2. Functional Genetic Information and Sequence Alignment

Question 1

How can we determine gene expression?

Answer 1

ChIP-seq
Western Blot
Polysomes
Mass spectrometry
Northern Blot
Microarray
RT-qPCR
RNA sequencing

Question 2

Transcriptomics (definition)

Answer 2

Measurement of gene expression by NGS of entire transcriptomes

Question 3

How do we quantify expression in RNA-sequencing experiments?

Answer 3

RPKM: reads per kilobase of transcript per million mapped reads
FPKM: fragments per kilobase per million reads mapped
TPM: transcripts per million transcripts

Question 4

What is the difference between a read and a fragment?

Answer 4

A read is the result of a single-read sequencing; it is a part of the gene sequenced in the forward direction
A fragment is the result of a paired-read sequencing; it includes a forward read and a reverse read to yield a more accurate sequencing

Question 5

What is baseline expression?

Answer 5

Baseline refers to where the gene is usually expressed

Question 6

What tools can we use to determine baseline expression?

Answer 6

RefSeqGene
UniProt
Expression Atlas
The Human Protein Atlas
GTEx portal

Question 7

What is studied in differential expression?

Answer 7

We compare gene expression across two or more states (healthy vs disease)

Question 8

In what aspects are ontologies connected?

Answer 8

Molecular function
Cellular components
Biological processes

Question 9

Ontology (definition)

Answer 9

a set of concepts and categories in a subject area or domain that shows their properties and the relations between them

Question 10

What do we use ontologies for?

Answer 10

To find tendencies, pathways or cellular components common to multiple genes

Question 11

Databases for gene ontology (GO) enrichment analysis:

Answer 11

G:profiler
Geneontology
Enrichr

Question 12

What is a Kegg pathway?

Answer 12

Map representing our knowledge of the molecular interaction, reaction and relation networks for metabolism, genetics, environmental information, cellular processes, organismal systems, human diseases and drug development

Question 13

What is homology in genes?

Answer 13

Two sequences are said to be homologous if they have evolved from a common ancestor. There are no degrees of homology, it’s yes or no

Question 14

What are paralogous genes?

Answer 14

two factors that came from some kind of gene duplication from the same organism and have evolved in parallel inside the same organism

Question 15

What are orthologous genes?

Answer 15

One single gene, coming from an ancestor, evolving differently in different species (mouse vs human hemoglobin)

Question 16

What is identity between two sequences?

Answer 16

The number of identical positions divided by the total number

Question 17

What is similarity between two sequences?

Answer 17

The number of identical positions + the number of similar positions (only possible in amino acid sequences, never nucleotides) divided by the total number

Question 18

How to use dot plots for pairwise sequence alignments?

Answer 18

On one axis plot one gene and on the other axis the other gene. Every time there is an identity between the two, plot a dot. In the end, if there is a 100% identity, you will see a straight line with a slope = 1 (y = x)

Question 19

Downsides to the dot plot approach for pairwise alignment?

Answer 19

Relies on visual analysis
Cannot distinguish between gaps and mismatches

Question 20

What is an S score?

Answer 20

It represents how good an alignment in through a dynamic approach

Question 21

How is an S raw score calculated?

Answer 21

Sum of all identities + Sum of all mismatches - Total gap penalty

Question 22

What is the total gap penalty?

Answer 22

Go is the gap opening penalty ( = 3) for the first nucleotide of a gap
Ge is the gap extension penalty ( = 1) for all nucleotides after the first in a gap
Gt = Go + (Ge * total number of gaps)

Question 23

What is the Jukes Cantor assumption?

Answer 23

We assume that all nucleotides appear with the same frequency, and we assign scores to identities, mismatches and gaps according to that assumption (high positive value for identities, low negative value for mismatches).

Question 24

What is the difference between local and global alignments?

Answer 24

In global alignments, sequences are aligned as a whole so we are forced to see both entire sequences
In local alignments, we find a high scoring subsequence; we only see the aligned fragments

Question 25

What is the PAM matrix?

Answer 25

The PAM matrix is based on the frequency that any one amino acid changes into any other in proteins that are closely related (>85% identity)

Question 26

What is the BLOSUM matrix?

Answer 26

The BLOSUM matrix is based on the frequency that any one amino acid changes into any other in individual domains of proteins that are not closely related

Question 27

What is the difference between probability and odds?

Answer 27

Probability is a measure of how likely an event is to happen.
Odds are the a ratio between the probability of event “a” happening compared to the probability of event “b” happening

Question 28

What is a log-odd?

Answer 28

It is the logarithmic transformation of odds that allows us to add probabilities instead of having to multiply them

Question 29

When would you use PAM30 or BLOSUM90 instead of PAM250 or BLOSUM45?

Answer 29

If we are working with sequences that are highly similar to each other we use PAM30 and BLOSUM90 (more stringent matrices than PAM250 and BLOSUM45)

Question 30

What is the default matrix for protein scoring and why?

Answer 30

BLOSUM62 because it’s good at detecting the majority of weak protein similarities

Question 31

What is a phylogenetic tree?

Answer 31

Diagrammatic representation of the relationship among sequences

Question 32

What is the difference between a rooted and unrooted tree?

Answer 32

Rooted tress branch out from LUCA or another common ancestor
Unrooted trees shows relationship between genes without showing which is the original group (oldest sequence)

Question 33

What is UPGMA (Unweighted Pair Group Method with Arithmetic Mean)

Answer 33

It is the simplest distance-matrix method that employs sequential clustering to build a rooted phylogenetic tree

Question 34

What is an informative site in an alignment?

Answer 34

Positions in the sequence where a different nucleotide is present in at least two sequences

Question 35

When we can make several phylogenetic trees, what do we consider the most plausible one and why?

Answer 35

The most plausible one is the simplest one - the one that requires the fewest number of changes to explain the data in the alignment. This is because we assume nature goes in a non-wasting manner.