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PATH30003- Frontiers in Human Disease > Lecture 19- Pathogen genomics > Flashcards

Lecture 19- Pathogen genomics Flashcards

1
Q

how do clinical and public health microbiology labs help?

A 
characterise pathogens (what is it, what drugs can be used)
investigate transmission and sources of infection (where did it come from, how can we stop its spread)
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2
Q

what can whole genome sequencing be used for? why is this better?

A

used for microbial diagnostics and surveillance

  • identify pathogens
  • predict drug resistance and virulence phenotypes
  • investigate transmission and sources of infection

better-

  • faster than huge barrage of traditional techniques
  • gives us more information (understand how resistance is spreading and how pathogens are moving in population)
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3
Q

how do we get the sequence data? 2 ways

A

isolated organism e.g. bacterial culture

culture-free approaches

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4
Q

explain the bacterial culture method of getting sequence data

A 
take sample you're interested in
culture the organism on agar plate
extract DNA from purified culture
put in sequencing e.g. illumina 
- sequence data represents only the cultured organism
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5
Q

explain the culture free approach to extracting sequence data

A

take sample of interest
directly extra DNA from it and sequencing everything we have (eliminate culturing step)
- sequence data represents the population of organisms int he sample (not just the organism we want)

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6
Q

what is the drawback to using a culture free approach to getting sequence data?

A

might be tricky to work out which organism is causing the problem
- sequence data contains population of organisms including human, bacteria, viruses, fungi, parasites

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7
Q

what does the sequence data look like?

A

millions of short overlapping sequences

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8
Q

2 things we can do with the sequence data

A
  1. align the sequences to a reference
  2. de novo assembly (try to piece together original original DNA sequence)
    * use these methods for downstream analysis
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9
Q

how to identify pathogens?

A

by aligning sequences to reference or de novo assembly of sequences
e.g. if E coli has a larger coverage than another bacteria when aligned to reference, the pathogen in the sample is more likely to be E.coli

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10
Q

what does the method of aligning to reference only work for?

A

purified cultures

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11
Q

why are metagenomic samples complex?

A

sequence data represents the population of organisms in the sample

  • human, bacteria, viruses, fungi, parasites
  • pathogens but also harmless commensal microbes
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12
Q

examples of identifying pathogens with metagenomic sequencing?

A

get the sequence data and align to malaria parasite genome ?

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13
Q

what can phenotype tell us?

A

observable characteristics

  • morphology
  • pathotype (e.g. HUS)
  • resistance to a specific antibiotic (treatment failure likely)
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14
Q

what can genotype tell us?

A

genetic basis for phenotype

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15
Q

what are 2 ways bacteria can become resistant?

A
  1. spontaneous mutation (protein changes shape, drug can no longer bind to protein)
  2. acquisition of resistance genes (move around between bacterial strains and spread resistance)
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16
Q

genotype for HUS phenotype?

A

shiga toxin genes (carried by E coli)

17
Q

genotype for resistance to fluoroquniolones

A

mutations in the gyrA or parC genes- spontaneous

18
Q

genotype for resistance to chloramphenicol

A

chloramphenicol acetyltransferase gene- acquisition

19
Q

how can we detect resistant genotype?

A

through aligning data to reference or de novo assembly

20
Q

is genotype always a good predictor of phenotype?

A

no- not 100% concordant

21
Q

why might there be a mismatch between genotype and phenotype?

A
  • antimicrobial resistance genes are there but might not be expressed
  • resistance proteins are being inhibited
  • unknown resistance mechanisms
22
Q

how to use phylogenetics to track outbreaks?

A
  • take samples from subset of people
  • sequence DNA of organism we think is causing disease
  • align DNA, find variations (SNPs)
  • assume that all these samples descended from recent common ancestor
  • model DNA substitution (probability for the mutations, transversion and transition rates)
  • take information and infer phylogeny
23
Q

what can phylogeny tell us?

A

how the samples are related to each other

how the pathogen is being spread e.g. between hospitals

24
Q

what do the tips of a phylogenic model represent?

A

the samples

25
Q

what do internal nodes represent? what do the numbers mean?

A

hypothetical ancestor
numbers from 0-1
- high values, strong support (more confidence that the node is correct)

26
Q

what do the horizontal branches mean?

A

amount of genetic change between samples

  • long branch, more genetic change
  • shorter branch, less genetic change
27
Q

what do the vertical branches mean?

A

nothing- just help connect the samples

28
Q

what does the root represent?

A

hypothetical ancestor of all samples

29
Q

example of WGS comparisons of samples from patients in US, Aus and NZ plus from heater-cooler units

A

the isolates were grouped together

  • very short branches separate clinical and heater-cooler unit samples
  • suggests heater-cooler unit as source of global outbreak
30
Q

global data sharing via?

A

GenomeTrakr

31
Q

what was GenomeTrakr used for initially?

A

sharing genomes for species that cause food-borne disease

- public health data sharing initiative started by US food and drug administration

32
Q

positives of GenomeTrackr?

A

publicly available
share genome data from labs around the world
can see how genome is moving around the world

33
Q

limitation of GenomeTrackr?

A

need data storage space in database

34
Q

challenges for using WGS in terms of identifying pathogens?

A
  • culture based approaches still not fast enough (delay for diagnosing samples)
  • culture free metagenomic samples are complex and suffer from contamination (human DNA)
35
Q

challenges for using WGS in terms of detecting resistance?

A

genotype may not accurately reflect phenotypes, particularly if there is a novel resistance mechanism

36
Q

challenges for using WGS in terms of tracking outbreaks?

A
  • need to combine genomic data with epidemiological information (e.g. geographical info, spatial info, patient/equipment movements)
  • large-scale comparisons have high data storage and computational requirements
37
Q

challenges for WGS in terms of understanding results?

A

need specialist training to run analyses and interpret results

PATH30003- Frontiers in Human Disease (20 decks)

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