Lesson 2

Question 1

What is the benefit of Oxford nanopore pacific biosciences when it comes to NGS?

Answer 1

They can produce long sequences→allows the entire sequence to be seen without worrying where the exons are and can allow for telomeric sequencing

Question 2

What are the disadvantages of pacific biosciences and Oxford nanopore?

Answer 2

Error rate is high, they have a lower throughput and they require high-quality RNA

Question 3

What specifically do illumina and microarrays focus on?

Answer 3

The poly-a structure of RNAs → huge enrichment of 3’ sequencing and less at the 5’ end

Question 4

In which scenario is having a single-handed sequence better?

Answer 4

When looking for differential expression

Question 5

What are the 2 types of replicates?

Answer 5

Biological and technical

Question 6

Describe biological replicates:

Answer 6

Parallel measurements of biologically distinct samples

Question 7

Describe technical replicates:

Answer 7

Repeated measurements of the same sample

Question 8

What are necessary to implement in order to avoid introducing bias?

Answer 8

Standard operational procedures

Question 9

What is a bias?

Answer 9

A systematic difference introduced to a study that alters the course of the results

Question 10

Describe the procedure for the creation of an array:

Answer 10

Used from a single batch and processed by one technician on the same day

Question 11

If large experiments are performed, how should the experimental procedures be arranged?

Answer 11

Orthogonalize every variable: analyze equal numbers of samples from 2 groups under assessment on each day of analysis

Question 12

What is normalization?

Answer 12

The removal of background and non-biological variation as much as possible

Question 13

What are the 2 key assumptions that normalization methods rely on?

Answer 13

1) the expression levels of most genes remain the same across replicate groups
2) different sample groups do not exhibit a meaningful difference in overall mRNA levels

Question 14

What is a spike-in?

Answer 14

Designed to bind to DNA molecules with a matching sequence known as a control probe→ a known quantity of RNA spike-ins is mixed with the sample and the degree of hybridization between the spike-ins and the central probes is used to normalize the hybridization measurements of the sample RNA

Question 15

Why is filtering performed when doing rna-seq experiments?

Answer 15

Reduces the impact of multiple testing ( multiple little t-tests introduces many false positives)

Question 16

What are the 2 steps of the filtering process?

Answer 16

1) identify and remove a set of genes that seems to generate uninformative signal
2) to do statistical testing only to genes which pass the filter

Question 17

What is the good of filtering?

Answer 17

To let the true differential expression emerge and get rid of the non-informative reads

Question 18

What are the 2 criteria of the initial step in filtering?

Answer 18

1) genes that are not expressed in any of the conditions of the experiment or those reporters on the array lack sensitivity to detect their expression
2) genes that are expressed and detectable but not differentially expressed in ALL conditions

Question 19

Describe the 1st step in filtering:

Answer 19

Do not compare samples (tumor vs healthy)→ take all samples without label: simply remove genes with low expression that are not differentially expressed on ALL conditions