Genome technologies excercises
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 this novel
organism?
Consider complete sequence of novel organism
Long read is better for novel organisms
Illumina long read (short read if you really don’t want mistakes)
PacBIO
ONT
You are working for a biotech company that commercializes proteins for industrial purposes. They
are interested in a bacterium that has a unique metabolic characteristic.
They suspect that the bacterium has an enzyme that catalyzes a reaction that converts a certain
cheap metabolite (that the bacterium takes up from the environment) into a valuable end product.
They suspect this because they identified several mutant strains that cannot do the conversion. The
company has an assay to detect the valuable end product. They want to sell this enzyme in the
future. They have a number of questions and ask your advice:
1) How can they prove their hypothesis that the reaction converting the cheap metabolite
into the valuable one is a one enzyme reaction?
2) How could they identify the enzyme?
3) How could they produce the enzyme in a cost efficient manner?
Find loss of function mutations in the enzyme that lead to not being able to do the transition
anymore
Do Genome sequencing of mutant strains and see how many variants you find (if its 1-> 1 enzyme
reaction , 2 -> likely 2 enzyme reaction)
Clone in vector and put in bacterium and mass produce enzyme of interest
(can also use mammalian cells but not ideal here because it is for industrial purposes so purity less an
issue (for medical better use mammalian cells))
, For the treatment of a disease, patients injected with a hormone. 10% of patients develop severe
side effects after the treatment.
How could you evaluate whether the gene encoding the receptor for that hormone could explain
the difference in effects observed in patients?
Sequence receptor of patients with side effects / look for rare variants
Look for rare variants because only 10% , can be common variant -> sequence and do association
analysis afterwards
Targeted gene sequencing:
Use Sanger sequencing
You are doing a PCR experiment in the lab, and you try to amplify a part if a collagen gene, as part
of DNA diagnostics for a new point mutation in a connective tissue disease. You ordered primers
and did a standard PCR reaction. However, you are getting several bands on the gel.
How would you proceed in order to make the PCR work? Start with easy measures, but also
mention more sophisticated solutions if the easy ones do not work.
Do gradient PCR where you test multiple temperatures at the same time
If this doesn’t work you can do Hot-start PCR where enzyme stays inactivated until the in…
temperature (no non-specific reactions at room temperature)
Touch-down PCR: start at high temperatures and go down
Nested PCR : 2 primer pairs (1 that amplifies larger fragment and then internal primers used to
amplify that one) most sophisticated so do this last
Easiest solution is to order new primers if you don’t think they will work
You obtained samples from three unrelated patients diagnosed with the same rare disease. You
performed exome sequencing to identify the disease-causing gene.
However, no gene was identified that has a mutation in all three patients. What are the possible
explanations?
Consider unrelated patients , same rare disease , exome sequencing done
Could be mutation in non-coding region / could be environmental
With exome sequencing difficult to find copy number variants / physician made a mistake in
diagnosing the disease
If it is missed in all 3 patients it is possible your exome sequencing kit doesn’t cover that coding part
of genome
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 this novel
organism?
Consider complete sequence of novel organism
Long read is better for novel organisms
Illumina long read (short read if you really don’t want mistakes)
PacBIO
ONT
You are working for a biotech company that commercializes proteins for industrial purposes. They
are interested in a bacterium that has a unique metabolic characteristic.
They suspect that the bacterium has an enzyme that catalyzes a reaction that converts a certain
cheap metabolite (that the bacterium takes up from the environment) into a valuable end product.
They suspect this because they identified several mutant strains that cannot do the conversion. The
company has an assay to detect the valuable end product. They want to sell this enzyme in the
future. They have a number of questions and ask your advice:
1) How can they prove their hypothesis that the reaction converting the cheap metabolite
into the valuable one is a one enzyme reaction?
2) How could they identify the enzyme?
3) How could they produce the enzyme in a cost efficient manner?
Find loss of function mutations in the enzyme that lead to not being able to do the transition
anymore
Do Genome sequencing of mutant strains and see how many variants you find (if its 1-> 1 enzyme
reaction , 2 -> likely 2 enzyme reaction)
Clone in vector and put in bacterium and mass produce enzyme of interest
(can also use mammalian cells but not ideal here because it is for industrial purposes so purity less an
issue (for medical better use mammalian cells))
, For the treatment of a disease, patients injected with a hormone. 10% of patients develop severe
side effects after the treatment.
How could you evaluate whether the gene encoding the receptor for that hormone could explain
the difference in effects observed in patients?
Sequence receptor of patients with side effects / look for rare variants
Look for rare variants because only 10% , can be common variant -> sequence and do association
analysis afterwards
Targeted gene sequencing:
Use Sanger sequencing
You are doing a PCR experiment in the lab, and you try to amplify a part if a collagen gene, as part
of DNA diagnostics for a new point mutation in a connective tissue disease. You ordered primers
and did a standard PCR reaction. However, you are getting several bands on the gel.
How would you proceed in order to make the PCR work? Start with easy measures, but also
mention more sophisticated solutions if the easy ones do not work.
Do gradient PCR where you test multiple temperatures at the same time
If this doesn’t work you can do Hot-start PCR where enzyme stays inactivated until the in…
temperature (no non-specific reactions at room temperature)
Touch-down PCR: start at high temperatures and go down
Nested PCR : 2 primer pairs (1 that amplifies larger fragment and then internal primers used to
amplify that one) most sophisticated so do this last
Easiest solution is to order new primers if you don’t think they will work
You obtained samples from three unrelated patients diagnosed with the same rare disease. You
performed exome sequencing to identify the disease-causing gene.
However, no gene was identified that has a mutation in all three patients. What are the possible
explanations?
Consider unrelated patients , same rare disease , exome sequencing done
Could be mutation in non-coding region / could be environmental
With exome sequencing difficult to find copy number variants / physician made a mistake in
diagnosing the disease
If it is missed in all 3 patients it is possible your exome sequencing kit doesn’t cover that coding part
of genome
