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Medical Microbiology > 7 WGS > Flashcards

7 WGS Flashcards

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Biology 101 flashcards USMLE® Step 1 flashcards
1
Q

what does the current paradigm used in diagnostic microbiology consist of

A
  • detection (or not) of the presence of a pathogen
  • identification
  • antibiotic susceptibility testing (and surveillance)
  • epidemiological typing (and surveillance)
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2
Q

what are the four main methods used in diagnostic microbiology

A
  • microscopy
  • culture
  • serology
  • molecular
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3
Q

how does most treatment start

A

empirically

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4
Q

what are the problems with methods in diagnostic microbiology

A
  • time consuming
  • expensive
  • labour-intensive
  • organism or sample-type specific
  • poor discrimination
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5
Q

why can diganostic microbiology be time-consuming

A

bacteria need time to grow

unusual organism – sent to reference lab

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6
Q

Genome

A

complete set of genes or genetic material present in cell or organism (DNA/RNA)
blueprint describing the characteristics of an organism

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7
Q

what does the genome information provide

A

record of ancestry revealing genetic relationships between members of the same species or more distant ones

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8
Q

Central dogma

A

information flow – DNA genes transcribed to mRNA then translated into protein

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9
Q

what are the genes like in bacterial genome

A

> Short intergenic regions
Little repeat / non-coding DNA
Introns are v. rare

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10
Q

how are genes organised in bacterial genome

A

operons

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11
Q

Bacterial genome organisation - Chromosome

A

Typically - a single circular chromosome (always DNA) which is negatively supercoiled in order to pack it in

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12
Q

what can chromosome of bacterial genome be like

A

Occasionally linear
Occasionally two unique circular
Can even be multiple and a mix of circular and linear

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13
Q

linear chromosome examples

A

Streptomyces

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14
Q

two unique circular chromosome example

A

Vibrio cholerae

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15
Q

multiple and a mix of circular and linear chromosome example

A

Agrobacterium tumefaciens

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16
Q

Plasmid replication

A

Independent autonomously replicating DNA molecule

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17
Q

what do plasmids encode for

A

‘non-essential’ genes, but relate to virulence (e.g. tetanus toxoid encoded by plasmid) or adaptive advantage

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18
Q

sequencing method example

A

sanger sequencing

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19
Q

sanger invention

A

Sanger came up with chain terminator – so DNA polymerase cannot add in next nucleotide – irreversibly stops DNA replication

20
Q

what is used in the sanger reaction

A

Labelled primer annealed to template DNA
Primer (radio labelled) extended by DNA polymerase until a ddNTP is incorporated terminating chain extension
Fragments separated by size on an electrophoresis gel and image collected via labelled primers
1 bp resolution

21
Q

Sanger sequencing improvements

A

Automated Sanger sequencing:
Fluorescent labels to replace radio-isotopes
Capillary gel electrophoresis

22
Q

Sequencing methods: Whole-Genome Shotgun Sanger Sequencing

A

Take the purified DNA
Random shearing
Selected certain size fragment
Clone into plasmid and transform into E. coli cells
Each E. coli has a different potion of the bacteria plasmid
Need thousands of colonies to cover all the bacterial plasmid
Plasmid prep
Sequence ‘insert’ in each clone using primers on either side
QC, assembly and annotation

23
Q

Sequence assembly

A

Assembly = reads are merged into a longer DNA sequence in the attempt to reconstruct the original DNA sequence

24
Q

what does sequencing generate

A

millions of reads

25
Q

Whole-Genome Shotgun Sanger Sequencing – Problems!!

A
  • Onerous colony picking and plasmid preparation
  • Expensive
  • Amplified templates generated in vivo, so genes toxic to E. coli not represented in sequence libraries
26
Q

Sequencing methods: High-throughput sequencing

A

platforms allow many millions of target DNA molecules to be sequenced simultaneously, resulting in substantial reductions in cost and in the time taken to produce a whole genome sequence

27
Q

what is amplified in high-throughput screening

A

amplification of individual clonal templates

based on chemistry not biology – DNA polymerase does the ‘work’ rather than E. coli

28
Q

High Throughput Sequencing – Problems!!

A
  • smaller read length
  • computational costs
  • cannot achieve accuracy in long repeat assembly
  • difficult to obtain finished genome
29
Q

Nanopore sequencing

A

One forms pore in membrane other is a DNA unwinding enzyme
Add DNA in and starts passing it through pore
As each base pair goes through form current

30
Q

what are the nanopores

A

MinION

GridION

31
Q

read-length of GridION

A

No limit to read-length, but typically 100s kb

32
Q

Single molecule sequencing - Problems!!

A
  • Higher error rate, but makes assembly much easier
33
Q

how are bacterial genomes generated

A

using a mix of ‘high- throughput’ sequencing (for accuracy) and ‘single molecule’ re- sequencing (to help assembly), but as accuracy improves single molecule sequencing will become the norm

34
Q

what is whole genome sequencing

A

the process of determining the complete sequence of an organism’s genome

35
Q

What clinicians need to know asap

A
  1. What organism is causing the infection?
  2. What drugs can be used to treat it?
  3. How is it related to other similar infections?
36
Q

what does WGS provide in detecting organism causing infection

A

WGS can provide (high resolution) identification of bacterial pathogen

37
Q

why is WGS not used to identify organisms

A

Still need to grow and isolate bacteria (24 h or longer)

38
Q

what drugs can be used to treat - WGD and detecting resistance

A

WGS can detect the presence of resistance genes & mutations

39
Q

what is necessary if using WGS for resistance detection

A

you need to know what you are looking for

40
Q

why is WGS not used for resistance detection

A
  • genetic basis of many resistance mechanisms is not known

- Need comprehensive and validated databases of clinically relevant genotype- phenotype correlations – not available yet

41
Q

what can WGS provide in infection detection

A

WGS can provide (v. high resolution) typing of a bacterial pathogen

42
Q

cons of determining how related it is to other similar infections

A

Unlikely to improve outcomes for those patients in real-time

43
Q

Whole genome sequencing: advantages over current methods

A
  • can be used for all bacteria due to ‘generic’ genome nature
  • better discriminate between pathogens, enable outbreaks to be resolved/ruled out quicker
  • provide description of wide range of clinically + epidemiological characteristics in one test
44
Q

Whole genome sequencing: potential problems

A

cost
time
data analysis - converting massive amount WGS yield

45
Q

benefits of WGS - working out how similar to related infection

A
  • determine the likely sources + transmission paths

- identify DNA sequences unique to outbreak strain - can be used to develop more rapid molecular assay

Medical Microbiology (13 decks)

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