Sequencing techniques Flashcards
(12 cards)
How does NanoPore work?
Not a sequencing by synthesis approach, instead a physical reading of the DNA.
We have a membrane and inside is a nanopore. If we push a single stranded DNA through the pore we will have a conductive force/electric force that should change when we push through the DNA strand. So we are looking at the change in current that is induced by the physical nucleotides thats going through the pore.
Each nanopore is connected to a sensory array chip that can measure the currency.
Essentially there is no biochemical reactions anymore, except for a HP motor. If we can connect the Watson and the Prick strand (the plus and the minus strand), to first push through the positive and then the negative strand. This already gives us both strands, so we have the complement sequence which improves the accuracy.
What is NanoPore?
Fourth generation sequencing. We are no longer sequencing by synthesis like the Illumina and PacBio, instead we are just reading the sequence through a pore.
What is Zero Mode Waveguide (ZMW)?
It is used in PacBio sequencing to observe the fluorescent labelling being cleaved off during synthesis.
How can we improve the accuracy of PacBio?
The benefit to this method is the length of the reads. The accuracy is quite low BUT if we circularize the molecule we can loop through the same molecule several times, meaning that we will get a higher accuracy. With 15 cycles we get 99.3% accuracy.
How does PacBio sequencing work?
Sequencing by synthesis. By labelling dNTPs with phosphor fluorescent, we can ensure that when a dNTP is bound, the fluorescence group will be released. Meaning that if we add different fluorescence labelling to each dNTP, we will be able to distinguish which nucleotide is being bound while the polymerase is working.
To observe the fluorescence we use the Zero Mode Waveguide (ZMW) which is physically linked to the polymerase. Here we get a very low signal to noise ratio, meaning that it can be hard to differentiate between noise and signal.
In comparison to illumina it is very slow meaning that the throughput is low. The machine is also very big and expensive.
The benefit to this method is the length of the reads. The accuracy is quite low BUT if we circularize the molecule we can loop through the same molecule several times, meaning that we will get a higher accuracy. With 15 cycles we get 99.3% accuracy.
So when we are doing this we can get long reads with a high accuracy. There is no bias in what gets more actively read, meaning that our noise is random and gets smaller with each cycle.
How does NanoPore work?
Not a sequencing by synthesis approach, instead a physical reading of the DNA.
We have a membrane and inside is a nanopore. If we push a single stranded DNA through the pore we will have a conductive force/electric force that should change when we push through the DNA strand. So we are looking at the change in current that is induced by the physical nucleotides thats going through the pore.
Each nanopore is connected to a sensory array chip that can measure the currency.
Essentially there is no biochemical reactions anymore, except for a HP motor. If we can connect the Watson and the Prick strand (the plus and the minus strand), to first push through the positive and then the negative strand. This already gives us both strands, so we have the complement sequence which improves the accuracy.
What is PacBio sequencing?
It is a sequencing by synthesis approach that can produce long reads with low bias. It utilizes fluorescent labelling of dNTPs.
How does the capillary electrophoresis in Sanger sequencing work?
In the capillary the fragments will be separated by size, meaning that small fragments pass through the electrophoresis device first. There is a laser that activates the fluorescent group and a detector to read them. This results in peaks we get out as a chromogram. The process is done at 55 degrees to avoid renaturation. The chromogram can then be analysed in computer models to get the full sequence.
Why is capillary electrophoresis in Sanger sequencing done at 55 degrees?
To keep the single stranded DNA from renaturation.
What are dNTPs and ddNTPs?
The nucleotides used to build DNA
dNTPS: (dATP, dTTP, dGTP, dCTP). The d stands for deoxy, meaning that the nucleotide is missing one OH group which means it can bind to the template strand.
ddNTP: means that its missing both OH groups and therefore the reaction stops when it is added.
What are the 4 primary sequencing techniques and which generation do they belong to?
First gen: Sanger
Second gen: Illumina
Third: PacBio
Fourth: Nanopore
How does Sanger sequencing work?
- Denature DNA by heating it to 95 degrees
- Anneal only 1 primer
- DNA polymerase will extend and amplify the fragment. This is done with both dNTPS and ddNTPs.
When adding a constant ratio of dNTPS and ddNTPS we will have equal chance of getting ddNTPs at all positions.
The general idea is that we dye the ddNTPs with fluorescent dye and we will have equal parts of sequences that ends at different places. Meaning that we have fragments of all lengths. Therefore, when we know how long the sequence is, we can see what nucleotide is at which position.
Now we do capillary electrophoresis.
In the capillary the fragments will be separated by size, meaning that small fragments pass through the electrophoresis device first. There is a laser that activates the fluorescent group and a detector to read them. This results in peaks we get out as a chromogram. The process is done at 55 degrees to avoid renaturation.
The chromogram can then be analysed in computer models to get the full sequence.
