Exam sets

Question 1

Explain the principle of DiGE. How to identify proteins in the subsequent step of different mass spectrometry techniques? Unlimited pages (Lavigne).

Answer 1

DIGE is a way to analyse differentially expressed genes by labelling with fluorescent dyes. We have 2 samples where we wish to separate and differentiate proteins. 3 samples are therefore prepared, sample A, sample B and sample A+B.
Each sample will be covalently added to a fluorescent dye, where it binds to the lysines. The dye needs to:
- Be excitable for different wavelengths so we only see one dye at a time
- Not affect pH or charge of the fragments
- Be photostable
When each sample has been dyed we add all three to the same 2D-PAGE gel.
2D PAGE gel works by first performing isoelectric focusing and then doing SDS PAGE.
Isoelectric focusing separates based on electric charge:
We place the sample on a pH strip and apply an electric charge. The sample contents will migrate until they reach their isoelectric point. Isoelectric point is the pH where the molecules net charge = 0.
The strip is denatured to unfold it and SDS anions are added. SDS works by binding 1 SDS to every 2 amino acids. In this way we make larger chains heavier than short ones. The strip is transversely added to the SDS-gel. We now apply an electric field where the proteins will separate based on molecular weight.

Now we have separated the proteins we have different choices of identifying the proteins:
- Protein mass fingerprinting
- MS/MS

Question 2

TMM normalization (Jelier, 1/3 page)

Answer 2

If we want to know change in gene expression in the same gene in different situations:
- Scaling factors (divide all counts by a constant)
The scaling factor reflects the size of the library between different runs.
We are always looking at the same gene, therefore we don’t need to normalize for gene length

If we have redistribution of reads:
- TMM: scaling factor normalization, based on weighed trimmed means of the log expression ratios between two conditions

Question 3

Mass spectrometry imaging and application in metabolomics (1/3 page)

Question 4

What is the purpose of the Burrows-Wheeler transform in read mapping. Don’t explain how the transform is attained (Faust, 1/3 page)

Answer 4

The purpose if to index the genome so we can access it quicker. It makes a fast look up algorithm.
IF we want to transform:
Build prefixes, sort alphabetically and the last column is the transformed.
BANANA
ANANA
NANA
ANA
NA
A
$

BANANA$
ANANA$B
NANA$BA
ANA$BAN
NA$BANA
A$BANAN
$BANANA

$BANANA
A$BANAN
ANA$BAN
ANANA$B
BANANA$
NA$BANA
NANA$BA

Question 5

Ribosome profiling and its applications (Lavigne, ½ page)

Answer 5

Is used to investigate translational control in the cell, meaning we can see what a cell is currently translating. It works by freezing the cell, lyse it, remove mRNA and purify. This leaves only the ribosome footprints. This is the ribosomal protected mRNAs that we can now do library prep:
- Ligate linkers
- Reverse transcribe into cDNA
- PCR amplification
Now we do sequencing, often illumina, and analyse the reads.

Question 6

CRISPR/Cas9 (Lavigne, ½ page)

Answer 6

CRISPR/Cas9 is a popular and relatively new gene editing technique. It uses the ‘immune system’ of bacteria when attacked by a virus. Make drawing. It’s used to knockout genes or inserting genes.

Question 7

Q4. (Lavigne, 1.5 page)What sequencing technique would you use and why (they don’t repeat). Explain library preparation for each.
A.To determine the full genome of a novel archaea
B.To resequence human genome for SNP analysis
C.To identify a megaplasmid 500 kb in a bacteria whose genome is 6 Mb
D.Genome-wide expression level of human parasite Trypanosoma <something> extracted from the bloodstream</something>

Question 8

Discuss Y2H. Emphasize the advantages and disadvantages of the technique and provide other 2H techniques that have been developed to overcome the disadvantages (no page limit).

Question 9

Compare TALEN and ZFN and compare with CRISPR.

Question 10

Discuss DIGE.

Question 11

Make an exercise about assembly with overlap.

Question 12

RNAseq: what is the problem with isoforms and two ways to solve it.

Question 13

In mass spectrometry, you use a library construct, why not a complete proteome library?

Question 14

4) Which sequencing technique would you use in the following four cases? Briefly explain. (questions were slightly different but essentially the same)

1. sequencing of a 200 kb plasmid with many repeats
2. resequencing of a human genome sequence for SNP analysis
3. you want to investigate the transcription of a bacterial species under stress
4. control of cloning in PUC vector

Answer 14

1. Long read: Nanopore or PacBio
2. Illumina for fast and accurate sequencing
3. RNA sequencing using illumina
4. Sanger: Sanger sequencing is often used for routine verification of cloning vectors like PUC vectors. It provides high accuracy for confirming the presence and correctness of inserted sequences and is cost-effective for sequencing relatively short DNA fragments, such as those in cloning vectors.

Question 15

Explain Illumina sequencing and Nanopore sequencing fully (from sampling until data analysis). Explain the differences between these sequencing techniques. Explain how these techniques can be complementary.(Lavigne) (unlimited pages)

Question 16

Explain the expertimental set-up and principles of Differential Gel Electrophoresis (DiGE) for quantitative proteomics.(Lavigne) (front + backside)

Question 17

In an RNAseq experiment, the power is often quite low. Explain this and what can be done to improve the power.(Jelier) (1/3 page)

Answer 17

• RNA seq
• Metabolomics
  Metabolomics is the study of metabolites (small molecules) produced by the microbiota and the host. By analyzing the metabolic profiles of fecal samples, researchers can infer changes in microbial activity and gut health, as different metabolites can indicate the presence and activity of specific microbial species and metabolic pathways.
• Metagenomics
  Metagenomic sequencing involves sequencing the entire genetic material from a microbial community in a sample.

Question 18

Q3C.Explain de Bruijn graph assembly for the following example. Reads: ACTT, CATG, GTCA, AAGT, TGAC. (Hint: You can use 4-mers for the assembly.)(Faust) (1/3 page)

Question 19

Explain different ways how you can remove the rRNA from your RNA sample for RNAseq.(Lavigne) (1/2 page)

Answer 19

rRNA depletion, mRNA selection

Question 20

Explain the advantages and disadvantages of ZFN and TALEN compared to CRISPR-Cas.(Lavigne) (1/2 page)

Question 21

Explain which interactomics techniques you would use for the following experiments. Each technique can be used only once.(Lavigne) (front + backside)
A) Determine which proteins interact with the bacterial DNA polymerase under specific conditions.
B) Determine the ligand of a bacterial protein with a hydrophobic part.
C) Determine the Michaelis-Menten kinetics of an enzyme and its ligand.
D) Assess the interaction of a small enzyme and its ligand in directional experimental evolution.