Behr Lectures Flashcards
List 2 factors that changed how sequencing was done
- cost of sequencing (dropped faster than cost of computing)
- cost of computing
How does the cost of NGS compare with old-gen sequencing?
Can sequence more bases per dollar with NGS
Sequencing is not useful unless it is paired with what process?
Bioinformatics
List 2 goals of sequencing in bacterial pathology
- Understand what makes a certain pathogen virulent
2. Determine the differences bw bacteria
Molecular epidemiology methods such as RFLP, RAPD, YATM have all been replaced by what?
Whole genome sequencing
List 3 advantages of WGS
- Less error-prone
- Better resolution
- Democratizes specific methods used to study pathogens (no more boutique labs)
What does performing genomics mean?
studying ALL (yes, EVERY SINGLE GENE) in an organism
Given an example of old gen sequencing
Sanger sequencing
What are the adaptors used in Illumina short-read sequencing used for?
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.
PacBio’s sequencing method is called:
SMRT = single molecule real-time sequencing
Define opportunistic pathogens
Organism that CAN cause disease under the right conditions; pathology is NOT part of its life cycle
Define professional pathogens
Pathology is part of its life cycle - necessary for it to survive as a species
Define obligate intracellular pathogens
A microorganism that requires the host to grow; cannot grow outside the host
Define host-associated pathogen
Depends on host for niche (Would not exist if humans were wiped out)
What is the Mtb complex (MTC)?
All the mycobacteria spp in the Mycobacteria genus that can cause disease in different hosts
T or F: all the species in the MTC have different 16S rRNA sequences. What is the significance?
F - means that they’re all the same species, but have adapted differently to cause disease in their specific host
What is another term for carrier state of Tb infection?
Latent infection
Are individuals with a latent tb infection infectious?
No
Are Mtb replicating in a tuberculous infection?
No
Which cells do Mtb infect?
Aveolar macrophages
How many ORFs does the RD1 region have?
9
Which method was used to discover the RD1 region?
Microarrays
What does the RD1 region do?
- It is what makes Mtb so virulent
- the genes code for proteins that make up the T7SS
Which operon is responsible for intracellular spread of Mtb?
RD1
If RD1 is knocked out in Mtb, would you expect the bacteria to be found in phagosomes or cytosol?
Phagosomes
Is RD1 a characteristic of a human-associated pathogen? Why/why not?
No, it has also been found in an environmental bacteria (M. kansasii)
What is 1-TbAd and what does it do?
An antacid that helps Mtb survive acidic conditions in lysosomes. Therefore, it is a virulence factor
What does the ability to cause disease rely on? (2)
- ability to establish infection
2. ability to persist in host
What are the advantages of Illumina? (3)
Sequence large quantities
High accuracy
Relatively low cost
What are the disadvantages of Illumina? (3)
Short reads only
Can’t assemble a complete genome de novo
Cannot resolve repetitive elements
Describe the PacBio SMRT method of sequencing
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
What are the advantages of SMRT (PacBio) sequencing?
Can sequence long reads
Can be used for genomic epidemiology
Can be used for bacterial pathogenesis
What are 2 disadvantages of SMRT (PacBio) sequencing?
Higher error rate than Illumina
Higher cost
