316
V. Mittard-Runte et al.
9.1 Sequence Data Generation
In the early days of genetics, scientists did not have the resources to look at more
than a few genes at a time. Nowadays microbial genome analysis is in a phase of
enormous growth. Since the publication of the first completely sequenced bacterial
genome Haemophilus influenzae (Fleischmann et al. 1995) in 1995 the genomes of
hundreds of bacteria have been sequenced. By January 2008 about 700 complete
genomes had been published and 3,250 ongoing genome projects were listed in the
Genomes OnLine Database (GOLD) [http://www.genomesonline.org]. Regarding
the latest GOLD reference from September 2007 (Liolios et al. 2008), the total number of recorded archaeal and bacterial projects was 1950 projects, while the advent
of new sequencing technology platforms such as pyrosequencing has significantly
contributed to the increase in the number of new microbial sequencing projects.
The GOLD site reports 134 projects using the 454 technology platform as part of a
Whole Genome Shotgun (WGS) sequencing project (Liolios et al. 2008).
9.1.1 Classical Genome Sequencing Approaches
Although several different sequencing techniques are available, the DNA sequencing method using chain termination inhibitors reported by Sanger et al. (1977) has
remained the basis for genome sequencing for more than 25 years.
9.1.1.1 The Sanger Method
Classical DNA sequencing is accomplished by the chain termination inhibitor
method (Sanger et al. 1977). This method essentially involves copying one strand of
a piece of DNA using a short DNA primer, which is complementary to the DNA
strand you want to sequence, an enzyme called DNA polymerase and the four
nucleotides. The Sanger method employs a mixture of normal nucleotides dNTPs
and special dideoxy-nucleotides (ddNTPs). These ddNTPs lack a hydroxyl-group at
the 3 carbon of the ribose sugar. This prevents new nucleotides from being added to
a DNA strand after a ddNTP has been incorporated. Thus, once a ddNTP is inserted
into a growing DNA strand, synthesis of that strand is stopped. In addition, different
fluorescent tags are attached to the four types of ddNTPs providing a means of identifying which ddNTP nucleotide has been incorporated (Prober et al. 1987). After
many repeated cycles of synthesis all the possible lengths of DNA are represented,
each piece of synthesized DNA containing a fluorescent label at its terminus.
Amplified DNA can then be separated according to size using gel electrophoresis. Early Sanger sequencing technology used vertical polyacrylamide gels to carry
out this electrophoresis step. These gels need to be prepared manually. Nowadays
capillary-electrophoretic sequencers, in which the fragments migrate through a resin
within an individual capillary, carry out this step of the process. The machine uses an
ultraviolet laser to detect the fluorescent labels that have been incorporated during
the polymerisation reaction. The most recent machines, such as the ABI3730XL,
V. Mittard-Runte et al.
9.1 Sequence Data Generation
In the early days of genetics, scientists did not have the resources to look at more
than a few genes at a time. Nowadays microbial genome analysis is in a phase of
enormous growth. Since the publication of the first completely sequenced bacterial
genome Haemophilus influenzae (Fleischmann et al. 1995) in 1995 the genomes of
hundreds of bacteria have been sequenced. By January 2008 about 700 complete
genomes had been published and 3,250 ongoing genome projects were listed in the
Genomes OnLine Database (GOLD) [http://www.genomesonline.org]. Regarding
the latest GOLD reference from September 2007 (Liolios et al. 2008), the total number of recorded archaeal and bacterial projects was 1950 projects, while the advent
of new sequencing technology platforms such as pyrosequencing has significantly
contributed to the increase in the number of new microbial sequencing projects.
The GOLD site reports 134 projects using the 454 technology platform as part of a
Whole Genome Shotgun (WGS) sequencing project (Liolios et al. 2008).
9.1.1 Classical Genome Sequencing Approaches
Although several different sequencing techniques are available, the DNA sequencing method using chain termination inhibitors reported by Sanger et al. (1977) has
remained the basis for genome sequencing for more than 25 years.
9.1.1.1 The Sanger Method
Classical DNA sequencing is accomplished by the chain termination inhibitor
method (Sanger et al. 1977). This method essentially involves copying one strand of
a piece of DNA using a short DNA primer, which is complementary to the DNA
strand you want to sequence, an enzyme called DNA polymerase and the four
nucleotides. The Sanger method employs a mixture of normal nucleotides dNTPs
and special dideoxy-nucleotides (ddNTPs). These ddNTPs lack a hydroxyl-group at
the 3 carbon of the ribose sugar. This prevents new nucleotides from being added to
a DNA strand after a ddNTP has been incorporated. Thus, once a ddNTP is inserted
into a growing DNA strand, synthesis of that strand is stopped. In addition, different
fluorescent tags are attached to the four types of ddNTPs providing a means of identifying which ddNTP nucleotide has been incorporated (Prober et al. 1987). After
many repeated cycles of synthesis all the possible lengths of DNA are represented,
each piece of synthesized DNA containing a fluorescent label at its terminus.
Amplified DNA can then be separated according to size using gel electrophoresis. Early Sanger sequencing technology used vertical polyacrylamide gels to carry
out this electrophoresis step. These gels need to be prepared manually. Nowadays
capillary-electrophoretic sequencers, in which the fragments migrate through a resin
within an individual capillary, carry out this step of the process. The machine uses an
ultraviolet laser to detect the fluorescent labels that have been incorporated during
the polymerisation reaction. The most recent machines, such as the ABI3730XL,