Next Generation Sequencing (NGS) is a general name for high throughput DNA sequencing technologies, which intend to lower the cost and higher the speed of DNA sequencing beyond what is possible with standard dye-terminator methods.

Method Pyrosequencing (454) Sequencing by synthesis (Illumina) Chain termination (Sanger sequencing)
Read length 700 bp 50 to 250 bp 400 to 900 bp
Accuracy 99.9% 98% 99.9%
Reads per run 1 million Upto 3 billion N/A
Time per run 24 hours 1 to 10 days, depending upon sequencer and specified read length 20 minutes to 3 hours
Cost per 1 million bases (in US $) 10$ $0.05 to $0.15 $2400
Advantages Long read size. Fast Potential for high sequence yield, depending upon sequencer model and desired application. Long individual reads. Useful for many applications
Disadvantages Runs are expensive. Homopolymer errors. Equipment can be very expensive. More expensive and impractical for larger sequencing projects.

Illumina sequencing technology based on adding four types of reversible terminator bases, each fluorescently labeled with a different color and attached with a blocking group. Those bases are compete for binding sites on template DNA to be sequenced and the non incorporated molecules are washed away. Then a laser is applied and remove the blocking group and the probe, and the process is repeated until full DNA molecule is sequenced. By fluorescently detection on every repeat the DNA is sequenced.


Roche 454 technology

AGTC cycle

Pyrosequencing. Template is attached to a bead surface, and a primer directs DNA synthesis, which involves addition of one deoxynucleoside triphosphate (dNTP) per sequencing cycle. One signal is generated per cycle by assaying the amount of pyrophosphate (PPi) released using a two-step reaction: Adenosine 5'-phosphosulfate (APS) and PPi is used to form ATP catalyzed by ATP-sulfurylase, and the ATP is used for production of light from luciferin oxidation catalyzed by luciferase.

The products of the reaction are released in solution and are contained within small wells to enable mapping of the signal to each template. The number of bases present in a homopolymeric run is decoded by quantifying the amount of PPi released in a reaction. Intensities are plotted with respect to the order of successful signal detections to create a 'flowgram'.

From: Bentley D, (2006) Current Opinions in Genetics and Development, 16:546-552
Ronaghi M.(2001), Genome Res. 11: 3-11