Exercises
1. You have 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 this novel bacterium?
You can use long read methods for de novo sequencing because of repetitive sequences. With short
reads, it is difficult to map the different short reads. But bacteria often don’t have repetitive
sequences so short read sequencing can be equally good. You can use whatever you have in the lab.
- Illumina: can be used
- PACBIO: can be used but very expensive
- IonProton:
- Nanopore: very error prone so not very suitable
- BioNano: for structural variation so not very suitable
- BGI sequencing:
2. There is a bacteria which makes out of a cheap metabolite into a valuable end product using one
enzyme. A: how can they prove their hypothesis that the reaction converting the cheap metabolite
into the valuable one is a one enzyme reaction? B: how could they produce the enzyme in a cost
efficient manner?
A: Sequence the bacterium that can produce it and a mutant strain that cannot produce it. Then look
for differences in the genome. You are interested in loss-of-function variants in the mutants that
make the bacterium unable to convert the metabolite to the product: frame-shift mutations,
deletions.. Since the genome is not that big it is possible to find the mutations in a single gene. Then
you have confirmed your hypothesis that it is just one enzyme. If it was a multiple-enzyme reaction
you would find mutations in several enzymes.
B: Expression cloning. Transformation of a bacteria (eg E. coli) with a plasmid that contains the gene
that encodes for this enzyme. You can also use the bacterium itself to do it and purify it but this
might be difficult if the bacterium doesn’t grow very easily in labs. Transformation of this bacterium
can also be more difficult than in E. coli in which it has been done uncountable times.
3. In a patient with a syndromic mental handicap, a spontaneous microdeletion is found on
chromosome 8q13 in your lab. Similar deletions have never been described in control individuals.
Yet in a lab in South-Africa a colleague reports exactly the same microdeletion in another patient.
Explain the most likely molecular mechanisms this microdeletion has occurred in either patient?
Non-allelic homologous recombination. Two sequences are recognised on the chromosome which
causes crossing over. Sequences are replaced over chromosomes.
Segmental duplications: pieces of the genome that are 99% identical can cause a misalignment. You
get a cross-over and get a duplication of the same site. It can also cause for a deletion. Explains why
there can be duplications or deletions in the same region in different people: the recurrent ones.
1. You have 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 this novel bacterium?
You can use long read methods for de novo sequencing because of repetitive sequences. With short
reads, it is difficult to map the different short reads. But bacteria often don’t have repetitive
sequences so short read sequencing can be equally good. You can use whatever you have in the lab.
- Illumina: can be used
- PACBIO: can be used but very expensive
- IonProton:
- Nanopore: very error prone so not very suitable
- BioNano: for structural variation so not very suitable
- BGI sequencing:
2. There is a bacteria which makes out of a cheap metabolite into a valuable end product using one
enzyme. A: how can they prove their hypothesis that the reaction converting the cheap metabolite
into the valuable one is a one enzyme reaction? B: how could they produce the enzyme in a cost
efficient manner?
A: Sequence the bacterium that can produce it and a mutant strain that cannot produce it. Then look
for differences in the genome. You are interested in loss-of-function variants in the mutants that
make the bacterium unable to convert the metabolite to the product: frame-shift mutations,
deletions.. Since the genome is not that big it is possible to find the mutations in a single gene. Then
you have confirmed your hypothesis that it is just one enzyme. If it was a multiple-enzyme reaction
you would find mutations in several enzymes.
B: Expression cloning. Transformation of a bacteria (eg E. coli) with a plasmid that contains the gene
that encodes for this enzyme. You can also use the bacterium itself to do it and purify it but this
might be difficult if the bacterium doesn’t grow very easily in labs. Transformation of this bacterium
can also be more difficult than in E. coli in which it has been done uncountable times.
3. In a patient with a syndromic mental handicap, a spontaneous microdeletion is found on
chromosome 8q13 in your lab. Similar deletions have never been described in control individuals.
Yet in a lab in South-Africa a colleague reports exactly the same microdeletion in another patient.
Explain the most likely molecular mechanisms this microdeletion has occurred in either patient?
Non-allelic homologous recombination. Two sequences are recognised on the chromosome which
causes crossing over. Sequences are replaced over chromosomes.
Segmental duplications: pieces of the genome that are 99% identical can cause a misalignment. You
get a cross-over and get a duplication of the same site. It can also cause for a deletion. Explains why
there can be duplications or deletions in the same region in different people: the recurrent ones.
