English > About genomes (glossary) > Second-generation DNA sequencers

About genomes (glossary)

Second-generation DNA sequencers

First-generation DNA sequencers are useful, as the accuracy of the sequencing obtained is high and a large number of bases can be read. However, when sequencing large DNA fragments cloned in Escherichia coli, the disadvantages of first-generation DNA sequencers are that sample preparation requires a lot of time and labor. In addition, when it may not possible to obtain a clone of a particular region, gaps in the sequence, called “clone gaps” can result.

To address these issues, second-generation DNA sequencers that utilize massively parallel processing of sequencing reactions were developed. In second-generation DNA sequencing, the DNA molecule is bound to the surface of a micro bead or a solid body to become the template for the sequencing reaction, so that the troublesome cloning process is no longer needed.

Second-generation DNA sequencing basically (1) uses gradual synthesis by a fluorescence-labeled nucleotide substrate, (2) detects pyrophosphoric acid that is released when DNA is synthesized, or (3) performs hybridization of sequence-specific, fluorescence-labeled oligonucleotides with a DNA template. Method (1) has been mainly used throughout the world. Recently, a device has been developed that uses a solid-type detection section to detect the change in hydrogen ion concentration during DNA synthesis to read base sequences (this device may be considered a type of third-generation DNA sequencer, although the reaction principle of this device is the same as that of the second method).

To provide the latest sequencing technology for the research community, the Advanced Genomics Center uses almost every type of DNA sequencer available. The Center not only provides information regarding the evaluation and operation methods of DNA sequencers but also performs DNA sequencing. The read length is shorter (100−several hundred bases) and the accuracy of sequencing is lower (below 99.9%) in the second-generation DNA sequencer than those from first-generation DNA sequencers (depending on the type of device). However, the latest second-generation model developed by Illumina, Inc., named the HiSeq-2000, can read approximately 3 billion molecules of DNA fragments and 650 billion bases when operated for 10 days, as it can read 100 bases from each sides of a DNA molecule.