts, allelic gene variants and single nucleotide polymorphisms
• higher dynamic range of signal
• requires less DNA/RNA as input (nanograms of materials are sufficient)
• higher reproducibility

As of 2025, we distinguish between second-generation sequencing (SGS, characterised by short-reads output) and third-generation sequencing (TGS, characterised by long-read output). Even though NGS principles described above apply to both, those sequencing methods differ in the technology used and/or research scopes.In this section, we will review the principles of SGS and TGS, and conclude with a sum up of the improvements NGS brought to DNA sequencing and, as a direct consequence, to life science research. For a more detailed review of the current trends in sequencing, see this review.