Behr Lectures

Question 1

List 2 factors that changed how sequencing was done

Answer 1

1. cost of sequencing (dropped faster than cost of computing)
2. cost of computing

Question 2

How does the cost of NGS compare with old-gen sequencing?

Answer 2

Can sequence more bases per dollar with NGS

Question 3

Sequencing is not useful unless it is paired with what process?

Answer 3

Bioinformatics

Question 4

List 2 goals of sequencing in bacterial pathology

Answer 4

1. Understand what makes a certain pathogen virulent

2. Determine the differences bw bacteria

Question 5

Molecular epidemiology methods such as RFLP, RAPD, YATM have all been replaced by what?

Answer 5

Whole genome sequencing

Question 6

List 3 advantages of WGS

Answer 6

1. Less error-prone
2. Better resolution
3. Democratizes specific methods used to study pathogens (no more boutique labs)

Question 7

What does performing genomics mean?

Answer 7

studying ALL (yes, EVERY SINGLE GENE) in an organism

Question 8

Given an example of old gen sequencing

Answer 8

Sanger sequencing

Question 9

What are the adaptors used in Illumina short-read sequencing used for?

Answer 9

Used as the complementary sequence that primers bind to to amplify a fragment. Reduces the need to find a different primer pair for every fragment (millions of fragments) present in tube.

Question 10

PacBio’s sequencing method is called:

Answer 10

SMRT = single molecule real-time sequencing

Question 11

Define opportunistic pathogens

Answer 11

Organism that CAN cause disease under the right conditions; pathology is NOT part of its life cycle

Question 12

Define professional pathogens

Answer 12

Pathology is part of its life cycle - necessary for it to survive as a species

Question 13

Define obligate intracellular pathogens

Answer 13

A microorganism that requires the host to grow; cannot grow outside the host

Question 14

Define host-associated pathogen

Answer 14

Depends on host for niche (Would not exist if humans were wiped out)

Question 15

What is the Mtb complex (MTC)?

Answer 15

All the mycobacteria spp in the Mycobacteria genus that can cause disease in different hosts

Question 16

T or F: all the species in the MTC have different 16S rRNA sequences. What is the significance?

Answer 16

F - means that they’re all the same species, but have adapted differently to cause disease in their specific host

Question 17

What is another term for carrier state of Tb infection?

Answer 17

Latent infection

Question 18

Are individuals with a latent tb infection infectious?

Answer 18

No

Question 19

Are Mtb replicating in a tuberculous infection?

Answer 19

No

Question 20

Which cells do Mtb infect?

Answer 20

Aveolar macrophages

Question 21

How many ORFs does the RD1 region have?

Answer 21

9

Question 22

Which method was used to discover the RD1 region?

Answer 22

Microarrays

Question 23

What does the RD1 region do?

Answer 23

• It is what makes Mtb so virulent

- the genes code for proteins that make up the T7SS

Question 24

Which operon is responsible for intracellular spread of Mtb?

Answer 24

RD1

Question 25

If RD1 is knocked out in Mtb, would you expect the bacteria to be found in phagosomes or cytosol?

Answer 25

Phagosomes

Question 26

Is RD1 a characteristic of a human-associated pathogen? Why/why not?

Answer 26

No, it has also been found in an environmental bacteria (M. kansasii)

Question 27

What is 1-TbAd and what does it do?

Answer 27

An antacid that helps Mtb survive acidic conditions in lysosomes. Therefore, it is a virulence factor

Question 28

What does the ability to cause disease rely on? (2)

Answer 28

1. ability to establish infection

2. ability to persist in host

Question 29

What are the advantages of Illumina? (3)

Answer 29

Sequence large quantities
High accuracy
Relatively low cost

Question 30

What are the disadvantages of Illumina? (3)

Answer 30

Short reads only
Can’t assemble a complete genome de novo
Cannot resolve repetitive elements

Question 31

Describe the PacBio SMRT method of sequencing

Answer 31

DNA is circularized and an enzyme walks around it to replicate it. A resolvase enzyme then opens up the dsDNA and sequences it 1 bp at a time

Question 32

What are the advantages of SMRT (PacBio) sequencing?

Answer 32

Can sequence long reads
Can be used for genomic epidemiology
Can be used for bacterial pathogenesis

Question 33

What are 2 disadvantages of SMRT (PacBio) sequencing?

Answer 33

Higher error rate than Illumina

Higher cost