320
V. Mittard-Runte et al.
9.1.2.1 Pyrosequencing or 454 Sequencing
The 454 Life Sciences
R
Corporation, USA has developed a scalable, highly parallel sequencing system. The 454 pyrosequencing method was described in detail by
Margulies and colleagues (2005). Briefly, the initial version of the apparatus (GS20)
used a novel fibre-optic slide of individual wells and was able to sequence 25 million bases, at 99% or better accuracy, in one 4-h run. The machine used an emulsion
method for DNA amplification (called emulsion polymerase chain reaction – short
emPCR) (Nakano et al. 2003) of bead-anchored oligonucleotides (Dressman et al.
2003). The technologies applied included pyrosequencing and an instrument for
sequencing by synthesis using a pyrosequencing protocol (Ronaghi et al. 1998)
optimized for solid support and picoliter-scale volumes (Margulies et al. 2005).
In pyrosequencing, each incorporation of a nucleotide by DNA polymerase results
in the release of pyrophosphate, which initiates a series of downstream reactions
that ultimately produce light by the firefly enzyme luciferase. The amount of light
produced is proportional to the number of nucleotides incorporated (Mardis 2008).
For more details regarding the utility, throughput, accuracy and robustness of the
454 system refer to the Margulies et al. publication (2005). The 454 pyrosequencing
method has experienced rapid growth since its partnership with Roche Diagnostics
and release of its GS20 sequencing machine in 2005 and the Genome Sequencer
FLX machine in 2007 (Table 9.1). The new GS FLX system Titanium series
generates up to one Million reads and 1 GB of data per one 10 h sequencing run.
For more detailed information and recent developments regarding the 454
sequencing technology and the Roche
R
GS FLX machine refer to the following
website http://www.454.com
9.1.2.2 Illumina
R
Sequencing Technology
Illumina
R
sequencing technology is based on massively parallel sequencing of
millions of fragments using a reversible terminator-based sequencing chemistry
(Ju et al. 2006). The technology relies on the attachment of randomly fragmented
genomic DNA to a planar, optically transparent surface, and solid phase amplification also called bridge amplification (Adams et al. 1997, Fedurco et al. 2006) to
create an ultra-high density sequencing flow cell with >50 million clusters, each
containing ∼1,000 copies of the same template. These dense clusters of dsDNA
are sequenced using reversible fluorescent dNTP terminators with removable fluorophores yielding about 1 GB of raw data (Table 9.1). This novel approach ensures
high accuracy and true base-by-base sequencing, eliminating sequence-context specific errors and enabling sequencing through homopolymers and short repetitive
sequences.
After completion of the first read, the templates can be regenerated in situ using
the Paired-End Module to enable a second > 36 bp read from the opposite end of the
fragment. This paired-end methodology leads to an increase of the yield to > 3 GB
of paired-end data.
V. Mittard-Runte et al.
9.1.2.1 Pyrosequencing or 454 Sequencing
The 454 Life Sciences
R
Corporation, USA has developed a scalable, highly parallel sequencing system. The 454 pyrosequencing method was described in detail by
Margulies and colleagues (2005). Briefly, the initial version of the apparatus (GS20)
used a novel fibre-optic slide of individual wells and was able to sequence 25 million bases, at 99% or better accuracy, in one 4-h run. The machine used an emulsion
method for DNA amplification (called emulsion polymerase chain reaction – short
emPCR) (Nakano et al. 2003) of bead-anchored oligonucleotides (Dressman et al.
2003). The technologies applied included pyrosequencing and an instrument for
sequencing by synthesis using a pyrosequencing protocol (Ronaghi et al. 1998)
optimized for solid support and picoliter-scale volumes (Margulies et al. 2005).
In pyrosequencing, each incorporation of a nucleotide by DNA polymerase results
in the release of pyrophosphate, which initiates a series of downstream reactions
that ultimately produce light by the firefly enzyme luciferase. The amount of light
produced is proportional to the number of nucleotides incorporated (Mardis 2008).
For more details regarding the utility, throughput, accuracy and robustness of the
454 system refer to the Margulies et al. publication (2005). The 454 pyrosequencing
method has experienced rapid growth since its partnership with Roche Diagnostics
and release of its GS20 sequencing machine in 2005 and the Genome Sequencer
FLX machine in 2007 (Table 9.1). The new GS FLX system Titanium series
generates up to one Million reads and 1 GB of data per one 10 h sequencing run.
For more detailed information and recent developments regarding the 454
sequencing technology and the Roche
R
GS FLX machine refer to the following
website http://www.454.com
9.1.2.2 Illumina
R
Sequencing Technology
Illumina
R
sequencing technology is based on massively parallel sequencing of
millions of fragments using a reversible terminator-based sequencing chemistry
(Ju et al. 2006). The technology relies on the attachment of randomly fragmented
genomic DNA to a planar, optically transparent surface, and solid phase amplification also called bridge amplification (Adams et al. 1997, Fedurco et al. 2006) to
create an ultra-high density sequencing flow cell with >50 million clusters, each
containing ∼1,000 copies of the same template. These dense clusters of dsDNA
are sequenced using reversible fluorescent dNTP terminators with removable fluorophores yielding about 1 GB of raw data (Table 9.1). This novel approach ensures
high accuracy and true base-by-base sequencing, eliminating sequence-context specific errors and enabling sequencing through homopolymers and short repetitive
sequences.
After completion of the first read, the templates can be regenerated in situ using
the Paired-End Module to enable a second > 36 bp read from the opposite end of the
fragment. This paired-end methodology leads to an increase of the yield to > 3 GB
of paired-end data.