322
V. Mittard-Runte et al.
subjected to another enzymatic method of sequencing called “sequencing by ligation”. The wavelength of fluorescence emission from each bead is detected by
a four-colour (e.g., red: adenine, green: cytosine, blue: guanine, yellow: thymine)
epifluorescence microscope, which follows each cycle of ligation. The “polony
sequencing” method is available as an open-source platform and has also been
licensed for further development to Agencourt/Applied Biosystems.
9.1.3.2 Sequencing-by-Hybridization
In contrast to the other methods described so far in this section, sequencing by
hybridization is a non-enzymatic method using the differential hybridization of target DNA to an array of immobilized oligonucleotide probes. This technique has
recently been used successfully in resequencing approaches.
Affymetrix
R
arrays were used for resequencing two Saccharomyces cerevisae
strains (Gresham et al. 2006) allowing the detection of approximately 30,000
known single-nucleotide polymorphisms (representing more than 90%) between the
two strains. Another study of five Escherichia coli strains was carried out using
NimbleGen resequencing arrays (Herring et al. 2006) in order to monitor spontaneous mutations that conveyed a selective growth advantage during adaptation to a
glycerol-based growth medium.
9.1.3.3 Nanopore Sequencing
Both Agilent and several academic research groups are developing nanopore
sequencing. The aim with this method is to sequence a single molecule of DNA
with no need for amplification by driving it through a small channel or nanopore
using an applied electric field. This technology is at the development stage and does
not yet seem to be capable of measuring single bases accurately.
9.1.4 Conclusion
The field of DNA sequencing is changing rapidly. We have therefore attempted to
provide a snapshot of the techniques now available. We have been concentrated
on established and widely used technologies, but nonetheless we have mentioned
emerging technologies even if these methods are probably far from becoming
“standard” sequencing technologies within the next years.
Whatever sequencing technology is used, the development of bioinformatic tools
which allow the analysis of the huge amount of sequencing data that will be
generated will continue to be a critical factor in the coming years.
The available budget for a particular study and the type of study that will be carried out (de novo, resequencing, metagenomics, gene expression via sequence tags,
etc) will also dictate which DNA sequencing technology will be used. A hybrid
strategy combining high coverage provided by the new generation of sequencing
V. Mittard-Runte et al.
subjected to another enzymatic method of sequencing called “sequencing by ligation”. The wavelength of fluorescence emission from each bead is detected by
a four-colour (e.g., red: adenine, green: cytosine, blue: guanine, yellow: thymine)
epifluorescence microscope, which follows each cycle of ligation. The “polony
sequencing” method is available as an open-source platform and has also been
licensed for further development to Agencourt/Applied Biosystems.
9.1.3.2 Sequencing-by-Hybridization
In contrast to the other methods described so far in this section, sequencing by
hybridization is a non-enzymatic method using the differential hybridization of target DNA to an array of immobilized oligonucleotide probes. This technique has
recently been used successfully in resequencing approaches.
Affymetrix
R
arrays were used for resequencing two Saccharomyces cerevisae
strains (Gresham et al. 2006) allowing the detection of approximately 30,000
known single-nucleotide polymorphisms (representing more than 90%) between the
two strains. Another study of five Escherichia coli strains was carried out using
NimbleGen resequencing arrays (Herring et al. 2006) in order to monitor spontaneous mutations that conveyed a selective growth advantage during adaptation to a
glycerol-based growth medium.
9.1.3.3 Nanopore Sequencing
Both Agilent and several academic research groups are developing nanopore
sequencing. The aim with this method is to sequence a single molecule of DNA
with no need for amplification by driving it through a small channel or nanopore
using an applied electric field. This technology is at the development stage and does
not yet seem to be capable of measuring single bases accurately.
9.1.4 Conclusion
The field of DNA sequencing is changing rapidly. We have therefore attempted to
provide a snapshot of the techniques now available. We have been concentrated
on established and widely used technologies, but nonetheless we have mentioned
emerging technologies even if these methods are probably far from becoming
“standard” sequencing technologies within the next years.
Whatever sequencing technology is used, the development of bioinformatic tools
which allow the analysis of the huge amount of sequencing data that will be
generated will continue to be a critical factor in the coming years.
The available budget for a particular study and the type of study that will be carried out (de novo, resequencing, metagenomics, gene expression via sequence tags,
etc) will also dictate which DNA sequencing technology will be used. A hybrid
strategy combining high coverage provided by the new generation of sequencing