All second-generation sequencing instruments utilize target molecule amplification techniques to create DNA colonies/clusters/nanoballs. The amplification step
increases sequencing signal intensity which reduces errors, at least at first. However,
simultaneous sequencing of clonally amplified DNA molecules is also the major
factor limiting the read length of second-generation instruments. Sequencing is
based on millions of synchronous elongation or ligation reactions at each DNA
cluster. However, the efficiency of any molecular reaction is never 100%. Consequently, during each round of elongation or ligation, some of the amplicons may not
be involved in a base incorporation or ligation event. This will cause these particular
amplicons to fall out of synch with their sister amplicons. While problems will be
minimal at first, those molecules that have become unsynchronized will create noise,
while molecular inefficiency in each additional cycle will lead to greater levels of
asynchronization. At some point, the noise created by asynchrony will make base
calling impossible (Buermans and den Dunnen 2014).
3.10 Third-Generation Sequencing
Third-generation sequencing is used to describe sequencing techniques that can
elucidate the sequence of a single (i.e., unamplified) DNA molecule.
Arguably the first third-generation machine was developed by Helicos. The
Helicos instrument utilized fluorescently tagged reversible terminator nucleotides
in a manner similar to Illumina machines. However, no amplification of the target
sequences was performed (Metzker 2010). The Helicos system had some powerful
features; e.g., easy sample preparation. However, Helicos went bankrupt in 2012
after selling only a few instruments (GenomeWeb News Staff Writer 2012). Of note,
a company called SeqLL has licensed technology from Helicos and appears to be
contemplating selling services and/or instruments (see www.seqll.com).
Pacific Biosciences (PacBio) single molecule, real-time (SMRT) sequencing is an
approach in which a powerful fluorescence microscope is used to “eavesdrop” on
DNA replication. The details of the PacBio approach are discussed in Table 1. The
PacBio SMRT technique was so captivating that the company rushed the instruments to market before many of the kinks had been worked out. This ultimately hurt
the company’s reputation, although PacBio has since worked hard to make improvements without overselling the capabilities of its machines. Greenleaf and Sidow
(2014) remarked upon the disappointing initial 2008 release of the first PacBio
machines and the critical improvement of the instruments since then by saying,
“Perhaps PacBio would have been well advised to have saved the fireworks it sent
into the Florida skies in 2008 for this year.”
Sequencing Plant Genomes
135
increases sequencing signal intensity which reduces errors, at least at first. However,
simultaneous sequencing of clonally amplified DNA molecules is also the major
factor limiting the read length of second-generation instruments. Sequencing is
based on millions of synchronous elongation or ligation reactions at each DNA
cluster. However, the efficiency of any molecular reaction is never 100%. Consequently, during each round of elongation or ligation, some of the amplicons may not
be involved in a base incorporation or ligation event. This will cause these particular
amplicons to fall out of synch with their sister amplicons. While problems will be
minimal at first, those molecules that have become unsynchronized will create noise,
while molecular inefficiency in each additional cycle will lead to greater levels of
asynchronization. At some point, the noise created by asynchrony will make base
calling impossible (Buermans and den Dunnen 2014).
3.10 Third-Generation Sequencing
Third-generation sequencing is used to describe sequencing techniques that can
elucidate the sequence of a single (i.e., unamplified) DNA molecule.
Arguably the first third-generation machine was developed by Helicos. The
Helicos instrument utilized fluorescently tagged reversible terminator nucleotides
in a manner similar to Illumina machines. However, no amplification of the target
sequences was performed (Metzker 2010). The Helicos system had some powerful
features; e.g., easy sample preparation. However, Helicos went bankrupt in 2012
after selling only a few instruments (GenomeWeb News Staff Writer 2012). Of note,
a company called SeqLL has licensed technology from Helicos and appears to be
contemplating selling services and/or instruments (see www.seqll.com).
Pacific Biosciences (PacBio) single molecule, real-time (SMRT) sequencing is an
approach in which a powerful fluorescence microscope is used to “eavesdrop” on
DNA replication. The details of the PacBio approach are discussed in Table 1. The
PacBio SMRT technique was so captivating that the company rushed the instruments to market before many of the kinks had been worked out. This ultimately hurt
the company’s reputation, although PacBio has since worked hard to make improvements without overselling the capabilities of its machines. Greenleaf and Sidow
(2014) remarked upon the disappointing initial 2008 release of the first PacBio
machines and the critical improvement of the instruments since then by saying,
“Perhaps PacBio would have been well advised to have saved the fireworks it sent
into the Florida skies in 2008 for this year.”
Sequencing Plant Genomes
135