OEFENINGEN SESSIE VERPLICHT 7/12/22
You discovered a novel bacterium of which the sequence is completely unknown. What NGS
technology would you prefer if you want to know the complete sequence of the novel
organisation:
-ik denk Illumina
Student: ‘nanopore advantage of long reads’
Opmerking: PacBio they share the long read capacity
Prof: ‘would Illumina be possible ’it does short sequences and if you have a lot of
repetitive sequences, you will have a problem with producing the whole genome .
- Ion current produces only short reads and has also high error rate especially with
these polymers
- Comparing PacBio with nanopore? Same reason, PacBio may be more expensive,
with nanopore is the error rate still some higher.
Lang probleem; vraag 2:
….. ‘how can they prove their hypothesis that the reaction converting the cheap metabolite
into the valuable one is a one enzyme reaction?
I think: by inactivating the enzyme they suspect that does it, and if the reaction doesn’t take
place any more than it has certainly something to do with it. And after that they inactivate
the other enzymes and if the conversion keeps happening it suggests the one enzyme
reaction.
‘prof’ in the mutant strains there are also the enzymes that can’t convert, so how can we use
these mutant strains compare if the enzyme is indeed a one enzyme reaction. So how can
we see it compare the strains of the mutant with healthy one so we do whole genome
sequencing on the strains
How can u be sure that it is indeed a one enzyme reaction? If all 5 mutant strains have a
mutation (not the same ) give the same mutation than it gives a strong indication that only
one gene can cause this abnormality in your 5 mutant strains
How could they identify the enzyme?
Clone this gene in a vector (expression vector) use a system that will produce this enzyme
for you. What host scan be used? Bacteria E. coli, yeasts (advantage: eukaryote cell, ), insect
cells, mammalian cells
, Vraag 3: imagine that for the treatment of a disease, patients are injected with a hormone.
However it turns out that about 10% of the patients develop severe adverse side effects
after the treatment. How could you evaluate whether the gene encoding the receptor for
the hormone could explain the difference in effects observed in the patients.
Antwoord: (van mij eumh miss weer knock-out model maken)
Antwoord prof/student: (student suggest sequencing) maybe as a first step you could
consider checking some of these preferential coding genes and see maybe in the extreme
10% maybe they will have let’s say an amino acid A while the other hand has a B amino acid
this gives in this case a very forward explanation. Then after this you can go sequencing
of this gene doesn’t mean whole genome sequencing you can go with the MIPs
(Molecular Inversion Probes) and the SNPs (Single Nucleotide Polymorphisms) to capture
only the gene encoding fort his receptor and then you sequence the receptor for this whole
cohort. (you could also go for the whole so you get an image of all the polymorphisms).
Vraag 4: in a patient with a syndromic mental handicap, a microdeletion is found on
chromosome 8q13 in your lab. Similar deletion have never been described in controls
individuals. Yet in a lab in South-Afrika, a colleague reports exactly the same microdeletion in
another patient. Explain the most likely molecular mechanism this deletion has occurred in
either patient.
(the fact that it’s not found in controls suggest that the microdeletion causes the disease)
Antwoord: it is a copy number variation in many cases the explanation is non homologous
recombination what can you find at the break points of the micro deletion.
During meiosis the homolog sequence will be put over each other, but if something goes
wrong or something is different then you will end up where the 2 homologous sequences
are positioned in another way. Could be that you end up with a chromosome that ends up
somewhere and goes un, instead of 3 repetitive sequences you end up with 4 repetitive
sequences and that a certain part of the human genome is duplicated.
So about the patients, unrelated, identical deletion and a crossover during meiosis that may
cause to be deletious.
Vraag 5: (follow-up question): the 2 patients mentioned above share various clinical
characteristics, e.g., show facial resemblance and have common abnormalities. A few years
later, many more patients that look very much like these 2 patients are described in the
literature. The deletion is now called the ABCD syndrome. In the deletion lie 6 genes.
Describe a strategy to demonstrate the contribution of the individual genes within the
deletion to the clinical characteristics of the disease.
You discovered a novel bacterium of which the sequence is completely unknown. What NGS
technology would you prefer if you want to know the complete sequence of the novel
organisation:
-ik denk Illumina
Student: ‘nanopore advantage of long reads’
Opmerking: PacBio they share the long read capacity
Prof: ‘would Illumina be possible ’it does short sequences and if you have a lot of
repetitive sequences, you will have a problem with producing the whole genome .
- Ion current produces only short reads and has also high error rate especially with
these polymers
- Comparing PacBio with nanopore? Same reason, PacBio may be more expensive,
with nanopore is the error rate still some higher.
Lang probleem; vraag 2:
….. ‘how can they prove their hypothesis that the reaction converting the cheap metabolite
into the valuable one is a one enzyme reaction?
I think: by inactivating the enzyme they suspect that does it, and if the reaction doesn’t take
place any more than it has certainly something to do with it. And after that they inactivate
the other enzymes and if the conversion keeps happening it suggests the one enzyme
reaction.
‘prof’ in the mutant strains there are also the enzymes that can’t convert, so how can we use
these mutant strains compare if the enzyme is indeed a one enzyme reaction. So how can
we see it compare the strains of the mutant with healthy one so we do whole genome
sequencing on the strains
How can u be sure that it is indeed a one enzyme reaction? If all 5 mutant strains have a
mutation (not the same ) give the same mutation than it gives a strong indication that only
one gene can cause this abnormality in your 5 mutant strains
How could they identify the enzyme?
Clone this gene in a vector (expression vector) use a system that will produce this enzyme
for you. What host scan be used? Bacteria E. coli, yeasts (advantage: eukaryote cell, ), insect
cells, mammalian cells
, Vraag 3: imagine that for the treatment of a disease, patients are injected with a hormone.
However it turns out that about 10% of the patients develop severe adverse side effects
after the treatment. How could you evaluate whether the gene encoding the receptor for
the hormone could explain the difference in effects observed in the patients.
Antwoord: (van mij eumh miss weer knock-out model maken)
Antwoord prof/student: (student suggest sequencing) maybe as a first step you could
consider checking some of these preferential coding genes and see maybe in the extreme
10% maybe they will have let’s say an amino acid A while the other hand has a B amino acid
this gives in this case a very forward explanation. Then after this you can go sequencing
of this gene doesn’t mean whole genome sequencing you can go with the MIPs
(Molecular Inversion Probes) and the SNPs (Single Nucleotide Polymorphisms) to capture
only the gene encoding fort his receptor and then you sequence the receptor for this whole
cohort. (you could also go for the whole so you get an image of all the polymorphisms).
Vraag 4: in a patient with a syndromic mental handicap, a microdeletion is found on
chromosome 8q13 in your lab. Similar deletion have never been described in controls
individuals. Yet in a lab in South-Afrika, a colleague reports exactly the same microdeletion in
another patient. Explain the most likely molecular mechanism this deletion has occurred in
either patient.
(the fact that it’s not found in controls suggest that the microdeletion causes the disease)
Antwoord: it is a copy number variation in many cases the explanation is non homologous
recombination what can you find at the break points of the micro deletion.
During meiosis the homolog sequence will be put over each other, but if something goes
wrong or something is different then you will end up where the 2 homologous sequences
are positioned in another way. Could be that you end up with a chromosome that ends up
somewhere and goes un, instead of 3 repetitive sequences you end up with 4 repetitive
sequences and that a certain part of the human genome is duplicated.
So about the patients, unrelated, identical deletion and a crossover during meiosis that may
cause to be deletious.
Vraag 5: (follow-up question): the 2 patients mentioned above share various clinical
characteristics, e.g., show facial resemblance and have common abnormalities. A few years
later, many more patients that look very much like these 2 patients are described in the
literature. The deletion is now called the ABCD syndrome. In the deletion lie 6 genes.
Describe a strategy to demonstrate the contribution of the individual genes within the
deletion to the clinical characteristics of the disease.
