Summary
Summary Next generation sequencing and analysis
- Course
- Institution
HC1 van het genome deel
[Show more]
2 reviews
By: KA26 • 4 year ago
By: thomasgrootnibbelink • 5 year ago
Seller
Follow
evelienfloor
Reviews received
Content preview
Bioinformatica & Genoomanalyse Evelien FloorNGS 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
The benefits of buying summaries with Stuvia:
Guaranteed quality through customer reviews
Stuvia customers have reviewed more than 700,000 summaries. This how you know that you are buying the best documents.
Quick and easy check-out
You can quickly pay through credit card or Stuvia-credit for the summaries. There is no membership needed.
Focus on what matters
Your fellow students write the study notes themselves, which is why the documents are always reliable and up-to-date. This ensures you quickly get to the core!
Frequently asked questions
What do I get when I buy this document?
You get a PDF, available immediately after your purchase. The purchased document is accessible anytime, anywhere and indefinitely through your profile.
Satisfaction guarantee: how does it work?
Our satisfaction guarantee ensures that you always find a study document that suits you well. You fill out a form, and our customer service team takes care of the rest.
Who am I buying these notes from?
Stuvia is a marketplace, so you are not buying this document from us, but from seller evelienfloor. Stuvia facilitates payment to the seller.
Will I be stuck with a subscription?
No, you only buy these notes for $4.33. You're not tied to anything after your purchase.
Can Stuvia be trusted?
4.6 stars on Google & Trustpilot (+1000 reviews)
53068 documents were sold in the last 30 days
Founded in 2010, the go-to place to buy study notes for 14 years now