Bioinformatica & Genoomanalyse Evelien Floor
NGS technology & primary data
processing
Sequencing technologies
Next generation sequencing
With NGS you first need to fragmentize the genomic DNA. Afterwards, adapters are added, and PCR
amplification of the library molecules is required to boost the signal within the sequencer.
Amplification is possible in two ways:
Emulsion based
o Water with primers and polymerase and oil are mixed which forms oil droplets filled
with the components. By increasing the temperature an amplification reaction will
start in the droplets. Each droplet contains less than one fragment of target DNA.
Local amplification on a carrier (chip, glass slide)
o First the genomic DNA will be randomly sheared into smaller pieces by nebulization
or sonication. The small fragments will be ligated with adapters, often followed by
PCR. The adapters contain binding sites for sequencing primers and are always the
same. The adapters bind to the primers on the surface and bridge amplification is
followed. Fragments have to be separated and sequenced one by one. Polonies (PCR
colonies) of the same molecule are generated to enhance the signal.
To detect the signal fluorophores attached to nucleotides are used. All four nucleotides are added
and bind to the sequence. The fluorophore attached to the nucleotide can be cleaved of to prevent
1
, Bioinformatica & Genoomanalyse Evelien Floor
mixed signals of the other nucleotides. After a cycle the block of the nucleotide is removed so a new
nucleotide can bind to the sequence.
Nowadays only two colors are used to speed up sequencing. Only green and red are used and in case
of an orange signal it means it is the other nucleotide. In case no signal is measured it is the
remaining nucleotide.
Ion torrent
When DNA polymerase incorporates a
nucleotide, it releases a proton. Ion torrent uses
small holes with ph sensors who measures the
release of protons. DNA is fragmentized and
coupled to a bead. Those beads are placed in the
well of the chip. Every 15 seconds a different
nucleotide is added to the wells, when a
nucleotide is incorporated the change in ph is
measured. When two of the same nucleotides
are incorporated two protons are released which
causes a higher increase in ph.
Nanoball sequencing
DNA nanoball sequencing is a high throughput
sequencing technology that is used to determine
the entire genomic sequence of an organism. The
method uses rolling circle replication to amplify
small fragments of genomic DNA into DNA
nanoballs. Fluorescent probes bind to
complementary DNA and the probes are then
ligated to anchor sequences bound to known
sequences on the DNA template. The base order is
determined via the fluorescence of the ligated and
bound probes.
Recap
Multitude of sequencing methods are
available
Library amplification methods:
o Emulsion PCR based
o Local amplification on a carrier
o Rolling circle amplification
Base-reading methods:
o Sequencing by synthesis: polymerase based
o Sequencing by ligation: ligase based
Detection of signals:
o Proton based (Ion torrent)
o Fluorescence
Sequencing technologies: single molecule
Third generation sequencing allows you to perform single molecule real time sequencing. This
technique generates very long reads and is useful for whole genome sequencing. However, a
disadvantage is that it makes a lot of errors.
2
NGS technology & primary data
processing
Sequencing technologies
Next generation sequencing
With NGS you first need to fragmentize the genomic DNA. Afterwards, adapters are added, and PCR
amplification of the library molecules is required to boost the signal within the sequencer.
Amplification is possible in two ways:
Emulsion based
o Water with primers and polymerase and oil are mixed which forms oil droplets filled
with the components. By increasing the temperature an amplification reaction will
start in the droplets. Each droplet contains less than one fragment of target DNA.
Local amplification on a carrier (chip, glass slide)
o First the genomic DNA will be randomly sheared into smaller pieces by nebulization
or sonication. The small fragments will be ligated with adapters, often followed by
PCR. The adapters contain binding sites for sequencing primers and are always the
same. The adapters bind to the primers on the surface and bridge amplification is
followed. Fragments have to be separated and sequenced one by one. Polonies (PCR
colonies) of the same molecule are generated to enhance the signal.
To detect the signal fluorophores attached to nucleotides are used. All four nucleotides are added
and bind to the sequence. The fluorophore attached to the nucleotide can be cleaved of to prevent
1
, Bioinformatica & Genoomanalyse Evelien Floor
mixed signals of the other nucleotides. After a cycle the block of the nucleotide is removed so a new
nucleotide can bind to the sequence.
Nowadays only two colors are used to speed up sequencing. Only green and red are used and in case
of an orange signal it means it is the other nucleotide. In case no signal is measured it is the
remaining nucleotide.
Ion torrent
When DNA polymerase incorporates a
nucleotide, it releases a proton. Ion torrent uses
small holes with ph sensors who measures the
release of protons. DNA is fragmentized and
coupled to a bead. Those beads are placed in the
well of the chip. Every 15 seconds a different
nucleotide is added to the wells, when a
nucleotide is incorporated the change in ph is
measured. When two of the same nucleotides
are incorporated two protons are released which
causes a higher increase in ph.
Nanoball sequencing
DNA nanoball sequencing is a high throughput
sequencing technology that is used to determine
the entire genomic sequence of an organism. The
method uses rolling circle replication to amplify
small fragments of genomic DNA into DNA
nanoballs. Fluorescent probes bind to
complementary DNA and the probes are then
ligated to anchor sequences bound to known
sequences on the DNA template. The base order is
determined via the fluorescence of the ligated and
bound probes.
Recap
Multitude of sequencing methods are
available
Library amplification methods:
o Emulsion PCR based
o Local amplification on a carrier
o Rolling circle amplification
Base-reading methods:
o Sequencing by synthesis: polymerase based
o Sequencing by ligation: ligase based
Detection of signals:
o Proton based (Ion torrent)
o Fluorescence
Sequencing technologies: single molecule
Third generation sequencing allows you to perform single molecule real time sequencing. This
technique generates very long reads and is useful for whole genome sequencing. However, a
disadvantage is that it makes a lot of errors.
2
